阅读说明：本技术 Mek抑制剂在减少car-t细胞耗竭和终末分化中的应用 (Use of MEK inhibitors to reduce CAR-T cell depletion and terminal differentiation ) 是由 黄河 王修健 韦聪 李侠 于 2021-05-27 设计创作，主要内容包括：本发明公开了MEK抑制剂在减少CAR-T细胞耗竭和终末分化中的应用。优选MEK抑制剂为曲美替尼。本发明还公开了一种CAR-T细胞体外培养方法,在CAR-T细胞体外培养的第6天起向培养基中加入MEK抑制剂。本发明经研究发现在CAR-T细胞培养过程中,加入MEK抑制剂处理,能够减少CAR-T细胞耗竭和终末分化,从而使CAR-T细胞体内持久性更好,抗肿瘤作用更强。(The invention discloses the use of a MEK inhibitor to reduce CAR-T cell depletion and terminal differentiation. Preferably the MEK inhibitor is trametinib. The invention also discloses an in vitro CAR-T cell culture method, and MEK inhibitors are added into the culture medium from the 6 th day of the in vitro culture of the CAR-T cells. According to research, the invention discovers that the CAR-T cell can be depleted and terminally differentiated by adding the MEK inhibitor in the CAR-T cell culture process, so that the CAR-T cell has better in-vivo durability and stronger anti-tumor effect.)

Use of a MEK inhibitor in reducing CAR-T cell depletion and terminal differentiation.

2. The use of claim 1, wherein the MEK inhibitor is trametinib, cobitinib or bimatinib.

3. Use according to claim 2, wherein trametinib is used in a concentration of 7.5 to 30 nmol/L.

4. Use according to claim 3, wherein trametinib is used in a concentration of 7.5 to 15 nmol/L.

5. Use according to claim 4, wherein trametinib is used in a concentration of 15 nmol/L.

6. The use of claim 1, wherein the MEK inhibitor is added to the culture medium from day 6 of the in vitro culture of the CAR-T cells.

7. A CAR-T cell in vitro culture method, wherein a MEK inhibitor is added to the culture medium from day 6 of the CAR-T cell in vitro culture.

8. The CAR-T cell in vitro culture method of claim 7, wherein said MEK inhibitor is trametinib, which is used at a concentration of 7.5-30 nmol/L.

9. The CAR-T cell in vitro culture method according to claim 8, wherein trametinib is used at a concentration of 7.5-15 nmol/L.

10. The CAR-T cell in vitro culture method according to claim 9, wherein trametinib is used at a concentration of 15 nmol/L.

Technical Field

The present invention relates to the field of biotechnology, in particular to the use of MEK inhibitors in reducing CAR-T cell depletion and terminal differentiation.

Background

Although the long-term survival rate of leukemia patients is obviously improved along with the advancement of medical technology, refractory/relapsed leukemia still is an international problem in the field of current hematological malignancy treatment, especially adult refractory/relapsed acute lymphocytic leukemia (R/R ALL), and the 5-year survival rate is only 5% -8%.

The CD19-targeted chimeric antigen receptor T (CD19-targeted polymeric antigen-modified T cell, CD19 CAR-T) cell therapy is an emerging revolutionary cellular immunotherapy method in the field of R/R ALL treatment, and brings new hopes for the treatment of R/R ALL. CAR-T cell therapy is a revolutionary cellular immunotherapy approach that utilizes genetic engineering techniques to genetically modify T cells to express chimeric antigen receptors that specifically recognize tumor antigens and mount immune attacks on tumor cells. Chimeric antigen receptors are composed of a single chain antibody variable domain that can recognize a specific tumor antigen extracellularly, a transmembrane domain, and an intracellular signaling domain, typically composed of CD3 ζ and the signaling domains of costimulatory molecules (including CD28, ICOS, 4-1BB, OX4O, CD27, and the like). CAR-T can be divided into primary CAR-T (intracellular signaling domain consisting of CD3 ζ only), secondary CAR-T (intracellular signaling domain consisting of CD3 ζ and one signaling domain of a costimulatory molecule) and tertiary CAR-T (intracellular signaling domain consisting of CD3 ζ and two signaling domains of a costimulatory molecule) according to the number of costimulatory signaling domains contained, and the most clinically used are the secondary CAR-T currently, whose intracellular signaling domain is usually composed of CD28 or 4-1BB costimulatory domain in tandem with CD3 ζ.

CAR-T cell therapy is currently widely used in the treatment of hematologic and solid tumors. Among them, CD19CAR-T cell therapy has attracted attention as a clinical therapeutic effect in B-cell derived R/R ALL of adults and children, and the complete remission rate reported by each unit internationally is between 60% and 93%. The FDA in the united states has approved 4 CD19CAR-T products for the treatment of B cell-derived refractory/relapsed leukemia or lymphoma.

The current international reported recurrence rate after CD19CAR-T treatment is between 30% and 60%. Depending on the condition of CD19 expression by leukemic cells at the time of relapse, relapses were classified as CD19 positive and negative relapses, with about 46.1% of patients developing CD19 positive relapses and 10% -20% developing CD19 negative relapses, based on CD19 positive relapses. Current studies indicate that CD 19-negative relapse is mainly associated with genetic mutation in CD19 of tumor cells, natural selection of CD 19-negative leukemia clones by the immune killing pressure of CAR-T, lineage switch of B lineage leukemia clones to myeloid lineage leukemia clones induced by the immune killing pressure of CAR-T, and the cytognawing effect of CAR-T on CD 19. While CD19 positive relapse is primarily associated with insufficient persistence of CAR-T cells in vivo.

Although the tumor killing ability of CAR-T with CD28 as co-stimulatory domain (28z CAR-T) was stronger than that of CAR-T with 4-1BB as co-stimulatory domain (4-1BBz CAR-T), it was more easily depleted and eventually differentiated into CD62L negative effector memory/effector T cells with less persistence in vivo than it was. Thus, patients receiving 28z CAR-T therapy are more susceptible to CD19 positive relapse, and it is important to reduce CD19 positive relapse and improve patient prognosis to try to prolong 28z CAR-T in vivo persistence without impairing its strong killing ability to tumors.

The reason for the poorer persistence of 28z CAR-T in vivo is that: the strength and speed of the activation signal transduction are higher than those of 4-1BBz CAR-T, and the faster and stronger activation signal transduction enables the activation signal transduction to be more easily exhausted and terminally differentiated, compared with the 4-1BBz CAR-T, the activation signal transduction is weaker in speed and strength, so the activation signal transduction is not easily exhausted and terminally differentiated, and the sustained anti-tumor effect can be exerted in vivo.

During the in vitro culture of CAR-T, Single-chain variable fragment ScFv of the CAR molecule can undergo spontaneous aggregation independent of ligand binding, which can cause CAR molecules to transmit sustained chronic activation signals (tonic signaling) intracellularly to trigger CAR-T depletion, apoptosis and terminal differentiation, impair CAR-T persistence and anti-tumor effects in vivo, and seek to reduce tonic signaling to facilitate long-term CAR-T maintenance in vivo. It has been shown that the higher the proportion of central memory T cells contained in CAR-T cells, the better the persistence in vivo and the stronger the antitumor effect.

