Immune-enhanced CAR-NK cell capable of overcoming tumor microenvironment and application of immune-enhanced CAR-NK cell

Abstract

The invention discloses an immune-enhanced CAR-NK cell capable of overcoming a tumor microenvironment and application of the immune-enhanced CAR-NK cell, the CAR-NK cell can modify a chimeric antigen receptor, the chimeric antigen receptor is PLL3.7-NKG2D-CAR and PLL3.7-TGF beta RII / IL21R-NKG2D-CAR carriers, the DNA sequence of each element of the carriers is as shown in SEQ ID NO: 1, and the connection sequence of each part of the chimeric antigen receptor is as shown in figure 3. The cell disclosed by the invention transmits an activation signal and activates an immune system by recognizing NKG2DL generated on the surface of a tumor cell, thereby playing a killing role on the tumor cell; meanwhile, an inhibition signal of TGF-beta in a tumor microenvironment is converted into an activation signal of the NK cells through a ligand TGF-beta R II extracellular domain, an IL-21R transmembrane region and an intracellular domain of the TGF-beta R II, so that the proliferation and survival capabilities of the NK cells in the tumor inhibition microenvironment are enhanced, the killing of the NK on the tumor cells is improved, and the anti-tumor function of the NKG2D targeted CAR-NK cells is enhanced; and a good treatment effect is finally achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor cells, and specifically relates to an immune-enhanced CAR-NK cell capable of overcoming a tumor microenvironment and an application thereof. Background Art

[0002] Chimeric antigen receptor T-cell (CAR-T) therapy has achieved good results in hematological malignancies, but CAR-T therapy has not yet achieved a breakthrough in the treatment of solid tumors. The biggest challenge is that solid tumors have an immunosuppressive tumor microenvironment (TME), which is characterized by the secretion of a variety of immunosuppressive factors, the most secreted of which is transforming growth factor (TGF-β). TGF-β can shape the structure of the tumor and inhibit the anti-tumor activity of immune cells, thereby producing an immunosuppressive microenvironment that blocks or weakens the efficacy of anti-tumor immunotherapy, thereby further promoting cancer progression. Therefore, inhibiting TGF-β signaling is considered to be one of the important ways to enhance the efficacy of immunotherapy, and it is of great significance to design and develop new cell therapy products based on this to overcome the immunosuppressive microenvironment of solid tumors. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide an immune-enhancing CAR-NK cell that overcomes the tumor microenvironment and its application, which is an engineered NK cell modified with a specific chimeric antigen receptor that targets NKG2DL and converts the inhibitory signal induced by TGF-β into an activation signal. The engineered cell transmits activation signals and activates the immune system by recognizing the NKG2DL produced on the surface of tumor cells, thereby playing a killing role on tumor cells; at the same time, the inhibitory signal of TGF-β in the tumor microenvironment is converted into an activation signal of NK cells through its ligand TGF-βRⅡ extracellular segment and IL-21R transmembrane region and intracellular segment, enhancing the proliferation and survival ability of NK cells in the tumor inhibitory microenvironment, thereby improving the killing of tumor cells by NK, thereby enhancing the anti-tumor function of NKG2D-targeted CAR-NK cells, and finally achieving a good therapeutic effect, with broad application prospects.

[0004] The objective of the present invention is achieved through the following technical solutions:

[0005] An immune-enhancing CAR-NK cell that overcomes a tumor microenvironment, wherein the CAR-NK cell is capable of modifying a chimeric antigen receptor, wherein the chimeric antigen receptor is a PLL3.7-NKG2D-CAR, a PLL3.7-TGFβR II / I L21 R-NKG2D-CAR vector, wherein the DNA sequence of each element of the vector is as shown in SEQ ID NO: 1, and the connection sequence of each part of the chimeric antigen receptor is as shown in Figure 3As shown, the CAR-NK cell is prepared by a method comprising the following steps:

[0006] (1) Construction of PLL3.7-NKG2D-CAR and PLL3.7-TGFβR II / I L21 R-NKG2D-CAR vectors;

[0007] (2) Preparation of bacteria containing chimeric antigen receptor expression vectors and extraction of chimeric antigen receptor expression plasmids targeting human NKG2DL;

[0008] (3) Virus packaging;

[0009] (4) Virus titer determination;

[0010] (5) Prepare NK cells with chimeric antigen receptors targeting human NKG2DL.

[0011] Wherein, the construction method of the PLL3.7-NKG2D-CAR and PLL3.7-TGFβR II / I L21 R-NKG2D-CAR vectors includes:

[0012] (1) Whole gene synthesis: CD8α signal peptide, NKG2D extracellular domain, CD8α hinge region, CD8α transmembrane domain, 4-1BB co-stimulatory signal transduction region and CD3ζ signal transduction domain, and the above sequences are connected in sequence to obtain a chimeric antigen receptor NKG2D-CAR expression cassette;

[0013] (2) After the whole gene synthesis of the chimeric antigen receptor expression cassette sequence, it was connected to the empty vector pLL3.7 through the NheI / EcoRI restriction site to obtain a chimeric antigen receptor expression vector. After the sequence was determined to be correct, it was named PLL3.7-NKG2D-CAR. Figure 1 As shown;

[0014] (3) Whole gene synthesis: self-cleaving T2A peptide, TGF-βRI I extracellular domain, IL-21R transmembrane domain, IL-21R intracellular domain, the above sequences are connected in sequence; EcoRI enzyme digestion is used to recover the linearized PLL3.7-NKG2D-CAR vector, and the vector is connected through the EcoRI enzyme cleavage site to obtain a chimeric antigen receptor expression vector. After the sequence is determined to be correct, it is named PLL3.7-TGFβRI I / IL21R-NKG2D-CAR. Figure 3 shown.

