Preparation method of natural killer cells and application of natural killer cells in disease treatment

Abstract

According to the method for efficiently preparing the natural killer cells, through combined application of optimized three-dimensional scaffold culture and tumor antigens, the amplification efficiency and killing activity of the NK cells are remarkably improved. The method is applied to treatment of diseases such as tumors and virus infection, and has remarkable clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of immune cell therapy, and in particular to a method for preparing natural killer cells and applications thereof in treating diseases such as tumors and viral infections. Background Art

[0002] Natural killer cells (NK cells) originate 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. NK cells are different from T and B cells. They are a type of lymphocyte that can non-specifically kill tumor cells and virus-infected cells without prior sensitization.

[0003] The exact origin of NK cells is still unclear. It is generally believed that they are directly derived from the bone marrow, and their development and maturation depend on the microenvironment of the bone marrow. In vitro experiments on mice and humans have shown that thymocytes can also induce NK cells when cultured in the presence of cytokines such as IL-2 in vitro. Mouse spleen can promote the differentiation of NK cells under the induction of IL-3 in vivo. NK cells are mainly distributed in peripheral blood, accounting for 5-10% of PBMC. There is also NK activity in lymph nodes and bone marrow, but the level is lower than that in peripheral blood. Since NK cells have some T cell differentiation antigens, such as 80-90% NK cells CD2+, 20-30% NK cells CD3+ (expressing CD3ζ chain), 30% NK cells CD8+ (α / α) and 75-90% NK cells CD38+, and NK cells have IL-2 medium affinity receptors, they can undergo a proliferation reaction under IL-2 stimulation, and activated NK cells can produce IFN-γ. Therefore, it is generally believed that NK cells and T cells are more closely related in development.

[0004] Since the killing activity of NK cells is not restricted by MHC and does not rely on antibodies, it is called natural killer activity. The cytoplasm of NK cells is rich and contains large azure granules. The content of granules is positively correlated with the killing activity of NK cells. The target cells of NK cells mainly include certain tumor cells (including some cell lines), virus-infected cells, certain self-tissue cells (such as blood cells), parasites, etc. Therefore, NK cells are important immune factors for the body's anti-tumor and anti-infection, and are also involved in type II hypersensitivity reactions and graft-versus-host reactions.

[0005] NK cells have become an important means of immunotherapy due to their unique anti-tumor and anti-viral properties. However, existing NK cell preparation technologies have problems such as low cell amplification efficiency and unstable activity, which limit their clinical application. The present invention provides an improved NK cell preparation method, which significantly improves the activity and amplification efficiency of NK cells and enhances their application effect in treating diseases such as tumors and viral infections. Summary of the invention

[0006] The present invention aims to provide a method for efficiently preparing NK cells, which significantly improves the proliferation rate and killing activity of NK cells by optimizing culture conditions and adding specific stimulating factors. The method can be widely used in the treatment of tumors, viral infections and autoimmune diseases, and has obvious innovation and clinical application value.

[0007] Therefore, the present invention discloses, on one hand, a monoclonal antibody against CD244 and CD137, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0008] In another aspect, the present invention further discloses a method for preparing NK cells, the method comprising the following steps:

[0009] (1) Isolation of PBMCs: PBMCs were isolated from peripheral blood using Ficoll-Paque density gradient centrifugation;

[0010] (2) Enrichment of NK cells: enriching CD56+ NK cells from the PBMCs separated in step (1) using an immunomagnetic bead method;

[0011] (3) NK cell expansion and activation: The NK cells enriched in step (2) are inoculated into a three-dimensional scaffold culture system at 5×105 cells / mL, and cultured in a serum-free medium containing the anti-CD244 and anti-CD137 monoclonal antibodies described in claim 1. 50% of the medium is replaced every 2 days. After continuous culture for 7 days, the NK cells can be collected.

[0012] Preferably, the three-dimensional scaffold in step (3) of the present invention is a disc with a diameter of 10 mm.

