CAR-T cell culture method and application thereof in tumor treatment

Abstract

The invention provides a CAR-T cell culture method and application of the CAR-T cell culture method in tumor treatment, and belongs to the technical field of biological medicine. Specifically, the method comprises the step of performing culture treatment on the CAR-T cells by adopting sphingolipid metabolites. The in-vitro culture treatment is carried out on the CAR-T cells by adopting the sphingolipid metabolite, so that the killing efficiency of the CAR-T cells can be remarkably improved, the growth speed of tumors can be slowed down, the lifetime of patients / animals can be prolonged, no extra metabolic burden is generated on the body, and the toxic and side effects are lower. In addition, the method does not carry out any modification on the cells, so that the unknown potential risk increased by the patient / animal due to the feedback of various modification adjuvants is reduced, and the safety is higher.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology. Specifically, the present application relates to a CAR-T cell culture method and its application in tumor treatment. Background Art

[0002] CAR T cells (Chimeric Antigen Receptor T cells) are genetically engineered T cells designed to recognize and kill cancer cells. The main process is to extract T cells from the patient's body and then transfer a special receptor, the chimeric antigen receptor (CAR), into these cells in the laboratory for modification. The modified T cells are amplified and then re-infused into the patient's body to recognize and attack cancer cells with specific antigens. Initially, the therapy was aimed at certain types of blood tumors, especially acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL). The unprecedented success of this therapy in treating B-cell malignancies led to its approval by the US Food and Drug Administration (FDA) in 2017. So far, eight CAR-T products, including Kymriah, have been approved by the US FDA for the treatment of blood system tumors.

[0003] Despite this, the development of CAR T-cell therapy still faces many challenges, such as T-cell exhaustion, poor persistence after reinfusion, and poor management of toxic side effects such as cytokine release syndrome. Summary of the invention

[0004] The present application aims to solve at least one of the technical problems in the related art to a certain extent. To this end, the present application proposes a method of applying sphingolipid metabolites to CAR-T cell culture to improve the killing ability of tumors and reduce toxic side effects.

[0005] In a first aspect, the present application proposes a CAR-T cell culture method. According to an embodiment of the present application, the aforementioned method includes: using sphingolipid metabolites to culture CAR-T cells.

[0006] Using sphingolipid metabolites to culture CAR-T cells in vitro can significantly increase the sustained killing efficiency of CAR-T cells, slow down tumor growth, and prolong the survival of patients / animals without causing additional metabolic burden on the body, and has lower toxic side effects. In addition, this method does not perform any modification on the cells themselves, thereby reducing the unknown potential risks of patients / animals due to the reinfusion of various modified adjuvants, and is therefore safer.

[0007] In some examples of the present application, the aforementioned sphingolipid metabolites include: Sphinganine (SA), 3-ketodihydrosphingosine (3KDS), dihydroceramide, ceramide, sphingosine (SO), sphingosine-1-phosphate (S1P) or at least one of their analogs. Using the aforementioned types of sphingolipid metabolites to treat CAR-T cells in vitro can increase the effector function of CAR-T cells to varying degrees, such as increased secretion of interferon γ (IFNγ), tumor necrosis factor α (TNFα), perforin, and granzyme B, thereby enhancing its anti-tumor efficacy. In some preferred examples of the present application, the aforementioned sphingolipid metabolites are dihydrosphingosine or its analogs. It has been proven that in vitro treatment of CAR-T cells with dihydrosphingosine or its analogues can effectively increase the ability to kill tumors. When re-infused into mice, tumor growth is significantly inhibited, effectively prolonging the survival time of mice.

[0008] In some examples of the present application, the aforementioned culture process includes: isolating peripheral blood mononuclear cells, and adjusting the density of mononuclear cells to (1-3)×10 6 cells / mL; a first culture is performed using a serum-free medium supplemented with IL-2, a T cell activating reagent is added to the serum-free medium, and the T cell activating reagent includes CD3 and CD28 antibodies; after the first culture, a virus containing a CAR structure is transduced to target antigens, and a second culture is performed; after the second culture, a predetermined concentration of sphingolipid metabolites is used for a third culture.

[0009] In some examples of the present application, the aforementioned serum-free medium can be selected from OPV serum-free medium.

[0010] In some examples of the present application, the concentration of the aforementioned IL-2 is selected from 80 IU / ml-120 IU / ml, optionally 80 IU / ml, 81 IU / ml, 82 IU / ml, 83 IU / ml, 84 IU / ml, 85 IU / ml, 86 IU / ml, 87 IU / ml, 88 IU / ml, 89 IU / ml, 90 IU / ml, 91 IU / ml, 92 IU / ml, 93 IU / ml, 94 IU / ml, 95 IU / ml, 96 IU / ml, 97 IU / ml, 98 IU / ml, 99 IU / ml, 100 IU / ml, 101 IU / ml, 102 IU / ml, 103 IU / ml, 104 IU / ml, 105 IU / ml, 106 IU / ml, 107 IU / ml, 108 IU / ml, 109 IU / ml, 110 IU / ml, 111 IU / ml, 112 IU / ml, 113 IU / ml, 114 IU / ml, 115 IU / ml, 116 IU / ml, 117 IU / ml, 118 IU / ml, 119 IU / ml or 120 IU / ml. In some preferred examples of the present application, the aforementioned IL-2 concentration is selected from 100 IU / ml. The aforementioned concentration of IL-2 can effectively promote T cell proliferation and survival.

