A method for producing car-t cells and use thereof

Abstract

The present invention provides a CAR-T cell production method and application thereof, which belongs to the field of tumor immunotherapy technology. The present invention first prepares CAR-T cells that overexpress Runx3, and then amplifies the CAR-T cells that overexpress Runx3 in a 1μM AKTi1 / 2 environment, thereby producing a new type of CAR-T cell. Wherein Runx3 overexpression can enhance the effector function of CAR-T cells and promote CAR-T cell tumor infiltration, but does not affect the maintenance of T cell stemness that depends on AKT inhibitors. The use of AKT inhibitors can save the problem of CAR-T cells not being able to survive continuously and amplify insufficiently in vivo. The two work together and complement each other, so the new CAR-T cells produced have better long-term survival, effect and solid tumor infiltration capabilities, and thus have a stronger comprehensive ability to overcome the dilemma of solid tumors.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of tumor immunotherapy, and particularly relates to a CAR-T cell production method and application thereof. BACKGROUND

[0002] CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy) is a kind of immunotherapy, and CAR-T technology has an impressive and excellent effect in the treatment of hematological tumors and lymphoma. However, in the treatment of solid tumors, CAR-T technology still faces many unique challenges. These unique challenges mainly include: lack of tumor cell specific targets, insufficient ability of CAR-T cells to survive and proliferate in vivo, low tumor infiltration ability, and immunosuppressive tumor microenvironment. In fact, scientists have gradually found some better CAR-T treatment targets, such as CLDN18.2 targets in gastric cancer and pancreatic cancer. However, even if there are available targets, only if the other three challenges are solved at the same time, can CAR-T treatment have a better effect on solid tumors.

[0003] Clinical studies have found that adoptive transfer of CAR-T cells with low differentiation and memory phenotype has stronger anti-tumor ability and better prognosis, because they have stronger ability to survive and proliferate in vivo. There have been many attempts to produce memory phenotype CAR-T cells and maintain T cell stemness, including using cytokines such as IL-7 and IL-15 to replace IL-2 which promotes conversion to effector phenotype, drug inhibition of PI3K-AKT pathway, activation of WNT pathway, etc. However, these methods can only promote the survival and proliferation of CAR-T cells in vivo, and cannot promote the tumor infiltration of CAR-T cells. Recent reports show that transcription factor Runx3 can promote the development of lymphocytes into tissue-retaining memory T cells. Overexpression of Runx3 in CAR-T cells promotes the development of tissue-retaining memory T cells, so that the tissue-retaining ability of lymphocytes can help CAR-T cells anchor in the tumor site, which may solve the problem of insufficient infiltration of effector lymphocytes in the tumor microenvironment. However, overexpression of Runx3 cannot solve the problem of insufficient survival and proliferation of CAR-T cells in vivo. SUMMARY

[0004] In order to solve the above technical problems, the application provides a CAR-T cell production method and application thereof. The application first prepares CAR-T cells overexpressing Runx3, and then amplifies the CAR-T cells overexpressing Runx3 in a 1 μM AKTi1 / 2 environment, thereby producing a new type of CAR-T cell. The overexpression of Runx3 can enhance the effector function of the CAR-T cell and promote the tumor infiltration of the CAR-T cell, but does not affect the maintenance of the T cell stemness dependent on the AKT inhibitor, and the use of the AKT inhibitor can save the problem that the CAR-T cell cannot survive and expand in vivo. The two are synergistic and complementary, so that the new type of CAR-T cell produced has better long-term survival, effector and solid tumor infiltration capacity, and thus has stronger comprehensive ability to overcome the difficulties of solid tumors. In addition, the method of the application is very suitable for the existing CAR-T cell production process and has great clinical application value.

[0005] In order to achieve the above purpose, the application adopts the following technical scheme:

[0006] The application provides a CAR-T cell production method, mainly including the following two steps: step one, preparing CAR-T cells overexpressing Runx3; and step two, amplifying the CAR-T cells overexpressing Runx3 in a 0.5-1.5 μM AKTi1 / 2 environment.

[0007] Preferably, the specific process of the step one is as follows: (1) preparing a CAR virus expression vector overexpressing Runx3; (2) producing a CAR virus overexpressing Runx3; (3) T cell separation and activation; and (4) virus infection of the activated T cells.

[0008] The application uses the strategy of combining Runx3 overexpression and drug inhibition of AKT to produce a new type of CAR-T cell which has better long-term survival, effector and solid tumor infiltration capacity, and can improve the curative effect of solid tumor CAR-T cell therapy.

[0009] The Runx3 gene is Runt-related transcription factor 3, and the nucleotide sequence of the human source in the NCBI database is NM_001031680, and the nucleotide sequence of the mouse source is NM_019732.

[0010] The AKT inhibitor is Akt Inhibitor VIII, also known as AKTi1 / 2, and the working concentration is 1 μM, and the molecular formula is C34H29N7O.

[0011] The application further provides application of the CAR-T cell production method in production of human CAR-T cells.

[0012] Preferably, the Runx3 is human Runx3, the CAR virus is CAR lentivirus, and the T cell is human peripheral blood lymphocyte.

