Intracellular structural domain, MSLN-targeted chimeric antigen receptor and application of MSLN-targeted chimeric antigen receptor

Abstract

The invention discloses an MSLN-targeted chimeric antigen receptor intracellular structural domain of which the amino acid sequence is shown as SEQ ID NO.11. The invention also discloses a nucleotide sequence of the intracellular structural domain, an MSLN-targeted chimeric antigen receptor, a recombinant lentiviral vector, an NK cell for expressing the chimeric antigen receptor and application of the intracellular structural domain in preparation of medicines for treating or preventing tumors. According to the present invention, after the ITSM sequence in the CD244 sequence of the CAR intracellular structural domain is optimized, the flow type immunofluorescence staining result shows that the expression of the CAR molecule in the NK cell has no significant influence, and the good killing effect on the MSLN positive ovarian cancer cell line is provided; when the NK cell expressing the chimeric antigen receptor is subjected to in-vitro function detection, the selected cell line is a cell line highly expressing an MSLN target spot outside a cell membrane, and the tumor killing effect evaluation of the NK cell expressing the chimeric antigen receptor is more scientific.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to an intracellular domain and its application in CAR-NK cells targeting MSLN. Background Art

[0002] Chimeric Antigen Receptor (CAR) immunocyte technology is a technology that, through in vitro gene modification, expresses the sequence of the variable region of a single-chain antibody (Single chain antibody variable fragments, scFvs) against a specific antigen on the surface of a patient's immune cells, such as T cells, NK cells, etc., and then targets and kills tumor cells. The basic design of CAR includes a tumor-associated antigen-binding region (usually the scFv segment derived from the antigen-binding region of an antibody), an extracellular hinge region, a transmembrane region, and an intracellular signaling region. The extracellular part of the cell membrane is the antigen-targeting part purified from a monoclonal antibody, which is composed of a single-chain variable fragment (scFv) (a fusion protein of the variable regions of the heavy and light chains), directly determining the targeting of drug treatment; the intracellular domain contains several signal transduction elements, which are activated after CAR binds to the antigen, initiating a series of signal transduction pathways within the cell. These pathways ultimately lead to the activation, proliferation, cytokine secretion of immune cells, and the killing of target cells.

[0003] CAR-modified natural killer cells (CAR-NK), as an emerging immunocyte therapy, have shown great potential in tumor treatment. Compared with CAR-T cells, CAR-NK cells have stronger safety, a lower risk of cytokine release syndrome (CRS), and a broader tumor-killing ability. However, the natural activation signal transduction mechanism of NK cells is significantly different from that of T cells, and the natural activation of NK cells depends on the dynamic balance between multiple co-stimulatory molecules and inhibitory receptors. Traditional CAR designs usually use co-stimulatory domains derived from T cells (such as CD28, 4-1BB), and these structures may not fully function in NK cells, or even lead to low signal transduction efficiency or cell dysfunction. Therefore, by introducing and optimizing NK cell-specific co-stimulatory domains, the persistence and anti-tumor activity of CAR-NK cells can be significantly enhanced.

[0004] CD244 (also known as 2B4) is a NK cell-specific co-stimulatory molecule. Its intracellular domain contains an immunoreceptor tyrosine-based switch motif (ITSM). This structure can bind to the adaptor protein SAP (SLAM-associated protein), which can transmit strong co-stimulatory signals and promote the activation, proliferation and cytotoxicity of NK cells. The CD244 molecule contains a total of 4 ITSM motifs. Studies have shown that the first and second ITSM motifs recruit SAP adaptor proteins through tyrosine phosphorylation after CD244 binds to the ligand CD48. SAP further binds to Fyn kinase and activates downstream signaling pathways (such as PI3K / AKT, MAPK / ERK), thereby promoting the activation, proliferation and cytotoxicity of NK cells. However, the functions of the third and fourth ITSMs are not yet very clear. Some researchers have found through sequence analysis that the third and fourth ITSM motifs tend to recruit phosphatases containing SH2 domains (such as SHP-1 and SHP-2). These phosphatases inhibit downstream signaling pathways through dephosphorylation, thereby transmitting inhibitory signals.

[0005] Based on this, the present invention optimizes and transforms the third and fourth ITSM intracellular domains of the CD244 molecule by amino acid replacement, and applies it to the in vitro functional evaluation of CAR-NK cell immunotherapy targeting MSLN, in order to improve the clinical efficacy of CAR-NK by relieving inhibitory signals. Summary of the invention

[0006] The purpose of the present invention is to make the chimeric antigen receptor targeting MSLN more efficiently and stably expressed in NK cells, and to improve the killing ability of CAR-NK cells targeting MSLN for tumor cells expressing MSLN on the surface.

