Application of cytarabine combined immune checkpoint inhibitor in glioma treatment

Abstract

The invention provides application of the cytarabine combined immune checkpoint inhibitor in glioma treatment, experiments prove that the cytarabine combined immune checkpoint inhibitor has a synergistic effect on glioma treatment, and the anti-tumor effect of the cytarabine can be enhanced by the cytarabine combined immune checkpoint inhibitor; the lifetime of the mouse is obviously prolonged, and a new strategy is provided for treating glioma.

Description

TECHNICAL FIELD

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

[0002] In recent years, immune checkpoint inhibitors have shown significant efficacy in a variety of solid tumors, but have not yet made breakthroughs in the treatment of glioma. Existing research shows that the effect of immune checkpoint inhibitors alone in the treatment of glioma is limited. For example, the prior art (Jing, L., 2023) discloses that PD-1 blockade does not provide survival benefit for patients with glioblastoma or hypermutated glioma; the prior art (CN117982636A) discloses that the growth of tumor tissue is not significantly inhibited in the case of using PD-1 antibody inhibitors (PD1) or Resiquimod (Resi) alone; the analysis result shows that the overall survival of mice is not significantly prolonged compared with the blank control group (NC) in the case of using PD-1 antibody inhibitors (PD1) or Resiquimod (Resi) alone. As for the combination of immune checkpoint inhibitors with other therapies, the effect is also not ideal, for example, the prior art (Michael Lim., 2022) discloses that the median overall survival of the Nivolumab+RT+TMZ group is 31.3, and the median overall survival of the placebo+RT+TMZ group is 33.0, and the Nivolumab combined with RT+TMZ treatment does not enhance the efficacy for glioblastoma, and even the effect is not as good as the placebo group.

[0003] Among the many subtypes of glioma, diffuse midline glioma caused by H3 K27 variation (H3-DMG) usually occurs in the thalamus, brainstem and spinal cord, and is a highly malignant tumor commonly seen in children and adolescents. This subtype of tumor has great differences in clinical biology and molecular genetic background from brain glioma, and most conventional treatment regimens are ineffective. 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 high morbidity have brought heavy economic and psychological burden to the patient's family and society. Because the location of this tumor is all super functional areas, surgery often cannot be completely resected, and even some patients cannot accept surgical treatment. Therefore, other treatment methods outside surgery have important value for improving the prognosis of patients.

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

[0005] In summary, the existing immunotherapy methods have insufficient effect on glioma, especially H3-DMG. There is an urgent need to develop new treatment strategies to break through the current treatment bottleneck and improve the quality of life and prolong the survival of patients.

[0006] References: [1] Jing, L., Qian, Z., Gao, Q.et al.Diffuse midline glioma treated with epigenetic agent-based immunotherapy.Sig Transduct Target Ther 8, 23 (2023). https: / / doi.org / 10.1038 / s41392-022-01274-7; [2] CN117982636A A combined drug product for preventing and / or treating brain glioma and combined pharmaceutical use; [3] Michael Lim, Michael Weller, Ahmed Idbaih.et al. Phase III trial of chemoradiotherapy with temozolomide plus nivolumab or placebo for newly diagnosed glioblastoma with methylated MGMT promoter, Neuro-Oncology, Volume 24, Issue 11, November 2022, Pages 1935-1949, https: / / doi.org / 10.1093 / neuonc / noac116. SUMMARY

[0007] In order to solve the technical problems in the prior art, the application provides application of cytarabine combined with an immune checkpoint inhibitor in glioma treatment, and experiments prove that the cytarabine combined with the immune checkpoint inhibitor has a synergistic effect on the treatment of glioma, the cytarabine combined with the immune checkpoint inhibitor can enhance the anti-tumor effect of cytarabine, and significantly prolong the survival period of mice, thereby providing a new strategy for treating glioma.

[0008] In order to achieve the above-mentioned purpose, the application adopts the following technical solutions: The application provides a pharmaceutical composition for treating spinal cord glioma, the pharmaceutical composition comprising cytarabine or a pharmaceutically acceptable salt thereof, and an immune checkpoint inhibitor.

[0009] Further, the spinal cord glioma is H3 K27 variant spinal cord glioma.

[0010] In the application, the cytarabine can be cytarabine itself, or a hydrate, a solvate or a crystalline form of cytarabine (hereinafter also referred to as a compound).

[0011] In the application, the pharmaceutically acceptable salt refers to acid salts formed with inorganic and / or organic acids and base salts formed with inorganic and / or organic bases. In addition, when the compound contains a basic moiety (for example, but not limited to, pyridine or imidazole) and an acidic moiety (for example, but not limited to, carboxylic acid), a zwitterion can be formed and the zwitterion is included in the pharmaceutically acceptable salt described in the application. Preferably, a pharmaceutically acceptable salt (i.e., non-toxic, physiologically acceptable) salt, but other salts are also useful. The pharmaceutically acceptable salt of the compound can be formed, for example, by reacting the compound with an amount of acid or base in a medium, such as a medium in which the salt is precipitated or an aqueous medium (lyophilized after reaction).

[0012] Specific pharmaceutically acceptable salts include those that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts (pharmaceutically acceptable salts) are well known in the art. The pharmaceutically acceptable salts of the compounds described in the application include salts derived from suitable inorganic and organic acids and inorganic and organic bases.

[0013] Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts of inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric and perchloric acid, or organic acids such as acetic, oxalic, maleic, tartaric, citric, succinic or malonic acid, or by using conventional methods of salt formation, for example, ion exchange methods. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, bisulfite, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, derived from appropriate bases include alkali, alkaline earth metal, ammonium salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium salts and the like.

[0014] In the present application, hydrate refers to a compound associated with water.

[0015] In the present application, solvate refers to a compound or its salt form associated with a solvent, usually formed from a solvolysis reaction. This physical association can include hydrogen bonding. Conventional solvents include methanol, ethanol, acetic acid, DMSO, THF, ether, and the like. In some cases, the solvate will be capable of isolation, for example, where one or more solvent molecules are incorporated into the crystal lattice of the solid state form. Solvates include solution phases and isolatable solvates.

[0016] In the present application, crystalline form refers to a crystalline form of a compound in a particular crystal packing arrangement. Different crystalline forms often have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability, and solubility. Recrystallization solvent, rate of crystallization, storage temperature, and other factors can cause one crystalline form to dominate. Various polymorphs of a compound can be prepared by crystallization under different conditions. In the present application, crystalline form also includes special crystal state such as amorphous and the like.

