CN114949000B

CN114949000B - Musk extract and application thereof in enhancing curative effect of CAR-T cells

Info

Publication number: CN114949000B
Application number: CN202210922616.9A
Authority: CN
Inventors: 钟晓松; 黄帅
Original assignee: Carrizi Beijing Life Technology Co ltd
Current assignee: Carrizi Beijing Life Technology Co ltd
Priority date: 2022-08-02
Filing date: 2022-08-02
Publication date: 2022-10-04
Anticipated expiration: 2042-08-02
Also published as: CN114949000A

Abstract

The invention provides a musk extract, a method for assisting and/or combining CAR-T cells in treating tumors and corresponding application, wherein the musk extract enhances the efficacy of CAR-T cell treatment and/or promotes CAR-T cells to enter the brain through the blood brain barrier so as to kill tumor tissues.

Description

Musk extract and application thereof in enhancing curative effect of CAR-T cells

Technical Field

The present invention relates to the field of cell therapy. In particular, the invention relates to a method for the adjuvant and/or combined CAR-T cell treatment of tumors by using musk extract and corresponding application.

Background

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in humans, with a high degree of malignancy in most cases. GBM poses a huge social and medical burden worldwide due to high morbidity, high mortality, and low cure rates, and is almost incurable by traditional therapeutic approaches, thus the overall life span of patients is far from ideal. In particular, the standardized treatment strategy currently available is the standard treatment protocol for maximal safety surgical resection of the tumor, simultaneous postoperative radiotherapy and chemotherapy, and subsequent six months of temozolomide-assisted chemotherapy. However, the mean survival of patients is only 14.6 months.

Therefore, there is a need to develop new methods of treating GBM to improve the prognosis of GBM patients.

A Chimeric Antigen Receptor (CAR) is an artificially synthesized molecule that directs the clearance of tumor-expressing immune effector cells (e.g., T cells, NK cells) by genetically engineered to express the CAR by specifically recognizing an antigen expressed on the surface of the tumor cell (Sampson JH, choi BD, sanchez-Perez L et al, EGFRvIII mCR-modified T-cell therapy with the ability to express both targeted and targeted antigenic activities of Cancer access 2014; 20 (4): 972-984). For example, chimeric antigen receptor T cells (CAR-T) are targeted directly to the surface antigen of tumor cells by a Chimeric Antigen Receptor (CAR) molecule on the T cell, which comprises an extracellular domain that recognizes the antigen at the N-terminus, for the purpose of recognizing and killing the tumor. When antigen-positive cells to which the CAR-T cells are directed are present, the CAR-T cells can recognize and kill these antigen-positive cells. CAR-T cell therapy has achieved some desirable effects for hematologic malignancies, e.g., CAR-T cell therapy targeting CD19 has cured or nearly cured some patients with leukemia or lymphoma. Therefore, it is actively desired to generalize it to the field of solid tumor therapy, such as glioma, but many attempts have not yet achieved sufficiently desirable results. The reasons for this are probably related to factors such as low migration efficiency of T cells to tumor tissues, insufficient survival and expansion of T cells, and immunosuppressive tumor microenvironment. In addition, gliomas may evade the killing effect of CAR-T cells by protection of the blood brain barrier. For example, if CAR-T is administered systemically, the number of cells that can reach the tumor local through the blood brain barrier is very limited; if the CAR-T cells are injected directly into the cerebrospinal fluid, for example, intracranially or intrathecally, this increases the difficulty of administration and carries unnecessary risks, such as intracranial infection and cytokine storm in the central nervous system.

There is an urgent need for a CAR-T cell potentiator, particularly for glioma-targeted CAR-T cell therapies.

Disclosure of Invention

According to the invention, the musk extract is utilized, and various key active ingredients including musk ketone, musk polypeptide and the like are reserved, so that the activity on CAR-T cells and the effect on entering the brain are researched. And surprisingly found that musk extracts have activity enhancing the efficacy of CAR-T cells in killing tumors and the ability of CAR-T cells to reach the tumor area (e.g. cross the blood brain barrier to glioma tissue), either as part of the former or in an independent relationship to the former.

