CN116790505A - Culture medium for CAR-T cell culture and application thereof - Google Patents
Culture medium for CAR-T cell culture and application thereof Download PDFInfo
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Abstract
The invention 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 basal culture medium and myrosin. According to the invention, through adding the geraniin into the culture medium, the central memory type T cell proportion of the CAR-T cells can be effectively improved, and the expression level of PD1 and LAG3 can be reduced. In addition, the geraniin can promote secretion of IL2, TNF and INFgamma and enhance the killing function of CAR-T cells. Experiments show that the capacity of D-CAR-T to secrete IL2, TNF and INFgamma is obviously higher than that of common CAR-T, and the D-CAR-T also has higher T after the action of killing CM Proportion and lower depletion levels.
Description
Technical Field
The invention relates to the fields of immunology, molecular biology and cell engineering, in particular to a culture medium for CAR-T cell culture and application thereof.
Background
In recent years, chimeric antigen receptor T cell (chimeric antigen receptor T cell, CAR-T) therapies have achieved breakthrough efficacy in the treatment of hematological tumors. However, factors such as tumor microenvironment inhibition, CAR-T cell depletion and terminal differentiation affect the therapeutic effect of CAR-T. Features of depleting CAR-T cells include reduced cytokine secretion, reduced proliferative capacity, and sustained high expression of various inhibitory receptors (including PD-1, TIM-3, LAG-3, etc.). Furthermore, studies have found that the proportion of memory-like CAR-T cells in CAR-T products is critical for the anti-tumor efficacy of CAR-T cells and patient prognosis. Although effector-based T cell products have greater cytotoxic potential, these cells also readily terminally differentiate and become dysfunctional (Ando, m., ito, m., srirat, t., kondo, T. & Yoshimura, a.memory T cell, exhaustion, and thermo-immune.immunol.med.43, 1-9 (2020)), while T cells with and maintaining a less differentiated phenotype (including memory T cells and precursor T cells) exhibit better therapeutic effects due to greater proliferative capacity and persistence.
Diosmetin (molecular formula C16H1206, CAS number 520-34-3) is a natural flavonoid, which is considered an active ingredient of Chinese herbal medicines and is widely found in natural plants and edible fruits. Flavonoid compounds exhibit various pharmacological activities such as antioxidant, anti-inflammatory, antibacterial, antitumor and antiviral activities. Studies have found that geraniin can inhibit Akt Ser473 phosphorylation levels, down-regulate Akt signaling pathways (Zhijie X, yuanliang Y, lingfang X, et al, radiosonisizing effect of diosmetin on radioresistant lung cancer cells via Akt signaling pathway. Plos One,2017, 12 (4): e 0175977.) whereas inhibition of Akt at the early stage of CAR-T production can increase CAR-T positive expression rates, memory phenotype and in vivo efficacy (Qing Z, jiage D, shichuo 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. America journal of cancer research,2019,9 (11): 2379-2396.).
Disclosure of Invention
In order to overcome the defects of the prior art, one of the purposes of the invention is to provide a culture medium for CAR-T cell culture.
The second object of the invention is to overcome the defects of CAR-T cell exhaustion and terminal differentiation in the existing research scheme, and provide a CAR-T cell preparation and culture method for improving the proportion of memory cells and reducing the exhaustion level, wherein the D-CAR-T (Diosmetin-CAR-T) cells prepared and cultured by the method have lower expression level of inhibition receptor, higher memory T cell occupation ratio and can secrete higher level of killing factor.
One of the purposes of the invention is realized by adopting the following technical scheme:
the invention provides a culture medium for culturing CAR-T cells, which comprises a basal culture medium and myrosin; the concentration of the geraniin is 1-4 mu M.
Preferably, the concentration of geraniin is 2-4 μm.
More preferably, the concentration of geraniin is 2 μm.
The basal medium comprises 87.9% of RPMI-1640 medium, 10% of fetal bovine serum, 1% of penicillin 100U/mL, 1% of streptomycin 100 mug/mL and 0.1% of interleukin 2 200U/mL according to volume percentage.
Interleukin (IL-2) plays an important role in the manufacturing process of CAR-T cells, can stimulate cell proliferation and maintain cell viability during the expansion phase, and is a common reagent for culturing CAR-T.
The invention provides application of the culture medium in culturing CAR-T cells.
The invention provides a construction and culture method of CAR-T cells for non-disease treatment, which comprises the following steps:
(1) T cells are separated from peripheral blood of a human body, and are transfected by 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.
Kit cytokine release and detection of cell T CM The experimental study such as ratio and cell depletion index detection is a non-disease treatment.
