Culture medium for car-t cell culture and application thereof

Abstract

The application discloses a culture medium for CAR-T cell culture and application thereof, and relates to the fields of immunology, molecular biology and cell engineering. The culture medium comprises a basic culture medium and dihydroeugenol. It is found in research that by adding dihydroeugenol in the culture medium, the proportion of central memory T cells of CAR-T cells can be effectively improved, and the expression levels of PD1 and LAG3 can be reduced. In addition, dihydroeugenol can also promote the secretion of IL2, TNF and INFgamma, and enhance the killing function of CAR-T cells. Experiments show that the ability of D-CAR-T to secrete IL2, TNF and INFgamma is significantly higher than that of ordinary CAR-T, and D-CAR-T also has a higher T CM cell proportion and a lower exhaustion level after performing the killing function.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunology, molecular biology and cell engineering, in particular to a culture medium for CAR-T cell culture and application thereof. BACKGROUND

[0002] In recent years, chimeric antigen receptor T cell (CAR-T) therapy has made a breakthrough in the treatment of hematological tumors. However, factors such as tumor microenvironment inhibition, CAR-T cell exhaustion and terminal differentiation affect the therapeutic effect of CAR-T. The characteristics of exhausted CAR-T cells include reduced cytokine secretion, reduced proliferation ability and sustained high expression of various inhibitory receptors (including PD-1, TIM-3, LAG-3, etc.). In addition, studies have found that the proportion of memory-like CAR-T cells in CAR-T products is crucial to the anti-tumor efficacy of CAR-T cells and the prognosis of patients. Although T cell products dominated by effector cells have greater cytotoxic potential, these cells are also prone to terminal differentiation and become dysfunctional (Ando, M., Ito, M., Srirat, T., Kondo, T. & Yoshimura, A. Memory T cell, exhaustion, and tumor immunity. Immunol. Med. 43, 1-9 (2020).), and T cells with and maintaining less differentiated phenotype (including memory T cells and precursor T cells) show better therapeutic effect due to stronger proliferation ability and persistence.

[0003] Diosmetin (molecular formula C16H1206, CAS number 520-34-3) is a natural flavonoid compound, which is considered as an active ingredient of Chinese herbal medicine and widely exists in natural plants and edible fruits. Flavonoids exhibit various pharmacological activities, such as antioxidant, anti-inflammatory, antibacterial, antitumor and antiviral activities. Studies have found that diosmetin can inhibit the Akt Ser473 phosphorylation level and down-regulate the Akt signaling pathway (Zhijie X, Yuanliang Y, Lingfang X, et al. Radiosensitizing effect of diosmetin on radioresistant lung cancer cells via Akt signaling pathway. Plos One, 2017, 12(4): e0175977.), and inhibition of Akt at the initial stage of CAR-T preparation can improve the CAR-T positive expression rate, memory phenotype and in vivo efficacy (Qing Z, Jiage D, Shishuo S, et al. Akt inhibition at the initial stage of CAR-T preparation enhances the CAR-positive expression rate, memory phenotype and in vivo efficacy. American journal of cancer research, 2019, 9(11): 2379-2396.). SUMMARY

[0004] In order to overcome the shortcomings of the prior art, one of the purposes of the present application is to provide a culture medium for CAR-T cell culture.

[0005] The second purpose of the present application is to overcome the shortcomings of CAR-T cell exhaustion and terminal differentiation in the prior research scheme, and to provide a CAR-T cell preparation and culture method for improving the proportion of memory cells and reducing the exhaustion level. The D-CAR-T (Diosmetin-CAR-T) cells prepared and cultured by the method have lower receptor expression level, higher proportion of memory T cells, and can secrete higher level of killing factors.

[0006] One of the purposes of the present application is achieved by adopting the following technical scheme:

[0007] The present application provides a culture medium for culturing CAR-T cells, which comprises a basic culture medium and diosmetin; the concentration of diosmetin is 1-4 μM.

[0008] Preferably, the concentration of diosmetin is 2-4 μM.

[0009] More preferably, the concentration of the artemisinic acid is 2 μM.