[0017] In the present invention, the immune checkpoint is capable of regulating T cell function in the immune system. T cells play a central role in cell-mediated immunity. Checkpoint proteins interact with specific ligands that send signals to T cells that essentially turn off or inhibit T cell function. Cancer cells exploit this system by expressing checkpoint proteins at high levels on their surface, thereby controlling T cells entering the tumor microenvironment that express checkpoint proteins on their surface, thereby suppressing the anti-cancer immune response. Therefore, inhibition of checkpoint proteins by agents referred to herein as "immune checkpoint inhibitors" will result in the restoration of T cell function and immune response to cancer cells.

[0018] Further, the immune checkpoint inhibitor is a biologic therapeutic agent or a small molecule.

[0019] Further, the immune checkpoint inhibitor is selected from a biologic therapeutic agent.

[0020] Further, the biologic therapeutic agent is selected from an antibody, a fusion protein, or a combination thereof.

[0021] In the present invention, the class of the antibody is not limited, including but not limited to an antigen-binding fragment, a monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody, or a multispecific antibody formed from at least two whole antibodies. In the present invention, the antigen-binding fragment includes but is not limited to a VHH, a Fab, a Fab', a (fab')2, or a Fv fragment.

[0022] In the present invention, the antibody can be a partially humanized antibody, or a fully human antibody.

[0023] In the present invention, the pharmaceutical composition can comprise one immune checkpoint inhibitor, or can comprise multiple immune checkpoint inhibitors.

[0024] Further, the immune checkpoint inhibitor includes but is not limited to a CTLA-4 inhibitor, a PDL1 inhibitor, a PDL2 inhibitor, a PD1 inhibitor, a TIGIT inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor, a BTLA inhibitor, a HVEM inhibitor, a TIM3 inhibitor, a GAL9 inhibitor, a LAG3 inhibitor, a VISTA inhibitor, a KIR inhibitor, a 2B4 inhibitor, a CD160 inhibitor, a CGEN-15049 inhibitor, a CHK 1 kinase inhibitor, a CHK2 kinase inhibitor, an A2aR inhibitor, an OX40 inhibitor, a B-7 family ligand inhibitor.

[0025] Further, the immune checkpoint inhibitor is selected from a CTLA-4 inhibitor, a PDL1 inhibitor, a PD1 inhibitor, a TIGIT inhibitor.

[0026] Further, the CTLA-4 inhibitor includes but is not limited to ipilimumab, tremelimumab, and cadonilimab.

[0027] Furthermore, the PDL1 inhibitors include, but are not limited to, sugemalimab, envorimab, adebelimumab, atezolizumab, durvalumab, and socarzolimumab.

[0028] Furthermore, the PD1 inhibitors include, but are not limited to, toripalimab, sintilimab, camrelizumab, tislelizumab, cepalimumab, penaplimab, slulimab, putelimab, pembrolizumab, nivolumab, and Bio X cell's anti-mouse PD-1 with catalog number BE0273.

[0029] Furthermore, the TIGIT inhibitors include, but are not limited to, vimbrolizumab, tireliumab, and anti-mouse TIGIT with the amino acid sequence shown in SEQ ID NO.1.

[0030] Furthermore, the pharmaceutical composition is selected from PD1 inhibitors and / or TIGIT inhibitors.

[0031] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0032] Furthermore, the pharmaceutically acceptable excipients include, but are not limited to, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, lubricants, surfactants, coating materials, colorants, pH adjusters, antioxidants, or antibacterial agents.

[0033] Furthermore, the diluents include, but are not limited to, lactose, sodium chloride, glucose, urea, starch, and water. The binders include, but are not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, alginate and alginate, xanthan gum, and hydroxypropylcellulose. The surfactants include, but are not limited to, sodium lauryl sulfate, glyceryl monostearate, and hexadecyl alcohol. The lubricants include, but are not limited to, zinc stearate, talc, calcium and magnesium stearate, polyethylene glycol, polyoxyethylene monostearate, monolaurate, and magnesium lauryl sulfate. The fillers include, but are not limited to, mannitol, xylitol, sorbitol, maltose, glucose, lactose, sucrose, dextrin, and starch. The disintegrants include, but are not limited to, crosylvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropylmethyl, crosylcarboxymethyl cellulose sodium, and soybean polysaccharides.

[0034] 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 containing cytarabine or a pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor, and the combination of single preparations is a combination containing cytarabine or a pharmaceutically acceptable salt thereof and a single preparation containing an immune checkpoint inhibitor.

[0035] Furthermore, in the combination of the two separate monotherapy formulations, the dosage forms of cytarabine or its pharmaceutically acceptable salts and immune checkpoint inhibitors can be the same or different.

[0036] Furthermore, the dosage forms of the single compound preparation or the combination of two separate single preparations include gastrointestinal dosage forms and non-gastrointestinal dosage forms.

[0037] Furthermore, the gastrointestinal drug delivery dosage forms include solutions, drops, tablets, capsules, granules, films, gels, powders, emulsions, suspensions, pills, suppositories, aerosols, sprays, powder sprays, patches, ointments, or creams.

[0038] Furthermore, the non-gastrointestinal dosage forms include injectable dosage forms, respiratory dosage forms, cavity dosage forms, mucosal dosage forms, and skin dosage forms.

[0039] Furthermore, the injectable dosage forms include, but are not limited to, various injectable drugs such as intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections; the respiratory dosage forms include, but are not limited to, sprays, aerosols, and powder inhalers; the cavity dosage forms include, but are not limited to, suppositories, aerosols, effervescent tablets, drops, and pills, for use in the rectum, vagina, urethra, nasal cavity, and ear canal; the mucosal dosage forms include, but are not limited to, eye drops, nasal drops, ointments, mouthwashes, sublingual tablets, adhesive tablets, and patches; and the skin dosage forms include, but are not limited to, topical solutions, lotions, liniments, ointments, plasters, pastes, and patches.

[0040] Furthermore, the dosage ratio of cytarabine or its pharmaceutically acceptable salt to immune checkpoint inhibitor in the pharmaceutical composition is 2:1 to 100:1.

[0041] Furthermore, the dosage ratio is 2:1 to 10:1.

[0042] Furthermore, the dosage ratio is 2:1 to 5:1.

