Method for separating and / or isolating exosomes

Abstract

The present invention relates to a method for separating and / or isolating exosomes from a sample, the method comprising the steps of i) providing a sample that is known to comprise or suspected of comprising exosomes; ii) contacting a first binding agent with the sample and allowing the binding agent to bind to exosomes, wherein the first binding agent comprises a) cinnamycin and fluoresceine isothiocyanate (FITC), wherein the FITC is covalently linked to cinnamycin, or b) a protein-tag linked to cinnamycin via a peptide linker, wherein the peptide linker comprises one or more proteolytic cleavage sites; iii) separating and / or isolating complexes of the first binding agent and exosomes from the sample. The present invention further relates to a binding agent to be used in the method and to kits comprising the binding agent and instructions for binding of said binding agent to exosomes in a sample.

Description

[0001] Therawis Diagnostics GmbH

[0002] 948-7 PCT

[0003] METHOD FOR SEPARATING AND / OR ISOLATING EXOSOMES

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to the field of exosomes and in particular to a method for separating or isolating or separating and isolating exosomes in a sample using a binding agent comprising cinnamycin. The present invention further pertains to kits comprising the binding agent and to the binding agent itself.

[0006] BACKGROUND OF THE INVENTION

[0007] Exosomes have first been reported in 1983 when culturing immature red blood cells with labeled transferrin receptors to trace the movement of the transferrin receptors from plasma membranes into the reticulocytes. It was observed that the labeled transferrin receptors were internalized within the reticulocytes, and then repackaged into small vesicles inside them (Harding 1983; Pan 1983). These vesicles were later termed “exosomes” (Johnstone 1989).

[0008] Exosomes belong to a large family of membrane vesicles referred to as extracellular vesicles (EVs), which generally include microvesicles (approx. 100 - 1,000 nm in diameter), apoptotic blebs (approx. 500 - 1,000 nm in diameter), and exosomes (approx. 30 - 150 nm in diameter) (Li 2017). Exosomes are thus the smallest type of extracellular microvesicles and are produced in inward budding multivesicular bodies (MVB) resulting in intra-luminal vesicles (ILV). If an MVB fuses with the cell surface (the plasma membrane), these ILVs are released as exosomes by exocytosis into the extracellular media. 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 which has an average thickness of about 5 nm. The lipid components of exosomes include ceramide, cholesterol, sphingolipids, and phosphoglycerides with long and saturated fatty-acyl chains. The outer surface of exosomes is rich in saccharide chains, such as mannose, polylactosamine, alpha-2,6 sialic acid, and N-linked glycans (summarized in Li 2017).

[0009] In addition to performing many biological functions, particularly cell-cell communication, cumulative evidence has suggested that several biological entities in exosomes like proteins and microRNAs are closely associated with the pathogenesis of most human malignancies (Li 2017). As stated above, exosomes are generated in various compartments of the cell, e.g. endosomes and Golgi apparatus. The different processes leading to the generation of exosomes are regulated by Rab GTPases. Among these GTPases, Rab4 seems to be involved in the recycling of exosomes from early endosomes, and Rab 11 regulates the slow transport from perinuclear recycling endosome compartment. Rab 11 is further proposed to regulate the transport of microvesicle endosomes to the plasma membrane and thus to exosome release (Blanc and Vidal 2018). It has been shown that overexpression of Rab 11 stimulates the exosome release in K562 cells, and the inhibition of Rab11 function decreases exosome release (Savina 1997).

[0010] Secretion of exosomes occurs from normal (thrombocytes, immune cells, etc.) and tumor cells. They can carry DNA, RNA, microRNA, proteins, and lipids. Exosome-associated proteins used as biomarkers today include tetraspanins (CD9, CD63, CD81), immune regulation molecules (HLA-G, MHCI / II), membrane transport and fusion proteins (Rab5). The molecular composition of exosomes is assumed to reflect the (patho-) physiological changes in their cell or tissue of origin (Jia 2017).

[0011] Viruses enter cells through the endocytic pathway. Viruses that enter through endocytosis can hijack and use exosomal pathways for their own benefit. Exosomes have several characteristics that are like some viruses. These characteristics include biogenesis, uptake by cells, and intercellular transfer of functional RNAs, mRNAs, and proteins. Virus-infected cells have been shown to secrete exosomes that vary from their viral counterparts but may comprise of viral RNAs and viral proteins (Crenshaw 2018). Such exosomes are referred to as “virosomes”. Thus, identification of exosomes released from cells upon viral infection (virosomes), and identification of viral proteins comprised in said virosomes will allow diagnosing virus infection with high sensitivity and independent of DNA or RNA analysis.

[0012] However, the separation, isolation, enrichment and detection of exosomes has proven to be complicated (van der Pol 2012; Thind 2016; Jia 2016). Due to the complexity of body fluids, physical separation of exosomes from cells and similar-sized particles turned out to be challenging. Methods have been applied to isolate exosomes such as immune-capturing with antibodies, ultracentrifugation, and precipitation with PEG6000, water deprivation etc. The isolation of exosomes using differential ultracentrifugation was found to result in co-isolation of proteins and other contaminants and incomplete separation of vesicles from lipoproteins. Combining ultracentrifugation with micro-filtration or a gradient was suggested to improve purity (Tauro 2012; van Deun 2014). Further, a single step isolation of extracellular vesicles by size-exclusion chromatography has been demonstrated to provide greater efficiency for recovering intact vesicles over centrifugation (Böing 2014), although a size-based technique alone will not be able to distinguish exosomes from other vesicle types. In addition, when applying these conventional methods, a mixed population of exosomes of different intracellular origin, further vesicles and lipoproteins are generally co-purified.

[0013] Aggregation of EVs through antibodies directed against certain marker molecules has been employed to study the phenotype of these particles by flow cytometry, or alternatively ultracentrifugation followed by immunoblot analysis (Willms 2016). However, such bulk analysis did not allow the study of individual EVs (Willms 2016), and thus, the information conveyed through EVs, e.g. mirroring the state of the donor cell, becomes lost. This has severely hampered the ability to study the use of EVs in detection of defined changes within cells, for example after a viral infection or cancerous disease.

[0014] 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.

[0015] WO 2019 / 099955 discloses a method for isolating exosomes from a cultivated placenta using affinity chromatography with binding agents against inter alia a Rab family GTPase.

[0016] WO 2021 / 209622 discloses a method for enriching exosomes, which method targets the extra-vesicular part of Rab4 and / or Rab 11 on the exosomal membrane for identifying and isolating exosomes.

[0017] WO 2024 / 223711 discloses a method for detecting and quantifying exosomes using a binding agent comprising a label and a phosphatidylethanolamine-binding lantibiotic, which binding agent specifically binds to phosphatidylethanolamine in the membrane of exosomes.

[0018] However, albeit progress has been made in the field, the remains a need for improved methods for separating and / or isolating exosomes from different types of samples.

[0019] SUMMARY OF THE INVENTION

[0020] In a first aspect, the present invention provides a method for separating and / or isolating exosomes from a sample, the method comprising the steps of:

[0021] i) providing a sample that is known to comprise or suspected of comprising exosomes; ii) contacting a first binding agent with the sample and allowing the binding agent to bind to exosomes, wherein the first binding agent comprises

[0022] a) cinnamycin and fluoresceine isothiocyanate (FITC), wherein the FITC is covalently linked to cinnamycin, or

[0023] b) a protein-tag linked to cinnamycin via a peptide linker, wherein the peptide linker comprises one or more proteolytic cleavage sites;

[0024] iii) separating and / or isolating complexes of the first binding agent and exosomes from the sample.

[0025] According to one embodiment, the peptide-linker comprises a TEV protease cleavage site. According to a preferred embodiment, the peptide-linker comprises a TEV protease cleavage site and a trypsin cleavage site.

[0026] According to one embodiment, a second binding agent is bound to the protein-tag or to the FITC of the first binding agent. According to a preferred embodiment, the second binding agent comprises a label. According to a further preferred embodiment, the label of the second binding agent comprises a magnetic bead.

[0027] According to an alternative embodiment, the method further comprises after step ii) a step of adding a second binding agent to the sample, wherein the second binding agent is capable of binding to the protein-tag or the FITC of the first binding agent. According to a preferred embodiment, the second binding agent comprises a label. According to a further preferred embodiment, the second binding agent comprises a magnetic bead.

[0028] According to one embodiment, the second binding agent comprises an antibody or antigen binding fragment thereof that specifically binds to the protein-tag or the FITC of the first binding agent.

[0029] According to one embodiment, the method further comprises after step iii) a step of releasing the complexes of the first binding agent and exosomes from the second binding agent. According to a preferred embodiment, the step of releasing the complexes of the first binding agent and exosomes from the second binding agent comprises proteolytically cleaving the complexes comprising the first binding agent and exosomes, preferably with trypsin.

[0030] According to yet another embodiment, the method further comprises adding a positive control, wherein the positive control comprises liposomes. According to a preferred embodiment, the liposomes comprise phosphatidylethanolamine and a proteolytically-cleavable peptide. According to a further embodiment, the method further comprises adding a negative control, wherein the negative control comprises liposomes comprising a trypsin-cleavable peptide that is different from the trypsin-cleavable peptide of the positive control, and wherein the liposomes does not comprise phosphatidylethanolamine.

[0031] 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. According to a preferred embodiment, the body fluid is selected from the group consisting of plasma, serum, ascites, cerebral fluid, bone marrow, urine, faeces and bronco-alveolar washing.

[0032] According to one embodiment, the sample is obtained from a subject known or suspected to suffer from a disease. According to a preferred embodiment, the disease is a virus disease, bacterial disease, autoimmune disease, inflammatory disease or cancer.

[0033] According to a further embodiment, the first binding agent has the structure:

[0034] i) FITC - cinnamycin,

[0035] ii) Strep-tag - peptide linker - cinnamycin,

[0036] iii) His-tag - peptide linker - cinnamycin, or

[0037] iv) Biotin - peptide linker - cinnamycin.

[0038] According to a preferred embodiment, the peptide linker comprises the sequence of SEQ ID NO: 2 or the sequence of SEQ ID NO: 7, most preferably the sequence of SEQ ID NO: 7.

[0039] According to another aspect, the present invention provides a binding agent comprising fluoresceine directly covalently coupled to the N-terminus of cinnamycin.

[0040] According to yet another aspect, the present invention provides a kit comprising: i) a first binding agent, wherein the first binding agent comprises

[0041] a) cinnamycin and fluoresceine isothiocyanate (FITC), wherein the FITC is covalently linked to cinnamycin, or

[0042] b) a protein-tag linked to cinnamycin via a peptide linker, wherein the peptide linker comprises one or more proteolytic cleavage sites; and ii) instructions for binding of said first binding agent to exosomes in a sample. According to one embodiment, the kit further comprises a second binding agent capable of binding to or bound to the first binding agent.

[0043] According to another embodiment, the second binding agent comprises a label. According to a preferred embodiment, the second binding agent comprises a magnetic bead.

[0044] Further aspects and embodiments of the present invention are derivable from the following detailed description and the examples. FIGURES

[0045] Figure 1: Schematic drawing of the FITC-cinnamycin binding agent; isothiocyanate is directly linked to N-terminus (Pos1) of cinnamycin.

[0046] Figure 2: A) Flow cytometry analysis of streptavidin signal of Strep-tag-peptide linker-cinnamycin construct incubated with exosomes in cell culture supernatant of breast cancer cell line SKBR-3 and streptavidin-atto488 (grey dots, SKBR-3). Controls: incubation with NaCl solution, NaCl+Triton and SKBR-3+Triton gave no signal. The same analysis by omitting streptavidin (white dots) or omitting Cinnamycin (black dots) gave no results. B) Concentration-dependent binding of FITC-cinnamycin to liposomes.

