Pharmaceutical composition for preventing or treating cancer, comprising FOXM1 inhibitor and immune checkpoint inhibitor

Abstract

The present invention relates to a composition for preventing, ameliorating or treating cancer, the composition comprising, as active ingredients, an FOXM1 inhibitor and an immune checkpoint inhibitor. The FOXM1 inhibitor of the present invention can inhibit the expression of PD-L1 in a cell membrane, inhibit the translocation of FOXM1 to the nucleus, reduce the proliferation and survival of cancer cells, and induce an increase in apoptosis, and thus has an effect of preventing, ameliorating or treating cancer. Furthermore, when administered in combination with an immune checkpoint inhibitor, the compound can effectively inhibit tumor growth compared to the FOXM1 inhibitor or immune checkpoint inhibitor component alone, and thus can be effectively used as a cancer prevention or treatment agent.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising a FOXM1 inhibitor and an immune checkpoint inhibitor as active ingredients. Background Art

[0002] Lung cancer is the second most common cancer in the world after breast cancer. Although cancer treatment technology has made great progress in the past, its mortality rate is still the highest among all cancers. Non-small cell lung cancer (NSCLC) accounts for 80-85% of all lung cancer patients. Although immune cells can infiltrate a large amount of NSCLC tissue, cytotoxic T lymphocytes in tumor tissue are functionally inhibited by multiple mechanisms, including abnormal overexpression of immune checkpoint proteins used by tumor cells.

[0003] As a form of cancer immunotherapy, immune checkpoint inhibitors (ICIs) are drugs that can block proteins called immune checkpoints expressed in immune cells such as T cells and cancer cells. Recently, immunotherapy using immune checkpoint inhibitors has greatly improved the treatment effect of various malignancies, including lung cancer. PD-L1 (Programmed Cell Death-ligand-1) is an important immune checkpoint molecule encoded by the CD274 gene and mediates the interaction between cancer cells and tumor-infiltrating T cells. PD-1 (programmed cell death-1) is expressed on the surface of T cells, while PD-L1 is expressed in normal tissues, thereby inhibiting the self-aggression of the immune system. However, overexpression of PD-L1 on the surface of cancer cells protects cancer cells from attack by immune cells. Therefore, ICIs such as anti-PD-1 or anti-PD-L1 antibodies that can inhibit the interaction between PD-1 and PD-L1 can reactivate the attack of immune cells on cancer cells, thereby improving the treatment effect of cancers such as lung cancer. However, the high cost of ICI therapy not only imposes a huge economic burden on cancer patients, but also has been reported to cause a variety of immune-related side effects in non-target body organs, including cardiac complications. In addition, there are reports that ICI tumor treatment effects are not significant in cases where the tumor size is large or the tumor growth rate is fast. Therefore, it is necessary to develop a new strategy or therapeutic agent that can solve the various problems of ICI.

[0004] FOXM1 (Forkhead box protein M1) is an important protein that regulates various processes related to the development of lung cancer tumors, including cell cycle progression, resistance to anticancer treatment, and metastasis. The function of FOXM1 is partly attributed to the ability of FOXM1 to move to the nucleus with various forkhead or DNA binding domains and bind to the regulatory regions of various target genes, which is crucial for the survival of cancer cells. As FOXM1 inhibitors, compounds such as thiostrepton (TST), salomycin A, RCM-1 (Robert Costa Memorial drug-1), and FDI-6 (Forkhead domain inhibitor-6) have been confirmed. Among them, TST, a natural thiazole antibiotic isolated from Streptomyces azureus, can inhibit the interaction between the forkhead domain of FOXM1 and target DNA. In addition, TST is used to treat mastitis induced by Gram-negative bacteria, and has been reported to be active against breast cancer.

[0005] In the above-mentioned background, the present inventors confirmed through research using FOXM1 inhibitors that FOXM1 inhibitors can not only significantly reduce the expression of PD-L1, but also inhibit the proliferation of tumor cells, thereby confirming that they have cancer prevention or treatment effects and completing the present invention. Summary of the invention

[0006] An object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising a FOXM1 inhibitor and an immune checkpoint inhibitor as active ingredients.

[0007] Another object of the present invention is to provide an anticancer adjuvant comprising a FOXM1 inhibitor and an immune checkpoint inhibitor as active ingredients.

[0008] Another object of the present invention is to provide a use of a composition comprising a FOXM1 inhibitor and an immune checkpoint inhibitor for preventing or treating cancer.

[0009] Another object of the present invention is to provide a use of a composition comprising a FOXM1 inhibitor and an immune checkpoint inhibitor for manufacturing a medicament for cancer prevention or treatment.

[0010] Another object of the present invention is to provide a method for preventing or treating cancer, comprising the step of administering a composition comprising a FOXM1 inhibitor and an immune checkpoint inhibitor to a subject.

[0011] Next, it will be described in detail. In addition, each description and implementation disclosed in the present application may also be applicable to each other description and implementation. That is, all combinations of the various elements disclosed in the present application are included within the scope of the present application. In addition, it should not be considered that the scope of the present application is limited to the specific description recorded in the following content.

[0012] As one aspect for achieving the above object, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising a FOXM1 inhibitor and an immune checkpoint inhibitor as active ingredients.

[0013] In the present invention, "FOXM1 (Forkhead box protein M1)" is a protein belonging to the transcription factor FOX family, which is known to be encoded by the FOXM1 gene and plays an important role in the cell cycle process.

[0014] In the present invention, "FOXM1 inhibitor" is a substance that inhibits FOXM1, and any substance that can inhibit or reduce the expression of FOXM1 can be used without restriction, specifically, it can be thiazolidinedione, diarylheptanoid, RCM-1, thiostrepton, honokiol, FDI-6, siomycin A, monensin, FOXM1 aptamer, peptide 9R-P201 or pharmaceutically acceptable salts thereof.

[0015] The thiazolidinedione compound is a type of heterocyclic compound composed of a 5-membered C3NS ring. Reports indicate that it is a pharmaceutical drug that can be used in the treatment of type 2 diabetes by lowering blood sugar levels. For example, it can be TFI-1 to TFI-10, preferably TFI-10, but it is not limited to this.

[0016] The transcription factor-10 (TFI-10) compound is a thiazolidinedione forkhead domain inhibitor, which refers to a substance with the structure of Chemical Formula 1 as the skeleton, namely (5Z)-5-[(3-methoxy-4-{[4-(trifluoromethyl)phenoxy]metal}phenyl)methylidene]-1,3-thiazolidine-2,4-dione ((5Z)-5-[(3-methoxy-4-{[4-(trifluoromethyl)phenoxy]methyl}phenyl)methylidene]-1,3-thiazolidine-2,4-dione).

[0017]

Chemical formula 1

[0018]

[0019] The diarylheptanoid is a general term for secondary metabolites of plants that are composed of two aromatic rings (aryl) connected by a 7-carbon chain (heptane) and have various substituents, and it is known that it has antioxidant activity.

[0020] The RCM-1 is a FOXM1 inhibitor known to block nuclear localization and increase proteasome degradation of FOXM1, and refers to a substance having a structure of Chemical Formula 2.

[0021]

Chemical formula 2

[0022]

[0023] Thiostrepton is a natural cyclic oligopeptide antibiotic derived from the thiopeptide series of various Streptomyces strains such as Streptomyces azureus and Streptomyces laurentii, and refers to a substance having a structure of Chemical Formula 3.

[0024]

Chemical formula 3

[0025]

[0026] Honokiol is a lignan isolated from the bark, seeds and leaves of trees belonging to the genus Magnolia. It is known that honokiol has high bioavailability because it easily crosses the blood-brain barrier and the blood-cerebrospinal fluid barrier. It refers to a substance having a structure of Chemical Formula 4.

[0027]

Chemical formula 4

[0028]

[0029] The FDI-6 is a FOXM1 inhibitor that induces transcriptional downregulation by directly binding to FOXM1, and refers to a substance having a structure of Chemical Formula 5.

[0030]

Chemical formula 5

[0031]

[0032] Siomycin A is a thiazole antibiotic that has been reported to downregulate the transcriptional activity of FOXM1 and even mRNA and protein expression, and refers to a substance having a structure of Chemical Formula 6.

[0033]

Chemical formula 6

[0034]

[0035] The monensin, also known as monensin, monensin A, cinnamycin, rumenin or rumensin, is a polyether antibiotic isolated from Streptomyces cinnamonensis and refers to a substance having a structure of Chemical Formula 7.

[0036]

Chemical formula 7

[0037]

[0038] The FOXM1 aptamer (FOXM1apt) is a general term for FOXM1-specific aptamers that target the FOXM1 DNA binding domain, and can be composed of, for example, the sequence of SEQ ID NO: 9, but is not limited thereto.

[0039] The peptide 9R-P201 is a peptide reported to inhibit the survival, proliferation and migration of cancer cells and induce apoptosis by downregulating the expression of FOXM1, and refers to a substance in which nine arginines are conjugated to P201 (peptide) (JianCui et al. Int J Pept Res Ther 20, 447-456 (2014)).

[0040] As an example, the FOXM1 inhibitor of the present invention can reduce the gene or protein level of FOXM1 or reduce the translocation of FOXM1 to the nucleus.

[0041] In the present invention, the FOXM1 inhibitor can be obtained from biological sources known in the art, synthesized by chemical means, or used as a commercial product.

[0042] In the present invention, FOXM1 inhibitors include any pharmaceutically or edible forms within the scope of having the same efficacy. The salts as described above as pharmaceutically or edible salts may be alkaline salts or acidic salts, and the alkaline salts may be used in the form of organic alkaline salts or inorganic alkaline salts, and may be selected from the group consisting of sodium salts, potassium salts, calcium salts, yttrium salts, magnesium salts, cesium salts, amine salts, ammonium salts, triethylamine salts, and pyridinium salts.

[0043] As the type of salt as mentioned above, acid salts are preferably acid addition salts formed by free acid. As free acid, inorganic acid and organic acid can be used, wherein as inorganic acid, hydrochloric acid, hydrobromic acid, sulfuric acid, sulfurous acid, phosphoric acid, diphosphoric acid and nitric acid can be used, and as organic acid, citric acid, acetic acid, maleic acid, malic acid, fumaric acid, gluconic acid, methanesulfonic acid, benzenesulfonic acid, camphorsulfonic acid, oxalic acid, malonic acid, glutaric acid, acetic acid, gluconic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, dandolinic acid, glutamic acid, citric acid, aspartic acid and stearic acid can be used, but it is not limited to this, and all salts formed by various inorganic acids and organic acids commonly used in the industry can be included.

[0044] In the present invention, "immune checkpoint inhibitor" as a form of cancer immunotherapy refers to a drug that can block proteins called immune checkpoints expressed in some immune cells such as T cells and cancer cells. Specifically, it can be PD-L1 inhibitors, anti-PD-L1 antibodies, PD-1 inhibitors, anti-PD-1 antibodies, CTLA inhibitors, anti-CTLA4 antibodies, anti-PD-L2 antibodies, LTF2 regulatory antibodies, anti-LAG3 antibodies, anti-A2aR antibodies, anti-TIGIT antibodies, anti-TIM-3 antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies, anti-VISTA antibodies, anti-CD47 antibodies, anti-BTLA antibodies, anti-KIR antibodies, anti-IDO antibodies or anti-4-1BB antibodies, but is not limited to these.

