Compound for activating and up-regulating body immune cell activity

Abstract

The invention discloses a compound for activating and up-regulating body immune cell activity, a composition for treating lung cancer as well as a preparation method and a use method of the compound. The composition comprises ophiopogonin-D and a PD-1 antibody, wherein the ophiopogonin-D is used as an effective component and is used for activating T cells and up-regulating expression of immune cell activation markers CD69 and CD25 and inducing the T cells to secrete TNF-alpha and IFN-gamma; and the anti-PD-1 antibody is used for cooperating with ophiopogonin-D to enhance the immune response of a host. The ophiopogonin-D disclosed by the invention is used for improving the immunocompetence of a body, realizing anti-lung cancer treatment and removing senescent cells, and can be used for effectively activating T cells and up-regulating the expression of immune cell activation markers CD69 and CD25, so that the immune response of a host is enhanced; the OP-D can be used for stimulating the production of interferon-gamma, interleukin-6 and chemotactic factor CXCL9-11.

Description

Technical Field

[0001] The present invention relates to the technical field of cancer immunotherapy, and in particular to a compound for activating and upregulating the activity of immune cells in the body, a composition for treating lung cancer, and a preparation method and a use method thereof. Background Art

[0002] Immunotherapy is a promising approach for cancer treatment, ushering in a new era of treatment. However, the efficacy of immunotherapy as a monotherapy for lung cancer patients remains limited due to the immunosuppressive nature of the solid tumor microenvironment (TME). To address this challenge, alternative drugs that can enhance host immunity are needed.

[0003] The emergence of immune checkpoint inhibitors (ICIs), such as programmed cell death protein-1 (PD-1) and cytotoxic T lymphocyte-associated protein-4 (CTLA-4), has ushered in a new era of anti-tumor therapy. In the past decade, ICIs, especially PD-1 axis inhibitors, have revolutionized lung cancer treatment and become an indispensable therapeutic tool. However, the clinical response rate of ICIs remains disappointingly low, with an average response rate of only 15% in lung cancer patients. Several factors contribute to this limited response, including tumor burden, microsatellite stability, programmed cell death ligand-1 (PD-L1) expression, and T cell infiltration. Resistance to ICIs treatment also poses a major challenge.

[0004] In addition, the immunosuppressive nature of the tumor microenvironment (TME) poses a major challenge to the efficacy of immunotherapy. The TME is a complex network of cells and molecules that not only supports tumor growth and survival, but also inhibits the function of immune cells through multiple mechanisms. For example, immune checkpoint molecules, stromal cells, and other factors in the TME can work together to limit the attack of immune cells on tumors, leading to immune escape, immune tolerance, or immune reversal phenomena.

[0005] To overcome these obstacles, researchers have been exploring new drugs and treatment strategies. Some strategies include the use of stimulator of interferon genes (STING) agonists, Toll-like receptor (TLR) agonists, and cellular immunotherapies such as chimeric antigen receptor T cell (CAR-T) therapy. These approaches aim to activate or enhance the immune system's response to tumors, but the complexity of solid tumors and the immunosuppressive nature of the TME limit the effectiveness of these treatments.

[0006] The present invention is proposed in this context, aiming to provide a new composition and treatment method, which combines the traditional Chinese medicine ingredient Ophiopogon japonicus saponin-D (OP-D) and PD-1 antibody to enhance the host's anti-tumor immune response and improve the efficacy of lung cancer treatment, especially for patients who do not respond well to existing immunotherapy. Summary of the invention

[0007] In view of the problems existing in the prior art, the present invention provides a compound for activating and up-regulating the activity of immune cells in the body, a composition for treating lung cancer, and a preparation method and a use method thereof.

