Application of combination of FXR agonist and PD1 / PD-L1 monoclonal antibody in preparation of anti-hepatoma drugs

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of an FXR agonist combined with a PD1 / PD-L1 monoclonal antibody in preparation of an anti-liver cancer medicine, in particular to application of obeticholic acid (OCA) combined with a Czodiasumab in treatment of liver cancer. The invention also provides an application of the nuclear receptors FXR and PD1 in liver cancer treatment, specifically, the nuclear receptors FXR and PD1 are taken as double targets, and the medicine for treating liver cancer is obtained. An FXR agonist OCA is used for activating FXR, and it is found that activation of FXR can regulate immune infiltration of T cells by promoting polarization of macrophage M1, so that growth of tumors is inhibited. In addition, expression of PD-L1 in liver cancer can be promoted by activating FXR, and a better tumor inhibition effect can be achieved by combining an FXR agonist OCA with a PD1 monoclonal antibody for immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the use of a FXR agonist combined with a PD1 monoclonal antibody in the preparation of an anti-liver cancer drug. Background Art

[0002] Primary liver cancer is one of the most common malignant tumors clinically. Local drug resistance, immune escape, and cancer metastasis of tumor cells are inseparable from the role of the tumor microenvironment (TME). TME includes cells of the innate and adaptive immune systems, such as granulocytes, lymphocytes, and macrophages. These cells participate in various immune responses and activities, contributing to the development and immune escape of tumors. The most prominent immune cell type in TME is macrophages. Macrophages can be divided into M1 type with immune activation and anti-tumor properties and M2 type that promotes tumor development. M1 macrophages release pro-inflammatory factors, such as TNF-α, IL-1, and IL-12, which have the function of enhancing T cell killing of tumor cells and participate in anti-tumor immunity, while the role of M2 macrophages is opposite to that of M1 macrophages. It releases anti-inflammatory factors, such as IL-10, TGF-β, and Arg-1, which inhibit T cell function, assist tumor cells in immune escape, and promote tumor growth. Currently, scientists are working on studying treatment options that target M2 macrophages or induce M2 macrophages to reprogram into M1 macrophages, in order to provide new ideas for tumor immunotherapy.

[0003] In the past few decades, surgery, radiotherapy and chemotherapy have been the main means of treating cancer. With the development of modern biotechnology, tumor immunotherapy has made breakthrough progress in clinical applications and has developed into another important standard treatment strategy. Tumor immunotherapy activates the body's own immune system to inhibit and kill tumor cells. It has the potential to treat many types of tumors, has low side effects and lasting responsiveness, and can substantially improve the patient's overall survival. It is a beginning for the cure of tumors. Among them, programmed death receptor-1 (PD-1) / programmed death ligand 1 (PD-L1) is one of the most promising targets in the field of immunotherapy. Liver cancer provides a particularly fertile ground for the use of immunotherapy because the underlying chronic disease can induce an immune-tolerant microenvironment, among which CD8 +T cell exhaustion is the reason why tumor cells can escape immune surveillance in it. In addition, PD-L1 expression is generally low in liver cancer, and too low PD-L1 expression affects the effect of immunotherapy. Therefore, although significant progress has been made in immunotherapy of liver cancer, only a few patients respond to immunotherapy. Sintilimab is a fully human IgG4 monoclonal antibody that binds to PD-1 and prevents the binding of PD-1 and PD-L1, thereby helping to restore anti-tumor T cell responses. The drug was jointly developed by Innovent Biologics and Eli Lilly and Company and was recently approved in China for the treatment of patients with relapsed or refractory classical Hodgkin lymphoma after ≥2 lines of systemic chemotherapy. Phase I / II development of sintilimab for solid tumors is underway in the United States, and the U.S. FDA accepted the investigational new drug application for sintilimab in January 2018.

[0004] Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily and is highly expressed in multiple tissues, including the liver, intestine, kidney, and adrenal gland. As a ligand-activated transcription factor, FXR regulates the expression of target genes involved in enterohepatic circulation and metabolic homeostasis. FXR is considered a negative regulator of tumors. FXR deficiency in mice leads to increased colon cell proliferation and spontaneous liver tumors. On the other hand, FXR is considered a regulator of inflammatory and immune responses in various diseases. OCA was successfully developed by Intercept Pharmaceuticals in the United States. It is the first drug developed in 20 years for the treatment of cholestatic liver disease. As a bile acid mimetic, it can activate FXR.