[0045] In the present invention, "active ingredient" refers to an ingredient that can exhibit the target activity alone or can exhibit activity together with a carrier that has no activity by itself.

[0046] In the present invention, "cancer (or tumor)" does not distinguish between primary cancer and metastatic cancer, and may be solid cancer or blood cancer. Specifically, the cancer may be lung cancer, pancreatic cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, gastric cancer, duodenal cancer, small intestine cancer, large intestine cancer, colon cancer, rectal cancer, breast cancer, ovarian cancer, prostate cancer, liver cancer, thyroid cancer, kidney cancer, uterine cancer, brain tumor, skin cancer, melanoma, malignant bone tumor, bladder cancer or blood cancer, but is not limited thereto. More specifically, the cancer may be lung cancer or colorectal cancer.

[0047] In the present invention, the term "prevention" refers to all actions that inhibit or delay the occurrence of cancer through the administration of the composition of the present invention, "improvement" refers to the effect of delaying cancer progression or alleviating cancer symptoms by applying the composition of the present invention, and "treatment" refers to all actions that completely inhibit cancer progression or improve symptoms, etc., through the administration of the composition of the present invention. Changes in a favorable direction.

[0048] The composition of the present invention, as an example, can reduce or inhibit the expression of PD-L1, reduce the proliferation and survival of cancer cells and induce an increase in cell apoptosis, and thus has the efficacy of preventing, improving or treating cancer.

[0049] The reduction in PD-L1 expression may refer to a reduction in the PD-L1 expression level in the cell membrane.

[0050] As one form for achieving the above-mentioned purpose, when the FOXM1 inhibitor of the present invention is a hydrophobic compound with low solubility in water, it can be provided in the form of being loaded on liposomes as a system for effectively delivering the hydrophobic compound to the tumor. Specifically, it can be loaded on the surface, interior or phospholipid bilayer of the liposome, but is not limited to this.

[0051] The liposome refers to a double-layer membrane structure composed of phospholipids, preferably, it can refer to a micelle structure.

[0052] The phospholipids constituting the liposomes may be at least one selected from the group consisting of 1,2-dioleoyl-sn-glycero-2-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methyl(polyethylene glycol)-5000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methyl(polyethylene glycol)-5000] ammonium salt, dioleoylphosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, dipalmitoylphosphatidylcholine and cholesterol hemisuccinate, but are not limited thereto.

[0053] The content ratio of the phospholipids constituting the liposome is not particularly limited, and the ratio can be freely set according to the size of the liposome to be produced.

[0054] As one embodiment for achieving the above purpose, the liposome of the present invention may be composed of DOPC, cholesterol and DSPE-PEG2000. Specifically, the molar ratio of each component may be 5 to 15, 3 to 13 and 1 to 12, but is not limited thereto.

[0055] The pharmaceutical composition of the present invention can be administered in combination with a FOXM1 inhibitor and an immune checkpoint inhibitor. Specifically, the pharmaceutical composition can be in the form of a mixed preparation of the FOXM1 inhibitor and the immune checkpoint inhibitor, or can be administered simultaneously or sequentially after the FOXM1 inhibitor and the immune checkpoint inhibitor are formulated separately, but is not limited thereto.

[0056] When a FOXM1 inhibitor and an immune checkpoint inhibitor are administered in combination, the administration concentration ratio of the FOXM1 inhibitor and the immune checkpoint inhibitor can be 1:0.01 to 1:1000, specifically, 1:0.01 to 1:500, 1:0.05 to 1:300, 1:0.1 to 1:200 or 1:1 to 1:100, more specifically, 1:0.01, 1:0.02, 1:0.03, 1:0.05, 1:0.08, 1:0.1, 1:0.3, 1:0.5, 1:0.7, 1:0.8, 1:1, 1:1.5, 1:2, 1:3, 1:5, 1:7, 1:8, 1:10, 1:25, 1:50, 1:100, 1:200 or 1:500, but is not limited thereto.

[0057] When the FOXM1 inhibitor loaded on the liposome and the immune checkpoint inhibitor are co-administered, the administration concentration ratio of the FOXM1 inhibitor loaded on the liposome and the immune checkpoint inhibitor can be 1:0.01 to 1:1000, specifically, 1:0.01 to 1:500, 1:0.05 to 1:300, 1:0.1 to 1:200 or 1:1 to 1:100, more specifically Generally speaking, it can be 1:0.01, 1:0.02, 1:0.03, 1:0.05, 1:0.08, 1:0.1, 1:0.3, 1:0.5, 1:0.7, 1:0.8, 1:1, 1:1.5, 1:2, 1:3, 1:5, 1:7, 1:8, 1:10, 1:25, 1:50, 1:100, 1:200 or 1:500, but it is not limited to these.

[0058] The pharmaceutical composition of the present invention can also include pharmaceutically acceptable carriers, excipients or diluents according to the general method. Pharmaceutically acceptable carriers belong to the known content of the industry according to their route of administration or dosage form. Specifically, the pharmacopoeias of various countries including the Korean Pharmacopoeia can be referred to. As carriers, excipients and diluents that can be included in the composition of the present invention, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talcum powder, magnesium stearate and mineral oil can be used as examples, but are not limited to this. In addition, carriers, excipients and diluents that can be included in the composition of the present invention can be non-natural carriers, but are not limited to this.

[0059] The pharmaceutical composition of the present invention can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols and external preparations, suppositories or sterile injection solutions according to general methods. Specifically, when preparing, commonly used diluents or excipients such as fillers, extenders, adhesives, wetting agents, disintegrants and surfactants can be used to manufacture. Solid preparations for oral administration include tablets, pills, powders, granules and capsules, and solid preparations as described above can be manufactured by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose and gelatin in the compound. In addition, lubricants such as magnesium stearate and talcum powder can also be used. Liquid preparations for oral administration include suspensions, oral liquids, emulsions and syrups, etc., and in addition to commonly used simple diluents, i.e. water and liquid paraffin, can also include multiple excipients such as wetting agents, sweeteners, spices and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate can be used. As the base of the suppository, Witepsol, polyethylene glycol, Tween 61, cocoa butter, lanolin, and glycerin gelatin can be used. The specific formulation method of the pharmaceutical composition belongs to the content known in the industry, for example, reference can be made to [Remington's Pharmaceutical Sciences (19th ed. 1995)] and the like. The said document is regarded as a part of this specification.

[0060] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The pharmaceutically effective amount refers to an amount that is sufficient to treat the disease without causing side effects according to a reasonable benefit / risk ratio applicable to medical treatment. The effective dose level can be determined based on factors including the patient's health status, the type and severity of the disease, the activity of the drug, sensitivity to the drug, the method of administration, the time of administration, the route of administration and the excretion rate, the treatment period, the drugs used in combination or simultaneously, and other factors known in the medical field. The dosage and frequency of administration are not intended to limit the scope of the present invention in all aspects.

[0061] The pharmaceutical composition of the present invention can be administered to mammals such as rats, dogs, cats, cows, horses, pigs and humans by various routes.

[0062] The pharmaceutical composition of the present invention can be used in any dosage form containing a FOXM1 inhibitor and an immune checkpoint inhibitor as active ingredients, and can be manufactured into an oral or semi-oral dosage form. Specifically, the mode of administration is not limited, including oral, rectal, nasal, topical (including cheek and sublingual), subcutaneous, vaginal, or suitable for parenteral (including intramuscular, subcutaneous and intravenous) administration or suitable for inhalation (inhalation) or insufflation (insufflation) administration.

[0063] As one aspect for achieving the above object, the present invention provides an anticancer adjuvant comprising a FOXM1 inhibitor and an immune checkpoint inhibitor as active ingredients.

[0064] The “FOXM1 inhibitor,” “immune checkpoint inhibitor,” “active ingredient” and “cancer” are as described above.

[0065] The anticancer adjuvant of the present invention refers to all forms used to enhance the anticancer effect of an anticancer agent or to inhibit or improve the side effects of an anticancer agent. The anticancer adjuvant of the present invention can be co-administered with various types of anticancer agents or anticancer adjuvants, and when co-administered, even if it is administered at a lower level than the general anticancer agent dosage, it can also show the same level of anticancer treatment effect, so that anticancer treatment can be performed more safely.

[0066] As the administration route of the anticancer adjuvant, it can be administered by any general route that can reach the target tissue. The anticancer adjuvant of the present invention can be administered by oral, rectal, nasal, topical (including cheek and sublingual), subcutaneous, vaginal or parenteral (including intramuscular, subcutaneous and intravenous) or inhalation or insufflation according to its purpose, but it is not limited to this. In addition, the anticancer adjuvant can be administered by any device that can move the active substance to the target cells.

[0067] Preferably, the anticancer adjuvant of the present invention may contain one or more carriers that are pharmaceutically acceptable in addition to the active ingredient for administration and be formulated into an anticancer adjuvant. Carriers, excipients or diluents that may be included in the anticancer treatment adjuvant of the present invention include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, but are not limited thereto.

[0068] As one aspect for achieving the above object, the present invention provides use of a composition comprising a FOXM1 inhibitor and an immune checkpoint inhibitor for preventing or treating cancer.

[0069] The “cancer”, “prevention”, “treatment”, “FOXM1 inhibitor”, “immune checkpoint inhibitor” and “composition” are as described above.

[0070] As one aspect for achieving the above object, the present invention provides use of a composition comprising a FOXM1 inhibitor and an immune checkpoint inhibitor for producing a drug for cancer prevention or treatment.

[0071] The “cancer”, “prevention”, “treatment”, “FOXM1 inhibitor”, “immune checkpoint inhibitor” and “composition” are as described above.

[0072] As one aspect for achieving the above object, the present invention provides a method for preventing or treating cancer, comprising the step of administering a composition comprising a FOXM1 inhibitor and an immune checkpoint inhibitor to a subject.

[0073] The “cancer”, “prevention”, “treatment”, “FOXM1 inhibitor”, “immune checkpoint inhibitor” and “composition” are as described above.

[0074] The pharmaceutical composition of the present invention comprising a FOXM1 inhibitor and an immune checkpoint inhibitor can more effectively inhibit tumor growth and induce cell apoptosis compared to a single FOXM1 inhibitor component or an immune checkpoint inhibitor component, thereby exhibiting the effect of preventing, improving or treating cancer, and can therefore be effectively used as a cancer prevention or treatment agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] FIG1 shows the results of confirming the downregulation of PD-L1 in FOXM1-knockdown lung adenocarcinoma (LUAD) cell lines. Figure 1aThe results are graphically presented as overall survival curves of lung adenocarcinoma patients with (red line) or without (blue line) simultaneous alterations in the expression of FOXM1 and PD-L1. Figure 1b The results are from immunohistochemical analysis of the expression levels of FOXM1 and PD-L1 in human lung cancer tissues and normal lung tissues. Figure 1c and 1d are the results of RNA-seq and Gene Ontology functional activation analysis performed on H1299 and PC9 cells transfected with siFOXM1. Figure 1e , 1f And 1g are the results of confirming the expression levels of FOXM1 and CD274 (PD-L1) when FOXM1 was knocked down, overexpressed, or treated with thiostrepton (TST) in H1299 and PC9 cells by semi-quantitative PCR.