[0015] Among them, the preparation of the bacterial strain containing the chimeric antigen receptor expression vector and the extraction of the chimeric antigen receptor expression plasmid targeting human NKG2DL:

[0016] Preparation of chimeric antigen receptor expression vector strains:

[0017] (1) Take out 100 μL of competent cell DH5α suspension from the -80°C refrigerator and thaw on ice;

[0018] (2) Add 0.1-1 μg of plasmid DNA (volume not exceeding 5 μL), mix gently, and place on ice for 5 minutes;

[0019] (3) Heat shock in a 42°C water bath for 90 seconds, followed by rapid cooling on ice for 5 minutes;

[0020] (4) Add 0.5 ml of LB liquid medium without resistance and culture at 37°C and 180 rpm for 30 min;

[0021] (5) Centrifuge at 5000 rpm for 4 min, remove part of the supernatant, retain 100-150 μl of culture medium, gently pipette the suspended bacteria, and then spread it on an LB plate containing antibiotics;

[0022] (6) Incubate overnight at 37°C;

[0023] (7) Pick a single clone and inoculate it into ampicillin-resistant LB medium and culture it at 37°C and 200 rpm for 9-12 hours;

[0024] (8) Add glycerol to the bacterial solution to a final concentration of 10% and store the bacteria in a -80°C refrigerator for later use;

[0025] Extraction of chimeric antigen receptor expression plasmid targeting human NKG2DL:

[0026] The DH5α strain containing the chimeric antigen receptor expression plasmid targeting human NKG2DL was cultured in large quantities in LB medium, and an endotoxin-free plasmid extraction kit was used for high-purity endotoxin-free extraction in preparation for lentiviral packaging.

[0027] Wherein, the virus packaging:

[0028] Preparation of 293T cells: 24 hours before virus packaging, 293T cells were digested with trypsin and re-plated on 10 cm plates. The cells were cultured in DMEM medium containing 10% FBS in a 37°C 5% CO2 incubator. Transfection was performed when the cell density reached 70-90%.

[0029] Plasmid transfection:

[0030] (1) Place plasmid, PE I, and DMEM medium at room temperature for 5 min;

[0031] (2) Take 450 μl of DMEM into a 1.5 ml EP tube, add 50 μl of PE I (1 μg / μl), mix well, and let stand at room temperature for 5 min;

[0032] (3) Take 5 μg of plasmid (pLL3.7-NKG2D-CAR, pLL3.7-TGFβR II / I L21 R-NKG2D-CAR), 5 μg of psPAX2, and 3 μg of pMD2.G, add DMEM to 500 μl, mix well, and let stand at room temperature for 5 min;

[0033] (4) adding the prepared PE I-DMEM solution of step (2) to the DMEM containing the plasmid obtained in step (3), mixing well, and standing at room temperature for 20 min to obtain a DNA / PE I mixture;

[0034] (5) Slowly drip 1 ml of DNA / PE I mixture into the 293T culture dish, mix gently, and incubate at 37°C incubator for 6-8 h;

[0035] (6) Discard the original culture medium, replace with fresh culture medium, and continue incubating in a 37°C incubator;

[0036] (7) After replacing the culture medium for 48 hours, the culture medium was collected, and then 10 ml of fresh culture medium was added to each dish to continue culturing. After 24 hours, the supernatant was collected again and mixed with the supernatant collected at 48 hours;

[0037] (8) Centrifugation at 4000 g for 10 min at 4°C to remove cell debris;

[0038] (9) filtering the obtained supernatant through a 0.45 μm filter;

[0039] (10) The filtered supernatant was transferred to an ultracentrifuge tube and centrifuged at 25,000 rpm for 2 h. The virus precipitate obtained after ultracentrifugation was resuspended in serum-free medium with 1% supernatant volume and gently pipetted until completely dissolved to obtain virus liquid. The virus liquids obtained using different vectors were named: LV-NKG2D-CAR, LV-TRⅡ / 21R-NKG2D-CAR;

[0040] (11) Aliquot each virus solution and store it in a -80°C refrigerator, and reserve 5-10 μl of virus concentrate for titer determination.

[0041] Wherein, the virus titer is determined:

[0042] (1) Digest and count 293T cells, prepare a cell suspension using DMEM medium containing 10% FBS, adjust the cell density to 4×105 / ml, and add 0.5 ml of the cell suspension to each well of a 24-well cell culture plate;

[0043] (2) The virus concentrate to be tested was added to a 24-well plate in different gradients, leaving a blank group as a control. The 24-well plate was placed in an incubator for incubation, and the positive rate of 293T cells was detected by flow cytometry after 48 hours;

[0044] (3) Centrifuge, resuspend, and adjust the cell density to 1×106 / ml. Add appropriate amount of antibody (0.3 μl each of APC-Anti-Human NKG2D and PE-Anti-Human TGF-βRⅡ) to 100 μl, gently pipette to mix, and place in a 4°C refrigerator to stain for 30 min in the dark.

[0045] (4) Wash once with PBS, resuspend, and perform flow cytometry analysis;

[0046] (5) Calculate the titer, titer (TU / ml) = (2*105*infection efficiency*1000) / virus volume (μl), after titer detection, pLL3.7-NKG2D-CAR, pLL3.7-TRⅡ

[0047] The titers of the two viruses / 21R-NKG2D-CAR were 1.1*108 and 0.42*108 respectively.