[0011] In some examples of the present application, the time of the first culture, the second culture, and the third culture is selected from 30 h-60 h, optionally 30 h, 36 h, 48 h or 60 h. In some preferred examples of the present application, the time of the first culture, the second culture, and the third culture is selected from 48 h.

[0012] In some examples of the present application, the predetermined concentration is selected from 3 μM-5 μM, and is optionally 3.1 μM, 3.2 μM, 3.3 μM, 3.4 μM, 3.5 μM, 3.6 μM, 3.7 μM, 3.8 μM, 3.9 μM, 4.0 μM, 4.1 μM, 4.2 μM, 4.3 μM, 4.4 μM, 4.5 μM, 4.6 μM, 4.7 μM, 4.8 μM, 4.9 μM or 5.0 μM. Culturing CAR-T cells in vitro with sphingolipid metabolites at the above concentrations can effectively kill tumors, inhibit tumor growth, and prolong the survival time of patients / animals.

[0013] It should be understood that the CAR-T cell culture processing step of the present application is a routine laboratory step. The difference from the existing CAR-T cell culture processing step is that CAR-T cells are cultured in vitro using sphingolipid metabolites, and the in vitro culture time and sphingolipid metabolite concentration are further optimized. After in vitro co-incubation (co-incubation of CAR-T cells and tumor cells), mouse tumor model and humanized mouse tumor model experimental verification, it has the following beneficial technical effects.

[0014] In the in vitro co-culture experiment, the co-culture of SA-treated CD8+T cells with tumor cells can observe a significant improvement in the tumor killing effect of CD8+T cells, which is mainly manifested in the significant proliferation of CD8+T cells and enhanced cytotoxicity (such as LDH release and cell apoptosis analysis), while the growth of tumor cells is significantly inhibited.

[0015] In the mouse tumor model experiment, after the reinfusion of SA-treated CAR-T cells (CD8+T cells), tumor growth slowed down, the survival rate increased, and the tumor volume and weight were significantly reduced, indicating that the tumor killing ability of SA-treated CD8+T cells was significantly improved and could effectively control the growth rate of the tumor.

[0016] In the humanized mouse tumor model experiment, SA-treated CAR-T cells were able to significantly reduce the fluorescence intensity of blood tumor cell lines (such as Raji cells) in mice and significantly prolong the survival time of mice. After the cells were transfused, the weight of the mice did not decrease significantly, indicating that the toxicity of SA-treated CAR-T cells was significantly reduced and would not cause additional burden on the body after transfusion.

[0017] The following exemplifies the steps of SA treating CAR-T cells in vitro: 1) Perform gradient centrifugation on peripheral blood to separate and count peripheral blood mononuclear cells (PBMCs).

[0018] 2) Take 2.5x10 7 PBMCs were sorted for CD4+CD8+ cells, and a total of 14 x10 cells were obtained. 6 indivual.

[0019] 3) Each group takes 1 x10 6 Adjust the cell density to 1 x10 6 The cells were cultured at 4% 4% paraformaldehyde and 100 IU / ml IL-2 were added to OPV serum-free medium, and T cell Transact (Miltenyibiotec 130-111-160) coated with magnetic beads containing CD3 and CD28 antibodies was used for stimulation and subsequent culture.

[0020] 4) After 48 h of activation, the cells were counted and transduced with a virus containing a CAR structure targeting CD19 antigen (10 MOI) based on the cell number.

[0021] 5) After continuing to culture for 48 hours, centrifuge at 300g and discard the supernatant; after resuspending, the cells were divided into two groups and the CART cells were treated with SA (4.2 μM).

[0022] 6) After that, the cell medium was changed every 48 h and cells were treated with SA (4.2 μM) until the day of treatment.

[0023] In the second aspect, the present application proposes a CAR-T cell. According to an embodiment of the present application, the aforementioned CAR-T cell is obtained by culture based on the method described in the first aspect. The CAR-T cells obtained by culture based on the method of the first aspect are used for the prevention and / or treatment of tumors, which can effectively improve the tumor killing efficiency, control the growth rate of tumors, reduce toxic side effects, and prolong the survival time of patients / animals.

