CAR-T Cell Culture Method and Its Application in Cancer Treatment

Abstract

The present application provides a method for culturing CAR-T cells and its application in tumor treatment, belonging to the field of biomedical technology. Specifically, the method includes culturing CAR-T cells with sphingolipid metabolites. Culturing CAR-T cells with sphingolipid metabolites in vitro can significantly increase the killing efficiency of CAR-T cells, slow down the tumor growth rate, extend the survival period of patients / animals, and will not impose an additional metabolic burden on the body, with lower toxic and side effects. In addition, this method does not perform any modification on the cells themselves, thus reducing the unknown potential risks for patients / animals due to the reinfusion of various modified adjuvants, and therefore has higher safety.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology. Specifically, this application relates to a method for culturing CAR-T cells and its application in tumor treatment. Background Art

[0002] CAR T cells (Chimeric Antigen Receptor T cells) are T cells genetically engineered to recognize and kill cancer cells. The main process involves extracting T cells from a patient and then transferring a special receptor, the chimeric antigen receptor (CAR), into these cells in the laboratory for modification. The modified T cells are expanded and then re-infused into the patient to recognize and attack cancer cells with specific antigens. Initially, this therapy was targeted 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 for market by the US Food and Drug Administration (FDA) in 2017. As of now, 8 CAR-T products including Kymriah have been approved by the US FDA for treating hematological malignancies.

[0003] Nevertheless, the development of CAR T cell therapy still faces many challenges, such as T cell exhaustion, poor persistence after re-infusion, and poor management of side effects such as cytokine release syndrome. Summary of the Invention

[0004] This application aims to solve at least one of the technical problems in the related art to some extent. For this purpose, this application proposes applying sphingolipid metabolites to the culture of CAR-T cells to improve the killing ability against tumors and reduce side effects.

[0005] In a first aspect, this application proposes a method for culturing CAR-T cells. According to an embodiment of this application, the aforementioned method includes: culturing and treating CAR-T cells with sphingolipid metabolites.

[0006] Culturing and treating CAR-T cells with sphingolipid metabolites in vitro can significantly increase the persistent killing efficiency of CAR-T cells, slow down the tumor growth rate, extend the survival period of patients / animals, and will not impose an additional metabolic burden on the body, with lower side effects. In addition, this method does not perform any modification on the cells themselves, thus reducing the unknown potential risks for patients / animals due to the re-infusion of various modified adjuvants, so the safety is higher.

[0007] In some examples of the present application, the foregoing sphingolipid metabolites include at least one of sphinganine (SA), 3-ketodihydrosphingosine (3KDS), dihydroceramide, ceramide, sphingosine (SO), sphingosine-1-phosphate (S1P), or analogs thereof. Treating CAR-T cells in vitro with the foregoing types of sphingolipid metabolites can increase the effector functions 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 their anti-tumor efficacy. In some preferred examples of the present application, the foregoing sphingolipid metabolite is sphinganine or an analog thereof. It has been verified that treating CAR-T cells in vitro with sphinganine or an analog thereof can effectively increase the killing ability against tumors. After being infused into mice, tumor growth is significantly inhibited, and the survival time of the mice is effectively prolonged.

[0008] In some examples of the present application, the foregoing culture treatment includes: isolating peripheral blood mononuclear cells and adjusting the density of mononuclear cells to (1-3)×10 6 cells / mL; performing a first culture using a serum-free medium supplemented with IL-2, wherein the serum-free medium is supplemented with a T cell activation reagent, and the T cell activation reagent includes anti-CD3 and anti-CD28 antibodies; after the first culture, transducing a virus containing a CAR construct to target a target antigen and performing a second culture; after the second culture, switching to a third culture using a sphingolipid metabolite at a predetermined concentration.

[0009] In some examples of the present application, the foregoing 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 concentration of the aforementioned IL-2 is selected from 100 IU / ml. The IL-2 at the aforementioned concentration can effectively promote the proliferation and survival of T cells.

[0011] In some examples of the present application, the time for the aforementioned first culture, second culture, and 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 for the aforementioned first culture, second culture, and third culture is selected from 48 h.

[0012] In some examples of the present application, the aforementioned predetermined concentration is selected from 3 μM - 5 μM, 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 the aforementioned concentration of sphingolipid metabolites 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:

[0018] 1) Perform gradient centrifugation on peripheral blood to separate and count peripheral blood mononuclear cells (PBMCs).

