PBMC-derived enhanced anti-tumor nk cells, methods of making and uses thereof

Abstract

The application provides PBMC-derived enhanced anti-tumor NK cells, a preparation method thereof and application thereof, belongs to the technical field of anti-tumor cells, and can solve the technical problems that the killing activity of the existing enhanced NK cells is not significant, the safety is insufficient, there is a potential risk of tumorigenicity, and an exogenous IL-12 needs to be added to cause side reactions on the human body. The PBMC-derived enhanced anti-tumor NK cells in the technical scheme can express HER2 and IL-12 simultaneously, target tumor cells through HER2, and enhance the anti-tumor ability through self-secretion of IL-12. The PBMC-derived enhanced anti-tumor NK cells are obtained by co-infecting NK cells obtained from PBMC with IL-12 lentivirus and HER2-CAR lentivirus, and then are prepared through detection and identification tests. The application can be applied to the preparation of anti-tumor drugs, in particular, the preparation of anti-solid tumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of anti-tumor cell technology, and particularly relates to an enhanced anti-tumor NK cell derived from PBMC, its preparation method and its application. Background Technology

[0002] Natural killer (NK) cells are important immune cells in the body, participating in a series of immune activities such as anti-tumor, anti-viral infection, and immune regulation. There are many cell sources for NK cells, such as peripheral blood mononuclear cells (PBMCs), umbilical cord blood mononuclear cells (UCBMCs), NK cell lines (NK-92, etc.), and induced pluripotent stem cells (iPSCs). Among them, NK cells derived from PBMCs have significant advantages over the other sources, mainly in the following aspects: (1) Peripheral blood sources are widely and reliably available. In addition to using the patient's own PBMC cells, NK cells from relatives' or other people's PBMCs used for reinfusion and treatment have no GvHD or immunosuppression; (2) NK cells derived from PBMCs are primary cells and do not have the potential risk of tumorigenesis, thus having high safety; (3) Compared with the other sources, NK cells derived from PBMCs highly express CD16 and NKG2D, exhibiting strong killing activity. Therefore, CAR modification of peripheral blood-derived NK cells (PBMC-NK cells) can enhance their ability to target and kill tumor cells. However, clinical studies have found that the survival period of NK cells after allogeneic infusion is relatively short, and their killing function is also limited in the absence of cytokine stimulation.

[0003] Based on the above problems, studies have shown that interleukin-12 (IL-12) can activate NK cells, promote their proliferation, and secrete tumor-killing factors (such as IFN-γ) to kill tumor cells. Therefore, constitutive secretion of IL-12 by engineered PBMC-derived CAR-NK cells is a safe and efficient strategy for treating solid tumors and enhancing the sustained killing effect of NK cells. However, no relevant reports have been found in the existing technology. Only some patents (WO2020051363A1) disclose methods for stimulating NK cells in vivo or in vitro with IL-12-targeting chimeric proteins to promote NK cell function. The limitations of this method are: (1) after IL-12 stimulation in vitro, IL-12 is removed before infusion therapy, and the effect on NK cells is not significant; (2) when IL-12 is infused in vivo, the IL-12-targeting chimeric protein can reach the tumor site through targeting, but a sufficient number of NK cells may not be able to reach the tumor site, thus leading to insignificant therapeutic effects.

[0004] Therefore, the current method of stimulating NK cells with IL-12-targeted chimeric proteins has not significantly enhanced the therapeutic effect on tumors. How to develop an enhanced anti-tumor NK cell with high safety, significant killing effect, no potential tumorigenic risk, and no need to add exogenous IL-12, thereby avoiding the side effects caused by systemic injection, is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] This invention addresses the technical problems of existing enhanced NK cell therapy, such as insignificant killing activity, insufficient safety, potential tumorigenic risks, and the need to add exogenous IL-12, which can cause side effects in the human body. It proposes an enhanced anti-tumor NK cell derived from PBMC, its preparation method, and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] Enhanced anti-tumor NK cells derived from PBMCs can simultaneously express HER2 and IL-12, targeting tumor cells through HER2 and enhancing their anti-tumor ability by secreting IL-12.

[0008] The enhanced anti-tumor NK cells derived from PBMCs were prepared by co-infecting PBMC-NK cells with IL-12 lentivirus and HER2-CAR lentivirus, followed by detection and identification experiments.

[0009] In one embodiment, the IL-12 lentivirus contains a synthetically produced IL-12 gene, which is obtained by linking the p35 subunit gene and the p40 subunit gene of the natural human IL-12 gene through a linker peptide gene. The nucleotide sequence of the synthetically produced IL-12 gene is shown in SEQ ID NO.1.

