CN115322968A

CN115322968A - CAR-T cell culture system, application and culture method

Info

Publication number: CN115322968A
Application number: CN202211149490.2A
Authority: CN
Inventors: 徐广贤; 王红霞; 官成浓; 蒋丹; 张爱君; 楚元奎; 张晓春
Original assignee: Dongguan Southeast Central Hospital; Ningxia Medical University
Current assignee: Dongguan Southeast Central Hospital; Ningxia Medical University
Priority date: 2022-09-21
Filing date: 2022-09-21
Publication date: 2022-11-11

Abstract

The invention belongs to the technical field of cellular immunity, and particularly relates to a CAR-T cell culture system, application and a culture method. The invention provides a culture system of CAR-T cells, compared with a basal culture system, spermidine can enhance the activity of the CAR-T cells in vivo and in vitro killing lymphoma cells and change the biological characteristics of the CAR-T cells, such as improvement of the proliferation capacity of the CAR-T cells, cytokine expression level and memory phenotype transformation. In the embodiment of the invention, after spermidine is supplemented into the basal culture system, the CAR-T cells are expanded, the proliferation capacity of the CAR-T cells is improved, the exhaustion state of the CAR-T cells is improved, the memory phenotype population is increased, and meanwhile, the secretion capacity of cytokines is increased, and the lysis capacity of lymphoma cells is increased. In addition, the survival of lymphoma mice is prolonged.

Description

CAR-T cell culture system, application and culture method

Technical Field

The invention belongs to the technical field of cellular immunity, and particularly relates to a CAR-T cell culture system, application and a culture method.

Background

CAR-T cell therapy is an adoptive cell therapy with significant success in treating hematologic malignancies. Several products have been approved by the FDA for the treatment of hematological malignancies such as B-cell acute lymphoblastic leukemia (B-ALL), large B-cell lymphoma (DLBCL), relapsed/refractory B-cell lymphoma (r/r LBCL), or relapsed/refractory multiple marrow (r/rMM), but drug resistance is still present in about 40% to 50% of patients. The reasons for failure of CAR-T cell therapy are complex and diverse. For example, the persistence of CAR-T cells in vivo is closely related to the prognosis of the patient; the clinical feedback of poorly differentiated CAR-T cells is superior to that of well differentiated CAR-T cells, which acquire killing capacity at the expense of losing self-renewal capacity; meanwhile, the therapeutic effect of the CAR-T cell is greatly influenced by the depletion of the CAR-T cell, and the depletion of the CAR-T cell is expressed by the reduction of self-renewal capacity; the expression of inhibitory receptors such as PD-1, CTLA-4, TIGIT and the like is up-regulated; and the molecular expression of the granulmycin B, perforin, IFN-gamma, TNF-alpha 18-21 and the like is reduced. Therefore, relieving the depletion state of the CAR-T cells becomes one of the problems to be solved urgently in clinical application.

Disclosure of Invention

The invention aims to provide a CAR-T cell culture system, application and a culture method, wherein the CAR-T cell culture system can obviously improve the CAR-T cell depletion level, and CAR-T cells (spd CAR-T) amplified by the CAR-T cell culture system are in a low differentiation state, have increased antigen-dependent proliferation capacity and IL-2 and IFN-gamma expression, and have improved lymphoma cell lysis capacity.

The invention provides a CAR-T cell culture system, which comprises a basal culture system and spermidine;

the basic culture system comprises: x-vivo serum-free culture system, penicillin, streptomycin and IL-2, wherein the basic culture system also comprises fetal bovine serum or autologous plasma.

Preferably, the concentration of penicillin in the basic culture system is 100U/ml, the concentration of streptomycin is 100 mu g/ml, the concentration of IL-2 is 200U/ml, and the final concentration of fetal bovine serum or autologous plasma is 10 percent of the basic culture system.

Preferably, the working concentration of spermidine is 0.5 to 500 μ M.

Preferably, the working concentration of spermidine is 0.5 to 5 μ M.

The invention also provides application of the culture system in preparation of a cell culture medium for improving in-vitro and in-vivo tumor killing capacity, proliferation capacity, cytokine expression level and memory phenotype transformation of CAR-T cells.

