Application of combination of ROR1 intervention agent and temozolomide in treatment of brain glioma

Abstract

The invention provides application of combination of an ROR1 intervention agent and temozolomide in treatment of brain glioma. Experiments prove that the intervention of glioma stem cells ROR1 can block the interaction between glioma stem cells and macrophages, inhibit the generation of endothelial-like stem cells and induce the normalization of glioma blood vessels. The ROR1 intervention agent is combined with temozolomide, so that the delivery efficiency of temozolomide can be improved, the killing effect of temozolomide is enhanced, and the treatment effect is finally improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to application of ROR1 intervention agent combined with temozolomide in treatment of brain glioma. BACKGROUND

[0002] Various targeted therapies for glioblastoma have shown good therapeutic prospects in cell and animal models, but most of the drugs are difficult to apply in clinical due to the existence of blood-brain barrier or due to the large side effects of the drugs. Further exploring safe, effective and suitable treatment options for the biological characteristics of glioblastoma is an important way to improve the clinical treatment effect.

[0003] Temozolomide (TMZ) is a standard chemotherapy drug for glioblastoma. However, the abnormal tortuous and unstable blood vessels of glioma cause uneven distribution of temozolomide in glioma, which cannot fully cover the tumor area. Glioma cells exist in an immunosuppressive microenvironment dominated by macrophages, and microenvironment cells can secrete specific cytokines to promote tumor cell growth and tumor angiogenesis, leading to malignant progression and treatment resistance of glioma. The existing single treatment method is limited by the above characteristics of glioma, and it is often difficult to achieve satisfactory efficacy. SUMMARY

[0004] In view of this, in order to make up for the shortcomings of the prior art, the present application is provided.

[0005] The present application provides, in a first aspect, use of an ROR1 intervention agent combined with temozolomide in the preparation of a drug for treating glioma.

[0006] In the present application, the intervention agent can be understood as any of the following substances or compounds: which can specifically silence, reduce, prevent and / or block the expression of a gene; or which can prevent the transcription of a gene, thus avoiding the formation of any transcription product of the gene; or which can promote the degradation of any transcription product of the gene (transcription product refers to RNA derived from gene transcription); or which can specifically reduce, prevent and / or block the expression of a protein-encoding gene; or which can inhibit the functional activity.

[0007] In some embodiments, the ROR1 intervention agent includes but is not limited to a substance that reduces the expression level of ROR1 or reduces the functional activity of ROR1.

[0008] The expression level of a protein or nucleic acid can be considered to be decreased when it is decreased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% (i.e., absence) relative to a reference value. The "reference value" can refer to the expression level of the protein or nucleic acid prior to administration of the intervention agent. The reference value can be an absolute value, a relative value, a value having an upper and / or lower limit, a range of values, an average value, a median, a mean, or a value expressed by reference to a control or reference value. The reference value can be based on a value obtained from a single sample, such as, for example, a value obtained from a study sample but obtained at an earlier time point. The reference value can be based on a large number of samples, such as, for example, values obtained in a population of samples.

[0009] The functional activity of a protein can be considered to be decreased when it is decreased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% (i.e., absence) relative to a reference value. The reference value can refer to the functional activity of the protein prior to administration of the intervention agent.

[0010] In some embodiments, the ROR1 intervention agent includes, but is not limited to, a reagent used for gene editing, a small nucleic acid drug, a protein inhibitor, a compound.

[0011] In some embodiments, the reagent used for gene editing includes, but is not limited to, a reagent used for zinc finger nuclease technology, transcription activator-like effector nuclease technology, CRISPR-Cas9, Cre-LoxP, FLP / FRT, R / RS, Gin / gix, Cin H / RS2, Par A / MRS, or phiC31.

[0012] In some embodiments, the small nucleic acid drug includes, but is not limited to, a reagent used for RNA interference, antisense oligonucleotide.

[0013] In the present application, RNA interference refers to a phenomenon of highly conserved in evolution, induced by double-stranded RNA, and efficient and specific degradation of homologous mRNA.

[0014] In some embodiments, the reagent used for RNA interference includes, but is not limited to, siRNA, shRNA.

[0015] In some embodiments, the reagent used by the RNA interference is selected from shRNA.

[0016] In some embodiments, the sequence of the shRNA is shown in SEQ ID NO. 1.

[0017] In the present application, a protein inhibitor refers to a substance that binds to some groups on the active center of a protein molecule, reduces or even eliminates the active function of the protein.

[0018] In some embodiments, the protein inhibitor includes but is not limited to an antibody.

[0019] In the present application, the type of the antibody is not limited, including but not limited to an antigen binding fragment, a monoclonal antibody, a polyclonal antibody, a human-derived antibody, a chimeric antibody, or a multispecific antibody formed from at least two complete antibodies. In the present application, the antigen binding fragment includes but is not limited to a VHH, a Fab, a Fab', a (Fab')2, or a Fv fragment.

[0020] In some embodiments, the antibody includes but is not limited to zilovertamab, NVG-111, BR111

[0021] In some embodiments, the antibody is selected from zilovertamab.

[0022] In the present application, the glioma includes a brain glioma and a spinal cord glioma.

[0023] In some embodiments, the glioma is selected from a brain glioma.

[0024] In the present application, the temozolomide can be temozolomide itself, or a hydrate, a pharmaceutically acceptable salt, a solvate, or a crystalline form of temozolomide (hereinafter also referred to as a compound).