[0048] Wherein, the preparation of NK cells with chimeric antigen receptors targeting human NKG2DL:

[0049] NK92 culture:

[0050] (1) Preparation of NK92-specific culture medium: incomplete culture medium: αMEM + 0.1 mM β-mercaptoethanol + 12.5% ​​HS and 12.5% ​​FBS at a final concentration. Add IL-2 (200 I U / ml) to the culture medium when using. Try to use it up within 7 days, otherwise you need to add IL-2 again;

[0051] (2) NK92 cells were revived by thawing in a 37°C water bath, centrifuging at 800 rpm for 5 min, discarding the cryopreservation solution, adding culture medium to resuspend, and culturing in a T25 culture flask with an initial density of 2-3*105 cells per ml;

[0052] (3) For passaging of NK92 cells, shake the culture flask to roughly mix the cells, count the cells, and perform a full or half medium change every 2-3 days at a cell density of 2-3*105 viable cells per ml. Determine the passaging time and frequency based on the cell density;

[0053] Lentivirus infection: Adjust the cell density of NK92 cells to 1×106 / ml, add 0.5 ml of cell suspension to each well of a 24-well cell culture plate, and infect NK92 cells with the two virus solutions obtained in 3.2 at an MOI of 20. After 24 hours, replace the solution to obtain different NK cells expressing chimeric antigen receptors targeting human NKG2DL antigen; they were named CAR-NK and TRⅡ / 21R-CAR-NK respectively;

[0054] Detection of chimeric antigen receptor targeting human NKG2DL (CAR-NKG2D) and TGFβRI I expression: During the culture process, NK92 cell CAR-NK, TRⅡ / 21R-CAR-NK, and NK92 cells not infected with the virus (named MOCK-NK) were taken 72 hours after virus infection, centrifuged, resuspended, and the cell density was adjusted to 1×106 / ml. An appropriate amount of antibody (0.3μl each of APC-Anti-Human NKG2D and PE-Ant i-Human TGF-βRⅡ) was added to 50μl, and the mixture was gently blown and placed in a 4°C refrigerator. The cells were stained for 30 minutes away from light, washed once with PBS, resuspended, and the positive rate was detected by flow cytometry.

[0055] Another object of the present invention is to provide an immune-enhanced CAR-NK cell that overcomes the tumor microenvironment and is used in anti-tumor treatment.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] The cells in the present invention transmit activation signals and activate the immune system by recognizing NKG2DL produced on the surface of tumor cells, thereby exerting a killing effect on tumor cells; at the same time, the inhibitory signal of TGF-β in the tumor microenvironment is converted into an activation signal of NK cells through its ligand TGF-βRⅡ extracellular segment and IL-21R transmembrane region and intracellular segment, thereby enhancing the proliferation and survival ability of NK cells in the tumor inhibitory microenvironment, thereby improving NK's killing of tumor cells, thereby enhancing the anti-tumor function of NKG2D-targeted CAR-NK cells, and ultimately achieving a good therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is the PLL3.7-NKG2D-CAR vector map of the present invention;

[0059] Figure 2 This is the PLL3.7-TGFβRI I / IL21R-NKG2D-CAR vector map of the present invention;

[0060] Figure 3 It is a schematic diagram of the structure of each component of the chimeric antigen receptor;

[0061] Figure 4 This is the vector enzyme digestion identification diagram;

[0062] Figure 5 This is a graph showing the results of detecting the expression of NKG2D and TGF-βRⅡ in chimeric antigen receptor NK cells;

[0063] Figure 6 This is a graph showing the results of the detection of the killing level of target cells by chimeric antigen receptor NK cells;

[0064] Figure 7 This is a graph showing the results of detecting the expression level of chimeric antigen receptor NK cell activation factor;

[0065] Figure 8 This is a graph showing the results of the chimeric antigen receptor NK cell exhaustion level test;

[0066] Fig. 9 This is a diagram showing the anti-tumor effect of chimeric antigen receptor NK cells in vivo. DETAILED DESCRIPTION

[0067] The following will describe the implementation methods of the present application in detail with the help of accompanying drawings and examples, so that the implementation process of how the present application uses technical means to solve technical problems and achieve technical effects can be fully understood and implemented accordingly.

[0068] The present invention provides an immune-enhancing CAR-NK cell that overcomes the tumor microenvironment. The CAR-NK cell can modify a chimeric antigen receptor, and the chimeric antigen receptor is a PLL3.7-NKG2D-CAR or PLL3.7-TGFβRI I / I L21 R-NKG2D-CAR vector.

[0069] 1. Construction of PLL3.7-NKG2D-CAR and PLL3.7-TGFβRI I / I L21 R-NKG2D-CAR vectors (for the convenience of expression, hereinafter referred to as NKG2D-CAR and TRⅡ / 21R-NKG2D-CAR, respectively).

[0070] 1.1 Vector map Figure 1-2 shown.

[0071] 1.2 Schematic diagram of the connection sequence of the various parts of the chimeric antigen receptor Figure 3 shown.

[0072] 1.3 The DNA sequences of the vector elements are shown in Table 1.

[0073] Table 1 Nucleotide sequences of plasmid elements

[0074]

[0075]

[0076]

[0077] 1.4 Construction method

[0078] (1) Full gene synthesis: CD8α signal peptide, NKG2D extracellular domain, CD8α hinge region, CD8α transmembrane domain, 4-1BB co-stimulatory signal transduction region and CD3ζ signal transduction domain. The above sequences are connected in sequence to obtain a chimeric antigen receptor NKG2D-CAR expression cassette.