The invention also provides application of the culture system in preparation of a culture medium for relieving the CAR-T cell exhaustion state.

The invention also provides a method for expanding CAR-T cells, comprising the steps of: transferring the constructed CAR-T cells into the culture system for amplification culture for 4d.

Preferably, the method comprises infecting T cells with lentivirus containing the target CAR sequence for 72h, and performing the amplification culture after positive verification.

Has the beneficial effects that: the invention provides a culture system of CAR-T cells, compared with a basal culture system, spermidine can enhance the activity of the CAR-T cells in vivo and in vitro killing lymphoma cells and change the biological characteristics of the CAR-T cells, such as improvement of the proliferation capacity of the CAR-T cells, cytokine expression level and memory phenotype transformation. In the present example, when spermidine was supplemented to the basal culture system, CAR-T cells were expanded, and their proliferative capacity was improved, depletion state was improved, and memory phenotype population was increased, and at the same time, cytokine secretion capacity was increased, and lymphoma cell lysis capacity was increased. In addition, the survival of lymphoma mice was prolonged.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 shows T cell apoptosis in the presence of varying concentrations of spermidine;

FIG. 2 is a graph showing flow cytometry detection of T cell proliferation at different concentrations of spermidine treatment;

FIG. 3 is a graph showing the detection of T cell proliferation by absolute counting method under different concentrations of spermidine;

FIG. 4 is the positive rate of CD19CAR-T cells;

FIG. 5 is the positive rate of Nb CAR-T cells;

FIG. 6 is a CD19CAR-T cell memory phenotype profile under spermidine treatment;

FIG. 7 is a picture of Nb CAR-T cell memory phenotype under spermidine treatment;

FIG. 8 is a graph of CD19CAR-T cell proliferation under spermidine treatment;

FIG. 9 is Nb CAR-T cell proliferation under spermidine treatment;

FIG. 10 shows IL-2 secretion from CD19CAR-T cells treated with spermidine;

FIG. 11 shows the secretion of IFN- γ from CD19CAR-T cells under spermidine treatment;

FIG. 12 is Nb CAR-T cell IL-2 secretion in spermidine treatment;

FIG. 13 is Nb CAR-T cell IFN- γ secretion under spermidine treatment;

FIG. 14 is CD19CAR-T cytotoxicity under spermidine treatment;

FIG. 15 is Nb CAR-T cytotoxicity under spermidine treatment;

FIG. 16 is the tumor burden on the ventral side of mice in each treatment group;

FIG. 17 shows dorsal tumor burden in mice of each treatment group;

FIG. 18 shows the body weight changes of mice in each treatment group;

FIG. 19 is a graph showing the body survival curves of mice in each treatment group;

figure 20 is a schematic of spermidine pre-treated CAR-T cells.

Detailed Description

the basic culture system comprises: the culture medium comprises an X-vivo serum-free culture system, penicillin, streptomycin and IL-2, wherein the basic culture system also comprises fetal bovine serum or autologous plasma.

In the basic culture system of the invention, the concentration of penicillin is preferably 100U/ml, the concentration of streptomycin is preferably 100 mu g/ml, the concentration of IL-2 is preferably 200U/ml, and the final concentration of fetal bovine serum or autologous plasma is preferably 10% of the basic culture system.

The working concentration of the spermidine is preferably 0.5-500. Mu.M, more preferably 0.5-5. Mu.M, and the change of the number of T cells is most significant and has statistical significance (p is less than 0.001) when compared with the spermidine concentration of 0. Mu.M, 0.5. Mu.M and 5. Mu.M.

In the embodiment of the invention, after the CAR-T cells are cultured for 4d by using the culture system, the TCM (CD 45RA-CCR7 +) memory population is increased, the memory cells still maintain the self-renewal capacity under the stimulation without tumor antigens, and the effector cells lose the renewal capacity; the proliferation capacity is obviously improved under the stimulation of Daudi cells, but the CAR-T cells do not proliferate under the stimulation of K562 cells; at a potent target ratio of 10, 1, 20, the spermidine-treated CAR-T cells secreted IL-2, both increased levels of ifn- γ, but the levels of cytokine secretion were not statistically significant under K562 stimulation; the cell lysis capacity is increased, and the clearing capacity of Daudi cells is obviously improved.