[0025] In the present 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 present application. A pharmaceutically acceptable salt is preferred (i.e., non-toxic, physiologically acceptable), 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 an acid or a base in a medium, such as a medium in which the salt is precipitated or an aqueous medium (lyophilized after the reaction).

[0026] Specific pharmaceutically acceptable salts include those that are within the scope of sound medical judgment and that are suitable for use with humans and lower animals with tissues being contacted with the pharmaceuticals without undue toxicity, incompatibility, allergic response, and the like, commensurate with a reasonable benefit / risk ratio. 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.

[0027] Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or the salts of organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid. Also included are salts formed when an acidic proton present in the parent compound is replaced by a cation process such as ion exchange procedures. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, besylate, benzoate, bisulfate, 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. Pharmaceutically acceptable salts derived from suitable bases include alkali metal, alkaline earth metal, ammonium salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic

[0028] In the present application, hydrates refer to compounds associated with water.

[0029] In the present application, solvates refer to forms of compounds or salts thereof associated with solvents, usually formed from solvolysis reactions. This physical association can include hydrogen bonding. Conventional solvents include, but are not limited to, water, methanol, ethanol, acetic acid, DMSO, THF, ether, and the like. In some instances, the solvates will be capable of isolation, for example, where one or more solvent molecules are incorporated into the crystal lattice of the solid state form of the compound. Solvates include both solution-phase solvates and isolatable solvates.

[0030] In the present application, crystalline forms refer to crystalline forms of compounds with specific crystal packing arrangements. 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 over another. Various polymorphs of a compound can be prepared by crystallization under different conditions. In the present application, crystalline forms also include special crystal states, such as amorphous, etc.

[0031] The second aspect of the present application provides a pharmaceutical composition for treating glioma, the pharmaceutical composition comprising a ROR1 intervention agent and temozolomide.

[0032] In some embodiments, the ROR1 intervention agent comprises, but is not limited to, a substance that reduces the expression level or functional activity of ROR1.

[0033] In some embodiments, the ROR1 intervention agent comprises, but is not limited to, a reagent used for gene editing, a small nucleic acid drug, a protein inhibitor, a compound.

[0034] In some embodiments, the reagent used for gene editing comprises, but is not limited to, a reagent used for zinc finger nuclease technology, transcription activator-like effector nucleases technology, CRISPR-Cas9, Cre-LoxP, FLP / FRT, R / RS, Gin / gix, Cin H / RS2, Par A / MRS or phiC31.

[0035] In some embodiments, the small nucleic acid drug comprises, but is not limited to, a reagent used for RNA interference, antisense oligonucleotide.

[0036] In some embodiments, the reagent used for RNA interference comprises, but is not limited to, siRNA, shRNA.

[0037] In some embodiments, the reagent used for RNA interference is selected from shRNA.

[0038] In some embodiments, the sequence of the shRNA is shown in SEQ ID NO. 1.

[0039] In some embodiments, the protein inhibitor comprises an antibody.

[0040] In some embodiments, the antibody is selected from zilovertamab.

[0041] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or adjuvant thereof.

[0042] "Pharmaceutically acceptable" refers to a composition that is physiologically acceptable and generally does not cause gastrointestinal disorders, dizziness, and the like allergic reactions or similar reactions when administered to a subject.

[0043] The pharmaceutically acceptable carrier and / or adjuvant of the present application includes any substance suitable for use in humans and / or mammals without excessive adverse side effects (e.g. toxicity, irritation, and allergic reaction), i.e. having a reasonable benefit / risk ratio. The pharmaceutically acceptable carrier and / or adjuvant is used as necessary to aid formulation stability or to enhance its activity or its bioavailability or to produce an acceptable taste or odor in the case of oral administration. The pharmaceutical composition thus formulated can administer the drug according to any appropriate administration mode known to those skilled in the art as necessary, using the pharmaceutical composition by administering a safe and appropriate dose of the pharmaceutical composition of the present application to the subject.

[0044] The pharmaceutical composition of the present application can include a pharmaceutically acceptable carrier and / or adjuvant including diluents, binders, surfactants, wetting agents, adsorption carriers, lubricants, and / or disintegrants. Among them, the diluents include but are not limited to lactose, sodium chloride, glucose, urea, starch, water; the binders include but are not limited to starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginic acid and alginic acid salt, xanthan gum, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose; the surfactants include but are not limited to polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfate, stearic acid monoglyceride, cetyl alcohol; the wetting agents include but are not limited to glycerol, starch; the adsorption carriers include but are not limited to starch, lactose, bentonite, silica gel, kaolin, soap clay; the lubricants include but are not limited to zinc stearate, glycerol monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearate fumarate, polyoxyethylene monostearate, monolauryl sucrose acid ester, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium dodecyl sulfate.

[0045] The appropriate dose of the pharmaceutical composition of the present application can be prescribed variously according to the method of formulation, the administration mode, the age, body weight, sex, disease state, diet, administration time, administration route, excretion rate, and reaction sensitivity of the patient, and the like, and a skilled doctor can easily determine the prescription and the desired therapeutically effective dose.

[0046] The pharmaceutical composition of the present application can be administered in any convenient pharmaceutical dosage form, including parenteral administration forms, enteral administration forms.

[0047] The non-gastrointestinal administration forms include injection administration forms, respiratory administration forms, cavity administration forms, mucosal administration forms, skin administration forms, and the gastrointestinal administration forms include tablets, granules, capsules, solutions, dry suspensions, powders, sustained-release preparations, effervescent tablets, emulsions, suspensions, syrups, drops, chewable tablets.

