Application of NKG7 in preparation of product for regulating and controlling killing effect of NK extracellular vesicles on tumors

Abstract

The invention provides application of NKG7 in preparation of a product for regulating and controlling the killing effect of NK extracellular vesicles on tumors, and belongs to the technical field of biomedicine. The NK cells can be promoted to secrete extracellular vesicles and the killing effect of the NK extracellular vesicles on tumors can be improved by a related method for increasing NKG7 expression in the NK cells. In-vivo and in-vitro tests prove that the killing effect of the NK cells on tumors can be improved by increasing NKG7 expression in the NK cells.

Description

[0001] This application is a divisional application with an application date of August 1, 2024, application number CN202411046131.3, and invention name “Application of reagents for detecting NKG7 in the preparation of a kit for predicting the efficacy and / or adverse reactions of tumor immunotherapy”. Technical Field

[0002] The present invention belongs to the field of biomedical technology, and in particular relates to the application of NKG7 in preparing products for regulating the killing effect of NK cell extracellular vesicles on tumors. Background Art

[0003] Natural killer (NK) cells are innate lymphoid cells that play a key role in preventing the development of tumor metastasis. NK cell-produced extracellular vesicles (NK-EVs), rich in cytotoxic proteins, cytokines, and miRNAs, can selectively kill tumor cells. However, the anti-tumor activity of NK cell EVs needs to be further improved. Summary of the Invention

[0004] The purpose of the present invention is to provide the use of NKG7 in the preparation of a product that regulates the killing effect of NK cell extracellular vesicles on tumors. Overexpression of NKG7 can enhance the killing effect of NK cell extracellular vesicles on tumors.

[0005] The present invention provides the use of NKG7 in preparing a product for regulating the killing effect of NK cell extracellular vesicles on tumors.

[0006] The present invention also provides the use of NKG7 or an agent that overexpresses NKG7 in the preparation of a product that promotes NK cells to secrete extracellular vesicles and / or enhances the tumor-killing effect of NK cell extracellular vesicles.

[0007] Preferably, said increasing the killing effect of NK cell extracellular vesicles on tumors includes increasing the content of killing proteins in NK cell extracellular vesicles.

[0008] Preferably, the product includes a medicine; and the dosage form of the medicine includes an injection.

[0009] Preferably, the tumor comprises a solid tumor or a lymphoblastic tumor.

[0010] Preferably, the solid tumor includes any one or more of lung cancer, esophageal cancer, gastric cancer, intestinal cancer, breast cancer, liver cancer, bile duct cancer, cervical cancer, breast cancer, skin cancer, kidney cancer, bladder cancer, nasopharyngeal cancer, hypopharyngeal cancer, tongue cancer and pancreatic cancer; the intestinal cancer includes colorectal cancer.

[0011] The present invention also provides a method for promoting NK cells to secrete extracellular vesicles and / or improving the tumor-killing effect of NK extracellular vesicles, comprising: overexpressing NKG7 in NK cells and then extracting NK extracellular vesicles.

[0012] The present invention also provides a NK cell extracellular vesicle extracted from NK cells overexpressing NKG7.

[0013] The present invention also provides the use of the NK cell extracellular vesicles described in the above scheme in the preparation of anti-tumor drugs.

[0014] Preferably, the tumor comprises a solid tumor or a lymphoblastic tumor.

[0015] The present invention provides the use of NKG7 in the preparation of products that regulate the anti-tumor activity of NK cell extracellular vesicles. Methods for increasing NKG7 expression in NK cells can promote NK cell secretion of extracellular vesicles and enhance the anti-tumor activity of NK cell extracellular vesicles. In vivo and in vitro studies have demonstrated that increasing NKG7 expression in NK cells can enhance the anti-tumor activity of NK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 The present invention provides a graph of the analysis results of immunohistochemical detection of NKG7 expression in local tumors of tumor patients and the relationship between its expression and the efficacy of immunotherapy; wherein, A: immunohistochemical detection of NKG7 protein expression in tumor tissues of patients with lung cancer, gastric cancer and esophageal cancer, 100× represents the original magnification of 100 times, scale = 200μm, 400× represents the original magnification of 400 times, scale = 50μm; (B, C) immunohistochemical detection of patients who respond to immunotherapy (Responder) and those who do not respond to immunotherapy Figure 1 shows the expression of NKG7 protein in tissues of nonresponders. (B) Immunohistochemistry results, 200× represents the original magnification of 200 times, scale bar = 100 μm, 400× represents the original magnification of 400 times, scale bar = 50 μm; (C) Immunohistochemistry score calculated from three randomly selected fields of view on each pathological section; (D) Receiver operating characteristic (ROC) curve for predicting immunotherapy efficacy using NKG7 expression levels in tumor tissues, ***P < 0.001.

[0018] Figure 2The present invention provides a graph showing the analysis results of soluble NKG7 levels in the plasma of tumor patients before and after treatment by ELISA, and the relationship between soluble NKG7 levels and the efficacy of immunotherapy; wherein, A: ELISA results for soluble NKG7 levels in the plasma of tumor patients before treatment (baseline); B: ELISA results for soluble NKG7 levels in the plasma of tumor patients after treatment, *P<0.05, ns indicates no statistically significant difference;

[0019] Figure 3 The present invention provides a graph of the analysis results of soluble NKG7 levels in the plasma of tumor patients before and after treatment by ELISA, as well as the relationship between soluble NKG7 levels and irAEs; wherein, A: a graph of the results of ELISA detection of soluble NKG7 levels in the plasma of tumor patients with different irAE types before (baseline) and after treatment; B: a graph of the ratio of soluble NKG7 in the plasma of tumor patients with different irAE types after immunotherapy to NKG7 in the plasma before treatment (baseline); C: a trend line graph of changes in soluble NKG7 levels in the plasma of patients without irAEs and patients with grade II-III skin-related irAEs before (baseline) and after treatment, *P<0.05, ns represents no significant statistical difference;

[0020] Figure 4 The present invention provides a graph of the analysis results of ELISA detection of exosomal NKG7 levels in the plasma of tumor patients before and after treatment, and the relationship between exosomal NKG7 levels and irAEs; wherein, A: ELISA detection results of exosomal NKG7 levels in the plasma of tumor patients with different irAE types before (baseline) and after treatment; B: trend line graph of changes in exosomal NKG7 levels in the plasma of patients with no irAEs and patients with cardiac-related irAEs before (baseline) and after treatment, *P<0.05, ns represents no significant statistical difference; C: ELISA detection results of exosomal NKG7 levels in the plasma of tumor patients with different irAE grades before (baseline) and after treatment;

[0021] Figure 5 The present invention provides a result diagram based on Bukhari single-cell data, which clearly shows that NKG7high CD803 Teff is the driving subpopulation of irAE; wherein, A: UMAP visualization result diagram of each cell subpopulation; B: expression diagram of each cell marker in different cell subpopulations; C: UMAP diagram of the proportion of CD803 Teff and the distribution of each cell subpopulation in patients with irAE and those without irAE at baseline and after treatment; D: comparison diagram of the changes in the proportion of each cell subpopulation before and after treatment in patients with irAE and those without irAE; E: result diagram of differentiating various types of irAE (immune arthritis, immune pneumonia, immune neuritis) from patients without irAE based on the proportion of CD803 Teff and its NKG7 expression;

[0022] Figure 6 The present invention provides a graph based on Zhu single cell data to verify that NKG7high CD803 Teff is the driving subpopulation of irAE; wherein, A: UMAP visualization result graph of each cell subpopulation; B: expression graph of each cell marker in different cell subpopulations; C: UMAP graph of the proportion of CD803 Teff and the distribution of each cell subpopulation after treatment in patients with irAE and those without irAE; D: comparison result graph of the proportion of each cell subpopulation after treatment in patients with irAE and those without irAE; E: result graph of differentiating patients with immune myocarditis from patients without irAE based on the proportion of CD803 Teff and their NKG7 expression;