[0043] Furthermore, the dosage ratio is 2:1.

[0044] Furthermore, the dosage of cytarabine is 5-75 mg / m². 2 every time.

[0045] Furthermore, the dosage of cytarabine is 30 mg / m². 2 every time.

[0046] Furthermore, cytarabine or its pharmaceutically acceptable salts and immune checkpoint inhibitors in the pharmaceutical composition can be administered sequentially, simultaneously, or alternately. Simultaneous administration refers to the concurrent administration of the two drugs. If not administered simultaneously, they are administered sequentially within a time frame so that both can be therapeutically effective within the same time frame. Therefore, sequential administration allows for the administration of one drug 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, or several hours after administering one drug, provided that the circulating half-life of the first administered drug allows for a simultaneously therapeutically effective amount of both. The time delay between administrations of the components will vary depending on the exact nature of the components, their interactions, and their respective half-lives.

[0047] The pharmaceutical compositions of the present invention can be used alone or in combination with surgery, radiotherapy, hormone therapy, chemotherapy and / or immunotherapy.

[0048] In this invention, chemotherapy refers to the treatment of tumors by administering chemotherapeutic agents. Chemotherapeutic agents are compounds or derivatives thereof that can interact with cancer cells, thereby reducing the proliferative state of cells and / or killing cells, for example by impairing cell division or DNA synthesis, or by effectively targeting rapidly dividing cells by disrupting DNA. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents (e.g., cyclophosphamide, ifosfamide); metabolic antagonists (e.g., methotrexate (MTX), 5-hydroxychloroquine, ... Fluorouracil or its derivatives); substituted nucleotides; substituted nucleosides; DNA demethylating agents (also known as antimetabolites), such as azacitidine; antitumor antibiotics (such as mitomycin, doxorubicin); plant-derived antitumor agents (such as vincristine, vindesine, paclitaxel, abraxane); cisplatin; carboplatin; etoposide. These agents may further include, but are not limited to, the anticancer agents trimethobenzoic acid ester (TMTX); temozolomide; raltitrexed; S-(4-nitrobenzyl)-6-thioinosine (NBMPR); 6-benzylguanidine (6-BG); nitrosoureas (rabinopyranosyl-N-methyl-N-nitrosourea (Aranose), carmustine (BCNU, BiCNU), chloramphenicol, ethylnitrosourea (ENU), fortimustine, lomustine (CCNU), nimustine, N-nitroso-N-methylurea (NMU), ranustine (MCNU), semustine, streptozotocin); cytarabine; camptothecin; and any of their therapeutic derivatives.

[0049] In this 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.

[0050] Furthermore, the pharmaceutical composition of the present invention may also include other drugs for treating gliomas. There are no particular limitations on these drugs, as long as they can produce a therapeutic effect on gliomas, they are all within the scope of protection 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, and brequina sodium. Sodium, bropirimine, busulfan, cactinomycin, Calusterone, caracemide, carbetimer, carboplatin, carmustine, carubicin hydrochloride, carzelesin, cedefinol, chlorambucil, ciromycin, cisplatin, and cladribine.

[0051] In this invention, treatment refers to the improvement, prevention, or reversal of a disease or condition or at least one identifiable symptom thereof. In some specific embodiments, treatment refers to the improvement, prevention, or reversal of at least one measurable physiological parameter associated with the disease or condition to be treated, which is not necessarily identifiable in or recognized by mammals. In some embodiments, treatment refers to the suppression or alleviation of a disease or its course, which may be physical, such as certain identifiable severe symptoms. The term "treatment" as used in this invention encompasses diseases in mammals, particularly humans, including: (a) preventing the onset of a disease or condition in individuals susceptible to the disease but not yet diagnosed with it; (b) suppressing a disease, such as halting its progression; or (c) alleviating a disease, such as reducing symptoms associated with the disease.

[0052] A 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 using cells or tissues of H3 K27 variant spinal cord glioma to construct the model.

[0053] Furthermore, the method for preparing a subcutaneous PDX model of H3 K27 variant spinal cord glioma includes: implanting an H3 K27 variant spinal cord glioma tissue block into a non-human animal.

[0054] In this 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 rodent class (e.g., mice, rats, squirrels, beavers, groundhogs, voles, hamsters, guinea pigs, and spiny guinea pigs), including any offspring derived therefrom. In a specific embodiment of the invention, the mammal is selected from mice.

[0055] Furthermore, the mice in question are immunodeficient mice.

[0056] Furthermore, the immunodeficient mice include, but are not limited to, BALB / c-nu, NIH-nu, NC-nu Swiss-nu, O3H-nu, and C57BL-nu.

[0057] Furthermore, the mice were BALB / c-nu nude mice.

[0058] Furthermore, the glioma tissue block was implanted into the axillary region of the mouse.

[0059] Furthermore, about one month after implantation, the tumor tissue began to proliferate stably.

[0060] Furthermore, the method for preparing the subcutaneous PDX model of H3 K27 variant spinal cord glioma includes: implanting a 5 mm³ tumor tissue block (from spinal cord glioma PDX_S01 and PDX_S29 tissues that have been stably passaged 3 times) into the right axillary region of mice. Approximately one month after implantation, the tumor tissue began to proliferate stably.

[0061] Furthermore, the method for preparing an in situ PDX model of H3 K27 variant spinal cord glioma includes: in situ implanting H3 K27 variant spinal cord glioma tissue blocks or cells into a non-human animal.

[0062] Furthermore, the non-human animal is selected from mice.

[0063] Furthermore, the mice in question are immunodeficient mice.

[0064] Furthermore, the immunodeficient mice include, but are not limited to, BALB / c-nu, NIH-nu, NC-nu Swiss-nu, O3H-nu, and C57BL-nu.

[0065] Furthermore, the mice were BALB / c-nu nude mice.

[0066] Furthermore, the glioma tissue block or cells are implanted in situ into the lumbar spine center.

[0067] Furthermore, the amount of glioma cells implanted is 5 × 10⁻⁶. 5 indivual.

[0068] Furthermore, the method for preparing the in situ PDX model of H3 K27 variant spinal cord glioma includes: anesthetizing 5-week-old female BALB / c-nu nude mice, and cutting open the dorsal skin to expose the thoracolumbar region of the spine. The first lumbar vertebra is determined based on the ribs of the 13th thoracic vertebra, which is typically located below the highest point of the spine in the prone position. The spine is stabilized using a stereotactic device, and 5 × 10⁵ PDX is injected 1 mm lateral to the center of the first lumbar vertebra. 5 H3 K27 variant spinal cord glioma PDC cells, volume 3 μl.

