CN119119276A - A method for preparing natural killer cells and use thereof in treating diseases - Google Patents
A method for preparing natural killer cells and use thereof in treating diseases Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
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Abstract
The invention provides a method for efficiently preparing natural killer cells, which remarkably improves the amplification efficiency and killing activity of NK cells through the combined application of optimized three-dimensional scaffold culture and tumor antigens. The method is applied to the treatment of diseases such as tumor, virus infection and the like, and has remarkable clinical application value.
Description
Technical Field
The invention relates to the field of immune cell treatment, in particular to a preparation method of natural killer cells and application thereof in treating diseases such as tumor, virus infection and the like.
Background
Natural killer cells (naturalkillercell, NK cells) are derived from bone marrow lymphoid stem cells, whose differentiation and development depend on the bone marrow and thymus microenvironment, and are predominantly distributed in bone marrow, peripheral blood, liver, spleen, lung and lymph nodes. NK cells, unlike T, B cells, are a class of lymphocytes that can nonspecifically kill tumor cells and virus-infected cells without prior sensitization.
The exact source of NK cells is not well understood and is generally thought to be derived directly from bone marrow, and its developmental maturation is dependent on the microenvironment of the bone marrow. In vitro experiments of mice and humans show that NK cells can be induced by culturing thymic cells in the presence of cytokines such as IL-2 in vitro. Spleen of mice can promote NK cell differentiation under induction of IL-3 in vivo. NK cells are mainly distributed in peripheral blood and account for 5-10% of PBMC, and NK activity is also found in lymph nodes and bone marrow, but the level is lower than that of peripheral blood. Since NK cells have partial T cell differentiation antigens, such as 80-90% of NK cell CD2+, 20-30% of NK cell CD3+ (expressing CD3 zeta chain), 30% of NK cell CD8+ (alpha/alpha) and 75-90% of NK cell CD38+, and NK cells have affinity receptors in IL-2, proliferation reaction can occur under the stimulation of IL-2, and IFN-gamma can be produced by activating NK cells, the NK cells are generally considered to have a more close relationship with T cells in development.
Since the killing activity of NK cells is not MHC-restricted and is not antibody dependent, it is called natural killing activity. NK cell cytoplasm is rich, contains larger azurophil particles, and the content of the particles is positively correlated with the killing activity of NK cells. The target cells of NK cells mainly comprise certain tumor cells (including partial cell lines), virus-infected cells, certain self-tissue cells (such as blood cells), parasites and the like, so that the NK cells are important immune factors for resisting tumor and infection of organisms and also participate in type II hypersensitivity and graft-versus-host reaction.
NK cells have become an important means of immunotherapy because of their unique antitumor and antiviral properties. However, the existing NK cell preparation technology has the problems of low cell expansion efficiency, unstable activity and the like, and limits the clinical application of the NK cell. The invention provides an improved NK cell preparation method, which obviously improves the activity and amplification efficiency of NK cells and improves the application effect of NK cells in treating diseases such as tumor, virus infection and the like.
Disclosure of Invention
The invention aims to provide a method for efficiently preparing NK cells, which can obviously improve the proliferation rate and the killing activity of the NK cells by optimizing culture conditions and adding specific stimulating factors. The method can be widely applied to the treatment of tumors, virus infection and autoimmune diseases, and has obvious innovation and clinical application value.
Accordingly, in one aspect, the invention discloses 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 as SEQ ID NO.3 and SEQ ID NO.4 respectively.
In still another aspect, the present invention also discloses a method for preparing NK cells, the method comprising the steps of:
(1) PBMC separation PBMC were separated from peripheral blood using Ficoll-Paque density gradient centrifugation;
(2) Enrichment of CD56+ NK cells from the PBMC isolated in step (1) using immunomagnetic beads method;
(3) NK cell expansion and activation, namely inoculating the NK cells enriched in the step (2) into a three-dimensional scaffold culture system at the rate of 5X 105 cells/mL, simultaneously culturing the NK cells by using a serum-free medium containing the monoclonal antibodies against CD244 and CD137 as set forth in claim 1, changing 50% of the medium every 2 days, and continuously culturing for 7 days to collect the NK cells.
