• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
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    • 专利摘要:
    The invention relates to a method for isolating exosomes from a biological fluid, comprising at least two successive affinity purification steps as follows: a) a first step using at least one generic ligand specific anti-ligand exosomes, to obtain a P population of exosomes, and b) a second step, applied to the P population of exosomes, using at least one anti-ligand specific for a ligand characteristic of an SP subpopulation. exosomes, to obtain said SP subpopulation of exosomes. as well as the applications of such a method.
    公开号:FR3014198A1
    申请号:FR1362021
    申请日:2013-12-03
    公开日:2015-06-05
    发明作者:Catherine Ott；Francois Mallet；Laurence Generenaz
    申请人:Biomerieux SA；Hospices Civils de Lyon HCL；
    IPC主号:
    专利说明:

    [0001] The present invention relates to a method of isolating exosomes from a biological sample, and the use of this method as a tool in the characterization of exosomes present in said sample, as well as in the diagnosis and prognosis of a pathology and / or the clinical stage of a pathology, but also in the follow-up of the evolution of a pathology, treated or not, in a human being or animal. Exosomes are membrane vesicles 40-120 nm in diameter, secreted by different cell types in vivo. Because of their origin and their biogenesis, the exosomes reflect the content and the normal or pathological physiological state of the cells from which they come. They are found in many biological fluids, such as blood, plasma, serum, urine, saliva, cerebrospinal fluid (CSF), lymph, bile, bronchoalveolar lavage, sperm, synovial fluid, amniotic fluid, breast milk, malignant ascites fluid, ... Their secretory process is a very active process for proliferating cells such as cancer cells. They contain nucleic and protein markers of tumor cells, from which they are secreted and are thus considered as reservoirs of potential new biomarkers of cancer. In this, their study is of growing interest to the scientific community. The major limitation for their study lies in obtaining purified and sufficiently enriched preparations, from the various biological fluids mentioned above, with the current techniques available in the state of the art. The literature highlights the complexity of the evaluation of exosomes because of contaminated preparations, in particular by proteins, microsomal fractions or organelles co-purified by techniques such as ultracentrifugation or nanofiltration, or preparations that are not very concentrated. in exosomes to be analyzed, resulting from immunological techniques of separation. To date, even the combination of these techniques has not been entirely satisfactory. Most of the work done to characterize exosomes is carried out on malignant cell cultures. They are subjected to exosome purification techniques that cells secrete during development, and proteomic profiles of isolated exosomes are determined. Thus, according to S. Mathivanan et al., Mol Cell Proteomics. 2010 Feb; 9 (2): 197-208, the authors isolated exosomes from a human colon carcinoma cell line, LIM1215. The culture medium was first subjected to a first series of ultracentrifugations to separate a population of particles of size 40-100 nm, which was then engaged in immunopurification with a humanized A33 antibody, specifically recognizing epithelial cells of the colon. 394 proteins have been identified, belonging to various protein categories, some of which are common to those isolated from cultures of human urine cell lines and culture of murine mast cell lines, and revealing a multifunctional role of exosomes. The importance of exosomes having been established, it is essential to have efficient and specific isolation techniques of exosomes, allowing at the same time to be reliable, usable routinely and not requiring large volumes of biological samples. The invention resides in an exosome isolation method which comprises two successive levels of separation which rely on the affinity separation technique. This sequential separation gives the process of the invention a high specificity. It also allows it to be applicable to any sample of a liquid or biological fluid. The method of the invention is a tool that can be used routinely. It overcomes the obstacles to which the skilled person has been confronted to this day, opening a generalized access to the characterization of exosomes.
    [0002] The method of the invention comprises at least the following two successive steps: a) a first affinity purification step, applied to a biological fluid, using at least one anti-ligand specific for a generic exosome ligand, to obtain a P population of exosomes, and b) a second affinity purification step, applied to the P population of exosomes, using at least one ligand-specific anti-ligand characteristic of an exosome SP subpopulation, to obtain said SP subpopulation of exosomes.
    [0003] Before explaining in detail the invention, certain terms used in the present text to characterize the invention are hereinafter defined. Exosomes belong to a fraction of nano-vesicles secreted by cells in biological fluids. Structurally, they are vesicles having a lipid bilayer comprising on the surface proteins and sugars. They are defined by their size ranging from 30 to 200 nm, more particularly by a size of at least 40 nm, or even at least 50 nm, and at most 150 nm, or even at most 120 nm and even not more than 100 nm. The terms biological fluid and biological fluid are used interchangeably. A biological fluid is produced by a human or animal, healthy or sick, diagnosed or not. It is collected or punctured in humans or animals, directly or indirectly. By indirectly, it is understood that cells or cell tissue which are cultured in a suitable medium in which said cells excrete exosomes and all or part of which will be removed can be taken from human or animal cells. to be subjected to the isolation process of the invention. By way of nonlimiting examples, mention may be made of cell supernatants, stool samples and bone marrow samples. By affinity purification technique, a technique based on a specific interaction or recognition between an exosomal ligand and an anti-ligand is understood. This recognition can be of immunological nature and lead to an immune complex, such as antigen / antibody interactions, epitope / antibody, epitope / paratope, antigen / paratope ..., it is called immunopurification. This recognition can also be of any other nature, for example covalent. Normal cell comprises a cell having no detectable markers or a detectable level of markers characteristic of an abnormal state of the cell. Included in this definition are stem cells, including mesenchymal, neural, and hematopoietic cells, which secrete exosomes that are characteristic of an undifferentiated state. In contrast, a cell is considered abnormal, when it has detectable marker (s), or a detectable level of markers, characteristic of a state different from that of a normal cell, and in particular a pathological state of the cell or a cell. state likely to evolve to a pathological state. In particular, this definition includes cancerous, adenomatous, infectious, inflammatory, immune-stimulated, burned cells, and more generally attacked by any type of stimulus. As indicated above, despite their very small size, exosomes have the advantage of accumulating a large number of proteins that constitute their traceability, and are the quintessence of the cells that excrete them, in particular, the nature of the cellular tissue, the condition normal or abnormal cell ... The method of the invention comprises a first affinity purification step for isolating a P population of exosomes, which is based on the existence of specific markers exosomes, likely to be present or exposed on the surface of these, at a stage of their development. These markers, which will also be called generic ligands, are antigens, receptors, growth factors, as well as any other particle or molecule, and any fraction thereof, capable of being specifically recognized by an anti-ligand. The first step thus uses at least one anti-ligand specifically recognizing at least one of the markers or ligands above. Depending on the specificity of the ligand for exosomes, and to improve the efficiency of this step, two or more anti-ligands may be used, each of which is specific for two or more generic ligands, respectively, these ligands. generics being specific to the exosomal population. According to a variant of the invention, the first step comprises only one purification, during which one or more anti-ligands mentioned above are used. According to another variant of the invention, the first step may comprise two or more consecutive substeps, each of them engaging one or more anti-ligands specific for one or more generic ligands, respectively. This first step a) can be applied to any biological fluid as defined above.