[0013] Preferably, in the step (1), A. the CAR adopts the composition of the third generation CAR, which is composed of human CD8 signal peptide, scFv fragment, human CD8 extracellular region, CD28 transmembrane region and intracellular segment, 4-1BB intracellular segment, CD3ζ intracellular segment, the nucleotide sequence is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2; B. the human Runx3 is connected with the CAR through P2A sequence, so as to achieve the purpose of co-overexpression of human Runx3 and CAR, the nucleotide sequence is shown as SEQ ID NO. 3, and the amino acid sequence is shown as SEQ ID NO. 4; C. the lentivirus expression vector skeleton is pLVX-EF1a-IRES-mCherry; D. the IRES-mCherry is replaced by CAR-P2A-RUNX3, so as to obtain the CAR lentivirus expression vector pLVX-EF1a-CA9CAR-P2A-Runx3 overexpressing human Runx3.

[0014] The CAR is anti-human CA9 CAR.

[0015] In the step (2), a three-plasmid system (pLVX, psPAX2, pMD2.G = 4:3:1) is adopted, and the calcium phosphate transfection method is used to co-transfect 293T cells, so as to produce lentivirus. The lentivirus supernatant is concentrated by the PEG6000 method.

[0016] In the step (3), human peripheral blood lymphocyte separation medium is used to separate human peripheral blood mononuclear cells from human peripheral blood. Dynabeads Human T-Activator CD3 / CD28 is used to activate T cells. The T cells are cultured in AIM-V medium containing 5% Immune Cell Serum Replacement, 100 IU / mL IL-2.

[0017] In the step (4), the T cell density is 1-3x10 6 / mL, the lentivirus MOI is 40:1, 8 μg / ml polybrene, 300 IU / mL IL-2, 32°C, 1200g centrifugal infection for 2 hours, 4 hours after liquid change.

[0018] Preferably, in the application, the specific process of step two is that when the T cells are expanded, the cell density is adjusted to 0.5x10 6 / mL, cultured in AIM-V medium with 5% Immune Cell Serum Replacement, 100 IU / mL IL-2, and supplemented with a final concentration of 1 μM AKTi-1 / 2, and the medium was changed every other day.

[0019] The method of step two can maintain T cell stemness while expanding CAR-T cells.

[0020] The application also provides an application of the CAR-T cell production method in producing mouse-derived CAR-T cells.

[0021] Preferably, the Runx3 is mouse Runx3, the CAR virus is a CAR retrovirus, and the T cells are mouse spleen T cells.

[0022] Preferably, in step (1): A. the CAR adopts the composition of the second-generation CAR, which is composed of a mouse CD8 signal peptide, a scFv fragment, a mouse CD8 extracellular region and transmembrane region, a CD28 intracellular segment, and a CD3ζ intracellular segment, with a Strep tag II added at the N-terminus for convenient detection, the nucleotide sequence being shown as SEQ ID NO. 9, and the amino acid sequence being shown as SEQ ID NO. 10; B. the mouse Runx3 is connected with the CAR through a P2A sequence, achieving the purpose of co-overexpression of the mouse Runx3 and the CAR, the nucleotide sequence being shown as SEQ ID NO. 11, and the amino acid sequence being shown as SEQ ID NO. 12; C. the retrovirus vector skeleton is pMSCV-IRES-GFP II (pMIGII); and D. the IRES-GFP is replaced with CAR-P2A-RUNX3 to obtain a CAR retrovirus expression vector pMIGII-CA9CAR-P2A-RUNX3 overexpressing mouse Runx3.

[0023] The CAR is an anti-human CA9 CAR.

[0024] In step (2), the retrovirus vector plasmid is transfected into PLAT-E cells by calcium phosphate transfection to produce retroviruses. The retrovirus supernatant is concentrated by ultrafiltration.

[0025] In step (3), the mouse spleen cells are activated by anti-mouse-CD3 (1 μg / mL) and anti-mouse-CD28 (0.5 μg / mL) antibodies. The activated T cells are enriched by Percoll density gradient centrifugation. The anti-mouse-CD19 antibody and MojoSort Mouse CD4 / CD8 Kit are used to isolate CD4+ T cells. TMStreptavidin Nanobeads remove CD19+ B cells. T cells are cultured in RPMI 1640 medium containing 10% FBS, 100 IU / mL IL-2.

[0026] The T cell density in step (4) is 1-3x10 6 IU / mL IL-2, 32℃, 2000g centrifugal infection for 2 hours, 0 up and 0 down, and the medium is changed after 4 hours.

[0027] Preferably, the specific process of step two in the application is: when the T cells are expanded, the cell density is adjusted to 0.5x10 6 IU / mL IL-2, and 1 μM AKTi-1 / 2 is added to the final concentration, and the medium is changed every other day.

[0028] The application also provides a method for detecting the memory phenotype and effector function of CAR-T cells, and the specific steps are: the CAR-T cells are stained with CD4, CD8 and CD62L surface proteins, and Granzyme B intracellular protein staining is performed, and then flow cytometry is used for detection.