In the invention, when spermidine is contained in the culture system, the change of the T cell function is induced, and the aging of the CAR-T cells is delayed, so that the CAR-T cell exhaustion state is relieved.

The invention also provides a method for expanding CAR-T cells, comprising the steps of: transferring the constructed CAR-T cells into the above culture system for amplification culture at 4d,37 deg.C, 5% CO ₂ 。

In the present invention, it is preferable to infect T cells with lentivirus containing the CAR sequence of interest for 72h, and perform the amplification culture after positive confirmation. The CAR sequence of interest is not specifically limited, but CD19CAR-T cells and Nb CAR-T (Nanobody based CAR-T cell) cells are exemplified in the examples, but they are not to be construed as the full scope of the invention. In the present example, the CD19CAR used was of the classical secondary structure, the scfv sequence of CD19 was derived from FMC63 antibody; CD19, CD20 nanobody sequences employed by Nb CAR-T cells have been disclosed in CN111574630A and CN 111574629A.

In order to further illustrate the present invention, the CAR-T cell culture system, application and culture method provided in the present invention are described in detail below with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.

Example 1

1.1 Effect of spermidine Pre-treatment on T cell apoptosis

According to the operation of the apoptosis kit instruction, the detailed operation steps are as follows:

(1) collecting the T cells of each group treated with spermidine at different concentrations (0. Mu.M, 0.5. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, 100. Mu.M, 500. Mu.M) for 4 days in a 15mL sterile centrifuge tube, adding 2mL PBS, centrifuging and washing at 1200rpm for 10min;

(2) resuspending the cell pellet with 1 × Binding buffer, and adjusting the cell density to 1 × 10 according to the counting result ⁶ /mL；

(3) Flow antibody staining. The chromosome line is 100 muL, according to the reagent instruction, 5 muL PE-AnnnexinV and 5 muL 7-AAD are added and mixed evenly. Meanwhile, setting a control tube: blank tubes (without any treatment), 7-AAD single-stained tubes, PE-AnnexinV single-stained tubes;

(4) incubating at room temperature in dark for 20-30 min, adding 400 μ L of 1 × Binding buffer, and performing computer analysis within 1 h.

1.2 Effect of spermidine Pre-treatment on T cell proliferation

CellTrace staining method for T cell proliferation determination. The specific operation steps are as follows:

(1) diluting 20 mu L of CellTrace mother liquor to working concentration (1 mu M) by adopting PBS, adding a corresponding volume of dye solution according to a cell counting result, resuspending a precipitate, and incubating for 20min in a dark place at 37 ℃;

(2) adding a serum-containing culture medium which is 4 to 5 times of the original dyeing system to stop dyeing (37 ℃ for 5 min); centrifuging to remove supernatant, performing resuspension precipitation by using a T cell complete culture medium preheated in advance, culturing for at least 1h in a cell culture box to fully combine a dye solution and cells, and performing flow detection on part of cells to detect whether dyeing is successful or not;

(3) after T cell staining, the cells were treated with spermidine at various final concentrations, in the order 0. Mu.M, 0.5. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, 100. Mu.M, 500. Mu.M. After 4d of culture, flow cytometry was used to analyze the proliferation of T cells at each concentration.

The results show that the apoptotic cell populations at 0. Mu.M, 0.5. Mu.M, 5. Mu.M, and 10. Mu.M respectively account for 6.62%,3.09%,3.35%, and 4.38% of the total population. At a spermidine concentration of 100. Mu.M, the cell status was very poor and apoptotic cells reached 95.5% (FIG. 1). In addition, when the concentration of spermidine is 0.5. Mu.M, 5. Mu.M, 10. Mu.M, T cells show obvious proliferation generations compared with the mother generation, and the proliferation population percentages are 76.5%,73.2%,71.6%, respectively. Similar to the results of the apoptosis experiment, T cells did not proliferate at 100. Mu.M. Meanwhile, the absolute count of T cells was performed 4 days after spermidine treatment, and the results are shown in fig. 2 and 3, in which the spermidine concentration was 0.5 μ M, and the change in T cell number was most significant at 5 μ M, compared to 0 μ M, and was statistically significant (p < 0.001).

