WO2024026694A1 - 麝香提取物及其增强car-t细胞疗效的应用 - Google Patents

麝香提取物及其增强car-t细胞疗效的应用 Download PDF

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WO2024026694A1
WO2024026694A1 PCT/CN2022/109798 CN2022109798W WO2024026694A1 WO 2024026694 A1 WO2024026694 A1 WO 2024026694A1 CN 2022109798 W CN2022109798 W CN 2022109798W WO 2024026694 A1 WO2024026694 A1 WO 2024026694A1
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car
cells
musk
extract
musk extract
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钟晓松
黄帅
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卡瑞济(北京)生命科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/55—Glands not provided for in groups A61K35/22 - A61K35/545, e.g. thyroids, parathyroids or pineal glands
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor

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  • the present invention relates to the field of cell therapy. Specifically, the present invention relates to a method and corresponding uses of musk extract to assist and/or combine with CAR-T cells to treat tumors.
  • GBM Glioblastoma
  • GBM is the most common and most aggressive primary malignant brain tumor in humans, and most cases are highly malignant. Due to high morbidity, high mortality, and low cure rate, GBM has caused a huge social and medical burden worldwide, and is almost impossible to cure with traditional treatment methods, so the overall survival of patients is very unsatisfactory.
  • the current standard treatment strategy is to maximize safety by surgical resection of the tumor, postoperative concurrent radiotherapy and chemotherapy, and subsequent six months of adjuvant chemotherapy with temozolomide.
  • the average survival time of patients is only 14.6 months.
  • Chimeric antigen receptor (Chimeric antigen receptor, CAR) is a synthetic molecule that specifically recognizes antigens expressed on the surface of tumor cells to guide immune effector cells genetically engineered to express CAR (e.g., T cells, NK cells) clear tumors (Sampson JH, Choi BD, Sanchez-Perez L et al., EGFRvIII mCAR-modified T-cell therapy cures mice with established intracerebralglioma and generates host immunity against tumor-antigen loss. Clinical cancer research:an official journal of the American Association for Cancer Research. 2014; 20(4):972-984).
  • CAR Chimeric antigen receptor
  • CAR-T chimeric antigen receptor T cells
  • CAR-T directly target the surface antigens of tumor cells through chimeric antigen receptor (CAR) molecules on T cells, thereby achieving the purpose of recognizing and killing tumors.
  • the N-terminus of the chimeric antigen receptor contains an extracellular domain that recognizes the antigen.
  • the CAR-T cells can recognize and kill these antigen-positive cells.
  • CAR-T cell therapy has achieved some satisfactory results.
  • CAR-T cell therapy targeting CD19 has cured or nearly cured some patients with leukemia or lymphoma.
  • gliomas can also rely on the protection of the blood-brain barrier to escape the killing effect of CAR-T cells. For example, if CAR-T is administered systemically, the number of cells that can pass through the blood-brain barrier and reach the tumor site is very limited; but if CAR-T cells are directly injected into the cerebrospinal fluid, such as intracranial injection or intrathecal injection, the number of cells will increase. It makes implementation difficult and brings unnecessary risks, such as intracranial infection and cytokine storm in the central nervous system.
  • the present invention uses musk extract, which retains a variety of key active ingredients including muscone and musk polypeptides, to study its activity on CAR-T cells and its effect on the brain. And unexpectedly found that musk extract has the activity of enhancing the efficacy of CAR-T cells in killing tumors, and has the activity of enhancing the ability of CAR-T cells to reach the tumor area (such as crossing the blood-brain barrier to reach glioma tissue), the latter It may be part of the former, or it may be independent of the former.
  • the present invention provides a musk extract, which is characterized in that it is prepared in the following manner: the musk powder is dissolved in sterile PBS or DMSO at a ratio of 1 mg of musk powder to 100 ⁇ L of solvent, Ultrasonicate for 1 hour, then centrifuge to take the supernatant, which is the musk extract.
  • the centrifugation conditions are 12,000 rpm for 5 min.
  • the content of muscone in the supernatant is not less than about 20 ⁇ g/mL. In a preferred embodiment, the content of muscone in the supernatant is no less than about 25 ⁇ g/mL, about 30 ⁇ g/mL, about 40 ⁇ g/mL, or about 50 ⁇ g/mL.
  • the present invention provides the use of the musk extract of the first aspect of the present invention for preparing a therapeutic efficacy enhancer for CAR-T cell therapy.
  • the CAR-T cell therapy targets hematological tumors.
  • the CAR-T cell therapy is directed against solid tumors.
  • the CAR-T cell therapy targets glioblastoma.
  • the target molecule of the chimeric antigen receptor of the CAR-T cell is IL-13R ⁇ , EphA2 or EGFRvIII (type III EGF deletion mutant receptor).
  • the efficacy enhancer promotes CAR-T cells to secrete cytokines to kill tumor cells. In other specific embodiments, the efficacy enhancer enhances the direct killing effect of CAR-T cells on target cells. In some preferred embodiments, the efficacy enhancer acts as a efficacy enhancer by enhancing the ability of CAR-T cells to reach the tumor area. In some more preferred embodiments, the efficacy enhancer promotes CAR-T cells to penetrate the blood-brain barrier so that more CAR-T cells reach the tumor (eg, glioblastoma) area in the brain, that is, the The effects of efficacy enhancers include at least increasing the number of CAR-T cells that penetrate the blood-brain barrier. In a preferred embodiment, the efficacy enhancer promotes the entry of CAR-T cells administered through systemic routes such as intravenously and intraperitoneally into the brain, thereby avoiding the side effects and/or risks of direct intracranial/intraventricular administration.
  • systemic routes such as intravenously
  • the present invention provides a composition comprising the musk extract of the first aspect of the present invention, said composition being used as a efficacy enhancer for CAR-T cell therapy.
  • the CAR-T cell therapy targets hematological tumors.
  • the CAR-T cell therapy is directed against solid tumors.
  • the CAR-T cell therapy targets glioblastoma.
  • the target molecule of the chimeric antigen receptor of the CAR-T cell is IL-13R ⁇ , EphA2 or EGFRvIII (type III EGF deletion mutant receptor).
  • the compositions act as efficacy enhancers by promoting cytokine secretion from CAR-T cells.
  • the composition acts as a therapeutic enhancer by enhancing the direct killing effect of CAR-T cells on target cells.
  • the composition acts as a efficacy enhancer by enhancing the ability of CAR-T cells to reach the tumor area.
  • the composition acts as a therapeutic agent by promoting the ability of CAR-T cells to penetrate the blood-brain barrier, allowing more CAR-T cells to reach the tumor (e.g., glioblastoma) area within the brain. Enhancers.
  • the composition promotes the entry of CAR-T cells administered via systemic routes such as intravenously and intraperitoneally into the brain, thereby avoiding the side effects and/or risks of direct intracranial/intraventricular administration.
