Application of cytarabine combined with immune checkpoint inhibitors in the treatment of glioma

Abstract

The present invention provides an application of cytarabine combined with an immune checkpoint inhibitor in the treatment of glioma. Experiments have proved that cytarabine combined with an immune checkpoint inhibitor has a synergistic effect in the treatment of glioma. Cytarabine combined with an immune checkpoint inhibitor can enhance the anti-tumor effect of cytarabine and significantly prolong the survival of mice, providing a new strategy for the treatment of glioma.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to the application of cytarabine combined with immune checkpoint inhibitors in the treatment of glioma. Background Art

[0002] Gliomas are the most common primary malignant tumors of the central nervous system, and they can occur anywhere in the central nervous system. The symptoms and prognosis of gliomas are mainly determined by the location of the glioma, the growth rate and its space-occupying effect. The symptoms are mainly manifested as limb dysfunction, epilepsy, visual field defects, headache and dizziness, nausea and vomiting, cognitive impairment and even coma, paralysis and death. At present, the standard treatment of gliomas includes surgery to remove the tumor to the greatest extent, as well as postoperative radiotherapy and TMZ chemotherapy. Surgery is the main means of treating gliomas. Many studies have confirmed the value of tumor resection range in improving overall survival, progression-free survival, malignant transformation time and epilepsy control. However, if the glioma infiltrates into the cortical and subcortical areas that are important for language, movement and neurocognitive functions, it is difficult to achieve a large total resection during surgery.

[0003] Glioma is divided into adult diffuse glioma, childhood diffuse low-grade glioma, childhood diffuse high-grade glioma, and localized astrocytic glioma. Diffuse midline glioma (H3-DMG) caused by H3 K27 mutation usually occurs in the thalamus, brainstem and spinal cord, and is a highly malignant tumor common in children and adolescents. The average median survival of this tumor is less than 1 year, and the five-year survival rate is less than 1%. Its high mortality and disability rates have brought heavy economic and psychological burdens to the patient's family and society. Because the location of this tumor is in the super-functional area, surgery often cannot completely remove it, and some patients cannot even undergo surgical treatment. Therefore, other treatment methods besides surgery are of great value in improving patient prognosis.

[0004] Radiotherapy is currently the only standard treatment option for H3-DMG other than surgery. However, at the current standard radiotherapy dose, the effect of radiotherapy on prolonging the survival of H3-DMG is still very limited. Increasing the radiotherapy dose is a potential way to improve patient survival, but the risk of damage to patient cognition and other functions will also increase sharply. Studies have shown that chemotherapy after radiotherapy, or radiotherapy combined with chemotherapy, cannot effectively improve the prognosis and survival of H3-DMG. These chemotherapy regimens include the use of cisplatin, etoposide, vincristine, or a combination of cisplatin, etoposide and ifosfamide, and even temozolomide, a first-line chemotherapy drug for glioblastoma.

[0005] Therefore, the development of new treatment options is urgently needed. Summary of the invention

[0006] In order to solve the technical problems existing in the prior art, the present invention provides the use of cytarabine combined with immune checkpoint inhibitors in the treatment of glioma. Experiments have proved that cytarabine combined with immune checkpoint inhibitors has a synergistic effect in the treatment of glioma. Cytarabine combined with immune checkpoint inhibitors can enhance the anti-tumor effect of cytarabine and significantly prolong the survival of mice, providing a new strategy for the treatment of glioma.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides a pharmaceutical composition, which comprises cytarabine or a pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor.

[0009] In the present invention, the cytarabine may be cytarabine itself, or a hydrate, a solvate or a crystal form of cytarabine (hereinafter also referred to as a compound).

[0010] In the present invention, the pharmaceutically acceptable salt refers to an acid salt formed with an inorganic and / or organic acid and a basic salt formed with an inorganic and / or organic base. In addition, when the compound contains a basic portion (such as but not limited to, pyridine or imidazole) and an acidic portion (such as but not limited to, carboxylic acid), zwitterions can be formed and the zwitterions are included in the pharmaceutically acceptable salts described in the present application. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, but other salts are also useful. The pharmaceutically acceptable salt of the compound can be formed, for example, by reacting the compound with a certain amount of acid or base in a medium, such as a medium in which the salt is precipitated or an aqueous medium (freeze-dried after the reaction).

[0011] Specific pharmaceutically acceptable salts include those salts that are suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reaction, etc., and are commensurate with a reasonable benefit or risk ratio within the scope of sound medical judgment. Pharmaceutically acceptable salts (pharmaceutically acceptable salts) are well known in the art. Pharmaceutically acceptable salts of the compounds described herein include salts derived from suitable inorganic and organic acids and inorganic and organic bases.

[0012] The example of pharmaceutically acceptable nontoxic acid addition salt is the salt formed with inorganic acid, for example hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or the salt formed with organic acid, for example acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid. Also include the salt formed using the conventional methods in the art, for example, ion exchange method.Other pharmaceutically acceptable salts include: adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, gluconate, glycerophosphate, hemisulfate, enanthate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, mesylate. Pharmaceutically acceptable salts derived from suitable bases include alkali metal, alkaline earth metal, ammonium salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium salts and the like.

[0013] In the present invention, a hydrate refers to a compound combined with water.

[0014] In the present invention, the solvate refers to a form of a compound or salt thereof that is combined with a solvent, usually formed by a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include methanol, ethanol, acetic acid, DMSO, THF, ether, etc. In some cases, the solvate will be able to separate, for example, when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid. Solvates include solvates in solution and separable solvates.

[0015] In the present invention, the crystalline form refers to the crystalline form of a compound with a specific crystal packing arrangement. Different crystalline forms usually have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardnesses, crystal shapes, photoelectric properties, stability and solubility. Recrystallization solvents, crystallization rates, storage temperatures and other factors may lead to a crystalline form being dominant. Various polymorphs of a compound can be prepared by crystallization under different conditions. In the present invention, crystalline form also includes special crystalline states, such as amorphous, etc.

[0016] In the present invention, immune checkpoints can regulate T cell function in the immune system. T cells play a central role in cell-mediated immunity. Checkpoint proteins interact with specific ligands, which send signals to T cells, essentially shutting down or inhibiting T cell function. Cancer cells use this system to express checkpoint proteins at high levels on their surfaces, thereby controlling T cells expressing checkpoint proteins on the surfaces of T cells entering the tumor microenvironment, thereby inhibiting anti-cancer immune responses. Therefore, inhibiting checkpoint proteins by agents referred to herein as "immune checkpoint inhibitors" will lead to the recovery of T cell function and immune responses to cancer cells.