Preferably, the CAR-T cells are present in the medium at a concentration of 1-10X 10 5 And each mL.
More preferably, the CAR-T cells are present in the medium at a concentration of 2X 10 5 And each mL.
Specifically, in the step (2), the culture is started from the 5 th to the 12 th days after the infection; the frequency of replacing the culture medium once is every 1-3 days.
Preferably, the transfection reagent in step (1) is a polybrene transfection reagent.
The invention discovers that the central memory type T cells (Central Memory T cell, T) of the CAR-T cells can be effectively improved by adding the geraniin into the culture medium CM ) The proportion decreases the PD1 and LAG3 depletion index. The cells treated with the geraniin still have higher T after killing function CM Ratio and lower PD1 expression level. In addition, the treatment of the geraniin can promote secretion of IL2, TNF and INFgamma and enhance the anti-tumor function of CAR-T cells.
Drawings
FIG. 1 is a schematic of CD19-CD28z-CAR-T cell T3 days after treatment with varying concentrations of diosmetin CM The ratio is increased; p < 0.0001 was expressed using one-way ANOVA analysis and the bangfrenni test.
FIG. 2 shows the levels of PD1 and LAG3 expression in CD19-CD28z-CAR-T cells after 3 days of treatment with varying concentrations of diosmetin; wherein, fig. 2A is a plot of PD1 expression levels using one-way ANOVA analysis and a tam black ni test, where p < 0.01 and p < 0.0001; FIG. 2B is a graph showing LAG3 expression level; non-parametric test using K independent samples, representing p < 0.05.
FIG. 3 shows the detection of T after 2 days of treatment with different concentrations of Pelargonium graveolens in CD19-CD28z-CAR-T cells co-cultured with Nalm6 tumor cells for 1 day CM A map of scale and depletion indicator lowering events; wherein A is T CM The ratio is increased; b is the expression level of PD 1; using one-way ANOVA analysis and the bangfranini test, p < 0.05, p < 0.01, p < 0.001, p < 0.0001.
FIG. 4 shows the effect of diosmetin on secretion of different cytokines after 3 days of CD19-CD28z-CAR-T cells treated with different concentrations of diosmetin and 1 day of co-culture with Nalm6 tumor cells; wherein A is the influence of the geraniin on the secretion of cell factor IL 2; b is the influence of the geraniin on the secretion of cytokines TNF; c is the influence of the geraniin on the secretion of the cytokine IFNgamma; both using one-way ANOVA analysis and the bangfrenni test, p < 0.05, p < 0.01, p < 0.001, p < 0.0001.
Detailed Description
The following detailed description of the invention is merely illustrative of the invention and is not intended to limit the scope of the invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In a first aspect, the invention provides a method for preparing a D-CAR-T cell, comprising culturing a conventional CAR-T cell under treatment with diosgenin to obtain a D-CAR-T cell having a higher proportion of T CM Lower depletion levels and greater cytokine secretion capacity.
According to the invention, the period of addition of the diosmetin can be selected within a wide range. Preferably, the myrcene is added continuously on days 5-12 of CAR-T cell culture, e.g., on day 7.
According to the invention, the time for which the CAR-T cells are cultured in diosmetin can also be selected within a wide range. Preferably, the CAR-T cells are cultured in the diosmetin for 72 hours, so that the CAR-T function can be remarkably improved.
According to the invention, the amount of diosmetin can be chosen within a wide range. Preferably, the amount of geraniin is such that its concentration in the culture medium is 1-8 μm, e.g. 2 μm.
According to the invention, the CAR-T cells to be treated may be any CAR-T cells in the art, which may be single target CAR-T cells and/or multi-target CAR-T cells. Preferably, the CAR-T cell is selected from the group consisting of a CD19-CAR-T cell, a CD20-CAR-T cell, a CD22-CAR-T cell, a CD20/CD19-CAR-T cell.
In a second aspect, the invention provides a D-CAR-T cell produced by a method as described above.
According to the invention, D-CAR-T cells, T in cells, are prepared using myrosinase CM The proportion of (2) is significantly increased and the depletion level is reduced; D-CAR-T cells secrete more cytokines including IL2, TNF and infγ during killing, and still have higher TCM ratios, lower depletion levels after killing.
In a third aspect, the invention provides the use of a D-CAR-T cell as described above in the preparation of a formulation for the treatment of a tumor.
In a fourth aspect, the invention provides the potential use of diosmetin in CAR-T clinical therapy, and any claim that uses diosmetin as an adjunct in CAR-T clinical therapy is encompassed by the invention.
Wherein the type of tumor to be treated may be selected according to different CAR-T cell types, which are well known to the person skilled in the art and will not be described in detail here.
