WO2021033973A1 - Composition pour inhiber une cellule suppressive dérivée de myéloïde comprenant un inhibiteur de mitf en tant que principe actif - Google Patents

Composition pour inhiber une cellule suppressive dérivée de myéloïde comprenant un inhibiteur de mitf en tant que principe actif Download PDF

Info

Publication number
WO2021033973A1
WO2021033973A1 PCT/KR2020/010480 KR2020010480W WO2021033973A1 WO 2021033973 A1 WO2021033973 A1 WO 2021033973A1 KR 2020010480 W KR2020010480 W KR 2020010480W WO 2021033973 A1 WO2021033973 A1 WO 2021033973A1
Authority
WO
WIPO (PCT)
Prior art keywords
mdsc
mitf
cancer
inhibitor
activity
Prior art date
Application number
PCT/KR2020/010480
Other languages
English (en)
Korean (ko)
Inventor
임종석
이아람
임지현
이명석
Original Assignee
숙명여자대학교산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 숙명여자대학교산학협력단 filed Critical 숙명여자대학교산학협력단
Publication of WO2021033973A1 publication Critical patent/WO2021033973A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • the present invention relates to a composition for inhibiting myeloid-derived suppressor cells containing a MITF (microphthalmia-associated transcription factor) inhibitor as an active ingredient, and specifically, a bone marrow-derived composition containing an MITF inhibitor as an active ingredient It relates to a composition for alleviating, treating, or preventing lowering of the immune response by inhibitory cells.
  • MITF microphthalmia-associated transcription factor
  • Myeloid cells originate from hematopoietic stem cells. These are the most common hematopoietic stem cells in our body, mainly in bone marrow and lymphatic tissues. Ultimately, they differentiate into macrophages, dendritic cells, and granulocytes, but they do not have a specific hierarchical structure, and myeloid cells with different degrees of differentiation are specifically distributed in various tissues and environments. It has the characteristics of being.
  • MDSCs Myeloid-derived suppressor cells
  • ROS reactive oxygen species
  • RNS reactive nitrogen species
  • ICI immune checkpoint inhibitors
  • CTL-4 cytotoxic T-lymphocyte-associated protein 4
  • PD-1 programmed cell death protein
  • P-L1 programmed cell death ligand 1
  • Immunosuppressive cells in TME that affect the efficacy of ICI treatment include Treg cells, MDSC, TH2 CD4 + cells, CAF (cancer-associated fibroblast) and M2 polarized tumor-associated macrophages (TAM).
  • Treg cells include Treg cells, MDSC, TH2 CD4 + cells, CAF (cancer-associated fibroblast) and M2 polarized tumor-associated macrophages (TAM).
  • TAM tumor-associated macrophages
  • MITF microphthalmia-associated transcription factor
  • LXR Liver X receptor
  • the present inventors have endeavored to develop a formulation that can alleviate the decrease in the immune response of MDSC through MDSC inhibition and improve the therapeutic effect of an anticancer immunotherapy agent.
  • MDSC is activated in the cancer cell microenvironment and the immune response is lowered.
  • the MITF inhibitor can be used to alleviate the decrease in the immune response of MDSC and to use anticancer immunotherapy. , Completed the present invention.
  • IRF4 Interferon regulatory factor 4
  • MDSC myeloid-derived suppressor cell
  • An object of the present invention is to alleviate and treat the reduction of immune response by bone marrow-derived suppressor cells, containing as an active ingredient a microphthalmia-associated transcription factor (MITF) inhibitor that inhibits myeloid-derived suppressor cells. Or to provide a preventive composition.
  • MITF microphthalmia-associated transcription factor
  • Another object of the present invention is to provide an anticancer adjuvant containing an MITF inhibitor as an active ingredient.
  • Another object of the present invention is to provide a method for inhibiting bone marrow-derived inhibitory cells, comprising administering a MITF inhibitor to an individual in need of inhibition of the bone marrow-derived inhibitory cells.
  • the present invention is a bone marrow-derived suppressor cell (myeloid-derived suppressor cell) containing a microphthalmia-associated transcription factor (MITF) inhibitor as an active ingredient alleviation, treatment or It provides a composition for prevention.
  • MITF microphthalmia-associated transcription factor
  • the present invention provides an anticancer adjuvant containing an MITF inhibitor as an active ingredient.
  • the present invention provides a method for inhibiting bone marrow-derived suppressor cells, comprising administering a MITF inhibitor to an individual in need of inhibition of the bone marrow-derived suppressor cells.
  • the present invention provides a method for alleviating or treating lowering of immune response by bone marrow-derived suppressor cells, comprising administering a MITF inhibitor to an individual.
  • the present invention provides a method for preventing lowering of immune response by bone marrow-derived suppressor cells, comprising administering a MITF inhibitor to an individual.
  • the present invention provides a method for treating cancer, comprising administering a MITF inhibitor and an anticancer agent to an individual.
  • the present invention provides a use of MITF for use as a composition for alleviating, treating, or preventing the decrease in immune response by bone marrow-derived suppressor cells.
  • the present invention provides the use of MITF for use as an anticancer adjuvant.
  • MDSC myeloid-derived suppressor cells
  • MITF microphthalmia-associated transcription factor
  • FIG. 1 is a diagram schematically illustrating a method of making a bone marrow-derived bone marrow-derived suppressor cell (MDSC) of a mouse.
  • MDSC bone marrow-derived bone marrow-derived suppressor cell
  • FIG. 2 is a diagram of MDSC after treatment with a tumor cell-conditioned medium (TCCM) with GM-CSF, which is a differentiation inducing factor of MDSC, to cells isolated from the bone marrow of a mouse according to an embodiment of the present invention. It is a diagram confirming changes in differentiation (Fig. 2A) and activity (Fig. 2B).
  • TCCM tumor cell-conditioned medium
  • GM-CSF a differentiation inducing factor of MDSC
  • FIG. 3 is a diagram of a gene (FIG. 3A) and a protein (FIG. 3B) of a microphthalmia-associated transcription factor (MITF) in MDSC after treatment of TCCM with GM-CSF in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention. ) This is a diagram confirming the change in expression.
  • MITF microphthalmia-associated transcription factor
  • FIG. 4 shows the differentiation of MDSC by treatment with GM-CSF and TCCM on cells isolated from the spleen of tumorigenic mice (FIG. 4A) or cells isolated from the bone marrow of mice (FIG. 4B) according to an embodiment of the present invention. It is a diagram confirming the change in inhibition of T cell proliferation of MDSC after inducing activity.
  • Fig. 5 is a diagram confirming changes in MDSC differentiation (Fig. 5A), activity (Fig. 5B) and MITF gene expression (Fig. 5C) in tumorigenic mice.
  • FIG. 6 is a drug that induces the activity of MDSC in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention, after treating IL-18 with GM-CSF for 96 hours (FIGS. 6A and 6B) or according to an embodiment of the invention, after inducing differentiation by treating cells isolated from the bone marrow of a mouse with GM-CSF, IL-18 was treated for 24 hours (Fig. 6C and Fig. 6D), and then differentiation of MDSC (Fig. 6A). And Figure 6C), is a diagram confirming the changes in the activity and gene expression of MITF ( Figure 6B and Figure 6D).
  • FIG. 7 is a drug that induces the activity of MDSC in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention.
  • FIG. 7A After treatment with IL-4 with GM-CSF, the differentiation of MDSC (FIG. 7A) and activity (FIG. 7B)
  • FIG. 7C It is a diagram confirming changes in gene expression (FIG. 7C) of MITF.
  • Figure 8 is a drug that induces the activity of MDSC in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention, after treatment with LPS with GM-CSF, differentiation of MDSC (Figure 8A), activity ( Figure 8B) And it is a diagram confirming the change in gene expression (FIG. 8C) of MITF.
