An enhanced anti-tumor NK cell, its preparation method and application

Abstract

This invention provides an enhanced anti-tumor NK cell, its preparation method, and its application. The preparation method includes: constructing an IL-12 lentiviral expression vector; constructing a CD19-CAR lentiviral expression vector; co-transfecting the vector with lentiviral packaging plasmids into 293T cells; collecting and concentrating the IL-12 lentivirus and CD19-CAR lentivirus separately; culturing and passaged NK-92 cells; co-infecting NK-92 cells with CD19-CAR lentivirus and IL-12 lentivirus at the same MOI; culturing and collecting the cells to obtain enhanced anti-tumor NK cells. These NK cells can not only recognize and promote the proliferation of autocrine IL-12 and secrete IFN-γ to enhance tumor killing, but also locally target and directly act on the tumor microenvironment, eliminating the need for exogenous IL-12 and avoiding adverse side effects caused by systemic injection.

Description

Technical Field

[0001] This invention belongs to the field of anti-tumor cell technology, specifically relating to an enhanced anti-tumor NK cell, its preparation method, and its application. Background Technology

[0002] Natural killer (NK) cells are important immune cells in the body, closely related to anti-tumor, anti-viral infection, and immune regulation. They are an important effector cell type used in adoptive cancer immunotherapy. Similar to T cells, NK cells can also be modified to express chimeric antigen receptors (CARs) to enhance anti-tumor activity. In recent years, CAR-NK therapy based on NK cells has also developed rapidly. Its unique advantages are mainly reflected in: fewer NK cell subsets, shorter in vivo survival time, and lower unpredictable risks; greater safety, with extremely low probability of cytokine storms and neurotoxicity; NK cells can exert extremely high cytotoxic activity without prior antigen sensitization and are not restricted by the major histocompatibility complex (MHC); due to the immune rejection mechanism, T cell therapy currently mostly uses autologous cells, while allogeneic NK cell-based tumor immunotherapy has not shown severe uncontrollable graft-versus-host rejection (GVHD), broadening the source of cells and providing feasibility for "off-the-shelf" cell immunotherapy.

[0003] CAR-NK cell therapy offers numerous advantages, but NK cells cannot proliferate and enhance their effector function in the absence of cytokine signaling. Interleukin-12 (IL-12) is a key regulator in cellular immune responses, capable of activating NK cells and promoting the secretion of large amounts of interferon-gamma (IFN-γ) to inhibit and kill tumor cells, exhibiting significant biological activity. However, systemic administration of IL-12 carries significant toxic side effects, and the concentration that may directly affect NK cells is not high. Therefore, constitutive secretion of IL-12 by engineered CAR-NK cells is an attractive strategy. Engineered cells can not only recognize and secrete IL-12 to promote NK cell proliferation and enhance tumor killing, but also avoid the adverse side effects caused by IL-12 due to the tumor-targeting effect of CAR-NK cells.

[0004] NK-92 cells are an NK cell line successfully isolated from peripheral blood lymphocytes of non-Hodgkin's lymphoma patients in 1992. They have been approved by the US FDA for clinical trials, and their safety and feasibility have been verified in multiple clinical trials. CD19 is a transmembrane glycoprotein belonging to the immunoglobulin superfamily, expressed in most cases of acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and B-cell lymphoma.

[0005] WO2020051363A1 discloses a composition and method for NK cell-based therapy. Specifically, IL-2-expressing or sensitized NK cells are stimulated in vivo or in vitro with a chimeric protein comprising a cancer cell-targeting portion and an IL-12 portion. Favorable results show that this chimeric protein can significantly reduce systemic toxicity and can also target and induce IFN-γ secretion. The step of contacting NK cells with the chimeric protein is performed ex vivo or in vivo. In vivo contact involves administering the chimeric protein at least 12 hours before, at least 12 hours after, or simultaneously with NK cells. However, this method has limitations: after in vitro IL-12 stimulation, IL-12 may be removed before infusion therapy, potentially resulting in insignificant NK cell efficacy; while in vivo infusion stimulation allows the IL-12-targeting chimeric protein to reach the tumor site through targeting, but a sufficient number of NK cells may not reach the tumor location, leading to insignificant therapeutic effects. CN108047332A discloses an anti-CD19 antibody or its antigen-binding fragment targeting CD19. The existing technology constructs Anti-CD19 CAR-NK cells obtained by culturing and expanding a monoclonal cell line, exhibiting stable characteristics and suitable for large-scale production. Anti-CD19 CAR-NK cells, targeting CD19 molecules, can specifically kill or cytotoxic lymphoma cells, and can be used as a therapeutic drug for lymphoma-like diseases in tumors with high CD19 expression. However, NK cells cannot expand and enhance their effector function in the absence of cytokine signaling. CD19-targeted CAR-NK cells may exhibit insufficient NK cell killing activity after targeting tumor cells due to a lack of cytokines, which is a common problem currently faced in NK cell therapy. Summary of the Invention

[0006] Based on the deficiencies of the existing technology, the first objective of this invention is to provide a method for preparing enhanced anti-tumor NK cells; the second objective of this invention is to provide enhanced anti-tumor NK cells prepared by this method; and the third objective of this invention is to provide the application of the enhanced anti-tumor NK cells in the preparation of anti-tumor drugs.

