Immune cell for up-regulating Nuak1 and application thereof

Abstract

The invention relates to an up-regulation Nuak1 immune cell and application thereof, and particularly provides an over-expression Nuak1 immune cell and application thereof in preparation of drugs for treating diseases. Specifically, up-regulation of the Nuak1 in immune cells can significantly improve the toxicity, proliferative activity and cytokine or GZMB secretion ability of the immune cells, and further has the effect of enhancing tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an immune cell that upregulates Nuak1 and its applications. Background Technology

[0002] The NUAK family mainly includes NUAK family SNF1-like kinase 1 (Nuak1) and NUAK family kinase 2 (Nuak2), which consist of an N-terminal catalytic domain, a ubiquitin-associated domain, and a C-terminal spacer sequence. In recent years, research on Nuak1 has deepened, revealing its important roles in biological processes such as cell adhesion, cancer cell invasion, embryonic development, cell proliferation and aging, neuronal polarity, and axonal branching.

[0003] Currently, inhibiting Nuak1 to treat tumors has become a common approach in cancer treatment. For example, Yu et al. (Yuhui Yu, Yongsheng Wang, Xiangying Xiao, Wei Cheng, Liqiang Hu, Weiyun Yao, Zhangxuan Qian, and Wei Wu. 2019. MiR-204 inhibits hepatocellular cancer drug resistance and metastasis through targeting NUAK1. Biochemistry and Cell Biology. 97(5):563-570.) found that the relative expression level of Nuak1 in hepatocellular carcinoma tissue was significantly higher than that in adjacent normal liver tissue, and downregulating Nuak1 expression could inhibit the growth and migration of hepatocytes. Nuak1 drives the dynamic rearrangement and epithelial-mesenchymal transition of hepatocellular carcinoma cells through an FYN-STAT3-dependent mechanism, thereby promoting the drug resistance of hepatocellular carcinoma cells to targeted drugs. Chen et al. (Chen D, Liu G, Xu N, You X, Zhou H, Zhao X, Liu Q. Knockdown of ARK5 Expression Suppresses Invasion and Metastasis of Gastric Cancer. Cell Physiol Biochem. 2017; 42(3):1025-1036.) found that high expression of Nuak1 is closely related to poor prognosis in gastric cancer patients. Nuak1 can induce epithelial-mesenchymal transition (EMT) and thus promote the progression of gastric cancer cells. By knocking down the expression of Nuak1 in gastric cancer cells, the in vivo metastasis of tumor cells was significantly inhibited. Chang et al. (Chang XZ, Yu J, Liu HY, Dong RH, Cao XC. ARK5 is associated with the invasive and metastatic potential of human breast cancer cells. J Cancer Res Clin Oncol. 2012 Feb; 138(2):247-54.) studied the role of Nuak1 in the development of breast cancer. They constructed an expression vector for human ARK5 (Nuak1) and transfected the expression vector into breast cancer cells. The results showed that breast cancer cells containing the Nuak1 expression vector exhibited higher adhesion and invasive abilities than the original breast cancer cells.

[0004] However, the invention unexpectedly discovered that upregulating Nuak1 expression, especially upregulating Nuak1 expression in immune cells, can enhance its therapeutic ability against tumors. This is of great significance for understanding the mechanism of action of Nuak1, the treatment of tumors, and the development of anti-tumor agents. Summary of the Invention

[0005] To fill the gaps in existing technologies, this invention upregulates Nuak1 in immune cells, significantly enhancing their activity and effector function, increasing the production of cytokines or GZMB, or reducing immune cell depletion. Furthermore, both in vivo and in vitro experiments have confirmed that immune cells overexpressing Nuak1 exhibit enhanced anti-tumor activity. The specific method is as follows:

[0006] In a first aspect, the present invention provides an immune cell that overexpresses Nuak1 and / or has a reagent that upregulates Nuak1.

[0007] The immune cells mentioned are primary cells derived from the subject, preferably primary cells derived from healthy individuals or cancer patients.

[0008] The immune cells mentioned are autologous or allogeneic.

[0009] The immune cells mentioned are human cells or non-human animal cells, such as leukocytes or peripheral blood mononuclear cells.

[0010] Preferably, the immune cells include, but are not limited to, one or more of lymphocytes, monocytes, macrophages, granulocytes, NK cells, mast cells, or dendritic cells.

[0011] More preferably, the immune cells are lymphocytes, such as T cells, B cells, or tumor-infiltrating lymphocytes.

[0012] In one specific embodiment of the present invention, the T cells include, but are not limited to, CD8. + T cells, CD4 + T cells, MAIT cells, DNT cells, NKT cells, regulatory T cells, CD28 + T cells or CD25 + At least one of the following in T cells.

[0013] In one specific embodiment of the present invention, the immune cells are CD8 cells. + T cells.

[0014] In one specific embodiment of the present invention, the immune cells are CD4 cells. + T cells.

[0015] The immune cells also include antigen receptors expressed on the surface of the immune cells, wherein the immune cells are capable of inducing cytotoxicity, proliferative activity and / or secreting cytokines or GZMB after the antigen receptors bind to the antigens.

[0016] The antigen receptor is either a chimeric antigen receptor (CAR) or a T-cell antigen receptor (TCR).

[0017] The antigen receptor specifically binds to one or more of the following antigens: receptor tyrosine kinase-like orphan receptor 1 (ROR1), human epidermal growth factor receptor 2 (Her2), L1-cell adhesion molecule (L1-CAM), B lymphocyte antigen CD19 (CD19), B lymphocyte antigen CD20 (CD20), leukocyte differentiation antigen 22 (CD22), carcinoembryonic antigen (CEA), hepatitis B surface antigen, folic acid receptor antibody, leukocyte differentiation antigen 23 (CD23), heat-stable antigen (CD24), leukocyte differentiation antigen 30 (CD30), sialic acid-binding immunoglobulin-like lectin 3 (CD33), leukocyte differentiation antigen 38 (CD38), CD276, C D44, Epidermal growth factor receptor (EGFR), Epidermal glycoprotein-2 (EGP-2), Epidermal protein-4 (EGP-4), Erythropoietin-producing hepatocyte receptor (EPHa2), Tyrosine kinase receptor 2 (ErbB2), Tyrosine kinase receptor 3 (ErbB3), Tyrosine kinase receptor 4 (ErbB4), Fructose-1,6-bisphosphatase (FBP), Fetal acetylcholine receptor, Disialotyl-ganglioside 2 (GD2), Disialotyl-ganglioside 3 (GD3), High molecular weight melanoma-associated antigen (HMW-MAA), Interleukin-22 receptor antibody (IL-22R), Kinase-containing receptor (kdr), Difucosylated oligosaccharide (Lewis) Y), L1 cell adhesion molecule, melanoma antigen family A1 (MAGE-A1), mesothelin, mucin 1 (MUC1), mucin 16 (MUC16), prostate stem cell antigen (PSCA), natural killer 2 group member D ligand (NKG2D ligand), human esophageal squamous cell carcinoma antigen 1 autoantibody (NY-ESO-1), T cell recognized melanoma antigen (MART-1), melanoma-associated antigen (gp100), tumor-fetoprotein antigen, tumor-associated glycoprotein 72 (TAG72), endothelial growth factor receptor 2 (VEGF-R2), carcinoembryonic antigen, prostate-specific antigen, prostate-specific membrane antigen (PSMA), estrogen receptor, progesterone receptor, erythropoiesis-promoting hepatocyte kinase receptor interactor B2 (ephrinB2), interleukin-3 receptor α chain (CD123), cleavage signal-1 (CS-1), hepatocyte growth factor receptor (c-Met), melanoma-associated antigen (MAGE) A3), cyclin A1, B cell maturation antigen (BCMA), and interleukin-12.