[0010] The base medium comprises, in terms of volume percentage, 87.9% of RPMI-1640 medium, 10% of fetal bovine serum, 1% of 100 U / mL of penicillin, 1% of 100 μg / mL of streptomycin and 0.1% of 200 U / mL of interleukin 2.

[0011] Interleukin (IL-2) plays an important role in the manufacturing process of CAR-T cells, can stimulate cell proliferation and maintain cell viability in the expansion stage, and is a common reagent for culturing CAR-T.

[0012] The application provides application of the medium in culturing CAR-T cells.

[0013] The application provides a CAR-T cell construction and culturing method for non-disease treatment purposes, comprising the following steps:

[0014] (1) T cells are separated from human peripheral blood, and the T cells are transfected by using a lentivirus containing a CAR gene expression sequence to prepare CAR-T cells;

[0015] (2) The CAR-T cells prepared in step (1) are cultured by using the medium.

[0016] Reagent kit cytokine release, detection of cell T CM In the case of experimental research on the proportion and detection of cell exhaustion indicators, etc., the CAR-T cells are non-disease treatment.

[0017] Preferably, the concentration of the CAR-T cells in the medium is 1-10×10 5 / mL.

[0018] More preferably, the concentration of the CAR-T cells in the medium is 2×10 5 / mL.

[0019] Specifically, in step (2), the culturing is started from the 5th to 12th day after infection; and the frequency of replacing the medium is once every 1-3 days.

[0020] Preferably, the transfection agent in step (1) is a polybrene transfection agent.

[0021] It is found in the research that by adding artemisinic acid in the medium, the Central Memory T cell (T CM) ratio, PD1 and LAG3 exhaustion indicators were reduced. After performing the killing function, the cells treated with Lariciresinol still had higher T CM proportion and lower PD1 expression levels. In addition, Lariciresinol treatment can also promote the secretion of IL2, TNF and INFγ, enhance the anti-tumor function of CAR-T cells. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The T cell proportion of CD19-CD28z-CAR-T cells treated with different concentrations of Lariciresinol for 3 days is shown in Figure 2A. The PD1 expression level of CD19-CD28z-CAR-T cells treated with different concentrations of Lariciresinol for 3 days is shown in Figure 2B. The LAG3 expression level of CD19-CD28z-CAR-T cells treated with different concentrations of Lariciresinol for 3 days is shown in Figure 2C. CM The proportion increased; single factor ANOVA analysis and Bonferroni test were used, **** means p<0.0001.

[0023] Figure 2 The PD1 and LAG3 expression levels of CD19-CD28z-CAR-T cells treated with different concentrations of Lariciresinol for 3 days are shown in Figure 3. Among them, Figure 2 A is the PD1 expression level, single factor ANOVA analysis and Tukey's test are used, ** means p<0.01, **** means p<0.0001; Figure 2 B is the LAG3 expression level; K independent sample non-parametric test is used, * means p<0.05.

[0024] Figure 3 The T cell proportion of CD19-CD28z-CAR-T cells treated with different concentrations of Lariciresinol for 2 days, and then co-cultured with Nalm6 tumor cells for 1 day is shown in Figure 4A. The PD1 and LAG3 exhaustion indicators of CD19-CD28z-CAR-T cells treated with different concentrations of Lariciresinol for 2 days, and then co-cultured with Nalm6 tumor cells for 1 day are shown in Figure 4B. CM The proportion increased; single factor ANOVA analysis and Bonferroni test were used, * means p<0.05, ** means p<0.01, *** means p<0.001, **** means p<0.0001. CM The proportion increased; single factor ANOVA analysis and Bonferroni test were used, * means p<0.05, ** means p<0.01, *** means p<0.001, **** means p<0.0001.

[0025] Figure 4 The influence of Lariciresinol on the secretion of different cytokines of CD19-CD28z-CAR-T cells treated with different concentrations of Lariciresinol for 3 days, and then co-cultured with Nalm6 tumor cells for 1 day is shown in Figure 5. Among them, A is the influence of Lariciresinol on the secretion of cytokine IL2; B is the influence of Lariciresinol on the secretion of cytokine TNF; C is the influence of Lariciresinol on the secretion of cytokine IFNγ; all use single factor ANOVA analysis and Bonferroni test, * means p<0.05, ** means p<0.01, *** means p<0.001, **** means p<0.0001. DETAILED DESCRIPTION

[0026] The following detailed description of the application is provided for the purpose of illustrating, not limiting, the present application. The specific conditions not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not specified by the manufacturer are all conventional products that can be purchased on the market.