[0019] 2) Take 2.5x10 7 PBMCs were sorted for CD4+CD8+ cells, and a total of 14 x10 cells were obtained. 6 indivual.

[0020] 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.

[0021] 4) After activation for 48 h, cells were counted and transduced with a virus containing the CAR construct targeting the CD19 antigen (10 MOI) according to the cell number.

[0022] 5) After continued culture for 48 h, the cells were centrifuged at 300 g and the supernatant was discarded; after resuspension, the cells were divided into 2 groups, and the CART cells were treated with SA (4.2 μM).

[0023] 6) Thereafter, the cell medium was changed every 48 h and maintained with SA (4.2 μM) treatment until the day of treatment.

[0024] In a second aspect, the present application provides a CAR-T cell. According to an embodiment of the present application, the aforementioned CAR-T cell is obtained by culturing based on the method described in the first aspect. The CAR-T cell obtained by culturing based on the method of the first aspect can effectively improve the tumor killing efficiency, control the growth rate of the tumor, reduce the toxic and side effects, and prolong the survival time of the patient / animal when used for the prevention and / or treatment of tumors.

[0025] In a third aspect, the present application provides a drug. According to an embodiment of the present application, the aforementioned drug comprises: the CAR-T cell described in the second aspect. The drug containing the aforementioned CAR-T cell can effectively improve the tumor killing efficiency, control the growth rate of the tumor, reduce the toxic and side effects, and prolong the survival time of the patient / animal when used for the prevention and / or treatment of tumors.

[0026] In some examples of the present application, the aforementioned drug further comprises: pharmaceutically acceptable excipients.

[0027] In some examples of the present application, the excipients include: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

[0028] In some examples of the present application, the drug is an injection.

[0029] It should be noted that the aforementioned drug includes combinations that are separated in time and / or space, as long as they can act together to achieve the purpose of the present application. For example, the components contained in the aforementioned drug can be administered to the subject as a whole, or separately administered to the subject. When the components contained in the drug are separately administered to the subject, the individual components can be administered to the subject simultaneously or sequentially.

[0030] 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 solution, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should be matched with the administration method, 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 adjuvants. The said drug should preferably be manufactured under aseptic conditions.

[0031] The effective amount of the active ingredient of the present application may vary depending on the mode of administration, the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by those of ordinary skill in the art according to various factors (such as through clinical trials). The foregoing factors include but are not limited to: the pharmacokinetic parameters of the said active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the patient's weight, the patient's immune status, the route of administration, etc. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be proportionally reduced.

[0032] In the fourth aspect, the present application proposes the use of the CAR-T cells described in the second aspect or the drug described in the third aspect in the prevention and / or treatment of tumors. Applying the foregoing CAR-T cells or drugs to the prevention and / or treatment of tumors can effectively improve the tumor killing efficiency, control the tumor growth rate, reduce the toxic and side effects, and prolong the survival time of patients / animals.

[0033] In some examples of the present application, the foregoing tumors include: melanoma, hematological tumors, liver cancer, and tumors treated by CAR-T cells targeting other antigens. It should be understood that tumors corresponding to the tumor antigens targeted by CAR are within the application scope of the present application, including but not limited to hematological tumors and solid tumors.

[0034] In the fifth aspect, the present application proposes a tumor treatment method. According to the embodiments of the present application, the foregoing method includes: administering an effective dose of CAR-T cells to a patient; and / or after administering an effective dose of CAR-T cells, administering SA and / or SA analogs by intravenous injection, wherein the foregoing CAR-T cells are obtained by culturing through the method of the first aspect. Adopting the CAR-T cell therapy can effectively improve the tumor killing efficiency, control the tumor growth rate, reduce the toxic and side effects, and prolong the survival time of patients / animals.

[0035] It can be understood that the tumor treatment method of the present application is the CAR-T cell therapy, which specifically includes processes such as T cell collection, gene modification, cell expansion, and reinfusion treatment.

[0036] In some examples of the present application, after administering an effective dose of CAR-T cells, the dose of SA and / or SA analogs administered by intravenous injection is 500 μg / kg - 700 μg / kg, optionally 500 μg / kg, 510 μg / kg, 520 μg / kg, 530 μg / kg, 540 μg / kg, 550 μg / kg, 560 μg / kg, 570 μg / kg, 580 μg / kg, 590 μg / kg, 600 μg / kg, 610 μg / kg, 620 μg / kg, 630 μg / kg, 640 μg / kg, 650 μg / kg, 660 μg / kg, 670 μg / kg, 680 μg / kg, 690 μg / kg or 700 μg / kg. It can be 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, etc. after administering the effective dose of CAR-T cells.

