Compositions comprising avenin i or specific analogs thereof or salts thereof

Abstract

A main object of the present invention is to provide a novel technique for activating T cell immune responses.SOLUTION: Solution to Problem The present inventors have found that the above problems can be solved by a compound represented by the general formula (I): wherein Sugar represents any monosaccharide, disaccharide, or trisaccharide; and R represents a hydrogen atom or an acyl group having 2 to 5 carbon atoms, or a salt thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a composition containing Arbenin I or a specific analogue thereof or a salt thereof, uses of the composition, and a method for treating a disease using Arbenin I or a specific analogue thereof or a salt thereof. [Background technology]

[0002] In recent years, immune checkpoint inhibitors have been developed as cancer immunotherapeutic drugs and have attracted considerable attention. Currently, immune checkpoint inhibitors widely used in clinical practice are anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies. PD-1 (Programmed Cell Death-1) is a receptor expressed on the surface of activated T cells. When PD-1 binds to its ligand, it suppresses T cell-mediated immune responses. In normal T cells, the interaction between PD-1 and PD-L1 suppresses excessive immune responses. On the other hand, some cancer cells and their surrounding cells express the PD-1 ligand, Programmed Cell Death-Ligand 1 (PD-L1), on their surface. These cells suppress T cell activity and avoid T cell attack by binding their own PD-L1 to PD-1 on the T cell surface. Anti-PD-1 and anti-PD-L1 antibodies inhibit the binding of PD-1 to PD-L1, thereby evading T cell suppression and promoting T cell activation, thereby enhancing attack on cancer cells. CTLA-4 (Cytotoxic T lymphocyte antigen-4) is also a receptor expressed on the surface of T cells, and when CTLA-4 binds to its ligand, it suppresses T cell activation. Anti-CTLA-4 antibodies inhibit the binding of CTLA-4 to its ligand, thereby promoting T cell activation and enhancing attack on cancer cells.

[0003] As mentioned above, immune checkpoint inhibitors, which have a fundamentally different mechanism from conventional anticancer drugs, have demonstrated remarkable therapeutic effects against various types of tumors. However, the efficacy of immune checkpoint inhibitors is partially limited by the dysfunction of T cells in the tumor microenvironment (Patent Document 1). For this reason, immune checkpoint inhibitors are ineffective in some patients. To address this issue, combination therapies combining molecularly targeted drugs with cancer immunotherapy have been investigated (Non-Patent Document 1). While such combination therapies have shown promising results against cancer subtypes expressing the target molecules of the molecularly targeted drugs, they have a major drawback: after several years of use, cancer cells may acquire drug resistance, leading to cancer recurrence. In light of this, the development of immune-enhancing agents that can convert the inactive immune state of the tumor microenvironment into an active one has recently been explored. In particular, TLR9 agonists and STING agonists are expected to indirectly activate T cells by stimulating antigen-presenting cells such as B cells, dendritic cells, and macrophages and inducing chemokines and cytokines. However, clinical application of these immune-enhancing agents remains challenging. One reason for this is that, to achieve effective antitumor effects, it is necessary to control the differentiation and activation of T cells in the right places, but it is difficult to control the differentiation and activity of T cells through other immune cells. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2024-045362 [Non-patent literature]

[0005] [Non-Patent Document 1] Zhu, S., Zhang, T., Zheng, L. et al. Combination strategies to maximize the benefits of cancer immunotherapy. J Hematol Oncol 14, 156 (2021). https: / / doi.org / 10.1186 / s13045-021-01164-5 Summary of the Invention [Problem to be solved by the invention]

[0006] In view of the above circumstances, the present inventors aimed to provide a novel technique for activating T cell immune responses. [Means for solving the problem]

[0007] The present inventors have discovered a compound of general formula (I): [ka] (wherein Sugar represents any monosaccharide, disaccharide, or trisaccharide; R represents a hydrogen atom or an acyl group having 2 to 5 carbon atoms) or a salt thereof, the inventors have found that the above-mentioned problems can be solved. Further improvements have been made, and the present disclosure has been completed.

[0008] The present disclosure includes, for example, the subject matter described in the following sections: Section 1. General formula (I): [ka] (wherein Sugar represents any monosaccharide, disaccharide, or trisaccharide; R represents a hydrogen atom or an acyl group having 1 to 5 carbon atoms) or a salt thereof. Section 2. General formula (I): [ka] (wherein Sugar represents any monosaccharide, disaccharide, or trisaccharide; R represents a hydrogen atom or an acyl group having 1 to 5 carbon atoms) or a salt thereof, for use in enhancing mitochondrial function in T cells. Section 3. General formula (I): [ka] (wherein Sugar represents any monosaccharide, disaccharide, or trisaccharide; R represents a hydrogen atom or an acyl group having 1 to 5 carbon atoms) or a salt thereof. Section 4. 1. A composition for use in the treatment of cancer, comprising: General formula (I): [ka] (wherein Sugar represents any monosaccharide, disaccharide, or trisaccharide; R represents a hydrogen atom or an acyl group having 1 to 5 carbon atoms) or a salt thereof, The composition, wherein the treatment comprises administration of an immune checkpoint inhibitor. Section 5. General formula (I): [ka] (wherein Sugar represents any monosaccharide, disaccharide, or trisaccharide; R represents a hydrogen atom or an acyl group having 1 to 5 carbon atoms) or a salt thereof, A composition used to enhance the antitumor effect of an immune checkpoint inhibitor. Section 6. The compound represented by the general formula (I) or a salt thereof is represented by the general formula (II): [ka] 6. The composition according to any one of items 1 to 5, which is a compound represented by the formula (wherein R represents a hydrogen atom or an acyl group having 1 or 2 carbon atoms) or a salt thereof. Section 7. For use in cancer immunotherapy, General formula (I): [ka] (wherein Sugar represents any monosaccharide, disaccharide, or trisaccharide; R represents a hydrogen atom or an acyl group having 1 to 5 carbon atoms) or a salt thereof. Section 8. For use in enhancing mitochondrial function in T cells General formula (I): [ka] (wherein Sugar represents any monosaccharide, disaccharide, or trisaccharide; R represents a hydrogen atom or an acyl group having 1 to 5 carbon atoms) or a salt thereof. Section 9. For use in MKK3 activation General formula (I): [ka] (wherein Sugar represents any monosaccharide, disaccharide, or trisaccharide; R represents a hydrogen atom or an acyl group having 1 to 5 carbon atoms) or a salt thereof. Section 10. For use in the treatment of cancer, including the administration of an immune checkpoint inhibitor, General formula (I): [ka] (wherein Sugar represents any monosaccharide, disaccharide, or trisaccharide; R represents a hydrogen atom or an acyl group having 1 to 5 carbon atoms) or a salt thereof. Section 11. For use in enhancing the antitumor effect of immune checkpoint inhibitors, General formula (I): [ka] (wherein Sugar represents any monosaccharide, disaccharide, or trisaccharide; R represents a hydrogen atom or an acyl group having 1 to 5 carbon atoms) or a salt thereof. Section 12. The compound represented by the general formula (I) or a salt thereof is represented by the general formula (II): [ka] 12. The compound or salt thereof according to any one of items 7 to 11, which is a compound represented by the formula (wherein R represents a hydrogen atom or an acyl group having 1 or 2 carbon atoms) or a salt thereof. Section 13. A method for cancer immunotherapy comprising administering to a subject in need thereof an effective amount of a compound of general formula (I): [ka] (wherein Sugar represents any monosaccharide, disaccharide, or trisaccharide; R represents a hydrogen atom or an acyl group having 1 to 5 carbon atoms) or a salt thereof. Section 14. A method for enhancing mitochondrial function in T cells, comprising administering to a subject an effective amount of a compound of general formula (I): [ka] (wherein Sugar represents any monosaccharide, disaccharide, or trisaccharide; R represents a hydrogen atom or an acyl group having 1 to 5 carbon atoms) or a salt thereof. Section 15. A method for activating MKK3, comprising administering to a subject an effective amount of a compound of general formula (I): [ka] (wherein Sugar represents any monosaccharide, disaccharide, or trisaccharide; R represents a hydrogen atom or an acyl group having 1 to 5 carbon atoms) or a salt thereof. Section 16. 1. A method for treating cancer, comprising administering to a subject in need thereof: administering an effective amount of an immune checkpoint inhibitor; and An effective amount of a compound of general formula (I): [ka] (wherein Sugar represents any monosaccharide, disaccharide, or trisaccharide; R represents a hydrogen atom or an acyl group having 1 to 5 carbon atoms) or a salt thereof. Section 17. A method for enhancing the anti-tumor effect of an immune checkpoint inhibitor, comprising administering to a subject in need thereof an effective amount of a compound of general formula (I): [ka] (wherein Sugar represents any monosaccharide, disaccharide, or trisaccharide; R represents a hydrogen atom or an acyl group having 1 to 5 carbon atoms) or a salt thereof. Section 18. The compound represented by the general formula (I) or a salt thereof is represented by the general formula (II): [ka] 18. The method according to any one of items 13 to 17, wherein the compound is a compound represented by the formula (wherein R represents a hydrogen atom or an acyl group having 1 or 2 carbon atoms) or a salt thereof. [Effects of the Invention]