Accordingly, in a first aspect, the present invention provides a musk extract characterised in that it is prepared by: dissolving Moschus powder in 100 μ L solvent per 1mg Moschus powder, dissolving in sterile PBS or DMSO, ultrasonic treating for 1 hr, centrifuging, and collecting supernatant to obtain Moschus extract. In a specific embodiment, the centrifugation conditions are 12000rpm for 5min. In a preferred embodiment, 1mg of musk powder is added into 100. Mu.L of sterile PBS (or DMSO), and the mixture is subjected to ultrasonic treatment for 1h by setting 90W power by using a conventional ultrasonic disruptor; then, 1mg of musk powder is added with 100. Mu.L PBS, ultrasonic treatment is carried out for 1h as before, centrifugation is carried out for 5min at 12000rpm, and supernatant extract is combined, namely 2 mg of musk powder, so that 200. Mu.L extract is obtained. In a specific embodiment, the level of muscone in the supernatant is not less than about 20 μ g/mL. In a preferred embodiment, the amount of muscone in the supernatant is no less than about 25. Mu.g/mL, about 30. Mu.g/mL, about 40. Mu.g/mL or about 50. Mu.g/mL.

In a second aspect, the present invention provides the use of a musk extract according to the first aspect of the invention for the preparation of a therapeutic effect enhancer of CAR-T cell therapy. In a specific embodiment, the CAR-T cell therapy is directed to a hematological tumor. In another specific embodiment, the CAR-T cell therapy is directed to a solid tumor. In a preferred embodiment, the CAR-T cell therapy is directed against a glioma. In a preferred embodiment, the target molecule of the chimeric antigen receptor of the CAR-T cell is IL-13 Ra, ephA2 or EGFRvIII (type III EGF deletion mutant receptor).

In some specific embodiments, the therapeutic efficacy-enhancing agent promotes the secretion of cytokines by CAR-T cells to kill tumor cells. In other specific embodiments, the therapeutic efficacy-enhancing agent enhances direct killing of the CAR-T cell against the target cell. In some preferred embodiments, the therapeutic efficacy-enhancing agent acts as a therapeutic efficacy-enhancing agent by enhancing the ability of CAR-T cells to reach the tumor region. In some more preferred embodiments, the therapeutic enhancer promotes the penetration of CAR-T cells across the blood brain barrier such that more CAR-T cells reach the tumor (e.g., glioma) region within the brain, i.e., the effect of the therapeutic enhancer includes at least increasing the number of CAR-T cells that penetrate the blood brain barrier. In preferred embodiments, the therapeutic efficacy-enhancing agent promotes entry of CAR-T cells into the cranium, administered by the systemic route, e.g., intravenously, intraperitoneally, etc., thereby avoiding the side effects and/or risks of direct intracranial/intracerebroventricular administration.

In a third aspect, the present invention provides a composition comprising a musk extract according to the first aspect of the invention for use as a therapeutic effect enhancer for CAR-T cell therapy. In a specific embodiment, the CAR-T cell therapy is directed to a hematologic tumor. In another specific embodiment, the CAR-T cell therapy is directed to a solid tumor. In a preferred embodiment, the CAR-T cell therapy is directed to a glioma. In a preferred embodiment, the target molecule of the chimeric antigen receptor of CAR-T cells is IL-13 Ra, ephA2 or EGFRvIII (type III EGF deletion mutant receptor).

In some specific embodiments, the composition acts as a therapeutic efficacy enhancer by promoting secretion of cytokines by CAR-T cells. In other specific embodiments, the composition acts as a therapeutic efficacy enhancer by enhancing the direct killing of target cells by CAR-T cells. In some preferred embodiments, the composition acts as a therapeutic efficacy enhancer by enhancing the ability of CAR-T cells to reach the tumor region. In some more preferred embodiments, the compositions act as therapeutic enhancers by promoting the ability of CAR-T cells to cross the blood brain barrier, allowing more CAR-T cells to reach the tumor (e.g., glioma) region in the brain. In preferred embodiments, the compositions promote entry of CAR-T cells into the cranium, administered by the systemic route, e.g., intravenously, intraperitoneally, etc., thereby avoiding the side effects and/or risks of direct intracranial/intracerebroventricular administration.

