Method for regulating activity of type-1 innate-like lymphocytes and application of type-1 innate-like lymphocytes in tumor treatment

Abstract

The invention belongs to the technical field of biology, and relates to a method for regulating the activity of type 1 innate-like lymphocytes and application of the method in tumor treatment. By determining the effect of the GPR34 on the 1-type innate-like lymphocyte in tumor immunity, theoretical support is provided for research on designing and preparing the medicine for treating the tumor-related diseases by taking the GPR34 as an action target, a new strategy is provided for treatment of the related diseases participated by the 1-type innate-like lymphocyte, and the application of the GPR34 on the 1-type innate-like lymphocyte is developed. Comprising but not limited to tumors, enteritis, obesity, diabetes, non-alcoholic fatty liver, cardiovascular and cerebrovascular diseases, enteritis, arthritis, cerebral apoplexy, demyelinating disease multiple sclerosis (MS), Alzheimer's disease (AD), glioma, asthma, autoimmune diseases, infectious diseases induced by bacteria, fungi or viruses.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for regulating the activity of type 1 innate-like lymphocytes and its application in tumors Background Art

[0002] The innate lymphoid cell (ILC) family includes natural killer cells (NK), ILC1, ILC2, ILC3 and lymphoid tissue inducer cells (LTi), which are involved in early immune responses to infectious pathogens, tissue repair, tumor progression, metabolic disorders, and inflammation regulation. Discovering the role of ILC populations in different diseases and analyzing their mechanisms will help us improve the paradigm of disease diagnosis and find new therapeutic targets.

[0003] Cancer-induced immunosuppression is essential for evading immune surveillance. Clinically, overcoming cancer-induced T cell immunosuppression by targeting multiple inhibitory receptors, including PD-1 and CTLA-4, has achieved encouraging results in various cancer types. However, approximately more than 70% of patients fail to maintain a sustained response to T cell-based therapies, indicating the need to develop novel therapies based on other immune cells. Group 1 innate lymphoid cells (ILCs), including NK cells and type 1 innate lymphoid cells (ILC1), are gaining increasing attention in tumor immunotherapy due to their cytotoxic capacity against tumor cells. NK cell-based therapies have achieved promising results in the treatment of hematological malignancies such as acute myeloid leukemia (AML), non-Hodgkin's lymphoma or multiple myeloma, but have encountered great challenges in immunotherapy against solid tumors such as melanoma, non-small cell lung cancer (NSCLC), breast cancer or colorectal cancer. Unlike NK cells that mainly circulate in the blood, ILC1s are largely tissue-resident cells, mainly distributed in the intestine, liver and other solid organs, and they infiltrate the reaction site at the early stage of the immune response, which suggests that they may play a more important monitoring role in solid tumors. In fact, some new studies have shown that ILC1s can infiltrate solid tumors at the early stage of the immune response and exert tumor-suppressing activity. Studies have found that when exposed to IL-15 produced by tumor cells, ILC1s can express granzyme A and produce cytotoxicity, thereby inhibiting the development of malignant tumors in humans and mice. ILC1s promote anti-tumor immunity in breast cancer models and autoimmune pathology in newborn mice by expressing granzyme C. In addition, it has recently been found that ILC1s can induce apoptosis and differentiation of LSCs, thereby controlling the development of AML. In addition, early studies on ILC1s showed that they lack strong cytolytic activity and can even promote tumor growth, indicating that their functions in tumors are very complex. Therefore, the role and mechanism of type 1 innate-like lymphocytes in tumor immunity and whether they can be manipulated to induce effective anti-tumor immune responses remain unclear.

[0004] In addition to tumors, type 1 innate-like lymphocytes are closely related to the development of many other diseases. It is reported that in patients with systemic lupus erythematosus (SLE), the proportion and number of ILC1s are significantly increased, and the levels of cytokines IL-12, IL-18, IL-25, IL-33, IL-23, IL-1β and IFN-γ are significantly increased, thereby increasing type 1 immune response and promoting the pathogenesis of SLE. Studies have shown that ILC1s are significantly increased in the inflamed intestine of patients with Crohn's disease and aggravate the development of enteritis by producing interferon-γ (IFN-γ). In addition, researchers have found that diet-induced obesity promotes the production of interleukin (IL)-12 in adipose tissue, and causes fat-resident ILC1s to selectively proliferate and accumulate in an IL-12 receptor and STAT4-dependent manner, and promotes polarization of inflammatory macrophages by producing IFN-γ to promote obesity-related insulin resistance. In addition, studies have found that liver ILC1s increase alcohol-induced fatty liver hepatitis through the production of IL17, and the consumption of ILC1s or the neutralization of IL17A can significantly reduce liver steatosis, inflammation and damage in alcohol-fed mice. It has been reported that liver ILC1s promote the onset of chronic hepatitis B by producing inflammatory cytokines. In addition, ILC1s play an important role in immune defense against various viral and bacterial pathogens. The above results show that ILC1s are involved in the occurrence and development of many diseases, including but not limited to tumors, enteritis, obesity, diabetes, non-alcoholic fatty liver, cardiovascular and cerebrovascular diseases, enteritis, arthritis, Alzheimer's disease, asthma, autoimmune diseases, bacterial, fungal or viral-induced infectious diseases, etc. Therefore, finding specific targets for manipulating type 1 innate-like lymphocytes and drugs designed for their targets has very important clinical significance and broad application prospects.

[0005] G protein-coupled receptors (GPCRs), also known as seven-transmembrane domain receptors, regulate many different physiological processes and have accessible drug sites on the cell surface. They have long been pharmacological targets of interest. According to statistics, among the clinical drugs approved by the US FDA, more than 30% of them use GPCRs as therapeutic targets. GPR34 is a G protein-coupled receptor belonging to the rhodopsin-like receptor class, which participates in a variety of physiological processes by activating downstream classical signaling cascades. Studies have reported that Gpr34 transcripts are mainly enriched in oligodendrocytes and glial cells in mice. Researchers have found that GPR34 deficiency in spinal microglia can aggravate neuropathic pain in mice. In addition, studies have shown that glial cells lacking Gpr34 have impaired phagocytic ability and increased DCs apoptosis. Researchers have found that inhibiting Gpr34 expression can inhibit the proliferation of liver cancer cells and cervical cancer cells. In addition, our laboratory found that apoptotic neutrophils can release lysophosphatidylserine (LysoPS) to act on the GPR34 receptor on ILC3s cells to induce ILC3s activation and IL-22 production, thereby promoting tissue repair. These results suggest that GPR34 plays a key role in regulating immune and inflammatory responses. However, the role of GPR34 in regulating tumor immunity has not been reported.

[0006] GPR34 needs to bind to its ligand LysoPS to function. LysoPS is an emerging lysophospholipid (LPL) mediator with rich biological effects. It is reported that LysoPS can enhance antigen-triggered mast cell degranulation and histamine release, inhibit T cell proliferation, stimulate fibroblast migration, and enhance the phagocytosis of apoptotic cells by macrophages. In addition, studies have found that LysoPS can also stimulate glucose flux in adipocytes and myoblasts, acting as a functional switch for cytochrome P450 activity. However, the role of the LysoPS-GPR34 signaling pathway in tumor immunity is still unclear, and whether the LysoPS-GPR34 signaling pathway is involved in regulating the function of type 1 innate-like lymphocytes and related disease processes is unknown. Summary of the invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a method for regulating the activity of type 1 innate-like lymphocytes and its application.

[0008] The present invention provides a substance with GPR34 as a target, and its use in preparing a drug for preventing and / or treating type 1 innate-like lymphocyte-related diseases.

[0009] In the present invention, the substance targeting GPR34 includes at least one of I to VI:

[0010] 1), an expression vector for knocking out or knocking down the Gpr34 gene;

[0011] II), a host cell or a viral vector containing I);

[0012] III), a nucleic acid molecule that interferes with the transcription of the Gpr34 gene;

[0013] IV), terminator or transposon of Gpr34 gene expression;

[0014] V), agents that inhibit the expression of the Gpr34 gene;

[0015] VI), inhibitors and / or antagonists of GPR34 protein function.