[0009] The present disclosure provides a novel technique for activating T cells. More specifically, the technique of the present disclosure can enhance the mitochondrial function of T cells. Furthermore, the present disclosure can provide a technique for enhancing the tumor-suppressing effect of immune checkpoint inhibitors, a technique for activating protein kinase MKK3, and the like. [Brief explanation of the drawings]

[0010] [Figure 1a] Test 2-1-1. IL-2 production levels of Jurkat hPD-1 cells under each culture condition shown in the figure (n = 3, biologically independent experiments). αPD-1 (EH12.2H7) shown in the figure is an anti-PD-1 neutralizing antibody. Statistical significance (* P < 0.05) was determined by a two-sided unpaired t test. [Figure 1b]Test 2-1-1. The results of FACS analysis of PD-L1 expression levels on the cell surface are shown. [Figure 1c] Test 2-1-1. The results of detecting exosomal PD-L1 in the culture supernatant of MDA-MB-231 cells and MCF7 cells by PD-L1 / CD9 exome ELISA analysis are shown. [Figure 1d] Test 2-1-1. The results of qPCR analysis of full-length PD-L1 and soluble PD-L1 mRNA levels are shown. The genomic structures of full-length PD-L1 and soluble PD-L1 are shown at the bottom of the figure. Each genotype can be detected using a specific primer set. The primer sets used to detect soluble PD-L1 are F1 / R1 (shown as S1 in the electrophoresis photograph at the top of the figure; the same applies below) and F1 / R2 (S2). The primer sets used to detect full-length PD-L1 are F2 / R3 (FL1) and F2 / R4 (FL2). [Figure 1e] Test 2-1-1. A schematic diagram of a cell culture model reproducing the cancer microenvironment is shown. IL-2 production was measured in Jurkat hPD-1 cells treated with PHA / PMA, MDA-MB-231 cell culture supernatant (conditional medium, CM), and anti-PD-1 antibody. CM contains soluble PD-L1 and exosomal PD-L1. [Figure 1f] Test 2-1-1. IL-2 production levels of Jurkat hPD-1 cells under each culture condition shown in the figure (n=3, biologically independent experiments). Statistical significance (*P < 0.05) was determined by two-sided unpaired t test. [Figure 1g] Test 2-1-2. Summary of natural product screening results. The horizontal axis shows cell viability (%), and the vertical axis shows IL-2 recovery (%). [Figure 2a] Test 2-1-2. Show the chemical structures of Arbenine I and its analogues. [Figure 2b]Test 2-1-2. IL-2 production levels after the addition of Arbenin I or its analogs are shown (n=3, biologically independent experiments). Statistical significance (*P < 0.05) was determined by a two-sided unpaired t test. [Figure 2c] Test 2-1-2. Cell viability (%) after the addition of Arbenin I or its analogs is shown (n=3, biologically independent experiments). Statistical significance (*P < 0.05) was determined by a two-sided unpaired t test. [Figure 2d] Test 2-2: Shows the effect of adding Arvenin I on IL-2 production in the presence or absence of PHA / PMA (n=2, biologically independent experiments). [Figure 2e] Test 2-2. The results of FACS analysis of PD-1 expression levels on the surface of Jurkat cells are shown. [Figure 3a] Experiment 2-3. Chemoproteomic analysis of reactive cysteines using 12C- or 13C-isotope-labeled cysteine-reactive iodoacetamide alkyne probes (IA-light / heavy probes) is shown. The IA-light or IA-heavy probes were reacted with lysates of Jurkat cells cultured in the absence or presence of arvenin I, respectively. After click reaction with the azobiotin-azide handle, the samples were mixed and subjected to streptavidin-agarose enrichment, on-resin trypsin digestion, and NaSO cleavage. The released probe-labeled peptides were sequenced by LC-MS / MS analysis. [Figure 3b] Test 2-3. Results of quantitative proteome analysis are shown. The left panel shows an enlarged view of the area enclosed by the dotted line in the right panel, where eight cysteines whose probe labeling was completely competed for by Arvenin I are mapped. Proteins containing six cysteines previously identified as reactive cysteines in the cysteinome database are highlighted in bold. [Figure 4a] Test 2-3. Results of gel shift assay. Anti-Flag antibody was used for detection. [Figure 4b] Experiment 2-4 shows the effect of Arvenin I on the phosphorylation levels of p38MAPK, FOXO1, and MKK3. The protein to be detected is listed after "IB:" ("P-" indicates phosphorylated protein). In this experiment, Jurkat hPD-1 cells were treated with DMSO or Arvenin I for 24 hours, and then stimulated with PHA and PMA for 10 minutes. Asterisks indicate nonspecific bands. [Figure 4c] Experiment 2-4. IL-2 production levels in Jurkat hPD-1 cells under each culture condition indicated in the figure. After treatment with DMSO, Arvenin I, or Arvenin I + SB203580 for 48 hours, IL-2 production in Jurkat hPD-1 cells was measured by IL-2 ELISA analysis. [Figure 4d] Experiment 2-4. Western blotting analysis of MKK3 expression in Jurkat MKK3 knockdown (KD) cells. In this experiment, Cas9-expressing Jurkat E6-1 cells were transfected with MKK3 gRNA or a negative control vector gRNA (denoted "vec" in the figure) using electroporation. [Figure 4e] Experiment 2-4 shows the phosphorylation level of p38MAPK in Jurkat MKK3 KD cells and vector control cells. In this experiment, cells were stimulated with PHA / PMA for 10 minutes to activate the p38MAPK pathway. [Figure 4f] Experiments 2-4 show IL-2 production levels in Jurkat MKK3 KD cells and vector control cells. IL-2 production was measured by IL-2 ELISA analysis (n = 3, biologically independent experiments). Statistical significance (*P < 0.05, **0.05 < P < 0.1) was determined by a two-sided unpaired t test. [Figure 4g] Test 2-4 shows the results of confirming MKK3 overexpression in Jurkat cells by Western blotting. [Figure 4h]Experiment 2-4. IL-2 production in Jurkat MKK3 wild-type (wt) cells and Jurkat MKK3 C227S cells was measured by IL-2 ELISA analysis (n = 3, biologically independent experiments). Statistical significance (*P < 0.05) was determined by a two-sided unpaired t test. [Figure 4i] Experiment 2-4. In vitro MKK3 kinase activity was assessed using His-p38KR (referred to as "p38KRmt" in this figure) and Flag-MKK3 (referred to simply as "MKK3" in this figure). Phosphorylation of His-p38KR was detected with phospho-p38 antibody (P-p38). Covalent modification of MKK3 (Arvenin I - MKK3) was verified by the mobility shift of the MKK3 band detected with anti-Flag antibody. [Figure 4j] Experiment 2-4 shows the results of evaluating MKK3 kinase activity when a constitutively active MEKK3 mutant (Myc-MEKK3CA) was added. Addition of Myc-MEKK3CA induced phosphorylation of His-p38KR in an MKK3-dependent manner, and addition of arvenin I further enhanced phosphorylation of His-p38KR. [Figure 5a] Test 2-5. The results of evaluating the effect of Arbenin I on cytokine (IFN-γ) production in primary CD8+ T cells are shown. The numbers below the graph indicate the concentration (μM) of added Arbenin I or DMSO (control). 0 μM on the far right of the graph indicates the absence of either Arbenin I or DMSO (non-treatment). [Figure 5b] Test 2-5. The effect of Arbenin I on cell proliferation in primary CD8+ T cells was evaluated by thymidine incorporation analysis. The vertical axis of the graph indicates the level of incorporated 3H-labeled thymidine, with higher values ​​indicating more active cell proliferation. The numbers below the graph indicate the concentration (μM) of added Arbenin I or DMSO (control). 0 μM on the far right of the graph indicates the absence of either Arbenin I or DMSO (non-treatment). [Figure 5c] Experiment 2-5. The effects of Arvenin I (250 nM) on mitochondrial energy production in primary CD8+ T cells were evaluated. The left panel shows the time course of oxygen consumption rates (OCR). The right panel shows basal respiration and spare respiratory capacity (SRC) calculated from OCR. *p< 0.05 (two-sided unpaired t test). Data are shown as mean ± SEM (n = 8-10). [Figure 5d] Experiment 2-5. Mitochondrial energy production was evaluated in the same manner as in Figure 5c, except that the p38 MAPK inhibitor SB203580 was added. *p < 0.05 (one-way ANOVA). Data are shown as mean ± SEM (n = 8-10). [Figure 5e] Experiment 2-5. The effect of Arvenin I (250 nM) on mitochondrial ROS levels in primary CD8+ T cells was evaluated. The CD8+ T cells were classified into high (left), intermediate (middle), and low (right) levels based on their intracellular mitochondrial ROS content, and the results are expressed as a percentage of all CD8+ T cells evaluated. *p < 0.05; **p < 0.01 (one-way ANOVA). [Figure 6a] Test 2-6. An overview of the in vivo test is shown. [Figure 6b] Experiment 2-6. Results from wild-type (WT) mice are shown. The vertical axis indicates tumor volume (mm3), and the horizontal axis indicates the number of days since MC38 cell inoculation. Statistical significance (* P < 0.05) was determined by a two-sided unpaired t test. Data are shown as mean ± SEM (n = 4). [Figure 6c]Experiment 2-6. Results from immunodeficient model mice (Rag2 KO mice) are shown. "ns" indicates that a two-sided unpaired t test did not reveal statistical significance (* P < 0.05). Data are shown as mean ± SEM (n = 4-5). [Figure 7] The structure of MKK3 predicted by the protein structure prediction program AlphaFold is shown. C227 is the cysteine ​​to which Arvenin I binds, and S218 and T222 are the sites phosphorylated by MEKK3, an upstream kinase of MKK3. L70, L72, A74, Y75, V77, V78, K93, and D190 are ATP-binding sites. DETAILED DESCRIPTION OF THE INVENTION

