KR20220095112A - Screening method for mdsc 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. Specifically, it was confirmed that MDSC is activated in a cancer cell microenvironment and the immune response is lowered, MITF is involved in the activation of the MDSC, and the MDSC can be inhibited using the MITF inhibitor such that the composition containing the MITF inhibitor as an active ingredient can be usefully used to alleviate the decrease in the immune response caused by MDSC and increase the efficiency of anti-cancer immunotherapy.

Description

Screening method for MDSC activity inhibitory drugs {SCREENING METHOD FOR MDSC INHIBITOR}

In order to prepare a composition for inhibiting myeloid-derived suppressor cell (MDSC), the present invention analyzes the MITF (microphthalmia-associated transcription factor) inhibitory effect in MDSC by a specific substance to prepare an MDSC activity inhibitory drug. The present invention relates to a method for screening, specifically, to a method for screening whether a specific substance is a gene expression inhibitor of MITF or an inhibitor of MITF protein activity, a method for screening an MDSC activity inhibitory drug, and a composition comprising the thus-selected MITF inhibitor.

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 (MDSCs) are cells with an immune response suppressing action among myeloid cell lineages, 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, inhibiting various processes from T cell proliferation to function. It is known to effectively inhibit T cells. Although the action of MDSC is beneficial in that it suppresses autoimmune response, if the immune suppressive environment continues due to the accumulation of MDSC, the living body is continuously exposed to allergens and/or viral infection, which may result in chronic inflammation. If the living body cannot 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 the research on the function and mechanism of action of these MDSCs, efforts to develop new cancer treatments through their control are accelerating. Typically, gemcitabine and 5-fluorouracil (5-fluorouracil, 5-FU) are known as chemotherapeutic agents that directly reduce 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 up to 80% of patients do not respond to these treatments, and it is estimated that the main cause is the cancer cell microenvironment, that is, immunosuppressive cells that make up the Tumor Micro-Environment (TME). The immunosuppressive cells in TME that affect the efficacy of ICI treatment are Treg cells, MDSCs, TH2 CD4 ⁺ cells, cancer-associated fibroblasts (CAFs) and M2-polarized tumor-associated macrophages (TAMs) are well known. 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, 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 increased compared to monotherapy. . 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 helix-loop-helix involved in the regulation of various types of lineage-specific pathways, including melanocytes, osteocytes and mast cells. leucine zipper) is a transcription factor. Lineage-specific refers to a gene or trait found only in a specific cell type. Thus, MITFs may be involved in the redesign of signaling cascades specifically required for the survival and physiological function of normal cell progenitors. In particular, MITF regulates the survival and differentiation of melanocytes and melanoma, and is known to be involved in the mechanism of melanin formation. 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.

[Patent Literature]

(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 literature]

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(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 (2018)

(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 GSK3β 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)

It is an object of the present invention to provide a composition for alleviating, treating or preventing the decrease in immune response caused 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 will do

Another object of the present invention is a method for inhibiting bone marrow-derived suppressor cells, comprising administering a gene expression inhibitor of microphthalmia-associated transcription factor (MITF) or an inhibitor of protein activity of MITF to an individual 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 screening MDSC activity inhibitory drugs by analyzing the inhibitory effect of microphthalmia-associated transcription factor (MITF) in MDSC by a specific substance.

Another object of the present invention is to provide a method for screening whether a specific substance is a gene expression inhibitor of MITF or a protein activity inhibitor of MITF, a method for screening an MDSC activity inhibitory drug, and a composition comprising the thus-selected MITF inhibitor.

The technical problem of the present invention is not limited to the technical problems mentioned above, and another technical problem not mentioned will be clearly understood by those skilled in the art from the following description.

In one embodiment of the present invention, the degree of MDSC activity change by analyzing the MITF gene expression or the MITF protein expression level in MDSC by a specific substance by any one method selected from qRT-PCR, Western blotting and flow cytometry (FACS) Provided is a method for predicting and selecting a drug that inhibits MDSC activity.

In one embodiment of the present invention, in the method for selecting a drug for inhibiting MDSC activity, the specific substance may be a gene expression inhibitor of MITF or a protein activity inhibitor of MITF.

In one embodiment of the present invention, the method for screening a drug for inhibiting MDSC activity comprises the steps of (a) preparing bone marrow cells; (b) inducing MDSC differentiation and activity from the bone marrow cells; (c) recovering the MDSC differentiated in step (b) and analyzing the expression level of the MITF gene or MITF protein; and (d) selecting an MDSC activity-inhibiting drug among the specific substances from the analysis result of (c).

In one embodiment of the present invention, the bone marrow cells used in the method for screening an MDSC activity inhibitory drug may be bone marrow cells obtained from a subject having a tumor.

In one embodiment of the present invention, in the method of screening for a drug inhibiting MDSC activity, in the step (b), an MDSC differentiation inducing factor and the specific substance are treated in a cancer cell conditioned medium, which is the test group medium, and the specific substance is included as a control medium Using an untreated medium, the expression level of the MITF gene or MITF protein can be compared.

In one embodiment of the present invention, in the method for screening MDSC activity inhibitory drugs, the expression level of the MITF gene or MITF protein in step (d) is qRT in the test group compared to the control group in the case of a direct MITF inhibitor when the qRT-PCR analysis is performed. -When the PCR measurement value is inhibited by 50% or more, in the case of a substance that inhibits MITF by an indirect route, if it is inhibited by 30% or more in the test group compared to the control group, the step of determining the substance as an MDSC activity inhibitory drug is additionally may include

In one embodiment of the present invention, the method for selecting a drug for inhibiting MDSC activity is 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, 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 It may be one or more selected from the group consisting of lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.

