WO2023096126A1 - Method for screening mdsc inhibitor - Google Patents

Method for screening mdsc inhibitor Download PDF

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WO2023096126A1
WO2023096126A1 PCT/KR2022/014211 KR2022014211W WO2023096126A1 WO 2023096126 A1 WO2023096126 A1 WO 2023096126A1 KR 2022014211 W KR2022014211 W KR 2022014211W WO 2023096126 A1 WO2023096126 A1 WO 2023096126A1
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mdsc
cancer
mitf
activity
mdscs
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Korean (ko)
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임종석
이아람
임지현
이명석
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(주)모임바이오
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • the present invention analyzes the inhibitory effect of microphthalmia-associated transcription factor (MITF) in MDSC by a specific substance to obtain a drug that inhibits MDSC activity. It relates to a method for screening, specifically, to a method for selecting whether a specific substance is an MITF gene expression inhibitor or a MITF protein activity inhibitor, a method for selecting a MDSC activity inhibitory drug, and a composition containing the MITF inhibitor thus selected.
  • MITF microphthalmia-associated transcription factor
  • Myeloid cells originate from hematopoietic stem cells. It is the most abundant hematopoietic stem cell in our body, and is mainly present in bone marrow and lymphoid tissue. Ultimately, they differentiate into macrophages, dendritic cells, and granulocytes, but these do not have a specific hierarchical structure, and myeloid cells with various degrees of differentiation are distributed in various ways specific to tissues and environments. has the characteristics of being
  • BACKGROUND Myeloid-derived suppressor cells are cells having immune response suppression activity among the myeloid cell lineage, and are a cell group that includes a very wide range of undifferentiated myeloid cells. MDSC activation is mediated through several transcription factors, and as a result, reactive oxygen species (ROS) or reactive nitrogen species (RNS) are produced, which inhibits various processes from T cell proliferation to function. It is known to effectively suppress T cells.
  • ROS reactive oxygen species
  • RNS reactive nitrogen species
  • ICI immune checkpoint inhibitors
  • CTL-4 cytotoxic T-lymphocyte-associated protein 4
  • PD-1 programmed cell death protein
  • P-L1 programmed cell death ligand 1
  • TME Tumor Micro-Environment
  • Treg cells, MDSC, TH2 CD4 + cells, cancer-associated fibroblasts (CAFs), and tumor-associated macrophages (TAMs) polarized to M2 are well known as immunosuppressive cells in the TME that affect the efficacy of ICI treatment.
  • CAFs cancer-associated fibroblasts
  • TAMs tumor-associated macrophages
  • MITF microphthalmia-associated transcription factor
  • LXR Liver X receptor
  • Patent Document 0001 International Patent Publication No. 2013-082591
  • Patent Document 0002 International Patent Publication No. 2011-116299
  • Patent Document 0003 Republic of Korea Patent No. 1902355
  • Patent Document 0004 Republic of Korea Patent No. 1826753
  • Non-Patent Document 0001 Gabrilovich DI, et al., Coordinated regulation of myeloid cells by tumours, Nat Rev Immunol. 12(4):253-68 (2012)
  • Non-Patent Document 0002 De Veirman K, et al., Myeloid-derived suppressor cells as therapeutic target in hematological malignancies, Front Oncol. 4:349 (2014)
  • Non-Patent Document 0003 Dufait I, et al., Signal transducer and activator of transcription 3 in myeloid-derived suppressor cells: an opportunity for cancer therapy. Oncotarget. 7(27):42698-42715 (2016)
  • Non-Patent Document 0004 Russell W Jenkins, et al., Mechanisms of resistance to immune checkpoint inhibitors. British Journal of Cancer. 118: 9-16 (2018)
  • Non-Patent Document 0005 Chae YK, et al., Current landscape and future of dual anti-CTLA4 and PD-1/PD-L1 blockade immunotherapy in cancer; Lessons learned from clinical trials with melanoma and non-small cell lung cancer (NSCLC), J Immunother Cancer. 6(1):39 (2018)
  • Non-Patent Document 0006 David Killock, et al., Targeting MDSCs with LXR agonists, Nature Reviews Clinical Oncology. 15: 200-201 (2016)
  • Non-Patent Document 0007 Nam S, et al., Interferon regulatory factor 4 (IRF4) controls myeloid-derived suppressor cell (MDSC) differentiation and function. J Leukoc Biol. 100(6):1273-1284 (2016)
  • Non-Patent Document 0008 Richard P. Tobin, et al., Effects of in vitro ATRA treatment on human MDSC expansion and function. Journal of Clinical Oncology. 35(7):125 (2017)
  • Non-Patent Document 0009 Song YC, et al., Berberine regulates melanin synthesis by activating PI3K/AKT, ERK and GSK3 ⁇ in B16F10 melanoma cells. Int J Mol Med. 35(4):1011-6 (2015)
  • Non-Patent Document 0010 Lim J, et al., Kazinol U inhibits melanogenesis through the inhibition of tyrosinase-related proteins via AMP kinase activation. Br J Pharmacol. 176(5):737-750 (2019)
  • An object of the present invention is to provide a composition for mitigating, treating or preventing a decrease in immune response by bone marrow-derived suppressor cells, containing a gene expression inhibitor of MITF (microphthalmia-associated transcription factor) or a protein activity inhibitor of MITF as an active ingredient. is to do
  • Another object of the present invention is a method for inhibiting bone marrow-derived suppressor cells, comprising administering a gene expression inhibitor of MITF (microphthalmia-associated transcription factor) or a protein activity inhibitor of MITF to a subject in need of inhibition of bone marrow-derived suppressor cells. is to provide
  • Another object of the present invention is to provide a method for selecting a drug that inhibits MDSC activity by analyzing the inhibitory effect of microphthalmia-associated transcription factor (MITF) in MDSC by a specific substance.
  • MITF microphthalmia-associated transcription factor
  • Another object of the present invention is to provide a method for selecting whether a specific substance is an MITF gene expression inhibitor or an MITF protein activity inhibitor, and a MDSC activity inhibitory drug, and a composition containing the MITF inhibitor thus selected.
  • MITF gene expression or MITF protein expression in MDSC by analyzing the level of MITF gene expression or MITF protein expression in MDSC by a specific substance by any one method selected from qRT-PCR, Western blotting and flow cytometry (FACS), the degree of MDSC activity change
  • FACS Western blotting and flow cytometry
  • the specific substance in the method for selecting a drug for inhibiting MDSC activity, may be an MITF gene expression inhibitor or an MITF protein activity inhibitor.
  • the method for screening MDSC activity inhibitory drugs comprises the steps of (a) preparing bone marrow cells; (b) inducing MDSC differentiation and activity from the bone marrow cells; (c) recovering the MDSCs differentiated in step (b) and analyzing the level of MITF gene or MITF protein expression; and (d) selecting a MDSC activity-inhibiting drug from among the specific substances from the analysis result of (c).
  • the bone marrow cells used in the screening method for drugs inhibiting MDSC activity may be bone marrow cells obtained from a subject having a tumor.
  • the method for screening MDSC activity inhibitory drugs is to treat MDSC differentiation inducing factors and the specific substance in the cancer cell condition medium, which is the test group medium in step (b), and include the specific substance as a control medium.
  • the level of expression of the MITF gene or MITF protein can be compared using the untreated medium.
  • the screening method for MDSC activity inhibitory drugs is such that the expression level of the MITF gene or MITF protein in the step (d) is qRT-PCR in the test group compared to the control group in the case of direct MITF inhibitors when qRT-PCR analysis is performed.
  • an additional step of determining the substance as an MDSC activity inhibitory drug can include
  • the screening method for MDSC activity inhibitory drugs is such that the tumor is breast cancer, liver cancer, stomach cancer, colon cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, Colon cancer, small intestine cancer, rectal cancer, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, lymphatic cancer, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, Parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, adenocarcinoma, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary CNS It may be at least one selected from the group consisting of lymph
  • the method for selecting a drug for inhibiting MDSC activity may include the steps of co-cultivating the MDSC and T cells and determining the degree of inhibition of T cell proliferation in order to screen for the drug for inhibiting MDSC activity.
  • the method for screening MDSC activity inhibitory drugs includes antisense nucleotides, aptamers, short hairpin RNA (shRNA), It may be at least one selected from the group consisting of small interfering RNA (siRNA), microRNA (miRNA), and ribozyme.
  • shRNA short hairpin RNA
  • siRNA small interfering RNA
  • miRNA microRNA
  • ribozyme ribozyme
  • the screening method for MDSC activity inhibitory drugs is a group consisting of compounds, peptides, peptide mimetics, substrate analogs, aptamers, and antibodies that specifically bind to the MITF protein activity inhibitor. It may be one or more selected from.
  • the specific substance in the method for screening MDSC activity inhibitory drugs, may be one or more selected from AMPK activity promoters, ML-329, MITF-gRNA cloned vectors, and anti-HIV agents.
  • composition comprising an MDSC activity inhibitory drug selected by a screening method for an MDSC activity inhibitory drug and restoring T cell proliferation.
  • the present invention alleviates the decrease in immune response by myeloid-derived suppressor cells (MDSC) containing a gene expression inhibitor of MITF (microphthalmia-associated transcription factor) or a protein activity inhibitor of MITF as an active ingredient, A composition for treatment or prevention is provided.
  • MDSC myeloid-derived suppressor cells
  • the present invention provides an anticancer adjuvant containing the composition as an active ingredient.
  • the present invention provides a method for inhibiting MDSC, comprising administering a gene expression inhibitor of MITF or a protein activity inhibitor of MITF to a subject in need of MDSC inhibition.
  • the present inventors have made efforts to identify factors that directly affect MDSC activity in order to develop a formulation capable of alleviating the decline in the immune response of MDSC caused by MDSC inhibition and improving the therapeutic effect of anti-cancer immunotherapeutic agents. It was found that MDSCs are activated in the environment and the immune response is lowered, and that MITF is involved in the activation of MDSCs. Therefore, based on the fact that MDSC can be inhibited using a MITF inhibitor, the present invention was completed by selecting a MITF inhibitor among a wide range of substances and revealing that it can be used as a MDSC activity inhibitory drug. From this, it is possible to more effectively identify active ingredients that can be used for alleviating the decline in the immune response of MDSC and for anticancer immunotherapy.
  • the present inventors found that suppressing MITF expression inevitably inhibits MDSC activity, and increasing MITF expression increases MDSC activity. Therefore, by analyzing the level of MITF expression by a specific substance, it is possible to predict the MDSC activation reaction result by a specific substance. In addition, from this, it is possible to select a MITF expression inhibitor that is expected to act most appropriately when administered in vivo to an individual whose MDSCs are activated or whose T cells are confirmed to be suppressed by the MDSC.
  • the patient can select an anti-cancer composition or an anti-cancer adjuvant composition containing the MITF expression inhibitor as an active ingredient that is expected to be most suitable for the patient before direct administration, and whether or not the administration composition is changed even if the treatment is in progress
  • an anti-cancer composition or an anti-cancer adjuvant composition containing the MITF expression inhibitor as an active ingredient that is expected to be most suitable for the patient before direct administration, and whether or not the administration composition is changed even if the treatment is in progress
  • new drug candidates can be considered and MDSC activation response results can be predicted, the basis for selecting the most appropriate MITF inhibitor considering the type or prognosis of cancer is laid.
  • compositions containing the selected MITF inhibitor as an active ingredient it can be usefully used to alleviate the decrease in immune response caused by MDSC and increase the efficiency of anti-cancer immunotherapy.
  • FIG. 1 is a diagram schematically illustrating a method for producing myeloid-derived suppressor cells (MDSC) derived from mouse bone marrow.
  • MDSC myeloid-derived suppressor cells
  • Figure 2 shows the results of MDSC after treating cells isolated from the bone marrow of a mouse with a tumor cell-conditioned medium (TCCM) in which cancer cells are cultured together with GM-CSF, a differentiation-inducing factor for MDSC, according to an embodiment of the present invention. It is a diagram confirming the differentiation (FIG. 2A) and activity (FIG. 2B) changes.
  • TCCM tumor cell-conditioned medium
  • Figure 3 shows the gene (Fig. 3A) and protein (Fig. 3B) of microphthalmia-associated transcription factor (MITF) in MDSC after treating cells isolated from bone marrow of mice with GM-CSF and TCCM according to an embodiment of the present invention. ) is a diagram confirming expression changes.
  • MITF microphthalmia-associated transcription factor
  • Figure 4 shows the differentiation and differentiation of MDSCs by treating cells isolated from the spleen of a tumor-forming mouse (FIG. 4A) or the cells isolated from the bone marrow of a mouse (FIG. 4B) with TCCM along with GM-CSF according to an embodiment of the present invention. After inducing activity, it is a diagram confirming the change in MDSC's T cell proliferation inhibition.
  • FIG. 5 is a diagram confirming changes in MDSC differentiation (FIG. 5A), activity (FIG. 5B), and MITF gene expression (FIG. 5C) in tumor-forming mice.
  • Figure 6 is a drug that induces the activity of MDSC in cells isolated from bone marrow of mice according to an embodiment of the present invention, after treating IL-18 and IL-10 together with GM-CSF for 96 hours (Figure 6A, Figure 6). 6B, FIG. 6E, and FIG. 6F) or after inducing differentiation by treating cells isolated from bone marrow of mice with GM-CSF according to an embodiment of the present invention, and then treating them with IL-18 for 24 hours (FIG. 6C and Fig. 6D), MDSC differentiation (Figs. 6A and 6C), activity, and MITF gene expression (Figs. 6B and 6D) confirming changes.
  • Figure 7 is a drug for inducing MDSC activity in cells isolated from the bone marrow of mice according to an embodiment of the present invention, treated with IL-4 together with GM-CSF, and then MDSC differentiation (Fig. 7A), activity (Fig. 7B) and MITF gene expression (FIG. 7C) confirming changes.
  • Figure 8 shows differentiation of MDSC after treating cells isolated from bone marrow of mice with lipopolysaccharide (LPS) as a drug inducing MDSC activity together with GM-CSF according to an embodiment of the present invention ( Figure 8A). , activity (FIG. 8B) and gene expression of MITF (FIG. 8C) confirming changes.
  • LPS lipopolysaccharide
  • FIG. 9 shows drugs inducing the activity of MDSC in cells isolated from mouse bone marrow according to an embodiment of the present invention, such as Simvastatin (Sim), Lovastatin (Lova), Provastatin (Prav), and Rovastatin. It is a diagram confirming the differentiation change of MDSC after treatment with subastatin (Rosuvastatin, Rosu) or atorvastatin (Atorvastatin, Ator) together with GM-CSF.
  • FIG. 10 shows drugs inducing the activity of MDSCs in cells isolated from mouse bone marrow according to an embodiment of the present invention, such as Simvastatin (Sim), Lovastatin (Lova), Provastatin (Prav), and Rovastatin.
  • Simvastatin Sim
  • Lovastatin Lovastatin
  • Prav Provastatin
  • Rovastatin Rovastatin
  • FIG. 11 shows that cells isolated from bone marrow of mice are treated with GM-CSF to induce differentiation according to an embodiment of the present invention, and then simvastatin (Sim) and lovastatin (Lovastatin, Lova) for 24 hours, and it is a diagram confirming the differentiation change of MDSC.
  • FIG. 12 shows that cells isolated from bone marrow of mice are treated with GM-CSF to induce differentiation according to an embodiment of the present invention, and then Simvastatin (Sim) and Lovastatin (Lovastatin, Lova) for 24 hours, confirming changes in MDSC activity (FIG. 12) and MITF gene expression (FIG. 12).
  • Sim Simvastatin
  • Lovastatin Lovastatin
  • Figure 13 shows the differentiation of MDSC after treatment with ATRA (All-trans retinoic acid) together with GM-CSF as a drug that inhibits the activity of MDSC to cells isolated from bone marrow of mice according to an embodiment of the present invention (Fig. 13A ), activity (FIG. 13B) and gene expression of MITF (FIG. 13C) confirming changes.
  • ATRA All-trans retinoic acid
  • FIG. 14 shows MDSC differentiation (FIG. 14A), MDSC activity, and MITF gene expression (FIG. 14B) after treatment of cells isolated from bone marrow of mice with GM-CSF as an MITF inducer in cells isolated from mouse bone marrow according to an embodiment of the present invention. and 14C), and MDSC suppression of T cell proliferation (FIG. 14D) confirming changes.
  • FIGS. 15A and 15B It is a diagram confirming the gene expression of MITF (FIG. 15C) when the change and 10 ⁇ M Berberine were treated together with GM-CSF.
  • FIG. 16 is a diagram confirming the distribution of MDSC expressing MITF in human lung cancer and head and neck cancer (H&N cancer) tissues.
  • FIG. 17 shows MDSC activity (FIG. 17A) and MITF gene expression (FIG. 17B) after treatment of cells isolated from mouse bone marrow with GM-CSF and ML-329 as an MITF inhibitor according to an embodiment of the present invention. This is the confirmation of the change.
  • FIG. 18 shows MDSC differentiation (FIG. 18A), activity, and MITF gene and protein expression ( 18B and 18C), a diagram confirming changes in MDSC inhibiting T cell proliferation (FIG. 18D).
  • FIG. 19 is a diagram confirming changes in ROS generation produced in MDSC after treatment with GM-CSF and TCCM of MDSC isolated from the spleen of a tumorigenic mouse according to an embodiment of the present invention with ML-329 or berberine (BBR) am.
  • FIG. 20 shows MITF protein expression (FIG. 20A), ROS generation (FIG. 20B), and A diagram confirming changes in T cell proliferation inhibition (FIG. 20C) and MITF protein expression (FIG. 20D) and T cell proliferation inhibition (FIG. 20E) in MDSCs overexpressing MITF according to an embodiment of the present invention. am.
  • FIG. 21 shows the reduction of MITF gene expression (FIG. 21A) after treatment of cells isolated from bone marrow of mice with MITF inhibitor, Nelfinavir, together with GM-CSF and TCCM according to an embodiment of the present invention.
  • Figure 22 is a diagram confirming the activity change of MDSC (FIG. 21B and FIG. 21C).
  • FIG. 22 is MDSC treated with ML-329 along with GM-CSF and TCCM administered to tumor-forming mice according to an embodiment of the present invention.
  • FIG. 22A changes in tumor volume in the tumor-forming mice
  • FIG. 22D changes in MDSC population in the tumor tissues of the tumor-forming mice
  • the present invention provides a method for selecting a drug that inhibits MDSC activity, which predicts and selects the degree of change in MDSC activity by analyzing the level of MITF gene expression or MITF protein expression in MDSC by a specific substance.
  • the present invention provides a composition for alleviating, treating, or preventing a decrease in immune response caused by myeloid-derived suppressor cells (MDSC), containing an MITF gene expression inhibitor or a MITF protein activity inhibitor as an active ingredient. do.
  • MDSC myeloid-derived suppressor cells
  • specific substance refers to a substance expected to change the gene expression or protein activity of MITF in MDSC, and is particularly known as a MITF gene expression inhibitor, a MITF protein activity inhibitor, an MDSC activity inhibitor, and an MDSC activity inhibitor. It includes known or expected substances and salts thereof.
  • the gene expression inhibitor of MITF is antisense nucleotide, aptamer, short hairpin RNA (shRNA), small interfering RNA (siRNA), micro It may be one or more selected from the group consisting of RNA (microRNA, miRNA) and ribozyme, but is not limited thereto.
  • MITF inhibitors include MITF gene expression inhibitors and MITF protein activity inhibitors, and inhibition of MDSC activity means that the effect of suppressing T cell activity shown by MDSCs disappears.
  • antisense nucleotide as defined in Watson-Crick base pairing, interferes with the flow of genetic information from DNA to protein by binding (hybridizing) to complementary nucleotide sequences of DNA, immature-mRNA or mature mRNA.
  • antisense nucleotides for their target sequences makes them exceptionally multifunctional. Since antisense nucleotides are long chains of monomeric units, they can be easily synthesized against the target RNA sequence.
  • a number of recent studies have demonstrated the usefulness of antisense nucleotides as a biochemical means to study target proteins (Rothenberg et al., J. Natl. Cancer Inst., 81:1539-1544, 1999).
  • shRNA refers to a single strand consisting of 50 to 60 nucleotides, and has a stem-loop structure in vivo . That is, shRNA is an RNA sequence that creates a tight hairpin structure to inhibit gene expression through RNA interference (RNAi). Long RNAs of 15-30 nucleotides complementary to both sides of the loop of 5-10 nucleotides are base-paired to form a double-stranded stem. shRNAs are generally transduced into cells via a vector containing a U6 promoter for expression and are usually passed on to daughter cells to allow inheritance of gene repression.
  • RNAi RNA interference
  • shRNA hairpin structure is cleaved by intracellular mechanisms to become siRNA, which then binds to RNA-induced silencing complex (RISC). These RISCs bind to and cleave mRNA.
  • shRNA is transcribed by RNA polymerase.
  • small interfering RNA refers to a short double-stranded RNA capable of inducing RNA interference through cleavage of a specific mRNA. It consists of a sense RNA strand having a sequence homologous to the mRNA of the target gene and an antisense RNA strand having a sequence complementary thereto. Since siRNA can suppress the expression of a target gene, it is provided as an efficient gene knock-down method or as a gene therapy method.
  • microRNA refers to a short non-coding RNA consisting of about 22 nucleotide sequences. It is known to function as a post-transcriptional regulator in the process of gene expression. By complementarily binding to target mRNAs having complementary nucleotide sequences, target mRNAs are degraded or translation into proteins is inhibited.
  • the MITF protein activity inhibitor may be at least one selected from the group consisting of compounds, peptides, peptide mimetics, substrate analogs, aptamers, and antibodies that specifically bind to the MITF protein, but is not limited thereto.
  • Peptidomimetics inhibit the activity of the MITF protein by inhibiting the binding domain of the MITF protein.
  • Peptidomimetics may be peptides or non-peptides, and may be composed of amino acids linked by non-peptide bonds, such as psi bonds.
  • Peptidomimetics can be novel small molecules that are structured similarly to the secondary structural properties of the MITF protein, can mimic the inhibitory properties of large molecules such as antibodies or water-soluble receptors, and can act with equivalent effects to natural antagonists.
  • the "aptamer” is a single-stranded DNA or RNA molecule, and is high in specific chemical or biological molecules by an evolutionary method using an oligonucleotide library called SELEX (systematic evolution of ligands by exponential enrichment). Oligomers that bind with affinity and selectivity can be separated and obtained. An aptamer can specifically bind to a target and modulate the activity of the target, such as blocking the function of the target through binding.
  • the "antibody” can specifically and directly bind to the MITF protein to effectively inhibit the activity of the MITF protein.
  • a polyclonal antibody or a monoclonal antibody may be used as an antibody specifically binding to the MITF protein.
  • An antibody specifically binding to the MITF protein may be prepared by a known method known to those skilled in the art, and a commercially known MITF antibody may be purchased and used.
  • the antibody may be prepared by injecting the immunogen MITF protein into an external host according to a conventional method known to those skilled in the art. External hosts include mammals such as mice, rats, sheep, and rabbits. Immunogens are injected intramuscularly, intraperitoneally or subcutaneously, and can be administered together with an adjuvant to increase antigenicity.
  • Antibodies can be isolated by collecting serum showing titer and specificity for the antigen formed by regularly drawing blood from an external host.
  • the "bone marrow-derived suppressor cells” or “MDSC” function to suppress immunity by inhibiting the activity of cytotoxic T lymphocytes and NK cells.
  • MDSC are highly increased in tumors or cancer patients, which significantly reduces the effectiveness of cancer vaccine administration, thereby neutralizing the efficacy of cancer vaccines.
  • ICI immune checkpoint inhibitors
  • MDSC used to select a drug inhibiting MDSC activity can be obtained by extracting bone marrow cells from a tumor-bearing individual and differentiating them.
  • the MDSC of the individual having the tumor may have a CD11b + Gr1 + PD-L1 + phenotype, but is not limited thereto.
  • the tumor is specifically breast cancer, liver cancer, stomach cancer, colon cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, proximal anal cancer, fallopian tube carcinoma , endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, lymph gland cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer , prostate cancer, adenocarcinoma, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma ,
  • the composition is preferably administered to a subject in need of inhibiting MDSC.
  • the present inventors can prepare mouse bone marrow-derived MDSC by treating cells isolated from mouse bone marrow with GM-CSF as an MDSC differentiation inducer.
  • the present inventors treated cells isolated from bone marrow of mice in a tumor cell-conditioned medium (TCCM) in which a mouse breast cancer cell line was cultured together with GM-CSF, a differentiation-inducing factor for MDSC. It was confirmed that the differentiation of MDSC was induced, the activity of MDSC was induced by TCCM, and the expression of the gene and protein of MITF increased in the MDSC in which the activity was induced. In addition, as a result of co-culture of MDSC and T cells whose activity was induced by TCCM, it was confirmed that the number of CD3 + CD8 + T cells was decreased by MDSC whose activity was induced. Through the above results, it was confirmed that MDSCs are activated in the cancer cell microenvironment and the immune response is lowered, and that MITF is involved in the activation of MDSCs as a target factor for the activation of MDSCs.
  • TCCM tumor cell-conditioned medium
  • the present inventors obtained MDSCs from the spleen and tumor regions of tumor-forming mice, and confirmed that MDSCs obtained from the tumor regions showed high activation and expression of MITF. In addition, it was confirmed that MDSC expressing MITF increased around lung cancer and head and neck cancer tissues. Through the above results, it was confirmed that MDSCs are activated in the cancer cell microenvironment, and that MITF is involved in the activation of the MDSCs.
  • MDSC differentiation inducing factors include glycoprotein hormones that regulate the proliferation and differentiation of hematopoietic progenitor cells and drugs that mimic their actions, and are directly purified from living organisms. It can be used or mass-produced using recombinant DNA technology.
  • erythropoietic factor (EPO) bone marrow growth factor (G-CSF)
  • GM-CSF granulocyte macrophage colony stimulating factor
  • MMF megakaryocyte growth factor
  • TPO platelet growth stimulating factor
  • IL-11 interleukin-11
  • the present inventors treated cells isolated from mouse bone marrow with drugs that induce MDSC activity, IL-18, IL-4, LPS or statins along with GM-CSF, an MDSC differentiation inducer.
  • drugs that induce MDSC activity IL-18, IL-4, LPS or statins along with GM-CSF
  • an MDSC differentiation inducer As a result, it was confirmed that MDSC differentiation was induced by GM-CSF, MDSC activity was induced by the MDSC activity-inducing drugs, and the expression of the MITF gene and protein was increased in the activity-induced MDSC.
  • the present inventors treated cells isolated from mouse bone marrow with IBMX as a MITF inducer along with GM-CSF, an MDSC differentiation inducer, and as a result, MDSC differentiation was induced by GM-CSF and MITF by IBMX. Expression increased, and at this time, it was confirmed that MDSC was activated.
  • cells isolated from mouse bone marrow or spleen were treated with GM-CSF, an MDSC differentiation-inducing factor, and berberine or ML-329 as a MITF inhibitor, MDSC differentiation was induced by GM-CSF. , it was confirmed that MITF expression was reduced by the MITF inhibitor, and at this time, the activity of MDSC was suppressed.
  • the present inventors used Nelfinavir, a type of anti-HIV agent, as a MITF inhibitor along with GM-CSF, a differentiation inducer of MDSC, in a cancer cell microenvironment for inducing MDSC activity in cells isolated from mouse bone marrow. As a result of the treatment, it was confirmed that MITF expression was reduced and MDSC activity was inhibited by nelfinavir.
  • MDSCs are activated in the cancer cell microenvironment, and that MITF is critically involved in the activation of MDSCs. Therefore, it was confirmed that MDSC can be inhibited by using an inhibitor of MITF, and thus, it is possible to select an inhibitor of MITF and predict MDSC activity therefrom.
  • This can be usefully used to develop a composition containing a MITF inhibitor as an active ingredient, and can also be usefully used to determine a drug when administration of an MDSC activity inhibitory drug is required. That is, the present invention can be usefully used to alleviate the decrease in immune response caused by MDSC and increase the efficiency of anti-cancer immunotherapy.
  • the effect of a specific substance on MDSC activity can be predicted by analyzing the effect on MITF gene expression or MITF protein.
  • bone marrow cells to induce MDSC differentiation are prepared.
  • the bone marrow cells may be extracted from a tumor-bearing individual, and the extracted bone marrow cells may be differentiated and/or activated by MDSC in a medium.
  • MDSC differentiation can be achieved by culturing bone marrow cells in a medium containing MDSC differentiation-inducing factors. Differentiation and activity induction can occur simultaneously or separately.
  • MDSC activation for analysis can be performed in a medium in which a cancer cell microenvironment is created, that is, TCCM.
  • a medium in which a cancer cell microenvironment is created and treated with a specific substance can be used as a test group, and a medium in which a specific substance is not treated can be used as a control group, or a cancer cell microenvironment is created in both the test group and the control group, and only the test group is treated with a specific substance.
  • the MDSCs of the test group may be recovered and then treated with a specific substance.
  • the effect of a specific substance on MDSC activity can be predicted by recovering MDSCs that have completed differentiation and activity induction and analyzing the level of expression of the MITF gene or MITF protein.
  • qRT-PCR can be used to analyze the level of MITF gene expression after recovery of MDSCs, which have been completely differentiated and activated.
  • iNOS, IL-10, TGF- ⁇ , etc. may be used as MDSC activity markers, but are not limited thereto.
  • MDSCs induced to differentiate and/or activate are recovered and total RNA is isolated using an appropriate solution, for example, TRIzol Reagent® Solution (Invitrogen). Then, qRT-PCR can be performed using the isolated total RNA.
  • Western blotting in order to analyze the level of MITF protein expression after recovering the MDSCs whose differentiation and induction of activity have been completed, Western blotting can be performed.
  • the cell lysate may be separated by electrophoresis.
  • Primary antibodies that can be used include, for example, anti-MITF antibodies and anti-actinin antibodies, and as secondary antibodies, HRP-conjugated secondary antibodies can be considered.
  • actinin can be used as a control protein.
  • a specific substance is a MITF inhibitor, that is, an MDSC activity inhibitor, or a MITF expression increasing agent, that is, an MDSC activity inducer. Predictable. Therefore, it can be expected that MDSC activity is inhibited by administration of an MDSC activity inhibitor, leading to recovery of T cell function in the body, and in the case of an MDSC activity increasing agent, T cell proliferation by MDSC can be expected to be inhibited. From this, a composition containing the MITF inhibitor as an active ingredient can be prepared.
  • the screening method for MDSC activity inhibitory drugs is the MDSC inhibitory drug when the expression level of the MITF gene or MITF protein is low in the test group compared to the control group, for example, when qRT-PCR analysis is used.
  • composition containing the MDSC activity inhibitory drug selected by the method of the present invention may be preferably formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration.
  • acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added if necessary.
  • composition of the present invention can be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant includes an excipient, a disintegrant, a sweetener, a binder, a coating agent, an expanding agent, and a lubricant , solubilizing agents such as lubricants or flavoring agents may be used.
