KR102498862B1 - Method for analyzing exosomes in liquid biopsy sample - Google Patents

Abstract

The present invention relates to a method for analyzing an exosome liquid biopsy sample, and the method for analyzing an exosome liquid biopsy sample according to an embodiment of the present invention includes exosomes containing exosomes secreted from a plurality of cells in the body of each of a normal group and a patient group. Preparing an exosome liquid biopsy sample containing the target exosome population by separating target exosomes related to a predetermined disease into subpopulations from the population sample (S100), target contained in the exosome liquid biopsy sample Measuring a quantitative signal for each of a plurality of biomarkers of the exosome (S200), and based on the signal value of the measured quantitative signal, according to the correlation between the plurality of biomarkers, exosomes of each of the normal group and the patient group and analyzing the liquid biopsy sample (S300).

Description

Exosome liquid biopsy sample analysis method {METHOD FOR ANALYZING EXOSOMES IN LIQUID BIOPSY SAMPLE}

The present invention relates to a method for analyzing an exosome liquid biopsy sample, and relates to a method for analyzing a liquid biopsy sample prepared by separating exosomes related to a specific disease, such as a cancer disease, from a population sample into subgroups, subgroups, or subgroups thereof. will be.

Recently, the concept of 'liquid biopsy' has been introduced to minimize the pain of testing for specific diseases such as cancer and degenerative diseases and implement early diagnosis. Liquid biopsy is a non-invasive method that extracts body fluids such as blood, saliva, urine, etc. relatively easily and analyzes cells related to specific diseases or components (peptides, proteins, nucleic acids, organelles, specific substances, etc.) leaked from them. way. Since it is possible to check the occurrence of a specific disease at an early stage through this, it is attracting attention as an alternative to conventional imaging diagnosis, biopsy, and blood test methods.

When diagnosing a specific disease, for example, cancer, using a liquid biopsy generally has the following advantages compared to a tissue biopsy. First, the use of bodily fluids during liquid biopsy is relatively easy and the risk of complications is reduced, compared to the potentially risky and expensive procedure of usually invasive surgical tissue collection. Second, liquid biopsy allows real-time continuous monitoring of cancer disease progression, whereas tissue biopsy only provides a snapshot of the disease of a single site when necessary and cannot monitor disease progression and treatment moment by moment. do. Third, it is difficult to fully understand the composition of tumors through tissue biopsy because of the variety of tumors. However, liquid biopsy collects biomaterials produced from tumor cells, enabling a more comprehensive analysis of the composition of tumors at the molecular level. do. Fourth, in tissue biopsy, the size of the sample is limited, so it becomes insufficient when divided and used for various diagnostic purposes, but in liquid biopsy, multiple samples can be collected from various body fluids as needed. Fifth, compared to the low accuracy of tissue biopsy, which limits the clinical usefulness of the test results, liquid biopsy provides relatively higher accuracy (positive sample measurement accuracy) and sensitivity (negative sample measurement accuracy) for clinical decision making. can provide definite results for

Circulating tumor cells (CTC), cell-free DNA, and exosomes are recognized for their potential as biomarkers for liquid biopsy in the field of cancer diagnosis. Circulating tumor cells are excellent in concentration, selectivity, and stability of isolated nucleic acids, but are difficult to use for early diagnosis of cancer because their distribution in the blood is extremely small. Cell-free DNA has a large distribution in blood, but it is difficult to concentrate and select, and the stability of the isolated nucleic acid is low. Moreover, due to the presence of a large amount of non-specific DNA unrelated to the target disease, overcoming the noise problem during analysis has become a major issue. Compared to these two biomarkers, exosomes have a high distribution in the blood, are easy to concentrate and select, and can separate nucleic acids in a stable state. However, since the size, density, and composition of protein and nucleic acid components contained in each exosome particle are extremely heterogeneous, separation into disease-related pure exosomes must be preceded.

Exosomes are attracting attention as a liquid biopsy biomarker. Exosomes (30 ~ 150 nm in size) secreted from cells in the human body in the form of microvesicles and reflecting the characteristics of the derived cells can be extracted from various body fluids such as blood, saliva, and urine. Since exosomes are endoplasmic reticulum composed of a lipid bilayer, they are not only stable in the body, but also free from attacks by proteases, RNases, and DNases. Therefore, studies to use exosomes as new biomarkers for the diagnosis of specific diseases such as cancer and degenerative diseases are increasing, and in fact, early diagnosis and accompanying diagnosis of cancer using components such as proteins and RNAs contained in exosomes as markers Targeted products are being developed.

Exosome-containing protein and RNA biomarkers that can be used for diagnosis in liquid biopsy for specific diseases, particularly cancer diseases, are as follows. First, looking at the exosome biosynthesis and process, first, micro-vesicles (MVEs), such as exosomes, bounce inside the cell, and at this time, proteins, mRNAs, and microRNAs (miRNAs) in the cytoplasm, including Forms small intracellular vesicles. Second, these inner vesicles are released as exosomes when MVEs fuse with the cell membrane. Thirdly, the released exosomes start migrating toward a destination determined by the attachment of specific ligands on their surface. Fourth, the exosomes that have reached the target cell are introduced through an endocytosis pathway or fused with the target cell membrane to release exosome components directly into the cytoplasm.

Protein components involved in the first step of exosome biosynthesis include hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), signal transducing adapter melecule (STAM1), tumor susceptability gene (TSG101), and CD81. As proteins involved in the second step of exosome release, RAB2B, RAB5A, RAB7, and RAB9A are known. Such protein molecules can be used not only as potential treatment targets in the development of therapeutic agents, but also as major targets in the development of detection and separation technologies for exosomes.

In addition, exosomes released into bodily fluids contain various cellular components such as double helix DNAs, RNAs, proteins, and lipid components. According to recent studies, exosome-loaded DNA molecules are representative of the genome as a whole and can also reflect the mutational status of tumor cells that released exosomes. Therefore, analysis of double helix DNA molecules in exosomes can be particularly useful for early detection of cancer or metastasis. In the case of exosome-containing proteins, their composition varies depending on the cell or tissue that released them, but most exosomes contain a general set of evolutionarily conserved protein molecules. The protein content in exosomes is important for the development of techniques for isolation and detection of exosomes and analysis methods. Furthermore, exosomal protein molecules can be used as biomarkers in the diagnosis and information gathering of other pathological conditions. In the case of exosome-containing RNA, after reporting on the difference in the content of cell-derived and exosome-derived mRNA and miRNA, and the functionality of exosome-containing miRNAs, the results of studies on several miRNAs that were simultaneously published are promising for exosomes. sparked an explosion of research. In fact, miRNAs are attracting attention as special targets for diagnosis as well as therapeutic development. In many reports using new detection technologies, miRNAs are present in almost all bodily fluids, and thus are emerging as potential biomarkers for analysis for diagnostic purposes. In fact, according to the analysis of various bodily fluid samples, a large number of different miRNAs have been found depending on the sample source.

Particularly of interest in cancer, exosomes are known to play a role in tumorigenesis, cancer metastasis, and drug resistance. Tumor cells, like other cells, secrete exosomes and other microvesicles in the microenvironment where tumors are generated, and these substances contribute to tumor cell migration during tumor progression and metastasis by promoting angiogenesis. Tumor cell-derived endoplasmic reticulum 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 the immune response. can In addition, transfer of tumor activity by exosomes secreted from tumor cells has also been reported.

However, since exosomes derived from all cells exist in the body fluid in a mixed form, there is a limit to accurately diagnosing specific diseases such as cancer with exosome samples in the form of bulk populations simply isolated from body fluids. . Moreover, since the concentration of exosomes derived from specific disease-related cells in body fluids is relatively low at the beginning of disease onset, separation and concentration of target exosomes from body fluids must be preceded for early diagnosis. Among existing exosome separation technologies, the density difference-based ultracentrifugation method is adopted as the gold standard separation process due to its high reproducibility and relatively stable process. However, it has the disadvantage of requiring expensive equipment and taking a long separation time. In order to compensate for these disadvantages, a method for separating exosomes by precipitation, using size exclusion chromatography, or using microfluidics as disclosed in the patent literature of the following prior literature, methods for separating according to size have been developed.

However, since these conventional exosome separation technologies provide all exosomes present in body fluids in the form of a single population, there is a limit to selectively detecting a very small amount of exosomes derived from specific cells such as cancer. In fact, exosomes released from cells contain various biochemical components (membrane proteins, internal proteins and RNA, etc.) in the exosome membrane or inside, so they are very heterogeneous in composition. In particular, it has been reported that certain exosomes among exosomes derived from cancer cells contain mRNA or miRNA involved in local environment creation in the early stages of angiogenesis and cancer metastasis. Moreover, as described above, it is known that the specific exosomes contain biochemical components involved in the process of inducing the death of immune cells or transforming normal cells into cancerous cells during cancer metastasis.

When diagnosing a specific disease such as cancer, it is very difficult to obtain high diagnostic accuracy when a bulk population containing all exosomes present in body fluids is used as a liquid biopsy sample. This is because sample matrix effects such as non-specific adhesion caused by non-specific exosomes negatively affect assay specificity and sensitivity. In order to overcome this analytical problem, multiple biomarkers (eg, multiple different miRNA species) known to be increased in body fluids of a specific disease patient may be simultaneously measured and used to determine whether or not a disease occurs. However, the synergistic effect by using multiple biomarkers is inevitably limited, because most of the exosome markers that are not specifically associated with a disease have fundamentally low disease specificity.

Accordingly, there is an urgent need for a solution to the problem of selectively isolating exosomes associated with the occurrence of specific diseases such as cancer and not using them for diagnosis of specific diseases.

KR 10-1807256 B1

The present invention is to solve the problems of the prior art described above, and one aspect of the present invention is to discover biomarkers related to specific diseases such as cancer, degenerative diseases, heart diseases, infectious diseases, etc., and to utilize them for diagnosis, exosomes Providing a method for analyzing exosome liquid biopsy samples in which liquid biopsy samples are prepared by isolating and recovering exosome subgroups, subgroups, or lower subpopulations in high yield and original state from the population, and analyzing the liquid biopsy samples is to do

Exosome liquid biopsy analysis method according to an embodiment of the present invention is to (a) extract target exosomes related to a predetermined disease from an exosome population sample containing exosomes secreted from a plurality of cells in the body of each of a normal person group and a patient group Separating into sub-populations, preparing an exosome liquid biopsy sample containing the target exosome population; (b) measuring quantitative signals for each of a plurality of biomarkers of the target exosomes contained in the exosome liquid biopsy sample; and (c) analyzing the exosome liquid biopsy samples of the normal population group and the patient group, respectively, according to the correlation between the plurality of biomarkers based on the measured value of the quantitative signal. .

In addition, in the exosome liquid biopsy analysis method according to an embodiment of the present invention, in the step (a), a first marker that specifically binds to a first separation marker of the first target exosome associated with the predetermined disease binding the capture material to a solid state anchoring member using a first reversible linker; Separating an exosome subset, which is a group of the first target exosomes, by reacting the exosome population sample with the first capture material coupled to the fixation member to capture a plurality of the first target exosomes step; and recovering the exosome subset by dissociating the first reversible linker so that the captured first target exosome is separated from the anchoring member.

In addition, in the exosome liquid biopsy analysis method according to an embodiment of the present invention, any one of magnetic force, gravity, and centrifugal force between the step of isolating the exosome subset and the step of recovering the exosome subset Using the above, concentrating the fixation member in which the first target exosomes are captured; may further include.

In addition, in the exosome liquid biopsy analysis method according to an embodiment of the present invention, in the step (a), a second target exosome in the exosome subset specifically binds to a second separation marker possessed coupling the capture material to the fixing member; and reacting the exosome subgroup sample containing the recovered exosome subpopulation with the second capture material coupled to the fixing member to capture a plurality of the second target exosomes, thereby capturing the second target exosomes. Separating a subpopulation of exosomes, which is a group of bits; may include.

In addition, in the exosome liquid biopsy analysis method according to an embodiment of the present invention, in the step of coupling the second capture material to the fixing member, the second capture material is bound to the fixing member by a second reversible linker. and recovering the exosome subset by dissociating the second reversible linker so that the captured second target exosome is separated from the fixation member.

In addition, in the exosome liquid biopsy analysis method according to an embodiment of the present invention, between the step of isolating the exosome subpopulation and the step of recovering the exosome subpopulation, any one of magnetic force, gravity, and centrifugal force Using the above, concentrating the fixation member in which the second target exosomes are captured; may further include.

In addition, in the exosome liquid biopsy analysis method according to an embodiment of the present invention, the step (c) is based on the correlation between the quantitative signal of any one of the plurality of biomarkers and the quantitative signal of the other. , the exosome liquid biopsy sample can be analyzed.

In addition, in the exosome liquid biopsy analysis method according to an embodiment of the present invention, in the step (c), the quantitative signal of any one of the plurality of biomarkers is used as a reference signal, and the quantitative signal of each of the remaining setting a number of parameters as a ratio of signals; and analyzing the exosome liquid biopsy sample according to the correlation between the two different parameters.

In addition, in the exosome liquid biopsy analysis method according to an embodiment of the present invention, the step of setting the parameter, the quantitative signal of any one of the plurality of biomarkers as the X-axis, and the quantitative signal of the other Coordinating the exosome liquid biopsy sample according to the measured values in a plurality of XY coordinate systems with a Y axis as a Y axis; calculating a slope of the exosome liquid biopsy sample of each of the normal group and the patient group; and setting the parameter using the biomarker constituting the XY coordinate system in which the difference between the slope of the exosome liquid biopsy sample of the normal group and the slope of the exosome liquid biopsy sample of the patient group is relatively large; can include

In addition, in the exosome liquid biopsy analysis method according to an embodiment of the present invention, in the step of analyzing the exosome liquid biopsy sample, the first parameter, which is any one of the plurality of parameters, is set as the X-axis, and the other parameter is converting the exosome liquid biopsy sample into coordinates according to parameter values calculated by substituting the measured values into the first parameter and the second parameter in a first XY coordinate system in which a Y axis is a second parameter; setting negative and positive regions in which the exosome liquid biopsy sample of the normal group is distributed in the first XY coordinate system; and a negative exosome liquid biopsy sample belonging to the negative or positive region among the coordinated exosome liquid biopsy samples according to the correlation between the two parameters in which at least one of the first parameter and the second parameter is different. Reanalyzing the; may include.

