CN111172115B - Method for efficiently amplifying NK cells in vitro - Google Patents

Abstract

The invention provides a method for amplifying NK cells by using gene-modified K562 trophoblasts, which comprises the steps of transfecting the K562 cells by using an IL-21, IL-27 and CD70 gene by using a lentivirus transfer system to obtain K562 engineering cells stably expressing IL-21, IL-27 and CD70 proteins, inactivating the cells, mixing the cells with PBMC (peripheral blood mononuclear cell) for culture, amplifying the NK cells in a serum-free culture medium in the presence of low-concentration IL-2, wherein the amplification multiple is about 800-1100, the effective purity of the NK cells reaches 91%, the secretion of IFN-gamma is 5 times of that of the NK cells cultured by a common method, and the killing rate of the NK cells to hepatoma cell HepG2 reaches 88.5% under the condition of an effective target ratio of 10.

Description

Method for efficiently amplifying NK cells in vitro

Technical Field

The invention belongs to the field of cell amplification, and particularly relates to a method for efficiently amplifying NK cells in vitro.

Background

Natural killer cells (NK cells) are a distinct lymphocyte population distinct from T, B cells. NK cells kill target cells with a rapid effect (immediate effect), without pre-sensitization, within 4 hours after mixing with target cells.

NK cells can not only directly kill target cells without Major Histocompatibility Complex (MHC) restriction, but also interfere virus replication and further activate phagocytes by secreting cytokines such as IFN-gamma and TNF-alpha, and can also kill tumor cells coated with specific IgG through antibody-dependent cell-mediated cytotoxicity (ADCC).

Therefore, NK cells have very important roles in anti-infection and anti-tumor therapy. However, NK cells, a rare cell subset among lymphocytes, account for only 5% -15% of human peripheral blood lymphocytes, and therefore need to be expanded in vitro and then reinfused into vivo. The clinical application of in vitro expanded NK cells has a problem of how to obtain a large amount of high-purity NK cells that can kill tumor cells.

Dunne J et al stimulate peripheral blood lymphocytes or cells isolated with magnetic beads to proliferate by IL-2 or IL-15. According to literature reports, the proliferation is about ten times, so that a large amount of Peripheral Blood Mononuclear Cells (PBMCs) need to be prepared. In addition, it is reported that NK cell culture is performed by a method of adding cell factors (IL-21, IL-15 and the like) in vitro, but the amplification capacity is only 200-500 times, the purity of NK cells is not stable, and the purity is distributed in 40% -80%, and the clinical requirement is difficult to meet. Imai C et al, cultured K562 cells and PBMC that integrated 4-1BBL gene and IL-21 gene, and obtained NK cells with greatly increased number and purity, but limited killing ability.

Disclosure of Invention

Aiming at the problems of lack of a method for efficiently amplifying NK cells and low killing capability at present, the invention provides a method for amplifying the NK cells by using genetically modified K562 trophoblasts, which can efficiently obtain the NK cells and has high purity and strong killing capability.

In order to achieve the purpose, the invention adopts the following technical scheme.

A K562 engineered cell expressing IL-21, IL-27 and CD70 proteins, wherein the IL-21, IL-27 and CD70 proteins are all expressed on the surface of the cell membrane.

A preparation method of the K562 engineering cell comprises the following steps:

and transfecting K562 cells by using a virus vector carrying IL-21, IL-27 and CD70 genes to obtain K562 engineering cells simultaneously expressing IL-21, IL-27 and CD70 proteins.

And signal peptides are added to the 5 'ends of the IL-21 and IL-27 gene sequences, and transmembrane region genes are added to the 3' ends of the IL-21 and IL-27 gene sequences.

The signal peptide is selected from signal peptides of OSM, VSV-G, trypsinogen-2, IL-2 and CD 8; preferably a CD8 signal peptide.