Preferably, the three-dimensional scaffold in the step (3) is a wafer with the diameter of 10 mm.
Preferably, the three-dimensional scaffold in step (3) of the present invention has a porosity of 90% and a pore size of 50-100 μm.
Preferably, the three-dimensional scaffold in step (3) of the present invention has a compression modulus of 200kPa.
Preferably, the serum-free medium in step (3) of the present invention has the formula:
(1) RPMI-1640, which accounts for 50% of the volume of the NK cell serum-free culture solution;
(2) L-glutamine 2mM;
(3) Glucose 25mM;
(4) Vitamin C100. Mu.M;
(5)IL-2100U/mL,IL-1550ng/mL,IL-2120ng/mL;
(6) Tumor antigen CEA 100ng/mL;
(7) anti-CD 244 and CD137 monoclonal antibody 20ng/mL.
In still another aspect, the invention also discloses an application of the monoclonal antibodies against CD244 and CD137 in preparing NK cells.
In still another aspect, the invention also discloses an application of the method in preparing NK cells.
The invention obviously improves the amplification efficiency and killing activity of NK cells through the combined application of the optimized three-dimensional scaffold culture and tumor antigen and serum-free culture medium. In vitro and in vivo experiments, the NK cells of the invention have better performance in the aspects of killing effect on tumor cells and tumor inhibition effect in a mouse tumor model. Therefore, the NK cells cultured by the method can be applied to the treatment of diseases such as tumors, virus infection and the like, and have remarkable clinical application value.
Drawings
FIG. 1 shows the SDS-PAGE detection result of a monoclonal antibody prepared according to the present invention, wherein 1 is a monoclonal antibody prepared according to the present invention.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. Reagents and materials used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 preparation of NK cells
1. Peripheral Blood Mononuclear Cell (PBMC) isolation
PBMC were isolated from peripheral blood of 3 healthy donors (20-35 years old, without infection or other disease, ethical requirements) using Ficoll-Paque density gradient centrifugation. About 60mL of peripheral blood was collected from each donor, and about 3X 10 8 PBMC were obtained per person after centrifugation.
Cell numbers were measured after isolation and the average PBMC recovery of 3 serum was 5 x 10 6 cells/mL blood, with cell viability exceeding 95%.
Enrichment of NK cells
The PBMC suspension was inoculated into a 6-well culture plate at a concentration of 1X 10 7 cells/mL, and after 2 hours of culture in a 37℃and 5% CO 2 environment, adherent cells were removed to obtain a non-adherent cell suspension.
Cd56+ NK cells were enriched using immunomagnetic beads. The proportion of beads was 50. Mu.L of CD56 beads (Miltenyi Biotec) per 1X 10 7 PBMC, and CD56+ NK cells were obtained after magnetic column sorting.
After separation, detecting the number of cells, wherein the purity of NK cells after enrichment is 85-90%, and the cell survival rate is 97%. This indicates that enriched NK cells are better.
NK cell expansion and activation
Enriched NK cells were inoculated at 5X 10 5 cells/mL into a three-dimensional scaffold culture system (its preparation method is shown in example 4) while culturing with serum-free medium, changing 50% of the medium every 2 days. After 7 days, the cells are observed by a microscope to be well attached on the three-dimensional bracket and proliferate in a spherical shape or a colony shape, and the apoptosis or decomposition phenomenon of the cells is not seen.
Wherein the formula of the serum-free medium is as follows:
(1) RPMI-1640, which accounts for 50% of the volume of the NK cell serum-free culture solution;
(2) L-glutamine 2mM;
(3) Glucose 25mM;
(4) Vitamin C100. Mu.M;
(5)IL-2100U/mL,IL-1550ng/mL,IL-2120ng/mL;
(6) Tumor antigen CEA 100ng/mL;
(7) Monoclonal antibodies against CD244 and CD137 (see, in particular, example 5) 20ng/mL.