[0029] The application also provides a method for detecting the effector function of mouse CAR-T cells, and the specific steps are: the mouse pancreatic cancer KPC cell line is overexpressed with human CA9, and then the mouse CAR-T is co-incubated with the KPC-hCA9 cell line at a ratio of 4:1, 2:1, 1:1, 1:2 for 14 hours, and the surviving KPC cells are adherent after 14 hours, the dead cells and T cells are resuspended and discarded, the CCK-8 reagent is used to detect the survival rate of KPC cells, and the killing efficiency of CAR-T cells is calculated.

[0030] The application also provides a method for detecting the anti-tumor ability of mouse CAR-T cells in vivo, and the specific steps are: 6-week-old nude mice are subcutaneously inoculated with tumor cells, and 1 week later, 5x10 6 CAR-T cells are injected into the tail vein, and the vernier caliper is used to measure the length and short diameter of the tumor every week, and the tumor volume is calculated (1 / 2x length x short diameter x short diameter), and the anti-tumor ability of CAR-T cells is compared.

[0031] Compared with the prior art, the application has the following beneficial effects:

[0032] (1) The application provides a combination of Runx3 overexpression and drug inhibition of AKT strategy, which can produce CAR-T cells with better long-term survival, effector and solid tumor infiltration ability, and can be effectively applied to the production of human and mouse-derived CAR-T cells.

[0033] (2) The present application proves that Runx3 overexpression and in vitro use of AKT inhibitor strategy can complement each other and synergistically produce CAR-T cells with better persistence, effect and solid tumor infiltration capacity.

[0034] (3) The present application proves that CAR-T cells produced by the combined strategy have stronger anti-tumor capacity in a mouse model.

[0035] (4) The present application proves that CAR-T cells produced by the combined strategy can survive for a long time in vivo and have better solid tumor infiltration capacity in a mouse model.

[0036] In summary, the present application provides a method for producing CAR-T cells with better long-term survival, effect and solid tumor infiltration capacity by combining Runx3 overexpression and drug inhibition of AKT strategy and its application. The novel CAR-T cells produced by the method can enhance the efficacy of solid tumor CAR-T cell therapy. The method has great clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The specific composition of the human CA9CAR and CA9CAR-RUNX3 genes in Example 1 is shown in Table 1.

[0038] Figure 2 The schematic diagram of the plasmid pLVX-EF1a-IRES-mCherry in Example 1 is shown in Figure 1.

[0039] Figure 3 The schematic diagram of the human CAR-T cell production process in Examples 1-4 is shown in Figure 2.

[0040] Figures 4-6 The flow cytometry analysis chart in Example 4 is shown in Figure 3.

[0041] Figure 7 The specific composition of the murine CA9CAR and CA9CAR-RUNX3 genes in Example 1 is shown in Table 2.

[0042] Figure 8 The schematic diagram of the plasmid pMESV-IRES-GFPII in Example 1 is shown in Figure 4.

[0043] Figure 9 The schematic diagram of the murine CAR-T cell production process in Examples 1 and 5-7 is shown in Figure 5.

[0044] Figures 10-12 The flow cytometry analysis chart in Example 7 is shown in Figure 6.

[0045] Figure 13 The flow cytometry analysis chart in Example 8 is shown in Figure 7.

[0046] Figure 14 Figure 9 shows the schematic diagram of CAR-T treatment in the subcutaneous tumor-bearing mouse model in Example 9;

[0047] Figure 15 Figure 10 shows the tumor volume change graph after CAR-T treatment in the subcutaneous tumor-bearing mouse model in Example 9;

[0048] Figure 16 Figure 11 shows the tumor size and tumor graph after CAR-T treatment in the subcutaneous tumor-bearing mouse model in Example 9;

[0049] Figure 17 Figure 12 shows the flow cytometry analysis graph in Example 9;

[0050] Figure 18 Figure 13 shows the schematic diagram of CAR-T (AKTi1 / 2 (1 μM) treated) treatment in the subcutaneous tumor-bearing mouse model in Example 9;

[0051] Figure 19 Figure 14 shows the tumor volume change graph after AKTi-treated CAR-T cell treatment in the subcutaneous tumor-bearing mouse model in Example 9;

[0052] Figure 20 Figure 15 shows the survival curve after AKTi-treated CAR-T cell treatment in the subcutaneous tumor-bearing mouse model in Example 9;

[0053] Figure 21 Figure 16 shows the recurrence graph after CAR-T treatment for 90 days in the subcutaneous tumor-bearing mouse model in Example 9;

[0054] Figure 22 Figure 17 shows the CAR-T cell survival graph after CAR-T treatment for 90 days in the subcutaneous tumor-bearing mouse model in Example 9. DETAILED DESCRIPTION

[0055] The following examples will further illustrate the specific steps and features of the application, which are only used for illustration and are not intended to limit the application. The methods used in the application are conventional methods in the art unless otherwise specified. The reagents and materials involved in the application are commercially available unless otherwise specified.

[0056] Wherein: the overall production process of human CAR-T cells is shown in Examples 1-4, and the schematic diagram is shown in Figure 3 The overall production process of mouse CAR-T cells is shown in Examples 1 and 5-7, and the schematic diagram is shown in Figure 9 .

