A reagent combination for efficiently activating NK cells and its application

Abstract

The present invention relates to a reagent combination for efficiently activating NK cells and its application. Specifically, the present invention provides a culture method using recombinant protein PVR and / or Nectin-2 as an activation reagent, activating and culturing NK cells in a container coated with an activation reagent, without the addition of serum and trophoblast cells, and can achieve efficient amplification of NK cells. The culture process of the present invention avoids high-risk materials, has high safety, is particularly suitable for the amplification and culture of iNK cells obtained by differentiation of iPSCs, and can meet the production needs of high-quality NK cell products.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a reagent combination for efficiently activating NK cells and an application thereof. Background Art

[0002] Immune cell therapy is to transplant or return normal or bioengineered immune cells to the patient, using these cells to enhance the immune killing function, thereby achieving the purpose of treating the disease. NK cells, the full name of which is Natural Killer cells, are important immune cells in the body and the first line of defense of the human immune system. They play an important role in the innate and acquired immune defenses against viral infections and tumors. The therapy of infusing NK cells cultured in vitro into the patient's body is called NK cell therapy.

[0003] Efficient and safe in vitro cultivation of NK cells is of great significance for NK cell therapy. In order to achieve better NK expansion, most of the existing NK expansion platforms use expansion methods with serum and trophoblast cells. For methods that require the addition of human or animal serum to the culture system, the source of serum is not only limited, but also the stability between batches is poor, and there are risks in clinical safety. From a regulatory perspective, although there is no explicit ban on the use of serum in cell production processes, its residue and safety assessment require additional attention.

[0004] The trophoblast cell expansion method requires the addition of trophoblast cells to the expansion of NK cells. Common trophoblast cells include chronic myeloid leukemia K562 cells (K562 cells are genetically modified to introduce 4-1BB ligands, membrane-bound IL-15, membrane-bound IL-21, etc., which can further enhance the NK cell induction activity), RPMI8866 and EBV-LCL, etc. These tumor cells have low expression of inhibitory signals and high expression of activation signals on their surfaces. Co-culturing NK cells with them can achieve NK cell activation and large-scale expansion. Compared with the non-trophoblast cell expansion method, the trophoblast cell expansion method can usually expand NK better, but trophoblast cells are tumor cells. Although they are inactivated by irradiation and other methods before use, the residues of tumor trophoblasts, including the residues of their degradation products, are safety issues that need to be considered during the development of cell therapy products.

[0005] Therefore, there is an urgent need in the art to develop a serum-free, trophoblast-free method that can efficiently expand NK cells. Summary of the invention

[0006] The purpose of the present invention is to provide a serum-free, trophoblast-free method for efficiently expanding NK cells, which uses PVR protein and Nectin-2 protein as activation reagents to achieve efficient and safe in vitro expansion of NK cells.

[0007] In a first aspect of the present invention, a method for expanding natural killer (NK) cells in vitro is provided, comprising the steps of:

[0008] (a) providing a cell culture container, wherein the solid surface in the cell culture container is coated with recombinant protein nectin-2 and / or PVR after being treated with a coating solution;

[0009] (b) Cultivating NK cells using the cell culture container in step (a).

[0010] In another preferred embodiment, the method does not require the addition of serum or trophoblastic cells.

[0011] In another preferred embodiment, the step (a) comprises: preparing a coating solution, wherein the coating solution comprises recombinant protein Nectin-2 and PVR; and coating the solid surface with the coating solution.

[0012] In another preferred embodiment, the concentration of Nectin-2 in the coating solution is 0.1-10 μg / ml; preferably 0.5-5 μg / ml; more preferably 1-3 μg / ml.

[0013] In another preferred embodiment, the concentration of PVR in the coating solution is 0.1-10 μg / ml; preferably 0.5-5 μg / ml; more preferably 1-3 μg / ml.

