Use of MDH1 expression inhibitor as or in the preparation of PD-L1 expression inhibitor

Abstract

The present invention belongs to the technical field of immunotherapy drugs, and specifically relates to the use of an MDH1 expression inhibitor as or in the preparation of a PD-L1 expression inhibitor. MDH1 expression inhibitors, especially ECH, are used in inhibiting the expression of PD-L1, which can significantly inhibit the expression of PD-L1 on the cell surface; and combined with anti-PD-1 / or anti-CTLA-4, it can more effectively reduce the tumor volume of the body, prolong the survival of the body, and enhance the efficacy of immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunotherapy drugs, and specifically relates to an application of an MDH1 expression inhibitor as or in the preparation of a PD-L1 expression inhibitor. Background Art

[0002] Malignant tumors are a major public problem worldwide and pose a serious threat to human physical and mental health. Under normal circumstances, the immune system can perform its immune surveillance function and promptly identify and eliminate cancerous tumor cells, but tumor cells can escape the surveillance of the immune system through various pathways.

[0003] In recent years, with the continuous breakthroughs in tumor immunology research, immune checkpoint blockade (ICB) therapy represented by PD-1 / PD-L1 has become a new means of cancer treatment after surgery, radiotherapy and chemotherapy. Immunotherapy targeting PD-1 and PD-L1 has been widely used in a variety of tumors such as melanoma, breast cancer, non-small cell lung cancer, and has achieved significant clinical effects, bringing new hope to patients. As the main ligand of PD-1, PD-L1 on tumor cells often interacts with PD-1 on the surface of T cells, inducing apoptosis of anti-tumor T cells and promoting tumor immune escape. Given the role of PD-1 and PD-L1 in T cell inhibition, they have become important targets for immunotherapy.

[0004] Therefore, an inhibitor that can target PD-L1 and inhibit PD-L1 expression is necessary. Summary of the invention

[0005] In view of the above problems, one of the objects of the present invention is to provide a new use of an MDH1 (malate dehydrogenase 1) expression inhibitor targeting PD-L1 and inhibiting PD-L1 expression. The MDH1 expression inhibitor can effectively inhibit PD-L1 expression.

[0006] In order to achieve the above purpose, the following technical solutions can be adopted:

[0007] In one aspect, the present invention provides a use of an MDH1 expression inhibitor as or in the preparation of a PD-L1 expression inhibitor.

[0008] Another aspect of the present invention provides a pharmaceutical composition comprising an MDH1 inhibitor.

[0009] In another aspect, the present invention provides an anti-tumor drug, which comprises any one of the above-mentioned pharmaceutical compositions and a pharmaceutically acceptable carrier.

[0010] In another aspect, the present invention provides a use of echinacoside as or in the preparation of an MDH1 expression inhibitor.

[0011] In another aspect, the present invention provides a use of echinacoside as or in the preparation of an inhibitor that inhibits PD-L1 expression in the MDH1-PD-L1 pathway.

[0012] The beneficial effects of the present invention include at least:

[0013] (1) MDH1 expression inhibitors, especially echinacoside (abbreviated as ECH), can significantly inhibit the expression of PD-L1 on the cell surface when used to inhibit the expression of PD-L1;

[0014] (2) MDH1 expression inhibitors, especially ECH, combined with anti-PD-1 / or anti-CTLA-4 can effectively reduce tumor volume, prolong survival, and enhance the efficacy of immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The expression levels of MDH1 and PD-L1 in human triple-negative breast cancer tissue samples;

[0016] Figure 2 This is a quantitative graph of the expression levels of MDH1 and PD-L1 in human triple-negative breast cancer tissue samples;

[0017] Figure 3 mRNA expression levels in tumor tissue samples from mice treated with anti-PD-1 or anti-CTLA-4;

[0018] Figure 4 The survival rate of breast cancer patients with different MDH1 expression levels;

[0019] Figure 5 This is the regulation and quantification of MDH1 on PD-L1 in human breast cancer cells;

[0020] Figure 6 This is the regulation and quantification diagram of MDH1 on PD-L1 in mouse adenocarcinoma cells;