    [0004] Beforehand, this liquid may have been treated. Thus, without departing from the scope of the invention, the biological fluid can be pre-treated by a physical separation step, by size, which makes it possible to isolate a fraction of the biological fluid containing no molecules or particles of a size greater than 800 nm, preferably 500 nm, which will then be subjected to the first step a).
    [0005] This physical separation step can be carried out by any appropriate technique such as those chosen from centrifugation and / or filtration techniques, such as series filtration, ultrafiltration, size exclusion chromatography, and combinations of these techniques. .
    [0006] At the end of the first step a), a population P of exosomes is obtained which is then engaged in the second step b) of the process of the invention. The second affinity purification step of the method of the invention making it possible to isolate, from the P population of exosomes, an SP subpopulation of exosomes, is based on the existence of specific markers specific to, or characteristics of, certain exosomes, said markers being capable of being present or exposed on the surface thereof, at a stage of their development. These markers, which will be called particular ligands, as opposed to generic ligands, are antigens, receptors, growth factors, as well as any other particle or molecule, and any fraction thereof, capable of being recognized specifically by an anti-ligand. The second step b) uses at least one anti-ligand specifically recognizing at least one of the particular markers or ligands above. Depending on the specificity of the ligand for exosomes, and to improve the efficiency of this step, two or more anti-ligands may be used, each of which is specific for two or more particular ligands, respectively. According to a variant of the invention, the second step b) comprises only one purification, during which one or more anti-ligands mentioned above are used. The purification of this second step b) can be carried out in several substeps involving one or more anti-ligands, contributing to the isolation of the same subpopulation SP of exosomes, which will therefore be more and more specific. According to another variant of the invention, the method of the invention comprises at least a third affinity purification step c), this step being applied to said SP subpopulation and using at least one specific ligand-specific antiligand an SP 'subpopulation of exosomes, the SP subpopulation being included in the SP subpopulation, to obtain said subpopulation SP'. Like the second step b), this third purification step c) may comprise substeps contributing to the isolation of the same SP 'subpopulation of exosomes.
    [0007] Of course, one or more additional consecutive affinity purification steps may further complement the process of the invention. The SP subpopulation and the SP 'subpopulation are special exosome populations. Depending on the particular anti-ligands used, the subpopulation SP or subpopulation SP 'may for example collect exosomes from the same organ, from the same cell tissue, or from the same type of cells. The original tissue or cells may be normal or abnormal.
    [0008] In practice and before an illustration of the implementation and the advantages of a method of the invention, in the examples that follow, the population P, resulting from the first step a), can be subjected to the second step b) of the method, in the presence of one or more anti-ligands specific for ligands characteristic of a pathological organ, for example a prostate tumor, for isolating an SP subpopulation of exosomes characteristic of a tumor of the prostate. prostate. In another embodiment, the population P may be subjected to the second step b), in the presence of one or more specific anti-ligands of ligands characteristic of an organ, for example of the prostate, without indication of the normal or pathological state of the organ. To refine the isolation process, this SP subpopulation can be subjected to a third step c) with anti-ligands specific for abnormal cell marker ligands, to allow the isolation of an SP 'subpopulation. exosomes characteristic of a prostate tumor. It can also be envisaged that this subpopulation SP 'is obtained by treating a population P resulting from the first step a), in a second step b) in the presence of anti-ligands specific for abnormal cell marker ligands to isolate an SP subpopulation of exosomes characteristic of abnormal cells; then this subpopulation SP is subjected to a third step c) with anti-ligands specific for ligands characteristic of the prostate. The generic ligand (s) and / or the characteristic ligand (s) may be chosen from polypeptides, proteins, antigens, receptors, enzymes, growth factors, glycolipids, polysaccharides, and the specific anti-ligands of said ligands. ligands are antibodies, antibody fragments such as F (ab ') 2 fragments, scFv, antibody analogs, lectins, aptamers, peptides.