[0011] Each embodiment included in the present disclosure will be described in more detail below. The present disclosure preferably includes, but is not limited to, a composition containing Arbenin I or a specific analogue thereof or a salt thereof, uses of the composition, and a method for treating a disease using Arbenin I or a specific analogue thereof or a salt thereof, and the present disclosure includes all that is disclosed herein and that can be recognized by a person skilled in the art. A composition included in the present disclosure containing Arbenin I or a specific analogue thereof or a salt thereof may be referred to as the "composition of the present disclosure."

[0012] 1. Arbenin I and certain of its analogues Arbenin I is the O-glycoside of cucurbitacin B (CuB), a steroidal natural compound, and has the following formula: [ka] Arbenin I is known to be contained in Cucurbitaceae plants such as Citrullus colocynthis. Specific analogs of Arbenin I encompassed by the present disclosure have the general formula (I): [ka] (wherein Sugar represents a group obtained by removing one hydrogen atom from any monosaccharide, disaccharide, or trisaccharide; R represents a hydrogen atom or an acyl group having 2 to 5 carbon atoms). Such compounds may be referred to as "analogs of the present disclosure."

[0013] In the above general formula (I), when R is an acyl group, the acyl group may be linear or branched. R is preferably a hydrogen atom or an acyl group having 2 to 4 carbon atoms, more preferably a hydrogen atom or an acyl group having 2 or 3 carbon atoms, still more preferably a hydrogen atom or an acetyl group, and particularly preferably an acetyl group.

[0014] In the above general formula (I), the sugar constituting the sugar is not particularly limited. Sugar may be, for example, a group formed by removing one hydrogen atom from a monosaccharide, disaccharide, or trisaccharide composed of one or more monosaccharides selected from the group consisting of glucose, galactose, mannose, ribose, deoxyribose, arabinose, xylose, rhamnose, fucose, and fructose. The monosaccharides described above may be L- or D-isomers, or a mixture of L- and D-isomers, with the enantiomer that is more abundant in nature being preferred. Therefore, sugar is preferably a group formed by removing one hydrogen atom from a monosaccharide, disaccharide, or trisaccharide composed of one or more monosaccharides selected from the group consisting of D-glucose, D-galactose, D-mannose, D-ribose, D-deoxyribose, L-arabinose, D-xylose, L-rhamnose, L-fucose, and D-fructose. The monosaccharides may be α-type, β-type, or a mixture of α-type and β-type, and are preferably the anomer that is more abundant in nature.

[0015] More specific examples of disaccharides include maltose, sucrose, lactose, cellobiose, trehalose, lactulose, cellobiose, isomaltose, isotrehalose, neotrehalose, turanose, palatinose, and mannobiose. More specific examples of trisaccharides include maltotriose, melezitose, raffinose, nigerotriose, and kestose.

[0016] Although not particularly limited, Sugar is preferably a group obtained by removing one hydrogen atom from a monosaccharide or disaccharide, more preferably a group obtained by removing one hydrogen atom from a monosaccharide, and even more preferably a group obtained by removing one hydrogen atom from glucose. The glucose is more preferably D-glucose, and even more preferably β-D-glucose. The hydrogen atom to be removed may be a hydrogen atom constituting any of the hydroxy groups possessed by the sugar. Sugar is most preferably a group obtained by removing a hydrogen atom from the hydroxy group directly bonded to the 1-carbon of β-D-glucose.

[0017] In one preferred embodiment of the analogs of the present disclosure, the analogs have the general formula (II): [ka] (wherein R represents a hydrogen atom or an acyl group having 2 or 3 carbon atoms). A more preferred embodiment of the analog of the present disclosure is a compound represented by the following formula: [ka] This compound has a structure in which R is a hydrogen atom and Sugar is a group formed by removing a hydrogen atom from the hydroxy group directly bonded to the 1-carbon atom of β-D-glucose in general formula (I), and is also called Arbenine III.

[0018] In the technology of the present disclosure, it is preferable to use Arbenin I and / or Arbenin III, and it is particularly preferable to use Arbenin I. When Arbenin I is used in the technology of the present disclosure, particularly excellent effects such as enhancing mitochondrial function in T cells, activating MKK3, suppressing tumors, and enhancing the effects of cancer immunotherapy are expected. These effects will be described later.

[0019] In the technology of the present disclosure, Arbenin I and the analogs of the present disclosure may optionally form pharmaceutically acceptable salts. Unless otherwise specified in the present disclosure, the descriptions of Arbenin I and / or the analogs of the present disclosure also apply to these salts.

[0020] Specific salts include, for example, alkali metal salts such as sodium salt, potassium salt, and lithium salt; alkaline earth metal salts such as calcium salt and magnesium salt; metal salts such as aluminum salt, iron salt, zinc salt, copper salt, nickel salt, and cobalt salt; inorganic salts such as ammonium salt; t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetrahydrofuran ... Examples of the amine salt include organic salts such as methylammonium salts and tris(hydroxymethyl)aminomethane salts; hydrohalogenated salts such as hydrofluoride, hydrochloride, hydrobromide and hydroiodide; inorganic salts such as nitrate, perchlorate, sulfate and phosphate; lower alkanesulfonates such as methanesulfonate, trifluoromethanesulfonate and ethanesulfonate; arylsulfonates such as benzenesulfonate and p-toluenesulfonate; organic salts such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate and maleate; and amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate and aspartate.

[0021] In the technology of the present disclosure, one or more of the above-mentioned Arbenin I, analogs of the present disclosure, and salts thereof can be used alone or in combination.

[0022] Arbenin I and analogs of the present disclosure can be prepared by conventionally known methods. For example, they can be prepared by organic chemical synthesis using any commercially available raw material compound as the starting material. More specifically, for example, cucurbitacin B can be used as the starting material, and any sugar can be added, or the analogs can be subjected to hydrolysis or transesterification to produce Arbenin I and analogs of the present disclosure. In addition, since some Arbenin I and analogs of the present disclosure are contained in plants of the Cucurbitaceae family, Arbenin I or analogs of the present disclosure can also be purified or extracted from such plants by known methods. Furthermore, Arbenin I or analogs of the present disclosure that are generally available on the market can be purchased and used in the technology of the present disclosure.

[0023] 2. Uses of the Technology of the Present Disclosure As shown in the Examples below, the present inventors have found that Arbenin I and analogs of the present disclosure can activate T cells. More specifically, they have found that Arbenin I and analogs of the present disclosure can enhance mitochondrial function in T cells by covalently binding to protein kinase MKK3 and activating MKK3. The present inventors have also found in vivo that Arbenin I and analogs of the present disclosure can have tumor-suppressing activity. Therefore, the technology of the present disclosure can be suitably used for at least one application selected from the group consisting of cancer immunotherapy, tumor suppression, T cell activation, enhancement of mitochondrial function in T cells, and MKK3 activation.