[0069] The third aspect of the present invention provides any of the following applications: (1) The use of the pharmaceutical composition described in the first aspect of the present invention in the preparation of a product for treating spinal cord glioma.

[0070] (2) The application of the pharmaceutical composition described in the first aspect of the present invention in in vitro inhibiting the growth of spinal cord glioma cells, promoting apoptosis of spinal cord glioma cells, and promoting DNA damage in spinal cord glioma cells.

[0071] (3) Application of the model prepared by the method described in the second aspect of the present invention in screening drugs for the treatment of H3 K27 variant glioma.

[0072] (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 variant spinal cord glioma.

[0073] (5) Application of the model prepared by the method described in the second aspect of the present invention in the study of the pathogenesis of H3 K27 variant spinal cord glioma.

[0074] Furthermore, the spinal cord glioma is an H3 K27M mutated spinal cord glioma.

[0075] The fourth aspect of the present invention provides any of the following methods: (1) A method for inhibiting the growth of spinal cord glioma cells and promoting apoptosis of spinal cord glioma cells in vitro without therapeutic purpose, the method comprising treating spinal cord glioma cells with the pharmaceutical composition described in the first aspect of the present invention.

[0076] (2) A method for screening drug candidates for the treatment of H3 K27 variant spinal cord glioma, the method comprising: a) Apply the reagent to be screened to the model prepared by the method described in the second aspect of the present invention; b) To test the therapeutic effect of the reagent to be screened on H3 K27 variant glioma.

[0077] (3) A method for evaluating the therapeutic effect of a drug for treating H3 K27 variant glioma, the method comprising: a) Applying the drug to a model prepared by the method described in the second aspect of the present invention; b) To test the therapeutic effect of the drug on the H3 K27 variant glioma.

[0078] (4) A method for studying the pathogenesis of H3 K27 variant glioma, wherein the method uses a model prepared by the method described in the second aspect of the present invention to study the pathogenesis of H3 K27 variant glioma.

[0079] Furthermore, the spinal cord glioma is an H3 K27M mutated spinal cord glioma.

[0080] In this invention, the method for screening drug candidates for treating H3 K27 variant glioma may involve administering varying amounts of the drug candidate (from no drug to amounts close to the upper limit of successful delivery to animals, e.g., within toxicity limits), and may include drug delivery via different formulations and routes. A single drug may be administered, or drugs may be combined in combinations of two or more drugs, particularly where the administration of the drug combination may result in a synergistic effect. The ability of the candidate drug to treat existing cancer can be evaluated by administering the candidate drug to a model prepared by the method described in the second aspect of this invention and assessing the regulation of the cancer phenotype. The regulation of the cancer phenotype can be assessed, for example, by evaluating the presence or absence of effects on, for example, tumor burden, tumor number, tumor size, metabolic activity of tumor cells, progression-free survival (PFS), overall survival (OFS), etc.

[0081] Drug candidates can be obtained from a wide variety of sources, including but not limited to synthetic, naturally occurring, or recombinant molecules, including small molecules, peptides, antibodies, or other polypeptides. For example, a variety of organic compounds and biomolecules, or libraries of natural compounds 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 known pharmacological agents can be chemically modified in a directed or random manner, such as by acylation, alkylation, esterification, amidation, etc., to generate structural analogs.

[0082] The fifth aspect of this invention provides the use of immune checkpoint inhibitors in the preparation of medicaments for improving the efficacy of cytarabine in the treatment of spinal cord glioma.

[0083] In this invention, "enhancement" refers to allowing a subject or tumor cell to improve its ability to respond to the treatments disclosed herein. For example, an enhanced response may include a response capability of 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. As used herein, "enhancement" may also refer to an increase in the number of subjects responding to treatments, such as combination therapies comprising chemotherapy, drug-resistant immune cells, and immune checkpoint inhibitors. For example, an enhanced response may refer to the total percentage of subjects responding 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.

[0084] In this 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 subjects are mammals, particularly primates, especially humans. For veterinary applications, a wide variety of subjects will be suitable, such as livestock like cattle, sheep, goats, cows, pigs, etc.; poultry like chickens, ducks, geese, turkeys, etc.; and domesticated animals, especially pets such as dogs and cats. For research applications, suitable subjects will be a wide variety of mammals, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and pigs, such as inbred pigs.

[0085] Furthermore, immune checkpoint inhibitors are biological therapeutics or small molecules.

[0086] Furthermore, immune checkpoint inhibitors are selected from biological therapeutic agents.

[0087] Furthermore, the biotherapeutic agent is selected from antibodies, fusion proteins, or combinations thereof.

[0088] In this invention, the type of antibody is not limited, and includes, but is not limited to, antigen-binding fragments, monoclonal antibodies, polyclonal antibodies, human antibodies, chimeric antibodies, or multispecific antibodies formed from at least two intact antibodies. In this invention, antigen-binding fragments include, but are not limited to, VHH, Fab, Fab', F(ab')2, or Fv fragments.

[0089] In this invention, the antibody may be a partially humanized antibody or a fully human antibody.

[0090] Furthermore, the immune checkpoint inhibitor can be one or more.

[0091] 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, CHK1 kinase inhibitors, CHK2 kinase inhibitors, A2aR inhibitors, OX40 inhibitors, and B-7 family ligand inhibitors.

[0092] Furthermore, the immune checkpoint inhibitor is selected from CTLA-4 inhibitors, PDL1 inhibitors, PD1 inhibitors, and TIGIT inhibitors.

[0093] Furthermore, the CTLA-4 inhibitors include, but are not limited to, ipilimumab, texilimumab, and canduniline.

[0094] Furthermore, the PDL1 inhibitors include, but are not limited to, sugemalimab, envorimab, adebelimumab, atezolizumab, durvalumab, and socarzolimumab.

[0095] Furthermore, the PD1 inhibitors include, but are not limited to, toripalimab, sintilimab, camrelizumab, tislelizumab, cepalimumab, penaprilimab, slulimab, putelizumab, pembrolizumab, and nivolumab, and the PD1 inhibitors include anti-mouse PD-1 with catalog number BE0273 produced by Bio X cell.

