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WO2022019649A1 - Composition anticancéreuse comprenant une cellule souche présentant une efficacité améliorée - Google Patents

Composition anticancéreuse comprenant une cellule souche présentant une efficacité améliorée Download PDF

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Publication number
WO2022019649A1
WO2022019649A1 PCT/KR2021/009416 KR2021009416W WO2022019649A1 WO 2022019649 A1 WO2022019649 A1 WO 2022019649A1 KR 2021009416 W KR2021009416 W KR 2021009416W WO 2022019649 A1 WO2022019649 A1 WO 2022019649A1
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cancer
stem cells
enhanced
preventing
anticancer efficacy
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Korean (ko)
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이명우
김대성
송주용
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Cellnlife Inc
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Cellnlife Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to an anticancer composition comprising stem cells with enhanced efficacy.
  • Stem cells are cells that have the ability to differentiate into all kinds of cells that make up the body, such as nerves, blood, and cartilage, if necessary, while remaining undifferentiated into specific cells. There are two main ways to obtain these stem cells. First, they are obtained from a fetus generated from a fertilized egg (embryonic stem cells), and secondly, stem cells (adult stem cells) stored in each part of our body as adults. is to recover Although there are differences in function, both embryonic and adult stem cells have the characteristic of being able to differentiate into various types of cells.
  • human mesenchymal stem cells can be derived from a variety of tissues and are strong candidates for cell-based transplantation or regenerative drug therapy. Characteristics of MSCs such as migration to damaged tissues, immunosuppressive function, self-renewal, and pluripotency open up the possibility of their therapeutic application.
  • MSCs mesenchymal stem cells
  • the present inventors have completed the present invention by developing a pharmaceutical composition for preventing or treating cancer containing stem cells with enhanced anticancer efficacy.
  • an object of the present invention is to provide a method for preparing a pharmaceutical composition for preventing or treating cancer, including stem cells with enhanced anticancer efficacy, and a composition prepared by the method.
  • Another object of the present invention PD-L1, CXCR7, PGES and two or more proteins selected from the group consisting of PGDS; Or a gene encoding it; to provide a pharmaceutical composition for preventing or treating cancer comprising stem cells having enhanced anticancer efficacy with increased expression.
  • Another object of the present invention is to provide a method for preventing or treating cancer, comprising the step of treating the pharmaceutical composition for preventing or treating cancer.
  • the present invention provides (a) PD-L1 or CXCR7 protein; or a gene encoding it; selecting stem cells with enhanced expression; (b) culturing the stem cells selected in step (a) to enable cell-to-cell interaction; And (c) treating the stem cells cultured in step (b) with a stimulant to prepare stem cells with enhanced anticancer efficacy; Provided is a method for preparing a red composition.
  • the present invention also provides a composition for preventing or treating cancer comprising stem cells with enhanced anticancer efficacy prepared by the above method.
  • the present invention provides two or more proteins selected from the group consisting of PD-L1, CXCR7, PGES and PGDS; Or a gene encoding it; provides a pharmaceutical composition for preventing or treating cancer comprising stem cells with enhanced anticancer efficacy with increased expression.
  • the present invention comprises the step of administering a composition for preventing or treating cancer comprising stem cells with enhanced anticancer efficacy to an individual in need thereof, wherein the stem cells with enhanced anticancer efficacy are (a) PD-L1 or CXCR7 protein; or a gene encoding it; selecting stem cells with enhanced expression; (b) culturing the stem cells selected in step (a) to enable cell-to-cell interaction; And (c) treating the stem cells cultured in step (b) with a stimulant; it provides a method for preventing or treating cancer, which is produced through.
  • a composition for preventing or treating cancer comprising stem cells with enhanced anticancer efficacy to an individual in need thereof, wherein the stem cells with enhanced anticancer efficacy are (a) PD-L1 or CXCR7 protein; or a gene encoding it; selecting stem cells with enhanced expression; (b) culturing the stem cells selected in step (a) to enable cell-to-cell interaction; And (c) treating the stem cells culture
  • the present invention includes; administering a composition for preventing or treating cancer comprising stem cells with enhanced anticancer efficacy to an individual in need thereof, wherein the stem cells with enhanced anticancer efficacy are PD-L1, CXCR7, two or more proteins selected from the group consisting of PGES and PGDS; Or a gene encoding it; the expression of which is enhanced, it provides a method for preventing or treating cancer.
