Cholesterol metabolite-based TCR-t cell preparation and immunotherapy

Abstract

The present application relates to the technical field of adoptive cell transfer therapy, and particularly, to a cholesterol metabolite-based TCR-T cell preparation and an immunotherapy. In the present application, 7-hydroxy-hydroxylated cholesterol is used for treating T cells, the level of TCR signals is reduced, the memory function of T cells is enhanced, the memory cell proportion of T cells is increased, and therefore, the continuity of killing tumors is improved.

Description

A TCR-T cell preparation and immunotherapy based on cholesterol metabolites Technical Field

[0001] The present application relates to the field of adoptive immune cell therapy, and in particular to a TCR-T cell preparation and immunotherapy based on cholesterol metabolites. Background Art

[0002] Adoptive Cell Transfer Therapy (ACT) involves collecting the patient's own cells, modifying them in vitro to enhance their targeting capabilities, and then amplifying them before returning them to the patient's body to control the tumor. Currently commonly used therapies include TCR-T therapy and chimeric antigen receptor T cell therapy (CAR-T). T cells carrying exogenous TCRs that recognize tumor antigens are called TCR-T, and T cells carrying CARs that target specific tumor antigens are called CAR-T cells. Adoptive cell therapy is widely used in the treatment of tumors, but there are still many limitations. For example, the sustainability of cell therapy products in clinical treatment needs to be improved, especially in the application of immunotherapy for solid tumors.

[0003] Current methods for addressing the poor persistence of T-cell therapy all have limitations: Clinically, the main approach currently used is to clear lymphocytes before infusion, using drugs such as cyclophosphamide (cy), fludarabine (flu), and bendamustine (ben) to remove existing suppressive cells to prolong the persistence of the infused cells, but this method has a significant impact on the patient's condition. Alternatively, a population of T cells with strong stemness, namely the TSCM (Stem-like central memory T cells) population, can be infused, but obtaining this population of cells requires a large number of autologous T cells, which increases the difficulty and cost of processing. The optimal solution for early intervention in T-cell persistence is currently being explored.

[0004] It may be a better approach to perform stemness modification during the in vitro expansion stage of cell therapy products to enhance long-term persistence. Summary of the Invention

[0005] In view of the above-mentioned shortcomings of the prior art, the purpose of this application is to provide a TCR-T cell preparation and immunotherapy based on cholesterol metabolites. The immunotherapy of this application is applied to the in vitro expansion stage of TCR-T cells.

[0006] To achieve the above-mentioned and other related purposes, the present application provides, in a first aspect, the use of 7-hydroxy-hydroxylated cholesterol in preparing a product, wherein the product has at least one of the following functions:

[0007] 1) Reduce TCR signaling levels;

[0008] 2) Increase the proportion of memory T cells;

[0009] 3) Improve the persistence of T cells in killing tumors;

[0010] 4) As TCR-T cell preparation.

[0011] In any embodiment of the present application, the TCR signal level includes the CD69 level and / or the costimulatory molecule ICOS level.

[0012] In any embodiment of the present application, the TCR signal level is reflected as the phosphorylation level of TCR downstream molecules; preferably, the downstream molecules include CD3ζ, ZAP70 or PLCγ1.

[0013] In any embodiment of the present application, the T cell is selected from a TCR-T cell.

[0014] In any embodiment of the present application, the TCR-T cell is suitable for binding to a tumor antigen or a viral antigen; preferably, the viral antigen is selected from one or more of HBV, HPV, EBV, CMV, HERV and HIV; the tumor antigen is selected from tumor-associated antigens and tumor-specific antigens; more preferably, the tumor antigen is selected from one or more of NY-ESO-I, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, Mesothelin, Wilm's tumor-1, PRAME, AFP, gp100, MART-1, TP53-R175H, TP53-R273Q, CEA, KRAS G12D and KRAS G12V.

[0015] In any embodiment of the present application, the cancer type treated by TCR-T cells is selected from bladder cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, biliary tract cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, or thyroid cancer.

[0016] In any embodiment of the present application, the memory cells include stem cell-like central memory T cells.

[0017] In any embodiment of the present application, the improvement of the persistence of T cell killing of tumors is reflected in improving the proliferation ability of T cells or reducing the degree of cell exhaustion of T cells; preferably, the reduction of the degree of cell exhaustion of T cells is reflected in reducing the PD-1 expression level of T cells.

[0018] In any embodiment of the present application, the TCR-T cell preparation is used to treat TCR-T cells in vitro.

[0019] The second aspect of the present application provides a method for preparing a TCR-T cell preparation, comprising adding a 7-hydroxy-hydroxylated cholesterol solution to a cyclodextrin compound solution to form a complex solution, and freeze-drying to obtain a TCR-T cell preparation.

[0020] In any embodiment of the present application, the solvent of the 7-hydroxy-hydroxylated cholesterol solution is selected from an organic solvent; the organic solvent is selected from benzene, toluene, xylene, pentane, hexane, octane, cyclohexane, cyclohexanone, toluene cyclohexanone, chlorobenzene, dichlorobenzene, dichloromethane, methanol, ethanol, isopropanol, ether, propylene oxide, methyl acetate, ethyl acetate, propyl acetate, acetone, methyl butyl ketone, methyl isobutyl ketone, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, acetonitrile, pyridine, or phenol; preferably, the organic solvent is ethanol.

