Tissue-specific extracellular vesicle isolation and verification

Abstract

A method of identifying a biomarker present in a defined category of tissue in an organism at an elevated level when compared to a level in at least one different category of tissue, wherein the biomarker is detectable on extracellular vesicles secreted from cells of the defined category of tissue. The method comprises the steps of: (i) identifying a candidate biomarker; (ii) providing a binding agent capable of specifically binding to the candidate biomarker identified in step i. (iii) detecting the presence of a candidate biomarker on extracellular vesicles obtained from a sample of defined categories of tissue by binding a binding agent to the candidate biomarker in the sample, where the candidate biomarker is not present on the extracellular vesicles obtained from the sample of defined categories of tissue, compared to the extracellular vesicles from at least one different category of tissue of the organism, extracellular vesicles from defined categories of tissue preferentially bind to a binding agent; and (iv) selecting the candidate biomarker as a biomarker present at an elevated level in the defined category of tissue. Also provided are methods of enriching a biological sample comprising a mixture of extracellular vesicles to generate an enriched fraction of extracellular vesicles from defined categories of tissue. Also provided are methods of analyzing the contents of extracellular vesicles in an enriched fraction of extracellular vesicles from defined categories of tissue.

Description

Technical Field

[0001] This invention relates to a method for identifying a biomarker present at an elevated level in a tissue of a defined category when compared to levels in at least one different category of tissues in an organism, wherein the biomarker is detectable on or in extracellular vesicles secreted from cells of the defined category of tissue. The invention also relates to methods for isolating or enriching biological samples composed of extracellular vesicles from cells of a defined category of tissue, methods for analyzing biological samples enriched in extracellular vesicles of a defined category of tissue, and methods for detecting one or more biomarkers on or in extracellular vesicles secreted from a defined category of tissue. Furthermore, the invention relates to the use of one or more biomarkers for generating enriched fractions of extracellular vesicles, the use of one or more binding agents for generating enriched fractions of extracellular vesicles, a kit for enriching a biological sample comprising a mixture of extracellular vesicles to generate enriched fractions of extracellular vesicles, and a method for manufacturing the kit thereof, and relates to enriched fractions of extracellular vesicles. Background Technology

[0002] Extracellular vesicles (EVs) are heterogeneous lipid bilayer-encapsulated particles, such as exosomes, microvesicles, ectosomes, oncosomes, and apoptotic bodies, which are naturally secreted by cells in tissues and circulate in the extracellular space of biological fluids such as blood. EVs play an important role in regulating cellular homeostasis and intercellular communication. Specifically, they function by (1) removing potentially erroneously produced or excessive components from cells (e.g., due to abnormal functions associated with existing diseases), and (2) carrying bioactive molecules to recipient cells, either locally or distantly (e.g., to prevent or spread disease).

[0003] Therefore, EVs are rich in biomarkers such as proteins, sugars, nucleic acid molecules, lipids, and metabolites, which reflect their molecular composition at the cellular level. Thus, EVs derived from specific tissues serve as a dynamic cyclic snapshot of tissue activity that can be obtained through liquid biopsy.

[0004] It is estimated that 99.8% of total EVs in the blood originate from cells naturally present in blood tissues, including but not limited to platelets, erythrocytes, and lymphocytes; 0.16% originate from adipose tissue; and only 0.03% originate from vital tissues, including but not limited to muscle, brain, skin, colon, lungs, liver, heart, or pancreas (Li et al., 2020). For example, it is estimated that only 0.0362% of EVs in the blood originate from the liver.

[0005] The isolation of enriched EV samples from a given tissue in biofluids is not trivial, but rather of concern in many clinical applications, from diagnosis to treatment.

[0006] Conventional methods for EV separation include ultracentrifugation, filtration, precipitation, and size-based microfluidic enrichment, and involve the batch separation of EVs based on their physical properties (i.e., size and / or density). However, these methods cannot distinguish the cellular origin of EVs in a sample.

[0007] More recent methods have described the separation of target populations of EVs using immune affinity-based capture techniques.

[0008] A study by Tauro et al. (2012) compared different capture methods for isolating exosomes from LIM1863 colorectal cancer cell culture medium, including ultracentrifugation, density gradient separation, and immunoaffinity capture using magnetic beads coated with anti-EpCAM (a universal surface marker on epithelial cells). They found that immunoaffinity capture was the most efficient separation method compared to differential centrifugation and density gradient methods.

[0009] Dias et al. (2024) have reported an electro-optical platform called Nano-Extracellular Omics Sensing for ultrasensitive detection of EV subtypes and their target proteins. This platform combines electrical detection (E-NEXOS) and optical detection (O-NEXOS) for the detection and characterization of EVs.

[0010] The platform was validated in vitro after separating EVs secreted from breast cancer cell lines MCF-7 and BT-474 using magnetic beads coated with anti-CD9 or anti-CD81 antibodies. O-NEXOS and E-NEXOS demonstrated higher sensitivity than currently available technologies and were compatible with EVs derived from biological fluids. The E-NEXOS and O-NEXOS detection methods are also disclosed in WO2019 / 211622, WO2021 / 084116, and WO2022 / 122768, which are incorporated herein by reference. Karimi et al. (2022) previously reported the use of O-NEXOS as a sensitive tool for the universal detection of EVs separated from serum and plasma samples using magnetic beads coated with antibodies against the tetraspan proteins CD63, CD81, or CD9 for the detection of CD41. + EV subtype. WO2021 / 260231 is incorporated herein by reference and discloses a two-step method for separating target EVs.

[0011] Sun et al. (2020) reported a method for isolating hepatocellular carcinoma (HCC) EVs. This method involves click chemistry-mediated EV capture followed by disulfide bond cleavage-driven EV release (referred to as an “EV click chip”), conjugated with an antibody mixture targeting three HCC-associated surface markers (EpCAM, ASGPR1, and CD147). The method was evaluated in artificial plasma samples incorporating EVs derived from the HCC cell line HepG2.

[0012] In vitro artificial plasma samples and cell culture systems are simplified systems for modeling the in vivo environment. However, such in vitro models cannot accurately simulate the in vivo environment and compromise the accuracy of the biological system being studied. Therefore, in vitro models cannot provide an accurate model for validating the presence of biomarkers at elevated levels in defined categories of tissues (e.g., liver, lung, etc.).

[0013] In a large study, Hoshino et al. (2020) analyzed 426 human plasma samples by mass spectrometry and identified that typical exosome markers in EVs derived from cell culture systems do not represent typical markers found in EVs derived from vivo.

[0014] The methodological sensitivity required to detect EV biomarkers from more complex in vivo systems (e.g., tissues from biofluids) is higher than that required to detect EV biomarkers from in vitro models. Therefore, existing techniques cannot determine the presence of identified biomarkers at elevated levels in EVs from specific tissues derived from biofluids.

[0015] Sun et al. (2023) further reported a method for isolating HCC-derived EVs from plasma obtained from HCC patients. This method involves incubating a sample with (1) HCC-associated antibodies against HCC-associated surface markers (EpCAM, CD147, GPC3, and ASGPR1) conjugated to trans-cyclooctene (TCO), (2) DNA-conjugated anti-CD63 and anti-CD9 antibodies for EV detection, and (3) methyltetraazine [mTz]-modified microbeads to covalently link the antibodies to the microbeads via click chemistry. The method is described as being intended for the early detection of HCC. This technique does not involve isolating EVs from whole tissue or from blood or blood derivatives derived from liver tissue; therefore, the tissue origin of the detected EVs is not confirmed, and some of the markers used are not specific to or enriched in the liver.

[0016] WO2023 / 074541 reports a method for selecting tissue-specific EV biomarkers and a purification method for purifying EVs based on their tissue of origin using the selected biomarkers. Tissue-specific EV biomarkers are identified using RNA sequencing data from the Human Protein Atlas (HPA) as a reference, whereby molecules expressed in a tissue-specific manner are found to be expressed at least four-fold in the target tissue compared to other tissues. The problem with WO2023 / 074541 is that its operation is based on the assumption that biomarkers identified using RNA sequencing data not only reflect protein concentrations in different tissues but also reflect which proteins are internalized onto EVs during their biogenesis and localized within or across the membrane of the secreted EVs for specific capture from the target tissue compared to other tissues. WO2023 / 074541 does not confirm that EVs from the target tissue are preferentially isolated or detected.

[0017] There remains a need for accurate methods to identify biomarkers present at elevated levels in tissues that define a class of organisms and to isolate EVs from complex samples for downstream applications. These downstream applications include, but are not limited to, collecting EVs from tissues that define a class of organisms from liquid biopsies, which are minimally invasive or non-invasive compared to conventional tissue biopsies.

[0018] Traditional tissue biopsies are not available for all patients, and liquid biopsies offer a promising alternative if information from tissues of defined categories can be obtained via blood or other biological fluids. Summary of the Invention

[0019] This invention stems from the recognition that biomarkers in EVs obtained from tissue samples of defined categories can be detected and validated using detection methods with sufficient sensitivity. Since there is no better representative of tissue-derived EVs in their in vivo environment than the tissue itself (tissue samples are a more accurate reflection of the in vivo environment), this invention enables the in vitro identification of biomarkers that can be sufficiently validated as being present at elevated levels in EVs from a specific category of tissue compared to at least one other tissue. This method achieves the selection of truly tissue-specific or enriched biomarkers in EVs derived from target tissues, thereby overcoming the lack of evidence present when translating proteomics data into EVs from information stored in bioinformatics databases. To date, there are no bioinformatics databases of tissue-specific biomarkers. Using this invention, such biomarkers can thus be identified and used to characterize EVs as originating from a specific category of tissue. Furthermore, these biomarkers can be used to isolate EVs from a specific category of tissue in complex biofluids containing a mixture of EVs derived from several or all tissues in an organism.

[0020] According to a first aspect of the invention, a method is provided for identifying a biomarker present in an organism at an elevated level compared to levels in at least one different type of tissue, in a tissue defining a specific category, wherein the biomarker is detectable on or in extracellular vesicles secreted from cells of the tissue defining the category, the method comprising the steps of: i. Identify candidate biomarkers; ii. Provide a binding agent that can specifically bind to the candidate biomarker identified in step i; iii. Detecting the presence of candidate biomarkers on extracellular vesicles obtained from samples of defined-category tissues by binding a binder to a candidate biomarker in a sample, wherein extracellular vesicles from defined-category tissues preferentially bind the binder compared to extracellular vesicles from at least one different-category tissue of the organism; iv. Select candidate biomarkers as those present at elevated levels in tissues within the defined category.

[0021] Preferably, step iii. includes using a device capable of operating at at least 1x10 7 Biomarkers / ml (preferably at least 1 x 10⁻⁶) 6 1x10 5 1x10 4 1x10 3 1x10 2 Techniques for detecting biomarkers at concentrations of 10 or 1 biomarker / ml are used to detect the presence of candidate biomarkers.

[0022] Alternatively, step iii. includes using a candidate biomarker at a rate of at least 1 x 10⁻⁶. 7 extracellular vesicles / ml (preferably at least 1 x 10⁻⁶) 6 1x10 5 1x10 4 1x10 3 1x10 2 The technique of detecting extracellular vesicles at concentrations of 10 or 1 extracellular vesicle / ml is used to detect the presence of candidate biomarkers.

[0023] Conveniently, step iii. further includes determining the concentration of biomarkers in the sample.

[0024] Advantageously, Step i. includes identifying multiple candidate biomarkers; Step ii. includes providing a variety of binding agents, each of which can specifically bind to the corresponding candidate biomarker identified in step i; Step iii. includes detecting the presence of each of the candidate biomarkers on extracellular vesicles obtained from samples of defined-category tissues by binding a binder to the corresponding candidate biomarker in the sample, wherein extracellular vesicles from defined-category tissues preferentially bind combinations of multiple binders compared to extracellular vesicles from at least one different category of tissue in the organism; and Step iv. involves selecting a combination of multiple candidate biomarkers as a group of biomarkers present at elevated levels in tissues of defined categories.

[0025] Advantageously, step iii. further includes contacting extracellular vesicles from at least one different class of tissues with a binder or each binder, and detecting the binding of extracellular vesicles from at least one different class of tissues to the binder or each binder.

[0026] Preferably, extracellular vesicles from tissues of a defined category preferentially bind to the binder or each binder, compared to extracellular vesicles from at least two different categories of tissues in an organism.

[0027] Conveniently, extracellular vesicles from tissues that define a specific category preferentially bind to the binder or each binder, compared to extracellular vesicles from all other categories of tissues in the organism.

[0028] Advantageously, step iii. further includes the step of detecting the presence of candidate biomarkers or each candidate biomarker on extracellular vesicles obtained from cultured cell lines representing the defined category of tissue.

[0029] Preferably, extracellular vesicles obtained from cultured cell lines representing defined categories of tissues preferentially bind to the binder compared to extracellular vesicles obtained from cultured cell lines representing at least one different category of tissue from an organism.

[0030] Conveniently, extracellular vesicles obtained from cultured cell lines are placed in plasma already containing extracellular vesicles.

[0031] Advantageously, the extracellular vesicles obtained from the tissue sample are in plasma that has been mixed with extracellular vesicles after they have been obtained from the tissue sample.

[0032] Preferably, step i includes the following steps: 1) Identify potential candidate biomarkers expressed in tissues that define the category; 2) Identify potential candidate biomarkers present in tissues of a defined category at higher levels than in at least one different category of tissues from an organism; 3) Identify potential candidate biomarkers as transmembrane or surface proteins; 4) Identify potential candidate biomarkers present in extracellular vesicles; or 5) Any combination of steps 1) through 4) performed in any order. And thus identify candidate biomarkers.

[0033] Conveniently, step i includes the following steps: 1) Identify potential candidate biomarkers expressed in tissues that define the category; 2) Identify potential candidate biomarkers from those identified in step 1) that exist at higher levels in the defined category of tissues than in at least one different category of tissues from the organism; 3) Identify potential candidate biomarkers as transmembrane or surface proteins from among the potential candidate biomarkers identified in step 2).

[0034] 4) Validate one candidate biomarker present in extracellular vesicles from the potential candidate biomarkers identified in step 3), or validate multiple candidate biomarkers present in extracellular vesicles from the potential candidate biomarkers identified in step 3).

[0035] Advantageously, step iii. further includes the step of grading and separating a precursor sample of a defined category of tissue rich in extracellular vesicles in order to provide a sample of the defined category of tissue.

[0036] Preferably, the candidate biomarker is a transmembrane or surface protein.

[0037] Alternatively, candidate biomarkers are cytoplasmic proteins.

[0038] Conveniently, candidate biomarkers are proteins with post-translational modifications.

[0039] Alternatively, candidate biomarkers are DNA molecules, RNA molecules, lipids, or post-translational modifications.

[0040] Advantageously, the binder or each binder includes an antibody, aptamer, lectin, lipid-binding protein or lipid-binding domain, or a DNA or RNA primer or probe.

[0041] Preferably, in step iii, the binder or each binder is provided attached to a substrate, preferably wherein the substrate comprises magnetic beads or nanoparticles, gold beads or nanoparticles, polystyrene beads, affinity chromatography columns, microplates, microfluidic channels, or biochips having surfaces made of gold, silicon oxide, glass, graphene, or polystyrene.

[0042] Conveniently, the tissue that defines the category is liver tissue, preferably wherein the cells that secrete extracellular vesicles from it are hepatocytes, Kupffer cells, stellate cells or resident dendritic cells of the liver.

[0043] Advantageously, the biomarkers present at elevated levels in the tissues defining the category, or each biomarker, are selected from ASGR1, ASGR2, TFR2, SLCO1B1, SLC38A3, TMEM56, UNC93A, SLC22A9, SLC2A2 and FXYD1 or combinations thereof, preferably wherein the biomarkers present at elevated levels in the tissues defining the category, or each biomarker, are selected from ASGR1, ASGR2, TFR2 and SLCO1B1 or combinations thereof.

[0044] Preferably, the biomarkers are present at elevated levels in tissues that define the category, or each biomarker consists of ASGR1, ASGR2, TFR2, and SLCO1B1.

[0045] Conveniently, the biomarker, or each biomarker being ASGR1 and the binding agent, or each binding agent being an anti-ASGR1 antibody or its antigen-binding fragment; the biomarker, or each biomarker being ASGR2 and the binding agent, or each binding agent being an anti-ASGR2 antibody or its antigen-binding fragment; the biomarker, or each biomarker being TFR2 and the binding agent, or each binding agent being an anti-TFR2 antibody or its antigen-binding fragment; the biomarker, or each biomarker being SLCO1B1 and the binding agent, or each binding agent being an anti-SLCO1B1 antibody or its antigen-binding fragment; the biomarker, or each biomarker being SLC38A3 and the binding agent, or each binding agent being an anti-SLC38A3 antibody or its antigen-binding fragment; the biomarker, or each biomarker being TMEM56 and the binding agent, or each binding agent being an anti-TMEM56 antibody or its antigen-binding fragment; the biomarker, or each biomarker being UNC93A and the binding agent, or each binding agent being an anti-UNC93A antibody or its antigen-binding fragment; the biomarker, or each... The biomarker is SLC22A9 and the binding agent is an anti-SLC22A9 antibody or its antigen-binding fragment; the biomarker is SLC2A2 and the binding agent is an anti-SLC2A2 antibody or its antigen-binding fragment; or the biomarker is FXYD1 and the binding agent is an anti-FXYD1 antibody or its antigen-binding fragment, or a combination thereof. Preferably, the biomarker is ASGR1 and the binding agent is an anti-ASGR1 antibody or its antigen-binding fragment; the biomarker is ASGR2 and the binding agent is an anti-ASGR2 antibody or its antigen-binding fragment; the biomarker is TFR2 and the binding agent is an anti-TFR2 antibody or its antigen-binding fragment; or the biomarker is SLCO1B1 and the binding agent is an anti-SLCO1B1 antibody or its antigen-binding fragment, or a combination thereof.

[0046] Advantageously, each binder is provided in proportion to 10%-50% of the total, wherein the total mixture of binders is equal to 100%, or wherein each binder is provided in equal proportion.

[0047] Preferably, the binder comprises a plurality of binders consisting of an anti-ASGR1 antibody or its antigen-binding fragment, an anti-ASGR2 antibody or its antigen-binding fragment, an anti-SLCO1B1 antibody or its antigen-binding fragment, and an anti-TFR2 antibody or its antigen-binding fragment, wherein the anti-ASGR1 antibody or its antigen-binding fragment is provided in a proportion of 30%-50% of the total, and each of the other antibodies or their antigen-binding fragments is provided in a proportion of 10%-30% of the total, wherein the total mixture of binders is equal to 100%.

[0048] Alternatively, the tissue that defines the category is blood tissue.

[0049] Advantageously, the biomarkers are specific to the tissue defining the category, or a combination of biomarkers is specific to the tissue defining the category.

[0050] Preferably, step iii. further includes the step of detecting the binding of the binder or each binder to the corresponding candidate biomarker on or in extracellular vesicles.

[0051] Conveniently, the tissue sample can be healthy or diseased tissue.

[0052] According to a second aspect of the present invention, a method for enriching a biological sample comprising a mixture of extracellular vesicles is provided, the method comprising the following steps: a) Identifying one or more biomarkers present in tissue at elevated levels by performing the method according to the first aspect of the invention; b) Capture extracellular vesicles in biological samples, wherein the extracellular vesicles exhibit the biomarkers identified in step a), or each of the biomarkers, to generate an enrichment fraction of extracellular vesicles from tissues of defined categories.

[0053] Advantageously, the extracellular vesicle mixture comprises extracellular vesicles from tissues of a defined category and extracellular vesicles from at least one different category of tissues of the organism.

[0054] Conveniently, in step b), one or more binding agents capable of specifically binding to a biomarker or each biomarker are used to capture extracellular vesicles.

[0055] Preferably, step b) further includes isolating extracellular vesicles of the biomarker or each biomarker captured using the biomarker or each biomarker, to demonstrate the biomarker or each biomarker identified in step a).

[0056] Conveniently, isolated extracellular vesicles are intact extracellular vesicles.

[0057] Advantageously, the method further includes the step of releasing the contents of the captured or isolated extracellular vesicles.

[0058] According to a third aspect of the present invention, a method for analyzing a biological sample comprising a mixture of extracellular vesicles is provided, the method comprising the following steps: a) Identifying biomarkers present in tissues at elevated levels by performing the method according to the first aspect of the invention; b) Capture extracellular vesicles in a biological sample, wherein the extracellular vesicles display the biomarkers identified in step a), or each biomarker, to generate an enrichment fraction of the extracellular vesicles; and c) Analyze the contents of extracellular vesicles in the enrichment fraction obtained in step b).

[0059] Conveniently, the extracellular vesicle mixture comprises extracellular vesicles from tissues of defined categories and extracellular vesicles from at least one different category of tissues in an organism.

[0060] Preferably, in step b), one or more binding agents capable of specifically binding to a biomarker or each biomarker are used to capture extracellular vesicles.

[0061] Advantageously, step b) further includes isolating extracellular vesicles of the biomarker or each biomarker captured using the biomarker or each biomarker identified in step a).

[0062] Preferably, the isolated extracellular vesicles are intact extracellular vesicles.

[0063] Conveniently, the method further includes the step of releasing the contents of the captured or isolated extracellular vesicles.

[0064] Advantageously, the biological sample is a biological fluid, preferably blood, urine, saliva, lymph, bile, cerebrospinal fluid, sputum, mucus, tears, bronchoalveolar lavage (BAL) fluid, earwax, sweat, feces, breast milk, tissue fluid, vaginal fluid, semen, gastric juice, vesicular fluid, or cystic fluid.

[0065] Preferably, step b) includes capturing extracellular vesicles in a biological sample using a binder or each binder.

[0066] According to a fourth aspect of the present invention, a method for enriching a biological sample comprising a mixture of extracellular vesicles is provided, the method comprising the following steps: Capture extracellular vesicles in biological samples, wherein the extracellular vesicles display one or more biomarkers, to generate an enrichment fraction of extracellular vesicles. The biomarkers, or each of the biomarkers, are selected from ASGR1, ASGR2, TFR2, SLCO1B1, SLC38A3, TMEM56, UNC93A, SLC22A9, SLC2A2 and FXYD1 or combinations thereof, preferably selected from ASGR1, ASGR2, TFR2 and SLCO1B1 or combinations thereof.

[0067] According to a fifth aspect of the present invention, a method for analyzing a biological sample comprising a mixture of extracellular vesicles is provided, the method comprising the following steps: a) Capturing extracellular vesicles in biological samples, wherein the extracellular vesicles display one or more biomarkers to generate an enrichment fraction of extracellular vesicles; and b) Analyze the contents of extracellular vesicles in the enrichment fraction obtained in step a). The biomarkers, or each of the biomarkers, are selected from ASGR1, ASGR2, TFR2, SLCO1B1, SLC38A3, TMEM56, UNC93A, SLC22A9, SLC2A2 and FXYD1 or combinations thereof, preferably selected from ASGR1, ASGR2, TFR2 and SLCO1B1 or combinations thereof.

[0068] Conveniently, the step of capturing extracellular vesicles includes capturing extracellular vesicles based on extracellular vesicles displaying one or more biomarkers, wherein one or more of the biomarkers consists of ASGR1, ASGR2, TFR2, and SLCO1B1.

[0069] Advantageously, one or more binding agents capable of specifically binding to a biomarker or each biomarker are used to capture extracellular vesicles.