[0016] In some embodiments, the nucleic acid molecule interfering with the transcription of the Gpr34 gene includes: an RNAi fragment targeting the Gpr34 gene, an siRNA fragment targeting the Gpr34 gene, or a CRISPR gRNA that promotes the degradation of the Gpr34 messenger RNA;

[0017] In some embodiments, the inhibitors and / or antagonists of GPR34 protein function include: GPR34 neutralizing antibodies, chemical small molecule inhibitors of GPR34.

[0018] In some specific embodiments, the neutralizing antibodies against GPR34 include chimeric antibodies, monoclonal antibodies, polyclonal antibodies, humanized antibodies, bispecific antibodies and multispecific antibodies.

[0019] In some specific embodiments, the chemical small molecule inhibitor of GPR34 is N-[((2E)-3-(4'-chloro[1,1'-biphenyl]-4-yl)-1-oxo-2-propen-1-yl]-O-(benzyl) or a structural analog thereof.

[0020] In the present invention, the related diseases in which the type 1 innate-like lymphocytes are involved include: at least one of tumors, obesity, depression, diabetes, cardiovascular and cerebrovascular diseases, enteritis, blood diseases, gout, arthritis, Alzheimer's disease, asthma, autoimmune diseases, and infectious diseases induced by bacteria, fungi and viruses.

[0021] In some embodiments, the tumor includes: solid tumors and / or non-solid tumors; the solid tumors include: cervical cancer, ovarian cancer, endometrial cancer, vulvar cancer, prostate cancer, parotid cancer, thyroid cancer, salivary gland cancer, colon cancer, lung cancer, fibrosarcoma, skin cancer, nasopharyngeal cancer, tongue cancer, oral cancer, laryngeal cancer, brain tumor, gallbladder cancer, kidney cancer, gum cancer, testicular cancer, penile cancer, multiple myeloma and / or malignant melanoma; the non-solid tumors include: leukemia and / or malignant lymphoma.

[0022] In the embodiments of the present invention, the prevention and / or treatment of the tumor includes: reducing tumor mass, reducing the number of metastatic lesions, improving the response to immunotherapy, inhibiting tumor growth and / or enhancing anti-tumor immunity.

[0023] In an embodiment of the present invention, the enhancement of anti-tumor immunity includes: increasing the proportion and number of ILC1s, enhancing the cytotoxicity, proliferation ability and activity of ILC1s, reducing the number or proportion of myeloid-derived suppressor cells, and increasing the number or proportion or killing function of toxic lymphocytes such as NK cells or T cells in tumors.

[0024] The present invention also provides a drug for treating type 1 innate-like lymphocyte-related diseases, characterized in that it comprises a substance targeting GPR34 and a pharmaceutically acceptable excipient;

[0025] The substance targeting GPR34 includes at least one of I to VI:

[0026] 1), an expression vector for knocking out or knocking down the Gpr34 gene;

[0027] II), a host cell or a viral vector containing I);

[0028] III), a nucleic acid molecule that interferes with the transcription of the Gpr34 gene;

[0029] IV), terminator or transposon of Gpr34 gene expression;

[0030] V), agents that inhibit the expression of the Gpr34 gene;

[0031] VI), inhibitors and / or antagonists of GPR34 protein function.

[0032] In the drug of the present invention, the nucleic acid molecule that interferes with the transcription of the Gpr34 gene includes: an RNAi fragment targeting the Gpr34 gene, an siRNA fragment targeting the Gpr34 gene, or a CRISPR gRNA that promotes the degradation of the Gpr34 messenger RNA.

[0033] In the medicine described in the present invention, the inhibitor and / or antagonist of the GPR34 protein function includes: GPR34 neutralizing antibodies and GPR34 small molecule compound inhibitors.

[0034] The drugs described in the present invention also include immunosuppressive molecule antibodies, small molecule compounds and nucleic acid inhibitors. In some embodiments, the immunosuppressive molecule antibodies are TIGIT antibodies, PD-L1 antibodies, CTLA-4 antibodies, LAG3 antibodies and / or PD-1 antibodies.

[0035] In some specific embodiments, the mass ratio of the TIGIT antibody to the GPR34-targeting substance is (0.05-2):(1-10).

[0036] In some embodiments, the drug comprises a TIGIT antibody and a GPR34 inhibitor, wherein the mass ratio of the TIGIT antibody to the GPR34 inhibitor is (0.05-2):(1-10). In some specific embodiments, the mass ratio of the TIGIT antibody to the GPR34 inhibitor is (0.05-1):(1-3). In some specific embodiments, the mass ratio of the TIGIT antibody to the GPR34 inhibitor is 0.05:1, 0.05:2, 0.05:3, 0.1:1, 0.1:2, 0.1:3, 0.5:1, 0.5:2, 0.5:3, 1:1, 1:2 or 1:3.

[0037] In the present invention, the drug is in the form of tablets, capsules, pills, lozenges, powders, syrups, elixirs, suspensions, solutions, emulsions or aerosols.

[0038] In some embodiments, the pharmaceutically acceptable excipients include at least one of the following types of excipients: diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, odor masking agents, colorants, anti-caking agents, humectants, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants and / or buffers.

[0039] The present invention also provides an application in a drug for treating and / or preventing type 1 innate-like lymphocyte-related diseases, which is administering the drug of the present invention. Specifically, the method in the drug for treating and / or preventing type 1 innate-like lymphocyte-related diseases comprises administering an effective amount of the drug as described above to a subject in need.

[0040] In the present invention, the administration method of the drug includes oral administration, inhalation, and parenteral administration. The parenteral administration includes subcutaneous injection, intramuscular injection, intravenous injection, or intradermal injection.

[0041] In an embodiment of the present invention, the dosage of the pharmaceutical composition is 0.5-30 mg / kg, 1-30 mg / kg, 2-30 mg / kg, 3-30 mg / kg, 4-30 mg / kg or more. In some embodiments, the dosage of the drug is, for example, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg or more.

[0042] The present invention achieves the following beneficial technical effects:

[0043] By determining the role of GPR34 in anti-tumor immunity, the present invention provides theoretical support for the design and preparation of drugs for treating type 1 innate-like lymphocyte-related diseases with GPR34 as the target, and provides a new strategy for treating tumors, obesity, depression, diabetes, cardiovascular and cerebrovascular diseases, enteritis, blood diseases, gout, arthritis, Alzheimer's disease, asthma, autoimmune diseases, and infectious diseases induced by bacteria, fungi and viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The expression of GPR34 on immune cells in Example 1 is shown, wherein a shows the expression of type 1 innate-like lymphocytes, B cells, CD8 + T cells, CD4 + a Result graph showing the expression level of GPR34 in T cells, dendritic cells, macrophages, monocytes and neutrophils; b Result graph showing the expression level of GPR34 in tumor-infiltrating immune cells in mouse tumor-bearing MC38 cells;

[0045] Figure 2The result graphs showing the changes of systemic Gpr34 knockout mice in different tumor models in Example 2 are as follows: in the MC38 tumor model, a shows that the tumor growth rate of systemic Gpr34 knockout mice is significantly slowed down compared with the control mice, and b shows that the tumor weight of systemic Gpr34 knockout mice is significantly reduced compared with the control mice; in the LLC tumor model, c shows that the tumor growth rate of systemic Gpr34 knockout mice is significantly slowed down compared with the control mice, and d shows that the tumor weight of systemic Gpr34 knockout mice is significantly reduced compared with the control mice; in the B16F10 tumor model, e shows that the tumor growth rate of systemic Gpr34 knockout mice is significantly slowed down compared with the control mice, and f shows that the tumor weight of systemic Gpr34 knockout mice is significantly reduced compared with the control mice;