Disclosure of Invention

The present invention addresses the above technical problems in the prior art with the in vitro culture of CAR-T cells and provides for the use of MEK inhibitors to reduce CAR-T cell depletion and terminal differentiation.

The invention provides, in a first aspect, the use of a MEK inhibitor in reducing CAR-T cell depletion and terminal differentiation.

Preferably, the MEK inhibitor is trametinib. The MEK inhibitor is an FDA approved MEK inhibitor represented by trametinib, such as trametinib (trametinib), cobinetinib (cobimetinib) or bimetinib (binimetinib).

More preferably, trametinib is used at a concentration of 7.5nmol/L to 30 nmol/L. More preferably, trametinib is used at a concentration of 7.5nmol/L to 15 nmol/L. Most preferably, trametinib is used at a concentration of 15 nmol/L.

Preferably, the MEK inhibitor is added to the culture medium from day 6 of the in vitro culture of the CAR-T cells.

The invention also provides an in vitro culture method of the CAR-T cells, wherein the MEK inhibitor is added into a culture medium from the 6 th day of the in vitro culture of the CAR-T cells. Preferably, the MEK inhibitor is trametinib, and the concentration of the used trametinib is 7.5 nmol/L-30 nmol/L. More preferably, trametinib is used at a concentration of 7.5nmol/L to 15 nmol/L. Most preferably, trametinib is used at a concentration of 15 nmol/L.

The addition of MEK inhibitor produces side effects of inhibiting the proliferation of CAR-T, since the effect of inhibiting proliferation and the effect of anti-depleting anti-differentiation are more significant at higher concentrations, 15nmol/L is preferred for balancing the inhibition of proliferation and anti-depleting differentiation, so the optimal concentration of the effect of trametinib is 15 nmol/L.

According to research, the CAR-T cell culture method disclosed by the invention has the advantages that in the CAR-T cell culture process, the MEK inhibitor is added for treatment, so that the CAR-T cell exhaustion and terminal differentiation can be reduced, the CAR-T cell in vivo durability is better, and the anti-tumor effect is stronger.

Drawings

FIG. 1 is a schematic representation of the composition of CAR fragments.

FIG. 2 is a schematic structural diagram of a CAR lentiviral vector carrying mCherry.

FIG. 3 is a flow cytometry assay of CAR-T cell surface CAR molecule expression in example 4.

FIG. 4 is a diagram showing the results of the detection of the GFP + luciferase + Nalm-6 cell line.

FIG. 5 is a graph of the results of the CAR-T cell killing rate measurements at different effective target ratios.

FIG. 6 is a bar graph of the results of flow assays of the effect of trametinib on differentiation of 28z CAR-T cells cultured in vitro, and the cell differentiation status after treatment with trametinib at D9 and D12 days. The test results were counted in 4 independent replicates, each test sample being provided by a different healthy donor. P < 0.05. NS, No significant, meaning No statistical difference.

FIG. 7 is a bar graph of the effect of trametinib on 28z CAR-T depletion and activation markers in vitro culture, flow assay results for activation and depletion markers after treatment with trametinib for Day9 and Day 12. The detection repetition times of all the markers are more than or equal to 4, and each detection sample is provided by different healthy donors. P < 0.05, P < 0.01, P < 0.001, P < 0.0001. NS, No significant, meaning No statistical difference.

FIG. 8 is a graph of the effect of trametinib on 28z CAR-T amplification. A: a fold line plot of 28zCAR-T amplification in a trametinib-containing culture system. B: 28z CAR-T was cultured in a trametinib-containing culture system for 3 days. C: 28z CAR-T was cultured in a trametinib-containing culture system for 6 days in expansion fold. The test results were counted in 4 independent replicates, each test sample being provided by a different healthy donor. P < 0.05, P < 0.01, NS, No significant, i.e. No statistical difference.

FIG. 9 is a graph of the results of in vitro culture of 28z CAR-T cell apoptosis by trametinib, A and B are bar charts of the effect of trametinib treatment at 3 concentrations for 3 days (day9) and 6 days (day12) on CAR-T apoptosis; the detection repetition times are more than or equal to 3, and each detection sample is provided by different healthy donors. P < 0.05, P < 0.01. NS, No significant, meaning No statistical difference.

FIG. 10 is a graph of the results of testing the effect of trametinib-containing culture systems on 28z CAR-T killing function in vitro. A: effect of trametinib-containing culture systems on killing function of CAR-T cultured for 3 days. B: effect of trametinib-containing culture systems on killing function of CAR-T by 6 days of culture. The test results were counted in 3 independent replicates, each test sample being provided by a different healthy donor. NS, No significant, meaning No statistical difference.

FIG. 11 is a graph of in vivo imaging of mice in groups as tested for the effect of trametinib-containing CAR-T in vitro culture systems on 28z CAR-T anti-tumor function in vivo.

FIG. 12 is a graph of the results of testing the effect of trametinib-containing CAR-T in vitro culture systems on 28z CAR-T anti-tumor function in vivo. A: survival curves for each group of mice. B: fold line plot of photon counts for each group of mice. P < 0.01.

Detailed Description

1. Experimental Material

HEK293T cells and ALL cell line Nalm-6 were introduced and stored by Shanghai cells of Chinese academy. Competent cells DH5 α were purchased from Nanjing Novowed Biotech Ltd. Peripheral blood T cells are derived from healthy donors. Lentiviral vectors employ a three plasmid system: psPAX2, pMD2.G and Lenti-EF1 α. B-NSG mice: 6-8 weeks old, purchased from Baiosaxituggu gene biotechnology Limited, Zhejiang university medicine safety evaluation research center SPF-level environmental routine breeding.

2. Laboratory apparatus

Flow cytometer Cytoflex (Beckman, usa); CytoFLEX LX flow cytometer (Beckman, usa); flow cytometric sorter (Beckman, usa); small animal Living body imager (Perkin-Elmer, USA).

3. Primary reagent

RPMI1640 medium (Corning, USA); dmem (high glucose) medium (Corning, usa); fetal Bovine Serum (FBS) (GIBCO, usa); ficoll lymphocyte separation (top grade Biotech, Inc., Tianjin); plasmid extraction kit (Life Biotechnology, USA); genomic DNA Purification Kit (Lifetech, CAT # K0512); PrimeScript^TMII 1st Strand cDNA Synthesis Kit (Takara, Japan, CAT # 6210A);Premix Ex Taq^TM(Tli RNaseH Plus), ROX Plus (Takara, Japan, CAT # RR42WR (LR. times.5));IL-2 (Peprotech, USA); anti-CD3/CD28 magnetic beads: clinical study grade Cat #40203D (Thermo corporation, usa); Bright-GloTM Luciferase Assay system (Promega corporation, USA, Cat: E2620); D-Luciferin Firefly, lotus salt (Perkin-Elmer, USA, Cat: # 122799); goat Anti-Mouse IgG, F (ab')₂fragment-specific antibodies (Jackson, USA, Cat: 115-; streptavidin FITC, (Biolegend, USA); polybrene (Sigma-Adrich, USA); polyethyleneimine hydrochloride (PEI) (Polysciences, usa); trametinib powder (selelck, usa); flow-through fluorescent antibodies: anti-human CD3(PE-cy7), anti-human CD19(APC), anti-human CD4(APC-cy7), anti-human CD8(PE-cy7), anti-human CD62L (PE), anti-human CD45RO (APC), anti-human CD25(APC), anti-human CD69(PE-cy7), anti-human PD-1(APC), anti-human TIM-3(PE), anti-human LAG-3(PE-cy7), anti-human FASL (PE), Annexin V (APC); PE, APC, PE-cy7, APC-cy7 isotype controls were purchased from Biolegend, USA. EasySep^TMHuman T Cell negative selection kit (Stem Cell inc., usa, CAT # 17951); 10 × Annexin V binding buffer (BD bioscience, CAT51-66121E, USA).