After 7 days of culture, NK cells were amplified about 10 times and cell purity reached 95% (NK cells cultured without using a three-dimensional scaffold were amplified about 5 times and cell purity was about 75% according to the above method), at which time NK cells could be collected for cryopreservation or experiment. The detection data of NK cells and different control groups at this time are as follows:
control group 1 NK cells cultured without adding tumor antigen CEA and monoclonal antibodies against CD244 and CD137 in serum-free medium were amplified about 5-fold, and cell purity reached 75%.
Control group 2 NK cells cultured by adding only tumor antigen CEA in serum-free culture medium are amplified about 6 times, and the cell purity reaches 85%.
Control group 3 NK cells cultured in serum-free medium with only monoclonal antibodies against CD244 and CD137 were amplified about 7-fold, and cell purity reached 86%.
The results show that the three-dimensional scaffold can remarkably improve the amplification efficiency and purity of NK cells. The tumor antigen CEA and the monoclonal antibodies against CD244 and CD137 in the serum-free medium also have good promotion effect on the amplification efficiency and purity of NK cells, and the effect of the combined application of the two groups is better!
Example 2 functional detection of NK cells
The killing activity of NK cells on K562 tumor cells was examined by LDH release method. After co-culturing the expanded NK cells with K562 cells at different potency target ratios (e=40:1, 20:1, 10:1, 5:1) for 4 hours, the release of LDH by cell lysis was determined. The experimental results are shown below:
when e=40:1, the killing rate is 91%;
when e=20:1, the killing rate is 85%;
When e=10:1, the killing rate is 77%;
e=5:1, the kill rate was 62%.
Control group 1 NK cells (e=10:1) cultured without addition of tumor antigen CEA and monoclonal antibodies against CD244 and CD137 in serum-free medium had a killing rate of only 42%.
Control group 2 NK cells (e=10:1) cultured with serum-free medium supplemented with tumor antigen CEA alone had a killing rate of 56%.
Control group 3 NK cells (e=10:1) cultured with serum-free medium supplemented with only monoclonal antibodies against CD244 and CD137 had a killing rate of 63%.
The above results show that NK cells prepared using the serum-free medium of the present invention have significantly improved killing activity and better effect than the addition of only the tumor antigen CEA and only the monoclonal antibodies against CD244 and CD 137.
EXAMPLE 3 treatment study of NK cells in mouse model
1. Tumor model establishment, wherein 6-8 week old male nude mice are selected, and each of the mice is injected with human colorectal cancer cells HCT116 (1X 10 6 cells/mouse) and subcutaneously planted on the back. After about 10 days of implantation, the tumor forms and reaches about 100mm 3. Mice were randomly divided into 3 groups of 10:
The experimental group, in which NK cells prepared by the invention were injected, each mouse was injected with 5X 10 6 cells once every 7 days for 3 consecutive weeks;
Control group 1, in which non-amplified NK cells were injected at the same frequency as that of the experimental group;
control group 2 an equal volume of PBS solution was injected.
2. Experimental results:
2.1 tumor volume change on day 21 after injection treatment, the tumor volume of the experimental group was reduced by 60% on average (initial volume 100mm 3 and final volume 40.+ -.10 mm 3), whereas the tumor volumes of control group 1 and control group 2 were increased by 120% (220.+ -.15 mm 3) and 180% (280.+ -.20 mm 3), respectively.
2.2 Survival rate of mice in the experimental group were 100% at 21 days, and the survival rates of the control group 1 and the control group 2 were 75% and 50%, respectively.
2.3 Toxicity detection, namely detecting the body weight, liver and kidney functions and blood biochemical indexes of all mice, wherein no obvious toxic or side effect is found in mice in an experimental group, the body weight is basically kept stable, and the liver and kidney functions are normal.
The conclusion is that the NK cells of the experimental group show remarkable anti-tumor effect and have no obvious toxicity, which indicates that the NK cells amplified by the method have good anti-tumor activity in vivo.