    [0009] By "antibody analogs" is meant biological and / or chemical compounds which possess the same binding capacities as antibodies or antibody fragments or similar binding capacities. In particular, the antibody analogs include small proteins which, like antibodies, are capable of binding to a biological target, thus making it possible to detect, capture or simply target it within an organism or an organism. biological sample. The fields of application of these antibody analogues are almost as large as those of the antibodies. By way of example, there may be mentioned NanofitinesTM, small proteins marketed by AFFILOGIC. More particularly, the generic ligand is chosen from the proteins of the family of tetraspanines, such as the tetraspanins CD63, CD9, CD81; proteins involved in adhesion such as lactadherin (or FG-8), ICAM-1; proteins involved in the transport and membrane fusion of the Rab-GTPase family, such as Rab5 and Rab7 and annexins; and major histocompatibility complex (CMF-1) molecules such as MI-11, MIL The ligand characteristic of a subpopulation is preferably selected from PSCA, annexin A3, PSMA, caveolin, B7H3, proteins of endogenous retroviral origin as envelope proteins. The invention also resides in a use of a method as described above for characterizing and / or quantifying exosomes. The method of the invention can also be applied to the diagnosis and prognosis of a pathology and / or the clinical stage of a pathology, but also in the follow-up of the evolution of a pathology, treated or not, in a patient. to be human or animal sick or the follow-up of the effect of the treatment of this pathology, in a human being or sick animal. The pathology may be chronic or acute, of infectious or non-infectious origin. In one use of the method, the pathology is adenoma, cancer, inflammation, sepsis, neurological disease such as Alzheimer's disease, Parkinson's disease, multiple sclerosis and prion diseases, or pathology of pregnancy like pre-eclampsia. The treatment can be a drug treatment, a radiotherapy or a transplant.
    [0010] The invention is illustrated in the following examples where it is applied to different biological fluids of healthy or sick patients, namely those suffering from prostate cancer, the anti-ligands used in the second stage being characteristic of the prostate. These examples refer to the following figures: FIG. 1 represents the detection of exosomes in ng / pl by ExoTEST Rab5 / CD63 sandwich assay in the immunopurified subpopulations by a method of the invention, with the generic marker CD63 and the markers specific PSMA (Protein Specific Membrane Antigen) and caveolin, from serum pools of healthy and diseased subjects. FIG. 2 represents the detection of exosomes in ng / pl by ExoTEST Rab5 / CD63 sandwich assay in immunopurified subpopulations by a method of the invention, with the generic marker CD63 and the markers AnxA3, PSCA (stem cell antigen). prostate) and B7H3 (tumor epithelial cell marker and prostate cancer), from CaP plasma pools of diseased subjects. Figure 3 is the result of a TEM observation of exosomes on beads of the CD63 / PSMA subpopulation isolated from pools of sera. Figure 4 is the result of a TEM observation of exosomes on beads of the CD63 / AnxA3 subpopulation isolated from pools of sera. Figure 5 is the result of MET observation of the exosomes of the CD63 / AnxA3 subpopulation isolated from pools of sera after elution (or desorption). Figure 6 shows the detection of exosomes in ng / pl by ExoTEST Rab5 / CD63 sandwich assay in immunopurified subpopulations by a method of the invention, with the generic marker CD63 and the markers PSMA, caveolin and AnxA3 for 2 P1 and P2 sera from subjects with prostate cancer, individually tested. Figure 7 shows the detection of exosomes in μg / μl by ExoTEST Rab5 / CD63 sandwich assay in immunopurified subpopulations by a method of the invention, with the generic marker CD63 and the markers CD9, PSMA, and AnxA3 for the CaP and EFS plasma pools.
    [0011] Figure 8 represents a comparison between the detection of exosomes in a plasma-derived CD63 / AnxA3 subpopulation (EFS and CaP) by the Nanosight technique and that performed by ExoTEST. Figure 9 represents a comparison between the detection of exosomes in a plasma-derived CD63 / PSMA subpopulation (EFS and CaP) by the Nanosight technique and that performed by ExoTEST. Example 1: Application of the method of the invention to a sample of blood (serum or plasma) or urine, to isolate exosomes characteristic of prostatic tumor cells 11 Material 1.1) Samples of biological fluids Samples of sick subjects Pool of CaP sera: The purification method was performed using a pool of 20 sera (V = 2.5m1) composed of 6 samples of prostate cancer patients at different stages of the disease with scores of Gleason measuring the aggressiveness of cancer cells, ranging from 6 to 9 (on a scale of 2 to 10). Individual sera CaP: A study of 2 individual sera from the above sample pool was carried out from a serum volume reduced to V = 1.2 ml, ie a test sample 2 times less important. These are 2 sera from patients (P1 and P2) with a Gleason score of 7 and 8, respectively. Plasma Pool CaP: The purification method was applied to a pool of plasmas (V = 2.5m1) consisting of 4 samples of patients with metastatic prostate cancer. CaP urine pool: 35 The purification method was applied to a urine pool of prostate cancer patients with a Gleason score of 7, after post-digital rectal examination (post-DRE) massage. (CaP) and patients with benign prostatic hyperplasia (BPH). Samples of healthy subjects Serum pool EFS: Serum samples from 6 healthy donors from the French Blood Establishment (EFS) made it possible to constitute an EFS serum pool (V = 2.5m1) as a control group for the study . EFS Plasma Pool: Plasma samples from 6 healthy EFS donors made it possible to construct an EFS pool (V = 2.5m1) as a control group for the study. 1.2) Antibodies used They are listed in Table 1 below. Table 1 Antibody Clone Target Type Marker anti-CD631 MX-49,129,5 Monoclonal mouse CD63 Generic anti-CD92 MEM-61 Monoclonal mouse CD9 Generic anti-PSM1 K1H7 Monoclonal mouse PSMA prostate cancer anti-PSCA3 5C2 Monoclonal mouse PSCA Anti-AnxA34 Prostate Cancer 13Al2G4 Mouse Monoclonal Annexin A3 Cancer Prostate Anti-B7H31 4396 Mouse Monoclonal B7H3 Aggressive Anti-Caveolin Cancer 'N-20 Polyclonal Rabbit Caveoline Cancer 1: Supplier Santa Cruz Technology 20 2: Novus Biological Provider 3: supplier Sigma Aldrich 4: produced by bioMérieux as described in FR2968767A1 2) Preparation of anti-ligands The abovementioned antibodies are coupled to magnetic beads coated with streptavidin (Dynabeads M-280 Streptavidin).
