Application of Vam6 (Vps39) as a target in drug screening, gene editing and disease treatment

Abstract

The present application discloses a screening method for tumor immunotherapy reagents targeting Vam6, which comprises administering a candidate reagent to iNKT cells, and treating the iNKT cells with and without the reagent with an iNKT cell activator, and measuring the expression level of Vam6 of the iNKT cells, wherein a decrease or increase in the expression level of Vam6 of the iNKT cells administered with the candidate reagent compared with the iNKT cells without the candidate reagent indicates that the candidate reagent is a potential tumor immunotherapy reagent targeting Vam6. The present application also discloses a method for screening reagents for enhancing the anti-tumor function of iNKT cells using the screening method, and a pharmaceutical composition for treating tumors, which comprises the screened candidate reagent, and a kit comprising the pharmaceutical composition.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology. Specifically, this invention relates to screening targeted drugs using Vam6 as a target, as well as these drugs, drugs for gene editing using Vam6 as a target, and the application of Vam6 gene-edited cells in the treatment of diseases. Background Technology

[0002] Current research has found that the development of many major diseases, including cancer, diabetes, autoimmune diseases, and infectious diseases, is related to the immune system, highlighting the immense potential of immunotherapy. The core of immunotherapy is to intervene in disease by correcting "malfunctions" or "out-of-control" states of the immune system. For example, immunotherapy for cancer, including PD-1 antibodies and CAR-T, CAR-NK, and CAR-NKT therapies, has become one of the most promising cancer treatment strategies. Its main purpose is to enhance the anti-tumor immune response in patients, thereby eliminating tumor cells. Therefore, developing new techniques for regulating immune cell function is crucial for developing novel immunotherapy strategies.

[0003] iNKT cells are a special type of immune cell discovered in recent years. Upon antigen stimulation, they respond rapidly, producing large amounts of Th1 (e.g., IFNγ) and Th2 (e.g., IL-4) cytokines, which in turn regulate downstream innate and adaptive immune cells, including dendritic cells (DCs), T cells, B cells, and NK cells. Therefore, iNKT cells are widely recognized as a bridge between innate and adaptive immunity. Their function is plastic, making them important immunomodulatory cells: promoting the Th1 response of iNKT cells can effectively promote the clearance of pathogenic microorganisms and inhibit tumor cell growth; inhibiting the Th1 response of iNKT cells can effectively suppress metabolic inflammation, type 2 diabetes, and autoimmune diseases (Bendelac et al., 2007).

[0004] In tumor immunotherapy, iNKT cells have demonstrated advantages such as fewer cytokine storm toxicities and lower graft-versus-host disease risk. Researchers have also found a positive correlation between clinical treatment outcomes and the number of iNKT cells producing IFN-γ in patients. In some unresponsive tumor patients, iNKT cell function was significantly suppressed. In mouse tumor models and clinical samples from liver cancer patients, we also found a significant reduction in IFN-γ production by iNKT cells in tumors. Restoring IFN-γ production in mouse tumor-bearing models significantly promoted the therapeutic effect of iNKT cells and inhibited tumor growth (Fu et al., 2020; Xie et al., 2016). Therefore, identifying targets that enhance the IFN-γ response of iNKT cells in tumors will be beneficial for developing new immunotherapy strategies. Summary of the Invention

[0005] The purpose of this application is to provide new gene editing sites and drug targets for regulating cell function and treating diseases.

[0006] This application discloses the use of Vam6 as a gene editing or drug target in regulating iNKT cell function and enhancing the efficacy of immunotherapy.

[0007] The inventors enhanced the iNKT cell-mediated anti-tumor effect by reducing the expression of Vam6 in iNKT cells, promoting iNKT cell proliferation and survival, and improving the IFN-γ response of iNKT cells, thereby effectively inhibiting the growth of tumors in mice.

[0008] Specifically, the inventors of this application have solved the technical problems in this field through the technical solutions described in the following items.

[0009] 1. A method for screening tumor immunotherapy reagents targeting Vam6, the method comprising the following steps:

[0010] Candidate reagents were administered to iNKT cells;

[0011] iNKT cells treated with candidate reagents and iNKT cells not treated with candidate reagents were treated with iNKT cell activators, such as CD1d-PBS57 tetramer.

[0012] The expression level of Vam6 protein in iNKT cells treated with and not treated with the candidate reagent was determined;

[0013] The decreased or increased expression level of Vam6 protein in iNKT cells treated with the candidate reagent compared to iNKT cells that had not been treated with the candidate reagent indicates that the candidate reagent is a potential immunotherapeutic agent targeting Vam6.

[0014] The iNKT cell activator described herein is capable of activating iNKT cells to promote the release of cytokines from iNKT cells, such as CD1d-PBS57 tetramer, α-galactosylceramide (α-GalCer, α-GC) and its analogues, ganglioside GD3, glycophosphatidylinositol, phosphatidylethanolamine, β-GalCer, iGb3 and its analogues 4-HO-iGb3 and 4-dh-iGb3, as well as other antigens that can be presented by CD1d, antigen-presenting cells carrying iNKT cell antigens, and chimeric receptors expressed on the surface of CAR-iNKT cells that recognize tumor antigens.

[0015] 2. The method described in Project 1, wherein the iNKT cells are unmodified wild-type iNKT cells or gene-edited iNKT cells, such as CAR-iNKT.

[0016] 3. The method of Project 1, wherein the expression level of Vam6 is determined by measuring the activity or protein amount of Vam6 or by measuring the level of transcripts of the Vam6 gene.