The transmembrane region is selected from the transmembrane regions of CD3epsilon, CD4, CD8, CD9, CD16, CD22, CD33, CD137, CTLA-4, PD-1 or LAG-3; preferably the CD8 transmembrane domain.

The virus vector can be adenovirus and lentivirus; preferably a lentivirus.

A method for amplifying NK cells by using the K562 engineering cells comprises the following steps:

(1) Irradiating and inactivating the K562 engineering cells to obtain K562 trophoblasts;

(2) Isolating and extracting mononuclear cells (PBMCs) in blood;

(3) Adding K562 feeder cells and IL-2 into mononuclear cells, culturing, adding K562 feeder cells again on day 7, culturing for 15 days, and collecting cells.

In the step (1), the inactivation step is as follows: the cells were irradiated with 100Gy dose of radiation for 20-30min.

In the step (2), the blood may be peripheral blood or umbilical cord blood.

In the step (3), the concentration of the mononuclear cell is 1.5X 10 ⁶ /mL。

In the step (3), the final concentration of the IL-2 is 200U/mL.

In the step (3), the number ratio of the K562 feeder cells to the mononuclear cells is 1:2-5 after the first addition, and the number ratio of the K562 feeder cells to the initial mononuclear cells is 1-10 after the second addition.

In the step (3), the culture conditions are 37 ℃ and 5% CO ₂ 100% humidity.

The invention has the following advantages:

the K562 nourishing cell membrane surface expressed protein obtained by the invention is closely related to the activation, proliferation and killing functions of NK cells: IL-21 is a traditional NK cell culture factor and can promote the proliferation and differentiation of NK cells and the production of IFN-gamma. IL-27 can induce cells to express IL-12R, cooperate with IL-12 to induce cells to produce IFN-gamma, and also can directly promote NK cells to secrete IFN-gamma. CD70 is a ligand of CD27, CD27 is expressed on the surface of NK cells to induce the activation of NK cells and the generation of IFN-gamma through the interaction with the ligand, and the NK cytotoxic activity can be enhanced through the induction of the generation of IFN-gamma.

The invention constructs the gene modified K562 trophoblast by a molecular cloning method, so that the gene modified K562 trophoblast can express IL-21, IL-27 and CD70 on the surface of a cell membrane simultaneously. The cell is mixed with PBMC for culture after being inactivated, the NK cell can be amplified in a serum-free culture medium in the presence of low-concentration IL-2, the amplification multiple is about 800-1100, the purity of the NK cell reaches 91%, the secretion of IFN-gamma is 5 times of that of the NK cell cultured by a common method, and the killing rate of the NK cell to hepatoma cell HepG2 reaches 88.5% under the condition of a target ratio of 10. Moreover, the serum in the serum-containing medium is mostly from bovine serum, has relatively complex components, is easy to introduce bovine pathogens, and is easy to cause immune response if bovine protein is adhered to the cell surface and is returned to the human body.

Drawings

FIG. 1 is a schematic structural diagram of the surface expression of IL-21, IL-27 and CD70 of a genetically modified K562 engineering cell;

FIG. 2 shows the expression of IL-21 (A), IL-27 (B) and CD70 (C) on the cell surface of the genetically modified K562 engineered cell;

FIG. 3 is a NK cell proliferation curve, wherein NK1 is NK cells cultured by the method of the present invention, and NK2 is NK cells cultured by the classical method;

FIG. 4 shows NK cell surface marker CD3 ^- 、CD16 ⁺ 、CD56 ⁺ Analyzing;

FIG. 5 shows the killing of the NK cells to hepatoma cell HepG2, wherein NK1 is the NK cells cultured by the method of the present invention, and NK2 is the NK cells cultured by the classical method;

FIG. 6 shows the secretion of NK cell cytokine IFN-. Gamma.in which NK1 is NK cell cultured by the method of the present invention and NK2 is NK cell cultured by the classical method.

Detailed Description

The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.

