[go: up one dir, main page]

WO2024258237A1 - Biopsie liquide basée sur une mesure locale d'un changement d'hétérogénéité d'exosomes - Google Patents

Biopsie liquide basée sur une mesure locale d'un changement d'hétérogénéité d'exosomes Download PDF

Info

Publication number
WO2024258237A1
WO2024258237A1 PCT/KR2024/008252 KR2024008252W WO2024258237A1 WO 2024258237 A1 WO2024258237 A1 WO 2024258237A1 KR 2024008252 W KR2024008252 W KR 2024008252W WO 2024258237 A1 WO2024258237 A1 WO 2024258237A1
Authority
WO
WIPO (PCT)
Prior art keywords
exosome
exosomes
heterogeneity
subpopulation
marker
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/KR2024/008252
Other languages
English (en)
Korean (ko)
Inventor
백세환
김택민
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sol Bio Corp
Original Assignee
Sol Bio Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sol Bio Corp filed Critical Sol Bio Corp
Priority claimed from KR1020240077631A external-priority patent/KR20240176084A/ko
Publication of WO2024258237A1 publication Critical patent/WO2024258237A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to a liquid biopsy based on the measurement of local exosome heterogeneity changes, and relates to a liquid biopsy technique for producing a liquid biopsy sample by separating exosomes related to a specific disease, such as cancer, from an exosome population sample into subpopulations with respect to the separation marker using a predetermined surface marker as a separation marker, and analyzing exosomes loaded with the same triple marker using two other surface markers in addition to the separation marker as capture and detection markers.
  • a specific disease such as cancer
  • Liquid biopsy is a non-invasive method that relatively easily extracts body fluids such as blood, saliva, and urine and analyzes cells related to certain diseases or components released from them (peptides, proteins, nucleic acids, organelles, specific substances, etc.). Since it can confirm the occurrence of certain diseases at an early stage, it is attracting attention as an alternative to existing testing methods such as imaging diagnosis, tissue examination, and blood test methods.
  • Liquid biopsy has the following advantages over tissue biopsy in diagnosing certain diseases, such as cancer.
  • tissue biopsy has a limited sample size, which can be insufficient when divided for multiple diagnostic purposes, whereas liquid biopsy allows for multiple collections of samples from various body fluids as needed.
  • liquid biopsy offers relatively higher accuracy (accuracy of measuring positive samples) and sensitivity (accuracy of measuring negative samples), and thus can provide reliable results for clinical decision-making.
  • CTCs circulating tumor cells
  • exosomes are recognized as potential biomarkers for liquid biopsy.
  • CTCs have excellent concentration and selectivity and stability of isolated nucleic acids, but their distribution in blood is extremely low, making them difficult to use for early diagnosis of cancer.
  • Cell-free DNA has a large distribution in blood, but it is difficult to concentrate and select, and the stability of isolated nucleic acids is low.
  • overcoming the noise problem during analysis is a major issue due to the presence of a large amount of non-specific DNA unrelated to the target disease.
  • exosomes have a large distribution in blood, are easy to concentrate and select, and can be separated into stable nucleic acids.
  • the size, density, and composition of proteins and nucleic acid components contained in each exosome particle are extremely heterogeneous, separation into disease-related pure exosomes must be performed first.
  • Exosomes are gaining attention as liquid biopsy biomarkers. Exosomes (30 to 150 nm in size) are secreted from cells in the human body in the form of microvesicles and reflect the characteristics of the cells from which they originated, and can be extracted from various body fluids such as blood, saliva, and urine. Since exosomes are vesicles made of a lipid bilayer, they are not only stable in the body, but also free from attacks by protease, RNase, and DNase. Therefore, research is increasing to use exosomes as new biomarkers for diagnosing specific diseases such as cancer and degenerative diseases, and in fact, products are being developed for the early diagnosis and companion diagnosis of cancer using components such as proteins and RNA contained in exosomes as markers.
  • exosomes are known to play a role in tumorigenesis, cancer metastasis, and drug resistance.
  • tumor cells Like other cells in the microscopic environment where tumors develop, tumor cells also secrete exosomes and other microvesicles, which contribute to tumor progression and tumor cell migration during metastasis by promoting angiogenesis.
  • Tumor cell-derived vesicles also contain molecules involved in immunosuppression, which can inactivate T lymphocytes or natural killer cells, or promote the differentiation of regulatory T lymphocytes or myeloid cells to suppress immune responses.
  • the transfer of tumor activity by exosomes secreted from tumor cells has also been reported.
  • exosomes derived from all cells exist in a mixed form in body fluids, it is difficult to accurately diagnose specific diseases such as cancer with an exosome sample in the form of a bulk population simply separated from body fluids.
  • concentration of exosomes derived from specific disease-related cells in body fluids is relatively very low, so separation and concentration of target exosomes from body fluids must be performed in advance for early diagnosis.
  • density difference-based ultracentrifugation has been adopted as the gold standard separation process due to its high reproducibility and relatively stable process.
  • it has the disadvantage of requiring expensive equipment and taking a long separation time.
  • methods have been developed to separate exosomes by precipitating them, to separate them by size using size exclusion chromatography, and to separate them by size using microfluidics.
  • exosome separation technologies provide all exosomes present in a body fluid as a single population, they have limitations in selectively detecting extremely small amounts of exosomes derived from specific cells such as cancer.
  • exosomes released from cells contain various biochemical components (membrane proteins, internal proteins, RNA, etc.) in the exosome membrane or interior, making them very heterogeneous in terms of component composition.
  • certain exosomes derived from cancer cells contain mRNA or miRNA that are involved in the local environmental composition in the early stages of angiogenesis and cancer metastasis.
  • the above-mentioned specific exosomes during cancer metastasis contain biochemical components that induce the death of immune cells or are involved in the process of converting normal cells into cancer cells.
  • the present invention is to solve the problems of the above-described prior art, and one aspect of the present invention is to provide a liquid biopsy method for discovering biomarkers related to specific diseases such as cancer, degenerative diseases, heart diseases, infectious diseases, etc., and utilizing them for diagnosis, by isolating and recovering a subpopulation, a hypopopulation, or a lower subpopulation of exosomes with high yield and in its original state from an exosome population for an isolation marker, and analyzing exosomes loaded with the same triple marker using two surface markers other than the isolation marker in the liquid biopsy sample as capture and detection markers.
  • specific diseases such as cancer, degenerative diseases, heart diseases, infectious diseases, etc.
  • a liquid biopsy comprises the steps of: (a) preparing an exosome liquid biopsy sample including the exosome subpopulation by separating and recovering first target exosomes having a first surface marker in common from an exosome population sample containing heterogeneous exosomes secreted from each of a plurality of cells in a human body into subpopulations; (b) measuring a quantitative signal for a third surface marker of second target exosomes belonging to the exosome subpopulation and having a second surface marker in common, thereby obtaining a triple marker signal; (c) calculating an exosome heterogeneity index as a ratio of the measured values of the double marker signal and the triple marker signal obtained by measuring the quantitative signal for the second surface marker of the first target exosomes; and (d) analyzing the exosome liquid biopsy sample based on the calculated exosome heterogeneity index.
  • steps (a) to (c) are sequentially performed for the exosome population samples of each of a normal group and a patient group with a specific disease, thereby calculating the exosome heterogeneity index for the normal group and the exosome heterogeneity index for the patient group, respectively, and in step (d), by comparing the respectively calculated exosome heterogeneity index for the normal group and the exosome heterogeneity index for the patient group, a biomarker related to the specific disease of the patient group can be selected.
  • steps (a) to (c) performed for selecting the biomarker are repeatedly performed for the exosome population sample of the disease screening subject, thereby calculating the exosome heterogeneity index for the disease screening subject, and in step (d), by comparing the calculated exosome heterogeneity index for the disease screening subject with the exosome heterogeneity index for the patient group, disease screening information for the disease screening subject can be generated.
  • the liquid biopsy according to an embodiment of the present invention, among the three surface markers possessed by the heterogeneous exosomes in the exosome population sample, at least one of a total of six selection paths is selected, in which one is selected as the first surface marker, another is selected as the second surface marker, and the remaining one is selected as the third surface marker, and by sequentially repeating steps (a) to (c), N (N is a natural number greater than or equal to 1 and less than or equal to 6) exosome heterogeneity indices can be calculated.
  • N exosome heterogeneity indices for the normal group and N exosome heterogeneity indices for the patient group are respectively calculated from the exosome population samples of the normal group and the patient group with a specific disease, and in the step (d), based on the N exosome heterogeneity indices for the normal group and the N exosome heterogeneity indices for the patient group respectively calculated, a selection path of a biomarker related to the specific disease of the patient group can be selected.
  • new indices are generated through arithmetic operations or functionalization of the N exosome heterogeneity indices for the normal group and the N exosome heterogeneity indices for the patient group, and based on the generated new indices, a selection path of a biomarker related to a specific disease of the patient group can be selected.
  • the exosome population sample of the disease screening subject is repeatedly performed from step (a) to step (c) according to the selection path performed for the selection path of the biomarker, thereby calculating the exosome heterogeneity index for the disease screening subject, and in step (d), the disease screening information for the disease screening subject can be generated by comparing the calculated exosome heterogeneity index for the disease screening subject with the exosome heterogeneity index for the patient group calculated according to the selection path performed for the selection path of the biomarker.
  • the exosome population sample of the disease screening subject is repeatedly performed from step (a) to step (c) according to the selection path performed for the selection path of the biomarker, thereby calculating the exosome heterogeneity index for the disease screening subject, and in step (d), the disease screening information for the disease screening subject can be generated by comparing the calculated new index for the disease screening subject with the new index for the patient group calculated according to the selection path performed for the selection path of the biomarker.
  • the step (a) may include: a step of binding a first specific binding material that specifically binds to the first surface marker to a solid-state fixation member using a first reversible linker; a step of reacting the exosome population sample with the first specific binding material bound to the fixation member to capture a plurality of the first target exosomes, thereby isolating an exosome subpopulation, which is a group of the first target exosomes; and a step of dissociating the first reversible linker so that the captured first target exosomes are separated from the fixation member, thereby recovering the exosome subpopulation.
  • the method may further include a step of concentrating the fixation member in which the first target exosome is captured using at least one of magnetic force, gravity, and centrifugal force.
  • the step (a) may include a step of binding a second specific binding material that specifically binds to the second surface marker possessed by the second target exosomes in the exosome subpopulation to the fixing member; and a step of reacting an exosome subpopulation sample containing the recovered exosome subpopulation with the second specific binding material bound to the fixing member to capture a plurality of the second target exosomes, thereby isolating an exosome subpopulation, which is a population of the second target exosomes.
  • the step of binding the second specific binding material to the fixing member in the step of binding the second specific binding material to the fixing member, the step of dissociating the second reversible linker so that the second specific binding material is bound to the fixing member by the second reversible linker and the captured second target exosome is separated from the fixing member, thereby recovering the exosome subpopulation may be further included.
  • the dual marker signal can be measured for the exosome population sample.
  • the liquid biopsy sample is separated and concentrated in the form of an exosome subpopulation, hypo-population, or lower subpopulation, the non-specific exosomes are removed, minimizing noise during analysis and maximizing specific exosome signals.
  • exosomes are used as biomarkers for specific diseases such as cancer, interference by non-specific exosomes due to exosome heterogeneity, which is a current problem, is minimized, so that information on the physiological characteristics and lineage of parent cells can be accurately identified from the liquid biopsy sample.