In a fourth aspect, the present invention provides a pharmaceutical composition comprising a musk extract according to the first aspect of the invention or a composition according to the third aspect of the invention, together with a suitable pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier renders the pharmaceutical composition suitable for administration by an intended route, such as, but not limited to, intravenous injection or local injection of a tumor, and the like. In some embodiments, the pharmaceutical composition of the invention is administered prior to the CAR-T cell. In some embodiments, the pharmaceutical composition of the invention is administered after administration of the CAR-T cells. In some embodiments, the pharmaceutical composition of the invention is administered concurrently with the CAR-T cells. In some embodiments, the pharmaceutical compositions of the invention further comprise CAR-T cells.

Thus, in some embodiments, the present invention provides the use of a musk extract according to the first aspect of the invention in combination with CAR-T cells for the preparation of a medicament for combating tumours.

In a fifth aspect, the present invention provides methods for preparing the musk extracts of the present invention and compositions comprising the musk extracts, comprising the steps of: dissolving Moschus powder 1mg in sterile PBS or DMSO 100 μ L, performing ultrasonic treatment for 1 hr, centrifuging, and collecting supernatant to obtain Moschus extract. In another embodiment, the present invention provides a method for preparing a pharmaceutical composition comprising the musk extract according to the invention, comprising the steps of: the musk extract or the composition containing the musk extract is provided, and a required medicinal carrier is added.

As used herein, the singular forms "a", "an", "the" and "the" may include more than one or one of the referenced item unless the context clearly dictates otherwise. As used herein, "about" is understood to mean in the range of-5% to +5% of the number mentioned. Moreover, all numerical ranges herein should be understood to include all integers or fractions within the range. The compositions disclosed herein may be free of any elements not specifically disclosed herein. Thus, disclosure of embodiments using the term "comprising" includes disclosure of embodiments "consisting essentially of and" consisting of the indicated components.

As used herein, the term "musk" (musk) is a dried product of the secretion of mature male body capsules of musk deer-like animals such as deer musk deer (moschus berezovski flerov), horse musk deer (m.silatus przewalssi.), and raw musk deer (m.moschefferus linaeus). According to records in traditional Chinese medicine book, musk is pungent in taste and warm in nature and enters heart meridian, brain meridian and blood system; has effects of relieving heart and tranquilizing, refreshing brain, dredging orifices, promoting blood circulation, dredging collaterals, relieving swelling and pain, dredging channels and collaterals, and penetrating muscle and bone, and can be used for treating fever coma, apoplexy, phlegm syncope, qi stagnation, sudden syncope, central aversion coma, amenorrhea, heart and abdomen pain, superficial infection, lymphoid tuberculosis, arthralgia, numbness, and has effects of inducing orifices, relieving spasm, and strengthening heart for central nervous system, respiratory circulation system, etc. Musk contains various chemical components, including macrocyclic ketones (3-methyl-cyclopentadecanone), nitrogen-containing heterocycles, steroids, polypeptides, fatty acids, etc. As used herein, musk is any of the usual forms of musk commercially available from any conventional source, e.g. musk powders available from drugstores, etc., unless otherwise specified.

Ultrasonic extraction is a method for extracting by using ultrasonic vibration, the mechanical action of ultrasonic enables a solvent to rapidly enter a solid substance, and organic components contained in the substance are dissolved in the solvent as completely as possible, so that a multi-component mixed extracting solution is obtained. The ultrasonic extraction can effectively extract the main active substance muscone in the musk.

The terms "tumor" and "cancer" are not mutually exclusive and are used interchangeably herein, encompassing solid tumors and hematological tumors, referring to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous (pre-cancer) and cancerous cells and tissues. In certain embodiments, cancers suitable for treatment by the antibodies of the invention include gastric or pancreatic cancers, including metastatic forms of those cancers.

The term "glioma" is short for glioma, also known as glioma. Gliomas are neoplastic diseases of the central nervous system, and refer to tumors that occur in the neuroectoderm and that arise from the carcinogenesis of neuronal or mesenchymal cells. Gliomas are the most common primary central nervous system tumors in the cranium, accounting for about half of all primary intracranial tumors. Glioblastoma is a relatively common type of glioma, also known as glioblastoma multiforme (GBM), and is the special name for the progression of astrocytomas (astrocytic tumors) to Grade IV (WHO international classification of diseases), with the highest degree of malignancy. The peak age of glioblastoma is generally between 50 and 60 years.