[0013] Preferably, the porosity of the three-dimensional scaffold in step (3) of the present invention is 90%, and the pore size is 50-100 μm.

[0014] Preferably, the compression modulus of the three-dimensional scaffold in step (3) of the present invention is 200 kPa.

[0015] Preferably, the formula of the serum-free medium in step (3) of the present invention is:

[0016] (1) RPMI-1640, which accounts for 50% of the volume of NK cell serum-free culture medium;

[0017] (2) L-glutamine 2 mM;

[0018] (3) Glucose 25 mM;

[0019] (4) Vitamin C 100 μM;

[0020] (5)IL-2100U / mL, IL-1550ng / mL, IL-2120ng / mL;

[0021] (6) Tumor antigen CEA 100 ng / mL;

[0022] (7) Monoclonal antibodies against CD244 and CD137, 20 ng / mL.

[0023] In another aspect, the present invention also discloses a use of the monoclonal antibody against CD244 and CD137 in preparing NK cells.

[0024] In another aspect, the present invention also discloses an application of the method in preparing NK cells.

[0025] The present invention significantly improves the amplification efficiency and killing activity of NK cells through the combined application of optimized three-dimensional scaffold culture, tumor antigens and serum-free culture medium. In in vitro and in vivo experiments, the NK cells of the present invention show good performance in terms of the killing effect on tumor cells and the tumor inhibition effect in mouse tumor models. Therefore, the NK cells cultured by this method can be used in the treatment of diseases such as tumors and viral infections, and have significant clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The SDS-PAGE test results of the monoclonal antibodies prepared by the present invention, wherein 1 is the monoclonal antibody prepared by the present invention. DETAILED DESCRIPTION

[0027] 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.

[0028] 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.

[0029] Example 1: Preparation of NK cells

[0030] 1. Peripheral Blood Mononuclear Cell (PBMC) Isolation

[0031] PBMCs were isolated from the peripheral blood of three healthy donors (20 to 35 years old, without infection or other diseases, and in compliance with ethical requirements) using Ficoll-Paque density gradient centrifugation. Approximately 60 mL of peripheral blood was collected from each donor, and approximately 3 × 10 8 PBMCs.

[0032] After separation, the number of cells was detected, and the average PBMC recovery rate of the three serum samples was 5×10 6 cells / mL blood, and the cell survival rate exceeded 95%.

[0033] 2. Enrichment of NK cells

[0034] PBMC suspension was added at 1×10 7 The cells were seeded at a concentration of 100 cells / mL in a 6-well culture plate and cultured at 37°C with 5% CO. 2 After culturing for 2 hours under the same environment, the adherent cells were removed to obtain a non-adherent cell suspension.

[0035] CD56+NK cells were enriched using the immunomagnetic bead method. The ratio of magnetic beads was: 1×10 7 PBMCs were sorted with 50 μL CD56 magnetic beads (Miltenyi Biotec) to obtain CD56+ NK cells.

[0036] After separation, the number of cells was detected, and the purity of the enriched NK cells was 85-90%, and the cell survival rate was 97%, which indicated that the enriched NK cells were of good quality.

[0037] 3. NK cell expansion and activation

[0038] The enriched NK cells were cultured at 5×10 5 The cells / mL were inoculated in a three-dimensional scaffold culture system (the preparation method of which is shown in Example 4), and cultured in a serum-free medium, with 50% of the medium replaced every 2 days. After 7 days, under microscopic observation, the cells were well attached to the three-dimensional scaffold and proliferated in spherical or colony forms, with no apoptosis or decomposition observed.

[0039] The formula of serum-free medium is as follows:

[0040] (1) RPMI-1640, which accounts for 50% of the volume of NK cell serum-free culture medium;

[0041] (2) L-glutamine 2 mM;

[0042] (3) Glucose 25 mM;

[0043] (4) Vitamin C 100 μM;

[0044] (5)IL-2100U / mL, IL-1550ng / mL, IL-2120ng / mL;

[0045] (6) Tumor antigen CEA 100 ng / mL;

[0046] (7) Monoclonal antibodies against CD244 and CD137 (see Example 5 for details) 20 ng / mL.