[0017] Further, immune checkpoint inhibitors are biological therapeutics or small molecules.

[0018] Further, the immune checkpoint inhibitor is selected from biological therapeutic agents.

[0019] Furthermore, the biotherapeutic agent is selected from antibodies, fusion proteins or a combination thereof.

[0020] In the present invention, the type of the antibody is not limited, including but not limited to antigen binding fragments, monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies or multispecific antibodies formed from at least two complete antibodies. In the present invention, antigen binding fragments include but are not limited to VHH, Fab, Fab', F(ab')2 or Fv fragments.

[0021] In the present invention, the antibody may be a partially humanized antibody or a fully human antibody.

[0022] In the present invention, the pharmaceutical composition may contain one immune checkpoint inhibitor or multiple immune checkpoint inhibitors.

[0023] Furthermore, the immune checkpoint inhibitors include but are not limited to CTLA-4 inhibitors, PDL1 inhibitors, PDL2 inhibitors, PD1 inhibitors, TIGIT inhibitors, B7-H3 inhibitors, B7-H4 inhibitors, BTLA inhibitors, HVEM inhibitors, TIM3 inhibitors, GAL9 inhibitors, LAG3 inhibitors, VISTA inhibitors, KIR inhibitors, 2B4 inhibitors, CD160 inhibitors, CGEN-15049 inhibitors, CHK 1 kinase inhibitors, CHK2 kinase inhibitors, A2aR inhibitors, OX40 inhibitors, and B-7 family ligand inhibitors.

[0024] Furthermore, the immune checkpoint inhibitor is selected from CTLA-4 inhibitors, PDL1 inhibitors, PD1 inhibitors, and TIGIT inhibitors.

[0025] Furthermore, the CTLA-4 inhibitors include but are not limited to ipilimumab, tesimumab, and cardunolimumab.

[0026] Furthermore, the PDL1 inhibitors include but are not limited to sugemalimab, envomalimab, adebelimumab, atezolizumab, durvalumab, and socazolimab.

[0027] Furthermore, the PD1 inhibitors include but are not limited to toripalimab, sintilimab, carrelizumab, tilelizumab, sepalizumab, penampalimab, srulimumab, putalizumab, pembrolizumab, and nivolumab.

[0028] Furthermore, the TIGIT inhibitors include but are not limited to vembrolizumab and tirelimumab.

[0029] Furthermore, the pharmaceutical composition is selected from PD1 inhibitors and / or TIGIT inhibitors.

[0030] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0031] Furthermore, the pharmaceutically acceptable excipients include but are not limited to diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, lubricants, surfactants, coating materials, colorants, pH regulators, antioxidants or antibacterial agents.

[0032] Further, the diluent includes but is not limited to lactose, sodium chloride, glucose, urea, starch, water, etc. The binder includes but is not limited to starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, alginic acid and alginate, xanthan gum, hydroxypropyl cellulose, etc. The surfactant includes but is not limited to sodium lauryl sulfate, stearic acid monoglyceride, hexadecanol, etc. The lubricant includes but is not limited to zinc stearate, talc, calcium and magnesium stearate, polyethylene glycol, polyoxyethylene monostearate, monolauric sucrose acid ester, magnesium lauryl sulfate, etc. The filler includes but is not limited to mannitol, xylitol, sorbitol, maltose, glucose, lactose, sucrose, dextrin, starch, etc. The disintegrant includes but is not limited to cross-linked vinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, soybean polysaccharide, etc.

[0033] Furthermore, the pharmaceutical composition is a single compound preparation or a combination of two separate single preparations. Specifically, the compound preparation is a compound preparation comprising cytarabine or its pharmaceutically acceptable salt and an immune checkpoint inhibitor, and the combination of single preparations is a combination of cytarabine or its pharmaceutically acceptable salt and a single preparation comprising an immune checkpoint inhibitor.

[0034] Furthermore, the dosage forms of cytarabine or its pharmaceutically acceptable salt and the immune checkpoint inhibitor in the combination of the two separate single preparations may be the same or different.

[0035] Furthermore, the dosage form of the single compound preparation or the combination of two separate single preparations includes a dosage form for enteral administration and a dosage form for parenteral administration.

[0036] Furthermore, the gastrointestinal dosage form includes solutions, drops, tablets, capsules, granules, films, gels, powders, emulsions, suspensions, pills, suppositories, aerosols, sprays, powder mists, patches, ointments or creams.

[0037] Furthermore, the non-intestinal administration dosage forms include injection dosage forms, respiratory tract administration dosage forms, cavity administration dosage forms, mucosal administration dosage forms, and skin administration dosage forms.

[0038] Furthermore, the injectable dosage forms include but are not limited to intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections and intracavitary injections; the respiratory tract administration dosage forms include but are not limited to sprays, aerosols, powder aerosols, etc.; the cavity administration dosage forms include but are not limited to suppositories, aerosols, effervescent tablets, drops, pills, etc., which are used in the rectum, vagina, urethra, nasal cavity, ear canal, etc.; the mucosal administration dosage forms include but are not limited to eye drops, nasal drops, eye ointments, gargles, sublingual tablets, adhesive tablets, patches, etc.; the skin administration dosage forms include but are not limited to external solutions, lotions, liniments, ointments, plasters, pastes, patches, etc.

[0039] Furthermore, the dosage ratio of cytarabine or its pharmaceutically acceptable salt to the immune checkpoint inhibitor in the pharmaceutical composition is 10:1 to 100:1.

[0040] Furthermore, the dosage ratio is 5:1 to 10:1.

[0041] Furthermore, the dosage ratio is 2:1.

[0042] Furthermore, the dosage of cytarabine is 5-75 mg / m 2 every time.

[0043] Furthermore, the dosage of cytarabine is 30 mg / m 2 every time.

[0044] Further, in the pharmaceutical composition, cytarabine or its pharmaceutically acceptable salt and immune checkpoint inhibitor can be administered sequentially, simultaneously or cross-administered. Wherein, simultaneously refers to the synchronous administration of two drugs. If not administered simultaneously, then administered sequentially within a time range so that the two can act therapeutically within the same time range. Therefore, sequential administration allows administration of another drug within 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours or several hours after providing a drug, provided that the circulating half-life of the first administered drug allows the two to simultaneously present a therapeutically effective amount. The time delay of administration between each component will vary according to the exact nature of the component, the interaction therebetween, and its respective half-life.

[0045] The pharmaceutical composition of the present invention can be used alone or in combination with surgery, radiotherapy, hormone therapy, chemotherapy and / or immune cell therapy.

