Liquid biopsy based on local measurement of exosome heterogeneity change

Abstract

The present invention relates to a liquid biopsy based on the local measurement of an exosome heterogeneity change. A liquid biopsy sample is prepared by separating exosomes related to a specific disease, such as cancer, from exosome population samples into a subpopulation for a separation marker by using a predetermined surface marker as the separation marker. In the liquid biopsy sample, two surface markers other than the separation marker are used as capture and detection markers to analyze exosomes loaded with the same triple markers.

Description

Liquid biopsy based on measurement of local exosome heterogeneity changes

[0001] The present invention relates to a liquid biopsy based on the measurement of local exosome heterogeneity changes, and relates to a liquid biopsy technique for preparing a liquid biopsy sample by separating exosomes related to a specific disease, such as cancer, from an exosome population sample into subpopulations with respect to the separation marker using a predetermined surface marker as a separation marker, and analyzing exosomes loaded with the same triple marker using two other surface markers in addition to the separation marker as capture and detection markers.

[0002] Recently, the concept of "liquid biopsy" has been introduced to minimize the pain of screening for certain diseases, such as cancer and degenerative diseases, and to facilitate early diagnosis. Liquid biopsy is a relatively simple, non-invasive method that extracts bodily fluids like blood, saliva, and urine and analyzes them for disease-related cells or components released from them (peptides, proteins, nucleic acids, organelles, specific substances, etc.). Because it allows for early detection of specific diseases, it is gaining attention as an alternative to existing diagnostic methods such as imaging, tissue biopsies, and blood tests.

[0003] Liquid biopsy, when used to diagnose certain diseases, such as cancer, generally offers the following advantages over tissue biopsy. First, compared to invasive surgical tissue collection, which is potentially risky and expensive, liquid biopsy utilizes bodily fluids relatively easily and reduces the risk of complications. Second, while tissue biopsies only provide a snapshot of a single area of ​​disease when needed and do not allow for continuous monitoring of disease progression and treatment, liquid biopsy allows for real-time, continuous monitoring of cancer progression. Third, while tissue biopsies often provide a complete understanding of tumor composition due to their diverse nature, liquid biopsies collect biomaterials produced by tumor cells, enabling a more comprehensive analysis of tumor composition at the molecular level. Fourth, while tissue biopsies have limited sample size, making them insufficient for multiple diagnostic purposes, liquid biopsies allow for multiple collections from various bodily fluids as needed. Fifth, compared to tissue biopsy, which has limited clinical utility of test results due to low accuracy, liquid biopsy offers relatively higher accuracy (accuracy of positive sample measurements) and sensitivity (accuracy of negative sample measurements), and thus can provide reliable results for clinical decision-making.

[0004] Circulating tumor cells (CTCs), cell-free DNA (CDNA), and exosomes are recognized as potential biomarkers for liquid biopsy in the field of cancer diagnosis. While CTCs offer excellent concentration and selectivity, and the stability of isolated nucleic acids, their extremely low distribution in blood makes them difficult to use for early cancer diagnosis. CTCs, while abundant in blood, are difficult to concentrate and select, and the stability of isolated nucleic acids is low. Furthermore, the presence of large amounts of non-specific DNA unrelated to the target disease presents a major challenge in analyzing noise. Compared to these two biomarkers, exosomes offer high concentration in blood, are easy to concentrate and select, and can be stably isolated. However, exosomes are extremely heterogeneous in size, density, and composition of proteins and nucleic acids, requiring prior separation of disease-specific exosomes.

[0005] Exosomes are attracting attention as liquid biopsy biomarkers. Exosomes (30-150 nm in size) are microvesicles secreted from human cells and reflect the characteristics of the cells they originate from. They can be extracted from various bodily fluids, including blood, saliva, and urine. Because exosomes are vesicles encased in a lipid bilayer, they are not only stable in the body but also free from attacks by proteases, RNases, and DNases. Therefore, research is increasing to utilize exosomes as novel biomarkers for the diagnosis of specific diseases, such as cancer and degenerative diseases. Indeed, products are being developed that utilize exosome components, such as proteins and RNA, as markers for the early diagnosis and companion diagnosis of cancer.

[0006] Specifically, exosomes are known to play a role in tumorigenesis, metastasis, and drug resistance, particularly in cancer. Like other cells in the tumor microenvironment, tumor cells secrete exosomes and other microvesicles, which contribute to tumor progression and metastasis by promoting angiogenesis. Tumor cell-derived vesicles also contain molecules involved in immunosuppression, which can inactivate T lymphocytes or natural killer cells, or promote the differentiation of regulatory T lymphocytes or myeloid cells to suppress immune responses. Furthermore, the transfer of tumor activity via exosomes secreted by tumor cells has also been reported.

[0007] However, because exosomes derived from all cells exist in a mixed form in bodily fluids, simply isolating a bulk population of exosomes from bodily fluids has limitations in accurately diagnosing specific diseases such as cancer. Moreover, in the early stages of disease onset, the concentration of exosomes derived from specific disease-related cells in bodily fluids is relatively low, so isolation and concentration of target exosomes from bodily fluids are necessary for early diagnosis. Among existing exosome isolation technologies, density difference-based ultracentrifugation is considered the gold standard due to its high reproducibility and relatively stable process. However, it has the disadvantage of requiring expensive equipment and taking a long time for separation. To overcome these shortcomings, methods such as exosome precipitation and separation, size exclusion chromatography, and microfluidics have been developed to separate exosomes by size.

[0008] However, since these existing exosome isolation technologies provide all exosomes present in bodily fluids as a single population, they have limitations in selectively detecting extremely small amounts of exosomes derived from specific cells, such as cancer cells. In fact, exosomes released from cells contain a variety of biochemical components (membrane proteins, endogenous proteins, and RNA, etc.) in the exosome membrane or interior, making them highly heterogeneous in terms of their composition. In particular, it has been reported that certain exosomes derived from cancer cells contain mRNA or miRNA that are involved in creating a local environment in the early stages of angiogenesis and cancer metastasis. Furthermore, as mentioned above, these specific exosomes are known to contain biochemical components that induce immune cell death or are involved in the process of converting normal cells to cancer cells during cancer metastasis.

[0009] When diagnosing specific diseases such as cancer, it is very difficult to achieve high diagnostic accuracy when using a bulk population containing all exosomes present in bodily fluids as a liquid biopsy sample. This is because sample matrix effects, such as non-specific adhesion caused by non-specific exosomes, negatively affect analytical specificity and sensitivity. To overcome this analytical challenge, multiple biomarkers known to be elevated in the bodily fluids of patients with specific diseases (e.g., multiple different protein markers present on the exosome surface) can be simultaneously measured to determine the presence of the disease, although with low specificity. However, the synergistic effect of using multiple biomarkers is generally limited, as most exosomal markers that are not specifically associated with diseases have fundamentally low disease specificity.

[0010] Accordingly, there is an urgent need for a solution to the problem of being unable to selectively isolate and analyze exosomes associated with the occurrence of specific diseases such as cancer and utilize them for the diagnosis of specific diseases.

[0011] The present invention is to solve the problems of the above-described prior art, and one aspect of the present invention is to provide a liquid biopsy method for isolating and recovering a subpopulation, a hypopopulation, or a lower subpopulation of exosomes for a separation marker from an exosome population with high yield and in its original state to prepare a liquid biopsy sample, and analyzing exosomes loaded with the same triple marker using two surface markers other than the separation marker as capture and detection markers in the liquid biopsy sample, in order to discover a biomarker related to a specific disease such as cancer, a degenerative disease, a heart disease, an infectious disease, etc. and to utilize the same for diagnosis.

[0012] A liquid biopsy according to an embodiment of the present invention comprises the steps of: (a) preparing an exosome liquid biopsy sample comprising the exosome subpopulation by separating and recovering first target exosomes having a first surface marker in common from an exosome population sample containing heterogeneous exosomes secreted from a plurality of cells in a human body into subpopulations; (b) obtaining a triple marker signal by measuring a quantitative signal for a third surface marker of second target exosomes belonging to the exosome subpopulation and having a second surface marker in common; (c) calculating an exosome heterogeneity index as a ratio of the measured values ​​of the double marker signal and the triple marker signal obtained by measuring the quantitative signal for the second surface marker of the first target exosomes; and (d) analyzing the exosome liquid biopsy sample based on the calculated exosome heterogeneity index.

[0013] In addition, in the liquid biopsy according to an embodiment of the present invention, steps (a) to (c) are sequentially performed for the exosome population samples of each of a normal group and a patient group with a specific disease, so as to calculate the exosome heterogeneity index for the normal group and the exosome heterogeneity index for the patient group, respectively, and in step (d), the calculated exosome heterogeneity index for the normal group and the exosome heterogeneity index for the patient group are compared to select a biomarker related to the specific disease of the patient group.

[0014] In addition, in the liquid biopsy according to an embodiment of the present invention, the exosome population sample of the disease screening subject is repeatedly performed from step (a) to step (c) performed for selecting the biomarker to calculate the exosome heterogeneity index for the disease screening subject, and in step (d), the exosome heterogeneity index for the disease screening subject is compared with the exosome heterogeneity index for the patient group to generate disease screening information for the disease screening subject.

[0015] In addition, in the liquid biopsy according to an embodiment of the present invention, among the three surface markers of the heterogeneous exosomes in the exosome population sample, at least one of a total of six selection paths is selected, in which one is selected as the first surface marker, another is selected as the second surface marker, and the remaining one is selected as the third surface marker, and steps (a) to (c) are sequentially repeated to produce N (N is a natural number greater than or equal to 1 and less than or equal to 6) exosome heterogeneity indices.

[0016] In addition, in the liquid biopsy according to an embodiment of the present invention, N exosome heterogeneity indices for the normal group and N exosome heterogeneity indices for the patient group are calculated for each of the exosome population samples of the normal group and the patient group with a specific disease, and in the step (d), based on the N exosome heterogeneity indices for the normal group and the N exosome heterogeneity indices for the patient group, a selection path of a biomarker related to a specific disease of the patient group can be selected.

[0017] In addition, in the liquid biopsy according to an embodiment of the present invention, in the step (d), new indices are generated through arithmetic operations or functionalization of the N exosome heterogeneity indices for the normal group and the N exosome heterogeneity indices for the patient group, and based on the generated new indices, a selection path of a biomarker related to a specific disease of the patient group can be selected.

[0018] In addition, in the liquid biopsy according to an embodiment of the present invention, the exosome population sample of the disease screening subject is repeatedly performed from step (a) to step (c) according to the selection path performed for the selection path selection of the biomarker, thereby calculating the exosome heterogeneity index for the disease screening subject, and in step (d), disease screening information for the disease screening subject can be generated by comparing the calculated exosome heterogeneity index for the disease screening subject with the exosome heterogeneity index for the patient group calculated according to the selection path performed for the selection path selection of the biomarker.

[0019] In addition, in the liquid biopsy according to an embodiment of the present invention, the exosome population sample of the disease screening subject is repeatedly performed from step (a) to step (c) according to the selection path performed for the selection path selection of the biomarker, thereby calculating the exosome heterogeneity index for the disease screening subject, and in step (d), the new index for the disease screening subject is compared with the new index for the patient group calculated according to the selection path performed for the selection path selection of the biomarker, thereby generating disease screening information for the disease screening subject.

[0020] In addition, in the liquid biopsy according to an embodiment of the present invention, the step (a) may include: a step of binding a first specific binding material that specifically binds to the first surface marker to a solid-state fixation member using a first reversible linker; a step of reacting the exosome population sample with the first specific binding material bound to the fixation member to capture a plurality of the first target exosomes, thereby isolating an exosome subpopulation, which is a group of the first target exosomes; and a step of dissociating the first reversible linker so that the captured first target exosomes are separated from the fixation member, thereby recovering the exosome subpopulation.

[0021] In addition, in the liquid biopsy according to an embodiment of the present invention, between the step of separating the exosome subpopulation and the step of recovering the exosome subpopulation, the method may further include a step of concentrating the fixing member in which the first target exosome is captured using at least one of magnetic force, gravity, and centrifugal force.

[0022] In addition, in the liquid biopsy according to an embodiment of the present invention, the step (a) may include a step of binding a second specific binding material that specifically binds to the second surface marker of the second target exosomes in the exosome subpopulation to the fixing member; and a step of reacting an exosome subpopulation sample containing the recovered exosome subpopulation with the second specific binding material bound to the fixing member to capture a plurality of the second target exosomes, thereby isolating an exosome subpopulation, which is a group of the second target exosomes.

[0023] In addition, in the liquid biopsy according to an embodiment of the present invention, in the step of binding the second specific binding material to the fixing member, the step of dissociating the second reversible linker so that the second specific binding material is bound to the fixing member by the second reversible linker and the captured second target exosome is separated from the fixing member, thereby recovering the exosome subpopulation may be further included.

[0024] In addition, in the liquid biopsy according to an embodiment of the present invention, between the step of separating the exosome subpopulation and the step of recovering the exosome subpopulation, the method may further include a step of concentrating the fixing member in which the second target exosome is captured using at least one of magnetic force, gravity, and centrifugal force.

[0025] Additionally, in the liquid biopsy according to an embodiment of the present invention, the dual marker signal can be measured for the exosome population sample.

[0026] The features and advantages of the present invention will become more apparent from the following detailed description based on the attached drawings.

[0027] Prior to this, the terms and words used in this specification and claims should not be interpreted in their usual or dictionary meanings, but should be interpreted in the sense and concept that conforms to the technical idea of ​​the present invention based on the principle that the inventor can appropriately define the concept of the term to explain his or her own invention in the best way.

[0028] According to the present invention, through exosome subpopulation separation for an exosome-specific first marker (separation marker) in a comprehensive supergroup including disease-related exosomes but having a relatively low target disease association, specific exosomes highly associated with the target disease can be concentrated and recovered to a level where they can be isolated later, and through capture of the exosome subpopulation for an exosome-specific second marker (capture marker) in the separation solution and detection for a captured exosome-specific third marker (detection marker), exosomes containing the same triple biomarker involved in the occurrence and spread of the target disease can be immunoanalyzed as a liquid biopsy target.

[0029] In addition, by isolating and recovering a subset of exosomes, a subset of exosomes, or a lower subset containing exosomes related to a specific disease from an exosome population sample containing exosomes secreted from a large number of cells in the body, and then preparing a liquid biopsy sample, and then performing a biomarker profiling analysis on the liquid biopsy sample, the composition ratio and mutual correlation of biomarkers such as proteins specific to a specific disease, especially the common abundance ratio of complex markers such as double and triple, can be discovered.

[0030] Since the liquid biopsy sample is separated and concentrated in the form of exosome subpopulations, hypopopulations, or lower subpopulations, the non-specific exosomes are removed, minimizing noise during analysis and maximizing specific exosome signals. As a result, when exosomes are used as biomarkers for specific diseases such as cancer, interference from non-specific exosomes due to exosome heterogeneity, which is a current problem, is minimized, allowing accurate identification of information on the physiological characteristics and lineage of parent cells from the liquid biopsy sample.

[0031] Furthermore, applying the present invention to disease diagnosis can significantly increase the diagnostic accuracy of specific diseases such as cancer, degenerative diseases, heart diseases, and infectious diseases, resulting in early clinical diagnosis, enabling diagnosis of diseases at an early stage. Furthermore, since it enables the acquisition of information on the progression or decline of disease stages depending on the treatment used, it can also be applied to companion diagnostics that provide personalized screening for individuals.

[0032] FIG. 1 is a flow chart of a liquid biopsy according to an embodiment of the present invention.

[0033] Figure 2 is a drawing illustrating a liquid biopsy process according to an embodiment of the present invention.

[0034] FIG. 3 is a drawing illustrating a triple marker signal measurement process of liquid biopsy according to an embodiment of the present invention.

[0035] FIG. 4 is a diagram illustrating a marker separation path for calculating an exosome heterogeneity index of liquid biopsy according to an embodiment of the present invention.

[0036] FIG. 5 is a schematic diagram of a device for producing exosome subpopulation and subpopulation samples according to an embodiment of the present invention.

[0037] FIGS. 6 to 9 are schematic diagrams illustrating the first reversible linker and the second reversible linker illustrated in FIG. 5 according to various embodiments.

[0038] Figures 10 to 16 are flowcharts of a method for preparing an exosome liquid biopsy sample according to an embodiment of the present invention.

