Use of pgc-1 alpha agonists in the preparation of anti-tumor drugs

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to application of PGC-1 alpha activator in preparation of anti-tumor drugs. The present application firstly finds that the PGC-1 alpha activator (such as ZLN005) alone can promote the mitochondrial function and IFN-gamma production of tumor-depleted and disabled iNKT cells, improve the proportion of iNKT and CD8 T cells in the tumor microenvironment, and then improve the tumor microenvironment and inhibit tumor growth. The anti-tumor effect of the PGC-1 alpha activator combined with the NKT cell antigen is more obvious. The present application provides a new scheme for tumor immunotherapy, and provides a new drug target for treatment of other iNKT cell dysfunction diseases, and has important clinical application prospect and value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and particularly relates to application of PGC-1 alpha agonist in preparation of anti-tumor drugs. BACKGROUND

[0002] Tumor immunotherapy, such as PD1 antibody and CAR-T, CAR-NK, CAR-NKT treatment, etc., has become the most promising tumor treatment strategy. The main purpose of immunotherapy is to enhance the anti-tumor immune response of immune cells in patients, so as to more efficiently clear tumor cells. However, the special microenvironment inside the tumor tissue will cause the abnormal function of anti-tumor immune cells, i.e. exhaustion, thereby inhibiting the killing of immune cells to tumor, leading to tumor immune escape. Therefore, finding a new method or drug to reverse or restore the exhausted immune cells is the key to developing new strategies for immunotherapy.

[0003] NKT cells are a kind of innate-like T lymphocytes connecting natural immunity and acquired immunity, and have important regulatory effects on the occurrence and development of various diseases such as autoimmune diseases, infections, metabolic diseases and tumors. In addition, NKT cells are also important candidate cells for immunotherapy, and have achieved preliminary clinical treatment effect. In tumor immunotherapy, NKT cells show the advantages of smaller cytokine storm side effects and lower risk of graft versus host. At the same time, researchers have found that the number of IFN-γ-producing NKT cells in the body of patients is positively correlated with the clinical treatment results. However, the function of NKT cells in some tumor patients who do not respond to NKT cell immunotherapy is significantly inhibited, indicating that NKT cell exhaustion is one of the main factors causing NKT cell tumor immunotherapy. Therefore, finding a target or drug to enhance the IFN-γ response of exhausted NKT cells in tumors will be beneficial to the development of new immunotherapy strategies.

[0004] ZLN005 is a new peroxisome proliferator-activated receptor coactivator (PGC-1 alpha) activator, which is a small benzimidazole compound with a molecular formula of C 17 H 18 N2, and the chemical structure is shown as formula I. As an agonist of PGC-1 alpha, it is involved in the regulation of various physiological processes such as mitochondrial function, so ZLN005 is also involved in the occurrence and development of various diseases. For example, ZLN005 protects cardiomyocytes from high glucose-induced cytotoxicity by promoting SIRT1 expression and autophagy, ZLN005 can also promote retinal pigment epithelial cell metabolism by inducing PGC-1 alpha, prevent oxidative damage, improve ischemia-induced neuronal damage, reduce blood glucose levels in diabetic mice, and relieve symptoms such as insulin resistance. However, the function of ZLN005 in enhancing the anti-tumor function of NKT cells and inhibiting tumor growth has not been found. SUMMARY

[0005] Therefore, the application provides an application of a PGC-1α agonist in preparing an antitumor drug.

[0006] To achieve the above-mentioned purposes, the application provides the following technical solutions.

[0007] The application of the PGC-1α agonist alone or in combination with the NKT cell antigen or the NKT cell in preparing an antitumor drug.

[0008] In the application, the PGC-1α agonist is at least one of ZLN005, ZLN005d4 and VPA. In some specific embodiments, the PGC-1α agonist is ZLN005. The chemical structure of ZLN005 is as follows:

[0009]

[0010] In the application, the NKT cell antigen is at least one of αGalCer, PBS57, αGC acC8, αGC acC20:2 and OCH. In some specific embodiments, the NKT cell antigen is αGalCer.