[0021] In any embodiment of the present application, the drug carrier is selected from a cyclodextrin compound; the cyclodextrin compound is selected from α-cyclodextrin or its derivatives, β-cyclodextrin or its derivatives, γ-cyclodextrin or its derivatives. Further, the α-cyclodextrin derivative is selected from α-hydroxypropyl cyclodextrin. The β-cyclodextrin derivative is selected from β-methylcyclodextrin, β-hydroxypropyl cyclodextrin, and β-sulfobutyl cyclodextrin; preferably, the β-cyclodextrin derivative is selected from β-methylcyclodextrin. The γ-cyclodextrin derivative is selected from γ-hydroxypropyl cyclodextrin.

[0022] In any embodiment of the present application, the solvent of the cyclodextrin compound solution is water.

[0023] In any embodiment of the present application, freeze-drying further includes re-dissolution to prepare a TCR-T cell preparation.

[0024] In any embodiment of the present application, the reconstituted solvent is a solvent compatible with the T cell culture system; preferably, the solvent compatible with the T cell culture system includes a combination of one or more of double-distilled water, PBS, and ethanol.

[0025] The third aspect of the present application provides a TCR-T cell preparation for reducing TCR signal levels, which is prepared by the aforementioned preparation method.

[0026] The fourth aspect of the present application provides a drug for reducing TCR signal levels, including an effective dose of a TCR-T cell preparation.

[0027] In any embodiment of the present application, the drug further includes a pharmaceutically acceptable carrier or excipient.

[0028] In a fifth aspect, the present application provides a method for reducing TCR signal levels, comprising contacting a subject with the aforementioned TCR-T cell preparation or the aforementioned drug.

[0029] In any embodiment of the present application, the subject is a mammalian cell.

[0030] In a sixth aspect, the present application provides a TCR-T cell with a reduced TCR signal level, wherein the TCR-T cell is a TCR-T cell obtained by treating T cells with the TCR-T cell preparation, and the T cell is a cell obtained by transfecting a viral expression vector containing TCR.

[0031] The seventh aspect of the present application provides an immune cell therapy for a disease, which comprises administering a therapeutically effective amount of the TCR-T cells to a subject.

[0032] Compared with the prior art, the present invention has the following advantages:

[0033] 1. This application discovered for the first time that treating T cells with the natural cholesterol metabolite 7a-HC can reduce TCR signaling levels, thereby enhancing the memory function of T cells. 7a-HC is an autologous metabolite and is safe and harmless.

[0034] 2. The present application found that 7a-HC can increase the proportion of memory T cells, thereby improving the persistence of tumor killing.

[0035] 3. This application uses 7a-HC to further improve the therapeutic effect of solid tumors in the treatment of HBV antigen-positive tumors. 7a-HC can regulate TCR signaling levels and can be widely used in TCR-T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1: a. Structural formulas of tested cholesterol precursors and hydroxylated cholesterol

[0037] Figure 1: b. Screening of cholesterol metabolites for their effects on the T cell activation marker CD69.

[0038] Figure 1: Effects of c.7a-HC on downstream signaling phosphorylation during T cell activation.

[0039] Figure 1: d. Toxicity of various cholesterol metabolites to T cells.

[0040] Figure 2: a. Effect of 7a-HC on ICOS levels in human PBMC-derived T cells.

[0041] Figure 2: Effect of b.7a-HC on the differentiation of human PBMC-derived T cell memory cell populations.

[0042] Figure 3: a. Schematic diagram of the experimental design of in vitro expansion of α-HBV-TCR-T cells and repeated liver tumor killing.

[0043] Figure 3: Effect of transient treatment with b.7a-HC on ICOS levels in α-HBV-TCR-T cells.

[0044] Figure 3: Effect of transient treatment with c.7a-HC on the differentiation of α-HBV-TCR-T cell memory cell population.

[0045] Figure 3: Effect of transient treatment with d.7a-HC on the killing function of α-HBV-TCR-T cells.

[0046] Figure 3: e. Changes in the number of α-HBV-TCR-T cells pretreated with 7a-HC under repeated liver tumor killing conditions.

[0047] Figure 3:f. PD-1 expression levels of α-HBV-TCR-T cells pretreated with 7a-HC in a repeated liver tumor killing context.

[0048] Figure 4: a. 7a-HC treatment scheme during the in vitro expansion stage of α-EBV-TCR-T cells.

[0049] Figure 4: Changes in the α-EBV-TCR-T cell memory population after transient treatment with b.7a-HC

[0050] Figure 4: c. α-EBV-TCR-T cell in vitro tumor killing experiment and functional detection.

[0051] Figure 5: a. Design of animal model for melanoma therapy using transient 7a-HC-treated TCR-T cells.

[0052] Figure 5: Effect of transient treatment with b.7a-HC on the memory of mouse TCR-T cells cultured in vitro.

[0053] Figure 5: Survival of TCR-T cells transiently treated with c.7a-HC after infusion into melanoma-bearing mice.

[0054] Figure 5: Control effect of TCR-T cells on mouse melanoma after transient treatment with d.7a-HC. DETAILED DESCRIPTION

[0055] In order to make the invention purpose, technical scheme and beneficial effects of this application clearer, the present application is further described below with reference to the examples. It should be understood that the examples are only used to explain this application and are not used to limit the scope of the application. Unless otherwise specified, the test methods used in the following examples are all conventional methods. People familiar with this technology can easily understand other advantages and effects of this application from the content disclosed in this description.