[0005] In the present invention, the applicant found that activating FXR in liver cancer can regulate the immune infiltration of T cells by promoting the polarization of macrophages M1, thereby inhibiting tumor growth. In addition, FXR agonist OCA combined with PD1 monoclonal antibody immunotherapy can have a better tumor inhibition effect. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides the use of FXR agonist combined with PD1 monoclonal antibody in the preparation of anti-liver cancer drugs.

[0007] The purpose of the present invention is to provide the use of an FXR agonist combined with a PD1 monoclonal antibody in the preparation of an anti-liver cancer drug; the FXR agonist may be obeticholic acid; and the PD1 monoclonal antibody may be sintilizumab.

[0008] Another object of the present invention is to provide an application of a FXR agonist combined with a PD1 monoclonal antibody in liver cancer, specifically to obtain a drug for treating liver cancer by using the nuclear receptor FXR and PD1 as dual targets.

[0009] The drugs for treating liver cancer targeting FXR include gene drugs that enhance or inhibit FXR expression, and chemical drugs that regulate the expression of FXR and its target genes through FXR ligands.

[0010] The drugs for treating liver cancer targeting PD1 include monoclonal antibodies and various molecular antagonists.

[0011] The gene medicine or chemical medicine includes a pharmaceutically acceptable carrier in a clinically acceptable form. The gene medicine contains an FXR expression vector or an interference vector; the chemical medicine contains a ligand that regulates FXR and a ligand that regulates FXR target gene.

[0012] The monoclonal antibody and various molecular antagonists include a pharmaceutically acceptable carrier in a clinically acceptable form. The monoclonal antibody and various molecular antagonists are drugs that target blocking the combination of PD1 and PD-L1.

[0013] The ligands of FXR include chenodeoxycholic acid (CDCA), obeticholic acid (OCA), GW4064, cholic acid (CA), deoxycholic acid (DCA), and the like.

[0014] The drugs targeting PD1 / PD-L1 include Pembrolizumab, Nivolumab, Cemiplimab, Toripalimab, Sintilimab, Camrelizumab, Atezolizumab, Avelumab, Durvalumab, etc. that are already on the market and those that will be on the market in the future.

[0015] The present invention has remarkable technical effects.

[0016] The present invention found that in liver cancer, the FXR agonist OCA can regulate the immune infiltration of T cells by promoting the polarization of macrophages M1, and the use of OCA combined with PD1 monoclonal antibody immunotherapy for liver cancer has a better tumor inhibition effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1FXR expression is negatively correlated with the malignancy of liver cancer, and positively correlated with immune infiltration; Figure A. Bioinformatics analysis of FXR (HR1H4) gene expression in different tumors and normal tissues, the red box shows the gene expression of FXR in liver cancer (red) and liver tissue (blue); Figure B. Bioinformatics analysis of FXR gene expression in liver tissue, adjacent tumors and liver cancer; Figure C. FXR gene expression in liver tissue and different grades of liver cancer in clinical samples; Figure D. Bioinformatics analysis of the correlation between FXR and immune cell infiltration; Figure E. Correlation analysis of FXR and T cell infiltration in clinical liver cancer samples; Figure F. Correlation analysis of FXR expression and M1 macrophage infiltration in clinical liver cancer samples.

[0018] Figure 2 The immune infiltration of liver cancer in FXR-deficient mice in the DEN liver cancer model is reduced; Figure A. Diagram of liver-induced tumors in the DEN liver cancer model; Figure B. Gene expression of immune infiltration and inflammatory factors in liver cancer in WT mice and FXR-KO mice in the DEN liver cancer model, *p<0.05; Figures C, D. Immunohistochemical analysis of T cell subset markers in the DEN liver cancer model, 200X, *p<0.05.

[0019] Figure 3 FXR agonist OCA can enhance T cell immune infiltration of liver cancer and inhibit tumor growth; Figure A. OCA tumor inhibition effect on tumor-bearing mice; Figure B. The difference in tumor weight of tumor-bearing mice in different treatment groups, *p<0.05; Figure C. The volume change trend of tumor growth in different treatment groups, *p<0.05; Figure D, E. CD45 in tumors of different treatment groups + T,CD4 + T and CD8 + Immunohistochemical staining and statistical analysis of T cell subsets, 400X, *p<0.05; F. Flow cytometry analysis of T cell subsets in tumors of different treatment groups, *p<0.05, **p<0.01.

