Amplification method of NK cells and application thereof

Abstract

The invention relates to a solid-phase carrier compound for amplifying NK cells, an amplification method of the NK cells and application of the NK cells, the solid-phase carrier compound is loaded with cell factors, and the cell factors comprise IL12, IL15, IL21, CD137L and CD16ab.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and in particular to a solid phase carrier complex for amplifying NK cells and a method for amplifying NK cells. Background Art

[0002] NK cells, also known as natural killer cells, are important immune cells in the human body, 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 lymphocytes that can non-specifically kill tumor cells and virus-infected cells without prior sensitization. Usually, according to their source, NK cells are divided into NK cells separated from peripheral blood, NK cells separated from umbilical cord blood, NK cells differentiated from pluripotent stem cells, etc. NK cells can achieve anti-tumor purposes through a variety of pathways, such as through ADCC, release of granzymes and perforins, etc. to exert their own anti-tumor effects. Currently, NK cells have been widely used in the process of tumor immunotherapy. However, NK cells also face the problem of short lifespan and unmet demand.

[0003] The NK cell preparation method disclosed in the prior art usually uses feeder cells, such as K562 cells. K562 cells are human chronic myeloid leukemia cells. The use of K562 cells can make NK cells obtain better amplification effects, but it is inevitable to bring potential risks in clinical treatment, thereby reducing the safety of NK cells. The methods for culturing NK cells without feeder cells disclosed in the prior art mostly use cytokines and immunomagnetic beads. Immunomagnetic beads (hereinafter referred to as magnetic beads) are based on immunology and have been widely used in the fields of immunoassay and cell separation. Magnetic beads are usually composed of core metal magnetic particles, polymer materials (such as polystyrene, polyvinyl chloride) wrapped in the outer layer of the core, and the outermost functional groups (such as -NH2, -COOH, -OH, -CHO). Under the action of magnetic adsorption, magnetic beads can separate substances that specifically bind to magnetic beads from the solution, which also plays an important role in NK cell amplification. Prior art CN105602899A discloses a method for inducing NK cell proliferation in peripheral blood mononuclear cells with interleukin 21 and 4-1BBL. The technical solution disclosed in the patent is to co-culture the initial NK cells with solid-phase cytokines to proliferate NK cells. The experimental results show that the lymphocyte population in unscreened human peripheral blood is directly stimulated by immobilized cytokines, and primary NK cells with killing activity are amplified in large quantities. However, the NK cell multiples amplified by this method are still low, which is difficult to meet the demand for NK cells in terms of quantity, and the NK cell purity is low, which will affect the clinical treatment effect.

[0004] In the process of amplifying NK cells using cytokines and immunomagnetic beads, in addition to interleukin 21 and 4-1BBL, commonly used cytokines include interleukin 15, interleukin 18, CD86, and MICA. When using cytokines to amplify NK cells, not only the amplification multiple of NK cells should be considered, but also the killing performance of the amplified NK cells should be considered.

[0005] How to increase the expansion multiple of NK cells while ensuring their safety and improving their killing performance is one of the main research directions in this field. Summary of the invention

[0006] The inventors of the present invention unexpectedly discovered in their research that when cytokines such as IL12, IL21, CD137L, IL15 and CD16ab are added to the culture medium, the amplification efficiency of NK cells can be greatly improved, and when these cytokines are loaded on a solid phase carrier, the amplification effect is better. Even more surprisingly, the NK cells obtained by this method have a stronger cell killing ability. Based on this, the present invention is completed.

[0007] In a first aspect, the present invention provides a solid phase carrier complex for amplifying NK cells, wherein the complex comprises a solid phase carrier and cytokines loaded on the solid phase carrier, and the cytokines comprise IL12, IL15, IL21, CD137L and CD16ab.

[0008] In some embodiments of the present invention, the solid phase carrier is a natural material or an artificial synthetic material. Preferably, the solid phase carrier is a magnetic bead or a glass bead. More preferably, the solid phase carrier is a magnetic bead.

[0009] In some embodiments of the present invention, the cytokines are loaded on the same solid phase carrier, such as magnetic beads of the same type and model.