[0046] In the present invention, chemotherapy refers to the administration of chemotherapeutic agents to treat tumors. Chemotherapeutic agents refer to compounds or derivatives thereof that can interact with cancer cells, thereby reducing the proliferation state of cells and / or killing cells, for example, by impairing cell division or DNA synthesis, or by effectively targeting rapidly dividing cells by damaging DNA. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents (e.g., cyclophosphamide, ifosfamide); metabolic antagonists (e.g., methotrexate (MTX), 5-fluorouracil or its derivatives); substituted nucleotides; substituted nucleosides; DNA demethylating agents (also called antimetabolites), such as azacitidine); antitumor antibiotics (e.g., mitomycin, doxorubicin); plant-derived antitumor agents (e.g., vincristine, vindesine, paclitaxel, Abraxane); cisplatin; carboplatin; etoposide. These agents may further include, but are not limited to, the anticancer agents trimethoprim-butyl ester (TMTX); temozolomide; raltitrexed; S-(4-nitrobenzyl)-6-thioinosine (NBMPR); 6-benzylguanidine (6-BG); nitrosoureas (rabinopyranosyl-N-methyl-N-nitrosourea (Aranose), carmustine (BCNU, BiCNU), chlorzoxazone, ethylnitrosourea (ENU), fordomustine, lomustine (CCNU), nimustine, N-nitroso-N-methylurea (NMU), ranimustine (MCNU), semustine, streptozotocin (streptozotocin)); cytarabine; camptothecin; and any therapeutic derivatives thereof.

[0047] In the present invention, immunotherapy includes but is not limited to chimeric antigen receptor T cell immunotherapy, chimeric antigen receptor NK cell immunotherapy, and tumor infiltrating lymphocyte therapy.

[0048] Furthermore, the pharmaceutical composition of the present invention may also include other drugs for treating gliomas. These drugs are not particularly limited. As long as they can produce a therapeutic effect on gliomas, they are within the protection scope of this application. These drugs include, but are not limited to, Acivicin, Aclarubicin, Acodazole Hydrochloride, AcrQnine, Adozelesin, Aldesleukin, Asparaginase, Asperlin, Azacitidine, Azetepa, Azotomycin, Batimastat, Benzodepa, Bicalutamide, Bisantrene Hydrochloride, Bisnafide Dimesylate, Bizelesin, Bleomycin Sulfate, Brequinar Sodium, Sodium), Bropirimine, Busulfan, Cactinomycin, Calusterone, Caracemide, Carbetimer, Carboplatin, Carmustine, Carubicin Hydrochloride, Carzelesin, Cedefingol, Chlorambucil, Cirolemycin, Cisplatin, Cladribine.

[0049] In the present invention, treatment refers to the improvement, prevention, or reversal of a disease or condition or at least one distinguishable symptom thereof. In certain specific embodiments, the treatment refers to the improvement, prevention, or reversal of at least one measurable physiological parameter associated with the disease or condition to be treated, and the parameter is not necessarily identifiable in mammals or recognized by mammals. In some embodiments, the treatment refers to the inhibition or mitigation of a disease or course of disease, and this inhibition or mitigation may be physical, such as certain distinguishable adverse symptoms. The "treatment" used in the present invention covers diseases in mammals, especially humans, including: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease. (b) Inhibiting the disease, such as blocking the development of the disease. Or (c) Alleviating the disease, such as alleviating the symptoms associated with the disease.

[0050] The second aspect of the present invention provides a method for preparing a PDC / subcutaneous PDX / orthotopic PDX model of H3 K27 variant spinal cord glioma, the method comprising constructing a model using cells or tissues of H3 K27 variant spinal cord glioma.

[0051] Furthermore, the method for preparing a subcutaneous PDX model of H3 K27 variant spinal cord glioma comprises: implanting a H3 K27 variant spinal cord glioma tissue block into a non-human animal.

[0052] In the present invention, non-human animals include non-human vertebrates, more preferably mammals, which refer to all members of the class Mammalia, such as domesticated livestock (e.g., cattle, horses, pigs), pets (e.g., dogs, cats), or rodents. The term "rodent" refers to any and all members of the phylogenetic class Rodentia (e.g., mice, rats, squirrels, beavers, woodchucks, hamsters, guinea pigs, and agouti), including any descendants of all descendants derived therefrom. In a specific embodiment of the present invention, the mammal is selected from mice.

[0053] Furthermore, the mice are immunodeficient mice.

[0054] Furthermore, the immunodeficient mice include but are not limited to BALB / c-nu, NIH-nu, NC-nu Swiss-nu, 03H-nu, and C57BL-nu.

[0055] Furthermore, the mouse is a BALB / c-nu nude mouse.

[0056] Furthermore, the glioma tissue block was implanted into the axillary region of the mouse.

[0057] Furthermore, about 1 month after implantation, the tumor tissue began to proliferate steadily.

[0058] Furthermore, the method for preparing a subcutaneous PDX model of H3 K27 variant spinal cord glioma comprises: 3 Tumor tissue pieces (derived from spinal cord glioma PDX_S01 and PDX_S29 tissues that have been stably passaged for 3 times) were implanted into the right axillary region of mice. About 1 month after implantation, the tumor tissue began to proliferate stably.

[0059] Furthermore, the method for preparing an in situ PDX model of H3 K27 variant spinal cord glioma comprises: implanting H3 K27 variant spinal cord glioma tissue blocks or cells in situ into a non-human animal.

[0060] Furthermore, the non-human animal is selected from mice.

[0061] Furthermore, the mice are immunodeficient mice.

[0062] Furthermore, the immunodeficient mice include but are not limited to BALB / c-nu, NIH-nu, NC-nu Swiss-nu, 03H-nu, and C57BL-nu.

[0063] Furthermore, the mouse is a BALB / c-nu nude mouse.

[0064] Furthermore, the glioma tissue block or cells are implanted in situ into the lumbar spine center.

[0065] Furthermore, the glioma cell implantation amount is 5×10 5 indivual.

[0066] Furthermore, the method for preparing an orthotopic PDX model of H3 K27 variant spinal cord glioma comprises: anesthetizing a 5-week-old female BALB / c-nu nude mouse, cutting the dorsal skin to expose the thoracolumbar part of the spine. The first lumbar vertebra is determined according to the ribs of the 13th thoracic vertebra, and the first lumbar vertebra is usually located below the highest point of the spine when the mouse is in a prone position. The spine is fixed using a stereotactic device, and 5×10 5 H3 K27 mutant spinal cord glioma PDC cells, volume 3μl.

[0067] The third aspect of the present invention provides any of the following applications:

[0068] (1) Use of the pharmaceutical composition described in the first aspect of the present invention in the preparation of a product for treating glioma.

