Application of combination of anti-fibrosis drug and PD1 / PD-L1 monoclonal antibody in preparation of anti-tumor drug

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of combination of an anti-fibrosis drug and a PD1 / PD-L1 monoclonal antibody in preparation of an anti-tumor drug, in particular to application of combination of pirfenidone (PFD), a credilalimab and radiation in treatment of liver cancer. The invention also provides an application of the anti-fibrosis and PD1 in liver cancer treatment, specifically, the anti-fibrosis and PD1 are taken as double targets, and the medicine for treating liver cancer is obtained through combined radiation. We find that radiation induces tumor fibrosis and prevents immune cells from infiltrating into tumor tissues, which is a key factor affecting the effect of radiotherapy combined with immunotherapy. The anti-fibrosis drug PFD reverses tumor fibrosis, and PDF is combined with radiation and immunotherapy to improve the immune infiltration capacity and tumor inhibition effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine. Background Art

[0002] Cancer is the second leading cause of death worldwide, with both cases and deaths increasing annually. According to the 2021 World Cancer Report released by the World Health Organization / International Agency for Research on Cancer (WHO / IARC), there were 18.1 million new cases of cancer and nearly 10 million deaths worldwide in 2021. Cancer treatments include surgery, chemotherapy, radiotherapy, and immunotherapy. According to the World Health Organization, 80% of patients with curable malignant tumors have received radiotherapy to varying degrees, with approximately half receiving curative radiotherapy.

[0003] Radiation therapy is categorized by therapeutic purpose into radical radiotherapy, adjuvant radiotherapy, palliative care, and salvage radiotherapy. Conventional radiotherapy equipment typically utilizes a small, repeated dose approach, driven by the physical and biological properties of electromagnetic waves such as gamma rays and X-rays. The energy of gamma rays and X-rays decays exponentially within the body, inevitably causing some damage to the skin and underlying normal tissues when treating tumors. This is why a small, repeated dose approach is used to minimize side effects. However, because cancer stem cells are radioresistant, conventional radiotherapy methods are ineffective in killing them, leading to the potential for recurrence after conventional radiotherapy. Tumor recurrence after radiotherapy is a major clinical challenge and a leading cause of cancer treatment failure. With the development of modern medicine, radiotherapy has entered an era of precision radiotherapy, with the widespread adoption of technologies such as stereotactic body radiotherapy (SBRT), image-guided radiotherapy (IGRT), and volumetric arc-modulated radiotherapy (VMAT). These new radiotherapy technologies, based on modern imaging diagnostics and computer technology, rely on advanced radiotherapy equipment to precisely focus radiation on tumor sites, minimizing the radiation dose to normal tissue and effectively protecting it, thereby increasing the therapeutic benefit ratio for tumors. This opens the door to the development of precision radiotherapy with low side effects and high efficacy, making single high-dose radiation therapy clinically feasible. However, a standardized treatment plan for high-dose radiation therapy has yet to be established, and a reliable theoretical basis is lacking.

[0004] Cancer immunotherapy has achieved breakthrough progress in clinical application and has become an important standard treatment strategy. Cancer immunotherapy activates the body's own immune system to inhibit and kill tumor cells, offering advantages such as low side effects and durable responses, and can substantially improve patients' overall survival. PD-1 / PD-L1 is one of the most promising targets in immunotherapy and has been widely used in clinical practice, significantly alleviating disease in some patients. However, immunotherapy alone does not benefit the majority of patients, and low overall response rates remain a pressing issue. Combining immunosuppressants with other therapies is currently a hot topic of research. Radiotherapy has been shown to synergize with immunotherapy through multiple mechanisms, including exposure to neoantigens, STING activation, upregulation of PD-L1, and enhanced tumor sensitivity to immunotherapy. Recent clinical trials combining radiotherapy with immunotherapy have also shown promising results. However, many challenges remain to be addressed before the clinical application of radiotherapy combined with PD-1 / PD-L1 inhibitors, such as radiation dose selection, tumor type, timing of combined therapy, and its impact on the tumor microenvironment. In short, as an emerging treatment option, radiotherapy combined with immunotherapy still requires a lot of basic and clinical research to verify.