[0048] In some embodiments, the administration forms are selected from injection administration forms.

[0049] The injection administration forms include, but are not limited to, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavity injections; the respiratory administration forms include, but are not limited to, sprays, aerosols, and powder sprays; the cavity administration forms include, but are not limited to, suppositories, aerosols, effervescent tablets, drops, dripping pills, and can be used for rectum, vagina, urethra, nasal cavity, and ear canal; the mucosal administration forms include, but are not limited to, eye drops, nose drops, eye ointments, gargles, sublingual tablets, sticking tablets, and film pasting; and the skin administration forms include, but are not limited to, external solution, lotion, liniment, ointment, plaster, paste, and patch.

[0050] In some embodiments, the pharmaceutical composition is a single complex preparation or a combination of two separate single preparations.

[0051] Specifically, the complex preparation is a complex preparation containing the ROR1 intervention agent and temozolomide, and the combination of single preparations is a combination of the ROR1 intervention agent and a single preparation containing temozolomide.

[0052] In some embodiments, the ROR1 intervention agent and temozolomide in the pharmaceutical composition can be administered sequentially, simultaneously, or alternately. Simultaneously means that the two drugs are administered at the same time. If not administered simultaneously, they are administered sequentially or alternately within a certain time range, so that both can function therapeutically within the same time range.

[0053] The pharmaceutical composition of the present application can be used alone or in combination with surgery, radiotherapy, hormone therapy, chemotherapy, and / or immune cell therapy.

[0054] In the present application, chemotherapy refers to the administration of a chemotherapeutic agent to treat a tumor. A chemotherapeutic agent refers to a compound or derivative thereof that can interact with cancer cells, thereby reducing the proliferative state of the cells and / or killing the 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 derivatives thereof); substituted nucleotides; substituted nucleosides; DNA demethylating agents (also known as antimetabolites), such as azacitidine); antitumor antibiotics (e.g., mitomycin, doxorubicin); antitumor agents of plant origin (e.g., vincristine, vindesine, paclitaxel, Abraxane); cisplatin; carboplatin; etoposide. These agents can further include, but are not limited to, the anticancer agent trimethoxybenzoic acid ester (TMTX); raltitrexed; S-(4-nitrobenzyl)-6-thioinosine (NBMPR); 6-benzylguanine (6-BG); nitrosoureas (rabinopyranosyl-N-methyl-N-nitrosourea (Aranose), carmustine (BCNU, BiCNU), chlorozotocin, ethylnitrosourea (ENU), fotemustine, lomustine (CCNU), nimustine, N-nitroso-N-methylurea (NMU), ranimustine (MCNU), semustine, streptozocin (streptozotocin)); cytarabine; camptothecin; and therapeutic derivatives of any of the same.

[0055] In the present application, immunotherapy includes, but is not limited to, chimeric antigen receptor T cell immunotherapy, chimeric antigen receptor NK cell immunotherapy, tumor infiltrating lymphocyte therapy.

[0056] Further, the pharmaceutical composition described in the present application can further comprise other drugs for treating glioma, which are not particularly limited as long as they can produce a therapeutic effect on glioma, and are within the scope of protection of the present application. These drugs include, but are not limited to, acivicin, aclarubicin, acodazole hydrochloride, acronine, adozelesin, aldesleukin, asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene hydrochloride, bisnafide dimesylate, bizelesin, bleomycin sulfate, brequinar sodium, bropirimine, busulfan, cactinomycin, calusterone, caracemide, carbetimer, carboplatin, carmustine, carubicin hydrochloride, carzelesin, cedefingol, chlorambucil, cirolemycin, cisplatin, cladribine.

[0057] In the present application, treatment refers to the improvement, prevention, or reversal of a disease or disorder or at least one discernible symptom thereof. In certain embodiments, the treatment refers to the improvement, prevention, or reversal of at least one measurable physiological parameter associated with the disease or disorder to be treated, which parameter need not necessarily be discernible to or recognized by the mammal. In some embodiments, the treatment refers to the inhibition or slowing of a disease or disease progression, which inhibition or slowing can be physical, such as the alleviation of some discernible adverse symptom. As used herein, "treatment" covers the treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or disorder from occurring in an individual that is predisposed or at risk to developing the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, e.g., arresting its development; or (c) relieving the disease, e.g., causing regression of the symptoms associated with the disease.

[0058] The third aspect of the present application provides any one of the following uses:

[0059] (1) use of a ROR1 intervention agent in the preparation of a medicament for improving the therapeutic effect of temozolomide monotherapy;

[0060] (2) use of a ROR1 intervention agent in the preparation of a medicament for improving the blood concentration of temozolomide;

[0061] (3) use of a ROR1 intervention agent, or a ROR1 intervention agent and temozolomide, in the preparation of a medicament for inducing vascular normalization of glioma;

[0062] In some embodiments, the inducing vascular normalization comprises reducing vascular leakage, improving vascular tight junctions;

[0063] (4) use of a ROR1 intervention agent, or a ROR1 intervention agent and temozolomide, in the preparation of a medicament for blocking the interaction between immunosuppressive macrophages and glioma stem cells.