  • composition for preventing or treating cancer containing stem cells with enhanced anticancer efficacy according to the present invention has an effect of inhibiting cell proliferation in various carcinomas, and thus can be variously used in the field of cancer prevention or treatment.
  • FIG. 1 is a diagram showing the results of confirming the factors whose expression is increased compared to the control group, naive MSC, in mesenchymal stem cells with enhanced anticancer efficacy prepared by the culturing method of the present invention through a microarray.
  • Figure 2 confirms the anticancer effect of mesenchymal stem cells with enhanced anticancer efficacy prepared by the culturing method of the present invention, and various cancer cell lines of naive MSC and MSC prepared by the culturing method according to the present invention (human acute T lymphocytes).
  • Anticancer effect by measuring the cell viability in a leukemia cell line (Jurkat), a human liver cancer cell line (HepG2, Hep3b), a human lung cancer cell line (A549), a human breast cancer cell line (MBA-MB-231), and a human colorectal cancer cell line (HCT116) shows the results of checking .
  • the present invention provides a method for preparing a pharmaceutical composition for preventing or treating cancer comprising stem cells with enhanced anticancer efficacy; And the composition prepared by the above method; provides.
  • the method for preparing a pharmaceutical composition for preventing or treating cancer comprising stem cells with enhanced anticancer efficacy includes (a) PD-L1 or CXCR7 protein; or a gene encoding it; selecting stem cells with enhanced expression; (b) culturing the stem cells selected in step (a) to enable cell-to-cell interaction; and (c) treating the stem cells cultured in step (b) with a stimulant to prepare stem cells with enhanced anticancer efficacy.
  • the present invention also provides a method for preventing or treating cancer, comprising administering the pharmaceutical composition for preventing or treating cancer to an individual in need thereof.
  • a stem cell refers to a cell having the ability to differentiate into two or more cells while having the ability to self-renew, and the stem cell is an adult stem cell, a pluripotent stem cell, an induced pluripotent stem cell or an embryonic stem cell. contains stem cells.
  • the stem cells preferably include embryonic stem cells or adult stem cells, stem cells with enhanced anticancer efficacy.
  • the adult stem cells are preferably derived from bone marrow, blood, skin, fat, brain, umbilical cord, umbilical cord blood, periodontal, amniotic membrane, chorion, decidua, placenta or Wharton's jelly.
  • the stem cells may be mesenchymal stem cells derived from human umbilical cord.
  • mesenchymal stem cells are undifferentiated stem cells isolated from human or mammalian tissues.
  • Mesenchymal stem cells can be derived from various tissues, and in particular, from one or more selected from the group consisting of bone marrow, blood, skin, fat, brain, umbilical cord, umbilical cord blood, periodontal, amniotic membrane, chorion, decidua, placenta, or Wharton's jelly. can come from Techniques for isolating stem cells from each tissue are already known in the art.
  • the mesenchymal stem cells are autologous, allogeneic or allogeneic bone marrow, blood, skin, fat, brain, umbilical cord, umbilical cord blood, periodontal, amniotic membrane, chorion, decidua, placenta or Wharton's jelly.
  • the mesenchymal stem cells may be derived from humans, fetuses, or mammals other than humans.
  • the mammals other than humans may be more preferably canines, felines, monkeys, cattle, sheep, pigs, horses, rats, mice or guinea pigs, and the origin is not limited thereto.
  • selection means selecting cells according to whether a certain protein or a gene encoding it is expressed or not and the amount of expression.
  • PD-L1 Programmed death-ligand 1 refers to a transmembrane protein encoded by the CD274 gene in humans, and suppresses the immune system in the treatment of pregnancy, tissue allograft, autoimmune disease and hepatitis. It plays an important role.
  • the PD-L1 transmits a signal that reduces the proliferation of antigen-specific T cells in the lymph node, and at the same time increases apoptosis.
  • CXCR7 C-X-C chemokine receptor type 7 refers to a chemokine receptor also known as ACKR3 (G-protein coupled receptor 159) and GPR159 (Atypical chemokine receptor 3).