[0010] The HER2-CAR lentivirus contains a synthetically produced HER2-CAR gene. The synthetically produced HER2-CAR gene is obtained by codon optimization after linking the sequences of the human CD8α signal peptide gene, the HER2-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 obtained from the NCBI database. The nucleotide sequence of the synthetically produced HER2-CAR gene is shown in SEQ ID NO.2.

[0011] In one embodiment, the HER2-CAR cell positivity rate of enhanced anti-tumor NK cells derived from PBMCs was above 13.2%, and the IL-12 and IFN-γ contents in the culture supernatant 72 h after lentiviral infection were above 99.7 ng / ml and above 16.5 ng / ml, respectively.

[0012] This invention also provides a method for preparing enhanced anti-tumor NK cells derived from PBMCs, comprising the following steps:

[0013] Construction of lentiviral expression vectors: The artificially synthesized IL-12 gene and the artificially synthesized HER2-CAR gene were ligated into lentiviral vector plasmid I and lentiviral vector plasmid II, respectively, to construct the IL-12 lentiviral expression vector and the HER2-CAR lentiviral expression vector.

[0014] Lentiviral expression vector packaging: The IL-12 lentiviral expression vector and HER2-CAR lentiviral expression vector were co-transfected with packaging plasmids into 293T cells. After being cultured for a period of time, the cells were collected and concentrated to obtain IL-12 lentivirus and HER2-CAR lentivirus.

[0015] Isolation and expansion of PBMC-NK cells: Peripheral blood was collected from healthy individuals, and NK cells were activated, induced, and expanded using NK induction medium supplemented with autologous plasma to obtain PBMC-NK cells;

[0016] Construction and identification of enhanced anti-tumor NK cells derived from PBMCs: PBMC-NK cells were co-infected with the IL-12 lentivirus and HER2-CAR lentivirus at a certain MOI value, and the cells were cultured and collected. After identification by cell positivity rate detection, cytokine secretion detection and in vitro killing assay, the enhanced anti-tumor NK cells derived from PBMCs were finally obtained.

[0017] In one embodiment, the lentiviral vector plasmid I and lentiviral vector plasmid II are selected from PGMLV plasmid, lenti plasmid, pCDH plasmid, pLVX plasmid or pRRLSIN plasmid.

[0018] In one embodiment, the titer of the IL-12 lentivirus is (1-10)×10⁻⁶. 8 TU / ml, the titer of HER2-CAR lentivirus is (1-10)×10 8 TU / ml.

[0019] In one embodiment, the NK cell isolation and expansion steps of PBMC-NK cells involve NK cell activation, induction, and expansion using NK induction medium supplemented with 1-10% autologous plasma.

[0020] In one embodiment, the IL-12 lentivirus and HER2-CAR lentivirus are co-infected with the PBMC-NK cells at a certain MOI value, wherein the MOI value of the IL-12 lentivirus and HER2-CAR lentivirus is 20-200.

[0021] This invention also provides the application of enhanced anti-tumor NK cells derived from PBMCs in the preparation of anti-tumor drugs.

[0022] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0023] 1. The enhanced anti-tumor NK cells derived from PBMCs proposed in this invention are obtained by introducing the IL-12 gene and HER2-CAR gene into PBMC-NK cells through a lentivirus-mediated method. After identification by combining HER2-CAR cell positivity rate, cytokine secretion detection and in vitro killing assay, these NK cells, obtained by expansion from PBMCs, can express HER2-CAR and self-secrete IL-12, enabling NK cells to directly target and kill tumors. At the same time, NK cells recognize self-secreted IL-12, promote their proliferation and secretion of killing factors to enhance the killing effect on tumors. This fundamentally solves the technical problem that existing enhanced NK cell therapy has insufficient killing activity and requires the addition of exogenous IL-12, which produces side effects on the human body.

[0024] 2. The enhanced anti-tumor NK cells derived from PBMCs proposed in this invention, wherein the NK cells isolated and expanded from peripheral blood have the characteristics of high safety, low cost, high killing activity, and no potential risk of tumorigenesis.