[0070] Preferably, the biomarker, or each biomarker is ASGR1 and the binding agent, or each binding agent is an anti-ASGR1 antibody or its antigen-binding fragment; the biomarker, or each biomarker is ASGR2 and the binding agent, or each binding agent is an anti-ASGR2 antibody or its antigen-binding fragment; the biomarker, or each biomarker is TFR2 and the binding agent, or each binding agent is an anti-TFR2 antibody or its antigen-binding fragment; the biomarker, or each biomarker is SLCO1B1 and the binding agent, or each binding agent is an anti-SLCO1B1 antibody or its antigen-binding fragment; the biomarker, or each biomarker is SLC38A3 and the binding agent, or each binding agent is an anti-SLC38A3 antibody or its antigen-binding fragment; the biomarker, or each biomarker is TMEM56 and the binding agent, or each binding agent is an anti-TMEM56 antibody or its antigen-binding fragment; the biomarker, or each biomarker is UNC93A and the binding agent, or each binding agent is an anti-UNC93A antibody or its antigen-binding fragment; the biomarker, or each... The biomarker is SLC22A9 and the binding agent is an anti-SLC22A9 antibody or its antigen-binding fragment; the biomarker is SLC2A2 and the binding agent is an anti-SLC2A2 antibody or its antigen-binding fragment; or the biomarker is FXYD1 and the binding agent is an anti-FXYD1 antibody or its antigen-binding fragment, or a combination thereof. Preferably, the biomarker is ASGR1 and the binding agent is an anti-ASGR1 antibody or its antigen-binding fragment; the biomarker is ASGR2 and the binding agent is an anti-ASGR2 antibody or its antigen-binding fragment; the biomarker is TFR2 and the binding agent is an anti-TFR2 antibody or its antigen-binding fragment; or the biomarker is SLCO1B1 and the binding agent is an anti-SLCO1B1 antibody or its antigen-binding fragment, or a combination thereof.

[0071] Conveniently, each binder is provided in proportions of 10% to 50% of the total, wherein the total mixture of binders is equal to 100%, or wherein each binder is provided in equal proportions.

[0072] Advantageously, the conjugate comprises a plurality of conjugates consisting of an anti-ASGR1 antibody or an antigen-binding fragment thereof, an anti-ASGR2 antibody or an antigen-binding fragment thereof, an anti-SLCO1B1 antibody or an antigen-binding fragment thereof, and an anti-TFR2 antibody or an antigen-binding fragment thereof, wherein the anti-ASGR1 antibody or an antigen-binding fragment thereof is provided in a proportion of 30%-50% of the total, and each of the other antibodies or antigen-binding fragments thereof is provided in a proportion of 10%-30% of the total, wherein the total mixture of conjugates is equal to 100%.

[0073] Preferably, the extracellular vesicle mixture comprises extracellular vesicles from tissues of a defined category and extracellular vesicles from at least one different category of tissues of the organism, and wherein the biomarker, or each biomarker, is present at an elevated level in the tissues of the defined category.

[0074] Conveniently, the method further includes isolating extracellular vesicles displaying the biomarker or each biomarker captured using the biomarker or each biomarker.

[0075] Advantageously, the isolated extracellular vesicles are intact extracellular vesicles.

[0076] Preferably, the method further includes the step of releasing the contents of the captured or isolated extracellular vesicles.

[0077] Conveniently, the biological sample is a biological fluid, preferably blood, urine, saliva, lymph, bile, cerebrospinal fluid, sputum, mucus, tears, bronchoalveolar lavage (BAL) fluid, earwax, sweat, feces, breast milk, tissue fluid, vaginal fluid, semen, gastric juice, vesicular fluid, or cystic fluid.

[0078] Advantageously, the step of capturing extracellular vesicles in a biological sample includes capturing extracellular vesicles in the biological sample using a binder or each binder.

[0079] According to a sixth aspect of the invention, there is provided the use of one or more biomarkers displayed on extracellular vesicles for generating enrichment fractions of extracellular vesicles, wherein the enrichment fractions of extracellular vesicles are secreted from tissues that define categories in an organism, wherein the biomarkers, or each biomarker, are selected from ASGR1, ASGR2, TFR2, SLCO1B1, SLC38A3, TMEM56, UNC93A, SLC22A9, SLC2A2 and FXYD1 or combinations thereof, preferably selected from ASGR1, ASGR2, TFR2 and SLCO1B1 or combinations thereof.

[0080] Preferably, the biomarkers consist of ASGR1, ASGR2, TFR2, and SLCO1B1.

[0081] Conveniently, enrichment fractions of extracellular vesicles can be enriched from biological samples containing mixtures of extracellular vesicles.

[0082] Advantageously, the biological sample is a biological fluid, preferably blood, urine, saliva, lymph, bile, cerebrospinal fluid, sputum, mucus, tears, bronchoalveolar lavage (BAL) fluid, earwax, sweat, feces, breast milk, tissue fluid, vaginal fluid, semen, gastric juice, vesicular fluid, or cystic fluid.

[0083] According to a seventh aspect of the invention, there is provided the use of one or more binding agents for generating enrichment fractions of extracellular vesicles, wherein the enrichment fractions of extracellular vesicles are secreted from tissues that define categories in an organism, wherein the binding agent or each binding agent is capable of specifically binding to a biomarker displayed on the extracellular vesicle, wherein the binding agent or each binding agent is selected from anti-ASGR1 antibody or its antigen-binding fragment, anti-ASGR2 antibody or its antigen-binding fragment, anti-TFR2 antibody or its antigen-binding fragment, anti-SLCO1B1 antibody or its antigen-binding fragment, anti-SLC38A3 antibody or its antigen-binding fragment, anti-TMEM56 antibody or its antigen-binding fragment, anti-UNC93A antibody or its antigen-binding fragment, anti-SLC22A9 antibody or its antigen-binding fragment, anti-SLC2A2 antibody or its antigen-binding fragment, and anti-FXYD1 antibody or its antigen-binding fragment, or combinations thereof, preferably selected from anti-ASGR1 antibody or its antigen-binding fragment, anti-ASGR2 antibody or its antigen-binding fragment, anti-TFR2 antibody or its antigen-binding fragment, or anti-SLCO1B1 antibody or its antigen-binding fragment, or combinations thereof.

[0084] Preferably, enrichment fractions of extracellular vesicles are enriched from biological samples containing a mixture of extracellular vesicles.

[0085] According to an eighth aspect of the invention, a method is provided for detecting one or more biomarkers on or in extracellular vesicles secreted from tissues of a defined category, wherein the biomarkers, or each biomarker, are selected from ASGR1, ASGR2, TFR2, SLCO1B1, SLC38A3, TMEM56, UNC93A, SLC22A9, SLC2A2 and FXYD1 or combinations thereof, preferably selected from ASGR1, ASGR2, TFR2 and SLCO1B1 or combinations thereof.

[0086] Conveniently, the method includes multiple biomarkers, including ASGR1, ASGR2, TFR2, and SLCO1B1.

[0087] Advantageously, the biomarker, or each biomarker, is detected by binding with one or more conjugates, wherein the biomarker, or each biomarker, is ASGR1 and the conjugate, or each conjugate, is an anti-ASGR1 antibody or its antigen-binding fragment; the biomarker, or each biomarker, is ASGR2 and the conjugate, or each conjugate, is an anti-ASGR2 antibody or its antigen-binding fragment; the biomarker, or each biomarker, is TFR2 and the conjugate, or each conjugate, is an anti-TFR2 antibody or its antigen-binding fragment; the biomarker, or each biomarker, is SLCO1B1 and the conjugate, or each conjugate, is an anti-SLCO1B1 antibody or its antigen-binding fragment; the biomarker, or each biomarker, is SLC38A3 and the conjugate, or each conjugate, is an anti-SLC38A3 antibody or its antigen-binding fragment; the biomarker, or each biomarker, is TMEM56 and the conjugate, or each conjugate, is an anti-TMEM56 antibody or its antigen-binding fragment; the biomarker, or each biomarker, is UNC93A and the conjugate, or each conjugate, is an anti-UNC93A antibody. Or its antigen-binding fragment, biomarker or each biomarker being SLC22A9 and the binding agent or each binding agent being an anti-SLC22A9 antibody or its antigen-binding fragment, biomarker or each biomarker being SLC2A2 and the binding agent or each binding agent being an anti-SLC2A2 antibody or its antigen-binding fragment, or biomarker or each biomarker being FXYD1 and the binding agent or each binding agent being an anti-FXYD1 antibody or its antigen-binding fragment, or combinations thereof, preferably wherein the biomarker or each biomarker is ASGR1 and the binding agent or each binding agent is an anti-ASGR1 antibody or its antigen-binding fragment, biomarker or each biomarker being ASGR2 and the binding agent or each binding agent being an anti-ASGR2 antibody or its antigen-binding fragment, biomarker or each biomarker being TFR2 and the binding agent or each binding agent being an anti-TFR2 antibody or its antigen-binding fragment, or biomarker or each biomarker being SLCO1B1 and the binding agent or each binding agent being an anti-SLCO1B1 antibody or its antigen-binding fragment, or combinations thereof.

[0088] Preferably, the biomarker or each biomarker is detected on or in extracellular vesicles secreted by tissues that define a category from a biological sample containing a mixture of extracellular vesicles.

[0089] According to a ninth aspect of the invention, a kit is provided for enriching a biological sample comprising a mixture of extracellular vesicles to generate an enrichment fraction of extracellular vesicles, wherein the enrichment fraction of extracellular vesicles is secreted from tissues of a defined class in an organism, wherein the kit comprises one or more binding agents capable of specifically binding to biomarkers present at elevated levels in tissues of a defined class, and wherein the binding agent, or each binding agent, is selected from anti-ASGR1 antibody or its antigen-binding fragment, anti-ASGR2 antibody or its antigen-binding fragment, anti-TFR2 antibody or its antigen-binding fragment, anti-SLC... O1B1 antibody or its antigen-binding fragment, anti-SLC38A3 antibody or its antigen-binding fragment, anti-TMEM56 antibody or its antigen-binding fragment, anti-UNC93A antibody or its antigen-binding fragment, anti-SLC22A9 antibody or its antigen-binding fragment, anti-SLC2A2 antibody or its antigen-binding fragment, and anti-FXYD1 antibody or its antigen-binding fragment, or combinations thereof, preferably selected from anti-ASGR1 antibody or its antigen-binding fragment, anti-ASGR2 antibody or its antigen-binding fragment, anti-TFR2 antibody or its antigen-binding fragment, or anti-SLCO1B1 antibody or its antigen-binding fragment, or combinations thereof.

[0090] Conveniently, the extracellular vesicle mixture comprises extracellular vesicles from tissues of defined categories and extracellular vesicles from at least one different category of tissues in an organism.

[0091] Advantageously, the biological sample is a biological fluid, preferably blood, urine, saliva, lymph, bile, cerebrospinal fluid, sputum, mucus, tears, bronchoalveolar lavage (BAL) fluid, earwax, sweat, feces, breast milk, tissue fluid, vaginal fluid, semen, gastric juice, vesicular fluid, or cystic fluid.

[0092] Preferably, the binder or each binder comprises an anti-ASGR1 antibody or its antigen-binding fragment, an anti-ASGR2 antibody or its antigen-binding fragment, an anti-SLCO1B1 antibody or its antigen-binding fragment, and an anti-TFR2 antibody or its antigen-binding fragment.

[0093] Conveniently, methods, uses, or kits may include multiple binding agents consisting of anti-ASGR1 antibody, anti-ASGR2 antibody, anti-SLCO1B1 antibody, and anti-TFR2 antibody.

[0094] Advantageously, the binder or each binder is provided attached to a substrate, preferably wherein the substrate comprises magnetic beads or nanoparticles, gold beads or nanoparticles, polystyrene beads, affinity chromatography columns, microplates, microfluidic channels, or biochips having surfaces made of gold, silicon oxide, glass, graphene, or polystyrene.

[0095] Preferably, each binder is provided in proportion to 10%-50% of the total, wherein the total mixture of binders is equal to 100%, or wherein each binder is provided in equal proportion.

[0096] Conveniently, uses, methods, or kits comprise a plurality of conjugates consisting of an anti-ASGR1 antibody or an antigen-binding fragment thereof, an anti-ASGR2 antibody or an antigen-binding fragment thereof, an anti-SLCO1B1 antibody or an antigen-binding fragment thereof, and an anti-TFR2 antibody or an antigen-binding fragment thereof, wherein the anti-ASGR1 antibody or an antigen-binding fragment thereof is provided in a proportion of 30%-50% of the total, and each of the other antibodies or antigen-binding fragments thereof is provided in a proportion of 10%-30% of the total, wherein the total mixture of conjugates is equal to 100%.

[0097] According to a tenth aspect of the invention, an enrichment fraction of extracellular vesicles is provided, the fraction being enriched with extracellular vesicles secreted by tissues of a defined class in an organism, the extracellular vesicles displaying one or more biomarkers selected from the following: ASGR1, ASGR2, TFR2, SLCO1B1, SLC38A3, TMEM56, UNC93A, SLC22A9, SLC2A2 and FXYD1 or combinations thereof (preferably ASGR1, ASGR2, TFR2 and SLCO1B1 or combinations thereof).

[0098] Advantageously, extracellular vesicles secreted by tissues that define categories in an organism exhibit multiple biomarkers, including ASGR1, ASGR2, TFR2, and SLCO1B1.

[0099] Preferably, the tissue used to define the category is liver tissue.

[0100] According to the eleventh aspect of the present invention, a method for manufacturing a kit according to the ninth aspect of the present invention is provided, wherein the method comprises a co-localizing binder or each binder.

[0101] definition The terms “biomarker” or “biological marker” are used interchangeably herein to refer to naturally occurring molecules, such as proteins, post-translational modifications on proteins, genes, nucleic acid molecules (e.g., DNA or RNA), epigenetic modifications on nucleic acid molecules, lipids or metabolites, or portions thereof, which are measurable indicators of a specific state. The term “candidate biomarker” is used interchangeably herein as a “candidate tissue-specific biomarker” or “tissue-enriched biomarker” selected for evaluation as a biomarker present at elevated levels in tissues of a defined category and which can be used to capture extracellular vesicles from tissues of a defined category.

[0102] As used herein, the term "present at elevated levels" refers to the presence of elevated biomarkers in tissues that define a category, relative to the presence of biomarkers in at least one different category of tissues.

[0103] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are modified residues or non-naturally occurring residues (e.g., artificial chemical mimics of corresponding naturally occurring amino acids), as well as naturally occurring amino acid polymers. In some embodiments, the phrase “transmembrane protein,” as used herein, refers to a class of integrated membrane proteins that span the entire plasma membrane. In some embodiments, the phrase “surface protein,” as used herein, refers to a class of integrated membrane proteins that are embedded in the plasma membrane and exposed on the outer side of the plasma membrane.

[0104] As used herein, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that have similar functions to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are modified post-translationally in the cell (e.g., hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine).

[0105] As used herein, the term "post-translational modification" refers to the biochemical modifications made to the amino acids in a protein after its biosynthesis. These post-translational modifications include, but are not limited to, acetylation, glycosylation, hydroxylation, lipidation, methylation, nitrosation, phosphorylation, proteolysis, and ubiquitination.

[0106] The terms “gene,” “polynucleotide,” and “nucleic acid molecule” are used interchangeably herein to refer to a polymer of multiple nucleotides. Nucleic acid molecules may include naturally occurring nucleic acids (i.e., DNA or RNA) or may include artificial nucleic acids such as peptide nucleic acids, morpholine and locked nucleic acids, as well as glycol nucleic acids and threonic acid.

[0107] As used in this article, the term "epigenetic modification" refers to the biochemical modification of nucleic acid molecules. These epigenetic modifications include, but are not limited to, DNA methylation.

[0108] As used in this article, the term "oligonucleotide" refers to a single-chain polymer of nucleotides.

[0109] As used herein, the term "nucleotide" refers to naturally occurring nucleotides and synthetic nucleotide analogues that are recognized by cellular enzymes. A nucleotide is the basic building block of nucleic acids (i.e., DNA or RNA) consisting of a sugar molecule attached to a phosphate group (deoxyribose in DNA or ribose in RNA) and a nitrogenous base. The bases used in DNA are adenine (A), cytosine (C), guanine (G), and thymine (T) in DNA, or uracil (U) in RNA. Double-stranded DNA and RNA are formed by two complementary nucleotide chains held together by hydrogen bonds between nucleotide pairs (G with C, and A with T or U). In this specification, the percentage of sequences "complementary" to another sequence (i.e., the target sequence) is determined using default parameters with sequencing methods such as the EMBOSS Needle pairwise sequence alignment (Rice et al., Trends Genet. 2000 Jun;16(6):276-7; Nucleic Acids Res. 2019 Jul 2;47(W1):W636-W641). Specifically, the EMBOSS Needle can be accessed online via the URL: https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / . Specifically, methods for determining oligonucleotide sequences that are 100% complementary to the target oligonucleotide sequence are known in the literature, for example using tools such as EMBOSSrevseq, which can be accessed online via the URL: https: / / www.bioinformatics.nl / cgi-bin / emboss / revseq. Sequencing methods such as pairwise sequence alignment using the EMBOSS Needle can then be used to determine the specific level of sequence identity between the complementary oligonucleotide sequence and the target oligonucleotide sequence.

[0110] As used in this article, the term "organism" refers to an individual animal, plant, or single-celled life form.

[0111] As used herein, the term "tissue" refers to a group or layer of cells that have similar structures and perform specific functions. An tissue may also contain groups or layers of different cells that work together to perform specific functions. The term "tissue" includes, but is not limited to, connective tissue (including, but not limited to, loose connective tissue, adipose tissue, dense fibrous connective tissue, elastic connective tissue, cartilage, bone tissue, and blood), epithelial tissue (including, but not limited to, simple squamous epithelium, stratified squamous epithelium, simple cuboidal epithelium, stratified cuboidal epithelium, simple columnar epithelium, stratified columnar epithelium, pseudostratified columnar epithelium, and transitional epithelium, as well as urothelial tissue), muscle tissue (including, but not limited to, cardiac tissue, smooth tissue, and skeletal tissue), neural tissue (including, but not limited to, neurons and glial cells), and liver tissue (including, but not limited to, hepatocytes, Kupffer cells, endothelial cells, and hepatic stellate cells). The phrase "defining category tissue" refers to tissue found or obtained in a specific organ or organ system. In one embodiment, the defining category tissue is liver tissue or blood tissue.

[0112] As used herein, the term "extracellular vesicle" refers to heterogeneous lipid bilayer-encapsulated particles naturally secreted by cells. EVs are non-replicating and circulate in the extracellular space of biological fluids. The term "extracellular vesicle" comprises various subtypes, including but not limited to exosomes, microvesicles, ectosomes, oncosomes, and apoptotic bodies. The term "extracellular vesicle" is also used interchangeably with the terms "extracellular nanoparticles" and "non-vesicular extracellular nanoparticles," which include, but are not limited to, superiors and exomers.

[0113] As used herein, the term "binding agent" refers to a naturally occurring or synthetic molecule capable of specifically binding to a target molecule on or within a target entity. In some embodiments, the term "binding agent" refers to a molecule capable of specifically binding to a candidate biomarker on or within an EV. The phrase "specifically binding" or "specifically binding" means that the binding agent preferentially binds to the target molecule compared to non-target molecules. The term "specifically binding" means that the antibody has a significantly higher affinity for its target peptide than for other related peptides. "significantly higher affinity" means that there is a substantial increase in affinity for the target peptide of the present invention compared to the affinity for other related peptides. In some embodiments, the affinity for the target peptide is at least 1.5 times, 2 times, 5 times, 10 times, 100 times, or 10 3 times, 10 4 times, 10 5 times, 10 6 Multiples or higher. In some implementations, the antibody binds with high affinity, with a dissociation constant of 10. -4M or lower, 10 -7 M or lower, 10 -9 M or lower; or binding with sub-nanomolar affinity (0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 nM or even less). For example, the binding specificity of the binder can be tested using an in vitro binding assay with purified protein.

[0114] The term "antigen-binding fragment" in antibody refers to a portion of the antibody that possesses antigen-binding activity, and includes Fab, F(ab')2, scFv, etc. The term also includes Fab', a monovalent fragment in the variable region of an antibody obtained by treating F(ab')2 under reducing conditions. However, the term is not limited to these molecules as long as the fragment has binding affinity for the antigen. Furthermore, these antigen-binding fragments include not only fragments obtained by treating the full-length molecule of the antibody protein with appropriate enzymes, but also proteins produced using genetically modified antibody genes in appropriate host cells.

[0115] As used herein, the term "capture" refers to the immobilization of a target entity relative to a substrate. In some embodiments, the term "capture" refers to the immobilization of an EV displaying a candidate biomarker in a sample (preferably a biological sample) using a binding agent.

[0116] As used herein, the term "biological sample" refers to a sample obtained from a living organism. In one embodiment, the biological sample is a tissue sample obtained by biopsy (e.g., tissue biopsy). In one embodiment, the biological sample (e.g., tissue biopsy material) is rapidly frozen. In an alternative embodiment, the biological sample is a biofluid. In one embodiment, the biological sample is a sample of whole blood, plasma, or serum. In some embodiments, the biological sample contains EV (extracellular EV). In embodiments where the biological sample is a biofluid, the biofluid contains circulating EV.

[0117] The terms “biofluid” or “biological fluid” are used interchangeably herein to refer to a liquid obtained from an organism, which helps transport cells, particles, vesicles and nutrients and remove waste from cells.

[0118] As used in this article, the term "plasma" refers to the liquid component of blood, excluding red blood cells, white blood cells, and platelets, in the presence of an anticoagulant.

[0119] As used in this article, the term "serum" refers to the liquid component of blood that does not include red blood cells, white blood cells, and platelets after the blood has clotted.

[0120] The terms plasma and serum can refer to either naturally occurring or artificially produced substances.

[0121] As used in this article, the term “artificial plasma” refers to a liquid formulated to mimic the properties of human plasma (Khan et al., 2020).

[0122] As used herein, the term "separation" refers to the separation of a target entity from a sample containing at least one other component. In some embodiments, the term "separation" refers to the separation of EVs displaying candidate biomarkers from a sample (preferably a biological sample) by using a binding agent to capture EVs.

[0123] As used herein, the term "enrichment fraction" refers to a fraction of the sample after separation that is primarily composed of the target entity. In some embodiments, the term "enrichment fraction" refers to a fraction of the sample (preferably a biological sample) after separation that is primarily composed of EVs.

[0124] The terms "detecting" or "detection" are used herein to refer to a measurable readout. In some embodiments, the term "detection" refers to a measurable readout of the presence of a candidate biomarker. In some embodiments, the level of detection of the presence of a candidate biomarker is quantified. In some embodiments, the presence of a candidate biomarker is detected with a desired sensitivity, defined by detecting the biomarker at a concentration in units of biomarker / ml. In some embodiments, the presence of a candidate biomarker is detected at a concentration in units of EV / mL. In some embodiments, the presence of a candidate biomarker is inferred from a signal output obtained using a technique for detection.

[0125] As used herein, the term "control" refers to a comparison against which a method can be evaluated. In one embodiment, the control is a negative control. In an alternative embodiment, the control is a positive control.

