CN110157680A - Cell Culture Method for Improving the Efficacy and Durability of Chimeric Antigen Receptor T Cells - Google Patents

Abstract

The present invention provides a cell culture method for improving the curative effect and duration of effect of chimeric antigen receptor T cells, by adding the tyrosine kinase inhibitor dasatinib (dasatinib) to reduce CAR-T cell activation signal transmission, and inhibit CAR -Terminal differentiation of T cells, increasing the ratio of naive T cells and central memory T cells in CAR-T cell preparations, while inhibiting the exhaustion tendency of CAR-T cells. The method of the present invention solves the problem of terminal differentiation and exhaustion tendency of CAR-T cells during in vitro culture. The method of the present invention has simple and easy cultivation process, low cost, and safe and reliable clinical application; the cultured CAR-T cells have a high proportion of initial T cells and central memory T cells, and avoid the exhaustion tendency of CAR-T cells, and have good repeatability; The cultured CAR-T cells show better therapeutic efficacy and persistence in vivo, and have a wide range of application and promotion value.

Description

Cell Culture Method for Improving the Efficacy and Durability of Chimeric Antigen Receptor T Cells

technical field

The invention belongs to the field of immunology and cell therapy research, and relates to a cell culture method for improving the curative effect and persistence of chimeric antigen receptor T cells. Central memory T cells, a culture method that reduces exhaustion of CAR-T cells.

Background technique

Chimeric antigen receptor modified T (chimeric antigen receptor modified T, CAR-T) cell immunotherapy has become the most promising treatment method in immune targeted therapy in recent years. Its working principle is to use genetic engineering technology to recognize a certain The antigen-binding part of the specific single-chain monoclonal antibody of a tumor antigen, the tyrosine activation motif of the T cell receptor and the co-stimulatory molecule are coupled into a chimeric protein in vitro, and T cells are transfected by viral vectors and other methods , so that T cells can specifically recognize tumor cells and transmit signals into the cells, causing the proliferation and activation of T cells to target and kill tumor cells by releasing perforin/granzymes, and are not affected by the major histocompatibility complex (MajorHistocompatibility Complex, MHC) restrictions. In recent years, CAR-T therapy has been applied to leukemia, lymphoma, multiple myeloma, glioma, melanoma, lung cancer, prostate cancer, pancreatic cancer and ovarian cancer. Among them, the research achievements in hematological malignancies are the most eye-catching. At present, clinical research on CAR-T cells targeting targets such as CD19, CD22, CD20, CD33, CD30, CD38, BCMA, CD138, and CD123 has been carried out. The most notable curative effect is mainly reflected in the application of B cell-derived malignant tumor diseases, such as acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), multiple myeloma (MM), B cell lymphoid Tumor (B-NHL) and so on. The internationally reported CD19-targeting CAR-T (CART19) cell therapy for refractory/relapsed B-ALL has a complete remission rate (CR) of 90%. The breakthrough of CAR-T cell therapy in the treatment of hematological tumors has brought new hope to this research field, especially when the traditional treatment fails or relapses, and the decision-makers of clinical treatment are helpless, CAR-T cell immunotherapy Be the best treatment option.

However, in patients receiving CD19-targeting CAR-T cell therapy, relapse has become the main obstacle to CAR-T cell therapy. About 30-50% of patients relapse after receiving CAR-T cell therapy, and most of the relapse occurs after receiving CAR-T cell therapy. CAR-T cell therapy within 1 year; most solid tumors are not effective in targeting CAR-T cell therapy. At present, it is believed that the main reason for the recurrence of primary disease after CD19-targeting CAR-T cell therapy is the loss of CAR-T cells in vivo, that is, the inability to maintain them in vivo for a long time; Disease survival is highly related to the maintenance time of CAR-T cells in vivo. The depletion of CAR-T cells is not only an important factor for tumor recurrence after CD19-targeted CAR-T cell therapy for hematologic malignancies, but also an important reason for the poor efficacy in the treatment of solid tumors. Therefore, the development of methods to improve the efficacy of CAR-T cells and prolong the maintenance of CAR-T cells in vivo is crucial to improving the efficacy of CAR-T cells in the treatment of tumor diseases, preventing disease recurrence and improving the long-term disease-free survival rate of patients.