[0025] 3. The enhanced anti-tumor NK cells derived from PBMC proposed in this invention have broad application prospects in the field of anti-tumor drug preparation, especially in the field of anti-solid tumor drug preparation. Attached Figure Description

[0026] Figure 1 A schematic diagram of the artificially synthesized IL-12 gene structure provided in an embodiment of the present invention;

[0027] Figure 2 This is a schematic diagram of the artificially synthesized HER2-CAR gene structure provided in an embodiment of the present invention;

[0028] Figure 3 This is a map of the IL-12 lentiviral expression vector provided in an embodiment of the present invention;

[0029] Figure 4 The HER2-CAR lentiviral expression vector map provided in the embodiments of the present invention;

[0030] Figure 5 The image is a fluorescence microscope photograph taken after 72 hours of infection of 293T cells with 0.1 μL of concentrated GFP control lentivirus provided in an embodiment of the present invention.

[0031] Figure 6 This is a schematic diagram of the flow cytometry results of PBMC-NK cells provided in an embodiment of the present invention;

[0032] Figure 7 This is a schematic diagram of the flow cytometry results of IL-12-HER2-CAR-PBMC-NK cells provided in an embodiment of the present invention;

[0033] Figure 8 The graph shows the content of IL-12 and IFN-γ in the supernatant of IL-12-HER2-CAR-PBMC-NK cells provided in the embodiments of the present invention.

[0034] Figure 9 Microscopic image (100×) of the killing test cells provided in the embodiments of the present invention;

[0035] Figure 10 This is a graph showing the IFN-γ content in the cell supernatant of the killing test provided in an embodiment of the present invention. Detailed Implementation

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] This invention provides an enhanced anti-tumor NK cell derived from PBMCs, which can simultaneously express HER2 and IL-12, target tumor cells through HER2, and enhance anti-tumor ability by secreting IL-12.

[0038] The enhanced anti-tumor NK cells derived from PBMCs were prepared by co-infecting PBMC-NK cells with IL-12 lentivirus and HER2-CAR lentivirus, followed by detection and identification experiments.

[0039] In one specific embodiment, the IL-12 lentivirus contains a synthetically produced IL-12 gene, which is obtained by linking the p35 subunit gene and the p40 subunit gene of the natural human IL-12 gene through a linker peptide gene. The nucleotide sequence of the synthetically produced IL-12 gene is shown in SEQ ID NO.1.

[0040] The HER2-CAR lentivirus contains a synthetically produced HER2-CAR gene. The synthetically produced HER2-CAR gene is obtained by codon optimization after linking the sequences of the human CD8α signal peptide gene, the HER2-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 obtained from the NCBI database. The nucleotide sequence of the synthetically produced HER2-CAR gene is shown in SEQ ID NO.2.

[0041] In one specific embodiment, the HER2-CAR cell positivity rate is above 13.2%, and the IL-12 and IFN-γ contents in the culture supernatant 72 h after lentiviral infection are above 99.7 ng / ml and above 16.5 ng / ml, respectively.

[0042] This invention also provides a method for preparing enhanced anti-tumor NK cells derived from PBMCs, comprising the following steps:

[0043] S1. Construction of lentiviral expression vectors: The artificially synthesized IL-12 gene and the artificially synthesized HER2-CAR gene were ligated into lentiviral vector plasmid I and lentiviral vector plasmid II, respectively, to construct the IL-12 lentiviral expression vector and the HER2-CAR lentiviral expression vector.

[0044] S2, Lentiviral expression vector packaging: The IL-12 lentiviral expression vector and HER2-CAR lentiviral expression vector were co-transfected with packaging plasmids into 293T cells. After being cultured for a period of time, they were collected and concentrated to obtain IL-12 lentivirus and HER2-CAR lentivirus.

[0045] S3. Isolation and expansion of PBMC-NK cells: Peripheral blood was collected from healthy individuals, and NK cells were activated, induced, and expanded using NK induction medium supplemented with autologous plasma to obtain PBMC-NK cells.

[0046] S4. Construction and identification of enhanced anti-tumor NK cells derived from PBMCs: The IL-12 lentivirus and HER2-CAR lentivirus were co-infected with the PBMC-NK cells at a certain MOI value. The cells were cultured and collected, and then identified by cell positivity rate detection, cytokine secretion detection and in vitro killing assay. Finally, the enhanced anti-tumor NK cells derived from PBMCs were obtained.

[0047] In one specific embodiment, the lentiviral vector plasmid I and lentiviral vector plasmid II are selected from PGMLV plasmid, lenti plasmid, pCDH plasmid, pLVX plasmid or pRRLSIN plasmid.

[0048] In one specific embodiment, the titer of the IL-12 lentivirus is (1-10)×10⁻⁶. 8 TU / ml, the titer of HER2-CAR lentivirus is (1-10)×10⁻⁶. 8 TU / ml.

