CN116790505A - A medium for CAR-T cell culture and its application - Google Patents

Abstract

The invention discloses a culture medium for CAR-T cell culture and its application, and relates to the fields of immunology, molecular biology and cell engineering. The culture medium includes basal culture medium and myerin. During the research, the present invention found that adding geranin to the culture medium can effectively increase the proportion of central memory T cells in CAR-T cells and reduce the expression levels of PD1 and LAG3. In addition, geranin can also promote the secretion of IL2, TNF, and INFγ, and enhance the killing function of CAR-T cells. Experiments have shown that the ability of D-CAR-T to secrete IL2, TNF, and INFγ is significantly higher than that of ordinary CAR-T, and D-CAR-T also has a higher T _CM ratio and a lower exhaustion level after exercising its killing function.

Description

A medium for CAR-T cell culture and its application

Technical field

The invention relates to the fields of immunology, molecular biology and cell engineering, and in particular to a culture medium for CAR-T cell culture and its application.

Background technique

In recent years, chimeric antigen receptor T cell (CAR-T) therapy has achieved breakthrough results in the treatment of hematological tumors. However, factors such as tumor microenvironment suppression, CAR-T cell exhaustion and terminal differentiation affect the therapeutic effect of CAR-T. The characteristics of exhausted CAR-T cells include reduced secretion of cytokines, reduced proliferation ability, and sustained high expression of multiple 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 patient prognosis. Although effector-based T cell products have greater cytotoxic potential, these cells are also susceptible to terminal differentiation and become dysfunctional (Ando, M., Ito, M., Srirat, T., Kondo , T. & Yoshimura, A. Memory Tcell, exhaustion, and tumor immunity. Immunol. Med. 43, 1-9 (2020).), while T cells that possess and maintain a less differentiated phenotype (including memory T cells and pre- Somatic T cells) show better therapeutic effects due to their strong proliferation ability and persistence.

Geranin (molecular formula C16H1206, CAS number 520-34-3) is a natural flavonoid compound, which is considered an active ingredient in Chinese herbal medicine and is widely found 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 phosphorylation level of Akt Ser473 and down-regulate the Akt signaling pathway (Zhijie X, Yuanliang Y, Lingfang 4): e0175977.), and inhibiting Akt at the initial stage of CAR-T preparation can improve the positive expression rate, memory phenotype and in vivo efficacy of CAR-T (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.).

Contents of the invention

In order to overcome the shortcomings of the existing technology, one of the objects of the present invention is to provide a culture medium for CAR-T cell culture.

The second purpose of the present invention is to overcome the shortcomings of CAR-T cell exhaustion and terminal differentiation in existing research programs, and to provide a CAR-T cell preparation and culture method that increases the proportion of memory cells and reduces the exhaustion level. This method The prepared and cultured D-CAR-T (Diosmetin-CAR-T) cells have lower inhibitory receptor expression levels, a higher proportion of memory T cells, and can secrete higher levels of killing factors.

One of the purposes of the present invention is achieved by adopting the following technical solutions:

The invention provides a culture medium for cultivating CAR-T cells, including a basic culture medium and geranin; the concentration of geranin is 1-4 μM.

Preferably, the concentration of geranin is 2-4 μM.

More preferably, the concentration of geranin is 2 μM.

In terms of volume percentage, the basal culture medium includes 87.9% volume ratio of RPMI-1640 culture medium, 10% volume ratio of fetal bovine serum, 1% volume ratio of 100 U/mL penicillin, 1% volume ratio of 100 μg/mL penicillin. Streptomycin and 0.1% volume ratio of 200 U/mL interleukin 2.

Interleukin (IL-2) plays an important role in the manufacturing process of CAR-T cells. It can stimulate cell proliferation and maintain cell vitality during the expansion stage. It is a commonly used reagent for culturing CAR-T cells.

The invention provides the application of the culture medium in culturing CAR-T cells.

The invention provides a method for constructing and cultivating CAR-T cells for non-disease treatment purposes, including the following steps:

(1) Isolate T cells from human peripheral blood, transfect the T cells with lentivirus containing the CAR gene expression sequence, and prepare CAR-T cells;

(2) Use the medium to culture the CAR-T cells prepared in step (1).

The kit is intended for non-disease treatment in the context of experimental studies such as cytokine release, detection of cell _TCM ratio, and detection of cell exhaustion indicators.

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

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

Specifically, in step (2), culture is started from the 5th to 12th day after infection; the frequency of replacing the culture medium is every 1 to 3 days.