  • Figure 9 is a drug that induces the activity of MDSC in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention, simvastatin (Sim), lovastatin (Lovastatin, Lova), provastatin (Pravastatin, Prav), as This is a diagram confirming the change in differentiation of MDSC after treatment with Suvastatin (Rosuvastatin, Rosu) or Atorvastatin (Ator) with GM-CSF.
  • Figure 10 is a drug for inducing the activity of MDSC in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention, after treatment with Simvastatin (Sim) or lovastatin (Lovastatin, Lova) with GM-CSF It is a diagram confirming changes in the activity of (Fig. 10A) and gene expression of MITF (Fig. 10B).
  • Figure 11 is a drug that induces the activity of MDSC after inducing differentiation by treating cells isolated from the bone marrow of a mouse with GM-CSF according to an embodiment of the present invention.
  • Simvastatin Sim
  • lovastatin Lovastatin, Lova
  • provastatin Pravastatin, Prav
  • rosuvastatin Rosu
  • atorvastatin Antor
  • Figure 12 is a drug that induces the activity of MDSC after inducing differentiation by treating cells isolated from the bone marrow of a mouse with GM-CSF according to an embodiment of the present invention, simvastatin (Sim) or lovastatin (Lovastatin, Lova) is a diagram confirming changes in the activity of MDSC (Fig. 12A) and gene expression of MITF (Fig. 12B) after treatment for 24 hours.
  • Sim simvastatin
  • lovastatin Lova
  • FIG. 13 shows the differentiation of MDSC after treatment with ATRA (All-trans retinoic acid) with GM-CSF as 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 (FIG. 13A ), activity (FIG. 13B) and gene expression of MITF (FIG. 13C) are confirmed.
  • ATRA All-trans retinoic acid
  • FIG. 14 shows the differentiation of MDSC (FIG. 14A ), activity of MDSC, and gene expression of MITF after treatment with GM-CSF with GM-CSF as a MITF inducer in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention (FIG. 14B And 14C), and T cell proliferation inhibition (FIG. 14D) of MDSC.
  • FIG. 15 shows gene expression of MITF (FIG. 15A) and differentiation of MDSC after treatment with GM-CSF with berberine as a MITF inhibitor in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention (FIG. 15B ). ) And activity (FIG. 15C) is a diagram confirming the change.
  • FIG. 16 is a diagram illustrating the distribution of MDSCs expressing MITF in lung cancer and head and neck cancer (H&N cancer) tissues.
  • FIG. 17 shows MDSC activity (FIG. 17A) and MITF gene expression (FIG. 17B) after treating ML-329 with GM-CSF as a MITF inhibitor in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention. It is a figure confirming the change.
  • FIG. 18 shows the differentiation of MDSC (FIG. 18A ), activity, and gene and protein expression of MITF after treating ML-329 with GM-CSF and TCCM in cells isolated from the bone marrow of a mouse according to an embodiment of the present invention ( 18B and 18C) is a diagram confirming the change in inhibition of T cell proliferation (FIG. 18D) of MDSC.
  • FIG. 19 is a diagram illustrating changes in ROS production of MDSC after treatment with ML-329 or berberine (BBR) together with GM-CSF and TCCM in MDSC isolated from the spleen of tumorigenic mice according to an embodiment of the present invention.
  • BBR berberine
  • FIG. 20 shows changes in protein expression of MITF (FIG. 20A), ROS generation of MDSC (FIG. 20B) and inhibition of T cell proliferation (FIG. 20C) in MDSC inhibiting MITF expression according to an embodiment of the present invention.
  • FIG. 20D a diagram confirming changes in protein expression of MITF
  • FIG. 20E a diagram confirming changes in protein expression of MDSC
  • Figure 21 is a method of administering MDSC treated with ML-329 together with GM-CSF and TCCM to tumor-forming mice according to an embodiment of the present invention (Figure 21A), tumor volume change in the tumor-forming mice ( Figure 21B and Fig. 21C)) and changes in the population of MDSCs (Fig. 21D) in the tumor tissues of the tumorigenic mice.
  • the present invention is a composition for alleviating, treating or preventing lowering of the immune response caused by bone marrow-derived suppressor cells (MDSC), containing an MITF (Microphthalmia-associated transcription factor) inhibitor as an active ingredient;
  • the "MITF inhibitor” may include, without limitation, a substance capable of achieving the purpose of inhibiting the activity of MDSC through inhibition of MITF expression or activity.
  • the MITF inhibitor may be a gene expression inhibitor of MITF or a protein activity inhibitor of MITF.
  • the gene expression inhibitor of MITF is an antisense nucleotide complementary to the mRNA of the MITF gene, an aptamer, a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro It may be one or more selected from the group consisting of RNA (microRNA, miRNA) and ribozyme, but is not limited thereto.
  • antisense nucleotide is, as defined by the Watson-Click base pair, binds (hybridizes) to the complementary nucleotide sequence of DNA, immature-mRNA, or mature mRNA to interfere with the flow of genetic information from DNA to protein.
  • the nature of antisense nucleotides specific to the target sequence makes them exceptionally multifunctional. Because antisense nucleotides are long chains of monomer units, they can be easily synthesized for target RNA sequences. Many recent studies have demonstrated the usefulness of antisense nucleotides as a biochemical means for studying target proteins (Rothenberg et al., J. Natl. Cancer Inst., 81:1539-1544, 1999).
  • antisense nucleotides can be considered as a new type of inhibitor, as there have been many recent advances in the field of oligonucleotide chemistry and nucleotide synthesis showing improved cell adhesion, target binding affinity and nuclease resistance.
  • shRNA refers to a nucleotide consisting of 50-60 single strands, and constitutes a stem-loop structure in vivo.
  • shRNA is an RNA sequence that makes a tight hairpin structure to suppress gene expression through RNA interference (RNAi).
  • RNAi RNA interference
  • the shRNA is generally transduced into cells through a vector containing the U6 promoter to be expressed, and is usually transferred to daughter cells to inhibit gene expression.
  • shRNA hairpin structure is cleaved by an intracellular mechanism to become siRNA and then binds to the RNA-induced silencing complex (RISC). These RISCs bind to and cleave mRNA.
  • shRNA is transcribed by RNA polymerase.
  • small interfering RNA refers to a short double-stranded RNA capable of inducing an RNA interference phenomenon through cleavage of a specific mRNA. It is composed of a sense RNA strand having a sequence homologous to the mRNA of the target gene and an antisense RNA strand having a sequence complementary thereto. Since siRNA can suppress the expression of a target gene, it is provided as an efficient gene knock-down method or as a gene therapy method.
  • microRNA refers to a short non-coding RNA consisting of about 22 base sequences. It is known to function as a post-transcriptional regulator in the process of gene expression. By binding complementarily to a target mRNA having a complementary nucleotide sequence, target mRNAs are degraded or translation into proteins is inhibited.
  • the protein activity inhibitor of MITF may be one or more selected from the group consisting of compounds, peptides, peptide mimetics, substrate analogs, aptamers, and antibodies that specifically bind to the MITF protein, but is not limited thereto.
  • peptide mimetics inhibit the activity of the MITF protein by inhibiting the binding domain of the MITF protein.
  • Peptidomimetics may be peptides or non-peptides, and may consist of amino acids linked by non-peptide bonds, such as psi bonds.
  • Peptide mimetics are structured similar to the secondary structural properties of MITF proteins and can mimic the inhibitory properties of large molecules such as antibodies or water-soluble receptors, and can be novel small molecules that can act with equal effects as natural antagonists.
  • the "aptamer” is a single-stranded DNA or RNA molecule, which is highly sensitive to specific chemical or biological molecules by an evolutionary method using an oligonucleotide library called SELEX (systematic evolution of ligands by exponential enrichment). It can be obtained by separating oligomers that bind with affinity and selectivity.