Example 2

Preparation of CD19CAR-T cells and Nb CAR-T cells

The scfv sequence of CD19 was derived from FMC63 antibody; CD19 and CD20 nano antibody sequences adopted by the Nb CAR-T cells (related sequences are published in Chinese patents CN111574630A and CN 111574629A). CD19CAR, nb CAR lentivirus particles infect primary T cells after preparation is complete. The specific steps of CAR-T cell preparation are as follows:

2.1 plates for T cell activation were prepared the day before T cell isolation, i.e., anti-human CD3/CD28 was diluted in sterile PBS to a final concentration of 1. Mu.g/mL in 24-well plates, 400. Mu.L of coating solution was added to each well.

2.2 isolation and purification of human peripheral blood CD3+ T cells

Collecting peripheral blood of a healthy person by using a clinical common heparin anticoagulation tube, and carrying out mononuclear cell separation operation within 2h, wherein the separation kit is an Tianjin common people peripheral mononuclear cell separation kit, and the separation step is as follows, and the operation condition is 20 +/-2 ℃.

Firstly, preparing 2 aseptic 15mL centrifuge tubes, adding 5mL separation solution into the centrifuge tubes, standing the centrifuge tubes in a centrifuge tube rack; then, gently mixing the collected peripheral blood, inclining the centrifugal tube added with the separating medium, slowly adding the mixed blood along the tube wall, and paying attention not to break the liquid level; then, the centrifugal tube is gently placed into a horizontal centrifuge for centrifugation, the centrifugation condition is 550g,30min and 20 ℃; after centrifugation, the liquid is divided into 4 layers, from top to bottom: a light yellow plasma layer, a ring-shaped milky single nucleus cell layer, a transparent separation liquid layer and a red blood cell layer. Collecting plasma layer, and slowly sucking leucoderma layer along tube wall; 10mL of PBS equilibrated at room temperature, 250g, was added to each tube, and washing was repeated 2 times to collect cell pellets, i.e., peripheral mononuclear cells.

The obtained mononuclear cells enter a T cell sorting process, a sorting reagent is a Meitian and whirly CD3 positive sorting kit, and all reagents are precooled at 4 ℃, and the method is simple:

collecting cells by using a precooled MACS buffer, and then carrying out resuspension counting on the cells in a 15mL sterile centrifuge tube; after adjusting the number of cells, resuspending the cells in 100. Mu.L of MACS buffer, adding 20. Mu.L of CD3 sorting magnetic beads into each tube, flicking and mixing uniformly, and then incubating in a refrigerator at 4 ℃ for 15min; after the incubation is finished, adding 1mL of MACS buffer to terminate the reaction, and centrifuging at 200g for 10min to collect cells; 500 μ L MACS buffer resuspends the cells, places the sorting column on a sorting rack, gently adds the cell suspension into the sorting column, avoids the generation of bubbles as much as possible during operation, at this time, labeled CD3 positive cells are left in the column, CD3 negative cells flow out of the column, and MACS buffer repeatedly washes the column three times; and (3) taking the sorting column out of the sorter, adding 1mL of MACS buffer, firmly pressing by adopting a configured piston, collecting the CD3 positive cells in a new sterile centrifuge tube, and centrifuging at 200g for 10min to collect cell precipitates to obtain the CD3 positive T cells.

Collecting cells before and after magnetic bead sorting, respectively, resuspending by 100 mu L PBS, adding 20 mu L PE Mouse Anti-Human CD3 flow antibody into each tube, flicking and uniformly mixing, incubating at room temperature in a dark place for 25min, adding 1mL PBS to wash and remove unbound antibody, discarding supernatant, precipitating by 500 mu L PBS resuspended cells in the flow tube, detecting the purity of T cells by an upper computer within 1h, and circling the gate by PE signals.