[0076] Figure 2 is the result of confirming the correlation between thiostrepton (TST)-mediated downregulation of FOXM1 and decreased cell proliferation, decreased survival, and increased cell apoptosis. This is the result of confirming the apoptotic effect of reducing FOXM1 by siFOXM1 transfection or thiostrepton treatment ( Figure 2a and 2b), FACS analysis of DNA content during PI staining ( Figure 2c and 2d), and FACS analysis of apoptotic cells after staining with Annexin V and PI ( Figure 2e and the results of 2f). Figure 2g and 2h are the results of confirming the protein expression levels of FOXM1, c-MYC, cyclin B1, cyclin E1, and cyclin D1 in cells knocked down by siFOXM1 or treated with thiostrepton.

[0077] FIG3 is the result of confirming the correlation between downregulation of FOXM1 by thiostrepton (TST) and reduced expression of PD-L1 on the cell membrane. H1299 cells ( Figures 3a to 3c ) and PC9 cells ( Figure 3b And 3d) fluorescence imaging results. Figure 3e And 3f is the result of quantitative analysis of PD-L1 fluorescence signal in the fluorescence image.

[0078] FIG4 is the result of confirming whether thiostrepton (TST) treatment can inhibit the translocation of FOXM1 to the nucleus. Figure 4a and 4b) and fluorescence imaging ( Figures 4c to 4f) The results of evaluating the changes in FOXM1 protein levels in the cytoplasmic and nuclear fractions of H1299 cells and PC9 cells treated with FOXM1 knockdown, overexpression, or TST.

[0079] Figure 5 was analyzed by immunoblotting ( Figure 5a and 5b) or fluorescence imaging ( Figures 5c to 5f ) The results confirmed whether the reduced expression of PD-L1 can be restored by overexpression of FOXM1.

[0080] FIG. 6 shows the results of confirming whether FOXM1 can bind to the promoter of the gene encoding PD-L1 and mediate the expression of PD-L1.

[0081] FIG. 7 shows the results of confirming whether thiostrepton (TST) can inhibit tumor growth and PD-L1 expression in vivo. Figures 7a to 7c The results are the results of confirming the timeline of tumor formation and thiostrepton administration performed in BALB / c mice and the corresponding tumor volume, body weight and expression level. Figure 7d The results are from the analysis of blood biochemical data of control mice and thiostrepton-treated mice. Figure 7e The results are obtained by performing hematoxylin & eosin (H&E) staining and TUNEL staining on tissue sections of the liver, kidney, and spleen of control mice and thiostrepton-treated mice, and analyzing the apoptotic ratio in the normal tissues as described above.

[0082] FIG. 8 shows the results of confirming the anti-tumor effect of thiostrepton (TST) and anti-4-1BB antibody combined administration in a syngeneic Lewis lung cancer (LLC-1) animal model. Figures 8a to 8c The results show that the timeline of tumor formation and drug administration performed in C57BL / 6 mice, as well as the corresponding tumor volume and body weight were confirmed. Figure 8d And 8e is Figure 8a TUNEL (Terminal deoxynucleotidyltransferase dUTP nick end labeling) analysis was performed on mice in the presence of 5% paraformaldehyde and 1% paraformaldehyde, and CD3 + The results of confirming T cell levels.

[0083] FIG. 9 shows the results of confirming the synergistic antitumor effect of thiostrepton (TST) and an immune checkpoint inhibitor in a syngeneic Lewis lung cancer (LLC-1) animal model using C57BL / 6 mice. Figure 9a It is about tumor formation in animal models and the timeline of drug administration. Figure 9b It is the result of confirming the anti-tumor synergistic effect when the combined administration treatment is compared with the case when thiostrepton (TST) and anti-4-1BB antibody are treated alone. Figure 9c is the result of confirming the anti-tumor synergistic effect when the combined administration treatment is compared with the case when thiostrepton (TST) and anti-PD-1 antibody are treated alone.

[0084] Fig.10 The results are from immunoblotting analysis to confirm the expression of FOXM1 and PD-L1 in human colorectal cancer cell lines treated with thiostrepton (TST).

[0085] Fig.11 The results confirmed the effects of suppressing PD-L1 expression and increasing cancer cell apoptosis when MC38 colorectal cancer cells were treated with thiostrepton (TST). Fig.11 a is the result of confirming the concentration-dependent reduction of FOXM1 and PD-L1 expression after thiostrepton (TST) treatment by immunoblotting analysis. Fig.11 b is the result of confirming the concentration-dependent decrease in PD-L1 expression on the surface of cancer cells after thiostrepton (TST) treatment by fluorescence imaging analysis. Fig.11 c is data showing the cell survival rate according to the thiostrepton (TST) treatment concentration in MC38 cancer cells. Fig.11 d is data showing graphically the cell survival rate according to the thiostrepton (TST) treatment concentration in normal cells, namely, primary human coronary artery smooth muscle cells (SMC). Fig.11 e is the result of analyzing the apoptosis rate of MC38 cells in the control group and the thiostrepton (TST)-treated group by flow cytometry.

[0086] Fig.12 This is the result of confirming the characteristics of liposomes (ThioLipo) loaded with thiostrepton. Fig.12 a The chemical structure of thiostrepton (TST) is illustrated. Fig.12 b and 12c are the size distribution measurement results of ThioLipos in aqueous solution and transmission electron microscopy (TEM) photos. Fig.12d is the result of evaluating the stability of ThioLipos by measuring the change in size distribution of ThioLipos based on time.

[0087] Fig.13 The results are from an analysis of the time-dependent changes in blood drug concentrations after intravenous administration of free thiostrepton (TST) and ThioLipo at the same dose.

[0088] FIG. 14 shows the results of confirming the anti-tumor synergistic effect of combined administration of ThioLipos and immune checkpoint inhibitors in a syngeneic colorectal cancer model. Fig.14a A graphical representation of the timeline of immune checkpoint inhibitors and ThioLipo administration in mice. Fig.14b Is it a good idea to use ThioLipo and anti-4-1BB in Fig.14a The results are graphically presented as tumor growth curves in mice administered with either drug alone or in combination, or with ThioLipo and anti-PD-1 antibodies administered alone or in combination. Fig.14c Yes Fig.14b The results are graphically presented as tumor growth curves of individual mice in each treatment group.

[0089] Fig.15 The results are obtained by quantitatively analyzing the expression of TUNEL, CD3, and PD-L1 by immunohistochemical staining of tumor sections of mice treated with ThioLipo and immune checkpoint inhibitors alone or in combination.

[0090] Figure 16 is the result of confirming that no side effects occur when ThioLipo and immune checkpoint inhibitors are administered alone or in combination. Fig.16a The results of TUNEL staining of sections of major organs (heart, kidney, liver, lung, and spleen) showed that no histopathological changes or increased apoptosis of normal cells were observed. Fig.16b The results of analyzing the blood biochemical data of mice showed that no side effects were observed.

[0091] Fig.17 The results confirmed whether FOXM1 and PD-L1 in lung adenocarcinoma (LUAD) and colorectal cancer cell lines can be downregulated by FDI-6. Fig.17 a is the result of observing whether the reduction in the expression of FOXM1 and PD-L1 is proportional to the concentration of FDI-6 treated in lung cancer cell lines by Western blotting. Fig.17 b is the result of observing whether the reduction in the expression of FOXM1 and PD-L1 is proportional to the concentration of FDI-6 treated with the colorectal cancer cell line by Western blotting.

[0092] Fig.18 The results confirmed whether FOXM1 and PD-L1 in lung cancer cells, colorectal cancer cells, and brain tumor cells could be downregulated by RCM1 treatment. Fig.18 a is the result of confirming whether the expression inhibition of FOXM1 and PD-L in lung cancer, colorectal cancer, and brain tumor cell lines is proportional to the RCM1 treatment concentration by Western blotting. Fig.18 b is the result of confirming, by fluorescent imaging, that the expression of PD-L1 on the surface of cancer cells was reduced when human lung cancer cell lines were treated with RCM1. DETAILED DESCRIPTION

[0093] Next, the present invention will be described in more detail by the following examples. However, these examples are only exemplary descriptions of the present invention, and the scope of the present invention is not limited by these examples.

[0094] Example 1: Confirmation of the correlation between FOXM1 and PD-L1 expression

[0095] 1.1. Analysis of FOXM1 and PD-L1 information in lung adenocarcinoma (LUAD)

[0096] (1) Dataset for analysis of FOXM1 and PD-L1 information in lung adenocarcinoma (LUAD)

[0097] To determine the role of FOXM1 and PD-L1 in LUAD prognosis, the TGCA (The Cancer Genome Atlas) LUAD Firehose Legacy (n=586), an open access resource for cancer genomes available in the cBioPortal, was used.

[0098] (2) Geneset Enrichment Analysis (GEA)

[0099] DEGs were listed in response to siFOXM1 knockdown, and significantly downregulated genes (less than 0.5-fold between NCsiRNA and siFOXM1 knockdown H1299 and PC9 cells) were fit for GSEA using the Enrichr database.

[0100] 1.2. Cell culture and transfection

[0101] H1299 and PC9 cells purchased from ATCC (Manassas, VA, USA) were cultured at 37°C and 5% CO2 using RPMI-1640 medium (#10-040-CV; Corning, New York, USA) supplemented with 10% human serum albumin (#35-010-CV; Corning) and a penicillin-streptomycin antibiotic cocktail (#15240062; ThermoFihser Scientific, Waltham, MA, USA).

[0102] Negative control (NC) or FOXM1 siRNA (siFOXM1) (QIAGEN, Hilden, Germany) at 5 nM was transfected into H1299 and PC9LUAD cells using Lipofectamine RNAiMAX (#13778150; Thermo Fisher Scientific). Human FOXM1 isoform b (pFLAG-FOXM1) expression plasmid or empty vector (pFLAG-CMV; mock) was transfected into H1299 and PC9 cells using Lipofectamine 2000 reagent (#11668019; Thermo Fisher Scientific) according to the manufacturer's guidelines.

[0103] 1.3. Reverse transcription and semi-quantitative PCR

[0104] Total RNA was extracted and purified using the RNeasy kit (#74004; QIAGEN) according to the manufacturer's operating procedures. mRNA reverse transcription was performed using the PrimeScript 1st Strand cDNA Synthesis Kit (#6110A; TaKaRa Bio, Otsu, Japan). Quantitative PCR was performed using primers specific to the CD274 and FOXM1 genes designed using Primer3 software. The β-actin gene was used as an internal control group for normalizing the expression of the target gene. The primer pair sequences used in the present invention are shown in Table 1.

[0105]

Table 1

[0106]

[0107] 1.4. Confirmation of the correlation between FOXM1 and PD-L1 expression in lung cancer cells

[0108] In order to investigate the clinical relevance of FOXM1 and PD-L1 in lung cancer patients, the survival curves of lung adenocarcinoma (LUAD) were analyzed in the TCGA (The Cancer Genome Atlas) dataset of cBioPortal. Compared with 443 patients with no change in FOXM1 and PD-L1 expression, the median overall survival of 70 patients with high expression of FOXM1 and PD-L1 was relatively lower (31.21 months vs. 50.20 months, p = 8.716 × 10 -4 , Figure 1a ).