[0069] (2) Use of the pharmaceutical composition described in the first aspect of the present invention to inhibit glioma cell growth, promote glioma cell apoptosis, and promote glioma cell DNA damage in vitro.

[0070] (3) Application of the model prepared by the method described in the second aspect of the present invention in screening drugs for treating H3 K27 mutant spinal cord glioma.

[0071] (4) Application of the model prepared by the method described in the second aspect of the present invention in evaluating the therapeutic effect of drugs for treating H3 K27 mutant spinal cord glioma.

[0072] (5) Application of the model prepared by the method described in the second aspect of the present invention in studying the pathogenesis of H3 K27 mutant spinal cord glioma.

[0073] Furthermore, the glioma includes brain glioma and spinal cord glioma.

[0074] Furthermore, the glioma is selected from spinal cord glioma.

[0075] Furthermore, the spinal cord glioma is a spinal cord glioma with H3 K27M mutation.

[0076] A fourth aspect of the present invention provides any of the following methods:

[0077] (1) An in vitro method for inhibiting the growth of glioma cells and promoting the apoptosis of glioma cells for non-therapeutic purposes, the method comprising treating glioma cells with the pharmaceutical composition described in the first aspect of the present invention.

[0078] (2) A method for screening drug candidates for treating H3 K27 mutant spinal cord glioma, the method comprising: a) applying the agent to be screened to the model prepared by the method described in the second aspect of the present invention;

[0079] b) detecting the therapeutic effect of the agent to be screened on H3 K27 variant spinal cord glioma.

[0080] (3) A method for evaluating the therapeutic effect of a drug for treating H3 K27 mutant spinal cord glioma, the method comprising: a) administering the drug to the model prepared by the method described in the second aspect of the present invention;

[0081] b) detecting the therapeutic effect of the drug on the H3 K27 variant spinal cord glioma.

[0082] (4) A method for studying the pathogenesis of H3 K27 variant spinal cord glioma, wherein the method uses the model prepared by the method described in the second aspect of the present invention to study the pathogenesis of H3 K27 variant spinal cord glioma.

[0083] Furthermore, the glioma includes brain glioma and spinal cord glioma.

[0084] Furthermore, the glioma is selected from spinal cord glioma.

[0085] Furthermore, the spinal cord glioma is a spinal cord glioma with H3 K27M mutation.

[0086] In the present invention, the method of screening for drug candidates for treating H3 K27 mutant spinal cord glioma may involve administering different amounts of drug candidates (from no drug to an amount of drug close to the upper limit of the amount that can be successfully delivered to an animal, for example, within the toxic limit), and may include delivering the drug in different formulations and routes. A single drug may be administered, or the drugs may be combined in a combination of two or more drugs, especially when the administration of the drug combination may result in a synergistic effect. The ability of a candidate agent to treat an existing cancer may be evaluated by administering the candidate drug to a model prepared by the method of the second aspect of the present invention and evaluating the regulation of the cancer phenotype. The regulation of the cancer phenotype may be evaluated, for example, by evaluating the presence or absence of an effect on, for example, tumor burden, tumor number, tumor size, metabolic activity of tumor cells, progression-free survival (PFS), overall survival (OFS), and the like.

[0087] Drug candidates can be obtained from a wide variety of sources, including but not limited to synthetic, naturally occurring or recombinantly produced molecules, including small molecules, peptides, antibodies or other polypeptides. For example, random and directed synthesis of a variety of organic compounds and biomolecules, or natural compound libraries in the form of bacterial, fungal, plant or animal extracts, or natural or synthetic libraries and compounds modified by conventional chemical, physical or biochemical means, or directed or random chemical modification of known pharmacological agents, such as acylation, alkylation, esterification, amidation, etc., to generate structural analogs.

[0088] The fifth aspect of the present invention provides the use of an immune checkpoint inhibitor in the preparation of a drug for improving the effect of cytarabine in treating glioma.

[0089] In the present invention, improving refers to allowing a subject or tumor cell to enhance its ability to respond to the treatment disclosed herein. For example, the improved response may include at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% or more of the responsiveness. As used herein, "improving" may also refer to increasing the number of subjects who respond to treatment, such as a combination therapy comprising chemotherapy, resistant immune active cells and immune checkpoint inhibitors. For example, the improved response may refer to the total percentage of subjects who respond to treatment, wherein the percentage is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% or more.

[0090] In the present invention, subjects include humans, mammals (e.g., cats, dogs, horses, etc.), living cells, and other living organisms. Living organisms can be as simple as, for example, a single eukaryotic cell or as complex as a mammal. Typical patients are mammals, particularly primates, and especially humans. For veterinary applications, a variety of subjects will be suitable, such as livestock such as cattle, sheep, goats, cows, pigs, etc.; poultry such as chickens, ducks, geese, turkeys, etc.; and domestic animals, particularly pets such as dogs and cats. For research applications, suitable subjects will be a variety of mammals, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and pigs, such as inbred pigs, etc.

[0091] Further, immune checkpoint inhibitors are biological therapeutics or small molecules.

[0092] Further, the immune checkpoint inhibitor is selected from biological therapeutic agents.

[0093] Furthermore, the biotherapeutic agent is selected from antibodies, fusion proteins or a combination thereof.

[0094] In the present invention, the type of the antibody is not limited, including but not limited to antigen binding fragments, monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies or multispecific antibodies formed from at least two complete antibodies. In the present invention, antigen binding fragments include but are not limited to VHH, Fab, Fab', F(ab')2 or Fv fragments.

[0095] In the present invention, the antibody may be a partially humanized antibody or a fully human antibody.

[0096] Furthermore, the immune checkpoint inhibitor may be one or more.

[0097] Furthermore, the immune checkpoint inhibitors include but are not limited to CTLA-4 inhibitors, PDL1 inhibitors, PDL2 inhibitors, PD1 inhibitors, TIGIT inhibitors, B7-H3 inhibitors, B7-H4 inhibitors, BTLA inhibitors, HVEM inhibitors, TIM3 inhibitors, GAL9 inhibitors, LAG3 inhibitors, VISTA inhibitors, KIR inhibitors, 2B4 inhibitors, CD160 inhibitors, CGEN-15049 inhibitors, CHK 1 kinase inhibitors, CHK2 kinase inhibitors, A2aR inhibitors, OX40 inhibitors, and B-7 family ligand inhibitors.

[0098] Furthermore, the immune checkpoint inhibitor is selected from CTLA-4 inhibitors, PDL1 inhibitors, PD1 inhibitors, and TIGIT inhibitors.

[0099] Furthermore, the CTLA-4 inhibitors include but are not limited to ipilimumab, tesimumab, and cardunolimumab.