[0126] As used herein, the terms "cancer" and "tumor" refer to the presence of cells in a subject exhibiting novel abnormalities and / or uncontrolled proliferation. In one embodiment, the cells have the ability to invade adjacent tissues and / or spread to other sites in the body (i.e., the cells are capable of metastasis). In one embodiment, cancer cells present as a tumor (i.e., an abnormal mass of tissue). As used herein, the term "tumor" includes both benign and malignant growths. In one embodiment, the cancer is liver cancer. In some embodiments, liver cancer is HCC.

[0127] As used in this article, the term "in vivo" in vivo (The Latin word for "in vivo") refers to an experiment performed in a complete, living organism. As used in this text, the term "in vitro" is used in the context of experiments conducted in vitro. in vitro(Latin for "in glass") refers to experiments conducted on biological material cultured or stored in an artificial container. As used in this article, the term "ex vivo" (or "in vitro")... ex vivo ("Outside the living body" in Latin) refers to experiments conducted on biological material taken from living organisms.

[0128] As used herein, in a preferred embodiment, each of the biomarkers ASGR1, ASGR2, TFR2, SLCO1B1, SLC38A3, TMEM56, UNC93A, SLC22A9, SLC2A2, or FXYD1 has an amino acid sequence corresponding to the corresponding Uniprot ID number shown in any of Tables 1 to 3, the corresponding amino acid sequences of which are incorporated herein by reference. Attached Figure Description

[0129] Embodiments of the present invention will now be described with reference to the following figures, wherein: Figure 1 This is a heatmap of protein expression profiles for 88 candidate biomarkers extracted from liver tissue from ProteomicsDB.

[0130] Figure 2 is a heatmap of protein expression profiles of 88 candidate biomarkers extracted from liver tissue from the Human Proteome Map.

[0131] Figure 3 This is a heatmap of protein expression profiles for 11 candidate biomarkers extracted from liver tissue from ProteomicsDB.

[0132] Figure 4 This is a heatmap of protein expression profiles for 11 candidate biomarkers extracted from liver tissue from the Human Proteome Map.

[0133] Figure 5 This is a graph summarizing the detection of CD63 in EVs isolated from HCC cell line HepG2, breast cancer cell line MCF-7 (1), breast cancer cell line BT-474 (2), or buffer control using antibodies targeting each of the 11 biomarkers as binding agents. ASGR1 (antibody 1; ab1) and ASGR1 (antibody 2; ab2) refer to two different antibodies targeting the ASGR1 biomarker.

[0134] Figure 6 This is a graph summarizing the detection of CD63 in EVs isolated from healthy liver tissue, HCC tissue, kidney tissue, lung tissue, or buffered controls using binders targeting each of 10 biomarkers.

[0135] Figure 7This is a graph summarizing the detection of CD63 in EVs isolated from human plasma samples derived from blood taken from five healthy individuals using binders and isotype controls of each of the targeted biomarkers ASGR1, ASGR2, TFR2, and SLCO1B1.

[0136] Figure 8 This is a graph summarizing the detection of CD63 in EVs isolated from human plasma from healthy individuals, HCC patients, two liver patients, and buffer controls using liver mixtures combining binders targeting ASGR1, ASGR2, TFR2, and SLCO1B1 in a final ratio of 4:2:2:2.

[0137] Figure 9 This is a graph summarizing the signal-to-noise ratio (SNR) of CD63 detection in EVs isolated from HCC tissue, cirrhotic tissue, healthy liver tissue, lung tissue, and kidney tissue using CD63-targeting binders.

[0138] Figure 10 This is a graph summarizing the total spectral counts of seven liver-specific proteins detected in EVs by mass spectrometry after collection from human plasma, separation using a mixture of binders targeting ASGR1, ASGR2, TFR2, and SLCO1B1, elution with glycine or DNase, or direct lysis with RIPA on magnetic beads.

[0139] Figure 11 This is a graph summarizing the GO cellular component analysis of genes detected in EVs by mass spectrometry after EVs were collected from human plasma, separated using a mixture of binders targeting ASGR1, ASGR2, TFR2, and SLCO1B1, eluted with glycine or DNase, or directly lysed on magnetic beads with RIPA.

[0140] Figure 12 This is a graph summarizing the log2 intensities of 13 classic EV markers detected in EVs by mass spectrometry after EVs were collected from human plasma, separated using a mixture of binders targeting ASGR1, ASGR2, TFR2, and SLCO1B1, and lysed using RIPA or SDC buffer.

[0141] Figure 13 is a graph summarizing the log2 intensities of 63 liver-specific biomarkers detected in EVs by mass spectrometry after collection from human plasma, separation using a mixture of binders targeting ASGR1, ASGR2, TFR2, and SLCO1B1, and lysis using RIPA or SDC buffer.

[0142] Figure 14 summarizes the process of collecting EVs from human plasma, separating them using a mixture of binders targeting ASGR1, ASGR2, TFR2, and SLCO1B1, and using RIPA ( Figure 14A ) or SDC buffer ( Figure 14B The figure shows the GO cellular component analysis of genes detected in EVs by mass spectrometry after lysis.

[0143] Figure 15 This graph summarizes the total number of miRNA IDs detected in EVs by RNA sequencing after collection from human plasma and isolation using either a binder targeting only ASGR1 or a mixture of binders targeting a combination of ASGR1, ASGR2, TFR2, and SLCO1B1 (TOP4). Two RNA library preparation methods were used for each in two independent runs: RealSeq (sequencing method 1) and NEXTFLEX v4 (sequencing method 2).

[0144] Figure 16 This paper presents a bioNatural Language Processing (BioNLP) analysis of RNA sequencing data obtained from EVs after collection from human plasma and separation of EVs using two different protocols. Group A was directly processed for RNA sequencing (Protocol A). Group B was separated using a mixture of binding agents targeting ASGR1, ASGR2, TFR2, and SLCO1B1 prior to processing for RNA sequencing (Protocol B). Detailed Implementation

[0145] A method for identifying biomarkers on EVs specific to defined tissue categories by isolating tissue-specific EVs from an organism. This invention provides a method for identifying biomarkers present in an organism at elevated levels compared to levels in at least one different type of tissue, within a defined category of tissue. The biomarkers are detectable on or in extracellular vesicles secreted by cells of the defined category of tissue. The method includes the following steps: v. To identify candidate biomarkers; vi. Provide a binding agent that can specifically bind to the candidate biomarker identified in step i; vii. Detecting the presence of candidate biomarkers on extracellular vesicles (EVs) obtained from samples of defined-category tissues by binding a binder to the candidate biomarker in the sample, wherein extracellular vesicles from defined-category tissues preferentially bind the binder compared to extracellular vesicles from at least one different category of tissue in the organism; and viii. Select candidate biomarkers as those present at elevated levels in tissues that define the category.

[0146] extracellular vesicles Extracellular vesicles are heterogeneous lipid bilayered granules naturally secreted by cells of all types. Extracellular vesicles play a role in removing potentially erroneously produced or excessive components from cells and / or in the local transport of cellular components or their transport via circulation to distant sites. Therefore, these extracellular vesicles are rich in proteins, nucleic acid molecules (e.g., DNA or RNA), lipids, and metabolites, thereby reflecting the molecular composition of their parent cell and / or the tissue class that defines their category. Thus, the proteins, nucleic acid molecules, lipids, and metabolites detectable on or within extracellular vesicles have potential use as candidate biomarkers for the cells and / or tissue classes from which EVs are secreted.

[0147] In some implementations, EVs are exosomes, microvesicles, extranuclear granules, oncosomes, or apoptotic bodies.

[0148] Candidate biomarkers In some embodiments, the candidate biomarker is a protein, DNA molecule, RNA molecule, lipid, complex sugar, or a portion of a protein, DNA molecule, RNA molecule, lipid, post-translational modification, or metabolite that is detectable on or within extracellular vesicles. In some embodiments, the candidate biomarker is detectable on the surface of extracellular vesicles. In embodiments where the candidate biomarker is a protein or a portion thereof, preferably, the candidate biomarker is a transmembrane protein, surface protein, or cytoplasmic protein or a portion thereof. Preferably, the candidate biomarker is a transmembrane or surface protein or a portion thereof. In embodiments where the candidate biomarker is a portion of a protein, preferably, the biomarker is an epitope on the protein. In embodiments where the candidate biomarker is a protein or a portion of a protein, the protein may have post-translational modifications, such as acetylation, glycosylation, hydroxylation, lipidation, methylation, nitrosation, phosphorylation, proteolysis, and ubiquitination. In some embodiments, the candidate biomarker is a post-translational modification on a protein. In some embodiments, the candidate biomarker is an epigenetic modification on a nucleic acid molecule, such as methylation.

[0149] In some embodiments, the candidate biomarker is a molecule present in the cytosol of extracellular vesicles. In embodiments where the candidate biomarker is a cytoplasmic molecule, the step of detecting the presence of the candidate biomarker includes a step of permeabilizing the membrane of the extracellular vesicle. Preferably, the permeabilization step includes using a detergent (such as Triton-X, NP-40, saponins, etc.), sonication, or electroporation.

[0150] binder A conjugate is one member of a binding pair capable of specifically binding to a candidate biomarker, which is the other member of the binding pair and is detectable on or in EVs secreted from cells of a defined category of tissue. In some embodiments, each conjugate may comprise an antibody, an antigen-binding fragment of an antibody (e.g., the Fab fragment of an F(ab')2 fragment), an aptamer, a lectin, a lipid-binding protein or lipid-binding domain, a DNA oligonucleotide, or an RNA oligonucleotide. In some embodiments, the conjugate is a DNA or RNA primer or probe. In embodiments where the conjugate is a DNA or RNA oligonucleotide, preferably, the candidate biomarker is also a DNA or RNA oligonucleotide, and the conjugate contains a region having a sequence complementary to the candidate biomarker. In some of these embodiments, the conjugate contains a region having a nucleic acid sequence 100% complementary to the nucleic acid sequence of the candidate biomarker. In some of these embodiments, the conjugate contains a region having a nucleic acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% complementary to the nucleic acid sequence of the candidate biomarker. Preferably, the binder or each binder comprises an antibody or antigen-binding fragment.

[0151] Antibodies (also known as immunoglobulins) consist of four polypeptide chains, including two identical light chains and two identical heavy chains, linked by non-covalent interactions and disulfide bonds to form a flexible Y-shaped structure. Each of the four chains has a variable region at its amino terminus that facilitates antigen binding and a constant region at its carboxyl terminus that determines the isotype. Based on the constant region structure and immune function, antibodies are classified into five major classes of immunoglobulins: IgM, IgG, IgG1a, IgD, and IgE.

[0152] The fragment antigen-binding (Fab) region consists of a constant domain and a variable domain from each of the heavy and light chains of the antibody. The variable region is further subdivided into hypervariable regions and framework regions. Each of the light and heavy chains contains three hypervariable loops (also known as complementarity-determining regions (CDRs)) and four framework regions. These six CDRs exhibit a highly variable amino acid composition and are involved in determining the antigen-binding specificity of the antibody.

[0153] In embodiments where the binder includes an antigen-binding fragment, preferably, the antigen-binding fragment is an F(ab')2 fragment, a Fab' fragment, a Fab fragment, or a variable region.

[0154] Antibodies are normally produced by B cells, which are part of the immune system, when an organism's immune system encounters a foreign molecule (usually a protein). Antibodies specific to a candidate biomarker can be generated through repeated immunization of suitable animals (such as rabbits, goats, donkeys, or sheep) with the candidate biomarker. Antigen-binding fragments can then be generated through antibody fragmentation (e.g., via enzyme-mediated antibody digestion).

[0155] In some embodiments, the binder, or each binder, is provided attached to a substrate. Preferably, the substrate is a structure or particles. In embodiments where the substrate is a structure, the structure is fixed relative to any surrounding sample. In embodiments where the substrate is a particle, the particles are mobile within the surrounding sample. Preferably, the substrate comprises magnetic beads or nanoparticles, gold beads or nanoparticles, polystyrene beads, affinity chromatography columns, microplates, microfluidic channels, or biochips having surfaces made of gold, silica, glass, graphene, or polystyrene. Most preferably, the substrate comprises magnetic beads. In a preferred embodiment, the binder comprises an antibody and the substrate comprises magnetic beads (i.e., antibody-labeled magnetic beads).

[0156] Method usage As described above, the present invention provides a method for identifying biomarkers on EVs specific to a defined category of tissue for the purpose of isolating tissue-specific EVs from an organism (e.g., from a biological fluid of an organism).

[0157] The presence of candidate biomarkers on EVs obtained from tissue samples of defined categories is detected by binding a binder to the candidate biomarkers in the sample. Specifically, EVs from tissues of defined categories preferentially bind to the binder compared to EVs from at least one different category of tissue in the organism due to the presence of the candidate biomarkers. A tissue of a defined category is tissue found or obtained in a specific organ or organ system. At least one different category of tissue from the organism refers to tissue that is not a tissue of a defined category.

[0158] In one implementation, a biological sample (e.g., liver tissue) is obtained from an individual to identify biomarkers present at elevated levels in tissues belonging to a defined category of the individual. Candidate biomarkers are selected using a method described in more detail below. The biological sample contains EVs secreted by cells from tissues belonging to the defined category. The biological sample is incubated with a binder (e.g., an antibody) capable of specifically binding to the candidate biomarker, wherein the binder is attached to a substrate such as magnetic beads.

[0159] Following the incubation period, candidate biomarkers present on EVs in the biological sample bind to a binder, causing the EVs presenting the candidate biomarkers on their surface to become attached to the substrate via the binder. Therefore, EVs presenting candidate biomarkers on their surface can be recovered from the biological sample, for example, by washing a sample containing EVs captured by magnetic beads in a magnetic field.

[0160] The binding of the binder to the candidate biomarker molecule was detected by recovering EVs from a biological sample. The recovered EVs were detected by an assay that was specific to global biomarkers present in EVs at elevated levels (e.g., biotinylated anti-CD63 antibodies), allowing the assay to bind to the global biomarkers on the recovered EVs. The assay could be performed using a reporter assay (e.g., a fluorescence assay, such as using horseradish peroxidase (HRP) conjugated with streptavidin). Detection of the signal by the reporter assay confirmed the presence of the candidate biomarker on the surface of EVs secreted from cells of a defined category of tissue.

[0161] The method is repeated in parallel or sequentially using biological samples from at least one different class of tissues from an organism, such that the biological samples contain EVs secreted by cells from the different class of tissues. A candidate biomarker is selected as the biomarker present at an elevated level in the defined class of tissue when the binder preferentially binds EVs from the defined class of tissue compared to EVs from at least one different class of tissue. Specifically, a candidate biomarker is selected as the biomarker present at an elevated level in the defined class of tissue when the signal detected by a reporting assay is higher in biological samples obtained from the defined class of tissue of an individual compared to biological samples from at least one different class of tissue.

[0162] In some implementations, a candidate biomarker is not selected as a biomarker present at an elevated level in the defined category of tissue when the signal detected by the reporting assay is at or below that in biological samples obtained from tissues of at least one different category of the individual.

[0163] In an embodiment where EVs from a defined category of tissue preferentially bind to the binder compared to EVs from at least one different category of tissue from an organism, the candidate biomarker is selected as the biomarker present at an elevated level in the defined category of tissue.

[0164] For example, in an implementation where the defined tissue category is liver tissue, EVs from liver tissue preferentially bind to the binder compared to EVs from non-liver tissues (such as kidney or breast tissue) because candidate biomarkers are present on liver-derived EVs. Therefore, candidate biomarkers are selected as those present at elevated levels in liver tissue.

[0165] In some implementations, the tissues that define the category are brain, breast, liver, lung, bladder, kidney, heart, gallbladder, pancreas, stomach, intestine, skin, or blood tissue. Preferably, the tissues that define the category are liver tissue or blood tissue.

[0166] In embodiments where the tissue category is defined as liver tissue, preferably, the cells secreting EVs are hepatocytes, Kupffer cells, stellate cells, or resident dendritic cells of the liver. Most preferably, the cells secreting EVs are hepatocytes.

[0167] In some implementations, the tissue that defines the category is blood tissue.

[0168] In some implementations, the step of detecting the presence of candidate biomarkers on EVs further includes the step of detecting the binding of the binder to the corresponding candidate biomarker on or in the extracellular vesicle (or the binding of each binder to it).

[0169] In some implementations, the method includes using a device capable of operating at at least 1x10 7 Biomarkers / ml, at least 1x10 6 At least 1x10 5 At least 1x10 4 At least 1x10 3 At least 1x10 2 A technique for detecting the presence of candidate biomarkers is employed, using a concentration of at least 10 or at least 1 biomarker / ml. Preferably, at least 1 x 10⁻⁶ biomarkers / ml is used. 6 At least 1x10 5 At least 1x10 4 At least 1x10 3 At least 1x10 2 At least 10 or at least 1 biomarker / ml. In some embodiments, the method includes using at least 1 x 10-1 biomarkers. 7 extracellular vesicles / ml (preferably at least 1 x 10⁻⁶) 6 1x10 5 1x10 4 1x10 3 1x10 2Techniques for detecting extracellular vesicles (EVs) at concentrations of 10 or 1 EV / ml are used to detect the presence of candidate biomarkers. For the avoidance of doubt, it should be understood that while biomarkers or EVs are detected at such concentrations, determining the concentration of the biomarker or EV is not necessary for these embodiments. A technique for detecting EVs, called O-NEXOS, is disclosed by reference in WO2022 / 122768, which is incorporated herein by reference. In some embodiments, this technique is used to detect the presence of a biomarker at a specified sensitivity. For example, the presence of a candidate biomarker can be detected using a reporter assay, such as horseradish peroxidase (HRP), an antibody-conjugated fluorophore, or E-NEXOS.

[0170] In some embodiments, the method further includes determining the concentration of a biomarker in the sample. The concentration of a biomarker in the sample can be determined by quantifying the number of biomarkers in the sample using a reporter assay (such as horseradish peroxidase (HRP) or an antibody-conjugated fluorophore) via a calibration curve.

[0171] In some implementations, the method is applicable to identifying a combination of multiple candidate biomarkers as biomarkers present at elevated levels in tissues of a defined category. The method includes the following steps: i. Identification of multiple candidate biomarkers; ii. Provide a variety of binding agents, each of which can specifically bind to the corresponding candidate biomarker identified in step i; iii. Detecting the presence of each of the candidate biomarkers on EVs obtained from samples of defined-category tissues by binding a binder to the corresponding candidate biomarker in the sample, wherein EVs from defined-category tissues preferentially bind combinations of multiple binders compared to EVs from at least one different category of tissue from the organism; and iv. Select a combination of multiple candidate biomarkers as a group of biomarkers present at elevated levels in tissues of defined categories.

[0172] In embodiments where the method includes identifying multiple binding agents, multiple binding agents refer to at least two, at least three, or at least four binding agents. Preferably, multiple candidate biomarkers refer to at least four binding agents.

[0173] In embodiments where the method includes identifying multiple candidate biomarkers, multiple candidate biomarkers refer to at least two, at least three, or at least four biomarkers. Preferably, multiple candidate biomarkers refer to at least four biomarkers. Preferably, the number of binders corresponds to the number of biomarkers.

[0174] The advantage of identifying multiple candidate biomarkers present at elevated levels in defined categories of tissues is that it increases the number of EVs obtainable from defined category tissue samples (particularly in biological fluids) by providing multiple binding agents capable of specifically binding to the respective candidate biomarkers. For example, components secreted into the bloodstream differ between individuals. Furthermore, as mentioned above, EVs are heterogeneous particles naturally secreted by cells in a dynamic manner within tissues. Tissues also contain multiple cell types. Therefore, EVs secreted from cells in defined category tissues can present different biomarkers present at elevated levels in defined category tissues. Thus, targeting multiple candidate biomarkers present at elevated levels in defined category tissues using multiple binding agents allows for the detection of heterogeneous EVs secreted from cells in defined category tissues.

[0175] In some embodiments, multiple binding agents are provided sequentially. For example, a first binding agent capable of specifically binding to a corresponding candidate biomarker on an EV is provided, and then the first binding agent is used to capture and separate the EV (as described below) to generate an enrichment fraction of EVs presenting the first candidate biomarker. Subsequently, a second binding agent capable of specifically binding to a corresponding candidate biomarker on an EV in the enrichment fraction of EVs is provided, and the second binding agent is used to capture and separate the EV to generate an enrichment fraction of EVs presenting the first and second candidate biomarkers. In embodiments where multiple binding agents are provided sequentially, another advantage for identifying multiple candidate biomarkers is that EVs from tissues of defined categories can be identified more accurately. For example, in the presence of first, second, and third categories of tissues, the first biomarker may be present on EVs from the first and second categories of tissues, and the second biomarker may be present on EVs from the second and third categories of tissues. Therefore, if a group of binding agents that bind to the first and second biomarkers is provided sequentially, this allows for the capture and separation of EVs from the second category of tissues from EVs from the first and third categories of tissues.

[0176] In some embodiments, the method further includes contacting EVs from at least one different category of tissue with a binder or each binder, and detecting the binding of EVs from at least one different category of tissue with the binder or each binder. For example, in an embodiment where the tissue category is defined as liver tissue, the method further includes contacting EVs from non-liver tissue (such as lung tissue, kidney tissue, or breast tissue) with the binder or each binder, and detecting the binding of EVs from non-liver tissue with the binder or each binder.

[0177] In some implementations, EVs from a defined category of tissue preferentially bind to the binder or each binder compared to EVs from at least two different categories of tissue from the organism. For example, in an implementation where the defined category of tissue is liver tissue, EVs from liver tissue preferentially bind to the binder or each binder compared to EVs from two non-liver tissues (such as kidney tissue and breast tissue) or from three non-liver tissues (such as lung tissue, kidney tissue, or breast tissue).

[0178] In some implementations, EVs from tissues of a defined category preferentially bind to the binder or each binder compared to EVs from all other categories of tissues in the organism.

[0179] In some embodiments, the method further includes the step of detecting the presence of candidate biomarkers, or each candidate biomarker, on EVs obtained from a cultured cell line representing a defined category of tissue. In some embodiments, extracellular vesicles obtained from a cultured cell line representing a defined category of tissue preferentially bind to the binding agent compared to extracellular vesicles obtained from a cultured cell line representing at least one different category of tissue from an organism. In some embodiments where the defined category of tissue is liver tissue, the cultured cell line is HepG2. In some embodiments, extracellular vesicles obtained from the HepG2 cell line preferentially bind to the binding agent compared to extracellular vesicles obtained from a cultured cell line representing non-liver tissue (e.g., a cultured cell line representing breast tissue, such as MCF-7 or BT-474).

[0180] In some implementations, EVs obtained from cultured cell lines are placed in plasma already doped with EVs.

[0181] In some implementations, the EV obtained from the tissue sample is in plasma that has been mixed with EV after EV has been obtained from the tissue sample.

[0182] In some embodiments, the tissue samples of the present invention are healthy tissue or diseased tissue. Preferably, the healthy tissue is liver tissue. Preferably, the diseased tissue is a disease associated with liver tissue, such as liver cancer or HCC. In some embodiments, the diseased tissue is cancerous tissue. One reason for obtaining tissue samples from diseased tissue is that cells in some diseased tissues are poorly differentiated and may not express typical biomarkers of healthy tissue. In some embodiments, the method is first performed on EVs obtained from a defined category of healthy tissue samples, and then the method is repeated on EVs obtained from the same category of diseased tissue samples. In this way, it is confirmed that the biomarker is characteristic of EVs from defined categories of tissue, whether healthy or diseased.