  • the adjuvant includes an excipient, a disintegrant, a sweetener, a binder, a coating agent, an expanding agent, and a lubricant , solubilizing agents such as lubricants or flavoring agents may be used.
  • composition of the present invention may be formulated into injection formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets by additionally adding diluents, dispersants, surfactants, binders, and lubricants.
  • injection formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets by additionally adding diluents, dispersants, surfactants, binders, and lubricants.
  • composition of the present invention can be administered through conventional intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes.
  • method can be administered.
  • the composition of the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type, severity, activity of the drug, It may be determined according to factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field.
  • the composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.
  • the effective amount of the composition according to the present invention may vary depending on the patient's age, sex, and body weight, and is generally 0.1 mg to 100 mg per 1 kg of body weight, more specifically 0.5 mg to 10 mg per day or every other day. Alternatively, it may be divided into 1 to 3 doses per day. However, since it may increase or decrease according to the route of administration, severity of disease, sex, weight, age, etc., the dosage is not intended to limit the scope of the present invention in any way.
  • the present invention relates to the reduction of immune response by myeloid-derived suppressor cells (MDSC) containing a gene expression inhibitor of MITF (microphthalmia-associated transcription factor) or a protein activity inhibitor of MITF as an active ingredient.
  • MDSC myeloid-derived suppressor cells
  • MITF microphthalmia-associated transcription factor
  • a protein activity inhibitor of MITF as an active ingredient.
  • an anti-cancer adjuvant containing a composition for relief, treatment or prevention as an active ingredient.
  • the gene expression inhibitor of MITF is antisense nucleotide, aptamer, short hairpin RNA (shRNA), small interfering RNA (siRNA), micro It may be one or more selected from the group consisting of RNA (microRNA, miRNA) and ribozyme, but is not limited thereto.
  • the MITF protein activity inhibitor may be at least one selected from the group consisting of compounds, peptides, peptide mimetics, substrate analogs, aptamers, and antibodies that specifically bind to the MITF protein, but is not limited thereto.
  • the anticancer adjuvant may be administered in combination with an anticancer agent, and the anticancer adjuvant suppresses the activity of MDSC to mitigate the decrease in immune response by MDSC, thereby exhibiting an anticancer adjuvant effect that significantly increases the effect of the anticancer agent.
  • the anticancer agent may be at least one selected from the group consisting of chemotherapeutic agents, targeted therapeutics, antibody therapeutics, immunotherapeutic agents, and hormone therapeutics, but is not limited thereto.
  • chemotherapeutic agents include, for example, antimetabolites (eg, folic acid, purine, and pyrimidine derivatives), alkylating agents (eg, nitrogen mustards, nitrosoureas, platinum, alkyl sulfonates, hydrazines, triazenes, aziridines, spindle inhibitors, cytotoxic agents, topoisomerase inhibitors and others) or hypomethylating agents (eg zebularine, isothiocyanate, azacitidine (5-azacytidine), 5-fluoro rho-2'-deoxycytidine, 5,6-dihydro-5-azacytidine and others), but are not limited thereto.
  • antimetabolites eg, folic acid, purine, and pyrimidine derivatives
  • alkylating agents eg, nitrogen mustards, nitrosoureas, platinum, alkyl sulfonates, hydrazines, triazenes, aziridines
  • the targeted therapeutic agent is an agent specific for a dysregulated protein of cancer cells, for example, a tyrosine kinase inhibitor such as Axitinib, Bosutinib, Cediranib , Dasatinib, Erlotinib, Imatinib, Gefitinib, Lapatinib, Lestaurtinib, Nilotinib , Semaxanib, Sorafenib, Sunitinib, and Vandetanib, or cyclin-dependent kinase inhibitors such as Alvocidib and Selicic Liv (Seliciclib), but is not limited thereto.
  • a tyrosine kinase inhibitor such as Axitinib, Bosutinib, Cediranib , Dasatinib, Erlotinib, Imatinib, Gefitinib, Lapatinib, Lestaurtinib, Nilotinib , Se
  • the antibody therapeutic is an antibody preparation that specifically binds to a protein on the surface of cancer cells, for example, Trastuzumab, Rituximab, Tositumomab, Cetuximab , Panitumumab, Alemtuzumab, Bevacizumab, Edrecolomab, or Gemtuzumab, but is not limited thereto.
  • the immunotherapeutic agent is an agent designed to induce the subject's own immune system to attack the tumor, for example, Ipilimumab, Avelumab, Nivolumab or Pembrolizumab. but is not limited thereto.
  • the hormone therapy is an agent that suppresses the growth of cancer by providing or blocking hormones in a specific cancer, for example, but is not limited thereto, such as tamoxifen or diethylstilbestrol.
  • an appropriate dosage of the anticancer agent is already well known in the art, it may be administered according to standards known in the art according to each patient's condition. Specific dosage determination is within the level of those skilled in the art, and its daily dosage is, for example, specifically 1 mg/kg/day to 10 g/kg/day, more specifically 10 mg/kg/day to 100 mg /kg/day may be, but is not limited thereto, and may vary depending on various factors such as the age, health condition, and complications of the subject to be administered.
  • the present inventors found that MDSCs are activated in the cancer cell microenvironment and the immune response is lowered, MITF is involved in the activation of the MDSCs, and the activity of MDSCs can be inhibited using the MITF inhibitor Since it was confirmed, the MITF inhibitor can be used as an anti-cancer adjuvant for anti-cancer immunotherapy.
  • the present invention provides a method for inhibiting MDSC, comprising administering a gene expression inhibitor of MITF or a protein activity inhibitor of MITF to a subject in need of MDSC inhibition.
  • the "myeloid-derived suppressor cell inhibition” or “MDSC inhibition” includes not only inhibiting the activity of MDSC but also reducing the number of MDSC. Reducing the number includes not only inhibiting the production of cells, but also killing or differentiating cells that have already been produced. In addition, any mechanism that is referred to as “inhibition" from a biological point of view is included.
  • the individual in need of MDSC inhibition may specifically be an individual having a tumor in need of MDSC inhibition, and the MDSC of the individual having the tumor may have a CD11b + Gr1 + PD-L1 + phenotype, It is not limited thereto.
  • the tumor is specifically breast cancer, liver cancer, stomach cancer, colon cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, proximal anal cancer, Fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, lymph gland cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, Penile cancer, prostate cancer, adenocarcinoma, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma It may
  • the present inventors found that MDSCs are activated in the cancer cell microenvironment and the immune response is lowered, MITF is involved in the activation of the MDSCs, and the activity of MDSCs can be inhibited using the MITF inhibitor Since it was confirmed, the MITF inhibitor can be administered to a subject in need of MDSC inhibition and used to treat MDSC-related diseases.
  • MDSC myeloid-derived suppressor cells
  • MDSC Myeloid-derived suppressor cells
  • 6- to 8-week-old Balb/c mice were Saeronbio. Inc. (Republic of Korea). Animal experiments were performed with the approval of the Institutional Ethical Committee of Sookmyung Women's University (SMWU-IACUC-1708-017-02). The femur bones of 6- to 8-week-old Balb/c mice were removed, bone marrow was separated from the bones, and red blood cells were removed by treatment with RBC lysis buffer (Sigma-Aldrich, St. Louis, MO) to obtain cells.
  • RBC lysis buffer Sigma-Aldrich, St. Louis, MO
  • the obtained bone marrow cells were cultured in a 24-well plate at a cell number of 5 ⁇ 10 5 cells/mL in RPMI medium (Invitrogen, Grand Island, NY) containing 10 ng/mL of granulocyte macrophage colony stimulating factor (GM-CSF) for 96 hours. During culture, the differentiation of MDSC was induced.
  • RPMI medium Invitrogen, Grand Island, NY
  • GM-CSF granulocyte macrophage colony stimulating factor
  • Tumor-forming mice were prepared by injecting breast cancer cell lines into Balb/c mice, and MDSCs were prepared from the tumor-forming mice.
  • mice 100 ⁇ l PBS containing 4T1 cells, a mouse breast cancer cell line, at a cell number of 5 ⁇ 10 5 cells/100 ⁇ l was subcutaneously injected into the right flank of 6- to 8-week-old Balb/c mice to form tumors.
  • the mice were bred in an animal laboratory at Sookmyung Women's University at a temperature of 23.5 ⁇ 1 °C and a humidity of 50 ⁇ 5% in a 12-hour light/dark cycle. After 2 weeks, the tumor, bone marrow and spleen were extracted and cells were isolated.
  • FACS Fluorescence-activated cell sorting
  • 4T1 cells a mouse breast cancer cell line
  • RPMI medium Invitrogen, Grand Island, NY
  • FBS fetal bovine serum
  • antibiotic-antimicrobial agent Invitrogen, Grand Island, NY
  • Bone marrow cells were obtained by the same method as described in ⁇ Example 1-1>, and the obtained TCCM and GM-CSF at a concentration of 10 ng/ml were placed in a 24-well plate at a cell number of 5 ⁇ 10 5 cells/ml. Differentiation of MDSC was induced by culturing for 96 hours in the prepared RPMI medium. As a control, MDSCs differentiated in RPMI medium containing GM-CSF were used.
  • the differentiated MDSCs were recovered, stained with fluorescently coupled anti-CD11b antibodies and anti-Gr1 antibodies, and subjected to FACS analysis (FIG. 2A).
  • the differentiation-induced MDSC was collected and subjected to qRT-PCR for MDSC activity markers iNOS, IL-10 and TGF- ⁇ .
  • the differentiated MDSCs were recovered and total RNA was isolated using TRIzol Reagent® Solution (Invitrogen) according to the manufacturer's procedure. Then, qRT-PCR was performed using the isolated total RNA.
  • RNA isolated was reverse transcribed using the M-MLV reverse transcription kit (Promega, Madison, WI), oligo-(dT) primers and dNTP (Bioneer, Daejeon, Republic of Korea) according to the manufacturer's procedure, and ABI Real-time Quantitative PCR was performed in a PCR 7500 system using primers for iNOS, IL-10, TGF- ⁇ and cyclophilin and SYBR Green PCR Master Mix (Applied Bosystems, Foster City, CA) according to the manufacturer's procedure. . At this time, cyclophilin was used as a control. Primers for iNOS, IL-10, TGF- ⁇ and cyclophilin were purchased from Bioneer. The experiment was repeated 3 times (Fig. 2B).
  • qRT-PCR for MITF was performed in the same manner as described in Example ⁇ 2-1> using MDSC recovered in Example ⁇ 2-1> to confirm the gene expression of MITF.
  • Primers for MITF were purchased from Bioneer (FIG. 3A).
  • Example ⁇ 2-1> Western blotting was performed using the MDSC recovered in Example ⁇ 2-1>.
  • the MDSCs recovered in Example ⁇ 2-1> were treated with a cell lysis buffer and dissolved.
  • the cell lysate was separated by electrophoresis on a 10% SDS-PAGE gel and transferred to a PVDF membrane.
  • HRP-conjugated secondary antibody is attached to the primary antibody attached to the membrane, and this is enhanced chemiluminescence technique (PicoEPD?
  • MITF was a target factor specifically expressed in MDSCs whose activity was induced by TCCM.
  • MDSCs are known to suppress the proliferation and function of T cells to lower the immune response. Therefore, in order to determine whether the immune response is reduced by MDSC in the cancer cell microenvironment, the degree of inhibition of T cell proliferation was confirmed using MDSC treated with a medium in which cancer cells were cultured.
  • mice Specifically, cells isolated from the spleen of tumorigenic mice using a MACS cell separation kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer's procedure were carried out in a 24-well plate at a cell count of 5 ⁇ 10 5 cells/ml. MDSC differentiation and activity were induced by culturing for 24 hours in RPMI medium containing TCCM obtained in Example ⁇ 2-1> and GM-CSF at a concentration of 10 ng/ml.
  • MACS cell separation kit Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
  • T cells were isolated by removing the spleens of 6- to 8-week-old Balb/c mice. Then, after staining with a fluorescently coupled anti-CD3 antibody, T cells were isolated using FACS. The isolated T cells were stained with 2.5 ⁇ M CFSE for 7 minutes to perform CFSE labeling. 5 ⁇ 10 CFSE-labeled CD3 + T cells of 5 cells were stimulated on plates coated with anti-CD3 monoclonal antibody and soluble anti-CD28 monoclonal antibody for 2 hours, and then cells isolated from the spleens of the tumorigenic mice. MDSC obtained by inducing differentiation and activity of (Fig. 4A) and MDSC recovered in Example ⁇ 2-1> (Fig.
  • MDSCs from tumorigenic mice were used to confirm their activity and changes in MITF gene expression.
  • Example ⁇ 1-2> FACS analysis was performed after separating cells from each of the spleen and tumor of tumor-forming mice in the same manner as described in Example ⁇ 1-2> above (FIG. 5A).
  • CD11b + Gr1 + MDSC were recovered using the FACS sorter, and qRT-PCR was performed in the same manner as described in Example ⁇ 2-1> (FIG. 5B).
  • a control the spleen of a tumor-forming mouse before sorting was used.
  • MDSCs are activated in the cancer cell microenvironment, and MITF is involved in the activation of the MDSCs.
  • tissue staining was performed at Seoul National University's Department of Pathology (Prof. Yoon Kyung Jeon) for each of 10 samples of lung cancer and head and neck cancer tissues and their corresponding cancer-free lymph node tissue samples.
  • the tissues were fixed in 4% formaldehyde solution, dehydrated in ethanol (70-100%) by concentration, and embedded in paraffin.
  • Tissues were sectioned with a microtome (thickness: 4 ⁇ m) and stained (H&E) with hematoxylin and eosin. Cross sections were observed under an optical microscope (Olympus, Japan) and photographed at ⁇ 400 magnification.
  • IHC immunohistochemistry
  • the slides were washed, the peroxidase reaction was developed with diaminobenzidine and peroxide, and aqua- Mounted on a mount (Aqua-Mount) and evaluated under an optical microscope (Olympus) at ⁇ 400 magnification
  • the number of MITF + /CD11b + /CD14 + MDSCs in a high power field (HPF) in cancer tissue and lymph node tissue was expressed as the average value of counts of triple positive cells in an arbitrary high-magnification field of three places, respectively.
  • MDSCs are activated around the cancer, and MITF is involved in the activation of the MDSCs.
  • Example ⁇ 1-1> IL-18 at a concentration of 10 or 50 ng/ml in a 24-well plate with a cell number of 5 ⁇ 10 5 cells/ml, and Differentiation of MDSC was induced by culturing for 96 hours in RPMI medium containing GM-CSF at a concentration of 10 ng/ml (FIGS. 6A and 6B).
  • MDSC differentiation was induced by culturing in RPMI medium containing GM-CSF for 96 hours, and IL-18 at a concentration of 10 or 50 ng/ml was added for 24 hours. treated (Fig. 6C and Fig. 6D).
  • MDSCs differentiated in RPMI medium containing GM-CSF were used.
  • the differentiated MDSCs were recovered and FACS analysis was performed in the same manner as described in Example ⁇ 2-1> to confirm MDSC differentiation (FIGS. 6A and 6C).
  • MDSCs from which the differentiation was induced were recovered and qRT-PCR was performed in the same manner as described in Examples ⁇ 2-1> and ⁇ 2-2> to confirm the activity of MDSCs and to confirm the gene expression of MITF. (FIGS. 6B and 6D).
  • IL-10 at a concentration of 5 ng/ml and 10 ng were added in a 24-well plate with a cell number of 5 ⁇ 10 5 cells/ml.
  • MDSC differentiation was induced by culturing for 96 hours in RPMI medium containing GM-CSF at a concentration of /ml (Fig. 6E and Fig. 6F).
  • Fig. 6E and Fig. 6F concentration of /ml
  • the differentiated MDSCs were collected and subjected to FACS analysis in the same manner as described in Example ⁇ 2-1> to confirm MDSC differentiation (FIG. 6E).
  • MDSCs from which the differentiation was induced were recovered and qRT-PCR was performed in the same manner as described in Examples ⁇ 2-1> and ⁇ 2-2> to confirm the activity of MDSCs and to confirm the gene expression of MITF. (FIG. 6F).
  • MDSC differentiation and activity were induced according to the method described in Example ⁇ 2-1>, but 10 ng/ml of IL-4 was treated as an activity-inducing drug.
  • the differentiated MDSCs were collected and subjected to FACS analysis in the same manner as described in Example ⁇ 2-1> to confirm MDSC differentiation (FIG. 7A).
  • MDSC differentiation and activity were induced according to the method described in Example ⁇ 2-1>, but 100 ng/ml of LPS was treated as an activity-inducing drug.
  • the differentiated MDSCs were collected and subjected to FACS analysis in the same manner as described in Example ⁇ 2-1> to confirm MDSC differentiation (FIG. 8A).
  • Simvastatin a statin drug, is known to induce the activation of MDSC by suppressing the expression of Interferon regulatory factor 4 (IRF4). Accordingly, after treatment with a statin-based drug as a drug inducing the activity of MDSC and inducing differentiation of MDSC, changes in gene expression of MITF were confirmed.
  • IRF4 Interferon regulatory factor 4
  • MDSC differentiation and activity were induced according to the method described in Example ⁇ 2-1>, but simvastatin (Sim, 0.5 or 1 ⁇ M) and lovastatin (Lovastatin, Lova, 0.5 or 1 ⁇ M) were used as the activity-inducing drugs.
  • ⁇ M simvastatin
  • lovastatin Lovastatin, Lova, 0.5 or 1 ⁇ M
  • provastatin Pravastatin, Prav, 5 or 10 ⁇ M
  • rosuvastatin Rosu, 0.5 or 1 ⁇ M
  • atorvastatin Atorvastatin, Ator, 0.05 or 0.1 ⁇ M
  • Example ⁇ 1-1> MDSC differentiation was induced by culturing in RPMI medium containing GM-CSF for 96 hours, and treated with the statin drug for 24 hours (FIG. 11 and Figure 12).
  • MDSCs were recovered and the differentiation of MDSCs (FIGS. 9 and 11) was confirmed in the same manner as described in Examples ⁇ 2-1> and ⁇ 2-2>.
  • MDSC activity FIGS. 10A and 12A
  • MITF gene expression FIGS. 10B and 12B
  • FIGS. 9 to 12 it was confirmed that MDSC differentiation was induced to a similar extent in both the control group and the statin-based drug-treated group (FIGS. 9 and 11).
  • the statin-based drug-treated group both when the statin-based drug was treated from the early stage of MDSC differentiation (FIG. 10) and when the statin-based drug was treated on already differentiated MDSC (FIG. 12), MDSC activation by the statin-based drug was induced (Figs. 10A and 12A), and it was confirmed that the gene expression of MITF (Figs. 10B and 12B) was increased compared to the control group.
  • All-trans retinoic acid is known to inhibit the activity of MDSC. Therefore, in order to investigate the correlation between MITF and MDSC, after treatment with ATRA as a drug that inhibits MDSC activity, changes in MITF expression were confirmed.
  • bone marrow cells were obtained by the same method as described in Example ⁇ 1-1>, and then ATRA (0.5 or 1 ⁇ M) and 10 ng/mL concentration were placed in a 24-well plate with a cell number of 5 ⁇ 10 5 cells/mL. Differentiation of MDSC was induced by culturing for 96 hours in RPMI medium containing GM-CSF of (FIG. 13).
  • the differentiated MDSCs were recovered and FACS analysis was performed in the same manner as described in Example ⁇ 2-1> to confirm the differentiation of MDSCs (FIG. 13A).
  • MDSCs treated with 1 ⁇ M of ATRA and induced to differentiate were recovered, and qRT-PCR was performed in the same manner as described in Examples ⁇ 2-1> and ⁇ 2-2> to confirm the activity of MDSCs ( Fig. 13B), MITF gene expression was confirmed (Fig. 13C).
  • IBMX induces the expression of MITF in melanoma cells. Accordingly, in order to investigate the effect of MITF expression or activity regulation on MDSC activity, changes in activity of MDSCs treated with IBMX as an inducer of MITF expression and changes in T cell proliferation by the MDSCs were confirmed.
  • bone marrow cells were obtained by the same method as described in Example ⁇ 1-1>, and IBMX at a concentration of 10 ⁇ M and GM at a concentration of 10 ng/mL were cultured in a 24-well plate at a cell number of 5 ⁇ 10 5 cells/mL.
  • Differentiation of MDSC was induced by culturing for 96 hours in RPMI medium containing -CSF.
  • MDSCs differentiated in RPMI medium containing GM-CSF were used.
  • the differentiated MDSCs were collected and subjected to FACS analysis in the same manner as described in Example ⁇ 2-1> to confirm MDSC differentiation (FIG. 14A).
  • MDSCs from which the differentiation was induced were recovered and subjected to qRT-PCR and Western blotting in the same manner as described in Examples ⁇ 2-1> and ⁇ 2-2> to confirm the activity of MDSCs (Fig. 14B and Fig. 14C), MITF gene expression and protein expression (Fig. 14C) were confirmed.
  • Berberine, Kazinol U, and the like are known to inhibit the expression or activity of MITF in melanocytes while being AMPK activators. Accordingly, in order to examine the effect of MITF expression or activity regulation on MDSC activity, changes in activity of MDSC treated with berberine at different concentrations as a MITF inhibitor were confirmed.
  • bone marrow cells were obtained by the same method as described in Example ⁇ 1-1>, and berberine at a concentration of 5 ⁇ M and GM at a concentration of 10 ng/mL were placed in a 24-well plate at a cell number of 5 ⁇ 10 5 cells/mL.
  • MDSC differentiation was induced by culturing in RPMI medium containing CSF and RPMI medium containing berberine at a concentration of 10 ⁇ M and GM-CSF at a concentration of 10 ng/mL for 96 hours, respectively.
  • MDSCs differentiated in RPMI medium containing GM-CSF were used as a control group.
  • Example ⁇ 2-1> the expression of MITF was confirmed by performing qRT-PCR in the same manner as described (Fig. 15C).
  • ML-329 inhibits the expression of MITF by suppressing TRPM-1 promoter activity in melanocytes. Accordingly, in order to investigate the effect of the regulation of MITF expression or activity on MDSC activity, changes in the activity of MDSCs treated with ML-329 as a MITF inhibitor and changes in T cell proliferation by the MDSCs were confirmed.
  • bone marrow cells were obtained by the same method as described in Example ⁇ 1-1>, and ML-329 (Cayman Chemical, Cayman Chemical, Ann Arbor, MI) and 10 ng/ml GM-CSF were cultured in RPMI medium for 96 hours to induce MDSC differentiation.
  • MDSCs differentiated in RPMI medium containing GM-CSF were used.
  • the MDSCs from which the differentiation was induced were recovered, and qRT-PCR was performed in the same manner as described in Examples ⁇ 2-1> and ⁇ 2-2> to confirm MDSC activity (FIG. 17A) and MITF expression. (FIG. 17B).
  • bone marrow cells were obtained by the same method as described in Example ⁇ 1-1>, and the number of cells at 5 ⁇ 10 5 cells/ml was 24 -96 hours in RPMI medium containing ML-329 (Cayman Chemical) at a concentration of 0.5, 1 or 2 ⁇ M in a well plate, TCCM obtained in Example ⁇ 2-1> and GM-CSF at a concentration of 10 ng/mL During culture, the differentiation of MDSC was induced. As a control group, MDSCs differentiated in RPMI medium containing GM-CSF were used.
  • Example ⁇ 2-1> FACS analysis was performed in the same manner as described in Example ⁇ 2-1> to confirm the differentiation of MDSCs (FIG. 18A), and qRT-PCR was performed to determine the number of MDSCs. The degree of activity was measured (FIG. 18B).
  • qRT-PCR and Western blotting were performed in the same manner as described in Example ⁇ 2-2> to confirm MITF expression (FIG. 18C).
  • the degree of inhibition of T cell proliferation was confirmed by the same method as described in Example ⁇ 2-3> using the obtained MDSCs (FIG. 18D).
  • FIGS. 17 and 18 in the case of the ML-329-treated group, it was confirmed that ML-329 suppressed MITF expression and inhibited MDSC activity (FIGS. 17A and 17B).
  • MDSC activity by TCCM was inhibited (FIGS. 18B and 18C)
  • MITF expression was inhibited (FIG. 18C)
  • T cell proliferation increased due to MDSC activity inhibition by ML-329. It was confirmed (FIG. 18D).
  • MDSCs are known to inhibit various processes from proliferation to function of T cells by producing reactive oxygen species (ROS) or reactive nitrogen species (RNS). Therefore, in order to examine the effect of the regulation of MITF expression or activity on MDSC activity, changes in ROS generation were confirmed in MDSC treated with berberine or ML-329 as a MITF inhibitor.
  • ROS reactive oxygen species
  • RNS reactive nitrogen species
  • MDSCs were isolated from the spleen of the tumorigenic mouse of Example ⁇ 1-2>, berberine at a concentration of 10 ⁇ M or ML-329 at a concentration of 1 ⁇ M, TCCM obtained in Example ⁇ 2-1> and 10 MDSC differentiation was induced by culturing for 48 hours in RPMI medium containing GM-CSF at a concentration of ng/ml.
  • the MDSC were collected and divided into 1 ⁇ 10 5 cells, and treated with LPS at a concentration of 100 ng/ml for 24 hours.
  • NAC N-acetyl-cysteine
  • DCF-DA was added and reacted at 37° C. for 30 minutes, and the degree of ROS generation was measured by flow cytometry.
  • RNA interference RNA interference
  • CRISPR CRISPR
  • sgRNA was added to the CRISPR vector, specifically the pLentiCRISPR-E vector, to the bone marrow cells at a cell number of 1 ⁇ 10 6 cells/ml.
  • MITF gRNA FW oligo 5'-CACCGTAAGGACTTCCATCGGCACC-3' (SEQ ID NO: 1)
  • MITF gRNA RV oligo 5'-AAACGGTGCCGATGGAAGTCCTTAC-3' (SEQ ID NO: 2).
  • non-target control gRNA was cloned into the pLentiCRISPR-E vector; non-target control sgRNA: 5'-CACCGGTATTACTGATATTGGTGGG-3' (SEQ ID NO: 3).
  • the cloned construct was transfected using lipofectamine according to the manufacturer's procedure. 24 hours after transfection, MDSC differentiation was induced by culturing for 72 hours in RPMI medium containing TCCM obtained in Example ⁇ 2-1> and GM-CSF at a concentration of 10 ng/ml.
  • MITF plasmid DNA (Addgene, Cambridge, MO) was transfected into bone marrow cells at a cell number of 1 ⁇ 10 6 cells/ml using Lipofectamine according to the manufacturer's procedure. 24 hours after transfection, MDSC differentiation was induced by culturing for 72 hours in RPMI medium containing GM-CSF at a concentration of 10 ng/ml. After 3 days of transfection, MDSCs were harvested and the expression of MITF (FIG. 20D) and the degree of T cell proliferation by MDSCs (FIG. 20E) were measured as described above.
  • the activity of MDSC can be inhibited by inhibiting the expression or activity of MITF.
  • Nelfinavir an HIV protease inhibitor
  • TCCM bone marrow cells
  • 24- MDSCs were cultured for 96 hours in RPMI medium containing 1, 5, or 10 ⁇ M of nelfinavir, TCCM obtained in Example ⁇ 2-1>, and 10 ng/ml of GM-CSF in a well plate. differentiation was induced.
  • MDSCs differentiated with RPMI medium containing TCCM without nelfinavir and GM-CSF at a concentration of 10 ng/ml were used. Then, the MDSCs from which the differentiation was induced were recovered, and qRT-PCR was performed in the same manner as described in Example ⁇ 2-2> to confirm MITF expression (FIG. 21A). Then, qRT-PCR was performed in the same manner as described in Example ⁇ 2-1> to measure the activity level of MDSC (FIG. 21B). In addition, in order to confirm the activity of MDSC, Western blotting was performed according to the method described in Example ⁇ 2-2> to confirm MDSC activity (FIG. 21C).
  • Example 7 it was confirmed that MDSC activity was inhibited by the MITF inhibitor. Therefore, in order to examine the effect of reducing MDSC activity in tumorigenic mice, after administering MDSC whose activity was reduced by the MITF inhibitor to tumorigenic mice, tumor growth changes were confirmed.
  • MDSCs were isolated from the spleens of tumor-forming mice prepared in the same manner as described in Example ⁇ 1-2>.
  • ML-329 at a concentration of 1 ⁇ M
  • TCCM obtained in Example ⁇ 2-1>
  • GM-CSF a concentration of 10 ng/mL were included in the same manner as described in Example ⁇ 7-2> above.
  • Differentiation of MDSC was induced by culturing for 48 hours with the prepared RPMI medium. After 48 hours, the MDSC + 4T1-luc (5 ⁇ 10 4 + 2.5 ⁇ 10 5 /100 ⁇ l) were subcutaneously injected into the left flank of the mouse.
  • the MDSC population in the tumor tissue was stained with anti-CD11b antibody, anti-Gr1 antibody, and anti-CD45 antibody, and FACS analysis was performed to confirm it (FIG. 22D).
  • a control group MDSC cultured in RPMI medium without ML-329 and containing TCCM and GM-CSF were injected.
  • Example 1> to ⁇ Example 8> MDSCs are activated in the cancer cell microenvironment and the immune response is lowered, MITF is involved in the activation of MDSC, and the activity of MDSC is inhibited using the MITF inhibitor. confirmed that it could be done.
  • MITF inhibitors among a wide range of substances can be selected and used as MDSC activity inhibitory drugs. It is possible to more effectively identify active ingredients that can be used for alleviating the decline in the immune response of MDSCs and for anticancer immunotherapy. In addition, from this, it is possible to select a MITF expression inhibitor that is expected to act most appropriately when administered in vivo to an individual whose MDSCs are activated or whose T cells are confirmed to be suppressed by the MDSC.
  • the patient can select an anti-cancer composition or an anti-cancer adjuvant composition containing the MITF expression inhibitor as an active ingredient that is expected to be most suitable for the patient before direct administration, and whether or not the administration composition is changed even if the treatment is in progress
  • an anti-cancer composition or an anti-cancer adjuvant composition containing the MITF expression inhibitor as an active ingredient that is expected to be most suitable for the patient before direct administration, and whether or not the administration composition is changed even if the treatment is in progress
  • new drug candidates can be considered and MDSC activation response results can be predicted, the basis for selecting the most appropriate MITF inhibitor considering the type or prognosis of cancer is laid.
  • composition containing the MITF inhibitor selected according to the present invention can be administered to a subject in need of inhibition of MDSC, such as a subject having a tumor, and can be usefully used to alleviate the decrease in immune response caused by MDSC.
  • composition containing the MITF inhibitor can be administered in combination with an anticancer agent, such as an anticancer immunotherapeutic agent, and can be usefully used as an adjuvant therapy to increase the efficiency of anticancer immunotherapy.