[0018] Overexpression of Nuak1 by immune cells includes induction of Nuak1 expression levels that are at least twice the level of Nuak1 expression levels before induction;

[0019] Preferably, the expression level of Nuak1 after induction is 2-5000 times that of Nuak1 before induction, for example, 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, 500 times, 1000 times, 2000 times, 3000 times, 3500 times, 4000 times, 4500 times, and 5000 times.

[0020] Preferably, the expression level is the mRNA expression level or the protein expression level.

[0021] The upregulation of Nuak1 includes upregulating the mRNA and / or protein expression levels of Nuak1, or upregulating the protein activity of Nuak1.

[0022] The reagents used to upregulate Nuak1 are selected from nucleic acids, peptides, antibodies, proteins, small molecule compounds, traditional Chinese medicines or their extracts.

[0023] The reagents for upregulating Nuak1 include vectors, such as viral or non-viral vectors; the vectors include the nucleotide sequence of Nuak1.

[0024] Preferably, the viral vector includes a lentiviral vector, adenovirus vector, poxvirus vector, herpes simplex virus vector, or retroviral vector.

[0025] In one specific embodiment of the present invention, the vector is a retroviral vector.

[0026] The Nuak1 mentioned refers to the Nuak1 gene or protein of a human or non-human animal, such as a mouse, lizard, gorilla, horse, beluga whale, or rhinoceros.

[0027] The amino acid sequence of Nuak1 includes SEQ ID NO:1 or has 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology with SEQ ID NO:1.

[0028] The nucleotide sequence of Nuak1 comprises SEQ ID NO:2 or a nucleotide sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology with SEQ ID NO:2.

[0029] The overexpression of Nuak1 includes increasing the promoter strength of the Nuak1 gene in immune cells and / or introducing the Nuak1 gene into immune cells.

[0030] A second aspect of the present invention provides a method for constructing the immune cells described in the first aspect, the method comprising adding a reagent that upregulates Nuak1 into the immune cells.

[0031] Preferably, the construction method includes preparing a Nuak1 overexpression vector and introducing the vector into immune cells.

[0032] The vectors mentioned include viral vectors or non-viral vectors;

[0033] Preferably, the viral vector includes a lentiviral vector, adenovirus vector, poxvirus vector, herpes simplex virus vector, or retroviral vector.

[0034] A third aspect of the present invention provides the use of a reagent for upregulating Nuak1 in immune cells, or the immune cells described in the first aspect, or the immune cells obtained by the construction method described in the second aspect, in the preparation of products for treating and / or preventing tumors.

[0035] The upregulation of Nuak1 in immune cells includes upregulating the mRNA and / or protein expression levels of Nuak1 in immune cells, or upregulating the protein activity of Nuak1 in immune cells.

[0036] The immune cells mentioned are primary cells derived from the subject, preferably primary cells derived from healthy individuals or cancer patients.

[0037] The immune cells mentioned are autologous or allogeneic.

[0038] The immune cells mentioned are human cells or non-human animal cells, such as leukocytes or peripheral blood mononuclear cells.

[0039] Preferably, the immune cells include, but are not limited to, one or more of lymphocytes, monocytes, macrophages, granulocytes, NK cells, mast cells, or dendritic cells.

[0040] More preferably, the immune cells are lymphocytes, such as T cells, B cells, or tumor-infiltrating lymphocytes.

[0041] The immune cells also include antigen receptors expressed on the surface of the immune cells, wherein the immune cells are capable of inducing cytotoxicity, proliferative activity and / or secreting cytokines or GZMB after the antigen receptors bind to the antigens.

[0042] The antigen receptor is either a chimeric antigen receptor (CAR) or a T-cell antigen receptor (TCR).

[0043] The reagents used to upregulate Nuak1 in immune cells are selected from nucleic acids, peptides, antibodies, proteins, small molecule compounds, traditional Chinese medicines or their extracts.

[0044] Preferably, the reagent that upregulates Nuak1 in immune cells includes hepatkinase B1.

[0045] The reagent that upregulates Nuak1 in immune cells includes a vector, such as a viral vector or a non-viral vector; the vector includes the nucleotide sequence of Nuak1.

[0046] Preferably, the viral vector includes a lentiviral vector, adenovirus vector, poxvirus vector, herpes simplex virus vector, or retroviral vector.

[0047] The tumors include those in which upregulating Nuak1 is beneficial for treatment; preferably, the tumors are solid tumors or hematologic malignancies.

[0048] More preferably, the tumors include leukemia, lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, indolent B-cell lymphoma, B-cell malignant tumors, colon cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer, epithelial cancer, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin's lymphoma, cervical cancer, colorectal cancer, glioblastoma, neuroblastoma, Ewing sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, and / or mesothelioma.

[0049] In a fourth aspect, the present invention provides a medicament comprising the immune cells described in the first aspect or a reagent that upregulates Nuak1 in immune cells.

[0050] The drug also includes pharmaceutically acceptable excipients.

[0051] The pharmaceutically acceptable excipients include, but are not limited to, one or more of the following: excipients, diluents, wetting agents, fillers, binders, lubricants, disintegrants, antioxidants, buffers, suspending agents, solubilizers, thickeners, stabilizers, flavoring agents, and preservatives.

[0052] The drug can be administered via any suitable route of administration, such as gastrointestinal (e.g., oral) or non-gastrointestinal (e.g., intravenous, intramuscular, subcutaneous, intradermal, intra-organ, intranasal, intraocular, intravenous, intracerebral, intrathecal, transdermal, rectal, etc.).

[0053] The drug can be any suitable dosage form, such as a gastrointestinal or non-gastrointestinal dosage form, preferably including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder inhalers, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, effervescent tablets, pellets, gels, etc.

[0054] The various dosage forms of the drug described in this invention can be prepared according to conventional pharmaceutical production methods.

[0055] The drug may contain 0.01-99.5% (specifically, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%) of the immune cells or the reagent that upregulates Nuak1 in the immune cells.

[0056] The drug can be for human or veterinary use.

[0057] A fifth aspect of the present invention provides a method for enhancing T cell activity, promoting T cell proliferation, enhancing the production of cytokines or GZMB, increasing T cell infiltration in tumors, or reducing T cell exhaustion, the method comprising upregulating Nuak1 in T cells; preferably, the method comprises adding a reagent for upregulating Nuak1.

[0058] Preferably, the cytokine is one or more of IFN-γ or TNF-α.

[0059] In a sixth aspect, the present invention provides a vector for overexpressing Nuak1, said vector comprising the nucleotide sequence of Nuak1.

[0060] Preferably, the vector includes a viral vector or a non-viral vector.

[0061] Preferably, the vector is a viral vector. More preferably, the viral vector includes a lentiviral vector, adenovirus vector, poxvirus vector, herpes simplex virus vector, or retroviral vector.

[0062] In one specific embodiment of the present invention, the vector is a retrovirus.

[0063] The amino acid sequence of Nuak1 includes SEQ ID NO:1 or an amino acid sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology with SEQ ID NO:1.

[0064] The nucleotide sequence of Nuak1 comprises SEQ ID NO:2 or a nucleotide sequence having 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology with SEQ ID NO:2.

[0065] A seventh aspect of the present invention provides a cell, tissue, or organ comprising the carrier described in the sixth aspect above.

[0066] An eighth aspect of the present invention provides a method for enhancing the cytotoxicity of immune cells or enhancing the infiltration of immune cells in tumors, the method comprising treating immune cells with a reagent that upregulates Nuak1 in the immune cells.

[0067] Preferably, the reagent upregulates the expression level of Nuak1 mRNA or protein in immune cells.

[0068] Preferably, the reagent enhances the activity of Nuak1 in immune cells.

[0069] The reagents are selected from nucleic acids, peptides, antibodies, proteins, small molecule compounds, traditional Chinese medicines or their extracts.

[0070] Preferably, the reagent includes a vector, such as a viral vector or a non-viral vector; the vector includes the nucleotide sequence of Nuak1.

