Genetically modified k562 cells and their use in the in vitro culture of nk cells

Abstract

The application discloses a genetically modified K562 cell, which comprises coding genes of CD48, CD137L, CD64, tCD19 and IL-21 in the genome, and a construction method thereof is as follows: (1) taking gene fragments of CD48, CD137L, tCD19 and CD64, connecting and inserting into a lentivirus expression vector, and infecting K562 cells to obtain K562 cells stably expressing CD48, CD137L, tCD19 and CD64; (2) taking a gene fragment of IL-21, connecting and inserting into a lentivirus expression vector, and infecting the K562 cells stably expressing the K562 cells to obtain K562 cells stably expressing CD48, CD137L, tCD19, CD64 and IL-21. The application also discloses an application of the genetically modified K562 cell as a trophoblast cell in culturing NK cells in vitro. The stably transfected cell strain constructed by the application can make NK cells proliferate rapidly, and the NK cells have high purity and strong killing power.

Description

Technical Field

[0001] This invention relates to a genetically modified K562 cell and its application in in vitro cultured NK cells, belonging to the field of NK cell culture technology. Background Technology

[0002] Adoptive cell therapy (ACT) involves isolating the patient's lymphocytes from the body, activating, expanding, culturing, selecting, or modifying them in vitro, and then reinfusing the tumor-killing lymphocytes back into the patient to control and eliminate tumors. It is currently one of the most promising treatment methods in the field of tumor immunotherapy. Natural killer cells (NK cells) are a third type of lymphocyte, distinct from T cells and B cells. They belong to the innate immune system and are widely distributed in peripheral blood, bone marrow, and tissues, accounting for approximately 10%–20% of the total number of peripheral blood lymphocytes and 5% of umbilical cord blood lymphocytes. NK cells possess a unique tumor recognition mechanism. They can directly kill target cells by releasing cytoplasmic granules containing perforin and granzymes, induce tumor cell apoptosis by releasing cytokines, and mediate ADCC to kill target cells. NK cells do not express antigen-specific recognition receptors and do not cause immune rejection, making them ideal allogeneic therapeutic vectors. NK cells modified with chimeric antigen receptors (CARs) exhibit stronger anti-tumor activity and do not cause GvHD or CRS. They also have advantages such as fewer toxic side effects and can be used as off-the-shelf cell therapy products, giving them a significant advantage in the field of cell therapy.

[0003] Currently, there are two main methods for in vitro expansion of NK cells. One method involves stimulating NK cell proliferation with cytokines such as IL-2, IL-12, IL-15, IL-18, and IL-21. However, the in vitro proliferation capacity of NK cells induced by these cytokines is generally limited, and the cost is high while the purity is low. The other method involves stimulating NK cell proliferation with feeder cells, such as the Wilms tumor cell line, autologous PBMCs, EBV-transformed lymphoblasts, and the chronic myeloid leukemia cell line K562. However, there is still room for improvement in the proliferation rate, and the resulting NK cells do not have strong cytotoxicity. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides a genetically modified K562 cell—a K562 cell that simultaneously expresses CD48, CD137L, CD64, tCD19, and IL-21, and applies it as a feeder cell for NK cells.

[0005] This invention is achieved through the following technical solution:

[0006] A genetically modified K562 cell whose genome contains the coding genes for CD48, CD137L, CD64, tCD19, and IL-21.

[0007] The method for constructing the genetically modified K562 cells includes the following steps:

[0008] (1) Take the gene fragments of CD48, CD137L, tCD19, and CD64, ligate them into a lentiviral expression vector, infect K562 cells, and obtain K562 cells that stably express CD48, CD137L, tCD19, and CD64.

[0009] (2) Take the IL-21 gene fragment, ligate it into a lentiviral expression vector, and infect the above K562 cells that stably express CD48, CD137L, tCD19, and CD64 to obtain K562 cells that stably express CD48, CD137L, tCD19, CD64, and IL-21.