  • PGES prostaglandin E2 synthase
  • PGE prostaglandin E2 synthase
  • PGDS Prostaglandin-D synthase refers to an enzyme belonging to the sigma class glutathione-S-transferase family.
  • the PGDS catalyzes the conversion of PGH2 to PGD2 (Prostaglandin-D), and is involved in prostanoid production in the immune system and mast cells.
  • the presence of PGDS is also used to identify the differentiation stage of human megakaryocytes.
  • IDO indoleamine 2,3-dioxygenase
  • IDO refers to a heme-containing enzyme encoded by the IDO 1 gene in humans. IDO limits T cell function and is involved in immune regulation through its ability to control mechanisms of immune tolerance.
  • CXCL9 (Chemokine (C-X-C motif) ligand 9), also known as gamma-interferon-induced monochitin, is a small cytokine belonging to the CXC chemokine.
  • the CXCL9 is a T-cell chemoattractant induced by gamma-interferon.
  • CXCL10 (Chemokine (CXC motif) ligand 10) is known as IP-10 (Interferon gamma-induced protein 10) or small-inducible cytokine B10, a protein of 8.7 kDa encoded by the CXCL10 gene in humans. it means.
  • CXCL10 is a small cytokine belonging to the C-X-C chemokine family.
  • the selected stem cells of step (a) are BD2 (Beta-defensin 2) or LL-37 (Cathelicidin antimicrobial peptides (CAMP) LL-37) protein; Or it is preferred that the expression of the gene encoding it is further enhanced.
  • BD2 Beta-defensin 2
  • LL-37 Cathelicidin antimicrobial peptides (CAMP) LL-37
  • culturing to enable the cell-to-cell interaction in step (b) maintains a state of 90% or more confluent when expressed in terms of culture density, but maintains the shape of the cells, It is preferable to be cultured at a level that does not overlap, and more preferably, after seeding at a density of 3,000 to 20,000 cells/cm 2 , it may be cultured for 3 to 5 days.
  • the stimulatory agent is preferably at least one selected from the group consisting of IFN- ⁇ , TNF- ⁇ and poly IC.
  • the stem cells of step (b) include one or more proteins selected from the group consisting of CXCR7 (C-X-C chemokine receptor type 7), PGES (prostaglandin E2 synthase), and PGDS (Prostaglandin-D synthase); Or the gene encoding it; it is preferred that the expression of the enhanced.
  • CXCR7 C-X-C chemokine receptor type 7
  • PGES prostaglandin E2 synthase
  • PGDS Prostaglandin-D synthase
  • the anticancer efficacy is enhanced stem cells (i) two or more proteins selected from the group consisting of PD-L1, CXCR7, PGES and PGDS; or a gene encoding it; (ii) IDO (indoleamine 2,3-dioxygenase), CXCL9 (Chemokine (CXC motif) ligand 9), CXCL10 (Chemokine (CXC motif) ligand 10), HLA-G (human leukocyte antigen G), ICAM1 (Intercellular Adhesion Molecule 1), VCAM1 (vascular cell adhesion molecule 1), IL18BP (Interleukin-18-binding protein), RARRES3 (Retinoic acid receptor responder protein 3), CCL8 (CC motif ligand 8) ), CCL13 (CC motif ligand 13), TRAIL (TNF-related apoptosis-inducing ligand), APRIL (A proliferation-inducing ligand),
  • IDO indo
  • TRAIL TRAIL, APRIL and BAFF proteins among the aforementioned factors; and a gene encoding it; is known in the art to be deeply related to anticancer function.
  • the steps (a) to (c) refer to selection, interaction, and stimulation steps, respectively, and each step is preferably performed sequentially.
  • the cancer is gastric cancer, breast cancer, lung cancer, liver cancer, blood cancer, acute leukemia, bone cancer ( bone cancer, pancreatic cancer, 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 ), one or more selected from the group consisting of bronchogenic cancer and bone marrow tumor, but is not limited thereto.
  • bone cancer bone cancer, pancreatic cancer, 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
  • the pharmaceutical composition for preventing or treating cancer comprising stem cells with enhanced anticancer efficacy prepared by the above method may be formulated in various forms according to a conventional method and used. For example, it can be formulated and used in the form of external preparations, suppositories and sterile injection solutions.