  • the present invention when the present invention is applied to disease diagnosis, it can significantly increase the diagnostic accuracy for specific diseases such as cancer, degenerative diseases, heart diseases, and infectious diseases, and can exhibit an early clinical diagnostic effect that allows diagnosis of diseases at an early stage of the disease. Furthermore, since it is possible to obtain information on the progression or decline of the disease depending on the treatment agent during disease treatment, it can also be applied to companion diagnosis that provides customized screening for individuals.
  • FIG. 1 is a flow chart of a liquid biopsy according to an embodiment of the present invention.
  • FIG. 3 is a drawing explaining a triple marker signal measurement process of liquid biopsy according to an embodiment of the present invention.
  • FIG. 4 is a diagram illustrating a marker separation path for calculating an exosome heterogeneity index of a liquid biopsy according to an embodiment of the present invention.
  • FIGS. 6 to 9 are schematic diagrams illustrating the first reversible linker and the second reversible linker illustrated in FIG. 5 according to various embodiments.
  • Figures 10 to 16 are flowcharts of a method for preparing an exosome liquid biopsy sample according to an embodiment of the present invention.
  • FIG. 17 is a diagram illustrating a novel sequential marker selection process (A) using Neutral Release technology in contrast to conventional methods, and a conceptual representation (B) of the resulting new approach.
  • Figure 20 is a diagram illustrating a sequential marker selection strategy for quantifying the triple marker positive subclass 'T' using three pan-exosome tetraspanin markers, namely CD9, CD63, and CD81.
  • Figure 21 is a diagram illustrating the process of quantifying the size of the triple marker positive subpopulation (T).
  • Figure 23 is a table showing a comprehensive experimental plan for quantifying exosome heterogeneity changes.
  • Figure 24 is a table comparing manual exosome magnetic separation and automated exosome magnetic separation conditions.
  • Figure 26 is a table showing the setting conditions for operation of the automatic exosome separation system.
  • Figure 27 shows the performance comparison results of automatic exosome separation using the Neutral Release protocol and manual separation.
  • Figure 28 shows the results of quantifying the changes in exosome heterogeneity for samples with various concentrations of serum containing cell line RWPE-1 exosomes (1.28 ⁇ 10 11 particles/mL).
  • Figure 29 is a graph showing the change in heterogeneity index according to concentration for exosome samples produced from RWPE-1 (1.28 ⁇ 10 11 particles/mL), LNCaP (1.18 ⁇ 10 11 particles/mL), and PC3 (2.53 ⁇ 10 11 particles/mL), respectively.
  • Figure 31 is a graph showing the results of quantifying the heterogeneity changes of dual marker-positive exosomes and triple marker-positive exosomes according to the stopover site (the region of the exosome subpopulation that has a specific dual marker in common), and the exosome heterogeneity change index.
  • Figure 33 is a diagram comparing sample classification performance based on local heterogeneity change indices using different third markers (CD81, CD151, TSPAN8).
  • Figure 34 shows the results of evaluating CD151 as a third marker at various stopover sites.
  • Figure 35 shows the results of measuring heterogeneity changes for CD151 at stopover site 1 (an exosome subpopulation region that shares dual markers of CD9 and CD63) using LNCaP exosome samples.
  • Figure 37 is a schematic illustrating a comprehensive demonstration of the potential use of specific stopover sites for cancer biomarker discovery.
  • Figure 39 is a table comparing the performance of three stopover sites in determining heterogeneity changes of the third marker.
  • Figure 40 is a graph showing the specificity of CD151 heterogeneity changes at stopover site 1 according to various tissues and cancer states.
  • FIG. 1 is a flow chart of a liquid biopsy according to an embodiment of the present invention
  • FIG. 2 is a drawing explaining a liquid biopsy process according to an embodiment of the present invention
  • FIG. 3 is a drawing explaining a triple marker signal measurement process of a liquid biopsy according to an embodiment of the present invention.
  • the liquid biopsy comprises the steps of: (S100) preparing an exosome liquid biopsy sample including the exosome subgroup by separating and recovering first target exosomes having a first surface marker (A) in common from an exosome population sample containing heterogeneous exosomes secreted from multiple cells in a human body; (S200) measuring a quantitative signal for a third surface marker of second target exosomes belonging to the exosome subgroup and having a second surface marker in common to obtain a triple marker signal; (S300) calculating a local exosome heterogeneity index (R) as a ratio of the measured values of the double marker signal and the triple marker signal obtained by measuring the quantitative signal for the second surface marker (B) of the first target exosome; and (S300) analyzing the exosome liquid biopsy sample based on the calculated exosome heterogeneity index (R). Includes step (S400).
  • the present invention relates to a liquid biopsy technique for preparing a liquid biopsy sample by separating exosomes related to a specific disease, such as cancer, from an exosome population sample into subpopulations with respect to the separation marker using a predetermined surface marker as a separation marker, and analyzing exosomes loaded with the same triple marker using two other surface markers in addition to the separation marker as capture and detection markers.
  • the liquid biopsy according to an embodiment of the present invention includes an exosome liquid biopsy sample preparation step (S100), a triple marker signal measurement step (S200), an exosome heterogeneity index calculation step (S300), and a sample analysis step (S400).
  • the exosome liquid biopsy sample preparation step (S100) prepares an exosome liquid biopsy sample by separating and recovering first target exosomes having a first surface marker (A) in common from an exosome population sample, which is an analysis sample to be analyzed, into subpopulations.
  • the exosome subpopulation includes a subpopulation, a subpopulation, and subpopulations below it.
  • the exosome subpopulation is a population of exosomes that belong to the exosome population and have at least one specific marker in common, and the exosome subpopulation is a population of exosomes that have at least one other marker in common as part of the exosome subpopulation.
  • the exosome subpopulation as one of the subpopulations below it is a population of exosomes that have at least another one or more other markers in common as part of the exosome subpopulation, and other subpopulations below it can be defined in this manner.
  • the exosome population sample includes exosomes secreted from multiple cells or tissues in the human body, and the exosomes here contain heterogeneous exosomes.
  • the present invention can utilize an immunoaffinity separation technique, which will be described later.
  • exosomes included in an exosome liquid biopsy sample have a first surface marker (A) in common, some of them may have a second surface marker (B) in addition to the first surface marker (A), and some of them may have a third surface marker (C) in addition to the first surface marker (A) and the second surface marker (B).
  • an exosome having a first surface marker (A) is referred to as a single-marker exosome
  • an exosome having the first and second surface markers (A, B) is referred to as a dual-marker exosome
  • an exosome having all of the first, second, and third surface markers (A, B, C) is referred to as a triple-marker exosome.
  • the first, second, and third surface markers (A, B, C) are predetermined disease-related biomarkers arranged on the surface of exosomes.
  • Changes in the biomarker composition distribution contained in exosomes in liquid biopsy samples can provide personalized diagnostic and therapeutic information for individual patients, and are also important as signs of accurate prognosis or differential diagnosis. Changes in exosome-derived biomarkers may not only indicate responses to more than a single condition, but may also be due to complex factors, as plasma exosomes are secreted by various cellular sources from both blood cells and surrounding tissues.
  • various proteins are heterogeneously distributed on the surface of exosomes in body fluids.
  • specific surface proteins associated with cancer are present in a complex manner in exosomes from normal individuals, although their expression ratios differ.
  • the types and expression levels of surface proteins such as tetraspanins (CD9, CD63, CD81, CD82, CD151, etc.), cell type-specific molecules such as histocompatibility complex (MHC) class-I and class-II, adhesion molecules, HSP60, and HSP70, may increase or decrease in relation to the characteristics of parent cells.
  • MHC histocompatibility complex
  • CD63 a tetraspanin-family exosome surface protein
  • exosome-derived proteins have been identified as potential biomarkers for a variety of diseases, including cancer, hepatitis, liver disease, central nervous system disease, and kidney disease. To date, hundreds of exosome proteins have been reported to be associated with specific diseases, and thus can be used as diagnostic or isolation targets in future product development.
  • markers associated with cancer include CD63, caveolin-1, TYRP2, VLA-4, HSP70, and HSP90 for melanoma; survivin for prostate cancer; and L1CAM, CD24, ADAM10, EMMPRIN, and claudin for ovarian cancer.
  • Markers associated with bladder cancer include EGF, resistin, and retinoic acid-induced protein 3; and PSA and PCA3 for prostate cancer.
  • CD81 is an exosomal protein marker associated with chronic hepatitis C in plasma.
  • CD81 an exosomal marker of the tetraspanin family, plays a crucial role in hepatitis C virus attachment and/or entry into cells.
  • the concentration of serum exosomal CD81 is increased in patients with chronic hepatitis C and is also associated with worsening inflammation and fibrosis. This suggests that exosomal CD81 may be a potential marker for the diagnosis and treatment response of hepatitis C.
  • Fetuin-A and ATF 3 are exosomal protein markers associated with acute kidney injury in urine
  • CD26, CD81, S1c3A1, and CD10 are markers associated with liver damage.
  • urinary exosomes proteins contained in urinary exosomes that can be obtained noninvasively are being explored for their potential diagnostic utility, especially for urinary tract diseases.
  • fetuin-A contained in urinary exosomes is increased in patients with acute kidney injury in the intensive care unit compared to healthy subjects.
  • ATF3 is also found only in exosomes isolated from patients with chronic kidney disease or patients with acute kidney injury compared to healthy subjects.
  • urinary exosomal proteins are also being investigated as potential biomarkers for bladder and prostate cancer. Our results showed that two types of prostate cancer biomarkers, PCA-3 and PSA, are present in exosomes isolated from urine of prostate cancer patients.
  • exosome-containing proteins identified in other body fluids are expected to be used as target platforms for the development of diagnostic assay systems in the future.
  • exosome markers have not been clearly characterized in relation to diseases and furthermore, have not been applied clinically.
  • the markers do not represent the state or progression of the disease on their own. It is predicted that the disease-related exosome markers will act on target cells in a complex manner with multiple and complex interactions among marker groups. In particular, it is thought that the types, numbers, and exosome locations of the involved markers are encoded in cancer cell-derived exosomes to evade the body's immune system surveillance.
  • the mapping method of exosome markers using a simple sandwich immunoassay has reached its technical limit in diagnosing target diseases with high accuracy. Therefore, a technology to simultaneously and in-depth immunoassay the compositional distribution of three or more multi-protein markers for exosomes in the same sample is essential to elucidate the correlation between exosome markers and specific diseases.
  • a technology to simultaneously and in-depth immunoassay the compositional distribution of three or more multi-protein markers for exosomes in the same sample is essential to elucidate the correlation between exosome markers and specific diseases.
  • the compositional ratio and correlation between pan-exosome tetraspanins CD9, CD63, and CD81
  • the compositional ratio and correlation can be easily measured, it will be possible to predict the source of the exosome, and thus, it will be possible to track abnormal changes such as diseases in specific tissues at an early stage through exosome liquid biopsy.
  • the exosome markers in the sample must be separated in advance.
  • the first target exosome contained in the exosome liquid biopsy sample may be an exosome constituting an exosome subpopulation separated and recovered for a specific one-type marker from an exosome population sample, an exosome subpopulation separated and recovered for two specific markers, or an exosome subpopulation separated and recovered for three specific markers.
  • the exosome liquid biopsy sample may be an exosome subpopulation sample separated as a population of first target exosomes having the first surface marker (A) in common, or an exosome subpopulation sample separated as a population of second target exosomes belonging to an exosome subpopulation and further having the second surface marker (B) in common.
  • the first target exosome is an exosome that has the first surface marker (B) in common
  • the second target exosome is an exosome that has the first surface marker (A) and the second surface marker (B) in common.
  • markers are generally defined using antibodies specific for each of two markers, i.e., capture markers and detection markers.
  • capture markers and detection markers i.e., antibodies specific for each of two markers.
  • a quantitative signal for the triple marker exosomes in the exosome liquid biopsy sample is measured.
  • a second target exosome i.e., a dual marker exosome, which has a second surface marker (B) in common is first separated from the exosome liquid biopsy sample.
  • the second target exosome can be separated by capturing using a capture antibody that specifically binds to the second surface marker (B).
  • a quantitative signal for the third surface marker (C) of the separated second target exosome is measured.
  • a detection antibody that specifically binds to the third surface marker (C) and an enzyme can be combined to measure the quantitative signal.
  • a representative detection method thereof is an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the quantitative signal measurement method is not necessarily limited to this, and there is no special limitation on the signal measurement method as long as the quantitative signal for the triple marker exosome can be measured.
