Application of PCA and PD-1 antibodies in drug combination for treating liver cancer

Abstract

The invention relates to the field of biological medicine, and discloses application of PCA and PD-1 antibodies in drug combination for treating liver cancer. It is found that the flora metabolite PCA can improve the tumor immunosuppression microenvironment and enhance the effect of the PD-1 monoclonal antibody in liver cancer treatment, so that the responsiveness of liver cancer to PD-1 monoclonal antibody immunotherapy is improved, the lifetime is prolonged, and prognosis is improved.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and more specifically, to the application of PCA and PD-1 antibodies in combination for the treatment of liver cancer. Background Art

[0002] Because early symptoms of liver cancer are often atypical, most patients are diagnosed in the advanced stage, resulting in a poor prognosis. Immune checkpoint inhibitors are a milestone in the treatment of malignant tumors, enhancing the body's immune response to tumors by restoring T cell function. PD-1 antibodies, also known as PD-1 inhibitors, are drugs that bind to the PD-1 receptor on the surface of T cells. By blocking the binding of PD-1 to PD-L1 or PD-L2, they relieve T cell inhibition, restore their activation and proliferation, and promote cytokine production. However, despite the approval of PD-1 antibodies for the treatment of various cancers, only a subset of liver cancer patients respond to PD-1 antibody therapy. Tumor immune evasion and immunosuppression are key factors in the body's immune system's inability to kill tumor cells. The tumor microenvironment can inhibit T cell metabolism and cytotoxicity, thereby reducing the immune cell's killing ability. Dysbiosis of the gut microbiome may mediate tumor immune evasion and reduce the efficacy of immune checkpoint inhibitors. Therefore, current consensus suggests that improving the tumor microenvironment is crucial for enhancing responsiveness to PD-1 antibodies.

[0003] P-Coumaric acid (PCA), also known as p-coumaric acid, is an endogenous and bacterial metabolite belonging to the phenolic acid class of organic acids. Phenolic acids have diverse pharmacological effects, including antiplatelet, antitumor, and immunomodulatory activities.

[0004] At present, there are no reports on the effects of PCA on improving the tumor immunosuppressive microenvironment and enhancing the efficacy of PD-1 antibodies in the treatment of liver cancer. Summary of the Invention

[0005] In view of this, the present invention aims to reveal the synergistic effect of PCA and PD-1 antibody in the treatment of liver cancer, and provide an application of PCA in the preparation of a drug for sensitizing PD-1 antibody to treat liver cancer.

[0006] In order to achieve the above-mentioned invention objectives, this application provides the following technical solutions:

[0007] In a first aspect, the present application provides the use of PCA in the preparation of drugs for reshaping the tumor immune microenvironment.

[0008] Furthermore, PCA acts on the tumor microenvironment to increase tumor-infiltrating CD8 + T cells, IFN-γ + CD8 +T cells, GZMB + CD8 + T cell content.

[0009] In a second aspect, the present application provides the use of PCA in the preparation of a drug for improving the anti-tumor effect of PD-1 antibodies.

[0010] Furthermore, PCA improves the anti-tumor effect of PD-1 antibodies by improving the tumor immunosuppressive microenvironment.

[0011] The third aspect is the application of PCA and PD-1 antibodies in the combined treatment of liver cancer.

[0012] In summary, this application has the following beneficial effects:

[0013] This application discovered that the microbial metabolite PCA can improve the tumor immunosuppressive microenvironment and sensitize the effect of PD-1 monoclonal antibody in the treatment of liver cancer, thereby increasing the responsiveness of liver cancer to PD-1 monoclonal antibody immunotherapy, prolonging survival, and improving prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 PCA is the effect of CD8 + Schematic diagram of the effects of IFN-γ and GZMB on T cell proliferation and effector indicators.

[0015] (Among them, AB. Flow cytometry detection of PCA against mouse CD8 + Effect of PCA on T cell proliferation. CE. Flow cytometry analysis of IFN-γ + CD8 + T cells and GzmB + CD8 + Effect of T cell ratio. ***P<0.001, n=5.)

[0016] Figure 2 Schematic diagram of the effect of PCA on tumor size and mass in hepatocellular carcinoma-bearing mice.

[0017] (A. Experimental flow chart. B. Comparison of tumor volume in each group of mice with orthotopic HCC models. C. Comparison of tumor weight in each group of mice with orthotopic HCC models. D. Gross images of the livers of mice in each group. E-F. Representative images of in vivo imaging and fluorescence value statistics of the orthotopic HCC models in mice in each group. Scale bar: 1 cm; **P < 0.01, ***P < 0.001, n = 5.)

[0018] Figure 3 PCA is an important factor in the regulation of tumor-infiltrating CD8 + T cells, IFN-γ + CD8 + T cells, GZMB+ CD8 + Schematic diagram of the effect of T cell content.

[0019] (A. Representative flow cytometry images of tumor-infiltrating lymphocytes in each group of mice. B. Statistical graph of local lymphocyte infiltration in the tumors of each group of mice. *P<0.05, **P<0.01, ***P<0.001, n=5.) DETAILED DESCRIPTION

[0020] The scheme and effects of the present application are further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the invention, rather than to limit the invention.