[0049] In one specific embodiment, in the isolation and expansion steps of PBMC-NK cells, NK cell activation, induction and expansion are performed using NK induction medium supplemented with 1-10% autologous plasma.

[0050] In one specific embodiment, the IL-12 lentivirus and HER2-CAR lentivirus are co-infected with the PBMC-NK cells according to a certain MOI value, wherein the MOI value of the IL-12 lentivirus and HER2-CAR lentivirus is 20-200.

[0051] This invention also provides the application of enhanced anti-tumor NK cells derived from PBMCs in the preparation of anti-tumor drugs.

[0052] To more clearly and in detail introduce the enhanced anti-tumor NK cells derived from PBMCs provided in the embodiments of the present invention, their preparation methods and applications, the following description will be based on specific embodiments.

[0053] Example 1

[0054] This embodiment provides a method for preparing enhanced anti-tumor NK cells derived from PBMCs, including the following steps:

[0055] (1) Construction of lentiviral expression vector: The IL-12 gene, namely IL-12P40 leader-IL-12P40-2A-IL-12P35, was artificially synthesized. Its nucleotide sequence is shown in SEQ ID NO.1, and its gene structure diagram is shown in... Figure 1 As shown; the artificially synthesized HER2-CAR gene, namely CD8αleader-HER2-scFv-CD8αHinge-CD8α-TM-4-1BB-CD3ζ, has the nucleotide sequence shown in SEQ ID NO.2, and its gene structure diagram is shown in... Figure 2 As shown, the artificially synthesized IL-12 gene was ligated into the PGMLV plasmid (Genomeditech) to construct the IL-12 lentiviral expression vector, namely PGMLV-IL-12; the artificially synthesized HER2-CAR gene was ligated into the lenti plasmid (Genomeditech) to construct the HER2-CAR lentiviral expression vector, namely lenti-HER2-CAR. The plasmid map is shown below. Figure 3-4 As shown;

[0056] (2) Lentiviral packaging and concentration:

[0057] Take 5 × 10⁵ 293T cells in logarithmic growth phase 6 Cells were seeded in 10cm cell culture dishes and incubated overnight until 90% confluence was achieved. Using Invitrogen's Lipofectamine 3000 reagent, a three-plasmid transfection system was used: lentiviral expression vector: psPAX2 (Addgene): pMD2.G (Addgene) in a 4:3:1 ratio, with a total transfection plasmid mass of 15μg. A transfection complex was prepared by mixing plasmid and transfection reagent at a 1:2 ratio, incubated at room temperature for 15 min, and then evenly added to the cell culture dishes. The cells were incubated at 37°C in a CO2 incubator. 48 h after transfection, the culture supernatant was collected, temporarily stored at 4°C, and the culture medium was replaced with fresh medium. Then, after 72 h, the culture supernatant was collected again and mixed with the 48 h supernatant. The mixture was incubated at 1800 rpm for 5 min, and the supernatant was collected and concentrated using Lenti-X Concentrator (Clontech).

[0058] (3) Titer determination: 293T cells were used for titer determination. Concentrated viral supernatant (HER2-CAR-GFP control lentivirus) was added to 293T cells in a gradient. The proportion of positive cells was measured 72 hours post-infection to determine the viral titer. The viral titer (TU / ml) of the lentivirus was determined based on the expression level of the fluorescent protein GFP in 293T cells. The specific steps are as follows:

[0059] (3-1) Day 1 Cell preparation: Seed 1.5 × 10⁶ cells into each well of a 96-well plate. 4 293 T cells;

[0060] (3-2) Day 2: Virus dilution and infection. Discard the cell supernatant from the previous day's plate. Then, add 100 μL of virus dilution buffer sequentially to each well of a 96-well cell plate, i.e., the AH-well virus volumes are 10 μL, 1 μL, 0.1 μL, 0.01 μL, 0.001 μL, 0.0001 μL, 0.00001 μL, and 0.000001 μL, respectively.

[0061] (3-3) Take photos under a fluorescence microscope on the fifth day (the results are as follows). Figure 5 (As shown) and perform titer calculation, such as Figure 5 The results showed that the positive rate of GFP in 0.1 μL wells of concentrated GFP control lentivirus was approximately 35-40%. Based on the formula Lentiviral titer (TU / ml) = (Number of cells at infection × % of positive cells) / Virus volume (ml), the calculated lentivirus titer was approximately 1 × 10⁻⁶. 8 TU / ml.