[0023] Figure 7 Result graphs of characteristic genes for determining NKG7high CD803 Teff cell subsets provided by the present invention; wherein, A: Volcano plot of differentially expressed genes between CD803 Teff and CD802 Tem in Bukhari data; B: Volcano plot of differentially expressed genes between CD803 Teff and CD804 Teff in Bukhari data; C: Volcano plot of differentially expressed genes between CD803 Teff and CD802 Tm in Zhu data; D: Volcano plot of differentially expressed genes between CD803Teff and CD804 Teff in Zhu data; E: Venn diagram of differentially expressed genes highly expressed in CD803 Teff; F: Receiver operating characteristic curve (ROC) result graph of each characteristic gene for distinguishing CD803 Teff from CD8 cells in Bukhari data; G: Receiver operating characteristic curve (ROC) result graph of each characteristic gene for distinguishing CD803 Teff from CD8 cells in Zhu data;

[0024] Figure 8 The present invention provides a result graph showing that an increase in the NKG7high CD803 Teff ratio can predict immune hypothyroidism; wherein, A: UMAP visualization result graph of each cell subset; B: expression graph of each cell marker in different cell subsets; C: trend graph of changes in CD803 Teff ratios in 4 patients at baseline and after 2 cycles of treatment; D: UMAP graph of the proportion of CD803 Teff and the distribution of each cell subset in patients with irAE and without irAE at baseline and after 2 cycles of treatment; E: trend graph of changes in CD803 Teff ratio and thyroid-stimulating hormone levels in 4 patients at baseline, 2 cycles of treatment, and subsequent cycles;

[0025] Figure 9Figure 3: Result of NKG7 knockdown inhibiting the killing effect of extracellular vesicles secreted by equal numbers of cells on tumor cells; A: Schematic diagram of an experiment in which NK extracellular vesicles secreted by equal numbers of cells (control group and stable NKG7 knockdown group) were collected, and the changes in extracellular vesicle characteristics were analyzed and the changes in killing effect were detected by co-culture with tumor cells 721.221; B: MTS method was used to detect the death rate of tumor cells after co-culture with YTS extracellular vesicles; C: MTS method was used to detect the death rate of tumor cells after co-culture with NK92MI extracellular vesicles; D: flow cytometry Detection of apoptosis of tumor cells after co-culture with YTS extracellular vesicles; (left) is the result of flow cytometry; (right) is the statistical result; E: Detection of apoptosis of tumor cells after co-culture with NK92MI extracellular vesicles by flow cytometry; (left) is the result of flow cytometry; (right) is the statistical result; AnnV-PI-, living cells; AnnV+PI-, early apoptotic cells; AnnV+PI+, late apoptotic cells; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0026] Figure 10 Figure 3 is a graph showing the results of the present invention's knockdown of NKG7 inhibiting the killing effect of equal amounts of extracellular vesicles on tumor cells; wherein, A: a schematic diagram of an experiment for collecting equal amounts of extracellular vesicles (control group and stable knockdown NKG7 group), analyzing changes in extracellular vesicle characteristics, and detecting changes in killing effects by co-culturing with tumor cells; B: MTS assay for detecting the death ratio of tumor cells after co-culture with NK cell extracellular vesicles; (left) YTS extracellular vesicles; (right) NK92MI extracellular vesicles; C: flow cytometry for detecting apoptosis of tumor cells after co-culture with NK cell extracellular vesicles; AnnV-PI-, living cells; AnnV+PI-, early apoptotic cells; AnnV+PI+, late apoptotic cells; D: statistical results of apoptosis of tumor cells after co-culture with NK cell extracellular vesicles; (left) YTS extracellular vesicles; (right) NK92MI extracellular vesicles; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0027] Figure 11The present invention provides a result diagram of overexpression of NKG7 promoting the secretion of extracellular vesicles by equal numbers of cells and promoting the killing effect of equal numbers of extracellular vesicles on tumor cells; wherein, A: NTA detection of the particle size and concentration of extracellular vesicles secreted by equal numbers of cells after overexpressing NKG7 in YTS cells with knockdown of NKG7, N=3; B: BCA protein quantitative detection of the total amount of extracellular vesicle protein secreted by equal numbers of cells after overexpressing NKG7 in YTS cells with knockdown of NKG7, N=3; C: Western Blot detection of the expression of extracellular vesicle marker proteins CD63, CD9 and Alix in cells and extracellular vesicles after overexpressing NKG7 in YTS cells with knockdown of NKG7; D: Western Blot detection of cytotoxic effector proteins Perforin and Granzyme B, Expression of FasL and TRAIL in cells and extracellular vesicles; E: Calcein assay for the death rate of tumor cells after co-culture with IFNγ or extracellular vesicles secreted by NK cells; F: MTS assay for the death rate of tumor cells after co-culture with an equal amount of extracellular vesicles secreted by NK cells; G: Flow cytometry assay for the apoptosis of tumor cells after co-culture with an equal amount of extracellular vesicles secreted by NK cells; AnnV-PI-, living cells; AnnV+PI-, early apoptotic cells; AnnV+PI+, late apoptotic cells; H: Statistical results of apoptosis of tumor cells after co-culture with an equal amount of extracellular vesicles secreted by NK cells; I: MTS assay for the death rate of tumor cells after co-culture with an equal amount of extracellular vesicles secreted by NK cells; J: Flow cytometry assay for the apoptosis of tumor cells after co-culture with an equal amount of extracellular vesicles secreted by NK cells; K: Statistical results of apoptosis of tumor cells after co-culture with an equal amount of extracellular vesicles secreted by NK cells; **P<0.01, ***P<0.001, ****P<0.0001;

[0028] Figure 12 The present invention provides an in vivo experimental verification that NKG7 promotes the killing effect of NK cell extracellular vesicles on tumors; wherein, A: animal experiment flow chart (721.221 tumor cell line was subcutaneously injected into NOG mice, and NK cell extracellular vesicles or PBS were injected into the tumor twice a week after tumor formation); B and C are display diagrams of subcutaneous tumor nodules in mice at the end of the experiment; D: statistical diagram of mouse tumor weight, N=6; E: mouse tumor volume growth curve, N=6; F: mouse body weight growth curve, N=6; G: HE staining of mouse tumor tissue, CD31 and Ki67 immunohistochemical staining; H: immunohistochemical score result diagram of 3 randomly selected fields of view for each pathological section, N=6, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns represents no significant statistical difference. DETAILED DESCRIPTION

[0029] The present invention provides the use of a reagent for detecting NKG7 in the preparation of a kit for predicting the efficacy and / or adverse reactions of tumor immunotherapy. Natural killer cell granule protein-7 (NKG7) was first described as an intrinsic membrane protein associated with the cytotoxic granules of natural killer (NK) cells. In the present invention, the NKG7 is preferably human NKG7. The human NKG7 detected in the present invention is preferably proteinNKG7isoform 1 [Homo sapiens], NCBI sequence number: NP_005592.1, UniProt number: Q16617. The present invention found that NKG7 can be used as a biomarker for the efficacy and adverse reactions of tumor (including solid tumor) immunotherapy. In the present invention, the tumor preferably includes a solid tumor. In one embodiment of the present invention, the solid tumor preferably includes any one or more of lung cancer, esophageal cancer, gastric cancer, intestinal cancer, breast cancer, liver cancer, tongue cancer and pancreatic cancer. In another embodiment of the present invention, the solid tumor preferably includes any one or more of lung cancer, gastric cancer, esophageal cancer, colorectal cancer, liver cancer, bile duct cancer, cervical cancer, breast cancer, skin cancer, kidney cancer, bladder cancer, nasopharyngeal cancer, hypopharyngeal cancer, and tongue cancer. In another embodiment of the present invention, the solid tumor preferably includes any one or more of lung cancer, gastric cancer, colon cancer, esophageal cancer, breast cancer, skin cancer, and bladder cancer.