In addition, in the exosome liquid biopsy analysis method according to an embodiment of the present invention, the biomarker is a predetermined disease-related biomarker arranged on the surface of the exosome and a predetermined disease-related biomarker eluted from the exosome. At least one or more of the markers may be included.

Features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings.

Prior to this, the terms or words used in this specification and claims should not be interpreted in a conventional and dictionary sense, and the inventor may appropriately define the concept of the term in order to explain his or her invention in the best way. It should be interpreted as a meaning and concept consistent with the technical idea of the present invention based on the principle that there is.

According to the present invention, from a sample of the exosome population containing exosomes secreted from a number of cells in the body, a subgroup of exosomes containing exosomes related to a specific disease, a subgroup, or a subpopulation thereof is separated and recovered, and liquid biopsy is performed. Biomarkers such as proteins and nucleic acids specific to a specific disease can be discovered by performing biomarker profiling analysis on the liquid biopsy sample after preparing the sample.

Since liquid biopsy samples separated and concentrated in the form of exosome subgroups, subgroups, or lower subgroups are analyzed, non-specific exosomes are removed to minimize noise during analysis and maximize specific exosome signals. As a result, when exosomes are used as biomarkers for specific diseases such as cancer, interference of non-specific exosomes due to the heterogeneity of exosomes, which is an existing problem, is minimized, and the physiological properties of parent cells from liquid biopsy samples You can accurately grasp information about characteristics and genealogy.

In addition, when the present invention is applied to disease diagnosis, the diagnostic accuracy for specific diseases such as cancer, degenerative diseases, heart diseases, and infectious diseases can be remarkably increased, thereby exhibiting an early clinical diagnosis effect capable of diagnosing diseases at an early stage. Furthermore, since it becomes possible to obtain information on the progress or decline of a stage according to the treatment during disease treatment, it can be applied to a companion diagnosis customized for each individual.

1 is a schematic diagram comparing a biomarker analysis process using a conventional liquid biopsy sample (left) and a biomarker analysis process using a liquid biopsy sample prepared according to the present invention (right).
2 is a flowchart of a method for analyzing an exosome liquid biopsy sample according to an embodiment of the present invention.
3 is a schematic configuration diagram of an apparatus for preparing an exosome liquid biopsy sample according to an embodiment of the present invention.
4 to 7 are configuration diagrams schematically showing the first reversible linker and the second reversible linker shown in FIG. 3 according to various embodiments.
8 to 14 are flowcharts of a method for preparing an exosome liquid biopsy sample according to an embodiment of the present invention.
15 is an experimental result comparing correlations between tumor suppression markers and tumor progression, promotion, and metastasis markers after exosome surface marker profiling analysis of exosome population analysis samples from a normal group and a malignant melanoma patient group, respectively.
16 shows the results of tumor progression, promotion, and tumor suppression markers after surface marker profiling analysis for the exosome subpopulation using the exosome subpopulations isolated from the exosome populations of the normal group and the malignant melanoma patient group as analysis samples. It is an experimental result comparing the correlation between metastasis markers with the normal sample group.
Figure 17 compares the correlation between tumor progression, promotion, and metastasis markers between a normal sample group and a cancer-positive sample group after profiling analysis of exosome surface markers for each of the exosome population analysis samples of the normal group and the malignant melanoma patient group. This is the result of an experiment.
18 shows correlations between tumor progression, promotion, and metastasis markers after surface marker profiling analysis of exosome subpopulations in the exosome subgroup analysis samples isolated from the exosome populations of the normal group and the malignant melanoma patient group, respectively. This is the result of the experiment comparing the sample group and the cancer-positive sample group.
19 shows the results of biomarker profiling analysis performed by applying newly defined parameters to the exosome population analysis samples of each of the normal group and the malignant melanoma patient group.
FIG. 20 shows the results of biomarker profiling analysis by applying newly defined parameters to samples for analysis of exosome subsets isolated from the exosome populations of a normal group and a malignant melanoma patient group, respectively.
21 shows the results of biomarker profiling analysis applying newly defined parameters to exosome population analysis samples (A) and exosome subpopulation analysis samples (B) isolated from each of the normal group and the malignant melanoma patient group. This is the result of synthesizing two-dimensional parameter analysis for
22 is a result of 2-dimensional parameter analysis on the results of subgroup biomarker profiling analysis to which newly defined parameters are applied to exosome subgroup analysis samples of each of a normal group and a malignant melanoma patient group.
FIG. 23 is a result of 2-dimensional parameter analysis on the results of biomarker profiling analysis using newly defined parameters for exosome subgroup analysis samples in each of a normal group and a prostate cancer patient group.
24 shows the results of analyzing prostate cancer miRNA biomarkers contained in exosome population, CD63+ subpopulation, and CD63+/CD81+ subpopulation samples of prostate cancer cell-derived cell line cultures.

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 adding reference numerals to components of each drawing in this specification, it should be noted that the same components have the same numbers as much as possible, even if they are displayed on different drawings. In addition, terms such as “first” and “second” are used to distinguish one component from another component, and the components are not limited by the terms. Hereinafter, in describing the present invention, detailed descriptions of related known technologies that may unnecessarily obscure the subject matter of the present invention will be omitted.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1 is a schematic diagram comparing a biomarker analysis process using a conventional liquid biopsy sample (left) and a biomarker analysis process using a liquid biopsy sample prepared according to the present invention (right).

The present invention separates exosomes in bodily fluids into subgroups and sequentially subgroups to prepare a liquid biopsy sample, and performs biomarker profiling analysis on the exosome subgroups to determine specific disease-related biopsies. It relates to technology that discovers markers and applies them to clinical early diagnosis.

As a diagnostic method for early confirmation of the occurrence of a specific disease, 'liquid biopsy' is an analysis of exosomes released from cells related to a specific disease by extracting bodily fluids such as blood, saliva, and urine. there is. A technique of using exosomes extracted from bodily fluids as a new biomarker for diagnosing specific diseases such as cancer, degenerative diseases, infectious diseases, and heart diseases has been recently studied. However, since exosomes derived from all cells are present in the body fluid in a mixed form, exosome samples in the form of a bulk population simply isolated from body fluids, as in the prior art shown on the left side of FIG. There is a limit to accurately diagnosing the same specific disease. It is quite difficult to find specific biomarker molecules contained in target exosomes in all bodily fluids provided as samples. The possibility of using exosome nanovesicles as biomarkers depends on how highly selected markers can be enriched during exosome isolation. If not concentrated, the target exosomes represent only a fraction of the total proteome/transcriptome in bodily fluid. As such, since it is necessary to classify contents such as biomarkers according to exosomes, enrichment of diagnostic markers in resources such as exosome-containing bodily fluids is helpful in finding biomarkers that are expressed relatively little to the extent that they are generally difficult to detect. Accordingly, a technology for isolating and enriching target exosomes is required. Conventional exosome separation techniques include separation methods using ultracentrifugation, precipitation separation, size exclusion chromatography, or microfluidics, but these conventional techniques are still used to treat all exosomes present in body fluids. Since the exosomes are provided in the form of a single population, there is a limit to selectively detecting a very small amount of target exosomes derived from specific cells such as cancer.

Therefore, as shown on the right side of FIG. 1, the present invention is a protein marker highly correlated with cancer on the surface of the exosome, for example, Caveolin 1 (Cav1) in the case of melanoma cancer, HER-2 / neu in the case of gastric cancer, and ovarian In the case of cancer, focusing on the fact that L1CAM exists, by adopting an immunoaffinity separation method for a specific marker, a specific disease-related exosome subpopulation and sequentially extended further subpopulations are intended to be isolated. That is, the target disease association is relatively low, but the exosome subpopulation for the exosome marker (eg, CD63 in the case of melanoma cancer) of a comprehensive upper population including disease-related exosomes is first isolated, and 1 Exosomes encapsulated with biomarkers (e.g., specific miRNAs) involved in the spread of target diseases through the isolation of exosome subpopulations for exosome-specific markers (e.g., Cav1 in the case of melanoma cancer) in a secondary isolation solution Omni can be prepared as a liquid biopsy sample in concentrated form.

Exosome profiling can provide personalized information for individual patients and is critical for accurate prognosis or differential diagnosis. In addition, changes in exosome-derived biomarkers can not only indicate a response to more than a single condition, but also exosomes in body fluids, for example, plasma, are secreted by various cellular resources from both blood cells and surrounding tissues.

During liquid biopsy, various proteins are heterogeneously distributed on the surface of exosomes in bodily fluids. In particular, specific surface proteins associated with cancer diseases are complexly present even in normal human exosomes, although the expression ratio is different. Tetraspanins (CD9, CD63, CD81, CD82, CD151, etc.), cell type-specific molecules such as histocompatibility complex (MHC) class-Ⅰ and class-Ⅱ, adhesion molecules, surface proteins such as HSP60, HSP70, etc. It is reported that it can increase or decrease in relation to the characteristics of For example, CD63, an exosome surface protein of the tetraspanin family, is a protein included in the lysosomal membrane of B cell-derived exosomes and has been reported to increase in samples from patients with several cancer diseases, including melanoma cancer. In addition, compared to CD81 and CD9 of the same series, the expression level of CD63 appears to change depending on the type of parent cell, and studies to use it as an optimal exosome marker for disease diagnosis have also been reported. Furthermore, for accurate quantitative analysis of exosome surface proteins, an invariant housekeeping marker unrelated to parental cell characteristics can be used for standardization purposes during quantification. For this standardization purpose, CD9 is expected to be more suitable than CD63 in the tetraspanin family.

Under this background, immunoaffinity separation into exosome subpopulations based on exosome surface proteins can provide the following advantages. First, unlike separation methods based on density, size, precipitation, etc., antigen-antibody attachment reaction is used, so the inclusion of impurities other than the target exosomes in the solution after separation is minimized. As described above, the smaller the content of impurities in the analysis sample at the time of diagnosis, the lower the sample maternal effect such as non-specific reaction, so the diagnostic sensitivity and specificity can be improved. Second, if immunoaffinity isolation is performed on the exosome markers of a comprehensive upper population that includes exosomes associated with a specific disease but has a relatively low disease association, the concentration of the specific disease-related exosomes in the separated subgroup can be increased. can Such an approach is useful when the concentration of disease-related exosomes in the exosome population is initially low enough to be difficult to detect in body fluids. In this case, the sensitivity can be improved when diagnosing a specific disease by using the separated subgroup as a diagnostic sample.

Furthermore, if it is possible to further separate the exosome subpopulations via specific exosome surface proteins associated with specific diseases such as cancer, high concentrations of specific disease markers contained in specific exosomes, such as mRNA and miRNA, etc. This subpopulation of exosomes can be provided as a diagnostic sample. In particular, it is known that exosomes released from cancer cells contain various kinds of RNAs that play an executive role in the formation of the local environment such as angiogenesis in the pre-metastatic stage of cancer and in the metastasis stage. being reported

Indeed, an increasing number of exosome-derived proteins have been identified as potential biomarkers for various diseases, including cancer, as well as hepatitis, liver, kidney, and degenerative diseases. Exosome proteins have been reported to be associated with specific diseases, and therefore can be used as diagnostic or separation targets in future product development. For example, there is CD81 as an exosomal protein marker associated with chronic hepatitis C in plasma, CD63, caveolin-1, TYRP2, VLA-4, HSP70, and HSP90 as markers associated with melanoma cancer, and prostate cancer and There is survivin as an associated marker, and L1CAM, CD24, ADAM10, EMMPRIN, and claudin as markers associated with ovarian cancer. In addition, there are fetuin-A and ATF 3 as exosomal protein markers associated with acute kidney injury in urine, CD26, CD81, S1c3A1, and CD10 as markers associated with liver damage, and EGF, resistin, and CD10 as markers associated with bladder cancer. There is retinoic acid-induced protein 3, and PSA and PCA3 are markers associated with prostate cancer. Furthermore, there are amyloid peptides as exosome peptide markers associated with Alzheimer's in brain tissue and Tau phosphorylated Thr181 as a marker associated with Alzheimer's in cerebrospinal fluid.

Tetraspanins, a type of skeletal membrane protein, are abundantly present in exosomes. For example, plasma CD63+ exosomes are significantly increased in melanoma cancer patients compared to healthy controls. CD81, another exosomal marker of the tetraspanin family, plays a critical role in hepatitis C virus attachment and/or entry into cells. In addition, the concentration of serum exosome-containing CD81 is elevated in patients with chronic hepatitis C and is also associated with exacerbation of inflammation and fibrosis. From this, it is suggested that exosome-containing CD81 can be a potential marker for hepatitis C diagnosis and treatment response. Moreover, many exosome-containing protein biomarkers have been reported to be potentially useful for the diagnosis of central nervous system diseases. EGFRvIII (glioblastoma-specific epidermal growth factor receptor vIII) was found in serum exosomes of glioblastoma patients, which may be useful for diagnostic detection of glioblastoma. In addition, exosome-containing amyloid peptides have been reported to accumulate in brain plaques of Alzheimer's disease patients. Tau protein phosphorylated at Thr-181, a well-established biomarker of Alzheimer's disease, is present in elevated concentrations in exosomes isolated from cerebrospinal fluid specimens of mild-symptomatic Alzheimer's patients. From these findings, the potential value of exosomes for the early diagnosis of Alzheimer's is highlighted. Currently, there are no diagnostic tests that can identify early Alzheimer's disease.

