CN109813913B

CN109813913B - Use of aromatic hydrocarbon receptor (AhR) for predicting immunotherapy effect

Info

Publication number: CN109813913B
Application number: CN201910098801.9A
Authority: CN
Inventors: 周光飚; 王桂珍; 张力
Original assignee: Cancer Hospital and Institute of CAMS and PUMC
Current assignee: Cancer Hospital and Institute of CAMS and PUMC
Priority date: 2019-01-31
Filing date: 2019-01-31
Publication date: 2021-11-09
Anticipated expiration: 2039-01-31
Also published as: CN109813913A

Abstract

The invention provides a new application of an aromatic hydrocarbon receptor (AhR) in predicting the effect of immunotherapy, and particularly provides an application of AhR serving as a target in developing, screening and/or preparing a medicament for treating tumors, an application of an AhR inhibiting, silencing and/or knocking out agent in preparing a medicament for treating tumors, and an application of an AhR level detecting agent in preparing a detection agent for predicting the effect of an immunodetection point inhibitor in treating tumors.

Description

Use of aromatic hydrocarbon receptor (AhR) for predicting immunotherapy effect

Technical Field

The invention relates to a new application of a transcription factor aromatic hydrocarbon receptor (AhR), in particular to an application of the aromatic hydrocarbon receptor in a detection method for predicting the treatment effect of an immunotherapy medicament and a target serving as an anti-cancer medicament, belonging to the field of medicines.

Background

An arene receptor (AhR) is a transcription factor, and plays an important role in regulating and controlling metabolism of compounds such as polycyclic aromatic hydrocarbons in the environment, organism immunity, rhythm, reproduction, oxidative stress and the like by regulating and controlling transcription of downstream genes. AhR is present in various tissues and cells such as lung, liver, kidney, placenta, tonsil, skin, and B lymphocyte of human body, and tryptophan metabolite, heme metabolite, arachidonic acid metabolite, etc. are endogenous ligands.

AhR is involved in the maintenance and stabilization of the immune system of the body. Although the AhR knockout mice do not die, their visceral tissues and immune system tend to be dysplastic, enlarged spleen, increased B cells, increased IFN γ and IL-12 production. AhR can modulate immune cell function in a ligand-dependent manner, such as CD8+ T cells, dendritic cells, Treg cells, macrophages, and the like. In dioxin treatment, AhR can inhibit CD8+ T cell activity caused by primary infection with virus, but has no effect on the activity of virus-specific memory CD8+ T cells. The AhR deletion results in the maturation disorder of langerhans dendritic cells, and the low expression of the co-stimulatory molecules CD40, CD80, CD24a, but the phagocytic capacity is higher. AhR can also affect the T cell-mediated immune response of Dendritic Cells (DCs). The AhR can change the distribution of Tregs and increase the proportion of Tregs in the spleen to inhibit immune response. The AhR can also be involved in regulating and controlling the synthesis and secretion of monocyte-macrophage cell factor, enhancing the sterilizing effect of the monocyte-macrophage and reducing the apoptosis of the monocyte-macrophage. AhR can enhance the ability of alveolar CD4+ T cells to resist viruses. AhR can up-regulate the expression of NF-kB and DNA binding capacity thereof, thereby enhancing inflammatory response. AhR can also regulate the expression of IL-1 beta, IL-6, TNF-alpha and the like so as to play a role in immune negative regulation. The AhR can enhance intracellular oxidative stress, promote proliferation of lung cancer cells, and protect lung adenocarcinoma cells against the redox reaction of tobacco particles. AhR mediates the environmental carcinogen polycyclic aromatic hydrocarbon-induced chemokine CXCL13 secreted by lung epithelial cells, which plays an important role in the development of lung cancer. However, the role of AhR in immunotherapy remains unclear.