[0030] The present invention also provides the use of a reagent for detecting NKG7 in tumor tissue, soluble NKG7 in plasma, and / or NKG7 in plasma exosomes in the preparation of a kit for predicting the efficacy and / or adverse reactions of tumor immunotherapy. The present invention also provides the use of a reagent for detecting soluble NKG7 in plasma in the preparation of a kit having any one or more of the functions described in ① to ③: ① predicting thyroid-related irAEs; ② predicting immune hypopituitarism; ③ predicting grade II-III skin-related irAEs. Experimental results showed that NKG7 is expressed in immune cells infiltrating tumor tissue (taking lung cancer, gastric cancer and esophageal cancer as examples) and is highly correlated with the efficacy of immunotherapy in patients. Immunotherapy is effective in patients with high NKG7 expression, and local NKG7 expression in tumors can be used as a prognostic marker for patients receiving immunotherapy; the level of soluble NKG7 in plasma is highly correlated with the efficacy and adverse reactions of patients receiving immunotherapy. A high level of soluble NKG7 in the patient's plasma before treatment indicates that immunotherapy is effective. A low level of soluble NKG7 in the patient's plasma before and after treatment indicates that the patient is more likely to develop thyroid-related irAEs. A high level of soluble NKG7 in the patient's plasma after treatment indicates that the patient is more likely to develop hypopituitarism. A significant increase in soluble NKG7 in the patient's plasma during treatment indicates that the patient is more likely to develop grade II-III skin-related irAEs. The level of plasma soluble NKG7 can be used as a predictive marker for the efficacy and adverse reactions of patients receiving immunotherapy.

[0031] The present invention also provides the use of a reagent for detecting exosomal NKG7 in plasma in the preparation of a kit for predicting cardiac-related irAEs and / or predicting grade III irAEs. Plasma exosomal NKG7 expression is highly correlated with the occurrence of immunotherapy-related adverse reactions in patients. Patients with high levels of exosomal NKG7 in plasma are more likely to develop cardiac-related immunotherapy adverse reactions and grade III immunotherapy adverse reactions. Plasma exosomal NKG7 expression can be used as a prognostic marker for adverse reactions in immunotherapy patients. In the present invention, the tumor preferably includes a solid tumor. In one embodiment of the present invention, the solid tumor preferably includes any one or more of lung cancer, esophageal cancer, gastric cancer, intestinal cancer, breast cancer, liver cancer, tongue cancer and pancreatic cancer. In another embodiment of the present invention, the solid tumor preferably includes any one or more of lung cancer, gastric cancer, esophageal cancer, colorectal cancer, liver cancer, bile duct cancer, cervical cancer, breast cancer, skin cancer, kidney cancer, bladder cancer, nasopharyngeal cancer, hypopharyngeal cancer and tongue cancer. In another embodiment of the present invention, the solid tumor preferably includes any one or more of lung cancer, gastric cancer, colon cancer, esophageal cancer, breast cancer, skin cancer and bladder cancer. 。

[0032] The present invention also provides the use of a reagent for detecting the proportion of NKG7high CD8+T cells in peripheral blood and / or detecting the expression of NKG7 in peripheral blood NKG7high CD8+T cells in the preparation of a kit for predicting tumor immune-related adverse reactions. The present invention also provides the use of a reagent for detecting the proportion of NKG7high CD8+T cells in peripheral blood and / or detecting the expression of NKG7 in peripheral blood NKG7high CD8+T cells in the preparation of a kit having any one or more of the functions described in a to c: a. Predicting immune myocarditis; b. Predicting immune arthritis; c. Predicting immune thyroiditis. The present invention found that NKG7high CD8+T cells are an irAE-driven subpopulation and identified their characteristic genes, which are NKG7. The proportion of NKG7high CD8+T cells and their NKG7 expression can distinguish between patients with irAE and those without irAE, especially immune myocarditis and immune arthritis. The present invention found that by monitoring the dynamic changes in the proportion of NKG7high CD8+T cells in peripheral blood at the treatment baseline and after 2 cycles of treatment, the occurrence of thyroid-related irAEs can be predicted early.

[0033] The present invention also provides the use of an agent that overexpresses NKG7 in the preparation of a product that improves the killing function of NK cells. The present invention also provides the use of an agent that overexpresses NKG7 in the preparation of a drug that enhances the efficacy of tumor immunotherapy. In the present invention, the tumor preferably includes a solid tumor. In one embodiment of the present invention, the solid tumor preferably includes any one or more of lung cancer, esophageal cancer, gastric cancer, intestinal cancer, breast cancer, liver cancer, tongue cancer and pancreatic cancer. In another embodiment of the present invention, the solid tumor preferably includes any one or more of lung cancer, gastric cancer, esophageal cancer, colorectal cancer, liver cancer, bile duct cancer, cervical cancer, breast cancer, skin cancer, kidney cancer, bladder cancer, nasopharyngeal cancer, hypopharyngeal cancer and tongue cancer. In another embodiment of the present invention, the solid tumor preferably includes any one or more of lung cancer, gastric cancer, colon cancer, esophageal cancer, breast cancer, skin cancer and bladder cancer.

[0034] To further illustrate the present invention, the application of the reagent for detecting NKG7 provided by the present invention in the preparation of a kit for predicting the efficacy and / or adverse reactions of tumor immunotherapy is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1

[0036] NKG7 is expressed in tumor-infiltrating immune cells and can predict the efficacy of immunotherapy in tumor patients. Immunotherapy is effective in patients with high NKG7 expression (H-scored>5).

[0037] 1 Experimental methods

[0038] (1) Research subjects and tissue sample collection

[0039] This study included 30 cancer patients who received immunosuppressive therapy at the First Affiliated Hospital of China Medical University from May 2018 to April 2023, including 14 lung cancer patients, 10 gastric cancer patients, and 6 esophageal cancer patients. The basic information, clinical course, treatment information, and results of the patients were collected from the medical records. The efficacy outcomes were evaluated by clinicians according to the Response Evaluation Criteria in Solid Tumours (RECIST) version 1.1. Patients whose best treatment response was complete response (CR) and partial response (PR) were defined as responders to immunotherapy (responders), and patients whose best treatment response was progressive disease (PD) and stable disease (SD) were defined as nonresponders to immunotherapy (nonresponders). The patients provided written informed consent before surgery and obtained approval from the hospital ethics committee (ethics approval number: Kelun Shen 2022

[216] ). The clinical data of the patients are summarized in Table 1.

[0040] Table 1 Clinical data of patients

[0041] feature Number of cases (%) gender male 25(83.3) female 5(16.7) Tumor type lung cancer 14(46.7) Gastric cancer 10(33.3) Esophageal cancer 6(20.0) Number of previous treatment lines 0 18(60.0) 1 10(33.3) 2 2(6.7)

[0042] Note: Median age (years / range): 64 years (44-75 years).

[0043] (2) Immunohistochemical staining

[0044] Each tumor tissue specimen was fixed in 10% formalin, embedded in paraffin, and serially sectioned. Paraffin sections were heated in an oven at 70°C for 4 hours and then deparaffinized using a gradient of xylene I, xylene II, anhydrous ethanol, 95% ethanol, and 75% ethanol. Finally, sections were immersed in distilled water. Sections were then placed in 0.01 M citrate buffer, autoclaved for 8 minutes, and allowed to cool naturally at room temperature. After rinsing with PBS, sections were treated with Solution A (hydrogen peroxide) for 10 minutes. Following another PBS rinse, sections were blocked with Solution B (superblocking buffer) for 1 hour. 200 μl of primary antibody (NKG7 antibody #84835S, Cell Signaling Technology, USA) diluted in PBS was added dropwise to the sections and incubated overnight at 4°C. The following day, sections were rinsed with PBS and treated with Solution C (primary antibody amplification buffer) for 10 minutes. After rinsing with PBS, sections were treated with Solution D (enzyme-labeled secondary antibody polymer) for 20 minutes. After rinsing with PBS, DAB staining solution was quickly added dropwise to the sections. After full color development, sections were observed under a microscope and the reaction was terminated by soaking in distilled water. Sections were counterstained with hematoxylin for 1 minute and rinsed with running water for 10 minutes. Sections were dehydrated in 75% ethanol, 95% ethanol, anhydrous ethanol I, and anhydrous ethanol II for 5 minutes each before removal. Sections were placed in xylene I for 10 minutes, then in xylene II for 10 minutes, and finally mounted with neutral gum. The entire tissue specimen was scanned at low magnification (10x) to assess staining, and three randomly selected fields of view were photographed at high magnification (20x). Protein expression was scored based on the percentage of positive cells and staining intensity. Staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). Positive cell frequency was defined as follows: 0 for ≤5%; 1 for 5-25%; 2 for 26-50%; 3 for 51-75%; and 4 for >75%. Histological score (H-score) = staining intensity score × positive cell frequency score. Scoring was performed by two independent pathologists, and ROC curves were drawn using GraphPad Prism software.