Abstract

The present invention relates to a composition comprising a microphthalmia-associated transcription factor (MITF) inhibitor as an active ingredient for inhibiting myeloid-derived suppressor cells (MDSC). Specifically, it was confirmed that an MDSC is activated in a cancer cell microenvironment to lower an immune response, and an MITF is involved in the activation of the MDSC, and the MDSC can be inhibited using the MITF inhibitor. Thus, a composition comprising the MITF inhibitor as an active ingredient can be advantageously used to alleviate a decrease in immune response caused by an MDSC and to increase an efficiency of anticancer immunotherapy.

Description

MDSC 활성 저해 약물의 선별 방법Method for screening MDSC activity inhibitory drugs
본 발명은 골수-유래 억제세포(MDSC; myeloid-derived suppressor cell) 저해용 조성물을 제조하기 위하여, 특정물질에 의한 MDSC에서의 MITF(microphthalmia-associated transcription factor) 억제효과를 분석하여 MDSC 활성 저해 약물을 선별하는 방법에 관한 것으로, 구체적으로 특정물질이 MITF의 유전자 발현 억제제 또는 MITF의 단백질 활성 억제제인지 선별하고, MDSC 활성 저해 약물을 선별하는 방법 및 이렇게 선별된 MITF 억제제를 포함하는 조성물에 관한 것이다.In order to prepare a composition for inhibiting myeloid-derived suppressor cells (MDSC), the present invention analyzes the inhibitory effect of microphthalmia-associated transcription factor (MITF) in MDSC by a specific substance to obtain a drug that inhibits MDSC activity. It relates to a method for screening, specifically, to a method for selecting whether a specific substance is an MITF gene expression inhibitor or a MITF protein activity inhibitor, a method for selecting a MDSC activity inhibitory drug, and a composition containing the MITF inhibitor thus selected.
골수성 세포(myeloid cells)는 조혈모줄기세포(hematopoietic stem cell)에서 기원한다. 이는 우리 몸에 가장 많이 존재하는 조혈모세포로, 골수 및 림프 조직에 주로 존재한다. 최종적으로는 대식세포(macrophage), 수지상세포(dendritic cell) 그리고 과립구(granulocyte)로 분화하나, 이들은 특정 계층 구조를 띄지 않고 다양한 단계의 분화도를 가진 골수성 세포가 조직과 환경에 특이적으로 다양하게 분포되는 특징을 가지고 있다.Myeloid cells originate from hematopoietic stem cells. It is the most abundant hematopoietic stem cell in our body, and is mainly present in bone marrow and lymphoid tissue. Ultimately, they differentiate into macrophages, dendritic cells, and granulocytes, but these do not have a specific hierarchical structure, and myeloid cells with various degrees of differentiation are distributed in various ways specific to tissues and environments. has the characteristics of being
골수-유래 억제세포(myeloid-derived suppressor cells, MDSC)는 골수성 세포 계통 중에서 면역반응 억제 작용을 가진 세포들로서, 매우 광범위한 미분화 골수성 세포를 포함하는 세포군이다. MDSC는 여러 전사 인자들을 통해 활성화가 매개되고, 그 결과 활성산소종(reactive oxygen species, ROS)이나 활성질소종(reactive nitrogen species, RNS)이 생산되어 T 세포의 증식에서부터 기능까지 다양한 과정을 저해하여 T 세포를 효과적으로 억제하는 것으로 알려져 있다. 이러한 MDSC의 작용이 자가면역반응을 억제하는 점에서는 유익하지만, MDSC의 축적으로 면역 억제 환경이 지속되면 생체가 지속적으로 알러젠 및/또는 바이러스 감염에 노출되고, 이를 인해 만성 염증이 나타날 수 있다. 생체가 효과적인 면역반응을 할 수 없게 되면 만성 염증 시 조직 손상이 일어나게 될 수 있다. 또한, MDSC는 암세포 미세환경(tumor microenvironment, TME)에서 종양 세포의 침입 및 전이를 보조하는 것이 확인되었다. BACKGROUND Myeloid-derived suppressor cells (MDSCs) are cells having immune response suppression activity among the myeloid cell lineage, and are a cell group that includes a very wide range of undifferentiated myeloid cells. MDSC activation is mediated through several transcription factors, and as a result, reactive oxygen species (ROS) or reactive nitrogen species (RNS) are produced, which inhibits various processes from T cell proliferation to function. It is known to effectively suppress T cells. Although the action of these MDSCs is beneficial in suppressing the autoimmune response, if the immunosuppressive environment continues due to the accumulation of MDSCs, the body is continuously exposed to allergens and / or viral infections, which can lead to chronic inflammation. If the living body cannot make an effective immune response, tissue damage may occur during chronic inflammation. In addition, MDSCs have been confirmed to assist tumor cell invasion and metastasis in the tumor microenvironment (TME).
이러한 MDSC의 기능과 작용 기전에 대한 연구를 바탕으로 최근에는 이들의 조절을 통해 새로운 암 치료법을 개발하고자 하는 노력이 가속화되고 있다. 대표적으로 젬시타빈(Gemcitabine) 및 5-플루오로우라실(5-fluorouracil, 5-FU)은 MDSC를 직접적으로 감소시키는 화학요법제로 알려져 있다.Based on studies on the function and mechanism of action of these MDSCs, efforts to develop new cancer therapies through their regulation have recently been accelerated. Representatively, gemcitabine and 5-fluorouracil (5-fluorouracil, 5-FU) are known as chemotherapeutic agents that directly reduce MDSC.
또한, MDSC의 빈도 및 기능이 최근 임상에서 항암면역치료제로 활발하게 사용되고 있는 면역관문억제제(immune checkpoint inhibitor, ICI)에 대한 환자의 내성에 기여하는 것이 알려지면서, MDSC의 중요성이 강조되고 있다. ICI는 암세포에 의한 T 세포의 억제를 막아줌으로써 암 환자의 항암 면역반응을 향상시킨다. 실제로 CTLA-4(cytotoxic T-lymphocyte-associated protein 4) 및 PD-1(programmed cell death protein) 및 PD-L1(programmed cell death ligand 1)을 표적으로 하는 다양한 항체들이 악성흑색종, 편평비소세포폐암 및 신장암 등의 치료 목적으로 허가된 바 있다. 그러나 최대 80%에 달하는 많은 환자들이 이러한 치료법에 반응하지 않는 것으로 알려져 있고, 그 주된 원인으로 암세포 미세환경, 즉, TME (Tumor Micro-Environment)를 구성하는 면역억제세포들 때문이라고 추정되고 있다. ICI 치료 효능에 영향을 주는 TME에서의 면역억제세포들은 Treg 세포, MDSC, TH2 CD4+ 세포, CAF(cancer-associated fibroblast) 및 M2로 분극된 TAM(tumor-associated macrophage)이 잘 알려져 있다. 특히, MDSC를 결핍시키면 항암면역반응이 증강되어 ICI에 대하여 내재되어 있던 저항성이 극복된다는 결과들이 보고되면서, MDSC를 ICI 치료에서의 중요한 예후인자로서 사용할 수 있음이 강조되고 있다.In addition, as it is known that the frequency and function of MDSCs contribute to patient resistance to immune checkpoint inhibitors (ICIs), which are actively used as anti-cancer immunotherapeutic agents in recent clinical trials, the importance of MDSCs is being emphasized. ICI enhances the anticancer immune response of cancer patients by preventing suppression of T cells by cancer cells. In fact, various antibodies targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein (PD-1) and programmed cell death ligand 1 (PD-L1) have been shown to treat malignant melanoma, squamous non-small cell lung cancer and It has been approved for the treatment of kidney cancer, etc. However, it is known that up to 80% of patients do not respond to these treatments, and it is estimated that the main cause is the immunosuppressive cells constituting the cancer cell microenvironment, that is, TME (Tumor Micro-Environment). Treg cells, MDSC, TH2 CD4 + cells, cancer-associated fibroblasts (CAFs), and tumor-associated macrophages (TAMs) polarized to M2 are well known as immunosuppressive cells in the TME that affect the efficacy of ICI treatment. In particular, as results have been reported that depletion of MDSCs enhances anticancer immune responses and overcomes inherent resistance to ICI, it is emphasized that MDSCs can be used as an important prognostic factor in ICI treatment.
한편, ICI에 대한 비반응 또는 낮은 반응성을 극복하기 위해 최근 다른 종류의 ICI와 조합하여 병용 치료하는 방법이 시도되고 있으며, 이 경우 단일치료(monotherapy)에 비해 임상적 이득이 의미 있게 높아짐이 보고되었다. 또한, 항-CTLA4와 항-PD-1의 병용, 항-PD-1과 IL-2R 작용제(agonist)의 병용, VEGF 억제제 또는 케모카인(chemokine) 조절제와 ICI의 병용, 항-CTLA4와 CTL 치료제의 병용 등이 임상 연구되고 있다. 나아가 방사선 치료와 함께 ICI를 투여하거나, ICI와 저농도의 다양한 기존 화학요법제 또는 표적 항암제를 병용하여 T 세포의 활성화와 종양으로의 침투를 유도함으로써, 치료반응을 높이고 항암면역반응의 효과를 상승시키기 위한 다양한 시도들도 이루어지고 있다. On the other hand, in order to overcome non-response or low reactivity to ICI, a combination treatment method in combination with other types of ICI has recently been attempted, and in this case, it has been reported that the clinical benefit is significantly higher than monotherapy. . In addition, combination of anti-CTLA4 and anti-PD-1, combination of anti-PD-1 and IL-2R agonist, combination of VEGF inhibitor or chemokine modulator and ICI, anti-CTLA4 and CTL treatment Combination use is being studied clinically. Furthermore, by inducing the activation of T cells and infiltration into tumors by administering ICI together with radiotherapy or using ICI in combination with various conventional chemotherapeutic agents or target anticancer agents at low concentrations, increasing the therapeutic response and enhancing the effect of anticancer immune response Various attempts are also being made.
MITF(microphthalmia-associated transcription factor)는 멜라닌 세포, 골 세포 및 비만 세포를 포함한 다양한 유형의 계통 특이적 경로 조절(lineage-specific pathway regulation)에 관여하는 헬릭스-루프-헬릭스 루신 지퍼(Helix-loop-helix leucine zipper) 전사인자이다. 계통 특이적이란, 특정 세포 유형에서만 발견되는 유전자나 형질을 의미한다. 따라서, MITF는 정상 세포 전구체의 생존 및 생리적 기능에 특별히 필요한 신호전달 캐스케이드의 재설계에 관여할 수 있다. 특히, MITF는 멜라닌 세포(Melanocyte)와 흑생종(Melanoma)의 생존과 분화를 조절하며 멜라닌 형성 기전에 관여하는 것으로 알려져 있다. MITF에 대한 연구 결과는 최근에 보고되기 시작했는데, LXR(Liver X receptor)이 활성화되면 MITF의 분해를 촉진하여 멜라닌 합성(Melanogenesis)이 저해된다는 것이 알려져 있으나, MDSC에서 MITF의 역할에 대해서는 아직 밝혀진 바 없다.MITF (microphthalmia-associated transcription factor) is a helix-loop-helix leucine zipper involved in lineage-specific pathway regulation in various types of cells including melanocytes, osteocytes and mast cells. leucine zipper) transcription factor. By lineage specific, we mean a gene or trait found only in a particular cell type. Thus, MITF may be involved in the reengineering of signaling cascades specifically required for the survival and physiological function of normal cell progenitors. In particular, MITF is known to regulate the survival and differentiation of melanocytes and melanomas and to be involved in melanogenesis. Research results on MITF have recently begun to be reported. It is known that activation of LXR (Liver X receptor) promotes the degradation of MITF and inhibits melanogenesis. However, the role of MITF in MDSC has not yet been clarified does not exist.
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본 발명의 목적은 MITF(microphthalmia-associated transcription factor)의 유전자 발현 억제제 또는 MITF의 단백질 활성 억제제를 유효성분으로 함유하는, 골수-유래 억제세포에 의한 면역반응 저하의 완화, 치료 또는 예방용 조성물을 제공하는 것이다.An object of the present invention is to provide a composition for mitigating, treating or preventing a decrease in immune response by bone marrow-derived suppressor cells, containing a gene expression inhibitor of MITF (microphthalmia-associated transcription factor) or a protein activity inhibitor of MITF as an active ingredient. is to do
본 발명의 다른 목적은 MITF(microphthalmia-associated transcription factor)의 유전자 발현 억제제 또는 MITF의 단백질 활성 억제제를 골수-유래 억제세포의 저해가 필요한 개체에 투여하는 단계를 포함하는, 골수-유래 억제세포 저해방법을 제공하는 것이다.Another object of the present invention is a method for inhibiting bone marrow-derived suppressor cells, comprising administering a gene expression inhibitor of MITF (microphthalmia-associated transcription factor) or a protein activity inhibitor of MITF to a subject in need of inhibition of bone marrow-derived suppressor cells. is to provide
본 발명의 다른 목적은 특정물질에 의한 MDSC에서의 MITF(microphthalmia-associated transcription factor) 억제효과를 분석하여 MDSC 활성 저해 약물을 선별하는 방법을 제공하는 것이다. Another object of the present invention is to provide a method for selecting a drug that inhibits MDSC activity by analyzing the inhibitory effect of microphthalmia-associated transcription factor (MITF) in MDSC by a specific substance.
*본 발명의 다른 목적은 특정물질이 MITF의 유전자 발현 억제제 또는 MITF의 단백질 활성 억제제인지 선별하고, MDSC 활성 저해 약물을 선별하는 방법 및 이렇게 선별된 MITF 억제제를 포함하는 조성물을 제공하는 것이다.* Another object of the present invention is to provide a method for selecting whether a specific substance is an MITF gene expression inhibitor or an MITF protein activity inhibitor, and a MDSC activity inhibitory drug, and a composition containing the MITF inhibitor thus selected.
본 발명의 기술적 과제는 이상에서 언급한 기술적 과제들로 제한되지 않으며, 언급되지 않은 또 다른 기술적 과제는 아래의 기재로부터 해당 기술 분야의 통상의 기술자에게 명확하게 이해될 수 있을 것이다.The technical problem of the present invention is not limited to the technical problems mentioned above, and other technical problems not mentioned will be clearly understood by those skilled in the art from the following description.
본 발명의 일 실시태양에서 특정물질에 의한 MDSC에서의 MITF 유전자 발현 또는 MITF 단백질 발현 정도를 qRT-PCR, 웨스턴 블롯팅 및 유세포 분석법 (FACS)으로부터 선택된 어느 하나의 방법으로 분석하여 MDSC 활성 변화 정도를 예측하고 선별하는, MDSC 활성 저해 약물의 선별 방법을 제공한다.In one embodiment of the present invention, by analyzing the level of MITF gene expression or MITF protein expression in MDSC by a specific substance by any one method selected from qRT-PCR, Western blotting and flow cytometry (FACS), the degree of MDSC activity change A method for predicting and selecting MDSC activity inhibitory drugs is provided.
본 발명의 일 실시태양에서, MDSC 활성 저해 약물의 선별 방법은 상기 특정물질이 MITF의 유전자 발현 억제제 또는 MITF의 단백질 활성 억제제일 수 있다.In one embodiment of the present invention, in the method for selecting a drug for inhibiting MDSC activity, the specific substance may be an MITF gene expression inhibitor or an MITF protein activity inhibitor.
본 발명의 일 실시태양에서, MDSC 활성 저해 약물의 선별 방법은 (a) 골수 세포를 준비하는 단계; (b) 상기 골수 세포로부터 MDSC 분화 및 활성을 유도하는 단계; (c) 상기 (b) 단계에서 분화된 MDSC를 회수하여 MITF 유전자 또는 MITF 단백질 발현 정도를 분석하는 단계; 및 (d) 상기 (c)의 분석결과로부터 상기 특정물질 중 MDSC 활성 저해 약물을 선별하는 단계를 포함할 수 있다.In one embodiment of the present invention, the method for screening MDSC activity inhibitory drugs comprises the steps of (a) preparing bone marrow cells; (b) inducing MDSC differentiation and activity from the bone marrow cells; (c) recovering the MDSCs differentiated in step (b) and analyzing the level of MITF gene or MITF protein expression; and (d) selecting a MDSC activity-inhibiting drug from among the specific substances from the analysis result of (c).
본 발명의 일 실시태양에서, MDSC 활성 저해 약물의 선별 방법에 사용되는 상기 골수 세포는 종양을 갖는 개체로부터 얻은 골수 세포일 수 있다.In one embodiment of the present invention, the bone marrow cells used in the screening method for drugs inhibiting MDSC activity may be bone marrow cells obtained from a subject having a tumor.
본 발명의 일 실시태양에서, MDSC 활성 저해 약물의 선별 방법은 상기 (b) 단계에서 시험군 배지인 암세포 조건 배지에 MDSC 분화 유도 인자 및 상기 특정물질을 처리하고, 대조군 배지로서 상기 특정물질이 포함되지 않은 배지를 사용하여, MITF 유전자 또는 MITF 단백질 발현 정도를 비교할 수 있다.In one embodiment of the present invention, the method for screening MDSC activity inhibitory drugs is to treat MDSC differentiation inducing factors and the specific substance in the cancer cell condition medium, which is the test group medium in step (b), and include the specific substance as a control medium. The level of expression of the MITF gene or MITF protein can be compared using the untreated medium.
본 발명의 일 실시태양에서, MDSC 활성 저해 약물의 선별 방법은, 상기 (d) 단계에서 MITF 유전자 또는 MITF 단백질의 발현 정도가 qRT-PCR 분석법 시행시 직접적인 MITF 저해 물질의 경우 대조군 대비 시험군에서 qRT-PCR 측정값이 50 % 이상 저해된 경우에, 간접적인 경로로 MITF를 저해하는 물질의 경우 대조군 대비 시험군에서 30 % 이상 저해된 경우에 해당 물질을 MDSC 활성 저해 약물로 결정하는 단계를 추가로 포함할 수 있다.In one embodiment of the present invention, the screening method for MDSC activity inhibitory drugs is such that the expression level of the MITF gene or MITF protein in the step (d) is qRT-PCR in the test group compared to the control group in the case of direct MITF inhibitors when qRT-PCR analysis is performed. -If the PCR measurement value is inhibited by more than 50%, in the case of a substance that inhibits MITF through an indirect route, if it is inhibited by more than 30% in the test group compared to the control group, an additional step of determining the substance as an MDSC activity inhibitory drug can include
본 발명의 일 실시태양에서, MDSC 활성 저해 약물의 선별 방법은 상기 종양이 유방암, 간암, 위암, 결장암, 폐암, 비소세포성폐암, 골암, 췌장암, 피부암, 두부 또는 경부암, 자궁경부암, 난소암, 대장암, 소장암, 직장암, 항문부근암, 나팔관암종, 자궁내막암종, 자궁경부암종, 질암종, 음문암종, 호지킨병, 식도암, 소장암, 임파선암, 방광암, 담낭암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 전립선암, 선암종, 만성 또는 급성 백혈병, 림프구 림프종, 방광암, 신장 또는 수뇨관 암, 신장세포 암종, 신장골반 암종, 중추신경계 종양, 1차 CNS 림프종, 척수 종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군으로부터 선택된 1종 이상인 것일 수 있다.In one embodiment of the present invention, the screening method for MDSC activity inhibitory drugs is such that the tumor is breast cancer, liver cancer, stomach cancer, colon cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, Colon cancer, small intestine cancer, rectal cancer, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, lymphatic cancer, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, Parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, adenocarcinoma, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary CNS It may be at least one selected from the group consisting of lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.
본 발명의 일 실시태양에서, MDSC 활성 저해 약물의 선별 방법은 상기 MDSC 활성 저해 약물을 선별하기 위하여, 보조적으로 상기 MDSC와 T 세포를 공동배양하고, T 세포 증식 억제 정도를 판단하는 단계를 포함할 수 있다.In one embodiment of the present invention, the method for selecting a drug for inhibiting MDSC activity may include the steps of co-cultivating the MDSC and T cells and determining the degree of inhibition of T cell proliferation in order to screen for the drug for inhibiting MDSC activity. can
본 발명의 일 실시태양에서, MDSC 활성 저해 약물의 선별 방법은 상기 MITF의 유전자 발현 억제제가 MITF 유전자의 mRNA에 상보적으로 결합하는 안티센스 뉴클레오타이드, 앱타머, 짧은 헤어핀 RNA(small hairpin RNA, shRNA), 작은 간섭 RNA(small interfering RNA, siRNA), 마이크로 RNA(microRNA, miRNA) 및 리보자임(ribozyme)으로 이루어진 군으로부터 선택된 1종 이상일 수 있다.In one embodiment of the present invention, the method for screening MDSC activity inhibitory drugs includes antisense nucleotides, aptamers, short hairpin RNA (shRNA), It may be at least one selected from the group consisting of small interfering RNA (siRNA), microRNA (miRNA), and ribozyme.
본 발명의 일 실시태양에서, MDSC 활성 저해 약물의 선별 방법은 상기 MITF의 단백질 활성 억제제가 MITF 단백질에 특이적으로 결합하는 화합물, 펩티드, 펩티드 모방체, 기질 유사체, 앱타머, 및 항체로 이루어진 군으로부터 선택된 1종 이상일 수 있다.In one embodiment of the present invention, the screening method for MDSC activity inhibitory drugs is a group consisting of compounds, peptides, peptide mimetics, substrate analogs, aptamers, and antibodies that specifically bind to the MITF protein activity inhibitor. It may be one or more selected from.
본 발명의 일 실시태양에서, MDSC 활성 저해 약물의 선별 방법은 상기 특정 물질이 AMPK 활성 촉진제, ML-329, MITF-gRNA 클로닝된 벡터, 항HIV제로부터 선택되는 1종 이상일 수 있다.In one embodiment of the present invention, in the method for screening MDSC activity inhibitory drugs, the specific substance may be one or more selected from AMPK activity promoters, ML-329, MITF-gRNA cloned vectors, and anti-HIV agents.
본 발명의 일 실시태양에서, MDSC 활성 저해 약물의 선별 방법에 의해 선별된 MDSC 활성 저해 약물을 포함하고, T 세포 증식을 회복시켜주는 조성물에 관한 것일 수 있다.In one embodiment of the present invention, it may relate to a composition comprising an MDSC activity inhibitory drug selected by a screening method for an MDSC activity inhibitory drug and restoring T cell proliferation.
본 발명은 MITF(microphthalmia-associated transcription factor)의 유전자 발현 억제제 또는 MITF의 단백질 활성 억제제를 유효성분으로 함유하는, 골수-유래 억제세포(myeloid-derived suppressor cell; MDSC)에 의한 면역 반응 저하의 완화, 치료 또는 예방용 조성물을 제공한다.The present invention alleviates the decrease in immune response by myeloid-derived suppressor cells (MDSC) containing a gene expression inhibitor of MITF (microphthalmia-associated transcription factor) or a protein activity inhibitor of MITF as an active ingredient, A composition for treatment or prevention is provided.
또한, 본 발명은 상기 조성물을 유효성분으로 함유하는, 항암 보조제를 제공한다.In addition, the present invention provides an anticancer adjuvant containing the composition as an active ingredient.
아울러, 본 발명은 MITF의 유전자 발현 억제제 또는 MITF의 단백질 활성 억제제를 MDSC의 저해가 필요한 개체에 투여하는 단계를 포함하는, MDSC 저해방법을 제공한다.In addition, the present invention provides a method for inhibiting MDSC, comprising administering a gene expression inhibitor of MITF or a protein activity inhibitor of MITF to a subject in need of MDSC inhibition.
기타 실시 예들의 구체적인 사항들은 상세한 설명 및 도면들에 포함되어 있다.Details of other embodiments are included in the detailed description and drawings.
본 발명자들은 MDSC 저해에 의한 MDSC의 면역반응 저하를 완화하고, 항암면역치료제의 치료 효과를 향상시킬 수 있는 제제를 개발하기 위해서 MDSC 활성에 직접적으로 영향을 미치는 인자를 밝혀내기 위하여 노력한 결과, 암세포 미세환경에서 MDSC가 활성화되어 면역반응이 저하되고, 상기 MDSC의 활성화에 MITF가 관여한다는 사실을 밝혀내었다. 따라서, MITF의 억제제를 이용하여 MDSC를 저해할 수 있음을 확인한 것에 기초하여, 광범위한 물질들 중 MITF 억제제를 선별하여 MDSC 활성 저해 약물로 사용할 수 있음을 밝힘으로써 본 발명을 완성하였다. 이로부터 MDSC의 면역반응 저하의 완화 및 항암 면역 치료에 이용할 수 있는 유효성분을 보다 효과적으로 특정할 수 있다.The present inventors have made efforts to identify factors that directly affect MDSC activity in order to develop a formulation capable of alleviating the decline in the immune response of MDSC caused by MDSC inhibition and improving the therapeutic effect of anti-cancer immunotherapeutic agents. It was found that MDSCs are activated in the environment and the immune response is lowered, and that MITF is involved in the activation of MDSCs. Therefore, based on the fact that MDSC can be inhibited using a MITF inhibitor, the present invention was completed by selecting a MITF inhibitor among a wide range of substances and revealing that it can be used as a MDSC activity inhibitory drug. From this, it is possible to more effectively identify active ingredients that can be used for alleviating the decline in the immune response of MDSC and for anticancer immunotherapy.
구체적으로, 본 발명자들은 MITF 발현을 억제하면 필연적으로 MDSC의 활성이 저해되고, MITF 발현 증가시 MDSC 활성도 증가함을 밝혀냈다. 따라서 특정물질에 의한 MITF 발현 정도를 분석하는 것에 의해, 특정물질에 의한 MDSC 활성 반응결과를 예측할 수 있게 되었다. 또한, 이로부터 MDSC가 활성화된 개체나 이에 의해 T 세포가 억제된 것으로 확인되는 개체에 대하여, 생체 내에 투여되는 경우 가장 적합하게 작용할 것으로 기대되는 MITF 발현 억제제를 선별해낼 수 있다. 또한, 환자는 직접 투여하기 전에 본인에게 가장 적합할 것으로 기대되는 MITF 발현 억제제를 유효성분으로 포함하는 항암 조성물 또는 항암 보조제 조성물을 선택할 수 있게 되며, 기존에 치료가 진행 중이었던 경우에도 투여 조성물 변경여부나 새로운 약물 후보군을 고려할 수 있으며, MDSC 활성 반응 결과를 예측할 수 있으므로, 암의 종류나 예후를 고려하여 가장 적절한 MITF 억제제를 선택할 수 있도록 하는 기틀을 마련한다.Specifically, the present inventors found that suppressing MITF expression inevitably inhibits MDSC activity, and increasing MITF expression increases MDSC activity. Therefore, by analyzing the level of MITF expression by a specific substance, it is possible to predict the MDSC activation reaction result by a specific substance. In addition, from this, it is possible to select a MITF expression inhibitor that is expected to act most appropriately when administered in vivo to an individual whose MDSCs are activated or whose T cells are confirmed to be suppressed by the MDSC. In addition, the patient can select an anti-cancer composition or an anti-cancer adjuvant composition containing the MITF expression inhibitor as an active ingredient that is expected to be most suitable for the patient before direct administration, and whether or not the administration composition is changed even if the treatment is in progress However, since new drug candidates can be considered and MDSC activation response results can be predicted, the basis for selecting the most appropriate MITF inhibitor considering the type or prognosis of cancer is laid.
또한, 상기 선별된 MITF 억제제를 유효성분으로 함유하는 조성물을 개발하여 MDSC에 의한 면역반응 저하를 완화하고, 항암 면역치료 효율을 증대하는데 유용하게 사용할 수 있다.In addition, by developing a composition containing the selected MITF inhibitor as an active ingredient, it can be usefully used to alleviate the decrease in immune response caused by MDSC and increase the efficiency of anti-cancer immunotherapy.
도 1은 마우스의 골수유래 골수-유래 억제세포(myeloid-derived suppressor cell, MDSC) 제작방법을 모식화한 도이다.1 is a diagram schematically illustrating a method for producing myeloid-derived suppressor cells (MDSC) derived from mouse bone marrow.
도 2는 본 발명의 일 실시예에 따라 마우스의 골수에서 분리한 세포에 MDSC의 분화 유도 인자인 GM-CSF와 함께 암세포를 배양한 배지(tumor cell-conditioned medium; TCCM)를 처리한 후 MDSC의 분화(도 2A) 및 활성(도 2B) 변화를 확인한 도이다.Figure 2 shows the results of MDSC after treating cells isolated from the bone marrow of a mouse with a tumor cell-conditioned medium (TCCM) in which cancer cells are cultured together with GM-CSF, a differentiation-inducing factor for MDSC, according to an embodiment of the present invention. It is a diagram confirming the differentiation (FIG. 2A) and activity (FIG. 2B) changes.
도 3은 본 발명의 일 실시예에 따라 마우스의 골수에서 분리한 세포에 GM-CSF와 함께 TCCM을 처리한 후 MDSC에서 MITF(microphthalmia-associated transcription factor)의 유전자(도 3A) 및 단백질(도 3B) 발현 변화를 확인한 도이다.Figure 3 shows the gene (Fig. 3A) and protein (Fig. 3B) of microphthalmia-associated transcription factor (MITF) in MDSC after treating cells isolated from bone marrow of mice with GM-CSF and TCCM according to an embodiment of the present invention. ) is a diagram confirming expression changes.
도 4는 본 발명의 일 실시예에 따라 종양형성 마우스의 비장에서 분리한 세포(도 4A) 또는 마우스의 골수에서 분리한 세포(도 4B)에 GM-CSF와 함께 TCCM을 처리하여 MDSC의 분화 및 활성을 유도한 후, MDSC의 T 세포 증식 억제 변화를 확인한 도이다.Figure 4 shows the differentiation and differentiation of MDSCs by treating cells isolated from the spleen of a tumor-forming mouse (FIG. 4A) or the cells isolated from the bone marrow of a mouse (FIG. 4B) with TCCM along with GM-CSF according to an embodiment of the present invention. After inducing activity, it is a diagram confirming the change in MDSC's T cell proliferation inhibition.
도 5는 종양 형성 마우스에서 MDSC의 분화(도 5A), 활성(도 5B) 및 MITF 유전자 발현(도 5C) 변화를 확인한 도이다.5 is a diagram confirming changes in MDSC differentiation (FIG. 5A), activity (FIG. 5B), and MITF gene expression (FIG. 5C) in tumor-forming mice.
도 6은 본 발명의 일 실시예에 따라 마우스의 골수에서 분리한 세포에 MDSC의 활성을 유도하는 약물로 IL-18 및 IL-10을 GM-CSF와 함께 96시간 처리한 후(도 6A, 도 6B, 도 6E, 및 도 6F) 또는 본 발명의 일 실시예에 따라 마우스의 골수에서 분리한 세포에 GM-CSF을 처리하여 분화를 유도한 이후 IL-18을 24시간 동안 처리한 후(도 6C 및 도 6D), MDSC의 분화(도 6A 및 도 6C), 활성 및 MITF의 유전자 발현(도 6B 및 도 6D) 변화를 확인한 도이다.Figure 6 is a drug that induces the activity of MDSC in cells isolated from bone marrow of mice according to an embodiment of the present invention, after treating IL-18 and IL-10 together with GM-CSF for 96 hours (Figure 6A, Figure 6). 6B, FIG. 6E, and FIG. 6F) or after inducing differentiation by treating cells isolated from bone marrow of mice with GM-CSF according to an embodiment of the present invention, and then treating them with IL-18 for 24 hours (FIG. 6C and Fig. 6D), MDSC differentiation (Figs. 6A and 6C), activity, and MITF gene expression (Figs. 6B and 6D) confirming changes.