In recent years, molecular targeted drugs have made remarkable progress in clinical application of patients with advanced non-small cell lung cancer (NSCLC), but the prognosis is still not satisfactory, and a new treatment method needs to be explored to obtain a new breakthrough in lung cancer treatment. In cancer immunotherapy, inhibition of the immune checkpoint pathway is considered to be one of the most promising therapeutic modalities, the mechanism of which is to release the T cell activity-inhibited state by inhibiting the relevant target (PD-1, PD-L1, CTLA-4) in the pathway, which activated T cells are able to attack and destroy tumor cells. PD-1/PD-L1 immunotherapy is a new anticancer immunotherapy which is currently spotlighted, aims to utilize the immune system of the human body to resist cancer, leads cancer cells to die by blocking a PD-1/PD-L1 signal channel, has the potential of treating various tumors, and is expected to substantially improve the Overall Survival (OS) of patients. Each large pharmaceutical huge head is also advancing its own project at the fire rate, investigating monotherapy and combination therapy for the treatment of various cancers to exploit the greatest clinical potential of this class of drugs. The PD-1/PD-L1 antibody is a broad spectrum drug, but not all patients respond to the PD-1/PD-L1 antibody. At present, 30% -40% of patients with malignant melanoma benefit from the treatment with PD-1/PD-L1 antibody, the response rate of non-small cell lung cancer is 20%, the initial data of liver cancer is also 20%, and renal cell carcinoma is about 20% to 30%.

In order to solve the problem that the effective rate of the PD-1/PD-L1 antibody is relatively low in the clinical application process, a new method for predicting the curative effect of the PD-1/PD-L1 antibody is urgently needed to be found so as to accurately screen effective cases. Meanwhile, the medicine for obviously enhancing the curative effect of the PD-1/PD-L1 antibody is also urgently needed in clinic.

Disclosure of Invention

An object of the present invention is to provide a detection technique for predicting the therapeutic effect of immunotherapy and a drug target that can significantly enhance the therapeutic effect of tumor immunotherapy.

The inventor of the present invention finds in research that AhR is a suitable target for treating various cancers, an AhR inhibitor can be used alone for treating cancers or used in combination with immunotherapy, and the AhR inhibitor and the immunotherapy have an obvious synergistic effect, so that the curative effect of the immunotherapy can be enhanced, the drug resistance of the immunotherapy can be overcome, a stronger therapeutic effect can be exerted, and a new therapeutic strategy is provided for treating tumors. The inventor further researches to find that the AhR expression level is related to the treatment effect of immunotherapy, the PD-1 antibody of a patient with high AhR expression has better curative effect, and the detection of the AhR expression level of a cancer tissue by an immunohistochemical method can be used as an important means for predicting the treatment effect of the immunotherapy.

Thus, in one aspect, the invention provides the use of AhR as a target in the development, screening and/or manufacture of a medicament for the treatment of a tumour.

In another aspect, the invention also provides the use of an agent that inhibits, silences and/or knocks AhR for the preparation of a medicament for the treatment of a tumor.

According to a specific embodiment of the present invention, the tumor includes, but is not limited to, lung cancer, cervical cancer, ovarian cancer, liver cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, melanoma, multiple myeloma, head and neck squamous cell carcinoma, prostate cancer, leukemia, lymphoma, brain tumor, and skin cancer.

According to a specific embodiment of the present invention, the tumor comprises an immunotherapy-sensitive and drug-resistant cancer.

In another aspect, the invention provides the use of an agent that detects AhR levels in the preparation of a test agent for predicting the efficacy of an immunodetection point inhibitor in treating a tumor.

According to a specific embodiment of the present invention, in the technical scheme of the present invention, the immune monitoring points comprise one or more of PD-1, PD-L1 and CTLA-4.

According to a specific embodiment of the present invention, the detection of AhR level comprises detecting AhR gene expression level, or detecting AhR protein expression level. The amount of AhR protein expressed in the patient's tissue can be measured by any method known in the art, including, but not limited to, immunohistochemistry or Western blot. The expression level and splicing cost of AhR at gene level can also be detected by polymerase chain reaction. Reagents for detecting AhR levels include, but are not limited to, detection reagents used in these detection methods.

According to a specific embodiment of the present invention, the treatment of tumor comprises treating tumor by inhibiting, silencing and/or knocking out AhR, or treating tumor by inhibiting, silencing and/or knocking out AhR in combination with immunotherapy.