[0096] Furthermore, the TIGIT inhibitors include, but are not limited to, vimbrolizumab, tireliumab, and anti-mouse TIGIT with the amino acid sequence shown in SEQ ID NO.1.

[0097] Furthermore, the spinal cord glioma is an H3 K27M mutated spinal cord glioma.

[0098] The advantages and beneficial effects of this invention are as follows: This invention provides the application of cytarabine combined with immune checkpoint inhibitors in the treatment of glioma. Experiments have demonstrated that cytarabine combined with immune checkpoint inhibitors has a synergistic effect on the treatment of glioma. Cytarabine combined with immune checkpoint inhibitors can enhance the anti-tumor effect of cytarabine and significantly prolong the survival time of mice, providing a new strategy for the treatment of glioma. Attached Figure Description

[0099] Figure 1 The results of intraperitoneal injection of cytarabine (Ara-C) on tumors are shown in Figure A, which shows the results of subcutaneous PDX tumor proliferation in mice after intraperitoneal injection of Ara-C; Figure B shows the data analysis of subcutaneous PDX tumor proliferation in mice after intraperitoneal injection of Ara-C.

[0100] Figure 2 These are the immunohistochemical results of PDX tumor tissue after Ara-C treatment. Figure A shows the immunohistochemical results of PDX tumor tissue after Ara-C treatment, and Figure B shows the immunohistochemical data analysis of PDX tumor tissue after Ara-C treatment.

[0101] Figure 3 These are the results of the PDC proliferation experiment after Ara-C treatment. Figure A shows the results of the PDC proliferation experiment after Ara-C treatment, and Figure B shows the analysis of the results of the PDC proliferation experiment after Ara-C treatment.

[0102] Figure 4 These are the experimental results of DNA damage in PDC cells after Ara-C treatment. Figure A shows the experimental results of DNA damage in PDC cells after Ara-C treatment; Figure B shows the analysis of the experimental results of DNA damage in PDC cells after Ara-C treatment.

[0103] Figure 5 The figures show the results of Ara-C treatment for PDX tumors in situ of the mouse spinal cord. Figure A shows the drug administration strategy; Figure B shows the tumor growth in the spinal cord monitored by biofluorescence after Ara-C treatment; Figure C shows the data analysis of tumor growth in the spinal cord monitored by biofluorescence after Ara-C treatment; and Figure D shows the survival results of mice after Ara-C treatment.

[0104] Figure 6 This is a diagram showing the staining results of β-galactosidase in senescent cells.

[0105] Figure 7 These are the results of the PBMC recruitment experiment. Figure A is a flowchart of the PBMC migration experiment; Figure B is a microscopic image of PBMC cells crossing the basal membrane of the upper chamber; Figure C is a count of PBMC cells crossing the basal membrane of the upper chamber in the lower chamber; and Figure D is a count of PBMC cells crossing the basal membrane of the upper chamber in the lower chamber.

[0106] Figure 8 The figures show the mRNA expression levels of inflammation-related factors and immune checkpoints in senescent cells. Figure A shows the mRNA expression levels of inflammation-related factors in senescent cells; Figure B shows the mRNA expression levels of immune checkpoints in SCA_S01 senescent cells; and Figure C shows the mRNA expression levels of immune checkpoints in SCA_S29 senescent cells.

[0107] Figure 9 The images show the results of treating H3 K27M mutant spinal cord gliomas in mice with Ara-C combined with immune checkpoint inhibitors. Figure A shows the dosing regimen; Figure B shows the growth of the tumor in the spinal cord after administration; Figure C shows the survival time of the mice after administration; Figure D shows the tumor growth after administration; and Figure E shows the weight change of the mice after administration.

[0108] Figure 10 A diagram illustrating the synergistic effect of Ara-C combined with immune checkpoint inhibitors in the treatment of H3 K27M mutant spinal cord gliomas in mice. Detailed Implementation

[0109] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0110] Example 1: Intraperitoneal injection of Ara-C for the treatment of H3 K27M mutant spinal cord glioma PDX 1. Experimental materials NSG mice, H3 K27M mutant spinal cord glioma PDX tissue, cytarabine (Ara-C), PBS, vernier calipers, and weight scale.

[0111] 2. Experimental Methods A subcutaneous SCA-PDX model was established using 6-week-old female BALB / c nude mice. A 5 mm³ volume... 3 Tumor tissue blocks (from stable, three-passaged spinal cord gliomas PDX_S01 and PDX_S29) were implanted into the right axillary region of mice. Approximately one month post-implantation, the tumor tissue began to proliferate stably, at which point the grouping experiment commenced. Control group mice received intraperitoneal injections of saline, while treatment group mice received intraperitoneal injections of 50 mg / kg Ara-C. Treatment continued for two weeks, once daily, with tumor volume and mouse weight monitored every 3–4 days.

[0112] 3. Experimental Results The results are as follows Figure 1 A, Figure 1 As shown in Figure B, intraperitoneal injection of Ara-C significantly inhibited the proliferation of subcutaneous PDX tumors in mice, and the tumor volume growth rate was significantly reduced in the treatment group.

[0113] Example 2 Immunohistochemistry of PDX tumor tissue after Ara-C treatment 1. Experimental materials Paraffin-embedded sections of H3 K27M mutant spinal cord glioma PDX tissue, hematoxylin, eosin, antigen retrieval solution, goat serum, specific primary antibody (H3 K27M, Ki67, cleaved-caspase 3), immunohistochemical secondary antibody, DAB kit.

[0114] 2. Experimental Methods Tumor tissue was first fixed in 4% paraformaldehyde, then dehydrated, and then 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 with sheep serum, sections were incubated overnight at 4°C with a specific primary antibody. The sections were then incubated with the corresponding secondary antibody at room temperature for 2 hours, followed by a diaminobenzidine (DAB) colorimetric reaction. The cell nuclei were then counterstained with hematoxylin. After dehydration, the sections were fixed with neutral resin. All images were acquired at 40x magnification using a NanoZoomer digital pathology slide scanner and analyzed using ImageJ software.

[0115] 3. Experimental Results The results are as follows Figure 2 A, Figure 2 As shown in Figure B, Ara-C treatment reduced Ki67 expression in PDX tumor tissue while increasing Cleaved-caspase 3 expression. This suggests that Ara-C exerts its anti-tumor effect by inhibiting tumor cell proliferation and promoting apoptosis.