[0014] In another preferred embodiment, the solid surface in the cell culture container is coated with the recombinant proteins Nectin-2 and PVR at the same time after being treated with the coating solution.

[0015] In another preferred embodiment, the ratio of Nectin-2 to PVR in the coating solution is (1:5) to (5:1).

[0016] In another preferred embodiment, the ratio of Nectin-2 to PVR in the coating solution is (1:2) to (2:1).

[0017] In another preferred embodiment, the solid surface in the cell culture container is further coated with CD16 antibody after being treated with the coating solution.

[0018] In another preferred embodiment, the concentration of CD16 antibody in the coating solution is 0.1-10 μg / ml; preferably 0.5-5 μg / ml; more preferably 1-3 μg / ml.

[0019] In another preferred embodiment, the Nectin-2 and PVR are dissolved in a buffer solution to prepare the coating solution.

[0020] In another preferred embodiment, the buffer is selected from the group consisting of phosphate buffer, carbonate buffer, or a combination thereof.

[0021] In another preferred embodiment, the buffer solution is DPBS solution.

[0022] In another preferred embodiment, the volume of the coating solution added to each square centimeter of the solid surface is 0.1-0.5 mL.

[0023] In another preferred embodiment, the volume of the coating solution added to each square centimeter of the solid surface is 0.15-0.35 mL.

[0024] In another preferred embodiment, the volume of the coating solution added to each square centimeter of the solid surface is 0.15-0.25 mL.

[0025] In another preferred embodiment, the protein successfully coated on the solid surface accounts for at least 70% of the total protein content in the coating solution; preferably at least 80%; more preferably at least 90%.

[0026] In another preferred embodiment, the content of Nectin-2 successfully coated on the surface of the solid phase carrier is 0.03-3 μg / cm 2 , the content of PVR is 0.03-3μg / cm 2 Preferably, the content of Nectin-2 is 0.05-2.5 μg / cm 2 , the content of PVR is 0.05-2.5μg / cm 2 More preferably, the content of Nectin-2 is 0.1-2 μg / cm 2 , the content of PVR is 0.1-2μg / cm 2 .

[0027] In another preferred embodiment, in step (a), the solid surface is a surface of a cell culture container, and the cell culture container is selected from the group consisting of a culture dish, a multi-well cell plate, a shake flask, or a combination thereof.

[0028] In another preferred embodiment, the solid phase surface is a magnetic bead surface.

[0029] In another preferred embodiment, the coating treatment conditions are: incubation at 4°C±1°C overnight, or incubation at 37°C±3°C for 2-4 hours.

[0030] In another preferred embodiment, the coating treatment conditions are: incubation at 4°C overnight, or incubation at 37°C for 2 hours.

[0031] In another preferred embodiment, the step (b) further comprises:

[0032] (b1) After the cell culture container in step (a) is coated, the coating solution is removed, and culture medium and NK cells to be cultured are immediately added to perform activation culture to obtain activated NK cells;

[0033] (b2) The NK cells activated in step (b1) are transferred to a culture container for dynamic expansion culture, and the expanded NK cells are harvested.

[0034] In another preferred embodiment, the culture media in step (b1) and step (b2) are the same or different.

[0035] In another preferred embodiment, the culture medium comprises a NK cell culture medium selected from the group consisting of: OptiVitro NK, CTS TM NK-Xpander, Irvine TM PRMIE-XV NK Cell CDM, or a combination thereof.

[0036] In another preferred embodiment, cytokines are also added to the culture medium.

[0037] In another preferred embodiment, the cytokine is selected from the following group: IL-2, IL-12, IL-15, IL-18, IL-21, or a combination thereof.

[0038] In another preferred embodiment, the concentrations of the cytokines are: IL-2 (100-2000 IU / ml), IL-12 (5-100 ng / ml), IL-15 (5-100 ng / ml), IL-18 (5-100 ng / ml) and IL-21 (10-200 ng / ml).