  • composition of the present invention may contain one or more known active ingredients having a preventive or therapeutic effect on cancer together with stem cells with enhanced anticancer efficacy.
  • composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive includes starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, and lactose.
  • the pharmaceutically acceptable additive includes starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, and lactose.
  • mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, Sucrose and the like may be used.
  • the pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.
  • composition of the present invention can be administered in various parenteral formulations during actual clinical administration, and when formulated, it is prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. It can be done, and suitable formulations known in the art are preferably those disclosed in the literature (Remington's Pharmaceutical Science, recently Mack Publishing Company, Easton PA).
  • the formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories.
  • Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
  • injectable esters such as ethyl oleate.
  • As the base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.
  • the dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration method, administration time and/or route of administration of the pharmaceutical composition, and the type and extent of a response to be achieved by administration of the pharmaceutical composition , various factors including the type of subject to be administered, age, weight, general health status, symptoms or severity of disease, sex, diet, excretion, components of drugs or other compositions used simultaneously or at the same time in the subject; It may vary depending on similar factors well known in the pharmaceutical field, and a person skilled in the art can easily determine and prescribe an effective dosage for a desired treatment.
  • the dosage of the pharmaceutical composition of the present invention is, for example, preferably administered at a concentration of 0.05 to 5 mg/kg, more preferably 0.1 to 0.4 mg/kg, more preferably 0.2 to 0.35 mg/kg , more preferably 0.25 mg/kg, but the dosage does not limit the scope of the present invention in any way.
  • the administration route and administration method of the pharmaceutical composition of the present invention may be each independent, and the method is not particularly limited, and any administration route and administration method as long as the pharmaceutical composition can reach the desired site. can follow
  • the pharmaceutical composition may be administered by parenteral administration.
  • the parenteral administration method includes, for example, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration, or subcutaneous administration.
  • composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the prevention or treatment of cancer.
  • the subject is a subject expected to develop cancer; affected individuals; or an individual who has been determined to be cured; may be, but is not limited thereto.
  • two or more proteins selected from the group consisting of PD-L1, CXCR7, PGES and PGDS; Or a gene encoding it; provides a pharmaceutical composition for preventing or treating cancer comprising stem cells with enhanced anticancer efficacy with increased expression.
  • the present invention also provides a method for preventing or treating cancer, comprising administering the composition for preventing or treating cancer to an individual in need thereof.
  • the anticancer efficacy is enhanced stem cells are additional selection factors BD-2 or LL-37 protein; Or the gene encoding it; expression may be further enhanced.
  • the stem cells with enhanced efficacy are IDO, CXCL9, CXCL10, HLA-G, ICAM1, VCAM1, IL18BP, RARRES3, CCL8, CCL13, TRAIL, APRIL, BAFF, HLA-DRA, CD74, GBP1 , at least one protein selected from the group consisting of GBP2, GBP4, GBP5, PGE2 and IFN-beta; Or it is preferred that the expression of the gene encoding it is further enhanced.
  • the subject is a subject expected to develop cancer; affected individuals; or an individual who has been determined to be cured; may be, but is not limited thereto.
  • the pharmaceutical composition for preventing or treating cancer according to this aspect has significantly improved anticancer efficacy compared to naive stem cells, and can be used in various ways in the field of prevention and treatment of cancer.
  • the umbilical cord tissue collected immediately after childbirth was washed before being transferred to the laboratory, and containing F-12 medium supplemented with transfer medium (50 IU/ml penicillin, 50 ⁇ g/ml streptomycin (purchased from Invitrogen))
  • transfer medium 50 IU/ml penicillin, 50 ⁇ g/ml streptomycin (purchased from Invitrogen)
  • the stem cells were extracted in a sterile flow hood.
  • the sample is transferred to a sterile stainless steel container, washed several times with PBS, cut into 2 cm lengths, transferred to a cell culture dish with a diameter of 10 cm, and additionally washed with 70% (v/v) ethanol. Infection treatment was carried out, and the solution was washed several times with PBS to which an antibiotic mixture (50 IU/ml penicillin, 50 ⁇ g/ml streptomycin (purchased from Invitrogen)) was added until the solution became clear.
