Novel medicine for enhancing T cell function

Abstract

The invention discloses a novel medicine for enhancing the function of a T cell (CD8 + T or CAR-T), and relates to an antisense oligonucleotide (ASO) for targeted inhibition of TMED4 of the CD8 + T or CAR-T cell. The invention also discloses a T cell culture treated by the ASO, and the T cell culture is used for inhibiting tumors. When the ASO is used for silencing the TMED4 gene, the treatment efficiency of tumor infiltrating T cells and CAR-T cells is effectively improved, the life cycle of the tumor infiltrating T cells and the CAR-T cells is effectively prolonged, and an effective target spot and an intervention medicine are provided for the curative effect of the solid tumor CAR-T cells.

Description

Technical Field

[0001] This invention pertains to, and more specifically, to, an enhancement method for T cells (CD8). + Novel drugs for T or CAR-T cell function. Background Technology

[0002] Data shows that the overall incidence of malignant tumors is currently showing a continuous and significant upward trend. Solid tumors account for a very high proportion of deaths caused by malignant tumors, approximately 90% of all malignant tumor deaths; among them, the five most common types with the highest incidence and mortality are lung cancer, liver cancer, stomach cancer, colorectal cancer, and esophageal cancer. Despite the existence of various prevention and treatment strategies, including cancer screening programs, surgery, chemotherapy, radiotherapy, immunotherapy, and targeted therapy, the number of new malignant tumor cases and overall deaths continues to show a serious upward trend year by year.

[0003] Therefore, actively developing novel and highly effective treatment methods is particularly important and urgent. Although the widely used small-molecule targeted drugs and antibody drugs have significantly prolonged the overall survival of some patients with solid tumors, it is still difficult to completely avoid the continued progression of tumors and recurrence after treatment in clinical practice. In fact, in recent years, there has been an increasing number of studies on establishing more effective immune cell-based treatments for malignant tumors by activating and expanding immune cells in vitro; a large amount of scientific experimental and clinical evidence has shown that chimeric antigen receptor T-cell (CAR-T) immunotherapy, as an emerging precision targeted therapy, has shown strong therapeutic potential and broad prospects in the fight against tumors. It is worth noting that the tumor microenvironment usually contains a variety of cell types with immunosuppressive capabilities, such as regulatory T cells and tumor-associated macrophages, as well as overexpressed cytokines with immunosuppressive activity, such as TGFβ, IL-10, and IL-4. This special immunosuppressive microenvironment can easily lead to insufficient migration and persistence of CAR-T cells infused into the body, significantly impaired cell function, and premature cell exhaustion, ultimately resulting in poor overall therapeutic efficacy.

[0004] T lymphocytes are a core component of the immune system, mainly divided into cytotoxic T cells (CD8+). + T cells), helper T cells (CD4) + T cells, regulatory T cells (Tregs), and memory T cells each play distinctly different roles in the immune response. CD8 + T cells and Treg cells play opposite roles in immune regulation; CD8 +T cells are primarily effector cells, responsible for directly killing virus-infected cells or tumor cells; they are the "attackers" of the immune system. Treg cells (regulatory T cells), on the other hand, are immunosuppressive cells that maintain immune tolerance by suppressing overactive immune responses, preventing autoimmune diseases; they act as the "brakes." CD8 + T cells and Treg cells differ significantly in cell phenotype and markers, CD8 + T cells express CD8 molecules, and some subsets (such as CD8+) + Treg cells may express KIRs, granzyme B (GZMB), etc.; Treg cells use CD4+. + For example, Treg cells highly express the transcription factor FOXP3, while CD8... + Treg cells may express inhibitory molecules such as FoxP3 and CTLA-4. CD8 + T cells and Treg cells have different developmental origins and regulatory mechanisms, CD8 + T primarily originates from CD8 in the thymus. + T cell precursors, which differentiate into effector cells; CD4 + Tregs are generated from CD4 in the thymus. + They differentiate from T cell precursors. Their mechanisms of action are also opposite; CD8... + T cells directly kill target cells by releasing perforin, granzymes, etc.; Treg cells suppress immune responses through contact inhibition (such as CTLA-4, LAG-3) or by secreting inhibitory cytokines (such as IL-10, TGF-β). In diseases, CD8... + Insufficient T cell function may lead to infection or tumor progression, while overactivation may trigger autoimmune diseases; Treg cell dysfunction or abnormality is closely related to autoimmune diseases (such as rheumatoid arthritis and multiple sclerosis).

