WO2023121394A1 - Anti-cancer composition comprising patient-derived exosome, and use thereof - Google Patents

Anti-cancer composition comprising patient-derived exosome, and use thereof Download PDF

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WO2023121394A1
WO2023121394A1 PCT/KR2022/021193 KR2022021193W WO2023121394A1 WO 2023121394 A1 WO2023121394 A1 WO 2023121394A1 KR 2022021193 W KR2022021193 W KR 2022021193W WO 2023121394 A1 WO2023121394 A1 WO 2023121394A1
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cancer
met
ascites
exo
oncogene
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PCT/KR2022/021193
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French (fr)
Korean (ko)
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이지연
형수진
임호영
강원기
박준오
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사회복지법인 삼성생명공익재단
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Priority claimed from KR1020220112968A external-priority patent/KR102564043B1/en
Application filed by 사회복지법인 삼성생명공익재단 filed Critical 사회복지법인 삼성생명공익재단
Publication of WO2023121394A1 publication Critical patent/WO2023121394A1/en

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    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
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    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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Definitions

  • the present invention relates to anticancer compositions comprising patient-derived exosomes and uses thereof.
  • Gastric cancer is the fifth most common malignancy worldwide and the third leading cause of cancer-related death, with a high incidence in East Asia.
  • the survival rate of gastric cancer is increasing with new surgical resection and anticancer drugs, gastric cancer patients with malignant ascites due to peritoneal metastasis are the most common cause of death in advanced gastric cancer, especially diffuse-type GC.
  • malignant ascites is closely related to highly malignant gastric cancer and poor prognosis, but effective treatment options are lacking.
  • Exosomes are nanometer-sized extracellular vesicles that are typically produced in most cell types and function as the body's natural intercellular transport system for proteins, nucleic acids, peptides, lipids, and various other molecules. These exosomes have a number of potential therapeutic uses and have already been studied as delivery vesicles for gene therapy, mRNA delivery, and delivery of short nucleic acids in a variety of settings.
  • exosomes have clear biological importance, and this property can be exploited to target the delivery of therapeutic agents to specific tissues to induce effective therapy.
  • the present inventors as a target strategy for personalized treatment based on exosome engineering, in particular, as a result of diligent efforts to develop an effective and safe treatment for tumor treatment in a subject, obtained from gastric cancer patient malignant ascites
  • gastric cancer-patient ascites-derived exosomes are engineered to suppress MET, an oncogene, and engineered into MET-amplified GC.
  • the present invention was completed by identifying that the anticancer effect was excellently achieved when exosomes (EXO MET depl. ) were treated in combination with a MET inhibitor and/or an anticancer agent.
  • one object of the present invention is (a) an oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); And (b) the oncogene inhibitor or anticancer agent; to provide a pharmaceutical composition for preventing or treating cancer comprising as an active ingredient.
  • another object of the present invention is (a) an oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); And (b) the oncogene inhibitor and anticancer agent; to provide a pharmaceutical composition for the prevention or treatment of cancer, containing as an active ingredient.
  • another object of the present invention is (a) an oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); and (b) to provide a pharmaceutical composition for a sensitizer for enhancing sensitivity to an anticancer agent, comprising the oncogene inhibitor as an active ingredient.
  • another object of the present invention is (a) an oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); And (b) the oncogene inhibitor or anticancer agent; to provide a food composition for preventing or improving cancer, containing as an active ingredient.
  • another object of the present invention is (a) an oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); And (b) the oncogene inhibitor and anticancer agent; to provide a food composition for the prevention or improvement of cancer containing as an active ingredient.
  • another object of the present invention is (a) an oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); And (b) the oncogene inhibitor or (c) the anticancer agent; to provide a method for treating cancer, comprising administering to a subject a composition for preventing or treating cancer comprising as an active ingredient .
  • another object of the present invention is (a) an oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); (b) the oncogene inhibitor; And (c) an anticancer agent; to provide a method for treating cancer, comprising the step of administering to a subject a pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient.
  • another object of the present invention is (a) an oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); And (b) a method for treating cancer comprising administering to a subject a pharmaceutical composition for a sensitizer for enhancing sensitivity to an anticancer agent, comprising the oncogene inhibitor as an active ingredient. is to provide
  • the present invention provides (a) an oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); And (b) the oncogene inhibitor and / or (c) anticancer agent; it provides a pharmaceutical composition for preventing or treating cancer, containing as an active ingredient.
  • oncogene refers to a genetic material that makes cancer cells, that is, a gene having the ability to induce cancer, and is also referred to as an oncogene or an oncogene.
  • the oncogene may include any oncogene known in the art as long as the object of the present invention can be achieved, for example, MET, EGFR, bFGF, aFGF, int2/FGF3, hst1/K- fgf/FGF4, FGF5, hst2/FGF6, KGF, AIGF, erbB1, erbB2/neu, ros, trkA, trkB, trkC, ret, kit, PDGFR, flt1, flt3, flk1/kdr, flk2, FGFR2/K-sam/ bek, KGFR, FGFR1/Nsam/flg/Cek1/bF, FGF3/Cek3, FGFR4, abl, src, yes, fyn, fgr, lyn, lck, hck, blk, csk, fps, fes, mas
  • the present invention targets the MET gene to treat or inhibit the growth of MET-overexpressed cancer, the cancer is the expression level of the MET gene; Alternatively, when the expression level of the protein thereof is measured, the expression level of the MET gene or protein is overexpressed.
  • overexpression used to describe cancer in which MET is overexpressed according to the present invention refers to the case where the expression level of MET is measured through a suitable expression assay, in a target cell for comparison (e.g., a normal gastric cell, which is a corresponding organ). ) means more than twice the MET expression level of ), and is used interchangeably with “MET-amplification” herein.
  • the (a) inhibitor for suppressing the expression level of a target oncogene or its protein used to secure patient-derived exosomes in which the expression level of an oncogene or its protein is suppressed is (b) an oncogene inhibitor
  • an oncogene inhibitor As the same as, as long as it can inhibit the expression level and / or expression level or activity of the target oncogene of the present invention, it may include any means known in the art, for example, siRNA (small interference RNA), shRNA (short hairpin RNA), miRNA (microRNA), ribozyme, DNAzyme, PNA (peptide nucleic acids), antisense oligonucleotide, antibody, aptamer, extract, and compound. It may be more than one, but is not limited thereto.
  • the inhibitor is an antisense oligonucleotide, aptamer, small interference RNA (siRNA), short hairpin RNA (shRNA), miRNA (microRNA) or compound that specifically binds to the mRNA of the gene, most preferably siRNA (small interference RNA), or a compound.
  • siRNA small interference RNA
  • shRNA short hairpin RNA
  • miRNA miRNA
  • siRNA small interference RNA
  • the inhibitors may include those that inhibit the expression of MET nucleotides or the activity of MET gene proteins, but are not limited thereto.
  • the inhibitor for inhibiting the expression of MET nucleotides is siRNA (small interference RNA), shRNA (short hairpin RNA), miRNA (microRNA), ribozyme, DNAzyme, PNA ( peptide nucleic acids) and at least one selected from the group consisting of antisense oligonucleotides, but is not limited thereto.
  • the inhibitor for inhibiting the expression of the MET protein is at least one selected from the group consisting of an antibody against MET, an aptamer, a compound that directly binds to and inhibits the activity of MET protein, and a natural extract. This includes, but is not limited to.
  • the siRNA is MET siRNA (siMET) represented by the nucleotide sequence of SEQ ID NO: 1.
  • the compound of the present invention is savolitinib, crizotinib (PF-02341066), capmatinib, NVP-BVU972, AMG 337, bozitinib, glumetinib ) and at least one selected from the group consisting of tepotinib, preferably savolitinib.
  • the compound may mean an anticancer agent that is a MET blocker, and any MET blocker may be applied as long as it can obtain the desired anticancer effect.
  • the (c) anticancer agent of the present invention may include any anticancer agent known in the art as long as it can achieve the object of the present invention, for example, ramucirumab, cisplatin ( cisplatin, carboplatin, oxaliplatin, paclitaxel, docetaxel, vincristine, vinorelbine, etoposide, methotrexate, thalidomide ( thalidomide) and bortezomib, and may be at least one selected from the group consisting of, preferably ramucirumab, but is not limited thereto.
  • ramucirumab cisplatin
  • carboplatin oxaliplatin
  • paclitaxel docetaxel
  • vincristine vinorelbine
  • etoposide methotrexate
  • thalidomide thalidomide
  • bortezomib bortezomib
  • Cancer the target disease of the present invention, has aggressive characteristics in which cells divide and grow in defiance of normal growth limits, invasive characteristics infiltrating surrounding tissues, and spreading to other parts of the body. It is a generic term for diseases caused by cells having metastatic characteristics. In the present specification, the cancer may also be used as the same meaning as a malignant tumor or malignant ascites.
  • the cancer of the present invention includes gastric cancer, breast cancer, lung cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, and skin cancer.
  • skin cancer head or neck cancer, cutaneous or intraocular melanoma, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer ( anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer (thyroid cancer), parathyroid cancer, renal cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchogenic cancer and bone marrow cancer ( bone marrow tumor), but is not limited thereto, but is preferably gastric cancer.
  • the patient-derived exosome is derived from malignant ascites of a cancer patient, and the patient-derived exosome may be autologous, allogenic or xenogenic. It can be, preferably autologous.
  • the present invention is a specific factor in exosomes derived from patient-derived cell lines (PDCs, Patient-Derived Cells) obtained from malignant ascites of gastric cancer patients, targeting the MET gene so that MET, an oncogene, is suppressed.
  • PDCs patient-derived cell lines
  • the gastric cancer-patient ascites-derived exosomes are engineered, and the engineered exosomes (EXO MET depl. ) are applied to the MET-amplified GC, MET inhibitors and/or anticancer drugs are used in combination to treat cancers in which MET is overexpressed or to suppress metastasis.
  • the expression "expression level of a gene; or measuring the expression level of a protein thereof" used herein means detecting a target to be detected within a corresponding sample.
  • the target to be detected is the mRNA and/or protein of the corresponding gene in the sample. That is, it is possible to determine whether the gene is expressed by detecting RNA, which is a transcription product of the target, or protein, which is a gene product.
  • RNA or protein can usually be performed by extracting RNA or protein from a sample and detecting RNA or protein in the extract. Detection of such RNA or protein may be measured by immunoassay methods, hybridization reactions, and amplification reactions, but is not limited thereto and can be easily performed using various techniques known in the art.
  • biological sample refers to any sample obtained from a subject in which the expression of the gene or protein of the present invention can be detected.
  • the biological sample is any one selected from the group consisting of saliva, biopsy, blood, skin tissue, liquid culture, feces and urine, but is not particularly limited thereto, It may be prepared by treatment by a method commonly used in the technical field of the present invention.
  • the agent for measuring the gene expression level may include an antisense oligonucleotide, a primer pair, or a probe that specifically binds to mRNA of the gene.
  • the agent for measuring the expression of the mRNA is selected from the group consisting of antisense oligonucleotides specific to the gene, primer pairs, probes, and combinations thereof. That is, detection of a nucleic acid may be performed by an amplification reaction using one or more oligonucleotide primers that hybridize to a nucleic acid molecule encoding a gene or a complement of the nucleic acid molecule.
  • mRNA detection using primers can be performed by amplifying the gene sequence using an amplification method such as PCR, and then confirming the amplification by a method known in the art.
  • the "probe” refers to a nucleic acid fragment such as RNA or DNA corresponding to a few bases to several hundred bases in length that can specifically bind to mRNA, and is labeled to confirm the presence or absence of a specific mRNA and the amount of expression.
  • the probe may be manufactured in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, an RNA probe, or the like. Selection of an appropriate probe and hybridization conditions can be appropriately selected according to techniques known in the art.
  • the "primer” is a nucleic acid sequence having a short free 3' hydroxyl group, capable of forming base pairs with a complementary template, and serving as a starting point for template strand copying.
  • Primers can initiate DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. PCR conditions and lengths of sense and antisense primers can be appropriately selected according to techniques known in the art.
  • the agent for measuring the expression level of the protein may include an antibody, peptide or nucleotide that specifically binds to the protein.
  • An agent for measuring the expression level of the protein refers to an antibody that specifically binds to the protein, and includes polyclonal antibodies, monoclonal antibodies, recombinant antibodies, and combinations thereof.
  • Such antibodies include polyclonal antibodies, monoclonal antibodies, recombinant antibodies and complete forms having two full-length light chains and two full-length heavy chains, as well as functional fragments of antibody molecules, such as Fab, F(ab' ), F(ab')2 and Fv.
  • Antibody production can be easily prepared using techniques well known in the field to which the present invention pertains, and commercially available antibodies can be used.
  • the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components.
  • a lubricant e.g., a talc, a kaolin, a kaolin, a kaolin, a kaolin, a kaolin, kaolin, kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, manni
  • the pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc. can be administered.
  • the suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, medical condition, food, administration time, administration route, excretion rate and reaction sensitivity, A ordinarily skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis.
  • the daily dosage of the pharmaceutical composition of the present invention is 0.0001-1000 mg/kg.
  • the pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. or it may be prepared by incorporating into a multi-dose container.
  • the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.
  • the present invention provides (a) a patient-derived exosome in which the expression level of the above-described oncogene or protein thereof is suppressed; And (b) the oncogene inhibitor and / or (c) anticancer agent; it provides a food composition for preventing or improving cancer containing.
  • composition of the present invention may be added to health functional foods for the purpose of preventing or improving cancer.
  • “functional health food” refers to food having bioregulatory functions such as cancer prevention and improvement, biodefense, immunity, and recovery after illness, and should be harmless to the human body when taken for a long time.
  • the composition of the present invention When the composition of the present invention is used as a food additive, the composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods.
  • the mixing amount of active ingredients may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment).
  • the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material during production of food or beverage.
  • the active ingredient may be used in an amount above the above range.
  • Examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and includes all health foods in a conventional sense.
  • the health beverage composition of the present invention may include various flavoring agents or natural carbohydrates as additional components, like conventional beverages.
  • the aforementioned natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame.
  • the proportion of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g per 100 ml of the composition of the present invention.
  • the composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages; and the like.
  • the composition of the present invention may include fruit flesh for preparing natural fruit juice, fruit juice beverages, and vegetable beverages. These components may be used independently or in combination. The ratio of these additives is not critical, but is generally selected in the range of 0.01 to 0.1 part by weight per 100 parts by weight of the composition of the present invention.
  • the present invention provides the above-described (a) oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); and (b) a pharmaceutical composition for a sensitizer for enhancing sensitivity to an anticancer agent of cancer, comprising the oncogene inhibitor as an active ingredient.
  • composition of the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient.
  • the pharmaceutical composition for the sensitizer of the present invention increases the sensitivity of cancer to radiation, chemotherapy or antibody anticancer agents.
  • the term “therapeutically effective amount” means that the amount of the inhibitor administered relieves to some extent one or more symptoms of cancer, the disease to be treated.
  • a pharmacologically effective amount is meant to (1) reverse the rate of cancer progression or (2) inhibit further progression of cancer to some extent, and (3) alleviate to some extent one or more symptoms associated with cancer. It means an amount having an effect of reducing (preferably, removing).
  • the term "pharmaceutically acceptable carrier” is commonly used in formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, including, but not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil; it is not going to be
  • the term "sensitizer” refers to a drug that increases the sensitivity of cancer cells to an anticancer drug (or radiation) or causes an anticancer drug (or radiation) to act specifically on cancer cells, thereby achieving a therapeutic effect even at a small dose. material that helps to achieve
  • composition of the present invention is administered in combination with an anticancer agent, and the combination administration is administered simultaneously (simultaneously), separately (separately) or sequentially (sequentially).
  • the sensitizer of the present invention is an anticancer adjuvant, which means any form for increasing the anticancer effect of an anticancer agent or suppressing or improving the side effects of an anticancer agent.
  • the anticancer adjuvant of the present invention can be administered in combination with various types of anticancer agents or anticancer adjuvants, and when administered in combination, even if the anticancer agent is administered at a lower dose than conventional anticancer agents, it can exhibit an equivalent level of anticancer treatment effect, which is safer anticancer treatment can be performed.
  • composition of the present invention when used in combination with existing anticancer agents, it is possible to exert a strong cell killing effect on cancer cells while greatly reducing the dose of existing anticancer agents that are highly toxic.
  • the pharmaceutical composition for a sensitizer of the present invention uses the above-described active ingredients, descriptions of common contents between the two are omitted in order to avoid excessive complexity of the present specification.
  • the present invention in addition, according to another aspect of the present invention, the above-described (a) oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); and (b) the oncogene inhibitor or (c) the anticancer agent; administering to a subject a composition for preventing or treating cancer comprising as an active ingredient, it provides a method for treating cancer.
  • the present invention provides a method for treating cancer, comprising administering to a subject a pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient.
  • the present invention in addition, according to another aspect of the present invention, the above-described (a) oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); And (b) a method for treating cancer comprising administering to a subject a pharmaceutical composition for a sensitizer for enhancing sensitivity to an anticancer agent, comprising the oncogene inhibitor as an active ingredient.
  • the present invention can be usefully used as a sensitizer that doubles the effect of not only patient-specific cancer-specific therapeutic agents but also other anti-cancer therapeutic agents.
  • Figure 1 shows the characteristics of exosomes isolated from ascites of cancer patients.
  • 1A is a schematic diagram of a patient's ascites including exosomes and cancer cells. Ascites obtained from four cancer patients was separated by centrifugation, and both the supernatant containing exosomes and the pellet containing cancer cells were collected.
  • Figure 1B shows the clinical information of each cancer patient including cancer type, MET amplification, sex and age. Ultracentrifugation was used to isolate pure exosomes from ascites.
  • 1C is a schematic diagram of an exosome isolation method.
  • FIG. 1D is a table of exosome concentration and size distribution of the isolated particles shown in Figure 1E.
  • 1F is a representative transmission electron microscopy (TEM) image showing the round cup-shaped morphology. Scale bar: 100 ⁇ m.
  • Figure 1G is a Western blot analysis of purified EXO Ascites showing multivesicular (MVB) biosynthetic proteins (eg Alix and TSG101) and tetraspanins (eg CD63 and CD9).
  • MVB multivesicular
  • 1H is a representative confocal image showing uptake of EXO Ascites into individual patient-derived cells (PDCs).
  • EXO Ascites labeled with PKH67 were applied to each PDC and incubated for 24 hours.
  • DAPI 4',6-diamidino-2-phenylindole; blue
  • EXO ascites were labeled with PKH67 (green).
  • Scale bar 10 ⁇ m.
  • Figure 2 shows the depletion of EXO Ascites according to gw4869 treatment.
  • Newly generated exosomes from cancer and stromal cells were depleted by treatment with gw4869, which blocks exosome formation by preventing intraluminal vesicle formation, to analyze the role of EXO Ascites in cancer progression.
  • Mixtures of PDCs and endothelial cells (ECs) were plated in 6-well culture plates and co-cultured in cell culture medium with increasing concentrations of gw4869 (0, 1, 10, 20 and 30 ⁇ M).
  • the cell culture fluid collected on day 3 was isolated using the exosome separation method and then analyzed using NTA, cell viability assay, western blotting, and the like.
  • the size distribution Fig.
  • FIG. 2A shows representative immunoblots of exosome specific markers (Alix, TSG101, CD9 and CD63) for exosomes (top) and PDC lysates (bottom) treated with 0 and 10 ⁇ M gw4869.
  • FIG 3 shows MET oncogenic driver identification in EXO Ascites .
  • MET biomarker
  • FIG. 3B shows representative immunohistochemistry (IHC) results for patient specimens showing MET amplified GCs (left) and MET non-amplified GCs (right) from four cancer patients.
  • Figure 3C shows the copy number of MET amplification in 4 cancer patients. Visualization of exosomal MET in each EXO Ascites was performed using the ExoViewTM R100 imaging platform.
  • Figure 3D is a representative fluorescence image (top) showing CD63 + -EXO Ascites captured using anti-MET (blue) and an enlarged view of the yellow dashed area (bottom).
  • Figure 4 shows the reduction of cancer progression through blocking exosome secretion using gw4869.
  • gw4869 was used to deplete exosomes in a tumor spheroid angiogenesis model using a three-dimensional (3D) spheroid chip.
  • Tumor spheroids preformed in a U-shaped 96-well plate were co-cultured with endothelial cells (EC) on a 3D tumor spheroid chip.
  • EC endothelial cells
  • PDC Patient-derived cells
  • FIG. 4A shows the effect of exosome depletion on cancer progression
  • Figure 4B shows a schematic of the process of validating a 3D tumor spheroid-angiogenesis model for the chip.
  • Figure 4B is a top view of the 3D tumor spheroid culture area and one well showing the extracellular matrix (light blue), EC area (red) and two "medium chamber” areas (orange).
  • FIG. 4C shows representative 3D tumor spheroid-angiogenesis images (EpCAM, red) and blood vessels (lectin, green) obtained after staining tumor spheroids.
  • Figure 4D shows representative confocal images of tumor spheroids (EpCAM, red) and blood vessels (lectin, green) counterstained with DAPI (blue).
  • Figure 4D shows quantification of cancer invasiveness and angiogenesis based on tumor spheroid images of MET amplified GC (Figure 4E) and MET non-amplified GC ( Figure 4F) models.
  • n 5, *p ⁇ 0.05, **p ⁇ 0.01 and ***p ⁇ 0.001 compared to control (repeated measures ANOVA with Tukey's multiple comparisons test). ns, there is no significant difference.
  • Scale bar 1 mm
  • FIG. 5 shows cancer progression results after treatment with various concentrations of a MET inhibitor (SVOL) or ramucirumab (RAM).
  • Varying concentrations of SVOL (0, 0.1, 1, 10 and 100 ⁇ M) were applied to tumor spheroid-angiogenesis models with and without MET gene alterations, showing cancer invasiveness (Fig. 5A) and angiogenesis (Fig. 5B).
  • Quantification of cultures is shown.
  • Cavities exhibiting cancer invasiveness (Fig. 5C) and angiogenesis (Fig. 5D) when various concentrations of RAM (0, 0.1, 1, 10 and 100 ⁇ M) were applied to tumor spheroid angiogenesis models with or without MET genetic alterations.
  • Quantification of cultures is shown.
  • n 3 ⁇ SEM. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001 compared to each value at 0 ⁇ M drug concentration.
  • FIG. 6 shows progressive enhancement of cancer invasiveness and angiogenesis by EXO Ascites .
  • EXO Ascites samples obtained from four patients were used as 3D tumor spheroid chips. Briefly, for 2 days pre-formed tumor spheroids were co-cultured with ECs on a 3D tumor spheroid chip. EXO Ascites with increasing concentrations (0, 10 2 , 10 4 , 10 6 and 10 8 EXO Ascites for each patient sample) were treated with cell culture medium containing 10 ⁇ M gw4869 on day 1 and set concentrations of EXO Ascites were immunized. It was kept until fixed for cytochemistry.
  • Figure 6A shows a schematic diagram of the role of EXO Ascites in cancer progression based on a tumor spheroid-angiogenesis model.
  • cancer spheroid models for all patients treated with EXO Ascites concentration of 0, 10 2 , 10 4 , 10 6 and 10 8
  • cancer invasiveness and angiogenesis were measured based on immunocytochemistry on day 6.
  • Fixed samples were immunostained with antibodies against EpCAM (red for cancer) and lectins (green for EC) and counterstained with DAPI (blue).
  • 6B is a representative 3D tumor spheroid-angiogenesis image staining with PKH67-EXOAscites (yellow), tumor spheroids (EpCAM, red), and blood vessels (lectin, green); Magnified images depict PKH67 labeled EXO Ascites with tumor cells (left) and blood vessels (right).
  • 6C-D shows representative confocal fluorescence images of co-culture showing changes in cancer growth and angiogenesis in EXO Ascites concentrations in cancer spheroids of MET amplified GC and MET non-amplified GC models.
  • Figures 6E and 6F show quantification of cancer invasiveness (Figure 6E) and angiogenesis (Figure 6F) of Figures 6C-D.
  • n 3 ⁇ SEM for images shown in C and D.
  • Scale bar 1 mm.
  • EXOAscites EXO Ascites in all patients induced cancer progression.
  • Scale bar 1 mm.
  • FIG. 7 shows the efficient intracellular delivery of MET-containing EXO Ascites to MET null GCs.
  • 7A shows a representative confocal image (top) showing EXOAscites capturing MET via the ExoViewTM R100 imaging platform and a schematic diagram of the transformation of MET-negative GC cells into MET-positive cancer cells by EXOAscites harboring MET. Scale bar: 5 ⁇ m.
  • Figure 7B shows representative confocal fluorescence images of MET null cancer cells with or without EXO Ascites capturing MET (red). Latent uptake of EXO Ascites with MET migrates to MET null GC (SNU-638) and induces promotion of MET expression (green). Scale bar: 100 ⁇ m.