[0024] The above-described effects of Arbenin I and analogs of the present disclosure can be confirmed by known methods and / or methods similar to those described in the present Examples, or by methods that can be easily derived by those skilled in the art from these methods. For example, the effect of activating T cells can be confirmed by the recovery of IL-2 production, which was reduced by PD-L1, by the addition of a test substance in a cell culture model reproducing the cancer microenvironment described in Example 2-1-1. The effect of activating MKK3 can be confirmed by the enhancement of phosphorylation of the MKK3 target protein p38MAPK in the presence of the test substance. The effect of enhancing T cell mitochondrial function can be confirmed by the increase in mitochondrial activity in T cells, more specifically, at least one selected from the group consisting of basal mitochondrial respiration, maximal respiration, and spare respiratory capacity (SRC), in the presence of the test substance. The tumor-suppressing effect can be confirmed in vivo by the suppression of tumor volume increase by the administration of the test substance. Furthermore, the effect can be confirmed in vitro by the suppression of tumor cell proliferation by the addition of the test substance.

[0025] Furthermore, the present inventors have found that Arbenin I and its analogs of the present disclosure can enhance the tumor-suppressing effect of immune checkpoint inhibitors. Therefore, the technology of the present disclosure can be suitably used in combination with immune checkpoint inhibitors and / or to enhance the tumor-suppressing effect of immune checkpoint inhibitors. Specific examples of immune checkpoint inhibitors include at least one selected from the group consisting of PD-1 inhibitors, PD-L1 inhibitors, and CTLA-4 inhibitors, and preferably at least one selected from the group consisting of anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies. The antibody may be a polyclonal or monoclonal antibody, preferably a monoclonal antibody. More specifically, examples of anti-PD-1 antibodies include nivolumab and pembrolizumab. Examples of anti-PD-L1 antibodies include atezolizumab, durvalumab, and avelumab. Examples of anti-CTLA-4 antibodies include ipilimumab and tremelimumab. Although not particularly limited, in the technology of the present disclosure, it is particularly preferred that Arbenin I and / or its analogs be used in combination with immune checkpoint inhibitors, particularly anti-PD-1 antibodies and / or anti-PD-L1 antibodies, and / or be used to enhance the tumor-suppressing effects of such antibodies.

[0026] Furthermore, Arbenin I and analogs of the present disclosure, which have the effect of activating T cells, are expected to enhance the effects of not only immune checkpoint inhibitors but also other cancer immunotherapies. Specific examples of other cancer immunotherapies include cancer vaccine therapy, T cell receptor (TCR) T cell (TCR-T) therapy, and chimeric antigen receptor (CAR) T cell (CAR-T) therapy. In the technology of the present disclosure, Arbenin I and / or analogs of the present disclosure can be used to enhance the effects of these cancer immunotherapies. In embodiments where the technology of the present disclosure is used in combination with cancer immunotherapies in which modified T cells are administered to a subject, such as TCR-T therapy and CAR-T therapy, Arbenin I and / or analogs of the present disclosure may be applied to the modified T cells before administration to a subject.

[0027] The technology of the present disclosure can be applied to, for example, humans and non-human mammals (e.g., rats, mice, rabbits, cows, pigs, dogs, cats, sheep, monkeys, etc.), with humans being preferred.

[0028] More specifically, subjects to which the technology of the present disclosure can be applied include subjects with cancer. As described above, there are no particular limitations on the cancers to which the technology of the present disclosure can be applied. As described in the Background Art section, the effectiveness of immune checkpoint inhibitors is partially limited by the dysfunction of T cells in the cancer microenvironment, and there are subjects for whom immune checkpoint inhibitors are ineffective. However, the technology of the present disclosure, which can activate T cells, can be suitably used for subjects who have not previously been able to fully enjoy the effects of immune checkpoint inhibitors, and for whom immune checkpoint inhibitors have not been effective.

[0029] In the present disclosure, the term "cancer" encompasses cancer, neoplasms, and malignant tumors. There are no particular limitations on the cancer to which the technology of the present disclosure is applied. Examples of cancer include gastric cancer, colorectal cancer (colon cancer, rectal cancer), esophageal cancer, liver cancer, pancreatic cancer, gallbladder cancer, malignant melanoma, kidney cancer, breast cancer, mesothelioma, bladder cancer, intraperitoneal disseminated cancer, lung cancer, soft tissue sarcoma, osteosarcoma, brain tumor, head and neck cancer, neuroblastoma, leukemia, lymphoma, prostate cancer, retinoblastoma, ciliary body tumor, basal cell carcinoma, squamous cell carcinoma, malignant soft tissue tumor, chondrosarcoma, ovarian cancer, uterine cancer, and cervical cancer.

[0030] The application method of the technology of the present disclosure is not particularly limited as long as the desired effect is achieved. For example, Arbenin I of the present disclosure or an analog thereof, or a composition containing the same may be administered by injection into or around a lesion, or by injection or infusion into a vein, an artery, an intraperitoneal cavity, an intramuscular cavity, a subcutaneous cavity, an intrapleural cavity, or the like, or by perfusion via a catheter, or may be administered orally.

[0031] The frequency of application of the technology of the present disclosure is not particularly limited. For example, it may be applied once or multiple times a day, once or multiple times a week, once or multiple times a month, or once or multiple times a year. The application period of the technology of the present disclosure is also not particularly limited, as long as the effects of the present disclosure are achieved. Furthermore, the dose of Arbenin I or an analog of the present disclosure is also not particularly limited, as long as the effects of the present disclosure are achieved. The application frequency, application period, and dose can be adjusted appropriately by those skilled in the art depending on the condition of the subject, the course of treatment, etc.

[0032] The technology of the present disclosure may be optionally combined with other pharmaceutical compositions and / or treatment methods, etc., that are applied to a subject with cancer. When the technology of the present disclosure is combined with other pharmaceutical compositions and / or treatment methods, etc., they may be applied to a subject simultaneously, or may be applied separately at any time. Furthermore, when the technology of the present disclosure is used in combination with other cancer immunotherapy, the technology of the present disclosure may be applied before, after, during, or simultaneously with the cancer immunotherapy.

[0033] In the technology of the present disclosure, Arbenin I or an analogue of the present disclosure may be mixed with a pharmaceutically acceptable base, carrier, excipient, diluent, solubilizer, emulsifier, preservative, pH adjuster, adjuvant, chelating agent, etc. These components may be used alone or in combination of two or more. That is, the present disclosure encompasses compositions containing Arbenin I and / or an analogue of the present disclosure. Such compositions may be referred to as "compositions of the present disclosure." The matters described with respect to Arbenin I and analogues of the present disclosure are applicable to compositions of the present disclosure, and the matters described with respect to compositions of the present disclosure are applicable to Arbenin I and analogues of the present disclosure.

[0034] Examples of preservatives include parabens such as methylparaben, ethylparaben, propylparaben, and butylparaben, sodium benzoate, phenoxyethanol, alkyldiaminoethylglycine hydrochloride, etc. The preservatives may be used alone or in combination of two or more.

[0035] Examples of pH adjusters include citric acid, phosphoric acid, malic acid, pyrophosphoric acid, lactic acid, tartaric acid, glycerophosphoric acid, acetic acid, nitric acid, and chemically possible salts thereof, as well as sodium hydroxide, potassium hydroxide, etc. pH adjusters can be used alone or in combination of two or more so that the pH of the composition of the present disclosure falls within the range of 4 to 8, preferably 5 to 7.

[0036] The method for preparing the composition of the present disclosure is not particularly limited as long as the effects of the present disclosure are achieved. For example, the composition can be prepared according to a method known in the art. More specifically, the composition can be prepared by mixing Arbenin I and / or an analogue of the present disclosure and other ingredients with sterile distilled water.

[0037] The present disclosure also encompasses a cancer treatment kit comprising Arbenin I and / or an analogue of the present disclosure and a cancer immunotherapy drug (preferably an immune checkpoint inhibitor, more preferably an anti-PD-1 antibody and / or an anti-PD-L1 antibody). Such a cancer treatment kit may be referred to as the "kit of the present disclosure." The matters described with respect to Arbenin I and an analogue of the present disclosure, and the composition of the present disclosure are incorporated by reference into the kit of the present disclosure, as appropriate, and the matters described with respect to the kit of the present disclosure are incorporated by reference into Arbenin I and an analogue of the present disclosure, and the composition of the present disclosure, as appropriate.

[0038] The timing of administration of Arbenin I and / or the analogs of the present disclosure and the cancer immunotherapy medicament contained in the kit of the present disclosure is not particularly limited as long as the desired effect is achieved. For example, all components may be administered simultaneously, or each component may be administered at a different time, or two or three components may be administered simultaneously with the remaining components administered at different times. When not all components are administered simultaneously, the interval between each administration is not limited as long as the desired effect is achieved, and the order of administration is also not limited. Furthermore, administration of any component may be repeated until the desired effect is achieved. Furthermore, the administration route of each component may be the same or different.