[0039] Figure 17 is a diagram illustrating a novel sequential marker selection process (A) using Neutral Release technology in contrast to conventional methods, and a conceptual representation (B) of the resulting new approach.

[0040] Figure 18 is a diagram illustrating the process of determining heterogeneity change indices (R1, R2, and R3) and the marker selection path.

[0041] Figure 19 is a diagram explaining the heterogeneity change pattern.

[0042] Figure 20 is a diagram illustrating a sequential marker selection strategy for quantifying triple marker positive subclass 'T' using three pan-exosome tetraspanin markers, namely CD9, CD63, and CD81.

[0043] Figure 21 is a diagram illustrating the process of quantifying the size of the triple marker positive subpopulation (T).

[0044] Figure 22 is a diagram illustrating a conventional sandwich immunoassay method for quantifying the size of a double marker positive subpopulation (D+T).

[0045] Figure 23 is a table showing a comprehensive experimental plan for quantifying exosome heterogeneity changes.

[0046] Figure 24 is a table comparing manual exosome magnetic separation and automatic exosome magnetic separation conditions.

[0047] Figure 25 is a diagram illustrating a liquid management system for automatic exosome magnetic separation using Neutral Release technology.

[0048] Figure 26 is a table showing the setting conditions for operation of the automatic exosome separation system.

[0049] Figure 27 shows the performance comparison results of automatic exosome separation using the Neutral Release protocol and manual separation.

[0050] Figure 28 shows the exosomes (1.28×10) of the cell line RWPE-1 in serum. 11 The results quantify the changes in exosome heterogeneity for samples with various concentrations of added particles / mL.

[0051] Figure 29 is RWPE-1 (1.28×10 11 particles / mL), LNCaP (1.18×10 11 particles / mL) and PC3 (2.53×10 11 This is a graph showing the change index of heterogeneity by concentration for each exosome sample produced from (100 particles / mL).

[0052] Figure 30 is a graph showing the exosome heterogeneity change index for various clinical samples.

[0053] Figure 31 is a graph showing the results of quantifying the heterogeneity changes of dual marker-positive exosomes and triple marker-positive exosomes according to the stopover site (the region of the exosome subpopulation that has a specific dual marker in common), and the exosome heterogeneity change index.

[0054] Figure 32 is a diagram illustrating a local heterogeneity change-based diagnostic algorithm that shows key factors that distinguish cancer samples from normal samples.

[0055] Figure 33 is a diagram comparing sample classification performance based on local heterogeneity change indices using different third markers (CD81, CD151, TSPAN8).

[0056] Figure 34 shows the results of evaluating CD151 as a third marker at various stopover sites.

[0057] Figure 35 shows the results of measuring heterogeneity changes for CD151 at stopover site 1 (an exosome subpopulation region that shares dual markers of CD9 and CD63) using LNCaP exosome samples.

[0058] Figure 36 shows the results characterizing the performance of CD151 at stopover site 1 for prostate cancer tracking.

[0059] Figure 37 is a schematic illustrating a comprehensive demonstration of the potential use of specific stopover sites for cancer biomarker discovery.

[0060] Figure 38 shows the local heterogeneity change index for prostate cancer samples and non-prostate cancer samples and the results of ROC analysis for prostate cancer samples.

[0061] Figure 39 is a table comparing the performance of three stopover sites in determining heterogeneity changes of the third marker.

[0062] Figure 40 is a graph showing the specificity of CD151 heterogeneity changes at stopover site 1 according to various tissues and cancer states.

[0063] The objects, specific advantages, and novel features of the present invention will become more apparent from the following detailed description and preferred embodiments taken in conjunction with the accompanying drawings. In this specification, when reference numerals are given to components in each drawing, it should be noted that, as far as possible, identical components are given the same numerals even if they are shown in different drawings. Furthermore, terms such as "first," "second," etc. are used to distinguish one component from another, and the components are not limited by these terms. In the following description of the present invention, detailed descriptions of related known technologies that may unnecessarily obscure the gist of the present invention will be omitted.

[0064] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the attached drawings.

[0065]

[0066] FIG. 1 is a flowchart of a liquid biopsy according to an embodiment of the present invention, and FIG. 2 is a diagram explaining a liquid biopsy process according to an embodiment of the present invention. In addition, FIG. 3 is a diagram explaining a triple marker signal measurement process of liquid biopsy according to an embodiment of the present invention.

[0067] As illustrated in FIGS. 1 to 3, the liquid biopsy according to an embodiment of the present invention comprises the steps of: preparing an exosome liquid biopsy sample including an exosome subpopulation by separating and recovering first target exosomes having a first surface marker (A) in common from an exosome population sample containing heterogeneous exosomes secreted from multiple cells in a human body into subpopulations; measuring a quantitative signal for a third surface marker of second target exosomes belonging to the exosome subpopulation and having a second surface marker in common, thereby obtaining a triple marker signal; calculating a local exosome heterogeneity index (R) as a ratio of the measured values ​​of the double marker signal and the triple marker signal obtained by measuring the quantitative signal for the second surface marker (B) of the first target exosomes; and analyzing the exosome liquid biopsy sample based on the calculated exosome heterogeneity index (R). Includes step (S400).

[0068]

[0069] The present invention relates to a liquid biopsy technique for preparing a liquid biopsy sample by separating exosomes related to a specific disease, such as cancer, from an exosome population sample into subpopulations for the separation marker using a predetermined surface marker as a separation marker, and analyzing exosomes loaded with the same triple marker using two surface markers other than the separation marker as capture and detection markers. The liquid biopsy according to an embodiment of the present invention includes an exosome liquid biopsy sample preparation step (S100), a triple marker signal measurement step (S200), an exosome heterogeneity index calculation step (S300), and a sample analysis step (S400).

[0070]

[0071] The exosome liquid biopsy sample preparation step (S100) prepares an exosome liquid biopsy sample by separating and recovering first target exosomes having a first surface marker (A) in common from an exosome population sample, which is an analysis sample to be analyzed, into subpopulations. Here, the exosome subpopulation includes a subpopulation, a sub-subpopulation, and sub-populations below it. The exosome subpopulation is a population of exosomes that belong to the exosome population and have at least one specific marker in common, and the exosome sub-subpopulation is a population of exosomes that have at least one other marker in common as part of the exosome subpopulation. As one of the subpopulations below it, the exosome sub-subpopulation is a population of exosomes that have at least another one or more other markers in common as part of the exosome subpopulation, and in this manner, other subpopulations below it can be defined. Meanwhile, the exosome population sample comprises exosomes secreted from multiple cells or tissues within the human body, each containing a heterogeneous exosome. To separate exosomes into subpopulations from this exosome modulator sample, the present invention utilizes an immunoaffinity separation technique, which will be described later.

[0072] Various exosomes included in an exosome liquid biopsy sample have a first surface marker (A) in common, some of which may have a second surface marker (B) in addition to the first surface marker (A), and some of which may have a third surface marker (C) in addition to the first surface marker (A) and the second surface marker (B). Hereinafter, an exosome having a first surface marker (A) is referred to as a single-marker exosome, an exosome having the first and second surface markers (A, B) is referred to as a dual-marker exosome, and an exosome having all of the first, second, and third surface markers (A, B, C) is referred to as a triple-marker exosome. The first, second, and third surface markers (A, B, C) are predetermined disease-related biomarkers arranged on the surface of the exosome.

[0073] Changes in the biomarker composition distribution of exosomes within liquid biopsy samples can provide personalized diagnostic and therapeutic information for individual patients, and are also important indicators of accurate prognosis or differential diagnosis. Changes in exosome-derived biomarkers may not only indicate a response to a single condition, but also reflect a complex mix of factors, as plasma exosomes are secreted by various cellular sources, including both blood cells and surrounding tissues.

[0074] During liquid biopsy, various proteins are heterogeneously distributed on the surface of exosomes in body fluids. In particular, specific surface proteins associated with cancer are present in a complex manner in exosomes from normal individuals, although the expression ratios vary. The types and expression levels of surface proteins, such as tetraspanins (CD9, CD63, CD81, CD82, CD151, etc.), cell type-specific molecules such as histocompatibility complex (MHC) class-Ⅰ and class-Ⅱ, adhesion molecules, HSP60, and HSP70, can increase or decrease in relation to the characteristics of the parent cell. For example, CD63, a tetraspanin-family exosome surface protein, is a protein contained in the lysosomal membrane of B cell-derived exosomes and is increased in samples from patients with several cancers, including melanoma.

[0075] Against this backdrop, immunoaffinity separation into exosome subpopulations based on exosome surface proteins can offer the following advantages. First, unlike separation methods based on density, size, and sedimentation, it utilizes antigen-antibody binding, minimizing the inclusion of impurities other than the target exosomes in the post-separation solution. As mentioned above, the lower the content of impurities in the diagnostic analysis sample, the lower the sample matrix effect, such as non-specific reactions, and thus the diagnostic sensitivity and specificity can be improved. Second, performing immunoaffinity separation on a comprehensive superset of exosome markers that includes exosomes associated with a specific disease but have a relatively low disease association can increase the concentration of disease-related exosomes within the separated subpopulation. This approach is useful when the concentration of disease-related exosomes within the exosome population is initially low enough to be difficult to detect in body fluids. In this case, using the separated subpopulation as a diagnostic sample can improve the sensitivity of diagnosing a specific disease.

[0076] Indeed, an increasing number of exosome-derived proteins are being identified as potential biomarkers for a variety of diseases, including cancer, hepatitis, liver disease, central nervous system disease, and kidney disease. To date, hundreds of exosome proteins have been reported to be associated with specific diseases, and thus could be utilized as diagnostic or isolation targets in future product development.

[0077] For example, markers associated with cancer include CD63, caveolin-1, TYRP2, VLA-4, HSP70, and HSP90 for melanoma; survivin for prostate cancer; and L1CAM, CD24, ADAM10, EMMPRIN, and claudin for ovarian cancer. Markers associated with bladder cancer include EGF, resistin, and retinoic acid-induced protein 3; and markers associated with prostate cancer include PSA and PCA3.

[0078] In relation to hepatitis and liver disease, CD81 is an exosomal protein marker associated with chronic hepatitis C in plasma. CD81, a tetraspanin-family exosomal marker, plays a crucial role in hepatitis C virus attachment and / or entry into cells. In addition, the concentration of serum exosomal CD81 is elevated in patients with chronic hepatitis C and is also associated with worsening inflammation and fibrosis. This suggests that exosomal CD81 may be a potential marker for the diagnosis and treatment response of hepatitis C. In addition, Fetuin-A and ATF-3 are exosomal protein markers associated with acute kidney injury in urine, and CD26, CD81, S1c3A1, and CD10 are markers associated with liver damage.

[0079] In relation to central nervous system diseases, many exosome-borne protein biomarkers have been reported to be potentially useful in the diagnosis of these diseases. EGFRvIII (glioblastoma-specific epidermal growth factor receptor vIII) has been detected in serum exosomes from glioblastoma patients, suggesting it may be useful for the diagnostic detection of glioblastoma. Furthermore, exosome-borne amyloid peptides have been reported to accumulate in brain plaques in patients with Alzheimer's disease. Tau protein phosphorylated at Thr-181, a well-established biomarker for Alzheimer's disease, was found at increased concentrations in exosomes isolated from cerebrospinal fluid samples of patients with mild Alzheimer's disease. These findings highlight the potential value of exosomes in the early diagnosis of Alzheimer's disease. Currently, there is no diagnostic test that can detect early-stage Alzheimer's disease.

[0080] Furthermore, in relation to kidney disease, proteins contained in urinary exosomes, which can be obtained noninvasively, are being explored for their potential diagnostic utility, particularly in urinary tract diseases. For example, fetuin-A contained in urinary exosomes is increased in patients with acute kidney injury in the intensive care unit compared to healthy individuals. ATF3 is also found only in exosomes isolated from patients with chronic kidney disease or acute kidney injury compared to healthy individuals. Furthermore, urinary exosomal proteins are being investigated as potential biomarkers for bladder and prostate cancer. Studies have shown that two prostate cancer biomarkers, PCA-3 and PSA, are present in exosomes isolated from the urine of prostate cancer patients. Overall, exosomal proteins identified in other body fluids are expected to serve as target platforms for the development of future diagnostic assay systems.

[0081] However, the aforementioned exosomal markers have not been clearly characterized in relation to disease and, furthermore, have not been applied clinically. One of the major reasons for this is that they do not independently represent the state or progression of the disease. It is predicted that disease-related exosomal markers will interact with target cells in a complex manner, with multiple marker groups interrelated. In particular, cancer cell-derived exosomes are expected to encode the type, number, and exosome locations of involved markers to evade immune surveillance.

[0082] To date, simple sandwich immunoassays for mapping exosome markers have reached technical limitations in accurately diagnosing target diseases. Therefore, technologies for simultaneous, in-depth immunoassay analysis of the compositional distribution of three or more multi-protein markers in exosomes within the same sample are essential for elucidating the correlation between exosome markers and specific diseases. For example, it has recently been reported that the compositional ratios and correlations among pan-exosome tetraspanins (CD9, CD63, and CD81), particularly the proportions of single, double, and triple complexes, vary significantly depending on cell type and origin. If these compositional ratios and correlations can be easily measured, it will be possible to predict the source of exosomes, enabling the early detection of abnormalities such as diseases in specific tissues through exosome liquid biopsy.

[0083] Currently, active research is being conducted using expensive, dedicated exosome analysis equipment to measure exosome composition and apply it to clinical applications, with published results. However, while exosome analysis on small samples is necessary at universities and research institutes, access to expensive equipment remains limited. Therefore, to expand research and development in the identification of disease-related exosome markers, individual laboratories must improve access to in-depth exosome analysis using general-purpose immunoassay methods and systems, such as ELISA.

[0084] In order to perform in-depth immunoassay for three or more markers, the exosome markers in the sample must be separated in advance. The first target exosomes contained in the exosome liquid biopsy sample may be exosomes constituting an exosome subpopulation separated and recovered for a specific one marker from an exosome population sample, an exosome subpopulation separated and recovered for two specific markers, or an exosome subpopulation separated and recovered for three specific markers. For example, the exosome liquid biopsy sample may be an exosome subpopulation sample separated into a population of first target exosomes that have a first surface marker (A) in common, or an exosome subpopulation sample separated into a population of second target exosomes that belong to an exosome subpopulation and further have a second surface marker (B) in common. Here, the first target exosome is an exosome that has a first surface marker (B) in common, and the second target exosome is an exosome that has a first surface marker (A) and a second surface marker (B) in common.

[0085] In immunoassays on exosome liquid biopsy samples, markers are generally defined using antibodies specific for two types, i.e., capture markers and detection markers. When a subpopulation exosome sample is used for analysis, simultaneous analysis of three markers is possible, when a subpopulation sample is used, four markers are possible, and when a sub-subpopulation sample is used, five markers are possible.

[0086] In order to obtain a sample in which exosomes are separated into subpopulations or subpopulations, it is necessary to be able to recover exosomes separated by an immunoaffinity method in a liquid phase. Details of preparing an exosome liquid biopsy sample in this regard are described below.

[0087]

[0088] In the triple marker signal measurement step (S200), a quantitative signal for triple marker exosomes in an exosome liquid biopsy sample is measured. To this end, in the case of an exosome liquid biopsy sample containing an exosome subpopulation, a second target exosome, i.e., a dual marker exosome, which has a second surface marker (B) in common is first isolated from the exosome liquid biopsy sample. The second target exosome can be captured and isolated using a capture antibody that specifically binds to the second surface marker (B). Then, a quantitative signal for the third surface marker (C) of the isolated second target exosome is measured. Here, the quantitative signal can be measured by combining a detection antibody that specifically binds to the third surface marker (C) with an enzyme. A representative detection method is the enzyme-linked immunosorbent assay (ELISA). However, the quantitative signal measurement method is not necessarily limited to this, and there is no special limitation on the signal measurement method as long as the quantitative signal for the triple marker exosome can be measured. Meanwhile, when using an exosome liquid biopsy sample including an exosome subpopulation, a second target exosome having a second surface marker (B) is already separated within the exosome liquid biopsy sample, so a separate second target exosome separation process may not be necessary.