In addition, the potential usefulness of proteins contained in urine exosomes obtained by non-invasive means in diagnosis is being explored, especially for urinary tract diseases. For example, urinary exosome-containing fetuin-A is increased in acute renal injury patients in intensive care units compared to normal subjects. ATF3 is also found only in exosomes isolated from patients with chronic kidney disease or acute renal impairment compared to normal subjects. Furthermore, urinary exosome proteins are being investigated for use as potential biomarkers for bladder and prostate cancer. According to the study results, two types of prostate cancer biomarkers, PCA-3 and PSA, are present in exosomes isolated from urine of prostate cancer patients. Overall, exosome-containing proteins identified in other bodily fluids are expected to be used as target platforms in the development of diagnostic analysis systems in the future.

In addition, protein markers highly correlated with cancer are present on the surface of exosomes, such as Caveolin 1 (Cav1) in melanoma cancer, HER-2/neu in gastric cancer, and L1CAM in ovarian cancer. It is reported. However, since the content of such specific exosomes is very low, especially in samples for early diagnosis, introduction of the above-described sequential iterative separation process is unavoidable in order to selectively isolate them.

2 is a flowchart of a method for analyzing an exosome liquid biopsy sample according to an embodiment of the present invention.

As shown in FIG. 2, the method for analyzing an exosome liquid biopsy sample according to an embodiment of the present invention, from an exosome population sample containing exosomes secreted from a plurality of cells in the body of each of the normal group and the patient group, Capturing target exosomes related to the disease and preparing an exosome liquid biopsy sample containing a population of the target exosomes (S100), according to a plurality of biomarkers of the target exosomes contained in the exosome liquid biopsy sample Measuring a quantitative signal (S200), and analyzing exosome liquid biopsy samples from each of the normal and patient groups according to the correlation between a plurality of biomarkers based on the signal value of the measured quantitative signal (S300 ).

In the exosome liquid biopsy sample preparation step (S100), as analysis samples to be analyzed, an exosome liquid biopsy sample of a normal group and an exosome liquid biopsy sample of a patient group separated from each of the exosome population samples of the normal group and the patient group are prepared do. Here, the exosome liquid biopsy samples of the normal group and the patient group are prepared separately from each exosome population sample in the same manner.

The exosome liquid biopsy sample contains a population of target exosomes captured from the exosome population sample and associated with a given disease. Specifically, an exosome subset, which is a group of exosomes belonging to the exosome population and having at least one specific assay target biomarker in common, and at least one other specific assay target biomarker in common as part of the exosome subset and an exosome subpopulation, which is a group of exosomes that belong to the exosome subpopulation and have at least one or more specific target biomarkers in common.

In order to clarify the understanding of the terms exosome population, subgroup, and subgroup used in the present invention, CD63 and Cav1, which are biomarkers associated with melanoma cancer, will be described as examples. The exosome population is a bulk population including exosomes secreted from a number of cells in the body. The exosome subpopulation is a part of the exosome population, and commonly includes a specific analysis target biomarker, and the exosome population having the CD63 marker, which is reported to increase in concentration in melanoma cancer samples, is the exosome subpopulation. can be used Since there are exosomes having the Cav1 marker among the exosome subpopulation having the CD63 marker, the exosome having the Cav1 marker can be isolated from the exosome subpopulation and used as the subgroup of exosomes. Conversely, exosomes having the Cav1 marker may be used as the exosome subgroup, and exosomes belonging to the exosome subgroup and having the CD63 marker may be used as the exosome subgroup.

Details of preparing an exosome liquid biopsy sample will be described later.

The quantitative signal measurement step (S200) is a process of measuring quantitative signals for each of a plurality of biomarkers of target exosomes contained in the exosome liquid biopsy sample. The biomarker may include at least one of a predetermined disease-related biomarker arranged on the surface of the exosome and a predetermined disease-related biomarker eluted from the exosome. A predetermined disease-related biomarker is a component that is relatively increased in body fluids compared to normal people when a specific disease occurs, and is involved in the biosynthesis of exosomes in cells, involved in the release of exosomes from cells, or exosomes by recipient cells. It may include proteins, mRNAs, tRNAs, miRNAs, long non-coding RNAs, DNAs, lipid components, sugar components, peptides, and other biochemical components involved in bituminous acceptance.

Quantitative signals for biomarkers can be measured using enzyme immunoassay (ELISA), but not necessarily limited thereto, and all known quantitative signal measurement methods can be used.

In the sample analysis step (S300), biomarker profiling analysis is performed on the exosome liquid biopsy sample. Based on the measured values of the quantitative signals measured for each of a plurality of biomarkers, analysis can be performed according to the correlation between the biomarkers. Here, an exosome liquid biopsy sample may be analyzed according to a two-dimensional correlation between one quantitative signal and another quantitative signal. For example, an exosome liquid biopsy sample can be coordinated and analyzed according to signal measurement values in an XY coordinate system in which the X-axis is the quantitative signal of any one of a plurality of biomarkers and the other quantitative signal is the Y-axis. there is. Here, a biomarker capable of diagnosing a disease in the patient group can be discovered by comparing the distribution pattern between the exosome liquid biopsy sample of the normal group and the exosome liquid biopsy sample of the patient group. In addition, it can be used to predict the stage of a disease such as cancer by confirming an increase in heterogeneity for the biomarker liver composition ratio.

Meanwhile, analysis may be performed by newly defining normalized parameters based on quantitative signals. Here, the parameter may be defined as a ratio of a quantitative signal of any one of a plurality of biomarkers as a reference signal and the reference signal to another quantitative signal. At this time, at least two or more parameters are set according to the number of quantitative signals of biomarkers other than the reference signal. For example, when quantitative signals are measured for 4 biomarkers 1 to 4, the signal of the first biomarker is used as a reference signal, and parameter 1 = (signal of the second biomarker) / (signal of the first biomarker) signal), parameter 2 = (third biomarker signal) / (first biomarker signal), parameter 3 = (fourth biomarker signal) / (first biomarker signal) can be set.

Here, as the biomarker used to set parameters, some of them may be selected from among a plurality of biomarkers for which quantitative signals are measured. For example, as in the two-dimensional correlation analysis between the quantitative signals, at least one or more of the quantitative signals of the X axis and the Y axis are coordinated in a plurality of XY coordinate systems with different exosome liquid biopsy samples, and the exosomes of the normal and patient groups, respectively. After calculating the gradient of a small body biopsy sample, parameters may be set by selecting a biomarker constituting an XY coordinate system having a relatively large difference in gradient. That is, a parameter may be set using a quantitative signal showing a significant difference by comparing the results of two-dimensional analysis between the quantitative signals of the exosome liquid biopsy samples of the normal group and the patient group.

If at least two or more parameters are set, analysis can be performed according to the correlation between the two different parameters. For example, in a first XY coordinate system in which one of the plurality of parameters, the first parameter, is the X axis, and the other, the second parameter, is the Y axis, the measured values are assigned to the first parameter and the second parameter According to the calculated parameter value, the exosome liquid biopsy sample can be coordinated and the distribution pattern can be compared.

Here, it is possible to set a negative region in which the exosome liquid biopsy samples of the normal group are distributed in the first XY coordinate system. If the exosome liquid biopsy samples of the patient group are distributed within the negative region, among the coordinated exosome liquid biopsy samples For the negative exosome liquid biopsy sample belonging to the negative region, reanalysis of the negative region distribution of the samples may be performed by constructing an XY coordinate system between at least one of the first parameter and the second parameter and other parameters. For example, positive accuracy (i.e., sensitivity) may be maximized by performing reanalysis of the negative region distribution between the first parameter and the third parameter and reanalysis of the negative region distribution between the third parameter and the fourth parameter individually or sequentially.

Similarly, a positive region in which exosome liquid biopsy samples of the patient group are distributed can be set in the first XY coordinate system. When the exosome liquid biopsy samples of the normal group are distributed within the positive region, For the positive exosome liquid biopsy sample belonging to the positive region, negative accuracy (ie, specificity) can be maximized by re-analyzing the positive region distribution of the samples according to the above process.

Hereinafter, the contents of manufacturing an exosome liquid biopsy sample will be described with reference to the manufacturing device and manufacturing method.

3 is a schematic configuration diagram of an apparatus for preparing an exosome liquid biopsy sample according to an embodiment of the present invention.

As shown in FIG. 3, the exosome subgroup liquid biopsy sample preparation device according to an embodiment of the present invention is a solid state fixing member 10 and exosomes 1 secreted from a plurality of cells. A first capture material 20 that captures the disease-related exosomes 1a by specifically binding to the first separation marker m1 possessed by the disease-related exosomes 1a, and the fixing member 10 and the first It may include a first reversible linker 30 that binds the capture material 20 in a separable manner.

In addition, the first capture material 20 is bound to the unbound fixing member 10, and the target exosome 1b in the exosome subpopulation S, which is a group of disease-related exosomes 1a, possesses 2 It may further include a second capture material 40 that specifically binds to the separation marker (m2) and captures the target exosome (1b).

In addition, as a means for coupling the second capture material 40 to the fixing member 10, a second reversible linker 50 may be further included.

The fixing member 10 is a solid state member, and the first reversible linker 30, the second capture material 40, and the second reversible linker 50 may be selectively bonded and fixed to its surface. The first reversible linker 30 is coupled and fixed on one fixing member 10 and then separated, and then the second capture material 40 or the second reversible linker 50 may be coupled and fixed. In addition, the fixing member 10 to which the first reversible linker 30 is fixed may be different from the fixing member 10 to which the second capture material 40 or the second reversible linker 50 is fixed. 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 fixing member 10 is not necessarily limited to the above, and if it is a material that can be combined with each of the first reversible linker 30, the second capture material 40, or the second reversible linker 50, there is no particular limitation does not exist.

The first capture material 20 and the second capture material 40 are materials that selectively capture predetermined exosomes 1 . The first capture material 20 and the second capture material 40 capture the exosome 1 by specifically binding to the separation marker m of the exosome 1. Here, the separation marker (m) is a biomarker that is relatively increased in body fluids compared to normal people when a specific disease occurs, and is a protein, nucleic acid, lipid component, sugar component, peptide, and Other biochemical components and the like may be included. Such a separate marker (m) may specifically bind to the first capture material 20 and the second capture material 40 through immunochemical interactions such as antigen-antibody reactions. The first capture material 20 and the second capture material 40 may be predetermined antibodies that react with the surface protein of the exosome 1 and the antigen-antibody.

Among the separation markers (m), the first separation marker (m1) that specifically binds to the first capture material (20) is present on the surface of the exosome (1a) associated with a predetermined disease. Therefore, from the bulk exosome population (B), which is a group of exosomes (1) isolated from a plurality of cells, the disease-related exosomes (1a) having the first separation marker (m1) are in the first capture material (20) They can be separated by capture. Here, the population of isolated disease-related exosomes (1a) becomes the exosome subpopulation (S). Among the disease-related exosomes (1a) in the exosome subset (S) thus generated, the target exosome (1b) having the second separation marker (m2) reacts specifically with the second separation marker (m2), thereby capturing it As a result, a subpopulation (SA) of exosomes is isolated within the subpopulation (S) of exosomes. Compared to the second separation marker (m2), the first separation marker (m1) on the exosome (1) shows relatively low accuracy when used for diagnosing a specific disease, but is present in a relatively high concentration in body fluid, and exo When classifying the quantitative system according to the markers of the Zom (1) group, it can have more comprehensive characteristics by being positioned at the top.

In the present invention, exosomes prepared by primary isolation, concentration, and recovery of an upper exosome surface protein marker (first separation marker)-positive exosome subpopulation (S) including a disease-specific marker predicted to have a low concentration in body fluid The exosome subpopulation (SA) recovered by using a liquid biopsy sample or performing secondary separation and enrichment for a disease-specific marker (second segregation marker) targeting the exosome subpopulation (S) is exosome It can be used as a liquid biopsy sample. Furthermore, in the present invention, after primary separation, concentration, and recovery of the exosome subpopulation (S) positive for the upper exosome surface protein marker (first separation marker) including a disease-specific marker predicted to have a low concentration in body fluid, , it is possible to recover the exosome subpopulation (SA) by sequentially separating and concentrating for a disease-specific marker (second separation marker). Here, 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, targeting the exosome subpopulation (SA) Alternatively, the subpopulation recovered through isolation and enrichment for another disease-specific marker may be used as an exosome liquid biopsy sample.

In liquid biopsy using exosomes in bodily fluids, switching from simply enriched bulk population exosome samples to using disease-relevant exosome samples that are selectively isolated for the disease being tested can improve diagnostic accuracy. . In the case of melanoma cancer, which is a skin cancer that occurs in melanin-producing cells, Caveolin 1 (Cav1), a surface protein of exosomes, is significantly increased in the blood of melanoma cancer patients compared to normal people, leading to Cav1-positive (Cav1+) exosome-based diagnosis The sensitivity is 68%, which is relatively higher than the diagnostic sensitivity of 43% for CD63-based diagnosis, another melanoma cancer marker. Selective isolation and concentration of target exosomes, such as Cav1+ exosomes, are required for clinical use through improved performance of liquid biopsy. Hard to come true.

Therefore, the present invention is a method of selectively isolating exosomes in body fluids, obtaining, as an example, subgroups or subgroups containing Cav1+ exosomes, and using them as test samples to increase the accuracy and sensitivity of melanoma cancer diagnosis. want to utilize Considering the yield and reproducibility of exosome isolation, Cav1+ exosomes were not targeted first, but the upper exosome population associated with cancer was selected as the primary isolation target. For example, CD63 protein, a house-keeping protein that is more abundant in exosomes than other surface proteins, is increased and expressed on the surface of exosomes in the occurrence of some cancers, including melanoma cancer. CD63+ exosome subgroup (S) was isolated using 1 as a separation marker (m1), and CD63+/Cav1+ exosome subpopulation from CD63+ exosome subgroup (S) using Cav1 as a second separation marker (m2) (SA) can be isolated.