[0011] In the application, the PGC-1α agonist alone can directly promote the mitochondrial function of the NKT cell and the production of IFN-γ, and enhance the tumor killing function of the NKT cell. The PGC-1α agonist in combination with the NKT cell antigen can improve the tumor microenvironment and inhibit the growth of tumor cells. The improvement of the tumor microenvironment includes promoting the production of IFN-γ and granzyme of immune cells, and / or increasing the proportion of infiltrated NKT, CD8 T and NK cells in the tumor microenvironment, and promoting the antitumor function of the immune cells.

[0012] In the application, the PGC-1α agonist alone or in combination with the NKT cell antigen can improve the mitochondrial function of the NKT cell.

[0013] In the application, the PGC-1α agonist alone or in combination with the NKT cell antigen can improve the antitumor function of the immune cell.

[0014] In the application, the NKT cell includes a wild NTK cell, an in-vitro amplified NTK cell or a modified NKT cell. The modified NKT cell can be a common type in the art, including but not limited to CAR-NKT and TCR-NKT.

[0015] In the application, the tumor includes at least one of colon cancer, melanoma, liver cancer, lung cancer and breast cancer. In some specific embodiments, the tumor is colon cancer, and specifically MC38 colon cancer.

[0016] The present application also provides a composition comprising a PGC-1a agonist and a NKT cell antigen. In some embodiments, the PGC-1a agonist is at least one of ZLN005, ZLN005d4, KL1333, VPA, Mogroside VI B, Ac-SVVVRT-NH2; the NKT cell antigen is at least one of aGalCer, PBS57, aGC acC8, aGC acC20:2, OCH, etc. In some specific embodiments, the PGC-1a agonist is ZLN005; the NKT cell antigen is aGalCer.

[0017] The present application first discovered that the combination of PGC-1a activators (such as ZLN005) and NKT cell antigens (such as aGalCer) or the treatment of NKT cells with PGC-1a activators alone can promote the mitochondrial function and IFN-γ production of tumor-depleted and dysfunctional NKT cells, improve the ratio of infiltrating NKT, CD8 T, and NK cells and anti-tumor function in the tumor microenvironment, and further improve the tumor microenvironment and inhibit tumor growth, providing a new solution for tumor immunotherapy and a new drug target for the treatment of other NKT cell dysfunction diseases, with important clinical application prospects and value. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Figure 1 shows the effect of ZLN005 on the mitochondrial function of dysfunctional and tumor-depleted NKT cells in Example 1 of the present application. Among them, Figure 1 A shows the expression of TMRM in dysfunctional NKT cells, Figure 1 B shows the expression of TMRM in tumor-depleted NKT cells;

[0019] Figure 2 Figure 2 shows the effect of ZLN005 on the IFN-γ production of dysfunctional NKT cells in Example 2 of the present application;

[0020] Figure 3 Figure 3 shows the effect of ZLN005 on the GzmB production of NKT cells in Example 3 of the present application;

[0021] Figure 4 Figure 4 shows that the combination of ZLN005 and aGalCer can significantly inhibit tumor growth in Example 4 of the present application; among them, Figure 4 A shows the treatment scheme, Figure 4 B to Figure 4 C shows the change of tumor volume and tumor, Figure 4 D shows the ratio of infiltrating NKT, NK, and CD8 T cells in the tumor microenvironment, Figure 4 E to Figure 4G shows the ability of tumor-infiltrating NKT, NK, and CD8 T cells to produce IFN-γ and GzmB. DETAILED DESCRIPTION

[0022] The present application provides the use of PGC-1α agonist in the preparation of anti-tumor drugs. Those skilled in the art can improve the process parameters according to the content of the present application. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The methods and applications of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0023] The experimental materials used in the following implementation examples are commercially available from regular biochemical reagent stores unless otherwise specified.

[0024] Wild-type C57BL / 6 mice: Nanjing Jiquan Pharmaceutical Biotechnology Co., Ltd.

[0025] Va14 Tg.Cxcr6 mice: Professor Albert Bendelac.

[0026] The NKT cells used in the examples were enriched and sorted from the livers of wild-type C57BL / 6 mice or Va14 Tg.Cxcr6 mice.

[0027] ZLN005: MCE, HY-17538.

[0028] Anti-mouse TCRβ antibody: Biolegend, 109222.

[0029] Mouse CD1d-PBS57 tetramer: donated by the National Institutes of Health Emory University Tetramer Core.

[0030] Anti-mouse IFN-γ antibody: Biolegend, 505841.

[0031] Anti-mouse CD45.2 antibody: Biolegend, 109828.