[0064] In the present application, improvement refers to allowing a subject or tumor cell to enhance its ability to respond to the treatment disclosed herein. For example, an improved response can include an ability to respond that 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. As used herein, "improvement" can also refer to an increase in the number of subjects that respond to temozolomide. For example, an improved response can refer to an increase in the total percentage of subjects that respond to the treatment, wherein the percentage increase 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.

[0065] In the present application, a subject includes a human, a mammal (e.g., a cat, a dog, a horse, etc.), a living cell, and other living organisms. A living organism can be as simple as a single eukaryotic cell or as complex as a mammal. A typical patient is a mammal, particularly a primate, and especially a human. For veterinary applications, a wide variety of subjects will be suitable, such as livestock, e.g., cattle, sheep, goats, cows, pigs, etc.; poultry, e.g., chickens, ducks, geese, turkeys, etc.; and domesticated animals, particularly pets such as dogs and cats, etc. For research applications, suitable subjects will be a wide variety of mammals, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and pigs, e.g., inbred pigs, etc.

[0066] The fourth aspect of the present application provides any one of the following methods:

[0067] (1) A method of inducing normalization of blood vessels in a glioma in vitro, the method comprising administering to the glioma a ROR1 intervention agent, or a ROR1 intervention agent and temozolomide;

[0068] In some embodiments, the inducing normalization of blood vessels comprises reducing vascular leakage, improving vascular tight junctions;

[0069] (2) A method of blocking the interaction of immunosuppressive macrophages with glioma stem cells in vitro, the method comprising administering to a system containing immunosuppressive macrophages with glioma stem cells a ROR1 intervention agent, or a ROR1 intervention agent and temozolomide.

[0070] The fifth aspect of the present application provides a method of constructing a glioma non-human animal model, the method comprising causing a non-human animal with a glioma to overexpress ROR1.

[0071] In some embodiments, the non-human animal is a mammal.

[0072] In some embodiments, the mammal includes but is not limited to mouse, rat, rabbit, dog, pig, monkey, sheep.

[0073] In some embodiments, the non-human animal is an immunodeficient non-human animal.

[0074] In some embodiments, the non-human animal is an immunodeficient mouse.

[0075] In some embodiments, the immunodeficient mouse includes but is not limited to BALB / c-nu, NIH-nu, NC-nu Swiss-nu, 03H-nu, C57BL-nu.

[0076] Further, the mouse is a BALB / c-nu nude mouse.

[0077] The sixth aspect of the present application provides any one of the following uses:

[0078] (1) Use of the glioma non-human animal model constructed by the method of the fifth aspect of the present application in screening drug candidates for treating glioma;

[0079] (2) Use of the glioma non-human animal model constructed by the method of the fifth aspect of the present application in evaluating the therapeutic effect of a drug for treating glioma;

[0080] (3) Use of the glioma non-human animal model constructed by the method of the fifth aspect of the present application in studying the pathogenesis of glioma.

[0081] In the present application, 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, libraries of natural compounds in the form of various organic compounds and biological molecules synthesized randomly and directionally, or 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 modifications of known pharmacological agents, such as acylation, alkylation, esterification, amidation, etc. to generate structural analogs.

[0082] The seventh aspect of the present application provides any one of the following methods:

[0083] (1) A method for screening drug candidates for treating glioma, the method comprising:

[0084] a) applying a test agent to be screened to a glioma non-human animal prepared by the method of the fifth aspect of the present application;

[0085] b) analyzing the therapeutic effect of the test agent to be screened and evaluating, selecting an agent that can significantly have a therapeutic effect as a drug candidate;

[0086] (2) A method for evaluating the efficacy of a drug for treating glioma, the method comprising:

[0087] a) applying a drug to a glioma non-human animal prepared by the method of the fifth aspect of the present application;

[0088] b) evaluating the therapeutic effect of the drug on the glioma;

[0089] (3) A method for studying the pathogenesis of glioma, the method being to use a glioma non-human animal prepared by the method of the fifth aspect of the present application to study the pathogenesis of glioma.

[0090] The eighth aspect of the present application provides a method for treating glioma, which comprises administering the pharmaceutical composition of the second aspect of the present application.

[0091] The present application has the advantages and beneficial effects:

[0092] The present application provides the application of ROR1 intervention agent combined with temozolomide in treating brain glioma. The present application proves through experiments that intervention of ROR1 of glioma stem cells can block the interaction between glioma stem cells and macrophages, inhibit endothelial-like stem cell generation, and induce glioma vascular normalization. ROR1 intervention agent combined with temozolomide can improve the delivery efficiency of temozolomide, enhance the killing effect of temozolomide, and ultimately improve the treatment effect. BRIEF DESCRIPTION OF DRAWINGS

[0093] Figure 1 is a result graph of the expression of ROR1 after endothelial differentiation of glioma stem cells, wherein, A is a flowchart of endothelial differentiation of glioma stem cells; B is a flowchart of screening ROR1; C is a result graph of the expression level of ROR1 being increased after endothelial differentiation of glioma stem cells; D is a result graph of the experiment of showing the co-localization of CD31, CD133 and ROR1 by multiple fluorescence staining of brain glioblastoma tissue section;

[0094] Figure 2 is an experimental result graph of the change of the tube formation ability of tumor stem cells after changing the expression of ROR1, wherein, A is an experimental result graph that the tube formation ability of glioma stem cells overexpressing ROR1 is higher than that of the control group cells; B is an experimental result graph that the tube formation ability of glioma stem cells with knockdown of ROR1 is lower than that of the control group cells;