[0089]

[0090] In the exosome heterogeneity index calculation step (S300), an exosome heterogeneity index (R) is generated based on the triple marker signal acquired in the triple marker signal measurement step (S200). In order to generate the exosome heterogeneity index (R), a measurement value of a dual marker signal is required as a variable other than the measurement value of the triple marker signal. The dual marker signal can be obtained by measuring the quantitative signal for the second surface marker (B) of the first target exosome.

[0091] Here, the dual marker signal can be measured for an exosome population sample. The exosome population sample can be reacted with a capture antibody that specifically binds to a first surface marker (A) to capture the first target exosomes, and then a detection antibody that specifically binds to a second surface marker (B) can be combined with an enzyme to measure the quantitative signal. In other words, the dual marker signal can be measured using an enzyme-linked immunosorbent assay (ELISA).

[0092] The exosome heterogeneity index (R) can be defined as the ratio of the measurement of the triple marker signal to the measurement of the dual marker signal.

[0093]

[0094] In the sample analysis step (S400), the exosome liquid biopsy sample is analyzed using the exosome heterogeneity index (R). The exosome heterogeneity index (R) represents the interrelationship between three surface markers (A, B, and C), and this interrelationship may exhibit different patterns depending on the specific disease. Therefore, based on the exosome heterogeneity index (R), biomarkers associated with specific diseases can be discovered and used for the detection of those diseases.

[0095] For example, the exosome population samples of a normal person group and a patient group with a specific disease are sequentially performed from the exosome liquid biopsy sample preparation step (S100) to the exosome heterogeneity index calculation step (S300), so that the exosome heterogeneity index (R) for the normal person group and the exosome heterogeneity index (R) for the patient group are calculated, respectively. Then, in the sample analysis step (S400), the calculated exosome heterogeneity index (R) for the normal person group and the exosome heterogeneity index (R) for the patient group are compared to select a biomarker related to the specific disease of the patient group. Here, if there is a clear difference between the exosome heterogeneity index (R) for the normal person group and the exosome heterogeneity index (R) for the patient group, it can be known that the exosome heterogeneity index (R) for the patient group is related to the specific disease of the patient group.

[0096] Based on this, disease screening information for a subject for disease screening can be generated. That is, from the exosome liquid biopsy sample preparation step (S100) performed for the biomarker selection to the exosome heterogeneity index calculation step (S300) are repeatedly performed for the exosome population sample of the subject for disease screening to calculate the exosome heterogeneity index (R) for the subject for disease screening, and then, in the sample analysis step (S400), the calculated exosome heterogeneity index (R) for the subject for disease screening is compared with the exosome heterogeneity index (R) for the patient group, thereby generating disease screening information for the subject for disease screening.

[0097]

[0098] FIG. 4 is a diagram illustrating a marker separation path for calculating an exosome heterogeneity index of a liquid biopsy according to an embodiment of the present invention. Referring to FIG. 4, the exosome heterogeneity index (R) may have different values ​​depending on the selection order of three surface markers (A, B, C). When one of the three surface markers (A, B, C) of heterogeneous exosomes in an exosome population sample is selected as the first surface marker, another is selected as the second surface marker, and the remaining one is selected as the third surface marker, a total of six different selection paths are created. By selecting at least one of them and sequentially repeating the steps from the exosome liquid biopsy sample preparation step (S100) to the exosome heterogeneity index calculation step (S300), N exosome heterogeneity indices (R) can be calculated (N is a natural number greater than or equal to 1 and less than or equal to 6). Here, since the N exosome heterogeneity indices (R) exhibit different characteristics depending on the selection path for the three surface markers (A, B, C), biomarkers related to a specific disease can be discovered based on the exosome heterogeneity indices (R) for each selection path, and these can be used for screening for the disease.

[0099] For example, for exosome population samples of a normal person group and a patient group with a specific disease, N exosome heterogeneity indices (R) for the normal person group and N exosome heterogeneity indices (R) for the patient group are respectively calculated. Then, in the sample analysis step (S400), based on the N exosome heterogeneity indices (R) for the normal person group and the N exosome heterogeneity indices (R) for the patient group, a selection path of a biomarker related to a specific disease of the patient group can be selected. Here, the biomarker selection path can be selected by considering the correlation between at least two or more indices among the N exosome heterogeneity indices (R).

[0100] Based on this, disease screening information for a subject for disease screening can be generated. For example, by repeatedly performing the exosome liquid biopsy sample preparation step (S100) to the exosome heterogeneity index calculation step (S300) according to the selection path performed in the biomarker selection path selection for the exosome population sample of the subject for disease screening, an exosome heterogeneity index (R) for the subject for disease screening can be generated, and the exosome heterogeneity index (R) for the subject for disease screening can be compared with the exosome heterogeneity index (R) for the patient group calculated according to the selection path performed in the biomarker selection path selection, thereby generating disease screening information for the subject for disease screening. At this time, in addition to the exosome heterogeneity index (R) for the patient group, the exosome heterogeneity index (R) for the normal group can also be compared with the exosome heterogeneity index (R) for the subject for disease screening.

[0101] Meanwhile, in selecting a selection path of a biomarker related to a specific disease of a patient group, in the sample analysis step (S400), based on the N exosome heterogeneity indices (R) for the normal group and the N exosome heterogeneity indices (R) for the patient group, the four arithmetic operations, i.e. addition, subtraction, multiplication, and division, may be performed between the N exosome heterogeneity indices (R) so that the contrast between the normal group and the patient group can be maximized, or a new index in which the exosome heterogeneity indices (R) are functionalized may be used.

[0102] In this case, for the exosome population sample of the disease screening subject, the exosome liquid biopsy sample preparation step (S100) to the exosome heterogeneity index calculation step (S300) are repeatedly performed according to the selection path performed for the biomarker selection path selection, thereby calculating the exosome heterogeneity index for the disease screening subject, and in the sample analysis step (S400), the new index for the disease screening subject is compared with the new index for the patient group calculated according to the selection path performed for the biomarker selection path selection, thereby generating disease screening information for the disease screening subject.

[0103]

[0104] Below, the preparation of an exosome liquid biopsy sample is described with reference to the manufacturing device and manufacturing method.

[0105]

[0106] FIG. 5 is a schematic diagram of a device for preparing exosome subpopulation and hypo-population samples according to an embodiment of the present invention. As illustrated in FIG. 5, the device for preparing exosome liquid biopsy samples according to an embodiment of the present invention may include a solid-state fixation member (10), a first specific binding material (20) that specifically binds to a first surface marker (m1) of a disease-related exosome (1a) among exosomes (1) secreted from a plurality of cells to capture the disease-related first target exosome (1a), and a first reversible linker (30) that releasably binds the fixation member (10) and the first specific binding material (20).

[0107] In addition, the first specific binding material (20) is bound to the unbound fixing member (10), and a second specific binding material (40) that specifically binds to a second surface marker (m2) of a second target exosome (1b) in an exosome subpopulation (S), which is a group of disease-related first target exosomes (1a), to capture the second target exosome (1b) may be further included.

[0108] Additionally, as a means for binding the second specific binding material (40) to the fixing member (10), a second reversible linker (50) may be further included.

[0109]

[0110] The fixing member (10) is a solid-state member, on the surface of which a first reversible linker (30), a second specific binding material (40), and a second reversible linker (50) can be selectively bonded and fixed. The first reversible linker (30) can be bonded and fixed onto one fixing member (10), then separated, and thereafter the second specific binding material (40) or the second reversible linker (50) can be bonded and fixed. In addition, the fixing member (10) to which the first reversible linker (30) is bonded and the fixing member (10) to which the second specific binding material (40) or the second reversible linker (50) is bonded may be different. The fixing member (10) may include at least one selected from the group consisting of a hydrogel, a magnetic bead, a latex bead, a glass bead, a nanometal structure, a porous membrane, and a non-porous membrane. However, the fixed member (10) is not necessarily limited to the above, and there is no special limitation as long as it is a material that can be combined with each of the first reversible linker (30), the second specific binding material (40), or the second reversible linker (50).

[0111]

[0112] The first specific binding material (20) and the second specific binding material (40) are materials that selectively capture a given exosome (1). The first specific binding material (20) and the second specific binding material (40) capture the exosome (1) by specifically binding to the surface marker (m) of the exosome (1). Here, the surface marker (m) is a biomarker that may relatively increase in body fluid compared to a normal person when a specific disease occurs, and may include proteins, nucleic acids, lipid components, sugar components, peptides, and other biochemical components present on the surface of the exosome (1). This surface marker (m) may specifically bind to the first specific binding material (20) and the second specific binding material (40) through immunochemical interactions such as antigen-antibody reactions. The first specific binding material (20) and the second specific binding material (40) may be a predetermined antibody that undergoes an antigen-antibody reaction with a surface protein of the exosome (1).

[0113] Among the surface markers (m), the first surface marker (m1) that specifically binds to the first specific binding material (20) exists on the surface of a predetermined disease-related first target exosome (1a). Therefore, from a bulk exosome population (B), which is a population of exosomes (1) separated from a plurality of cells, the disease-related first target exosome (1a) having the first surface marker (m1) can be separated by being captured by the first specific binding material (20). Here, the population of separated disease-related first target exosomes (1a) becomes an exosome subpopulation (S).

[0114] Among the disease-related exosomes (1a) within the exosome subpopulation (S) thus generated, the second target exosome (1b) having the second surface marker (m2) reacts specifically with the second specific binding material (40) and is captured, thereby separating the exosome subpopulation (SA) within the exosome subpopulation (S). The first surface marker (m1) on the exosome (1) exhibits relatively low accuracy when used for diagnosing a specific disease compared to the second surface marker (m2), but exists in a relatively high concentration in body fluids and is positioned at a higher level when quantitatively systematically classifying the exosome (1) population according to markers, thereby allowing it to have more comprehensive characteristics.

[0115] In the present invention, an exosome liquid biopsy sample prepared by first separating, concentrating, and recovering an exosome subpopulation (S) positive for a disease-specific marker (a first surface marker) that is predicted to have a low concentration in body fluids may be used, or an exosome subpopulation (SA) recovered by second separating and concentrating the exosome subpopulation (S) for a disease-specific marker (a second surface marker) may be used as an exosome liquid biopsy sample.

[0116] Furthermore, in the present invention, the number of repetitions of the separation, concentration, and recovery processes that can be sequentially performed for different exosome surface protein markers can be extended to two or more times depending on the purpose, and a subpopulation recovered through separation and concentration for another disease-specific marker from the exosome subpopulation (SA) can be used as an exosome liquid biopsy sample.

[0117] In liquid biopsy using exosomes in body fluids, switching from simply concentrated bulk exosome samples to disease-relevant exosome samples selectively isolated for the disease being examined may improve diagnostic accuracy.

[0118]

[0119] Meanwhile, the first specific binding material (20) can be fixed to the fixing member (10) and then separated via the first reversible linker (30). The second specific binding material (40) can be directly fixed to the fixing member (10) without separation, or can be fixed to the fixing member (10) and then separated by introducing the second reversible linker (50).

[0120]

[0121] Among the reversible linkers, the first reversible linker (30) detachably binds the first specific binding material (20) to the fixing member (10), and the other second reversible linker (50) detachably binds the second specific binding material (40) to the fixing member (10). Here, the first specific binding material (20) is not bound to the fixing member (10) to which the second specific binding material (40) is fixed. That is, the second specific binding material (40) is fixed to the fixing member (10) to which the first specific binding material (20) is not bound through the second reversible linker (50). The first reversible linker (30) and the second reversible linker (50) may be made of the same material and structure, or may be implemented differently, and the specific materials and structures will be described later.

[0122]

[0123] Exosome immunoaffinity isolation under mild conditions is possible using a reversible linker based on a ligand-binder attachment reaction. The reversible linker initially immobilizes a specific exosome (1)-specific binding material (antibody) on an immobilizing member (10), but after capturing the exosome (1) in the sample on the immobilizing member (10), it dissociates through a change in reaction conditions, allowing the captured exosome (1) to be recovered. For example, in addition to sugar-sugar receptor binding reactions through hydrophilic and hydrophobic bonds, ion-ion or ion-ion receptor binding reactions, substrate-enzyme reactions, antigen-antibody reactions, nucleic acid conjugation reactions, hormone-cell receptor reactions, and ligand-nanostructure reactions, polyhistidine-tag reactions, biotin-avidin reactions, and attachment reactions using chelators, among others, can be used as the reversible linker. Such attachment reactions can be dissociated by modulating the affinity between the binder and the ligand, such as through structural changes in the binder after binding or competitive reactions with the binder. Therefore, using a reversible linker, it is possible to immunologically capture exosomes (1) in a sample, and then isolate and recover second target exosomes (1b) under mild conditions, without using harsh conditions such as acidic pH.

[0124] In addition, any process that enables separation and recovery using physical, biological, and chemical properties such as density, size, solubility, membrane permeability, molecular structure, and magnetism can be used as an immunoaffinity separation process using the above-mentioned reversible linker based on the attachment reaction. In particular, by using a specially designed immunoaffinity separation process such as chromatography or magnetic separation, it is possible to isolate and recover trace amounts of exosomes in body fluids by controlling the affinity between the binder and the ligand within the linker. For example, by using the 'switchable attachment reaction' of a recognition material that recognizes the structural change of the binder within the linker induced by ligand attachment, it is possible to isolate and recover exosomes through the immunoseparation process simply depending on the presence or absence of the ligand. Furthermore, by introducing a competitive attachment reaction method using a ligand-like structure that competitively reacts with the ligand for the binder in the ligand-binder reaction during immunoseparation, it is possible to isolate and recover exosomes depending on the presence or absence of the competitive attachment reaction.

[0125]

[0126] Hereinafter, the first reversible linker (30) and the second reversible linker (50) described above will be described in detail. The reversible linker can be implemented according to the first to fourth embodiments below, and the first reversible linker (30, 30a to 30d) and the second reversible linker (50, 50a to 50d) can be formed of any one of them. FIGS. 6 to 9 are schematic diagrams illustrating the first reversible linker and the second reversible linker illustrated in FIG. 5 according to various embodiments.

[0127]

[0128] As illustrated in FIG. 6, the reversible linker (30a, 50a) according to the first embodiment of the present invention may include a ligand (31a), a binder (33a), and a recognition material (35a).

[0129] The ligand (31a) is a substance that is detachably bonded to the binder (33a), and may include at least one selected from the group consisting of sugar molecules, ions, substrates, antigens, peptides, vitamins, growth factors, and hormones.

[0130] The binder (33a) is a material that undergoes a conformation change by an attachment reaction with the ligand (31a). The binder (33a) may include at least one selected from the group consisting of a sugar-binding protein, an ion-binding protein, an enzyme, an antibody, an aptamer, a cell receptor, and a nanostructure. Accordingly, the binder (33a) and the ligand (31a) may form a binder-ligand pair, such as a sugar-binding protein-sugar molecule pair, an ion-binding protein-ion pair, an enzyme-substrate pair, an antigen-antibody pair, an aptamer-ligand pair, a cell receptor-ligand pair, a nanostructure-ligand pair, etc., and the most representative examples of the binder (33a) and the ligand (31a) include calcium binding protein (CBP) and calcium ions. However, the binder (33a) and the ligand (31a) are not necessarily limited to the above materials, and any material that can detachably bond to each other and cause a structural change may be used. In the reversible linker according to the first embodiment, the binder (33a) is polymerized with the first specific binding material (20) and / or the second specific binding material (40) to form a binder-specific binding material polymer (C).

[0131] The recognition material (35a) is a substance that specifically binds to the binder (33a) whose structure has changed by binding to the ligand (31a), and may include at least one selected from the group consisting of antibodies, protein receptors, cell receptors, aptamers, enzymes, nanoparticles, nanostructures, and heavy metal chelators. However, since the above substance is only an example of the recognition material (35a), the scope of the present invention should not be limited thereto. For example, when explaining the binding reaction between the ligand (31a), the binder (33a), and the recognition material (35a), a calcium ion (ligand) first binds to a calcium binding protein (binder), causing a structural change in the calcium binding protein, and an antigen-antibody reaction occurs between the calcium binding protein whose structure has changed and the antibody (recognition material), so that they can bind to each other. In the reversible linker according to the first embodiment, the recognition material (35a) is fixed to the surface of the fixing member (10).