[0046] Figure 3 The results of investigating the changes in different tumor models of mice with specific knockout of Gpr34 gene on NCR1 cells in Example 3 are shown: in the MC38 tumor model, a shows that compared with the control mice, the tumor growth rate of mice with specific knockout of Gpr34 gene on NCR1 cells was significantly slowed down, b shows that compared with the control mice, the tumor weight of mice with specific knockout of Gpr34 gene on NCR1 cells was significantly reduced, g shows that compared with the control mice, the proportion and number of ILC1s infiltrating in the tumor of mice with specific knockout of Gpr34 gene on NCR1 cells were significantly increased, and the proliferation ability was enhanced, h shows that compared with the control mice, the proportion and number of ILC1s infiltrating in the tumor of mice with specific knockout of Gpr34 gene on NCR1 cells were significantly increased, and the proliferation ability was enhanced, h shows that compared with the control mice, the proportion of ILC1s infiltrating in the tumor of mice with specific knockout of Gpr34 gene on NCR1 cells was significantly increased, and the proliferation ability was enhanced, The effector function of ILC1s infiltrating in the tumors of mice with Gpr34 knockout was significantly enhanced; in the B16F10 tumor model, c showed that the tumor growth rate of mice with Gpr34 knockout on NCR1 cells was significantly slowed down compared with control mice, and d showed that the tumor weight of mice with Gpr34 knockout on NCR1 cells was significantly reduced compared with control mice; in the LLC tumor model, e showed that the tumor growth rate of mice with Gpr34 knockout on NCR1 cells was significantly slowed down compared with control mice, and f showed that the tumor weight of mice with Gpr34 knockout on NCR1 cells was significantly reduced compared with control mice;

[0047] Figure 4The results of investigating the effect of tumor-derived LysoPS on ILC1s activity in Example 4 are shown in Figure 4: a shows that in different tumor models, the concentration of LysoPS in tumor interstitial fluid was significantly increased compared with serum; b shows that compared with the control group, interleukin 12 / 15 / 18 can significantly induce ILC1s cytokine production, while 5 micromolar concentration of LysoPS can inhibit ILC1s cytokine production and this process depends on GPR34; in the MC38 tumor model, c shows that compared with the control mice, the tumor growth rate of mice in the Abhd16a gene expression knockdown group was significantly slowed down, and d shows that compared with the control mice, the Abhd16a gene expression knockdown group The tumor weight of mice in the knockdown group was significantly reduced, e showed that compared with the control mice, the effector function of ILC1s infiltrating in the tumor of mice in the knockdown group of Abhd16a gene expression was significantly enhanced; in the B16F10 tumor model, f showed that compared with the control mice, the tumor growth rate of mice in the knockdown group of Abhd16a gene expression was significantly slowed down; g showed that compared with the control mice, the tumor weight of mice in the knockdown group of Abhd16a gene expression was significantly reduced; in the LLC tumor model, h showed that compared with the control mice, the tumor growth rate of mice in the knockdown group of Abhd16a gene expression was significantly slowed down; i showed that compared with the control mice, the tumor weight of mice in the knockdown group of Abhd16a gene expression was significantly reduced;

[0048] Figure 5 The result graphs of exploring the therapeutic effect of GPR34 inhibitors in tumor models in Example 5 are shown: in the MC38 tumor model, a shows that compared with the control mice, the tumor growth rate of the inhibitor-treated mice was significantly slowed; b shows that compared with the control mice, the tumor weight of the inhibitor-treated mice was significantly reduced; g shows that compared with the control mice, the proportion and number of ILC1s infiltrating in the tumors of the inhibitor-treated mice were significantly increased and the proliferation capacity was enhanced; h shows that compared with the control mice, the effector function of ILC1s infiltrating in the tumors of the inhibitor-treated mice was significantly enhanced; in the B16F10 tumor model, c shows that compared with the control mice, the tumor growth rate of the inhibitor-treated mice was significantly slowed; d shows that compared with the control mice, the tumor weight of the inhibitor-treated mice was significantly reduced; in the LLC tumor model, e shows that compared with the control mice, the tumor growth rate of the inhibitor-treated mice was significantly slowed; f shows that compared with the control mice, the tumor weight of the inhibitor-treated mice was significantly reduced;

[0049] Figure 6The results of exploring the therapeutic effect of GPR34 inhibitor combined with TIGIT antibody in tumor models in Example 6 are shown: in the B16F10 tumor model, a shows that compared with the control mice, the inhibitor and TIGIT antibody alone effectively inhibited the growth of the tumor, and the inhibitor combined with TIGIT antibody treatment was more effective, and the tumor growth rate was further slowed down; in the MC38 tumor model, b shows that compared with the control mice, the growth rate of the tumor did not change when TIGIT antibody was treated alone, the inhibitor alone effectively slowed the growth of the tumor, and the inhibitor combined with TIGIT antibody treatment could better inhibit the tumor growth rate, and c shows that compared with the control mice, TIGIT antibody alone Treatment did not affect the survival time of mice, but treatment with the inhibitor alone could prolong the survival time of mice, and treatment with the inhibitor combined with TIGIT antibody could further prolong the survival time of mice; in the LLC tumor model, d showed that compared with control mice, treatment with TIGIT antibody alone slightly slowed down the growth rate of tumors, and treatment with the inhibitor alone effectively slowed down the growth of tumors, and treatment with the inhibitor combined with TIGIT antibody could better inhibit the growth rate of tumors; e showed that compared with control mice, treatment with TIGIT antibody alone did not affect the survival time of mice, but treatment with the inhibitor alone could prolong the survival time of mice, and treatment with the inhibitor combined with TIGIT antibody could further prolong the survival time and survival rate of mice;

[0050] Data statistical analysis: Unless otherwise specified in the figure captions, the data used in this article were analyzed using GraphPadPrism (software version 8) using two-tailed unpaired Student's t-test; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, NS: no significant difference. DETAILED DESCRIPTION

[0051] The present invention provides a method for regulating the activity of type 1 innate-like lymphocytes and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0052] In this study, we found that GPR34 was highly expressed on type 1 innate-like lymphocytes and less expressed on other immune cells. More importantly, we found that tumor-derived LysoPS inhibited the antitumor activity of ILC1s through GPR34, while deleting the expression of GPR34 in ILC1s or antagonizing GPR34 with chemical inhibitors enhanced antitumor immunity and suppressed tumor growth. Therefore, this study shows that manipulating type 1 innate-like lymphocytes to restore their function can induce effective antitumor immunity, suggesting that type 1 innate-like lymphocyte-based immunotherapy has potential value.

[0053] The reagents and consumables used in the present invention are all common commercial products and can be purchased in the market.

[0054] GPR34 small molecule inhibitor: purchased from Shanghai Taoshu Biotechnology Co., Ltd., molecular formula: C31H26ClNO4, structure see the figure below.

[0055]

[0056] Experimental animals: Wild-type C57BL / 6J mice were purchased from Shanghai Slake Laboratory Animal Co., Ltd. Systemic or conditional knockout mice of Gpr34 were obtained by technicians from the Animal Experiment Center of the School of Life Sciences of the University of Science and Technology of China using Crispr-Cas9 technology. + / + Genetic mice were used as experimental control mice, Gpr34 - / - The genetic mice were Gpr34 gene-deficient mice. All experimental animals were kept in SPF facilities.

[0057] The present invention will be further described below in conjunction with embodiments:

[0058] Example 1. GPR34 is highly expressed in ILC1s cells.

[0059] 1. Culture of mouse tumor cell line MC38: Mouse tumor cell line MC38 was cultured in DMEM medium + 10% FBS and placed in an incubator at 37°C and 5% CO2 for passage and growth. Cells in logarithmic growth phase were washed with PBS and digested with 0.25% trypsin. Cells were collected with PBS to prepare cell suspension and injected subcutaneously into each mouse. The number of cells was MC38 (1×10 6 / Only).