[0056] After extensive research, the inventors of this application discovered an immunotherapy based on cholesterol metabolites.

[0057] On the one hand, the present application provides the use of 7-hydroxy-hydroxylated cholesterol in preparing a product, the product having at least one of the following functions:

[0058] 1) Reduce TCR signaling levels;

[0059] 2) Increase the proportion of memory T cells;

[0060] 3) Improve the persistence of T cells in killing tumors;

[0061] 4) As TCR-T cell preparation.

[0062] This application unexpectedly discovered, through screening of multiple cholesterol metabolites, that 7-hydroxy-hydroxycholesterol (7a-HC) is the most effective in reducing TCR signaling levels. As a natural product, 7a-HC is non-toxic to cells. 7a-HC, short for 7-alpha-hydroxycholesterol, is a natural product in which cholesterol undergoes catalytic substitution of a hydroxyl group at the 7-position. It is the rate-limiting product in the bile acid synthesis pathway under physiological conditions.

[0063] In the use provided in the present application, the TCR signal level includes the CD69 level and / or the costimulatory molecule ICOS level.

[0064] In the use provided in the present application, the TCR signal level is reflected as the phosphorylation level of TCR downstream molecules; preferably, the downstream molecules include CD3z, ZAP70 or PLCg1.

[0065] In the use provided in the present application, the T cells are selected from TCR-T cells.

[0066] In any embodiment of the present application, the TCR-T cell is suitable for binding to a tumor antigen or a viral antigen; preferably, the viral antigen is selected from one or more of HBV, HPV, EBV, CMV, HERV, and HIV; the tumor antigen is selected from a screened and identified tumor-associated antigen (TAA) and a tumor-specific antigen (TSA); more preferably, the tumor antigen is selected from one or more of NY-ESO-1, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, Mesothelin, Wilm's tumor-1, PRAME, AFP, gp100, MART-1, TP53-R175H, TP53-R273Q, CEA, KRAS G12D, or KRAS G12V. The 7a-HC of the present application can be widely used in TCR-T anti-tumor or anti-viral scenarios.

[0067] In the uses provided herein, the cancer type treated by TCR-T cells is selected from bladder cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, biliary tract cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, or thyroid cancer. The present application uses 7a-HC to further improve the therapeutic effect of solid tumors in the treatment of HBV antigen-positive tumors. 7a-HC can be widely used in TCR-T cells to regulate TCR signaling levels.

[0068] In the use provided herein, memory cells include stem-like central memory T cells. Stem-like central memory T cells (SCM) are CD62L and CD45RA double-positive T cells with the strongest stemness indication.

[0069] In the uses provided in the present application, improving the persistence of T cells in killing tumors is reflected in improving the proliferation ability of T cells or reducing the degree of cell exhaustion of T cells; preferably, reducing the degree of cell exhaustion of T cells is reflected in reducing the PD-1 expression level of T cells.

[0070] In the use provided in the present application, the TCR-T cell preparation is used to treat TCR-T cells in vitro.

[0071] On the other hand, the present application provides a method for preparing a TCR-T cell preparation, comprising adding a 7-hydroxy-hydroxylated cholesterol solution to a cyclodextrin compound solution to form a complex solution, and freeze-drying to obtain a TCR-T cell preparation.

[0072] In the preparation method provided herein, the solvent of the 7-hydroxy-hydroxylated cholesterol solution is selected from an organic solvent. Specifically, the organic solvent is selected from benzene, toluene, xylene, pentane, hexane, octane, cyclohexane, cyclohexanone, toluene-cyclohexanone, chlorobenzene, dichlorobenzene, dichloromethane, methanol, ethanol, isopropanol, ether, propylene oxide, methyl acetate, ethyl acetate, propyl acetate, acetone, methyl butyl ketone, methyl isobutyl ketone, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, acetonitrile, pyridine, or phenol. In a specific embodiment of the present application, the organic solvent is ethanol.

[0073] In the preparation method provided in the present application, cyclodextrin compound (CD for short) is a general term for a series of cyclic oligosaccharides produced by amylose under the action of cyclodextrin glucosyltransferase produced by Bacillus, usually containing 6 to 12 D-pyranose units. It mainly includes molecules containing 6, 7, and 8 glucose units, which are called alpha-, beta- and gamma-cyclodextrins, respectively. Cyclodextrin compounds can crystallize well in aqueous solution and alcohol-water solution; they have no fixed melting point, begin to decompose when heated to about 200°C, and have good thermal stability; they are not hygroscopic, but easily form various stable hydrates. Cyclodextrin can effectively increase the solubility and dissolution rate of some poorly water-soluble drugs in water.

[0074] In some embodiments, the cyclodextrin compound is selected from α-cyclodextrin or its derivatives, β-cyclodextrin or its derivatives, γ-cyclodextrin or its derivatives. Furthermore, the α-cyclodextrin derivative is selected from α-hydroxypropyl cyclodextrin. The β-cyclodextrin derivative is selected from β-methylcyclodextrin, β-hydroxypropyl cyclodextrin, and β-sulfobutyl cyclodextrin. The γ-cyclodextrin derivative is selected from γ-hydroxypropyl cyclodextrin. In a specific embodiment of the present application, the cyclodextrin compound is Mb-CD (β-methylcyclodextrin). The solvent of the cyclodextrin compound solution is water.