[0132] Meanwhile, when exosomes (1) are captured by the first specific binding material (20) and / or the second specific binding material (40) coupled to the fixed member (10) via a reversible linker (30, 50), the captured exosomes (1a, 1b) can be recovered by dissociating the reversible linker through a change in reaction conditions. In the case of the reversible linker (30a, 50a) according to the first embodiment, the binder (33a) whose structure has changed can be dissociated when it is restored to its original structure. For example, when an antibody (recognition material) binds to a calcium-binding protein (binder) whose structure has changed due to the attachment of a calcium ion (ligand) and the antibody captures an exosome (1), by adding a recovery solution that does not contain calcium ions and desorbing calcium ions from the calcium-binding protein, the structure of the calcium-binding protein can be restored and the calcium-binding protein and antibody can be separated.

[0133]

[0134] With reference to FIG. 7, the reversible linker (30b, 50b) according to the second embodiment of the present invention may include a ligand (31b), a binder (33b), and a recognition material (35b).

[0135] The ligand (31b), binder (33b), and recognition material (35b) of the reversible linker (30b, 50b) according to the second embodiment correspond to the ligand (31a), binder (33a), and recognition material (35a) of the reversible linker (30a, 50a) according to the first embodiment, respectively, and the following description focuses on the differences.

[0136] In the reversible linker (30a, 50a) according to the first embodiment, the binder (33a) is polymerized with the specific binding material (20, 40) to form a polymer (C), and the recognition material (35a) is fixed to the surface of the fixing member (10), whereas in the case of the reversible linker (30b, 50b) according to the second embodiment, the binder (33b) is fixed to the surface of the fixing member (10), and the recognition material (35b) is polymerized with the specific binding material (20, 40) to form a recognition material-specific binding material polymer (C). Other than that, it is the same as the reversible linker (30a, 50a) according to the first embodiment, and a detailed description thereof is omitted.

[0137] As an example of a reversible linker (30, 50) according to the first and second embodiments that enables the recovery of exosome subpopulations or subpopulations in a sample under mild conditions, a recognition material (35a, 35b), such as an antibody, that specifically recognizes a structural change caused by binding of a binder (33a, 33b), such as a calcium binding protein, to a ligand (31a, 31b), such as a calcium ion, that specifically reacts therewith may be used. This binder-recognition material reaction, such as an antigen-antibody reaction, is described as a 'switch-like reversible binding' because it rapidly binds or dissociates depending on the presence or absence of a ligand (31a, 31b), such as a calcium ion, and is similar to the principle of turning a light on and off by operating a switch.

[0138]

[0139] As illustrated in FIG. 8, the reversible linker (30c, 50c) according to the third embodiment of the present invention may include a ligand (31c) and a binder (33c).

[0140] The ligand (31c) of the reversible linker (30c, 50c) according to the third embodiment is polymerized with the first specific binding material (20) and / or the second specific binding material (40) to form a specific binding material-ligand polymer (C).

[0141] The binder (33c) of the reversible linker (30c, 50c) according to the third embodiment is fixed to the surface of the fixing member (10), and captures the exosomes (1a, 1b) to the fixing member (10) by binding to a specific binding material-ligand polymer (C) that specifically binds to the exosomes (1a, 1b). The binder (33c) binds to the ligand (31c), and when a competitive reaction ligand (32) that competes with the ligand (31c) is added, the competitive reaction ligand (32) is attached. As a result, the exosomes (1a, 1b) can be separated from the fixing member (10) and recovered as the ligand (31c) is detached. Here, the ligand (31c) may include at least one selected from the group consisting of vitamins, sugar molecules, ions, substrates, antigens, amino acids, peptides, nucleic acids, growth factors, and hormones, such as His-tag (polyhistidine-tag), biotin, etc. In addition, the binder (33c) may include at least one selected from the group consisting of divalent metal ions, avidins, sugar-binding proteins, ion-binding proteins, enzymes, antibodies, aptamers, protein receptors, cell receptors, and nanostructures. The competitive ligand (32) may include at least one selected from the group consisting of vitamins, sugar molecules, ions, substrates, antigens, amino acids, peptides, nucleic acids, growth factors, and hormones, such as imidazole, biotin, etc.

[0142] For example, His-stack can be used as a ligand (31c), a divalent metal ion can be used as a binder (33c), and imidazole can be used as a competitive ligand (32). His-stack is an amino acid motif consisting of at least six histidine (His) residues in a protein, and is usually located at the N-terminus or C-terminus of the protein, and is mainly used for purification after expression of a recombinant protein. Here, the protein labeled with His-stack can be captured using the affinity for nickel or cobalt, which are divalent ions bound to a chelator on a Sepharose / agarose resin, and then recovered by adding a recovery solution containing imidazole.

[0143]

[0144] With reference to FIG. 9, the reversible linker (30d, 50d) according to the fourth embodiment of the present invention may include a ligand (31d) and a binder (33d).

[0145] The ligand (31d) and binder (33d) of the reversible linker (30d, 50d) according to the fourth embodiment correspond to the ligand (31c) and binder (33c) of the reversible linker (30c, 50c) according to the third embodiment, respectively, and the differences will be mainly explained below.

[0146] In the reversible linker (30c, 50c) according to the third embodiment, the ligand (31c) is polymerized with the specific binding material (20, 40), and the binder (33c) is fixed to the surface of the fixing member (10), whereas in the reversible linker (30d, 50d) according to the fourth embodiment, the ligand (31d) is fixed to the surface of the fixing member (10), and the binder (33d) is polymerized with the specific binding material (20, 40) to form the specific binding material-binder polymer (C). In addition, the ligand (31d) is similarly bound to the binder (33d), but is separated from the binder (33d) by a competitive reaction with the competitive reaction ligand (32).

[0147]

[0148] Meanwhile, the exosome liquid biopsy sample manufacturing device may further include a reactor (not shown). The reactor may accommodate a fixing member (10) therein. At this time, when an exosome (1) sample is added to the reactor, disease-related exosomes (1a) are captured by the fixing member (10) via the first reversible linker (30) and the first specific binding material (20) therein, and an exosome subpopulation (S) is separated, and the first reversible linker (30) is dissociated, and the exosome subpopulation (S) is recovered. In addition, the recovered exosome subpopulation (S) sample is added to the reactor, and a second target exosome (1b) is captured by the fixing member (10) using the second reversible linker (50) and the second specific binding material (40), and an exosome subpopulation (SA) is separated. The separated exosome subpopulation (SA) is recovered as the second reversible linker (50) dissociates. Here, the exosome subpopulation (S) and / or the exosome subpopulation (SA) can be concentrated by concentrating the fixation member (10) using at least one of magnetic force, gravity, and centrifugal force. For example, when a magnetic bead is used as the fixation member (10), the fixation member (10) bound to the disease-related exosomes (1a) and / or the second target exosomes (1b) is captured using a magnet, and the supernatant is removed and the magnetic beads are removed, thereby concentrating the exosome subpopulation (S) and / or the exosome subpopulation (SA).

[0149]

[0150] Below, a method for preparing a liquid biopsy sample using the exosome liquid biopsy sample preparation device described above is described. The exosome liquid biopsy sample preparation device has been described above, and any overlapping details will be omitted or described only briefly.

[0151] Figures 10 to 16 are flowcharts of a method for preparing an exosome liquid biopsy sample according to an embodiment of the present invention.

[0152] With reference to FIG. 10, the method for preparing an exosome liquid biopsy sample according to the first embodiment of the present invention may include a step (S110) of binding a first specific binding material that specifically binds to a first surface marker of a disease-related first target exosome among exosomes secreted from a plurality of cells to a solid-state fixation member using a first reversible linker, a step (S120) of reacting a sample solution containing a population of exosomes with the first specific binding material bound to the fixation member to capture the disease-related first target exosomes, thereby isolating an exosome subpopulation, which is a population of disease-related first target exosomes, and a step (S130) of dissociating the first reversible linker so that the captured disease-related first target exosomes are separated from the fixation member, thereby recovering the exosome subpopulation.

[0153] Here, a bulk exosome population or body fluid, which is a group of exosomes secreted from multiple cells, can be used as a sample solution, and after first separating and recovering a subpopulation of exosomes, this can be provided as a liquid biopsy sample.

[0154]

[0155] In addition, as shown in FIG. 11, in order to prepare a liquid biopsy sample by separating and recovering an exosome subpopulation from an exosome subpopulation, the method may further include a step (S140) of binding a second specific binding material that specifically binds to a second surface marker of a second target exosome in the exosome subpopulation to a fixing member, and a step (S150) of reacting an exosome subpopulation solution containing the recovered exosome subpopulation with the second specific binding material bound to the fixing member to capture the second target exosomes, thereby isolating the exosome subpopulation, which is a population of the second target exosomes. Here, the second specific binding material may be bound to the fixing member by a second reversible linker.

[0156] First, to isolate and recover exosome subpopulations, a first specific binding material and a first reversible linker are reacted with a fixing member (S110). At this time, the first specific binding material is bound to the fixing member via the first reversible linker.

[0157] Next, by adding a sample solution, the first specific binding material is reacted (S120). Here, the first specific binding material specifically binds to the first surface marker of the disease-related first target exosome contained in the sample solution. As a result, a subset of exosomes targeting the disease-related first target exosome are captured by the fixation member and separated from the sample solution.

[0158] After the exosome subpopulation is separated, the first reversible linker is dissociated (S130). At this time, the disease-related first target exosome is separated from the fixation member, allowing the exosome subpopulation to be recovered.

[0159] Once the disease-related exosomes are separated from the fixation member, the fixation member is recycled or a new fixation member is used to bind a second specific binding material to the fixation member (S140). At this time, the second specific binding material may be directly bound to the fixation member or may be bound to the fixation member using a second reversible linker.

[0160] Next, the exosome subpopulation solution containing the recovered exosome subpopulation is reacted with a second specific binding material (S150). At this time, since the second specific binding material specifically binds to the second surface marker of the second target exosomes in the exosome subpopulation, the second target exosomes are fixed to the fixing member. As a result, an exosome subpopulation, which is a collection of the second target exosomes, is separated. In this way, the exosome subpopulation that is not recovered but is fixed to the fixing member can be provided as a liquid biopsy sample. That is, when an analysis of the biomarker is performed directly without recovering the exosomes after the biomarker for diagnosing a specific disease is separated in the form of a subpopulation, or when an analysis of the eluted biomarker is performed by dissolving the exosomes, in the last separation step of the sequential multiplex immunoaffinity separation process, only the biomarker can be used as a sample without recovering the subpopulation exosomes.

[0161]

[0162] Hereinafter, the present invention will be described in more detail with specific examples relating to the first reversible linker and the second reversible linker.

[0163] Fig. 12 illustrates a method for producing an exosome subpopulation liquid biopsy sample using the first reversible linker (30a) and the second reversible linker (50a) according to the first embodiment described above. Referring to Fig. 10, first, a recognition material (35a) is fixed to the surface of a fixing member (10), and a ligand solution containing a ligand (31a), a binder (33a), and a first specific binding material (20) are reacted. At this time, the binder (33a) and the first specific binding material (20) form a binder-first specific binding material polymer (C), and the recognition material (35a) binds to the polymer (C). Here, the ligand (31a) contained in the ligand solution binds to the binder (33a), causing a structural change in the binder (33a), and the recognition material (35a) specifically recognizes and binds to the binder (33a) with a changed structure. Next, a sample solution containing a population of bulk exosomes (1) is reacted. At this time, the disease-related first target exosome (1a) having the first surface marker (m1) that specifically binds to the first specific binding material (20) is captured by the fixing member (10), thereby isolating the exosome subpopulation (S). When the exosome subpopulation (S) is separated, a recovery solution is added. The recovery solution is a ligand-free solution that does not contain a ligand (31a). When the recovery solution is added, the ligand (31a) bound to the binder (33a) is detached, and as a result, the changed structure of the binder (33a) is restored to its original state, and the bond between the binder (33a) and the recognition material (35a) is dissociated. Therefore, the polymer (C) to which the disease-related first target exosome (1a), which is the target of the exosome subpopulation (S), is bound is separated from the recognition material (35a), and the exosome subpopulation (S) can be recovered.

[0164] Next, the disease-related exosome (1a) is separated and the fixing member (10) to which the recognition material (35a) is fixed is recycled to react with the second reversible linker (50a) and the second specific binding material (40). At this time, the second reversible linker (50a) shares the recognition material (35a) of the first reversible linker (30a), and compared to the first reversible linker (30a), the first specific binding material (20) is replaced by the second specific binding material (40) to bind to the fixing member (10). When the recovered exosome subpopulation (S) solution is added here, the second specific binding material (40) specifically binds to the second surface marker (m2) of the second target exosome (1b) in the exosome subpopulation (S) solution, thereby isolating the exosome subpopulation (SA). At this time, since the exosome subset (S) includes the binder (33a) of the first reversible linker (30a), the recognition material (35a) bound to the fixing member (10) and the binder (33a) of the first reversible linker (30a) can react with each other, so after reacting the second reversible linker (50a) and the second specific binding material (40), the remaining attachment site on the recognition material (35a) bound to the fixing member (10) can be blocked with an appropriate reactive component such as the binder (33a).

[0165] As an example, a method for isolating CD63+ / Cav1+ exosome subpopulation (SA) according to the present invention having a first reversible link and a second reversible link according to the first embodiment is described. An antibody (reversible recognition material) is immobilized on a solid (fixing member) surface (Bead surface), and a calcium binding protein (CBP, binder) is polymerized through biotin-streptavidin linkage with a specific antibody (first specific binding material) for CD63 exosome surface protein (CBP-capture antibody polymer). Calcium ions (e.g., >10 mM Ca) are added to the immobilized reversible recognition material. 2 +) is added to the solution containing the CBP-capture antibody polymer, the polymer is fixed to the solid surface by the 'switch on' recognition reaction. When the bulk exosome sample prepared in the same solution is sequentially added, the exosomes containing the CD63 marker react with the capture antibody on the solid surface and are captured. After washing with the same solution, when the exosome recovery solution from which calcium has been removed is added, the calcium ions bound to CBP are desorbed and the 'switch off' state is entered, so that the captured exosomes can be recovered into the solution in a state bound to the capture antibody. As a result, the CD63+ exosome subpopulation is separated and recovered, and the concentration of the exosomes is also possible depending on the volume ratio of the sample solution and the recovery solution used. Additionally, the solid surface can be regenerated and recycled for the separation of other samples.

[0166] For sequential exosome subpopulation isolation, a reversible recognition material is immobilized on a regenerated or new solid surface, and a specific antibody against the Cav1 exosome surface protein, which is highly associated with a specific disease, is polymerized with CBP, and then the CBP-capture antibody polymer is immobilized on the solid surface through a 'switch-on' recognition reaction in the same manner as above. While maintaining the same state, the CD63+ exosome subpopulation recovered after the first separation is added as a second separation sample, and exosomes containing CD63 and Cav1 markers (CD63+ / Cav1+) react with the capture antibody on the solid surface and are captured. After washing with the same solution, the addition of a calcium-free exosome recovery solution switches the 'switch-off' state, allowing the captured exosomes to be recovered into the solution. As a result, the CD63+ / Cav1+ exosome subpopulation is isolated and recovered through the second separation.

[0167] Meanwhile, if recovery after separation of the exosome subpopulation is not required, the subpopulation can be separated by directly immobilizing the capture antibody on a solid surface without introducing a second reversible linker. In this case, immunoassay is mainly performed on the captured exosomes, or the exosomes are immediately dissolved while still captured to extract and perform molecular tests on the nucleic acids contained within the exosomes.

[0168]

[0169] Fig. 13 illustrates a method for preparing an exosome subpopulation liquid biopsy sample using the first reversible linker (30c) and the second reversible linker (50c) according to the third embodiment described above. Referring to Fig. 13, a ligand (31c) is polymerized with a first specific binding material (20) that specifically reacts with the first surface marker (m1) of a disease-related first target exosome (1a), and the ligand-specific binding material polymer is fixed using the affinity with a binder (33c) bound to a fixing member (10), and then an exosome (1) sample is added to capture and isolate an exosome subpopulation (S), and the exosome subpopulation (S) is recovered using a recovery solution containing a competitive reaction ligand (32).