[0057] Example 1 Construction of recombinant plasmid

[0058] 1. Construction of human CA9 CAR lentiviral vector

[0059] The human CA9 CAR sequence (hCA9 CAR) is composed of a human CD8a signal peptide, an anti-human CA9 scFv, a human CD8a hinge region and transmembrane region, a CD28 signal, a 4-1BB signal, and a CD3 zeta cytoplasmic signal, as shown in the schematic diagram Figure 1 The nucleotide sequence is shown in SEQ ID NO. 1 in Table 1, and the amino acid sequence is shown in SEQ ID NO. 2 in Table 1.

[0060] The hCA9 CAR gene fragment is inserted into a pLVX-EF1a-IRES-mCherry lentiviral expression vector (item number 3039, Shanghai Huwu Biotechnology) through EcoRI and MluI cloning sites, as shown in the schematic diagram Figure 2 , to obtain a recombinant plasmid pLVX-EF1a-hCA9CAR.

[0061] Table 1 Human CA9 CAR sequence

[0062]

[0063]

[0064] 2. Construction of human CA9 CAR-RUNX3 lentiviral vector

[0065] The human CA9 CAR-RUNX3 sequence is composed of hCA9 CAR, P2A, and human Runx3 cDNA, as shown in the schematic diagram Figure 1 The nucleotide sequence is shown in SEQ ID NO. 3 in Table 2, and the amino acid sequence is shown in SEQ ID NO. 4 in Table 2.

[0066] Using primers CAR-F1 (SEQ ID NO. 5) and P2A-R1 (SEQ ID NO. 6), and pLVX-EF1a-hCA9CAR as a template, a gene fragment hCA9CAR-P2A is amplified using high-fidelity enzyme 2xPhanta Flash Master Mix (Dye Plus) (Vazyme) at an annealing temperature of 55°C.

[0067] The hCA9CAR-P2A gene fragment is inserted into a pLVX-EF1a-IRES-mCherry lentiviral expression vector through EcoRI and BamHI cloning sites, to obtain an intermediate plasmid pLVX-EF1a-hCA9CAR-P2A-IRES-mCherry.

[0068] The primer Runx3-F1 (SEQ ID NO. 7) and Runx3-R1 (SEQ ID NO. 8) were used to amplify the gene fragment hRunx3 from the human RUNX3 (NM_001031680) cDNA clone (Cat. No. G166907, UbiBiosci) using high-fidelity enzyme 2x PhantaFlash Master Mix (Dye Plus) (Vazyme) with an annealing temperature of 55°C.

[0069] The hRunx3 gene fragment was inserted into the intermediate plasmid pLVX-EF1a-hCA9CAR-P2A-IRES-mCherry through the BamHI and MluI cloning sites to obtain the recombinant plasmid pLVX-EF1a-hCA9CAR-P2A-hRunx3.

[0070] Table 2 Human CA9CAR-RUNX3 and primer sequences used

[0071]

[0072]

[0073] 3. Construction of mouse CA9CAR retroviral vector

[0074] The mouse CA9CAR sequence (mCA9CAR) consists of a mouse CD8a signal peptide, an anti-human CA9 scFv, a mouse CD8a hinge region and transmembrane region, a CD28 signal, and a CD3ζ cytoplasmic signal, as shown in the schematic diagram Figure 7 The nucleotide sequence is shown in SEQ ID NO. 9 in Table 3, and the amino acid sequence is shown in SEQ ID NO. 10 in Table 3, which was synthesized by GenScript Biotech Co., Ltd.

[0075] The mCA9CAR gene fragment was inserted into the pMSCV-IRES-GFP II (pMIGII) retroviral expression vector (Cat. No. 52107, Addgene) through the EcoRI and BamHI cloning sites, as shown in the schematic diagram Figure 8 , to obtain the recombinant plasmid pMIGII-mCA9CAR-IRES-GFP.

[0076] Table 3 Mouse CA9CAR sequence

[0077]

[0078] 4. Construction of mouse CA9CAR-RUNX3 retroviral vector

[0079] The mouse CA9 CAR-RUNX3 sequence is composed of mCA9 CAR, P2A, and mouse Runx3 cDNA, as shown in the schematic diagram Figure 7 The sequence is shown as SEQ ID NO. 11 in Table 4, and the amino acid sequence is shown as SEQ ID NO. 12 in Table 4.

[0080] The primer CAR-F2 (SEQ ID NO. 13) and P2A-R2 (SEQ ID NO. 14) were used to amplify the gene fragment mCA9 CAR-P2A from the template pMIGII-mCA9CAR-IRES-GFP using high-fidelity enzyme 2xPhanta Flash Master Mix (Dye Plus) (Vazyme) at an annealing temperature of 55°C.

[0081] The mCA9 CAR-P2A gene fragment was inserted into the pMIGII retrovirus expression vector through EcoRI and BamHI cloning sites to obtain the intermediate plasmid pMIGII-mCA9CAR-P2A-IRES-GFP.

[0082] The primer Runx3-F2 (SEQ ID NO. 15) and Runx3-R2 (SEQ ID NO. 16) were used to amplify the gene fragment mRunx3 from the template mouse Runx3 (NM_019732) cDNA clone (catalog number G129415, Youbao Biological) using high-fidelity enzyme 2xPhanta Flash Master Mix (Dye Plus) (Vazyme) at an annealing temperature of 55°C.

