PD-L1 expression inhibitors for tumor cells, combination drugs for treating cancer, and anticancer drugs

Abstract

The present invention provides PD-L1 expression inhibitors for tumor cells, combination drugs for treating cancer, and anticancer agents. [Solution] A PD-L1 expression inhibitor for tumor cells, containing brusatol as an active ingredient.

Description

[Technical Field]

[0001] The disclosures in this application relate to inhibitors of PD-L1 expression in tumor cells, combination drugs for treating cancer, and anticancer agents. [Background technology]

[0002] In recent years, cancer immunotherapy has been implemented as a treatment method for cancer. Unlike conventional treatments such as surgery, radiation therapy, and drug therapy with anti-cancer drugs and molecular targeted drugs, cancer immunotherapy works by acting on the immune surveillance mechanism that the malignant tumor patient possesses, thereby strengthening the immune response to the malignant tumor and inhibiting or treating its progression.

[0003] Cancer cells are eliminated by activated cytotoxic T cells. However, as shown in Figure 1, it is known that PD-L1 on the surface of cancer cell membranes binds to PD-1 expressed on the surface of cytotoxic T cells, thereby suppressing the activity of cytotoxic T cells. Among the drugs used in cancer immunotherapy, anti-PD-1 antibodies (Opdivo, Keytruda, etc.) that act on PD-1 (programmed cell death-1) expressed on the surface of activated cytotoxic T cells are known as cancer immune checkpoint inhibitors. When anti-PD-1 antibodies bind to PD-1 expressed on the surface of cytotoxic T cells, the binding of PD-L1 on the surface of cancer cell membranes and PD-1 expressed on the surface of cytotoxic T cells is suppressed. As a result, the inactivation of cytotoxic T cells by cancer cells is suppressed, and cancer cells can be eliminated by cytotoxic T cells.

[0004] Other drugs used in cancer immunotherapy include anti-PD-L1 antibodies (such as Bavencio, Tecentriq, and Imfinzi) that act on PD-L1, a ligand for PD-1. Anti-PD-L1 antibodies bind to PD-L1 expressed on the membrane surface of cancer cells, thereby suppressing the binding of PD-L1 on the cancer cell membrane surface to PD-1 expressed on the membrane surface of cytotoxic T cells. As a result, the inactivation of cytotoxic T cells by cancer cells is suppressed, allowing cytotoxic T cells to eliminate cancer cells.

[0005] Among cancer immunotherapies, immune checkpoint inhibitor therapy has few side effects and offers groundbreaking therapeutic effects, but it has the problem that it is not effective for all patients. Therefore, the development of combination drugs that are effective with cancer immunotherapy is underway.

[0006] Patent Document 1 discloses that, as an example of an adjunct agent for cancer immunotherapy, a UBL3 inhibitor containing a statin compound or a pharmaceutically acceptable salt thereof as an active ingredient functions as an enhancer of the effect of an anticancer agent. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2024-028182 [Overview of the project] [Problems that the invention aims to solve]

[0008] As described in Patent Document 1 above, UBL3 inhibitors containing statin compounds or pharmaceutically acceptable salts thereof as active ingredients are known to function as enhancers of anticancer drug effects. However, cells utilize various survival signals to ensure their own survival. Therefore, even with the same objective of enhancing the effects of anticancer drugs, it is desirable to develop pharmaceutical compositions that exert their effects through different mechanisms.

[0009] The disclosures in this application are made to solve the above-mentioned problems. Through diligent research, the inventors have newly discovered that (1) brusatol has the function of suppressing the expression of PD-L1 in tumor cells, and (2) therefore, tumor cells in which PD-L1 expression is suppressed become more susceptible to the immune response of cytotoxic T cells.

[0010] In other words, the purpose of the disclosure in this application is to provide PD-L1 expression inhibitors for tumor cells, combination drugs for treating cancer, and anticancer agents. [Means for solving the problem]

[0011] The disclosures in this application relate to inhibitors of PD-L1 expression in tumor cells, combination drugs for treating cancer, and anticancer agents, as described below.