    [0012] A step of biotinylation of the antibodies is carried out previously with the commercial kit, One-step Antibody Biotinylation, marketed by Miltenyi Biotec, according to the supplier's recommendations. A volume of 150 μl of MP-280 beads (ie 108 beads) are removed and then washed for 5 minutes with 500 μl of buffer + 0.5% Tween. The tube is placed on a magnetic support to eliminate the supernatant. 500 μl of the anti-human CD63, anti-PSMA, anti-AnxA3, anti-caveolin or anti-CD9 monoclonal antibodies diluted to a concentration of 20 μg / ml in buffer + 0.05% Tween are added and incubated for 30 minutes. with rotary stirring. For blocking free sites, a 10 mM biotin solution is added and incubated for 30 minutes with rotary shaking. The beads are then washed 5 times for 5 minutes with 500 μl buffer + 0.5% Tween. Streptavidin MP-280 / biotinylated antibody conjugate is ready to be contacted with the blood sample. 3) Pre-treatment of the sample The serum or plasma sample is subjected to an ultracentrifugation pretreatment consisting of two differential centrifugations and filtration as follows.
    [0013] The sample (volume of 2.5 ml) is centrifuged at 500 g for 10 minutes at + 4 ° C to eliminate blood cells and cell debris then at 16500 g for 20 minutes at + 4 ° C to remove microparticles and body apoptotic fluid. A 0.45 μm filtration step is performed for removal of extracellular vesicles and protein aggregates larger than 450 nm. The urine sample is also subjected to a preliminary treatment of differential centrifugations, and then filtration on 0.45 pm. The urine is then concentrated 5X on Vivaspin 20 (cut off 10 kD, Vivasciences). 4) Application of the first step a) of immunopurification of the process of the invention to obtain a population P For this step, it is a generic tetraspanine ligand that is targeted. The anti-ligand used is an anti-tetraspanin antibody, more specifically an anti-CD63.
    [0014] The first immunopurification with anti-tetrasparanine CD63 is carried out in 2 incubations of the pre-treated blood sample. In a first step, a batch is made by incubating a volume of 1.25 ml of sample with the biotinylated streptavidin beads / anti-BD63 conjugate for 3 hours at room temperature with rotary stirring. The remaining volume of 1.25 ml of pretreated serum is incubated batchwise with the bioconjugate overnight with rotary stirring at room temperature. 5 washes of 5 minutes are made with 500 μl buffer + 0.5% Tween. The final elution step is carried out by adding 100 μl of elution buffer (0.2 M glycine, HCl, pH 2.2 + 1 mg / ml of BSA) after 2 minutes of incubation with the bioconjugate by vortexing. slightly a few seconds. To a first elution volume E1 of 100 μl is added 14 μl of neutralization buffer (2M Tris, pH 9.5). A second elution + identical neutralization E2 is performed. The eluates El and E2 are mixed and a final elution volume of 228 μl is stored at -80 ° C. until analysis.
    [0015] Under the same conditions, this immunopurification step was carried out with the anti-tetrasparan antibody, anti-CD81, on the pools of sera of sick patients (CaP). The CaP and HBP urine pools are subjected to this first step of immunopurification with anti-tetrasparanine CD63. 5) Application of the second step b) of immunopurification of the method of the invention to obtain an SP subpopulation For this step, the particular ligands are PSMA, caveolin and annexin A3.
    [0016] The anti-ligands used are the anti-PSMA, anti-caveolin and anti-annexin A3 antibodies. This second specific immunopurification step is carried out from the elution fractions P CD63 and P CD81, respectively, obtained in 4). A volume of 205 μl elution P is adjusted to a final volume of 1200 μl with buffer and fractionated three times 400 μl before being contacted with bioconjugate beads / anti-biotinylated mAb mAbs, beads / anti-Ab. biotinylated scavenger and beads / anti-annexin A3 biotinylated for 3 hours at room temperature with rotary stirring. 5 washes of 5 minutes are carried out with 500 μl buffer + 0.5% Tween. In the same way as for the population P, the elution is carried out twice with a final elution volume obtained of 228 μl stored at -80 ° C. until analysis.
    [0017] The following SP sub-populations are obtained: CD63 / PSMA, CD63 / caveolin, CD63 / AnxA3, CD81 / PSMA, CD81 / PSCA, CD81 / AnxA3. The CaP and HBP urine pools are subjected to the second immunopurification step with the anti-PSMA antibody.