[0021] Figure 7 The effects of different inhibitors on PD-L1 expression;

[0022] Figure 8 The expression of PD-L1 on the surface of ECH and mouse breast cancer cell EMT6;

[0023] Fig. 9 This is a flowchart of mouse experimental operation;

[0024] Fig.10 This is the tumor growth and survival curve of the EMT6 breast cancer mouse model;

[0025] Fig.11It is the tumor growth curve and survival curve of CT26 colon cancer model;

[0026] Fig.12 Flow cytometry analysis and statistical results for the EMT6 breast cancer mouse model;

[0027] Fig.13 Flow cytometry analysis and statistical results of CT26 colon cancer model;

[0028] Fig.14 The results of routine blood tests after ECH inhibitor and combined immunotherapy;

[0029] in, Figure 6 In, ①: sgCtrl; ②: sgMdh1-1 ③: sgMdh1-2; Fig.10 In, ①: WT; ②WT+αCTLA-4: ③: ECH; ④: ECH+αCTLA-4; Fig.11 Among them, ①: WT; ②WT+αPD-1: ③: ECH; ④: ECH+αPD-1. DETAILED DESCRIPTION

[0030] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0031] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0032] On the one hand, the present invention provides an application of an MDH1 expression inhibitor as or in the preparation of a PD-L1 expression inhibitor. Specifically, MDH1 is a key enzyme in the tricarboxylic acid cycle. It was originally found to be highly expressed in tissues with high energy demands, such as the heart and skeletal muscle. Its deficiency can lead to abnormal neurodevelopment. Existing studies have found that the expression of MDH1 can promote the occurrence and development of a variety of tumors, but its role in tumor immunity is still unclear. The present invention has concluded through research that in the histopathological staining sections of patients with triple-negative breast cancer in clinical practice, the expression of MDH1 in tumor tissue is positively correlated with the expression of PD-L1, and through the analysis of the TISMO and Kaplan-Meier online databases, it was found that MDH1 expression is high, the immunotherapy effect is poor, and the survival rate of cancer patients is low; that is, MDH1 promotes the occurrence and development of tumors by promoting PD-L1 expression.

[0033] From the above, we can know that the MDH1-PD-L1 pathway is a new signaling pathway that regulates PD-L1. In this way, the expression of PD-L1 can be precisely inhibited by inhibiting the expression of MDH1. The binding of the T cell immune checkpoint PD-1 and PD-L1 will be blocked, and the T cell killing of the tumor will not stagnate, thereby continuing to kill the tumor.

[0034] Furthermore, the above-mentioned MDH1 expression inhibitor can be echinacoside, or a CRISPR-Cas reagent that targets the knockout / knockdown of the MDH1 expression gene.

[0035] Specifically, echinacoside can significantly inhibit the expression of MDH1, that is, it can be applied in the expression of MDH1, and then inhibit the expression of PD-L1 through the above-mentioned MDH1-PD-L1 pathway; it can be verified from the examples that echinacoside can significantly downregulate the expression of PD-L1 on the surface of tumor cells, and the use of ECH in mice can significantly inhibit tumor growth and enhance the efficacy of ICB, and it has no toxic side effects and is highly safe.

[0036] It should be noted that, as mentioned above, the MDH1-PD-L1 pathway is a new signaling pathway that regulates PD-L1. Therefore, in addition to using echinacoside to inhibit the expression of PD-L1, gene editing tools can also be used to knock out or knock down MDH1 and inhibit the expression of PD-L1. In particular, the CRISPR-Cas reagents currently used for gene editing can accurately knock down / or knock out the MDH1 expression gene, and it is quick and convenient.

[0037] Another aspect of the present invention provides a pharmaceutical composition, which includes an MDH1 inhibitor. Specifically, based on the inhibitory effect of the above-mentioned MDH1 inhibitor on PD-L1, a pharmaceutical composition for inhibiting PD-L1 expression containing the above-mentioned MDH1 inhibitor can be prepared, which has a significant inhibitory effect on PD-L1 expression.