[0012] (1) A PD-L1 expression inhibitor for tumor cells, containing brusatol as the active ingredient. (2) The expression inhibitor according to (1) above, wherein the brusatol suppresses the expression of SQSTM1 in the tumor cells, thereby suppressing the expression of PD-L1 in the tumor cells. (3) The expression inhibitor described in (1) or (2) above, wherein the expression inhibitor functions as an anticancer agent. (4) An expression inhibitor described in any one of (1) to (3) above, used in combination therapy with anticancer drugs. (5) The expression inhibitor according to (4) above, wherein the anticancer agent is an anti-PD-1 antibody and / or cisplatin. (6) A combination drug for treating cancer, said combination drug is The first pharmaceutical product contains one of the expression inhibitors described in (1) to (4) above as an active ingredient, A Class II pharmaceutical product containing an anticancer drug as an active ingredient, Concomitant medications, including those mentioned above. (7) The combination drug according to (6) above, wherein the anticancer agent is an anti-PD-1 antibody and / or cisplatin. (8) An anticancer drug containing brusatol as an active ingredient. [Effects of the Invention]

[0013] Bursatol can inhibit the expression of PD-L1 in tumor cells. Therefore, tumor cells with inhibited PD-L1 expression become more susceptible to the immune response of cytotoxic T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] [Figure 1] This figure is to explain that PD-L1 on the surface of cancer cell membrane inhibits the activity of cytotoxic T cells by binding to PD-1 expressed on the membrane surface of cytotoxic T cells. [Figure 2] Figure 2 shows that the cells established in Experimental Example 1 overexpress SQSTM1. Figure 2A is an immunoblot photograph, Figure 2B is a graph showing the measurement results of CD274 (PD-L1) mRNA, and Figure 2C is a graph showing the results of analyzing the expression level of CD274 (PD-L1) protein by flow cytometry. [Figure 3] Figure 3 is a graph showing the measurement results of CD152 (CTLA4) mRNA in the cells established in Experimental Example 1. [Figure 4] Figure 4A is an immunoblot photograph showing that the expression of SQSTM1 is inhibited in SQSTM1-deficient MOC2-sgSQSTM1 cells established in Experimental Example 2, and Figure 4B is a graph showing the results of analysis of PD-L1 expressed on the surface of the established SQSTM1-deficient MOC2-sgSQSTM1 cells. [Figure 5A] Figure 5A is a diagram showing the experimental schedule for investigating tumor immune evasion by SQSTM1 in Experimental Example 3. [Figure 5B] Figure 5B is a graph showing the measurement results of tumor volume in the experiment of Experimental Example 3. [Figure 5C] Figure 5C shows photographs and a graph showing the results of measuring the number of CD8+ cytotoxic T cells in the experiment of Experimental Example 3. [Figure 6]Figures 6A and 6B are immunoblot images showing the results of an experiment to confirm the SQSTM1 inhibitory function of brusatol in Example 1. [Figure 7] Figures 7A to 7C are graphs showing the results of flow cytometry analysis of the PD-L1 protein expression level on the cell surface of each cell treated with brusatol in Example 1. [Figure 8A] Figure 8A shows the experimental schedule for confirming the combined effect of the anti-PD-1 antibody and brusatol in Example 2. [Figure 8B] Figure 8B is a graph showing the measurement results of tumor volume and tumor weight in the experiment of Example 2. [Figure 8C] Figure 8C is a graph showing the results of the experiment in Example 2, which examined the percentage of cytotoxic T cells infiltrating the tumor. [Figure 9A] Figure 9A shows the experimental schedule for confirming the combined effect of the anti-PD-1 antibody and brusatol in Example 3. [Figure 9B] Figure 9B is a graph showing the measurement results of tumor volume and tumor weight in the experiment of Example 3. [Figure 9C] Figure 9C is a graph showing the results of the experiment in Example 3, which examined the percentage of cytotoxic T cells infiltrating the tumor. [Figure 10] Figure 10 shows photographs and graphs illustrating the results of measuring the number of CD8+ cytotoxic T cells in the experiment of Example 3. [Figure 11] Figure 11 is a graph showing the measurement results of tumor volume and tumor weight when an anti-PD-L1 antibody was used instead of an anti-PD-1 antibody in Example 4. [Modes for carrying out the invention]

[0015] The following describes the PD-L1 expression inhibitors for tumor cells and combination drugs for treating cancer disclosed in this application.

[0016] (Example of an agent for suppressing PD-L1 expression in tumor cells) The PD-L1 expression inhibitor for tumor cells according to this embodiment (hereinafter sometimes simply referred to as "expression inhibitor") contains brusatol as an active ingredient.