[0095] Figure 3 is the influence of changing the expression of ROR1 on glioma in vivo, wherein, A is an experimental result graph that the tumor growth rate of nude mice overexpressing ROR1 is higher than that of the control group mice; B is an experimental result graph that the tumor growth rate of nude mice with knockdown of ROR1 is lower than that of the control group mice; C is an experimental result graph that the tumor growth rate of nude mice overexpressing ROR1 is higher than that of the control group mice; D is an experimental result graph that the survival period of nude mice overexpressing ROR1 is shorter; E is an experimental result graph that the tumor growth rate of nude mice with knockdown of ROR1 is lower than that of the control group mice; F is an experimental result graph that the survival period of nude mice with knockdown of ROR1 is longer; G is an experimental result graph of the influence of changing the expression of ROR1 on the integrity of glioma blood vessels;

[0096] Figure 4Figure 1 shows the experimental results of how the membrane receptor ROR1 of glioma stem cells promotes endothelial-like differentiation by binding to its own secreted WNT5A and WNT5A secreted by myeloid cells. Figure A shows the protein-protein interaction network between ROR1 and WNT5A; Figure B shows the verification results of the protein-protein interaction between ROR1 and WNT5A; Figure C shows the molecular simulation results of the docking between ROR1 and WNT5A; Figure D shows the results of the CO-IP experiment; Figure E shows the expression of WNT5A in myeloid cells by single-cell sequencing of glioma blastoma; Figure F shows the expression of WNT5A in tumor cells, myeloid cells, T cells, and glial cells; Figure G shows the protein expression of WNT5A in tumor-associated macrophages induced by Western blot analysis of glioma stem cells GSC-1, GSC-2, and THP-1 and peripheral blood mononuclear cells; Figure H shows the experimental results of immunofluorescence detection of the co-localization of WNT5A with CD68 and CD133, respectively.

[0097] Figure 5 This is a diagram showing the experimental results of ROR1 intervention combined with temozolomide synergistically delaying glioma growth;

[0098] Figure 6 This is a graph showing the experimental results of ROR1 intervention combined with temozolomide synergistically prolonging the survival of glioma patients;

[0099] Figure 7 These are experimental results of ROR1 intervention combined with temozolomide for synergistic treatment of glioma. Figure A shows the experimental results of ROR1 intervention combined with temozolomide increasing temozolomide blood concentration; Figure B shows the experimental results of ROR1 intervention combined with temozolomide enhancing the killing effect of temozolomide; Figure C shows the experimental results of ROR1 intervention combined with temozolomide improving tumor vascular tightness; and Figure D shows the experimental results of ROR1 intervention combined with temozolomide reducing tumor vascular leakage. Detailed Implementation

[0100] 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.

[0101] Example 1: RNA sequencing before and after glioma stem cell induction for endothelial-like differentiation

[0102] 1. Experimental materials

[0103] Glioma stem cells, accutase, ECM medium, Matrigel, Trizol reagent (Sigma-Aldrich).

[0104] 2. Experimental methods

[0105] Two glioma stem cell lines (BNI2-4, GSC-1; BNI22-1, GSC-2) were digested into single cells using accutase and inoculated into Matrigel pre-coated culture plates to promote stem cell adhesion. Endothelial differentiation medium was used, and the medium was changed every 2-3 days Figure 1 A). Then the RNA of glioma stem cells and glioma endothelial-like stem cells was extracted using trizol for sequencing, and the differentially expressed genes before and after differentiation of glioma stem cells were analyzed by Deseq2. The up-regulated differential genes of the two glioma endothelial-like stem cell lines were intersected, and after screening according to the correlation of genes with tumor grade and patient prognosis, the potential driver factor ROR1 of endothelial-like differentiation of glioma stem cells was obtained Figure 1 B). Western blot was used to confirm that the expression of ROR1 protein increased after endothelial-like (GDEC) differentiation of glioma stem cells (GSC). CD31, CD133, and ROR1 triple fluorescence staining was performed on brain glioblastoma tissue sections.

[0106] 3. Experimental results

[0107] The experimental results are shown in Figure 1 , the expression of ROR1 increased at the RNA Figure 1 B), protein Figure 1 C) levels after endothelial-like differentiation of glioma stem cells, and multiple fluorescence staining of brain glioblastoma tissue sections showed co-localization of CD31, CD133, and ROR1 Figure 1 D).

[0108] Example Two: Intervention of Glioma Stem Cell ROR1 in vitro tube formation experiment

[0109] 1. Experimental materials

[0110] Glioma stem cells, Matrigel (Matrigel, Corning, #356234), cell culture medium, overexpression and knockdown ROR1 lentiviral vector, puromycin