[0079] (2) After the whole gene synthesis of the chimeric antigen receptor expression cassette sequence, it was connected to the empty vector pLL3.7 through the Nhe I / EcoRI restriction site to obtain a chimeric antigen receptor expression vector. After the sequence was determined to be correct, it was named PLL3.7-NKG2D-CAR. Figure 1 shown.

[0080] (3) Whole gene synthesis: self-cleaved T2A peptide, TGF-βRI I extracellular domain, IL-21R transmembrane domain, IL-21R intracellular domain, the above sequences are connected in sequence; EcoRI enzyme digestion is used to recover the linearized PLL3.7-NKG2D-CAR vector, and the vector is connected through the EcoRI enzyme cleavage site to obtain a chimeric antigen receptor expression vector. After the sequence is determined to be correct, it is named PLL3.7-TGFβRI I / IL21R-NKG2D-CAR. Figure 2 shown.

[0081] (4) Vector enzyme digestion identification diagram Figure 4 shown.

[0082] 2. Preparation of strains containing chimeric antigen receptor expression vectors and extraction of chimeric antigen receptor expression plasmids targeting human NKG2DL

[0083] 2.1 Preparation of chimeric antigen receptor expression vector strains

[0084] (1) Take out 100 μL of competent cell DH5α suspension from the -80°C refrigerator and thaw on ice;

[0085] (2) Add 0.1-1 μg of plasmid DNA (volume not exceeding 5 μL), mix gently, and place on ice for 5 minutes;

[0086] (3) Heat shock in a 42°C water bath for 90 seconds, followed by rapid cooling on ice for 5 minutes;

[0087] (4) Add 0.5 ml of LB liquid medium without resistance and culture at 37°C and 180 rpm for 30 min;

[0088] (5) Centrifuge at 5000 rpm for 4 min, remove part of the supernatant, retain 100-150 μl of culture medium, gently pipette the suspended bacteria, and then spread it on an LB plate containing antibiotics;

[0089] (6) Incubate overnight at 37°C;

[0090] (7) Pick a single clone and inoculate it into ampicillin-resistant LB medium and culture it at 37°C and 200 rpm for 9-12 hours;

[0091] (8) Add glycerol to the bacterial solution to a final concentration of 10% and store the bacteria in a -80°C refrigerator for later use.

[0092] 2.2 Extraction of chimeric antigen receptor expression plasmid targeting human NKG2DL

[0093] The DH5α strain containing the chimeric antigen receptor expression plasmid targeting human NKG2DL was cultured in large quantities in LB medium and extracted with high purity and endotoxin-free plasmid extraction kit (Beijing Tiangen Biochemical Technology Co., Ltd.) in preparation for lentiviral packaging (for the specific operation process, see the instructions of the Tiangen Plasmid Extraction Kit).

[0094] 3. Virus packaging

[0095] 3.1293T Cell Preparation

[0096] 24 h before virus packaging, 293T cells were digested with trypsin and re-plated on 10 cm plates. The cells were cultured in DMEM medium containing 10% FBS in a 37°C 5% CO2 incubator. Transfection was performed when the cell density reached 70-90%.

[0097] 3.2 Plasmid transfection

[0098] (1) Place plasmid, PE I, and DMEM medium at room temperature for 5 min;

[0099] (2) Take 450 μl of DMEM and place it in a 1.5 ml EP tube. Add 50 μl of PE I (1 μg / μl) and mix well. Let stand at room temperature for 5 min.

[0100] (3) Take 5 μg of plasmid (pLL3.7-NKG2D-CAR, pLL3.7-TGFβR II / I L21 R-NKG2D-CAR), 5 μg of psPAX2, and 3 μg of pMD2.G, add DMEM to 500 μl, mix well, and let stand at room temperature for 5 min;

[0101] (4) adding the prepared PE I-DMEM solution of step (2) to the DMEM containing the plasmid obtained in step (3), mixing well, and standing at room temperature for 20 min to obtain a DNA / PE I mixture;

[0102] (5) Slowly drip 1 ml of DNA / PE I mixture into the 293T culture dish, mix gently, and incubate at 37°C incubator for 6-8 h;

[0103] (6) Discard the original culture medium, replace with fresh culture medium, and continue incubating in a 37°C incubator;

[0104] (7) After replacing the culture medium for 48 hours, the culture medium was collected, and then 10 ml of fresh culture medium was added to each dish to continue culturing. After 24 hours, the supernatant was collected again and mixed with the supernatant collected at 48 hours;

[0105] (8) Centrifugation at 4000 g for 10 min at 4°C to remove cell debris;

[0106] (9) filtering the obtained supernatant through a 0.45 μm filter;

[0107] (10) The filtered supernatant was transferred to an ultracentrifuge tube and centrifuged at 25,000 rpm for 2 h. The virus precipitate obtained after ultracentrifugation was resuspended in 1% supernatant volume of serum-free culture medium and gently blown until completely dissolved to obtain virus liquid. The virus liquids obtained using different vectors were named: LV-NKG2D-CAR, LV-TRⅡ / 21R-NKG2D-CAR.

[0108] (11) Aliquot each virus solution and store it in a -80°C refrigerator, and reserve 5-10 μl of virus concentrate for titer determination.

[0109] 4. Virus Titer Determination

[0110] (1) Digest and count 293T cells, prepare a cell suspension using DMEM medium containing 10% FBS, adjust the cell density to 4×10 5 / ml, and add 0.5 ml of the cell suspension to each well of a 24-well cell culture plate.