[0007] The technical solution adopted by the present invention to solve its technical problem is: a chimeric antigen receptor intracellular domain CAR-CD244-4ITSM-F targeting MSLN, the intracellular domain sequence optimization site is the tyrosine (Y) mutation of the 4th ITSM domain in the chimeric antigen receptor intracellular domain CD244 molecule to phenylalanine (F), the optimized structure is named CAR-CD244-4ITSM-F, and its amino acid sequence is shown in SEQ ID NO.11.

[0008] The present invention also provides a nucleotide sequence encoding the above-mentioned intracellular domain CAR-CD244-4ITSM-F, as shown in SEQ ID NO.12.

[0009] The present invention also provides a chimeric antigen receptor targeting MSLN, wherein the chimeric antigen receptor comprises the above-mentioned intracellular domain CAR-CD244-4ITSM-F.

[0010] Specifically, the above-mentioned chimeric antigen receptor comprises a single-chain variable region of an anti-MSLN antibody, a CD8 hinge region, a CD244 transmembrane region, a CD244 intracellular domain CD244-4ITSM-F, and an intracellular co-stimulatory domain CD3ζ chain, which are sequentially spliced from the N-terminus to the C-terminus.

[0011] Specifically, the amino acid sequence of the above-mentioned single-chain variable region of the anti-MSLN antibody is as shown in SEQ ID NO.1; the amino acid sequence of the CD8 hinge region is as shown in SEQ ID NO.3; the amino acid sequence of the CD244 transmembrane region is as shown in SEQ ID NO.5; the amino acid sequence of the CD244 intracellular domain is as shown in SEQ ID NO.11; the amino acid sequence of the CD3ζ is as shown in SEQ ID NO.13. The nucleotide sequence of the above-mentioned single-chain variable region of the anti-MSLN antibody is as shown in SEQ ID NO.2; the nucleotide sequence of the CD8 hinge region is as shown in SEQ ID NO.4; the nucleotide sequence of the CD244 transmembrane region is as shown in SEQ ID NO.6; the nucleotide sequence of the CD244 intracellular domain is as shown in SEQ ID NO.12; the nucleotide sequence of the CD3ζ is as shown in SEQ ID NO.14.

[0012] Specifically, the amino acid sequence of the above-mentioned chimeric antigen receptor is as shown in SEQ ID NO.19; the nucleotide sequence of the chimeric antigen receptor is as shown in SEQ ID NO.20.

[0013] The present invention also provides a recombinant lentiviral vector, which is based on the pTK-EF1α-Kan vector and contains the coding nucleotide shown in SEQ ID NO.20.

[0014] The present invention also provides a natural killer cell (CAR-NK) expressing a chimeric antigen receptor, and the CAR-NK immune cell is obtained by transfecting the NK cells of a mammal with the above-mentioned chimeric antigen receptor.

[0015] Specifically, the immune cells of humans or other mammals are selected from umbilical cord blood, apheresis blood, peripheral blood or NK cells derived from iPSCs.

[0016] The chimeric antigen receptor, recombinant lentiviral vector or NK cell provided by the present invention can be used to prepare a drug for treating or preventing tumors. The tumor is ovarian cancer or other diseases with MSLN as a therapeutic target. The disease with MSLN as a therapeutic target is preferably ovarian cancer.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects: After the intracellular domain CAR-CD244-4ITSM-F sequence targeting MSLN provided by the present invention is optimized, the results of flow cytometry immunofluorescence staining show that the expression of the CAR molecule in NK cells is not significantly affected, while it can play a better killing effect on MSLN-positive ovarian cancer cells.

[0018] The NK cells expressing chimeric antigen receptors provided by the present invention are obtained by in vitro inducing and activating the isolated peripheral blood mononuclear cells for 10-12 days to obtain NK cells with higher purity for transducing lentivirus expressing chimeric antigen receptors. The original immune cells will not affect the anti-tumor effect of the NK cells expressing chimeric antigen receptors after transfection. When performing in vitro functional detection on the NK cells expressing chimeric antigen receptors, the selected cell line is a cell line with high expression of MSLN target outside the cell membrane, which is more scientific for evaluating the anti-tumor effect of the NK cells expressing chimeric antigen receptors. Brief Description of the Drawings

[0019] Figure 1 It is a schematic diagram of the results of the active titers of lentiviruses packaged with three CAR plasmids, CD244-ITSM-WT, CD244-3ITSM-F, and CD244-4ITSM-F. Figure A is the flow cytometry detection result, and Figure B is the significance quantification diagram; Figure 2 It is a schematic diagram of the transfection efficiency results of three CAR-NK cells, CD244-ITSM-WT, CD244-3ITSM-F, and CD244-4ITSM-F; Figure 3 It is a schematic diagram of the killing results of three CAR-NK cells on cell line A2780 (A) and a schematic diagram of the killing results of three CAR-NK cells on negative cell line OVCAR8 (B); Figure 4 It is a schematic diagram of the results of cytokine IFN-γ release after in vitro co-incubation of three CAR-NK cells with cell line A2780 (A) and a schematic diagram of the results of cytokine IFN-γ release after in vitro co-incubation of three CAR-NK cells with cell line OVCAR8 (B). Detailed Embodiments