[0109] In addition, to confirm whether FOXM1, as a potential transcription factor (TF) regulating the expression of PD-L1, is associated with the poor prognosis of LUAD patients, lung cancer tissues and corresponding normal tissues were immunostained with antibodies against FOXM1 and PD-L1, and the expression of FOXM1 and PD-L1 was compared. As a result, FOXM1 and PD-L1 were upregulated in LUAD tissues compared with adjacent normal tissues ( Figure 1b ).

[0110] RNA-seq analysis of H1299 and PC9 cells in which FOXM1 was knocked down by siRNA revealed that among the 67 differentially expressed genes (DEGs) expressed less than 0.5 times in the two cell lines, the CD274 gene encoding PD-L1 was speculated to be a target of FOXM1 ( Figure 1c ). In order to confirm the biological functions of the genes downregulated after knockdown of FOXM1 in H1299 and PC9 cells, gene set enrichment analysis (GSEA) was performed on the downregulated genes (67 in total) using the Enrichr database. The target genes were significantly activated by positive transcriptional regulation of RNA polymerase II and cell cycle regulation ( Figure 1d ), indicating that FOXM1 can also contribute to CD274 regulation. The encoded PD-L1 protein plays an important role in the immune escape of lung cancer cells. After knocking down FOXM1 using siRNA, the expression of PD-L1 was confirmed, and the PD-L1 transcription level was significantly reduced in both cell lines ( Figure 1e ).

[0111] In addition, the results of evaluating the transcriptional level of PD-L1 after overexpression of FOXM1 using pFLAG-FOXM1 isoform b, the major isoform expressed under physiological conditions, showed that overexpression of FOXM1 significantly increased PD-L1 mRNA expression in H1299 and PC9 cells ( Figure 1f ).

[0112] The potential effect of FOXM1 on PD-L1 expression in NSCLC cells was evaluated using FOXM1 inhibition strategies other than siRNA knockdown. Various FOXM1 inhibitors including the FOXM1 inhibitory natural substance TST were tested in H1299 and PC9 cells, with TST being the most effective inhibitor that significantly reduced FOXM1 levels.

[0113] To induce an increase in PD-L1 expression, we analyzed the PD-L1 transcription level in cells treated with TST in the presence of IFN-γ. The results showed that TST-induced reduction of FOXM1 induced a concentration-dependent decrease in PD-L1 mRNA expression in both cell lines ( Figure 1g ).

[0114] Example 2: Confirmation of the effect of FOXM1 inhibition on cells

[0115] 2.1. Cell analysis methods

[0116] (1) Analysis of cell proliferation

[0117] H1299 and PC9 cells were propagated in 96-well plates and treated with 5 μM TST or transfected with siFOXM1 at a density of 3,000 cells / well in 6-well plates. For cell proliferation, 4-[3-(4-iodophenyl)-2-(4-nitro-phenyl)-2H-5-tetrazolinyl]-1,3-benzenesulfonate (WST-1, #MK400, TaKaRaBio) was added for 2 hours at 37°C and recorded at the specified time, and the absorbance was measured at 450nm. All experiments were repeated 4 times.

[0118] (2) Analysis of cell cycle distribution

[0119] H1299 and PC9 cells were enzymatically dissociated using TrypLE (#12605-010; Thermo Fisher Scientific) and then centrifuged at 2000×g for 2 minutes and collected, then resuspended in PBS and fixed with 70% ethanol and stored at 4°C overnight. The fixed cells were pelleted and washed twice with PBS, then resuspended in FxCyclePI (propidium iodide) / RNase staining solution (#F10797; Thermo Fisher Scientific) and cultured at 37°C in a dark room for 30 minutes. Flow cytometric analysis was performed using a FACS-LSR Fortessa flow cytometer (BD Biosciences, Santa Clara, CA, USA).

[0120] (3) Analysis of cell apoptosis (Annexin V / PI staining)

[0121] H1299 and PC9 cells (1×10 5 Cells were cultured in 6-well plates and transfected with siFOXM1 or NCsiRNA, or treated with 5 μM TST or dimethyl sulfoxide (DMSO) and stained using fluoresceinisothiocyanate (FITC) Annexin V Apoptosis Detection Kit I (#556547; BD Biosciences). To reduce the side effects caused by staining, cells were analyzed within 1 hour and flow cytometric analysis was performed using a FACSVerse cell analyzer (BD Biosciences).

[0122] 2.2. Cell growth inhibition and apoptosis induction based on FOXM1 inhibition

[0123] Time-course experiments were performed over 24, 48, and 72 hours to confirm the effects of FOXM1 on cell proliferation and survival in NSCLC. In response to siRNA-mediated knockdown and treatment with 5 μM TST, a time-dependent decrease in cell proliferation was observed in both cell lines ( Figure 2a as well as Figure 2b). In addition, the results of analyzing the protein content of cells treated with siFOXM1 or TST using flow cytometry showed that FOXM1 knockdown by siFOXM1 or inhibition by TST increased the lower G0 / G1 cell population and reduced the S phase cell population in a concentration-dependent manner compared with the negative control (NC) siRNA or dimethyl sulfoxide (DMSO) treatment groups ( Figure 2c as well as Figure 2d ). Compared with the DMSO-treated group, the two cell lines treated with TST showed an increase in the G2 cell population due to a decrease in the S phase cell population. This confirmed that the reduction of FOXM1 changes the cell cycle process in NSCLC cells and plays an important role in the G1-S phase and G2-M phase transitions. In addition, it was confirmed that the apoptotic cell population (Annexin V / PI-positive cells) increased significantly in a time-dependent manner in cells with reduced FOXM1 by siRNA or TST treatment ( Figure 2e and 2f).

[0124] Western blot analysis was used to further confirm the effect of reduced FOXM1 expression on c-MYC, cyclin B1, cyclin E1, and cyclin D1 in both cell lines. The protein levels of c-MYC, cyclin B1, and cyclin D1 were significantly reduced after FOXM1 expression was reduced by siRNA or TST treatment ( Figure 2g and 2h).

[0125] 2.3. Downregulation of PD-L1 on lung cancer cell membranes based on FOXM1 reduction

[0126] PD-L1 is a transmembrane protein, and to evaluate whether the effect of FOXM1 knockdown by siRNA or TST treatment to reduce total PD-L1 protein levels can be observed on the cell membrane phenotype, immunocytochemistry (ICC) analysis was performed. In addition, the results of membrane PD-L1 enrichment were evaluated by ICC analysis of H1299 and PC9 cells transfected with siFOXM1 or NCsiRNA for 72 hours, and PD-L1 membrane expression in H1299 control cells was significantly higher than that in siFOXM1 transfected cells ( Figure 3a ). FOXM1 downregulation reduced PD-L1 expression by 67.4% ( Figure 3e ), while PC9 cells transfected with siFOXM1 reduced PD-L1 expression by 81.68% through downregulation of FOXM1 ( Figure 3b and 3f).

[0127] Considering that total PD-L1 protein levels in lung cancer cells can be reduced by TST treatment in the presence of IFNγ, the effect of TST on cell membrane PD-L1 enrichment was investigated. IFNγ-enhanced (IFNγ(+)) membrane PD-L1 levels were observed in both cell lines. Figure 3c and 3d). PD-L1 was reduced on the cell membrane of H1299 cells and PC9 cells treated with TST ( Figure 3c and 3d), while FOXM1 downregulation reduced PD-L1 levels by 87.23% and 85.82% in H1299 and PC9 cells, respectively, compared with the dimethyl sulfoxide (DMSO) control group ( Figure 3e and 3f).

[0128] Example 3: Confirmation of the FOXM1 inhibitory effect based on TST

[0129] Protein analysis

[0130] (1) Nuclear and cytoplasmic fractionation

[0131] To confirm the localization of the protein in the cell, H1299 and PC9 cell pellets were lysed for 10 minutes using buffer A (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol (DTT) and 0.05% IGEPAL CA-630 (pH 7.9)). The buffer A lysate was centrifuged at 1,000 x g for 10 minutes at 4°C to collect the nuclear fraction. After removing the cytoplasmic fraction, buffer B containing 5 mM HEPES, 0.2 mM EDTA, 1.5 mM MgCl2, 0.5 mM DTT and 26% glycerol (v / v) (pH 7.9) was added to the nuclear fraction and lysed on ice for 30 minutes.

[0132] (2) Immunoblotting analysis of protein expression

[0133] Cells were lysed in RIPA buffer supplemented with complete mini protease inhibitor cocktail (#04693124001; Roche, Basel, Switzerland) and kept on ice for 30 minutes. Equal amounts of protein were evenly absorbed and analyzed by electrophoresis (SDS-PAGE, sulfate-polyacrylamide gel electrophoresis), followed by transfer to (PVDF, polyvinylidene difluoride) membrane (#IPVH00010; Millipore, Billerica, MA, USA) at 100 V for 1 hour. The target proteins were investigated using primary antibodies specific for FOXM1 (#sc-271746), cyclin E1 (#sc-481), α-tubulin (#sc-8035), and β-actin (#sc-47778) (Santa Cruz Biotechnology, Texas, USA); PD-L1 (#13684S), cyclin D1 (#2978S), cyclin B1 (#4138S), c-MYC (#5605S), and poly (ADP-ribose) polymerase (PARP, #9542S; Cell Signaling Technology, Beverly, MA, USA). As horseradish peroxidase-conjugated secondary antibodies specific for the primary antibodies, Pico-enhanced chemiluminescent substrate (#34577; Thermo Fisher Scientific) was used for protein detection.

[0134] (3) Immunocytochemistry (ICC)

[0135] Cells were fixed in 4% paraformaldehyde (#30525-89-4; FUJIFILM Wako Chemical, Wako, Japan) for 15 minutes and blocked with 1% bovine serum albumin (BSA) in PBS-0.01% Tween-20 (PBS-T) to reduce nonspecific antibody binding. Next, cells were incubated overnight with anti-PD-L1 antibody (#14598382; eBioscience, San Diego, CA, USA) diluted 1:200 and anti-FOXM1 antibody (#sc-271746; Santa Cruz Biotechnology) diluted 1:50, followed by 1 hour of incubation with Alexa Fluor 488-labeled secondary antibody (#A11029; Thermo Fisher Scientific). Cell nuclei were stained with Hoechst 33342. After incubation, cells were washed with PBS and mounted on slides using ProlongGlass Antifade mounting medium (#P36982; Thermo Fisher Scientific). Confocal microscopy was performed using a Zeiss 730 Meta microscope (Carl Zeiss, Oberkochen, Germany) and analyzed using Axiovision software (Carl Zeiss).