[0047] Figure 3: Concentration-dependent binding of FITC-cinnamycin to liposomes containing PE and packed with protein X.

[0048] Figure 4: Peptide X encapsulated in liposomes is detected in a concentration dependent manner by mass spectrometry.

[0049] Figure 5: Flow cytometry results of FITC-Cinnamycin construct and anti-FITC antibody beads use for purifying exosomes from cell culture supernatant.

[0050] Figure 6: Mass spectrometry analysis of exosome enrichment using capturing with cinnamycin-peptide (TEV cleavage site and Biotin binding moiety) added to Streptavidin coated microtiter plates, subsequent washing and release of cinnamycin bound exosomes by TEV protease cleavage versus the corresponding plasma. X-axis shows proteins which have been identified only in plasma, y-axis shows proteins found only in the enriched samples using the method of the invention. The diagonal shows proteins which are detected in both, plasma and the samples prepared by the method of the invention.

[0051] Figure 7: Mass spectrometry analysis of exosome enrichment using the method of the invention and identification of the non-human, non-animal-derived peptide exclusively detected in samples prepared using a protease-containing release buffer. The following samples were analyzed: Plasma+CIN. A: plasma incubated with the binding agent without subsequent washing; NaCl. B: workflow performed using NaCl solution instead of plasma; Plasma 1-4. B: plasma samples without first binding agent and without release buffer; Plasma+CIN. B: plasma incubated with binding agent but without TEV protease-containing release buffer; Plasma+CIN+TEV_B: samples obtained using the method of the invention; Plasma Control: untreated (neat) plasma. SEQUENCES

[0052] SEQ ID NO: 1 (cinnamycin):

[0053] ARQAAAFGPFXFVADGNXL, wherein X denotes a-aminobutyric acid

[0054] SEQ ID NO: 2 (trypsin-cleavable peptide linker):

[0055] AGGRAVNEALRGGAK

[0056] SEQ ID NO: 3 (strep-tag):

[0057] WMHPQFER

[0058] SEQ ID NO: 4 (strep-tag II):

[0059] WSHPQFEK

[0060] SEQ ID NO: 5 (positive control):

[0061] DTDILAAFRTFQGPPHGIQVERDDENVNSQPFMRSQAETGEIK

[0062] SEQ ID NO: 6 (negative control):

[0063] DNGLLLHIHRNHGMHFRESTLGFVDLLR

[0064] SEQ ID NO: 7 (TEV protease and trypsin protease cleavable peptide linker): AGGGSGGGENLYFQGAVNEALR

[0065] SEQ ID NO: 8 (TEV protease cleavage site):

[0066] ENLYFQ G

[0067] SEQ ID NO: 9 (HRV 3C protease cleavage site):

[0068] LEVLFQ GP

[0069] SEQ ID NO: 10 (thrombin cleavage site):

[0070] LVPR GS

[0071] SEQ ID NO: 11 (Factor Xa cleavage site):

[0072] lEGFQ SEQ ID NO: 12 (enterokinase cleavage site):

[0073] DDDD K

[0074] SEQ ID NO: 13: (non-human, non-animal peptide):

[0075] AVNEALR

[0076] DETAILED DESCRIPTION OF THE INVENTION

[0077] Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may 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 invention 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.

[0078] Preferably, the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)” (Leuenberger 1995).

[0079] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0080] In the following, the elements of the present invention will be described. These elements are listed with specific embodiments; however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments 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 disclosed by the description of the present application unless the context indicates otherwise.

[0081] The practice of the present invention will employ, unless otherwise indicated, conventional methods of biochemistry, cell biology, and immunology techniques which are explained in the literature in the field (c, e.g., Molecular Cloning: A Laboratory Manual, 2ndEdition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0082] 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 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 although in some embodiments such other member, integer or step or group of members, integers or steps may be excluded, i.e. the subject-matter consists in the inclusion of a stated member, integer or step or group of members, integers or steps.

[0083] The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0084] In the following, some definitions of terms frequently used in this specification are provided. These terms will, in each instance of its use, in the remainder of the specification have the respectively defined meaning and preferred meanings.

[0085] The term "subject" as used herein refers to an individual, such as a human, a non-human primate (e.g. chimpanzees and other apes and monkey species); farm animals, such as birds, fish, cattle, sheep, pigs, goats and horses; domestic mammals, such as dogs and cats; laboratory animals including rodents, such as mice, rats and guinea pigs. The term does not denote 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 suffering from or suspected of having one or more diseases. A subject suffering from or suspected of having one or more diseases is also referred to as a patient.

[0086] The term "sample" as used herein refers to biological material obtained from a subject. A sample can be obtained from any suitable tissue or biological fluid such as nipple aspirate, blood, serum, plasma, ascites, cerebral fluid, bone marrow, urine, faeces or bronco-alveolar washing. A 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 the group consisting of blood, such as full blood, (blood) plasma, serum, ascites, cerebral fluid, bone marrow, urine, faeces and bronco-alveolar washing. A particularly preferred sample is blood or plasma. Preferably, said sample is provided in a state selected from the group consisting of natural, frozen, lyophilized, preserved, embedded, and all possible combinations thereof. Methods for obtaining samples from a subject are well known to those skilled in the art. The sample can be pre-treated by methods known in the art for bringing the sample into a state that allows carrying out the steps of the methods of the present invention. For example, if the sample is in a solid, semi-solid, or essentially solid or semi-solid state, the sample can be suspended and / or solubilized. The sample can be further pre-treated by removing e.g. cellular debris and larger cellular components. Such methods include but are not limited to centrifugation techniques, particularly ultrafiltration. According to one embodiment, the sample is centrifuged at around 10,000 to 15,000 g, preferably about 12,000 g for separating larger solid particles such as cellular debris and maintaining EVs in the supernatant. The sample after pre-treatment and before applying the methods of the present invention preferably does not comprise microvesicles larger than 1,000 nm in diameter, more preferably not larger than 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 150 nm in diameter. Most preferably, the sample after pre-treatment and before applying the methods of the present invention does not comprise microvesicles having a diameter of more than 100 nm. It is to be understood that the diameter of such microvesicles refers to the average diameter of a plurality or a population of such microvesicles. The sample can be further pre-treated by enriching the exosome concentration in the sample. Suitable methods include size- and / or densitycentrifugation. These methods are well known to the person of ordinary skill in the art. However, such pre-treatment and / or enriching in exosomes is not necessary and the methods of the invention can be performed directly on the sample. Thus, according to one embodiment of the invention, the method does not comprise any purification step of the exosomes contained in the sample in advance to the claimed method, i.e. no pre-purification, such as by ultracentrifugation, density centrifugation, fractionation and / or the use of markers for microvesicles or exosomes (e.g. Rab4 and / or Rab11), markers for tumors, or viral markers, that may bind to all or a subfraction of the exosomes contained in said sample. The method of the invention preferably also does not require any previous exosome enrichment using agents that bind to an exosome or to an exosome sub-population. According to one embodiment, the exclusion of a pre-purification step includes an exclusion of cellular lysis and / or removing of cellular debris in the sample. According to an alternative embodiment, the exclusion of a pre- purification step does not include an exclusion of cellular lysis and / or removing of cellular debris in the sample. In accordance with the present invention, the method can thus be directly performed on a respective sample, such as on plasma.

[0087] The present invention provides an innovative and optimized procedure that enables to separate and / or isolate single EVs and specifically exosomes from samples such as human blood plasma.

[0088] In the experiments leading to the present invention it was found that FITC can be directly covalently coupled to cinnamycin without an intervening linker between FITC and cinnamycin, and that this compound (also referred to as binding agent or first binding agent herein) can be used for reliably separating, isolating or separating and isolating a significantly large portion of exosomes in a sample, and not just a fraction or subpopulation thereof.

[0089] Therefore, the present invention provides a method for separating and / or isolating exosomes from a sample, the method comprising the steps of

[0090] i) providing a sample that is known to comprise or suspected of comprising exosomes;

[0091] ii) contacting a first binding agent with the sample and allowing the binding agent to bind to exosomes;

[0092] iii) separating and / or isolating complexes of the first binding agent and exosomes from the sample.

[0093] The sample is as defined above and is preferably selected from the group consisting of but not limited to a cell culture supernatant, a cell preparation obtained by lysing and / or centrifuging cells, or a body fluid. According to a preferred embodiment, the sample is a body fluid selected from the group consisting of but not limited to plasma, serum, ascites, cerebral fluid, bone marrow, urine, faeces and bronco-alveolar washing. The sample is preferably obtained from a subject known to or suspected of suffering from a disease. According to a preferred embodiment, the sample is obtained from a subject suffering from one or more of a virus disease, a bacterial disease, an autoimmune disease, an inflammatory disease or cancer.

[0094] According to the present invention, the sample is contacted with a first binding agent under conditions allowing the first binding agent to bind to exosomes contained in the sample. The conditions allowing the first binding agent to bind to exosomes contained in the sample can include incubating the first binding agent and the sample at room temperature. The duration of this incubation can extend between several minutes such as about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 minutes or more such as about 20, 25, 30, 40, 50 or 60 minutes to several hours. In the experiments leading to the present invention an incubation time of about 30 minutes at room temperature for the first binding agent on a plasma sample or on a cell culture supernatant sample provided satisfying yields of exosomes. However, the skilled person may readily determine other parameters more suitable for a binding of the first binding agent to exosomes contained in the sample. According to a preferred embodiment, the incubation of the first binding agent and the sample is performed at room temperature for about 30 minutes. The incubation may further include a movement of the sample to which the first binding agent has been added, such as an initial movement or mixing by inverting or vortexing the vessel containing the sample, or a continuous movement during the incubation time such as a movement on a shaker. A preferred protocol for allowing the first binding agent to bind to exosomes contained in the sample includes incubating the first binding agent with the sample for 30 minutes at room temperature on a shaker.

[0095] The first binding agent in accordance with the present invention comprises cinnamycin. The cinnamycin of the first binding agent is either covalently linked to fluoresceine isothiocyanate (FITC), or it is linked to a protein-tag via a peptide linker.

[0096] In the case of the FITC being covalently linked to cinnamycin, the FITC is directly linked or coupled to the N-terminus of cinnamycin via a covalent bond. Directly linked or coupled means that there are not intervening linkers or chemical structures between cinnamycin and FITC, that are not derived from FITC or cinnamycin. Without wishing to be bound by any theory, FITC reacts with free primary amine groups of proteins to form covalent bonds. The isothiocyanate (N=C=S) group is reactive towards nucleophilic sites, and FITC reacts with N-terminal amines or with the amino group of lysine. The primary amine attacks the electrophilic carbon of the isothiocyanate group, forming a thiourea bond connecting FITC and the protein. Since cinnamycin has only one N-terminus free amino group, Fluorescein is attached at the N-terminus (Position 1) of cinnamycin only. A schematic drawing of this direct covalent linkage or coupling is shown in Fig. 1, in which the carbon of the NCS group of isothiocyanate is directly linked or coupled to the N-terminal amine group of the first position amino acid of cinnamycin.