[0071] Preferably, the viral vector includes a lentiviral vector, adenovirus vector, poxvirus vector, herpes simplex virus vector, or retroviral vector.

[0072] In one specific embodiment of the present invention, the vector is a retroviral vector.

[0073] A ninth aspect of the present invention provides a method for treating and / or preventing tumors, the method comprising administering to a subject an effective amount of the immune cells described in the first aspect above, or immune cells obtained by the construction method described in the second aspect, or a drug described in the fourth aspect, or a carrier described in the sixth aspect, or a reagent that upregulates Nuak1.

[0074] The administration method can be any suitable route of administration, such as gastrointestinal administration (e.g., oral) or non-gastrointestinal administration (e.g., intravenous, intramuscular, subcutaneous, intradermal, intra-organ, intranasal, intraocular, infusion, intracerebral, intrathecal, transdermal, rectal, etc.).

[0075] Preferably, the method includes: (1) obtaining immune cells from a patient; (2) treating the immune cells with a reagent that induces overexpression of the Nuak1 gene; and (3) administering the immune cells to the patient.

[0076] In a tenth aspect, the present invention provides a method for enhancing the cytotoxicity of immune cells, the method comprising adding a reagent that upregulates Nuak1. Preferably, the immune cells overexpress Nuak1.

[0077] The "method" described in this invention can be for therapeutic or non-therapeutic purposes.

[0078] The "primary cells" mentioned in this invention are cells that have been taken out of the body and cultured, including at least first-generation cells and cells that have been passaged up to 50 times, preferably cells that have been passaged up to 10 times.

[0079] The "overexpression" described in this invention refers to upregulating gene expression to a level higher than the natural expression level or causing a gene that was not originally expressed to begin expressing itself. This "upregulation" can be achieved by enhancing regulatory elements to upregulate gene expression, or by further introducing a certain number of coding sequences, etc.

[0080] The term "tumor" as used in this invention can refer to any undesirable cell proliferation (or any disease that manifests as undesirable cell proliferation), vegetation, or an increased tendency or risk of undesirable cell proliferation, vegetation, or tumor. It can be benign or malignant, and can be primary or secondary (metastatic). A vegetation can be any abnormal growth or proliferation of cells and can be located in any tissue. Examples of tissues include the adrenal glands, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, mammary glands, cecum, central nervous system (including or excluding the cerebrum), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelial cells), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidneys, lacrimal glands, larynx, liver, lungs, lymph nodes, lymphoblasts, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary glands, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testes, thymus, thyroid gland, tongue, tonsils, trachea, uterus, vulva, and leukocytes.

[0081] The term "pharmaceutically acceptable" as used in this invention refers to the biological activity and characteristics of the active substances in the applied product that neither significantly stimulate the organism nor inhibit it.

[0082] The term "treatment" as used in this invention refers to slowing down, interrupting, preventing, controlling, stopping, reducing, mitigating, or reversing a sign, symptom, disorder, condition, or the progression or severity of a disease after it has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0083] The term "prevention" as used in this invention refers to all behaviors that suppress or delay specific symptoms of stress by applying the products described in this invention.

[0084] The term "effective amount" as used in this invention refers to the amount or dose of the product of this invention that provides the desired treatment or prevention after being administered to a patient or organ in one or more doses.

[0085] The "subject" described in this invention can be a human or a non-human mammal. The non-human mammal can be a wild animal, a zoo animal, an economically important animal, a pet, a laboratory animal, etc. Preferably, the non-human mammal includes, but is not limited to, pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels), or monkeys.

[0086] The term "homology" as used in this invention refers to the ability of those skilled in the art, when using amino acid or nucleotide sequences, to adjust the sequence according to actual working needs, while ensuring structural or functional similarity to known sequences, so that the sequence used, compared to sequences obtained by existing technologies, has (including but not limited to) at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%. %, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% of the sameness.

[0087] The abbreviation and full name of this application are shown in Table 1.

[0088] Table 1

[0089]

[0090] Attached Figure Description

[0091] Figure 1 The process of constructing an experimental system for Nuak1 overexpression.

[0092] Figure 2 Infection efficiency graph of Nuak1 overexpression group and control group.

[0093] Figure 3 Relative mRNA expression levels in the Nuak1 overexpression group and the control group.

[0094] Figure 4 Statistical graph of CD25 expression levels in the Nuak1 overexpression group and the control group.

[0095] Figure 5 Statistical graph of CD69 expression levels in the Nuak1 overexpression group and the control group.

[0096] Figure 6 Statistical graph of expression levels of granulamycin B (GZMB) in the Nuak1 overexpression group and the control group.

[0097] Figure 7 Statistical graph of IFN-γ expression levels in the Nuak1 overexpression group and the control group.

[0098] Figure 8 : Statistical chart of TNF-α expression levels in the Nuak1 overexpression group and the control group.

[0099] Figure 9 Proliferation curves of T cells in the Nuak1 overexpression group and the control group 48 hours after retrovirus transfection.

[0100] Figure 10 : Statistical graph of Ki-67 expression levels in the Nuak1 overexpression group and the control group.

[0101] Figure 11 : Statistical chart of TIM-3 expression levels in the Nuak1 overexpression group and the control group.

[0102] Figure 12 : Statistical chart of PD-1 expression levels in the Nuak1 overexpression group and the control group.

[0103] Figure 13Flowchart of in vitro experiments demonstrating the effects of Nuak1 overexpression on T cell activity and function.

[0104] Figure 14 Flow cytometry plot of the killing ability of Nuak1-overexpressing T cells against tumor cells in in vitro experiments.

[0105] Figure 15 Statistical graph of tumor cell mortality rates in the Nuak1 overexpression group and the control group (empty vector).

[0106] Figure 16 Flowchart of T-cell adoptive infusion experiment in B16-F10-OVA tumor-bearing mouse model.

[0107] Figure 17 : Graph showing changes in tumor area in mice.

[0108] Figure 18 : A graph showing changes in tumor volume in mice.

[0109] Figure 19 : Graph showing changes in tumor weight in mice.

[0110] Figure 20 The number of CD45.1 cells per gram of tumor tissue in the Nuak1 overexpression group and the control group was significantly higher. + A comparison chart of cell counts.

[0111] Figure 21 : Statistical graph of IFN-γ expression levels in the Nuak1 overexpression group and the control group in in vivo experiments.

[0112] Figure 22 : Statistical chart of TNF-α expression levels in the Nuak1 overexpression group and the control group in in vivo experiments.

[0113] Figure 23 : Statistical chart of TIM-3 expression levels in the Nuak1 overexpression group and the control group in in vivo experiments.

[0114] Figure 24 : Statistical chart of LAG-3 expression levels in the Nuak1 overexpression group and the control group in in vivo experiments.

[0115] Figure 25 Comparison of appearance between wild-type and Nuak1 knockout mice.

[0116] Figure 26 Morphological comparison of spleens in wild-type and Nuak1 knockout mice.

[0117] Figure 27 Comparison of thymus morphology between wild-type and Nuak1 knockout mice.

[0118] Figure 28Figure showing the weight changes of mice of different sexes 7 weeks after Nuak1 knockout.

[0119] Figure 29 : mRNA expression levels of CD4 and CD8 in wild-type and Nuak1 knockout groups.

[0120] Figure 30 At 4 weeks, CD4+ levels in lymph nodes were observed in wild-type and Nuak1 knockout mice. + T and CD8 + A scale diagram of T.

[0121] Figure 31 At 4 weeks, CD4+ levels in the spleen of wild-type and Nuak1 knockout mice were significantly higher than normal. + T and CD8 + A scale diagram of T.

[0122] Figure 32 At 4 weeks, CD4+ in the thymus of wild-type and Nuak1 knockout mice + T and CD8 + A scale diagram of T.