[0062] (4) Isolation and expansion of PBMC-NK cells: Peripheral blood from healthy individuals was added to a lymphocyte separation tube (Dakco), centrifuged to collect the upper layer of autologous plasma, and inactivated at 56°C for 30 min for later use. Then, the lower layer of PBMCs and separation solution were poured into a new centrifuge tube, and RPMI 1640 medium (Gibco) was added for washing twice. A small number of cells were taken for flow cytometry detection. Following the Dakco NK induction medium kit (Dakco, 6813523), NK cell induction medium supplemented with 10% autologous plasma was used to incubate the cells at a rate of 3 × 10⁻⁶ cells / year. 6 Cells were activated and cultured at a density of [cell density] / ml, and then analyzed by flow cytometry on Day 12. The flow cytometry analysis employed PE anti-human CD56 Antibody (Biolegend, 362507) and Alexa [analyzer]. Cells were labeled with 488 anti-human CD3 Antibody (Biolegend, 300319), and the proportion of CD56+ NK cells was 84.2% on day 12. Figure 6As shown), cells were cryopreserved using serum-free cryopreservation solution (Dakow). Figure 6 The left image shows the flow cytometry results of PBMC-NK cells cultured using the NK induction medium kit on Day 0, and the right image shows the flow cytometry results of the CD3 / CD56 cell ratio on Day 12.

[0063] (5) Construction of enhanced anti-tumor NK cells derived from PBMCs:

[0064] (5-1) After resuscitation, the viability of PBMC-NK cells was >90%. They were cultured in NK cell induction medium (Dacowed) for 2-4 days, followed by lentiviral infection. PBMC-NK cells were collected by centrifugation and prepared into 5×10⁶ cells / days. 5 Add 2 ml of cell suspension per well to a 6-well plate, and set up blank wells (PBMC-NK cells), control wells (PBMC-NK cells infected with HER2-CAR lentivirus alone), and experimental wells (PBMC-NK cells infected with IL-12 lentivirus and HER2-CAR lentivirus co-infected).

[0065] (5-2) Thaw the lentivirus solution stored at -80℃ in an ice bath. Add 200 μL of IL-12 lentivirus and HER2-CAR lentivirus to the experimental wells at an MOI of 20. Add 200 μL of HER2-CAR lentivirus to the control wells and 200 μL of complete culture medium to the blank wells. Add polybrene at a final concentration of 8 μg / ml. Mix well and incubate at 37℃ in a 5% CO2 incubator. After 72 h, replenish the medium, passage, and detect the cells according to the cell density. The cells obtained from the experimental wells are IL-12 secretory HER2-CAR-PBMC-NK cells (i.e., enhanced anti-tumor NK cells derived from PBMC, IL-12-HER2-CAR-PBMC-NK).

[0066] Example 2

[0067] This embodiment provides a flow cytometry assay for detecting IL-12-HER2-CAR-PBMC-NK cells, specifically:

[0068] (1) Collect IL-12-HER2-CAR-PBMC-NK cells infected with lentivirus for 72 h, and use uninfected PBMC-NK cells as a negative control;

[0069] (2) Washing cells: Wash cells once with pre-cooled 0.1% BSA-PBS, centrifuge at 1800 rpm for 8 min at 4℃, and discard the supernatant;

[0070] (3) Staining and incubation: Resuspend the cells in 100 μL of 0.1% BSA-PBS, add 5 μL of FITC-Labeled HumanHer2 Protein (ACRO), mix well, and incubate at 4°C in the dark for 15 min.

[0071] (4) Washing: Wash twice with 0.1% BSA-PBS, centrifuge at 1800 rpm for 8 min, discard the supernatant and resuspend the cells in 200 μl of 0.1% BSA-PBS;

[0072] (5) Flow cytometer detection.

[0073] See test results Figure 7 :from Figure 7 The results show that the positivity rate of IL-12-HER2-CAR-PBMC-NK cells infected with lentivirus for 72 hours was approximately 13.2%.

[0074] Example 3

[0075] This embodiment provides an ELISA assay for detecting cytokines in IL-12-HER2-CAR-PBMC-NK cells, specifically as follows:

[0076] Culture supernatants of IL-12-HER2-CAR-PBMC-NK cells infected with lentivirus for 72 hours were collected for the detection of IL-12 and IFN-γ levels. The experimental procedure was performed according to the instructions of the Human IL-12(P70) Precoated ELISA Kit (Dacowi; 1111202) and the Human IFN-γ Precoated ELISA Kit (Dacowi; 1111002).