[0017] Brusatol is a compound represented by the following formula (1) and is a component of the herbal medicine Bruceae Fructus. Brusatol is known to have the function of promoting the ubiquitination and degradation of Nrf2 (nuclear factor erythrocyte-derived 2-like 2), and to upregulate the expression of multiple ribosomal components and control the function of macromolecular complexes. [ka]

[0018] In the expression inhibitor of the embodiment, brusatol suppresses the expression of PD-L1 in tumor cells. More specifically, brusatol suppresses the expression of PD-L1 in tumor cells by suppressing the expression of SQSTM1 (Sequestosome 1, also known as p62) in tumor cells. The mechanism of action by which brusatol suppresses PD-L1 expression via the suppression of SQSTM1 expression in tumor cells is a newly discovered mechanism of action by the present inventors.

[0019] Tumor cells in which PD-L1 expression is suppressed become more susceptible to cytotoxic T cell immune responses. Therefore, the expression inhibitor according to this embodiment can be used as an anticancer agent on its own. Furthermore, the expression inhibitor according to this embodiment can also be used in combination therapy with anticancer agents. Examples of anticancer agents used in combination therapy include immune checkpoint inhibitors and chemotherapeutic agents.

[0020] Examples of immune checkpoint inhibitors include anti-PD-1 antibodies and anti-PD-L1 antibodies. Examples of anti-PD-1 antibodies include nivolumab (Opdivo) and pembrolizumab (Keytruda). Examples of anti-PD-L1 antibodies include avelumab (Bavencio), atezolizumab (Tecentriq), and durvalumab (Imfinzi). As shown in the examples described later, no synergistic effect was obtained when anti-PD-L1 antibodies were used in combination with brusatol, but no negative results were obtained from the combination, so there is no particular problem with using them together. On the other hand, a significant synergistic effect was obtained when anti-PD-1 antibodies were used in combination with brusatol. This is thought to be because brusatol suppresses PD-L1 expression by suppressing the expression of SQSTM1 in tumor cells, and by blocking PD-1 expressed on T cells, it suppresses T cell inactivation, resulting in a high synergistic effect. Therefore, anti-PD-1 antibodies are a preferred example of immune checkpoint inhibitors to be used in combination with brusatol. In addition to anti-PD-1 antibodies and / or anti-PD-L1 antibodies, CTLA-4 inhibitors and / or anti-LAG-3 antibodies may be optionally added in combination. Examples of anti-CTLA-4 inhibitors include ipilibumab (Yervoy). Examples of anti-LAG-3 antibodies include relatrimab.

[0021] Examples of chemotherapeutic agents include platinum-based drugs such as cisplatin, carboplatin, oxaliplatin, and nedaplatin, and molecularly targeted therapies such as kinase inhibitors. When using the expression inhibitor according to this embodiment in combination with an anticancer drug, one or more agents selected from the immune checkpoint inhibitors and chemotherapeutic agents exemplified above may be used in combination.

[0022] The expression inhibitor according to the embodiment is considered useful for tumor cells (cancer cells) that express SQSTM1(p62) at a high rate compared to normal cells. Such tumor cells (cancer cells) can be identified using public databases or experiments. While not limited to these, cancer types that express SQSTM1(p62) at a high rate include head and neck cancer (HNSCC), malignant melanoma (Melanoma), hepatocellular carcinoma (HCC), pancreatic cancer (PDAC), breast cancer (TNBC), lung cancer (NSCLC), prostate cancer (PCs), head and neck cancer (HNSCC), small cell lung cancer (non-NE type), colorectal cancer (CRC), glioblastoma (GBM), and others.

[0023] The expression inhibitor according to this embodiment may contain brusatol as an active ingredient, and depending on the dosage form, may also contain a pharmacologically acceptable carrier. Examples of carriers include excipients, disintegrants or disintegration aids, binders, lubricants, coatings, dyes, diluents, bases, solubilizers or solubilizers, isotonic agents, pH adjusters, stabilizers, propellants, and adhesives.

[0024] The brusatol contained in the expression inhibitor disclosed in this application varies depending on the patient's weight, age, disease severity, etc., and is not particularly limited. The dosage should be determined by the physician as appropriate.

[0025] The expression inhibitors disclosed in this application can be administered orally or parenterally. The form of administration is not particularly limited, but oral administration is standard for brusatol, for example. Formulations for parenteral administration may include sterile aqueous or non-aqueous solutions, suspensions, emulsions, etc. Examples of non-aqueous diluents include propylene glycol, polyethylene glycol, vegetable oils, e.g., olive oil, and organic ester compositions, e.g., ethyl oleate. Aqueous carriers may include water, alcoholic aqueous solutions, emulsions, suspensions, saline solutions, and buffering media. Parenteral carriers may include sodium chloride solution, ringer's dextrose, dextrose, sodium chloride, ringer's lactate, and binding oils, etc. Intravenous carriers may include, for example, liquid supplements, nutrients, and electrolytes (e.g., those based on ringer's dextrose). The expression inhibitors may further include preservatives and other additives, e.g., antimicrobial compounds, antioxidants, chelating agents, and inert gases, etc.