  • Figure 8 shows the strong therapeutic effect of engineered EXO MET depl in the MET amplification GC model.
  • engineered MET-depleted exosomes were added to a 3D tumor spheroid-angiogenesis model.
  • siMET was transfected into cultured PDCs using Lipofectamine and incubated for 48 hours before collecting the conditioned media.
  • EXO MET depl was isolated by ultracentrifugation (FIG. 8A).
  • 8B shows representative immunoblots of MET protein of exosomes treated with or without siMET in MET amplified GCs or MET non-amplified GCs. Gapdh was used as a loading control.
  • FIG. 8C shows a schematic diagram of the therapeutic effect of EXO MET depl in a 3D tumor spheroid-angiogenesis model.
  • Gw4869, EXO MET depl , EXO Ascites , SVOL and/or RAM were added to the cultures on day 1 and their concentrations were maintained throughout the experiment.
  • Figure 8D shows representative 3D tumor spheroid-angiogenesis images obtained after staining tumor spheroids (EpCAM, red) and blood vessels (lectin, green). Cancer invasiveness and angiogenesis were reduced by EXO MET depl treatment.
  • Figure 8E shows representative confocal images showing tumor spheroids (EpCAM, red) and blood vessels (lectin, green) counterstained with DAPI (blue) in tumor spheroid MET-amplified and non-MET-amplified GC models.
  • Figures 8F and 8G show the quantification of cancer invasiveness and angiogenesis in the MET amplified GC model ( Figure 8F) and the MET non-amplified GC model ( Figure 8G) based on the tumor spheroid images in Figure 8E.
  • n 5, ##p ⁇ 0.01, ###p ⁇ 0.001, +p ⁇ 0.05, +++p ⁇ 0.001, and *p ⁇ 0.05 compared to control, EXO Ascites or EXO MET depl (Tukey's multiple comparisons Repeated measures ANOVA with test). ns, there is no significant difference. A gw4869 treated sample was used as a control. Scale bar: 1 mm.
  • Specimen collection and related clinical data used in this example were approved by the Samsung Seoul Hospital Institutional Review Board (IRB# 2021-09-052). All patients participating in this study provided written informed consent prior to enrollment and sample collection. This example was performed according to the principles of the Helsinki Declaration and Korean Good Clinical Practice guidelines.
  • Exosomes were isolated from the ascites of four GC patients by various sequential centrifugation steps with some modifications to methods known in the art. Briefly, after filtering the ascites with a 30 ⁇ m pore filter (Millipore), centrifugation was performed at 300 g for 15 minutes to collect pellets and supernatants, respectively. Pellets containing cancer cells were used to create cancer spheroids in further experiments. 2000g of supernatant ascites was centrifuged to discard debris, and 10,000g of ascites was further purified by centrifugation at 4°C for 30 minutes. Thereafter, 50 ml of ascites was ultracentrifuged at 110,000 g and 4° C.
  • proteinase K (1mg/ml, 0.05 volume per exosome) was incubated with EXO Ascites at 37°C for 30 minutes, followed by heat inactivation at 60°C for 20 minutes. The exosome samples were then ultracentrifuged at 110,000g for 70 min at 4°C. Proteinase K treated exosomes were used for all in vitro assays.
  • NTA Nanoparticle tracking analysis
  • Nanoparticle concentration and size distribution were measured with a NanoSight LM10 (NanoSight Technology, Malvern, UK) system equipped with a blue 488 nm laser and a high-sensitivity Scientific Complementary Metal-Oxide Semiconductor (sCMOS) camera. During the analysis, the temperature was set and kept constant at 25°C. Images were measured three times at camera setting 13 with an acquisition time of 30 seconds and a detection threshold setting of 10. Acquired images were analyzed using NTA analysis software (version 3.4 build 3.4.003).
  • EXO Ascites or PDC lysates were lysed in lysis buffer (150 mM NaCl, 1 mM MgCl 2 , 1 mM EDTA, 10% glycerol, 20 mM HEPES [PH 7.4] 1% Triton x-100) with complete protease inhibitors (Invitrogen). Protein concentration was measured using BCA protein reagent (Pierce Biotechnology). Dissolved EXOAscites (20ug) or PDC lysates (10ug) were separated by SDS-polyacrylamide gel electrophoresis (PAGE) gel (using 12% gel) and blotted onto a nitrocellulose membrane with 0.2 ⁇ m pore size (Whatman) for 60 min.
  • lysis buffer 150 mM NaCl, 1 mM MgCl 2 , 1 mM EDTA, 10% glycerol, 20 mM HEPES [PH 7.4] 1% Triton x-100
  • Membranes were blocked with 5% skim milk (BD Bioscience) in TBST buffer (sigma) for 1 hour on a shaker at room temperature and then treated with primary antibodies overnight at 4°C. After washing three times with TBST buffer, the membrane was incubated with the corresponding IgG-horseadish peroxidase (HRP) secondary antibody for 1 hour at room temperature. Visualization of the membrane was determined by the ECL system (Invitrogen).
  • HRP IgG-horseadish peroxidase
  • Ascites fluid from patients with advanced GC was cultured according to methods known in the art. Briefly, cells isolated from ascites were supplemented with 10% fetal bovine serum (gibco), 1% penicillin/streptomycin (Gibco), 0.5 ⁇ g/ml hydrocortisone (Sigma), 5 ⁇ g/ml insulin (PeproTech) and They were cultured in RPMI 1640 medium (gibco) supplemented with 5 ng/ml epidermal growth factor (PeproTech). The culture medium was changed every 3 days, and the PDCs were maintained in a 37°C incubator. Passage 1-2 PDCs were used to derive the cancer-spheroid model.
  • EXO Ascites were labeled with the PKH 67 or 26 Fluorescent Cell Linker Kit (Sigma) according to the manufacturer's instructions with slight modifications. Briefly, 2.5 ul dye solution was diluted with 100 ul solution C and incubated with 2 x 10 7 particles of EXO Ascites for 15 min at room temperature. Filtered PBS was added to the mixture, followed by ultracentrifugation at 100,000 for 1 hour and the pellet resuspended in filtered PBS.
  • PDC, gastric cancer cell line (SNU-638), and PDC and ECs plated on coverslips were treated with PKH 67 or PKH26 and cultured for 48 hours.
  • Fluorescent cells labeled with EXO Ascites were analyzed with the CQ1 system (Yokogawa Electric Corporation, Tokyo, Japan).
  • Samples were fixed with 4% paraformaldehyde for 20 minutes at room temperature and then treated with 0.1% Triton X-100 followed by 3% bovine serum albumin (BSA; Millipore, Burlington, MA, USA). Fixed samples were stained with primary antibodies (Table 1) in 1% BSA overnight at 4°C. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; Life Technologies, Carlsbad, CA, USA) for 1 hour. Image acquisition was performed by the CQ1 system.
  • BSA bovine serum albumin
  • PDCs (6 ⁇ 10 4 cells/ml) and ECs (1 ⁇ 10 4 cells/ml) were cultured on day 0 in cell culture medium containing 10% exosome-depleted FBS (gibco). ), were treated with different concentrations of gw4869 (0, 1, 10, 20, 30 ⁇ M, respectively). After 72 hours, cell conditioned medium was collected under different concentrations of gw4869 and depleted nanoparticles were isolated using the differential ultracentrifugation method as described above.
  • the viability of the cultured cells was determined by a live and dead cell staining kit (abcam) according to the manufacturer's instructions. Briefly, cell mixtures containing PDCs and ECs were cultured in 6-well plates and treated with different gw4869 concentrations (0, 1, 10, 20 and 30 ⁇ M) as mentioned above for 72 hours. Then, the cells were washed with PBS and treated with a combination of 5 ul calcein-AM and 20 ul propidium iodide solution in a 37° C. incubator. Samples were washed three times with PBS and examined under a confocal microscope (LSM 700). Live and dead cells were counted in 5 random confocal images and analyzed via ImageJ software (version 2.1.0/1.53c).
  • endothelial cells (1 x 10 5 cells/ml) were plated on one side of the hydrogel interface and the other side of the hydrogel interface was filled with culture medium.
  • Cells were maintained in EGM2 medium (Lonza, Walkersville, MD, USA) containing 5 ⁇ g/ml insulin (PeproTech, Rocky Hill, NJ, USA).
  • EGM2 medium Licos, Walkersville, MD, USA
  • sovolitinib (SVOL, MET inhibitor), or ramucirumab (RAM) each factor was added to the medium and the concentration was maintained throughout the culture until fixed for analysis.
  • the spheroid intensity core region means the intensity level of the sample stained in the initial region of the cancer spheroid.
  • the average intensity level of the protein of interest in each bar experiment was normalized to the average intensity level of the control group. At least three independent experiments were analyzed in each condition.
  • EXO Ascites labeled with PKH67 (green) were plated on one chamber of a 3D tumor spheroid chip pre-incubated with cancer spheroid-angiogenesis for 24 hours.
  • Live imaging of green-expressing EXO Ascites in bright-field images was performed with a CQ1 system equipped with a high-sensitivity sCMOS camera and a 37 °C chamber using a 20x objective with a fluorescence filter (excitation at 488 nm, emission at 525/50 nm). It became.
  • Time-lapse movies with full chip area and cropped enlarged images of EXO Ascites (green) homing to cells at 60-minute intervals for a total of 9 hours were acquired with CQ1 software.
  • EXOAscites were tracked in each time-lapse sequence using the ImageJ plugin TrackMate. Fluorescent exosomes were captured using the Analyze Particles function after automatically setting a size threshold. Exosome cellular uptake was evaluated by calculating the exosome fluorescence intensity as a ratio to the cell area. TrackMate also labeled each sample and provided tracking information including displacement. Based on the above information, the homing function evaluated the displacement of labeled exosomes into cells.
  • EXO MET depl of MET amplified GC and MET non-amplified GC each PDC (1x10 6 cells/10ml) was cultured in a 100 mm 2 dish and siMET (20 ⁇ M, 5ul, final concentration 10Nm, Ambion ®, Assay ID 8700, SEQ ID NO: 1; GCACTAGCAAAGTCCGAGAtt). Cultured cells and medium were collected after 72 hours and medium was changed every 2 days. Each PDC lysate from MET amplified GC and MET non-amplified GC was lysed for further analysis for western blotting analysis. EXO MET depl was isolated from the cultured conditioned medium (50 ml) through an ultracentrifugation method.
  • EXO MET depl was treated with proteinase K and then removed by ultracentrifugation. si control was used as a transfection control.
  • FIG. 7 in order to compare the cancer progression between EXO Ascites and EXO MET depl , the particle concentration of conditioned exosomes was analyzed by NTA before constructing EXO MET depl . 10 6 particles/chip each of EXO Ascites or EXO MET depl were treated in this medium and maintained during cultivation.
  • FFPE formalin-fixed, paraffin-embedded
  • fluorescently labeled antibodies (tetraspanin Ab/dye 1:600, MET antibody 1:300, and PD-L1 antibody 1:200) were diluted in solution A and blocking solution, respectively.
  • Exosomes captured on the chip were scanned with ExoVeiw R-100 through nScan software (NanoView Bioscience), and data were analyzed through NanoViewer 2.9 software (NanoView Bioscience). At least six randomly selected regions from each EXO Ascites were quantified.
  • Fig. 1A The clinical signs of all patients were mesenchymal-epithelial transition factor (MET, NCBI Gene ID: 4233) amplification in half (2/4 cases), and the PD-L1 protein binding positivity score (CPS) had various values as follows: Shown: 0 to 10. The age of the patients was 45-56 years, and the male to female ratio was 1:3 (Fig. 1B).
  • MET mesenchymal-epithelial transition factor
  • CPS PD-L1 protein binding positivity score
  • Exosomes were isolated from a 50ml ascites sample (EXOAscites) by high-speed centrifugation, and the isolated EXO Ascites were confirmed through nanoparticle tracking analysis (NTA), Western blotting, transmission electron microscopy (TEM) and confocal microscopy. (Fig. 1D-H).
  • NTA nanoparticle tracking analysis
  • TEM transmission electron microscopy
  • Fig. 1D-H The NTA of particles isolated from all four EXO Ascites samples showed a size distribution consistent with the known size distribution of exosomes, with average sizes of 75.2 ⁇ 9.2 nm, 149.3 ⁇ 3.6 nm, 87.1 ⁇ 11.6 nm and 138.3 ⁇ 4.2 nm. .
  • the concentrations of the separated particles were 140 ⁇ 10 8 , 138 ⁇ 10 8 , 55.6 ⁇ 10 8 and 260 ⁇ 10 8 particles/ml, respectively (FIGS. 1D and E).
  • Immunoblot analysis of the isolated particles showed that exosomal markers such as multivesicular body (MVB) biosynthetic proteins (Alix and TSG101) and tetraspanins (CD63 and CD9) were enriched in particles isolated from all ascites (Fig. 1G). .
  • the typical cup-shaped morphology of EXO Ascites was observed through TEM (FIG. 1F). Exosomes are known to be powerful carriers that effectively deliver cargo to target cells.
  • EXO Ascites were incubated with PKH67-labeled EXO Ascites (green) for 24 hours, EXO Ascites clearly migrated to PDC in all cases and green fluorescence was evenly distributed around cell nuclei (blue). distributed (Fig. 1H).
  • exosomes in the cancer microenvironment, we first depleted exosome production in an in vitro cancer model including PDCs and endothelial cells (ECs).
  • gw4869 a common pharmacological agent for inhibition of exosome formation, which blocked both ceramide-mediated internal budding of MVBs and secretion of mature exosomes from MVBs.
  • mixtures of PDC associated with ascites and EC were cultured with medium containing gw4869 at concentrations of 0, 1, 10, 20 and 30 ⁇ M. Nanoparticles were separated from the collected cell condition medium through standard ultracentrifugation, and the size distribution and concentration of the separated nanoparticles were measured through NTA.
  • the maximum abundance of isolated particles in the 100-250 nm size range decreased rapidly when the concentration of gw4869 increased in all cancer models for all clinical samples.
  • a gradual decrease in the expression levels of exosomal markers such as Alix, TSG101, CD9 and CD63 was observed as the concentration of gw4869 increased, but the levels of PDC lysates remained constant regardless.
  • the concentration of isolated particles at 10 ⁇ M gw4869 was substantially reduced by nearly 5.0-fold, 74.8-fold, 5.5-fold, and 55.5-fold, respectively, in all clinical samples (Fig. 2B).
  • the present inventors next evaluated whether treatment with gw4869 affects the viability of cells constituting the cancer microenvironment.
  • Mixtures of PDC and EC were treated with various concentrations of gw4869 for 24 hours, followed by live and dead assays. There was no appreciable change in either condition in the live and dead cell images (Fig. 2E). More than 95% of the cell mixtures from all clinical samples survived regardless of the gw4869 concentration, indicating that gw4869 did not affect the viability of the cells (Fig. 2F).
  • 10 ⁇ M gw4869 has the potential to block the production and secretion of exosomes generated in the cancer microenvironment without causing cell damage.
  • the present inventors confirmed whether MET, an oncogenic factor reflecting the patient's clinical characteristics, was expressed in EXO Ascites .
  • Immunoblot results showed that MET protein was expressed in EXO Ascites from both patients (pt1 and pt4), respectively, whereas low expression of MET protein was observed in all PDC lysates (Fig. 3A).
  • Example 4 Possibility of reducing cancer invasiveness and angiogenesis by gw4869 treatment in 3D tumor spheroid-angiogenesis model
  • PDCs isolated from ascites were cultured with fibroblasts for 2 days to form tumor spheroids in 96-well plates (Fig. 4A).
  • Preformed tumor spheroids were injected into the middle channel of the chip and ECs were plated into the side channels of the chip ( Figure 4B).
  • SVOL savolitinib
  • RAM ramucirumab
  • tumor spheroid-angiogenic models were treated with 10 ⁇ M gw4869, 1 ⁇ M SVOL and/or 1 ⁇ M RAM. Metastasis of the tumor microenvironment by gw4869 treatment was determined by levels of EpCAM protein (red), a cancer invasive marker, and lectin protein (green), which indicates angiogenesis (Fig. 4C). Cancer invasiveness and angiogenesis were greatly reduced after gw4869 treatment in tumor spheroids of MET-amplified and non-MET-amplified GC models (Fig. 4D-F).
  • EpCAM and lectin in the MET amplified and non-MET amplified GC models by gw4869 treatment were 2.9, 1.7, 5.6 and 1.7 times lower than those in the control group, respectively (Fig. 4E and F).
  • a synergistic effect was observed with reduced EpCAM and lectin expression levels in both models in response to gw4869 co-treatment with SVOL and RAM (Fig. 4D-F).
  • the EpCAM level was 7.8 times lower and the lectin level was 3.3 times lower in the MET amplified GC model, and the EpCAM level was 3.3 times lower and the lectin level was 2.4 times lower in the MET non-amplified GC model.
  • the co-cultures were then treated with PKH-67 labeled EXO Ascites (0, 10 2 , 10 4 , 10 6 and 10 8 particles) with 10 ⁇ M gw4869 on day 1 to reduce the effect of exosomes secreted from PDCs and ECs. /chip) were treated with increasing concentrations (Fig. 6A). Cancer growth (red) and angiogenesis (green) were investigated on day 6 using EXO Ascites (yellow) labeled with PKH-67 (Fig. 6B). A 3D tumor spheroid model without gw4869 was used as a negative control (Fig. 6C).
  • EXO Ascites were preferentially delivered.
  • PKH67-labeled EXO Ascites were plated on PDC or EC and the number of cells containing EXO Ascites was counted.
  • the potential orientation of EXO Ascites was observed in the PDC, and most showed green fluorescence indicated by EXO Ascites , whereas the number of ECs with EXO Ascites was quite low.
  • EXO Ascites resulted in a dose-dependent enhancement of cancer progression (FIG. 6D-F).
  • PKH-67 labeled EXO Ascites gradually concentrated into tumor spheroids and were high in both GC models.
  • Immunocytochemistry results showed that the expression of both EpCAM protein and lectin protein gradually increased with the addition of a large number of EXO Ascites , but the saturation level of EXO Ascites for cancer progression differed between EXO Ascites samples (Fig. 6E and F). ).
  • EXO Ascites were delivered to 3D tumor spheroid models to promote cancer invasiveness.
  • EXO Ascites labeled with PKH67 was injected into one chamber of the chip, and flow was induced to the opposite chamber of the chip according to the volume difference of the cell culture medium.
  • EXO Ascites location on the 3D tumor spheroid model was determined through live cell imaging.
  • a time-lapse sequence of EXO Ascites movement on the chip showed that the distribution of PKH67-labeled EXOAscites (green) spread over the entire chip over time.
  • an exosome delivery system containing cargo is useful for managing cell functions by regulating the expression level of a target protein, enabling exosome-based cancer drug delivery.
  • Example 7 Therapeutic effect of engineered EXO MET depl on tumor spheroids obtained from MET amplified GC models
  • MET-amplified GCs were transfected with siMET to completely silence MET, whereas MET-non-amplified GCs could not be detected due to the absence of MET protein.
  • tumor spheroids co-cultured with ECs were treated with EXO MET depl , and the MET inhibitor savolitinib (SVOL) and the anticancer drug Lamu A synergistic effect was confirmed by co-treatment with ramucirumab (RAM) (FIG. 8C).
  • EXO Ascites of MET-amplified and non-MET-amplified GCs promoted cancer invasiveness and angiogenesis in a tumor spheroid angiogenesis model (FIG. 8E-G).
  • Cancer progression in tumor spheroids of the MET amplified GC model was substantially inhibited by EXO MET depl treatment isolated from MET amplified GCs compared to EXO Ascites ; Cancer invasiveness and angiogenesis were reduced 1.6-fold and 2.3-fold, respectively (Fig. 8E-G).
  • no change was observed when the MET unamplified GC model was treated with EXO MET depl separated from the MET unamplified GC.

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Abstract

The present invention relates to an anti-cancer composition comprising a patient-derived exosome, and the use thereof. The present invention may be utilized in the use of a sensitizer which doubles the effect of a patient-specific cancer-specific therapeutic agent as well as other anti-cancer therapeutic agents.

Description

환자-유래 엑소좀을 포함하는 항암 조성물 및 이의 용도Anti-cancer composition comprising patient-derived exosomes and uses thereof
본 발명은 환자-유래 엑소좀을 포함하는 항암 조성물 및 이의 용도에 관한 것이다.The present invention relates to anticancer compositions comprising patient-derived exosomes and uses thereof.
위암(GC)은 전 세계적으로 다섯 번째로 흔한 악성 종양이며 암 관련 사망의 세 번째 주요 원인이며 동아시아에서 높은 발병률을 보인다. 새로운 외과적 절제술과 항암제로 위암의 생존율이 증가하고 있으나, 복막전이로 인한 악성 복수가 있는 위암 환자는 진행성 위암, 특히, 미만형 위암(diffuse-type GC)에서 가장 흔한 사망 원인이다. 더욱이, 악성 복수는 고도 악성 위암 및 불량한 예후와 밀접한 관련이 있으나 효과적인 치료 옵션이 부족하다. Gastric cancer (GC) is the fifth most common malignancy worldwide and the third leading cause of cancer-related death, with a high incidence in East Asia. Although the survival rate of gastric cancer is increasing with new surgical resection and anticancer drugs, gastric cancer patients with malignant ascites due to peritoneal metastasis are the most common cause of death in advanced gastric cancer, especially diffuse-type GC. Moreover, malignant ascites is closely related to highly malignant gastric cancer and poor prognosis, but effective treatment options are lacking.
엑소좀은 전형적으로 대부분의 세포 유형에서 생산되고 인체의 자연적인 단백질, 핵산, 펩타이드, 지질, 및 다양한 다른 분자의 세포 간 운반 시스템으로 기능하는 나노미터-크기의 세포외 소포이다. 이러한 엑소좀은 다수의 잠재적 치료 용도를 가지며 이미 유전자 치료, mRNA 전달, 및 다양한 설정의 짧은 핵산의 전달을 위한 전달 소포로 연구되어 왔다.Exosomes are nanometer-sized extracellular vesicles that are typically produced in most cell types and function as the body's natural intercellular transport system for proteins, nucleic acids, peptides, lipids, and various other molecules. These exosomes have a number of potential therapeutic uses and have already been studied as delivery vesicles for gene therapy, mRNA delivery, and delivery of short nucleic acids in a variety of settings.
따라서, 세포-세포 소통의 맥락에서 엑소좀은 명백한 생물학적 중요성을 가지며, 이러한 특성은 특정 조직에 치료제의 전달을 표적화하여 효과적인 치료를 유도하는 데 이용될 수 있다.Thus, in the context of cell-cell communication, exosomes have clear biological importance, and this property can be exploited to target the delivery of therapeutic agents to specific tissues to induce effective therapy.
상술한 상황 하에서, 본 발명자들은 엑소좀 공학에 기반한 개인맞춤화 치료를 위한 표적 전략으로서, 특히, 대상에서의 종양 치료를 위한 효과적이고 안전한 치료법을 개발하기 위하여 예의 노력한 결과, 위암 환자 악성 복수에서 획득한 환자-유래 세포주(PDCs, Patient-Derived Cells)로부터 유래된 엑소좀 내 특정 인자로서 발암유전자인 MET이 억제되도록 위암-환자 복수 유래 엑소좀을 조작하고, MET-증폭 GC에 상기 엔지니어링(engineering)한 엑소좀(EXOMET depl.)을, MET 저해제 및/또는 항암제와 함께 병용처리했을 때, 항암 효과가 우수하게 달성됨을 규명함으로써, 본 발명을 완성하였다.Under the above circumstances, the present inventors, as a target strategy for personalized treatment based on exosome engineering, in particular, as a result of diligent efforts to develop an effective and safe treatment for tumor treatment in a subject, obtained from gastric cancer patient malignant ascites As a specific factor in exosomes derived from patient-derived cell lines (PDCs, Patient-Derived Cells), gastric cancer-patient ascites-derived exosomes are engineered to suppress MET, an oncogene, and engineered into MET-amplified GC. The present invention was completed by identifying that the anticancer effect was excellently achieved when exosomes (EXO MET depl. ) were treated in combination with a MET inhibitor and/or an anticancer agent.
따라서, 본 발명의 일 목적은 (a) 발암 유전자 또는 이의 단백질 발현 수준이 억제된 환자-유래 엑소좀(exosome); 및 (b) 상기 발암 유전자 저해제 또는 항암제;를 유효성분으로 포함하는, 암의 예방 또는 치료용 약학적 조성물을 제공하는 데 있다.Accordingly, one object of the present invention is (a) an oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); And (b) the oncogene inhibitor or anticancer agent; to provide a pharmaceutical composition for preventing or treating cancer comprising as an active ingredient.