4. Solution preparation

(1) RPMI1640 complete medium: RPMI1640 + 10% FBS + 1% streptomycin lividans;

(2) DMEM (high sugar) complete medium: DMEM (high sugar) medium + 10% FBS + 1% streptomycin qing;

(3) FBS: carrying out water bath at 56 ℃ for 30min before use, cooling at room temperature, subpackaging into 50ml centrifuge tubes, and storing at-20 ℃;

(4) transfection reagent PEI solution: 50mg of linearized polyethyleneimine hydrochloride (polyethyleneimine, Linear, mw-25000) was accurately weighed, transferred to a 50mL centrifuge tube, added with 50mL of ddH2O, and placed in a 80 ℃ water bath tank to be sufficiently dissolved. Adjusting pH to 7.0, filtering with 0.22um filter membrane in ultra-clean bench for sterilization, subpackaging in sterile 1.5ml of LEP tube, and storing at-20 deg.C;

(5) preparing an IL-2 solution: 1 piece of 500. mu.g of the IL-2 lyophilized powder was dissolved in 1mL of 100mM acetic acid, and 49mL of PBS containing 0.1% BSA was further added thereto and stored at 1X 10⁶IU/ml, subpackaging and storing at-80 ℃;

(6) goat anti-mouse IgG, F (ab')₂Fragment-specific antibody configuration: dissolving the lyophilized powder of the antibody with 500 μ L of sterile ddH2O to a concentration of 1.5mg/ml, packaging into 100 μ L of EP tube, and storing at-80 deg.C;

(7)Bright-Glo^TMpreparing a Luciferase Assay reaction solution: 100ml of Bright-Glo was added at room temperature^TMAdding Luciferase Assay Buffer into 1 visual Bright-GloTM Luciferase Assay Substrate, fully dissolving, and subpackaging into 15ml centrifuge tubes for preservation at-80 ℃;

(8) preparing a trametinib solution for in-vitro culture: dissolving trametinib powder in DMSO to prepare a stock solution with the concentration of 10mmol/L, subpackaging and storing at-80 ℃. Diluting the mixture into different required concentrations by using DMSO before use, and ensuring that the final concentration of the DMSO in all experiments is one thousandth;

(9) preparing luciferin injection for imaging of small animals: before use, the total required amount is calculated according to 3 mg/mouse, D-Luciferin Firefoy with corresponding mass is weighed, podassium salt powder is dissolved in DPBS with corresponding volume, the prepared concentration is 15mg/ml, and the solution is filtered by a 0.22um filter for use.

(10)1 × Annexin V binding buffer: adding a proper volume of 10 × Annexin V binding buffer into 9 times of volume of ddH before use₂The O was diluted to 1 Xsolution and stored in a refrigerator at 4 ℃.

Example 1 construction of CAR-carrying Lentiviral vectors

1. Synthesis of CAR Gene fragments according to Gene design of interest

Based on the variable region sequences of heavy and light chains of a murine antibody targeting CD19 (clone number FMC63, https:// www.sciencedirect.com/science/article/pii/S0161589097001442), a second generation CAR sequence was constructed with CD28 and CD3 zeta as T cell activation signals, and the 3' end of the CAR was linked to red fluorescent protein Mchery via 2A polypeptide. The composition schematic diagram of the CAR fragment is shown in figure 1, the CAR fragment (the sequence is shown in SEQ ID No. 1) is directly subjected to whole-gene synthesis, and the synthesized fragment is used as a PCR amplification template.

2. Construction of CAR Lentiviral vectors

(1) The synthesized fragment is used as a template for PCR amplification, a CAR-mcherry fragment is amplified by using a primer sequence in the following table 1, and meanwhile EcoRI enzyme cutting sites and XbaI enzyme cutting sites are respectively introduced into two ends of the sequence through PCR for construction of a lentiviral expression vector.

TABLE 1 CAR cloning primers

Primer name	5 '-3' sequence	Primer cleavage site
			CAR Mcherry F	ttcgaattcgccgccaccatggcctt	EcoRI cleavage site gaattc
CAR Mcherry R	cggtctagattactacttgtacagctcgtcc	XbaI cleavage site tctaga

(2) The PCR reaction system was configured as follows:

TABLE 2 PCR reaction System

Composition (I)	Dosage of
		Ex Taq HS(Takara，RR006)	1μL
10×Buffer	5μL
		dNTP(25mM)	2μL
CAR F(10μM)	1μL
		CAR R(10μM)	1μL
PCR template	1μL
		Sterile water	39μL
Total volume	50μL

(3) After a PCR reaction system is prepared, a PCR instrument is set according to the following program: pre-denaturation at 98 ℃ for 5 min; denaturation at 98 deg.C for 1min, annealing at 60 deg.C for 2.5min, and extension at 72 deg.C for 1min for 30 cycles; final extension at 72 ℃ for 10 min.

(4) After the PCR reaction, the PCR product was analyzed by electrophoresis on a 1% agarose gel, and the desired fragment was recovered by gel.

(5) And carrying out double digestion on the lentivirus expression vector Lenti-EF1 alpha and the PCR product fragment recovered by the gel by using EcoRI-XbaI, carrying out agarose gel electrophoresis, recovering the digested vector and the PCR product fragment, and carrying out T4 DNA ligase connection. The attachment system is shown in table 3 below:

TABLE 3 cloning of the ligation systems

Reagent	Dosage of
		CAR fragments	5μL
Lenti-EF1α	200ng
		10×T4 buffer	2μL
T4 DNA ligase	1μL
		Sterile water	The volume is fixed to 20 mu L

(6) The T4 DNA ligation system was placed at 16 ℃ for overnight ligation, 10. mu.L of the resulting DNA was transformed into E.coli competent stbl3 cells, coated with ampicillin-resistant LB plates, and then the LB plates were placed at 37 ℃ for overnight culture, and on the following day, about 5mL of monoclonal cells were picked and subjected to small-scale shake culture, and plasmid DNA was extracted using a plasmid miniprep kit and subjected to Sanger sequencing. The structure of the lentiviral vector is shown in FIG. 2.