[0100] Furthermore, the PDL1 inhibitors include but are not limited to sugemalimab, envomalimab, adebelimumab, atezolizumab, durvalumab, and socazolimab.

[0101] Furthermore, the PD1 inhibitors include but are not limited to toripalimab, sintilimab, carrelizumab, tilelizumab, sepalizumab, penampalimab, srulimumab, putalizumab, pembrolizumab, and nivolumab.

[0102] Furthermore, the TIGIT inhibitors include but are not limited to vembrolizumab and tirelimumab.

[0103] Furthermore, the glioma includes brain glioma and spinal cord glioma.

[0104] Furthermore, the glioma is selected from spinal cord glioma.

[0105] Furthermore, the spinal cord glioma is a spinal cord glioma with H3 K27M mutation.

[0106] The advantages and beneficial effects of the present invention are as follows: the present invention provides an application of cytarabine combined with an immune checkpoint inhibitor in the treatment of gliomas, and experiments have proved that cytarabine combined with an immune checkpoint inhibitor has a synergistic effect in the treatment of gliomas. Cytarabine combined with an immune checkpoint inhibitor can enhance the anti-tumor effect of cytarabine and significantly prolong the survival of mice, providing a new strategy for the treatment of gliomas. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] Figure 1 These are the results of the effect of cytarabine (Ara-C) on tumors after intraperitoneal injection. Figure A is the result of subcutaneous PDX tumor proliferation in mice after intraperitoneal injection of Ara-C; Figure B is the data analysis of subcutaneous PDX tumor proliferation in mice after intraperitoneal injection of Ara-C.

[0108] Figure 2 These are the immunohistochemistry results of PDX tumor tissue after Ara-C treatment, where Figure A is the immunohistochemistry results of PDX tumor tissue after Ara-C treatment; Figure B is the immunohistochemistry data analysis diagram of PDX tumor tissue after Ara-C treatment.

[0109] Figure 3 These are the results of the PDC proliferation experiment after Ara-C treatment, wherein Figure A is the results of the PDC proliferation experiment after Ara-C treatment; Figure B is the analysis diagram of the results of the PDC proliferation experiment after Ara-C treatment.

[0110] Figure 4 These are the results of the DNA damage experiment on PDC cells after Ara-C treatment, wherein Figure A is the results of the DNA damage experiment on PDC cells after Ara-C treatment; Figure B is the analysis diagram of the results of the DNA damage experiment on PDC cells after Ara-C treatment.

[0111] Figure 5 These are the results of Ara-C treatment of mouse spinal cord orthotopic PDX tumors, where Figure A is a diagram of the dosing strategy; Figure B is a diagram of the growth of tumors in the spinal cord monitored by bioluminescence after Ara-C treatment; Figure C is a data analysis diagram of the growth of tumors in the spinal cord monitored by bioluminescence after Ara-C treatment; and Figure D is a diagram of the survival results of mice after Ara-C treatment.

[0112] Figure 6 This is the result of β-galactosidase staining of senescent cells.

[0113] Figure 7 These are the results of the PBMC recruitment experiment, where A is the flow chart of the PBMC migration experiment; B is a picture of PBMC cells passing through the bottom membrane of the upper chamber under a microscope; C is a picture of the PBMC cell count passing through the bottom membrane of the upper chamber in the lower chamber; and D is a picture of the PBMC cell count passing through the bottom membrane of the upper chamber in the lower chamber.

[0114] Figure 8 Figure 1 is the result graph of mRNA expression levels of inflammatory factors and immune checkpoints in senescent cells. Figure A is the result graph of mRNA expression levels of inflammatory factors in senescent cells; Figure B is the result graph of mRNA expression levels of immune checkpoints in SCA_S01 senescent cells; Figure C is the result graph of mRNA expression levels of immune checkpoints in SCA_S29 senescent cells.

[0115] Fig. 9 These are the results of Ara-C combined with immune checkpoint inhibitors in the treatment of spinal H3 K27M mutant spinal glioma in mice, where Figure A is the dosing regimen; Figure B is the growth of tumors in the spinal cord of mice after dosing; Figure C is the survival results of mice after dosing; Figure D is the tumor growth results of mice after dosing; and Figure E is the weight change results of mice after dosing.

[0116] Fig.10 This is a diagram showing the synergistic effect analysis of Ara-C combined with immune checkpoint inhibitors in the treatment of mouse spinal cord H3 K27M mutant spinal glioma. DETAILED DESCRIPTION

[0117] The disclosure above generally describes the present invention. A more complete understanding can be obtained with reference to the following specific embodiments. The purpose of describing these embodiments is only for illustration and is not intended to limit the scope of the present invention. Form changes and equivalent substitutions are considered to be circumstances that may suggest or confer convenience. Although specific terms are used in this article, the purpose of these terms is descriptive rather than limiting.

[0118] Example 1 Intraperitoneal injection of Ara-C to treat H3 K27M mutant spinal cord glioma PDX

[0119] 1. Experimental Materials

[0120] NSG mice, H3 K27M mutant spinal glioma PDX tissues, cytarabine (Ara-C), PBS, vernier calipers, and body weight scale.

[0121] 2. Experimental methods

[0122] The subcutaneous SCA-PDX model was established using 6-week-old female BALB / c nude mice. 3 Tumor tissue blocks (derived from spinal cord glioma PDX_S01 and PDX_S29 tissues that have been stably passaged for 3 times) were implanted into the right axillary region of mice. About 1 month after implantation, the tumor tissue began to proliferate stably, and the grouping experiment began at this time. The control group mice were intraperitoneally injected with normal saline, and the treatment group mice were intraperitoneally injected with 50mg / kg Ara-C. The treatment was continued for 2 weeks, once a day, and the tumor volume and mouse weight were monitored every 3 to 4 days.

[0123] 3. Experimental results

[0124] The results are as follows Figure 1 A. Figure 1 As shown in B, intraperitoneal injection of Ara-C can significantly inhibit the proliferation of subcutaneous PDX tumors in mice, and the growth rate of tumor volume in the treatment group is significantly reduced.

[0125] Example 2 Immunohistochemistry of PDX tumor tissue after Ara-C treatment

[0126] 1. Experimental Materials

[0127] H3 K27M mutant spinal glioma PDX tissue paraffin-embedded sections, hematoxylin, eosin, antigen retrieval solution, goat serum, specific primary antibodies (H3 K27M, Ki67, cleaved-caspase3), immunohistochemical secondary antibodies, DAB kit.