Preferably, the transfection agent in step (1) is polybrene transfection agent.

During the research, the present invention found that by adding geranin to the culture medium, the proportion of central memory T cells (Central Memory T cells, T _CM ) of CAR-T cells can be effectively increased and the PD1 and LAG3 depletion indicators can be reduced. Cells treated with geranin still had a higher T _{to CM} ratio and a lower PD1 expression level after exercising their killing function. In addition, geranin treatment can also promote the secretion of IL2, TNF and INFγ, and enhance the anti-tumor function of CAR-T cells.

Description of the drawings

Figure 1 shows the improvement in the _TCM ratio of CD19-CD28z-CAR-T cells after treatment with different concentrations of myrogen for 3 days; using one-way ANOVA analysis and Bonferroni test, **** means p<0.0001.

Figure 2 shows the expression levels of PD1 and LAG3 in CD19-CD28z-CAR-T cells after treatment with different concentrations of geranin for 3 days. Figure 2A shows the expression level of PD1, using one-way ANOVA analysis and Tamheny test. ** represents p＜0.01, **** represents p＜0.0001; Figure 2B shows the LAG3 expression level; K independent samples non-parametric test is used, * represents p＜0.05.

Figure 3 shows the reduction in T _CM ratio and depletion index detected after CD19-CD28z-CAR-T cells were treated with different concentrations of geranin for 2 days and then co-cultured with Nalm6 tumor cells for 1 day; where A is T _CM Ratio increase; B is PD1 expression level; using one-way ANOVA analysis and Bonferroni test, * represents p<0.05, ** represents p<0.01, *** represents p<0.001, **** represents p <0.0001.

Figure 4 shows the effect of geranin on the secretion of different cytokines after CD19-CD28z-CAR-T cells were treated with geranin at different concentrations for 3 days and then co-cultured with Nalm6 tumor cells for 1 day; among them, A is geranium. The effect of geranin on the secretion of cytokine IL2; B is the effect of geranin on the secretion of cytokine TNF; C is the effect of geranin on the secretion of cytokine IFNγ; all used one-way ANOVA analysis and Bonferroni test, * indicates p<0.05, ** means p<0.01, *** means p<0.001, **** means p<0.0001.

Detailed ways

Specific embodiments of the present invention are described in detail below. The specific embodiments described here are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If the specific conditions are not specified in the examples, the conditions should be carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.

In a first aspect, the present invention provides a method for preparing D-CAR-T cells, which method includes culturing conventional CAR-T cells under the treatment of geranin to obtain D-CAR-T cells, wherein the D- CAR-T cells have a higher proportion of _TCM , lower exhaustion levels and stronger cytokine secretion capacity.

According to the present invention, the adding period of the geranin can be selected within a wide range. Preferably, the geranin is added continuously on the 5th to 12th day of CAR-T cell culture, for example, on the 7th day.

According to the present invention, the time for culturing the CAR-T cells in geranin can also be selected within a wide range. Preferably, the CAR-T cells are cultured in geranin for 72 hours, which can significantly improve the CAR-T function.

According to the present invention, the dosage of geranin can be selected within a wide range. Preferably, in the culture medium, the amount of geranin used is such that its concentration is 1-8 μM, for example, 2 μM.

According to the present invention, 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, and CD20/CD19-CAR-T cells.

In a second aspect, the present invention provides D-CAR-T cells prepared as described above.

According to the present invention, the proportion of _TCM in the D-CAR-T cells prepared using geranin is significantly increased and the exhaustion level is reduced; the D-CAR-T cells secrete more cytokines during the killing process, including IL2, TNF and INFγ, and after exercising its killing function, it still has a higher TCM ratio and a lower depletion level.

In a third aspect, the present invention provides the use of the above-mentioned D-CAR-T cells in preparing preparations for treating tumors.

In the fourth aspect, the present invention provides the potential application value of geranin in CAR-T clinical treatment. The rights to use geranin as an auxiliary agent in any CAR-T clinical treatment should be included in this patented invention.

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

Hereinafter, the present invention will be described in detail through examples.

CBAFlex Set kit was purchased from BD Company of the United States.

Bright-GloTM Luciferase Assay system was purchased from Promega Company of the United States.

HEK293T cells and ALL cell line Nalm6 were introduced and maintained by the Shanghai Institute of Cell Biology, Chinese Academy of Sciences.