[0170] Next, a second specific binding material (40) and a ligand (31c) that specifically react to the second surface marker (m2) of the second target exosome (1b) are polymerized, and after fixing the binder (33c) and the polymer (C) bound to the fixing member (10), an exosome subpopulation (S) solution is added to capture and isolate the exosome subpopulation (SA). At this time, since the exosome subset (S) includes the ligand (31c) of the first reversible linker (30c), the binder (33c) bound to the fixing member (10) and the ligand (31c) of the first reversible linker (30c) can react with each other, so after reacting the second reversible linker (50c) and the second specific binding material (40), the remaining attachment site on the binder (33c) bound to the fixing member (10) can be blocked with an appropriate reactive component such as the ligand (31c).

[0171] As an example, a method for isolating a CD63+ / Cav1+ exosome subpopulation according to the present invention having a first reversible link and a second reversible link according to the third embodiment is described. A binder (nickel ion) is immobilized on a solid surface (bead surface), and a ligand (His-stacking) is polymerized with a specific antibody for a CD63 exosome (1) surface protein as a first surface marker (m1) (His-stacking-capture antibody polymer). When the His-stacking-capture antibody polymer is added to the immobilized nickel ion, the polymer is immobilized on the solid surface by a binder-ligand reaction. When a bulk exosome sample prepared in the same solution is added, CD63+ exosomes react with the capture antibody on the solid surface and are captured. After washing with the solution, an exosome recovery solution containing a high concentration of a competitive ligand (imidazole) is added. This competitive reaction detaches the nickel ion-bound His-stacs, allowing the captured exosomes to be recovered into the solution. As a result, the CD63+ exosome subpopulation is separated and recovered, and the solid surface is regenerated, allowing it to be recycled for the separation of the next sample.

[0172] The above separation process is used for sequential isolation of the CD63+ / Cav1+ exosome subpopulation from the CD63+ exosome subpopulation recovered above. In this case, the ligand (His-Stack) polymerizes with an antibody specific for the Cav1 exosome surface protein as a second surface marker. After the polymer is immobilized on the solid surface through a binder-ligand reaction, the CD63+ exosome subpopulation sample is added, and the CD63+ / Cav1+ exosomes react with the capture antibody on the solid surface and are captured. After washing, the CD63+ / Cav1+ exosomes bound to Con A can be recovered into the solution by adding an exosome recovery solution containing a high concentration of a competitive ligand (imidazole). As a result, the CD63+ / Cav1+ exosome subpopulation is separated and recovered.

[0173]

[0174] FIG. 14 illustrates a method for preparing an exosome subpopulation liquid biopsy sample using a first reversible linker (30a) according to the first embodiment described above, and a second reversible linker (50c) according to the third embodiment. Referring to FIG. 14 , as described above in FIG. 10 , an exosome subpopulation (S) can be isolated and recovered using the first reversible linker (30a) according to the first embodiment, and as described above in FIG. 13 , an exosome subpopulation (SA) can be isolated using the second reversible linker (50c) according to the third embodiment. At this time, the exosome subpopulation (S) contains the binder (33a) of the first reversible linker (30a), but when separating the exosome subpopulation (SA), a binder (33c) different from the binder (33a) according to the first embodiment is bound to the fixing member (10) in the third embodiment, that is, a second reversible linker (50c) that operates with a different mechanism from the first reversible linker (30a) is used, so the blocking process described above is unnecessary.

[0175]

[0176] FIG. 15 illustrates a method for preparing an exosome subpopulation liquid biopsy sample using the first reversible linker (30c) according to the third embodiment described above, and the second reversible linker (50a) according to the first embodiment. Referring to FIG. 15, after isolating and recovering an exosome subpopulation (S) using the first reversible linker (30c) according to the third embodiment described above in FIG. 13, an exosome subpopulation (SA) can be isolated using the second reversible linker (50a) according to the first embodiment described above in FIG. 10. At this time, the exosome subpopulation (S) contains the ligand (31c) of the first reversible linker (30c), but when separating the exosome subpopulation (SA), a second reversible linker (50a) distinct from the first reversible linker (30c) is adopted, so the blocking process described above is unnecessary.

[0177]

[0178] Figure 16 is a flowchart of a method for preparing an exosome subpopulation liquid biopsy sample according to another embodiment of the present invention.

[0179] With reference to FIG. 16, a method for preparing an exosome subpopulation liquid biopsy sample according to another embodiment of the present invention may further include a step (S160) of recovering an exosome subpopulation. Here, the second specific binding material is bound to a fixing member by a second reversible linker, and by dissociating the second reversible linker, the captured second target exosomes are separated from the fixing member, thereby recovering the exosome subpopulation.

[0180] In addition, a step of concentrating the exosome subpopulation (S125) may be further included. The exosome subpopulation may be concentrated after adding the sample solution and before adding the recovery solution. Specifically, the fixation member combined with the disease-related exosomes of the exosome subpopulation is concentrated using at least one of magnetic force, gravity, and centrifugal force. For example, a magnetic bead may be used as the fixation member, and the fixation member may be captured to a predetermined spatial region within a mixed solution containing a ligand solution and a sample solution by magnetic force, and then a portion of the mixed solution (e.g., a supernatant) that does not contain the fixation member may be removed, thereby concentrating the exosome subpopulation.

[0181] In the same manner, the exosome subpopulation can also be concentrated by concentrating the fixation member combined with the second target exosome using at least one of magnetic force, gravity, and centrifugal force (S155).

[0182] Below, the present invention is described in more detail with experimental examples.

[0183]

[0184] Experimental example

[0185] 1. Overview

[0186] Exosomes released from cells and tissues in bodily fluids (e.g., peripheral blood) are highly heterogeneous, limiting their utility as biomarkers for disease diagnosis and therapeutic drug monitoring. Therefore, exosome isolation is necessary prior to sample analysis. Conventionally, exosome isolation was performed based on exosome density, size, solubility, surface markers, and other factors, resulting in exosome samples with reduced heterogeneity.

[0187] While numerous exosomal biomarkers have been reported to distinguish between patient samples with specific diseases, such as cancer, and healthy individuals, the practical application of exosomal biomarker-based diagnostic technologies remains limited. This stems from the dynamic nature of exosomal heterogeneity, which can vary significantly depending on various factors, including constitutional status, disease state, sample preparation, and separation techniques. In these circumstances, sample-dependent marker detection cannot maintain consistency.

[0188] To overcome limitations in accurate cancer diagnosis, issues related to exosome heterogeneity must be addressed, for example, by separating exosome samples from body fluids into subpopulations. However, conventional step-by-step separation based on size, density, and solubility does not significantly improve the issues mentioned in the analysis of early-stage cancer samples. While attempts can be made to isolate exosomes based on surface markers, current immunoassay techniques cannot continuously and repeatedly isolate multiple markers from the same exosome.

[0189] The present invention proposes an innovative exosome separation technique capable of sequential, multi-step separation of exosomes into subpopulations or smaller subpopulations. This technique, termed "Neutral Release," is fundamentally based on reversible antigen-antibody binding, enabling exosomes to be recovered in an aqueous phase under mild conditions. Furthermore, the recovered exosomes can be further separated into subpopulations that share two biomarkers. One of the tetraspanins (e.g., CD9, CD63, and CD81) can be used as a primary marker for separating exosome subpopulations from the bulk population, and sequential separation of the markers can reduce exosome heterogeneity.

[0190] To achieve this goal, we conducted a study to track changes in exosome heterogeneity depending on the cell source producing target exosomes within a sample. Exosomes play a crucial role in the mutual regulation of tumor and immune cells in the tumor microenvironment. In certain cases, the composition of immune cell-derived exosomes in body fluids can dramatically increase, leading to greater changes in exosome heterogeneity in specific exosome subpopulations compared to non-cancerous conditions. Here, we sequentially selected surface markers to identify subpopulations containing exosomes associated with the immune response. These subpopulations were determined through differential analysis of local heterogeneity changes in clinical samples between healthy donors and cancer patients. This approach can serve as the basis for an analytical model for biomarker discovery by eliminating background noise caused by changes in exosome heterogeneity.

[0191]

[0192] 2. Materials

[0193] A monoclonal antibody that specifically recognizes the unique structure of calcium binding protein (CBP) that changes when calcium ions bind to CBP was produced by the present inventors. In addition, a mutated glucose-galactose binding protein (GGBP) was selected as CBP, and this protein was produced from Escherichia coli (E. coli) and purified. Casein (sodium salt form, extract from bovine milk), bovine serum albumin (BSA), Tween-20, ProClin300, trehalose, ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA), biotin, human serum, and L-cysteine ​​were purchased from Sigma-Aldrich (St. Louis, MO, USA). Magnetic beads (Dynabeads M-280 tosyl-activated, 2.8 νm in diameter), streptavidin (SA), sulfosuccinimidyl-6-[biotinamido]-6-hexanamido hexanoate (NHS-LC-LC-biotin), RPMI 1640, Dulbecco's modified eagle medium (DMEM), exosome-depleted fetal bovine serum (FBS), penicillin / streptomycin, keratinocyte SFM supplied in separate packaging with recombinant EGF (rEGF) and bovine pituitary extract (BPE), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), dithiothreitol (DTT), and Zeba Spin Desalting Column, 2 mL, (Zeba Column) were supplied by Thermo Fisher Scientific (Rockford, IL, USA). Anti-CD9 antibody (clone HI9a), anti-CD63 antibody (clone H5C6), anti-CD81 antibody (clone 5A6), anti-CD151 antibody (clone 50-6), and anti-TSPAN8 antibody (clone TAL69) were purchased from BioLegend (San Diego, CA, USA). Streptavidin-poly-horseradish peroxidase 20 (HRP20) polymerization and strep-poly HRP polymerization stabilizer were purchased from Fitzgerald (Acton, MA, USA).Antibody diluents (HAMA Blocker) and tetramethyl benzidine-HK (TMB-HK) were purchased from Abcam (Cambridge, UK) and Mossbio (Pasadena, Maryland, USA), respectively. Ultracel 100 kDa ultrafiltration discs and Amicon Ultra-4 Centrifugal Filters (10 kDa MWCO; Amicon Filter) were purchased from Merck / Millipore (Burlington, MA, USA). StabilCoat Immunoassay Stabilizer, microwell plates, and FBS were purchased from Surmodics (Eden Prairie, MN, USA), Corning (Somerville, MA, USA), and Cytiva (Marlborough, MA, USA), respectively.

[0194] Human prostate cancer cell lines LNCaP and PC-3, human colon cancer cell lines HT-29 and HCT116, and human embryonic kidney cell line HEK293 were purchased from the Korea Cell Line Bank (KCLB) in Seoul, South Korea. The prostate epithelial cell line RWPE-1 was provided by the American Type Culture Collection (ATCC), Manassas, USA. Human serum clinical samples from healthy individuals and prostate cancer patients were obtained from Precision for Medicine (PM), Maryland, USA, and human sera from malignant melanoma patients and colon cancer patients were obtained from iSpecimen (ISP), Massachusetts, USA.

[0195]

[0196] 3. Method

[0197] 3-1. Exosome production

[0198] Cell culture and exosome production. After filling RPMI 1640 medium supplemented with 10% FBS and 1% penicillin / streptomycin in a 100 mm diameter culture dish, LNCaP (2Х10 6 cells), PC-3 cells (1Х10 6 cells), HT-29 (2Х10 6 cells), and HCT116 (2Х10 6 Cells) were seeded on each culture dish and cultured for 72 hours in an incubator maintained at 37°C and 5% CO2. After culture, the medium was washed, replaced with the same medium (containing 10% exosome-depleted FBS), and cultured again for 48 hours under the same conditions. HEK293 cells (1Х10 6 RWPE-1 cells (1Х10) were cultured in keratinocyte SFM medium (supplemented with 0.05 mg / ml BPE and 5 ng / ml rEGF). 6 cells) were seeded and cultured. The culture was performed for 72 hours under the same conditions as LNCaP and PC-3, and after washing, only rEGF was added to the same medium and cultured for an additional 48 hours.

[0199]

[0200] Preparation of exosome sample stock. After exosome production, the medium was collected from each dish. To pellet the cells, centrifugation was performed at 1,000 g for 10 min, and the supernatant was filtered through a 0.2 μm syringe filter. The filtered medium (approximately 1,000 mL) was concentrated via ultrafiltration. The medium was placed in an Amicon stirred cell (400 mL capacity; Merck / Millipore, Burlington, MA, USA) containing a membrane disk (Ultracel 100 kDa) and concentrated under nitrogen gas pressure of 10 psi until the volume was reduced to approximately 40 mL or less. To replace the remaining medium, 20 mM Tris-HCl, pH 7.4, and 140 mM NaCl (TBS; 100 mL) were added and concentrated again. Exosomes were obtained when the final volume reached ≤18 mL. Further filtration through a 0.2 μm filter was performed and reconstituted with TBS containing trehalose (final 0.95%) and Proclin300 (final 1%) to a final volume of 18 mL. The exosomes were aliquoted, rapidly frozen, and stored at -80°C until use. The exosome concentration was measured by nanoparticle tracking analysis at Yoon Idea Lab Co. (Seoul, Korea).

[0201]

[0202] 3-2. Preparation of components for exosome isolation

[0203] Preparation of immunomagnetic beads. Tosyl-activated magnetic beads (11 mL at 30 mg / mL) were magnetically separated according to the manufacturer's protocol and washed with 100 mM phosphate buffer (pH 7.4, 100 mM PB; 48 mL). After removing the supernatant, anti-CBP antibody (clone 36F; 16.5 mL at 0.4 mg / mL) diluted in 100 mM PB and 3 M ammonium sulfate (11 mL) were sequentially added to the beads. The beads were incubated on a roll mixer at room temperature for 20 h to chemically bind the antibody to the bead surface. The beads were then washed three times with 100 mM PB, and the remaining surface was blocked for 1 h in a solution containing 10 mM phosphate buffer (pH 7.4), 140 mM NaCl (PBS), 0.5% casein, and 0.03% Proclin300 (Casein-PBS). After blocking, the beads were washed three times with Casein-PBS, magnetically separated from the solution, and reconstituted in Casein-PBS to a final concentration of 31.5 mg / mL. The beads were stored at 4°C until use.

[0204]

[0205] Polymerization of SA and CBP. CBP (1 mg / mL, 550 μL) was prepared in phosphate buffer (PBS), and SA (5 mg / mL, 490 μL) was prepared in 10 mM phosphate buffer (pH 7.4, 10 mM PB). The sulfhydryl groups of CBP were activated with DTT (final concentration 10 mM) diluted in 10 mM PB for 1 h at room temperature. Simultaneously, SA was activated by reaction with SMCC (≥5 molar) diluted in DMSO for 30 min at room temperature. The two protein components were combined and polymerized at room temperature for 2 h. After polymerization, the remaining functional groups were inactivated by sequential addition of L-cysteine ​​(final concentration 2.22 mM) and CaCl2 (final concentration 2 mM) diluted in deionized water. After reaction at room temperature for 1 h, CBP-SA polymer was prepared at a final concentration of 0.25 mg / mL. Casein (final 0.5%) and Proclin300 (final 0.03%) were added, aliquoted, rapidly frozen, and stored at -80°C until use.

[0206]

[0207] Biotinylation of antibodies specific for tetraspanins. Antibodies (0.5 mg / mL, 560 μL) specific for CD9 (clone HI9a), CD63 (clone H5C6), or CD81 (clone 5A6) in phosphate-buffered saline (PBS) were mixed with a 5 molar excess of NHS-LC-LC-C-biotin dissolved in DMOS and incubated at room temperature for 1 hour. Each biotinylated antibody was aliquoted and stored at 4°C until use. Casein (final concentration 0.05%) and Proclin 300 (final concentration 0.03%) were then added.

[0208]

[0209] 3-3. Exosome subpopulation separation in manual mode

[0210] Sample preparation. The sample used in this experiment was human male serum donated from cancer patients and healthy individuals, containing cell line exosomes mixed with sample binding buffer or Sigma serum. Sigma serum was used as a control to simulate clinical samples. Before isolation, the clinical sample was centrifuged at 1,200 g for 20 minutes at 4°C. After removing the precipitate, the solution was centrifuged again at 10,000 g for 1 hour at 4°C. The supernatant was filtered through a 0.2 μm filter and stored at 4°C for use within 1 hour. For long-term storage, it was rapidly frozen at -80°C.