  • an antibiotic mixture 50 IU/ml penicillin,
  • the umbilical cord tissue was incised to separate Wharton's jelly (the matrix of the umbilical cord) from the blood vessels and other internal elements of the umbilical cord, the blood vessels were removed to separate the Wharton's jelly.
  • the isolated Wharton's jelly was cut into small pieces (0.5 cm ⁇ 0.5 cm). The culture of the isolated tissue was performed according to cell culture conditions suitable for the extraction of mesenchymal stem cells.
  • the explanted tissue was impregnated with 5 ml of MEM- ⁇ (Minimum essential medium-alpha, Gibco) with 10% FBS, 10% FBS and 1% antibiotics-antimycotic. It was cultured at a temperature of 37° C. in a carbon dioxide cell incubator. At this time, the medium was replaced every 3 or 4 days, and the outgrowth of cells was monitored with an optical microscope. Elongating cells were trypsinized (0.125% trypsin/0.05% EDTA) for further expansion and cryopreservation (MEM- ⁇ , 10% FBS), and the medium was changed every 3 or 4 days. The outgrowth of cells from the explanted tissue was monitored by light microscopy.
  • MEM- ⁇ Minimum essential medium-alpha, Gibco
  • the cell pellet was resuspended in medium (MEM- ⁇ (Gibco), 10% FBS, 1% Antibiotics-antimycotic) and counted, and inoculated into T75 tissue culture flasks. The medium was changed every 3 or 4 days. Cell growth and clonogenesis were monitored by light microscopy. At about 90% confluence, cells were sub-cultured as described above.
  • Human acute T lymphocytic leukemia cell line Jurkat
  • human liver cancer cell line HepG2, Hep3b
  • human lung cancer cell line A549
  • human breast cancer cell line MAA-MB-231
  • human colorectal cancer cell line HCT116
  • Example 2 S-I-S (Selection-Interaction-Stimulation) culture method for producing mesenchymal stem cells with enhanced efficacy
  • mesenchymal stem cells isolated from the human umbilical cord tissue of Example 1 cells with enhanced expression of PD-L1 and CXCR7 proteins were selected. Specifically, the mesenchymal stem cells of Example 1 were cultured confluently to enable cell-to-cell interaction. An experiment was performed to confirm the expression of PD-L1 and CXCR7 proteins in cultured mesenchymal stem cells. In addition, the expression of BD-2 and LL-37 proteins was confirmed as additional selection factors.
  • Example 1-2 Among the mesenchymal stem cells isolated and cultured in Example 1-2, cells with enhanced expression of PD-L1, CXCR7, BD-2 and LL-37 proteins were identified and selected, and these were used in the next experiment.
  • the selected PD-L1, CXCR7, BD-2 and LL-37 protein expression-enhanced mesenchymal stem cells were subcultured. Specifically, the mesenchymal stem cells were put into the mesenchymal stem cell culture medium and tissue culture flask of Example 1-2, and cultured at 37° C., 5% CO 2 in an incubator. At this time, bFGF was added to the culture medium. During subculture, mesenchymal stem cells of each generation were frozen. In addition, experiments were performed to confirm the morphology and cell proliferation rate of mesenchymal stem cells during subculture and freezing.
  • mesenchymal stem cells with enhanced expression of PD-L1, CXCR7, BD-2 and LL-37 proteins maintained the characteristics of mesenchymal stem cells even during subculture, and maintained or increased the cell proliferation rate.
  • mesenchymal stem cells with enhanced expression of PD-L1, CXCR7, BD-2 and LL-37 proteins maintained the characteristics of mesenchymal stem cells even when frozen and thawed, and maintained or increased the cell proliferation rate.
  • mesenchymal stem cells selected in the selection step of Example 2-1 cells with enhanced expression of CXCR7, PGES and PGDS proteins were selected in the additional selection step.
  • 'culture to enable interaction between cells' refers to maintaining a state of 90% or more confluent when expressed in terms of culture density, but maintaining the shape of the cells and culturing at a level where cells do not overlap. do.
  • the mesenchymal stem cells selected in the selection step of Example 2-1 were seeded at a density of 3,000 to 20,000 cells/cm 2 to enable cell-to-cell interaction, and then harvested after culturing for 3 to 5 days. An experiment was performed to confirm the expression of CXCR7, PGES and PGDS proteins in the harvested mesenchymal stem cells.
