WO2019240559A1 - Method for enhancing cell viability of anticancer virus-introduced mesenchymal stem cells - Google Patents

Method for enhancing cell viability of anticancer virus-introduced mesenchymal stem cells Download PDF

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WO2019240559A1
WO2019240559A1 PCT/KR2019/007276 KR2019007276W WO2019240559A1 WO 2019240559 A1 WO2019240559 A1 WO 2019240559A1 KR 2019007276 W KR2019007276 W KR 2019007276W WO 2019240559 A1 WO2019240559 A1 WO 2019240559A1
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stem cells
mesenchymal stem
cancer
virus
anticancer
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Korean (ko)
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라정찬
김은영
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주식회사 알바이오
주식회사 네이처셀
라정찬
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2529/00—Culture process characterised by the use of electromagnetic stimulation
    • C12N2529/10—Stimulation by light
    • C—CHEMISTRY; METALLURGY
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
    • C12N2760/18451—Methods of production or purification of viral material

Definitions

  • the present invention relates to a method for producing mesenchymal stem cells containing an anticancer virus with improved cell viability and a cell therapy agent for treating cancer containing the stem cells prepared by the above method.
  • the present invention relates to the production of anticancer stem cell therapeutics having excellent activity and improved survival of mesenchymal stem cells by prolonging the replication time of anticancer viruses by introducing them into natural products and preventing the virus from lysing stem cells. .
  • Stem cells are cells that have the ability of self-replication and differentiate into two or more cells. Totipotent stem cells, pluripotent stem cells, and multipotent stem cells can be classified as a multipotent stem cell.
  • Pluripotent stem cells are pluripotent cells that can develop into a complete individual. Cells up to 8 cell stages after fertilization of eggs and sperm have these properties. If you transplant it into a single, complete entity.
  • Pluripotent stem cells are cells that can develop into a variety of cells and tissues derived from ectoderm, mesoderm, and endoderm.Inner cell mass located inside the blastocyst after 4-5 days of fertilization. They are called embryonic stem cells and differentiate into a variety of other tissue cells but do not form new life.
  • Multipotent stem cells are stem cells that can only differentiate into cells specific to the tissues and organs in which they are contained. In addition to growth and development, it is involved in maintaining homeostasis of adult tissues and inducing regeneration in tissue damage. Tissue-specific pluripotent cells are collectively called mesenchymal stem cells.
  • Mesenchymal stem cells (Rebecca SY Wong, et al., J Biomed Biotechnol 24: 2011, 2011) have been used for cell-based treatment in a variety of disease states, such as heart disease, osteoplastic insufficiency and spinal cord injury. It is becoming.
  • cancer is characterized by "uncontrolled cell growth,” and this abnormal cell growth forms a mass of cells called tumors that penetrate into surrounding tissues and, in severe cases, metastasize to other organs of the body. Sometimes. Academia is also called neoplasia.
  • Methods for treating cancer include surgery, radiation therapy, and chemotherapy for administering anticancer agents.
  • Bi-specific T-cell Engager (Bite), CAR-T (Chemeric antigen receptor T-cell or NK cells), anti-virus (Oncolytic Virus) from Immun checkpoint inhibitor
  • Numerous studies have been conducted for cancer conquest using various platforms such as.
  • the present inventors have a small amount of side effects, because the specific reaction to cancer only, and the anti-cancer efficacy of the anti-cancer virus is introduced as a result of the efforts to develop cancer treatments using stem cells, the anti-cancer virus introduced into the mesenchymal stem cells
  • the treatment with the following natural products prolongs the replication time of the anticancer virus and prevents the virus from lysing the stem cells, thereby improving the survival rate and survival of the stem cells and confirming that an anticancer stem cell therapy with excellent activity can be prepared.
  • the present invention has been completed.
  • An object of the present invention is to extend the replication time of oncolytic viruses through the treatment of natural substances in the mesenchymal stem cells introduced with oncolytic viruses, preventing the virus from lysing the stem cells, stem cells
  • the present invention comprises the steps of (a) preparing mesenchymal stem cells in a pellet; (b) infecting an anticancer virus to the mesenchymal stem cells in the pellet state; (c) centrifuging the mesenchymal stem cells infected with the anticancer virus; And (d) provides a method for producing mesenchymal stem cells containing an anti-cancer virus improved cell viability comprising the step of obtaining the mesenchymal stem cells into which the anti-cancer virus is introduced.
  • the present invention also provides a cell therapy agent for treating cancer containing mesenchymal stem cells prepared by the above method as an active ingredient.
  • the present invention also provides a method for treating cancer comprising administering to the subject a mesenchymal stem cell prepared by the above method.
  • the present invention also provides the use of mesenchymal stem cells prepared by the above method for use in the treatment of cancer.
  • the present invention also provides a cell therapy comprising mesenchymal stem cells prepared by the above method for use in the treatment of cancer.
  • the present invention also provides the use of mesenchymal stem cells prepared by the above method for the manufacture of a medicament for the treatment of cancer.
  • 1 is a fluorescence micrograph confirming MVeGFP by FITC fluorescence after measles virus infection in adipose derived stem cells.
  • Figure 2 confirms CD46, CD150, nectin-4 expression by FACS in cancer cell lines.
  • Figure 3 confirms the ability of MVeGFP killing in cancer cell lines and stem cells by the CPE (cytopathic effect) assay.
  • Figure 4 confirms the ability of MVeGFP killing in 1000TCID50 / ml breast cancer cell line by CPE assay.
  • Figure 6 confirms the measles virus infection in the flask and pellets of the CPE degree.
  • Figure 7 shows the CPE after varying the time and concentration of measles virus infection in pelleted cells.
  • Figure 8a confirms the survival rate of the virostem not cultured after measles virus infection.
  • Figure 8b confirms the survival rate of virostem cultured for 4 days after measles virus infection.
  • the anti-cancer virus is introduced into the mesenchymal stem cells, and then treated with natural products to prolong the replication time of the anti-cancer virus and prevent the virus from lysing the stem cells.
  • the anticancer stem cell therapy with excellent activity was prepared by improving the survival rate and survival time.
  • stem cells infected with the measles virus prepared by the method of the present invention was confirmed that the period of 80% survival was significantly increased.
  • the present invention is a point of consistency, (a) preparing mesenchymal stem cells in a pellet; (b) infecting an anticancer virus to the mesenchymal stem cells in the pellet state; (c) centrifuging the mesenchymal stem cells infected with the anticancer virus; And (d) relates to a method for producing mesenchymal stem cells containing an anti-cancer virus improved cell viability comprising the step of obtaining the mesenchymal stem cells into which the anti-cancer virus is introduced.
  • the mesenchymal stem cells of the step (a) is preferably cultured in a medium containing aspirin, the concentration of the aspirin is preferably 0.1mM to 1mM, but is not limited thereto.
  • the medium containing the aspirin preferably further comprises vitamin C, but is not limited thereto.
  • the medium is preferably DMEM or K-SFM containing 5-10% FBS and NAC (N-acetyl Cystein), and more preferably calcium, rEGF, insulin and hydrocortisone.
  • FBS N-acetyl Cystein
  • the present invention is not limited thereto.
  • the mesenchymal stem cells of the step (a) is preferably pretreated with vitamin C, but is not limited thereto.
  • mesenchymal stem cells with improved cancer cell proliferation inhibitory ability was prepared. That is, it may be a stem cell having anticancer function prepared by culturing fat-derived mesenchymal stem cells cultured by pretreatment with vitamin C in a medium containing aspirin, and fat-derived mesenchyme in a medium containing vitamin C and aspirin.
  • Stem cells may be prepared by culturing stem cells, and may also be anti-cancer function prepared by culturing fat-derived mesenchymal stem cells pre-treated with vitamin C in a medium containing vitamin C and aspirin.
  • Branches may be stem cells. We named all of these cells "Angel-Stem Cells" (WO / 2018/021879).
  • the degree of anti-cancer virus infection in the pellet state and the flask state of the stem cells was analyzed, and it was confirmed that the measles virus infection degree was excellent in the stem cell in the pellet state.
  • the mesenchymal stem cells of the step (b) is preferably 1 x 10 5 ⁇ 1 x 10 6 cells, more preferably 1 x 10 5 ⁇ 3 x 10 5 cells, most preferably Preferably 2 x 10 5 cells, but is not limited thereto.
  • the mesenchymal stem cells may be derived from a tissue selected from the group consisting of fat, uterus, bone marrow, muscle, placenta, umbilical cord blood, urine, hair follicles and skin.
  • stem cell used in the present invention refers to a cell having the ability of self-replicating and differentiating into two or more cells, and "adult stem cell” refers to each organ of the embryo during development. Refers to stem cells appearing in the stage of formation or adulthood.
  • meenchymal stem cell used in the present invention is an undifferentiated stem cell isolated from human or mammalian tissue, and may be derived from various tissues.
  • umbilical cord-derived mesenchymal stem cells umbilical cord blood-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, muscle-derived mesenchymal stem cells, nerve-derived mesenchymal stem cells, skin-derived mesenchymal stem cells , Amnion derived mesenchymal stem cells and placental derived mesenchymal stem cells, and techniques for isolating stem cells from each tissue are already known in the art.
  • fat-derived stem cell used in the present invention is an undifferentiated stem cell isolated from adipose tissue, and the separation method may be as follows.
  • the suspension containing fat suspended in physiological saline obtained from liposuction and then treated with trypsin of the stem cell layer attached to the culture vessel such as a flask and recovered, or scraped with a scraper to directly suspended in a small amount of physiological saline
  • Adipose-derived mesenchymal stem cells can be separated by a method such as recovery.
  • adult tissue-derived adult stem cells or "fatty tissue-derived mesenchymal stem cells” are undifferentiated adult stem cells isolated from adipose tissue, and may be abbreviated herein as “fat stem cells”. This can be obtained through conventional methods known in the art.
  • a conventional medium known in the art to be suitable for culturing stem cells may be used.
  • DMEM Denssion Medium
  • Keratinocyte-SFM Keratinocyte serum free medium
  • IMSC Iscove's Modified Dulbecco's Medium
  • F12 Nutrient Mixture F-12
  • DMEM / F12 Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12
  • Adipose stem cell culture medium may be supplemented with an additive that promotes proliferation of the undifferentiated phenotype of adipose stem cells while inhibiting differentiation.
  • the medium generally contains neutral buffers (such as phosphates and / or high concentrations of bicarbonate) and protein nutrients (such as serum, such as FBS, serum substitutes, albumin, or essential and non-essential amino acids, such as glutamine) in isotonic solutions. can do.
  • lipids fatty acids, cholesterol, HDL or LDL extracts of serum
  • other components found in most preservative media of this kind such as insulin or transferrin, nucleosides or nucleotides, pyruvate salts, any ionized form or salt
  • Sugar sources such as glucose, selenium, glucocorticoids such as hydrocortisone and / or reducing agents such as ⁇ -mercaptoethanol.
  • the medium also contains anti-clumping agents, such as those sold by Invitrogen (Cat # 0010057AE), with the aim of preventing the cells from adhering to each other, adhering to the vessel wall, or forming too large a bundle. It can be beneficial to do so.
  • anti-clumping agents such as those sold by Invitrogen (Cat # 0010057AE)
  • the medium for obtaining or culturing the adipose stem cells used in one embodiment of the present invention is a basal medium selected from the group consisting of DMEM, Defined Keratinocyte-SFM, ⁇ -MEM, IMDM, F12 and DMEM / F12, L It is preferable to contain aspirin in the medium composition for mesenchymal stem cell culture containing ascorbic acid 2-phosphate (vitamin C), fetal bovine serum and N-acetyl-L-cysteine.
  • vitamin C ascorbic acid 2-phosphate
  • fetal bovine serum fetal bovine serum
  • N-acetyl-L-cysteine N-acetyl-L-cysteine
  • the medium is 0.05-1 mM ascorbic acid 2-phosphate, 2-20% fetal bovine serum, 0.2-20 mM N-acetyl-L-cysteine and 0.1 to It may be characterized by containing 1mM aspirin, but is not limited thereto.
  • the anticancer virus is preferably a measles virus, more preferably MV (Edmonston strain), but is not limited thereto.
