Culture medium for NK cell in-vitro amplification and NK cell in-vitro amplification method

Abstract

According to the NK cell in-vitro amplification culture medium and the NK cell in-vitro amplification method provided by the invention, by innovatively using the composite growth factor, the extracellular matrix protein, the beta-cyclodextrin and the combination of the Anti-NKG2D antibody and the Anti-NKG2D-KIR3DL2 antibody, the proliferation efficiency and the functional activity of the NK cells are remarkably improved. The improvements not only solve the defects in the prior art, but also provide a reliable solution for clinical application and basic research of the NK cells.

Description

Technical Field

[0001] The present invention relates to the field of immune cell culture, and specifically to a culture medium for in vitro expansion of natural killer (NK) cells and a corresponding expansion method. The culture medium is particularly suitable for improving the proliferation ability of NK cells in vitro and optimizing their functional activity, providing support for cell immunotherapy and related research. Background Art

[0002] Natural killer (NK) cells are important immune cells that can recognize and kill tumor cells and virus-infected cells. However, during in vitro culture, NK cells often face problems such as difficulty in expansion and reduced activity due to their stringent requirements for the culture environment. Therefore, the development of a new culture medium and optimized expansion method is of great significance for the research and clinical application of NK cells. Summary of the invention

[0003] The purpose of the present invention is to provide a novel culture medium and method for in vitro expansion of NK cells to improve the proliferation efficiency and cell activity of NK cells. By using the culture medium and method provided by the present invention, the number and function of NK cells can be significantly increased to meet clinical and scientific research needs.

[0004] Therefore, in one aspect, the present invention discloses a culture medium for in vitro expansion of NK cells, the culture medium comprising the following components:

[0005] (1) Basic culture medium: RPMI-1640 culture medium containing 10% fetal bovine serum;

[0006] (2) Growth factors: human recombinant interleukin-2, concentration of 1000 IU / mL; human recombinant interleukin-15, concentration of 10 ng / mL;

[0007] (3) Other additives: L-glutamine, concentration is 2 mM.

[0008] In another aspect, the present invention further discloses a culture medium for in vitro expansion of NK cells, the culture medium comprising the following components:

[0009] (1) Basic culture medium: RPMI-1640 culture medium containing 10% fetal bovine serum;

[0010] (2) Growth factors: human recombinant interleukin-2, concentration of 1000 IU / mL; human recombinant interleukin-15, concentration of 10 ng / mL;

[0011] (3) Extracellular matrix protein: recombinant human fibronectin, concentration of 100 μg / mL;

[0012] (4) Other additives: L-glutamine, concentration is 2 mM.

[0013] In another aspect, the present invention further discloses a culture medium for in vitro expansion of NK cells, the culture medium comprising the following components:

[0014] (1) Basic culture medium: RPMI-1640 culture medium containing 10% fetal bovine serum;

[0015] (2) Growth factors: human recombinant interleukin-2, concentration of 1000 IU / mL; human recombinant interleukin-15, concentration of 10 ng / mL;

[0016] (3) Extracellular matrix protein: recombinant human fibronectin, concentration of 100 μg / mL;

[0017] (4) Other additives: L-glutamine, concentration is 2 mM; β-cyclodextrin, concentration is 0.1%.

[0018] In another aspect, the present invention further discloses a culture medium for in vitro expansion of NK cells, the culture medium comprising the following components:

[0019] (1) Basic culture medium: RPMI-1640 culture medium containing 10% fetal bovine serum;

[0020] (2) Growth factors: human recombinant interleukin-2, concentration of 1000 IU / mL; human recombinant interleukin-15, concentration of 10 ng / mL;

[0021] (3) Special additives: Anti-NKG2D antibody, concentration of 5 μg / mL;

[0022] (4) Extracellular matrix protein: recombinant human fibronectin, concentration of 100 μg / mL;

[0023] (5) Other additives: L-glutamine, concentration is 2 mM; β-cyclodextrin, concentration is 0.1%.

[0024] In another aspect, the present invention further discloses a culture medium for in vitro expansion of NK cells, the culture medium comprising the following components:

[0025] (1) Basic culture medium: RPMI-1640 culture medium containing 10% fetal bovine serum;

[0026] (2) Growth factors: human recombinant interleukin-2, concentration of 1000 IU / mL; human recombinant interleukin-15, concentration of 10 ng / mL;

[0027] (3) Special additives: Anti-NKG2D-KIR3DL2 antibody, concentration of 5 μg / mL;

[0028] (4) Extracellular matrix protein: recombinant human fibronectin, concentration of 100 μg / mL;

[0029] (5) Other additives: L-glutamine, concentration is 2 mM; β-cyclodextrin, concentration is 0.1%.