The term "CAR-T" or "CAR T" refers to a T lymphocyte that transduces and expresses a Chimeric Antigen Receptor (CAR). The chimeric antigen receptor refers to one or more sets of polypeptides that, when in an immune effector cell, provide the cell with specificity for a target cell (typically a cancer cell) and have intracellular signal production. Typically, a CAR comprises at least an extracellular binding region, a transmembrane region, and an intracellular signaling region. Exemplary CAR construction methods and/or transduction methods of CAR-T cells are described, for example, in chinese patent publication No. CN 114014941A.

The term "pharmaceutical composition" refers to a mixture of a musk extract useful in the present invention as an active ingredient with a pharmaceutically acceptable carrier, as the case may be. The pharmaceutical composition facilitates administration of the active ingredient to a patient.

As used herein, 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, solvent or encapsulating material, involved in carrying or transporting a compound useful in the present invention within or to a patient so that it may perform its intended function. Typically, such constructs may be carried to, or transported from, one organ or portion of the body to another organ or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the musk extract described herein, and not injurious to the patient. Some examples of 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, ethyl 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; buffering agents such as magnesium hydroxide and aluminum hydroxide; a surfactant; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol; phosphate buffer; and other non-toxic compatible materials used in pharmaceutical formulations. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like, that are compatible with the activity of the compounds of the present invention and that are physiologically acceptable to a patient. Supplementary active compounds may also be incorporated into the compositions. The "pharmaceutically acceptable carrier" may further include pharmaceutically acceptable salts of the 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.

In some embodiments, the pharmaceutical composition is formulated for intravenous injection. In some embodiments, the pharmaceutical composition comprises a musk extract formulated for intravenous injection. In some embodiments, the pharmaceutical composition comprises a musk extract and CAR-T cells formulated for intravenous injection. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include saline, bacteriostatic water, or fluid to the extent that easy injection is achieved. 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 can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. 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.

As used herein, "treating" refers to slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

As used herein, "prevention" includes inhibition of the occurrence or development of a disease or disorder or a symptom of a particular disease or disorder. In some embodiments, subjects with a family history of cancer are candidates for a prophylactic regimen. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in a subject at risk for cancer.

As used herein, "efficacy-enhancing agent" or "efficacy-enhancing agent" means an agent capable of enhancing the therapeutic effect of an existing therapy, wherein an existing therapy may refer to any therapy already used or known in the art, preferably a therapy directed to cancer, more preferably CAR-T cell therapy. Enhanced therapeutic effect refers to a slower, more interrupted, more retarded, more alleviated, more stopped, less advanced, or more reversed progression or severity of an existing symptom, disorder, condition, or disease in a patient receiving a therapeutic effect-enhancing agent, or, in short, achieving or substantially achieving a better course or outcome than a patient treated with an existing therapy without a therapeutic effect-enhancing agent.

All patents, patent applications, publications, technical and/or academic articles, and other references cited or mentioned herein are incorporated by reference in their entirety to the extent allowed by law.

Drawings

FIG. 1 shows the composition of Moschus extract by HPLC analysis. The horizontal axis of the graph is a time axis, and the vertical axis is a light absorption intensity unit (Absorbance unit).

Figure 2 shows a histogram statistical comparison of proliferation of CAR11-3 CAR-T cells by CCK-8 assay after treatment with various concentrations of musk extract.

FIG. 3 shows the cytokine IFN-. Gamma.levels in the culture supernatants of PMBC cells and CAR11-3 CAR-T cells, each cultured alone or in combination with U87, U251, after treatment with graded concentrations (0, 15, 60 and 150. Mu.g/mL) of musk extract.

FIG. 4 shows the direct killing of target cells by CAR11-3 cells after the CAR11-3 cells co-cultured with the target cells U87 for 24h treatment with musk extract, followed by aspiration of unbound CAR11-3 cells from the supernatant, followed by observation under a mirror and photography. 4X, 10X and 20X represent objective lens multiples of 4 times, 10 times and 20 times. The solvent control is an extraction solvent control.