  • Measles virus is a malignant tumor (Msaouel P et al., Curr Pharm Biotechnol . 13 (9): 1732-41, 2012), cancer stem cell (CSC) (Fang Huang et al., World J Gastroenterol . 22 (35): 7999-8009, 2016), lung cancer (Zhao D et al., Oncol Rep . 29 (1): 199-204, 2013; Ong HT et al., J Hepatol . 59 (5): 999-1006, 2013), Blood tumors (D Grote et al., Blood . 97 (12): 3746-54, 2001), ovarian cancer (Zhou S et al., Cancer Lett .
  • CSC cancer stem cell
  • the anticancer virus of step (b) may be characterized in that the attenuated measles vaccine virus.
  • the attenuated virus may be a commercially available one, or may be used by attenuating and further attenuating a wild type virus or a commercially available attenuated virus.
  • MVeGFP a measles virus tagged with GFP
  • the anticancer virus of step (b) is preferably 1 x 10 5 ⁇ 1 x 10 7 TCID50, more preferably 1 x 10 5 ⁇ 5 x 10 6 TCID50, most preferably 1 x 10 6 TCID50, but is not limited thereto.
  • step (b) the infection of step (b) is preferably performed for 30 minutes to 2 hours at 36 ⁇ 37 °C, more preferably 30 minutes, but is not limited thereto.
  • the natural product is preferably an anticancer agent or an antioxidant, but is not limited thereto.
  • the anticancer agent is preferably aspirin or paclitaxel
  • the antioxidant is preferably alder extract or vitamin C, but is not limited thereto.
  • Treatment of these natural products with stem cells infected with measles virus or measles virus can increase the replication time of the virus, thereby prolonging the time for the virus to lyse the cells.
  • Stem cells infected with the measles virus of the present invention can be infected with stem cells by pretreatment of the measles vaccine virus itself with natural products to prevent the infected virus from lysing the cells, or the natural products can be treated to the stem cells infected with measles virus.
  • UV light irradiation may be further performed.
  • the time when the virus-infected mesenchymal stem cells survive more than 80% is only 48 hours (Mader EK et al., Clin Cancer Res . 1; 15; (23): 7246-55, 2009).
  • the time when the survival rate of mesenchymal stem cells is 80% or more is increased. Increased over 7 days.
  • the mesenchymal stem cells of the step (d) is preferably not further cultured after the anticancer virus is introduced, but is not limited thereto.
  • the present invention relates to a cell therapy agent for treating cancer containing mesenchymal stem cells prepared by the above method as an active ingredient.
  • the present invention relates to a method for treating cancer comprising administering to the subject a mesenchymal stem cell prepared by the above method.
  • the present invention relates to mesenchymal stem cells prepared by the above method for use in the treatment of cancer.
  • the present invention relates to a cell therapeutic agent comprising mesenchymal stem cells prepared by the above method for use in the treatment of cancer.
  • the present invention relates to mesenchymal stem cells prepared by the above method for the manufacture of a medicament for the treatment of cancer.
  • the anticancer stem cell therapy prepared by the method of the present invention can be administered in the form of a substantial product.
  • the anticancer stem cell therapeutic agent of the present invention can be produced without infecting the anticancer virus to the stem cells in the pellet state and then centrifuged, and further undergoing a culture step.
  • the cancer is lung cancer, blood tumor. Ovarian cancer, myeloma, breast cancer, brain cancer, rectal cancer, colon cancer, colorectal adenocarcinoma, osteosarcoma or cancer stem cells, but is not limited thereto.
  • the anticancer effect of the anticancer virus was excellent in breast cancer cells, and the effects were the same for both the estrogen-dependent breast cancer cell line (MCF-7) and the estrogen-independent breast cancer cell line (MDA-MB-231).
  • the mesenchymal stem cells may be derived from a tissue selected from the group consisting of fat, uterus, bone marrow, muscle, placenta, umbilical cord blood, urine, hair follicles and skin.
  • Cancer is characterized by uncontrolled cell growth, which results in the formation of cell masses called tumors that infiltrate surrounding tissues and, in severe cases, metastasize to other organs of the body.
  • anticancer includes not only the treatment of cancer diseases, ie, inhibiting the proliferation of cancer cells or cancer stem cells, or killing cancer cells or cancer stem cells, but also the prevention of cancer diseases, that is, increasing resistance to cancer before the onset of cancer. It is interpreted as. Therefore, the terms “cancer prevention or treatment” or “inhibition of cancer proliferation” and the term “anticancer” are used interchangeably herein.
  • cancer cells include cells with abnormal cell growth due to genetic modification during normal cell proliferation and growth, and cells with aggressive other organ mobility, which may be referred to as an ex.
  • cancer stem cells are known to exist in tumors and are thought to be caused by abnormal metastasis of genetic information of normal stem cells. Cancer stem cells are maintained and proliferated due to the presence of a microenvironment, a niche for their survival, and it is known that normal cells, immune-related cells, or differentiated cancer cells existing around them affect these properties.
  • cell therapeutic injection product or “cell therapeutic agent” refers to a parenteral administration containing stem cells for injection of a defect of a tissue, that is, injected into or near a defect in the form of an injection to correct a defect. It means a pharmaceutical composition that can be.
  • treating reverses, alleviates, inhibits, or prevents the disease or condition to which the term applies, or one or more symptoms of the disease or condition, Means that.
  • treatment refers to the act of treating when “treating” is defined as above.
  • treatment or “therapy” of cancer includes one or more of the following:
  • Cancer is an intractable chronic disease that, even if treated with surgery, radiation, and chemotherapy, in many cases does not heal fundamentally, suffers the patient, and ultimately leads to death. Surgery, chemotherapy and radiation therapy over the last few decades have not been the ultimate solution to cancer despite many advances.
  • the stem cells infected with the measles virus prepared by the method of the present invention is significantly increased to 7 days or more, showing a 80% survival rate, and can be administered in the form of a substantial stem cell therapeutic product.
  • the present inventors named the anticancer stem cell therapy combining the anticancer virus and the stem cells as "ViroSTEM.”
  • Human adipose tissue obtained from abdominal fat by liposuction was isolated and washed with PBS.
  • the tissue was chopped and digested for 2 hours at 37 ° C using DMEM medium containing collagenase type1 (1 mg / ml). After washing with PBS and centrifuged for 5 minutes at 1000rpm. The supernatant was suctioned and the pellet remaining on the bottom was washed with PBS, and then centrifuged at 1000 rpm for 5 minutes. Debris was removed by filtering on a 100 ⁇ m mesh, washed with PBS, and then cultured in DMEM medium containing 10% FBS, 2 mM NAC, and 0.2 mM ascorbic acid.
  • Adipose tissue-derived multipotent mesenchymal stem cells were isolated by subculture with alternating Keratinocyte-SFM medium every two days.
  • Adipose tissue-derived mesenchymal stem cells isolated as described above are stem cells cultured in a medium containing vitamin C, that is, vitamin C pre-treated mesenchymal stem cells.
  • Example 2 Cultivation of Mesenchymal Stem Cells in Aspirin-Containing Medium
  • Stem cells cultured in a medium containing vitamin C of Example 1 were inoculated in a 96-well cell culture plate at a concentration of 1 ⁇ 10 4 cells / plate, and then cultured overnight to attach and stabilize.
  • 0.5 mM of aspirin was added to Keratinocyte-SFM containing RKCM-N medium containing 5% FBS, 2 mM NAC, 0.2 mM ascorbic acid, 0.09 mM calcium, 5 ng / ml rEGF, 5 ug / ml insulin and 74 ng / ml Hydrocortisone. After exchange with a medium added at the concentration of and incubated for 24 hours.
  • Adipose tissue-derived mesenchymal stem cells obtained by culturing as described above were named Angel-Stem Cells (WO / 2018/021879) and were found to have excellent anticancer effects through direct and indirect co-culture with cancer cells. .
  • Example 1 Infection of adipose derived stem cells isolated in Example 1 or Example 2 with MVeGFP (GFP tagged measles virus) was confirmed. After purchasing Measles-GFP sold by Limanis, MVeGFP was amplified and titer was calculated to quantify MVeGFP, and the amplification method and titer identification method were established for the culture field of the present invention.
  • MVeGFP GFP tagged measles virus
  • the measles vaccine virus (10 6 TCID50) was infected by gently shaking every 30 minutes at 37 ° C in adipose derived stem cells (2X10 5 cells) in pellet or flask state. Stem cell infection of the measles vaccine virus obtained by culturing at 37 °C was confirmed by fluorescence microscopy (Fig. 1).
  • the killing ability of measles virus in various cancer cell lines can be measured by CD46, CD150 and nectin-4. Since the expression of three surface markers can be indirectly confirmed that the ability to kill due to measles virus is increased, the surface markers of each cancer cell line were confirmed by FACS assay (FIG. 2).
  • CPE cytopathic effect
  • Breast cancer cell lines were selected as cancer cell lines with high anti-cancer effect by infecting cancer cell lines with MVeGFP, and breast cancer cell lines (MCF-7: estrogen-dependent cell line and MDA-MB-231: estrogen-independent cell line) at 1000TCID50 / ml. Breast cancer killing ability of MVeGFP was confirmed (FIG. 4). As a result, when progressed to 1000TCID50 / ml, the positive control group showed CPE from day 2 and nearly 80% of CPE was formed on day 3, and both MCF-7 and MDA-MB-231 started to form CPE from day 3 It was.
  • MVeGFP infection in breast cancer cells was established by FACS assay to quantitatively determine the extent of infection (FIG. 5).
  • Measles virus was used after measles vaccine virus or after attenuating the measles virus virus once again.
  • the cells were infected in a flask or pellet state, and then the degree of CPE was confirmed.
  • the degree of infection was high in the cellular environment of the pellet state (FIG. 6), and the degree of infection was increased by giving a physical change by centrifugation after infection.
  • Example 6 Extending the survival time of stem cells infected with measles virus by natural product treatment
  • Virostem a mesenchymal stem cell incorporating measles virus, increased the survival by treating vitamin C or aspirin alone or in combination with antioxidants.
  • Example 7 Improvement of anticancer effect of measles virus infected stem cells by natural product treatment
  • the method for producing mesenchymal stem cells containing the anticancer virus according to the present invention extends the replication time of the anticancer virus through the treatment of natural substances and prevents the virus from dissolving the stem cells, thereby improving the viability and survival of the stem cells.
  • Stem cells containing the anticancer virus with improved activity can be prepared, and the anticancer stem cell therapy thus prepared has a markedly improved shelf life, which is very useful in medicine and industry.

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Abstract

The present invention relates to a cell therapy product comprising virus-introduced stem cells for treatment of cancer. More particularly, an anticancer virus is introduced into mesenchymal stem cells, followed by treatment with a natural substance to prolong the replication time of the anticancer virus and prevent the virus from lysing the stem cells, thereby creating stem cells that contain the anticancer virus having excellent activity and improve in the viability and duration of life of the mesenchymal stem cells. The anticancer stem cell product thus prepared has a remarkably enhanced expiry date and is industrially very valuable.

Description

항암 바이러스가 도입된 중간엽 줄기세포의 세포 생존능을 향상시키는 방법How to Improve Cell Viability of Mesenchymal Stem Cells Introduced with Anticancer Virus
본 발명은 세포 생존능이 향상된 항암 바이러스를 함유하는 중간엽 줄기세포의 제조방법 및 상기 방법으로 제조된 줄기세포를 함유하는 암 치료용 세포치료제에 관한 것으로, 더욱 자세하게는 중간엽 줄기세포에 항암 바이러스를 도입한 다음 천연물로 처리하여 항암 바이러스의 복제시간을 연장시키고, 바이러스가 줄기세포를 용해시키는 것을 방지함으로써, 중간엽 줄기세포의 생존율 및 생존기간이 향상된 활성이 우수한 항암 줄기세포치료제의 제조에 관한 것이다.The present invention relates to a method for producing mesenchymal stem cells containing an anticancer virus with improved cell viability and a cell therapy agent for treating cancer containing the stem cells prepared by the above method. The present invention relates to the production of anticancer stem cell therapeutics having excellent activity and improved survival of mesenchymal stem cells by prolonging the replication time of anticancer viruses by introducing them into natural products and preventing the virus from lysing stem cells. .
줄기세포 (stem cell)란 자기 복제 능력을 가지면서 두 개 이상의 세포로 분화하는 능력을 갖는 세포를 말하며, 만능 줄기세포 (totipotent stem cell), 전분화능 줄기세포 (pluripotent stem cell), 다분화능 줄기세포 (multipotent stem cell)로 분류할 수 있다.Stem cells are cells that have the ability of self-replication and differentiate into two or more cells. Totipotent stem cells, pluripotent stem cells, and multipotent stem cells can be classified as a multipotent stem cell.