[0037] In some examples of the present application, the aforementioned effective dose of CAR-T cells can be determined based on the patient's body weight, tumor severity or tumor stage.

[0038] Additional aspects and advantages of the present application will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] 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 in the following description are only some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0040] Figure 1A Schematic diagram of the treatment process of SA-treated CD19-CART cells in a Raji (human Burkitt's lymphoma cell) humanized animal model provided by an embodiment of the present application;

[0041] Figure 1B Schematic diagram of the survival rate analysis results provided by an embodiment of the present application;

[0042] Figure 1C Schematic diagram of the body weight analysis results of tumor-bearing mice after CART treatment provided by an embodiment of the present application;

[0043] Figure 1D Schematic diagram of the comparison results of the area under the body weight change curve provided by an embodiment of the present application;

[0044] Figure 2ASchematic diagram of the killing effect treatment process of SA-treated OT1 CD8+ T cells on tumor cells provided by an embodiment of the present application;

[0045] Figure 2B Schematic diagram of the flow cytometry results of the Dmso control group and the SA treatment group provided by an embodiment of the present application;

[0046] Figure 2C Schematic diagram of the killing effect of CD8+ T cells in the Dmso control group and the SA treatment group on melanoma provided by an embodiment of the present application;

[0047] Figure 3A Schematic diagram of the tumor killing ability treatment process of SA-treated OT1 CD8+ T cells in a melanoma animal model provided by an embodiment of the present application;

[0048] Figure 3B Schematic diagram of the tumor volume change results provided by an embodiment of the present application;

[0049] Figure 3C Schematic diagram of the tumor weight comparison results of the blank control group, the Dmso control group and the SA treatment group provided by an embodiment of the present application;

[0050] Figure 4A Schematic diagram of the release results of SA and its structural analogs treatment effector factors provided by an embodiment of the present application;

[0051] Figure 4B Schematic diagram of the release comparison results of SA and its structural analogs treatment effector factors provided by an embodiment of the present application;

[0052] Figure 4C Schematic diagram of the release comparison results of perforin treated with SA and its structural analogs provided by an embodiment of the present application;

[0053] Figure 4D Schematic diagram of the release comparison results of granzyme B treated with SA and its structural analogs provided by an embodiment of the present application. Detailed implementation manners

[0054] The embodiments of the present invention are described in detail below, and the examples of the embodiments are shown in the drawings. Wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the drawings are exemplary and are intended to explain the present invention and should not be construed as limiting the present invention.

[0055] In the process of describing the present invention, the relevant terms in this article are explained and illustrated. These explanations and illustrations are only for the convenience of understanding the solution and should not be regarded as a limitation on the protection solution of the present invention.

[0056] In this text, the term "comprising" or "including" is an open-ended expression, that is, it includes the content specified in the present invention, but does not exclude other aspects of the content.

[0057] In this text, the terms "optionally", "optional" or "option" generally mean that the subsequent described event or condition can but does not necessarily occur, and this description includes the cases where the event or condition occurs, as well as the cases where the event or condition does not occur.

[0058] In the present application, "CAR-T cells" are a type of specifically anti-tumor immune effector cells genetically engineered. These cells are derived from T lymphocytes of patients or healthy donors, and by expressing chimeric antigen receptors (CARs) on their cell surfaces, they can specifically recognize target antigens on the surface of tumor cells that are independent of MHC presentation, and directly activate the killing mechanism after recognition, including releasing perforin, granzyme and cytokines, thereby precisely killing tumor cells. Without genetic modification, T cells recognize antigens on the surface of tumor cells through the T cell receptor (TCR), and these antigens are presented on MHC-I molecules after being processed by abnormal proteins inside the tumor cells. When the TCR binds to the MHC-I / antigen complex, the T cells are activated and exert cytotoxic effects, releasing perforin and granzyme to kill tumor cells. By genetically engineering the method of introducing the CAR gene capable of recognizing tumor antigens into T cells, the T cells have obtained the ability to directly recognize and kill tumor cells.

[0059] In the present application, "sphingolipid metabolites" refer to various derivatives produced through the metabolic pathways of sphingolipids (such as sphingomyelin, ceramide, glycosphingolipid) in cells, including dihydrosphingosine, 3-ketodihydrosphingosine, dihydroceramide, ceramide, sphingosine, sphingosine-1-phosphate or their analogs.