[0008] Among them, Ophiopogon japonicus saponin-D (OP-D) is an immunologically active substance extracted from Ophiopogon japonicus. Studies have shown that cooking Ophiopogon japonicus and ginseng in a specific ratio can promote the proliferation of bone marrow and peripheral blood cells, increase the thymus index, and reduce chemotherapy-induced bone marrow suppression. In addition, as a vaccine adjuvant, OP-D can enhance the antibody response to antigens. Under antigen stimulation, OP-D can significantly increase the range of Th1 cytokines (IFN-γ, IL-12p40), Th2 cytokines (IL-4), and Th17 cytokines (IL-17A) in mouse spleen cells.

[0009] This study aims to explore the potential of OP-D as an immunotherapeutic agent to improve anti-tumor efficacy in the treatment of lung cancer and to examine its potential mechanisms for stimulating the immune system.

[0010] In order to achieve the above object, the present invention provides a compound for activating and up-regulating the activity of immune cells in the body, characterized in that the compound is ophiopogon saponin-D, which is used to induce the expression of immune cell activation markers CD25 and CD69; the structural formula of the ophiopogon saponin-D is as follows:

[0011]

[0012] The present invention also provides a composition for treating lung cancer, which comprises ophiopogon saponin-D and PD-1 antibody; wherein ophiopogon saponin-D is used as an effective ingredient to activate and upregulate the activity of the body's immune cells; and the anti-PD-1 antibody is used to synergize with ophiopogon saponin-D to enhance the host's immune response.

[0013] Preferably, the ophiopogon saponin-D is extracted from the root of the genus Ophiopogon, and the PD-1 antibody is a monoclonal antibody that specifically binds to programmed cell death protein-1.

[0014] Preferably, when activating T cells, the ophiopogon saponin-D activates T cells and upregulates the expression of immune cell activation markers CD69 and CD25, induces T cells to secrete cytokines interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α), and can stimulate the production of interleukin-6 and chemokines CXCL9-11.

[0015] Preferably, the ophiopogonin-D activates immune cells via the STING signaling pathway;

[0016] Preferably, the ophiopogonin-D can induce calcium influx and nuclear translocation of nuclear factor of activated T cells (NFAT) and STAT5.

[0017] The present invention provides a method for evaluating the effect of ophiopogon saponin-D (OP-D) on host immune cell function, the method comprising the following steps:

[0018] Step S1: co-culturing the host's immune cells with Ophiopogon japonicus saponin-D;

[0019] Step S2: measuring the expression levels of CD69 and CD25 in the immune cells after co-culture;

[0020] Step S3: Measure the changes in calcium ion concentration in immune cells after treatment with Ophiopogon japonicus saponin-D and the nuclear translocation of NFAT and STAT5.

[0021] The present invention provides a method for preparing a composition, characterized in that it comprises the following steps:

[0022] Step a: extracting ophiopogonin-D from the root of ophiopogon japonicus;

[0023] Step b: producing or purchasing PD-1 antibodies;

[0024] Step c: Mix OP-D and PD-1 antibodies in a specific ratio to form a composition.

[0025] The present invention also provides a method of using the composition for treating lung cancer, the method comprising the following steps:

[0026] Step X1: combining OP-D (5-10 mg / kg) and anti-PD-1 antibody (10 mg / kg) to form a composition required for treatment;

[0027] Step X2: administering the composition to a patient by intravenous injection, subcutaneous injection or intramuscular injection to enhance the patient's immune response to the tumor;

[0028] Step X3: Monitor the patient's immune response and tumor growth to evaluate treatment efficacy.

[0029] Preferably, the specific ratio of OP-D and anti-PD-1 antibody in step X1 is determined according to the expression levels of immune cell activation markers CD69 and CD25 in the patient.

[0030] In the present invention, the immunologically active substance Ophiopogon japonicus saponin-D (OP-D) extracted from the root of Ophiopogon japonicus has been identified as a potential candidate drug for enhancing antigen-specific immune responses and synergizing with anti-tumor immunotherapy. In vitro experiments have shown that OP-D can activate T cells and upregulate the expression of immune cell activation markers: CD69 and CD25, induce T cells to secrete IFN-γ and TNF-α, and in addition, OP-D can stimulate the production of cytokines IL-6 and chemokine molecules CXCL9-11. In vivo experiments have shown that OP-D has a significant synergistic effect when combined with anti-PD-1 therapy. This combination enhances the host's immune response and creates an immune microenvironment that inhibits tumor growth. Overall, the research results of the present invention highlight the potential of OP-D as an immune response enhancer and a promising supplement to anti-tumor immunotherapy, paving the way for better treatment outcomes for lung cancer.