[0123] Figure 33 CD4+ in lymph nodes of wild-type and Nuak1 knockout mice + Effector T cells in T cells (T cells) eff ) and naïve T cells The proportion of change.

[0124] Figure 34 CD4 in the spleen of wild-type and Nuak1 knockout mice + Effector T cells in T cells (T cells) eff ) and naïve T cells The proportion of change.

[0125] Figure 35 CD8+ in lymph nodes of wild-type and Nuak1 knockout mice + Effector T cells in T cells (T cells) eff ), naïve T cells and central memory T cells (T cm ( ) proportional changes.

[0126] Figure 36 CD8+ in the spleen of wild-type and Nuak1 knockout mice + Effector T cells in T cells (T cells) eff ), naïve T cells and central memory T cells (T cm ( ) proportional changes.

[0127] Figure 37Nuak1 knockout group vs. control group CD4 + Statistical chart of CD25 expression levels in T cells.

[0128] Figure 38 Nuak1 knockout group vs. control group CD4 + Statistical chart of CD69 expression levels in T cells.

[0129] Figure 39 Nuak1 knockout group vs. control group CD8 + Statistical chart of CD25 expression levels in T cells.

[0130] Figure 40 Nuak1 knockout group vs. control group CD8 + Statistical chart of CD69 expression levels in T cells.

[0131] Figure 41 Nuak1 knockout group vs. control group CD4 + Statistical chart of IFN-γ expression levels in T cells.

[0132] Figure 42 Nuak1 knockout group vs. control group CD4 + Statistical chart of TNF-α expression levels in T cells.

[0133] Figure 43 Nuak1 knockout group vs. control group CD4 + Statistical chart of GZMB expression levels in T cells.

[0134] Figure 44 Nuak1 knockout group vs. control group CD8 + Statistical chart of IFN-γ expression levels in T cells.

[0135] Figure 45 Nuak1 knockout group vs. control group CD8 + Statistical chart of TNF-α expression levels in T cells.

[0136] Figure 46 Nuak1 knockout group vs. control group CD8 + Statistical chart of GZMB expression levels in T cells.

[0137] Figure 47 CD4 counts in the Nuak1 knockout group and the control group + T cell proliferation curve.

[0138] Figure 48 CD8 counts in the Nuak1 knockout group and the control group + T cell proliferation curve.

[0139] Figure 49Nuak1 knockout group vs. control group CD4 + Statistical chart of Ki-67 expression levels in T cells.

[0140] Figure 50 Nuak1 knockout group vs. control group CD8 + Statistical chart of Ki-67 expression levels in T cells.

[0141] Figure 51 Nuak1 knockout group vs. control group CD4 + Statistical chart of TIM-3 expression levels in T cells.

[0142] Figure 52 Nuak1 knockout group vs. control group CD4 + Statistical chart of PD-1 expression levels in T cells.

[0143] Figure 53 Nuak1 knockout group vs. control group CD8 + Statistical chart of TIM-3 expression levels in T cells.

[0144] Figure 54 Nuak1 knockout group vs. control group CD8 + Statistical chart of PD-1 expression levels in T cells.

[0145] Figure 55 Survival curves of Nuak1 knockout B16-F10 tumor-bearing mice.

[0146] Figure 56 Flowchart for constructing the Nuak1 knockout B16-F10 tumor-bearing mouse model.

[0147] Figure 57 The effect of Nuak1 knockout on tumor size.

[0148] Figure 58 The effect of Nuak1 knockout on tumor volume.

[0149] Figure 59 The effect of Nuak1 knockout on tumor weight.

[0150] Figure 60 Nuak1 knockout group vs. control group CD8 + Statistical chart of CD25 expression levels in T cells.

[0151] Figure 61 Nuak1 knockout group vs. control group CD8 + Statistical chart of CD69 expression levels in T cells.

[0152] Figure 62 Nuak1 knockout group vs. control group CD8 +Statistical chart of IFN-γ expression levels in T cells.

[0153] Figure 63 Nuak1 knockout group vs. control group CD8 + Statistical chart of GZMB expression levels in T cells.

[0154] Figure 64 Schematic diagram of point mutation in Nuak1-212A.

[0155] Figure 65 Figure: Effect of Nuak1 point mutation on CD69 expression level in OT-I cells.

[0156] Figure 66 Figure: Effect of Nuak1 point mutation on GZMB expression level in OT-I cells.

[0157] Figure 67 Without CD3 and CD28 antibody activation, the Nuak1 knockout group and the control group had different CD8 values. + Statistical graph of ROS expression levels in T cells.

[0158] Figure 68 When CD3 and CD28 antibodies are activated, the Nuak1 knockout group and the control group have different CD8 values. + Statistical graph of ROS expression levels in T cells.

[0159] Figure 69 Map of Nuak1 overexpression retroviral plasmid vectors. Detailed Implementation

[0160] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0161] The experimental methods involved in the examples are as follows:

[0162] 1. Preparation of Nuak1 overexpression retroviral plasmid vector

[0163] 1) Primer Design: Based on the obtained Nuak1 cDNA sequence (its nucleic acid sequence is shown in NM_001004363.2, as shown in SEQ ID NO: 2, and its amino acid sequence is shown in SEQ ID NO: 1), forward primers (primer-F) and reverse primers (primer-R) were designed at the N-terminus and C-terminus, respectively. The primers must contain the corresponding restriction enzyme sites and have an overlap of more than 15 bp with the target gene fragment. After design, the primers were sent to a biotechnology company for synthesis.

[0164] 2) Obtaining the target fragment: Using the above primers, polymerase chain reaction (PCR) amplification was performed with mouse cDNA as a template. The product was subjected to agarose gel electrophoresis, and the corresponding size bands were cut and purified by gel recovery to obtain the target fragment of the Nuak1 gene.

[0165] 3) Enzyme digestion of the target fragment and vector: Use restriction endonucleases BglII and EcoRI, reaction buffer corresponding to the digestion sites, and plasmid vector or the target fragment obtained in the above steps, preparing the digestion reaction system according to an appropriate ratio. Perform the digestion reaction at 37°C for 1 hour, or at 4°C overnight. Perform agarose gel electrophoresis on the digested plasmid vector, cutting the corresponding size bands for gel purification to obtain the linearized vector. If the target fragment is digested, agarose gel electrophoresis is not required; DNA purification can be performed directly to obtain the digested target fragment.

[0166] 4) Ligation of the target fragment with the linearized vector: Prepare the reaction system by mixing the enzyme-digested target fragment, linearized vector, T4 DNA ligase, and ligation reaction buffer obtained in the above steps in an appropriate ratio. Incubate at 16°C for 30 minutes to carry out the ligation reaction and obtain the ligation product.

[0167] 5) Transformation of competent cells: The above ligation product was thoroughly mixed with 30 μL of competent cells, placed on ice for 30 minutes, then heat-shocked at 42°C for 90 seconds, and placed on ice for another 2 minutes. 1 mL of antibiotic-free LB medium was then added, and the mixture was incubated at 37°C in a shaker for 1 hour. After centrifugation, the supernatant was discarded, and the bacterial pellet was resuspended in 50 μL of medium. The pellet was then spread onto LB agar plates containing ampicillin resistance and incubated upside down at 37°C for 12-16 hours.

[0168] 6) Plasmid identification: Single colonies from LB agar plates were picked and placed in LB liquid medium containing antibiotic resistance. Using the bacterial culture as a template, PCR was performed using specific primers. Single clones identified as positive by agarose gel electrophoresis were sent to a biotechnology company for Sanger sequencing, and the sequencing results were compared to verify accuracy.

[0169] 7) Bacterial cell preservation and plasmid extraction: After confirming that the sequencing results match the target cDNA sequence, the corresponding bacterial culture was stored in 30% glycerol at -80°C. It was then expanded using 300 mL of ampicillin-resistant LB liquid medium. Plasmids were extracted using a plasmid extraction kit (plasmid vector map shown). Figure 69 (As shown) Extraction, used for subsequent experiments.