Polyethylene imide (PEI) was purchased from Polysciences Company in the United States.

Penicillin-streptomycin mixture (100X) was purchased from Beijing Solebao Technology Co., Ltd.

RPMI-1640 culture medium was purchased from Coming Company in the United States.

DMEM (High Glucose) medium was purchased from Coming Company in the United States.

Fetal bovine serum (FBS) was purchased from GIBCO, USA.

Ficoll lymphocyte separation solution was purchased from Tianjin Haoyang Biological Products Technology Co., Ltd.

IL-2 was purchased from Peprotech Company of the United States.

Plasmids: CD28z, psPAX2 and pMD2.G were deposited by the Institute of Hematology, Zhejiang University.

anti-CD3/CD28 magnetic beads: clinical research grade, purchased from Thermo Company of the United States, CAT#40203D.

Polybrene was purchased from Sigma-Adrich Company in the United States.

Flow cytometry fluorescent antibodies: anti-human CD62L(PE), anti-human CD45RO(APC), anti-humanPD-1(APC), anti-human LAG-3(PE-cy7); PE, APC, PE-cy7, Isotype controls were purchased from Biolegend Company of the United States.

EasySep ^TM human T cell negative selection kit was purchased from Stem Cell Company of the United States, CAT#17951.

Example 1: Virus Preparation

1. Use DMEM complete medium to culture 293T cells. The DMEM complete medium includes DMEM (HighGlucose) medium, 10% FBS by volume, 100 U/ml penicillin, and 100 μg/ml streptomycin. When the density of 293T reaches 60%-70%, change the medium, add 5 ml of new DMEM complete medium, and culture for 30 minutes before proceeding to the next step;

2. Configure the plasmid public system. The specifications are as follows: add 7.5 μg of the 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 to each 10cm culture dish. Prepare the DNA mixture in the order of DMEM (High Glucose) medium, plasmid, and PEI;

3. After letting it stand for 20 minutes, add the common system evenly into the petri dish according to the required volume of each dish, shake it crosswise 2-3 times and then place it in a 37°C incubator;

4. Change the medium after 6-8 hours and add 10ml DMEM complete medium;

5. Collect the first batch of viruses 48 hours after adding the plasmid, store it at 4°C, and add 10 ml DMEM complete medium to the culture dish;

6. Collect the second batch of viruses 72 hours after adding the plasmid;

7. Set the centrifugation parameters to 400 × g, 10 minutes, and centrifuge to remove cell debris;

8. After filtering with 0.45μm yellow filter membrane, ultracentrifuge, and the centrifuge parameters are set to 25000rpm, 2h, 4℃;

9. Pour off the supernatant, add RPMI 1640 culture medium to concentrate 100-200 times, let it stand for 2 hours in a refrigerator at 4 degrees Celsius, and aliquot into 200 μl EP tubes.

Example 2: Preparation of CAR-T

1. Take 10 mL of peripheral blood from healthy adults into a blood collection tube containing EDTA, use a dropper to transfer the blood to a 50 mL centrifuge tube, add an equal volume of PBS solution to the blood, and mix;

2. With the ratio of blood volume: Ficoll lymphocyte separation solution volume = 1:1, first add the separation solution into a new 15mL centrifuge tube, and then add the blood mixed with PBS gently with a dropper;

3. Centrifuge, set the speed to 400×g, the duration to 25 minutes, and adjust the parameters to increase by 4 and decrease by 0;

4. Use a pipette to suck out the white coat layer composed of peripheral blood mononuclear cells in the middle of the centrifuge tube, and suck it into a new centrifuge tube;

5. Dilute to 15 mL with PBS and wash, centrifuge at a speed of 300 × g, for 7 minutes, and the parameters are up 9 down 9;

6. After centrifugation, discard the supernatant, add 5 mL of PBS to rinse, count, and transfer the cells to a flow tube;

7. Use EasySep ^TM human T cell negative selection kit, add the isolation cocktail according to the standard amount of the kit, and leave it at room temperature for 5 minutes;

8. Pre-shake the rapid spheres in the kit for 30 seconds;

9. Add rapid spheres beads according to the standard amount of the kit, add the total volume to 2.5mL, mix well, and let stand at room temperature for 3 minutes;

10. Place the flow tube into a small magnetic stand, let it stand for 1 minute, and then pour the obtained T cells into a new 15mL centrifuge tube;

11. Centrifuge at 300 × g for 5 minutes, remove the supernatant and resuspend the cells in 1 mL of basic medium, and count;