As mentioned above, in the technical solution of the present invention, the tumor includes, but is not limited to, lung cancer, cervical cancer, ovarian cancer, liver cancer, esophageal cancer, stomach cancer, colon cancer, rectal cancer, melanoma, multiple myeloma, head and neck squamous cell carcinoma, prostate cancer, leukemia, lymphoma, brain tumor, and skin cancer. In some specific embodiments, the tumor comprises an immunotherapy-sensitive and drug-resistant cancer.

In another aspect, the invention provides a pharmaceutical composition for treating a tumor, the pharmaceutical composition comprising an agent that inhibits, silences and/or knocks out AhR, and further comprising an immune checkpoint antibody. In some specific embodiments, the immune monitoring point antibody is preferably a PD-1 and/or PD-L1 antibody. The experimental result shows that the AhR inhibitor and the PD-1/PD-L1 antibody have good combined effect. Specifically, the AhR inhibitor can be mixed with appropriate auxiliary agents and prepared into any suitable form of tablets, injection and the like, or the AhR inhibitor and the PD-1/PD-L1 antibody are prepared into compound medicines, so that a novel method is provided for immunotherapy of cancers.

In the specific embodiment of the invention, AhR plays a key role in increasing PD-L1 caused by smoking and promoting lung cancer, evidence that AhR can be used as a new target for cancer treatment is provided, and the expression level of AhR in the tissues of a patient can be used for well predicting the treatment effect of the patient on tumor immunotherapy, and the AhR plays an important application value role in predicting the tumor immunotherapy effect.

Drawings

FIGS. 1A to 1D show the results of experiments in which tobacco and BaP promote the expression of PD-L1 at the cellular level. In FIG. 1A, tobacco extract was used to treat H460 cells and 16HBE cells, and real-time PCR was used to detect expression of PDL 1. FIG. 1B, tobacco extract treated H460 cells and 16HBE cells, and flow analysis detected the expression of PDL 1. FIG. 1C, different concentrations of BaP treated H460 cells and 16HBE cells, real-time PCR detected expression of PDL 1. FIG. 1D, 5 μ MBaP treated H460 cells and 16HBE cells, cells harvested at different time periods, real-time PCR to detect expression of PDL 1.

FIGS. 2A-2C show the results of experiments in which tobacco and BaP promote expression of PD-L1 at an in vivo level in animals. In FIG. 2A, A/J mice treated with air/tobacco at different time stages were immunohistochemically examined for PDL1 expression in mouse lung tissue specimens. FIG. 2B immunohistochemistry of A/J mice treated with BaP/corn oil at various time periods examined mouse lung tissue sections for PDL1 expression. FIG. 2C, immunohistochemistry and immunofluorescence measures the expression and localization of PDL1 and TTF1 in mouse lung specimen tissues.

FIGS. 3A-3B show the experimental results of high expression of PD-L1 in smoking lung cancer patients, negatively correlated with the prognosis of survival in lung cancer patients. In FIG. 3A, the Westernblot analysis of cancer tissue (T) from 62 specimens of lung cancer patients and the corresponding paracarcinoma tissue (N) from the same patient was performed using anti-PDL 1 and Actin antibodies, and the expression levels of 10 specimens are shown, with the numbers corresponding to the patient numbers. FIG. 3B shows that SPSS software is used to perform Kaplan-Meier survival analysis and long-rank test on the survival prognosis relationship between PDL1 high expression and lung cancer patients, and p is less than 0.05.

Fig. 4A to 4B show the experimental results that knocking out AhR can slow down the occurrence of BaP-induced lung cancer and the expression of PD-L1. In FIG. 4A, BaP/corn oil treated mice of different genotypes C57 (AhR)^+/+、AhR^+/-、 AhR^-/-) Pulmonary micct (top), HE staining results (middle) and immunohistochemistry were performed to examine the expression of PDL1 in lung tissues of BaP/corn oil-treated mice of different genotypes C57 (bottom). FIG. 4B, immunofluorescence assay of AhR and PDL1 expression and localization in tissues from 3 lung cancer patients.

