KR102352126B1 - Composition for inhibiting myeloid-derived suppressor cells comprising MITF inhibitor - Google Patents

Abstract

The present invention relates to a composition for inhibiting myeloid-derived suppressor cells (MDSC) containing a microphthalmia-associated transcription factor (MITF) inhibitor as an active ingredient. is lowered, MITF is involved in the activation of the MDSC, and it was confirmed that MDSC can be inhibited using the MITF inhibitor. And it can be usefully used to increase the efficiency of anticancer immunotherapy.

Description

Composition for inhibiting myeloid-derived suppressor cells comprising MITF inhibitor

The present invention relates to a composition for inhibiting myeloid-derived suppressor cells containing a microphthalmia-associated transcription factor (MITF) inhibitor as an active ingredient, and specifically to a bone marrow-derived composition containing an MITF inhibitor as an active ingredient. It relates to a composition for alleviating, treating or preventing the decrease in immune response caused by suppressor cells.

Myeloid cells originate from hematopoietic stem cells. These are the most abundant hematopoietic stem cells in our body and are mainly present in bone marrow and lymphoid tissues. Finally, they differentiate into macrophages, dendritic cells, and granulocytes, but these do not show a specific hierarchical structure and myeloid cells with various levels of differentiation are specifically and diversely distributed in tissues and environments. has the characteristics to be

Myeloid-derived suppressor cells (MDSC) are cells with an immune response suppressing action among myeloid cells, and are a cell group including 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, inhibiting various processes from T cell proliferation to function. It is known to effectively inhibit T cells. Although the action of MDSCs is beneficial in that it suppresses autoimmune responses, if the immune suppressive environment continues due to the accumulation of MDSCs, the living body is continuously exposed to allergens and/or viral infections, which may result in chronic inflammation. When the body is unable to make an effective immune response, tissue damage may occur during chronic inflammation. In addition, it was confirmed that MDSC aids in invasion and metastasis of tumor cells in the cancer cell microenvironment (TME).

Based on these studies on the function and mechanism of action of MDSCs, efforts to develop new cancer therapies through their control are accelerating. Representative gemcitabine (Gemcitabine) and 5-fluorouracil (5-fluorouracil, 5-FU) is known as a chemotherapeutic agent that directly reduces MDSC.

In addition, as the frequency and function of MDSCs are known to contribute to patient resistance to immune checkpoint inhibitors (ICIs), which are currently actively used as anticancer immunotherapeutic agents in clinical practice, the importance of MDSCs is being emphasized. ICI improves the anticancer immune response of cancer patients by preventing the 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 be effective against 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 many patients, up to 80%, do not respond to these treatments, and it is estimated that the main cause is the immunosuppressive cells constituting the TEM. ^{Treg cells, MDSCs, TH2 CD4 +} cells, cancer-associated fibroblasts (CARs) and M2-polarized tumor associated macrophages (TAMs) are well known as immunosuppressive cells in TME that affect the efficacy of ICI treatment. In particular, it is emphasized that MDSC can be used as an important prognostic factor in the treatment of ICI as the results of overcoming the inherent resistance to ICI by enhancing the anticancer immune response when MDSC is deficient have been reported.

On the other hand, to overcome non-response or low reactivity to ICI, recent attempts have been made to combine treatment with other types of ICI, and in this case, clinical benefit is significantly increased compared to monotherapy. has been reported In addition, anti-CTLA4 and anti-PD-1 combination, anti-PD-1 and IL-2R agonist combination, VEGF inhibitor or chemokine modulator and ICI combination, anti-CTLA4 and CTL therapeutic agent The combination is being studied clinically. Furthermore, by administering ICI together with radiation therapy or by using ICI and various conventional chemotherapeutic agents or targeted anticancer agents in combination with radiation therapy to induce T cell activation and invasion into the tumor, increase the therapeutic response and the effect of the anticancer immune response. Various attempts are being made for this.

MITF (microphthalmia-associated transcription factor) is a transcription factor containing a helix-loop-helix leucine zipper. It controls the survival and differentiation of melanocytes and melanomas, and melanin. It is known to be involved in the formation mechanism. Research results on MITF have started to be reported recently. It is known that the activation of LXR (Liver X receptor) promotes the degradation of MITF and inhibits melanogenesis, but the role of MITF in MDSC has not yet been elucidated. none.

Accordingly, the present inventors have made efforts to develop a formulation capable of alleviating the decrease in the immune response of MDSC through inhibition of MDSC and improving the therapeutic effect of an anticancer immunotherapeutic agent. Based on the confirmation that MITF is involved in the activation of MDSC and that MDSC can be inhibited using the MITF inhibitor, it is revealed that the MITF inhibitor can be used for alleviating the decrease in the immune response of MDSC and for anticancer immunotherapy, The present invention was completed.

International Patent Publication No. 2013-082591 International Patent Publication No. 2011-116299 Republic of Korea Patent No. 1902355 Republic of Korea Patent No. 1826753

Gabrilovich DI, et al., Coordinated regulation of myeloid cells by tumours, Nat Rev Immunol. 12(4):253-68 (2012) De Veirman K, et al., Myeloid-derived suppressor cells as therapeutic target in hematological malignancies, Front Oncol. 4:349 (2014) 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) Russell W Jenkins, et al., Mechanisms of resistance to immune checkpoint inhibitors. British Journal of Cancer. 118: 9-16 (2018) 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) David Killock, et al., Targeting MDSCs with LXR agonists, Nature Reviews Clinical Oncology. 15: 200-201　 (2018) 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) 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) Song YC, et al., Berberine regulates melanin synthesis by activating PI3K/AKT, ERK and GSK3β in B16F10 melanoma cells. Int J Mol Med. 2015 Apr;35(4):1011-6 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)

SUMMARY OF THE INVENTION It is an object of the present invention to contain, as an active ingredient, a microphthalmia-associated transcription factor (MITF) inhibitor that inhibits myeloid-derived suppressor cells, alleviation and treatment of decreased immune response caused by bone marrow-derived suppressor cells. Or to provide a composition for prevention.