[0017] 4. A method for screening Vam6-targeting inhibitors to enhance the anti-tumor function of iNKT cells, comprising treating iNKT cells with a candidate reagent screened by the method described in any one of items 1-3, then using the iNKT cells for tumor treatment, or using iNKT cells for tumor treatment in the presence of the candidate reagent, and judging changes in the anti-tumor function of iNKT cells, wherein the improvement in the anti-tumor function of iNKT cells treated with the candidate reagent compared with iNKT cells not treated with the candidate reagent indicates that the candidate reagent is a potential reagent for enhancing the anti-tumor function of iNKT cells.

[0018] 5. The method described in Project 4, wherein the iNKT cells are unmodified wild-type iNKT cells or gene-edited iNKT cells, such as CAR-iNKT.

[0019] 6. Reagents screened using any one of the methods in items 1-3 or any one of the methods in items 4-5.

[0020] 7. Use of the reagents described in Project 6 in the preparation of drugs for treating tumors.

[0021] 8. The use of Vam6 as a target in methods for screening immunotherapy agents.

[0022] 9. A pharmaceutical composition for treating tumors, comprising the reagents described in item 6, an iNKT cell activator, and a pharmaceutically acceptable carrier, wherein the iNKT cell activator is capable of activating iNKT cells to promote the release of cytokines from iNKT cells, such as CD1d-PBS57 tetramer, α-galactosylceramide (α-GalCer, α-GC) and analogues thereof, ganglioside GD3, glycophosphatidylinositol, phosphatidylethanolamine, β-GalCer, iGb3 and analogues 4-HO-iGb3 and 4-dh-iGb3, and other antigens that can be presented by CD1d, antigen-presenting cells carrying iNKT cell antigens, and chimeric receptors expressing tumor antigens on the surface of CAR-iNKT cells.

[0023] 10. A kit for treating tumors comprising the pharmaceutical composition described in item 9, and wild-type iNKT cells or gene-edited iNKT cells, such as CAR-iNKT, optionally modified to reduce Vam6 expression levels, wherein the pharmaceutical composition and the modified iNKT cells are placed in separate containers.

[0024] 11. The use of Item 7, the pharmaceutical composition of Item 9, or the kit of Item 10, wherein the tumor includes solid tumors and non-solid tumors, including but not limited to: malignant tumors of the nasal cavity and sinuses, nasopharyngeal carcinoma, oral cancer, laryngeal cancer, salivary gland tumors, intracranial tumors, thyroid cancer, tongue cancer, lung cancer, esophageal cancer, cardia cancer, breast cancer, mediastinal tumors, gastric cancer, colorectal cancer, sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, biliary tract cancer, small bowel tumors, kidney cancer, prostate cancer, bladder cancer, malignant tumors of the testis, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, fibrous histiocytoma, rhabdomyosarcoma, synovial sarcoma, melanoma, osteosarcoma, Ewing's sarcoma, lymphoma, multiple myeloma, leukemia, preferably, the tumor is melanoma.

[0025] In this article, immunotherapeutic drugs (or reagents) that target Vam6 are compounds that have a regulatory effect on Vam6 expression, wherein the compounds inhibit or overexpress Vam6 gene transcription or expression, including but not limited to Vam6 targeting inhibitors and compounds that promote Vam6 expression.

[0026] Determining whether a drug can regulate Vam6 gene transcription or expression can also be done using existing techniques. For example, cells that normally express Vam6 can be provided, cultured in the presence of the test drug or a vector carrying the test drug, and the changes in Vam6 transcription or protein expression levels can be detected.

[0027] Furthermore, the method for screening tumor immunotherapy drugs in vitro may optionally include:

[0028] Candidate drugs are screened for in vivo and in vitro effects to identify drugs that enhance iNKT response. These effects can be determined by measuring the production of IL-4 or IFN-γ.

[0029] Antitumor experiments are conducted on candidate screening drugs to identify those that enhance the antitumor function of iNKT cells. These experiments may involve using iNKT cells treated with the screening drug for antitumor experiments, or using iNKT cells in the presence of the screening drug for antitumor experiments, with the antitumor function of iNKT cells assessed by tumor growth rate. The iNKT cells mentioned above include gene-edited iNKT cells, such as, but not limited to, CAR-iNKT cells.

[0030] The drug can be administered by any suitable method, including, for example, oral, intravenous, parenteral, transdermal, subcutaneous, intravaginal, intranasal, mucosal, sublingual, local, or rectal administration, and any combination thereof. In some embodiments, the subject includes mammals such as humans, monkeys, horses, cattle, dogs, cats, mice, rats, and pigs. In some embodiments, the route of administration includes intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, or other non-enteric routes of administration, such as by injection or infusion. In some embodiments, the route of injection includes, but is not limited to, intravenous, intramuscular, intraarterial, intradural, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. In some embodiments, administration can be made via local, epidermal, or mucosal routes.

[0031] This invention discloses the role of Vam6 in regulating iNKT cell activity. In some embodiments, regulating iNKT cell activity includes inhibiting or activating iNKT cell function. In some embodiments, regulating iNKT cell activity includes promoting or inhibiting iNKT cell survival. The iNKT cells mentioned above include gene-edited iNKT cells, such as, but not limited to, CAR-iNKT.