FIG. 5 shows the direct killing of target cells by CAR11-3 cells recorded by microscopic observation and photography after 24h treatment of the CAR11-3 cell and target cell U251 coculture system with musk extract after aspiration of unbound CAR11-3 cells from the supernatant. 4X, 10X and 20X represent the objective lens multiples of 4 times, 10 times and 20 times. The solvent control is an extraction solvent control.

FIG. 6 shows IFN-. Gamma.secretion levels in supernatants after treatment of non-transduced PBMC and CAR-V3 cells (co-cultured and non-co-cultured with NALM-6 target cells, respectively) with graded concentrations of musk extract (0, 15, 60 and 150. Mu.g/mL) for 24 h. NT = untransduced PBMC cells. V3= CAR-V3 cells. N6= adult type B acute lymphoblastic leukemia NALM-6 cell line.

FIG. 7 shows luciferase-expressing stable transfection of brain glioma cell lines into the tail vein after in situ tumorigenesis in mouse brain 10 ⁷ CAR-T cells were simultaneously intraperitoneally injected with musk extract at a dose of 100 mg musk extract/kg mouse body weight, and compared before and after treatment (day 0 vs day 6) by fluorescence imaging of the small animals in vivo.

Detailed Description

The invention will now be described with reference to the following examples. These examples are provided for illustrative purposes only, and the present invention is not limited to these examples, but encompasses all variations that are apparent based on the teachings provided herein.

Example 1 preparation of Musk extract

Accurately weighing Moschus powder (Beijing century Tan Hospital) 5 mg, subpackaging, adding sterile PBS (or DMSO) 100 μ L into 1mg, and performing ultrasound for 1h with 90W power by conventional ultrasonicator; taking 1mg powder, adding 100 μ L PBS, ultrasonic treating for 1 hr, centrifuging at 12000rpm for 5min, and mixing supernatant extractive solutions, i.e. 2 mg Moschus powder to obtain 200 μ L extractive solution (i.e. 10 mg/ml) with muscone content of 30 μ g/ml. Filtering with pinhole type microporous sterile filter membrane, subpackaging, sealing at low temperature, and storing for use.

Identifying the main components of Moschus extract by high performance liquid chromatography, and detecting by 254nm ultraviolet absorption.

TABLE 1 peaks in HPLC chromatogram (FIG. 1)

Name (R)	Retention time (the fourth of 8230; minute)	Area of	% area	Height
					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

Referring to fig. 1 and table 1, the peak time was from 6.2 minutes to 19.1 minutes, and the active substance separated. The content of the components of the substance with the retention time of about 14 minutes is up to about 61.57 percent. It is demonstrated that most of the key active substances can be effectively retained by the extraction method in the patent.

Example 2 Musk extract toxicity assay for CAR-T cells in vitro

CAR-T cell preparation

Chimeric antigen receptor-modified (CAR) CAR-T cells targeting IL-13R α 2 (CAR 11-3) were prepared according to the methods described in Xu C, bai Y, an Z, hu Y, zhang C, zhong X. IL-13R α 2 humanized scFv-based CAR-T cells exhibit therapeutic activity against glioblastoma. Mol Ther Oncolytics. 20224. Or in the Chinese patent application published as CN 114014A.

Cell culture and processing

CAR11-3 CAR-T cells in X-VIVO containing 10% FBS (Biosera) and IL-2 (Beijing Shunlu pharmaceutical) ^TM 15 (Lonza) amplification in complete medium. The cells were placed in an environment containing 5% carbon dioxide at 37 ℃. And (5) stably culturing.

Cytotoxicity assessment

Cytotoxicity assays were performed by CCK-8 cell proliferation/toxicity assay kit (Solambio CA 1210): cultured CAR11-3 CAR-T cells were counted, plated in 96-well plates, and treated for 72 h with different concentration gradients of musk extract, i.e. 0, 15, 30, 60, 120 and 240 μ g/mL of extract (0 μ g/mL group as extraction solvent control, see below). Thereafter, 20. Mu.L of CCK-8 reagent was added to each well according to the manufacturer's instructions, and after incubation at 37 ℃ for 3h, the absorbance at OD 450nm was determined.

The results are shown in figure 2, and the musk extract with different concentrations has no obvious toxicity to CAR 11-3T cells, which indicates that the musk extract has no toxic and side effects on the growth and proliferation of CAR-T cells.