Meanwhile, the first capture material 20 may be fixed to the fixing member 10 via the first reversible linker 30 and then separated. The second capture material 40 may be directly bonded and fixed to the fixing member 10 without separation, or may be fixed to the fixing member 10 and then separated by introduction of the second reversible linker 50 .

One of the reversible linkers, the first reversible linker 30, detachably binds the first capture material 20 to the fixing member 10, and the other second reversible linker 50 binds the second capture material ( 40) is detachably coupled to the fixing member 10. Here, the first capture material 20 is not coupled to the fixing member 10 to which the second capture material 40 is fixed. That is, the second capture material 40 is fixed through the second reversible linker 50 on the fixing member 10 to which the first capture material 20 is uncoupled. 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 from each other, and specific materials and structures will be described later.

Immunoaffinity isolation of exosomes under mild conditions is possible using a ligand-binder attachment-based reversible linker. The reversible linker initially serves to immobilize the specific exosome (1) capture material (antibody) on the fixation member (10), but after capturing the exosome (1) in the sample on the fixation member (10), the reaction conditions It is dissociated through change, allowing the recovery of the captured exosomes (1). For example, sugar-sugar receptor binding reactions through hydrophilic and hydrophobic bonds, interionic or ion-ion receptor binding reactions, substrate-enzyme reactions, antigen-antibody reactions, nucleic acid conjugation reactions, hormone-cell receptor reactions, and ligand- In addition to the nanostructure reaction, a polyhistidine-tag reaction, a biotin-avidin reaction, and an attachment reaction using a chelator may be used as the reversible linker. Such attachment reactions can be dissociated through adjustment of the affinity between the binder and the ligand of the linker, such as a change in the structure of the binder after binding or a competitive reaction to the binder. Therefore, if a reversible linker is used, it is possible to separate and recover the target exosome (1b) under mild conditions after immunologically capturing the exosome (1) in the sample without using harsh conditions such as acidic pH.

In addition, with the immunoaffinity separation process using the above-mentioned attachment reaction-based reversible linker, all the separation and recovery enable separation and recovery using physical, biological, and chemical properties such as density, size, solubility, membrane permeability, molecular structure, and magnetism. process can be used. In particular, using a specially designed immunoaffinity separation process such as chromatography or magnetic separation, it is possible to separate and recover trace amounts of exosomes in body fluids by adjusting the affinity between the binder and the ligand in the linker. For example, using the ‘switch attachment reaction’ of a recognition material that recognizes the structural change of the binder in the linker caused by ligand attachment, it is possible to separate and recover exosomes through an immunoisolation process depending on the presence or absence of a ligand. In addition, when a competitive attachment reaction method using a ligand-like structure that competes with a ligand for a binder in a ligand-binder reaction during immunoisolation is introduced, it is possible to separate and recover exosomes depending on the presence or absence of a competitive attachment reaction.

Hereinafter, the aforementioned first reversible linker 30 and the second reversible linker 50 will be described in detail. The reversible linker may be implemented according to the following first to fourth embodiments, and the first reversible linker (30, 30a to 30d) and the second reversible linker (50, 50a to 50d) may be formed of any one of them there is. 4 to 7 are configuration diagrams schematically showing the first reversible linker and the second reversible linker shown in FIG. 3 according to various embodiments.

As shown in FIG. 4 , the reversible linkers 30a and 50a according to the first embodiment of the present invention may include a ligand 31a, a binder 33a, and a recognition material 35a.

The ligand 31a is a material 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. .

The binder 33a is a material that causes a conformational change by an attachment reaction with the ligand 31a. The binder 33a may include at least one selected from the group consisting of sugar-binding proteins, ion-binding proteins, enzymes, antibodies, aptamers, cell receptors, and nanostructures. Accordingly, the binder 33a and the ligand 31a may be 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 A binder-ligand pair such as a pair, a nanostructure-ligand pair, etc. may be formed, and examples of the most representative binder 33a and 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 may be any material as long as they are detachably bonded to each other to cause a structural change. In the reversible linker according to the first embodiment, the binder 33a is polymerized with the first capture material 20 and/or the second capture material 40 to form a binder-capture material polymer (C).

The recognition material 35a is a material that specifically binds to the binder 33a whose structure is changed by binding to the ligand 31a, and includes antibodies, protein receptors, cell receptors, aptamers, enzymes, nanoparticles, nanostructures, and It may include any one or more selected from the group consisting of heavy metal chelators. However, since the material is only an example of the recognition material 35a, the scope of the present invention should not necessarily be limited thereto. As an example, describing the binding reaction between the ligand 31a, the binder 33a, and the recognition material 35a, a calcium ion (ligand) is first bonded to a calcium-binding protein (binder), resulting in a structural change of the calcium-binding protein. An antigen-antibody reaction occurs between the calcium-binding protein and the antibody (recognition material), whose structure is changed, 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 .

On the other hand, when the exosome 1 is captured by the first capture material 20 and/or the second capture material 40 coupled to the fixation member 10 via the reversible linkers 30 and 50, the reaction conditions By dissociating the reversible linker through change, the captured exosomes (1a, 1b) can be recovered. In the case of the reversible linkers 30a and 50a according to the first embodiment, the structure-changed binder 33a may be dissociated when the original structure is restored. For example, when an antibody (recognition material) is bound to a calcium-binding protein (binder) whose structure is changed by attaching calcium ions (ligand), and the antibody captures the exosome (1), recovery without calcium ions By adding a solution to desorb 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.

Referring to FIG. 5 , the reversible linkers 30b and 50b according to the second embodiment of the present invention may include a ligand 31b, a binder 33b, and a recognition material 35b.

The ligand 31b of the reversible linkers 30b and 50b according to the second embodiment, the binder 33b, and the recognition material 35b are respectively ligands 31a of the reversible linkers 30a and 50a according to the first embodiment , the binder 33a, and the recognition material 35a, respectively. Hereinafter, differences will be mainly described.

In the reversible linkers 30a and 50a according to the first embodiment, the binder 33a is polymerized with the capture materials 20 and 40 to form a polymer (C), and the recognition material 35a is used to form a polymer of the fixing member 10. On the other hand, in the case of the reversible linkers 30b and 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 attached to the capture material 20, 40 ) to form a recognition material-capture material polymer (C). Other than that, since it is the same as the reversible linkers 30a and 50a according to the first embodiment, a detailed description is omitted.

As an example of the reversible linkers 30 and 50 according to the first and second embodiments, which enable the recovery of exosome subpopulations or subpopulations in a sample under mild conditions, binders 33a and 33b such as calcium binding proteins are used. Recognition materials 35a and 35b such as antibodies that specifically recognize structural changes caused by binding to ligands 31a and 31b such as calcium ions that react specifically may be used. The binder-recognition material reaction, such as the antigen-antibody reaction, is rapidly combined or dissociated depending on the presence or absence of ligands such as calcium ions (31a, 31b), similar to the principle of turning on and off by manipulating a switch. It is described as 'switch-like reversible binding'.

As shown in FIG. 6 , the reversible linkers 30c and 50c according to the third embodiment of the present invention may include a ligand 31c and a binder 33c.

The ligand 31c of the reversible linkers 30c and 50c according to the third embodiment is polymerized with the first capture material 20 and/or the second capture material 40 to form a capture material-ligand polymer (C) .

The binder 33c of the reversible linkers 30c and 50c according to the third embodiment is fixed to the surface of the fixing member 10, and the capture material-ligand polymer (C ), the exosomes 1a and 1b are captured by the fixing member 10. The binder 33c binds to the ligand 31c, and when the ligand 32 for competitive reaction with the ligand 31c is added, the ligand 32 for competition reaction is attached. Due to this, as the ligand 31c is desorbed, the exosomes 1a and 1b may be separated from the fixing member 10 and recovered. Here, the ligand 31c is selected from the group consisting of His-tag (His-tag, polyhistidine-tag), vitamins such as biotin, sugar molecules, ions, substrates, antigens, amino acids, peptides, nucleic acids, growth factors, and hormones. Any one or more may be included. In addition, the binder 33c includes 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. can do. The competitive reaction ligand 32 may include at least one selected from the group consisting of vitamins such as imidazole and biotin, sugar molecules, ions, substrates, antigens, amino acids, peptides, nucleic acids, growth factors, and hormones. can

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

Referring to FIG. 7 , the reversible linkers 30d and 50d according to the fourth embodiment of the present invention may include a ligand 31d and a binder 33d.

The ligand 31d of the reversible linkers 30d and 50d according to the fourth embodiment and the binder 33d are the ligands 31c and the binder 33c of the reversible linkers 30c and 50c according to the third embodiment, respectively. Corresponds to, and the following will mainly explain the differences.

In the reversible linkers 30c and 50c according to the third embodiment, the ligand 31c is polymerized with the capture materials 20 and 40 and the binder 33c is fixed to the surface of the fixing member 10, while the fourth embodiment In the case of the reversible linkers 30d and 50d according to the example, the ligand 31d is fixed to the surface of the fixing member 10, and the binder 33d is polymerized with the capture materials 20 and 40 to form a capture material-binder polymer ( C) form Otherwise, the ligand 31d is similarly bonded to the binder 33d, but is separated from the binder 33d by a competitive reaction with the ligand 32.

Meanwhile, the exosome liquid biopsy sample preparation device may further include a reactor (not shown). The reactor may house the fixing member 10 therein. At this time, when the exosome (1) sample is added to the reactor, the disease-related exosome (1a) is attached to the fixing member (10) via the first reversible linker (30) and the first capture material (20) therein The exosome subpopulation (S) is separated by capture, and the exosome subpopulation (S) is recovered by dissociation of the first reversible linker (30). In addition, the recovered exosome subgroup (S) sample is added to the reactor, and the target exosomes (1b) are captured by the fixing member (10) using the second reversible linker (50) and the second capture material (40). An exosome hypopopulation (SA) is isolated. The separated exosome subpopulation (SA) is recovered as the second reversible linker 50 dissociates. Here, by concentrating the fixing member 10 using at least one of magnetic force, gravity, and centrifugal force, the exosome subpopulation (S) and/or the exosome subpopulation (SA) may be enriched. For example, when magnetic beads are used as the fixing member 10, the upper layer in a state in which the fixing member 10 associated with the disease-related exosome 1a and/or the target exosome 1b is captured using a magnet Exosome subpopulations (S) and/or exosome subpopulations (SA) can be enriched by removing the liquid and removing the magnetic beads.

Hereinafter, a liquid biopsy sample preparation method using the above-described exosome liquid biopsy sample preparation device will be described. Since the exosome liquid biopsy sample manufacturing apparatus has been described above, descriptions of overlapping items will be omitted or only briefly described.

8 to 14 are flowcharts of a method for preparing an exosome subpopulation liquid biopsy sample according to an embodiment of the present invention.

Referring to FIG. 8, in the method for preparing an exosome liquid biopsy sample according to the first embodiment of the present invention, among exosomes secreted from a plurality of cells, a specific disease-related exosome has a first separation marker that specifically binds A step of coupling the first capture material to the fixation member in a solid state using a first reversible linker (S110), a sample solution containing a group of exosomes, and the first capture material bound to the fixation member Separating an exosome subset, which is a group of disease-related exosomes, by reacting to capture disease-related exosomes (S120), and dissociating the first reversible linker so that the captured disease-related exosomes are separated from the anchoring member and recovering the exosome subset (S130).

Here, a bulk exosome population or body fluid, which is a group of exosomes secreted from a plurality of cells, may be used as a sample solution, and the exosome subset may be primarily separated and recovered, and then provided as a liquid biopsy sample.

In addition, as shown in FIG. 9, in order to separate and recover the exosome subpopulation from the exosome subpopulation to prepare a liquid biopsy sample, a second marker that specifically binds to a second separation marker possessed by the target exosomes in the exosome subpopulation binding the capture material to the fixing member (S140), and reacting the exosome subgroup solution containing the recovered exosome subpopulation with the second capture material bound to the fixation member to capture the target exosomes , Separating a subpopulation of exosomes that is a group of target exosomes (S150) may be further included. Here, the second capture material may be coupled to the fixing member by a second reversible linker.

First, in order to separate and recover the exosome subset, the first capture material and the first reversible linker are reacted with the fixation member (S110). At this time, the first capture material is coupled to the fixing member via the first reversible linker.

Next, by adding a sample solution, it reacts with the first capture material (S120). Here, the first capture material specifically binds to the first separation marker of disease-related exosomes contained in the sample solution. Thereby, subpopulations of exosomes targeted for disease-related exosomes are entrapped in the fixation member and separated from the sample solution.

After the exosome subpopulation is isolated, the first reversible linker is dissociated (S130). At this time, since disease-related exosomes are separated from the fixed member, exosome subsets can be recovered.

When the disease-related exosomes are separated from the fixation member, the fixation member is recycled or a second capture material is coupled to the fixation member using a new fixation member (S140). In this case, the second capture material may be directly coupled to the fixing member or may be coupled to the fixing member using a second reversible linker.

Next, the exosome subgroup solution containing the recovered exosome subgroup is reacted with the second capture material (S150). At this time, since the second capture material specifically binds to the second separation marker of the target exosomes in the exosome subset, the target exosomes are fixed to the fixing member. As a result, a subpopulation of exosomes, which is a set of target exosomes, is isolated. Instead of being recovered in this way, it may be provided as a liquid biopsy sample in the form of a subpopulation of exosomes immobilized on a fixation member. That is, when a biomarker for diagnosis of a specific disease is separated in the form of a subgroup and then the analysis of the biomarker is performed immediately without recovering the exosome or the analysis of the biomarker eluted by dissolving the exosome is performed, sequential multiplexing In the final separation step of the immunoaffinity separation process, only the biomarker can be directly used as a sample without recovering the subpopulation exosomes.