  • a second target exosome having a second surface marker (B) is already separated within the exosome liquid biopsy sample, and therefore a separate second target exosome separation process may not be necessary.
  • the exosome heterogeneity index (R) is generated based on the triple marker signal acquired in the triple marker signal measurement step (S200).
  • a measurement value of a dual marker signal is required as a variable other than the measurement value of the triple marker signal.
  • the dual marker signal can be obtained by measuring a quantitative signal for the second surface marker (B) of the first target exosome.
  • the dual marker signal can be measured for the exosome population sample.
  • a capture antibody that specifically binds to the first surface marker (A) is reacted to capture the first target exosome, and then a detection antibody that specifically binds to the second surface marker (B) and an enzyme are combined to measure the quantitative signal. That is, the dual marker signal can be measured using an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the exosome heterogeneity index (R) can be defined as the ratio of the measurement of the triple marker signal to the measurement of the dual marker signal.
  • the exosome liquid biopsy sample is analyzed using the exosome heterogeneity index (R).
  • the exosome heterogeneity index (R) is an index that shows the interrelationship between three surface markers (A, B, C), and the interrelationship may show different aspects depending on the disease. Accordingly, based on the exosome heterogeneity index (R), a biomarker related to a specific disease can be discovered and used to detect the disease.
  • the exosome population samples of each of a normal group and a patient group with a specific disease are sequentially performed from the exosome liquid biopsy sample preparation step (S100) to the exosome heterogeneity index calculation step (S300), so as to calculate the exosome heterogeneity index (R) for the normal group and the exosome heterogeneity index (R) for the patient group, respectively.
  • the sample analysis step (S400) the calculated exosome heterogeneity index (R) for the normal group and the exosome heterogeneity index (R) for the patient group are compared, so as to select a biomarker related to the specific disease of the patient group.
  • the exosome heterogeneity index (R) for the normal group is related to the specific disease of the patient group.
  • disease screening information for a subject of disease screening can be generated. That is, from the exosome liquid biopsy sample preparation step (S100) performed for selecting the biomarker to the exosome heterogeneity index calculation step (S300) are repeatedly performed for the exosome population sample of the subject of disease screening to calculate the exosome heterogeneity index (R) for the subject of disease screening, and then, in the sample analysis step (S400), the calculated exosome heterogeneity index (R) for the subject of disease screening can be compared with the exosome heterogeneity index (R) for the patient group to generate disease screening information for the subject of disease screening.
  • FIG. 4 is a diagram illustrating a marker separation path for calculating an exosome heterogeneity index of a liquid biopsy according to an embodiment of the present invention.
  • the exosome heterogeneity index (R) may have different values depending on the selection order of three surface markers (A, B, C).
  • the three surface markers (A, B, C) of heterogeneous exosomes in an exosome population sample is selected as the first surface marker, another one is selected as the second surface marker, and the remaining one is selected as the third surface marker, a total of six different selection paths are created.
  • N is a natural number greater than or equal to 1 and less than or equal to 6
  • exosome heterogeneity indices (R) can be calculated.
  • the N exosome heterogeneity indices (R) exhibit different characteristics depending on the selection path for the three surface markers (A, B, C)
  • biomarkers related to specific diseases can be discovered based on the exosome heterogeneity indices (R) for each selection path, and these can be used for the diagnosis of the disease.
  • N exosome heterogeneity indices (R) for the normal group and N exosome heterogeneity indices (R) for the patient group are respectively calculated.
  • a selection path of a biomarker related to a specific disease of the patient group can be selected.
  • the biomarker selection path can be selected by considering the correlation between at least two or more indices among the N exosome heterogeneity indices (R).
  • disease screening information for a subject for disease screening can be generated. For example, by repeatedly performing the exosome liquid biopsy sample preparation step (S100) to the exosome heterogeneity index calculation step (S300) according to the selection path performed for the biomarker selection path selection for the exosome population sample of the subject for disease screening, an exosome heterogeneity index (R) for the subject for disease screening is calculated, and by comparing the calculated exosome heterogeneity index (R) for the subject for disease screening with the exosome heterogeneity index (R) for the patient group calculated according to the selection path performed for the biomarker selection path selection, disease screening information for the subject for disease screening can be generated.
  • the exosome heterogeneity index (R) for the patient group can also be compared with the exosome heterogeneity index (R) for the subject for disease screening.
  • the sample analysis step (S400) based on the N exosome heterogeneity indices (R) for the normal group and the N exosome heterogeneity indices (R) for the patient group, the four basic operations, i.e. addition, subtraction, multiplication, and division, may be performed between the N exosome heterogeneity indices (R) so that the contrast between the normal group and the patient group can be maximized, or a new indice in which the exosome heterogeneity indices (R) are functionalized may be used.
  • the exosome liquid biopsy sample preparation step (S100) to the exosome heterogeneity index calculation step (S300) are repeatedly performed according to the selection path performed in the selection path selection of the biomarker, targeting the exosome population sample of the disease screening subject, to calculate the exosome heterogeneity index for the disease screening subject, and in the sample analysis step (S400), the calculated new index for the disease screening subject is compared with the new index for the patient group calculated according to the selection path performed in the selection path selection of the biomarker, so that disease screening information for the disease screening subject can be generated.
  • FIG. 5 is a schematic diagram illustrating a device for preparing exosome subpopulation and hypo-subpopulation samples according to an embodiment of the present invention.
  • the device for preparing an exosome liquid biopsy sample according to an embodiment of the present invention may include a solid-state fixation member (10), a first specific binding material (20) that specifically binds to a first surface marker (m1) of a disease-related exosome (1a) among exosomes (1) secreted from a plurality of cells to capture the disease-related first target exosome (1a), and a first reversible linker (30) that releasably binds the fixation member (10) and the first specific binding material (20).
  • first specific binding material (20) is bound to the unbound fixing member (10), and a second specific binding material (40) that specifically binds to a second surface marker (m2) of a second target exosome (1b) in an exosome subpopulation (S), which is a group of disease-related first target exosomes (1a), to capture the second target exosome (1b) may be further included.
  • a second specific binding material (40) that specifically binds to a second surface marker (m2) of a second target exosome (1b) in an exosome subpopulation (S), which is a group of disease-related first target exosomes (1a), to capture the second target exosome (1b) may be further included.
  • a second reversible linker (50) may be further included as a means for binding the second specific binding material (40) to the fixing member (10).
  • This fixing member (10) can 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.
  • the fixed member (10) is not necessarily limited to the above, and there is no special limitation as long as it is a material that can be combined with each of the first reversible linker (30), the second specific binding material (40), or the second reversible linker (50).
  • the first specific binding material (20) and the second specific binding material (40) are materials that selectively capture a given exosome (1).
  • the first specific binding material (20) and the second specific binding material (40) capture the exosome (1) by specifically binding to a surface marker (m) of the exosome (1).
  • the surface marker (m) is a biomarker that may relatively increase in body fluid compared to a normal person when a specific disease occurs, and may include proteins, nucleic acids, lipid components, sugar components, peptides, and other biochemical components present on the surface of the exosome (1).
  • This surface marker (m) may specifically bind to the first specific binding material (20) and the second specific binding material (40) through immunochemical interactions such as antigen-antibody reactions.
  • the first specific binding material (20) and the second specific binding material (40) may be specific antibodies that perform an antigen-antibody reaction with surface proteins of exosomes (1).
  • the first surface marker (m1) that specifically binds to the first specific binding material (20) exists on the surface of a predetermined disease-related first target exosome (1a). Therefore, from a bulk exosome population (B), which is a population of exosomes (1) separated from a plurality of cells, the disease-related first target exosome (1a) having the first surface marker (m1) can be separated by being captured by the first specific binding material (20). Here, the population of separated disease-related first target exosomes (1a) becomes an exosome subpopulation (S).
  • the second target exosome (1b) having the second surface marker (m2) reacts specifically with the second specific binding material (40) and is captured, thereby separating the exosome subpopulation (SA) within the exosome subpopulation (S).
  • the first surface marker (m1) on the exosome (1) exhibits relatively low accuracy when used for diagnosing a specific disease compared to the second surface marker (m2), but exists in a relatively high concentration in body fluids, and is positioned at a high level when quantitatively systematically classifying the exosome (1) population according to markers, thereby allowing it to have more comprehensive characteristics.
  • an exosome liquid biopsy sample is prepared by first separating, concentrating, and recovering an exosome subpopulation (S) positive for a disease-specific marker (a first surface marker) predicted to have a low concentration in a body fluid, or an exosome subpopulation (SA) recovered by second separating and concentrating the exosome subpopulation (S) for a disease-specific marker (a second surface marker) can be used as an exosome liquid biopsy sample.
  • S exosome subpopulation
  • SA exosome subpopulation
  • the number of repetitions of the separation, concentration, and recovery processes that can be sequentially performed for different exosome surface protein markers can be extended to two or more times depending on the purpose, and a subset of the exosome subpopulation (SA) recovered through separation and concentration for another disease-specific marker can be used as an exosome liquid biopsy sample.
  • SA exosome subpopulation
  • first specific binding material (20) can be fixed to the fixing member (10) and then separated via the first reversible linker (30).
  • the second specific binding material (40) can be directly fixed to the fixing member (10) without separation, or can be fixed to the fixing member (10) and then separated by introducing the second reversible linker (50).
  • the first reversible linker (30) detachably binds the first specific binding material (20) to the fixing member (10), and the other second reversible linker (50) detachably binds the second specific binding material (40) to the fixing member (10).
  • the first specific binding material (20) is not bound to the fixing member (10) to which the second specific binding material (40) is fixed. That is, the second specific binding material (40) is fixed on the fixing member (10) to which the first specific binding material (20) is not bound through the second reversible linker (50).
  • the first reversible linker (30) and the second reversible linker (50) may be formed of the same material and structure, or may be implemented differently, and the specific material and structure will be described later.
  • Exosome immunoaffinity separation under mild conditions is possible using a reversible linker based on a ligand-binder attachment reaction.
  • the reversible linker initially immobilizes a specific exosome (1)-specific binding material (antibody) on a fixing member (10), but after capturing the exosome (1) in the sample on the fixing member (10), it dissociates through a change in reaction conditions, allowing the captured exosome (1) to be recovered.
  • ion-ion or ion-ion receptor binding reactions in addition to sugar-sugar receptor binding reactions through hydrophilic and hydrophobic bonds, ion-ion or ion-ion receptor binding reactions, substrate-enzyme reactions, antigen-antibody reactions, nucleic acid conjugation reactions, hormone-cell receptor reactions, and ligand-nanostructure reactions, polyhistidine-tag, biotin-avidin reactions, and attachment reactions using chelators, in particular, can be used as the reversible linker.
  • Such attachment reactions can be dissociated by adjusting the affinity between the binder and the ligand of the linker, such as a change in the binder structure after binding or a competitive reaction for the binder. Therefore, by using a reversible linker, it is possible to separate and recover the second target exosome (1b) under mild conditions after immunologically capturing the exosome (1) in the sample without using harsh conditions such as acidic pH.
  • any process that enables separation and recovery using physical, biological, and chemical properties such as density, size, solubility, membrane permeability, molecular structure, and magnetism can be used as an immunoaffinity separation process using the above-mentioned reversible linker based on the attachment reaction.