Example 3 Musk extract enhances CAR11-3 CAR-T cell Activity in vitro

Cell culture and processing

Human brain glioma U87 cells (ATCC HTB-14) and U251 cells (shanghai cell bank of chinese academy of sciences) were cultured in DMEM medium (Invitrogen) containing 10% FBS and the dual antibody to streptomycin; human Peripheral Blood Mononuclear Cells (PBMC) and IL-13R alpha 2-targeting chimeric antigen receptor modified (CAR) CAR-T cells (CAR 11-3) in X-VIVO containing 10% FBS and IL-2 ^TM 15. Amplification in complete medium. All cells were placed in an environment containing 5% carbon dioxide at 37 ℃. And (5) stably culturing.

Cell viability assessment

PBMC were cultured separately and CAR11-3 cells were prepared, counted and co-cultured with U87 and U251 in 12 well plates (non co-culture controls, respectively) with lymphocyte plating of 5 x10 ^ 5/well and tumor cell plating of 10^ 5/well. And treated simultaneously with different concentrations of musk extracts (0, 15, 60 and 150. Mu.g/mL). After 24h of treatment, cell supernatants were harvested by centrifugation and the concentration levels of interferon-gamma (IFN-. Gamma.) were determined. Results as shown in figure 3, IFN- γ levels were unchanged (< 1000 pg/mL) in each group of musk extract treated PBMCs, whether or not co-cultured with target tumor cells. In each group of CAR11-3 (including non-co-cultured and co-cultured groups), the musk extract was effective in increasing IFN-gamma secretion of CAR-T cells and was dose-dependent; after coculture treatment of CAR11-3 cells with target cells U87 and U251, in particular in each group of musk extract treated CAR11-3 cells with U87 cells, the level of cellular IFN- γ was further increased with statistical significance (./p <0.05 and/p <0.01 n = 3. As a target protein of CAR11-3-T cells, the expression level of IL-13R alpha on the cell surface of U87 is higher than that of U251, and the IFN-gamma concentration is increased to a higher degree than that of CAR11-3 and U251 co-culture group after the same concentration of musk extract is used for the treatment of CAR11-3 and U87 co-culture. The musk extract can specifically act on CAR-T cells, promote the CAR-T cells to secrete IFN-gamma in a dose-dependent manner, and increase the killing capacity of CAR-T.

In addition, CAR-T binding to target cells and levels of killing were also observed under the microscope. CAR11-3 was co-cultured with target cells U87 or U251 and treated with 100 μ g/mL musk; 24 After h, unbound CAR-T cells from the supernatant were aspirated, observed under a mirror and photographed. Bright field images at different magnifications are shown in fig. 4 (U87) and fig. 5 (U251). As can be seen from the images, for both target cells, the musk extract significantly increased the number of CAR-T cells bound, promoting the vesiculation of the target cells, compared to the solvent control group.

The results show that the musk extract can specifically act on CAR-T cells, enhance the direct killing capability and the cytokine secretion capability of the CAR-T cells, simultaneously can not activate or enhance unrelated lymphocytes, and ensures the safety while enhancing the effect.

Example 4 Musk extract enhances multiple CAR-T cell activities in vitro

CAR-T cell preparation

The targeting CD19 CAR-T (CAR-V3) is a product on the market for treating large B lymphoma, and can be prepared according to the method described in Chinese patent publication No. CN 113278652B.

Cell culture and processing

Adult type B acute lymphoblastic leukemia NALM-6 cells (N6) (ATCC CRL-3273) were cultured in RPMI-1640 complete medium (Gibco); PBMC, CAR-V3 (if necessary) in X-VIVO containing 10% FBS and IL-2 ^TM 15. Amplification in complete medium. All cells were placed in an environment containing 5% carbon dioxide at 37 ℃. And (5) stably culturing.

Evaluation of cellular Activity

Non-transduced PBMC and CAR-V3 cells (co-cultured and non-co-cultured groups, respectively) were treated with graded concentrations of Moschus extract (0, 15, 60 and 150 μ g/mL) for 24h, and IFN- γ secretion in the supernatant was measured, and the results are shown in FIG. 6, in which the Moschus extract specifically increased the IFN- γ release amount of CAR-V3, and further increased IFN- γ levels after co-culturing CAR-V3 with adult type B acute lymphoblastic leukemia NALM-6 cells (N6 cells). The musk extract also has an enhancing effect on CAR-T cells targeting tumors in the blood system, and the CAR-T cell activity is increased.

