CN115990254A - Application of MDH1 expression inhibitor in serving as or preparing PD-L1 expression inhibitor - Google Patents

Abstract

The invention belongs to the technical field of immunotherapeutic drugs, and particularly relates to an application of an MDH1 expression inhibitor in serving as or preparing a PD-L1 expression inhibitor. MDH1 expression inhibitors, especially ECH, are applied to inhibit PD-L1 expression, and can obviously inhibit cell surface PD-L1 expression; and can be combined with anti-PD-1 and/or anti-CTLA-4 to reduce tumor volume, prolong life of organism and enhance therapeutic effect of immunotherapy.

Description

Application of MDH1 expression inhibitor as or preparation of PD-L1 expression inhibitor

technical field

The invention belongs to the technical field of immunotherapy drugs, and in particular relates to the application of an MDH1 expression inhibitor as or preparation of a PD-L1 expression inhibitor.

Background technique

As a major public problem worldwide, malignant tumors seriously threaten human physical and mental health. Under normal circumstances, the immune system can perform the immune surveillance function to identify and eliminate cancerous tumor cells in time, but tumor cells can escape the surveillance of the immune system through various channels.

In recent years, with continuous breakthroughs in tumor immunity research, immune checkpoint blockade (ICB) therapy represented by PD-1/PD-L1 has become a new method of cancer treatment after surgery, radiotherapy and chemotherapy. Immunotherapy targeting PD-1 and PD-L1 has been widely used in various tumors such as melanoma, breast cancer, and 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 to induce apoptosis of anti-tumor T cells and promote tumor immune escape. Given their role in T cell suppression, PD-1 and PD-L1 have become important targets for immunotherapy.

Therefore, an inhibitor that can target PD-L1 and inhibit PD-L1 expression is necessary.

Contents of the invention

In response to the above problems, one of the purposes of the present invention is to provide a new application of an MDH1 (malate dehydrogenase 1) expression inhibitor targeting PD-L1 and inhibiting the expression of PD-L1. The MDH1 expression inhibitor can effectively inhibit PD-L1 Express.

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

One aspect of the present invention provides an application of an MDH1 expression inhibitor as or preparation of a PD-L1 expression inhibitor.

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

Another aspect of the present invention provides an anti-tumor drug, which includes any one of the above-mentioned pharmaceutical compositions and a pharmaceutically acceptable carrier.

Another aspect of the present invention provides an application of echinacoside as or preparation of an MDH1 expression inhibitor.

Another aspect of the present invention provides an application of echinacoside as or in the preparation of an inhibitor targeting MDH1-PD-L1 pathway to inhibit the expression of PD-L1.

The beneficial effects of the present invention at least include:

(1) MDH1 expression inhibitors, especially echinacoside (referred to as Echinacoside or ECH), are used to inhibit the expression of PD-L1, which can significantly inhibit the expression of PD-L1 on the cell surface;

(2) MDH1 expression inhibitors, especially ECH, combined with anti-PD-1/or anti-CTLA-4, can effectively reduce the tumor volume of the body, prolong the survival of the body, and enhance the efficacy of immunotherapy.

Description of drawings

Figure 1 shows the expression levels of MDH1 and PD-L1 in human triple-negative breast cancer tissue samples;

Figure 2 is a quantitative diagram of the expression levels of MDH1 and PD-L1 in human triple-negative breast cancer tissue samples;

Figure 3 is the mRNA expression level of tumor tissue samples of mice treated with anti-PD-1 or anti-CTLA-4;

Figure 4 shows the survival rate of breast cancer patients with different MDH1 expression levels;

Figure 5 is a graph showing the regulation and quantification of MDH1 on PD-L1 in human breast cancer cells;

Figure 6 is a graph showing the regulation and quantification of MDH1 on PD-L1 in mouse adenocarcinoma cells;

Figure 7 is the effect of different inhibitors on the expression of PD-L1;