  • the aptamer can specifically bind to a target and modulate the activity of the target, for example, it can block the function of the target through binding.
  • the "antibody” can effectively inhibit the activity of the MITF protein by specifically and directly binding to the MITF protein.
  • a polyclonal antibody or a monoclonal antibody may be used as an antibody that specifically binds to the MITF protein.
  • Antibodies that specifically bind to the MITF protein may be prepared by known methods known to those skilled in the art, and commercially known MITF antibodies may be purchased and used.
  • the antibody can be prepared by injecting an immunogen MITF protein into an external host according to a conventional method known to those skilled in the art. External hosts include mammals such as mice, rats, sheep, and rabbits. Immunogens are injected by intramuscular, intraperitoneal or subcutaneous injection, and can generally be administered with an adjuvant to increase antigenicity.
  • Antibodies can be isolated by collecting serum showing specific titer and antigen specificity by regularly collecting blood from an external host.
  • the MITF inhibitor may be ML-329 and/or an AMPK activator.
  • the ML-329 is a compound represented by the following [Formula 1], and inhibits the expression of the MITF gene or the activity of the MITF protein, such as transcriptional activity:
  • the AMPK activity promoter may be berberine or Kazinol U, but is not limited thereto.
  • the AMPK activity promoter inhibits the expression of the MITF gene or the activity of the MITF protein, such as transcriptional activity.
  • the "bone marrow-derived suppressor cell” or “MDSC” functions to suppress immunity by inhibiting the activity of cytotoxic T lymphocytes and NK cells. Although it has a pure function of suppressing unnecessary excessive immune reactions such as autoimmunity, there is also a dysfunctional function that causes or worsens disease by suppressing immunity when an immune response is required, or interferes with appropriate treatment.
  • MDSC is highly increased in tumor or cancer patients, which negates the efficacy of cancer vaccines by significantly reducing the effectiveness of cancer vaccine administration. In addition, it contributes to the patient's resistance to the Imuune Checkpoint Inhibitor (ICI), which is used as an anticancer immunotherapy, reducing the effectiveness of the anticancer immunotherapy.
  • ICI Imuune Checkpoint Inhibitor
  • the MDSC of the tumor-bearing individual may have a phenotype of CD11b + Gr1 + PD-L1 + , but is not limited thereto.
  • the tumor is specifically breast cancer, skin or intraocular melanoma, liver cancer, gastric cancer, colon cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer , Anal cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, lymph node cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue Sarcoma, urethral cancer, penile cancer, prostate cancer, adenocarcinoma, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem nerve It may be a glioma or
  • the composition is preferably administered to a subject in need of inhibiting MDSC.
  • the present inventors produced a mouse bone marrow-derived MDSC by treating cells isolated from the bone marrow of a mouse with GM-CSF as a factor for inducing differentiation of MDSC.
  • the present inventors treated the cells isolated from the bone marrow of mice with GM-CSF, which is a differentiation-inducing factor of MDSC, and a tumor cell-conditioned medium (TCCM) cultured with a mouse breast cancer cell line. It was confirmed that the differentiation of MDSC was induced, the activity of MDSC was induced by TCCM, and the expression of the MITF gene and protein was increased in the MDSC in which the activity was induced. In addition, as a result of co-culturing the MDSC and T cells in which the activity was induced by the TCCM, it was confirmed that the number of CD3 + CD8 + T cells was decreased by the MDSC in which the activity was induced. Through the above results, it was confirmed that MDSC was activated in the cancer cell microenvironment to lower the immune response, and that MITF was involved in the activation of MDSC as a target factor for the activation of MDSC.
  • GM-CSF which is a differentiation-inducing factor of MDSC
  • TCCM tumor cell-
  • the present inventors obtained MDSCs from the spleen and tumor sites of tumorigenic mice, and confirmed that high activation and expression of MITF were observed in MDSCs obtained from the tumor sites. In addition, it was confirmed that MDSCs expressing MITF were 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.
  • the present inventors treated drugs that induce MDSC activity, IL-18, IL-4, LPS, or statin-based drugs together with GM-CSF, which is a differentiation-inducing factor of MDSC, in cells isolated from the bone marrow of mice.
  • GM-CSF which is a differentiation-inducing factor of MDSC
  • the present inventors treated IBMX as a MITF inducer together with GM-CSF, which is a differentiation inducing factor of MDSC, to cells isolated from the bone marrow of mice.
  • GM-CSF which is a differentiation inducing factor of MDSC
  • IBMX a differentiation inducing factor of MDSC
  • the present inventors confirmed that MDSC is activated in the cancer cell microenvironment to lower the immune response, and MITF is involved in the activation of MDSC, and that the MITF inhibitor can be used to inhibit MDSC, so that the MITF inhibitor
  • the composition containing as an active ingredient can be usefully used to alleviate the decrease in immune response caused by MDSC and to increase the efficiency of anticancer immunotherapy.
  • composition of the present invention can be preferably formulated as a pharmaceutical composition, including at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.
  • compositions formulated as liquid solutions, sterilization and biocompatible saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as necessary.
  • composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvants include excipients, disintegrants, sweetening agents, binders, coating agents, expanding agents, lubricants.
  • the adjuvants include excipients, disintegrants, sweetening agents, binders, coating agents, expanding agents, lubricants.
  • a solubilizing agent such as a lubricant or a flavoring agent may be used.
  • composition of the present invention may be formulated as an injectable formulation such as an aqueous solution, suspension, emulsion, pill, capsule, granule or tablet by additionally adding a diluent, a dispersant, a surfactant, a binder and a lubricant.
  • injectable formulation such as an aqueous solution, suspension, emulsion, pill, capsule, granule or tablet by additionally adding a diluent, a dispersant, a surfactant, a binder and a lubricant.
  • it can be preferably formulated according to disease or ingredient using a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.
  • composition of the present invention is a conventional intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, nasal, inhaled, topical, rectal, oral, intraocular or intradermal route. It can be administered in a manner.
  • the composition of the present invention is administered in a pharmaceutically effective amount.
  • 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 the type of disease, severity, drug activity, Sensitivity to drugs, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field.
  • composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and this can be easily determined by a person skilled in the art.
  • the effective amount of the composition according to the present invention may vary depending on the age, sex, and body weight of the patient, and generally 0.1 mg to 100 mg per 1 kg of body weight, more specifically 0.5 mg to 10 mg daily or every other day Alternatively, it can be administered in 1 to 3 times a day. However, since it may increase or decrease according to the route of administration, the severity of the disease, sex, weight, age, etc., the dosage amount does not limit the scope of the present invention in any way.
  • the present invention contains an MITF inhibitor as an active ingredient, anticancer adjuvant;
  • a method of treating cancer comprising administering to the subject an MITF inhibitor and an anticancer agent;
