Method for amplifying NK (Natural Killer) cells by taking NK92 cells as trophoblast cells

Abstract

The invention belongs to the field of immunology, and particularly relates to amplification of NK cells, in particular to a method for amplifying NK cells by using NK92 cells as trophoblast cells, which specifically uses NK92 cells secreting IL-21, IL-15 and IL-18 as trophoblast cells and PBMC as seed cells to directionally amplify and activate natural killer cells, and comprises the following steps: transfecting an NK92 cell strain through lentivirus; the NK92 cell strain capable of stably expressing IL-21, IL-15 and IL-18 is obtained; then obtaining monoclonal cells of NK92 cells through flow sorting and limit dilution methods, finally culturing and amplifying, preparing NK92 trophoblasts through irradiation, and mixing the NK92 trophoblasts with PBMC (peripheral blood mononuclear cells) for culturing so as to prepare the NK cells. According to the method, the NK cells can be efficiently amplified and activated, and the activated NK cells can improve the immunity of the organism, so that tumor and virus infection is resisted. The invention has wide application in medicine.

Description

Technical Field

[0001] The present invention relates to the technical field of immunology, and in particular to a method for expanding NK cells by using NK92 cells as trophoblast cells. Background Art

[0002] NK cells, also known as natural killer cells, are derived from bone marrow lymphoid stem cells. Their differentiation and development depend on the bone marrow and thymus microenvironment. They are mainly distributed in the bone marrow, peripheral blood, liver, spleen, lungs and lymph nodes. They can produce proinflammatory cytokines and kill virus-infected cells or cancer cells. In the treatment of advanced leukemia patients 20 years ago, NK cell-mediated immunotherapy has become a safe and effective therapy. With the passage of time, the deepening of medical research and the development of medical technology, the anti-cancer research on NK cells has grown exponentially and has become the main field of innovation in immunotherapy. However, NK cells only account for 5-10% of lymphocytes in the blood. In cell therapy, sufficient cell number and cell purity are important factors for NK to kill tumor cells, but the proliferation of NK cells is very weak, and it is impossible to provide a sufficient number of cells for administration, which has become the biggest problem in its application in clinical practice. Therefore, seeking an effective method to obtain sufficient number and purity of NK cells in vitro is still a hot topic that scholars are committed to studying.

[0003] NK cell-based adoptive immunotherapy requires a large number of cells. Autologous or allogeneic NK cells for transplantation can be obtained from different sources: peripheral blood mononuclear cells (PBMC), umbilical cord blood (UCB), bone marrow (BM), induced pluripotent stem cells (iPSC) and cell lines. The expansion of large numbers of highly pure and active NK cells is a key factor for their use in treatment. NK cell expansion can be based on purified or enriched NK cells or from single blood products (such as PBMCs) as starting material. Subsequently, the expanded NK cells are purified in the final stage, usually by magnetic sorting to eliminate other immune cells. The most commonly used trophoblast cells are irradiated K562 cells expressing IL-15 and 4-1BBL. IL-15 is a pleiotropic cytokine that promotes NK and CD8 + T cell survival, proliferation and cytotoxicity, 4-1BBL can also regulate NK cell activation and signal transduction. Currently, K562 cells are used to amplify NK cells, and high-purity, large quantities and good reproducibility of NK cells have been obtained. However, the original cells of K562 cells are a kind of malignant tumor cells of the hematopoietic system with multidirectional differentiation potential, which can spontaneously differentiate into identifiable progenitor cells of the erythroid, granulocyte and monocytic lineages. There are ethical risks in clinical application. In addition, the currently prepared NK cells have the disadvantage of weak killing ability, which also limits the application of NK cells.

[0004] NK92 cells are human NK cell lines that were isolated from the peripheral blood of a 51-year-old man by the National Institutes of Health (NIH) in 1982. NK92 cells are an immortal cell line with highly efficient cytotoxicity and stable immune properties. Several NK92 clinical trials have been conducted, and it has been proven that irradiated NK92 cell lines are safe and reliable. However, there are no reports on the use of NK92 cells as trophoblast cells to expand NK cells. Summary of the invention

[0005] To solve the above technical problems, the present invention provides a new NK expansion method, which uses engineered NK92 cells as trophoblast cells and PBMC as seed cells to expand and activate natural killer cells, thereby achieving high-efficiency and high-purity preparation of NK cells and simplifying subsequent purification steps.

[0006] The present invention claims a method for expanding NK cells using NK92 cells as trophoblast cells, comprising the following steps: using NK92 cells expressing interleukin 15, interleukin 21 and interleukin 18 as trophoblast cells and peripheral blood mononuclear cells as seed cells to expand NK cells.