[0007] The objective of this invention is achieved through the following technical means:

[0008] On one hand, the present invention provides a method for preparing enhanced anti-tumor NK cells, which includes the following steps:

[0009] The artificially synthesized IL-12 gene was linked to a lentiviral vector plasmid to construct an IL-12 lentiviral expression vector.

[0010] The artificially synthesized CD19-CAR gene was linked to a lentiviral vector plasmid to construct a CD19-CAR lentiviral expression vector.

[0011] The IL-12 lentiviral expression vector and the CD19-CAR lentiviral expression vector were co-transfected with lentiviral packaging plasmids into 293T cells and cultured. Cell supernatants rich in IL-12 lentiviral particles and CD19-CAR lentiviral particles were collected, respectively. After concentration, IL-12 lentivirus and CD19-CAR lentivirus were collected, respectively.

[0012] NK-92 cells were cultured and passaged, and CD19-CAR lentivirus and IL-12 lentivirus were co-infected with NK-92 cells at the same MOI value. Cells were cultured and collected to obtain enhanced anti-tumor NK cells.

[0013] In the above preparation method, the CD19-CAR lentivirus and IL-12 lentivirus can be co-infected with NK-92 cells at the same MOI value in the following ways: CD19-CAR lentivirus and IL-12 lentivirus are co-infected with NK-92 cells at the same time; or CD19-CAR lentivirus is first infecting NK-92 cells, and then IL-12 lentivirus is infecting NK-92 cells after a certain interval (e.g., one week); or IL-12 lentivirus is first infecting NK-92 cells, and then CD19-CAR lentivirus is infecting NK-92 cells after a certain interval (e.g., one week).

[0014] In the above preparation method, preferably, the lentiviral vector plasmid includes the PGMLV plasmid.

[0015] In the above preparation method, preferably, the IL-12 gene is obtained by linking the P40 subunit gene and the P35 subunit gene of the natural IL-12 gene through a linker peptide gene.

[0016] In the above preparation method, preferably, the nucleotide sequence of the IL-12 gene is as shown in SEQ ID NO: 1; and its amino acid sequence is as shown in SEQ ID NO: 2.

[0017] In the above preparation method, preferably, the CD19-CAR gene is obtained by codon optimization after linking the human CD8α signal peptide gene, the CD19-targeting single-chain antibody gene, the human CD8α hinge region gene, the human CD8α transmembrane region gene, the 4-1BB intracellular region gene, and the human CD3ζ intracellular signal peptide gene.

[0018] In the above preparation method, preferably, the nucleotide sequence of the CD19-CAR gene is shown in SEQ ID NO: 3; and its amino acid sequence is shown in SEQ ID NO: 4.

[0019] In the above preparation method, preferably, the titer of the IL-12 lentivirus is (1-5)×10⁻⁶. 7 TU / ml; the titer of the CD19-CAR lentivirus is (0.5~2)×10⁻⁶. 7 TU / ml.

[0020] In the above preparation method, preferably, the complete culture medium used for the culture of passaged NK-92 cells and the culture of NK-92 cells infected with lentivirus includes: Alpha MEM medium and Alpha MEM medium supplemented with 10% to 15% horse serum, 10% to 15% fetal bovine serum, 0.1 mM β-mercaptoethanol, 0.02 mM folic acid and 50 to 200 U / ml IL-2.

[0021] In the above preparation method, preferably, it is prepared according to 1.5 × 10 5 Cells were cultured at a density of 1 cell / ml, passaged every 2-3 days, and cultured in a 37°C, 5% CO2 incubator.

[0022] In the above preparation method, preferably, CD19-CAR lentivirus and IL-12 lentivirus are used to infect NK-92 cells at the same MOI, wherein the MOI value is 10 to 100.

[0023] In the above preparation method, preferably, 6-10 μg / ml of polybrene is added during the culture of NK-92 cells infected with lentivirus to improve the lentivirus infection efficiency.

[0024] In the above preparation method, preferably, the culture conditions for lentivirus-infected NK-92 cells are cultured in a 37°C, 5% CO2 incubator.

[0025] On the other hand, the present invention also provides an enhanced anti-tumor NK cell, which is prepared by the above-described preparation method.

[0026] Furthermore, the present invention also provides the application of the above-mentioned enhanced anti-tumor NK cells in the preparation of therapeutic anti-tumor drugs.

[0027] The beneficial effects of this invention are:

[0028] The enhanced anti-tumor NK cells constructed by the method of this invention can express targeted CAR proteins while auto-secreting IL-12, enabling them to directly target and kill tumors. They can not only recognize auto-secreting IL-12 and promote its proliferation and secrete IFN-γ to enhance the killing effect on tumors, thus continuously enhancing the anti-tumor activity of NK cells, but also the local targeted secretion of these NK cells directly acts on the tumor microenvironment, enhancing the cytokine and cytotoxic responses against tumor cells. This eliminates the need for the addition of exogenous IL-12 and avoids the adverse side effects caused by systemic injection. Attached Figure Description

[0029] Figure 1 This is a map of the IL-12 lentiviral expression vector (PGMLV-IL-12) constructed in Example 1 of this invention.

[0030] Figure 2 This is a map of the CD19-CAR lentiviral expression vector (PGMLV-CD19-CAR) constructed in Example 1 of this invention.

[0031] Figure 3 Microscopic comparison images (40×) of NK-92 cells, CD19-CAR-NK-92 cells, and CD19-CAR-IL-12-NK-92 cells in Example 1 of this invention.