Another object of the present invention is to provide a method for inhibiting bone marrow-derived suppressor cells, comprising administering an MITF inhibitor to a subject in need of inhibition of bone marrow-derived suppressor cells.

In order to achieve the object of the present invention, the present invention comprises the step of administering a microphthalmia-associated transcription factor (MITF) inhibitor to a subject in need of inhibition of myeloid-derived suppressor cells, bone marrow-derived A method for inhibiting suppressor cells is provided.

The present inventors found that in the cancer cell environment, myeloid-derived suppressor cells (MDSC) are activated, the immune response is lowered, and MITF (microphthalmia-associated transcription factor) is involved in the activation of the MDSC, and the MITF inhibitor Since it was confirmed that MDSC can be inhibited using

1 is a diagram schematically illustrating a method for manufacturing a mouse bone marrow-derived bone marrow-derived suppressor cell (MDSC).
2 is a view of MDSC after treatment with a tumor cell-conditioned medium (TCCM) in which cells isolated from the bone marrow of a mouse are cultured with GM-CSF, a differentiation inducing factor of MDSC, according to an embodiment of the present invention; It is a diagram confirming differentiation (FIG. 2A) and activity (FIG. 2B) changes.
3 is a microphthalmia-associated transcription factor (MITF) gene (FIG. 3A) and protein (FIG. 3B) in MDSC after TCCM treatment with GM-CSF in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention; ) is a diagram confirming the expression change.
4 is a view confirming the change in the inhibition of T cell activity of MDSC treated with TCCM together with GM-CSF in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention.
5 is a diagram confirming the differentiation (FIG. 5A), activity (FIG. 5B) and MITF gene expression (FIG. 5C) changes of MDSC in tumorigenic mice.
6 is a drug inducing the activity of MDSC in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention. After treatment with IL-18 with GM-CSF, the differentiation of MDSC (FIG. 6A), activity (FIG. 6B), and a diagram confirming the expression change of the gene (FIG. 6C) and protein (FIG. 6D) of MITF.
7 is a drug inducing MDSC activity in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention, after treatment with IL-4 with GM-CSF, differentiation of MDSC ( FIG. 7A ), activity ( FIG. 7A ) 7B) and MITF gene expression (FIG. 7C) is a diagram confirming the change.
8 is a drug inducing MDSC activity in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention, and LPS is treated with GM-CSF, followed by MDSC differentiation (FIG. 8A), activity (FIG. 8B) and MITF gene expression (FIG. 8C) is a diagram confirming the change.
9 is a drug inducing MDSC activity in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention. Simvastatin (Simvastatin, Sim), lovastatin (Lovastatin, Lova), It is a diagram confirming the differentiation change of MDSC after treatment with GM-CSF with suvastatin (Rosuvastatin, Rosu), or atorvastatin (Atorvastatin, Ator).
10 is a drug inducing MDSC activity in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention. Simvastatin (Simvastatin, Sim), lovastatin (Lovastatin, Lova), After treatment with suvastatin (Rosuvastatin, Rosu), or atorvastatin (Ator) together with GM-CSF, the activity of MDSC (Fig. 10A), and MITF gene (Fig. 10B) and protein (Fig. 10C) expression changes is a confirmation of
11 is a drug inducing MDSC activity after inducing differentiation by treating cells isolated from the bone marrow of a mouse according to an embodiment of the present invention, simvastatin (Simvastatin, Sim), lovastatin (Lovastatin, Lova), provastatin (Pravastatin, Prav), rosuvastatin (Rosuvastatin, Rosu), or atorvastatin (Atorvastatin, Ator) after treatment for 24 hours is a diagram confirming the differentiation change of MDSC.
12 is a drug inducing MDSC activity after inducing differentiation by treating cells isolated from the bone marrow of a mouse according to an embodiment of the present invention, simvastatin (Simvastatin, Sim) or lovastatin (Lovastatin, Lova) is a diagram confirming changes in MDSC activity (FIG. 12A) and MITF gene expression (FIG. 12B) after treatment for 24 hours.
13 is a drug that inhibits MDSC activity in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention. After treatment with GM-CSF, ATRA (All-trans retinoic acid), MDSC differentiation (FIG. 13A) ), activity (FIG. 13B), and MITF gene expression (FIG. 13C) is a view confirming the change.
Figure 14 shows the differentiation (FIG. 14A), activity (FIG. 14B), and MITF gene expression of MDSC after treating IBMX with GM-CSF as an MITF inducer in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention; (FIG. 14C) is a diagram confirming the change.
15 is MITF gene expression (FIG. 15A), MDSC differentiation (FIG. 15B) after treatment with GM-CSF with berberine as an MITF inhibitor in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention; ) and activity (FIG. 15C) confirming the change.

Hereinafter, the present invention will be described in more detail.

The present invention provides a composition for alleviating, treating or preventing the decrease in immune response caused by myeloid-derived suppressor cells (MDSC), which contains an MITF (Microphthalmia-associated transcription factor) inhibitor as an active ingredient.

In the present invention, the "MITF inhibitor" may include, without limitation, a substance capable of achieving the purpose of inhibiting the activity of MDSC through inhibition of MITF expression or activity. Specifically, the MITF inhibitor may be a gene expression inhibitor of MITF or a protein activity inhibitor of MITF.