[0007] Furthermore, the amino acid sequence of the interleukin 21 is shown as SEQ ID NO.1, the amino acid sequence of the interleukin 18 is shown as SEQ ID NO.2, and the amino acid sequence of the interleukin 15 is shown as SEQ ID NO.3.

[0008] Furthermore, the interleukin 15, interleukin 21 and interleukin 18 are connected via a 2A peptide; preferably, the 2A peptide comprises a T2A peptide; the amino acid sequence of the T2A peptide is shown in SEQ ID NO.4.

[0009] Furthermore, the preparation of NK92 cells expressing interleukin 15, interleukin 21 and interleukin 18 comprises the following steps: inserting the interleukin 15, interleukin 21 and interleukin 18 encoding gene sequences linked to the 2A peptide into an expression vector, and transferring the recombinant vector into NK92 cells.

[0010] Furthermore, the expression vector includes but is not limited to a viral vector (such as a lentiviral vector, a retroviral vector, etc.).

[0011] Furthermore, the ratio of the number of trophoblast cells to seed cells is 1-10:1.

[0012] Furthermore, the method specifically comprises the following steps:

[0013] S1. preparing engineered NK92 cells expressing interleukin 15, interleukin 21 and interleukin 18, and irradiating the engineered NK92 cells to obtain trophoblast cells;

[0014] S2. Co-culture peripheral blood mononuclear cells and trophoblast cells of S1 to obtain NK cells.

[0015] Furthermore, in step S1, the irradiation includes but is not limited to irradiation with γ rays, α rays, β rays, X-rays, etc.

[0016] Furthermore, in step S1, the irradiation dose is 20-100 Gy.

[0017] Furthermore, after the co-culture in step S2, the method further includes a step of separating NK cells.

[0018] Furthermore, the NK cells are separated by surface markers, and the sorting methods include but are not limited to flow sorting.

[0019] Furthermore, in step S2, the co-culture is to culture the mixed cells at 35-38° C. and a carbon dioxide concentration of 1%-10% for 10-18 days (preferably 12-16 days, more preferably 13-15 days).

[0020] The second object of the present invention is to provide NK cells prepared by the above method. The activity of the NK cells obtained by the present invention is much higher than that of the NK cells amplified by the prior art, and the purity and safety are also higher.

[0021] The third object of the present invention is to provide the use of the above-mentioned NK cells in the preparation of immune drugs.

[0022] Furthermore, the immune drug is used to treat tumors, viral infections, autoimmune diseases, post-transplant rejection, graft-versus-host disease, etc.

[0023] Furthermore, the tumor includes but is not limited to solid tumors.

[0024] Furthermore, the solid tumors include but are not limited to pancreatic cancer, melanoma, liver cancer, gastric cancer, intestinal cancer, colon cancer, etc.

[0025] By means of the above scheme, the present invention has at least the following advantages:

[0026] (1) The present invention prepares NK cells by using specially modified engineered NK92 cells as trophoblast cells, which can achieve explosive proliferation of NK cells in a short period of time. The NK cell expansion multiple after 14 days of in vitro expansion culture is as high as 3300 times, and the purity of the obtained NK cells reaches more than 95%, and the repeatability of the expansion multiple is good; at the same time, compared with other engineered trophoblast cells and NK cells prepared by cytokines, the activity of the obtained NK cells is higher, and the killing ability of the obtained NK cells for tumor cells is greater than 80%, and the cytotoxic effect is not reduced due to the increase in the number of passages.

[0027] (2) The NK cells obtained by the method of the present invention can be used to treat various diseases such as malignant tumors, viral infections, autoimmune diseases, post-transplant rejection, and graft-versus-host disease, and comprehensively solve the problems of standardization and scale expansion of high-purity NK cells. The prepared NK cells are suitable for long-term cryopreservation and can be used immediately after recovery, which is safe and convenient.

[0028] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a description of the preferred embodiments of the present invention in conjunction with detailed drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to make the contents of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings.

[0030] Figure 1 It is a schematic diagram of the engineering transformation of the present invention.

[0031] Figure 2 It is the purity of NK cells prepared by the present invention.

[0032] Figure 3 This is the morphology of NK cells under a microscope.

[0033] Figure 4 The killing ability of NK cells prepared by different methods. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0035] The scheme of the present invention is as follows:

[0036] By transfecting the NK92 cell line with lentivirus, a NK92 cell line that stably expresses IL-21, IL-15, and IL-18 was obtained; then, a monoclonal clone of NK92 cells was obtained by flow cytometry and limiting dilution method, and finally, NK92 trophoblast cells were prepared by irradiation after culture and amplification, and then mixed with PBMC cells for culture to achieve the preparation of NK cells.