[0027] In the first aspect, the present application provides a method for preparing D-CAR-T cells, which comprises culturing conventional CAR-T cells in the presence of diterpenes to obtain D-CAR-T cells, wherein the D-CAR-T cells have a higher proportion of T CM , lower exhaustion level and stronger cytokine secretion capacity.

[0028] According to the present application, the addition time of diterpenes can be selected within a wide range. Preferably, the diterpenes are added continuously on days 5-12 of CAR-T cell culture, for example, on day 7.

[0029] According to the present application, the culturing time of CAR-T cells in diterpenes can also be selected within a wide range. Preferably, the culturing time of CAR-T cells in diterpenes is 72 h, which can significantly improve the function of CAR-T.

[0030] According to the present application, the amount of diterpenes can be selected within a wide range. Preferably, the amount of diterpenes in the culture medium is such that the concentration is 1-8 μM, for example, 2 μM.

[0031] According to the present application, the CAR-T cells to be treated can be any CAR-T cells in the art, which can be single-target CAR-T cells and / or multi-target CAR-T cells. Preferably, the CAR-T cells are selected from CD19-CAR-T cells, CD20-CAR-T cells, CD22-CAR-T cells, CD20 / CD19-CAR-T cells.

[0032] In the second aspect, the present application provides D-CAR-T cells prepared by the above method.

[0033] According to the present application, the D-CAR-T cells prepared using diterpenes have a significantly increased proportion of T CM cells in the cells, a reduced exhaustion level; the D-CAR-T cells secrete more cytokines, including IL2, TNF and INFγ, during the killing process, and have a higher proportion of TCM and a lower exhaustion level after performing the killing function.

[0034] In the third aspect, the present application provides the use of the above D-CAR-T cells in the preparation of tumor treatment preparations.

[0035] In a fourth aspect, the present application provides the potential application value of the dimeric proanthocyanidin in CAR-T clinical treatment. Any CAR-T clinical treatment using the dimeric proanthocyanidin as an auxiliary medicament for treatment shall be included in the present application.

[0036] The type of the tumor to be treated can be selected according to different CAR-T cell types, which are well known to those skilled in the art and will not be described in detail here.

[0037] Hereinafter, the present application will be described in detail through examples.

[0038] The CBA Flex Set kit was purchased from BD Company, USA.

[0039] The Bright-GloTM Luciferase Assay system was purchased from Promega Company, USA.

[0040] The HEK293T cells and the ALL cell line Nalm6 were introduced and preserved by Shanghai Cell Institute, Chinese Academy of Sciences.

[0041] The polyethyleneimine acid salt (PEI) was purchased from Polysciences Company, USA.

[0042] The streptomycin mixture (100X) was purchased from Solabio Technology Co., Ltd., Beijing.

[0043] The RPMI-1640 medium was purchased from Coming Company, USA.

[0044] The DMEM (High Glucose) medium was purchased from Coming Company, USA.

[0045] The fetal bovine serum (FBS) was purchased from GIBCO Company, USA.

[0046] The Ficoll lymphocyte separation medium was purchased from Haoyang Biotech Co., Ltd., Tianjin.

[0047] The IL-2 was purchased from Peprotech Company, USA.

[0048] The plasmids CD28z, psPAX2 and pMD2.G were preserved by Hematology Institute, Zhejiang University.

[0049] The anti-CD3 / CD28 magnetic beads were of clinical research level and were purchased from Thermo Company, USA, CAT#40203D.

[0050] The polybrene was purchased from Sigma-Adrich Company, USA.

[0051] Flow cytometry antibodies: anti-human CD62L (PE), anti-human CD45RO (APC), anti-human PD-1 (APC), anti-human LAG-3 (PE-cy7); PE, APC, PE-cy7, isotype control were purchased from Biolegend, USA.