[0032] Figure 4 This is a comparison of the Western blot results of NK-92 cells and CD19-CAR-IL-12-NK-92 cells in Example 2 of the present invention.

[0033] Figure 5 This is a comparison chart of the IL-12 and IFN-γ content in the supernatant of NK-92 cells, CD19-CAR-NK-92 cells and CD19-CAR-IL-12-NK-92 cells after culturing for 48 hours in Example 3 of the present invention.

[0034] Figure 6 This is a graph showing the killing rate of CD19-CAR-NK-92 cells and CD19-CAR-IL-12-NK-92 cells against Raji cells in Example 4 of the present invention (where the vertical axis represents the killing effect and the horizontal axis represents the effector-to-target ratio at different levels). Detailed Implementation

[0035] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0036] Example 1:

[0037] 1. Construction of lentiviral expression vector:

[0038] IL-12 lentiviral expression vectors and CD19-CDR lentiviral expression vectors were constructed using the Genomeditech lentiviral vector plasmid PGMLV (catalog number: GM-7772), as detailed below:

[0039] (1) Construction of IL-12 lentiviral expression vector:

[0040] First, the IL-12 gene was obtained by linking the P35 and P40 subunits of the natural IL-12 gene using a linker peptide gene. Its gene structure is as follows: Its nucleotide sequence is shown in SEQ ID NO: 1; its amino acid sequence is shown in SEQ ID NO: 2. This ligation process was commissioned to a gene synthesis company and synthesized using conventional genetic engineering methods.

[0041] Secondly, the artificially synthesized IL-12 gene was ligated into the PGMLV-plasmid to construct the IL-12 lentiviral expression vector, namely PGMLV-IL-12. This ligation process was commissioned to a gene synthesis company and synthesized using conventional genetic engineering methods.

[0042] The map of the IL-12 lentiviral expression vector (PGMLV-IL-12) is as follows: Figure 1 As shown.

[0043] (2) Construction of CD19-CAR lentiviral expression vector:

[0044] First, based on the sequence information of the human CD8α signal peptide gene, CD19-targeting single-chain antibody gene, human CD8α hinge region gene, human CD8α transmembrane region gene, 4-1BB intracellular region gene, and human CD3ζ intracellular signal peptide gene retrieved from the NCBI website database, these genes were linked and codons optimized to obtain the CD19-CAR gene, whose gene structure is as follows: The nucleotide sequence of the CD19-CAR gene is shown in SEQ ID NO: 3; its amino acid sequence is shown in SEQ ID NO: 4. This ligation process was commissioned to a gene synthesis company and synthesized using conventional genetic engineering methods.

[0045] Secondly, the artificially synthesized CD19-CAR gene was ligated into the PGMLV plasmid to construct the CD19-CAR lentiviral expression vector, namely PGMLV-CD19-CAR. This ligation process was commissioned to a gene synthesis company and synthesized using conventional genetic engineering methods.

[0046] The map of the CD19-CAR lentiviral expression vector (PGMLV-CD19-CAR) is as follows: Figure 2 As shown.

[0047] 2. Packaging and titer determination of lentiviruses:

[0048] (1) 293T cells were used as packaging cells for lentivirus. These cells are adherent epithelioid cells. The cells were cultured in DMEM (containing 10% FBS) to grow and proliferate to form a monolayer for later use.

[0049] (2) One day before transfection, passage the cells into 10cm culture dishes at an appropriate ratio. When the cells reach 70%–80% confluency, prepare for transfection. 1–2 hours before transfection, replace the culture medium with fresh medium (12ml / 10cm dish). Use sterile 1.5ml EP tubes or 15ml centrifuge tubes. The lentiviral expression vectors are PGMLV-IL-12 and PGMLV-CD19-CAR, which are packaged separately. The transfection system is shown in Table 1 below:

[0050] Table 1:

[0051] Reagent Name Specification DMEM 1ml Lentiviral expression vector 10μg Lenti-HG Mix (Genomeditech) 10 μl (10 μg) HG transgene reagent(Genomeditech) 60μl

[0052] Taking IL-12 lentivirus packaging as an example:

[0053] Specifically, the following steps were taken: A high-purity, endotoxin-free IL-12 lentiviral vector (PGMLV-IL-12) and its auxiliary packaging vector plasmid (Lenti-HG Mix (Genomeditech)) were extracted and co-transfected into 293T cells using HG transgene reagent (Genomeditech). After incubation at room temperature for 15-20 minutes, the mixture was evenly added to culture dishes that had been pre-treated, and then placed in a CO2 incubator. 10-12 hours after transfection, 100×Enhancing buffer (100 μl / dish) was evenly added to promote transfection. 18-20 hours after transfection, the cell culture medium was carefully aspirated and discarded into a waste container filled with sterilizing solution, and then 15 ml of fresh cell culture medium was added for continued culturing.

[0054] Collection and concentration of IL-12 lentivirus: After culturing in a different medium for 48 hours, the cell supernatant was aspirated into a 50 ml centrifuge tube and centrifuged at 4°C, 4500 g for 5 min. The supernatant was filtered through a 0.22 μm filter and transferred to a new centrifuge tube. Finally, the filtrate was transferred in batches to a concentration device and centrifuged at 4°C, 4500 g for 10 min. The lower layer of liquid was discarded into a waste container filled with disinfectant. The final centrifugation was performed at 4°C, 4500 g for 20 min. The liquid visible at the top of the filter at this point is the concentrated IL-12 lentivirus solution. The IL-12 lentivirus was aliquoted and stored at -80°C.