More specifically, the MITF gene expression inhibitor is an antisense nucleotide complementary to the mRNA of the MITF gene, an aptamer, a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro It may be one or more selected from the group consisting of RNA (microRNA, miRNA) and ribozyme, but is not limited thereto.

The "antisense nucleotide" binds (hybridizes) to the complementary base sequence of DNA, immature-mRNA or mature mRNA, as defined in Watson-Click base pairing, and interferes with the flow of genetic information from DNA to protein. The nature of antisense nucleotides that are specific for a target sequence makes them exceptionally versatile. Since antisense nucleotides are long chains of monomeric units, they can be readily synthesized for the target RNA sequence. Many recent studies have demonstrated the usefulness of antisense nucleotides as biochemical means for studying 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 nucleotide synthesis exhibiting improved cell adsorption, target binding affinity and nuclease resistance, the use of antisense nucleotides can be considered as a new type of inhibitor.

The "short hairpin RNA (small hairpin RNA)" or "shRNA" refers to a single strand consisting of 50 to 60 nucleotides, and forms a stem-loop structure in vivo. That is, shRNA is an RNA sequence that makes a tight hairpin structure for suppressing gene expression through RNA interference (RNAi). A long RNA of 15-30 nucleotides is complementary to both sides of a loop of 5-10 nucleotides to form a double-stranded stem. For general expression, shRNA is transduced into a cell through a vector containing a U6 promoter, and is usually transferred to daughter cells so that gene expression inhibition is inherited. The shRNA hairpin structure is cleaved by an intracellular mechanism to become siRNA and then binds to RISC (RNA-induced silencing complex). These RISCs bind to and cleave mRNA. shRNA is transcribed by RNA polymerase.

The “small interfering RNA” or “siRNA” refers to a short double-stranded RNA capable of inducing RNA interference through specific mRNA cleavage. It is composed of a sense RNA strand having a sequence homologous to the mRNA of a target gene and an antisense RNA strand having a sequence complementary thereto. Since siRNA can inhibit the expression of a target gene, it is provided as an efficient gene knock-down method or as a gene therapy method.

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 complementary binding to a target mRNA having a complementary base sequence, degradation of the target mRNA or translation into a protein is inhibited.

In addition, the protein activity inhibitor of MITF may be at least one selected from the group consisting of a compound that specifically binds to the MITF protein, a peptide, a peptidomimetic, a substrate analog, an aptamer, and an antibody, but is not limited thereto.

The "peptide mimetics" inhibits the activity of the MITF protein by inhibiting the binding domain of the MITF protein. A peptidomimetic may be peptide or non-peptide, and may consist of amino acids joined by non-peptide bonds, such as psi bonds. In addition, "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 sieve The peptidomimetic is 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 be a novel small molecule that can act with an effect equivalent to that of a natural antagonist.

The "aptamer" is a single-stranded DNA or RNA molecule, and is high in a specific chemical molecule or biological molecule by an evolutionary method using an oligonucleotide library called SELEX (systematic evolution of ligands by exponential enrichment). It can be obtained by isolating an oligomer that binds with affinity and selectivity. Aptamers can specifically bind to a target and modulate the activity of the target, eg, block 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. As the antibody specifically binding to the MITF protein, a polyclonal antibody or a monoclonal antibody may be used. The antibody specifically binding to the MITF protein may be prepared by a known method known to those skilled in the art, or a commercially known MITF antibody may be purchased and used. The antibody can 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. The immunogen is injected by an intramuscular, intraperitoneal or subcutaneous injection method, and can generally be administered together with an adjuvant to increase antigenicity. Antibodies can be isolated by routinely collecting blood from an external host and collecting sera showing a titer and antigen specificity formed.

In addition, the MITF inhibitor may be an AMPK activity promoter (activator), specifically, the AMPK activity promoter may be berberine or Kazinol U, but is not limited thereto. The AMPK activity promoter suppresses the expression of the MITF gene or the activity of the MITF protein, for example, transcriptional activity.

In the present invention, the "bone marrow-derived suppressor cells" or "MDSCs" function to suppress immunity by inhibiting the activity of cytotoxic T lymphocytes and NK cells. It has a positive function of suppressing an unnecessary excessive immune response, such as autoimmunity, but it also has a negative function of suppressing immunity in situations where an immune response is required to cause or aggravate a disease or prevent an appropriate treatment. For example, MDSCs are highly elevated in tumor or cancer patients, which significantly reduces the effectiveness of cancer vaccine administration, thereby neutralizing the efficacy of cancer vaccines. In addition, it contributes to the patient's resistance to the immune checkpoint inhibitor (ICI), which is used as an anticancer immunotherapeutic agent, thereby reducing the effectiveness of the anticancer immunotherapeutic agent. In this situation, specifically, if the number of MDSCs in a tumor-bearing individual is effectively reduced or the activity of MDSCs is effectively inhibited, it is possible to prevent a decrease in the immune response caused by MDSCs to increase the efficacy of a cancer vaccine or anticancer immunotherapeutic agent, and to treat cancer will be able to perform smoothly and effectively.

The MDSC of an individual having the tumor may have a phenotype of CD11b ⁺ Gr1 ⁺ PD-L1 ⁺ , but is not limited thereto.

The tumor is specifically breast cancer, skin or intraocular melanoma, 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 , perianal cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin’s disease, esophageal cancer, small intestine cancer, lymph adenocarcinoma, bladder cancer, gallbladder cancer, endocrine adenocarcinoma, 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 nerve It may be a glioma or a pituitary adenoma, but is not limited thereto.

In the present invention, the composition is preferably administered to a subject in need of inhibiting MDSC.

In a specific example of the present invention, the present inventors prepared mouse bone marrow-derived MDSCs by treating GM-CSF as an MDSC differentiation inducing factor in cells isolated from mouse bone marrow.