  • the present invention provides a pharmaceutical composition comprising the musk extract of the first aspect of the present invention or the composition of the third aspect of the present invention, in addition to a suitable pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier makes the pharmaceutical composition suitable for administration by the intended route, such as, but not limited to, intravenous injection or local tumor injection, and the like.
  • pharmaceutical compositions of the invention are administered prior to CAR-T cells.
  • pharmaceutical compositions of the invention are administered following administration of CAR-T cells.
  • pharmaceutical compositions of the invention are administered simultaneously with CAR-T cells.
  • the pharmaceutical compositions of the invention further comprise CAR-T cells.
  • the present invention provides the use of the musk extract of the first aspect of the present invention in combination with CAR-T cells for the preparation of anti-tumor drugs.
  • the present invention provides a method for preparing the musk extract of the present invention and a composition containing the musk extract, comprising the following steps: Dissolve 1 mg of musk powder in 100 ⁇ L of sterile PBS or DMSO , ultrasonic for 1 hour, centrifuge and take the supernatant, which is the musk extract.
  • the invention provides a method for preparing a pharmaceutical composition comprising the musk extract of the invention, comprising the steps of: providing the musk extract of the invention, or a composition comprising the musk extract , and add the required pharmaceutically acceptable carrier.
  • musk refers to the scent sac secreted by mature males of musk deer species such as Moschus berezovskii flerov, M. sifanicus gravwalssi. and M. moschiferus linaeus. Dry products. It is recorded in traditional Chinese medicine classics that musk is pungent and warm in nature, and enters the heart meridian and brain meridians. It has the effects of calming the mind and calming the mind, refreshing the brain, unblocking the orifices, activating blood circulation and dredging meridians, reducing swelling and relieving pain, opening meridians and penetrating muscles and bones. It is mainly used to treat fever.
  • musk used in this article is any common form of musk that can be commercially obtained from any conventional channels, for example, musk powder purchased in pharmacies, etc.
  • Ultrasonic extraction is a method that uses ultrasonic vibration for extraction.
  • the mechanical action of ultrasound causes the solvent to quickly enter the solid material and dissolve the organic components contained in the material into the solvent as completely as possible, thereby obtaining multi-component mixed extraction. liquid.
  • Ultrasonic extraction can effectively extract muscone, the main active substance in musk.
  • tumor and cancer are not mutually exclusive and are used interchangeably herein, encompass both solid and hematological tumors, and refer to all neoplastic cell growth and proliferation, whether malignant or benign, and All pre-cancerous and cancerous cells and tissues.
  • cancers suitable for treatment by the antibodies of the invention include gastric or pancreatic cancer, including metastatic forms of those cancers.
  • glioma is short for glioma, also known as glioma.
  • Glioblastoma is a tumor disease of the central nervous system, which refers to tumors that occur in the neuroectoderm and originate from the canceration of neuronal cells or mesenchymal cells.
  • Glioma is the most common intracranial primary central nervous system tumor, accounting for approximately half of all intracranial primary tumors.
  • Glioblastoma is a relatively common type of glioma, also known as glioblastoma multiforme (GBM). It is an astrocytic tumor that has progressed to Grade IV stage (WHO International Classification of Diseases). The special name of law), which is the most malignant.
  • the peak age of glioblastoma incidence is generally between 50 and 60 years old.
  • CAR-T or “CAR T” refers to T lymphocytes that are transduced and express a chimeric antigen receptor (CAR).
  • the chimeric antigen receptor refers to one or more groups of polypeptides that, when in immune effector cells, provide the cells with specificity for target cells (usually cancer cells) and have intracellular signal generation .
  • a CAR includes at least an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain.
  • Exemplary CAR construction methods and/or CAR-T cell transduction methods are described in, for example, Chinese Patent Publication No. CN114014941A.
  • composition refers to a mixture of the musk extract useful in the present invention as the active ingredient and a pharmaceutically acceptable carrier (as appropriate). Pharmaceutical compositions facilitate the administration of active ingredients to patients.
  • the term "pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener Agents, solvents or encapsulating materials involved in carrying or transporting useful compounds of the present invention into or into a patient so that they can perform their intended function.
  • a pharmaceutically acceptable material, composition or vehicle such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener Agents, solvents or encapsulating materials involved in carrying or transporting useful compounds of the present invention into or into a patient so that they can perform their intended function.
  • a pharmaceutically acceptable material, composition or vehicle such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener Agents, solvents or encapsulating materials involved in carrying or transporting useful compounds of the present invention into or into a
  • materials that can be used as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethanol Base cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and Soybean oil; glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and Aluminum hydroxide; surfactants; alginic acid; pyrogen-free water; isotonic sa
  • pharmaceutically acceptable carrier also includes any and all coatings, antibacterial and antifungal agents, absorption delaying agents, and the like that are compatible with the activity of the compounds of the invention and physiologically acceptable to the patient. Supplementary active compounds can also be incorporated into the compositions. "Pharmaceutically acceptable carriers” may further include pharmaceutically acceptable salts of compounds useful in the present invention. Other ingredients that may be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, Pa.), which is incorporated herein by reference. .
  • compositions are formulated for intravenous injection.
  • the pharmaceutical composition comprises a musk extract formulated for intravenous injection.
  • the pharmaceutical composition comprises musk extract and CAR-T cells formulated for intravenous injection.
  • Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (water-soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • suitable carriers include physiological saline, bacteriostatic water, or fluids formulated to facilitate injection. Injectable compositions must be stable under the conditions of manufacture and storage and must be protected from contamination by microorganisms such as bacteria and fungi.
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (eg, glycerin, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.
  • polyols eg, glycerin, propylene glycol, liquid polyethylene glycol, etc.
  • suitable mixtures thereof for example, water, ethanol, polyols (eg, glycerin, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.
  • Proper flowability can be maintained, for example, by using coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.
  • the action of microorganisms is prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.
  • isotonic agents such as sugars, polyols such as mannitol, sorbitol, sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • treating means slowing, interrupting, retarding, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
  • prevention includes the inhibition of the onset or progression of a disease or condition or symptoms of a particular disease or condition.
  • subjects with a family history of cancer are candidates for a preventive regimen.
  • prevention refers to the administration of a drug before signs or symptoms of cancer occur, particularly in a subject at risk for cancer.
  • efficacy enhancer or “efficacy enhancer” means an agent that can enhance the therapeutic effect of an existing therapy, where the existing therapy can refer to any therapy that has been used or known in the art, preferably a therapy for cancer. , CAR-T cell therapy is more preferred. The efficacy is enhanced.
  • the patients who have accepted the efficacy enhancer have slowed, more interrupted, more blocked, more alleviated, more stopped, reduced or a greater reversal of the progression or severity of an existing symptom, disorder, condition or disease, or fewer side effects, a better treatment experience, a higher quality of life, or simply, achieved or substantially achieved than without treatment Efficacy enhancers provide better treatment courses or outcomes for patients treated with existing therapies.
  • Figure 1 shows the component map obtained by HPLC analysis of musk extract.
  • the horizontal axis of the graph is the time axis, and the vertical axis is the absorption intensity unit (Absorbance unit).