[0183] Further steps in identifying candidate biomarkers In some implementations, the step of identifying candidate biomarkers includes the following steps: 1) Identify potential candidate biomarkers expressed in tissues that define the category; 2) Identify potential candidate biomarkers present in tissues of a defined category at higher levels than in at least one different category of tissues from an organism; 3) Identify potential candidate biomarkers as transmembrane or surface proteins; 4) Identify potential candidate biomarkers present in extracellular vesicles; or 5) Any combination of steps 1) through 4) performed in any order. And thus identify candidate biomarkers.

[0184] In some implementations, the step of identifying candidate biomarkers includes the following steps: 1) Identify potential candidate biomarkers expressed in tissues that define the category; 2) Identify potential candidate biomarkers from those identified in step 1) that exist at higher levels in the defined category of tissues than in at least one different category of tissues from the organism; 3) Identify potential candidate biomarkers as transmembrane or surface proteins from among the potential candidate biomarkers identified in step 2); and 4) Validate one candidate biomarker present in EV from the potential candidate biomarkers identified in step 3), or validate multiple candidate biomarkers present in EV from the potential candidate biomarkers identified in step 3).

[0185] In some implementations, prior to verifying the candidate biomarkers, the step of identifying candidate biomarkers further includes identifying potential candidate biomarkers from those identified in step 3) by further ranking the expression of potential candidate biomarkers present at higher levels in tissues of a defined category compared to expression in at least one different category of tissues from an organism (step 3b).

[0186] In some implementations, steps 1) through 3) and optional 3b) are performed using bioinformatics database searches and selections. Examples of bioinformatics databases include, but are not limited to, UniProt, ProteomicsDB, and HumanProteome Map.

[0187] In some implementations, information such as protein mRNA expression and protein expression profiles is used to identify potential candidate biomarkers expressed in a defined category of tissue and present in that tissue at a higher level than in at least one different category of tissue from the organism. For example, if a potential candidate biomarker is known to be expressed in a defined category of tissue, it is identified in step 1). If a potential candidate biomarker identified in step 1) is known to be expressed at a high level in a defined category of tissue and is known to be expressed at a low or intermediate level in at least one different category, it is then identified in step 2). Preferably, step 2) includes identifying potential candidate biomarkers present in a defined category of tissue at a higher level than in all other different categories of tissue from the organism from the potential candidate biomarkers identified in step 1). For example, if a potential candidate biomarker identified in step 1) is known to be expressed at a high level in a defined category of tissue and is known to be expressed at a low or intermediate level in all other categories of tissue, it is then identified in step 2).

[0188] In some embodiments, information such as subcellular localization is used to identify potential candidate biomarkers as transmembrane or surface proteins. In some embodiments, if a potential candidate biomarker is known to be located or partially located in the cell membrane, it is identified in step 3).

[0189] In some embodiments, step 4) includes evaluating potential candidate biomarkers using cell-based assays (e.g., artificial plasma systems, in vitro cultured cell lines, and / or ex vivo tissue samples). For example, in some embodiments, the validation step includes evaluating potential candidate biomarkers using cultured cell lines from defined-category tissues. EVs are secreted from the cultured cell lines. If the binder or each binder is observed to bind to the potential candidate biomarker or each potential candidate biomarker on the EV secreted from the cultured cell lines from defined-category tissues at a desired sensitivity threshold, the potential candidate biomarker passes the validation step. If the binder or each binder is observed not to bind to the potential candidate biomarker or each potential candidate biomarker on the EV secreted from the cultured cell lines from defined-category tissues at a desired sensitivity threshold, the potential candidate biomarker fails the validation step.

[0190] The advantage of performing steps 1) through 3) is that it limits the list of potential candidate biomarkers to those that are more likely to exist at elevated levels in tissues of the defined category within the organism. Specifically, step 1) limits the list of potential candidate biomarkers to those expressed in tissues of the defined category. Step 2) limits the list of potential candidate biomarkers to those existing at higher levels in tissues of the defined category than in tissues from at least one different category within the organism. Step 3) limits the list of potential candidate biomarkers to those that are transmembrane or surface proteins. Transmembrane or surface proteins are advantageous to the method of the present invention because they are exposed on the outer side of the EV's plasma membrane and are therefore available for binding by binders or each type of binder without permeation of the EV plasma membrane.

[0191] The advantage of performing step 4) is that it confirms the presence of potential candidate biomarkers on the surface of the EV and their availability for binding with the binder. In vitro artificial plasma systems and cultured cell lines provide simplified models of the in vivo environment. In contrast, ex vivo tissue samples are derived from living organisms and therefore accurately reflect the in vivo environment.

[0192] In some embodiments, step 3) further includes the step of fractionating the precursor sample rich in EVs into fractions of a defined category of tissue, in order to provide a sample of the defined category of tissue. For example, the precursor sample can be fractionated using size exclusion chromatography.

[0193] In some implementations, the biomarkers are specific to the tissue defining the category. That is, the biomarkers are present only in the target tissue and not in non-target tissues. In other implementations, combinations of biomarkers are specific to the tissue defining the category.

[0194] Although the above implementation has been described as including all steps 1) through 4), it should be understood that in alternative implementations, only one of steps 1), 2), 3), or 4) is performed in order to select candidate biomarkers. In alternative implementations, more than one step of steps 1) through 4) is performed, but in an alternative order as listed above.

[0195] In embodiments where the tissue category is defined as liver tissue, preferably, the biomarker is ASGR1, ASGR2, TFR2, SLCO1B1, SLC38A3, TMEM56, UNC93A, SLC22A9, SLC2A2, or FXYD1, or a combination thereof. Most preferably, the biomarker is ASGR1, ASGR2, TFR2, or SLCO1B1, or a combination thereof.

[0196] As used herein, in a preferred embodiment, the biomarker has an amino acid sequence corresponding to the corresponding Uniprot ID number shown in any of Tables 1 to 3.

[0197] In embodiments where the biomarker is ASGR1, the binding agent is preferably an anti-ASGR1 antibody. In embodiments where the biomarker is ASGR2, the binding agent is preferably an anti-ASGR2 antibody. In embodiments where the biomarker is TFR2, the binding agent is preferably an anti-TFR2 antibody. In embodiments where the biomarker is SLCO1B1, the binding agent is preferably an anti-SLCO1B1 antibody. In embodiments where the biomarker is SLC38A3, the binding agent is preferably an anti-SLC38A3 antibody. In embodiments where the biomarker is TMEM56, the binding agent is preferably an anti-TMEM56 antibody. In embodiments where the biomarker is UNC93A, the binding agent is preferably an anti-UNC93A antibody. In embodiments where the biomarker is SLC22A9, the binding agent is preferably an anti-SLC22A9 antibody. In embodiments where the biomarker is SLC2A2, the binding agent is preferably an anti-SLC2A2 antibody. In embodiments where the biomarker is FXYD1, the binding agent is preferably an anti-FXYD1 antibody.

[0198] In embodiments that include multiple binders, each binder is preferably provided in a proportion of 10%-50% of the total, wherein the total mixture of binders is equal to 100%.

[0199] In embodiments comprising a plurality of binding agents consisting of anti-ASGR1 antibody, anti-ASGR2 antibody, anti-SLCO1B1 antibody, and anti-TFR2 antibody, the anti-ASGR1 antibody is preferably provided at a proportion of 30%-50% of the total, and each of the other antibodies is preferably provided at a proportion of 10%-30% of the total, wherein the total mixture of binding agents is equal to 100%. In some embodiments, the anti-ASGR1 antibody is preferably provided at a proportion of 40% of the total, and each of the anti-ASGR2 antibody, anti-SLCO1B1 antibody, and anti-TFR2 antibody is preferably provided at a proportion of 20% of the total of the four antibodies (this is equivalent to a final ratio of 4:2:2:2).

[0200] It should be understood that in some implementations, the binder may be an antigen-binding fragment of the antibody.

[0201] A method for enriching biological samples containing EV mixtures The present invention also provides a method for enriching biological samples containing EV mixtures, the method comprising the following steps: a) Identify one or more biomarkers present at elevated levels in tissues (such as tissues with defined categories) by performing the methods described above; b) Capture EVs in biological samples, wherein extracellular vesicles display the biomarkers identified in step a), or each of the biomarkers, to generate an enrichment fraction of EVs from tissues that define the category.

[0202] The present invention also provides a method for enriching biological samples containing EV mixtures, the method comprising the following steps: EVs are captured from biological samples, where EVs exhibit one or more biomarkers, to generate enrichment fractions of EVs. The biomarkers, or each of the biomarkers, are selected from ASGR1, ASGR2, TFR2, SLCO1B1, SLC38A3, TMEM56, UNC93A, SLC22A9, SLC2A2 and FXYD1 or combinations thereof, preferably selected from ASGR1, ASGR2, TFR2 and SLCO1B1 or combinations thereof.

[0203] In some implementations, the EV displays two or more, three or more, or four or more biomarkers.

[0204] In some embodiments, the step of capturing extracellular vesicles includes capturing extracellular vesicles based on extracellular vesicles displaying one or more biomarkers, and one or more of the biomarkers consists of ASGR1, ASGR2, TFR2, and SLCO1B1.

[0205] In some embodiments, the biomarker, or each biomarker, is present at elevated levels in the tissues of a defined category. In some embodiments, the biomarker, or each biomarker, present at elevated levels in the tissues of a defined category is identified using the identification methods disclosed herein.

[0206] In some implementations, one or more binders are used to capture EVs, which are capable of binding specifically to biomarkers displayed on the EVs or to each biomarker.

[0207] In some embodiments, the biomarker, or each biomarker being ASGR1 and the binding agent, or each binding agent being an anti-ASGR1 antibody or its antigen-binding fragment; the biomarker, or each biomarker being ASGR2 and the binding agent, or each binding agent being an anti-ASGR2 antibody or its antigen-binding fragment; the biomarker, or each biomarker being TFR2 and the binding agent, or each binding agent being an anti-TFR2 antibody or its antigen-binding fragment; the biomarker, or each biomarker being SLCO1B1 and the binding agent, or each binding agent being an anti-SLCO1B1 antibody or its antigen-binding fragment; the biomarker, or each biomarker being SLC38A3 and the binding agent, or each binding agent being an anti-SLC38A3 antibody or its antigen-binding fragment; the biomarker, or each biomarker being TMEM56 and the binding agent, or each binding agent being an anti-TMEM56 antibody or its antigen-binding fragment; the biomarker, or each biomarker being UNC93A and the binding agent, or each binding agent being an anti-UNC93A antibody or its antigen-binding fragment; the biomarker... The biomarker or each biomarker is SLC22A9 and the binding agent or each binding agent is an anti-SLC22A9 antibody or its antigen-binding fragment; the biomarker or each biomarker is SLC2A2 and the binding agent or each binding agent is an anti-SLC2A2 antibody or its antigen-binding fragment; or the biomarker or each biomarker is FXYD1 and the binding agent or each binding agent is an anti-FXYD1 antibody or its antigen-binding fragment; or a combination thereof, preferably, wherein the biomarker or each biomarker is ASGR1 and the binding agent or each binding agent is an anti-ASGR1 antibody or its antigen-binding fragment; the biomarker or each biomarker is ASGR2 and the binding agent or each binding agent is an anti-ASGR2 antibody or its antigen-binding fragment; the biomarker or each biomarker is TFR2 and the binding agent or each binding agent is an anti-TFR2 antibody or its antigen-binding fragment; or the biomarker or each biomarker is SLCO1B1 and the binding agent or each binding agent is an anti-SLCO1B1 antibody or its antigen-binding fragment; or a combination thereof.

[0208] In some embodiments, the binder or each binder is provided attached to a substrate, preferably wherein the substrate includes magnetic beads or nanoparticles, gold beads or nanoparticles, polystyrene beads, affinity chromatography columns, microplates, microfluidic channels, or biochips having surfaces made of gold, silicon dioxide, glass, graphene, or polystyrene.

[0209] In embodiments that include multiple binders, each binder is preferably provided in a proportion of 10%-50% of the total, wherein the total mixture of binders is equal to 100%.

[0210] In embodiments comprising a plurality of binding agents consisting of anti-ASGR1 antibody, anti-ASGR2 antibody, anti-SLCO1B1 antibody, and anti-TFR2 antibody, the anti-ASGR1 antibody is preferably provided at a proportion of 30%-50% of the total, and each of the other antibodies is preferably provided at a proportion of 10%-30% of the total, wherein the total mixture of binding agents is equal to 100%. In some embodiments, the anti-ASGR1 antibody is preferably provided at a proportion of 40% of the total, and each of the anti-ASGR2 antibody, anti-SLCO1B1 antibody, and anti-TFR2 antibody is preferably provided at a proportion of 20% of the total of the four antibodies (this is equivalent to a final ratio of 4:2:2:2).

[0211] It should be understood that in some implementations, the binder may be an antigen-binding fragment of the antibody.

[0212] Preferably, the biological sample is a tissue sample or a biological fluid. In an embodiment where the biological sample is a biological fluid, the biological fluid comprises a circulating EV. Preferably, the biological fluid is blood, urine, saliva, lymph, bile, cerebrospinal fluid, sputum, mucus, tears, bronchoalveolar lavage (BAL) fluid, earwax, sweat, feces, breast milk, tissue fluid, vaginal fluid, semen, gastric juice, vesicular fluid, or cystic fluid. Most preferably, the biological fluid is blood, urine, cerebrospinal fluid, or mucus.

[0213] In some embodiments, the EV mixture comprises extracellular vesicles from a defined category of tissue and EVs from at least one different category of tissue in the organism. For example, in an embodiment where the defined category of tissue is liver tissue, preferably, when the biological sample is a biofluid, the EV mixture may comprise EVs from liver tissue and EVs from at least one other category of tissue (such as breast tissue).

[0214] In some embodiments, the step of capturing EVs in a biological sample further includes isolating extracellular vesicles exhibiting the biomarkers identified in the steps or methods disclosed herein for identifying biomarkers, which are captured using the biomarkers or each biomarker.

[0215] In some implementations, the isolated extracellular vesicles are intact. This allows for further downstream processing of the intact extracellular vesicles, such as determining the presence of other biomarkers on or within the extracellular vesicles.

[0216] In some embodiments, the method for enriching biological samples further includes the step of releasing the contents of captured or isolated extracellular vesicles. For example, the contents of isolated extracellular vesicles can be released by lysis of the EV membrane. This allows for further downstream processing of the aggregated contents of captured or isolated extracellular vesicles.

[0217] In some embodiments, the step of capturing extracellular vesicles (EVs) in a biological sample includes using a binding agent or each type of binding agent. For example, in some embodiments, the binding agent is provided on a substrate (e.g., magnetic beads), and the biological sample is brought into contact with the substrate. The binding agent binds to the extracellular vesicles that present the biomarker, thereby “capturing” the extracellular vesicles. The unbound biological sample is then washed away, leaving the captured extracellular vesicles bound to the substrate by the binding agent. In some specific embodiments, the captured extracellular vesicles are then released from the binding agent to provide an enriched fraction of extracellular vesicles or completely isolated extracellular vesicles.

[0218] An advantage of performing a method for enriching a biological sample containing a mixture of EVs is that it generates an enrichment fraction of EVs present at elevated levels in tissues of a defined category. For example, in an embodiment where the defined category of tissue is liver tissue, the method is capable of generating a fraction of EVs present at elevated levels in liver tissue from a mixture of EVs (e.g., EVs from non-liver tissues of the organism).

[0219] It should be understood that once a candidate biomarker or each candidate biomarker has been identified using the identification methods disclosed herein, the biomarker can be used without performing the identification steps in the method for enriching a biological sample containing a mixture of EVs.

[0220] A method for analyzing biological samples containing EV mixtures The present invention also provides a method for analyzing biological samples containing EV mixtures, the method comprising the following steps: a) Identifying one or more biomarkers present at elevated levels in tissues (such as tissues of defined categories) by performing the method of the present invention for identifying biomarkers present at elevated levels in tissues of defined categories of organisms; b) Capture EVs in a biological sample, wherein extracellular vesicles display the biomarkers identified in step a), or each of the biomarkers, to generate an enrichment fraction of EVs; and c) Analyze the contents of the EV in the enrichment fraction obtained in step b).

[0221] The present invention also provides a method for analyzing biological samples containing EV mixtures, the method comprising the following steps: a) Capturing EVs in biological samples, wherein the EVs display one or more biomarkers, to generate enrichment fractions for the EVs; and b) Analyze the contents of the EVs in the enrichment fractions obtained in step a). The biomarkers, or each of the biomarkers, are selected from ASGR1, ASGR2, TFR2, SLCO1B1, SLC38A3, TMEM56, UNC93A, SLC22A9, SLC2A2 and FXYD1 or combinations thereof, preferably selected from ASGR1, ASGR2, TFR2 and SLCO1B1 or combinations thereof.

[0222] In some implementations, the EV displays two or more, three or more, or four or more biomarkers.

[0223] In some embodiments, the step of capturing extracellular vesicles includes capturing extracellular vesicles based on extracellular vesicles displaying one or more biomarkers, and one or more of the biomarkers consists of ASGR1, ASGR2, TFR2, and SLCO1B1.

[0224] In some embodiments, the biomarker, or each biomarker, is present at elevated levels in the tissues of the defined category. In some embodiments, the biomarker, or each biomarker, present at elevated levels in the tissues of the defined category is identified using the identification methods disclosed herein.

[0225] In some implementations, one or more binders are used to capture EVs, which are capable of binding specifically to biomarkers displayed on the EVs or to each biomarker.

[0226] In some embodiments, the biomarker, or each biomarker being ASGR1 and the binding agent, or each binding agent being an anti-ASGR1 antibody or its antigen-binding fragment; the biomarker, or each biomarker being ASGR2 and the binding agent, or each binding agent being an anti-ASGR2 antibody or its antigen-binding fragment; the biomarker, or each biomarker being TFR2 and the binding agent, or each binding agent being an anti-TFR2 antibody or its antigen-binding fragment; the biomarker, or each biomarker being SLCO1B1 and the binding agent, or each binding agent being an anti-SLCO1B1 antibody or its antigen-binding fragment; the biomarker, or each biomarker being SLC38A3 and the binding agent, or each binding agent being an anti-SLC38A3 antibody or its antigen-binding fragment; the biomarker, or each biomarker being TMEM56 and the binding agent, or each binding agent being an anti-TMEM56 antibody or its antigen-binding fragment; the biomarker, or each biomarker being UNC93A and the binding agent, or each binding agent being an anti-UNC93A antibody or its antigen-binding fragment; the biomarker... The biomarker or each biomarker is SLC22A9 and the binding agent or each binding agent is an anti-SLC22A9 antibody or its antigen-binding fragment; the biomarker or each biomarker is SLC2A2 and the binding agent or each binding agent is an anti-SLC2A2 antibody or its antigen-binding fragment; or the biomarker or each biomarker is FXYD1 and the binding agent or each binding agent is an anti-FXYD1 antibody or its antigen-binding fragment; or a combination thereof, preferably, wherein the biomarker or each biomarker is ASGR1 and the binding agent or each binding agent is an anti-ASGR1 antibody or its antigen-binding fragment; the biomarker or each biomarker is ASGR2 and the binding agent or each binding agent is an anti-ASGR2 antibody or its antigen-binding fragment; the biomarker or each biomarker is TFR2 and the binding agent or each binding agent is an anti-TFR2 antibody or its antigen-binding fragment; or the biomarker or each biomarker is SLCO1B1 and the binding agent or each binding agent is an anti-SLCO1B1 antibody or its antigen-binding fragment; or a combination thereof.

[0227] In some embodiments, the binder or each binder is provided attached to a substrate, preferably wherein the substrate includes magnetic beads or nanoparticles, gold beads or nanoparticles, polystyrene beads, affinity chromatography columns, microplates, microfluidic channels, or biochips having surfaces made of gold, silicon dioxide, glass, graphene, or polystyrene.

[0228] In embodiments that include multiple binders, each binder is preferably provided in a proportion of 10%-50% of the total, wherein the total mixture of binders is equal to 100%.

[0229] In embodiments comprising a plurality of binding agents consisting of anti-ASGR1 antibody, anti-ASGR2 antibody, anti-SLCO1B1 antibody, and anti-TFR2 antibody, the anti-ASGR1 antibody is preferably provided at a proportion of 30%-50% of the total, and each of the other antibodies is preferably provided at a proportion of 10%-30% of the total, wherein the total mixture of binding agents is equal to 100%. In some embodiments, the anti-ASGR1 antibody is preferably provided at a proportion of 40% of the total, and each of the anti-ASGR2 antibody, anti-SLCO1B1 antibody, and anti-TFR2 antibody is preferably provided at a proportion of 20% of the total of the four antibodies (this is equivalent to a final ratio of 4:2:2:2).

[0230] It should be understood that in some implementations, the binder may be an antigen-binding fragment of the antibody.

[0231] Preferably, the biological sample is a tissue sample or a biological fluid. In an embodiment where the biological sample is a biological fluid, the biological fluid comprises a circulating EV. Preferably, the biological fluid is blood, urine, saliva, lymph, bile, cerebrospinal fluid, sputum, mucus, tears, bronchoalveolar lavage (BAL) fluid, earwax, sweat, feces, breast milk, tissue fluid, vaginal fluid, semen, gastric juice, vesicular fluid, or cystic fluid. Most preferably, the biological fluid is blood, urine, cerebrospinal fluid, or mucus.

[0232] In some embodiments, the EV mixture comprises EVs from a defined category of tissue and EVs from at least one different category of tissue from the organism. For example, in an embodiment where the defined category of tissue is liver tissue, preferably, when the biological sample is a biofluid, the EV mixture may comprise EVs from liver tissue and EVs from at least one other category of tissue (such as breast tissue).

[0233] In some embodiments, the step of capturing EVs in a biological sample further includes separating EVs of the biomarkers or each biomarker identified in the steps or methods for identifying biomarkers disclosed herein, which are captured using the biomarkers or each biomarker.

[0234] In some implementations, the step of capturing EVs in biological samples includes using a binder or each binder.

[0235] Examples of extracellular vesicle (EV) contents include proteins, DNA, RNA, lipids, post-translational modifications, or metabolite contents. By performing analytical methods on biological samples, EV contents (such as proteins, DNA, RNA, lipids, post-translational modifications, or metabolite contents) can be present at elevated levels in defined tissues within an organism. Furthermore, in cases where biological samples are obtained from individuals in a specific demographic group, risk group, or patient group, associations can be established between EV contents and individuals within the study group. For example, if a biological sample is obtained from a liver cancer patient, and the defined tissue is liver tissue, the EV contents can be used to search for and identify biomarkers for liver cancer.

[0236] In some implementations, the step of analyzing the contents of an EV includes pyrolyzing the EV to release its contents.

[0237] In some implementations, the contents of the EV are analyzed by mass spectrometry and / or RNA sequencing.

[0238] In some implementations, the EV is eluted intact for use in downstream applications that require intact EV, such as in biomarker quantification or EV therapy (given the immunomodulatory and anti-inflammatory properties of EVs obtained from certain tissues, such as umbilical cord, bone marrow, and adipose tissue) (Zheng et al., 2021; Weng et al., 2021).