[0111] (2) The virus concentrate to be tested was added to a 24-well plate in different gradients, leaving a blank group as a control, and the 24-well plate was placed in an incubator for incubation. After 48 hours, the positive rate of 293T cells was detected by flow cytometry.

[0112] (3) Centrifuge, resuspend, and adjust the cell density to 1×106 / ml. Add appropriate amount of antibodies (0.3 μl each of APC-Anti-Human NKG2D and PE-Anti-Human TGF-βRⅡ) to 100 μl, gently pipette to mix, and place in a 4°C refrigerator to stain for 30 min in the dark.

[0113] (4) Wash once with PBS, resuspend, and perform flow cytometry analysis.

[0114] (5) Calculate the titer, titer (TU / ml) = (2*105*infection efficiency*1000) / virus volume (μl).

[0115] The titers of the two viruses / 21R-NKG2D-CAR were 1.1*108 and 0.42*108 respectively.

[0116] 5. Preparation of NK cells expressing chimeric antigen receptor targeting human NKG2DL

[0117] 5.1 NK92 cell culture

[0118] (1) Preparation of NK92-specific culture medium. Incomplete culture medium: αMEM + 0.1 mM β-mercaptoethanol + 12.5% ​​HS and 12.5% ​​FBS at a final concentration. Add IL-2 (200 I U / ml) to the culture medium when using it. Try to use it up within 7 days, otherwise you need to add IL-2 again.

[0119] (2) NK92 cell recovery: After thawing in a 37°C water bath, centrifuge at 800 rpm for 5 min, discard the cryopreservation solution, add culture medium to resuspend, and culture in a T25 culture flask with an initial density of 2-3*105 cells per ml.

[0120] (3) Passaging of NK92 cells. Shake the culture flask to roughly mix the cells, count the cells, and perform a full or half medium change every 2-3 days at a cell density of 2-3*105 viable cells per ml. Determine the passaging time and frequency based on the cell density.

[0121] 5.2 Lentiviral infection

[0122] The cell density of NK92 cells was adjusted to 1×106 / ml, and 0.5 ml of cell suspension was added to each well of a 24-well cell culture plate. The NK92 cells were infected with the two virus solutions obtained in 3.2 at an MOI of 20. The solution was replaced after 24 hours to obtain different NK cells expressing chimeric antigen receptors targeting human NKG2DL antigen; they were named CAR-NK and TRⅡ / 21R-CAR-NK, respectively.

[0123] 5.3 Detection of chimeric antigen receptor targeting human NKG2DL (CAR-NKG2D) and TGFβR II expression:

[0124] (1) During the culture process, NK92 cells CAR-NK, TRⅡ / 21R-CAR-NK, and NK92 cells not infected with the virus (named MOCK-NK) were taken 72 hours after virus infection, centrifuged, resuspended, and the cell density was adjusted to 1×106 / ml. An appropriate amount of antibody (0.3μl each of APC-Anti-Human NKG2D and PE-Anti-Human TGF-βRⅡ) was added to 50μl, and the mixture was gently pipetted and placed in a 4℃ refrigerator for staining for 30 minutes in the dark. Wash once with PBS, resuspend, and detect the positive rate by flow cytometry.

[0125] (2) Results Figure 5 The results showed that NKG2D and TGFβRI I were expressed in MOCK-NK, CAR-NK and TRⅡ / 21R-CAR-NK, with the positive rates being 11%, 96.3%, 95.8% and 22.9%, 29.5%, 46.2%, respectively.

[0126] 6. In vitro co-culture to detect the tumor killing effect of NK cells with chimeric antigen receptor targeting human NKG2DL, the target cell is MGC803-Luciferase

[0127] (1) NK92 cells (CAR-NK and TRⅡ / 21R-CAR-NK) infected with the virus 72 hours later, NK92 cells not infected with the virus, and NKG2DL+ and TGF-β+ tumor cells MGC803-Luciferase were taken, counted, and the cell density was adjusted. The cells were co-cultured in a 96-well low-adsorption cell culture plate in an incubator at a ratio of effector cells to target cells of 1.25:1, 2.5:1, and 5:1, i.e., 2×104 / well of MGC803-Luciferase cells and 2.5×104 / well, 5×104 / well, and 1×105 / well of NK92 cells were added, respectively. Three replicate wells were set for each effector-target ratio. The control cells were MGC803-Luciferase without effector cells. The final volume of the culture medium was 200 μl.

[0128] (2) After 4 h of co-culture, carefully pipette the cell suspension from the 96-well wells and detect the expression level of luciferase using a multifunctional microplate reader according to the instructions of the Firefly Luciferase Reporter Gene Assay Kit (Shanghai Biotech Co., Ltd.). Calculate the killing efficiency. The statistical results of cell killing are shown in Table 1. Figure 6 .

[0129] (3) The results showed that in the presence of CAR-NK and TRⅡ / 21R-CAR-NK, the killing ratio of the target cell MGC803-Luciferase increased significantly with the increase of the effector-target ratio. The killing efficiency of MOCK-NK against MGC803-Luciferase at the three effector-target ratios was 16.99%, 28.5%, and 37.7%, respectively; the killing efficiency of CAR-NK against MGC803-Luciferase was 22.68%, 46.12%, and 56.4%, respectively; the killing efficiency of TRⅡ / 21R-CAR-NK against MGC803-Luciferase was 49.68%, 64.4%, and 74.21%, respectively. At different effector-target ratios, the killing efficiency of TRⅡ / 21R-CAR-NK group was better than that of CAR-NK group, and that of CAR-NK group was better than that of MOCK-NK group. Statistical analysis showed that the difference in killing efficiency between the two groups was extremely significant (P < 0.001). This shows that both types of chimeric antigen receptor NK cells can kill target cells MGC803-Luciferase very well, and TRⅡ / 21R-CAR-NK is significantly better than CAR-NK.