[0005] The tumor microenvironment (TME) is a complex ecosystem interwoven with tumor cells, infiltrating immune cells, stromal cells, and non-cellular components. It has garnered significant attention due to its crucial role in tumor immunosuppression, local drug resistance, metastasis, and response to targeted therapies. Unfavorable components of the TME can prevent effective lymphocyte priming, reduce immune cell infiltration, and inhibit effector cell function, potentially leading to treatment failure. Studies have shown that radiation, in addition to inducing cell death through DNA damage, can modulate the immune system and the tumor microenvironment in a dose-dependent manner. For example, radiation induces a local inflammatory response that enhances tumor-specific T cell infiltration but also induces PD-L1 expression, significantly impairing radiation-induced antitumor immunity. The concept of radiation-induced PD-L1 expression and subsequent blockade has broadened the application of PD-1 / PD-L1 inhibitors and demonstrated their effectiveness in combination with radiation as an anti-tumor therapy. The complexity of the TME and the varying radiation modalities may contribute to the diverse effects of radiation on the TME.

[0006] In this study, the applicants discovered that high-dose radiation induces tumor fibrosis, hindering the infiltration of immune cells into tumor tissue, a key factor affecting the efficacy of radiotherapy combined with immunotherapy. The anti-fibrotic drug PFD reversed tumor fibrosis, while PDF combined with high-dose radiation and immunotherapy enhanced immune infiltration and tumor suppression. Summary of the Invention

[0007] In response to the problems existing in the prior art, the present invention provides the use of an anti-fibrosis drug combined with PD1 / PD-L1 monoclonal antibody in the preparation of an anti-tumor drug.

[0008] The purpose of the present invention is to provide the use of an anti-fibrosis drug combined with a PD1 / PD-L1 monoclonal antibody in the preparation of an anti-tumor drug; the anti-fibrosis drug may be pirfenidone; the PD1 monoclonal antibody may be sintilimab; and the radiation may be SBRT.

[0009] Another object of the present invention is to provide the use of anti-fibrosis drugs combined with PD1 / PD-L1 monoclonal antibodies in the treatment of radiation-induced tumor fibrosis, specifically to obtain drugs for treating liver cancer by combining anti-fibrosis drugs with radiation and PD1 / PD-L1 monoclonal antibodies.

[0010] The anti-fibrosis drugs for treating liver cancer include clinical first-line drugs pirfenidone, nintedanib and traditional Chinese medicine preparations.

[0011] The drugs for treating liver cancer targeting PD1 / PD-L1 include monoclonal antibodies and various molecular antagonists.

[0012] The radiation mentioned above refers to clinically used radiotherapy options, including stereotactic body radiation therapy (SBRT), image-guided radiation therapy (IGRT), volumetric arc modulated radiation therapy (VMAT), conventional radiotherapy, and hypofractionated radiotherapy.

[0013] The radiation mentioned above refers to radiotherapy schemes used in clinical practice, including γ-rays, X-rays, protons, heavy ions and other radiotherapy methods.

[0014] The monoclonal antibodies and various molecular antagonists include pharmaceutically acceptable carriers 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.

[0015] 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.

[0016] The present invention has significant technical effects.

[0017] The present inventors have discovered that the anti-fibrosis drug PFD improves the immunotherapy effect by reversing tumor fibrosis and promoting immune infiltration. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1SBRT combined with sintilimab treatment showed no additive effect and no temporal differences; A. Schematic diagram of the tumor-bearing mouse model; B. Images of tumor formation (tumors in the control and sintilimab groups grew rapidly, approaching animal ethics limits before treatment; therefore, the control and sintilimab-bearing mice were treated in advance); C. Statistical analysis of tumor weights: *P < 0.05, **P < 0.01 compared with 15 Gy; D. Tumor growth curve; E. Survival curve of tumor-bearing mice; F. Analysis of liver function and biochemical parameters in tumor-bearing mice. N ≥ 5.