도 7은 본 발명의 일 실시예에 따라 마우스의 골수에서 분리한 세포에 MDSC의 활성을 유도하는 약물로 IL-4를 GM-CSF와 함께 처리한 후 MDSC의 분화(도 7A), 활성(도 7B) 및 MITF의 유전자 발현(도 7C) 변화를 확인한 도이다.Figure 7 is a drug for inducing MDSC activity in cells isolated from the bone marrow of mice according to an embodiment of the present invention, treated with IL-4 together with GM-CSF, and then MDSC differentiation (Fig. 7A), activity (Fig. 7B) and MITF gene expression (FIG. 7C) confirming changes.
도 8은 본 발명의 일 실시예에 따라 마우스의 골수에서 분리한 세포에 MDSC의 활성을 유도하는 약물로 지질다당류 (LPS; lipopolysaccharide)를 GM-CSF와 함께 처리한 후 MDSC의 분화(도 8A), 활성(도 8B) 및 MITF의 유전자 발현(도 8C) 변화를 확인한 도이다.Figure 8 shows differentiation of MDSC after treating cells isolated from bone marrow of mice with lipopolysaccharide (LPS) as a drug inducing MDSC activity together with GM-CSF according to an embodiment of the present invention (Figure 8A). , activity (FIG. 8B) and gene expression of MITF (FIG. 8C) confirming changes.
도 9는 본 발명의 일 실시예에 따라 마우스의 골수에서 분리한 세포에 MDSC의 활성을 유도하는 약물로 심바스타틴(Simvastatin, Sim), 로바스타틴(Lovastatin, Lova), 프로바스타틴(Provastatin, Prav), 로수바스타틴(Rosuvastatin, Rosu), 또는 아토바스타틴(Atorvastatin, Ator)을 GM-CSF와 함께 처리한 후 MDSC의 분화 변화를 확인한 도이다.9 shows drugs inducing the activity of MDSC in cells isolated from mouse bone marrow according to an embodiment of the present invention, such as Simvastatin (Sim), Lovastatin (Lova), Provastatin (Prav), and Rovastatin. It is a diagram confirming the differentiation change of MDSC after treatment with subastatin (Rosuvastatin, Rosu) or atorvastatin (Atorvastatin, Ator) together with GM-CSF.
도 10은 본 발명의 일 실시예에 따라 마우스의 골수에서 분리한 세포에 MDSC의 활성을 유도하는 약물로 심바스타틴(Simvastatin, Sim), 로바스타틴(Lovastatin, Lova), 프로바스타틴(Provastatin, Prav), 로수바스타틴(Rosuvastatin, Rosu), 또는 아토바스타틴(Atorvastatin, Ator)을 GM-CSF와 함께 처리한 후 MDSC의 활성(도 10A) 및 MITF의 유전자 발현(도 10B) 변화를 확인한 도이다.10 shows drugs inducing the activity of MDSCs in cells isolated from mouse bone marrow according to an embodiment of the present invention, such as Simvastatin (Sim), Lovastatin (Lova), Provastatin (Prav), and Rovastatin. It is a diagram confirming changes in MDSC activity (FIG. 10A) and MITF gene expression (FIG. 10B) after treatment of Rosuvastatin (Rosu) or Atorvastatin (Ator) with GM-CSF.
도 11은 본 발명의 일 실시예에 따라 마우스의 골수에서 분리한 세포에 GM-CSF를 처리하여 분화를 유도한 이후, MDSC의 활성을 유도하는 약물로 심바스타틴(Simvastatin, Sim), 로바스타틴(Lovastatin, Lova)을 24시간 동안 처리한 후 MDSC의 분화 변화를 확인한 도이다.FIG. 11 shows that cells isolated from bone marrow of mice are treated with GM-CSF to induce differentiation according to an embodiment of the present invention, and then simvastatin (Sim) and lovastatin (Lovastatin, Lova) for 24 hours, and it is a diagram confirming the differentiation change of MDSC.
도 12는 본 발명의 일 실시예에 따라 마우스의 골수에서 분리한 세포에 GM-CSF를 처리하여 분화를 유도한 이후, MDSC의 활성을 유도하는 약물로 심바스타틴(Simvastatin, Sim) 및 로바스타틴(Lovastatin, Lova) 을 24시간 동안 처리한 후 MDSC의 활성(도 12) 및 MITF의 유전자 발현(도 12) 변화를 확인한 도이다.FIG. 12 shows that cells isolated from bone marrow of mice are treated with GM-CSF to induce differentiation according to an embodiment of the present invention, and then Simvastatin (Sim) and Lovastatin (Lovastatin, Lova) for 24 hours, confirming changes in MDSC activity (FIG. 12) and MITF gene expression (FIG. 12).
도 13은 본 발명의 일 실시예에 따라 마우스의 골수에서 분리한 세포에 MDSC의 활성을 억제하는 약물로 ATRA(All-trans retinoic acid)를 GM-CSF와 함께 처리한 후 MDSC의 분화(도 13A), 활성(도 13B) 및 MITF의 유전자 발현(도 13C) 변화를 확인한 도이다.Figure 13 shows the differentiation of MDSC after treatment with ATRA (All-trans retinoic acid) together with GM-CSF as a drug that inhibits the activity of MDSC to cells isolated from bone marrow of mice according to an embodiment of the present invention (Fig. 13A ), activity (FIG. 13B) and gene expression of MITF (FIG. 13C) confirming changes.
도 14는 본 발명의 실시예에 따라 마우스의 골수에서 분리한 세포에 MITF 유도제로 IBMX를 GM-CSF와 함께 처리한 후 MDSC의 분화(도 14A), MDSC의 활성 및 MITF의 유전자 발현(도 14B 및 14C), 및 MDSC의 T 세포 증식 억제(도 14D) 변화를 확인한 도이다.14 shows MDSC differentiation (FIG. 14A), MDSC activity, and MITF gene expression (FIG. 14B) after treatment of cells isolated from bone marrow of mice with GM-CSF as an MITF inducer in cells isolated from mouse bone marrow according to an embodiment of the present invention. and 14C), and MDSC suppression of T cell proliferation (FIG. 14D) confirming changes.
도 15는 본 발명의 일 실시예에 따라 마우스의 골수에서 분리한 세포에 MITF 억제제로 5 μM 및 10 μM의 베르베린(Berberine)을 GM-CSF와 함께 처리한 후 MDSC의 분화 (도 15A 및 15B) 변화 및 10 μM의 베르베린(Berberine)을 GM-CSF와 함께 처리한 경우의 MITF의 유전자 발현(도 15C)을 확인한 도이다.15 shows MDSC differentiation after treating cells isolated from bone marrow of mice with 5 μM and 10 μM Berberine as an MITF inhibitor together with GM-CSF according to an embodiment of the present invention ( FIGS. 15A and 15B ) It is a diagram confirming the gene expression of MITF (FIG. 15C) when the change and 10 μM Berberine were treated together with GM-CSF.
도 16은 인간의 폐암 및 두경부암(H&N cancer) 조직에서 MITF를 발현하는 MDSC의 분포를 확인한 도이다.16 is a diagram confirming the distribution of MDSC expressing MITF in human lung cancer and head and neck cancer (H&N cancer) tissues.
도 17은 본 발명의 일 실시예에 따라 마우스의 골수에서 분리한 세포에 MITF 억제제로 ML-329를 GM-CSF와 함께 처리한 후 MDSC의 활성(도 17A) 및 MITF의 유전자 발현(도 17B) 변화를 확인한 도이다.17 shows MDSC activity (FIG. 17A) and MITF gene expression (FIG. 17B) after treatment of cells isolated from mouse bone marrow with GM-CSF and ML-329 as an MITF inhibitor according to an embodiment of the present invention. This is the confirmation of the change.
도 18은 본 발명의 일 실시예에 따라 마우스의 골수에서 분리한 세포에 ML-329를 GM-CSF 및 TCCM과 함께 처리한 후 MDSC의 분화(도 18A), 활성 및 MITF의 유전자 및 단백질 발현(도 18B 및 도 18C), MDSC의 T 세포 증식 억제(도 18D) 변화를 확인한 도이다.FIG. 18 shows MDSC differentiation (FIG. 18A), activity, and MITF gene and protein expression ( 18B and 18C), a diagram confirming changes in MDSC inhibiting T cell proliferation (FIG. 18D).
도 19는 본 발명의 일 실시예에 따라 종양형성 마우스의 비장에서 분리한 MDSC에 ML-329 또는 베르베린(BBR)을 GM-CSF 및 TCCM과 함께 처리한 후 MDSC에서 만들어지는 ROS 생성 변화를 확인한 도이다.19 is a diagram confirming changes in ROS generation produced in MDSC after treatment with GM-CSF and TCCM of MDSC isolated from the spleen of a tumorigenic mouse according to an embodiment of the present invention with ML-329 or berberine (BBR) am.
도 20은 본 발명의 일 실시예에 따라 RNA간섭(RNA interference, RNAi) 및 CRISPR 기술을 이용하여 MITF 발현을 억제한 MDSC에서 MITF의 단백질 발현(도 20A), MDSC의 ROS 생성(도 20B) 및 T 세포 증식 억제(도 20C) 변화를 확인하고, 본 발명의 일 실시예에 따라 MITF를 과발현한 MDSC에서 MITF의 단백질 발현(도 20D) 및 MDSC의 T 세포 증식 억제(도 20E) 변화를 확인한 도이다.20 shows MITF protein expression (FIG. 20A), ROS generation (FIG. 20B), and A diagram confirming changes in T cell proliferation inhibition (FIG. 20C) and MITF protein expression (FIG. 20D) and T cell proliferation inhibition (FIG. 20E) in MDSCs overexpressing MITF according to an embodiment of the present invention. am.
도 21은 본 발명의 일 실시예에 따라 마우스의 골수에서 분리한 세포에 MITF 억제제로 넬피나비르(Nelfinavir)를 GM-CSF 및 TCCM과 함께 처리한 후, MITF의 유전자 발현 저하(도 21A)를 확인하고, MDSC의 활성 변화(도 21B 및 도 21C)를 확인한 도이다.도 22는 본 발명의 일 실시예에 따라 종양 형성 마우스에 ML-329를 GM-CSF 및 TCCM과 함께 처리한 MDSC를 투여하는 방법(도 22A), 상기 종양 형성 마우스에서 종양 부피 변화(도 22B 및 도 22C) 및 상기 종양 형성 마우스의 종양 조직에서 MDSC의 population 변화(도 22D)를 확인한 도이다.FIG. 21 shows the reduction of MITF gene expression (FIG. 21A) after treatment of cells isolated from bone marrow of mice with MITF inhibitor, Nelfinavir, together with GM-CSF and TCCM according to an embodiment of the present invention. Figure 22 is a diagram confirming the activity change of MDSC (FIG. 21B and FIG. 21C). FIG. 22 is MDSC treated with ML-329 along with GM-CSF and TCCM administered to tumor-forming mice according to an embodiment of the present invention. (FIG. 22A), changes in tumor volume in the tumor-forming mice (FIGS. 22B and 22C), and changes in MDSC population in the tumor tissues of the tumor-forming mice (FIG. 22D).
아래에서는 첨부한 도면을 참조하여 본 발명의 실시 예에 대하여 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있도록 상세히 설명한다. 그러나 본 발명은 여러 가지 상이한 형태로 구현될 수 있으며 여기에서 설명하는 실시 예에 한정되지 않는다. Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein.
명세서 및 청구범위 전체에서, 어떤 부분이 어떤 구성 요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.Throughout the specification and claims, when a certain component is said to "include", it means that it may further include other components without excluding other components unless otherwise stated.
본 발명은 특정물질에 의한 MDSC에서의 MITF 유전자 발현 또는 MITF 단백질 발현 정도를 분석하여 MDSC 활성 변화 정도를 예측하고 선별하는, MDSC 활성 저해 약물의 선별 방법을 제공한다.The present invention provides a method for selecting a drug that inhibits MDSC activity, which predicts and selects the degree of change in MDSC activity by analyzing the level of MITF gene expression or MITF protein expression in MDSC by a specific substance.
본 발명은 MITF의 유전자 발현 억제제 또는 MITF의 단백질 활성 억제제를 유효성분으로 함유하는, 골수-유래 억제세포(myeloid-derived suppressor cell; MDSC)에 의한 면역 반응 저하의 완화, 치료 또는 예방용 조성물을 제공한다.The present invention provides a composition for alleviating, treating, or preventing a decrease in immune response caused by myeloid-derived suppressor cells (MDSC), containing an MITF gene expression inhibitor or a MITF protein activity inhibitor as an active ingredient. do.
본 발명에서 "특정 물질"이란, MDSC에서 MITF의 유전자 발현 또는 단백질 활성을 변화시킬 것으로 기대되는 물질을 지칭하며, 특히, MITF 유전자 발현 억제제, MITF 단백질 활성 억제제, MDSC 활성 저해제, MDSC 활성 억제제로 공지되거나 기대되는 물질 및 그의 염을 포함한다. In the present invention, "specific substance" refers to a substance expected to change the gene expression or protein activity of MITF in MDSC, and is particularly known as a MITF gene expression inhibitor, a MITF protein activity inhibitor, an MDSC activity inhibitor, and an MDSC activity inhibitor. It includes known or expected substances and salts thereof.
본 발명에서, 상기 MITF의 유전자 발현 억제제는 MITF 유전자의 mRNA에 상보적으로 결합하는 안티센스 뉴클레오타이드, 앱타머, 짧은 헤어핀 RNA(small hairpin RNA, shRNA), 작은 간섭 RNA(small interfering RNA, siRNA), 마이크로 RNA(microRNA, miRNA) 및 리보자임(ribozyme)으로 이루어진 군으로부터 선택된 1종 이상일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the gene expression inhibitor of MITF is antisense nucleotide, aptamer, short hairpin RNA (shRNA), small interfering RNA (siRNA), micro It may be one or more selected from the group consisting of RNA (microRNA, miRNA) and ribozyme, but is not limited thereto.
본 발명에서 MITF 억제제는 MITF 유전자 발현 억제제 및 MITF 단백질 활성 억제제를 포함하는 의미이며, MDSC 활성 저해는 MDSC가 보여주는 T 세포의 활성 억제효과가 사라짐을 의미한다In the present invention, MITF inhibitors include MITF gene expression inhibitors and MITF protein activity inhibitors, and inhibition of MDSC activity means that the effect of suppressing T cell activity shown by MDSCs disappears.
상기 "안티센스 뉴클레오타이드"는 왓슨-클릭 염기쌍에 정의된 바에 따라, DNA, 미성숙-mRNA 또는 성숙된 mRNA의 상보적 염기서열에 결합(혼성화)하여 DNA에서 단백질로서 유전정보의 흐름을 방해하는 것이다. 표적 서열에 특이성이 있는 안티센스 뉴클레오타이드의 성질은 그것들을 예외적으로 다기능이 되도록 한다. 안티센스 뉴클레오타이드는 모노머 단위의 긴 사슬이기 때문에 이들은 표적 RNA 서열에 대해 쉽게 합성될 수 있다. 최근 많은 연구들은 표적 단백질을 연구하기 위한 생화학적 수단으로 안티센스 뉴클레오타이드의 유용성을 증명하였다(Rothenberg et al., J. Natl. Cancer Inst., 81:1539-1544, 1999). 올리고뉴클레오타이드 화학 및 향상된 세포흡착, 표적결합 친화도 및 뉴클레아제 내성을 나타내는 뉴클레오타이드 합성 분야에서 최근 많은 진보가 있었으므로 안티센스 뉴클레오타이드의 사용은 새로운 형태의 억제제로 고려될 수 있다.The "antisense nucleotide", as defined in Watson-Crick base pairing, interferes with the flow of genetic information from DNA to protein by binding (hybridizing) to complementary nucleotide sequences of DNA, immature-mRNA or mature mRNA. The specific nature of antisense nucleotides for their target sequences makes them exceptionally multifunctional. Since antisense nucleotides are long chains of monomeric units, they can be easily synthesized against the target RNA sequence. A number of recent studies have demonstrated the usefulness of antisense nucleotides as a biochemical means to study target proteins (Rothenberg et al., J. Natl. Cancer Inst., 81:1539-1544, 1999). Since many recent advances have been made in the field of oligonucleotide chemistry and the synthesis of nucleotides that exhibit improved cell adhesion, target binding affinity and nuclease resistance, the use of antisense nucleotides can be considered as a new type of inhibitor.
상기 "짧은 헤어핀 RNA(small hairpin RNA)" 또는 "shRNA"는 단일 가닥으로 50-60개로 구성된 뉴클레오타이드를 의미하며, in vivo에서 스템-루프(stem-loop) 구조를 이루고 있다. 즉, shRNA는 RNA간섭(RNA interference; RNAi)을 통해 유전자 발현을 억제하기 위한 타이트한 헤어핀 구조를 만드는 RNA 서열이다. 5-10개의 뉴클레오타이드의 루프 부위 양쪽으로 상보적으로 15-30개의 뉴클레오타이드의 긴 RNA가 염기쌍을 이루어 이중가닥의 스템을 형성한다. shRNA는 일반적으로 발현되도록 하기 위하여 U6 프로모터를 포함하는 벡터를 통해 세포 내로 형질도입되며 대개 딸세포로 전달되어 유전자 발현억제가 유전되도록 한다. shRNA 헤어핀 구조는 세포 내 기작에 의하여 절단되어 siRNA가 된 후 RISC(RNA-induced silencing complex)에 결합한다. 이들 RISC는 mRNA에 결합하여 이를 절단한다. shRNA는 RNA 폴리머레이즈(polymerase)에 의해 전사된다.The "short hairpin RNA" or "shRNA" refers to a single strand consisting of 50 to 60 nucleotides, and has a stem-loop structure in vivo . That is, shRNA is an RNA sequence that creates a tight hairpin structure to inhibit gene expression through RNA interference (RNAi). Long RNAs of 15-30 nucleotides complementary to both sides of the loop of 5-10 nucleotides are base-paired to form a double-stranded stem. shRNAs are generally transduced into cells via a vector containing a U6 promoter for expression and are usually passed on to daughter cells to allow inheritance of gene repression. The shRNA hairpin structure is cleaved by intracellular mechanisms to become siRNA, which then binds to RNA-induced silencing complex (RISC). These RISCs bind to and cleave mRNA. shRNA is transcribed by RNA polymerase.
상기 "작은 간섭 RNA(small interfering RNA)" 또는 "siRNA"는 특정 mRNA의 절단(cleavage)을 통하여 RNA간섭 현상을 유도할 수 있는 짧은 이중사슬 RNA를 의미한다. 타겟 유전자의 mRNA와 상동인 서열을 가지는 센스 RNA 가닥과 이와 상보적인 서열을 가지는 안티센스 RNA 가닥으로 구성된다. siRNA는 타겟 유전자의 발현을 억제할 수 있기 때문에 효율적인 유전자 넉다운(knock-down) 방법으로서 또는, 유전자치료(gene therapy)의 방법으로 제공된다.The "small interfering RNA" or "siRNA" refers to a short double-stranded RNA capable of inducing RNA interference through cleavage of a specific mRNA. It consists of a sense RNA strand having a sequence homologous to the mRNA of the target gene and an antisense RNA strand having a sequence complementary thereto. Since siRNA can suppress the expression of a target gene, it is provided as an efficient gene knock-down method or as a gene therapy method.
상기 "마이크로 RNA(microRNA)" 또는 "miRNA"는 약 22개의 염기서열로 이루어진 짧은 non-coding RNA를 의미한다. 유전자의 발현 과정에서 전사 후 조절인자(post-transcriptional regulator)로서 기능을 한다고 알려져 있다. 상보적인 염기 서열을 가진 표적(target) mRNA에 상보적으로 결합함으로써 표적 mRNA들을 분해시키거나 단백질로 번역되는 것을 억제한다.The "microRNA" or "miRNA" refers to a short non-coding RNA consisting of about 22 nucleotide sequences. It is known to function as a post-transcriptional regulator in the process of gene expression. By complementarily binding to target mRNAs having complementary nucleotide sequences, target mRNAs are degraded or translation into proteins is inhibited.
또한, 상기 MITF의 단백질 활성 억제제는 MITF 단백질에 특이적으로 결합하는 화합물, 펩티드, 펩티드 모방체, 기질 유사체, 앱타머, 및 항체로 이루어진 군으로부터 선택된 1종 이상일 수 있으나, 이에 제한되는 것은 아니다.In addition, the MITF protein activity inhibitor may be at least one selected from the group consisting of compounds, peptides, peptide mimetics, substrate analogs, aptamers, and antibodies that specifically bind to the MITF protein, but is not limited thereto.
상기 "펩티드 모방체(peptide mimetics)"는 MITF 단백질의 결합 도메인을 억제하여 MITF 단백질의 활성을 억제하는 것이다. 펩티드 모방체는 펩티드 또는 비펩티드일 수 있고, psi 결합과 같은, 비펩티드 결합에 의해 결합된 아미노산으로 구성될 수 있다. 또한, "구조적으로 강제된(conformationally constrained)" 펩티드, 사이클릭 모방체(cyclic mimetics), 적어도 하나의 엑소사이클릭 도메인(exocyclic domain), 결합 부분(결합 아미노산) 및 활성 부위를 포함하는 사이클릭 모방체일 수 있다. 펩티드 모방체는 MITF 단백질의 이차구조 특성과 유사하게 구조화되고 항체 또는 수용성 수용체와 같은 거대한 분자의 억제 특성을 모방할 수 있으며, 천연의 길항제와 동등한 효과로 작용할 수 있는 신규한 소분자일 수 있다.The "peptide mimetics" inhibit the activity of the MITF protein by inhibiting the binding domain of the MITF protein. Peptidomimetics may be peptides or non-peptides, and may be composed of amino acids linked by non-peptide bonds, such as psi bonds. Also included are "conformationally constrained" peptides, cyclic mimetics, cyclic mimetics comprising at least one exocyclic domain, a binding moiety (binding amino acid) and an active site. can be chained Peptidomimetics can be novel small molecules that are structured similarly to the secondary structural properties of the MITF protein, can mimic the inhibitory properties of large molecules such as antibodies or water-soluble receptors, and can act with equivalent effects to natural antagonists.
상기 "앱타머(aptamer)"는 단일 사슬 DNA 또는 RNA 분자로서, SELEX(systematic evolution of ligands by exponential enrichment)라 불리는 올리고 뉴클레오타이드(oligonucleotide) 라이브러리를 이용한 진화적인 방법에 의해 특정 화학 분자나 생물학적 분자에 높은 친화력과 선별력을 갖고 결합하는 올리고머를 분리하여 수득할 수 있다. 앱타머는 표적에 특이적으로 결합하고 표적의 활성을 조정할 수 있는데, 예컨대, 결합을 통하여 표적의 기능을 차단할 수 있다.The "aptamer" is a single-stranded DNA or RNA molecule, and is high in specific chemical or biological molecules by an evolutionary method using an oligonucleotide library called SELEX (systematic evolution of ligands by exponential enrichment). Oligomers that bind with affinity and selectivity can be separated and obtained. An aptamer can specifically bind to a target and modulate the activity of the target, such as blocking the function of the target through binding.
상기 "항체"는 MITF 단백질에 특이적이고 직접적으로 결합하여 MITF 단백질의 활성을 효과적으로 억제할 수 있다. 상기 MITF 단백질에 특이적으로 결합하는 항체로는 폴리클로날(polyclonal) 항체 또는 모노클로날(monoclonal) 항체를 사용할 수 있다. 상기 MITF 단백질에 특이적으로 결합하는 항체는 당업자에게 알려진 공지의 방법으로 제작하여도 무방하며, 상업적으로 알려진 MITF 항체를 구입하여 사용할 수 있다. 상기 항체는 당업자에게 알려진 종래 방법에 따라 면역원인 MITF 단백질을 외부 숙주에 주사함으로써 제조될 수 있다. 외부 숙주는 마우스, 랫트, 양, 토끼와 같은 포유동물을 포함한다. 면역원은 근육 내, 복강 내 또는 피하 주사방법으로 주사되며, 일반적으로 항원성을 증가시키기 위한 보조제(adjuvant)와 함께 투여할 수 있다. 외부 숙주로부터 정기적으로 혈액을 채취하여 형성된 역가 및 항원에 대한 특이성을 보이는 혈청을 수거하여 항체를 분리할 수 있다.The "antibody" can specifically and directly bind to the MITF protein to effectively inhibit the activity of the MITF protein. As an antibody specifically binding to the MITF protein, a polyclonal antibody or a monoclonal antibody may be used. An antibody specifically binding to the MITF protein may be prepared by a known method known to those skilled in the art, and a commercially known MITF antibody may be purchased and used. The antibody may be prepared by injecting the immunogen MITF protein into an external host according to a conventional method known to those skilled in the art. External hosts include mammals such as mice, rats, sheep, and rabbits. Immunogens are injected intramuscularly, intraperitoneally or subcutaneously, and can be administered together with an adjuvant to increase antigenicity. Antibodies can be isolated by collecting serum showing titer and specificity for the antigen formed by regularly drawing blood from an external host.
본 발명에서, 상기 "골수-유래 억제세포" 또는 "MDSC"는 세포독성 T 림프구 (cytotoxic T lymphocyte), NK 세포의 활성을 저해함으로써 면역을 억제하는 기능을 한다. 자가면역과 같이 불필요한 과도한 면역반응을 억제하는 순기능이 있지만, 면역반응이 필요한 상황에서 면역을 억제하여 질병을 발생시키거나 악화시키거나 또는 적절한 치료를 방해하는 역기능도 있다. 예컨대, MDSC는 종양 또는 암 환자에서 많이 증가되어 있는데, 이는 암 백신 투여의 효과를 현저히 감소시킴으로써 암 백신의 효능을 무력화시킨다. 또한, 항암면역 치료제로 사용되고 있는 면역관문억제제(Imuune Checkpoint Inhibitor, ICI)에 대한 환자의 내성에 기여하여 항암면역치료제의 효율을 떨어뜨린다. 이러한 상황, 구체적으로 종양을 갖는 개체의 MDSC의 수를 효과적으로 감소시키거나, MDSC의 활성을 효과적으로 억제시킨다면 MDSC에 의한 면역반응 저하를 막아 암 백신 또는 항암 면역 치료제의 효능을 증대시킬 수 있고, 암 치료를 원활하고 효과적으로 수행할 수 있게 될 것이다. In the present invention, the "bone marrow-derived suppressor cells" or "MDSC" function to suppress immunity by inhibiting the activity of cytotoxic T lymphocytes and NK cells. Although there is a positive function of suppressing an unnecessary excessive immune response such as autoimmunity, there is also an adverse function of causing or exacerbating a disease by suppressing immunity in a situation where an immune response is required or preventing proper treatment. For example, MDSC are highly increased in tumors or cancer patients, which significantly reduces the effectiveness of cancer vaccine administration, thereby neutralizing the efficacy of cancer vaccines. In addition, it contributes to patient resistance to immune checkpoint inhibitors (ICI) used as anticancer immunotherapeutic agents, thereby reducing the effectiveness of anticancer immunotherapeutic agents. In this situation, specifically, if the number of MDSCs of a tumor-bearing individual is effectively reduced or the activity of MDSCs is effectively inhibited, the reduction of the immune response by MDSCs can be prevented to increase the efficacy of cancer vaccines or anticancer immunotherapeutic agents, and cancer treatment can be carried out smoothly and effectively.
본 발명에서 MDSC 활성 저해 약물을 선별하기 위하여 사용되는 MDSC는 종양을 가진 개체로부터 골수세포를 추출하여 이를 분화시켜서 얻어질 수 있다. In the present invention, MDSC used to select a drug inhibiting MDSC activity can be obtained by extracting bone marrow cells from a tumor-bearing individual and differentiating them.
상기 종양을 갖는 개체의 MDSC는 표현형이 CD11b+Gr1+PD-L1+인 것일 수 있으나, 이에 제한되는 것은 아니다.The MDSC of the individual having the tumor may have a CD11b + Gr1 + PD-L1 + phenotype, but is not limited thereto.
상기 종양은 구체적으로 유방암, 간암, 위암, 결장암, 폐암, 비소세포성폐암, 골암, 췌장암, 피부암, 두부 또는 경부암, 자궁경부암, 난소암, 대장암, 소장암, 직장암, 항문부근암, 나팔관암종, 자궁내막암종, 자궁경부암종, 질암종, 음문암종, 호지킨병, 식도암, 소장암, 임파선암, 방광암, 담낭암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 전립선암, 선암종, 만성 또는 급성 백혈병, 림프구 림프종, 방광암, 신장 또는 수뇨관 암, 신장세포 암종, 신장골반 암종, 중추신경계 종양, 1차 CNS 림프종, 척수 종양, 뇌간 신경교종 또는 뇌하수체 선종일 수 있으나, 이에 제한되는 것은 아니다.The tumor is specifically breast cancer, liver cancer, stomach cancer, colon cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, proximal anal cancer, fallopian tube carcinoma , endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, lymph gland cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer , prostate cancer, adenocarcinoma, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma , but is not limited thereto.
본 발명에서, 상기 조성물은 MDSC를 저해할 필요가 있는 개체에 투여하는 것이 바람직하다.In the present invention, the composition is preferably administered to a subject in need of inhibiting MDSC.
본 발명의 구체적인 실시예에서, 본 발명자들은 마우스의 골수에서 분리한 세포에 MDSC의 분화 유도 인자로 GM-CSF를 처리하여 마우스 골수유래 MDSC를 제작할 수 있다.In a specific embodiment of the present invention, the present inventors can prepare mouse bone marrow-derived MDSC by treating cells isolated from mouse bone marrow with GM-CSF as an MDSC differentiation inducer.
또한, 본 발명자들은 마우스의 골수에서 분리한 세포를 MDSC의 분화 유도 인자인 GM-CSF와 함께 마우스 유방암 세포주를 배양한 배지(tumor cell-conditioned medium; TCCM)에서 처리한 결과, GM-CSF에 의해 MDSC의 분화가 유도되고, TCCM에 의해 MDSC의 활성이 유도되며, 상기 활성이 유도된 MDSC에서 MITF의 유전자 및 단백질 발현이 증가하는 것을 확인하였다. 또한, 상기 TCCM에 의해 활성이 유도된 MDSC와 T 세포를 공동배양한 결과, 활성이 유도된 MDSC에 의해 CD3+CD8+ T 세포 수가 감소하는 것을 확인하였다. 상기의 결과를 통해 암세포 미세환경에서 MDSC가 활성화되어 면역반응이 저하되고, MITF가 상기 MDSC의 활성화의 표적 인자로서 MDSC의 활성화에 관여하는 것을 확인하였다.In addition, the present inventors treated cells isolated from bone marrow of mice in a tumor cell-conditioned medium (TCCM) in which a mouse breast cancer cell line was cultured together with GM-CSF, a differentiation-inducing factor for MDSC. It was confirmed that the differentiation of MDSC was induced, the activity of MDSC was induced by TCCM, and the expression of the gene and protein of MITF increased in the MDSC in which the activity was induced. In addition, as a result of co-culture of MDSC and T cells whose activity was induced by TCCM, it was confirmed that the number of CD3 + CD8 + T cells was decreased by MDSC whose activity was induced. Through the above results, it was confirmed that MDSCs are activated in the cancer cell microenvironment and the immune response is lowered, and that MITF is involved in the activation of MDSCs as a target factor for the activation of MDSCs.