[0239] It should be understood that once a candidate biomarker or each candidate biomarker has been identified using the identification methods disclosed herein, the biomarker can be used without performing further identification steps in the methods, particularly the identification steps in methods for analyzing biological samples containing EV mixtures.

[0240] Use of one or more biomarkers or one or more binding agents to generate enrichment fractions of EVs The present invention also provides the use of one or more biomarkers displayed on an EV for generating enrichment fractions of the EV, wherein the enrichment fractions of the EV are secreted from tissues that define categories in an organism, wherein the biomarkers, or each biomarker, are selected from ASGR1, ASGR2, TFR2, SLCO1B1, SLC38A3, TMEM56, UNC93A, SLC22A9, SLC2A2 and FXYD1 or combinations thereof, preferably selected from ASGR1, ASGR2, TFR2 and SLCO1B1 or combinations thereof.

[0241] In some implementations, two or more, three or more, or four or more biomarkers are used to generate enrichment fractions of EVs.

[0242] In some implementations, the biomarkers consist of ASGR1, ASGR2, TFR2, and SLCO1B1.

[0243] In some embodiments, the biomarker, or each biomarker, is present at elevated levels in tissues of a defined category. In some embodiments, the biomarker, or each biomarker, present at elevated levels in tissues of a defined category is identified using the identification methods disclosed herein. The present invention also provides the use of one or more binders for generating enrichment fractions of EVs, wherein the enrichment fractions of EVs are secreted from tissues of defined categories in an organism, wherein the binder or each binder is capable of specifically binding to a biomarker displayed on the EV, wherein the binder or each binder is selected from anti-ASGR1 antibody or its antigen-binding fragment, anti-ASGR2 antibody or its antigen-binding fragment, anti-TFR2 antibody or its antigen-binding fragment, anti-SLCO1B1 antibody or its antigen-binding fragment, anti-SLC38A3 antibody or its antigen-binding fragment, anti-TMEM56 antibody or its antigen-binding fragment, anti-UNC93A antibody or its antigen-binding fragment, anti-SLC22A9 antibody or its antigen-binding fragment, anti-SLC2A2 antibody or its antigen-binding fragment, and anti-FXYD1 antibody or its antigen-binding fragment, or combinations thereof, preferably selected from anti-ASGR1 antibody or its antigen-binding fragment, anti-ASGR2 antibody or its antigen-binding fragment, anti-TFR2 antibody or its antigen-binding fragment, or anti-SLCO1B1 antibody or its antigen-binding fragment, or combinations thereof.

[0244] In some implementations, two or more, three or more, or four or more binders are used to generate enrichment fractions of EVs.

[0245] In some embodiments, the binder, or each binder, comprises an anti-ASGR1 antibody or its antigen-binding fragment, an anti-ASGR2 antibody or its antigen-binding fragment, an anti-SLCO1B1 antibody or its antigen-binding fragment, and an anti-TFR2 antibody or its antigen-binding fragment. In a preferred embodiment, the multiple binders comprise an anti-ASGR1 antibody, an anti-ASGR2 antibody, an anti-SLCO1B1 antibody, and an anti-TFR2 antibody.

[0246] It should be understood that the binder, or each binder, is capable of specifically binding to the corresponding biomarker displayed on the EV. In some embodiments, the biomarker, or each biomarker, is present at elevated levels in the tissue of the defined category. In some embodiments, the biomarker, or each biomarker, present at elevated levels in the tissue of the defined category is identified using the identification methods disclosed herein.

[0247] In some embodiments, the binder or each binder is provided attached to a substrate, preferably wherein the substrate includes magnetic beads or nanoparticles, gold beads or nanoparticles, polystyrene beads, affinity chromatography columns, microplates, microfluidic channels, or biochips having surfaces made of gold, silicon dioxide, glass, graphene, or polystyrene.

[0248] In embodiments that include multiple binders, each binder is preferably provided in a proportion of 10%-50% of the total, wherein the total mixture of binders is equal to 100%.

[0249] In embodiments comprising a plurality of binding agents consisting of anti-ASGR1 antibody, anti-ASGR2 antibody, anti-SLCO1B1 antibody, and anti-TFR2 antibody, the anti-ASGR1 antibody is preferably provided at a proportion of 30%-50% of the total, and each of the other antibodies is preferably provided at a proportion of 10%-30% of the total, wherein the total mixture of binding agents is equal to 100%. In some embodiments, the anti-ASGR1 antibody is preferably provided at a proportion of 40% of the total, and each of the anti-ASGR2 antibody, anti-SLCO1B1 antibody, and anti-TFR2 antibody is preferably provided at a proportion of 20% of the total of the four antibodies (this is equivalent to a final ratio of 4:2:2:2).

[0250] It should be understood that in some implementations, the binder may be an antigen-binding fragment of the antibody.

[0251] In some embodiments, the enrichment fraction of EVs is enriched from a biological sample containing an EV mixture. In a preferred embodiment, the EV mixture contains EVs from a defined category of tissue and EVs from at least one different category of tissue in the organism. For example, in an embodiment where the defined category of tissue is liver tissue, preferably, in the case of a biological fluid, the EV mixture may contain EVs from liver tissue and EVs from at least one other category of tissue (such as breast tissue).

[0252] Preferably, the biological sample is a tissue sample or a biological fluid. In an embodiment where the biological sample is a biological fluid, the biological fluid comprises a circulating EV. Preferably, the biological fluid is blood, urine, saliva, lymph, bile, cerebrospinal fluid, sputum, mucus, tears, bronchoalveolar lavage (BAL) fluid, earwax, sweat, feces, breast milk, tissue fluid, vaginal fluid, semen, gastric juice, vesicular fluid, or cystic fluid. Most preferably, the biological fluid is blood, urine, cerebrospinal fluid, or mucus.

[0253] Preferably, the tissue defining the category is liver tissue. In embodiments where the tissue defining the category is liver tissue, preferably, the cells secreting EVs therefrom are hepatocytes, Kupffer cells, stellate cells, or resident dendritic cells of the liver. Most preferably, the cells secreting EVs therefrom are hepatocytes.

[0254] A method for detecting one or more biomarkers on or in EVs The present invention also provides a method for detecting one or more biomarkers on or in EVs secreted from tissues of a defined category, wherein the biomarkers or each biomarker is selected from ASGR1, ASGR2, TFR2, SLCO1B1, SLC38A3, TMEM56, UNC93A, SLC22A9, SLC2A2 and FXYD1 or combinations thereof, preferably selected from ASGR1, ASGR2, TFR2 and SLCO1B1 or combinations thereof.

[0255] In some implementations, two or more, three or more, or four or more biomarkers are detected on or in EVs secreted from tissues of defined categories.

[0256] In some implementations, the biomarkers consist of ASGR1, ASGR2, TFR2, and SLCO1B1.

[0257] In some embodiments, the biomarker, or each biomarker, is present at elevated levels in the tissues of a defined category. In some embodiments, the biomarker, or each biomarker, present at elevated levels in the tissues of a defined category is identified using the identification methods disclosed herein.

[0258] In some embodiments, the biomarker, or each biomarker, is detected by binding with one or more conjugates, wherein the biomarker, or each biomarker, is ASGR1 and the conjugate, or each conjugate, is an anti-ASGR1 antibody or its antigen-binding fragment; the biomarker, or each biomarker, is ASGR2 and the conjugate, or each conjugate, is an anti-ASGR2 antibody or its antigen-binding fragment; the biomarker, or each biomarker, is TFR2 and the conjugate, or each conjugate, is an anti-TFR2 antibody or its antigen-binding fragment; the biomarker, or each biomarker, is SLCO1B1 and the conjugate, or each conjugate, is an anti-SLCO1B1 antibody or its antigen-binding fragment; the biomarker, or each biomarker, is SLC38A3 and the conjugate, or each conjugate, is an anti-SLC38A3 antibody or its antigen-binding fragment; the biomarker, or each biomarker, is TMEM56 and the conjugate, or each conjugate, is an anti-TMEM56 antibody or its antigen-binding fragment; the biomarker, or each biomarker, is UNC93A and the conjugate, or each conjugate, is an anti-UNC93A antibody. A. Antibody or its antigen-binding fragment thereof, biomarker or each biomarker being SLC22A9 and the binding agent or each binding agent being an anti-SLC22A9 antibody or its antigen-binding fragment thereof, biomarker or each biomarker being SLC2A2 and the binding agent or each binding agent being an anti-SLC2A2 antibody or its antigen-binding fragment thereof, or biomarker or each biomarker being FXYD1 and the binding agent or each binding agent being an anti-FXYD1 antibody or its antigen-binding fragment thereof, or combinations thereof, preferably wherein the biomarker or each biomarker is ASGR1 and the binding agent or each binding agent is an anti-ASGR1 antibody or its antigen-binding fragment thereof, biomarker or each biomarker being ASGR2 and the binding agent or each binding agent being an anti-ASGR2 antibody or its antigen-binding fragment thereof, biomarker or each biomarker being TFR2 and the binding agent or each binding agent being an anti-TFR2 antibody or its antigen-binding fragment thereof, or biomarker or each biomarker being SLCO1B1 and the binding agent or each binding agent being an anti-SLCO1B1 antibody or its antigen-binding fragment thereof, or combinations thereof.

[0259] In embodiments that include the detection of two or more biomarkers, the biomarkers are detected by the combination of two or more binding agents. In embodiments that include the detection of three or more biomarkers, the biomarkers are detected by the combination of three or more binding agents. In embodiments that include the detection of four or more biomarkers, the biomarkers are detected by the combination of four or more binding agents.

[0260] In some embodiments, the binder, or each binder, comprises an anti-ASGR1 antibody or its antigen-binding fragment, an anti-ASGR2 antibody or its antigen-binding fragment, an anti-SLCO1B1 antibody or its antigen-binding fragment, and an anti-TFR2 antibody or its antigen-binding fragment. In a preferred embodiment, the multiple binders comprise an anti-ASGR1 antibody, an anti-ASGR2 antibody, an anti-SLCO1B1 antibody, and an anti-TFR2 antibody.

[0261] It should be understood that the binder, or each binder, is capable of specifically binding to the corresponding biomarker displayed on the EV. In some embodiments, the biomarker, or each biomarker, is present at elevated levels in the tissue of the defined category. In some embodiments, the biomarker, or each biomarker, present at elevated levels in the tissue of the defined category is identified using the identification methods disclosed herein.

[0262] In some embodiments, the binder or each binder is provided attached to a substrate, preferably wherein the substrate includes magnetic beads or nanoparticles, gold beads or nanoparticles, polystyrene beads, affinity chromatography columns, microplates, microfluidic channels, or biochips having surfaces made of gold, silicon dioxide, glass, graphene, or polystyrene.

[0263] In embodiments that include multiple binders, each binder is preferably provided in a proportion of 10%-50% of the total, wherein the total mixture of binders is equal to 100%.

[0264] In embodiments comprising a plurality of binding agents consisting of anti-ASGR1 antibody, anti-ASGR2 antibody, anti-SLCO1B1 antibody, and anti-TFR2 antibody, the anti-ASGR1 antibody is preferably provided at a proportion of 30%-50% of the total, and each of the other antibodies is preferably provided at a proportion of 10%-30% of the total, wherein the total mixture of binding agents is equal to 100%. In some embodiments, the anti-ASGR1 antibody is preferably provided at a proportion of 40% of the total, and each of the anti-ASGR2 antibody, anti-SLCO1B1 antibody, and anti-TFR2 antibody is preferably provided at a proportion of 20% of the total of the four antibodies (this is equivalent to a final ratio of 4:2:2:2).

[0265] It should be understood that in some implementations, the binder may be an antigen-binding fragment of the antibody.

[0266] In some implementations, biomarkers or each biomarker are detected on or in EVs secreted by tissues that define categories from a biological sample containing a mixture of EVs.

[0267] In a preferred embodiment, the EV mixture comprises EVs from a defined category of tissue and EVs from at least one different category of tissue from the organism. For example, in an embodiment where the defined category of tissue is liver tissue, preferably, when the biological sample is a biofluid, the EV mixture may comprise EVs from liver tissue and EVs from at least one other category of tissue (such as breast tissue).

[0268] Preferably, the biological sample is a tissue sample or a biological fluid. In an embodiment where the biological sample is a biological fluid, the biological fluid comprises a circulating EV. Preferably, the biological fluid is blood, urine, saliva, lymph, bile, cerebrospinal fluid, sputum, mucus, tears, bronchoalveolar lavage (BAL) fluid, earwax, sweat, feces, breast milk, tissue fluid, vaginal fluid, semen, gastric juice, vesicular fluid, or cystic fluid. Most preferably, the biological fluid is blood, urine, cerebrospinal fluid, or mucus.

[0269] Preferably, the tissue defining the category is liver tissue. In embodiments where the tissue defining the category is liver tissue, preferably, the cells secreting EVs therefrom are hepatocytes, Kupffer cells, stellate cells, or resident dendritic cells of the liver. Most preferably, the cells secreting EVs therefrom are hepatocytes.

[0270] A kit for enriching biological samples containing a mixture of EVs to generate enrichment fractions of EVs. The present invention also provides a kit for enriching a biological sample containing a mixture of EVs to generate an enrichment fraction of EVs, wherein the enrichment fraction of EVs is secreted from tissues of a defined category in the organism, wherein the kit comprises one or more binding agents capable of specifically binding to biomarkers present at elevated levels in tissues of a defined category, and wherein the binding agent or each binding agent is selected from anti-ASGR1 antibody or its antigen-binding fragment, anti-ASGR2 antibody or its antigen-binding fragment, anti-TFR2 antibody or its antigen-binding fragment, anti-SLCO1B1 antibody or its antigen-binding fragment, anti-SLC38A3 antibody or its antigen-binding fragment, anti-TMEM56 antibody or its antigen-binding fragment, anti-UNC93A antibody or its antigen-binding fragment, anti-SLC22A9 antibody or its antigen-binding fragment, anti-SLC2A2 antibody or its antigen-binding fragment, and anti-FXYD1 antibody or its antigen-binding fragment, or combinations thereof, preferably selected from anti-ASGR1 antibody or its antigen-binding fragment, anti-ASGR2 antibody or its antigen-binding fragment, anti-TFR2 antibody or its antigen-binding fragment, or anti-SLCO1B1 antibody or its antigen-binding fragment, or combinations thereof.

[0271] In some implementations, the kit contains two or more, three or more, or four or more binding agents that are specifically capable of binding to biomarkers present at elevated levels in tissues of defined categories.

[0272] In some embodiments, the binder, or each binder, comprises an anti-ASGR1 antibody or its antigen-binding fragment, an anti-ASGR2 antibody or its antigen-binding fragment, an anti-SLCO1B1 antibody or its antigen-binding fragment, and an anti-TFR2 antibody or its antigen-binding fragment. In a preferred embodiment, the multiple binders comprise an anti-ASGR1 antibody, an anti-ASGR2 antibody, an anti-SLCO1B1 antibody, and an anti-TFR2 antibody.

[0273] It should be understood that the binders included in the kit, or each binder, are specifically capable of binding to the corresponding biomarkers displayed on the EV. In some embodiments, the biomarkers, or each biomarker, are present at elevated levels in tissues of a defined category. In some embodiments, the biomarkers, or each biomarker, present at elevated levels in tissues of a defined category are identified using the identification methods disclosed herein.

[0274] In some embodiments, the binder or each binder is provided attached to a substrate, preferably wherein the substrate includes magnetic beads or nanoparticles, gold beads or nanoparticles, polystyrene beads, affinity chromatography columns, microplates, microfluidic channels, or biochips having surfaces made of gold, silicon dioxide, glass, graphene, or polystyrene.

[0275] In embodiments that include multiple binders, each binder is preferably provided in a proportion of 10%-50% of the total, wherein the total mixture of binders is equal to 100%.

[0276] In embodiments comprising a plurality of binding agents consisting of anti-ASGR1 antibody, anti-ASGR2 antibody, anti-SLCO1B1 antibody, and anti-TFR2 antibody, the anti-ASGR1 antibody is preferably provided at a proportion of 30%-50% of the total, and each of the other antibodies is preferably provided at a proportion of 10%-30% of the total, wherein the total mixture of binding agents is equal to 100%. In some embodiments, the anti-ASGR1 antibody is preferably provided at a proportion of 40% of the total, and each of the anti-ASGR2 antibody, anti-SLCO1B1 antibody, and anti-TFR2 antibody is preferably provided at a proportion of 20% of the total of the four antibodies (this is equivalent to a final ratio of 4:2:2:2).

[0277] It should be understood that in some implementations, the binder may be an antigen-binding fragment of the antibody.

[0278] In a preferred embodiment, the EV mixture comprises EVs from a defined category of tissue and EVs from at least one different category of tissue from the organism. For example, in an embodiment where the defined category of tissue is liver tissue, preferably, when the biological sample is a biofluid, the EV mixture may comprise EVs from liver tissue and EVs from at least one other category of tissue (such as breast tissue).

[0279] Preferably, the biological sample is a tissue sample or a biological fluid. In an embodiment where the biological sample is a biological fluid, the biological fluid comprises a circulating EV. Preferably, the biological fluid is blood, urine, saliva, lymph, bile, cerebrospinal fluid, sputum, mucus, tears, bronchoalveolar lavage (BAL) fluid, earwax, sweat, feces, breast milk, tissue fluid, vaginal fluid, semen, gastric juice, vesicular fluid, or cystic fluid. Most preferably, the biological fluid is blood, urine, cerebrospinal fluid, or mucus.

[0280] Preferably, the tissue defining the category is liver tissue. In embodiments where the tissue defining the category is liver tissue, preferably, the cells secreting EVs therefrom are hepatocytes, Kupffer cells, stellate cells, or resident dendritic cells of the liver. Most preferably, the cells secreting EVs therefrom are hepatocytes.

[0281] The present invention also provides a method for manufacturing the kit disclosed herein, wherein the method includes a co-localizing binder or each binder.

[0282] An enrichment level of an EV The present invention also provides an enrichment fraction of EVs enriched with EVs secreted by tissues of a defined class in an organism, the EVs exhibiting one or more biomarkers selected from the following: ASGR1, ASGR2, TFR2, SLCO1B1, SLC38A3, TMEM56, UNC93A, SLC22A9, SLC2A2 and FXYD1, or combinations thereof (preferably ASGR1, ASGR2, TFR2 and SLCO1B1, or combinations thereof).

[0283] In some implementations, EVs secreted by tissues that define categories within an organism exhibit two or more, three or more, or four or more biomarkers.

[0284] In some implementations, the EV display of tissue secretion that defines categories from an organism includes multiple biomarkers such as ASGR1, ASGR2, TFR2, and SLCO1B1.

[0285] In some implementations, biomarkers, or each biomarker, are present at elevated levels in tissues that define a category.

[0286] Preferably, the tissue defining the category is liver tissue. In embodiments where the tissue defining the category is liver tissue, preferably, the cells secreting EVs therefrom are hepatocytes, Kupffer cells, stellate cells, or resident dendritic cells of the liver. Most preferably, the cells secreting EVs therefrom are hepatocytes.

[0287] In some implementations, enrichment fractions of EVs are enriched from biological samples containing a mixture of EVs.

[0288] Preferably, the biological sample is a tissue sample or a biological fluid. In an embodiment where the biological sample is a biological fluid, the biological fluid comprises a circulating EV. Preferably, the biological fluid is blood, urine, saliva, lymph, bile, cerebrospinal fluid, sputum, mucus, tears, bronchoalveolar lavage (BAL) fluid, earwax, sweat, feces, breast milk, tissue fluid, vaginal fluid, semen, gastric juice, vesicular fluid, or cystic fluid. Most preferably, the biological fluid is blood, urine, cerebrospinal fluid, or mucus.

[0289] In some implementations, enrichment fractions of EVs are generated by performing the enrichment method for biological samples containing EV mixtures as described in detail above, or after using one or more biomarkers or one or more binding agents as described in detail above.

[0290] In some implementations, enrichment fractions of EVs are generated, and the contents of the EVs are subsequently analyzed by performing the analytical methods for biological samples described in detail above.

[0291] Detailed Implementation Plan In a specific embodiment, the present invention provides a method for identifying a biomarker present in liver tissue of an individual at an elevated level compared to levels in at least one of lung, kidney, or breast tissue. The biomarker is detectable on the surface of EVs secreted from cells of liver tissue. The method includes the following steps: i. Identify candidate biomarkers; ii. Provide an antibody that can specifically bind to the candidate biomarker identified in step i; iii. Detecting the presence of candidate biomarkers on EVs obtained from an individual's liver tissue sample by binding an antibody to a candidate biomarker in the sample, wherein EVs from liver tissue preferentially bind the antibody compared to EVs from at least one of the individual's lung, kidney, or breast tissue; and iv. Select candidate biomarkers as those present at elevated levels in liver tissue.

[0292] In some implementations, the method is applicable to identifying a combination of multiple candidate biomarkers present in liver tissue at elevated levels compared to those in at least one of lung, kidney, or breast tissue. The method includes the following steps: i. Identify four candidate biomarkers; ii. Provide four antibodies, each of which can specifically bind to the corresponding candidate biomarker identified in step i; iii. Detection of the presence of each of the candidate biomarkers on EVs obtained from an individual's liver tissue sample by binding antibodies to the corresponding candidate biomarkers in the sample, wherein EVs from liver tissue preferentially bind to a combination of multiple antibodies compared to at least one EV from the lung, kidney, or breast tissue of the organism; and iv. Select these four candidate biomarkers as a combination of biomarkers present at elevated levels in liver tissue.

[0293] In some embodiments, the four antibodies are provided in a mixture in equal proportions. In other embodiments, the four antibodies are provided in a mixture in unequal proportions, for example, a mixture of the four antibodies in a final ratio of 4:2:2:2.

[0294] In some implementations, antibodies, or each antibody, are provided by attaching to magnetic beads.

[0295] One embodiment includes a method for enriching a blood sample containing an EV mixture taken from an individual, the method comprising the following steps: a) Identify biomarkers present in liver tissue at elevated levels by performing the methods described above; b) Capture EVs in a blood sample, wherein the EVs represent the biomarkers identified in step a), or each of the biomarkers, to generate an enrichment fraction of EVs derived from liver tissue.

[0296] One embodiment includes a method for enriching a blood sample containing an EV mixture taken from an individual, the method comprising the following steps: EVs are captured from blood samples, where EVs exhibit elevated levels in liver tissue, to generate enrichment fractions of EVs derived from liver tissue. The biomarkers, or each biomarker, are selected from ASGR1, ASGR2, TFR2, SLCO1B1, SLC38A3, TMEM56, UNC93A, SLC22A9, SLC2A2, and FXYD1.

[0297] In some embodiments, the step of capturing EVs in a blood sample displaying a biomarker or each biomarker further includes using the biomarker or each biomarker to isolate the captured extracellular vesicle EVs derived from liver tissue.

[0298] One implementation includes a method for analyzing a blood sample containing an EV mixture taken from an individual, the method comprising the following steps: a) Identifying biomarkers present at elevated levels in liver tissue by performing the method of the present invention; b) Capture EVs from a blood sample, wherein the EVs represent the biomarkers identified in step a), or each of the biomarkers, to generate an enrichment fraction of EVs derived from liver tissue; and c) Analyze the contents of the EV in the enrichment fraction obtained in step b).