[0020] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Although the representative embodiments of the present invention have been described in detail, those of ordinary skill in the technical field to which the present invention belongs will understand that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0021] The intracellular domain of the present invention and its application effect in CAR-NK cells targeting MSLN are studied through specific embodiments below. Example 1

[0022] Obtaining lentiviral vectors of chimeric antigen receptors targeting MSLN (pTK-EF1α-CAR-CD244-ITSM-WT, pTK-EF1α-CAR-CD244-3ITSM-F, pTK-EF1α-CAR-CD244-4ITSM-F).

[0023] 1. Obtain CAR-CD244-ITSM-WT (wild type), CAR-CD244-3ITSM-F (mutant 1), and CAR-CD244-4ITSM-F (mutant 2).

[0024] Research shows that the intracellular domain of CD244 is crucial for the proliferation and anti-tumor function of NK cells. The tyrosine phosphorylation site of the ITSM motif in the intracellular domain of CD244 activates the downstream signal transduction pathway by binding to SAP, thereby enhancing the cytotoxicity and proliferation ability of NK cells and playing a key role in regulating the immune response. To further improve the function of CAR-NK cells, in this example, the tyrosine (Y) in the 3rd or 4th ITSM motif in the intracellular domain sequence of wild-type CD244 in CAR-CD244 was mutated to phenylalanine (F), and they were named CAR-CD244-3ITSM-F and CAR-CD244-4ITSM-F respectively.

[0025] 2. Obtain the complete CAR structure sequences of CAR-CD244-ITSM-WT (wild type), CAR-CD244-3ITSM-F (mutant 1), and CAR-CD244-4ITSM-F (mutant 2).

[0026] Use OverlapPCR to amplify using scFv and CD8hinge-CD244TM+CD244ICD-CD3ζ as templates respectively to obtain sequences with restriction enzyme sites Eco R I and BamThe MSLN-CAR-CD244-WT fragment of HI, and then by designing site-directed mutagenesis primers, the MSLN-CAR-CD244-3ITSM-F fragment and the MSLN-CAR-CD244-4ITSM-F fragment were obtained by OverlapPCR amplification technology. Among them, the sequences of anti-MSLN SCFV, CD8hinge, CD244TM, CD244-ITSM-WT, CD244-3ITSM-F, CD244-4ITSM-F, and CD3ζ are shown in the following table (chimeric antigen receptor structure sequence information).

[0027] 。

[0028] The complete CAR structure amino acid sequence of CAR-CD244-WT is shown in SEQ ID NO.15, and the nucleotide sequence is shown in SEQ ID NO.16.

[0029] The complete CAR structure amino acid sequence of CAR-CD244-3ITSM-F is shown in SEQ ID NO.17, and the nucleotide sequence is shown in SEQ ID NO.18.

[0030] The complete CAR structure amino acid sequence of CAR-CD244-4ITSM-F is shown in SEQ ID NO.19, and the nucleotide sequence is shown in SEQ ID NO.20.

[0031] 3. The plasmid pTK-EF1α-Kan was constructed by the method in the patent ZL201611246081.9. The plasmid pTK-EF1α-Kan was digested with Eco R I and Bam H I restriction endonucleases. The products were subjected to 0.8% agarose gel electrophoresis, and the gel was cut and recovered and placed in an Eppendorf tube. The corresponding fragments were recovered using the agarose gel recovery kit of Axygen Company, and the purity and concentration of the products were determined.

[0032] 4. Add the fragments to an Eppendorf tube at a molar ratio of 1:2, add Exnase ligase (Vazyme) and homologous recombinase 5×CE buffer, and react at 37 °C for 0.5 h. Take out 10 μL of the ligation solution and add it to 100 μL of DH5α competent cells. Incubate on ice for 30 min, then heat shock at 42 °C for 90 s. After completion, add 500 μL of soc medium and culture at 37 °C and 220 rpm for 2 h. After 2 h, centrifuge the Eppendorf tube at 4000 g for 1 min to remove 400 μL of excess liquid. Spread the remaining liquid on an LB plate and culture at 37 °C for 12 h. Pick a single colony from the plate and inoculate it into 5 mL of LB liquid medium and culture at 37 °C and 220 rpm for 12 h.