Fig. 5A to 5B show experimental results that the AhR inhibitor significantly inhibited the growth of mouse lung cancer and had a good combined therapeutic effect with PD-L1 antibody. In the figure 5A, LLC cells are inoculated to the tail vein of a C57 mouse, and micro-CT and HE staining are used for detecting the influence of ANF on the tumor volume of the mouse after in vivo administration. FIG. 5B, C57 mice tail vein inoculated with LLC cells, micro-CT and HE staining to examine the effect of ANF and PDL1 antibody combination on tumor volume in mice after in vivo administration.

Fig. 6A to 6B show the experimental results of the AhR inhibitor having significant anti-cancer activity in the mouse solid tumor model for treating PD-L1 antibody sensitivity and PD-L1 antibody drug resistance. In FIG. 6A, MC38 was subcutaneously inoculated into C57 mice, and tumor tissues were taken out after the experiment and photographed to show the tumor size inhibitory effect of ANF and PDL1 antibodies. Fig. 6B, Ag104Ld, subcutaneously inoculated B6C3F1 mice, tumor tissues were taken out of the mice after the experiment was completed and photographed to show the tumor size inhibitory effect of ANF and PDL1 antibody.

FIG. 7 shows the experimental results that the AhR expression level in clinical patient specimens well predicts the therapeutic effect of PD-1 antibody. In the figure, PR: partial response (partial response); SD: stable disease condition (stable disease); PD: disease progression (progressive disease).

Detailed Description

The following detailed description is provided for the purpose of illustrating the embodiments and the advantageous effects thereof, and is not intended to limit the scope of the present disclosure. In the examples, the experimental methods without specifying the specific conditions were conventional methods and conventional conditions well known in the art, or were operated according to the conditions suggested by the instrument manufacturer.

Example 1

The effect of tobacco exposure on PD-L1 expression is not currently reported. In the present invention, tobacco extract (CES) was prepared using a smoke generator, and the smoke was dissolved in 50mL of 1640 medium without serum. Tobacco extract (CES) and tobacco carcinogen benzopyrene (BaP) (sigma) were used to treat normal lung epithelial 16HBE cells and non-small cell lung cancer H460 cells. The results are shown in FIGS. 1A to 1D. In FIG. 1A, tobacco extract was used to treat H460 cells and 16HBE cells, and real-time PCR was used to detect expression of PDL 1. FIG. 1B, tobacco extract treated H460 cells and 16HBE cells, and flow analysis detected the expression of PDL 1. FIG. 1C, different concentrations of BaP treated H460 cells and 16HBE cells, real-time PCR detected expression of PDL 1. FIG. 1D, 5 μ MBaP treated H460 cells and 16HBE cells, cells harvested at different time periods, real-time PCR to detect expression of PDL 1. The results show that the mRNA level and the protein level of PD-L1 are obviously increased after the tobacco extract is treated, and the expression level of PD-L1 is positively correlated with the treatment time and the treatment dosage of the tobacco extract. The BaP treated cells were consistent with tobacco extracts. The above results indicate that tobacco extract and BaP promote expression of PD-L1 at the cellular level, with the degree of increase being positively correlated with the time and dosage of tobacco extract treatment.

In order to further verify smoking and BaP-caused changes at an in vivo level, experiments are carried out on A/J mice, and the mice are exposed to smoke generated by a smoke generator smoking the smoke to simulate human smoking, wherein 12 cigarettes are smoked each day (like human smoking, each cigarette is smoked intermittently for 3 minutes, then clean air is returned for 15 minutes), the mice are exposed for 5 days each week for 3 to 24 weeks continuously, then mouse lung tissues are taken, and the PD-L1 condition is detected by a real-time quantitative RT-PCR, immunohistochemistry and Western blot method. The results are shown in FIGS. 2A to 2C. In FIG. 2A, A/J mice treated with air/tobacco at different time stages were immunohistochemically examined for PDL1 expression in mouse lung tissue specimens. FIG. 2B immunohistochemistry of A/J mice treated with BaP/corn oil at various time periods examined mouse lung tissue sections for PDL1 expression. FIG. 2C, immunohistochemistry and immunofluorescence measures the expression and localization of PDL1 and TTF1 in mouse lung specimen tissues. As a result, the expression of PD-L1 can be obviously increased by smoking; in addition, when mice were treated with BaP (each dose was 100mg/kg body weight twice a week for 5 consecutive weeks) and then lung tissues of the mice were examined for the expression of PD-L1, BaP treatment was found to significantly increase the expression of PD-L1 in lung tissues of the mice.