[0039] 3. Possible mechanism Without wishing to be bound by any theory, it is presumed that the technology of the present disclosure, Arbenin I and its analogs of the present disclosure, activate T cells and thereby exert tumor-suppressing effects through the following mechanism: Arbenin I and its analogs of the present disclosure covalently bind to Cys227 of the protein kinase MKK3, changing the conformation of MKK3. This makes it easier for MEKK3, an upstream kinase of MKK3, to phosphorylate MKK3. Activated MKK3, phosphorylated by MEKK3, enhances mitochondrial energy production in T cells through the phosphorylation of p38 MAPK. T cells that have obtained sufficient energy can be activated and exert tumor-suppressing effects.

[0040] Based on this presumed mechanism, it is reasonably predicted that the technology of the present disclosure will be effective against all types of cancer, and that the efficacy of all types of cancer immunotherapy will be enhanced by combining it with the technology of the present disclosure.

[0041] In this specification, the term "comprising" includes "essentially consisting of" and "consisting of" in addition to "containing." Furthermore, the present disclosure includes all arbitrary combinations of the constituent elements described in this specification.

[0042] Furthermore, the various characteristics (numerical values, properties, structures, functions, etc.) described in each embodiment of the present disclosure may be combined in any way to specify the subject matter encompassed by the present disclosure. In other words, the present disclosure encompasses all subject matter consisting of all combinations of the combinable characteristics described herein. [Example]

[0043] Hereinafter, the embodiments of the present disclosure will be described more specifically with reference to examples, but the embodiments of the present disclosure are not limited to the following examples.

[0044] 1. Materials and Methods 1-1. Cell culture Jurkat cells, modified Jurkat cells, MDA-MB-231, and MC38 cells were maintained under standard culture conditions (37°C, 5% CO) in Roswell Park Menorial Institute 1640 medium (RPMI 1640, Gibco, #11875119) supplemented with 10% (v / v) fetal bovine serum (FBS, Biowest) and 1% (v / v) penicillin-streptomycin (Nacalai Tesque, #26253-84). HEK293 cells were maintained under standard culture conditions (37°C, 5% CO) in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, #11995073) supplemented with 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin. In most experiments, the medium was changed to RPMI containing 1% (v / v) FBS and 1% (v / v) penicillin-streptomycin mixture.

[0045] 1-2. Cell culture model of T cell inactivation in a cancer-competitive environment For the coculture of Jurkat hPD-1 cells and MDA-MB-231 cells, each cell was washed twice with RPMI containing 1% (v / v) FBS and 1% (v / v) penicillin-streptomycin. Jurkat hPD-1 cells were added to wells containing MDA-MB-231 cells at a ratio of 1:2 (Jurkat cells:MDA-MB-231 cells). The cells were cocultured for 48 hours with or without 3 μg / ml PHA (Sigma, #L9017) and 2 ng / ml PMA (Fujifilm Wako, #168-23593). After 48 hours of incubation, the supernatant was collected.

[0046] In a cell culture model reproducing the tumor microenvironment, we used MDA-MB-231 cell culture supernatant (conditional medium, sometimes abbreviated as CM) containing soluble PD-L1 (sPD-L1) and PD-L1-positive exosomes (exoPD-L1). Jurkat hPD-1 cells were cultured at a density of 2.5 × 10 in RPMI containing 1% (v / v) FBS and 1% (v / v) penicillin-streptomycin. 4Cells were plated in 96-well plates at a density of 1000 ng / mL and stimulated with 3 μg / mL PHA and 2 ng / mL PMA for 48 hours. For compound screening, PHA / PMA-stimulated Jurkat hPD-1 cells were cultured in RPMI containing 70% CM and each compound was added. After 48 hours of culture, the supernatant was collected. Secreted IL-2 concentrations in the medium were measured using an IL-2-specific ELISA kit (R&D systems, #D2050) according to the recommended protocol. Cell viability was also measured using Cell counting kits-8 (Dojindo, #341-07624) according to the recommended protocol.

[0047] 1-3. Proteome labeling of reactive cysteine ​​residues Isotopically labeled IA-probes (IA-heavy and IA-light) were synthesized and used to prepare proteome samples. Prior to proteome labeling with the IA probes, Jurkat cells were treated with DMSO (vehicle) or 4 μM avernine I in the presence of 0.3 μg / ml PHA and 2 ng / ml PMA for 24 hours. Lysates of DMSO-treated and avernine I-treated Jurkat cells were then labeled with IA-light and IA-heavy, respectively, as test samples. As a control, lysates of DMSO-treated Jurkat cells were labeled with IA-light or IA-heavy. Specifically, the soluble fraction (2 mg / ml protein) of each cell lysate was reacted with 100 μM IA-heavy or IA-light in 2 ml of 1× PBS containing ×1 / 100 protease inhibitor cocktail (Nacalai Tesque, #25955) at 25°C for 60 minutes.

[0048] After the reaction, each sample underwent copper-catalyzed azide-alkyne cycloaddition (CuAAC, sometimes referred to as click reaction) using 100 μM azobiotin azide (Sigma), 5 mM sodium ascorbate, 2 mM THPTA (tris(3-hydroxypropyltriazolylmethyl)amine, Sigma, #762342), and 1 mM CuSO4 at 25°C for 2 h. The tagged proteins were precipitated by adding three volumes of cold acetone, collected by centrifugation (6,500 x g, 4 min), and washed twice with cold methanol. The probe-labeled samples were mixed (DMSO vs. arvenin I for test samples, DMSO vs. DMSO for control samples) and solubilized in 1 ml of 1x PBS containing 1.2% SDS by sonication and heating (80°C, 5 min). The sample was diluted with 5 ml of 1x PBS and incubated overnight at 4°C with 100 μl of streptavidin agarose beads (Thermo Fisher Scientific). The beads were washed with 0.2% SDS / 1x PBS (1x), 1x PBS (3x), and water (3x) and then suspended in 500 μl of 6 M urea / 1x PBS. The suspension was treated with 10 mM DTT at 65°C for 15 minutes and 10 mM iodoacetamide at 37°C for 30 minutes. The beads were pelleted by centrifugation (1,400 g, 2 minutes) and subjected to on-bead trypsin digestion with sequencing-grade modified trypsin (Promega, #V5111) in 200 μl of 1x PBS containing 1.0 mM calcium chloride. After washing with 1× PBS (×3) and water (×3), tryptic peptides were cleaved with Na2S2O4 (25 mM ×2 and 50 mM ×1, ​​Sigma, #157953) at 25°C for 1 h.

[0049] 1-4. Quantitative chemoproteomic analysis of reactive cysteine ​​residues Proteome samples for mass spectrometry were fractionated into eight fractions using an SDB-SCX stage chip (GL Science) according to published methods. Each fraction was dried under vacuum and dissolved in an aqueous solution containing 2% acetonitrile and 0.1% formic acid. Mass spectra were acquired using an LTQ-Orbitrap Velos Pro (Thermo Fisher Scientific) coupled to a nanoflow UHPLC system (ADVANCE UHPLC, AMR Inc.) equipped with an Advanced Captive Spray SOURCE (AMR Inc.). The fractionated peptides were loaded onto a C18 trap column (L-column C18, 5 μm particle size, 0.3 × 5 mm, CELI) and fractionated on an analytical column (L-column C18, 3 μm particle size, 0.075 × 150 mm, CELI) using a linear gradient from 5% B (0 min) to 35% B (155 min). The column was then washed with 90% B and re-equilibrated with 5% B at a flow rate of 300 nL / min. The total run time was 180 min. The solvent composition was Buffer A: 100% H2O, 0.1% formic acid (Thermo Fisher Scientific), Buffer B: 100% acetonitrile (Thermo Fisher Scientific).

[0050] The mass spectrometer was programmed to perform 11 consecutive scans. The first scan was a full-scan MS scan from 350 to 2,000 m / z using the Orbitrap at a resolution of 60,000. Scans 2 through 11 were automated data-dependent MS / MS scans using the ion trap analyzer of the 10 most intense ion signals from the first precursor scan. For MS / MS spectra, the normalized collision energy of the ion trap was set to 35% CID, and the exclusion time for molecules in the same m / z range was set to 90 seconds. Raw data files were processed using the SequestHT algorithm node (Thermo Fisher Scientific) in Proteome Discoverer 2.4 software. The precursor mass tolerance was set to 10 ppm, and the product tolerance was set to 0.8 Da. The following dynamic modifications were configured: methionine oxidation, protein N-terminal acetylation, cysteine ​​carbamidomethylation, and cysteine ​​IA-Light / Heavy labeling. For quantification of IA labeling, the following settings were used in the Quan channel: IA-Light was cysteine ​​+393.170, and IA-Heavy was cysteine ​​+369.190.