[0045] (3) Statistical analysis

[0046] Statistical analysis was performed using GraphPad Prism software (Version 8.0.2). All mean values ​​were calculated from at least three independent experiments and presented as mean ± standard deviation. Differences were analyzed using the Student's t-test. P values ​​less than 0.05 were considered statistically significant.

[0047] 2 Experimental results

[0048] The present invention collected tumor tissue specimens from 30 tumor patients who were treated with immunotherapy and performed immunohistochemical staining analysis. The immunohistochemical results showed that NKG7 was mainly expressed in tumor-infiltrating immune cells (see Figure 1A in ), and its expression level was significantly correlated with the efficacy of immunotherapy (P<0.05) (see Figure 1 B and Figure 1 C), patients with high NKG7 expression are effective in immunotherapy. The ROC curve results show that local NKG7 expression in tumors is a good marker for predicting the efficacy of immunotherapy. When the H-score = 5 is used to define high and low NKG7 expression, the sensitivity of predicting the effectiveness of immunotherapy is 73.3% and the specificity is 86.7%; when the H-score = 2.5 is used to define high and low NKG7 expression, the sensitivity of predicting the effectiveness of immunotherapy is 100% and the specificity is 60% (see Figure 1 D) in.

[0049] 3 Experimental Conclusions

[0050] The present study found that NKG7 is expressed in immune cells infiltrating tumor tissue in patients with lung, gastric, and esophageal cancers and is highly correlated with the efficacy of immunotherapy. Patients with high NKG7 expression (H-scored > 5) are more likely to respond to immunotherapy. NKG7 expression in local tumor tissue can serve as a prognostic marker for patients undergoing immunotherapy.

[0051] Example 2

[0052] The level of soluble NKG7 in the plasma of tumor patients before immunotherapy can predict the efficacy of immunotherapy.

[0053] 1 Experimental methods

[0054] (1) Patients and tissue samples

[0055] The patient plasma included in the present invention was obtained from the First Affiliated Hospital of China Medical University. The patients provided written informed consent before the samples were collected, and the Ethics Committee of the First Affiliated Hospital of China Medical University was approved (Ethics Approval Number: Kelun Shen 2022

[216] ). The present invention included 87 cancer patients who received single-agent immunosuppressant treatment at the First Affiliated Hospital of China Medical University from January 2018 to December 2020, including 46 lung cancer patients, 6 gastric cancer patients, 6 esophageal cancer patients, 8 intestinal cancer patients, 5 gynecological cancer patients and 16 other tumor patients (breast cancer, liver cancer, tongue cancer, pancreatic cancer, etc.). The basic information, clinical course, treatment information and results of the patients were collected from the medical records. The efficacy outcomes were evaluated by clinicians according to the 1.1 version of RECIST. Patients with the best treatment response of CR and PR were defined as responders to immunotherapy, and patients with the best treatment response of PD and SD were defined as non-responders to immunotherapy. The clinical data of the patients are summarized in Table 2.

[0056] Table 2 Clinical data of patients

[0057] feature Number of cases (%) gender male 54(62.1) female 33(37.9) Tumor type lung cancer 46(52.9) Gastric cancer 6(6.9) Esophageal cancer 6(6.9) Bowel cancer 8(9.2) Gynecological tumors 5(5.7) Other tumors 16(18.4) Number of previous treatment lines 0 17(19.5) 1 44(50.6) 2 17(19.5) ≥3 9(10.3) ICI type PD1 inhibitors 81(93.1) PDL1 inhibitors 6(6.9)

[0058] Note: Median age (years / range): 60 years (30-76 years).

[0059] Plasma samples were collected from patients before and after treatment (at the time of the first efficacy evaluation after 2 cycles of medication). Whole blood was collected in venous blood collection tubes containing EDTA. The blood samples in the blood collection tubes were centrifuged at 3,000 rpm at 4°C for 10 minutes within 1 hour. The upper plasma phase was then carefully transferred to a new tube and stored in a -80°C refrigerator.

[0060] (2)ELISA

[0061] The expression of soluble NKG7 in patient plasma was detected by enzyme-linked immunosorbent assay (ELISA) using the Human NKG7 ELISA Detection Kit (Assay Genie #HUEB1938). 100 μl of plasma or the standard in the kit was added to each well and kept at room temperature for 2 hours. After the liquid was completely discarded, 100 μL of detection reagent A was added to each well and incubated at 37°C for 1 hour. Each well was washed three times with wash buffer, and then 100 μl of detection reagent B was added and incubated at 37°C for 1 hour. Then, each well was washed five times with wash buffer. 90 μl of substrate was added to each well and incubated at 37°C for 20 minutes. 50 μl of stop solution was added to each well, and the results were immediately measured by measuring the absorbance at 450 nm using the Bio-RADi Mark.

[0062] (3) Statistical analysis

[0063] See Example 1

[0064] 2 Experimental results

[0065] Clinically, it is difficult to obtain pathological samples from cancer patients, and invasive procedures bring additional pain to patients. In order to develop a clinically feasible detection method, the present invention collected plasma samples from 87 cancer patients who were treated with immunotherapy, and analyzed the correlation between the expression level of soluble NKG7 in the patient's plasma and the efficacy of immunotherapy by ELISA. Figure 2 A and Figure 2 The results in B showed that patients with a complete or partial response to immunotherapy had higher soluble NKG7 expression in plasma before treatment compared with patients who did not respond to immunotherapy, while patients who did not respond to immunotherapy had lower soluble NKG7 expression in plasma before treatment.

[0066] 3 Experimental Conclusions

[0067] The present invention found that the level of plasma soluble NKG7 is highly correlated with the efficacy of immunotherapy in patients. High levels of soluble NKG7 in patients' plasma before treatment indicate that immunotherapy is effective. The level of plasma soluble NKG7 can be used as a predictive marker for the efficacy of immunotherapy in patients.

[0068] Example 3

[0069] The level of soluble NKG7 in plasma can predict the occurrence of different types of adverse immune reactions in patients receiving immunotherapy.

[0070] 1 Experimental methods

[0071] (1) Patients and tissue samples

[0072] The present invention included 142 patients who received immunotherapy for lung cancer, gastric cancer, esophageal cancer and other cancers (colorectal cancer, liver cancer, bile duct cancer, cervical cancer, breast cancer, skin cancer, kidney cancer, bladder cancer, nasopharyngeal cancer, hypopharyngeal cancer, tongue cancer) before and after treatment to detect the level of soluble NKG7. Plasma was obtained from the First Affiliated Hospital of China Medical University. The patients provided written informed consent before sampling, and the ethics committee of the First Affiliated Hospital of China Medical University was approved. Patients diagnosed with malignant tumors and receiving immunotherapy. Patients had histologically confirmed cancer. Further eligibility criteria were an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, adequate hematological and biochemical values, and no known hypersensitivity reaction to PD-1 monoclonal antibodies. Patients who had previously received drugs targeting the immune checkpoint pathway (including drugs targeting PD-1, PD-L1 or PD-L2 or CTLA-4) were excluded. All patients at the First Affiliated Hospital of China Medical University received immunotherapy according to standard protocols. All patients' clinical information was retrieved from the hospital information system. The severity of irAEs (irAEs) was assessed according to the Common Terminology Criteria for Adverse Events (CTCAEs). The type and grade of immune adverse reactions are summarized in Table 3.

[0073] Table 3 Data on the types and grades of adverse immune reactions in patients

[0074]

[0075]

[0076] (2)ELISA

[0077] See Example 2.

[0078] (3) Statistical analysis

[0079] Statistical analysis was performed using R software (R-4.1.0). Differences were analyzed using the Wilcoxon rank-sum test. P values ​​less than 0.05 were considered statistically significant.