The efficacy and persistence of CAR-T cells in vivo are significantly related to the differentiation stage and exhaustion state of the cell preparations obtained during in vitro culture. During in vitro culture, spontaneous and continuous activation signals are generated due to the mutual aggregation of CAR molecules on the surface of CAR-T cells, which can not only make CAR-T cells differentiate to the terminal stage, but also cause exhaustion, especially when the CD28 co-stimulatory structure is used. domain CAR-T cells. The differentiation stages of CAR-T cells are divided into: naive T cells, stem central memory T cells, central memory T cells, effector memory T cells, and effector T cells. Naive T cells and central memory T cells in CAR-T cell preparations show long-term survival in vivo, while CAR-T cells differentiated to terminal stages such as effector memory and effectors cannot last in vivo; at the same time, depletion of CAR-T T cells continuously express high levels of inhibitory receptors such as PD1, TIM3, and LAG3, have low proliferation ability, low cytokine release ability, and are prone to apoptosis, which severely limits the persistent effector function of CAR-T in vivo. However, at present, there is no method to solve the terminal differentiation and exhaustion tendency of CAR-T cells during in vitro culture.

Contents of the invention

The object of the present invention is to provide a kind of cell culture method that improves curative effect and action persistence of chimeric antigen receptor T cells, realizes by following specific steps:

1. Peripheral Blood Mononuclear Cell Preparation

Peripheral blood samples were taken, anticoagulated with heparin, and peripheral blood mononuclear cells were separated and prepared using human lymphocyte separation medium.

2. CD3(+) T cell enrichment and T cell activation

Use anti-CD3/CD28 magnetic beads, mix well with peripheral blood mononuclear cells and bind to CD3(+) T cells, enrich CD3(+) T cells with a magnetic frame, and at the same time use the magnetic beads bound to the surface Anti-CD3/CD28 antibodies activate CD3(+) T cells.

3. Transfection of T cells with CAR lentivirus targeting CD19

The prepared lentivirus carrying CD19-targeting CAR was transfected into CD3(+) T cells activated by anti-CD3/CD28 magnetic beads according to MOI=10.

4. Adding the tyrosine kinase inhibitor dasatinib to expand the culture of CAR-T cells

The GFP+CAR-T cells cultured for 3-5 days were taken, and the expression of CAR molecules was detected by flow cytometry. After confirming that the CAR-T cells were successfully prepared, the CAR-T cells were divided into two groups and cultured continuously for 9 days: respectively, with addition and without The CAR-T cells were cultured in the medium supplemented with the tyrosine kinase inhibitor dasatinib. Divided into two groups: experimental group and control group, Dasatinib 30nmol/L experimental group

(RPMI1640+10%FBS+IL-2200U/ml+dasatinib 30nM), control group: equal volume DMSO control group

(RPMI1640+10%FBS+IL-2200U/ml+equal volume of DMSO).

5. Detection of CAR-T cell subsets

On the 9th day of culture, samples were taken to label fluorescent antibodies CD45RO and CD62L, and CAR-T cell subsets were detected by flow cytometry; the differentiation of CAR-T cells cultured in step (4) can continue to maintain the naive T cells and central memory T cells. At the cell stage, it effectively inhibits the differentiation of CAR-T cells to the downstream and terminal stages.

6. Detection of surface molecules related to CAR-T cell exhaustion

On the 9th day of culture, samples were taken labeled with fluorescent antibodies PD1, TIM3, and LAG3, and the surface molecules related to CAR-T cell exhaustion were detected by flow cytometry; PD1, TIM3, and LAG3 effectively inhibited the exhaustion tendency of CAR-T cells.

7. Evaluate the efficacy and persistence of CAR-T cells treated with dasatinib

The ALL-NSG mouse model was prepared and divided into the following groups: ① control group (CAR-T cells treated with DMSO injected into the tail vein), and ② experimental group (CAR-T cells cultured with dasatinib 30 nM injected into the tail vein) for testing; The mice were imaged every week using a small animal in vivo imager, and the difference in tumor burden between the two groups was compared; the proportion of CAR-T cells was detected by flow cytometry every week; the death time of the mice in each group was recorded, and the survival curve was drawn. The CAR-T cells cultured in step (4) can be used to treat mice with acute lymphoblastic leukemia, and better curative effect and survival period can be obtained.