[0170] 2. Preparation of retroviruses

[0171] 1) Cell Expansion Culture: HEK293FT cells were expanded one day before plasmid transfection. Cells with rapid proliferation were selected and digested with 0.25% trypsin until they could be easily blown off the culture dish wall. The digestion process was terminated with twice the volume of DMEM complete medium. The cells were gently resuspended and transferred to appropriately sized centrifuge tubes. Centrifuged at 800 rpm for 5 minutes, the supernatant was discarded, and the cell pellet was resuspended with an appropriate volume of DMEM transfection medium. The cells were then expanded into 10cm cell culture dishes, with 7 mL of DMEM transfection medium added to each dish. The culture density was ideally 60-70% after 12-16 hours.

[0172] 2) Plasmid transfection: The transfection system for one dish of HEK293FT cells contained 20 μg of the target plasmid, 6 μg of the helper plasmid pCL-Eco, and sterile double-distilled water filtered through a 0.22 μm filter, bringing the volume to 450 μL. Slowly add 50 μL of CaCl2 solution to the above system by rotating and mix well. Then, add 500 μL of 2x HBSS reagent by rotating and mix well, allowing it to stand for 10-15 minutes. Gently add the liquid dropwise to the cell culture medium, rotating to distribute it evenly. After 8-10 hours, discard the culture medium and add 7 mL of room temperature DMEM transfection medium. Incubate at 37°C.

[0173] 3) Virus Concentration and Collection: 48 hours after plasmid transfection, observe the GFP fluorescence intensity of the cells using a laser microscope. If the proportion of GFP-positive cells is high and the fluorescence intensity is strong, virus collection can proceed. Collect the culture medium from the above plasmid transfection system into a centrifuge tube, centrifuge at 500g for 5 minutes, collect the supernatant, and filter it through a 0.45µm filter. Place the filtered liquid in a 100k ultrafiltration centrifuge tube and centrifuge at 2500rpm for 0.5 hours or more until the virus volume in the collection tube is concentrated to 500µL. Collect the virus in a centrifuge tube for subsequent experiments or store it at -80℃. If the HEK293FT cells that have undergone culture medium collection show good GFP fluorescence positivity, add 7mL of DMEM transfection medium again, and repeat the same concentration procedure for virus collection after 24 hours.

[0174] 3. Isolation and culture of primary mouse T lymphocytes

[0175] 1) Immunological organ harvesting: Mice aged 6-8 weeks were euthanized. After soaking the carcass in 75% alcohol for 5 minutes, it was fixed on a dissecting board. Subcutaneous inguinal lymph nodes, axillary lymph nodes, etc., were harvested using sterile instruments. The mice were then laparotomized, and the spleen, thymus, and mesenteric lymph nodes attached to the mesentery were harvested. Each immunological organ was placed in PBS solution containing 2% streptomycin-penicillin (double antibody).

[0176] 2) T cell isolation: The procedure was performed in a clean bench. Immune organs were rinsed three times with sterile PBS solution. Lymph nodes or spleens were placed in a 70μm cell filter in 1mL PBS solution and gently ground using a 1mL syringe inner tube until no large tissue fragments were visible. The filter was rinsed with PBS solution, and the cell suspension was transferred to a 15mL centrifuge tube and centrifuged at 500g for 5 minutes. The supernatant was discarded. The lymph node or thymocyte pellet was resuspended in an appropriate volume of primary culture medium. The spleen cell pellet was resuspended in 1mL of erythrocyte lysis buffer, incubated at room temperature for 5 minutes, and then 10mL of PBS solution was added to terminate the reaction. The pellet was centrifuged at 500g for 5 minutes, the supernatant was discarded, and the spleen cell pellet was resuspended in an appropriate volume of primary culture medium.

[0177] 3) T cell culture: Filter the cell suspension through a 70μm cell filter, transfer the filtered suspension to a T25 cell culture flask, add 5-10mL of primary culture medium and mix well, and add an appropriate concentration of OVA peptide or anti-CD3 / anti-CD28 antibody according to the mouse strain to stimulate T cells, and culture in a 37℃ cell culture incubator.

[0178] 4. Mouse primary T lymphocyte retroviral infection

[0179] 1) Preparation of primary cells before infection: OT-I mice were used, and their inguinal and mesenteric lymph nodes were collected. T cells were isolated and cultured following the steps described above. After culturing in primary culture medium supplemented with 5 μg / mL OVA peptide for 24 hours, rmIL-2 was added to the culture flask to a final concentration of 1 ng / mL to improve infection efficiency. After rmIL-2 treatment for 4 hours, the cell pellet was collected by centrifugation at 500g for 5 minutes. The supernatant was discarded, and the cell pellet was resuspended in primary culture medium and counted.

[0180] 2) Retroviral infection of T cells: Calculate the concentration of the T cell suspension, take a 24-well plate, and add 1.3 × 10⁻⁶ cells to each well. 6Add the virus produced by two cells to each well. Bring each well to a final volume of 900 μL with primary culture medium, then add 0.9 μL of 10 mg / mL polybrene and mix well. Seal the 24-well plate with sealing film and centrifuge at 2500 rpm for 1.5 hours at 30°C.

[0181] 3) Virus-infected T cell culture: After centrifugation, carefully remove the supernatant from the wells and slowly add approximately 1 mL of primary culture medium containing 1 ng / mL rmIL-2 to each well, maintaining the integrity of the cell aggregation at the bottom of the plate. Incubate the infected cells in a 37°C cell culture incubator. After 24 hours, replenish the medium with primary culture medium containing rmIL-2, using a larger well plate or cell culture flask as needed. Collect T cells 48 hours after viral infection for infection efficiency testing and other required experimental procedures.

[0182] 5. Flow cytometry

[0183] For all experiments using flow cytometry, the following staining procedure was followed:

[0184] 1) After centrifuging the pretreated cells at 500g for 5 minutes and discarding the supernatant, resuspend and wash with 100μL of 0.5% BSA-PBS, centrifuge again at 500g for 5 minutes and discard the supernatant.

[0185] 2) Dilute the required fluorescent antibody in 0.5% BSA-PBS at an appropriate ratio, and dilute the cell pellet obtained in the previous step with 40 μL per well.

[0186] 3) After staining the cells in a 4°C dark environment for 30 minutes, wash them twice by centrifugation and resuspending with 0.5% BSA-PBS.

[0187] 4) If only cell membrane surface staining is involved, resuspend the washed cell pellet in 100 μL PBS solution and transfer it to a flow cytometer for immediate use.

[0188] 5) If intracellular staining (such as cytokines) or intracellular staining (such as transcription factors) is required, after the surface staining is washed away, the cells should be treated as described in the instructions using BD Cell Fixation and Infiltration Solution or Thermo Foxp3 / Transcription Factor Staining Reagent, and then washed with the corresponding buffer wash.

[0189] 6) After completing the cell membrane or nuclear membrane puncture treatment, dilute the required fluorescent antibody at an appropriate ratio using the corresponding buffer wash and perform cell staining. After staining at 4°C in the dark for 1 hour, centrifuge and resuspend twice with the corresponding buffer wash. Resuspend the washed cell pellet in 100 μL PBS solution and transfer it to a flow cytometry tube.

[0190] 7) If flow cytometry staining involves flow cytometry sorting, all reagents used in the process should be sterile. After staining, resuspend the cells in PBS solution containing 2-3% streptomycin-penicillin (double antibody), and use primary culture medium containing 2-3% streptomycin-penicillin (double antibody) in the corresponding receiving tubes.

[0191] 6. RNA extraction

[0192] 1) Count the required number of cells, taking 1×10⁻⁶. 6 Centrifuge each cell at 500g for 5 minutes, discard the supernatant, resuspend in 100μL PBS solution, and add 300μL TRIZOL reagent and mix well.