또한, 본 발명의 다른 목적은 (a) 발암 유전자 또는 이의 단백질 발현 수준이 억제된 환자-유래 엑소좀(exosome); 및 (b) 상기 발암 유전자 저해제 및 항암제;를 유효성분으로 포함하는, 암의 예방 또는 치료용 약학적 조성물을 제공하는 데 있다.In addition, another object of the present invention is (a) an oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); And (b) the oncogene inhibitor and anticancer agent; to provide a pharmaceutical composition for the prevention or treatment of cancer, containing as an active ingredient.
또한, 본 발명의 또 다른 목적은 (a) 발암 유전자 또는 이의 단백질 발현 수준이 억제된 환자-유래 엑소좀(exosome); 및 (b) 상기 발암 유전자 저해제를 유효성분으로 포함하는, 항암제에 대한 민감성 증진을 위한 감작제(sensitizer)용 약학적 조성물을 제공하는 데 있다.In addition, another object of the present invention is (a) an oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); and (b) to provide a pharmaceutical composition for a sensitizer for enhancing sensitivity to an anticancer agent, comprising the oncogene inhibitor as an active ingredient.
또한, 본 발명의 또 다른 목적은 (a) 발암 유전자 또는 이의 단백질 발현 수준이 억제된 환자-유래 엑소좀(exosome); 및 (b) 상기 발암 유전자 저해제 또는 항암제;를 유효성분으로 포함하는, 암의 예방 또는 개선용 식품 조성물을 제공하는 데 있다.In addition, another object of the present invention is (a) an oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); And (b) the oncogene inhibitor or anticancer agent; to provide a food composition for preventing or improving cancer, containing as an active ingredient.
또한, 본 발명의 또 다른 목적은 (a) 발암 유전자 또는 이의 단백질 발현 수준이 억제된 환자-유래 엑소좀(exosome); 및 (b) 상기 발암 유전자 저해제 및 항암제;를 유효성분으로 포함하는, 암의 예방 또는 개선용 식품 조성물을 제공하는 데 있다. In addition, another object of the present invention is (a) an oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); And (b) the oncogene inhibitor and anticancer agent; to provide a food composition for the prevention or improvement of cancer containing as an active ingredient.
또한, 본 발명의 또 다른 목적은 (a) 발암 유전자 또는 이의 단백질 발현 수준이 억제된 환자-유래 엑소좀(exosome); 및 (b) 상기 발암 유전자 저해제 또는 (c) 항암제;를 유효성분으로 포함하는 암의 예방 또는 치료용 조성물을, 대상(subject)에게 투여하는 단계를 포함하는, 암의 치료 방법을 제공하는 데 있다.In addition, another object of the present invention is (a) an oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); And (b) the oncogene inhibitor or (c) the anticancer agent; to provide a method for treating cancer, comprising administering to a subject a composition for preventing or treating cancer comprising as an active ingredient .
또한, 본 발명의 또 다른 목적은 (a) 발암 유전자 또는 이의 단백질 발현 수준이 억제된 환자-유래 엑소좀(exosome); (b) 상기 발암 유전자 저해제; 및 (c) 항암제;를 유효성분으로 포함하는, 암의 예방 또는 치료용 약학적 조성물을, 대상(subject)에게 투여하는 단계를 포함하는, 암의 치료 방법을 제공하는 데 있다.In addition, another object of the present invention is (a) an oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); (b) the oncogene inhibitor; And (c) an anticancer agent; to provide a method for treating cancer, comprising the step of administering to a subject a pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient.
또한, 본 발명의 또 다른 목적은 (a) 발암 유전자 또는 이의 단백질 발현 수준이 억제된 환자-유래 엑소좀(exosome); 및 (b) 상기 발암 유전자 저해제;를 유효성분으로 포함하는, 항암제에 대한 민감성 증진을 위한 감작제(sensitizer)용 약학적 조성물을, 대상(subject)에게 투여하는 단계를 포함하는, 암의 치료 방법을 제공하는 데 있다.In addition, another object of the present invention is (a) an oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); And (b) a method for treating cancer comprising administering to a subject a pharmaceutical composition for a sensitizer for enhancing sensitivity to an anticancer agent, comprising the oncogene inhibitor as an active ingredient. is to provide
본 명세서에서 사용한 용어는 단지 설명을 목적으로 사용된 것으로, 한정하려는 의도로 해석되어서는 안된다. 단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한, 복수의 표현을 포함한다. 본 명세서에서, "포함하다" 또는 "가지다" 등의 용어는 명세서 상에 기재된 특징, 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것이 존재함을 지정하려는 것이지, 하나 또는 그 이상의 다른 특징들이나 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것들의 존재 또는 부가 가능성을 미리 배제하지 않는 것으로 이해되어야 한다.The terms used herein are used for descriptive purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "include" or "have" are intended to designate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features It should be understood that the presence or addition of numbers, steps, operations, components, parts, or combinations thereof is not precluded.
다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 실시예가 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥 상 가지는 의미와 일치하는 의미를 가지는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다.Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiment belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in the present application, they should not be interpreted in an ideal or excessively formal meaning. don't
이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명의 일 양태에 따르면, 본 발명은 (a) 발암 유전자 또는 이의 단백질 발현 수준이 억제된 환자-유래 엑소좀(exosome); 및 (b) 상기 발암 유전자 저해제 및/또는 (c) 항암제;를 유효성분으로 포함하는, 암의 예방 또는 치료용 약학적 조성물을 제공한다.According to one aspect of the present invention, the present invention provides (a) an oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); And (b) the oncogene inhibitor and / or (c) anticancer agent; it provides a pharmaceutical composition for preventing or treating cancer, containing as an active ingredient.
본 명세서에서 용어 "발암 유전자(oncogene)"는 암세포를 만드는 유전물질, 즉, 암을 유발시키는 능력을 가진 유전자를 의미하며, 암유전자 또는 종양유전자라고도 한다.As used herein, the term "oncogene" refers to a genetic material that makes cancer cells, that is, a gene having the ability to induce cancer, and is also referred to as an oncogene or an oncogene.
상기 발암 유전자는 본 발명의 목적을 달성할 수 있는 한, 당업계에 공지된 임의의 발암 유전자를 포함할 수 있으며, 예를 들어, MET, EGFR, bFGF, aFGF, int2/FGF3, hst1/K-fgf/FGF4, FGF5, hst2/FGF6, KGF, AIGF, erbB1, erbB2/neu, ros, trkA, trkB, trkC, ret, kit, PDGFR, flt1, flt3, flk1/kdr, flk2, FGFR2/K-sam/bek, KGFR, FGFR1/Nsam/flg/Cek1/bF, FGF3/Cek3, FGFR4, abl, src, yes, fyn, fgr, lyn, lck, hck, blk, csk, fps, fes, mas, gsp, gip, H-ras, K-ras, N-ras, mos, raf, A-raf1, B-raf, rel, ski, sno, c-Myc, N-myc, L-myc, max, myb, A-myb, B-myb, fos fos, fosB, fra1, fra2, jun, junB, junD, jif1, ets1, ets2, erg, elk1, Spi1/PU.1, fli1, GABPa, elf1, SAP1, db1, ect2, bcl1, bcl2, bcl3, bcl6, bcr, pml, pbx1/prf, PIK3CA, all1/mll, aml1, dec, ews, tls/fus 및 tel로 이루어진 군으로부터 선택된 1 종 이상일 수 있고, 바람직하게는 MET이나, 이에 한정되지 않는다.The oncogene may include any oncogene known in the art as long as the object of the present invention can be achieved, for example, MET, EGFR, bFGF, aFGF, int2/FGF3, hst1/K- fgf/FGF4, FGF5, hst2/FGF6, KGF, AIGF, erbB1, erbB2/neu, ros, trkA, trkB, trkC, ret, kit, PDGFR, flt1, flt3, flk1/kdr, flk2, FGFR2/K-sam/ bek, KGFR, FGFR1/Nsam/flg/Cek1/bF, FGF3/Cek3, FGFR4, abl, src, yes, fyn, fgr, lyn, lck, hck, blk, csk, fps, fes, mas, gsp, gip, H-ras, K-ras, N-ras, mos, raf, A-raf1, B-raf, rel, ski, sno, c-Myc, N-myc, L-myc, max, myb, A-myb, B-myb, fos fos, fosB, fra1, fra2, jun, junB, junD, jif1, ets1, ets2, erg, elk1, Spi1/PU.1, fli1, GABPa, elf1, SAP1, db1, ect2, bcl1, bcl2 , bcl3, bcl6, bcr, pml, pbx1/prf, PIK3CA, all1/mll, aml1, dec, ews, tls/fus, and tel may be one or more selected from the group consisting of, preferably MET, but not limited thereto. don't
본 발명의 일 구현예에 따르면, 본 발명은 MET 유전자를 타겟으로 하여 MET가 과발현된 암을 치료하거나 성장을 억제시키며, 상기 암은 MET 유전자의 발현 수준; 또는 이의 단백질의 발현 수준을 측정한 경우 MET 유전자 또는 단백질의 발현 수준이 과발현되는 것이다. According to one embodiment of the present invention, the present invention targets the MET gene to treat or inhibit the growth of MET-overexpressed cancer, the cancer is the expression level of the MET gene; Alternatively, when the expression level of the protein thereof is measured, the expression level of the MET gene or protein is overexpressed.
본 발명의 MET가 과다 발현된 암을 표현하기 위해 사용된 용어 "과발현(overexpression)"은 적합한 발현 분석법을 통해 MET의 발현 수준을 측정한 경우에 있어서, 비교 대상 세포(예컨대, 해당 기관인 정상 위 세포)의 MET 발현 수준에 비하여 2 배 이상인 것을 의미하며, 본 명세서에서 "MET-증폭"과 혼용된다.The term "overexpression" used to describe cancer in which MET is overexpressed according to the present invention refers to the case where the expression level of MET is measured through a suitable expression assay, in a target cell for comparison (e.g., a normal gastric cell, which is a corresponding organ). ) means more than twice the MET expression level of ), and is used interchangeably with "MET-amplification" herein.
또한, 상기 (a) 발암 유전자 또는 이의 단백질 발현 수준이 억제된 환자-유래 엑소좀(exosome)을 확보하기 위해서 이용되는 타겟 발암 유전자 또는 이의 단백질 발현 수준을 억제시키는 억제제는, (b) 발암 유전자 저해제와 동일한 것으로서, 본 발명의 타겟 발암 유전자의 발현 수준 및/또는 단백질의 발현 수준 또는 활성을 억제할 수 있는 한, 당업계에 공지된 임의의 수단을 포함할 수 있으며, 예를 들어, siRNA(small interference RNA), shRNA(short hairpin RNA), miRNA(microRNA), 리보자임(ribozyme), DNAzyme, PNA(peptide nucleic acids), 안티센스 올리고뉴클레오타이드, 항체, 앱타머, 추출물 및 화합물로 이루어진 군으로부터 선택된 1 종 이상일 수 있으나, 이에 한정되지 않는다.In addition, the (a) inhibitor for suppressing the expression level of a target oncogene or its protein used to secure patient-derived exosomes in which the expression level of an oncogene or its protein is suppressed is (b) an oncogene inhibitor As the same as, as long as it can inhibit the expression level and / or expression level or activity of the target oncogene of the present invention, it may include any means known in the art, for example, siRNA (small interference RNA), shRNA (short hairpin RNA), miRNA (microRNA), ribozyme, DNAzyme, PNA (peptide nucleic acids), antisense oligonucleotide, antibody, aptamer, extract, and compound. It may be more than one, but is not limited thereto.
바람직하게는, 상기 억제제는 상기 유전자의 mRNA에 특이적으로 결합하는 안티센스 올리고뉴클레오티드, 압타머, siRNA(small interference RNA), shRNA(short hairpin RNA), miRNA(microRNA) 또는 화합물이며, 가장 바람직하게는 siRNA(small interference RNA), 또는 화합물이다.Preferably, the inhibitor is an antisense oligonucleotide, aptamer, small interference RNA (siRNA), short hairpin RNA (shRNA), miRNA (microRNA) or compound that specifically binds to the mRNA of the gene, most preferably siRNA (small interference RNA), or a compound.
본 발명에 있어서, 상기 억제제는 MET의 뉴클레오티드의 발현 또는 MET 유전자 단백질의 활성을 억제하는 것을 모두 포함할 수 있으며, 이에 제한되지 않는다.In the present invention, the inhibitors may include those that inhibit the expression of MET nucleotides or the activity of MET gene proteins, but are not limited thereto.
본 발명에 있어서, MET의 뉴클레오티드의 발현을 억제하는 억제제는, MET의 mRNA에 대한 siRNA(small interference RNA), shRNA(short hairpin RNA), miRNA(microRNA), 리보자임(ribozyme), DNAzyme, PNA(peptide nucleic acids) 및 안티센스 올리고뉴클레오타이드로 이루어진 군으로부터 선택된 1 종 이상이 포함되며, 이에 제한되지 않는다.In the present invention, the inhibitor for inhibiting the expression of MET nucleotides is siRNA (small interference RNA), shRNA (short hairpin RNA), miRNA (microRNA), ribozyme, DNAzyme, PNA ( peptide nucleic acids) and at least one selected from the group consisting of antisense oligonucleotides, but is not limited thereto.
또한, 본 발명에 있어서, MET 단백질의 발현을 억제하는 억제제는, MET 에 대한 항체, 앱타머, MET의 단백질에 직접적으로 결합하여 그 활성을 억제하는 화합물 및 천연 추출물로 이루어진 군으로부터 선택된 1 종 이상이 포함되며, 이에 제한되지 않는다.In addition, in the present invention, the inhibitor for inhibiting the expression of the MET protein is at least one selected from the group consisting of an antibody against MET, an aptamer, a compound that directly binds to and inhibits the activity of MET protein, and a natural extract. This includes, but is not limited to.
본 발명의 바람직한 구현예에 따르면, 상기 siRNA는 서열번호 1의 염기서열로 표시되는 MET siRNA(siMET)이다. According to a preferred embodiment of the present invention, the siRNA is MET siRNA (siMET) represented by the nucleotide sequence of SEQ ID NO: 1.
본 발명의 상기 화합물은 사볼리티닙(savolitinib), 크리조티닙(crizotinib)(PF-02341066), 카프마티닙(capmatinib), NVP-BVU972, AMG 337, 보지티닙(bozitinib), 글루메티닙(glumetinib) 및 테포티닙(tepotinib)으로 이루어진 군으로부터 선택된 1 종 이상이고, 바람직하게는 사볼리티닙(savolitinib이다.The compound of the present invention is savolitinib, crizotinib (PF-02341066), capmatinib, NVP-BVU972, AMG 337, bozitinib, glumetinib ) and at least one selected from the group consisting of tepotinib, preferably savolitinib.
본 발명에서 상기 화합물은 MET 차단제인 항암제를 의미할 수 있으며, 본 발명에서 목적으로 하는 항암 효과를 얻을 수 있는 한, 어떠한 MET 차단제도 적용할 수 있다.In the present invention, the compound may mean an anticancer agent that is a MET blocker, and any MET blocker may be applied as long as it can obtain the desired anticancer effect.
또한, 본 발명의 상기 (c) 항암제는, 본 발명의 목적을 달성할 수 있는 한, 당업계에 공지된 임의의 항암제를 포함할 수 있으며, 예를 들어, 라무시루맙(ramucirumab), 시스플라틴(cisplatin), 카보플라틴(carboplatin), 옥살리플라틴(oxaliplatin), 파클리탁셀(paclitaxel), 도세탁셀(docetaxel), 빈크리스틴(vincristine), 비노렐빈(vinorelbine), 에토포사이드(etoposide), 메토트렉세이트(methotrexate), 탈리도마이드(thalidomide) 및 보테조밉(bortezomib)으로 이루어진 군으로부터 선택된 1 종 이상일 수 있고, 바람직하게는 라무시루맙(ramucirumab)이나, 이에 한정되지 않는다.In addition, the (c) anticancer agent of the present invention may include any anticancer agent known in the art as long as it can achieve the object of the present invention, for example, ramucirumab, cisplatin ( cisplatin, carboplatin, oxaliplatin, paclitaxel, docetaxel, vincristine, vinorelbine, etoposide, methotrexate, thalidomide ( thalidomide) and bortezomib, and may be at least one selected from the group consisting of, preferably ramucirumab, but is not limited thereto.
본 발명의 대상 질환인 "암(cancer)"은 세포가 정상적인 성장 한계를 무시하고 분열 및 성장하는 공격적(aggressive) 특성, 주위 조직에 침투하는 침투적(invasive) 특성, 및 체내의 다른 부위로 퍼지는 전이적(metastatic) 특성을 갖는 세포에 의한 질병을 총칭하는 의미이다. 본 명세서에서 상기 암은 악성 종양(malignant tumor) 또는 악성 복수(malignant ascites)와 동일한 의미로도 사용될 수 있다."Cancer", the target disease of the present invention, has aggressive characteristics in which cells divide and grow in defiance of normal growth limits, invasive characteristics infiltrating surrounding tissues, and spreading to other parts of the body. It is a generic term for diseases caused by cells having metastatic characteristics. In the present specification, the cancer may also be used as the same meaning as a malignant tumor or malignant ascites.
본 발명의 상기 암은 위암(gastric cancer), 유방암(breast cancer), 폐암(lung cancer), 간암(liver cancer), 혈액암(blood cancer), 뼈암(bone cancer), 췌장암(pancreatic cancer), 피부암(skin cancer), 머리 또는 목암(head or neck cancer), 피부 또는 안구 흑색종(cutaneous or intraocular melanoma), 자궁육종(uterine sarcoma), 난소암(ovarian cancer), 직장암(rectal cancer), 항문암(anal cancer), 대장암(colon cancer), 난관암(fallopian tube carcinoma), 자궁내막암(endometrial carcinoma), 자궁경부암(cervical cancer), 소장암(small intestine cancer), 내분비암(endocrine cancer), 갑상선암(thyroid cancer), 부갑상선암(parathyroid cancer), 신장암(adrenal cancer), 연조직종양(soft tissue tumor), 요도암(urethral cancer), 전립선암(prostate cancer), 기관지암(bronchogenic cancer) 및 골수암(bone marrow tumor)으로 이루어진 군에서 선택된 1 종 이상이며, 이에 한정되는 것은 아니나, 바람직하게는 위암이다.The cancer of the present invention includes gastric cancer, breast cancer, lung cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, and skin cancer. (skin cancer), head or neck cancer, cutaneous or intraocular melanoma, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer ( anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer (thyroid cancer), parathyroid cancer, renal cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchogenic cancer and bone marrow cancer ( bone marrow tumor), but is not limited thereto, but is preferably gastric cancer.
또한, 본 발명의 바람직한 구현예에 따르면, 상기 환자-유래 엑소좀은 암 환자의 악성 복수로부터 유래된 것으로서, 상기 환자-유래 엑소좀은 자가(autologous), 동종(allogenic) 또는 이종(xenogenic)일 수 있으며, 바람직하게는 자가(autologous)이다.In addition, according to a preferred embodiment of the present invention, the patient-derived exosome is derived from malignant ascites of a cancer patient, and the patient-derived exosome may be autologous, allogenic or xenogenic. It can be, preferably autologous.
따라서, 본 발명은 위암 환자 악성 복수에서 획득한 환자-유래 세포주(PDCs, Patient-Derived Cells)로부터 유래된 엑소좀 내 특정 인자로서 발암유전자인 MET이 억제되도록, MET 유전자를 타겟으로 하여 상기 MET 유전자의 발현 수준 또는 상기 MET 단백질의 발현 또는 활성을 억제시키는 억제제를 이용하여 위암-환자 복수 유래 엑소좀을 조작하고, MET-증폭 GC에 상기 엔지니어링(engineering)한 엑소좀(EXOMET depl.)을, MET 저해제 및/또는 항암제와 함께 병용처리하여 MET가 과발현된 암을 치료하거나 또는 전이를 억제시킨다.Therefore, the present invention is a specific factor in exosomes derived from patient-derived cell lines (PDCs, Patient-Derived Cells) obtained from malignant ascites of gastric cancer patients, targeting the MET gene so that MET, an oncogene, is suppressed. Using an inhibitor that suppresses the expression level or the expression or activity of the MET protein, the gastric cancer-patient ascites-derived exosomes are engineered, and the engineered exosomes (EXO MET depl. ) are applied to the MET-amplified GC, MET inhibitors and/or anticancer drugs are used in combination to treat cancers in which MET is overexpressed or to suppress metastasis.
본 명세서에서 특별한 언급이 없는 한, 본 명세서에서 사용되는 표현 " 유전자의 발현 수준; 또는 이의 단백질의 발현 수준 측정"은 해당 시료 내에서 검출하고자 하는 대상을 검출하는 것을 의미한다. 본 발명에서는, 그 검출하고자 하는 대상은 시료 내 해당 유전자의 mRNA 및/또는 단백질이다. 즉, 타겟의 전사 산물인 RNA 또는 유전자 산물인 단백질을 검출함으로써 상기 유전자의 발현 여부를 확인할 수 있다.Unless otherwise specified herein, the expression "expression level of a gene; or measuring the expression level of a protein thereof" used herein means detecting a target to be detected within a corresponding sample. In the present invention, the target to be detected is the mRNA and/or protein of the corresponding gene in the sample. That is, it is possible to determine whether the gene is expressed by detecting RNA, which is a transcription product of the target, or protein, which is a gene product.
RNA 또는 단백질의 검출은 통상적으로는 시료부터 RNA 또는 단백질을 추출하여, 추출물 중의 RNA 또는 단백질을 검출함으로써 실시할 수 있다. 이러한 RNA 또는 단백질의 검출은 면역분석학적 방법, 하이브리드화 반응 및 증폭반응에 의해 측정될 수 있으나, 이에 제한되지 않고 당업계에 공지된 다양한 기술을 이용하여 용이하게 실시될 수 있다.Detection of RNA or protein can usually be performed by extracting RNA or protein from a sample and detecting RNA or protein in the extract. Detection of such RNA or protein may be measured by immunoassay methods, hybridization reactions, and amplification reactions, but is not limited thereto and can be easily performed using various techniques known in the art.
본 명세서에서 사용되는 용어 "생물학적 시료"란 본 발명의 유전자 또는 단백질의 발현이 검출될 수 있는 대상 개체로부터 얻어지는 모든 시료를 의미한다. The term "biological sample" as used herein refers to any sample obtained from a subject in which the expression of the gene or protein of the present invention can be detected.
본 발명의 바람직한 구현예에 따르면, 상기 생물학적 시료는 타액(saliva), 생검(biopsy), 혈액, 피부 조직, 액체 배양물, 분변 및 소변으로 이루어진 군에서 선택된 어느 하나이며, 특별히 이에 제한되지 않고, 본 발명의 기술분야에서 통상적으로 사용되는 방법으로 처리하여 준비될 수 있다.According to a preferred embodiment of the present invention, the biological sample is any one selected from the group consisting of saliva, biopsy, blood, skin tissue, liquid culture, feces and urine, but is not particularly limited thereto, It may be prepared by treatment by a method commonly used in the technical field of the present invention.
본 발명에서, 상기 유전자 발현 수준을 측정하는 제제는 상기 유전자의 mRNA에 특이적으로 결합하는 안티센스 올리고뉴클레오티드, 프라이머 쌍 또는 프로브를 포함할 수 있다.In the present invention, the agent for measuring the gene expression level may include an antisense oligonucleotide, a primer pair, or a probe that specifically binds to mRNA of the gene.
상기 mRNA의 발현 여부를 측정하는 제제는 상기 유전자에 특이적인 안티센스 올리고뉴클레오티드, 프라이머 쌍, 프로브 및 이들의 조합으로 이루어진 군에서 선택된다. 즉, 핵산의 검출은 유전자를 암호화하는 핵산 분자 또는 상기 핵산 분자의 상보물에 하이브리드화되는 하나 이상의 올리고뉴클레오타이드 프라이머를 사용하는 증폭반응에 의해 수행될 수 있다.The agent for measuring the expression of the mRNA is selected from the group consisting of antisense oligonucleotides specific to the gene, primer pairs, probes, and combinations thereof. That is, detection of a nucleic acid may be performed by an amplification reaction using one or more oligonucleotide primers that hybridize to a nucleic acid molecule encoding a gene or a complement of the nucleic acid molecule.
예컨대, 프라이머를 이용한 mRNA의 검출은 PCR과 같은 증폭 방법을 사용하여 유전자 서열을 증폭한 다음 당 분야에 공지된 방법으로 증폭 여부를 확인함으로써 수행될 수 있다.For example, mRNA detection using primers can be performed by amplifying the gene sequence using an amplification method such as PCR, and then confirming the amplification by a method known in the art.