[0299] One implementation includes a method for analyzing a blood sample containing an EV mixture taken from an individual, the method comprising the following steps: a) Capture EVs from blood samples, wherein EVs exhibit one or more biomarkers present at elevated levels in liver tissue, to generate an enrichment fraction of EVs derived from liver tissue; and b) Analyze the contents of the EVs in the enrichment fractions obtained in step a). The biomarkers, or each biomarker, are selected from ASGR1, ASGR2, TFR2, SLCO1B1, SLC38A3, TMEM56, UNC93A, SLC22A9, SLC2A2, and FXYD1.

[0300] In some implementations, the step of capturing EVs in a blood sample includes using an antibody, or each antibody, such that the antibody can specifically bind to the biomarker.

[0301] One embodiment includes the use of one or more biomarkers for generating an enriched fraction of EV from a blood sample containing a mixture of EVs taken from an individual, wherein the enriched fraction of EVs is secreted from cells of liver tissue, wherein the biomarker or each biomarker is selected from ASGR1, ASGR2, TFR2, SLCO1B1, SLC38A3, TMEM56, UNC93A, SLC22A9, SLC2A2, and FXYD1.

[0302] One embodiment includes the use of one or more antibodies for generating an enriched fraction of EVs from a blood sample containing a mixture of EVs taken from an individual, wherein the enriched fraction of EVs is secreted from cells of liver tissue, wherein the antibody or each antibody is selected from anti-ASGR1 antibody, anti-ASGR2 antibody, anti-TFR2 antibody, anti-SLCO1B1 antibody, anti-SLC38A3 antibody, anti-TMEM56 antibody, anti-UNC93A antibody, anti-SLC22A9 antibody, anti-SLC2A2 antibody, and anti-FXYD1 antibody.

[0303] In some implementations, the use includes a variety of antibodies consisting of anti-ASGR1 antibody, anti-ASGR2 antibody, anti-SLCO1B1 antibody, and anti-TFR2 antibody.

[0304] One embodiment includes a method for detecting one or more biomarkers on or in EVs secreted by cells from liver tissue, wherein the biomarkers or each biomarker is selected from ASGR1, ASGR2, TFR2, SLCO1B1, SLC38A3, TMEM56, UNC93A, SLC22A9, SLC2A2, and FXYD1.

[0305] One embodiment includes a kit for enriching a blood sample containing a mixture of EVs taken from an individual to generate an enriched fraction of EVs, wherein the enriched fraction of EVs is secreted from cells of liver tissue, wherein the kit contains one or more antibodies capable of specifically binding to biomarkers present in elevated levels in liver tissue, and wherein the antibody, or each antibody, is selected from anti-ASGR1 antibody, anti-ASGR2 antibody, anti-TFR2 antibody, anti-SLCO1B1 antibody, anti-SLC38A3 antibody, anti-TMEM56 antibody, anti-UNC93A antibody, anti-SLC22A9 antibody, anti-SLC2A2 antibody, and anti-FXYD1 antibody.

[0306] In some implementations, the kit contains a variety of antibodies, including anti-ASGR1 antibody, anti-ASGR2 antibody, anti-SLCO1B1 antibody, and anti-TFR2 antibody.

[0307] One embodiment includes a method of manufacturing a kit for enriching a blood sample containing a mixture of EVs taken from an individual to generate an enriched fraction of EVs, wherein the enriched fraction of EVs is secreted from cells of liver tissue, wherein the kit comprises one or more antibodies capable of specifically binding to biomarkers present in elevated levels in liver tissue, wherein the antibodies, or each antibody, are selected from anti-ASGR1 antibody, anti-ASGR2 antibody, anti-TFR2 antibody, anti-SLCO1B1 antibody, anti-SLC38A3 antibody, anti-TMEM56 antibody, anti-UNC93A antibody, anti-SLC22A9 antibody, anti-SLC2A2 antibody, and anti-FXYD1 antibody, and wherein the method includes co-localization antibodies, or each antibody One implementation includes an enrichment fraction of EVs derived from liver tissue, which is enriched with EVs secreted by cells from the liver tissue, and these EVs display one or more biomarkers selected from ASGR1, ASGR2, TFR2, SLCO1B1, SLC38A3, TMEM56, UNC93A, SLC22A9, SLC2A2, and FXYD1.

[0308] In some implementations, enrichment fractions of EVs are enriched from blood samples containing a mixture of EVs taken from an individual.

[0309] In some variations of the above implementation scheme, the biomarker, or each biomarker, consists of ASGR1, ASGR2, TFR2, and SLCO1B1.

[0310] In some variations of the above embodiments, the biomarker or each biomarker is ASGR1 and the antibody or each antibody is an anti-ASGR1 antibody, the biomarker or each biomarker is ASGR2 and the antibody or each antibody is an anti-ASGR2 antibody, the biomarker or each biomarker is TFR2 and the antibody or each antibody is an anti-TFR2 antibody, or the biomarker or each biomarker is SLCO1B1 and the antibody or each antibody is an anti-SLCO1B1 antibody.

[0311] In some variations of the above embodiments, the four antibodies are provided in a mixture in equal proportions. In other embodiments, the four antibodies are provided in a mixture in unequal proportions, for example, a mixture of the four antibodies in a final ratio of 4:2:2:2.

[0312] In some variations of the above implementation scheme, antibodies, or each antibody, are provided by being attached to magnetic beads.

[0313] In some variations of the above implementation scheme, the EV mixture comprises EVs derived from liver tissue and EVs derived from lung, kidney, or breast tissue.

[0314] Example The present invention will now be described in detail with reference to embodiments.

[0315] Example 1: Biomarkers for identifying liver tissue 1-1) Identification of potential candidate biomarkers for liver tissue Use bioinformatics database search and selection to identify liver-specific biomarkers.

[0316] mRNA and protein expression profiles from The Human Proteins Atlas database (last accessed November 2021) were used to retrieve and obtain a preliminary ranking of hepatocyte-specific markers.

[0317] Four different lists of biomarkers were obtained by combining four search terms, and then these lists were merged to produce a total of 412 candidate biomarkers: 1) Proteins that are enriched or elevated at the transcript level in the liver and not annotated as having high or moderate protein expression in other tissues; 2) Proteins that are considered to be highly expressed with high specificity for hepatocytes and not annotated as having high or intermediate protein expression in other tissues besides the liver are annotated as having high protein expression in hepatocytes. 3) Proteins that are specifically highly expressed in hepatocytes and not annotated as having high protein expression in other tissues besides the liver are annotated as having high protein expression in hepatocytes. 4) Proteins that are considered to be expressed with high specificity and moderate expression in hepatocytes and not annotated as having high or moderate protein expression in other tissues besides the liver are annotated as having moderate protein expression in hepatocytes.

[0318] UniProt was used to retrieve the subcellular localization of these proteins. Proteins labeled as located in the membrane were selected for subsequent steps. A total of 88 candidate biomarkers were followed up (Table 1).

[0319] Table 1. Top 88 candidate biomarkers

[0320] A heatmap with protein expression profiles was generated from a preliminary list of 88 membrane proteins extracted from ProteomicsDB and Human Proteome Map following UniProt subcellular localization annotation. Figure 1 (and Figure 2).

[0321] Expression profiles from ProteomicsDB and Human Proteome Map, along with information previously obtained from The HumanProtein Atlas (last accessed November 2021), were used for further tissue-specific protein sequencing. A total of 34 candidate biomarkers were followed up (Table 2).

[0322] Table 2. Top 34 candidate biomarkers

[0323] 1-2) Validate potential candidate biomarkers in liver tissue Materials and methods: Collecting EVs from cultured cells In a concentration of 5% CO2 and 37 o The HCC cell line HepG2 was grown in Advanced-DMEM growth medium supplemented with 5% (v / v) FBS and 4 mM glutaMAX under a humidified atmosphere at C. After reaching 70% confluence in T175 flasks, the cells were gently washed three times with PBS and cultured in FBS-free Advanced-DMEM medium.

[0324] After 48 hours, the conditioned medium was collected and centrifuged at 300 g for 5 min, followed by centrifugation at 3000 g and 4 min. o Centrifuge again at C for 10 min to remove cell debris and other large contaminants. Then, filter the conditioned medium through a 0.22 μm PES filter and concentrate to 500 μL using an Amicon Ultra-15 100K MWCO centrifuge filter. Following the supplier's instructions, EVs were finally separated by size exclusion chromatography using an IZON qEV original / 70 nm column on an automated fractionating collector (AFC).

[0325] Collect the first three fractions corresponding to a total elution volume of 1.5 mL, combine them, aliquot them, and store them at -80°C. o C, until further use.

[0326] EVs collected from tissue samples Frozen liver tissue was obtained from a dedicated biobank. To prepare tissue for EV isolation, 2 mL of Advanced-DMEM was added to 5 small pieces of 0.2 mg tissue. Collagenase D and DNase I were added to final concentrations of 2 mg / mL and 40 U / mL, respectively, and the mixture was incubated at 37°C. o Incubate at C with gentle rotation for 30 min. To remove large debris, the solution was filtered through a 70 μM filter, and the filter was washed with 20 mL of sterile PBS. Furthermore, a series of centrifugations at 500, 3000, and 10000 g were performed at 10, 20, and 30 min, respectively, discarding any precipitates formed at each step. Finally, the sample was filtered through a 0.22 μM filter to remove large vesicles and other contaminants, and concentrated to 1 mL using an Amicon Ultra-15 100K MWCO centrifugal filter.

[0327] According to the supplier's instructions, EVs were separated by size exclusion chromatography using an Izon qEV GEN2 35 nm column on an automated fraction collector (AFC).

[0328] The first two fractions corresponding to a total elution volume of 2.4 mL were collected, combined, aliquoted, and stored at -80°C. o C, until further use.

[0329] The separated EVs were characterized by nanoparticle tracking analysis. The number and size distribution of extracellular vesicle formulations were determined using nanoparticle tracking analysis (NTA) with a NanoSight NS300 system (Malvern) equipped with a 488 nm laser and a high-sensitivity scientific CMOS camera.

[0330] Following the manufacturer's recommendations, the samples were diluted to an acceptable concentration in particle-free PBS. The samples were analyzed at a constant flow rate, and 3 × 60 seconds of video were captured at camera level 12. The data were analyzed using NTA 3.4 software at a detection threshold of 7.

[0331] Preparation of antibody-labeled magnetic beads Using Dynabeads TMThe antibody conjugation kit conjugates magnetic beads (Dynabeads® M-270 epoxy beads) to the following antibodies: anti-CD63 antibody (orb506484, Biorbyt; anti-ABCB11 (sc-74500, Santa Cruz Biotechnology); anti-ABCB11 antibody (STJ190611, St John's Laboratory); anti-ABCB4 antibody (ABIN6388963, antibodies-online.com); anti-ABCB4 antibody (CSB-PA001050LA01HU, Cusabio); anti-ASGR1 antibody (NBP1-60150, Novus Biologicals); anti-ASGR1 antibody (MAB43941-100, R&D Systems); anti-ASGR1 antibody (ab254262, Abcam); anti-ASGR1 antibody (sc-52623, Santa Cruz Biotechnology); and anti-TFR2 antibody (MAB3120, Novus). Biologicals); Anti-TFR2 antibody (ab13579, Abcam); Anti-GHR antibody (STJ97268, St John's Laboratory); Anti-RNF130 antibody (orb1291, Biorbyt); Anti-SLC17A4 antibody (sc-135562, Santa Cruz Biotechnology); Anti-SLC2A2 antibody (orb33144, Biorbyt); Anti-SLC2A2 (CSB-PA13329A0Rb, Cusabio); Anti-SLC2A2 antibody (sc-518022, Santa Cruz Biotechnology); Anti-AQP9 antibody (ABIN2746270, antibodies-online.com); Anti-ASGR2 antibody (orb757191, Biorbyt); Anti-ASGR2 antibody (ab200196, Abcam); Anti-SLC10A1 antibody (sc-518115, Santa Cruz Biotechnology); Biotechnology); Anti-SLCO1B1 antibody (CSB-PA896932LA01HU, Cusabio); Anti-SLCO1B1 antibody (sc-271157, Santa Cruz Biotechnology); Anti-SLCO1B1 antibody (STJ94587, St. John's Laboratory); Anti-SLC13A5 antibody (CSB-PA768239LA01HU, Cusabio);Anti-SLC13A5 antibody (sc-293277, Santa Cruz Biotechnology); Anti-SLC38A4 antibody (sc-515125, Santa Cruz Biotechnology); Anti-SLC38A3 antibody (sc-398982, Santa Cruz Biotechnology); Anti-SLC38A3 antibody (orb37057, Biorbyt); Anti-SLC22A9 antibody (CSB-PA811607LA01HU, Cusabio); Anti-CLEC4G antibody (MAB2947, R&D Systems); Anti-CLEC4G antibody (sc-65478, Santa Cruz Biotechnology); Anti-CLEC4G antibody (CSB-PA744269LA01HU, Cusabio); Anti-FXYD1 antibody (sc-393415, Santa Cruz Biotechnology); Anti-ELFN1 antibody (MAB10644, R&D Systems). Systems); Anti-LRRC3 antibody (MAB5039, R&D Systems); Anti-PTP4A1 antibody (sc-130354, Santa Cruz Biotechnology); Anti-TMEM56 antibody (CSB-PA021714LA01HU, Cusabio); Anti-UNC93A antibody (sc-390157, Santa Cruz Biotechnology); Anti-UNC93A antibody (CSB-PA773046LA01HU, Cusabio); Anti-RTP3 antibody (CSB-PA887106LA01HU, Cusabio); Anti-SLC17A2 antibody (CSB-PA004099, Cusabio); Anti-HEPCAM antibody (anti-sc-515637, Santa Cruz Biotechnology); Anti-ABCC6 antibody (ab167564, Abcam); or Anti-APOE antibody (sc-13521, Santa Cruz Biotechnology).

[0332] In short, wash the magnetic beads in LoBind microcentrifuge tubes (Eppendorf) under constant rotation according to the kit instructions and at 37°C. o Incubate overnight at C. The amount of capture antibody used per reaction is 10 μg / mg magnetic beads. Tween® 20 (0.05%) was added to the HB and LB wash buffers to improve stringency, as recommended by the supplier. The magnetic beads were washed with a DynaMag-rotating magnetic rack (Invitrogen).

[0333] Detection of EVs captured by antibody-labeled magnetic beads After separating EVs by differential centrifugation, ultrafiltration, and size exclusion chromatography, cell culture-derived EVs were normalized to 7 × 10⁻⁶ in PBS. 8 The concentration was calculated as parts per mL, and tissue-derived EVs were normalized to 1 × 10⁻⁶ in 20% Assay Defender (Candor Bioscience). 10 Concentration per part / mL.

[0334] Cell-derived EVs and tissue-derived EVs were captured in separate reactions using magnetic beads pre-coated with the following antibodies: anti-CD63 antibody; anti-ABCB1 antibody; anti-ABCB11 antibody; anti-ABCB4 antibody; anti-ABCB4 antibody; anti-ASGR1 antibody; anti-ASGR1 antibody; anti-ASGR1 antibody; anti-ASGR1 antibody; anti-TFR2 antibody; anti-TFR2 antibody; anti-GHR antibody; anti-RNF130 antibody; anti-SLC17A4 antibody; anti-SLC2A2 antibody; anti-SLC2A2 antibody; anti-SLC2A2 antibody; anti-AQP9 antibody; anti-ASGR2 antibody; anti-ASGR2 antibody; anti-SLC10A1 antibody; anti-SLCO1 antibody. Antibody B; anti-SLCO1B1 antibody; anti-SLCO1B1 antibody; anti-SLC13A5 antibody; anti-SLC13A5 antibody; anti-SLC38A4 antibody; anti-SLC38A3 antibody; anti-SLC38A3 antibody; anti-SLC22A9 antibody; anti-CLEC4G antibody; anti-CLEC4G antibody; anti-CLEC4G antibody; anti-FXYD1 antibody; anti-ELFN1 antibody; anti-LRRC3 antibody; anti-PTP4A1 antibody; anti-TMEM56 antibody; anti-UNC93A antibody; anti-UNC93A antibody; anti-RTP3 antibody; anti-SLC17A2 antibody; anti-HEPCAM antibody; anti-ABCC6 antibody; or anti-APOE antibody.

[0335] Pre-coated magnetic beads with anti-CD63 antibody prepared according to Examples 1-2) were used as a control. The samples were mixed every 30 min, and after 12 hours of incubation, the EVs captured by the magnetic beads were recovered by washing the samples in a magnetic field using KingFisher.

[0336] For all experiments, biotinylated anti-CD63 antibody was used for signal detection. The biotinylated anti-CD63 antibody was incubated with EVs captured by magnetic beads at 10 ng / μL for 60 min. Then, the magnetic beads were washed, and 200 μL of 1:6000 diluted Poly-HRP strep was incubated with the sample for 30 min. After washing, 100 μL of QuantaBlu was added... TMAdd to each sample and incubate for 40 minutes. Stop the reaction by adding 100 μL of stop solution, and transfer 190 μL of each sample to an F-shaped 96-well plate for signal measurement at Ex / Em = 320 / 405 nm.

[0337] result: EVs were collected from HEPG-2 HCC cell cultures and HCC tissue samples, and then captured and isolated by targeting the top 34 candidate biomarkers identified in the previous steps (Tables 3 and 4, respectively).

[0338] Table 3.

[0339] Table 4.

[0340] Considering the functional importance of biomarkers in the liver, candidate biomarkers that exhibit a signal-to-noise ratio greater than 2.5 and a signal-to-noise difference greater than 0.5 OD in EVs derived from liver tissue or liver cell lines were followed up (Table 5).

[0341] Table 5. Top 11 candidate biomarkers

[0342] After capturing EVs collected from HCC cell lines and HCC tumor tissues, heatmaps of protein expression profiles for 11 proteins showing high signal intensity were extracted from ProteomicsDB and HumanProteome Map. Figure 3 and Figure 4 ).

[0343] 1-3) Detection of candidate biomarkers Materials and methods: Collecting EVs from cultured cells HCC cell lines HepG2 (ATCC, catalog number HB-8065) ​​and breast cancer cell lines MCF-7 (ATCC, catalog number HTB-22) and BT-474 (ATCC, catalog number HTB-20) were cultured according to Examples 1-2 and EVs were collected.

[0344] EVs collected from tissue samples According to Examples 1-2), frozen lung, kidney, and liver tissues were obtained from a specialized biobank and EVs were collected.

[0345] EVs collected from human blood plasma Frozen human plasma was obtained from whole blood in the K2EDTA Vacutainer (Cambridgebioscience). The human plasma was obtained with ethical consent and derived from a pool of healthy, paid volunteers (Research Donors). Whole blood from patients with cirrhosis and HCC in the K2EDTA Vacutainer was collected with ethical consent by the BIOBANCO-Molecular Medicine Institute / Santa Maria Hospital and UCL Biobank - Royal Free London NHS Foundation Trust (RFL B-ERC).

[0346] Human plasma was slowly thawed on ice and divided into two 2 mL portions. One portion was then inoculated with BT-474EV to achieve a final concentration of 1.67E9 portions / mL. Then, it was subjected to treatment at 3000 g and 4... o Centrifugation at C for 15 min followed by centrifugation at 10000 g and 4 o Centrifugation at C for an additional 20 min to remove any cell debris, large particles, or aggregates was performed on both doped and undoped samples equally. Samples were then filtered using a 0.22 μm PES filter and processed in 1.8 mL fractions per sample using an Izon qEV270 nm column at AFC via size exclusion chromatography. As with cell culture formulations, the first three fractions of elution from each sample, corresponding to a total of 5.4 mL / sample, were collected and combined.

[0347] The separated EVs were characterized by nanoparticle tracking analysis. The particle number and size distribution of MCF-7, BT-474 and extracellular vesicle formulations were determined according to Examples 1-2.

[0348] Preparation of antibody-labeled magnetic beads Antibody-labeled magnetic beads were prepared according to Examples 1-2.

[0349] Detection of EVs captured by antibody-labeled magnetic beads After EVs were separated by differential centrifugation, ultrafiltration, and size exclusion chromatography, EVs from various cell cultures were normalized to 7 × 10⁻⁶ in PBS. 8 The concentration was calculated as parts per mL, and tissue-derived EVs were normalized to 1 × 10⁻⁶ in 20% Assay Defender. 10 Concentration per part / mL. Dilute the isolated plasma EVs in 20% Assay Defender.

[0350] Cell-derived EVs were captured using magnetic beads pre-coated with the following antibodies prepared according to Examples 1-2): anti-ASGR1 antibody, anti-TFR2 antibody, anti-SLC2A2 antibody, anti-ASGR2 antibody, anti-SLCO1B1 antibody, anti-SLC22A9 antibody, anti-SLC38A3 antibody, anti-CLEC4G antibody, anti-FXYD1 antibody, anti-TMEM56 antibody, or anti-UNC93A antibody.

[0351] Tissue-derived EVs were captured using magnetic beads pre-coated with the following antibodies prepared according to Examples 1-2): anti-ASGR1 antibody, anti-TFR2 antibody, anti-SLC2A2 antibody, anti-ASGR2 antibody, anti-SLCO1B1 antibody, anti-SLC22A9 antibody, anti-SLC38A3 antibody, anti-CLEC4G antibody, anti-FXYD1 antibody, anti-TMEM56 antibody, or anti-UNC93A antibody.

[0352] The isolated plasma EVs were captured in separate reactions of each antibody-pre-coated magnetic bead formulation or mixed together with magnetic beads prepared according to Examples 1-2) in a ratio of 4:2:2:2 ASGR1:ASGR2:TRF1:SLCO1B1 and incubated in a KingFisher Flex system.

[0353] result: Detection of candidate biomarkers in cultured cells EVs were collected from HCC and breast cancer cell cultures and then captured and isolated by targeting the top 11 candidate biomarkers identified in previous bioinformatics steps (Table 5).

[0354] like Figure 5 As shown, capturing EVs using magnetic beads pre-coated with anti-ASGR1 antibody (ab1), anti-ASGR2 antibody, anti-TFR2 antibody, anti-SLCO1B1 antibody, anti-SLC38A3 antibody, anti-TMEM56 antibody, anti-UNC93A antibody, anti-SLC22A9 antibody, anti-SLC2A2 antibody, or anti-FXYD1 antibody resulted in higher signal intensity in HCC cell lines compared to breast cancer cell lines 1 (MCF-7) and 2 (BT-474) and the buffer control. In contrast, no significant differences were observed for EVs isolated from different cell cultures using magnetic beads pre-coated with anti-CLEC4G antibody or a second anti-ASGR1 antibody (ab2).

[0355] Candidate biomarkers showing higher signals for EVs isolated from HCC cell lines were validated compared to non-target breast cancer cell lines 1 and 2 and buffer control, and continued to be detected in tissue samples.

[0356] Those biomarkers that did not result in any significant difference in CD63 surface marker signaling on EVs between HCC cell lines, breast cancer cell lines 1 and 2, and buffer control were not validated.

[0357] Detection of candidate biomarkers in tissue samples EVs were collected from various frozen human tissue samples. EVs were then captured and isolated by targeting different candidate biomarkers identified from the aforementioned cell culture experiments.

[0358] like Figure 6 As shown, capturing EVs using magnetic beads pre-coated with anti-ASGR1 antibody (ab1), anti-ASGR2 antibody, anti-TFR2 antibody, or anti-SLCO1B1 antibody resulted in higher signal intensity from healthy liver and HCC tissues compared to kidney, lung, and buffer controls. In contrast, no significant differences were observed for EVs isolated from different tissue types using magnetic beads pre-coated with anti-SLC38A3 antibody, anti-TMEM56 antibody, anti-UNC93A antibody, anti-SLC22A9 antibody, anti-SLC2A2 antibody, or anti-FXYD1 antibody.