[0116] Example 3: PDC proliferation experiment after Ara-C treatment 1. Experimental materials H3 K27M mutant 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 dishes, Hoechst 33342 staining solution, and antifluorescence quenching aqueous mounting medium.

[0117] 2. Experimental Methods After treating cells with Ara-C (1 μM) or DMSO (Sigma, D2650, 1 μM) for 24 h, 1 × 10⁻⁶ cells were added. 5Cells were seeded into confocal culture dishes pre-treated with poly-L-lysine and cultured overnight to allow cell adhesion to the bottom. EDU staining was performed using the EdU-594 cell proliferation assay kit (C0078S, Beyotime). Cells were cultured in medium containing 10 μM EDU for 4 hours and then labeled. After labeling, cells were fixed with 4% paraformaldehyde solution, and the nuclei were stained with Hoechst 33342 solution. After washing the culture dishes with PBS to remove residual staining reagent, the bottom of the confocal culture dishes was mounted onto a glass slide using an anti-fluorescence quenching aqueous mounting medium. Cell labeling was then observed under fluorescence microscopy at wavelengths of 594 nm and 460 nm, and the labeled cells were quantified.

[0118] 3. Experimental Results The results are as follows Figure 3 A, Figure 3 As shown in Figure B, Ara-C significantly reduced the proliferation rate of PDC cells, and the number of EDU-labeled cell nuclei decreased significantly.

[0119] Example 4: DNA damage experiment in PDC cells after Ara-C treatment 1. Experimental materials 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), Alexa Fluor 594 goat anti-rabbit secondary antibody (Lab, Y1007), 4',6-diamidinyl-2-phenylindole (DAPI; Solarbio Life Sciences), confocal culture dishes, and anti-fluorescence quenching aqueous mounting medium.

[0120] 2. Experimental Methods PDC cells were seeded in confocal culture dishes pretreated with poly-L-lysine and cultured overnight to allow cell adhesion to the bottom. Cells were treated with Ara-C (1 μM) or DMSO for 24 hours. Cells were then fixed with 4% paraformaldehyde for 15 minutes and blocked with bovine serum albumin (BSA) (Solarbio, A8010) antigen for 1 hour. Subsequently, cells were incubated overnight at 4°C with the specific primary antibody gamma-H2AX (1:250, abcam, 81299). Cells were then incubated with the Alexa Fluor 594-bound secondary antibody (1:500; Lab, Y1007) at room temperature for 1 hour, and the nuclei were stained with 4',6-diamidinyl-2-phenylindole (DAPI; Solarbio Life Sciences). The confocal dish bottom was mounted onto a slide using an antifluorescence-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.

[0121] 3. Experimental Results The results are as follows Figure 4 A, Figure 4 As shown in Figure B, Ara-C treatment significantly increased the expression of γ-H2AX in the cell nucleus, indicating cellular DNA damage.

[0122] Example 5: Ara-C Treatment of Orthotopic PDX Tumors in Mouse Spinal Cord 1. Experimental materials H3 K27M mutant spinal cord glioma patient-derived tumor cell line SCA_S01, 5-week-old female BALB / c-Nude nude mice, mouse experimental equipment such as scissors, microsyringes, stereotactic apparatus, tribromoethanol anesthetic, luciferin potassium (HY-12591B, MCE), and Ara-C.

[0123] 2. Experimental Methods Five-week-old female BALB / c-Nude nude mice were anesthetized, and the dorsal skin was cut to expose the thoracolumbar region of the spine. The first lumbar vertebra was located based on the ribs of the 13th thoracic vertebra, which, in the prone position, is typically located below the highest point of the spine. The spine was stabilized using a stereotactic apparatus, and 5 × 10⁵ ppm was injected 1 mm lateral to the center of the first lumbar vertebra. 5SCA_S01 luciferase-overexpressing PDC cells, 3 μl in volume. Intramedullary tumor growth was monitored weekly using biofluorescence on an 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 assigned to two groups: a control group received intraperitoneal injection of PBS, and a treatment group received 50 mg / kg of Ara-C. Administration was once daily for two weeks. Mice were observed daily, and euthanasia was performed if progressive neurological deficits such as limb paralysis or significant decline in weight and mobility occurred.

[0124] 3. Experimental Results The results are as follows Figure 5 As shown in A-5D, Ara-C treatment inhibited the rate of intramedullary tumor proliferation in mice and prolonged their survival.

[0125] Example 6: β-galactosidase staining in senescent cells 1. Experimental materials β-galactosidase (β-gal) staining assay 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.

[0126] 2. Experimental Methods Cellular senescence was quantified using a β-galactosidase (β-gal) staining assay kit (Cat# G1580, Solarbio). 5000 cells were seeded in poly-L-lysine-pre-coated confocal dishes and treated with Ara-C (1 μM) for 0, 1, 3, and 5 days. At the end of the culture period, cells were fixed and incubated overnight at 37°C in a CO2-independent environment with β-gal staining solution at pH 6.0. The intensity and distribution of the blue staining were observed and quantified under a microscope to indicate cellular senescence.

[0127] 3. Experimental Results The results are as follows Figure 6 As shown, with the duration of Ara-C treatment, the expression of β-galactosidase in PDC cells gradually increased, and the cell morphology became larger, exhibiting characteristics of senescent cells.

[0128] Example 7: PBMC Recruitment Experiment 1. Experimental materials Peripheral blood mononuclear cells (PBMCs), 24-well cell culture plates with 3.0 μm pore membranes (Cat# 3415, Corning Incorporated), 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.

[0129] 2. Experimental Methods Peripheral blood mononuclear cell (PBMC) migration assays were performed using 24-well cell culture plates (Cat# 3415, Corning Incorporated) with 3.0 μm pore membranes. 3 × 10⁶ cells were cultured in each well. 5 A suspension of PBMCs was added to the upper chamber at a volume of 100 μL. 700 μL of conditioned medium for PDC cells treated with DMSO or Ara-C was added to the lower chamber. After co-culturing for 24 h, the number of PBMCs migrating across the membrane to the lower chamber was assessed. The number of PBMCs crossing the bottom membrane of the upper chamber was counted under a microscope at five different fields of view.

[0130] 3. Experimental Results The results are as follows Figure 7 As shown in A-7D, the conditioned medium for senescent cells can recruit more PBMCs to migrate from the upper chamber to the lower chamber, indicating that the secretion of cytokines and chemokines by senescent cells can recruit immune cells.