상기 "프로브"는 mRNA외 특이적으로 결합을 이룰 수 있는 짧게는 수 염기 내지 길게는 수백 염기에 해당하는 RNA 또는 DNA 등의 핵산 단편을 의미하며 라벨링되어 있어서 특정 mRNA의 존재 유무, 발현양을 확인할 수 있다. 프로브는 올리고뉴클레오타이드(oligonucleotide) 프로브, 단쇄 DNA(single strand DNA) 프로브, 이중쇄DNA(double strand DNA) 프로브, RNA 프로브 등의 형태로 제작될 수 있다. 적절한 프로브의 선택 및 혼성화 조건은 당해 기술 분야에 공지된 기술에 따라 적절히 선택할 수 있다.The "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to a few bases to several hundred bases in length that can specifically bind to mRNA, and is labeled to confirm the presence or absence of a specific mRNA and the amount of expression. can The probe may be manufactured in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, an RNA probe, or the like. Selection of an appropriate probe and hybridization conditions can be appropriately selected according to techniques known in the art.
상기 "프라이머"는 짧은 자유 3-말단 수산화기(free 3' hydroxyl group)를 가지는 핵산 서열로 상보적인 템플레이트(template)와 염기쌍을 형성할 수 있고 템플레이트 가닥 복사를 위한 시작 지점으로서 작용하는 짧은 핵산서열을 말한다. 프라이머는 적절한 완충용액 및 온도에서 중합반응을 위한 시약(즉, DNA 폴리머라제 또는 역전사효소) 및 상이한 4 가지의 뉴클레오사이드 트리포스페이트의 존재 하에서 DNA 합성을 개시할 수 있다. PCR 조건, 센스 및 안티센스 프라이머의 길이는 당업계에 공지된 기술에 따라 적절히 선택될 수 있다.The "primer" is a nucleic acid sequence having a short free 3' hydroxyl group, capable of forming base pairs with a complementary template, and serving as a starting point for template strand copying. say Primers can initiate DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. PCR conditions and lengths of sense and antisense primers can be appropriately selected according to techniques known in the art.
본 발명에서, 상기 단백질의 발현 수준을 측정하는 제제는 상기 단백질에 특이적으로 결합하는 항체, 펩타이드 또는 뉴클레오티드를 포함할 수 있다.In the present invention, the agent for measuring the expression level of the protein may include an antibody, peptide or nucleotide that specifically binds to the protein.
상기 단백질의 발현 수준을 측정하는 제제는 상기 단백질에 대하여 특이적으로 결합하는 항체를 의미하고, 다클론 항체, 단클론 항체, 재조합 항체 및 이들의 조합을 모두 포함한다.An agent for measuring the expression level of the protein refers to an antibody that specifically binds to the protein, and includes polyclonal antibodies, monoclonal antibodies, recombinant antibodies, and combinations thereof.
상기 항체는 다클론 항체, 단클론 항체, 재조합 항체 및 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 가지는 완전한 형태뿐만 아니라 및 항체 분자의 기능적인 단편, 예를 들어, Fab, F(ab'), F(ab')2및 Fv를 모두 포함한다. 항체 생산은 본 발명이 속하는 분야에 널리 공지된 기술을 이용하여 용이하게 제조할 수 있고, 제조되어 상업적으로 판매되는 항체를 이용할 수 있다.Such antibodies include polyclonal antibodies, monoclonal antibodies, recombinant antibodies and complete forms having two full-length light chains and two full-length heavy chains, as well as functional fragments of antibody molecules, such as Fab, F(ab' ), F(ab')2 and Fv. Antibody production can be easily prepared using techniques well known in the field to which the present invention pertains, and commercially available antibodies can be used.
본 발명의 약학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences (19th ed., 1995)에 상세히 기재되어 있다.The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
본 발명의 약학적 조성물은 경구 또는 비경구로 투여할 수 있고, 비경구 투여인 경우에는 정맥내 주입, 피하주입, 근육 주입, 복강 주입, 경피 투여 등으로 투여할 수 있다.The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc. can be administered.
본 발명의 약학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 보통으로 숙련된 의사는 소망하는 치료 또는 예방에 효과적인 투여량을 용이하게 결정 및 처방할 수 있다. 본 발명의 일 구현예에 따르면, 본 발명의 약학적 조성물의 1일 투여량은 0.0001-1000 ㎎/㎏이다.The suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, medical condition, food, administration time, administration route, excretion rate and reaction sensitivity, A ordinarily skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to one embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.0001-1000 mg/kg.
본 발명의 약학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. or it may be prepared by incorporating into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.
또한, 본 발명의 다른 양태에 따르면, 본 발명은 (a) 상술한 발암 유전자 또는 이의 단백질 발현 수준이 억제된 환자-유래 엑소좀(exosome); 및 (b) 상기 발암 유전자 저해제 및/또는 (c) 항암제;를 포함하는 암의 예방 또는 개선용 식품 조성물을 제공한다.In addition, according to another aspect of the present invention, the present invention provides (a) a patient-derived exosome in which the expression level of the above-described oncogene or protein thereof is suppressed; And (b) the oncogene inhibitor and / or (c) anticancer agent; it provides a food composition for preventing or improving cancer containing.
본 발명의 조성물은 암의 예방 또는 개선을 목적으로 건강기능식품에 첨가될 수 있다. 본 발명에서, "건강기능식품"이란, 암의 예방 및 개선, 생체방어, 면역, 병후의 회복 등 생체조절 기능을 가지는 식품을 말하는 것으로, 장기적으로 복용하였을 때 인체에 무해해야한다.The composition of the present invention may be added to health functional foods for the purpose of preventing or improving cancer. In the present invention, "functional health food" refers to food having bioregulatory functions such as cancer prevention and improvement, biodefense, immunity, and recovery after illness, and should be harmless to the human body when taken for a long time.
본 발명의 조성물을 식품 첨가물로 사용할 경우, 상기 조성물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합양은 사용 목적 (예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조 시에 본 발명의 조성물은 원료에 대하여 15중량% 이하, 바람직하게는 10 중량% 이하의 양으로 첨가된다. 그러나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다.When the composition of the present invention is used as a food additive, the composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods. The mixing amount of active ingredients may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material during production of food or beverage. However, in the case of long-term intake for the purpose of health and hygiene or health control, it may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.
상기 식품의 종류에는 특별한 제한은 없다. 상기 물질을 첨가할 수 있는 식품의 예로는 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 수프, 음료수, 차, 드링크제, 알코올 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강 식품을 모두 포함한다.There is no particular limitation on the type of food. Examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and includes all health foods in a conventional sense.
본 발명의 건강음료 조성물은 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 포함할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토오스, 수크로오스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 ml 당 일반적으로 약 0.01 내지 10 g, 바람직하게는 약 0.01 내지 0.1g 이다.The health beverage composition of the present invention may include various flavoring agents or natural carbohydrates as additional components, like conventional beverages. The aforementioned natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g per 100 ml of the composition of the present invention.
상기 외에 본 발명의 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산 음료에 사용되는 탄산화제 등을 포함할 수 있다. 그 밖에 본 발명의 조성물은 천연 과일주스, 과일주스 음료 및 야채 음료의 제조를 위한 과육을 포함할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 조성물 100 중량부 당 0.01 내지 0.1 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages; and the like. In addition, the composition of the present invention may include fruit flesh for preparing natural fruit juice, fruit juice beverages, and vegetable beverages. These components may be used independently or in combination. The ratio of these additives is not critical, but is generally selected in the range of 0.01 to 0.1 part by weight per 100 parts by weight of the composition of the present invention.
또한, 본 발명의 또 다른 양태에 따르면, 본 발명은 상술한 (a) 발암 유전자 또는 이의 단백질 발현 수준이 억제된 환자-유래 엑소좀(exosome); 및 (b) 상기 발암 유전자 저해제를 유효성분으로 포함하는, 암의 항암제에 대한 민감성 증진을 위한 감작제(sensitizer)용 약학적 조성물을 제공한다.Further, according to another aspect of the present invention, the present invention provides the above-described (a) oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); and (b) a pharmaceutical composition for a sensitizer for enhancing sensitivity to an anticancer agent of cancer, comprising the oncogene inhibitor as an active ingredient.
본 발명의 조성물은 상기 유효성분 외에 약학적으로 허용되는 담체를 포함할 수 있다.The composition of the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient.
본 발명의 상기 감작제용 약학적 조성물은 방사선, 화학항암제 또는 항체항암제에 대한 암의 민감성을 증가시킨다.The pharmaceutical composition for the sensitizer of the present invention increases the sensitivity of cancer to radiation, chemotherapy or antibody anticancer agents.
본 명세서에서 사용된 용어, "약학적 유효량(therapeutically effective amount)"은 투여되는 억제제의 양이 치료하고자 하는 질환인 암의 하나 또는 그 이상의 증상을 어느 정도 경감하는 것을 의미한다. 따라서, 약학적 유효량은, (1) 암의 진행속도를 역전시키거나 (2) 암의 그 이상의 진행을 어느 정도 금지시키게 하는 것을 의미하며, (3) 암과 관련된 하나 또는 그 이상의 증상을 어느 정도 경감(바람직하게는, 제거)하는 효과를 가지는 양을 의미한다.As used herein, the term “therapeutically effective amount” means that the amount of the inhibitor administered relieves to some extent one or more symptoms of cancer, the disease to be treated. Thus, a pharmacologically effective amount is meant to (1) reverse the rate of cancer progression or (2) inhibit further progression of cancer to some extent, and (3) alleviate to some extent one or more symptoms associated with cancer. It means an amount having an effect of reducing (preferably, removing).
본 명세서에서 사용된 용어, "약학적으로 허용되는 담체"는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다.As used herein, the term "pharmaceutically acceptable carrier" is commonly used in formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, including, but not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil; it is not going to be
본 명세서에서 사용된 용어, "감작제"는 항암 약물(또는 방사선)에 대한 암세포의 감수성을 증가시키거나, 항암 약물(또는 방사선)이 암세포 특이적으로 작용하도록 하여 적은 투여량으로도 치료 효과를 달성할 수 있도록 돕는 물질을 말한다.As used herein, the term "sensitizer" refers to a drug that increases the sensitivity of cancer cells to an anticancer drug (or radiation) or causes an anticancer drug (or radiation) to act specifically on cancer cells, thereby achieving a therapeutic effect even at a small dose. material that helps to achieve
따라서, 본 발명의 조성물은 항암제와 병용 투여하며, 상기 병용 투여는 동시에(simultaneous), 별도로(separate) 또는 순차적(seqeuntial)으로 투여된다.Therefore, the composition of the present invention is administered in combination with an anticancer agent, and the combination administration is administered simultaneously (simultaneously), separately (separately) or sequentially (sequentially).
즉, 본 발명의 감작제는 항암 보조제로서 항암제의 항암효과를 증대시키거나 항암제의 부작용을 억제 또는 개선시키기 위한 모든 형태를 의미한다. 본 발명의 항암 보조제는 다양한 종류의 항암제 또는 항암 보조제와 병용투여될 수 있으며, 병용투여시 통상적인 항암제의 투여량보다 낮은 수준으로 항암제를 투여하더라도 동등한 수준의 항암치료효과를 나타낼 수 있으므로 보다 안전한 항암치료를 수행할 수 있다.That is, the sensitizer of the present invention is an anticancer adjuvant, which means any form for increasing the anticancer effect of an anticancer agent or suppressing or improving the side effects of an anticancer agent. The anticancer adjuvant of the present invention can be administered in combination with various types of anticancer agents or anticancer adjuvants, and when administered in combination, even if the anticancer agent is administered at a lower dose than conventional anticancer agents, it can exhibit an equivalent level of anticancer treatment effect, which is safer anticancer treatment can be performed.
기존에 항암치료에 사용하고 있는 화학 항암제들은 정상세포에 독성을 일으키고 부작용이 커서 그 투여량을 줄이는 것은 당업계의 오래된 과제이다. 또한, 항암화학요법제에 대한 암세포 내성은 종양학에서 중요한 문제이다.It is a long-standing task in the art to reduce the dosage of chemical anticancer drugs that are conventionally used for anticancer treatment because they are toxic to normal cells and have large side effects. In addition, resistance of cancer cells to chemotherapeutic agents is an important problem in oncology.
따라서, 본 발명의 조성물을 기존에 사용하고 있는 항암제들과 병용하는 경우, 독성이 강한 기존 항암제들의 투여량을 크게 줄이면서도 암세포에 대한 강력한 세포사 효과를 발휘할 수 있다.Therefore, when the composition of the present invention is used in combination with existing anticancer agents, it is possible to exert a strong cell killing effect on cancer cells while greatly reducing the dose of existing anticancer agents that are highly toxic.
본 발명의 감작제용 약학적 조성물은 상술한 유효성분을 이용하기 때문에, 이 둘 사이에 공통된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다.Since the pharmaceutical composition for a sensitizer of the present invention uses the above-described active ingredients, descriptions of common contents between the two are omitted in order to avoid excessive complexity of the present specification.
또한, 본 발명의 또 다른 양태에 따르면, 본 발명은, 상술한 (a) 발암 유전자 또는 이의 단백질 발현 수준이 억제된 환자-유래 엑소좀(exosome); 및 (b) 상기 발암 유전자 저해제 또는 (c) 항암제;를 유효성분으로 포함하는 암의 예방 또는 치료용 조성물을, 대상(subject)에게 투여하는 단계를 포함하는, 암의 치료 방법을 제공한다.In addition, according to another aspect of the present invention, the present invention, the above-described (a) oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); and (b) the oncogene inhibitor or (c) the anticancer agent; administering to a subject a composition for preventing or treating cancer comprising as an active ingredient, it provides a method for treating cancer.
또한, 본 발명의 또 다른 양태에 따르면, 본 발명은, 상술한 (a) 발암 유전자 또는 이의 단백질 발현 수준이 억제된 환자-유래 엑소좀(exosome); (b) 상기 발암 유전자 저해제; 및 (c) 항암제;를 유효성분으로 포함하는, 암의 예방 또는 치료용 약학적 조성물을, 대상(subject)에게 투여하는 단계를 포함하는, 암의 치료 방법을 제공한다.In addition, according to another aspect of the present invention, the present invention, the above-described (a) oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); (b) the oncogene inhibitor; And (c) an anticancer agent; It provides a method for treating cancer, comprising administering to a subject a pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient.
또한, 본 발명의 또 다른 양태에 따르면, 본 발명은, 상술한 (a) 발암 유전자 또는 이의 단백질 발현 수준이 억제된 환자-유래 엑소좀(exosome); 및 (b) 상기 발암 유전자 저해제;를 유효성분으로 포함하는, 항암제에 대한 민감성 증진을 위한 감작제(sensitizer)용 약학적 조성물을, 대상(subject)에게 투여하는 단계를 포함하는, 암의 치료 방법을 제공한다. In addition, according to another aspect of the present invention, the present invention, the above-described (a) oncogene or a patient whose protein expression level is suppressed-derived exosome (exosome); And (b) a method for treating cancer comprising administering to a subject a pharmaceutical composition for a sensitizer for enhancing sensitivity to an anticancer agent, comprising the oncogene inhibitor as an active ingredient. provides
본 발명의 방법은 상술한 조성물을 이용하므로, 공통된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다.Since the method of the present invention uses the above-described composition, the description of common details is omitted in order to avoid excessive complexity in the present specification.
본 발명은 환자 맞춤형 암-특이 치료제 뿐만 아니라 다른 항암 치료제의 효과를 배가시키는 감작제 용도로서 유용하게 사용될 수 있다.The present invention can be usefully used as a sensitizer that doubles the effect of not only patient-specific cancer-specific therapeutic agents but also other anti-cancer therapeutic agents.
도 1은 암 환자의 복수에서 분리된 엑소좀의 특성을 보여준다. 도 1A는 엑소좀 및 암세포를 포함한 환자의 복수 개략도이다. 4명의 암 환자로부터 획득한 복수를 원심분리를 통해 분리하여 엑소좀을 포함하는 상등액과 암세포를 포함하는 펠렛을 모두 수집하였다. 도 1B는 암 유형, MET 증폭, 성별 및 연령을 포함한 각 암 환자의 임상 정보를 보여준다. 복수에서 순수한 엑소좀을 분리하기 위해 초원심분리 방법을 사용했다. 도 1C는 엑소좀 분리 방법의 개략도이다. 각 환자의 복수 샘플 50ml에서 분리한 엑소좀 펠릿을 0.22μm 여과된 인산염 완충 식염수로 재현탁하고, 나노입자 추적 분석(NTA)을 통해 EXOAscites 농도를 측정했다. 도 1D는 도 1E에 표시된 단리된 입자의 엑소좀 농도 및 크기 분포 표이다. 도 1F는 둥근 컵 모양의 형태를 보여주는 대표적인 투과 전자 현미경(TEM) 이미지이다. 스케일 바: 100 μm. 도 1G는 다소포체(MVB) 생합성 단백질(예: Alix 및 TSG101) 및 테트라스파닌(예: CD63 및 CD9)을 나타내는 정제된 EXOAscites의 웨스턴 블롯 분석 결과이다. 도 1H는 각 환자 유래 세포(PDC)로의 EXOAscites 흡수를 보여주는 대표적인 공초점 이미지이다. PKH67로 표지된 EXOAscites를 각 PDC에 적용하고 24시간 동안 인큐베이션하였다. 고정된 샘플의 시각화에는 DAPI(4',6-diamidino-2-phenylindole; 파란색)로 염색하고 PKH67(녹색)로 EXOAscites를 표지하였다. 스케일 바: 10 μm.Figure 1 shows the characteristics of exosomes isolated from ascites of cancer patients. 1A is a schematic diagram of a patient's ascites including exosomes and cancer cells. Ascites obtained from four cancer patients was separated by centrifugation, and both the supernatant containing exosomes and the pellet containing cancer cells were collected. Figure 1B shows the clinical information of each cancer patient including cancer type, MET amplification, sex and age. Ultracentrifugation was used to isolate pure exosomes from ascites. 1C is a schematic diagram of an exosome isolation method. Exosome pellets isolated from 50 ml of ascites samples from each patient were resuspended in 0.22 μm filtered phosphate buffered saline, and the concentration of EXO Ascites was measured by nanoparticle tracking assay (NTA). Figure 1D is a table of exosome concentration and size distribution of the isolated particles shown in Figure 1E. 1F is a representative transmission electron microscopy (TEM) image showing the round cup-shaped morphology. Scale bar: 100 μm. Figure 1G is a Western blot analysis of purified EXO Ascites showing multivesicular (MVB) biosynthetic proteins (eg Alix and TSG101) and tetraspanins (eg CD63 and CD9). 1H is a representative confocal image showing uptake of EXO Ascites into individual patient-derived cells (PDCs). EXO Ascites labeled with PKH67 were applied to each PDC and incubated for 24 hours. For visualization of fixed samples, DAPI (4',6-diamidino-2-phenylindole; blue) was stained and EXO ascites were labeled with PKH67 (green). Scale bar: 10 μm.
도 2는 gw4869 처리에 따른 EXOAscites의 고갈을 보여준다. 암 및 기질 세포에서 새로 생성된 엑소좀을 관강내 소포 형성을 막아 엑소좀 생성을 차단하는 gw4869 처리를 통해 고갈되어 암 진행에서 EXOAscites의 역할을 분석했다. PDC와 내피 세포(EC)의 혼합물을 6웰 배양 플레이트에 플레이팅하고 gw4869(0, 1, 10, 20 및 30 μM)의 농도를 증가시키면서 세포 배양 배지에서 공동 배양했다. 3일째에 채취한 세포 배양액을 엑소좀 분리법을 이용하여 분리한 후 NTA, cell viability assay, western blotting 등을 이용하여 분석하였다. gw4869(0,1,10, 20 및 30 μM)의 농도에 따른 분리된 입자의 크기 분포(도 2A) 및 평균 농도(도 2B)를 보여준다. n = 3 ± 평균의 표준 오차(SEM). 도 2C는 0 및 10 μM gw4869로 처리된 엑소좀(상단) 및 PDC 용해물(하단)에 대한 엑소좀 특이적 마커(Alix, TSG101, CD9 및 CD63)의 대표적인 면역블롯을 보여준다. 도 2D는 0 및 10 μM gw4869 처리로 인한 감소된 엑소좀 생성의 정량화를 보여준다. 10 μM gw4869로 처리하면 엑소좀 생합성 및 분비가 크게 감소하였다. n = 3 ± SEM. *p < 0.05, ** p < 0.01 0 μM gw4869에서 각 값과 비교. gw4869 처리가 세포 생존에 영향을 미치는지 여부를 확인하기 위해 3일째에 칼세인-아세톡시메틸(녹색) 및 프로피디움 요오드화물(PI, 적색) 이중 염색을 통해 혼합 세포의 생존력을 결정했다. gw4869의 설정 농도는 세포 생존력 분석까지 유지되었다. 도 2E는 대표적인 공초점 이미지 및 도 2F는 살아있는 세포의 정량화를 보여준다; n = 3 ± SEM. 각 p 값은 0 μM gw4869에서 각 값과 비교. gw4869는 세포 생존력에 영향을 미치지 않았다.Figure 2 shows the depletion of EXO Ascites according to gw4869 treatment. Newly generated exosomes from cancer and stromal cells were depleted by treatment with gw4869, which blocks exosome formation by preventing intraluminal vesicle formation, to analyze the role of EXO Ascites in cancer progression. Mixtures of PDCs and endothelial cells (ECs) were plated in 6-well culture plates and co-cultured in cell culture medium with increasing concentrations of gw4869 (0, 1, 10, 20 and 30 μM). The cell culture fluid collected on day 3 was isolated using the exosome separation method and then analyzed using NTA, cell viability assay, western blotting, and the like. The size distribution (Fig. 2A) and average concentration (Fig. 2B) of the isolated particles according to the concentration of gw4869 (0, 1, 10, 20 and 30 μM) are shown. n = 3 ± standard error of the mean (SEM). Figure 2C shows representative immunoblots of exosome specific markers (Alix, TSG101, CD9 and CD63) for exosomes (top) and PDC lysates (bottom) treated with 0 and 10 μM gw4869. Figure 2D shows the quantification of reduced exosome production due to 0 and 10 μM gw4869 treatment. Treatment with 10 μM gw4869 greatly reduced exosome biosynthesis and secretion. n = 3 ± SEM. *p < 0.05, **p < 0.01 compared to each value at 0 μM gw4869. To determine whether gw4869 treatment affects cell viability, viability of mixed cells was determined on day 3 by calcein-acetoxymethyl (green) and propidium iodide (PI, red) double staining. The set concentration of gw4869 was maintained until the cell viability assay. Figure 2E shows representative confocal images and Figure 2F quantification of live cells; n = 3 ± SEM. Each p value compared to each value at 0 μM gw4869. gw4869 did not affect cell viability.
도 3은 EXOAscites에서 MET 발암성 드라이버 식별을 보여준다. 암 침습성 및 혈관신생과 관련된 EXOAsscites의 내용을 추가로 조사하기 위해 EXOAscites에서 하나의 후보 바이오마커(MET)를 스크리닝했다. PDC 용해물(패시지 0) 및 EXOAscites 모두에서 MET 단백질의 발현 수준을 웨스턴 블롯팅을 통해 측정하였다. 도 3A는 PDC 용해물(상단) 및 EXOAscites(하단)에 대한 MET의 면역블롯을 보여준다. β-액틴 및 폰소 S는 각각 PDC 용해물 및 EXOAscites에 대한 로딩 대조군으로 사용되었다. 도 3B는 4명의 암 환자에서 MET 증폭 GC(왼쪽) 및 MET 비증폭 GC(오른쪽)를 나타내는 환자 표본에 대한 대표적인 면역조직화학(IHC) 결과를 보여준다. 도 3C는 4명의 암 환자에서 MET 증폭의 카피 수를 보여준다. 각 EXOAscites에서 엑소좀 MET의 시각화는 ExoView쪠 R100 이미징 플랫폼을 사용하여 수행되었다. 도 3D는 항-MET(파란색)를 사용하여 캡처한 CD63+-EXOAscites를 보여주는 대표적인 형광 이미지(상단) 및 노란색 점선 영역의 확대도(하단)이다. 도 3E는 총 CD63+ 및 CD81+ 입자의 정량화 및 도 3F는 각 환자의 EXOAscites와 함께 항-MET(파란색)를 사용하여 캡처된 CD63+-EXOAscites이다. 스케일 바: 5, 1 μm, n = 6 ± SEM. EXOAscites에서 엑소좀 MET 단백질의 특징은 4명의 환자 모두의 임상병리학적 데이터를 잘 반영한다.Figure 3 shows MET oncogenic driver identification in EXO Ascites . To further investigate the content of EXO Ascites related to cancer invasiveness and angiogenesis, we screened one candidate biomarker (MET) from EXO Ascites . The expression level of MET protein in both PDC lysate (passage 0) and EXO Ascites was measured by Western blotting. Figure 3A shows immunoblots of MET for PDC lysate (top) and EXOAscites (bottom). β-Actin and Ponso S were used as loading controls for PDC lysate and EXO Ascites , respectively. 3B shows representative immunohistochemistry (IHC) results for patient specimens showing MET amplified GCs (left) and MET non-amplified GCs (right) from four cancer patients. Figure 3C shows the copy number of MET amplification in 4 cancer patients. Visualization of exosomal MET in each EXO Ascites was performed using the ExoView™ R100 imaging platform. Figure 3D is a representative fluorescence image (top) showing CD63 + -EXO Ascites captured using anti-MET (blue) and an enlarged view of the yellow dashed area (bottom). Figure 3E is quantification of total CD63 + and CD81 + particles and Figure 3F is CD63 + -EXO Ascites captured using anti-MET (blue) along with EXO Ascites from each patient. Scale bar: 5, 1 μm, n = 6 ± SEM. The characteristics of the exosomal MET protein in EXO Ascites reflect well the clinicopathological data of all four patients.