[0080] 2 Experimental results

[0081] The side effects of immune checkpoint inhibitors are different from other anti-tumor drugs and may occur in any organ or tissue in the body, such as the skin, digestive organs, respiratory organs, thyroid gland and pituitary gland. In particular, irAEs above level III can affect organ function, reduce the quality of life of patients, lead to discontinuation of treatment, and may even be fatal. The present invention collected plasma samples from 142 tumor patients who received immunotherapy before and after treatment, and analyzed the correlation between the level of soluble NKG7 in the patient's plasma and immunotherapy-related adverse reactions by ELISA. Figure 3 The results of A in Figure 2 showed that the levels of soluble NKG7 in the plasma of patients with thyroid-related irAEs at baseline and after treatment were significantly lower than those in patients without irAEs, and the levels of soluble NKG7 in the plasma of patients with hypopituitarism after treatment were significantly higher than those in patients without irAEs (see Figure 3 In addition, the present invention found that after receiving immunotherapy, the level of soluble NKG7 in the plasma of patients with grade II-III skin-related irAEs was significantly increased compared with the baseline (see Figure 3 B and Figure 3 C). These results suggest that elevated plasma soluble NKG7 levels are a good predictive marker for grade II-III skin-related irAEs.

[0082] 3 Experimental Conclusions

[0083] The present invention found that the level of plasma soluble NKG7 is highly correlated with adverse reactions in patients. Low levels of soluble NKG7 in patient plasma before and after treatment indicate that patients are more likely to develop thyroid-related irAEs, while high levels of soluble NKG7 in patient plasma after treatment indicate that hypopituitarism is more likely to occur. In addition, a significant increase in soluble NKG7 levels during treatment indicates that patients are more likely to develop grade II-III skin-related irAEs. Therefore, the level of plasma soluble NKG7 can be used as a predictive marker for different types of adverse immune reactions.

[0084] Example 4

[0085] The level of exosomal NKG7 in plasma can predict the occurrence of cardiac-related and grade III adverse reactions in patients receiving immunotherapy.

[0086] 1 Experimental methods

[0087] (1) Patients and tissue samples

[0088] This study included plasma samples collected before and after treatment from 27 patients undergoing immunotherapy for lung, gastric, colon, esophageal, breast, skin, and bladder cancers to measure exosomal NKG7 levels. Plasma sampling procedures are described in Example 3. Data on the types and levels of adverse immune reactions in these patients are summarized in Table 4.

[0089] Table 4 Data on the types and grades of adverse immune reactions in patients

[0090] Classification Number of cases Types of irAEs Thyroid-related irAEs 10 Skin-related irAEs 5 Cardiac-related irAEs 3 Liver damage 2 Lung-related irAEs 1 Hypopituitarism 1 No irAEs 5 irAE grade G1 10 G2 5 G3 7 No irAEs 5

[0091] (2) Isolation and purification of plasma exosomes

[0092] Remove the frozen plasma and slowly thaw it at 4°C. Centrifuge at 5000 rpm at 4°C for 15 minutes to remove cell debris, and transfer the supernatant to a new EP tube. To remove fibrinogen, add 3.2 μL of thrombin (611 U / mL, EXOQ5TM-1, SBI) per 0.4 mL of plasma. After incubation at room temperature for 10 minutes, centrifuge at 10000 rpm at 4°C for 10 minutes. Fibrin particles can be seen at the bottom of the tube. Transfer the serum sample supernatant to a new centrifuge tube and add 100 μl of ExoQuick to every 400 μl of supernatant. TM Mix well with Exosome Precipitation Solution (EXOQ5TM-1, SBI) and incubate at 4°C for 30 minutes. Centrifuge at 1500g for 30 minutes at room temperature, discard the supernatant, and resuspend the pellet in lysis buffer.

[0093] (3)ELISA

[0094] See Example 2.

[0095] (4) Statistical analysis

[0096] See Example 3.

[0097] 2 Experimental results

[0098] The present invention collected plasma samples from 27 cancer patients before and after immunotherapy, and analyzed the correlation between the level of exosomal NKG7 in the patient's plasma and irAE by ELISA. The results showed that the expression of exosomal NKG7 was detected in the plasma of 3 patients with cardiac-related irAE after treatment, and its level was significantly increased compared with the baseline (see the results). Figure 4 A and Figure 4 The expression of exosomal NKG7 in the plasma of patients with other types of irAE and those without irAE was extremely low (see the results in Figure 4The results show that the level of exosomal NKG7 in plasma is a good predictive marker for adverse reactions to cardiac-related immunotherapy. In addition, more abundant exosomal NKG7 was detected in the plasma of patients with grade III irAE after treatment, while the overall expression of exosomal NKG7 in the plasma of patients without irAE and patients with grade I-II irAE was extremely low (see the results). Figure 4 C). The results indicate that the level of exosomal NKG7 in plasma is a predictive marker for grade III adverse reactions to immunotherapy.

[0099] 3 Experimental Conclusions

[0100] The present invention found that plasma exosomal NKG7 levels were highly correlated with the occurrence of cardiac-related adverse reactions to immunotherapy and grade III adverse reactions to immunotherapy in patients. Patients whose plasma exosomal NKG7 was detected after immunotherapy were more likely to experience cardiac-related adverse reactions to immunotherapy and grade III adverse reactions to immunotherapy. Plasma exosomal NKG7 levels can serve as a prognostic marker for cardiac-related and grade III adverse reactions to immunotherapy in patients receiving immunotherapy.

[0101] Example 5

[0102] NKG7high CD8+ T cells are the driving subset of irAEs

[0103] 1 Experimental methods

[0104] (1) Data collection: The present invention collected Bukhari (GSE216329) and Zhu (GSE180045) single-cell transcriptome sequencing data. The Bukhari dataset contains single-cell data of peripheral T cells from 15 patients receiving immunotherapy at baseline and after treatment. Among them, 6 patients developed irAEs, while 9 patients did not develop irAEs. The Zhu dataset contains peripheral blood single-cell data from 13 patients after immunotherapy. Among them, 10 patients developed irAEs, while 3 patients did not develop irAEs.

[0105] (2) Data analysis: The present invention performs quality control based on the number of genes and mitochondrial ratio to filter out cells that do not meet the requirements. The Seurat R package and the PCA algorithm are used to perform cell dimensionality reduction clustering on the two single-cell data, and then the results are visualized using the UMAP (Uniform Manifold Approximation and Projection) algorithm.

[0106] (3) T cell subset annotation: Based on the recognized cell markers of T cell subsets (naive state: TCF7, CCR7, LEF1, SELL; memory state: IL7R, ANXA1, GZMK, CXCR3; effector state: NKG7, GZMH, CCL5, CST7; proliferation state: MKI67), the present invention annotates T cells into 9 different subsets, namely CD401 Tn, CD402 Tm, CD403, CD404 Treg, CD801Tn, CD802 Tem, CD803 Teff, CD804 Teff and Tprolif. Since the Zhu dataset contains other immune cells besides T cells, the present invention first annotates the T cell population based on the expression of T cell marker genes CD3D and CD3E, and then annotates the T cell subsets according to the above method.

[0107] 2 Experimental results:

[0108] In the Bukhari dataset, the present invention annotated a CD803 Teff cell subset characterized by high expression of NKG7 and other cytotoxic genes (see Figure 5 A and Figure 5 The UMAP scatter plot showed that the overall proportion of NKG7highCD803 Teff cells in irAE patients increased after treatment compared with baseline (10.3% vs 12.9%), while the proportion of NKG7highCD803 Teff cells in patients without irAEs decreased slightly after treatment compared with baseline (12.4% vs 12%) (see Figure 5 The present invention further calculated the difference between the changes in the proportion of each cell subset after treatment and at baseline for each patient, and the results showed that the changes in the proportion of NKG7high CD803Teff cells in patients with irAE were significantly higher than those in patients without irAE (see Figure 5 In addition, the present invention found that the proportion of NKG7high CD803 Teff cell subsets and the expression of NKG7 were significantly higher in patients with immune arthritis compared with patients without irAE. However, this difference was not significant in immune pneumonia and immune neuritis (see Figure 5 E).

