Application of thymol in enhancing the efficacy of anti-pd-1 antibody in treating lung adenocarcinoma

Abstract

The application discloses application of thymol in enhancing the curative effect of anti-PD-1 antibody in treating lung adenocarcinoma. The application first identifies the small-molecule drug thymol targeting IL4I1 expression, and first finds that the thymol targeting IL4I1 can block the AHR signal pathway, so as to reshape the activated immune microenvironment of lung adenocarcinoma. In addition, it is also first found that the thymol can significantly enhance the curative effect of the anti-PD-1 antibody for lung adenocarcinoma, and can provide a new scheme and strategy for the immunotherapy of lung adenocarcinoma patients in clinic, and has a great application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to the application of thymol in enhancing the efficacy of anti-PD-1 antibody in treating lung adenocarcinoma. BACKGROUND

[0002] Lung cancer is the malignant tumor with the highest mortality rate in the world, and its morbidity and mortality rates are the highest among malignant tumors in China. Lung adenocarcinoma (LUAD) is the most common pathological type of lung cancer. Immune checkpoint inhibitors, represented by anti-PD-1 antibody, are the most promising new method for the treatment of tumors. Currently, a variety of anti-PD-1 antibodies have been approved for the first-line treatment of patients with advanced lung adenocarcinoma. However, the results of clinical trials are not optimistic, and most patients still cannot benefit from anti-PD-1 antibody treatment. Studies have found that the clinical response rate of lung adenocarcinoma patients to anti-PD-1 antibody drugs is less than 20%. Therefore, further development of combination therapy to improve the efficacy of anti-PD-1 antibody in lung adenocarcinoma is an important problem in clinical practice.

[0003] The tryptophan (Trp) / aryl hydrocarbon receptor (AHR) pathway is an important metabolic immune pathway in tumors. Trp in tumor cells is in a high metabolic state, and its metabolites can activate AHR. The activated AHR enters the nucleus and promotes the formation of tumor suppressive immune microenvironment by regulating the expression of downstream target genes. Because the Trp / AHR pathway can promote tumor immune escape through various mechanisms such as inhibiting T cell activation and promoting the infiltration of suppressive immune cells, targeting this pathway is an important strategy for tumor immunotherapy.

[0004] IL4I1 belongs to the L-amino-acid oxidase (LAAO) family, which can catalyze the decomposition of phenylalanine to produce phenylpyruvic acid. Previous studies have found that IL4I1 can promote the progression of malignant tumors such as ovarian cancer and lymphoma by regulating the function of various immune cells. A study published in CELL in 2020 found that IL4I1 is a key enzyme for the activation of the Trp / AHR pathway in glioma and melanoma, which can promote the immune escape of glioma by activating the Trp / AHR pathway. In a contemporaneous study, the inventors identified IL4I1 as an important new target for enhancing the efficacy of anti-PD-1 antibody in lung adenocarcinoma. Knocking down IL4I1 can remodel the activated immune microenvironment of lung adenocarcinoma by blocking the Trp / AHR pathway, thereby significantly enhancing the efficacy of anti-PD-1 antibody. Therefore, developing small molecule drugs targeting IL4I1 is an important strategy and direction for enhancing the efficacy of anti-PD-1 antibody in lung adenocarcinoma. However, there is currently no report on small molecule drugs effectively targeting IL4I for the treatment of tumors.

[0005] Thymol's chemical name is 5-methyl-2-isopropylphenol, and its molecular formula is C63-C62 ... 10 H 14 Thymol, also known as thymol, is a monoterpene phenol isolated from the traditional Chinese medicines thyme and musk. Thymol is commonly used in the production of fragrances and indicators, and is also frequently used for dermatophytes and tinea. Previous studies have found that thymol possesses a wide range of biological and therapeutic activities, such as antifungal, antileishmaniasis, and antiviral activities. However, there are currently no reports on the role of thymol in the tumor immune microenvironment and tumor immunotherapy, nor is there in vivo experimental evidence to suggest its role in lung adenocarcinoma, and its effects on IL4I1 and its downstream AHR pathway remain unclear.

[0006] Anti-PD-1 antibodies represent one of the most promising new treatment methods for lung adenocarcinoma, but the current clinical response rate to anti-PD-1 antibody drugs in lung adenocarcinoma patients remains below 20%. Therefore, there is an urgent need to further develop new combination therapy regimens to improve the efficacy of anti-PD-1 antibodies in the treatment of lung adenocarcinoma. Summary of the Invention

[0007] In view of this, the present invention aims to overcome the deficiencies in the prior art and proposes the application of thymol in enhancing the efficacy of anti-PD-1 antibody therapy for lung adenocarcinoma.