[0037] Example 1 Expansion of NK cells

[0038] (1) Genetic engineering of NK92 cells

[0039] ① The expression sequences of IL-21, IL-18, and IL-15 were sequentially connected through T2A and constructed into the lentiviral expression vector PLVX-EF1a-puro to obtain the lentiviral vector PLVX-EF1a-NK92. The combination order of each element in the constructed lentiviral expression vector is as follows: Figure 1 shown.

[0040] The amino acid sequences of each element in the constructed expression vector are as follows:

[0041] IL-21 (SEQ ID NO.1):

[0042] MRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

[0043] IL-18 (SEQ ID NO. 2):

[0044] MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENLESDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

[0045] IL-15 (SEQ ID NO.3):

[0046] MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0047] T2A (SEQ ID NO.4):

[0048] EGRGSLLTCGDVEENPG

[0049] ② Lentivirus preparation

[0050] The specific experimental steps are as follows:

[0051] S1, prepare a 15cm dish, inoculate 5*10 6 293T cells (purchased from ATCC) were added with complete culture medium (DMEM high glucose, 10% FBS, double antibody), placed in a 37°C, 5% CO2 incubator, and cultured overnight.

[0052] S2. Take out 100μM PEI and lentiviral packaging plasmids (PLVX-EF1a-NK92, pGP, pVSVG) from the refrigerator, thaw at room temperature, and mix thoroughly by pipetting up and down. Take out PBS or HBSS buffer and warm to room temperature. Take 2mL PBS to one well of a 6-well plate, add 10μg PLVX-EF1a-NK92, 4μg pGP, and 2μg pVSVG respectively, mix thoroughly by pipetting up and down, add 18μL 100μM PEI, and immediately mix by pipetting up and down, and let stand at room temperature for 10 minutes.

[0053] S3. Add the above DNA / PEI complex dropwise into a 15 cm culture dish, gently shake the dish and mix thoroughly. Place the dish in a 37°C, 5% CO2 incubator, and after 6 to 8 hours of culture, remove the culture medium containing the transfection reagent and replace it with fresh complete culture medium.

[0054] After continuous culture for 48 hours, collect the supernatant of the culture medium containing the virus in the culture dish, filter it with a 0.45μm filter membrane, add 20% volume of 50% PEG6000 solution, incubate at 4 degrees for 2 hours, transfer to a centrifuge tube, balance it, and centrifuge it at 3000xg at 4℃ for 0.5 hours. After the centrifugation, carefully remove the liquid in the centrifuge tube in the biosafety cabinet, add 500μL PBS buffer to resuspend the precipitate, and store the virus at -80℃.

[0055] ③ Lentivirus infection of NK92 cells

[0056] The specific experimental steps are as follows:

[0057] S1. Adjust the density of NK92 cells to 1*10 6 cells / mL, cytokines (IL-2 with a final concentration of 300 U / mL) were added, and culture was continued for 48 hours.

[0058] S2. Calculate the required amount of virus according to MOI = 20. The calculation formula is as follows: Required amount of virus (mL) = (MOI * number of cells) / virus titer.

[0059] S3. After taking out the virus from the -80℃ refrigerator, quickly melt it in a 37℃ water bath. Add the above calculated amount of virus to the six-well plate, add polybrene with a final concentration of 6μg / mL, mix thoroughly, seal the four sides of the six-well plate with sealing film, and centrifuge at 800xg for 1 hour.

[0060] S4. After centrifugation, remove the sealing film and place the six-well plate in an incubator at 37°C and 5% CO2 for 24 hours.

[0061] S5. Centrifuge at 250 x g for 10 minutes, remove the culture medium supernatant containing the virus, resuspend the cell pellet with fresh culture medium, transfer the cells to a new six-well plate, and continue culturing for 3-6 days for use.

[0062] ④NK92 cell monoclonal selection

[0063] S1. Take the above NK92 cells transfected with lentivirus and adjust the density to 1*10 6 cells / mL, added 1ug / ml of puromycin, and cultured continuously for 2 weeks.

[0064] S2. Collect the cells after Puro screening, dilute the cell suspension to a very low density, and inoculate it into a 96-well plate until a single cell appears in the well. Continue to culture until clones are formed.

[0065] S3. After the obtained monoclonal cells are cultured and expanded in large quantities, genetically engineered NK92 cells are harvested.

[0066] (2) Expansion of NK cells

[0067] The genetically engineered NK92 cells obtained in step (1) are irradiated (γ rays, 50 Gy) to serve as trophoblast cells.

[0068] Peripheral blood mononuclear cells were isolated from venous blood, resuspended in NK cell complete medium X-VIVO-15, and divided into two parts:

[0069] One portion was added with the above trophoblast cells, mixed evenly (the ratio of trophoblast cells to peripheral blood mononuclear cells was 5:1), and cultured in an incubator at 37°C and 5% CO2; culture medium X-VIVO-15 was added every 2-3 days, and culture was continued until the 14th day to obtain NK cells expanded by NK92 cells (experimental group);

[0070] One portion was added with IL15, IL18, and IL-21 cytokines and cultured for 14 days, and the remaining steps were the same as above.