By detecting the expression level of PD-L1 in the cancer tissues of 62 patients, the expression of PD-L1 in patients with advanced lung cancer (stages III and IV) is obviously higher than that in early patients (stages I and II); the survival time of the patients was analyzed by the Kaplan-Meier method, and the results are shown in FIGS. 3A to 3B. In FIG. 3A, the Westernblot analysis of cancer tissue (T) from 62 specimens of lung cancer patients and the corresponding paracarcinoma tissue (N) from the same patient was performed using anti-PDL 1 and Actin antibodies, and the expression levels of 10 specimens are shown, with the numbers corresponding to the patient numbers. FIG. 3B shows that SPSS software is used to perform Kaplan-Meier survival analysis and long-rank test on the survival prognosis relationship between PDL1 high expression and lung cancer patients, and p is less than 0.05. As a result, the survival time of the lung cancer patient with high PD-L1 expression is obviously shorter than that of the lung cancer patient with low PD-L1 expression (p <0.05), which indicates that the high PD-L1 expression is inversely related to the survival prognosis of the lung cancer patient.

In order to evaluate the effect of AhR in PD-L1 increase caused by smoking and lung cancer induction, the invention utilizes AhR knockout mice (Jackson Laboratory), after reasonable grouping, treatment with carcinogen BaP (BaP 100mg/kg, twice a week for 8 weeks), and detection with small animal computed tomography (Micro-CT), lung tissue HE staining, immunohistochemistry and the like, and the results are shown in FIGS. 4A-4B. In FIG. 4A, BaP/corn oil treated mice of different genotypes C57 (AhR)^+/+、AhR^+/-、AhR^-/-) Pulmonary micct (top), HE staining results (middle) and immunohistochemistry were performed to examine the expression of PDL1 in lung tissues of BaP/corn oil-treated mice of different genotypes C57 (bottom). FIG. 4B, immunofluorescence assay of AhR and PDL1 expression and localization in tissues from 3 lung cancer patients. The result shows that the knockout of AhR can obviously reduce the up-regulation of PD-L1 expression caused by BaP; wild type mice developed significant tumors 6 months after BaP gavage, whereas AhR knockout mice did not develop tumors. Through immunofluorescence technology, it is found that AhR and PD-L1 in lung cancer tissues of patients are jointly highly expressed on lung cancer cells.

Mouse lung cancer cell line LLC cells were injected into immune system-normal C57 mice via tail vein, and then treated with AhR inhibitors ANF and CH223191, and the results are shown in fig. 5A-5B. In the figure 5A, C57 mice tail vein inoculated with LLC cells, micro-CT and HE staining to detect the effect of ANF on tumor volume in mice after in vivo administration. FIG. 5B, C57 mice tail vein inoculated with LLC cells, micro-CT and HE staining to examine the effect of ANF and PDL1 antibody combination on tumor volume in mice after in vivo administration. The result shows that the AhR inhibitor can obviously inhibit the development of lung cancer, which indicates that the AhR inhibitor has obvious effect of resisting lung cancer; in addition, the AhR inhibitor and the anti-PD-L1 antibody are combined for application, so that the anti-lung cancer effect of the PD-L1 antibody can be obviously enhanced, and the AhR inhibitor and the anti-PD-L1 antibody have good combined effect. These results indicate that AhR is a suitable target for lung cancer treatment, and that AhR inhibitors have significant synergistic effects with immunotherapy, providing a new therapeutic strategy for lung cancer treatment.