[0010] In some embodiments, the cytokines are loaded on the same solid phase carrier, for example, 1, 2, 3, 4 or 5 cytokines are loaded on the same magnetic bead. In other words, one magnetic bead is loaded with 1, 2, 3, 4 or 5 cytokines.

[0011] In other embodiments, the cytokine is not loaded on the same solid phase carrier. In some embodiments, IL12, IL15, IL21, CD137L and CD16ab are loaded on different solid phase carriers respectively. In other embodiments, IL12, IL15, IL21, CD137L and CD16ab are loaded on different numbers of solid phase carriers in different combinations, for example, one magnetic bead is loaded with 1 factor, and another magnetic bead is loaded with 4 factors; or one magnetic bead is loaded with 2 factors, and another magnetic bead is loaded with 3 factors. In a specific embodiment, IL12, IL21 and CD137L are loaded on the same solid phase carrier A, IL15 and CD16ab are loaded on another solid phase carrier B, and A and B are different individuals of the same solid phase carrier.

[0012] In other embodiments of the present invention, the cytokines are loaded onto different types of solid phase carriers, such as magnetic beads and glass beads.

[0013] In some embodiments, the cytokine is loaded on 2, 3, 4 or 5 solid phase carriers. For example, IL12, IL15, IL21, CD137L and CD16ab are loaded on 5 different solid phase carriers respectively. Those skilled in the art can select different types of solid phase carriers as needed and make different combinations without special restrictions.

[0014] In other embodiments, IL12, IL21 and CD137L are loaded on the same solid phase carrier I, and IL15 and CD16ab are loaded on another solid phase carrier II.

[0015] In some embodiments of the present invention, the molar ratio of IL12, IL21, CD137L, IL15 and CD16ab is 1:0.1-5:0.1-7:0.1-6:0.1-5, preferably 1:0.5-5:0.5-7:0.5-6:0.5-5, more preferably 1:1-5:1-7:1-6:1-5. The molar amount referred to here refers to the molar ratio of IL12, IL21, CD137L, IL15 and CD16ab added when preparing the complex.

[0016] In some embodiments of the present invention, the NK cells are NK cells separated from peripheral blood, or NK cells differentiated from progenitor cells derived from bone marrow, umbilical cord blood or peripheral blood, or NK cells differentiated by induction of pluripotent stem cells.

[0017] Herein, proteins include naturally occurring proteins and protein variants that retain their inherent properties. In some embodiments of the present invention, IL12 is IL12 protein or a modified IL12 protein or a variant thereof; IL21 is IL21 protein or a modified IL21 protein or a variant thereof; CD137L is CD137L protein or a modified CD137L protein or a variant thereof; IL15 is IL15 protein or a modified IL15 protein or a variant thereof, preferably, IL15 is a fusion protein of IL15 and IL15Ra; and / or CD16ab is an anti-human CD16 specific antibody or antigen-binding fragment, including but not limited to a monoclonal antibody, a bispecific antibody, a multispecific antibody, a scFv, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, and a Fv fragment.

[0018] In a second aspect, the present invention provides a method for preparing the complex described in the first aspect, comprising: contacting the cytokine with the solid phase carrier, wherein the cytokine and the solid phase carrier respectively carry a pairing part that can bind to each other through affinity. Pairings that can bind to each other known in the art can be used in the present invention. This binding can be reversible or irreversible, is one of the non-covalent interactions known in nature, and has good tolerance.

[0019] In some embodiments of the present invention, the pairing part capable of binding to each other by affinity is an affinity tag and a binding fragment thereof, wherein the affinity tag is selected from His tag, glutathione sulfhydryl transferase (GST), S-peptide, ZZ domain, albumin binding domain (ABD), HA, Myc, FLAGTM, maltose binding protein (MBP), calmodulin binding peptide (CBP), SUMO, biotin, streptococcal protein G (Protein G) and Staphylococcus aureus protein A (Protein A). For example, the affinity tag and the binding fragment thereof are biotin and avidin, and the avidin is selected from avidin, streptavidin, NeutrAvidinTM and avidin-like, preferably streptavidin. For example, the affinity tag and the binding fragment thereof are tags Protein A and Fc. The term "Fc" or "Fc part" used herein is well known to those skilled in the art.