Figure 8 shows the expression of PD-L1 on the surface of ECH and mouse breast cancer cells EMT6;

Fig. 9 is a flow chart of mouse experiment operation;

Figure 10 is a graph showing the tumor growth and survival curves of the EMT6 breast cancer mouse model;

Figure 11 is the tumor growth curve and survival curve of the CT26 colon cancer model;

Figure 12 is the flow cytometric analysis and statistical results of the EMT6 breast cancer mouse model;

Figure 13 is the flow cytometric analysis and statistical results of the CT26 colon cancer model;

Figure 14 shows the blood routine detection after inhibitor ECH and combined immunotherapy;

Among them, in Figure 6, ①: sgCtrl; ②: sgMdh1-1; ③: sgMdh1-2; in Figure 10, ①: WT; ②WT+αCTLA-4: ③: ECH; ④: ECH+αCTLA-4; in Figure 11, ①: WT; ②WT+αPD-1: ③: ECH; ④: ECH+αPD-1.

Detailed ways

The examples given are for better description of the present invention, but the content of the present invention is not limited to the examples given. Therefore, non-essential improvements and adjustments to the implementation by those skilled in the art based on the content of the invention above still fall within the protection scope of the present invention.

The terminology used herein is for describing particular embodiments only and is not intended to limit the present disclosure. Expressions in the singular include expressions in the plural unless there are clearly different meanings in the context. As used herein, it should be understood that terms such as "comprising", "having", "comprising" 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 it is 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" as the case may be.

One aspect of the present invention provides an application of an MDH1 expression inhibitor as or 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 requirements, such as the heart and skeletal muscle. Its deficiency can lead to abnormal neurodevelopment; existing studies have found that the expression of MDH1 It can promote the occurrence and development of various tumors, but its role in tumor immunity is still unclear. The present invention has obtained through research that in the histopathologically stained sections of patients with triple-negative breast cancer clinically, the expression of MDH1 in tumor tissue is positively correlated with the expression of PD-L1, and it is found through the analysis of TISMO and Kaplan-Meier online database that the expression of MDH1 High, the effect of immunotherapy is poor, and the survival rate of cancer patients is low; that is, MDH1 promotes the occurrence and development of tumors by promoting the expression of PD-L1.

It can be known from the above 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 T cell immune checkpoint PD-1 and PD The combination of -L1 will be blocked, and the killing of tumors by T cells will not be stagnant, so as to continue to kill tumors.

Further, the above-mentioned MDH1 expression inhibitor may be echinacoside, or a CRISPR-Cas reagent targeting knockout/knockdown of MDH1 expression gene.

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 known from the verification of the examples that echinacoside It can significantly down-regulate 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, with no toxic side effects and high safety.

It should be noted that, as mentioned above, the MDH1-PD-L1 pathway is a new signaling pathway that regulates PD-L1, so 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 to inhibit the expression of PD-L1, especially the CRISPR-Cas reagents currently used for gene editing can accurately knock down/or knock out MDH1 expression genes, and it is convenient and quick.

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

Further, the above pharmaceutical composition also includes one or both of anti-PD-1 or anti-CTLA-4. Specifically, the above-mentioned MDH1 inhibitors can be used in combination with PD-1 antibodies and other immune checkpoint antibodies, such as anti-PD-1 targeting PD-1 or anti-CTLA-4 targeting CTLA-4, which can The synergistic effect inhibits tumor growth, significantly prolongs the survival period of mice, and has no significant toxic and side effects, further enhancing the effect of inhibiting tumor growth.

Further, the above-mentioned MDH1 inhibitor may be echinacoside. As mentioned above, echinacoside had a significant inhibitory effect on MDH1 expression as well as PD-L1 expression. It should be understood that, in addition to echinacoside, other agents that can achieve the same effect of inhibiting MDH1 expression can also be selected as the above-mentioned MDH1 inhibitor.