[0024] In the third aspect, the present application proposes a drug. According to an embodiment of the present application, the aforementioned drug includes: the CAR-T cells described in the second aspect. The drug containing the aforementioned CAR-T cells, used for the prevention and / or treatment of tumors, can effectively improve the tumor killing efficiency, control the growth rate of tumors, reduce toxic side effects, and prolong the survival time of patients / animals.

[0025] In some examples of the present application, the aforementioned drug further includes: a pharmaceutically acceptable excipient.

[0026] In some examples of the present application, the auxiliary material includes: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

[0027] In some examples of the present application, the drug is an injection.

[0028] It should be noted that the aforementioned drugs include combinations separated in time and / or space, as long as they can work together to achieve the purpose of the present application. For example, the ingredients contained in the aforementioned drugs can be administered to the subject as a whole, or separately. When the ingredients contained in the drug are administered to the subject separately, each ingredient can be administered to the subject simultaneously or sequentially.

[0029] The drug of the present application contains a safe and effective amount of the active ingredient (CAR-T cells) of the present application and pharmaceutically acceptable excipients. Such excipients include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the drug preparation should match the mode of administration, and the dosage form of the drug of the present application is an injection. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. The drug is preferably manufactured under sterile conditions.

[0030] The effective amount of the active ingredient of the present application may vary with the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The aforementioned factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment situation, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0031] In the fourth aspect, the present application proposes the use of the CAR-T cells described in the second aspect or the drugs described in the third aspect in the prevention and / or treatment of tumors. The application of the aforementioned CAR-T cells or drugs in the prevention and / or treatment of tumors can effectively improve the tumor killing efficiency, control the growth rate of tumors, reduce toxic side effects, and prolong the survival time of patients / animals.

[0032] In some examples of the present application, the aforementioned tumors include: melanoma, blood tumors, liver cancer, and tumors treated by CAR-T cells targeting other antigens. It should be understood that tumors corresponding to tumor antigens targeted by CAR are within the scope of application of the present application, including but not limited to blood tumors and solid tumors.

[0033] In the fifth aspect, the present application proposes a method for treating tumors. According to an embodiment of the present application, the aforementioned method comprises: administering an effective dose of CAR-T cells to a patient; and / or administering SA and / or SA analogs by intravenous injection after administering an effective dose of CAR-T cells, wherein the aforementioned CAR-T cells are obtained by culturing the method of the first aspect. The use of CAR-T cell therapy can effectively improve the efficiency of tumor killing, control the growth rate of tumors, reduce toxic side effects, and prolong the survival time of patients / animals.

[0034] It can be understood that the tumor treatment method of the present application is CAR-T cell therapy, which specifically includes: T cell collection, gene modification, cell expansion, re-infusion therapy and other processes.

[0035] In some examples of the present application, after administration of an effective dose of CAR-T cells, the dose of SA and / or SA analogs administered by intravenous injection is 500μg / kg-700μg / kg, optionally 500μg / kg, 510μg / kg, 520μg / kg, 530μg / kg, 540μg / kg, 550μg / kg, 560μg / kg, 570μg / kg, 580μg / kg, 590μg / kg, 600μg / kg, 610μg / kg, 620μg / kg, 630μg / kg, 640μg / kg, 650μg / kg, 660μg / kg, 670μg / kg, 680μg / kg, 690μg / kg or 700μg / kg. It is understood that the administration time can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days after the administration of an effective dose of CAR-T cells.

[0036] In some examples of the present application, the aforementioned effective dose of CAR-T cells can be determined based on the patient's weight, tumor severity, or tumor stage.

[0037] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0039] Figure 1A A schematic diagram of the treatment process of SA-treated CD19-CART cells in a Raji (human Burkitt's lymphoma cell) humanized animal model provided in one embodiment of the present application; Figure 1B A schematic diagram of the survival rate analysis results provided in one embodiment of the present application; Figure 1C A schematic diagram of the weight analysis results of tumor-bearing mice after CART treatment provided in one embodiment of the present application; Figure 1D A schematic diagram of the comparison results of the area under the weight change curve provided by an embodiment of the present application; Figure 2A A schematic diagram of the process of treating the killing effect of SA on OT1 CD8+T cells in vitro on tumor cells provided in one embodiment of the present application; Figure 2B A schematic diagram of flow cytometry results of a Dmso control group and a SA treatment group provided in one embodiment of the present application; Figure 2C A schematic diagram of the killing effect of CD8+T cells on melanoma in the Dmso control group and the SA treatment group provided in one embodiment of the present application; Figure 3A A schematic diagram of a processing flow for the tumor killing ability of SA-treated OT1 CD8+T cells in a melanoma animal model provided in one embodiment of the present application; Figure 3B A schematic diagram of tumor volume change results provided in one embodiment of the present application; Figure 3C A schematic diagram of the comparison results of tumor weights of a blank control group, a Dmso control group, and a SA-treated group provided in one embodiment of the present application; Figure 4A A schematic diagram of the release results of effector factors treated with SA and its structural analogs provided in one embodiment of the present application; Figure 4B A schematic diagram showing the comparative results of the release of effector factors treated with SA and its structural analogs provided in one embodiment of the present application; Figure 4C A schematic diagram showing the comparative results of perforin release treated by SA and its structural analogs provided in one embodiment of the present application; Figure 4D This is a schematic diagram of the comparative results of granzyme B release after treatment with SA and its structural analogs provided in one embodiment of the present application. DETAILED DESCRIPTION

[0040] Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0041] In the process of describing the present invention, the relevant terms in this document are explained and illustrated. These explanations and illustrations are only for the convenience of understanding of the scheme and cannot be regarded as limitations on the protection scheme of the present invention.