In addition, the present inventors treated the cells isolated from the bone marrow of the mouse with a tumor cell-conditioned medium (TCCM) culturing a mouse breast cancer cell line together with GM-CSF, which is a factor for inducing MDSC differentiation, by GM-CSF. It was confirmed that differentiation of MDSC was induced, MDSC activity was induced by TCCM, and gene and protein expression of MITF was increased in the MDSC induced by the activity. In addition, as a result of co-culturing the TCCM-induced MDSCs and T cells, it was confirmed that the number of ^{CD3 +} CD8 ^{+ T cells decreased by the activity-induced MDSCs.} Through the above results, it was confirmed that MDSCs were activated in the cancer cell environment and the immune response was lowered, and that MITF was involved in the activation of MDSCs as a target factor for the activation of MDSCs.

In addition, the present inventors obtained MDSCs from the spleen and tumor sites of tumorigenic mice, and confirmed that MDSCs obtained from the tumor sites showed high activation and expression of MITF. Through the above results, it was confirmed that MDSC was activated in the cancer cell environment, and that MITF was involved in the activation of the MDSC.

In addition, the present inventors treated the cells isolated from the bone marrow of the mouse together with GM-CSF, a differentiation inducing factor of MDSC, drugs that induce MDSC activity, IL-18, IL-4, LPS, or statin-based drugs As a result, it was confirmed that the differentiation of MDSC was induced by GM-CSF, the activity of MDSC was induced by the MDSC activity-inducing drugs, and the gene and protein expression of MITF 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) as a drug that inhibits MDSC activity together with GM-CSF, a factor for inducing MDSC differentiation, the differentiation of MDSCs by GM-CSF was inhibited. induced, the activity of MDSC was inhibited by the MDSC activity inhibitory drug, and it was confirmed that the gene expression of MITF was decreased in the MDSC in which the activity was suppressed. Through the above results, it was confirmed that MITF was involved in the activation of MDSC.

In addition, the present inventors treated cells isolated from the bone marrow of mice with IBMX as an MITF inducer together with GM-CSF, a differentiation inducing factor of MDSC. As a result, MDSC differentiation was induced by GM-CSF, and Expression is increased, and it was confirmed that MDSC was activated at this time. On the other hand, as a result of treating the cells isolated from the bone marrow of mice with berberine as an MITF inhibitor together with GM-CSF, a differentiation inducing factor of MDSC, MDSC differentiation was induced by GM-CSF, but the MITF inhibitor MITF expression was decreased, and it was confirmed that the activity of MDSC was suppressed at this time. Through the above results, it was confirmed that the activity of MDSC can be inhibited by using an inhibitor of MITF.

Therefore, the present inventors confirmed that MDSC is activated in the cancer cell environment, thereby reducing the immune response, MITF is involved in the activation of MDSC, and can inhibit MDSC using the MITF inhibitor, so the MITF inhibitor is effective The composition containing as a component can be usefully used to alleviate the decrease in immune response caused by MDSC and to increase the efficiency of anticancer immunotherapy.

The composition of the present invention may be preferably formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration.

In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection, 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 bacteriostats may be added as needed.

In addition, the composition of the present invention may 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, a swelling agent, and a lubricant. , a solubilizing agent such as a lubricant or flavoring agent may be used.

In addition, the composition of the present invention may be formulated into an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets by additionally adding diluents, dispersants, surfactants, binders and lubricants. Furthermore, by using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA by an appropriate method in the art, it can be preferably formulated according to the disease or component.

In addition, the compositions of the present invention may be administered via 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 at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, drug activity, and type of the patient's disease; Sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs and other factors well known in the medical field may be determined. 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 singly or multiple times. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by a person skilled in the art.

Specifically, the effective amount of the composition according to the present invention may vary depending on the age, sex, and weight of the patient, and generally 0.1 mg to 100 mg per kg body weight, more specifically 0.5 mg to 10 mg per kg body weight, administered daily or every other day Or it can be administered in divided doses 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, the severity of the disease, sex, weight, age, etc., the dosage is not intended to limit the scope of the present invention in any way.

In addition, the present invention provides an anticancer adjuvant containing an MITF inhibitor as an active ingredient.

In the present invention, the MITF inhibitor may be a gene expression inhibitor of MITF or a protein activity inhibitor of MITF.

More specifically, the MITF gene expression inhibitor is an antisense nucleotide complementary to the mRNA of the MITF gene, an aptamer, a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro It may be one or more selected from the group consisting of RNA (microRNA, miRNA) and ribozyme, but is not limited thereto.

The protein activity inhibitor of MITF may be at least one selected from the group consisting of a compound that specifically binds to the MITF protein, a peptide, a peptidomimetic, a substrate analog, an aptamer, and an antibody, but is not limited thereto.

In addition, the MITF inhibitor may be an AMPK activity promoter (activator), specifically, the AMPK activity promoter may be berberine or Kazinol U, but is not limited thereto.

In the present invention, the anti-cancer adjuvant may be administered in combination with an anti-cancer agent, and the anti-cancer adjuvant suppresses the activity of MDSC to alleviate the decrease in immune response caused by MDSC, thereby exhibiting an anti-cancer adjuvant effect that significantly increases the effect of the anti-cancer agent. can

The anticancer agent may be one or more selected from the group consisting of chemotherapeutic agents, targeted therapeutics, antibody therapeutics, immunotherapeutics, and hormone therapeutics, but is not limited thereto.

Such chemotherapeutic agents include, for example, antimetabolites (eg, folic acid, purine, and pyrimidine derivatives), alkylating agents (eg, nitrogen mustards, nitrosoureas, platinum, alkyl sulfonates, hydrazine, triazines, aziridine, 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).