만능 줄기세포 (totipotent stem cell)는 하나의 완전한 개체로 발생해 나갈 수 있는 만능의 성질을 가진 세포로 난자와 정자의 수정 이후 8세포기까지의 세포가 이러한 성질을 가지며, 이 세포를 분리하여 자궁에 이식하면 하나의 완전한 개체로 발생해 나갈 수 있다. Pluripotent stem cells are pluripotent cells that can develop into a complete individual. Cells up to 8 cell stages after fertilization of eggs and sperm have these properties. If you transplant it into a single, complete entity.
전분화능 줄기세포 (pluripotent stem cell)는 외배엽, 중배엽, 내배엽 유래의 다양한 세포와 조직으로 발생할 수 있는 세포로서, 수정 4~5일 후 나타나는 배반포 (blastocyst)의 안쪽에 위치한 내세포괴 (inner cell mass)에서 유래하며, 이를 배아 줄기세포라 하며 다양한 다른 조직 세포로 분화되지만 새로운 생명체를 형성하지는 못한다. Pluripotent stem cells are cells that can develop into a variety of cells and tissues derived from ectoderm, mesoderm, and endoderm.Inner cell mass located inside the blastocyst after 4-5 days of fertilization. They are called embryonic stem cells and differentiate into a variety of other tissue cells but do not form new life.
다분화능 (또는 다능성) 줄기세포 (multipotent stem cell)는 이 세포가 포함되어 있는 조직 및 기관에 특이적인 세포로만 분화할 수 있는 줄기세포로서, 태아기, 신생아기 및 성체기의 각 조직 및 장기의 성장과 발달은 물론 성체조직의 항상성 유지와 조직손상시 재생을 유도하는 기능에 관여하고 있으며 조직 특이적 다능성 세포들을 총칭하여 중간엽 줄기세포라 한다.Multipotent stem cells are stem cells that can only differentiate into cells specific to the tissues and organs in which they are contained. In addition to growth and development, it is involved in maintaining homeostasis of adult tissues and inducing regeneration in tissue damage. Tissue-specific pluripotent cells are collectively called mesenchymal stem cells.
중간엽 줄기세포 (Rebecca S. Y. Wong, et al., J Biomed Biotechnol 24:2011, 2011)는 심장 질환, 골형성 부전증 및 척수 손상과 같은 다양한 질병 상태에서 세포 기반 치료에 사용되어 왔으며, 그 결과가 주목되고 있다.Mesenchymal stem cells (Rebecca SY Wong, et al., J Biomed Biotechnol 24: 2011, 2011) have been used for cell-based treatment in a variety of disease states, such as heart disease, osteoplastic insufficiency and spinal cord injury. It is becoming.
한편, "암"이란 '제어되지 않은 세포성장'으로 특징지어지며, 이러한 비정상적인 세포성장에 의해 종양 (tumor)이라고 불리는 세포 덩어리가 형성되어 주위의 조직으로 침투하고 심한 경우에는 신체의 다른 기관으로 전이되기도 한다. 학문적으로는 신생물 (neoplasia)이라고도 불린다.On the other hand, "cancer" is characterized by "uncontrolled cell growth," and this abnormal cell growth forms a mass of cells called tumors that penetrate into surrounding tissues and, in severe cases, metastasize to other organs of the body. Sometimes. Academia is also called neoplasia.
암을 치료하기 위한 방법으로는 수술요법, 방사선 요법 및 항암제를 투여하는 화학요법 등이 있으며, 현재 종양면역요법 (cancer immunotherapy) 개발을 위해 전세계적으로 많은 연구들이 진행되고 있다. 면역체크포인트 억제제 (Immune checkpoint inhibitor)로부터 이중 특이적 T 세포 관여 항체 (Bi-specific T-cell Engager, Bite), CAR-T (Chemeric antigen receptor T-cell or NK cells), 항바이러스 (Oncolytic Virus) 등의 다양한 플랫폼을 활용하여 암정복을 위한 수많은 연구들이 수행되고 있다. Methods for treating cancer include surgery, radiation therapy, and chemotherapy for administering anticancer agents. Currently, many studies are being conducted around the world to develop cancer immunotherapy. Bi-specific T-cell Engager (Bite), CAR-T (Chemeric antigen receptor T-cell or NK cells), anti-virus (Oncolytic Virus) from Immun checkpoint inhibitor Numerous studies have been conducted for cancer conquest using various platforms such as.
특히, 항암 바이러스 (Oncolytic virus)를 연구하는 전문가들이 주장하는 가장 큰 매력은 바로 기존 치료제와는 다르게 바이러스가 살아있다는 것이다. 이와 함께, 바이러스는 그 자체로의 유전자를 소유하기 때문에 스스로 번식할 수 있어 주사된 부위 외에 인근에 있는 암세포에까지 감염을 일으켜 파괴할 수 있다는 점이 매력이다. 물론, 임상에서 항암 바이러스가 상용화되기 위해서는 스스로 증식된 바이러스 자체가 각종 질병을 유발한다는 원인이라는 고정관념을 극복해야만 하는 큰 장애물을 넘어야 하겠지만, 암 환자에 있어 단순한 생명연장이 아닌 완치의 목적을 위해 항바이러스 (Oncolytic virus) 기술의 발전 필요성이 매우 크다.In particular, the biggest attraction of experts studying oncolytic viruses is that they are alive, unlike conventional therapies. In addition, since the virus possesses its own genes, it can multiply itself and can infect and destroy nearby cancer cells in addition to the injected site. Of course, in order to commercialize anti-cancer virus in the clinic, it must overcome the big obstacle that must overcome the stereotype that the self-proliferating virus itself causes various diseases. There is a great need for the development of oncolytic virus technology.
아울러, 기존의 암 치료 방법들은 치료시 부작용이 항암 분야의 주요한 장애물이 되어 왔으며, 치료 후 재발의 위험성으로부터 자유로울 수 없다. 또한, 암으로부터 자유로울 수 없는 환경, 예를 들어 가족력이 있거나 여러 질병으로 인해 면역력이 저하된 사람에게 암을 예방할 수 있는 대안책이 필요하다. In addition, conventional cancer treatment methods have been a major obstacle in the anti-cancer field of treatment, and cannot be free from the risk of recurrence after treatment. In addition, there is a need for an alternative that can prevent cancer in an environment that cannot be free from cancer, such as a person with a family history or reduced immunity due to various diseases.
이에, 본 발명자들은 암에만 특정하게 반응하기 때문에 부작용이 적고, 항암 효능은 우수한 항암 바이러스가 도입된 줄기세포를 이용하여 암 치료제를 개발하기 위해 예의 노력한 결과, 중간엽 줄기세포에 항암 바이러스를 도입한 다음 천연물로 처리하면 항암 바이러스의 복제시간이 연장되고, 바이러스가 줄기세포를 용해시키는 것을 방지함으로써, 줄기세포의 생존율 및 생존기간이 향상되어 활성이 우수한 항암 줄기세포치료제를 제조할 수 있음을 확인하고, 본 발명을 완성하게 되었다.Therefore, the present inventors have a small amount of side effects, because the specific reaction to cancer only, and the anti-cancer efficacy of the anti-cancer virus is introduced as a result of the efforts to develop cancer treatments using stem cells, the anti-cancer virus introduced into the mesenchymal stem cells The treatment with the following natural products prolongs the replication time of the anticancer virus and prevents the virus from lysing the stem cells, thereby improving the survival rate and survival of the stem cells and confirming that an anticancer stem cell therapy with excellent activity can be prepared. The present invention has been completed.
발명의 요약Summary of the Invention
본 발명의 목적은 항암 바이러스 (oncolytic viruses)가 도입된 중간엽 줄기세포에 천연물질 처리를 통해 항암 바이러스 (oncolytic viruses)의 복제시간은 연장시키고, 바이러스가 줄기세포를 용해시키는 것을 방지함으로써, 줄기세포의 생존능 및 생존기간을 향상시킨 항암 바이러스를 함유하는 중간엽 줄기세포의 제조방법 및 상기 방법으로 제조된 중간엽 줄기세포를 유효성분으로 함유하는 암 치료용 세포치료제를 제공하는데 있다.An object of the present invention is to extend the replication time of oncolytic viruses through the treatment of natural substances in the mesenchymal stem cells introduced with oncolytic viruses, preventing the virus from lysing the stem cells, stem cells To provide a method for producing mesenchymal stem cells containing an anticancer virus with improved viability and survival time and a cell therapy for cancer treatment containing mesenchymal stem cells prepared by the above method as an active ingredient.
상기 목적을 달성하기 위하여, 본 발명은 (a) 중간엽 줄기세포를 펠렛 (pellet) 상태로 제조하는 단계; (b) 상기 펠렛 상태의 중간엽 줄기세포에 항암 바이러스를 감염시키는 단계; (c) 상기 항암 바이러스가 감염된 중간엽 줄기세포를 원심분리하는 단계; 및 (d) 상기 항암 바이러스가 도입된 중간엽 줄기세포를 수득하는 단계를 포함하는 세포 생존능이 향상된 항암 바이러스를 함유하는 중간엽 줄기세포의 제조방법을 제공한다.In order to achieve the above object, the present invention comprises the steps of (a) preparing mesenchymal stem cells in a pellet; (b) infecting an anticancer virus to the mesenchymal stem cells in the pellet state; (c) centrifuging the mesenchymal stem cells infected with the anticancer virus; And (d) provides a method for producing mesenchymal stem cells containing an anti-cancer virus improved cell viability comprising the step of obtaining the mesenchymal stem cells into which the anti-cancer virus is introduced.
본 발명은 또한, 상기 방법으로 제조된 중간엽 줄기세포를 유효성분으로 함유하는 암 치료용 세포치료제를 제공한다.The present invention also provides a cell therapy agent for treating cancer containing mesenchymal stem cells prepared by the above method as an active ingredient.
본 발명은 또한, 상기 방법으로 제조된 중간엽 줄기세포를 개체에 투여하는 단계를 포함하는 암 치료방법을 제공한다.The present invention also provides a method for treating cancer comprising administering to the subject a mesenchymal stem cell prepared by the above method.
본 발명은 또한, 암 치료에 사용하기 위한 상기 방법으로 제조된 중간엽 줄기세포의 용도를 제공한다.The present invention also provides the use of mesenchymal stem cells prepared by the above method for use in the treatment of cancer.
본 발명은 또한, 암 치료에 사용하기 위한 상기 방법으로 제조된 중간엽 줄기세포를 포함하는 세포치료제를 제공한다.The present invention also provides a cell therapy comprising mesenchymal stem cells prepared by the above method for use in the treatment of cancer.
본 발명은 또한, 암 치료용 약제의 제조를 위한 상기 방법으로 제조된 중간엽 줄기세포의 용도를 제공한다.The present invention also provides the use of mesenchymal stem cells prepared by the above method for the manufacture of a medicament for the treatment of cancer.
도 1은 지방 유래 줄기세포에 홍역 바이러스 감염 후 MVeGFP를 FITC 형광으로 확인한 형광 현미경 사진이다.1 is a fluorescence micrograph confirming MVeGFP by FITC fluorescence after measles virus infection in adipose derived stem cells.
도 2는 암 세포주에서 CD46, CD150, nectin-4 발현을 FACS로 확인한 것이다.Figure 2 confirms CD46, CD150, nectin-4 expression by FACS in cancer cell lines.
도 3은 암 세포주 및 줄기세포에서의 MVeGFP 사멸능력을 CPE (cytopathic effect) assay로 확인한 것이다.Figure 3 confirms the ability of MVeGFP killing in cancer cell lines and stem cells by the CPE (cytopathic effect) assay.
도 4는 1000TCID50/ml의 유방암 세포주에서 MVeGFP 사멸 능력을 CPE assay로 확인한 것이다.Figure 4 confirms the ability of MVeGFP killing in 1000TCID50 / ml breast cancer cell line by CPE assay.
도 5는 MCF7 유방암 세포주에 MVeGFP를 감염시켜 FACS로 감염정도를 정량한 것이다.5 is infected with MVeGFP in MCF7 breast cancer cell line to quantify the degree of infection by FACS.
도 6은 플라스크 및 펠렛 상태의 세포에서 홍역 바이러스 감염을 CPE 정도로 확인한 것이다.Figure 6 confirms the measles virus infection in the flask and pellets of the CPE degree.