[0032] Anti-mouse CD8α antibody: Biolegend, 155022.

[0033] Anti-mouse NK1.1 antibody: Biolegend, 108749.

[0034] Anti-mouse GzmB antibody: Biolegend, 372208.

[0035] aGalCer: Avanti Polar Lipids, 86700.

[0036] TMRM: Sigma, T5428.

[0037] The application is further described below in conjunction with examples:

[0038] Example 1 ZLN005 restores mitochondrial function and glucose uptake capacity of anergized and exhausted NKT cells

[0039] (1) The mice were treated as follows:

[0040] Group 1: The mice were subcutaneously inoculated with 500,000 MC38 tumor cells, and the subcutaneous tumors were allowed to grow for about 3 weeks.

[0041] Group 2: In vivo experiment, the mice were intraperitoneally injected with 3 μg of aGalCer, and anergized NKT cell model was induced for 1 week.

[0042] (2) For tumor samples, the tumor was taken down to an appropriate size and cut into pieces as much as possible with an ophthalmic scissors. 5 ml of collagenase II with a concentration of 2 mg / ml was added, and the mixture was digested in a 37°C shaking bed at 200 rpm for 30 minutes. After filtering through a mesh and grinding, the mixture was resuspended in 40% Percoll, 70% Percoll was added to the lower layer, and after density gradient centrifugation (6 for ascending speed, 2 for descending speed, 2000 rpm, 20 minutes), the intermediate layer lymphocytes were extracted.

[0043] (3) For anergized mice, the mouse liver was removed, ground, and the suspension was resuspended in 40% Percoll, 70% Percoll was added to the lower layer, and after density gradient centrifugation (6 for ascending speed, 2 for descending speed, 2000 rpm, 20 minutes), the intermediate layer liver lymphocytes were extracted.

[0044] (4) The exhausted (tumor) and anergized NKT cells obtained above were each divided into two groups and added to a 96-well plate, one group was treated with ZLN005 (20 μM) or DMSO overnight; wherein ZLN005 was dissolved with DMSO.

[0045] (5) After step (4) treatment, the cells were collected, and flow cytometry antibodies (anti-TCRβ) and CD1d-PBS57 tetramer were added for staining for 45 minutes on ice in the dark.

[0046] (6) For TMRM detection, the above cells were added with TMRM dye in Hanks buffer and placed in a 37°C incubator for 20 minutes.

[0047] (7) Flow cytometry was used to detect the expression of TMRM in NKT cells in the control group and the ZLN005 treatment group, and the results are shown in Figure 1, data points are the results of 3 replicates.

[0048] ZLN005 treatment can significantly increase the level of TMRM of exhausted and anergic NKT cells, Figure 1 , indicating that ZLN005 can promote the mitochondrial function of exhausted and anergic NKT cells.

[0049] Example 2 ZLN005 can restore the ability of anergic NKT cells to produce IFN-γ

[0050] (1) Plate 96-well plates with 10 μg / ml anti-CD3 and anti-CD28 antibodies at 4°C overnight.

[0051] (2) For the induction of anergic mice, remove the mouse liver, grind the suspension in 40% Percoll, add 70% Percoll to the lower layer, and extract the liver lymphocytes in the middle layer after density gradient centrifugation (6 up, 2 down, 2000 rpm, 20 minutes). Add flow cytometry antibody staining (45 minutes on ice in the dark), and sort out TCRβ + CD1d-PBS57 tetramer + cells, and sort the cells into the pretreated 96-well plates in (1), 5 x 10 4 cells per well.

[0052] The first group is anergic NKT cells, DMSO treatment, control group.

[0053] The second group is anergic NKT cells, ZLN005 (20 μM) treatment.

[0054] (3) After 24 hours of treatment in step (2), flow cytometry analysis is used to detect the proportion of NKT cells producing IFN-γ intracellularly. The results are shown in Figure 2 .

[0055] The results show that the ability of anergic NKT cells to produce IFN-γ is significantly enhanced after ZLN005 treatment, indicating that ZLN005 can promote the function of anergic (exhausted) NKT cells.

[0056] Example 3 ZLN005 does not affect the ability of NKT cells to produce granzyme B

[0057] (1) Plate 96-well plates with 10 μg / ml anti-CD3 and anti-CD28 antibodies at 4°C overnight.