    [0018] EXAMPLE 2 Techniques for Detecting Exosomes Purified by the Invention Process 1) Immunodetection of Exosomes by ELISA, ExoTEST® (HansaBiomed supplier) In order to detect the presence of exosomes in purified fractions by the process of the invention described above, the ExoTEST test marketed by HansaBiomed was used. This is a sandwich ELISA microplate assay that uses in the capture phase a monoclonal Ab directed against Rab5 protein (Rab GTPase family) and detection of a monoclonal anti-CD63 mAb. The sandwich format allows the specific capture of exosomes by reducing the detection of contaminating proteins. In addition, the test allows the quantification of exosomes from biological samples and purified and enriched exosome preparations, thanks to the presence of a calibration standard included in the kit. Sampling of samples was performed according to the supplier's recommendations. 2) Detection of exosomes by the physical method, NTA (Nanoparticle Trackinq Analysis) NanoSight sells an analytical instrument, the LM10-HS, to measure and characterize all types of nanoparticles with a size between 10 nm and 1 μm, within a polydisperse sample. Using a 405 nm laser, the nanoparticles are excited and their Brownian motion is followed by an optical microscope and filmed by a camera. The software supplied with the device (Nanosight 2.0) provides an analysis of the size and concentration of the different particles present in the sample. A detection threshold was determined from measurements made from 6 injections of buffer PBS1X previously filtered twice over 0.22 μm. This threshold corresponds to the average of the values + 3 standard deviation is 0.42.108 particles / mL. At the beginning of each manipulation, the quality of the PBS1X buffer, extemporaneously filtered at 0.1 μm) used for the dilution of the samples is checked. The value of the blank must not exceed the detection limit.
    [0019] The immunopurified fractions by the process according to the invention are diluted 1 / 50th in PBS1X for analysis. The fractions resulting from the pre-treatment by ultracentrifugation [cf. Example 1, 3)] are diluted 1: 500 in PBS1X. For each sample, the coefficient of variation (CV) is calculated on the concentration and size measurements (mode and mean) obtained after 5 to 6 injections. These measurements make it possible to evaluate the reproducibility of the NTA analysis of the samples. 3) Detection of Exosomes by Transmission Electron Microscopy (TEM) Direct observation of suspended exosomes after negative staining is carried out by transmission electron microscopy. Exosomes coupled to the beads are observed (b) and exosomes alone after elution.
    [0020] EXAMPLE 3 Application to the Detection of Immunopurified Exosomes Deriving from a Serum Pool According to the Invention The different detection techniques described in Example 2 are applied in the present example to the CD63 / AnxA3 and CD63 exosome subpopulations. / PSMA, from the pool of sera, of Example 1 after treatments 4) and 5) of Example 1. 1) Detection with ExoTEST ° From a pool of sera of patients with cancer of the CaP prostate and a pool of healthy donors (EFS), the detection of circulating exosomes isolated by generic and specific sequential immunoaffinity is performed using the ExoTEST RabS / CD63 sandwich ELISA assay. A test portion corresponding to 1/4 of the volume of each exosomal isolation fraction obtained is assayed by ExoTEST =).
    [0021] Figures 1 and 2 indicate the concentration of exosomes in ng / pl obtained for the different markers tested during the second immunopurification. FIG. 1 shows the detection of serum exosomes in the following fractions: The CD63 / PSMA population corresponds to a subpopulation of exosomes resulting from a double immunopurification process according to the invention, by application of a step a) CaP serum pool (prostate cancer) and EFS pool (healthy), using an anti-ligand directed against the CD63 generic ligand, followed by a step b) to the population thus isolated, using an anti-ligand directed against the specific PSMA prostate cancer marker; The CD63 / Caveacoline population corresponds to a subpopulation of exosomes resulting from a double immunopurification process according to the invention, by application of a step a) of the CaP serum pool process (subjects suffering from a cancer of the prostate), using an anti-ligand directed against the generic ligand CD63, then application of a step b) to the population thus isolated, using an anti-ligand directed against the specific marker of prostate cancer caveolin. Higher exosome concentrations are detected for the pool of CaP sera from prostate cancer compared to the pool of 25 sera from healthy donors. FIG. 2 shows the detection of serum exosomes in the following fractions: The CD63 / AnxA3, CD63 / PSCA and CD63 / B7H3 subpopulations of exosomes are obtained after a double immunopurification process according to FIG. the invention, by applying a step a) of the CaP serum pool (prostate cancer) method, using an anti-ligand directed against the CD63 generic ligand and then applying a step b) to the thus isolated population, using an anti-ligand directed against the specific markers AnxA3, PSCA and B7H3, respectively. The specificity of the isolation method of the invention was verified by Luminex AnxA3 sandwich immunoassay of the CD63 / AnxA3 subpopulation. This assay is very sensitive, it has a calculated detection limit of 1.1 μg / ml. By this assay, an AnxA3 concentration of 50 μg / mL was measured in the CD63 / AnxA3 subpopulation. The following table 2 gives the concentration values of the 5 exosomes detected by ExoTEST ° on the CD81 / PSMA, CD81 / PSCA, CD81 / AnxA3 subpopulations: Table 2 CD81 / PSMA sub-population CD81 / PSCA CD81 / AnxA3 763 concentration , 44 615.33 690.02 exosomes (ng / μl) 2) Detection by NTA The analysis of the SP CD63 / AnxA3 and SP CD63 / PSMA SP serum subpopulations from the pool of CaP serum by NTA indicates the concentration of exosomes as well as the size of the majority and mean peak in the purified samples shown in Table 3 below. Table 3 SP Particles CV% Mean peak size (nm) CV% Peak size CV% X109 / m1 maximum (nm) CD63 / AnxA3 16.4 24 125 1.7 92 4.5 CD63 / PSMA 24.6 30 77 A good reproducibility of the size distribution profiles is observed, with for each of the markers studied predominant peak sizes between 90 and 100 nm and consistent with the size described for the exosomes. 