[0031] The technical solution of the present invention has the following beneficial effects:

[0032] The present invention can enhance immune response: Ophiopogon japonicus saponin-D (OP-D) in the composition can effectively activate T cells and upregulate the expression of immune cell activation markers CD69 and CD25, thereby enhancing the host's immune response.

[0033] The present invention can promote the production of cytokines: OP-D can stimulate the production of interferon-γ (IFN-γ), interleukin-6 (IL-6) and chemokine CXCL9-11, which are crucial for regulating immune responses.

[0034] The present invention can activate the STING signaling pathway: OP-D activates immune cells through the STING signaling pathway, which plays a core role in the innate immune response of cells to pathogens and tumor cells.

[0035] The present invention can promote calcium influx and nuclear translocation of transcription factors: calcium influx and nuclear translocation of NFAT and STAT5 induced by OP-D help activate T cells and enhance their recognition and response to tumor cells.

[0036] The present invention can synergistically enhance efficacy: When OP-D is used in combination with PD-1 antibodies, OP-D shows a synergistic effect and may improve the efficacy of immune checkpoint inhibitors, especially for lung cancer patients who do not respond well to PD-1 antibody treatment alone.

[0037] The compositions and treatment methods of the present invention provide a new treatment option for lung cancer patients, especially for those patients who need more effective treatment options.

[0038] The present invention is easy to prepare and administer: the preparation method of the composition is simple and cost-effective, and the administration method (intravenous injection, subcutaneous injection or intramuscular injection) provides convenience for clinical application.

[0039] The present invention provides new insights and treatment methods for the field of cancer immunotherapy by combining traditional Chinese medicine ingredients with modern immunotherapy technology; by combining the traditional Chinese medicine ingredient Ophiopogon japonicus saponin-D (OP-D) and PD-1 antibody, it is hoped to enhance the host's anti-tumor immune response and improve the efficacy of lung cancer treatment, especially for those patients who do not respond well to existing immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1a It is a schematic diagram of the structure of OP-D of the present invention;

[0041] Figure 1b Schematic diagram of the effect of OP-D of the present invention on the expression of CD69, a T cell activation marker, (n=3, *P<0.05, ****P<0.0001), wherein n=3 means the experiment was repeated three times, and the P value represents a statistically significant difference;

[0042] Figure 1c Schematic diagram of OP-D of the present invention enhancing the expression of T cell activation markers and CD25 (n=3, *P<0.05, ****P<0.0001);

[0043] Figure 2a This is a schematic diagram of the secretion level of TNF-α by macrophages of the present invention;

[0044] Figure 2b CD4 + Schematic diagram of the secretion level of TNF-α by T cells;

[0045] Figure 2c CD8 + Schematic diagram of IFN-γ secretion levels by T cells;

[0046] Figure 3a The Ca2+ concentration in OP-D Kasumi cells treated for 15 min and 30 min is shown in Table 1. 2+ Schematic diagram of level rise;

[0047] Figure 3b This is a schematic diagram showing that OP-D of the present invention upregulates NFAT1 in total protein and nuclear protein of Kasumi cells;

[0048] Figure 3cThis is a schematic diagram showing that the STAT5 phosphorylation level of T cells significantly increased after OP-D treatment of the present invention;

[0049] Figure 3d This is a schematic diagram of the increase in phosphorylation expression of the transcription factor stat5 in Jurkat cells caused by OPD of the present invention;

[0050] Figure 4a It is a schematic diagram of the volcano analysis of differentially expressed transcripts after OP-D treatment of Kasumi cells of the present invention, (n=3, **P<0.01, ****P<0.0001);