도 7은 펠렛 상태의 세포에서 홍역 바이러스 감염 시간 및 농도를 다르게 한 후 CPE 정도로 확인한 것이다.Figure 7 shows the CPE after varying the time and concentration of measles virus infection in pelleted cells.
도 8a는 홍역 바이러스 감염 후 배양하지 않은 바이로스템의 생존율을 확인한 것이다.Figure 8a confirms the survival rate of the virostem not cultured after measles virus infection.
도 8b는 홍역 바이러스 감염 후 4일 동안 배양한 바이로스템의 생존율을 확인한 것이다.Figure 8b confirms the survival rate of virostem cultured for 4 days after measles virus infection.
도 9는 바이로스템의 유효기간을 qPCR로 확인한 것이다.9 confirms the validity period of the virostem by qPCR.
발명의 상세한 설명 및 바람직한 구현예Detailed Description of the Invention and Preferred Embodiments
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술 분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로, 본 명세서에서 사용된 명명법은 본 기술 분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.
본 발명에서는 중간엽 줄기세포에 항암 바이러스를 도입한 다음 천연물로 처리하여 항암 바이러스의 복제시간 (replication time)을 연장시키고, 바이러스가 줄기세포를 용해시키는 것을 방지함으로써, 항암 바이러스가 감염된 중간엽 줄기세포의 생존율 및 생존기간을 향상시켜 활성이 우수한 항암 줄기세포치료제를 제조하였다. 이에, 본 발명의 방법으로 제조된 홍역 바이러스가 감염된 줄기세포는 80% 생존율을 나타내는 기간이 현저히 증가된 것을 확인하였다.In the present invention, the anti-cancer virus is introduced into the mesenchymal stem cells, and then treated with natural products to prolong the replication time of the anti-cancer virus and prevent the virus from lysing the stem cells. The anticancer stem cell therapy with excellent activity was prepared by improving the survival rate and survival time. Thus, stem cells infected with the measles virus prepared by the method of the present invention was confirmed that the period of 80% survival was significantly increased.
따라서, 본 발명은 일관점에서, (a) 중간엽 줄기세포를 펠렛 (pellet) 상태로 제조하는 단계; (b) 상기 펠렛 상태의 중간엽 줄기세포에 항암 바이러스를 감염시키는 단계; (c) 상기 항암 바이러스가 감염된 중간엽 줄기세포를 원심분리하는 단계; 및 (d) 상기 항암 바이러스가 도입된 중간엽 줄기세포를 수득하는 단계를 포함하는 세포 생존능이 향상된 항암 바이러스를 함유하는 중간엽 줄기세포의 제조방법에 관한 것이다.Therefore, the present invention is a point of consistency, (a) preparing mesenchymal stem cells in a pellet; (b) infecting an anticancer virus to the mesenchymal stem cells in the pellet state; (c) centrifuging the mesenchymal stem cells infected with the anticancer virus; And (d) relates to a method for producing mesenchymal stem cells containing an anti-cancer virus improved cell viability comprising the step of obtaining the mesenchymal stem cells into which the anti-cancer virus is introduced.
본 발명에 있어서, 상기 (a) 단계의 중간엽 줄기세포는 아스피린을 함유하는 배지에서 배양된 것이 바람직하며, 상기 아스피린의 농도는 0.1mM 내지 1mM인 것이 바람직하나, 이에 한정되는 것은 아니다.In the present invention, the mesenchymal stem cells of the step (a) is preferably cultured in a medium containing aspirin, the concentration of the aspirin is preferably 0.1mM to 1mM, but is not limited thereto.
본 발명에 있어서, 상기 아스피린을 함유하는 배지는 비타민 C를 추가로 포함하는 것이 바람직하나, 이에 한정되는 것은 아니다.In the present invention, the medium containing the aspirin preferably further comprises vitamin C, but is not limited thereto.
본 발명에 있어서, 상기 배지는 5~10% FBS 및 NAC (N-acetyl Cystein)을 함유하는 DMEM 또는 K-SFM인 것이 바람직하며, 추가로 칼슘, rEGF, 인슐린 및 하이드로코티손을 함유하는 것이 더욱 바람직하나, 이에 한정되는 것은 아니다.In the present invention, the medium is preferably DMEM or K-SFM containing 5-10% FBS and NAC (N-acetyl Cystein), and more preferably calcium, rEGF, insulin and hydrocortisone. However, the present invention is not limited thereto.
본 발명에 있어서, 상기 (a) 단계의 중간엽 줄기세포는 비타민 C로 전처리된 것이 바람직하나, 이에 한정되는 것은 아니다.In the present invention, the mesenchymal stem cells of the step (a) is preferably pretreated with vitamin C, but is not limited thereto.
본 발명의 일 실시예에서는 비타민 C 함유 배지에서 배양된 지방 유래 중간엽 줄기세포를 아스피린을 함유하는 배지에서 배양하여, 개선된 암세포 증식 억제능을 가지는 중간엽 줄기세포를 제조하였다. 즉, 비타민 C를 전처리하여 배양한 지방 유래 중간엽 줄기세포를 아스피린을 함유하는 배지에서 배양하여 제조된 항암 기능을 가지는 줄기세포일 수 있으며, 비타민 C와 아스피린을 함께 함유하는 배지에서 지방 유래 중간엽 줄기세포를 배양하여 제조된 항암 기능을 가지는 줄기세포일 수 있으며, 또한, 비타민 C를 전처리하여 배양한 지방 유래 중간엽 줄기세포를 비타민 C와 아스피린을 함께 함유하는 배지에서 배양하여 제조된 항암 기능을 가지는 줄기세포일 수 있다. 본 발명자들은 이러한 세포를 모두 "Angel-Stem Cell" (WO/2018/021879)로 명명하였다.In one embodiment of the present invention by culturing fat-derived mesenchymal stem cells cultured in a vitamin C-containing medium in aspirin-containing medium, mesenchymal stem cells with improved cancer cell proliferation inhibitory ability was prepared. That is, it may be a stem cell having anticancer function prepared by culturing fat-derived mesenchymal stem cells cultured by pretreatment with vitamin C in a medium containing aspirin, and fat-derived mesenchyme in a medium containing vitamin C and aspirin. Stem cells may be prepared by culturing stem cells, and may also be anti-cancer function prepared by culturing fat-derived mesenchymal stem cells pre-treated with vitamin C in a medium containing vitamin C and aspirin. Branches may be stem cells. We named all of these cells "Angel-Stem Cells" (WO / 2018/021879).
본 발명의 일 실시예에서는 상기 줄기세포의 펠렛 상태 및 플라스크 상태에서의 항암 바이러스 감염 정도를 분석하였으며, 펠렛 상태의 줄기세포에서 홍역 바이러스 감염 정도가 우수한 것을 확인하였다. In one embodiment of the present invention, the degree of anti-cancer virus infection in the pellet state and the flask state of the stem cells was analyzed, and it was confirmed that the measles virus infection degree was excellent in the stem cell in the pellet state.
특히, 감염 정도를 높이기 위해서는 홍역 바이러스를 감염시킨 줄기세포를 원심분리하여 물리적 변화를 주는 것이 바람직하다.In particular, in order to increase the degree of infection, it is preferable to centrifuge the stem cells infected with the measles virus to give physical changes.
본 발명에 있어서, 상기 (b) 단계의 중간엽 줄기세포는 1 x 105 ~ 1 x 106 세포인 것이 바람직하며, 더욱 바람직하게는 1 x 105 ~ 3 x 105 세포인 것이며, 가장 바람직하게는 2 x 105 세포인 것이나, 이에 한정되는 것은 아니다.In the present invention, the mesenchymal stem cells of the step (b) is preferably 1 x 10 5 ~ 1 x 10 6 cells, more preferably 1 x 10 5 ~ 3 x 10 5 cells, most preferably Preferably 2 x 10 5 cells, but is not limited thereto.
본 발명에 있어서, 상기 중간엽 줄기세포는 지방, 자궁, 골수, 근육, 태반, 제대혈, 소변, 모낭 및 피부로 구성된 군에서 선택된 조직 유래인 것을 특징으로 할 수 있다.In the present invention, the mesenchymal stem cells may be derived from a tissue selected from the group consisting of fat, uterus, bone marrow, muscle, placenta, umbilical cord blood, urine, hair follicles and skin.
본 발명에서 사용하는 용어 "줄기세포(stem cell)"이란 자기 복제 능력을 가지면서 두 개 이상의 세포로 분화하는 능력을 갖는 세포를 말하며, "성체 줄기세포"는 발생과정이 진행되어 배아의 각 장기가 형성되는 단계 혹은 성체단계에서 나타나는 줄기세포를 의미한다.The term "stem cell" used in the present invention refers to a cell having the ability of self-replicating and differentiating into two or more cells, and "adult stem cell" refers to each organ of the embryo during development. Refers to stem cells appearing in the stage of formation or adulthood.
본 발명에서 사용하는 용어 "중간엽 줄기세포"는 인간 또는 포유류의 조직으로부터 분리해 낸 미분화된 줄기세포로서, 다양한 조직에서 유래할 수 있다. 특히, 제대 유래 중간엽 줄기세포, 제대혈 유래 중간엽 줄기세포, 골수 유래 중간엽 줄기세포, 지방 유래 중간엽 줄기세포, 근육 유래 중간엽 줄기세포, 신경 유래 중간엽 줄기세포, 피부 유래 중간엽 줄기세포, 양막 유래 중간엽 줄기세포 및 태반 유래 중간엽 줄기세포일 수 있으며, 각 조직에서 줄기세포를 분리하는 기술은 당해 업계에 이미 공지되어 있다.The term "mesenchymal stem cell" used in the present invention is an undifferentiated stem cell isolated from human or mammalian tissue, and may be derived from various tissues. In particular, umbilical cord-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, muscle-derived mesenchymal stem cells, nerve-derived mesenchymal stem cells, skin-derived mesenchymal stem cells , Amnion derived mesenchymal stem cells and placental derived mesenchymal stem cells, and techniques for isolating stem cells from each tissue are already known in the art.
본 발명에서 사용하는 용어 "지방 유래 줄기세포"란 지방조직에서 분리해 낸 미분화 줄기세포로, 그 분리 방법은 예를 들어 다음과 같을 수 있다. 즉, 지방흡입술로부터 얻어지는 생리 식염수에 부유된 지방 함유 suspension을 배양한 다음, 플라스크 등 배양용기에 부착된 줄기세포 층을 트립신으로 처리한 다음 회수하거나, 스크래퍼로 긁어서 소량의 생리 식염수에 부유되는 것을 직접 회수하거나 하는 방법 등을 통해 지방 유래 중간엽 줄기세포를 분리할 수 있다.The term "fat-derived stem cell" used in the present invention is an undifferentiated stem cell isolated from adipose tissue, and the separation method may be as follows. In other words, the suspension containing fat suspended in physiological saline obtained from liposuction, and then treated with trypsin of the stem cell layer attached to the culture vessel such as a flask and recovered, or scraped with a scraper to directly suspended in a small amount of physiological saline Adipose-derived mesenchymal stem cells can be separated by a method such as recovery.
이러한, "지방 조직 유래 성체 줄기세포" 또는 "지방 조직 유래 중간엽 줄기세포"는 지방 조직으로부터 분리해 낸 미분화된 성체 줄기세포로서, 본 명세서에서는 축약하여 "지방 줄기세포"라고 지칭하기도 한다. 이는 당업계에 공지된 통상의 방법을 통해 수득할 수 있다.Such "adult tissue-derived adult stem cells" or "fatty tissue-derived mesenchymal stem cells" are undifferentiated adult stem cells isolated from adipose tissue, and may be abbreviated herein as "fat stem cells". This can be obtained through conventional methods known in the art.
상기 지방 줄기세포의 획득에 사용되는 배지로서는 당업계에서 줄기세포 배양에 적합하다고 알려져 있는 통상적인 배지를 사용할 수 있는데, 바람직하게는 DMEM (Dulbecco's modified Eagle medium) 또는 Keratinocyte-SFM (Keratinocyte serum free medium)을 사용할 수 있으며, IMDM (Iscove's Modified Dulbecco's Medium), a-MEM (Alpha Modification of Eagle's Medium), F12 (Nutrient Mixture F-12) 및 DMEM/F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) 배지를 혼합한 배지도 사용할 수 있으나, 이에 한정되는 것은 아니다.As a medium used for obtaining the adipose stem cells, a conventional medium known in the art to be suitable for culturing stem cells may be used. Preferably, DMEM (Dulbecco's modified Eagle medium) or Keratinocyte-SFM (Keratinocyte serum free medium) You can use IMSC (Iscove's Modified Dulbecco's Medium), a-MEM (Alpha Modification of Eagle's Medium), F12 (Nutrient Mixture F-12), and DMEM / F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) Mixed media may also be used, but is not limited thereto.