[0032] As is known in the art, promoting iNKT cell responses can effectively facilitate the clearance of pathogenic microorganisms and inhibit tumor cell growth; inhibiting iNKT cell responses can effectively prevent metabolic inflammation, type 2 diabetes, and suppress autoimmune diseases. In some embodiments, iNKT cell responses and iNKT cell-mediated anti-tumor immunity are promoted by reducing Vam6 expression. This invention provides iNKT cells with enhanced or reduced Vam6 expression to regulate iNKT cell responses, including but not limited to iNKT cells treated with Vam6-targeting reagents, gene-edited iNKT cells, and / or in vitro expanded iNKT cells.

[0033] It has been found that activating iNKT cells can enhance anti-tumor immunity. This invention relates to a method for enhancing the anti-tumor effect of iNKT cells by restoring their IFNγ response in subjects suffering from diseases such as tumors, the method comprising administering gene-edited iNKT cells and / or gene-edited in vitro expanded iNKT cells and an iNKT cell activator, thereby obtaining a synergistic anti-tumor effect.

[0034] In some embodiments, the present invention provides therapeutic pharmaceutical compositions and / or kits. The pharmaceutical compositions comprise the Vam6-targeting agent and iNKT cell activators described herein, and pharmaceutically acceptable carriers, wherein the iNKT cell activators are capable of activating iNKT cells to promote the release of cytokines from iNKT cells, such as CD1d-PBS57 tetramer, α-galactosylceramide (α-GalCer, α-GC) and analogues thereof, ganglioside GD3, glycophosphatidylinositol, phosphatidylethanolamine, β-GalCer, iGb3 and its analogues 4-HO-iGb3 and 4-dh-iGb3, and other antigens that can be presented by CD1d, antigen-presenting cells carrying iNKT cell antigens, and chimeric receptors expressing tumor antigens on the surface of CAR-iNKT cells. The compositions may also comprise other iNKT cell activity-regulating cells or molecules and / or iNKT cells (including gene-edited iNKT cells and gene-edited in vitro expanded iNKT cells).

[0035] In some embodiments, the kit may comprise a container, suitable of which include, for example, a bottle or syringe. The container contains the composition described herein and optionally modified iNKT cells, such as gene-edited iNKT cells, e.g., CAR-iNKT, wherein the pharmaceutical composition and the modified iNKT cells are placed in separate containers.

[0036] In some embodiments, at least one component of the composition is a Vam6-targeting agent, and may also include an iNKT cell activator and / or iNKT cells (including gene-edited iNKT cells and gene-edited in vitro expanded iNKT cells). In some embodiments, the composition and / or kit may include a first container containing a Vam6 inhibitor, a second container containing other compositions, and optionally more containers containing even more other compositions. In some embodiments, the kit may include a first container containing a Vam6 inhibitor, a second container containing an iNKT cell activator and / or iNKT cells (including gene-edited iNKT cells and gene-edited in vitro expanded iNKT cells), and optionally more containers containing even more other compositions. In some embodiments, the kit may also include a container containing pharmaceutical buffer, diluent, and carrier. In some embodiments, the kit may also include other materials such as filters, needles, and syringes.

[0037] In some embodiments, iNKT cell activators include any agent capable of activating iNKT cells (e.g., promoting the release of various cytokines such as IFN-γ and IL-4 from iNKT cells). In some embodiments, iNKT cell activators suitable for use in the present invention include CD1d-PBS57 tetramer, iNKT cell antigens including, for example, α-galactosylceramide (α-GalCer, α-GC) and analogues thereof, ganglioside GD3, glycophosphatidylinositol, phosphatidylethanolamine, β-GalCer, iGb3 and analogues 4-HO-iGb3 and 4-dh-iGb3, and other antigens that can be presented by CD1d. In some embodiments, iNKT cell activators suitable for use in the present invention include antigen-presenting cells carrying iNKT cell antigens, such as, but not limited to, dendritic cells. In some embodiments, iNKT cell activators suitable for use in the present invention include chimeric receptors (CARs) expressing tumor antigens on the surface of CAR-iNKT cells. In some implementations, one or more iNKT cell activators, or in combination with other substances, may be used to regulate the activity of iNKT cells, such as antibodies or fusion proteins, disease-associated antigens, cytokines, antigen-presenting cells, etc.

[0038] In some embodiments, the present invention provides a method for treating a subject's disease, the method comprising: administering a Vam6-targeting agent and / or an iNKT cell activator and / or iNKT cells (including gene-edited iNKT cells and gene-edited in vitro expanded iNKT cells) to the subject, thereby treating the subject's disease, such as cancer. In some embodiments, other substances may be further combined to modulate iNKT cell activity, such as disease-associated antigens, antigen-presenting cells, cytokines such as IL-4, IL-10, TGFβ, IFN-γ, IL-2, IL-18, IL-12, or IL-15, or adhesion molecules such as integrin, selectin, and ICAM. In some embodiments, the subject includes mammals such as humans, monkeys, horses, cattle, dogs, cats, mice, rats, and pigs.

[0039] In some embodiments, the compositions and / or kits of the present invention further comprise other components, such as antigen-presenting dendritic cells (DCs). In some embodiments, the compositions and / or kits of the present invention may include co-stimulatory molecules, such as B7 signaling molecules, allowing for effective and prolonged activation of iNKT cells. The compositions of the present invention may also comprise additional adjuvant components, such as LPS, TLR9 agonists like CPGODNS, TLR7 / 8 agonists, cytokines and growth factors such as IL12 and IL2.

[0040] As is known to those skilled in the art, it is possible to determine whether a composition can modulate the activity of iNKT cells by appropriate methods, such as by measuring the production of IL-4 or IFN-γ after administration of the test compound or composition to an animal, such as a mouse. For example, the production of IL-4 or IFN-γ or iNKT cell proliferation can be measured to determine the activation or response of iNKT cells.