[0051] Figure 4b This is a schematic diagram of GO pathway enrichment analysis of up-regulated genes in Kasumi cells of the present invention;

[0052] Figure 4c The protein interaction network diagram and TOP20 core nodes and their frequency statistics table analyzed by RNA sequencing in the present invention;

[0053] Figure 5a This is a diagram of the animal experiment strategy and timetable of the present invention;

[0054] Figure 5b This is a schematic diagram showing that OP-D of the present invention has no effect on the body weight of mice;

[0055] Figure 5c Schematic diagram of 6 groups of tumor tissues of the present invention;

[0056] Figure 5d This is a schematic diagram showing that the tumor weight of the anti-PD-1 plus OP-DH group of the present invention was significantly inhibited (n=5,

[0057] *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001);

[0058] Figure 5e This is a schematic diagram showing that the tumor size of the anti-PD-1 plus OP-DH group of the present invention was significantly inhibited (n=5,

[0059] *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). DETAILED DESCRIPTION

[0060] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0061] Reference Figures 1a to 5eThe present invention provides a compound for activating and up-regulating the activity of immune cells in the body, the compound is ophiopogon saponin-D, which is used to induce the expression of immune cell activation markers CD25 and CD69; the structural formula of the ophiopogon saponin-D is as follows:

[0062]

[0063] The present invention also provides a composition for treating lung cancer, the composition comprising ophiopogon japonicus saponin-D (OP-D) and PD-1 antibody;

[0064] Among them, Ophiopogon japonicus saponin-D is used as an active ingredient to activate and upregulate the activity of the body's immune cells;

[0065] Anti-PD-1 antibody is used to work synergistically with Ophiopogon japonicus saponin-D to enhance the host's immune response.

[0066] The Ophiopogon japonicus saponin-D is extracted from the root of Ophiopogon japonicus, a traditional Chinese medicine material that has been found to have multiple pharmacological effects. PD-1 antibody is a monoclonal antibody that specifically binds to programmed cell death protein-1. PD-1 is an immune checkpoint molecule mainly expressed on the surface of T cells. It can inhibit the activity of T cells after binding to its ligand PD-L1.

[0067] When activating T cells, the ophiopogon saponin-D activates T cells and upregulates the expression of immune cell activation markers CD69 and CD25, inducing T cells to secrete cytokines interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α), and can also stimulate the production of interleukin-6 (IL-6) and chemokine CXCL9-11. Among them, cytokines interferon-γ (IFN-γ) and interleukin-6 (IL-6) are important cytokines that participate in regulating immune responses. PD can also promote the production of chemokines CXCL9 and CXCL11, which contribute to the migration and positioning of immune cells.

[0068] The ophiopogon saponin-D activates immune cells through the STING signaling pathway; the ophiopogon saponin-D can cause calcium influx and nuclear translocation of nuclear factor of activated T cells (NFAT) and STAT5.

[0069] The present invention provides a method for evaluating the effect of ophiopogon saponin-D (OP-D) on host immune cell function, the method comprising the following steps:

[0070] Step S1: Collect immune cells from the host, such as PBMCs (peripheral blood mononuclear cells) obtained by peripheral blood sampling; co-culture the immune cells with a specific concentration of Ophiopogon japonicus saponin-D under in vitro conditions, in a cell culture plate or culture bottle containing appropriate culture medium and cell growth factors.

[0071] Step S2: After a certain period of culture, the expression levels of immune cell surface activation markers CD69 and CD25 are measured by flow cytometry or other molecular biology techniques (such as qPCR, ELISA or immunofluorescence staining). The upregulation of these markers generally indicates that T cells have been activated.