[0211]

[0212] Preparation of magnetic beads immobilizing reversible binders. CBP-SA polymer (200 μL, concentration 1 μg / mL) was added to immunomagnetic beads (total concentration 0.15 mg / mL) and mixed in a shaker at room temperature for 1 h to bind (step 1; see (A) of Figure 24). After removing the supernatant by magnetic separation, the beads were washed with a washing solution. The washing solution was TBS (350 μL) containing 10 mM CaCl2, 0.1% Tween-20, and 0.03% ProClin300, vortexed for 15–20 s, and then magnetically separated (step 2). Biotinylated antibodies specific for tetraspanins (200 μL, concentration 1 μg / mL) were diluted in binding buffer TBS (containing 2% BSA, 10 mM CaCl2, 0.1% Tween-20, and 0.03% ProClin300), added to the beads bound to the CBP-SA polymer, and reacted by mixing on a shaker under the same conditions (step 3). The beads were washed, and the supernatant was removed by magnetic separation (step 4). A biotin solution (200 μL, concentration 2 μg / mL) diluted in binding buffer was added to the beads to saturate the remaining SA binding sites, and the beads were reacted on a shaker for 30 min (step 5). The beads were washed, and the supernatant was removed to obtain functional immunomagnetic beads for reversible exosome isolation (step 6).

[0213]

[0214] Exosome subpopulation isolation. 100 μL of sample binding buffer (TBS) (containing 2% BSA, 20 mM CaCl2, 0.2% Tween-20, 0.03% ProClin300, and 25% HAMA blocker) and the prepared exosome sample (100 μL) were sequentially added to functionalized immunomagnetic beads and incubated on a shaker for 2 h (step 7). The beads were washed with washing solution (500 μL), and the supernatant was removed by magnetic separation. This process was repeated twice (steps 8–10). This separated the target exosome subpopulation in a form captured by the magnetic beads.

[0215]

[0216] Exosome recovery. An elution solution (200 μL) containing 500 mM NaCl, 0.5% casein, 0.1% Tween-20, 0.03% ProClin300, 2 mM EDTA, and 0.01% phenol red in TBS was prepared. Exosome-bound beads were added to the elution solution and incubated on a shaker for 30 minutes (step 11). The supernatant was collected in a new tube by magnetic separation. The isolated exosomes were used immediately or stored by flash freezing at -80°C.

[0217]

[0218] 3-4. Exosome subpopulation separation in automated mode

[0219] Reagent dispensing. Exosome isolation was performed in automated mode using a sample purification system (Nextractor NX-32N; Genolution, Seoul, Korea). This system uses a 96-deep well plate. Six reagents were dispensed into wells in the same row, and exosome isolation was performed in the row direction (see Figure 24(B) and Figure 25(A)). In the first row, anti-CBP antibody-coated immunomagnetic beads (200 μL; concentration 0.75 mg / mL) premixed with CBP-SA polymer (final concentration 1 μg / mL) were added to each well. In the second row, biotinylated antibody for exosome isolation (200 μL; concentration 1 μg / mL) diluted in binding buffer was loaded. In the third column, a biotin solution (200 μL; concentration 2 μg / mL) prepared in binding buffer was loaded. In the fourth column, sample binding buffer (100 μL) was dispensed, followed by the previously pretreated samples (100 μL each). In the fifth column, a wash solution (1 mL) was added. In the sixth column, an elution solution (200 μL) was added.

[0220]

[0221] Exosome isolation procedure. A 96-deep well plate loaded with reagents was placed in the plate rack of a sample purification system (Nextractor NX-32N), and the magnetic rod bundle was covered with a cover strip (refer to Figure S5C) (see (B) and (C) of Figure 25). The exosome isolation procedure was performed at room temperature according to the system's preset program. Upon completion of the exosome isolation process from Steps 1 to 6, the 96-deep well plate was removed from the system, and each isolated exosome sample was transferred to a new tube. The isolated exosome subpopulations were immediately used for subsequent analysis or stored by rapid freezing at -80°C.

[0222]

[0223] 3-5. Exosome analysis

[0224] Preparation of antibody-coated plates. Anti-CD9 antibodies (2.5 μg / mL; clone HI9a), anti-CD81 antibodies (2.5 μg / mL; clone 5A6), and anti-CD63 antibodies (1.0 μg / mL; clone H5C6) were diluted in 100 mM carbonate buffer (pH 9.6, ELISA coating buffer). Each antibody solution (100 μL) was transferred to microwells and incubated at 4°C for 16 h. The antibody-coated microwells were washed three times with PBS containing 0.1% Tween-20 (300 μL per well) using a microplate washer (Wellwash™Versa Microplate Washer; Thermo Fisher Scientific, Rockford, IL, USA). The microwells were then filled with StabilCoat Immunoassay Stabilizer (200 μL per well) and incubated at room temperature for 2 h. After removing the stabilizer solution, the microwells were dried for 24 h in a clean room with less than 30% humidity, sealed in aluminum pouches, and stored at 4°C.

[0225]

[0226] Biotinylation of detection antibodies. Biotinylation was performed on the antibodies used for detection in the immunoassay: anti-CD9 antibody (1.0 mg / mL; clone 5A6), anti-CD63 antibody (1.0 mg / mL; clone H5C6), anti-CD81 antibody (1.0 mg / mL; clone 5A6), anti-CD151 antibody (1.0 mg / mL; clone 50-6), and anti-TSPAN8 antibody (1.0 mg / mL; clone TAL69). The buffer of each antibody solution (300 μL each) was replaced with PBS using a Zeba column. Each antibody was then conjugated with a 20 molar excess of NHS-LC-LC-biotin dissolved in DMSO and incubated in a vortex shaker at room temperature for 1 hour. After removing unbound biotin using a desalting column washed with PBS, the biotinylated antibody was stored in Casein-PBS containing 0.1% Tween-20 at 4°C until use.

[0227]

[0228] Immunoassay procedure. A standard sandwich immunoassay for various target exosome markers was performed by combining appropriate capture antibody-coated microwell plates with corresponding biotinylated detection antibodies. Analytical samples included cell line exosomes diluted in media, buffer, or Sigma serum, and clinical serum samples pretreated as described previously. Samples (100 μL) were added separately to the microwells and incubated on a shaker at room temperature for 3 hours. The wells were washed three times with 270 μL of PBS containing 0.1% Tween-20. Biotinylated detection antibodies (100 μL; concentration 0.1 μg / mL) diluted in TBS (dilution buffer) containing 0.5% casein, 0.1% Tween-20, and 1% ProClin300 were transferred to the wells and incubated on a shaker for 1 hour. After washing the wells three times, SA-poly HRP20 (100 μL; concentration 66.67 ng / mL) was added to each well and incubated for 1 h under the same conditions. The wells were washed again, and substrate solution (200 μL TMB-HK) was added for signal generation. After 15 min, the reaction was stopped by adding 0.5 M sulfuric acid solution (50 μL), and the signal was measured at 450 nm absorbance using a microplate reader (Synergy H4; BioTek Inc., Winooski, VT, USA).

[0229]

[0230] 3-6. Performance characterization procedure using exosome samples

[0231] Selection of clinical samples. To compare test variables associated with prostate cancer, serum samples were obtained from healthy male donors and male donors with various cancers, including prostate cancer, colon cancer, or malignant melanoma. All healthy samples were collected from Caucasian donors aged 40 years or older who tested negative for HIV, HBsAg, and HCV. Cancer samples were collected from Caucasian donors aged 52–80 years for prostate cancer, 43–69 years for colon cancer, and 50–81 years for malignant melanoma (except for one patient aged 35 years). All cancer samples were confirmed to have the corresponding diagnosis.

[0232]

[0233] Quantification of specific dual-marker-positive exosomes. To quantify dual-marker-positive exosomes, pretreated serum was diluted appropriately in dilution buffer to prevent optical density saturation. Samples were analyzed using a sandwich immunoassay using the correct combination of antibody-coated plates and biotinylated antibodies, as described above for the exosome immunoassay. Dilution buffer was used as a control sample and analyzed under identical conditions. All immunoassays were performed in duplicate, and the average of the optical density replicates for each sample was subtracted from the control to obtain the "D+T" value, which is used to determine the index of local heterogeneity.

[0234]

[0235] Quantification of exosomes containing a third marker. To quantify the third marker, "T," within a specific dual-marker-positive exosome subpopulation, "D+T," subpopulation samples were appropriately diluted with dilution buffer and subjected to a sandwich immunoassay using antibody-coated plates and biotinylated antibodies as capture and detection reagents. Other conditions were identical to those for quantifying dual-marker-positive exosomes.

[0236]

[0237] Calculation of indices for changes in local exosome heterogeneity. Each indices R or Rc for changes in local exosome heterogeneity of a third marker were determined by dividing the quantified value of the third marker, "T," by the quantified value of the dual marker-positive exosomes, "D+T." The reciprocal, 1 / R, was used for plotting to ensure significant digits.

[0238]

[0239] Scaling procedure. Potential errors arising from performing experiments on different dates were compensated for by scaling the test values ​​to the standard value. LNCaP exosomes were used as the standard sample and analyzed three times using the same method as the test sample. The average 1 / Rs was calculated for the standard, and scaling was performed by dividing the 1 / Rs of the test sample by the value of the standard sample. This scaling was applied only to measurements where pan-exosome tetraspanins were used as a third marker.

[0240]

[0241] Data plotting and performance characterization. The values ​​of a pair of indices, R and Rc, were plotted on the corresponding axes of an XY plot using Excel. Based on the sample distribution on the plot, the cutoff values ​​for each plot were used to determine false negative results and resolution. For performance comparison, data from different sample groups for a single parameter were plotted together using Excel's box-and-whisker plot function.

[0242]

[0243] Receiver operating characteristic (ROC) curve analysis. A preclinical test was conducted using CD151 as a third marker. This was performed only at stopover site 1 (an exosome subpopulation region that shares the dual markers CD9 and CD63) and included four groups of samples: 18 healthy individuals, 18 prostate cancer samples, 12 malignant melanoma samples, and 12 colon cancer samples. The paired indices R and Rc were measured as described above, and the data from the prostate cancer group (positive) were compared with the other three groups (negative), and the ROC curve was used to evaluate the discriminatory ability based on the parameter '1 / R + 1 / Rc'. Briefly, the entire data range was divided into 23 intervals, and the number of negative or positive samples in each interval was recorded. For each interval sorted in ascending order, the cumulative counts were calculated, and the false positive rate (FPR) and true positive rate (TPR) were determined. In a plot of '1-Specificity' (FPR) versus 'Sensitivity' (TPR), the area under the curve (AUC) value was calculated to assess the discrimination between the prostate cancer group and the non-prostate cancer group.

[0244]

[0245] 4. Results

[0246] 4-1. Definition of Terms

[0247] We developed Neutral Release, a novel immunomagnetic separation technology utilizing a reversible linker, which allows for the isolation of exosome subpopulations based on exosome surface markers, recovery of single marker-positive exosomes in a liquid phase, and use of the isolated exosomes as samples for sequential immunoisolation of other markers. The target markers are exosome surface markers such as pan-exosome tetraspanins (CD9, CD63, and CD81).

[0248]

[0249] FIG. 17 is a diagram illustrating a novel sequential marker selection process (A) using Neutral Release technology in contrast to conventional methods, and a conceptual representation (B) of the resulting new approach; FIG. 18 is a diagram illustrating the process of determining heterogeneity change indices (R1, R2 and R3) and the marker selection pathway; FIG. 19 is a diagram illustrating heterogeneity change patterns; and FIG. 20 is a diagram illustrating a sequential marker selection strategy for quantifying triple marker positive subclass 'T' when using three pan-exosome tetraspanin markers, namely CD9, CD63 and CD81. Figure 21 is a diagram illustrating the process of quantifying the size of the triple marker positive subpopulation (T), Figure 22 is a diagram illustrating the conventional sandwich immunoassay method for quantifying the size of the double marker positive subpopulation (D+T), Figure 23 is a table illustrating a comprehensive experimental plan for quantifying exosome heterogeneity changes, Figure 24 is a table comparing the conditions of manual exosome magnetic separation and automated exosome magnetic separation, Figure 25 is a diagram illustrating a liquid management system for automated exosome magnetic separation using Neutral Release technology, Figure 26 is a table illustrating the setting conditions for the operation of the automated exosome separation system, Figure 27 is a performance comparison result of automated exosome separation using Neutral Release protocol and manual separation, Figure 28 is a diagram illustrating the results of the performance comparison of automated exosome separation using Neutral Release protocol and manual separation after irradiation of serum with cell line RWPE-1 exosomes (1.28×1011 The results of quantifying the change in exosome heterogeneity for samples with various concentrations added (1.28×10 particles / mL). Figure 29 shows the change in exosome heterogeneity for RWPE-1 (1.28×10 11 particles / mL), LNCaP (1.18×10 11 particles / mL) and PC3 (2.53×10 11particles / mL), FIG. 30 is a graph showing the index of exosome heterogeneity change according to concentration for each exosome sample produced from various clinical samples, FIG. 31 is a graph showing the results of quantifying the heterogeneity change of dual marker-positive exosomes and triple marker-positive exosomes according to the stopover site (the region of the exosome subpopulation that has a specific dual marker in common), and showing the index of exosome heterogeneity change, FIG. 32 is a diagram illustrating a diagnostic algorithm based on local heterogeneity change showing the main factors that distinguish cancer samples from normal samples. Figure 33 is a diagram comparing the sample classification performance based on local heterogeneity change indices using different third markers (CD81, CD151, TSPAN8), Figure 34 is the results of evaluating CD151 as a third marker at various stopover sites, Figure 35 is the results of measuring heterogeneity change for CD151 at stopover site 1 (an exosome subpopulation region that has dual markers of CD9 and CD63 in common) using LNCaP exosome samples, Figure 36 is the results of characterizing the performance of CD151 at stopover site 1 for prostate cancer tracking, Figure 37 is a diagram illustrating a comprehensive demonstration of the potential use of specific stopover sites for cancer biomarker discovery, Figure 38 is the results of local heterogeneity change indices for prostate cancer samples and non-prostate cancer samples and ROC analysis for prostate cancer samples. Figure 39 is a table comparing the performance of three stopover sites in determining heterogeneity changes of the third marker, and Figure 40 is a graph showing the specificity of CD151 heterogeneity changes of stopover site 1 according to various tissues and cancer states.

[0250]

[0251] Dual-Marker Selection. Using Neutral Release technology, a subpopulation of exosomes labeled with a specific antigen A, such as CD9-positive (CD9+), can be immunomagnetically separated in a liquid phase. Subsequently, an immunoassay can be designed to quantify a triple-positive ('A+B+C+') exosome subpopulation using antibodies specific for capture marker B (e.g., CD63) and detection marker C (e.g., CD81) (see Figure 17(A)). If a hierarchical correlation exists between a specific exosome subpopulation and its corresponding marker, that relationship can quantify the composition of marker C in the 'A+B+' exosome subpopulation.

[0252]

[0253] Changes in exosome heterogeneity. During successive selections, exosome heterogeneity within a sample gradually changes. This change depends on the markers selected and the order in which they are selected. This change in heterogeneity can be influenced by exosome properties determined by their cellular origin (see Figure 17B). The parameters that describe this heterogeneity change can be complex but can be quantitatively expressed using detection antibodies directed against a third marker, such as marker C present in 'A+B+' exosomes. This local heterogeneity change can be defined as the ratio of the signal from the 'A+B+C+' subpopulation to the signal from the 'A+B+' subpopulation within a subpopulation. If this change is associated with the origin of the target exosomes, it can be used to diagnose diseases characterized by changes in cellular properties.

[0254] Here, the local heterogeneity of the tested samples can be quantified using the index R. This index R is defined as the size of the triple marker-positive subpopulation after the third selection divided by the size of the double marker-positive subpopulation after the second selection. Here, the subpopulation sizes can be determined by measuring the immunoassay signals for the triple (T) and double (D+T) marker-positive subpopulations, respectively. If the selection order rotates clockwise among the three markers (A, B, and C), the three indices (R1, R2, and R3) can be experimentally determined (see Figure 18). The sizes of the triple marker-positive subpopulations quantified separately may differ. This discrepancy stems from the use of different sandwich immunoassays that utilize different capture and detection antibody pairs. These different assay conditions are indicated by the prime (') and double prime (“) symbols.