[0005] CD8 + T cells, as key cells in the immune system, can recognize tumor-specific antigens presented by MHC class I molecules (MHC-I) through their surface T cell receptors (TCRs), thereby effectively killing cancer cells. Therefore, they play an indispensable core role in the body's tumor clearance process. However, many types of cancer evade the body's immune surveillance by actively downregulating MHC-I molecule expression, thereby blocking CD8... +T cells recognize tumor cells and exert cytotoxic effects. To overcome this significant limitation, chimeric antigen receptor (CAR)-T cell therapy has emerged. CAR-T cells are genetically engineered to directly recognize relevant antigens on the surface of tumor cells in a manner independent of MHC molecule presentation, enabling them to effectively kill tumor cells even when facing tumor cells with low or absent MHC-I expression.

[0006] The above strategies have achieved remarkable results in the treatment of B-cell malignancies, but due to immunosuppressive factors in the solid tumor microenvironment and T-cell exhaustion, which are common problems during treatment, their application in the field of solid tumor treatment still needs further improvement and optimization. Summary of the Invention

[0007] The purpose of this invention is to provide a method for enhancing T cells (CD8). + Novel drugs that enhance the function of T cells or chimeric antigen receptor T cells (CAR-T).

[0008] In a first aspect of the invention, the use of an antisense oligonucleotide (ASO) for preparing a composition that enhances T cell function is provided; said antisense oligonucleotide targets and inhibits TMED4 of T cells, said T cells being CD8. + T cells or chimeric antigen receptor T cells (CAR-T).

[0009] In one or more embodiments, the T cell function is CD8. + The tumor-killing function of T cells or chimeric antigen receptor T cells.

[0010] In one or more embodiments, the T cell is not a Treg cell.

[0011] In one or more embodiments, the composition comprises a pharmaceutical composition or a culture medium.

[0012] In one or more embodiments, the antisense oligonucleotide is a modified, performance-enhanced antisense oligonucleotide.

[0013] In one or more embodiments, the antisense oligonucleotide includes (but is not limited to) modifications selected from the group consisting of: 2'-methoxyethyl (2'-MOE) modification, methoxy modification, fluorinated modification, thiomodification, cholesterol modification, alkyl modification, locked nucleic acid modification, or a phosphothioester group linking adjacent nucleosides or between a nucleoside and a target ligand.

[0014] In one or more embodiments, the antisense oligonucleotide targets a conserved CDS sequence that is homologous to human and mouse genes in the TMED4 gene.

[0015] In one or more embodiments, the conserved human-mouse homologous CDS sequences are based on: mouse transcript: NM_134020.1; human transcript: NM_182547.4; NM_001303058.2; NM_001303059.2; NM_001303060.2; NM_001303061.2 or NM_001303062.2.

[0016] In one or more embodiments, the antisense oligonucleotide is chemically synthesized.

[0017] In one or more embodiments, the modification of the antisense oligonucleotide is a full-chain modification.

[0018] In one or more embodiments, the sequence of the antisense oligonucleotide is: AACACCTCTTTCTGTT.

[0019] In one or more embodiments, the antisense oligonucleotide is modified with 2'-methoxyethyl (2'-MOE), and the structure of the 2'-methoxyethyl modified antisense oligonucleotide is as follows: .

[0020] In another aspect of the present invention, an antisense oligonucleotide for enhancing T cell function is provided, wherein the structure of the 2'-methoxyethyl modified antisense oligonucleotide is as follows: .

[0021] In another aspect of the invention, a method for enhancing T cell function is provided, comprising: treating the T cells, wherein the T cells are CD8+, with an antisense oligonucleotide. + T cells or chimeric antigen receptor T cells (CAR-T); the antisense oligonucleotide targets and inhibits TMED4 of T cells; preferably, the antisense oligonucleotide is the aforementioned antisense oligonucleotide.