[0072] Step S3: Measure the changes in calcium ion concentration in immune cells after OP-D treatment, and the nuclear translocation of NFAT and STAT5. Measure the changes in calcium ion concentration in immune cells after OP-D treatment, which can be achieved by fluorescent probes or other calcium ion indicators. Detect the nuclear translocation of NFAT and STAT5, which is usually done by Western blotting, immunofluorescence staining or flow cytometry. Nuclear translocation of NFAT and STAT5 is a key signaling event for T cell activation and immune response.

[0073] The purpose of this method is to determine whether Ophiopogon japonicus saponin-D can effectively activate the host's immune cells, especially T cells, and evaluate its effects on the immune system by measuring the expression of immune cell activation markers and the activation of key signaling pathways. This method can provide important experimental data for further pharmacological research and clinical applications.

[0074] The present invention provides a method for preparing a composition, characterized in that it comprises the following steps:

[0075] Step a: extracting ophiopogonin-D from the root of ophiopogon japonicus;

[0076] Further, appropriate root materials of Ophiopogon japonicus are selected, and the root materials are cleaned, dried and crushed;

[0077] Extract OP-D from the crushed roots using appropriate solvents and extraction techniques (e.g., hot water extraction, alcohol extraction, or ultrasound-assisted extraction);

[0078] OP-D is purified from the extract by filtration, concentration and purification steps including column chromatography, crystallization or membrane separation techniques.

[0079] Step b: producing or purchasing PD-1 antibodies;

[0080] For self-production, it is necessary to use bioengineering technology to prepare monoclonal antibodies that specifically bind to PD-1, including cell culture, antibody production and purification processes; when purchasing, it is necessary to obtain the produced PD-1 antibodies from qualified suppliers to ensure their quality and specificity.

[0081] Step c: combining OP-D (5-10 mg / kg) and anti-PD-1 antibody (10 mg / kg) to form a composition required for treatment;

[0082] During the mixing process, ensure the sterility of the environment to avoid contamination;

[0083] During the entire preparation process, quality control is carried out at multiple stages, including quality inspection of raw materials, purity and activity testing of intermediates, and stability and efficacy evaluation of the final product; the prepared composition is packaged into appropriate containers and stored according to its stability requirements, which usually requires a light-proof, low-temperature and dry environment.

[0084] The present invention also provides a method of using the composition for treating lung cancer, the method comprising the following steps:

[0085] Step X1: combining OP-D and anti-PD-1 antibody in a specific ratio to form a composition required for treatment;

[0086] Step X2: administering the composition to a patient by intravenous injection, subcutaneous injection or intramuscular injection to enhance the patient's immune response to the tumor;

[0087] Step X3: Monitor the patient’s immune response and tumor growth to assess treatment efficacy;

[0088] Furthermore, the patient's immune response is regularly monitored, including but not limited to the expression levels of immune cell activation markers such as CD69 and CD25; the tumor growth can be evaluated through imaging examinations (such as CT, MRI or PET scans) and measurement of tumor markers; the treatment plan can be adjusted according to the monitoring results, and if necessary, the drug dosage or administration frequency can be changed.

[0089] Preferably, the specific ratio of OP-D and anti-PD-1 antibody in step X1 is determined according to the expression levels of immune cell activation markers CD69 and CD25 in the patient.

[0090] Embodiment 1:

[0091] OP-D effectively induces lymphocyte activation:

[0092] The immunosuppressive TME largely determines the efficacy of current therapies. Small molecule drugs have great potential to penetrate into the TME and reactivate exhausted immune cells. In order to quickly find effective drugs to enhance host immunity and achieve cancer eradication, we used lymphocyte activation markers CD69 and CD25 for screening. Figure 1b-Figure 1cAs shown, OP-D effectively increased the levels of CD25 in Kasumi T cells and CD69 in Jurkat cells, induced the proliferation and activation of innate immune cells, and enhanced T cell activity.

[0093] We further investigated the immune activation effects of OP-D in vitro and in vivo. Immune cells isolated from the spleen of C57BL / 6J mice were cultured with OP-D. It significantly enhanced the expression of TNF-α in macrophages and CD4+ T cells and the expression of IFN-γ in CD8+ T cells ( Figure 2a to Figure 2c The results provide evidence that OP-D can effectively stimulate the host's immune response.