[0109] In the Zhu dataset, the present invention also annotated NKG7high CD803 Teff cells. Similar to the Bukhari dataset, this group of cells highly expressed NKG7 and other cytotoxic genes (see Figure 6 A and Figure 6B in the figure). The UMAP scatter plot shows that the overall proportion of NKG7high CD803 Teff cells in irAE patients after treatment is higher than that in patients without irAE (9.2% vs 12.7%). The present invention further calculated the proportion of each cell subset after treatment for each patient, and the results showed that the proportion of NKG7high CD803 Teff cells in irAE patients after treatment was higher than that in patients without irAE (see Figure 6 C and Figure 6 In addition, the present invention found that the proportion of NKG7high CD803 Teff cell subsets and the expression of NKG7 in patients with immune myocarditis were significantly higher than those without irAE (see Figure 6 E).

[0110] 3 Experimental Conclusions

[0111] This study identified the NKG7high CD8+ T cell subset as a driver of irAEs. An elevated proportion of NKG7high CD8+ T cells and high NKG7 expression can indicate the development of irAEs, particularly immune myocarditis and immune arthritis.

[0112] Example 6

[0113] Identification of signature genes of the CD803 Teff subset

[0114] 1 Experimental methods

[0115] (1) Gene differential expression analysis: The present invention used wilcox.test to perform gene differential expression analysis. In the two datasets of Bukhari et al. and Zhu et al., CD803 Teff was compared with other CD8 T cells (CD803 Teff vs. CD802 Tm, CD803 Teff vs. CD804 Teff), and volcano plots were drawn. The threshold was set to logFC value > 0.5 and adj. P value < 0.05. By taking the intersection of the differentially expressed genes in CD803 Teff cells, the present invention further identified the characteristic genes of CD803 Teff.

[0116] (2) Receiver operating characteristic (ROC) curve analysis: Based on the two data sets in Example 4, the present invention used the “pROC” R package to draw the ROC curve of each characteristic gene to identify the CD803 Teff subpopulation from CD8 T cells and calculated its corresponding area under the curve (AUC).

[0117] 2 Experimental results

[0118] Such as the volcano map ( Figure 7 A~ Figure 7As shown in D), the present invention obtains genes with high expression of CD803 Teff subgroup relative to CD802 and CD804 from the Bukhari and Zhu datasets. By further screening the intersection of these genes, the present invention finally determines the characteristic genes of 6 CD803 Teff subgroups including NKG7 (see Figure 7 E).

[0119] Based on the ROC curve (see Figure 7 F and Figure 7 G), the present invention determined that the AUC values ​​of NKG7 in the Bukhari and Zhu datasets were both at the forefront, which were 0.971 and 0.821, respectively.

[0120] 3 Experimental Conclusions

[0121] The present invention found that high expression of NKG7 is the most significant feature of the NKG7high CD8+ T cell subset (i.e., the CD803 Teff cell subset shown in the figure), which provides a key marker for the identification of this cell subset.

[0122] Example 7

[0123] NKG7high CD8+ T cells can predict thyroid-related irAEs caused by immunotherapy

[0124] 1 Experimental methods

[0125] (1) Clinical sample collection: Peripheral blood samples from four patients receiving immunotherapy were collected at baseline and after two cycles of treatment for single-cell sequencing. Two of these patients developed immune hypothyroidism during subsequent treatment, while the other two patients did not experience any irAEs. The patients provided written informed consent before the samples were collected, and the study was approved by the Ethics Committee of the First Affiliated Hospital of China Medical University.

[0126] (2) Data analysis: Same as Example 4.

[0127] (3) Cell annotation: Same as Example 4.

[0128] 2 Experimental results:

[0129] The present invention annotates the NKG7high CD803 Teff cell subpopulation, which is characterized by high expression of NKG7 and other cytotoxic genes (see Figure 8 A and Figure 8 The trend graph shows that, unlike patients without irAE (P1, P2), the proportion of NKG7high CD803 Teff cells in two patients with immune hypothyroidism (P3, P4) after two cycles of treatment was significantly higher than that at baseline (see Figure 8C in Figure 1). The UMAP scatter plot showed that the overall proportion of NKG7high CD803 Teff cells in patients with irAEs increased after 2 cycles of treatment compared with baseline (35% vs 42.3%), while the overall proportion of NKG7high CD803 Teff cells in patients without irAEs decreased after 2 cycles of treatment compared with baseline (47.1% vs 41.8%) (see Figure 1). Figure 8 In addition, the present invention found that the change in the ratio of NKG7high CD803Teff was consistent with the change trend of thyroid stimulating hormone (TSH) in peripheral blood (see Figure 8 E).

[0130] 3 Experimental Conclusions

[0131] The present invention found that the proportion of NKG7high CD8+ T cells in patients with immune hypothyroidism increased compared to baseline after two cycles of immunotherapy. Therefore, by monitoring the increase in the proportion of NKG7high CD8+ T cells, the occurrence of thyroid-related irAEs can be indicated early.

[0132] Example 8

[0133] In vitro experiments verified that NKG7 promotes the killing effect of NK cells on tumors

[0134] 1 Experimental methods

[0135] (1) Establishment of NK cell lines with stable knockdown and overexpression of NKG7

[0136] NKG7 short hairpin RNA (shRNA) and negative control lentivirus were purchased from Heyuan Biotechnology Co., Ltd. (Shanghai, China). The sequences are as follows:

[0137] shNKG7-1: 5'-GCACCGATTTCTGGTTTGA-3' (SEQ ID NO. 1);

[0138] shNKG7-2: 5'-GGGTCTCAGCTATCCTCTT-3' (SEQ ID NO. 2);

[0139] NKG7 overexpression (OE) and negative control lentivirus were purchased from Heyuan Biotechnology Co., Ltd. (Shanghai, China). NK cell line YTS or NK92MI cells were cultured at 1×10 5The cells were seeded at a density of 1000 cells / well in a low-adhesion 6-well plate, and 8 μg / mL polybrene was added to improve the transfection efficiency. After adding the lentivirus and mixing well (MOI = 10 for YTS cells and MOI = 100 for NK92MI cells), the plate was sealed with a sealing film. The cells were centrifuged at 360g for 2 hours at 32°C. After the centrifugation, they were placed in an incubator and continued to be cultured. After 4 hours, 2 mL of complete culture medium was added. 48 hours after transfection, when the cells were in good condition, puromycin or blasticidin was used to screen the stably transfected cells according to the resistance tag contained in the lentiviral vector. The transfection efficiency was verified by Western Blot and subsequent experiments were carried out.

[0140] (2) Extracellular vesicle extraction

[0141] Inactivated fetal bovine serum and horse serum were centrifuged at 100,000 g for 18 hours at 4°C to prepare extracellular vesicle-depleted fetal bovine serum and horse serum. After washing with PBS, the cells were cultured in culture medium prepared from extracellular vesicle-depleted fetal bovine serum and horse serum, and the cell supernatant was collected after 48 hours. The cell supernatant was then centrifuged at 300 g for 10 minutes at 4°C to remove dead cells and collect the cell supernatant. The cell supernatant was then centrifuged at 2000 g for 10 minutes at 4°C to remove cell debris and collect the cell supernatant. The cell supernatant was filtered through a 0.22 μm pore size filter and the filtrate was collected in an ultracentrifuge tube. The filtrate was centrifuged at 100,000 g for 2 hours at 4°C to enrich extracellular vesicles, and the supernatant was discarded. The precipitate was dissolved in PBS and washed again by centrifugation at 100,000 g for 2 hours at 4°C to remove impurities. The PBS was discarded, and the white precipitate at the bottom of the ultracentrifuge tube was the extracellular vesicles. Resuspend the extracellular vesicles in an appropriate volume of PBS or lysis buffer for subsequent electron microscopy observation, particle size analysis, protein quantification, Western Blot, co-culture with tumor cells, in vivo animal experiments, or store in a -80°C freezer.

[0142] (3) Target cell killing rate determination (MTS method)

[0143] The MHCI class I molecule-negative lymphoblastoid tumor cell line 721.221 was collected as target cells, fully resuspended into a single cell suspension, and 1×10 4The cell suspension was seeded into a 96-well plate at a density of 10 cells / well. Cells or extracellular vesicles from different treatment groups were added, with three replicates per group. An equal amount of PBS was co-cultured with tumor cells as a negative control well. A blank control well containing only complete culture medium was also set up. The cells were co-cultured at 37°C and 5% CO2 for 24 hours. After the co-culture, 40 μL of MTS reagent (Promega, G3581) was added to each well and incubated at 37°C and 5% CO2 for 4 hours. After incubation, the 96-well plate was shaken on a horizontal shaker for 10 minutes to thoroughly mix the cells. The absorbance was measured at 490 nm using a microplate reader.