[0041] In some embodiments, the treatments described in this invention comprise altering the natural clinical course of the treated individual and may include prevention or improvement of clinicopathological levels. Treatments include, but are not limited to, preventing disease onset or recurrence, alleviating symptoms, eliminating direct or indirect pathological consequences of the disease, reducing the rate of disease progression, improving or mitigating the disease state, and improving prognosis. In some embodiments, the medicaments of this invention are used to treat and / or prevent related diseases, and / or to delay the onset of disease or slow its progression.

[0042] In some embodiments, for the prevention or treatment of disease, the appropriate dosage of the medicament of the present invention can be determined by factors such as the type of disease to be treated, the severity and progression of the disease, whether the medicament is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history, and response to the medicament. The medicament can be administered once or multiple times separately, or by continuous infusion. The progress of treatment can be monitored using conventional techniques and assays. Attached Figure Description

[0043] Figure 1 and Figure 2 Corresponding to Example 1. Through Figure 1 A confirms Vam6 + / - Vam6 expression is decreased in mouse T cells. Figure 1 B displays Vam6 + / - After activation and stimulation in vitro, iNKT cells can produce more Th1 cytokine IFN-γ and Th2 cytokine IL-4 compared to wild-type iNKT cells.

[0044] Figure 2 A shows the process of constructing bone marrow chimeric mice and the flow cytometry gate strategy for iNKT cells. CD45.1 iNKT cells are Vam6 cells. + / + iNKT cells, CD45.2 iNKT cells are Vam6 + / - iNKT cells, Figure 2 B displays CD45.2Vam6 + / - iNKT cells, after in vivo stimulation, compared to CD45.1Vam6 + / + iNKT cells can produce more Th1 cytokine IFN-γ and Th2 cytokine IL-4. Figure 1 and Figure 2 Experimental results demonstrate that reducing Vam6 expression can enhance the immune response of iNKT cells.

[0045] Figure 3 Corresponding to Example 2. Vam6 + / - iNKT and Vam6 + / + After iNKT cells were cultured overnight in vitro, flow cytometry antibody staining was used to detect the iNKT cell proliferation index Ki67 (…). Figure 3 A), apoptosis markers PI and Annexin V (A), Figure 3 B), and the anti-apoptotic marker Bcl-2 ( Figure 3 C) The results showed that, regardless of whether or not activation stimulation was experienced, Vam6 + / - iNKT cell proliferation capacity Figure 3 A) and anti-apoptotic ability ( Figure 3 C) All are stronger than Vam6 + / + iNKT cells, while the proportion of late apoptotic cells ( Figure 3 B) Significantly lower than Vam6 + / + iNKT cells. Specifically, reducing Vam6 expression can inhibit iNKT cell apoptosis and enhance iNKT cell proliferation.

[0046] Figure 4 Corresponding to Example 3. From Figure 4A confirms in vitro amplification of Vam6 + / - Vam6 expression in iNKT cells is lower than that in vitro expanded Vam6. + / + iNKT cells, Figure 4 B shows that in vitro amplification of Vam6 + / - iNKT cells, after activation stimulation in vitro, showed a greater effect compared to in vitro expansion of Vam6 cells. + / + iNKT cells can produce more cytokine IFN-γ. Figure 4 C represents the construction process of a mouse B16F10 melanoma lung metastasis model, which involves in vitro amplification of Vam6... + / - iNKT cells and in vitro expansion of Vam6 + / + After iNKT cells were injected into tumor-bearing mice, the metastasis of tumors to the lungs of the mice was observed. Figure 4 The D image documented the lung metastasis of melanoma in mice. Figure 4 E is a statistical graph showing the number of melanoma nodules in the lungs. Figure 4 F represents the survival curve of tumor-bearing mice. Figure 4 The results demonstrated that the in vitro amplified Vam6 + / - iNKT cells compared to in vitro expanded Vam6 + / + iNKT cells can produce more IFN-γ, and after being transfused into tumor-bearing mice, they can better inhibit tumor migration and significantly prolong the lifespan of tumor-bearing mice.

[0047] Figure 5 Corresponding to Example 4. Figure 5 A represents the procedure for constructing a subcutaneous tumor model in wild-type mice, which involves in vitro amplification of Vam6... + / - iNKT cells and in vitro expansion of Vam6 + / + After iNKT cells were injected into tumor-bearing mice, subcutaneous tumor growth was observed. Figure 5 B. Photographs to record the size of the subcutaneous tumor in the tumor-bearing mouse. Figure 5 C is a statistical chart of subcutaneous tumor weight. Figure 5 D represents the growth curve of subcutaneous tumors in mice. Figure 5 A-5D results showed that the in vitro amplified Vam6 + / - iNKT cells compared to in vitro expanded Vam6 + / + iNKT cells can better inhibit the growth of subcutaneous tumors. Figure 5 E is Jα18 - / - Procedure for constructing a mouse subcutaneous tumor model. This involves using in vitro expanded Vam6... + / - iNKT cells and in vitro expansion of Vam6 + / + iNKT cells were injected into tumor-bearing Jα18 cells. - / -After in vivo administration to mice, α-GC was injected into the mice to activate expanded iNKT cells of tumor-bearing mice. Flow cytometry was used to detect the intracellular IFN-γ expression level of the expanded iNKT cells. Figure 5 The F results showed that the in vitro amplified Vam6 + / - iNKT cells compared to in vitro expanded Vam6 + / + iNKT cells can produce more IFN-γ, indicating that the in vitro expanded Vam6 + / - iNKT cells showed stronger anti-tumor activity in mice than in vitro expanded Vam6 cells. + / + iNKT cells. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0049] The following examples are provided to better understand the present invention, but are not limited to the specific embodiments described below. The terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0050] Wild-type mice: purchased from Nanjing Jicui Biotechnology Co., Ltd.