[0051] 1-5. Preparation of cell lysates for Western blotting For gel shift assays, HEK 293 cells transfected with epitope tag plasmids were treated for 24 hours under standard culture conditions (37°C, 5% CO2) with DMSO or Arvenin I at the concentrations indicated in the corresponding figures. Cells were lysed by sonication in 1x PBS containing 1 / 100 protease inhibitor cocktail. To analyze the effect of Arvenin I on the status of each protein in Jurkat hPD-1 cells, Jurkat hPD-1 cells were treated with DMSO or 4 μM Arvenin I under standard culture conditions (37°C, 5% CO2) for 24 hours. Each Jurkat hPD-1 cell sample was stimulated with 3 μg / ml PHA and 2 ng / ml PMA for 10 minutes under standard culture conditions (37°C, 5% CO2). Cells were harvested and lysed for 10 minutes on ice in a lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 / 100 protease inhibitor cocktail, and 1 / 100 phosphatase inhibitor. The cell lysates were centrifuged (14,000 g, 20 minutes). The total protein concentration in the soluble cell lysates was measured by Bradford protein assay, and each soluble cell lysate was adjusted to the same concentration.

[0052] 1-6. In vitro MKK3 kinase analysis Recombinant Flag-MKK3 wt and Myc-MEKK3CA were produced by expression in HEK293 cells and purified by immunoprecipitation. Recombinant His-p38KR mutant was produced in E. coli and purified with Ni-NTA agarose. Flag-MKK3 wt was treated with DMSO or 4 μM Arvenin I in kinase buffer at 25°C for 1 hour. Flag-MKK3 wt was reacted with Myc-MEKK3CA and His-p38KR in the presence or absence of ATP at 30°C for 10 minutes. The reaction was stopped by adding SDS sample buffer and then boiled at 95°C for 5 minutes.

[0053] 1-7. Western blotting For gel electrophoresis, each sample was loaded onto an 8% or 10% acrylamide SDS gel and transferred to a nitrocellulose membrane (Amersham Protoran NC0.45, 0.45 μm, Cytiva) using a Mini Trans-Blot system (Bio-Rad). The membrane was blocked for 30 minutes with 1x PBS containing 0.1% (w / v) Tween-20 and 3% (w / v) skim milk (blocking buffer), washed with 1x PBS containing 0.1% (w / v) Tween-20 (PBST), and incubated overnight at 4°C with primary antibodies in blocking buffer or Can Get Signal Immunoreaction Enhancer Solution #1 (Toyobo, #NKB-101). After washing with PBST, the membrane was probed with horseradish peroxidase-conjugated secondary antibodies in blocking buffer or Can Get Signal Immunoreaction Enhancer Solution #2 for 1 hour at room temperature. The membrane was washed with PBST and then treated with ECL Western blotting detection reagent (Cytiva). Protein bands were visualized using an ImageQuant LAS 500 (GE Healthcare).

[0054] 1-8. IFN-γ ELISA analysis The IFN-γ concentration in the culture supernatant was measured using ELISA MAX Deluxe Set Mouse IFN-γ (Biolegend) according to the recommended protocol.

[0055] 1-9. Thymidine incorporation analysis 3 The H-labeled thymidine solution was diluted with RPMI (RPMI medium containing 10% FCS, 1% (v / v) penicillin-streptomycin mixed solution, 50 μM 2-mercaptoethanol, L-glutamine, sodium pyruvate, and NEAA) and added to the cells. The cells were then incubated at 37°C in a humidified incubator with 5% CO for 4 hours. After incubation, the cells were transferred to a 96-well filter plate and scintillation buffer was added.3 H-thymidine incorporation was measured in a Microbeta2 microplate counter (PerkinElmer, # 2450-0120).

[0056] 1-10. Analysis of mitochondrial activity The isolated cells were seeded onto XFe96 plates (3 × 10 per well). 5 Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using an XFe96 extracellular flux analyzer (Seahorse Bioscience). Oligomycin, FCCP, and rotenone / antimycin A were used as per the XFe Cell Mito Stress Test Kit (Seahorse Bioscience). Basal respiration was defined as (the last respiratory rate measurement before oligomycin addition) minus (the non-mitochondrial respiratory rate after rotenone / antimycin A addition). Maximum OCR was defined as (the first rate measurement after FCCP addition) minus (the non-mitochondrial respiratory rate). Spare respiratory capacity (SRC) was calculated by subtracting basal respiration from maximum OCR.

[0057] 1-11. Analysis of mitochondrial ROS levels First CD8 + T cells were stimulated with anti-CD3 and anti-CD28 antibody-coated beads for 2 hours in the presence or absence of Arvenin I, and then mitochondrial ROS levels were analyzed using MitoSOX (Mitochondrial Superoxide Indicator) (Thermo Fisher Scientific) according to the recommended protocol.

[0058] 1-12. Mouse treatment model C57BL / 6N WT mice were obtained at 6 weeks of age from Charles River Laboratories Japan or Shimizu Laboratory Supplies Japan. The RAG2- / - (RAG2 NO) inbred mouse strain was maintained under specific pathogen-free conditions at the Institute for Experimental Animals, Graduate School of Medicine, Kyoto University. MC38 cells (5 × 10 5 Mice were injected intradermally (100 μl) into the right flank (day 0). From day 10 to day 22 after inoculation, Arbenin I was administered intraperitoneally every 3 days at a dose of 6 mg / kg, and anti-PD-L1 monoclonal antibody was administered intraperitoneally every 6 days at a dose of 30 μg. Tumor volume was calculated using digital caliper measurements using the formula π × (length × width × height) / 6.

[0059] 1-13. Antibodies The antibodies used in this example are shown in the table below. [Table 1]

[0060] 2. Results 2-1. Search for T cell activators 2-1-1. Development of cell culture models that reproduce the cancer microenvironment We first developed a cell culture model that mimics the tumor microenvironment in which T cells are suppressed through PD-1 / PD-L1 interaction. Specifically, we stably transfected Jurkat cells, derived from human helper T cells, with an expression vector encoding PD-1. This cell line is designated Jurkat hPD-1 cells. Stimulation of the resulting cell line with phorbol 12-myristate 13-acetate (PMA) and phytohemagglutinin (PHA) activated the TCR pathway and led to the production of IL-2, a cytokine marker of T cell activation (Figure 1a). IL-2 was detected by ELISA. PMA is an activator of protein kinase C (PKC), and PHA is a lectin that induces T lymphocyte cell division.

[0061] Jurkat hPD-1 cells were inactivated by co-culture with MDA-MB-231 cells, a human breast cancer cell line that expresses high levels of PD-L1, resulting in reduced IL-2 production (Fig. 1a, b). The reduced IL-2 production was restored by the addition of an anti-PD-1 neutralizing antibody (Fig. 1a). These results confirmed that PD-1 / PD-L1 interaction suppresses IL-2 production.

[0062] Furthermore, soluble PD-L1 and exosomal PD-L1 were present in the culture supernatant of MDA-MB-231 cells (Fig. 1c, d). These findings confirmed that MDA-MB-231 cells secrete soluble and exosomal PD-L1. When Jurkat hPD-1 cells were cultured in the culture supernatant of MDA-MB-231 cells (conditional medium, sometimes abbreviated as CM) containing these secreted PD-L1 molecules, the effects on Jurkat hPD-1 cells observed in the coculture with MDA-MB-231 cells were reproduced (Fig. 1e, f). Therefore, the present inventors established a cell culture model in which Jurkat hPD-1 cells were cultured in the CM containing secreted PD-L1 molecules, reproducing the tumor microenvironment in which T cells are suppressed via PD-1 / PD-L1 interaction.

[0063] 2-1-2. Compound screening Next, we used the cell culture model developed in 2-1-1 to screen for small molecules that activate T cells, using the ability to restore IL-2 production from Jurkat hPD-1 cells as an indicator. First, we screened 397 endogenous metabolites, but no significant hits were found that restored IL-2 production (data not shown). Next, we screened 232 natural products, and identified arbenin I (cucurbitacin B 2-O-β-D-glucoside, CuBg) as a potential hit (Figure 1g). Arbenin I is the O-glycoside of the steroidal natural product cucurbitacin B (CuB) (Figure 2a). Arbenin I is a natural component found in Cucurbitaceae plants, and several analogs exist naturally (Figure 2a). Arbenin III, which has a structure in which the acetyl group of arbenin I is replaced with a hydroxyl group, was also identified as a hit in this screening (Figure 1g).

[0064] 2-1-3. Structure-activity relationships Based on the above results, the inventors further evaluated the T cell activation activity of Arbenin I and its analogs. Specifically, Arbenin I, CuB (which lacks the glucose moiety of Arbenin I, i.e., the aglycone of Arbenin I), Arbenin III, CuD (the aglycone of Arbenin III), and Arbenin II (which has a saturated double bond (Michael acceptor) in Arbenin I, where Michael addition can occur), were added to the cell culture model and the recovery of IL-2 production was evaluated (Figures 2a and 2b). In this test, Arbenin I and Arbenin III also restored IL-2 production (Figure 2b). On the other hand, CuB and CuD failed to restore IL-2 production (Figure 2b). Furthermore, Arbenin II showed no detectable ability to restore IL-2 production (Figure 2b). Arbenin I, Arbenin II, and Arbenin III did not exhibit significant cytotoxicity at 4 μM (Figure 2c).