[0097] In the case of the cinnamycin being linked to a protein-tag via a linker, the linker is a peptide linker which comprises one or more proteolytic or protease cleavage sites, such as one or more trypsin cleavage sites and / or one or more TEV (tobacco etch virus) protease cleavage sites that allow later cleavage of the exosome-cinnamycin complex from the protein tag. Therefore, in accordance with the present invention, the peptide linker comprises one or more, preferably two cleavage sites. According to one embodiment, the peptide linker comprising two trypsin cleavage site has the amino acid sequence AGGR*AVNEALR*GGAK (SEQ ID NO: 2), wherein "*" denotes the trypsin cleavage site. According to a particularly preferred embodiment of the present invention, the peptide linker comprises a first proteolytic cleavage site, preferably a TEV protease cleavage site, and a second different proteolytic cleavage site, preferably a trypsin cleavage site. The second cleavage site is preferably C-terminal of the first cleavage site, which allows removal of large parts of the peptide linker from the exosome if a respective second protease that is active on the second cleavage site is allowed to cleave the peptide linker. The skilled person can choose other peptide linkers with other cleavage sites depending on the intended use in the context of the present invention. The present invention is therefore not limited to any specific type of peptide linker and / or a specific type of proteolytic cleavage site. Exemplary cleavage sites comprise but are not limited to TEV protease cleavage sites (preferably having the recognition sequence of SEQ ID NO: 8), trypsin cleavage sites (preferably cleaving after Lysine (K) or Arginine (R) residues in a peptide, see e.g. above with respect to SEQ ID NO: 2), HRV 3C protease cleavage sites (preferably having the recognition sequence of SEQ ID NO: 9), thrombin cleavage sites (preferably having the recognition sequence of SEQ ID NO: 10), Factor Xa cleavage sites (preferably having the recognition sequence of SEQ ID NO: 11), and enterokinase cleavage sites (preferably having the recognition sequence of SEQ ID NO: 12). A particularly preferred peptide linker comprises a TEV protease cleavage site and a trypsin cleavage site preferably C-terminally of the TEV protease cleavage site. Such a peptide linker preferably has the amino acid sequence as set forth in SEQ ID NO: 7.

[0098] If the exosomes separated and / or isolated by the method of the present invention shall be subsequently analyzed using e.g. mass spectrometry, linkers other than serine-glycine linkers are usually recommended since serine-glycine linkers are difficult to detect in mass spectrometry. The present invention therefore provides in one embodiment a linker having a peptide sequence that can be detected in mass spectrometry since it does not consist of glycine and serine residues only. The linker preferably comprises a section of amino acids that resemble a sequence that cannot be found in humans, preferably not in humans and other mammals, more preferably not in humans and in other animals. According to one embodiment, this section or sequence comprises between 5 and 10 amino acids, or between 6 and 9 amino acids such as 7 or 8 amino acids. According to a particularly preferred embodiment, this section or sequence comprises seven amino acids, most preferably the sequence as set forth in SEQ ID NO: 13. According to a preferred embodiment, the first binding agent has the structure of FITC-cinnamycin, or protein-tag - linker - cinnamycin.

[0099] The protein-tag can be any protein tag known to the skilled person and is not particularly limited. A preferred protein tag is a Biotin-tag, a strep-tag or a His-tag. The term " Biotin" as used herein generally refers to 5-[(3aS,4S,6aR)-2-Oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoic acid also known as vitamin B7 or vitamin H. The term "strep-tag" as used herein generally refers to protein tags known under the name strep-tag and includes strep-tag having a sequence as set forth in SEQ ID NO: 3, as well as strep-tag II having a sequence as set forth in SEQ ID NO: 4. A His-tag (also called hexahistidine tag or His 6-Tag) is well known to the skilled person and commonly includes six histidine residues. According to a preferred embodiment, the protein-tag is Biotin, strep-tag or strep-tag II. It will be appreciated that the skilled person can readily select other protein-tags known in the art and that the present invention is not limited to the specific protein-tags discussed herein.

[0100] According to one embodiment, the first binding agent has the structure of Biotin-linker-cinnamycin, strep-tag - linker - cinnamycin, or His-tag - linker - cinnamycin. According to one embodiment, the first binding agent has the structure of strep-tag II - peptide-linker -cinnamycin, more preferably of SEQ ID NO: 4 - SEQ ID NO: 2 - cinnamycin. According to an alternative preferred embodiment, the first binding agent has the structure of Biotin or strep-tag - linker - cinnamycin, more preferably of Biotin or strep-tag - SEQ ID NO: 7 - cinnamycin, such as Biotin - SEQ ID NO: 7 - cinnamycin, or SEQ ID NO: 3 - SEQ ID NO: 7 - cinnamycin, or SEQ ID NO: 4 - SEQ ID NO: 7 - cinnamycin. A particularly preferred emnbodiment of the first binding agent is Biotin - SEQ ID NO: 7 - cinnamycin.

[0101] In the peptide linker identified in SEQ ID NO: 7, the Ala in position 1 is preferably P-Alanin. The peptide linker identified in SEQ ID NO: 7 is preferably attached or coupled to the cinnamycin via an iso-thiocyanate group (e.g. as exemplarily shown in Fig. 1).

[0102] According to one embodiment, the exosomes are enriched in the sample prior to being contacted with the first binding agent e.g. based on size and / or density. Such enrichment of exosomes can be performed e.g. by density centrifugation or ultra-centrifugation using e.g. a Pancoll gradient (PAN-Biotech GmbH). Other suitable gradients include but are not limited to Ficoll and Ficoll-Paque (both GE Healthcare), and Biocoll (Biochrom GmbH). Methods of performing ultra-centrifugation are well known to the person of skill in the art and are disclosed for example in Li et al., 2017, which is herein incorporated by reference. The term "agent" as used herein denotes a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials. The term "binding agent" as used herein pertains to any agent capable of specifically and / or selectively binding to a specific biological structure.

[0103] When contacting the first binding agent with the sample under conditions allowing the first binding agent to bind to exosomes contained in the sample, complexes of the first binding agent and of exosomes in the sample will be build. These complexes can be subsequently separated or isolated or separated and isolated from the sample.

[0104] According to one embodiment, in the method of the present invention, a second binding agent is bound to the first binding agent before it is contacted with the sample in step ii). This approach will lead to the formation of a binding complex consisting of the first binding agent and the second binding agent, and the complex of both binding agents is brought into contact with the sample in step ii) of the method of the invention. The second binding agent preferably binds to the FITC of the first binding agent. The second binding agent can be, for example, an antigen binding agent such as an antibody or an antigen binding fragment of an antibody, binding to e.g. the FITC of the first binding agent.

[0105] According to a preferred embodiment, a second binding agent as described herein and which is capable of binding to the protein-tag or to the FITC of the first binding agent is bound to a substrate such as a solid support, e.g. a plate or a bead. This allows immobilizing the exosomes via the first binding agent to the substrate comprising the second binding agent.

[0106] The second binding agent preferably additionally comprises a label which can be selected from the group consisting of an enzymatic label, a fluorochrome, a magnetic label such as magnetic beads, and a peptide label as disclosed herein. According to a preferred embodiment, the label is a magnetic or paramagnetic bead, more preferably a magnetic bead. In these cases, the label of the second binding agent can also serve as a substrate or solid support.

[0107] In certain embodiments, the label is attached by one or more spacer arms of various lengths to reduce potential steric hindrance. Such spacers can be, for example, chemical spacers or amino acid spacers.

[0108] If the second binding agent is an antigen binding agent, it "specifically binds" a target antigen when the dissociation constant (Ka) is < 10-7M. The binding agent specifically binds its antigen with "high affinity" when the Ka is < 5 x 10-9M, and with "very high affinity" when the Ka is < 5 x 10-10M, preferably when the Ka is < 5 x 10-11M. If the binding agent is an antigen binding agent, it is "selective" when it binds to one target more tightly than it binds to a second target.

[0109] According to a preferred embodiment, the second binding agent is an anti-FITC antibody or antigen binding fragment thereof preferably further comprising a magnetic bead as label. According to a particularly preferred embodiment, the second binding agent binds to the protein-tag of the first binding agent. In case Biotin, strep-tag or strep-tag II is used as protein tag in the first binding agent, the second binding agent preferably comprises streptavidin or Strep-Tactin as binding partner.

[0110] According to an alternative embodiment of the present invention, the method further comprises after step ii) a step of adding a second binding agent to the sample, which second binding agent is capable of binding to the protein-tag or to the FITC of the first binding agent. According to one embodiment of the present invention, the sample comprising the first binding agent is contacted with the second binding agent under conditions allowing the second binding agent to bind to the protein-tag or to the FITC of the first binding agent contained in the sample. The conditions allowing the second binding agent to bind to the first biding agent contained in the sample include incubating the second binding agent and the sample at a temperature and for a duration as set forth above for the incubation of the first binding agent with the sample, however, the skilled person may readily determine other parameters more suitable for a binding of the second binding agent to the first binding agent. According to a preferred embodiment, the incubation of the second binding agent and the first binding agent in the sample is performed at room temperature for about 30 minutes. The incubation may further include a movement of the sample to which the second binding agent has been added, such as an initial movement or mixing by inverting or vortexing the vessel containing the sample, or a continuous movement during the incubation time such as a movement on a shaker. A preferred protocol for allowing the second binding agent to bind to the first binding agent contained in the sample includes incubating the second binding agent with the sample for 30 minutes at room temperature on a shaker.

[0111] Alternatively, the second binding agent as described herein can be immobilized or bound to a substrate such as a plate or a bead, or it can be immobilized on a surface such as the surface of a well or micro-well.

[0112] The second binding agent can be, for example, an antigen binding agent such as an antibody or an antigen binding fragment of an antibody, binding to e.g. the FITC or to the peptide-tag of the first binding agent. Alternatively, in case the protein-tag is Biotin or a strep- tag, the second binding agent can be streptavidin, Strep-Tactin or Strep-Tactin XT which binds to the strep-tag of the first binding agent with high affinity. If the protein-tag is a His-tag, the second binding agent may comprise Ni2+and / or Co2+ions for binding to the His-tag, preferably in a complex form such as in complex with nitrilotriacetic acid (NTA).

[0113] The second binding agent may additionally comprise a label which can be selected from the group consisting of an enzymatic label, a fluorochrome, a magnetic label such as magnetic beads, and a peptide label as disclosed herein. Examples of peptide labels include but are not limited to biotin and avi din / streptavidin and other labels such as His-tag, FLAG-tag and Strep-tag. Commonly used enzymatic labels, fluorochromes, and magnetic labels can be used in the context of the present invention without any specific limitation thereto. According to a particularly preferred embodiment, the label is a fluorochrome (also termed fluorophore). The term "fluorochrome" and likewise "fluorophore" as used herein refers to a non-protein fluorescent chemical compound that can re-emit light upon light excitation. The fluorochrome is preferably selected from the group consisting of but not limited to xanthene derivatives such as fluorescein and its derivatives, rhodamine, Oregon green, eosin, and Texas red; cyanine derivatives such as cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine; squaraine derivatives and ring-substituted squaraines, including Seta and Square dyes; squaraine rotaxane derivatives such as See Tau dyes; naphthalene derivatives; coumarin derivatives; oxadiazole derivatives such as pyridyl oxazole, nitrob enzoxadi azole and benzoxadiazole; anthracene derivatives such as anthraquinones, 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 proflavin, acridine orange, and acridine yellow; arylmethine derivatives such as auramine, crystal violet, and malachite green; tetrapyrrole derivatives such as porphin, phthalocyanine, and bilirubin; and dipyrromethene derivatives such as BODIPY, and aza-BODIPY. Preferred fluorochromes have a size of between 200 and 1,000 Dalton. A preferred fluorochrome is Atto 488. According to an alternatively preferred embodiment, the label is a magnetic or paramagnetic bead, more preferably a magnetic bead.

[0114] In certain embodiments, the label is attached by one or more spacer arms of various lengths to reduce potential steric hindrance. Such spacers can be, for example, chemical spacers or amino acid spacers.

[0115] If the second binding agent is an antigen binding agent, it "specifically binds" a target antigen when the dissociation constant (Ka) is < 10-7M. The binding agent specifically binds its antigen with "high affinity" when the Kd is < 5 x 10-9M, and with "very high affinity" when the Kd is < 5 x 10-10M, preferably when the Kd is < 5 x 10-11M.