[0193] 2) Add 60 μL of CHCl3 to the above system, shake vigorously for 15 seconds to mix thoroughly, and let stand at room temperature for 2-3 minutes. Centrifuge at 12000g for 15 minutes at 4℃.

[0194] 3) After centrifugation, the liquid separates into layers. Transfer the aqueous phase to a new EP tube without RNase, add 0.5 times the volume of isopropanol, mix thoroughly, and incubate at 4°C for 30 minutes. After incubation, centrifuge at 12000 rpm for 10 minutes at 4°C.

[0195] 4) After centrifugation, discard the supernatant and resuspend the precipitate in 1 mL of 75% ethanol, then mix well. Centrifuge at 7500 g for 5 minutes at 4°C. After centrifugation, discard the supernatant and place the precipitate in a clean bench with the lid off. Dry for 10-15 minutes, then resuspend the precipitate in 20-30 μL LEPC (DNase / RNase free) water to dissolve it. Measure the concentration for subsequent experiments.

[0196] 7. Construction of mouse tumor model

[0197] 1) After expanding the culture of B16-F10-OVA cells (melanoma), trypsin digestion was performed. The cells were centrifuged at 500g for 5 minutes, the supernatant was discarded, and the cells were resuspended in PBS and washed twice. The cell pellet was resuspended in PBS and counted. Based on the counting results, the cell concentration was adjusted to 2-3 × 10⁻⁶ cells / year. 6 per milliliter.

[0198] 2) One day prior to the event, anesthetize female C57BL / 6 mice aged 6-8 weeks and shave the hair on their backs. Administer a subcutaneous injection of the adjusted cell suspension onto the right side of the back of each mouse, with a volume of 100 μL.

[0199] 3) One week after tumor cell inoculation, tumor morphology is observed. Mice with subcutaneous tumor bulges and relatively regular shapes (round or oval) are considered to have successfully constructed a tumor model.

[0200] 8. T-cell adoptive infusion

[0201] 1) From day 8 to day 10 after subcutaneous inoculation of tumor cells into mice, the tumors were observed, and tumor-bearing mice with similar tumor sizes were randomly divided into two groups.

[0202] 2) Removing dead cells from T cells infected with retroviruses was achieved by gradient density centrifugation using Ficoll reagent (2500 rpm ramp 3, depress 0, centrifugation 20 min). The intermediate layer of cells at the phase boundary was aspirated and placed in a new 15 ml centrifuge tube pre-filled with 10 ml of PBS. The cell suspension was centrifuged at 500 g for 5 min, the supernatant was discarded, and the cells were resuspended in PBS and washed twice. The cell pellet was adjusted to an appropriate concentration using PBS counting and then reinfused into mice via the orbital vein, with each mouse receiving 2 × 10⁶ cells / mL. 5 T cells.

[0203] 3) Starting from the day of intravenous infusion as day 0, use calipers to measure the length and width of the tumor, and measure it every 2 days to statistically analyze the tumor size and plot the growth curve.

[0204] 9. Isolation of tumor-infiltrating T lymphocytes

[0205] 1) Analyze tumor growth curves until differences appear between groups, then euthanize the cadavers. After soaking the cadavers in 75% alcohol, fix them on a dissection board, remove subcutaneous tumors using sterile instruments, and place them in PBS solution for later use.

[0206] 2) After photographing and weighing the tumor, take about 0.2 grams of tumor tissue, cut it into small pieces in the tumor digestion solution, and digest it in a shaker at 37°C and 200 rpm for 30 minutes. Add EDTA solution to a final concentration of 0.5 mM to terminate the digestion reaction. Grind the tumor tissue block on a 70 μmol filter and filter it. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 500 g for 5 minutes.

[0207] 3) After centrifugation, discard the supernatant and resuspend the cell pellet in 4 mL of 40% Percoll reagent in a 15 mL centrifuge tube. Using a Pasteur tube, slowly add 3 mL of 40% Percoll reagent to the bottom of the centrifuge tube, avoiding disruption of the cell layer. Perform gradient density centrifugation (2500 rpm ramp 6, descending 2, centrifugation 25 min). After centrifugation, aspirate the cells from the intermediate layer at the boundary between the two phases and place them in a new 15 mL centrifuge tube pre-filled with 10 mL of PBS solution. Centrifuge the cell suspension at 500 g for 5 min, discard the supernatant, resuspend in PBS solution, wash, and proceed with subsequent experiments.

[0208] 10. Detection of T lymphocyte activation indicators and cytokine levels

[0209] (1) Nuak1 overexpressing cells:

[0210] 1) After OT-I T cells are infected with retroviruses for 48 hours (or after TILs cells are isolated), collect the cell suspension, centrifuge at 500g for 5 minutes, discard the supernatant, resuspend in PBS solution and wash once, then resuspend in primary cell culture medium to adjust the appropriate concentration, and add 3×10⁻⁶ cells / well to a 96-well U-bottom cell plate. 5 Each cell.

[0211] 2) Centrifuge at 500g for 5 minutes, and resuspend the cell pellet in primary cell culture medium containing a protein transport inhibitor (1x) and OVA peptide at a final concentration of 1 μg / ml. Incubate the cells at 37°C for 4 hours before cell staining and subsequent flow cytometry analysis.

[0212] (2) Nuak1 cKO cells:

[0213] After T cells were isolated from immune organs, they were stimulated and cultured for 48 hours in primary culture medium containing 1 μg / ml anti-CD3 and 1 μg / ml anti-CD28. The stimulation source used for subsequent incubation was changed from OVA peptide to 1 μg / ml anti-CD3 and 1 μg / ml anti-CD28. The rest of the operation was the same as above.

[0214] 11. Detection of T lymphocyte exhaustion indicators

[0215] (1) Nuak1 overexpressing cells:

[0216] 48 hours after retroviral infection of OT-I T cells (or after TILs cell isolation), collect the cell suspension, centrifuge at 500g for 5 minutes, discard the supernatant, resuspend in PBS solution and wash once, then resuspend in primary cell culture medium to adjust the appropriate concentration, and add 5×10⁻⁶ cells / mL to a 96-well U-bottom cell plate. 5 Cells. Cell staining and subsequent flow cytometry analysis can be performed without additional stimulation.

[0217] (2) Nuak1 cKO cells:

[0218] After T cells were isolated from immune organs, they were stimulated and cultured for 72 h with primary culture medium containing a final concentration of 1 μg / ml anti-CD3 and 1 μg / ml anti-CD28. Subsequent procedures were the same as above.

[0219] 12. T lymphocyte proliferation curve labeling and proliferation index detection

[0220] (1) Nuak1 overexpressing cells:

[0221] T cells were isolated from the immune organs of OT-I mice and counted. The cell suspension was diluted to 4 × 10⁶ cells / mL with preheated PBS. 6 Cells per milliliter. CTV-labeled dye was added to the cell suspension at a ratio of 1:1000 and incubated at 37°C for 30 minutes. The reaction was terminated by adding 5 times the volume of culture medium, centrifuged at 500g for 5 minutes, resuspended in primary culture medium containing 5 μg / m³ LOVA peptide, and incubated at 37°C for 24 hours before routine retroviral infection. Flow cytometry was used to detect the CTV curve and the Ki-67 proliferation index 48 hours after infection.

[0222] (2) Nuak1 cKO cells:

[0223] After isolating T cells from immune organs, the staining procedure was the same as above. After staining, the cells were cultured in primary culture medium containing 1 μg / ml anti-CD3 and 1 μg / ml anti-CD28 for 72 h, and subsequent procedures were the same as above.