Example 4 preparation and inspection of three-dimensional scaffolds
1. Preparation of three-dimensional scaffolds
(1) Gelatin solution preparation 2g gelatin was dissolved in 100mL deionized water at 50 ℃ and stirred well until completely dissolved. Gold nanoparticles (Gold nanoparticles,10nm, final concentration of 1% w/v) were added and stirring continued for 30 min to ensure uniform dispersion.
(2) The preparation of the chitosan solution comprises dissolving 2g of chitosan in 100mL of 1% glacial acetic acid solution, and stirring at room temperature until the chitosan is completely dissolved to form the chitosan solution. Mixing chitosan solution and gelatin solution according to a ratio of 1:1, and stirring uniformly.
(3) Pouring the mixed solution into a special silica gel mold to form a uniform film. Frozen at 4 ℃ for 24 hours to be completely solidified.
(4) And (3) freeze drying, namely drying the frozen bracket in a freeze dryer for 48 hours. This step forms a three-dimensional porous structure with high porosity.
(5) Crosslinking treatment, namely crosslinking treatment is carried out on the dried stent by using 0.1% glutaraldehyde aqueous solution, and the stent is soaked for 2 hours to improve the mechanical strength of the stent. Wash with ethanol 3 times for 10 minutes each to remove excess crosslinker. The stent was rinsed again with deionized water and dried at 37 ℃.
(6) Final shaping and sterilization after drying the scaffolds, cutting into dimensions suitable for experimental use (scaffolds of the invention are discs of 10mm diameter). Sterilizing with gamma rays or ultraviolet rays for later use.
2. Inspection of three-dimensional scaffolds
(1) Porosity analysis, namely analyzing the pore structure of the three-dimensional scaffold by a Scanning Electron Microscope (SEM), wherein the porosity reaches 90%, and the pore size is 50-100 mu m, so that the three-dimensional scaffold is suitable for cell attachment and proliferation. Experimental results show that the addition of the gold nanoparticles enables the pore structure of the scaffold to be more uniform, and the interaction between cells and the scaffold is improved.
(2) And (3) mechanical strength test, namely using a material testing instrument to test the compression strength of the three-dimensional bracket. The results showed that the mechanical strength of the scaffolds with gold nanoparticles added was about 20% higher than that of the scaffolds without addition. The compression modulus of the bracket is 200kPa, and the bracket is suitable for in vitro cell culture and transplantation.
(3) Cell proliferation and attachment experiments NK cells were cultured on a three-dimensional scaffold, and the attachment rate and proliferation capacity of the cells were examined. Cell proliferation was measured 3, 5 and 7 days after culture using the CCK-8 method. The experimental result shows that the cell attachment rate is 85%, and the proliferation multiple of the cells is 6 times after 7 days of culture and is obviously higher than 3 times proliferation under the traditional two-dimensional culture condition.
(4) And the biocompatibility test shows that the scaffold has no obvious toxicity to cells under different concentrations and has good biocompatibility through the detection of the cytotoxicity of the scaffold by an MTT method.
Example 5 preparation and testing of monoclonal antibodies against CD244 and CD137
1. Preparation of monoclonal antibodies
The polypeptide (with the amino acid sequence shown as SEQ ID NO. 1) of the CD244 protein (with the positions aa 70-aa 80 and the amino acid sequence SLLIKAAQQQD) and the polypeptide (with the amino acid sequence shown as SEQ ID NO. 2) of the CD137 protein (with the positions aa 122-aa 132 and the amino acid sequence SVLVNGTKERD) are designed and synthesized. And coupling the polypeptide with KLH, and mixing the polypeptide with the KLH according to the equal mass ratio to prepare the immunogen. After emulsification of the immunogen, 6 week old mice were immunized 3-4 times, 0.1 mg/time/mouse, 2-3 weeks apart. After eligibility using ELISA, spleen cells were mixed with myeloma cells at a ratio of 8:1, and cell fusion was mediated with PEG. The fused cells are inoculated in a 96-well plate, the HAT culture medium is used for screening hybridoma cells, and antibody activity is detected after the cells are cloned and grown. The antibody secretion positive clones were detected using ELISA, and high affinity hybridoma cells were selected for further expansion culture. Purifying the antibody by using a protein A/G affinity chromatographic column, filtering, sterilizing and subpackaging the purified antibody, and storing at-80 ℃ for later use.