[0077] Specifically: Add 100 μL of Cytokine standard to the standard wells, 100 μL of sample to the sample wells, and 100 μL of Dilution buffer R (1×) to the blank control wells in the pre-coated microplate. Then add 50 μL of Biotinylated antibody to each well, mix well, cover with a sealing film, and incubate at room temperature for 2-3 hours. Remove the liquid from the wells, add 300 μL of 1× Washing buffer, discard the liquid from the wells, and repeat 4 times. Add 100 μL of Streptavidin-HRP to each well, cover with a sealing film, and incubate at room temperature for 20 minutes. Remove the liquid from the wells, add 300 μL of 1× Washing buffer, discard the liquid from the wells, and repeat 4 times. Add 100 μL of TMB to each well, incubate at room temperature in the dark, and stop the reaction by adding 100 μL of Stopsolution according to the intensity of the color in the well (dark blue). Within 10 minutes after termination, use an MD SpectraMax M2 microplate reader at a wavelength of 450 nm to read and process the data.

[0078] See test results Figure 8 :from Figure 8 The results show that the contents of IL-12 and IFN-γ in the culture supernatant of IL-12-HER2-CAR-PBMC-NK cells were 99.7 ng / ml and 16.5 ng / ml, respectively, which were significantly higher than those in the culture supernatant of the control group. The results indicate that the cells were successfully constructed and have enhanced anti-tumor potential.

[0079] Example 4

[0080] This embodiment provides an in vitro cell killing assay of enhanced anti-tumor NK cells, specifically:

[0081] PBMC-NK cells, HER2-CAR-PBMC-NK cells, and IL-12-HER2-CAR-PBMC-NK cells were mixed with target cells (SK-BR3 breast cancer cells and MGC-803 human gastric cancer cells) at an effector-target ratio of 5:1. Effector cell control wells, target cell control wells, and effector cell-target cell killing wells were set up. The cells were incubated at 37°C and 5% CO2 for 24 hours. After incubation, the cells were photographed under a microscope, and the supernatant was collected to detect the expression level of IFN-γ. The IFN-γ ELISA detection was performed according to the instructions of the Human IFN-γ Precoated ELISA Kit (Dakow; 1111002), and the specific method was the same as in Example 3.

[0082] See test results Figure 9-10 :from Figure 9The results show that IL-12-HER2-CAR-PBMC-NK cells have the most significant killing activity against SK-BR3 cells, which is significantly higher than that of the PBMC-NK and HER2-PBMC-NK groups.

[0083] The IFN-γ content in the supernatant of the lethality test was as follows Figure 10 And as shown in Table 1:

[0084] Table 1. IFN-γ content in the supernatant of the lethality test

[0085]

[0086]

[0087] As shown in the table above, the IFN-γ content in the effector cell-SKBR3 killing group, effector cell spontaneous release group, effector cell-MGC803 killing group, and effector cell spontaneous release group were 2319.1 pg / ml, 1501.7 pg / ml, 5345.0 pg / ml, and 1408.0 pg / ml, respectively. The IFN-γ content in the PBMC-NK group and the HER2-CAR-PBMC-NK group was significantly lower than that in the IL-12-HER2-CAR-PBMC-NK group.