[0038] Furthermore, the pharmaceutical composition also includes one or both of anti-PD-1 or anti-CTLA-4. Specifically, the MDH1 inhibitor can be used in combination with PD-1 antibodies and antibodies of other immune checkpoints, such as anti-PD-1 targeting PD-1 or anti-CTLA-4 targeting CTLA-4, to synergistically inhibit tumor growth, significantly prolong the survival of mice, and have no significant toxicity, further enhancing the effect of inhibiting tumor growth.

[0039] Furthermore, the MDH1 inhibitor may be echinacoside. As described above, echinacoside has a significant inhibitory effect on MDH1 expression and PD-L1 expression. It should be understood that, in addition to echinacoside, the MDH1 inhibitor may also include other agents that can achieve the same inhibitory effect on MDH1 expression.

[0040] On the other hand, the present invention provides an anti-tumor drug, which includes any one of the above-mentioned pharmaceutical compositions and a pharmaceutically acceptable carrier. Specifically, the above-mentioned pharmaceutical composition can be added with a pharmaceutically acceptable carrier to prepare an anti-tumor drug, and the pharmaceutically acceptable carrier is known in the art and can be selected according to the dosage form of the drug; in addition, the above-mentioned anti-tumor drug can be combined with surgery, chemotherapy and other methods for tumor treatment; it should also be noted that immune checkpoints have a broad spectrum for tumors, so the above-mentioned anti-tumor drug can target tumors known in the art, such as breast cancer, colon cancer, melanoma, breast cancer or non-small cell lung cancer.

[0041] In another aspect, the present invention provides a use of echinacoside as or in the preparation of an inhibitor that targets the MDH1-PD-L1 pathway and inhibits PD-L1 expression. As described above, the present invention is based on the newly discovered MDH1-PD-L1 pathway, and echinacoside has a significant inhibitory effect on MDH1, thereby significantly inhibiting the expression of PD-L1 in the MDH1-PD-L1 pathway, thereby effectively inhibiting tumor cell growth.

[0042] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0043] Example 1 Immunohistochemical detection of MDH1 and PD-L1 expression in triple-negative breast cancer tissues

[0044] (1) Collect tumor tissue specimens from patients with triple-negative breast cancer and prepare paraffin sections; (2) Dewax and hydrate the paraffin sections; (3) Repair with antigen repair solution at high temperature for 20 minutes, and then wash with TBST three times, each time for 5 minutes; (4) Inactivate endogenous peroxidase for 20 minutes, and then wash with TBST three times, each time for 5 minutes; (5) Add goat serum for blocking for 30 minutes; (6) Add diluted MDH1 working solution and incubate overnight at 4°; (7) After overnight, warm up at room temperature for 30 minutes, and wash with TBST three times, each time for 5 minutes; (8) Add biotin-labeled secondary antibody and incubate for 15 minutes, and then wash with TBST three times, each time for 5 minutes; (9) Add avidin working solution and incubate for 15 minutes, and then wash with TBST three times, each time for 5 minutes; (10) Add DBA diluent to develop color and terminate the reaction; then wash with TBST three times, 5 minutes each time; (11) Stain in hematoxylin for 10 seconds and soak in running water to turn blue for 5 minutes; (12) Dehydrate and make it transparent, air-dry and then seal and scan the slides; (13) Immunohistochemistry score (Hscore): Randomly select 5 high-power fields and calculate the average score; Positive cell percentage: 0-5%, 0 point; 6-25%, 1 point; 26-50%, 2 points; 51-75%, 3 points; more than 75%, 4 points; Staining intensity: no staining 0 point, light yellow 1 point, brown yellow 2 points, brown brown 3 points; Final score = staining intensity score * positive cell percentage score; MDH1 score greater than 4 points is considered as high expression of MDH1, and less than or equal to 4 points is considered as low expression of MDH1.

[0045] The results are as follows Figure 1 and Figure 2 As shown in Figure 2, in triple-negative breast cancer tissues, the expression of MDH1 is positively correlated with PD-L1 ( Figure 1 ), according to the immunohistochemical scoring results of 180 triple-negative breast cancer tissue samples, the expression of MDH1 was positively correlated with PD-L1, and the results were statistically significant, **P<0.01( Figure 2 ).