In one embodiment of the present invention, the method for selecting a drug for inhibiting MDSC activity may include co-culturing the MDSC and T cells and determining the degree of inhibition of T cell proliferation in order to select the drug for inhibiting MDSC activity. can

In one embodiment of the present invention, the method for selecting a drug for inhibiting MDSC activity comprises an antisense nucleotide, an aptamer, a short hairpin RNA (small hairpin RNA, shRNA), wherein the gene expression inhibitor of the MITF is complementary to the mRNA of the MITF gene; It may be at least one selected from the group consisting of small interfering RNA (siRNA), microRNA (miRNA), and ribozyme.

In one embodiment of the present invention, the method for screening a drug for inhibiting MDSC activity comprises a compound in which the protein activity inhibitor of MITF specifically binds to MITF protein, a peptide, a peptidomimetic, a substrate analogue, an aptamer, and an antibody. It may be at least one selected from

In one embodiment of the present invention, in the method for selecting a drug for inhibiting MDSC activity, the specific substance may be at least one selected from AMPK activity promoter, ML-329, MITF-gRNA cloned vector, and anti-HIV agent.

In one embodiment of the present invention, it may relate to a composition comprising the MDSC activity inhibitory drug selected by the method for selecting the MDSC activity inhibitory drug, and for restoring T cell proliferation.

The present invention contains a gene expression inhibitor of MITF (microphthalmia-associated transcription factor) or a protein activity inhibitor of MITF as an active ingredient, myeloid-derived suppressor cell (MDSC) for alleviating the decrease in immune response by, A composition for treatment or prevention is provided.

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

In addition, the present invention provides a method for inhibiting MDSC, comprising administering an MITF gene expression inhibitor or MITF protein activity inhibitor to an individual in need of MDSC inhibition.

Specific details of other embodiments are included in the detailed description and drawings.

The present inventors have made efforts to discover factors that directly affect MDSC activity in order to develop a formulation capable of alleviating the decrease in the immune response of MDSC caused by MDSC inhibition and improving the therapeutic effect of an anticancer immunotherapeutic agent. It has been found that MDSCs are activated in the environment and the immune response is lowered, and that MITF is involved in the activation of the MDSCs. Therefore, based on the confirmation that MDSC can be inhibited using an MITF inhibitor, the present invention was completed by selecting an MITF inhibitor from a wide range of substances and revealing that it can be used as an MDSC activity inhibitory drug. From this, it is possible to more effectively specify active ingredients that can be used for alleviating the decrease in the immune response of MDSC and for anticancer immunotherapy.

Specifically, the present inventors found that when MITF expression is suppressed, the activity of MDSC is inevitably inhibited, and when MITF expression is increased, MDSC activity is also increased. Therefore, by analyzing the level of MITF expression by a specific substance, it became possible to predict the MDSC activity reaction result by a specific substance. In addition, it is possible to select an MITF expression inhibitor, which is expected to act most suitably when administered in vivo, to an individual in which MDSC is activated or an individual in which T cells are confirmed to be inhibited thereby. In addition, before direct administration, the patient can select an anti-cancer composition or anti-cancer adjuvant composition containing an MITF expression inhibitor as an active ingredient, which is expected to be most suitable for him or her, and whether to change the administration composition even if treatment is in progress However, a new drug candidate group can be considered and the result of MDSC activity can be predicted, thus laying the foundation for selecting the most appropriate MITF inhibitor considering the type and prognosis of cancer.

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 to increase the anticancer immunotherapy efficiency.