[0224] 13. In vitro tumor cell killing detection of T lymphocytes

[0225] 1) Trypsin digest B16-F10-OVA cells, collect the cell suspension, centrifuge at 500g for 5 minutes, and resuspend in PBS solution for washing once. Resuspend the cell pellet in DMEM complete medium, count and adjust to an appropriate cell concentration, and take 1×10⁶ cells. 4 One B16-F10-OVA cell was placed in a 48-well plate. The 48-well plate was pre-cultured in a 37°C cell culture incubator for 4 hours until the cells adhered.

[0226] 2) Collect OT-I T cells infected with retroviruses for 48 h, centrifuge at 500g for 5 minutes, wash once with PBS solution, and resuspend in primary culture medium. Count the cells and adjust to an appropriate cell concentration. After B16-F10-OVA cells adhere, remove the DMEM medium, add T cell suspension at a ratio of T cells to B16-F10-OVA cells of 1:1, and incubate with tumor cells in a 37°C cell culture incubator.

[0227] 3) After co-incubation for 24 hours, the supernatant in the well plate was collected and the adherent cells were digested. After centrifugation at 500g for 5 minutes, the cells were washed with PBS solution and transferred to a 96-well U-bottom cell plate. The cells were stained and then subjected to subsequent flow cytometry analysis.

[0228] Example 1: Effects of Nuak1 on T cell activation and function

[0229] A Nuak1-overexpressing retrovirus was constructed, and OT-I T cells activated by OVA peptide for 24 h were infected with the Nuak1-overexpressing retrovirus. The effects of Nuak1 overexpression on various T cell parameters were detected 48 h after infection. Figure 1 Infection efficiency tests on the overexpression system showed that both the Nuak1 overexpression group and the control group had high infection efficiency. Figure 2 Meanwhile, RNA was extracted and qPCR was performed, and the results showed a significant increase in the relative level of Nuak1 mRNA. Figure 3 This indicates that the retroviral system overexpressing Nuak1 is effective.

[0230] To assess the effect of Nuak1 overexpression on T cell activation and effector function, retrovirus-infected OT-I T cells were activated using OVA peptides, and changes in the levels of typical activation markers CD25 and CD69 on the T cell surface were detected. Figure 4 and Figure 5 Simultaneously, the levels of indicators characterizing T cell effector function, such as cytokines IFN-γ, TNF-α, and globular mycotoxin B (GZMB), were detected. Figures 6-8 Following Nuak1 overexpression, all of the above indicators increased in T cells, suggesting that Nuak1 overexpression leads to an increase in CD8+. + T cell activation and effector function were enhanced. Simultaneously, CTV staining of T cells and proliferation curves 48 hours after viral infection showed that the Nuak1 overexpression group proliferated more actively than the control group. Figure 9 ), and an increase in its Ki-67 level was also observed. Figure 10 The above results indicate that overexpression of Nuak1 can enhance CD8. + The proliferative capacity of T cells was also examined. Furthermore, the exhaustion-related marker TIM-3 was found to be elevated in T cells overexpressing Nuak1. Figure 11 ) and PD-1 ( Figure 12 The decrease in all values ​​suggests that overexpression of Nuak1 may protect CD8. + T cells are less likely to enter a state of exhaustion.

[0231] Example 2: Antitumor effect of Nuak1 overexpression

[0232] To investigate whether the alterations in T cell activity and function caused by Nuak1 overexpression can affect CD8 cells... + The anti-tumor function of T cells was demonstrated through both in vitro and in vivo experiments.

[0233] In vitro, OT-I T cells overexpressing Nuak1 were co-cultured with B16-OVA cells, and the proportion of tumor cell death was detected after 24 hours. Figure 13 The results showed that Nuak1 overexpression enhanced the killing ability of T cells against tumor cells in vitro. Figure 14 and Figure 15 ).

[0234] In vivo, T-cell adoptive infusion experiments were conducted on a B16-F10-OVA tumor-bearing mouse model, and changes in tumor size were continuously monitored. Figure 16 As can be seen, compared with the control group, the Nuak1 overexpression group showed slower tumor progression. Figure 17 Its size and weight are also smaller. Figure 18 and Figure 19 This means that Nuak1-overexpressing T cells have a stronger anti-tumor ability in vivo.

[0235] Further analysis of tumor-infiltrating lymphocytes (TILs) was performed, calculating CD45.1 per gram of tumor in both groups. + The comparison of cell (i.e., TILs) showed that the Nuak1 overexpression group exhibited a higher level of TILs. Figure 20 This suggests that its overexpression may enhance CD8. + T cell migration and infiltration. TILs were isolated and cytokine levels were detected. Results showed that the Nuak1 overexpression group exhibited higher levels of IFN-γ and TNF-α released by T cells. Figure 21 and Figure 22 This reflects the enhanced effect of its function. The levels of TIM-3 and LAG-3 in TILs of the Nuak1 overexpression group were lower than those in the control group (…). Figure 23 and Figure 24 This suggests that overexpression of Nuak1 may reduce the gradual depletion of TILs in the tumor microenvironment.

[0236] Based on the above results, overexpression of Nuak1 affects CD8. + T cells have many positive effects in their anti-tumor function.

[0237] Example 3: Effects of Nuak1 deficiency on basal development and T cell development and differentiation in mice

[0238] A recombination system based on cre-loxp was constructed to be used in CD4 + A conditional knockout mouse model of Nuak1 knockout in T cells was established. Appearance and immune organ morphology were observed in mature (7-8 weeks old) WT mice and Nuak1 cKO mice, and no significant differences were observed. Figures 25-27The body weights of the two groups of mice were counted separately according to sex, and no significant difference was found. Figure 28 RNA was extracted from isolated T cells and analyzed by qPCR. The results showed a relative decrease in mRNA levels in Nuak1cKO mice. Figure 29 This indicates that the conditional knockout mouse model was successfully constructed.

[0239] To determine whether Nuak1 deficiency affects T cell positive and negative selection and lineage differentiation during T cell development, the development of T cells in the lymph nodes, spleen, and thymus of WT mice and Nuak1 cKO mice was analyzed. It was observed that at 8 weeks of age, Nuak1 cKO did not significantly affect the development of T cells in the lymph nodes, spleen, and thymus, and CD4+ was not significantly affected. + / CD8 + The proportion of / DP T cells was not significantly different between the WT mice and the mice. Figures 30-32 CD4 was also detected in the lymph nodes and spleen of 4-week-old WT and Nuak1 cKO mice. + Effector T cells in T cells (T cells) eff ) and naïve T cells The proportion of changes showed that neither of the two indicators changed significantly. Figure 33 and Figure 34 Similarly, CD8 detection + Effector T cells in T cells (T cells) eff ), naïve T cells and central memory T cells (T cm The proportions of ) did not show significant differences between groups. Figure 35 and Figure 36 ).

[0240] In summary, Nuak1 on CD4 + Conditional knockout in T cells did not have a significant impact on basal development or T cell development and differentiation in mice.

[0241] Example 4: Effects of Nuak1 knockout on T cell activation and effector function

[0242] To assess the impact of Nuak1 knockout on T cell activation and effector function, T cells from a Nuak1 knockout mouse model were isolated and activated using CD3 and CD28 antibodies. Changes in the levels of CD25 and CD69, typical activation markers on the T cell surface, were then analyzed. It was observed that regardless of CD4... + Still CD8 + In T cells, all T cell activation markers decreased after Nuak1 knockout. Figures 37-40Under the same activation conditions, the levels of indicators characterizing T cell effector function, such as cytokines IFN-γ, TNF-α, and granzyme B (GZMB), were detected. Figures 41-46 CD4 after Nuak1 is knocked out. + With CD8 + All of the above-mentioned indicators decreased in T cells, therefore, after Nuak1 knockout or knockdown, T cell activation and effector function were weakened. Furthermore, after T cells were isolated and stained with CTV, and activated with CD3 and CD28 antibodies for 48 hours, proliferation curves showed that compared with the control group, the Nuak1 cKO group had significantly lower CD4+ levels. + With CD8 + T cell proliferation was significantly inhibited. Figure 47 and Figure 48 Furthermore, a significant decrease in Ki-67 levels was observed. Figure 49 and Figure 50 The above results indicate that knockout of Nuak1 significantly reduces CD8. + The proliferative capacity of T cells was also assessed. Furthermore, in conditionally knocked-out Nuak1 T cells, exhaustion-related markers TIM-3 and PD-1 were significantly elevated. Figures 51-54 This suggests that Nuak1 may provide some protection for T cells, and that this protection disappears when it is knocked out, making T cells more susceptible to exhaustion.