[0111] 2. Experimental methods

[0112] Glioma stem cells were digested into single cells at 1 x 10^ 5Cells were seeded in 24-well plates at a density of 1-5 x 10 cells per well and cultured for 16-24 hours to reach 40%-60% confluence. According to the MOI value, 1-5 μL of lentivirus was added to the well plate, and after 8-12 hours of culture in the culture medium, the complete culture medium was replaced and the cells were cultured for another 48-72 hours. After 72 hours, puromycin was added for screening (2 μg / mL), and the medium was replaced every 2-3 days for 7-14 days until all untransfected cells died, thereby obtaining glioma stem cells overexpressing or knocking down ROR1. The shRNA sequence for knocking down ROR1 was SEQ ID NO. 15'-GCAAGATCAAATCCCATGATT-3', and the control shRNA sequence was SEQ ID NO. 25'-CCTAAGGTTAAGTCGCCCTCG-3'. Matrigel was transferred from -20°C to the 4°C refrigerator overnight for thawing (kept in a liquid state to avoid repeated freezing and thawing). The gun head, 96-well plate, and workbench were pre-cooled to prevent Matrigel from solidifying in advance. The Matrigel was gently mixed with the pre-cooled gun head (to avoid air bubbles). 100 μL of Matrigel was added to each well of the 48-well plate to ensure a smooth liquid surface. The Matrigel was incubated at 37°C for 30 minutes to 1 hour to completely solidify (to form a uniform gel layer). The induced glioma stem cells were digested into a single-cell suspension with Accutase. After centrifugation (1000 rpm, 5 minutes), the cells were resuspended in the endothelial culture medium, counted, and adjusted to a density of about 3-5 x 10 4 Cells were seeded in 24-well plates at a density of 1-5 x 10 cells per well and cultured for 16-24 hours to reach 40%-60% confluence. According to the MOI value, 1-5 μL of lentivirus was added to the well plate, and after 8-12 hours of culture in the culture medium, the complete culture medium was replaced and the cells were cultured for another 48-72 hours. After 72 hours, puromycin was added for screening (2 μg / mL), and the medium was replaced every 2-3 days for 7-14 days until all untransfected cells died, thereby obtaining glioma stem cells overexpressing or knocking down ROR1. The shRNA sequence for knocking down ROR1 was SEQ ID NO. 15'-GCAAGATCAAATCCCATGATT-3', and the control shRNA sequence was SEQ ID NO. 25'-CCTAAGGTTAAGTCGCCCTCG-3'. Matrigel was transferred from -20°C to the 4°C refrigerator overnight for thawing (kept in a liquid state to avoid repeated freezing and thawing). The gun head, 96-well plate, and workbench were pre-cooled to prevent Matrigel from solidifying in advance. The Matrigel was gently mixed with the pre-cooled gun head (to avoid air bubbles). 100 μL of Matrigel was added to each well of the 48-well plate to ensure a smooth liquid surface. The Matrigel was incubated at 37°C for 30 minutes to 1 hour to completely solidify (to form a uniform gel layer). The induced glioma stem cells were digested into a single-cell suspension with Accutase. After centrifugation (1000 rpm, 5 minutes), the cells were resuspended in the endothelial culture medium, counted, and adjusted to a density of about 3-5 x 10

[0113] 3. Experimental results

[0114] The experimental results are shown in Figure 2 Fig. 3A, the tube formation ability of glioma stem cells overexpressing ROR1 was higher than that of the control cells (A), and the tube formation ability of glioma stem cells knocking down ROR1 was lower than that of the control cells (B). Figure 2 Figure 2

[0115] Example Three: In vivo experiment after intervention of ROR1 of glioma stem cells

[0116] 1. Experimental materials

[0117] Glioma patient-derived brain tumor stem cells, 6-week-old female BALB / c -Nude mice, scissors, microsyringe, stereotactic instrument, mouse experimental instruments, trichloroethanol anesthetic, luciferin potassium salt (D-Luciferin potassium, HY-12591B, MCE), and fluorescent dye dextran.

[0118] 2. Experimental method

[0119] ​​Glioma stem cells in the logarithmic growth phase were harvested, washed with PBS, digested, centrifuged, resuspended, and stored on ice for later use. Female nude mice (weighing 16-20 grams) were intraperitoneally injected with 45 mg / kg of 2% sodium pentobarbital for 6-8 weeks. After the anesthetic took effect, the mice were fixed to a stereotactic apparatus using ear rods and upper tooth fixation plates. The scalp was transversely incised along the line connecting the bilateral eye fissures to expose the scalp.

[0120] Drill a hole 1.5 mm from the midline on the left side of the anterior fontanelle, horizontally positioned. Using a 10 μL microsyringe, aspirate 3 μL of cell suspension (containing 3 × 10⁻⁶ cells / mL). 5 (One cell) was fixed in a stereotactic instrument and aligned with the drill hole. The needle was slowly advanced vertically to 3.3 mm and withdrawn 0.5 mm, and the cell suspension was slowly injected, pausing for 1 minute after each 1 μL injection. After injection, the needle was slowly withdrawn after a 5-minute pause. The bone window was sealed with bone wax, and the skull was closed layer by layer. On days 7 and 18 after tumor implantation, in vivo imaging was performed using a small animal fluorescence imaging system, and the fluorescence signal values ​​were recorded. The weight of the mice was monitored, and if cachexia occurred, they were anesthetized and euthanized. Two hours before euthanasia, fluorescein was injected via the tail vein, followed by fixation with paraformaldehyde and tissue section fluorescence staining.

[0121] 3. Experimental Results

[0122] Experimental results are as follows Figure 3 As shown, nude mice overexpressing ROR1 had a higher tumor growth rate, shorter survival time, and more severe tumor vascular leakage than control mice; nude mice with knocked-down ROR1 had a lower tumor growth rate, longer survival time, better tumor vascular integrity, and less leakage than control mice.

[0123] Example 4: Blocking the binding of microenvironment-derived WNT5A to ROR1 in glioma stem cells

[0124] 1. Experimental materials

[0125] Glioma stem cells, myeloid cells, paraffin-embedded sections of tumor tissue from glioma patients, Co-IP kit, PBS, RIPA lysis buffer.