[0039] In another preferred embodiment, the concentrations of the cytokines are: IL-2 (150-1000 IU / ml), IL-12 (10-50 ng / ml), IL-15 (5-50 ng / ml), IL-18 (5-50 ng / ml) and IL-21 (10-100 ng / ml).

[0040] In another preferred embodiment, in step (b1), the activated culture is static culture.

[0041] In another preferred embodiment, the activation culture is performed for 2 to 10 days; preferably, 3 to 8 days; more preferably, 4 to 6 days.

[0042] In another preferred embodiment, in step (b1), culture fluid and cytokines are added to the culture medium every 2 to 3 days.

[0043] In another preferred embodiment, in step (b2), the expansion culture is three-dimensional cell culture or dynamic culture on a shaker.

[0044] In another preferred embodiment, in step (b2), culture fluid and cytokines are added to the culture medium every 1 to 3 days.

[0045] In another preferred embodiment, the NK cells are iNK cells differentiated from induced pluripotent stem cells (iPSCs).

[0046] In another preferred embodiment, the NK cells are engineered NK cells, such as CAR-NK cells.

[0047] In another preferred embodiment, the NK cells cultured by the method have one or more of the following characteristics:

[0048] (1) Amplification factor ≥ 150 times; preferably ≥ 200 times; more preferably ≥ 300 times;

[0049] (2) Purity ≥ 97%; preferably ≥ 98%; more preferably ≥ 99%;

[0050] (3) When the effector-target ratio (E:T) is (1:1) to (3:1), the cytotoxicity to target cells is ≥70%, preferably ≥80%, and more preferably ≥90%.

[0051] In another preferred embodiment, the target cells are tumor cells.

[0052] In another preferred embodiment, the target cells are K562 cells.

[0053] In the second aspect of the present invention, a use of a recombinant protein combination is provided, wherein the recombinant protein combination is a combination of Nectin-2 and PVR, and is used to prepare an activation reagent, wherein the activation reagent is coated into a solid phase carrier and is used for activation and expansion of NK cells.

[0054] In another preferred embodiment, the activation reagent comprises: (i) an effective amount of Nectin-2 and PVR; and (ii) a diluent or a carrier.

[0055] In another preferred embodiment, the concentration of Nectin-2 in the activation reagent is 0.1-10 μg / ml; preferably 0.5-5 μg / ml; more preferably 1-3 μg / ml.

[0056] In another preferred embodiment, the concentration of PVR in the activation reagent is 0.1-10 μg / ml; preferably 0.5-5 μg / ml; more preferably 1-3 μg / ml.

[0057] In another preferred embodiment, the ratio of Nectin-2 to PVR in the activation reagent is (1:5) to (5:1).

[0058] In another preferred embodiment, the ratio of Nectin-2 to PVR in the activation reagent is (1:2) to (2:1).

[0059] In another preferred embodiment, the diluent or carrier is selected from the following group: phosphate buffer, carbonate buffer, or a combination thereof.

[0060] In another preferred embodiment, the diluent or carrier is DPBS solution.

[0061] In the third aspect of the present invention, a recombinant protein-coated solid phase carrier is provided, the surface of the solid phase carrier is coated with recombinant protein Nectin-2 and recombinant protein PVR, and the solid phase carrier is used to activate and culture NK cells.

[0062] In another preferred embodiment, the solid phase carrier is a cell culture container or magnetic beads.

[0063] In another preferred embodiment, the cell culture container is selected from the following group: a culture dish, a multi-well cell plate, a shake flask, or a combination thereof.

[0064] In a fourth aspect of the present invention, a kit for activating cultured NK cells is provided, the kit comprising a solid phase carrier coated with the recombinant protein as described in the third aspect of the present invention.

[0065] In another preferred embodiment, the kit further comprises an instruction manual, wherein the instruction manual describes a method for activating and culturing NK cells using a solid phase carrier coated with the recombinant protein.