[0051] Jα18 - / - Mice: A gift from Professor Sun Rui of the University of Science and Technology of China

[0052] Vα14Tg.cxcr6 mice

[0053] Percoll cell separation medium: GE Healthcare, 17-0891-01

[0054] RIPA lysis buffer: beyotime, P0013B

[0055] SDS-PAGE protein loading buffer (5×): beyotime, P0015

[0056] Foxp3 / transcription factor staining buffer: eBioscience, 00-5523-00; PE-CY7 anti-mouse TCRβ antibody: Biolegend, 109222

[0057] BV421 anti-mouse IL-4 antibody: Biolegend, 504119

[0058] BV510 anti-mouse IFN-γ antibody: Biolegend, 505841

[0059] PE anti-mouse CD45.1 antibody: Biolegend, 110708

[0060] PerCP / Cy5.5 anti-mouse CD45.2 antibody: Biolegend, 109828

[0061] Anti-mouse Bcl-2 antibody: BioLegend, 633508

[0062] PI and Annexin V reagent kit: BioLegend, 640914

[0063] Anti-mouse Ki67 antibody: BD Pharmingen, 556026

[0064] Anti-mouse Vam6 antibody: Thermo Fisher, PA5-21104

[0065] Mouse IFN-γ Detection Kit: BD, 558296

[0066] Mouse IL-4 Detection Kit: BD, 558298

[0067] CD1d-PBS57 tetramer: provided by the Tetramer Research Center at Emory University, National Institutes of Health.

[0068] Since Vam6 gene knockout mice are homozygous lethal, Vam6 was used in the following examples. + / - Mice and littermate control Vam6 + / + Mice.

[0069] Example 1: Reducing Vam6 expression can enhance the iNKT cell immune response.

[0070] 1. Vam6 + / - iNKT cells enhance the immune response after in vitro stimulation.

[0071] (1) Treat 96-well plates with polylysine and spread 1 μg / mL CD1d-PBS57 tetramer at 37°C for 1 hour.

[0072] (2) Remove Vam6 + / + and Vam6 + / - Mouse spleen and liver were used. Spleen was ground and then enriched with magnetic beads to collect T lymphocytes. Cells were treated with cell lysis buffer (RIPA lysis buffer: SDS-PAGE protein loading buffer (5×) = 4:1) and heated in a metal bath at 100℃ for 20 minutes. Western blotting was performed, and the expression level of Vam6 protein in the cell lysis buffer was detected using anti-mouse Vam6 antibody. Results are shown below. Figure 1 A. After grinding the liver, the suspension was subjected to density gradient centrifugation in 40% Percoll / 70% Percoll (volume percentage) (up 6, down 2, 2000 rpm, 20 min). Intermediate liver lymphocytes were extracted, stained with flow cytometry antibodies (on ice, protected from light for 45 min), and TCRβ were separated by flow cytometry. + CD1d-PBS57 tetramer + Cells were sorted and added to a 96-well plate pretreated with (1), with 5 × 10⁶ cells per well. 4 Each cell.

[0073] Group 1, Vam6 + / + iNKT cells, wells of PBS-coated plates, negative control group.

[0074] Group 2, Vam6 + / - iNKT cells, wells of PBS-coated plates, negative control group.

[0075] Group 3, Vam6 + / + iNKT cells, CD1d-PBS57 tetramer plated in wells.

[0076] Group 4, Vam6 + / - iNKT cells, CD1d-PBS57 tetramer plated in wells.

[0077] (3) After 48 hours of treatment in step (2), cell supernatant was collected, and the IFN-γ and IL-4 in the supernatant were detected according to the kit instructions using an IFN-γ detection kit and an IL-4 detection kit. Results are shown below. Figure 1 B.

[0078] Under the action of CD1d-PBS57 tetramer, iNKT cells are activated and produce large amounts of Th1 cytokine IFN-γ and Th2 cytokine IL-4. Compared with wild-type iNKT cells, Vam6-deficient iNKT cells can produce more cytokines IFN-γ and IL-4.

[0079] 2. Vam6 + / - iNKT cells enhance the immune response after in vivo stimulation.

[0080] (1) Day 1, Jα18 - / - CD45.2 mice were irradiated (irradiation dose: 10 Gy).

[0081] (2) The next day, Vam6 + / + CD45.1 mouse bone marrow cells and Vam6 + / - CD45.2 mouse bone marrow cells were mixed in a 1:1 ratio and intravenously injected into irradiated recipient mice, with each mouse receiving 1 × 10⁻⁶ cells.6 Each cell.

[0082] (3) Seven weeks later, recipient mice were intraperitoneally injected with iNKT cell-specific antigen α-GC.

[0083] Group 1, the negative control group, each mouse was injected intraperitoneally with 100 μL of PBS.

[0084] The second group, the positive control group, received an intraperitoneal injection of 2 μg α-GC (dissolved in 100 μL PBS) in each mouse.