  • Figure 2 shows the bar chart statistical comparison of the proliferation of CAR11-3 CAR-T cells measured by CCK-8 after treatment with different concentrations of musk extract.
  • Figure 3 shows the culture supernatant of PMBC cells and CAR11-3 CAR-T cells cultured alone or co-cultured with U87 and U251 after being treated with musk extract at gradient concentrations (0, 15, 60 and 150 ⁇ g/mL). levels of the cytokine IFN- ⁇ .
  • Figure 4 shows the CAR11-3 cells and target cell U87 co-culture system. After being treated with musk extract for 24 hours, the unbound CAR11-3 cells in the supernatant were aspirated, and then the CAR11-3 cells were observed under the microscope and photographed. Direct killing of target cells.
  • 4X, 10X, and 20X represent objective lens magnifications of 4x, 10x, and 20x.
  • the solvent control is the extraction solvent control.
  • Figure 5 shows the co-culture system of CAR11-3 cells and target cell U251. After being treated with musk extract for 24 hours, the unbound CAR11-3 cells in the supernatant were aspirated, and then the CAR11-3 cells were observed under the microscope and photographed. Direct killing of target cells.
  • 4X, 10X, and 20X represent objective lens magnifications of 4x, 10x, and 20x.
  • the solvent control is the extraction solvent control.
  • Figure 6 shows the gradient concentration of musk extract (0, 15, 60 and 150 ⁇ g/mL) treating untransduced PBMC and CAR-V3 cells (co-culture group and non-co-culture group with NALM-6 target cells respectively) After 24 hours, the secretion level of IFN- ⁇ in the supernatant.
  • NT non-transduced PBMC cells.
  • V3 CAR-V3 cells.
  • N6 Adult B acute lymphoblastic leukemia NALM-6 cell line.
  • Figure 7 shows that after the luciferase-expressing glioma stably transfected cell line was established in situ in the mouse brain, 10 7 CAR-T cells were injected through the tail vein, and at the same time, 100 mg musk extract/kg The dosage of mouse body weight was injected intraperitoneally with musk extract, and comparisons were made between before and after treatment (day 0 vs day 6) through in vivo fluorescence imaging of small animals.
  • musk powder Precisely weigh 5 mg of musk powder (Beijing Millennium Monument Hospital) into separate packages. Take 1 mg and add 100 ⁇ L of sterile PBS (or DMSO). Use an ultrasonic disruptor to set the power of 90W for 1 hour. Take another 1 mg of powder and add 100 ⁇ L of PBS as before. Ultrasonicate for 1 hour, then centrifuge at 12000 rpm for 5 minutes and combine the supernatant extracts, i.e. 2 mg of musk powder, to obtain a total of 200 ⁇ L of extract (i.e. 10 mg/ml). The content of muscone is 30 ⁇ g/ml. Use pinhole microporous sterile filter membrane for filtration. After packaging, seal and store at low temperature for later use.
  • High-performance liquid chromatography was used to identify the main components of the musk extract, and detection of 254nm ultraviolet absorption was used.
  • the peak time starts from 6.2 minutes to 19.1 minutes, and there are peaks of active substances separated. Substances with a retention time of about 14 minutes have the highest component content of about 61.57%. It shows that using the extraction method in this patent, most key active substances can be effectively retained.
  • Example 2 Determination of in vitro toxicity of musk extract to CAR-T cells
  • CAR11-3 CAR-T cells were expanded in X–VIVO TM 15 (Lonza) complete medium containing 10% FBS (Biosera) and IL-2 (Beijing Shuanglu Pharmaceutical). Cells were placed in an environment containing 5% carbon dioxide at 37°C. Stable cultivation.
  • Cytotoxicity was measured using the CCK-8 cell proliferation/toxicity detection kit (Solarbio CA1210): the cultured CAR11-3 CAR-T cells were counted and spread on a 96-well plate, and different concentration gradients of musk extract were used, namely 0, 15 , 30, 60, 120 and 240 ⁇ g/mL extraction solutions (0 ⁇ g/mL group is the extraction solvent control, the same below), treated for 72 hours. Then, according to the manufacturer's instructions, add 20 ⁇ L of CCK-8 reagent to each well, incubate at 37°C for 3 hours, and measure the absorbance value at OD 450 nm.
  • Human glioma U87 cells (ATCC HTB-14) and U251 cells (Shanghai Cell Bank, Chinese Academy of Sciences) were cultured in DMEM medium (Invitrogen) containing 10% FBS and penicillin-streptomycin antibodies; human peripheral blood mononuclear cells (PBMC) and chimeric antigen receptor-modified (CAR) CAR-T cells targeting IL-13R ⁇ 2 (CAR11-3) were expanded in X–VIVO TM 15 complete medium containing 10% FBS and IL-2. All cells were placed in an environment containing 5% carbon dioxide at 37°C. Stable cultivation.
  • DMEM medium Invitrogen
  • PBMC peripheral blood mononuclear cells
  • CAR chimeric antigen receptor-modified CAR-T cells targeting IL-13R ⁇ 2
  • Isolate and culture PBMC and prepare CAR11-3 cells After counting, they are co-cultured with U87 and U251 in 12-well plates (non-co-culture control groups are set up respectively).
  • the number of lymphocytes plated is 5*10 ⁇ 5/well, and the number of tumor cells is 5*10 ⁇ 5/well.
  • the number of boards is 10 ⁇ 5/hole.
  • concentrations of musk extract (0, 15, 60 and 150 ⁇ g/mL) for treatment. After 24 hours of treatment, the cell supernatant was collected by centrifugation, and the concentration level of interferon- ⁇ (IFN- ⁇ ) was measured. The results are shown in Figure 3.
  • the IFN- ⁇ level did not change ( ⁇ 1000 pg/mL) regardless of whether it was co-cultured with target tumor cells or not.
  • the expression level of IL-13R ⁇ on the surface of U87 cells is higher than that on the surface of U251 cells.
  • the concentration of IFN- ⁇ increased. Higher than the CAR11-3 and U251 co-culture group.
  • CAR11-3 was co-cultured with target cells U87 or U251, and treated with 100 ⁇ g/mL musk. After 24 hours, the unbound CAR-T cells in the supernatant were aspirated, observed under a microscope and photographed. Bright field images at different magnifications are shown in Figure 4 (U87) and Figure 5 (U251). It can be seen from the image that for both target cells, compared with the solvent control group, musk extract can significantly increase the number of bound CAR-T cells and promote the vesicles of the target cells.
  • musk extract can specifically act on CAR-T cells, enhancing their direct killing ability and ability to secrete cytokines. At the same time, it will not activate or enhance irrelevant lymphocytes. It enhances the effect while ensuring safety.
  • CD19-targeting CAR-T (CAR-V3) is a marketed product for the treatment of large B lymphoma and can also be prepared according to the method described in Chinese Patent Publication No. CN113278652B.