Another aspect of 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. 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 It can be combined with surgery, chemotherapy, etc. for tumor treatment; it should also be noted that immune checkpoints have a broad spectrum for tumors, so the above-mentioned anti-tumor drugs can be targeted to treat tumors known in the art, such as breast cancer, colon cancer , melanoma, breast cancer, or non-small cell lung cancer.

Another aspect of the present invention provides an application of echinacoside as or in the preparation of an inhibitor targeting the MDH1-PD-L1 pathway to inhibit the expression of PD-L1. As mentioned above, the present invention is based on the newly discovered MDH1-PD-L1 pathway, and echinacoside has a significant inhibitory effect on MDH1, so that it can significantly inhibit the expression of PD-L1 in the MDH1-PD-L1 pathway, thereby effectively inhibiting Tumor cells grow.

In order to better understand the present invention, the content of the present invention will be further clarified below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

Example 1 Immunohistochemical detection of MDH1 and PD-L1 expression in triple-negative breast cancer tissue

(1) Collect tumor tissue samples from patients with triple-negative breast cancer and make paraffin sections; (2) Dewax and hydrate the paraffin sections; (3) Repair with antigen retrieval solution at high temperature for 20 minutes, then wash with TBST three times, each time 5 minutes; (4) After inactivation with endogenous peroxidase for 20 minutes, wash 3 times with TBST for 5 minutes each time; (5) Add goat serum dropwise to block for 30 minutes; (6) Add diluted Incubate the MDH1 working solution overnight at 4°; (7) rewarm at room temperature for 30 minutes after overnight, wash with TBST for 3 times, 5 minutes each time; (8) add biotin-labeled secondary antibody for 15 minutes, and then use Wash 3 times with TBST, 5 minutes each time; (9) Add avidin working solution dropwise and incubate for 15 minutes, then wash 3 times with TBST, 5 minutes each time; (10) Add DBA diluent to develop color, and terminate the reaction; Then wash with TBST 3 times, 5 minutes each time; (11) stain in hematoxylin for 10 seconds, and soak in running water for 5 minutes to return to blue; (12) dehydrate and transparent, seal and scan after air drying; (13) immunohistochemical score (Hscore): Randomly select 5 high-powered fields of view and calculate the average score; percentage of positive cells: 0-5%, 0 points; 6-25%, 1 point; 26-50%, 2 points; 51-75%, 3 points ; more than 75%, 4 points; staining intensity: no coloring 0 points, light yellow 1 point, brown yellow 2 points, brown 3 points; final score = staining intensity score * positive cell percentage score; MDH1 score greater than 4 points is evaluated as High expression of MDH1, less than or equal to 4 points for low expression of MDH1.

The results are shown in Figures 1 and 2. In triple-negative breast cancer tissues, the expression of MDH1 was positively correlated with PD-L1 (Figure 1). According to the results of immunohistochemical scoring of 180 triple-negative breast cancer tissue samples, the expression of MDH1 It was positively correlated with PD-L1, and the result was statistically significant, **P<0.01 (Figure 2).

Example 2 Relationship between Database Prediction of MDH1 Expression and Immunotherapy Efficacy and Survival Rate

In the TISMO database (http://tismo.cistrome.org), the tumor tissue samples of anti-PD-1 or anti-CTLA-4 treated mice were divided into baseline group (Baseline), responder group (Responders) and non-response group (Non-responders), and mRNA sequencing was carried out, and the expression levels of MDH1 in the response group and the baseline group, the non-responder group and the baseline group were compared respectively; the results are shown in Fig. The mRNA expression level of MDH1 in the response group was higher than that of the baseline group; compared with the baseline group, the mRNA expression level of MDH1 in the response group was lower than that of the baseline group.