In addition, the culture plate required for virus infection of T cells on the next day is prepared, namely, a 48-well cell culture plate is adopted

(20. Mu.g/mL), anti-human CD3/CD28 (5. Mu.g/mL) in a 200. Mu.L system.

2.3 Lentiviral infection of T cells

The following day, the virus-infected plate medium was discarded, rinsed 1 time with PBS, and 500. Mu.L of 2% BSA was added to block non-specific binding. According to the MOI value, adding corresponding volume of CD19CAR or Nb CAR lentivirus into each well, horizontally centrifuging at 2000g and 2h to ensure that the virus is in

Is in close contact with the culture plate under the action of the pressure.

After the T cells are activated for 48 hours, the cells can be observed to be transparent, smooth and mellow under an inverted microscope, and the phenomenon of conglomeration growth appears, which indicates that the T cells are fully activated. Collecting cells, and resuspending and counting the complete T cell culture system to 1X 10 ⁶ mL, 200. Mu.L of cell suspension was added to the virus-infected plate and centrifuged at 1000g and 1h.

After centrifugation is finished, observing the cell state under a microscope, then gently placing the cell state into an incubator for culture, and after 24 hours, supplementing an equal volume of T cell complete culture system into each hole.

2.4 after the lentivirus infects the T cells for 72h, primarily observing the CAR-T cell positive rate under a fluorescence microscope, and further quantitatively detecting the CAR-T cell positive rate of each group by using flow cytometry.

The flow cytometry results are shown in FIGS. 4 and 5, and the positive rate of the CAR-T cells in each group is over 50%, which indicates that the CAR-T cells are successfully prepared.

Example 3

Expansion culture of CD19CAR-T cells, nb CAR-T cells

The positive rate of CD19CAR-T cells, nb CAR-T cells, as shown in example 2 were expanded for 4 days in the culture system provided by the present invention. The medium was changed every 2 days according to CAR-T cell proliferation.

The culture system is as follows: the X-vivo serum-free culture system is supplemented with 10% final concentration of fetal bovine serum or autologous plasma, penicillin (100U/ml), streptomycin (100. Mu.g/ml), IL-2 (200U/ml) and spermidine at a working concentration of 0.5. Mu.M, 5. Mu.M, to obtain spermidine-pretreated CAR-T cells.

Example 4

Examination of the memory phenotype and proliferation potency of CAR-T cells pretreated with spermidine in example 3

4.1 flow cytometry detection of CAR-T cell memory phenotype after spermidine Pre-treatment

(1) Collecting various groups of CAR-T cells treated by spermidine with different concentrations for 4d in a 15mL sterile centrifuge tube, adding 2mL PBS for centrifugal washing at 1200rpm for 10min;

PBS resuspend cells, adjust cell density to 1X 10 ⁶ /mL；

Sucking 100 mu L of cell suspension into a 1.5mL sterile centrifuge tube for staining, adding 5 mu L of BB700Rat Anti-Human CCR7 (CD 197) and 5 mu L of APC Mouse Anti-Human CD45RA into each tube, and gently and uniformly mixing;

incubating at room temperature in a dark place for 20-30 min, and adding 500 mu L PBS to wash the unbound antibody;

300 μ L PBS resuspended cell pellet, filtered through 300 Mo cell filter screen in 5mL flow tube, and tested on machine within 1 h.

As a result, as shown in FIGS. 6 and 7, the memory cells remained self-renewing without the stimulation of the tumor antigen, while the effector cells lost the renewal ability. CD19CAR-T cells and Nb CAR-T cells TCM memory population are increased after spermidine pretreatment, tn is CD45RA + CCR7L +; TCM, CD45RA-CCR7+; TEM is CD45RA-CCR7-; TEMRA CD45RA + CCR7-.

4.2 flow cytometry detection of CAR-T cell proliferation after spermidine pretreatment

Proliferation assay methods as in example 1, CD19CAR-T cells and groups of Nb CAR-T cells were studied under spermidine treatment.

The proliferation ability test results are shown in fig. 8 and fig. 9, compared with the Mock group, the proliferation ability of the CAR-T cells pretreated by spermidine was significantly improved under the stimulation of Daudi cells after 4 days, and the CAR-T cells did not proliferate under the stimulation of K562 cells.