  • N6 Adult B acute lymphoblastic leukemia NALM-6 cells (N6) (ATCC CRL-3273) were cultured in RPMI-1640 complete medium (Gibco); PBMC, CAR-V3 (if required) were cultured in 10% FBS and IL-2 Proliferated in X–VIVO TM 15 complete medium. All cells were placed in an environment containing 5% carbon dioxide at 37°C. Stable cultivation.
  • Untransduced PBMC and CAR-V3 cells (co-culture group and non-co-culture group respectively) were treated with gradient concentrations of musk extract (0, 15, 60 and 150 ⁇ g/mL) for 24 hours, and then IFN in the supernatant was measured. - ⁇ secretion.
  • the results are shown in Figure 6.
  • Musk extract can specifically increase the release of IFN- ⁇ from CAR-V3, which has a positive effect on CAR-V3 and adult B acute lymphoblastic leukemia NALM-6 cells (N6 cells). ), the level of IFN- ⁇ further increased. It shows that musk extract also has an enhancing effect on CAR-T cells targeting hematological tumors and increases the activity of CAR-T cells.
  • Human glioma U251 cells were cultured in DMEM medium containing 10% FBS and penicillin-streptomycin; human peripheral blood mononuclear cells (PBMC) and chimeric antigen receptor-modified CAR11- targeting IL-13R ⁇ 2 3 CAR-T cells were expanded in X–VIVO TM 15 complete medium containing 10% FBS and IL-2. All cells were cultured stably in an environment containing 5% carbon dioxide at 37°C.
  • PBMC peripheral blood mononuclear cells
  • the instrument was placed above the skull of anesthetized and fixed mice. After drilling, the injection needle was inserted into the brain tissue to inject the cell suspension to construct an intracranial orthotopic model of glioma mice. Five mice were set up as 5 Parallel experimental groups. One week later, the mice were anesthetized with oxygen-inflated isoflurane, and each mouse was intraperitoneally injected with 200 ⁇ L of fluorescein substrate (Solarbio D9390). Imaging was performed using a small animal in vivo fluorescence imager to confirm by fluorescence imaging. Tumor cells form tumors in situ.
  • mice After all five mice were confirmed to have tumors, 10 7 CAR 11-3 CAR-T cells were injected into each mouse through the tail vein. At the same time, a dosage of 100 mg musk extract/kg mouse body weight was injected intraperitoneally. For musk extract, the day of injection was counted as day 0. After 7 days of treatment (that is, day 6), in vivo fluorescence imaging of small animals was performed again.

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Abstract

本发明提供了一种麝香提取物，及其辅助和/或联合CAR-T细胞治疗肿瘤的方法和相应的用途，其中所述麝香提取物增强CAR-T细胞治疗的功效和/或促进CAR-T细胞通过血脑屏障进入脑中杀伤肿瘤组织。

Description

麝香提取物及其增强CAR-T细胞疗效的应用 技术领域
本发明涉及细胞治疗领域。具体地，本发明涉及一种麝香提取物辅助和/或联合CAR-T细胞治疗肿瘤的方法和相应的用途。
背景技术
胶质母细胞瘤(Glioblastoma，GBM)是人类最常见、最具侵袭性的原发性恶性脑肿瘤，大多数病例恶性程度比较高。由于高发病率、高死亡率和低治愈率，GBM在世界范围内造成了巨大的社会和医疗负担，而且几乎无法用传统的治疗方法治愈，因此患者总体生存期很不理想。具体而言，目前现有的标准化治疗策略，是最大安全限度的手术切除肿瘤，术后同步放疗和化疗，以及随后六个月的替莫唑胺辅助化疗的标准治疗方案。然而，患者平均生存期仅为14.6个月。
因此，有必要开发治疗GBM的新方法，以改善GBM患者的预后。