[0060] 2. Establishment of tumor model: Select 8-week-old female mice, and after general anesthesia with ether, shave the hair centered on the right posterior armpit of the mouse, wipe and disinfect with alcohol cotton balls, lift the central skin with sharp tweezers in the left hand, and slowly inject 100μl of MC38 tumor cell suspension 0.3 cm below the lifted skin with a 31-gauge needle syringe in the right hand. When a subcutaneous balloon-like bulge is seen, relax the sharp tweezers and remove the needle, and gently press the puncture site with a sterile cotton ball for 10 seconds. During the whole process of establishing the model, awakening should be prevented, and leakage of tumor cell suspension at the injection site should also be prevented.

[0061] 3. Isolation of liver lymphocytes: Cut the liver into small pieces (sesame size), then transfer to a 50ml centrifuge tube filled with digestion solution (0.5mg / ml IV collagenase + 2% FBS + 0.005% DNase I + 10ml RPMI 1640 culture medium), place in a 37°C shaker, and digest at 250rpm for 45min; Use a vortex to shake and mix the digested liver tissue, then add 20ml 1×PBS to stop, filter through a 200-mesh steel mesh after mixing (no grinding is required), and centrifuge (2200rpm, 10min); Carefully aspirate the supernatant, add 5ml 40% Percoll solution to resuspend and transfer to a centrifuge tube, and centrifuge at 2300rpm for 20min according to the procedure of 4 up and 1 down; Carefully aspirate the supernatant with a vacuum pump, add 1ml 1×PBS (containing 0.2% FBS) to resuspend and filter into a centrifuge tube, and place on ice to prepare for the next experiment.

[0062] 4. Isolation of spleen lymphocytes: Grind the spleen with 1×PBS containing 0.2% FBS, and then filter it with a 200-mesh steel mesh; centrifuge the filtrate (2000rpm, time is 10min); remove the supernatant, add red blood cell lysis buffer to blow the cells apart, terminate the reaction after 5min and centrifuge; remove the supernatant, add 1ml 1×PBS (containing 0.2% FBS) to resuspend the cells, and place on ice in preparation for subsequent experiments.

[0063] 5. Isolation of tumor lymphocytes: Cut the tumor into small pieces (sesame-sized), then transfer to a 50ml centrifuge tube containing digestion solution (1mg / ml IV collagenase + 2% FBS + 0.005% DNase I + 5ml RPMI 1640 culture medium), place in a 37°C shaker, and digest at 250rpm for 30min; Use a vortex to shake and mix the digested tumor tissue, then add 20ml 1×PBS to stop, filter through a 200-mesh steel mesh after mixing (no grinding is required), and centrifuge (2200rpm, 10min); Carefully aspirate the supernatant, add 5ml 40% Percoll solution to resuspend and transfer to a centrifuge tube, and centrifuge at 2300rpm for 20min according to the procedure of 4 up and 1 down; Carefully aspirate the supernatant with a vacuum pump, add 1ml 1×PBS (containing 0.2% FBS), resuspend and filter into a centrifuge tube, and place on ice to prepare for the next experiment.

[0064] 6. Collect lymphocytes from different organs, label them with relevant antibodies, and use flow cytometry to detect the expression of GPR34 protein: Use rat serum to block the isolated lymphocytes, and perform antibody staining after 30 minutes; add antibody labeling buffer (30μl / sample), mix well, and label at 4℃ in the dark; add 1ml 1×PBS to wash away unlabeled antibodies, and centrifuge at 3500rpm for 5min; aspirate the supernatant, add 1×PBS to resuspend the cells, and detect on the flow cytometer.

[0065] Figure 1 The results of experiment a in Figure 1 showed that flow cytometry was used to detect the expression of GPR34 on type 1 innate-like lymphocytes and other immune cells in the liver and spleen. The results showed that GPR34 was highly expressed on type 1 innate-like lymphocytes, but less expressed on B cells, CD8+T cells, CD4+T cells, dendritic cells, macrophages, monocytes and neutrophils.

[0066] Figure 1 The results of experiment b in Figure 2 showed that flow cytometry was used to detect the expression of GPR34 on lymphocytes infiltrating the tumor, and it was found that GPR34 was highly expressed on type 1 innate-like lymphocytes, but not on B cells, CD8+T cells, CD4+T cells, dendritic cells, macrophages, monocytes and neutrophils.

[0067] The above experimental results show that GPR34 is highly expressed on type 1 innate-like lymphocytes.

[0068] Example 2. Systemic Gpr34 knockout mice significantly slowed tumor growth in a tumor model

[0069] 1. Culture of mouse tumor cell line LLC / MC38 / B16F10: Culture mouse tumor cell line LLC / MC38 / B16F10 with DMEM medium + 10% FBS, and place in an incubator at 37°C and 5% CO2 for subculture. Cells in logarithmic growth phase were washed with PBS and digested with 0.25% trypsin, and cells were collected with PBS to prepare cell suspension, which was subcutaneously injected into each mouse. The number of cells was LLC (1×10 6 / pcs), MC38(1×10 6 / pcs) and B16F10(5×10 5 / Only).

[0070] 2. Establishment of tumor model: 8-week-old female C57BL / 6J mice were selected and anesthetized with ether. The hair was shaved from the right posterior axilla of the mice. After disinfection with alcohol cotton balls, the central skin was lifted with sharp forceps in the left hand. 100 μl of LLC, MC38 and B16F10 tumor cell suspension was slowly injected into the area 0.3 cm below the lifted skin with a 31-gauge needle syringe in the right hand. When a subcutaneous balloon-like bulge was seen, the sharp forceps were relaxed and the needle was removed. The puncture site was gently pressed for 10 seconds with a sterile cotton ball. Awakening should be prevented throughout the establishment of the model, and leakage of tumor cell suspension at the injection site should also be prevented.

[0071] 1. Systemic knockout of Gpr34 in mice significantly slowed tumor growth in the MC38 tumor model

[0072] 1. Day 0: Prepare 8-week-old Gpr34 mice matched for age and sex + / + and Gpr34 - / - Mouse, in which Gpr34 + / + The mice were control mice from the same parent. After general anesthesia with ether, the hair was shaved from the right posterior axilla of the mouse, and after disinfection with alcohol cotton balls, the central skin was lifted with sharp forceps in the left hand, and MC38 (1×10 6 / ) 100μl of tumor cell suspension was injected into the rat. When a subcutaneous balloon-like bulge was observed, the forceps were loosened and the needle was removed. The puncture site was gently pressed for 10 seconds with a sterile cotton ball. The rat should be prevented from waking up during the whole process of model establishment, and leakage of tumor cell suspension at the injection site should also be prevented.

[0073] 2. On the 7th day, use a vernier caliper to measure the length and width of the mouse tumor, and measure once every 7 days.

[0074] 3. On day 35, the mice were sacrificed, the tumors were removed, the tumor weight was measured, and the tumor cells were isolated. Flow cytometry was used to detect the changes in lymphocytes.

[0075] Figure 2The results in a showed that in the MC38 tumor model, the growth rate of tumors in mice with systemic Gpr34 knockout was significantly slowed compared with that in wild-type mice.

[0076] Figure 2 The results in b showed that in the MC38 tumor model, the tumor weight of mice with systemic Gpr34 knockout was significantly reduced compared with that of wild-type mice.

[0077] 2. Systemic Gpr34 knockout mice significantly slowed tumor growth in LLC tumor model

[0078] 1. Day 0: Prepare 8-week-old Gpr34 mice matched for age and sex + / + and Gpr34 - / - Mouse, in which Gpr34 + / + The mice were control mice from the same parent. After general anesthesia with ether, the hair was shaved from the right posterior axilla of the mouse, and after disinfection with alcohol cotton balls, the central skin was lifted with sharp forceps in the left hand, and LLC (1×10 6 / ) 100μl of tumor cell suspension was injected into the rat. When a subcutaneous balloon-like bulge was observed, the forceps were loosened and the needle was removed. The puncture site was gently pressed for 10 seconds with a sterile cotton ball. The rat should be prevented from waking up during the whole process of model establishment, and leakage of tumor cell suspension at the injection site should also be prevented.