Example 2 transformation, amplification and extraction of CAR-carrying lentiviral vector plasmids

1. Transformation of plasmids

(1) DH5 alpha competent cells were removed from the freezer at-80 ℃ and placed on ice to be thawed slowly; (2) mixing competent cells, subpackaging 10 μ L/tube into sterile 200 μ L EP tube, adding 1 μ L CAR-carrying vector plasmid, and ice-cooling for 20 min; (3) thermally shocking in a water bath at 42 deg.C for 60s, transferring to ice, and standing for 5 min; (4) coating the transformed competent cells in a solid LB culture dish containing ampicillin; (5) the plates were inverted and incubated in an incubator at 37 ℃ for 16-24 hours.

2. Bacterial amplification

(1) Taking a 15mL centrifuge tube, and adding 4mL LB culture medium containing ampicillin; (2) using a sterile 10-mu L gun head, picking single colony clone growing in a solid LB culture dish, putting the colony clone into the centrifuge tube, and performing shake culture for 4-6 hours (37 ℃, 200 rpm); (3) after 4-6 hours, if flocculent suspended bacteria appear in the centrifuge tube, the bacteria are transferred to a 500ml conical flask containing LB culture medium containing ampicillin, and the shaking table is continued to shake and culture for 12-16 hours.

3. Plasmid extraction (big drawer)

(1)500mL of bacterial liquid is subpackaged into a sterile centrifuge bottle, 4000g of bacteria is centrifuged for 10min, supernatant is discarded, and precipitated bacteria are collected; (2) adding 10ml of R3 solution containing RNase to resuspend the pellet; (3) adding 10ml Lysis Buffer (L7), mixing, standing at room temperature for 5min, and lysing thallus; (4) adding 10ml Precipitation Buffer (N3), mixing, centrifuging at room temperature 12000g for 10 min; (5) collecting supernatant suspension, and placing the supernatant suspension in a column for filtration; (6) adding 60ml of Wash Buffer (W8) to the column, and washing the plasmid adsorbed in the column; (7) placing the column on a new sterile 50ml centrifuge tube, adding 15ml of Elution Buffer (E4) for Elution, and obtaining plasmid suspension in the centrifuge tube; (8) adding 10.5ml isopropanol, mixing, and centrifuging at 4 deg.C (12000g for 30 min); (9) discarding the supernatant, adding 5ml 70% ethanol, and centrifuging at 4 deg.C (12000g, 10 min); (10) after discarding the supernatant, the supernatant was naturally dried for 10min, and according to the amount of the obtained plasmid, an appropriate volume of ddH2O was added to resuspend the plasmid, and the DNA concentration was determined to adjust the concentration to 500-1000 ng/. mu.L and stored at-20 ℃.

Example 3 packaging with CAR lentivirus

1. Recovery and culture of HEK293T cells

(1) Taking out HEK293T cells from a liquid nitrogen tank, and quickly putting the HEK293T cells into a water bath kettle at 37 ℃ for unfreezing; (2) after the cells are unfrozen, the cells in the tube are frozen and stored in a super clean workbenchThe suspension was transferred to a 15mL centrifuge tube and 5mL of DMEM (high sugar) complete medium was added to the centrifuge tube. Uniformly mixing the cell suspension, placing the cell suspension in a centrifuge, setting a centrifugation parameter of 100g, and centrifuging for 8 minutes at room temperature; (3) after centrifugation is finished, the supernatant is discarded, 10mL of DMEM (high-sugar) complete culture medium is added, the resuspended cells are slightly blown and beaten, then the cells are transferred to a 10cm culture dish, and the cells are uniformly mixed and placed at 37 ℃ and 5% CO₂Culturing in an incubator overnight; (4) observing the cell morphology and the confluence degree every day under an inverted microscope, and if the cell confluence degree is less than 80%, returning the cells to the incubator for continuous culture; degree of cell confluence>80%, carrying out cell subculture and expanded culture; (5) subculturing: discarding the medium in the dish, adding 5mL PBS to gently rinse the cells once to remove residual medium, adding 1mL 0.05% pancreatin, gently shaking the dish to ensure that all cell surfaces are rinsed with pancreatin, placing the dish at 37 deg.C and 5% CO₂The culture box is taken out for 2 minutes and placed under an inverted microscope for observation until the cell shape becomes round or is separated from the bottom of the culture dish; (6) adding 5mL of DMEM (high-sugar) complete culture medium into the culture dish, uniformly mixing, transferring the cells into a 15mL centrifuge tube, and centrifuging for 5 minutes at 300 g; (7) centrifuging, discarding supernatant, reselecting DMEM (high-sugar) complete culture medium, and standing at 37 deg.C and 5% CO₂And continuously carrying out amplification culture in the incubator.

2. Lentiviral packaging

(1) HEK293T cells cultured as described above were used for virus packaging at 60-70% cell confluence; (2) calculating the required dosage of each plasmid, and configuring a PEI/DNA complex (calculated by a 10ml system on a 10cm culture dish): high-glucose DMEM medium without serum and streptomycin (200 μ L) + CAR (7.5 μ g) + psPAX2(5.625 μ g) + pmd2.g (1.875 μ g) + PEI (45 μ g) mixed by gentle shaking and then allowed to stand at room temperature for 20 minutes; (3) the DNA/PEI complex is added dropwise into a 10cm culture dish, and the culture dish is gently shaken and mixed evenly. Placing the culture dish at 37 ℃ and 5% CO₂An incubator, after culturing for 6-8 hours, absorbing and discarding the culture medium containing the transfection reagent, and replacing the culture medium with a fresh DMEM (high-sugar) complete culture medium; (4) after 48 hours, collecting the culture medium in the culture dish, and replacing fresh DMEM (high-sugar) complete culture medium for continuous culture; (5) culture in the Petri dish was collected again for 72 hoursMixing the culture medium with the culture medium collected for 48 hours, centrifuging for 5 minutes at 400g, discarding cell debris in the culture medium, and filtering with a 0.45um filter membrane; (6) transferring the collected culture medium into an ultracentrifuge tube (25 ml/tube), accurately weighing and balancing on an electronic balance, and centrifuging for 2 hours at 60000g and 4 ℃ by using a Beckman ultracentrifuge; (7) after the centrifugation is finished, the liquid in the centrifuge tube is discarded in a clean bench, 100 mu L of RPMI1640 medium without FBS and streptomycin is added (the added volume is concentrated according to 250 times proportion), the sediment in the centrifuge tube is covered by the medium, and the centrifuge tube is soaked in a refrigerator at 4 ℃ for overnight; (8) the next day, the pellet was resuspended by repeatedly pipetting with a pipette. Subpackaging with 100 μ L/tube, and storing the virus suspension at-80 deg.C.

3. Lentiviral titer determination

(1) 293T cells were plated at 2X 10⁵Inoculate/well in 6-well plates overnight; (2) observing the form of 293 cells in a six-hole plate the next day, taking one hole of cells after confirming that the cells grow well, digesting the cells with pancreatin, and counting; (3) thawing 20 μ L of a subpackaged concentrated virus solution, mixing uniformly, and preparing a virus diluent (containing polybrene of 6 μ g/mL) in an EP tube; (4) the virus concentrate was diluted 10-fold to 200 μ L final volume and mixed well and virus dilutions were made at the following concentrations:

wells 1: 100. mu.L of 10-fold diluted virus solution + 400. mu.L of cell culture medium (containing 10. mu.L of concentrated virus solution);

wells 2: 30. mu.L of 10-fold diluted virus solution + 470. mu.L of cell culture medium (containing 3. mu.L of concentrated virus solution);

wells 3: 10. mu.L of 10-fold diluted virus solution + 490. mu.L of cell culture medium (1. mu.L containing concentrated virus solution);

10. mu.L of the 10-fold diluted virus solution was added to 90. mu.L of the cell culture solution and diluted 10-fold again.