[0019] Figure 2 SBRT combined with Sintilimab treatment did not further enhance the infiltration of T cells and macrophages; A, B. CD8 + 、CD4 + Immunohistochemical staining of T cells and statistical analysis, compared with the MOCK group, *P<0.05, **P<0.01, ***P<0.001, Scale bar=100 μm. C. CD8 and CD4 gene expression, *P < 0.05, **P < 0.01 compared with the MOCK group, ##P < 0.01 compared with 15 Gy; D, E. Immunohistochemical staining and statistical analysis of M1 / M2 macrophage markers, *P < 0.05, **P < 0.01, ***P < 0.001 compared with the MOCK group, Scale bar = 100 μm; F. M1 macrophage marker gene expression, *P < 0.05, **P < 0.01, ***P < 0.001 compared with the MOCK group, #P < 0.05, ##P < 0.01 compared with 15 Gy; G. M2 macrophage marker gene expression, *P < 0.05, **P < 0.01 compared with the MOCK group, #P < 0.05 compared with 15 Gy. N ≥ 5.

[0020] Figure 3SBRT induced tumor fibrosis and impeded immune infiltration. A. Sirius red staining, Scale bar = 200 μm; B, C. Immunohistochemical staining and statistical analysis of α-SMA, a marker of tumor fibrosis, compared with the MOCK group, **P < 0.01, ***P < 0.001, Scale bar = 100 μm; D. Gene expression of tumor fibrosis markers, compared with the MOCK group, *P < 0.05, **P < 0.01; E, F. Statistical analysis of immune cells in fibrous and tumor tissue areas. Tumor area group: compared with the MOCK group, *P < 0.05, **P < 0.01, ***P < 0.001; Fiber area group: compared with the MOCK group, #P < 0.05, ##P < 0.01, ###P < 0.001, Scale bar = 100 μm. N ≥ 4.

[0021] Figure 4 PFD reversed radiation-induced tumor fibrosis; A. Sirius red staining, Scale bar = 200 μm; B, C. Immunohistochemical staining for α-SMA and statistical analysis. **P < 0.01 compared with the MOCK group, #P < 0.05 compared with the 15 Gy group, Scale bar = 100 μm. D. Gene expression of fibrosis markers. *P < 0.05, **P < 0.01 compared with the MOCK group, #P < 0.05 compared with the 15 Gy group. N ≥ 5.

[0022] Figure 5 SBRT combined with sintilimab and PFD treatment significantly inhibited tumor growth; A. Schematic diagram of the tumor-bearing mouse model; B. Images of tumor growth in tumor-bearing mice in different treatment groups (tumors in the control and PFD groups grew faster, and their tumor sizes approached animal ethics limits before treatment; therefore, the mice in the control and PFD groups were sacrificed prematurely and could not be photographed together); C. Statistical analysis of tumor weights in tumor-bearing mice in different treatment groups. *P < 0.05 compared with the 15 Gy group; D. Tumor growth curves in tumor-bearing mice in different treatment groups; E. Survival curves in tumor-bearing mice in different treatment groups; F. Analysis of liver function and biochemical indicators in tumor-bearing mice in different treatment groups. N ≥ 5. DETAILED DESCRIPTION

[0023] The present invention is described in detail below with reference to specific embodiments.

[0024] Example 1.

[0025] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0026] 12-week-old wild-type C57BL / 6N male mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.; methylcellulose was purchased from Taosu, with the product catalog number 9004-67-5; pirfenidone (PFD) was purchased from the hospital; sintilimab was purchased from the hospital; ALB, ALT, AST, Urea and other detection kits were purchased from Fuji Corporation of Japan; CD4 antibody was purchased from abcam, with the product catalog number ab183685; CD8 antibody was purchased from abcam, with the product catalog number ab209775; CD206 antibody was purchased from CST, with the product catalog number 24595T; CD86 antibody was purchased from CST, with the product catalog number 19589S; α-SMA antibody was purchased from CST, with the product catalog number 19245T; immunohistochemistry secondary antibody kit was purchased from Zhongshan Jinqiao, PV-9001.