[0026] (Embodiment of combination therapy) Next, we will describe embodiments of combination drugs for treating cancer. Combination drugs are, A first pharmaceutical product comprising any of the expression inhibitors according to the above embodiment as an active ingredient, • Contains a Class II pharmaceutical product containing an anticancer drug as an active ingredient.

[0027] The first pharmaceutical agent is the same as the expression inhibitor according to the embodiment. Therefore, a detailed explanation of the first pharmaceutical agent is omitted to avoid repetition.

[0028] The anticancer agent included in the second pharmaceutical product has already been described in the embodiment of the expression inhibitor, so a detailed description is omitted. The second pharmaceutical product may contain a pharmacologically acceptable carrier similar to that of the first pharmaceutical product, depending on the administration method. The second pharmaceutical product can be administered orally or parenterally. The second pharmaceutical product may contain a carrier similar to that of the first pharmaceutical product, depending on the administration method. Furthermore, other drugs may be optionally added as concomitant agents, provided that they do not impair the concomitant effect. Examples of other drugs, though not limited to these, include xCT inhibitors such as sulfasalazine and elastin, and ferroptosis inducers such as sorafenib, which are anticancer agents with xCT inhibitory activity.

[0029] The first drug is used to suppress the expression level of PD-L1 in tumor cells, thereby making it more difficult for tumor cells to evade the immune system. Therefore, it is desirable to administer the first drug before or simultaneously with the second drug. If the first drug is administered before the second drug, the timing should be set within a range that does not impair the combined effect. It is also possible to set independent administration schedules for both drugs and administer each drug to the target according to the aforementioned schedule. The number of administrations for both drugs can be set arbitrarily, and they can be administered as a single dose or multiple times.

[0030] The dosages of the first and second medications will vary depending on the patient's weight, age, and the severity of the disease, and are not particularly limited. The dosage should be determined by the physician as appropriate.

[0031] (Implementation of anticancer drug) The anticancer agent according to the embodiment contains brusatol as an active ingredient. The anticancer agent according to the embodiment is substantially the same as the PD-L1 expression inhibitor for tumor cells according to the embodiment. In other words, if you replace "PD-L1 expression inhibitor for tumor cells" with "anticancer agent" in the embodiment, it becomes a description of the anticancer agent according to the embodiment. Therefore, to avoid redundancy, a detailed description of the anticancer agent according to the embodiment is omitted. Furthermore, the anticancer agent according to the embodiment can also be used in place of the expression inhibitor for the first pharmaceutical product according to the embodiment described above.

[0032] Examples are provided below to specifically illustrate the embodiments disclosed in this application. These examples are solely for illustrative purposes and are not intended to limit or restrict the technical scope disclosed in this application. [Examples]

[0033] [Confirmation of PD-L1 expression regulation via SQSTM1(p62)] <Experimental Example 1> (1) Establishment of SQSTM1 overexpressing cells SQSTM1 overexpressing cells were generated using the human small cell lung cancer cell line DMS273 (Sigma-Aldrich, Cat #95062830). The obtained DMS273 cells were maintained at 37°C and under 5% CO2 in RPMI 1640 medium (Nacalai) supplemented with 10% fetal bovine serum. EGFP (mock) and cDNA encoding human SQSTM1 were obtained from the DNASU Plasmid Repository (Arizona State University) and individually subcloned into the pLEX_307 lentiviral vector (Addgene plasmid #41392). DMS273 cells stably expressing SQSTM1 and EGFP (mock) were established by the aforementioned lentiviral infection.

[0034] Next, the established cells were cultured and immunoblot analysis was performed using a standard method. For the SQSTM1 antibody, Rabbit monoclonal anti SQSTM1 (p62, MBL, Cat #PM045) was used. The results are shown in Figure 2A. As is clear from Figure 2A, no increase in SQSTM1 expression was observed in DMS273 cells into which EGFP (control) cDNA was introduced. On the other hand, an increase in SQSTM1 expression was confirmed in DMS273 cells into which SQSTM1 cDNA was introduced.