[0066] In another preferred embodiment, the method comprises the following steps:

[0067] (S1) providing NK cells to be cultured;

[0068] (S2) Activating and culturing the NK cells using the solid phase carrier coated with the recombinant protein.

[0069] In a fifth aspect of the present invention, a method for preparing a solid phase carrier coated with a recombinant protein as described in the third aspect of the present invention is provided, characterized in that the method comprises the steps of:

[0070] (Y1) preparing a coating solution, wherein the coating solution comprises recombinant protein Nectin-2 and PVR;

[0071] (Y2) coating the solid surface with the coating solution.

[0072] In another preferred embodiment, the concentration of Nectin-2 in the coating solution is 0.1-10 μg / ml; preferably 0.5-5 μg / ml; more preferably 1-3 μg / ml.

[0073] In another preferred embodiment, the concentration of PVR in the coating solution is 0.1-10 μg / ml; preferably 0.5-5 μg / ml; more preferably 1-3 μg / ml.

[0074] In another preferred embodiment, the ratio of Nectin-2 to PVR in the coating solution is (1:5) to (5:1).

[0075] In another preferred embodiment, the ratio of Nectin-2 to PVR in the coating solution is (1:2) to (2:1).

[0076] In another preferred embodiment, the content of the coating solution added to the surface of the solid phase carrier per square centimeter is 0.1-0.5 mL; preferably 0.15-0.35 mL; more preferably 0.15-0.25 mL.

[0077] In another preferred embodiment, the protein successfully coated on the surface of the solid carrier accounts for at least 70% of the total protein content in the coating solution; preferably at least 80%; more preferably at least 90%.

[0078] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0079] The beneficial effects of the present invention include:

[0080] (1) The NK cell activation and expansion method of the present invention can obtain NK cells with a high expansion multiple, and the highest expansion multiple can reach nearly 400 times.

[0081] (2) The NK cells obtained by the NK cell activation and expansion method of the present invention have high purity and high cell killing activity.

[0082] (3) The NK cell activation and expansion method of the present invention is serum-free and trophoblast-free, thus avoiding the risk of introduction of animal-derived factors brought by materials and increasing the safety of the product.

[0083] (4) The NK cell activation and expansion method of the present invention has a simple process flow and reduces the cost of quality control;

[0084] (5) The NK cell activation and expansion method of the present invention is universal and applicable to NK cells from different sources, especially iPSC-derived iNK cells, and the effect is consistent and stable in different batches of iNK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 The expansion rate of NK cells in culture with different activation protocols is shown.

[0086] Figure 2 The expansion rates of different activation protocols at day 12 of culture are shown.

[0087] Figure 3 The results show the cytotoxic activity of NK cells activated and expanded by different schemes against K562 tumor cells at different effector-target ratios.

[0088] Figure 4 The expression of marker proteins of NK cells activated and expanded by different flow cytometry assays is shown.

[0089] Figure 5 The expansion rate of NK cells after activation and expansion in Example 4 during the culture process is shown.

[0090] Figure 6 The results show the killing activity of NK cells activated and expanded in Example 4 against K562 tumor cells at different effector-target ratios.

[0091] Figure 7 The expression of the marker proteins of the activated and expanded NK cells in Example 4 is shown.

[0092] Figure 8 The expression of activation receptors and inhibitory receptors on the surface of NK cells after activation and expansion in Example 4 is shown. DETAILED DESCRIPTION

[0093] After extensive and in-depth research, the inventors have developed a culture method using recombinant proteins PVR and Nectin-2 as activation reagents, and NK cells are activated and cultured in a container coated with the activation reagent. The culture method of the present invention is serum-free and does not require the addition of trophoblast cells, and can achieve efficient amplification of NK cells, which is significantly better than the commonly used methods in the field. The culture process of the present invention avoids high-risk materials and has high safety, and is particularly suitable for iNK cells differentiated from iPSCs. The NK cells finally obtained have high purity and strong killing activity, which meet the production of high-quality NK cell products.