Example 5

CAR-T cytotoxicity assay under spermidine pretreatment

5.1 enzyme-linked immunosorbent assay (ELISA) detection of IL-2 in CAR-T cells of each group

And (3) co-culturing the CAR-T cells and K562 cells (not expressing CD19 and CD20 molecules and serving as negative control target cells) or Daudi cells (highly expressing CD19 and CD20 molecules and serving as positive control target cells) for 24 hours respectively according to corresponding effect-target ratios, collecting cell supernatants, and detecting the IL-2, IFN-gamma level in the culture supernatants by adopting an ELISA method.

All required reagents were pre-equilibrated to room temperature prior to performing the formal experiments, operating according to LEGEND MAXTM Human IFN-. Gamma.IL-2 ELISA Kit reagent instructions.

Results as shown in fig. 10 and fig. 11, the levels of IL-2, ifn- γ, secreted by spermidine-treated CD19CAR-T cells were both increased at an effective target ratio of 10, 1, 20. Cytokine secretion levels under K562 stimulation were not statistically significant. Similarly, nb CAR-T cells also possessed this property (fig. 12 and 13).

5.2 Lactate Dehydrogenase (LDH) assay for in vitro cytotoxicity of various groups of CAR-T cells.

The culture volume of the 96-well culture plate is 100 mu L, and the number of tumor cells is 1 multiplied by 10 ⁵ cells, each group of at least 3 multiple holes, the specific detection process is as follows:

(1) tumor cells were collected, washed several times with PBS, and resuspended to 2×10 ⁶ cells/mL,50 μ L per well of 96-well plate (except effector cell spontaneous wells, volume corrected wells and background wells);

(2) and collecting each group of CAR-T cells which are well expanded and cultured for about one week, and determining the number of the CAR-T cells in each group according to the effective target ratio. For example: the number of target cells is 1X 10 ⁵ cells, when the E: T is 20 ⁶ cells, so CAR-T cells were diluted 4X 10 ⁷ cells/mL, 50. Mu.L/well;

(3) according to the initial design of the well distribution, adding the corresponding volume of culture system or lysine buffer, horizontal centrifuge, 250g, centrifugation 5min,37 ℃,5% ₂ Culturing for 18h;

(4) collecting cell culture supernatant 45min before, taking out the culture plate, adding lysine buffer into the maximum lysis hole of the target cell, and fully lysing the target cell;

(5) when collecting the supernatant, horizontally centrifuging the detection plate for 250g and 5min, and transferring the supernatant to a new marked 96-hole detection plate by using a line gun, wherein each hole is 50 mu L;

(6) adding 50 mu L of the mixture into each hole, balancing to room temperature CytoTox 96 Reagent, and reacting for 30min at room temperature in a dark place;

(7) adding 50 mu L of stop solution into each hole, and reading the absorbance value of the sample at 490nm within 1h by using a micropore plate reading instrument;

calculating the cytotoxicity:

Cytolisis％＝(Experiment-Effector Spontaneous-Target Spontaneous)/(Target Maxium-Target Spontaneous)×100

the results are shown in fig. 14 and fig. 15, the cell lysis capacity of the spermidine-pretreated group is increased, wherein the killing rate of the CD19CAR-T cells is obviously improved under the action of 5 μ M concentration, and the killing rate reaches 80% when the effective target ratio is 5. However, no significant killing was observed when the target cells were K562 cells, regardless of spermidine pretreatment (fig. 14). Furthermore, daudi cell clearance was significantly improved compared to control spermidine-pretreated Nb CAR-T cells (fig. 15).

Example 6

Small animal in vivo imaging detection of CAR-T cell in vivo anti-tumor effect

The animal experiments related to the invention are all developed after ethical examination of animal welfare. The serial numbers of the animal experiment certificates are as follows: GDY2104005. NCG mice used in the experiments were purchased from Guangdong Yaokang Biotechnology Ltd (license number: SYXK (Yue) 2019-0213).