Hereinafter, the present invention will be described in more detail with reference to specific examples of the first reversible linker and the second reversible linker.

FIG. 10 shows a method for preparing an exosome subpopulation liquid biopsy sample using the first reversible linker 30a and the second reversible linker 50a according to the above-described first embodiment. Referring to FIG. 8 , first, the recognition material 35a is fixed to the surface of the fixing member 10, and the ligand solution containing the ligand 31a, the binder 33a, and the first capture material 20 are reacted. At this time, the binder 33a and the first capture material 20 form a binder-first capture material polymer (C), and the recognition material 35a is bonded to the polymer (C). Here, the ligand 31a contained in the ligand solution is bound to the binder 33a, causing a structural change of the binder 33a, and the recognition material 35a specifically recognizes and binds the binder 33a whose structure has changed. do. Next, the sample solution containing the bulk exosome (1) population is reacted. At this time, while the disease-related exosomes (1a) having the first separation marker (m1) that specifically binds to the first capture material (20) are captured by the fixing member (10), the exosome subset (S) is separated do. When the exosome subpopulation (S) is separated, a recovery solution is added. The recovery solution is a ligand-free solution that does not contain the ligand 31a. When the recovery solution is added, the ligand 31a bound to the binder 33a As a result, the changed structure of the binder 33a is restored to its original state, and the coupling between the binder 33a and the recognition material 35a is released. Accordingly, the polymer (C) to which the disease-related exosome (1a), which is the target of the exosome subset (S), is separated from the recognition material (35a), and the exosome subset (S) can be recovered.

Next, the disease-related exosome 1a is separated, and the fixing member 10 to which the recognition material 35a is fixed is recycled, and the second reversible linker 50a and the second capture material 40 are reacted. At this time, the second reversible linker (50a) shares the recognition material (35a) of the first reversible linker (30a), and compared with the first reversible linker (30a), the first capture material (20) to the second capture material ( 40) to be coupled to the fixing member 10. When the recovered exosome subgroup (S) solution is added here, the second capture material (40) is specific for the second separation marker (m2) of the target exosome (1b) in the exosome subgroup (S) solution , the exosome subpopulation (SA) is isolated. At this time, since the exosome subgroup (S) includes the binder 33a of the first reversible linker 30a, the recognition material 35a coupled to the fixing member 10 and the binder of the first reversible linker 30a Since (33a) can react with each other, after reacting the second reversible linker (50a) and the second capture material (40), the remaining attachment site on the recognition material (35a) bound to the fixing member (10) is replaced by a binder (33a). ) may be blocked with an appropriate reactive component such as the like.

As an example, the method for isolating the CD63+/Cav1+ exosome subpopulation (SA) according to the present invention having the first reversible link and the second reversible link according to the first embodiment is described. (Fixed member) It is immobilized on the surface (Bead surface), and the calcium-binding protein (CBP, binder) is polymerized through a specific antibody (first capture material) for CD63 exosome surface protein and biotin-streptavidin linkage (CBP-capture antibody polymer). When ^a CBP-capturing antibody polymer dissolved in a solution containing calcium ions (eg, >10 mM Ca ²⁺ ) is added to the fixed reversible recognition material, the polymer is fixed on the solid surface by a 'switch-on' recognition reaction. When bulk exosome samples prepared in the same solution are 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, if the calcium-free exosome recovery solution is added, the calcium ion bound to CBP is desorbed and becomes a 'switch off' state. can As a result, the CD63+ exosome subset is separated and recovered, and exosomes can be enriched according to 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.

For the sequential isolation of exosome subpopulations, a reversible recognition material is immobilized on a regenerated or new solid surface, and a specific antibody for the Cav1 exosome surface protein, which is highly related to a specific disease, is polymerized with CBP, and then, as above, 'switch' Through the 'on' recognition reaction, the CBP-capture antibody polymer is immobilized on the solid surface. When the CD63+ exosome subset recovered after the first separation is added as a second separation sample while maintaining the same state, the 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, when the exosome recovery solution from which calcium is removed is added, the 'switch off' state is established, so the captured exosomes can be recovered into the solution. As a result, the CD63+/Cav1+ exosome subset is separated and recovered through secondary separation.

On the other hand, if recovery after isolation of the exosome subpopulation is not required, the capture antibody may be directly immobilized on a solid surface to isolate the subpopulation without introduction of a second reversible linker. In this case, mainly for the captured exosomes An immunoassay is performed, or the nucleic acid contained in the exosome is extracted and molecularly tested by dissolving it while being captured.

FIG. 11 shows 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 above-described third embodiment. Referring to FIG. 11, the ligand 31c is polymerized with the first capture material 20 that specifically reacts with the first isolation marker m1 of the disease-related exosome 1a, and binds to the fixation member 10 After immobilizing the ligand-capture material polymer using affinity with the binder (33c), the exosome (1) sample is added to capture and separate the exosome subpopulation (S), and the recovery containing the competing reaction ligand (32) The solution is used to recover the exosome subpopulation (S).

Next, the second capture material 40 that specifically reacts to the second separation marker m2 of the target exosome 1b and the ligand 31c are polymerized, and the binder 33c bound to the fixing member 10 ) and the polymer (C) thereof, the exosome subgroup (S) solution may be added to capture and isolate the exosome subgroup (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 of the first reversible linker 30c ( Since 31c) can react with each other, after reacting the second reversible linker 50c and the second capture material 40, the remaining attachment site on the binder 33c bound to the fixing member 10 is formed with the ligand 31c, etc. It can be blocked with the same appropriate reactive component.

As an example, describing the method for isolating the CD63+/Cav1+ exosome subgroup according to the present invention having the first reversible link and the second reversible link according to the third embodiment, a binder (nickel ion) is applied to a solid surface (Bead surface ), and the ligand (Histag) polymerizes with a specific antibody against the surface protein of CD63 exosome (1) as the first separation marker (m1) (Histag-capture antibody polymer). When Histack-capture antibody polymer is added to the immobilized nickel ion, the polymer is fixed on the solid surface by a binder-ligand reaction. When the prepared bulk exosome sample is added to the same solution, CD63+ exosomes react with the capture antibody on the solid surface and are captured. After washing with the solution, when an exosome recovery solution containing a high-concentration competitive ligand (imidazole) is added, the histac bound to nickel ions is desorbed by the competitive reaction, so the captured exosomes can be recovered into the solution. As a result, the CD63+ exosome subset is separated and recovered, and the solid surface is regenerated and can be recycled for the isolation of the next sample.

The above separation process is used for the sequential isolation of the CD63+/Cav1+ exosome subpopulation from the CD63+ exosome subpopulation recovered above. In this case, the ligand (hi-stack) polymerizes with an antibody specific for the Cav1 exosome surface protein as a second segregation marker. After the polymer is immobilized on the solid surface through a binder-ligand reaction, when a CD63+ exosome subgroup sample is added, the CD63+/Cav1+ exosome reacts with the capture antibody on the solid surface and is captured. After washing, when an exosome recovery solution containing a high concentration of competing ligand (imidazole) is added, CD63+/Cav1+ exosomes bound to Con A can be recovered into the solution. As a result, the CD63+/Cav1+ exosome subpopulation is isolated and recovered.

12 illustrates a method for preparing an exosome subgroup liquid biopsy sample using the first reversible linker 30a according to the first embodiment and the second reversible linker 50c according to the third embodiment. Referring to FIG. 12, as described above in FIG. 10, the exosome subset (S) is isolated and recovered using the first reversible linker 30a according to the first embodiment, and as described above in FIG. 11, the third The exosome subset (SA) may be isolated using the second reversible linker 50c according to the embodiment. At this time, the exosome subpopulation (S) includes the binder 33a of the first reversible linker 30a, but when the exosome subpopulation (SA) is isolated, it reacts with the binder 33a according to the first embodiment Since another binder 33c is coupled to the fixing member 10 in the third embodiment, that is, the second reversible linker 50c that operates by a different mechanism from the first reversible linker 30a is used, the aforementioned blocking process is unnecessary.

FIG. 13 shows a method for preparing an exosome subgroup liquid biopsy sample using the first reversible linker 30c according to the third embodiment and the second reversible linker 50a according to the first embodiment. Referring to FIG. 13, after isolating and recovering the exosome subpopulation (S) using the first reversible linker 30c according to the third embodiment as described above in FIG. 11, as described above in FIG. 10, the first An exosome subset (SA) can be isolated using the second reversible linker 50a according to the first embodiment. At this time, the exosome subgroup (S) includes the ligand 31c of the first reversible linker 30c, but when the exosome subpopulation (SA) is isolated, a second reversible linker 30c and the second Since the reversible linker 50a is employed, the aforementioned blocking process is unnecessary.

14 is a flow chart of a method for preparing an exosome subpopulation liquid biopsy sample according to another embodiment of the present invention.

Referring to FIG. 14 , the method for preparing a subpopulation of exosomes liquid biopsy sample according to another embodiment of the present invention may further include a step of recovering the subgroup of exosomes (S160). Here, the second capture material is coupled to the anchoring member by a second reversible linker, and the captured target exosomes are separated from the anchoring member by dissociating the second reversible linker, thereby recovering a subset of exosomes. Analysis of biomarkers may be performed on the recovered exosomes or biomarkers eluted by dissolving the exosomes may be analyzed.

In addition, a step of enriching the exosome subset (S125) may be further included. After adding the sample solution, the exosome subpopulations can be concentrated before adding the recovery solution. Specifically, by using any one or more of magnetic force, gravity, and centrifugal force, the exosome subgroup is enriched with a fixed member associated with the target disease-related exosomes. For example, a magnetic bead is used as a fixing member, the fixing member is captured in a predetermined spatial region through magnetic force in a mixed solution in which a ligand solution and a sample solution are mixed, and then a part of the mixed solution containing no fixing member ( For example, by removing the supernatant), the exosome subpopulation can be enriched.

In the same way, by using any one or more of magnetic force, gravity, and centrifugal force, the fixation member associated with the target exosomes may be enriched, thereby enriching the exosome subpopulation (S155).

As described above, biomarker profiling analysis for subgroups of exosomes is difficult to realize with conventional immunoisolation techniques that use harsh conditions such as acidic pH when recovering exosomes after immunostaining for exosome surface proteins. Therefore, it is difficult to use existing technologies such as chromatographic separation, magnetic separation, fluorescence-activated cell sorting (FACS), and microfluidic separation for sequential immunoisolation two or more times. In fact, in addition to the simple one-time immunoisolation method based on exosome surface proteins, it is still only a rudimentary exosome separation step, such as fractionation based on exosome particle size, density, and solubility, and continuous immunoaffinity separation process. The isolation of exosome subgroups and the diagnosis of specific diseases based on subgroup biomarker profiling have not yet been reported.

According to the present invention, profiling analysis of a specific exosome subgroup, subgroup, and subgroup biomarkers in a sample through two or more sequential iterative isolations enables early diagnosis of specific diseases such as cancer. . This is because harsh conditions such as acidic pH or a surfactant are not used to dissociate the specific exosomes trapped in the immobilization member, thereby solving the fundamental problem of exosome damage and yield reduction. Therefore, as the existing technology overcomes the problems of difficult preservation of original form and low recovery rate when recovering exosome subpopulations, isolation of target exosome subpopulations associated with specific diseases through sequential iterative isolation and diagnosis of specific diseases based on biomarker profiling It becomes possible.

Furthermore, as described above, if it is possible to separate exosome subgroups, subgroups, and subgroups thereof through the medium of specific exosome surface proteins associated with specific diseases such as cancer, it is possible to treat specific diseases at high concentrations contained in specific exosomes. It is possible to provide nucleic acid biomarkers, such as mRNA and miRNA, for liquid biopsy samples. In particular, as it is known that miRNAs containing exosomes can be stably detected in circulating plasma or serum as they are protected from degradation by RNase, they are emerging as ideal biomarkers for application in clinical diagnosis. Examples of miRNAs (miRs) found in exosomes in plasma include miR-21, -141, -200a, -200c, -203, -205, and -214 associated with ovarian cancer, and miR-214 associated with lung cancer. 17, -3p, -21, -20b, -223, -301, and let-7f, miR-141 and miR-375 associated with prostate cancer, miR-21 associated with breast cancer, and cardiovascular disease There are miR-1 and miR-133a associated, and miR-122 and miR-155 associated with alcoholic liver disease. In addition, examples of miRNAs found in exosomes in urine include miR-29c and CD2APmRNA associated with renal fibrosis.

Most of the studies on exosome-derived miRNAs have focused on cancer diagnosis. According to the study results, specific miRNAs are found in similar concentrations in ovarian cancer biopsy specimens and serum exosomes isolated from the same ovarian cancer patients. However, these exosome-containing miRNAs were not found in normal subjects, suggesting that exosome-derived miRNAs can be used as potential diagnostic markers in biopsy data collection. Early detection and diagnosis of prostate cancer can be performed using prostate-specific antigen (PSA). However, PSA-based tests exhibit low diagnostic specificity and high false positive rates, which may lead to overtreatment of prostate cancer. Therefore, it is required to develop new markers that exhibit higher diagnostic accuracy when diagnosing prostate cancer. According to several studies, increased levels of miR-141 and miR-375 as miRNA (miR) markers are associated with tumor progression in prostate cancer. Since exosome-derived miRNAs are RNase-resistant in serum or plasma specimens, exosome-derived miR-141 and miR-375 can be valuable markers for prostate cancer diagnosis.

The miRNAs contained in exosomes also have potential as biomarkers in the diagnosis of squamous cell cancer (ESCC). According to the study results, exosome-derived miR-21 concentration is elevated in serum from ESCC patients compared to serum from patients with benign tumors without systemic inflammation, and its concentration is clearly associated with tumor progression and aggressiveness. Moreover, exosome-derived miRNAs also have potential as diagnostic biomarkers for cardiovascular disease and renal fibrosis.