[0033] 5. Extract the plasmids using the Axygen miniprep kit to obtain plasmids pTK-EF1α-CAR-CD244-ITSM-WT, pTK-EF1α-CAR-CD244-3ITSM-F, and pTK-EF1α-CAR-CD244-4ITSM-F. After sending them to Tsingke Biotechnology (Wuhan) Co., Ltd. for first-generation sequencing and verifying that they are correct, preserve the Escherichia coli DH5α strains containing plasmids pTK-EF1α-CAR-CD244-ITSM-WT, pTK-EF1α-CAR-CD244-3ITSM-F, and pTK-EF1α-CAR-CD244-4ITSM-F. Example 2

[0034] Preparation and sequencing of plasmids.

[0035] 1. Preparation of plasmids. Inoculate the Escherichia coli DH5α strains containing plasmids pTK-EF1α-CAR-CD244-ITSM-WT, pTK-EF1α-CAR-CD244-3ITSM-F, and pTK-EF1α-CAR-CD244-4ITSM-F into 250 mL of LB culture medium containing 100 μg / mL kanamycin respectively, and culture overnight at 37 °C and 220 rpm. Centrifuge the culture solution at 6000 g for 20 min at 4 °C, and discard the supernatant.

[0036] Take out Buffer P1 in the Endo Free plasmid mega kit (Qiagen), add 120 mL of pre-cooled Buffer P1 to the centrifuged Escherichia coli pellet, cover the centrifuge bottle cap, and shake the centrifuge bottle vigorously to completely disperse the Escherichia coli pellet in Buffer P1.

[0037] Add 120 mL of Buffer P2 to the centrifuge bottle, cover the bottle cap, place it on the roller mixer, gradually increase the speed to 50 rpm, mix thoroughly, and then let it stand at room temperature for 5 min.

[0038] Add 120 mL of Buffer P3 to the centrifuge bottle, cover the bottle cap, place it on the roller mixer, gradually increase the speed to the maximum speed of the roller mixer, 70 rpm, mix thoroughly until it becomes a white, non-viscous, fluffy mixture. Centrifuge at 9000 g for 15 min at 4 °C.

[0039] Pour 50 mL of Buffer FW into the QIA filter Cartridge, pour the supernatant obtained by centrifugation into the QIA filter Cartridge, and gently stir to mix. Filter the mixture into the corresponding labeled glass bottle.

[0040] Add 20 mL of Buffer ER to each glass bottle, invert it up and down 6 times to mix, and incubate at -20 °C for 30 min.

[0041] Place the labeled mega column on the corresponding rack, add 35 mL of Buffer QBT to each mega column to equilibrate, and let it drain by gravity.

[0042] Pour all the liquid in the glass bottle into the corresponding labeled mega column in batches. After the liquid in the column has drained, add 200 mL of Buffer QC to each mega column in batches for washing. After the liquid in the column has drained, pour the waste liquid in the waste liquid collection tray into a 50 mL clean centrifuge tube.

[0043] Then add 40 mL of Buffer QN to each mega column, collect the eluate using a 50 mL clean centrifuge tube, invert it up and down 6 times to mix, and aliquot 20 mL into another clean and labeled 50 mL centrifuge tube.

[0044] Add 14 mL of isopropanol (at room temperature) to each 50 mL centrifuge tube, invert it up and down 6 times to mix. Centrifuge at 15000 g for 50 min at 4 °C.

[0045] Aspirate the supernatant completely in the laminar flow hood, add 3.5 mL of Endotoxin-free water to each tube for rinsing, and do not disperse the bottom precipitate. Centrifuge at 15000 g for 30 min at 4 °C. Preheat the Buffer TE in the Endo Free plasmid mega kit in the oven.

[0046] Aspirate the supernatant after centrifugation inside the laminar flow hood and dry it inside the laminar flow hood (volatilize the residual absolute ethanol, for about 10 minutes).

[0047] Take out Buffer TE from the oven, add 1 mL of Buffer TE to each tube inside the laminar flow hood, pipette 10 times, and then place it in an oven at 65 °C. Continuously tap the tube wall during this period to completely dissolve the precipitate. Centrifuge at 4000 g for 1 minute at 4 °C to spin the liquid on the tube wall to the bottom of the tube, and then pipette and mix well.

[0048] Transfer all the liquid to endotoxin-free, pyrogen-free, and nuclease-free EP tubes with corresponding labels inside the laminar flow hood. Aspirate 2 μL and measure the plasmid concentration with a micro-spectrophotometer, and label it on the corresponding EP tubes to obtain plasmids pTK-EF1α-CAR-CD244-ITSM-WT, pTK-EF1α-CAR-CD244-3ITSM-F, and pTK-EF1α-CAR-CD244-4ITSM-F.

[0049] 2. Sequencing of the target gene. Take 20 μL (500 ng) of plasmid DNA respectively and send it for external sequencing. According to the original seed sequence, check whether the target gene of the product obtained from plasmid production has changed. Under a stable process, the target gene will not change during the fermentation culture amplification process of the working seed, and it can be used for the production and correct protein expression in the next step. Example 3

[0050] Preparation and live titer detection of lentiviral vectors pTK-EF1α-CAR-CD244-ITSM-WT, pTK-EF1α-CAR-CD244-3ITSM-F, and pTK-EF1α-CAR-CD244-4ITSM-F.