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.
Figure 4 shows the differentiation and differentiation of MDSCs by treating cells isolated from the spleen of a tumorigenic mouse (FIG. 4A) or cells isolated from the bone marrow of a mouse (FIG. 4B) with GM-CSF according to an embodiment of the present invention; After inducing the activity, it is a diagram confirming the change in the inhibition of T cell proliferation of MDSC.
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 MDSC activity in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention, after treatment with IL-18 and IL-10 with GM-CSF for 96 hours (FIG. 6A, FIG. 6A, FIG. 6B, 6E, and 6F) or after inducing differentiation by treatment with GM-CSF in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention and then IL-18 treatment for 24 hours (FIG. 6C) and Fig. 6D), MDSC differentiation (Fig. 6A and Fig. 6C), activity and MITF gene expression (Fig. 6B and Fig. 6D) confirming changes.
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. After treatment with GM-CSF, lipopolysaccharide (LPS; lipopolysaccharide), differentiation of MDSC (FIG. 8A) , is a diagram confirming the activity (FIG. 8B) and gene expression of MITF (FIG. 8C).
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 (Atorvastatin, Ator) with GM-CSF, the activity of MDSC ( FIG. 10A ) and the gene expression of MITF ( FIG. 10B ) were confirmed.
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) is a diagram confirming the differentiation change of MDSC after treatment for 24 hours.
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) and lovastatin (Lovastatin, Lova) is a diagram confirming changes in MDSC activity (FIG. 12) and MITF gene expression (FIG. 12) after treatment for 24 hours.
13 is a drug that inhibits the activity of MDSC in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention. ), activity (FIG. 13B) and MITF gene expression (FIG. 13C) is a diagram confirming the change.
14 is a MDSC differentiation (FIG. 14A), MDSC activity and MITF gene expression (FIG. 14B) 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; and 14C), and a diagram confirming the change in T cell proliferation inhibition of MDSC (FIG. 14D).
Figure 15 shows the differentiation of MDSCs after treatment with GM-CSF at 5 μM and 10 μM as an MITF inhibitor in cells isolated from the bone marrow of a mouse 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 of berberine were treated together with GM-CSF.
16 is a diagram confirming the distribution of MDSC expressing MITF in human lung and head and neck cancer (H&N cancer) tissues.
Figure 17 is an MDSC activity (FIG. 17A) and MITF gene expression (FIG. 17B) after treatment with GM-CSF with ML-329 as an MITF inhibitor in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention This is a diagram that confirms the change.
Figure 18 is MDSC differentiation (Figure 18A), activity and MITF gene and protein expression ( 18B and 18C), is a view confirming the change in the inhibition of T cell proliferation of MDSC (FIG. 18D).
19 is a diagram confirming the change in ROS production made in MDSC after treatment with GM-CSF and TCCM with ML-329 or berberine (BBR) in MDSC isolated from the spleen of a tumorigenic mouse according to an embodiment of the present invention to be.
20 is a protein expression of MITF in MDSC that suppressed MITF expression using RNA interference (RNAi) and CRISPR technology according to an embodiment of the present invention (FIG. 20A), ROS generation of MDSC (FIG. 20B) and Confirming the change in T cell proliferation inhibition (FIG. 20C), the protein expression of MITF in MDSC overexpressing MITF according to an embodiment of the present invention (FIG. 20D) and T cell proliferation inhibition of MDSC (FIG. 20E) confirming the change to be.
Figure 21 is after treatment with GM-CSF and TCCM with nelfinavir as an MITF inhibitor in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention, the decrease in gene expression of MITF (FIG. 21A) It is a view confirming the change in activity of MDSC ( FIGS. 21B and 21C ). FIG. 22 is MDSC treated with GM-CSF and TCCM in tumor-forming mice according to an embodiment of the present invention. Methods (FIG. 22A), changes in tumor volume in the tumor-forming mice (FIGS. 22B and 22C), and changes in the population of MDSCs in the tumor tissues of the tumor-forming mice (FIG. 22D) are confirmed.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily implement them with reference to the accompanying drawings. However, the present invention may be implemented in several different forms and is not limited to the embodiments described herein.

Throughout the specification and claims, when a part "includes" a certain element, it means that other elements may be further included, rather than excluding other elements, unless otherwise stated.

The present invention provides a method for screening MDSC activity inhibitory drugs, which predicts and selects the degree of MDSC activity change by analyzing the MITF gene expression or MITF protein expression level in MDSC by a specific substance.

The present invention provides a composition for alleviating, treating or preventing the decrease in immune response caused by myeloid-derived suppressor cells (MDSC), containing an MITF gene expression inhibitor or MITF protein activity inhibitor as an active ingredient do.

In the present invention, "specific substance" refers to a substance expected to change the gene expression or protein activity of MITF in MDSC, in particular, known as MITF gene expression inhibitor, MITF protein activity inhibitor, MDSC activity inhibitor, MDSC activity inhibitor substances and salts thereof.

In the present invention, the MITF gene expression inhibitor is an antisense nucleotide complementary to the mRNA of the MITF gene, an aptamer, a short 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.

In the present invention, MITF inhibitor is meant to include MITF gene expression inhibitor and MITF protein activity inhibitor, and inhibition of MDSC activity means that the inhibitory effect of T cell activity shown by MDSC disappears.

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 in general, it may 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 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 Imuune 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 increase the efficacy of a cancer vaccine or anticancer immunotherapeutic agent by preventing a decrease in the immune response by MDSC, and to treat cancer will be able to perform smoothly and effectively.

In the present invention, the MDSC used to select the MDSC activity inhibitory drug can be obtained by extracting bone marrow cells from a tumor-bearing individual and differentiating them.

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, 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 glioma or 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 embodiment of the present invention, the present inventors can prepare 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 mouse bone marrow in a tumor cell-conditioned medium (TCCM) culturing a mouse breast cancer cell line together with GM-CSF, a differentiation inducing factor of MDSC, 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 microenvironment to lower the immune response, 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. In addition, it was confirmed that MDSC expressing MITF was increased around lung cancer and head and neck cancer tissues. Through the above results, it was confirmed that MDSC was activated in the cancer cell microenvironment, and that MITF was involved in the activation of the MDSC.

In the present invention, "MDSC differentiation inducing factor" includes glycoprotein hormones regulating the proliferation and differentiation of hematopoietic progenitor cells and drugs that mimic their actions. or mass-produced using recombinant DNA technology may be used. Exemplarily, erythropoietin (EPO), bone marrow growth factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), megakaryocyte growth factor (MGF), platelet growth promoting factor (TPO), interleukin-11 ( IL-11) and the like, and preferably GM-CSF, but is not limited thereto.

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 MDSC differentiation was induced by GM-CSF, MDSC activity was induced by the MDSC activity-inducing drugs, and 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 cells isolated from the bone marrow or spleen of mice with berberine or ML-329 as an MITF inhibitor together with GM-CSF, which is an MDSC differentiation inducing factor, MDSC differentiation was induced by GM-CSF. , It was confirmed that the MITF expression was decreased by the MITF inhibitor, and the activity of MDSC was inhibited at this time. 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 MDSC pre-treated with ML-329 to the tumor-forming mouse prepared in an embodiment of the present invention, it was confirmed that the immune response was enhanced, tumor growth was suppressed, and the degree of MDSC invasion at the tumor site was weakened. did Through the above results, it was confirmed that the activity of MDSC can be inhibited by using an inhibitor of MITF.