[0085] (4) Four hours later, mice were sacrificed, and spleen lymphocytes were extracted. The cell surface was labeled with flow cytometry antibodies TCRβ, CD45.1, CD45.2, and CD1d-PBS57 tetramer, and incubated on ice for 45 minutes. After surface labeling, cells were fixed and permeabilized using Foxp3 / transcription factor staining buffer, and then intracellular IL-4 and IFN-γ proteins were labeled with flow cytometry antibodies IL-4 and IFN-γ, incubated at room temperature for 60 minutes. CD45.1 + TCRβ + CD1d-PBS57 tetramer + The cell is Vam6 + / + iNKT cells, CD45.2 + TCRβ + CD1d-PBS57 tetramer + The cell is Vam6 + / - iNKT cells, experimental flowchart see [link / details] Figure 2 A, Statistical analysis of flow cytometry results can be found in [reference]. Figure 2 B.

[0086] Following in vivo injection of α-GC, mouse iNKT cells were activated, producing large amounts of the Th1 cytokine IFN-γ and the Th2 cytokine IL-4. This contrasts with wild-type CD45.1. + Compared to iNKT cells, Vam6 + / - CD45.2 + The proportion of IFN-γ and IL-4 positivity was higher in iNKT cells, consistent with in vitro results.

[0087] Example 2: Reducing Vam6 expression in iNKT cells can inhibit apoptosis and promote cell survival.

[0088] 1. Mouse Vam6 + / - iNKT cells show enhanced Ki67 expression.

[0089] (1) Treat 96-well plates with polylysine, and then lay 2 μg / mL CD1d-PBS57 tetramer at 37°C for 1 hour.

[0090] (2) Remove Vam6 + / + and Vam6 + / - Mouse spleens were ground and then enriched with magnetic beads to collect T lymphocytes. The enriched cells were added to a 96-well plate pretreated with (1), with approximately 5 × 10⁶ cells per well. 5 Each cell.

[0091] Group 1, Vam6 + / + iNKT cells, wells of PBS-coated plates, negative control group.

[0092] Group 2, Vam6 + / - iNKT cells, wells of PBS-coated plates, negative control group.

[0093] Group 3, Vam6 + / + iNKT cells, CD1d-PBS57 tetramer plated in wells.

[0094] Group 4, Vam6 + / - iNKT cells, CD1d-PBS57 tetramer plated in wells.

[0095] (3) After 18 hours, collect the cells treated in step (2), and label the cell surface with flow cytometry TCRβ antibody and CD1d-PBS57 tetramer, then incubate on ice for 45 minutes. After surface labeling, fix the cells permeabilized with Foxp3 / transcription factor staining buffer, and detect the intracellular Ki67 expression level using flow cytometry anti-mouse Ki67 antibody. The TCRβ expression level in lymphocytes was also measured. + CD1d-PBS57 tetramer + The cells are iNKT cells. Results are shown below. Figure 3 A.

[0096] Regardless of whether or not CD1d-PBS57 tetramer stimulation was performed, Vam6 + / - Ki67 expression in iNKT cells was higher than that in Vam6 cells. + / + iNKT cells, indicating Vam6 + / - iNKT cells have a stronger proliferative capacity.

[0097] 2. Mouse Vam6 + / - iNKT cells have enhanced anti-apoptotic ability.

[0098] (1) Treat 96-well plates with polylysine and spread 2 μg / mL CD1d-PBS57 tetramer at 37°C for 1 hour.

[0099] (2) Remove Vam6 + / + and Vam6 + / - Mouse spleens were ground and then enriched with magnetic beads to collect T lymphocytes. The enriched cells were added to a 96-well plate pretreated with (1), with approximately 5 × 10⁶ cells per well.5 Each cell.

[0100] Group 1, Vam6 + / + iNKT cells, wells of PBS-coated plates, negative control group.

[0101] Group 2, Vam6 + / - iNKT cells, wells of PBS-coated plates, negative control group.

[0102] Group 3, Vam6 + / + iNKT cells, CD1d-PBS57 tetramer plated in wells.

[0103] Group 4, Vam6 + / - iNKT cells, CD1d-PBS57 tetramer plated in wells.

[0104] (3) After 18 hours, collect the cells treated in step (2), and label the cell surface with flow cytometry TCRβ antibody and CD1d-PBS57 tetramer, then incubate on ice for 45 minutes. After surface labeling, perform combined staining with PI and Annexin V, and detect the fluorescence intensity by flow cytometry. The results are shown in [Figure 1]. Figure 3 B; The cell surface was labeled with TCRβ antibody and CD1d-PBS57 tetramer by flow cytometry and incubated on ice for 45 minutes. After surface labeling, the cells were fixed and permeabilized using Foxp3 / transcription factor staining buffer, and the expression level of the anti-apoptotic protein Bcl-2 in the cells was detected by flow cytometry. The results are shown in the figure. Figure 3 C.

[0105] Regardless of whether or not CD1d-PBS57 tetramer stimulation was performed, Vam6 + / - The proportion of late-stage apoptotic iNKT cells (PI) + Annexin V + All are lower than Vam6 + / + iNKT cells, while Vam6 + / - Bcl-2 expression levels in iNKT cells were higher than those in Vam6 cells. + / + iNKT cells, indicating Vam6 + / - iNKT cells have a stronger anti-apoptotic ability.

[0106] Example 3: Reducing Vam6 expression in in vitro expanded iNKT cells can promote IFN-γ production and significantly inhibit melanoma metastasis.

[0107] 1. In vitro amplification of Vam6 + / - Enhanced iNKT cell immune response

[0108] (1) Take Vα14Tg.cxcr6×Vam6+ / + and Vα14Tg.cxcr6×Vam6 + / - Mouse spleens were ground and resuspended in RPMI-1640 medium containing 10% fetal bovine serum. 50 mM β-mercaptoethanol, 100 ng / mL α-GC, and 200 IU / mL IL-2 were added, and the cells were incubated at 37°C for 3 days. The cell suspension was collected, centrifuged, and resuspended in fresh RPMI-1640 medium containing 10% fetal bovine serum. 50 mM β-mercaptoethanol and 200 IU / mL IL-2 were added, and the cells were incubated at 37°C for another 4 to 7 days.