[0047] After 7 days of culture, NK cells expanded about 10 times, and the cell purity reached 95% (according to the above method, NK cells cultured without 3D scaffolds expanded about 5 times, and the cell purity was about 75%). At this time, NK cells can be collected for freezing or experiment. The test data of NK cells and different control groups at this time are as follows:

[0048] Control group 1: NK cells cultured in serum-free medium without adding tumor antigen CEA and monoclonal antibodies against CD244 and CD137 expanded about 5 times, and the cell purity reached 75%.

[0049] Control group 2: NK cells cultured in serum-free medium with only tumor antigen CEA expanded about 6 times, and the cell purity reached 85%.

[0050] Control group 3: NK cells cultured in serum-free medium supplemented with only anti-CD244 and CD137 monoclonal antibodies expanded by about 7 times, and the cell purity reached 86%.

[0051] The above results show that the three-dimensional scaffold can significantly improve the expansion efficiency and purity of NK cells. Tumor antigen CEA and monoclonal antibodies against CD244 and CD137 in serum-free culture medium also have a good promoting effect on the expansion efficiency and purity of NK cells, and the effect is better when the two groups are used in combination!

[0052] Example 2: Functional detection of NK cells

[0053] The killing activity of NK cells against K562 tumor cells was detected by LDH release method. After the expanded NK cells were co-cultured with K562 cells at different effector-target ratios (E=40:1, 20:1, 10:1, 5:1) for 4 hours, the amount of LDH released by cell lysis was measured. The experimental results are shown below:

[0054] When E = 40:1, the killing rate is 91%;

[0055] When E = 20:1, the killing rate is 85%;

[0056] When E = 10:1, the killing rate is 77%;

[0057] When E=5:1, the killing rate is 62%.

[0058] Control group 1: The killing rate of NK cells (E=10:1) cultured in serum-free medium without adding tumor antigen CEA and monoclonal antibodies against CD244 and CD137 was only 42%.

[0059] Control group 2: NK cells cultured in serum-free medium with only tumor antigen CEA (E=10:1) had a killing rate of 56%.

[0060] Control group 3: NK cells cultured in serum-free medium with only anti-CD244 and CD137 monoclonal antibodies added (E=10:1) had a killing rate of 63%.

[0061] The above results show that the killing activity of NK cells prepared using the serum-free culture medium of the present invention is significantly improved, and is better than the effect of adding only tumor antigen CEA and only adding monoclonal antibodies against CD244 and CD137.

[0062] Example 3: NK cell therapy study in mouse models

[0063] 1. Tumor model establishment: 6-8 week old male nude mice were selected and injected with human colorectal cancer cells HCT116 (1×10 6 cells / mouse) and implanted subcutaneously on the back. About 10 days after implantation, a tumor formed and reached a size of about 100 mm. 3 The mice were randomly divided into 3 groups, 10 mice in each group:

[0064] Experimental group: 5×10 NK cells prepared by the present invention were injected into each mouse. 6 cells, injected every 7 days for 3 weeks;

[0065] Control group 1: Injection of unexpanded NK cells, the injection frequency is the same as the experimental group;

[0066] Control group 2: Injected with an equal volume of PBS solution.

[0067] 2. Experimental results:

[0068] 2.1 Changes in tumor volume: On the 21st day after injection, the tumor volume of the experimental group decreased by an average of 60% (the initial volume was 100 mm 3 The final volume is 40±10mm 3 ), while the tumor volumes of control group 1 and control group 2 increased by 120% (220±15mm 3 ) and 180% (280 ± 20mm 3 ).

[0069] 2.2 Survival rate: The 21-day survival rate of mice in the experimental group was 100%, while the survival rates of control group 1 and control group 2 were 75% and 50%, respectively.

[0070] 2.3 Toxicity test: The body weight, liver and kidney function, and blood biochemical indexes of all mice were tested. No obvious toxic side effects were found in the experimental group mice. The body weight remained basically stable, and the liver and kidney functions were normal.