[0052] EasySep TM Human T cell negative selection kit was purchased from Stem Cell, USA, CAT# 17951.

[0053] Example 1: Virus preparation

[0054] 1. 293T cells were cultured with DMEM complete medium, which included DMEM (High Glucose) medium, 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin. When the density of 293T reached 60%-70%, the medium was replaced with 5 ml of fresh DMEM complete medium, and the next step was performed after 30 min of culture;

[0055] 2. The plasmid common system was configured, with a specification of 7.5 μg of target plasmid (CD28z), 5.625 μg of psPAX2 plasmid, 1.875 μg of pMD2.G plasmid, 45 μl of PEI solution, and 200 μl of DMEM (High Glucose) medium per 10 cm dish. The DNA mixture was configured in the order of DMEM (High Glucose) medium, plasmid, and PEI;

[0056] 3. After 20 min of standing, the common system was uniformly added to the culture dish according to the required volume per dish, and then placed in a 37°C incubator after cross shaking for 2-3 times;

[0057] 4. The medium was replaced with 10 ml of DMEM complete medium after 6-8 h;

[0058] 5. The first batch of virus was collected 48 h after adding the plasmid, and stored at 4°C. 10 ml of DMEM complete medium was added to the culture dish;

[0059] 6. The second batch of virus was collected 72 h after adding the plasmid;

[0060] 7. The centrifugation parameters were set to 400 x g, 10 min, and the cells were centrifuged to remove cell debris;

[0061] 8. After filtration with a 0.45 μm yellow filter membrane, ultracentrifugation was performed with a centrifuge parameter setting of 25000 rpm, 2 h, and 4°C;

[0062] 9. Discard the supernatant, concentrate 100-200 times with RPMI 1640 medium, and stand in a 4-degree refrigerator for 2 h, and then distribute into 200-μl EP tubes.

[0063] Example 2: Preparation of CAR-T

[0064] 1. Take 10 mL of peripheral blood from a healthy adult into an EDTA-containing blood collection tube, and transfer the blood into a 50-mL centrifuge tube with a dropper, and add an equal volume of PBS solution to the blood, and mix well;

[0065] 2. Add the separation liquid to a new 15-mL centrifuge tube in a ratio of blood volume:Ficoll lymphocyte separation liquid volume = 1:1, and then gently add the blood mixed with PBS using a dropper;

[0066] 3. Centrifuge at a speed of 400 x g for 25 min, and set the parameters to rise 4 and fall 0;

[0067] 4. Use a pipette to suck the white membrane layer composed of peripheral blood mononuclear cells in the middle part of the centrifuge tube, and suck it into a new centrifuge tube;

[0068] 5. Dilute to 15 mL with PBS, centrifuge at a speed of 300 x g for 7 min, and set the parameters to rise 9 and fall 9;

[0069] 6. Discard the supernatant after centrifugation, add 5 mL of PBS for washing, count, and transfer the cells to a flow tube;

[0070] 7. Use the EasySep TM Human T cell negative selection kit, add the isolation cocktail according to the standard amount of the kit, and stand at room temperature for 5 min;

[0071] 8. Pre-oscillate the rapid spheres in the kit for 30 s;

[0072] 9. Add the rapid spheres beads according to the standard amount of the kit, supplement the total volume to 2.5 mL, mix well, and stand at room temperature for 3 min;

[0073] 10. Place the flow tube in a small magnetic stand, stand for 1 min, and then pour the obtained T cells into a new 15-mL centrifuge tube;

[0074] 11. Centrifuge at a speed of 300 x g for 5 min, discard the supernatant, and resuspend the cells with 1 mL of basic medium, and count;

[0075] 12. Take anti-CD3 / CD28 magnetic beads, and calculate the amount in a ratio of magnetic beads:cells = 3:1;

[0076] 13. Absorb the magnetic beads into a new 50 mL centrifuge tube, add 5 mL RPMI-1640 medium to wash the magnetic beads, and place the centrifuge tube on the magnetic stand for 1 min. Remove the waste liquid with a gun, and wash twice;

[0077] 14. After adding 1 mL of cells to the 50 mL tube mixed with the magnetic beads, transfer to the bottom of a T25 culture flask, and shake for 20 min on a shaker to allow the T cells and magnetic beads to fully contact;