Wells 4: 30. mu.L of 100-fold diluted virus solution + 470. mu.L of cell culture medium (containing 0.3. mu.L of concentrated virus solution);

wells 5: 10. mu.L of 100-fold diluted virus solution + 490. mu.L of cell culture medium (containing 0.1. mu.L of concentrated virus solution);

wells 6: 500 μ L cell culture medium (control);

(5) discarding the original cell culture solution in each hole of the 6-hole plate, and adding the prepared virus diluent with each concentration; (6) after 6 hours, carefully add 2.5ml of fresh medium to each well along the walls of the well; (7) digesting 293 cells in each well after 72 hours, washing, and detecting the proportion of Mcherry positive cells in each well in a flow mode; (8) calculating the virus titer: the number of virus particles/ml (number of 293T cells per well × Mcherry positive cell ratio/volume of virus stock (ml) per well at the time of adding virus solution).

Example 4 preparation of CAR-T

1. Isolation of healthy donor Peripheral Blood Mononuclear Cells (PBMC).

(1) Collecting 10-20ml of peripheral blood of a healthy donor; (2) peripheral blood was diluted with an equal volume of PBS; (3) taking a 15ml centrifuge tube, adding 5ml of ficoll separating medium, and slowly adding 10ml of diluted blood sample to the upper part of the ficoll separating medium along the tube wall by using a pipette gun to avoid mixing the ficoll separating medium and the blood sample; (4) setting the centrifuge as 400g, setting the rotating speed to be increased to 4 gears and the rotating speed to be decreased to 0 gear, and centrifuging for 30 minutes at room temperature; (5) after the centrifugation is finished, the flocculent mononuclear cell layer at the interface of the serum and the ficoll separating medium is gently sucked and transferred to a new centrifuge tube, and the cells are washed for 2 times by PBS.

2. Using EasySep^TMHuman T Cell negative selection kit T cells were isolated from PBMC (Stem Cell Co., USA, CAT #17951)

(1) The obtained mononuclear cells were counted, the cells were resuspended in PBS containing 2% FBS, and the cell concentration was adjusted to 5X 10⁷And/ml. (2) The samples were transferred to a 5ml sterile flow tube, 50 μ L of the isolated antibody combination was added per ml of sample and mixed well and incubated for 5min at room temperature. (3) Shaking RapidSpheres^TM30s, 40. mu.L RapidSpheres per ml of sample^TMAnd mixed well and incubated at room temperature for 30 s. (4) An amount of PBS containing 2% FBS was added to a total volume of 2.5 mL. (5) Placing the sterile flow tube with the sample into easy Sep^TMThe magnetic pole (StemCell, Canada, Catalog #18000) was left to stand at room temperature for 3 min. (6) A new 15ml centrifuge tube was taken and the magnetic pole and associated sterile flow tube were poured to pour the cell suspension in the sterile flow tube into the 15ml centrifuge tube. The cell suspension containing the enrichmentThe T cell (the cell is marked with anti-human CD3 PE-cy7 flow antibody, and the purity of the detection by a flow cytometer is more than 90 percent). (7) Setting the centrifuge parameter as 300g, 5min, centrifuging and precipitating T cell mass.

3. Activation of T cells Using anti CD3/CD28 Dynabeads

(1) Counting the T cells obtained, resuspending in RPMI1640 complete medium containing IL-2(200IU/ml), adjusting the cell concentration to 10⁷And/ml. (2) Washing anti-CD3/CD28 magnetic beads (clinical research grade: Cat #40203D, Thermo) with 1% BSA/PBS solution for 2 times; the washing method comprises the following steps: placing 5ml of 1% BSA/PBS solution into a 50ml centrifuge tube, adding the amount of anti-CD3/CD28 magnetic beads required after calculation, fully and uniformly mixing, placing on a magnetic frame, standing for 1 minute, attaching the magnetic beads to the walls of the centrifuge tubes on two sides, sucking and discarding the 1% BSA/PBS solution. The washing was repeated 1 time. (3) And (3) according to the condition that the purity of the T cells is 95 percent and the ratio of the magnetic beads to the CD3(+) T cells is 3: 1, fully mixing the washed anti-CD3/CD28 magnetic beads with the T cells, transferring the mixture into a T25 cell culture bottle, and slightly shaking the mixture for 30 minutes by a shaking table to ensure that the magnetic beads are fully combined with the CD3(+) T cells. (4) The CD3(+) T cells bound to the magnetic beads were resuspended in complete medium in RPMI1640 containing IL-2(200IU/ml) and the cell concentration was adjusted to 1X 10⁶/ml，37℃、5％CO₂And (5) culturing for 24 hours in a saturated humidity incubator.

4. Lentiviral infection of T cells

(1) The CD3(+) T cells bound to the magnetic beads were centrifuged and counted, resuspended in complete medium RPMI1640 containing IL-2(200IU/ml), adjusted to a cell concentration of 4X 10⁶Each well was inoculated at 500. mu.L/well in 12-well plates. (2) The required amount of virus was calculated according to MOI ═ 3. The calculation formula is as follows: required amount of virus (MOI × number of cells)/viral titer. (3) The virus was removed from the freezer at-80 ℃ and rapidly thawed in a 37 ℃ water bath. Adding the amount of the virus obtained by the calculation into a 12-well plate, reserving a hole of T cells without adding the virus as a T cell control (untraduced T) not infected with CAR, adding polybrene with a final concentration of 6 mu g/mL into each hole of the T cells, fully mixing, placing at 37 ℃ and 5% CO₂The culture chamber (2) was supplemented with RPMI1640 complete medium containing IL-2(200IU/ml) to 2ml after 6 to 8 hours, and the culture was continued for 24 hours. (4) After 24 hours, centrifuge at 300g5 minutes, remove virus-containing medium supernatant, resuspend the cell pellet with fresh IL-2(200IU/ml) in RPMI1640 complete medium, transfer the cells to a six-well plate or flask, after which time the medium turns yellow or the cell density exceeds 2.5X 10⁶At/ml, cells were replated and cultured in fresh RPMI1640 medium containing IL-2(200 IU/ml). (5) 4 days after virus infection, the CAR-T cells in the culture flask were blown up using a 5ml pipette and transferred to a 50ml centrifuge tube, placed on a magnetic rack and left to stand for 1min, after the magnetic beads were adsorbed to the tube wall, the cell suspension was transferred to a new centrifuge tube, centrifuged and the complete medium RPMI1640 containing fresh IL-2(200IU/ml) was added to continue the expansion culture. (6) A portion of the cells were taken and the expression of the cell surface CAR molecule was detected using flow cytometry.

