A method for preparing NK cells and their application in the treatment of malignant tumors

Abstract

The present invention discloses a method for preparing NK cells, the method comprising: (1) separation and purification of NK cells; (2) preliminary expansion of NK cells under a hypoxic environment; (3) co-culture of NK cells; and (4) continuous expansion and harvesting of NK cells. The NK cells prepared by the method show high cell purity and activity, and exhibit significant anti-tumor effects in a mouse model for treating malignant tumors. The method of the present invention has the advantages of high efficiency and reliability, and provides a good reference for the research and clinical application of NK cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for preparing natural killer cells (NK cells) and application thereof in the treatment of malignant tumors. Background Art

[0002] Natural killer cells (NK cells) are a type of immune cells with powerful cytotoxic effects that can specifically recognize and kill tumor cells or infected cells. In recent years, NK cells have received widespread attention in tumor immunotherapy. Existing NK cell preparation methods usually involve isolating NK cells from peripheral blood or inducing the proliferation and differentiation of NK cells through hematopoietic stem cells. However, these methods have problems such as low cell purity, low proliferation efficiency, and long in vitro expansion cycle, which limit the widespread application of NK cells in clinical treatment. Summary of the invention

[0003] The present invention provides a novel method for preparing NK cells and explores its application in treating malignant tumors. Through improved cell culture and induction technology, the method of the present invention can efficiently prepare high-purity and high-activity NK cells and can play an excellent role in treating malignant tumors.

[0004] Therefore, the present invention discloses a method for preparing NK cells, which comprises the following steps:

[0005] (1) Isolation and purification of NK cells: First, PBMCs were isolated from peripheral blood using Ficoll density gradient centrifugation. The isolated PBMCs were then suspended in RPMI medium and NK cells were purified using immunomagnetic beads.

[0006] (2) Preliminary expansion of NK cells in a hypoxic environment: The NK cells purified in step (1) were inoculated in a serum-free medium at a concentration of 1×10^6 cells / mL, and placed in a hypoxic incubator at 5% O2 and 37°C for culture; 50% of the serum-free medium was replaced every 2 days to maintain the NK cell concentration at about 2×10^6 cells / mL, and the culture was continued for 5 days;

[0007] (3) Co-culture of NK cells: adding tumor-associated antigen stimulation to the NK cells cultured in step (2) at a concentration of 10 μg / mL, and increasing the IL-2 concentration in the serum-free culture medium to 50 ng / mL, wherein the tumor-associated antigen is a total protein mixed with MUC1 protein, CEA protein, HER2 protein, etc. in a mass ratio; continuing co-culture under hypoxic conditions for 3 days, and collecting NK cells;

[0008] (4) Continuous expansion and harvesting of NK cells: Continue to culture the NK cells after co-culture in step (3) under hypoxia, and at the same time increase the IL-2 concentration in the serum-free culture medium to 100 ng / mL; replace 50% of the culture medium every 2 days to maintain the NK cell concentration at about 2×10^6 cells / mL, continue to culture for 7 days, collect the NK cells, i.e., the prepared NK cells, and freeze them for later use.

[0009] Preferably, the immunomagnetic beads of the present invention label antibodies that simultaneously bind to CD56 and CD16, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody that simultaneously binds to CD56 and CD16 are as shown in SEQ ID NO.3 and SEQ ID NO.4.

[0010] Preferably, the antibody that simultaneously binds to CD56 and CD16 of the present invention specifically binds to the CD56 protein at aa323-aa340, and its amino acid sequence is EQVTLTCEASGDPIPSIT.

[0011] Preferably, the antibody that simultaneously binds to CD56 and CD16 of the present invention specifically binds to the CD16 protein at aa21-aa38, and its amino acid sequence is DSVTLKCQGAYSPEDNST.