[0042] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0043] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0044] In this application, "CAR-T cells" are specific anti-tumor immune effector cells modified by genetic engineering. This cell is derived from T lymphocytes of patients or healthy donors. By expressing chimeric antigen receptors (CAR) on their cell surfaces, it can specifically recognize targeted antigens on the surface of tumor cells that are independent of MHC presentation, and directly activate the killing mechanism after recognition, including the release of perforin, granzyme and cytokines, thereby accurately killing tumor cells. In the absence of genetic modification, T cells recognize antigens on the surface of tumor cells through T cell receptors (TCRs). These antigens are processed by abnormal proteins inside tumor cells and presented on MHC-I molecules. When TCR binds to the MHC-I / antigen complex, T cells are activated and exert cytotoxic effects, releasing perforin and granzyme to kill tumor cells. The CAR gene that can recognize tumor antigens is introduced into T cells through genetic engineering methods, so that T cells acquire the ability to directly recognize and kill tumor cells.

[0045] In the present application, "sphingolipid metabolites" refer to various derivatives produced by the metabolic pathways of sphingolipid substances (such as sphingomyelin, ceramide, sphingolipids) in cells, including dihydrosphingosine, 3-ketodihydrosphingosine, dihydroceramide, ceramide, sphingosine, sphingosine-1-phosphate or their analogs.

[0046] In the present application, "pharmaceutically acceptable excipients" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. In addition to any conventional excipients that are incompatible with the CAR-T cells of the present application, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of consideration of the present application.

[0047] In the present application, "administration" refers to the introduction of a predetermined amount of a substance into a patient by a suitable method. The antibody or antigen binding fragment, recombinant protein, multispecific antibody, conjugate or pharmaceutical composition of the present invention can be administered by any common route as long as it can reach the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous injection, etc., but the present invention is not limited to these exemplified modes of administration. Preferably, the composition of the present invention is administered by intravenous or subcutaneous injection.

[0048] In the present application, "treatment" refers to the use of the term to refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, including: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, mitigate or inhibit an individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0049] In the present application, "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0050] The embodiments of the present application will be described in more detail below, and the examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present application, and should not be construed as limiting the present application. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained commercially.

[0051] Example 1: SA-treated CD19-CART cells showed stronger tumor killing effect in Raji (human Burkitt's lymphoma cells) humanized animal model Isolation of PBMCs The T cells used in this example are derived from human peripheral blood, and peripheral blood mononuclear cells (PBMC) are isolated using the following steps and methods: 1. Laboratory preparation: Before entering the laboratory, the air conditioner should be turned on half an hour in advance. The biological safety cabinet needs to be disinfected with ultraviolet light for 30 minutes before use. The table top should be wiped with 75% alcohol before use. After the biological safety cabinet is turned on for 20 minutes, it can be operated only after it is confirmed to be operating normally. 2. Dilute the donor's fresh anticoagulated blood with an equal volume of tissue diluent and mix thoroughly. 3. Add 5 ml of human peripheral lymphocyte separation medium (Solarbio, Cat. No. P8610) to a 15 ml centrifuge tube and use a Pasteur pipette to carefully spread the diluted donor whole blood on top of the separation medium.

[0052] 4. Centrifuge at room temperature, horizontal rotor 1000g, centrifuge for 30 minutes. After centrifugation, there will be obvious stratification: the top layer is the plasma layer, and the middle layer is the separation liquid layer. Pipette the white film layer (lymphocytes) between the two layers into a new centrifuge tube, and resuspend the white film layer cells in 10ml PBS. After the cells are fully mixed, centrifuge at 500g for 10 minutes at room temperature.

[0053] 5. Resuspend the separated PBMC in PBS and adjust the cell concentration to 10 8 / ml, every 10 7 Add 10 μl CD8 nanobeads (Genescript, L00864) to each cell, mix thoroughly with the cells and incubate at 4°C for 15 minutes. Add 2 ml PBS to the incubated cell suspension, mix thoroughly, centrifuge at 500g at room temperature for 10 minutes, and remove the supernatant. Resuspend the precipitated cells in 500 μl PBS.