[0078] 15. Add 5 mL of basic medium, and incubate in a 37°C incubator for 24 h;

[0079] 16. After 24 h, count the CAR-T cells on day 1, and prepare a T cell infection system. Prepare 1.5-2) x 10 6 cells per well, and 500 μL of the system. The system includes T cells, virus (CD28z) prepared in Example 1, polybrene transfection agent, and basic medium. The CAR structure includes a single-chain variable fragment (clone FMC63) specific for human CD19, followed by a CD8a leader peptide, followed by a CD8 hinge, a CD28 costimulatory domain, and a CD3z intracellular region connected to a P2A-mCherry sequence. The specific sequence is shown in SEQ ID NO. 1. Resuspend the T cells in basic medium, and use a volume of virus that is 3-4 times the volume of T cells. Use 0.25 μL of polybrene transfection agent, and supplement IL-2 and FBS at a concentration of 200 U / mL IL-2 and 10% FBS by volume. The remaining volume in the system is supplemented with basic medium. The basic medium includes 87.9% RPMI-1640 medium, 10% fetal bovine serum, 1% penicillin at 100 U / mL, 1% streptomycin at 100 μg / mL, and 0.1% interleukin 2 at 200 U / mL (interleukin 2 is added to promote long-term proliferation of T cells in vitro).

[0080] 17. After 6-8 h, supplement 1.5 mL of basic medium, and replace the medium after 24 h. Thereafter, use basic medium at a concentration of 1 x 10 6 / mL for incubation;

[0081] 18. On day 7, cells were seeded in 6-well plates and divided into control and experimental groups. The control group was treated with the same volume of DMSO to dissolve geraniol. The experimental groups were treated with three concentrations of geraniol: 1 μM, 2 μM, and 4 μM. Each group had three replicates, with 4 mL per well. The basal medium was changed every 3 days after drug addition, and the corresponding amount of geraniol was added to the experimental groups. The CAR-T cells treated with geraniol were called D-CAR-T cells, thus obtaining control group CAR-T cells and experimental group D-CAR-T cells.

[0082] Example 3: Flow cytometry detection of CAR-T cell subset distribution

[0083] 1. Take 5×10 from the control group and the D-CAR-T group respectively. 5 One cell was transferred to a flow cytometry tube, centrifuged, and the parameters were set to 350×g for 5min.

[0084] 2. Remove the supernatant, add 1 mL of PBS to wash, and centrifuge again at 350×g for 5 min.

[0085] 3. After removing the supernatant, add 100 μL of PBS to each tube, then add 0.5 μL each of anti-human CD62L (PE) and anti-human CD45RO (APC), and incubate at room temperature in the dark for 15 min.

[0086] 4. Add 1 mL of PBS to wash the antibody, centrifuge at 350×g for 5 min, and discard the supernatant;

[0087] 5. After resuspending in 300 μL PBS, the cells were analyzed by flow cytometry; double positivity for CD62L and CD45RO was used as the T cell line. CM Phenotypic criteria, recording T CM Percentage.

[0088] The test results showed that as the concentration of geraniol increased, T CM The proportion is constantly increasing, and the T in each D-CAR-T group is... CM The percentage was significantly higher than that of the control group. See the results below. Figure 1 Flow cytometry analysis showed that geraniol can effectively increase the T cells in CAR-T cells. CM The percentage.

[0089] In Example 2, the experimental group D-CAR-T cells and the control group prepared in step 18 were centrifuged and counted 48 hours after drug administration, and 5×10⁻⁶ cells were collected from each group. 5 Each cell line was seeded back into a 6-well plate, and 5 × 10⁶ Nalm⁶ cells were added to each well at a 1:1 effector-to-target ratio. 5After 24h culture, flow cytometry was performed (steps 1-5), and the results are shown in Figure 3 As shown in FIG. 2B, the CD19-CD28z-CAR-T cells treated with D-kaempferol for 48h, after killing the tumor for 24h, the D-CAR-T cells after killing function CM The ratio was higher than that of the untreated group.