2. Monoclonal antibody assay
(1) The concentration of the purified monoclonal antibody was measured, and the BCA kit assay result was 2.56mg/ml.
(2) The purified monoclonal antibody is subjected to SDS-PAGE detection, and the result is shown in figure 1, and the purity of the SDS-PAGE of the monoclonal antibody can reach more than 95%.
(3) The RNA extracted from the hybridoma cells is sequenced by a third party service company (e.g., nanjin Style) to detect the amino acid sequences of the heavy and light chain variable regions of the monoclonal antibody. The detection result shows that the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4.
(4) ELISA detection is carried out on the binding activity of the prepared monoclonal antibody and commercial antibody to target protein, firstly, recombinant human CD244 protein (ab 185248) and recombinant human CD137 protein (ab 287936) are respectively coated on an ELISA plate at the concentration of 1 mug/ml, and then, three antibodies are diluted to 1 mug/ml and are respectively added into the ELISA plate, and detection is carried out by using the corresponding secondary antibodies. The results are shown in Table 1, and the higher OD values of the monoclonal antibodies of the invention against CD244 and CD137 indicate that the monoclonal antibodies have better binding activity than commercial monoclonal antibodies.
TABLE 1 antibody Activity assay results (OD 450 nm)
| Group of | CD244 protein coating | CD137 protein coating |
| Monoclonal antibodies of the invention | 2.12、2.09 | 1.98、2.01 |
| Anti-CD244 antibody (ab 313993) | 1.34、1.41 | 0.101、0.087 |
| Anti-CD137antibody(ab89085) | 0.097、0.095 | 1.45、1.38 |
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (8)
1. The monoclonal antibody for resisting CD244 and CD137 is 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 as SEQ ID NO.3 and SEQ ID NO.4 respectively.
2. A method of preparing NK cells, comprising the steps of:
(1) PBMC separation PBMC were separated from peripheral blood using Ficoll-Paque density gradient centrifugation;
(2) Enrichment of CD56+ NK cells from the PBMC isolated in step (1) using immunomagnetic beads method;
(3) NK cell expansion and activation, namely inoculating the NK cells enriched in the step (2) into a three-dimensional bracket culture system at the concentration of 5 multiplied by 10 5 cells/mL, simultaneously culturing the NK cells by using a serum-free culture medium containing the monoclonal antibodies against CD244 and CD137 as set forth in claim 1, changing 50% of the culture medium every 2 days, and continuously culturing for 7 days to collect the NK cells.
3. The method of claim 2, wherein the three-dimensional scaffold in step (3) is a 10mm diameter disc.
4. The method according to claim 2, wherein the three-dimensional scaffold in step (3) has a porosity of up to 90% and a pore size of 50-100 μm.
5. The method of claim 2, wherein the three-dimensional scaffold in step (3) has a compression modulus of 200kPa.
6. The method of claim 2, wherein the serum-free medium of step (3) is formulated as follows:
(1) RPMI-1640, which accounts for 50% of the volume of the NK cell serum-free culture solution;
(2) L-glutamine 2mM;
(3) Glucose 25mM;
(4) Vitamin C100. Mu.M;
(5)IL-2100U/mL,IL-1550ng/mL,IL-2120ng/mL;
(6) Tumor antigen CEA 100ng/mL;
(7) anti-CD 244 and CD137 monoclonal antibody 20ng/mL.
7. Use of the monoclonal antibodies against CD244 and CD137 according to claim 1 for the preparation of NK cells.
8. Use of the method of claim 2 for the preparation of NK cells.
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