  • the use of MITF inhibitors for use as anticancer adjuvants is provided.
  • the MITF inhibitor may be a gene expression inhibitor of MITF or a protein activity inhibitor of MITF.
  • the gene expression inhibitor of MITF is an antisense nucleotide complementary to the mRNA of the MITF gene, an aptamer, a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a micro It may be one or more selected from the group consisting of RNA (microRNA, miRNA) and ribozyme, but is not limited thereto.
  • the protein activity inhibitor of MITF may be one or more selected from the group consisting of compounds, peptides, peptide mimetics, substrate analogs, aptamers, and antibodies that specifically bind to MITF protein, but is not limited thereto.
  • the MITF inhibitor may be ML-329 and/or an AMPK activator, and specifically, the AMPK activation promoter may be berberine or kazinol U, but is not limited thereto. .
  • the anticancer adjuvant may be administered in combination with an anticancer agent, and the anticancer adjuvant suppresses the activity of MDSC to alleviate the decrease in the immune response caused by MDSC, thereby significantly increasing the effect of the anticancer agent. I can.
  • the anticancer agent may be one or more selected from the group consisting of chemotherapeutic agents, targeted therapeutic agents, antibody therapeutic agents, immunotherapy agents, and hormone therapeutic agents, but is not limited thereto.
  • the chemotherapeutic agent is, for example, an antagonist (e.g., folic acid, purine, and pyrimidine derivatives), an alkylating agent (e.g., nitrogen mustard, nitrosourea, platinum, alkyl sulfonate, hydrazine, triazene, Aziridines, spindle inhibitors, cytotoxic agents, topoisomerase inhibitors and others) or hypomethylating agents (e.g. zebulaline, isothiocyanate, azacytidine (5-azacytidine), 5-fluoro Rho-2'-deoxycytidine, 5,6-dihydro-5-azacytidine and others), but are not limited thereto.
  • an antagonist e.g., folic acid, purine, and pyrimidine derivatives
  • an alkylating agent e.g., nitrogen mustard, nitrosourea, platinum, alkyl sulfonate, hydrazine, triazen
  • the targeted therapeutic agent is an agent specific for an unregulated protein of cancer cells, for example, a tyrosine kinase inhibitor such as Axitinib, Bosutinib, and Cediranib. , Dasatinib, Erlotinib, Imatinib, Gefitinib, Lapatinib, Lestaurtinib, Nilotinib , Semaxanib, Sorafenib, Sunitinib, and Vandetanib, or cyclin-dependent kinase inhibitors such as Alvocidib and Celishic There is a rib (Seliciclib), but is not limited thereto.
  • a tyrosine kinase inhibitor such as Axitinib, Bosutinib, and Cediranib.
  • 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, and Cetuximab. , Panitumumab, Alemtuzumab, Bevacizumab, Edrecolomab, or Gemtuzumab, but are not limited thereto.
  • the immunotherapeutic agent is an agent designed to induce the subject's own immune system to attack a tumor.
  • Ipilimumab, Avelumab, Nivolumab, or Pembrolizumab is used. However, it is not limited thereto.
  • the hormone therapy is an agent that inhibits the growth of cancer by providing or blocking a hormone in a specific cancer, for example, tamoxifen or diethylstilbestrol, but is not limited thereto.
  • the appropriate dosage of the anticancer agent is already well known in the art, it may be administered according to a standard known in the art according to the condition of each patient.
  • the specific dosage determination 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 age, health status, and complications of the target to be administered.
  • the present inventors have activated MDSC in the microenvironment of cancer cells to lower the immune response, and MITF is involved in the activation of the MDSC, and the MITF inhibitor can be used to inhibit the activity of MDSC. As confirmed, the MITF inhibitor can be used as an anticancer adjuvant in anticancer immunotherapy.
  • the present invention provides a method for inhibiting MDSC, comprising administering a MITF inhibitor to an individual in need of inhibition of MDSC.
  • MITF inhibitor and the MDSC are the same as the description of the composition, and the specific description uses the above, and hereinafter, only the configuration specific to the MDSC inhibition method will be described.
  • the "bone marrow-derived suppressor cell inhibition” or “MDSC inhibition” includes not only inhibiting the activity of MDSC, but also reducing the number of MDSCs. Reducing the number includes not only inhibiting the production of cells, but also killing already produced cells or differentiating them into other cells. In addition, all mechanisms referred to as “inhibition" from a biological point of view are included.
  • the individual requiring inhibition of MDSC may be an individual having a tumor that requires inhibition of MDSC, and the MDSC of the individual having the tumor may have a phenotype of CD11b + Gr1 + PD-L1 + , It is not limited thereto.
  • the tumor is specifically breast cancer, skin or intraocular melanoma, liver cancer, gastric cancer, colon cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer , Rectal cancer, anal muscle 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 cancer, thyroid cancer, parathyroid cancer, adrenal cancer , Soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, adenocarcinoma, chronic or acute leukemia, lymphocyte lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary CNS lymphoma, spinal cord tumor, It may be a brains
  • the present inventors have activated MDSC in the microenvironment of cancer cells to lower the immune response, and MITF is involved in the activation of the MDSC, and the MITF inhibitor can be used to inhibit the activity of MDSC. As confirmed, the MITF inhibitor can be administered to an individual in need of inhibition of MDSC and used for treatment of MDSC-related diseases.
  • MDSC Myeloid-derived suppressor cells
  • Balb / c mice were Saeronbio. It was purchased from Inc. (Republic of Korea). Animal experiments were performed with the approval of the Institutional Ethical Committee of Sookmyung Women's University (SMWU-IACUC-1708-017-02). The thigh bones of 6 to 8-week-old Balb/c mice were excised to separate the bone marrow in the bone, and then treated with RBC lysis buffer (Sigma-Aldrich, St. Louis, MO) to remove red blood cells to obtain cells.
  • RBC lysis buffer Sigma-Aldrich, St. Louis, MO
  • the obtained bone marrow cells were cultured in a 24-well plate at a cell number of 5 ⁇ 10 5 cells/ml in RPMI medium (Invitrogen, Grand Island, NY) containing 10 ng/ml of GM-CSF for 96 hours to differentiate MDSC.
  • RPMI medium Invitrogen, Grand Island, NY
  • GM-CSF GM-CSF
  • mice were injected with a breast cancer cell line to prepare tumor-forming mice, and MDSCs were prepared from the tumor-forming mice.
  • tumors were formed by subcutaneous injection of 100 ⁇ l PBS containing 4T1 cells, which is a mouse breast cancer cell line, to a cell number of 5 ⁇ 10 5 cells/100 ⁇ l to the right flank to 6 to 8-week-old Balb/c mice.
  • the mice were bred in the animal laboratory of Sookmyung Women's University at a temperature of 23.5 ⁇ 1 °C and a humidity of 50 ⁇ 5% in a 12-hour light/dark cycle. After 2 weeks, the tumor, bone marrow and spleen were respectively excised and cells were isolated.
  • FACS Fluorescence-acitivated cell sorting
  • the mouse breast cancer cell line 4T1 cells 10% fetal bovine serum (FBS), 100 units of RPMI medium (Invitrogen, Grand Island, NY) containing 10 ml of 5% CO 2 , 37 °C 3 under conditions Incubated for days to bring the cells to 80% saturation. Then, the culture medium was recovered and concentrated with a 3000 NMWL (nominal molecular weight limit) centrifugal filter (Merck Milipore, Billerica, MA) at 3000 ⁇ g for 20 minutes in a centrifuge maintained at 4°C, and concentrated with a TCCM (tumor cell-conditioned medium). was obtained.
  • FBS fetal bovine serum
  • RPMI medium Invitrogen, Grand Island, NY
  • the obtained TCCM and GM-CSF at a concentration of 10 ng/ml were included in a 24-well plate with a cell number of 5 ⁇ 10 5 cells/ml. It was cultured in RPMI medium for 96 hours to induce differentiation of MDSC. As a control, MDSC induced differentiation with RPMI medium containing GM-CSF was used.
  • the MDSCs induced differentiation were recovered, stained with fluorescence-conjugated anti-CD11b antibody and anti-Gr1 antibody, and then FACS analysis was performed (FIG. 2A).