[0131] Example 8: Q-PCR detection of mRNA expression of inflammation-related factors and immune checkpoints in senescent cells 1. Experimental materials Trizol reagent (Sigma-Aldrich), Ara-C (1 μM, MedChemExpress, HY-13605), H3 K27M mutant spinal cord 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).

[0132] 2. Experimental Methods Total RNA was extracted from PDC cells treated with Ara-C (1 μM) at different time points using Trizol reagent (Sigma-Aldrich). 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 a first-strand cDNA synthesis mix kit (Cat# F0202, LABLEAD). Quantitative polymerase chain reaction (qPCR) of the target gene was performed using SYBR Green Master Mix (Cat# R0202, LABLEAD). PCR amplification and detection of the target gene were performed using a Quant Studio 5 Flex Real-Time PCR system.

[0133] 3. Experimental Results The results are as follows Figure 8 As shown in A-8C, Ara-C treatment increased the expression of inflammation-related factors and multiple immune checkpoint mRNAs in PDC cells.

[0134] CD274 and PDCD1LG2 are ligands of PD-1, while PVR and NECTIN2 are ligands of TIGIT. As shown in the figure, during the D0 stage of Ara-C treatment, the expression of PD-1 and TIGIT ligands was relatively low, while after Ara-C treatment, the expression of PD-1 and TIGIT ligands increased significantly. This suggests that after Ara-C treatment, subsequent treatment with PD-1 or TIGIT antibodies can enhance their efficacy and improve the therapeutic effect of cytarabine or PD-1 and TIGIT antibodies.

[0135] Example 9: Treatment of mouse spinal cord H3 K27M mutant glioma with Ara-C combined with immune checkpoint inhibitors 1. Experimental materials Five-week-old female C57BL / 6J mice, CT-2A H3 K27M-Luciferase cells, mouse experimental equipment including scissors, microsyringes, and stereotactic apparatus, tribromoethanol anesthetic, luciferin potassium (D-Luciferin potassium, HY-12591B, MCE), Ara-C, anti-mouse PD-1 antibody (Bio X cell, BE0273), and anti-mouse TIGIT antibody (Buchang Pharmaceutical, sequence SEQ ID NO.1: EVQLVESGGGLVQPGGSLRLSCAASGYKYGVYSMGWFRQAPGKGLEGVSAICSGGRTTYSDSVKGRFTISRDNSNQILYLQMNSLRAEDTAVYYCAARPLWTGDCDLSSSWYKTWGQGTLVTVSS)).

[0136] 2. Experimental Methods Five-week-old female C57BL / 6J mice were anesthetized, and the skin and muscles of the neck were carefully dissected to expose the thoracic vertebrae. The 7th cervical vertebra was identified by the prominent spinous process of the 2nd thoracic vertebra. The mice were fixed on a stereotactic apparatus, and a 3 μl volume of 5×10⁻⁶ tissue was used. 5CT-2A H3 K27M-Luciferase cells were injected into a flat bone surface above the high spinous process. Intraspinal tumor growth was monitored using the IVIS SpectrumIn Vivo imaging system and every five days using Live Image software (version 4.0; PerkinElmer). One week post-surgery, when the intramedullary tumor reached a stable proliferative state, mice were randomly divided into five treatment groups: Group 1 (control group) received intraperitoneal injections of PBS daily; Group 2 (Ara-C treatment group) received intraperitoneal injections of Ara-C 50 mg / kg daily; Group 3 (anti-PD1 and anti-TIGIT combination therapy group) received intraperitoneal injections of PBS daily, followed by 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 / day, anti-PD1 10 mg / kg / 3 days, every 3 days; Group 5 (cytarabine + anti-PD1 and anti-TIGIT combination therapy): cytarabine 50 mg / kg / day, anti-PD1 10 mg / kg / 3 days, anti-TIGIT 10 mg / kg / 3 days. Treatment continued for 6 days. Mice were monitored daily, and euthanized if any progressive neurological deficits (such as limb paralysis) or significant loss of weight and mobility were observed.

[0137] 3. Experimental Results (1) The results are as follows Figure 9 As shown in A-9E, Ara-C combined with a PD-1 antibody enhances the antitumor effect of Ara-C monotherapy. Dual blockade therapy with Ara-C combined with both PD-1 and TIGIT antibodies further enhances the antitumor effect and significantly prolongs the survival time of mice. However, the combined use of PD-1 and TIGIT antibodies has no therapeutic effect. Figure 9 Group C, anti-PD1+anti-TIGIT).

[0138] (2) Survival period proves that Ara-C combined with PD1 antibody, Ara-C combined with PD1 antibody and TIGIT antibody have synergistic effects. Survival period is the "hard endpoint" and gold standard for evaluating the efficacy of anti-tumor drugs. It directly reflects whether the therapy can make patients live longer, which is the ultimate goal of tumor treatment.

[0139] Demonstration of the synergistic effect of Ara-C combined with PD1 antibody: PD1 antibody monotherapy cannot improve glioma survival (as disclosed in the prior art, the efficacy can be considered zero), this application Figure 9C indicates that the technical effect achieved by combining PD1 antibody with cytarabine (Group IV) is better than the effect of cytarabine alone (Group II). In other words, this application has achieved a technical effect of 0+1>1, which is a synergistic effect.

[0140] Evidence of the synergistic effect of Ara-C combined with PD1 antibody and TIGIT antibody: This application Figure 9 C indicates that there was no significant difference in overall survival between group III (anti-PD1 + anti-TIGIT) and group I (PBS) (denoted as ns in the figure, signifying a statistically insignificant difference; therefore, the scientific conclusion is clear and unique: anti-PD1 + anti-TIGIT is ineffective in treating spinal glioma), while group II (Ara-C) showed a significant difference compared to group I. Group V (Ara-C + anti-PD1 + anti-TIGIT) showed an extremely significant difference compared to group I. If the combined group effect were merely a simple summation of the individual effects of the two groups, the difference between group V and group I should be the same as that between group II (significant difference). However, the actual difference was extremely significant, indicating that the combined group effect transcends simple summation, proving the existence of a synergistic effect.