[0130] 7. Flow cytometry detection of chimeric antigen receptor NK cell activation cytokine expression levels

[0131] (1) NK92 cells (CAR-NK and TRⅡ / 21R-CAR-NK) and NKG2DL+ and TGF-β+ tumor cells MGC803 were taken 72 hours after virus infection, counted, and the cell density was adjusted. The effector cells and target cells were co-cultured in a common 96-well cell culture plate in an incubator at a ratio of 5:1, that is, 2×104 MGC803-Luciferase cell suspension and 1×105 NK92 cells were added / well, and 3 replicate wells were set. The control cells were NK92 cells that were not infected with the virus. The final volume of the culture medium was 200 μl.

[0132] (2) Add 1 μL of 10 μg / mL Golgi inhibitor (BFA) to each well, gently shake to disperse the Golgi inhibitor in the cell suspension, and place in the incubator for 4 h.

[0133] (3) After incubation, transfer each group of samples to a centrifuge tube and wash twice with FACS buffer.

[0134] (4) Add 200 μL of membrane permeabilization fixative to suspend the cells and incubate them in a 4°C refrigerator overnight.

[0135] (5) After permeabilization overnight, add FACS buffer to wash the cells, discard the supernatant after centrifugation, add 100 μL FACS buffer to suspend the cells, add 0.3 μL each of PEcy7-anti-Human GzmB and APC-anti-Human I FN-γ flow cytometry antibodies, and gently tap the tube wall to mix.

[0136] (6) Incubate the cells at 4°C in the dark for half an hour and then add FACS buffer to wash away excess antibodies.

[0137] (7) Add 300 μL FACS buffer to resuspend the cells and perform flow cytometry analysis.

[0138] Results Figure 7 As can be seen from the figure, the expression levels of activation cytokines GzmB and IFNγ of the two chimeric antigen receptor NK cells CAR-NK and TRⅡ / 21R-CAR-NK were higher than those of the control MOCK-NK, which is consistent with Figure 5 The killing results are consistent.

[0139] 8. Flow cytometry detection of chimeric antigen receptor NK cell depletion

[0140] (1) As described above, chimeric antigen receptor NK cells (CAR-NK, TRⅡ / 21R-CAR-NK), MOCK-NK cells and target cells MGC803 were plated at an effector-target ratio of 4:1 and co-cultured for 5 days.

[0141] (2) On the fifth day of co-culture, each group of samples was transferred to an EP tube, the cells were washed with FACS buffer, and the supernatant was discarded.

[0142] (3) Resuspend the cells in 100 μl FACS buffer, add 0.3 μl PE-anti-human PD-1 and APC-anti-human TIM-3 flow cytometry antibodies to each group, and gently tap the tube wall to mix.

[0143] (4) Stain for 30 min at 4°C in the dark, wash with FACS buffer to remove excess antibody, resuspend cells in 300 μl FACS buffer, and analyze by flow cytometry.

[0144] (5) Results Figure 8As can be seen from the figure, after 5 days of co-culture with target cells, NK cells were exhausted due to continuous activation. Compared with MOCK-NK cells, the proportion of PD-1 and TIM-3 positive cells in the CAR-NK group increased; while the proportion of PD-1 and TIM-3 positive cells in the TRⅡ / 21R-CAR-NK group decreased significantly compared with the CAR-NK group, and was lower than that in the MOCK-NK group. This indicates that co-expression of TGF-βRⅡECD / IL-21RTM&ICD reduces the exhaustion of CAR-NK cells, which is conducive to the more lasting anti-tumor effect of CAR-NK cells.

[0145] 9. Animal Experimentation

[0146] NSG mice were used, and the tumor cells used were MGC803-Luc i, which stably expressed firefly luciferase, and 1.5×106 / mouse were injected subcutaneously. The effector cells injected for treatment were chimeric antigen receptor NK cells (CAR-NK, TRⅡ / 21R-CAR-NK), and the control group was a group of NK92 (MOCK-NK) cells that were not infected with the virus. After the model was successfully established, 1×107 / mouse effector cells were injected into the tail vein every 7 days. Every 7 days, the IVI S in vivo imaging system was used to take pictures and images to show the tumor growth. The mice were weighed, and the survival of the mice was observed and recorded. The results are shown in Fig. 9 .

[0147] As can be seen from Figure A, the fluorescent area of ​​the tumor of mice injected with CAR-NK and TRⅡ / 21R-CAR-NK is reduced compared with the previous time, indicating that the tumor cells are significantly reduced. As can be seen from the survival curve of NSG mice in Figure B, the survival rate of the TRⅡ / 21R-CAR-NK treatment group is 100% at 55 days; the survival rate of the CAR-NK treatment group is 80%; the death of the MOCK-NK treatment group began on the 22nd day, and all died on the 38th day. Figure C shows the photos of the subcutaneous tumors of mice separated after the end of the experiment.

[0148] In summary, compared with the three, the therapeutic effect of the TRⅡ / 21R-CAR-NK treatment group was significantly better than that of the CAR-NK treatment group, and the CAR-NK treatment group was better than that of the MOCK-NK treatment group. The results showed that the chimeric antigen receptor NK cell CAR-NK expressing NKG2D, TRⅡ / 21R-CAR-NK can target NKG2DL and effectively kill tumor cells expressing NKG2DL. After co-expression of the TGFβR II extracellular segment with the IL-21R transmembrane and intracellular segments, the immunosuppressive effect of TGF-β in the tumor was reversed, the activation, anti-exhaustion and killing functions of CAR-NK were promoted, the anti-tumor activity of CAR-NK was enhanced, the therapeutic effect was more obvious, and the purpose of treating tumors could be achieved.