[0243] Example 5: Effects of Nuak1 gene knockout on tumors

[0244] Using a knockout mouse model, a Nuak1 cKO B16-F10 tumor-bearing mouse model was constructed. The survival of these tumor-bearing mice was tracked, and survival curves were plotted. It was observed that compared to WT mice, Nuak1 cKO mice experienced faster disease progression and shorter survival times. Figure 55 ).

[0245] Monitoring Nuak1 cKO in B16-F10 tumor-bearing mouse model ( Figure 56 The tumor size changes were observed, and it can be seen that the Nuak1 knockout group showed more rapid tumor progression compared to the control group. Figure 57 Its volume and weight are also larger. Figure 58 and Figure 59 This means that after conditional knockout of Nuak1, the anti-tumor ability of T cells in mice also decreased.

[0246] Tumor-infiltrating lymphocytes (TILs) were isolated and activated with CD3 and CD28 antibodies. Changes in CD25 and CD69 levels in T cells were then analyzed, and both were observed to decrease in the Nuak1 cKO group. Figure 60 and Figure 61 This indicates a decrease in its activation level in the tumor microenvironment. Furthermore, the results of cytokine and globular mycotoxin B (GZMB) detection showed a decrease in the levels of IFN-γ and GZMB released by T cells in the Nuak1 cKO group. Figure 62 and Figure 63 This is consistent with the weakening of its effect.

[0247] In summary: Nuak1 vs CD8 + T cells have multiple beneficial effects on anti-tumor function and may play a positive role in rescuing T cells from exhaustion. When CD4 expression is lost, CD4... + and CD8 + The activation, proliferation, and function of T cells were significantly impaired, suggesting the important regulatory role played by Nuak1.

[0248] Example 6: The effect of phosphorylation site mutations on Nuak1 function

[0249] Nuak1 is a member of the AMPK family and has a similar structural domain to AMPK. Sequence alignment revealed that the critical phosphorylation site 183 of AMPK has a similar structure to the critical phosphorylation site 212 of Nuak1. Therefore, in this embodiment, a mutant of Nuak1, Nuak1-212A, was constructed. The construction process is described below. Figure 64 .

[0250] Four hours after OT-I cells were infected with Nuak1 and Nuak1-212A, Nuak1 function-related factors were detected. The results showed that, compared with Nuak1, the mutant Nuak1-212A had lower CD69 (… Figure 65 ) and GZMB ( Figure 66 The levels of ) were not significantly different, and the above results show that Nuak1 and AMPK have different functional sites.

[0251] Example 7: Effect of Nuak1 knockout on ROS levels

[0252] Detecting the knockout of Nuak1 on CD8 + ROS levels in T cells, in the absence of CD3 and CD28 antibody activation, Nuak1 knockout significantly reduced ROS levels. Figure 67 After activation with CD3 and CD28 antibodies for 72 hours, the knockout group was still significantly lower than the non-knockout group. Figure 68 When AMPK is knocked out, ROS levels are significantly increased, indicating that although Nuak1 and AMPK have similar domains, their effects on cells are significantly different, or even opposite.

[0253] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. An immune cell, characterized in that, The immune cells overexpress Nuak1 and / or have reagents that upregulate Nuak1.

2. The immune cells according to claim 1, characterized in that, The immune cells are primary cells derived from the subject, preferably primary cells derived from tumor patients.

3. The immune cells according to claim 1, characterized in that, The immune cells mentioned are human cells, such as leukocytes or peripheral blood mononuclear cells; Preferably, the immune cells include lymphocytes, monocytes, macrophages, granulocytes, NK cells, mast cells, or dendritic cells. More preferably, the immune cells are lymphocytes, such as T cells, B cells, or tumor-infiltrating lymphocytes.

4. The immune cells according to claim 3, characterized in that, The T cells mentioned include CD8 + T cells, CD4 + T cells, MAIT cells, DNT cells, NKT cells, regulatory T cells, CD28 + T cells or CD25 + T cells.

5. The immune cells according to claim 1, characterized in that, The immune cells also include antigen receptors expressed on the surface of the immune cells, wherein the immune cells are capable of inducing cytotoxicity, proliferative activity and / or secreting cytokines or GZMB after the antigen receptors bind to the antigens.

6. The immune cells according to claim 5, characterized in that, The antigen receptor is either a chimeric antigen receptor (CAR) or a T-cell antigen receptor (TCR).

7. The immune cells according to claim 5 or 6, characterized in that, The antigen receptor specifically binds to one or more of the following antigens: ROR1, Her2, L1-CAM, CD19, CD20, CD22, CEA, hepatitis B surface antigen, folate receptor antibody, CD23, CD24, CD30, CD33, CD38, CD276, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, ErbB3, ErbB4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R, kdr, Lewis Y, L1 cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gp100, tumor-fetal antigen, TAG72, VEGF-R2, carcinoembryonic antigen, prostate-specific antigen, PSMA, estrogen receptor, progesterone receptor, ephrin B2, CD123, CS-1, c-Met, MAGE A3, cyclin A1, BCMA, and interleukin-12.

8. The immune cells according to claim 1, characterized in that, Overexpression of Nuak1 by immune cells includes induction of Nuak1 expression levels that are at least twice the level of Nuak1 expression levels before induction; Preferably, the expression level is the mRNA expression level or the protein expression level.

9. The immune cells according to claim 1, characterized in that, The upregulation of Nuak1 includes upregulating the mRNA and / or protein expression levels of Nuak1 or upregulating the protein activity of Nuak1.

10. The immune cells according to claim 1, characterized in that, The reagents used to upregulate Nuak1 are selected from nucleic acids, peptides, antibodies, proteins, small molecule compounds, traditional Chinese medicines, or their extracts.

11. The immune cells according to claim 1, characterized in that, The reagents used to upregulate Nuak1 include vectors, such as viral vectors or non-viral vectors. The vector includes the nucleotide sequence of Nuak1; Preferably, the viral vector includes a lentiviral vector, adenovirus vector, poxvirus vector, herpes simplex virus vector, or retroviral vector.

12. The immune cells according to claim 1, characterized in that, The Nuak1 mentioned refers to the Nuak1 gene or protein of humans or non-human animals.

13. The use of a reagent that upregulates Nuak1 in immune cells or the immune cells according to any one of claims 1-12 in the preparation of products for the treatment and / or prevention of tumors.

14. The application according to claim 13, characterized in that, The tumors include those in which upregulating Nuak1 is beneficial for treatment; preferably, the tumors are solid tumors or hematologic malignancies. More preferably, the tumors include leukemia, lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, indolent B-cell lymphoma, B-cell malignant tumors, colon cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer, epithelial cancer, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin's lymphoma, cervical cancer, colorectal cancer, glioblastoma, neuroblastoma, Ewing sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, and / or mesothelioma.

15. A drug, characterized in that, The drug comprises any of the immune cells or reagents that upregulate Nuak1 in immune cells as described in any one of claims 1-12.

16. A method for enhancing T cell activity, promoting T cell proliferation, increasing the production of cytokines or GZMB, improving T cell infiltration in tumors, and reducing T cell exhaustion, characterized in that, The method includes upregulating Nuak1 in T cells; preferably, the method includes adding a reagent that upregulates Nuak1.

Images