[0075] In the preparation method provided herein, the concentration of 7a-HC in the 7-hydroxy-hydroxylated cholesterol solution, based on the total volume of the 7a-HC solution, can be, for example, 5 to 10 mg / mL; specifically, it can be 5 to 6 mg / mL, 6 to 8 mg / mL, or 8 to 10 mg / mL, etc.

[0076] In the preparation method provided herein, the cyclodextrin compound solution needs to be preheated before use, for example, to 80 degrees Celsius. The concentration of the cyclodextrin compound can be, for example, 5-10% (w / v) based on the total volume of the cyclodextrin compound solution; specifically, 5-6% (w / v), 6-7% (w / v), or 7-10% (w / v).

[0077] In a specific embodiment of the present application, a 7-hydroxy-hydroxylated cholesterol solution is added dropwise to a cyclodextrin compound solution, and after the solution is clarified, it is returned to room temperature, the original solvent is removed by freeze-drying, and the TCR-T cell preparation is prepared by redissolving. The redissolved solvent is a solvent compatible with the T cell culture system. In some embodiments, the solvent compatible with the T cell culture system includes a suitable solvent such as double distilled water, PBS, and ethanol. In a specific embodiment of the present application, the solvent compatible with the T cell culture system is double distilled water. Based on the total volume of the TCR-T cell preparation, the concentration of 7a-HC during redissolution can be 1 to 10 mg / mL; specifically, it can be 1 to 3 mg / mL, 3 to 8 mg / mL, or 8 to 10 mg / mL, etc.

[0078] On the other hand, the present application provides a TCR-T cell preparation that reduces the TCR signal level, which is prepared by the aforementioned preparation method.

[0079] On the other hand, the present application provides a drug for reducing TCR signal levels, comprising an effective dose of the aforementioned TCR-T cell preparation.

[0080] The effective dose refers to the dose at which a drug is effective. A drug must be absorbed by the body at a certain dose to reach a certain concentration, and only then will its effects manifest. If the dose is too low, the effective concentration in the body cannot be achieved, and the drug will not exert its effective effect. However, if the dose is too high, exceeding a certain limit, the drug's effects may change qualitatively, potentially causing varying degrees of toxicity to the body. Therefore, to ensure the drug's effective effects while avoiding adverse reactions, it is necessary to strictly control the dosage range.

[0081] The medicine provided in this application also includes pharmaceutically acceptable carriers or excipients.

[0082] "Pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when appropriately administered to an animal or a human.

[0083] "Pharmaceutically acceptable carriers or excipients" should be compatible with the TCR-T cell formulation, meaning they can be blended with it without significantly reducing the efficacy of the pharmaceutical composition under normal circumstances. Specific examples of substances that can serve as pharmaceutically acceptable carriers or excipients include sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols, such as propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; colorants; flavorings; tableting agents, stabilizers, antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffer. These materials are used as needed to aid in the stability of the formulation or to help increase the activity or its bioavailability.

[0084] In the TCR-T cell preparation or drug provided in the present application, the TCR-T cell preparation or drug reduces the phosphorylation level of TCR downstream molecules, thereby reducing the TCR signal level, and can increase the proportion of memory cells of T cells, thereby improving the sustainability of tumor killing.

[0085] On the other hand, the present application provides a method for reducing the level of TCR signaling, comprising contacting a subject with the aforementioned TCR-T cell preparation or drug. The main target is mammalian cells. Among them, mammals are, for example, rodents, artiodactyls, perissodactyls, lagomorphs, primates, etc. Primates are, for example, monkeys, apes or Homo sapiens. The mammalian cells are specifically T cells; preferably, they are TCR-T cells. In some embodiments, the cancer type treated by TCR-T cells is selected from bladder cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, biliary tract cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer or thyroid cancer.

[0086] The use of the TCR-T cell preparation or drug of the present application is specifically to perform multiple in vitro transient pretreatments during TCR-T amplification. Transient pretreatment specifically refers to adding TCR-T cell preparations or drugs to a T cell suspension in the absence of serum, and washing them away after incubation. The incubation time can be, for example, 5 to 60 minutes, for example, 10 to 40 minutes; specifically, it can be 10 to 20 minutes, 20 to 30 minutes or 30 to 40 minutes, etc. The working concentration of the TCR-T cell preparation or drug can be, for example, 0.1 to 10 ng / μL, specifically 0.1 to 0.2 ng / μL, 0.2 to 0.4 ng / μL, 0.4 to 0.6 ng / μL, 0.6 to 0.8 ng / μL, 0.8 to 1.0 ng / μL, 1 to 2 ng / μL, 2 to 5 ng / μL, or 5 to 10 ng / μL, etc. The working concentration here refers to the concentration when in contact with the T cell suspension.

[0087] On the other hand, the present application provides a TCR-T cell with a reduced TCR signal level, wherein the TCR-T cell is a TCR-T cell obtained by treating T cells with the TCR-T cell preparation, and the T cell is a cell obtained by transfecting a viral expression vector containing TCR.

[0088] In certain embodiments of the present invention, the treatment concentration of the TCR-T cell preparation is 0.1-10 ng / μL; and / or the treatment time is 5-60 min; and / or the treatment temperature is 35-38 degrees Celsius; and / or the number of treatments is one or more. The treatment concentration refers to the concentration at the time of contact with T cells, based on the final volume of the system.