[0035] Next, we performed RT-qPCR analysis of CD274 (PD-L1) mRNA abundance in DMS273 cells stably expressing EGFP (mock) and SQSTM1. For comparison, we also performed RT-qPCR analysis of CD152 (CTLA4) mRNA abundance. The specific procedure was as follows: Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen) and subjected to RT using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics). Quantitative PCR analysis was performed using the Thermal Cycler Dice Real Time System (Takara Bio). The amplification protocol consisted of an initial incubation at 95°C for 2 minutes, followed by 40 incubation cycles at 95°C for 30 seconds and 60°C for 30 seconds. Dissociation curve analysis was then performed to confirm specificity. The following FW primer and RV primer sets were used for qPCR analysis. The measurement results were normalized by the ACTB mRNA abundance. <CD274(PD-L1)> ·FW:5'-TGGCATTTGCTGAACGCATTT-3'(Sequence ID 1) ·RV:5'-TGCAGCCAGGTCTAATTGTTTT-3'(Sequence ID 2) <actb> ·FW:5'-AGGCACCAGGGCGTGAT-3'(Sequence ID 3) ·RV:5'-GCCCACATAGGAATCCTTCTGAC-3'(Sequence ID 4) <CD152(CTLA4)> ·FW:5'-CATGATGGGGAATGAGTTGACC-3'(Sequence ID 5) ·RV:5'-TCAGTCCTTGGATAGTGAGGTTC-3'(Sequence ID 6)

[0036] Figure 2B shows the measurement results for CD274 (PD-L1), and Figure 3 shows the measurement results for CD152 (CTLA4). Note that Figures 2B and 3 represent the average values ​​obtained from four independent experiments.

[0037] Next, the expression level of CD274 (PD-L1) protein in DMS273 cells stably expressing EGFP (mock) and SQSTM1 was analyzed by flow cytometry. The measurement results are shown in Figure 2C. Specifically, established cultured cells were exposed to cell dissociation buffer (Life Technologies) to dissociate, and the resulting single-cell suspension was incubated with APC anti-human CD274 (Biolegend Cat #374514) at 4°C for 60 minutes. Next, the cells were washed with PBS and flow cytometry analysis was performed using an Attune Acoustic Focusing Cytometer (Life Technologies). Figure 2C shows the average values ​​obtained from four independent experiments.

[0038] As shown in Figures 2B and 2C, we confirmed that the expression levels of PD-L1 mRNA and protein increased in DMS273 cells into which SQSTM1 cDNA was introduced. Furthermore, as shown in Figure 3, no increase in the expression level of CTLA4, one of the immune checkpoint molecules, was observed in DMS273 cells into which SQSTM1 cDNA was introduced.

[0039] Based on these results, we confirmed that SQSTM1 specifically promotes the expression (mRNA, protein) of PD-L1, an immune checkpoint molecule.

[0040] <Experimental Example 2> To investigate the role of SQSTM1 in the immune evasion capabilities of cancer cells, SQSTM1-deficient MOC2-sgSQSTM1 cells were established using the mouse oral squamous cell carcinoma cell line MOC2 (kerafast, Cat #EWL002-FP), which is rich in SQSTM1. The specific procedure was as follows: Synthesized sgScr (control) and sgSqstm1 were cloned into the lentiCRISPR v2 vector (Addgene plasmid #52961). Subsequently, MOC2 cells were infected with the cloned lentivirus to establish MOC2 cells with SQSTM1 knockout and control cells. The synthesized sgScr and sgSqstm1 sequences are as follows. <sgscr> ·5'-CACCGGGCCCGCATAGGATATC-3'(Sequence ID 7) ·5'-ACGTGACACGTTCGGAGAATT-3'(Sequence ID 8) <sgsqstm1> ·5'-CACCGCCAGGCGCACTACCGCGGTG-3'(Sequence ID 9) ·5'-AAACCACCGCGGTAGTGCGCCTGGC-3'(Sequence ID 10)

[0041] The established cells were cultured and immunoblot analysis was performed using a standard method. Rabbit monoclonal anti SQSTM1 (p62, MBL, Cat #PM045) was used as the SQSTM1 antibody. Figure 4A shows the results of the immunoblot analysis.

[0042] Next, we analyzed PD-L1 expressed on the cell surface. Specifically, we cultured the established cells (#1 shown in Figure 4A) in or without IFN-γ (100 ng / ml, PeproTech) and then exposed them to cell dissociation buffer (Life Technologies) to dissociate them. The resulting single-cell suspension was incubated with APC anti-mouse CD274 (Biolegend Cat #124311) at 4°C for 60 minutes. Next, we washed the cells with PBS and performed flow cytometry analysis using an Attune Acoustic Focusing Cytometer (Life Technologies). Figure 4B shows the results of the flow cytometry analysis.