[0027] The present invention relates to an experimental result detection method.

[0028] 1. Tumor-bearing mouse model.

[0029] In each experiment, 4-6 week old C57BL / 6N male mice were selected and 6×10 6 After the Hepa1-6 cells were subcutaneously injected into the groin of mice, the mice were maintained in the original environment and the tumor growth of the mice was observed every day. When the tumor grew to 0.8×0.8 cm in size, the mice with relatively uniform tumor size were randomly divided into groups. (1) SBRT combined with PD-1 monoclonal antibody experiment: The experiment was divided into 6 groups: MOCK group, 15Gy group, Sin group (intraperitoneal injection, 10 mg / kg / 2d), 15Gy+Sin group (Sintilimab injection was given 3 days after 15Gy radiation), 15Gy-Sin group (15Gy radiation and Sintilimab injection were performed at the same time), Sin+15Gy group (15Gy radiation was given 3 days after Sintilimab injection); ( 2) SBRT combined with PD-1 monoclonal antibody and anti-fibrosis therapy: The experiment was divided into 6 groups: MOCK group, PFD group (pirfenidone, gavage, 500 mg / kg / d), 15Gy group, 15Gy+Sin group (15Gy radiation and Sintilimab one day later), 15Gy+PFD group (15Gy radiation and PFD one day later), 15Gy+PFD+Sin group (15Gy radiation and PFD and Sintilimab one day later). Tumor size was measured regularly. When the tumor of the mice in the control group grew to 1500mm, 3Mice in the experimental group were killed at 12 days after irradiation. Serum was collected and tested for liver function-related indicators using a biochemical analyzer. Tumors were removed, weighed, and photographed. Tumor tissues were fixed or frozen in a -80°C medical refrigerator for subsequent experiments. Real-time fluorescence quantitative PCR (qRT-PCR) and immunohistochemistry (IHC) staining were performed. In the mouse survival experiment, after the mice were treated in different groups, they continued to be maintained in the original environment. The growth of the mouse tumor was observed and recorded every day. When the tumor grew to about 1500mm 3 When the size reached 400 μg / cm2, the mice were sacrificed and considered dead.

[0030] 2. Radiotherapy of tumor-bearing mice.

[0031] Mice requiring radiotherapy were anesthetized with tribromoethanol (250 mg / kg). After the mice entered a deep anesthesia state, a phantom design was performed on the mice and a CT scan was performed. The target area was irradiated based on the CT scan image and a radiotherapy plan was prepared. Radiation was performed using the image-guided gamma-ray stereotactic radiotherapy system developed by Xi'an Dayi Group. The mice were placed on the treatment bed for image-guided γ-ray stereotactic radiotherapy.

[0032] 3. Detection of ALB, ALT, AST and TP-D levels in plasma.

[0033] Eyeballs of mice treated with different methods were removed and blood was collected. The blood was placed in a heparinized centrifuge tube and centrifuged at 6000 rpm at 4°C for 15 minutes. Plasma was then collected and immediately assayed for ALB, ALT, and AST levels using a Fuji Biochemical Analyzer.

[0034] 4. Immunohistochemistry.