12. Take anti-CD3/CD28 magnetic beads and calculate the dosage based on the ratio of magnetic beads: cells = 3:1;

13. Absorb the magnetic beads into a new 50mL centrifuge tube, add 5mL of RPMI-1640 culture medium to clean the magnetic beads, place the centrifuge tube on a magnetic stand and let it stand for 1 minute, use a gun to absorb the waste liquid, and wash twice;

14. Add 1mL of cells into a 50mL tube and mix with the magnetic beads, then transfer to the bottom of the T25 culture flask and stand on the shaker for 20 minutes to fully contact the T cells and magnetic beads;

15. Add 5 mL of basic medium and culture in a 37°C incubator for 24 hours;

16. After 24 hours, it is counted as the first day of CAR-T cells. Configure the T cell infection system according to (1.5~2)×10 ⁶ T cells per well and 500 μL system per well. The system contains T cells, Example 1 Prepared virus (CD28z), polybrene transfection agent, and minimal culture medium. The CAR structure includes: a human CD19-specific single-chain variable fragment (clone FMC63), preceded by a CD8a leader peptide, followed by a CD8 hinge, a CD28 costimulatory domain, and a CD3z intracellular region connected to the P2A-mCherry sequence. , the specific sequence is shown in SEQ ID NO.1. Resuspend the T cells in basic culture medium. Use a virus volume so that the amount of virus is 3 to 4 times the amount of T cells. Use 0.25 μL of polybrene transfection agent to supplement IL-2 at a concentration of 200 U/mL IL-2 and 10% FBS by volume. 2 and FBS, and the remaining volume in the system is made up with basic medium. Among them, the basic culture medium includes 87.9% volume ratio of RPMI-1640 medium, 10% volume ratio of fetal bovine serum, 1% volume ratio of 100 U/mL penicillin, 1% volume ratio of 100 μg/mL streptomycin, and 0.1% volume ratio of 200 U/mL interleukin 2 (interleukin 2 is added to promote long-term sustained proliferation of T cells in vitro).

17. Supplement 1.5 mL of basic medium after 6 to 8 hours, change the medium after 24 hours, and then culture with basic medium at a concentration of 1×10 ⁶ /mL;

18. On the 7th day, the cells were seeded in a 6-well plate and divided into a control group and an experimental group. The same volume of DMSO that dissolved geranin was added to the control group. The experimental group was 1 μM geranin, 2 μM geranin, and geranin. There were three treatment concentrations of 4 μM, and 3 duplicate wells were set up in each group, with 4 mL in each well. After adding the drug, the basic culture medium was changed every 3 days, and a corresponding amount of geranin was added to the experimental group. The CAR-T cells treated with geranin were called D-CAR-T cells, and the control group was obtained. CAR-T and experimental group D-CAR-T cells.

Example 3: Detection of subpopulation distribution of CAR-T cells by flow cytometry

1. Take 5×10 ⁵ cells from the control group and D-CAR-T group respectively, transfer them to the flow tube, centrifuge, and set the parameters to 350×g, 5 min;

2. Remove the supernatant, add 1mL PBS for washing, and centrifuge again. The parameters are set to 350×g, 5min;

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

4. Add 1mL PBS to wash the antibody, centrifuge, set the parameters to 350×g, 5min, and remove the supernatant;

5. Add 300 μL of PBS to resuspend, and run the flow cytometer for detection; use CD62L and CD45RO double positivity as the phenotypic standard of T _CM , and record the proportion of T _CM .

The test results found that as the concentration of myrogenin increased, the proportion of T _CM continued to increase, and the proportion of T _CM in each D-CAR-T group was significantly higher than that of the control group. The results are shown in the flow cytometry analysis diagram in Figure 1. The results show that geranin can effectively increase the percentage of T _CM in CAR-T cells.

For the D-CAR-T cells in the experimental group and the control group prepared in step 18 in Example 2, 48 hours after adding the drug, the cells in each group were centrifuged and counted. 5×10 ⁵ cells were seeded back into the 6-well plate, and at the same time, cells in each group were added to each well. Add 5 × 10 ⁵ Nalm6 cells at an effect-to-target ratio of 1:1. After culturing for 24 hours, perform a flow cytometry experiment (same as steps 1 to 5). The experimental results are shown in Figure 3A. CD19- cells treated with germin for 48 hours After CD28z-CAR-T cells killed tumors for 24 hours, that is, after exercising their killing function, the _TCM ratio of D-CAR-T cells was higher than that of the untreated group.