[0089] The TCR-T cell is suitable for binding to tumor antigens or viral antigens; the specific antigen type is not specifically limited in the present invention. Preferably, the viral antigen is selected from one or more of HBV, HPV, EBV, CMV, HERV, and HIV; the tumor antigen is selected from tumor-associated antigens and tumor-specific antigens; more preferably, the tumor antigen is selected from one or more of NY-ESO-I, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, Mesothelin, Wilm's tumor-1, PRAME, AFP, gp100, MART-1, TP53-R175H, TP53-R273Q, CEA, KRAS G12D, or KRAS G12V.

[0090] In certain embodiments of the present invention, the cancer type treated by the TCR-T cells is selected from bladder cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, biliary tract cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer or thyroid cancer.

[0091] The viral expression vector is selected from retrovirus, lentivirus, adenovirus or adeno-associated virus.

[0092] On the other hand, the present application provides an immune cell therapy for a disease, which comprises administering a therapeutically effective amount of the TCR-T cells to a subject.

[0093] "Subjects" include, but are not limited to, animals, preferably mammals; the mammals are preferably rodents, artiodactyls, perissodactyls, lagomorphs, primates, etc. The mammals include, for example, humans, non-human primates (e.g., monkeys), mice, pigs, cows, goats, rabbits, rats, guinea pigs, hamsters, horses, monkeys, sheep, or other non-human mammals; non-mammals include, for example, non-mammalian vertebrates, such as birds (e.g., chickens or ducks) or fish, and non-mammalian invertebrates. The subject can be a human, such as a patient with a weakened immune system or cancer.

[0094] "Cancer" in this application refers to any medical condition mediated by the growth, proliferation or metastasis of tumor or malignant cells, and causes solid tumors and non-solid tumors such as leukemia. "Tumor" in this invention refers to the physical substance of tumor and / or malignant cells.

[0095] "Treatment" or "treatment" of a condition includes preventing or alleviating the condition, reducing the rate of onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, causing complete or partial reversal of a condition, curing a condition, or some combination thereof. With respect to cancer, "treat" or "treatment" may refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination thereof. With respect to tumors, "treat" or "treatment" includes eliminating all or part of a tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor progression, or some combination thereof.

[0096] Such cancers are, for example, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer; leukemia, lymphoma, myeloma, mycoses fungoids, Merkel cell carcinoma and other hematological malignancies, such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary effusion lymphoma.

[0097] As used herein, a "therapeutically effective amount" or "effective dose" refers to a dose or concentration of a drug that is effective in treating a disease or condition associated with an antigen. For example, with respect to the use of an antibody or antigen-binding fragment thereof disclosed herein, a therapeutically effective amount is a dose or concentration at which the antibody or antigen-binding compound can eliminate all or part of a tumor, inhibit or slow tumor growth, inhibit the growth or proliferation of cells that mediate a cancerous condition, inhibit tumor cell metastasis, alleviate any symptoms or markers associated with a tumor or cancerous condition, prevent or delay the progression of a tumor or cancerous condition, or some combination thereof.

[0098] Specifically, when administered to a subject, the dosage varies depending on the patient's age and weight, disease characteristics and severity, and route of administration. The results of animal experiments and various circumstances may be referenced, and the total dosage cannot exceed a certain range.

[0099] In some embodiments, the methods described herein may further comprise co-administration with other compounds or other cancer treatment regimens known in the art.

[0100] Other cancer treatment options may include, but are not limited to, surgery, radiation therapy, chemotherapy, toxin therapy, cryotherapy, cancer vaccines (e.g., HPV vaccine, hepatitis B vaccine), and gene therapy, and any combination thereof.

[0101] The immunotherapy of the present invention includes but is not limited to adoptive cell therapy, derivation of stem cells and / or dendritic cells, blood transfusion, lavage, etc.

[0102] The present application is further described below by way of examples, but the scope of the present application is not limited thereby.

[0103] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention are all based on the

[0104] Conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related fields are used and are well described in the literature.

[0105] Example 1

[0106] 7a-HC inhibits T cell signaling activation

[0107] In the early stage, various cholesterol metabolites (including cholesterol synthesis precursors and hydroxylated cholesterol, the structural formula and name are shown in Figure 1a) were used to identify the effects of these metabolites on T cell signaling function. In order to enhance the water solubility of cholesterol metabolites, various cholesterol metabolites were first dissolved in ethanol to prepare various cholesterol metabolite solutions with a concentration of 10 mg / mL, and then formed into complexes with cyclodextrin compounds. Various cholesterol metabolite solutions were added dropwise to a 5% (w / v) Mb-CD (β-methylcyclodextrin) aqueous solution preheated at 80 degrees Celsius. After the solution was clarified, it was returned to room temperature, the original solvent was removed by freeze-drying, and it was re-dissolved to 1 mg / mL with sterile double-distilled water to prepare various cholesterol metabolite treatment solutions. Mice were taken CD8 + T cells were subjected to transient metabolite treatment, that is, to a density of 1*10 6 Various cholesterol metabolite treatment solutions were added to a T cell suspension of 100 cells / mL to a final concentration of 2 ng / μL. Metabolites were washed away after 20 minutes. Cells were then stimulated with an α-CD3 antibody for 2 hours, and surface CD69 expression was measured by flow cytometry to reflect activation levels.