[0088] This shows that the IFN-γ content in the IL-12-HER2-CAR-PBMC-NK group increased significantly during the killing process, further indirectly proving the tumor-killing potential of IL-12-HER2-CAR-PBMC-NK cells. SEQUENCE LISTING <110> Kanglitai Biopharmaceutical (Qingdao) Co., Ltd. <120> PBMC-targeted enhanced anti-tumor NK cells, their preparation methods and applications <130> 20220210 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 1479 <212> DNA <213> Artificially synthesized IL-12 gene <400> 1 gccaccatgt gtcaccagca gttggtcatc tcttggtttt ccctggtttt tctggcatct 60 cccctcgtgg ccatatggga actgaagaaa gatgtttatg tcgtagaatt ggattggtat 120 ccggatgccc ctggagaaat ggtggtcctc acctgtgaca cccctgaaga agatggtatc 180 acctggacct tggaccagag cagtgaggtc ttaggctctg gcaaaaccct gaccatccaa 240 gtcaaagagt ttggagatgc tggccagtac acctgtcaca aaagggcga ggttctaagc 300 cattcgctcc tgctgcttca caaaaaggaa gatggaattt ggtccactga tattttaaag 360 gaccagaaag aacccaaaaa taagaccttt ctaagatgcg aggccaagaa ttatctgga 420 cgtttcacct gctggtggct gacgacaatc agtactgatt tgacattcag tgtcaaaagc 480 agcagaggct cttctgaccc ccaaggggtg acgtgcggag ctgctacact ctctgcagag 540 600 tgcccagctg ctgaggagag tctgcccatt gaggtcatgg tggatgccgt tcacaagctc 660 aagtgaaa actacaccag cagcttcttc atcagggaca tcatcaaacc tgacccaccc 720 aagaacttgc agctgaagcc attaaagaat tctcggcagg tggaggtcag ctgggagtac 780 cctgacacct ggagtactcc acattcctac ttctccctga cattctgcgt tcaggtccag 840 ggcaagagca agagagaaaa gaaagataga gtcttcacgg acaagacctc agccacggtc atctgccgca aaaatgccag cattagcgtg cgggcccagg accgctacta tagctcatct tggagcgaat gggcatctgt gccctgcagg ggcggaggcg gaagcggagg cggaggagc 1020 ggcggtggcg gcagcagaaa cctccccgtg gccactccag acccaggaat gttcccatgc 1080 cttcaccact cccaaaacct gctgagggcc gtcagcaaca tgctccagaa ggccagacaa actctaga tttacccttg cacttctga gagattgatc atgagata cacaaaagat aaaaccagca cagtggaggc ctgtttacca ttggaatta ccaagaatga gagttgccta aattccagag agacctcttt cataactat gggagttgcc tggcctccag aaagacctct tttatgatgg ccctgtgcct tagtagtatt tatgaagact tgaagatgta ccaggtggag ttcaagacca tgaatgcaaa gcttctgatg gatcctaaga ggcagatctt tctagatcaa aacatgctgg cagttattga tgagctgatg caggccctga atttcaacag tgagactgtg ccacaaaaat cctcccttga agaaccggat ttttataaaa ctaaaatcaa gctctgcata 1560 cttcttcatg ctttcagaat tcgggcagtg actattgata gagtgatgag ctatctgaat 1620 gcttcctaa 1629 <210> 2 <211> 1629 <212> DNA <213> Artificial synthetic HER2-CAR gene <400> 2 gccaccatgg ctctgcccgt gaccgccctg ctgctccctc tggccctgct cctgcacgcc 60 gctagacccg acatccagat gacccagagc cccagcagcc tgtcagcttc tgtgggcgac 120 agagtgacaa tcacctgcag agcctcccag gacgtgaaca ccgccgtggc ctggtaccag 180 cagaaacctg gcaaagcccc caagctgctg atctacagcg ccagcttcct gtacagcggc 240 gtgcctagcc ggttctccgg cagcagaagc ggaacagatt ttacactgac catcagcagc 300 ctacagcctg aagacttcgc cacatactac tgccagcaac actacaccac ccctcctaca 360 ttcggccagg gcaccaaggt ggaaatcaag ggcggaggcg gatctggagg cggcggctct 420 ggcggaggag gctccgaggt gcagctggtt gagagcggcg gaggcctggt gcagcctggc 480 ggatctctga gactgagctg cgccgccagc ggatttaaca tcaaggacac atacatccac 540 tgggtgcggc aggccccagg aaagggcctg gaatgggtcg ccagaatcta tcctactaac 600 ggctacacca gatacgccga cagcgtcaag ggtagattta ccattagcgc cgacaccagc 660 aagaacacag cctacctgca aatgaacagc ctgcgggctg aagataccgc cgtgtactac 720 tgtagcagat ggggagggga tggcttctac gccatggact actggggcca aggaaccctg 780 gtgaccgtgt ccagcaccac aacccctgcc cctagacctc caacaccggc tcctaccatc 840 gccagccagc ccctgagcct gcggcctgag gcctgcagac ctgctgctgg cggcgccgtg 900 cacaccagag gcctggactt cgcctgcgac attacatct gggcccctct ggccggcacc 960 tgtggcgtgc tgctgctgag cctggttatc acactgtact gcaatcacag aaacaagaga 1020 ggccggaaaa agctgctgta catcttcaag cagcccttca tgcggccagt gcagaccacg 1080 caggaggaag atggctgtag ctgccggttc cccgaggaag aggaaggcgg ctgcgagctg 1140 cgcgtgaaat tcagcaggtc cgccgatgcc cctgcctacc agcagggaca gaaccagctg 1200 tataatgagc tgaacctggg cagacgcgag gaatacgacg tgctggacaa gagaaggggc 1260 agagatcctg agatgggcgg caagcctaga cggaaatc ctcaggaggg cctgtacaac 1320 gagctgcaga aggacaagat ggccgaggcc tatagcgaga tcggcatgaa gggcgagcgg 1380 agacggggca agggccacga cggcctgtac caagcctgt ctaccgccac caaagataca 1440 tacgacgctc tgcatatgca ggctctgccc cccagataa 1479