[0109] (2) Collect the cells cultured in (1), add cell lysis buffer (RIPA lysis buffer: SDS-PAGE protein loading buffer (5×) = 4:1) and heat in a metal bath at 100℃ for 20 minutes. Load the cells into a Western blotting table and detect the expression level of Vam6 protein using anti-mouse Vam6 antibody. Results are shown below. Figure 4 A.

[0110] (3) Treat 96-well plates with polylysine, and lay 1 μg / mL CD1d-PBS57 tetramer at 37°C for 1 hour.

[0111] (4) Take the cells cultured in (1) and add them to the 96-well plate of the pretreatment, 5 × 10⁶ cells per well. 4 Each group contains 10 cells and has 3-4 replicates.

[0112] Group 1, Vam6 + / + iNKT cells, wells of PBS-coated plates, negative control group.

[0113] Group 2, Vam6 + / - iNKT cells, wells of PBS-coated plates, negative control group.

[0114] Group 3, Vam6 + / + iNKT cells, CD1d-PBS57 tetramer plated in wells.

[0115] Group 4, Vam6 + / - iNKT cells, CD1d-PBS57 tetramer plated in wells.

[0116] (5) After 24 hours of treatment with cells from step (4), cell supernatant was collected, and IFN-γ in the supernatant was detected by flow cytometry using an IFN-γ detection kit. Results are shown below. Figure 4 B.

[0117] Similar to primary cells, in vitro expanded iNKT cells, after activation in vitro, produce large amounts of the cytokine IFN-γ, which interacts with in vitro expanded Vam6. + / +Compared to iNKT cells, in vitro expansion of Vam6 + / - iNKT cells can produce more cytokine IFN-γ.

[0118] 2. In vitro amplification of Vam6 + / - iNKT cells have enhanced anti-tumor capabilities.

[0119] 5×10 4 One melanoma B16F10 cell was intravenously injected into each wild-type mouse. Twenty-four hours later, each tumor-bearing mouse was intraperitoneally injected with 1 × 102 melanoma B16F10 cells. 6 One in vitro expanded iNKT cell.

[0120] Group 1, the negative control group, each mouse was injected intraperitoneally with 100 μL of PBS.

[0121] In the second group, each mouse was injected intraperitoneally with Vam6. + / + iNKT cells (dissolved in 100 μL PBS).

[0122] In the third group, each mouse was injected intraperitoneally with Vam6. + / - iNKT cells (dissolved in 100 μL PBS).

[0123] The survival rate of mice was then recorded daily. After 19 or 21 days, mice in each group were randomly sacrificed, and lung tumor nodules were photographed and counted. The experimental procedure is described below. Figure 4 C, lung image (see) Figure 4 D, Statistical analysis of the number of pulmonary nodules can be found in [reference needed]. Figure 4 E, mouse survival rate is shown in Figure 4 F.

[0124] In vitro amplification of Vam6 + / + and Vam6 + / - iNKT cells can inhibit the migration of B16F10 tumors and improve the survival rate of mice to some extent. Among them, in vitro expansion of Vam6 + / - iNKT cells have a more significant anti-cancer effect.

[0125] Example 4: Reducing the expression level of Vam6 in in vitro expanded iNKT cells can significantly inhibit the growth rate of subcutaneous tumors.

[0126] 1. In vitro amplification of Vam6 + / - iNKT cells have a stronger inhibitory effect on subcutaneous tumors.

[0127] (1) Melanoma B16F10 cells were injected subcutaneously into wild-type mice, with each mouse receiving 5 × 10⁶ tumor cells. 5 indivual.

[0128] (2) Ten days after the injection of tumor cells, the size of the subcutaneous tumor in the mice was measured with calipers every other day.

[0129] (3) Twelve days after tumor cell injection, Vam6 amplification was injected adjacent to the tumor in recipient mice. + / + iNKT cells or expanded Vam6 + / - iNKT cells or an equal volume of PBS.

[0130] (4) After 20 days, photograph and record the size of the subcutaneous tumor, and weigh and record the tumor weight. See the experimental procedure below. Figure 5 A, Tumor image (see image) Figure 5 B, Tumor weight statistics are shown in [the original text]. Figure 5 C, tumor growth rate is shown in... Figure 5 D.

[0131] The results showed that injection of expanded iNKT cells significantly inhibited subcutaneous tumor growth in mice. In particular, compared with Vam6... + / + Compared to the group, Vam6 + / - The size and weight of subcutaneous tumors in the control group were significantly reduced, and their growth rate was significantly slowed, indicating that in mice, compared with the amplified Vam6... + / + iNKT cells, expanded Vam6 + / - iNKT cells can better inhibit the growth of subcutaneous tumors.

[0132] 2. In vitro amplification of Vam6 within the tumor + / - Enhanced iNKT cell immune response

[0133] In addition, to detect the immune response capacity of the transferred expanded iNKT cells in vivo, we examined the immune response capacity of tumor-infiltrating expanded iNKT cells.

[0134] (1) B16F10 cells were injected into Jα18 - / - Subcutaneous injection of 5 × 10⁶ tumor cells into each mouse (iNKT cell-deficient mouse). 5 indivual.