[0090] Example 4: Flow cytometry detection of CAR-T cell exhaustion index

[0091] 1. 5x10 5 cells were taken from the control group and the D-CAR-T group, respectively, and transferred to a flow tube, centrifuged, and the parameters were set to 350xg for 5min;

[0092] 2. The supernatant was removed, and 1ml of PBS was added for washing, and the parameters were set to 350xg for 5min;

[0093] 3. After removing the supernatant, 100μL of PBS was added to each tube, and 0.5μL of anti-human PD-1 (APC) and anti-human LAG-3 (PE-cy7) was added, and incubated at room temperature for 15min in the dark;

[0094] 4. The antibody was washed with 1mL of PBS, and the parameters were set to 350xg for 5min, and the supernatant was removed;

[0095] 5. After resuspension with 300μL of PBS, the flow cytometer was used for detection. PD1 and LAG3 were used as the standard for exhaustion phenotype.

[0096] The results showed that as the concentration of kaempferol increased, the exhaustion index decreased. Figure 2 The statistical chart shows the changes in PD1 and LAG3 indexes.

[0097] The experimental group D-CAR-T cells prepared in step 18 of Example 2 and the control group were centrifuged and counted after 48h of drug addition, and 5x10 5 cells were taken back to a 6-well plate, and Nalm6 cells 5x10 5 cells were added to each well at a ratio of 1:1, and cultured for 24h, and then flow cytometry was performed (steps 1-5), and the results are shown in Figure 3 As shown in FIG. 2B, the CD19-CD28z-CAR-T cells treated with D-kaempferol for 48h, after killing the tumor for 24h, the D-CAR-T cells after killing function

[0098] The above results show that kaempferol can effectively reduce the exhaustion index of CAR-T cells.

[0099] Example 5: Cytokine release detected by CBA Flex Set kit

[0100] 1. Centrifuge cells of control group and D-CAR-T (CD19-CD28z-CAR-T) group at 300xg for 5 min.

[0101] 2. Take the supernatant and complete the experiment according to the operation steps of CBA Flex Set kit. The results are shown in the following table. CAR-T cells treated with Lariciresinol can release more cytokines (including IL2, TNF and INFy), suggesting the enhancement of tumor killing ability. Figure 4 ​

Claims

1. A culture medium for culturing CAR-T cells, characterized by, consists of a basic medium and a dihydroeugenol; the concentration of the dihydroeugenol is 1-4 μM. The basic medium is 10% (volume ratio) fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin and 200 U / ml interleukin 2, and the rest is RPMI 1640 medium.

2. The medium of claim 1, wherein, The concentration of the dihydroeugenol is 2-4 μM.

3. The medium of claim 1, wherein, The concentration of the dihydroeugenol is 2 μM.

4. The medium of claim 1, wherein, The basic medium comprises, in terms of volume percentage, 87.9% (volume ratio) RPMI-1640 medium, 10% (volume ratio) fetal bovine serum, 1% (volume ratio) 100 U / mL penicillin, 1% (volume ratio) 100 μg / mL streptomycin and 0.1% (volume ratio) 200 U / mL interleukin 2.

5. The medium according to any one of claims 1-4 for use in culturing CAR-T cells.

6. A method for constructing and culturing CAR-T cells for non-disease treatment purposes, characterized in that, The method comprises the following steps: (1) isolating T cells from human peripheral blood, and transfecting the T cells with a lentivirus containing a CAR gene expression sequence to prepare CAR-T cells; (2) culturing the CAR-T cells prepared in step (1) using the medium according to any one of claims 1-4.

7. The method of constructing and culturing CAR-T cells according to claim 6, wherein, The concentration of the CAR-T cells in the culture medium is 1-10 x 10 5 cells / mL.

8. The method of constructing and culturing CAR-T cells according to claim 7, wherein, The concentration of the CAR-T cells in the culture medium is 2 x 10 5 cells / mL.

9. The method of constructing and culturing CAR-T cells according to claim 6, wherein, In step (2), the culturing starts from the 5th to 12th day after successful transfection; the frequency of replacing the medium is once every 1-3 days.

10. The method of constructing and culturing CAR-T cells according to claim 9, wherein, The transfection agent in step (1) is a polybrene transfection agent.

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