[0116] If the binding agent is an antigen binding agent, it is "selective" when it binds to one target more tightly than it binds to a second target.

[0117] According to a preferred embodiment, the second binding agent is a) in the case of the first binding agent comprising no protein tag an anti-FITC antibody or antigen binding fragment thereof, b) in the case of the first binding agent comprising Biotin or a strep-tag, a streptavidin, Strep-Tactin or Streptactin XT molecule, or c) in the case of the first binding agent comprising a His-tag a molecule comprising one or more divalent cations such as Ni2+and / or Co2+, optionally in the form of a complex with nitrilotriacetic acid (NTA) such as Ni-NTA or Co-NTA. These second binding agents preferably further comprise a magnetic bead as label.

[0118] The conditions allowing binding of the first binding agent to the sample and / or the second binding agent to the first binding agent depend in particular on the type of binding agent(s) used and on the type of sample. The conditions can be easily determined by a person of ordinary skill in the art.

[0119] After the complexes of first binding agent, second binding agent and exosomes have been build, the exosomes can be separated and / or isolated via the label on the second binding agent. For example, if the label is a fluorescent label, the exosomes can be purified e.g. via a cell sorter such as a FACS. If the label is a magnetic or paramagnetic bead, the exosomes can be separated and / or isolated by applying a magnetic field. Further methods of isolating and / or separating molecules via an attached label are known to the skilled person. These isolation and / or separation steps can be accompanied by one or more washing steps, which are also known to the skilled person. If the complex of exosome, first binding agent and second binding agent is immobilized on a solid support (e.g. on the surface of a well or a plate), the support can be washed in one or more steps to remove residues of the sample.

[0120] According to one embodiment of the present invention, the method further comprises after step iii) a step of releasing the complexes of the first binding agent and exosomes from the second binding agent. In cases where the first binding agent comprises a proteolytically cleavable peptide linker, one preferred way of releasing the complexes consisting of the first binding agent and exosomes from the second binding agent is by attacking the linker e.g. by proteolytic cleavage of the peptide linker. A preferred enzyme for this purpose is TEV protease or trypsin. Thus, in cases where the linker is a peptide linker comprising one or more trypsin cleavage sites, the releasing of the complexes of the first binding agent and exosomes from the second binding agent is by subjecting the complex of first binding agent, second binding agent and exosomes to an enzymatic trypsin digest, resulting in cleavage of the peptide linker at one or more positions, thereby separating the second binding agent from the complexes of first binding agent and exosomes.

[0121] A first proteolytic digestion step aims at separating the exosome bound via the first binding agent to the second binding agent from the second binding agent. In cases where the linker is a peptide linker comprising one or more first cleavage sites (such as one or more TEV protease cleavage sites), the releasing of the complexes of the first binding agent and exosomes from the second binding agent is by subjecting the complex of first binding agent, second binding agent and exosome to an enzymatic digest (such as a digest using TEV protease), resulting in cleavage of the peptide linker at one or more positions, thereby separating the second binding agent from the complexes of exosome and cinnamycin of the first binding agent. In cases where the linker is a peptide linker comprising two different proteolytic cleavage sites, the exosomes can be released by subjecting the complex of first binding agent, second binding agent and exosomes to a first enzymatic digest (such as a TEV protease digest), resulting in cleavage of the peptide linker at one position, thereby separating the second binding agent from the complexes of exosome and cinnamycin. Residues of the peptide linker can then be further removed by a second enzymatic digest (such as a trypsin digest), which preferably removes all, essentially all or parts of the remaining peptide linker from the cinnamycin of the first binding agent. The skilled person will readily envisage other proteolytic enzymes in case other cleavage sites are present in the peptide linker, some examples of which are disclosed herein. The present invention is therefore not particularly limited to the embodiment of a proteolytic trypsin digest of a peptide linker comprising one or more trypsin cleavage sites. According to a particularly preferred embodiment, the peptide linker comprises one first cleavage site (such as a TEV protease cleavage site) and a further different second cleavage site (such as a trypsin cleavage site). This allows a particularly preferred release of the exosome and the cinnamycin of the first binding agent from the complex of exosome, first binding agent and second binding agent, and subsequently allows removal of parts or all of the peptide linker of the complex of cinnamycin and exosome.

[0122] According to one embodiment, before releasing the complexes of the first binding agent and exosomes from the second binding agent by way of e.g. a proteolytic cleavage, the complexes are separated from the remains of the sample and any unbound binding agents in the sample by standard methods such as washing and / or centrifugation or by isolation / separation via the second binding agent as disclosed herein. One way of removing unbound binding agents and sample residues is by way of ultrafiltration, e.g. between 20,000 and 60,000 rpm, preferably at 30,000 rpm. The supernatant after such centrifugation comprises the unbound binding agents and other unbound material, whereas the complexes of second binding agent, first binding agent and exosomes are present in the pellet, which can be further used in subsequent steps such as the releasing of the complexes of the first binding agent and exosomes from the second binding agent as described herein. To this end, the pellet is preferably resuspended in a suitable buffer such as PBS (pH 7.4) or 0.9% NaCl.

[0123] The exosomes bound to the first binding agent or to the cinnamycin of the first binding agent with partial or full removal of the peptide linker can be further prepared for subsequent analysis such as mass spectrometry or protein analysis by eluting the bound exosomes with acid, biotin or imidazole.

[0124] A workflow in accordance with the present invention comprises:

[0125] 1. incubate sample with the first binding agent;

[0126] 2. add second binding agent and incubate;

[0127] 3. remove unbound molecules or isolate exosomes via label on second binding agent;

[0128] 4. optionally further preparation for subsequent analysis such as mass spectrometry.

[0129] A particularly preferred embodiment of the present invention pertains to the following method:

[0130] A method for separating and / or isolating exosomes from a sample, the method comprising the steps of:

[0131] i) providing a sample that is known to comprise or suspected of comprising exosomes;

[0132] ii) contacting a first binding agent with the sample and allowing the binding agent to bind to exosomes, wherein the first binding agent comprises a protein-tag linked to cinnamycin via a peptide linker, wherein the peptide linker comprises one or more proteolytic cleavage sites;

[0133] iii) separating and / or isolating complexes of the first binding agent and exosomes from the sample.

[0134] The peptide linker of the first binding agent preferably has two different proteolytic cleavage sites. According to a preferred embodiment, the peptide linker of the first binding agent comprises a TEV protease cleavage site and a trypsin cleavage site. According to a particularly preferred embodiment, the trypsin cleavage site is C-terminally of the TEV protease cleavage site.

[0135] According to one embodiment, the first binding agent has the structure of Biotin-tag, strep-tag or strep-tag II–linker–cinnamycin. According to a preferred embodiment, the first binding agent has the structure of Biotin-tag, strep-tag or strep-tag II – SEQ ID NO: 2 or 7 – cinnamycin. According to a more preferred embodiment, the first binding agent has the structure of Biotin-tag, strep-tag or strep-tag II – SEQ ID NO: 7 – cinnamycin. According to the most preferred embodiments, the first binding agent has the structure of SEQ ID NO: 3 – SEQ ID NO: 7 – cinnamycin, SEQ ID NO: 4 – SEQ ID NO: 7 – cinnamycin or Biotin – SEQ ID NO: 7 – cinnamycin.

[0136] The method preferably further comprises a step iv) of contacting the complex of first binding agent bound to exosome with a second binding agent that binds to the protein-tag of the first binding agent. According to a preferred embodiment, the second binding agent is immobilized or bound to a substrate such as a plate or a bead, or it is immobilized on a surface such as the surface of a well or micro-well. According to a particularly preferred embodiment, the second binding agent is bound to the surface of a well. The second binding agent may have the structure as defined herein. According to a preferred embodiment, the second binding agent is streptavidin or an antigen binding agent against the protein-tag or against FITC such as an antibody or the binding portion of an antibody. According to a further preferred embodiment, the second binding agent is immobilized on a support such as a bead or a plate. One or more washing steps may follow such a step iv), which can also be described as removing unbound molecules.

[0137] The method preferably further comprises one or more, preferably two proteolytic digestion steps after step iv). A first proteolytic digestion step aims at separating the exosome bound via the first binding agent to the second binding agent from the second binding agent. This first proteolytic digestion takes place on the peptide linker of the first binding agent which comprises a first proteolytic cleavage site, preferably a TEV protease cleavage site. Accordingly, this step includes the addition of a first proteolytic enzyme (preferably TEV protease) to the complex of exosome, first binding agent and second binding agent. The cleavage of the peptide linker leaves a first part of the peptide linker bound to the second binding agent, and a second part still connected to the cinnamycin of the first binding agent bound to the exosome. If the second binding agent is bound to or immobilized on a solid support, the released exosome bound to the cinnamycin is preferably separated from the second binding agent.

[0138] For certain subsequent analysis steps, it may be advantageous to remove most, essentially all or all of the remaining peptide linker attached to the cinnamycin of the first binding agent. Thus, according to a particularly preferred embodiment, the method of the invention further comprises a second proteolytic digestion step aimed at removing most, essentially all or all of the remaining peptide linker bound to the cinnamycin of the first binding agent after the first proteolytic digestion step. According to a particularly preferred embodiment, the peptide linker to this end comprises a second proteolytic cleavage site, preferably a trypsin cleavage site, which is preferably C-terminal of the first proteolytic cleavage site and N-terminal of the cinnamycin. This second proteolytic digestion step thus releases most, essentially all or all of the remaining peptide linker attached to the cinnamycin. This released peptide may be detected in a subsequent analysis such as mass spectrometry and may serve as positive control indicating the successful separation of the remaining peptide linker from the cinnamycin. A separation step separating the released exosome bound to the cinnamycin from the remainder of the peptide linker may follow.

[0139] A particularly preferred embodiment of the present invention thus pertains to a method for separating and / or isolating exosomes from a sample, the method comprising the steps of:

[0140] i) providing a sample that is known to comprise or suspected of comprising exosomes;

[0141] ii) contacting a first binding agent with the sample and allowing the binding agent to bind to exosomes, wherein the first binding agent has the structure of protein-tag - peptide linker - cinnamycin, preferably of Biotin or strep-tag or strep-tag II - SEQ ID NO: 7 -cinnamycin;

[0142] iii) separating and / or isolating complexes of the first binding agent and exosomes from the sample;

[0143] iv) contacting the complex of first binding agents bound to exosomes with a second binding agent that binds to the Biotin or strep-tag or strep-tag II of the first binding agent, wherein the second binding agent is immobilized on a solid support;

[0144] v) contacting TEV protease with the complex of first binding agent - exosome - second binding agent immobilized on the solid support and allowing the protease to cleave the peptide linker of the first binding agent at the TEV protease cleavage site; vi) separating the complex comprising the exosome released from the second binding agent;

[0145] vii) optionally contacting trypsin protease with the complex comprising the exosome and the cinnamycin of the first binding agent and allowing the protease to cleave the remainder of the peptide linker of the first binding agent at the trypsin cleavage site, and separating the complex comprising the exosome and the cinnamycin from the cleaved peptide linker.

[0146] Further embodiments for the preferred methods are the same as the embodiments disclosed with respect to the alternative methods disclosed herein.

[0147] An alternative workflow in accordance with the present invention comprises:

[0148] 1. incubate sample with the first binding agent;

[0149] 2. add second binding agent or add mixture of 1. to second binding agent optionally immobilized on a solid support, and incubate;

[0150] 3. remove unbound molecules;

[0151] 4. release complex of exosome bound to first binding agent from the second binding agent by proteolytic cleavage of the peptide linker of the first binding agent;

[0152] 5. optionally perform a second proteolytic digest to remove parts or all of the peptide linker;

[0153] 4. optionally further preparation for subsequent analysis such as mass spectrometry.