5. Flow cytometry detection of CAR-T cell ratio

(1) Preparing a cell suspension: collecting CAR-T cells prepared and amplified in vitro, centrifuging, washing, counting, and collecting 3 × 10 cells⁵Individual cells were added to the flow tube and the cells were resuspended in a single cell suspension using 100. mu.L of PBS buffer. (2) Labeling the antibody: add 1.5. mu.L of Goat Anti-Mouse IgG, F (ab')2fragment specific antibody to the corresponding flow sample tube and incubate at 4 ℃ for 30min in the dark. (3) Washing: adding 1ml PBS buffer solution into each tube, mixing uniformly, centrifuging for 5min at 300g at normal temperature, and removing supernatant. (4) Add 100. mu.L PBS to the flow tube, mix well, add streptavidin FITC 1.5. mu.L, incubate 30min at 4 ℃ in the dark. (5) Washing: adding 1ml PBS buffer solution into each tube, mixing uniformly, centrifuging for 5min at 300g at normal temperature, and removing supernatant. (6) Detection and analysis: the labeled cells were resuspended using 500. mu.L of PBS buffer and tested on a flow cytometer. (7) The data were analyzed by the FlowJo X10.0.7 r2 software, and the results are expressed as positive cell ratio, and the ratio of CAR-T cells is RFP/CAR double positive cell ratio.

As shown in FIG. 3, the virus was added at MOI-3 to infect T cells, the infection efficiency was 50% or more, the expression of CAR molecule on the cell surface was good, and the detection of murine scFv by using Goat Anti-Mouse IgG, F (ab')2 fragment-specific antibody was excellent.

Example 5 flow sorting of CAR-T cells

(1) T cells4-5 days after infection with the CAR-bearing lentivirus, cells were harvested after flow assay to determine cell surface CAR expression. (2) Flow cytometric sorting buffer (PBS containing 2% FBS and 2% streptomycin) the CAR-T cells were resuspended at a cell concentration of 3-5X 10⁷And (3) ml. (3) And (3) loading the cell on a machine, selecting and collecting Mcherry positive cells in a culture solution containing 40% FBS and 2% streptomycin RPMI1640 under a sterile environment, and taking a small amount of cells after sorting to perform retest to determine whether the sorting purity is greater than 90%. (4) The sorted CAR-T cells were incubated at 37 ℃ with 5% CO₂Incubate in a saturated humidity incubator for 20 min. (5) Setting centrifugation parameter at 200g, centrifuging for 20min, discarding supernatant, suspending cells with 2% streptomycin and IL-2200IU/ml RPMI1640 complete culture medium, adjusting cell concentration to 0.5-1 × 10⁶ml is placed at 37 ℃ and 5% CO₂And (5) continuing culturing in a saturated humidity incubator.

EXAMPLE 6 Nalm-6 cell line culture and cryopreservation

(1) Conventional culture of tumor cells with RPMI1640 complete culture medium at 37 deg.C and 5% CO₂Culturing in a saturated humidity incubator, changing the culture medium for passage once in 2-3 days, and taking the cells in the logarithmic growth phase for experiment. (2) Freezing and storing cells: taking cell strain in logarithmic growth phase, centrifuging for 5min at 300g, discarding supernatant, adding freezing medium (prepared from 90% FBS + 10% DMSO), and adjusting density to 1 × 10⁷and/mL. Subpackaging according to 1ml cell suspension/cryopreservation tube, and sealing the cryopreservation tube with a sealing film. The cryopreservation tube is placed in a cell cryopreservation box, placed in a refrigerator at minus 80 ℃ for 24 hours, and then transferred into liquid nitrogen.

Example 7 establishment of Nalm-6 cell line overexpressing luciferase and GFP

(1) The Nalm-6 cell line is cultured in RPMI1640 complete culture medium at 37 deg.c and 5% CO₂A saturated humidity incubator. (2) The pHIV-Luc-ZsGreen lentiviral vector plasmid (cat # 39196) was purchased from Addgene. (3) Plasmids were transformed, bacteria amplified, plasmids extracted, and stored in a freezer at-20 ℃ as described above. (4) The virus was packaged as described above and stored in a freezer at-80 ℃. (5) Taking Nalm-6 cell strain in logarithmic growth phase, adjusting cell concentration to 4 x 10⁶The virus was inoculated into a 12-well plate at 500. mu.L/well, and the amount of the virus required was calculated at an MOI of 3. (6) Disease of additionAfter the toxicity, polybrene with the final concentration of 6 mug/mL is added, the mixture is fully mixed and placed at 37 ℃ and 5% CO₂The culture was continued for 24 hours in an incubator (2 ml) supplemented with RPMI1640 complete medium. (7) After 24 hours, the cells were centrifuged at 300g for 5 minutes, the virus-containing medium supernatant was removed, the cell pellet was resuspended in fresh medium, the cells were transferred to a six-well plate or flask, and incubation was continued for 3-6 days. (8) The proportion of GFP positive Nalm-6 cells was determined by flow cytometry. (9) And (3) sorting and collecting GFP positive Nalm-6 cells by a flow cytometry sorter, ensuring that the proportion of the GFP positive Nalm-6 cells is more than 90%, culturing for a long time, and freezing and storing part of the cells.

As shown in FIG. 4, Nalm-6 cell lines overexpressing GFP and luciferase showed green fluorescence indicating successful transfection.

Example 8 evaluation of killing function of CAR-T cells in vitro

(1) The cultured CAR-T and untraduced T cells were harvested, centrifuged, resuspended in complete medium RPMI1640 containing IL-2(200IU/ml), and adjusted to a cell concentration of 2X 10⁵And/ml. (2) The cultured luciferase (+) Nalm-6 cells were collected, centrifuged, resuspended in complete medium RPMI1640 containing IL-2(200IU/ml), and the cell concentration was adjusted to 2X 10⁵And/ml. (3) mu.L of luciferase (+) Nalm-6 cells were added to a round bottom 96 well plate. (4) Adding corresponding amount of effector cells (CAR-T and T cells) into round-bottom 96-well plate at effective target ratio of 1: 1, 0.5: 1, and 0.25: 1, respectively, sucking into 100 μ L, 50 μ L, and 25 μ L, respectively, placing at 37 deg.C and 5% CO₂Was cultured in the incubator of (1) for 8 hours. (5) Round-bottom 96-well plates were centrifuged at 400g for 10min, the supernatant discarded, and 50. mu.L PBS was added to each well. (6) After mixing, cells were transferred to black opaque 96-well plates. (7) 50 μ L of Bright-Glo per well^TMAnd (3) a Luciferase Assay System, uniformly mixing and keeping out of the sun for 2min, and detecting the fluorescence value of each hole by using an enzyme labeling instrument. (8) The killing rate calculation method comprises the following steps: the killing rate (%) - (T-cell well fluorescence value-CART-cell well fluorescence value)/T-cell well fluorescence value × 100%.

As a result: CAR-T has the obvious function of killing Nalm-6 cell strain in vitro. And the killing effect becomes stronger as the target ratio increases (fig. 5).