[0035] Specific experimental procedures: ① Baking: Place paraffin-embedded tissue sections in an oven for half an hour. ② Dewaxing and hydration: Place tissue sections in xylene for ten minutes, remove and place in fresh xylene for ten minutes, repeat once, then remove and place in 95% ethanol for five minutes, remove and place in 85% ethanol for five minutes, remove and place in 75% ethanol for five minutes, then place in single-distilled water for five minutes each, repeat twice. ③ Catalase removal: Add 3% hydrogen peroxide solution dropwise, let stand at room temperature for 15 minutes, and wash three times with PBS. ④ Antigen retrieval: Microwave method. Place sections in antigen retrieval solution and microwave on high for three minutes, then stop. Start a 15-minute timer and microwave on medium-high for 20 seconds every two minutes until the 15-minute mark is reached. Allow the antigen retrieval solution to cool naturally at room temperature. Wash three times with PBS, each for 5 minutes. ⑤ Blocking: Wipe the edge of the tissue slide dry, add 5% BSA to each tissue section, and place in a humidified chamber for blocking for 30 minutes; then wash three times with PBS, 5 minutes each time. ⑥ Incubation of primary antibody: According to the ratio in the instructions, dilute the primary antibody with 5% BSA, add 50 microliters of antibody to each tissue section, place in a humidified chamber, and incubate in the refrigerator at 4°C overnight; the next day, remove the humidified chamber, leave at room temperature for 30 minutes, observe to avoid drying, and wash three times with PBS. ⑦ Incubation of secondary antibody: Wipe the edge of the tissue slide dry, add secondary antibody to each tissue section, incubate at 37°C for 20 minutes, and then wash three times with PBS, 5 minutes each time. ⑧ DAB color development (protect from light): Prepare the color development solution in advance; add the color development solution to each tissue slide and incubate at room temperature for ten minutes in the dark; pay attention to observe the specific color development under the microscope and adjust the color development time appropriately; after the color development is completed, rinse with running water. 9. Counterstaining: Counterstain the slides 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, remove quickly, and rinse with tap water for 10 minutes. Dehydration and clearing: Filter through 70% ethanol, 80% ethanol, 95% ethanol, and anhydrous ethanol, one time for 5 minutes each; then filter through xylene I and xylene II, one time for 10 minutes each. 10. Mounting: Mount with a neutral resin; be careful to avoid air bubbles, then lay flat to air dry. Observe and photograph under a microscope.

[0036] 5. Fluorescence quantitative PCR was used to detect the expression of T cell and macrophage marker molecules.

[0037] (1) Extract RNA.

[0038] ① Quickly freeze the removed tumor tissue in liquid nitrogen and store in a freezer at -80°C until ready for use. ② Remove the frozen tissue, add Tri-Regent, and lyse the cells using a tissue disruptor. ③ Add BCP layered cell / tissue lysis buffer and centrifuge at 12,000g for 15 minutes at 4°C. Transfer the supernatant to a new EP tube. ④ Add 2-protocol (isopropanol) (2-protocol:supernatant = 1:1), mix up and down more than 10 times, and let stand for 10 minutes to allow for full RNA extraction. Centrifuge at 12,000g for 10 minutes at 4°C. ⑤ Discard the supernatant, add 1ml of 75% DEPC ethanol, and wash the RNA once, gently pipetting. Centrifuge at 7,500g for 5 minutes at 4°C. ⑥ Aspirate the supernatant and dry on a laminar flow hood for 10 minutes. Dissolve the RNA in PCR water, place in a 55°C water bath for 10 minutes, and store in a -20°C refrigerator.

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

[0040] ① Add samples in the following order: H2O—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 the cDNA to the PCR tube, mix thoroughly, and centrifuge. Place the PCR tube in a PCR instrument and incubate at 42°C for 90 minutes, followed by 95°C for 5 minutes. After the reaction is complete, store the cDNA in a -20°C refrigerator.

[0045] (3)real-time PCR.