[0051] 1. Preparation of lentiviral vectors. Inoculate 0.4×10 6 number of 293T-S cells into a cell culture shake flask (Hyperflask), add 200 mL of F medium (100 mL of OPM-293 CD05 Medium, 100 mL of SMM 293 T-II medium), and place it on an orbital shaker in a cell culture incubator at 37.0 °C and 5.0% CO 2 for shaking culture (130 rpm). Passage 293T-S cells at a cell density of (4.0 ± 1.0)×10 5 cells / mL. Two days after passage, when the cell density reaches (2.0 ± 0.5)×10 6When the cell density reaches 1×10⁶ cells / mL, it can be used for lentivirus packaging. First, prepare the transfection mixture (taking 100 mL of cultured 293TS cells as an example). Add 100 µg of plasmids (CAR expression plasmid: pMDLg / pRRE:pRSV-Rev:pMD2.G = 12:10:6:5, i.e., 36 µg:30 µg:18 µg:15 µg) into a 50 mL centrifuge tube containing 5 mL of complete medium for 293T-S cells, mix well, and let it stand at room temperature for 5 min. Take another 50 mL centrifuge tube, add 5 mL of complete medium for 293T-S cells, and then add 300 µL of PEI solution (1 g / L), mix well, and let it stand at room temperature for 5 min.

[0052] Dropwise add the PEI solution diluted with complete medium for 293T-S cells to the plasmid mixture solution diluted with complete medium for 293T-S cells, shake and mix well while adding, and then mix well by pipetting and let it stand at room temperature for 30 min. Use a 10 mL pipette to dropwise add 10 mL of the PEI / plasmid mixture to a cell culture flask containing 100 mL of cultured 293T-S cells, mix well, and then place it in a cell culture incubator shaker for culture. After culturing for 24 h, add 5 mL of OPM-CHO PFF06 medium to each flask of cells, and continue culturing for 24 h before collecting the lentivirus.

[0053] Centrifuge the collected supernatant at 4000 rpm (or 3000 g) for 30 min, then add cryonase enzyme (Takara) to the centrifuged supernatant and place it at 4°C. After 6 h, filter the lentivirus supernatant through a 0.22 µm filter membrane, and centrifuge at 30000 g at 4°C for 2.5 h. Remove the supernatant, add 1 mL of serum-free DMEM medium to resuspend the precipitate. After resuspension, take 20 µL for virus activity titer detection, and aliquot the remaining lentivirus concentrate and store it at -80°C for later use.

[0054] 2. Detection of lentiviral vector activity titer.

[0055] Principle: FMC63-APC is labeled with a fluorophore, and FMC63-APC can specifically bind to the ScFv in CAR. The fluorescence signal detected by flow cytometry indirectly reflects the expression of CAR in 293T cells.

[0056] Method: 293T cells cultured in a T75 culture flask adherently, after trypsin digestion and resuspension with medium, adjust the cell density to 5×10 5 cells / mL. Add 2 mL to each well of the surface-treated 6-well plate, that is, a total of 5×10 5293T cells. Then, 10 µL of polybrene solution (0.8 µg / mL) was added to each well to make the final concentration 4 µg / µL. The lentivirus was diluted with serum-free DMEM medium at a ratio of 1:100. After dilution, the lentivirus was added to the 6-well plate at 1, 2, 5, 10, 20, and 50 µL per well respectively, and one well was not added with lentivirus as a negative control. After mixing, it was placed in an incubator at 37 °C and 5% CO 2 2 for cell culture. Three days after cell culture, the cells were taken out, and the cells in each well were digested with 0.5 mL of Versene digestive solution for 10 - 15 min. 2 mL of flow cytometry buffer (PBS + 2% FBS) was added to each well of the 6-well plate, and the cells were pipetted and mixed well to disperse the cells into single cells. 0.5 mL was taken for flow cytometry to detect the proportion of CAR+ cells. The flow cytometry gating was performed using the negative control group to analyze the proportion of CAR+ cells in different lentivirus addition groups. The lentivirus addition volume corresponding to the proportion of CAR+ cells of 10% - 20% was selected, and the lentivirus activity titer was calculated according to the following formula.

[0057] Lentivirus activity titer (Tu / mL) = (number of cells × 10 3 × percentage of CAR+ cells) / virus volume (µL) Currently, the activity titer of the current lentivirus concentrate is in the range of 1×10 7 ~10×10 7 (TU / mL). The detection and analysis results are shown in Figure 1 and the following table (detection and analysis results of lentivirus activity titer). It shows that each lentivirus vector can obtain a high activity titer and can be used for the preparation of chimeric antigen receptor immune cells in the follow-up.