[0108] As shown in Figures 1:a and 1:b, the results showed that T cells treated with 7a-HC showed lower CD69 levels than those treated with other metabolites (Figure 1b). CD4 + When examining protein samples, it was found that with the cross-linking stimulation of α-CD3 / CD28 antibodies, the signals of phosphorylated proteins downstream of the TCR signal (p-CD3ζY142, CST#67748; p-ZAP70 Y319, CST#2717; p-PLCγ1Y783, CST#2821) increased significantly more slowly and with lower intensity when treated with 7a-HC compared to the control group, indicating that 7a-HC can reduce the phosphorylation level of TCR downstream during activation (Figure 1c), indicating that it can significantly inhibit T cell signal activation. Subsequently, Annexin-V staining was used to detect T cell apoptosis after treatment with the above-mentioned cholesterol metabolites. As shown in Figure 1:d, it was found that transient treatment with 7a-HC had no obvious toxic effect on T cells.

[0109] Example 2

[0110] 7a-HC is beneficial for the formation of memory T cell populations

[0111] It is generally believed that lower TCR signals are conducive to the formation of memory T cell populations. Based on the T cell signal inhibitory function of 7a-HC, 7a-HC is used to treat T cells to achieve the purpose of enhancing the memory cell population.

[0112] Human PBMC-derived T cells were transiently treated with 7a-HC every three days. TCR signaling and memory cell differentiation were assessed before the third treatment. ICOS, a marker of T cell signaling, was identified. Surface staining of T cells with an ICOS antibody, followed by flow cytometry analysis, revealed that T cells treated with both concentrations (1 ng / μL and 2 ng / μL) exhibited lower levels of the costimulatory molecule ICOS, as shown in Figure 2a, reflecting lower TCR signaling. T cells positive for both CD62L and CD45RA, representing the most stem-like memory T cell population, known as stem-like central memory (SCM), were significantly increased in 7a-HC-treated T cells, as shown in Figure 2b.

[0113] Example 3

[0114] 7a-HC is beneficial for the sustained killing of liver tumors expressing HBV antigens

[0115] In order to study whether the above treatment method can demonstrate stronger anti-tumor function in TCR-T cell therapy, this example constructed an in vitro TCR-T cell expansion and repeated killing system to simulate the clinical cell therapy scenario, as shown in Figure 3: a. Hepatocellular carcinoma is a common tumor with a high incidence in China. Its main cause is long-term infection with hepatitis B virus HBV. Therefore, HBV can be used as a target for TCR-T treatment of liver cancer. This example uses TCR-T (α-HBV-TCR-T, structure is TRAC+T2A+TRBC, amino acid sequence is shown in SEQ ID NO.1) cells that recognize hepatitis B antigens as a research model to simulate the killing of HBV-positive tumor cells.

[0116] To prepare these cells, HEK 293FT cells were used to produce α-HBV-TCR lentivirus. After ultracentrifugation, the virus was enriched and then infected with PBMC cells that had been activated with α-CD3 / CD28 Dynabeads (Gibco, #11161D) for 48 hours. After 24 hours, the virus and Dynabeads were removed. Subsequently, the cells were expanded in vitro for 12 days in X-VIVO 15 medium (Lonza, #02-060Q) to obtain sufficient cell numbers to simulate the expansion phase of clinical TCR-T production. During this period, the T cells were transiently treated with 7a-HC every 3 days, following the same steps as in Example 1.

[0117] Before the third treatment, antibody staining was used to detect the level of ICOS, a T cell signal intensity marker, and memory cell differentiation markers (CCR7 and CD45RA) on the surface of T cells treated with 7a-HC by flow cytometry. It was found that cells treated with 7a-HC had lower ICOS expression and more stem cell-like central memory T cells (CCR7 + CD45RA + ), as shown in Figures 3b and 3c. Because 7a-HC can inhibit TCR signaling, the killing ability of TCR-T cells was tested three days after transient 7a-HC treatment by measuring the intensity of intracellular luciferase release in target cells, which reflects the lytic and killing ability of TCR-T cells against target cells. The killing ability of TCR-T cells pretreated with 7a-HC at this stage was not significantly different from that of the control group, as shown in Figure 3d. After 12 days of culture, TCR-T cells were co-cultured with Huh7 hepatoma cell lines expressing HBV antigens at a 2:1 ratio, marking the first round of killing. Three days after co-culture, a new batch of tumor cells was added, marking the second round of killing. This process was repeated to simulate a continuous killing scenario in vivo. T cell counts were measured every three days during this process. In the later stages of repeated killing, 7a-HC-pretreated T cells exhibited a certain proliferation advantage and maintained a certain level of proliferation even when the cell number in the control group began to decrease, indicating a stronger persistence of killing, as shown in Figure 3e. In addition, through α-PD-1 flow cytometry surface staining, the results showed that TCR-T cells pretreated with 7a-HC showed lower PD-1 expression levels in the fifth round of killing, suggesting that the degree of exhaustion of these cells may be lower, as shown in Figure 3: f.