[0094] Embodiment 2:

[0095] OP-D induces calcium influx and nuclear translocation of nuclear factor of activated T cells (NFAT) and STAT5:

[0096] Intracellular Ca 2+ Signaling plays a crucial role in maintaining the activation and proliferation of immune cells. 2+ Activate calcineurin, promote nuclear translocation of NFAT and then start the expression of immune function genes. 2+ Effects of changes in Ca levels 2+ The indicator fluo-3 / AM was used as the experimental object, and the results showed that OP-D rapidly increased intracellular Ca within 30 minutes. 2+ concentration( Figure 3a In addition, we observed that both the total protein level and nuclear translocation of NFAT1 were significantly enhanced after OP-D treatment ( Figure 3b These results highlight the role of OP-D in regulating intracellular Ca 2+ The levels and ability to activate NFAT1 suggest its potential role in regulating immune cell function and immune responses.

[0097] As the main downstream of NFAT1, the STAT5 axis plays a vital role in the development of immune cells. After receiving activation signals, STAT5 is phosphorylated (p-STAT5) and translocated to the nucleus, inducing the transcription of a series of target genes, including CD25 and CD69. Figure 3c As shown, treatment with OP-D resulted in a significant upregulation of p-STAT5 in Jurkat T cells, suggesting that OP-D plays a potential role in regulating immune cell function and immune responses through NFAT1-STAT5 signaling.

[0098] Embodiment 3:

[0099] Proteomic analysis revealed that STING signaling is responsible for immune cell activation initiated by OP-D:

[0100] To explore the signaling pathways targeted by OP-D in immune cell activation, the present invention performed RNA sequencing analysis to identify a set of differentially expressed genes. Figure 4a As shown, 194 upregulated genes and 89 downregulated genes were identified after OP-D treatment. Protein-protein interaction analysis showed that the core nodes were mainly composed of cytokines and chemokines, such as interleukin-1, interleukin-10, and chemokine-8, which promote the pro-inflammatory and anti-tumor effects of immune cells ( Figure 4b ). Through GO enrichment analysis, we found that cytokine-mediated signaling pathways and Erk1 / 2 cascades were significantly enriched ( Figure 4c ), OP-D significantly enhanced the secretion of a series of pro-inflammatory cytokines.

[0101] Embodiment 4:

[0102] OP-D strongly enhances the anti-tumor effect of αPD-1 antibody in vivo:

[0103] To examine the potential benefits of combined OP-D and αPD-1 therapy in an in vivo lung cancer model, we performed experiments using a mouse lung transplant model. Mice were treated with OP-D, αPD-1 alone, or the combination therapy ( Figure 5a ); Figure 5a The mice were divided into six groups: control group (injected with PBS), anti-PD-1 group (injected with 10 mg / kg anti-PD-1), OP-DL group (injected with 5 mg / kg OP-D), OP-DH group (injected with 10 mg / kg OP-D), and anti-PD-1 plus OP-DL or OP-DH. Treatment was performed every three days from day 0 to day 18; tumor tissues, spleen cells, and peripheral blood cells were obtained on day 18. Importantly, all treatment regimens did not lead to significant weight loss in mice, indicating that OP-D has a good safety profile ( Figure 5b ). In terms of tumor weight and volume, high-dose OP-D treatment had a significant inhibitory effect on tumor growth ( Figure 5c-5e ), among which the combined group had the most obvious effect, suggesting that there is a potential synergistic effect between OP-D and αPD-1 treatment. OP-D alone can effectively inhibit the tumor volume of lung cancer and significantly enhance the efficacy of αPD-1. Animal experiments have shown that OP-D can sensitize anti-PD-1 antibody therapy.