[0079] 2. On the 8th day, use a vernier caliper to measure the length and width of the mouse tumor, and measure once every 7 days.

[0080] 3. On day 27, the mice were sacrificed, the tumors were removed, the tumor weight was measured, and the tumor cells were isolated. Flow cytometry was used to detect the changes in lymphocytes.

[0081] Figure 2 The results in c showed that in the LLC tumor model, the growth rate of tumors in mice with systemic Gpr34 knockout was significantly slowed compared with that in wild-type mice.

[0082] Figure 2 The results in (d) showed that in the LLC tumor model, the tumor weight of mice with systemic Gpr34 knockout was significantly reduced compared with that of wild-type mice.

[0083] 3. Systemic Gpr34 knockout mice significantly slowed tumor growth in the B16F10 tumor model

[0084] 1. Day 0: Prepare 8-week-old Gpr34 mice matched for age and sex + / + and Gpr34 - / - Mouse, in which Gpr34 + / +The mice were control mice from the same parent. After general anesthesia with ether, the hair was shaved from the right posterior axilla of the mouse, and after disinfection with alcohol cotton balls, the central skin was lifted with sharp forceps in the left hand, and B16F10 (5×10 5 / ) 100μl of tumor cell suspension was injected into the rat. When a subcutaneous balloon-like bulge was observed, the forceps were loosened and the needle was removed. The puncture site was gently pressed for 10 seconds with a sterile cotton ball. The rat should be prevented from waking up during the whole process of model establishment, and leakage of tumor cell suspension at the injection site should also be prevented.

[0085] 2. On the 6th day, use a vernier caliper to measure the length and width of the mouse tumor, and measure once every 7 days.

[0086] 3. On day 16, the mice were sacrificed, the tumors were removed, the tumor weight was measured, and the tumor cells were isolated. Flow cytometry was used to detect the changes in lymphocytes.

[0087] Figure 2 The results in e showed that in the B16F10 tumor model, the growth rate of tumors in mice with systemic Gpr34 knockout was significantly slowed compared with that in wild-type mice.

[0088] Figure 2 The results in f showed that in the B16F10 tumor model, the tumor weight of mice with systemic Gpr34 knockout was significantly reduced compared with that of wild-type mice.

[0089] The above experimental results show that systemic knockout of Gpr34 can effectively alleviate tumor growth.

[0090] Example 3. NCR1-specific knockout of Gpr34 mice significantly slowed down tumor growth in a tumor model

[0091] 1. NCR1-specific knockout of Gpr34 mice significantly slowed tumor growth in the MC38 tumor model

[0092] 1. Day 0: Prepare 8-week-old Gpr34 mice matched for age and sex fl / fl and Gpr34 fl / fl Ncr1 cre Mouse, in which Gpr34 fl / fl The mice were control mice from the same parent. After general anesthesia with ether, the hair was shaved from the right posterior axilla of the mouse, and after disinfection with alcohol cotton balls, the central skin was lifted with sharp forceps in the left hand, and MC38 (1×10 6 / ) 100μl of tumor cell suspension was injected into the rat. When a subcutaneous balloon-like bulge was observed, the forceps were loosened and the needle was removed. The puncture site was gently pressed for 10 seconds with a sterile cotton ball. The rat should be prevented from waking up during the whole process of model establishment, and leakage of tumor cell suspension at the injection site should also be prevented.

[0093] 2. On the 7th day, use a vernier caliper to measure the length and width of the mouse tumor, and measure once every 5 days.

[0094] 3. On day 32, the mice were sacrificed, the tumors were removed, the tumor weight was measured, and the tumor cells were isolated. Flow cytometry was used to detect the changes in lymphocytes.

[0095] Figure 3 The results in a showed that in the MC38 tumor model, the tumor growth rate in NCR1-specific Gpr34 knockout mice was significantly slowed compared with that in wild-type mice.

[0096] Figure 3 The results in b showed that in the MC38 tumor model, the tumor weight of NCR1-specific Gpr34 knockout mice was significantly reduced compared with that of wild-type mice.

[0097] Figure 3 The results in g showed that in the MC38 tumor model, compared with wild-type mice, the proportion and number of infiltrating ILC1s in tumors of NCR1-specific Gpr34 knockout mice were significantly increased and their proliferation ability was enhanced.

[0098] Figure 3 The results in h showed that in the MC38 tumor model, the effector function of ILC1s infiltrating in the tumors of NCR1-specific Gpr34 knockout mice was significantly enhanced compared with that of wild-type mice.

[0099] 2. NCR1-specific knockout of Gpr34 mice significantly slowed tumor growth in the B16F10 tumor model

[0100] 1. Day 0: Prepare 8-week-old Gpr34 mice matched for age and sex fl / fl and Gpr34 fl / fl Ncr1 cre Mouse, in which Gpr34 fl / fl The mice were control mice from the same parent. After general anesthesia with ether, the hair was shaved from the right posterior axilla of the mouse, and after disinfection with alcohol cotton balls, the central skin was lifted with sharp forceps in the left hand, and B16F10 (5×10 5 / ) 100μl of tumor cell suspension was injected into the rat. When a subcutaneous balloon-like bulge was observed, the forceps were loosened and the needle was removed. The puncture site was gently pressed for 10 seconds with a sterile cotton ball. The rat should be prevented from waking up during the whole process of model establishment, and leakage of tumor cell suspension at the injection site should also be prevented.

[0101] 2. On the 7th day, use a vernier caliper to measure the length and width of the mouse tumor, and measure once every 5 days.

[0102] 3. On day 17, sacrifice the mice, remove the tumors, and weigh the tumors.

[0103] Figure 3 The results in c showed that in the B16F10 tumor model, the tumor growth rate in NCR1-specific Gpr34 knockout mice was significantly slowed compared with that in wild-type mice.

[0104] Figure 3 The results in (d) showed that in the B16F10 tumor model, the tumor weight of NCR1-specific Gpr34 knockout mice was significantly reduced compared with that of wild-type mice.

[0105] 3. NCR1-specific knockout of Gpr34 mice significantly slowed tumor growth in LLC tumor model

[0106] 1. Day 0: Prepare 8-week-old Gpr34 mice matched for age and sex fl / fl and Gpr34 fl / fl Ncr1 cre Mouse, in which Gpr34 fl / fl The mice were control mice from the same parent. After general anesthesia with ether, the hair was shaved from the right posterior axilla of the mouse, and after disinfection with alcohol cotton balls, the central skin was lifted with sharp forceps in the left hand, and LLC (1×10 6 / ) 100μl of tumor cell suspension was injected into the rat. When a subcutaneous balloon-like bulge was observed, the forceps were loosened and the needle was removed. The puncture site was gently pressed for 10 seconds with a sterile cotton ball. The rat should be prevented from waking up during the whole process of model establishment, and leakage of tumor cell suspension at the injection site should also be prevented.

[0107] 2. On the 7th day, use a vernier caliper to measure the length and width of the mouse tumor, and measure once every 5 days.

[0108] 3. On day 22, sacrifice the mice, remove the tumors, and weigh the tumors.

[0109] Figure 3 The results in e showed that in the LLC tumor model, the tumor growth rate in NCR1-specific Gpr34 knockout mice was significantly slowed compared with that in wild-type mice.

[0110] Figure 3 The results in f showed that in the LLC tumor model, the tumor weight of NCR1-specific Gpr34 knockout mice was significantly reduced compared with that of wild-type mice.

[0111] The above experimental results show that NCR1-specific knockout of Gpr34 can effectively alleviate tumor growth.