[0030] Preferably, the amino acid sequences of the heavy chain variable region and the light chain variable region of the Anti-NKG2D-KIR3DL2 antibody of the present invention are shown as SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0031] Preferably, the Anti-NKG2D-KIR3DL2 antibody of the present invention specifically binds to the NKG2D protein at aa99-aa109, and its amino acid sequence is GSILSPNLLTI.

[0032] Preferably, the Anti-NKG2D-KIR3DL2 antibody of the present invention specifically binds to the KIR3DL2 protein at aa510-aa520, and its amino acid sequence is GSFFLYSKLTV.

[0033] On the other hand, the present invention also discloses a use of the culture medium in in vitro expansion of NK cells.

[0034] The present invention proposes for the first time to add recombinant human fibronectin (100 μg / mL) to the NK cell in vitro expansion culture medium to enhance the adhesion and survival rate of NK cells. This innovative component significantly improves the proliferation efficiency and functional activity of NK cells. The present invention also introduces β-cyclodextrin (0.1%) to stabilize the activity of growth factors, ensure that the growth factors can continue to be effective during long-term culture, and solve the problem of growth factor degradation during the culture process. The combined application of the two has achieved good results.

[0035] In addition, NKG2D is an important activating receptor that participates in the recognition and attack of tumor cells by NK cells. Anti-NKG2D antibodies can regulate the recognition of NKG2D ligands by NK cells. The present invention enhances the cytotoxicity of NK cells by adding Anti-NKG2D antibodies to the culture medium. And when Anti-NKG2D-KIR3DL2 antibodies are added, the cytotoxicity of NK cells can be further enhanced; this is mainly because Anti-NKG2D-KIR3DL2 antibodies can also block the binding of KIR3DL2 to MHC class I molecules, thereby relieving the inhibition of NK cells.

[0036] Therefore, the NK cell in vitro expansion medium provided by the present invention has a good culture effect, can not only provide NK cell culture titer, but also enhance the killing effect of NK cells. Therefore, the culture medium has a wider range of uses and can be used for in vitro expansion of NK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Anti-NKG2D-KIR3DL2 antibody SDS-PAGE detection results.

[0038] Figure 2 Western blot detection results of NKG2D protein and KIR3DL2 protein using Anti-NKG2D-KIR3DL2 antibody. DETAILED DESCRIPTION

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0040] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0041] Example 1: Preparation of culture medium for in vitro expansion of NK cells

[0042] (1) Basic culture medium: RPMI-1640 culture medium containing 10% fetal bovine serum;

[0043] (2) Growth factors: human recombinant interleukin-2, concentration of 1000 IU / mL; human recombinant interleukin-15, concentration of 10 ng / mL;

[0044] (3) Other additives: L-glutamine, concentration is 2 mM.

[0045] After stirring and mixing, sterilize with a 220 nm membrane and store at 4°C for later use.

[0046] Example 2: Preparation of culture medium for in vitro expansion of NK cells

[0047] (1) Basic culture medium: RPMI-1640 culture medium containing 10% fetal bovine serum;

[0048] (2) Growth factors: human recombinant interleukin-2, concentration of 1000 IU / mL; human recombinant interleukin-15, concentration of 10 ng / mL;

[0049] (3) Extracellular matrix protein: recombinant human fibronectin, concentration of 100 μg / mL;

[0050] (4) Other additives: L-glutamine, concentration is 2 mM.

[0051] After stirring and mixing, sterilize with a 220 nm membrane and store at 4°C for later use.

[0052] Recombinant human fibronectin was from Zhihetai Ke, RP10015.

[0053] Example 3: Preparation of culture medium for in vitro expansion of NK cells

[0054] (1) Basic culture medium: RPMI-1640 culture medium containing 10% fetal bovine serum;

[0055] (2) Growth factors: human recombinant interleukin-2, concentration of 1000 IU / mL; human recombinant interleukin-15, concentration of 10 ng / mL;

[0056] (3) Extracellular matrix protein: recombinant human fibronectin, concentration of 100 μg / mL;

[0057] (4) Other additives: L-glutamine, concentration is 2 mM; β-cyclodextrin, concentration is 0.1%.

[0058] After stirring and mixing, sterilize with a 220 nm membrane and store at 4°C for later use.