[0046] ① Dilute the cDNA sample. ② Serial dilution of the standard curve sample: Take a diluted cDNA sample and serially dilute it to a final concentration of 6 ng / μl, 0.6 ng / μl, 0.06 ng / μl, 0.006 ng / μl, and 0.0006 ng / μl. ③ Prepare a mixture of β-actin, CD206, CD4, CD8, and CD86 genes. ④ Loading: Using an 8-well tube array for real-time PCR, add 15 μl / well of the mix → 5 μl / well of the diluted cDNA sample. After loading, vortex the 8-well tube array or 96-well plate on a vortex mixer to mix thoroughly. After centrifugation, turn on the 7500 Real-Time PCR System and place the 8-well plate in the machine for testing. ⑤ Analysis of results: Divide the value of each well by the corresponding internal reference value (β-actin) before comparing the results of the relevant experimental groups.

[0047] 5. Sirius red staining.

[0048] Mouse tumor specimens were fixed in 4% paraformaldehyde for 2 days, dehydrated, and paraffin-embedded. Tissue sections were then sliced, baked, dewaxed, and hydrated. Using a paintbrush, the tissue was circled and immediately stained with Sirius Red solution for 1 hour. Dehydrated, transparent, and mounted. Observed and photographed under an upright microscope.

[0049] The specific experimental process of the present invention is as follows.

[0050] 1. SBRT combined with sintilimab treatment showed no additive effect and no temporal difference

[0051] Tumor-bearing mice with uniform tumor size were randomly divided into 6 groups: MOCK group, 15Gy group, Sin group, 15Gy+Sin group (Sintilimab was injected 3 days after 15Gy radiation), 15Gy-Sin group (Sintilimab was injected 2 hours before 15Gy radiation), and Sin+15Gy group (15Gy radiation was performed 3 days after Sintilimab injection). Sintilimab was injected intraperitoneally (10 mg / kg) once every two days, and radiotherapy was performed with a single 15Gy SBRT. Tumor size was measured regularly, and the mice were killed 12 days after radiation treatment ( Figure 1 A). Compared with the 15Gy group, the tumor growth in the 15Gy combined with Sintilimab treatment group was more significantly inhibited ( Figure 1 BD). However, in the mouse survival experiment, the 15Gy combined with Sintilimab treatment group did not significantly improve the survival rate of mice compared with the 15Gy group ( Figure 1E). It is worth noting that there was no significant difference between the three treatment groups with different time sequences of administration before and after radiation. In addition, analysis of serum biochemical indicators showed that the different treatment groups did not cause significant toxic side effects on the liver function of mice ( Figure 1 F). This indicates that the effect of radiotherapy combined with immunotherapy is more pronounced in the early stages of treatment. As radiotherapy alters the tumor microenvironment, the effect of immunotherapy is suppressed.

[0052] SBRT combined with sintilimab did not further enhance immune infiltration

[0053] In the tumor-bearing mouse model, qRT-PCR and IHC experiments were used to detect T cell and macrophage infiltration in tumor tissues. IHC results showed that compared with the control group, 15Gy radiation or Sintilimab treatment significantly promoted the immune infiltration of CD8+, CD4+ T cells and M1 (marker molecule CD86) and M2 (marker molecule CD206) macrophages, while 15Gy radiation combined with Sintilimab treatment did not significantly increase the infiltration compared with the 15Gy group, and there was no significant difference between the three treatment groups with different time sequences of administration before and after radiation ( Figure 2 A, B, D, E). qRT-PCR experiments also showed similar results ( Figure 2 C, F, G). These results indicate that combined therapy did not further enhance immune infiltration compared with radiation therapy alone.

[0054] SBRT induces tumor tissue fibrosis and hinders immune infiltration

[0055] Pathological observation of tumor tissue after 15Gy radiation revealed that radiation induced tumor fibrosis, which was also confirmed by Sirius red staining ( Figure 3 A). qRT-PCR and IHC results showed that the expression of tumor fibrosis-related genes COL1A2, MMP2, TGF-β, and protein α-SMA was significantly increased in the 15Gy group and the 15Gy combined with immunotherapy group, but there was no significant difference between them. In addition, immunotherapy alone did not induce fibrosis in tumor tissue ( Figure 3 BD). By distinguishing and counting immune cells in fibrous tissue and tumor tissue areas, it was found that CD8 + T, CD4 + T cells, M1 and M2 macrophages were obviously accumulated at the junction of fibrous tissue and tumor tissue ( Figure 3 E,F). These results suggest that radiation-induced fibrosis impedes immune infiltration.