[0020] Figure 4 FXR can promote the immune infiltration of M1 macrophages; Figure A. Gene expression of M1 macrophage marker molecules in tumors of tumor-bearing mice, *p<0.05; Figure B. Expression of M1 macrophage marker molecule CD86 in tumors of tumor-bearing mice, *p<0.05; Figure C. Gene expression of M1 macrophage marker molecules in DEN liver cancer model, *p<0.05, **p<0.01.

[0021] Figure 5FXR activation can promote the polarization of M1 macrophages; gene expression of M1 macrophage markers and inflammatory factors in mouse macrophage RAW264.7 after different treatments, NC group vs SOCA group *p<0.05, NC group vs GS group # p<0.05, ### p<0.001.

[0022] Figure 6 Activation of FXR can promote the expression of PD-L1 in liver cancer cells; Figure A. Bioinformatics analysis of the correlation between FXR and PD-L1 (CD274); Figure B. Correlation between FXR and PD-L1 in clinical liver cancer samples; Figure C. Expression of PD-L1 in liver cancer cell lines after addition of OCA, *p<0.05; Figure D. Expression of PD-L1 in Hepa1-6 cell line and tumors of tumor-bearing mice, ***p<0.001; Figure E. Flow cytometry analysis of PD-L1 expression in tumors of different treatment groups; Figure F. Statistical results of flow cytometry analysis of PD-L1 expression, **p<0.01; Figure G. Immunohistochemical analysis of PD-L1 expression in tumors of different treatment groups, 400X; Figure H. Statistical results of immunohistochemical analysis of PD-L1 expression, **p<0.01.

[0023] Figure 7 FXR agonist combined with anti-PD-1 drug (Sintilimab) can further inhibit tumor growth; Figure A. Tumor inhibition effect of different treatment groups on tumor-bearing mice; Figure B. Difference in tumor weight of tumor-bearing mice in different treatment groups; Figure C. Volume change trend of tumor growth in different treatment groups, *p<0.05, **p<0.01; Figure D. Contents of liver function-related indicators AST, ALT, ALB and Urea in plasma of different treatment groups. DETAILED DESCRIPTION

[0024] The present invention is described in detail below with reference to specific embodiments.

[0025] Example 1.

[0026] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0027] 12-week-old wild-type C57BL / 6N male mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. C57BL / 6N FXR-deficient mice were purchased from the laboratory of City of Hope Medical Center, USA; obeticholic acid (OCA) was purchased from Taosu, product catalog number 459789-99-2; methylcellulose was purchased from Taosu, product catalog number 9004-67-5; N-nitrosodiethylamine (DEN) was purchased from Sigma, product catalog number 55-18-5; Sintilimab was purchased from the hospital; lipopolysaccharide (LPS) was purchased from Sigma, product catalog number 297-473-0; immune interferon (IFN-γ) was purchased from Sigma, product catalog number 32160405; ALB, ALT, AS T, Urea and other detection kits: purchased from Fuji Company, Japan; CD3 antibody: purchased from abcam, product catalog number is ab11089; CD4 antibody: purchased from abcam, product catalog number is ab183685; CD8 antibody: purchased from abcam, product catalog number is ab209775; CD45 antibody: purchased from Proteintech, product catalog number is 20103-1-AP; CD86 antibody: purchased from CST, product catalog number is 19589; PD-L1 antibody: purchased from CST, product catalog number is 13684; Immunohistochemistry secondary antibody kit: purchased from Zhongshan Jinqiao, PV-9001.

[0028] The present invention relates to an experimental result detection method.

[0029] 1. Establishment of experimental animal models.

[0030] (1) DEN liver cancer model: Two-week-old wild-type C57BL / 6N male mice (MOCK) and FXR-deficient mice (FXR-KO) were intraperitoneally injected with 25 mg / kg DEN and killed 40 weeks later. The livers were fixed and cryopreserved for subsequent experiments.