[0020] In a third aspect, the present invention provides a composition comprising the solid phase carrier complex as described in the first aspect, or a solid phase carrier complex prepared by the method described in the second aspect.

[0021] In a fourth aspect, the present invention provides a method for expanding NK cells, comprising contacting the solid phase carrier complex described in the first aspect, the solid phase carrier complex prepared by the method described in the second aspect, or the composition described in the third aspect with NK cells, and culturing them together. In some embodiments, the cytokine can be directly added to the culture medium without using a solid phase carrier.

[0022] In a fifth aspect, the present invention provides a method for enhancing the killing ability of NK cells, comprising contacting NK cells with the solid phase carrier complex described in the first aspect and culturing them together. In some embodiments, the cytokine can be directly added to the culture medium without using a solid phase carrier.

[0023] In some embodiments of the present invention, the NK cells are NK cells separated from peripheral blood, or NK cells differentiated from progenitor cells derived from bone marrow, umbilical cord blood or peripheral blood, or NK cells differentiated by induction of pluripotent stem cells.

[0024] In other embodiments of the present invention, the method further comprises adding cytokine IL2 when culturing NK cells. Preferably, the amount of IL2 is 10 IU-2500 IU / mL, and more preferably, the amount is 1000 IU / mL.

[0025] In a sixth aspect, the present invention provides NK cells prepared according to the method described in the fourth aspect or the fifth aspect, and use of the obtained NK cells in preparing drugs for treating tumors.

[0026] In a seventh aspect, the present invention provides a method for killing tumor cells in vitro, comprising contacting tumor cells with NK cells of the present invention, and the method is not used for diagnosis and treatment of diseases.

[0027] In a specific embodiment of the present invention, cytokines IL12, IL15, IL21, CD137L and CD16ab are specifically loaded onto a solid phase carrier, which is more effective in amplifying NK cells than directly adding the corresponding cytokines and has stronger cytotoxicity.

[0028] The present invention particularly optimizes the dosage of each cytokine. During the preparation of the solid phase carrier, the dosage ratio of IL12, IL21, CD137L, IL15 and CD16ab is preferably 1:1-5:1-7:1-6:1-5, and more preferably 1:1:1:1:1, based on the added molar amount, to achieve better amplification multiples and cytotoxicity.

[0029] The present invention loads cytokines IL12, IL15, IL21, CD137L and CD16ab on a solid phase carrier to prepare a multi-cytokine loaded solid phase carrier complex, and uses this solid phase carrier complex to amplify NK cells, achieving amazing amplification multiples and cytotoxicity. And more preferably, when the dosage ratio of IL12, IL21, CD137L, IL15 and CD16ab added is 1:0.1-5:0.1-2:1-4:0.1-5, its amplification effect and cytotoxicity are better. The solid phase carrier complex for amplifying NK cells and the method for amplifying NK cells provided by the present invention can obtain a larger amplification multiple and stronger cytotoxicity, further meet the requirements for the number of NK cells in clinical practice, and promote the application of NK cells in tumor treatment drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The image shows the morphology of NK cells during expansion, taken on Day 14. Scale bar: 200 μm.

[0031] Figure 2 The figure shows the killing effect of NK cells on K562 cells after different treatments and expansion. The vertical axis indicates the proportion of K562 cells that still maintain a good state after co-culture to the total K562 cells. The lower the percentage, the more cells are killed and the stronger the killing ability of NK cells. DETAILED DESCRIPTION

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as understood by one of ordinary skill in the art.

[0033] Although the numerical ranges and parameter approximations shown in the broad scope of the present invention, the numerical values ​​shown in the specific embodiments are recorded as accurately as possible. However, any numerical value is bound to contain a certain error, which is caused by the standard deviation in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, the range of "1 to 10" recorded should be considered to include any and all sub-ranges between the minimum value 1 and the maximum value 10 (including the endpoints); that is, all sub-ranges starting with a minimum value of 1 or greater, such as 1 to 6.1, and sub-ranges ending with a maximum value of 10 or less, such as 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0034] It should also be noted that, as used in this specification, singular forms include plural forms of their referents unless clearly and unambiguously limited to one referent. The term "or" can be used interchangeably with the term "and / or" unless the context clearly indicates otherwise.