[0359] Candidate biomarkers showing higher signals for EVs isolated from liver-derived tissue samples (healthy tissue and HCC tissue) compared to non-target tissue types and buffer controls have been validated and continue to be used for detection in plasma samples.

[0360] Those biomarkers that did not result in any significant differences in CD63 surface marker signaling on EVs between liver tissue, HCC tissue, kidney tissue, lung tissue, and buffered controls were not validated.

[0361] Detection of candidate biomarkers in plasma samples EVs were collected from human plasma derived from blood taken from healthy individuals. EVs were then captured and isolated by targeting different candidate biomarkers identified in the aforementioned tissue sample experiments.

[0362] like Figure 7 As shown, capturing EVs using magnetic beads pre-coated with anti-ASGR1 antibody (ab1), anti-ASGR2 antibody, anti-TFR2 antibody, or anti-SLCO1B1 antibody resulted in at least one detectable signal in plasma samples. Differences in biomarker expression were also observed between individuals.

[0363] Based on these results, ASGR1, ASGR2, TFR2, and SLCO1B1 were identified as candidate biomarkers for liver tissue.

[0364] EVs collected from human plasma were then captured and separated using a binding mixture validated in the previous steps. This mixture contained magnetic beads pre-coated with anti-ASGR1, anti-ASGR2, anti-SLCO1B1, and anti-TFR2 antibodies in a final ratio of 4:2:2:2.

[0365] like Figure 8 As shown, compared with the isotype control, the antibody mixture using the targeted biomarkers ASGR1, ASGR2, TFR2 and SLCO1B1 captured EVs and consistently detected higher signals in human plasma from individuals with different phenotypes (healthy, HCC and liver disease).

[0366] Validation of the detection method EVs were collected from different tissues and then captured and isolated by targeting the cell surface antigen CD63. Biotinylated anti-CD63 antibodies were used for signal detection.

[0367] like Figure 9 As shown, the signal-to-noise ratio (SNR) of EVs captured by anti-CD63-labeled magnetic beads, detected using biotinylated anti-CD63 antibody, was comparable across different tissue types. This indicates that CD63 expression was similar in EVs obtained from different tissues.

[0368] This result suggests that any differences in the detection signal observed after capturing EVs using magnetic beads labeled with antibodies targeting other biomarkers depend on their differential capture of EVs.

[0369] Example 2: Mass spectrometry validation of the method for isolating liver-derived EVs Materials and methods: Collecting EVs from human blood plasma Frozen human plasma was obtained according to Examples 1-3, and EVs were collected.

[0370] Isolation of hepatocyte-derived EV 5.4 mL of separated plasma EVs were diluted in 20% Assay Defender (Candor Bioscience) and captured with 300 μL of a binding agent mixture selected in Examples 1-3 (a mixture of magnetic beads pre-coated with anti-ASGR1 antibody, anti-ASGR2 antibody, anti-SLCO1B1 antibody, and anti-TFR2 antibody in a final ratio of 4:2:2:2). o Incubate at C for 16 hours, and mix every 30 minutes in KingFisher.

[0371] EVs captured by antibody-labeled magnetic beads were recovered, and other uncaptured EVs and plasma particles were washed away with KingFisher in a magnetic field. Finally, the captured EVs were lysed for protein or RNA separation.

[0372] Preparation of antibody-oligo-labeled magnetic beads The antibodies selected in Examples 1-3) were functionalized with DBCO-PEG4-NHS ester. 20 μg of antibody was incubated with 1.54 nmol DBCO in 100 μL PBS at room temperature for 1 hour. Free DBCO was washed using a 12 kDa D-tube dialyzer. The DBCO-functionalized antibody was then mixed with 8 μg of oligo poly(A)-azide via a 4-diol dialyzer. o Incubate overnight at C for conjugation. Remove free oligos using a 40kDA Zeba column. Then label the magnetic beads (Dynabeads® Oligo(dT)25) with oligo-antibody at a ratio of 1 mg magnetic beads to 10 μL oligo-antibody. Prepare the oligo-magnetic beads according to the kit instructions.

[0373] Elution of intact extracellular vesicles After separating the EVs, 300 μL of 0.2 M glycine at pH 2.5 was added to the MB at room temperature and incubated for 10 min, retaining the supernatant. This step was repeated once. Then, at 70 °C... o The magnetic beads were incubated in 300 μL of 0.05% T20 in Milli-Q solution for 5 min at C. All the resulting supernatants were then combined.

[0374] If oligo magnetic beads are used for EV separation, then 7.5 U of DNase I is added to 300 μL of 2.5 mM MgCl2 along with the magnetic beads and incubated at 37°C. o Incubate at C for 1 hour to wash away EVs.

[0375] Pyrolysis of captured EVs After separation, 1x RIPA was added to lyse EVs bound to antibody-labeled magnetic beads, or added to the eluted EVs, and the mixture was kept at 4°C. o C lasted for 1 hour, then at 95 o 10 min at C, 1 min at HIFU (100% amplitude), 2 x 2 min at 30 Hz in a tissue homogenizer with glass beads, followed by 95 Hz. oAn additional heating step of 10 min is performed at C. Alternatively, the sample is pyrolyzed using the commercially available buffer sodium deoxycholate (SDC). The sample is then mixed in a shaker at 1000 rpm and heated at 95°C. o Heating at C for 10 min and obtaining the pyrolysis product.

[0376] Sample preparation for mass spectrometry The protein was precipitated in trichloroacetic acid (5% TCA) and dissolved in 10 mM Tris / 2 mM CaCl2 at pH 8.2, followed by precipitation with TCEP and chloroacetamide at 30 °C. o Reduction and alkylation were performed at C for 30 min. The mixture was then treated with trypsin (100 ng / μL in 10 mM HCl) at 37°C. o Protein digestion was performed overnight at C. The digested sample was dissolved in 12 μL ddH2O + 0.1% formic acid and transferred to an autosampler vial for liquid chromatography-mass spectrometry (LC-MS / MS). The sample was injected onto a M-class UPLC-coupled Fusion Lumos mass spectrometer (Thermo) or onto a timsTOF-SCP (Bruker). Data were processed in data-dependent acquisition (DDA) format.

[0377] Protein identification and quantification were performed using two search engine platforms: Mascot (Matrixscience) and MaxQuant (MQ).

[0378] Mass spectrometry analysis For data analysis using Fusion Lumos, total spectrum counts were obtained using Scaffold 5 with the following stringency: total spectrum count > 1; protein false detection rate (FDR): 1%; minimum peptide count / protein: 2; peptide FDR: 0.1%. Proteins identified using these cutoff values ​​were matched against a list of 412 liver-specific proteins obtained from The Human Protein Atlas in Examples 1-1 (Table 1) to evaluate the efficiency of our method in capturing liver proteins in complex samples. The Gene Ontology Resource Perform GO enrichment analysis.

[0379] For data analysis acquired using timsTOF-SCP, LFQ intensity data were obtained from MaxQuant output files. Identified protein groups with quantifiable LFQ intensity data were matched against our list of 412 liver proteins and 16 proteins known to be essential or relevant to EV and exosome biogenesis (i.e., ATP1A1, BSG, CAV1, CAV2, CD47, CD63, CD81, CD9, CLTC, ITGB1Bp, LAMP1, LGALS3BP, PDCD6IP, SDCBP, SLC3A2, and TSG101).

[0380] Use RStudio to plot the LFQ intensity values ​​of the Log2 transformation for protein groups matching two lists. Identified protein groups with quantifiable LFQ intensity data are used for... DAVID Functional enrichment was performed using CC (cellular component) GO terms with high statistical significance (Benjamini-Hochberg adjusted p-value < 1E-10). Revigo Used to generate a ThreeMap graph.

[0381] result: Orbitrap Quality Analyzer EVs collected from human plasma were captured and separated using a mixture of binders selected in Examples 1-3. As described above, the captured EVs were eluted using (1) glycine, (2) RIPA, or (3) DNase, followed by processing of the lysate and analysis by mass spectrometry using Fusion Lumos.

[0382] like Figure 10 As shown, a total of seven liver-specific biomarkers were detected by mass spectrometry: ASGR1, HPR, ARG1, CAT, ASGR2, C4B, and F2.

[0383] In addition, such as Figure 11 As shown, regardless of the lysis protocol, EVs were identified as enriched after annotating the genes identified by mass spectrometry using GO cell component analysis.

[0384] These results indicate that liver-derived EVs were enriched after the above-described separation method.

[0385] TIMS-TOF quality technology EVs collected from human plasma were captured and separated using a mixture of binders selected in Examples 1-3. As described above, the captured EVs were lysed using either (1) RIPA or (2) SDC buffer, and the lysates were subsequently processed and analyzed by timsTOF-SCP mass spectrometry.

[0386] like Figure 12 As shown, among the 16 proteins searched, a total of 13 classic EV markers were detected by mass spectrometry.

[0387] As shown in Figure 13, a total of 63 liver-specific biomarkers were detected by mass spectrometry.

[0388] Furthermore, as shown in Figure 14, regardless of the lysis protocol, components annotated as originating from extracellular exosomes were identified as enriched after annotating protein groups identified by mass spectrometry using GO cell component analysis.

[0389] These results further demonstrate that liver-derived EVs were enriched after the above-described separation method.

[0390] Example 3: RNA sequencing validation of liver-derived EV isolation method Materials and methods: Collecting EVs from human blood plasma According to Examples 1-3), frozen human plasma was obtained from healthy volunteers, patients with cirrhosis, and patients with HCC, and EVs were collected.

[0391] Isolation of hepatocyte-derived EV 5.4 mL of isolated plasma EVs were diluted in 20% Assay Defender (Candor Bioscience) and captured with 300 μL of magnetic beads pre-coated with anti-ASGR1 antibody only (prepared according to Examples 1-2), or captured with 300 μL of a mixture of binders selected in Examples 1-3 (TOP4) (a mixture of magnetic beads pre-coated with anti-ASGR1 antibody, anti-ASGR2 antibody, anti-SLCO1B1 antibody, and anti-TFR2 antibody in a final ratio of 4:2:2:2). For RNA analysis, 0.75 mL of isolated plasma EVs were used in 20% Assay Defender with 35 μL of magnetic beads coated with the aforementioned antibodies. o Incubate at C for 16 hours, and mix every 30 minutes in KingFisher.

[0392] EVs captured by antibody-labeled magnetic beads were recovered, while other uncaptured EVs and plasma particles were washed away with KingFisher in a magnetic field. Finally, RNA was isolated from the captured EVs.

[0393] RNA was isolated from the captured EVs. After separation, 600 μL of PBS was added to the EVs bound to antibody-labeled magnetic beads, and the qEV RNA extraction kit from IZON was used for RNA separation according to the supplier's instructions. In short, the sample was lysed for 20 min, and then the antibody-labeled magnetic beads were removed from the solution. The RNA was precipitated with ethanol and loaded onto a spinning column. The column was washed twice, and then the RNA was eluted with 50 μL of elution buffer. The sample was incubated at 45°C. o Rapidly vacuum-dry at C and resuspend in 10 μL of nuclease-free water, and store at -80°C if not used immediately. o C below.

[0394] Construction of small RNA library Small RNA libraries were prepared using the PerkinElmer NEXTFLEX V4 kit, following the manufacturer's instructions. The starting material consisted of 4 μL of isolated RNA as described above, with 1 μL of tRNA / yRNA blocking agent added. Library construction quality was assessed using a high-sensitivity D1000 DNA ScreenTape assay on a TapeStation 4200, while concentrations were measured using a Qubit dsDNA high-sensitivity assay.

[0395] RNA sequencing and data analysis Sequencing was performed on a NextSeq 550 or NextSeq 2000 Illumina instrument at 1 x 75 or 1 x 100 cycles, respectively. Small RNA reads were QC and annotated via the miRge3.0 (Wickham, 2016) pipeline with the following tags activated: -on human -ie -ai -spl and parameters supplied for appropriate adapter removal. Post-annotation processing was performed using Rstudio (Patil and Halushka, 2021) (for presence / absence analysis, differential expression analysis, and results visualization) and Python version 3 (for correlation investigation). Presence and absence analysis was performed using simple set operations. Differential expression was performed using DESeq2 with settings similar to those supplied for miRge3.0 differential expression analysis. Correlation investigation was performed using custom BioNLP Python code, and results were visualized using ggplot2 (R Core Team, 2023).

[0396] result: RNAseq was used to compare the isolation of liver-derived EVs using ASGR1 binding agent versus a mixture of binding agents. EVs collected from human plasma obtained from healthy volunteers were pooled in equal volumes and then captured and separated using magnetic beads labeled with anti-ASGR1 antibody or a mixture of binding agents selected in Examples 1-3. Following EV and small RNA segregation, samples were evaluated in two independent runs using two different RNA library preparation methods: RealSeq (sequencing method 1) and NEXTFLEX v4 (sequencing method 2). The total number of miRNA IDs obtained was analyzed.

[0397] like Figure 15 As shown, regardless of the RNA sequencing method used, separating EVs using a mixture of binders targeting ASGR1, ASGR2, SLCO1B1, and TFR2 (TOP4) resulted in a higher number of annotated miRNA IDs detected in small samples compared to using binders that only target ASGR1.

[0398] These results demonstrate that using a mixture of binders offers advantages in capturing and identifying miRNAs from liver-derived EVs compared to using binders targeting a single biomarker, ASGR1, alone.

[0399] Comparison of liver-derived EV isolation with previous methods Plasma was obtained from 8 HCC patients, 8 cirrhosis patients, and 4 healthy volunteers. EVs were then collected and combined in two equal volumes. Group A underwent direct RNA sequencing (Protocol A). Meanwhile, Group B was further captured and isolated using a mixture of binding agents selected in Examples 1-3 (Protocol B) prior to RNA sequencing.

[0400] like Figure 16 As shown, BioNLP analysis of RNA sequencing data revealed that, compared to scheme A, scheme B significantly enriched annotated miRNA information related to all key search terms (extracellular vesicles (EV), liver, cirrhosis, cancer, and HCC).

[0401] These results demonstrate that the separation method of the present invention effectively enriches the populations of EVs derived from healthy livers and EVs derived from the livers of HCC patients.

[0402] The results also indicate that, compared to previous separation methods, capturing liver-derived EVs using the separation method of the present invention provides a way to obtain additional information from EVs.

[0403] References: Chang et al. (2019). Role of miR-16-5p in the proliferation and metastasis of hepatocellular carcinoma. European Review for Medical and Pharmacological Sciences , 23(1), 137-145. Dias et al. (2024). An electro-optical technology for the ultrasensitive detection of small extracellular vesicle sub-populations and their protein epitope counts. ISCIENCE . Hoshino et al. (2020). Extracellular Vesicle and Particle Biomarkers Define Multiple Human Cancers. Cell , 182(4):1044-1061. Karimi et al. (2022). Tetraspanins distinguish separate extracellular vesicle subpopulations in human serum and plasma - Contributions of platelet extracellular vesicles in plasma samples. Journal of Extracellular Vesicles ,11, e12213. Khan et al. (2020). Artificial Blood: The History and Current Perspectives of Blood Substitutes. Discoveries (Craiova) , 8(1), e104. Lekchnov et al. (2018). Searching for the Novel Specific Predictors of Prostate Cancer in Urine: The Analysis of 84 miRNA Expression. International Journal of Molecular Sciences , 19(12, 4088. Li et al. (2020). EV-origin: Enumerating the tissue-cellular origin of circulating extracellular vesicles using exLR profile. Computational and Structural Biotechnology Journal , 18, 2851-285. Lightbody et al. (2021). Induction of microRNA hsa-let-7d-5p, and repression of HMGA2, contribute protection against lipid accumulation in macrophage ‘foam’ cells. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids , 1866(11), 159005. Moirangthem et al. (2023). Extracellular vesicle-mediated miR-126-3p transfer contributes to inter-cellular communication in the liver tumor microenvironment. International Journal of Oncology , 62(2), 31. Patil and Halushka (2021). miRge3. 0: a comprehensive microRNA and tRF sequencing analysis pipeline. NAR Genomics and Bioinformatics , 3(3). R Core Team (2023). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria . Available from: https: / / www.R-project.org / . Sun et al. (2020). Purification of HCC-specific extracellular vesicles on nanosubstrates for early HCC detection by digital scoring. Nature Communications , 11, 4489. Sun et al. (2023). HCC EV ECG score: An extracellular vesicle‐based protein assay for detection of early‐stage hepatocellular carcinoma. Hepatology , 77(3), 774-788. Tauro et al. (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. Weng, Z. (2021). Therapeutic roles of mesenchymal stem cell-derived extracellular vesicles in cancer. Journal of Hematology & Oncology , 14, 136. Wickham, H. (2016). ggplot2: Elegant Graphics for Data Analysis .Springer-Verlag, New York. Zheng, Q. (2021). The Unique Immunomodulatory Properties of MSC-Derived Exosomes in Organ Transplantation. Frontiers in Immunology , 12:659621.

Claims

1. A method for identifying a biomarker present in an organism at an elevated level compared to levels in at least one different category of tissue, in a tissue defining a category, wherein the biomarker is detectable on or in extracellular vesicles secreted from cells of the tissue defining the category, the method comprising the steps of: i. Identify candidate biomarkers; ii. Provide a binding agent that is specifically bound to the candidate biomarker identified in step i; iii. Detecting the presence of the candidate biomarker on extracellular vesicles obtained from the sample of the defined category of tissue by binding the binder to the candidate biomarker in the sample, wherein extracellular vesicles from the defined category of tissue preferentially bind the binder compared to extracellular vesicles from at least one different category of tissue of the organism; iv. Select the candidate biomarkers as biomarkers present at elevated levels in the defined categories of tissues.

2. The method of claim 1, wherein step iii. comprises using a device capable of operating at least 1x10 7 The technique for detecting the biomarker at a concentration of at least 1 x 10⁻⁶ biomarkers / ml is used to detect the presence of the candidate biomarker, wherein the concentration is preferably at least 1 x 10⁻⁶. 6 1x10 5 1x10 4 1x10 3 1x10 2 10 or 1 biomarker / ml.

3. The method of claim 1, wherein step iii. comprises using a biomarker capable of being applied at least 1x10⁻¹⁰ using the candidate biomarker. 7 The presence of the candidate biomarker is detected by a technique that measures extracellular vesicles at a concentration of at least 1 x 10⁻⁶ / ml, wherein the concentration is preferably at least 1 x 10⁻⁶ / ml. 6 1x10 5 1x10 4 1x10 3 1x10 2 10 or 1 extracellular vesicle / ml.

4. The method according to any one of the preceding claims, wherein step iii. further comprises determining the concentration of the biomarker in the sample.

5. The method according to any one of the preceding claims, wherein: Step i. includes identifying multiple candidate biomarkers; Step ii. includes providing a variety of binding agents, each of which can specifically bind to the corresponding candidate biomarker identified in step i; Step iii. includes detecting the presence of each of the candidate biomarkers on extracellular vesicles obtained from the sample of the defined category of tissue by binding the binding agent to the corresponding candidate biomarker in the sample, wherein extracellular vesicles from the defined category of tissue preferentially bind the combination of the multiple binding agents compared to extracellular vesicles from the at least one different category of tissue of the organism; and Step iv. includes selecting the multiple candidate biomarkers as a combination of biomarkers present at elevated levels in the defined categories of tissues.

6. The method according to any one of the preceding claims, wherein step iii. further comprises contacting extracellular vesicles from the at least one different type of tissue with the binder or each binder and detecting the binding of the extracellular vesicles from the at least one different type of tissue to the binder or each binder.

7. The method according to any one of the preceding claims, wherein extracellular vesicles from the defined category of tissue preferentially bind the binding agent or each binding agent compared to extracellular vesicles from at least two different categories of tissues of the organism.

8. The method according to any one of the preceding claims, wherein extracellular vesicles from the defined category of tissue preferentially bind the binding agent or each binding agent compared to extracellular vesicles from all other categories of tissues of the organism.

9. The method according to any one of the preceding claims, wherein step iii. further comprises the step of detecting the presence of the candidate biomarker or each candidate biomarker on extracellular vesicles obtained from a cultured cell line representing the defined category of tissue.

10. The method of claim 9, wherein the extracellular vesicles obtained from the cultured cell line representing the defined category of tissue preferentially bind to the binding agent compared to those obtained from the cultured cell line representing at least one different category of tissue from the organism.

11. The method according to claim 9 or 10, wherein the extracellular vesicles obtained from the cultured cell line are in plasma containing the extracellular vesicles.

12. The method according to any one of the preceding claims, wherein the extracellular vesicles obtained from the tissue sample are in plasma containing the extracellular vesicles after the extracellular vesicles have been obtained from the tissue sample.

13. The method according to any one of the preceding claims, wherein step i. comprises the following steps: 1) Identify potential candidate biomarkers expressed in tissues within the defined categories; 2) Identify potential candidate biomarkers present in the defined category of tissues at higher levels than in at least one different category of tissues from the organism; 3) Identify potential candidate biomarkers as transmembrane or surface proteins; 4) Identify potential candidate biomarkers present in extracellular vesicles; or 5) Any combination of steps 1) through 4) performed in any order. And thus identify candidate biomarkers.

14. The method according to any one of the preceding claims, wherein step i. comprises the following steps: 1) Identify potential candidate biomarkers expressed in tissues within the defined categories; 2) Identify potential candidate biomarkers from those identified in step 1) that are present in the defined category of tissue at a higher level than in at least one different category of tissue from the organism; 3) Identify potential candidate biomarkers as transmembrane or surface proteins from among the potential candidate biomarkers identified in step 2); 4) Validate one candidate biomarker present in extracellular vesicles from the potential candidate biomarkers identified in step 3), or validate multiple candidate biomarkers present in extracellular vesicles from the potential candidate biomarkers identified in step 3).

15. The method according to any one of the preceding claims, wherein step iii. further comprises the step of grading and separating a precursor sample of the defined category of tissue rich in extracellular vesicles in order to provide the sample of the defined category of tissue.

16. The method according to any one of the preceding claims, wherein the candidate biomarker is a transmembrane or surface protein.

17. The method according to any one of claims 1 to 15, wherein the candidate biomarker is a cytoplasmic protein.

18. The method according to any one of the preceding claims, wherein the candidate biomarker is a protein having post-translational modifications.

19. The method according to any one of claims 1 to 15, wherein the candidate biomarker is a DNA molecule, RNA molecule, lipid, or post-translational modification.

20. The method according to any one of the preceding claims, wherein the binding agent or each binding agent comprises an antibody, aptamer, lectin, lipid-binding protein or lipid-binding domain, or a DNA or RNA primer or probe.

21. The method according to any one of the preceding claims, wherein the binder or each binder described in step iii. is provided attached to a substrate, preferably wherein the substrate comprises magnetic beads or nanoparticles, gold beads or nanoparticles, polystyrene beads, affinity chromatography columns, microplates, microfluidic channels, or biochips having a surface made of gold, silicon oxide, glass, graphene, or polystyrene.

22. The method according to any one of the preceding claims, wherein the tissue defining the category is liver tissue, preferably wherein the cells secreting the extracellular vesicles therefrom are hepatocytes, Kupffer cells, stellate cells or resident dendritic cells of the liver.