[0058] . Example 4

[0059] Preparation of CAR-CD244-ITSM-WT-NK, CAR-CD244-3ITSM-F-NK, and CAR-CD244-4ITSM-F-NK cells.

[0060] 1. Preparation of CAR-NK cell preparation.

[0061] In this study, an NK cell culture kit produced by Shenzhen Jiake Biotechnology Co., Ltd. was used to culture human peripheral blood NK cells. The cell culture method was carried out according to the instructions provided by the manufacturer. The main operation process is as follows.

[0062] (1) On the first day: 4 mL of PBS was added to a T75 culture flask, 0.2 mL of each of the coating reagents JV-1 and JV-2 in the kit was added and mixed well, and it was placed at 37 °C and 5.0% CO 2In the incubator, coat for 2 hours. Take 10 mL of anticoagulated peripheral blood and transfer it to a 15 mL centrifuge tube. Centrifuge at 800×g for 15 min at room temperature. Take the supernatant plasma, place it in a water bath at 56°C for 30 min; then let it stand at -20°C for 10 min; centrifuge at 1,100×g for 15 min at 4°C, and store the supernatant plasma at 4°C for later use. Add PBS to the centrifuged blood cells to make 10 mL, and mix well. Then add it to a 50 mL centrifuge tube containing 5 mL of human lymphocyte separation solution, centrifuge at 800×g for 15 min at room temperature, with slow acceleration and deceleration. Take the cell layer, add 5 mL of NK medium without any factors, centrifuge at 800×g for 10 min at room temperature, discard the supernatant, and collect peripheral blood mononuclear cells (PBMC). The isolated PBMC is made into an 8 mL cell suspension with NK medium without any factors, add 0.1 mL of each of JV-3-1 to 5, and then add 0.4 mL of JV-R-plus. Add the mixed cell suspension to a T75 culture flask from which the coating solution has been discarded, add 1 mL of plasma, and place it in an incubator at 37°C and 5.0% CO 2 Cultivate in the incubator.

[0063] (2)On the 4th day: Prepare NK cell proliferation medium and NK cell activation medium. The proliferation medium is 0.2 mL of JV-5 added to every 200 mL of cell factor-free NK cell medium; the NK cell activation medium is 0.1 mL of JV-4 added to 50 mL of NK cell proliferation medium. After the medium is prepared, store it at 2 - 8°C for later use. Add 8 mL of NK activation medium to the cell culture flask, add 1 mL of plasma, add 0.4 mL of JV-R-plus, and 0.2 mL of JV-6.

[0064] (3)On the 6th day: Add 14 mL of NK activation medium, add 1 mL of plasma, add 0.4 mL of JV-R-plus, and 0.2 mL of JV-7.

[0065] (4)On the 8th day: Transfer the cells to 1 T175 cell culture flask, add 1 mL of plasma, and add the remaining 28 mL of NK cell activation medium.

[0066] (5)On the 10th day: Supplement 40 mL of NK cell proliferation medium.

[0067] (6)On the 12th - 16th day: Depending on the cell growth status, supplement NK cell proliferation medium and adjust the cell density to 1 - 2×10 6 cells / mL. Detect the purity of NK cells by flow cytometry and prepare for lentiviral transduction to prepare CAR-NK cells.

[0068] (7)Lentiviral transduction: Take cells cultured for 12 - 16 days, with a density of 1.5 - 2.5×10 64 - 10 mL of primary NK cells cultured at [number] cells / mL were added to the completed-coated T25 cell culture flask. Calculate the volume of each lentivirus to be added according to the multiplicity of infection (MOI) of 10, and add the lentivirus to the cell culture flask and mix well. Incubate at 37 °C in a 5% CO 2 cell culture incubator. After 24 h, supplement the NK cell proliferation medium, and the volume of the supplemented medium is 2 times the original culture volume. Count the cells every 2 - 4 days, and supplement the NK cell proliferation medium to control the cell density at 1.0 - 2.0×10 6 cells / mL.

[0069] 2. Detection of transduction efficiency of CAR-CD244-ITSM-WT-NK, CAR-CD244-3ITSM-F-NK, and CAR-CD244-4ITSM-F-NK cells.

[0070] Three days after the NK cells were transduced with lentivirus, the proportion of CAR+ cells in the CAR-NK cells was detected by flow cytometry to evaluate the transduction efficiency of the lentivirus on the NK cells. In this study, recombinant human MSLN protein was used to label the scFv against MSLN on the cell surface. The recombinant protein carried a biotin tag and could bind to streptavidin labeled with PE fluorophore to fluorescently label the CAR+ cells. The specific operation is as follows: Take 2 - 5×10 5 cells and add them to a 5 mL round-bottom flow cytometry tube. Add 1 mL of flow cytometry Buffer and wash 3 times, then add 100 μL of flow cytometry Buffer to resuspend the cells. Add 1 μL of biotin-labeled human MSLN recombinant protein to each group of cells, and incubate at 2 - 8 °C in the dark for 30 min. Wash 3 times with flow cytometry Buffer, and add 100 μL of flow cytometry Buffer to resuspend the cells. Add 1 μL of PE-fluorescently labeled streptavidin to each group of cells, and incubate at 2 - 8 °C in the dark for 30 min. After briefly shaking and mixing evenly, detect with a flow cytometer.