In addition, the present inventors used nelfinavir, a type of anti-HIV agent, as an MITF inhibitor together with GM-CSF, a factor for inducing MDSC differentiation, in a cancer cell microenvironment for inducing MDSC activity in cells isolated from the bone marrow of a mouse. As a result of the treatment, it was confirmed that the expression of MITF was decreased by nelfinavir, and the MDSC activity was inhibited.

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, using an inhibitor of MITF, it was confirmed that MDSC could be inhibited, and thus, it was possible to select an MITF inhibitor and predict MDSC activity therefrom. This can be usefully used to develop a composition containing an MITF inhibitor as an active ingredient, and can 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 to increase the efficiency of anticancer immunotherapy.

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, to prepare bone marrow cells to induce MDSC differentiation, bone marrow cells can be extracted from a tumor-bearing individual, and the extracted bone marrow cells can differentiate and/or activate MDSCs in a medium. MDSC differentiation can be achieved by culturing bone marrow cells in a medium containing MDSC differentiation inducing factors. Differentiation and induction of activity may occur simultaneously or separately. MDSC activation for analysis can be made in a medium in which the cancer cell microenvironment is formed, that is, TCCM. can For example, a cancer cell microenvironment is created as a test group and a medium treated with a specific substance and a medium not treated with a specific substance can be used as a control group, or both the test group and the control group create a cancer cell microenvironment and only the test group is treated with a specific substance You may. Alternatively, after using a medium in which the cancer cell microenvironment is formed as a test group and a medium not having a cancer cell microenvironment as a control group, MDSCs from 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 a person skilled in the art may appropriately design them if necessary.

It is possible to predict the effect of a specific substance on MDSC activity by recovering MDSC that has undergone differentiation and activity induction and analyzing the expression level of MITF gene or MITF protein.

Specifically, in the present invention, qRT-PCR can be used to analyze the level of MITF gene expression after recovery of MDSCs that have undergone differentiation and induction of activity. In the case of using qRT-PCR, iNOS, IL-10, TGF-β, etc. may be used as MDSC activity markers, but the present invention is not limited thereto. MDSCs that induced differentiation and/or activity are recovered and total RNA is isolated using an appropriate solution, for example, TRIzol Reagent® Solution (Invitrogen). Thereafter, qRT-PCR can be performed using the isolated total RNA.

In the present invention, after MDSCs that have undergone differentiation and activity induction are recovered, Western blotting may be performed to analyze the level of MITF protein expression. As an exemplary method, the recovered MDSC may be lysed by treatment with an appropriate solution, for example, a cell lysis buffer, and then separated by electrophoresis of the cell lysate. Primary antibodies that can be used include, for example, anti-MITF antibodies and anti-actinin antibodies, and HRP-conjugated secondary antibodies may be considered as secondary antibodies. In this case, actinin may be used as a control protein.

By comparing the results of the test group and the control group, it can be determined whether a specific substance is an MITF inhibitor, that is, an MDSC activity inhibitor, or an MITF expression increaser, that is, an MDSC activity inducer. predictable. Therefore, it can be expected that MDSC activity is inhibited by administration of an MDSC activity inhibitor, etc., leading to recovery of T cell function in the body. From this, it is possible to prepare a composition comprising the MITF inhibitor as an active ingredient.

In the present invention, the screening method of the MDSC activity inhibitory drug is when the expression level of the MITF gene or MITF protein is, for example, using qRT-PCR analysis, the qRT-PCR measurement value in the test group compared to the control group is low when the MDSC inhibitor drug can be decided with 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 compared to the control group, in the case of a substance that inhibits MITF by an indirect route, it is preferable in the test group compared to the control group may be 30% or more of inhibition, but is not limited to this range, and those skilled in the art can determine the degree of inhibition in a necessary range.

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 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 contains a gene expression inhibitor of MITF (microphthalmia-associated transcription factor) or an inhibitor of protein activity of MITF as an active ingredient, myeloid-derived suppressor cell (MDSC) for reducing immune response by It provides an anticancer adjuvant containing a composition for alleviation, treatment or prevention as an active ingredient.

In the present invention, the MITF gene expression inhibitor is an antisense nucleotide complementary to the mRNA of the MITF gene, an aptamer, a short 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 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 the 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 MDSCs are activated in the cancer cell microenvironment and the immune response is lowered, MITF is involved in the activation of MDSCs, and the MITF inhibitors can be used to inhibit the activity of MDSCs. Therefore, 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 a gene expression inhibitor of MITF or an inhibitor of protein activity of MITF to an individual in need of MDSC inhibition.

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 refers to the above content, and in the following, only the composition specific to the MDSC inhibition method will be described. do.

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, 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 gland cancer, 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 glioma or pituitary adenoma However, the present invention is not limited thereto.

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 MDSCs, and the MITF inhibitors can be used to inhibit the activity of MDSCs. , it can be used to treat MDSC-related diseases by administering the MITF inhibitor to an individual in need of inhibition of MDSC.