[0154] An alternative workflow in accordance with the present invention comprises:

[0155] 1. incubate sample with the first binding agent bound to the second binding agent; 2. isolate exosomes via label on second binding agent;

[0156] 3. optionally further preparation for subsequent analysis such as mass spectrometry.

[0157] A preferred protocol for separating and / or isolating exosomes from a sample is shown below:

[0158] 1. incubate sample with the first binding agent for 30 minutes at room temperature and continuous shaking;

[0159] 2. add second binding agent and incubate for 30 minutes at room temperature and continuous shaking;

[0160] 3. capturing of second binding agent via its label, e.g. capturing magnetic beads of the second binding agent;

[0161] 4. washing step, preferably with PBS; 5. optionally treatment of positive and / or negative control, e.g. by adding SDC; 6. optionally further analysis such as mass spectrometry.

[0162] It will be appreciated that washing steps can be performed at any step of the method of the invention as described herein, as will be readily recognized by the skilled person.

[0163] The method of the invention allows isolating exosomes without isolating any other microvesicles in any substantial amounts. Thus, according to one embodiment, the method of the invention isolates essentially no microvesicles other than exosomes. According to a preferred embodiment, the method of the invention isolates no microvesicles other than exosomes.

[0164] In accordance with one embodiment, the method of the invention further comprises the step of using a positive control. The positive control can be added at step ii) of the method to the sample which comprises complexes of the first binding agent and exosomes. Alternatively, the positive control can be added to a retained sample of the sample provided in step i) of the method of the invention, which then follows the same method steps and protocol which the sample provided in step i) follows. According to the invention, the positive control comprises liposomes. The liposomes are preferably modified to comprise phosphatidylethanolamine on their surface and a proteolytically-cleavable peptide in its lumen. The liposomes containing the peptides can be added to the sample, and the intraluminal peptide will become accessible after perforation of the liposome, e.g. by SDC treatment. The proteolytically-cleavable peptide comprises at least one proteolytic cleavage site, such as 1, 2, 3 or more cleavage sites. According to a preferred embodiment, the proteolytically-cleavable peptide comprises three proteolytic cleavage sites. A preferred enzyme for cleavage is trypsin. In accordance with this preferred embodiment, the proteolytically-cleavable peptide is a trypsin-cleavable peptide and comprises three trypsin cleavage sites. The peptide is preferably a non-human and non-animal peptide which is detectable in analytical methods such as in mass spectrometry. By using non-human and non-animal peptides, it can be ensured that the analyze peptide derives from the liposome of the positive or negative control. According to a particularly preferred embodiment, the liposome of the positive control comprises PE and the peptide according to SEQ ID NO: 5.

[0165] The first binding agent will also bind and co-purify the PE-containing liposomes of the positive control. Mass spectrometry analysis of the isolated and / or separated exosomes also including the co-purified liposomes of the positive control will provide sensitivity that the purification process worked because the mass spectrometry peptide profile will also identify the Positive Control (PC)-peptides within the PE-liposomes. The proteolytical digest of the positive control will generate smaller peptides detectable by mass spectrometry analysis.

[0166] In accordance with one embodiment, the method of the invention further comprises the step of using a negative control. The negative control can be added at step ii) of the method to the sample which comprises complexes of the first binding agent and exosomes. Alternatively, the negative control can be added to a retained sample of the sample provided in step i) of the method of the invention or to the sample used in the positive control workflow as described above. Alternatively, the negative control can be added to a further retained sample of the sample provided in step i) of the method of the invention, which then follows the same method steps and protocol which the sample provided in step i) follows. According to the invention, the negative control comprises liposomes. The liposomes are preferably modified to comprise a proteolytically-cleavable peptide in its lumen. The negative control preferably does not comprise PE on its surface to avoid binding of the first binding agent. The proteolytically-cleavable peptide differs by way of its amino acid sequence and / or number of proteolytic cleavage sites from the proteolytically-cleavable peptide of the positive control. According to a preferred embodiment, the proteolytically-cleavable peptide of the negative control comprises two proteolytic cleavage sites. A preferred enzyme for cleavage is trypsin. In accordance with this preferred embodiment, the proteolytically-cleavable peptide of the negative control is a trypsin-cleavable peptide and comprises two trypsin cleavage sites. The peptide is preferably a non-human and non-animal peptide which is detectable in analytical methods such as in mass spectrometry. According to a particularly preferred embodiment, the liposome of the negative control comprises PE and the peptide according to SEQ ID NO: 6.

[0167] Liposomes of the negative control have no phosphatidylethanolamine (PE) on their surface, and their lumen contains a proteolytically cleavable peptide preferably different from that of the positive control. The non-PE containing liposomes of the negative control should not be bound by the first biding agent and should be removed when the complexes of first binding agent and exosomes are separated from the remaining of the sample and any unbound binding agents in the sample as described herein. Mass spectrometry analysis of the purified exosomes which should not include any liposomes of the negative control since the mass spectrometry peptide profile should not identify the negative control peptides after proteolytic cleavage.

[0168] In accordance with the present invention, if a positive and / or negative control as described herein is used, the exosomes bound to the first binding agent can be prepared for subsequent analysis such as mass spectrometry or protein analysis by eluting the bound exosomes with acid, biotin or imidazole, and by adding sodium deoxycholate (SDC) followed by tryptic digest.

[0169] In accordance with a preferred embodiment of the method of the invention, the method comprises the steps of:

[0170] i) providing a sample that is known to comprise or suspected of comprising exosomes;

[0171] ii) contacting a first binding agent with the sample and allowing the first binding agent to bind to exosomes in the sample, wherein the first binding agent has the structure of a) FITC - cinnamycin, wherein the FITC is covalently linked to the N-terminus of cinnamycin, or b) SEQ ID NO: 4 - SEQ ID NO: 2 - cinnamycin, or c) Biotin - SEQ ID NO: 7 - cinnamycin;

[0172] iii) contacting a second binding agent comprising anti-FITC antibody and magnetic beads or Strep-tag and magnetic beads with the sample comprising complexes of the first binding agent and exosomes;

[0173] iv) separating and / or isolating complexes of the first binding agent and exosomes. This particularly preferred method may optionally comprise the use of a positive and / or negative control as described herein above. Details to the individual steps of the preferred method are described herein above and apply likewise.

[0174] This particularly preferred method may optionally comprise the step of separating complexes of the first binding agent, second binding agent and exosomes from unbound binding agents, and / or releasing the complexes of the first binding agent and exosomes from the second binding agent.

[0175] In accordance with a particularly preferred embodiment of the method of the invention, the method comprises the steps of:

[0176] i) providing a sample that is known to comprise or suspected of comprising exosomes;

[0177] ii) contacting a first binding agent with the sample and allowing the first binding agent to bind to exosomes in the sample, wherein the first binding agent has the structure of protein-tag - peptide linker - cinnamycin, preferably Biotin or strep-tag or strep-tag II – SEQ ID NO: 7 – cinnamycin;

[0178] iii) contacting a second binding agent with the sample comprising complexes of the first binding agent and exosomes, or adding complexes of the first binding agent and exosomes to a solid support comprising the second binding agent; iv) optionally washing the solid support;

[0179] v) releasing complexes comprising the exosomes from the second binding agent preferably by one or more proteolytic digests, preferably by a TEV protease digest;

[0180] vi) optionally purifying the released complexes comprising the exosomes from the second binding agent.

[0181] This particularly preferred method may optionally comprise the use of a positive and / or negative control as described herein above. Details to the individual steps of the preferred method are described herein above and apply likewise. A specific workflow for this particularly preferred embodiment is disclosed in the following:

[0182] 1. Contact first binding agent with sample known to comprise or suspected of comprising exosomes;

[0183] 2. Incubate on a shaker (e.g. 30 minutes, 21°C, 450 rpm);

[0184] 3. Add mixture of 2. to a solid support comprising immobilized second binding agents specifically binding first binding agents;

[0185] 4. Incubate solid support (e.g. 30 minutes, 21°C, 400 rpm);

[0186] 5. Remove supernatant and wash solid support (e.g. multiple times) with washing solution; remove residual liquid;

[0187] 6. Add first protease and incubate (e.g. 30 minutes; 30°C; gentle shaking);

[0188] 7. Collect supernatant;

[0189] 8. optionally add second protease and incubate; collect supernatant.

[0190] In case magnetic beads are used as label on the second binding agent, the complexes of first binding agent, second binding agent and exosomes can be separated and / or isolated using any method known in the art for separating / isolating magnetic beads from a sample or solution, such as by applying a magnetic field.

[0191] When magnetic beads are used and a magnetic field is applied for separating the complexes of the first binding agent, second binding agent and exosomes, this magnetic pulldown is preferably followed by a washing step. During the magnetic pull-down, the magnetic beads accumulate at the side of the vessel at which the magnetic field is applied. This can be for example one side of the vessel or the bottom of the vessel. The washing step can include removing the supernatant, i.e. the solution covering the side of the vessel at which the magnetic field is applied. The magnetic beads held by the magnetic field can be resuspended in a buffer or in NaCl solution. This washing procedure can be repeated several times. After the final resuspension, the complexes of first binding agent, second binding agent and exosomes can be used in further analytic methods.

[0192] According to the present invention, the isolated and / or separated exosomes can be further detected and / or quantified by flow cytometry and / or super-resolution microscopy. The term "flow cytometry" as used herein refers to technique used to detect and measure physical and chemical characteristics of a population of cells or particles. Flow cytometry is a sorting technique that rapidly analyzes single cells but also other particles as they flow past single or multiple detectors while suspended in solution. Cell sorting in general describes the process of purifying or enriching cell populations based on the presence or absence of specific physical characteristics. Using flow cytometry, exosomes in the sample to which the first binding agent is bound can be identified based on the FITC label of the first binding agent and can be separated from other components in the sample. Flow cytometry is an established method in the art and the person of ordinary skill will readily identify various possible flow cytometers and respective methods for putting the invention into practice. Flow cytometers that can be used in this context are commercially available and well established in the field of cell biology. A preferred flow cytometry technique is fluorescence flow cytometry such as fluorescence-activated cell sorting (FACS). Any suitable flow cytometer or cell sorter can be used in the context of the invention.

[0193] Any of the methods described herein may further comprise the step of comparing the quantity and / or type of exosomes, preferably of disease related exosomes, in the sample of the subject (e.g. a subject known to or suspected of suffering from a disease) with the quantity and / or type of similar exosomes known to be present in a sample of a control such as a healthy subject. An increase in the quantity of the exosomes in the sample of the subject known or suspected to suffer from a disease may e.g. be indicative of the presence or stage of the disease. Exosomes of subjects suffering from a disease are known to comprise peptides associated with the disease such as viral peptides and antigens as well as cancer peptides and antigens. A disease to be detected or monitored according to the invention is preferably cancer. In such methods, comparing the quantity of exosomes may comprise applying CD mapping and t-SNE analysis, NTA Tracking, and using a Zetasizer by e.g. applying counting beads with flow cytometry.