[0055] Similarly, CD19-CAR lentivirus was obtained through packaging.

[0056] (3) Determination of lentivirus titer:

[0057] 293T cells were cultured to the logarithmic growth phase, and the virus was diluted using cell culture medium containing 10% FBS. After trypsin digestion and cell counting, cells were divided at a ratio of 2 × 10⁶ cells / well. 5 Cells were seeded in 12-well plates and incubated overnight at 37°C until they reached a confluence density of 20-40%. One to two hours before infection, the culture medium was replaced with fresh medium (0.91 ml per well) containing 1 μg / ml polybrene. On the second day, for transfection of the 12-well plates, the lentivirus solution, stored at -80°C, was thawed on ice and serially diluted 10-fold with cell culture medium containing 10% FBS. 90 μl of the diluted lentivirus solution was added to each well, and the plates were incubated overnight at 37°C. A known viral titer was used as a control. On the third day, the lentivirus-containing culture medium was removed, and 1 ml of complete culture medium was added. Four days later, RNA was extracted for RT-qPCR. Cell samples with different viral loads were compared, and the titer was determined by comparing the Ct values ​​of the control and experimental groups.

[0058] The test results show that the lentivirus packaging in this embodiment can achieve a titer of 3 × 10⁻⁶. 7 IL-12 lentivirus at TU / ml and titer of 1×10 7 CD19-CAR lentivirus at TU / ml.

[0059] 3. Preparation of enhanced anti-tumor NK cells:

[0060] (1) Culture and passage of NK-92 cells: NK-92 cells were cultured in complete medium (Alpha MEM + 12.5% ​​horse serum + 12.5% ​​fetal bovine serum + 0.1 mM β-mercaptoethanol + 0.02 mM folic acid + 100 U / ml IL-2) at a concentration of 1.5 × 10⁻⁶ cells / mL. 5Subculture at a cell / ml density every 2-3 days, and culture in a 37℃, 5% CO2 incubator.

[0061] (2) Lentiviral infection: NK-92 cells were collected by centrifugation and prepared into 5×10⁻⁶ cells. 5 Cell suspension was prepared at 0.1 ml / well in 24-well plates. Three wells were prepared: blank (NK-92 cells), control (NK-92 cells infected with CD19-CAR lentivirus alone), and experimental (NK-92 cells infected with both IL-12 lentivirus and CD19-CAR lentivirus). Lentiviral solution stored at -80°C was thawed on ice. At an MOI of 20, 100 μL of CD19-CAR lentivirus was added to the control wells, and 33.3 μL of IL-12 lentivirus and 100 μL of CD19-CAR lentivirus were added to the experimental wells. The remaining cells were topped with 1 ml of complete culture medium, and polybrene (to enhance lentivirus infection efficiency) was added at a final concentration of 8 μg / ml. The mixture was then incubated at 37°C in a 5% CO2 incubator. After 48 hours, 2 ml of fresh complete culture medium was added. Cells were then cultured for an additional 4 days before being used for subsequent experimental studies. The cells infected with lentiviruses were named CD19-CAR-NK-92 cells and CD19-CAR-IL-12-NK-92 cells (i.e., enhanced anti-tumor NK cells).

[0062] See cell micrograph of this enhanced anti-tumor NK cell. Figure 3 As shown, by Figure 3 It can be seen that CD19-CAR-IL-12-NK-92 cells are similar to CD19-CAR-NK-92 cells and NK-92 cells, exhibiting cluster growth.

[0063] Example 2: Western blotting (WB) analysis of proteins

[0064] Total protein was extracted from cells, and the expression of CD19-CAR in cells was detected at the protein level. In addition to the expression of endogenous CD3ζ (16KD), exogenous CD3ζ (54KD) was also expressed, i.e., CD19-CAR expression. The specific procedure is as follows:

[0065] (1) Extraction of total cellular protein: NK-92 cells and CD19-CAR-IL-12-NK-92 cells were collected (the number of cells was approximately 2 × 10⁻⁶). 6Centrifuge the cells, resuspend in 1 ml PBS, and transfer to a 1.5 ml centrifuge tube. Centrifuge at 1000 rpm for 5 min, wash 3 times, and discard the supernatant. Refer to the manufacturer's instructions (Beyotime-RIPA lysis buffer-P0013B, take an appropriate amount of RIPA lysis buffer, add PMSF a few minutes before use to make the final PMSF concentration 1 mM), add 250 μl of the prepared RIPA lysis buffer to each tube on ice, gently pipette 2-3 times, and gently tap with your finger to mix thoroughly several times until there is no obvious cell pellet. Centrifuge at 12000g, 4℃ for 5 min, aliquot the supernatant into pre-chilled centrifuge tubes (30 μl / tube), and store at -80℃ or for subsequent experiments.

[0066] (2) WB process:

[0067] ① Electrophoresis. Add 5 μl of protein marker and 20 μl of sample to each well. Apply the upper gel at 80V for 30 min and the lower gel at 120V for about 120 min.

[0068] ② Transfer. After electrophoresis, activate the PVDF membrane in methanol for 5 minutes, then equilibrate it in transfer buffer for 15 minutes. After electrophoresis, peel the gel from the gel plate. Rinse thoroughly with water and equilibrate in transfer buffer for about 10 minutes. Assemble the transfer unit starting from the negative electrode, placing 3 sheets of filter paper, the gel, the PVDF membrane, and 3 sheets of filter paper in the clamp in sequence. After the transfer unit is assembled, transfer at 100V for 60–90 minutes.