The targeted therapeutic agent is an agent specific for a protein that is not regulated in 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 lib (Seliciclib), but is not limited thereto.

The antibody therapeutic agent 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.

The immunotherapeutic agent is an agent designed to induce the subject's own immune system to attack a tumor. For example, Ipilimumab, Avelumab, Nivolumab, or Pembrolizumab. However, the present invention is not limited thereto.

The hormone therapeutic agent is an agent that inhibits cancer growth by providing or blocking hormones in a specific cancer, for example, tamoxifen (Tamoxifen) or diethylstilbestrol (diethylstilbestrol), but is not limited thereto.

Since the appropriate dosage of the anticancer agent is already well known in the art, it may be administered according to the standards known in the art according to the condition of each patient. Determination of specific dosages is within the level of those skilled in the art, and the daily dosage thereof is, for example, specifically 1 mg/kg/day to 10 g/kg/day, more specifically 10 mg/kg/day to 100 mg It may be /kg/day, 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.

In a specific embodiment of the present invention, the present inventors found that MDSC is activated in a cancer cell environment, thereby lowering the immune response, MITF is involved in the activation of the MDSC, and the MITF inhibitor can be used to inhibit the activity of MDSC. As it was confirmed, the MITF inhibitor can be used as an anticancer adjuvant for anticancer immunotherapy.

In addition, the present invention provides a method for inhibiting MDSC, comprising administering an MITF inhibitor to a subject in need of inhibition of MDSC.

The detailed description of the MITF inhibitor and MDSC is the same as the description of the composition, and the detailed description will refer to the above content, and only the composition specific to the MDSC inhibitory method will be described below.

In the present invention, the "inhibition of bone marrow-derived suppressor cells" or "MDSC inhibition" includes not only inhibiting the activity of MDSCs, but also reducing the number of MDSCs. Reducing the number includes not only inhibiting the production of cells, but also killing or differentiating cells that have already been formed into other cells. In addition, all mechanisms that are referred to as “inhibition” from a biological point of view are included.

In the present invention, the individual in need of MDSC inhibition may be an individual having a tumor that specifically requires MDSC inhibition, and the MDSC of the individual having the tumor may have a phenotype of CD11b ⁺ Gr1 ⁺ PD-L1 ⁺ , However, the present invention is not limited thereto.

In addition, the tumor is specifically breast cancer, skin or intraocular melanoma, 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, lymph adenocarcinoma, bladder cancer, gallbladder cancer, endocrine adenocarcinoma, 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, It may be, but is not limited to, a brainstem glioma or a pituitary adenoma.

In a specific embodiment of the present invention, the present inventors found that MDSC is activated in a cancer cell environment, thereby lowering the immune response, MITF is involved in the activation of the MDSC, and the MITF inhibitor can be used to inhibit the activity of MDSC. As it has been confirmed, the MITF inhibitor can be administered to an individual in need of MDSC inhibition and used to treat MDSC-related diseases.

Hereinafter, the present invention will be described in detail by way of Examples.

However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

<Example 1> Bone marrow-derived suppressor cells (myeloid-derived suppressor cells, MDSC) production

<1-1> Production of mouse bone marrow-derived MDSC

As shown in the schematic diagram of FIG. 1, myeloid-derived suppressor cells (MDSC) were prepared from the bone marrow (BM) of Balb/c mice.

Specifically, 6 to 8-week-old Balb/c mice were tested in Saeronbio. It was purchased from Inc. (Korea). Animal experiments were performed with the approval of the Institutional Ethical Committee of Sookmyung Women's University (SMWU-IACUC-1708-017-02). After removing the femur from 6 to 8-week-old Balb/c mice, the bone marrow was isolated from the bones, and then treated with RBC lysis buffer (Sigma-Aldrich, St. Louis, MO) to remove red blood cells to obtain cells. Differentiation of MDSCs by culturing the obtained bone marrow cells in RPMI medium (Invitrogen, Grand Island, NY) containing GM-CSF at a concentration of 10 ng/ml in a 24-well plate at a cell number ^{of 5×10 5 cells/ml for 96 hours} was induced.

<1-2> MDSC production in tumorigenic mice

Balb/c mice were injected with a breast cancer cell line to prepare tumorigenic mice, and MDSCs were prepared from the tumorigenic mice.

Specifically, a tumor was formed by subcutaneously injecting 100 μl PBS containing ^{4T1 cells, a mouse breast cancer cell line, at a cell count of 5×10 5} cells/100 μl to the right flank in 6 to 8-week-old Balb/c mice. The mice were bred in the animal laboratory of 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. Two weeks later, the tumor, bone marrow, and spleen were removed, respectively, and cells were isolated. Then, after staining using a fluorescence-conjugated anti-CD11b antibody, an anti-Gr1 antibody, and an MDSC activity marker, PD-L1 antibody, FACS (Fluorescence-acitivated cell sorting) analysis was performed to confirm the differentiation and activity of MDSCs. did. The antibody was purchased from eBioscience (San Diago, CA).

<Example 2> Confirmation of decreased immune response by MDSC in cancer cell environment

<2-1> Confirmation of MDSC activity in cancer cell environment

In order to examine whether the immune response is lowered by MDSC in the cancer cell environment, the activity of MDSC treated with the cancer cell culture medium was checked.