[0012] Preferably, the formula of the serum-free medium in step (2) of the present invention is:

[0013] (1) Basal culture medium: RPMI-1640, which accounts for 50% of the volume of NK cell serum-free culture medium;

[0014] (2) Amino acids: L-glutamine 2 mM;

[0015] (3) Glucose: 25 mM;

[0016] (4) Vitamins: Vitamin C 100 μM;

[0017] (5) Chemical small molecules: metformin 1mM;

[0018] (6) Cytokines: IL-2 20 ng / mL, IL-1 5 50 ng / mL, IL-2 1 20 ng / mL;.

[0019] (7) Taurine: 100 μM.

[0020] In another aspect, the present invention also discloses an application of the method in preparing NK cells.

[0021] In another aspect, the present invention also discloses a use of the antibody that simultaneously binds to CD56 and CD16 in NK cell purification.

[0022] In another aspect, the present invention also discloses an application of the serum-free culture medium in NK cell culture.

[0023] The present invention achieves efficient expansion and functional enhancement of NK cells by optimizing the culture conditions, metabolic regulation and immunomodulatory strategies of NK cells, especially showing significant improvement in anti-tumor killing activity. The experimental results show that this method has strong innovation and clinical application prospects, and provides a good reference and research direction for the cultivation of NK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results of SDS-PAGE detection of antibodies binding to CD56 and CD16 at the same time, where 1 is the antibody. DETAILED DESCRIPTION

[0025] 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.

[0026] 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.

[0027] Example 1: Isolation and purification of NK cells

[0028] 1. Extract 20 mL of peripheral blood from healthy volunteers (with their consent and in compliance with ethical requirements) and separate peripheral blood mononuclear cells (PBMCs) using Ficoll density gradient centrifugation, as briefly described below:

[0029] Collect 10-20mL of peripheral blood from the donor, add an anticoagulant (such as EDTA or heparin) to prevent coagulation, and preheat the Ficoll-Paque density gradient solution to room temperature. Dilute the collected peripheral blood with sterile PBS in a 1:1 ratio and mix gently to avoid bubbles. In a 50mL centrifuge tube, add 15mL of Ficoll-Paque density gradient solution and carefully add the diluted blood sample slowly along the wall of the tube above the Ficoll to ensure that the interface between the two is clearly layered and avoid mixing. Place the 50mL centrifuge tube in a centrifuge and centrifuge at 400×g for 30 minutes at room temperature, slowly raising and lowering the speed. Gently aspirate the PBMC layer (the white fuzzy layer between plasma and Ficoll) with a sterile pipette and transfer it to a new 15mL centrifuge tube. Add 1×PBS or RPMI-1640 medium to the centrifuge tube, mix the PBMC suspension, centrifuge at 300×g for 10 minutes, remove the supernatant, and repeat the wash twice to ensure that the Ficoll and plasma residues are removed. Count the number of PBMCs and assess cell viability using trypan blue staining or other cell viability assays.

[0030] 2. The separated PBMCs were suspended in RPMI medium and NK cells were purified using immunomagnetic beads (labeled with antibodies that bind to both CD56 and CD16, as shown in Example 5). A brief description is as follows:

[0031] Count PBMCs and adjust PBMCs to an appropriate concentration (usually 1×10 7 In a biosafety cabinet, resuspend PBMC in an appropriate amount of PBS solution (containing 0.5% bovine serum albumin or 2% fetal bovine serum), ensure that the cells are evenly suspended, and add an appropriate amount of immunomagnetic beads (usually 1×10 7PBMC plus 10-20μL magnetic bead solution), mix gently to avoid bubbles. Incubate at 4°C for 15-30 minutes to ensure that the antibody fully binds to CD56 and CD16 on the surface of NK cells. After labeling, wash the cells with PBS 1-2 times, centrifuging at 300×g for 10 minutes each time to remove unbound magnetic beads and antibodies. Finally, resuspend the cells in 500-1000μL PBS. Install the MACS magnetic field separator and place the MACS separation column in the magnetic field. Use the balance solution (PBS, containing 0.5% bovine serum albumin or 2% fetal bovine serum) to pre-rinse the MACS separation column (usually use about 3mL of PBS), avoid bubbles, and ensure that the column is fully wetted. Slowly add the labeled cell suspension to the MACS separation column, ensure that the cells pass through the magnetic column, and slowly rinse with PBS (2-3mL). CD56+CD16+NK cells will be captured by magnetic beads and remain in the column, while unlabeled cells (such as T cells, B cells, etc.) will flow out, and the effluent will be collected as the negative separation group. Remove the MACS column from the magnetic field separator, immediately rinse the column with fresh PBS, use the plunger to flush the magnetic bead-labeled CD56+CD16+NK cells out of the column, and collect them in a new centrifuge tube. Use a cell counting plate to count the number of purified NK cells and assess cell viability (such as using trypan blue staining).