[0071] (3) Purity testing

[0072] Results Figure 2 ,from Figure 2 It can be seen that the purity of NK cells cultured with trophoblast cells reaches 96%, while the purity of NK cells cultured with cytokines is only 71%.

[0073] Example 2 Verification of the killing ability of NK cells

[0074] The lysis ability of NK cells prepared by different methods in Example 1 on target cells was detected. The specific experimental steps are as follows:

[0075] S1. Adjust the target cells (K562) to the logarithmic growth phase and pass them twice before the experiment.

[0076] S2. Resuspend the target cells in complete medium and adjust the cell density to 5*10 5 / mL, take a new 96-well plate and inoculate the target cells at 100μL / well. Add 100μL of sterile water to each of the unused wells around the 96-well plate to prevent the water in the middle experimental well from evaporating. Place the plate in a 5% CO2 37℃ incubator and culture overnight.

[0077] S3. Collect the NK cells prepared above by centrifugation and resuspend them in serum-free X-VIVO-15 medium. Take out the 96-well plate from the incubator, completely aspirate the medium in the wells, gently wash the cells with sterile PBS, and then add NK cells according to different E / T ratios, and make up the final volume to 100 μL / well. Maxi lysis and Mini lysis are inoculated with the same number of target cells, but without NK cells. Place the well plate in a 5% CO2 37°C incubator and culture for 6 hours.

[0078] S4. After the culture is completed, take the well plate out of the incubator, add the lysis solution in the LDH detection kit to the Maxi lysis wells, completely lyse the target cells, centrifuge the 96-well plate at 1200xg at room temperature for 5 minutes, gently take out the plate, transfer 50μL from each well to another new 96-well plate, add LDH detection reagent, and use an enzyme reader to read the OD value.

[0079] The target cell lysis percentage is calculated as follows:

[0080] lysis % = (OD eachwell -OD minilysis ) / (OD maxilysis )x100%

[0081] Results Figure 3 .from Figure 3 It can be seen that the NK cells prepared by the present invention have a better ability to kill tumor cells K562.

[0082] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for expanding NK cells using NK92 cells as trophoblast cells, characterized in that: The method comprises the following steps: using NK92 cells expressing interleukin 15, interleukin 21 and interleukin 18 as trophoblast cells and peripheral blood mononuclear cells as seed cells to amplify NK cells.

2. The method according to claim 1, characterized in that The amino acid sequence of the interleukin 21 is shown in SEQ ID NO.1, the amino acid sequence of the interleukin 18 is shown in SEQ ID NO.2, and the amino acid sequence of the interleukin 15 is shown in SEQ ID NO.

3.

3. The method according to claim 1, characterized in that The interleukin 15, interleukin 21 and interleukin 18 are connected via a 2A peptide; Preferably, the 2A peptide comprises a T2A peptide; The amino acid sequence of the T2A peptide is shown in SEQ ID NO.

4.

4. The method according to claim 1, characterized in that: The quantitative ratio of the trophoblast cells to the seed cells is 1-10:

1.

5. The method according to claim 1, characterized in that The method specifically comprises the following steps: S1. preparing engineered NK92 cells expressing interleukin 15, interleukin 21 and interleukin 18, and irradiating the engineered NK92 cells to obtain trophoblast cells; S2. Co-culture peripheral blood mononuclear cells and trophoblast cells of S1 to obtain NK cells.

6. The method according to claim 5, characterized in that In step S1, at least one of the following features is included: (1) Preparing NK92 cells expressing interleukin 15, interleukin 21 and interleukin 18 comprises the following steps: Insert the interleukin-15, interleukin-21 and interleukin-18 coding gene sequences linked to the 2A peptide into an expression vector, and transfer the recombinant vector into NK92 cells; (2) The irradiation includes irradiation with one or more of gamma rays, alpha rays, beta rays, and X-rays; (3) The irradiation dose is 20-100 Gy.

7. The method according to claim 5, characterized in that In step S1, at least one of the following features is included: (1) After the co-culture, the step of isolating NK cells is also included; (2) separating the NK cells by surface markers; (3) In step S2, the co-culture is to culture the mixed cells at 35-38° C. and a carbon dioxide concentration of 1%-10% for 10-18 days.

8. NK cells prepared by the method according to any one of claims 1-7.

9. Use of the NK cells according to claim 8 in the preparation of immune drugs.

10. The use according to claim 9, characterized in that: The immune medicine is used to treat tumors, viral infections, autoimmune diseases, post-transplant rejection or graft-versus-host disease.

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