  • the differentiation-induced MDSC was recovered and qRT-PCR was performed for iNOS, IL-10, and TGF- ⁇ , which are MDSC activity markers.
  • the MDSC induced differentiation was 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.
  • MITF microphthalmia-associated transcription factor
  • qRT-PCR for MITF was performed in the same manner as described in Example ⁇ 2-1> using the MDSC recovered in Example ⁇ 2-1> to confirm the gene expression of MITF.
  • Primers for MITF were purchased from Bioneer (Fig. 3A).
  • Example ⁇ 2-1> Western blotting was performed using the MDSC recovered in Example ⁇ 2-1> to confirm the protein expression of MITF.
  • the MDSC recovered in Example ⁇ 2-1> was treated with a cell lysis buffer and lysed. Cell lysates were separated by electrophoresis with 10% SDS-PAGE gel, and transferred to a PVDF membrane. Then, after reacting by treating an anti-MITF antibody and an anti-actinin antibody with a primary antibody, an HRP-conjugated secondary antibody is attached to the primary antibody attached to the membrane, and this is enhanced chemiluminescence (PicoEPDTM Western Reagent kit).
  • MITF is a target factor specifically expressed in MDSCs induced by TCCM.
  • MDSC is known to lower the immune response by inhibiting the proliferation and function of T cells. Accordingly, in order to find out whether the immune response is lowered by MDSC in the cancer cell microenvironment, the degree of inhibition of T cell proliferation was confirmed using MDSC treated with a culture medium in which cancer cells were cultured.
  • mice Specifically, cells isolated from the spleen of tumorigenic mice according to the manufacturer's procedure using a MACS cell separation kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) were separated in a 24-well plate with a cell number of 5 ⁇ 10 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.
  • MACS cell separation kit Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
  • T cells In addition, in order to obtain T cells, the spleens of 6 to 8 week old Balb/c mice were excised and the cells were isolated. Then, after staining using a fluorescently-conjugated anti-CD3 antibody, T cells were isolated using FACS. The separated T cells were stained with 2.5 ⁇ M CFSE for 7 minutes to perform CFSE labeling. 5 ⁇ 10 5 CFSE-labeled CD3 + T cells were stimulated for 2 hours on a plate coated with an anti-CD3 monoclonal antibody and a soluble anti-CD28 monoclonal antibody, and then the cells isolated from the spleen of the tumorigenic mice MDSC obtained by inducing differentiation and activity (FIG.
  • Example ⁇ 2-1> (FIG. 4B) and MDSC recovered in Example ⁇ 2-1> (FIG. 4B) were put in 5 ⁇ 10 5 cells or 10 ⁇ 10 5 cells, and for 3 days Cultured. After 3 days of culture, staining was performed using a fluorescently-conjugated anti-CD8 antibody, and then FACS analysis was performed to measure the degree of proliferation of CD8 + T cells.
  • the antibody was purchased from eBioscience.
  • MDSC is activated in the cancer cell microenvironment to lower the immune response
  • MITF is involved in the activation of MDSC as a target factor for the activation of the MDSC.
  • MDSC in tumorigenic mice was used to confirm its activity and changes in gene expression of MITF.
  • qRT-PCR was performed in the same manner as described in Example ⁇ 2-2> using the recovered CD11b + Gr1 + MDSC to confirm the gene expression of MITF (Fig. 5C).
  • MDSC is activated in the cancer cell microenvironment, and that MITF is involved in the activation of the MDSC.
  • tissue staining was performed on each of 10 samples of tissues around lung cancer and head and neck cancer tissues and correspondingly non-cancer lymph node tissues at Seoul National University Pathology Department (Prof. Yoon Kyung Jeon). Specifically, after fixing the tissue in 4% formaldehyde solution, it was dehydrated in ethanol (70-100%) by concentration and embedded in paraffin. The tissue was sectioned with a microtome (thickness 4 ⁇ m) and stained (H&E) with hematoxylin and eosin. The cross section was observed with an optical microscope (Olympus, Japan) and photographed at x400 magnification.
  • tissue sections were blocked with 3% normal horse serum diluted in PBS for 30 minutes. Block the sections and dilute the appropriate dilution factor (1:100) with CD11b, CD14, MITF antibodies (anti-mouse monoclonal primary antibody (Cat.No 790-4367), Ventana Medical Systems, Oro Valley, AZ) overnight at 4°C. ). After washing the slides with PBS, the avidin-biotin-peroxidase complex (ABC; Vector Laboratories, Burlingame, CA) was washed.
  • ABS avidin-biotin-peroxidase complex
  • the slides were washed, and the peroxidase reaction was developed with diaminobenzidine and peroxide, and aqua- Mounted on a mount (Aqua-Mount) and evaluated under a light microscope (Olympus) ⁇ 400.
  • HPF high power field
  • Example ⁇ 1-1> After obtaining bone marrow cells by the same method as described in Example ⁇ 1-1>, IL-18 at a concentration of 10 or 50 ng/ml in a 24-well plate with a cell number of 5 ⁇ 10 5 cells/ml, And 10 ng/ml of GM-CSF-containing RPMI medium was cultured for 96 hours to induce differentiation of MDSC (FIGS. 6A and 6B ).
  • the differentiation of MDSC was induced by culturing with RPMI medium containing GM-CSF for 96 hours, and IL-18 at a concentration of 10 or 50 ng/ml was added for 24 hours. Treated (Fig. 6C and Fig. 6D).
  • MDSC induced differentiation with RPMI medium containing GM-CSF was 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).
  • Example ⁇ 2-1> the differentiation and activity of MDSC were induced, but 10 ng/ml of IL-4 was treated with a drug that induces the activity.
  • the differentiation-induced MDSC was recovered and FACS analysis was performed in the same manner as described in Example ⁇ 2-1> to confirm the differentiation of MDSC (Fig. 7A).
  • Example ⁇ 2-1> the differentiation and activity of MDSC were induced, but 100 ng/ml of LPS was treated with a drug that induces the activity.
  • 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. 8A).
  • Simvastatin a statin-based drug, is known to induce the activity of MDSC by inhibiting the expression of IRF4 (Interferon regulatory factor 4). Accordingly, a statin drug was treated with a drug that induces the activity of MDSC, and after inducing the differentiation of MDSC, the change in gene expression of MITF was confirmed.
  • the differentiation and activity of MDSC are induced, but as drugs that induce the activity, simvastatin (Sim, 0.5 or 1 ⁇ M), lovastatin (Lovastatin, Lova, 0.5 or 1 ⁇ M), Provastatin (Pravastatin, Prav, 5 or 10 ⁇ M), Rosuvastatin (Rosu, 0.5 or 1 ⁇ M), or atorvastatin (Atorvastatin, Ator, 0.05 or 0.1 ⁇ M) were treated (Fig. 9 And FIG. 10).
  • Example ⁇ 1-1> it was cultured with RPMI medium containing GM-CSF for 96 hours to induce differentiation of MDSC, and the statin drug was treated for 24 hours (Fig. 11 and Fig. 12).
  • MDSC was recovered, and the differentiation of MDSCs (Figs. 9 and 11) was confirmed by the same method as described in Examples ⁇ 2-1> and ⁇ 2-2>.
  • the activity of MDSC (FIGS. 10A and 12A) was confirmed in the simvastatin or lovastatin 1 ⁇ M-treated group, and gene expression of MITF (FIGS. 10B and 12B) was confirmed.
  • ATRA all-trans retinoic acid
  • Example ⁇ 1-1> After obtaining bone marrow cells by the same method as described in Example ⁇ 1-1>, ATRA (0.5 or 1 ⁇ M) and 10 ng/ml in a 24-well plate with a cell number of 5 ⁇ 10 5 cells/ml The differentiation of MDSC was induced by incubating for 96 hours in RPMI medium containing GM-CSF at a concentration (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).
  • IBMX induces the expression of MITF in melanoma cells. Accordingly, in order to examine the effect of the expression or regulation of MITF on MDSC activity, changes in the activity of MDSC treated with IBMX as an MITF expression inducing agent and changes in T cell proliferation by the MDSC were confirmed.
  • Example ⁇ 1-1> after obtaining bone marrow cells by the same method as described in Example ⁇ 1-1>, in a 24-well plate with a cell number of 5 ⁇ 10 5 cells/ml, IBMX at a concentration of 10 ⁇ M and a concentration of 10 ng/ml The differentiation of MDSC was induced by incubating for 96 hours in RPMI medium containing GM-CSF. As a control, MDSC induced differentiation with RPMI medium containing GM-CSF was 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).