Specifically, 4T1 cells, a mouse breast cancer cell line, were treated with 10 ml of RPMI medium (Invitrogen, Grand Island, NY) containing 10% fetal bovine serum (FBS) and 100 units of antibiotics (Invitrogen, Grand Island, NY) _{under 5% CO 2} , 3 Cells were allowed to reach 80% confluency by incubation for one day. Then, the culture medium was recovered and concentrated with a 3000 NMWL (nominal molecular weight limit) centrifugal filter (Merck Milipore, Billerica, MA) at 3000 g for 20 minutes in a centrifuge maintained at 4° C. to obtain TCCM (tumor cell-conditioned medium). obtained. After the bone marrow cells were obtained in the same manner as in <Example 1>, the obtained TCCM and GM-CSF at a concentration of 10 ng/ml were contained in a 24-well plate at a cell number of ^{5×10 5 cells/ml.} Differentiation of MDSCs was induced by incubation in RPMI medium for 96 hours. As a control, MDSCs induced to differentiate in RPMI medium containing GM-CSF were used.

In order to confirm the differentiation of MDSCs, the differentiation-induced MDSCs were recovered and stained with fluorescence-conjugated anti-CD11b and anti-Gr1 antibodies, followed by FACS analysis (FIG. 2A).

In addition, in order to confirm the activity of MDSC, the differentiation-induced MDSC was recovered and qRT-PCR was performed on iNOS, IL-10 and TGF-β, which are MDSC activity markers. The differentiation-induced 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 according to the manufacturer's procedure was reverse transcribed using the M-MLV reverse transcription kit (Promega, Madison, WI), oligo-(dT) primer, and dNTP (Bioneer, Korea), and in the ABI Real-time PCR 7500 system. Quantitative PCR was performed according to the manufacturer's procedure using primers for iNOS, IL-10, TGF-β and cyclophilin and SYBR Green PCR Master Mix (Applied Bosystems, Foster City, CA). In this case, cyclophilin was used as a control. Primers for iNOS, IL-10, TGF-β and cyclophilin were purchased from Bioneer. The experiment was repeated three times (FIG. 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 (GM-CSF) and the TCCM treatment group (GM+TCCM) ( FIG. 2A ). On the other hand, in the case of the TCCM-treated group (GM+TCCM), the expression of iNOS, IL-10 and TGF-β, which are active markers of MDSC, was higher than that of the control group, so it was confirmed that the activity of MDSC was induced by TCCM (Fig. 2B). ).

<2-2> Confirmation of increased MITF expression of MDSC activated in cancer cell environment

Changes in gene and protein expression of microphthalmia-associated transcription factor (MITF) were confirmed in MDSC induced by TCCM of Example <2-1>.

Specifically, in order to confirm the gene expression of MITF, qRT-PCR was performed on MITF using the MDSC recovered in Example <2-1> in the same manner as in Example <2-1>. . Primers for MITF were purchased from Bioneer (FIG. 3A).

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 MDSC recovered in Example <2-1> was treated with a cell lysis buffer and lysed. Cell lysates were separated by electrophoresis on a 10% SDS-PAGE gel and transferred to a PVDF membrane. Then, after reacting with the primary antibody by treating the anti-MITF antibody and the anti-actinin antibody, an HRP-conjugated secondary antibody is attached to the primary antibody attached to the membrane, and this enhanced chemiluminescence (PicoEPD™ Western Reagent kit) , ELPIS-Biotech, Daejeon, Republic of Korea) was used for analysis with the LAS-3000 imaging system (FUJIFILM Corporation, Tokyo, Japan). Actinin was used as a control protein ( FIG. 3B ).

As a result, as shown in FIG. 3, in the case of the TCCM treatment group (GM+TCCM), the gene expression (FIG. 3A) and protein expression (FIG. 3B) of MITF were increased compared to the control group (GM-CSF). It was confirmed that MITF is a target factor specifically expressed in MDSCs whose activity is induced by TCCM.

<2-3> Confirmation of decreased immune response by MDSC in cancer cell environment

MDSC is known to decrease the immune response by suppressing the proliferation and function of T cells. Therefore, in order to find out whether the immune response is reduced by MDSC in the cancer cell environment, the degree of inhibition of T cell activity was confirmed using MDSC treated with a medium in which cancer cells were cultured.

Specifically, the spleens of 6 to 8-week-old Balb/c mice were extracted and cells were isolated. Then, after staining using a fluorescence-conjugated 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. 5×10 ⁵ CFSE-labeled CD3 ⁺ T cells were stimulated on plates coated with anti-CD3 monoclonal antibody and soluble anti-CD28 monoclonal antibody for 2 hours, and then recovered in Example <2-1>. MDSCs were put into 5×10 ⁵ cells or 10×10 ⁵ cells, and cultured for 3 days. After 3 days of culture, the cells were stained using a fluorescence-conjugated anti-CD8 antibody, and then the degree of proliferation of ^{CD8 + T cells was measured by performing FACS analysis.} The antibody was purchased from eBioscience.

As a result, as shown in Figure 4, when the TCCM-treated group (GM+TCCM) and T cells were co-cultured, the number of CD3 ⁺ CD8 ⁺ T cells decreased compared to the case of co-culture with the control group (GM-CSF), so TCCM It was confirmed that the activity-induced MDSC inhibited T cell proliferation.

From the above results, it can be seen that MDSCs are activated in the cancer cell environment to lower the immune response, and MITF is involved in the activation of MDSCs as a target factor for the activation of MDSCs.

<Example 3> Confirmation of activity and MITF expression in MDSC of tumor-forming mice

MDSC is activated in tumorigenic mice, and in order to find out whether MITF is involved in the activation of MDSC, its activity and gene expression changes of MITF were confirmed using MDSC in tumorigenic mice.

Specifically, in order to confirm the activity of MDSC, cells were isolated from each of the spleen and tumor of a tumor-forming mouse in the same manner as that described in Example <1-2>, and then FACS analysis was performed (FIG. 5A). In addition, CD11b ⁺ Gr1 ⁺ MDSC were recovered with the FACS sorter, and qRT-PCR was performed in the same manner as in Example <2-1> (FIG. 5B). As a control group, the spleen of tumor-forming mice before sorting was used.