[0071] Conclusion: The NK cells in the experimental group showed significant anti-tumor effects without obvious toxicity, indicating that the NK cells amplified by the method of the present invention have good anti-tumor activity in vivo.

[0072] Example 4: Preparation and testing of three-dimensional scaffolds

[0073] 1. Preparation of three-dimensional scaffolds

[0074] (1) Preparation of gelatin solution: Dissolve 2 g of gelatin in 100 mL of deionized water at 50°C and stir until completely dissolved. Add gold nanoparticles (10 nm, final concentration 1% w / v) and continue stirring for 30 minutes to ensure uniform dispersion.

[0075] (2) Preparation of chitosan solution: Dissolve 2 g of chitosan in 100 mL of 1% glacial acetic acid solution and stir at room temperature until completely dissolved to form a chitosan solution. Mix the chitosan solution with the gelatin solution in a ratio of 1:1 and stir evenly.

[0076] (3) Casting and molding: Pour the mixed solution into a special silicone mold to form a uniform film. Freeze at 4°C for 24 hours until it is completely solidified.

[0077] (4) Freeze drying: The frozen scaffold is dried in a freeze dryer for 48 hours. This step forms a three-dimensional porous structure with high porosity.

[0078] (5) Cross-linking treatment: The dried scaffolds were cross-linked using a 0.1% glutaraldehyde aqueous solution and soaked for 2 hours to improve the mechanical strength of the scaffolds. The scaffolds were washed with ethanol three times, 10 minutes each time, to remove excess cross-linking agent. The scaffolds were rinsed again with deionized water and dried at 37°C.

[0079] (6) Final molding and sterilization: After the stent is dried, it is cut into a size suitable for experimental use (the stent of the present invention is a disc with a diameter of 10 mm), sterilized using gamma rays or ultraviolet rays, and set aside.

[0080] 2. Inspection of three-dimensional stents

[0081] (1) Porosity analysis: The pore structure of the three-dimensional scaffold was analyzed by scanning electron microscopy (SEM). The porosity reached 90% and the pore size was 50-100 μm, which is suitable for cell attachment and proliferation. The experimental results show that the addition of gold nanoparticles makes the pore structure of the scaffold more uniform and enhances the interaction between cells and the scaffold.

[0082] (2) Mechanical strength test: The three-dimensional scaffold was subjected to a compression strength test using a material testing instrument. The results showed that the mechanical strength of the scaffold with gold nanoparticles added was about 20% higher than that of the scaffold without gold nanoparticles. The compression modulus of the scaffold was 200 kPa, which is suitable for in vitro cell culture and transplantation.

[0083] (3) Cell proliferation and adhesion experiment: NK cells were cultured on three-dimensional scaffolds to detect cell adhesion rate and proliferation ability. The CCK-8 method was used to measure cell proliferation 3, 5, and 7 days after culture. The experimental results showed that the cell adhesion rate was 85%. After 7 days of culture, the cell proliferation was 6 times, which was significantly higher than the 3-fold proliferation under traditional two-dimensional culture conditions.

[0084] (4) Biocompatibility test: The cytotoxicity of the scaffold was detected by the MTT method. The results showed that the scaffold had no significant toxicity to cells at different concentrations and had good biocompatibility.

[0085] Example 5: Preparation and testing of monoclonal antibodies against CD244 and CD137

[0086] 1. Preparation of monoclonal antibodies

[0087] The CD244 protein (amino acid sequence as shown in SEQ ID NO.1) polypeptide (position aa70-aa80, amino acid sequence SLLIKAAQQQD) and CD137 protein (amino acid sequence as shown in SEQ ID NO.2) polypeptide (position aa122-aa132, amino acid sequence SVLVNGTKERD) were designed and synthesized. The above polypeptides were then coupled with KLH and mixed in equal mass ratios to prepare immunogens. After the immunogen was emulsified, 6-week-old mice were immunized 3-4 times, 0.1 mg / time / mouse, with an interval of 2-3 weeks each time. After the ELISA test was qualified, spleen cells were taken and mixed with myeloma cells in a ratio of 8:1, and cell fusion was mediated by PEG. The fused cells were inoculated in a 96-well plate, and hybridoma cells were screened using HAT culture medium. After the cell clones grew, the antibody activity was detected. Antibody secretion positive clones were detected using ELISA, and high-affinity hybridoma cells were selected for further amplification and culture. The antibody was purified by protein A / G affinity chromatography column, and the purified antibody was sterilized by filtration and stored at -80°C for future use.