[0069] ③ Sealing. After the transfer, fix the PVDF membrane with methanol for 3 minutes. Then seal it with pre-prepared 5% skim milk on a shaker for 1 hour.

[0070] ④ Incubate with primary antibody. After blocking, incubate with primary antibody overnight at 4°C. The primary antibody is Mouse anti-human CD3ζ (BD, catalog number 551034), which is diluted 1:1000 with 5% skim milk. Rinse the overnight PVDF membrane with prepared 1×TBST buffer three times, 10 min each time.

[0071] ⑤ Incubate with secondary antibody. The secondary antibody, Goat anti-Mouse HRP (R&D, catalog number HAF007), is diluted 1:1000 with milk-PBST. Incubate at room temperature for 45 minutes, then discard the antibody. Wash three times with PBST for 5 minutes each time.

[0072] ⑥ Exposure: Prepare the exposure substrate according to the ratio of solution A to solution B = 1:1. After removing the film and slightly drying it, place it on a thin plastic film. Add the freshly prepared exposure substrate, react in the dark for 2 minutes, and then take a picture.

[0073] See the experimental results. Figure 4 The expression of CD19-CAR in cells was detected at the protein level. Figure 4 It can be seen that, in addition to the expression of endogenous CD3ζ (16KD), there is also the expression of exogenous CD3ζ (54KD), which proves the expression of CD19-CAR in CD19-CAR-IL-12-NK-92 cells.

[0074] Example 3: ELISA detection of cytokine levels

[0075] After culturing NK-92 cells, CD19-CAR-NK-92 cells, and CD19-CAR-IL-12-NK-92 cells for 48 hours, the culture supernatant was collected, and the levels of IL-12 and IFN-γ in the supernatant were detected using an ELISA kit. The specific procedure is as follows:

[0076] NK-92 cells, CD19-CAR-NK-92 cells, and CD19-CAR-IL-12-NK-92 cells were cultured in complete medium (Alpha MEM medium + 12.5% ​​horse serum + 12.5% ​​fetal bovine serum + 0.1 mM β-mercaptoethanol + 0.02 mM folic acid + 100 U / ml IL-2) at a concentration of 1.5 × 10⁻⁶ cells / mL. 5 After culturing cells / ml for 48 hours, the levels of IL-12 and IFN-γ in the supernatant were detected. The kits used were the Human IL-12 (P70) Precoated ELISA Kit (catalog number: 1111202) and the Human IFN-γ Precoated ELISA Kit (catalog number: 1111002) from Dakowei Biotechnology Co., Ltd.

[0077] See the experimental results. Figure 5 ,Depend on Figure 5 It can be seen that the levels of IL-12 (1.4 ng / ml) and IFN-γ (3.4 ng / ml) in the supernatant of CD19-CAR-IL-12-NK-92 cells after 48 h of culture were much higher than those in NK-92 cells and CD19-CAR-NK-92 cells (both below the detection limit of 6.25 pg / ml, close to 0).

[0078] Example 4: Detection of in vitro cytotoxic activity of cells

[0079] CD19-CAR-NK-92 cells and CD19-CAR-IL-12-NK-92 cells were mixed with Raji cells at different effector-to-target ratios (10:1, 5:1, 2.5:1, and 1:1) and seeded into 96-well cell culture plates. The wells were divided into the following groups: blank control wells (cell-free culture medium wells), effector cell control wells, target cell control wells, sample maximum enzyme activity control wells (target cell lysis wells), and experimental wells (effector cell and target cell wells). The total volume of each well was 200 μl, and the plates were incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0080] This experiment used the LDH cytotoxicity assay kit (catalog number C0016) from Beyotime Biotechnology Co., Ltd. One hour before the predetermined detection time, the 96-well plate was removed from the cell culture incubator. The LDH release reagent provided in the kit was added to the "Sample Maximum Enzyme Activity Control Well," at a volume of 10% of the original culture medium. After adding the LDH release reagent, the plate was repeatedly pipetted to mix thoroughly, and then the plate was returned to the incubator for further incubation. After the co-incubation time was reached, the 96-well plate was removed and centrifuged at 1000 rpm for 5 min using a multi-well centrifuge. 120 μl of the supernatant from each well was then added to the corresponding well of a new 96-well plate, and the samples were immediately analyzed.

[0081] The sample determination steps are as follows:

[0082] (1) Add 60 μl of LDH detection working solution to each well.

[0083] (2) Mix well, place the 96-well plate on a shaker at 100 rpm, and incubate at room temperature in the dark for 30 min. Then measure the absorbance at 490 nm using a microplate reader.

[0084] (3) The absorbance values ​​of each well should be reduced by the absorbance values ​​of the background blank control well.

[0085] (4) Kill rate (target cell lysis rate) (%) = (experimental group - target cell control well - effector cell control well) / (sample maximum enzyme activity control well - target cell control well) × 100%.