In addition, in order to confirm the gene expression of MITF ^{, qRT-PCR was performed using the recovered CD11b +} Gr1 ⁺ MDSC in the same manner as in Example <2-2> (FIG. 5C).

As a result, as shown in FIG. 5, MDSCs obtained from the spleen and tumor sites of tumorigenic mice took an activated form compared to the control group ( FIGS. 5A and 5B ), and it was confirmed that the gene expression of MITF was increased ( Figure 5C). In particular, it was confirmed that MDSCs obtained from the tumor site were more activated than those obtained from the spleen, and the expression of MITF was significantly higher.

From the above results, it can be seen that MDSC is activated in the cancer cell environment, and MITF is involved in the activation of the MDSC.

<Example 4> Confirmation of increased MITF expression in MDSC induced activity

In order to examine the correlation between MITF and MDSC, MITF expression change was confirmed after treatment with a drug that induces MDSC activity.

<4-1> Confirmation of increased MITF expression in IL-18-induced MDSC

After IL-18 was treated with a drug inducing MDSC activity and differentiation of MDSC was induced, changes in MITF gene and protein expression were confirmed.

Specifically, after obtaining bone marrow cells in the same manner as in <Example 1>, IL-18 at a concentration of 10 or 50 ng/ml in a 24-well plate at a cell number of ^{5×10 5 cells/ml, and 10} Differentiation of MDSCs was induced by culturing in RPMI medium containing ng/ml concentration of GM-CSF for 96 hours. As a control, MDSCs induced to differentiate in RPMI medium containing GM-CSF were used.

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 (FIG. 6A).

In addition, the differentiation-induced MDSCs were recovered and qRT-PCR was performed in the same manner as those described in Examples <2-1> and <2-2> to confirm the activity of MDSCs (FIG. 6B), and MITF Gene expression was confirmed (FIG. 6C).

In addition, the differentiation-induced MDSC was recovered and Western blotting was performed in the same manner as in Example <2-2> to confirm the protein expression of MITF (FIG. 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 treatment group ( FIG. 6A ). On the other hand, in the case of the IL-18 treatment group, it was confirmed that the activity of MDSC increased (FIG. 6B), MITF gene expression (FIG. 6C), and protein expression increased (FIG. 6D) in a concentration-dependent manner.

<4-2> Confirmation of increased MITF expression in MDSC induced by IL-4

After IL-4 was treated with a drug that induces MDSC activity and differentiation of MDSC was induced, a change in MITF gene expression was confirmed.

Specifically, the differentiation and activity of MDSC was induced according to the method described in Example <3-1>, but 10 ng/ml of IL-4 was treated with an activity-inducing drug.

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 (FIG. 7A).

In addition, by recovering the differentiation-induced MDSC, the activity of MDSC was confirmed in the same manner as in Examples <2-1> and <2-2> (FIG. 7B), and the gene expression of MITF was confirmed ( 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-14 treatment group ( FIG. 7A ). On the other hand, in the case of the IL-4 treatment group, the activation of MDSC was induced by IL-4 (FIG. 7B), and it was confirmed that the gene expression of MITF was increased compared to the control group (FIG. 7C).

<4-3> Confirmation of increased MITF expression in LPS-induced MDSC

After treating LPS with a drug that induces MDSC activity and inducing differentiation of MDSC, the change in MITF gene expression was confirmed.

Specifically, the differentiation and activity of MDSC was induced according to the method described in Example <3-1>, but 100 ng/ml of LPS was treated with an activity-inducing drug.

The differentiation-induced MDSCs were recovered and FACS analysis was performed in the same manner as in Example <2-1> to confirm the differentiation of MDSCs (FIG. 8A).

In addition, by recovering the differentiation-induced MDSC, activity of MDSC was confirmed in the same manner as in Examples <2-1> and <2-2> (FIG. 8B), and gene expression of MITF was confirmed ( Figure 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 treatment group ( FIG. 8A ). On the other hand, in the case of the LPS-treated group, it was confirmed that the activation of MDSC was induced by LPS (FIG. 8B), and the gene expression of MITF was increased compared to the control group (FIG. 8C).

<4-4> Confirmation of increase in MITF expression in MDSC induced by statin-based drugs

Simvastatin, a statin-based drug, is known to induce MDSC activity by suppressing the expression of IRF4 (Interferon regulatory factor 4). Accordingly, statin-based drugs were treated with a drug inducing MDSC activity, and MDSC differentiation was induced, and then gene and protein expression changes of MITF were confirmed.

Specifically, inducing differentiation and activity of MDSC according to the method described in Example <3-1>, simvastatin (Sim, 0.5 or 1 μM), lovastatin (Lovastatin, Lova, 0.5 or 1) as a drug inducing activity μM), provastatin (Pravastatin, Prav, 5 or 10 μM), rosuvastatin (Rosuvastatin, Rosu, 0.5 or 1 μM), or atorvastatin (Atorvastatin, Ator, 0.05 or 0.1 μM) (Fig. 9) and Fig. 10).

In addition, according to the method described in Example <1-1>, the differentiation of MDSCs was induced by culturing for 96 hours in RPMI medium containing GM-CSF, and the statin drugs were treated for 24 hours (Fig. 11 and 12).

Then, the MDSCs were recovered and the differentiation (FIGS. 9 and 11) and activity (FIGS. 10A and 12A) of MDSCs was confirmed in the same manner as those described in Examples <2-1> and <2-2>. and MITF gene expression ( FIGS. 10B and 12B ) and protein expression ( FIG. 10C ) were confirmed.