[0118] Example 4

[0119] 7a-HC is beneficial for sustained killing of blood tumors expressing EBV antigens

[0120] EBV is a virus with an infection rate of over 95% in the Chinese population. Many malignant tumors and diseases show a strong correlation with EBV, including nasopharyngeal carcinoma and gastric cancer, which are highly prevalent in southern my country, as well as a variety of malignant lymphoma diseases. EBV-related diseases are usually treated with traditional radiotherapy, chemotherapy and other treatments, which have relatively obvious side effects and the risk of recurrence. In recent years, the emerging adoptive cell therapy has shown great potential in the treatment of virus-induced tumors, but there are still defects such as the short maintenance time of T cells in the body and insufficient disease control ability. There is an urgent need to develop a regulatory method to promote the long-term function of T cells. This embodiment aims to construct TCR-T cells targeting EBV latent protein, and on the basis of Example 3, simplify the processing steps as much as possible to establish a reasonable and effective TCR-T sterol regulatory system to promote the persistence and therapeutic effect of TCR-T cells targeting EBV.

[0121] At different stages after primary PBMC thawing, cells were transiently treated with 7a-HC for 20 minutes in a 37°C water bath to a final concentration of 1 ng / μL. Group 1 included cells treated immediately after thawing, followed by stimulation with α-CD3 / CD28 and lentiviral transfection with α-EBV-TCR (structure: TRAC+T2A+TRBC, amino acid sequence shown in SEQ ID NO. 2); Group 2: cells were stimulated with α-CD3 / CD28 and lentiviral transfection with α-EBV-TCR after thawing, and then, on day 3, the virus and α-CD3 / CD28 stimulation were removed and treated with 1 ng / μL 7a-HC; Group 3: cells were stimulated and transfected as in Group 2 after thawing, and then treated with 1 ng / μL 7a-HC on day 7 of thawing; Group 4: cells were stimulated and transfected as in Group 2 after thawing, and then treated with 2 ng / μL 7a-HC on day 7 of thawing. The control group received no treatment, as shown in Figure 4:a.

[0122] On the tenth day after recovery, the memory cell differentiation markers (CCR7 and CD45RA) were detected, and it was found that the α-EBV-TCR-T cells in the second and third groups treated with 7a-HC had more stem cell-like central memory T cells (CCR7 and CD45RA) than those in the control group, both CD4 and CD8 T cells. + CD45RA + ), indicating that the treatment methods for the second and third groups are more appropriate, as shown in Figure 4: b.

[0123] Subsequently, the second and third treatment groups were subjected to in vitro tumor killing, targeting myeloid hematologic malignancies transfected with EBV antigens. After two repeated killings, the a-EBV-TCR-T cells transiently treated with 7a-HC in both groups showed a strong proliferation advantage, as shown in Figure 4:c.

[0124] Example 5

[0125] 7a-HC is beneficial in controlling melanoma progression in a mouse model

[0126] To further validate the transient effects of 7a-HC on TCR-T cells, mice inoculated with B16 melanoma cells expressing the OVA antigen were infused with OT-I TCR-T cells that specifically recognize OVA and that had been treated in vitro with 7a-HC to establish an adoptive transfer therapy model. The OT-I-OVA system is a publicly available TCR system from The Jackson Lab, where the OT-I mice were derived.

[0127] The in vitro treatment process of OT-I T cells is shown in Figure 5: a. The spleen cells of OT-I mice were first activated with OVA 257-264 peptide, then the OVA peptide was withdrawn and treated with 7a-HC every 2 days at a final concentration of 1 ng / μL in a 37°C water bath for 20 minutes. The memory cell population was detected on the sixth day. - CD62L + The cell population has the strongest memory transcription factor TCF1, and the proportion of this group of cells was significantly expanded after 7a-HC treatment, as shown in Figure 5:b.

[0128] Subsequently, CD45.1 treated with 7a-HC + OT-I T cells were infused into mice bearing B16 melanoma. Nine days after infusion, the survival of circulating exogenous T cells was assessed. The proportion of T cells treated with 7a-HC was significantly increased compared to the control group, as shown in Figure 5c.

[0129] After the 7a-HC-treated OT-I T cells were reinfused, the tumor control effect was significantly enhanced and the survival time of the mice was effectively prolonged, as shown in Figure 5:d (wherein, PBS is phosphate buffered saline, used as a control).

[0130] In summary, this application has discovered for the first time that treating T cells with 7a-HC, a natural cholesterol metabolite, can reduce TCR signaling levels, thereby enhancing the memory function of T cells. 7a-HC is an autologous metabolite and is safe and harmless. This application has found that 7a-HC can increase the proportion of memory T cells, thereby improving the persistence of tumor killing. This application uses 7a-HC to further improve the therapeutic effect of solid tumors in the treatment of HBV antigen-positive tumors. 7a-HC can be widely used in TCR-T cells to regulate TCR signaling levels.

[0131] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit this application. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical concepts disclosed herein shall be covered by the claims of this application.

Claims

1. Use of 7-hydroxy-hydroxylated cholesterol in the preparation of a product, wherein the product has at least one of the following functions: 1) Reduce TCR signaling levels; 2) Increase the proportion of memory T cells; 3) Improve the persistence of T cells in killing tumors; 4) As TCR-T cell preparation.

2. The use according to claim 1, characterized in that The TCR signal level includes the CD69 level and / or the costimulatory molecule ICOS level.

3. The use according to claim 1, characterized in that The TCR signal level is reflected in the phosphorylation level of TCR downstream molecules; preferably, the downstream molecules include CD3ζ, ZAP70 or PLCγ1.