[0032] The method of coupling monoclonal antibodies to magnetic beads is briefly described as follows: Weigh an appropriate amount of 1μm magnetic beads (such as 100μL magnetic bead suspension), wash the magnetic beads 3 times with MES buffer (pH 5.0) to ensure that impurities in the storage solution are removed, separate the magnetic beads with a magnetic stand each time and discard the supernatant. Resuspend the magnetic beads in 200μMES buffer. Add 40μLEDC (10mg / mL) and 40μL NHS (5mg / mL) to the magnetic bead suspension, mix gently, and incubate at room temperature for 30 minutes, gently rotating to mix to ensure uniform reaction. Slowly add the monoclonal antibody solution (about 100μg antibody, 1-2mg / mL) to the activated magnetic bead mixture, gently rotate and incubate at room temperature for 2 hours to promote the coupling of the antibody to the magnetic bead surface. After the coupling is completed, add 40μL 1M Tris-HCl solution (pH 7.4) to terminate the cross-linking reaction and incubate for 15 minutes. Wash the magnetic beads at least 3 times with PBS buffer (pH 7.4) to ensure that unbound antibodies and cross-linking agent residues are removed. Separate the magnetic beads with a magnetic stand and discard the supernatant each time. After washing, resuspend the magnetic beads in an appropriate amount of PBS or other storage buffer (such as PBS containing 0.05% NaN3), usually resuspended in the original volume (such as 100 μL). Dispense the coupled antibody magnetic bead suspension into EP tubes and store at 4°C for future use.

[0033] 3. Detect CD56 and CD16 markers by flow cytometry to ensure that the purity of the separated NK cells reaches more than 90%. After testing, the purity of CD56+CD16+NK cells is 95.8%.

[0034] 4. It should be noted that the immunomagnetic beads currently popular in the market only label and bind to CD56 antibodies. When this study began to use this magnetic bead for NK cell separation, the purity of the obtained CD56+CD16+NK cells was only 86%.

[0035] Example 2: Initial expansion of NK cells in a hypoxic environment

[0036] 1. The NK cells separated in Example 1 were inoculated in serum-free culture medium at a concentration of 1×10^6 cells / mL.

[0037] The formula of serum-free medium is as follows:

[0038] (1) Basal culture medium: RPMI-1640, which accounts for 50% of the volume of NK cell serum-free culture medium;

[0039] (2) Amino acids: L-glutamine 2 mM;

[0040] (3) Glucose: 25 mM;

[0041] (4) Vitamins: Vitamin C 100 μM;

[0042] (5) Chemical small molecules: metformin 1mM;

[0043] (6) Cytokines: IL-2 20 ng / mL, IL-1 5 50 ng / mL, IL-2 1 20 ng / mL;.

[0044] (7) Taurine: 100 μM.

[0045] 2. Place the NK cells in a hypoxic incubator at 5% O2 and 37°C for culture.

[0046] 3. Replace 50% serum-free medium every 2 days, maintain the NK cell concentration at about 2×10^6 cells / mL, and continue culturing for 5 days. After 5 days of culture, the cell proliferation multiple is 5 times, and the CD56 and NKG2D markers are detected by flow cytometry, and the proportion of positive cells reaches 93.5%.

[0047] 4. It should be noted that when cultured in the above method using normal oxygen content, the cell proliferation multiple is 6 times, but the proportion of positive cells detected by flow cytometry for CD56 and NKG2D markers is only 76.6%. This shows that under hypoxic conditions, the proliferation of NK cells is affected, but they are closer to the tumor microenvironment, so their surface markers are higher.