또한, 본 발명자들은 종양 형성 마우스의 비장 및 종양 부위에서 MDSC를 획득하였고, 상기 종양 부위에서 얻은 MDSC에서 높은 활성화 및 MITF의 발현이 나타나는 것을 확인하였다. 또한, 폐암 및 두경부암 조직 주변에서 MITF를 발현하는 MDSC가 증가되어 있는 것을 확인하였다. 상기의 결과를 통해 암세포 미세환경에서 MDSC가 활성화되고, 상기 MDSC의 활성화에 MITF가 관여하는 것을 확인하였다.In addition, the present inventors obtained MDSCs from the spleen and tumor regions of tumor-forming mice, and confirmed that MDSCs obtained from the tumor regions showed high activation and expression of MITF. In addition, it was confirmed that MDSC expressing MITF increased around lung cancer and head and neck cancer tissues. Through the above results, it was confirmed that MDSCs are activated in the cancer cell microenvironment, and that MITF is involved in the activation of the MDSCs.
본 발명에서 "MDSC 분화 유도 인자"는 조혈전구세포(hematopoietic progenitor cell)의 증식과 분화를 조절하는 당단백질 호르몬과 이들의 작용을 모방한 약물들을 포함하며, 생체로부터 유래한 걸 직접적으로 정제한 것을 사용하거나, 재조합 DNA 기술을 이용해 대량 생산된 것을 사용할 수 있다. 예시적으로, 적혈구형성인자 (EPO), 골수성장인자(G-CSF), 과립구대식구집락자극인자(GM-CSF), 거핵구성장인자(MGF), 혈소판증식촉진인자(TPO), 인터루킨-11(IL-11) 등을 들 수 있으며, 바람직하게는 GM-CSF를 사용할 수 있으나, 이에 제한되지 않는다. In the present invention, "MDSC differentiation inducing factors" include glycoprotein hormones that regulate the proliferation and differentiation of hematopoietic progenitor cells and drugs that mimic their actions, and are directly purified from living organisms. It can be used or mass-produced using recombinant DNA technology. Illustratively, erythropoietic factor (EPO), bone marrow growth factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), megakaryocyte growth factor (MGF), platelet growth stimulating factor (TPO), interleukin-11 ( IL-11) and the like, and preferably GM-CSF may be used, but is not limited thereto.
또한, 본 발명자들은 마우스의 골수에서 분리한 세포에 MDSC의 분화 유도 인자인 GM-CSF와 함께 MDSC 활성을 유도하는 약물들, IL-18, IL-4, LPS 또는 스타틴(Statin) 계열 약물들을 처리한 결과, GM-CSF에 의해 MDSC의 분화가 유도되고, 상기 MDSC 활성 유도 약물들에 의해 MDSC의 활성이 유도되며, 상기 활성이 유도된 MDSC에서 MITF의 유전자 및 단백질 발현이 증가한 것을 확인하였다. 또한, 마우스의 골수에서 분리한 세포에 MDSC의 분화 유도 인자인 GM-CSF와 함께 MDSC 활성을 억제하는 약물로 ATRA(all-trans retinoic acid)를 처리한 결과, GM-CSF에 의해 MDSC의 분화가 유도되고, 상기 MDSC 활성 저해 약물에 의해 MDSC의 활성이 억제되며, 상기 활성이 억제된 MDSC에서 MITF의 유전자 발현이 감소한 것을 확인하였다. 상기의 결과를 통해 MDSC의 활성화에 MITF가 관여하는 것을 확인하였다.In addition, the present inventors treated cells isolated from mouse bone marrow with drugs that induce MDSC activity, IL-18, IL-4, LPS or statins along with GM-CSF, an MDSC differentiation inducer. As a result, it was confirmed that MDSC differentiation was induced by GM-CSF, MDSC activity was induced by the MDSC activity-inducing drugs, and the expression of the MITF gene and protein was increased in the activity-induced MDSC. In addition, as a result of treating cells isolated from mouse bone marrow with ATRA (all-trans retinoic acid), a drug that inhibits MDSC activity, along with GM-CSF, an MDSC differentiation-inducing factor, MDSC differentiation by GM-CSF induced, the MDSC activity was inhibited by the MDSC activity inhibitory drug, and it was confirmed that the MITF gene expression was reduced in the activity-inhibited MDSC. Through the above results, it was confirmed that MITF is involved in MDSC activation.
또한, 본 발명자들은 마우스의 골수에서 분리한 세포에 MDSC의 분화 유도 인자인 GM-CSF와 함께 MITF 유도제로 IBMX를 처리한 결과, GM-CSF에 의해 MDSC의 분화가 유도되고, IBMX에 의해 MITF의 발현이 증가하며, 이때 MDSC가 활성화되는 것을 확인하였다. 반면, 마우스의 골수 또는 비장에서 분리한 세포에 MDSC의 분화 유도 인자인 GM-CSF와 함께 MITF 억제제로 베르베린(Berberine) 또는 ML-329를 처리한 결과, GM-CSF에 의해 MDSC의 분화가 유도되나, 상기 MITF 억제제에 의해 MITF 발현이 감소하고, 이때 MDSC의 활성이 억제되는 것을 확인하였다. 또한, MDSC에서 MITF 발현을 억제한 결과, MDSC의 활성이 억제되는 것을 확인하였다. 아울러, 본 발명의 일 실시예에서 제작한 종양 형성 마우스에 ML-329를 사전 처리한 MDSC를 투여한 결과, 면역반응이 증진되어 종양 성장이 억제되고 종양 부위에서 MDSC의 침입 정도가 약화되는 것을 확인하였다. 상기의 결과를 통해 MITF의 억제제를 이용하여 MDSC의 활성을 저해할 수 있음을 확인하였다.In addition, the present inventors treated cells isolated from mouse bone marrow with IBMX as a MITF inducer along with GM-CSF, an MDSC differentiation inducer, and as a result, MDSC differentiation was induced by GM-CSF and MITF by IBMX. Expression increased, and at this time, it was confirmed that MDSC was activated. On the other hand, when cells isolated from mouse bone marrow or spleen were treated with GM-CSF, an MDSC differentiation-inducing factor, and berberine or ML-329 as a MITF inhibitor, MDSC differentiation was induced by GM-CSF. , it was confirmed that MITF expression was reduced by the MITF inhibitor, and at this time, the activity of MDSC was suppressed. In addition, as a result of suppressing MITF expression in MDSC, it was confirmed that the activity of MDSC was suppressed. In addition, as a result of administering MDSCs pre-treated with ML-329 to tumorigenic mice prepared in one embodiment of the present invention, it was confirmed that the immune response was enhanced, tumor growth was suppressed, and the degree of invasion of MDSCs at the tumor site was attenuated. did Through the above results, it was confirmed that the activity of MDSC can be inhibited using an inhibitor of MITF.
또한, 본 발명자들은 마우스의 골수에서 분리한 세포에 MDSC 활성유도를 위한 암세포 미세환경에서, MDSC의 분화 유도 인자인 GM-CSF와 함께 MITF 억제제로 항HIV제의 일종인 넬피나비르(Nelfinavir)를 처리한 결과, 넬피나비르에 의해 MITF의 발현이 감소하고, MDSC 활성이 저해되는 것을 확인하였다.In addition, the present inventors used Nelfinavir, a type of anti-HIV agent, as a MITF inhibitor along with GM-CSF, a differentiation inducer of MDSC, in a cancer cell microenvironment for inducing MDSC activity in cells isolated from mouse bone marrow. As a result of the treatment, it was confirmed that MITF expression was reduced and MDSC activity was inhibited by nelfinavir.
이와 같이, 본 발명자들에 의해, 암세포 미세환경에서 MDSC가 활성화되며, MDSC의 활성화에 MITF가 결정적으로 관여한다는 점이 밝혀졌다. 따라서, MITF의 억제제를 이용하면, MDSC를 저해할 수 있음을 확인하였으며, 따라서, MITF 억제제를 선별하고 그로부터 MDSC 활성을 예측할 수 있게 되었다. 이는 MITF 억제제를 유효성분으로 함유하는 조성물을 개발하는데 유용하게 사용될 수 있으며, MDSC 활성 저해 약물의 투여가 필요한 경우에, 약물을 결정하는 데에도 유용하게 사용될 수 있다. 즉, 본 발명은 MDSC에 의한 면역반응 저하를 완화하고, 항암 면역치료 효율을 증대하는데 유용하게 사용할 수 있다. As such, it was found by the present inventors that MDSCs are activated in the cancer cell microenvironment, and that MITF is critically involved in the activation of MDSCs. Therefore, it was confirmed that MDSC can be inhibited by using an inhibitor of MITF, and thus, it is possible to select an inhibitor of MITF and predict MDSC activity therefrom. This can be usefully used to develop a composition containing a MITF inhibitor as an active ingredient, and can also be usefully used to determine a drug when administration of an MDSC activity inhibitory drug is required. That is, the present invention can be usefully used to alleviate the decrease in immune response caused by MDSC and increase the efficiency of anti-cancer immunotherapy.
본 발명에서 특정물질이 MDSC 활성에 미칠 영향을 MITF 유전자 발현 또는 MITF 단백질에 미치는 영향을 분석하여 예측할 수 있다. 이를 위하여, MDSC 분화를 유도할 골수 세포를 준비하는데, 골수 세포는 종양을 가진 개체로부터 추출될 수 있으며, 추출된 골수 세포를 배지에서 MDSC 분화 및/또는 활성화시킬 수 있다. MDSC 분화는 MDSC 분화 유도 인자가 포함된 배지에서 골수세포를 배양함으로써 이루어질 수 있다. 분화 및 활성 유도는 동시에 또는 개별적으로 이루어질 수 있다. 분석을 위한 MDSC 활성화는 암세포 미세환경이 조성된 배지, 즉, TCCM에서 이루어질 수 있으며, 분석하려는 대상인 특정물질의 영향을 알기 위해, TCCM에 특정물질을 처리된 배지를 이용할 수 있거나 후속적으로 처리할 수 있다. 예를 들어서, 시험군으로서 암세포 미세환경이 조성되고 특정물질이 처리된 배지와 대조군으로 그렇지 않은 배지를 사용할 수 있거나, 또는 시험군과 대조군 모두 암세포 미세환경을 조성하고, 시험군에만 특정물질을 처리할 수도 있다. 또는 시험군으로서 암세포 미세환경이 조성된 배지와 대조군으로 그렇지 않은 배지를 사용한 후, 시험군의 MDSC를 회수한 후 특정물질을 처리할 수도 있다. 다양한 예시를 들었으나, 본 발명의 시험군과 대조군은 이에 한정되지 않으며, 필요에 따라 통상의 기술자가 적절히 설계할 수 있다.In the present invention, the effect of a specific substance on MDSC activity can be predicted by analyzing the effect on MITF gene expression or MITF protein. To this end, bone marrow cells to induce MDSC differentiation are prepared. The bone marrow cells may be extracted from a tumor-bearing individual, and the extracted bone marrow cells may be differentiated and/or activated by MDSC in a medium. MDSC differentiation can be achieved by culturing bone marrow cells in a medium containing MDSC differentiation-inducing factors. Differentiation and activity induction can occur simultaneously or separately. MDSC activation for analysis can be performed in a medium in which a cancer cell microenvironment is created, that is, TCCM. can For example, a medium in which a cancer cell microenvironment is created and treated with a specific substance can be used as a test group, and a medium in which a specific substance is not treated can be used as a control group, or a cancer cell microenvironment is created in both the test group and the control group, and only the test group is treated with a specific substance. You may. Alternatively, after using a medium with a cancer cell microenvironment as a test group and a medium without a cancer cell microenvironment as a control group, the MDSCs of the test group may be recovered and then treated with a specific substance. Although various examples have been given, the test group and the control group of the present invention are not limited thereto, and may be appropriately designed by a person skilled in the art if necessary.
분화 및 활성 유도가 완료된 MDSC를 회수하여 MITF 유전자 또는 MITF 단백질 발현 정도를 분석하여 특정물질이 MDSC 활성에 미치는 영향을 예측할 수 있다 The effect of a specific substance on MDSC activity can be predicted by recovering MDSCs that have completed differentiation and activity induction and analyzing the level of expression of the MITF gene or MITF protein.
구체적으로, 본 발명에서 분화 및 활성유도가 완료된 MDSC를 회수한 후, MITF 유전자 발현 정도를 분석하기 위해서, qRT-PCR를 사용할 수 있다. qRT-PCR을 사용하는 경우, MDSC의 활성 마커로서 iNOS, IL-10 및 TGF-β 등을 사용할 수 있으나, 이에 제한되지 않는다. 분화 및/또는 활성을 유도한 MDSC를 회수하고 적절한 용액, 예를 들면, TRIzol Reagent® Solution(Invitrogen)을 사용하여 총 RNA를 분리한다. 그 후, 분리한 총 RNA를 이용하여 qRT-PCR을 수행할 수 있다.Specifically, in the present invention, qRT-PCR can be used to analyze the level of MITF gene expression after recovery of MDSCs, which have been completely differentiated and activated. In the case of using qRT-PCR, iNOS, IL-10, TGF-β, etc. may be used as MDSC activity markers, but are not limited thereto. MDSCs induced to differentiate and/or activate are recovered and total RNA is isolated using an appropriate solution, for example, TRIzol Reagent® Solution (Invitrogen). Then, qRT-PCR can be performed using the isolated total RNA.
본 발명에서 분화 및 활성 유도가 완료된 MDSC를 회수한 후, MITF 단백질 발현 정도를 분석하기 위해서, 웨스턴 블럿팅을 수행할 수 있다. 예시적인 방법으로, 회수한 MDSC에 적절한 용액, 예를 들면, 세포 용해 버퍼를 처리하여 용해한 후, 세포 용해물을 전기영동하여 분리할 수 있다. 사용될 수 있는 일차 항체로는 예를 들어서, 항-MITF 항체 및 항-액티닌(actinin) 항체를 들 수 있으며, 이차 항체로는 HRP-접합 이차 항체를 고려할 수 있다. 이 때, 대조 단백질로 액티닌을 사용할 수 있다. In the present invention, in order to analyze the level of MITF protein expression after recovering the MDSCs whose differentiation and induction of activity have been completed, Western blotting can be performed. As an exemplary method, after dissolving the recovered MDSCs by treating them with an appropriate solution, for example, a cell lysis buffer, the cell lysate may be separated by electrophoresis. Primary antibodies that can be used include, for example, anti-MITF antibodies and anti-actinin antibodies, and as secondary antibodies, HRP-conjugated secondary antibodies can be considered. At this time, actinin can be used as a control protein.
시험군과 대조군의 결과를 비교하여, 특정물질이 MITF 억제제, 즉, MDSC 활성 억제제인지, 또는 MITF 발현 증가제, 즉, MDSC 활성 유도제인지를 판단할 수 있으며, 이로부터 체내 투입시 MDSC 활성 반응을 예측할 수 있다. 따라서, MDSC 활성 억제제 투여 등에 의해 MDSC의 활성이 억제되어, 체내 T 세포 기능의 회복으로 이어질 것을 기대할 수 있으며, MDSC 활성 증가제의 경우, MDSC에 의한 T 세포 증식을 억제할 것을 기대할 수 있다. 이로부터, MITF 억제제를 유효성분으로 포함하는 조성물을 제조할 수 있다. By comparing the results of the test group and the control group, it is possible to determine whether a specific substance is a MITF inhibitor, that is, an MDSC activity inhibitor, or a MITF expression increasing agent, that is, an MDSC activity inducer. Predictable. Therefore, it can be expected that MDSC activity is inhibited by administration of an MDSC activity inhibitor, leading to recovery of T cell function in the body, and in the case of an MDSC activity increasing agent, T cell proliferation by MDSC can be expected to be inhibited. From this, a composition containing the MITF inhibitor as an active ingredient can be prepared.
본 발명에서 MDSC 활성 저해 약물의 선별 방법은, MITF 유전자 또는 MITF 단백질의 발현 정도가 예를 들어, qRT-PCR 분석법을 이용하는 경우, 대조군 대비 시험군에서 qRT-PCR 측정값이 낮은 경우에 MDSC 저해약물로 결정할 수 있다. 예를 들어, 직접적인 MITF 저해 물질의 경우 대조군 대비 시험군에서 qRT-PCR 측정값이 바람직하게는 50 % 이상 저해된 경우에, 간접적인 경로로 MITF를 저해하는 물질의 경우 대조군 대비 시험군에서 바람직하게는 30 % 이상 저해된 경우일 수 있으나, 이 범위에 한정되는 것은 아니며, 통상의 기술자는 필요한 범위에서 저해의 정도를 결정할 수 있다.In the present invention, the screening method for MDSC activity inhibitory drugs is the MDSC inhibitory drug when the expression level of the MITF gene or MITF protein is low in the test group compared to the control group, for example, when qRT-PCR analysis is used. can be determined by For example, in the case of a direct MITF inhibitor, when the qRT-PCR measurement value is preferably inhibited by 50% or more in the test group versus the control group, in the case of a substance that inhibits MITF through an indirect route, the test group versus the control group preferably may be inhibited by 30% or more, but is not limited to this range, and a person skilled in the art may determine the degree of inhibition within a necessary range.
본 발명의 방법으로 선별된 MDSC 활성 저해 약물을 포함하는 조성물은 투여를 위해서 상기 기재한 유효성분 이외에 추가로 약제학적으로 허용 가능한 담체를 1종 이상 포함하여 약제학적 조성물로 바람직하게 제제화할 수 있다. The composition containing the MDSC activity inhibitory drug selected by the method of the present invention may be preferably formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration.
액상 용액으로 제제화되는 조성물에 있어서 허용 가능한 약제학적 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사용액, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다.In compositions formulated as liquid solutions, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added if necessary.
또한, 상기 본 발명의 조성물은 상기 유효 성분 이외에 약제학적으로 적합하고 생리학적으로 허용되는 보조제를 사용하여 제조될 수 있으며, 상기 보조제로는 부형제, 붕해제, 감미제, 결합제, 피복제, 팽창제, 윤활제, 활택제 또는 향미제 등의 가용화제를 사용할 수 있다.In addition, the composition of the present invention can be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant includes an excipient, a disintegrant, a sweetener, a binder, a coating agent, an expanding agent, and a lubricant , solubilizing agents such as lubricants or flavoring agents may be used.
또한, 본 발명의 조성물은 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 더 나아가 해당분야의 적절한 방법으로 Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA에 개시되어 있는 방법을 이용하여 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.In addition, the composition of the present invention may be formulated into injection formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets by additionally adding diluents, dispersants, surfactants, binders, and lubricants. Furthermore, using a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field, it can be preferably formulated according to the disease or component.
또한, 본 발명의 조성물은 정맥내, 동맥내, 복강내, 근육내, 동맥내, 복강내, 흉골내, 경피, 비측내, 흡입, 국소, 직장, 경구, 안구내 또는 피내 경로를 통해 통상적인 방식으로 투여할 수 있다. 본 발명의 조성물은 약제학적으로 유효한 양으로 투여한다. 본 발명에서, "약제학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.In addition, the composition of the present invention can be administered through conventional intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. method can be administered. The composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type, severity, activity of the drug, It may be determined according to factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.
구체적으로, 본 발명에 따른 조성물의 유효량은 환자의 나이, 성별, 체중에 따라 달라질 수 있으며, 일반적으로는 체중 1 kg 당 0.1 mg 내지 100 mg, 보다 구체적으로 0.5 mg 내지 10 mg을 매일 또는 격일 투여하거나 1일 1 내지 3회로 나누어 투여할 수 있다. 그러나 투여 경로, 질환의 중증도, 성별, 체중, 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 제한하는 것은 아니다.Specifically, the effective amount of the composition according to the present invention may vary depending on the patient's age, sex, and body weight, and is generally 0.1 mg to 100 mg per 1 kg of body weight, more specifically 0.5 mg to 10 mg per day or every other day. Alternatively, it may be divided into 1 to 3 doses per day. However, since it may increase or decrease according to the route of administration, severity of disease, sex, weight, age, etc., the dosage is not intended to limit the scope of the present invention in any way.
또한, 본 발명은 MITF(microphthalmia-associated transcription factor)의 유전자 발현 억제제 또는 MITF의 단백질 활성 억제제를 유효성분으로 함유하는, 골수-유래 억제세포(myeloid-derived suppressor cell; MDSC)에 의한 면역 반응 저하의 완화, 치료 또는 예방용 조성물을 유효성분으로 함유하는, 항암 보조제를 제공한다.In addition, the present invention relates to the reduction of immune response by myeloid-derived suppressor cells (MDSC) containing a gene expression inhibitor of MITF (microphthalmia-associated transcription factor) or a protein activity inhibitor of MITF as an active ingredient. Provided is an anti-cancer adjuvant containing a composition for relief, treatment or prevention as an active ingredient.
본 발명에서, 상기 MITF의 유전자 발현 억제제는 MITF 유전자의 mRNA에 상보적으로 결합하는 안티센스 뉴클레오타이드, 앱타머, 짧은 헤어핀 RNA(small hairpin RNA, shRNA), 작은 간섭 RNA(small interfering RNA, siRNA), 마이크로 RNA(microRNA, miRNA) 및 리보자임(ribozyme)으로 이루어진 군으로부터 선택된 1종 이상일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the gene expression inhibitor of MITF is antisense nucleotide, aptamer, short hairpin RNA (shRNA), small interfering RNA (siRNA), micro It may be one or more selected from the group consisting of RNA (microRNA, miRNA) and ribozyme, but is not limited thereto.
상기 MITF의 단백질 활성 억제제는 MITF 단백질에 특이적으로 결합하는 화합물, 펩티드, 펩티드 모방체, 기질 유사체, 앱타머, 및 항체로 이루어진 군으로부터 선택된 1종 이상일 수 있으나, 이에 제한되는 것은 아니다.The MITF protein activity inhibitor may be at least one selected from the group consisting of compounds, peptides, peptide mimetics, substrate analogs, aptamers, and antibodies that specifically bind to the MITF protein, but is not limited thereto.
본 발명에서, 상기 항암 보조제는 항암제와 병용 투여될 수 있고, 상기 항암 보조제는 MDSC의 활성을 억제하여 MDSC에 의한 면역반응 저하를 완화함으로써, 항암제의 효과를 유의적으로 상승시키는 항암 보조 효과를 나타낼 수 있다.In the present invention, the anticancer adjuvant may be administered in combination with an anticancer agent, and the anticancer adjuvant suppresses the activity of MDSC to mitigate the decrease in immune response by MDSC, thereby exhibiting an anticancer adjuvant effect that significantly increases the effect of the anticancer agent. can
상기 항암제는 화학치료제, 타겟화된 치료제, 항체 치료제, 면역치료제 및 호르몬 치료제로 이루어진 군으로부터 선택된 1종 이상일 수 있으나, 이에 제한되는 것은 아니다.The anticancer agent may be at least one selected from the group consisting of chemotherapeutic agents, targeted therapeutics, antibody therapeutics, immunotherapeutic agents, and hormone therapeutics, but is not limited thereto.
상기 화학치료제는 예를 들어, 대사길항물질(예를 들어, 폴산, 푸린, 및 피리미딘 유도체), 알킬화제(예를 들어, 질소 머스타드, 니트로소우레아, 백금, 알킬 설포네이트, 히드라진, 트리아젠, 아지리딘, 방추체 저해제, 세포독성제, 토포이소머라제 억제제 및 기타) 또는 저메틸화제(예를 들어, 제불라린, 이소티오시아네이트, 아자시티딘(5-아자시티딘), 5-플루오로-2'-데옥시시티딘, 5,6-디하이드로-5-아자시티딘 및 기타)가 있으나 이에 제한되는 것은 아니다.Such chemotherapeutic agents include, for example, antimetabolites (eg, folic acid, purine, and pyrimidine derivatives), alkylating agents (eg, nitrogen mustards, nitrosoureas, platinum, alkyl sulfonates, hydrazines, triazenes, aziridines, spindle inhibitors, cytotoxic agents, topoisomerase inhibitors and others) or hypomethylating agents (eg zebularine, isothiocyanate, azacitidine (5-azacytidine), 5-fluoro rho-2'-deoxycytidine, 5,6-dihydro-5-azacytidine and others), but are not limited thereto.
상기 타겟화된 치료제는 암 세포의 조절되지 않는 단백질에 대해 특이적인 제제로 예를 들어, 티로신 키나제 억제제, 예를 들어 악시티니브(Axitinib), 보수티니브(Bosutinib), 세디라니브(Cediranib), 다사티니브(Dasatinib), 에르로티니브(Erlotinib), 이마티니브(Imatinib), 게피티니브(Gefitinib), 라파티니브(Lapatinib), 레스타우르티니브(Lestaurtinib), 닐로티니브(Nilotinib), 세막사니브(Semaxanib), 소라페니브(Sorafenib), 수니티니브(Sunitinib), 및 반데타니브(Vandetanib), 또는 시클린-의존성 키나제 억제제, 예를 들어 알보시디브(Alvocidib) 및 셀리시크리브(Seliciclib)가 있으나 이에 제한되는 것은 아니다.The targeted therapeutic agent is an agent specific for a dysregulated protein of cancer cells, for example, a tyrosine kinase inhibitor such as Axitinib, Bosutinib, Cediranib , Dasatinib, Erlotinib, Imatinib, Gefitinib, Lapatinib, Lestaurtinib, Nilotinib , Semaxanib, Sorafenib, Sunitinib, and Vandetanib, or cyclin-dependent kinase inhibitors such as Alvocidib and Selicic Liv (Seliciclib), but is not limited thereto.
상기 항체 치료제는 암 세포의 표면 상에서 단백질에 특이적으로 결합하는 항체 제제로 예를 들어, 트라스투주맙(Trastuzumab), 리툭시맙(Rituximab), 토시투모맙(Tositumomab), 세툭시맙(Cetuximab), 파니투무맙(Panitumumab), 알렘투주맙(Alemtuzumab), 베바시주맙(Bevacizumab), 에드레콜로맙(Edrecolomab) 또는 겜투주맙(Gemtuzumab)이 있으나 이에 제한되는 것은 아니다.The antibody therapeutic is an antibody preparation that specifically binds to a protein on the surface of cancer cells, for example, Trastuzumab, Rituximab, Tositumomab, Cetuximab , Panitumumab, Alemtuzumab, Bevacizumab, Edrecolomab, or Gemtuzumab, but is not limited thereto.
상기 면역 치료제는 종양을 공격하기 위해 피검체의 자체 면역계를 유도하도록 설계된 제제로 예를 들어, 이필리루맙(Ipilimumab), 아벨루맙(Avelumab), 니볼루맙(Nivolumab) 또는 펨브롤리주맙(Pembrolizumab)이 있으나 이에 제한되는 것은 아니다.The immunotherapeutic agent is an agent designed to induce the subject's own immune system to attack the tumor, for example, Ipilimumab, Avelumab, Nivolumab or Pembrolizumab. but is not limited thereto.
상기 호르몬 치료제는 특정 암에서 호르몬을 제공하거나 차단함으로써 암의 성장을 억제하는 제제로 예를 들어, 타목시펜(Tamoxifen) 또는 디에틸스틸베스테롤(diethylstilbestrol)이 있으나 이에 제한되는 것은 아니다.The hormone therapy is an agent that suppresses the growth of cancer by providing or blocking hormones in a specific cancer, for example, but is not limited thereto, such as tamoxifen or diethylstilbestrol.
상기 항암제의 적절한 투여량은 이미 당업계에 널리 알려져 있으므로, 각 환자의 상태에 따라 당업계에 알려진 기준에 의해 투여할 수 있다. 구체적인 투여량 결정은 당업자의 수준 내에 있으며, 이의 1일 투여 용량은 예를 들어 구체적으로는 1 mg/kg/일 내지 10 g/kg/일, 더 구체적으로는 10 mg/kg/일 내지 100 mg/kg/일이 될 수 있으나, 이에 제한되지 않으며, 투여하고자 하는 대상의 연령, 건강 상태, 합병증 등 다양한 요인에 따라 달라질 수 있다.Since an appropriate dosage of the anticancer agent is already well known in the art, it may be administered according to standards known in the art according to each patient's condition. Specific dosage determination is within the level of those skilled in the art, and its daily dosage is, for example, specifically 1 mg/kg/day to 10 g/kg/day, more specifically 10 mg/kg/day to 100 mg /kg/day may be, but is not limited thereto, and may vary depending on various factors such as the age, health condition, and complications of the subject to be administered.
본 발명의 구체적인 실시예에서, 본 발명자들은 암세포 미세환경에서 MDSC가 활성화되어 면역반응이 저하되고, 상기 MDSC의 활성화에 MITF가 관여하며, 상기 MITF의 억제제를 이용하여 MDSC의 활성을 억제할 수 있음을 확인하였으므로, 상기 MITF 억제제를 항암 면역치료의 항암 보조제로 이용할 수 있다.In a specific embodiment of the present invention, the present inventors found that MDSCs are activated in the cancer cell microenvironment and the immune response is lowered, MITF is involved in the activation of the MDSCs, and the activity of MDSCs can be inhibited using the MITF inhibitor Since it was confirmed, the MITF inhibitor can be used as an anti-cancer adjuvant for anti-cancer immunotherapy.
또한, 본 발명은 MITF의 유전자 발현 억제제 또는 MITF의 단백질 활성 억제제를 MDSC의 저해가 필요한 개체에 투여하는 단계를 포함하는, MDSC 저해방법을 제공한다.In addition, the present invention provides a method for inhibiting MDSC, comprising administering a gene expression inhibitor of MITF or a protein activity inhibitor of MITF to a subject in need of MDSC inhibition.
상기 MITF의 유전자 발현 억제제 또는 MITF의 단백질 활성 억제제, 및 MDSC에 대한 구체적인 설명은 상기 조성물에 대한 설명과 동일한 바, 구체적인 설명은 상기 내용을 원용하고, 이하에서는 MDSC 저해방법에 특유한 구성에 대해서만 설명하도록 한다.The specific description of the gene expression inhibitor of MITF or the protein activity inhibitor of MITF, and MDSC is the same as the description of the composition, and the specific description uses the above content, and hereinafter, only the configuration specific to the MDSC inhibition method will be described. do.