Example 9 cell subpopulation detection

(1) The mcherry positive CAR-T cells and untraduced T cells cultured for 6 days were taken, resuspended in rpml 640 complete medium containing human IL-2200IU/mL and counted using a full-automatic cell counter at 3 × 10⁵Inoculating to 6-well plate, adding equal volume of trametinib and DMSO with different concentrations as control, culturing at 37 deg.C in 5% CO2 saturated humidity incubator, centrifuging every 3 days, changing liquid, adding trametinib again, and counting to obtain 1 × 10⁶The CAR-T cells were re-dosed and cultured. (2) Grouping experiments: CAR-T + trametinib 7.5nmol/L group, CAR-T + trametinib 15nmol/L group, CAR-T + trametinib 30nmol/L group, CAR-T + isovolumetric DMSO group, and untraduced T + isovolumetric DMSO group. (3) Collecting time points: flow cytometry assays were performed at day 3 and day 6 after dosing (equivalent to in vitro culture at day9 and day12), respectively. (4) Flow cytometry detection operation steps: firstly, sample collection and processing: uniformly mixing each group of cell suspension in the six-hole plate, counting, respectively sucking a proper amount of cell suspension for each group, centrifugally washing, adding the cell suspension into a flow tube according to 3-4 multiplied by 10^5 cells/tube, and re-suspending the cells by using 100 mu L of PBS buffer solution; ② labeling the antibody: adding 1.5 μ L of corresponding fluorescence labeled CD4, CD8, CD45RO and CD62L antibody into corresponding flow sample tubes, and incubating at 4 ℃ in dark for 30 min; washing: adding 1ml PBS buffer solution into each tube, uniformly mixing, centrifuging for 5min at 300g at normal temperature, and removing supernatant; and fourthly, detection and analysis: the labeled cells were resuspended in 500. mu.L PBS buffer, examined on a flow cytometer, analyzed using FlowJo X10.0.7 r2 software, gated on mCherry positive CAR-T as the assay target, and the ratio of each subpopulation was analyzed. (5) Each subpopulation of cells is 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 shown to scale.

As a result: we added trametinib at day 6 of 28z CAR-T in vitro culture and continued to culture 28z CAR-T to day12, and then samples were taken at day9 and day12 for flow assay. We have found that: the 28z CAR-T subgroup was predominantly TCM and TEM, whether on day9 or day 12. At day9, the effect of trametinib in reducing the terminal differentiation of 28z CAR-T was not significant since 28z CAR-T had not differentiated downstream, but by day12, terminal differentiation of 28z CAR-T occurred with the drive of tonic signaling compared to untraduced T, and the effect of trametinib in reducing terminal differentiation of 28z CAR-T was demonstrated, as was the significant increase in the proportion of TCM-treated 28z CAR-T cells, as was the significant decrease in the proportion of TEM, and this increase and decrease in TEM were evident upon low concentration of trametinib (7.5nM and 15nM) and exhibited a dose-dependent effect, i.e., the degree of change increased with increasing concentration of trametinib in the culture system (fig. 6).

Example 10 detection of cell activation and depletion associated surface molecules

(1) Experimental groupings, cell cultures, sample collection time points, and flow cytometry sample preparation procedures were all identical to those of example 9. (2) Samples, fluorescently labeled antibodies labeled CD25, CD69, PD-1, TIM-3, and LAG-3, and corresponding isotype control antibodies were collected as described above, washed, resuspended, and assayed by flow cytometry. (3) And (3) data analysis: data were analyzed using FlowJo X10.0.7 r2 software, gated on mcherry-positive CAR-T as the assay target, and the results are presented as positive cell fraction. (4) The activation markers are expressed as positive ratio of CD25 to CD 69; the markers associated with depletion are expressed as positive ratios of PD-1, TIM-3, LAG-3.

As a result: we added trametinib on day 6 of 28z CAR-T in vitro culture and continued the culture to day12, then samples were taken on days 9 and 12 for flow assays. We have found that: on both day9 and day12, trametinib was able to reduce the extent of CAR-T activation, reducing the expression of tonic signaling-driven depletion-associated inhibitory molecules (PD-1, TIM-3, LAG-3). And this decrease in depletion and activation markers was evident upon treatment with lower concentrations of trametinib (7.5nM and 15nM) and exhibited a dose-dependent effect, i.e. the extent of change increased with increasing concentration of trametinib in the culture system (figure 7).

Example 11 CAR-T cell Absolute number counting and proportional detection

(1) Taking the mcherry positive CAR-T fine cultured for 6 daysCells and untraduced T cells, resuspended in complete medium RPMll640 containing human IL-2200IU/mL and counted using a full-automatic cell counter at 3X 10⁵Perwell was inoculated into 6-well plates and equal volumes of trametinib and DMSO at different concentrations were added as controls, 5% CO at 37 ℃₂Culturing in a saturated humidity incubator, centrifuging every 3 days, changing the liquid, adding trametinib again, counting and detecting the proportion of CAR-T cells; each group is respectively reserved with 1 multiplied by 10⁶The CAR-T cells were re-dosed and cultured. (2) The experimental groups are CAR-T + trametinib 7.5nmol/L group, CAR-T + trametinib 15nmol/L group, CAR-T + trametinib 30nmol/L group, CAR-T + isometric DMSO group, and untraduced T + isometric DMSO group. (3) The cell counting and flow cytometry detection comprises the following specific operation steps: firstly, collecting time points: sampling and detecting at 3 days and 6 days (equivalent to 9 days and 12 days of in vitro culture) after dosing; collecting and processing samples: after mixing the cell suspensions in the six-well plate, respectively sucking the cell suspensions into 15ml centrifuge tubes, centrifuging for 5min at 300g, and then discarding the supernatant. 1ml of RPMI1640 containing IL-2200IU/ml was added to each tube to resuspend the cell pellet and mix well, then 15. mu.L of cell suspension was aspirated from each tube, and diluted 1: 1 with 15. mu.L of 0.8% Trypan blue. Adding the diluted cell suspension into a cell counting plate, counting the number of living cells for three times by using a Count Star full-automatic cell counter, and taking an average value; additional groups were aspirated with appropriate amounts of CAR-T cells labeled with Goat Anti-Mouse IgG, F (ab')₂fragment-specific antibodies; thirdly, sample detection: flow cytometry was used to measure the ratio of mcherry and CAR and fluorescence intensity in the samples, and FlowJo X10.0.7 r2 software was used to analyze the ratio of mcherry/CAR dicationic CAR-T cells in the samples. Fourthly, the calculation method: CAR-T cell expansion fold ═ number of viable cells × CAR-T cell ratio/number of naive cells.

As a result: trametinib inhibits proliferation of 28z CAR-T cells cultured in vitro, and this proliferation-inhibiting effect appears dose-dependent, i.e., the higher the concentration of trametinib in the culture system, the more pronounced it inhibits proliferation of 28z CAR-T cells. In addition, the effect of trametinib on inhibiting 28z CAR-T cell proliferation was more pronounced with prolonged in vitro culture time. Exhibited greater differences in fold expansion of 28z CAR-T cells between the trametinib-treated and DMSO-treated groups at day12 than at day9 (fig. 8A, B, C). Since we added trametinib at day 6 of 28z CAR-T in vitro culture, this reduction in expansion fold did not affect the clinical availability of sufficient numbers of 28z CAR-T cells. Moreover, the data of examples 9 and 10 show that trametinib significantly reduces over-activation, depletion, apoptosis and terminal differentiation of 28z CAR-T at low concentrations (7.5nM and 15nM), whereas trametinib does not have a strong effect on inhibiting 28z CAR-T amplification at low concentrations, so to balance the effects of 28z CAR-T amplification and trametinib anti-depletion and terminal differentiation, we propose to add trametinib at a concentration of 15nM to the 28z CAR-T culture system.