The method of the present invention solves the difficult problem of terminal differentiation and exhaustion tendency of CAR-T cells in the in vitro culture process, by adding the tyrosine kinase inhibitor dasatinib (dasatinib) to reduce the activation signal transmission of CAR-T cells, and inhibit CAR - The terminal differentiation of T cells increases the ratio of naive T cells and central memory T cells in CAR-T cell products, and at the same time inhibits the tendency of CAR-T cells to be exhausted, achieving more significant curative effect and longer maintenance time in vivo CAR-T cell products. The method established by the present invention solves the difficult problem of terminal differentiation and exhaustion tendency of CAR-T cells in the in vitro culture process, and the CAR-T cell activation signal transmission is reduced by adding the tyrosine kinase inhibitor dasatinib, and the CAR-T cells are inhibited. The terminal differentiation of T cells increases the ratio of initial T cells and central memory T cells in CAR-T cell products, and at the same time inhibits the exhaustion tendency of CAR-T cells, and obtains CARs with more significant curative effect and longer maintenance time in vivo - T cell preparations. The inventive method has the following characteristics: (1) The culture process is simple and easy, the cost is low, and the clinical application is safe and reliable; (2) The proportion of the initial T cells and central memory T cells in the cultured CAR-T cells is high, and CAR-T cells are avoided. The cell exhaustion tends to be reproducible; (3) The cultured CAR-T cells show better therapeutic efficacy and persistence in vivo in the leukemia mouse model, and have a wide range of application and promotion values. The present invention aims to establish a method system to prevent CAR-T cells from terminal differentiation during culture, inhibit CAR-T cell exhaustion, and obtain high-quality CAR-T cells with high initial T cells and central memory T cells The product can not only improve the curative effect of CAR-T cells, but also reduce the recurrence after CAR-T treatment and improve the long-term disease-free survival rate of patients.

Description of drawings

Figure 1. During the culture of CAR-T cells in vitro, due to the mutual aggregation of CAR molecules, the continuous activation of CAR-T cells results in the differentiation of some CAR-T cells to the effector stage, which limits their persistence in the body; during the culture process The addition of dasatinib can significantly reduce the downstream differentiation of CAR-T cells, keeping the cells in the initial and central memory stages.

Figure 2. Due to the mutual aggregation of CAR molecules, the continuous activation of CAR-T cells leads to the exhaustion of some CAR-T cells, which limits the clinical efficacy of CAR-T cells; the addition of dasatinib during the culture process can reverse CAR-T cells The depletion tendency of the cells maintains low levels of expression of PD1, TIM3, and LAG3, and high-quality CAR-T cell products are obtained.

Figure 3. After tail vein injection of CAR-T cells cultured with dasatinib 30nmol/L to treat acute lymphoblastic leukemia mice, compared with the control group, the tumor burden of the mice was significantly lower than that of the control group.

Figure 4. Acute lymphoblastic leukemia mice treated with 30nmol/L of dasatinib-cultured CAR-T cells had a median survival of 55 days, while the median survival of the control group was 43 days, suggesting that CAR-T cells cultured with dasatinib were added. T cells significantly prolong survival in mice with acute lymphoblastic leukemia.

Figure 5. One week after the injection of CAR-T cells into the mice, flow cytometry detected the proportion of CAR-T cells in the peripheral blood nucleated cells of the mice. The results showed that the CAR- The proportion of T cells was significantly higher than that of the control group, suggesting that the CAR-T cells treated with 30nmol/L dasatinib were more durable in vivo.

Detailed ways

The present invention will be further described in conjunction with the accompanying drawings and embodiments.