[0060] In the present application, "pharmaceutically acceptable excipients" can include any solvents, solid excipients, diluents or other liquid excipients, etc., suitable for the specific target dosage form. Except for the scope where any conventional excipients 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 uses are also within the scope considered in the present application.

[0061] In the present application, "administering" refers to introducing a predetermined amount of a substance into a patient by a suitable means. 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 intended tissue. Various modes of administration are contemplated, including intraperitoneal, intravenous, intramuscular, subcutaneous injection, etc., but the present invention is not limited to the exemplified modes of administration. Preferably, the composition of the present invention is administered by intravenous injection or subcutaneous injection.

[0062] In the present application, "treatment" is used to refer to obtaining the desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or 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 disorder in an individual who is susceptible to the disease but has not been diagnosed with the disease; (b) inhibiting a disease, such as arresting the progression of the disease; or (c) alleviating a disease, such as reducing the symptoms associated with the disease. "Treatment" as used herein covers any administration of a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the disease of the individual, including but not limited to administering a drug containing the compound described herein to an individual in need thereof.

[0063] In the present application, "effective amount" or "effective dose" refers to an amount that can produce a function or activity in humans and / or animals and is acceptable to humans and / or animals.

[0064] Embodiments of the present application will be described in more detail below, and 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 explain the present application and should not be construed as limiting the present application. Reagents or instruments not indicated by the manufacturer can be obtained as conventional products through commercial purchase.

[0065] Example 1: SA-treated CD19-CART cells showed stronger tumor killing effect in a Raji (human Burkitt's lymphoma cell) humanized animal model

[0066] Isolate PBMC

[0067] The T cells used in this example were derived from human peripheral blood, and peripheral blood mononuclear cells (PBMC) were isolated using the following steps and methods:

[0068] 1. Preparation of the laboratory: Before entering the laboratory, the air conditioner in the laboratory should be turned on half an hour in advance. The biosafety cabinet needs to be disinfected with ultraviolet light for 30 minutes before use, and the workbench should be wiped with 75% alcohol before use. After the biosafety cabinet is turned on for 20 minutes, it can be determined that it is operating normally before work can be carried out.

[0069] 2. Dilute the fresh anticoagulated blood of the donor with an equal volume of tissue diluent to dilute the whole blood, and mix well.

[0070] 3. Add 5 ml of human peripheral lymphocyte separation solution (Solarbio, Cat.No.P8610) to a 15 ml centrifuge tube, and carefully spread the diluted donor whole blood on top of the separation solution with a Pasteur pipette.

[0071] 4. Centrifuge at room temperature, 1000 g with a horizontal rotor for 30 minutes. After centrifugation, there will be an obvious stratification: the top layer is the plasma layer, and the middle layer is the separation solution layer. Aspirate the white film layer (lymphocytes) between the two layers into a new centrifuge tube, and resuspend the white film layer cells with 10 ml of PBS. Mix the well-mixed cells, and centrifuge at 500 g at room temperature for 10 minutes.

[0072] 5. Resuspend the separated PBMC with PBS and adjust the cell concentration to 10 8 cells / ml. Add 10 μl of CD8 nanobeads (Genescript, L00864) to every 10 7 cells, mix well with the cells and incubate at 4°C for 15 minutes. Continue to add 2 ml of PBS to the incubated cell suspension, mix well, centrifuge at 500 g at room temperature for 10 minutes, and remove the supernatant. Resuspend the precipitated cells in 500 μl of PBS.

[0073] 6. Place the gL cell sorting column (Genescript, D00008) on the magnetic sorting rack (Genescript, D00012) and pre-wash the sorting column with 3 ml of PBS. Add the cells obtained in step 5 to the sorting column and let the liquid pass through the sorting column completely by gravity. After all the cell suspension has passed through the sorting column, wash the column 3 times with 3 ml of PBS to fully remove the cells not labeled with magnetic beads.

[0074] 7. Remove the sorting column from the magnetic rack and place it on a new centrifuge tube. Add 5 ml of PBS to the sorting column and quickly wash the magnetic bead-labeled cells into the centrifuge tube with a plunger. In the obtained cell suspension, take a small amount of cells for counting, and label the CD8+ T cells with percp-cy5.5 fluorescein-labeled anti-human CD8a antibody, and detect the purity of CD8+ T cells using a Fortessa flow cytometer. Usually, the purity is about >95%.