[0043] As shown in Figure 4A, SQSTM1-deficient MOC2-sgSqstm1 cells (#1, #2, #3) were established using the CRISPR / Cas9 system. Furthermore, as is clear from Figure 4B, SQSTM1 deficiency significantly reduced the cell surface abundance of PD-L1 in sgScrMOC2 cells and sgScrMOC2 cells treated with IFN-γ. These results confirm that suppression of SQSTM1 expression suppresses PD-L1 expression. The reason for treating the cells with IFN-γ was to reproduce in vitro the situation in which IFN-γ enhances PD-L1 expression in vivo.

[0044] [Regarding tumor immunity evasion using SQSTM1] <Experimental Example 3> The results from Experimental Examples 1 and 2 suggest a correlation between SQSTM1 expression and PD-L1 expression. Increased SQSTM1 expression leads to increased PD-L1 expression, which may promote tumor immunity evasion in MOC2 cells. Therefore, to investigate the role of SQSTM1 in the tumorigenic potential of MOC2 cells, mouse experiments were conducted using the MOC2 cells established in Experimental Example 2 according to the following procedure. Figure 5A shows the experimental schedule. Specifically, 2.0x10 5 One MOC2-sgScr cell (control) and one MOC2-sgSqstm1 cell (#1 in Figure 4A) were transplanted subcutaneously into the backs of immunocompetent C57BL / 6J mice (Jackson Laboratory) and immunodeficient BALB / c nu / nu mice (CLEA JAPAN), respectively. The long and short diameters of the subcutaneous tumors were measured over time, and the mice were euthanized on day 21. Tumor volume was calculated as (long diameter) × (short diameter). 2 It was calculated using ×0.5236.

[0045] Figure 5B shows the results of tumor volume measurement. As shown in Figure 5B, in immunocompetent mice C57BL / 6J, the tumor volume formed by MOC2-sgSqstm1 cells was significantly smaller than that formed by MOC2-sgScr cells (control). However, in immunodeficient BALB / c nu / nu mice, the tumor volume formed by MOC2-sgSqstm1 cells was considerably larger than that formed by MOC2-sgScr cells. From these results, it is thought that SQSTM1 expression conferred the ability of MOC2 cells to evade cytotoxic T cell immunity through upregulation of PD-L1 expression, thereby promoting tumorigenesis.

[0046] Next, to investigate the role of SQSTM1 in the immune evasion ability of MOC2 cells, MOC2-sgScr cells and MOC2-sgSqstm1 cells were transplanted into syngeneic mice (C57BL / 6J), and then CD8 + The number of cytotoxic T cells was measured. Specifically, subcutaneous tumors formed by transplantation of MOC2-sgScr cells and MOC2-sgSqstm1 cells were immunohistochemically stained with a rabbit monoclonal antibody against CD8α (Rabbit monoclonal anti CD8α, Cell Signaling Technologies, Cat #98941) (dilution 1:500; #98941, Cell Signaling Technology). Five different regions of approximately 200 cells were imaged to assess CD8. + The number of cytotoxic T cells was evaluated.

[0047] The results are shown in Figure 5C. As is clear from Figure 5C, knockdown of SQSTM1 is associated with CD8 in subcutaneous tumors. + The population of cytotoxic T cells was increased. These results suggest that SQSTM1 is involved in tumor development by promoting the immune evasion ability of tumor cells, while knocking down SQSTM1 suppresses the immune evasion ability of tumor cells and CD8 + We confirmed that the cells become more susceptible to attack by cytotoxic T cells.

[0048] [Confirmation of Brusatol's SQSTM1 inhibitory function] <Example 1> In addition to the aforementioned MOC2, mouse melanoma cells B16F10 (RIKEN Bioresource Center, Cat #RCB2630) and cisplatin-resistant human advanced small cell lung cancer cell lines (DMS273-CPr and SBC5-CPr) were used as tumor cells. DMS273-CPr and SBC5-CPr were established by exposing DMS273 and SBC5 (Japan Collection of Research Bioresources Cell Bank, Cat #JCRB0819) to progressively increasing concentrations of cisplatin. All cells were maintained at 37°C and less than 5% CO2 in RPMI 1640 medium (Nacalai) supplemented with 10% fetal bovine serum.