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. Inc. (Republic of Korea) was purchased. 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. The obtained bone marrow cells were treated with RPMI medium (Invitrogen, Grand Island, NY) containing granulocyte macrophage colony stimulating factor (GM-CSF) at a concentration of 10 ng/ml in a 24-well plate at a cell number of 5Х10 ⁵ cells/ml for 96 hours. MDSC differentiation 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 5Х10 ⁵ cells/100 μl to the right flank of 6-8 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 microenvironment

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

In order to determine whether the immune response is lowered by MDSCs in the cancer cell microenvironment, the activity of MDSCs 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) for 20 minutes at 3000 Хg in a centrifuge maintained at 4° C. to conditioned medium for cancer cells. (TCCM; tumor cell-conditioned medium) was obtained. After obtaining bone marrow cells in the same manner as described in <Example 1-1>, the obtained TCCM and GM-CSF at a concentration of 10 ng/ml were included in a 24-well plate at a cell number of 5Х10 ⁵ cells/ml. Differentiation of MDSCs was induced by culturing for 96 hours in an RPMI medium. 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 M-MLV reverse transcription kit (Promega, Madison, WI), oligo-(dT) primer and dNTP (Bioneer, Daejeon, Republic of Korea), and ABI Real-time 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 the PCR 7500 system. . 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 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 (RPMI medium containing 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), 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. Induction was confirmed (FIG. 2B).

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

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, the primary antibody is treated with an anti-MITF antibody and an anti-actinin antibody and reacted, and then an HRP-conjugated secondary antibody is attached to the primary antibody attached to the membrane, and this enhanced chemiluminescence technique (PicoEPD?? Western Reagent kit, ELPIS-Biotech, Daejeon, Korea) was used for analysis with the LAS-3000 imaging system (FUJIFILM, Tokyo, Japan). 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 an anti-Arg1 antibody and an anti-pSTAT3 antibody as a primary antibody.

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 increased compared to the control group (GM-CSF), 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> Confirmation of decreased immune response by MDSC in cancer cell microenvironment

MDSC is known to decrease the immune response by suppressing the proliferation and function of T cells. Accordingly, in order to examine 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.

Specifically, cells isolated from the spleen of tumorigenic mice using the MACS cell separation kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer's procedure were performed in a 24-well plate at a cell number of 5Х10 ⁵ cells/ml. The differentiation and activity of MDSCs 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.

In addition, in order to obtain T cells, the spleens of 6 to 8-week-old Balb/c mice were removed and the 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. 5Х10 CFSE-labeled CD3 ⁺ T cells of ⁵ cell numbers were stimulated for 2 hours on plates coated with anti-CD3 monoclonal antibody and soluble anti-CD28 monoclonal antibody, and then cells isolated from the spleen of the tumorigenic mice. The MDSCs obtained by inducing the differentiation and activity of (FIG. 4A) and the MDSCs recovered in Example <2-1> (FIG. 4B) were put into 5Х10 ⁵ cells or 10Х10 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 FIG. 4 , when MDSCs (GM+TCCM) and T cells of the TCCM-treated group were co-cultured, compared to the case of co-culture with MDSCs of the control group (GM-CSF), CD3 ⁺ CD8 ⁺ T cells. As the number decreased, it was confirmed that MDSC activity-induced by TCCM inhibited T cell proliferation.

From 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.

<Example 3> Confirmation of MDSC activity and MITF expression in tumorigenic 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 change of MITF were confirmed using MDSC from 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 using 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 microenvironment, and MITF is involved in the activation of the MDSC.

<Example 4> Confirmation of MDSC and MITF expression in tumor tissue

MDSC is activated in tumor tissue, and in order to examine whether MITF is involved in the activation of MDSC, expression changes of MDSC and MITF in tumor tissue were checked.

Specifically, tissue staining was performed at Seoul National University's Department of Pathology (Prof. Yoon Kyung Jeon) for each of 10 samples of lymph node tissue surrounding lung and head and neck cancer tissues and corresponding cancer-free lymph nodes. Specifically, the tissue was fixed in 4% formaldehyde solution, dehydrated in ethanol (70-100%) according to concentration, and embedded in paraffin. The tissue was sectioned with a microtome (thickness 4 μm) and stained with hematoxylin and eosin (H&E). The cross-section was observed with an optical microscope (Olympus, Japan) and photographed at a magnification of Х400. For IHC (immunohistochemistry), tissue sections were blocked with 3% normal horse serum diluted with PBS for 30 minutes. Sections were blocked and mixed with CD11b, CD14, MITF antibody (anti-mouse monoclonal primary antibody (Cat. No 790-4367), Ventana Medical Systems, Oro Valley, AZ) overnight at 4°C at the appropriate dilution factor (1:100 dilution). ) was reacted. After washing the slides with PBS, the avidin-biotin-peroxidase complex (ABC; Vector Laboratories, Burlingame, CA) was washed. The slides were washed and the peroxidase reaction was run with diaminobenzidine and peroxide, and aqua- It was mounted on a mount (Aqua-Mount) and evaluated under an optical microscope (Olympus) at Х400 magnification The number of MDSCs with MITF ⁺ /CD11b ⁺ /CD14 ⁺ in the High Power Field (HPF) in cancer tissues and lymph node tissues. is expressed as the average value of the triple-positive cells counted in three random high-magnification fields, respectively.

As a result, as shown in FIG. 16 , it was confirmed that while the distribution of MDSC stained with MITF in the tissue surrounding the tumor was clear, MDSC stained with MITF was hardly observed in the tissue of the lymph node region without a tumor.

From the above results, it can be seen that MDSCs are activated around the cancer, and MITF is involved in the activation of the MDSCs.

<Example 5> Confirmation of increase in 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.

<5-1> Confirmation of increased MITF expression in IL-18-induced MDSC

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

Specifically, after obtaining bone marrow cells in the same manner as described in Example <1-1>, IL-18 at a concentration of 10 or 50 ng/ml in a 24-well plate at a cell number of 5Х10 ⁵ cells/ml, and Differentiation of MDSCs 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 for 96 hours in RPMI medium containing GM-CSF, and IL-18 at a concentration of 10 or 50 ng/ml was administered for 24 hours. treated (FIGS. 6C and 6D). 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 ( FIGS. 6A and 6C ).