[0075] Preparation of CD19 CART cells

[0076] 1. CD8+ T cell culture and activation: Aspirate approximately 1x10 6 cells of purified CD8+ T cells, centrifuge at 500 g for 5 minutes at 4°C, and discard the supernatant. Resuspend the cells using OptiVitro® Serum-Free T Cell Medium (ExCell Bio, TE000-N022) containing IL-2 (100 IU / ml), and adjust the final cell density to 1x10 6 cells / ml. Stimulate and activate the T cells using magnetic beads coated with CD3 / CD28 antibodies (Miltenyi biotec, 130-111-160).

[0077] 2. After 48 hours of CD8+ T cell activation, mix the cells, take 20 μl for counting, and transduce 10 MOI of lentivirus containing the CAR structure targeting the FMC63 epitope of CD19 protein according to the cell number (CART plasmid: pCDH-EF1a-FMC63-BBz-EGFP; lentiviral packaging plasmids: pMDLg-pRRE, pMD2.G, pRSV-Rev).

[0078] SA treatment of CD19 CART cells

[0079] 1. 48 hours after lentiviral transduction of CD8+ T cells, aspirate 20 μl of cells for counting, and detect the proportion of EGFP-positive cells in the cells by Fortessa flow cytometer to determine the CAR transduction efficiency.

[0080] 2. Centrifuge the cell suspension after counting at 500 g for 5 minutes at 4°C, and discard the supernatant. Resuspend the cells using OptiVitro® Serum-Free T Cell Medium containing IL-2 (100 IU / ml) and dilute to 0.5x10 6 cells / ml.

[0081] 3. Divide the diluted cells into two equal parts, and add DMSO or SA (4.2 μM) for treatment respectively. Then change the cell medium every 48 hours, and add DMSO or SA (4.2 μM) for treatment each time when changing the medium until day 15. Harvest 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.

[0082] Determination of the validity of experimental results

[0083] Reference Figures 1A - 1D , the effect of CD19-CART cells in controlling lymphoma is significant, and it can significantly improve the survival time of tumor-bearing mice. From Figure 1BAs a result, it can be seen that the CD19-CART cells treated with SA have a more significant killing effect on Raji lymphoma, not only prolonging the survival time of tumor-bearing mice, but also significantly increasing the survival rate of tumor-bearing mice. At the same time, we detected the body weight of tumor-bearing mice. Figure 1C and Figure 1D The results suggest that compared with the gradual decrease in the body weight of tumor-bearing mice after infusion of untreated CD19-CART cells, the body weight of tumor-bearing mice treated with SA-treated CD19-CART cells did not show a significant decrease after infusion, indicating that the infusion treatment with SA-treated CD19-CART cells has better safety. The addition of the natural metabolite SA not only does not impose an additional burden on the body, but may also improve the toxic side effects during the CD19-CART cell treatment process.

[0084] 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 experiments

[0085] Isolation and purification of CD8+ T cells from C57 / BL6 mice

[0086] In this example, the T cells are derived from the spleen of OT1 mice (OT1 is a TCR transgenic mouse, and its CD8+ T cells can specifically recognize the 257-264 amino acid antigen epitope of ovalbumin OVA presented by MHC-1, which is generally used to study the effector function of specific CD8+ T cells). The purified CD8+ T cells are isolated using the following steps and methods.

[0087] 1. Preparation of the laboratory: Half an hour before entering the laboratory, the air conditioner in the laboratory should be turned on in advance. The biosafety cabinet needs to be disinfected with ultraviolet light for 30 minutes before use. Wipe the workbench with 75% alcohol before use. After the biosafety cabinet is turned on for 20 minutes, it can be determined that it is operating normally before work can be carried out.

[0088] 2. After decapitating and sacrificing the mice, take 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 lysate, lyse at room temperature for 1 minute, add 10ml 1% FBS 1640, and centrifuge at 500g for 5 minutes.

[0089] 3. After centrifugation, discard the supernatant, resuspend the cells in MACS buffer (PBS + 0.5% FBS + 2mM EDTA), and resuspend every 1x10 7 cells with 40μl MACS buffer. Every 1x10 7Add 10 μl of purified antibody mixture (Militenyi, 130 - 104 - 075) to the cells and incubate at 4°C for 5 minutes. Add 3 ml of MACS buffer, centrifuge to remove the supernatant, resuspend the cells in MACS buffer, and add 80 μl of MACS buffer per 1x10 7 cells. Add 20 μl of Anti - Biotin Microbeads (Militenyi, 130 - 104 - 075) per 1x10 7 cells and incubate at 4°C for 10 minutes.