[0086] See the experimental results. Figure 6 ,Depend on Figure 6 It can be seen that the killing activity of CD19-CAR-IL-12-NK-92 cells against Raji cells is significantly enhanced compared to CD19-CAR-NK-92 cells, and the killing rate gradually increases with the increase of the effector-target ratio. sequence list <110> Kanglitai Pharmaceutical Co., Ltd. <120> An enhanced anti-tumor NK cell, its preparation method and application <130> GAI21CN1890 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 1629 <212> DNA <213> Artificial Sequence <220> <223> IL-12 gene <220> <221> CDS <222> (1)..(1629) <400> 1 gcc acc atg tgt cac cag cag ttg gtc atc tct tgg ttt tcc ctg gtt 48 Ala Thr Met Cys His Gln Gln Leu Val Ile Ser Trp Phe Ser Leu Val 1 5 10 15 ttt ctg gca tct ccc ctc gtg gcc ata tgg gaa ctg aag aaa gat gtt 96 Phe Leu Ala Ser Pro Leu Val Ala Ile Trp Glu Leu Lys Lys Asp Val 20 25 30 tat gtc gta gaa ttg gat tgg tat ccg gat gcc cct gga gaa atg gtg 144 Tyr Val Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val 35 40 45 gtc ctc acc tgt gac acc cct gaa gaa gat ggt atc acc tgg acc ttg 192 Val Leu Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu 50 55 60 gac cag agc agt gag gtc tta ggc tct ggc aaa acc ctg acc atc caa 240 Asp Gln Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln 65 70 75 80 gtc aaa gag ttt gga gat gct ggc cag tac acc tgt cac aaa gga ggc 288 Val Lys Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly 85 90 95 gag gtt cta agc cat tcg ctc ctg ctg ctt cac aaa aag gaa gat gga 336 Glu Val Leu Ser His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly 100 105 110 att tgg tcc act gat att tta aag gac cag aaa gaa ccc aaa aat aag 384 Ile Trp Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys 115 120 125 acc ttt cta aga tgc gag gcc aag aat tat tct gga cgt ttc acc tgc 432 Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys 130 135 140 tgg tgg ctg acg aca atc agt act gat ttg aca ttc agt gtc aaa agc 480 Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser 145 150 155 160 agc aga ggc tct tct gac ccc caa ggg gtg acg tgc gga gct gct aca 528 Ser Arg Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr 165 170 175 ctc tct gca gag aga gtc aga ggg gac aac aag gag tat gag tac tca 576 Leu Ser Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser 180 185 190 gtg gag tgc cag gag gac agt gcc tgc cca gct gct gag gag agt ctg 624 Val Glu Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu 195 200 205 ccc att gag gtc atg gtg gat gcc gtt cac aag ctc aag tat gaa aac 672 Pro Ile Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn 210 215 220 tac acc agc agc ttc ttc atc agg gac atc atc aaa cct gac cca ccc 720 Tyr Thr Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro 225 230 235 240 aag aac ttg cag ctg aag cca tta aag aat tct cgg cag gtg gag gtc 768 Lys Asn Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val 245 250 255 agc tgg gag tac cct gac acc tgg agt act cca cat tcc tac ttc tcc 816 Ser Trp Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser 260 265 270 ctg aca ttc tgc gtt cag gtc cag ggc aag agc aag aga gaa aag aaa 864 The Three Phe Cys Val Gln Val Gln Gly Lys Served By Lys Arg Glu Lys 275 280 285 gat aga gtc ttc acg gac aag acc tca gcc acg gtc atc tgc cgc aaa 912 Asp Arg Val Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys 290,295,300 aat gcc agc att agc gtg cgg gcc cag gac cgc tac tat agc tca tct 960 Asn Only Serve Ile Ser Val Arg Only Gln Asp Arg Tyr Tyr Ser Ser Ser 305 310 315 320 tgg agc gaa tgg gca tct gtg ccc tgc agg ggc gga ggc gga agc gga 1008 Trp Ser Glu Trp Ala Ser Val Pro Cys Arg Gly Gly Gly Gly Ser Gly 325 330 335 ggc gga gga agc ggc ggt ggc ggc agc aga aac ctc ccc gtg gcc act 1056 Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Asn Leu Pro Val Ala Thr 340 345 350 cca gac cca gga atg ttc cca tgc ctt cac cac tcc caa aac ctg ctg 1104 Pro Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu 355 360 365 agg gcc gtc agc aac atg ctc cag aag gcc aga caa act cta gaa ttt 1152 Arg Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe 370 375 380 tac cct tgc act tct gaa gag att gat cat gaa gat atc aca aaa gat 1200 Tyr Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp 385 390 395 400 aaa acc agc aca gtg gag gcc tgt tta cca ttg gaa tta acc aag aat 1248 Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn 405 410 415 gag agt tgc cta aat tcc aga gag acc tct ttc ata act aat ggg agt Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser 420 425 430 tgc ctg gcc tcc aga aag acc tct ttt atg atg gcc ctg tgc ctt agt 1344 Cys Leu Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser 435 440 445 agt att tat gaa gac ttg aag atg tac cag cag gtg gag ttc aag acc atg 1392 Served With Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met 450 455 460 aat gca aag ctt ctg atg gat cct aag agg cag atc ttt cta gat caa 1440 Asn Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln 465 470 475 480 aac atg ctg gca gtt att gat gag ctg atg cag gcc ctg aat ttc aac 1488 Asn With Leu Val Ile Asp Glue With Gln Ala Leu Asn Phe Asn 485,490,495 agt gag act gtg cca aaa tcc tcc ctt gaa gaa ccg gat ttt tat 1536 Ser Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr 500 505 510 aaa act aaa atc aag ctc tgc ata ctt ctt cat gct ttc aga att cgg 1584 Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg 515 520 525 gca gtg act att gat aga gtg atg agc tat ctg aat gct tcc taa 1629 Ala Val Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser 530 535 540 <210> 2 <211> 542 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 2 Ala Thr Met Cys His Gln Gln Leu Val Ile Ser Trp Phe Ser Leu Val 1 5 10 15 Phe Leu Ala Ser Pro Leu Val Ala Ile Trp Glu Leu Lys Lys Asp Val 20 25 30 Tyr Val Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val 35 40 45 Val Leu Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu ]50 55 60 Asp Gln Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln 65 70 75 80 Val Lys Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly 85 90 95 Glu Val Leu Ser His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly 100 105 110 Ile Trp Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys 115 120 125 Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys 130 135 140 Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser 145 150 155 160 Ser Arg Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr 165 170 175 Leu Ser Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser 180 185 190 Val Glu Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu 195 200 205 Pro Ile Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn 210 215 220 Tyr Thr Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro 225 230 235 240 Lys Asn Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val 245 250 255 Ser Trp Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser 260 265 270 Leu Thr Phe Cys Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys 275 280 285 Asp Arg Val Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys 290 295 300 Asn Ala Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser 305 310 315 320 Trp Ser Glu Trp Ala Ser Val Pro Cys Arg Gly Gly Gly Gly Ser Gly 325 330 335 Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Asn Leu Pro Val Ala Thr 340 345 350 Pro Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu 355 360 365 Arg Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe 370 375 380 Tyr Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp 385 390 395 400 Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn 405 410 415 Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser 420 425 430 Cys Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser 435 440 445 Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met 450 455 460 Asn Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln 465 470 475 480 Asn Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn 485 490 495 Ser Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr 500 505 510 Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg 515 520 525 Ala Val Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser 530 535 540 <210> 3 <211> 1479 <212> DNA <213> Artificial sequence <220> <223> CD19-CAR gene <220> <221> CDS <222> (1)..