[0059] It should be specially noted here that this study also cultured NK cells in a culture medium with only β-cyclodextrin added but not recombinant human fibronectin, and found that when only β-cyclodextrin was added, the proliferation of NK cells was slightly better than that of the culture medium of Example 1 (compared with the proliferation rate of 55.6% of the culture medium of Example 1, the proliferation rate of NK cells was 65.6%), but far less effective than the combination of the two (see Example 6 of the present invention).

[0060] Example 4: Preparation of culture medium for in vitro expansion of NK cells

[0061] (1) Basic culture medium: RPMI-1640 culture medium containing 10% fetal bovine serum;

[0062] (2) Growth factors: human recombinant interleukin-2, concentration of 1000 IU / mL; human recombinant interleukin-15, concentration of 10 ng / mL;

[0063] (3) Special additives: Anti-NKG2D antibody, concentration of 5 μg / mL;

[0064] (4) Extracellular matrix protein: recombinant human fibronectin, concentration of 100 μg / mL;

[0065] (5) Other additives: L-glutamine, concentration is 2 mM; β-cyclodextrin, concentration is 0.1%.

[0066] After stirring and mixing, sterilize with a 220 nm membrane and store at 4°C for later use.

[0067] Anti-NKG2D antibody was from abcam (ab302907).

[0068] Example 5: Preparation of culture medium for in vitro expansion of NK cells

[0069] (1) Basic culture medium: RPMI-1640 culture medium containing 10% fetal bovine serum;

[0070] (2) Growth factors: human recombinant interleukin-2, concentration of 1000 IU / mL; human recombinant interleukin-15, concentration of 10 ng / mL;

[0071] (3) Special additives: Anti-NKG2D-KIR3DL2 antibody (see Example 7 for details), with a concentration of 5 μg / mL;

[0072] (4) Extracellular matrix protein: recombinant human fibronectin, concentration of 100 μg / mL;

[0073] (5) Other additives: L-glutamine, concentration is 2 mM; β-cyclodextrin, concentration is 0.1%.

[0074] After stirring and mixing, sterilize with a 220 nm membrane and store at 4°C for later use.

[0075] Example 6: Cultivation and detection of NK cells

[0076] (1) NK cells were isolated from the peripheral blood of healthy volunteers (with their consent and in compliance with ethical requirements) and purified using Ficoll density gradient centrifugation and magnetic bead separation.

[0077] (2) NK cells were inoculated into the culture media of Examples 1 to 5 at a concentration of 1×10^6 cells / mL, and cultured in an incubator at 37° C. and 5% CO 2 .

[0078] (3) The culture media prepared in Examples 1 to 5 were replaced every 3 days.

[0079] (4) On the 7th day, samples were taken for analysis to detect their proliferation efficiency and killing activity.

[0080] Proliferation efficiency detection: After the culture is completed, NK cells are collected and washed twice with PBS. Antibodies (such as CD56-PE, CD16-APC, etc.) are added to label the surface antigens of NK cells and incubated for 30 minutes at 4°C in the dark. Wash the cells twice with PBS to remove free antibodies. Use flow cytometry to detect cell proliferation. Use flow cytometer software (such as FlowJo or FCS Express) to analyze the data.

[0081] The results show (as shown in Table 1) that the proliferation efficiency of the culture medium prepared in Examples 3 to 5 is the highest, and there is no significant difference, followed by Example 2, and Example 1 is the worst. In addition, the proliferation rates of the culture media of Examples 2 to 5 are significantly different from those of Example 1.

[0082] Table 1 Cell proliferation detection results (proliferation rate %)

[0083]

[0084]

[0085] Killing activity detection: The specific killing activity of the five NK cells cultured above on K562 cells was detected using the LDH release method. The results showed (as shown in Table 2) that the cells cultured in the medium of Example 5 had the best effect, followed by the medium of Example 4, then Examples 3 and 2, and the worst was Example 1. Compared with the killing activity of Example 1, Examples 2 to 5 all had significant differences.