  • a specially designed immunoaffinity separation process such as chromatography or magnetic separation is used, it is possible to separate and recover a trace amount of exosomes in a body fluid by controlling the affinity between the binder and the ligand in the linker.
  • a ‘switchable attachment reaction’ of a recognition material that recognizes the structural change of the binder in the linker induced by the attachment of the ligand is used, it is possible to separate and recover exosomes through the immunoseparation process only depending on the presence or absence of the ligand.
  • a competitive attachment reaction method using a ligand-like structure that competitively reacts with the ligand for the binder in the ligand-binder reaction during immunoseparation is introduced, it is possible to separate and recover exosomes depending on the presence or absence of the competitive attachment reaction.
  • FIGS. 6 to 9 are schematic diagrams illustrating the first reversible linker and the second reversible linker illustrated in FIG. 5 according to various embodiments.
  • the reversible linker (30a, 50a) may include a ligand (31a), a binder (33a), and a recognition material (35a).
  • the ligand (31a) is a substance that is releasably bound 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 undergoes a conformation change by an attachment reaction with the ligand (31a).
  • the binder (33a) may include at least one selected from the group consisting of a sugar-binding protein, an ion-binding protein, an enzyme, an antibody, an aptamer, a cell receptor, and a nanostructure.
  • the binder (33a) and the ligand (31a) may form a binder-ligand pair, such as a sugar-binding protein-sugar molecule pair, an ion-binding protein-ion pair, an enzyme-substrate pair, an antigen-antibody pair, an aptamer-ligand pair, a cell receptor-ligand pair, a nanostructure-ligand pair, and the like.
  • a binder-ligand pair such as a sugar-binding protein-sugar molecule pair, an ion-binding protein-ion pair, an enzyme-substrate pair, an antigen-antibody pair, an aptamer-ligand pair, a cell receptor-ligand pair, a nanostructure-ligand pair, and the like.
  • Representative examples of the binder (33a) and the ligand (31a) include calcium binding protein (CBP) and calcium ion.
  • the binder (33a) and the ligand (31a) are not necessarily limited to the above materials, and any material may be used as long as it is a material that can be releasably bonded to each other and causes a structural change.
  • the binder (33a) is polymerized with the first specific binding material (20) and/or the second specific binding material (40) to form a binder-specific binding material polymer (C).
  • the recognition material (35a) is a substance that specifically binds to the binder (33a) whose structure has been changed by binding to the ligand (31a), and may include at least one selected from the group consisting of antibodies, protein receptors, cell receptors, aptamers, enzymes, nanoparticles, nanostructures, and heavy metal chelators.
  • the recognition material (35a) since the above substance is only an example of the recognition material (35a), the scope of the present invention should not be limited thereto.
  • a calcium ion (ligand) first binds to a calcium binding protein (binder), causing a structural change in the calcium binding protein, and an antigen-antibody reaction occurs between the calcium binding protein whose structure has been changed and the antibody (recognition material), so that they can bind to each other.
  • the recognition material (35a) is fixed to the surface of the fixing member (10).
  • the captured exosomes (1a, 1b) can be recovered by dissociating the reversible linker through a change in reaction conditions.
  • the binder (33a) whose structure has been changed can be dissociated when it is restored to its original structure.
  • an antibody recognition material
  • a calcium-binding protein binding protein
  • the structure of the calcium-binding protein can be restored and the calcium-binding protein and antibody can be separated.
  • the reversible linker (30b, 50b) may include a ligand (31b), a binder (33b), and a recognition material (35b).
  • the ligand (31b), binder (33b), and recognition material (35b) of the reversible linker (30b, 50b) according to the second embodiment correspond to the ligand (31a), binder (33a), and recognition material (35a) of the reversible linker (30a, 50a) according to the first embodiment, respectively, and the following description focuses mainly on the differences.
  • the binder (33a) is polymerized with the specific binding material (20, 40) to form a polymer (C), and the recognition material (35a) is fixed to the surface of the fixing member (10), whereas in the case of the reversible linker (30b, 50b) according to the second embodiment, the binder (33b) is fixed to the surface of the fixing member (10), and the recognition material (35b) is polymerized with the specific binding material (20, 40) to form a recognition material-specific binding material polymer (C).
  • the recognition material (35b) is polymerized with the specific binding material (20, 40) to form a recognition material-specific binding material polymer (C).
  • a recognition material such as an antibody, that specifically recognizes a structural change caused by binding of a binder (33a, 33b), such as a calcium binding protein, to a ligand (31a, 31b), such as a calcium ion, that specifically reacts therewith can be used.
  • This binder-recognition material reaction such as an antigen-antibody reaction
  • a 'switch-like reversible binding' because it rapidly binds or dissociates depending on the presence or absence of a ligand (31a, 31b), such as a calcium ion, and is similar to the principle of turning a light on and off by operating a switch.
  • the reversible linker (30c, 50c) may include a ligand (31c) and a binder (33c).
  • the ligand (31c) of the reversible linker (30c, 50c) according to the third embodiment is polymerized with the first specific binding material (20) and/or the second specific binding material (40) to form a specific binding material-ligand polymer (C).
  • the binder (33c) of the reversible linker (30c, 50c) is fixed to the surface of the fixing member (10), and captures the exosome (1a, 1b) to the fixing member (10) by binding to the specific binding material-ligand polymer (C) that specifically binds to the exosome (1a, 1b).
  • the binder (33c) binds to the ligand (31c), and when a competitive reaction ligand (32) that competes with the ligand (31c) is added, the competitive reaction ligand (32) is attached.
  • the ligand (31c) is detached, the exosome (1a, 1b) can be separated from the fixing member (10) and recovered.
  • the ligand (31c) may include at least one selected from the group consisting of vitamins, sugar molecules, ions, substrates, antigens, amino acids, peptides, nucleic acids, growth factors, and hormones, such as His-tag (polyhistidine-tag), biotin, etc.
  • the binder (33c) may include at least one selected from the group consisting of divalent metal ions, avidins, sugar-binding proteins, ion-binding proteins, enzymes, antibodies, aptamers, protein receptors, cell receptors, and nanostructures.
  • the competitive ligand (32) may include at least one selected from the group consisting of vitamins, sugar molecules, ions, substrates, antigens, amino acids, peptides, nucleic acids, growth factors, and hormones, such as imidazole, biotin, etc.
  • His-stack can be used as a ligand (31c), a divalent metal ion can be used as a binder (33c), and imidazole can be used as a competitive ligand (32).
  • His-stack is an amino acid motif consisting of at least 6 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.
  • the protein labeled with His-stack can be captured by utilizing the affinity for nickel or cobalt, which are divalent ions bound to a chelator on a Sepharose/agarose resin, and then recovered by adding a recovery solution containing imidazole.
  • the reversible linker (30d, 50d) may include a ligand (31d) and a binder (33d).
  • the ligand (31d) and the binder (33d) of the reversible linker (30d, 50d) according to the fourth embodiment correspond to the ligand (31c) and the binder (33c) of the reversible linker (30c, 50c) according to the third embodiment, respectively, and the following description focuses mainly on the differences.
  • the ligand (31c) is polymerized with the specific binding material (20, 40), and the binder (33c) is fixed to the surface of the fixing member (10), whereas in the reversible linker (30d, 50d) according to the fourth embodiment, the ligand (31d) is fixed to the surface of the fixing member (10), and the binder (33d) is polymerized with the specific binding material (20, 40) to form the specific binding material-binder polymer (C).
  • the ligand (31d) is similarly bound to the binder (33d), but is separated from the binder (33d) by a competitive reaction with the competitive reaction ligand (32).
  • the exosome liquid biopsy sample manufacturing device may further include a reactor (not shown).
  • the reactor may accommodate a fixing member (10) therein.
  • disease-related exosomes (1a) are captured by the fixing member (10) via the first reversible linker (30) and the first specific binding material (20) therein, and an exosome subpopulation (S) is separated, and the first reversible linker (30) is dissociated, and the exosome subpopulation (S) is recovered.
  • the recovered exosome subpopulation (S) sample is added to the reactor, and a second target exosome (1b) is captured by the fixing member (10) using the second reversible linker (50) and the second specific binding material (40), and an exosome subpopulation (SA) is separated.
  • the separated exosome subpopulation (SA) is recovered when the second reversible linker (50) is dissociated.
  • the exosome subpopulation (S) and/or the exosome subpopulation (SA) can be concentrated by concentrating the fixation member (10) using at least one of a magnetic force, a gravity, and a centrifugal force.
  • the fixation member (10) when a magnetic bead is used as the fixation member (10), the fixation member (10) combined with the disease-related exosomes (1a) and/or the second target exosomes (1b) is captured using a magnet, and the supernatant is removed and the magnetic beads are removed, thereby concentrating the exosome subpopulation (S) and/or the exosome subpopulation (SA).
  • a magnetic bead used as the fixation member (10)
  • the fixation member (10) combined with the disease-related exosomes (1a) and/or the second target exosomes (1b) is captured using a magnet, and the supernatant is removed and the magnetic beads are removed, thereby concentrating the exosome subpopulation (S) and/or the exosome subpopulation (SA).
  • Figures 10 to 16 are flowcharts of a method for preparing an exosome liquid biopsy sample according to an embodiment of the present invention.
  • the method for preparing an exosome liquid biopsy sample may include a step (S110) of binding a first specific binding material that specifically binds to a first surface marker of a disease-related first target exosome among exosomes secreted from a plurality of cells to a solid-state fixation member using a first reversible linker, a step (S120) of reacting a sample solution containing a population of exosomes with the first specific binding material bound to the fixation member to capture the disease-related first target exosomes, thereby isolating an exosome subpopulation, which is a population of disease-related first target exosomes, and a step (S130) of dissociating the first reversible linker so that the captured disease-related first target exosomes are separated from the fixation member, thereby recovering the exosome subpopulation.
  • the bulk exosome population which is a group of exosomes secreted from multiple cells, or the body fluid can be used as a sample solution, and then the exosome subpopulation can be separated and recovered first, and then provided as a liquid biopsy sample.
  • the method may further include a step (S140) of binding a second specific binding material that specifically binds to a second surface marker of a second target exosome in the exosome sub-population to a fixing member, and a step (S150) of reacting an exosome sub-population solution containing the recovered exosome sub-population with the second specific binding material bound to the fixing member to capture the second target exosomes, thereby separating the exosome sub-population, which is a population of the second target exosomes.
  • the second specific binding material may be bound to the fixing member by a second reversible linker.
  • a first specific binding material and a first reversible linker are reacted with a fixed member (S110). At this time, the first specific binding material is bound to the fixed member via the first reversible linker.
  • the first specific binding material is reacted with the first specific binding material (S120).
  • the first specific binding material specifically binds to the first surface marker of the disease-related first target exosome contained in the sample solution.
  • an exosome subpopulation targeting the disease-related first target exosome is captured by the fixing member and separated from the sample solution.
  • the first reversible linker is dissociated (S130). At this time, the disease-related first target exosome is separated from the anchoring member, so that the exosome subpopulation can be recovered.
  • the fixation member is recycled, or a new fixation member is used to bind a second specific binding material onto the fixation member (S140).
  • the second specific binding material may be directly bound to the fixation member, or may be bound to the fixation member using a second reversible linker.
  • the exosome sub-population solution containing the recovered exosome sub-population is reacted with the second specific binding material (S150).
  • the second specific binding material specifically binds to the second surface marker of the second target exosome in the exosome sub-population, the second target exosome is fixed to the fixing member.
  • the exosome sub-population which is a collection of the second target exosomes, is separated. In this way, the exosome sub-population that is not recovered and is fixed to the fixing member can be provided as a liquid biopsy sample.
  • FIG. 12 illustrates a method for producing an exosome subpopulation liquid biopsy sample using the first reversible linker (30a) and the second reversible linker (50a) according to the first embodiment described above.
  • a recognition material (35a) is fixed to the surface of a fixing member (10), and a ligand solution containing a ligand (31a), a binder (33a), and a first specific binding material (20) are reacted.
  • the binder (33a) and the first specific binding material (20) form a binder-first specific binding material polymer (C)
  • the recognition material (35a) binds to the polymer (C).
  • the ligand (31a) contained in the ligand solution binds to the binder (33a), causing a structural change in the binder (33a), and the recognition material (35a) specifically recognizes and binds to the binder (33a) whose structure has been changed.