  • FIG. 14 it was confirmed that the differentiation of MDSC was induced in both the control group and the IBMX-treated group to a similar degree (FIG. 14A).
  • the IBMX treatment group it was confirmed that the gene and protein expression of MITF increased compared to the control group (FIG. 14C), and the activity of MDSC increased (FIG. 14B and FIG. 14C).
  • the activity of MDSC activity was increased to inhibit T cell proliferation (FIG. 14D).
  • Berberine, Kazinol U, and the like are known to inhibit the expression or activity of MITF in melanocytes while being AMPK activators. Accordingly, in order to examine the effect of the regulation of MITF expression or activity on MDSC activity, changes in the activity of MDSC treated with berberine as an MITF inhibitor were confirmed.
  • Example ⁇ 1-1> After bone marrow cells were obtained by the same method as described in Example ⁇ 1-1>, 10 ⁇ M berberine and 10 ng/ml concentration in a 24-well plate with a cell number of 5 ⁇ 10 5 cells/ml The differentiation of MDSC was induced by incubating for 96 hours in RPMI medium containing GM-CSF. As a control, MDSC induced differentiation with RPMI medium containing GM-CSF was used.
  • the MDSC induced differentiation was recovered, and qRT-PCR was performed in the same manner as described in Example ⁇ 2-2> to confirm the expression of MITF (FIG. 15A).
  • FIG. 15 in the case of the berberine-treated group, it was confirmed that the expression of MITF was suppressed by berberine (FIG. 15A), and the differentiation of MDSC was somewhat reduced (FIG. 15B). In addition, in the case of the berberine treatment group, it was confirmed that the activity of MDSC was significantly inhibited compared to the control group (FIG. 15C).
  • ML-329 is known to suppress the expression of MITF by inhibiting the TRPM-1 promoter activity in melanocytes. Accordingly, in order to examine the effect of the regulation of MITF expression or activity on MDSC activity, changes in the activity of MDSC treated with ML-329 as an MITF inhibitor and changes in T cell proliferation by the MDSC were confirmed.
  • Example ⁇ 1-1> After bone marrow cells were obtained by the same method as described in Example ⁇ 1-1>, ML-329 (Cayman Chemical) at a concentration of 0.5 or 1 ⁇ M in a 24-well plate with a cell number of 5 ⁇ 10 5 cells/ml. , Ann Arbor, MI) and 10 ng/ml of GM-CSF-containing RPMI medium was cultured for 96 hours to induce differentiation of MDSC. As a control, MDSC induced differentiation with RPMI medium containing GM-CSF was used. Then, the differentiation-induced MDSC was recovered and qRT-PCR was performed in the same manner as described in Examples ⁇ 2-1> and ⁇ 2-2> to confirm the activity of MDSC (Fig. 17A) and MITF expression. (Fig. 17B).
  • bone marrow cells were obtained by the same method as described in Example ⁇ 1-1>, and then the number of cells was 5 ⁇ 10 5 cells/ml.
  • a 24-well plate 96 with RPMI medium containing 0.5, 1 or 2 ⁇ M concentration of ML-329 (Cayman Chemical), TCCM obtained in Example ⁇ 2-1>, and 10 ng/mL concentration of GM-CSF. Differentiation of MDSC was induced by incubation for a period of time. As a control, MDSC induced differentiation with RPMI medium containing GM-CSF was used.
  • Figs. 17 and 18 in the case of the ML-329 treatment group, it was confirmed that MITF expression was inhibited by ML-329 and the activity of MDSC was inhibited (FIGS. 17A and 17B ).
  • MDSC activity by TCCM is inhibited (FIGS. 18B and 18C)
  • MITF expression is inhibited (FIG. 18C)
  • T cell proliferation is increased by inhibition of MDSC activity by ML-329. It was confirmed (Fig. 18D).
  • 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 the regulation of MITF expression or activity on MDSC activity, changes in ROS production were confirmed in MDSC treated with berberine or ML-329 as an MITF inhibitor.
  • ROS reactive oxygen species
  • RNS reactive nitrogen species
  • MDSC was isolated from the spleen of the tumorigenic mouse of Example ⁇ 1-2>, berberine at a concentration of 10 ⁇ M or ML-329 at a concentration of 1 ⁇ M, TCCM and 10 obtained in Example ⁇ 2-1>
  • the differentiation of MDSC was induced by incubating for 48 hours in RPMI medium containing GM-CSF at a concentration of ng/ml.
  • the MDSCs were collected and dispensed at 1 ⁇ 10 5 cells, and LPS was treated at a concentration of 100 ng/ml for 24 hours.
  • the ROS inhibitor NAC N-acetyl-cysteine
  • DCF-DA was added and reacted at 37° C. for 30 minutes, and the degree of ROS production was measured by flow cytometry.
  • ROS production by MDSC was inhibited in the case of berberine or ML-329 treatment group, and ROS was strongly inhibited when ROS inhibitor was treated together.
  • RNA interference RNA interference
  • CRISPR CRISPR
  • a CRISPR vector specifically pLentiCRISPR-E vector
  • pLentiCRISPR-E vector was added to the bone marrow cells of 1 ⁇ 10 6 cells/ml.
  • the sgRNA was cloned; MITF gRNA FW oligo: 5'-CACCGTAAGGACTTCCATCGGCACC-3' (SEQ ID NO: 1), MITF gRNA RV oligo: 5'-AAACGGTGCCGATGGAAGTCCTTAC-3' (SEQ ID NO: 2).
  • non-target control gRNA was cloned into the pLentiCRISPR-E vector; non-target control sgRNA: 5'-CACCGGTATTACTGATATTGGTGGG-3' (SEQ ID NO: 3).
  • the cloned construct was transfected using lipofectamine according to the manufacturer's procedure. After 24 hours of transfection, the differentiation of MDSC was induced by incubating with RPMI medium containing TCCM obtained in Example ⁇ 2-1> and 10 ng/ml of GM-CSF for 72 hours. 72 hours after transfection, MDSC was recovered, and the method described in ⁇ 2-2> and Western blotting were performed to confirm the expression of MITF and MDSC activity (FIG. 20A). In addition, the degree of ROS production by MDSC was measured by the same method as described above (Fig. 20B), and the degree of T cell proliferation by MDSC was measured by the same method as described in Example ⁇ 2-3> ( Figure 20C).
  • MITF plasmid DNA (Addgene, Cambridge, MO) was transfected into bone marrow cells having a cell number of 1 ⁇ 10 6 cells/ml using lipofectamine according to the manufacturer's procedure. After 24 hours of transfection, the differentiation of MDSC was induced by incubating for 72 hours in RPMI medium containing 10 ng/ml of GM-CSF. Three days after transfection, MDSCs were recovered and the expression of MITF (FIG. 20D) and the degree of T cell proliferation by MDSC (FIG. 20E) were measured as described above.
  • FIG. 20A it was confirmed that MITF expression and MDSC activity were inhibited in MDSC using MITF shRNA and CRISPR technology (FIG. 20A), and ROS generation by MDSC was inhibited by inhibition of MITF expression ( 20B), it was confirmed that T cell inhibition by MDSC was alleviated (FIG. 20C). On the other hand, it was confirmed that in the MDSC overexpressing MITF, contrary results appeared (FIGS. 20D and 20E).
  • Example 7> it was confirmed that the MDSC activity was inhibited by the MITF inhibitor. Accordingly, in order to examine the effect of the decrease in MDSC activity in tumorigenic mice, changes in tumor growth were confirmed after administering MDSCs whose activity was decreased by MITF inhibitors to tumorigenic mice.
  • MDSC was 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>, 1 ⁇ M concentration of ML-329, TCCM obtained in Example ⁇ 2-1>, and 10 ng/mL concentration of GM-CSF were included. The differentiation of MDSC was induced by incubating for 48 hours with the prepared RPMI medium. After 48 hours, the MDSC + 4T1-luc (5 ⁇ 10 4 + 2.5 ⁇ 10 5 /100 ⁇ l) was injected subcutaneously on the left flank of the mouse.
  • the composition containing the MITF inhibitor can be administered to an individual in need of inhibition of MDSC, such as a tumor-bearing individual, and can be usefully used to alleviate the decrease in immune response caused by MDSC.
  • the composition containing the MITF inhibitor can be used in combination with an anticancer agent, such as an anticancer immunotherapy agent, to increase the efficiency of anticancer immunotherapy.
  • 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, wherein the composition containing the MITF inhibitor as an active ingredient
  • MDSC myeloid-derived suppressor cells
  • MITF microphthalmia-associated transcription factor