[0022] In one or more embodiments, the T cell function is CD8. + Tumor-killing function of T cells or chimeric antigen receptor T cells (CAR-T).

[0023] In one or more embodiments, the method for enhancing T cell function is an in vitro method, which involves treating T cell cultures.

[0024] In one or more embodiments, the method for enhancing T cell function is a non-therapeutic method.

[0025] In another aspect of the invention, a T cell culture with enhanced function is provided, which is prepared by the method described above; preferably, the T cells are CD8 cells. + T cells or chimeric antigen receptor T cells (CAR-T); preferably, the enhanced function is enhanced tumor killing function.

[0026] In another aspect of the invention, a composition (pharmaceutical composition or culture medium) or kit (pharmaceutical kit) for enhancing T cell function is provided, comprising the said antisense oligonucleotide for enhancing T cell function; wherein the T cells are CD8. + T cells or chimeric antigen receptor T cells (CAR-T).

[0027] In another aspect of the invention, a pharmaceutical composition or kit for inhibiting tumors is provided, comprising the T cell culture and a pharmaceutically acceptable carrier or excipient.

[0028] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Attached Figure Description

[0029] Figure 1 Construction of an adoptive transfer model of ASO-treated T cells.

[0030] Figure 2 Model construction for ASO-Tmed4 combined with CAR-T therapy for tumors.

[0031] Figure 3 ASO-Tmed4 treatment significantly downregulated the expression level of TMED4 protein in T cells.

[0032] Figure 4 ASO-Tmed4 treatment significantly downregulated the expression level of TMED4 protein in CAR-T cells.

[0033] Figure 5 Tumor growth curves of MC38 tumor-bearing mice transplanted with wild-type or Tmed4 knockdown T cells; the horizontal axis is in "days".

[0034] Figure 6 Quantitative analysis of tumor weight in MC38 tumor-bearing mice transplanted with wild-type or Tmed4 knockdown T cells.

[0035] Figure 7 Tumor growth curves of MC38-CLDN18.2 tumor-bearing mice transfected with ASO CAR-T; the horizontal axis is in "days".

[0036] Figure 8 Survival curves of MC38-CLDN18.2 tumor-bearing mice transfected with ASO CAR-T; the horizontal axis is in "days".

[0037] Figure 9 A schematic diagram of the CLDN18.2 CAR construct. Detailed Implementation

[0038] This invention discloses for the first time a novel type of enhancing T cell (CD8) + The invention discloses a biomolecule that functions as a T cell or chimeric antigen receptor T cell (CAR-T, preferably CAR-T cell) biomolecule. This biomolecule is an antisense oligonucleotide (ASO) that targets and inhibits T cell TMED4, and the ASO exhibits a highly significant enhancing effect. The invention also discloses a T cell culture treated with the ASO for tumor suppression. This invention utilizes ASO to silence the TMED4 protein, significantly improving the therapeutic efficiency of tumor-infiltrating T cells and CAR-T cells, effectively prolonging their survival, and providing an effective target and intervention for the therapeutic efficacy of CAR-T cells in solid tumors.

[0039] The transmembrane emp24 domain protein (TMED) family participates in immune regulation and tumor progression by modulating the endoplasmic reticulum-Golgi transport pathway. Recent research by the inventors indicates that TMED4 is a CD8... + Key regulatory factors of T or CAR-T anti-tumor immunity.

[0040] In previous techniques, TMED4 regulated ROS homeostasis through the IRE1α-XBP1 signaling axis, maintaining Foxp3 stability and immunosuppressive function in regulatory T (Treg) cells. TMED4 further promotes ROS homeostasis in Treg cells through NRF2-related antioxidant responses, ensuring the persistence of Foxp3 expression and inhibitory effects. In mouse models of tumors and autoimmune diseases, Treg cells lacking TMED4 lead to overactivation of effector T cells. A comparison was made between wild-type and Tmed4-specific knockout mice in Treg cells (…). Tmed4 ΔTreg The phenotype of ). Tmed4 ΔTreg Mouse models show that the absence of Tmed4 It can damage Treg cell function and trigger autoinflammatory symptoms.