嵌合抗原受体(Chimeric antigen receptor，CAR)是一种人工合成的分子，其通过特异性识别肿瘤细胞表面表达的抗原来引导经基因工程化以表达CAR的免疫效应细胞(例如，T细胞、NK细胞)清除肿瘤(Sampson JH,Choi BD,Sanchez-Perez L等人,EGFRvIII mCAR-modified T-cell therapy cures mice with established intracerebralglioma and generates host immunity against tumor-antigen loss.Clinical cancer research:an official journal of the American Association for Cancer Research.2014；20(4):972-984)。例如，嵌合抗原受体T细胞(CAR-T)是通过T细胞上的嵌合抗原受体(CAR)分子直接靶向肿瘤细胞的表面抗原，从而达到识别和杀伤肿瘤的目的，其中所述嵌合抗原受体的N端包含识别抗原的胞外域。当所述CAR-T细胞针对的抗原阳性细胞存在时，CAR-T细胞能识别并杀伤这些抗原阳性细胞。对于血液系统恶性肿瘤，CAR-T细胞疗法已经得到了一些比较理想的效果，例如靶向CD19的CAR-T细胞疗法已经治愈或近乎治愈了一些白血病或淋巴瘤患者。因此，人们积极地希望将其推广到实体瘤治疗领域，例如胶质瘤，但是经过多次尝试，仍然没有获得足够理想的结果。究其原因，可能与T细胞向肿瘤组织的迁移效率低、T细胞的存活及扩增不足、免疫抑制的肿瘤微环境等因素有关。此外，胶质瘤还可借助血脑屏障的保护，逃避CAR-T细胞的杀伤作用。例如，如果将CAR-T全身给药，能够通过血脑屏障到达肿瘤局部的细胞数量非常有限；而如果直接将CAR-T细胞注射到脑脊液中，例如颅内注射或鞘内注射，则增大了实施难度，并带来了不必要的风险，例如颅内感染和中枢神经系统中的细胞因子风暴。
迫切地需要一种CAR-T细胞的增效剂，尤其是对于靶向胶质瘤的CAR-T细胞疗法。
发明内容
本发明利用麝香提取物，其中保留了麝香酮和麝香多肽等在内的多种关键活性成分，研究对CAR-T类细胞的活性和入脑的作用。并出人意料地发现，麝香提取物具有增强CAR-T细胞杀伤肿瘤的疗效的活性，并且具有增强CAR-T细胞到达肿瘤区域(例如跨过血脑屏障到达胶质瘤组织)能力的活性，后者可能是作为前者的一部分，也可能与前者是独立的关系。
因此，在第一方面，本发明提供了一种麝香提取物，其特征在于，通过以下方式制备：将麝香粉末按每1mg麝香粉末溶于100μL溶剂的比例，溶于无菌PBS或DMSO中，超声1小时，随后离心取上清，即为麝香提取物。在一个具体的实施方案中，离心条件为12000rpm离心5min。在一个优选的实施方案中，取1mg麝香粉末加入100μL无菌PBS(或DMSO)，常规用超声破碎仪设置90W功率进行超声1h；再取1mg麝香粉末，加100μL PBS，如前超声1h，随后12000rpm离心5min并合并上清提取液，即2mg麝香粉末共得200μL提取液。在一个具体的实施方案中，上清液中的麝香酮的含量不低于约20μg/mL。在一个优选的实施方案中，上清液中的麝香酮的含量不低于约25μg/mL、约30μg/mL、约40μg/mL或约50μg/mL。
在第二个方面，本发明提供了本发明第一方面的麝香提取物的用途，用于制备CAR-T细胞疗法的疗效增强剂。在一个具体的实施方案中，所述CAR-T细胞疗法针对血液系统肿瘤。在另一个具体的实施方案中，所述CAR-T细胞疗法针对实体瘤。在一个优选的实施方案中，所述CAR-T细胞疗法针对胶质细胞瘤。在一个优选的实施方案中，所述CAR-T细胞的嵌合抗原受体的靶分子是IL-13Rα、EphA2或EGFRvIII(III型EGF缺失突变受体)。
在一些具体的实施方案中，所述疗效增强剂促进CAR-T细胞分泌细胞因子杀伤肿瘤细胞。在另一些具体的实施方案中，所述疗效增强剂增强CAR-T细胞对靶细胞的直接杀伤作用。在一些优选的实施方案中，所述疗效增强剂增强CAR-T细胞到达肿瘤区域的能力而作为疗效增强剂。在一些更优选的实施方案中，所述疗效增强剂促进CAR-T细胞透过血脑屏障而使得更多CAR-T细胞到达脑内肿瘤(例如，胶质细胞瘤)区域，即，所述疗效增强剂的作用至少包括增加透过血脑屏障的CAR-T细胞的数量。在优选的实施方案中，所述疗效增强剂促进通过静脉、腹腔等全身性途径施用的CAR-T细胞进入颅内，从而避免了直接颅内/脑室内给药的副作用和/或风险。
在第三个方面，本发明提供了一种组合物，其包含本发明第一方面的麝香提取物，所述组合物用作CAR-T细胞疗法的疗效增强剂。在一个具体的实施方案中，所述CAR-T细 胞疗法针对血液系统肿瘤。在另一个具体的实施方案中，所述CAR-T细胞疗法针对实体瘤。在一个优选的实施方案中，所述CAR-T细胞疗法针对胶质细胞瘤。在一个优选的实施方案中，所述CAR-T细胞的嵌合抗原受体的靶分子是IL-13Rα、EphA2或EGFRvIII(III型EGF缺失突变受体)。
在一些具体的实施方案中，所述组合物通过促进CAR-T细胞分泌细胞因子而作为疗效增强剂。在另一些具体的实施方案中，所述组合物通过增强CAR-T细胞对靶细胞的直接杀伤作用而作为疗效增强剂。在一些优选的实施方案中，所述组合物通过增强CAR-T细胞到达肿瘤区域的能力而作为疗效增强剂。在一些更优选的实施方案中，所述组合物通过促进CAR-T细胞透过血脑屏障而使得更多CAR-T细胞到达脑内肿瘤(例如，胶质细胞瘤)区域的能力而作为疗效增强剂。在优选的实施方案中，所述组合物促进通过静脉、腹腔等全身性途径施用的CAR-T细胞进入颅内，从而避免了直接颅内/脑室内给药的副作用和/或风险。
在第四个方面，本发明提供了药物组合物，其包含本发明第一方面的麝香提取物或本发明第三方面的组合物，另外还包含适当的可药用载体。所述可药用载体使得所述药物组合物适合于通过预期的途径给药，例如，但不限于，静脉注射或肿瘤局部注射等等。在一些实施方案中，本发明的药物组合物先于CAR-T细胞施用。在一些实施方案中，本发明的药物组合物在CAR-T细胞施用之后施用。在一些实施方案中，本发明的药物组合物与CAR-T细胞同时施用。在一些实施方案中，本发明的药物组合物进一步包含CAR-T细胞。
因而，在一些实施方案中，本发明提供了本发明第一方面的麝香提取物与CAR-T细胞联合用于制备抗肿瘤的药物的用途。
在第五个方面，本发明提供了用于制备本发明的麝香提取物以及包含所述麝香提取物的组合物的方法，包括以下步骤：按每1mg麝香粉末溶于100μL无菌PBS或DMSO中，超声1小时，离心取上清，即为麝香提取物。在另一个实施方案中，本发明提供了用于制备包含本发明的麝香提取物的药物组合物的方法，包括以下步骤：提供本发明的麝香提取物，或包含所述麝香提取物的组合物，并添加所需的可药用载体。
如在本文中所用的，单数形式“一个”、“一种”、“该”和“所述”可以包括多于一个或一种的被指代物，除非上下文另外明确规定。如本文所用，“约”应理解为是指处在该所提及数字的-5％至+5％的范围内。此外，本文中的所有数值范围都应理解为包含该范围内的所有整数或分数。本文所公开的组合物可以不含本文未具体公开的任何要素。因此，使用术语“包括/包含”的实施方案的公开内容包括“基本上由所指明的组分组成”和“由所指明的组分组成” 的实施方案的公开内容。