[0136] 3.2. Confirmation of the inhibitory effect of TST on FOMX1 translocation to the nucleus

[0137] Nuclear translocation of FOXM1 is a prominent feature in various cancers, so after investigating the enrichment of FOXM1 in the nucleus of H1299 and PC9 cells transfected with pFLAG-FOXM1, siFOXM1 knockdown or TST (5μM) treatment was performed in the presence of INF-γ (20ngmL-1). FOXM1 is present in the nucleus and cytoplasm, but overexpression of FOXM1 increases the translocation of cytoplasmic FOXM1 to the nucleus. siFOXM1-mediated FOXM1 inhibition or TST treatment reduced FOXM1 protein levels and nuclear translocation in H1299 and PC9 cells ( Figure 4a as well as Figure 4b). Compared with mock transfection, overexpression of FOXM1 in H1299 cells restored 48.85% and 84.33% of FOXM1 levels in the case of siFOXM1 and TST treatment, respectively. In PC9 cells, the recovery values ​​were 30.95% and 57.56% in the case of siFOXM1 and TST treatment, respectively. Immunocytochemistry (ICC) analysis showed that FOXM1 was mainly confined to the nucleus of H1299 cells transfected with pFLAG-FOXM1 and PC9 cells transfected with NCsiRNA compared with siFOXM1-transfected cells ( Figure 4c and 4d). Similarly, in pFLAG-FOXM1-transfected H1299 cells and PC9 cells, FOXM1 expression was mainly confined to the nucleus compared with TST-treated cells, which inhibited FOXM1 expression and nuclear translocation. pFLAG-FOXM1-mediated FOXM1 overexpression was significantly reduced to 54.22% in H1299 cells (p<0.01) and 35% in PC9 cells (p<0.001) by siRNA-mediated knockdown. In TST-treated cells, FOXM1 overexpression was significantly reduced to 63.7% in H1299 cells (p<0.05) and 44% in PC9 cells (p<0.01).

[0138] Example 4: Identification of the PD-L1 expression regulation mechanism based on FOXM1

[0139] 4.1. Confirmation of PD-L restoration based on FOXM1 overexpression

[0140] The restoration of PD-L1 expression was confirmed by inducing overexpression of FOXM1 in FOXM1 knockdown cell lines. Specifically, H1299 and PC9 cells were transfected with NCsiRNA or siFOXM1 and then transfected with pFLAG-FOXM1 or empty vector for 72 hours. As a result, it was confirmed by western blot analysis and confocal imaging of cells that the PD-L1 downregulation induced by siFOXM1 was not restored in mock-transfected cells, but was restored in pFLAG-FOXM1-transfected cells. FOXM1 restored PD-L1 expression to 55.9% and 51.8% (on the cell membranes of H1299 and PC9, respectively). Figure 5a , 5cSimilarly, the results of an investigation into whether the reduced PD-L1 expression in H1299 and PC9 cells treated with TST (5 μM) for 48 hours in the presence of INFγ could be restored showed that the reduced PD-L1 expression in FOXM1 knockdown cells was restored by FOXM1 overexpression, and the reduced PD-L1 expression in H1299 and PC9 cells by TST treatment was also restored. Western blotting ( Figure 5b ) and confocal microscopy ( Figure 5e 5f) confirmed that FOXM1 restored PD-L1 expression to 38.7% and 46% on the cell membrane of H1299 and PC9 cells, respectively. In addition, the increase in PD-L1 levels caused by pFLAG-FOXM1 overexpression (mimicking the malignant tumor mass) was significantly reduced to 52.6% (p<0.05) in H1299 cells and 59.2% (p<0.01) in PC9 cells by siRNA treatment, and significantly reduced to 41.9% (p<0.01) in H1299 cells and 68.0% (p<0.05) in PC9 cells by TST treatment. Taken together, these results further support the hypothesis that PD-L1 is a downstream target of FOXM1 and can be positively regulated by FOXM1, which means that PD-L1 levels can be precisely regulated by inhibiting FOXM1.

[0141] 4.2. Confirmation of the FOXM1-based PD-L1 expression regulation mechanism

[0142] (1) Chromatin immunoprecipitation (ChIP)

[0143] To cross-link proteins to DNA, H1299 and PC9 cells were treated with 0.7% formaldehyde at 37°C for 10 minutes. Cross-linking was quenched by adding glycine to the culture medium at a final concentration of 0.125 M for 5 minutes. ~500 bp DNA fragments were obtained by sonicating the cells for 5 minutes with a cycle of 30 seconds on / 30 seconds off using a Bioruptor Next Gen sonicator (Diagenode, Denville, NJ, USA). Unwanted nonspecific components were removed by previously removing the sonicated cell lysate containing genomic DNA fragments. ChIP was performed by rotating overnight at 4°C using an antibody to FOXM1 (#sc-271746, Santa Cruz Biotechnology), followed by various washing steps, reverse cross-linking, and protein degradation using proteinase K (#P2308; Sigma-Aldrich, St., St. Louis, MO, USA). The immunoprecipitated DNA fragments were purified using the phenol-chloroform method, and the purified DNA pellets were resuspended in TE buffer (pH 8.0) and used in PCR. Primers specifically detecting the FOXM1 binding region were designed using the ChIP-PCR Primer3 tool (Table 1).

[0144] (2) Dual luciferase assay

[0145] The pGL3 plasmid containing the 2 kb promoter of CD274 (#107003; Addgene, Watertown, MA, USA) was provided by Julian Downward laboratory (Coelho MA, de Came Trecesson S, Rana S, Zecchin D, Moore C, Molina-Areas M, East P, Spencer-Dene B, Nye E, Barnouin K, Snijders AP, Lai WS, Blackshear PJ, Downward J, Immunity 2017, Vol. 47, p. 1083).

[0146] H1299 and PC9 cells were co-transfected with the pGL3 2kb promoter. Cells containing CD274 or control pGL3-basic plasmids and pGL4.70hRluc Renilla luciferase vector (Promega, Madison, WI, USA) were transfected with siFOXM1 or treated with TST in the presence of interferon-γ (IFNγ). Cells were harvested 48 hours after transfection and dual-luciferase assays were performed according to the manufacturer's protocol (#E1910; Promega), and at least four independent biological replicates were performed to obtain reliable results.

[0147] (3) Regulating PD-L1 expression by directly binding FOXM1 to the promoter In order to clarify the mechanism by which FOXM1 induces PD-L1 expression, ChIP-seq data from the ENCODE3 database obtained from the UCSC genome browser (Human assembly Dec 2013, GRCh38 / hg38) were analyzed. In K562 (a human immortalized myeloid leukemia cell line), FOXM1 enrichment was confirmed in the promoter region with a peak signal approximately 167 bp upstream of the CD274 transcription start site, and the TAAAC FOXM1 common DNA-binding domain (DBD) was detected not only in two distant regions (-1988 bp and -2310 bp) from the CD274 transcription start site, but also in the vicinity (approximately 142 bp) ( Figure 6a ).

[0148] Based on the in silico data described above, the CD274 promoter region of lung cancer cells was postulated as a potential target of the FOXM1 transcription factor, and ChIP-PCR analysis was performed using an anti-FOXM1 antibody, but excluding the FOXM1-CD274 DNA complex. The CD274 promoter region adjacent to the putative FOXM1 binding site (~167 nt) was detected by semi-quantitative PCR in H1299 and PC9 cells, in contrast to the dramatic reduction in FOXM1 binding to the CD274 promoter observed in the respective control and siFOXM1-transfected cells and in cells treated with TST. pFLAG-FOXM1-transfected cells showed increased FOXM1 binding to the CD274 promoter compared to mock-transfected cells, suggesting that FOXM1 can bind directly to the CD274 promoter ( Figure 6b ). Therefore, FOXM1 directly regulates PD-L1 transcription by directly binding to the promoter, and targeting FOXM1 can terminate its mediated regulation of PD-L1 expression.

[0149] In addition, to confirm whether the reduction of FOXM1 binding by siRNA or TST treatment can significantly reduce CD274 transcriptional activity, a dual luciferase reporter assay was performed using a CD274 promoter-luciferase construct. Consistent with the ChIP-PCR results, the reduction of FOXM1 by siRNA significantly reduced the relative luciferase activity to 40% in H1299 cells (p<0.001) and to 42% in PC9 cells (p<0.01) ( Figure 6c Similarly, inhibition of FOXM1 by TST in the presence of INFγ reduced relative luciferase activity to 53% in H1299 cells (p<0.001) and to 66% in PC9 cells (p<0.001), indicating that CD274 promoter activity was also significantly reduced ( Figure 6d ).

[0150] The results described above demonstrate that the translocation of FOXM1 to the nucleus can positively regulate the expression of PD-L1 in both H1299 and PC9 cell lines by directly binding to the CD274 promoter. This proposes a new model for FOXM1-mediated regulation of PD-L1 expression in lung cancer, in which FOXM1 can regulate PD-L1 expression by directly binding to the CD274 promoter, thereby inducing PD-L1 transcriptional activation (Figure 6g). Because FOXM1-induced PD-L1 upregulation can lead to immune escape of lung cancer cells, targeting FOXM1 using TST, a natural FOXM1 inhibitor that can eliminate immune escape and reduce tumor growth, is expected to become a new therapeutic strategy for PD-L1-mediated immune escape in lung cancer cells.

[0151] Example 5: Confirmation of FOXM1 inhibitory effect in vivo

[0152] Animal models

[0153] (1) Xenograft tumor model

[0154] All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of the National Cancer Center Research Institute, Korea (NCC-21-619). TST dissolved in DMSO was injected intraperitoneally into mice (n=6) at a dose of 17 mg / kg every 2 days before euthanasia and sacrifice. Tumor volume and body weight of mice were measured every other day. Tumor volume (V) was calculated according to AB 2 / 2 was calculated (A and B are long diameter and short diameter (mm) respectively). Two days after the last injection of TST, all mice were killed, and tumor tissues were collected for immunohistochemistry (IHC) staining of FOXM1 and PD-L1, and the tissues were fixed in 10% neutral formalin solution and embedded in paraffin blocks. Tissue microarray (TMA) sections were made with a diameter of 4 mm for each sample, and cut into a thickness of 4 μM for IHC using anti-FOXM1 (#20459S) or anti-PD-L1 (#13684S) (Cell Signaling Technology) antibodies. Autostainer Link48 (Dako, Agilent Technologies, Santa Clara, CA, USA) was used for analysis.

[0155] IHC staining was performed using anti-rabbit-horseradish peroxidase (HRP) antibody (K4003, EnVision+HRPLabelled Polymer Anti-Rabbit, Dako, Agilent Technologies, Carpinteria, CA, USA) and 3,3'-diaminobenzidine (DAB) (K3468, DAB+Chromogen, Dako, Agilent Technologies, SantaClara, CA, USA) as a matrix, and sections were control stained with hematoxylin (K8008, EnVision FLEX Hematoxylin, Dako, Agilent Technologies Singapore, Singapore). In order to analyze the toxicity of the test drugs in vivo, serum biochemical analysis was performed using 4 xenograft mice in each group (Table 2). On day 9, serum samples were collected from mice in the control and TST-treated groups. In addition, the vital organs (liver, spleen, and kidney) of the mice were collected and dissected, and H&E staining was performed to monitor the histopathological changes. To analyze apoptosis in response to test drugs, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis (ApopTag Plus Proxidase In Situ kit, EMD Milipore, Temecula, CA, USA) was also performed on the sectioned tissues.