[0094] On this basis, the present invention has been completed.

[0095] definition

[0096] 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.

[0097] As used herein, NK cells refer to natural killer cells, which are important immune cells of the body and core cells of innate immunity. In the present invention, the source of the NK cells is not limited, for example, it can include NK cells derived from peripheral blood or NK cells differentiated from iPCS.

[0098] As used herein, the terms "NK cells differentiated from iPSC", "iPSC-NK cells" or "iNK" are used interchangeably and are produced by induced pluripotent stem cells (iPSC) in vitro through specific culture and induced differentiation. The preparation process includes inducing iPSC to form hematopoietic progenitor cells, and then further inducing differentiation into NK cells. NK cells differentiated from iPSC can be expanded on a large scale, do not rely on specific donors, can provide a more sufficient source of cells, and reduce production costs.

[0099] As used herein, the term "activation culture" refers to the addition of an activation agent that has an activation effect on NK cells at the initial stage of in vitro culture of NK cells to fully activate the proliferation ability, killing ability, etc. of NK cells.

[0100] As used herein, the term "expansion culture" refers to removing the activation agent after the activation culture of NK cells, and continuing to culture the NK cells under conditions without the activation agent, so that the NK cells can be expanded in large quantities to obtain a sufficient number of NK cells.

[0101] As used herein, the term "trophoblast cells" is also called "Feeder cells". When added during the culture of NK cells, it can promote the proliferation and activation of NK cells and enhance their killing activity. Trophoblast cells are usually tumor cells.

[0102] As used herein, the term "recombinant protein" refers to a protein obtained using recombinant DNA or recombinant RNA technology. The target gene is first obtained through gene cloning or chemical synthesis technology, connected to a suitable expression vector, and then introduced into a specific host cell to express a functional protein molecule using the genetic system of the host cell.

[0103] As used herein, the term "coating" is a treatment method for fixing a specific substance on a solid surface or the surface of a cell culture vessel.

[0104] NK cell activation reagent

[0105] The activation of NK cells depends on the abnormal expression of MHC molecules and the activation of NK cell surface ligands. There are a variety of activating and inhibitory receptors on the surface of NK cells. These receptors mediate multiple signal pathways by binding to corresponding ligands, respectively realizing the activation and inhibition of NK cells. NK cells need to be fully activated to induce tumor cell killing. Activated NK cells can be amplified in large quantities with high purity and high cytotoxicity.

[0106] In the present invention, an activation reagent for in vitro culture of NK cells is provided, and the reagent comprises: recombinant PVR protein, Nectin-2 protein or a combination thereof.

[0107] PVR protein, also known as poliovirus receptor (CD155, ECL-5), is a member of the immunoglobulin superfamily and has many important biological functions. The accession number of human PVR protein is P15151.

[0108] Nectin-2 protein, also known as nectin cell adhesion molecule 2, also known as CD112 and PVRL2, is an important immunoglobulin-like cell adhesion molecule. The accession number of human nectin-2 protein is Q92692.

[0109] In the present invention, coating Nectin-2, PVR protein or a combination thereof on the surface of a cell culture container to culture NK cells can significantly activate the proliferation ability of NK cells and improve the expansion rate of NK cells during the culture process.

[0110] In the present invention, Nectin-2 and PVR proteins can be used alone as NK cell activation agents or in combination. The combination of Nectin-2 and PVR has a synergistic effect on the activation of NK cells, that is, the effect of the combined use is higher than the sum of the effects of Nectin-2 protein alone or PVR protein alone on NK cell activation.

[0111] In addition, the present invention also found that Nectin-2 and PVR proteins have a significant effect on the proliferation of NK cells only in the coated state, but have no significant activation effect in the solution state. Therefore, Nectin-2 and PVR proteins can be coated on the surface of cell culture containers, such as culture dishes and multi-well plates, for subsequent NK cell culture; Nectin-2 and PVR proteins can also be coated on the surface of magnetic beads for NK cell culture.