[0041] In stark contrast to what has been observed in Treg cells, this invention discloses the ability to specifically target and suppress T cells (CD8). + T cells treated with TMED4 (or CAR-T) ASO showed enhanced killing activity.

[0042] Animal experiments show that CD8 + T cells Tmed4 The deletion enhanced the animal's anti-tumor response and promoted CD8. + T cell proliferation and infiltration enhance antigen-specific killing ability and reduce terminal exhaustion. Pharmacological inhibition of Tmed4 using antisense oligonucleotides (ASO) can further enhance CD8. + T-cell-mediated tumor control. This is a surprising effect.

[0043] This invention also discloses CAR-T cells treated with specific ASOs, which exhibit excellent tumor-killing activity. CD8 + T cells are distinctly different immune cells from Treg cells. This is due to the CD8+ receptor antagonist. + T cells are central to anti-tumor immunity, and CD8 is also the main cell component of CAR-T cells. + This invention uses antisense oligonucleotide (ASO) technology to silence the TMED4 protein level in CAR-T cells, revealing a novel drug to enhance the killing function of CAR-T cells.

[0044] In the embodiments of the present invention, antisense oligonucleotides (ASO) were synthesized artificially, and their function was demonstrated using Western blotting, animal model construction and subcutaneous tumor inoculation and monitoring in experimental animal science, and CAR-T cell construction in cell biology.

[0045] Antisense oligonucleotide (ASO) synthesis: ASO is an oligonucleotide that can induce gene silencing. Generally, ASOs are 15-22 nucleotides in length and are designed to be anticomplementary to target RNA, blocking RNA transcription, splicing, translation, or stability, thereby achieving therapeutic goals by inhibiting the expression of specific genes.

[0046] In a particularly preferred embodiment, the ASO is a modified, performance-enhanced antisense oligonucleotide; more preferably, the antisense oligonucleotide includes (but is not limited to) modifications selected from the group consisting of: 2'-methoxyethyl (2'-MOE) modification, methoxy modification, fluorinated modification, thiomodification, cholesterol modification, alkyl modification, locked nucleic acid modification, or a phosphothioester group linking adjacent nucleosides or between a nucleoside and a target ligand.

[0047] Preferably, the synthesis of ASO is carried out in three steps. First, antisense oligonucleotides are synthesized by adding nucleotide molecules one by one to a solid-phase support by adding protecting and active groups, and then purified and identified to ensure quality and purity. Second, Affinity Plus locked nucleic acids or 2'-MOE modifications are used at both ends of the antisense oligonucleotides to increase the stability of the nucleic acid and the binding affinity to the target RNA. Finally, natural chemical linkages (NCLs) are used as an efficient strategy for synthesizing CPP-ASO conjugates.

[0048] The ASO-targeted inhibition of T cells (CD8) of this invention + TMED4 (T or CAR-T) has precise targeting and ideal inhibitory effects.

[0049] Western blotting is a technique used in molecular biology to separate proteins from cells or tissues via electrophoresis. The proteins or peptides on the solid support act as antigens, triggering an immune reaction with corresponding antibodies. Further reactions occur with enzyme- or isotope-labeled secondary antibodies. The protein components in the gel are then detected by substrate color development or autoradiography. This technique is the most commonly used protein research method in molecular biology, widely applied to gene expression studies at the protein level, antibody activity detection, and early disease diagnosis.

[0050] Animal Model Construction and Subcutaneous Tumor Inoculation and Monitoring: Animal models, using animals as research subjects, are one of the tools in disease simulation, drug screening, gene therapy, and other research. In disease treatment and drug screening, animal models are a primary method for preliminary screening and testing of new drugs. In medical and life science research, animal models are widely used because they can provide physiological and behavioral characteristics similar to humans, helping scientists understand the development and treatment of human diseases. Mouse subcutaneous tumors are one of the important methods for studying tumor growth, invasion, metastasis, and drug efficacy. By monitoring differences in tumor growth volume, the effectiveness of drug treatment can be assessed.