Example 1

1. Isolation of Mononuclear Cells

(1) Collect 10-20ml of peripheral blood;

(2) Peripheral blood was diluted with an equal volume of PBS;

(3) Take a 15ml centrifuge tube, add 5ml of lymphocyte separation solution, and use a pipette gun to slowly add 10ml of the diluted blood sample to the upper layer of the separation reagent along the tube wall to avoid mixing of the separation reagent and the blood sample;

(4) Set the centrifuge to 400G, set the speed up to 4th gear, and the speed down to 0 gear, and centrifuge at room temperature for 30 minutes;

(5) After centrifugation, gently aspirate and transfer the mononuclear cell layer at the interface between the serum and the separation reagent to a new centrifuge tube, and wash the cells twice with PBS.

2. CD3(+) T cell enrichment and T cell activation

(1) Count the obtained mononuclear cells, resuspend in RPMI 1640 complete medium, and adjust the cell concentration to 10 ⁷ /ml;

(2) Wash anti-CD3/CD28 magnetic beads twice with 0.1% BSA/PBS solution; washing method: take 5-10ml of 0.1%BSA/PBS solution and place it in a 15ml centrifuge tube, add the required anti- For CD3/CD28 magnetic beads, mix well and place on the magnetic stand for 1 minute. The magnetic beads are attached to the walls of the centrifuge tube on both sides, and the 0.1% BSA/PBS solution is discarded by suction. Repeat washing once;

(3) According to magnetic beads: CD3(+) T cells = 3:1, fully mix the washed anti-CD3/CD28 magnetic beads and mononuclear cells, and transfer to a culture bottle (select the size of the culture bottle depending on the liquid volume) , put on a shaker and shake gently for 20 minutes to fully combine the magnetic beads with CD3(+) T cells;

(4) Transfer the cell suspension of magnetic beads and PBMC to a centrifuge tube, place it on a magnetic stand and let it stand for 1 minute. The CD3(+) T cells bound to the magnetic beads are attached to the walls of the centrifuge tube on both sides. Cell suspension without magnetic beads in the tube;

(5) Resuspend CD3(+) T cells bound to magnetic beads in RPMI 1640 complete medium containing IL-2 (200IU/ml), adjust the cell concentration to 1×10 ⁶ /ml, 37°C, 5% CO ₂ Cultivate in a saturated humidity incubator for 24 hours.

3. Carrying CAR lentivirus to transfect T cells

(1) Centrifuge and count CD3(+) T cells bound to magnetic beads, resuspend in RPMI 1640 complete medium containing IL-2 (200IU/ml), adjust the cell concentration to 4×10 ⁶ /ml, press 500ul/well Inoculated in 12-well plate;

(2) In this experiment, the lentivirus carrying the GFP/mCherry and CAR target genes was used, and the required amount of virus was calculated according to MOI=10. The calculation formula is as follows: required virus amount = (MOI × cell number) / virus titer;

(3) After the virus was taken out from the -80°C refrigerator, it was quickly melted in a 37°C water bath. Add the amount of virus calculated above to a 12-well plate, add polybrene with a final concentration of 5 μg/mL, mix well and place it in an incubator at 37°C with 5% CO ₂ , and supplement with IL- 2 (200IU/ml) RPMI 1640 complete medium to 2ml, continue to culture for 24 hours;

(4) Centrifuge at 800rpm for 8 minutes, remove the medium supernatant containing the virus, resuspend the cell pellet with fresh medium, transfer the cells to a six-well plate or culture flask, and continue to culture for 3-5 days;

(5) Use a 5ml pipette gun to blow the CAR-T cells in the culture flask, transfer the cells into a 50ml centrifuge tube, place them on a magnetic stand for 1min, the magnetic beads are adsorbed to the tube wall, and transfer the cell suspension to a new tube. In a centrifuge tube, centrifuge and add fresh RPMI 1640 complete medium containing IL-2 (200IU/ml) to continue the expansion culture.

4. Adding the tyrosine kinase inhibitor dasatinib to expand the culture of CAR-T cells

(1) Take the CAR-T cells cultured for 3-5 days, resuspend the cells in RPMI l640 complete medium containing human IL-2 200IU/mL, count them with an automatic cell counter, and count them at 5×10 ⁵ /mL Wells were inoculated into 12-well plates;

(2) Experimental grouping treatment: the experimental group was Dasatinib 30nmol/L group, equal volume DMSO control group; cultivated in a 37°C, 5% _CO2 saturated humidity incubator, centrifuged to change the medium every 3 days, and added Dasatinib again tinib and DMSO.