도 4는 gw4869를 이용한 엑소좀 분비 차단을 통한 암 진행 감소를 보여준다. 엑소좀이 암 진행에 미치는 영향을 조사하기 위해 gw4869를 사용하여 3차원(3D) 스페로이드 칩을 사용하여 종양 스페로이드 혈관신생 모델에서 엑소좀을 고갈시켰다. U자형 96웰 플레이트에 미리 형성된 종양 스페로이드를 3D 종양 스페로이드 칩의 내피 세포(EC)와 공동 배양했다. MET 증폭이 있는 경우와 없는 경우의 환자 유래 세포(PDC)를 사용했다. 두 가지 유형의 종양 스페로이드, 즉, MET 증폭이 있거나 없는 경우 1일차에 gw4869(10μM), 사볼리티닙(SVOL, MET 억제제, 1μM) 및/또는 라무시루맙(RAM, 1μM)으로 처리되었다. 각 제제의 농도는 배양 동안 유지되었다. 도 4A는 암 진행에 대한 엑소좀 고갈의 효과 및 도 4B는 칩에 대한 3D 종양 스페로이드-혈관신생 모델을 확인하는 과정의 개략도를 보여준다. 도 4B에서 3D 종양 스페로이드 배양 영역 및 세포외 기질(하늘색), EC 영역(빨간색) 및 2개의 "배지 챔버" 영역(주황색)을 보여주는 하나의 웰의 평면도이다. EC는 점차적으로 3D 종양 스페로이드를 향해 이동했다. 고정 샘플을 EpCAM(암의 경우, 빨간색) 및 렉틴(EC의 경우, 녹색)에 대한 항체로 면역염색하고 6일째에 DAPI(파란색)로 대조염색했다. 도 4C는 종양 스페로이드를 염색한 후 획득한 대표적인 3D 종양 스페로이드-혈관신생 이미지 (EpCAM, 빨간색) 및 혈관(렉틴, 녹색)을 보여준다. 도 4D는 DAPI(파란색)로 대조 염색된 종양 스페로이드(EpCAM, 빨간색) 및 혈관(렉틴, 녹색)의 대표적인 공초점 이미지를 보여준다. 도 4D에 나타낸 MET 증폭 GC(도 4E) 및 MET 비증폭 GC(도 4F) 모델의 종양 스페로이드 이미지를 기반으로 하는 암 침습성 및 혈관신생의 정량화를 보여준다. 대조군과 비교하여 n = 5, * p < 0.05, ** p < 0.01 및 ***p < 0.001(Tukey의 다중 비교 테스트를 사용한 반복 측정 ANOVA). ns, 큰 차이는 없다. 스케일 바: 1mmFigure 4 shows the reduction of cancer progression through blocking exosome secretion using gw4869. To investigate the effect of exosomes on cancer progression, gw4869 was used to deplete exosomes in a tumor spheroid angiogenesis model using a three-dimensional (3D) spheroid chip. Tumor spheroids preformed in a U-shaped 96-well plate were co-cultured with endothelial cells (EC) on a 3D tumor spheroid chip. Patient-derived cells (PDC) with and without MET amplification were used. Two types of tumor spheroids, i.e., with or without MET amplification, were treated on day 1 with gw4869 (10 μM), sabolitinib (SVOL, a MET inhibitor, 1 μM), and/or ramucirumab (RAM, 1 μM). The concentration of each agent was maintained during incubation. Figure 4A shows the effect of exosome depletion on cancer progression and Figure 4B shows a schematic of the process of validating a 3D tumor spheroid-angiogenesis model for the chip. In Figure 4B is a top view of the 3D tumor spheroid culture area and one well showing the extracellular matrix (light blue), EC area (red) and two "medium chamber" areas (orange). ECs gradually migrated towards 3D tumor spheroids. Fixed samples were immunostained with antibodies against EpCAM (red for cancer) and lectins (green for EC) and counterstained with DAPI (blue) on day 6. Figure 4C shows representative 3D tumor spheroid-angiogenesis images (EpCAM, red) and blood vessels (lectin, green) obtained after staining tumor spheroids. Figure 4D shows representative confocal images of tumor spheroids (EpCAM, red) and blood vessels (lectin, green) counterstained with DAPI (blue). Figure 4D shows quantification of cancer invasiveness and angiogenesis based on tumor spheroid images of MET amplified GC (Figure 4E) and MET non-amplified GC (Figure 4F) models. n = 5, *p < 0.05, **p < 0.01 and ***p < 0.001 compared to control (repeated measures ANOVA with Tukey's multiple comparisons test). ns, there is no significant difference. Scale bar: 1 mm
도 5는 다양한 농도의 MET 억제제(SVOL) 또는 라무시루맙(RAM) 치료 후 암 진행 결과를 보여준다. 다양한 농도의 SVOL(0, 0.1, 1, 10 및 100 μM)을 MET 유전자 변경이 있거나 없는 종양 스페로이드-혈관신생 모델에 적용했을 때 암 침습성(도 5A) 및 혈관신생(도 5B)을 나타내는 공동 배양물의 정량화를 보여준다. 다양한 농도의 RAM(0, 0.1, 1, 10 및 100 μM)이 MET 유전자 변경의 유무에 관계없이 종양 스페로이드 혈관신생 모델에 적용될 때 암 침습성(도 5C) 및 혈관신생(도 5D)을 나타내는 공동 배양물의 정량화를 보여준다. n = 3 ± SEM. *p < 0.05, ** p < 0.01, *** p < 0.001은 0 μM 약물 농도에서 각 값과 비교.5 shows cancer progression results after treatment with various concentrations of a MET inhibitor (SVOL) or ramucirumab (RAM). Varying concentrations of SVOL (0, 0.1, 1, 10 and 100 μM) were applied to tumor spheroid-angiogenesis models with and without MET gene alterations, showing cancer invasiveness (Fig. 5A) and angiogenesis (Fig. 5B). Quantification of cultures is shown. Cavities exhibiting cancer invasiveness (Fig. 5C) and angiogenesis (Fig. 5D) when various concentrations of RAM (0, 0.1, 1, 10 and 100 μM) were applied to tumor spheroid angiogenesis models with or without MET genetic alterations. Quantification of cultures is shown. n = 3 ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 compared to each value at 0 μM drug concentration.
도 6은 EXOAscites에 의한 암 침습성과 혈관신생의 점진적인 향상을 보여준다. 암 진행에서 EXOAscites의 역할을 측정하기 위해 4명의 환자로부터 얻은 EXOAscites 샘플을 3D 종양 스페로이드 칩으로 사용했다. 간략하게는, 2일 동안 미리 형성된 종양 스페로이드는 3D 종양 스페로이드 칩에서 EC와 공동 배양되었다. 농도가 증가하는 EXOAscites(각 환자 샘플에 대해 0, 102, 104, 106 및 108 EXOAscites)를 1일째에 10μM gw4869가 포함된 세포 배양 배지로 처리하고 EXOAscites의 설정 농도를 면역세포화학을 위한 고정까지 유지했다. gw4869 또는 EXOAscites가 없는 조건을 대조군으로 사용했다. 도 6A는 종양 스페로이드-혈관신생 모델을 기반으로 하는 암 진행에서 EXOAscites의 역할에 대한 개략도를 보여준다. EXOAscites(0, 102, 104, 106 및 108의 농도)로 처리한 모든 환자에 대한 암 스페로이드 모델에서 암 침습성과 혈관신생은 6일째의 면역세포화학에 기초하여 측정되었다. 고정 샘플은 EpCAM(암의 경우 빨간색) 및 렉틴(EC의 경우 녹색)에 대한 항체로 면역염색되었고 DAPI(파란색)로 대조염색하였다. 도 6B는 PKH67-EXOAscites(노란색), 종양 스페로이드(EpCAM, 빨간색) 및 혈관(렉틴, 녹색)으로 염색한 대표적인 3D 종양 스페로이드-혈관신생 이미지이고; 확대된 이미지는 종양 세포(왼쪽) 및 혈관(오른쪽)으로 PKH67 표지된 EXOAscites를 묘사한다. 도 6C-D는 MET 증폭 GC 및 MET 비증폭된 GC 모델의 암 스페로이드에서 EXOAscites 농도 중 암 성장 및 혈관신생의 변화를 보여주는 공동 배양의 대표적인 공초점 형광 이미지를 보여준다. 도 6E 및 도 6F는 도 6C-D의 암 침습성(도 6E) 및 혈관신생(도 6F)의 정량화를 보여준다. C 및 D에 표시된 이미지의 경우 n = 3 ± SEM. 스케일 바: 1mm. #p < 0.05, ## p < 0.01 및 ### p < 0.001, 10μM gw4869와 비교. *p < 0.05, ** p < 0.01, *** p < 0.001은 0개의 입자/ml EXOAscites에서 각 값과 비교. 모든 환자의 EXOAscites는 암 진행을 유도했다. 스케일 바: 1mm.Figure 6 shows progressive enhancement of cancer invasiveness and angiogenesis by EXO Ascites . To determine the role of EXO Ascites in cancer progression, EXO Ascites samples obtained from four patients were used as 3D tumor spheroid chips. Briefly, for 2 days pre-formed tumor spheroids were co-cultured with ECs on a 3D tumor spheroid chip. EXO Ascites with increasing concentrations (0, 10 2 , 10 4 , 10 6 and 10 8 EXO Ascites for each patient sample) were treated with cell culture medium containing 10 μM gw4869 on day 1 and set concentrations of EXO Ascites were immunized. It was kept until fixed for cytochemistry. Conditions without gw4869 or EXO Ascites were used as controls. Figure 6A shows a schematic diagram of the role of EXO Ascites in cancer progression based on a tumor spheroid-angiogenesis model. In cancer spheroid models for all patients treated with EXO Ascites (concentrations of 0, 10 2 , 10 4 , 10 6 and 10 8 ), cancer invasiveness and angiogenesis were measured based on immunocytochemistry on day 6. Fixed samples were immunostained with antibodies against EpCAM (red for cancer) and lectins (green for EC) and counterstained with DAPI (blue). 6B is a representative 3D tumor spheroid-angiogenesis image staining with PKH67-EXOAscites (yellow), tumor spheroids (EpCAM, red), and blood vessels (lectin, green); Magnified images depict PKH67 labeled EXO Ascites with tumor cells (left) and blood vessels (right). 6C-D shows representative confocal fluorescence images of co-culture showing changes in cancer growth and angiogenesis in EXO Ascites concentrations in cancer spheroids of MET amplified GC and MET non-amplified GC models. Figures 6E and 6F show quantification of cancer invasiveness (Figure 6E) and angiogenesis (Figure 6F) of Figures 6C-D. n = 3 ± SEM for images shown in C and D. Scale bar: 1 mm. #p < 0.05, ## p < 0.01 and ### p < 0.001 compared to 10 μM gw4869. *p < 0.05, ** p < 0.01, *** p < 0.001 compared to each value at 0 particles/ml EXOAscites. EXO Ascites in all patients induced cancer progression. Scale bar: 1 mm.
도 7은 MET를 포함하는 EXOAscites의 MET null GC로의 효율적인 세포 내 전달을 보여준다. 도 7A는 ExoView쪠 R100 이미징 플랫폼을 통해 MET를 캡처하는 EXOAscites를 보여주는 대표적인 공초점 이미지(상단) 및 MET를 보유하는 EXOAscites에 의한 MET 음성 GC 세포의 MET 양성 암 세포로의 변환의 개략도를 보여준다. 스케일 바: 5μm. 도 7B는 MET(빨간색)을 캡처하는 EXOAscites가 있거나 없는 MET null 암 세포의 대표적인 공초점 형광 이미지를 보여준다. MET가 있는 EXOAscites의 잠재적인 흡수는 MET null GC(SNU-638)로 이동하고 MET 발현의 촉진을 유도한다(녹색). 스케일 바: 100μm.Figure 7 shows the efficient intracellular delivery of MET-containing EXO Ascites to MET null GCs. 7A shows a representative confocal image (top) showing EXOAscites capturing MET via the ExoView™ R100 imaging platform and a schematic diagram of the transformation of MET-negative GC cells into MET-positive cancer cells by EXOAscites harboring MET. Scale bar: 5 μm. Figure 7B shows representative confocal fluorescence images of MET null cancer cells with or without EXO Ascites capturing MET (red). Latent uptake of EXO Ascites with MET migrates to MET null GC (SNU-638) and induces promotion of MET expression (green). Scale bar: 100 μm.
도 8은 MET 증폭 GC 모델에서 조작된 EXOMET depl의 강력한 치료 효과를 보여준다. 엑소좀에서 MET와 관련된 암 침습성 및 혈관신생을 확인하기 위해 조작된 MET-고갈 엑소좀을 3D 종양 스페로이드-혈관신생 모델에 추가했다. siMET를 리포펙타민을 사용하여 배양된 PDC에 형질감염시키고, 컨디셔닝된 배지를 수집하기 전에 48시간 동안 인큐베이션하였다. EXOMET depl은 초원심분리에 의해 분리되었다(도 8A). 도 8B는 MET 증폭 GC 또는 MET 비증폭 GC에서 siMET로 처리되거나 처리되지 않은 엑소좀의 MET 단백질의 대표적인 면역블롯을 보여준다. Gapdh는 로딩 컨트롤로 사용되었다. MET는 MET 증폭 GC 모델에서만 완전히 고갈되었다. 도 8C는 3D 종양 스페로이드-혈관 신생 모델에서의 EXOMET depl의 치료 효과에 대한 개략도를 보여준다. Gw4869, EXOMET depl, EXOAscites, SVOL 및/또는 RAM은 1일째에 배양물에 추가되었고, 이들의 농도는 실험 내내 유지되었다. 도 8D는 종양 스페로이드(EpCAM, 빨간색) 및 혈관(렉틴, 녹색)을 염색한 후 획득한 대표적인 3D 종양 스페로이드-혈관신생 이미지를 보여준다. 암 침습성과 혈관신생은 EXOMET depl 치료에 의해 감소되었다. 도 8E는 종양 스페로이드 MET 증폭 및 비 MET 증폭 GC 모델에서 DAPI(파란색)로 대조 염색된 종양 스페로이드(EpCAM, 빨간색) 및 혈관(렉틴, 녹색)을 보여주는 대표적인 공초점 이미지를 보여준다. 도 8F 및 도 8G는 도 8E의 종양 스페로이드 이미지를 기반으로 한 MET 증폭 GC 모델(도 8F) 및 MET 비증폭 GC 모델(도 8G)의 암 침습성 및 혈관신생의 정량화를 보여준다. n = 5, 대조군, EXOAscites 또는 EXOMET depl과 비교하여 ##p < 0.01, ###p < 0.001, +p < 0.05, +++p < 0.001, 및 * p < 0.05(Tukey의 다중 비교 테스트를 사용한 반복 측정 ANOVA). ns, 큰 차이는 없다. gw4869 처리된 샘플을 대조군으로 사용했다. 스케일 바: 1mm.Figure 8 shows the strong therapeutic effect of engineered EXO MET depl in the MET amplification GC model. To confirm MET-associated cancer invasiveness and angiogenesis in exosomes, engineered MET-depleted exosomes were added to a 3D tumor spheroid-angiogenesis model. siMET was transfected into cultured PDCs using Lipofectamine and incubated for 48 hours before collecting the conditioned media. EXO MET depl was isolated by ultracentrifugation (FIG. 8A). 8B shows representative immunoblots of MET protein of exosomes treated with or without siMET in MET amplified GCs or MET non-amplified GCs. Gapdh was used as a loading control. MET was completely depleted only in the MET-amplified GC model. Figure 8C shows a schematic diagram of the therapeutic effect of EXO MET depl in a 3D tumor spheroid-angiogenesis model. Gw4869, EXO MET depl , EXO Ascites , SVOL and/or RAM were added to the cultures on day 1 and their concentrations were maintained throughout the experiment. Figure 8D shows representative 3D tumor spheroid-angiogenesis images obtained after staining tumor spheroids (EpCAM, red) and blood vessels (lectin, green). Cancer invasiveness and angiogenesis were reduced by EXO MET depl treatment. Figure 8E shows representative confocal images showing tumor spheroids (EpCAM, red) and blood vessels (lectin, green) counterstained with DAPI (blue) in tumor spheroid MET-amplified and non-MET-amplified GC models. Figures 8F and 8G show the quantification of cancer invasiveness and angiogenesis in the MET amplified GC model (Figure 8F) and the MET non-amplified GC model (Figure 8G) based on the tumor spheroid images in Figure 8E. n = 5, ##p < 0.01, ###p < 0.001, +p < 0.05, +++p < 0.001, and *p < 0.05 compared to control, EXO Ascites or EXO MET depl (Tukey's multiple comparisons Repeated measures ANOVA with test). ns, there is no significant difference. A gw4869 treated sample was used as a control. Scale bar: 1 mm.
이하, 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the examples are only for explaining the present invention in more detail, and those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention It will be self-evident for
실험 재료 및 방법Experimental Materials and Methods
환자 등록patient registration
본 실시예에 사용된 검체 수집 및 관련 임상 데이터는 삼성서울병원 기관심사위원회(IRB# 2021-09-052)의 승인을 받았다. 이 연구에 참여한 모든 환자는 등록 및 표본 수집 전에 서면 동의서를 제공했다. 본 실시예는 Helsinki Declaration 및 Korean Good Clinical Practice 가이드라인의 원칙에 따라 수행되었다.Specimen collection and related clinical data used in this example were approved by the Samsung Seoul Hospital Institutional Review Board (IRB# 2021-09-052). All patients participating in this study provided written informed consent prior to enrollment and sample collection. This example was performed according to the principles of the Helsinki Declaration and Korean Good Clinical Practice guidelines.
차등 초원심분리(differential Ultracentrifugation)에 의한 엑소좀 정제Exosome Purification by Differential Ultracentrifugation
엑소좀은 당업계에 공지된 방법에 일부 수정을 가한 다양한 연속 원심분리 단계에 의해 GC 환자 4명의 복수에서 분리되었다. 간략하게는, 복수를 30μm pore filter(Millipore)로 여과한 후, 300g에서 15분간 원심분리하여 각각 펠릿과 상층액을 수집하였다. 암세포를 포함하는 펠릿은 추가 실험에서 암 스페로이드를 만드는 데 사용하였다. 상층액 복수를 2000g 원심분리하여 잔해물을 버리고, 10,000g을 4℃에서 30분간 원심분리하여 복수를 추가로 정제하였다. 그 후 복수 50ml를 110,000g, 4℃에서 70분간 초원심분리(Optima MAX-XP, Beckman Coulter, SW32 Ti 로터)한 후, 펠렛을 10ml 여과된 PBS로 재현탁하고 0.22μm 필터로 여과하였다. 그런 다음 여과된 상층액을 다시 4℃에서 110,000g에서 70분간 초원심분리하였다. 상층액을 버리고 엑소좀 펠릿을 1ml 여과된 PBS에 재현탁시켰다. PBS-재현탁-EXO Ascites를 사용하여 EXOAscites의 특성을 분석하였다. 엑소좀 상층액에서 가용성 단백질을 제거하기 위해 proteinase K(1mg/ml, exosome에 대해 0.05 volume)를 EXOAscites와 함께 37℃에서 30분간 배양한 후, 60℃에서 20분간 열 불활성화시켰다. 그런 다음 엑소좀 샘플을 4℃에서 70분 동안 110,000g에서 초원심분리하였다. Proteinase K 처리된 엑소좀은 모든 시험관 내 분석에 사용되었다.Exosomes were isolated from the ascites of four GC patients by various sequential centrifugation steps with some modifications to methods known in the art. Briefly, after filtering the ascites with a 30 μm pore filter (Millipore), centrifugation was performed at 300 g for 15 minutes to collect pellets and supernatants, respectively. Pellets containing cancer cells were used to create cancer spheroids in further experiments. 2000g of supernatant ascites was centrifuged to discard debris, and 10,000g of ascites was further purified by centrifugation at 4°C for 30 minutes. Thereafter, 50 ml of ascites was ultracentrifuged at 110,000 g and 4° C. for 70 minutes (Optima MAX-XP, Beckman Coulter, SW32 Ti rotor), and then the pellet was resuspended in 10 ml filtered PBS and filtered through a 0.22 μm filter. Then, the filtered supernatant was subjected to ultracentrifugation at 110,000g at 4°C for 70 minutes. The supernatant was discarded and the exosome pellet was resuspended in 1ml filtered PBS. The properties of EXO Ascites were analyzed using PBS-resuspended-EXO Ascites . To remove soluble proteins from the exosome supernatant, proteinase K (1mg/ml, 0.05 volume per exosome) was incubated with EXO Ascites at 37°C for 30 minutes, followed by heat inactivation at 60°C for 20 minutes. The exosome samples were then ultracentrifuged at 110,000g for 70 min at 4°C. Proteinase K treated exosomes were used for all in vitro assays.
NTA(Nanoparticle tracking analysis)Nanoparticle tracking analysis (NTA)
나노 입자 농도 및 크기 분포는 청색 488nm 레이저와 고감도 sCMOS(Scientific Complementary Metal-Oxide Semiconductor) 카메라가 장착된 NanoSight LM10(NanoSight Technology, Malvern, UK) 시스템으로 측정되었다. 분석하는 동안 온도는 25℃로 일정하게 설정되어 유지되었다. 이미지는 30초의 획득 시간 및 10의 감지 임계값 설정으로 카메라 설정 13에서 세 번 측정되었다. 획득한 이미지는 NTA 분석 소프트웨어(버전 3.4 빌드 3.4.003)를 사용하여 분석되었다.Nanoparticle concentration and size distribution were measured with a NanoSight LM10 (NanoSight Technology, Malvern, UK) system equipped with a blue 488 nm laser and a high-sensitivity Scientific Complementary Metal-Oxide Semiconductor (sCMOS) camera. During the analysis, the temperature was set and kept constant at 25°C. Images were measured three times at camera setting 13 with an acquisition time of 30 seconds and a detection threshold setting of 10. Acquired images were analyzed using NTA analysis software (version 3.4 build 3.4.003).
투과전자현미경(TEM)Transmission Electron Microscopy (TEM)
10 ul의 엑소좀을 동일한 부피의 4% 파라포름알데히드 용액(T&I)과 혼합한 다음, 그리드(TED PELLA Inc.)에 10분 동안 두었다. 그리드를 PBS로 1회 세척한 후, 1% 글루타르알데히드(sigma)로 5분 동안 고정하였다. 그 후 샘플을 증류수(10회, 각 2분)로 헹구고 실온에서 1분 동안 uranyl acetate와 함께 배양하였다. 엑소좀의 시각화는 Tecnai T20 투과 전자 현미경으로 촬영하였다.10 ul of exosomes were mixed with an equal volume of 4% paraformaldehyde solution (T&I) and then placed on a grid (TED PELLA Inc.) for 10 minutes. After washing the grid once with PBS, it was fixed with 1% glutaraldehyde (sigma) for 5 minutes. Then, the samples were rinsed with distilled water (10 times, 2 minutes each) and incubated with uranyl acetate for 1 minute at room temperature. Visualization of exosomes was taken with a Tecnai T20 transmission electron microscope.