Example 4: Detection of exhaustion indicators of CAR-T cells by flow cytometry

1. Take 5×10 ⁵ cells from the control group and D-CAR-T group respectively, transfer them to the flow tube, centrifuge, and set the parameters to 350×g, 5 min;

2. Remove the supernatant, add 1ml PBS to wash, and centrifuge again. The parameters are set to 350×g, 5min;

3. After removing the supernatant, add 100 μL PBS to each tube, then add 0.5 μL each of antibodies anti-human PD-1 (APC) and anti-human LAG-3 (PE-cy7), and incubate at room temperature in the dark for 15 minutes;

4. Add 1mL PBS to wash the antibody, centrifuge, set the parameters to 350×g, 5min, and remove the supernatant;

5. Add 300 μL PBS to resuspend, and then run the flow cytometer for detection. PD1 and LAG3 were used as phenotypic standards for depletion.

The test results found that as the concentration of myramine increased, the depletion index continued to decrease. The statistical graph in Figure 2 shows the changes in PD1 and LAG3 indicators.

For the D-CAR-T cells in the experimental group and the control group prepared in step 18 in Example 2, 48 hours after adding the drug, the cells in each group were centrifuged and counted. 5×10 ⁵ cells were seeded back into the 6-well plate, and at the same time, cells in each group were added to each well. Add 5 × 10 ⁵ Nalm6 cells at an effect-to-target ratio of 1:1. After culturing for 24 hours, perform a flow cytometry experiment (same as steps 1 to 5). The experimental results are shown in Figure 3B. After treatment with different concentrations of geranin for 48 hours, CD19-CD28z-CAR-T cells kill tumors for 24 hours, that is, the PD1 index in the D-CAR-T cells after exercising their killing function decreases.

The above results show that geranol can effectively reduce the exhaustion indicators of CAR-T cells.

Example 5: CBA Flex Set kit detects cytokine release

1. Centrifuge the cells in the control group and D-CAR-T (CD19-CD28z-CAR-T) group, and set the parameters to 300 × g, 5 minutes;

2. Take the supernatant and complete the experiment according to the operating steps of the CBA Flex Set kit. The results are shown in Figure 4. CAR-T cells treated with geranin can release more cytokines (including IL2, TNF and INFγ), indicating an increase in tumor killing ability.

Claims (10)

1. A culture medium for culturing CAR-T cells, characterized in that it consists of a basic culture medium and geranin; the concentration of geranin is 1-4 μM. The basic culture medium is: 10% fetal bovine serum by volume, 100 U/ml penicillin, 100 μg/ml streptomycin, and 200 U/ml interleukin 2, with the remainder being RPMI 1640 medium. 2. The culture medium according to claim 1, wherein the concentration of myrogen is 2-4 μM. 3. The culture medium according to claim 1, wherein the concentration of myrogen is 2 μM. 4. The culture medium of claim 1, wherein, in terms of volume percentage, the basal culture medium includes 87.9% volume ratio of RPMI-1640 culture medium, 10% volume ratio of fetal bovine serum, 1% volume ratio of fetal bovine serum. 100 U/mL penicillin, 1% volume ratio of 100 μg/mL streptomycin, and 0.1% volume ratio of 200 U/mL interleukin 2. 5. Application of the culture medium according to any one of claims 1 to 4 in culturing CAR-T cells. 6. A method for constructing and cultivating CAR-T cells for non-disease treatment purposes, which is characterized by including the following steps: (1) Isolate T cells from human peripheral blood, transfect the T cells with lentivirus containing the CAR gene expression sequence, and prepare CAR-T cells; (2) Use the medium described in any one of claims 1 to 4 to culture the CAR-T cells prepared in step (1). 7. The method for constructing and culturing CAR-T cells according to claim 6, wherein the concentration of the CAR-T cells in the culture medium is 1-10×10 ⁵ cells/mL. 8. The method for constructing and culturing CAR-T cells according to claim 7, wherein the concentration of the CAR-T cells in the culture medium is 2×10 ⁵ cells/mL. 9. The construction and culture method of CAR-T cells as claimed in claim 6, characterized in that in step (2), culture is started from the 5th to 12th day after successful transfection; the medium is replaced once. The frequency is every 1 to 3 days. 10. The method for constructing and culturing CAR-T cells according to claim 9, wherein the transfection agent in step (1) is a polybrene transfection agent.