In addition to miRNAs, exosome-containing mRNAs also have potential utility as biomarkers in clinical diagnosis. Indeed, urinary exosome samples from prostate cancer patients contain mRNA of PCA3 and mRNA of TMPRSS2, which is the product of a v-ets avian erythroblastosis virus E26 oncogene homolog (ERG) fusion chromosomal rearrangement. Similarly, diagnostic and prognostic markers of renal ischemia/reperfusion injury or antidiuretic hormone action (eg, aquaporin-1 and -2) were also found in urinary exosomes.

As described above, specific diseases to which the exosome subpopulation and subgroup samples prepared using the immunoaffinity separation technology that can be performed under mild conditions can be applied for diagnosis, and exosome-derived proteins and nucleic acids that can be used as isolation and diagnostic markers are listed below. presented in

A number of exosome-derived breast cancer markers have recently been clinically identified, such as CD24 and EpCAM. In addition to breast cancer, exosomal proteins serve as a useful resource of biomarkers for many other cancers. In several studies, one type of inhibitor of apoptosis (IAP) protein was detected in the plasma-derived exosomes of normal people and prostate cancer patients, but the relative content of exosome-containing survivin was significantly higher in the plasma of prostate cancer patients. appeared to be higher. Urinary exosomes, which are easily accessible by non-invasive means, are useful for developing biomarkers for prostate and bladder cancer. Protein profiling (label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis) of urinary exosomes from normal subjects and bladder cancer patients identified proteins contained in urinary exosomes as potential biomarkers for bladder cancer. made sure For example, two known prostate cancer markers, PCA3 and TMPRSS2-ERG, are also present in urinary exosomes taken from prostate cancer patients. Cell surface proteoglycans (protein-linked polysaccharides) are not found in benign pancreatic disease but have been shown to be present in exosomes isolated by FACS from serum of patients with early and late stages of pancreatic cancer. Exosomes isolated from plasma of ovarian cancer patients by ultracentrifugation contained TGF-1 and MAGE 3/6, but these two proteins were not present in exosomes obtained from patients with benign tumors.

In addition to proteins, miRNAs (miRs) and mRNAs were identified as major components of exosome loading at the beginning of the analysis of nucleic acids isolated from exosomes. Further studies have shown that exosomes contain different types of RNAs, including transfer RNAs (tRNAs) and long non-coding RNAs. In addition to RNA species, fragments of single-stranded and double-stranded DNAs have been identified as being present in exosomes. Among exosome-derived nucleic acids, exosome miRNAs are receiving the most attention as cancer diagnostic biomarkers as they secure stability from RNase-dependent degradation. Examples of exosomal nucleic acid biomarkers discovered in preclinical and clinical studies include miR-21 and miR-1246 as exosome nucleic acids in plasma applied to breast cancer diagnosis, and exosome nucleic acids in serum applied in ovarian cancer diagnosis and screening steps. There are miR-21, miR-141, miR-200a, miR-200c, miR-200b, miR-203, miR-205, and miR-214, and let-7a as exosome nucleic acid in serum applied to the diagnosis of colorectal cancer; There are miR-1229, miR-1246, miR-150, miR-21, miR-223, and miR-23a, and miR-141 and miR-375 are nucleosomal nucleic acids in plasma and serum applied to prostate cancer diagnosis, MiR-17-5p and miR-21 are nucleosomal nucleic acids in serum and urine applied to the diagnosis of pancreatic cancer.

In summary, cancer cell-derived exosomes contribute to cancer progression by enhancing the cell-to-cell transport of substances including proteins, lipids, and nucleic acids in the tumorigenic microenvironment. Exosomes are highly valuable as minimally invasive biomarkers for early detection, diagnosis, and prognosis because exosome-loaded components reflect altered states from the original cancer. In fact, since the stability of exosomes is maintained in bodily fluids, exosome-based diagnosis provides higher sensitivity and specificity than conventional tissue biopsy or other liquid biopsy biomarkers.

In addition, exosome markers can be easily obtained from most bodily fluids, and recent advances in exosome isolation technology have made exosome-based diagnostics cost-effective and labor-efficient. However, a major hurdle to overcome in the field of exosome diagnostics is to develop optimal methods for isolating pure exosome populations. Although microvesicles themselves can be useful diagnostic tools for cancer detection, techniques that separate the two major extracellular vesicles into pure exosomes and other populations of microvesicles allow only comparative studies. Another challenge is to understand the mechanisms that regulate the heterogeneity of cancer exosomes in which cancer-derived exosome loaded components become different and thus affect the reproducibility of diagnostic results. Despite its importance, future efforts combining next-generation sequencing of exosomal RNAs and DNAs, proteomic analysis of exosome surface proteins, and immunoaffinity-based capture technologies will advance exosome-based cancer diagnosis to the next level. step will be reached.

Hereinafter, the present invention will be described in more detail through specific examples. Specific examples are only for exemplifying the present invention, and the scope of the present invention is not limited by the examples.

ingredient

A monoclonal antibody that specifically recognizes the unique structure of CBP, which is changed when calcium ions attach to calcium adhesion protein (CBP), has been produced by the present inventors. In addition, a mutated glucose-galactose binding protein (GGBP) was selected as CBP and this protein was coli ; E. coli ) and then purified. Sodium chloride, Trizma, casein (sodium salt form, extract from bovine milk), 3,3',5,5'-tetramethylbenzidine (TMB), sodium dodecyl sulfate (SDS), bovine serum albumin (BSA), and human serum are It was purchased from Sigma (St. Louis, MO, USA). Calcium chloride dehydrate and potassium chloride were supplied from Daejeong (Siheung, Korea). Sulfosuccinimidyl-6-[biotinamido]-6-hexanamido hexanoate (NHS-LC-LC-biotin), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), N-succinimidyl 3-(2-pyridyldithio)- Propionate (SPDP), dithiotheritol (DTT), maleimide, streptavidin (SA), and horseradish peroxidase (HRP) were purchased from Thermo Fisher Scientific (Rockford, IL, USA). Anti-CD63 antibody (Cat. No. 353014), anti-CD81 antibody (Cat. No. 349501), and anti-CD151 antibody (Cat. No. 350404) were supplied by BioLegend (San Diego, CA, USA), and anti-survivin antibody (Cat. No. NB500-201) was supplied by Novus Biologicals (Centennial, CO, USA). Monoclonal antibodies specific for CD9 (Cat. No. MAB1880) or Caveolin-1 (Cav1; Cat. No. MAB5736) were supplied by R&D Systems (Minneapolis, MN, USA). Malignant melanoma or prostate cancer-positive patient samples (serum or plasma) were purchased from Discovery Life Sciences (Huntsville, AL, USA) or Promeddx (Norton, MA, USA). Magnetic beads (Dynabeads M-280 Tosylactivated, 2.8 mm in diameter) and streptavidin-poly-HRP20 were supplied by Invitrogen (Carlsbad, CA, USA) and Fitzgerald (Acton, MA, USA), respectively. Other reagents were also used for analytical products.

Example 1: CD63+ exosome subpopulation sample preparation

1.1 Polymerization between SA and CBP

CBP (400 μg) dissolved in 10 mM phosphate buffer (pH 7.4; PB) was mixed with 20 molar SMCC and reacted at room temperature for 1 hour to activate. SA (2 mg) dissolved in PB was reacted with 5 molar amounts of SPDP for 1 hour, and then chemically reduced with a 10 mM DTT solution containing 5 mM ethylenediaminetetraacetic acid (EDTA) at 37 °C for 1 hour to remove sulfhydryl groups. expressed Each activated component was separated by removing excess reagent through a Sephadex G-15 gel filtration column (volume of 10 ml). Two activated proteins, maleimide-CBP and thilolated-SA, were mixed in a 1:5 molar ratio and reacted at room temperature for 4 hours. Then, the final solution containing the polymer between SA and CBP (CBP-SA) was stored at 4°C in a well-sealed container until use.

1.2 Biotinylation of anti-CD63 antibody

A biotinylated antibody was prepared by reacting the succinimidyl-ester portion of NHS-LC-LC-biotin with an amine group on an antibody molecule. Briefly, the anti-CD63 antibody dissolved in PB solution (PBS) containing 140 mM NaCl was mixed with 20 molar NHS-LC-LC-biotin dissolved in dimethyl sulfoxide (DMSO) for 2 hours at room temperature. reacted during Unreacted reagents in the mixture were removed through size-exclusion chromatography using a Sephadex G-15 column equilibrated with PBS. The purified polymer was dialyzed twice with PBS, concentrated to a concentration of 1 mg/ml using Vivaspin 500, and then stored at 4°C until use.

1.3 Pretreatment of clinical samples

Clinical samples (serum or plasma; 200 μl) were centrifuged at low speed (1,200 g) at 4° C. for 20 minutes to remove precipitates, and then centrifuged again at high speed (10,000 g) at the same temperature for 30 minutes to remove precipitates. The clinical sample supernatant obtained therefrom was filtered through an HM syringe filter (Cat. No. SC13P020SL; 0.2 μm) to prepare a pretreated clinical sample (population sample).

1.4 Immunomagnetic Bead Manufacturing

For magnetic enrichment, an antibody specific to CBP was chemically bonded to the activated tosyl moiety on the solid surface of the magnetic bead with the amine group on the antibody, and then immobilized. After washing three times with Tris-HCl buffer solution, the remaining surface was blocked with Casein-PBS. The beads (total 1 mg) were magnetically separated from the solution, and then the CBP-SA polymer diluted with Ca ²⁺ (10 mM)-containing BSA-Tris attachment solution ('Ca ^{2 +} switch on ^' condition) (5 μg/ml). ml; 1 ml) was added and reacted for 1 hour on a shaker. After washing the beads with the BSA-free attachment solution, the beads were sequentially reacted with biotinylated anti-CD63 antibody (3 μg/ml; 1 ml) diluted in the BSA-Tris attachment solution under the same conditions, followed by magnetic separation.

1.5 CD63 positive (CD63+) exosome isolation

Recovery of CD63-positive (CD63+) exosomes and regeneration of the beads were performed using the magnetic immune beads prepared above. In order to isolate the ^{CD63 +} exosome subpopulation, which is a kind of exosome marker, clinical samples ( ^serum or plasma; 200 μl) was diluted 5 times and added to the magnetic immune beads. After reacting on a shaker for 1 hour, magnetic separation was performed, ^and after adding unbonded components by injecting an adhesion solution containing Ca ²⁺ , washing was performed through magnetic separation. Then, by injecting 20 mM Tris-HCl (pH 7.4) buffer solution ('Ca ²⁺ switch off' condition) containing only 500 mM NaCl, CBP-antibody-exosome conjugates (CD63+ exosomes) attached to magnetic beads subgroup) were recovered through magnetic separation. Then, 100 mM sodium acetate buffer solution (pH 4.5) containing 500 mM NaCl, 5% (v/v) Tween-20, and 0.02% (w/v) thimerosal was supplied to the magnetic beads, and from the magnetic beads The magnetic beads were regenerated by washing thoroughly until there was no protein leakage.

Example 2: Exosome surface biomarker profiling analysis

2.1 Enzyme Immunoassay (ELISA)

To characterize the CD63+ exosome subpopulation isolated through magnetic separation in the above, biomarker profiling analysis present on the surface of the exosome was performed. To this end, antibodies specific to each biomarker were immobilized in each well of the microtiter plate, and sandwich enzyme immunoassay for each target biomarker (CD9, CD81, Cav1, and survivin) was performed using this. As target biomarkers, CD81, Cav1, and survivin, which are exosome biomarkers known to be involved in tumor progression, promotion, or metastasis, and CD9, which is known to be a tumor suppressor, were selected according to the type of cancer disease. First, antibodies (5 μg/ml; 100 μl) specific to each biomarker were added to the wells, fixed at 37° C. for 1 hour, and then washed with deionized water. The remaining surface was treated with a 20 mM Tris-HCl (pH 7.4) buffer solution containing 0.5% casein under the same conditions as above. CD63+ exosome subpopulation samples isolated through magnetic separation as described above or, as a control, population samples before isolation were added to wells fixed with antibodies specific to each biomarker and reacted at 37° C. for 10 hours. After washing, streptavidin-poly-HRP20 (66 ng/ml; 100 μl) was added and reacted at room temperature for 1 hour. After washing again, HRP substrate solution (0.05 M acetate buffer containing 0.003% hydrogen peroxide and 100 μg/ml TMB, pH 5.1; 200 μl) was added and reacted for 15 minutes, followed by sulfuric acid solution (2 M, 50 μl). The reaction was stopped by further addition. The color development in each well was quantified with an ELISA reader (Synergy H4, BioTek Inc.; Winooski, VT, USA) at 450 nm absorbance.

2.2 Correlation analysis between biomarkers

As described above, an immunoassay for CD81, Cav1, survivin, and CD9 was performed using the exosome population obtained after pretreatment of malignant melanoma-positive clinical samples (serum or plasma) and normal human samples (serum) as analysis samples. , In this case, since the exosome population sample is reacted with the solid-phase capture antibody specific to each biomarker and the signal proportional to the remaining immunocomplex is measured after separation, quantitative analysis is performed on the subgroup containing each biomarker. 15 is an experimental result comparing correlations between tumor suppression markers and tumor progression, promotion, and metastasis markers after profiling analysis of exosome surface markers for exosome population analysis samples from each of a normal group and a malignant melanoma patient group. From the quantitative signals obtained through immunoassay, the distribution of the analyzed samples was displayed on a two-dimensional graph to obtain the correlation between the CD9 signal, a tumor suppression marker, and the CD81, Cav1, and survivin signals, which are tumor progression, promotion, or metastasis markers. . With reference to this, when the exosome population was used as an analysis sample, it was found that data between cancer-positive samples and normal samples were mixed and distributed without clear correlation. For reference, when exosome population samples are used as analysis samples, signal values of Cav1 and survivin are very low in most samples, so it is difficult to secure reliability for correlation analysis.