3) Detection by TEM A morphological characterization of vesicles isolated by transmission electron microscopy was performed. a) Observation of the Exosomes Fixed on the Beads: The vesicles captured on the beads (Dynabeads® M-280 streptavidin) conjugated with the anti-Anxa3 and anti-PSMA biotinylated antibodies are directly observed. Thus, after a first immunopurification of serum exosomes using anti-CD63, the eluate is incubated with beads coated with either anti-AnxA3 or anti-PSMA, washed and taken up in PBS. The result of this observation is illustrated by the snapshots below of Figure 3 (Figures 3A and 3B) for SP CD63 / PSMA and Figure 4 (Figures 4A and 4B) for SP CD63 / AnxA3, on which we visualize the capture of small vesicles on the surface of the magnetic beads. b) Observation of exosomes alone after elution: A volume of 7.5 ml of the sera pools was subjected to a double immunopurification according to the invention using the anti-CD63 antibodies for the first step and anti-AnxA3 and anti-PSMA , respectively, for the second step. Each of the SP, CD63 / AnxA3 and CD63 / PSMA subpopulations was eluted with 2 x 60 μl of 0.2M glycine buffer, pH 2.5, and then neutralized. The result of this observation is illustrated in Figure 5 (Figures 5A and 5B). The "cup-shaped" morphology and size ranging from 50 to 120 nm of the observed vesicles are typical of the exosomes, demonstrating the effectiveness of the method of the invention. EXAMPLE 4 Application to the Detection of Immunopurified Exosomes Derived from Individual Serums According to the Invention The ExoTEST detection technique described in Example 2 is applied in the present example to the CD63 / AnxA3 and CD63 exosome subpopulations. / PSMA, derived from individual CaP sera (diseased subjects) 1.1) of Example 1 after treatments 4) and 5) of Example 1. Figure 5 shows the exosome concentration obtained for the individual sera P1 and P2 from pool of sera from patients with prostate cancer. The PSMA, Caveolin and AnxA3 populations of exosomes are obtained after a double immunopurification process according to the invention, by applying a step a) of the method to the sera P1 and P2, using an anti-ligand. directed against the CD63 generic ligand, then application of a step b) to the thus isolated population, using an anti-ligand directed against specific markers PSMA, Cavéoline and AnxA3, respectively.
    [0022] EXAMPLE 5 Application to the Detection of Immunopurified Exosomes Deriving from a Plasma Pool According to the Invention The ExoTES-C and NTA detection techniques described in Example 2 are applied in the present example to subpopulations CD63 / AnxA3 and CD63 / PSMA exosomes from the pool of CaP plasmas (diseased subjects) and the pool of EFS plasmas (healthy subjects), from Example 1 after treatments 4) and 5) of Example 1. 1) Detection with ExoTES-C From a plasma pool of patients with prostate cancer and a pool of healthy donors (EFS), the detection of circulating exosomes isolated by the procedure of the The invention is carried out using the ExoTEST RabS / CD63 sandwich ELISA assay. The different exosomal isolation fractions obtained are diluted 1/2 in PBS1X and assayed by ExoTEST. FIG. 6 indicates the concentration of exosomes in ng / μl obtained in the following fractions: The CD63 / PSMA population corresponds to a subpopulation of exosomes resulting from a double immunopurification process according to the invention, by application of step a) of the CaP plasma pool (subjects with prostate cancer) and the pool of EFS plasmas (healthy subjects), using an anti-ligand directed against the CD63 generic ligand, then application of a step b) to the population thus isolated, using an anti-ligand directed against the specific PSMA prostate cancer marker; The CD63 / AnxA3 population corresponds to a subpopulation of exosomes resulting from a double immunopurification process according to the invention, by application of a step a) of the CaP plasma pool method (subjects suffering from cancer of the prostate) and the pool of EFS plasmas (healthy subjects), using an anti-ligand directed against the generic ligand CD63, then application of a step b) to the population thus isolated, using an anti-ligand directed against the specific marker Annexin A3 prostate cancer; Higher exosome concentrations were observed for the CaP pool compared to the healthy donor pool for the PSMA marker. Interestingly, a difference in exosome concentration was observed between the pool of healthy donor plasmas and that of the CaP pool for the AnxA3 marker, the former being greater than the second. 2) Detection by NTA Analysis of the CD63 and AnxA3 and CD63 / PSMA SPs from the CaF plasma pool and plasma pool EFS by NTA indicates the exosome concentration as well as the size of the majority and average peak in the cells. purified samples shown in Table 4 below. Table 4 SP Particles CV% Mean peak size (nm) CV% Peak size CV% X109 / m1 maximum (nm) CD63 / AnxA3 EFS 3.1 10.8 140 32.4 108 14.1 CD63 / AnxA3 CaP 2.7 12.8 124 2.8 85 7.3 CD63 / PSMA 8.5 34.8 163 19.9 122 15.5 EFS CD63 / PSMA 14.2 15.9 176 7.5 133 14.4 CaP The size of the majority peaks is consistent with the described size of the exosomes. Figures 7 and 8 illustrate a comparison between the two ExoTEST and NTA techniques for CD63 / AnxA3 SP and SP CD63 / PSMA subpopulations respectively. There is a match between these two detection techniques. Example 6: Application to the detection of immunopurified exosomes from a urine pool according to the invention The immunopurified fractions above are assayed by ExoTEST ELISA assay. The results are shown in Table 5 below.