[0149] The present invention designs and develops for the first time an engineered NK cell modified with a specific chimeric antigen receptor that targets NKG2DL and converts the inhibitory signal induced by TGF-β into an activation signal.

[0150] For the first time, the inhibitory signal of TGF-β in the tumor microenvironment was converted into an activation signal for NK cells through the extracellular segment of its ligand TGF-βRⅡ and the transmembrane region and intracellular segment of IL-21R, thereby enhancing the survival ability of NK cells in the tumor inhibitory microenvironment, improving the killing efficiency of NK against tumor cells, and enhancing the anti-tumor activity of NKG2D-targeted CAR-NK cells.

[0151] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are within the scope of the technical solution of the present invention.

Claims

1. An immune-enhancing CAR-NK cell that overcomes the tumor microenvironment, characterized in that: The CAR-NK cell can modify a chimeric antigen receptor, the chimeric antigen receptor is a PLL3.7-NKG2D-CAR, PLL3.7-TGFβRI I / IL21 R-NKG2D-CAR vector, the DNA sequence of each element of the vector is shown in SEQ ID NO: 1, the connection sequence of each part of the chimeric antigen receptor is shown in FIG3, and the CAR-NK cell is prepared by a method comprising the following steps: (1) Construction of PLL3.7-NKG2D-CAR and PLL3.7-TGFβRI I / IL21 R-NKG2D-CAR vectors; (2) Preparation of bacteria containing chimeric antigen receptor expression vectors and extraction of chimeric antigen receptor expression plasmids targeting human NKG2DL; (3) Virus packaging; (4) Virus titer determination; (5) Prepare NK cells with chimeric antigen receptors targeting human NKG2DL.

2. The immune-enhancing CAR-NK cell for overcoming the tumor microenvironment according to claim 1, characterized in that: The construction method of the PLL3.7-NKG2D-CAR and PLL3.7-TGFβRI I / IL21 R-NKG2D-CAR vectors comprises: (1) Whole gene synthesis: CD8α signal peptide, NKG2D extracellular domain, CD8α hinge region, CD8α transmembrane domain, 4-1BB co-stimulatory signal transduction region and CD3ζ signal transduction domain, and the above sequences are connected in sequence to obtain a chimeric antigen receptor NKG2D-CAR expression cassette; (2) After the whole gene was synthesized, the sequence of the chimeric antigen receptor expression cassette was connected to the empty vector pLL3.7 through the NheI / EcoRI restriction site to obtain a chimeric antigen receptor expression vector. After the sequence was determined to be correct, it was named PLL3.7-NKG2D-CAR, as shown in Figure 1; (3) Whole gene synthesis: self-cleaving T2A peptide, TGF-βRII extracellular domain, IL-21R transmembrane domain, IL-21R intracellular domain, the above sequences are connected in sequence; EcoRI enzyme digestion is used to recover the linearized PLL3.7-NKG2D-CAR vector, and the vector is connected through the EcoRI enzyme cleavage site to obtain a chimeric antigen receptor expression vector. After the sequence is determined to be correct, it is named PLL3.7-TGFβRI I / IL21R-NKG2D-CAR, as shown in Figure 3.

3. The immune-enhanced CAR-NK cell for overcoming the tumor microenvironment according to claim 1, characterized in that: The preparation of bacterial strains containing chimeric antigen receptor expression vectors and extraction of chimeric antigen receptor expression plasmids targeting human NKG2DL: Preparation of chimeric antigen receptor expression vector strains: (1) Take out 100 μL of competent cell DH5α suspension from the -80°C refrigerator and thaw on ice; (2) Add 0.1-1 μg of plasmid DNA (volume not exceeding 5 μL), mix gently, and place on ice for 5 minutes; (3) Heat shock in a 42°C water bath for 90 seconds, followed by rapid cooling on ice for 5 minutes; (4) Add 0.5 ml of LB liquid medium without resistance and culture at 37°C and 180 rpm for 30 min; (5) Centrifuge at 5000 rpm for 4 min, remove part of the supernatant, retain 100-150 μl of culture medium, gently pipette the suspended bacteria, and then spread it on an LB plate containing antibiotics; (6) Incubate overnight at 37°C; (7) Pick a single clone and inoculate it into ampicillin-resistant LB medium and culture it at 37°C and 200 rpm for 9-12 hours; (8) Add glycerol to the bacterial solution to a final concentration of 10% and store the bacteria in a -80°C refrigerator for later use; Extraction of chimeric antigen receptor expression plasmid targeting human NKG2DL: The DH5α strain containing the chimeric antigen receptor expression plasmid targeting human NKG2DL was cultured in large quantities in LB medium, and an endotoxin-free plasmid extraction kit was used for high-purity endotoxin-free extraction in preparation for lentiviral packaging.