[0054] 6. Place the gL cell sorting column (Genescript, D00008) on a magnetic sorting stand (Genescript, D00012) and pre-rinse the column with 3 ml PBS. Add the cells obtained in step 5 to the sorting column and allow the liquid to pass through the sorting column completely by gravity. After the cell suspension has completely passed through the sorting column, rinse the column 3 times with 3 ml PBS to fully remove the cells not labeled by the magnetic beads.

[0055] 7. Remove the sorting column from the magnetic stand and place it on a new centrifuge tube. Add 5 ml of PBS to the sorting column and use the plunger to quickly flush the magnetic bead-labeled cells into the centrifuge tube. Take a small amount of cells from the obtained cell suspension for counting, and label CD8+T cells with percp-cy5.5 fluorescently labeled anti-human CD8a antibodies. Use Fortessa flow cytometer to detect the purity of CD8+T cells, which is usually about >95%.

[0056] CD19 CART cell preparation 1. CD8+T cell culture and activation: Absorb about 1x10 purified CD8+T cells 6 , 500g, 4℃, centrifuge for 5 minutes, discard the supernatant. Resuspend the cells in OptiVitro® T cell serum-free medium (TE000-N022) containing IL-2 (100 IU / ml) and adjust the final cell density to 1x10 6 T cells were stimulated and activated using magnetic beads coated with CD3 / CD28 antibodies (Miltenyi biotec, 130-111-160).

[0057] 2. After CD8+ T cells were activated for 48 hours, the cells were mixed and 20 μl was taken for counting. According to the number of cells, 10 MOI of lentivirus containing CAR structure targeting CD19 protein FMC63 antigen epitope was transduced (CART plasmid: pCDH-EF1a-FMC63-BBz-EGFP lentiviral packaging plasmid: pMDLg-pRRE, pMD2.G, pRSV-Rev).

[0058] SA treatment of CD19 CART cells 1. 48 hours after CD8+ T cell viral transduction, aspirate 20 μl of cells for counting and use Fortessa flow cytometer to detect the proportion of EGFP-positive cells in the cells to determine the CAR transduction efficiency.

[0059] 2. Centrifuge the cell suspension after counting at 500g, 4°C for 5 minutes and discard the supernatant. Resuspend the cells and dilute to 0.5x10 in OptiVitro® T Cell Serum-Free Medium containing IL-2 (100 IU / ml). 6 Pieces / ml.

[0060] 3. Divide the diluted cells into two equal parts and treat them with DMSO or SA (4.2μM) respectively. After that, change the cell medium every 48h, and add DMSO or SA (4.2μM) every time the medium is changed until the 15th day, collect the cells for counting, and detect the proportion of CAR-positive cells by flow cytometry. The cells are used for subsequent functional and molecular experiments.

[0061] Determination of the validity of experimental results refer to Figure 1A-Figure 1D CD19-CART cells have a significant effect in controlling lymphoma and can significantly improve the survival time of tumor-bearing mice. Figure 1B The results show that CD19-CART cells treated with SA have a more significant killing effect on Raji lymphoma, which not only prolongs the survival time of tumor-bearing mice, but also significantly improves the survival rate of tumor-bearing mice. At the same time, we tested the weight of tumor-bearing mice. Figure 1C and Figure 1D The results suggest that compared with the gradual decrease in weight of tumor-bearing mice after infusion of untreated CD19-CART cells, there was no significant decrease in weight of tumor-bearing mice after infusion of SA-treated CD19-CART cells, indicating that SA-treated CD19-CART cell infusion therapy is safer, and the addition of the natural metabolite SA not only does not impose additional burden on the body, but also may improve the toxic side effects during CD19-CART cell therapy.

[0062] Example 2: SA treatment can significantly enhance the effector function of OT-1 CD8+ T cells and enhance the killing effect on tumor cells in vitro Isolation and purification of CD8+ T cells from C57 / BL6 mice In this example, T cells were derived from the spleen of OT1 mice (OT1 is a TCR transgenic mouse, whose CD8+ T cells can specifically recognize the 257-264 amino acid antigen epitope of ovalbumin OVA presented by MHC-1, and are generally used to study the effector function of specific CD8+ T cells). The following steps and methods were used to separate and obtain purified CD8+ T cells. 1. Laboratory preparation: Before entering the laboratory, the air conditioner should be turned on half an hour in advance. The biological safety cabinet needs to be disinfected with ultraviolet light for 30 minutes before use. The table top should be wiped with 75% alcohol before use. After the biological safety cabinet is turned on for 20 minutes, it can be operated only after it is confirmed to be operating normally.

[0063] 2. After killing the mouse by cervical dislocation, remove the spleen, grind the spleen on a 70μm cell sieve, rinse the cell sieve with pre-cooled 1640 containing 1% FBS, collect the cells in a 50ml centrifuge tube, and centrifuge at 500g for 5 minutes. After centrifugation, discard the supernatant, add 1ml ACK red blood cell lysis buffer, lyse at room temperature for 1 minute, add 10ml 1% FBS 1640, and centrifuge at 500g for 5 minutes.