[0008] To achieve the above objectives, the technical solution created by this invention is implemented as follows:

[0009] As a first aspect of the invention, the use of thymol and anti-PD-1 antibody in combination in the preparation of a medicament for the prevention and / or treatment of lung adenocarcinoma is proposed.

[0010] Preferably, the thymol targets IL4I1 to exert its effect in preventing and / or treating lung adenocarcinoma.

[0011] Preferably, the drug performs one or more of the following functions:

[0012] 1) It inhibits the growth of lung adenocarcinoma by blocking the entry of AHR into the nucleus in lung adenocarcinoma cells;

[0013] 2) It inhibits the expression of downstream target genes of AHR in lung adenocarcinoma cells, thereby inhibiting the growth of lung adenocarcinoma;

[0014] 3) Inhibiting the activity of the AHR pathway in lung adenocarcinoma cells can inhibit the growth of lung adenocarcinoma;

[0015] 4) It regulates the immune microenvironment of lung adenocarcinoma to inhibit its growth;

[0016] 5) Enhanced CD8 +Effect of T cell killing activity on lung adenocarcinoma;

[0017] 6) Effect of sensitizing lung adenocarcinoma to anti-PD-1 antibody efficacy.

[0018] As a second aspect of the present application, a combination drug for preventing and / or treating lung adenocarcinoma is proposed, the combination drug comprising thymol and an anti-PD-1 antibody.

[0019] Preferably, the combination drug enhances the efficacy of lung adenocarcinoma anti-PD-1 antibody by inhibiting or reducing the expression and / or activity of IL4I1.

[0020] Preferably, the combination drug has one or more of the following effects:

[0021] 1) Effect of blocking AHR nuclear entry in lung adenocarcinoma cells to inhibit lung adenocarcinoma growth;

[0022] 2) Effect of inhibiting AHR downstream target gene expression in lung adenocarcinoma cells to inhibit lung adenocarcinoma growth;

[0023] 3) Effect of inhibiting AHR pathway activity in lung adenocarcinoma cells to inhibit lung adenocarcinoma growth;

[0024] 4) Effect of regulating lung adenocarcinoma immune microenvironment to inhibit lung adenocarcinoma growth;

[0025] 5) Effect of enhancing CD8 + Effect of T cell killing activity on lung adenocarcinoma;

[0026] 6) Effect of sensitizing lung adenocarcinoma to anti-PD-1 antibody efficacy.

[0027] Compared with the prior art, the present application has the following advantages:

[0028] (1) The present application first discovers that thymol treatment can significantly enhance the therapeutic effect of anti-PD-1 antibody in lung adenocarcinoma, and the tumor is almost completely inhibited and the survival of mice is significantly prolonged, indicating that thymol combined with anti-PD-1 antibody is a new strategy for lung adenocarcinoma combined immunotherapy;

[0029] (2) The present application first discovers that thymol can significantly inhibit the expression of IL4I1, a new target for tumor immunotherapy, and the activation of its downstream AHR pathway;

[0030] (3) The present application first discovers that thymol targeting IL4I1 can significantly reshape the activated immune microenvironment of lung adenocarcinoma. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1Thymol inhibits IL4I1 expression in lung adenocarcinoma cells. (A. Inhibition efficiency of thymol on IL4I1 mRNA level in LLC cells under different concentration gradients; B. Inhibition efficiency of thymol on IL4I1 protein level in LLC cells under different time gradients; C. Inhibition efficiency of thymol on IL4I1 mRNA level in A549 cells under different concentration gradients.)

[0032] Figure 2 Thymol inhibits AHR pathway in lung adenocarcinoma cells. (A. Effect of 5 mM thymol on IL4I1 and AHR levels in LLC cells; B-C. Effect of 5 mM thymol on AHR levels in cytoplasm and nucleus of LLC cells and gray scale quantitative analysis; D. Effect of 5 mM thymol on expression of AHR downstream target genes in LLC cells; E. Effect of 20 mM thymol on IL4I1 and AHR levels in A549 cells; F-G. Effect of 20 mM thymol on AHR levels in cytoplasm and nucleus of A549 cells and gray scale quantitative analysis; H. Effect of 20 mM thymol on expression of AHR downstream target genes in A549 cells.)

[0033] Figure 3 Effect of thymol on anti-PD-1 antibody treatment of lung adenocarcinoma. (A. Mode diagram of thymol and / or anti-PD-1 antibody treatment of orthotopic lung adenocarcinoma mice; B. Small animal live imaging detection of tumor size of four groups of mice; C. Fluorescence quantitative statistics of small animal live imaging of four groups of mice; D. Survival analysis of four groups of mice; E. Expression difference of IL4I1 protein in tumors of control group and thymol treatment group mice; F. Expression difference of IL4I1 mRNA in tumors of control group and thymol treatment group mice; G. Immunohistochemical staining of IL4I1 in tumors of control group and thymol treatment group mice and quantitative statistical analysis of H score.)