[0135] (2) Twelve days after tumor cell injection, Vam6 amplification was injected adjacent to the tumor in recipient mice. + / + iNKT cells or expanded Vam6 + / - iNKT cells.

[0136] (3) Fourteen days later, recipient mice were intraperitoneally injected with α-GalCer or an equal volume of PBS. Subcutaneous tumors were removed, and the tumors were ground up. The suspension was then subjected to density gradient centrifugation (6 rpm, 2000 rpm, 20 min) in 40% Percoll / 70% Percoll (volume percentage). Intermediate lymphocytes were extracted, and the cell surface was labeled with TCRβ antibody and CD1d-PBS57 tetramer by flow cytometry and incubated on ice for 45 min. After surface labeling, the cells were fixed and permeabilized using Foxp3 / transcription factor staining buffer. IFN-γ in tumor-infiltrating and amplified iNKT cells was then detected by flow cytometry. + Cell proportion, TCRβ in tumor lymphocytes + CD1d-PBS57 tetramer + The cells are tumor-infiltrating and expanded iNKT cells. The experimental procedure is as follows: Figure 5 E. Results showed that Vam6 amplified intratumorally after antigen stimulation. + / - iNKT cells have a stronger ability to produce IFN-γ, see Figure 5 F, consistent with the results for subcutaneous tumor size.

[0137] The above experiments demonstrate that, in mice, compared to amplified Vam6... + / + iNKT cells, expanded Vam6 + / - iNKT cells have a stronger anti-tumor effect.

[0138] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0139] References

[0140] Bendelac, A., PBSavage, and L. Teyton. 2007. The Biology of NKT Cells. Annual Review of Immunology. 25: 297-336.

[0141] Fu,S.,K.He,C.Tian,H.Sun,C.Zhu,S.Bai,J.Liu,Q.Wu,D.Xie,T.Yue,Z. Shen,Q.Dai,X.Yu,S.Zhu,G.Liu,R.Zhou,S.Duan,Z.Tian,T.Xu,H.Wang,andL.Bai.2020.Impaired lipid biosynthesis hinders anti-tumor efficacy ofintratumoral iNKT cells.Nat Commun.11:438.

[0142] Xie,D.,S.Zhu,and L.Bai.2016.Lactic acid in tumor microenvironmentscauses dysfunction of NKT cells by interfering with mTOR signaling.Sci ChinaLife Sci.59:1290-1296.

Claims

1. A method for screening a tumor immunotherapy agent targeting Vam6, the method comprising the steps of: administering a candidate agent to iNKT cells; treating the candidate agent-administered iNKT cells and the candidate agent- unadministered iNKT cells with an iNKT cell activator; determining the expression level of Vam6 in the candidate agent-administered iNKT cells and the candidate agent-unadministered iNKT cells; wherein a decreased expression level of Vam6 in the candidate agent-administered iNKT cells compared to the candidate agent-unadministered iNKT cells indicates that the candidate agent is a potential immunotherapy agent targeting Vam6, wherein the iNKT cell activator is capable of activating iNKT cells to promote cytokine release by iNKT cells.

2. The method of claim 1, wherein the iNKT cell activator is CD1d-PBS57 tetramer, a-galactosylceramide (a-GalCer, a-GC) and its analogs, ganglioside GD3, glycosylphosphatidylinositol, phosphatidylethanolamine, b-GalCer, iGb3 and its analogs 4-HO-iGb3 and 4-dh-iGb3, antigen presenting cells carrying iNKT cell antigens, and chimeric receptors expressing on the surface of CAR-iNKT recognizing tumor antigens.

3. The method of claim 1, wherein the iNKT cells are unmodified wild-type iNKT cells or genetically edited iNKT cells.

4. The method of claim 3, wherein the iNKT cells are CAR-iNKT.

5. The method of claim 1, wherein the expression level of Vam6 is determined by measuring the activity or protein amount of Vam6 or measuring the level of Vam6 gene transcript.

6. A method for screening an agent that enhances iNKT cell anti-tumor function by targeting Vam6, comprising treating iNKT cells with a candidate agent screened by the method of any one of claims 1-5, and then using the iNKT cells for tumor treatment in the presence of the candidate agent or using the iNKT cells for tumor treatment in the absence of the candidate agent, and determining the change in the anti-tumor function of the iNKT cells, wherein an improved anti-tumor function of the iNKT cells treated with the candidate agent compared to the iNKT cells not treated with the candidate agent indicates that the candidate agent is a potential agent that enhances iNKT cell anti-tumor function.

7. The method of claim 6, wherein the iNKT cells are unmodified wild-type iNKT cells or genetically edited iNKT cells.

8. The method of claim 7, wherein the iNKT cells are CAR-iNKT.

9. Use of Vam6 as a target in a method of screening for an immunotherapeutic agent, said method being a method of administering a candidate agent to iNKT cells, treating the iNKT cells administered with the candidate agent with an iNKT cell activator, and determining the expression level of Vam6 in the iNKT cells administered with the candidate agent and in the iNKT cells not administered with the candidate agent, wherein the immunotherapeutic agent decreases the expression level of Vam6 in iNKT cells; or a method of screening for an immunotherapeutic agent using the method of any one of claims 1 to 5, and then using the iNKT cells treated with the candidate agent for tumor treatment or using the iNKT cells in the presence of the candidate agent for tumor treatment, and determining the change in the anti-tumor function of the iNKT cells, wherein the anti-tumor function of the iNKT cells treated with the immunotherapeutic agent is improved compared to the anti-tumor function of the iNKT cells not treated with the immunotherapeutic agent.

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