    [0023] Table 5 CD63 / PSMA IP2 ELISA ExoTest [exosomes] ng / ml Pool Urine HBP 513 Pool Urine CaP 1366 A two-fold higher concentration of exosomes is detected for the CaP pool versus the HBP pool. Urinary exosomes with PSMA surface prostate marker are thus twice as numerous in the urine of patients with CaP. Example 7: Specific Assay for Total Prostate Specific Antigen (tPSA) Label The tPSA was assayed in the CaP and EFS serum pools as well as purified exosomal fractions to verify the quality of the exosomal fractions obtained after double immunopurification. For this purpose, an adaptation of the TPSA VIDAS sandwich assay using capture of the 12C11C3 monoclonal antibody and detection of the biotinylated 11E5C6 antibody was done in Luminex format. The technique allows from microspheres in suspension to detect and quantify several biomolecules in the same low volume sample with high sensitivity. Magnetic beads 5.6 μm in diameter, having a spectral address based on their red / infrared content served as a support for the assay. 9 μg of 12C11C3 capture antibody was grafted onto the surface of the magnetic beads (Bio-Rad, Bio-Plex Pro Magnetic COOH Beads Amine Coupling Kit) according to the supplier's instructions.
    [0024] For the determination of serum and plasma pools, samples are diluted 1: 5 in TBST buffer. For the purified exosomal fractions, the tPSA assay is performed with 1/8 of the volume of the elosomal fraction eluted.
    [0025] The samples are incubated in a 96-well plate (Bio-Rad, 171025001) in the presence of 5000 beads coupled to the capture antibody for 2 hours at 37 ° C., 650 rpm, and protected from light. Between each step, the wells are washed 3 times with 0.05% TBST. Detection is performed with 100 μl of biotinylated secondary antibody 11E5C6 at a concentration of 0.005 μg / ml for 1 hour at 37 ° C with shaking. Immune complex is revealed by incubation of 100 μl of phycoerythrin-streptavidin (RPE) solution at a concentration of 2 μg / ml (Dako) for 30 minutes at 37 ° C with shaking. The final step consists in resuspending the immune complexes in 100 μl of TBS for a flow fluorimetry analysis carried out by the Bio-Plex 200 (Bio-Rad) automaton. Each ball will undergo a double excitation by a red laser (633 nm) for its identification and a green laser (532 nm) for quantification of the analyte by measuring the fluorescent conjugate. The analytical detection limit of the tPSA assay developed on Luminex achieves an excellent sensitivity of 1.1 pq / m 2 of tPSA. The CaP serum assay results are shown in Table 6 below. Table 6 [tPSA] in ng / ml purified Pool Fraction IP2 serums PSMA AnxA3 CaP 2.98 0 0 The results of the Luminex tPSA assay show that the serum tPSA marker is detected at a concentration of 2.98 ng / ml in the pool of CaP serum. After purification of the serum pool, the tPSA marker is not or very little detected in the purified exosomal fractions. This result indicates the quality of the purified exosomal fraction obtained by the method of invention, with the removal of the soluble tPSA protein marker from the purified preparations. Example 8: Repeatability and Reproducibility of the Process The repeatability, ie the intra-series variability, and the reproducibility, that is to say the inter-series and inter-day variability, of the isolation process of the invention have been studied over 4 days. The assay is performed for the isolation of CD63 / AnxA3 and CD63 / PSMA subpopulations of exosomes from a pool of serum from diseased patients. Immunodetection of the exosomes is done by ExoTEST and the results are shown in Table 7 below. Table 7 15 Ligand Statistic Repeatability Reproducibility AnxA3 Standard Deviation 17.95 27.78 CV (%) 4.87 7.54 MAW Standard Deviation 129.89 94.80 CV (%) 24.63 18.62 the ligand AnxA3 a very good repeatability, with a CV of 4.87%. For this marker, inter-day reproducibility also indicates a very good CV of 7.5%.
    [0026] Slightly larger but perfectly acceptable variations are observed for the specific PSMA marker. 25
    权利要求:
    Claims (17)
    [0001]
    REVENDICATIONS1. A method for isolating exosomes from a biological fluid, characterized in that it comprises at least two successive affinity purification steps as follows: a) a first step using at least one ligand-specific anti-ligand generic exosomes, to obtain a P population of exosomes, and b) a second step, applied to the P population of exosomes, using at least one anti-ligand specific for a ligand characteristic of an SP subpopulation. exosomes to obtain said SP subpopulation of exosomes.
    [0002]
    2. Method according to claim 1, characterized in that the second step uses at least two anti-ligands, each of them being specific for a ligand respectively characteristic of a subpopulation SP1 and SP2.
    [0003]
    3. Method according to claim 1 or 2, characterized in that the first step uses at least two anti-ligands, each of them being specific for a respectively generic ligand of the exosomes.
    [0004]
    4. Method according to any one of claims 1 to 3, characterized in that it comprises at least a third step c) of affinity purification, this step being applied to said subpopulation SP and using at least one anti- ligand-specific ligand characteristic of an SP 'subpopulation of exosomes, the SP subpopulation being included in the SP subpopulation, to obtain said SP' subpopulation.
    [0005]
    5. Method according to claim 4, characterized in that the third step uses at least two anti-ligands, each of them being specific for a ligand respectively characteristic of a subpopulation SP'1 and SP'2.
    [0006]
    6. Method according to any one of claims 1 to 5, characterized in that the generic ligand of the exosomes and / or the ligand or characteristics of an SP or SP 'subpopulation of exosomes are chosen from the ligands present on the surface of exosomes.
    [0007]
    7. A method according to any one of claims 1 to 6, wherein the subpopulation of exosomes are subpopulations of exosomes from the same organ.
    [0008]
    8. Method according to any one of claims 1 to 7, characterized in that the subset or subpopulations of exosomes are subpopulations of exosomes from the same tissue.
    [0009]
    9. Method according to any one of claims 1 to 8, characterized in that the subset or subpopulations of exosomes are subpopulations of exosomes from the same type of cells.