[0049] Next, MOC2 was cultured for 4 hours in medium supplemented with brusatol (Sigma-Aldrich, Cat #SML1868) at final concentrations of 0 nM, 250 nM, and 500 nM. B16F10 was cultured for 4 hours in medium supplemented with brusatol at final concentrations of 0 nM, 500 nM, and 750 nM. DMS273-CPr and SBC5-CPr were cultured for 4 hours in medium supplemented with cisplatin (Nichi-Iko Pharmaceutical, Cat #874291) at final concentrations of 0 μM, 25 μM, and 50 μM, and brusatol at a final concentration of 500 nM. After culturing, immunoblot analysis was performed using the same procedure as in Experimental Example 1.

[0050] The results are shown in Figures 6A and 6B. As shown in Figure 6A, we confirmed that brusatol (hereinafter sometimes referred to as "Bru" in the figures) treatment effectively suppressed SQSTM1 expression in a concentration-dependent manner in MOC2 and B16F10 melanoma cells that highly expressed SQSTM1.

[0051] Furthermore, as shown in Figure 6B, in cisplatin-resistant DMS273-CPr cells and SBC5-CPr cells, the expression level of SQSTM1 was significantly suppressed when brusatol was used in combination with cisplatin. These results confirm that brusatol can effectively suppress SQSTM1 expression even in cisplatin-resistant cells. In other words, it was confirmed that combination therapy with cisplatin and brusatol is useful for cancer patients for whom cisplatin is ineffective.

[0052] [Suppression of PD-L1 expression via suppression of SQSTM1(p62) expression by brusatol] Next, MOC2 cells, B16F10 cells, and DMS273 cells cultured in or without IFN-γ (100 ng / ml) were treated with brusatol added to a final concentration of 500 nM for 8 hours, and then flow cytometry analysis was performed using the same procedure as in Experimental Example 2. The results are shown in Figures 7A to 7C. As is clear from Figures 7A to 7C, brusatol treatment suppressed PD-L1 expression in B16F10 melanoma cells (Figure 7A), MOC2 (Figure 7B), and DMS273 (Figure 7C). Furthermore, when each cell was treated with IFN-γ, brusatol treatment also suppressed PD-L1 expression in each cell.

[0053] The results shown in Figures 6 and 7 confirm that brusatol suppresses PD-L1 expression via the suppression of SQSTM1(p62) expression. Furthermore, brusatol treatment was confirmed to suppress PD-L1 expression even in an IFN-γ-rich inflammatory tumor environment.

[0054] [Confirmation of the combined effect of anti-PD-1 antibody and brusatol] <Example 2> Figure 8A shows the experimental schedule. 2.5 × 10 5 B16F10 cells were subcutaneously injected into C57BL / 6J mice. Following the subcutaneous injection, the following treatments were initiated. (1) Anti-PD-1 antibody (BioXcell) monotherapy: 200 μg / mouse. Subcutaneous injection, starting 6 days after administration, every 2 days for 5 doses. (2) Brusatol monotherapy: 0.67 mg / kg. Administer daily from day 6 to day 34 after subcutaneous injection. (3) Combined administration of anti-PD-1 antibody and brusatol: A combination of (1) and (2) above. (4) Control (Vehicle): Saline solution was administered instead of anti-PD-1 antibody and brusatol.

[0055] After subcutaneous injection of B16F10 cells, the longest and shortest diameters of the subcutaneous tumors were measured over time. On day 35, the mice were euthanized and the tumors were removed. Tumor volume was calculated as (longest diameter) × (shortest diameter). 2 The tumor weight was calculated using a multiplier of 0.5236, and the weight of the excised tumor was measured. The results are shown in Figure 8B. As is clear from Figure 8B, antitumor effects were confirmed in both cases of administration of anti-PD-1 antibody alone and administration of brusatol alone. However, it was confirmed that co-administration of anti-PD-1 antibody and brusatol produced a significantly greater antitumor effect compared to administration of either drug alone.