Hereinafter, the present invention will be described in detail by way of examples.
The CBAFlex Set kit was purchased from BD company in the United states.
Bright-GloTM Luciferase Assay system is available from Promega corporation, USA.
HEK293T cells and ALL cell line Nalm6 are introduced and stored by Shanghai cells of the national academy of sciences.
Polyethyleneimine (PEI) is available from Polysciences, usa.
The green streptomycin mixture (100X) was purchased from Beijing Soy Bao technology Co.
RPMI-1640 medium was purchased from Coming, USA.
DMEM (High Glucose) medium was purchased from corning corporation, usa.
Fetal Bovine Serum (FBS) was purchased from GIBCO, usa.
Ficoll lymphocyte isolates were purchased from Sedan, inc. of the ocean biologicals science and technology Co.
IL-2 is available from Peprotech, inc. of America.
Plasmid: CD28z, psPAX2 and pmd2.G were saved by university of Zhejiang, hematopathy institute.
anti-CD3/CD28 magnetic beads: clinical study grade, CAT #40203D, available from Thermo corporation of united states.
polybrene was purchased from Sigma-Adrich company, usa.
Flow fluorescent antibody: anti-human CD62L (PE), anti-human CD45RO (APC), anti-human PD-1 (APC), anti-human LAG-3 (PE-cy 7); PE, APC, PE-cy7, isotype control were purchased from Biolegend, usa.
EasySep TM Human T Cell negative selection kit was purchased from Stem Cell company, usa, cat#17951.
Example 1: virus preparation
1. 293T cells were cultured using DMEM complete medium, which includes DMEM (High Glucose) medium, 10% FBS by volume, 100U/ml penicillin, 100 μg/ml streptomycin. Changing liquid when the 293T density reaches 60% -70%, adding 5ml of new DMEM complete culture medium, culturing for 30min, and performing the next step;
2. a plasmid public system was prepared, with the specification of 7.5. Mu.g of the plasmid of interest (CD 28 z), 5.625. Mu.g of the psPAX2 plasmid, 1.875. Mu.g of the pMD2.G plasmid, 45. Mu.l of PEI solution, and 200. Mu.l of DMEM (High Glucose) medium were added to each 10cm dish. Preparing a DNA mixture according to the sequence of DMEM (High Glucose) culture medium, plasmid and PEI;
3. standing for 20min, taking a public system according to the required volume of each dish, uniformly dripping the public system into the culture dish, shaking the culture dish for 2-3 times in a cross manner, and then placing the culture dish into a 37 ℃ incubator;
4. changing the liquid after 6-8 hours, and adding 10ml of DMEM complete culture medium;
5. collecting the first batch of virus after adding the plasmid for 48 hours, storing at 4 ℃, and adding 10ml of DMEM complete medium into a culture dish;
6. collecting a second batch of viruses after the plasmid is added for 72 hours;
7. setting the centrifugation parameters to 400 Xg for 10min, centrifuging, and removing cell debris;
8. after filtration with a 0.45 μm yellow filter membrane, ultracentrifugation was performed with centrifuge parameters set at 25000rpm, 2h, 4 ℃;
9. pouring out the supernatant, adding RPMI 1640 culture medium, concentrating 100-200 times, standing in a refrigerator at 4 deg.C for 2 hr, and packaging into 200 μl EP tube.