본 발명에서, 상기 "골수-유래 억제세포 저해" 또는 "MDSC 저해"는 MDSC의 활성을 억제시키는 것뿐만 아니라, MDSC의 수를 감소시키는 것까지도 포함한다. 수를 감소시키는 것은 세포의 생성을 억제하는 것뿐만 아니라, 이미 생성된 세포를 사멸시키거나 다른 세포로 분화시키는 것도 포함한다. 그 외에도 생물학적 관점에서 "저해"라고 지칭되고 있는 모든 매커니즘이 포함된다.In the present invention, the "myeloid-derived suppressor cell inhibition" or "MDSC inhibition" includes not only inhibiting the activity of MDSC but also reducing the number of MDSC. Reducing the number includes not only inhibiting the production of cells, but also killing or differentiating cells that have already been produced. In addition, any mechanism that is referred to as "inhibition" from a biological point of view is included.
본 발명에서, 상기 MDSC의 저해가 필요한 개체는 구체적으로 MDSC의 저해가 필요한 종양을 갖는 개체일 수 있고, 상기 종양을 갖는 개체의 MDSC는 표현형이 CD11b+Gr1+PD-L1+인 것일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the individual in need of MDSC inhibition may specifically be an individual having a tumor in need of MDSC inhibition, and the MDSC of the individual having the tumor may have a CD11b + Gr1 + PD-L1 + phenotype, It is not limited thereto.
또한, 상기 종양은 구체적으로 유방암, 간암, 위암, 결장암, 폐암, 비소세포성폐암, 골암, 췌장암, 피부암, 두부 또는 경부암, 자궁경부암, 난소암, 대장암, 소장암, 직장암, 항문부근암, 나팔관암종, 자궁내막암종, 자궁경부암종, 질암종, 음문암종, 호지킨병, 식도암, 소장암, 임파선암, 방광암, 담낭암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 전립선암, 선암종, 만성 또는 급성 백혈병, 림프구 림프종, 방광암, 신장 또는 수뇨관 암, 신장세포 암종, 신장골반 암종, 중추신경계 종양, 1차 CNS 림프종, 척수 종양, 뇌간 신경교종 또는 뇌하수체 선종일 수 있으나, 이에 제한되는 것은 아니다.In addition, the tumor is specifically breast cancer, liver cancer, stomach cancer, colon cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, proximal anal cancer, Fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, lymph gland cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, Penile cancer, prostate cancer, adenocarcinoma, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma It may be, but is not limited thereto.
본 발명의 구체적인 실시예에서, 본 발명자들은 암세포 미세환경에서 MDSC가 활성화되어 면역반응이 저하되고, 상기 MDSC의 활성화에 MITF가 관여하며, 상기 MITF의 억제제를 이용하여 MDSC의 활성을 억제할 수 있음을 확인하였으므로, 상기 MITF 억제제를 MDSC의 저해가 필요한 개체에 투여하여 MDSC 관련 질환 치료에 이용할 수 있다.In a specific embodiment of the present invention, the present inventors found that MDSCs are activated in the cancer cell microenvironment and the immune response is lowered, MITF is involved in the activation of the MDSCs, and the activity of MDSCs can be inhibited using the MITF inhibitor Since it was confirmed, the MITF inhibitor can be administered to a subject in need of MDSC inhibition and used to treat MDSC-related diseases.
이하, 본 발명을 실시예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by examples.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.However, the following examples are only to illustrate the present invention, and the content of the present invention is not limited to the following examples.
<실시예 1> 골수-유래 억제세포(myeloid-derived suppressor cells, MDSC) 제작<Example 1> Production of myeloid-derived suppressor cells (MDSC)
<1-1> 마우스 골수유래 MDSC 제작<1-1> Production of mouse bone marrow-derived MDSC
Balb/c 마우스의 골수(bone marrow, BM)로부터 도 1의 모식도와 같이 골수-유래 억제세포(myeloid-derived suppressor cells, MDSC)를 제작하였다.Myeloid-derived suppressor cells (MDSC) were prepared from the bone marrow (BM) of Balb/c mice as shown in the schematic diagram of FIG. 1 .
구체적으로, 6 내지 8주령 된 Balb/c 마우스는 Saeronbio. Inc.(Republic of Korea)에서 구입하였다. 동물실험은 Institutional Ethical Committee of Sookmyung Women's University (SMWU-IACUC-1708-017-02)의 승인을 받아 수행하였다. 6 내지 8주령 된 Balb/c 마우스의 넙적다리뼈를 적출하여 뼈 안의 골수를 분리한 뒤, RBC lysis buffer(Sigma-Aldrich, St. Louis, MO)를 처리하여 적혈구를 제거하여 세포를 얻었다. 얻어진 골수세포를 5Х105 cells/㎖의 세포 수로 24-웰 플레이트에서 10 ng/㎖ 농도의 과립구 대식세포 콜로니 자극 인자(GM-CSF)가 포함된 RPMI 배지(Invitrogen, Grand Island, NY)로 96시간 동안 배양하여 MDSC의 분화를 유도하였다.Specifically, 6- to 8-week-old Balb/c mice were Saeronbio. Inc. (Republic of Korea). Animal experiments were performed with the approval of the Institutional Ethical Committee of Sookmyung Women's University (SMWU-IACUC-1708-017-02). The femur bones of 6- to 8-week-old Balb/c mice were removed, bone marrow was separated from the bones, and red blood cells were removed by treatment with RBC lysis buffer (Sigma-Aldrich, St. Louis, MO) to obtain cells. The obtained bone marrow cells were cultured in a 24-well plate at a cell number of 5Х10 5 cells/mL in RPMI medium (Invitrogen, Grand Island, NY) containing 10 ng/mL of granulocyte macrophage colony stimulating factor (GM-CSF) for 96 hours. During culture, the differentiation of MDSC was induced.
<1-2> 종양 형성 마우스에서의 MDSC 제작<1-2> Construction of MDSCs in tumor-forming mice
Balb/c 마우스에 유방암 세포주를 주사하여 종양 형성 마우스를 제작하고, 상기 종양 형성 마우스로부터 MDSC를 제작하였다.Tumor-forming mice were prepared by injecting breast cancer cell lines into Balb/c mice, and MDSCs were prepared from the tumor-forming mice.
구체적으로 6 내지 8주령 된 Balb/c 마우스에 마우스 유방암 세포주인 4T1 세포를 5Х105 cells/100 ㎕ 의 세포 수로 포함하는 100 ㎕ PBS를 오른쪽 옆구리 쪽에 피하 주사하여 종양을 형성하였다. 상기 마우스는 12시간 명/암 주기에서 온도 23.5 ± 1℃, 습도 50 ± 5%의 숙명여자대학교 동물 실험실에서 사육하였다. 2주 뒤 종양, 골수 및 비장을 각각 적출하여 세포를 분리하였다. 그 다음, 형광 결합된 항-CD11b 항체, 항-Gr1 항체 및 MDSC 활성 마커인 PD-L1 항체를 이용하여 염색한 후, FACS(Fluorescence-acitivated cell sorting) 분석을 수행하여 MDSC의 분화 및 활성을 확인하였다. 상기 항체는 eBioscience (San Diago, CA)에서 구입하였다.Specifically, 100 μl PBS containing 4T1 cells, a mouse breast cancer cell line, at a cell number of 5Х10 5 cells/100 μl was subcutaneously injected into the right flank of 6- to 8-week-old Balb/c mice to form tumors. The mice were bred in an animal laboratory at Sookmyung Women's University at a temperature of 23.5 ± 1 °C and a humidity of 50 ± 5% in a 12-hour light/dark cycle. After 2 weeks, the tumor, bone marrow and spleen were extracted and cells were isolated. Then, after staining using fluorescently coupled anti-CD11b antibody, anti-Gr1 antibody, and MDSC activity marker PD-L1 antibody, FACS (Fluorescence-activated cell sorting) analysis was performed to confirm MDSC differentiation and activity. did The antibody was purchased from eBioscience (San Diago, CA).
<실시예 2> 암세포 미세환경에서 MDSC에 의한 면역반응 저하 확인<Example 2> Confirmation of reduced immune response by MDSC in cancer cell microenvironment
<2-1> 암세포 미세환경에서 MDSC 활성 확인<2-1> Confirmation of MDSC activity in cancer cell microenvironment
암세포 미세환경에서 MDSC에 의해 면역반응이 저하되는지 알아보기 위하여, 암세포를 배양한 배지를 처리한 MDSC의 활성을 확인하였다.In order to examine whether the immune response is lowered by MDSC in the cancer cell microenvironment, the activity of MDSC treated with a medium in which cancer cells were cultured was confirmed.
구체적으로, 마우스 유방암 세포주인 4T1 세포를 10% 소태아혈청(FBS), 100 unit 항생제-항균제를 함유하는 RPMI 배지(Invitrogen, Grand Island, NY) 10 ml로 5% CO2, 37℃ 조건 하에서 3일 동안 배양하여 세포가 80% 포화 상태가 되도록 하였다. 그 다음, 배양 배지를 회수하고 4℃로 유지된 원심분리기에서 3000 Хg로 20분간 3000 NMWL(명목분자량 한계(nominal molecular weight limit)) 원심필터 (Merck Milipore, Billerica, MA)로 농축하여 암세포 조건 배지(TCCM; tumor cell-conditioned medium)을 획득하였다. 상기 <실시예 1-1>에서 개시된 방법과 동일한 방법으로 골수세포를 얻은 후, 5Х105 cells/㎖의 세포 수로 24-웰 플레이트에서 상기 획득한 TCCM 및 10 ng/㎖ 농도의 GM-CSF가 포함된 RPMI 배지에서 96시간 동안 배양하여 MDSC의 분화를 유도하였다. 대조군으로 GM-CSF가 포함된 RPMI 배지에서 분화를 유도한 MDSC를 이용하였다.Specifically, 4T1 cells, a mouse breast cancer cell line, were cultured in 10 ml of RPMI medium (Invitrogen, Grand Island, NY) containing 10% fetal bovine serum (FBS) and 100 units of an antibiotic-antimicrobial agent (Invitrogen, Grand Island, NY) in 5% CO 2 , 3 under 37°C conditions. Days of incubation allowed the cells to reach 80% confluency. Then, the culture medium was recovered and concentrated with a centrifugal filter (Merck Milipore, Billerica, MA) at 3000 NMWL (nominal molecular weight limit) for 20 minutes at 3000 Хg in a centrifuge maintained at 4 ° C. (TCCM; tumor cell-conditioned medium) was obtained. Bone marrow cells were obtained by the same method as described in <Example 1-1>, and the obtained TCCM and GM-CSF at a concentration of 10 ng/ml were placed in a 24-well plate at a cell number of 5Х10 5 cells/ml. Differentiation of MDSC was induced by culturing for 96 hours in the prepared RPMI medium. As a control, MDSCs differentiated in RPMI medium containing GM-CSF were used.
MDSC의 분화를 확인하기 위하여, 상기 분화 유도한 MDSC를 회수하여 형광 결합된 항-CD11b 항체 및 항-Gr1 항체로 염색한 후, FACS 분석을 수행하였다(도 2A).In order to confirm the differentiation of MDSCs, the differentiated MDSCs were recovered, stained with fluorescently coupled anti-CD11b antibodies and anti-Gr1 antibodies, and subjected to FACS analysis (FIG. 2A).
또한, MDSC의 활성을 확인하기 위하여, 상기 분화 유도한 MDSC를 회수하여 MDSC의 활성 마커인 iNOS, IL-10 및 TGF-β에 대한 qRT-PCR을 수행하였다. 상기 분화 유도한 MDSC를 회수하고 TRIzol Reagent® Solution(Invitrogen)을 사용하여 제조사의 절차에 따라 총 RNA를 분리하였다. 그 다음, 분리한 총 RNA를 이용하여 qRT-PCR을 수행하였다. M-MLV 역전사 키트(Promega, Madison, WI)와 oligo-(dT) 프라이머와 dNTP(Bioneer, Daejeon, Republic of Korea)를 사용하여 제조사의 절차에 따라 분리한 총 RNA를 역전사하고, ABI Real-time PCR 7500 시스템에서 iNOS, IL-10, TGF-β 및 시클로필린(cyclophilin)에 대한 프라이머 및 SYBR Green PCR Master Mix (Applied Bosystems, Foster City, CA)를 이용하여 제조사의 절차에 따라 정량적 PCR을 수행하였다. 이때 대조군으로 시클로필린을 사용하였다. iNOS, IL-10, TGF-β 및 시클로필린에 대한 프라이머는 Bioneer에서 구입하였다. 상기 실험은 3번 반복 수행하였다(도 2B).In addition, in order to confirm the activity of MDSC, the differentiation-induced MDSC was collected and subjected to qRT-PCR for MDSC activity markers iNOS, IL-10 and TGF-β. The differentiated MDSCs were recovered and total RNA was isolated using TRIzol Reagent® Solution (Invitrogen) according to the manufacturer's procedure. Then, qRT-PCR was performed using the isolated total RNA. Total RNA isolated was reverse transcribed using the M-MLV reverse transcription kit (Promega, Madison, WI), oligo-(dT) primers and dNTP (Bioneer, Daejeon, Republic of Korea) according to the manufacturer's procedure, and ABI Real-time Quantitative PCR was performed in a PCR 7500 system using primers for iNOS, IL-10, TGF-β and cyclophilin and SYBR Green PCR Master Mix (Applied Bosystems, Foster City, CA) according to the manufacturer's procedure. . At this time, cyclophilin was used as a control. Primers for iNOS, IL-10, TGF-β and cyclophilin were purchased from Bioneer. The experiment was repeated 3 times (Fig. 2B).
그 결과, 도 2에 나타낸 바와 같이, 대조군(GM-CSF가 포함된 RPMI 배지) 및 TCCM 처리군(GM+TCCM)에서 모두 유사한 정도로 MDSC의 분화가 유도됨을 확인하였다(도 2A). 반면, TCCM 처리군(GM+TCCM)의 경우 MDSC의 활성 마커로 iNOS, IL-10 및 TGF-β을 측정한 모든 경우에서, MDSC의 활성이 대조군에 비해 높게 나타나므로 TCCM에 의해 MDSC의 활성이 유도됨을 확인하였다(도 2B).As a result, as shown in FIG. 2, it was confirmed that MDSC differentiation was induced to a similar degree in both the control group (RPMI medium containing GM-CSF) and the TCCM-treated group (GM+TCCM) (FIG. 2A). On the other hand, in the case of the TCCM-treated group (GM+TCCM), in all cases where iNOS, IL-10, and TGF-β were measured as MDSC activity markers, MDSC activity was higher than that of the control group, indicating that MDSC activity was increased by TCCM. It was confirmed that it was induced (Fig. 2B).
<2-2> 암세포 미세환경에서 활성화된 MDSC의 MITF 발현 증가 확인<2-2> Confirmation of increased MITF expression of MDSC activated in cancer cell microenvironment
상기 실시예 <2-1>의 TCCM에 의해 활성이 유도된 MDSC에서 MITF(microphthalmia-associated transcription factor)의 유전자 및 단백질 발현 변화를 확인하였다.In the MDSCs whose activity was induced by TCCM of Example <2-1>, changes in the gene and protein expression of MITF (microphthalmia-associated transcription factor) were confirmed.
구체적으로, MITF의 유전자 발현을 확인하기 위하여 상기 실시예 <2-1>에서 회수한 MDSC를 이용하여 상기 실시예 <2-1>에 기재된 방법과 동일한 방법으로 MITF에 대한 qRT-PCR을 수행하였다. MITF에 대한 프라이머는 Bioneer에서 구입하였다(도 3A). Specifically, qRT-PCR for MITF was performed in the same manner as described in Example <2-1> using MDSC recovered in Example <2-1> to confirm the gene expression of MITF. . Primers for MITF were purchased from Bioneer (FIG. 3A).
또한, MITF의 단백질 발현을 확인하기 위하여 상기 실시예 <2-1>에서 회수한 MDSC를 이용하여 웨스턴 블럿팅을 수행하였다. 이를 위해 상기 실시예 <2-1>에서 회수한 MDSC에 세포 용해 버퍼를 처리하여 용해하였다. 세포 용해물을 10% SDS-PAGE 겔로 전기영동하여 분리하고, PVDF 멤브레인에 옮겼다. 그 다음, 일차 항체로 항-MITF 항체 및 항-액티닌(actinin) 항체를 처리하여 반응시킨 후, 상기 막에 붙은 일차 항체에 HRP-접합 이차 항체를 붙이고, 이를 증강 화학발광기법(PicoEPD쪠 Western Reagent kit, ELPIS-Biotech, 대전, 대한민국)을 이용하여 LAS-3000 영상 시스템(FUJIFILM 사, 도쿄, 일본)으로 분석하였다. 액티닌은 대조 단백질로 사용하였다(도 3B). 또한, MDSC의 활성을 확인하기 위하여 일차 항체로 항-Arg1 항체 및 항-pSTAT3 항체를 처리하여 상기에 기재된 바와 같이 웨스턴 블럿팅을 수행하였다.In addition, in order to confirm the protein expression of MITF, Western blotting was performed using the MDSC recovered in Example <2-1>. To this end, the MDSCs recovered in Example <2-1> were treated with a cell lysis buffer and dissolved. The cell lysate was separated by electrophoresis on a 10% SDS-PAGE gel and transferred to a PVDF membrane. Then, after treating and reacting the anti-MITF antibody and the anti-actinin antibody with the primary antibody, HRP-conjugated secondary antibody is attached to the primary antibody attached to the membrane, and this is enhanced chemiluminescence technique (PicoEPD? Western It was analyzed with a LAS-3000 imaging system (FUJIFILM, Tokyo, Japan) using a Reagent kit, ELPIS-Biotech, Daejeon, Korea. Actinin was used as a control protein (Fig. 3B). In addition, in order to confirm the activity of MDSC, Western blotting was performed as described above by treating anti-Arg1 antibody and anti-pSTAT3 antibody as primary antibodies.
그 결과, 도 3에 나타낸 바와 같이, TCCM 처리군(GM+TCCM)의 경우 대조군(GM-CSF)에 비해 MITF의 유전자 발현(도 3A) 및 단백질 발현(도 3B)이 증가하고, Arg1 및 STAT3 인산화가 증가하는 것으로 나타나므로, MITF가 TCCM에 의해 활성이 유도된 MDSC에서 특이적으로 발현되는 표적 인자임을 확인하였다.As a result, as shown in FIG. 3, in the case of the TCCM-treated group (GM+TCCM), the gene expression (FIG. 3A) and protein expression (FIG. 3B) of MITF increased compared to the control group (GM-CSF), and Arg1 and STAT3 Since phosphorylation was shown to increase, it was confirmed that MITF was a target factor specifically expressed in MDSCs whose activity was induced by TCCM.
<2-3> 암세포 미세환경에서 MDSC에 의한 면역반응 저하 확인<2-3> Confirmation of reduced immune response by MDSC in cancer cell microenvironment
MDSC는 T 세포의 증식 및 기능을 억제하여 면역반응을 저하하는 것으로 알려져 있다. 이에 암세포 미세환경에서 MDSC에 의해 면역반응이 저하되는지 알아보기 위하여, 암세포를 배양한 배지를 처리한 MDSC를 이용하여 T 세포 증식 억제 정도를 확인하였다.MDSCs are known to suppress the proliferation and function of T cells to lower the immune response. Therefore, in order to determine whether the immune response is reduced by MDSC in the cancer cell microenvironment, the degree of inhibition of T cell proliferation was confirmed using MDSC treated with a medium in which cancer cells were cultured.
구체적으로, 종양형성 마우스의 비장에서 MACS cell separation kit(Miltenyi Biotec GmbH, Bergisch Gladbach, Germany)를 이용하여 제조사의 절차에 따라 분리한 세포를 5Х105 cells/㎖의 세포 수로 24-웰 플레이트에서 상기 실시예 <2-1>에서 획득한 TCCM 및 10 ng/㎖ 농도의 GM-CSF가 포함된 RPMI 배지로 24시간 동안 배양하여 MDSC의 분화 및 활성을 유도하였다.Specifically, cells isolated from the spleen of tumorigenic mice using a MACS cell separation kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer's procedure were carried out in a 24-well plate at a cell count of 5Х10 5 cells/ml. MDSC differentiation and activity were induced by culturing for 24 hours in RPMI medium containing TCCM obtained in Example <2-1> and GM-CSF at a concentration of 10 ng/ml.
또한, T 세포를 얻기 위해, 6 내지 8주령 된 Balb/c 마우스의 비장을 적출하여 세포를 분리하였다. 그 다음, 형광 결합된 항-CD3 항체를 이용하여 염색한 후, FACS를 이용하여 T 세포를 분리하였다. 분리한 T 세포를 2.5 μM CFSE로 7 분간 염색하여 CFSE 표지(Labeling)을 수행하였다. 5Х105 개의 세포수의 CFSE 표지된 CD3+ T 세포를 항-CD3 단일 클론 항체 및 가용성 항-CD28 단일 클론 항체로 코팅한 플레이트에서 2시간 동안 자극한 후, 상기 종양형성 마우스의 비장에서 분리한 세포의 분화 및 활성을 유도하여 획득한 MDSC(도 4A) 및 상기 실시예 <2-1>에서 회수한 MDSC(도 4B)를 5Х105 세포 수 또는 10Х105 세포 수로 넣어주고, 3일 동안 배양하였다. 배양 3일 후 형광 결합된 항-CD8 항체를 이용하여 염색한 후, FACS 분석을 수행하여 CD8+ T 세포의 증식 정도를 측정하였다. 상기 항체는 eBioscience사에서 구입하였다. In addition, to obtain T cells, cells were isolated by removing the spleens of 6- to 8-week-old Balb/c mice. Then, after staining with a fluorescently coupled anti-CD3 antibody, T cells were isolated using FACS. The isolated T cells were stained with 2.5 μM CFSE for 7 minutes to perform CFSE labeling. 5Х10 CFSE-labeled CD3 + T cells of 5 cells were stimulated on plates coated with anti-CD3 monoclonal antibody and soluble anti-CD28 monoclonal antibody for 2 hours, and then cells isolated from the spleens of the tumorigenic mice. MDSC obtained by inducing differentiation and activity of (Fig. 4A) and MDSC recovered in Example <2-1> (Fig. 4B) were added at 5Х10 5 cells or 10Х10 5 cells and cultured for 3 days. After 3 days of culture, after staining with a fluorescently coupled anti-CD8 antibody, FACS analysis was performed to measure the proliferation of CD8 + T cells. The antibody was purchased from eBioscience.
그 결과, 도 4에 나타낸 바와 같이, TCCM 처리군의 MDSC(GM+TCCM)와T 세포를 공동배양할 경우, 대조군(GM-CSF)의 MDSC와 공동배양한 경우에 비해 CD3+CD8+ T 세포 수가 감소하므로, TCCM에 의해 활성이 유도된 MDSC가 T 세포 증식을 억제함을 확인하였다.As a result, as shown in Figure 4, when the MDSC (GM + TCCM) and T cells of the TCCM treatment group were co-cultured, compared to the case of co-culture with the MDSC of the control group (GM-CSF), CD3 + CD8 + T cells Since the number decreased, it was confirmed that MDSCs whose activity was induced by TCCM inhibited T cell proliferation.
상기 결과를 통해 암세포 미세환경에서 활성화된 MDSC가 면역반응을 저하하고, MITF가 상기 MDSC의 활성화의 표적 인자로서 MDSC의 활성화에 관여하는 것을 알 수 있다.Through the above results, it can be seen that activated MDSC in the cancer cell microenvironment lowers the immune response, and MITF is involved in the activation of MDSC as a target factor for the activation of the MDSC.
<실시예 3> 종양 형성 마우스에서의 MDSC의 활성 및 MITF 발현 확인<Example 3> Confirmation of MDSC activity and MITF expression in tumor-forming mice
종양 형성 마우스에서 MDSC가 활성화되고, 상기 MDSC의 활성화에 MITF가 관여하는지 알아보기 위하여, 종양 형성 마우스의 MDSC를 이용하여 이의 활성 및 MITF의 유전자 발현 변화를 확인하였다.In order to determine whether MDSCs are activated in tumorigenic mice and whether MITF is involved in the activation of MDSCs, MDSCs from tumorigenic mice were used to confirm their activity and changes in MITF gene expression.
구체적으로, MDSC의 활성을 확인하기 위하여 상기 실시예 <1-2>에 개시된 방법과 동일한 방법으로 종양 형성 마우스의 비장 및 종양 각각에서 세포를 분리한 후 FACS 분석을 수행하였다(도 5A). 또한, 상기 FACS 분류기(sorter)로 CD11b+Gr1+ MDSC를 회수하여 상기 실시예 <2-1>에 기재된 방법과 동일한 방법으로 qRT-PCR을 수행하였다(도 5B). 대조군으로는 분리(sorting)하기 전의 종양 형성 마우스의 비장을 이용하였다.Specifically, in order to confirm the activity of MDSC, FACS analysis was performed after separating cells from each of the spleen and tumor of tumor-forming mice in the same manner as described in Example <1-2> above (FIG. 5A). In addition, CD11b + Gr1 + MDSC were recovered using the FACS sorter, and qRT-PCR was performed in the same manner as described in Example <2-1> (FIG. 5B). As a control, the spleen of a tumor-forming mouse before sorting was used.
또한, MITF의 유전자 발현을 확인하기 위하여, 상기 회수한 CD11b+Gr1+ MDSC를 이용하여 상기 실시예 <2-2>에 기재된 방법과 동일한 방법으로 qRT-PCR을 수행하였다(도 5C). In addition, to confirm the gene expression of MITF, qRT-PCR was performed using the recovered CD11b + Gr1 + MDSC in the same manner as described in Example <2-2> (FIG. 5C).
그 결과, 도 5에 나타낸 바와 같이, 종양 형성 마우스의 비장 및 종양 부위에서 얻은 MDSC는 대조군에 비해 활성화된 형태를 띠고(도 5A 및 도 5B), MITF의 유전자 발현이 증가해 있음을 확인하였다(도 5C). 특히, 종양 부위에서 얻은 MDSC가 비장 부위에서 얻은 것에 비해 활성화가 더 많이 되어 있고, MITF의 발현이 현저히 높게 나타나는 것을 확인하였다.As a result, as shown in FIG. 5, it was confirmed that MDSCs obtained from the spleen and tumor region of tumor-forming mice were activated compared to the control group (FIG. 5A and FIG. 5B), and the gene expression of MITF was increased ( Figure 5C). In particular, it was confirmed that MDSCs obtained from the tumor area were more activated than those obtained from the spleen area, and the expression of MITF was remarkably high.
상기 결과를 통해 암세포 미세환경에서 MDSC가 활성화되고, 상기 MDSC의 활성화에 MITF가 관여하는 것을 알 수 있다.Through the above results, it can be seen that MDSCs are activated in the cancer cell microenvironment, and MITF is involved in the activation of the MDSCs.
<실시예 4> 종양 조직에서 MDSC 및 MITF 발현 확인<Example 4> Confirmation of MDSC and MITF expression in tumor tissue
종양 조직에서 MDSC가 활성화되고, 상기 MDSC의 활성화에 MITF가 관여하는지 알아보기 위하여, 종양 조직에서 MDSC 및 MITF의 발현 변화를 확인하였다.In order to determine whether MDSCs are activated in tumor tissues and whether MITF is involved in the activation of MDSCs, changes in the expression of MDSCs and MITFs in tumor tissues were confirmed.
구체적으로, 폐암과 두경부암 조직 주변과 그에 상응하는 암이 없는 림프절 부위 조직 10 샘플 각각에 대하여 서울대학교 병리학 교실(Prof. Yoon Kyung Jeon)에서 조직염색을 수행하였다. 구체적으로, 조직을 4 % 포름 알데히드 용액에 고정시킨 후, 농도별 에탄올(70-100 %)에서 탈수시키고 파라핀에 끼워 넣었다. 조직을 마이크로톰으로 절편화하고(두께 4 μm) 헤마톡실린(hematoxylin)과 에오신(eosin)으로 염색(H&E)하였다. 광학 현미경(Olympus, Japan)으로 단면을 관찰하고 Х400 배율로 촬영하였다. IHC(immunohistochemistry)의 경우 조직 절편을 PBS로 희석한 3% 정상 말 혈청으로 30 분간 차단하였다. 절편을 블로킹하고 4℃에서 밤새 CD11b, CD14, MITF 항체(항-마우스 단일클론 일차항체(Cat. No 790-4367), Ventana Medical Systems, Oro Valley, AZ)와 함께 적절한 희석 배수(1:100 희석)에서 반응시켰다. 슬라이드를 PBS로 세척한 후, 아비딘-비오틴-퍼옥시다제 복합체 (ABC; Vector Laboratories, Burlingame, CA를 세척하였다. 슬라이드를 세척하고, 퍼옥시다아제 반응을 디아미노벤지딘 및 퍼옥사이드로 전개시키고, 아쿠아-마운트(Aqua-Mount)에 장착하여 광학 현미경 (Olympus) 하에서 Х400 배율로 평가하였다. 암조직과 림프절 조직에서 고배율시야(High Power Field, HPF)내에서 MITF+/CD11b+/CD14+인 MDSC의 수는 삼중양성인 세포들을 각각 세 군데 임의의 고배율시야내에서 count된 것의 평균값으로 표현하였다.Specifically, tissue staining was performed at Seoul National University's Department of Pathology (Prof. Yoon Kyung Jeon) for each of 10 samples of lung cancer and head and neck cancer tissues and their corresponding cancer-free lymph node tissue samples. Specifically, the tissues were fixed in 4% formaldehyde solution, dehydrated in ethanol (70-100%) by concentration, and embedded in paraffin. Tissues were sectioned with a microtome (thickness: 4 μm) and stained (H&E) with hematoxylin and eosin. Cross sections were observed under an optical microscope (Olympus, Japan) and photographed at Х400 magnification. For immunohistochemistry (IHC), tissue sections were blocked with 3% normal horse serum diluted in PBS for 30 minutes. Sections were blocked and incubated at appropriate dilution (1:100 dilution) with CD11b, CD14, MITF antibodies (anti-mouse monoclonal primary antibody (Cat. No 790-4367), Ventana Medical Systems, Oro Valley, AZ) overnight at 4°C. ) was reacted at. After washing the slides with PBS, an avidin-biotin-peroxidase complex (ABC; Vector Laboratories, Burlingame, Calif.) was washed. The slides were washed, the peroxidase reaction was developed with diaminobenzidine and peroxide, and aqua- Mounted on a mount (Aqua-Mount) and evaluated under an optical microscope (Olympus) at Х400 magnification The number of MITF + /CD11b + /CD14 + MDSCs in a high power field (HPF) in cancer tissue and lymph node tissue was expressed as the average value of counts of triple positive cells in an arbitrary high-magnification field of three places, respectively.
그 결과, 도 16에 나타낸 바와 같이, 종양 주변 조직에서 MITF로 염색된 MDSC의 분포가 뚜렷한 반면, 종양이 없는 림프절 부위 조직에서는 MITF로 염색된 MDSC가 거의 관찰되지 않음을 확인하였다. As a result, as shown in FIG. 16, it was confirmed that the distribution of MITF-stained MDSCs was clear in the tissue surrounding the tumor, whereas MITF-stained MDSCs were hardly observed in tumor-free lymph node tissue.