Example 2

To further study the role of AhR inhibitors in the development of other cancers, in this example, C57 mice were inoculated with a mouse colon cancer cell line MC38 with high PD-L1 expression, and after 4 days of inoculation, AhR antagonist ANF treatment was given, and tumor tissues of the mice were taken out and photographed after the experiment. The results, see fig. 6A, show that AhR inhibitors significantly inhibited the growth of MC38 in mice and had a good combination effect with anti-PD-L1 antibody.

A mouse fibrosarcoma cell line Ag104ld which is highly expressed by PD-L1 and resistant to a PD-L1 antibody is selected to be inoculated into a B6C3F1 mouse, an AhR inhibitor is given for treatment in combination with the PD-L1 antibody 4 days after inoculation, and tumor tissues of the mouse are taken out and photographed after the experiment is finished. Results referring to fig. 6B, it was found that the combination of AhR inhibitor and PD-L1 antibody reversed the resistance of PD-L1 antibody, significantly inhibiting the growth of Ag104ld cells in mice. These results indicate that AhR has therapeutic effects on both PD-L1 antibody-sensitive and PD-L1 antibody-resistant cancers, and can reverse PD-L1 antibody resistance.

Example 3

This example investigates the role of AhR expression levels in predicting the therapeutic effect of immunotherapy. Selecting a lung cancer specimen of a patient treated by the PD-1 antibody, and detecting the relation between the expression level of AhR and the curative effect of the PD-1 antibody by an immunohistochemical method. The specific detection method comprises the following steps:

1) dewaxing: preheating in an oven at 60 ℃ for 30min, and then respectively putting into fresh dimethylbenzene I, II and III for 10 min;

2) hydration: 5 minutes respectively in the absolute ethyl alcohol I, II and II, and 5 minutes respectively in 95 percent, 85 percent and 75 percent of ethyl alcohol;

3) washing with PBS for 1 time;

4) placing the slices in a citric acid buffer solution for restoration, boiling for 5 minutes with high fire in a microwave oven, and boiling for 15 minutes with medium and low fire;

5) cooling to room temperature for about 1 h;

6) and (3) sealing: dropwise adding confining liquid, incubating at 37 ℃ for 30 minutes, and removing redundant liquid;

7) a first antibody: adding primary antibody covering tissues with proper concentration, and standing overnight at 4 ℃;

8) PBS wash 3 times, 10 minutes each time;

9) inactivation of endogenous peroxidase: 3 percent of H2O2 is dripped, and the mixture is kept stand for 10 minutes at room temperature;

10) PBS wash 1 time, 10 minutes each time;

11) secondary antibody: adding enzyme-labeled secondary antibody to cover the tissue, and incubating at 37 ℃ for 90 minutes;

12) PBS wash 3 times, each for 5 minutes;

13) color development: adding the prepared DAB color developing solution, standing for 1-5 minutes at room temperature, and stopping observing the color at proper time;

14) and (3) stopping color development: washing with tap water for 3 minutes;

15) counterdyeing: adding hematoxylin, standing at room temperature for 2 minutes, and washing with tap water for 20 minutes;

16) and (3) dehydrating: 75%, 85%, 95% and 3 minutes each in absolute ethanol I & II & III;

17) and (3) transparency: xylene I & II & III each for 3 minutes;

18) the appropriate amount of the blocking agent was used for blocking, and observed under a microscope and photographed.

See FIG. 7 for results. The results show that: the PD-1 antibody of the patient with high AhR expression has better curative effect (PR + SD), while the PD-1 antibody of the patient with low AhR expression has poor curative effect (PD). These results indicate that the detection of AhR expression in clinical patients can be an important means for detecting antibody efficacy in immunodetection sites.

The foregoing is directed to embodiments of the present invention, and it is understood that various modifications and improvements can be made by those skilled in the art without departing from the spirit of the invention.

Claims

1. Use of an agent for detecting AhR levels in the preparation of a test agent for predicting the efficacy of an immunodetection point inhibitor in treating a tumour;

wherein the immunodetection point inhibitor is a PD-1 antibody;

the tumor is lung cancer;

the AhR level is the expression level of AhR in a lung cancer tissue specimen.

2. The use of claim 1, wherein the reagent for detecting AhR levels comprises a reagent for detecting AhR levels using immunohistochemistry.