[0051] T-cell adoptive infusion: T-cell adoptive infusion refers to the process of activating, expanding, and culturing the donor's own T cells in vitro to enhance their targeted killing function, and then reinfusing them into the recipient's body, thereby enhancing the recipient's anti-tumor ability and prolonging the recipient's survival period.

[0052] CAR-T cell adoptive delivery: CAR-T cell therapy involves genetically modifying T cells to express specific chimeric antigen receptors (CARs), enabling the T cells to recognize and attack tumor cells. The complete CAR-T cell manufacturing process includes key steps such as T cell isolation and collection, CAR structure design, CAR gene transduction into T cells, in vitro expansion of CAR-T cells, CAR-T cell reinfusion, and in vivo effects and monitoring. Target specificity and the T cell lifecycle are crucial factors determining the ultimate success of CAR-T cell therapy.

[0053] This invention also provides enhancement of T cells (CD8) + Compositions (pharmaceutical compositions or culture media) or kits that enhance T cell function (or CAR-T) include an effective amount of the aforementioned ASO that enhances T cell function.

[0054] The present invention also provides a pharmaceutical composition or kit for inhibiting tumors, comprising the aforementioned T cells (CD8). + (T or CAR-T) cultures, and pharmaceutically acceptable carriers or excipients.

[0055] As used herein, the term "effective amount" or "effective dose" means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals, as used herein.

[0056] As used herein, a "pharmaceuticalally acceptable" ingredient is a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents.

[0057] This invention also provides a method for enhancing T cells (CD8). + A kit for T-cell or CAR-T cell therapy or tumor suppression, wherein the kit includes: an effective amount of the ASO of the present invention, or the T-cell culture thereof. More preferably, the kit also includes: an instruction manual to guide clinicians in administering the medication correctly and appropriately.

[0058] For ease of administration, the ASO is formulated as a single dosage form and included in the kit. "Single dosage form" refers to a dosage form prepared for a single dose, including but not limited to various liquids (such as injections), solids (such as tablets), capsules, and sustained-release formulations. Furthermore, the ASO can also be individually dispensed into different containers for mixing and application as needed.

[0059] Although specific embodiments of the present invention provide dosing regimens for animals such as mice, it should be understood that converting animal dosages to human dosages is readily achievable by those skilled in the art, for example, using the Meeh-Rubner formula: Meeh-Rubner formula: A=k (W² / 3) / 10,000. Where A is the body surface area, expressed in meters. 2 Calculations; W is body weight, expressed in grams; K is a constant that varies depending on the animal species, for example, but not limited to: mice and rats 9.1, guinea pigs 9.8, rabbits 10.1, cats 9.9, dogs 11.2, monkeys 11.8, and humans 10.6. It should be understood that dosage conversions may vary depending on the drug and clinical circumstances, and are based on the assessment of an experienced pharmacist.

[0060] The invention will be better understood from the following examples. However, those skilled in the art will understand that the specific methods and results are merely for illustrating the invention and not for limiting it. Experimental methods in the following examples that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Science Press, or according to the manufacturer's recommendations.

[0061] Example 1: Synthesis and Verification of ASO-Tmed4

[0062] Based on the sequence: AACACCTCTTTCTGTT (SEQ ID NO: 1), a specific 2'-MOE full-chain modified antisense oligonucleotide (ASO-Tmed4) was prepared, with the sequence: ; in, ( ) represents a thiophosphate bond; ( / i2MOEr / ) represents 2'-O-(2-methoxyethyl) uniform modification.

[0063] According to ASO (ASO-control), the sequence is: .

[0064] Using GeticoFect 3000 as the transfection reagent, the cells were incubated with 75 μM control or TMED4 knockdown antisense oligonucleotides (ASO) at room temperature for 20 min, then added to activated T cell (CD8+ OT-I T cell) culture medium without antibiotics. After transfection for 48 hours, the silencing effect of TMED4 protein level was analyzed by Western blotting.

[0065] The results showed that ASO targeting TMED4 effectively silenced TMED4 expression in T cells. Figure 3 ).

[0066] Meanwhile, ASO targeting TMED4 can also effectively silence TMED4 expression in CAR-T cells (prepared in Example 3). Figure 4 ).