[0194] Additional analysis of markers such as proteins, peptides and / or antigens in addition to those associated with a disease (such as viral or cancer peptides and / or antigens) may allow localizing the disease to a specific organ or tissue. For example, a disease related exosome may additionally carry or comprise one or more markers for a specific organ or tissue such as cardiac troponin for the heart. Thus, additionally identifying in a population of disease related exosomes organ or tissue specific markers or markers associated with a group of tissues or organs allows associating disease related exosomes to a tissue or an organ, thereby associating the disease to the tissue or organ. Therefore, for further investigating the separated and / or isolated exosomes of a sample, further surface markers and / or the content of the exosomes can be analyzed. Such further surface markers include but are not limited to amyloid-beta, 14-3-3 protein, Actin, ADAM 10, Alix, alpha-Enolase, alpha-Synuclein, Aminopeptidase N, Annexin 5 A, Annexin A2, AP-1, ATP citrate lyase, ATPase, Basigin, Caveolin-1, Clathrin, Claudin-1, Cofilin- 1, EGFR, Ep-CAM, ICAM, HLA-ABC, prostate specific antigen, Rab-14, Rab-7, Syndecan, Tumor-Associated Glycoprotein, Tetraspanin-8, TsglOl, vacuolar-sorting protein 35, CD2, CD3, CD5, CD8, CD9, CDlla, CDllb, CDllc, CD13, CD29, CD37, CD41, CD44, CD49d, CD49f, CD62L, CD63, CD68, CD80, CD81, CD86, CD90, CD 142, CD 146, CD 163, CD 192, and CD202b.

[0195] Specific examples of markers that may 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 endothelium, Enolase-2 (ENO2) and neuron specific enolase (NSE) for the brain or neuro tissues, Asialoglycoproteinreceptor 1 (ASGR-1) for the liver, and Aquaporin 6 for the kidney. These markers are particularly suitable for associating a virus infection with a specific organ or tissue as origin of virus replication, more preferably for associating a SARS-CoV virus or Influenza A / B virus infection with a specific organ or tissue, and most preferably a SARS-CoV 2 virus infection.

[0196] Analyzing the content of the exosome may include lysis of the exosomes. The content to be analyzed include but are not limited to peptides, proteins, microRNA, DNA, and / or RNA such as mRNA. Proteins to be analyzed typically include but are not limited to platelet derived growth factor receptor, lactadherin, transmembrane proteins and lysosome associated membrane protein-2B, membrane transport and fusion proteins like annexins, flotillins, GTPases, heat shock proteins, tetraspanins, proteins involved in multivesicular body biogenesis, as well as lipid-related proteins and phospholipases. The analysis of the contents may include DNA mutation analysis, RNA expression, DNA methylation quantification and / or protein expression, as well as fluorescence flow cytometry. In case of analyzing nucleic acids, these can be for example quantified to identify their profiles by methods known in the field and involving for example RT-PCR. Exosomes which are released from virus infected cells contain Rabll which allows virosome identification and applying a specific second antibody directed against a specific viral protein for diagnosing and monitoring viral diseases. The analysis of the exosome contents and surface proteins and peptides is not limited to detecting and monitoring cancer or viral infections but may be generally used for detecting or characterizing many medical conditions and diseases.

[0197] According to a further aspect, the present invention provides a binding agent suitable for performing any of the methods of the present invention as described herein. Thus, the present invention provides a binding agent comprising fluoresceine directly covalently linked to cinnamycin. In accordance with the present invention, the fluoresceine is directly covalently linked to the N-terminus of cinnamycin as described herein and as schematically shown in Fig.

[0198] 1. According to a preferred embodiment, the binding agent comprises a peptide linker linking a peptide-tag comprising one or more proteolytic cleavage sites to the cinnamycin. The peptide linker is as described herein above and preferably comprises the amino acid sequence as set forth in SEQ ID NO: 2 or 7. According to a particularly preferred embodiment, the binding agent comprises or consists of from N-terminus to C-terminus: (i) a protein-tag (preferably Biotin, strep-tag or strep-tag II), attached to (ii) a peptide linker as described herein and comprising a first proteolytic cleavage site (preferably a TEV protease cleavage site), a second proteolytic cleavage site which is different from the first proteolytic cleavage site (preferably a trypsin cleavage site) and which is located C-terminally from the first proteolytic cleavage site, preferably a peptide linker as set forth in SEQ ID NO: 7, attached to (iii) cinnamycin.

[0199] According to one embodiment, the binding agent further comprises a second binding agent bound to the protein-tag or the FITC of the first binding agent. The second binding agent is as described herein and preferably comprises a label as described herein. The label is preferably a magnetic bead. According to a particularly preferred embodiment, the second binding agent is an anti-FITC antibody comprising a magnetic bead. According to a further preferred embodiment, the second binding agent is a magnetic bead coated with streptavidin.

[0200] According to a further aspect, the present invention provides a kit comprising a first binding agent as described herein. The first binding agent may comprise a) cinnamycin and fluoresceine isothiocyanate, wherein the FITC is covalently linked to cinnamycin, or b) a protein-tag linked to cinnamycin via a peptide linker, wherein the peptide linker comprises one or more proteolytic cleavage sites. The first binding agent is as described herein. The kit further comprises instructions for binding of said first binding agent to exosomes in a sample. The kit is particularly suitable for performing any of the methods of the present invention as described herein. According to one embodiment, the kit further comprises a second binding agent capable of binding to the first binding agent, and preferably to the FITC or to the protein-tag of the first binding agent. The second binding agent is as described herein. In accordance with one embodiment, the second binding agent comprises a label, preferably a magnetic bead. According to one embodiment, the second binding agent is bound to the first binding agent. The kit may further comprise the positive and / or negative control as described herein above.

[0201] The invention is further described by way of the following examples which are to be construed as merely illustrative and not limitative of the scope of the invention.

[0202] EXAMPLES

[0203] Devices and reagents

[0204] In the examples of the invention, the following devices and reagents have been used:

[0205] FACS device:

[0206] Sony SP6800 Spectral Analyzer; Sony SA3800 Spectral Analyzer

[0207] Pancoll gradient:

[0208] PAN Biotech, Density: 1.077 g / ml

[0209] Centrifuges:

[0210] Heraeus Multifuge X3 R (Cat.no.: 75004515; Serial number: 41615170 Thermo Scientific) Heraeus Fresco 17 (Cat.no. 75002420; Serial number: 41284997; Thermo Scientific) Optima LE-80K (Beckman Coulter)

[0211] Antibodies:

[0212] Anti-FITC antibody conjugated magnetic beads, Miltenyi Biotec: Order number: 130-048-701. Component: MicroBeads conjugated to monoclonal anti-FITC Isomer-1 antibody (isotype: mouse IgG1). Anti-FITC MicroBeads are supplied in a solution containing stabilizer and 0.05% sodium azide.

[0213] Further Reagents:

[0214] Triton-X100 (PanReac AppliChem)

[0215] Counting Beads (Count Bright Absolute Counting Beads, ThermoFisher, Cat. No.: #C36950, Lot.: 2466363)

[0216] NaCl (0.9%; sterile solution; Braun)

[0217] RPMI (+) L-Glutamine cell culture medium (Ref. no: 21875-034; Gibco)

[0218] DMEM Medium high glucose (Cat.no.: D5796; 500 ml; Sigma Life Science) ProTEVPlus; Promega: #V6101; Lot.: 652269

[0219] Streptavidin coated clear 96 well plate with superblock blocking buffer; Pierce: #15124; Lot.: 3212456

[0220] Wash Buffer for Streptavidin Plate: 25mM Trizema Base (121,14 g / mol, Sigma Aldrich: #336028; Lot.: 361328); 150 mM sodium chloride in IL H2O; pH 7.4

[0221] Nanomag CLD beads; size 500 nm; magnetic beads; Micromod; #05-19-502

[0222] Example 1: Construct binding to exosomes and Streptavidin Atto488 - UZ - Flow cytometry

[0223] The first binding agent in this experiment is MS II: Strep-tag (SEQ ID NO: 3) - peptide linker (SEQ ID NO: 2) - cinnamycin, having 4.77 kDa and a purity of 90% after HPLC.

[0224] Experimental workflow:

[0225] 1. 1 ml SKBR-3 cell culture supernatant (breast cancer cell line) or 1 ml NaCl were incubated with 1 pg MS II construct;

[0226] 2. Incubation for 30 minutes on shaker; 300 rpm; 21°C to mix gently all components; 3. 1.5 pg of streptavidin - atto488 (second binding agent) was added to samples;

[0227] 4. Samples were incubated for 30 minutes on shaker, 300 rpm; 21°C in the dark;

[0228] 5. Ultracentrifugation was performed for 01:30h; 10°C; 30.000 rpm;

[0229] 6. 1 ml supernatant was removed and stored for further analysis;

[0230] 7. Samples were placed up-side down onto a paper towel to remove excess supernatant for 5 min;

[0231] 8. Pellet was resuspended in 200 pl NaCl;

[0232] 9. Incubate part of the samples as control with 1% Triton for 1 hour; 450 rpm, 21°C and protect samples from light (180 pl supernatant + 20pl 10% Triton; 18 pl eluate + 2 pl 10% Triton; use equivalent amount of NaCl instead of Triton);

[0233] 10. Dilute eluate samples 1:10 with NaCl containing counting beads (CountBright™ Absolute Counting Beads; Thermo Fisher, USA, used for generating standard curve) and add counting beads to supernatant samples;

[0234] 11. Measure samples on spectral analyzer SA3800 from Sony.

[0235] Results are shown in Figure 2A. The MS II cinnamcyin-StrepTag construct incubated with exosomes in cell culture supernatant of the breast cancer cell line SKBR-3 and streptavidin - atto488 as second binding agent results in significant binding as shown by means of flow cytometry analysis (grey dots, SKBR-3). As a controls, the sample was incubated with NaCl solution. NaCl + Triton and SKBR-3 + Triton solubilize the exosomes resulting in no signal. The same analysis by omitting streptavidin (white dots) or omitting cinnamycin (black dots) gave no results, demonstrating the specificity of analysis.

[0236] In conclusion, the MS II construct is capable of binding to exosomes and to streptavidin.

[0237] Example 2: Cinnamycin-FITC binding to phosphatidylethanolamine positive liposomes Liposomes with phosphatidylethanolamine incorporated in their surface were tested for Cinnamycin-FITC binding. Figure 2B shows the binding of Cinnamycin-FITC in a concentration dependent manner through flow cytometry using counting beads to determine the number of liposomes in the sample.

[0238] Liposome characteristics:

[0239] Nr. Liposome composition size: Z- total Lipid Vesicles amount average concentration per pL

[0240] 1 KW211102-H 5% Cholesterol, 117 nm 7.95 mM 2.5 x101050 pL 5% DMPE + / - 3.7 nm

[0241]

[0242] Workflow:

[0243] 1. Pre-dilution of liposomes 1:10 with PBS- NaCl (210mM);

[0244] 2. Staining of samples with different concentrations Cinnamycin-FITC and incubation for 30 minutes in the dark at 21 °C;

[0245] 3. Final dilution of all samples 1: 100 with NaCl; liposomes were diluted with PBS-NaCl; 4. Analysis by means of flow cytometry on a SA 3800 machine (Sony Biotechnologies, Japan).

[0246] Example 3: Liposomes packaged with Protein X, Cinnamycin-FITC binding by flow cytometry

[0247] Phosphatidylethanolamine positive liposomes which also contain Sulforhodamine (Fluorochome) in their lumen were tested for binding of Cinnamycin-FITC. The experiment shows that Cinnamycin-FITC binds to PE-positive liposomes in a concentration manner. Figure 3 shows the of Cinnamycin-FITC and Sulforhodamine signals (double positive events) analyzed by flow cytometry. Protein X is as set forth in SEQ ID NO: 5. Liposome characteristics:

[0248] Composition with 71% dipalmitoylphosphatidylcholine (DPPC), 19% 1,2-dipalmitoyl-sn-glycero-3-phosphoglycero (DPPG), 5% cholesterol, 5% phosphatidylethanolamine Size average of liposomes: 121 nm

[0249] Composition contains 10 mM Sulforhodamine

[0250] Workflow:

[0251] 1. Serial dilution of samples (1:2; 1:4; 1:8, 1:16, 1:32) with PBS-NaCl (210 mM);

[0252] 2. Staining of samples with 10 ng / pl Cinnamycin-FITC and incubation 30 min in the dark at RT;

[0253] 3. Addition of counting beads and measurement using the S A3800 spectral analyzer.