Example 12 Annexin V flow assay

(1) Cell culture, experimental grouping and sample collection time points were consistent with example 9. (2) After collecting samples as described in example 9, adding them to flow tubes and washing out the residual medium by centrifugation in PBS, 100. mu.L of 1 × Annexin V binding buffer was added to each tube to resuspend the cells. (3) Add 1.5. mu.L Annexin V APC per tube and incubate at 4 ℃ for 30min in the dark. (4) After incubation, 500. mu.L of 1 × Annexin V binding buffer was added directly and the cells were mixed and tested on a flow cytometer. The results were expressed as the proportion of Annexin V positive cells, analyzed using FlowJo X10.0.7 r2 software.

As a result: we added trametinib at day 6 of 28z CAR-T in vitro culture and continued to culture 28z CAR-T to day12, and then samples were taken at day9 and day12 for flow assay. We have found that: on day9, 28z CAR-T did not undergo significant apoptosis compared to untraduced T, when the proportion of apoptotic cells in the group treated with trametinib was slightly increased on the contrary, but there was no statistical difference (FIG. 9A). However, by day12, significant apoptosis occurred in 28z CAR-T versus untraduced T under the drive of tonic signaling, at which time the proportion of 28z CAR-T in the trametinib-treated group versus 28z CAR-T apoptotic cells in the DMSO-treated group was reduced, and this reduction was evident at low concentrations of trametinib (7.5nM and 15nM), with statistical differences (FIG. 9B).

Example 13 multifunctional microplate reader assay to analyze the Effect of in vitro addition of trametinib culture on CAR-T killing function

(1) Grouping experiments: CAR-T + trametinib 15nmol/L group, CAR-T + equal volume DMSO group. (2) Cell culture and sample collection time points were consistent with example 11. (3) Collecting the cultured CAR-T cells, untransfected T cells and luciferase (+) Nalm-6 cells, and inoculating the cells in a round bottom 96-well plate according to the configured cell concentration and the effective-target ratio of 0.25: 1, 0.5: 1 and 1: 1; when the CAR-T and Nalm-6 cells are cultured in a 96-well plate, trametinib is not added, and the cells are placed at 37 ℃ and 5% CO₂Was cultured in an incubator for 16 hours. (4) After 16 hours, the cells were centrifuged, transferred, PBS and Bright-GloTM Luciferase Assay System were added, and fluorescence values of each well were measured with a microplate reader, according to the procedure described in example 8. (5) The killing rate calculation method comprises the following steps: the killing rate (%) - (T-cell well fluorescence value-CART-cell well fluorescence value)/T-cell well fluorescence value × 100%.

As a result: the in vitro killing function of 28z CAR-T was not affected by the culture system containing trametinib. Since it was observed that the trametinib-containing 28z CAR-T culture system could alter the ratio of CD8+ T cells and CD4+ T cells in 28z CAR-T, we further evaluated whether this culture system would affect the killing function of 28z CAR-T in vitro. We added trametinib at 6 days of 28z CAR-T in vitro culture and continuously cultured CAR-T to 12 days, then samples were taken at 9 days and 12 days and co-cultured with Nalm-6 cells with luciferase for 16 hours at effective target ratios of 0.25: 1, 0.5: 1 and 1: 1 respectively to perform in vitro killing experiments, and no trametinib was added during in vitro killing. We have found that: on either day9 or day12, the in vitro culture system containing trametinib did not affect the killing function of 28z CAR-T at each effect-target ratio (figure 10).

Example 14 ALL-NSG mice the efficacy of trametinib-treated CAR-T cells was evaluated in vivo.

(1) CAR-T cell preparation: untraduced T cells and Mchery-carrying CAR-T cells were prepared, 6 days of culture of the CAR-T cells were taken, and the proportion of CAR-T cells was measured by flow cytometry. Divided into 3 groups for culturing a first CAR-T + trametinib 15nM group, and a second CAR-T + DMSO with the same volumeGroup (③ untraduced T + isovolumetric DMSO group), the medium was changed every 3 days and the drug was added again, and the culture was continued for 9 days. (2) ALL-NSG mouse model preparation: NSG mice 6-8 weeks old were housed in the SPF-level animal research center. Taking a luciferase (+) Nalm-6 cell strain in logarithmic growth phase, and preparing the cell concentration to 5 multiplied by 10⁶Per ml, in a 1X 10⁶Rat tail vein injection, total volume 200 μ L per mouse. After 5 days, the small animal living body imaging instrument detects the tumor load, the tumor load is randomly divided into 3 groups according to the fluorescence intensity, the average fluorescence intensity of the 3 groups is adjusted without obvious difference, and different processed CAR-T cells are injected into tail veins on the next day. (3) Grouping experiments: the experiment was divided into 3 groups of 4 mice each, namely (i) tail vein injection of CAR-T cells treated with trametinib 15nM for 9 days, ((ii) tail vein injection of CAR-T cells treated with an equal volume of DMSO for 9 days, ((iii) tail vein injection of untraduced T cells treated with an equal volume of DMSO for 9 days. (4) Tail vein injection of CAR-T cells: according to 3X 10⁶CAR-T cells/mouse and calculating the total cell amount required for each mouse based on the CAR-T cell ratio, collecting cultured CAR-T cells, centrifuging, resuspending in PBS at a concentration of 1.5 × 10⁷CAR-T cells/ml; the total cell mass of each group of mice transfused into the body was adjusted to be uniform using untraduced T cells, and each prepared cell suspension was inoculated into NSG mice by tail vein injection in a volume of 200 μ L/mouse. (5) Observation of curative effect and survival: imaging mice by using a small animal living body imaging instrument every week, and comparing the tumor load difference of three groups of mice; secondly, comparing and observing the hair color and the activity state of each group of mice; recording the death time of each group of mice, and drawing a survival curve.

We added 15nM trametinib at day 6 of CAR-T in vitro culture for continuous culture of CAR-T from day9 to day12, and then returned CAR-T cells to ALL tumor mice at day 12. The change of the tumor load of the mice is detected by live imaging of the mice at regular intervals, and a survival curve of the mice is drawn. We have found that: the 28z CAR-T after trametinib in vitro treatment has longer duration in vivo due to the richer TCM, and can exert better antitumor effect in vivo (fig. 11, fig. 12A, B). Wherein the median survival time of mice infused with DMSO-treated untraduced T cells was 27 days, the median survival time of mice infused with trametinib-treated 28z CAR-T was 62.5 days, and the median survival time of mice infused with DMSO-treated 28z CAR-T was 41 days, and the survival times of these three groups of mice were statistically different from each other, indicating that the in vitro CAR-T culture system containing trametinib can improve the in vivo efficacy of 28z CAR-T cells.

Sequence listing

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