In addition, as described above, after pretreatment of the two sample groups, malignant melanoma-positive and normal, exosome subpopulations were isolated and recovered for the CD63 biomarker using the reversible immunoisolation system using the 'switchable adhesion reaction' described above. Population analysis samples were prepared, and immunoassays were performed for four types of biomarkers. As a singularity in immunoassay, the exosomes containing each biomarker present in the exosome subpopulation react with each specific antibody immobilized on the solid phase, and then are separated and generate a signal proportional to that from the remaining immune complex, so that the specific subgroup It is possible to perform profiling analysis on . Figure 16 shows the exosome surface marker profiling analysis for the exosome subpopulation using the exosome subgroup isolated from the exosome population of each of the normal group and the malignant melanoma patient group as analysis samples, and then tumor progression and promotion compared to tumor suppressor markers It is an experimental result comparing the correlation between , and metastasis markers with the normal sample group. From the quantitative signals obtained through enzyme immunoassay, the distribution of the analyzed samples is displayed on a two-dimensional graph to obtain the correlation between the CD9 signal, a tumor suppression marker, and the CD81, Cav1, and survivin signals, which are markers of tumor progression, promotion, or metastasis, respectively. did In the first graph depicting the CD81 signal, a tumor progression marker, against the CD9 signal, a tumor suppression marker, it can be seen that the composition ratio of the two markers in each sample is kept constant because the normal samples showed a linear proportional relationship between the two markers. On the other hand, malignant melanoma-positive samples showed an inversely proportional relationship between the two markers, from which it was found that the composition ratio of CD81 to CD9 was different for each cancer-positive sample.

Comparing the two results between these different sample groups, it is predicted that the heterogeneity of the composition ratio between surface markers of exosomes increases in the occurrence of malignant melanoma. In particular, as the cancer stage progressed from stage 1 to stage 4, the CD81 signal increased, whereas the CD9 signal decreased, increasing the CD81/CD9 signal ratio. Therefore, the increase in exosome heterogeneity for marker liver composition ratio was correlated with the stage of cancer. predicted to be related.

In the second and third graphs showing the CD9 signal versus the Cav1 or survivin signal from the results of the exosome subgroup immunoassay, the distribution of cancer samples also seems to deviate from the linear proportional relationship between the two markers shown in normal samples. However, the increase in heterogeneity as described above was not evident.

Figure 17 compares the correlation between tumor progression, promotion, and metastasis markers between a normal sample group and a cancer-positive sample group after profiling analysis of exosome surface markers for each of the exosome population analysis samples of the normal group and the malignant melanoma patient group. As an experimental result, immunoassay was performed for each biomarker on exosome population analysis samples obtained after pretreatment of melanoma cancer-positive clinical samples (serum or plasma) and normal human samples (serum) as described above, and from the results This is the result of analyzing the correlation between CD81, Cav1, and survivin, which are tumor progression, promotion, or metastasis markers. For reference, when the exosome population was used as the analysis sample, as shown in FIG. 15, the data between cancer-positive samples and normal samples were mixed and distributed without any clear correlation, and in most samples, the signal values of Cav1 and survivin were very low, so there was no correlation. It was not easy to secure the reliability of the relationship analysis.

18 shows correlations between tumor progression, promotion, and metastasis markers after surface marker profiling analysis of exosome subpopulations in the exosome subgroup analysis samples isolated from the exosome populations of the normal group and the malignant melanoma patient group, respectively. As a result of the experiment comparing the sample group and the cancer-positive sample group, the two sample groups, malignant melanoma-positive and normal, were pretreated, and then separated for CD63 using the above-described reversible immunoisolation system, and the exosome subset was recovered. In the same manner as in, after immunoassay for each exosome subpopulation biomarker, correlation analysis was performed for three types of tumor progression, promotion, or metastasis markers. In the first graph plotting the CD81 signal versus the Cav1 signal from the immunoassay results, normal samples showed an approximately linear proportional relationship between the two markers. On the other hand, cancer-positive samples showed an inverse relationship between the two markers, showing that the Cav1 signal was higher than the CD81 signal in the early stage of cancer disease and reversed in the late stage.

In the second and third graphs plotting the survivin signal versus the CD81 or Cav1 signal from the immunoassay results, the above phenomenon did not appear clearly. However, in the third graph depicting the Cav1 signal versus the survivin signal, the distribution of cancer samples seems to deviate from the linear proportional relationship between the two markers shown in the normal samples.

Example 3: Parameter setting and analysis for initial diagnosis of disease using biomarker profiling analysis results

3.1 Diagnosis parameter setting

Since the concentration of exosomes is different between samples and the concentration changes according to the sample dilution ratio, it is difficult to directly compare the signal difference for each biomarker between samples obtained from the immunoassay results. In order to alleviate this problem, two or more new parameters were newly defined for each of the diagnostic target diseases, normalized by taking the signal ratio of one biomarker to another biomarker. Accordingly, normalized parameters 1, 2, and 3 were set by taking the ratio of signals for CD81, Cav1, and survivin, which are tumor progression, promotion, or metastasis markers, to signals for CD9, a tumor suppressor marker. Here, parameter 1 = CD81 signal/CD9 signal, parameter 2 = Cav1 signal/CD9 signal, and parameter 3 = survivin signal/CD9 signal.

3.2 2D parameter analysis

After converting the profiling analysis results for exosomes containing each biomarker in the sample into each parameter as defined above, two-dimensional analysis was performed by selecting the optimal two parameters in pairs, and using the results, normal subjects To distinguish between the sample and the target disease sample, two parameter values were set up, and the discriminative power for target disease negative and positive samples, that is, positive accuracy (sensitivity) and negative accuracy (specificity), was determined. For reference, parameter values of the partition line dividing the target disease sample and the normal sample may be changed according to analysis conditions.

19 shows the results of biomarker profiling analysis performed by applying newly defined parameters to the exosome population analysis samples of each of the normal group and the malignant melanoma patient group. This is the result of biomarker profiling analysis by applying newly defined parameters to the exosome subgroup analysis sample isolated from the bom population.

In the biomarker profiling results for the exosome population sample before immunoisolation (see FIG. 19), the value for parameter 3 was too small, regardless of whether it was a normal person sample or a cancer patient sample, so that the difference in profiling between normal and cancer patient samples proved difficult to ascertain. This is because, as described in FIGS. 15 and 17 above, the immunoassay results for survivin were too small in all population samples.

Next, in the biomarker profiling results for the exosome subpopulation samples obtained after immunoisolation and recovery (see FIG. 20), the values of the three parameters were sufficiently high to be comparable between samples. In particular, since the values for parameter 3 increased remarkably, profiling analysis for the selected exosome biomarker becomes possible when an appropriate exosome subpopulation is isolated and recovered and used as an analysis sample.

Such an effect is made possible by the effect of reducing the analysis observation window and the synergistic effect of concentrating the target concentration because the target exosome is separated and recovered and used as an analysis sample. Comparing the observation window size, exosome species released from many different cells are mixed in the body fluid used as the analysis sample, so the exosome subpopulation containing the target exosome is separated from the body fluid and recovered and used as the analysis sample This has the effect of narrowing the observation window in a desirable direction by excluding unrelated exosome species from disease-related exosome species. In this case, when diagnosing a disease, interference caused by non-specific reactions of unrelated biomarkers can be minimized. Furthermore, when the concentration process is used in the separation into the subgroups described above, the concentration of the target exosomes is increased to obtain the effect of recovery in high yield. Therefore, according to the present invention, when the exosome subset is used as an immunoassay sample for diagnosis, the interference effect caused by non-specific exosomes is minimized, and at the same time, the target exosome concentration is increased, thereby obtaining a positive synergistic effect on the accuracy of the profiling analysis. .

21 is a newly defined exosome population analysis sample (FIG. 21(A)) and an exosome subgroup analysis sample (FIG. 21(B)) isolated from each of a normal group and a malignant melanoma patient group. This is the result of synthesizing the two-dimensional parameter analysis for the biomarker profiling analysis results with parameters applied.

In order to investigate the effect of using the exosome subpopulation recovered after immunoisolation as a diagnostic sample, first, the profiling analysis results for subgroups containing each biomarker in the exosome population sample before isolation (FIGS. 15 to 18) Reference) was converted to each parameter as defined in FIGS. 19 to 20, and first, a two-dimensional analysis was performed on parameters 1 and 2 (see FIG. 21 (A)). As a result, it was found that the two-dimensional distributions of normal and cancer samples were mixed with no difference between the sample groups.

Next, on the profiling results for subgroups in subgroup samples obtained through recovery after immunoseparation, two-dimensional analysis was performed on parameters 1 and 2 after converting to parameters in the same manner as above (Fig. 21 (B ) reference). As a result, it was found that normal samples were distributed in a limited area of the two parameter values (parameter 1 < about 3.2; parameter 2 < about 0.9) (refer to the dotted line in the graph), and in the case of the malignant melanoma sample used, that compartment It was found to be distributed in the upper or outer regions. However, one normal sample (N52) was located outside the compartment, which is considered to be caused by an analysis error or an unknown sample at the time of analysis. Therefore, when the present invention is used, a normalization effect occurs through sequential immunoisolation, enabling selective analysis of disease-related subgroup exosome biomarkers, and through this, discrimination against cancer disease negative and positive samples is improved. appeared. For reference, the parameter values of the partition line dividing the cancer sample and the normal sample are shown as examples, and the values may change depending on the analysis conditions.

Furthermore, it was shown that performing a two-dimensional analysis of parameters 1 and 2 could predict the stage of malignant melanoma disease. In particular, looking at the analysis results of the samples of malignant melanoma I, II, and IV patients, the value of parameter 1 of each sample increased proportionally as the stage progressed. Notably, the parameter 1 values of melanoma cancer stage I and II samples were distributed in the range of normal sample values, but the parameter 2 values of these samples were found to be higher than the range of values for normal samples. This is predicted to be because the Cav1 marker, which regulates the value of parameter 2, is involved in tumor suppression in the early stage of cancer development and is highly expressed. Therefore, it is judged that not only the stage of malignant melanoma can be predicted through the two-dimensional analysis of parameters 1 and 2, but also early diagnosis in the early stage of cancer occurrence. From this, through the selection and analysis of exosome subpopulation multiple markers related to a specific cancer disease, it is possible to solve the problem of cancer diagnosis in the existing cancer stage or metastasis stage.

22 is a result of 2-dimensional parameter analysis on the results of subgroup biomarker profiling analysis to which newly defined parameters are applied to exosome subgroup analysis samples of each of a normal group and a malignant melanoma patient group. In addition to the two-dimensional analysis of parameters 1 and 2 for the exosome subpopulation profiling analysis results (see the left side of FIG. 22), parameters 1 and 3 (see the middle of FIG. 22) and parameters 2 and 3 (see the middle of FIG. 22) 2-dimensional analysis was performed on (see right). As a result of the two-dimensional analysis of parameters 1 and 3 (refer to the center of FIG. 22), samples of normal subjects and samples of malignant melanoma patients were distributed in the graph without distinction. Therefore, it is judged that the analysis of parameters 1 and 3 cannot distinguish between malignant melanoma samples and normal samples. On the other hand, the analysis of parameters 2 and 3 (see the right side of FIG. 22) showed that normal samples and malignant melanoma patient samples could be distinguished. 0.7; parameter 3 < about 2.5) (refer to the dotted line compartment in the graph), and in the case of the malignant melanoma sample used, it was found to be distributed on or outside the compartment. Therefore, two-dimensional analysis of parameters 2 and 3 through exosome subpopulation profiling analysis made it possible to distinguish malignant melanoma samples from normal samples.

3.3 Parameter Panel Composition

In order to maximize the sensitivity and specificity of diagnosis according to the target disease, two or more parameters were formed into a panel and used for early diagnosis of the disease. As described above, a two-dimensional analysis was performed on each parameter pair, and a parameter panel was formed by selecting a parameter pair that provides the maximum discriminative power for the target disease negative and positive samples. If necessary, one or more types of parameters were sequentially applied to the first configured parameter panel to determine the final parameter panel maximizing the accuracy of distinguishing between negative and positive and a diagnostic algorithm using the same.

FIG. 23 is a result of 2-dimensional parameter analysis on the results of biomarker profiling analysis using newly defined parameters for exosome subgroup analysis samples in each of a normal group and a prostate cancer patient group. In the case of prostate cancer, CD151 was added as an exosome surface marker, and a total of 5 biomarkers (CD9, CD81, Cav1, survivin, and CD151) were profiled for CD63+ exosome subgroup samples through the same process as described above Ring analysis was performed. Similar to the above, by taking the ratio of the signals for any two biomarkers, the normalized parameters 4, 5, 6, and 7, respectively, were newly defined. Here, parameter 4 = CD9 signal/Cav1 signal, parameter 5 = CD151 signal/CD9 signal, parameter 6 = survivin signal/CD9 signal, and parameter 7 = Cav1 signal/CD81 signal.

For the results of exosome subpopulation profiling performed after subgroup immunoisolation and recovery of prostate cancer and normal subjects, a two-dimensional analysis was first performed on parameters 4 and 5. As a result of the two-dimensional analysis of parameters 4 and 5 (see the top of FIG. 23 ), most of the normal samples were found to be distributed in a limited range of the values of the two parameters (under practical conditions, parameter 4 < about 3.3; parameter 5 < about 1.5). (see the dotted line in the graph), and most of the prostate cancer samples appeared to be distributed outside that compartment. When these two parameters were used, positive accuracy, that is, sensitivity of 80% and negative accuracy, that is, specificity of 100% were shown under the analysis conditions.