[0086] Table 2 Detection results of NK cell killing activity

[0087]

[0088] Example 7: Preparation and testing of Anti-NKG2D-KIR3DL2 antibody

[0089] (1) Preparation of Anti-NKG2D-KIR3DL2 Antibody

[0090] By comparing the amino acid sequences of NKG2D protein (SEQ ID NO.1) and KIR3DL2 protein (SEQ ID NO.2), two peptides were designed and synthesized (NKG2D protein aa99-aa109: GSILSPNLLTI, KIR3DL2 protein aa510-aa520: GSFFLYSKLTV). The two peptides were conjugated to KLH respectively using a classic conjugation method (such as the succinimide method). The conjugation ratio is generally 1:1 for peptide:KLH. After the two peptide-KLH conjugates were mixed in equal proportions and mixed with complete Freund's adjuvant in a ratio of 1:1, they were subcutaneously injected into mice, with 50 μg of conjugate injected into each mouse. Each time, the peptide-KLH conjugate and incomplete Freund's adjuvant were injected subcutaneously at intervals of 2-3 weeks, and the subcutaneous injection was performed in the same way. A total of 4 immunizations were performed. Two weeks after the completion of the immunization, serum was collected to detect the antibody titer. The specific antibody titers of antiserum against peptides were detected by ELISA of NKG2D protein and KIR3DL2 protein, and mice with high titers in both groups were selected for cell fusion.

[0091] Take spleen cells: 3 days after immunization, take the spleen of the mouse and prepare spleen cell suspension using a cell screen. Mix spleen cells and myeloma cells in a ratio of 10:1, and use PEG solution (37°C, 40%) for cell fusion. After dilution, add HAT selective medium and incubate at 37°C, 5% CO2, avoiding light. After 7-10 days, select the grown hybridoma cells and screen the cell lines that produce specific anti-peptide antibodies by ELISA. The positive hybridoma cells screened were subjected to ELISA to verify the polypeptide binding activity of the antibody. The positive cells were cloned by limiting dilution to obtain a stable monoclonal cell line. The monoclonal antibody was purified using a protein A / G column. After purification, a total of one Anti-NKG2D-KIR3DL2 antibody was harvested, filtered through a 220nm filter, and stored at -80°C for later use.

[0092] (2) Testing of Anti-NKG2D-KIR3DL2 Antibodies

[0093] ① The concentration of the purified antibody was tested using BCA, and the concentration was 3.15 mg / ml. The expression yield of the antibody in ascites was converted to 2.5 mg / ml after purification, indicating that the antibody has a high expression yield, which provides a good foundation for its large-scale application.

[0094] ② Use SDS-PAGE to detect the purity of the antibody. The results are as follows Figure 1 As shown, its SDS-PAGE purity can reach more than 95%.

[0095] ③Use this antibody to perform western blot detection on NKG2D protein and KIR3DL2 protein. The results are as follows Figure 2 As shown, the antibody can specifically bind to both NKG2D protein and KIR3DL2 protein, indicating that the antibody has good specificity and sensitivity.

[0096] ④ The amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody were detected. The results showed that the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody were shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The specific detection method is briefly described as follows:

[0097] Total RNA was extracted from mouse hybridoma cells secreting monoclonal antibodies to ensure that the RNA purity and quality met the requirements. The extracted RNA was reverse transcribed into cDNA using reverse transcriptase and used as a template for PCR amplification. Specific primers were designed for the variable regions of the heavy chain (VH) and light chain (VL) of mouse antibodies and used for PCR amplification, respectively. The VH and VL regions were amplified using the designed primers. The PCR reaction conditions included 95°C pre-denaturation, 58°C annealing, and 72°C extension for a total of 35 cycles. The PCR products were detected by 1.5% agarose gel electrophoresis, and the expected amplification product sizes of the heavy chain and light chain were approximately 350 bp and 320 bp, respectively. The purified PCR products were sent for Sanger sequencing to obtain the sequences of VH and VL. The sequences were compared and analyzed using the IgBLAST or IMGT database to confirm the variable region sequences of the heavy chain and light chain.

[0098] Heavy chain variable region (VH) primers:

[0099] Forward primer: 5'-SARGTNMAGCTGSAGSAGTC-3',

[0100] Reverse primer: 5′-GGGGAGGAGACGGTGACTGAGG-3′.

[0101] Light chain variable region (VL) primers:

[0102] Forward primer: 5'-CAGTCTGGTGGAGGTGG-3',

[0103] Reverse primer: 5′-GCTCAGCTTGGTCCCAG-3′.