[0064] 3. After centrifugation, discard the supernatant and resuspend the cells in MACS buffer (PBS + 0.5% FBS + 2mM EDTA) at 1x10 7 Add 40 μl MACS buffer to each cell and resuspend. 7 Add 10 μl purified antibody mixture (Militenyi, 130-104-075) to each cell and incubate at 4°C for 5 minutes. Add 3 ml MACS buffer, centrifuge to remove the supernatant, and resuspend the cells in MACS buffer. 7 Add 80 μl MACS buffer to each cell. 7 Add 20 μl Anti-Biotin Microbeads (Militenyi, 130-104-075) to each cell and incubate at 4°C for 10 minutes.

[0065] 4. Place the gL cell sorting column (Genescript, D00008) on a magnetic sorting stand (Genescript, D00012) and pre-rinse the sorting column with 3 ml MACS buffer. Add the cells obtained in step 3 to the sorting column and allow the liquid to flow completely through the sorting column into the collection tube by gravity. After the cell suspension has completely passed through the sorting column, rinse the sorting column once with 3 ml MACS buffer. At this time, the cells in the collection tube are the purified CD8+T cells.

[0066] 5. Take a small amount of cells from the obtained cell suspension for counting, and label the CD8+T cells with anti-mouse CD8a antibody labeled with percp-cy5.5 fluorescein. Use BD Fortessa flow cytometer to detect the purity of CD8+T cells. Usually the purity is about >95%.

[0067] SA treatment of OT1 CD8+ T cells 1. Use PBS containing concentrations of Anti-CD3 / 28 antibodies to pre-coat a 96-well plate (round bottom, tissue culture treated) at 37°C for 2 hours, so that the working concentration of Anti-CD3 antibody in the culture medium is 5μg / ml and the working concentration of Anti-CD28 antibody is 2μg / ml.

[0068] 2. Use complete culture medium (1640+10% FBS+double antibody+non-essential amino acids+β-mercaptoethanol) to dilute the purified CD8+T cells and adjust the cell concentration to 1x10 6 / ml, 10ng / ml IL2 was added to the cells, and the cells were treated with DMSO or SA (4.2μM) respectively, and the cells were added to a 96-well plate at 200μl / well.

[0069] 3. Stimulate and treat cells for 48 hours Co-culture of SA-treated OT1 CD8+ T cells with melanoma B16-OVA 1. Use DMEM complete medium (DMEM + 10% FBS + double antibody) + Blasticidin (used to maintain the expression of OVA in B16 cells) to culture B16-OVA cells (melanoma, ovalbumin gene modified, can be specifically recognized by OT1 CD8+T cells). When the cells grow to a confluence of 70%-80%, digest, centrifuge, and resuspend the cells in 1640 complete medium to adjust the cell concentration to 1x10 5 Cells were added into a 96-well round-bottom plate at 100 μl / well.

[0070] 2. After B16-OVA cells have grown for 18 hours, take OT1CD8+T cells treated with DMSO or SA for 48 hours from the 96-well plate, centrifuge at 500g for 5 minutes, remove the supernatant, and resuspend the cells in fresh 1640 complete medium to adjust the cell density to 2x10 5 The cells were added to a 96-well plate cultured with B16-OVA cells at 100 μl / well for co-culture. 3. After 24 hours of co-culture, BD Fortessa flow cytometer was used to detect tumor cell death and T cell effector factor release. Cell death and death staining strategy: LIVE / DEAD™ Fixable Red (Invitrogen, L23102), CD45BV785, CD8 Percp-cy5.5, detect LIVE / DEAD positive cells in CD45 negative tumor cells, which are tumor cells killed by T cells. Cell effector factor release detection strategy: PMA+Ionomycin+BFA was used to stimulate cells for 4 hours and then antibodies were used to stain the cells. The staining strategy is as follows: LIVE / DEAD™ Fixable Red, CD8 Percp-cy5.5, CD44 FITC, PD1 BV785, IFNγPE / CY7, TNFαPE, Perforin APC, Granzyme B APC / CY7, IL-2 BV421, flow cytometry was used to detect the release of effector factors in the CD8+CD44+PD1+ subpopulation.

[0071] Killing result detection refer to Figure 2A-2C From the results of co-culture of OT1 CD8+T cells and melanoma B16-OVA, the killing effect of OT1 CD8+T cells on tumors was significantly increased after SA treatment. Figure 2B Taking the median-to-target ratio of 2:1 as an example, compared with melanoma B16-OVA, the proportion of tumor cells decreased from 60% in the DMSO group to about 20%; Figure 2C The results are a concrete presentation of the killing effect of OT1 CD8+T cells. The killing effect of OT1 CD8+T cells on melanoma continues to increase with the increase of the effector-target ratio.