EXAMPLE 1 construction of genetically modified K562 trophoblasts

(1) Construction of lentiviral vectors: the target gene is synthesized according to the sequence of CD8 asp-IL 21-CD 8 TM-T2A-CD 8 asp-IL 27-CD 8 TM-IRES-CD 70, the sequence is shown in SEQ ID NO:1, and a SpeI enzyme cutting site (actagt) is added at the 5 'end and a NotI enzyme cutting site (gcggccgc) is added at the 3' end. After double enzyme digestion, the double enzyme digested lentivirus backbone plasmid (pHR-mCheery) is transferred to obtain the lentivirus backbone plasmid containing IL-21, IL-27 and CD70 genes. The 5 'ends of IL-21 and IL-27 sequences are respectively added with a signal peptide (MALPVTALLLPLALLLHAARP) gene of CD8, and the 3' ends are respectively added with a transmembrane region gene of CD8, so that the two proteins can be anchored on a cell membrane; CD70, a transmembrane protein per se, can be anchored to the cell membrane;

(2) The backbone plasmid and packaging plasmid were mixed at 0.89 (mass ratio) to 0.11, and cotransfected to 293FT cells, and the supernatant containing the viral particles was collected 48 hours after transfection;

(3) Infecting K562 cells (purchased from Kunming cell Bank of Chinese academy) in logarithmic growth phase with virus, and screening monoclonal cells simultaneously expressing IL-21, IL-27 and CD70 proteins as shown in FIG. 1 according to limiting dilution method;

(4) And performing amplification culture on the screened monoclonal antibody clone cells, and then identifying the stability of the expressed IL-21, IL-27 and CD70 proteins. The expression levels of IL-21, IL-27 and CD70 on the surface of the engineering cells cultured for 14 days are 90.1%, 88.0% and 59.6% respectively, as shown in FIG. 2;

(5) Irradiating cells with 100Gy dose of radioactive rays for 20-30min to inactivate the cells;

(6) The inactivated cells are washed by centrifugation for 1 time, and added with cell freezing medium according to the proportion of 1 multiplied by 10 ⁷ Perml and 4X 10 ⁷ and/mL 2 specifications are subpackaged and frozen to obtain the genetically modified K562 trophoblast for culturing NK cells.

Example 2 culture and characterization of NK cells

(1) Collecting fresh blood 30-50mL from healthy volunteers on day 0, separating Peripheral Blood Mononuclear Cells (PBMC) with lymphocyte separation solution, washing for 2 times, and collecting 4 × 10 ⁷ The cell density was adjusted to 1.5X 10 by using a culture medium (551-H3, TAKARA) for each cell ⁶ /mL；

(2) 1X 10 portions were added in proportion to the amount of PBMC 1:4 ⁷ K562 feeder cells/mL and IL-2 at a final concentration of 200U/mL (spring harbor); 37 ℃ and 5% of CO ₂ Culturing cells under the condition of 100% humidity, and observing the cell state to replenish liquid in time; on day 7, 4 × 10 was added in proportion to the starting PBMC number 1 ⁷ the/mL K562 feeder cells are used for continuously activating NK cells and are subjected to bagging operation; culturing until day 15, and collecting cells NK1;

with reference to Zhang H. (Activating signaling inhibition signaling in CD137L/IL-15 activated natural killer cells J Immunother 2011 (34): 187-195), NK cells (NK 2) obtained by co-culturing classical K562 engineered cells expressing IL-21 and CD137L on a membrane with PBMC cells were constructed as a control.