상기 결과를 통해 암 주변에서 MDSC가 활성화되고, 상기 MDSC의 활성화에 MITF가 관여하는 것을 알 수 있다.Through the above results, it can be seen that MDSCs are activated around the cancer, and MITF is involved in the activation of the MDSCs.
<실시예 5> 활성을 유도한 MDSC에서 MITF 발현 증가 확인<Example 5> Confirmation of an increase in MITF expression in MDSCs whose activity was induced
MITF와 MDSC의 상관성을 알아보기 위하여, MDSC의 활성을 유도하는 약물을 처리한 후 MITF 발현 변화를 확인하였다.To examine the correlation between MITF and MDSC, changes in MITF expression were confirmed after treatment with a drug that induces MDSC activity.
<5-1> IL-18로 활성 유도한 MDSC에서 MITF 발현 증가 확인<5-1> Confirmation of an increase in MITF expression in MDSCs activated with IL-18
MDSC의 활성을 유도하는 약물로 IL-18을 처리하고 MDSC의 분화를 유도한 후, MITF의 유전자 발현 변화를 확인하였다.After treating IL-18 with a drug that induces MDSC activity and inducing differentiation of MDSC, changes in MITF gene expression were confirmed.
구체적으로, 상기 실시예 <1-1>에서 개시된 방법과 동일한 방법으로 골수세포를 얻은 후, 5Х105 cells/㎖의 세포 수로 24-웰 플레이트에서 10 또는 50 ng/㎖ 농도의 IL-18, 및 10 ng/㎖ 농도의 GM-CSF가 포함된 RPMI 배지로 96시간 동안 배양하여 MDSC의 분화를 유도하였다(도 6A 및 도 6B). 또한 상기 실시예 <1-1>에 기재된 방법에 따라 GM-CSF가 포함된 RPMI 배지로 96시간 동안 배양하여 MDSC의 분화를 유도하고, 10 또는 50 ng/㎖ 농도의 IL-18을 24시간 동안 처리하였다(도 6C 및 도 6D). 대조군으로 GM-CSF가 포함된 RPMI 배지로 분화를 유도한 MDSC를 이용하였다.Specifically, after obtaining bone marrow cells by the same method as described in Example <1-1>, IL-18 at a concentration of 10 or 50 ng/ml in a 24-well plate with a cell number of 5Х10 5 cells/ml, and Differentiation of MDSC was induced by culturing for 96 hours in RPMI medium containing GM-CSF at a concentration of 10 ng/ml (FIGS. 6A and 6B). In addition, according to the method described in Example <1-1>, MDSC differentiation was induced by culturing in RPMI medium containing GM-CSF for 96 hours, and IL-18 at a concentration of 10 or 50 ng/ml was added for 24 hours. treated (Fig. 6C and Fig. 6D). As a control group, MDSCs differentiated in RPMI medium containing GM-CSF were used.
상기 분화 유도한 MDSC를 회수하여 상기 실시예 <2-1>에 기재된 방법과 동일한 방법으로 FACS 분석을 수행하여 MDSC의 분화를 확인하였다(도 6A 및 도 6C).The differentiated MDSCs were recovered and FACS analysis was performed in the same manner as described in Example <2-1> to confirm MDSC differentiation (FIGS. 6A and 6C).
또한, 상기 분화 유도한 MDSC를 회수하여 상기 실시예 <2-1> 및 <2-2>에 기재된 방법과 동일한 방법으로 qRT-PCR을 수행하여 MDSC의 활성을 확인하고, MITF의 유전자 발현을 확인하였다(도 6B 및 도 6D).In addition, the MDSCs from which the differentiation was induced were recovered and qRT-PCR was performed in the same manner as described in Examples <2-1> and <2-2> to confirm the activity of MDSCs and to confirm the gene expression of MITF. (FIGS. 6B and 6D).
그 결과, 도 6에 나타낸 바와 같이, 대조군 및 IL-18 처리군 모두 유사한 정도로 MDSC의 분화가 유도됨을 확인하였다(도 6A 및 도 6C). 반면, IL-18 처리군의 경우 MDSC의 활성이 증가하고, MITF의 유전자 발현이 증가한 것을 확인하였다(도 6B 및 도 6D).As a result, as shown in FIG. 6, it was confirmed that MDSC differentiation was induced to a similar degree in both the control group and the IL-18-treated group (FIGS. 6A and 6C). On the other hand, in the case of the IL-18 treatment group, it was confirmed that MDSC activity increased and MITF gene expression increased (FIGS. 6B and 6D).
<5-2> IL-10으로 분화 및 활성 유도한 MDSC에서 MITF 발현 증가 확인<5-2> Confirmation of increased MITF expression in MDSCs induced to differentiate and activate with IL-10
MDSC의 활성을 유도하는 약물로 IL-10을 처리하고 MDSC의 분화를 유도한 후, MITF의 유전자 발현 변화를 확인하였다.After treating IL-10 with a drug that induces MDSC activity and inducing differentiation of MDSC, changes in MITF gene expression were confirmed.
구체적으로, 상기 실시예 <1-1>에서 개시된 방법과 동일한 방법으로 골수세포를 얻은 후, 5Х105 cells/㎖의 세포 수로 24-웰 플레이트에서 5 ng/㎖ 농도의 IL-10, 및 10 ng/㎖ 농도의 GM-CSF가 포함된 RPMI 배지로 96시간 동안 배양하여 MDSC의 분화를 유도하였다(도 6E 및 도 6F). 대조군으로 GM-CSF가 포함된 RPMI 배지로 분화를 유도한 MDSC를 이용하였다.Specifically, after obtaining bone marrow cells by the same method as described in Example <1-1>, IL-10 at a concentration of 5 ng/ml and 10 ng were added in a 24-well plate with a cell number of 5Х10 5 cells/ml. MDSC differentiation was induced by culturing for 96 hours in RPMI medium containing GM-CSF at a concentration of /ml (Fig. 6E and Fig. 6F). As a control group, MDSCs differentiated in RPMI medium containing GM-CSF were used.
상기 분화 유도한 MDSC를 회수하여 상기 실시예 <2-1>에 기재된 방법과 동일한 방법으로 FACS 분석을 수행하여 MDSC의 분화를 확인하였다(도 6E).The differentiated MDSCs were collected and subjected to FACS analysis in the same manner as described in Example <2-1> to confirm MDSC differentiation (FIG. 6E).
또한, 상기 분화 유도한 MDSC를 회수하여 상기 실시예 <2-1> 및 <2-2>에 기재된 방법과 동일한 방법으로 qRT-PCR을 수행하여 MDSC의 활성을 확인하고, MITF의 유전자 발현을 확인하였다(도 6F).In addition, the MDSCs from which the differentiation was induced were recovered and qRT-PCR was performed in the same manner as described in Examples <2-1> and <2-2> to confirm the activity of MDSCs and to confirm the gene expression of MITF. (FIG. 6F).
그 결과, 도 6에 나타낸 바와 같이, 대조군 및 IL-10 처리군 모두 유사한 정도로 MDSC의 분화가 유도됨을 확인하였다(도 6E). 반면, IL-10 처리군의 경우 MDSC의 활성이 증가하고, MITF의 유전자 발현이 증가한 것을 확인하였다(도 6F).As a result, as shown in FIG. 6, it was confirmed that MDSC differentiation was induced to a similar degree in both the control and IL-10 treated groups (FIG. 6E). On the other hand, in the case of the IL-10 treatment group, it was confirmed that MDSC activity increased and MITF gene expression increased (FIG. 6F).
<5-3> IL-4로 활성 유도한 MDSC에서 MITF 발현 증가 확인<5-3> Confirmation of increased MITF expression in IL-4-induced MDSC
MDSC의 활성을 유도하는 약물로 IL-4를 처리하고 MDSC의 분화를 유도한 후, MITF의 유전자 발현 변화를 확인하였다.After treating IL-4 with a drug that induces MDSC activity and inducing differentiation of MDSC, changes in MITF gene expression were confirmed.
구체적으로, 상기 실시예 <2-1>에 기재된 방법에 따라 MDSC의 분화 및 활성을 유도하되, 활성을 유도하는 약물로 10 ng/㎖의 IL-4를 처리하였다.Specifically, MDSC differentiation and activity were induced according to the method described in Example <2-1>, but 10 ng/ml of IL-4 was treated as an activity-inducing drug.
상기 분화 유도한 MDSC를 회수하여 상기 실시예 <2-1>에 기재된 방법과 동일한 방법으로 FACS 분석을 수행하여 MDSC의 분화를 확인하였다(도 7A).The differentiated MDSCs were collected and subjected to FACS analysis in the same manner as described in Example <2-1> to confirm MDSC differentiation (FIG. 7A).
또한, 상기 분화 유도한 MDSC를 회수하여 상기 실시예 <2-1> 및 <2-2>에 기재된 방법과 동일한 방법으로 MDSC의 활성을 확인하고(도 7B), MITF의 유전자 발현을 확인하였다(도 7C).In addition, the MDSCs from which the differentiation was induced were recovered, and the activity of MDSCs was confirmed in the same manner as described in Examples <2-1> and <2-2> (FIG. 7B), and the gene expression of MITF was confirmed ( Figure 7C).
그 결과, 도 7에 나타낸 바와 같이, 대조군 및 IL-4 처리군 모두 MDSC의 분화가 유도됨을 확인하였다(도 7A). 반면, IL-4 처리군의 경우 IL-4에 의해 MDSC의 활성화가 유도되고(도 7B), 대조군과 비교하여 MITF의 유전자 발현이 증가한 것을 확인하였다(도 7C).As a result, as shown in FIG. 7, it was confirmed that MDSC differentiation was induced in both the control group and the IL-4 treatment group (FIG. 7A). On the other hand, in the case of the IL-4 treatment group, MDSC activation was induced by IL-4 (FIG. 7B), and it was confirmed that the gene expression of MITF increased compared to the control group (FIG. 7C).
*<5-4> LPS로 활성 유도한 MDSC에서 MITF 발현 증가 확인 * <5-4> Confirmation of increased MITF expression in MDSC activated by LPS
MDSC의 활성을 유도하는 약물로 LPS를 처리하고 MDSC의 분화를 유도한 후, MITF의 유전자 발현 변화를 확인하였다.After treating LPS with a drug that induces MDSC activity and inducing differentiation of MDSC, changes in MITF gene expression were confirmed.
구체적으로, 상기 실시예 <2-1>에 기재된 방법에 따라 MDSC의 분화 및 활성을 유도하되, 활성을 유도하는 약물로 100 ng/㎖의 LPS를 처리하였다.Specifically, MDSC differentiation and activity were induced according to the method described in Example <2-1>, but 100 ng/ml of LPS was treated as an activity-inducing drug.
상기 분화 유도한 MDSC를 회수하여 상기 실시예 <2-1>에 기재된 방법과 동일한 방법으로 FACS 분석을 수행하여 MDSC의 분화를 확인하였다(도 8A).The differentiated MDSCs were collected and subjected to FACS analysis in the same manner as described in Example <2-1> to confirm MDSC differentiation (FIG. 8A).
또한, 상기 분화 유도한 MDSC를 회수하여 상기 실시예 <2-1> 및 <2-2>에 기재된 방법과 동일한 방법으로 MDSC의 활성을 확인하고(도 8B), MITF의 유전자 발현을 확인하였다(도 8C).In addition, the MDSCs from which the differentiation was induced were recovered, and the activity of MDSCs was confirmed in the same manner as described in Examples <2-1> and <2-2> (FIG. 8B), and the gene expression of MITF was confirmed ( Figure 8C).
그 결과, 도 8에 나타낸 바와 같이, 대조군 및 LPS 처리군 모두 MDSC의 분화가 유도됨을 확인하였다(도 8A). 반면, LPS 처리군의 경우 LPS에 의해 MDSC의 활성화가 유도되고(도 8B), 대조군과 비교하여 MITF의 유전자 발현이 증가한 것을 확인하였다(도 8C).As a result, as shown in FIG. 8, it was confirmed that MDSC differentiation was induced in both the control group and the LPS-treated group (FIG. 8A). On the other hand, in the case of the LPS-treated group, MDSC activation was induced by LPS (FIG. 8B), and it was confirmed that the MITF gene expression increased compared to the control group (FIG. 8C).
<5-5> 스타틴(Statin) 계열 약물로 활성 유도한 MDSC에서 MITF 발현 증가 확인<5-5> Confirmation of an increase in MITF expression in MDSCs induced by statin drugs
스타틴(Statin) 계열 약물인 심바스타틴(Simvastatin)은 IRF4(Interferon regulatory factor 4)의 발현을 억제하여 MDSC의 활성을 유도하는 것으로 알려져 있다. 이에 MDSC의 활성을 유도하는 약물로 스타틴 계열 약물을 처리하고 MDSC의 분화를 유도한 후, MITF의 유전자 발현 변화를 확인하였다.Simvastatin, a statin drug, is known to induce the activation of MDSC by suppressing the expression of Interferon regulatory factor 4 (IRF4). Accordingly, after treatment with a statin-based drug as a drug inducing the activity of MDSC and inducing differentiation of MDSC, changes in gene expression of MITF were confirmed.
구체적으로, 상기 실시예 <2-1>에 기재된 방법에 따라 MDSC의 분화 및 활성을 유도하되, 활성을 유도하는 약물로 심바스타틴(Sim, 0.5 또는 1 μM), 로바스타틴(Lovastatin, Lova, 0.5 또는 1 μM), 프로바스타틴(Pravastatin, Prav, 5 또는 10 μM), 로수바스타틴(Rosuvastatin, Rosu, 0.5 또는 1 μM), 또는 아토바스타틴(Atorvastatin, Ator, 0.05 또는 0.1 μM) 을 처리하였다(도 9 및 도 10).Specifically, MDSC differentiation and activity were induced according to the method described in Example <2-1>, but simvastatin (Sim, 0.5 or 1 μM) and lovastatin (Lovastatin, Lova, 0.5 or 1 μM) were used as the activity-inducing drugs. μM), provastatin (Pravastatin, Prav, 5 or 10 μM), rosuvastatin (Rosu, 0.5 or 1 μM), or atorvastatin (Atorvastatin, Ator, 0.05 or 0.1 μM) were treated (FIG. 9). and Figure 10).
또한, 상기 실시예 <1-1>에 기재된 방법에 따라 GM-CSF가 포함된 RPMI 배지로 96시간 동안 배양하여 MDSC의 분화를 유도하고, 상기 스타틴 계열 약물을 24시간 동안 처리하였다(도 11 및 도 12). In addition, according to the method described in Example <1-1>, MDSC differentiation was induced by culturing in RPMI medium containing GM-CSF for 96 hours, and treated with the statin drug for 24 hours (FIG. 11 and Figure 12).
그 다음, 상기 MDSC를 회수하여 상기 실시예 <2-1> 및 <2-2>에 기재된 방법과 동일한 방법으로 MDSC의 분화(도 9 및 도 11)를 확인하였다. 또한, 심바스타틴 또는 로바스타틴 1 μM 처리군에서 MDSC의 활성(도 10A 및 도 12A)을 확인하고, MITF의 유전자 발현(도 10B 및 도 12B)을 확인하였다. Then, the MDSCs were recovered and the differentiation of MDSCs (FIGS. 9 and 11) was confirmed in the same manner as described in Examples <2-1> and <2-2>. In addition, MDSC activity (FIGS. 10A and 12A) and MITF gene expression (FIGS. 10B and 12B) were confirmed in the simvastatin or lovastatin 1 μM treated group.
그 결과, 도 9 내지 도 12에 나타낸 바와 같이, 대조군 및 스타틴 계열 약물 처리군 모두 유사한 정도로 MDSC의 분화가 유도됨을 확인하였다(도 9 및 도 11). 반면, 스타틴 계열 약물 처리군의 경우 MDSC의 분화 초기부터 스타틴 계열 약물 처리한 경우(도 10) 및 이미 분화된 MDSC에 스타틴 계열 약물을 처리한 경우(도 12) 모두 스타틴 계열 약물에 의해 MDSC의 활성화가 유도되고(도 10A 및 도 12A), 대조군과 비교하여 MITF의 유전자 발현(도 10B 및 도 12B)이 증가한 것을 확인하였다.As a result, as shown in FIGS. 9 to 12, it was confirmed that MDSC differentiation was induced to a similar extent in both the control group and the statin-based drug-treated group (FIGS. 9 and 11). On the other hand, in the case of the statin-based drug-treated group, both when the statin-based drug was treated from the early stage of MDSC differentiation (FIG. 10) and when the statin-based drug was treated on already differentiated MDSC (FIG. 12), MDSC activation by the statin-based drug was induced (Figs. 10A and 12A), and it was confirmed that the gene expression of MITF (Figs. 10B and 12B) was increased compared to the control group.
상기 결과를 통해 활성이 유도된 MDSC에서 MITF의 발현이 증가한 것을 확인하였으므로, MDSC의 활성화에 MITF가 관여하는 것을 알 수 있다.Through the above results, it was confirmed that the expression of MITF was increased in MDSCs whose activity was induced, indicating that MITF is involved in the activation of MDSCs.
<실시예 6> 활성을 억제한 MDSC에서 MITF 발현 감소 확인<Example 6> Confirmation of reduction of MITF expression in MDSC whose activity was suppressed
ATRA(all-trans retinoic acid)는 MDSC의 활성을 억제하는 것으로 알려져 있다. 이에 MITF와 MDSC의 상관성을 알아보기 위하여, MDSC의 활성을 억제하는 약물로 ATRA를 처리한 후 MITF 발현 변화를 확인하였다.All-trans retinoic acid (ATRA) is known to inhibit the activity of MDSC. Therefore, in order to investigate the correlation between MITF and MDSC, after treatment with ATRA as a drug that inhibits MDSC activity, changes in MITF expression were confirmed.
구체적으로, 상기 실시예 <1-1>에 개시된 방법과 동일한 방법으로 골수세포를 얻은 후, 5Х105 cells/㎖의 세포 수로 24-웰 플레이트에서 ATRA(0.5 또는 1 μM) 및 10 ng/㎖ 농도의 GM-CSF가 포함된 RPMI 배지로 96시간 동안 배양하여 MDSC의 분화를 유도하였다(도 13).Specifically, bone marrow cells were obtained by the same method as described in Example <1-1>, and then ATRA (0.5 or 1 μM) and 10 ng/mL concentration were placed in a 24-well plate with a cell number of 5Х10 5 cells/mL. Differentiation of MDSC was induced by culturing for 96 hours in RPMI medium containing GM-CSF of (FIG. 13).
상기 분화 유도한 MDSC를 회수하여 상기 실시예 <2-1>에 기재된 방법과 동일한 방법으로 FACS 분석을 수행하여 MDSC의 분화를 확인하였다(도 13A).The differentiated MDSCs were recovered and FACS analysis was performed in the same manner as described in Example <2-1> to confirm the differentiation of MDSCs (FIG. 13A).
또한, 상기 ATRA 1 μM을 처리하고 분화 유도한 MDSC를 회수하여 상기 실시예 <2-1> 및 <2-2>에 기재된 방법과 동일한 방법으로 qRT-PCR을 수행하여 MDSC의 활성을 확인하였다(도 13B), MITF의 유전자 발현을 확인하였다(도 13C).In addition, MDSCs treated with 1 μM of ATRA and induced to differentiate were recovered, and qRT-PCR was performed in the same manner as described in Examples <2-1> and <2-2> to confirm the activity of MDSCs ( Fig. 13B), MITF gene expression was confirmed (Fig. 13C).
그 결과, 도 13에 나타낸 바와 같이, 대조군 및 ATRA 처리군 모두 유사한 정도로 MDSC의 분화가 유도됨을 확인하였다(도 13A). 반면 ATRA 처리군의 경우 ATRA에 의해 MDSC의 활성화가 억제되고(도 13B), 대조군과 비교하여 MITF의 유전자 발현이 감소한 것을 확인하였다(도 13C).As a result, as shown in FIG. 13, it was confirmed that MDSC differentiation was induced to a similar degree in both the control group and the ATRA-treated group (FIG. 13A). On the other hand, in the case of the ATRA-treated group, it was confirmed that the activation of MDSC was inhibited by ATRA (FIG. 13B), and the gene expression of MITF was decreased compared to the control group (FIG. 13C).
상기 결과를 통해 활성이 억제된 MDSC에서 MITF의 발현이 감소한 것을 확인하였으므로, MDSC의 활성화에 MITF가 관여하는 것을 알 수 있다.Through the above results, it was confirmed that the expression of MITF was reduced in the MDSCs whose activity was suppressed, and thus it can be seen that MITF is involved in the activation of MDSCs.
<실시예 7> MITF 조절에 의한 MDSC 활성 변화 확인<Example 7> Confirmation of MDSC activity change by MITF control
<7-1> MITF 유도제에 의한 MDSC의 활성 증가 확인<7-1> Confirmation of MDSC activity increase by MITF inducer
흑색종 세포에서 IBMX가 MITF의 발현을 유도하는 것이 알려져 있다. 이에, MITF의 발현 또는 활성 조절이 MDSC 활성에 미치는 영향을 알아보기 위하여, MITF 발현 유도제로 IBMX를 처리한 MDSC의 활성 변화 및 상기 MDSC에 의한 T 세포 증식 변화를 확인하였다. It is known that IBMX induces the expression of MITF in melanoma cells. Accordingly, in order to investigate the effect of MITF expression or activity regulation on MDSC activity, changes in activity of MDSCs treated with IBMX as an inducer of MITF expression and changes in T cell proliferation by the MDSCs were confirmed.
구체적으로, 상기 실시예 <1-1>에서 개시된 방법과 동일한 방법으로 골수세포를 얻은 후, 5Х105 cells/㎖의 세포 수로 24-웰 플레이트에서 10 μM 농도의 IBMX 및 10 ng/㎖ 농도의 GM-CSF가 포함된 RPMI 배지로 96시간 동안 배양하여 MDSC의 분화를 유도하였다. 대조군으로 GM-CSF가 포함된 RPMI 배지로 분화를 유도한 MDSC를 이용하였다.Specifically, bone marrow cells were obtained by the same method as described in Example <1-1>, and IBMX at a concentration of 10 μM and GM at a concentration of 10 ng/mL were cultured in a 24-well plate at a cell number of 5Х10 5 cells/mL. Differentiation of MDSC was induced by culturing for 96 hours in RPMI medium containing -CSF. As a control group, MDSCs differentiated in RPMI medium containing GM-CSF were used.
상기 분화 유도한 MDSC를 회수하여 상기 실시예 <2-1>에 기재된 방법과 동일한 방법으로 FACS 분석을 수행하여 MDSC의 분화를 확인하였다(도 14A).The differentiated MDSCs were collected and subjected to FACS analysis in the same manner as described in Example <2-1> to confirm MDSC differentiation (FIG. 14A).
또한, 상기 분화 유도한 MDSC를 회수하여 상기 실시예 <2-1> 및 <2-2>에 기재된 방법과 동일한 방법으로 qRT-PCR 및 웨스턴 블럿팅을 수행하여 MDSC의 활성을 확인하고(도 14B 및 도 14C), MITF의 유전자 발현 및 단백질 발현(도 14C)을 확인하였다.In addition, the MDSCs from which the differentiation was induced were recovered and subjected to qRT-PCR and Western blotting in the same manner as described in Examples <2-1> and <2-2> to confirm the activity of MDSCs (Fig. 14B and Fig. 14C), MITF gene expression and protein expression (Fig. 14C) were confirmed.
또한, 상기 분화 유도한 MDSC를 회수하여 실시예 <2-3>에 기재된 방법과 동일한 방법으로 T 세포 증식 억제 정도를 확인하였다(도 14D).In addition, the differentiation-induced MDSCs were recovered and the degree of inhibition of T cell proliferation was confirmed by the same method as described in Example <2-3> (FIG. 14D).
그 결과, 도 14에 나타낸 바와 같이, 대조군 및 IBMX 처리군 모두 유사한 정도로 MDSC의 분화가 유도됨을 확인하였다(도 14A). 반면, IBMX 처리군의 경우 대조군과 비교하여 MITF의 유전자 및 단백질 발현이 증가하고(도 14C), MDSC의 활성이 증가하는 것을 확인하였다(도 14B 및 도 14C). 또한, IBMX 처리군의 경우 MDSC의 활성이 증가하여 T 세포 증식이 억제되는 것을 확인하였다(도 14D).As a result, as shown in FIG. 14, it was confirmed that MDSC differentiation was induced to a similar degree in both the control group and the IBMX-treated group (FIG. 14A). On the other hand, in the case of the IBMX-treated group, compared to the control group, it was confirmed that the gene and protein expression of MITF increased (FIG. 14C) and the activity of MDSC increased (FIGS. 14B and 14C). In addition, in the case of the IBMX-treated group, it was confirmed that MDSC activity was increased and T cell proliferation was suppressed (FIG. 14D).
<7-2> MITF 억제제에 의한 MDSC 활성 저해 확인<7-2> Confirmation of inhibition of MDSC activity by MITF inhibitors
베르베린(Berberine), 카지놀 U(Kazinol U) 등은 AMPK 활성촉진제(AMPK activator)이면서 멜라닌 세포에서 MITF의 발현 또는 활성을 억제하는 것이 알려져 있다. 이에 MITF의 발현 또는 활성 조절이 MDSC 활성에 미치는 영향을 알아보기 위하여, MITF 억제제로 농도를 달리한 베르베린을 처리한 MDSC의 활성 변화를 확인하였다.Berberine, Kazinol U, and the like are known to inhibit the expression or activity of MITF in melanocytes while being AMPK activators. Accordingly, in order to examine the effect of MITF expression or activity regulation on MDSC activity, changes in activity of MDSC treated with berberine at different concentrations as a MITF inhibitor were confirmed.
구체적으로, 상기 실시예 <1-1>에 개시된 방법과 동일한 방법으로 골수세포를 얻은 후, 5Х105 cells/㎖의 세포 수로 24-웰 플레이트에서 5 μM 농도의 베르베린 및 10 ng/㎖ 농도의 GM-CSF가 포함된 RPMI 배지와 10 μM 농도의 베르베린 및 10 ng/㎖ 농도의 GM-CSF가 포함된 RPMI 배지에서 각각 96시간 동안 배양하여 MDSC의 분화를 유도하였다. 대조군으로 GM-CSF가 포함된 RPMI 배지로 분화를 유도한 MDSC를 이용하였다.Specifically, bone marrow cells were obtained by the same method as described in Example <1-1>, and berberine at a concentration of 5 μM and GM at a concentration of 10 ng/mL were placed in a 24-well plate at a cell number of 5Х10 5 cells/mL. MDSC differentiation was induced by culturing in RPMI medium containing CSF and RPMI medium containing berberine at a concentration of 10 µM and GM-CSF at a concentration of 10 ng/mL for 96 hours, respectively. As a control group, MDSCs differentiated in RPMI medium containing GM-CSF were used.
또한, 상기 분화 유도한 MDSC를 회수하여 상기 실시예 <2-1>에 기재된 방법과 동일한 방법으로 FACS 분석을 수행하여 MDSC의 분화를 확인하였다(도 15A 및 도 15B).In addition, the differentiation-induced MDSCs were recovered and FACS analysis was performed in the same manner as described in Example <2-1> to confirm the differentiation of MDSCs (FIGS. 15A and 15B).
또한, 상기 분화 유도한 MDSC를 회수하여 상기 실시예 <2-1>에 기재된 방법과 동일한 방법으로 qRT-PCR을 수행하여 MDSC의 활성을 확인하였고, 상기 분화 유도한 MDSC를 회수하여 상기 실시예 <2-2>에 기재된 방법과 동일한 방법으로 qRT-PCR을 수행하여 MITF의 발현을 확인하였다 (도 15C)In addition, the MDSCs induced to differentiate were recovered and qRT-PCR was performed in the same manner as described in Example <2-1> to confirm the activity of MDSCs. 2-2>, the expression of MITF was confirmed by performing qRT-PCR in the same manner as described (Fig. 15C).
그 결과, 도 15에 나타낸 바와 같이, 베르베린 처리군의 경우 MDSC의 분화가 다소 감소한 것을 확인하였다(도 15A 및 도 15B). 또한, 기대한 바와 같이, 베르베린 처리군의 경우 대조군과 비교하여 MDSC의 활성이 유의적으로 저해되는 것을 확인하였으며, MITF의 발현도 억제된 것을 확인하였다 (도 15C).As a result, as shown in FIG. 15, it was confirmed that the differentiation of MDSC was slightly reduced in the case of the berberine-treated group (FIGS. 15A and 15B). In addition, as expected, it was confirmed that MDSC activity was significantly inhibited in the berberine-treated group compared to the control group, and MITF expression was also inhibited (FIG. 15C).
또한, ML-329는 멜라닌 세포에서 TRPM-1 프로모터 활성을 억제하여 MITF의 발현을 억제하는 것이 알려져 있다. 이에 MITF의 발현 또는 활성 조절이 MDSC 활성에 미치는 영향을 알아보기 위하여, MITF 억제제로 ML-329를 처리한 MDSC의 활성 변화 및 상기 MDSC에 의한 T 세포 증식 변화를 확인하였다.In addition, it is known that ML-329 inhibits the expression of MITF by suppressing TRPM-1 promoter activity in melanocytes. Accordingly, in order to investigate the effect of the regulation of MITF expression or activity on MDSC activity, changes in the activity of MDSCs treated with ML-329 as a MITF inhibitor and changes in T cell proliferation by the MDSCs were confirmed.
구체적으로, 상기 실시예 <1-1>에 개시된 방법과 동일한 방법으로 골수세포를 얻은 후, 5Х105 cells/㎖의 세포 수로 24-웰 플레이트에서 0.5 또는 1 μM 농도의 ML-329(Cayman Chemical, Ann Arbor, MI) 및 10 ng/㎖ 농도의 GM-CSF가 포함된 RPMI 배지로 96시간 동안 배양하여 MDSC의 분화를 유도하였다. 대조군으로 GM-CSF가 포함된 RPMI 배지로 분화를 유도한 MDSC를 이용하였다. 그 다음, 상기 분화 유도한 MDSC를 회수하여 상기 실시예 <2-1> 및 <2-2>에 기재된 방법과 동일한 방법으로 qRT-PCR을 수행하여 MDSC의 활성(도 17A) 및 MITF 발현을 확인하였다(도 17B).Specifically, bone marrow cells were obtained by the same method as described in Example <1-1>, and ML-329 (Cayman Chemical, Cayman Chemical, Ann Arbor, MI) and 10 ng/ml GM-CSF were cultured in RPMI medium for 96 hours to induce MDSC differentiation. As a control group, MDSCs differentiated in RPMI medium containing GM-CSF were used. Then, the MDSCs from which the differentiation was induced were recovered, and qRT-PCR was performed in the same manner as described in Examples <2-1> and <2-2> to confirm MDSC activity (FIG. 17A) and MITF expression. (FIG. 17B).