[0035] In the present invention, some quantities are represented by nen, where n is a specific number, for example, 2e4 represents 2×10 4 . Example

[0036] Below in conjunction with specific examples, some preferred embodiments and aspects of the present invention are further described, which should not be construed as limiting the scope thereof. The methods, steps, etc. not specifically described in the following examples are all methods and steps known to those skilled in the art, or are operated according to the instructions of the commercially available products. The various experimental reagents used in the examples are all commercially available products, and the same products used in different processes or experimental steps are from the same source. Example 1: Preparation of solid support complex

[0037] In this example, the affinity between different paired molecules is used to load cytokines onto solid phase carrier magnetic beads. The magnetic beads used in this example are Dynabeads_M280_Streptavidin (purchased from ThermoFisher, item number 11206D) and Dynabeads Protein A (purchased from Thermo Fisher, item number 10002D).

[0038] 1.1Beads loaded with cytokines IL12, IL21 and CD137L

[0039] The cytokine contains a biotin tag, which is ideally located at an inactive position of the cytokine or at another position that does not affect the activity. The surface of the beads is modified with streptavidin, and the cytokine is coated on the surface of the beads through the binding of biotin and streptavidin.

[0040] The cytokines containing biotin tags in this embodiment are IL12 (purchased from Acro, item number IL2-H8210), IL21 (purchased from Acro, item number IL1-H82F7) and CD137L (purchased from Acro, item number 41L-H82F9). The streptavidin modified Beads is Dynabeads_M280_Streptavidin (purchased from Thermo Fisher, item number 11206D), with a specification of 10 mg / mL and a binding capacity of 200 pmol biotinylated peptide per mg. The biotinylated peptides described herein refer to cytokines containing biotin tags, such as IL12, IL21 and CD137L.

[0041] For specific coating operation steps, please refer to the instruction manual of Dynabeads_M280_Streptavidin. The implementation steps include 1) washing beads; 2) coating beads: incubating the factors with beads to coat the factors on the beads; 3) recovering the beads, resuspending them in a specific buffer to make the target specifications, and storing them for future use. Here, the target specification is 4e7 beads / mL; the specific buffer is PBS / BSA (PBS, pH 7.4; 0.01% [w / v] BSA) or PBST (PBS pH 7.4; 0.01% [v / v] Tween TM -20), where Tween TM -20 content can be increased to 0.1%.

[0042] The amount of cytokines used when coating Beads can be adjusted according to experimental needs. For example, when the amounts of IL12, IL21 and CD137L are equimolar, it is recorded as B12 / 21 / 137L-1 / 1 / 1; or when the amounts of IL12, IL21 and CD137L are unequal molar, for example, 1:1:7, it is recorded as B12 / 21 / 137L-1 / 1 / 7. Here, B is the abbreviation of Beads.

[0043] 1.2Beads loaded with cytokines IL15 and CD16ab

[0044] The binding of cytokines to magnetic beads can also be carried out by other methods known in the prior art that are not dependent on biotin-streptavidin. For example, the cytokines are coated on beads using the binding ability of Protein A to Ig Fc. Cytokines are coated on the surface of beads modified by Protein A through the binding of Fc to Protein A. Here, the cytokine contains Fc; the Fc is generally located at the C-terminus of the cytokine peptide chain or other positions that do not affect the activity. When the cytokine is an antibody, it generally contains Fc itself, does not require additional Fc, and may also contain a set Fc. When the cytokine is a non-antibody peptide chain, Fc can also play the role of purification, stabilization factor, etc.