[0104] ⑤ ELISA was used to detect the binding activity of the antibody and the commercial antibody, and the results are shown in Table 3. The antibody has better binding activity than the commercial monoclonal antibody. The specific method is briefly described as follows:

[0105] Dilute NKG2D protein (ab281815) and KIR3DL2 protein (ab223025) to 1 μg / mL with carbonate buffer for coating. Add 100 μL of antigen solution to each well and incubate at 4°C overnight. The next day, wash the ELISA plate 3-5 times with PBST, and let it stand for 1-2 minutes each time. Add 200 μL of PBST solution containing 1% BSA to each well and incubate at 37°C for 2 hours. Dilute the monoclonal antibodies to be tested to 1 mg / ml, and then dilute them 10,000 times and 20,000 times, add 100 μL to each well, and incubate at 37°C for 1 hour. Repeat the plate washing step and wash 3-5 times. Dilute the HRP-labeled secondary antibody to an appropriate concentration (such as 1:5000), add 100 μL to each well, and incubate at 37°C for 0.5 hours. Wash again 3-5 times. Add 100 μL of TMB substrate solution to each well and incubate at 37°C for 10 minutes in the dark. Add 50 μL of 2M sulfuric acid to each well to terminate the color reaction, and the color changes from blue to yellow. Use an enzyme reader to read the OD value at a wavelength of 450 nm.

[0106] Table 3 Anti-NKG2D-KIR3DL2 antibody activity detection results (OD450nm)

[0107]

[0108] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A culture medium for in vitro expansion of NK cells, characterized in that: The culture medium comprises the following components: (1) Basic culture medium: RPMI-1640 culture medium containing 10% fetal bovine serum; (2) Growth factors: human recombinant interleukin-2, concentration of 1000 IU / mL; human recombinant interleukin-15, concentration of 10 ng / mL; (3) Other additives: L-glutamine, concentration is 2 mM.

2. A culture medium for in vitro expansion of NK cells, characterized in that: The culture medium comprises the following components: (1) Basic culture medium: RPMI-1640 culture medium containing 10% fetal bovine serum; (2) Growth factors: human recombinant interleukin-2, concentration of 1000 IU / mL; human recombinant interleukin-15, concentration of 10 ng / mL; (3) Extracellular matrix protein: recombinant human fibronectin, concentration of 100 μg / mL; (4) Other additives: L-glutamine, concentration is 2 mM.

3. A culture medium for in vitro expansion of NK cells, characterized in that: The culture medium comprises the following components: (1) Basic culture medium: RPMI-1640 culture medium containing 10% fetal bovine serum; (2) Growth factors: human recombinant interleukin-2, concentration of 1000 IU / mL; human recombinant interleukin-15, concentration of 10 ng / mL; (3) Extracellular matrix protein: recombinant human fibronectin, concentration of 100 μg / mL; (4) Other additives: L-glutamine, concentration is 2 mM; β-cyclodextrin, concentration is 0.1%.

4. A culture medium for in vitro expansion of NK cells, characterized in that: The culture medium comprises the following components: (1) Basic culture medium: RPMI-1640 culture medium containing 10% fetal bovine serum; (2) Growth factors: human recombinant interleukin-2, concentration of 1000 IU / mL; human recombinant interleukin-15, concentration of 10 ng / mL; (3) Special additives: Anti-NKG2D antibody, concentration of 5 μg / mL; (4) Extracellular matrix protein: recombinant human fibronectin, concentration of 100 μg / mL; (5) Other additives: L-glutamine, concentration is 2 mM; β-cyclodextrin, concentration is 0.1%.

5. A culture medium for in vitro expansion of NK cells, characterized in that: The culture medium comprises the following components: (1) Basic culture medium: RPMI-1640 culture medium containing 10% fetal bovine serum; (2) Growth factors: human recombinant interleukin-2, concentration of 1000 IU / mL; human recombinant interleukin-15, concentration of 10 ng / mL; (3) Special additives: Anti-NKG2D-KIR3DL2 antibody, concentration of 5 μg / mL; (4) Extracellular matrix protein: recombinant human fibronectin, concentration of 100 μg / mL; (5) Other additives: L-glutamine, concentration is 2 mM; β-cyclodextrin, concentration is 0.1%.

6. The culture medium according to claim 5, characterized in that The amino acid sequences of the heavy chain variable region and the light chain variable region of the Anti-NKG2D-KIR3DL2 antibody are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

7. The culture medium according to claim 5, characterized in that The Anti-NKG2D-KIR3DL2 antibody specifically binds to the NKG2D protein at aa99-aa109, and its amino acid sequence is GSILSPNLLTI.

8. The culture medium according to claim 5, characterized in that The Anti-NKG2D-KIR3DL2 antibody specifically binds to the KIR3DL2 protein at aa510-aa520, and its amino acid sequence is GSFFLYSKLTV.

9. Use of the culture medium as claimed in any one of claims 1 to 5 in the in vitro expansion of NK cells.

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