[0048] 5. At the same time, using conventional RPMI-1640 medium containing 10% FBS (containing IL-2500IU / mL, IL-1550ng / mL, IL-2120ng / mL), hypoxic culture was performed according to the above method, and it was found that its proliferation multiple was 4.2, and the CD56 and NKG2D markers were detected by flow cytometry, and the positive cell ratio reached 83.1%. This shows that the serum-free medium of the present invention has a good culture effect.

[0049] 6. In addition, the effect of adding "metformin" to serum-free culture medium was compared. This study cultured the two groups of culture media separately and tested them after culture. It was found that the proliferation effects of the two groups of culture media were basically the same, both 5 times. However, the NK cells cultured in the culture medium without metformin were tested by flow cytometry for CD56 and NKG2D markers, and the proportion of positive cells was only 86.8%, which shows that metformin has the ability to promote NK cell differentiation and can enhance the production of NK cell surface markers.

[0050] Example 3: Co-culture of NK cells

[0051] 1. Tumor-associated antigen (TAA) was added to the cultured NK cells in Example 2 for stimulation at a concentration of 10 μg / mL, and the IL-2 concentration in the serum-free culture medium was increased to 50 ng / mL, wherein TAA was a total protein mixed with MUC1 protein, CEA protein, HER2 protein, etc. in equal mass ratios.

[0052] 2. Continue to co-culture under hypoxic conditions for 3 days, collect NK cells and perform killing activity test.

[0053] 3. LDH release assay showed that the killing rate of amplified NK cells against K562 cells reached 86%, and the killing rate against HCT116 cells was 80%. Compared with NK cells not co-cultured, the killing rate was significantly improved (the killing rate of NK cells not co-cultured against K562 cells reached 76%, and the killing rate against HCT116 cells was 73%). This shows that co-culture with TAA can significantly provide NK cell killing activity.

[0054] 4. It should be noted that the IL-2 concentration was increased to 50 ng / mL in this culture. When the IL-2 concentration was not increased, the killing rate of the expanded NK cells on K562 cells reached 82%, and the killing rate on HCT116 cells was 76%. This shows that the concentration of IL-2 provided in this step helps to improve the killing activity of NK cells.

[0055] Example 4: Continuous Expansion and Harvesting of NK Cells

[0056] 1. The NK cells co-cultured in Example 3 were continued to be cultured under hypoxia, and at the same time, the IL-2 concentration in the serum-free culture medium was increased to 100 ng / mL, and the hypoxia culture was continued to ensure that the NK cells maintained high activity.

[0057] 2. Replace 50% of the culture medium every 2 days to maintain the NK cell concentration at about 2×10^6 cells / mL, and continue culturing for 7 days. Collect NK cells for cryopreservation (the cryopreservation solution is 90% FBS + 10% DMSO; use the cryopreservation solution to dilute the NK cells to 1×10^6 cells / mL). 7 cells / mL, put the cryovial containing the cell suspension into a programmed cooling box, place it at -80°C overnight, and transfer it to a liquid nitrogen tank for long-term storage on the second day), or directly perform treatment or function and activity testing.

[0058] 3. The test results showed that the proliferation multiple of NK cells reached 8 times, and the killing rate detected by LDH release method was 93% for K562 cells and 88% for HCT116 cells.

[0059] 4. It should be noted that the IL-2 concentration was increased to 100 ng / mL in this culture. When the IL-2 concentration was not increased, the proliferation of NK cells was 6.8 times, the killing rate of K562 cells reached 86%, and the killing rate of HCT116 cells was 82%. This shows that the concentration of IL-2 provided in this step helps to further improve the killing activity of NK cells.

[0060] Example 5: Preparation and testing of antibodies that bind to both CD56 and CD16

[0061] 1. Preparation of Antibodies that Bind Both CD56 and CD16

[0062] First, the amino acid sequences of CD56 protein (SEQ ID NO.1) and CD16 protein (SEQ ID NO.2) were compared and analyzed, and two peptides were designed and synthesized (CD56 protein aa323-aa340: EQVTLTCEASGDPIPSIT, CD16 protein aa21-aa38: DSVTLKCQGAYSPEDNST).