  • a sample solution containing a bulk exosome (1) population is reacted.
  • the disease-related first target exosome (1a) having the first surface marker (m1) that specifically binds to the first specific binding material (20) is captured to the fixing member (10), thereby separating the exosome subpopulation (S).
  • S exosome subset
  • a recovery solution is added.
  • the recovery solution is a ligand-free solution that does not contain a ligand (31a).
  • the ligand (31a) bound to the binder (33a) is detached, and as a result, the changed structure of the binder (33a) is restored to its original state, and the bond between the binder (33a) and the recognition material (35a) is dissociated. Accordingly, the polymer (C) to which the disease-related first target exosome (1a), which is the target of the exosome subset (S), is bound is separated from the recognition material (35a), and the exosome subset (S) can be recovered.
  • the disease-related exosome (1a) is separated and the fixing member (10) to which the recognition material (35a) is fixed is recycled to react with the second reversible linker (50a) and the second specific binding material (40).
  • the second reversible linker (50a) shares the recognition material (35a) of the first reversible linker (30a), and, compared to the first reversible linker (30a), the first specific binding material (20) is replaced by the second specific binding material (40) to bind to the fixing member (10).
  • the second specific binding material (40) specifically binds to the second surface marker (m2) of the second target exosome (1b) in the exosome subpopulation (S) solution, thereby isolating the exosome subpopulation (SA).
  • the exosome subset (S) includes the binder (33a) of the first reversible linker (30a), the recognition material (35a) bound to the fixing member (10) and the binder (33a) of the first reversible linker (30a) can react with each other, so after reacting the second reversible linker (50a) and the second specific binding material (40), the remaining attachment site on the recognition material (35a) bound to the fixing member (10) can be blocked with an appropriate reactive component such as the binder (33a).
  • SA CD63+/Cav1+ exosome subpopulation
  • the CBP-capture antibody polymer dissolved in a solution containing calcium ions e.g., >10 mM Ca 2+
  • the polymer is immobilized on the solid surface by a 'switch on' recognition reaction.
  • exosomes including the CD63 marker are captured by reacting with the capture antibody on the solid surface.
  • the exosome recovery solution from which calcium has been removed is added, the calcium ions bound to CBP are detached and the 'switched off' state is entered, so that the captured exosomes can be recovered into the solution in a state bound to the capture antibody.
  • the CD63+ exosome subpopulation is separated and recovered, and exosomes can also be concentrated depending on the volume ratio of the sample solution and recovery solution used.
  • the solid surface can be regenerated and reused for the separation of other samples.
  • a reversible recognition material is immobilized on a regenerated or new solid surface, and a specific antibody against Cav1 exosome surface protein, which is highly associated with a specific disease, is polymerized with CBP, and then the CBP-capture antibody polymer is immobilized on the solid surface through a 'switch on' recognition reaction in the same manner as above.
  • CBP CBP-capture antibody polymer
  • the subpopulation can be separated by directly immobilizing the capture antibody on the solid surface without introducing a second reversible linker.
  • the captured exosomes are mainly subjected to immunoassay analysis, or the exosomes are immediately lysed while captured to extract the nucleic acids contained within the exosomes and perform molecular tests.
  • FIG. 13 illustrates a method for producing an exosome subpopulation liquid biopsy sample using the first reversible linker (30c) and the second reversible linker (50c) according to the third embodiment described above.
  • a ligand (31c) is polymerized with a first specific binding material (20) that specifically reacts with a first surface marker (m1) of a disease-related first target exosome (1a), and the ligand-specific binding material polymer is fixed using the affinity with a binder (33c) bound to a fixing member (10), and then an exosome (1) sample is added to capture and isolate an exosome subpopulation (S), and the exosome subpopulation (S) is recovered using a recovery solution containing a competitive reaction ligand (32).
  • a second specific binding material (40) that specifically reacts to the second surface marker (m2) of the second target exosome (1b) and a ligand (31c) are polymerized, and after fixing the binder (33c) and the polymer (C) bound to the fixing member (10), an exosome subpopulation (S) solution is added to capture and isolate the exosome subpopulation (SA).
  • the exosome subset (S) includes the ligand (31c) of the first reversible linker (30c)
  • the binder (33c) bound to the fixing member (10) and the ligand (31c) of the first reversible linker (30c) can react with each other, so after reacting the second reversible linker (50c) and the second specific binding material (40), the remaining attachment site on the binder (33c) bound to the fixing member (10) can be blocked with an appropriate reactive component such as the ligand (31c).
  • a method for isolating a CD63+/Cav1+ exosome subpopulation according to the present invention having a first reversible link and a second reversible link according to the third embodiment is described.
  • a binder nickel ion
  • a ligand his-stack
  • m1 first surface marker
  • CD63+ exosomes are captured by reacting with the capture antibody on the solid surface.
  • an exosome recovery solution containing a high concentration of competitive ligand imidazole
  • the His-stacs bound to the nickel ions are detached by the competitive reaction, so that the captured exosomes can be recovered into the solution.
  • the CD63+ exosome subpopulation is separated and recovered, and the solid surface is regenerated and can be recycled for the separation of the next sample.
  • the above separation process is used for sequential separation of the CD63+/Cav1+ exosome subpopulation from the CD63+ exosome subpopulation recovered above in the same manner.
  • the ligand Histax
  • the ligand polymerizes with an antibody specific for the Cav1 exosome surface protein as a second surface marker.
  • the CD63+ exosome subpopulation sample is added, and the CD63+/Cav1+ exosomes are captured by reacting with the capture antibody on the solid surface.
  • FIG. 14 illustrates a method for preparing an exosome subpopulation liquid biopsy sample using the first reversible linker (30a) according to the first embodiment described above, and the second reversible linker (50c) according to the third embodiment.
  • the exosome subpopulation (S) can be separated and recovered using the first reversible linker (30a) according to the first embodiment, and as described above in FIG. 13, the exosome subpopulation (SA) can be separated using the second reversible linker (50c) according to the third embodiment.
  • the exosome sub-population (S) contains the binder (33a) of the first reversible linker (30a), but when separating the exosome sub-population (SA), a binder (33c) different from the binder (33a) according to the first embodiment is bound to the fixing member (10) according to the third embodiment, that is, a second reversible linker (50c) that operates by a different mechanism from the first reversible linker (30a) is used, so the blocking process described above is unnecessary.
  • FIG. 15 illustrates a method for preparing an exosome subpopulation liquid biopsy sample using the first reversible linker (30c) according to the third embodiment described above, and the second reversible linker (50a) according to the first embodiment.
  • an exosome subpopulation (SA) can be isolated using the second reversible linker (50a) according to the first embodiment described above in FIG. 10.
  • the exosome subpopulation (S) contains the ligand (31c) of the first reversible linker (30c), but when separating the exosome subpopulation (SA), a second reversible linker (50a) distinct from the first reversible linker (30c) is adopted, so the blocking process described above is unnecessary.
  • a method for preparing an exosome subpopulation liquid biopsy sample may further include a step (S160) of recovering an exosome subpopulation.
  • the second specific binding material is bound to a fixing member by a second reversible linker, and by dissociating the second reversible linker, the captured second target exosomes can be separated from the fixing member, thereby recovering the exosome subpopulation.
  • a step of concentrating the exosome subpopulation may be further included.
  • the exosome subpopulation may be concentrated after adding the sample solution and before adding the recovery solution.
  • the fixation member combined with the exosome related to the target disease of the exosome subpopulation is concentrated using at least one of magnetic force, gravity, and centrifugal force.
  • a magnetic bead is used as the fixation member, and the fixation member is captured to a predetermined space area through magnetic force in a mixed solution in which a ligand solution and a sample solution are mixed, and then a portion of the mixed solution that does not contain the fixation member (for example, a supernatant) is removed, thereby concentrating the exosome subpopulation.
  • the exosome subpopulation can also be enriched by concentrating the fixation member bound to the second target exosome using one or more of magnetic force, gravity, and centrifugal force (S155).
  • Exosomes in body fluids e.g., peripheral blood
  • body fluids e.g., peripheral blood
  • exosomes were separated based on the density, size, solubility, surface markers, and other factors of exosomes to obtain exosome samples with reduced heterogeneity.
  • exosome biomarkers Although numerous exosome biomarkers have been reported to distinguish between patient samples with specific diseases such as cancer and healthy samples, there are limitations in applying exosome biomarker-based diagnostic technologies to the field. This is due to the dynamic nature of exosome heterogeneity, which can vary greatly depending on various factors such as constitutional status, disease status, sample preparation, and separation technology. In such a situation, sample-dependent marker detection cannot maintain consistency.
  • the present invention proposes an innovative exosome separation technology capable of sequential multi-step separation of exosomes into subpopulations or lower subpopulations.
  • the technology named 'Neutral Release', is based on reversible antigen-antibody binding, and can recover exosomes in an aqueous phase under mild conditions. Furthermore, the recovered exosomes can be further separated into subpopulations having two biomarkers in common.
  • One of the tetraspanins e.g., CD9, CD63 and CD81
  • CD9, CD63 and CD81 can be used as a primary marker for separating exosome subpopulations from the bulk population, and the heterogeneity of exosomes can be reduced through sequential separation of the markers.
  • exosome heterogeneity changes were assessed for their expression levels in the cell source producing the target exosome in the sample.
  • Exosomes play an important role in the mutual regulation of tumor and immune cells in the tumor microenvironment, and in certain cases, the composition of exosomes derived from immune cells increases rapidly in body fluids, which may result in greater changes in exosome heterogeneity in specific exosome subpopulations compared to non-cancerous conditions.
  • surface markers were sequentially selected to search for subpopulations containing exosomes associated with immune response. These subpopulations were determined through differential analysis of local heterogeneity changes in clinical samples between two groups of healthy donors and cancer patients. This approach can serve as the basis for an analysis model for biomarker discovery by removing background noise caused by changes in exosome heterogeneity.
  • GGBP glucose-galactose binding protein
  • Magnetic beads (Dynabeads M-280 tosyl-activated, 2.8 ⁇ m in diameter), streptavidin (SA), sulfosuccinimidyl-6-[biotinamido]-6-hexanamido hexanoate (NHS-LC-LC-biotin), RPMI 1640, Dulbecco's modified eagle medium (DMEM), exosome-depleted fetal bovine serum (FBS), penicillin/streptomycin, keratinocyte SFM supplied in separate packaging with recombinant EGF (rEGF) and bovine pituitary extract (BPE), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), dithiothreitol (DTT), and Zeba Spin Desalting Column, 2 mL, (Zeba Column) was purchased from Thermo Fisher Scientific (Rockford, IL; Anti-
  • Streptavidin-poly-horseradish peroxidase 20 (HRP20) polymerization and strep-poly HRP polymerization stabilizer were purchased from Fitzgerald (Acton, MA, USA).
  • Antibody diluent (HAMA Blocker) and tetramethyl benzidine-HK (TMB-HK) were purchased from Abcam (Cambridge, UK) and Mossbio (Pasadena, Maryland, USA), respectively.
  • Ultracel 100 kDa ultrafiltration discs and Amicon Ultra-4 Centrifugal Filters (10 kDa MWCO; Amicon Filter) were purchased from Merck/Millipore (Burlington, MA, USA).
  • StabilCoat Immunoassay Stabilizer, microwell plates, and FBS were purchased from Surmodics (Eden Prairie, MN, USA), Corning (Somerville, MA, USA), and Cytiva (Marlborough, MA, USA), respectively.
  • LNCaP (2 ⁇ 10 6 cells), PC-3 cells (1 ⁇ 10 6 cells), HT-29 (2 ⁇ 10 6 cells), and HCT116 ( 2 ⁇ 10 6 cells ) were seeded into 100 mm diameter culture dishes containing RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin, respectively, and cultured for 72 h in an incubator maintained at 37°C and 5% CO 2. After culture, the medium was washed and replaced with the same medium (containing 10% exosome-depleted FBS), and then cultured again under the same conditions for 48 h.
  • HEK293 cells (1 ⁇ 10 6 cells) were cultured under the same conditions as above, except that DMEM was used as the basal medium.
  • RWPE-1 cells (1 ⁇ 10 6 cells) were seeded and cultured in keratinocyte SFM medium (supplemented with 0.05 mg/ml BPE and 5 ng/ml rEGF). The culture was performed for 72 h under the same conditions as LNCaP and PC-3, and after washing, the cells were cultured for an additional 48 h in the same medium except for rEGF.
  • the beads were then washed three times with 100 mM PB and their residual surfaces were blocked for 1 h in a solution containing 10 mM phosphate buffer (pH 7.4) and 140 mM NaCl (PBS), 0.5% casein, and 0.03% Proclin300 (Casein-PBS). After blocking, the beads were washed three times with Casein-PBS, magnetically separated from the solution, and reconstituted with Casein-PBS to a final concentration of 31.5 mg/mL. The beads were stored at 4°C until use.