[0045] In this embodiment, the cytokine IL15 containing Fc is IL15 protein or a fusion protein of IL15 and IL15Ra, the latter of which is generally more likely to activate the IL15 signaling pathway and has stronger biological activity. In this embodiment, the preferred cytokine IL15 is a fusion protein of IL15 and IL15Ra with an Fc tag (purchased from Seafrom, item number GMP-TL801), and the Fc contained is human IgG1. Factor CD16ab is an anti-human CD16 specific antibody (purchased from Seafrom, item number GMP-TL201), and the Fc contained is human IgG1. Protein A modified Beads is Dynabeads Protein A (purchased from Thermo Fisher, item number 10002D), with a specification of 30 mg / mL and a binding capacity of 100 μl Dynabeads (3 mg) can separate about 25-30 μg of human IgG from a sample containing 20-200 μg IgG / ml. For specific coating operation steps, refer to the instruction manual of Dynabeads Protein A.

[0046] The amount of cytokines used when coating Beads can be adjusted according to experimental needs. For example, when the amount of IL15 and CD16ab used is equimolar, it is recorded as B15 / 16ab-1 / 1; or when the amount of IL15 and CD16ab used is unequal molar, such as 6:1, it is recorded as B15 / 16ab-6 / 1. Here, B is the abbreviation of Beads.

[0047] The Beads-cytokine products prepared above were detected by flow cytometry. Beads were treated as "cells", and the flow cytometry staining and detection were performed in the same manner as conventional flow cytometry detection protocols in the art. The corresponding commercial antibodies were used to detect IL12, IL15 and IL21, and CD137 protein and CD16 protein were used to detect the coating of CD137L and CD16ab, respectively. The experimental results showed that a clear and narrow signal peak could be detected, indicating that the corresponding factors had been successfully loaded on the surface of the Beads. Example 2: Expansion of NK cells

[0048] The beads prepared in Example 1 were used to amplify NK cells, and the amplification multiple of NK cells was detected.

[0049] 2.1 Isolation of NK cells

[0050] The NK cells used in this example were obtained from the peripheral blood of healthy people. 50-100 mL of peripheral blood was collected and peripheral blood mononuclear cells (PBMC) were isolated within 2 hours. NK cell isolation was performed using EasySep TMHuman NK Cell Isolation Kit (purchased from Stem Cell, catalog number 17955) was used and the steps were followed according to the product manual.

[0051] 2.2 Expansion of NK cells using beads

[0052] In this example, the beads prepared in Example 1 were used to amplify NK cells, and a control group was used without adding beads. Culture medium (purchased from Miltenyi Biotec, catalog number 130-114-429). Cytokine IL2 (purchased from Sansheng Pharmaceutical) was optionally added to all groups, with an amount of 10 IU-2500 IU / mL, preferably 1000 IU / mL. There is no particular limitation on IL2 in the specific embodiments of the present invention.

[0053] The specific expansion and culture steps are as follows:

[0054] 1) Take NK cells and inoculate them in a culture plate, with an initial cell density of 4e5-2e6 / mL, preferably 1e6 / mL. After fully dispersing the magnetic beads, add beads at a ratio of Beads:Cells=1:1, and add equal amounts of beads loaded with IL-12 / 15 / 21 / CD137L factors and beads loaded with IL15 / CD16ab factors, i.e., the number of both types of beads is 50% of the number of cells. Gently mix the beads / cells. Culture in a 37°C, 5% CO2 incubator. This is recorded as Day 0.

[0055] 2) On Day 3, add an equal volume of culture medium.

[0056] 3) Day 5: Gently resuspend and mix the cells and add fresh culture medium. The amount of culture medium added is based on the cell count results, and the cell density is readjusted to 4e5-2e6 / mL, preferably 8e5-1e6 / mL.

[0057] 4) Thereafter, depending on the cell growth status, the culture medium is replenished every 1-3 days according to the cell number.

[0058] The control group #4 was set up, and the corresponding factors were directly added on Day 0, with the dosage of 10 ng / mL each of factors 12 / 21 / 137L and 10 ng / mL each of factors IL15 / CD16ab. The blank control group #5 did not add the above cytokines.