[0126] 2. Experimental Methods

[0127] The strongest ligand WNT5A that binds to ROR1 is predicted using String database. After adding WNT5A in the culture medium of glioma stem cells, the endothelial phenotype molecules are detected by WB. The combination of ROR1 and WNT5A in glioma stem cells is verified by Co-IP: the glioma stem cells are cultured, washed twice with PBS after reaching 80% confluence, and collected by scraping or trypsin digestion. Pre-cooled RIPA lysis buffer is added, and lysis is performed on ice for 30 minutes. Centrifuged at 4°C, 12000 rpm for 15 minutes, and the supernatant (protein sample) is taken. The protein concentration is determined by BCA method, and adjusted to 1-2 μg / μL. Take 100-200 μg of protein, add 20 μL of Protein A / G magnetic beads, and rotate at 4°C for 1 hour. Centrifugation to take the supernatant (remove non-specific binding proteins).

[0128] Take 500-1000 μg of protein, add 1-5 μg of IP antibody (optimize the concentration), and incubate at 4°C for 2-4 hours or overnight. Add 20-50 μL of Protein A / G magnetic beads, and rotate at 4°C for 1-2 hours. Follow the same steps, and use the same species IgG instead of IP antibody for negative control. Wash the magnetic beads 3-5 times with 1 mL of lysis buffer (5 minutes each time, rotate at 4°C). Wash with PBS or TBS for the last time, remove the detergent (avoid interference with WB). Add Loading Buffer, boil at 95°C for 5 minutes, and centrifuge to take the supernatant. Then perform WB detection and development. Collect myeloid cell and glioma stem cell proteins, and detect the protein expression level of WNT5A by WB. Use glioblastoma tissue sections, and detect the co-localization of WNT5A with CD68 and CD133 by immunofluorescence.

[0129] 3、Experimental results

[0130] The experimental results are shown in Figure 4 The membrane receptor ROR1 of glioma stem cells binds to WNT5A secreted by itself and WNT5A secreted by myeloid cells to promote endothelial-like differentiation. WNT5A in the glioma microenvironment mainly comes from myeloid cells.

[0131] Example Five: Targeted intervention of ROR1 combined with temozolomide for treating orthotopic glioma in mice

[0132] 1、Experimental materials

[0133] Glioma patient-derived brain tumor stem cells, 6-week-old female BALB / c-Nude nude mice, scissors, microsyringe, stereotaxic instrument, mouse experimental instruments, trichloroethanol anesthetic, luciferin potassium salt (D-Luciferin potassium, HY-12591B, MCE), luciferin dye dextran, temozolomide (MCE, HY-17364), electron microscope fixing solution, VLS-101 (MCE, HY-P99956).

[0134] 2. Experimental method

[0135] (1) Glioma stem cells (1 x 10 5 BNI2-4 cells) were mixed with macrophages (2 x 10 5 THP-1 cells) and then implanted into the brain of mice in situ. The specific operation steps were the same as those in Example Three Experimental Method. Five days after implantation, tumor began to proliferate stably, and the control group was intervened, the ROR1 targeting drug or temozolomide single drug intervention, and the ROR1 targeting drug combined with temozolomide intervention were performed, respectively. The control group of mice was injected with PBS through the tail vein and the abdominal cavity, the temozolomide single drug treatment group was injected with 5mg / kg of temozolomide through the abdominal cavity on the 19th, 20th and 21st days after implantation, the ROR1 targeting drug single drug group was injected with 2.5mg / kg of VLS-101 through the tail vein on the 7th, 14th and 21st days after implantation, and the combination treatment group was injected with 2.5mg / kg of VLS-101 through the tail vein on the 7th, 14th and 21st days after implantation, and 5mg / kg of temozolomide through the abdominal cavity on the 19th, 20th and 21st days after implantation.

[0136] The growth rate of mouse tumor was monitored by in vivo fluorescence imaging, and the survival period of mice was monitored. At the end of the experiment, part of the mouse brain tissue was fixed for sampling using paraformaldehyde.

[0137] (2) Glioma stem cells were mixed with macrophages and then implanted into the brain of mice in situ. The control group was intervened, the single drug intervention, and the ROR1 targeting drug combined with temozolomide intervention were performed, respectively. The growth rate of mouse tumor was monitored by in vivo fluorescence imaging, and the survival period of mice was monitored. At the end of the experiment, part of the mouse brain tissue was fixed for sampling using paraformaldehyde, and fluorescence staining of tissue sections was performed. Part of the mouse tumor was ground, and the concentration of temozolomide in the tumor was detected by high performance liquid chromatography. The tumor was cut into 1mm 3 left and right sizes, and then fixed with electron microscope fixing solution for transmission electron microscopy imaging to observe the changes of endothelial cell tight junctions.

[0138] 3. Experimental results

[0139] The experimental results are as follows: Figure 5 , Figure 6 , Figure 7As shown, the combined targeting ROR1 and temozolomide treatment significantly delayed tumor growth compared with control treatment, ROR1 targeting treatment alone, and temozolomide treatment alone Figure 5 , and improved the survival of tumor-bearing mice Figure 6 . The combined targeting ROR1 and temozolomide treatment reduced tumor vascular leakage, enhanced the killing effect of temozolomide and intratumoral drug concentration, and improved tumor vascular tight junctions compared with temozolomide treatment alone Figure 7 . The tumor shrinkage rate was 60% in the temozolomide monotherapy group, 36% in the ROR1 targeting monotherapy group, and 99.9% in the combined treatment group. The Q value calculated using the King's formula was 1.34.