[0083] The mRunx3 gene fragment was inserted into the recombinant plasmid pMIGII-CAR-P2A-IRES-GFP through BamHI and SalI cloning sites to obtain the recombinant plasmid pMIGII-mCA9CAR-P2A-mRunx3.

[0084] Table 4 Mouse CA9 CAR-RUNX3 and primer sequences used

[0085]

[0086]

[0087]

[0088] Example 2 Lentivirus production, concentration, and titer detection

[0089] Reagent preparation:

[0090] 2.5M CaCl2solution: Weigh 13.875g of anhydrous calcium chloride, add 40ml of ultrapure water to dissolve, cool to room temperature, constant volume to 50ml, 0.22μm filter membrane, 4℃ storage for standby.

[0091] 2xHBS solution: Weigh NaCl 16.3g, KCl 0.74g, Na2HPO40.214g, Glucose 2.4g, HEPES 10g, add 900ml of ultrapure water to dissolve, adjust pH to 7.05, constant volume to 1000ml, 0.22μm filter membrane, 4℃ storage for standby.

[0092] 40% PEG6000: Weigh NaCl 8.755g, PEG6000 40g dissolved in 100Ml ddH2O, autoclaved, 4℃ storage for standby.

[0093] HEK293T(ATCC) cells were cultured and expanded with 10% FBS, 4mM Glutamin DMEM (hereinafter referred to as DMEM complete medium). 293T cells were cultured in 10cm dishes, with a confluence of 70-90% for transfection. Before transfection, the medium was changed, and chloroquine was added at a final concentration of 25μM.

[0094] Prepare 2 15ml centrifuge tubes, one with 500μL 2xHBS, the other with 400μL sterile ddH2O, 10μg vector plasmid (such as pLVX-EF1a-hCA9CAR-P2A-hRunx3), 7.5μg packaging plasmid psPAX2, 2.5μg envelope plasmid pMD2.G, 50μL 2.5M CaCl2, blow and mix evenly. Slowly add DNA-CaCl2 to 2xHBS, vortex mix, continue mixing for 30s after adding.

[0095] Add the DNA-calcium phosphate mixture evenly to the culture medium of 293T cells and mix well, incubate at 37℃ with 5% CO2 for 6-8h. Collect the virus-containing supernatant after 48h, add 10mL DMEM complete medium. Virus supernatant was stored at 4℃. Virus supernatant was collected again at 60h and 72h.

[0096] The collected virus supernatant was centrifuged at 1000g for 10min, the supernatant was filtered with a 0.45μm filter, and collected in a sterile 50mL centrifuge tube. Add 1 / 4 volume of 40% PEG6000 to the virus supernatant and mix well, 4℃ overnight. Centrifuge at 2500g for 45min, discard the supernatant, centrifuge again for 2min, and discard the supernatant again. Dissolve the precipitate with 1xPBS and store at -80℃.

[0097] Lentivirus titer test: HEK293T cells (2x105 / mL) were seeded in 96-well cell culture plates, 104per well, 50 μL / well. The culture medium contained 8 μg / mL of polybrene. Each well was supplemented with 50 μL of culture medium containing 1 μL, 0.1 μL, 0.01 μL, 0.001 μL of virus concentrate, and mixed. Cultured at 37°C, 5% CO2, for 72 h. Then the cells were recovered by trypsin digestion, and the infection positive rate of HEK293T cells was detected by flow cytometry.

[0098] Flow cytometry detection of infection positive rate: CA9 CAR-expressing cells were first labeled with recombinant human CA9-Fc protein (Yiqiao Shenzhou), then labeled with PE-anti-human-Fc antibody (biolegend) secondary antibody, and finally detected in the PE channel.

[0099] The group with an infection rate of about 10%-30% was used to calculate the virus titer, and the virus titer (TU / mL) = infection positive rate x 10 4 x 1000 / volume of virus added (μL). Generally, the concentration of lentivirus titer was about 0.1-10x10^7 TU / mL.

[0100] Example 3 Isolation, activation and lentivirus transduction of human peripheral blood T lymphocytes

[0101] Healthy human peripheral blood was diluted 1:1 with 1xPBS in a 15 mL centrifuge tube, and 1 / 2 volume of human peripheral blood lymphocyte separation medium (Solabio) was added to the bottom, centrifuged at 600g for 20 min, and the 3rd rise and the 1st fall. The middle layer of human peripheral blood mononuclear cells PBMC was taken, washed with PBS twice, and cultured in AIM-V medium (Gibco) containing 5% Immune Cell Serum Replacement (Gibco) and 100 IU / mL IL-2 (Novoprotein), and activated PBMC with Dynabeads Human T-Activator CD3 / CD28 (Gibco) 1:1 for 24 h.

[0102] Activated PBMC were collected after 24 h, removed magnetic beads by gradient centrifugation with human peripheral blood lymphocyte separation medium, and washed PBMC twice with PBS.

[0103] 1x10 6 PBMC were added to a 24-well plate, and an appropriate amount of lentivirus (MOI = 40:1) and 8ug / ml polybrene were added, centrifuged at 1200g, 32°C, 0 rise and 0 fall for 2h, then cultured for 4h, and then the T cells were transferred to a 6-well plate for expansion.