[0031] (2) Tumor-bearing mouse model: Mouse liver cancer cell line Hepa1-6 5×10 6 The mice were injected subcutaneously. When the tumor grew to 0.5×0.5cm in size, four groups were set up, one as control (MOCK), one fed with FXR agonist OCA 10mg / kg (OCA), one intraperitoneally injected with Sintilimab 10mg / kg (Sintilimab), and one fed with OCA and injected with Sintilimab at the same time (OCA+Sintilimab). The drug was given every two days, and the tumor volume was measured. The mice were killed after 12 days, and the tumor, blood and spleen were taken.

[0032] 2. Detection of ALB, ALT, AST and urea levels in plasma.

[0033] The eyeballs of mice treated with different methods were removed to collect blood, which was then placed in a centrifuge tube containing heparin and centrifuged at 6000 rpm for 15 minutes at 4°C to collect plasma. The plasma contents of ALB, ALT, AST, urea, etc. were immediately tested using a Fuji Biochemical Analyzer.

[0034] 3. Immunohistochemistry.

[0035] Specific experimental operation process: ① Baking: Put the paraffin-embedded tissue slices in the oven for half an hour. ② Dewaxing and hydration: Put the tissue slices in xylene for ten minutes; take them out and put them in new xylene for ten minutes; repeat once; then take the tissue slices out and put them in 95% ethanol solution (the following percentages are all volume percentage concentrations without special instructions) for five minutes; take the tissue slices out and put them in 85% ethanol solution for five minutes; then take the tissue slices out and put them in 75% ethanol solution for five minutes; then put them in single distilled water for five minutes each time, repeat twice. ③ Removal of catalase: Add 3% hydrogen peroxide solution, let it stand at room temperature for 15 minutes, and wash with PBS solution three times. ④ Antigen repair: microwave method. Put the slices in the antigen repair solution, put them in the microwave, microwave high for three minutes, and then stop; start timing for 15 minutes, and perform medium-high heat for 20 seconds every two minutes until the end of 15 minutes; leave it at room temperature to allow the antigen repair solution to cool naturally;. Then wash with PBS solution 3 times, 5min / time. ⑤ Blocking: wipe the edge of the slide tissue dry, add 5% BSA solution to each tissue section, place in a wet box for blocking for 30 minutes; then wash 3 times with PBS solution, 5min / time. ⑥ Incubate the primary antibody: dilute the primary antibody with 5% BSA solution according to the proportion in the instructions, add 50 microliters of antibody to each tissue section, place in a wet box, and incubate in a refrigerator at 4℃ overnight; take out the wet box the next day, place at room temperature for 30min, observe to avoid dry slices, and wash 3 times with PBS solution. ⑦ Incubate the secondary antibody: wipe the edge of the slide tissue dry, add secondary antibody to each tissue section, incubate at 37℃ for 20min, and then wash 3 times with PBS solution, 5min / time. ⑧ DAB color development (avoid light): prepare the color development solution in advance; add the color development solution to each slide tissue, incubate at room temperature for ten minutes away from light; pay attention to observe the specific color development under the microscope and adjust the color development time; after the color development is completed, rinse with running water. ⑨Re-staining: Re-stain the slide in hematoxylin for five minutes, observe the color change, and rinse with tap water for 10 minutes; Differentiation: Soak in 1% hydrochloric acid alcohol for 2 seconds, take out quickly, and rinse with tap water for 10 minutes. Dehydration and transparency: Pass through 70% ethanol solution, 80% ethanol solution, 95% ethanol solution and anhydrous ethanol solution in sequence, once each, 5 minutes each time; then pass through xylene I and xylene II in sequence, once each, 10 minutes each time. ⑩Seal: Seal with neutral resin; be careful to avoid bubbles, then lay flat to let it dry naturally. Observe and take pictures under a microscope.

[0036] 4. Fluorescence quantitative PCR was used to detect the expression of T cell and macrophage marker molecules and inflammation-related genes.

[0037] (1) Extract RNA.

[0038] ① Quickly freeze the liver, tumor and other tissues removed in liquid nitrogen and store them in a refrigerator at -80 degrees Celsius for later use. ② Take the frozen tissue, add tri-regent, and use a tissue disruptor to lyse the cells. ③ Add BCP layered cell / tissue lysis buffer, centrifuge at 12000g for 15min at 4℃, and transfer the supernatant to a new EP tube. ④ Add 2-protocal (isopropanol) (2-protocal: supernatant = volume ratio 1:1), mix up and down for more than 10 times, and place for 10min to allow RNA to fully precipitate. Centrifuge at 12000g for 10min at 4℃. ⑤ Discard the supernatant, add 1ml of 75% DEPC ethanol solution, wash the RNA once, and blow it up gently. Centrifuge at 7500g for 5min at 4℃. ⑥ Aspirate the supernatant and place it on a clean bench to dry for 10min. 1.7 Add PCR water to dissolve the RNA, place it in a 55℃ water bath for 10min, and store it in a -20℃ refrigerator.