5. Cell differentiation detection during CAR-T cell culture

(1) On the 9th day of culture, samples were taken for flow cytometry testing;

①Sample collection and processing: Mix the CAR-T cells of each group in the ^six -well plate, absorb an appropriate amount of cell suspension, centrifuge and wash, add 0.5-1×106 cells/tube to the flow tube, and use 100ul PBS buffer resuspended cells;

②Labeled antibodies: Add 2.5ul corresponding fluorescently labeled CD45RO and CD62L antibodies to the corresponding flow cytometry sample tubes, and incubate at 4°C in the dark for 30 minutes;

③Washing: Add 2mL PBS buffer solution to each tube, mix well, centrifuge at room temperature for 5min, and discard the supernatant. Repeat 2 times;

④ Detection and analysis: Use 500ul PBS buffer to resuspend the labeled cells, run the flow cytometer to detect, use Flowj7.6 software to analyze, set a gate with GFP positive CAR-T as the analysis object, and analyze the proportion of each subgroup .

(2) Cell subgroups are defined as: killer T cells CD8+, helper T cells CD4+, naive T cells CD45RO-CD62L+, central memory T cells CD45RO+CD62L+, effector memory T cells CD45RO+CD62L-, effector T cells CD45RO +CD62L-, all expressed as ratios.

(3) Compared with the conventional culture method (control group), this culture method can significantly reduce the downstream differentiation of CAR-T cells and keep the cells in the initial and central memory stages. The results are shown in Figure 1.

6. Detection of cell exhaustion-related surface molecules during CAR-T cell culture

(1) On the 9th day of culture, samples were taken for flow cytometry testing;

(2) Collect samples according to the aforementioned method, label PD1, TIM3, LAG3 fluorescently-labeled antibodies and isotype control antibodies, resuspend after washing, and detect by flow cytometry;

(3) Data analysis: use Flowjo 7.6 software to analyze data, set a gate with GFP-positive CAR-T as the analysis object, and the results are expressed in the proportion of positive cells;

(4) Exhaustion-related markers are represented by the positive ratio of PD1, TIM3, and LAG3. Compared with the conventional culture method (control group), this culture method can reverse the exhaustion tendency of CAR-T cells and maintain low levels of expression of PD1, TIM3, and LAG3 in cells. The results are shown in Figure 2.

7. In vivo evaluation of the efficacy and persistence of CAR-T cells treated with dasatinib in ALL-NSG mice

(1) CAR-T cell preparation: Prepare CAR-T cells carrying mCherry, take the CAR-T cells cultured for 3-5 days, and measure the proportion of CAR-T cells by flow cytometry. Divided into two groups for culture ①control group (adding equal volume of DMSO) ②experimental group (adding dasatinib 30nM), the medium was replaced every 3 days and drugs were added again, and the culture continued for 9 days.

(2) ALL-NSG mouse model preparation: 4-5 week old NSG mice were bred in SPF level animal research center. Take the luciferase (+) Nalm6 cell line in the logarithmic growth phase, prepare the cell concentration to 1×10 ⁶ /200ul, inject 1×10 ⁶ /rat tail vein, and inject a total volume of 200ul to each mouse. Five days later, the small animal in vivo imager was used to detect the tumor burden, and they were randomly divided into two groups according to the fluorescence intensity. There was no significant difference in the average fluorescence intensity between the two groups after adjustment. The next day, the CAR-T cells with different treatments were injected into the tail vein.

(3) Experimental grouping: The experiment was divided into two groups, with 5 mice in each group, respectively ① control group (CAR-T cells treated with tail vein injection of DMSO), ② experimental group (tail vein injection of CAR-T cells).

(4) Tail vein injection of CAR-T cells: Calculate the total amount of cells required for each mouse according to 1×10 ⁶ CAR-T cells/mouse and the proportion of CAR-T cells, collect the cultured CAR-T cells and centrifuge them in PBS Resuspend, and configure the concentration of 1×10 ⁶ CAR-T cells/200ul; inoculate the prepared CAR-T cell suspensions of the control group and the experimental group into NSG mice by tail vein injection, and the injection volume is 200ul/ mouse.