[0065] These results suggest that the glycoside structure and Michael acceptor shared by Arbenin I and Arbenin III may be important for T cell activation. On the other hand, Arbenin III, which has a structure in which the acetyl group of Arbenin I is replaced with a hydroxy group, also maintained its T cell activation activity, suggesting that the T cell activation activity can be maintained even if the structure of the acetyl group moiety is modified to some extent.

[0066] 2-2. Analysis of Arbenin I activity We analyzed the activity of Arbenin I in the presence or absence of PHA / PMA. In the presence of PHA and PMA, Arbenin I restored IL-2 production in a concentration-dependent manner (Fig. 2d). However, in the absence of PHA and PMA, Arbenin I did not detectably restore IL-2 production (Fig. 2d). These results suggest that Arbenin I does not activate T cells independently, but rather enhances PHA / PMA-induced T cell activation.

[0067] Since PHA and PMA upregulated PD-1 expression on the cell surface of Jurkat cells, we investigated the possibility that Arvenin I might upregulate PD-1 expression. However, treatment with Arvenin I had no detectable effect on cell surface PD-1 expression (Fig. 2e).

[0068] 2-3. Chemoproteomic analysis of covalent binding targets of Arvenin I Recently, it has been reported that proteins involved in T cell immune responses tend to have reactive cysteine ​​residues. Furthermore, as described in 2-1-3, this example suggests that the Michael acceptor shared by Arbenin I and Arbenin III may be important for their T cell activation. Based on the above, the inventors speculate that the Michael acceptors of Arbenin I and Arbenin III activate T cells by forming covalent bonds with cysteine ​​residues in target proteins. To verify this possibility, the following chemoproteomic analysis was performed (Figure 3a).

[0069] In this analysis, 12 C or 13 We utilized a 1C-isotope-labeled cysteine-reactive iodoacetamide alkyne probe (IA light / heavy probe). The IA light probe was reacted with lysates of Jurkat cells treated with PHA and PMA in the absence of Arvenin I. The IA heavy probe was reacted with lysates of Jurkat cells treated with PHA and PMA in the presence of Arvenin I. After click reaction of each sample with the azobiotin-azide handle, the samples containing the IA light probe and the samples containing the IA heavy probe were mixed and subjected to streptavidin-agarose enrichment, on-resin trypsin digestion, and NaSO cleavage. The released probe-labeled peptides were sequenced by LC-MS / MS analysis.

[0070] This quantitative proteomic analysis mapped the cysteine ​​residues of eight proteins whose labeling was completely competed by Arbenin I (Figure 3b). Of these eight cysteines, six were mapped as reactive cysteines in the cysteinome database (https: / / backuslab.shinyapps.io / cysdb / ). The proteins containing these cysteines are shown in bold in Figure 3b. We noted that Cys310 of AKT (protein kinase B) and Cys227 of MKK3 (mitogen-activated protein kinase kinase 3) are located near the enzyme active site. Furthermore, given the numerous reports supporting the importance of AKT and MKK3 in regulating immune responses, we considered AKT and MKK3 to be promising target proteins of Arbenin I.

[0071] To verify the direct reaction of Arvenin I with the cysteine ​​residues of AKT and MKK3, we performed a gel shift assay (Fig. 4a). The gel shift assay allows the evaluation of the molecular weight change of modified proteins by measuring their electrophoretic mobility on an SDS-PAGE gel. Specifically, the gel shift assay was performed as follows: HEK293 cells were transfected with expression vectors encoding Flag-tagged wild-type AKT or MKK3 (Flag-AKT wt or Flag-MKK3 wt) and then treated with various concentrations of Arvenin I. The electrophoretic mobility of the Flag-AKT wt and Flag-MKK3 wt bands on the SDS-PAGE gel was slightly delayed by treatment with a low concentration of Arvenin I (Fig. 4a, upper panel). In contrast, in mutants in which the corresponding cysteine ​​residues were replaced with serine residues (Flag-AKT C310S and Flag-MKK3 C227S), no band shift was observed even when treated with a high concentration (30 μM) of Arbenin I (Figure 4a, middle). Similarly, no band shift was observed with Arbenin II, a derivative in which the Michael acceptor double bond is saturated (Figure 4a, bottom). These results suggest that covalent bonds are formed between the Michael acceptor of Arbenin I and specific cysteine ​​residues in AKT and MKK3.

[0072] 2-4. Activation of MKK3 by Arvenin I AKT and MKK3 are redox-sensitive protein kinases involved in numerous biological processes, particularly cell proliferation and energy metabolism. AKT phosphorylates many downstream factors, such as GSK3β and FOXO1, while MKK3 specifically phosphorylates p38MAPK. To examine whether Arvenin I alters the phosphorylation status of these downstream factors, we performed blotting analysis using site-specific phosphoantibodies (Figure 4b). Arvenin I significantly increased the phosphorylation of p38MAPK at Thr180 / Tyr182 in Jurkat hPD-1 cells, both in the presence and absence of PHA and PMA. In contrast, Arvenin I had no detectable effect on the phosphorylation of FOXO1 at Ser256, which has been reported to be involved in the metabolic activation of T cells. Given the significant effect of Arvenin I on p38MAPK phosphorylation, we focused on MKK3 as a major target protein of Arvenin I.

[0073] To confirm the involvement of p38MAPK activation in Arvenin I-induced T cell activation, we used the p38MAPK inhibitor SB203580. Addition of SB203580 to the cell culture model developed in 2-1-1 dose-dependently abolished the effect of Arvenin I in restoring IL-2 production (Figure 4c).

[0074] To confirm the role of MKK3 in IL-2 production in Jurkat cells, we generated Jurkat MKK3 knockdown cells using CRISPR / Cas9-mediated genome engineering. In the resulting Jurkat MKK3 knockdown cells, MKK3 protein levels were reduced to approximately 50% of the control level (Fig. 4d). Knockdown of MKK3 attenuated PHA / PMA-induced p38 MAPK phosphorylation and IL-2 production (Fig. 4e, f). Furthermore, Jurkat cells were stably engineered to overexpress wild-type MKK3 (Jurkat MKK3 wt cells) or its C227S mutant (Jurkat MKK3 C227S cells) (Fig. 4g). Overexpression of wild-type MKK3 enhanced IL-2 production, which was further enhanced by arvenin I (Fig. 4h). In contrast, overexpression of the C227S mutant had only a limited effect on IL-2 levels (Fig. 4h). These results support the hypothesis that the covalent interaction of Arbenin I with MKK3 activates its ability to phosphorylate p38 MAPK, thereby activating IL-2 production.

[0075] To investigate how the covalent binding of Arbenin I to MKK3 activates MKK3, we next performed in vitro analysis using a His-tagged inactive recombinant mutant of p38MAPK (His-p38KR) as a substrate for Flag-MKK3 (Fig. 4i). Phosphorylation of His-p38KR was observed in a Flag-MKK3-dependent manner (Fig. 4i, top). Surprisingly, however, the addition of Arbenin I, even at a high concentration of 30 μM, did not enhance His-p38KR phosphorylation (Fig. 4i, top). Western blotting of Flag-MKK3 revealed an upward shift in the band in the presence of Arbenin I (Fig. 4i, bottom), confirming the covalent modification of Flag-MKK3 by Arbenin I in vitro. These results suggest that additional factors, in addition to the covalent modification of MKK3 by Arbenin I, are required to enhance the ability of MKK3 to phosphorylate p38MAPK.

[0076] MKK3 is phosphorylated and activated by a group of upstream kinases known as MAP3Ks. Among these, MEKK3 is well known to play an important role in T cell activation and differentiation. Indeed, when a constitutively active MEKK3 mutant (Myc-MEKK3CA) was added to a system containing Flag-MKK3 and His-p38KR, MKK3-dependent phosphorylation of His-p38KR was enhanced (Fig. 4j, top panel). Furthermore, the addition of Arvenin I further enhanced His-p38KR phosphorylation (Fig. 4j, top panel). These results suggest that covalent binding of Arvenin I to Cys227 of MKK3 renders MKK3 susceptible to MEKK3 activation, thereby inducing hyperphosphorylation of p38MAPK. This hypothesis was supported by blotting results showing that phosphorylation of MKK3 increased in the presence of Arvenin I in Jurkat hPD-1 cells (Fig. 4b, second row from the bottom).