[0255] Measuring different R values ​​for a sample allows monitoring the properties collectively determined by the three markers expressed in individual exosomes. As described above, the dual-marker-positive exosome subpopulation serves as the basis for the marker composition and allows for the measurement of a third marker present in the same exosomes. The composition ratio of this third marker may vary from sample to sample, depending on the characteristics of the exosomes (see Figure 19). Under normal conditions, exosomes produced by individual cells can be assumed to have a uniform surface marker composition. This assumption is based on observations of exosome heterogeneity in body fluids, which arises from the mixing of numerous exosome populations secreted from different cells and tissues.

[0256] First, we developed an experimental system to test the hypothesis of local heterogeneity changes between exosome samples. Our goal was to determine whether heterogeneity change indices (R indices) are uniquely associated with each exosome sample derived from a specific cell line and can clearly characterize each cell. We then applied this concept to more complex clinical samples, such as serum from healthy individuals and cancer patients, to evaluate the effectiveness of a liquid biopsy approach based on local heterogeneity changes for cancer diagnosis. Furthermore, we tested the same concept to confirm its potential use in multiple cancer screening.

[0257]

[0258] 4-2. Quantification of changes in local exosome heterogeneity

[0259] Exosome selection strategy. Quantification of 'T' can be planned in multiple paths through three different exosome subpopulation regions, designated as stopover sites, 'D1+T', 'D2+T', and 'D3+T' (see Figure 20 (A)). These triple markers selected for the experiment are the pan-exosome tetraspanins CD9, CD63, and CD81, corresponding to markers A, B, and C, respectively. The initial selection marker passing through each stopover site can be determined by selecting any one of two different markers, excluding the final marker. For example, if 'D1+T' is determined as the stopover site, the initial selection marker should be either CD9 or CD63, and the selection path ends at CD81. In general, the quantification of 'T' can be designed to be approached either clockwise (defined as path 1) or counterclockwise (defined as path 2) of Fig. 20.

[0260] Additionally, as illustrated in (B) of Fig. 20, the degree of heterogeneity change of a specific sample is determined based on the initial marker selection (indicated as 'scheme') and subsequent marker arrangement (indicated as 'route'). According to a preset method, six heterogeneity change indices, such as R1 to R3 and R1c to R3c (where 'c' indicates a counterclockwise direction), can be determined to quantify the degree of exosome heterogeneity change.

[0261]

[0262] Experimental approach. As described above, to determine the sizes of the 'T' and 'D+T' subpopulations, we designed immunoisolation and immunoassays to sequentially select triple-pan exosomal tetraspanin markers and dual exosomal markers (see Figures 21 and 22). The heterogeneity index was ultimately determined by rotating the three markers, CD9, CD63, and CD81, clockwise or counterclockwise (see Figure 23 for the comprehensive experimental plan).

[0263] To determine the size of 'T', we designed an immunoisolation and immunoassay protocol that sequentially selects triple-pan exosomal tetraspanin markers (CD9, CD63, and CD81) (see Figures 17 and 21). Neutral Release was used in the first selection step to isolate exosomes based on the first target marker A (e.g., CD9). After separation, the exosome subpopulation in solution (e.g., CD9+ exosomes) was used as a sample for the second selection. The second selection was performed in microtiter plate wells with capture antibodies specific for marker B (e.g., CD63) immobilized on their inner surface. Here, the captured exosomes contained two target markers (e.g., CD9+CD63+), and analysis was performed for marker C (e.g., CD81) using a detection antibody linked to a tracer such as a signal-generating enzyme.

[0264] Next, the size of 'D+T' was quantified by immunoblotting for the first two of the pan-exosome tetraspanin markers (see Figure 22). The first marker (e.g., CD9) and the second marker (e.g., CD63) were selected by capture and detection antibodies, respectively, in microtiter plate wells. The heterogeneity change index R1 was determined as the size of 'T' divided by 'D1+T'. Similarly, the other indices R2 and R3 were measured by rotating the three markers clockwise (see Figure 20(A) and Figure 23).

[0265] Using Neutral Release technology, exosomes were recovered intact and efficiently by isolating exosome subpopulations in a liquid phase. However, the manual magnetic separation process involves 11 complex and labor-intensive steps (see Figure 24(A)), which can lead to problems in yield and subsequent immunoassays, especially when processing large volumes of samples, such as in clinical trials. To address these issues, an automated magnetic separation system was introduced. Here, the Neutral Release protocol was simplified to a total of six steps (see Figure 24(B) and Figure 25). Nevertheless, the performance of the automated separation system was nearly equivalent, as presented in the "Separation Performance Comparison" section below.

[0266]

[0267] Automation of the 'Neutral Release' process. An automated magnetic separation system was introduced based on the Neutral Release protocol (see Figure 25 (A)). For accurate and consistent exosome isolation, a commercial device (Nextractor NX-32N; Genolution; Seoul, Korea) was used (see Figures 25 (B) and 26). This device can process up to 32 exosome samples and isolate each subpopulation in a liquid phase. As described above, the exosome subpopulations were used in subsequent sandwich immunoassays to select the second and third markers (see Figure 21).

[0268]

[0269] Comparison of separation performance. We tested the performance of automated exosome separation using a six-step Neutral Release protocol and compared it with an 11-step manual separation. To this end, test samples (10 μg PC-3 exosomes / mL Sigma serum) were prepared by spiking exosomes from PC-3 cell lines in Sigma serum. Typically, during the isolation of the CD63+ exosome subpopulation, the exosome sample was reacted with anti-CD63 antibodies releasably attached to magnetic beads (see step IV in Figure 25(A)). Unbound exosome samples were then collected for subsequent exosome binding analysis. A portion of the collected exosomes was simultaneously analyzed with the original sample using a homogeneous sandwich immunoassay using anti-CD63 antibodies for capture and detection. Each experiment was repeated five times under each separation condition. The ratio of bound exosomes was calculated as a percentage (represented by the dotted line) from the signal for the unbound sample compared to the original sample (see the bar chart in (A) of Figure 27). The bound fraction under automated separation conditions (94.3–95.2%) was almost identical to that under manual conditions (93.2–95.3%).

[0270] After separation, the exosome subpopulation recovered in the liquid phase was used as a sample for downstream immunoassays using a capture antibody for CD9 and a detection antibody for CD81 (see Figure 27 (B)). The average results of five replicate experiments show that automated separation, despite requiring significantly fewer steps, did not affect the exosome recovery rate compared to manual separation.

[0271]

[0272] Quantification of heterogeneity changes. To validate the exosome heterogeneity change model (see Figure 17), experiments were conducted using exosomes produced from the benign prostate epithelial cell line RWPE-1. To mimic diverse clinical specimens, RWPE-1 exosomes were added to Sigma serum pools at various ratios. These ratios represent large population samples collected at various stages of disease progression. As depicted in Figure 20, according to the 'scheme' for marker selection, the bulk exosome sample was separated once and twice to produce exosome subpopulations and their subpopulations, respectively. Next, the sizes of each exosome population, 'D+T' and 'T', were measured and quantified using pre-designated detection markers (see Figure 28 (A) and (B)). The results showed that the signal level generally exhibited a response pattern proportional to the RWPE-1 exosome dose.

[0273] The two signals representing the sizes of the subgroups of 'D+T' and 'T' were used to determine the heterogeneity change indices R1 to R3 and R1c to R3c, respectively, as shown in Fig. 23. The results were plotted using the inverse of the R and Rc indices (see (C) in Fig. 28). The plot showed that two pairs of indices based on the same stopover site (see (A) in Fig. 20), for example, 1 / R1 and 1 / R1c, showed similar response levels and patterns. The three pairs of indices for each stopover site showed significantly different patterns with respect to response and distribution. This indicates that the heterogeneity change is dependent on the stopover site.

[0274]

[0275] Tracking the Cellular Origin of Exosomes. To trace the cellular origin of exosomes based on changes in exosome heterogeneity, we conducted experiments analyzing exosome samples collected from various cell lines associated with cancer characteristics. As a control, we produced exosomes from the normal prostate cell line RWPE-1 and two different prostate cancer cell lines, LNCaP and PC3. These exosomes were added to serum at various dilutions to prepare bulk exosome samples. Following the experimental design shown in Figure 23, a total of six indices, R1 to R3 and R1c to R3c, were calculated for each exosome sample.

[0276] For exosomes from each cell line, the values ​​of the 1 / R and 1 / Rc indices were plotted pairwise against exosome concentration (see Figure 29). When comparing the three pairs of indices across different cell lines, the third pair, 1 / R3 and 1 / R3c, showed a clear difference in concentration response. Compared to RWPE-1 exosomes (see Figure 29 (A)), the response to LNCaP exosomes was significantly increased (see Figure 29 (B)), while the response to PC3 exosomes was significantly decreased (see Figure 29 (C)). This indicates that local heterogeneity changes at stopover site 3 are sensitive to exosome source. In contrast, the changes in the first and second pairs of indices across different cell lines were minimal. Overall, the local exosome heterogeneity change index clearly distinguishes cell origin with respect to cancer characteristics, suggesting the potential for further clinical sample testing for cancer cell tracking.

[0277]

[0278] 4-3. Clinical Application: Cancer Cell Tracking

[0279] Performance control factors. We investigated whether heterogeneity change indices, R and Rc, were useful in distinguishing clinical sample groups between healthy individuals and patients with various cancers. Specifically, we tested each scheme, focusing on route dependency, which determines the clockwise (route 1) or counterclockwise (route 2) direction of approach to 'T' (see Figure 20). We tested and compared the performance of the two routes in classifying the two different sample groups. Twenty-four samples (six per group) from healthy individuals and patients with various cancers (prostate cancer, malignant melanoma, or colon cancer) were analyzed according to the aforementioned protocol to determine the 1 / R and 1 / Rc indices. Additionally, we used LNCaP exosomes as a standard sample to adjust for inter-experimental variation through scaling.

[0280] For each route, three XY plots were generated using two of the three heterogeneity indices, respectively. A single cutoff line was set on each axis to represent a specificity of 83.3% (correctly diagnosing five out of six healthy samples) in each plot (see (A) and (B) in Fig. 30). Under these conditions, the resolution for 18 positive samples (six samples for each cancer type) was determined for each heterogeneity indices. For route 1, the order of good resolution was 1 / R3 > 1 / R1 = 1 / R2, and for route 2, the order of good resolution was 1 / R3c > 1 / R2c > 1 / R1c, and the false negative (FN) rate was significantly lower for route 2 than for route 1 (see the FN values ​​indicated in each plot). For each route, the indicators R3 or R3c consistently showed the best resolution when CD63 was measured as a third marker within the CD9+CD81+ subpopulation as a stopover site. To confirm the dependence on the stopover site, we further rearranged the data collected along each route to examine the classification performance of the indicators for the stopover site (see Figure 30(C)). As expected, the best performance (only 1 FN out of 18 positive samples) was observed at stopover site 3, which used CD63 as the third marker and was based on the CD9+CD81+ subpopulation. These results allowed us to determine the parameters controlling the performance as follows: 1) the direction of approach to the third marker: route 2 > route 1, and 2) the stopover site as the entry point to the marker: site 3 > site 2 > site 1. Here, the resolution was consistent regardless of the cancer type.

[0281]

[0282] Performance Evaluation of Stopover Sites. To evaluate the classification performance of the heterogeneity index, we used a single cancer sample, focusing on prostate cancer, to verify the role of stopover sites. Clinical samples (12 serum samples each from healthy individuals and prostate cancer patients) were tested, and the results were plotted for each stopover site (see Figure 31). For each stopover site, a sandwich immunoassay was performed, sequentially reacting specific capture and detection antibodies to quantify the dual marker region, [D+T]. The antibody pairs were then reacted in reverse order to determine [D+T]c. These quantified values ​​were plotted on a graph ('Doubles'; see Figure 31 (A)). For the third marker, the same stopover site, 'D+T' was approached clockwise ([T]) or counterclockwise ([T]c) to quantify the marker ('Triples'; see Figure 31 (B)). Finally, the heterogeneity index was calculated as the ratio of the values ​​quantified above ('[D+T]' / 'T'), and scaled using LNCaP exosomes as a standard sample as above, and then 1 / R for each stopover site. Scaled and 1 / Rc, Scaled Plotted for indicator pairs ('Doubles / Triples (1 / R)'; see (C) of Fig. 31).

[0283] Looking at the 'Doubles' plot in (A) of Figure 31, it shows a nearly linear distribution of samples regardless of the stopover site, which means that the existing sandwich immunoassay measuring 'D+T' cannot distinguish between different exosome-derived cells. However, the 'Triples' plot in (B) of Figure 31 shows different distributions for each sample depending on the three stopover sites. While the samples are linearly distributed for stopover site 1, they are relatively distributed above or below the diagonal line for stopover sites 2 and 3.

[0284] Additionally, for each clinical sample, we computed scaled exosome heterogeneity change indices 1 / R and 1 / Rc relative to the LNCaP exosome sample and compared those indices (e.g., 1 / R1, Scaled Contrast 1 / R1c, Scaled ) were plotted (see (C) of Fig. 31). As a result, regardless of the stopover site used, most normal samples (9 to 10 out of 12 samples) formed clusters, and cancer samples were distributed away from or around the normal clusters, showing 100% sensitivity. The distribution direction of cancer samples with respect to the normal sample clusters showed uniquely different classification patterns depending on the stopover site.

[0285]

[0286] Key factors in sample classification. The linearly correlated subgroup [D+T] vs. [D+T]c plot is not suitable for classifying sample groups, suggesting that subgroup marker concentrations alone are not sufficient for classifying sample groups (see (A) in Figure 32). On the other hand, the ratio of the third marker-positive subgroup 'T' to the dual marker-positive subgroup 'D+T' may be an important factor in sample classification (see (B) in Figure 32). This suggests that the ratio of the third marker may change with disease onset, i.e., that local heterogeneity may vary. Therefore, it can be seen that the subgroup containing the third marker itself may act as a disease biomarker.

[0287]

[0288] 4-4. Expanding the concept of specific cancer detection

[0289] Additional subpopulation markers. As shown in Figures 30 and 31, 1 / R3c appears to be the optimal single index for detecting various cancers. However, when a sample is tested positive, it can be difficult to identify the specific cancer that actually occurred. To address this issue, we explored additional subpopulation markers that could represent a single cancer, such as prostate cancer. Various exosomal markers have the potential to distinguish between cancer and healthy samples.

[0290] In preliminary experiments, several markers were examined, including CD151, CD82, Tetraspanin 8 (TSPAN8), CD147, and EpCAM. Among these candidates, CD151 and TSPAN8 emerged as suitable third markers detectable in the 'D+T' region by immunoassay (see Figure 33).

[0291] In the local heterogeneity change plot for CD81 as a third marker, the cutoff line showed a characteristic pattern, in which the local changes in cancer samples increased or decreased along both the X and Y axes compared to the normal samples (see (A) in Figure 33). Due to this characteristic pattern of heterogeneity change, the normal samples formed a distinct cluster in a limited area, while the prostate cancer samples were distributed scattered outside the cluster boundaries. Consequently, a double-crossing cutoff line was set on the graph to distinguish the two sample groups. In contrast, in the local change plots for the other two markers, CD151 and TSPAN8, the samples from different groups were distributed in a diagonal shape in each graph (see (B) and (C) in Figure 33). In such cases, the two sample groups were separated by a single-crossing cutoff line, as is typically observed. In the heterogeneity change, such changes may indicate unique differences in the local composition of the third marker based on each designated subpopulation, which may provide insights into the onset or progression of the disease.

[0292] CD81, a target marker used to measure local heterogeneity, is present on the surface of exosomes. The presence of CD81+ exosomes in the tumor microenvironment is associated with several aspects of cancer progression, including tumor growth, angiogenesis, and invasion, and mediates communication between cancer cells and surrounding stromal cells. CD151 and TSPAN8, transmembrane proteins of the tetraspanin family, are components of exosomes and facilitate interactions between cancer-initiating cells and their surroundings. CD151 has been noted for its potential role in cancer progression and metastasis. TSPAN8 has also been reported to be highly expressed in prostate cancer samples compared to control samples. The role of each cancer-related marker can be further supported by the results of the present analysis, which is based on local heterogeneity changes of each marker.