The cell proliferation curve is shown in figure 3, the effect of K562 cells expressing IL-21, IL-27 and CD70 on the membrane surface on promoting the growth of NK is obviously stronger than that of classical K562 engineering cells, the NK cells enter a logarithmic growth phase on day 7, and the cell proliferation multiple reaches 1156 times when the culture is finished on day 15; and the classical K562 engineering cells enter a logarithmic growth phase on day 7, and the proliferation multiple is 562 times at the end of the 15 th culture. Flow analysis of NK cells at the end of culture, as shown in FIG. 4-a, CD3 ^- CD16 ⁺ CD56 ⁺ The proportion of cells was 91.1%, and FIG. 4-b is CD3 cells from NK after coculture with classical K562 cells ^- CD16 ⁺ CD56 ⁺ The proportion of cells was 64.5%.

Example 3 in vitro killing assay of NK cells against tumor cell lines

Mixing HepG2 cells (hepatoma cell lines) were seeded individually on xCELLigence RTCA S16 cell proliferation plates (ACEA) at 1X 10 cells per well ⁵ Respectively, placing the plate in a cell culture chamber (37 deg.C 5% ₂ ) The cultured NK cells are inoculated and cultured for 14 days according to the following steps of E: T =1:1, 3:1, 6:1 and 10. The plate was returned to the RTCA Station, incubated for 24 hours, and 100. Mu.L of supernatant was aspirated from each well and stored at-80 ℃ for future use. The results are shown in fig. 5, and the NK1 cells specifically killed HepG2 cells, with killing rates of 1:1, 3:1, 6:1, and 10 averaging 21.5%, 47.5%, 68.5%, and 88.5% when the effective target ratios were 1:1, 3:1, 6:1, and 10. The killing rate increases with increasing effective target ratio. The specific killing ratio of NK2 in the control group is 15.1%, 30.5%, 49.8% and 70.2%, and the two groups are paired for t test P<0.05, with differences.

Example 4 measurement of secreted factor content in NK cell culture supernatant

Taking the supernatant fluid left by the in vitro killing test, detecting the content of the secretion factor by an ELISA method according to the instruction (human IFN-gamma ELISA KIT, biolegend Cat.: 430104): 1. Add blocking solution 200. Mu.L/well, block for 1 hour at room temperature, wash plate 3 times. The standard and the sample to be tested are diluted, 100 mul of the diluted standard and the sample to be tested are added into each well, incubated for 2 hours at room temperature, and the plate is washed for 3 times. The diluted detection antibody was added at 100. Mu.L/well, incubated at room temperature for 1 hour, and the plate was washed 3 times. Diluted HRP was added at 100. Mu.L/well, incubated at room temperature for 30min, and the plate was washed 4 times. Adding prepared color developing solution into 100 mu L/hole, and incubating for 30min at room temperature. Stop solution was added at 100. Mu.L/well to terminate the reaction. Absorbance at 450nm was measured in each well.

The results are shown in FIG. 6, and the average secretion amounts of IFN-. Gamma.are 0.77ng/mL, 3.02ng/mL, 4.81 ng/mL, 6.66 ng/mL for NK1 group effective target ratios of 1:1, 3:1, 6:1 and 10; the average secretion of IFN-gamma of 1:1, 3:1, 6:1 and 10 for the control NK2 group effective target ratios were 0.17ng/mL, 0.61ng/mL, 0.96 ng/mL, 1.37ng/mL; NK cells (NK 1) cultured by K562 feeder cells obtained in example 1 secrete a great amount of IFN-gamma cytokines to kill cells after stimulation, the secretion amount is far larger than that of NK cells (NK 2) cultured by classical K562 engineering cells, and the concentration of IFN-gamma is in positive correlation with the effect-target ratio.