6.1 mouse blood tumor model construction

After the mice are adaptively raised in the barrier for one week, the diet, activity and the like of the mice are observed, so that the mice are in the optimal state, and the molding is started.

(1) Recovering Daudi-luc cells, and carrying out passage for 2-3 times to ensure the activity of the tumor cells in a proliferation stage;

(2) tumor cells were collected, washed with PBS, and counted at 5X 10 ⁶ /mL；

(3) Disinfecting tail veins of mice by using an alcohol cotton ball, wiping the tail veins repeatedly to fully expose the tail veins to cause congestion, and selecting any vein on the left side and the right side for injection, wherein each vein is 200 mu L;

(4) after one week of molding, the in vivo imaging is used for detecting whether the mouse molding is successful.

6.2 injection of CAR-T cells

After the pre-experiment verifies that the mouse model is successfully made, a formal experiment is carried out, the steps are the same as 6.1, after one week, tail vein injection is carried out, each group of CAR-T cells with good in-vitro amplification state is carried out for about 10 days, and meanwhile, PBS with the same volume is injected to serve as a control group.

6.3 Small animal Living body imaging

(1) Taking out the mice, weighing the body weight to determine the volume of the D-fluorescein potassium salt;

(2) after about 10min of substrate injection, carrying out inhalation type anesthesia by using isoflurane for 3min, and then detecting the fluorescence value;

(3) the mouse is continuously anesthetized in the process of obtaining the image, the parameters are set to be automatic exposure, and the image is stored after the exposure is finished.

In vivo imaging results of small animals as shown in fig. 16 to 19, the ventral and dorsal tumor burden was significantly reduced and significantly improved in the Nb CAR-T group compared to the PBS group (fig. 16, fig. 17); and CAR-T cell treatment did not cause significant weight loss in mice, which were euthanized when the mice had loose hair and rapidly lost weight (figure 18). Statistics were performed on all mice in the PBS group that died 23 days after Daudi-Luc injection. Surprisingly, the overall survival of mice treated with spermidine-pretreated bipecific Nb CAR-T cells was improved, while tumor burden was not statistically significant (figure 19). Spermidine-pretreated CAR-T cells were cytotoxic in vitro and in vivo better than the untreated group.

The invention adopts the results of a small-molecule drug spermidine for CAR-T cell pretreatment. 4 days after spermidine pretreatment of CAR-T cells, the T cells are observed to reverse to memory phenotype, and the anti-apoptosis capability and proliferation capability are increased, thereby contributing to the in vivo and in vitro anti-tumor effect of the CAR-T cells, and the specific molecular mechanism of the CAR-T cells needs to be further researched. It is postulated that spermidine may promote the development of CAR-T cell effector functions by modulating the expression levels of genes involved in CAR-T cell proliferation, autophagy or cell survival, among other relevant genes. The underlying principle of spermidine action is shown in figure 20.

Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

Claims

1. A culture system for CAR-T cells comprising a basal culture system and spermidine;

2. The culture system of claim 1, wherein the concentration of penicillin in the basal culture system is 100U/ml, the concentration of streptomycin is 100 μ g/ml, the concentration of IL-2 is 200U/ml, and the final concentration of fetal bovine serum or autologous plasma is 10% of the basal culture system.

3. The culture system according to claim 1, wherein the working concentration of spermidine is 0.5-500 μ M.

4. The culture system according to claim 1 or 3, wherein the working concentration of spermidine is between 0.5 and 5 μ M.

5. Use of a culture system according to any one of claims 1 to 4 for the preparation of a cell culture medium for enhancing the tumor killing capacity, proliferation capacity, cytokine expression level and transformation of a memory phenotype of CAR-T cells in vitro and in vivo.

6. Use of a culture system according to any one of claims 1 to 4 in the preparation of a medium for alleviating a CAR-T cell depletion state.

7. A method of expanding CAR-T cells, comprising the steps of: transferring the constructed CAR-T cells into a culture system according to any one of claims 1 to 4 for amplification culture for 4d.

8. The amplification method according to claim 7, which comprises infecting T cells with lentivirus containing the target CAR sequence for 72h, and performing the amplification culture after positive verification.