[0046] Example 2 Database predicts the relationship between MDH1 expression and immunotherapy efficacy and survival rate

[0047] In the TISMO database (http: / / tismo.cistrome.org), tumor tissue samples of mice treated with anti-PD-1 or anti-CTLA-4 were divided into a baseline group, a responder group, and a non-responder group. mRNA sequencing was performed to compare the expression levels of MDH1 in the responder group and the baseline group, and in the non-responder group and the baseline group. The results are as follows: Figure 3As shown, compared with the baseline group, the mRNA expression level of MDH1 in the non-response group was higher than that in the baseline group; compared with the baseline group, the mRNA expression level of MDH1 in the response group was lower than that in the baseline group.

[0048] The survival rates of breast cancer patients with different MDH1 expression levels were analyzed from the Kaplan-Meier database. Figure 4 As shown, among breast cancer patients, those with high MDH1 expression levels had a lower survival rate than those with low MDH1 expression levels, and the result was statistically significant.

[0049] Example 3 Verification of the specific mechanism by which MDH1 downregulates PD-L1

[0050] CRISPR-Ca9 technology was used to knock out MDH1 in human breast cancer cells (MDA-MB-231), and two different sites were knocked out, named sgMDH1-1 group and sgMDH1-2 group;

[0051] CRISPR-Ca9 technology was used to knock out MDH1 in mouse breast cancer cells (MDA-MB-231) at two different sites, named sgMdh1-1 group and sgMdh1-2 group;

[0052] In MDH1-knockout human breast cancer cells (sgMDH1-1 and sgMDH1-2) or mouse breast cancer cells (sgMdh1-1 and sgMdh1-2), cell surface PD-L1 expression was detected in the presence of IFN-γ (IFN-γ(+)) and without IFN-γ (IFN-γ(-)) (the presence of IFN-γ stimulates PD-L1 expression), and the correlation with MDH1 expression was analyzed by GSEA;

[0053] The results of the human breast cancer cell line with MDH1 knockout were as follows Figure 5 As shown, compared with the control group (sgCtrl), in human breast cancer cells, the expression of PD-L1 was reduced after knocking out MDH1 in the basal state and under IFN-γ stimulation;

[0054] The results of the MDH1 knockout mouse breast cancer cell test are as follows Figure 6 As shown, compared with the control group (sgCtrl), in human breast cancer cells, the expression of PD-L1 was reduced after MDH1 knockout in the basal state and IFN-γ stimulated state; the results were statistically significant, **P<0.01, ***p<0.001.

[0055] Example 4 Inhibition of Echinacoside on Tumor Cell PD-L1

[0056] The drug Echinacoside, Efaproxiral, Droperidol and Stachyose powder were diluted with water to a concentration of 10 μm. After reacting with MDA-MB-231 cells for 24 hours, flow staining was performed to detect the expression of PD-L1 on the cell surface and the drug with the strongest inhibitory effect was screened. The results are as follows Figure 7 As shown, among the four inhibitors, ECH had the most significant downregulation effect on PD-L1;

[0057] The screened drug Echinacoside was tested in the EMT6 cell line. The results are as follows Figure 8 As shown, Echinacoside can downregulate the expression of PD-L1 on the surface of mouse breast cancer cells EMT6.

[0058] Example 5 Analysis of the effect of Echinacoside inhibitor combined with immunotherapy

[0059] In the embodiment of the present invention, the mouse experiment operation process is as follows Fig. 9 As shown, anti-PD-1 (also known as αPD-1) and anti-CLTA-4 (also known as αCLTA-4) immunotherapy were performed on approximately the 7th, 11th, and 14th days, respectively, and mouse tumors were removed around the 21st day. The specific steps are as follows:

[0060] (1) The mice were divided into control group (WT), monoclonal antibody group (WT+anti-PD-1 / WT+anti-CTLA-4), drug inhibitor group (ECH), and combination treatment group (ECH+anti-PD-1 / ECH+anti-CTLA-4b);

[0061] (2) 4 × 10 EMT6 breast cancer cells and CT26 colon cancer wild-type cells were injected subcutaneously around the third nipple tissue of ventral balb / c mice aged 6-8 weeks. 5 Each group has 20 pieces;