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 MDSC, and to confirm the gene expression of MITF (FIGS. 6B and 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 ( FIGS. 6A and 6C ). On the other hand, in the case of the IL-18 treatment group, it was confirmed that the activity of MDSC was increased and the gene expression of MITF was increased ( FIGS. 6B and 6D ).

<5-2> Confirmation of increased MITF expression in MDSCs differentiated and activated by IL-10

After treatment with IL-10 with a drug inducing MDSC activity and inducing differentiation of MDSC, a change in MITF gene expression was confirmed.

Specifically, after obtaining bone marrow cells in the same manner as described in Example <1-1>, IL-10 at a concentration of 5 ng/ml, and 10 ng in a 24-well plate at a cell number of 5Х10 ⁵ cells/ml Differentiation of MDSCs was induced by culturing for 96 hours in RPMI medium containing GM-CSF at a concentration of /ml ( FIGS. 6E and 6F ). 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. 6E).

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 MDSC, and to confirm the gene expression of MITF (FIG. 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 group and the IL-10 treatment group ( FIG. 6E ). On the other hand, in the case of the IL-10 treatment group, it was confirmed that the activity of MDSC was increased and the gene expression of MITF was increased (FIG. 6F).

<5-3> Confirmation of increased expression of MITF 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, MDSC differentiation and activity were induced according to the method described in Example <2-1>, and 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-4 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).

<5-4> 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 <2-1>, 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).

<5-5> Confirmation of increased 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 the gene expression change of MITF was confirmed.

Specifically, inducing differentiation and activity of MDSC according to the method described in Example <2-1>, 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) was treated (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 of MDSCs ( FIGS. 9 and 11 ) was confirmed in the same manner as those described in Examples <2-1> and <2-2>. In addition, in the simvastatin or lovastatin 1 μM treatment group, the activity of MDSC ( FIGS. 10A and 12A ) was confirmed, and the gene expression of MITF ( FIGS. 10B and 12B ) was 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 of MITF ( FIGS. 10B and 12B ) was increased compared to the control group.

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

<Example 6> Confirmation of reduced expression of MITF 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 <1-1>, ATRA (0.5 or 1 μM) and 10 ng/ml concentration in a 24-well plate at a cell number of 5Х10 ⁵ cells/ml MDSC differentiation was induced by culturing for 96 hours in RPMI medium containing GM-CSF (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, MDSCs treated with 1 μM of ATRA and 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 ( 13B), the gene expression of MITF 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).

Since it was confirmed that the expression of MITF was decreased in the MDSCs whose activity was suppressed through the above results, it can be seen that MITF is involved in the activation of MDSCs.

<Example 7> Confirmation of MDSC activity change by MITF regulation

<7-1> Confirmation of increased activity of MDSC by MITF inducer

It is known that IBMX induces the expression of MITF in melanoma cells. Accordingly, in order to examine the effect of MITF expression or activity regulation on MDSC activity, changes in the activity of MDSCs treated with IBMX as an MITF expression inducer and changes in T cell proliferation by the MDSCs were confirmed.

Specifically, after obtaining bone marrow cells in the same manner as described in Example <1-1>, 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 5Х10 ⁵ cells/ml -CSF-containing RPMI medium was cultured for 96 hours to induce differentiation of MDSCs. 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 and Western blotting were performed in the same manner as those described in Examples <2-1> and <2-2> to confirm the activity of MDSCs (FIG. 14B). and Fig. 14C), gene expression and protein expression of MITF (Fig. 14C) were confirmed.

In addition, the degree of inhibition of T cell proliferation was confirmed in the same manner as in Example <2-3> by recovering the differentiation-induced MDSC (FIG. 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 treatment group ( FIG. 14A ). On the other hand, in the case of the IBMX-treated group, it was confirmed that the gene and protein expression of MITF increased ( FIG. 14C ), and the activity of MDSC was increased compared to the control group ( FIGS. 14B and 14C ). In addition, in the case of the IBMX-treated group, it was confirmed that the activity of MDSC was increased to suppress T cell proliferation ( FIG. 14D ).

<7-2> 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 MDSCs treated with berberine at different concentrations with an MITF inhibitor were confirmed.

Specifically, after obtaining bone marrow cells by the same method as described in Example <1-1>, berberine at a concentration of 5 μM and GM at a concentration of 10 ng/ml in a 24-well plate at a cell number of 5Х10 ⁵ cells/ml The differentiation of MDSCs was induced by culturing in RPMI medium containing -CSF and RPMI medium containing 10 μM berberine and 10 ng/ml GM-CSF for 96 hours, respectively. As a control, MDSCs induced to differentiate in RPMI medium containing GM-CSF were used.

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 ).

In addition, the differentiation-induced MDSCs 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>, qRT-PCR was performed in the same manner as described above to confirm the expression of MITF (FIG. 15C)

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

In addition, ML-329 is known to suppress the expression of MITF by suppressing the TRPM-1 promoter activity in melanocytes. Accordingly, in order to examine the effect of MITF expression or activity regulation on MDSC activity, the change in the activity of MDSC treated with ML-329 as an MITF inhibitor and the change in T cell proliferation by the MDSC were confirmed.

Specifically, after obtaining bone marrow cells ⁱⁿ the same manner as described in Example <1-1>, ML-329 (Cayman Chemical, Ann Arbor, MI) and 10 ng/ml of GM-CSF were cultured in RPMI medium for 96 hours to induce differentiation of MDSCs. As a control, MDSCs induced to differentiate in RPMI medium containing GM-CSF were used. Then, 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 MDSC activity (FIG. 17A) and MITF expression. (FIG. 17B).