(5) Curative effect and survival observation:

①The mice were imaged with a small animal in vivo imager every week, and the difference in tumor burden between the two groups was compared;

②The proportion of mCherry(+)CAR-T cells in mice was detected by flow cytometry every week;

③Record the death time of mice in each group, and draw the survival curve;

(6) Compared with the control group, the tumor burden of the CAR-T cells obtained by this culture method was significantly lower than that of the control group. The results are shown in Figure 3. The CAR-T cells cultured by this method can significantly prolong the survival period of acute lymphoblastic leukemia mice, and the results are shown in Figure 4.

One week after the injection of CAR-T cells into the mice, the proportion of CAR-T cells in the peripheral blood nucleated cells of the mice was detected by flow cytometry, and the proportion of CAR-T cells cultured with 30nmol/L of dasatinib was significantly higher In the control group, the results are shown in Figure 5.

Claims (4)

1. a kind of cell culture processes for improving Chimeric antigen receptor T cell curative effect and duration of action, which is characterized in that pass through Following steps are realized:

(1) prepared by peripheral blood mononuclear cells: taking periphery blood specimen, anticoagulant heparin is separated using human lymphocyte separating liquid and made Standby peripheral blood mononuclear cells；

(2) enrichment of CD3 (+) T cell and t cell activation:

It is given after mixing well with peripheral blood mononuclear cells and be incorporated into CD3 (+) T cell using anti-CD3/CD28 magnetic bead Magnetic frame is enriched with CD3 (+) T cell, and activates CD3 (+) T using the anti-CD3/CD28 antibody for being incorporated in magnetic bead surfaces simultaneously Cell；

(3) the CAR slow-virus transfection T cell of targeting CD19 is carried: by the slow virus of the CAR of the carrying targeting CD19 prepared According to CD3 (+) T cell of MOI=10 transfection anti-CD3/CD28 magnetic bead activation；

(4) addition tyrosine kinase inhibitor Dasatinib expands culture CAR-T cell

The CAR-T cell of culture 3-5 days is taken, the expression of flow cytometer detection CAR molecule will after confirmation CAR-T cell is successfully prepared 2 groups of CAR-T cell point is continuous respectively to be cultivated 9 days；

(5) CAR-T cell subsets detects: culture samples this mark fluorescent antibody CD45RO, CD62L, flow cytometer inspection on the 9th day Survey CAR-T cell subsets；

(6) detection of CAR-T cell depletion relevant surfaces molecule

It cultivates and samples within the 9th day this mark fluorescent antibody PD1, TIM3, LAG3, flow cytomery CAR-T cell depletion is related Surface molecular；

(7) the CAR-T cell curative effect and persistence handled through Dasatinib is assessed

Divide control group and experimental group, the CAR-T cell that control group DMSO is handled, experimental group is cultivated with Dasatinib 30nM CAR-T cell compares two groups of tumor load differences by imager, by Flow cytometry CAR-T cell proportion, draws Survivorship curve.

2. a kind of cell culture for improving Chimeric antigen receptor T cell curative effect and duration of action according to claim 1 Method, which is characterized in that step (4) it is described culture be respectively using addition with do not add tyrosine kinase inhibitor Dasatinib Culture solution culture CAR-T cell.

3. a kind of cell culture for improving Chimeric antigen receptor T cell curative effect and duration of action according to claim 1 Method, which is characterized in that described point 2 groups of step (4) are experimental group and experimental group, and experimental group is Dasatinib 30nmol/L group, Control group is isometric DMSO group.

4. a kind of cell culture for improving Chimeric antigen receptor T cell curative effect and duration of action according to claim 2 Method, which is characterized in that the culture solution of the addition Dasatinib is that RPMI1640+10%FBS+IL-2 200U/ml+ reaches sand For Buddhist nun 30nM, the culture solution for not adding Dasatinib is that RPMI1640+10%FBS+IL-2 200U/ml+ is isometric DMSO。