[0254] Example 4: Liposomes packaged with Protein X, mass spectrometry profiles Positive and negative controls serve as specificity and negativity determination. Liposomes with phosphatidylethanolamine (PE) on their surface and packaged with a nonanimal, non-human peptide (Protein X, SEQ ID NO: 5) added to the sample can be detected in the sample by mass spectrometry analysis. Figure 4 demonstrates that the peptide is detected in a concentration dependent manner by mass spectrometry. In Fig. 4, small dots represent proteins that are not Protein X, big dots represent proteins that are Protein X. The peptide detection is linear according to the concentration of liposomes / Protein X added.

[0255] Preparation of samples for proteomic analysis

[0256] Suspension of liposome-packaged or liposome-free proteins (total volume of 50 pL, concentrations: 0.01, 0.1, 1, and 10 ng / pL for liposome packaged, lOng / pL for liposome-free samples) was dissolved in 200 pl sodium deoxy cholate (SDC) lysis buffer (4% SDC in 100 mM Tris-HCl pH 8.5), and heat-inactivated (95°C for 5 min). Preparations were sonicated (4°C, 15 x 30 sec on and 30 sec off setting), and 1 / 10 volume of reduction / alkylation buffer (100 mM TCEP and 400 mM 2-chloroacetamide, pH 7-8) was added prior to incubation for 5 min at 45°C. Subsequently, samples were subjected to proteolytic digestion overnight (37°C, 16 h, 1000 rpm) using LysC (1:50 w / w, enzyme / protein, Wako) and trypsin (1:50 w / w, enzyme / protein, Promega). The samples were prepared for SDB-RPS stage tipping by adding 5 volumes of loading buffer (1% TFA in isopropanol). Peptides were desalted with 2-layer SDB-RPS stage tips. Columns were equilibrated with 100 pl loading buffer at room temperature and loaded with the samples, washed twice with 100 pl washing buffer I (1% TFA in isopropanol) and washing buffer II (0.2% TFA, 5% acetonitrile in isopropanol). 60 pl of freshly prepared SDB-RPS elution buffer was used to elute peptides (1% NH40H, 60% acetonitrile). The eluates were spin-evaporated (45°C, vacuum), and finally resuspended in 7 pl MS of loading buffer (2% acetonitrile, 0.3% TFA). 5 pl of peptide solution was used for LC-MS / MS analysis.

[0257] LC-MS / MS measurement

[0258] Samples were measured on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) coupled online to a Vanquish Neo UHPLC-System (Thermo Fisher Scientific). The liquid chromatography setup consisted of a 75 pm x 45 cm analytical column, packed in-house with Reprosil Pur C18-AQ 1.9 pm particles (Dr. Maisch GmbH). Peptides were separated using a 60 min gradient at a flow rate of 300 nl / min, and a binary buffer system consisting of buffer A (0.1 % V / V formic acid in water) and buffer B (80 % V / V acetonitrile, 0.1 % V / V formic acid in water). The following gradient was used: 5-30% (35 min), 30-95% (10 min), wash out at 95% for 5 min, readjustment to 5% in 5 min, and kept at 5% for 5 min. The mass spectrometer was operated in the positive ionization mode, the spray voltage was set to 2.5 kV, funnel RF level at 60, and heated capillary at 250 °C. Full scan MS1 spectra were recorded from 300 to 1,650 m / z at 60k resolution using an automatic gain control (AGC) target value of 3E6 and a max injection time (IT) of 20 msec. The top 15 peptide precursors were selected for higher energy collisional dissociation (HCD) with 27 % normalized collision energy. The precursor isolation window was set to 1.4 m / z with 15 k MS2 resolution, an AGC target value of 1E5 and a max IT of 25 msec. Only precursors with charge state 2 to 6 were selected and the dynamic exclusion was set to 20 sec.

[0259] Protein quantification

[0260] Protein content of the measured samples was quantified using MaxQuant (version 2.4.9.0), with label-free quantification (LFQ), iBAQ calculation, and match between runs enabled. Synthetic protein sequences were used alongside Uniprot human reviewed canonical isoforms as search basis. Visualization of peptide intensities and protein iBAQ values as a measure of protein abundances in the samples was performed using R (version 4.3.3). Example 5: FITC-Cinnamycin construct with anti-FITC antibody beads purify exosomes from cell culture supernatant

[0261] Lot no. Company /

[0262] Reagent / product Concentration Comment / Cat.no Reference

[0263] Cinnamycin-FITC Batch 9 Self-labelled; 500 ng / pl Source for July 2024 Cinnamycin:

[0264] Sigma Triton X-100 solution N. A. AppliChem 100% N. A.

[0265] NaCl 0.9% Braun 0,9%

[0266] Sample name Anti FITC beads MicroBeads

[0267] Size 50 nm

[0268] Cat.no / Lot.no 130048701 / 5240207344

[0269] Concentration / binding capacity N. A.

[0270] Company Miltenyi

[0271]

[0272] Experimental workflow

[0273] 1. Use 0.5 ml reaction tubes and 100 pl of SKBR-3 cell culture supernatant or NaCl and add 2 pl of Cinnamycin-FITC stock solution and incubate at RT for 30 min protected from light

[0274] 2. Add 20 pl of anti-FITC beads to each sample and incubate samples at 4°C for 30 minutes protected from light

[0275] 3. Magnetic pull down and wash step: place reaction tubes in magnet for 10 minutes, cover with aluminum foil to protect from light

[0276] 4. Remove first supernatant carefully and discard

[0277] 5. Remove reaction tubes from magnet and add 100 pl NaCl

[0278] 6. Place reaction tubes in magnet and perform magnetic pulldown / wash steps two more times

[0279] 7. Resuspend beads in 100 pl NaCl and continue with sample analysis using the spectral analyzer flow cytometer SA-3800 (Sony Biotechnologies)

[0280] 8. For controls: perform Triton incubation by preparing samples as followed:

[0281] EV control samples: 180 pl NaCl / SKBR-3 ZKSN + 20 pl Triton or 10 pl NaCl. Incubate samples for 1 hour; 450 rpm on a shaker at 21 °C followed by sample analysis by flow cytometry. Results are shown in Fig. 5. Exosomes in cell culture supernatant of the breast cancer cell line SKBR-3 are detected by Cinnamycin-FITC which are captured by anti-FITC antibody coated beads (far left upper panel denoted SKBR-3). The others (upper panels SKBR-3+T, NaCl, and NaCl+T) are control either with NaCl solution instead of cell culture supernatant or treatment with Triton which solubilizes the exosomes gave not results by means of flow cytometry. Lower panel unstained controls, no signal.

[0282] It can be concluded that Cinnamycin-FITC detects exosomes which are efficiently captured by anti-FITC antibody beads and thereby purified.

[0283] Example 6: Microtiter Assay

[0284] Magnetic beads-based assay for isolating exosomes from biological fluids such as cell culture supernatant and human plasma. The first binding agent comprises (from C-terminus to N-terminus) cinnamycin coupled to a trypsin cleavage site followed by a non-human, nonanimal designed six amino acid peptide as internal standard, followed by a TEV protease cleavage site and biotin. This first binding agent was incubated with plasma and then added to streptavidin coated microtiter plates which were prepared by washing three times with 200 pl Tris-NaCl wash buffer per well (Streptavidin coated clear 96 well plate with superblock blocking buffer; Pierce: #15124; Lot.: 3212456). After washing three times, release buffer containing TEV protease was added, incubated for 30 minutes and the supernatant processed for mass spectrometry analysis. This concept avoids non-specific binding contamination of the exosome preparation.

[0285] The workflow for this microtiter assay was as follows:

[0286] 1. For each sample, mix 50 pl plasma with 150 pl NaCl;

[0287] 2. Add 1 pg first binding agent (Cinnamycin-peptide linker-Biotin) (2 pl) to 200 pl of pre-diluted plasma;

[0288] 3. Incubate samples on a shaker for 30 minutes, 21°C; 450 rpm;

[0289] 4. Prepare microtiter plate with streptavidin coating;

[0290] 5. Wash well-plate three times with 200 pl Tris-NaCl wash buffer per well, remove residual liquid;

[0291] 6. Add stained samples to the wells;

[0292] 7. Incubate plate for 30 minutes; 21°C; 400 rpm;

[0293] 8. Prepare TEV protease mix: 5 pl TEV Buffer; 1 pl DTT; 2 pl enzyme; 992 µl nuclease-free H2O, add TEV protease before applying the mix to the plate; 9. Remove supernatant and wash plate three times with 200 pl NaCl per well, remove residual liquid;

[0294] 11. Add 100 pl of TEV enzyme mix or NaCl as control to samples;

[0295] 12. Incubate samples for 30 minutes at 30°C and gentle shaking;

[0296] 13. Collect supernatant and retain it for MS analysis.

[0297] Example 7: Bead Assay

[0298] Magnetic beads-based assay for isolating exosomes from biological fluids such as cell culture supernatant and human plasma. The first binding agent comprises (from C-terminus to N-terminus) cinnamycin coupled to a trypsin cleavage site followed by a non-human, nonanimal designed six amino acid peptide as internal standard, followed by a TEV protease cleavage site and biotin. This first binding agent was incubated with plasma and then added to streptavidin coated beads. After washing three times, release buffer containing TEV protease was added, incubated for 30 minutes and the supernatant processed for mass spectrometry analysis. This concept avoids non-specific binding contamination of the exosome preparation.

[0299] The workflow for this bead assay was as follows:

[0300] 1. Prepare micro-beads coated with streptavidin (10 pl of beads were washed three times using magnetic pulldown for removing bead storage medium);

[0301] 2. Prepare plasma samples: use 200 pl plasma and 800 pl NaCl for one sample; 3. Stain sample with 4 pg of the first binding agent construct: stock concentration: 500 ng / pl — > 8 pl first binding agent construct per 200 pl plasma;

[0302] 4. Add the first binding agent construct to the samples and incubate for 1 hour at 21 °C at 450 rpm on a shaker;

[0303] 5. Prepare 2 ml reaction tubes and add 10 pl of washed beads to each tube;

[0304] 6. Add 1000 pl sample to the beads;

[0305] 7. Incubate the samples for 1 hour at 21 °C and at 450 rpm on a shaker;

[0306] 8. Prepare TEV protease mix: 5 pl TEV Buffer; 1 pl DTT; 2 pl enzyme; 992 pl nuclease-free H2O (add TEV protease immediately before applying the mix);

[0307] 9. Place the reaction tubes on DynaMag Spin magnet and allow beads to collect at tube wall;

[0308] 10. Remove and discard the supernatant;

[0309] 11. Wash bead samples at least three times with 500 pl NaCl using magnetic pulldown between washes; 12. Add 100 pl of either NaCl (control) or the TEV enzyme mix to the samples; 13. Incubate all samples at 30°C for 30 minutes with gentle shaking;

[0310] 14. Place the tubes back on the Dynaspin magnet and allow beads to settle for at least 5 minutes;

[0311] 15. Collect the supernatant;

[0312] 16. Optional second enzymatic digest using trypsin protease;

[0313] 17. Prepare sample for MS analysis.

[0314] Figure 6 shows the results of the mass spectrometry analysis. The scatter plot compares proteins detected exclusively in plasma (x-axis) with proteins detected exclusively in samples prepared using the method of the invention (y-axis). MS-peptide denotes the residual linker peptide remaining after sequential protease digestion, first released from streptavidin binding by TEV protease and subsequently from the exosome-cinnamycin complex by trypsin digestion. The plot depicts relative protein abundances in exosome-enriched samples versus plasma, demonstrating up to a 1,000-fold enrichment of exosomal proteins. As shown, the method efficiently purifies exosomes with high abundance of established exosome marker proteins.