지방 줄기세포 배양용 배지는 지방 줄기세포의 미분화된 표현형의 증식을 촉진하면서 분화는 억제하는 첨가제로 보충될 수 있다. 또한, 배지는 일반적으로, 등장액 중의 중성 완충제(예컨대 인산염 및/또는 고농도 중탄산염) 및 단백질 영양분(예를 들면 혈청, 예컨대 FBS, 혈청 대체물, 알부민, 또는 필수 아미노산 및 비필수 아미노산, 예컨대 글루타민)을 함유할 수 있다. 나아가, 지질(지방산, 콜레스테롤, 혈청의 HDL 또는 LDL 추출물) 및 이 종류의 대부분의 보존액 배지에서 발견되는 기타 성분(예컨대 인슐린 또는 트랜스페린, 뉴클레오시드 또는 뉴클레오티드, 피루빈산염, 임의의 이온화 형태 또는 염인 당원, 예컨대 글루코스, 셀레늄, 글루코코르티코이드, 예컨대 히드로코르티존 및/또는 환원제, 예컨대 β-메르캅토에탄올)을 함유할 수 있다.Adipose stem cell culture medium may be supplemented with an additive that promotes proliferation of the undifferentiated phenotype of adipose stem cells while inhibiting differentiation. In addition, the medium generally contains neutral buffers (such as phosphates and / or high concentrations of bicarbonate) and protein nutrients (such as serum, such as FBS, serum substitutes, albumin, or essential and non-essential amino acids, such as glutamine) in isotonic solutions. can do. Furthermore, lipids (fatty acids, cholesterol, HDL or LDL extracts of serum) and other components found in most preservative media of this kind (such as insulin or transferrin, nucleosides or nucleotides, pyruvate salts, any ionized form or salt) Sugar sources such as glucose, selenium, glucocorticoids such as hydrocortisone and / or reducing agents such as β-mercaptoethanol.
또한, 배지는 세포가 서로 유착하거나, 용기벽에 유착하거나, 너무 큰 다발을 형성하는 것을 방지할 목적으로, 항응집제 (anti-clumping agent), 예컨대 Invitrogen이 판매하는 것들(Cat # 0010057AE)을 포함하는 것이 유익할 수 있다.The medium also contains anti-clumping agents, such as those sold by Invitrogen (Cat # 0010057AE), with the aim of preventing the cells from adhering to each other, adhering to the vessel wall, or forming too large a bundle. It can be beneficial to do so.
특히, 본 발명의 일 구체예에서 사용되는 지방 줄기세포를 수득 또는 배양하기 위한 배지는 DMEM, Defined Keratinocyte-SFM, α-MEM, IMDM, F12 및 DMEM/F12로 구성된 군에서 선택되는 기본 배지, L-아스코르브산 2-인산 (비타민 C), 우태아혈청 및 N-아세틸-L-시스테인(N-acetyl-L-cysteine)을 함유하는 중간엽 줄기세포 배양을 위한 배지 조성물에 아스피린을 함유하는 것이 바람직하나, 이에 한정되는 것은 아니다.In particular, the medium for obtaining or culturing the adipose stem cells used in one embodiment of the present invention is a basal medium selected from the group consisting of DMEM, Defined Keratinocyte-SFM, α-MEM, IMDM, F12 and DMEM / F12, L It is preferable to contain aspirin in the medium composition for mesenchymal stem cell culture containing ascorbic acid 2-phosphate (vitamin C), fetal bovine serum and N-acetyl-L-cysteine. However, the present invention is not limited thereto.
본 발명에 있어서, 상기 배지는 0.05~1 mM의 아스코르브산 2-인산, 2~20% 우태아혈청, 0.2~20mM의 N-아세틸-L-시스테인(N-acetyl-L-cysteine) 및 0.1 내지 1mM 아스피린을 함유하는 것을 특징으로 할 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the medium is 0.05-1 mM ascorbic acid 2-phosphate, 2-20% fetal bovine serum, 0.2-20 mM N-acetyl-L-cysteine and 0.1 to It may be characterized by containing 1mM aspirin, but is not limited thereto.
본 발명에 있어서, 상기 항암 바이러스는 홍역 바이러스 (measles virus)인 것이 바람직하며, 더욱 바람직하게는 MV (Edmonston strain)인 것이나, 이에 한정되는 것은 아니다.In the present invention, the anticancer virus is preferably a measles virus, more preferably MV (Edmonston strain), but is not limited thereto.
홍역 바이러스는 악성 종양 (Msaouel P et al., Curr Pharm Biotechnol. 13(9):1732-41, 2012), CSC (cancer stem cell) (Fang Huang et al., World J Gastroenterol. 22(35):7999-8009, 2016), 폐암 (Zhao D et al., Oncol Rep. 29(1):199-204, 2013; Ong HT et al., J Hepatol. 59(5):999-1006, 2013), 혈액종양 (D Grote et al., Blood. 97(12):3746-54, 2001), 난소암 (Zhou S et al., Cancer Lett. 318(1):14-25, 2012), 골수종 (Peng KW, et al. Blood. 98(7):2002-2007, 2001), 유방암 (McDonald CJ et al., Breast Cancer Res Treat. 99(2):177-84, 2006), 뇌암 (Phuong LK, et al., Cancer Res. 63(10):2462-2469, 2003), 직장 결장 선암 (Nicolas Boisgerault et al., Biomed Res Int. 11, 2013), 골육종 (Evidio Domingo Musibay et al., Cancer Gene Ther. 21(11): 483-490, 2014)에 항암효능이 있다고 알려져 있다. 이러한 항암 바이러스를 태운 줄기세포가 암에 특정하게 반응하기 때문에 부작용은 줄어들고 항암 효능은 증가되는 효과를 유도할 수 있으며, 또한 최종 목표인 건강 수명 연장에도 그 역할을 할 수 있을 것이라 생각한다. Measles virus is a malignant tumor (Msaouel P et al., Curr Pharm Biotechnol . 13 (9): 1732-41, 2012), cancer stem cell (CSC) (Fang Huang et al., World J Gastroenterol . 22 (35): 7999-8009, 2016), lung cancer (Zhao D et al., Oncol Rep . 29 (1): 199-204, 2013; Ong HT et al., J Hepatol . 59 (5): 999-1006, 2013), Blood tumors (D Grote et al., Blood . 97 (12): 3746-54, 2001), ovarian cancer (Zhou S et al., Cancer Lett . 318 (1): 14-25, 2012), myeloma (Peng KW, et al. Blood . 98 (7): 2002-2007, 2001) , Breast Cancer (McDonald CJ et al., Breast Cancer Res Treat . 99 (2): 177-84, 2006), brain cancer (Phuong LK, et al., Cancer Res . 63 (10): 2462-2469, 2003), colorectal adenocarcinoma (Nicolas Boisgerault et al., Biomed Res Int . 11, 2013), osteosarcoma (Evidio Domingo Musibay et al., Cancer Gene Ther . 21 (11): 483-490, 2014). Stem cells carrying these anticancer viruses specifically respond to cancer, which can lead to less side effects and increased anticancer efficacy, and may also play a role in prolonging healthy lifespan.
본 발명에 있어서, 상기 (b) 단계의 항암 바이러스는 약독화 홍역백신 바이러스인 것을 특징으로 할 수 있다. 상기 약독화 바이러스는 상용화된 것을 사용할 수도 있으며, 야생형 바이러스 또는 상용화된 약독화 바이러스를 약독화 및 추가 약독화시켜 사용할 수도 있다.In the present invention, the anticancer virus of step (b) may be characterized in that the attenuated measles vaccine virus. The attenuated virus may be a commercially available one, or may be used by attenuating and further attenuating a wild type virus or a commercially available attenuated virus.
본 발명의 일 실시예에서는, GFP가 tagging된 홍역 바이러스인 MVeGFP를 사용하였다.In one embodiment of the present invention, MVeGFP, a measles virus tagged with GFP, was used.
본 발명에 있어서, 상기 (b) 단계의 항암 바이러스는 1 x 105 ~ 1 x 107 TCID50인 것이 바람직하며, 더욱 바람직하게는 1 x 105 ~ 5 x 106 TCID50인 것이고, 가장 바람직하게는 1 x 106 TCID50인 것이나, 이에 한정되는 것은 아니다.In the present invention, the anticancer virus of step (b) is preferably 1 x 10 5 ~ 1 x 10 7 TCID50, more preferably 1 x 10 5 ~ 5 x 10 6 TCID50, most preferably 1 x 10 6 TCID50, but is not limited thereto.
본 발명에 있어서, 상기 (b) 단계의 감염은 36~37℃에서 30분~2시간 동안 수행되는 것이 바람직하며, 더욱 바람직하게는 30분인 것이나, 이에 한정되는 것은 아니다.In the present invention, the infection of step (b) is preferably performed for 30 minutes to 2 hours at 36 ~ 37 ℃, more preferably 30 minutes, but is not limited thereto.
본 발명에 있어서, 상기 천연물은 항암제 또는 항산화제인 것이 바람직하나, 이에 한정되는 것은 아니다.In the present invention, the natural product is preferably an anticancer agent or an antioxidant, but is not limited thereto.
또한, 상기 항암제는 아스피린 또는 파클리탁셀인 것이 바람직하며, 상기 항산화제는 오리나무 추출액 또는 비타민 C인 것이 바람직하나, 이에 한정되는 것은 아니다.In addition, the anticancer agent is preferably aspirin or paclitaxel, and the antioxidant is preferably alder extract or vitamin C, but is not limited thereto.
홍역 바이러스 또는 홍역 바이러스가 감염된 줄기세포에 상기 천연물을 처리하면, 바이러스의 복제시간 (replication time)을 증가시킬 수 있어서 바이러스가 세포를 용해시키는 시간을 연장시킬 수 있다. Treatment of these natural products with stem cells infected with measles virus or measles virus can increase the replication time of the virus, thereby prolonging the time for the virus to lyse the cells.
본 발명의 홍역 바이러스가 감염된 줄기세포는 감염된 바이러스가 세포를 용해시키는 것을 방지하기 위하여 홍역백신 바이러스 자체를 천연물로 전처리하여 줄기세포에 감염시키거나, 또는 홍역 바이러스가 감염된 줄기세포에 천연물을 처리할 수 있으며, 이렇게 약독화된 홍역백신 바이러스가 감염된 줄기세포의 생존기간 및 생존능을 향상시키기 위해, 추가로 UV 광조사를 할 수 있다. Stem cells infected with the measles virus of the present invention can be infected with stem cells by pretreatment of the measles vaccine virus itself with natural products to prevent the infected virus from lysing the cells, or the natural products can be treated to the stem cells infected with measles virus. In addition, in order to improve the survival and viability of stem cells infected with the attenuated measles vaccine virus, UV light irradiation may be further performed.
일반적으로 중간엽 줄기세포에 홍역 바이러스를 1.0 MOI로 감염시킬 경우, 바이러스 감염된 중간엽 줄기세포의 생존율이 80% 이상인 시간은 48시간에 불과하다 (Mader EK et al., Clin Cancer Res. 1;15(23):7246-55, 2009). 하지만, 본 발명의 천연물 처리에 의해 복제시간이 연장된 홍역 바이러스를 줄기세포에 감염시키거나, 또는 홍역 바이러스가 감염된 줄기세포에 천연물 처리를 할 경우, 중간엽 줄기세포의 생존율이 80% 이상인 시간이 7일 이상으로 증가하였다.In general, when the mesenchymal stem cells are infected with measles virus at 1.0 MOI, the time when the virus-infected mesenchymal stem cells survive more than 80% is only 48 hours (Mader EK et al., Clin Cancer Res . 1; 15; (23): 7246-55, 2009). However, when infecting stem cells with measles virus whose replication time is extended by the natural product treatment of the present invention, or when natural products are treated with the measles virus-infected stem cells, the time when the survival rate of mesenchymal stem cells is 80% or more is increased. Increased over 7 days.