Example 2: preparation of CAR-T
1. Taking 10mL of peripheral blood of a healthy adult into an EDTA-containing blood collection tube, transferring the blood into a 50mL centrifuge tube by using a dropper, adding PBS (phosphate buffered saline) solution with the same volume as the blood, and uniformly mixing;
2. adding the separating liquid into a new 15mL centrifuge tube at the ratio of the volume of the blood to the volume of the Ficoll lymphocyte separating liquid=1:1, and lightly adding the blood mixed with PBS by using a dropper;
3. centrifuging, setting the rotating speed to 400 Xg, setting the duration to 25min, and adjusting the parameters to be increased by 4 and decreased by 0;
4. sucking a white membrane layer consisting of peripheral blood mononuclear cells at the middle part of the centrifuge tube by using a pipetting gun, and sucking the white membrane layer into a new centrifuge tube;
5. diluting with PBS to 15mL, washing, centrifuging at 300 Xg for 7min with 9-9;
6. after centrifugation, the supernatant was discarded, and 5mL of PBS was added for washing, counted, and the cells were transferred to a flow tube;
7. using EasySep TM The human T cell negative selection kit is added with isolation cocktail according to the standard quantity of the kit and is placed for 5min at room temperature;
8. pre-shaking rapid spheres in the kit for 30s;
9. adding rapid spheres beads according to the standard amount of the kit, supplementing the total volume to 2.5mL, uniformly mixing, and standing at room temperature for 3min;
10. placing the flow tube into a small magnetic rack, standing for 1min, and then pouring the obtained T cells into a new 15mL centrifuge tube;
11. centrifuging at 300 Xg for 5min, removing supernatant, re-suspending cells with 1mL of basic culture medium, and counting;
12. taking anti-CD3/CD28 magnetic beads, and calculating the dosage according to the proportion of the magnetic beads to the cells=3:1;
13. sucking the magnetic beads to a new 50mL centrifuge tube, adding 5mL RPMI-1640 culture medium to clean the magnetic beads, standing the centrifuge tube with a magnetic rack for 1min, sucking the waste liquid with a gun, and washing twice;
14. adding 1mL of cells into a 50mL tube, mixing with magnetic beads, transferring to the bottom of a T25 culture flask, standing on a shaking table, and shaking for 20min to enable the T cells to be fully contacted with the magnetic beads;
15. supplementing 5mL of basic culture medium, and culturing for 24 hours at 37 ℃;
16. on day 1 after 24 hours, as CAR-T cells, a T cell infection system was prepared in a manner of 1.5 to 2X 10 per well 6 Each T cell was configured in a 500. Mu.L per well system comprising T cells, virus prepared in example 1 (CD 28 z), polybrene transfection reagent, minimal medium. Wherein the CAR structure comprises: the human CD 19-specific single-chain variable fragment (clone FMC 63), preceded by a CD8a leader peptide, followed by a CD8 hinge, a CD28 co-stimulatory domain and an intracellular region of CD3z linked to a P2A-mCherry sequence, is shown in SEQ ID NO. 1. T cells were resuspended in minimal medium at a virus volume such that the virus was 3-4 times the T cell volume, polybrene transfection agent was 0.25 μl, IL-2 and FBS were made up at 200U/mL IL-2 and 10% FBS by volume, the remaining volume in the system was made up with minimal medium. Wherein the minimal medium comprises 87.9% by volume of RPMI-1640 medium, 10% by volume of fetal bovine serum, 1% by volume of penicillin 100U/mL, 1% by volume of streptomycin 100 μg/mL, and 0.1% by volume of interleukin 2 200U/mL (interleukin 2 is added to promote long-term continuous proliferation of T cells in vitro).
17. After 6 to 8 hours, 1.5mL of basic culture medium is added, after 24 hours, the liquid is changed, and after that, the basic culture medium is used for 1X 10 6 Culturing at a concentration of/mL;
18. cells were seeded on a 6-well plate on day 7 and divided into a control group and an experimental group, DMSO was added to the control group to dissolve the same volume of diosmetin, and the experimental group was three treatment concentrations of diosmetin 1. Mu.M, diosmetin 2. Mu.M, and diosmetin 4. Mu.M, each group was set up with 3 duplicate wells, each well being 4mL. And (3) changing the basic culture medium every 3 days after dosing, adding a corresponding amount of geraniin into the experimental group, and calling the CAR-T cells treated by the geraniin as D-CAR-T cells to obtain CAR-T cells of the control group and D-CAR-T cells of the experimental group.
Example 3: detection of the sub-population distribution of CAR-T cells by flow cytometry
1. From the control group and the D-CAR-T group, 5X 10 was taken, respectively 5 Transferring the individual cells into a flow tube, centrifuging, and setting parameters to be 350 Xg and 5min;
2. removing the supernatant, adding 1mL of PBS for cleaning, and centrifuging again, wherein the parameters are set to be 350 Xg and 5min;
3. after removing the supernatant, 100. Mu.L of PBS was added to each tube, and then 0.5. Mu.L of each of the antibodies anti-human CD62L (PE) and anti-human CD45RO (APC) was added, and incubated at room temperature for 15min in the absence of light;
4. adding 1mL PBS to wash the antibody, centrifuging, setting the parameters to be 350 Xg and 5min, and removing the supernatant;
5. adding 300 mu L of PBS for resuspension, and performing on-machine detection by a flow cytometer; double positives of CD62L and CD45RO as T CM Record T CM The duty cycle.
The detection result shows that as the concentration of the myrosinin increases, T CM The duty ratio is continuously increased, and T of each D-CAR-T group CM The ratio was significantly higher than the control. The results are shown in the flow chart of FIG. 1, and the results show that the geraniin can effectively improve T in CAR-T cells CM Is a percentage of (c).