(1479) <400> 3 gcc acc atg gcc tta cca gtg acc gcc ttg ctc ctg ccg ctg gcc ttg 48 Ala Thr Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu 1 5 10 15 ctg ctc cac gcc gcc agg ccg gaa att gtg atg acc cag tca ccc gcc 96 Leu Leu His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala 20 25 30 act ctt agc ctt tca ccc ggt gag cgc gca acc ctg tct tgc aga gcc 144 Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala 35 40 45[[ID=z8]] tcc caa gac atc tca aaa tac ctt aat tgg tat caa cag aag ccc gga 192 Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly 50 55 60 cag gct cct cgc ctt ctg atc tac cac acc agc cgg ctc cat tct gga 240 Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly 65 70 75 80 atc cct gcc agg ttc agc ggt agc gga tct ggg acc gac tac acc ctc 288 Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu 85 90 95 act atc agc tca ctg cag cca gag gac ttc gct gtc tat ttc tgt cag 336 Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln 100 105 110 caa ggg aac acc ctg ccc tac acc ttt gga cag ggc acc aag ctc gag 384 Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu 115 120 125 att aaa ggt gga ggt ggc agc gga gga ggt ggg tcc ggc ggt gga gga 432 Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 agc cag gtc caa ctc caa gaa agc gga ccg ggt ctt gtg aag cca tca 480 Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser 145 150 155 160 gaa act ctt tca ctg act tgt act gtg agc gga gtg tct ctc ccc gat 528 Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp 165 170 175 tac ggg gtg tct tgg atc aga cag cca ccg ggg aag ggt ctg gaa tgg 576 Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 180 185 190 to gga gtg to tgg ggc tct gag act act tac tac tct tca tcc ctc 624 Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Ser Ser Leu 195 200 205 aag tca cgc gtc acc atc tca aag gac aac tct aag aat cag gtg tca 672 Light Ser Arg Val Thr Ile Ser Light Asp Asn Ser Light Asn Gln Val Ser 210 215 220 ctg aaa ctg tca tct gtg acc gca gcc gac acc gcc gtg tac tat tgc 720 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 225 230 235 240 gct aag cat tac tat tat ggc ggg agc tac gca atg gat tac tgg gga 768 Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly 245 250 255 cag ggt act ctg gtc acc gtg tcc agc acc acg acg cca gcg ccg cga 816 Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg 260 265 270 cca cca aca ccg gcg ccc acc atc gcg tcg cag ccc ctg tcc ctg cgc 864 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 275 280 285 cca gag gcg tgc cgg cca gcg gcg ggg ggc gca gtg cac acg agg ggg 912 Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly 290 295 300 ctg gac ttc gcc tgt gat atc tac atc tgg gcg ccc ttg gcc ggg act 960 Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr 305 310 315 320 tgt ggg gtc ctt ctc ctg tca ctg gtt atc acc ctt tac tgc aac cac 1008 How Does Gly Go To Lion Ser Lion Goes To Thr Lion Tyr How To Asn His 325 330 335 agg aac aaa cgg ggc aga aag aaa ctc ctg tat ata ttc aaa caa cca 1056 Arg Asn Lys Arg Gly Arg Lys Leu Tyr Ile Phe Lys Gln Pro 340 345 350 ttt atg aga cca gta caa act caa gag gaa gat ggc tgt agc tgc 1104 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 355 360 365 cga ttt cca gaa gaa gaa gaa gga gga gga tgt gaa ctg aga gtg aag ttc 1152 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 370 375 380 agc agg agc gca gac gcc ccc gcg tac cag cag ggc cag aac cag ctc 1200 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Glyn Glyn Asn Gln Leu 385 390 395 400 tat aac gag ctc aat cta gga cga aga gag gag tac gat gtt ttg gac 1248 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 405 410 415 aag aga cgt ggc cgg gac cct gag atg ggg gga aag ccg aga agg aag 1296 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 420 425 430 aac cct cag gaa ggc ctg tac aat gaa ctg cag aaa gat aag atg gcg 1344 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 435 440 445 gag gcc tac agt gag att ggg atg aaa ggc gag cgc cgg agg ggc aag 1392 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 450 455 460 ggg cac gat ggc ctt tac cag ggt ctc agt aca gcc acc aag gac acc 1440 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 465 470 475 480 tac gac gcc ctt cac atg cag gcc ctg ccc cct cgc taa 1479 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 4 <211> 492 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 4 Ala Thr Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu 1 5 10 15 Leu Leu His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala 20 25 30 Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala 35 40 45 Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly 50 55 60 Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly 65 70 75 80 Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu 85 90 95 Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln 100 105 110 Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu 115 120 125 Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser 145 150 155 160 Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp 165 170 175 Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 180 185 190 Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Ser Ser Leu 195 200 205 Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser 210 215 220 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 225 230 235 240 Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly 245 250 255 Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg 260 265 270 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 275 280 285 Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly 290 295 300 Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr 305 310 315 320 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His 325 330 335 Arg Asn Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 340 345 350 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 355 360 365 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 370 375 380 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 385 390 395 400 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 405 410 415 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 420 425 430 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 435 440 445 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 450 455 460 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 465 470 475 480 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490