[0077] 2-5. Enhancement of mitochondrial energy production by Arvenin I Since the above test revealed that Arbenin I has the ability to enhance the p38MAPK pathway in Jurkat cells, we next investigated the effect of Arbenin I on primary CD8 cells isolated from the spleen and lymph nodes of young mice. + The following test was performed using T cells. First, primary CD8 T cells were stimulated with anti-CD3 antibody and anti-CD28 antibody-coated beads. + The effects of Arbenin I on cytokine production and cell proliferation in T cells were evaluated. High concentrations of Arbenin I were toxic and reduced IFN-γ production in primary T cells (Fig. 5a, b), whereas submicromolar concentrations of Arbenin I increased IFN-γ production and cell proliferation without toxicity (Fig. 5a, b). From these results, the maximum tolerated dose of Arbenin I for primary T cells was determined to be 250 nM.

[0078] Next, we investigated the primary CD8 + We evaluated the effect of Arvenin I (250 nM) on mitochondrial energy production in T cells. Specifically, primary CD8 +T cells were stimulated with anti-CD3 and anti-CD28 antibody-coated beads for 2 hours in the presence or absence of Arbenin I. Oxygen consumption rates (OCR) were measured at basal and subsequent treatment with oligomycin (which inhibits oxidative phosphorylation-dependent ATP synthesis), carbonylcyanide-4(trifluoromethoxy)phenylhydrazone (FCCP, which uncouples ATP synthesis from the electron transport chain), and rotenone plus antimycin A (which inhibit electron transport complexes I and III, respectively). Basal respiration and spare respiratory capacity (SRC) were calculated from the measured OCR. In the presence of Arbenin I, mitochondrial basal respiration, maximal OCR, and spare respiratory capacity increased (Figure 5c). Particularly noteworthy was the effect of Arbenin I on SRC (Figure 5c, right), indicating additional ATP production in response to acute energy demands, such as those observed in the tumor microenvironment. The enhancement of SRC by Arbenin I addition was abolished by the addition of the p38MAPK inhibitor SB203580 (Fig. 5d), suggesting that the enhancement of SRC by Arbenin I addition occurs via activation of the p38MAPK pathway.

[0079] The p38MAPK signaling pathway has been reported to promote oxidative phosphorylation and increase mitochondrial ROS generation. Mitochondrial ROS is also recognized as an important factor in initiating T cell activation. As shown in Figure 5e, primary CD8 T cells were upregulated by CD3 and CD28. + T cell activation increased the proportion of T cells with high mitochondrial ROS levels and decreased the proportion of T cells with low mitochondrial ROS levels. Furthermore, the addition of Arbenin I enhanced the effects of CD3 and CD28 stimulation (Fig. 5e). These results are consistent with the effect of Arbenin I on enhancing mitochondrial energy production (Fig. 5c). These results suggest that Arbenin I enhances mitochondrial energy production in T cells via activation of the p38 MAPK pathway.

[0080] 2-6. Activation of antitumor immunity by Arbenin I We investigated the effect of Arbenin I in combination with anti-PD-L1 antibody on the anti-tumor activity in vivo. Specifically, wild-type (WT) mice were inoculated with MC38 colon cancer cells and administered Arbenin I at a dose of 6 mg / kg every 3 days and anti-PD-L1 antibody at a dose of 30 μg every 6 days from day 10 to day 22 after inoculation (Fig. 6a). Tumor volume was closely monitored up to day 26 after inoculation with MC38 colon cancer cells.

[0081] As a result, tumor growth was suppressed by approximately 40% in mice administered Arbenin I alone (Arbenin I group) compared to the control group, an effect similar to that in mice administered anti-PD-L1 antibody alone (anti-PD-L1 group) (Figure 6b). Furthermore, tumor growth was suppressed by approximately 70% in mice administered Arbenin I in combination with anti-PD-L1 antibody (anti-PD-L1 + Arbenin I group) (Figure 6b). These results suggest that Arbenin I exerts tumor-suppressing effects alone and that its combination with drugs that inhibit PD-1 / PD-L1 interaction can enhance the anti-tumor effects of these drugs.

[0082] Furthermore, we performed a similar study using immunodeficient mouse models (Rag2 KO mice) instead of WT mice. As a result, the tumor-suppressing effect of Arbenin I was not confirmed in Rag2 KO mice (Fig. 6c). These results suggest that Arbenin I suppresses tumor growth by enhancing the immune response.

[0083] 3. Discussion This example demonstrates that Arbenin I and its analogs covalently bind to MKK3, activating MKK3 and thereby enhancing mitochondrial function in T cells. While numerous kinase inhibitors that act directly on kinases have been known, direct kinase activators are relatively rare, with only a limited number of kinases, such as AMPK, AKT, and PKC, known to act directly on these kinases. Arbenin I, evaluated in this example, is believed to be the first small molecule activator of MKK3 discovered. Therefore, the technology of the present disclosure is expected to be useful as a tool for activating MKK3, for example, in cell biological research. Furthermore, Arbenin I is believed to be the first covalent kinase activator discovered.

[0084] Cys227 of MKK3, the specific reactive residue labeled by Arbenin I, is not shared by the other six MKK homologs. In the MKK3 structure predicted by the protein structure prediction program AlphaFold, Cys227 is exposed and is close to Ser218 and Tyr222, which are phosphorylated by MEKK3, the upstream kinase of MKK3 (Figure 7). Considering this and the results of this example, we speculate that covalent binding of Arbenin I to MKK3 induces a conformational change in MKK3, making it more susceptible to phosphorylation by MEKK3.

[0085] Furthermore, this example suggests that Arbenin I exerts excellent tumor-suppressing effects both alone and in combination with immune checkpoint inhibitors in vivo. As described in the Background Art section, the efficacy of immune checkpoint inhibitors is partially limited by impaired T cell function in the tumor microenvironment, and there are some subjects for whom immune checkpoint inhibitors are ineffective. Given the newly discovered effect of Arbenin I and its analogs in enhancing T cell mitochondrial function in the present disclosure, it is expected that excellent tumor-suppressing effects can be achieved by administering Arbenin I or its analogs alone or in combination with immune checkpoint inhibitors, even in subjects who have not previously benefited fully from immune checkpoint inhibitors or for whom immune checkpoint inhibitors have not been effective. In addition, the in vivo test in this example evaluated the tumor-suppressing effect on MC38 colon cancer cells. MC38 colon cancer cells are a cell line that forms subcutaneous tumors upon subcutaneous inoculation and is commonly used in tumor analysis. Therefore, based on the common general knowledge in the art and the effect of enhancing T cell mitochondrial function demonstrated in this example, it is reasonably predicted that the technology disclosed herein will be effective not only against colon cancer but also against all types of cancer.

[0086] Furthermore, based on the effect of enhancing mitochondrial function of T cells demonstrated in this example, it is reasonably predicted that the technology of the present disclosure will enhance the effects of cancer immunotherapies not only when used in combination with immune checkpoint inhibitors but also when used in combination with other cancer immunotherapies, such as cancer vaccine therapy, T cell receptor (TCR) T cell (TCR-T) therapy, and chimeric antigen receptor (CAR) T cell (CAR-T) therapy.

Claims

1. General formula (I): 【Chemistry 1】 (wherein Sugar represents any monosaccharide, disaccharide, or trisaccharide; R represents a hydrogen atom or an acyl group having 1 to 5 carbon atoms) or a salt thereof.

2. General formula (I): 【Chemistry 2】 (wherein Sugar represents any monosaccharide, disaccharide, or trisaccharide; R represents a hydrogen atom or an acyl group having 1 to 5 carbon atoms) or a salt thereof.

3. General formula (I): 【Transformation 3】 (wherein Sugar represents any monosaccharide, disaccharide, or trisaccharide; R represents a hydrogen atom or an acyl group having 1 to 5 carbon atoms) or a salt thereof.

4. 1. A composition for use in the treatment of cancer, comprising: General formula (I): 【Chemistry 4】 (wherein Sugar represents any monosaccharide, disaccharide, or trisaccharide; R represents a hydrogen atom or an acyl group having 1 to 5 carbon atoms) or a salt thereof, The composition, wherein the treatment comprises administration of an immune checkpoint inhibitor.

5. General formula (I): 【Transformation 5】 (wherein Sugar represents any monosaccharide, disaccharide, or trisaccharide; R represents a hydrogen atom or an acyl group having 1 to 5 carbon atoms) or a salt thereof, A composition used to enhance the antitumor effect of an immune checkpoint inhibitor.

6. The compound represented by the general formula (I) or a salt thereof is represented by the general formula (II): 【Transformation 6】 6. The composition according to claim 1, which is a compound represented by the formula: (wherein R represents a hydrogen atom or an acyl group having 1 or 2 carbon atoms) or a salt thereof.

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