[0156] (2) Syngeneic tumor model using mouse lung cancer cell line LLC-1

[0157] All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of the National Cancer Center, Korea (NCC-22-675B). 5 ) were subcutaneously transplanted into 5-week-old C57BL / 6N mice (n=6 per group) (OrientBio, Seoul, Korea) and left for 10 days to form a volume of approximately 150 mm 3 Tumors. Next, 17mg / kg dose of TST and 0.2mg / dose of anti-4-1BB antibody were injected into mice at the time points indicated by black arrows (day 1, day 3, day 5, and day 7). The tumor volume and body weight of mice were measured every other day. All mice were killed on the 9th day and tumor tissues were collected, then fixed and embedded in paraffin using formalin, and then sliced ​​for IHC. In addition, in order to analyze apoptosis as a reaction to TST and anti-4-1BB antibody treatment, TUNEL analysis was performed on the sliced ​​tissue.

[0158] CD3 in tumor sections + The assessment of T cells was performed as described in a previous paper (Huang Y, Lin C, Kao HK, Hung SY, Ko HJ, Huang YC, Chang YL, Chang KP, Cancer Manag Res 2020, 12, 8275). + T-cells were detected by anti-CD3 antibody (ab16669, Abeam, Cambridge, UK) according to the manufacturer's instructions. + The positive signal intensity of T-cells was analyzed by computer image analysis using ImageJ software (LOCI, University of Wisconsin), while CD3 + Signal intensity was calculated as the relative ratio of total positive area / total surface area. Pathological images were acquired using an Aperio VERSA slide scanning system (Leica Biosystems Imaging, Vista, CA, USA). Three independent and intact computer microscopic fields of each tissue sample were captured at 100X magnification using Aperio Imagescope software (Leica Biosystems Imaging, Vista, CA, USA), and the entire microscopic field was used for CD3 + The signal intensity of the cells was analyzed.

[0159] The in vivo synergistic effect between TST and anti-4-1BB antibody treatment groups was determined by calculating the CDI (drug interaction coefficient), where CDI values ​​of 1, <0.7, =1, and >1 represent synergy, significant synergy, additivity, and antagonism, respectively.

[0160] In all statistical analyses, the χ 2 Differences between groups were assessed using the Student's t-test. Statistical significance was set at p < 0.05, and deviations are presented using standard deviations.

[0161] 5.2. Tumor Growth Inhibition Based on FOXM1 Inhibition and Regulation of PD-LE Expression

[0162] To investigate the effects of TST, a FOXM1 inhibitor, on tumor growth and PD-L1 expression in vivo, human H1299 or PC9 cells (5 × 10 6 When the average size of xenograft tumor mass reached approximately 170 mm 3 At 17 mg / kg, mice were intraperitoneally injected with TST 4 times every two days. Figure 7a ). As a result of regular monitoring of tumor volume during inhibitor treatment, a significant reduction in tumor volume was observed in the TST-treated group compared with the control group. No difference in body weight was observed between the two groups after 9 days ( Figure 7b Two days after the last TST treatment, mice were killed and tumor tissues were collected, followed by sectioning and staining to analyze changes in FOXM1 and PD-L1 expression levels. Immunohistochemical analysis confirmed that FOXM1 and PD-L1 were overexpressed in lung tumors of control mice, but were significantly downregulated in TST-treated tumors ( Figure 7c ). FOXM1 was mainly confined to the nuclei of control tumor cells, but showed weak immunopositivity in the nuclei of TST-treated tumor cells. The above results show that PD-L1 was positive in the control tumors of H1299 and PC9 xenografts along with FOXM1 expression. In contrast, PD-L1 expression was significantly reduced by TST treatment ( Figure 7c ).

[0163] In addition, the potential toxicity of TST in vivo was evaluated by performing serum biochemical analysis. The blood biomarker levels in the TST-treated group did not change significantly compared with the DMSO control group ( Figure 7dIn addition, the results of hematoxylin and eosin (H&E) staining showed that no significant histological changes of important organs were observed in the TST-treated group ( Figure 7e ). TUNEL analysis of tissue sections confirmed that TST treatment did not affect cell apoptosis in normal tissues compared with the DMSO control group tissues ( Figure 7e ). In summary, the results described above indicate that TST-mediated FOXM1 inhibition can suppress immune escape and effectively reduce tumor growth.

[0164]

Table 2

[0165] Blood biomarker differentiation Control group (n=4) TST treatment group (n=4) ALT(U / L) 32.00±1.87 31.25±4.21 AST(U / L) 72.00±9.30 82.75±8.58 ALP(U / L) 115.00±10.42 91.50±15.04 Glu (mg / dL) 381.75±22.86 312.00±58.73 BUN (mg / dL) 20.93±2.87 19.70±1.11 Crea (mg / dL) 0.03±0.04 0.00±0.00 T-Bill (mg / dL) 1.10±0.05 0.10±0.00 B-chol (mg / dL) 104.00±10.70 107.50±3.20 TG (mg / dL) 103.75±9.07 116.00±14.88 TP (mg / dL) 4.93±0.18 4.93±0.08 Alb (mg / dL) 3.25±0.21 3.28±0.08 Glo(mg / dL) 1.68±0.06 1.65±0.09 A / G Ratio 1.95±0.19 1.99±0.13

[0166] Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; Glu, glucose; BUN, blood urea nitrogen; Crea, creatinine; T-ill, total bilirubin; T-chol, total cholesterol; TG, triglyceride; TP, total protein; Alb, serum albumin; Glo, serum globulin; A / G ratio, serum albumin / serum globulin ratio.

[0167] Example 6: Confirmation of the synergistic anti-tumor effect of FOXM1 inhibitors and immune checkpoint inhibitors when administered in combination

[0168] To evaluate the therapeutic potential of TST in cancer immunotherapy, we used the LLC-1 syngeneic tumor model ( Figure 8a Lewis lung cancer (LLC-1) mouse lung cancer cells (5×10 5 ) were injected subcutaneously into C57BL / 6N mice (n=6 per group) to form a volume of 150 mm over 10 days. 3 On days 1, 3, 5, and 7, mice were injected with 17 mg / kg TST and 0.2 mg / mouse anti-4-1BB antibody. When anti-4-1BB antibody or TST was used alone to treat tumor-bearing mice, tumor growth was inhibited by 38% and 48%, respectively, compared with the control group ( Figure 8b In particular, the combination of anti-4-1BB antibody and TST inhibited tumor growth by 65% ​​in tumor-bearing mice compared with the control group, showing a significantly improved therapeutic effect in inhibiting tumor growth (p<0.001). No difference in body weight was observed between the experimental groups after 9 days ( Figure 8c ).

[0169] Next, in order to investigate apoptosis in the tumor, TUNEL staining was performed on the tumor sections on the 9th day. The results showed that the number of apoptotic tumor cells in the anti-4-1BB treatment group (p<0.05) and the TST treatment group (p<0.05) was 1.38 times and 2.46 times higher than that in the control group (Figure 8D). The combined administration of TST and anti-4-1BB antibodies increased the number of apoptotic LLC-1 tumor cells to 4.92 times and 2.76 times (CDI=0.5) compared with the anti-4-1BB antibody treatment group (p<0.001) or the TST treatment group (p<0.001). In addition, when immunohistochemical staining of CD3 was performed in tumor sections, CD3 in tumors treated with anti-4-1BB antibodies (p<0.05) and TST (p<0.05) was significantly higher than that in the control group. + Compared with the control group, the number of T cells increased by 1.28 and 1.32 times ( Figure 8d Compared with the anti-4-1BB antibody-treated group (p<0.001) or the TST-treated group (p<0.001), the combined administration of TST and anti-4-1BB antibody increased the expression of CD3 + The frequency of T-cells (CDI = 0.49) also increased by 2.72 and 2.63 times, respectively ( Figure 8e ). The above results show that the combined administration of TST and anti-4-1BB antibody has a significant synergistic effect on immunogenicity and anti-tumor activity. In particular, the combined administration of TST and anti-4-1BB antibody significantly delayed tumor growth and induced T cells to the tumor area in the immune-resistant LLC1 lung tumor model. In addition, when the tumor size was smaller (i.e., ~80mm 3 ), the anti-4-1BB antibody and TST administration groups showed 36.6% and 63.1% tumor growth inhibition effects, respectively, while the combined administration group of TST and anti-4-1BB antibody showed a 78% tumor growth inhibition effect, indicating that a synergistic (CDI = 0.94) anti-tumor effect can be obtained by combined administration ( Figure 9a Moreover, the anti-PD-1 antibody (0.2 mg / mouse) and TST administration groups showed 38.7% and 63.1% tumor growth inhibition effects, respectively, while the combined administration group of TST and anti-PD-1 antibody showed a 78.4% tumor growth inhibition effect, indicating that a synergistic (CDI = 0.95) anti-tumor effect can be obtained through combined administration ( Figure 9a ). The results described above indicate that the combined administration of TST and immune checkpoint inhibitors is a clinically meaningful strategy that can inhibit tumor growth and enhance the efficacy of immunotherapy.

[0170] Example 7: Confirmation of the effect of TST on colorectal cancer cell lines

[0171] Analysis of cell apoptosis, survival rate and protein expression

[0172] (1) Reagents and cell cultures used in the experiment

[0173] Dimethyl sulfoxide (DMSO) and TST (Sigma-Aldrich, St. Louis, MO, USA). Amicon centrifugal filter device (MWCO 50K, UFC805024, Merck Millipore). Sephadex G25 gel filtration column (PD-10; #17-0851-01, GE Healthcare). Anti-PD-1 (clone: ​​RMP1-14, BP0146) and anti-4-1BB (clone: ​​17B5, BE0296) antibodies (BioXcell). Anti-CD3 antibody (ab16669, Abcam). Anti-PD-L1 antibody (13684, Cell Signaling Technology).

[0174] Human colorectal cancer cell lines (HCT116, HCT8, and SW480) and primary human coronary artery smooth muscle cells (SMC) were purchased from the American Type Culture Collection (Manassas, VA, USA), while the MC38 mouse colorectal adenocarcinoma cell line was provided by Dr. Bum-Kyu Choi (National Cancer Center, Goyang, South Korea). Mc38 cells were cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. SW480 and HCT8 cells were cultured in RPMI (Roswell Park Memorial Institute) medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. HCT116 cells were maintained in McCoy's 5A medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. The cells were maintained at 37°C in a humidified incubator with 5% CO2.

[0175] (2) Immunoblotting

[0176] Protein concentration was determined using a bicinchoninic acid kit (Cat. 23225; Thermo Fisher Scientific, Carlsbad, CA, USA) for immunoblotting analysis. Proteins were separated by SDS-PAGE electrophoresis. Primary antibodies to FOXM1 (ab207298, Abcam), PD-L1 (14-5982-82, Invitrogen), and glyceraldehyde-3-phosphate dehydrogenase (sc-25778, Santa Cruz Biotechnology) were added and incubated overnight at 4°C. After binding of appropriate secondary antibodies conjugated to horseradish peroxidase to the washed membranes, protein bands were detected using a chemiluminescent kit (Thermo Fisher Scientific, Carlsbad, CA, USA).