  • CBP (1 mg/mL, 550 ⁇ L) was prepared in PBS, and SA (5 mg/mL, 490 ⁇ L) was prepared in 10 mM phosphate buffer (pH 7.4, 10 mM PB). Sulfhydryl groups of CBP were activated with DTT (final 10 mM) diluted in 10 mM PB at room temperature for 1 h. Simultaneously, SA was activated by reacting with SMCC ( ⁇ 5 molar) diluted in DMSO at room temperature for 30 min. The two protein components were combined and polymerized at room temperature for 2 h.
  • SMCC ⁇ 5 molar
  • Biotinylation of antibodies specific to tetraspanins Antibodies ( 0.5 mg/ mL , 560 ⁇ L) specific for CD9 (clone HI9a), CD63 (clone H5C6), or CD81 (clone 5A6) in PBS were mixed with 5 molar excess of NHS-LC-LC-C-biotin dissolved in DMOS and reacted at room temperature for 1 h. Each biotinylated antibody was aliquoted and stored at 4°C until use. Casein (final concentration 0.05%) and Proclin300 (final concentration 0.03%) were added at this time.
  • Sample preparation The sample used in this experimental example was human male serum, donated from cancer patients and healthy individuals, and contained cell line exosomes mixed with sample binding buffer or Sigma serum.
  • Sigma serum was used as a control to simulate clinical samples.
  • the clinical sample was centrifuged at 1,200 g for 20 minutes at 4°C. After removing the precipitate, the solution was centrifuged again at 10,000 g for 1 hour at 4°C. The supernatant was filtered through a 0.2 ⁇ m filter, and stored at 4°C for use within 1 hour, or rapidly frozen at -80°C for long-term storage.
  • Biotinylated antibodies specific for tetraspanins were diluted in binding buffer TBS (containing 2% BSA, 10 mM CaCl 2 , 0.1% Tween-20, and 0.03% ProClin300), added to the beads coupled with CBP-SA polymers, and reacted by mixing on a shaker under the same conditions (step 3). The beads were washed, and the supernatant was removed by magnetic separation (step 4). A biotin solution (200 ⁇ L, concentration 2 ⁇ g/mL) diluted in binding buffer was added to the beads to saturate the remaining SA binding sites, and reacted on a shaker for 30 min (step 5). The beads were washed, and the supernatant was removed to obtain functional immunomagnetic beads for reversible exosome isolation (step 6).
  • TBS containing 2% BSA, 10 mM CaCl 2 , 0.1% Tween-20, and 0.03% ProClin300
  • Exosome Subpopulation isolation Sample binding buffer TBS (containing 2% BSA, 20 mM CaCl 2 , 0.2% Tween-20, 0.03% ProClin300, and 25% HAMA blocker) (100 ⁇ L) and the prepared exosome sample (100 ⁇ L) were sequentially added to the functional immunomagnetic beads and incubated for 2 h on a shaker (step 7). The beads were washed with washing solution (500 ⁇ L), and the supernatant was removed by magnetic separation, and this process was repeated twice (steps 8–10). This isolated the target exosome subpopulation in a form captured by the magnetic beads.
  • TBS containing 2% BSA, 20 mM CaCl 2 , 0.2% Tween-20, 0.03% ProClin300, and 25% HAMA blocker
  • Exosome recovery Prepare an elution solution (200 ⁇ L) containing 500 mM NaCl, 0.5% casein, 0.1% Tween-20, 0.03% ProClin300, 2 mM EDTA, and 0.01% phenol red in TBS. Add the exosome-bound beads to the elution solution and incubate on a shaker for 30 min (step 11). Collect the supernatant into a new tube by magnetic separation. The isolated exosomes were used immediately or stored by rapid freezing at -80°C.
  • Exosome isolation in automated mode was performed using a sample purification system (Nextractor NX-32N; Genolution, Seoul, Korea). This system uses a 96-deep well plate. Six reagents were dispensed into wells in the same row, and exosome isolation was performed in the row direction (see Fig. 24(B) and Fig. 25(A)). In the first row, anti-CBP antibody-coated immunomagnetic beads (200 ⁇ L; concentration 0.75 mg/mL) premixed with CBP-SA polymer (final concentration 1 ⁇ g/mL) were added to each well.
  • biotinylated antibodies for exosome isolation 200 ⁇ L; concentration 1 ⁇ g/mL diluted in binding buffer were loaded.
  • biotin solution 200 ⁇ L; concentration 2 ⁇ g/mL prepared in binding buffer was loaded.
  • sample binding buffer 100 ⁇ L was dispensed, and then the previously pretreated samples (each 100 ⁇ L) were added.
  • wash solution (1 mL) was added.
  • elution solution 200 ⁇ L
  • Exosome isolation procedure The 96-deep well plate loaded with reagents was placed in the plate rack of the sample purification system (Nextractor NX-32N), and the magnetic rod bundle was covered with a cover strip (refer to Figure S5C) (see (B) and (C) in Fig. 25). The exosome isolation procedure was performed at room temperature according to the preset program of the system. Upon completion of the exosome isolation process from Step 1 to Step 6, the 96-deep well plate was removed from the system, and each isolated exosome sample was transferred to a new tube. The isolated exosome subpopulations were immediately used for subsequent analyses or stored by rapid freezing at -80°C.
  • Anti-CD9 antibody 2.5 ⁇ g/mL; clone HI9a
  • anti-CD81 antibody 2.5 ⁇ g/mL; clone 5A6
  • anti-CD63 antibody 1.0 ⁇ g/mL; clone H5C6
  • the antibody-coated microwells were washed three times with PBS containing 0.1% Tween-20 (300 ⁇ L per well) using a microplate washer (WellwashTMVersa Microplate Washer; Thermo Fisher Scientific, Rockford, IL, USA). The microwells were then filled with StabilCoat Immunoassay Stabilizer (200 ⁇ L per well) and incubated for 2 h at room temperature. After removing the stabilizer solution, the microwells were dried for 24 h in a clean room with less than 30% humidity, sealed in aluminum pouches, and stored at 4°C.
  • Biotinylation of detection antibodies was performed on antibodies used for detection in the immunoassay: anti-CD9 antibody (1.0 mg/mL; clone 5A6), anti-CD63 antibody (1.0 mg/mL; clone H5C6), anti-CD81 antibody (1.0 mg/mL; clone 5A6), anti-CD151 antibody (1.0 mg/mL; clone 50-6), and anti-TSPAN8 antibody (1.0 mg/mL; clone TAL69).
  • the buffer of each antibody solution 300 ⁇ L each) was replaced with PBS using a Zeba Column.
  • Immunoassay procedure A standard sandwich immunoassay for various target exosome markers was performed by combining appropriate capture antibody-coated microwell plates with corresponding biotinylated antibodies for detection.
  • the assay samples included cell line exosomes diluted in media, buffer, or Sigma serum and clinical serum samples pretreated as described previously. Samples (100 ⁇ L) were added separately to the microwells and incubated on a shaker at room temperature for 3 h. The wells were washed three times with phosphate-buffered saline (PBS) containing 0.1% Tween-20 (270 ⁇ L).
  • PBS phosphate-buffered saline
  • Biotinylated detection antibodies 100 ⁇ L; concentration 0.1 ⁇ g/mL diluted in TBS (dilution buffer) containing 0.5% casein, 0.1% Tween-20, and 1% ProClin300 were transferred to the wells and incubated on a shaker for 1 h. After washing the wells three times, SA-poly HRP20 (100 ⁇ L; concentration 66.67 ng/mL) was added to each well and reacted under the same conditions for 1 h. The wells were washed again, and the substrate solution (200 ⁇ L TMB-HK) was added for signal generation.
  • TBS dilution buffer
  • SA-poly HRP20 100 ⁇ L; concentration 66.67 ng/mL
  • Serum samples from healthy individuals and male donors with various cancers, including prostate cancer, colon cancer, or malignant melanoma were purchased to compare test variables associated with prostate cancer. All healthy individual samples were obtained from white donors aged 40 years or older who tested negative for HIV, HBsAg, and HCV. Cancer samples were obtained from white donors aged 52–80 years for prostate cancer, 43–69 years for colon cancer, and 50–81 years for malignant melanoma (except one patient aged 35 years). All cancer samples were confirmed to have the corresponding diagnosis.
  • Quantification of specific dual marker -positive exosomes Quantification of specific dual marker -positive exosomes .
  • pretreated serum was diluted appropriately in dilution buffer to prevent optical density saturation.
  • the analyzed samples were subjected to sandwich immunoassay using the correct combination of antibody-coated plates and biotinylated antibodies as described above for exosome immunoassay.
  • the dilution buffer was used as a control sample and analyzed under the same conditions. All immunoassays were performed in duplicate, and the average of the repeated measurements of optical density for each sample was subtracted from the control to obtain the 'D+T' value, which is used to determine the index of local heterogeneity change.
  • indices for changes in local exosome heterogeneity were determined by dividing the quantified value of the third marker 'T' by the quantified value of the dual marker-positive exosomes 'D+T'. The reciprocal, 1/R, was used for plotting for significant digits.
  • the entire data range was divided into 23 intervals, and the number of negative or positive samples in each interval was recorded.
  • the cumulative counts were calculated for each interval sorted in ascending order, and the false positive rate (FPR) and true positive rate (TPR) were determined.
  • FPR false positive rate
  • TPR true positive rate
  • AUC area under the curve
  • FIG. 17 is a diagram illustrating a novel sequential marker selection process (A) using Neutral Release technology as contrasted with conventional methods, and a conceptual representation of the resulting new approach (B),
  • FIG. 18 is a diagram illustrating the process of determining heterogeneity variation indices (R1, R2 and R3) and the marker selection pathway
  • FIG. 19 is a diagram illustrating heterogeneity variation patterns
  • FIG. 20 is a diagram illustrating a sequential marker selection strategy for quantifying triple marker positive subclass 'T' when utilizing pan-exosome tetraspanin three markers, namely CD9, CD63 and CD81.
  • FIG. 21 is a diagram illustrating the process of quantifying the size of the triple marker-positive subpopulation (T),
  • FIG. 28 is the result of quantifying exosome heterogeneity changes for samples with various concentrations in which cell line RWPE-1 exosomes (1.28 ⁇ 10 11 particles/mL) were added to serum.
  • FIG. 29 is a graph showing the concentration-dependent heterogeneity change index for exosome samples produced from RWPE-1 (1.28 ⁇ 10 11 particles/mL), LNCaP (1.18 ⁇ 10 11 particles/mL), and PC3 (2.53 ⁇ 10 11 particles/mL)
  • FIG. 30 is a graph showing the exosome heterogeneity change index for various clinical samples, FIG.
  • FIG. 31 is a graph showing the results of quantifying the heterogeneity change of dual marker-positive exosomes and triple marker-positive exosomes according to stopover sites (exosome subpopulation regions having specific dual markers in common), and showing the exosome heterogeneity change index
  • FIG. 32 is a diagram explaining a local heterogeneity change-based diagnostic algorithm showing the main factors that distinguish cancer samples from normal samples.
  • FIG. 33 is a diagram comparing the sample classification performance based on the local heterogeneity change index using different third markers (CD81, CD151, TSPAN8)
  • FIG. 34 is the results of evaluating CD151 as a third marker at various stopover sites
  • a subpopulation of exosomes tagged with a specific antigen A such as CD9 positive (CD9+) can be immunomagnetically separated in the liquid phase.
  • a specific antigen A such as CD9 positive (CD9+)
  • an immunoassay can be designed to quantify the triple positive ('A+B+C+') exosome subpopulation (see (A) in Figure 17). If a hierarchical relationship exists between a specific exosome subpopulation and its marker, that relationship can quantify the composition of marker C in the 'A+B+' exosome subpopulation.
  • the heterogeneity change can be influenced by the properties of the exosomes determined by their cellular origin (see (B) in Figure 17).
  • the parameters representing the heterogeneity change can be complex, but can be quantitatively expressed using a detection antibody for a third marker, such as marker C present in 'A+B+' exosomes.
  • This local heterogeneity change can be defined as the ratio of the 'A+B+C+' subpopulation signal to the 'A+B+' subpopulation signal within the subpopulation. If this change is related to the origin of the target exosomes, it can be used to diagnose diseases characterized by changes in cellular properties.
  • the local heterogeneity change of the tested sample can be quantified using the index R.
  • This index R is defined as the size of the triple marker-positive subpopulation after the third selection divided by the size of the double marker-positive subpopulation after the second selection.
  • the sizes of the subpopulations can be determined by measuring the immunoassay signals for the triple (T) and double (D+T) marker-positive subpopulations, respectively.
  • the order of selection is rotated clockwise among the three markers (A, B and C)
  • the three indices (R1, R2 and R3) can be experimentally determined (see Fig. 18).
  • the sizes of the triple marker-positive subpopulations, each quantified separately, can be different. This discrepancy is due to the use of different sandwich immunoassays using different capture and detection antibody pairs. These different assay conditions are indicated by the prime (') and double prime (“) symbols.
  • Measuring different R values for a sample allows monitoring the properties collectively determined by the three markers expressed in individual exosomes.
  • the dual marker positive exosome subpopulation serves as the basis for the marker composition and allows for the measurement of a third marker present in the same exosomes.
  • the composition ratio of the third marker can vary from sample to sample depending on the properties of the exosomes (see Figure 19).
  • exosomes produced by individual cells can be assumed to have a uniform surface marker composition. This assumption is based on the observation of exosome heterogeneity in body fluids, which is caused by mixing of numerous exosome populations secreted from different cells and tissues.
  • Exosome selection strategy Quantification of 'T' can be planned in several paths through three different exosome subpopulation regions of the double markers, named as stopover sites, 'D1+T', 'D2+T', and 'D3+T' (see (A) in Fig. 20).
  • These triple markers selected for the experiment are pan-exosome tetraspanins, CD9, CD63, and CD81, which correspond to the A, B, and C markers, respectively.
  • the initial selection marker passing through each stopover site can be determined by selecting any one of the two different markers except the final marker. For example, if 'D1+T' is determined as the stopover site, the initial selection marker should be CD9 or CD63, and the selection path ends at CD81.
  • the quantification of 'T' can be designed to be approached in either the clockwise (defined as path 1) or counterclockwise (defined as path 2) direction of Fig. 20.