    [0010]
    10. Method according to any one of claims 1 to 9, characterized in that the subpopulation or subpopulations of exosomes are subpopulations of exosomes from tissue or healthy cells or tissue or abnormal cells.
    [0011]
    11. The method of claim 10, characterized in that the abnormal tissue or cells are tumorous.
    [0012]
    12. Method according to any one of claims 1 to 11, characterized in that the ligand is characteristic of an SP subset of exosomes from the prostate.
    [0013]
    13. Method according to any one of claims 1 to 12, characterized in that, prior to the first step a), the biological fluid is treated by a physical separation step, by size, which isolates a fraction of the biological fluid containing no molecules or particles larger than 800 nm, preferably 500 nm, which will be subjected to the first step a).
    [0014]
    14. Use of a method according to any one of claims 1 to 13 for characterizing and / or quantifying exosomes.
    [0015]
    15. Use according to claim 14, for the diagnosis and prognosis of a pathology and / or the clinical stage of a pathology, and for monitoring the evolution of a pathology, treated or not, or for monitoring the effectiveness of treatment of a pathology, in a human or animal.
    [0016]
    16. Use according to claim 15, characterized in that the pathology is chronic or acute, of infectious or non-infectious origin.
    [0017]
    17. Use according to claim 15 or 16, characterized in that the treatment is a medicinal treatment, a radiotherapy or a transplant.
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    同族专利:
    公开号 | 公开日
    FR3014198B1|2017-03-03|
    CN105934670A|2016-09-07|
    ES2701250T3|2019-02-21|
    CN105934670B|2018-10-30|
    WO2015082839A1|2015-06-11|
    EP3077813A1|2016-10-12|
    EP3077813B1|2018-10-24|
    US20160370265A1|2016-12-22|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2012162563A2|2011-05-24|2012-11-29|The Regents Of The University Of California|Method for exosomal biomarker detection by electric field-induced release and measurement|
    WO2013022995A2|2011-08-08|2013-02-14|Caris Life Sciences Luxembourg Holdings, S.A.R.L.|Biomarker compositions and methods|
    WO2013041913A1|2011-09-22|2013-03-28|Universidad De Los Andes|Method for monitoring, diagnosis and/or prognosis of acute kidney injury in early stage|
    FR2788780B1|1999-01-27|2001-03-30|Ap Cells Inc|PROCESS FOR THE PREPARATION OF MEMBRANE VESICLES|
    WO2009015357A1|2007-07-25|2009-01-29|University Of Louisville Research Foundation, Inc.|Exosome-associated microrna as a diagnostic marker|
    US20130196355A1|2008-01-25|2013-08-01|Hansabiomed Ou|Method and a kit to quantify and qualify exosomes for diagnosis of prostate cancer and prostate hyperplasia|
    US10545149B2|2008-10-06|2020-01-28|Morehouse School Of Medicine|Detection of HIV-related proteins in urine|
    US20140113310A9|2008-12-05|2014-04-24|Myriad Genetics, Incorporated|Cancer detection markers|
    WO2010141862A2|2009-06-05|2010-12-09|Mayo Foundation For Medical Education And Research|Methods and materials for isolating exosomes|
    FR2968767B1|2010-12-08|2012-12-21|Biomerieux Sa|METHOD AND KIT FOR IN VITRO DIAGNOSIS OF PROSTATE CANCER|
    CN103197066B|2013-03-07|2015-12-23|美国纳米材料创新有限公司|A kind of immunoliposome biochip, its preparation method and the application in biological detection thereof|CN108697740A|2015-11-18|2018-10-23|佐治亚大学研究基金会|The outer vesica of nerve cell|
    EP3455371A1|2016-05-09|2019-03-20|Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt |Tissue-specific exosomes as biomarkers|
    CN107446879B|2017-06-27|2020-10-23|华南农业大学|Method for separating and purifying different exosome subgroups|
    CN110133272A|2018-02-09|2019-08-16|北京大学|Method for the excretion body in astroglia source to be enriched with or detected from biological fluid|
    WO2020194968A1|2019-03-26|2020-10-01|株式会社カネカ|Method for producing extracellular membrane vesicle-binding carrier|
    KR102323360B1|2019-12-02|2021-11-09|솔바이오 주식회사|Apparatus and method for preparing a liquid biopsy sample with an exosome subpopulation or the lower|
    WO2021112464A1|2019-12-02|2021-06-10|솔바이오 주식회사|Preparation device and preparation method for exosome liquid biopsy sample and method for analyzing exosome liquid biopsy sample prepared thereby|
    CN111394296A|2020-04-01|2020-07-10|丰能医药科技（上海）有限责任公司|Separation method of single exosome and nucleic acid sequencing method|
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    优先权:
    申请号 | 申请日 | 专利标题
    FR1362021A|FR3014198B1|2013-12-03|2013-12-03|METHOD FOR ISOLATING EXOSOMES|FR1362021A| FR3014198B1|2013-12-03|2013-12-03|METHOD FOR ISOLATING EXOSOMES|
    PCT/FR2014/053143| WO2015082839A1|2013-12-03|2014-12-03|Method for isolating exosomes|
    EP14824058.3A| EP3077813B1|2013-12-03|2014-12-03|Method for isolating exosomes|
    ES14824058T| ES2701250T3|2013-12-03|2014-12-03|Isolation procedure of exosomes|
    CN201480066123.3A| CN105934670B|2013-12-03|2014-12-03|Method for detaching excretion body|
    US15/100,931| US20160370265A1|2013-12-03|2014-12-03|Method for isolating exosomes|
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