[0293] Preliminary experimental results showed that CD151 as a third marker not only performed as well as CD81 at stopover site 1, but also provided a simpler classification pattern than CD81 (compare (A) and (B) in Figure 33). The same test targeting CD151 at each of the three stopover sites was performed on different clinical samples under the same conditions (not scaled for comparison; see (A) in Figure 34). Two groups of samples (12 samples in each group) from healthy individuals and prostate cancer patients were satisfactorily distinguished using stopover site 1, resulting in a resolution of 87.5% (21 out of 24 samples were distinguished based on the cutoff line). However, stopover sites 2 and 3 were difficult to utilize for their intended purpose due to the extremely low signal in the immunoassay used to detect CD151 (see raw data with an optical density (OD) of less than 0.1 in Figure 34 (B)). Note that LNCaP-based scaling was not applied when determining CD151 heterogeneity changes because the LNCaP standard signal was highly volatile due to low signals of OD <0.1 (see Figure 35).

[0294]

[0295] Preclinical Experiments. A preclinical experiment was conducted for CD151 as a third marker at stopover site 1 using groups of healthy subjects and prostate cancer samples (18 samples each). Samples were isolated and analyzed under identical conditions. The cutoff lines obtained in the preliminary experiments were plotted and the resolution was evaluated (see Figures 36(A) and 37(A)). Here, a resolution of 86.1% was achieved, distinguishing 31 of the 36 samples, demonstrating consistent performance of CD151 as a third marker at the pre-designated stopover site.

[0296] To evaluate the tracking ability of CD151 as a third marker for various cancer cells, two additional groups of cancer samples (12 samples each) were acquired from patients with malignant melanoma and colon cancer. The 1 / R and 1 / Rc indices measured for the additional cancer samples were plotted together with the prostate cancer data (see also Figure 36(B) and Figure 37(A)). Here, both additional cancer groups were distributed in the same area as the normal group, indicating that they could be distinguished from prostate cancer samples. The resolution between malignant melanoma and prostate cancer was 90% (27 out of 30 samples distinguished), and between colon cancer and prostate cancer was 83.3% (25 out of 30 samples distinguished). By simultaneously comparing the data from the other sample groups for the parameter combining the two indices, i.e., '1 / R + 1 / R1c', we confirmed that prostate cancer was detected in a specific manner (see Figure 36(C) and Figure 37(A)). The analysis of the receiver operating characteristic curve showed that the area under the curve (AUC) was 0.880, indicating that prostate cancer samples could be strongly distinguished from normal samples as well as other cancer samples (see Figure 38).

[0297]

[0298] 5. Discussion

[0299] The present invention proposes a novel approach to measure local heterogeneity of a third marker within a specific exosome subpopulation, utilizing the concept of a stopover site (an exosome subpopulation region that shares a specific double marker). This approach provides an alternative algorithm for liquid biopsy (see Figure 32). As shown in Figure 31(C), the heterogeneity within the normal group samples is significantly less than that within the cancer group. This indicates a stable exosome composition in healthy samples. In contrast, the high heterogeneity observed in the cancer group reflects either the production of new exosomes from cancer cells or an active immune response.

[0300] Diagnostic approaches based on changes in local exosome heterogeneity differ from conventional methods that focus on target marker concentration. These methods can result in significant variations in target marker concentration due to substantial heterogeneity within bulk exosomes. Sequential exosome separation at the subpopulation level can mitigate these variations, making it a practical approach for discovering biomarkers for cancer diagnosis. Tetraspanins, used as separation markers, play a role in distinguishing exosome subpopulations in human serum and plasma. Therefore, controlling exosome heterogeneity through tetraspanin-based separation is essential for utilizing exosomes as biomarkers for early cancer detection.

[0301] Stopover sites, defined as pan-exosomal tetraspanins such as CD9, CD63, and CD81, serve as entry points for mutation detection. At the subpopulation level, various combinations of these markers are generally known to influence tumor growth, angiogenesis, and invasion, but may vary depending on the cancer type. Despite the somewhat complex nature of defining the role of stopover sites in terms of local heterogeneity, the present invention has revealed that stopover sites possess unique properties that enable the identification of cancer-specific patterns (see Figures 37 and 39).

[0302] The main characteristics of the stopover site according to the present invention and the diagnostic performance using the same are as follows. First, at stopover site 2 (CD63+CD81+), using CD9 as a third marker and using a cutoff parameter based on the ratio of the two 1 / R2 and 1 / R2c indices showed excellent performance in distinguishing prostate cancer and colon cancer from normal samples, with a resolution of 93.3% (28 out of 30 samples; see (B) of Figure 37). However, this method could not distinguish malignant melanoma samples from normal samples. At stopover site 3 (CD9+CD81+), using a single indices, 1 / R3c, prostate and malignant melanoma samples could be distinguished from normal samples, with a resolution of 93.3% (28 out of 30 samples; see (C) of Figure 37). However, colon cancer could not be selectively detected. Therefore, the lack of selectivity at individual points can be compensated for by cross-checking the results at the two stopover sites.

[0303] At stopover site 1 (CD9+CD63+), pan-exosomal tetraspanins alone were insufficient for selective detection of cancer samples due to the highly variable direction of sample distribution (see Figure 30(C)). However, certain other cancer markers showed promise for specific cancer detection. In particular, CD151 was evaluated as a valid third marker that could distinguish prostate cancer samples from colon cancer, malignant melanoma, and healthy controls (see Figure 37(A)). By combining the two indices along each axis of the plot and using them as a single cutoff parameter, a resolution of up to 88.3% (53 out of 60 samples) for prostate cancer was achieved.

[0304] CD151, a tetraspanin, has been associated with various cancers, including prostate cancer. CD151 expression is elevated in prostate cancer cells compared to normal prostate tissue. Elevated CD151 levels are associated with more aggressive forms of prostate cancer and poorer clinical outcomes. Initially considered a biomarker for prostate cancer aggressiveness and metastatic potential, recent studies suggest that CD151 may act as an inhibitor of prostate cancer progression. One possible explanation for this discrepancy lies in the dependence of CD151 subpopulations and their functions. This hypothesis explains why CD151, as a third marker within the CD9+CD63+ subpopulation, selectively differentiates prostate cancer from other cancers (see Figure 37 (A)). Furthermore, it suggests that markers such as CD151 may differentially influence tumor growth and invasion in different cancer types, depending on specific subpopulation interactions.

[0305] To support the above hypothesis, we tested whether CD151 up-regulation at stopover site 1, i.e., the CD9+CD63+ subpopulation, was prostate tissue-specific and associated with tumorigenesis. We found that CD151 heterogeneity at stopover site 1, as measured by the parameters 1 / R1 and 1 / R1c, and particularly 1 / R1c, increased proportionally with the concentration of exosomes extracted from a normal prostate tissue-derived cell line (RWPE-1) (see Figure 40(A)). Furthermore, this heterogeneity was significantly amplified when exosomes from a prostate cancer cell line (LNCaP) were used as a sample. LNCaP is a human prostate cancer cell line derived from a lymph node metastatic lesion that expresses a mutated androgen receptor, indicating that prostate tissue characteristics are maintained. In contrast, this increase was completely absent when measured in exosomes from PC3, a human prostate cancer cell line derived from a bone metastasis. Unlike LNCaP cells, PC3 cells do not express androgen receptor or prostate-specific antigen (PSA), indicating a loss of typical prostate tissue characteristics. These results indicate that the 1 / R1c index is particularly responsive to exosomes derived from prostate tissue, and is even more responsive to cancer development within that tissue. However, this response can be completely lost when metastases occur in other tissues.

[0306] Next, we tested the 1 / R1 index response to exosomes derived from tissues other than prostate, such as kidney and colon, regardless of cancer presence. The response to exosomes from tissues such as embryonic kidney (HEK293) and cancerous colon (HT29) was found to be inversely dose-dependent (see Figure 40 (B)). HT29 is a human colon adenocarcinoma cell line derived from a colon cancer patient. However, while a somewhat dose-dependent response was observed with exosomes from HCT116, the concentration-response pattern was not as consistent as that of RWPE-1. HCT116 is a human colon cancer cell line commonly used as a model for aggressive colon cancer due to its high tumorigenic potential.

[0307] The association between prostate cancer and CD9- and CD63-positive exosome subpopulations has been extensively investigated. CD9 and CD63 are tetraspanins found on the surface of exosomes, each associated with unique aspects of prostate cancer biology. CD9- and CD63-positive exosomes are elevated in the circulation and body fluids of prostate cancer patients compared to healthy individuals, suggesting their involvement in cancer progression and metastasis. While there are no direct reports of interactions between CD9+CD63+ exosomes and CD151, understanding the individual roles of these markers will likely elucidate their potential interactions and their broader impact on disease progression.

[0308] By measuring the composition of a third marker within specific exosome subpopulations, an innovative cancer diagnostic algorithm can be introduced that focuses on changes in local exosome heterogeneity. The pan-exosome tetraspanins CD9, CD63, and CD81 are used for sequential isolation, forming the basis for quantifying unique target markers within these subpopulations. At the subpopulation level, different combinations of these three markers exhibit different selectivity for cancer, despite their common association with tumor growth, angiogenesis, and invasion (see Figure 39). For example, stopover sites containing CD81 could serve as entry points for mutation detection, while the third marker could indicate tissue type or immune response mechanisms (see Figures 37 (B) and (C)). Consequently, exosome stopover sites, i.e., specific exosome subpopulations, can be used as biomarkers.

[0309]

[0310] 6. Conclusion

[0311] The present invention provides a novel and reproducible cancer detection method that monitors localized heterogeneity changes at the subpopulation level, focusing on exosome surface protein markers in bodily fluid samples such as serum. The invention is based on the idea that cytopathic effects, such as those due to cancer, lead to changes in exosome composition compared to normal conditions. These changes can be quantified using indicators derived from a third marker within a specific exosome subpopulation. The invention utilizes a "stopover site" defined by a combination of pan-exosomal tetraspanins, such as CD9, CD63, and CD81, which serves as an entry point for analyzing mutation patterns and identifying cancer-specific markers. Because specific tetraspanin combinations offer varying levels of selectivity for different tumors, the diagnostic method according to the invention holds great promise. This approach, which focuses on subpopulation heterogeneity, could represent an innovative alternative to conventional liquid biopsy techniques and could be of significant use in early cancer screening. The present invention can be utilized to improve the accuracy of cancer diagnosis by focusing on specific target cancers and tissues. While initially focused on prostate cancer, the invention can be broadly applied to various types of cancer. This innovative model will provide a versatile diagnostic technology for various cancer types while effectively addressing the complexities of heterogeneity, thereby bringing about a paradigm shift in early cancer diagnosis.

[0312]

[0313] Although the present invention has been described in detail through specific examples and experimental examples, this is intended to specifically explain the present invention, and the present invention is not limited thereto, and it is clear that modifications and improvements can be made by those skilled in the art within the technical spirit of the present invention.

[0314] All simple modifications or changes of the present invention fall within the scope of the present invention, and the specific scope of protection of the present invention will be made clear by the appended claims.

[0315] The present invention is a liquid biopsy method that is capable of producing a liquid biopsy sample by isolating and recovering an exosome subpopulation, a hypopopulation, or a lower subpopulation for a separation marker from an exosome population with high yield and in its original state, and analyzing exosomes loaded with the same triple marker using two surface markers other than the separation marker as capture and detection markers in the liquid biopsy sample, and is recognized as having industrial applicability.

Claims

1. (a) A step of preparing an exosome liquid biopsy sample comprising the exosome subpopulation by isolating and recovering a first target exosome having a first surface marker in common from an exosome population sample containing heterogeneous exosomes secreted from multiple cells in a human body; (b) a step of obtaining a triple marker signal by measuring a quantitative signal for a third surface marker of a second target exosome that belongs to the exosome subgroup and has a second surface marker in common; (c) a step of calculating a local exosome heterogeneity index as a ratio of the measured values ​​of the dual marker signal and the triple marker signal obtained by measuring the quantitative signal for the second surface marker of the first target exosome; and (d) a liquid biopsy comprising a step of analyzing the exosome liquid biopsy sample based on the produced exosome heterogeneity index; 2. In claim 1, By sequentially performing steps (a) to (c) for the exosome population samples of each of the normal group and the patient group with a specific disease, the exosome heterogeneity index for the normal group and the exosome heterogeneity index for the patient group are calculated, respectively. In the above step (d), a liquid biopsy is performed to select a biomarker related to a specific disease of the patient group by comparing the exosome heterogeneity index for the normal group and the exosome heterogeneity index for the patient group, which are respectively calculated.

3. In claim 2, By repeatedly performing steps (a) to (c) performed for selecting the biomarker for the exosome population sample of the disease screening subject, the exosome heterogeneity index for the disease screening subject is calculated, In the step (d) above, a liquid biopsy that generates disease screening information for the disease screening subject by comparing the exosome heterogeneity index for the disease screening subject and the exosome heterogeneity index for the patient group.

4. In claim 1, Among the three surface markers of the heterologous exosomes in the exosome population sample, at least one of a total of six selection paths is selected, in which one is selected as the first surface marker, another is selected as the second surface marker, and the remaining one is selected as the third surface marker, and steps (a) to (c) are sequentially repeated, Liquid biopsy that produces N (where N is a natural number greater than or equal to 1 and less than or equal to 6) exosome heterogeneity indices.

5. In claim 4, For each of the above exosome population samples of the normal group and the patient group with a specific disease, N exosome heterogeneity indices for the normal group and N exosome heterogeneity indices for the patient group are calculated, respectively. In the step (d) above, a liquid biopsy for selecting a selection path of a biomarker related to a specific disease of the patient group based on the N exosome heterogeneity indices for the normal group and the N exosome heterogeneity indices for the patient group, respectively.

6. In claim 5, In the step (d), a liquid biopsy that generates new indices through arithmetic operations or functionalization of N exosome heterogeneity indices for the normal group and N exosome heterogeneity indices for the patient group, and selects a selection path of a biomarker related to a specific disease of the patient group based on the generated new indices.

7. In claim 5, For the exosome population sample of the disease screening subject, the steps (a) to (c) are repeatedly performed according to the selection path performed for the selection path of the biomarker to produce the exosome heterogeneity index for the disease screening subject, A liquid biopsy that generates disease screening information for the subject of the disease screening by comparing the exosome heterogeneity index for the subject of the disease screening calculated in step (d) with the exosome heterogeneity index for the patient group calculated according to the selection path performed for the selection path of the biomarker.

8. In claim 6, For the exosome population sample of the disease screening subject, the steps (a) to (c) are repeatedly performed according to the selection path performed for the selection path of the biomarker to produce the exosome heterogeneity index for the disease screening subject, A liquid biopsy that generates disease screening information for the subject of the disease screening by comparing the new indicator for the subject of the disease screening produced in step (d) with the new indicator for the patient group produced according to the selection path performed for the selection path of the biomarker.

9. In claim 1, Step (a) above, A step of binding a first specific binding material that specifically binds to the first surface marker to a solid state fixing member using a first reversible linker; A step of reacting the exosome population sample with the first specific binding material bound to the fixing member to capture a plurality of the first target exosomes, thereby isolating an exosome subpopulation, which is a group of the first target exosomes; and A liquid biopsy comprising a step of dissociating the first reversible linker so that the captured first target exosomes are separated from the fixing member, thereby recovering the exosome subpopulation.

10. In claim 9, Between the step of isolating the exosome subpopulation and the step of recovering the exosome subpopulation, A liquid biopsy further comprising a step of concentrating the fixing member in which the first target exosome is captured using at least one of magnetic force, gravity, and centrifugal force.

11. In claim 9, Step (a) above, A step of binding a second specific binding material that specifically binds to the second surface marker of the second target exosome within the exosome subpopulation to the fixing member; and A liquid biopsy comprising a step of reacting an exosome subpopulation sample containing the recovered exosome subpopulation with the second specific binding material bound to the fixing member to capture a plurality of the second target exosomes, thereby isolating an exosome subpopulation, which is a population of the second target exosomes.

12. In claim 11, In the step of binding the second specific binding material to the fixing member, the second specific binding material is bound to the fixing member by a second reversible linker, A liquid biopsy further comprising a step of dissociating the second reversible linker to separate the captured second target exosomes from the fixing member, thereby recovering the exosome subpopulation.

13. In claim 12, Between the step of isolating the exosome subpopulation and the step of recovering the exosome subpopulation, A liquid biopsy further comprising a step of concentrating the fixing member in which the second target exosome is captured using at least one of magnetic force, gravity, and centrifugal force.

14. In claim 1, The above double marker signal is, Liquid biopsy measured on the above exosome population sample.

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