如本文所用，术语“麝香”(musk)是鹿科林麝(Moschusberezovskii flerov)、马麝(M.sifanicus przewalssi.)和原麝(M.moschiferus linaeus.)等麝类动物成熟雄体香囊分泌物的干燥品。传统中医药典籍中记载，麝香味辛、性温，入心经脑经血分；具有镇心安神、醒脑、通诸窍、活血通络、消肿止痛开经络和透肌骨功效,主治热病神昏、中风痰厥、气郁暴厥、中恶昏迷、经闭、心腹暴痛、痈肿瘰疬、痹痛麻木，对中枢神经、呼吸循环等系统具有开窍解痉,强心等作用。麝香含有多种化学成分,其中包括大环酮类(麝香酮，即3-甲基环十五烷酮(3-methyl-cyclopentade))、含氮杂环类、甾体类化合物、多肽类和脂肪酸等。如无特别说明，本文所用的麝香是可以从任何常规渠道商购获得的任何常见形式的麝香，例如，药店中购得的麝香粉末，等等。
超声提取是利用超声波振动的方法进行提取的方法，超声的机械作用使得溶剂快速地进入固体物质中，将其物质所含的有机成分尽可能完全地溶于溶剂之中，从而得到多成分混合提取液。超声提取能够有效地提取出麝香中主要的活性物质麝香酮。
术语“肿瘤”和“癌症”在本文中并不互相排斥且可互换地使用，涵盖实体瘤和血液肿瘤，指所有赘生性(neoplastic)细胞生长和增殖，无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。在某些实施方案中，适合于通过本发明的抗体来治疗的癌症包括胃癌或胰腺癌，包括那些癌症的转移性形式。
术语“胶质细胞瘤”是神经胶质瘤的简称，也称为胶质瘤。胶质细胞瘤是一种中枢神经系统的肿瘤疾病，是指是发生于神经外胚层的肿瘤，其源自神经元细胞或间质细胞的癌变。胶质瘤是颅内最常见的原发性中枢神经系统肿瘤，约占所有颅内原发肿瘤的一半。胶质母细胞瘤是胶质瘤中比较常见的一种类型，又称多形性胶质母细胞瘤(GBM)，是星形细胞瘤(astrocytic tumors)进展到Grade IV阶段(WHO国际疾病分类法)的专门名称，恶性程度最高。胶质母细胞瘤的发病高峰年龄一般为50—60岁。
术语“CAR-T”或“CAR T”是指转导并表达了嵌合抗原受体(CAR)的T淋巴细胞。所述嵌合抗原受体是指一组或多组多肽，当其在免疫效应细胞中时，给所述的细胞提供针对靶细胞(通常是癌细胞)的特异性，并且具有细胞内信号产生。通常，CAR包括至少一个胞外结合区、跨膜区和胞内信号区。示例性的CAR构建方法和/或CAR-T细胞的转导方法记载于例如中国专利公开号CN114014941A之中。
术语“药物组合物”是指，本发明中有用的麝香提取物作为有效成分与可药用载体(根据情况而定)的混合物。药物组合物有助于有效成分向患者的施用。
如本文所用，术语“可药用载体”是指药学上可接受的材料、组合物或载体，例如液体或固体填充剂，稳定剂，分散剂，悬浮剂，稀释剂，赋形剂，增稠剂，溶剂或包封材料，涉及在患者体内或向患者体内携带或运输本发明有用的化合物，使其可以发挥其预期的功能。通常，这种构建物可以被携带至、或从一个器官或身体的一部分运输至另一个器官或身体的一部分。在与制剂的其他成分(包括本发明所述的麝香提取物)相容且对患者无害的意义上，每种载体必须是“可接受的”。可用作药学上可接受的载体的材料的一些实例包括：糖，例如乳糖，葡萄糖和蔗糖；淀粉，如玉米淀粉和马铃薯淀粉；纤维素及其衍生物，如羧甲基纤维素钠，乙基纤维素和醋酸纤维素；粉末黄蓍胶；麦芽；明胶；滑石；赋形剂，如可可脂和栓剂蜡；油，如花生油，棉籽油，红花油，芝麻油，橄榄油，玉米油和豆油；二醇，如丙二醇；多元醇，如甘油，山梨糖醇，甘露糖醇和聚乙二醇；酯类，如油酸乙酯和月桂酸乙酯；琼脂；缓冲剂，如氢氧化镁和氢氧化铝；表面活性剂；海藻酸；无热原水；等渗盐水；林格氏溶液；乙醇；磷酸盐缓冲液；和药物制剂中使用的其他无毒相容物质。如本文所用，“可药用载体”还包括与本发明化合物的活性相容的且对于患者是生理学上可接受的任何和所有包衣、抗细菌和抗真菌剂、和吸收延迟剂等。增补性活性化合物也可以掺入组合物中。“可药用载体”可以进一步包括可用于本发明的化合物的药学上可接受盐。可以包括在本发明的药物组合物中的其他成分是本领域已知的并且描述于例如Remington's Pharmaceutical Sciences(Genaro，Ed.，Mack Publishing Co.，1985，Easton，PA)，其通过引用并入本文。
在一些实施方案中，配制药物组合物用于静脉内注射。在一些实施方案中，药物组合物包含配制用于静脉内注射的麝香提取物。在一些实施方案中，药物组合物包含配制用于静脉内注射的麝香提取物和CAR-T细胞。适于注射使用的药物组合物包括无菌水溶液(水溶性的)或分散体、和用于即时制备无菌可注射溶液或分散体的无菌粉末。对于静脉内施用，合适的载体包括生理盐水，抑菌水，或达到易于注射程度的流体。可注射组合物在制造和储存条件下必须是稳定的，并且必须防止微生物如细菌和真菌的污染。载体可以是溶剂或分散介质，含有例如水，乙醇，多元醇(例如，甘油，丙二醇和液体聚乙二醇等)，以及它们的合适混合物。例如，可以通过使用诸如卵磷脂的涂层、在分散体的情况下通过维持所需的颗粒尺寸、和通过使用表面活性剂，来保持适当的流动性。通过各种抗细菌剂和抗真菌剂，例如对羟基苯甲酸酯，氯丁醇，苯酚，抗坏血酸，硫柳汞等，可以防止微生物的作用。在许多情况下，优选在组合物中包含等渗剂，例如糖，多元醇如甘露醇，山梨糖醇，氯化钠。通过在组合物中包括延迟吸收的试剂，例如单硬脂酸铝和明胶，可以实现可注射组合物的延长吸收。
用于本文时，“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转已存在的症状、 病症、病况或疾病的进展或严重性。
用于本文时，“预防”包括对疾病或病症或特定疾病或病症的症状的发生或发展的抑制。在一些实施方案中，具有癌症家族病史的受试者是预防性方案的候选。通常，在癌症的背景中，术语“预防”是指在癌症的病征或症状发生前，特别是在具有癌症风险的受试者中发生前的药物施用。
用于本文时，“疗效增强剂”或“功效增强剂”意指能够增强已有疗法的治疗效果的试剂，其中已有疗法可以指任何本领域已经使用或知晓的疗法，优选针对癌症的疗法，更优选CAR-T细胞疗法。疗效得到增强，是指相比于没有接受疗效增强剂的已有疗法治疗的患者，在接受了疗效增强剂的患者中更加减缓、更加中断、更加阻滞、更加缓解、更加停止、更加降低、或更加逆转已存在的症状、病症、病况或疾病的进展或严重性，或者副作用更少、治疗体验更好、生存质量更高，或者简而言之，取得了或者基本上取得了比没有接受疗效增强剂的已有疗法治疗的患者更好的治疗过程或结局。
本文引用或提及的所有专利，专利申请，出版物，技术文献和/或学术文章以及其他参考文献在法律允许的范围内通过引用而整体并入本文。
附图说明
图1所示为对麝香提取物进行HPLC分析得到的成分图谱。图谱横轴为时间轴，纵轴为吸光强度单位(Absorbance unit)。
图2所示为不同浓度的麝香提取物处理后的CAR11-3 CAR-T细胞通过CCK-8测定增殖情况的柱形图统计对比。
图3所示为各自单独培养或与U87、U251共同培养的PMBC细胞和CAR11-3 CAR-T细胞经梯度浓度(0，15，60和150μg/mL)的麝香提取物处理后，培养上清中细胞因子IFN-γ水平。
图4所示为CAR11-3细胞与靶细胞U87共培养体系经麝香提取物处理24h后，吸走上清未结合的CAR11-3细胞，随后置于镜下观察和拍照记录的CAR11-3细胞对靶细胞的直接杀伤情况。4X、10X、20X代表物镜倍数为4倍、10倍、20倍。溶剂对照为提取溶剂对照。