[0090] 4. Place the gL cell sorting column (Genescript, D00008) on the magnetic sorting rack (Genescript, D00012) and pre - rinse the sorting column with 3 ml of MACS buffer. Add the cells obtained in step 3 to the sorting column and let the liquid flow completely through the sorting column by gravity into the collection tube. After all the cell suspension has passed through the sorting column, rinse the sorting column once with 3 ml of MACS buffer. The cells in the collection tube are the purified CD8+ T cells.

[0091] 5. In the obtained cell suspension, take a small amount of cells for counting, and label the CD8+ T cells with percp - cy5.5 fluorescein - labeled anti - mouse CD8a antibody. Use a BD Fortessa flow cytometer to detect the purity of CD8+ T cells, and usually the purity is about >95%.

[0092] SA treatment of OT1 CD8+ T cells

[0093] 1. Pre - coat a 96 - well plate (round bottom, tissue culture - treated) with PBS containing the concentration of Anti - CD3 / 28 antibodies at 37°C for 2 hours, so that the working concentration of Anti - CD3 antibody in the medium is 5 μg / ml and the working concentration of Anti - CD28 antibody is 2 μg / ml.

[0094] 2. Dilute the purified CD8+ T cells with complete medium (1640 + 10% FBS + double antibodies + non - essential amino acids + β - mercaptoethanol), adjust the cell concentration to 1x10 6 cells / ml, add 10 ng / ml IL2 to the cells, and treat the cells with DMSO or SA (4.2 μM) respectively. Add the cells to the 96 - well plate at 200 μl / well.

[0095] 3. Stimulate and treat the cells for 48 hours

[0096] Co - culture of SA - treated OT1 CD8+ T cells with melanoma B16 - OVA

[0097] 1. Culture B16-OVA cells (melanoma, ovalbumin gene-modified, specifically recognizable by OT1 CD8+ T cells) using DMEM complete medium (DMEM + 10% FBS + penicillin-streptomycin) + Blasticidin (used to maintain the expression of OVA in B16 cells). When the cell confluence reaches 70% - 80%, digest the cells, centrifuge them, and resuspend the cells using 1640 complete medium. Adjust the cell concentration to 1x10 5 cells / ml, and add the cells into a 96-well round-bottom plate at 100 μl / well.

[0098] 2. After the B16-OVA cells have grown for 18 hours, aspirate the OT1 CD8+ T cells treated with DMSO or SA in the 96-well plate for 48 hours, centrifuge at 500g for 5 minutes, discard the supernatant, resuspend the cells using fresh 1640 complete medium, and adjust the cell density to 2x10 5 cells / ml. Add the cells into the 96-well plate containing the cultured B16-OVA cells at 100 μl / well for co-culture.

[0099] 3. After 24 hours of cell co-culture, use a BD Fortessa flow cytometer to detect the viability of tumor cells and the release of T cell effector factors. Tumor cell viability staining strategy: LIVE / DEAD™ Fixable Red (Invitrogen, L23102), CD45 BV785, CD8 Percp-cy5.5. Detect the LIVE / DEAD-positive cells in the CD45-negative population of tumor cells. This population of cells is the tumor cells killed by T cells. Cell effector factor release detection strategy: Stimulate the cells with PMA + Ionomycin + BFA for 4 hours and then stain the cells with antibodies. The staining strategy is as follows: LIVE / DEAD™ Fixable Red, CD8 Percp-cy5.5, CD44 FITC, PD1 BV785, IFNγ PE / CY7, TNFα PE, Perforin APC, Granzyme B APC / CY7, IL-2 BV421. Use flow cytometry to detect the release of effector factors in the CD8+CD44+PD1+ subset.

[0100] Detection of killing results

[0101] Reference Figures 2A - 2C From the results of the co-culture of OT1 CD8+ T cells and melanoma B16-OVA, it can be seen that the killing effect of OT1 CD8+ T cells treated with SA on tumors is significantly increased. Figure 2BTaking the medium effector-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 result is a concrete manifestation of the killing effect of OT1 CD8+ T cells. The killing effect of OT1 CD8+ T cells on melanoma continuously increases with the increase of the effector-to-target ratio.

[0102] Example 3: OT-1 CD8+ T cells treated with SA showed stronger tumor killing ability in a melanoma animal model

[0103] Subcutaneous tumor implantation in C57 / BL6 mice

[0104] 1. Culture B16-OVA cells using DMEM complete medium (DMEM + 10% FBS + double antibiotics) + Blasticidin. When the cell confluence reaches 70% - 80%, digest the cells, centrifuge them, and wash the cells 2 times with 1640 medium to remove the residual serum. After the last wash, resuspend the cells, count them, and adjust the cell concentration to 1x10 6 cells / ml using 1640 medium.