As a result, as shown in FIGS. 9 to 12 , it was confirmed that MDSC differentiation was induced to a similar degree in both the control group and the statin-based drug treatment group ( FIGS. 9 and 11 ). On the other hand, in the case of the statin-based drug-treated group, statin-based drug treatment from the initial stage of differentiation of MDSC (FIG. 10) and the already differentiated MDSC treated with statin-based drug (FIG. 12) Activation of MDSC by statin-based drug was induced ( FIGS. 10A and 12A ), and it was confirmed that the gene expression ( FIGS. 10B and 12B ) and protein expression ( FIG. 10C ) of MITF was increased compared to the control group.

Since it was confirmed that the expression of MITF increased in the activity-induced MDSC through the above results, it can be seen that MITF is involved in the activation of MDSC.

<Example 5> Confirmation of reduction in MITF expression in MDSCs with suppressed activity

All-trans retinoic acid (ATRA) is known to inhibit the activity of MDSC. Accordingly, in order to examine the correlation between MITF and MDSC, MITF expression change was confirmed after treatment with ATRA with a drug that inhibits MDSC activity.

Specifically, after obtaining bone marrow cells in the same manner as described in <Example 1>, ATRA (0.5 or 1 μM) and 10 ng/ml concentration of ATRA (0.5 or 1 μM) and 10 ng/ml in a 24-well plate at a cell number of ^{5×10 5 cells/ml} Differentiation of MDSCs was induced by culturing in RPMI medium containing GM-CSF for 96 hours (FIG. 13).

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 (FIG. 13A).

In addition, the differentiation-induced MDSCs were recovered and qRT-PCR was performed in the same manner as those described in Examples <2-1> and <2-2> to confirm the activity of MDSCs (FIG. 13B), MITF Gene expression was confirmed (FIG. 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).

From the above results, it was confirmed that the expression of MITF was decreased in the MDSC whose activity was suppressed, so it can be seen that MITF is involved in the activation of MDSC.

<Example 6> Confirmation of MDSC activity change by MITF regulation

<6-1> Confirmation of increase in MDSC activity by MITF inducer

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, the change in activity of MDSC treated with IBMX as an MITF expression inducer was confirmed.

Specifically, after obtaining bone marrow cells in the same manner as described in <Example 1>, IBMX at a concentration of 10 μM and GM- at a concentration of 10 ng/ml in a 24-well plate at a cell number of ^{5×10 5 cells/ml} Differentiation of MDSCs was induced by culturing in RPMI medium containing CSF for 96 hours. As a control, MDSCs induced to differentiate in RPMI medium containing GM-CSF were used.

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 (FIG. 14A).

In addition, the differentiation-induced MDSCs were recovered and qRT-PCR was performed in the same manner as those described in Examples <2-1> and <2-2> to confirm the activity of MDSCs (FIG. 14B), and MITF Gene expression was confirmed ( FIG. 14C ).

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 treatment group ( FIG. 14A ). On the other hand, in the case of the IBMX-treated group, it was confirmed that the gene expression of MITF increased (FIG. 14C), and the activity of MDSC was increased compared to the control group (FIG. 14B).

<6-2> Confirmation of inhibition of MDSC activity by MITF inhibitor

Berberine (Berberine), Kazinol U (Kazinol U) and the like are known to inhibit the expression or activity of MITF in melanocytes while being an AMPK activator. Accordingly, in order to examine the effect of MITF expression or activity regulation on MDSC activity, changes in the activity of MDSC treated with berberine as an MITF inhibitor were confirmed.

Specifically, after obtaining bone marrow cells by the same method as described in <Example 1>, berberine at a concentration of 10 μM and GM- at a concentration of 10 ng/ml in a 24-well plate at a cell number of ^{5×10 5 cells/ml} Differentiation of MDSCs was induced by culturing in RPMI medium containing CSF for 96 hours. As a control, MDSCs induced to differentiate in RPMI medium containing GM-CSF were used.

The differentiation-induced MDSCs were recovered and qRT-PCR was performed in the same manner as in Example <2-2> to confirm the expression of MITF (FIG. 15A).

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 (FIG. 15B).

In addition, the differentiation-induced MDSCs were recovered and qRT-PCR was performed in the same manner as in Example <2-1> to confirm the activity of MDSCs (FIG. 15C).

As a result, as shown in FIG. 15 , in the case of the berberine-treated group, it was confirmed that the expression of MITF was suppressed by berberine ( FIG. 15A ), and the differentiation of MDSCs was somewhat reduced ( FIG. 15B ). In addition, in the case of the berberine-treated group, it was confirmed that the activity of MDSC was significantly inhibited compared to the control group (FIG. 15C).

From the above results, it can be seen that the activity of MDSC can be inhibited by suppressing the expression or transcriptional activity of MITF.

Through the <Example 1> to <Example 6>, MDSC is activated in the cancer cell environment, the immune response is lowered, MITF is involved in the activation of the MDSC, and the MITF inhibitor is used to inhibit the activity of MDSC. Since it has been confirmed that the MITF inhibitor can be administered to an individual in need of inhibition of MDSC, for example, an individual having a tumor, it can be usefully used to alleviate the decrease in immune response caused by MDSC. In addition, the composition containing the MITF inhibitor can be effectively used as an adjuvant therapy to increase the anticancer immunotherapy efficiency by co-administration with an anticancer agent, for example, an anticancer immunotherapeutic agent.

Claims (2)

Berberine, an AMPK activator as an MITF (microphthalmia-associated transcription factor) inhibitor, is administered to individuals with breast cancer except for humans who need inhibition of myeloid-derived suppressor cells (MDSC). A method of alleviating the decrease in immune response caused by MDSC, comprising the steps.
The method of claim 1, wherein the MDSCs are activated by MITF.

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