웨스턴 블로팅 분석Western blotting analysis
EXOAscites 또는 PDC 용해물은 완전한 프로테아제 억제제(Invitrogen)와 함께 용해 완충액(150mM NaCl, 1mM MgCl2, 1mM EDTA, 10% 글리세롤, 20mM HEPES [PH 7.4] 1% 트리톤 x-100)으로 용해되었다. BCA 단백질 시약(Pierce Biotechnology)을 사용하여 단백질 농도를 측정하였다. 용해된 EXOAscites(20ug) 또는 PDC 용해물(10ug)을 SDS-폴리아크릴아미드 겔 전기영동(PAGE) 겔(12% 겔 사용)로 분리하고 60분 동안 기공 크기가 0.2μm인 니트로셀룰로오스 막(Whatman)으로 옮겼다. TBST 완충액(sigma) 중 5% 탈지유(BD Bioscience)로 실온에서 진탕기 상에서 1시간 동안 막을 차단한 후, 4℃에서 1차 항체로 밤새 처리하였다. TBST 완충액으로 3회 세척한 후, 막을 상응하는 IgG-horseadish peroxidase(HRP) 2차 항체와 함께 실온에서 1시간 동안 인큐베이션하였다. 멤브레인의 시각화는 ECL 시스템(Invitrogen)에 의해 결정되었다.EXO Ascites or PDC lysates were lysed in lysis buffer (150 mM NaCl, 1 mM MgCl 2 , 1 mM EDTA, 10% glycerol, 20 mM HEPES [PH 7.4] 1% Triton x-100) with complete protease inhibitors (Invitrogen). Protein concentration was measured using BCA protein reagent (Pierce Biotechnology). Dissolved EXOAscites (20ug) or PDC lysates (10ug) were separated by SDS-polyacrylamide gel electrophoresis (PAGE) gel (using 12% gel) and blotted onto a nitrocellulose membrane with 0.2 μm pore size (Whatman) for 60 min. moved to Membranes were blocked with 5% skim milk (BD Bioscience) in TBST buffer (sigma) for 1 hour on a shaker at room temperature and then treated with primary antibodies overnight at 4°C. After washing three times with TBST buffer, the membrane was incubated with the corresponding IgG-horseadish peroxidase (HRP) secondary antibody for 1 hour at room temperature. Visualization of the membrane was determined by the ECL system (Invitrogen).
환자-유래 세포(Patient-derived cell, PDC) 배양Patient-derived cell (PDC) culture
진행성 GC 환자의 복수액은 당업계에 공지된 방법에 따라 배양되었다. 간략하게는, 복수에서 분리된 세포를 10% 소태아혈청(gibco), 1% 페니실린/스트렙토마이신(Gibco), 0.5㎍/ml의 하이드로코르티손(Sigma), 5㎍/ml의 인슐린(PeproTech) 및 5ng/ml의 표피 성장 인자(PeproTech)가 보충된 RPMI 1640 배지(gibco)에서 배양하였다. 배양 배지는 3일마다 교체하였고, PDC는 37℃ 인큐베이터에서 유지하였다. 암-스페로이드 모델을 유도하기 위해 계대 1~2 PDC를 사용하였다.Ascites fluid from patients with advanced GC was cultured according to methods known in the art. Briefly, cells isolated from ascites were supplemented with 10% fetal bovine serum (gibco), 1% penicillin/streptomycin (Gibco), 0.5 μg/ml hydrocortisone (Sigma), 5 μg/ml insulin (PeproTech) and They were cultured in RPMI 1640 medium (gibco) supplemented with 5 ng/ml epidermal growth factor (PeproTech). The culture medium was changed every 3 days, and the PDCs were maintained in a 37°C incubator. Passage 1-2 PDCs were used to derive the cancer-spheroid model.
엑소좀을 사용한 PKH67 또는 26 표지PKH67 or 26 labeling using exosomes
정제된 EXOAscites는 약간 수정된 제조업체의 지침에 따라 PKH 67 또는 26 형광 세포 링커 키트(Sigma)로 표지되었다. 간략하게는, 2.5 ul 염료 용액을 100 ul 용액 C로 희석하고 2 x 107 입자의 EXOAscites와 함께 실온에서 15분 동안 인큐베이션하였다. 혼합물에 여과된 PBS를 첨가한 다음, 100,000에서 1시간 동안 초원심분리하고 펠렛을 여과된 PBS로 재현탁시켰다.Purified EXO Ascites were labeled with the PKH 67 or 26 Fluorescent Cell Linker Kit (Sigma) according to the manufacturer's instructions with slight modifications. Briefly, 2.5 ul dye solution was diluted with 100 ul solution C and incubated with 2 x 10 7 particles of EXO Ascites for 15 min at room temperature. Filtered PBS was added to the mixture, followed by ultracentrifugation at 100,000 for 1 hour and the pellet resuspended in filtered PBS.
PDC, 위암 세포주(SNU-638), 커버슬립에 도금된 PDC, ECs에 PKH 67 또는 PKH26을 처리하고 48시간 동안 배양하였다. EXOAscites로 표지된 형광 세포는 CQ1 시스템(Yokogawa Electric Corporation, Tokyo, Japan)으로 분석되었다.PDC, gastric cancer cell line (SNU-638), and PDC and ECs plated on coverslips were treated with PKH 67 or PKH26 and cultured for 48 hours. Fluorescent cells labeled with EXO Ascites were analyzed with the CQ1 system (Yokogawa Electric Corporation, Tokyo, Japan).
항체 및 시약Antibodies and reagents
웨스턴 블로팅, 면역세포화학, 면역조직화학 및 엑소좀 시각화에 사용된 항체 목록은 표 1에 정렬하였다.Lists of antibodies used for Western blotting, immunocytochemistry, immunohistochemistry and exosome visualization are listed in Table 1.
[표 1][Table 1]
Figure PCTKR2022021193-appb-img-000001
Figure PCTKR2022021193-appb-img-000001
면역세포화학immunocytochemistry
샘플을 실온에서 20분 동안 4% 파라포름알데히드로 고정한 후 0.1% Triton X-100으로 처리한 다음 3% 소 혈청 알부민(BSA; Millipore, Burlington, MA, USA)으로 처리하였다. 고정된 샘플은 4℃에서 밤새 1% BSA의 1차 항체(표 1)로 염색되었다. 핵은 4',6-diamidino-2-phenylindole(DAPI; Life Technologies, Carlsbad, CA, USA)로 1시간 동안 염색되었다. 이미지 획득은 CQ1 시스템에 의해 수행되었다.Samples were fixed with 4% paraformaldehyde for 20 minutes at room temperature and then treated with 0.1% Triton X-100 followed by 3% bovine serum albumin (BSA; Millipore, Burlington, MA, USA). Fixed samples were stained with primary antibodies (Table 1) in 1% BSA overnight at 4°C. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; Life Technologies, Carlsbad, CA, USA) for 1 hour. Image acquisition was performed by the CQ1 system.
도 6D에서 반전된 이미지를 얻기 위해 형광색을 흑백 이미지로 반전시킨 다음 ImageJ 소프트웨어(버전 2.1.0/1.53c)를 통해 암 스페로이드 및 혈관 신생의 강도를 측정하였다.In order to obtain an inverted image in Fig. 6D, the fluorescence color was inverted into a black and white image, and then the intensity of cancer spheroids and angiogenesis was measured through ImageJ software (version 2.1.0/1.53c).
대조군으로서의 엑소좀 고갈 분석Exosome depletion assay as a control
세포로부터 분비된 엑소좀의 고갈을 조사하기 위해, 10% 엑소좀-고갈 FBS(gibco)를 함유한 세포 배양 배지에서 0일째에, PDC(6Х104 cells/ml)와 ECs(1Х104 cells/ml)의 세포 혼합물에, 상이한 농도의 gw4869(각각 0, 1, 10, 20, 30 μM)를 처리하였다. 72 시간 후, 상이한 gw4869 농도 하에서 세포 조건 배지를 수집하고, 상기 언급된 바와 같이 차등 초원심분리 방법을 사용하여 고갈된 나노입자를 분리하였다.To investigate the depletion of exosomes secreted from cells, PDCs (6Х10 4 cells/ml) and ECs (1Х10 4 cells/ml) were cultured on day 0 in cell culture medium containing 10% exosome-depleted FBS (gibco). ), were treated with different concentrations of gw4869 (0, 1, 10, 20, 30 μM, respectively). After 72 hours, cell conditioned medium was collected under different concentrations of gw4869 and depleted nanoparticles were isolated using the differential ultracentrifugation method as described above.
세포 생존력 측정Cell viability measurement
배양된 세포의 생존력은 제조사의 지침에 따라 살아있는 세포 및 죽은 세포 염색 키트(abcam)에 의해 결정되었다. 요약하면, PDC 및 EC를 포함하는 세포 혼합물을 6웰 플레이트에서 배양하고 위에서 언급한 바와 같이 상이한 gw4869 농도(0,1, 10, 20 및 30μM)로 72시간 동안 처리하였다. 그런 다음 세포를 PBS로 세척한 다음 37℃ 인큐베이터에서 5 ul 칼세인-AM 및 20 ul 요오드화 프로피디움 용액의 조합으로 처리하였다. 샘플을 PBS로 3회 세척하고 공초점 현미경(LSM 700)으로 검사하였다. 5개의 무작위 공초점 이미지에서 살아있는 세포와 죽은 세포를 계산하고 ImageJ 소프트웨어(버전 2.1.0/1.53c)를 통해 분석하였다.The viability of the cultured cells was determined by a live and dead cell staining kit (abcam) according to the manufacturer's instructions. Briefly, cell mixtures containing PDCs and ECs were cultured in 6-well plates and treated with different gw4869 concentrations (0, 1, 10, 20 and 30 μM) as mentioned above for 72 hours. Then, the cells were washed with PBS and treated with a combination of 5 ul calcein-AM and 20 ul propidium iodide solution in a 37° C. incubator. Samples were washed three times with PBS and examined under a confocal microscope (LSM 700). Live and dead cells were counted in 5 random confocal images and analyzed via ImageJ software (version 2.1.0/1.53c).
암 스페로이드 칩에서 3D 암 스페로이드 혈관신생 모델의 형성Formation of a 3D cancer spheroid angiogenesis model on a cancer spheroid chip
3D 암-혈관신생 모델을 구축하기 전에, PDC(6Х104 cells/ml), 폐 섬유아세포(5Х102 cells/ml) 및 1% Matrigel(Corning Inc., Corning, NY, USA)의 혼합물을 96웰 플레이트(Sumitomo Bakelite, Osaka, Japan)에 도말하고 배양하여 2-3일 동안 암 스페로이드를 생성하였다. PDC 스페로이드를 종양-온-칩에 도입하기 위해, 당업계에 공지된 방법에 따라 진행하였다. 요약하면, PDC 스페로이드를 함유하는 피브린 하이드로겔을 3D 미세유체 칩에 로딩하고, 겔 가교를 유도하기 위해 실온에서 5분 동안 인큐베이션하였다. 그런 다음 내피 세포(1 x 105 cells/ml)를 하이드로겔 경계면의 한 면에 플레이팅하고 하이드로겔 경계면의 다른 면을 배양 배지로 채웠다. 세포는 5 μg/ml 인슐린(PeproTech, Rocky Hill, NJ, USA)이 포함된 EGM2 배지(Lonza, Walkersville, MD, USA)에서 유지되었다. EXOAscites, sovolitinib(SVOL, MET inhibitor) 또는 ramucirumab(RAM) 처리를 위해 각 인자를 배지에 첨가하고 분석을 위해 고정될 때까지 배양 내내 농도를 유지하였다.Before constructing a 3D cancer-angiogenesis model, a mixture of PDC (6Х10 4 cells/ml), lung fibroblasts (5Х10 2 cells/ml) and 1% Matrigel (Corning Inc., Corning, NY, USA) was mixed in 96 wells. Plates (Sumitomo Bakelite, Osaka, Japan) were plated and cultured to generate cancer spheroids for 2-3 days. In order to introduce PDC spheroids into tumor-on-chip, it proceeded according to methods known in the art. Briefly, fibrin hydrogels containing PDC spheroids were loaded onto a 3D microfluidic chip and incubated for 5 min at room temperature to induce gel crosslinking. Then, endothelial cells (1 x 10 5 cells/ml) were plated on one side of the hydrogel interface and the other side of the hydrogel interface was filled with culture medium. Cells were maintained in EGM2 medium (Lonza, Walkersville, MD, USA) containing 5 μg/ml insulin (PeproTech, Rocky Hill, NJ, USA). For treatment with EXO Ascites , sovolitinib (SVOL, MET inhibitor), or ramucirumab (RAM), each factor was added to the medium and the concentration was maintained throughout the culture until fixed for analysis.
암 침습성 및 혈관신생 측정Cancer invasiveness and angiogenesis measurement
EpCAM 및 렉틴 형광 신호를 평가하기 위해 염색된 공동 배양 샘플을 순차적으로 처리하고 동일한 노출 및 이득 설정을 사용하여 단일 세션에서 공초점 현미경 이미지를 획득하였다. EpCAM 또는 렉틴 강도에 대한 임계값을 설정하여 각각 암 침습성 또는 혈관신생을 분석하였다. 암 침습성은 다음과 같이 정의되었다:To evaluate EpCAM and lectin fluorescence signals, stained co-culture samples were processed sequentially and confocal microscopy images were acquired in a single session using the same exposure and gain settings. Thresholds for EpCAM or lectin intensity were set to analyze cancer invasiveness or angiogenesis, respectively. Cancer invasiveness was defined as:
Figure PCTKR2022021193-appb-img-000002
.
Figure PCTKR2022021193-appb-img-000002
.
스페로이드 intensitytotal이 분산 성장 스페로이드 영역에서 염색된 샘플의 강도 수준인 경우, 스페로이드 intensitycore 영역은 암 스페로이드의 초기 영역에서 염색된 샘플의 강도 수준을 의미하였다. 각 막대 실험에서 관심 단백질의 평균 강도 수준은 대조군의 평균 강도 수준으로 정규화되었다. 각 조건에서 최소 3개의 독립적인 실험을 분석했다.If the spheroid intensity total is the intensity level of the sample stained in the distributed growth spheroid region, the spheroid intensity core region means the intensity level of the sample stained in the initial region of the cancer spheroid. The average intensity level of the protein of interest in each bar experiment was normalized to the average intensity level of the control group. At least three independent experiments were analyzed in each condition.
라이브 EXOAscites 이미징Live EXO Ascites Imaging
PKH67(녹색)으로 표지된 EXOAscites는 24시간 동안 암 스페로이드-혈관형성과 함께 사전 배양된 3D 종양 스페로이드 칩의 한쪽 챔버에 플레이팅되었다. 명시야 이미지의 녹색-발현 EXOAscites의 라이브 이미징은 형광 필터(488 nm에서 여기, 525/50 nm에서 방출)가 있는 20x 대물 렌즈를 사용하여 37℃ 챔버 및 고감도 sCMOS 카메라가 장착된 CQ1 시스템으로 수행되었다. 총 9시간 동안 60분 간격으로 세포로 호밍하는(homing) EXOAscites(녹색)의 전체 칩 영역 및 자른 확대 이미지가 있는 타임랩스 동영상을 CQ1 소프트웨어로 획득하였다.EXO Ascites labeled with PKH67 (green) were plated on one chamber of a 3D tumor spheroid chip pre-incubated with cancer spheroid-angiogenesis for 24 hours. Live imaging of green-expressing EXO Ascites in bright-field images was performed with a CQ1 system equipped with a high-sensitivity sCMOS camera and a 37 °C chamber using a 20x objective with a fluorescence filter (excitation at 488 nm, emission at 525/50 nm). It became. Time-lapse movies with full chip area and cropped enlarged images of EXO Ascites (green) homing to cells at 60-minute intervals for a total of 9 hours were acquired with CQ1 software.
EXOAscites가 세포로 호밍하는 것을 평가하기 위해 ImageJ의 플러그인 TrackMate를 사용하여 각 타임랩스 시퀀스에서 EXOAscites를 추적하였다. 형광 엑소좀은 자동으로 크기 임계값을 설정한 다음 Analyze Particles 기능을 사용하여 캡처하였다. 엑소좀 세포 흡수는 엑소좀 형광 강도를 세포 면적에 대한 비율로 계산하여 평가하였다. 또한 TrackMate는 각 샘플에 레이블을 지정하고 변위를 포함한 추적 정보를 제공하였다. 위의 정보를 기반으로 귀소 기능은 표지된 엑소좀의 세포 내로의 변위를 평가하였다.To assess the homing of EXO Ascites into cells, EXOAscites were tracked in each time-lapse sequence using the ImageJ plugin TrackMate. Fluorescent exosomes were captured using the Analyze Particles function after automatically setting a size threshold. Exosome cellular uptake was evaluated by calculating the exosome fluorescence intensity as a ratio to the cell area. TrackMate also labeled each sample and provided tracking information including displacement. Based on the above information, the homing function evaluated the displacement of labeled exosomes into cells.
조작된(engineered) 엑소좀의 생산Production of engineered exosomes
MET 증폭 GC 및 MET 비증폭 GC의 EXOMET depl을 구축하기 위해 각 PDC(1x106 cells/10ml)를 100mm2 접시에서 배양하고 리포펙타민 시약을 사용하여 siMET(20μM, 5ul, 최종 농도 10Nm, Ambion®, Assay ID 8700, 서열번호 1; GCACTAGCAAAGTCCGAGAtt)로 형질감염시켰다. 배양된 세포 및 배지는 72시간 후에 수집되었고 배지는 2일마다 교체되었다. MET 증폭 GC 및 MET 비증폭 GC에서 얻은 각 PDC 용해물은 웨스턴 블롯팅 분석을 위한 추가 분석을 위해 용해되었다. 초원심분리 방법을 통해 배양된 조건 배지(50 ml)로부터 EXOMET depl을 분리했다. 3D 종양 스페로이드 모델에서의 EXOMET depl의 효능을 검증하기 위해, 분리된 EXOMET depl의 가용성 단백질을 proteinase K로 처리한 후 초원심분리 단계로 제거했다. si control을 형질감염 대조군으로 사용하였다. 도 7에서, EXOAscites와 EXOMET depl간의 암 진행을 비교하기 위해, EXOMET depl을 구축하기 전에 컨디셔닝된 엑소좀의 입자 농도를 NTA로 분석완료하였다. 106개 입자/칩의 EXOAscites 또는 EXOMET depl 각각을 이 배지에서 처리하고 배양하는 동안 유지하였다.To construct EXO MET depl of MET amplified GC and MET non-amplified GC, each PDC (1x10 6 cells/10ml) was cultured in a 100 mm 2 dish and siMET (20μM, 5ul, final concentration 10Nm, Ambion ®, Assay ID 8700, SEQ ID NO: 1; GCACTAGCAAAGTCCGAGAtt). Cultured cells and medium were collected after 72 hours and medium was changed every 2 days. Each PDC lysate from MET amplified GC and MET non-amplified GC was lysed for further analysis for western blotting analysis. EXO MET depl was isolated from the cultured conditioned medium (50 ml) through an ultracentrifugation method. To verify the efficacy of EXO MET depl in a 3D tumor spheroid model, the soluble protein of isolated EXO MET depl was treated with proteinase K and then removed by ultracentrifugation. si control was used as a transfection control. In FIG. 7 , in order to compare the cancer progression between EXO Ascites and EXO MET depl , the particle concentration of conditioned exosomes was analyzed by NTA before constructing EXO MET depl . 10 6 particles/chip each of EXO Ascites or EXO MET depl were treated in this medium and maintained during cultivation.
MET 및 PD-L1의 면역조직화학(IHC)Immunohistochemistry (IHC) of MET and PD-L1
모든 환자 조직에서 MET 단백질 수준을 분석하기 위해 모든 환자의 포르말린 고정 파라핀 내장(FFPE) 조직 블록에서 얻은 슬라이드를, MET(SP44; Ventana Medical system, Tucson, AZ) 항체와 함께 배양하였다. 항-MET 항체로 염색된 슬라이드는 당업계에 공지된 방법에 따라 GC에 대한 MET 점수 시스템으로 평가되었다. PD-L1 발현 수준의 측정을 위해 FFPE 조직 블록의 IHC 분석은 앞서 설명한 PD-L1 22C3 pharmDx 분석(Agilent Technologies)을 사용하여 수행되었다. PD-L1 CPS는 PD-L1 염색된 세포(종양, 림프구, 대식세포)의 수를 합산하고 그 결과를 생존 가능한 종양 세포의 총 수로 나누어 100을 곱하여 이전에 설명한 대로 계산하였다.Slides from formalin-fixed, paraffin-embedded (FFPE) tissue blocks from all patients were incubated with MET (SP44; Ventana Medical system, Tucson, AZ) antibody to analyze MET protein levels in all patient tissues. Slides stained with anti-MET antibodies were evaluated with the MET scoring system for GC according to methods known in the art. IHC analysis of FFPE tissue blocks for measurement of PD-L1 expression levels was performed using the previously described PD-L1 22C3 pharmDx assay (Agilent Technologies). PD-L1 CPS was calculated as previously described by summing the number of PD-L1 stained cells (tumor, lymphocyte, macrophage) and dividing the result by the total number of viable tumor cells and multiplying by 100.
MET 및 PD-L1+ 엑소좀의 정량화Quantification of MET and PD-L1+ exosomes
MET 또는 PD-L1 + EXOAscites의 추가적인 생물학적 특성은 ExoView R-100 시스템(NanoView Bioscience, Boston, MA, USA)을 사용하여 특성화되었다. 요약하면, 각 엑소좀 샘플 용액 35 ul를 CD 63 Ab, CD 81 Ab 고정 어레이 스팟으로 구성된 엑소뷰 테트라스파닌 칩(NanoView Bioscience, Boston, MA, USA)에 떨어뜨리고 밤새 배양하였다. 배양 후 CD63/ALEXA 647, CD 81/ALEXA 555 및 Dye conjugated anti-MET 또는 anti-PDL1과 같은 여러 표지제 혼합물을 처리하여 표적 단백질 및 공동국소화(colocalization) 정보를 측정하였다. 이 경우, 형광 표지된 항체(테트라스파닌 Ab/dye 1:600, MET 항체 1:300 및 PD-L1 항체 1:200)를 용액 A 및 차단 용액에 각각 희석하였다. 칩에 포착된 엑소좀은 nScan 소프트웨어(NanoView Bioscience)를 통해 ExoVeiw R-100으로 스캔되었고, 데이터는 NanoViewer 2.9 소프트웨어(NanoView Bioscience)를 통해 분석되었다. 각 EXOAscites에서 무작위로 선택된 6개 이상의 영역이 정량화되었다.Additional biological properties of MET or PD-L1 + EXO Ascites were characterized using the ExoView R-100 system (NanoView Bioscience, Boston, MA, USA). Briefly, 35 ul of each exosome sample solution was dropped onto an ExoView tetraspanin chip (NanoView Bioscience, Boston, MA, USA) composed of CD 63 Ab and CD 81 Ab fixed array spots and incubated overnight. After incubation, target proteins and colocalization information were measured by treatment with a mixture of various labels such as CD63/ALEXA 647, CD 81/ALEXA 555, and Dye conjugated anti-MET or anti-PDL1. In this case, fluorescently labeled antibodies (tetraspanin Ab/dye 1:600, MET antibody 1:300, and PD-L1 antibody 1:200) were diluted in solution A and blocking solution, respectively. Exosomes captured on the chip were scanned with ExoVeiw R-100 through nScan software (NanoView Bioscience), and data were analyzed through NanoViewer 2.9 software (NanoView Bioscience). At least six randomly selected regions from each EXO Ascites were quantified.
통계 분석statistical analysis
모든 통계 분석은 Prism(GraphPad Software, USA)을 사용하여 수행되었다. 모든 데이터의 정규화는 Shapiro-Wilk 테스트에 의해 평가되었다. 모든 통계 데이터는 평균 ± SEM으로 표시된다. #, ##, ### 또는 *, ** 및 ***는 각각 p < 0.05, 0.01 및 0.001을 나타낸다. N은 통계적으로 유의한 차이가 없음을 나타낸다. All statistical analyzes were performed using Prism (GraphPad Software, USA). Normalization of all data was assessed by the Shapiro-Wilk test. All statistical data are presented as mean ± SEM. #, ##, ### or *, ** and *** indicate p < 0.05, 0.01 and 0.001, respectively. N indicates no statistically significant difference.