The experimental group D-CAR-T cells prepared in step 18 of example 2 and the control group were centrifuged at 48 hours after the administration of the drug, and 5X 10 cells were collected 5 The individual cells were seeded back into 6-well plates while 1:1 effective target ratio 5X 10 addition of Nalm6 cells 5 After culturing for 24h, carrying out a flow experiment (the same with steps 1-5), wherein the experimental result is shown in figure 3A, CD19-CD28z-CAR-T cells treated by myrosin for 48h, killing tumors for 24h, namely D-CAR-T cells T after killing function CM The ratio was higher than in the untreated group.
Example 4: detection of CAR-T cell depletion index by flow cytometry
1. From the control group and the D-CAR-T group, 5X 10 was taken, respectively 5 Individual cells, transfer to flowIn the tube, centrifuging, and setting parameters to be 350 Xg and 5min;
2. removing the supernatant, adding 1ml PBS for cleaning, and centrifuging again, wherein the parameters are set to be 350 Xg and 5min;
3. after removing the supernatant, 100. Mu.L of PBS was added to each tube, and then 0.5. Mu.L of each of the antibodies anti-human PD-1 (APC) and anti-human LAG-3 (PE-cy 7) was added, and incubated at room temperature for 15min in the absence of light;
4. adding 1mL PBS to wash the antibody, centrifuging, setting the parameters to be 350 Xg and 5min, and removing the supernatant;
5. after adding 300. Mu.L PBS for resuspension, the flow cytometer was used for detection. PD1 and LAG3 were used as phenotypic criteria for depletion.
The detection result shows that the depletion index is continuously reduced along with the increase of the concentration of the myrosinase. The statistical graph of fig. 2 shows the changes in PD1 and LAG3 indices.
The experimental group D-CAR-T cells prepared in step 18 of example 2 and the control group were centrifuged at 48 hours after the administration of the drug, and 5X 10 cells were collected 5 The individual cells were seeded back into 6-well plates while 5X 10 Nalm6 cells were added to each well at a 1:1 effective target ratio 5 After culturing for 24 hours, a flow experiment (the same steps 1-5) is carried out, the experimental result is shown in figure 3B, CD19-CD28z-CAR-T cells treated by the geraniin with different concentrations for 48 hours, and after tumor killing for 24 hours, the PD1 index in the D-CAR-T cells after killing function is reduced.
The results show that the myrosin can effectively reduce the depletion index of the CAR-T cells.
Example 5: CBA Flex Set kit for detecting cytokine release
1. Cells of the control group and the D-CAR-T (CD 19-CD28 z-CAR-T) group were centrifuged and the parameters were set at 300 Xg for 5min;
2. the supernatant was taken and the experiment was completed according to the procedure of the CBA Flex Set kit. The results are shown in FIG. 4, where the myrosinase treated CAR-T cells released more cytokines (including IL2, TNF and INFγ), suggesting an increase in tumor killing capacity.
Claims (10)
1. A culture medium for culturing CAR-T cells, comprising a minimal medium and diosmetin; the concentration of the geraniin is 1-4 mu M.
The basic culture medium is as follows: 10% by volume of fetal bovine serum, 100U/ml of penicillin, 100. Mu.g/ml of streptomycin and 200U/ml of interleukin 2, the balance being RPMI 1640 medium.
2. The medium of claim 1, wherein the concentration of geraniin is 2-4 μm.
3. The medium of claim 1, wherein the concentration of geraniin is 2 μm.
4. The culture medium of claim 1, wherein the basal medium comprises, in volume percent, 87.9% RPMI-1640 medium, 10% fetal bovine serum, 1% penicillin 100U/mL, 1% streptomycin 100 μg/mL, and 0.1% interleukin 2 200U/mL.
5. Use of the medium of any one of claims 1-4 for culturing CAR-T cells.
6. A method for constructing and culturing CAR-T cells for the purpose of non-disease treatment, comprising the steps of:
(1) T cells are separated from peripheral blood of a human body, and are transfected by 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 of any one of claims 1-4.
7. The method of constructing and culturing CAR-T cells according to claim 6, wherein the CAR-T cells are present in the medium at a concentration of 1 to 10 x 10 5 And each mL.
8. The method of constructing and culturing CAR-T cells according to claim 7The method is characterized in that the concentration of the CAR-T cells in the culture medium is 2X 10 5 And each mL.
9. The method for constructing and culturing CAR-T cells according to claim 6, wherein in the step (2), the culture is started from the 5 th to 12 th days after the successful transfection; the frequency of replacing the culture medium once is every 1-3 days.
10. The method of claim 9, wherein the transfection reagent in step (1) is polybrene transfection reagent.
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