[0112] Example 4. Tumor-derived LysoPS inhibits the anti-tumor activity of ILC1 via GPR34

[0113] 1. The concentration of LysoPS in tumor interstitial fluid is significantly higher than that in serum

[0114] 1. On day 0, 8-week-old C56BL / 6J mice matched in age and sex were prepared. After general anesthesia with ether, the hair was shaved from the right posterior axilla of the mice. After disinfection with alcohol cotton balls, the central skin was lifted with sharp forceps in the left hand, and MC38 (1×10 6 / pcs), B16F10(5×10 5 / ), LLC(1×10 6 / ) 100μl of tumor cell suspension was injected into the rat. When a subcutaneous balloon-like bulge was observed, the forceps were loosened and the needle was removed. The puncture site was gently pressed for 10 seconds with a sterile cotton ball. The rat should be prevented from waking up during the whole process of model establishment, and leakage of tumor cell suspension at the injection site should also be prevented.

[0115] 2. When the tumor volume grows to 1000 cubic millimeters, sacrifice the mice, collect the serum and tumor interstitial fluid of the tumor-bearing mice, and use mass spectrometry to detect the concentration of LysoPS.

[0116] Figure 4 The results in a showed that in different tumor models, the concentration of LysoPS in tumor interstitial fluid was significantly increased compared with serum.

[0117] 2. LysoPS inhibits ILC1 activity in a GPR34-dependent manner

[0118] 1. Isolation of liver lymphocytes: Cut the liver into small pieces (sesame-sized), then transfer to a 50ml centrifuge tube containing digestion solution (0.5mg / ml IV collagenase + 2% FBS + 0.005% DNase I + 10ml RPMI 1640 culture medium), place in a 37°C shaker, and digest at 250rpm for 45min; Use a vortex to shake and mix the digested liver tissue, then add 20ml 1×PBS to terminate, filter through a 200-mesh steel mesh after mixing (no grinding is required), and centrifuge (2200rpm, 10min); Carefully aspirate the supernatant, add 5ml 40% Percoll solution to resuspend, and transfer to a centrifuge tube, according to the procedure of 4 up and 1 down, centrifuge at 2300rpm for 20min; Carefully aspirate the supernatant with a vacuum pump, add 1ml 1×PBS (containing 0.2% FBS) to resuspend and filter into a centrifuge tube, and place on ice to prepare for the next experiment.

[0119] 2. Flow Cytometry Sorting of Gpr34 + / + and Gpr34 - / - Mouse liver ILC1s cells were treated with LysoPS (5 μM) for 1 hour in advance and then stimulated with IL-12 (20 ng / ml), IL-15 (50 ng / ml) and IL-18 (100 ng / ml) for 12 hours before the cells were collected.

[0120] 3. Three hours before collecting cells, add a Golgi protein transport inhibitor, then collect and wash the cells, and stain the cell surface molecules and intracellular IFN-γ, Granzyme C, Granzyme B and Perforin. Flow cytometry was used to detect the percentages of IFN-γ, GzmC, GzmB and Perforin in ILC1s cells.

[0121] Figure 4 The results in b show that compared with the control group, interleukin 12 / 15 / 18 can significantly induce ILC1s cytokine production, while 5 micromolar concentration of LysoPS can inhibit ILC1s cytokine production and this process is dependent on GPR34. 2. Inhibiting LysoPS production can effectively alleviate tumor growth

[0122] 1. CRISPR-Cas9 mediated knockout of Abhd16a gene in tumor cells. The sgRNA sequence targeting mouse Abhd16a was designed using the CRISPR online design tool. The sgRNA sequence was cloned into the lentiCRISPR v2 vector digested with Esp3I (NEB), and then transfected into HEK293T cells to generate sgRNA-encoding CRISPR lentivirus. The packaged lentivirus was then used to infect LLC / MC38 / B16F10 tumor cells, which were then cultured and grown in an incubator at 37°C and 5% CO2. Polyclonal cells were screened with puromycin (2.5 μg / ml for B16F10 cells, 5 μg / ml for MC38 cells, and 2.5 μg / ml for LLC cells) for 1 week, and the absence of ABHD16a protein was analyzed by Western blotting.

[0123] 2. Establishment of tumor model: 8-week-old female C57BL / 6J mice were selected and anesthetized with ether. The hair was shaved from the right posterior axilla of the mice. After disinfection with alcohol cotton balls, the central skin was lifted with sharp forceps in the left hand. The right hand used a 31-gauge needle syringe to slowly inject 100 μl of the control group and LLC, MC38 and B16F10 tumor cell line suspensions with knockdown of Abhd16A gene expression into the 0.3 cm below the lifted skin. When a subcutaneous balloon-like bulge was seen, the sharp forceps were relaxed and the needle was removed. The puncture site was gently pressed for 10 seconds with a sterile cotton ball. Awakening should be prevented throughout the establishment of the model, and leakage of the tumor cell suspension at the injection site should also be prevented.

[0124] 3. On day 5 (MC38) / 7 (B16F10) / 9 (LLC), use a vernier caliper to measure the length and width of the mouse tumors every 4-5 days.

[0125] 4. When the tumor size reaches about 1000 cubic millimeters, sacrifice the mouse, then remove the mouse tumor, weigh the tumor, separate the tumor cells, and use flow cytometry to detect changes in lymphocytes.

[0126] Figure 4 The results in c showed that in the MC38 tumor model, the tumor growth rate of mice in the Abhd16a gene expression knockdown group was significantly slowed down compared with the control mice.

[0127] Figure 4 The results in (d) showed that compared with the control mice, the tumor weight of mice in the Abhd16a gene expression knockdown group was significantly reduced.

[0128] Figure 4 The results in e showed that compared with control mice, the effector function of ILC1s infiltrating in the tumors of mice in the Abhd16a gene expression knockdown group was significantly enhanced.

[0129] Figure 4 The results in f showed that in the B16F10 tumor model, the tumor growth rate of mice in the Abhd16a gene expression knockdown group was significantly slowed down compared with the control mice.

[0130] Figure 4 The results in g showed that compared with the control mice, the tumor weight of mice in the Abhd16a gene expression knockdown group was significantly reduced.

[0131] Figure 4 The results in the figure showed that in the LLC tumor model, the tumor growth rate of mice in the Abhd16a gene expression knockdown group was significantly slowed down compared with the control mice.

[0132] Figure 4 The results in Fig. i showed that compared with the control mice, the tumor weight of mice in the Abhd16a gene expression knockdown group was significantly reduced.

[0133] The above experimental results show that tumor-derived LysoPS inhibits the anti-tumor activity of ILC1 through GPR34. Example 5. GPR34 inhibitors can effectively alleviate tumor growth

[0134] 1. GPR34 inhibitors can effectively alleviate the growth of MC38 tumors

[0135] 1. On day 0, 8-week-old C56BL / 6J mice matched in age and sex were prepared. After general anesthesia with ether, the hair was shaved from the right posterior axilla of the mice. After disinfection with alcohol cotton balls, the central skin was lifted with sharp forceps in the left hand, and MC38 (1×10 6 / ) 100μl of tumor cell suspension was injected into the rat. When a subcutaneous balloon-like bulge was observed, the forceps were loosened and the needle was removed. The puncture site was gently pressed for 10 seconds with a sterile cotton ball. The rat should be prevented from waking up during the whole process of model establishment, and leakage of tumor cell suspension at the injection site should also be prevented.

[0136] 2. On the 5th day, mice with tumors of the same size were selected and randomly divided into four groups, with 8 mice in each group. The specific grouping treatments were as follows:

[0137] Group 1: Starting from the 5th day, 100 μl of control reagent was intraperitoneally injected every day until the end of the experiment, i.e. the control group.

[0138] Group 2: Starting from the 5th day, 100 μl of GPR34 inhibitor (injection volume of 10 mg / kg) was intraperitoneally injected every day until the end of the experiment.

[0139] Group 3: Starting from the 5th day, 100 μl of GPR34 inhibitor (injection volume of 25 mg / kg) was intraperitoneally injected every day until the end of the experiment.