[0144] Target cell killing rate = (average absorbance value of PBS group - average absorbance value of treatment group) / (average absorbance value of PBS group - average absorbance value of blank control group).

[0145] (4) Detection of target cell apoptosis by flow cytometry

[0146] The Annexin V-FITC Apoptosis Detection Kit (Invitrogen, BMS500FI-300) was used as described in the instructions. After treating tumor cells 721.221 with different groups of NK cell extracellular vesicles, the tumor cells were washed once with PBS. The tumor cells were then plated at 1×10 5 Cells were resuspended in 200 μL of 1× Binding Buffer at a density of 10 cells / mL. To 195 μL of cell suspension, 5 μL of Annexin V-FITC was added, mixed, and incubated at room temperature in the dark for 10 minutes. Cells were washed with 200 μL of 1× Binding Buffer and resuspended in 190 μL of 1× Binding Buffer. Finally, 10 μL of Propidium Iodide was added and analyzed using a BD Accuri C6 flow cytometer. Results were analyzed using FlowJo software (Version 10.4).

[0147] (5) NTA detection of extracellular vesicle concentration and diameter

[0148] The concentration and size distribution of extracellular vesicles obtained by ultracentrifugation were analyzed using a Malvern Zetasizer Nano ZS instrument (Malvern Instruments, Malvern, UK) according to the manufacturer's instructions.

[0149] (6) Calcein-AM release experiment:

[0150] 7860 cells were cultured at 1×10 4Cells were seeded per well of a 96-well plate and Calcein-AM (1 μM, 30 min) was added after adherence. After washing once with PBS, cells were added with each treatment (control group vs. IFNγ group vs. NK-EVs group vs. IFNγ+NK-EVs group). Supernatants were collected after 24 and 48 h of culture, and fluorescence intensity was measured and the mortality rate was calculated:

[0151] (7) Protein immunoblotting

[0152] Cells were pre-washed with cold PBS and lysed on ice for 5 minutes using an appropriate amount of protein lysis buffer (1% Triton X-100, 50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 10 mM EDTA, 100 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 2 μg / mL Laprotinin). The cell lysate was further sonicated in an ice-water mixture using 150 W power, 30% amplitude, 3 seconds on, 3 seconds off, and repeated three times. After sonication, the cell lysate was centrifuged at 13,000 rpm for 25 minutes at 4°C, and the supernatant was aspirated to obtain cellular protein. Protein quantification was performed using the Coomassie Brilliant Blue method. The volume was made up with lysis buffer, and the protein sample was thoroughly mixed with 3× loading buffer. The sample was then boiled in a 95°C thermostat for 5-10 minutes to fully denature the protein.

[0153] The cooled protein sample was subjected to protein electrophoresis by SDS-PAGE. After electrophoresis, the separation gel was transferred to a transfer device and the protein was transferred to a PVDF membrane (Merke Millipore, IPVH00010) at 80V constant voltage for 120 minutes. After the transfer was completed, the PVDF membrane was soaked in TBST buffer (10mM Tris-Cl pH 7.4, 150mM NaCl, 0.1% Tween-20) containing 5% skim milk and blocked with slow shaking at room temperature for 40 minutes to 1 hour. The PVDF membrane was cut according to the pre-designed conditions and incubated in the primary antibody diluted in TBST buffer containing 5% skim milk at an appropriate ratio. It was incubated with slow shaking at room temperature for more than 6 hours or at 4°C overnight. After the incubation, it was washed with TBST buffer (10 minutes / time, 4 times in a row). After washing, the PVDF membrane was placed in a secondary antibody labeled with horseradish peroxidase (1:2000) diluted in TBST buffer and incubated at room temperature for 40 minutes. The membrane was then washed again with TBST buffer (10 minutes each time, four times in a row). Finally, Western Lightning Plus-ECL reagent (PerkinElmer, NEL104001EA) was used to image the membrane using chemiluminescence on an ECL luminometer.

[0154] (8) BCA protein concentration determination

[0155] Follow the instructions of the BCA protein concentration assay kit (enhanced) (Biyuntian, P0010). Take 1.2mL of protein standard preparation solution and add it to a tube of protein standard. After fully dissolving, prepare a 25mg / mL protein standard solution. Dilute the standard with PBS to concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5mg / mL according to the instructions. The remaining protein standards are divided and stored in a -20℃ refrigerator. According to the number of samples, add 1 volume of BCA reagent B to 50 volumes of BCA reagent A to prepare an appropriate amount of BCA working solution, mix thoroughly, and use within 24 hours at room temperature. Add 20μL of standards and samples of different concentrations to a 96-well plate, and set up 3 replicates for each standard and sample. Add 200μL of BCA working solution to each well and place at 37℃ for 20 minutes. After completion, place the 96-well plate on a horizontal shaker for 5 minutes to thoroughly mix. Measure the absorbance at 570 nm using a microplate reader. Calculate the protein concentration of the sample based on the standard curve and the sample dilution ratio.

[0156] (9) Statistical analysis

[0157] Statistical analysis was performed using GraphPad Prism software (Version 8.0.2). All mean values ​​were calculated from at least three independent experiments, and data are presented as mean ± standard deviation. Differences were analyzed using the Student's t-test. A p-value of less than 0.05 was considered statistically significant.

[0158] 2 Experimental results

[0159] The present invention analyzes the changes in the killing effect of extracellular vesicles secreted by equal amounts of NK cells on tumor cells after knockdown of NKG7 ( Figure 9 MTS assay showed that after knocking down NKG7, the killing effect of extracellular vesicles of equal numbers of NK cells on tumor cells was significantly reduced ( Figure 9 B and C in Figure 1). Flow cytometry showed that after knocking down NKG7, apoptosis of tumor cells co-cultured with NK cell YTS and NK92MI extracellular vesicles was significantly reduced ( Figure 9 D and E in

[0160] In order to clearly exclude the influence of the number of extracellular vesicles and determine whether knocking down NKG7 affects the function of NK cell extracellular vesicles, the present invention further analyzed the changes in the killing effect of equal amounts of extracellular vesicles secreted by NK cells YTS and NK92MI after knocking down NKG7 ( Figure 10 A in Figure 1). MTS assay results showed that at different NK cell extracellular vesicle concentrations, after knocking down NKG7, the killing effect of NK cell extracellular vesicles on tumor cells was significantly reduced ( Figure 10 B in Figure 1). Flow cytometry results showed that at different concentrations of NK cell extracellular vesicles, knockdown of NKG7 significantly reduced tumor cell apoptosis ( Figure 10 These results indicate that knocking down NKG7 not only reduces the secretion of NK cell extracellular vesicles, but also inhibits the tumor-killing function of NK cell extracellular vesicles.

[0161] To clarify the regulation of NKG7 on NK cell extracellular vesicle secretion, we overexpressed NKG7 in the NKG7 knockdown NK cell line YTS for a reversion experiment. NTA analysis results showed that compared with NKG7 knockdown YTS cells, the number of NK cell extracellular vesicle particles secreted by the same number of cells increased significantly after NKG7 overexpression ( Figure 11 A in the figure), BCA protein quantification results showed that the total protein content of NK cell extracellular vesicles was significantly increased ( Figure 11 At the same time, Western Blot results showed that after overexpression of NKG7, the expression of extracellular vesicle marker proteins CD63, CD9 and Alix in extracellular vesicles secreted by equal numbers of cells increased significantly ( Figure 11 In addition, Western Blot results showed that after overexpression of NKG7, the expression of Granzyme B, FasL and TRAIL in NK cell extracellular vesicles was significantly increased ( Figure 11 (D) The results showed that overexpression of NKG7 promoted the secretion of extracellular vesicles by NK cells and increased the content of killer proteins in the vesicles.