[0062] (3) Starting from the fifth day after inoculation, the volume of the mouse was measured every three days. Volume = 0.5 × length × width 2 ;

[0063] (4) Starting from the fifth day after inoculation, the inhibitor ECH was administered to the drug inhibitor group and the combination treatment group once every three days at a dose of 20 mg / kg by intragastric administration;

[0064] (5) When the mouse volume reaches 100 mm 3 (It should be noted that the volume is measured from the fifth day, and generally the volume reaches 100mm on the seventh day. 3 , so in Fig. 9The monoclonal antibody group and the combination treatment group were vaccinated with anti-CTLA-4 and anti-PD-1 (anti-CTLA-4 for EMT6 breast cancer mice and anti-PD-1 for CT26 colon cancer mice), 100ug for each mouse, once every three days, intraperitoneally, for a total of three injections, 10 mice in each group;

[0065] (6) After measuring for five time points (i.e., after 5 vaccinations with anti-CTLA-4 and anti-PD-1), half of the mice were killed, tumor tissues were extracted for photography, and single-cell suspensions of tumor tissues were extracted for subsequent analysis;

[0066] (7) Continue to observe the survival time of the remaining mice until the tumor volume reaches 2000mm 3 If the longest diameter of the tumor exceeded 20 mm, the mouse was considered dead.

[0067] The data during the test are as follows Figures 10 to 13 As shown:

[0068] Fig.10 (Among them, Fig.10 A is the tumor growth curve of the EMT6 breast cancer mouse model. Fig.10 B is the survival curve of the EMT6 breast cancer mouse model). Compared with other groups, the inhibitor ECH combined with anti-CTLA-4 treatment can significantly reduce the tumor volume of the EMT6 breast cancer mouse model, prolong the survival of the EMT6 breast cancer mouse model, and enhance the efficacy of immunotherapy;

[0069] Fig.11 (Among them, Fig.11 A is the tumor growth curve of CT26 colon cancer model. Fig.11 B is the survival curve of the CT26 colon cancer model). Compared with other groups, the inhibitor ECH combined with anti-PD-1 treatment can significantly reduce the tumor volume of the CT26 colon cancer model, prolong the survival of the CT26 colon cancer model, and enhance the efficacy of immunotherapy;

[0070] Fig.12 (Among them, Fig.12 A is the flow cytometry result of EMT6 breast cancer mouse model. Fig.12 B is the statistical result of flow cytometry analysis of EMT6 breast cancer mouse model). Compared with other groups, the inhibitor ECH combined with anti-CTLA-4 treatment can significantly improve CTL infiltration and increase IFN-γ + CD8 + T cell ratio, enhancing the efficacy of immunotherapy;

[0071] Fig.13 (Among them, Fig.13 A is the flow cytometry result of EMT6 breast cancer mouse model. Fig.13 B is the statistical result of flow cytometry analysis of EMT6 breast cancer mouse model). Compared with other groups, the inhibitor ECH combined with anti-PD-1 treatment can significantly improve CTL infiltration and increase IFN-γ + CD8 + T cell ratio, enhancing the efficacy of immunotherapy.

[0072] Example 6 Safety Analysis of Inhibitor Echinacoside and Combined Immunotherapy

[0073] In the steps of Example 5, blood was collected from the eyeballs of mice before they were killed. About 500ul of fresh blood was collected from each mouse and collected in anticoagulant tubes for routine blood tests. The results were as follows: Fig.14 The following are the blood routine results of mice. Compared with other groups, the inhibitor ECH combined with anti-CTLA-4 treatment has an effect on the blood cells of mice ( Fig.14 A) Leukocytes ( Fig.14 B) Hemoglobin ( Fig.14 C) Platelets ( Fig.14 D) and neutrophils ( Fig.14 E) No significant effect.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A pharmaceutical composition, characterized in that Including echinacoside and anti-CTLA-4 antibodies.

2. An anti-tumor drug, characterized in that: The invention comprises the pharmaceutical composition according to claim 1 and a pharmaceutically acceptable carrier.

3. Use of the pharmaceutical composition according to claim 1 in preparing a drug for treating breast cancer.

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