[0063] Secondly, the two segments of polypeptides were mixed in equal mass ratios and then fully mixed and emulsified with an equal volume of complete Freund's adjuvant (CFA), wherein the mass of the mixed polypeptide was 0.2 mg / ml and injected subcutaneously or intraperitoneally into mice. Immunization was performed once every 2 weeks, and the polypeptide was mixed with incomplete Freund's adjuvant (IFA) and injected into mice (3 consecutive injections). The last immunization used the polypeptide not mixed with the adjuvant and injected through the tail vein (100 μg).

[0064] Finally, 3-4 days after the final immunization, the immunized mice were sacrificed and the spleens were isolated. The spleens were ground using sterile PBS, and the splenocytes were filtered and collected, counted, and adjusted to a concentration of 1 × 10 7 cells / mL. Mix spleen cells with SP2 / 0 or NS0 myeloma cells (ratio of 5:1). Add PEG (37°C, slowly drip) to promote cell fusion. Add RPMI-1640 medium to terminate the reaction and collect the cells. Inoculate the fused cells in HAT medium and incubate in a 37°C 5% CO2 incubator. Replace the HAT medium every 3 days, and transfer to a 24-well plate for continued culture after clones appear. Use ELISA to detect whether the supernatant contains antibodies against the target polypeptide, take positive clones, and continue to expand and culture. Amplify the positive clone cells to produce sufficient antibodies, collect the culture supernatant or obtain antibodies by the ascites method, and purify the antibodies through a Protein G column.

[0065] After the preparation and purification by the above method, a purified antibody was obtained, which was sterilized by filtering through a 220 nm filter membrane and stored at -80°C for future use.

[0066] 2. Testing of antibodies that bind to both CD56 and CD16

[0067] (1) The concentration of the purified antibody was detected using a BCA test kit, and the result was 2.15 mg / ml. The ascites expression yield of the antibody was calculated based on the ascites volume before purification and the total antibody quantity after purification. After conversion, the expression level can reach 1.78 mg / ml. This indicates that the antibody has a high expression yield, providing a good foundation for its large-scale application.

[0068] (2) The purity of the antibody was tested using the SDS-PAGE method. The results were as follows: Figure 1 As shown, its SDS-PAGE purity can reach more than 95%.

[0069] (3) RNA was extracted from hybridoma cells to detect the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody. The results showed that the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody were as shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0070] (4) The ELISA method was used to detect the binding activity of the antibody and the commercial antibody to the target protein. The results are shown in Table 1. When the concentrations of the three antibodies were all 0.5 mg / ml, the OD values ​​detected after dilution 10,000 times were higher than those of the antibody of the present invention to CD56 protein and CD16 protein, indicating that the antibody has better binding activity than the commercial monoclonal antibody.

[0071] Table 1 Antibody activity test results (OD450nm)

[0072] Group CD56 protein coating CD16 protein coating Antibodies of the present invention 1.78 1.67 Anti-CD56 antibody (ab220360) 1.12 0.076 Anti-CD16 antibody (ab308605) 0.089 1.21

[0073] Example 6: NK cell therapy study in mouse models

[0074] 1. Establishment of Mouse B16 Melanoma Model

[0075] Tumor cell inoculation: 1×10 6 B16 melanoma cells were suspended in 100 μL PBS and inoculated subcutaneously into the right back or axilla of C57BL / 6 mice.

[0076] Tumor growth monitoring: After tumor inoculation, the long and short diameters of the tumor were measured every 2-3 days using an electronic caliper, the tumor volume was calculated (formula: V = (L × W2) / 2), and the changes in tumor volume were recorded.

[0077] 2. NK cell injection: The NK cells (1×10 6 Cells / mouse) were injected into the C57BL / 6 mice modeled above through the tail vein, and a control group injected with PBS was set up, with 10 mice in each group. The mice were injected once every 7 days for 3 consecutive times and followed up until the 28th day after the first treatment.