[0128] 2. Experimental methods

[0129] Tumor tissues were first fixed in 4% paraformaldehyde, then dehydrated and embedded in paraffin to prepare tissue sections. Hematoxylin-eosin (HE) staining was used to confirm the pathological diagnosis. For immunohistochemical analysis, tissue sections were dewaxed and boiled in Tris buffer for antigen retrieval. After blocking the sections with goat serum, they were incubated with specific primary antibodies at 4°C overnight. The sections were then incubated with the corresponding secondary antibodies at room temperature for 2 hours, followed by diaminobenzidine (DAB) color development. The nuclei were then counterstained with hematoxylin. The sections were dehydrated and fixed with neutral resin. All images were acquired at 40× magnification using a NanoZoomer digital pathology slide scanner and analyzed using ImageJ software.

[0130] 3. Experimental results

[0131] The results are as follows Figure 2 A. Figure 2 As shown in B, Ara-C treatment can reduce the expression of Ki67 in PDX tumor tissues, while increasing the expression of Cleaved-caspase3, suggesting that Ara-C can inhibit tumor cell proliferation and promote apoptosis to play an anti-tumor effect.

[0132] Example 3 PDC proliferation experiment after Ara-C treatment

[0133] 1. Experimental Materials

[0134] H3 K27M mutation spinal cord glioma patient-derived tumor cell lines SCA_S01 and SCA_S29, Ara-C (1 μM, MedChemExpress, HY-13605), DMSO, EDU staining kit (C0078S, Beyotime), 4% paraformaldehyde, PBS, confocal culture dish, Hoechst33342 staining solution, anti-fluorescence quenching aqueous mounting medium.

[0135] 2. Experimental methods

[0136] After treating cells with Ara-C (1 μM) or DMSO (Sigma, D2650, 1 μM) for 24 h, 1×10 5Cells were seeded into confocal culture dishes that had been pre-treated with poly-L-lysine and cultured overnight to allow the cells to adhere to the bottom of the dish. EdU-594 cell proliferation detection kit (C0078S, Beyotime) was used for EDU staining detection. The cells were placed in a culture medium containing 10 μM EDU and cultured for 4 hours to label the cells. After labeling, the cells were fixed with 4% paraformaldehyde solution, and then the cell nuclei were stained with Hoechst 33342 solution. After washing the culture dish with PBS to remove the residual staining reagent, the bottom of the confocal culture dish was mounted on a slide using an anti-fluorescence quenching aqueous mounting medium. Afterwards, the cell labeling was observed under a fluorescence microscope with wavelengths of 594nm and 460nm, and the labeled cells were quantified.

[0137] 3. Experimental results

[0138] The results are as follows Figure 3 A. Figure 3 As shown in B, Ara-C significantly reduced the proliferation rate of PDC cells and the number of cell nuclei labeled with EdU decreased significantly.

[0139] Example 4 DNA damage experiment of PDC cells after Ara-C treatment

[0140] 1. Experimental Materials

[0141] H3 K27M mutant spinal cord glioma patient-derived tumor cell lines SCA_S01 and SCA_S29, Ara-C (1 μM, MedChemExpress, HY-13605), DMSO, 4% paraformaldehyde, PBS, bovine serum albumin (BSA) (Solarbio, A8010), gamma-H2AX (abcam, 81299), AlexaFluor 594 goat anti-rabbit secondary antibody (Lab, Y1007), 4',6-diamidino-2-phenylindole (DAPI; Solarbio Life Sciences), confocal culture dish, anti-fluorescence quenching aqueous mounting medium.

[0142] 2. Experimental methods

[0143] PDC cells were seeded in confocal culture dishes pre-treated with poly-L-lysine and cultured overnight to allow cells to adhere to the bottom of the dish. Cells were treated with Ara-C (1 μM) or DMSO for 24 hours. The cells were then fixed with 4% paraformaldehyde for 15 minutes and blocked with bovine serum albumin (BSA) (Solarbio, A8010) antigen for 1 hour. Subsequently, the cells were incubated with the specific primary antibody gamma-H2AX (1:250, abcam, 81299) at 4°C overnight. Alexa Fluor 594-conjugated secondary antibodies (1:500; Lab, Y1007) were incubated for 1 hour at room temperature, and cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; Solarbio LifeSciences). The confocal dish bottom was mounted on a slide using an anti-fluorescence quenching aqueous mounting medium. The mounted samples were then imaged using a Zeiss Axio Observer Z1 confocal microscope. Immunopositive cells were manually counted using ImageJ (v1.51) software.

[0144] 3. Experimental results

[0145] The results are as follows Figure 4 A. Figure 4 As shown in B, Ara-C treatment can significantly increase the expression of γ-H2AX in the nucleus, indicating cellular DNA damage.

[0146] Example 5 Ara-C treatment of orthotopic PDX tumors in the mouse spinal cord

[0147] 1. Experimental Materials

[0148] H3 K27M mutant spinal cord glioma patient-derived tumor cell line SCA_S01, 5-week-old female BALB / c-Nude nude mice, scissors, micro-syringes, stereotaxic apparatus and other mouse experimental equipment, tribromoethanol anesthetic, fluorescein potassium salt (D-Luciferin potassium, HY-12591B, MCE), Ara-C.

[0149] 2. Experimental methods

[0150] Five-week-old female BALB / c-Nude mice were anesthetized and the dorsal skin was cut to expose the thoracolumbar spine. The first lumbar vertebra was identified based on the ribs of the 13th thoracic vertebra, which was usually located below the highest point of the spine when the mouse was in a prone position. The spine was fixed using a stereotaxic apparatus and 5×10 5SCA_S01 luciferase-overexpressing PDC cells were placed in a volume of 3 μl. The growth of intramedullary tumors was monitored weekly using bioluminescence on the IVIS Spectrum In Vivo imaging system and quantified using Live Image software (version 4.0; Living Image; PerkinElmer). Two weeks after surgery, mice with intramedullary tumors were randomly divided into two groups: the control group was intraperitoneally injected with PBS, and the treatment group was injected with a dose of 50 mg / kg of Ara-C. The drug was administered once a day for two weeks. The mice were observed daily and euthanized if progressive neurological deficits such as limb paralysis or a significant decrease in body weight and mobility occurred.

[0151] 3. Experimental results

[0152] The results are as follows Figure 5 As shown in A-5D, Ara-C treatment inhibited the proliferation rate of intramedullary tumors in mice and prolonged the survival of mice.

[0153] Example 6 β-galactosidase staining of senescent cells

[0154] 1. Experimental Materials

[0155] β-galactosidase (β-gal) staining detection kit (Cat#G1580, Solarbio), H3 K27M mutant spinal cord glioma patient-derived tumor cell lines SCA_S01 and SCA_S29, Ara-C (1 μM, MedChemExpress, HY-13605), DMSO, PBS, cell culture medium.