[0140] Group 4: Starting from the 5th day, 100 μl of GPR34 inhibitor (injection volume of 50 mg / kg) was intraperitoneally injected every day until the end of the experiment.

[0141] 3. On day 30, sacrifice the mice, remove the tumors, and weigh the tumors.

[0142] Figure 5 The results in a showed that in the MC38 tumor model, the growth rate of tumors in the inhibitor-treated mice was significantly slowed compared with that in the control mice.

[0143] Figure 5 The results in b showed that in the MC38 tumor model, the tumor weight of mice in the inhibitor treatment group was significantly reduced compared with that of mice in the control group.

[0144] Figure 5 The results in g showed that in the MC38 tumor model, compared with the control group mice, the proportion and number of ILC1s infiltrating in the tumors of the inhibitor-treated group mice increased significantly, and their proliferation ability was enhanced.

[0145] Figure 5 The results in h showed that in the MC38 tumor model, the effector function of ILC1s infiltrating in the tumors of mice in the inhibitor treatment group was significantly enhanced compared with that of mice in the control group.

[0146] 2. GPR34 inhibitors can effectively alleviate the growth of B16F10 tumors

[0147] 1. On day 0, 8-week-old C56BL / 6J mice matched in age and sex were prepared. After general anesthesia with ether, the hair was shaved from the right posterior axilla of the mice. After disinfection with alcohol cotton balls, the central skin was lifted with sharp forceps in the left hand, and B16F10 (5×10 5 / ) 100μl of tumor cell suspension was injected into the rat. When a subcutaneous balloon-like bulge was observed, the forceps were loosened and the needle was removed. The puncture site was gently pressed for 10 seconds with a sterile cotton ball. The rat should be prevented from waking up during the whole process of model establishment, and leakage of tumor cell suspension at the injection site should also be prevented.

[0148] 2. On the 7th day, mice with tumors of the same size were selected and randomly divided into two groups, with 9 mice in each group. The specific grouping treatments were as follows:

[0149] Group 1: Starting from the 7th day, 100 μl of control reagent was intraperitoneally injected every day until the end of the experiment, i.e. the control group.

[0150] Group 2: Starting from the 7th day, 100 μl of GPR34 inhibitor (injection volume of 25 mg / kg) was intraperitoneally injected every day until the end of the experiment, ie, the inhibitor-treated group.

[0151] 3. On day 17, sacrifice the mice, remove the tumors, and weigh the tumors.

[0152] Figure 5 The results in c showed that in the B16F10 tumor model, the growth rate of tumors in the inhibitor-treated mice was significantly slowed compared with that in the control mice.

[0153] Figure 5 The results in (d) showed that in the B16F10 tumor model, the tumor weight of mice in the inhibitor treatment group was significantly reduced compared with that of mice in the control group.

[0154] 3. GPR34 inhibitors can effectively alleviate LLC tumor growth

[0155] 1. On day 0, 8-week-old C56BL / 6J mice matched in age and sex were prepared. After general anesthesia with ether, the hair was shaved from the right posterior axilla of the mice. After disinfection with alcohol cotton balls, the central skin was lifted with sharp forceps in the left hand, and LLC (1×10 6 / ) 100μl of tumor cell suspension was injected into the rat. When a subcutaneous balloon-like bulge was observed, the forceps were loosened and the needle was removed. The puncture site was gently pressed for 10 seconds with a sterile cotton ball. The rat should be prevented from waking up during the whole process of model establishment, and leakage of tumor cell suspension at the injection site should also be prevented.

[0156] 2. On the 6th day, mice with tumors of the same size were selected and randomly divided into two groups, with 6 mice in each group. The specific grouping treatments were as follows:

[0157] Group 1: Starting from the 6th day, 100 μl of control reagent was intraperitoneally injected every day until the end of the experiment, i.e. the control group.

[0158] Group 2: Starting from the 6th day, 100 μl of GPR34 inhibitor (injection volume of 25 mg / kg) was intraperitoneally injected every day until the end of the experiment, ie, the inhibitor-treated group.

[0159] 3. On day 31, sacrifice the mice, remove the tumors, and weigh the tumors.

[0160] Figure 5 The results in Figure 4 showed that in the LLC tumor model, the growth rate of tumors in the inhibitor-treated mice was significantly slowed compared with that in the control mice.

[0161] Figure 5The results in f showed that in the LLC tumor model, the tumor weight of mice in the inhibitor treatment group was significantly reduced compared with that of mice in the control group.

[0162] The above experimental results show that GPR34 inhibitors can effectively alleviate tumor growth.

[0163] Example 6. GPR34 inhibitor combined with TIGIT antibody can effectively alleviate tumor growth

[0164] 1. GPR34 inhibitor combined with TIGIT antibody can effectively alleviate the growth of B16F10 tumors

[0165] 1. On day 0, 8-week-old C56BL / 6J mice matched in age and sex were prepared. After general anesthesia with ether, the hair was shaved from the right posterior axilla of the mice. After disinfection with alcohol cotton balls, the central skin was lifted with sharp forceps in the left hand, and B16F10 (5×10 5 / ) 100μl of tumor cell suspension was injected into the rat. When a subcutaneous balloon-like bulge was observed, the forceps were loosened and the needle was removed. The puncture site was gently pressed for 10 seconds with a sterile cotton ball. The rat should be prevented from waking up during the whole process of model establishment, and leakage of tumor cell suspension at the injection site should also be prevented.

[0166] 2. On the 4th day, mice with tumors of the same size were selected and randomly divided into four groups, with 7 mice in each group. The specific grouping treatments were as follows:

[0167] Group 1: Starting from the third day, 100 μl of control reagent was intraperitoneally injected every day until the end of the experiment, i.e. the control group.

[0168] Group 2: Starting from the 4th day, 100 μl of TIGIT antibody (injection volume 200 μg) was intraperitoneally injected every day, once every 3 days, for a total of 4 injections, namely the TIGIT antibody treatment group.

[0169] Group 3: Starting from the 3rd day, 100 μl of GPR34 inhibitor (injection volume 25 mg / kg) was intraperitoneally injected every day until the end of the experiment, ie, the inhibitor-treated group.

[0170] Group 4: Starting from the 3rd day, 100 μl of GPR34 inhibitor (injection volume of 25 mg / kg) was intraperitoneally injected every day until the end of the experiment. In addition, 100 μl of TIGIT antibody (injection volume of 200 μg) was intraperitoneally injected starting from the 4th day, once every 3 days, for a total of 4 injections, i.e. the combined treatment group.

[0171] Figure 6The results in a showed that in the B16F10 tumor model, compared with the control group mice, treatment with the inhibitor and TIGIT antibody alone effectively inhibited tumor growth, and the effect of the inhibitor combined with TIGIT antibody treatment was more significant, and the tumor growth rate was further slowed down.

[0172] 2. GPR34 inhibitor combined with TIGIT antibody can effectively alleviate the growth of MC38 tumors

[0173] 1. On day 0, 8-week-old C56BL / 6J mice matched in age and sex were prepared. After general anesthesia with ether, the hair was shaved from the right posterior axilla of the mice. After disinfection with alcohol cotton balls, the central skin was lifted with sharp forceps in the left hand, and MC38 (1×10 6 / ) 100μl of tumor cell suspension was injected into the rat. When a subcutaneous balloon-like bulge was observed, the forceps were loosened and the needle was removed. The puncture site was gently pressed for 10 seconds with a sterile cotton ball. The rat should be prevented from waking up during the whole process of model establishment, and leakage of tumor cell suspension at the injection site should also be prevented.

[0174] 2. On the 5th day, mice with tumors of the same size were selected and randomly divided into four groups, with 8 mice in each group. The specific grouping treatments were as follows:

[0175] Group 1: Starting from the 5th day, 100 μl of control reagent was intraperitoneally injected every day until the end of the experiment, i.e. the control group.