[0067] Example 2: Adoptive transfer model of ASO-treated T cells

[0068] CD8 + OT-I T cells: CD8 cells derived from OT-I mice (purchased from Shanghai Southern Model Biotechnology Co., Ltd.) + T cells.

[0069] Wild-type mice were subcutaneously inoculated with 1 × 10 6 MC38 tumor cells, 2×10⁸ intravenously infused after day 9. 6 A control (Ctrl) or Tmed4 knockdown antisense oligonucleotide (ASO) treatment (transfection) of CD8 + OT-I T cells. Infuse wild-type or Tmed4 knockdown CD8 cells every 2-3 days. + The volume of OT-I T cells in MC38 tumor-bearing mice was measured and tumor growth curves were plotted. Tumors were collected and weighed on day 20, and the tumor weight was quantitatively analyzed. The procedure is as follows: Figure 1 .

[0070] Transplantation of wild-type or Tmed4 knockdown of CD8 + Tumor growth curves of MC38 tumor-bearing mice carrying T cells are shown below. Figure 5 Transplantation of wild-type or Tmed4 knockdown of CD8 + Tumor weight quantification analysis of T cells in MC38 tumor-bearing mice, such as Figure 6 .

[0071] The results showed that silencing TMED4 expression in T cells by ASO significantly slowed tumor growth and enhanced the anti-tumor ability of mice.

[0072] Example 3: Preparation of mouse CAR-T cells targeting CLDN18.2

[0073] (1) CLDN18.2 CAR Construction

[0074] A second-generation mouse CLDN18.2 CAR was prepared, comprising CD8α as a signal peptide, CLDN18.2 scFv (reference PMID: 30203099) as an antigen-binding domain, and a myc tag for detection. This structure further incorporates CD8α as a hinge and transmembrane domain, 4-1BB (CD137) as a co-stimulatory domain, and CD3ζ for intracellular signal transduction. The complete CAR sequence was inserted into an MSCV retroviral vector to facilitate stable gene transfer.

[0075] A schematic diagram of the CLDN18.2 CAR construct is shown below. Figure 9 As shown.

[0076] (2) Reversing virus production

[0077] Reverse virus particles were produced by transfecting Eco cells with the CLDN18.2 construct. Eco cells were seeded in 10 cm culture dishes and transfected after 20 hours of culture. The medium was replaced with 7.5 mL of fresh medium before transduction. The transfection complex was prepared by mixing the pCL-Eco packaging plasmid and the CLDN18.2 plasmid with linear polyethyleneimine (PEI). After incubation at room temperature for 25 minutes, the transfection mixture was added to the cells. Six hours after transfection, the medium was replaced with 10 mL of pre-warmed medium containing sodium butyrate to enhance viral yield. Forty-eight hours after transfection, the viral supernatant was collected, centrifuged to remove cell debris, aliquoted, and stored at -80°C.

[0078] (3) CAR-T cell viral transduction

[0079] 24 hours prior to transduction, Dynabeads™ Mouse T-Activator CD3 / CD28 was administered in R10 medium (RPMI 1640 supplemented with 10% fetal bovine serum and β-mercaptoethanol) at a rate of 1×10⁻⁶. 6 Spleen cells from WT and Tmed4-KO mice were activated at a density of cells / mL and 80 U / mL IL-2 was added. Untreated 24-well plates were coated with RetroNectin for 2 hours, followed by overnight incubation at 4°C. 1 mL of viral supernatant was added to each well, and the plates were centrifuged at 2,000 g for 3 hours at 32°C to enhance viral adsorption. The activated T cells were resuspended in complete RPMI medium containing IL-2 and β-mercaptoethanol at a density of 1×10⁻⁶ cells / mL. 6 Inoculate virus-coated wells at a density of cells / mL. Centrifuge at 32°C and 600 g for 30 minutes for infection, then transfer the culture plate to an incubator for overnight culture.

[0080] Forty-eight hours later, the infection efficiency of CLDN18.2 CAR was assessed, and in vitro amplification was continued by replacing the culture medium with complete medium containing IL-2 (80 U / mL), IL-7 (10 ng / mL), and IL-15 (10 ng / mL).