실시예 1. 암 환자의 복수에서 분리된 엑소좀의 특성화Example 1. Characterization of exosomes isolated from ascites of cancer patients
본 발명자들은 복수로부터의 엑소좀의 분비를 조사하기 위해 IV기 GC를 가진 4명의 환자를 무작위로 선택하고 각 환자의 50ml 복수를 엑소좀 특성화에 대해 분석하였다(도 1A). 모든 환자의 임상 징후는 절반(2/4 사례)에서 중간엽-상피 전이 인자(MET, NCBI Gene ID:4233) 증폭이 있었고, PD-L1 단백질 결합 양성 점수(CPS)는 다음과 같은 다양한 값을 보였다: 0~10. 환자의 연령은 45~56세이며 남녀비는 1:3이었다(도 1B). 50ml의 복수 샘플(EXOAscites)에서 고속 원심분리를 통해 엑소좀을 분리하고, 나노입자 추적 분석(NTA), 웨스턴 블롯팅, 투과전자현미경(TEM) 및 공초점 현미경을 통해 분리된 EXOAscites를 확인하였다(도 1D-H). 4개의 EXOAscites 샘플 모두에서 분리된 입자의 NTA는 엑소좀의 알려진 크기 분포와 일치하는 크기 분포를 나타냈으며 평균 크기는 75.2 ± 9.2 nm, 149.3 ± 3.6 nm, 87.1 ± 11.6 nm 및 138.3 ± 4.2 nm였다. 분리된 입자의 농도는 각각 140 Х 108, 138 Х 108, 55.6 Х 108 및 260 Х 108 입자/ml이었다(도 1D 및 E). 분리된 입자의 면역블롯 분석은 MVB(multivesicular body) 생합성 단백질(Alix 및 TSG101) 및 테트라스파닌(CD63 및 CD9)과 같은 엑소좀 마커가 모든 복수에서 분리된 입자에 풍부함을 보여주었다(도 1G). 또한 TEM을 통해 EXOAscites의 전형적인 컵 모양의 형태를 관찰하였다(도 1F). 엑소좀은 카고(cargo)를 표적 세포로 효과적으로 전달하는 강력한 운반체로 알려져 있다. 이에, 본 발명자들은 분리된 EXOAscites의 세포 내로의 전달 효율을 관찰하였다. 복수에서 얻은 환자 유래 세포(PDC)를 PKH67-표지 EXOAscites(녹색)와 함께 24시간 동안 배양했을 때, 모든 경우에 EXOAscites가 PDC로 명확하게 이동하고 녹색 형광이 세포 핵(파란색) 주위에 고르게 분포되었다(도 1H). 이러한 데이터는 위암 환자의 복수에서 채취한 EXOAscites가 각 환자의 복수에서 얻은 암세포에 정확하게 전달되는 높은 농도의 카고를 가지고 있음을 보여준다.We randomly selected 4 patients with stage IV GC to investigate secretion of exosomes from ascites and analyzed 50 ml ascites from each patient for exosome characterization (Fig. 1A). The clinical signs of all patients were mesenchymal-epithelial transition factor (MET, NCBI Gene ID: 4233) amplification in half (2/4 cases), and the PD-L1 protein binding positivity score (CPS) had various values as follows: Shown: 0 to 10. The age of the patients was 45-56 years, and the male to female ratio was 1:3 (Fig. 1B). Exosomes were isolated from a 50ml ascites sample (EXOAscites) by high-speed centrifugation, and the isolated EXO Ascites were confirmed through nanoparticle tracking analysis (NTA), Western blotting, transmission electron microscopy (TEM) and confocal microscopy. (Fig. 1D-H). The NTA of particles isolated from all four EXO Ascites samples showed a size distribution consistent with the known size distribution of exosomes, with average sizes of 75.2 ± 9.2 nm, 149.3 ± 3.6 nm, 87.1 ± 11.6 nm and 138.3 ± 4.2 nm. . The concentrations of the separated particles were 140 Х 10 8 , 138 Х 10 8 , 55.6 Х 10 8 and 260 Х 10 8 particles/ml, respectively (FIGS. 1D and E). Immunoblot analysis of the isolated particles showed that exosomal markers such as multivesicular body (MVB) biosynthetic proteins (Alix and TSG101) and tetraspanins (CD63 and CD9) were enriched in particles isolated from all ascites (Fig. 1G). . In addition, the typical cup-shaped morphology of EXO Ascites was observed through TEM (FIG. 1F). Exosomes are known to be powerful carriers that effectively deliver cargo to target cells. Thus, the present inventors observed the delivery efficiency of the isolated EXO Ascites into cells. When patient-derived cells (PDC) obtained from ascites were incubated with PKH67-labeled EXO Ascites (green) for 24 hours, EXO Ascites clearly migrated to PDC in all cases and green fluorescence was evenly distributed around cell nuclei (blue). distributed (Fig. 1H). These data show that EXO Ascites collected from the ascites of gastric cancer patients has a high concentration of cargo that is accurately delivered to the cancer cells obtained from each patient's ascites.
실시예 2. gw4869 처리를 통한 엑소좀 생성의 고갈Example 2. Depletion of exosome production through gw4869 treatment
본 발명자들은 암 미세 환경에서 엑소좀의 역할을 추가로 조사하기 위해 먼저 PDC 및 내피 세포(EC)를 포함한 시험관 내 암 모델에서 엑소좀 생성을 고갈시켰다. 본 발명자들은 엑소좀 형성 억제를 위한 일반적인 약리학적 제제인 gw4869를 사용했는데, 이는 MVB의 세라마이드 매개 내부 신진과 MVB에서 성숙한 엑소좀의 분비를 모두 차단하였다. 0일에 복수 및 EC와 관련된 각 PDC의 혼합물을 0, 1, 10, 20 및 30 μM 농도의 gw4869를 함유하는 배지로 배양하였다. 수집된 세포 조건 배지에서 나노입자를 표준 초원심분리를 통해 분리하고, 분리된 나노입자의 크기 분포 및 농도를 NTA를 통해 측정하였다. 도 2A 및 B에 나타낸 바와 같이, 모든 임상 샘플에 대한 모든 암 모델에서 gw4869의 농도가 증가할 때 100-250 nm 크기 범위에서 분리된 입자의 최대 존재비가 급격히 감소하였다. gw4869의 농도가 증가함에 따라 Alix, TSG101, CD9 및 CD63과 같은 엑소좀 마커의 발현 수준의 점진적인 감소가 관찰되었으나, PDC 용해물의 수준은 상관없이 일정하게 유지되었다. 특히, 10μM gw4869에서 분리된 입자의 농도는 모든 임상 샘플에서 각각 5.0배, 74.8배, 5.5배, 55.5배 가까이 실질적으로 감소하였다(도 2B). 10 μM gw4869가 있는 엑소좀에서는 엑소좀 마커가 검출되지 않은 반면, 모든 임상 샘플의 PDC 용해물은 모든 조건에서 엑소좀 마커를 일관되게 발현하였다(도 2C). 상기 데이터는 10 μM gw4869를 사용했을때 분리된 엑소좀 풍부도의 극적인 감소를 보여주었고, 엑소좀 마커의 발현 수준은 gw4869가 없는 조건보다 낮았다(도 2D).To further investigate the role of exosomes in the cancer microenvironment, we first depleted exosome production in an in vitro cancer model including PDCs and endothelial cells (ECs). We used gw4869, a common pharmacological agent for inhibition of exosome formation, which blocked both ceramide-mediated internal budding of MVBs and secretion of mature exosomes from MVBs. On day 0, mixtures of PDC associated with ascites and EC were cultured with medium containing gw4869 at concentrations of 0, 1, 10, 20 and 30 μM. Nanoparticles were separated from the collected cell condition medium through standard ultracentrifugation, and the size distribution and concentration of the separated nanoparticles were measured through NTA. As shown in Figures 2A and B, the maximum abundance of isolated particles in the 100-250 nm size range decreased rapidly when the concentration of gw4869 increased in all cancer models for all clinical samples. A gradual decrease in the expression levels of exosomal markers such as Alix, TSG101, CD9 and CD63 was observed as the concentration of gw4869 increased, but the levels of PDC lysates remained constant regardless. In particular, the concentration of isolated particles at 10 μM gw4869 was substantially reduced by nearly 5.0-fold, 74.8-fold, 5.5-fold, and 55.5-fold, respectively, in all clinical samples (Fig. 2B). No exosome markers were detected in exosomes with 10 μM gw4869, whereas PDC lysates from all clinical samples consistently expressed exosome markers in all conditions (Fig. 2C). The data showed a dramatic decrease in the abundance of isolated exosomes when 10 μM gw4869 was used, and the expression levels of exosome markers were lower than in the condition without gw4869 (Fig. 2D).
본 발명자들은 다음으로 gw4869를 사용한 치료가 암 미세 환경을 구성하는 세포의 생존력에 영향을 미치는지 여부를 평가하였다. PDC와 EC의 혼합물에 대해 다양한 농도의 gw4869로 24시간 처리한 후, 라이브 및 데드 분석을 수행하였다. 살아 있는 세포와 죽은 세포 이미지에서 어떤 조건에서도 눈에 띄는 변화가 없었다(도 2E). 모든 임상 샘플에서 얻은 세포 혼합물의 95% 이상이 gw4869 농도에 관계없이 생존하여 gw4869가 세포의 생존력에 영향을 미치지 않았음을 나타낸다(도 2F). 이러한 결과는 10μM gw4869가 세포 손상을 일으키지 않으면서 암 미세 환경에서 생성된 엑소좀의 생성과 분비를 차단할 수 있는 잠재력을 가지고 있음을 나타낸다.The present inventors next evaluated whether treatment with gw4869 affects the viability of cells constituting the cancer microenvironment. Mixtures of PDC and EC were treated with various concentrations of gw4869 for 24 hours, followed by live and dead assays. There was no appreciable change in either condition in the live and dead cell images (Fig. 2E). More than 95% of the cell mixtures from all clinical samples survived regardless of the gw4869 concentration, indicating that gw4869 did not affect the viability of the cells (Fig. 2F). These results indicate that 10 μM gw4869 has the potential to block the production and secretion of exosomes generated in the cancer microenvironment without causing cell damage.
실시예 3. 암 환자의 복수에서 발암 유전자 MET의 식별Example 3. Identification of the oncogene MET in ascites of cancer patients
본 발명자들은 환자의 임상적 특징을 반영한 발암성 인자인 MET가 EXOAscites에서 발현되는지 여부를 확인하였다. Immunoblot 결과는 MET 단백질이 각각 환자(pt1 및 pt4) 모두에서 EXOAscites에서 발현된 반면, 모든 PDC 용해물에서 MET 단백질의 낮은 발현이 관찰되었음을 보여주었다(도 3A). 모든 환자에서 MET 증폭 GC 및 MET 비증폭 GC에 대한 면역조직화학(IHC) 데이터 및 MET 복제수와 일치하였고(MET 유전자 복제수: pt1: 7.1, pt2: -, pt3: -, pt4: 25, pt2 및 3은 MET 변경을 감지하지 못함), 상기 결과는 EXOAscites가 PDC 용해물이 아닌 임상 특징을 정확하게 반영한다는 것을 나타낸다(도 3B 및 C). EXOAscites에서 MET 단백질 수준의 추가 확인은 엑소좀 영상 분석을 통해 달성되었다(도 3D). MET(파란색)에 대한 항체로 면역염색된 분리된 EXOAscites를 CD63 또는 CD81로 미리 코팅된 ExoView쪠 칩에 플레이팅하였다. 도 3E에 나타낸 CD63+-EXOAscites 또는 CD81+-EXOAscites 결과의 검증을 통해, pt1 및 pt4의 EXOAscites 샘플에서 MET+-EXOAscites 존재비를 결정할 수 있었다. EXOAscites에 대한 웨스턴 블롯팅 데이터(도 3A, 하단 참조)와 일치하는 상당한 수의 MET+-EXOAscites가 pt1 및 pt4-EXOAscites 샘플에서 관찰되었다(도 3D 및 F).The present inventors confirmed whether MET, an oncogenic factor reflecting the patient's clinical characteristics, was expressed in EXO Ascites . Immunoblot results showed that MET protein was expressed in EXO Ascites from both patients (pt1 and pt4), respectively, whereas low expression of MET protein was observed in all PDC lysates (Fig. 3A). In all patients, immunohistochemical (IHC) data and MET copy number for MET amplified GC and MET non-amplified GC were consistent (MET gene copy number: pt1: 7.1, pt2: -, pt3: -, pt4: 25, pt2 and 3 did not detect MET alteration), the results indicate that EXO Ascites accurately reflect clinical features but not PDC lysates (Figures 3B and C). Further confirmation of MET protein levels in EXO Ascites was achieved through exosome image analysis (Fig. 3D). Isolated EXO Ascites immunostained with an antibody against MET (blue) were plated on ExoView™ chips pre-coated with CD63 or CD81. Through verification of the results of CD63 + -EXO Ascites or CD81 + -EXO Ascites shown in FIG. 3E, it was possible to determine the abundance of MET + -EXO Ascites in the EXO Ascites samples of pt1 and pt4. A significant number of MET + -EXO Ascites were observed in the pt1 and pt4-EXO Ascites samples (Figs. 3D and F) consistent with the Western blotting data for EXO Ascites (Fig. 3A, bottom).
실시예 4. 3D 종양 스페로이드-혈관신생 모델에서 gw4869 처리에 의한 암 침습성 및 혈관신생 감소 가능성Example 4. Possibility of reducing cancer invasiveness and angiogenesis by gw4869 treatment in 3D tumor spheroid-angiogenesis model
본 발명자들은 종양 미세 환경에서 엑소좀의 분비를 차단하는 것이 종양 상태를 변경하는지 여부를 조사하였다. 환자 유래 세포(PDC)와 기질 세포(EC 및 섬유아세포)로 구성된 암 미세 환경을 3D 미세 유체 칩에서 시험관 내에서 gw4869로 처리하였다. 칩에 3D 종양 미세 환경을 구성하기 전에 복수에서 분리된 PDC를 섬유아세포와 함께 2일 동안 배양하여 96웰 플레이트에서 종양 스페로이드를 형성하였다(도 4A). 미리 형성된 종양 스페로이드를 칩의 중간 채널에 주입하고 EC를 칩의 측면 채널에 도금하였다(도 4B). We investigated whether blocking the secretion of exosomes in the tumor microenvironment would alter the tumor status. A cancer microenvironment composed of patient-derived cells (PDCs) and stromal cells (ECs and fibroblasts) was treated with gw4869 in vitro on a 3D microfluidic chip. Before constructing a 3D tumor microenvironment on the chip, PDCs isolated from ascites were cultured with fibroblasts for 2 days to form tumor spheroids in 96-well plates (Fig. 4A). Preformed tumor spheroids were injected into the middle channel of the chip and ECs were plated into the side channels of the chip (Figure 4B).
다양한 농도(0, 0.1, 1, 10, 100 μM savolitinib[SVOL]; 0, 0.1, 1, 10, 100 μM ramucirumab[RAM])에서 종양 세포와 혈관을 분석하여 각 약물의 최적 농도는 1μM SVOL 및 1μM RAM이 선택되었다(도 5). SVOL 처리는 MET 증폭 GC에서 침습성과 혈관신생의 점진적인 감소를 유도한 반면, RAM 처리는 어느 모델에서도 침습성을 크게 변화시키지 않았다(도 5A 및 5C). Invasiveness는 non-MET-amplified GC에서 변화가 없었으나, RAM 농도가 증가했을 때 MET 상태와 상관없이 혈관신생은 유의하게 감소하였다(도 5C 및 D). 이러한 결과는 SVOL이 MET 증폭 GC의 암 침습성에 주로 영향을 미치는 반면 RAM은 두 모델에서 혈관 신생을 조절함을 나타낸다.Tumor cells and blood vessels were analyzed at various concentrations (0, 0.1, 1, 10, 100 μM savolitinib [SVOL]; 0, 0.1, 1, 10, 100 μM ramucirumab [RAM]), and the optimal concentration of each drug was 1 μM SVOL and 1 μM RAM was selected (FIG. 5). SVOL treatment induced a gradual decrease in invasiveness and angiogenesis in MET-amplified GCs, whereas RAM treatment did not significantly alter invasiveness in either model (Figures 5A and 5C). Invasiveness was not changed in non-MET-amplified GC, but angiogenesis was significantly reduced when RAM concentration was increased regardless of MET status (Fig. 5C and D). These results indicate that SVOL primarily affects the cancer invasiveness of MET-amplified GCs, while RAM regulates angiogenesis in both models.
1일째에 종양 스페로이드-혈관신생 모델은 10μM gw4869, 1μM SVOL 및/또는 1μM RAM으로 처리되었다. gw4869 처리에 의한 종양 미세환경의 전이는 암 침습성 마커인 EpCAM 단백질(적색) 및 혈관신생을 나타내는 렉틴 단백질(녹색)의 수준에 의해 결정되었다(도 4C). MET 증폭 및 MET 비증폭 GC 모델의 종양 스페로이드에서 gw4869 처리 후 암 침습성과 혈관 신생이 크게 감소하였다(도 4D-F). gw4869 처리에 의해 MET 증폭 및 MET 비증폭 GC 모델의 EpCAM 및 렉틴 발현 수준은 대조군보다 각각 2.9, 1.7, 5.6 및 1.7배 낮았다(도 4E 및 F). 특히, SVOL 및 RAM을 사용한 gw4869 공동 처리에 대한 반응으로 두 모델에서 EpCAM 및 렉틴 발현 수준이 감소하는 상승 효과가 관찰되었다(도 4D-F). 대조군에 비해, MET 증폭 GC 모델에서는 EpCAM 수준이 7.8배, 렉틴 수준이 3.3배 낮았고, MET 비증폭 GC 모델에서는 EpCAM 수준이 3.3배, 렉틴 수준이 2.4배 낮았다. On day 1, tumor spheroid-angiogenic models were treated with 10 μM gw4869, 1 μM SVOL and/or 1 μM RAM. Metastasis of the tumor microenvironment by gw4869 treatment was determined by levels of EpCAM protein (red), a cancer invasive marker, and lectin protein (green), which indicates angiogenesis (Fig. 4C). Cancer invasiveness and angiogenesis were greatly reduced after gw4869 treatment in tumor spheroids of MET-amplified and non-MET-amplified GC models (Fig. 4D-F). The expression levels of EpCAM and lectin in the MET amplified and non-MET amplified GC models by gw4869 treatment were 2.9, 1.7, 5.6 and 1.7 times lower than those in the control group, respectively (Fig. 4E and F). In particular, a synergistic effect was observed with reduced EpCAM and lectin expression levels in both models in response to gw4869 co-treatment with SVOL and RAM (Fig. 4D-F). Compared to the control group, the EpCAM level was 7.8 times lower and the lectin level was 3.3 times lower in the MET amplified GC model, and the EpCAM level was 3.3 times lower and the lectin level was 2.4 times lower in the MET non-amplified GC model.
따라서, 상기 결과는 특정 엑소좀 분비를 억제하는 것이 암 진행을 억제한다는 것을 나타내며, 이는 특정 엑소좀 분비를 억제하는 제제가 암 치료제로서의 가능성이 있음을 시사한다.Therefore, the above results indicate that inhibiting specific exosome secretion inhibits cancer progression, suggesting that an agent that inhibits specific exosome secretion has potential as a cancer therapeutic agent.
실시예 5. 3D 종양 스페로이드 혈관 신생 모델에서 EXOAscites로 인한 암 진행의 점진적 촉진Example 5. Gradual Acceleration of Cancer Progression by EXO Ascites in 3D Tumor Spheroid Angiogenesis Model
본 발명자들은, 반대로, 암 침습성 및 혈관신생에서 EXOAscites의 역할을 평가하기 위해 MET 증폭 GC 모델이 있거나 없는 종양 스페로이드에서 다양한 EXOAscites 농도를 처리하였다. 본 실시예에서 각각 pt2(MET 비증폭 GC) 및 pt4(MET 증폭 GC)의 EXOAscites를 사용했으며 각 EXOAscites는 종양 스페로이드-혈관신생 모델과 관련하여 추가되었다. 도 4A 및 B에서 설명한 바와 같이, MET 증폭 및 MET 비증폭이 있는 미리 형성된 종양 스페로이드를 칩의 중앙 채널에 도금한 다음 칩의 한쪽 채널에 EC를 추가하였다(도 6A). 그런 다음 공동 배양물은 PDC 및 EC에서 분비되는 엑소좀의 효과를 줄이기 위해, 1일째에 10 μM gw4869와 함께 PKH-67 표지된 EXOAscites(0, 102, 104, 106 및 108 입자/칩)의 농도를 증가시키면서 처리되었다(도 6A). PKH-67로 표지된 EXOAscites(노란색)를 사용하여 6일째에 암 성장(빨간색) 및 혈관신생(녹색)을 조사하였다(도 6B). gw4869가 없는 3D 종양 스페로이드 모델을 음성 대조군으로 사용하였다(도 6C).Conversely, we treated tumor spheroids with and without a MET-amplified GC model with various concentrations of EXO Ascites to evaluate the role of EXO Ascites in cancer invasiveness and angiogenesis. In this example, EXO Ascites of pt2 (MET non-amplified GC) and pt4 (MET-amplified GC), respectively, were used and each EXO Ascites was added in relation to the tumor spheroid-angiogenesis model. As described in Figures 4A and B, preformed tumor spheroids with MET amplification and non-MET amplification were plated on the central channel of the chip, then ECs were added to one channel of the chip (Figure 6A). The co-cultures were then treated with PKH-67 labeled EXO Ascites (0, 10 2 , 10 4 , 10 6 and 10 8 particles) with 10 μM gw4869 on day 1 to reduce the effect of exosomes secreted from PDCs and ECs. /chip) were treated with increasing concentrations (Fig. 6A). Cancer growth (red) and angiogenesis (green) were investigated on day 6 using EXO Ascites (yellow) labeled with PKH-67 (Fig. 6B). A 3D tumor spheroid model without gw4869 was used as a negative control (Fig. 6C).
본 발명자들은 먼저 EXOAscites가 우선적으로 전달되는 세포 유형을 식별하였다. PKH67-표지 EXOAscites를 PDC 또는 EC로 플레이팅하고 EXOAscites를 포함하는 세포의 수를 계산하였다. 그 결과, EXOAscites의 잠재적인 방향성이 PDC에서 관찰되었으며, 대부분이 EXOAscites로 표시된 녹색 형광을 보인 반면, EXOAscites가 있는 EC의 수는 상당히 낮았다. 이러한 결과는 EXOAscites가 해당 출처에 우선적으로 전달됨을 보여준다.We first identified the cell type to which EXO Ascites are preferentially delivered. PKH67-labeled EXO Ascites were plated on PDC or EC and the number of cells containing EXO Ascites was counted. As a result, the potential orientation of EXO Ascites was observed in the PDC, and most showed green fluorescence indicated by EXO Ascites , whereas the number of ECs with EXO Ascites was quite low. These results show that EXO Ascites are preferentially delivered to the corresponding source.
EXOAscites를 사용한 치료는 암 진행의 용량 의존적 향상으로 이어졌다(도 6D-F). 놀랍게도, PKH-67 표지된 EXOAscites(노란색)는 점차적으로 종양 스페로이드로 집중되었고 두 GC 모델 모두에서 높게 나타났다. Immunocytochemistry 결과는 EpCAM 단백질과 렉틴 단백질 모두의 발현이 많은 수의 EXOAscites를 추가함에 따라 점차적으로 증가했지만 암 진행에 대한 EXOAscites의 포화 수준은 EXOAscites 샘플 사이에서 달랐다는 것을 보여주었다(도 6E 및 F). 모든 샘플에 대해 EXOAscites의 102개 입자/칩이 MET 증폭 및 MET 비증폭 GC 모델에 추가되었을 때 침습성 및 혈관신생 증가를 포함하여 상당히 향상된 암 진행이 관찰되었다(도 6D-F). Treatment with EXO Ascites resulted in a dose-dependent enhancement of cancer progression (FIG. 6D-F). Surprisingly, PKH-67 labeled EXO Ascites (yellow) gradually concentrated into tumor spheroids and were high in both GC models. Immunocytochemistry results showed that the expression of both EpCAM protein and lectin protein gradually increased with the addition of a large number of EXO Ascites , but the saturation level of EXO Ascites for cancer progression differed between EXO Ascites samples (Fig. 6E and F). ). Significantly enhanced cancer progression, including increased invasiveness and angiogenesis, was observed when 10 2 particles/chip of EXO Ascites were added to the MET-amplified and non-MET-amplified GC models for all samples (FIG. 6D-F).
따라서, 상기 결과는 EXOAscites가 암 침습성과 혈관신생의 가속화에 필수적인 역할을 한다는 것을 뒷받침하였다.Thus, the above results supported that EXO Ascites play an essential role in accelerating cancer invasiveness and angiogenesis.
또한, 본 발명자들은 EXOAscites가 3D 종양 스페로이드 모델에 전달되어 암 침습성을 촉진하는 방법을 조사하였다. PKH67로 표지된 EXOAscites를 칩의 챔버 한쪽에 주입하여 세포 배양 배지의 부피 차이에 따라 칩의 반대쪽 챔버로의 흐름을 유도하였다. 3D 종양 스페로이드 모델에 대한 EXOAscites 위치는 라이브 세포 이미징을 통해 결정되었다. 칩에서 EXOAscites 움직임의 시간 경과 시퀀스는 PKH67로 표지된 EXOAscites(녹색) 분포가 시간이 지남에 따라 전체 칩에 퍼져 있음을 보여주었다. 또한, 고배율 이미지는 PKH67로 표지된 EXOAscites의 초기 흡수가 2시간에 시작되고 세포의 녹색 형광이 점차적으로 증가함을 보여주었다. 6시간 동안 30분마다 엑소좀의 흡수를 분석한 배경 영역의 녹색 형광 비율과 비교하여 세포 영역의 녹색 형광 비율이 상대적으로 높았다. 구체적으로, 세포 영역으로 endo-cytolyz된 개별 EXOAscites가 자동으로 표시되었을 때, 약 46개의 EXOAscites가 9시간에 걸쳐 세포에 농축되었다. In addition, we investigated how EXO Ascites were delivered to 3D tumor spheroid models to promote cancer invasiveness. EXO Ascites labeled with PKH67 was injected into one chamber of the chip, and flow was induced to the opposite chamber of the chip according to the volume difference of the cell culture medium. EXO Ascites location on the 3D tumor spheroid model was determined through live cell imaging. A time-lapse sequence of EXO Ascites movement on the chip showed that the distribution of PKH67-labeled EXOAscites (green) spread over the entire chip over time. In addition, high-magnification images showed that the initial uptake of PKH67-labeled EXO Ascites started at 2 h and the green fluorescence of the cells gradually increased. Compared to the green fluorescence ratio of the background area analyzed for exosome uptake every 30 minutes for 6 hours, the green fluorescence ratio of the cell area was relatively high. Specifically, when individual EXO Ascites that were endo-cytolyzed into the cell area were automatically labeled, about 46 EXO Ascites were concentrated in the cells over 9 hours.