[0078] 3. Monitoring of treatment effects: Measure the tumor volume with an electronic caliper every 2-3 days and record the tumor growth curve. Compare the NK cell treatment group and the control group to evaluate the tumor growth inhibition effect. At the same time, record the survival time of each mouse to evaluate the effect of NK cell treatment on extending the survival of mice.

[0079] 4. Results

[0080] 4.1 Tumor inhibition effect: The tumor volume of mice injected with NK cells was significantly smaller than that of the control group. The tumor of the control group grew to 500±89mm on the 28th day of follow-up. 3 , while the tumor volume of the NK cell treatment group was 200±45mm 3 .

[0081] 4.2 Prolonged survival: NK cell therapy significantly prolonged the survival of tumor-bearing mice. The average survival of mice in the control group was 20 days (i.e., 10 / 10 died within 28 days of tracking), while all mice in the NK cell treatment group survived within 28 days of tracking; at the same time, this study continued to track the mice, and 5 / 10 mice survived within the 50th day of tracking.

[0082] In addition, liver cancer and breast cancer models were prepared using the same method as above, and the NK cells prepared by the present invention were used for treatment. The results were basically consistent with the above results, and good therapeutic effects were achieved. This shows that the NK cells prepared by the present invention have a wider clinical applicability and better clinical application effect.

[0083] 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 method for preparing NK cells, characterized in that: The method comprises the following steps: (1) Isolation and purification of NK cells: First, PBMCs were isolated from peripheral blood using Ficoll density gradient centrifugation. The isolated PBMCs were then suspended in RPMI medium and NK cells were purified using immunomagnetic beads. The immunomagnetic beads label the antibody that binds to CD56 and CD16 simultaneously, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody that binds to CD56 and CD16 simultaneously are as shown in SEQ ID NO.3 and SEQ ID NO.4; The antibody that simultaneously binds to CD56 and CD16 specifically binds to the CD56 protein at aa323-aa340, and its amino acid sequence is EQVTLTCEASGDPIPSIT; The antibody that simultaneously binds to CD56 and CD16 specifically binds to the CD16 protein at aa21-aa38, and its amino acid sequence is DSVTLKCQGAYSPEDNST; (2) Preliminary expansion of NK cells in a hypoxic environment: The NK cells purified in step (1) were inoculated in serum-free culture medium at a concentration of 1×10^6 cells / mL and cultured in a hypoxic incubator at 5% O2 and 37°C; 50% serum-free culture medium was replaced every 2 days to maintain the NK cell concentration at about 2×10^6 cells / mL, and the culture was continued for 5 days; The formula of the serum-free medium is: Basic culture medium: RPMI-1640, which accounts for 50% of the volume of NK cell serum-free culture medium; Amino acids: L-glutamine 2 mM; Glucose: 25 mM; Vitamins: Vitamin C 100 µM; Chemical small molecules: metformin 1 mM; Cytokines: IL-2 20 ng / mL, IL-15 50 ng / mL, IL-21 20 ng / mL; Taurine: 100 µM; (3) Co-culture of NK cells: After culture in step (2), tumor-associated antigens were added to stimulate the NK cells at a concentration of 10 μg / mL, and the IL-2 concentration in the serum-free culture medium was increased to 50 ng / mL. The tumor-associated antigens were total proteins mixed in equal weight ratios of MUC1 protein, CEA protein, and HER2 protein. The co-culture was continued under hypoxic conditions for 3 days, and the NK cells were collected. (4) Continuous expansion and harvesting of NK cells: Continue to culture the NK cells after co-culture in step (3) under hypoxia, and at the same time increase the IL-2 concentration in the serum-free culture medium to 100 ng / mL; replace 50% of the culture medium every 2 days to maintain the NK cell concentration at around 2×10^6 cells / mL. Continue culturing for 7 days, collect the NK cells, i.e., the prepared NK cells, and freeze them for later use.

2. Use of the method according to claim 1 in preparing NK cells.

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