[0104] Example 4 In vitro expansion of human CAR-T cells and maintenance of T cell stemness

[0105] At the time of T cell expansion, the cell concentration was adjusted to 5x10 6 / mL, cultured in AIM-V medium with 5% Immune Cell Serum Replacement, 100 IU / mL IL-2, and 1 μΜ AKTi-1 / 2 was added to the experimental group, and no AKTi-1 / 2 was added to the control group. After 5 days of expansion, the CAR-T cell status was detected by flow cytometry.

[0106] Surface protein flow detection: collect cells, resuspend cells with 100 μΐ^of 2% FBS-PBS, then incubate with recombinant human CA9-Fc protein (Sino Biological); wash cells with 2% FBS-PBS, then incubate with secondary antibody PE-anti-human-Fc antibody (biolegend) and other surface protein antibodies, such as CD4, CD8, CD62L; wash cells with 2% FBS-PBS, then detect on the machine.

[0107] Intracellular protein flow detection: after T cells are stained with surface proteins and washed, fix and permeabilize with fixation / permeabilization solution kit (Cat. No. 555028, BD), then stain with intracellular antibodies, such as Granzyme B. The detection results are shown in Figures 4 to 6 .

[0108] The results show that: 1 μΜ AKTi-1 / 2 treatment can help maintain the percentage of CAR-T cells Figure 4 ), and maintain the CD62L+ memory phenotype of CAR-T cells Figure 5 ). 1 μΜ AKTi-1 / 2 treatment inhibits Granzyme B expression, while Runx3 overexpression promotes Granzyme B expression Figure 6 .

[0109] Example 5 Production and concentration of retrovirus

[0110] PLAT-E cells were cultured and expanded in DMEM with 10% FBS, 4 mM Glutamin. PLAT-E cells were cultured in 10 cm dishes, and the confluence was 70-90% for transfection. Before transfection, the medium was changed, and chloroquine was added at a final concentration of 25 μΜ.

[0111] Prepare two 15 ml centrifuge tubes, one with 500 μL 2xHBS, the other with 400 μL sterile ddH2O, 20 μg vector plasmid (e.g. pMIGII-CAR-P2A-RUNX3), 50 μL 2.5 M CaCl2, mix well by pipetting. Slowly add the DNA-CaCl2 mixture to the 2xHBS while vortexing, continue mixing for 30 s after the addition is complete.

[0112] Add the DNA-calcium phosphate mixture to the culture medium of PLAT-E cells and mix well, incubate at 37 °C in 5% CO2 for 6-8 h, change the medium. Collect the virus-containing supernatant after 48 h, add new 10 mL DMEM complete medium, and store the virus supernatant at 4 °C. Collect the virus supernatant again at 60 h and 72 h.

[0113] Centrifuge the collected virus supernatant at 1000 g for 10 min, filter the supernatant with a 0.45 μm filter, and collect it in a sterile 50 mL centrifuge tube. Add the virus supernatant to an Amicon Ultra-15 (catalog number UFC910024, Millipore), and centrifuge at 4000 g for 20 min to concentrate it 10-20 times. Store the concentrated retrovirus at 4 °C for later use.

[0114] Example 6 Mouse T cell activation, enrichment and retroviral transduction

[0115] Take the spleen of an 8-week-old BALB / c mouse, crush it in a 70 μm filter, and collect the filtered spleen cells. Remove the red blood cells with red blood cell lysis solution. Adjust the cell density to 5 x 10 6 / mL with RPMI 1640 medium containing 10% FBS, add 1 ug / mL anti-mouse-CD3 (catalog number 100302, Biolegend), 0.5 ug / mL anti-mouse-CD28 (catalog number 102116, Biolegend), 100 IU / mL IL-2 (Novoprotein), and incubate at 37 °C in 5% CO2 for 24 h.

[0116] Prepare 100% Percoll: 231.25 ml Percoll, 36 ml 10xPBS, 3.26 ml 7.5% (wt / vol) NaHCO3. 60% and 30% (vol / vol) Percoll are obtained by diluting 100% Percoll with RPMI-1640 medium.

[0117] Resuspend the activated spleen cells in a 15 mL centrifuge tube with 30% Percoll, slowly add 60% Percoll to the bottom, centrifuge at 800 g for 20 min, 3 up and 1 down. Collect the activated lymphocytes in the middle layer and wash them once with PBS.

[0118] Activated lymphocytes were adjusted to 1x10 8 / mL, 10 ug / mL biotin anti-mouse-CD19 (Cat# 115504, Biolegend) was added and incubated at 4°C for 30 min, then centrifuged at 400g for 5 min and the supernatant was discarded. 20ul / mL Streptavidin Nanobeads (Cat# 480016, Biolegend) was added and incubated at 4°C for 30 min. TM CD19+ B cells were labeled with magnetic beads, then removed by MojoSort TM Magnet.

[0119] 6-well plates were added with 3-6x10 6 Activated T cells were added with 1.5 mL concentrated retrovirus and 6 ug / mL polybrene, centrifuged at 2000g, 32°C, 2h, then changed the medium and transferred the T cells to a 10cm dish for expansion.