[0039] (2) RNA is reverse transcribed into cDNA.

[0040] ① Sample addition order: H 2 O—RNA—oligdT, mix thoroughly, centrifuge, and place the PCR tube in a PCR instrument at 65°C for 10 minutes.

[0041] ②Reaction system, see Table 1:

[0042] Table 1 PCR reaction system.

[0043]

[0044] After mixing, add to the above PCR tube, mix well, and centrifuge. Put the PCR tube into the PCR instrument, 42℃ for 90min, 95℃ for 5min. After the reaction is completed, store the cDNA in a -20℃ refrigerator.

[0045] (3) real-time PCR.

[0046] ① Dilute the cDNA sample. ② Samples for the standard curve are diluted in multiples: Take out a diluted cDNA sample and dilute it in multiples, with the final concentrations of 6ng / μl, 0.6ng / μl, 0.06ng / μl, 0.006ng / μl, and 0.0006ng / μl. ③ Prepare a mixture of genes such as β-actin, FXR, CD3, CD4, CD8, and CD45. ④ Loading: Use 8 rows of tubes dedicated to real-time PCR, and the order of adding liquid is mix 15μl / well→diluted cDNA sample 5μl / well. After loading, place the 8 rows of tubes or 96-well plate dedicated to PCR on a vortex oscillator to shake and mix, centrifuge; turn on the 7500real time PCR system, and place the 8 rows of tubes and wells dedicated to PCR in the machine for detection. ⑤ Result analysis: Divide the value of each well by the corresponding internal reference value (β-actin), and then compare the relevant experimental groups.

[0047] 4. Streaming analysis

[0048] The tumor tissue was soaked in PBS solution, the surrounding blood clots, fat and necrotic tissue were removed, the PBS solution was washed once, and the tissue was cut into 1 mm 3 The cells were immersed in RPMI-1640 serum-free medium solution containing collagenase IV (200 U / mL; Invitrogen) and DNase I (40 U / mL; Sigma-Aldrich) and digested in a water bath at 37°C for 45 min. After digestion, the supernatant was filtered with a cell mesh with a pore size of 70 μm, and the cells were washed with RPMI-1640 medium (containing 2 mM EDTA) solution, centrifuged at 1500 r for 5 min, the supernatant was discarded, and the cells were resuspended in PBS solution.

[0049] Add 20 μl of cell suspension (5-10x10 5 cells / tube), and washed once with PBS solution.

[0050] Add fluorescent labeled antibody FITC anti-mouse CD3 (BioLegend, Cat.100203), double stain with PE anti-mouse CD4 (BioLegend, Cat.100407) and PE anti-mouse CD8 (BioLegend, Cat.100707), set up single staining tubes at the same time, and add the same amount of isotype control reagent as the antibody to the control tube. Incubate on ice in the dark for 15-20 minutes. Wash twice with PBS solution, resuspend in 500μL FACS buffer, and detect by flow cytometry (BeckmanCoulter, CytoFLEXS).

[0051] 5. Cell experiments

[0052] (1) Cell culture

[0053] Take the frozen mouse macrophage RAW264.7 cells out of liquid nitrogen and quickly thaw them in a 37°C water bath. Add 1 ml of 1640 complete medium (containing a mixture of 10% FBS and 1% penicillin-streptomycin) to the cryotube and centrifuge at 800 rpm for 5 min. Discard the supernatant, resuspend the cell pellet with 1 ml of culture medium, then add the cell suspension to a 6 cm culture dish, mix gently, and place at 37°C with 5% CO. 2 The cells were cultured in an air incubator for three passages and were stable before use.