[0056] 4. PFD reversed SBRT-induced tumor fibrosis

[0057] Sirius red staining revealed that PFD reversed tumor fibrosis (4A). qRT-PCR and IHC results showed that PFD downregulated the expression of α-SMA, a marker protein for post-irradiation fibrosis, and related genes COL1A2, MMP2, and MMP9 ( Figure 4 BD). This indicates that PFD can reverse tumor fibrosis.

[0058] 5. Anti-fibrosis + radiation + immunotherapy is a more effective treatment for liver cancer

[0059] Tumor-bearing mice were divided into 6 groups: MOCK group, PFD group (pirfenidone, gavage, 500 mg / kg / d), 15Gy group, 15Gy+Sin group (15Gy irradiation 1 day followed by Sintilimab), 15Gy+PFD group (15Gy irradiation 1 day followed by PFD), and 15Gy+PFD+Sin group (15Gy irradiation 1 day followed by PFD and Sintilimab). Tumor size was measured regularly. When the tumors in the control group grew to 1500 mm, the tumors in the control group were treated with Sintilimab. 3 The mice in the experimental group were killed 12 days after irradiation ( Figure 5 A) Serum was collected and tested for liver function related indicators using a biochemical analyzer. The tumor was removed, weighed, and photographed. The tumor tissue was fixed or frozen in a -80°C medical refrigerator for subsequent experiments. Real-time fluorescence quantitative PCR (qRT-PCR) and immunohistochemistry (IHC) were performed. In the mouse survival experiment, after the mice were treated in different groups, they continued to be maintained in the original environment. The growth of the mouse tumor was observed and recorded every day. When the tumor grew to about 1500mm 3 When the size reached 400 μg / cm2, the mice were sacrificed and considered dead.

[0060] In a tumor-bearing mouse model, we found that the use of PFD alone had a certain tumor-suppressing effect. In radiation therapy or radiation combined with immunotherapy, the addition of PFD could more significantly inhibit tumor growth, especially in the 15Gy+PFD+Sin triple combination group ( Figure 5 BE). In addition, analysis of serum biochemical indicators showed that the drug alone or combined with radiation had no significant toxic side effects on the liver function of mice ( Figure 5 F). Therefore, we believe that combined PFD can enhance the effect of high-dose radiation, more significantly inhibit tumor growth and improve overall survival rate. Anti-fibrosis combined with high-dose radiation may be a new strategy for tumor treatment.

Claims

1. Application of anti-fibrosis drugs combined with PD1 / PD-L1 monoclonal antibodies in the preparation of anti-tumor drugs.

2. The use according to claim 1, characterized in that Specifically, anti-fibrosis drugs are combined with PD1 / PD-L1 monoclonal antibodies and radiation to obtain drugs for treating liver cancer.

3. The use according to claim 2, characterized in that The anti-fibrosis drugs include the clinical first-line drugs pirfenidone, nintedanib or traditional Chinese medicine preparations.

4. The use according to claim 2, characterized in that The drugs for treating liver cancer targeting PD1 / PD-L1 include monoclonal antibodies and various molecular antagonists.

5. The use according to claim 2, characterized in that The radiation includes stereotactic body radiation therapy (SBRT), image-guided radiation therapy (IGRT), volumetric arc modulated radiation therapy (VMAT), radiotherapy or hypofractionated radiotherapy.

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 5, characterized in that The radiation includes gamma-ray, X-ray, proton or heavy ion radiotherapy.

8. The use according to claim 6, characterized in that The drugs targeting PD1 include Pembrolizumab, Nivolumab, Cemiplimab, Toripalimab, Sintilimab, Camrelizumab, Atezolizumab, Avelumab or Durvalumab.

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