[0010] (3) After expanding the culture of K562 cells obtained in step (2), the culture medium is irradiated with 100 Gy of γ rays. The culture medium can then be used as a feeder cell for in vitro culture of NK cells.

[0011] The application of the genetically modified K562 cells as feeder cells in the in vitro culture of NK cells.

[0012] A method for culturing NK cells in vitro: The culture medium containing K562 cells with the above-mentioned gene modification is placed together with NK cells in a culture medium, and 200 U / mL of IL-2 is added. The cells are cultured for 7 to 21 days, and fresh culture medium is added every one day, along with 200 U / mL of IL-2.

[0013] Furthermore, the ratio of genetically modified K562 cells to NK cells was 2:1.

[0014] This invention utilizes complete or functional gene fragments of CD48, CD137L, CD64, CD19, and IL-21 to construct expression vectors, package lentiviruses, and then infect K562 cells with the lentiviruses to achieve stable expression of each target protein in K562 cells. Gamma irradiation is then used to deactivate the target protein while preserving its activity. The selected stable cell line K562-CD48-CD137L-tCD19-CD64-mbIL21 (1D8-IL21) is mixed with NK cells for in vitro expansion, while simultaneously supplementing with the cytokine IL-2. During the process, AOPI technology is used to detect NK cell proliferation, flow cytometry is used to detect NK cell purity, luciferase is used to detect cytotoxicity, and ELISA kits are used to detect cytokine secretion. The effects of the feeder cells of this invention on NK cell proliferation, cytotoxicity, purity, and cytokine secretion are tested. The results showed that the stable cell line K562-CD48-CD137L-tCD19-CD64-mbIL21 (1D8-IL21) constructed in this invention can rapidly proliferate NK cells, and the NK cells have high purity and strong killing power. Attached Figure Description

[0015] Figure 1 Diagram of K562-CD48-CD137L-tCD19-CD64-mbIL21 trophoblast cells.

[0016] Figure 2 Schematic diagram of flow cytometry results for trophoblast cells.

[0017] Figure 3 Schematic diagram of NK cell proliferation curve.

[0018] Figure 4 Schematic diagram of NK cell purity test results.

[0019] Figure 5 : Schematic diagram of lethality performance results. Detailed Implementation

[0020] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0021] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0022] Example 1: Preparation of trophoblast cells

[0023] 1.1 Construction of feeder cell lines (model as follows) Figure 1 (As shown)

[0024] The coding sequences of CD48, CD137L, CD19, and CD64 were queried in NCBI. CD19 required intracellular truncation (excluding the intracellular signal transduction region). CD48, CD137L, tCD19, and CD64 gene fragments were synthesized using gene synthesis methods. Different restriction enzyme sites were then introduced at the beginning and end of the fragments, which were ligated into a lentiviral expression vector. The lentivirus was then packaged using Lipofectamine 2000 transfection reagent and the packaging plasmid PMD2G / psPAX2 and used to infect K562 cells. Flow cytometry was used to detect the expression of each target after K562 cell infection. Monoclonal cells that simultaneously expressed each target were selected using a limiting dilution method, ensuring stable expression of CD48, CD137L, tCD19, and CD64 in K562 cells.

[0025] A fragment of the mbIL21 gene, which stably expresses IL-21 on the cell membrane, was introduced into the K562-CD48-CD137L-tCD19-CD64 cell line. The expressed IL-21 (mbIL21) consists of a GM-CSF signal peptide, IL-21, an IgG4 hinge region, an IgG4 Fc region, and a CD4 transmembrane region. mbIL21 was stably expressed in the K562-CD48-CD137L-tCD19-CD64 cell line via lentiviral infection, and flow cytometry was used to confirm the expression of all targets. A stable K562-CD48-CD137L-tCD19-CD64-mbIL21 cell line was obtained, with a positive expression rate of over 90% for all targets. The detection results are as follows: Figure 2 As shown, this indicates that the feeder cell line was successfully constructed.