[0034] Figure 4 Regulatory effect of thymol on immune microenvironment of lung adenocarcinoma. (A. Ratio of CD8 + T cells, GZMB + CD8 + T cells, Treg cells, macrophages and M2 type macrophages in tumors of control group and thymol treatment group mice; B. Immunohistochemical staining and statistical analysis of CD8 + T cells in tumors of control group and thymol treatment group mice; C. Immunohistochemical staining and statistical analysis of GZMB + T cells in tumors of control group and thymol treatment group mice.) DETAILED DESCRIPTION

[0035] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which this invention pertains. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0036] The invention will be described in detail below with reference to specific embodiments.

[0037] Example 1: Thymol significantly inhibits the expression of IL4I1 in lung adenocarcinoma cells.

[0038] The experimental methods are as follows: LLC cells were treated with DMSO, 0.1 μM, 1 μM, 5 μM, and 10 μM thymol, respectively. After 24 h, RNA was extracted from the cells and subjected to reverse transcription and qRT-PCR to detect changes in IL4I1 mRNA levels. LLC cells were treated with 5 μM thymol for 24 h, 48 h, and 72 h, respectively, and total cellular protein was extracted. Changes in IL4I1 protein levels were detected by Western blot. A549 cells were treated with DMSO, 5 μM, 10 μM, 20 μM, and 50 μM thymol, respectively. After 24 h, RNA was extracted from the cells and subjected to reverse transcription and qRT-PCR to detect changes in IL4I1 mRNA levels.

[0039] Experimental results are as follows Figure 1 As shown, by Figure 1 It can be seen that the inhibitory efficiency of thymol on IL4I1 in LLC cells is concentration-dependent and time-dependent, with the optimal inhibitory concentration being 5 μM. 5 μM thymol can inhibit the expression of endogenous IL4I1 in LLC cells to 12.66%. Figure 1 (AB). The inhibitory efficiency of thymol on IL4I1 in A549 cells was also concentration-dependent, with the optimal inhibitory concentration being 20 μM. 20 μM thymol could inhibit the expression of endogenous IL4I1 in A549 cells to 33.31% (AB). Figure 1 C). This indicates that thymol can significantly inhibit the expression of IL4I1 in lung adenocarcinoma cells.

[0040] Example 2: Thymol inhibits the activation of the AHR pathway in lung adenocarcinoma cells.

[0041] The experimental method is as follows: LLC cells are treated with 5 μM thymol, and total protein is extracted after 48 h. The total AHR and IL4I1 protein levels are detected by western blot. LLC cells are treated with 5 μM thymol, and cell pellets are collected after 48 h. Cytosolic protein and nuclear protein are extracted using a nuclear and cytoplasmic extraction kit. The expression of AHR in cytosolic protein and nuclear protein is detected by western blot. LLC cells are treated with 5 μM thymol, and total RNA is extracted after 48 h. The expression of AHR downstream target genes is detected by reverse transcription and qRT-PCR. A549 cells are treated with 20 μM thymol, and the total AHR and IL4I1 protein levels, the expression of AHR in cytosolic protein and nuclear protein, and the expression of AHR downstream target genes in A549 cells are detected by the above methods. Therefore, thymol can effectively inhibit the activation of the AHR pathway in lung adenocarcinoma cells.

[0042] The experimental results are shown in Figure 2 As can be seen from Figure 2 It can be seen that 5 μM thymol can significantly inhibit the expression of IL4I1 in LLC cells and significantly inhibit the nuclear entry of AHR and the expression of its downstream target genes (Il6, Il10, Il22, Il1b, Arg1, Serpinb2, Tiparp, Mmp13, Cyp1b1) (A-D). Figure 2 20 μM thymol can significantly inhibit the expression of IL4I1, the nuclear entry of AHR, and the expression of AHR downstream target genes in A549 cells (E-H). Figure 2

[0043] Example 3 Thymol targeting IL4I1 significantly enhances the therapeutic effect of anti-PD-1 antibody in lung adenocarcinoma mice

[0044] The experimental method is as follows: LLC cells with luciferase are used to construct a lung orthotopic tumor model. C57BL / 6 mice are sterilized with iodophor on the right chest, and LLC cells mixed with an equal volume of Matrigel gel are implanted into the right middle lobe of the lung through percutaneous puncture with a 1 mL needle, and 1 × 10 6 After 5 days of tumor implantation, the mice are randomly divided into four groups: a control group, a thymol treatment group, an anti-PD-1 antibody (CD279) treatment group, and a thymol combined with anti-PD-1 antibody treatment group (5 mice in each group). Anti-PD-1 antibody 10 mg / kg is injected intraperitoneally once a week. Thymol 75 mg / kg is injected intraperitoneally every other day. The tumor size is detected by small animal live imaging 21 days after tumor implantation. The survival period of the mice is observed in the same way by constructing a lung adenocarcinoma mouse treatment model (7 mice in each group).