Claims

1. A method for preparing enhanced anti-tumor NK cells, comprising the following steps: The artificially synthesized IL-12 gene was linked to a lentiviral vector plasmid to construct an IL-12 lentiviral expression vector. The artificially synthesized CD19-CAR gene was linked to a lentiviral vector plasmid to construct a CD19-CAR lentiviral expression vector. The IL-12 lentiviral expression vector and the CD19-CAR lentiviral expression vector were co-transfected with lentiviral packaging plasmids into 293T cells and cultured. Cell supernatants rich in IL-12 lentiviral particles and CD19-CAR lentiviral particles were collected, respectively. After concentration, IL-12 lentivirus and CD19-CAR lentivirus were collected, respectively. NK-92 cells were cultured and passaged, and CD19-CAR lentivirus and IL-12 lentivirus were co-infected with NK-92 cells at the same MOI value. Cells were cultured and collected to obtain enhanced anti-tumor NK cells. The CD19-CAR gene is obtained by codon optimization after linking the human CD8α signal peptide gene, the CD19-targeting single-chain antibody gene, the human CD8α hinge region gene, the human CD8α transmembrane region gene, the 4-1BB intracellular region gene, and the human CD3ζ intracellular signal peptide gene.

2. The preparation method according to claim 1, wherein, The lentiviral vector plasmid includes the PGMLV plasmid.

3. The preparation method according to claim 1, wherein, The IL-12 gene is obtained by linking the P40 and P35 subunit genes of the natural IL-12 gene using a linker peptide gene.

4. The preparation method according to claim 3, wherein, The nucleotide sequence of the IL-12 gene is shown in SEQ ID NO: 1; its amino acid sequence is shown in SEQ ID NO:

2.

5. The preparation method according to claim 1, wherein, The nucleotide sequence of the CD19-CAR gene is shown in SEQ ID NO: 3; its amino acid sequence is shown in SEQ ID NO:

4.

6. The preparation method according to claim 1, wherein, The titer of the IL-12 lentivirus was (1~5)×10⁻⁶. 7 TU / ml; the titer of the CD19-CAR lentivirus is (0.5~2)×10⁻⁶. 7 TU / ml.

7. The preparation method according to claim 1, wherein, The complete culture media used for the culture of passaged NK-92 cells and the culture of NK-92 cells infected with lentivirus included: Alpha MEM medium and Alpha MEM medium supplemented with 10%~15% horse serum, 10%~15% fetal bovine serum, 0.1mM β-mercaptoethanol, 0.02mM folic acid and 50-200U / ml IL-2.

8. The preparation method according to claim 7, wherein, Passage NK-92 cells at a rate of 1.5 × 10⁻⁶ 5 Cells were cultured at a density of 1 cell / ml, passaged every 2-3 days, and cultured in a 37°C, 5% CO2 incubator.

9. The preparation method according to claim 1, wherein, CD19-CAR lentivirus and IL-12 lentivirus were used to infect NK-92 cells with the same MOI, where the multiplicity of infection (MOI) ranged from 10 to 100.

10. The preparation method according to claim 1, wherein, During the culture of NK-92 cells infected with lentivirus, 6~10μg / ml of polybrene was also added.

11. The preparation method according to claim 10, wherein, The culture conditions for lentivirus-infected NK-92 cells were 37°C in a 5% CO2 incubator.

12. An enhanced anti-tumor NK cell, which is prepared by the preparation method according to any one of claims 1 to 11.

13. The use of the enhanced anti-tumor NK cells of claim 12 in the preparation of therapeutic anti-tumor drugs, wherein the tumor is selected from acute lymphoblastic leukemia, chronic lymphocytic leukemia and B-cell lymphoma.

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