[0120] Example 7: In vitro expansion of mouse CAR-T cells and maintenance of T cell stemness

[0121] During T cell expansion, the cell density was adjusted to 0.5x10 6 / mL, and cultured in 10% FBS, 100 IU / mL IL-2 RPMI 1640 medium, and the experimental group was added with a final concentration of 1 μM AKTi-1 / 2, and the control group was not added. After 4 days of expansion, the CAR-T cell status was detected by flow cytometry.

[0122] Surface protein flow detection: collect cells, resuspend cells with 100 μL 2% FBS-PBS, incubate flow cytometry antibodies FITC anti-strep tag II (Cat# A01736, Genscript) and other surface antibodies such as CD4 (Cat# 100414, Biolegend), CD8 (Cat# 100722, Biolegend), CD62L (Cat# 104408, Biolegend), CD44 (Cat# 103044, Biolegend); after washing the cells with 2% FBS-PBS, detect them on the machine.

[0123] Intracellular protein flow detection: after T cells were stained with surface proteins and washed, fix and permeabilize with fixation / permeabilization solution kit (Cat# 555028, BD), then stain intracellular antibodies such as Granzyme B (Cat# 396413, Biolegend).

[0124] Flow cytometry showed that Runx3 overexpression and AKTi1 / 2 (1 μM) treatment were beneficial to the survival and proliferation of mouse CAR-T cells and had a synergistic effect ( Figure 10 AKTi1 / 2 (1 μM) treatment is beneficial to maintain the CD62L+ memory phenotype of mouse CAR-T cells ( Figure 11 AKTi1 / 2 (1 μM) treatment inhibited GranzymeB expression, while Runx3 overexpression promoted GranzymeB expression ( Figure 12 ).

[0125] Example 8 In vitro detection of the killing ability of mouse CAR-T cells

[0126] The hCA9-overexpressing mouse pancreatic cancer KPC cell line was derived by infecting a conventional KPC cell line with a retrovirus that overexpresses hCA9. The hCA9-overexpressing KPC cell line was labeled with APC anti-hCA9 antibody, purified by flow cytometry, and hCA9-expressing KPC cell clones were isolated by limiting dilution.

[0127] Single clone selection by limiting dilution: Approximately 100 cells were evenly plated in a 96-well plate. After one week, single clones were selected and passaged to 24-well plates for expansion. After expansion in the 24-well plates, the cell clones were labeled with APC anti-hCA9 antibody and then screened for KPC cell clones that expressed 100% hCA9 by flow cytometry.

[0128] After 14 hours of co-culture of CAR-T cells and hCA9-KPCs at varying ratios, surviving KPCs adhered to the wall. T cells were resuspended and discarded. No T cells were added to the control group.

[0129] CCK-8 kit was used to detect KPC cell viability: RMPI 1640 medium containing 10% CCK-8 was added to each well, and a well without cells was used as a blank control. The cells were cultured at 37°C for 1 hour, and the OD450 was measured using a microplate reader. Cytolysis rate = 100-(OD450 每孔 -OD450 空白 ) / (OD450 对照 -OD450 空白 )×100.

[0130] The results showed that AKTi1 / 2 (1 μM) treatment inhibited the killing ability of CAR-T cells, while Runx3 overexpression enhanced the killing ability of CAR-T cells ( Figure 13 ).

[0131] Example 9 Detection of the anti-tumor ability of novel CAR-T cells in tumor-bearing mice

[0132] 6-8 weeks old nude mice were subcutaneously loaded with 1×106 / 50μl KPC-hCA9 cells, wait for one week until the tumor is the size of a grain of rice, and inject an appropriate amount (5x10 6 )CAR-T cells. The long and short diameters of the tumor were measured weekly using a vernier caliper and the tumor volume was calculated (1 / 2 x long diameter x short diameter x short diameter). The diagram is shown in the figure. Figure 14 shown.

[0133] One week after treatment, the lymph nodes and tumors of the mice were collected, weighed, and single-cell suspensions were prepared. Flow cytometry was used to detect CAR-T cell survival and tumor infiltration ability.

[0134] The results showed that CAR-T cells overexpressing Runx3 treated with AKTi1 / 2 (1 μM) had better anti-tumor ability (Appendix Figure 15-16 AKTi1 / 2 (1 μM) treatment is beneficial to the lymph node return, survival and proliferation of CAR-T cells, and Runx3 overexpression is beneficial to the tumor infiltration of CAR-T cells ( Figure 17 ).

[0135] 6-8 weeks old nude mice were subcutaneously loaded with 1×10 6 / 50μl KPC-hCA9 cells. After one week, the tumor was the size of a grain of rice, and an appropriate amount of AKTi1 / 2 (1μM)-treated CAR-T cells were reinfused into the tail vein. The long and short diameters of the tumor were measured weekly using a vernier caliper and the tumor volume was calculated (1 / 2 x long diameter x short diameter x short diameter). When the tumor diameter was greater than 1.5cm or the volume was greater than 1 cubic centimeter, the mouse was euthanized. The mouse survival curve was calculated. The schematic diagram is shown in the figure. Figure 18 shown.