[0071] The detection results of the transduction efficiency of CAR-CD244-ITSM-WT-NK, CAR-CD244-3ITSM-F-NK, and CAR-CD244-4ITSM-F-NK cells are as Figure 2 shown. The results show that CAR-NK cells were successfully prepared, and the expression efficiencies of CAR in the three groups of cells were 23.32%, 23.24%, and 23.15% respectively, with no significant difference among them. This result indicates that the optimized intracellular domain has no obvious effect on the expression of the chimeric antigen receptor. Example 5

[0072] In vitro functional detection of CAR-CD244-ITSM-WT-NK, CAR-CD244-3ITSM-F-NK, and CAR-CD244-4ITSM-F-NK.

[0073] 1. In vitro tumor killing assay. The in vitro tumor killing functions of CAR-CD244-ITSM-WT-NK, CAR-CD244-3ITSM-F-NK, and CAR-CD244-4ITSM-F-NK cells were detected using the calcein assay.

[0074] The target cells were the OVCAR8 tumor cell line (MSLN-positive cell line) and A2780 (MSLN-negative cell line). An appropriate amount of target cells was taken, and calcein-acetoxymethyl ester (Calcein-AM) was added to a final concentration of 25 μM in a cell suspension of 1×10 6 / mL (PBS, 5% fetal bovine serum), and incubated in an incubator for 30 min. At room temperature, after washing twice, the cells were resuspended to 0.5×10 5 / mL. 0.5×10 5 / mL cells were added to each well of a 96-well plate, and CAR-CD244-ITSM-WT-NK, CAR-CD244-3ITSM-F-NK, and CAR-CD244-4ITSM-F-NK cells were added at effector-to-target ratios of 25:1, 5:1, and 1:1, respectively, and incubated at 37°C for 2 - 3 hours. After incubation, the supernatant was taken, the fluorescence intensity of calcein in it was measured, and the percentage of target cell lysis was calculated based on the spontaneous release control and the maximum release control.

[0075] The in vitro killing and lysis results of CAR-CD244-ITSM-WT-NK, CAR-CD244-3ITSM-F-NK, and CAR-CD244-4ITSM-F-NK cells against the A2780 cell line that does not express MSLN are shown in Figure 3 (A), and the in vitro killing and lysis results against the OVCAR8 cell line with high MSLN expression are shown in Figure 3 (B). The results showed that there was no significant difference in the lysis ability between the CAR-CD244-3ITSM-F-NK group (Y mutated to F in the third ITSM motif) and CAR-CD244-ITSM-WT-NK (wild type) cells. However, the cell-targeted lysis ability of the CAR-CD244-4ITSM-F-NK group (Y mutated to F in the fourth ITSM motif) was significantly improved, indicating its in vitro killing function against the OVCAR8 cell line. From the above in vitro tumor killing results, it can be seen that CAR-CD244-4ITSM-F-NK cells constructed by preferentially mutating the intracellular domain are used for the treatment of ovarian cancer.

[0076] 2. In vitro cytokine detection. Effector cells (CAR-CD244-ITSM-WT-NK, CAR-CD244-3ITSM-F-NK, CAR-CD244-4ITSM-F-NK) and target cells were co-cultured at an effector-to-target ratio of 5:1. First, each type of ovarian cancer target cell was digested and counted, and according to the counting results, the target cells were diluted to 1.0×10 5 cells / mL with RPMI-1640 medium containing 10% fetal bovine serum. Each type of effector cell was counted, and according to the counting results, the effector cells were diluted to 5.0×10 5 cells / mL with NK cell proliferation medium. A U-bottom 96-well plate was taken, and 100 μL of the diluted target cells and effector cells were added to each well. The U-bottom 96-well plate was placed in a 37°C, 5% CO 2 2 cell incubator and cultured for 24 h. After the culture was completed, the U-bottom 96-well plate was centrifuged at 300×g at room temperature for 5 min, and 120 μL of the supernatant was aspirated with a pipette (avoiding aspirating the bottom cell precipitate) and transferred to a 1.5 mL EP tube. The collected co-culture supernatant was diluted at a ratio of 1:10, and then cytokine detection was performed according to the ELISA kit instructions. Finally, the absorbance value at 450 nm was measured using an enzyme-linked immunosorbent assay reader. The absorbance value measured minus the absorbance value at the 0 point of the standard curve was used as the abscissa, and the concentration values of different concentrations of IFN-γ standard products were used as the ordinate to plot the standard curve. Using the regression equation, after substituting the absorbance values of each sample to be measured and then multiplying by the dilution factor, the concentration (pg / mL) of IFN-γ in each sample could be calculated.