Example 5 in vivo Activity assay of Musk extract

In situ glioma animal experiments

Human brain glioma U251 cells were cultured in DMEM medium containing 10% fbs and the dual antibody to streptomycin; human Peripheral Blood Mononuclear Cells (PBMC) and IL-13R alpha 2 targeting chimeric antigen receptor modified CAR11-3 CAR-T cells in X-VIVO containing 10% FBS and IL-2 ^TM 15. Amplification in complete medium. All cells were stably cultured in an atmosphere containing 5% carbon dioxide at 37 ℃.

A stable transfected cell line containing a brain glioma expressing luciferase was constructed using U251 cells according to the method described in Xu C, bai Y, an Z, hu Y, zhang C, zhong X, IL-13R α 2 manipulated scFv-based CAR-T cells exhibit therapeutic activity against gliobastoms. 20224-443. Or Chinese patent publication No. CN 104130977A. After sufficient cells were harvested by culture, 10. Mu.L of 2X 10-containing solution was injected into each mouse ⁵ And (2) stably transfecting a cell suspension of tumor cells of luciferase, sucking the cell suspension by using a sterilized injection needle, placing the cell suspension above the skull of an anesthetized and fixed mouse by using a small animal brain positioning instrument, drilling a hole, and then inserting the injection needle into brain tissues to inject the cell suspension, thereby constructing an intracranial in-situ model of the glioma mouse, and setting 5 mice as 5 parallel experimental groups. After 1 week, mice were anesthetized with oxygen-blown isoflurane and 200. Mu.L of fluorescein substrate per mouse was intraperitoneally injected(Solarbio D9390), imaging was performed using a small animal in vivo fluorescence imager to confirm tumor cell in situ tumorigenesis by fluorescence imaging.

After 5 mice all confirmed tumor formation, each mouse was injected via tail vein with 10 injections ⁷ CAR11-3 CAR-T cells were administered, and the musk extract was intraperitoneally injected at a dose of 100 mg musk extract/kg mouse body weight, day 0. Small animal in vivo fluorescence imaging was performed again 7 days after treatment (i.e., day 6).

The images obtained by two fluorescence imaging are compared, and the result is shown in fig. 7, so that the mouse tumor fluorescence signal is obviously reduced and even disappears after 7 days of CAR11-3 cell injection, which indicates that the tumor focus is obviously reduced. Thus, in vivo experiments suggest that musk can promote the entry of intravenously injected CAR-T cells into the brain, enhancing targeting of the CAR-T cells to tumor cells in the brain.

The published patent and non-patent documents referred to and/or referred to in the above examples are incorporated by reference herein in their entirety.

Claims

1. Use of a musk extract for the preparation of a therapeutic effect enhancer of CAR-T cell therapy, characterized in that the musk extract is prepared by: dissolving Moschus powder in sterile PBS or DMSO at a ratio of 1 mg/100 μ L solvent, performing ultrasonic treatment for 1 hr, centrifuging, and collecting supernatant to obtain Moschus extract; in the musk extract, the content of musk ketone is not less than 20 mu g/mL.

2. The use of claim 1, wherein the CAR-T cell therapy is directed against a hematological tumor.

3. The use of claim 1, wherein the CAR-T cell therapy is directed against a solid tumor.

4. The use of claim 3, wherein the solid tumor is a glioma.

5. The use of claim 1, wherein the CAR-T cell therapy is directed against a glioma, and wherein the effect of the therapeutic effect enhancer comprises at least an increase in the number of CAR-T cells that cross the blood brain barrier.

6. A pharmaceutical composition comprising a musk extract and CAR-T cells, together with a pharmaceutically acceptable carrier, wherein the musk extract is prepared by: dissolving Moschus powder in sterile PBS or DMSO at a ratio of 1 mg/100 μ L solvent, performing ultrasonic treatment for 1 hr, centrifuging, and collecting supernatant to obtain Moschus extract; in the musk extract, the content of musk ketone is not less than 20 mu g/mL.