[0054] (2) M1 polarization model of RAW264.7 cells

[0055] RAW264.7 cells were plated and induced to polarize to M1 with LPS and IFN-γ for 24 hours to establish the M1 polarization model. At the same time, DMSO (NC), 2μM OCA (OCA) and 5μM GS (GS) were added to the culture medium for 24 hours to collect RNA, and the gene expression of M1 macrophage marker molecules and inflammatory factors was detected by fluorescence quantitative PCR.

[0056] The specific experimental process of the present invention is as follows.

[0057] 1. FXR expression is negatively correlated with the malignancy of liver cancer and positively correlated with immune infiltration:

[0058] First, we logged into the TIMER bioinformatics database to analyze the correlation between FXR and liver cancer malignancy and immune infiltration. We found that the expression of FXR in liver cancer was significantly lower than that in normal liver tissue and was negatively correlated with the malignancy of liver cancer ( Figure 1 A, B). In addition, the expression of FXR is related to CD4 + , CD8 + , the infiltration of immune cells such as NK, DC, and B was positively correlated ( Figure 1 D). To verify the above results, we also performed quantitative PCR on 50 pairs of clinical liver cancer samples. The results were consistent with the previous analysis, showing that FXR expression was negatively correlated with the malignancy of liver cancer ( Figure 1 C), and positively correlated with T cell immune infiltration ( Figure 1 E), and positively correlated with M1 macrophage immune infiltration ( Figure 1 F). Therefore, we speculate that FXR, as a negative regulator of liver cancer, may be related to immune infiltration.

[0059] 2. The immune infiltration of liver cancer in FXR-deficient mice was reduced in the DEN liver cancer model.

[0060] Two-week-old wild-type C57BL / 6N male mice (MOCK) and FXR-deficient mice (FXR-KO) were intraperitoneally injected with 25 mg / kg DEN and killed 40 weeks later. The livers were fixed and frozen for subsequent experiments. The expression of surface marker molecules and inflammatory molecule genes of T cells and M1 macrophages was detected by fluorescence quantitative PCR. Immunohistochemistry was used to detect T cell immune infiltration.

[0061] The results showed that in the DEN liver cancer model, the incidence of liver cancer in FXR-KO mice was much higher than that in WT mice ( Figure 2 A). Quantitative PCR and immunohistochemistry revealed that the T cell immune infiltration in the liver of WT mice was significantly higher than that of FXR-KO mice, and the expression of related inflammatory molecules such as TNF-a, INF-r, MCP-1, IL-6, and IL-10 also showed the same trend ( Figure 2 BD). The above results once again verified that FXR can be used as a negative regulatory factor for liver cancer, and the increased incidence of liver cancer in FXR-KO mice is related to the decreased immune infiltration ability.

[0062] 3. Activating FXR can enhance T cell immune infiltration of liver cancer and inhibit tumor growth.

[0063] Mouse hepatocellular carcinoma cell line Hepa1-6 5×10 6 The mice were injected subcutaneously. When the tumor grew to 0.5×0.5cm in size, two groups were set up, one as a control (MOCK) and the other fed with FXR agonist OCA 10mg / kg (OCA). The drug was given every two days, and the volume of the tumor was measured. The mice were killed after 12 days, and the tumor and blood were taken. Fluorescence quantitative PCR was used to detect the expression of M1 macrophage surface marker molecules and inflammatory molecule genes, and immunohistochemistry was used to detect the expression of T cell subsets and M1 macrophage surface marker molecules.

[0064] The results showed that the FXR agonist OCA effectively inhibited the growth potential of tumors ( Figure 3 AC). Immunohistochemistry and flow cytometry analysis also confirmed that activation of FXR promoted the immune infiltration of T cells, CD3 + CD4 + and CD3 + CD8 + The level of T cells increased significantly ( Figure 3 DF). Therefore, we believe that the FXR agonist OCA inhibits tumor growth by promoting immune infiltration of tumors.

[0065] 4. Activation of FXR promotes M1 polarization of macrophages

[0066] In the tumor-bearing mouse model, the immune infiltration of M1 macrophages was significantly enhanced in the OCA group ( Figure 4 A, B), while in the DEN liver cancer model, the M1 macrophage immune infiltration in the liver of FXR-KO mice was significantly lower than that of WT mice ( Figure 4 C).