[0056] Next, the proportion of cytotoxic T cells infiltrating tumor cells extracted from mice was examined. Specifically, the tumor was dissected with scissors, 5 mL of Accumax (Nakalai) was added to a 15 mL tube containing tumor fragments, and the mixture was incubated on a shaker at 200 rpm at 37°C for 1 hour. The cell suspension was centrifuged at 460×g for 5 minutes, the cell pellet was collected, the supernatant was discarded, the cells were resuspended in 1 mL of ACK lysis buffer, and the suspension was allowed to stand on ice for 1 minute. After centrifugation at 460×g for 5 minutes at 4°C, the supernatant was discarded, the cell pellet was resuspended in 600 μL of DMEM containing 10% FBS supplemented with Protein Transport Inhibitor Cocktail (1:500; 00-4980-93, Thermo Fisher) and Cell Stimulation Cocktail (1:500; 00-4970-93, Thermo Fisher), and the cells were incubated at 37°C under 5% CO2 for 3 hours. Cell surface antigens were labeled with 1 μg / mL antibodies (PerCP anti mouse CD45, Biolegend, Cat #103129; PE / Cyanine7 anti mouse CD8a, Biolegend, Cat #100722; APC anti mouse CD3e) at 4°C for 30 minutes, followed by washing with ice-cold staining buffer. The cells were fixed with IC fixation buffer (Thermo Fischer Scientific) at 4°C for 24 hours, and permeabilized with Permeabilization Buffer (Thermo Fischer Scientific). Intracellular antigens were labeled with 1 μg / mL antibodies (Biolegend, Cat #100312; PE anti mouse IFN-γ, Biolegend, Cat #505807; FITC anti human / mouse Granzyme B, Biolegend, Cat #515403) at 4°C for 30 minutes, followed by washing with ice-cold staining buffer. Flow cytometry analysis was performed to examine the proportion of cytotoxic T cells infiltrating the tumor.

[0057] The results are shown in Figure 8C. As shown in Figure 8C, administration of bursatole reduces IFN-γ in B16F10 tumor cells + and ZGMB + This significantly increased the population of cytotoxic T cells. These results confirm that administration of brusatol suppressed SQSTM1 levels and PD-L1 expression in B16F10 tumor cells, thereby activating anti-tumor immunity in cytotoxic T cells.

[0058] <Example 3> Next, the experiment was conducted in the same manner as in Example 2, except that MOC2 was used as the tumor cell instead of B16F10 as in Example 2, and the experimental schedule shown in Figure 9A was followed. In addition, the excised tumor cells were used in the same procedure as in Experimental Example 3 to test for CD8 + Immunochemical staining of cytotoxic T cells was performed.

[0059] Figure 9B shows the results of tumor volume and tumor weight measurements, and Figure 9C shows the results of flow cytometry analysis. Figure 10 also shows the results of CD8 immunochemical staining. + The results of measuring the number of cytotoxic T cells are shown. As is clear from Figures 9B, 9C, and 10, when MOC2 was used instead of B16F10, antitumor effects were observed in both cases of anti-PD-1 antibody monotherapy and brusatol monotherapy, similar to Example 2. However, it was confirmed that combination therapy produced a significantly greater antitumor effect compared to monotherapy.

[0060] <Example 4> The experiment was conducted using the same procedure as in Example 3, except that an anti-PD-L1 antibody (Selleck) was used instead of the anti-PD-1 antibody used in Example 3. Figure 11 shows the results for tumor volume and tumor weight. As is clear from Figure 11, when anti-PD-L1 antibody and brusatol were administered together, no negative effects were observed, but no synergistic effects were observed either. Therefore, there is no problem with administering anti-PD-L1 antibody and brusatol together, but it was confirmed that anti-PD-1 antibody is preferable as an immune checkpoint inhibitor to be used in combination with brusatol. [Industrial applicability]

[0061] The expression inhibitor containing brusatol as an active ingredient disclosed in this application exhibits a novel mechanism of action, suppressing PD-L1 expression in tumor cells. Therefore, the expression inhibitor can be used as a standalone anticancer agent, or as an enhancer of the effects of other anticancer agents. Consequently, it is useful in the discovery of anticancer drugs. < / sgscr> < / actb>

Claims

1. A PD-L1 expression inhibitor for tumor cells, containing brusatol as the active ingredient.

2. The expression inhibitor according to claim 1, wherein the brusatol suppresses the expression of PD-L1 in the tumor cells by suppressing the expression of SQSTM1 in the tumor cells.

3. The expression inhibitor according to claim 1 or 2, wherein the expression inhibitor functions as an anticancer agent.

4. An expression inhibitor according to claim 1 or 2, used in combination therapy with anticancer drugs.

5. The expression inhibitor according to claim 4, wherein the anticancer agent is an anti-PD-1 antibody and / or cisplatin.

6. A combination drug for treating cancer, said combination drug is A first pharmaceutical product comprising the expression inhibitor described in claim 1 or 2 as an active ingredient, A second-class pharmaceutical product containing an anticancer drug as an active ingredient, Concomitant medications, including those mentioned above.

7. The combination drug according to claim 6, wherein the anticancer agent is an anti-PD-1 antibody and / or cisplatin.

8. An anticancer drug containing brusatol as its active ingredient.

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