[0315] Figure 7 shows the mass spectrometry analysis of exosome enrichment using the methods of the invention and the identification of a non-human, non-animal-derived peptide exclusively detected in samples prepared according to the invention using a TEV proteasecontaining release buffer. This peptide was not detectable in any control samples, demonstrating the specificity of the assay format. The MS peptide serving as an internal control was released only when the complex of first binding agent and exosome was immobilized on streptavidin-coated plates or beads and co-released with the exosomes. Specifically, exosomes were captured using a construct of cinnamycin, peptide linker containing a TEV protease cleavage site, and a biotin-binding moiety, immobilized on streptavidin-coated microtiter plates. After washing, cinnamycin-bound exosomes were released by TEV protease cleavage. The x-axis in Fig. 7 shows proteins identified exclusively in plasma, whereas the y-axis shows proteins detected only in samples enriched using the method of the invention. The diagonal indicates proteins detected in both plasma and enriched samples. ITEMS OF THE INVENTION

[0316] The present invention also pertains to the following items:

[0317] Item 1: A method for separating and / or isolating exosomes from a sample, the method comprising the steps of:

[0318] i) providing a sample that is known to comprise or suspected of comprising exosomes;

[0319] ii) contacting a first binding agent with the sample and allowing the binding agent to bind to exosomes, wherein the first binding agent comprises

[0320] a) cinnamycin and fluoresceine isothiocyanate (FITC), wherein the FITC is covalently linked to cinnamycin, or

[0321] b) a protein-tag linked to cinnamycin via a peptide linker, wherein the peptide linker comprises one or more proteolytic cleavage sites;

[0322] iii) separating and / or isolating complexes of the first binding agent and exosomes from the sample.

[0323] Item 2: The method according to item 1, wherein a second binding agent is bound to the protein-tag or the FITC of the first binding agent, preferably wherein the second binding agent comprises a label, more preferably a magnetic bead.

[0324] Item 3: The method according to item 1, further comprising after step ii) a step of adding a second binding agent to the sample, wherein the second binding agent is capable of binding to the protein-tag or the FITC of the first binding agent, preferably wherein the second binding agent comprises a label, more preferably a magnetic bead.

[0325] Item 4: The method according to item 2 or 3, wherein the second binding agent comprises an antibody or antigen binding fragment thereof that specifically binds to the proteintag or the FITC of the first binding agent.

[0326] Item 5: The method according to any one of items 2 to 4, further comprising after step iii) a step of releasing the complexes of the first binding agent and exosomes from the second binding agent.

[0327] Item 6: The method according to item 5, wherein the releasing the complexes of the first binding agent and exosomes from the second binding agent comprises proteolytically cleaving the complexes comprising the first binding agent and exosomes, preferably with trypsin. Item 7: The method according to any one of items 1 to 6, further comprising adding a positive control, wherein the positive control comprises liposomes, wherein the liposomes preferably comprise phosphatidylethanolamine and a proteolytically-cleavable peptide.

[0328] Item 8: The method according to any one of items 1 to 7, further comprising adding a negative control, wherein the negative control comprises liposomes comprising a trypsin-cleavable peptide that is different from the trypsin-cleavable peptide of the positive control, and wherein the liposomes does not comprise phosphatidylethanolamine.

[0329] Item 9: The method of any one of items 1 to 8, wherein the sample is a cell culture supernatant, a cell preparation obtained by lysing and / or centrifuging cells, or a body fluid, preferably wherein the body fluid is selected from the group consisting of plasma, serum, ascites, cerebral fluid, bone marrow, urine, faeces and bronco-alveolar washing.

[0330] Item 10: The method according to any one of items 1 to 9, wherein the sample is obtained from a subject known or suspected to suffer from a disease, preferably wherein the disease is a virus disease, bacterial disease, autoimmune disease, inflammatory disease or cancer.

[0331] Item 11: The method according to any one of items 1 to 10, wherein the first binding agent has the structure:

[0332] i) FITC - cinnamycin,

[0333] ii) Strep-tag - peptide linker - cinnamycin, or

[0334] iii) His-tag - peptide linker - cinnamycin.

[0335] preferably wherein the peptide linker comprises the sequence AGGRAVNEALRGGAK (SEQ ID NO: 2).

[0336] Item 12: A binding agent comprising fluoresceine directly covalently coupled to the N-terminus of cinnamycin.

[0337] Item 13: A kit comprising:

[0338] i) a first binding agent, wherein the first binding agent comprises

[0339] a) cinnamycin and fluoresceine isothiocyanate (FITC), wherein the FITC is covalently linked to cinnamycin, or

[0340] b) a protein-tag linked to cinnamycin via a peptide linker, wherein the peptide linker comprises one or more proteolytic cleavage sites; and ii) instructions for binding of said first binding agent to exosomes in a sample. Item 14: The kit according to item 13, further comprising a second binding agent capable of binding to or bound to the first binding agent. Item 15: The kit according to item 14, wherein the second binding agent comprises a label, preferably a magnetic bead.

[0341] LITERATURE

[0342] Böing AN, van der Pol E, Grootemaat AE, Coumans FA, Sturk A, Nieuwland R (2014). Single-step isolation of extracellular vesicles by size-exclusion chromatography. Journal of Extracellular Vesicles. 3: 23430.

[0343] Blanc L and Vidal M. New insights into the function of Rab GTPases in the context of exosomeal secretion. Small GTPases 2018. Vol. 9, NOS 1-2, 95-106.

[0344] Harding C, Heuser J, Stahl P. Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes. J. Cell Biol. 1983;97:329-339.

[0345] Hench IB, Hench J, Tolnay M. Liquid Biopsy in Clinical Management of Breast, Lung, and Colorectal Cancer. Frontiers in Medicine 2018. Vol. 5, Article 9.

[0346] Jia et al. Exosome: emerging biomarker in breast cancer. Oncotarget. 2017; 8:41717-41733.

[0347] Johnstone RM, Bianchini A, Teng K. Reticulocyte maturation and exosome release: transferrin receptor containing exosomes shows multiple plasma membrane functions. Blood 1989;74: 1844-1851.

[0348] Leuenberger HGW, Nagel B and Kölbl H. eds. 1995. Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0349] Li P, Kaslan M, Lee SH, Yao J and Gao Z. Progress in Exosome Isolation Techniques. Theranosics 2017. Vol. 7 (3): 789-804.

[0350] Makino A, Baba T, Fujimoto K, Umeda M, Matsuzaki K, Kobayashi T. Cinnamycin (Ro 09-0198) Promotes Cell Binding and Toxicity by Inducing Transbilayer Lipid Movement. J Biol Chem. 2003, 278(5): 3204-3209.

[0351] McAndrews K and Kallun R. Mechanisms associated with biogenesis of exosomes in cancer. Molecular Cancer 2019. DOI.org / 10.1186 / s12943-019-0963-9.

[0352] Pan BT, Johnstone RM. Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor. Cell 1983;33:967-978.

[0353] Savina A, Vidal M, Colombo MI. The exosome pathwayin K562 cells is regulated by Rabll. J Cell Sci 2002;115:2505-15; PMID: 12045221. Tauro BJ, Greening DW, Mathias RA, Ji H, Mathivanan S, Scott AM, Simpson RJ (February 2012). "Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes". Methods. 56 (2): 293–304.

[0354] Thind A, Wilson C (2016). " Exosomal miRNAs as cancer biomarkers and therapeutic targets". Journal of Extracellular Vesicles. 5: 31292.

[0355] van der Pol E, Böing AN, Harrison P, Sturk A, Nieuwland R (July 2012). "Classification, functions, and clinical relevance of extracellular vesicles". Pharmacological Reviews. 64 (3): 676–705.

[0356] Van Deun J, Mestdagh P, Sormunen R, Cocquyt V, Vermaelen K, Vandesompele J, Bracke M, De Wever O, Hendrix A (2014). " The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling". Journal of Extracellular Vesicles. 3: 24858.

[0357] Vidal M, Mangeat P, Hoekstra D. Aggregation reroutesmolecules from a recycling to a vesicle-mediated secre-tion pathway during reticulocyte maturation. J Cell Sci 1997; 110(16): 1867-77; PMID:9296387.

Claims

CLAIMS1. A method for separating and / or isolating exosomes from a sample, the method comprising the steps of:i) providing a sample that is known to comprise or suspected of comprising exosomes;ii) contacting a first binding agent with the sample and allowing the binding agent to bind to exosomes, wherein the first binding agent comprisesa protein-tag linked to cinnamycin via a peptide linker, wherein the peptide linker comprises one or more proteolytic cleavage sites;iii) separating and / or isolating complexes of the first binding agent and exosomes from the sample.

2. The method according to claim 1, wherein the peptide linker comprises a TEV protease cleavage site, preferably a TEV protease cleavage site and a trypsin cleavage site.

3. The method according to claim 1 or 2, wherein a second binding agent is bound to the protein-tag of the first binding agent, preferably wherein the second binding agent comprises a label, more preferably a magnetic bead.

4. The method according to claim 1 or 2, further comprising after step ii) a step of adding a second binding agent to the sample, wherein the second binding agent is capable of binding to the protein-tag of the first binding agent, preferably wherein the second binding agent comprises a label, more preferably a magnetic bead.

5. The method according to claim 3 or 4, wherein the second binding agent comprises an antibody or antigen binding fragment thereof that specifically binds to the protein-tag of the first binding agent.

6. The method according to any one of claims 3 to 5, further comprising after step iii) a step of releasing the complexes of the first binding agent and exosomes from the second binding agent.

7. The method according to claim 6, wherein the releasing the complexes of the first binding agent and exosomes from the second binding agent comprises proteolytically cleaving the complexes comprising the first binding agent and exosomes, preferably with trypsin.

8. The method according to any one of claims 1 to 7, further comprising adding a positive control, wherein the positive control comprises liposomes, wherein the liposomes preferably comprise phosphatidylethanolamine and a proteolytically-cleavable peptide.

9. The method according to any one of claims 1 to 8, further comprising adding a negative control, wherein the negative control comprises liposomes comprising a trypsin-cleavable peptide that is different from the trypsin-cleavable peptide of the positive control, and wherein the liposomes do not comprise phosphatidylethanolamine.

10. The method of any one of claims 1 to 9, wherein the sample is a cell culture supernatant, a cell preparation obtained by lysing and / or centrifuging cells, or a body fluid, preferably wherein the body fluid is selected from the group consisting of plasma, serum, ascites, cerebral fluid, bone marrow, urine, faeces and bronco-alveolar washing.

11. The method according to any one of claims 1 to 10, wherein the sample is obtained from a subject known or suspected to suffer from a disease, preferably wherein the disease is a virus disease, bacterial disease, autoimmune disease, inflammatory disease or cancer.

12. The method according to any one of claims 1 to 11, wherein the first binding agent has the structure:i) Strep-tag - peptide linker - cinnamycin,ii) His-tag - peptide linker - cinnamycin, oriii) Biotin - peptide linker - cinnamycin,preferably wherein the peptide linker comprises the sequence SEQ ID NO: 2 or SEQ ID NO: 7, most preferably wherein the peptide linker comprises the sequence of SEQ ID NO: 7.

13. A kit comprising:i) a first binding agent, wherein the first binding agent comprisesa protein-tag linked to cinnamycin via a peptide linker, wherein the peptide linker comprises one or more proteolytic cleavage sites; and ii) instructions for binding of said first binding agent to exosomes in a sample.

14. The kit according to claim 13, further comprising a second binding agent capable of binding to or bound to the first binding agent.

15. The kit according to claim 14, wherein the second binding agent comprises a label, preferably a magnetic bead.

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