[0177] (3) Analysis of cell apoptosis (Annexin V analysis)

[0178] MC38 cells (8.0×10 4 Cells were seeded in 6-well plates and cultured for 24 hours. Cells were harvested after being treated with TST (5 μM) for 48 hours and then analyzed by FACS-Verse using BD FITC Annexin V Apoptosis Detection Kit I (Cat No. 556547; BD Biosciences, San Diego, CA, USA) according to the manufacturer's operating procedures.

[0179] (4) Analysis of cell survival rate

[0180] MC38 cells and SMCs were plated in 96-well plates at 3 × 10 3 After treating the cells with different concentrations of TST dissolved in the culture medium for 48 hours, WST-1 reagent (MK400; Takara) was added at a concentration of 10 μL / well and cultured for 1 hour, and the absorbance was measured at 450 nm for further analysis.

[0181] (5) Analysis of PD-L1 expression during TST treatment

[0182] To confirm the dose-dependent downregulation effect of FOXM1 inhibitor TST on PD-L1 levels, live cell imaging of PD-L1 expression was performed in MC38 cells. M38 cells were cultured in Lab-TekII and treated with different concentrations of TST (0, 1, 5 and 10 μM) for 48 hours. Next, after culturing the cells with anti-PD-L1 antibodies (14-5982-82; eBioscience, San Diego, CA, USA), they were combined with secondary antibodies labeled with Alexa Fluor 594 (A21209; Thermo Fisher, Waltham, MA, USA), and the nuclei were stained with Hoechst33342 dye (ThermoFisher, Waltham, MA, USA). After washing the cells three times with PBS (pH 7.4, 10mM, 136mM NaCl), the culture medium was replaced with a new complete culture medium. Fluorescence images of cell nuclei (λex 359 nm, λem 457 nm) and PD-L1 (λex 590 nm, λem 618 nm) were acquired using a confocal scanning laser microscope (LSM780; Carl Zeiss, Oberkochen, Germany).

[0183] 7.2. Confirmation of the effect of TST on PD-L1 expression in colorectal cancer cells

[0184] The results of analyzing the effect of TST on PD-L1 expression in human and mouse colorectal cancer cells showed that in human colorectal cancer cells (HCT8, HCT116, and SW480), when treated with 5μM TST, the levels of FOXM1 and PD-L1 were significantly reduced ( Fig.10 ). In MC38 mouse colorectal cancer cells, the expression levels of FOXM1 and PD-L1 also showed a dose-dependent decrease after TST treatment ( Fig.11 a). In the confocal fluorescence image of immunocytochemical analysis of PD-L1 expression, a strong fluorescence signal was shown on the surface of cancer cells ( Fig.11 b) In the plasma membrane, PD-L1 levels showed a dose-dependent decrease after TST treatment. As FOXM1 directly binds to the PD-L1 promoter in the nucleus and selectively upregulates PD-L1 expression, TST significantly reduced PD-L1 expression in human and mouse colorectal cancer cells through TST-mediated FOXM1 inhibition.

[0185] In vitro cytotoxicity tests of TST were performed on MC38 cancer cells and SMC normal cells. The median inhibitory concentration (IC50 ) was 3.8μM. In contrast, no significant apoptosis was observed in normal SMC cells. Flow cytometry analysis confirmed that TST treatment can significantly increase apoptosis in MC38 cancer cells ( Fig.11 e).

[0186] Example 8: Confirmation of the anti-tumor effect of ThioLipo

[0187] 8.1. Manufacturing and Characterization of ThioLipos

[0188] (1) Method for manufacturing ThioLipos

[0189] Cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) were purchased from Avanti Polar Lipids (Alabama, USA). DOPC, cholesterol and DSPE-PEG2000 were added to a round-bottom flask at a molar ratio of 55, 40 and 5, respectively. TST was dissolved in chloroform at a concentration of 1.5 mg / mL, and 1 mL of the solution was added to the lipid sample. The solution was dried using a rotary evaporator, leaving a thin lipid film. PBS (1 mL) was added to the round-bottom flask and ultrasonicated for 5 minutes in a bath ultrasonicator at 25°C. After removing the lipids from the flask, the solution was transferred to an ampoule and ultrasonicated for 20 minutes using a probe ultrasonicator. TST and other impurities that were not captured were removed by centrifugation at 600 x g for 5 minutes at 25°C. Free TST was further removed using a PD-10 size exclusion column. The size distribution (Z-average) and polydispersity index were characterized using dynamic light scattering (DLS; Malvern Zetasizer). The concentration of TST encapsulated in the nanoliposomes was analyzed by dissolving the nanoliposomes in DMSO and measuring the absorbance at 300 nm using a UV-Vis spectrophotometer (DU730, Beckman Coulter, Brea, CA). For transmission electron microscopy (TEM) observation of ThioLipos, a grid (Quantifoil, R1.2 / 1.3, 200mesh, EMS) was formed on a hydrophilic surface using a glow discharge system (PELCO easiGlow, Tedpella) and 4 μL of sample was added to the grid, and then the imprinting process was performed for 1.5 seconds at a humidity and temperature of 100% and 4°C, respectively. Next, the sample was frozen for vitrification in liquid nitrogen using a Vitrobot Mark IV (FEI). The sample was analyzed at 120 kV using a Talos L120C (FEI) at the Nano Bio-Imaging Center (Seoul University, South Korea).

[0190] (2) Find out the characteristics of ThioLipos

[0191] TST is a hydrophobic compound with low solubility in water. As a system for effectively delivering the hydrophobic drug described above to tumors, liposome encapsulation ( Fig.12a). The hydrodynamic size of the produced ThioLipos is 192nm, and the TEM image of ThioLipos shows a circular single-layer structure ( Fig.12 c). The stability of ThioLipos was then tested. No significant changes in size distribution were observed within 15 days ( Fig.12 d). The encapsulation efficiency of TST in nanoliposomes was 63±3%, and the ratio of TST to lipid in nanoliposomes was 0.37±0.018.

[0192] In addition, we investigated whether the blood concentration of TST could be increased in vivo in the form of ThioLipos. Due to the hydrophobic nature of TST, free TST was injected intraperitoneally, while LipoThio was injected intravenously. Since there is no clear and sensitive method for detecting TST in blood and other tissues, the TST concentration was analyzed using TripleTOF5600 equipped with ultra-high performance liquid chromatography. Mice treated with ThioLipo had a significant increase in TST concentration in their blood after 24 hours ( Fig.13 ).

[0193] 8.2. Confirmation of ThioLipo's effect

[0194] (1) Syngeneic colorectal cancer model

[0195] All animal experiments were approved by the Institutional Animal Care and Use Committee of the National Cancer Center, Korea (NCC-21-615). MC38 cells (5 × 10 5 The tumor size reached approximately 150 mm. 3Mice were randomly assigned to treatment groups. Mice were treated with anti-4-1BB (10 mg / kg, n=10) and anti-PD-1 (10 mg / kg, n=9) antibodies by intraperitoneal injection on day 0 and every 2 days. ThioLipo was administered intravenously through the tail vein at a dose of 10 mg TST equivalent (equiv.) / kg on day 0 and every 2 days (n=11). Mice in the anti-4-1BB+ThioLipo group (n=10) were actively treated with anti-4-1BB antibody (10 mg / kg, intraperitoneal injection) and Thio-Lipo (10 mg TST equivalent / kg, intravenous injection) every 2 days on day 0, while mice in the anti-PD-1+ThioLipo group (n=8) were treated with anti-PD-1 antibody (10 mg / kg, intraperitoneal injection) and Thio-Lipo (10 mg TST equivalent / kg, intravenous injection) on day 0 and every 2 days. The control group mice (n=9) were intravenously injected with PBS solution (100 μL) on day 0 and every 2 days. The antibody and ThioLipos were treated 4 times in total, and the tumor volume and body weight of the mice were measured every day until day 8, and the tumor volume (V) was calculated by the formula AB 2 / 2 for calculation (A and B are the long diameter, and B are the long diameter and short diameter, respectively (mm)). The anti-tumor synergistic effect of combined administration is determined by calculating the drug interaction coefficient (CDI). CDI is calculated using the following formula: CDI = AB / (A × B), where AB is the ratio of tumor size between the control group and the combined drug group, and A and B are the ratio of tumor size between the control group and the single treatment group. CDI values ​​<1, <0.7, =1 and >1 represent synergy, significant synergy, additivity and antagonism, respectively.

[0196] On the 8th day, mice were euthanized and tumor tissues and normal vital organs (heart, lung, kidney, spleen and liver) were collected and then fixed in 4% formaldehyde solution for immunohistochemistry (IHC) and histopathological analysis. The tissues were paraffin-embedded and sectioned for staining. According to the DAB-chromogenic discovery study standard procedure, IHC analysis was performed on the CD3 (1:150, ab16669; Abcam) and PD-L1 (1:100, 13684; Cell Signaling Technology) levels in tumor tissues in paraffin sections using Ventana Roche Discovery XT Immunostainer (Mannheim, Germany). Antigen retrieval was initiated by epitope heat-induced label removal (unmarking) during the period when the slides were immersed (immersed) according to the manufacturer's operating instructions. After the sections were incubated with the primary antibody at 37°C, the secondary antibody was applied at room temperature. According to the diaminobenzidine development method enhanced by copper, after using DAB colorimetric detection kit to detect, hematoxylin was used to carry out 4 minutes of counterstaining. Next, the next section was dehydrated manually and removed in dimethylbenzene, and then sealed on a cover glass. In addition, TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) analysis was performed in order to assess apoptosis in the treatment group. Tissue sections were stained with hematoxylin and eosin (H&E) and histopathological analysis was performed, and the imaging of stained tissue sections was performed using Vectra Polaris system (Akoya Biosciences). Tissue sections were stained using TUNEL assay kit, so that apoptosis in the normal tissue of the treatment group was analyzed and compared with the control group.

[0197] (2) Serum biochemical analysis

[0198] To analyze the potential toxicity of each treatment, serum was collected from mice in each group on the last day of the experiment. Albumin (ALB), creatinine (CREA), total protein (TP), blood urea nitrogen (BUN), glucose (GLU), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatine kinase (CPK), total bilirubin (T-Bil), triglyceride (TG), phosphate (PHOS), calcium (CA), blood cholesterol (CHO) resin, and albumin / globulin ratio (A / G) were measured by TNPbio (Gwangju, Gyeonggi-do, Korea).

[0199] In all statistical analyses, differences between groups were assessed using Student's t-test. Statistical significance was set at p < 0.05, and data are presented as mean ± standard error.

[0200] (3) Confirm the efficacy of combined administration of immune checkpoint inhibitors and ThioLipos

[0201] Tumor size reaches 150mm in syngeneic colorectal cancer model 3 Treatment started on day 0 ( Fig.14a ). ThioLipos was administered intravenously, while the antibody therapeutic was administered intraperitoneally. Each treatment group was treated with ThioLipo (10 mg of TST equivalent / kg), anti-4-1BB antibody (10 mg / kg) or anti-PD-1 antibody (10 mg / kg). When anti-4-1BB antibody, anti-PD-1 antibody or ThioLipo was treated alone, tumor volume was reduced by 24% (p<0.05), 39% (p<0.05) and 31% (p<0.05) respectively compared with the control group ( Fig.14b and 14c).