[0072] Example 3: SA-treated OT-1 CD8+ T cells showed stronger tumor killing ability in melanoma animal model C57 / BL6 mice bearing subcutaneous tumors 1. Use DMEM complete medium (DMEM+10% FBS+antibody)+Blasticidin to culture B16-OVA cells. When the cells grow to a confluence of 70%-80%, digest and centrifuge. Wash the cells twice with 1640 medium to remove residual serum. After the last wash, resuspend the cells, count them, and adjust the cell concentration to 1x10 using 1640 medium. 6 Pieces / ml.

[0073] 2. Use isoflurane gas anesthesia machine (Rayward, R530IE) to anesthetize C57 / BL6 mice (isoflurane, Rayward R510), and adjust the concentration to 1%-1.5% to maintain the anesthesia state of the mice. Draw 200μl of B16-OVA tumor cell suspension (2x10 5 cells), puncture into the upper right lower groin area of ​​the mouse, slowly push the syringe to allow the cell suspension to enter the subcutaneous tissue, withdraw the syringe, turn off the anesthesia vaporizer, and keep the mouse breathing in pure oxygen for 5 minutes to facilitate rapid awakening of the mouse.

[0074] 3. Observe and measure tumor growth every day from the 4th day until the 8th day.

[0075] SA treatment of OT1 CD8+ T cells 1. Use PBS containing concentrations of Anti-CD3 / 28 antibodies to pre-coat a 96-well plate (round bottom, tissue culture treated) at 37°C for 2 hours, so that the working concentration of Anti-CD3 antibody in the culture medium is 5μg / ml and the working concentration of Anti-CD28 antibody is 2μg / ml.

[0076] 2. Use complete culture medium (1640+10% FBS+double antibody+non-essential amino acids+β-mercaptoethanol) to dilute the purified CD8+T cells and adjust the cell concentration to 1x10 6 10 ng / ml IL2 was added to the cells, and the cells were treated with DMSO or SA (4.2 μM) respectively, and the cells were added to a 96-well plate at 200 μl / well.

[0077] 3. The treatment of step 1 and step 2 lasts for 48 hours.

[0078] Adoptive transfer of OT1 CD8+ T cells into tumor-bearing mice 1. Tumor-bearing mice were randomly divided into 3 groups according to tumor size: 1. The control group was not injected with cells; 2. The DMSO group was injected with DMSO-treated OT1 cells; 3. The SA group was injected with SA-treated OT1 cells.

[0079] 2. Take OT1 CD8+ T cells treated with DMSO or SA for 48 hours from the 96-well plate, centrifuge at 500g for 5 minutes, remove the supernatant, and wash the cells twice with 1640 medium to remove residual serum. After the last wash, resuspend the cells, count them, and adjust the cell concentration to 4x10 using 1640 medium. 6 Pieces / ml.

[0080] 3. Place the tumor-bearing mouse in a holder and draw 200 μl of OT1 CD8+ T cells (8x10 5 cells), puncture into the mouse tail vein, slowly push the syringe to allow the cell suspension to enter the mouse body, withdraw the syringe, and use an alcohol cotton ball to press the mouse tail for 2 minutes to stop bleeding.

[0081] 4. Measure the tumor volume of mice on days 8, 10, 12, 14, 16, and 18 after T cell transfer, and record and create a tumor growth curve.

[0082] 5. Ten days after the tumor-bearing mice were transplanted with OT1 CD8+T cells, the mice were killed, the subcutaneous tumors were isolated, and the tumor weights were measured.

[0083] Tumor killing result detection: refer to Figure 3A-3C ,Depend on Figure 3B The statistical tumor growth curve shows that compared with the DMSO-treated OT1 CD8+T cell-treated group, the SA-treated OT1 CD8+T cells showed a stronger inhibitory effect on tumor growth, and the tumor growth rate was significantly slowed down. There was a significant statistical difference between the two groups. Ten days after adoptive transfer, the mice were killed and the tumor weights were compared ( Figure 3C ) It can also be clearly seen that the tumor weight was reduced and the growth was significantly controlled.

[0084] Example 4: Effects of SA and its structural analogs on the in vitro tumor killing effect of OT-1 CD8+ T cells Isolation and purification of CD8+ T cells from C57 / BL6 mice In this example, T cells were derived from the spleen of OT1 mice (OT1 is a TCR transgenic mouse, whose CD8+ T cells can specifically recognize the 257-264 amino acid antigen epitope of ovalbumin OVA presented by MHC-1, and are generally used to study the effector function of specific CD8+ T cells). The following steps and methods were used to separate and obtain purified CD8+ T cells, which were the same as the separation and purification of CD8+ T cells from C57 / BL6 mice in Example 2.

[0085] SA and its structural analogs treat OT1 CD8+T cells 1. Use PBS containing concentrations of Anti-CD3 / 28 antibodies to pre-coat a 96-well plate (round bottom, tissue culture treated) at 37°C for 2 hours, so that the working concentration of Anti-CD3 antibody in the culture medium is 5μg / ml and the working concentration of Anti-CD28 antibody is 2μg / ml.