The survival rate of breast cancer patients with different MDH1 expression levels was analyzed from the Kaplan-Meier database. The results are shown in Figure 4. Among breast cancer patients, the survival rate of patients with high MDH1 expression levels is lower than that of patients with low MDH1 expression levels. The results are statistically significant. learning meaning.

Example 3 Verification of the specific mechanism of MDH1 down-regulating PD-L1

Using CRISPR-Ca9 technology to knock out MDH1 in human breast cancer cells (MDA-MB-231), knock out two different sites respectively, named sgMDH1-1 group and sgMDH1-2 group;

Using CRISPR-Ca9 technology to knock out MDH1 in mouse breast cancer cells (MDA-MB-231), knock out two different sites respectively, named sgMdh1-1 group and sgMdh1-2 group;

In MDH1-knockout human breast cancer cells (sgMDH1-1 and sgMDH1-2) or mouse breast cancer cells (sgMdh1-1 and sgMdh1-2), IFN-γ (IFN-γ(+)) and no In the case of adding IFN-γ (IFN-γ(-)) (the presence of IFN-γ stimulates the expression of PD-L1), the expression of PD-L1 on the cell surface was detected, and the situation related to the expression of MDH1 was analyzed by GSEA;

The detection results of MDH1-knockout human breast cancer cells are shown in Figure 5. Compared with the control group (sgCtrl), in human breast cancer cells, in the basal state and the state stimulated by IFN-γ, after knocking out MDH1, PD - decreased expression of L1;

The detection results of MDH1-knockout mouse breast cancer cells are shown in Figure 6. Compared with the control group (sgCtrl), in human breast cancer cells, in the basal state and the state stimulated by IFN-γ, after knocking out MDH1, PD The expression of -L1 decreased; the results were statistically significant, **P<0.01, ***p<0.001.

Example 4 Echinacoside inhibits tumor cell PD-L1

Dilute the powders of Echinacoside, Efaproxiral, Droperidol and Stachyose with water at a concentration of 10um; after reacting with MDA-MB-231 cells for 24 hours, perform flow staining to detect the expression of PD-L1 on the cell surface, and screen out the drugs with the strongest inhibitory effect , the results are shown in Figure 7, among the four inhibitors, ECH has the most obvious down-regulation effect on PD-L1;

The screened drug Echinacoside was verified in the EMT6 cell line, and the results are shown in Figure 8. Echinacoside can down-regulate the expression of PD-L1 on the surface of mouse breast cancer cells EMT6.

Example 5 Analysis of the Effect of Inhibitor Echinacoside Combined with Immunotherapy

In the embodiment of the present invention, the mouse experimental operation process is shown in Figure 9, and anti-PD-1 (also called αPD-1) and anti-CLTA-4 (also called αCLTA-4) immunotherapy, the mouse tumors were taken around the 21st day, and the specific steps were as follows:

(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 combined treatment group (ECH+anti -PD-1/ECH+anti-CTLA-4b);

(2) balb/c healthy mice of 6-8 weeks were subcutaneously injected with 4* ¹⁰⁵ EMT6 breast cancer and CT26 colon cancer wild line cells around the third pair of papillae on the ventral side, 20 in each group;

(3) From the 5th day after inoculation, measure the mouse volume every three days, volume=0.5*length*width ² ;

(4) From the 5th day after inoculation, the drug inhibitor group and the combined treatment group were given the inhibitor ECH once every three days, the dosage was 20 mg/kg, and the injection method was intragastric administration;

(5) When the volume of the mouse reaches 100 mm ³ (it should be noted that the volume is measured from the fifth day, and generally the volume reaches 100 mm ³ on the 7th day, so the drug administration is marked on the 7th day in Figure 9), the single EMT6-injected breast cancer mice and CT26-injected colon cancer mice in the anti-group and combined treatment groups were inoculated with anti-CTLA-4 and anti-PD-1 (the mice injected with EMT6 breast cancer were inoculated with anti-CTLA-4 and injected with CT26 Colon cancer mice were inoculated with anti-PD-1), 100ug per mouse, once every three days, intraperitoneal injection, a total of three injections, 10 mice in each group;