Precise analysis was performed on prostate cancer-positive samples (P7, P10, P13, and P18) located in the above-mentioned compartments (see bottom of FIG. 23). As described above, when using two parameters (ie, parameters 4 and 5), the clinical sensitivity was limited to 80% (see the lower left of FIG. 23), and a two-dimensional analysis was performed for parameters 4 and 6 by adding parameter 6. (ie, 3-parameter analysis), negative samples stayed in the negative region, while P13 among positive samples moved to the positive region (refer to the bottom center of FIG. 23). From this, the sensitivity was found to rise to 85%. Furthermore, when a two-dimensional analysis (i.e., four-parameter analysis) is performed on parameters 6 and 7 by adding parameter 7, negative samples still remain in the negative region, while P7 and P18 among positive samples move to the positive region. (see lower right of FIG. 23). In the end, the clinical sensitivity increased by 95% when analyzing the four parameters, and the clinical performance for the final prostate cancer sample showed a sensitivity of 95% and specificity of 100%.

Furthermore, two-dimensional analysis of parameters 4 and 5 was shown to be able to predict prostate cancer disease stage. In particular, looking at the analysis results of the patient's samples according to the stage number (roman numeral in parentheses next to the sample number) of prostate cancer set by the clinical judgment criteria (see the upper part of FIG. 23), based on the dividing line defined above In the lower right corner, 9 types of prostate cancer-positive samples were distributed, of which 8 types were stage I and stage II positive samples, and one type of stage IV sample was also included. Based on the partition line, 7 types of prostate cancer-positive samples were distributed in the upper left corner, of which 5 types were stage III and stage IV positive samples, and two types of stage I and II samples were also included. Overall, each sample decreased in inverse proportion to the value of parameter 4 as the stage progressed, and at the same time increased in proportion to the value of parameter 5 (see arrow in FIG. 23). Notably, early prostate cancer samples (stage I and II samples) were distributed in the lower right corner, that is, a region in which the value of parameter 4 was particularly high and the value of parameter 5 was low. Therefore, it is judged that not only can the stage of prostate cancer be predicted through sequential two-dimensional analysis of two or more standardized biomarkers, but also early diagnosis in the early stage of cancer occurrence.

Example 4: Exosome miRNA biomarker profiling analysis

4.1 Cell line culture and exosome sample preparation

DU145, LNCaP, and PC3 cell lines, which are generally known as prostate cancer-derived cell lines, were selected, and after culturing each cell line, the culture medium was collected to prepare an exosome population sample as in clinical samples. As described above, CD63+ subpopulation exosome samples were prepared from the cell line culture exosome population using the 'switchable reversible adhesion reaction'. Sequentially, CD63+/CD81+ subpopulation samples were prepared from the CD63+ subpopulation exosomes using a magnetic immunoisolation method using anti-CD81 antibody-immobilized magnetic beads. As a control, the kidney cell-derived HEK293 cell line was selected, and exosome samples of each population, subgroup, and subgroup were prepared through the same procedure as described above.

More specifically, HEK293 cell line derived from human embryonic kidney cells and LNCaP, DU145, and PC3 cell lines derived from prostate cancer were purchased from Korea Cell Line Bank (Seoul, Korea). Each cell line was cultured for 34-39 hours in a CO ₂ incubator using an optimal medium containing 10% fetal bovine serum (FBS) and antibiotics, that is, DMEM medium containing HEPES for HEK293 and RPMI 1640 medium for prostate cancer-derived cell lines. did Thereafter, the medium of each cell line was replaced with a medium containing 5% exosome-free FBS, and cultured under the same conditions. In each culture medium, the cells were separated by centrifugation and then the supernatant was collected. The separated supernatant was centrifuged at low speed to remove the precipitate, and then centrifuged at high speed again to remove the precipitate. The supernatant of the clinical sample obtained therefrom was filtered through an HM syringe filter (0.2 μm pore size) to prepare an exosome population sample.

Using the same immunomagnetic isolation method as in Example 1, CD63+ subpopulation exosome samples were prepared from the cell line culture medium exosome population. Sequentially, the anti-CD81 antibody was chemically immobilized on the magnetic beads according to the procedure provided by the magnetic bead manufacturer, and CD63+/CD81+ subgroup samples were prepared from CD63+ subgroup exosomes through immunomagnetic separation using the anti-CD81 antibody.

4.2 Exosome miRNA extraction, cDNA synthesis, and real-time PCR

As target nucleic acid biomarkers, four types of prostate cancer-related miRNAs were selected: miR-17, miR-21, miR-221, and miR-375. After extracting miRNA markers from each exosome sample, cDNA was synthesized through a reverse transcription reaction, and the product was sensitive to the C _t (cycle threshold) value for each sample as a signal through an amplification process using a polymerase chain reaction. was measured. All procedures were performed using commercially available kits. The signals for each measured miRNA marker are signal ratios between the two markers for standardization, i.e. miR-21 signal/miR-17 signal, miR-221 signal/miR-17 signal, and miR-375 signal/miR-17 signal ratio. were obtained, respectively.

In detail, three types of exosome samples, that is, population, subgroup, and subgroup samples, were prepared for each cell line, and miRNAs contained in each sample were extracted using the miRNeasy Micro Kit (QIAGEN, 217084) . Each step for this was performed according to the instructions provided by the manufacturer. The extracted miRNA was quantified using a spectroscopy instrument (ND-2000; NanoDrop Technologies, Wilmington, DE, USA). As a next step, miRNA (2 ng) extracted from each exosome sample was subjected to reverse transcription using the TaqMan Advanced miRNA cDNA Synthesis kit (Applied Biosystems, Grand Island, NY, USA) according to the manufacturer's instructions. It was performed on the μl reaction volume scale. Then, miRNA amplification reaction was performed, and real-time PCR reaction was carried out quantitatively. For quantitative PCR, TaqMan microRNA Assay kits (Applied Biosystems) for target miRNAs (miR-17, miR-21, miR-221, and miR-375) were used, CFX 384 (Bio-Rad Laboratories, CA, USA). It was quantified using the instrument. The miRNA in each sample was measured repeatedly, and each Ct value was normalized to miR-17 and then scaled to the signal ratio of each Ct value in the exosome population of the HEK293 cell line selected as a control. change) was calculated.

4.3 Analysis of exosomal miRNA biomarkers in samples

Changes in signal ratios between the three miRNAs defined as described above were monitored for the exosome population, subpopulation, and subpopulation samples prepared from each cell line, and the results are shown in FIG. 24 . 24 shows the results of analyzing prostate cancer miRNA biomarkers contained in exosome population, CD63+ subpopulation, and CD62+/CD81+ subpopulation samples of prostate cancer cell-derived cell line cultures. For easy comparison, all signal ratios were scaled for each signal ratio in the exosome population of the HEK293 cell line selected as a control, and the fold change was calculated and plotted based on this.

First of all, in the case of the miR-21 signal/miR-17 signal ratio (see upper part of FIG. 24), all three prostate cancer cell lines compared to the control HEK293 cell line significantly increased in the exosome population and subgroup samples, but did not increase in the subgroup. or not measured. In particular, this fold increase was remarkably observed in the DU145 cell line, indicating a specific association with the miR-21 biomarker. As another peculiarity, since miR-21 was not measured in CD63+/CD81+ subpopulation exosomes of all the selected cell lines, an inverse correlation between exosome subpopulation surface markers and miRNAs contained in exosomes is predicted.

Next, in the case of the miR-221 signal/miR-17 signal ratio (see middle of FIG. 24 ), the signal ratio in the DU145 cell line was similar to that in the control cell line population regardless of the exosome population. Signal ratios in LNCaP and PC3 cell lines showed no fold increase in the population and subpopulation, whereas significantly higher levels were observed in the subpopulation exosomes. This result is opposite to the case of the miR-21 signal/miR-17 signal ratio, and showed a proportional correlation between the exosome subpopulation surface marker and the miRNA contained in the exosome.

Finally, in the case of the miR-375 signal/miR-17 signal ratio (see bottom of Figure 24), no fold increase was observed in all exosome samples in DU145 and LNCaP, similar to the control group, but fold increase was observed in the PC3 cell line. The increase was observed, especially in subpopulation samples. Since the PC3 cell line was derived from prostate cancer with a relatively high degree of malignancy, miR-375 contained in CD63+/CD81+ subpopulation exosomes is expected to be used as a marker associated with cancer progression. From this, it is expected that if the exosome subpopulation is used as an analysis sample for a specific miRNA, a specific biomarker can be selected according to the type of cell.

Eventually, using the present invention, proteins and miRNAs present in exosome subpopulations related to cancer diseases such as malignant melanoma and prostate cancer are selected as biomarkers, converted into standardized parameters, and used for analysis, each cancer disease It is judged that it is possible to discriminate between negative and positive samples, and furthermore, early diagnosis of cancer disease. According to the present invention, it is predicted that a parameter or a combination of parameters effective for diagnosis determination may change depending on the type of cancer disease. For reference, in the graphs of FIGS. 22 and 23, each parameter value for determining whether cancer disease is negative or positive may change according to analysis conditions, so the value itself has no absolute meaning, and is two-dimensional (in some cases, one-dimensional). The relative distribution of cancer patient samples to normal samples in the graph obtained through parameter analysis is meaningful.

Although the present invention has been described in detail through specific examples and experimental examples, this is for explaining the present invention in detail, the present invention is not limited thereto, and within the technical spirit of the present invention, those skilled in the art It is clear that modification or improvement is possible by the person.

All simple modifications or changes of the present invention belong to the scope of the present invention, and the specific protection scope of the present invention will be clarified by the appended claims.

1: exosome 1a: disease-related exosome
1b: target exosome 10: anchoring member
20: first capture material 30: first reversible linker
31a to 31d: ligand 32: competitive reaction ligand
33a to 33d: binder 35a, 35b: recognition material
40: second capture material 50: second reversible linker
m: separation marker m1: first separation marker
m2: first segregation marker B: bulk exosome population
S: exosome subpopulation SA: exosome hypopopulation
C: polymer

Claims (11)

(a) from the exosome population sample containing exosomes secreted from a plurality of cells in the body of each of the normal and patient groups, target exosomes related to a given disease are separated into subpopulations, and the target exosomes population is included Preparing an exosome liquid biopsy sample;
(b) measuring quantitative signals for each of a plurality of biomarkers of the target exosomes contained in the exosome liquid biopsy sample; and
(c) analyzing the exosome liquid biopsy samples of each of the normal group and the patient group according to the correlation between a plurality of biomarkers based on the measured value of the quantitative signal;
In step (c),
Setting a plurality of parameters as a ratio of each of the quantitative signals to the reference signal with a quantitative signal of any one of the plurality of biomarkers as a reference signal; and
Analyzing the exosome liquid biopsy sample according to the correlation between the two different parameters,
Setting the parameters,
Coordinates the exosome liquid biopsy sample according to the measured values in a plurality of XY coordinate systems in which the quantitative signal of any one of the plurality of biomarkers is the X-axis and the quantitative signal of the other is the Y-axis step;
calculating a slope of the exosome liquid biopsy sample of each of the normal group and the patient group; and
setting the parameter using the biomarker constituting the XY coordinate system in which the difference between the slope of the exosome liquid biopsy sample of the normal group and the slope of the exosome liquid biopsy sample of the patient group is relatively large; Exosome liquid biopsy sample analysis method comprising.
The method of claim 1,
In step (a),
binding a first capture material that specifically binds to a first separation marker possessed by the first target exosome associated with the predetermined disease to a solid-state fixation member using a first reversible linker;
Separating an exosome subset, which is a group of the first target exosomes, by reacting the exosome population sample with the first capture material coupled to the fixation member to capture a plurality of the first target exosomes step; and
and recovering the exosome subset by dissociating the first reversible linker so that the captured first target exosome is separated from the fixation member.
The method of claim 2,
Between isolating the exosome subset and recovering the exosome subset,
The exosome liquid biopsy sample analysis method further comprising: concentrating the fixing member in which the first target exosomes are captured using at least one of magnetic force, gravity, and centrifugal force.
The method of claim 2,
In step (a),
binding a second capture material that specifically binds to a second separation marker possessed by a second target exosome in the exosome subset to the fixing member; and
The second target exosomes are captured by reacting the exosome subgroup sample containing the recovered exosome subgroup with the second capture material coupled to the fixing member to capture a plurality of the second target exosomes A method for preparing an exosome liquid biopsy sample comprising the steps of isolating an exosome subpopulation, which is a group of.
The method of claim 4,
In the step of coupling the second capture material to the fixing member, the second capture material is coupled to the fixing member by a second reversible linker;
The method of analyzing an exosome liquid biopsy sample, further comprising dissociating the second reversible linker so that the captured second target exosomes are separated from the fixation member, and recovering the exosome subpopulation.
The method of claim 5,
Between isolating the exosome subpopulation and recovering the exosome subpopulation,
The exosome liquid biopsy sample analysis method further comprising: concentrating the fixing member in which the second target exosomes are captured using at least one of magnetic force, gravity, and centrifugal force.
delete delete delete The method of claim 1,
The step of analyzing the exosome liquid biopsy sample,
By substituting the measured values into the first parameter and the second parameter in a first XY coordinate system in which one of the plurality of parameters, the first parameter, is the X axis and the other second parameter is the Y axis, Coordinating the exosome liquid biopsy sample according to the calculated parameter value;
setting negative and positive regions in which the exosome liquid biopsy sample of the normal group is distributed in the first XY coordinate system; and
Reanalyzing the exosome liquid biopsy sample belonging to the negative or positive region among the coordinated exosome liquid biopsy samples according to the correlation between the two parameters in which at least one of the first parameter and the second parameter is different Exosome liquid biopsy analysis method comprising the step of doing.
The method of claim 1,
The biomarker is
An exosome liquid biopsy analysis method comprising at least one of a predetermined disease-related biomarker arranged on the surface of the exosome and a predetermined disease-related biomarker eluted from the exosome.

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