  • IFN- ⁇ , TNF- ⁇ , or polyIC Polyinosinic:polycytidylic acid, Poly I:C
  • the gene expression of mesenchymal stem cells ie, mesenchymal stem cells cultured by S-I-S culture method
  • the results of analysis of genes with increased expression in mesenchymal stem cells treated with immune stimulants are shown in FIG. 1 .
  • the genes encoding the PD-L1, TRAIL, APRIL and BAFF proteins are denoted as CD274, TNFSF10, TNFSF13 and TNFSF13B, respectively.
  • the mesenchymal stem cells stimulated with the immune stimulator increased the expression of the gene encoding TRAIL (TNFSF10), the gene encoding APRIL (TNFSF13) and the gene encoding BAFF (TNFSF13B), which are anticancer function-related factors.
  • TNFSF10 the gene encoding TRAIL
  • APRIL the gene encoding APRIL
  • BAFF the gene encoding BAFF
  • Example 3 Evaluation of anticancer function of stem cells with enhanced efficacy prepared by the culture method of Example 2
  • Alamar Blue analysis was performed.
  • the Alamar Blue assay is a modified form of the MTT assay. After treating living cells with a compound that is degraded by a specific enzyme, the relative number of living cells after drug treatment is confirmed by measuring the fluorescence intensity of the product that is released as the compound is decomposed.
  • the cell growth inhibitory effect on the cancer cell line of the mesenchymal stem cells with enhanced efficacy prepared by the culture method of Example 2 was confirmed.
  • the mesenchymal stem cells with enhanced efficacy are mesenchymal stem cells (MSC SIS CP (I) ) cultured by the culture method of Example 2 and pretreated with IFN- ⁇ in the stimulant treatment step.
  • the cancer cell lines used in this experiment were human acute T lymphocytic leukemia cell line (Jurkat), human liver cancer cell line (HepG2, Hep3b), human lung cancer cell line (A549), human breast cancer cell line (MBA-MB-231), and human colorectal cancer cell line (HCT116). )to be.
  • 4.0 ⁇ 10 4 cancer cells per well were dispensed in a 24-well plate, and mesenchymal stem cells with enhanced efficacy prepared by the culturing method of Example 2, which inhibited growth with MMC 0.4 mm pores (Corning Inc.) were plated at a density of 1 ⁇ 10 4 cells in the inserted transwell, and cultured at 37° C. in a humid atmosphere containing 5% CO 2 for 72 hours.
  • a 24-well plate After adding Alamar Blue reagent corresponding to 1/10 of the amount of the culture solution to the cell culture solution filled in each well, the plate was incubated in an incubator for 2 hours. To evenly react the cells in each well, shake the plate slowly and measure the fluorescence intensity at 600 nm while irradiating light at a wavelength of 570 nm with a Fluorescence Microplate Reader (Molecular Devices Corp.) to determine the cell viability. Confirmed. As a control, naive MSCs that were not subjected to the S-I-S culture method were used. The results of confirming the cell viability are shown in FIG. 2 .
  • MSC SIS mesenchymal stem cell
  • mesenchymal stem cells with enhanced anticancer efficacy through the culturing method of the present invention, and confirmed their anticancer efficacy against various carcinomas such as acute T lymphocytic leukemia, liver cancer, lung cancer, breast cancer and colorectal cancer did Therefore, mesenchymal stem cells with enhanced anticancer efficacy can be used in various ways in the field of cancer treatment and prevention.

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Abstract

La présente invention concerne une composition anticancéreuse comprenant des cellules souches présentant une efficacité améliorée. Une composition, comprenant des cellules souches présentant une efficacité anticancéreuse améliorée, pour la prévention ou le traitement du cancer selon la présente invention a pour effet d'inhiber la croissance cellulaire de divers carcinomes, ce qui permet de trouver des applications dans les domaines prophylactiques et thérapeutiques du cancer.
PCT/KR2021/009416 2020-07-22 2021-07-21 Composition anticancéreuse comprenant une cellule souche présentant une efficacité améliorée Ceased WO2022019649A1 (fr)

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