(6) After measuring five time points (that is, after inoculating anti-CTLA-4 and anti-PD-15 times), half of the mice were sacrificed, tumor tissues were extracted for photographing, and single-cell suspensions of tumor tissues were extracted for subsequent analysis ;

(7) Continue to observe the survival time of the remaining mice until ^the tumor volume of the mouse reaches 2000 mm or the longest diameter of the tumor exceeds 20 mm, and the mouse is judged as dead;

The data situation during the test is shown in Figure 10 to Figure 13:

In Figure 10 (wherein, Figure 10A is the tumor growth curve of the EMT6 breast cancer mouse model, and Figure 10B is the survival curve of the EMT6 breast cancer mouse model), compared with other groups, the inhibitor ECH combined with anti-CTLA-4 treatment It 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;

In Figure 11 (wherein, Figure 11A is the tumor growth curve of the CT26 colon cancer model, and Figure 11B 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 CT26 The tumor volume of the colon cancer model was increased, and the survival period of the CT26 colon cancer model was prolonged, and the efficacy of immunotherapy was enhanced;

In Figure 12 (wherein, Figure 12A is the flow cytometry result of the EMT6 breast cancer mouse model, and Figure 12B is the statistical result of the flow cytometry analysis of the EMT6 breast cancer mouse model), compared with other groups, the inhibitor ECH combined with anti-CTLA-4 Treatment can significantly increase CTL infiltration, increase the proportion of IFN-γ ⁺ CD8 ⁺ T cells, and enhance the efficacy of immunotherapy;

In Figure 13 (wherein, Figure 13A is the flow cytometry result of the EMT6 breast cancer mouse model, and Figure 13B is the statistical result of the flow cytometry analysis of the EMT6 breast cancer mouse model), compared with other groups, the inhibitor ECH combined with anti-PD-1 Treatment can significantly increase CTL infiltration, increase the proportion of IFN-γ ⁺ CD8 ⁺ T cells, and enhance the efficacy of immunotherapy.

Example 6 Inhibitor Echinacoside and combined immunotherapy safety analysis

In the steps of Example 5, blood was taken from the eyeballs before the mice were killed, and about 500ul of fresh blood was taken from each mouse, which was collected in anticoagulant tubes for blood routine testing; the results are shown in Figure 14, which is the mouse blood routine As a result, compared with other groups, inhibitor ECH combined with anti-CTLA-4 treatment had significant effects on blood cells (Figure 14A), leukocytes (Figure 14B), hemoglobin (Figure 14C), platelets (Figure 14D) and neutrophils in mice. (FIG. 14E) No significant effect.

Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be carried out Modifications or equivalent replacements without departing from the spirit and scope of the technical solution of the present invention shall fall within the scope of the claims of the present invention.

Claims (8)

1. The application of MDH1 expression inhibitor as or preparation of PD-L1 expression inhibitor. 2. The application according to claim 1, wherein the MDH1 expression inhibitor is echinacoside, or a CRISPR-Cas reagent targeting knockout/knockdown of the MDH1 expression gene. 3. A pharmaceutical composition comprising an MDH1 inhibitor. 4. The pharmaceutical composition according to claim 3, further comprising one or both of anti-PD-1 and anti-CTLA-4. 5. The pharmaceutical composition according to claim 3 or 4, characterized in that the MDH1 inhibitor is echinacoside. 6. The antitumor drug, characterized in that it comprises any one of the pharmaceutical compositions of claims 3 to 5 and a pharmaceutically acceptable carrier. 7. The application of echinacoside as or preparation of MDH1 expression inhibitor. 8. The application of echinacoside as or in the preparation of an inhibitor targeting MDH1-PD-L1 pathway to inhibit the expression of PD-L1.

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