[0077] The results of IFN-γ secretion after incubation of CAR-CD244-ITSM-WT-NK, CAR-CD244-3ITSM-F-NK, and CAR-CD244-4ITSM-F-NK cells with MSLN-negative A2780 cell line and MSLN-high-expressing OVCAR8 tumor cell line in vitro are shown in Figure 4 A and B, which are consistent with the tumor-killing results.

[0078] The results showed that the IFN-γ secreted by CAR-CD244-4ITSM-F-NK cells was significantly higher than that of the CAR-CD244-ITSM-WT-NK and CAR-CD244-3ITSM-F-NK groups, further indicating that the tyrosine mutation to phenylalanine in the fourth ITSM of the CD244 intracellular domain could significantly enhance the in vitro killing function of MSLN-targeted CAR-NK cells against ovarian cancer cells.

[0079] The embodiments of this patent are similar to the content disclosed in the inventor's prior patent application document CN117964736A. However, the prior application involves T cells, which are another molecule in the intracellular domain of CAR-T. This patent application involves NK cells, and the molecules of the two are different, with completely different structures. The modified molecule CD244 in this patent application is a molecule unique to NK cells and cannot be used for T cells. Compared with the prior application that modified the CD28 molecule with hydrophobic amino acids, this patent application is a phosphorylation modification, and the modification ideas are also different.

[0080] The above examples are only illustrative of the present invention and do not constitute a limitation on the protection scope of the present invention. Any design identical or similar to the present invention falls within the protection scope of the present invention.

Claims

1. A chimeric antigen receptor intracellular domain CAR-CD244-4ITSM-F targeting MSLN, characterized in that: The amino acid sequence of the intracellular domain CAR-CD244-4ITSM-F is shown in SEQ ID NO.

11.

2. A nucleotide sequence encoding the intracellular domain CAR-CD244-4ITSM-F according to claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

12.

3. A chimeric antigen receptor targeting MSLN, characterized in that: The chimeric antigen receptor comprises the intracellular domain CAR-CD244-4ITSM-F as described in claim 1.

4. The chimeric antigen receptor targeting MSLN according to claim 3, characterized in that: The chimeric antigen receptor comprises an anti-MSLN antibody single-chain variable region, a CD8 hinge region, a CD244 transmembrane region, a CD244 intracellular domain CD244-4ITSM-F, and an intracellular co-stimulatory domain CD3ζ chain, which are sequentially spliced ​​from the N-terminus to the C-terminus.

5. The chimeric antigen receptor targeting MSLN according to claim 4, characterized in that: The amino acid sequence of the single-chain variable region of the anti-MSLN antibody is shown in SEQ ID NO.1; the amino acid sequence of the CD8 hinge region is shown in SEQ ID NO.3; the amino acid sequence of the CD244 transmembrane region is shown in SEQ ID NO.5; the amino acid sequence of the CD244 intracellular domain is shown in SEQ ID NO.11; the amino acid sequence of CD3ζ is shown in SEQ ID NO.13; the nucleotide sequence of the single-chain variable region of the anti-MSLN antibody is shown in SEQ ID NO.2; the nucleotide sequence of the CD8 hinge region is shown in SEQ ID NO.4; the nucleotide sequence of the CD244 transmembrane region is shown in SEQ ID NO.6; the nucleotide sequence of the CD244 intracellular domain is shown in SEQID NO.12; the nucleotide sequence of CD3ζ is shown in SEQ ID NO.

14.

6. The chimeric antigen receptor targeting MSLN according to claim 4, characterized in that: The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.19; the nucleotide sequence of the chimeric antigen receptor is shown in SEQ ID NO.

20.

7. A recombinant lentiviral vector, characterized in that: The recombinant lentiviral vector uses the pTK-EF1α-Kan vector as a skeleton and contains the nucleotide described in claim 6.

8. A NK cell expressing a chimeric antigen receptor, characterized in that: The CAR-NK cells are obtained by transfecting NK cells of a mammal with the chimeric antigen receptor according to any one of claims 3 to 6.

9. The NK cell expressing a chimeric antigen receptor according to claim 8, characterized in that: The NK cells are selected from NK cells derived from umbilical cord blood, single blood collection, peripheral blood or IPSC.

10. Use of the chimeric antigen receptor according to any one of claims 3 to 6, the recombinant lentiviral vector according to claim 7, or the NK cell according to any one of claims 8 to 9 in the preparation of a drug for treating or preventing tumors.

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