  • the degree of heterogeneity change of a specific sample is determined based on the initial marker selection (indicated as 'scheme') and the subsequent marker arrangement (indicated as 'route').
  • six heterogeneity change indices such as R1 to R3 and R1c to R3c (where 'c' means counterclockwise), can be determined to quantify the degree of exosome heterogeneity change.
  • 'T' To determine the size of 'T', we designed an immunoisolation and immunoassay protocol that allows sequential selection of triple pan exosome tetraspanin markers (CD9, CD63, and CD81) (see Figs. 17 and 21). Neutral Release was used in the first selection step to isolate exosomes based on the first target marker A (e.g., CD9). After separation, the exosome subpopulation in solution (e.g., CD9+ exosomes) was used as the sample for the second selection. The second selection was performed in microtiter plate wells whose inner surface was immobilized with capture antibodies specific for marker B (e.g., CD63). Here, the captured exosomes contained two target markers (e.g., CD9+CD63+) and were assayed for marker C (e.g., CD81) using a detection antibody linked to a tracer such as a signal-generating enzyme.
  • a detection antibody linked to a tracer such as a signal-generating
  • the size of 'D+T' was quantified by immunoassay for the first two of the pan exosome tetraspanin markers (see Fig. 22).
  • the first marker e.g., CD9
  • the second marker e.g., CD63
  • the heterogeneity change index R1 was determined as the size of 'T' divided by 'D1+T'.
  • the other indices R2 and R3 were measured by rotating the three markers clockwise (see Fig. 20(A) and Fig. 23).
  • test samples (10 ⁇ g PC-3 exosomes/mL Sigma serum) were prepared by spiking exosomes from PC-3 cell line into Sigma serum.
  • the exosome sample is reacted with anti-CD63 antibody releasably attached to magnetic beads (see step IV in (A) of Figure 25).
  • Unbound exosome samples were then collected for subsequent exosome binding analysis. A portion of the collected samples was simultaneously analyzed with the original sample using a homogeneous sandwich immunoassay using anti-CD63 antibody for capture and detection.
  • the two signals representing the sizes of the subgroups of 'D+T' and 'T' were used to determine the heterogeneity change indices R1 to R3 and R1c to R3c, respectively, as shown in Fig. 23.
  • the results were plotted using the inverse of the R indices and the Rc indices (see (C) in Fig. 28).
  • the plot showed that a pair of two indices based on the same stopover site (see (A) in Fig. 20), for example, 1/R1 and 1/R1c, showed similar response levels and patterns.
  • the three pairs of indices according to each stopover site showed significantly different patterns with respect to response and distribution. This indicates that the heterogeneity change is dependent on the stopover site.
  • Exosomes To track the cellular origin based on exosome heterogeneity changes, we performed experiments to analyze exosome samples collected from various cell lines associated with cancer characteristics. As a control, exosomes were produced from two different prostate cancer cell lines, LNCaP and PC3, in addition to the normal prostate cell line RWPE-1. These exosomes were added to serum at various dilutions to prepare bulk exosome samples. Then, a total of six indices, R1 to R3 and R1c to R3c, were derived for each exosome sample according to the experimental design shown in Fig. 23.
  • indicator R3 or R3c consistently showed the best resolution when CD63 was measured as the third marker within the CD9+CD81+ subpopulation as a stopover site.
  • the data collected along each route to investigate the classification performance of the indicators with respect to the stopover site (see Fig. 30(C)).
  • the best performance was observed for stopover site 3 based on the CD9+CD81+ subpopulation with CD63 as the third marker.
  • the same stopover site 'D+T' was approached clockwise ([T]) or counterclockwise ([T]c) and quantified ('Triples'; see Fig. 31 (B)).
  • the heterogeneity index was calculated as the ratio of the values quantified above ('[D+T]'/'T'), scaled using LNCaP exosomes as a standard sample as above, and plotted for pairs of indices 1/R, Scaled and 1/Rc, Scaled for each stopover site ('Doubles/Triples (1/R)'; see Fig. 31(C)).
  • the 'Doubles' plot in (A) of Fig. 31 shows a sample distribution that is arranged almost linearly regardless of the stopover site, which means that the conventional sandwich immunoassay measuring 'D+T' cannot distinguish different exosome-originating cells.
  • the 'Triples' plot in (B) of Fig. 31 shows different distributions for each sample depending on the three stopover sites. While the samples are distributed linearly for stopover site 1, they are distributed relatively above or below the diagonal line for stopover sites 2 and 3.
  • the subgroup [D+T] vs. [D+T]c plot with a linear relationship is not suitable for classifying the sample groups, which suggests that the subgroup marker concentrations alone are not sufficient to classify the sample groups (see (A) in Figure 32).
  • the ratio of the third marker-positive subgroup 'T' to the dual marker-positive subgroup 'D+T' may be an important factor for sample classification (see (B) in Figure 32). This suggests that the ratio of the third marker may change with the onset of the disease, i.e., the local heterogeneity changes may be different. Therefore, it can be seen that the subgroup containing the third marker itself can act as a disease biomarker.
  • CD151 CD82
  • TSPAN8 Tetraspanin 8
  • CD147 CD147
  • EpCAM EpCAM
  • the cutoff line showed a characteristic pattern, which is that the local changes of cancer samples increased or decreased in both X and Y axes compared to normal samples (see (A) in Figure 33). Due to this characteristic pattern of heterogeneity change, normal samples formed a distinct cluster in a limited area, while prostate cancer samples were distributed scattered outside the cluster boundary. As a result, a double-crossing cutoff line was set in the graph to distinguish the two sample groups. In contrast, in the local change plots of the other two markers, CD151 and TSPAN8, the samples of different groups were distributed in a diagonal shape in each graph (see (B) and (C) in Figure 33).
  • the two sample groups are separated by a single-crossing cutoff line, as is commonly observed.
  • heterogeneity change such changes may uniquely differ in the local composition of the third marker based on each designated subgroup, which may provide insight into the disease onset or progression.
  • CD81 is a target marker used to measure local heterogeneity changes and is present on the surface of exosomes.
  • the presence of CD81+ exosomes in the tumor microenvironment is associated with several aspects of cancer progression, such as tumor growth, angiogenesis, and invasion, and mediates communication between cancer cells and surrounding stromal cells.
  • CD151 and TSPAN8, which are transmembrane proteins of the tetraspanin family, are components of exosomes and promote interactions between cancer-initiating cells and their surroundings.
  • CD151 has been noted to play a potential role in cancer progression and metastasis.
  • TSPAN8 has also been reported to be abundantly expressed in prostate cancer compared to control samples. The role of each cancer-related marker can be further supported by the results of the analysis according to the present invention based on the local heterogeneity changes of the marker.
  • stopover sites 2 and 3 were difficult to utilize for the intended purpose due to the extremely low signal in the immunoassay used to detect CD151 (see raw data with optical density (OD) less than 0.1 in (B) in Fig. 34).
  • OD optical density
  • B optical density
  • Fig. 34 we did not apply LNCaP-based scaling when determining CD151 heterogeneity changes because the LNCaP standard signal was highly volatile due to low signals of OD ⁇ 0.1 (see Figure 35).
  • the present invention proposes a novel approach to measure local heterogeneity changes of a third marker within a specific exosome subpopulation by using the concept of stopover sites (exosome subpopulation regions that share specific dual markers).
  • This approach provides an alternative algorithm for liquid biopsy (see Fig. 32).
  • the heterogeneity changes within the normal group samples are significantly less than those within the cancer group. This indicates that the exosome composition in the healthy samples is stable.
  • the high heterogeneity observed in the cancer group reflects either the production of new exosomes from cancer cells or an active immune response.
  • the diagnostic approach based on local exosome heterogeneity changes differs from the conventional method that focuses on the concentration of target markers.
  • the target marker concentration can vary significantly due to substantial heterogeneity changes within the bulk exosome. Sequential separation of exosomes into subpopulations can alleviate these changes, which can be utilized as a practical method for discovering biomarkers for cancer diagnosis.
  • Tetraspanins which are used as separation markers, play a role in distinguishing exosome subpopulations in human serum and plasma. Therefore, controlling the heterogeneity of exosomes through tetraspanin marker-based separation is essential for utilizing exosomes as biomarkers for early cancer detection.
  • the main characteristics of the stopover site according to the present invention and the diagnostic performance using the same are as follows. First, at stopover site 2 (CD63+CD81+), using CD9 as the third marker and using the cutoff parameter based on the ratio of the two 1/R2 and 1/R2c indices showed excellent performance in distinguishing prostate cancer and colon cancer from normal samples, with a resolution of 93.3% (28 out of 30 samples; see (B) of Fig. 37). However, this method failed to distinguish malignant melanoma samples from normal samples.
  • pan exosomal tetraspanins alone were insufficient to selectively detect cancer samples due to the highly variable direction of sample distribution (see (C) in Figure 30).
  • certain other cancer markers showed promise for specific cancer detection.
  • CD151 was evaluated as a valid third marker that could distinguish prostate cancer samples from colon cancer, malignant melanoma, and healthy samples (see (A) in Figure 37). By combining the two indices along each axis of the plot and using them as a single cutoff parameter, the resolution for prostate cancer was achieved up to 88.3% (53 out of 60 samples).
  • LNCaP is a human prostate cancer cell line derived from a lymph node metastatic lesion that expresses a mutated androgen receptor, indicating that prostate tissue characteristics are maintained. In contrast, this increase was not observed at all when measured in exosomes from PC3, a human prostate cancer cell line derived from a bone metastasis. Unlike LNCaP, PC3 cells do not express androgen receptor or prostate-specific antigen (PSA), indicating a loss of typical prostate tissue characteristics.
  • PSA prostate-specific antigen
  • HT29 is a human colon adenocarcinoma cell line derived from a colon cancer patient.
  • HCT116 is a human colon cancer cell line that is commonly used as a model for aggressive colon cancer due to its high tumorigenic potential.
  • pan-exosome tetraspanins CD9, CD63 and CD81 are used for sequential isolation, forming the basis for quantifying the unique target markers present within them. Different combinations of these three markers at the subpopulation level exhibit different selectivity for cancer, despite their common association with tumor growth, angiogenesis and invasion (see Figure 39).
  • the stopover site containing CD81 could act as an entry point for mutation detection, while the third marker could indicate tissue type or immune response mechanism (see Figure 37 (B) and (C)). Consequently, the exosome stopover site, i.e., specific exosome subpopulation, can be used as a biomarker.
  • the present invention provides a novel and reproducible cancer detection method that monitors local heterogeneity changes at the subpopulation level, focusing on exosome surface protein markers in body fluid samples such as serum.
  • the invention is based on the idea that cytopathic effects, such as those due to cancer, induce changes in exosome composition when compared to normal conditions. These changes can be quantified using indicators derived from a third marker within a specific exosome subpopulation.
  • the invention uses a "stopover site" defined by a combination of pan-exosome tetraspanins, such as CD9, CD63 and CD81, which serves as an entry point for analyzing mutation patterns and identifying cancer-specific markers.
  • the diagnostic method according to the invention can be very useful.
  • This method which focuses on subpopulation-level heterogeneity, can be an innovative alternative to conventional liquid biopsy techniques and has great potential for early cancer detection.
  • the present invention can be utilized to improve the accuracy of cancer diagnosis by focusing on specific target cancers and tissues. Although initially focused on prostate cancer, the present invention can be widely applied to various types of cancer. This innovative model will bring about a paradigm shift in early cancer diagnosis by effectively resolving the complexity of heterogeneity while providing a versatile diagnostic technology for various types of cancer.
  • the present invention is a liquid biopsy method for isolating and recovering an exosome subpopulation, a hypopopulation, or a lower subpopulation from an exosome population with a high yield and in its original state for a separation marker, thereby preparing a liquid biopsy sample, and analyzing exosomes loaded with the same triple marker using two surface markers other than the separation marker as capture and detection markers in the liquid biopsy sample, which is recognized to have industrial applicability.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention concerne une biopsie liquide basée sur la mesure locale d'un changement d'hétérogénéité d'exosomes. Un échantillon de biopsie liquide est préparé par séparation d'exosomes associés à une maladie spécifique, telle que le cancer, à partir d'échantillons de population d'exosomes en une sous-population pour un marqueur de séparation en utilisant un marqueur de surface prédéterminé en tant que marqueur de séparation. Dans l'échantillon de biopsie liquide, deux marqueurs de surface autres que le marqueur de séparation sont utilisés en tant que marqueurs de capture et de détection pour analyser des exosomes chargés avec les mêmes marqueurs triples.
PCT/KR2024/008252 2023-06-14 2024-06-14 Biopsie liquide basée sur une mesure locale d'un changement d'hétérogénéité d'exosomes Pending WO2024258237A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20230075957 2023-06-14
KR10-2023-0075957 2023-06-14
KR10-2024-0077631 2024-06-14
KR1020240077631A KR20240176084A (ko) 2023-06-14 2024-06-14 국소적 엑소좀 이질성 변화 측정 기반 액체생검