4. The immune-enhancing CAR-NK cell for overcoming the tumor microenvironment according to claim 1, characterized in that: The virus packaging: Preparation of 293T cells: 24 hours before virus packaging, 293T cells were digested with trypsin and re-plated on 10 cm plates. The cells were cultured in DMEM medium containing 10% FBS in a 37°C 5% CO2 incubator. Transfection was performed when the cell density reached 70-90%. Plasmid transfection: (1) Place plasmid, PEI, and DMEM medium at room temperature for 5 min; (2) Take 450 μl of DMEM into a 1.5 ml EP tube, add 50 μl of PEI (1 μg / μl), mix well, and let stand at room temperature for 5 min; (3) Take 5 μg of plasmid (pLL3.7-NKG2D-CAR, pLL3.7-TGFβ RI I / IL21 R-NKG2D-CAR), 5 μg psPAX2, 3 μg pMD2.G, add DMEM to 500 μl, mix well, and stand at room temperature for 5 min; (4) adding the prepared PEI-DMEM solution of step (2) to the DMEM containing the plasmid obtained in step (3), mixing well, and standing at room temperature for 20 minutes to obtain a DNA / PEI mixture; (5) Slowly drip 1 ml of DNA / PEI mixture into the 293T culture dish, mix gently, and incubate at 37°C for 6-8 h. (6) Discard the original culture medium, replace with fresh culture medium, and continue incubating in a 37°C incubator; (7) After replacing the culture medium for 48 hours, the culture medium was collected, and then 10 ml of fresh culture medium was added to each dish to continue culturing. After 24 hours, the supernatant was collected again and mixed with the supernatant collected at 48 hours; (8) Centrifugation at 4000 g for 10 min at 4°C to remove cell debris; (9) filtering the obtained supernatant through a 0.45 μm filter; (10) The filtered supernatant was transferred to an ultracentrifuge tube and centrifuged at 25,000 rpm for 2 h. The virus precipitate obtained after ultracentrifugation was resuspended in serum-free medium with 1% supernatant volume and gently pipetted until completely dissolved to obtain virus liquid. The virus liquids obtained using different vectors were named: LV-NKG2D-CAR, LV-TRⅡ / 21R-NKG2D-CAR; (11) Aliquot each virus solution and store it in a -80°C refrigerator, and reserve 5-10 μl of virus concentrate for titer determination.

5. The immune-enhanced CAR-NK cell for overcoming the tumor microenvironment according to claim 1, characterized in that: The virus titer determination: (1) Digest and count 293T cells, prepare a cell suspension using DMEM medium containing 10% FBS, adjust the cell density to 4×105 / ml, and add 0.5 ml of the cell suspension to each well of a 24-well cell culture plate; (2) The virus concentrate to be tested was added to a 24-well plate in different gradients, leaving a blank group as a control. The 24-well plate was placed in an incubator for incubation, and the positive rate of 293T cells was detected by flow cytometry after 48 hours; (3) Centrifuge, resuspend, and adjust the cell density to 1×106 / ml. Add appropriate amount of antibody (0.3 μl each of APC-Anti-HumanNKG2D and PE-Anti-Human TGF-βRⅡ) to 100 μl, gently pipette to mix, and place in a 4°C refrigerator to stain for 30 min in the dark. (4) Wash once with PBS, resuspend, and perform flow cytometry analysis; (5) Calculate the titer, titer (TU / ml) = (2*105*infection efficiency*1000) / virus volume (μl). After titer detection, the titers of pLL3.7-NKG2D-CAR and pLL3.7-TRⅡ / 21R-NKG2D-CAR were 1.1*108 and 0.42*108, respectively.

6. The immune-enhanced CAR-NK cell for overcoming the tumor microenvironment according to claim 1, characterized in that: The method for preparing NK cells with chimeric antigen receptors targeting human NKG2DL: NK92 culture: (1) Preparation of NK92-specific culture medium, incomplete culture medium: αMEM + 0.1 mM β-mercaptoethanol + 12.5% ​​HS and 12.5% ​​FBS at a final concentration. Add IL-2 (200 IU / ml) to the culture medium when using. Try to use it up within 7 days, otherwise IL-2 needs to be added again; (2) NK92 cells were revived by thawing in a 37°C water bath, centrifuging at 800 rpm for 5 min, discarding the cryoprotectant, adding culture medium to resuspend, and culturing in a T25 culture flask with an initial density of 2-3*105 cells per ml; (3) For passaging of NK92 cells, shake the culture flask to roughly mix the cells, count the cells, and perform a full or half medium change every 2-3 days at a cell density of 2-3*105 viable cells per ml. Determine the passaging time and frequency based on the cell density; Lentivirus infection: Adjust the cell density of NK92 cells to 1×106 / ml, add 0.5 ml of cell suspension to each well of a 24-well cell culture plate, and infect NK92 cells with the two virus solutions obtained in 3.2 at an MOI of 20. After 24 hours, replace the solution to obtain different NK cells expressing chimeric antigen receptors targeting human NKG2DL antigen; they were named CAR-NK and TRⅡ / 21R-CAR-NK respectively; Detection of chimeric antigen receptor targeting human NKG2DL (CAR-NKG2D) and TGFβRI I expression: During the culture process, NK92 cell CAR-NK, TRⅡ / 21R-CAR-NK, and NK92 cells not infected with the virus (named MOCK-NK) were taken 72 hours after virus infection, centrifuged, resuspended, and the cell density was adjusted to 1×106 / ml. An appropriate amount of antibody (0.3μl each of APC-Anti-Human NKG2D and PE-Anti-Human TGF-βRⅡ) was added to 50μl, and the mixture was gently blown and placed in a 4°C refrigerator. The cells were stained for 30 minutes away from light, washed once with PBS, resuspended, and the positive rate was detected by flow cytometry.

7. Use of the immune-enhanced CAR-NK cells that overcome the tumor microenvironment according to any one of claims 1-6 in anti-tumor.

Images