[0140] The above description of the embodiments is only for the purpose of understanding the method of the present application and its core idea. It should be noted that, for those skilled in the art, without departing from the principles of the present application, a number of improvements and modifications can be made to the present application, and these improvements and modifications will also fall within the scope of protection of the claims of the present application.

Claims

1. Application of ROR1 intervention combined with temozolomide in the preparation of drugs for the treatment of glioma.

2. The application according to claim 1, characterized in that, The ROR1 intervention includes substances that reduce the expression level or functional activity of ROR1.

3. The application according to claim 2, characterized in that, The ROR1 intervention agents include reagents used in gene editing, small nucleic acid drugs, protein inhibitors, and compounds; Preferably, the reagents used in the gene editing include those used in zinc finger nuclease technology, transcription activator-like effector nuclease technology, CRISPR-Cas9, Cre-LoxP, FLP / FRT, R / RS, Gin / gix, Cin H / RS2, Par A / MRS, or phiC31. Preferably, the small nucleic acid drug includes reagents used for RNA interference and antisense oligonucleotides; Preferably, the reagents used for RNA interference include siRNA and shRNA; Preferably, the reagent used for RNA interference is selected from shRNA; Preferably, the sequence of the shRNA is shown in SEQ ID NO.1; Preferably, the protein inhibitor comprises an antibody; Preferably, the antibody is selected from zilovertamab.

4. The application according to claim 1, characterized in that, The glioma was selected from brain gliomas.

5. A pharmaceutical composition for treating glioma, characterized in that, The pharmaceutical composition comprises an ROR1 intervention agent and temozolomide; Preferably, the ROR1 intervention includes substances that reduce the expression level or functional activity of ROR1; Preferably, the ROR1 intervention agent includes reagents used in gene editing, small nucleic acid drugs, protein inhibitors, and compounds; Preferably, the reagents used in the gene editing include those used in zinc finger nuclease technology, transcription activator-like effector nuclease technology, CRISPR-Cas9, Cre-LoxP, FLP / FRT, R / RS, Gin / gix, Cin H / RS2, Par A / MRS, or phiC31. Preferably, the small nucleic acid drug includes reagents used for RNA interference and antisense oligonucleotides; Preferably, the reagents used for RNA interference include siRNA and shRNA; Preferably, the reagent used for RNA interference is selected from shRNA; Preferably, the sequence of the shRNA is shown in SEQ ID NO.1; Preferably, the protein inhibitor comprises an antibody; Preferably, the antibody is selected from zilovertamab; Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipients; Preferably, the glioma is selected from brain gliomas.

6. Any of the following applications: (1) Application of ROR1 intervention in the preparation of drugs that improve the efficacy of temozolomide monotherapy; (2) Application of ROR1 intervention in the preparation of drugs that increase temozolomide blood concentration; (3) The use of ROR1 intervention agents, or ROR1 intervention agents and temozolomide in the preparation of drugs that induce glioma vascular normalization; Preferably, the induction of vascular normalization includes reducing vascular leakage and improving vascular tightness. (4) The use of ROR1 intervention agents, or ROR1 intervention agents and temozolomide in the preparation of drugs that block the interaction between immunosuppressive macrophages and glioma stem cells.

7. Any one of the following methods: (1) A method for in vitro induction of glioma vascular normalization, characterized in that, The method includes administering a ROR1 intervention agent, or a ROR1 intervention agent and temozolomide, to the glioma; Preferably, the induction of vascular normalization includes reducing vascular leakage and improving vascular tightness. (2) A method for blocking the interaction between immunosuppressive macrophages and glioma stem cells in vitro, characterized in that the method includes administering ROR1 intervention agent, or ROR1 intervention agent and temozolomide, to a system containing immunosuppressive macrophages and glioma stem cells.

8. A method for constructing a non-human animal model of glioma, characterized in that, The method includes overexpressing ROR1 in non-human animals with gliomas; Preferably, the non-human animal is a mammal; Preferably, the mammals include mice, rats, rabbits, dogs, pigs, monkeys, and sheep; Preferably, the non-human animal is an immunodeficient non-human animal; Preferably, the non-human animal is an immunodeficient mouse.

9. Any of the following applications: (1) The application of the non-human animal model of glioma constructed by the method of claim 8 in screening drug candidates for the treatment of glioma; (2) The application of the non-human animal model of glioma constructed by the method of claim 8 in evaluating the therapeutic effect of drugs for treating glioma; (3) Application of the non-human animal model of glioma constructed by the method described in claim 8 in the study of the pathogenesis of glioma.

10. Any one of the following methods: (1) A method for screening drug candidates for the treatment of glioma, characterized in that, The method includes: a) Apply the screening reagent to non-human animals with gliomas prepared by the method of claim 8; b) Analyze and evaluate the therapeutic effects of the reagents to be screened, and select reagents that have significant therapeutic effects as drug candidates; (2) A method for evaluating the efficacy of a drug for treating glioma, characterized in that the method comprises: a) Applying the drug to a non-human animal with a glioma prepared by the method of claim 8; b) Evaluate the therapeutic effect of the drug on the glioma; (3) A method for studying the pathogenesis of glioma, characterized in that the method uses non-human animals with gliomas prepared by the method described in claim 8 to study the pathogenesis of gliomas.

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