Sequence listing

<110> Qilu cell therapy engineering Co., ltd, shandong province

<120> method for efficiently amplifying NK cells in vitro

<130> 20200109

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 2952

<212> DNA

<213> Artificial Sequence

<220>

<223> IL 21-IL 27-CD70

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ctgaaaaatt atgtgaatga cttggtccct gaatttctgc cagctccaga agatgtagag 180

acaaactgtg agtggtcagc tttttcctgc tttcagaagg cccaactaaa gtcagcaaat 240

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tccacaaatg cagggagaag acagaaacac agactaacat gcccttcatg tgattcttat 360

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cgaccaccaa caccggcgcc caccatcgcg tcgcagcccc tgtccctgcg cccagaggcg 540

tgccggccag cggcgggggg cgcagtgcac acgagggggc tggacttcgc ctgtgatatc 600

tacatctggg cgcccttggc cgggacttgt ggggtccttc tcctgtcact ggttatcacc 660

ctttactgct gagccgaggg aaggggttct ctcctgacct gcggcgatgt ggaggagaac 720

cccggaccca tggccttacc agtgaccgcc ttgctcctgc cgctggcctt gctgctccac 780

gccgccaggc cgatgggcca gacggcaggc gaccttggct ggcggctcag cctgttgctg 840

cttcccttgc tcctggttca agctggtgtc tggggattcc caaggccccc agggaggccc 900

cagctgagcc tgcaggagct gcggagggag ttcacagtca gcctgcatct cgccaggaag 960

ctgctctccg aggttcgggg ccaggcccac cgctttgcgg aatctcacct gccaggagtg 1020

aacctgtacc tcctgcccct gggagagcag ctccctgatg tttccctgac cttccaggcc 1080

tggcgccgcc tctctgaccc ggagcgtctc tgcttcatct ccaccacgct tcagcccttc 1140

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gtacacatcc aggtgacgct ggccatctgc tcctccacga cggcctccag gcaccacccc 2700

accaccctgg ccgtgggaat ctgctctccc gcctcccgta gcatcagcct gctgcgtctc 2760

agcttccacc aagggctttt tggattttgg aactggggac tcaaagtcaa gtgcttctta 2820

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gccctacaaa gctggaggaa tcatcattgt tcccatttca cagatgagga aaacagaggc 2940

gtaaaccgtt ga 2952

Claims (9)

1. A preparation method of K562 engineering cells for expressing IL-21, IL-27 and CD70 proteins is characterized by comprising the following steps:

transfecting K562 cells by using a virus vector carrying IL-21, IL-27 and CD70 genes to obtain K562 engineering cells for simultaneously expressing IL-21, IL-27 and CD70 proteins;

the 5 'ends of the IL-21 and IL-27 gene sequences are both added with signal peptides, and the 3' ends are both added with transmembrane region genes; the signal peptide is a CD8 signal peptide; the transmembrane region is a CD8 transmembrane region.

2. The method of claim 1, wherein the viral vector is selected from the group consisting of adenovirus and lentivirus.

3. The method according to claim 1, wherein the viral vector is a lentivirus.

4. A K562 engineered cell expressing IL-21, IL-27 and CD70 proteins obtained by the production method according to any one of claims 1 to 3.

5. A method for expanding NK cells by using the K562 engineered cell of claim 4, comprising the steps of:

(1) Irradiating and inactivating the K562 engineering cells to obtain K562 trophoblasts;

(2) Separating and extracting mononuclear cells from blood;

(3) Adding K562 feeder cells and IL-2 into mononuclear cells, culturing, adding K562 feeder cells again on day 7, culturing for 15 days, and collecting cells.

6. The method according to claim 5, wherein the concentration of the mononuclear cells in the step (3) is 1.5X 10 ⁶ /mL。

7. The method according to claim 5, wherein in step (3), the final concentration of IL-2 is 200U/mL.

8. The method according to claim 5, wherein in step (3), the number ratio of the first addition of K562 feeder cells to mononuclear cells is 1:2-5, and the second addition of K562 feeder cells to the initial mononuclear cells is 1.

9. The method according to claim 5, wherein in step (1), the inactivation step is: irradiating cells with 100Gy radiation for 20-30min;

in the step (2), the blood can be peripheral blood or umbilical cord blood;

in the step (3), the culture conditions are 37 ℃ and 5% CO ₂ 100% humidity.

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