In addition, in order to examine the effect of ML-329 on the induction of MDSC activity by TCCM, bone marrow cells were obtained in the same manner as in Example <1-1>, and the cell number of 5Х10 ⁵ cells/ml was 24 - 96 hours in RPMI medium containing 0.5, 1 or 2 μM concentration of ML-329 (Cayman Chemical), TCCM obtained in Example <2-1> and GM-CSF of 10 ng/ml concentration in a well plate MDSC differentiation was induced. As a control, MDSCs induced to differentiate in RPMI medium containing GM-CSF were used. Then, 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. 18A), and qRT-PCR was performed to The degree of activity was measured (FIG. 18B). In addition, qRT-PCR and Western blotting were performed in the same manner as in Example <2-2> to confirm MITF expression (FIG. 18C). In addition, the degree of inhibition of T cell proliferation was confirmed in the same manner as in Example <2-3> using the MDSC obtained above (FIG. 18D).

As a result, as shown in FIGS. 17 and 18 , in the case of the ML-329 treatment group, it was confirmed that MITF expression was suppressed and the activity of MDSC was inhibited by ML-329 ( 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 was increased by inhibition of MDSC activity by ML-329. was confirmed (FIG. 18D).

In addition, MDSC is known to inhibit various processes from proliferation to function of T cells by making reactive oxygen species (ROS) or reactive nitrogen species (RNS). Accordingly, in order to examine the effect of MITF expression or activity regulation on MDSC activity, ROS generation changes were confirmed in MDSCs treated with berberine or ML-329 as an MITF inhibitor.

Specifically, by separating MDSC 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 and 10 obtained in Example <2-1> Differentiation of MDSCs was induced by culturing in RPMI medium containing ng/ml concentration of GM-CSF for 48 hours. The MDSCs were collected and divided into 1Х10 ⁵ cells, and LPS was treated at a concentration of 100 ng/ml for 24 hours. At this time, the ROS inhibitor NAC (N-acetyl-cysteine) 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.

As a result, as shown in FIG. 19 , it was confirmed that ROS production by MDSC was inhibited in the berberine or ML-329 treatment group, and ROS was strongly inhibited when treated with the ROS inhibitor.

In addition, after suppressing the expression of MITF in MDSC using RNA interference (RNAi) and CRISPR technology, ROS generation and T cell proliferation changes by MDSC were confirmed.

Specifically, after obtaining bone marrow cells in the same manner as described in Example <1-1>, the following sgRNA was added to the CRISPR vector, specifically the pLentiCRISPR-E vector, into bone marrow cells having 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, the control group cloned the following non-target control gRNA 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 MITF expression and MDSC activity were confirmed by performing the method described in <2-2> and Western blotting (FIG. 20A). In addition, the degree of ROS generation by MDSCs was measured in the same manner as described above (FIG. 20B), and the degree of T cell proliferation by MDSCs was measured in the same manner as in Example <2-3> ( Figure 20C).

In addition, as a control group, the degree of T cell proliferation by MDSC overexpressing MIFT was measured. Specifically, MITF plasmid DNA (Addgene, Cambridge, MO) was transfected into bone marrow cells having 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. Three days after transfection, MDSCs were recovered and the expression of MITF (FIG. 20D) and the degree of T cell proliferation by MDSCs (FIG. 20E) were measured as described above.

As a result, as shown in FIG. 20, it was confirmed that the expression of MITF and MDSC activity were suppressed in MDSC using MITF shRNA and CRISPR technology (FIG. 20A), and ROS production by MDSC was suppressed by suppression of MITF expression ( Fig. 20B), it was confirmed that the inhibition of T cells by MDSC was alleviated (Fig. 20C). On the other hand, it was confirmed that contradictory results appeared in MDSCs overexpressing MITF ( FIGS. 20D and 20E ).

Through the above results, it can be seen that the activity of MDSC can be inhibited by inhibiting the expression or activity of MITF.

In addition, nelfinavir, an HIV protease inhibitor, is an 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 in the same manner as in Example <1-1>, and the cell number of 5Х10 ⁵ cells/ml was 24- In a well plate, 1, 5 or 10 μM concentration of nelfinavir, TCCM obtained in Example <2-1>, and 10 ng/ml of MDSC were cultured in RPMI medium containing GM-CSF for 96 hours. Differentiation was induced. As a control, MDSC inducing differentiation in RPMI medium containing TCCM without nelfinavir and GM-CSF at a concentration of 10 ng/ml was used. Then, the differentiation-induced MDSCs were recovered and qRT-PCR was performed in the same manner as 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 degree of MDSC activity (FIG. 21B). In addition, in order to confirm the activity of MDSC, Western blotting was performed by the method described in Example <2-2> to confirm MDSC activity (FIG. 21C).

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)

<Example 8> Immune response enhancement effect by lowering MDSC activity in tumor-forming mice

It was confirmed that the MDSC activity was inhibited by the MITF inhibitor through <Example 7>. Therefore, in order to examine the effect of the lowered MDSC activity in tumorigenic mice, tumor growth changes were confirmed after administration of MDSCs whose activity was reduced by the MITF inhibitor to tumorigenic mice.