[0081] Example 4: A model of adoptive infusion of ASO-treated CAR-T cells into tumors

[0082] Wild-type mice were subcutaneously inoculated with 1 × 10 6 A new MC38 tumor cell line, MC38-CLDN18.2, overexpressing tight junction protein 18.2 (Cdaudin18.2) was established. On day 9, mice were sacrificed and spleen cells were selected for T cell activation. Twenty-four hours later, the activated T cells were transfected with CAR virus, and CAR expression efficiency was measured after 48 hours. With good CAR expression efficiency, the prepared CAR-T cells were divided into two groups: a control group (ASO) and a TMED4-targeted knockdown group (ASO). On day 14, wild-type tumor-bearing mice were evenly divided into three groups according to tumor size: a non-infusion group (control treatment with only PBS); a CAR-T-ASO-Control group (2 × 10⁶ cells / mL infused via tail vein); and a CAR-T-ASO-Control group. 6 ASO-control CAR-T cells; CAR-T-ASO-TMED4 group, i.e., 2 × 10⁶ cells were infused via tail vein. 6 ASO-TMED4-CART cells. Tumor volume was measured every 2-3 days, and the tumor volume was calculated as: length Width Width 0.52; when the tumor volume reaches 1500 mmHg 3 Tumor-bearing mice were declared dead, and tumor growth curves and mouse survival curves were plotted based on tumor growth volume. The procedure is as follows: Figure 2 .

[0083] The results showed that CAR-T cells treated with ASO targeting TMED4 exhibited significantly stronger tumor control capabilities, and the tumor volume was significantly smaller than that of the control group and the non-infusion group. Figure 7 The survival rate of mice was also significantly improved. Figure 8 ).

[0084] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. Use of antisense oligonucleotides for preparing compositions that enhance T cell function; wherein the antisense oligonucleotides target and inhibit TMED4 of T cells, and the T cells are CD8+. + T cells or chimeric antigen receptor T cells.

2. The use as described in claim 1, characterized in that, The T cells described are CD8. + The tumor-killing function of T cells or chimeric antigen receptor T cells.

3. The use as described in claim 1, characterized in that, The antisense oligonucleotide is a modified, performance-enhanced antisense oligonucleotide; More preferably, the antisense oligonucleotide includes modifications selected from the group consisting of: 2'-methoxyethyl modification, methoxy modification, fluorinated modification, thiomodification, cholesterol modification, alkyl modification, locked nucleic acid modification, or a phosphothioester group linking adjacent nucleosides or between a nucleoside and a target ligand.

4. The use as described in claim 3, characterized in that, The sequence of the antisense oligonucleotide is: AACACCTCTTTCTGTT; Preferably, the antisense oligonucleotide is modified with 2'-methoxyethyl, and the structure of the 2'-methoxyethyl modified antisense oligonucleotide is as follows: 。 5. An antisense oligonucleotide for enhancing T cell function, wherein the structure of the 2'-methoxyethyl modified antisense oligonucleotide is as follows: 。 6. A method for enhancing T cell function, comprising: The T cells, which are CD8, are treated with antisense oligonucleotides. + T cells or chimeric antigen receptor T cells; The antisense oligonucleotide targets and inhibits TMED4 on T cells; preferably, the antisense oligonucleotide is the antisense oligonucleotide of claim 5.

7. The method as described in claim 6, characterized in that, The T cells described are CD8. + The tumor-killing function of T cells or chimeric antigen receptor T cells.

8. A T cell culture having an enhancing function, prepared by the method of claim 6 or 7; preferably, the T cells are CD8+. + T cells or chimeric antigen receptor T cells; preferably, the enhanced function is enhanced tumor killing function.

9. A composition or kit for enhancing T cell function, comprising the antisense oligonucleotide for enhancing T cell function as described in claim 5; wherein the T cells are CD8+. + T cells or chimeric antigen receptor T cells.

10. A pharmaceutical composition or kit for inhibiting tumors, comprising the T cell culture of claim 8, and a pharmaceutically acceptable carrier or excipient.

Images