[0086] 2. Use complete culture medium (1640+10% FBS+double antibody+non-essential amino acids+β-mercaptoethanol) to dilute the purified CD8+T cells and adjust the cell concentration to 1x10 6 / ml, added 10ng / ml IL2 to the cells, and treated the cells with DMSO, SA (4.2μM), 3-keto Sphinganine (4.2μM), C16 Dihydroceramide (50nM), C18 Ceramide (50nM), Sphingosine (4.2μM), and Sphingosine-1-P (5μM), respectively, and added the cells into a 96-well plate at 200μl / well.

[0087] 3. Stimulate and treat cells for 48 hours Co-culture of OT1 CD8+T cells treated with SA and its structural analogs with melanoma B16-OVA 1. Use DMEM complete medium (DMEM + 10% FBS + double antibody) to culture B16-OVA cells (melanoma, ovalbumin gene modified, can be specifically recognized by OT1 CD8+T cells). When the cells grow to a confluence of 70%-80%, digest, centrifuge, and resuspend the cells in 1640 complete medium to adjust the cell concentration to 1x10 5 Cells were added into a 96-well round-bottom plate at 100 μl / well.

[0088] 2. After B16-OVA cells have grown for 18 hours, take out OT1 CD8+ T cells treated with DMSO, SA or its analogs for 48 hours from the 96-well plate, centrifuge at 500g for 5 minutes, remove the supernatant, and resuspend the cells in fresh 1640 complete medium to adjust the cell density to 2x10 5 The cells were added to a 96-well plate cultured with B16-OVA cells at 100 μl / well for co-culture. 3. After 24 hours of co-culture, BD Fortessa flow cytometer was used to detect tumor cell death and T cell effector factor release. Cell death and death staining strategy: LIVE / DEAD™ Fixable Red (Invitrogen, L23102), CD45BV785, CD8 Percp-cy5.5, detect LIVE / DEAD positive cells in CD45 negative tumor cells, which are tumor cells killed by T cells. Cell effector factor release detection strategy: PMA+Ionomycin+BFA was used to stimulate cells for 4 hours and then antibodies were used to stain the cells. The staining strategy is as follows: LIVE / DEAD™ Fixable Red, CD8 Percp-cy5.5, CD44 FITC, PD1 BV785, IFNγPE / CY7, TNFαPE, Perforin APC, Granzyme B APC / CY7, IL-2 BV421, flow cytometry was used to detect the release of effector factors in the CD8+CD44+PD1+ subpopulation.

[0089] Judgment of the killing results of OT1 CD8+T cells treated with SA and its structural analogs: In this example, flow cytometry was used to analyze Figure 4A and Figure 4B It can be seen that after the activated OT1 CD8+T cells were treated with small molecule compounds such as dihydroceramide and sphingosine (SO), the proportion of effector CD8+T cells that simultaneously expressed IFNγ and TNFα increased significantly, indicating that the effector function of immune cells was enhanced and the killing effect on tumor cells was more significant. Figure 4C This suggests that the number of CD8+T cells secreting perforin increased significantly after treatment with small molecule compounds such as 3-ketodihydrosphingosine (3KDS) and ceramide. Figure 4D SA and its structural analogs had no significant effect on the production of CD8+T cell granzyme B. IFNγ, TNFα and perforin are the main cytokines for CD8+T cells to exert effector functions, and increased secretion is crucial for enhancing the tumor killing effect of CD8+T cells.

[0090] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0091] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0092] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A CAR-T cell culture method, characterized in that: include: CAR-T cells were cultured and treated with sphingolipid metabolites; The sphingolipid metabolites include at least one of dihydrosphingosine, 3-ketodihydrosphingosine, dihydroceramide, ceramide, sphingosine, and sphingosine-1-phosphate.

2. The method according to claim 1, characterized in that The sphingolipid metabolite is dihydrosphingosine.

3. The method according to claim 1 or 2, characterized in that: The culture process comprises: Isolate peripheral blood mononuclear cells and adjust the mononuclear cell density to (1-3)×10 6 cells / mL; A serum-free medium supplemented with IL-2 is used for the first culture, wherein a T cell activating reagent is added to the serum-free medium, and the T cell activating reagent includes CD3 and CD28 antibodies; After the first culture, transducing a virus containing a CAR structure to target the target antigen, and performing a second culture; After the second culture, a third culture is performed using a predetermined concentration of sphingolipid metabolites.

4. The method according to claim 3, characterized in that The concentration of IL-2 is selected from 80 IU / ml-120 IU / ml.

5. The method according to claim 4, characterized in that The time of the first culture, the second culture and the third culture is selected from 30-60 hours.

6. The method according to claim 4 or 5, characterized in that The predetermined concentration is selected from 3 μM-5 μM.

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