[0045] ​At 21 days after tumor inoculation, the tumor tissues of the control and thymol treatment groups of mice were stripped out, a part was put into Trizol solution for tissue disruption, RNA and total protein extraction, and the expression of IL4I1 mRNA and protein levels in the tumor was detected by qRT-PCR and western blot; the other part was put into 4% paraformaldehyde solution for fixation, dehydration, embedding, sectioning, etc., and the expression of IL4I1 in the tumor was detected by immunohistochemistry.

[0046] The experimental results are shown in Figure 3 As can be seen from Figure 3 It can be seen that: thymol monotherapy can significantly inhibit tumor progression and prolong the survival of mice; compared with thymol alone or anti-PD-1 antibody alone treatment group, the tumor of the combination treatment group of mice is almost completely inhibited, and the survival of the mice is significantly prolonged Figure 3 A-D). In addition, the protein expression and mRNA expression of IL4I1 in the tumor of the thymol treatment group of mice are significantly lower than those of the control group Figure 3 E-F). The immunohistochemical results also show that the staining intensity of IL4I1 in the tumor of the thymol treatment group of mice is significantly lower than that of the control group Figure 3 G). Therefore, it is proved that thymol targeting IL4I1 expression can significantly inhibit lung adenocarcinoma progression and enhance the efficacy of anti-PD-1 antibody.

[0047] Example 4 Thymol remodeling of lung adenocarcinoma activated immune microenvironment

[0048] The experimental method is as follows: LLC cells with luciferase are used to construct a lung orthotopic tumor model, and the mice are treated with control or thymol (75 mg / kg intraperitoneal injection, once every other day). At 21 days after tumor inoculation, the tumor tissues of the mice were stripped out, a part of the tissues was ground and added with 0.1% trypsin and 0.2 μg / mL collagenase, and after mixing uniformly by blowing, it was placed in a 37℃ shaking bed for 2-4h, and then the red blood cells were lysed and washed, and then the cell suspension was filtered into a single cell suspension with a 40 μm nylon filter, and then a certain amount of membrane flow cytometry antibody was added to the flow cytometry tube according to the recommended concentration of the antibody instruction, the cells were resuspended, and incubated on ice for 30 min. For intracellular factor staining, the cells were treated with flow cytometry fixing solution and flow cytometry membrane breaking solution, and then stained with intracellular factors, and incubated on ice for 30 min. After staining, it was washed twice, and then analyzed by flow cytometry. CD8 +The T cell staining scheme was: CD45-APC, CD3-FITC, CD8-PE / CY7, GZMB-PE; the Treg cell staining scheme was: CD45-APC, CD4-PE, CD25-FITC; and the macrophage staining scheme was: CD45-PE, CD11b-APC, F4 / 80-APC / CY7 and CD206-FITC. Another part of the tumor tissue was placed in a 4% paraformaldehyde solution, and after fixation, dehydration, embedding and sectioning, immunohistochemical staining of CD8 and GZMB was performed.

[0049] The experimental results are shown in Figure 4 As can be seen from Figure 4 Compared with the control group mice, the number of CD8 + T cells and the number of CD8 + T cells expressing GZMB in the tumors of the thymol treatment group mice were significantly increased, while the proportion of Treg cells and M2 type macrophages was significantly decreased Figure 4 (A). In addition, immunohistochemical staining also found that the infiltration of CD8 + T cells and GZMB + T cells in the tumors of the thymol treatment group mice was significantly higher than that of the control group mice Figure 4 (B-C). It was proved that thymol can promote the formation of an activated immune microenvironment in lung adenocarcinoma.

[0050] In summary, the present application first identified the small molecule drug thymol targeting IL4I1 expression, and first found that thymol targeting IL4I1 can block the AHR signaling pathway, thereby remodeling the activated immune microenvironment of lung adenocarcinoma. In addition, it was also first found that thymol can significantly enhance the efficacy of anti-PD-1 antibody for lung adenocarcinoma, which can provide a new scheme and strategy for the immunotherapy of lung adenocarcinoma patients in clinic, and has great application prospect.

[0051] The above only describes the preferred embodiments of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A combination drug for treating lung adenocarcinoma, characterized in that: The active ingredients of the combined medication are thymol and anti-PD-1 antibody.

2. The use of thymol and anti-PD-1 antibody as active ingredients in the preparation of drugs for the treatment of lung adenocarcinoma.

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