본 발명의 일 실시예에서는, 항암 바이러스가 도입된 중간엽 줄기세포인 "바이로스템"의 세포 생존율을 감염 직후와 배양 후에 확인하였다. 그 결과, 바이로스템의 세포 생존율은 추가 배양하지 않는 것이 더 우수하였다.In one embodiment of the present invention, the cell viability of "Byrostem", which is a mesenchymal stem cell into which anticancer virus was introduced, was confirmed immediately after infection and after culture. As a result, cell viability of Virostem was better not further cultured.
따라서, 본 발명에 있어서, 상기 (d) 단계의 중간엽 줄기세포는 항암 바이러스가 도입된 후 추가로 배양을 하지 않는 것이 바람직하나, 이에 한정되는 것은 아니다.Therefore, in the present invention, the mesenchymal stem cells of the step (d) is preferably not further cultured after the anticancer virus is introduced, but is not limited thereto.
본 발명은 다른 관점에서, 상기 방법으로 제조된 중간엽 줄기세포를 유효성분으로 함유하는 암 치료용 세포치료제에 관한 것이다.In another aspect, the present invention relates to a cell therapy agent for treating cancer containing mesenchymal stem cells prepared by the above method as an active ingredient.
본 발명은 또 다른 관점에서, 상기 방법으로 제조된 중간엽 줄기세포를 개체에 투여하는 단계를 포함하는 암 치료방법에 관한 것이다.In another aspect, the present invention relates to a method for treating cancer comprising administering to the subject a mesenchymal stem cell prepared by the above method.
본 발명은 또 다른 관점에서, 암 치료에 사용하기 위한 상기 방법으로 제조된 중간엽 줄기세포에 관한 것이다.In another aspect, the present invention relates to mesenchymal stem cells prepared by the above method for use in the treatment of cancer.
본 발명은 또 다른 관점에서, 암 치료에 사용하기 위한 상기 방법으로 제조된 중간엽 줄기세포를 포함하는 세포치료제에 관한 것이다.In another aspect, the present invention relates to a cell therapeutic agent comprising mesenchymal stem cells prepared by the above method for use in the treatment of cancer.
본 발명은 또 다른 관점에서, 암 치료용 약제의 제조를 위한 상기 방법으로 제조된 중간엽 줄기세포에 관한 것이다.In another aspect, the present invention relates to mesenchymal stem cells prepared by the above method for the manufacture of a medicament for the treatment of cancer.
일반적으로 중간엽 줄기세포에 홍역 바이러스를 1.0 MOI로 감염시킬 경우, 바이러스 감염된 중간엽 줄기세포의 생존율이 80% 이상인 시간은 48시간에 불과하다 (Mader EK et al., Clin Cancer Res. 1;15(23):7246-55, 2009). 즉, 바이러스가 줄기세포를 용해시키기 때문에 바이러스가 감염된 줄기세포의 생존기간이 짧다. 이런 이유로 항암 바이러스에 감염된 줄기세포를 세포치료제로 완제품하여 제품화하는 것이 불가능하다.In general, when the mesenchymal stem cells are infected with measles virus at 1.0 MOI, the time when the virus-infected mesenchymal stem cells survive more than 80% is only 48 hours (Mader EK et al., Clin Cancer Res . 1; 15; (23): 7246-55, 2009). That is, since the virus dissolves the stem cells, the survival time of the virus-infected stem cells is short. For this reason, it is impossible to commercialize stem cells infected with an anticancer virus as a finished product for cell therapy.
하지만, 본 발명의 천연물 처리에 의해 복제시간이 연장된 홍역 바이러스를 줄기세포에 감염시키거나, 또는 홍역 바이러스가 감염된 줄기세포에 천연물을 처리할 경우, 항암 바이러스를 함유하는 중간엽 줄기세포의 생존율이 80% 이상인 시간이 7일 이상으로 증가하였다. 따라서, 본 발명의 방법으로 제조된 항암 줄기세포치료제는 실질적 제품형태로 투여가 가능하다.However, when infecting the measles virus prolonged replication time by the natural products of the present invention to the stem cells, or when treated with natural products to the stem cells infected with measles virus, the survival rate of the mesenchymal stem cells containing the anticancer virus More than 80% of the time increased to more than seven days. Thus, the anticancer stem cell therapy prepared by the method of the present invention can be administered in the form of a substantial product.
특히, 본 발명의 항암 줄기세포치료제는 펠렛 상태의 줄기세포에 항암 바이러스를 감염시켜 원심분리한 다음, 추가로 배양 단계를 거치지 않고 제품화할 수 있다.In particular, the anticancer stem cell therapeutic agent of the present invention can be produced without infecting the anticancer virus to the stem cells in the pellet state and then centrifuged, and further undergoing a culture step.
본 발명에 있어서, 상기 암은 폐암, 혈액종양. 난소암, 골수종, 유방암, 뇌암, 직장암, 결장암, 직결장 선암, 골육종 또는 암줄기세포인 것이 바람직하나, 이에 한정되는 것은 아니다.In the present invention, the cancer is lung cancer, blood tumor. Ovarian cancer, myeloma, breast cancer, brain cancer, rectal cancer, colon cancer, colorectal adenocarcinoma, osteosarcoma or cancer stem cells, but is not limited thereto.
본 발명의 일 실시예에서는 유방암 세포에서 항암 바이러스의 항암 효과가 우수하였으며, 에스트로겐 의존성 유방암 세포주 (MCF-7) 및 에스트로겐 비의존성 유방암 세포주 (MDA-MB-231) 모두 효과가 동일하였다.In an embodiment of the present invention, the anticancer effect of the anticancer virus was excellent in breast cancer cells, and the effects were the same for both the estrogen-dependent breast cancer cell line (MCF-7) and the estrogen-independent breast cancer cell line (MDA-MB-231).
본 발명에 있어서, 상기 중간엽 줄기세포는 지방, 자궁, 골수, 근육, 태반, 제대혈, 소변, 모낭 및 피부로 구성된 군에서 선택된 조직 유래인 것을 특징으로 할 수 있다.In the present invention, the mesenchymal stem cells may be derived from a tissue selected from the group consisting of fat, uterus, bone marrow, muscle, placenta, umbilical cord blood, urine, hair follicles and skin.
"암"이란 제어되지 않은 세포성장으로 특징지어지며, 이러한 비정상적인 세포성장에 의해 종양(tumor)이라고 불리는 세포 덩어리가 형성되어 주위의 조직으로 침투하고 심한 경우에는 신체의 다른 기관으로 전이되기도 한다."Cancer" is characterized by uncontrolled cell growth, which results in the formation of cell masses called tumors that infiltrate surrounding tissues and, in severe cases, metastasize to other organs of the body.
"항암"이라는 용어는 암 질환의 치료 즉, 암세포 또는 암줄기세포의 증식을 억제하거나 암세포 또는 암줄기세포를 죽이는 것뿐만 아니라, 암 질환의 예방, 즉 암 발병 이전에 암에 대한 저항력을 높이는 것도 함께 포함하는 것으로 해석된다. 따라서, 본 명세서에서 "암 예방 또는 치료" 또는 "암의 증식 억제"라는 용어와 '항암'이라는 용어는 혼재하여 사용되었다.The term "anticancer" includes not only the treatment of cancer diseases, ie, inhibiting the proliferation of cancer cells or cancer stem cells, or killing cancer cells or cancer stem cells, but also the prevention of cancer diseases, that is, increasing resistance to cancer before the onset of cancer. It is interpreted as. Therefore, the terms "cancer prevention or treatment" or "inhibition of cancer proliferation" and the term "anticancer" are used interchangeably herein.
본 발명에 있어서, "암세포"는 정상세포의 증식 및 생장기전에 유전적인 변형으로 인해 비정상적인 세포생장을 하며, 전이라고 지칭할 수 있는 공격적인 타 기관 이동성을 지니는 세포를 포함한다. 또한, "암줄기세포"는 종양 내 존재하는 것으로 알려져 있으며 정상 줄기세포의 유전적인 정보의 비정상적인 전이로 인해 발생하는 것으로 생각되고 있다. 암줄기세포는 그들이 생존하기 위한 미세환경, 니쉬(niche)의 존재로 인해 유지, 증식되며 주변에 존재하는 정상 세포, 면역관련 세포 또는 분화된 암세포가 이들 특성 유지와 증식에 영향을 미치는 것으로 알려져 있다. In the present invention, "cancer cells" include cells with abnormal cell growth due to genetic modification during normal cell proliferation and growth, and cells with aggressive other organ mobility, which may be referred to as an ex. In addition, "cancer stem cells" are known to exist in tumors and are thought to be caused by abnormal metastasis of genetic information of normal stem cells. Cancer stem cells are maintained and proliferated due to the presence of a microenvironment, a niche for their survival, and it is known that normal cells, immune-related cells, or differentiated cancer cells existing around them affect these properties.
본 발명에서 용어 "세포치료제 주사제품" 또는 "세포치료제"란 조직의 결함을 치료하기 위해 줄기세포를 함유하여 비경구투여, 즉 주사의 형태로 결함부위 또는 그 인접부위에 주사되어, 결함을 교정할 수 있는 약학 조성물을 의미한다.In the present invention, the term "cell therapeutic injection product" or "cell therapeutic agent" refers to a parenteral administration containing stem cells for injection of a defect of a tissue, that is, injected into or near a defect in the form of an injection to correct a defect. It means a pharmaceutical composition that can be.
"치료하는"이란 용어는, 달리 언급되지 않는 한, 상기 용어가 적용되는 질환 또는 질병, 또는 상기 질환 또는 질병의 하나 이상의 증상을 역전시키거나, 완화시키거나, 그 진행을 억제하거나, 또는 예방하는 것을 의미한다.The term “treating”, unless stated otherwise, reverses, alleviates, inhibits, or prevents the disease or condition to which the term applies, or one or more symptoms of the disease or condition, Means that.
본원에서 사용된 바와 같이, "치료"란 용어는 "치료하는"이 상기와 같이 정의될 때 치료하는 행위를 말한다.As used herein, the term "treatment" refers to the act of treating when "treating" is defined as above.
따라서, 포유 동물에 있어서 암의 "치료" 또는 "치료요법"은 하기의 하나 이상을 포함한다:Thus, in mammals, "treatment" or "therapy" of cancer includes one or more of the following:
(1) 암의 성장을 저해함, 즉, 그 발달을 저지시킴,(1) inhibits the growth of cancer, that is, inhibits its development,
(2) 암의 확산을 예방함, 즉, 전이를 예방함,(2) preventing the spread of cancer, ie preventing metastasis,
(3) 암을 경감시킴, 즉, 암의 퇴행을 야기시킴,(3) relieve cancer, ie cause cancer to regress,
(4) 암의 재발을 예방함, 및(4) prevent the recurrence of cancer, and
(5) 암의 증상을 완화함(palliating).(5) Palliating the symptoms of cancer.
암은 수술, 방사선 및 화학요법으로 치료를 하더라도 많은 경우에 근본적인 치유가 되지 못하고 환자에게 고통을 주며 궁극적으로는 죽음에 이르게 하는 난치성 만성질환이다. 지난 수십 년간의 수술요법, 화학요법(항암제치료), 방사선요법 등은 많은 발전에도 불구하고 암에 대한 궁극적인 해결책이 되지 못하고 있다.Cancer is an intractable chronic disease that, even if treated with surgery, radiation, and chemotherapy, in many cases does not heal fundamentally, suffers the patient, and ultimately leads to death. Surgery, chemotherapy and radiation therapy over the last few decades have not been the ultimate solution to cancer despite many advances.
본 발명에서는 약독화 홍역 바이러스에 항암제 또는 항산화제를 처리하여 복제시간 (replication time)을 연장시킨 후 줄기세포에 감염시키거나, 또는 약독화 홍역 바이러스를 줄기세포에 감염시켜 항암제 또는 항산화제를 처리함으로써 바이러스 복제시간을 연장시키고, 바이러스가 줄기세포를 용해시키는 것을 또한 억제하였다. 따라서, 본 발명의 방법으로 제조된 홍역 바이러스가 감염된 줄기세포는 80% 생존율을 나타내는 기간이 7일 이상으로 현저히 증가되어, 실질적 줄기세포치료제 제품형태로 투여가 가능하다. 이러한 항암 바이러스와 줄기세포를 접목시킨 항암 줄기세포치료제를 본 발명자들은 "바이로스템 (ViroSTEM)"이라고 명명하였다.In the present invention, by treating the attenuated measles virus with an anticancer agent or an antioxidant to prolong replication time and then infecting the stem cells, or by attenuating the attenuated measles virus to the stem cells to treat an anticancer or antioxidant agent. Prolonged viral replication time and also inhibited the virus from lysing stem cells. Therefore, the stem cells infected with the measles virus prepared by the method of the present invention is significantly increased to 7 days or more, showing a 80% survival rate, and can be administered in the form of a substantial stem cell therapeutic product. The present inventors named the anticancer stem cell therapy combining the anticancer virus and the stem cells as "ViroSTEM."