[0104] As can be seen from the above examples, the present invention demonstrates that OP-D can effectively enhance immune recognition and activate innate immune cells and T cells in the tumor microenvironment both in vivo and in vitro. In addition, when combined with αPD-1 treatment, OP-D significantly increased tumor infiltration of lymphocytes, thereby improving the response rate of immunotherapy and enhancing anti-tumor response. Importantly, even at high doses (10 mg / kg), OP-D did not significantly affect the body weight of animals, indicating its safety. Therefore, combining OP-D therapy with ICI has great prospects for improving the clinical efficacy of immunotherapy.

[0105] Interestingly, the present study found that OP-D can transiently increase the Ca2+ concentration in immune cells, accompanied by increased NFAT1 expression and its translocation to the nucleus, indicating that the calcium / NFAT1 signaling pathway is involved in OP-D-induced innate immune cell activation. In short, this new combination therapy is expected to become an alternative therapy to improve treatment outcomes, especially for patients with non-small cell lung cancer who do not respond to immunomodulatory therapy.

[0106] This invention presents an innovative approach to combine the extraction of immunologically active substances from traditional Chinese medicine with immune checkpoint blockade, providing valuable insights into cancer immunotherapy; overall, continued research and clinical studies will bring us closer to realizing the full therapeutic potential of OP-D in the fight against lung cancer.

[0107] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification and drawings under the inventive concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A compound for activating and upregulating the activity of immune cells in the body, characterized in that: The compound is ophiopogon saponin-D, which is used to induce the expression of immune cell activation markers CD25 and CD69; the structural formula of ophiopogon saponin-D is as follows:

2. A composition for treating lung cancer, characterized in that: The composition comprises ophiopogon saponin-D and PD-1 antibody; wherein ophiopogon saponin-D is used as an effective ingredient to activate and upregulate the activity of the body's immune cells; and the anti-PD-1 antibody is used to synergize with ophiopogon saponin-D to enhance the host's immune response.

3. The composition for treating lung cancer according to claim 2, characterized in that: When activating T cells, the ophiopogon saponin-D activates T cells and upregulates the expression of immune cell activation markers CD69 and CD25, inducing T cells to secrete cytokines interferon-γ and tumor necrosis factor α, and can also stimulate the production of interleukin-6 and chemokine CXCL9-11.

4. The composition for treating lung cancer according to claim 3, characterized in that: The ophiopogon saponin-D activates immune cells through the STING signaling pathway.

5. The composition for treating lung cancer according to claim 4, characterized in that: The ophiopogon saponin-D can induce calcium influx and nuclear translocation of activated T cell nuclear factor and STAT5.

6. A method for evaluating the effect of Ophiopogon japonicus saponin-D on host immune cell function, characterized in that: The method comprises the following steps: Step S1: co-culturing the host's immune cells with Ophiopogon japonicus saponin-D; Step S2: measuring the expression levels of CD69 and CD25 in the immune cells after co-culture; Step S3: Measure the changes in calcium ion concentration in immune cells after treatment with Ophiopogon japonicus saponin-D and the nuclear translocation of NFAT and STAT5.

7. A method for preparing the composition according to any one of claims 2 to 5, characterized in that The following steps are involved: Step a: chemically synthesizing or extracting ophiopogonin-D from the root of ophiopogon japonicus plants; Step b: producing or purchasing PD-1 antibodies; Step c: Mix 5-10 mg / kg of OP-D and 10 mg / kg of PD-1 antibody to form a composition.

8. A method of using the composition according to any one of claims 2 to 5 for treating lung cancer, characterized in that: The method comprises the following steps: Step X1: combining OP-D and anti-PD-1 antibody in a specific ratio to form a composition required for treatment; Step X2: administering the composition to a patient by intravenous injection, subcutaneous injection or intramuscular injection to enhance the patient's immune response to the tumor; Step X3: Monitor the patient's immune response and tumor growth to evaluate treatment efficacy.

9. The method of using the composition according to claim 8, characterized in that: The specific ratio of OP-D and anti-PD-1 antibody in step X1 is determined based on the expression levels of immune cell activation markers CD69 and CD25 in the patient's body.

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