[0059] The aggregation and expansion characteristics of NK cells were significantly enhanced in the presence of beads, and the expansion multiples were also significantly increased. Taking the number of cells on Day 0 as the base number 1, the number of NK cells at different time points was calculated to obtain the expansion multiples. The specific results are shown in Table 1.

[0060] Table 1 NK cell expansion fold under stimulation of different beads

[0061] Comparing group #4 with group #5, see Table 2, it can be seen that adding IL12, IL21, CD137L, IL15 and CD16ab to the culture medium can significantly promote NK cell proliferation.

[0062] Table 2 Amplification fold of group #4 compared to group #5

[0063] Comparing Group #1, 2, 3 with Group #5, see Table 3, it can be seen that when the solid phase carrier is used to load the cytokines, the expansion multiple of NK cells is further significantly increased, and the effect is unexpected.

[0064] Table 3 Amplification folds of groups #1, 2, 3 compared to group #5

[0065] It is worth noting that simply increasing the amount of a certain cytokine cannot increase the expansion multiple of NK cells, see groups #2 and #3, where the amount of IL15 in group #2 is 6 times that of group #1, and the amount of CD137L in group #3 is 7 times that of group #1.

[0066] Observe the morphology of NK cells during proliferation under a microscope, see Figure 1 When the Beads-based amplification scheme was used, NK cells showed more cell aggregation in morphology. When the Beads-based amplification scheme was not used, the cell morphology aggregation was significantly weaker and the morphology was poor. Example 3: NK cell killing ability detection

[0067] The inventors unexpectedly discovered that NK cells prepared by the method of the present invention have enhanced cell killing ability (also referred to as cytotoxicity herein).

[0068] The different NK cells obtained by amplification of scheme #1-5 in Example 2 were co-cultured with K562 cells in vitro for 24 hours, and the culture medium was K562 complete medium (RPMI 1640 + 10% FBS). Then flow cytometry was used to detect the level of cell apoptosis and evaluate the killing efficiency of NK cells on K562 cells. The co-culture ratio was 0.5:1 and 1:1 for NK cells: K562 cells. Annexin V-APC (Biolegend, catalog number 640920) and Zombie NIR (Biolegend, catalog number 423105) were used to detect apoptosis of K562 cells. During the process of cells being killed by NK cells, their cell membranes will change. In the early stage, phosphatidylserine (PS) inside the cell membrane will be exposed and recognized by Annexin V antibodies; in the later stage, the integrity of the cell membrane will be damaged, and more Zombie NIR dyes will enter the cells. Therefore, cells that are Annexin V negative and Zombie NIR negative (indicated as Annexin V- / NIR- in the figure) are surviving cells that maintain a better state. The experimental results are as follows Figure 2 As shown, NK cells amplified using Beads prepared in Example 1 had significantly enhanced cytotoxicity, and the survival rate of K562 was significantly lower than that of control group #4 and blank control group #5, among which group #1 showed stronger cytotoxicity, which is very attractive for applications that require enhanced NK cell toxicity, such as tumor treatment, and shows good application potential.

[0069] All disclosures and patents mentioned in this application are incorporated herein by reference. Without departing from the scope and spirit of the present invention, multiple modifications and variants of the described method and composition of the present invention are apparent to those skilled in the art. Although the present invention has been described by specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. In fact, those multiple variants of the described mode for implementing the present invention that are apparent to those skilled in the relevant art are intended to be included in the scope of the appended claims.

Claims

1. A solid phase carrier complex for amplifying NK cells, wherein the complex comprises a solid phase carrier and cytokines loaded on the solid phase carrier, and the cytokines comprise IL12, IL15, IL21, CD137L and CD16ab.

2. The complex according to claim 1, wherein the solid phase carrier is a natural material or an artificial synthetic material, preferably, the solid phase carrier is magnetic beads or glass beads, and more preferably, the solid phase carrier is magnetic beads. The complex according to claim 1 or 2, wherein the cytokines are loaded on the same solid phase carrier. The complex according to claim 3 , wherein the cytokines are loaded on the same solid phase carrier.