[0105] 2. Anesthetize C57 / BL6 mice using an isoflurane gas anesthesia machine (RWD, R530IE) (isoflurane, RWD R510), and adjust the concentration to 1% - 1.5% to maintain the anesthesia state of the mice. Use a syringe to aspirate 200 μl of B16-OVA tumor cell suspension (2x10 5 cells), puncture into the area above the right lower groin of the mouse, slowly push the syringe to let the cell suspension enter the subcutaneous tissue, withdraw the syringe, turn off the anesthesia evaporator, and keep the mouse breathing in pure oxygen for 5 minutes to facilitate the rapid awakening of the mouse.

[0106] 3. Observe and measure the tumor growth situation every day from the 4th day until the 8th day.

[0107] SA treatment of OT1 CD8+ T cells

[0108] 1. Coat a 96-well plate (round bottom, tissue culture treated) with PBS containing a concentration of Anti-CD3 / 28 antibodies in advance at 37°C for 2 hours, so that the working concentration of Anti-CD3 antibody in the medium is 5 μg / ml and the working concentration of Anti-CD28 antibody is 2 μg / ml.

[0109] 2. Dilute the purified CD8+ T cells using complete medium (1640 + 10% FBS + double antibiotics + non-essential amino acids + β-mercaptoethanol), and adjust the cell concentration to 1x10 6Add 10 ng / ml IL2 to the cells, and treat the cells with DMSO or SA (4.2 μM) respectively. Add the cells to a 96-well plate at 200 μl / well.

[0110] 3. The treatments in Step 1 and Step 2 last for 48 hours.

[0111] Adoptive transfer of OT1 CD8+ T cells into tumor-bearing mice

[0112] 1. Randomly divide the tumor-bearing mice into 3 groups according to tumor size: 1. Control group, no cell injection; 2. DMSO group, inject OT1 cells treated with DMSO; 3. SA group, inject OT1 cells treated with SA.

[0113] 2. Aspirate OT1 CD8+ T cells treated with DMSO or SA for 48 hours from the 96-well plate, centrifuge at 500 g for 5 minutes, discard the supernatant, wash the cells 2 times with 1640 medium to remove residual serum. After the last wash, resuspend the cells, count them, and adjust the cell concentration to 4x10 6 cells / ml with 1640 medium.

[0114] 3. Place the tumor-bearing mice in a fixator, aspirate 200 μl of OT1 CD8+ T cells (8x10 5 cells) with a syringe, puncture into the mouse tail vein, slowly push the syringe to let the cell suspension enter the mouse body, withdraw the syringe, and press the mouse tail with an alcohol cotton ball for 2 minutes to stop bleeding.

[0115] 4. Measure the tumor volume of the mice on the 8th, 10th, 12th, 14th, 16th, and 18th days after T cell transfer, record and make a tumor growth curve.

[0116] 5. Sacrifice the mice 10 days after adoptive transfer of OT1 CD8+ T cells, isolate the subcutaneous tumors, and weigh the tumor weights.

[0117] Detection of tumor killing results:

[0118] Refer to Figures 3A - 3C , from Figure 3B The statistical tumor growth curve shows that compared with the OT1 CD8+ T cell treatment group treated with DMSO, the OT1 CD8+ T cells treated with SA show a stronger effect of inhibiting tumor growth, the tumor growth rate is significantly slowed down, and there is a significant statistical difference between the two groups; 10 days after adoptive transfer, the mice are sacrificed, and comparing the tumor weights ( Figure 3C ) can also clearly show that the tumor weight is reduced and the growth is significantly controlled.

[0119] Example 4: Effects of SA and Its Structural Analogs on the Anti-tumor Effect of OT-1 CD8+T Cells in vitro

[0120] Isolation and Purification of CD8+T Cells from C57 / BL6 Mice

[0121] In this example, T cells were derived from the spleens of OT1 mice (OT1 is a TCR transgenic mouse, and its CD8+T cells can specifically recognize the 257-264 amino acid epitope of ovalbumin OVA presented by MHC-1, and are generally used to study the effector functions of specific CD8+T cells). The purified CD8+T cells were isolated using the following steps and methods, which were the same as the isolation and purification of C57 / BL6 mouse CD8+T cells in Example 2.