[0162] This study further evaluated the killing effect of interferon (IFNγ), NK extracellular vesicles (NK-EV), and interferon combined with NK extracellular vesicles (++) on tumor cells 7860. Compared with the control group, the death rate of tumor 7860 cells increased significantly after 48 hours of NK-EVs treatment, while the combination of IFNγ did not further increase the death rate of tumor cells ( Figure 11 E). The present invention shows that NK-EVs have the effect of directly killing tumor cells.

[0163] In addition, the present invention overexpressed NKG7 in the NK cell line YTS in which NKG7 was stably knocked down, and collected equal amounts of NK extracellular vesicles secreted by the cells and co-cultured them with tumor cells 721.221. Compared with YTS cells in which NKG7 was knocked down, the killing effect of NK extracellular vesicles on tumor cells was significantly increased after overexpression of NKG7 ( Figure 11 F in Figure 3). Flow cytometry results showed that overexpression of NKG7 increased apoptosis in tumor cells co-cultured with NK cell extracellular vesicles ( Figure 11G and H). The results showed that overexpression of NKG7 promoted the killing effect of extracellular vesicles secreted by equal numbers of cells on tumor cells.

[0164] The present invention further overexpressed NKG7 in the NK cell line YTS with stable NKG7 knockdown, and then collected an equal amount of extracellular vesicles and co-cultured them with tumor cells 721.221. The results of MTS cell viability assay at different concentrations of NK cell extracellular vesicles showed that compared with YTS cells with NKG7 knockdown, the killing effect of NK cell extracellular vesicles on tumor cells was significantly increased after overexpression of NKG7 ( Figure 11 Flow cytometry results showed that after overexpression of NKG7, the apoptosis of tumor cells co-cultured with NK cell extracellular vesicles increased significantly at different concentrations of NK cell extracellular vesicles ( Figure 11 J and K in Figure 1). The results showed that overexpression of NKG7 promoted the killing effect of equal amounts of extracellular vesicles on tumor cells.

[0165] 3 Experimental Conclusions

[0166] The present invention demonstrates that overexpression of NKG7 significantly promotes the secretion of extracellular vesicles from NK cells and enhances the loading of cytotoxic effector molecules within these vesicles. The present invention also demonstrates that increasing NKG7 expression in NK cells can enhance the anti-tumor activity of NK cells.

[0167] Example 9

[0168] In vivo experiments verified that NKG7 promotes the killing effect of NK cells on tumors

[0169] 1 Experimental methods

[0170] (1) Construction of subcutaneous transplanted tumor model in severely immunodeficient mice

[0171] Animal research was approved by the Biological and Medical Ethics Committee of Northeastern University (ethics approval number NEU-EC-2023A077S). Twenty-four 4- to 6-week-old female severely immunodeficient NOG mice (NOD.Cg-Prkdcscid Il2rgtm1Sug / ShiJic, lacking T, B, and NK cells) were purchased from Beijing Weitonglihua Company and maintained in a specific pathogen-free environment. After adaptive feeding, 3 × 10 6 721.221 tumor cells were injected into the armpits of mice. After palpable tumors appeared, the mice were randomly divided into 4 groups, with 6 mice in each group. In the negative control group, PBS was injected into the tumor of mice twice a week; in the control group, 1×10 7The NK cell extracellular vesicles produced by YTS cells in the control group were injected into the tumor; experimental group 1 (equal amount of cells group): 1×10 7 NKG7 knockdown group NK cell extracellular vesicles produced by NK cells were injected into the tumor; Experimental group 2 (equal amount of extracellular vesicles group): 1×10 7 An equivalent number of NK cell extracellular vesicles (EVs) from the NKG7 knockdown group and EVs from the control group were injected intratumorally. Tumor volume was measured twice weekly using a vernier caliper and mice were weighed. Three weeks after subcutaneous injection of tumor cells, all mice were sacrificed by cervical dislocation according to the standards of our center's Animal Care Committee. Tumor size was photographed and recorded, and differences in tumor size and growth curves between groups were statistically compared. Tumor tissues were then collected for hematoxylin-eosin (HE) staining and immunohistochemistry.

[0172] (2) Immunohistochemistry

[0173] See Example 1

[0174] (3) Statistical analysis

[0175] See Example 1

[0176] 2 Experimental results

[0177] To clarify whether NKG7 can promote the killing effect of NK cell extracellular vesicles on tumors in vivo, the present invention injected NK cell target cells 721.221 tumor cells into severely immunodeficient NOG mice subcutaneously. After palpable tumors appeared, three groups of differently treated NK cell extracellular vesicles or equal amounts of PBS were injected into the tumor twice a week. The treatment groups were: control group NK cell extracellular vesicles (shNC EVs), knockdown NKG7 group extracellular vesicles with the same amount of extracellular vesicles as the control group (shNKG7 EVs-equal amount EVs), and knockdown NKG7 group extracellular vesicles secreted by the same amount of cells as the control group (shNKG7 EVs-equal amount cells). After the experiment, the tumor burden of the mice was evaluated, and it was found that the tumor volume and weight of the mice were in the following order from large to small: PBS treatment group > shNKG7 EVs-equal amount cell treatment group > shNKG7 EVs-equal amount EVs treatment group > shNC EVs treatment group ( Figure 12 B in Figure 12 C in the figure). The results show that after knocking down NKG7, the inhibitory effect of NK cell extracellular vesicles on tumor cells was reduced. Due to the reduction in the number of extracellular vesicles, the inhibitory effect of NK cell extracellular vesicles secreted by the same number of cells on tumor cells was further reduced after knocking down NKG7. Similarly, HE staining showed that compared with the control group, NK cell extracellular vesicles after knocking down NKG7 caused less tumor necrosis ( Figure 12E in Figure 1). Immunohistochemistry results showed that after knockdown of NKG7, the expression of CD31 in tumors treated with NK cell extracellular vesicles was higher, indicating that its inhibitory effect on angiogenesis was reduced ( Figure 12 F in Figure 3). No significant changes were observed in the expression of Ki67, the tumor proliferation index. This may be because the tumor cells are lymphoma cell lines, which generally have high Ki67 expression, and no significant differences were observed ( Figure 12 These results indicate that knockdown of NKG7 in NK cells reduced the cytotoxicity of EVs against tumor cells, and that the EVs secreted by NKG7 knockdown cells further reduced their cytotoxicity due to the reduced number of EVs. NKG7 can promote the cytotoxicity of EVs against tumors in vivo.

[0178] 3 Experimental Conclusions

[0179] The present invention found that NKG7 in vivo can promote the killing effect of NK cell extracellular vesicles on tumors.

[0180] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creative work, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Application of NKG7 in the preparation of products that regulate the tumor-killing effect of NK cell extracellular vesicles.

2. Use of NKG7 or an agent that overexpresses NKG7 in the preparation of a product that promotes NK cells to secrete extracellular vesicles and / or enhances the tumor-killing effect of NK cell extracellular vesicles.

3. The use according to claim 2, characterized in that The method of increasing the killing effect of NK cell extracellular vesicles on tumors includes increasing the content of killing proteins in NK cell extracellular vesicles.

4. The use according to claim 2, characterized in that The product includes a medicine; the dosage form of the medicine includes an injection.

5. The use according to claim 1 or 2, characterized in that: The tumor includes a solid tumor or a lymphoblastic tumor.

6. The use according to claim 5, characterized in that The solid tumors include any one or more of lung cancer, esophageal cancer, gastric cancer, intestinal cancer, breast cancer, liver cancer, bile duct cancer, cervical cancer, breast cancer, skin cancer, kidney cancer, bladder cancer, nasopharyngeal cancer, hypopharyngeal cancer, tongue cancer and pancreatic cancer; the intestinal cancer includes colorectal cancer.

7. A method for promoting NK cells to secrete extracellular vesicles and / or enhancing the tumor-killing effect of NK cell extracellular vesicles, characterized in that: include: NKG7 was overexpressed in NK cells, and NK cell extracellular vesicles were extracted.

8. A NK cell extracellular vesicle, characterized in that Extracted from NK cells overexpressing NKG7.

9. Use of the NK cell extracellular vesicles according to claim 8 in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that The tumor includes a solid tumor or a lymphoblastic tumor.

Images