Publications (1)

Publication Number Publication Date
WO2024258237A1 true WO2024258237A1 (fr) 2024-12-19

Family

ID=93852448

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2024/008252 Pending WO2024258237A1 (fr) 2023-06-14 2024-06-14 Biopsie liquide basée sur une mesure locale d'un changement d'hétérogénéité d'exosomes

Country Status (1)

Country Link
WO (1) WO2024258237A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20200102453A (ko) * 2017-12-22 2020-08-31 위스콘신 얼럼나이 리서어치 화운데이션 암 바이오마커 포획을 위해 나노가공된 표면
KR102291105B1 (ko) * 2019-03-04 2021-08-23 주식회사 엑소퍼트 엑소좀에 의한 인공지능 기반의 액체생검을 이용한 암 진단 정보 제공 방법 및 시스템
KR20220004645A (ko) * 2019-03-27 2022-01-11 주노 다이어그노스틱스, 인크. 최적화된 초저 부피 액체 생검 방법, 시스템 및 장치
KR102498862B1 (ko) * 2020-06-29 2023-02-13 솔바이오 주식회사 엑소좀 액체생검 샘플 분석방법

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20200102453A (ko) * 2017-12-22 2020-08-31 위스콘신 얼럼나이 리서어치 화운데이션 암 바이오마커 포획을 위해 나노가공된 표면
KR102291105B1 (ko) * 2019-03-04 2021-08-23 주식회사 엑소퍼트 엑소좀에 의한 인공지능 기반의 액체생검을 이용한 암 진단 정보 제공 방법 및 시스템
KR20220004645A (ko) * 2019-03-27 2022-01-11 주노 다이어그노스틱스, 인크. 최적화된 초저 부피 액체 생검 방법, 시스템 및 장치
KR102498862B1 (ko) * 2020-06-29 2023-02-13 솔바이오 주식회사 엑소좀 액체생검 샘플 분석방법

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YU, D. ET AL.: "Exosomes as a new frontier of cancer liquid biopsy", MOLECULAR CANCER, vol. 21, 2022, pages 1 - 33, XP055968906, DOI: 10.1186/s12943-022-01509-9 *

Similar Documents

Publication Publication Date Title
WO2021112464A1 (fr) Dispositif de préparation et procédé de préparation d'un échantillon de biopsie liquide d'exosomes et procédé d'analyse d'échantillon de biopsie liquide d'exosomes préparé par celui-ci
JP6096813B2 (ja) 乳癌診断用マルチバイオマーカーセット、その検出方法、及びそれに対する抗体を含む乳癌診断キット
KR100620297B1 (ko) ＮＴ―ｐｒｏＢＮＰ 측정용 분석 샌드위치 테스트
Hiraoka et al. Expression of CD163 in the liver of patients with viral hepatitis
WO2015182580A1 (fr) Procédé de détection de métastase du cancer colorectal
WO2015126030A1 (fr) Nouvelle méthode pour le diagnostic d'un cancer de l'estomac
WO2005121795A1 (fr) Procédé de dosage immunoenzymatique ayant une réaction non spécifique inhibée et réactif pour celui-ci
WO2017008388A1 (fr) Biomarqueur des protéines pancréatiques, ses utilisations, puce de détection dudit biomarqueur, et dispositif de détection
WO2020080891A1 (fr) Procédé de prédiction d'une récidive d'un cancer du sein
Liu et al. Increased Concentrations of Apo AI and Apo A-II Fragments in the Serum of Patients With Hepatocellular Carcinoma by Magnetic Beads–Assisted MALDI-TOF Mass Spectrometry
WO2020241999A1 (fr) Système et procédé de séparation par affinité mettant en oeuvre une réaction d'adhérence de type substituable
WO2024258237A1 (fr) Biopsie liquide basée sur une mesure locale d'un changement d'hétérogénéité d'exosomes
Li et al. Establishment and application of an immunoassay for the simultaneous detection of IgG and its subtype IgG4 autoantibodies against M-type phospholipase A2 receptor
JP5674339B2 (ja) 可溶性インターロイキン−２受容体の測定方法及び測定用試薬
KR101207797B1 (ko) 다중렉틴을 이용한 체액 유래 단백질 동정 방법 및 이 방법에 의하여 탐지된 간암 바이오마커
US20230160864A1 (en) Methods of measuring and purifying extracellular vesicles
WO2011046309A2 (fr) Marqueur de diagnostic pour carcinome hépatocellulaire comprenant des auto-anticorps anti-fasn et composition de diagnostic pour carcinome hépatocellulaire comprenant des antigènes de celui-ci
WO2020171346A1 (fr) Composition immunologique pour le diagnostic du cancer du poumon à l'aide d'un complexe auto-anticorps-antigène, procédé de diagnostic du cancer du poumon l'utilisant, et kit de diagnostic du cancer du poumon le comprenant
WO2023058885A1 (fr) Biomarqueur de diagnostic pour le cancer et utilisation associée
WO2022216113A1 (fr) Méthode de traitement du cancer à l'aide d'un inhibiteur de point de contrôle immunitaire
WO2016093567A1 (fr) Biomarqueur pour le diagnostic de l'hépatome et son utilisation
WO2015037907A1 (fr) Trousse et méthode de diagnostic, de pronostic ou de surveillance d'une maladie du foie par mesure de la quantité d'ast
KR102323361B1 (ko) 스위치 성 부착반응을 이용한 친화 분리 시스템 및 방법
WO2016175393A1 (fr) Marqueurs pour diagnostiquer l'angiosténose et leur utilisation
KR101311718B1 (ko) 대장암 진단용 단백질 마커 ｒｐｅ-스폰딘 및 이에 대한 항체를 포함하는 대장암 진단 키트

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24823753

Country of ref document: EP

Kind code of ref document: A1