따라서, 상기 결과는 PDC로의 EXOAscites의 향성이 암 침습성과 혈관신생에 영향을 미칠 가능성이 있음을 보여준다.Thus, the above results show that the orientation of EXO Ascites to PDC has the potential to affect cancer invasiveness and angiogenesis.
실시예 6. MET 단백질을 포함하는 EXOAscites의 MET- GC로의 전달Example 6. Transfer of EXO Ascites Containing MET Protein to MET - GC
많은 선행 연구에서 카고를 포함하는 엑소좀의 전달 시스템이 표적 단백질의 발현 수준을 조절하여 세포 기능을 관리하는 데 유용하여 엑소좀 기반의 암 치료 약물 전달이 가능하다고 보고된 바 있다.In many previous studies, it has been reported that an exosome delivery system containing cargo is useful for managing cell functions by regulating the expression level of a target protein, enabling exosome-based cancer drug delivery.
이에, 본 발명자들은, MET를 카고로 포함하는 EXOAscites의 전달 능력을 관찰하기 위해, MET를 포함하는 EXOAscites(pt4의 EXOAscites 사용)를 MET-(MET null) GC 세포에 적용하였다(도 7A). 공초점 현미경 데이터에서 EXOAscites(빨간색)의 발암성 MET가 표적 세포에 전달되었고 MET 단백질(녹색)이 암세포의 세포질에서 강하게 발현된 반면, MET를 운반하는 EXOAscites가 없을 때 MET 단백질의 발현이 관찰되지 않았다는 것을 보여주었다(도 7B). Therefore, in order to observe the delivery ability of EXO Ascites containing MET as a cargo, the present inventors applied MET-containing EXO Ascites (using pt4 EXO Ascites ) to MET- ( MET null) GC cells (FIG. 7A ). Confocal microscopy data showed that oncogenic MET from EXO Ascites (red) was delivered to target cells and MET protein (green) was strongly expressed in the cytoplasm of cancer cells, whereas MET protein expression was observed in the absence of MET-carrying EXO Ascites . showed that it did not (Fig. 7B).
이러한 발견들은 표적 세포로 전달된 엑소좀의 발암성 MET가 표적 세포에서 MET 단백질의 발현 수준을 증가시킨다는 전제를 뒷받침하였다.These findings supported the premise that oncogenic MET from exosomes delivered to target cells increased the expression level of MET protein in target cells.
실시예 7. MET 증폭 GC 모델로부터 획득된 종양 스페로이드에 대한 조작된 EXOMET depl의 치료 효과 Example 7. Therapeutic effect of engineered EXO MET depl on tumor spheroids obtained from MET amplified GC models
본 발명자들은 EXOAscites에 의해 촉진되는 암 진행이 EXOAscites에서 주로 MET와 관련되는지 여부를 조사하였다. MET-고갈 엑소좀(MET-depleted exosomes)을 구축하기 위해, MET-증폭 GC 모델(pt4) 및 MET-비증폭 GC 모델(pt2)에서 배양된 PDC를 48시간 동안 Lipofectamine을 사용하여 siMET(Ambion®, Assay ID 8700, 서열번호 1)로 형질감염시키고 세포-조건 배지를 수집하여 MET가 고갈된 엑소좀(EXOMET depl)을 분리하였다(도 8A). We investigated whether cancer progression promoted by EXO Ascites was mainly related to MET in EXO Ascites . To construct MET-depleted exosomes, PDC cultured in the MET-amplified GC model (pt4) and the MET-non-amplified GC model (pt2) were incubated with siMET (Ambion®) using Lipofectamine for 48 h. , Assay ID 8700, SEQ ID NO: 1) and cell-conditioned medium was collected to isolate MET-depleted exosomes (EXO MET depl ) (FIG. 8A).
한편, 본 발명자들은 PDC 조건 배지에서 수집한 엑소좀(EXOcell media)의 MET 발현 수준을 보고하고 이를 EXOAscites의 것과 비교한 결과, 복수 및 PDC 조건 배지에서 획득한 엑소좀의 동일한 MET 발현 패턴이 모든 환자에서 관찰되었다. 또한, 복수의 PDC 용해물과 유사한 시험관 내 PDC 용해물은 각 환자의 임상 특징을 반영하지 않았다. On the other hand, the present inventors reported the MET expression level of exosomes (EXO cell media ) collected from PDC-conditioned medium and compared it to that of EXO Ascites , and as a result, the same MET expression pattern of exosomes obtained from ascites and PDC-conditioned medium observed in all patients. In addition, in vitro PDC lysates similar to multiple PDC lysates did not reflect the clinical characteristics of each patient.
이에, 본 발명자들은 MET 증폭 및 MET 비증폭 GC의 세포-조건 배지로부터 획득한 엑소좀 내 MET를 억제(침묵)시켜 두 가지 유형의 조작된 엑소좀을 구축하였다. Accordingly, we constructed two types of engineered exosomes by suppressing (silencing) MET in exosomes obtained from cell-conditioned media of MET-amplified and non-MET-amplified GCs.
도 8B에 나타낸 바와 같이, MET 증폭 GC에서는 siMET로 형질감염시켜 MET를 완전히 침묵시킨 반면, MET 단백질이 없기 때문에 MET 증폭되지 않은 GC에서는 이를 검출할 수 없었다. 종양 스페로이드-혈관 신생 모델에서의 EXOMET depl의 효과를 확인하기 위해, EC와 공동 배양된 종양 스페로이드를 EXOMET depl로 처리하였고, MET 저해제인 사볼리티닙(savolitinib, SVOL) 및 항암제인 라무시루맙(ramucirumab, RAM)를 공동처리하여 상승적 효과를 확인하였다(도 8C). 상기 종양 스페로이드 혈관 신생 결과와 유사하게, EXOMET depl에 의한 암 침습성과 혈관 신생을 EpCAM 및 렉틴 발현 수준에 따라 각각 분석하였다(도 8D). 조작된 엑소좀의 효과를 분석하기 위해 종양 스페로이드-혈관신생 모델에서 생성된 엑소좀은 초기에 10 μM gw4869로 고갈되었고 gw4869 농도는 배양 전반에 걸쳐 유지되었다.As shown in Fig. 8B, MET-amplified GCs were transfected with siMET to completely silence MET, whereas MET-non-amplified GCs could not be detected due to the absence of MET protein. To confirm the effect of EXO MET depl in the tumor spheroid-angiogenesis model, tumor spheroids co-cultured with ECs were treated with EXO MET depl , and the MET inhibitor savolitinib (SVOL) and the anticancer drug Lamu A synergistic effect was confirmed by co-treatment with ramucirumab (RAM) (FIG. 8C). Similar to the tumor spheroid angiogenesis results, cancer invasiveness and angiogenesis by EXO MET depl were analyzed according to EpCAM and lectin expression levels, respectively (Fig. 8D). To analyze the effect of engineered exosomes, exosomes generated in the tumor spheroid-angiogenesis model were initially depleted with 10 μM gw4869 and the gw4869 concentration was maintained throughout the culture.
그 결과, MET 증폭 및 MET 비증폭 GC의 EXOAscites는 종양 스페로이드 혈관 신생 모델에서 암 침습성과 혈관 신생을 촉진하였다(도 8E-G). MET 증폭 GC 모델의 종양 스페로이드에서 암 진행은, EXOAscites와 비교하여 MET 증폭 GC에서 분리된 EXOMET depl 처리에 의해 실질적으로 억제되었고; 암 침습성과 혈관신생은 각각 1.6배와 2.3배 감소하였다(도 8E-G). 반면, MET 비증폭 GC 모델을 MET 비증폭 GC로부터 분리된 EXOMET depl로 처리했을 때 변화가 관찰되지 않았다. 또한, MET 비증폭 GC 모델에서 EXOAscites와 EXOMET depl 간의 암 침습성 또는 혈관신생에서 유의한 차이가 관찰되지 않았으며, 이는 엑소좀 MET가 MET 증폭 GC 모델에서 암 진행을 촉진하는 잠재적 발암 인자로 작용했음을 나타낸다. As a result, EXO Ascites of MET-amplified and non-MET-amplified GCs promoted cancer invasiveness and angiogenesis in a tumor spheroid angiogenesis model (FIG. 8E-G). Cancer progression in tumor spheroids of the MET amplified GC model was substantially inhibited by EXO MET depl treatment isolated from MET amplified GCs compared to EXO Ascites ; Cancer invasiveness and angiogenesis were reduced 1.6-fold and 2.3-fold, respectively (Fig. 8E-G). On the other hand, no change was observed when the MET unamplified GC model was treated with EXO MET depl separated from the MET unamplified GC. In addition, no significant difference was observed in cancer invasiveness or angiogenesis between EXO Ascites and EXO MET depl in the non-MET amplified GC model, indicating that exosome MET acts as a potential oncogenic factor promoting cancer progression in the MET amplified GC model. indicates that
또한, EXOMET depl에 MET 저해제인 사볼리티닙(savolitinib, SVOL) 및 항암제인 라무시루맙(ramucirumab, RAM)를 공동처리했을 때 MET 증폭 GC 모델에서 암 침습성과 혈관신생에 대한 상승적 치료 효과가 관찰되었다.In addition, when EXO MET depl was co-treated with the MET inhibitor savolitinib (SVOL) and the anticancer drug ramucirumab (RAM), a synergistic therapeutic effect on cancer invasion and angiogenesis was observed in a MET-amplified GC model It became.
MET 증폭 GC 모델에서 EXOMET depl의 것과 비교하여, EpCAM(3.5배) 및 렉틴(2.0배)의 단백질 수준은 EXOMET depl/SVOL/RAM에 의해 유의하게 억제되었다(도 8E-G). Compared to those of EXO MET depl in the MET-amplified GC model, the protein levels of EpCAM (3.5-fold) and lectin (2.0-fold) were significantly suppressed by EXO MET depl /SVOL/RAM (Fig. 8E-G).
종합하면, 상기 결과는, 환자로부터 유래되고 MET를 표적으로 하여 MET가 고갈된 엑소좀(EXOMET depl)과, MET 저해제 및/또는 항암제와의 병용처리가 MET-증폭된 GC에 강력한 치료 효과를 발휘함을 입증하므로, 엑소좀 공학에 기반한 표적 치료가 가능하여 개인 맞춤 의학에 대한 경로를 제공할 수 있음을 시사한다.Taken together, these results suggest that the combination of patient-derived exosomes targeting MET and depleted of MET (EXO MET depl ), MET inhibitors, and/or anticancer drugs has a potent therapeutic effect on MET-amplified GCs. Therefore, it suggests that targeted therapy based on exosome engineering is possible, providing a route to personalized medicine.
이상으로, 본 발명내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의해 정의된다고 할 것이다.In the above, specific parts of the present invention have been described in detail, and for those skilled in the art, these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. It will be clear. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (20)

  1. (a) 발암 유전자 또는 이의 단백질 발현 수준이 억제된 환자-유래 엑소좀(exosome); 및 (b) 상기 발암 유전자 저해제 또는 (c) 항암제;를 유효성분으로 포함하는, 암의 예방 또는 치료용 약학적 조성물.(a) patient-derived exosomes in which the expression level of an oncogene or its protein is suppressed; And (b) the oncogene inhibitor or (c) anticancer agent; containing as an active ingredient, a pharmaceutical composition for preventing or treating cancer.
  2. 제1항에 있어서,According to claim 1,
    상기 발암 유전자는 MET, EGFR, bFGF, aFGF, int2/FGF3, hst1/K-fgf/FGF4, FGF5, hst2/FGF6, KGF, AIGF, erbB1, erbB2/neu, ros, trkA, trkB, trkC, ret, kit, PDGFR, flt1, flt3, flk1/kdr, flk2, FGFR2/K-sam/bek, KGFR, FGFR1/Nsam/flg/Cek1/bF, FGF3/Cek3, FGFR4, abl, src, yes, fyn, fgr, lyn, lck, hck, blk, csk, fps, fes, mas, gsp, gip, H-ras, K-ras, N-ras, mos, raf, A-raf1, B-raf, rel, ski, sno, c-Myc, N-myc, L-myc, max, myb, A-myb, B-myb, fos fos, fosB, fra1, fra2, jun, junB, junD, jif1, ets1, ets2, erg, elk1, Spi1/PU.1, fli1, GABPa, elf1, SAP1, db1, ect2, bcl1, bcl2, bcl3, bcl6, bcr, pml, pbx1/prf, PIK3CA, all1/mll, aml1, dec, ews, tls/fus 및 tel로 이루어진 군으로부터 선택된 1 종 이상인 것을 특징으로 하는,The oncogenes include MET, EGFR, bFGF, aFGF, int2/FGF3, hst1/K-fgf/FGF4, FGF5, hst2/FGF6, KGF, AIGF, erbB1, erbB2/neu, ros, trkA, trkB, trkC, ret, kit, PDGFR, flt1, flt3, flk1/kdr, flk2, FGFR2/K-sam/bek, KGFR, FGFR1/Nsam/flg/Cek1/bF, FGF3/Cek3, FGFR4, abl, src, yes, fyn, fgr, lyn, lck, hck, blk, csk, fps, fes, mas, gsp, gip, H-ras, K-ras, N-ras, mos, raf, A-raf1, B-raf, rel, ski, sno, c-Myc, N-myc, L-myc, max, myb, A-myb, B-myb, fos fos, fosB, fra1, fra2, jun, junB, junD, jif1, ets1, ets2, erg, elk1, Spi1 /PU.1, fli1, GABPa, elf1, SAP1, db1, ect2, bcl1, bcl2, bcl3, bcl6, bcr, pml, pbx1/prf, PIK3CA, all1/mll, aml1, dec, ews, tls/fus and tel Characterized in that at least one selected from the group consisting of
    암의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating cancer.
  3. 제1항에 있어서, According to claim 1,
    상기 발암 유전자 저해제는, siRNA(small interference RNA), shRNA(short hairpin RNA), miRNA(microRNA), 리보자임(ribozyme), DNAzyme, PNA(peptide nucleic acids), 안티센스 올리고뉴클레오타이드, 항체, 앱타머, 추출물 및 화합물로 이루어진 군으로부터 선택된 1 종 이상인 것을 특징으로 하는,The oncogene inhibitors include small interference RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozyme, DNAzyme, peptide nucleic acids (PNA), antisense oligonucleotides, antibodies, aptamers, and extracts. And characterized in that at least one selected from the group consisting of compounds,
    암의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating cancer.
  4. 제3항에 있어서, According to claim 3,
    상기 siRNA는 서열번호 1의 염기서열로 표시되는 MET siRNA인 것을 특징으로 하는, Characterized in that the siRNA is MET siRNA represented by the nucleotide sequence of SEQ ID NO: 1,
    암의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating cancer.
  5. 제3항에 있어서,According to claim 3,
    상기 화합물은, 사볼리티닙(savolitinib), 크리조티닙(crizotinib)(PF-02341066), 카프마티닙(capmatinib), NVP-BVU972, AMG 337, 보지티닙(bozitinib), 글루메티닙(glumetinib) 및 테포티닙(tepotinib)으로 이루어진 군으로부터 선택된 1 종 이상인 것을 특징으로 하는,The compound is savolitinib, crizotinib (PF-02341066), capmatinib, NVP-BVU972, AMG 337, bozitinib, glumetinib and Characterized in that at least one selected from the group consisting of tepotinib,
    암의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating cancer.
  6. 제1항에 있어서,According to claim 1,
    상기 (c) 항암제는, 라무시루맙(ramucirumab), 시스플라틴(cisplatin), 카보플라틴(carboplatin), 옥살리플라틴(oxaliplatin), 파클리탁셀(paclitaxel), 도세탁셀(docetaxel), 빈크리스틴(vincristine), 비노렐빈(vinorelbine), 에토포사이드(etoposide), 메토트렉세이트(methotrexate), 탈리도마이드(thalidomide) 및 보테조밉(bortezomib)으로 이루어진 군으로부터 선택된 1 종 이상인 것을 특징으로 하는,The (c) anticancer agent, ramucirumab, cisplatin, carboplatin, oxaliplatin, paclitaxel, docetaxel, vincristine, vinorelbine ( characterized in that at least one selected from the group consisting of vinorelbine, etoposide, methotrexate, thalidomide and bortezomib,
    암의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating cancer.
  7. 제1항에 있어서,According to claim 1,
    상기 암은 위암(stomach cancer), 유방암(breast cancer), 폐암(lung cancer), 간암(liver cancer), 혈액암(blood cancer), 뼈암(bone cancer), 췌장암(pancreatic cancer), 피부암(skin cancer), 머리 또는 목암(head or neck cancer), 피부 또는 안구 흑색종(cutaneous or intraocular melanoma), 자궁육종(uterine sarcoma), 난소암(ovarian cancer), 직장암(rectal cancer), 항문암(anal cancer), 대장암(colon cancer), 난관암(fallopian tube carcinoma), 자궁내막암(endometrial carcinoma), 자궁경부암(cervical cancer), 소장암(small intestine cancer), 내분비암(endocrine cancer), 갑상선암(thyroid cancer), 부갑상선암(parathyroid cancer), 신장암(adrenal cancer), 연조직종양(soft tissue tumor), 요도암(urethral cancer), 전립선암(prostate cancer), 기관지암(bronchogenic cancer) 및 골수암(bone marrow tumor)으로 이루어진 군으로부터 선택된 1 종 이상인 것을 특징으로 하는,The cancer includes stomach cancer, breast cancer, lung cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, and skin cancer. ), head or neck cancer, cutaneous or intraocular melanoma, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer , colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer ), parathyroid cancer, adrenal cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchogenic cancer and bone marrow tumor ) Characterized in that at least one selected from the group consisting of,
    암의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating cancer.
  8. 제7항에 있어서,According to claim 7,
    상기 암은 MET-과발현된 암인 것을 특징으로 하는,Characterized in that the cancer is a MET-overexpressed cancer,
    암의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating cancer.
  9. 제1항에 있어서,According to claim 1,
    상기 환자-유래 엑소좀은 암 환자의 악성 복수로부터 유래된 것을 특징으로 하는,Characterized in that the patient-derived exosome is derived from malignant ascites of a cancer patient,
    암의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating cancer.
  10. 제1항에 있어서,According to claim 1,
    상기 환자-유래 엑소좀은 자가(autologous), 동종(allogenic) 또는 이종(xenogenic)인 것을 특징으로 하는,Characterized in that the patient-derived exosome is autologous, allogenic or xenogenic,
    암의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating cancer.
  11. (a) 발암 유전자 또는 이의 단백질 발현 수준이 억제된 환자-유래 엑소좀(exosome); (b) 상기 발암 유전자 저해제; 및 (c) 항암제;를 유효성분으로 포함하는, 암의 예방 또는 치료용 약학적 조성물.(a) patient-derived exosomes in which the expression level of an oncogene or its protein is suppressed; (b) the oncogene inhibitor; And (c) anti-cancer agent; containing as an active ingredient, a pharmaceutical composition for the prevention or treatment of cancer.
  12. (a) 발암 유전자 또는 이의 단백질 발현 수준이 억제된 환자-유래 엑소좀(exosome); 및 (b) 상기 발암 유전자 저해제;를 유효성분으로 포함하는, 항암제에 대한 민감성 증진을 위한 감작제(sensitizer)용 약학적 조성물.(a) patient-derived exosomes in which the expression level of an oncogene or its protein is suppressed; and (b) the oncogene inhibitor.
  13. 제12항에 있어서, According to claim 12,
    상기 감작제용 약학적 조성물은 방사선, 화학항암제 또는 항체항암제에 대한 암의 민감성을 증가시키는 것을 특징으로 하는,Characterized in that the pharmaceutical composition for the sensitizer increases the sensitivity of cancer to radiation, chemotherapy or antibody anticancer agents,
    항암제에 대한 민감성 증진을 위한 감작제(sensitizer)용 약학적 조성물.A pharmaceutical composition for a sensitizer for enhancing sensitivity to an anticancer agent.
  14. 제12항에 있어서,According to claim 12,
    상기 항암제에 대한 민감성 증진을 위한 감작제(sensitizer)용 약학적 조성물은 항암제와 병용 투여하는 것을 특징으로 하는, Characterized in that the pharmaceutical composition for a sensitizer for enhancing sensitivity to the anticancer agent is administered in combination with the anticancer agent,
    항암제에 대한 민감성 증진을 위한 감작제(sensitizer)용 약학적 조성물.A pharmaceutical composition for a sensitizer for enhancing sensitivity to an anticancer agent.
  15. 제14항에 있어서,According to claim 14,
    상기 병용 투여는 동시에(simultaneous), 별도로(separate) 또는 순차적(seqeuntial)으로 투여되는 것을 특징으로 하는,Characterized in that the combined administration is administered simultaneously, separately or sequentially,
    항암제에 대한 민감성 증진을 위한 감작제(sensitizer)용 약학적 조성물.A pharmaceutical composition for a sensitizer for enhancing sensitivity to an anticancer agent.
  16. (a) 발암 유전자 또는 이의 단백질 발현 수준이 억제된 환자-유래 엑소좀(exosome); 및 (b) 상기 발암 유전자 저해제 또는 (c) 항암제;를 유효성분으로 포함하는, 암의 예방 또는 개선용 식품 조성물.(a) patient-derived exosomes in which the expression level of an oncogene or its protein is suppressed; And (b) the oncogene inhibitor or (c) anticancer agent; containing as an active ingredient, a food composition for preventing or improving cancer.
  17. (a) 발암 유전자 또는 이의 단백질 발현 수준이 억제된 환자-유래 엑소좀(exosome); (b) 상기 발암 유전자 저해제; 및 (c) 항암제;를 유효성분으로 포함하는, 암의 예방 또는 개선용 식품 조성물.(a) patient-derived exosomes in which the expression level of an oncogene or its protein is suppressed; (b) the oncogene inhibitor; And (c) an anticancer agent; containing as an active ingredient, a food composition for preventing or improving cancer.
  18. (a) 발암 유전자 또는 이의 단백질 발현 수준이 억제된 환자-유래 엑소좀(exosome); 및 (b) 상기 발암 유전자 저해제 또는 (c) 항암제;를 유효성분으로 포함하는 암의 예방 또는 치료용 조성물을, 대상(subject)에게 투여하는 단계를 포함하는, 암의 치료 방법.(a) patient-derived exosomes in which the expression level of an oncogene or its protein is suppressed; and (b) the oncogene inhibitor or (c) the anticancer agent; administering to a subject a composition for preventing or treating cancer comprising as an active ingredient, a cancer treatment method.
  19. (a) 발암 유전자 또는 이의 단백질 발현 수준이 억제된 환자-유래 엑소좀(exosome); (b) 상기 발암 유전자 저해제; 및 (c) 항암제;를 유효성분으로 포함하는, 암의 예방 또는 치료용 약학적 조성물을, 대상(subject)에게 투여하는 단계를 포함하는, 암의 치료 방법.(a) patient-derived exosomes in which the expression level of an oncogene or its protein is suppressed; (b) the oncogene inhibitor; And (c) an anti-cancer agent; A method for treating cancer comprising the step of administering to a subject a pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient.
  20. (a) 발암 유전자 또는 이의 단백질 발현 수준이 억제된 환자-유래 엑소좀(exosome); 및 (b) 상기 발암 유전자 저해제;를 유효성분으로 포함하는, 항암제에 대한 민감성 증진을 위한 감작제(sensitizer)용 약학적 조성물을, 대상(subject)에게 투여하는 단계를 포함하는, 암의 치료 방법.(a) patient-derived exosomes in which the expression level of an oncogene or its protein is suppressed; And (b) a method for treating cancer comprising administering to a subject a pharmaceutical composition for a sensitizer for enhancing sensitivity to an anticancer agent, comprising the oncogene inhibitor as an active ingredient. .
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