23. The method of claim 22, wherein the biomarker present at elevated levels in the tissues of the defined category, or each biomarker, is selected from ASGR1, ASGR2, TFR2, SLCO1B1, SLC38A3, TMEM56, UNC93A, SLC22A9, SLC2A2, and FXYD1, or combinations thereof, preferably wherein the biomarker present at elevated levels in the tissues of the defined category, or each biomarker, is selected from ASGR1, ASGR2, TFR2, and SLCO1B1, or combinations thereof.

24. The method of claim 23, wherein the biomarkers present at elevated levels in the tissues of the defined category, or each biomarker comprises ASGR1, ASGR2, TFR2, and SLCO1B1.

25. The method of claim 23 or 24, wherein the biomarker or each biomarker is ASGR1 and the binding agent or each binding agent is an anti-ASGR1 antibody or its antigen-binding fragment; the biomarker or each biomarker is ASGR2 and the binding agent or each binding agent is an anti-ASGR2 antibody or its antigen-binding fragment; the biomarker or each biomarker is TFR2 and the binding agent or each binding agent is an anti-TFR2 antibody or its antigen-binding fragment; or the biomarker or each biomarker is SLCO1. B1, wherein the binding agent or each binding agent is an anti-SLCO1B1 antibody or its antigen-binding fragment; wherein the biomarker or each biomarker is SLC38A3 and the binding agent or each binding agent is an anti-SLC38A3 antibody or its antigen-binding fragment; wherein the biomarker or each biomarker is TMEM56 and the binding agent or each binding agent is an anti-TMEM56 antibody or its antigen-binding fragment; wherein the biomarker or each biomarker is UNC93A and the binding agent or each binding agent is an anti-UNC93A antibody or its antigen-binding tablet. The biomarker, or each biomarker being SLC22A9, and the binding agent, or each binding agent being an anti-SLC22A9 antibody or its antigen-binding fragment; the biomarker, or each biomarker being SLC2A2, and the binding agent, or each binding agent being an anti-SLC2A2 antibody or its antigen-binding fragment; or the biomarker, or each biomarker being FXYD1, and the binding agent, or each binding agent being an anti-FXYD1 antibody or its antigen-binding fragment; or combinations thereof, preferably, wherein the biomarker, or each biomarker being ASGR 1. The binding agent or each binding agent is an anti-ASGR1 antibody or its antigen-binding fragment, the biomarker or each biomarker is ASGR2 and the binding agent or each binding agent is an anti-ASGR2 antibody or its antigen-binding fragment, the biomarker or each biomarker is TFR2 and the binding agent or each binding agent is an anti-TFR2 antibody or its antigen-binding fragment, or the biomarker or each biomarker is SLCO1B1 and the binding agent or each binding agent is an anti-SLCO1B1 antibody or its antigen-binding fragment, or a combination thereof.

26. The method of claim 25, wherein each binder is provided in proportion to 10%-50% of the total, wherein the total mixture of binders is equal to 100%, or wherein each binder is provided in equal proportion.

27. The method of claim 25 or 26, wherein the binding agent comprises a plurality of binding agents consisting of the anti-ASGR1 antibody or its antigen-binding fragment, the anti-ASGR2 antibody or its antigen-binding fragment, the anti-SLCO1B1 antibody or its antigen-binding fragment, and the anti-TFR2 antibody or its antigen-binding fragment, wherein the anti-ASGR1 antibody or its antigen-binding fragment is provided in a proportion of 30%-50% of the total, and each of the other antibodies or their antigen-binding fragments is provided in a proportion of 10%-30% of the total, wherein the total mixture of the binding agents is equal to 100%.

28. The method according to any one of claims 1 to 21, wherein the tissue defining the category is blood tissue.

29. The method according to any one of the preceding claims, wherein the biomarker is specific to the tissue defining the category, or wherein the combination of the biomarkers is specific to the tissue defining the category.

30. The method according to any one of the preceding claims, wherein step iii. further comprises the step of detecting the binding of the binding agent or each binding agent to the corresponding candidate biomarker on or in extracellular vesicles.

31. The method according to any one of the preceding claims, wherein the tissue sample is healthy tissue or diseased tissue.

32. A method for enriching a biological sample containing a mixture of extracellular vesicles, the method comprising the following steps: a) Identifying one or more biomarkers present in the tissue at elevated levels by performing the method of any one of the preceding claims; and b) Capture extracellular vesicles in the biological sample, wherein the extracellular vesicles exhibit the biomarkers identified in step a), or each of the biomarkers, to generate an enrichment fraction of extracellular vesicles from the defined category of tissue.

33. The method of claim 32, wherein the extracellular vesicle mixture comprises extracellular vesicles from the defined category of tissues and extracellular vesicles from at least one different category of tissues of the organism.

34. The method of claim 32 or 33, wherein in step b), one or more binding agents capable of specifically binding to the biomarker or each biomarker are used to capture the extracellular vesicles.

35. The method according to any one of claims 32 to 34, wherein step b) further comprises isolating the extracellular vesicles of the biomarker or each biomarker captured using the biomarker or each biomarker and demonstrating the biomarker or each biomarker identified in step a).

36. The method of claim 35, wherein the isolated extracellular vesicle is an intact extracellular vesicle.

37. The method according to any one of claims 32 to 36, wherein the method further comprises the step of releasing the contents of the captured or separated extracellular vesicles.

38. A method for analyzing a biological sample comprising a mixture of extracellular vesicles, the method comprising the following steps: a) Identifying one or more biomarkers present in the tissue at elevated levels by performing the method of any one of claims 1 to 31; b) Capture extracellular vesicles in the biological sample, wherein the extracellular vesicles display the biomarkers identified in step a), or each of the biomarkers, to generate an enrichment fraction of the extracellular vesicles; and c) Analyze the contents of extracellular vesicles in the enrichment fraction obtained in step b).

39. The method of claim 37, wherein the extracellular vesicle mixture comprises extracellular vesicles from the defined category of tissues and extracellular vesicles from at least one different category of tissues of the organism.

40. The method of claim 38 or 39, wherein in step b), one or more binding agents capable of specifically binding to the biomarker or each biomarker are used to capture the extracellular vesicles.

41. The method according to any one of claims 38 to 40, wherein step b) further comprises isolating the extracellular vesicles of the biomarker or each biomarker captured using the biomarker or each biomarker and demonstrating the biomarker or each biomarker identified in step a).

42. The method of claim 41, wherein the isolated extracellular vesicles are intact extracellular vesicles.

43. The method according to any one of claims 38 to 42, wherein the method further comprises the step of releasing the contents of the captured or isolated extracellular vesicles.

44. The method according to any one of claims 32 to 43, wherein the biological sample is a biological fluid, preferably blood, urine, saliva, lymph, bile, cerebrospinal fluid, sputum, mucus, tears, bronchoalveolar lavage (BAL) fluid, earwax, sweat, feces, breast milk, tissue fluid, vaginal fluid, semen, gastric juice, vesicular fluid, or cystic fluid.

45. The method according to any one of claims 32 to 44, wherein step b) comprises capturing extracellular vesicles in the biological sample using the binder or each binder.

46. ​​A method for enriching a biological sample comprising a mixture of extracellular vesicles, the method comprising the following steps: Extracellular vesicles in the biological sample are captured, wherein the extracellular vesicles exhibit one or more biomarkers to generate an enrichment fraction of the extracellular vesicles. The biomarker, or each biomarker, is selected from ASGR1, ASGR2, TFR2, SLCO1B1, SLC38A3, TMEM56, UNC93A, SLC22A9, SLC2A2 and FXYD1 or combinations thereof, preferably selected from ASGR1, ASGR2, TFR2 and SLCO1B1 or combinations thereof.

47. A method for analyzing a biological sample comprising a mixture of extracellular vesicles, the method comprising the steps of: a) Capturing extracellular vesicles in the biological sample, wherein the extracellular vesicles display one or more biomarkers to generate an enrichment fraction of the extracellular vesicles; and b) Analyze the contents of extracellular vesicles in the enriched fraction obtained in step a). The biomarker, or each biomarker, is selected from ASGR1, ASGR2, TFR2, SLCO1B1, SLC38A3, TMEM56, UNC93A, SLC22A9, SLC2A2 and FXYD1 or combinations thereof, preferably selected from ASGR1, ASGR2, TFR2 and SLCO1B1 or combinations thereof.

48. The method of claim 46 or 47, wherein the step of capturing extracellular vesicles comprises capturing extracellular vesicles based on the extracellular vesicles displaying one or more biomarkers, and wherein the one or more biomarkers comprises ASGR1, ASGR2, TFR2, and SLCO1B1.

49. The method according to any one of claims 46 to 48, wherein one or more binding agents capable of specifically binding to the biomarker or each biomarker are used to capture the extracellular vesicles.

50. The method according to any one of claims 46 to 49, wherein the biomarker or each biomarker is ASGR1 and the binding agent or each binding agent is an anti-ASGR1 antibody or its antigen-binding fragment; the biomarker or each biomarker is ASGR2 and the binding agent or each binding agent is an anti-ASGR2 antibody or its antigen-binding fragment; the biomarker or each biomarker is TFR2 and the binding agent or each binding agent is an anti-TFR2 antibody or its antigen-binding fragment; the biomarker or each biomarker is SL. CO1B1 and the binding agent or each binding agent is an anti-SLCO1B1 antibody or its antigen-binding fragment, the biomarker or each biomarker is SLC38A3 and the binding agent or each binding agent is an anti-SLC38A3 antibody or its antigen-binding fragment, the biomarker or each biomarker is TMEM56 and the binding agent or each binding agent is an anti-TMEM56 antibody or its antigen-binding fragment, the biomarker or each biomarker is UNC93A and the binding agent or each binding agent is an anti-UNC93A antibody or its antigen-binding fragment. The biomarker or each biomarker is SLC22A9 and the conjugate or each conjugate is an anti-SLC22A9 antibody or its antigen-binding fragment; the biomarker or each biomarker is SLC2A2 and the conjugate or each conjugate is an anti-SLC2A2 antibody or its antigen-binding fragment; or the biomarker or each biomarker is FXYD1 and the conjugate or each conjugate is an anti-FXYD1 antibody or its antigen-binding fragment; or a combination thereof, preferably, wherein the biomarker or each biomarker is ASG. R1 and the binding agent or each binding agent is an anti-ASGR1 antibody or its antigen-binding fragment, the biomarker or each biomarker is ASGR2 and the binding agent or each binding agent is an anti-ASGR2 antibody or its antigen-binding fragment, the biomarker or each biomarker is TFR2 and the binding agent or each binding agent is an anti-TFR2 antibody or its antigen-binding fragment, or the biomarker or each biomarker is SLCO1B1 and the binding agent or each binding agent is an anti-SLCO1B1 antibody or its antigen-binding fragment, or a combination thereof.

51. The method of claim 50, wherein each binder is provided in proportion to 10%-50% of the total, wherein the total mixture of binders is equal to 100%, or wherein each binder is provided in equal proportion.

52. The method of claim 50 or 51, wherein the binding agent comprises a plurality of binding agents consisting of the anti-ASGR1 antibody or its antigen-binding fragment, the anti-ASGR2 antibody or its antigen-binding fragment, the anti-SLCO1B1 antibody or its antigen-binding fragment, and the anti-TFR2 antibody or its antigen-binding fragment, wherein the anti-ASGR1 antibody or its antigen-binding fragment is provided in a proportion of 30%-50% of the total, and each of the other antibodies or their antigen-binding fragments is provided in a proportion of 10%-30% of the total, wherein the total mixture of the binding agents is equal to 100%.

53. The method according to any one of claims 46 to 52, wherein the extracellular vesicle mixture comprises extracellular vesicles from tissues of a defined category and extracellular vesicles from at least one different category of tissues of the organism, and wherein the biomarker or each biomarker is present at an elevated level in the tissues of the defined category.

54. The method according to any one of claims 46 to 53, wherein the method further comprises isolating the extracellular vesicles displaying the biomarker or each biomarker captured using the biomarker or each biomarker.

55. The method of claim 54, wherein the isolated extracellular vesicle is an intact extracellular vesicle.

56. The method according to any one of claims 46 to 55, wherein the method further comprises the step of releasing the contents of the captured or isolated extracellular vesicles.

57. The method according to any one of claims 46 to 56, wherein the biological sample is a biological fluid, preferably blood, urine, saliva, lymph, bile, cerebrospinal fluid, sputum, mucus, tears, bronchoalveolar lavage (BAL) fluid, earwax, sweat, feces, breast milk, tissue fluid, vaginal fluid, semen, gastric juice, vesicular fluid, or cystic fluid.

58. The method according to any one of claims 46 to 57, wherein the step of capturing extracellular vesicles in the biological sample comprises capturing extracellular vesicles in the biological sample using the binding agent or each binding agent.

59. Use of one or more biomarkers displayed on extracellular vesicles for generating enrichment fractions of extracellular vesicles, wherein the enrichment fractions of extracellular vesicles are secreted from tissues that define a class in an organism, wherein the biomarkers, or each biomarker, are selected from ASGR1, ASGR2, TFR2, SLCO1B1, SLC38A3, TMEM56, UNC93A, SLC22A9, SLC2A2 and FXYD1 or combinations thereof, preferably selected from ASGR1, ASGR2, TFR2 and SLCO1B1 or combinations thereof.

60. The use according to claim 59, wherein the biomarker comprises ASGR1, ASGR2, TFR2 and SLCO1B1.

61. The use according to claim 59 or 60, wherein an enrichment fraction of the extracellular vesicles is enriched from a biological sample comprising a mixture of extracellular vesicles.

62. The use according to claim 61, wherein the extracellular vesicle mixture comprises extracellular vesicles from the defined category of tissues and extracellular vesicles from at least one different category of tissues of the organism.

63. The use according to claim 61 or 62, wherein the biological sample is a biological fluid, preferably blood, urine, saliva, lymph, bile, cerebrospinal fluid, sputum, mucus, tears, bronchoalveolar lavage (BAL) fluid, earwax, sweat, feces, breast milk, tissue fluid, vaginal fluid, semen, gastric juice, vesicular fluid, or cystic fluid.

64. Use of one or more binding agents for generating enriched fractions of extracellular vesicles, wherein the enriched fractions of extracellular vesicles are secreted from tissues of a defined class in an organism, wherein the binding agent or each binding agent is capable of specifically binding to a biomarker displayed on the extracellular vesicles, wherein the binding agent or each binding agent is selected from anti-ASGR1 antibody or its antigen-binding fragment, anti-ASGR2 antibody or its antigen-binding fragment, anti-TFR2 antibody or its antigen-binding fragment, anti-SLCO1B1 antibody or its antigen-binding fragment, anti-SLC38A3 antibody or its antigen-binding fragment, anti-TMEM56 antibody or its antigen-binding fragment, anti-UNC93A antibody or its antigen-binding fragment, anti-SLC22A9 antibody or its antigen-binding fragment, anti-SLC2A2 antibody or its antigen-binding fragment, and anti-FXYD1 antibody or its antigen-binding fragment, or combinations thereof, preferably selected from anti-ASGR1 antibody or its antigen-binding fragment, anti-ASGR2 antibody or its antigen-binding fragment, anti-TFR2 antibody or its antigen-binding fragment, or anti-SLCO1B1 antibody or its antigen-binding fragment, or combinations thereof.

65. The use according to claim 64, wherein the enrichment fraction of the extracellular vesicles is enriched from a biological sample containing a mixture of extracellular vesicles.

66. A method for detecting one or more biomarkers on or in extracellular vesicles secreted from a defined category of tissue, wherein the biomarkers or each biomarker is selected from ASGR1, ASGR2, TFR2, SLCO1B1, SLC38A3, TMEM56, UNC93A, SLC22A9, SLC2A2 and FXYD1 or combinations thereof, preferably selected from ASGR1, ASGR2, TFR2 and SLCO1B1 or combinations thereof.

67. The method of claim 66, wherein the method comprises a plurality of biomarkers, the plurality of biomarkers including ASGR1, ASGR2, TFR2 and SLCO1B1.

68. The method of claim 66 or 67, wherein the biomarker or each biomarker is detected by binding with one or more conjugates, wherein the biomarker or each biomarker is ASGR1 and the conjugate or each conjugate is an anti-ASGR1 antibody or an antigen-binding fragment thereof, wherein the biomarker or each biomarker is ASGR2 and the conjugate or each conjugate is an anti-ASGR2 antibody or an antigen-binding fragment thereof, wherein the biomarker or each biomarker is TFR2 and the conjugate or each conjugate is an anti-TFR2 antibody or an antigen-binding fragment thereof. The biomarker, or each biomarker being SLCO1B1, and the binding agent, or each binding agent being an anti-SLCO1B1 antibody or its antigen-binding fragment; the biomarker, or each biomarker being SLC38A3, and the binding agent, or each binding agent being an anti-SLC38A3 antibody or its antigen-binding fragment; the biomarker, or each biomarker being TMEM56, and the binding agent, or each binding agent being an anti-TMEM56 antibody or its antigen-binding fragment; the biomarker, or each biomarker being UNC93A, and the binding agent, or each binding agent... The biomarker is an anti-UNC93A antibody or its antigen-binding fragment, wherein the biomarker or each biomarker is SLC22A9 and the binding agent or each binding agent is an anti-SLC22A9 antibody or its antigen-binding fragment, wherein the biomarker or each biomarker is SLC2A2 and the binding agent or each binding agent is an anti-SLC2A2 antibody or its antigen-binding fragment, or the biomarker or each biomarker is FXYD1 and the binding agent or each binding agent is an anti-FXYD1 antibody or its antigen-binding fragment, or a combination thereof, preferably wherein the biomarker or each biomarker is an anti-UNC93A antibody or its antigen-binding fragment, ... The biomarker is ASGR1 and the binding agent or each binding agent is an anti-ASGR1 antibody or its antigen-binding fragment; the biomarker or each biomarker is ASGR2 and the binding agent or each binding agent is an anti-ASGR2 antibody or its antigen-binding fragment; the biomarker or each biomarker is TFR2 and the binding agent or each binding agent is an anti-TFR2 antibody or its antigen-binding fragment; or the biomarker or each biomarker is SLCO1B1 and the binding agent or each binding agent is an anti-SLCO1B1 antibody or its antigen-binding fragment; or a combination thereof.

69. The method according to claims 66 to 68, wherein the biomarker or each biomarker is detected on or in extracellular vesicles secreted by tissues that define a category from a biological sample comprising a mixture of extracellular vesicles.

70. A kit for enriching a biological sample comprising a mixture of extracellular vesicles to generate an enrichment fraction of extracellular vesicles, wherein the enrichment fraction of extracellular vesicles is secreted from tissues of a defined class in an organism, wherein the kit comprises one or more binding agents capable of specifically binding to biomarkers present at elevated levels in the tissues of the defined class, and wherein the binding agent or each binding agent is selected from anti-ASGR1 antibody or its antigen-binding fragment, anti-ASGR2 antibody or its antigen-binding fragment, anti-SLCO1B1 antibody or its antigen-binding fragment, anti-SLC38A3 antibody or its antigen-binding fragment, anti-TMEM56 antibody or its antigen-binding fragment, anti-UNC93A antibody or its antigen-binding fragment, anti-SLC22A9 antibody or its antigen-binding fragment, anti-SLC2A2 antibody or its antigen-binding fragment, and anti-FXYD1 antibody or its antigen-binding fragment, or combinations thereof, and optionally, wherein the kit comprises anti-TFR2 The antibody or antigen-binding fragment thereof preferably comprises an anti-ASGR1 antibody or antigen-binding fragment thereof, an anti-ASGR2 antibody or antigen-binding fragment thereof, or an anti-SLCO1B1 antibody or antigen-binding fragment thereof, or a combination thereof, and optionally, the kit comprises an anti-TFR2 antibody or antigen-binding fragment thereof.

71. The use according to claim 65, the method according to claim 69, or the kit according to claim 70, wherein the extracellular vesicle mixture comprises extracellular vesicles from the defined category of tissues and extracellular vesicles from at least one different category of tissues of the organism.

72. The use, method, or kit according to any one of claims 65 and 69 to 71, wherein the biological sample is a biological fluid, preferably blood, urine, saliva, lymph, bile, cerebrospinal fluid, sputum, mucus, tears, bronchoalveolar lavage (BAL) fluid, earwax, sweat, feces, breast milk, tissue fluid, vaginal fluid, semen, gastric juice, vesicular fluid, or cystic fluid.

73. The use, method, or kit according to any one of claims 64, 65, and 68 to 72, wherein the binder or each binder comprises the anti-ASGR1 antibody or its antigen-binding fragment, the anti-ASGR2 antibody or its antigen-binding fragment, the anti-SLCO1B1 antibody or its antigen-binding fragment, and the anti-TFR2 antibody or its antigen-binding fragment.

74. The use, method, or kit according to any one of claims 64, 65, and 68 to 73, wherein the method, use, or kit comprises a plurality of binding agents consisting of the anti-ASGR1 antibody, the anti-ASGR2 antibody, the anti-SLCO1B1 antibody, and the anti-TFR2 antibody.

75. The use, method, or kit according to any one of claims 64, 65, and 68 to 74, wherein the binder or each binder is provided attached to a substrate, preferably wherein the substrate comprises magnetic beads or nanoparticles, gold beads or nanoparticles, polystyrene beads, affinity chromatography columns, microplates, microfluidic channels, or biochips having a surface made of gold, silicon oxide, glass, graphene, or polystyrene.

76. The use, method, or kit according to any one of claims 64, 65, and 68 to 75, wherein each binder is provided in a proportion of 10% to 50% of the total, wherein the total mixture of binders is equal to 100%, or wherein each binder is provided in equal proportion.

77. The use, method, or kit according to any one of claims 64, 65, and 68 to 74, wherein the use, method, or kit comprises a plurality of binders consisting of the anti-ASGR1 antibody or its antigen-binding fragment, the anti-ASGR2 antibody or its antigen-binding fragment, the anti-SLCO1B1 antibody or its antigen-binding fragment, and the anti-TFR2 antibody or its antigen-binding fragment, wherein the anti-ASGR1 antibody or its antigen-binding fragment is provided in a proportion of 30% to 50% of the total, and each of the other antibodies or their antigen-binding fragments is provided in a proportion of 10% to 30% of the total, wherein the total mixture of binders is equal to 100%.

78. An enrichment fraction of extracellular vesicles, said fraction enriched with extracellular vesicles secreted by tissues of defined categories in an organism, said extracellular vesicles displaying one or more biomarkers selected from ASGR1, ASGR2, TFR2, SLCO1B1, SLC38A3, TMEM56, UNC93A, SLC22A9, SLC2A2 and FXYD1 or combinations thereof, preferably selected from ASGR1, ASGR2, TFR2 and SLCO1B1 or combinations thereof.

79. The enrichment fraction of extracellular vesicles according to claim 78, wherein the extracellular vesicles secreted by tissues that define categories from an organism exhibit multiple biomarkers including ASGR1, ASGR2, TFR2, and SLCO1B1.

80. The use, method, kit, or enrichment fraction of extracellular vesicles according to any one of claims 53 to 79, wherein the tissue defining the category is liver tissue.

81. A method of manufacturing a kit according to any one of claims 70 to 77 and 80, wherein the method comprises co-locating the binder or each binder.

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