4. The use according to claim 1, wherein The T cells are selected from TCR-T cells.

5. The use according to claim 4, characterized in that The TCR-T cell is suitable for binding to a tumor antigen or a viral antigen; preferably, the viral antigen is selected from one or more of HBV, HPV, EBV, CMV, HERV and HIV; the tumor antigen is selected from tumor-associated antigens and tumor-specific antigens; more preferably, the tumor antigen is selected from one or more of NY-ESO-I, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, Mesothelin, Wilm's tumor-1, PRAME, AFP, gp100, MART-1, TP53-R175H, TP53-R273Q, CEA, KRAS G12D or KRAS G12V.

6. The use according to claim 4, characterized in that The cancer type treated by the TCR-T cells is selected from bladder cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, biliary tract cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, or thyroid cancer.

7. The use according to claim 1, characterized in that The memory cells include stem cell-like central memory T cells.

8. The use according to claim 1, characterized in that The improvement of the persistence of T cell killing of tumors is reflected in improving the proliferation ability of T cells or reducing the degree of cell exhaustion of T cells; preferably, the reduction of the degree of cell exhaustion of T cells is reflected in reducing the PD-1 expression level of T cells.

9. The use according to claim 1, characterized in that The TCR-T cell preparation is used for treating TCR-T cells in vitro.

10. A method for preparing a TCR-T cell preparation, comprising adding a 7-hydroxy-hydroxylated cholesterol solution to a cyclodextrin compound solution to form a complex solution, and freeze-drying the solution to obtain the TCR-T cell preparation.

11. The preparation method according to claim 10, characterized in that The solvent of the 7-hydroxy-hydroxylated cholesterol solution is selected from an organic solvent; preferably, the organic solvent is selected from benzene, toluene, xylene, pentane, hexane, octane, cyclohexane, cyclohexanone, toluene-cyclohexanone, chlorobenzene, dichlorobenzene, dichloromethane, methanol, ethanol, isopropanol, ether, propylene oxide, methyl acetate, ethyl acetate, propyl acetate, acetone, methyl butyl ketone, methyl isobutyl ketone, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, acetonitrile, pyridine, or phenol; more preferably, the organic solvent is ethanol.

12. The preparation method according to claim 10, wherein The cyclodextrin compound is selected from α-cyclodextrin or its derivatives, β-cyclodextrin or its derivatives, γ-cyclodextrin or its derivatives; and / or the solvent of the cyclodextrin compound solution is water.

13. The preparation method according to claim 12, wherein The α-cyclodextrin derivative is selected from α-hydroxypropyl cyclodextrin; And / or, the β-cyclodextrin derivative is selected from β-methylcyclodextrin, β-hydroxypropylcyclodextrin, β-sulfobutylcyclodextrin; preferably, the β-cyclodextrin derivative is selected from β-methylcyclodextrin. And / or, the γ-cyclodextrin derivative is selected from γ-hydroxypropyl cyclodextrin.

14. The preparation method according to claim 10, wherein The method further includes re-dissolving after freeze-drying to prepare a TCR-T cell preparation.

15. The method according to claim 14, wherein The reconstituted solvent is a solvent compatible with the T cell culture system; preferably, the solvent compatible with the T cell culture system includes a combination of one or more of double-distilled water, PBS, and ethanol.

16. A TCR-T cell preparation for reducing TCR signal levels, prepared by the preparation method according to any one of claims 10 to 15.

17. A drug for reducing TCR signal levels, comprising an effective dose of the TCR-T cell preparation according to claim 16.

18. A method for reducing TCR signaling levels, comprising contacting a subject with the TCR-T cell preparation of claim 16 or the medicament of claim 17.

19. The method of claim 18, wherein the subject is a mammalian cell.

20. A TCR-T cell with reduced TCR signaling level, characterized in that The TCR-T cells are TCR-T cells obtained by treating T cells with the TCR-T cell preparation according to claim 16, and the T cells are cells obtained by transfecting a viral expression vector containing TCR.

21. The TCR-T cell according to claim 20, characterized in that The treatment concentration of the TCR-T cell preparation is 0.1-10 ng / μL; and / or the treatment time is 5-60 min; and / or the treatment temperature is 35-38 degrees Celsius; and / or the treatment times are one or more.

22. The TCR-T cell according to claim 20, characterized in that The TCR-T cell is suitable for binding to a tumor antigen or a viral antigen; preferably, the viral antigen is selected from one or more of HBV, HPV, EBV, CMV, HERV and HIV; the tumor antigen is selected from tumor-associated antigens and tumor-specific antigens; more preferably, the tumor antigen is selected from one or more of NY-ESO-I, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, Mesothelin, Wilm's tumor-1, PRAME, AFP, gp100, MART-1, TP53-R175H, TP53-R273Q, CEA, KRAS G12D or KRAS G12V.

23. The TCR-T cell according to claim 20, characterized in that The cancer type treated by the TCR-T cells is selected from bladder cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, biliary tract cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer or thyroid cancer.

24. An immune cell therapy for a disease, characterized in that: The immune cell therapy comprises administering a therapeutically effective amount of the TCR-T cell of claim 20 to a subject.

25. The immune cell therapy according to claim 24, characterized in that The subject is a patient suffering from any one or more of the following cancers: bladder cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, biliary tract cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer or thyroid cancer.

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