[0067] Mouse macrophages RAW264.7 were plated and induced to polarize to M1 with LPS and IFN-γ for 24 hours to establish an M1 polarization model. At the same time, DMSO (NC), FXR agonist 2μM OCA (OCA) and FXR antagonist 5μM GS (GS) were added to the culture medium for 24 hours to collect RNA, and the gene expression of M1 macrophage marker molecules and inflammatory factors was detected by fluorescence quantitative PCR. The results showed that FXR agonist OCA promoted the M1 polarization of RAW264.7 cells, while FXR antagonist GS inhibited the M1 polarization of RAW264.7 cells ( Figure 5 ).

[0068] 5. Activation of FXR promotes the expression of PD-L1 in liver cancer cells

[0069] Analysis of the TIMER bioinformatics database found that FXR and PD-L1 expression were positively correlated ( Figure 6 A), and the same results were obtained in clinical sample testing ( Figure 6 B). Activation of FXR in cells also promotes the expression of PD-L1 ( Figure 6 C). In addition, compared with cell lines cultured in vitro, tumors in tumor-bearing mice expressed PD-L1 better ( Figure 6 D). The tumors of tumor-bearing mice were cut into pieces and digested into single cells with collagenase IV. The PD-L1 antibody was labeled and the expression of PD-L1 in different treatment groups was detected by flow cytometry. The results showed that the expression of PD-L1 in the OCA group was significantly increased ( Figure 6 E, F), immunohistochemical analysis also reached the same conclusion ( Figure 6 G). This indicates that activation of FXR can promote the expression of PD-L1, which is beneficial for the effect of PD-1 monoclonal antibody.

[0070] 6. FXR agonists combined with sintilimab can further inhibit tumor growth

[0071] Mouse hepatocellular carcinoma cell line Hepa1-6 5×10 6The mice were injected subcutaneously. When the tumor grew to 0.5×0.5cm in size, four groups were set up, one as a control (MOCK), one fed with FXR agonist OCA 10mg / kg (OCA), one intraperitoneally injected with Sintilimab 10mg / kg (Sintilimab), and one fed with OCA and injected with Sintilimab at the same time (OCA+Sintilimab). The drug was given every two days, and the volume of the tumor was measured. After 12 days, the mice were killed, and the tumor and blood were taken. The levels of ALB, ALT, AST and urea in plasma were detected by biochemical analyzer.

[0072] The results showed that both the OCA group and the Sintilimab group could significantly inhibit tumor growth, and the OCA + Sintilimab combination group showed a superposition effect, which could more significantly inhibit tumor growth ( Figure 7 AC). In addition, OCA and sintilimab had no obvious toxic side effects on the liver function of mice ( Figure 7 D).

Claims

1. Application of FXR agonist combined with PD1 monoclonal antibody in the preparation of drugs for the treatment of liver cancer.

2. The use according to claim 1, characterized in that Specifically, the nuclear receptors FXR and PD1 are used as dual targets to obtain a drug for treating liver cancer.

3. The use according to claim 2, characterized in that Drugs targeting FXR for the treatment of liver cancer include gene drugs that enhance or inhibit FXR expression, and chemical drugs that regulate the expression of FXR and its target genes through FXR ligands.

4. The use according to claim 2, characterized in that: Drugs targeting PD1 for the treatment of liver cancer include monoclonal antibodies and various molecular antagonists.

5. The use according to claim 3, characterized in that The gene medicine or chemical medicine comprises a pharmaceutically acceptable carrier in a clinically acceptable form; the gene medicine contains an FXR expression vector or an interference vector; the chemical medicine contains a ligand regulating FXR and a ligand regulating FXR target gene.

6. The use according to claim 4, characterized in that The monoclonal antibodies and various molecular antagonists include a pharmaceutically acceptable carrier in a clinically acceptable form; the monoclonal antibodies and various molecular antagonists are drugs that target and block the binding of PD1 and PD-L1.

7. The use according to claim 1, characterized in that The ligands of FXR include chenodeoxycholic acid (CDCA), obeticholic acid (OCA), GW4064, cholic acid (CA), and deoxycholic acid (DCA).

8. The use according to claim 1, characterized in that Drugs targeting PD1 include the already marketed Pembrolizumab, Nivolumab, Cemiplimab, Toripalimab, Sintilimab, Camrelizumab, Atezolizumab, Avelumab, and Durvalumab.

9. The use according to claim 1, characterized in that Application of obeticholic acid (OCA) combined with sintilimab in the preparation of drugs for the treatment of liver cancer.

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