[0112] NK cell activation and culture method of the present invention

[0113] At present, the production of NK cells mostly uses serum or trophoblastic cells, which not only has problems with consistency and stability between batches of materials, but also has safety risks such as material residues and the introduction of animal-derived factors. In view of this shortcoming, the present invention has developed a serum-free and trophoblast-free NK expansion process, which avoids the risk of introducing animal-derived factors brought by materials, and does not use trophoblastic cells (tumor cells), which can minimize the unfavorable factors in process production complexity and quality cost control, as well as concerns about product supervision and safety.

[0114] The present invention provides a method for activating and culturing NK cells using recombinant protein Nectin-2 and PVR as activation reagents.

[0115] The recombinant proteins Nectin-2 and PVR of the present invention can only exert activation effects in a coated state, so a coating solution containing both Nectin-2 and PVR proteins is first prepared, in which the concentration of Nectin-2 is 0.1-10 μg / ml; preferably 0.5-5 μg / ml; more preferably 1-3 μg / ml; the concentration of PVR is 0.1-10 μg / ml; preferably 0.5-5 μg / ml; more preferably 1-3 μg / ml.

[0116] Preferably, the ratio of Nectin-2 to PVR in the coating solution is (1:5) to (5:1); more preferably (1:2) to (2:1). In a preferred embodiment, the ratio of Nectin-2 to PVR is 1:2, 1:1 or 2:1, all of which have a higher activation level.

[0117] After coating the surface of the cell culture container with the coating solution, NK professional culture medium is added to culture NK cells. In the present invention, the type of NK culture medium is not limited, preferably a serum-free NK culture medium. Preferably, a cytokine combination is added to the culture medium, such as IL-2 (100-2000 IU / ml), IL-12 (5-100 ng / ml), IL-15 (5-100 ng / ml), IL-18 (5-100 ng / ml) and IL-21 (10-200 ng / ml).

[0118] Preferably, the NK cell activation culture process of the present invention mainly includes two stages: an activation culture stage with the addition of nectin-2 and PVR, and an expansion culture stage without the addition of an activator.

[0119] In the activation culture stage, NK cells are inoculated into a container coated with Nectin-2 and PVR and statically activated in a culture medium. In a preferred embodiment, this stage lasts for 5 days, and an equal volume of culture medium and cytokines are added on the 3rd day. Then, the expansion culture stage is entered.

[0120] In the expansion culture stage, no activation reagent is added, and NK cells are expanded and cultured using three-dimensional cell culture and / or on a shaker. The culture medium used in this stage is the same as the activation stage. This process lasts for 10 to 12 days, and the culture medium and cytokines are supplemented every 1 to 3 days depending on the cell growth. NK cells are harvested.

[0121] Optionally, the harvested NK cells are tested for expansion multiple, purity, cell killing activity, etc.

[0122] The activation culture method of the present invention can harvest NK cells with high expansion multiples, high purity and high cell killing activity, and no serum and trophoblast cells are added in the process, so the process is simple and safe.

[0123] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and fractions are weight percentages and weight fractions.

[0124] Materials and Methods

[0125] 1. Materials

[0126] 1. Culture medium

[0127] In the embodiments of the present invention, a serum-free NK-specific culture medium, OptiVitro NK culture medium, is used, and no trophoblast cells are added during the culture process.

[0128] 2. NK cells

[0129] In the embodiment of the present invention, iNK cells obtained by differentiation of reprogrammed iPSC cells are used as a source of NK cells for expansion and culture.

[0130] 3. Activation Reagents

[0131] In the embodiments of the present invention, recombinant PVR protein and / or recombinant Nectin-2 protein are used as NK cell activators, and CD16 monoclonal antibody (mAb-CD16) is used as a control activator. The recombinant proteins and antibodies are commercially available, recombinant PVR protein (ACRO, catalog number: CD5-H5251), recombinant Nectin-2 protein (ACRO, catalog number: PV2-H5253), mAb-CD16 (Excellbio, catalog number: BA0112).