실시예Example
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .
실시예 1: 인간 지방조직 유래 중간엽 줄기세포의 분리 및 배양Example 1 Isolation and Culture of Human Adipose Tissue-derived Mesenchymal Stem Cells
지방흡입술(Liposuction)에 의해 복부지방으로부터 얻어진 인간 지방조직을 분리하여 PBS로 세척하였다. 조직을 잘게 자른 후 collagenase type1 (1mg/ml)을 첨가한 DMEM 배지를 이용하여 37℃에서 2시간 동안 digestion하였다. PBS로 세척 후 1000rpm에서 5분간 원심분리 하였다. 상층액은 suction하고 바닥에 남은 펠렛은 PBS로 세척한 후, 1000rpm으로 5분간 원심분리하였다. 100㎛ mesh에 필터링하여 debris를 제거하고 PBS로 세척한 후, 10% FBS, 2mM NAC, 0.2mM ascorbic acid를 함유하는 DMEM 배지에 배양하였다.Human adipose tissue obtained from abdominal fat by liposuction was isolated and washed with PBS. The tissue was chopped and digested for 2 hours at 37 ° C using DMEM medium containing collagenase type1 (1 mg / ml). After washing with PBS and centrifuged for 5 minutes at 1000rpm. The supernatant was suctioned and the pellet remaining on the bottom was washed with PBS, and then centrifuged at 1000 rpm for 5 minutes. Debris was removed by filtering on a 100 μm mesh, washed with PBS, and then cultured in DMEM medium containing 10% FBS, 2 mM NAC, and 0.2 mM ascorbic acid.
하룻밤 지난 후 부착되지 않은 세포들은 PBS로 세척하고, RKCM-N 배지인 5% FBS, 2mM NAC, 0.2mM ascorbic acid, 0.09mM calcium, 5ng/ml rEGF, 5㎍/ml 인슐린 및 74ng/ml Hydrocortisone를 함유한 Keratinocyte-SFM 배지를 2일마다 교체하면서 계대배양하여 지방조직 유래 다분화능 중간엽 줄기세포를 분리하였다.After overnight, non-attached cells were washed with PBS and washed with RKCM-N medium, 5% FBS, 2 mM NAC, 0.2 mM ascorbic acid, 0.09 mM calcium, 5 ng / ml rEGF, 5 μg / ml insulin and 74 ng / ml Hydrocortisone. Adipose tissue-derived multipotent mesenchymal stem cells were isolated by subculture with alternating Keratinocyte-SFM medium every two days.
상기와 같이 분리한 지방조직 유래 중간엽 줄기세포는 비타민 C를 함유하는 배지에서 배양한 줄기세포로, 즉 비타민 C 전처리 중간엽 줄기세포이다.Adipose tissue-derived mesenchymal stem cells isolated as described above are stem cells cultured in a medium containing vitamin C, that is, vitamin C pre-treated mesenchymal stem cells.
실시예 2: 아스피린 함유 배지에서 중간엽 줄기세포의 배양Example 2: Cultivation of Mesenchymal Stem Cells in Aspirin-Containing Medium
실시예 1의 비타민 C를 함유하는 배지에서 배양한 줄기세포를 96-웰 세포배양 플레이트에 1x104 cells/plate 농도로 접종한 후, 하룻밤 배양하여 부착 및 안정화시킨다. 그 다음, RKCM-N 배지인 5% FBS, 2mM NAC, 0.2mM ascorbic acid, 0.09mM calcium, 5ng/ml rEGF, 5㎍/ml 인슐린 및 74ng/ml Hydrocortisone를 함유한 Keratinocyte-SFM에 아스피린을 0.5mM의 농도로 첨가한 배지로 교환 후, 24시간 동안 배양하였다. Stem cells cultured in a medium containing vitamin C of Example 1 were inoculated in a 96-well cell culture plate at a concentration of 1 × 10 4 cells / plate, and then cultured overnight to attach and stabilize. Next, 0.5 mM of aspirin was added to Keratinocyte-SFM containing RKCM-N medium containing 5% FBS, 2 mM NAC, 0.2 mM ascorbic acid, 0.09 mM calcium, 5 ng / ml rEGF, 5 ug / ml insulin and 74 ng / ml Hydrocortisone. After exchange with a medium added at the concentration of and incubated for 24 hours.
상기와 같이 배양하여 수득한 지방조직 유래 중간엽 줄기세포는 Angel-Stem Cell (WO/2018/021879)로 명명하였으며, 암세포와의 직접 공배양 및 간접 공배양을 통해 항암 효과가 우수한 것을 확인할 수 있었다.Adipose tissue-derived mesenchymal stem cells obtained by culturing as described above were named Angel-Stem Cells (WO / 2018/021879) and were found to have excellent anticancer effects through direct and indirect co-culture with cancer cells. .
실시예 3: 줄기세포에 홍역 바이러스 감염 확인Example 3 Identification of Measles Virus Infection on Stem Cells
실시예 1 또는 실시예 2에서 분리한 지방 유래 줄기세포에 MVeGFP (GFP가 tagging된 홍역 바이러스)의 감염을 확인하였다. Limanis사에서 판매하는 Measles-GFP를 구매한 후 MVeGFP를 증폭하고 titer를 계산하여 MVeGFP를 정량하고, 본 발명의 배양 field에 맞는 증폭 방법 및 titer 확인 방법을 구축하였다 Infection of adipose derived stem cells isolated in Example 1 or Example 2 with MVeGFP (GFP tagged measles virus) was confirmed. After purchasing Measles-GFP sold by Limanis, MVeGFP was amplified and titer was calculated to quantify MVeGFP, and the amplification method and titer identification method were established for the culture field of the present invention.
구체적으로 살펴보면, 펠렛 (Pellet) 또는 플라스크 (flask) 상태의 지방 유래 줄기세포 (2X105 세포)에 홍역백신 바이러스(106 TCID50)를 37℃에서 30분마다 gently shake 하면서 감염시켰다. 37℃에서 배양하여 얻어진 홍역백신 바이러스의 줄기세포 감염여부를 형광 현미경으로 확인하였다 (도 1). Specifically, the measles vaccine virus (10 6 TCID50) was infected by gently shaking every 30 minutes at 37 ° C in adipose derived stem cells (2X10 5 cells) in pellet or flask state. Stem cell infection of the measles vaccine virus obtained by culturing at 37 ℃ was confirmed by fluorescence microscopy (Fig. 1).
실시예 4: 암 세포주에서 홍역 바이러스의 사멸 능력 확인Example 4 Confirmation of the Killing Capacity of Measles Virus in Cancer Cell Lines
다양한 암 세포주에서 홍역 바이러스의 사멸 능력은 CD46, CD150, nectin-4로 측정할 수 있다. 3개의 표면 마커의 발현이 있으면 홍역 바이러스로 인한 사멸 능력이 높아진다고 간접적으로 확인할 수 있으므로, FACS assay로 각 암 세포주의 표면 마커를 확인하였다 (도 2). The killing ability of measles virus in various cancer cell lines can be measured by CD46, CD150 and nectin-4. Since the expression of three surface markers can be indirectly confirmed that the ability to kill due to measles virus is increased, the surface markers of each cancer cell line were confirmed by FACS assay (FIG. 2).
그 다음, CPE (cytopathic effect)로 암세포 사멸을 확인하였다 (도 3). CPE는 암세포를 24-well plate에 2X105 cells/well로 도말한 다음 TCIE50 홍역 바이러스로 2시간 동안 감염시킨 후, 바이러스 접종물을 제거하였다. 세포 배양배지 1ml을 추가하여 96시간 동안 감염시킨 다음 PBS로 2회 세척하고, 남아있는 세포를 4% 포름알데하이드로 10분간 상온에서 고정하였다. 다시 PBS로 세척 후, 0.1% crystal violet solubilized in 2% EtOH-DW로 염색하고 DW로 2회 세척한 다음 air dry하고 사진 촬영하였다.Next, cancer cell death was confirmed by the cytopathic effect (CPE) (FIG. 3). CPE plated cancer cells in a 24-well plate at 2 × 10 5 cells / well, then infected with TCIE50 measles virus for 2 hours, and then removed the virus inoculum. 1 ml of cell culture medium was added and infected for 96 hours, washed twice with PBS, and the remaining cells were fixed at room temperature for 10 minutes with 4% formaldehyde. After washing with PBS again, stained with 0.1% crystal violet solubilized in 2% EtOH-DW, washed twice with DW, air dried and photographed.
암 세포주에 MVeGFP를 감염시켜 항암효과 확률이 높은 암 세포주로 유방암 세포주를 선정하고, 1000TCID50/ml에서의 유방암 세포주 (MCF-7: 에스트로겐 의존성 세포주 및 MDA-MB-231: 에스트로겐 비의존성 세포주) CPE로 MVeGFP의 유방암 사멸 능력을 확인하였다 (도 4). 그 결과, 1000TCID50/ml로 진행하였을 때 양성 대조군은 2일차부터 CPE를 나타내고 3일차에 거의 80% 이상이 CPE를 형성하였고, MCF-7 및 MDA-MB-231 모두 3일차부터 CPE를 형성하기 시작하였다. Breast cancer cell lines were selected as cancer cell lines with high anti-cancer effect by infecting cancer cell lines with MVeGFP, and breast cancer cell lines (MCF-7: estrogen-dependent cell line and MDA-MB-231: estrogen-independent cell line) at 1000TCID50 / ml. Breast cancer killing ability of MVeGFP was confirmed (FIG. 4). As a result, when progressed to 1000TCID50 / ml, the positive control group showed CPE from day 2 and nearly 80% of CPE was formed on day 3, and both MCF-7 and MDA-MB-231 started to form CPE from day 3 It was.
또한, 유방암세포에 MVeGFP를 감염시켜 감염정도를 정량적으로 확인할 수 있는 방법을 FACS assay로 구축하였다 (도 5).In addition, MVeGFP infection in breast cancer cells was established by FACS assay to quantitatively determine the extent of infection (FIG. 5).
실시예 5: 바이로스템의 감염 스펙 확인Example 5 Confirmation of Infection Specification of Virostem
실시예 3에서 제조한 홍역 바이러스가 도입된 줄기세포를 "바이로스템 (ViroSTEM)"으로 명명하였다. 홍역 바이러스는 홍역백신 바이러스를 사용하거나, 홍역백신 바이러스를 다시 한번 약독화시킨 다음 사용하였다. Stem cells into which the measles virus prepared in Example 3 were introduced were named "ViroSTEM". Measles virus was used after measles vaccine virus or after attenuating the measles virus virus once again.
사람에서 안전한 홍역백신 바이러스 및 줄기세포 수 (Myo clinic의 세포수 및 홍역 바이러스 농도 참고)를 토대로 감염 환경, 감염 시간 및 농도 시험을 통해 바이로스템 (ViroSTEM)의 감염 스펙을 설정하였다 (표 1). 줄기세포에 홍역 바이러스 감염 정도를 높이고자 감염 환경을 다양하게 변화시켜 확인하였다.Based on the safe measles vaccine virus and stem cell numbers in humans (see Myo clinic cell count and measles virus concentration), the infection specification of ViroSTEM was established through the infection environment, infection time and concentration test (Table 1). . To increase the degree of measles virus infection in stem cells, the infection environment was changed in various ways.
먼저, 세포를 플라스크 또는 펠렛 상태에서 감염을 진행한 후, CPE 정도를 확인하였다. 그 결과, 펠렛 상태의 세포 환경에서 감염 정도가 높았으며 (도 6), 감염 후 원심분리로 물리적 변화를 주어 감염 정도를 높일 수 있었다.First, the cells were infected in a flask or pellet state, and then the degree of CPE was confirmed. As a result, the degree of infection was high in the cellular environment of the pellet state (FIG. 6), and the degree of infection was increased by giving a physical change by centrifugation after infection.