5. The complex according to claim 3, wherein the cytokines are not loaded on the same solid phase carrier, for example, IL12, IL15, IL21, CD137L and CD16ab are loaded on different solid phase carriers, respectively. For another example, IL12, IL21 and CD137L are loaded on the same solid phase carrier A, and IL15 and CD16ab are loaded on another solid phase carrier B. The complex according to claim 1 or 2 , wherein the cytokines are loaded on different solid phase carriers. The complex according to claim 6 , wherein the cytokine is loaded on 2, 3, 4 or 5 solid phase carriers.

8. The complex according to claim 7, wherein IL12, IL15, IL21, CD137L and CD16ab are loaded on five different solid phase carriers respectively.

9. The complex according to claim 7, wherein IL12, IL21 and CD137L are loaded on the same solid phase carrier I, and IL15 and CD16ab are loaded on another solid phase carrier II.

10. The complex according to any one of claims 1 to 9, wherein the molar ratio of IL12, IL21, CD137L, IL15 and CD16ab is 1:0.1-5:0.1-7:0.1-6:0.1-5, preferably 1:0.5-5:0.5-7:0.5-6:0.5-5, more preferably 1:1-5:1-7:1-6:1-5.

11. The complex according to any one of claims 1 to 10, wherein the NK cells are NK cells separated from peripheral blood, or NK cells differentiated from progenitor cells derived from bone marrow, umbilical cord blood or peripheral blood, or NK cells differentiated by induction of pluripotent stem cells.

12. According to the complex according to any one of claims 1 to 11, wherein IL12 is IL12 protein or a modified IL12 protein or a variant thereof; IL21 is IL21 protein or a modified IL21 protein or a variant thereof; CD137L is CD137L protein or a modified CD137L protein or a variant thereof; IL15 is IL15 protein or a modified IL15 protein or a variant thereof, preferably, IL15 is a fusion protein of IL15 and IL15Ra; and / or CD16ab is an anti-human CD16 specific antibody or antigen-binding fragment.

13. A method for preparing a composite according to any one of claims 1 to 12, comprising: The cytokine is contacted with the solid phase carrier, wherein the cytokine and the solid phase carrier respectively carry a pairing part that can bind to each other through affinity.

14. The method according to claim 13, wherein the pairing parts capable of binding to each other by affinity are affinity tags and binding fragments thereof, and the affinity tags are selected from His tags, glutathione S-transferase (GST), S-peptide, ZZ domain, albumin binding domain (ABD), HA, Myc, FLAGTM, maltose binding protein (MBP), calmodulin binding peptide (CBP), SUMO, biotin, streptococcal protein G (Protein G) and Staphylococcus aureus protein A (Protein A).

15. The method according to claim 14, wherein the affinity tag and its binding fragment are biotin and avidin, and the avidin is selected from avidin, streptavidin, neutravidin (NeutrAvidinTM) and avidin-like, preferably streptavidin. The method according to claim 14 , wherein the affinity tag and its binding fragment are tags Protein A and Fc.

17. A composition comprising the solid phase carrier complex according to any one of claims 1 to 12, or a solid phase carrier complex prepared by the method according to any one of claims 13 to 16.

18. A method for expanding NK cells, comprising: The solid phase carrier complex described in any one of claims 1 to 12, the solid phase carrier complex prepared by the method described in any one of claims 13 to 16, or the composition described in claim 17 is contacted with NK cells and cultured together.

19. A method for enhancing the killing ability of NK cells, comprising contacting NK cells with the solid phase carrier complex described in any one of claims 1-12 and culturing them together.

20. The method according to claim 18 or 19, wherein the NK cells are NK cells separated from peripheral blood, or NK cells differentiated from progenitor cells derived from bone marrow, cord blood or peripheral blood, or NK cells differentiated by induction of pluripotent stem cells.

21. The method according to any one of claims 18-20, wherein cytokine IL2 is optionally added, preferably, the amount of IL2 is 10 IU-2500 IU / mL, more preferably, the amount is 1000 IU / mL.

22. A NK cell with enhanced cell-killing ability, prepared according to the method according to any one of claims 18-21.

23. Use of the NK cells according to claim 22 in the preparation of drugs for treating tumors.

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