图5所示为CAR11-3细胞与靶细胞U251共培养体系经麝香提取物处理24h后，吸走上清未结合的CAR11-3细胞，随后置于镜下观察和拍照记录的CAR11-3细胞对靶细胞的直接杀伤情况。4X、10X、20X代表物镜倍数为4倍、10倍、20倍。溶剂对照为提取溶剂对照。
图6所示为梯度浓度麝香提取物(0，15，60和150μg/mL)分别处理未转导PBMC和CAR-V3细胞(分别设与NALM-6靶细胞共培养组和非共培养组)24h之后，上清中IFN-γ的分泌量水平。NT＝未转导的PBMC细胞。V3＝CAR-V3细胞。N6＝成人B型急性淋巴细胞白血病NALM-6细胞系。
图7所示为表达荧光素酶的脑胶质瘤稳定转染细胞系在小鼠脑内原位成瘤后，经尾静脉注射10 7个CAR-T细胞，同时按照100mg麝香提取物/kg小鼠体重的给药量经腹腔注射麝香提取物，通过小动物活体荧光成像对治疗前后(第0天vs第6天)进行对比。
具体实施方式
现在参考以下实施例描述本发明。提供这些实施例仅用于举例说明的目的，本发明不局限于这些实施例，而是涵盖基于本文提供的教导而显而易见的所有变化。
实施例1.麝香提取物制备
精密称取麝香粉末(北京世纪坛医院)5mg分装，取1mg加100μL无菌PBS(或DMSO)，常规用超声破碎仪设置90W功率进行超声1h；再取1mg粉末，加100μL PBS，如前超声1h，随后12000rpm离心5min并合并上清提取液，即2mg麝香粉末共得200μL提取液(即10mg/ml)，麝香酮的含量为30μg/ml。采用针孔式微孔无菌滤膜过滤，分装后，低温封口储藏备用。
利用高效液相色谱仪对麝香提取物的主要成分进行鉴定，利用254nm紫外吸收的检测。
表1 HPLC图谱(附图1)中各个峰
名称 保留时间(第…分钟) 面积 ％面积 高度
1 6.212 4077 4.89 542
2 7.723 2704 3.24 480
3 14.249 51330 61.57 8221
4 14.61 11938 14.32 2463
5 17.223 4653 5.58 838
6 17.863 4539 5.44 928
7 18.143 129 0.15 -63
8 18.436 2328 2.79 599
9 19.144 1676 2.01 293
参见图1及表1所示，出峰时间从6.2分钟开始至19.1分钟之间，均有活性物质分离出峰。保留时间在14分钟左右的物质，成分含量最高约占61.57％。说明利用本专利中的提取方法，可有效保留多数关键活性物质。
实施例2.麝香提取物对CAR-T细胞体外毒性测定
CAR-T细胞制备
按照Xu C,Bai Y,An Z,Hu Y,Zhang C,Zhong X.IL-13Rα2 humanized scFv-based CAR-T cells exhibit therapeutic activity against glioblastoma.Mol Ther Oncolytics.2022；24:443-451.或公开号为CN114014941A的中国专利申请中所记载的方法，制备靶向IL-13Rα2的嵌合抗原受体修饰(CAR)的CAR-T细胞(CAR11-3)。
细胞培养和处理
CAR11-3 CAR-T细胞在含10％FBS(Biosera)和IL-2(北京双鹭药业)的X–VIVO TM15(Lonza)完全培养基中扩增。细胞置于37℃含5％二氧化碳的环境中。稳定培养。
b胞毒性评估
通过CCK-8细胞增殖/毒性检测试剂盒(Solarbio CA1210)进行细胞毒性测定：将培养的CAR11-3CAR-T细胞计数后铺96孔板，利用不同的浓度梯度的麝香提取物，即0，15，30，60，120和240μg/mL的提取液(0μg/mL组为提取溶剂对照，下文同)，处理72h。之后按照制造商说明书，每孔加入20μL的CCK-8试剂，37℃孵育3h后，测定OD 450nm处吸光值。
结果如图2所示，不同浓度的麝香提取物对CAR11-3T细胞，均无明显毒性，说明麝香提取物对CAR-T细胞的生长、增殖无毒副作用。
实施例3.麝香提取物在体外增强CAR11-3 CAR-T细胞活性
细胞培养和处理
人脑胶质瘤U87细胞(ATCC HTB-14)和U251细胞(中科院上海细胞库)用含10％FBS和青链霉素双抗的DMEM培养基(Invitrogen)培养；人外周血单个核细胞(PBMC)和靶向IL-13Rα2的嵌合抗原受体修饰(CAR)的CAR-T细胞(CAR11-3)在含10％FBS和IL-2的X–VIVO TM15完全培养基中扩增。所有细胞置于37℃含5％二氧化碳的环境中。稳定培养。
细胞活性评估
分离培养PBMC和制备CAR11-3细胞，计数后分别与U87和U251在12孔板进行共培养(分别设非共培养对照组)，其中淋巴细胞铺板数量为5*10^5/孔，肿瘤细胞铺板数量为10^5/孔。并同步利用不同浓度麝香提取物(0，15，60和150μg/mL)进行处理。处理24h后离心收取细胞上清，测定其中γ-干扰素(IFN-γ)的浓度水平。结果如图3示，在麝香提取物处理的PBMC各组，无论是否与靶肿瘤细胞共培养，IFN-γ水平均无变化(<1000 pg/mL)。而在CAR11-3的各组(包括非共培养和共培养组)中，麝香提取物均能有效增加CAR-T细胞的IFN-γ的分泌，且呈剂量依赖性；CAR11-3细胞与靶细胞U87和U251共培养处理后，特别是在麝香提取物处理的CAR11-3细胞与U87细胞共培养各组，细胞IFN-γ的水平进一步增加，具有统计学意义(*p<0.05和**p<0.01；n＝3)。作为CAR11-3-T细胞的靶蛋白，IL-13Rα在U87细胞表面的表达水平高于U251细胞表面水平，相同浓度麝香提取物对CAR11-3与U87共培养处理后，IFN-γ浓度增加程度高于CAR11-3与U251共培养组。上述说明，麝香提取物能特异选择作用CAR-T细胞，剂量依赖性的促进CAR-T细胞分泌IFN-γ，增加CAR-T的杀伤能力。
此外，还在显微镜下观察了CAR-T与靶细胞结合及杀伤水平。将CAR11-3与靶细胞U87或U251共培养，并用100μg/mL麝香处理；24h后，吸走上清未结合的CAR-T细胞，置于镜下观察并拍照。不同放大倍数下的明场图像示于图4(U87)及图5(U251)。从图像中可以看出，对于两种靶细胞，与溶剂对照组相比，麝香提取物均可显著增加结合的CAR-T细胞数目，促进靶细胞的囊泡化。
上述结果表明，麝香提取物能特异地作用于CAR-T细胞，增强其直接杀伤能力和分泌细胞因子能力，同时不会活化或增强无关淋巴细胞，在增强效应的同时也保证了安全性。
实施例4.麝香提取物在体外增强多种CAR-T细胞活性
CAR-T细胞制备
靶向CD19 CAR-T(CAR-V3)是治疗大B淋巴瘤的上市产品，也可按照中国专利公开号CN113278652B中所记载的方法制备获得。
细胞培养和处理
成人B型急性淋巴细胞白血病NALM-6细胞(N6)(ATCC CRL-3273)用RPMI-1640完全培养基(Gibco)培养；PBMC、CAR-V3(如需要)在含10％FBS和IL-2的X–VIVO TM15完全培养基中扩增。所有细胞置于37℃含5％二氧化碳的环境中。稳定培养。
细胞活性评估
利用梯度浓度的麝香提取物(0，15，60和150μg/mL)，分别处理未转导PBMC和CAR-V3细胞(分别设共培养组和非共培养组)24h，之后测定上清中IFN-γ的分泌量，结果如图6所显示，麝香提取物可特异性增加CAR-V3的IFN-γ的释放量，对CAR-V3与成人B型急性淋巴细胞白血病NALM-6细胞(N6细胞)共培养后，IFN-γ的水平进一步增加。说明麝香提取物对靶向血液系统肿瘤的CAR-T细胞也具有增强作用，增加CAR-T细胞活力。