Specifically, MDSCs were isolated from the spleen of tumor-forming mice prepared by the same method as described in Example <1-2>. Then, in the same manner as in Example <7-2>, 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. Differentiation of MDSCs was induced by culturing for 48 hours with the used RPMI medium. After 48 hours, the MDSC + 4T1-luc (5Х10 ⁴ + 2.5Х10 ⁵ /100 μl) was injected subcutaneously into the left flank of the mouse. Tumor volume was continuously measured with a vernier caliper until the end of the experiment (tumor volume = a ² Хb/2 mm ³ ) (a = tumor width, b = tumor height) (Fig. 22B), and IVIS (In vivo imaging system) analysis was performed (FIG. 22C). In addition, after 2 weeks, the population of MDSCs in the tumor tissue was confirmed by staining with anti-CD11b antibody, anti-Gr1 antibody, and anti-CD45 antibody, followed by FACS analysis ( FIG. 22D ). For the control group, MDSC cultured in RPMI medium containing ML-329 and TCCM and GM-CSF was injected.

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, thereby enhancing the immune response in tumor-forming mice, suppressing tumor growth, and reducing tumor site It was confirmed that the degree of invasion of MDSC was weakened ( FIGS. 22B to 22D ).

Through the <Example 1> to <Example 8>, MDSC is activated in the cancer cell microenvironment, and 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 confirmed that it can be done.

Based on this, it was revealed that MITF inhibitors can be selected from a wide range of substances and can be used as MDSC activity inhibitory drugs. It is possible to more effectively specify active ingredients that can be used for alleviating the decrease in the immune response of MDSC and for anticancer immunotherapy. In addition, it is possible to select an MITF expression inhibitor, which is expected to act most suitably when administered in vivo, to an individual in which MDSC is activated or an individual in which T cells are confirmed to be inhibited thereby. In addition, before direct administration, the patient can select an anti-cancer composition or anti-cancer adjuvant composition containing an MITF expression inhibitor as an active ingredient, which is expected to be most suitable for him or her, and whether to change the administration composition even if treatment is in progress However, a new drug candidate group can be considered and the result of MDSC activity can be predicted, thus laying the foundation for selecting the most appropriate MITF inhibitor considering the type and prognosis of cancer.

The composition comprising the MITF inhibitor selected by the present invention can be usefully used to alleviate the decrease in immune response caused by MDSC by administering the composition to an individual in need of inhibition of MDSC, for example, an individual having a tumor. 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.

Although embodiments of the present invention have been described above with reference to the accompanying drawings, the present invention is not limited to the above embodiments and may be manufactured in various different forms, and those of ordinary skill in the art to which the present invention pertains It will be understood that the present invention may be embodied in other specific forms without changing the spirit or essential features of the present invention. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> SOOKMYUNG WOMEN'S UNIVERSITY Research & Business Development Foundation <120> SCRENNING METHOD FOR MDSC INHIBITOR <130> DPC210262 <150> KR 10-2020-0186826 <151> 2020-12-29 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> MITF gRNA FW oligo <400> 1 caccgtaagg acttccatcg gcacc 25 <210> 2 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> MITF gRNA RV oligo <400> 2 aaacggtgcc gatggaagtc cttac 25 <210> 3 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> control sgRNA <400> 3 caccggtatt actgatattg gtggg 25

Claims (12)

MDSC activity that predicts and selects the degree of MDSC activity change by analyzing the MITF gene expression or MITF protein expression level in MDSC by a specific substance by any one method selected from qRT-PCR, Western blotting and flow cytometry (FACS) Methods for screening inhibitors. The method of claim 1, wherein the specific substance is a gene expression inhibitor of MITF or a protein activity inhibitor of MITF. The method according to claim 1, comprising the following steps:
(a) preparing bone marrow cells;
(b) inducing MDSC differentiation and activity from the bone marrow cells;
(c) recovering the differentiation and activity-induced MDSC in step (b) and analyzing the expression level of the MITF gene or MITF protein; and
(d) selecting a drug for inhibiting MDSC activity among the specific substances from the analysis result of (c) The method according to claim 3, wherein the bone marrow cells are bone marrow cells obtained from a tumor-bearing individual. The MITF gene or A screening method for MDSC activity-inhibiting drugs that compares the expression level of MITF protein. The method according to claim 3, wherein the expression level of the MITF gene or MITF protein in step (d) is a direct MITF inhibitor when the RT-PCR analysis is performed. In the case of a substance that inhibits MITF by an indirect route, when it is inhibited by 30% or more in the test group compared to the control group, the method further comprising the step of determining the substance as an MDSC activity inhibitory drug . 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, 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 adenocarcinoma, 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 A method for screening a drug that inhibits MDSC activity, which is at least one selected from the group consisting of adenomas. According to claim 1, wherein in order to select the MDSC activity inhibitory drug, adjuvantly co-culturing the MDSC and T cells, and determining the degree of inhibition of T cell proliferation, the screening method of the MDSC activity inhibitory drug. The method of claim 2, wherein the MITF gene expression inhibitor is an antisense nucleotide complementary to the mRNA of the MITF gene, an aptamer, a short hairpin RNA (small hairpin RNA, shRNA), a small interfering RNA (small interfering RNA, siRNA) ), microRNA (microRNA, miRNA), and at least one selected from the group consisting of ribozyme, a method for screening MDSC activity inhibitory drugs. According to claim 2, wherein the protein activity inhibitor of MITF is at least one selected from the group consisting of a compound that specifically binds to MITF protein, a peptide, a peptidomimetic, a substrate analogue, an aptamer, and an antibody, MDSC activity inhibitory drug screening method. The method of claim 1, wherein the specific substance is at least one MDSC activity inhibitor selected from AMPK activity promoter, ML-329, MITF-gRNA cloned vector, and anti-HIV agent. A composition comprising an MDSC activity inhibitory drug selected by the selection method of claim 1, and inhibiting T cell proliferation.

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