[0132] NK cell activation culture program

[0133] Scheme 1. PVR+Nectin-2 coated activated NK cells

[0134] (1) Preparation of coating solution: Using DPBS as diluent, dissolve PVR and Nectin-2 recombinant proteins in DPBS to prepare coating solutions with the following concentration ratios: Nectin-2 (1.5 μg / ml) + PVR (1.5 μg / ml), Nectin-2 (1 μg / ml) + PVR (2 μg / ml), Nectin-2 (2 μg / ml) + PVR (1 μg / ml), Nectin-2 (1 μg / ml) + PVR (1 μg / ml). Add the coating solution to the culture dish on the NTC surface, cover the surface with the liquid, and incubate overnight at 4°C.

[0135] (2) Resuspend the iPSC-derived iNK cells to a density of 2-10E5 using serum-free NK-specific culture medium, and add IL-2 (concentration 200 IU / ml), IL-12 (concentration 20 ng / ml), IL-15 (concentration 10 ng / ml), IL-18 (concentration 5 ng / ml), and IL-21 (concentration 10 ng / ml) to the culture medium.

[0136] (3) Take out the culture plate that has been coated overnight, remove the coating solution, add the iNK cell suspension with cytokines to the coated culture plate, incubate in a 37°C, 5% CO2 incubator for 3 days, and activate by static activation.

[0137] (4) After 3 days, add the same volume of fresh culture medium as in the previous culture system, and continue to add IL-2 (concentration 200 IU / ml), IL-12 (concentration 20 ng / ml), IL-15 (concentration 10 ng / ml), IL-18 (concentration 5 ng / ml), and IL-21 (concentration 10 ng / ml), and then let it rest for activation for another 2 days.

[0138] (5) After a total of 5 days of activation, the activated iNK cells were transferred to a shake flask. At the same time, fresh culture medium was added to dilute the activated cells to a density of 2~10E5, and IL-2 (concentration 200IU / ml), IL-12 (concentration 20ng / ml), IL-15 (concentration 10ng / ml), IL-18 (concentration 5ng / ml), and IL-21 (concentration 10ng / ml) were added. The shake flask was placed in a shaker and cultured dynamically at 45rpm.

[0139] (6) The culture medium and IL-2 (concentration 200 IU / ml), IL-12 (concentration 20 ng / ml), IL-15 (concentration 10 ng / ml), IL-18 (concentration 5 ng / ml), and IL-21 (concentration 10 ng / ml) were then added every 1 to 3 days and culture was continued for 10 to 15 days. After washing and freezing, the expanded NK cells were obtained.

[0140] Scheme 2. Activation and expansion of NK cells by PVR or Nectin-2 alone

[0141] The coating solution in step (1) was prepared as a coating solution containing only PVR or only Nectin-2 using the method of Scheme 1. The concentrations of the coating solutions were: PVR (0.5 μg / ml), PVR (1 μg / ml), PVR (2 μg / ml), PVR (3 μg / ml), Nectin-2 (0.5 μg / ml), Nectin-2 (1 μg / ml), Nectin-2 (2 μg / ml), Nectin-2 (3 μg / ml). The other steps were the same as Scheme 1.

[0142] Protocol 3. Activation and expansion of NK cells by soluble PVR+Nectin-2 in culture medium

[0143] The method of Scheme 1 was adopted, and the coating solution in step (1) was replaced with DPBS without any protein. Nectin-2 (1.5 μg / ml) + PVR (1.5 μg / ml) proteins were directly added to the culture medium in step (2), so that the two proteins were dissolved in the culture medium to activate iNK in a non-solid-bound form. The other steps were the same as Scheme 1.