实施例5.麝香提取物体内活性测定
原位脑胶质瘤动物实验
人脑胶质瘤U251细胞用含10％FBS和青链霉素双抗的DMEM培养基培养；人外周血单个核细胞(PBMC)和靶向IL-13Rα2的嵌合抗原受体修饰的CAR11-3 CAR-T细胞在含10％FBS和IL-2的X–VIVO TM15完全培养基中扩增。所有细胞置于37℃含5％二氧化碳的环境中，稳定培养。
按照Xu C,Bai Y,An Z,Hu Y,Zhang C,Zhong X.IL-13Rα2 humanized scFv-based CAR-T cells exhibit therapeutic activity against glioblastoma.Mol Ther Oncolytics.2022；24:443-451.或中国专利公开号CN104130977A中所记载的方法，利用U251细胞构建含表达荧光素酶的脑胶质瘤稳定转染细胞系。培养收取足量细胞后，按照每只小鼠注射10μL含2x10 5个稳定转染荧光素酶的肿瘤细胞的细胞悬液，将消毒后的进样针吸取细胞悬液后，利用小动物脑定位仪，置于麻醉并固定的小鼠颅骨上方，钻孔后将进样针刺入脑组织中注射细胞悬液，从而构建胶质瘤小鼠颅内原位模型，设置5只小鼠作为5组平行实验组。1周后，用氧气吹气的异氟烷麻醉小鼠，按照每只小鼠腹腔注射200μL的荧光素底物(Solarbio D9390)，利用小动物活体荧光成像仪进行成像，从而通过荧光显像确认肿瘤细胞原位成瘤。
5只小鼠均确认成瘤后，每只小鼠经尾静脉注射10 7个CAR 11-3 CAR-T细胞，同时，按照100mg麝香提取物/kg小鼠体重的给药量，经腹腔注射麝香提取物，注射当日计为第0天。处理7天后(即第6天)再次进行小动物活体荧光成像。
将两次荧光成像的图像进行对比，结果如图7所示，可见CAR 11-3细胞注射7天后，小鼠肿瘤荧光信号显著减小甚至消失，表明肿瘤病灶明显减小。因此，体内实验说明麝香可以促进经静脉注射的CAR-T细胞入脑，增强CAR-T细胞脑内靶向肿瘤细胞。
以上实施例中参考和/或提及的已公开的专利及非专利文献均通过引用的方式以其全文并入本文之中。

Claims (13)

  1. 一种麝香提取物，其特征在于，通过以下方式制备：将麝香粉末按每1mg麝香粉末溶于100μL溶剂的比例，溶于无菌PBS或DMSO中，超声1小时，随后离心取上清，即为麝香提取物。
  2. 如权利要求1所述的麝香提取物，其特征在于，其中麝香酮的含量不低于20μg/mL。
  3. 权利要求1所述的麝香提取物的用途，其特征在于，用于制备CAR-T细胞疗法的疗效增强剂。
  4. 如权利要求3所述的用途，其特征在于，所述CAR-T细胞疗法针对血液系统肿瘤。
  5. 如权利要求3所述的用途，其特征在于，所述CAR-T细胞疗法针对实体瘤。
  6. 如权利要求5所述的用途，其特征在于，所述实体瘤是胶质细胞瘤。
  7. 如权利要求3所述的用途，其特征在于，所述CAR-T细胞疗法针对胶质细胞瘤，并且所述疗效增强剂的作用至少包括增加透过血脑屏障的CAR-T细胞的数量。
  8. 权利要求1所述的麝香提取物与CAR-T细胞的用途，其特征在于，联合用于制备抗肿瘤的药物。
  9. 组合物，其特征在于，包含权利要求1所述的麝香提取物。
  10. 药物组合物，其特征在于，包含权利要求1所述的麝香提取物或权利要求9所述的组合物，以及可药用载体。
  11. 如权利要求10所述的药物组合物，其特征在于，进一步包含CAR-T细胞。
  12. 用于制备权利要求1所述的麝香提取物的方法，其特征在于，包括以下步骤：将麝香粉末按每1mg麝香粉末溶于100μL溶剂的比例，溶于无菌PBS或DMSO中，超声1小时，随后离心取上清。
  13. 用于制备权利要求10所述的药物组合物的方法，其特征在于，包括以下步骤：提供权利要求1所述的麝香提取物，或权利要求9所述的组合物；并添加所需的可药用载体。
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Citations (1)

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CN1834234A (zh) * 2006-03-30 2006-09-20 上海大学 麝香水溶性提取物提高神经干细胞电穿孔转染效率的方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1834234A (zh) * 2006-03-30 2006-09-20 上海大学 麝香水溶性提取物提高神经干细胞电穿孔转染效率的方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MA, WEI: "Experimental Study on the Effect of Musk on Promoting Cell Proliferation and Apoptosis of Lung Adenocarcinoma Cells in Vitro", JOURNAL OF SICHUAN OF TRADITIONAL CHINESE MEDICINE, vol. 34, no. 11, 15 November 2016 (2016-11-15), China , pages 48 - 51, XP009552790, ISSN: 1000-3649 *
QI, NA; DUAN, WENJUAN; LI, YAJING; KANG, SHUANGMING; ZHANG, SHANGQIAN; ZHOU, XIANTAI; MO, FUQUAN; FENG, JIANFANG: "Research Progress on the Pharmacological Action of Muscone", MODERNIZATION OF TRADITIONAL CHINESE MEDICINE AND MATERIA MEDICA-WORLD SCIENCE AND TECHNOLOGY, vol. 22, no. 8, 31 August 2020 (2020-08-31), CN , pages 3042 - 3047, XP009552838, ISSN: 1674-3849, DOI: 10.11842/wst.20181223004 *
ZHENG, MIN; ZHANG, LAN: "Current Situation and Development Trend of CAR-T in Anti-tumor Research", JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCE), vol. 54, no. 11, 30 November 2016 (2016-11-30), XP009520597, DOI: 10.6040/j.issn.1671-7554.0.2016.835 *

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