[0122] Treatment of OT1 CD8+T Cells with SA and Its Structural Analogs

[0123] 1. Coat a 96-well plate (round bottom, tissue culture-treated) with PBS containing a concentration of Anti-CD3 / 28 antibodies in advance at 37°C for 2 hours, so that the working concentration of Anti-CD3 antibody in the culture medium is 5 μg / ml and the working concentration of Anti-CD28 antibody is 2 μg / ml.

[0124] 2. Dilute the purified CD8+T cells with complete medium (1640 + 10% FBS + double antibodies + non-essential amino acids + β-mercaptoethanol), adjust the cell concentration to 1x10 6 cells / ml, add 10 ng / ml IL2 to the cells, and treat the cells with DMSO, SA (4.2 μM), 3-keto Sphinganine (4.2 μM), C16 Dihydroceramide (50 nM), C18 Ceramide (50 nM), Sphingosine (4.2 μM), and Sphingosine-1-P (5 μM) respectively. Add the cells to the 96-well plate at 200 μl / well.

[0125] 3. Stimulate and treat the cells for 48 hours

[0126] Co-culture of OT1 CD8+T Cells Treated with SA and Its Structural Analogs with Melanoma B16-OVA

[0127] 1. Culture B16-OVA cells (melanoma, ovalbumin gene-modified, which can be specifically recognized by OT1 CD8+T cells) with DMEM complete medium (DMEM + 10% FBS + double antibodies). When the cell confluence reaches 70%-80%, digest, centrifuge, and resuspend the cells with 1640 complete medium, and adjust the cell concentration to 1x105 cells / ml, and add the cells into a 96-well round-bottom plate at 100 μl / well.

[0128] 2. After the B16-OVA cells have grown for 18 hours, aspirate the OT1 CD8+ T cells treated with DMSO, SA or its analogues for 48 hours from the 96-well plate, centrifuge at 500 g for 5 minutes, discard the supernatant, resuspend the cells with fresh complete 1640 medium, and adjust the cell density to 2×10 5 cells / ml, and add the cells into the 96-well plate for culturing B16-OVA cells at 100 μl / well for co-culture.

[0129] 3. After 24 hours of cell co-culture, use a BD Fortessa flow cytometer to detect the viability of tumor cells and the release of T cell effector factors. Strategy for staining cell viability: LIVE / DEAD™ Fixable Red (Invitrogen, L23102), CD45 BV785, CD8 Percp-cy5.5, detect the LIVE / DEAD positive cells in the CD45-negative population of tumor cells, and this population of cells is the tumor cells killed by T cells. Strategy for detecting the release of cell effector factors: Stimulate the cells with PMA + Ionomycin + BFA for 4 hours and then stain the cells with antibodies. The staining strategy is as follows: LIVE / DEAD™ Fixable Red, CD8 Percp-cy5.5, CD44 FITC, PD1 BV785, IFNγ PE / CY7, TNFα PE, Perforin APC, Granzyme B APC / CY7, IL-2 BV421, and use flow cytometry to detect the release of effector factors in the CD8+CD44+PD1+ subset.

[0130] Judgment of the killing results of OT1 CD8+ T cells treated with SA and its structural analogues:

[0131] In this example, through flow cytometry analysis Figure 4A and Figure 4B it can be seen that after activated OT1 CD8+ T cells are treated with small molecule compounds such as dihydroceramide and sphingosine (SO), the proportion of effector CD8+ T cells that simultaneously express IFNγ and TNFα increases significantly, indicating that the effector function of immune cells is enhanced and the killing effect on tumor cells is more significant. Figure 4C It is indicated that the number of CD8+ T cells secreting perforin increases significantly after treatment with small molecule compounds such as 3-ketodihydrosphingosine (3KDS) and ceramide. Figure 4D, Treatment with SA and its structural analogues had no significant effect on the production of granzyme B in CD8+ T cells. IFNγ, TNFα, and perforin are all major cytokines for CD8+ T cells to exert effector functions, and increased secretion is crucial for enhancing the tumor-killing effect of CD8+ T cells.

[0132] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include at least one of such features. In the description of the present invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0133] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection 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 a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0134] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to 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; Wherein, the sphingolipid metabolite is dihydrosphingosine.

2. The method according to claim 1, 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.

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

4. The method according to claim 3, characterized in that The time of the first culture, the second culture and the third culture is selected from 30-60 hours.

5. The method according to any one of claims 2 to 4, characterized in that: The predetermined concentration is selected from 3 μM-5 μM.

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