[0136] The results showed that overexpression of Runx3 enhanced the short-term anti-tumor ability of CAR-T cells treated with AKTi1 / 2 (1 μM). Figure 19 ) and long-term anti-tumor ability ( Figure 20-21 After 90 days of CAR-T treatment, a large number of CAR-T cells still survived in the surviving mice ( Figure 22 ), further indicating that CAR-T cells treated with AKTi1 / 2 (1 μM) have the ability to survive long-term in vivo.

[0137] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for producing CAR-T cells, characterized in that: It mainly includes the following two steps: Step 1: Preparation of CAR-T cells overexpressing Runx3; Step 2: Expand CAR-T cells overexpressing Runx3 in the presence of 0.5-1.5 μM AKTi1 / 2; The CAR-T cells include human CAR-T cells and mouse CAR-T cells, and the step 1 includes a process of preparing a CAR viral expression vector that overexpresses Runx3; When preparing a CAR viral expression vector for overexpressing Runx3 in human CAR-T cells, the CAR adopts the composition of a third-generation CAR, consisting of a human CD8 signal peptide, a scFv fragment, a human CD8 extracellular region, a CD28 transmembrane region and intracellular segment, a 4-1BB intracellular segment, and a CD3ζ intracellular segment. The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

2. The viral expression vector backbone is pLVX-EF1a-IRES-mCherry. When preparing a CAR viral expression vector for overexpressing Runx3 in mouse-derived CAR-T cells, the CAR adopts the composition of a second-generation CAR, consisting of a mouse CD8 signal peptide, a scFv fragment, the mouse CD8 extracellular and transmembrane regions, the CD28 intracellular segment, and the CD3ζ intracellular segment. Strep tag II is added to the N-terminus for easy detection. The nucleotide sequence is shown in SEQ ID NO.9, and the amino acid sequence is shown in SEQ ID NO.

10. The viral expression vector backbone is pMSCV-IRES-GFP II (pMIGII). Both human Runx3 and CAR and mouse Runx3 and CAR are connected through the P2A sequence.

2. The method for producing CAR-T cells according to claim 1, wherein: The specific process of step one is: (1) Preparation of a CAR viral expression vector overexpressing Runx3; (2) Production of CAR virus overexpressing Runx3; (3) T cell isolation and activation; (4) Virus infection activates T cells.

3. Use of the CAR-T cell production method according to claim 2 in producing human CAR-T cells.

4. The use according to claim 3, characterized in that The Runx3 is human Runx3, the CAR virus is a CAR lentivirus, and the T cells are human peripheral blood lymphocytes.

5. The use according to claim 4, characterized in that In step (1): A.CAR adopts the third-generation CAR composition, consisting of human CD8 signal peptide, scFv fragment, human CD8 extracellular region, CD28 transmembrane region and intracellular segment, 4-1BB intracellular segment, and CD3ζ intracellular segment. The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

2. B. Human Runx3 is linked to CAR via the P2A sequence to achieve the purpose of co-overexpression of human Runx3 and CAR. The nucleotide sequence is shown in SEQ ID NO. 3, and the amino acid sequence is shown in SEQ ID NO. 4; C. The backbone of the lentiviral expression vector is pLVX-EF1a-IRES-mCherry; D. IRES-mCherry was replaced with CAR-P2A-RUNX3 to obtain the CAR lentiviral expression vector pLVX-EF1a-CA9CAR-P2A-Runx3 that overexpresses human Runx3.

6. The use according to claim 5, characterized in that The specific process of step 2 is as follows: when T cells are expanded, the cell density is adjusted to 0.5x10 6 / mL, cultured in AIM-V medium containing 5% Immune Cell Serum Replacement and 100 IU / mL IL-2, and added with AKTi-1 / 2 at a final concentration of 1 μM, and the medium was changed every other day.

7. Use of the CAR-T cell production method according to claim 2 in producing mouse-derived CAR-T cells.

8. The use according to claim 7, characterized in that The Runx3 is mouse Runx3, the CAR virus is CAR retrovirus, and the T cells are mouse spleen T cells.

9. The use according to claim 8, characterized in that In step (1): A.CAR uses a second-generation CAR composition consisting of a mouse CD8 signal peptide, a scFv fragment, the mouse CD8 extracellular and transmembrane regions, the CD28 intracellular segment, and the CD3ζ intracellular segment. Strep tag II is added to the N-terminus for easy detection. The nucleotide sequence is shown in SEQ ID NO. 9, and the amino acid sequence is shown in SEQ ID NO.

10. B. Mouse Runx3 and CAR are linked via a P2A sequence to achieve co-overexpression of mouse Runx3 and CAR. The nucleotide sequence is shown in SEQ ID NO.11, and the amino acid sequence is shown in SEQ ID NO.

12. C. The retroviral vector backbone is pMSCV-IRES-GFP II (pMIGII); D. IRES-GFP was replaced by CAR-P2A-RUNX3 to obtain the CAR retroviral expression vector pMIGII-CA9CAR-P2A-RUNX3 that overexpresses mouse Runx3.

10. The use according to claim 9, characterized in that The specific process of step 2 is as follows: when T cells are expanded, the cell density is adjusted to 0.5x10 6 / mL, cultured in RMPI 1640 medium containing 10% FBS and 100 IU / mL IL-2, and added with AKTi-1 / 2 at a final concentration of 1 μM, and the medium was changed every other day.

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