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne une composition pour inhiber des cellules myéloïdes suppressives (MDSC) dérivées de myéloïde (MDSC) comprenant un inhibiteur du facteur de transcription associé à la microphtalmie (MITF) en tant que principe actif. En particulier, il a été confirmé qu'une MDSC est activée dans un microenvironnement de cellule cancéreuse pour abaisser une réponse immunitaire, et un MITF est impliqué dans l'activation de la MDSC, la MDSC pouvant être inhibée à l'aide de l'inhibiteur de MITF. Ainsi, une composition comprenant l'inhibiteur de MITF en tant que principe actif peut être efficacement utilisée pour atténuer une diminution de la réponse immunitaire provoquée par une MDSC et pour augmenter l'efficacité d'une immunothérapie anticancéreuse.
PCT/KR2020/010480 2019-08-22 2020-08-07 Composition pour inhiber une cellule suppressive dérivée de myéloïde comprenant un inhibiteur de mitf en tant que principe actif WO2021033973A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2019-0103340 2019-08-22
KR1020190103340A KR20210023306A (ko) 2019-08-22 2019-08-22 Mitf 억제제를 유효성분으로 함유하는 골수-유래 억제세포 저해용 조성물

Publications (1)

Publication Number Publication Date
WO2021033973A1 true WO2021033973A1 (fr) 2021-02-25

Family

ID=74660311

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2020/010480 WO2021033973A1 (fr) 2019-08-22 2020-08-07 Composition pour inhiber une cellule suppressive dérivée de myéloïde comprenant un inhibiteur de mitf en tant que principe actif

Country Status (2)

Country Link
KR (3) KR20210023306A (fr)
WO (1) WO2021033973A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20220095112A (ko) * 2020-12-29 2022-07-06 숙명여자대학교산학협력단 Mdsc 활성 저해 약물의 선별 방법

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2547366A4 (fr) 2010-03-18 2013-08-07 Univ Colorado State Res Found Agents d'inhibition des cellules myéloïdes suppressives
US9550992B2 (en) 2011-12-02 2017-01-24 University Of South Florida Compositions and methods for modulating myeloid derived suppressor cells
WO2015182798A1 (fr) 2014-05-28 2015-12-03 주식회사 카엘젬백스 Composition pour inhiber des cellules suppressives dérivées de myéloïde (mdsc)
KR101902355B1 (ko) 2015-11-09 2018-10-01 서울대학교산학협력단 데시타빈 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 골수유래억제세포 저해용 조성물