[0026] 1.2 Application and treatment of feeder cell lines

[0027] After expanding the culture of the above K562-CD48-CD137L-tCD19-CD64-mbIL21 cells, they were irradiated with 100 Gy of γ rays and then cryopreserved in liquid nitrogen for later use.

[0028] Example 2: Screening of NK cells

[0029] 2.1 Mononuclear cells derived from umbilical cord blood were separated using lymphocyte separation medium, and then cell counts were performed. The cells were centrifuged at 300g for 10 minutes.

[0030] 2.2 Resuspend cells in buffer, 40 μL / 10 7 10 cells; per 107 Add 10 μL of NK Cell Biotin-Antibody Cocktail to each cell; mix well, incubate at 2–8°C for 5 minutes; repeat every 10 cells. 7 Add 30 μL of buffer to each cell.

[0031] 2.3 per 10 7 Add 20 μL of NK cell microbead cocktail to each cell; mix well and incubate at 2–8 °C for 10 minutes.

[0032] 2.4 Place the sorting column on the sorting magnet and rinse the column with an appropriate amount of buffer solution.

[0033] 2.5 After the buffer solution has been used up, add the cell suspension to the column and collect the effluent (sorted NK cells); wash the column once with an appropriate volume of buffer solution and collect the effluent.

[0034] Example 3: Expansion of NK cells

[0035] The NK cells obtained from the sorting process were resuspended in NK-MACS medium (with an additional 10% FBS). Irradiated feeder cells (prepared in Example 1) were taken, resuspended in the same NK medium, mixed with the NK cells at a ratio of 2:1, and supplemented with 200 U / mL IL-2. Culture was continued, with fresh medium (containing 200 U / mL IL-2) added every one day. Cell proliferation was counted using AOPI during culture to monitor the cell proliferation curve. At the end of culture, the purity of the collected cells was detected by flow cytometry after labeling with CD56 / CD3 antibody.

[0036] Proliferation curves as follows Figure 3 As shown, the cell purity test results are as follows: Figure 4 As shown. The feeder cells (1D8-mbIL21) constructed in Example 1 can increase the proliferation of NK cells by more than 1000 times, which is superior to commercial feeder cells (PC). The proportion of CD56+CD3- cells in NK cells is about 99%.

[0037] Example 4: NK cell killing experiment

[0038] Using Raji cells stably transfected with Luciferase as target cells, healthy NK cells were resuspended in culture medium and seeded with target cells Raji-Luciferase at ratios of 1:1, 5:1, and 10:1 into 96-well plates and co-cultured for 4 hours. Bright-Glo TM A luciferase assay kit was used to detect Luciferase release levels in target cells and to assess NK cell killing ability. Results are as follows: Figure 5As shown, compared with NK cells expanded from commercially available trophoblast cells (PCs), the NK cells expanded from the trophoblast cells K562-CD48-CD137L-tCD19-CD64-mbIL21 (1D8-mbIL21) constructed in this invention have the same ability to kill target cells.

[0039] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A method for culturing NK cells in vitro, characterized in that: Culture medium containing genetically modified K562 cells was placed in the same culture medium as NK cells at a ratio of 2:

1. IL-2 was added at a concentration of 200 U / mL. The cells were cultured for 21 days, with fresh culture medium and IL-2 added every other day. The proportion of CD56+CD3- cells in the resulting NK cells was 99%. The genetically modified K562 cells were constructed using the following method: (1) Take the gene fragments of CD48, CD137L, tCD19 and CD64, ligate them into a lentiviral expression vector, infect K562 cells, and obtain K562 cells that stably express CD48, CD137L, tCD19 and CD64; (2) Take the IL-21 gene fragment, ligate it into a lentiviral expression vector, and infect the above K562 cells that stably express CD48, CD137L, tCD19 and CD64 to obtain K562 cells that stably express CD48, CD137L, tCD19, CD64 and IL-21.

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