[0021] Example 1: PCA increases CD8 + T cell proliferation and cytotoxicity

[0022] The mice used in this experiment were CD8 + The isolation and extraction of T cells was performed according to the instructions of Biolegend's Mouse CD8 Tcell Isolation Kit. First, the spleen single cell suspension after erythrocyte lysis was filtered through a 70 μm filter, centrifuged at 300 g for 5 minutes, and then filtered using MojoSort TM Buffer was used to adjust the cell density to 1×10 8 Take 100 μL of cell suspension, add 10 μL of Biotin-Antibody Cocktail (incubate on ice for 15 min) and 10 μL of Streptavidin Nanobeads (incubate on ice for 15 min), and add MojoSort TM Buffer was added to 3.5 mL and magnetic separation was performed for 5 minutes to collect the purified CD8 + T cells. After centrifugation, resuspend in PBS to 5×10 6 / mL, CFSE staining (10μM, room temperature in the dark for 10min) and washing three times to remove excess dye. The cells were resuspended in T cell culture medium containing 10% FBS, 100U / mL IL-2, 2mM L-glutamine and other ingredients, and inoculated into 96-well plates pre-coated with anti-CD3 / CD28 antibodies (2μg / mL each) and cultured for 48 hours for pre-activation. + T cells were co-cultured with 0.2 mM or 0.5 mM PCA for 72 hours, and the proliferation capacity was assessed by flow cytometry to detect CFSE fluorescence intensity, or the expression of IFN-γ and GZMB was detected to analyze the effector function. +T cell proliferation, and this promoting effect was obviously concentration-dependent. In addition, 0.2mM or 0.5mM PCA promoted CD8 + T cells expressed higher levels of IFN-γ and GZMB.

[0023] Example 2: Combination of PCA and PD-1 Antibody Significantly Reduces Tumor Volume and Weight in Tumor-Bearing Mice

[0024] This study evaluated the effect of PCA combined with PD-1 monoclonal antibody on the progression of liver cancer in wild-type C57BL / 6 mice (SPF grade, male, 6 weeks old, weighing 16g-22g). In situ hepatocellular carcinoma modeling was performed using Hepa1-6 cells in the logarithmic growth phase. After washing with PBS and digesting with 0.25% trypsin, the cells were centrifuged and counted, and the cell density was adjusted to 1×10 with an equal ratio of sterile PBS and Matrix gel. 8 Cells / mL, stored on ice until use. After anesthesia, the upper abdomen of the mice was shaved and disinfected, and the mice were fixed in the supine position on the operating table. A transverse incision was made along the lower edge of the costal arch to expose the left lateral lobe of the liver. 10 μL of cell suspension was slowly injected with a microsyringe. After pressing with a cotton swab to stop bleeding, the abdominal cavity was interrupted and sutured and disinfected with iodine. Tumor-bearing C57BL / 6 mice were randomly divided into four groups, namely the control group, the PCA gavage treatment group (100 mg / kg / day), the PD-1 monoclonal antibody treatment group (10 mg / kg, q3d, intraperitoneal injection), and the PCA and PD-1 monoclonal antibody combination treatment group. 14 days after tumor implantation, bioluminescence imaging was performed using an IVIS system: filter-sterilized D-luciferin potassium salt (150 mg / kg) was injected intraperitoneally, and the mice were anesthetized and taken in the supine position. Subsequently, the mice were killed by cervical dislocation, and the liver was removed to measure the tumor volume (V = 0.5 × L × W 2 The tumors were weighed (V represents tumor volume, L represents the long diameter of the tumor, and W represents the short diameter of the tumor). Both PCA and PD-1 monoclonal antibody treatment inhibited liver cancer progression. However, the combination of PCA and PD-1 monoclonal antibody inhibited liver cancer growth to a greater extent, confirming that PCA supplementation in mice can enhance the therapeutic effect of PD-1 monoclonal antibody on in situ liver cancer.

[0025] Example 3: PCA increases tumor infiltrating CD8 + T cells, IFN-γ + CD8 + T cells, GZMB + CD8 + T cell content

[0026] The liver cancer tumor tissue in Example 2 was minced into 1 mm pieces in DMEM medium. 3After sizing, add 2 mL of enzymatic digestion solution containing 5% fetal bovine serum, 1 mg / mL collagenase IV and 200 U / mL DNase I, and shake at 37°C, 200 rpm for 45 minutes. The digested suspension was filtered through a 100 μm filter into a 50 mL centrifuge tube, and after adding 10 mL of DMEM medium, centrifuged at 1500 rpm and 4°C for 5 minutes, the supernatant was discarded and resuspended with 5 mL of medium and filtered again. After repeated centrifugation and washing, a single cell suspension was prepared with PBS and counted. Take 100 μL (about 1×10 6 The cells were suspended in a PBS-treated, live-death stain and surface antibody labeling (25 minutes at room temperature). After washing with PBS, 200 μL of fixation / permeabilization buffer was added and incubated in the dark for 40 minutes. After washing with permeabilization buffer, intracellular antibodies were added and incubated in the dark for 30 minutes at room temperature. Finally, the cells were resuspended in PBS and analyzed by a three-laser flow cytometer. Compared with the control group, PCA treatment increased the expression of local CD8 + Compared with PD-1 monoclonal antibody treatment, PCA combined with PD-1 monoclonal antibody treatment had a significant effect on the infiltration level of T cells. + The present invention also found that after PCA combined with PD-1 monoclonal antibody treatment, CD8 + T cell function was significantly enhanced, and the expressions of IFN-γ and GZMB were significantly upregulated.

[0027] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. Application of PCA in the preparation of drugs that reshape the tumor immune microenvironment.

2. The use according to claim 1, characterized in that PCA acts on the tumor microenvironment and increases tumor-infiltrating CD8 + T cells, IFN-γ + CD8 + T cells, GZMB + CD8 + T cell content.

3. Application of PCA in the preparation of drugs to enhance the anti-tumor effect of PD-1 antibodies.

4. The use according to claim 3, characterized in that PCA improves the anti-tumor effect of PD-1 antibodies by improving the tumor immunosuppressive microenvironment.

5. Application of PCA and PD-1 antibody in the combined treatment of liver cancer.

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