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
INES HAMMAMI;JINGKUI CHEN;FREDERIC MURSCHEL;VINCENZO BRONTE;GREGORY DE CRESCENZO;MARIO JOLICOEUR: "Immunosuppressive activity enhances central carbon metabolism and bioenergetics in myeloid-derived suppressor cells in vitro models", BMC CELL BIOLOGY, BIOMED CENTRAL, LONDON, GB, vol. 13, no. 1, 4 July 2012 (2012-07-04), GB, pages 18, XP021115087, ISSN: 1471-2121, DOI: 10.1186/1471-2121-13-18 *
SONG YOUNG CHAN; LEE YONGHEE; KIM HYEONG MI; HYUN MOO YEOL; LIM YUN YOUNG; SONG KYE YONG; KIM BEOM JOON: "Berberine regulates melanin synthesis by activating PI3K/AKT, ERK and GSK3 beta in B16F10 melanoma cells", INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE, SPANDIDOS PUBLICATIONS, GR, vol. 35, no. 4, 31 March 2015 (2015-03-31), GR, pages 1011 - 1016, XP009513538, ISSN: 1107-3756, DOI: 10.3892/ijmm.2015.2113 *
WEIDONG LI, SAUD SHAKIR M., YOUNG MATTHEW R., CHEN GUOHONG, HUA BAOJIN: "Targeting AMPK for cancer prevention and treatment", ONCOTARGET, vol. 6, no. 10, 20 March 2015 (2015-03-20), pages 7365 - 7378, XP055219090, DOI: 10.18632/oncotarget.3629 *
WIEDEMANN GABRIELA M, AITHAL CELINA, KRAECHAN ANGELINA, HEISE CONSTANZE, CADILHA BRUNO L, ZHANG JIN, DUEWELL PETER, BALLOTTI ROBER: "Microphthalmia-Associated Transcription Factor (MITF) Regulates Immune Cell Migration into Melanoma", TRANSLATIONAL ONCOLOGY, NEOPLASIA PRESS, UNITED STATES, vol. 12, no. 2, 1 February 2019 (2019-02-01), United States, pages 350 - 360, XP055782105, ISSN: 1936-5233, DOI: 10.1016/j.tranon.2018.10.014 *
ZHU, T. ET AL.: "Berberine increases doxorubicin sensitivity by suppressing STAT3 in lung cancer", THE AMERICAN JOURNAL OF CHINESE MEDICINE, vol. 43, no. 7, 2015, pages 1 - 16 *
ZOU KUN, LI ZHAO, ZHANG YONG, ZHANG HAO-YUE, LI BO, ZHU WEI-LIANG, SHI JI-YE, JIA QI, LI YI-MING: "Advances in the study of berberine and its derivatives: a focus on anti-inflammatory and anti-tumor effects in the digestive system", ACTA PHARMACOLOGICA SINICA, NATURE PUBLISHING GROUP, GB, vol. 38, no. 2, 1 February 2017 (2017-02-01), GB, pages 157 - 167, XP055782100, ISSN: 1671-4083, DOI: 10.1038/aps.2016.125 *

Also Published As

Publication number Publication date
KR102352127B1 (ko) 2022-01-14
KR20210023306A (ko) 2021-03-04
KR20210075056A (ko) 2021-06-22
KR102352126B1 (ko) 2022-01-14
KR20210075055A (ko) 2021-06-22

Similar Documents

Publication Publication Date Title
WO2023096126A1 (fr) Procédé de criblage d'inhibiteur de mdsc
US10934550B2 (en) Immunotherapy of cancer
WO2018164518A1 (fr) Biomarqueur du cancer her2-positif et traitement anti-her2 et son utilisation
WO2017022962A1 (fr) Composition pour la prévention ou le traitement d'une maladie immunitaire, comprenant un inhibiteur de la rip kinase en tant que principe actif
WO2011052883A9 (fr) Procédé d'activation d'une cellule tueuse naturelle par l'ajustement de l'expression du gène socs2
WO2018199593A1 (fr) Anticorps bispécifique se liant à her3 et cd3
WO2021033973A1 (fr) Composition pour inhiber une cellule suppressive dérivée de myéloïde comprenant un inhibiteur de mitf en tant que principe actif
WO2021158091A2 (fr) Composition comprenant un inhibiteur de chimiokine, un inhibiteur de facteur de stimulation de colonie et un agent d'immunothérapie anticancéreuse pour la prévention ou le traitement du cancer et la polythérapie
WO2019132547A1 (fr) Composition pharmaceutique permettant de prévenir ou de traiter la métastase du cancer vers le poumon, contenant un inhibiteur de chi3l1 à titre de principe actif
WO2019194586A1 (fr) Nouvelle cible pour lutter contre le cancer et renforcer l'immunité
WO2021172889A1 (fr) Procédé pour fournir des informations pour diagnostiquer ou prédire le pronostic d'un cancer par mesure du niveau d'expression de tim-3 dans des cellules cd11b+
WO2017171373A2 (fr) Composition pour réduire la résistance à un agent ciblant l'egfr
WO2012111900A1 (fr) Procédé de traitement du cancer du sein par réduction de l'expression de l'arnm du translocateur 2 de nucléotides à adénine
WO2021002735A1 (fr) Antigène de surface cellulaire d'un lymphocyte t et ses différentes utilisations
WO2014157965A1 (fr) Composition pour le traitement ou l'inhibition de la métastase de cancers qui comprend un inhibiteur de l'expression de p34 ou un inhibiteur de l'activité p34 comme principe actif
WO2018155890A1 (fr) Petit arni asymétrique permettant d'inhiber l'expression d'un gène cible de perte de cheveux masculine
WO2021137506A1 (fr) Composition pour inhiber la croissance de cellules souches cancéreuses, contenant un inhibiteur de wdr34 et utilisation correspondante
WO2011118994A9 (fr) Gène nlk en tant que marqueur pour le diagnostic du cancer du foie et en tant qu'agent thérapeutique correspondant
Fu et al. Anti-tumor necrosis factor receptor 2 antibody combined with anti-Pd-L1 therapy exerts robust antitumor effects in breast cancer
WO2011087343A2 (fr) Composition destinée à traiter un cancer lié à une infection par un papillomavirus humain
Nakayama et al. NFκB and TGFβ contribute to the expression of PTPN3 in activated human lymphocytes
WO2023101505A1 (fr) Composition pour inhiber des points de contrôle immunitaires comprenant un inhibiteur de wnk3 en tant que principe actif
WO2023200257A1 (fr) Vésicules extracellulaires isolées à partir de cellules souches, et leurs utilisations
WO2023033506A1 (fr) Composition d'immunothérapie contre le cancer contenant un inhibiteur d'interaction de sting et trim29 comme ingrédient actif
WO2023200256A1 (fr) Composition comprenant un mutant il-32 thêta et son utilisation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20854900

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20854900

Country of ref document: EP

Kind code of ref document: A1