[0156] 2. Experimental methods

[0157] Cell senescence was quantified using a β-galactosidase (β-gal) staining detection kit (Cat#G1580, Solarbio). 5000 cells were seeded into confocal dishes pre-coated with poly-lysine and treated with Ara-C (1 μM) for 0, 1, 3, and 5 days. At the end of the culture period, the cells were fixed and incubated overnight with β-gal staining solution at pH 6.0 at 37°C in a CO2-independent environment. The intensity and distribution of blue were observed and quantified under a microscope to indicate cell senescence.

[0158] 3. Experimental results

[0159] The results are as follows Figure 6 As shown, with the Ara-C treatment time, the expression of β-galactosidase in PDC cells gradually increased, and the cell morphology became larger, showing the characteristics of senescent cells.

[0160] Example 7 PBMC recruitment experiment

[0161] 1. Experimental Materials

[0162] Peripheral blood mononuclear cells (PBMC), 24-well cell culture plates with 3.0 μm pore membrane (Cat#3415, Corning), H3 K27M mutant spinal cord glioma patient-derived tumor cell lines SCA_S01 and SCA_S29, Ara-C (1 μM, MedChemExpress, HY-13605), DMSO, cell culture medium.

[0163] 2. Experimental methods

[0164] Peripheral blood mononuclear cell (PBMC) migration assay was performed using a 24-well cell culture plate with a 3.0 μm pore membrane (Cat#3415, Corning). 5 The suspension of PBMCs was added to the upper chamber in a volume of 100 μL. 700 μL of conditioned medium from PDC cells treated with DMSO or Ara-C was added to the lower chamber. After 24 h of co-culture, the number of PBMCs that migrated across the membrane to the lower chamber was detected. The number of PBMC cells that passed through the bottom membrane of the upper chamber was counted under a microscope in 5 different fields of view.

[0165] 3. Experimental results

[0166] The results are as follows Figure 7 As shown in A-7D, the conditioned medium of senescent cells can recruit more PBMCs to migrate from the upper chamber to the lower chamber, indicating that senescent cells secrete cytokines and chemokines that can recruit immune cells.

[0167] Example 8 Q-PCR detection of aging cell inflammation-related factors and immune checkpoint mRNA expression

[0168] 1. Experimental Materials

[0169] Trizol reagent (Sigma-Aldrich), Ara-C (1 μM, MedChemExpress, HY-13605), H3 K27M mutant spinal glioma patient-derived tumor cell lines SCA_S01 and SCA_S29, cDNA synthesis Mix kit (Cat#F0202, LABLEAD), SYBR Green Master Mix kit (Cat#R0202, LABLEAD).

[0170] 2. Experimental methods

[0171] Trizol reagent (Sigma-Aldrich) was used to extract total RNA from PDC cells treated with Ara-C (1 μM) for different time periods. The concentration and purity of the extracted RNA were determined using a Nano Drop 2000 spectrophotometer. For complementary DNA (cDNA) synthesis, 2 μg of total RNA was reverse transcribed using the first-strand cDNA synthesis mix kit (Cat#F0202, LABLEAD). SYBR Green Master Mix (Cat#R0202, LABLEAD) was used for quantitative polymerase chain reaction (qPCR) detection of target genes. Quant Studio 5Flex Real-Time PCR system was used for PCR amplification and detection of target genes.

[0172] 3. Experimental results

[0173] The results are as follows Figure 8 As shown in A-8C, after Ara-C treatment, the mRNA expressions of inflammatory-related factors and multiple immune checkpoints in PDC cells were increased.

[0174] Example 9 Ara-C combined with immune checkpoint inhibitors to treat mouse spinal cord H3 K27M mutation spinal glioma

[0175] 1. Experimental Materials

[0176] 5-week-old female C57BL / 6J mice, CT-2AH3 K27M-Luciferase cells, scissors, microinjectors, stereotaxic apparatus and other mouse experimental instruments, tribromoethanol anesthetic, fluorescein potassium salt (D-Luciferin potassium, HY-12591B, MCE), Ara-C, anti-mouse PD-1 (Bio X cell, BE0273), anti-mouse TIGIT (Buchang Pharmaceutical).

[0177] 2. Experimental methods

[0178] After anesthetizing 5-week-old female C57BL / 6J mice, the neck skin and muscles were carefully separated to expose the upper thoracic vertebrae of the spine. The seventh cervical vertebra was identified based on the protruding spinous process of the second thoracic vertebra. The mice were fixed on a stereotaxic instrument and 5×10 5CT-2AH3K27M-Luciferase cells were injected into the flat bone surface above the high spinous processes. The growth of tumors in the spinal cord was monitored every five days using the IVIS Spectrum InVivo imaging system and Live Image software (version 4.0; Living Image; PerkinElmer). One week after surgery, when the intramedullary tumor reached a stable proliferation state, the mice were randomly divided into 5 treatment groups: Group 1 (control group) was intraperitoneally injected with PBS every day; Group 2 (Ara-C treatment group) was intraperitoneally injected with Ara-C 50 mg / kg every day; Group 3 (anti-PD1 and anti-TIGIT combined group) was intraperitoneally injected with PBS every day, anti-PD1 10 mg / kg every 3 days, and anti-TIGIT 10 mg / kg every 3 days; Group 4 (Ara-C + anti-PD1): Ara-C 50 mg / kg / d, anti-PD1 10 mg / kg / 3d, once every 3 days; Group 5 (cytarabine + anti-PD1 and anti-TIGIT combined): cytarabine 50 mg / kg / d, anti-PD1 10 mg / kg / 3d, anti-TIGIT 10 mg / kg / 3d. The treatment was continued for 6 days. Mice were monitored daily and euthanized if any progressive neurological deficits (e.g., limb paralysis) or significant loss of body weight and mobility were observed.

[0179] 3. Experimental results

[0180] (1) The results are as follows Fig. 9 As shown in A-9E, Ara-C combined with PD1 inhibitor can enhance the anti-tumor effect of Ara-C alone, and Ara-C combined with PD1 and TIGIT dual blockade therapy can further enhance the anti-tumor effect and significantly prolong the survival of mice.