또한, TCCM에 의한 MDSC 활성 유도에 있어서 ML-329의 효과를 알아보기 위하여, 상기 실시예 <1-1>에 개시된 방법과 동일한 방법으로 골수세포를 얻은 후, 5Х105 cells/㎖의 세포 수로 24-웰 플레이트에서 0.5, 1 또는 2 μM 농도의 ML-329(Cayman Chemical), 상기 실시예 <2-1>에서 획득한 TCCM 및 10 ng/㎖ 농도의 GM-CSF가 포함된 RPMI 배지로 96시간 동안 배양하여 MDSC의 분화를 유도하였다. 대조군으로 GM-CSF가 포함된 RPMI 배지로 분화를 유도한 MDSC를 이용하였다. 그 다음, 상기 분화 유도한 MDSC를 회수하여 상기 실시예 <2-1>에 기재된 방법과 동일한 방법으로 FACS 분석을 수행하여 MDSC의 분화를 확인하고(도 18A), qRT-PCR을 수행하여 MDSC의 활성 정도를 측정하였다(도 18B). 또한, 상기 실시예 <2-2>에 기재된 방법과 동일한 방법으로 qRT-PCR 및 웨스턴 블럿팅을 수행하여 MITF 발현을 확인하였다(도 18C). 또한, 상기에서 획득한 MDSC를 이용하여 상기 실시예 <2-3>에 기재된 방법과 동일한 방법으로 T 세포 증식 억제 정도를 확인하였다(도 18D).In addition, in order to examine the effect of ML-329 on the induction of MDSC activity by TCCM, bone marrow cells were obtained by the same method as described in Example <1-1>, and the number of cells at 5Х10 5 cells/ml was 24 -96 hours in RPMI medium containing ML-329 (Cayman Chemical) at a concentration of 0.5, 1 or 2 µM in a well plate, TCCM obtained in Example <2-1> and GM-CSF at a concentration of 10 ng/mL During culture, the differentiation of MDSC was induced. As a control group, MDSCs differentiated in RPMI medium containing GM-CSF were used. Then, the differentiation-induced MDSCs were recovered, FACS analysis was performed in the same manner as described in Example <2-1> to confirm the differentiation of MDSCs (FIG. 18A), and qRT-PCR was performed to determine the number of MDSCs. The degree of activity was measured (FIG. 18B). In addition, qRT-PCR and Western blotting were performed in the same manner as described in Example <2-2> to confirm MITF expression (FIG. 18C). In addition, the degree of inhibition of T cell proliferation was confirmed by the same method as described in Example <2-3> using the obtained MDSCs (FIG. 18D).
그 결과, 도 17 및 도 18에 나타낸 바와 같이, ML-329 처리군의 경우 ML-329에 의해 MITF 발현이 억제되고 MDSC의 활성이 저해되는 것을 확인하였다(도 17A 및 도 17B). 또한, ML-329 처리군의 경우 TCCM에 의한 MDSC 활성이 저해되고(도 18B 및 도 18C), MITF 발현이 저해되며(도 18C), ML-329에 의한 MDSC 활성 저해로 T 세포 증식이 증가하는 것을 확인하였다(도 18D).As a result, as shown in FIGS. 17 and 18, in the case of the ML-329-treated group, it was confirmed that ML-329 suppressed MITF expression and inhibited MDSC activity (FIGS. 17A and 17B). In addition, in the case of the ML-329 treatment group, MDSC activity by TCCM was inhibited (FIGS. 18B and 18C), MITF expression was inhibited (FIG. 18C), and T cell proliferation increased due to MDSC activity inhibition by ML-329. It was confirmed (FIG. 18D).
또한, MDSC는 활성산소종(reactive oxygen species, ROS)이나 활성질소종(reactive nitrogen species, RNS)을 만들어 T 세포의 증식에서부터 기능까지 다양한 과정을 저해하는 것으로 알려져 있다. 이에, MITF의 발현 또는 활성 조절이 MDSC 활성에 미치는 영향을 알아보기 위하여, MITF 억제제로 베르베린 또는 ML-329를 처리한 MDSC에서 ROS 생성 변화를 확인하였다.In addition, MDSCs are known to inhibit various processes from proliferation to function of T cells by producing reactive oxygen species (ROS) or reactive nitrogen species (RNS). Therefore, in order to examine the effect of the regulation of MITF expression or activity on MDSC activity, changes in ROS generation were confirmed in MDSC treated with berberine or ML-329 as a MITF inhibitor.
구체적으로, 상기 실시예 <1-2>의 종양형성 마우스의 비장에서 MDSC를 분리하여 10 μM 농도의 베르베린 또는 1 μM 농도의 ML-329, 상기 실시예 <2-1>에서 획득한 TCCM 및 10 ng/㎖ 농도의 GM-CSF가 포함된 RPMI 배지로 48시간 동안 배양하여 MDSC의 분화를 유도하였다. 상기 MDSC를 수집하여 1Х105 세포 수로 분주하고, LPS를 100 ng/ml 농도로 24시간 동안 처리하였다. 이때 ROS 저해제인 NAC(N-acetyl-cysteine)을 처리/무처리하였다. 24시간 후에 DCF-DA를 넣어 37℃에서 30분간 반응하고, 유세포분석기로 ROS 생성 정도를 측정하였다. Specifically, MDSCs were isolated from the spleen of the tumorigenic mouse of Example <1-2>, berberine at a concentration of 10 μM or ML-329 at a concentration of 1 μM, TCCM obtained in Example <2-1> and 10 MDSC differentiation was induced by culturing for 48 hours in RPMI medium containing GM-CSF at a concentration of ng/ml. The MDSC were collected and divided into 1Х10 5 cells, and treated with LPS at a concentration of 100 ng/ml for 24 hours. At this time, NAC (N-acetyl-cysteine), a ROS inhibitor, was treated/untreated. After 24 hours, DCF-DA was added and reacted at 37° C. for 30 minutes, and the degree of ROS generation was measured by flow cytometry.
그 결과, 도 19에 나타낸 바와 같이, 베르베린 또는 ML-329 처리군의 경우 MDSC에 의한 ROS 생성이 저해되는 것을 확인하였고, ROS 저해제를 함께 처리한 경우 ROS가 강력하게 저해되는 것을 확인하였다.As a result, as shown in FIG. 19, it was confirmed that ROS generation by MDSC was inhibited in the case of the berberine or ML-329 treatment group, and ROS was strongly inhibited when the ROS inhibitor was also treated.
아울러, RNA간섭(RNA interference, RNAi) 및 CRISPR 기술을 이용하여 MDSC에서 MITF의 발현을 억제한 후, MDSC에 의한 ROS 생성 및 T 세포 증식 변화를 확인하였다.In addition, after inhibiting the expression of MITF in MDSCs using RNA interference (RNAi) and CRISPR technology, ROS generation and T cell proliferation changes by MDSCs were confirmed.
구체적으로, 상기 실시예 <1-1>에 개시된 방법과 동일한 방법으로 골수세포를 얻은 후, 1Х106 cells/㎖의 세포 수의 골수세포에 CRISPR 벡터, 구체적으로 pLentiCRISPR-E 벡터에 다음의 sgRNA를 클로닝하였다; MITF gRNA FW oligo: 5'-CACCGTAAGGACTTCCATCGGCACC-3'(서열번호 1), MITF gRNA RV oligo: 5'-AAACGGTGCCGATGGAAGTCCTTAC-3'(서열번호 2). 또한, 대조군은 pLentiCRISPR-E 벡터에 다음의 non-target control gRNA를 클로닝하였다; non-target control sgRNA: 5'-CACCGGTATTACTGATATTGGTGGG-3'(서열번호 3). 상기 클로닝한 컨스트럭트를 리포펙타민(lipofectamine)을 이용하여 제조사의 절차에 따라 형질감염시켰다. 형질감염 24시간 후 상기 실시예 <2-1>에서 획득한 TCCM 및 10 ng/㎖ 농도의 GM-CSF가 포함된 RPMI 배지로 72시간 동안 배양하여 MDSC의 분화를 유도하였다. 형질감염 72시간 후, MDSC를 회수하여 상기 <2-2>에 기재된 방법과 웨스턴 블럿팅을 수행하여 MITF의 발현 및 MDSC 활성을 확인하였다(도 20A). 또한, 상기에 기재된 방법과 동일한 방법으로 MDSC에 의한 ROS 생성 정도를 측정하고(도 20B), 상기 실시예 <2-3>에 기재된 방법과 동일한 방법으로 MDSC에 의한 T 세포 증식 정도를 측정하였다(도 20C).Specifically, after obtaining bone marrow cells by the same method as described in Example <1-1>, the following sgRNA was added to the CRISPR vector, specifically the pLentiCRISPR-E vector, to the bone marrow cells at a cell number of 1Х10 6 cells/ml. cloned; MITF gRNA FW oligo: 5'-CACCGTAAGGACTTCCATCGGCACC-3' (SEQ ID NO: 1), MITF gRNA RV oligo: 5'-AAACGGTGCCGATGGAAGTCCTTAC-3' (SEQ ID NO: 2). In addition, as a control, the following non-target control gRNA was cloned into the pLentiCRISPR-E vector; non-target control sgRNA: 5'-CACCGGTATTACTGATATTGGTGGG-3' (SEQ ID NO: 3). The cloned construct was transfected using lipofectamine according to the manufacturer's procedure. 24 hours after transfection, MDSC differentiation was induced by culturing for 72 hours in RPMI medium containing TCCM obtained in Example <2-1> and GM-CSF at a concentration of 10 ng/ml. 72 hours after transfection, MDSCs were recovered and subjected to Western blotting and the method described in <2-2> above to confirm MITF expression and MDSC activity (FIG. 20A). In addition, the degree of ROS generation by MDSC was measured by the same method as described above (FIG. 20B), and the degree of T cell proliferation by MDSC was measured by the same method as described in Example <2-3> ( Figure 20C).
또한, 비교군으로 MIFT가 과발현된 MDSC에 의한 T 세포 증식 정도를 측정하였다. 구체적으로, 상기 1Х106 cells/㎖의 세포 수의 골수세포에 MITF plasmid DNA(Addgene, Cambridge, MO)를 리포펙타민을 이용하여 제조사의 절차에 따라 형질감염시켰다. 형질감염 24시간 후 10 ng/㎖ 농도의 GM-CSF가 포함된 RPMI 배지로 72시간 동안 배양하여 MDSC의 분화를 유도하였다. 형질감염 3일 후, MDSC를 회수하여 상기와 같이 MITF의 발현(도 20D) 및 MDSC에 의한 T 세포 증식 정도(도 20E)를 측정하였다. In addition, as a comparison group, the degree of T cell proliferation by MIFT-overexpressing MDSCs was measured. Specifically, MITF plasmid DNA (Addgene, Cambridge, MO) was transfected into bone marrow cells at a cell number of 1Х10 6 cells/ml using Lipofectamine according to the manufacturer's procedure. 24 hours after transfection, MDSC differentiation was induced by culturing for 72 hours in RPMI medium containing GM-CSF at a concentration of 10 ng/ml. After 3 days of transfection, MDSCs were harvested and the expression of MITF (FIG. 20D) and the degree of T cell proliferation by MDSCs (FIG. 20E) were measured as described above.
그 결과, 도 20에 나타낸 바와 같이, MITF shRNA 및 CRISPR 기술을 이용하여 MDSC에서 MITF의 발현 및 MDSC 활성이 억제되는 것을 확인하였고(도 20A), MITF 발현 억제로 MDSC에 의한 ROS 생성이 억제되고(도 20B), MDSC에 의한 T 세포 저해가 완화되는 것을 확인하였다(도 20C). 반면, MITF가 과발현된 MDSC에서는 상반된 결과가 나타나는 것을 확인하였다(도 20D 및 도 20E).As a result, as shown in FIG. 20, it was confirmed that MITF expression and MDSC activity were suppressed in MDSC using MITF shRNA and CRISPR technology (FIG. 20A), and ROS generation by MDSC was suppressed by suppressing MITF expression ( 20B), it was confirmed that T cell inhibition by MDSC was alleviated (FIG. 20C). On the other hand, it was confirmed that MDSCs in which MITF was overexpressed showed conflicting results (FIGS. 20D and 20E).
상기의 결과를 통해 MITF의 발현 또는 활성을 억제하여 MDSC의 활성을 저해할 수 있음을 알 수 있다.Through the above results, it can be seen that the activity of MDSC can be inhibited by inhibiting the expression or activity of MITF.
또한, HIV 프로테아제 저해제인 넬피나비르(Nelfinavir)는 MITF 억제제로서, MITF 유전자 발현을 억제할 수 있다. 이에 TCCM에 의한 MDSC 활성 유도에 있어서 넬피나비르의 효과를 알아보기 위하여, 상기 실시예 <1-1>에 개시된 방법과 동일한 방법으로 골수세포를 얻은 후, 5Х105 cells/㎖의 세포 수로 24-웰 플레이트에서 1, 5 또는 10 μM 농도의 넬피나비르, 상기 실시예 <2-1>에서 획득한 TCCM 및 10 ng/㎖ 농도의 GM-CSF가 포함된 RPMI 배지로 96시간 동안 배양하여 MDSC의 분화를 유도하였다. 대조군으로 넬피나비르가 포함되지 않은 TCCM 및 10 ng/㎖ 농도의 GM-CSF가 포함된 RPMI 배지로 분화를 유도한 MDSC를 이용하였다. 그 다음, 상기 분화 유도한 MDSC를 회수하여 상기 실시예 <2-2>에 기재된 방법과 동일한 방법으로 qRT-PCR을 수행하여 MITF 발현을 확인하였다(도 21A). 그 다음, 상기 실시예 <2-1>에 기재된 방법과 동일한 방법으로 qRT-PCR을 수행하여 MDSC의 활성 정도를 측정하였다(도 21B). 또한, MDSC의 활성을 확인하기 위하여 상기 실시예 <2-2>에 기재된 방법으로 웨스턴 블럿팅을 수행하여 MDSC 활성을 확인하였다(도 21C).In addition, Nelfinavir, an HIV protease inhibitor, is a MITF inhibitor and can suppress MITF gene expression. Therefore, in order to examine the effect of nelfinavir on the induction of MDSC activity by TCCM, bone marrow cells were obtained by the same method as described in Example <1-1>, and then 24- MDSCs were cultured for 96 hours in RPMI medium containing 1, 5, or 10 μM of nelfinavir, TCCM obtained in Example <2-1>, and 10 ng/ml of GM-CSF in a well plate. differentiation was induced. As a control, MDSCs differentiated with RPMI medium containing TCCM without nelfinavir and GM-CSF at a concentration of 10 ng/ml were used. Then, the MDSCs from which the differentiation was induced were recovered, and qRT-PCR was performed in the same manner as described in Example <2-2> to confirm MITF expression (FIG. 21A). Then, qRT-PCR was performed in the same manner as described in Example <2-1> to measure the activity level of MDSC (FIG. 21B). In addition, in order to confirm the activity of MDSC, Western blotting was performed according to the method described in Example <2-2> to confirm MDSC activity (FIG. 21C).
그 결과, 도 21에 나타낸 바와 같이, 넬피나비르 처리군의 경우 넬피나비르에 의해 MITF 발현이 억제되었고, TCCM에 의한 MDSC의 활성도 저해되는 것을 확인하였다(도 21) As a result, as shown in FIG. 21, in the case of the nelfinavir-treated group, it was confirmed that MITF expression was suppressed by nelfinavir, and the activity of MDSC by TCCM was also inhibited (FIG. 21).
<실시예 8> 종양 형성 마우스에서 MDSC 활성 저하에 의한 면역반응 증진 효과 확인<Example 8> Confirmation of immune response enhancement effect by reducing MDSC activity in tumor-forming mice
상기 <실시예 7>을 통해 MITF 억제제에 의해 MDSC 활성이 저해되는 것을 확인하였다. 이에, 종양 형성 마우스에서 MDSC 활성 저하에 의한 효과를 알아보기 위하여, 종양 형성 마우스에 MITF 억제제에 의해 활성이 저하된 MDSC를 투여한 후 종양 성장 변화를 확인하였다.Through <Example 7>, it was confirmed that MDSC activity was inhibited by the MITF inhibitor. Therefore, in order to examine the effect of reducing MDSC activity in tumorigenic mice, after administering MDSC whose activity was reduced by the MITF inhibitor to tumorigenic mice, tumor growth changes were confirmed.
구체적으로, 상기 실시예 <1-2>에 기재된 방법과 동일한 방법으로 제작한 종양 형성 마우스의 비장에서 MDSC를 분리하였다. 그 다음, 상기 실시예 <7-2>에 기재된 방법과 동일한 방법으로 1 μM 농도의 ML-329, 상기 실시예 <2-1>에서 획득한 TCCM 및 10 ng/㎖ 농도의 GM-CSF가 포함된 RPMI 배지로 48시간 동안 배양하여 MDSC의 분화를 유도하였다. 48시간 후 상기 MDSC + 4T1-luc(5Х104 + 2.5Х105/100 μl)를 함께 마우스의 왼쪽 옆구리 쪽의 피하에 주입하였다. 실험 종료 시점까지 지속적으로 vernier caliper로 종양 부피를 측정하였고(종양 부피 = a2Хb/2 mm3)(a = 종양 너비, b = 종양 높이)(도 22B), IVIS(In vivo imaging system) 분석을 수행하였다(도 22C). 또한, 2주 뒤에 종양 조직에서 MDSC의 population을 항-CD11b 항체, 항-Gr1 항체, 항-CD45 항체를 이용하여 염색한 후, FACS 분석을 수행하여 확인하였다(도 22D). 대조군은 ML-329가 불포함되고, TCCM 및 GM-CSF가 포함된 RPMI 배지로 배양한 MDSC를 주입하였다.Specifically, MDSCs were isolated from the spleens of tumor-forming mice prepared in the same manner as described in Example <1-2>. Next, ML-329 at a concentration of 1 μM, TCCM obtained in Example <2-1> and GM-CSF at a concentration of 10 ng/mL were included in the same manner as described in Example <7-2> above. Differentiation of MDSC was induced by culturing for 48 hours with the prepared RPMI medium. After 48 hours, the MDSC + 4T1-luc (5Х10 4 + 2.5Х10 5 /100 μl) were subcutaneously injected into the left flank of the mouse. Until the end of the experiment, the tumor volume was continuously measured with a vernier caliper (tumor volume = a 2 Хb/2 mm 3 ) (a = tumor width, b = tumor height) (FIG. 22B), IVIS (In vivo imaging system) analysis was performed (FIG. 22C). In addition, after 2 weeks, the MDSC population in the tumor tissue was stained with anti-CD11b antibody, anti-Gr1 antibody, and anti-CD45 antibody, and FACS analysis was performed to confirm it (FIG. 22D). As a control group, MDSC cultured in RPMI medium without ML-329 and containing TCCM and GM-CSF were injected.
그 결과, 도 22에 나타낸 바와 같이, ML-329를 사전에 처리한 MDSC의 경우 ML-329에 의해 MDSC 활성이 저하되고, 이로 인해 종양 형성 마우스에서 면역반응이 증진되어 종양 성장이 억제되고 종양 부위에서 MDSC의 침입 정도가 약화되는 것을 확인하였다(도 22B 내지 도 22D).As a result, as shown in FIG. 22, in the case of MDSC treated with ML-329 in advance, MDSC activity was lowered by ML-329, and as a result, the immune response was enhanced in tumor-forming mice, suppressing tumor growth, and tumor site It was confirmed that the degree of invasion of MDSC was weakened (FIGS. 22B to 22D).
상기 <실시예 1> 내지 <실시예 8>을 통해 암세포 미세환경에서 MDSC가 활성화되어 면역반응이 저하되고, 상기 MDSC의 활성화에 MITF가 관여하며, 상기 MITF의 억제제를 이용하여 MDSC의 활성을 억제할 수 있음을 확인하였다. Through <Example 1> to <Example 8>, MDSCs are activated in the cancer cell microenvironment and the immune response is lowered, MITF is involved in the activation of MDSC, and the activity of MDSC is inhibited using the MITF inhibitor. confirmed that it could be done.
이에 기초하여, 광범위한 물질들 중 MITF 억제제를 선별하여 MDSC 활성 저해 약물로 사용할 수 있음을 밝혔다. MDSC의 면역반응 저하의 완화 및 항암 면역 치료에 이용할 수 있는 유효성분을 보다 효과적으로 특정할 수 있다. 또한, 이로부터 MDSC가 활성화된 개체나 이에 의해 T 세포가 억제된 것으로 확인되는 개체에 대하여, 생체 내에 투여되는 경우 가장 적합하게 작용할 것으로 기대되는 MITF 발현 억제제를 선별해낼 수 있다. 또한, 환자는 직접 투여하기 전에 본인에게 가장 적합할 것으로 기대되는 MITF 발현 억제제를 유효성분으로 포함하는 항암 조성물 또는 항암 보조제 조성물을 선택할 수 있게 되며, 기존에 치료가 진행 중이었던 경우에도 투여 조성물 변경여부나 새로운 약물 후보군을 고려할 수 있으며, MDSC 활성 반응 결과를 예측할 수 있으므로, 암의 종류나 예후를 고려하여 가장 적절한 MITF 억제제를 선택할 수 있도록 하는 기틀을 마련한다.Based on this, it was revealed that MITF inhibitors among a wide range of substances can be selected and used as MDSC activity inhibitory drugs. It is possible to more effectively identify active ingredients that can be used for alleviating the decline in the immune response of MDSCs and for anticancer immunotherapy. In addition, from this, it is possible to select a MITF expression inhibitor that is expected to act most appropriately when administered in vivo to an individual whose MDSCs are activated or whose T cells are confirmed to be suppressed by the MDSC. In addition, the patient can select an anti-cancer composition or an anti-cancer adjuvant composition containing the MITF expression inhibitor as an active ingredient that is expected to be most suitable for the patient before direct administration, and whether or not the administration composition is changed even if the treatment is in progress However, since new drug candidates can be considered and MDSC activation response results can be predicted, the basis for selecting the most appropriate MITF inhibitor considering the type or prognosis of cancer is laid.
본 발명에 의해 선별된 MITF 억제제를 포함하는 조성물을 MDSC의 저해가 필요한 개체, 예컨대 종양을 갖는 개체에 투여하여 MDSC에 의한 면역반응 저하를 완화하는데 유용하게 사용할 수 있다. 또한, 상기 MITF 억제제를 포함하는 조성물을 항암제, 예컨대 항암면역치료제와 병용 투여하여 항암 면역치료 효율을 증대하는 보조요법으로 유용하게 사용할 수 있다.The composition containing the MITF inhibitor selected according to the present invention can be administered to a subject in need of inhibition of MDSC, such as a subject having a tumor, and can be usefully used to alleviate the decrease in immune response caused by MDSC. In addition, the composition containing the MITF inhibitor can be administered in combination with an anticancer agent, such as an anticancer immunotherapeutic agent, and can be usefully used as an adjuvant therapy to increase the efficiency of anticancer immunotherapy.
이상 첨부된 도면을 참조하여 본 발명의 실시 예들을 설명하였으나, 본 발명은 상기 실시 예들에 한정되는 것이 아니라 서로 다른 다양한 형태로 제조될 수 있으며, 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시 예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.Although embodiments of the present invention have been described with reference to the accompanying drawings, the present invention is not limited to the above embodiments and can be manufactured in various different forms, and those skilled in the art to which the present invention belongs It will be understood that the present invention may be embodied in other specific forms without changing its spirit or essential characteristics. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

Claims (12)

  1. 특정물질에 의한 MDSC에서의 MITF 유전자 발현 또는 MITF 단백질 발현 정도를 qRT-PCR, 웨스턴 블롯팅 및 유세포 분석법 (FACS)으로부터 선택된 어느 하나의 방법으로 분석하여 MDSC 활성 변화 정도를 예측하고 선별하는, MDSC 활성 저해 약물의 선별 방법.MDSC activity, which predicts and selects the degree of change in MDSC activity by analyzing the level of MITF gene expression or MITF protein expression in MDSC by a specific substance by any one method selected from qRT-PCR, Western blotting and flow cytometry (FACS) Methods for screening inhibitory drugs.
  2. 제1항에 있어서, 상기 특정물질은 MITF의 유전자 발현 억제제 또는 MITF의 단백질 활성 억제제인, MDSC 활성 저해 약물의 선별 방법.The method of claim 1, wherein the specific substance is a MITF gene expression inhibitor or a MITF protein activity inhibitor.
  3. 제1항에 있어서, 다음 단계를 포함하는 MDSC 활성 저해 약물의 선별 방법.The method according to claim 1, comprising the following steps.
    (a) 골수 세포를 준비하는 단계;(a) preparing bone marrow cells;
    (b) 상기 골수 세포로부터 MDSC 분화 및 활성을 유도하는 단계;(b) inducing MDSC differentiation and activity from the bone marrow cells;
    (c) 상기 (b) 단계에서 분화 및 활성 유도된 MDSC를 회수하여 MITF 유전자 또는 MITF 단백질 발현 정도를 분석하는 단계; 및(c) recovering the MDSCs differentiated and activated in step (b) and analyzing the level of expression of the MITF gene or MITF protein; and
    (d) 상기 (c)의 분석결과로부터 상기 특정물질 중 MDSC 활성 저해 약물을 선별하는 단계(d) selecting a MDSC activity inhibitory drug from the specific substance from the analysis result of (c)
  4. 제3항에 있어서, 상기 골수 세포는 종양을 갖는 개체로부터 얻은 골수 세포인 MDSC 활성 저해 약물의 선별 방법. The method of claim 3, wherein the bone marrow cells are bone marrow cells obtained from a subject having a tumor.
  5. 제3항에 있어서, 상기 (b) 단계에서 시험군 배지인 암세포 조건 배지에 MDSC 분화 유도 인자 및 상기 특정물질을 처리하고, 대조군 배지로서 상기 특정물질이 포함되지 않은 배지를 사용하여, MITF 유전자 또는 MITF 단백질 발현 정도를 비교하는 MDSC 활성 저해 약물의 선별 방법.The method of claim 3, wherein in the step (b), MDSC differentiation inducing factor and the specific substance are treated with the cancer cell condition medium, which is the test group medium, and the medium without the specific substance is used as the control medium, and the MITF gene or A method for selecting drugs that inhibit MDSC activity by comparing the level of MITF protein expression.
  6. 제3항에 있어서, 상기 (d) 단계에서 MITF 유전자 또는 MITF 단백질의 발현 정도가 RT-PCR 분석법 시행시 직접적인 MITF 저해 물질의 경우 대조군 대비 시험군에서 qRT-PCR 측정값이 50 % 이상 저해된 경우에, 간접적인 경로로 MITF를 저해하는 물질의 경우 대조군 대비 시험군에서 30 % 이상 저해된 경우에, 해당 물질을 MDSC 활성 저해 약물로 결정하는 단계를 추가로 포함하는, MDSC 활성 저해 약물의 선별 방법.The method of claim 3, wherein the expression level of the MITF gene or the MITF protein in step (d) is inhibited by 50% or more in the qRT-PCR measurement value in the test group compared to the control group in the case of a direct MITF inhibitor when the RT-PCR assay is performed In the case of a substance that inhibits MITF by an indirect route, if the substance is inhibited by 30% or more in the test group compared to the control group, a method for selecting a drug that inhibits MDSC activity, further comprising determining the substance as an MDSC activity inhibitory drug .
  7. 제3항에 있어서, 상기 종양은 유방암, 간암, 위암, 결장암, 폐암, 비소세포성폐암, 골암, 췌장암, 피부암, 두부 또는 경부암, 자궁경부암, 난소암, 대장암, 소장암, 직장암, 항문부근암, 나팔관암종, 자궁내막암종, 자궁경부암종, 질암종, 음문암종, 호지킨병, 식도암, 소장암, 임파선암, 방광암, 담낭암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 전립선암, 선암종, 만성 또는 급성 백혈병, 림프구 림프종, 방광암, 신장 또는 수뇨관 암, 신장세포 암종, 신장골반 암종, 중추신경계 종양, 1차 CNS 림프종, 척수 종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군으로부터 선택된 1종 이상인 것인, MDSC 활성 저해 약물의 선별 방법.The method of claim 3, wherein the tumor is breast cancer, liver cancer, stomach cancer, colon cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, or near the anus. Cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, lymph gland cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethra Cancer, penile cancer, prostate cancer, adenocarcinoma, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma and pituitary gland At least one selected from the group consisting of adenomas, a method for screening MDSC activity inhibitory drugs.
  8. 제1항에 있어서, 상기 MDSC 활성 저해 약물을 선별하기 위하여, 보조적으로 상기 MDSC와 T 세포를 공동배양하고, T 세포 증식 억제 정도를 판단하는 단계를 포함하는, MDSC 활성 저해 약물의 선별 방법.According to claim 1, In order to select the MDSC activity inhibitory drug, co-culture of the MDSC and T cells as an auxiliary, comprising the step of determining the degree of T cell proliferation inhibition, MDSC activity inhibitory drug screening method.
  9. 제2항에 있어서, 상기 MITF의 유전자 발현 억제제는, MITF 유전자의 mRNA에 상보적으로 결합하는 안티센스 뉴클레오타이드, 앱타머, 짧은 헤어핀 RNA(small hairpin RNA, shRNA), 작은 간섭 RNA(small interfering RNA, siRNA), 마이크로 RNA(microRNA, miRNA) 및 리보자임(ribozyme)으로 이루어진 군으로부터 선택된 1종 이상인, MDSC 활성 저해 약물의 선별 방법.The method of claim 2, wherein the MITF gene expression inhibitor is an antisense nucleotide, an aptamer, a short hairpin RNA (shRNA), a small interfering RNA (siRNA) that complementarily binds to the mRNA of the MITF gene ), micro RNA (microRNA, miRNA) and ribozyme (ribozyme) a selection method of at least one selected from the group consisting of MDSC activity inhibitory drug.
  10. 제2항에 있어서, 상기 MITF의 단백질 활성 억제제는 MITF 단백질에 특이적으로 결합하는 화합물, 펩티드, 펩티드 모방체, 기질 유사체, 앱타머, 및 항체로 이루어진 군으로부터 선택된 1종 이상인, MDSC 활성 저해 약물의 선별 방법.The drug according to claim 2, wherein the MITF protein activity inhibitor is at least one selected from the group consisting of compounds, peptides, peptidomimetics, substrate analogs, aptamers, and antibodies that specifically bind to the MITF protein. selection method.
  11. 제1항에 있어서, 상기 특정물질은 AMPK 활성 촉진제, ML-329, MITF-gRNA 클로닝된 벡터, 항HIV제로부터 선택되는 1종 이상인 MDSC 활성 저해 약물의 선별 방법.The method of claim 1, wherein the specific substance is one or more selected from AMPK activity promoters, ML-329, MITF-gRNA cloned vectors, and anti-HIV agents.
  12. 제1항의 선별 방법에 의해 선별된 MDSC 활성 저해 약물을 포함하고, T 세포 증식을 억제하는 조성물.A composition comprising an MDSC activity inhibitor selected by the screening method of claim 1 and inhibiting T cell proliferation.
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