Claims

1. Enhanced anti-tumor NK cells derived from PBMCs, characterized in that, It can simultaneously express HER2 and IL-12, target tumor cells through HER2, and enhance anti-tumor ability by secreting IL-12. The enhanced anti-tumor NK cells derived from PBMCs were prepared by co-infecting NK cells from PBMCs with IL-12 lentivirus and HER2-CAR lentivirus, and then subjected to detection and identification experiments. The IL-12 lentivirus contains a synthetically produced IL-12 gene, which is obtained by linking the p35 and p40 subunit genes of the natural human IL-12 gene through a linker peptide gene. The nucleotide sequence of the synthetically produced IL-12 gene is shown in SEQ ID NO.

1. The HER2-CAR lentivirus contains a synthetically produced HER2-CAR gene. The synthetically produced HER2-CAR gene is obtained by codon optimization after linking the sequences of the human CD8α signal peptide gene, the HER2-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 obtained from the NCBI database. The nucleotide sequence of the synthetically produced HER2-CAR gene is shown in SEQ ID NO.

2.

2. The enhanced anti-tumor NK cells derived from PBMCs according to claim 1, characterized in that, The HER2-CAR cell positivity rate was above 13.2%, and the IL-12 and IFN-γ levels in the culture supernatant 72 h after lentiviral infection were above 99.7 ng / ml and 16.5 ng / ml, respectively.

3. The method for preparing enhanced anti-tumor NK cells derived from PBMCs according to claim 1 or 2, characterized in that, Includes the following steps: Construction of lentiviral expression vectors: The artificially synthesized IL-12 gene and the artificially synthesized HER2-CAR gene were ligated into lentiviral vector plasmid I and lentiviral vector plasmid II, respectively, to construct the IL-12 lentiviral expression vector and the HER2-CAR lentiviral expression vector. Lentiviral expression vector packaging: The IL-12 lentiviral expression vector and HER2-CAR lentiviral expression vector were co-transfected with packaging plasmids into 293T cells. After being cultured for a period of time, the cells were collected and concentrated to obtain IL-12 lentivirus and HER2-CAR lentivirus. Isolation and expansion of PBMC-NK cells: Peripheral blood was collected from healthy individuals, and NK cells were activated, induced, and expanded using NK induction medium supplemented with autologous plasma to obtain PBMC-NK cells; Construction and identification of enhanced anti-tumor NK cells derived from PBMCs: PBMC-NK cells were co-infected with IL-12 lentivirus and HER2-CAR lentivirus at a certain MOI value, and the cells were cultured and collected. After identification by HER2-CAR cell positivity rate detection, cytokine secretion detection and in vitro killing assay, the enhanced anti-tumor NK cells derived from PBMCs were finally obtained.

4. The method for preparing enhanced anti-tumor NK cells derived from PBMCs according to claim 3, characterized in that, The lentiviral vector plasmid I and lentiviral vector plasmid II are selected from PGMLV plasmid, lenti plasmid, pCDH plasmid, pLVX plasmid or pRRLSIN plasmid.

5. The method for preparing enhanced anti-tumor NK cells derived from PBMCs according to claim 3, characterized in that, The titer of the IL-12 lentivirus is (1-10)×10⁻⁶. 8 TU / ml, the titer of HER2-CAR lentivirus is (1-10)×10 8 TU / ml.

6. The method for preparing enhanced anti-tumor NK cells derived from PBMCs according to claim 3, characterized in that, In the isolation and expansion of PBMC-NK cells, NK cell activation, induction and expansion were performed using NK induction medium supplemented with 1-10% autologous plasma.

7. The method for preparing enhanced anti-tumor NK cells derived from PBMCs according to claim 3, characterized in that, The IL-12 lentivirus and HER2-CAR lentivirus were co-infected with the PBMC-NK cells at a certain MOI value, wherein the MOI value of the IL-12 lentivirus and HER2-CAR lentivirus was 20-200.

8. The use of enhanced anti-tumor NK cells derived from PBMCs as described in claim 1 or 2 in the preparation of anti-breast cancer drugs.

Images