[0181] (2) The anti-tumor effects of Ara-C combined with PD1 inhibitors, Ara-C combined with PD1 inhibitors and TIGIT inhibitors were evaluated by the two-drug interaction index (CDI). The CDI calculation formula is: CDI = AB / (A*B), AB is the ratio of the tumor signal size of the two-drug combination group to the control group, and A or B is the ratio of the tumor signal size of each drug alone group to the control group. When CDI is less than 1, it means that the two drugs have a synergistic therapeutic effect. The results are as follows Fig.10 As shown: When Ara-C is combined with PD1 inhibitors and TIGIT inhibitors for anti-tumor effects, the CDI value is 0.52242, indicating that Ara-C combined with PD1 inhibitors and TIGIT inhibitors has a significant synergistic effect.

[0182] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises cytarabine or a pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor.

2. The pharmaceutical composition according to claim 1, characterized in that The immune checkpoint inhibitors include CTLA-4 inhibitors, PDL1 inhibitors, PDL2 inhibitors, PD1 inhibitors, TIGIT inhibitors, B7-H3 inhibitors, B7-H4 inhibitors, BTLA inhibitors, HVEM inhibitors, TIM3 inhibitors, GAL9 inhibitors, LAG3 inhibitors, VISTA inhibitors, KIR inhibitors, 2B4 inhibitors, CD160 inhibitors, CGEN-15049 inhibitors, CHK 1 kinase inhibitors, CHK2 kinase inhibitors, A2aR inhibitors, OX40 inhibitors and / or B-7 family ligand inhibitors; Preferably, the immune checkpoint inhibitor is selected from PD1 inhibitors and / or TIGIT inhibitors; Preferably, the PDL1 inhibitors include sugemalimab, envomalimab, adebelimumab, atezolizumab, durvalumab, and socazolimab; Preferably, the TIGIT inhibitors include vembrolizumab and tirelimumab.

3. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition also includes pharmaceutically acceptable excipients; Preferably, the pharmaceutically acceptable excipients include diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, lubricants, surfactants, coating materials, colorants, pH regulators, antioxidants or antibacterial agents.

4. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition is a single compound preparation or a combination of two separate single preparations; Preferably, the dosage forms of cytarabine or a pharmaceutically acceptable salt thereof and the immune checkpoint inhibitor in the combination of the two separate single-ingredient preparations are the same or different; Preferably, the dosage form of the single compound preparation or the combination of two separate single preparations includes solutions, drops, tablets, capsules, granules, films, gels, powders, emulsions, suspensions, pills, suppositories, aerosols, sprays, powder mists, patches, ointments or creams.

5. The pharmaceutical composition according to claim 1, characterized in that The dosage ratio of cytarabine or its pharmaceutically acceptable salt to the immune checkpoint inhibitor in the pharmaceutical composition is 10:1 to 100:1; Preferably, the dosage ratio is 5:1 to 10:1; Preferably, the dosage ratio is 2:1; Preferably, the dosage of cytarabine is 5-75 mg / m 2 every time; Preferably, the dosage of cytarabine is 30 mg / m 2 every time; Preferably, cytarabine or a pharmaceutically acceptable salt thereof and the immune checkpoint inhibitor in the pharmaceutical composition can be administered sequentially, simultaneously or cross-administered.

6. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition also includes other drugs for treating glioma.

7. A method for preparing a PDC / subcutaneous PDX / orthotopic PDX model of H3 K27 variant spinal cord glioma, characterized in that: The method includes constructing a model using cells or tissues of H3 K27 mutant spinal cord glioma.

8. Any of the following applications: (1) Use of the pharmaceutical composition according to any one of claims 1 to 6 in the preparation of a product for treating glioma; (2) Use of the pharmaceutical composition according to any one of claims 1 to 6 for inhibiting glioma cell growth, promoting glioma cell apoptosis, and promoting glioma cell DNA damage in vitro; (3) Use of the model prepared by the method of claim 7 in screening drugs for treating H3 K27 mutant spinal cord gliomas; (4) Use of the model prepared by the method of claim 7 in evaluating the therapeutic effect of drugs for treating H3 K27 mutant spinal cord glioma; (5) Application of the model prepared by the method of claim 7 in studying the pathogenesis of H3 K27 mutant spinal cord glioma; Preferably, the glioma includes brain glioma and spinal cord glioma; Preferably, the glioma is selected from spinal cord glioma; Preferably, the spinal cord glioma is a spinal cord glioma with H3 K27M mutation.

9. Any of the following methods: (1) A method for inhibiting the growth of glioma cells and promoting apoptosis of glioma cells in vitro without therapeutic purposes, characterized in that: The method comprises treating glioma cells with the pharmaceutical composition according to any one of claims 1 to 6; (2) A method for screening drug candidates for treating H3 K27 variant spinal cord glioma, characterized in that the method comprises: a) applying the agent to be screened to the model prepared by the method of claim 7; b) detecting the therapeutic effect of the agent to be screened on H3 K27 variant spinal cord glioma; (3) A method for evaluating the therapeutic effect of a drug for treating H3 K27 variant spinal cord glioma, characterized in that the method comprises: a) administering the drug to the model prepared by the method of claim 7; b) detecting the therapeutic effect of the drug on the H3 K27 variant spinal cord glioma; (4) A method for studying the pathogenesis of H3 K27 variant spinal cord glioma, characterized in that the method uses the model prepared by the method of claim 7 to study the pathogenesis of H3 K27 variant spinal cord glioma; Preferably, the glioma includes brain glioma and spinal cord glioma; Preferably, the glioma is selected from spinal cord glioma; Preferably, the spinal cord glioma is a spinal cord glioma with H3 K27M mutation.

10. The use of immune checkpoint inhibitors in the preparation of drugs for improving the effect of cytarabine in treating gliomas; Preferably, the immune checkpoint inhibitors include CTLA-4 inhibitors, PDL1 inhibitors, PDL2 inhibitors, PD1 inhibitors, TIGIT inhibitors, B7-H3 inhibitors, B7-H4 inhibitors, BTLA inhibitors, HVEM inhibitors, TIM3 inhibitors, GAL9 inhibitors, LAG3 inhibitors, VISTA inhibitors, KIR inhibitors, 2B4 inhibitors, CD160 inhibitors, CGEN-15049 inhibitors, CHK 1 kinase inhibitors, CHK2 kinase inhibitors, A2aR inhibitors, OX40 inhibitors and / or B-7 family ligand inhibitors; Preferably, the immune checkpoint inhibitor is selected from PD1 inhibitors and / or TIGIT inhibitors; Preferably, the PDL1 inhibitors include sugemalimab, envomalimab, adebelimumab, atezolizumab, durvalumab, and socazolimab; Preferably, the TIGIT inhibitors include vebotulimab and tirelimumab; Preferably, the glioma includes brain glioma and spinal cord glioma; Preferably, the glioma is selected from spinal cord glioma; Preferably, the spinal cord glioma is a spinal cord glioma with H3 K27M mutation.

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