[0176] Group 2: Starting from the 5th day, 100 μl of TIGIT antibody (injection volume 200 μg) was intraperitoneally injected every day, once every 4 days, for a total of 5 injections, namely the TIGIT antibody treatment group.

[0177] Group 3: Starting from the 3rd day, 100 μl of GPR34 inhibitor (injection volume 25 mg / kg) was intraperitoneally injected every day until the end of the experiment, ie, the inhibitor-treated group.

[0178] Group 4: Starting from the 3rd day, 100 μl of GPR34 inhibitor (injection volume of 25 mg / kg) was intraperitoneally injected every day until the end of the experiment. In addition, 100 μl of TIGIT antibody (injection volume of 200 μg) was intraperitoneally injected starting from the 5th day, once every 4 days, for a total of 5 injections, i.e. the combined treatment group.

[0179] Figure 6 The results in b show that in the MC38 tumor model, compared with the control group mice, the growth rate of the tumor did not change when treated with TIGIT antibody alone, the inhibitor alone effectively slowed the growth of the tumor, and the inhibitor combined with TIGIT antibody treatment could better inhibit the tumor growth rate.

[0180] Figure 6 The results in c showed that in the MC38 tumor model, compared with the control group mice, treatment with TIGIT antibody alone did not affect the survival time of mice, treatment with inhibitor alone could prolong the survival time of mice, and treatment with inhibitor combined with TIGIT antibody could further prolong the survival time of mice.

[0181] 3. GPR34 inhibitors combined with TIGIT antibodies can effectively alleviate LLC tumor growth

[0182] 1. On day 0, 8-week-old C56BL / 6J mice matched in age and sex were prepared. After general anesthesia with ether, the hair was shaved from the right posterior axilla of the mice. After disinfection with alcohol cotton balls, the central skin was lifted with sharp forceps in the left hand, and LLC (1×10 6 / ) 100μl of tumor cell suspension was injected into the rat. When a subcutaneous balloon-like bulge was observed, the forceps were loosened and the needle was removed. The puncture site was gently pressed for 10 seconds with a sterile cotton ball. The rat should be prevented from waking up during the whole process of model establishment, and leakage of tumor cell suspension at the injection site should also be prevented.

[0183] 2. On the third day, mice with tumors of the same size were selected and randomly divided into four groups, with 9 mice in each group. The specific grouping treatments were as follows:

[0184] Group 1: Starting from the third day, 100 μl of control reagent was intraperitoneally injected every day until the end of the experiment, i.e. the control group.

[0185] Group 2: Starting from the 6th day, 100 μl of TIGIT antibody (injection volume 200 μg) was injected intraperitoneally once every 4 days for a total of 5 injections, namely the TIGIT antibody treatment group.

[0186] Group 3: Starting from the 3rd day, 100 μl of GPR34 inhibitor (injection volume 25 mg / kg) was intraperitoneally injected every day until the end of the experiment, ie, the inhibitor-treated group.

[0187] Group 4: Starting from the 3rd day, 100 μl of GPR34 inhibitor (injection volume 25 mg / kg) was intraperitoneally injected every day until the end of the experiment. In addition, 100 μl of TIGIT antibody (injection volume 200 μg) was intraperitoneally injected starting from the 6th day, once every 4 days, for a total of 5 injections, i.e. the combined treatment group.

[0188] Figure 6The results in d showed that in the LLC tumor model, compared with the control group mice, treatment with TIGIT antibody alone slightly slowed the tumor growth rate, treatment with inhibitor alone effectively slowed tumor growth, and treatment with inhibitor combined with TIGIT antibody could better inhibit tumor growth rate.

[0189] Figure 6 The results in the e showed that in the LLC tumor model, compared with the control group mice, TIGIT antibody treatment alone did not affect the survival time of mice, inhibitor treatment alone could prolong the survival time of mice, and inhibitor combined with TIGIT antibody treatment could further prolong the survival time and survival rate of mice.

[0190] The above experimental results show that GPR34 inhibitors combined with TIGIT antibodies can effectively alleviate tumor growth.

[0191] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of substances targeting GPR34 in the preparation of drugs for preventing and / or treating diseases related to type 1 innate-like lymphocytes.

2. The use according to claim 1, characterized in that: The substance targeting GPR34 includes at least one of I to VI: 1), an expression vector for knocking out or knocking down the Gpr34 gene; II), a host cell or a viral vector containing I); III), a nucleic acid molecule that interferes with the transcription of the Gpr34 gene; IV), terminator or transposon of Gpr34 gene expression; V), agents that inhibit the expression of the Gpr34 gene; VI), inhibitors and / or antagonists of GPR34 protein function.

3. The use according to claim 2, characterized in that: The nucleic acid molecule interfering with the transcription of the Gpr34 gene includes: an RNAi fragment targeting the Gpr34 gene, an siRNA fragment targeting the Gpr34 gene, or a CRISPR gRNA that promotes the degradation of the Gpr34 messenger RNA; The inhibitors and / or antagonists of GPR34 protein function include: GPR34 neutralizing antibodies, chemical small molecule inhibitors of GPR34 and / or polypeptide inhibitors of GPR34, preferably tyrosine, N-[((2E)-3-(4'-chloro[1,1'-biphenyl]-4-yl)-1-oxo-2-propen-1-yl]-O-(benzyl) or their structural analogs.

4. The use according to any one of claims 1 to 3, characterized in that: The related diseases in which the type 1 innate-like lymphocytes are involved include: at least one of tumors, obesity, depression, diabetes, cardiovascular and cerebrovascular diseases, enteritis, blood diseases, gout, arthritis, Alzheimer's disease, asthma, autoimmune diseases, and infectious diseases induced by bacteria, fungi and viruses.

5. The use according to claim 4, characterized in that: The tumors include: solid tumors and / or non-solid tumors; the solid tumors include: cervical cancer, ovarian cancer, endometrial cancer, vulvar cancer, prostate cancer, parotid cancer, thyroid cancer, salivary gland cancer, colon cancer, lung cancer, fibrosarcoma, skin cancer, nasopharyngeal cancer, tongue cancer, oral cancer, laryngeal cancer, brain tumor, gallbladder cancer, kidney cancer, gum cancer, testicular cancer, penile cancer, multiple myeloma and / or malignant melanoma; The non-solid tumors include: leukemia and / or malignant lymphoma.

6. The use according to claim 5, characterized in that: The prevention and / or treatment of the tumor includes: reducing tumor mass, reducing the number of metastatic foci, improving the effect of immunotherapy, inhibiting tumor growth and / or enhancing anti-tumor immunity.

7. The use according to claim 6, characterized in that: The enhancement of anti-tumor immunity includes: increasing the proportion and number of ILC1s, enhancing the cytotoxicity, proliferation ability and activity of ILC1s, reducing the number or proportion of myeloid-derived suppressor cells, and increasing the number or proportion or killing function of NK cells or T cell toxic lymphocytes in tumors.

8. A drug for treating type 1 innate-like lymphocyte-related diseases, characterized in that: including a substance targeting GPR34 and a pharmaceutically acceptable excipient; The substance targeting GPR34 includes at least one of I to VI: 1), an expression vector for knocking out or knocking down the Gpr34 gene; II), a host cell or a viral vector containing I); III), a nucleic acid molecule that interferes with the transcription of the Gpr34 gene; IV), terminator or transposon of Gpr34 gene expression; V), agents that inhibit the expression of the Gpr34 gene; VI), inhibitors and / or antagonists of GPR34 protein function.

9. The drug according to claim 8, characterized in that It also includes immunosuppressive molecule antibodies, small molecule compounds and nucleic acid inhibitors.

10. The drug according to claim 9, characterized in that The immunosuppressive molecule antibody is PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG3 antibody and / or TIGIT antibody; the mass ratio of the TIGIT antibody to the substance targeting GPR34 is (0.05-2): (1-10).

Images