[0058] (2) Control and induced disabled mice, remove the mouse liver, grind the suspension in 40% Percoll, add 70% Percoll to the lower layer, extract the middle layer liver lymphocytes after density gradient centrifugation (6, 2, 2000 rpm, 20 min). Add flow cytometry antibody staining (on ice, avoid light for 45 min), use flow cytometry to sort TCRβ + CD1d-PBS57 tetramer + Cells, sort cells into (1) pretreated 96-well plates, 5x10 4 cells per well.

[0059] (3) After 24 hours of treatment in step (2), flow cytometry analysis was used to detect the proportion of NKT cells producing GzmB intracellularly. The results are shown in Figure 3 .

[0060] The results show that ZLN005 does not affect the level of GzmB produced by NKT cells, indicating that ZLN005 is not involved in regulating the production of NKT cell granzyme.

[0061] Example 4 ZLN005 promotes NKT cell-mediated anti-tumor function and inhibits tumor growth

[0062] (1) Subcutaneously inoculate 500,000 MC38 tumor cells into mice.

[0063] (2) After 7 days, inject 3 μg of αGalCer into the abdominal cavity of the mice and treat the mice with ZLN005 (15 mg / kg) by gavage every 2 days. The groups are as follows:

[0064] The first group, the control group, injects PBS into the abdominal cavity of the mice and gavages the solvent.

[0065] The second group, the control group, injects PBS into the abdominal cavity of the mice and gavages ZLN005.

[0066] The third group, the control group, injects αGalCer into the abdominal cavity of the mice and gavages the solvent.

[0067] The fourth group, injects αGalCer into the abdominal cavity of the mice and gavages ZLN005, combined treatment.

[0068] Subsequently, measure and record the tumor size of the mice every 2 days, and the experimental procedure is shown in Figure 4 A. The results of tumor volume and weight statistics are shown in Figure 4 B and Figure 4 C.

[0069] (3) In order to detect the effect of ZLN005 on the proportion and function of infiltrating immune cells in the tumor microenvironment, we detected the proportion and function of intratumoral NKT, NK and CD8 T cells after 4 weeks of treatment in tumor-bearing mice. The results are shown inFigure 4 D-4G.

[0070] The results show that ZLN005 treatment can promote NKT cell treatment group to infiltrate more NKT and CD8 T cells, and promote NKT, NK and CD8 T cells to produce more IFN-γ. It is shown that ZLN005 can significantly increase the anti-tumor function of immune cells and inhibit cell growth.

[0071] The results of the above examples show that ZLN005 can promote tumor-depleted NKT and disabled NKT cell mitochondrial function and IFN-γ production; promote NKT cell-mediated anti-tumor effect, improve the proportion of infiltrating NKT, CD8 T cells in the tumor microenvironment, and increase the anti-tumor function of NKT, CD8 T, NK cells, thereby inhibiting tumor growth. These results show that ZLN005 plays an important role in reversing the function of exhausted NKT cells to improve the tumor microenvironment and inhibit tumor growth, and also provides a new theoretical basis for the preparation of related drugs.

[0072] The above are only preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. Use of a PGC-1α agonist alone or in combination with an NKT cell antigen in the preparation of an anti-tumor drug. The PGC-1α agonist is ZLN005. The NKT cell antigen is αGalCer. The tumor is colon cancer.

2. Use according to claim 1, characterized in that, The combination of a PGC-1α agonist and an NKT cell can promote the production of IFN-γ by NKT cells and enhance anti-tumor function.

3. Use according to claim 1, characterized in that, The combination of a PGC-1α agonist and an NKT cell antigen can improve the tumor microenvironment and inhibit tumor cell growth.

4. Use according to claim 3, characterized in that, The improvement of the tumor microenvironment includes: promoting the production of IFN-γ and / or GzmB by immune cells, and / or increasing the proportion and function of infiltrating NKT, CD8 T, and NK cells in the tumor microenvironment.

5. The use according to claim 1, characterized in that, The PGC-1α agonist alone or in combination with an NKT cell antigen improves the mitochondrial function of NKT cells.

6. The use according to any one of claims 1 to 5, characterized in that, The NKT cells include isolated wild-type NTK cells, in vitro expanded NTK cells, or engineered NKT cells; the engineered NKT cells include CAR-NKT or TCR-NKT.

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