그 다음 펠렛 상태의 세포 환경에서 감염 시간 및 농도를 다르게 한 후, CPE 정도를 확인하였다 (도 7). 그 결과, 중간엽 줄기세포 수는 2 x 105, 홍역 바이러스 농도는 1 x 106 TCID50에서 가장 감염 정도가 우수한 것을 알 수 있었다.Then, after varying the infection time and concentration in the cell environment of the pellet state, the degree of CPE was confirmed (FIG. 7). As a result, the number of mesenchymal stem cells was 2 x 10 5 , measles virus concentration was 1 x 10 6 TCID50 was the most excellent degree of infection.
또한, 감염 시간을 다르게 하여 홍역 바이러스를 감염시킨 줄기세포의 생존율을 감염 직후와 감염 4일 후에 분석하였다. 그 결과, 감염 시간은 30분이 가장 효과적이였으며, 홍역 바이러스 감염 후 배양 단계를 거치지 않는 것이 감염 정도가 우수하였다 (도 8a 및 8b).In addition, survival rates of stem cells infected with measles virus at different infection times were analyzed immediately after and 4 days after infection. As a result, the infection time was the most effective 30 minutes, it was excellent in the degree of infection that does not go through the culture step after the measles virus infection (Figs. 8a and 8b).
이렇게 설정된 바이로스템 (ViroSTEM)의 감염 스펙은 하기 표 1에 나타내었다.The infection specification of ViroSTEM thus set is shown in Table 1 below.
Figure PCTKR2019007276-appb-T000001
Figure PCTKR2019007276-appb-T000001
실시예 6: 천연물 처리에 의한 홍역 바이러스가 감염된 줄기세포의 생존시간 연장Example 6: Extending the survival time of stem cells infected with measles virus by natural product treatment
홍역 바이러스가 도입된 중간엽 줄기세포인 바이로스템은 항산화제인 비타민 C 또는 아스피린을 단독 또는 함께 처리하여 생존 기간을 증가시켰다. Virostem, a mesenchymal stem cell incorporating measles virus, increased the survival by treating vitamin C or aspirin alone or in combination with antioxidants.
그 결과, 항산화제 또는 아스피린이 전혀 처리되지 않은 대조군에 비해 항산화제 또는 아스피린이 처리된 줄기세포의 생존시간이 현저히 증가한 것을 확인하였다. 아무런 처리도 하지 않을 경우, 세포 생존율이 80% 이상인 시간은 48시간 정도이지만, 항산화제 또는 아스피린을 처리하면 홍역 바이러스가 감염된 항암 줄기세포 치료제의 세포 생존율이 80% 이상인 시간은 7일 이상으로 증가하였다. As a result, it was confirmed that the survival time of stem cells treated with antioxidants or aspirin was significantly increased compared to the control group not treated with antioxidants or aspirin at all. In the absence of any treatment, the cell survival rate was more than 80% for 48 hours, but treatment with antioxidants or aspirin increased the cell survival rate of the measles virus-infected anticancer stem cell therapy to more than 7 days. .
실시예 7: 천연물 처리에 의한 홍역 바이러스가 감염된 줄기세포의 항암 효과 향상Example 7: Improvement of anticancer effect of measles virus infected stem cells by natural product treatment
실시예 3에서 제조한 "바이로스템 (ViroSTEM)"에 천연물을 처리한 후 항암효과를 확인하였다.The anti-cancer effect was confirmed after treating the natural product to "ViroSTEM" prepared in Example 3.
다양한 암세포 또는 암줄기세포가 각각 부착 배양된 세포배양 접시에 "바이로스템 (ViroSTEM)"을 추가 접종하여 암세포에 대한 "바이로스템 (ViroSTEM)"의 항암 효과를 확인하였다.The anti-cancer effect of "ViroSTEM" on cancer cells was confirmed by additionally inoculating "ViroSTEM" in a cell culture dish in which various cancer cells or cancer stem cells were attached and cultured.
그 결과, "바이로스템 (ViroSTEM)"의 세포 생존율이 80% 이상 유지되는 기간인 1시간 내지 7일 동안의 항암효과는 천연물이 처리되지 않은 대조군 줄기세포 치료제에 비해 우수한 것을 확인할 수 있었다.As a result, it was confirmed that the anticancer effect during 1 hour to 7 days, which is a period in which cell viability of "ViroSTEM" is maintained at 80% or more, was superior to that of the control stem cell treatment not treated with natural products.
실시예 8: 바이로스템의 안정성 (stability)Example 8 Stability of Virostem
항암 줄기세포 Angel-Stem Cell에 홍역 바이러스를 감염시킨 "바이로스템 (ViroSTEM)"의 안정성을 시험하고자, 줄기세포에 MVeGFP를 2시간 감염시킨 후 냉장 보관하였다. 냉장 상태에서 바이로스템 내의 홍역 바이러스가 얼마나 유지되는지를 qPCR로 정량하였다.To test the stability of "ViroSTEM" infected with measles virus in the anti-cancer stem cell Angel-Stem Cell, stem cells were infected with MVeGFP for 2 hours and then refrigerated. The qPCR quantified how much measles virus remained in virostem in refrigerated condition.
그 결과, 바이로스템 (ViroSTEM)은 냉장 상태에서 1주일까지 바이로스템 내에서 항암 바이러스가 유지되고 있는 것을 확인할 수 있었다 (도 9).As a result, ViroSTEM was confirmed that the anti-cancer virus is maintained in the virostem for one week in the refrigerated state (Fig. 9).
본 발명에 따른 항암 바이러스를 함유하는 중간엽 줄기세포의 제조방법은 천연물질 처리를 통해 항암 바이러스의 복제시간은 연장시키고, 바이러스가 줄기세포를 용해시키는 것을 방지함으로써, 줄기세포의 생존능 및 생존기간을 향상시킨 활성이 우수한 항암 바이러스를 함유한 줄기세포를 제조할 수 있고, 이렇게 제조된 항암 줄기세포치료제는 유효기간이 현저히 향상되어 의학 및 산업적으로 매우 유용하다.The method for producing mesenchymal stem cells containing the anticancer virus according to the present invention extends the replication time of the anticancer virus through the treatment of natural substances and prevents the virus from dissolving the stem cells, thereby improving the viability and survival of the stem cells. Stem cells containing the anticancer virus with improved activity can be prepared, and the anticancer stem cell therapy thus prepared has a markedly improved shelf life, which is very useful in medicine and industry.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 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 의하여 정의된다고 할 것이다.Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (18)

  1. 다음 단계를 포함하는 세포 생존능이 향상된 항암 바이러스를 함유하는 중간엽 줄기세포의 제조방법:Method for producing mesenchymal stem cells containing anticancer virus with improved cell viability, including the following steps:
    (a) 중간엽 줄기세포를 펠렛 (pellet) 상태로 제조하는 단계;(a) preparing mesenchymal stem cells in a pellet state;
    (b) 상기 펠렛 상태의 중간엽 줄기세포에 항암 바이러스를 감염시키는 단계;(b) infecting an anticancer virus to the mesenchymal stem cells in the pellet state;
    (c) 상기 항암 바이러스가 감염된 중간엽 줄기세포를 원심분리하는 단계; 및(c) centrifuging the mesenchymal stem cells infected with the anticancer virus; And
    (d) 상기 항암 바이러스가 도입된 중간엽 줄기세포를 수득하는 단계.(d) obtaining mesenchymal stem cells into which the anticancer virus is introduced.
  2. 제1항에 있어서, 상기 (a) 단계의 중간엽 줄기세포는 아스피린을 함유하는 배지에서 배양된 것을 특징으로 하는 제조방법.The method of claim 1, wherein the mesenchymal stem cells of step (a) are cultured in a medium containing aspirin.
  3. 제2항에 있어서, 상기 배지는 비타민 C을 추가로 함유하는 것을 특징으로 하는 제조방법.The method of claim 2, wherein the medium further contains vitamin C.
  4. 제1항에 있어서, 상기 (a) 단계의 중간엽 줄기세포는 비타민 C로 전처리된 것을 특징으로 하는 제조방법.The method of claim 1, wherein the mesenchymal stem cells of step (a) are pretreated with vitamin C.
  5. 제1항에 있어서, 상기 (b) 단계의 중간엽 줄기세포는 1 x 105 ~ 1 x 106 세포인 것을 특징으로 하는 제조방법.The method according to claim 1, wherein the mesenchymal stem cells of step (b) are 1 x 10 5 to 1 x 10 6 cells.
  6. 제1항에 있어서, 상기 중간엽 줄기세포는 지방, 자궁, 골수, 근육, 태반, 제대혈, 소변, 모낭 및 피부로 구성된 군에서 선택된 조직 유래인 것을 특징으로 하는 제조방법.The method of claim 1, wherein the mesenchymal stem cells are derived from a tissue selected from the group consisting of fat, uterus, bone marrow, muscle, placenta, umbilical cord blood, urine, hair follicles and skin.
  7. 제1항에 있어서, 상기 항암 바이러스는 홍역 바이러스 (measles virus)인 것을 특징으로 하는 제조방법.The method of claim 1, wherein the anticancer virus is a measles virus.
  8. 제1항에 있어서, 상기 (b) 단계의 항암 바이러스는 1 x 105 ~ 1 x 107 TCID50인 것을 특징으로 하는 제조방법.The method according to claim 1, wherein the anticancer virus of step (b) is 1 x 10 5 to 1 x 10 7 TCID50.
  9. 제1항에 있어서, 상기 (b) 단계의 항암 바이러스는 약독화 항암 바이러스인 것을 특징으로 하는 제조방법.The method of claim 1, wherein the anticancer virus of step (b) is an attenuated anticancer virus.
  10. 제1항에 있어서, 상기 (b) 단계의 감염은 36~37℃에서 30분 동안 수행되는 것을 특징으로 하는 제조방법.The method of claim 1, wherein the infection of step (b) is carried out for 30 minutes at 36 ~ 37 ℃.
  11. 제1항에 있어서, 상기 (c) 단계의 항암 바이러스가 감염된 중간엽 줄기세포에 천연물을 처리하는 것을 특징으로 하는 제조방법.The method according to claim 1, wherein the anticancer virus of step (c) is treated with natural products to the mesenchymal stem cells.
  12. 제11항에 있어서, 상기 천연물은 항암제 또는 항산화제인 것을 특징으로 하는 제조방법.The method of claim 11, wherein the natural product is an anticancer agent or an antioxidant.
  13. 제12항에 있어서, 상기 항암제는 아스피린 또는 파클리탁셀인 것을 특징으로 하는 제조방법.The method of claim 12, wherein the anticancer agent is aspirin or paclitaxel.
  14. 제12항에 있어서, 상기 항산화제는 오리나무 추출액 또는 비타민 C인 것을 특징으로 하는 제조방법.The method according to claim 12, wherein the antioxidant is alder extract or vitamin C.
  15. 제1항에 있어서, 상기 (d) 단계의 중간엽 줄기세포는 항암 바이러스가 도입된 후 추가로 배양을 하지 않는 것을 특징으로 하는 제조방법.The method of claim 1, wherein the mesenchymal stem cells of step (d) are not cultured further after the anticancer virus is introduced.
  16. 제1항의 방법으로 제조된 중간엽 줄기세포를 유효성분으로 함유하는 암 치료용 세포치료제.Cell therapy for cancer treatment containing mesenchymal stem cells prepared by the method of claim 1 as an active ingredient.
  17. 제16항에 있어서, 상기 암은 폐암, 혈액종양. 난소암, 골수종, 유방암, 뇌암, 직장암, 결장암, 직결장 선암, 골육종 또는 암줄기세포인 것을 특징으로 하는 세포치료제.The method of claim 16, wherein the cancer is lung cancer, blood tumor. Cell therapy for ovarian cancer, myeloma, breast cancer, brain cancer, rectal cancer, colon cancer, colorectal adenocarcinoma, osteosarcoma or cancer stem cells.
  18. 제16항에 있어서, 상기 중간엽 줄기세포는 지방, 자궁, 골수, 근육, 태반, 제대혈, 소변, 모낭 및 피부로 구성된 군에서 선택된 조직 유래인 것을 특징으로 하는 세포치료제.The cell therapeutic agent according to claim 16, wherein the mesenchymal stem cells are derived from a tissue selected from the group consisting of fat, uterus, bone marrow, muscle, placenta, umbilical cord blood, urine, hair follicle and skin.
PCT/KR2019/007276 2018-06-15 2019-06-17 Method for enhancing cell viability of anticancer virus-introduced mesenchymal stem cells WO2019240559A1 (en)

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