[0141] (3) Synergistic effects were assessed using the drug interaction index (CDI), a widely accepted and commonly used computational model in this field. The antitumor effects of Ara-C combined with PD-1 antibody, Ara-C combined with PD-1 antibody, and TIGIT antibody were assessed using the CDI. The CDI calculation formula is: CDI = AB / (A * B), where AB is the ratio of the tumor signal size of the combined drug group to the control group, and A or B is the ratio of the tumor signal size of the single drug group to the control group. A CDI less than 1 indicates a synergistic therapeutic effect between the two drugs. Results are as follows: Figure 10 As shown.

[0142] according to Figure 10 Data calculations show that A = 0.27932, B = 0.80237, AB = 0.11709, and CDI = 0.52242, indicating a significant synergistic effect. Furthermore, Bootstrap analysis revealed that in 10,000 bootstraps, the number of times CDI ≥ 1 was 0 (p < 0.0001), demonstrating statistically significant synergy.

[0143] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A pharmaceutical composition for treating spinal cord glioma, characterized by, The pharmaceutical composition comprises cytarabine or a pharmaceutically acceptable salt thereof, an immune checkpoint inhibitor; Preferably, the spinal cord glioma is H3 K27 variant spinal cord glioma.

2. The pharmaceutical composition of claim 1, wherein, The immune checkpoint inhibitor comprises a CTLA-4 inhibitor, a PDL1 inhibitor, a PDL2 inhibitor, a PD1 inhibitor, a TIGIT inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor, a BTLA inhibitor, a HVEM inhibitor, a TIM3 inhibitor, a GAL9 inhibitor, a LAG3 inhibitor, a VISTA inhibitor, a KIR inhibitor, a 2B4 inhibitor, a CD160 inhibitor, a CGEN-15049 inhibitor, a CHK 1 kinase inhibitor, a CHK2 kinase inhibitor, an A2aR inhibitor, an OX40 inhibitor, and / or a B-7 family ligand inhibitor; Preferably, the immune checkpoint inhibitor is selected from a PD1 inhibitor and / or a TIGIT inhibitor; The immune checkpoint inhibitor comprises a biological therapeutic agent, a small molecule; Preferably, the biological therapeutic agent is selected from an antibody, a fusion protein, or a combination thereof; Preferably, the PD1 inhibitor comprises teprotumumab, sintilimab, camrelizumab, toripalimab, seviprotumab, pembrolizumab, nivolumab, and / or atezolizumab. Preferably, the TIGIT inhibitor comprises vibostolimab and / or tiragolumab.

3. The pharmaceutical composition of claim 1, wherein, The pharmaceutical composition further comprises a pharmaceutically acceptable excipient; Preferably, the pharmaceutically acceptable excipient comprises a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an emulsifying agent, a co-solvent, a solubilizer, an osmotic pressure regulator, a lubricant, a surfactant, a coating material, a coloring agent, a pH regulator, an antioxidant, or a bacteriostatic agent.

4. The pharmaceutical composition of claim 1, wherein, The pharmaceutical composition is a single complex 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 preparations are the same or different; Preferably, the dosage form of the single complex preparation or the combination of the two separate single preparations comprises a solution, a drop, a tablet, a capsule, a granule, a film, a gel, a powder, an emulsion, a suspension, a dripping pill, a suppository, an aerosol, a spray, a powder spray, a patch, an ointment, or a cream.

5. The pharmaceutical composition of claim 1, wherein, The dose ratio of cytarabine or a pharmaceutically acceptable salt thereof and the immune checkpoint inhibitor in the pharmaceutical composition is 2:1 to 100:1; Preferably, the dose ratio is 2:1 to 10:1; Preferably, the dose ratio is 2:1 to 5:1; Preferably, the dose ratio is 2:

1. Preferably, the dosage of said cytarabine is 5-75 mg / m 2 per dose; Preferably, the dosage of said cytarabine is 30 mg / m 2 per dose; Preferably, cytarabine or a pharmaceutically acceptable salt thereof and the immune checkpoint inhibitor in the pharmaceutical composition can be administered sequentially, simultaneously, or alternately.

6. The pharmaceutical composition of claim 1, wherein, The pharmaceutical composition further comprises other drugs for treating glioma.

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

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

9. Any one of the following methods: (1) A method for inhibiting growth of spinal cord glioma cells and promoting apoptosis of spinal cord glioma cells in vitro for non-therapeutic purposes, characterized in that, The method comprises treating spinal cord glioma cells with the pharmaceutical composition of any one of claims 1-6; (2) A method for screening drug candidates for treating H3 K27 variant spinal cord glioma, characterized in that the method comprises: a) applying a test agent to be screened to the model prepared by the method of claim 7; b) detecting the therapeutic effect of the test 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) applying a drug to the model prepared by the method of claim 7; b) detecting the therapeutic effect of the drug on 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 is to study the pathogenesis of H3 K27 variant spinal cord glioma using the model prepared by the method of claim 7. Preferably, the spinal cord glioma is H3 K27 variant spinal cord glioma.

10. Use of an immune checkpoint inhibitor in the preparation of a medicament for improving the effect of cytarabine in treating spinal cord glioma; Preferably, the immune checkpoint inhibitor comprises a CTLA-4 inhibitor, a PDL1 inhibitor, a PDL2 inhibitor, a PD1 inhibitor, a TIGIT inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor, a BTLA inhibitor, a HVEM inhibitor, a TIM3 inhibitor, a GAL9 inhibitor, a LAG3 inhibitor, a VISTA inhibitor, a KIR inhibitor, a 2B4 inhibitor, a CD160 inhibitor, a CGEN-15049 inhibitor, a CHK 1 kinase inhibitor, a CHK2 kinase inhibitor, an A2aR inhibitor, an OX40 inhibitor, and / or a B-7 family ligand inhibitor; Preferably, the immune checkpoint inhibitor is selected from a PD1 inhibitor and / or a TIGIT inhibitor; Preferably, the immune checkpoint inhibitor comprises a biological therapeutic agent or a small molecule; Preferably, the biological therapeutic agent is selected from an antibody, a fusion protein, or a combination thereof. Preferably, the PD1 inhibitor comprises tislelizumab, sintyrosimab, carotuximab, tiraguliximab, seviprotmab, pembrolizumab, pidilizumab, sluvirumab, utomilumab, pembrolizumab, nivolumab; Preferably, the TIGIT inhibitor comprises vibostolimab, tiraguliximab; Preferably, the spinal cord glioma is H3 K27 variant spinal cord glioma.

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