[0202] In cases with smaller tumor size (80 mm 3 ), it is expected that a single ICI treatment can achieve better therapeutic effects, but in order to confirm the effect of ICI and ThioLipos combination therapy in a fast-growing tumor model, we conducted a double-blind study in a tumor size of 150 mm 3In particular, the combination therapy of anti-4-1BB+ThioLipo and anti-PD-1+ThioLipo showed synergistic antitumor effects, thereby showing 56% (p<0.001) and 74% (p<0.001) tumor growth inhibition in tumor-bearing mice, respectively, compared with the control group. The CDI values ​​calculated for the anti-4-1BB+ThioLipos group and the anti-PD-1+ThioLipo group were 0.83 and 0.61, respectively, and no body weight differences were observed between the treatment groups.

[0203] In addition, the enhanced therapeutic effect of ThioLipos was analyzed by staining tumor sections using a TUNEL assay kit and IHC on day 8 ( Fig.15 ). Regarding cell apoptosis in tumors, compared with the control group, the ThioLipos-treated group (P<0.05), the anti-4-1BB-treated group (p<0.001), and the anti-PD-1-treated group increased by 3.2 times, 8.1 times, and 7.7 times, respectively. Cell apoptosis in the anti-4-1BB+ThioLipos and anti-PD-1+ThioLipos groups increased by 2.4 times and 2.7 times, respectively, compared with the anti-4-1BB-treated group (p<0.001) and the anti-PD-1-treated group (p<0.01). In IHC staining of tumor sections, CD3 + The number of T cells increased by 3.8-fold, 3.4-fold and 4.7-fold in the ThioLipo-treated group (p<0.01), the anti-4-1BB-treated group (p<0.001) and the anti-PD-1-treated group (p<0.001), respectively. + T cells increased by 2.3 times and 3.4 times compared with the anti-4-1BB treatment group (P<0.01) and the anti-PD-1 treatment group (P<0.001), respectively. In addition, in order to determine the changes in PD-L1 levels, IHC staining of PD-L1 protein was performed in tumor sections. As expected, PD-L1 levels in tumor sections were significantly downregulated in tumors treated with ThioLipos (47.7% inhibition compared with the control group, p<0.001). The PD-L1 level in the combined drug group was also significantly lower than that in the ICI treatment group. Although the expression of PD-L1 increased when treated with anti-PD-1, the combination treatment of anti-PD-1 antibodies and ThioLipos significantly inhibited PD-L1 levels in tumor tissues.

[0204] In order to confirm the side effects of each treatment group on normal tissues in vivo, i.e., the side effects of the treatment, at the end of the experiment, vital organs (heart, spleen, lung, kidney, and liver) were collected and sliced, and then the tissue slices were stained with hematoxylin-eosin (H&E) and TUNEL, and blood samples of mice were collected and analyzed. As mentioned above, no weight difference was observed between the treatment groups. In the TUNEL staining images of normal tissue sections, no increase in apoptosis was shown in all treatment groups compared with the control group ( Fig.16a In addition, no histopathological changes were observed in the H&E staining images of normal tissue sections. In serum biochemical analysis, the treated group also showed no signs of toxic effects compared with the control group ( Fig.16b ).

[0205] Example 9: Confirmation of the effects of FDI-6 and RCM1 on lung cancer, colorectal cancer and brain tumor cell lines

[0206] Analysis of cell apoptosis, survival rate and protein expression

[0207] (1) Reagents and cell cultures used in the experiment

[0208] Dimethyl sulfoxide (DMSO), FDI-6 (Sigma-Aldrich, St. Louis, MO, USA, CASNumber: 313380-27-7), RCM1 (R&D Systems, Minneapolis, MN55413, USA, CASNo: 339163-65-4), Amicon centrifugal filter device (MWCO 50K, UFC805024, Merck Millipore), Sephadex G25 gel filtration column (PD-10; #17-0851-01, GE Healthcare), anti-PD-1 (clone: ​​RMP1-14, BP0146), anti-4-1BB (clone: ​​17B5, BE0296) antibodies (BioXcell), anti-CD3 antibody (ab16669, Abeam) and anti-PD-L1 antibody (13684, Cell Signaling Technology).

[0209] Human lung cancer cell lines (H1299, PC9), colorectal cancer cell lines (HCT116, SW480) and brain tumor cell lines (U118) were purchased from American Type Culture Collection (Manassas, VA, USA). H1299, PC9, U118 and SW480 cells were cultured in RPMI (Roswell Park Memorial Institute) medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, while HCT116 cells were cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. The cells were maintained at 37° C. in a humidified incubator with 5% CO2.

[0210] (2) Immunoblotting

[0211] Protein concentration was determined using a bicinchoninic acid kit (Cat. 23225; Thermo Fisher Scientific, Carlsbad, CA, USA) for immunoblotting analysis, and proteins were separated by SDS-PAGE electrophoresis. Primary antibodies to FOXM1 (ab207298, Abcam), PD-L1 (14-5982-82, Invitrogen), and glyceraldehyde-3-phosphate dehydrogenase (sc-25778, Santa Cruz Biotechnology) were added and incubated overnight at 4°C. After washing, appropriate secondary antibodies conjugated to horseradish peroxidase were bound to the membrane, and protein bands were detected using a chemiluminescent kit (Thermo Fisher Scientific, Carlsbad, CA, USA).

[0212] (3) Analysis of PD-L1 expression during RCM1 treatment in lung cancer cell lines

[0213] To confirm the dose-dependent downregulation effect of the FOXM1 inhibitor, RCM1, on PD-L1 expression, live cell imaging of PD-L1 expression was performed in the lung cancer cell line, H1299 cells. H1299 cells were cultured in Lab-TekII and treated with RCM1 (10 μM) for 48 hours. Next, after the cells were cultured with anti-PD-L1 antibodies (14-5982-82; eBioscience, San Diego, CA, USA), they were combined with secondary antibodies labeled with Alexa Fluor 594 (A21209; Thermo Fisher, Waltham, MA, USA), and the cell nuclei were stained with Hoechst33342 dye (ThermoFisher, Waltham, MA, USA). After washing the cells three times with PBS (pH 7.4, 10 mM, 136 mM NaCl), the culture medium was replaced with a new complete culture medium. Fluorescence images of cell nuclei (λex 359 nm, λem 457 nm) and PD-L1 (λex 590 nm, λem 618 nm) were acquired using a confocal scanning laser microscope (LSM780; Carl Zeiss, Oberkochen, Germany).

[0214] 9.2. Confirmation of the effects of FDI-6 and RCM1 on PD-L1 expression in lung cancer, colorectal cancer, and brain tumor cells

[0215] The results of analyzing the effects of FDI-6 and RCM1 on PD-L1 expression in lung cancer, colorectal cancer, and brain tumor cell lines showed that the expression of FOXM1 and PD-L1 was significantly reduced in lung cancer cell lines (PC9) and colorectal cancer cell lines (SW480) when treated with FDI-6 ( Fig.17 a and 17b). In addition, when RCM1 was used to treat lung cancer cell lines (H1299), colorectal cancer cell lines (HCT116), and brain tumor cell lines (U118), the expression of FOXM1 and PD-L1 was also significantly reduced ( Fig.18 a). Confocal fluorescence imaging analysis also confirmed that the level of PD-L1 expressed on the surface of cancer cells decreased after treatment with RCM1 ( Fig.18 b) Similar to the fact that FOXM1 directly binds to the PD-L1 promoter in the nucleus and selectively upregulates PD-L1 expression, it was determined that FOXM1 inhibitors, namely FDI-6 and RCM1, can reduce PD-L1 expression in human lung cancer, colorectal cancer and brain tumor cells via inhibition of FOXM1.

[0216] Through the above description, practitioners in the technical field to which the present invention belongs should understand that the present invention can be implemented in other specific forms without changing its technical ideas or essential features. Therefore, the embodiments counted in the above content should be understood in all aspects only for exemplary purposes and not for limitation. The scope of the present invention should not be interpreted based on the detailed description, but should be interpreted as the scope of the present invention includes all changes or deformation forms that can be derived from the meaning and scope of the claims and their equivalent concepts.

Claims

1. A pharmaceutical composition for preventing or treating cancer, The active ingredients include a FOXM1 inhibitor and an immune checkpoint inhibitor.

2. The pharmaceutical composition according to claim 1, The FOXM1 inhibitor is at least one selected from the group consisting of thiazolidine dione, diarylheptane, RCM-1, thiostrepton, honokiol, FDI-6, siomycin A, monensin, FOXM1ap t and peptide 9R-P201.

3. The pharmaceutical composition according to claim 1, The immune checkpoint inhibitor is at least one selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA4 antibody, anti-PD-L2 antibody, LTF2 regulatory antibody, anti-LAG3 antibody, anti-A2 aR antibody, anti-TIGIT antibody, anti-TIM-3 antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-VIS TA antibody, anti-CD47 antibody, anti-BTLA antibody, anti-KIR antibody, anti-IDO antibody and anti-4-1BB antibody.

4. The pharmaceutical composition according to claim 1, The composition reduces the expression of PD-L 1.

5. The pharmaceutical composition according to claim 1, The FOXM1 inhibitor is loaded into liposomes.

6. The pharmaceutical composition according to claim 1, The pharmaceutical composition is for combined administration of a FOXM1 inhibitor and an immune checkpoint inhibitor.

7. The pharmaceutical composition according to claim 6, The pharmaceutical composition is in the form of a mixed preparation of the FOXM1 inhibitor and the immune checkpoint inhibitor, or the FOXM1 inhibitor and the immune checkpoint inhibitor are formulated separately and then administered simultaneously or sequentially.

8. The pharmaceutical composition according to claim 1, The cancer is lung cancer, pancreatic cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, gastric cancer, duodenal cancer, small intestine cancer, large intestine cancer, colon cancer, rectal cancer, breast cancer, ovarian cancer, prostate cancer, liver cancer, thyroid cancer, kidney cancer, uterine cancer, brain tumor, skin cancer, melanoma, malignant bone tumor, bladder cancer or blood cancer.

9. An anticancer adjuvant, The active ingredients include a FOXM1 inhibitor and an immune checkpoint inhibitor.

10. The anticancer adjuvant according to claim 9, The FOXM1 inhibitor is at least one selected from the group consisting of thiazolidine dione, diarylheptane, RCM-1, thiostrepton, honokiol, FDI-6, siomycin A, monensin, FOXM1ap t and peptide 9R-P201.

11. The anticancer adjuvant according to claim 9, The immune checkpoint inhibitor is at least one selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA4 antibody, anti-PD-L2 antibody, LTF2 regulatory antibody, anti-LAG3 antibody, anti-A2 aR antibody, anti-TIGIT antibody, anti-TIM-3 antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-VIS TA antibody, anti-CD47 antibody, anti-BTLA antibody, anti-KIR antibody, anti-IDO antibody and anti-4-1BB antibody.

12. Use of a composition, Used to prevent or treat cancer, including FOXM1 inhibitors and immune checkpoint inhibitors.

13. Use of a composition, Used to manufacture cancer prevention or treatment agents, including FOXM1 inhibitors and immune checkpoint inhibitors.

14. A method for preventing or treating cancer, The method comprises administering to a subject a composition comprising a FOXM1 inhibitor and an immune checkpoint inhibitor.

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