[0088] 2. Testing of monoclonal antibodies

[0089] (1) The concentration of the purified monoclonal antibody was tested, and the result of the BCA kit test was 2.56 mg / ml.

[0090] (2) The purified monoclonal antibody was subjected to SDS-PAGE detection. The results were as follows Figure 1 As shown, the SDS-PAGE purity of the monoclonal antibody can reach more than 95%.

[0091] (3) Entrust a third-party service company (such as Nanjing GenScript) to sequence the RNA extracted from the hybridoma cells to detect the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody. The test results show that the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0092] (4) The binding activity of the prepared monoclonal antibodies and commercial antibodies to the target protein was tested by ELISA. First, the recombinant human CD244 protein (ab185248) and the recombinant human CD137 protein (ab287936) were coated with ELISA plates at a concentration of 1 μg / ml, and then the three antibodies were diluted to 1 μg / ml and added to the ELISA plates, respectively, and detected using the corresponding secondary antibodies. The results are shown in Table 1. The OD values ​​of the monoclonal antibodies of the present invention for CD244 and CD137 are higher, indicating that the monoclonal antibodies have better binding activity than the commercial monoclonal antibodies.

[0093] Table 1 Antibody activity test results (OD450nm)

[0094] Group CD244 protein coating CD137 protein coating Monoclonal antibodies of the present invention 2.12、2.09 1.98、2.01 Anti-CD244 antibody (ab313993) 1.34、1.41 0.101、0.087 Anti-CD137 antibody (ab89085) 0.097、0.095 1.45、1.38

[0095] 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 monoclonal antibody against CD244 and CD137, characterized in that: The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

2. A method for preparing NK cells, characterized in that: The method comprises the following steps: (1) Isolation of PBMCs: PBMCs were isolated from peripheral blood using Ficoll-Paque density gradient centrifugation; (2) Enrichment of NK cells: enriching CD56+ NK cells from the PBMCs separated in step (1) using an immunomagnetic bead method; (3) NK cell expansion and activation: The NK cells enriched in step (2) were cultured at a rate of 5×10 5 The cells / mL are inoculated in a three-dimensional scaffold culture system, and cultured with a serum-free culture medium containing the monoclonal antibodies against CD244 and CD137 according to claim 1, and 50% of the culture medium is replaced every 2 days. After continuous culture for 7 days, NK cells can be collected.

3. The method according to claim 2, characterized in that The three-dimensional scaffold in step (3) is a disc with a diameter of 10 mm.

4. The method according to claim 2, characterized in that: The porosity of the three-dimensional scaffold in step (3) is up to 90%, and the pore size is 50-100 μm.

5. The method according to claim 2, characterized in that: The compression modulus of the three-dimensional scaffold in step (3) is 200 kPa.

6. The method according to claim 2, characterized in that The formula of the serum-free medium in step (3) is: (1) RPMI-1640, which accounts for 50% of the volume of NK cell serum-free culture medium; (2) L-glutamine 2 mM; (3) Glucose 25 mM; (4) Vitamin C 100 μM; (5)IL-2100U / mL, IL-1550ng / mL, IL-2120ng / mL; (6) Tumor antigen CEA 100 ng / mL; (7) Monoclonal antibodies against CD244 and CD137, 20 ng / mL.

7. Use of the monoclonal antibody against CD244 and CD137 as claimed in claim 1 in preparing NK cells.

8. Use of the method according to claim 2 in preparing NK cells.

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