[0144] Scheme 4. mAb-CD16 coated activated NK cells

[0145] Activating NK cells with CD16 antibodies is a commonly used activation method in the art. The method of Scheme 1 was used, and the coating solution in step (1) was replaced with a coating solution containing mAb-CD16 (3 μg / ml). The other steps were the same as Scheme 1.

[0146] Example 1: Comparison of the effects of different culture schemes on activating and expanding NK cells

[0147] With other conditions being the same, iNK cells were activated and expanded using methods from Schemes 1 to 4, and a negative control group was set up to culture without adding activation proteins or antibodies (No-activation group).

[0148] During the expansion and culture process of each group, the cell growth status was recorded, the cell count was performed on the 4th, 6th, 8th, 10th and 12th days, and the NK cell expansion curve was drawn. The results are shown in Figure 1 shown.

[0149] The ability of NK cells to expand is very poor without adding activation reagent (No-activated) or without coating activation solution (Dissolved Nectin-2 and PVR in medium); the culture scheme in which recombinant PVR or Nectin-2 protein is coated on the culture plate can activate NK and expand it in large quantities, and the activated NK expansion ability is better than CD16 monoclonal antibody. Moreover, the combined use is better than the use of recombinant PVR or recombinant Nectin-2 alone. In addition, the experimental results unexpectedly found that recombinant PVR and Nectin-2 proteins can only activate NK cells when coated on the culture plate, and cannot activate NK cells when added only to the culture medium. Recombinant PVR and recombinant Nectin-2 proteins can achieve a high level of activation effect when combined in a ratio of 1:2, 1:1 or 2:1.

[0150] Figure 2 The bar graph shows the results on the 12th day of culture to more intuitively demonstrate the comparison of the total expansion multiples of NK cell culture under different schemes. The results show that the scheme of the present invention using a combination of recombinant PVR protein and recombinant Nectin-2 protein to coat and culture NK cells can increase the expansion effect after activation by about 50% compared with CD16 monoclonal antibody.

[0151] In addition, the effect of PVR+Nectin-2 coating was better than the sum of the effects of PVR or Nectin-2 coating alone, indicating that PVR and Nectin-2 proteins have a synergistic effect on the activation of NK cells.

[0152] Example 2: Comparison of the killing effects of NK cells cultured in different schemes

[0153] After 12 days of expansion, NK cells were collected and co-cultured with tumor cells K562 cells at a certain ratio, and the ratio of NK cells to K562 cells (i.e., E:T) was 0.5:1, 1.7:1, and 5:1, respectively. The starting cell amount of K562 was 3e5 / ml, and the proportion of K562 live cells (ANNEXIN V- / 7AAD-) was detected by flow cytometry after 12 hours of co-culture, so as to calculate the killing ratio of NK cells.

[0154] The results of NK cell killing effect on target cells (Annexin V-7AAD-surviving target cells) are as follows Figure 3 The results showed that NK cells activated by recombinant PVR and recombinant Nectin-2 proteins at different concentrations had high killing activity against K562 tumor cells. When E:T=1.7:1, the killing efficiency could reach more than 95%. There was no significant difference in the killing ability of NK cells obtained by different culture schemes.

[0155] Example 3: Purity of NK cells cultured in different protocols

[0156] The expression of the iconic surface proteins of NK cells is: CD45 positive, CD56 positive and CD3 negative. The expression of the iconic proteins of NK cells on the 12th day of amplification, including CD45, CD56 and CD3, was detected by flow cytometry to detect the purity of NK.

[0157] The results are as follows Figure 4 As shown. The flow cytometry results showed that no matter whether mAb-CD16 or Nectin-2+PVR was used, the obtained NK cells would highly express immune cell marker proteins CD45 and CD56, with the positive rates of CD45 and CD56 both above 99%, and almost no T cell marker protein CD3 was expressed. The above results indicate that the present invention adopts the method of activating and culturing NK cells by coating with Nectin-2+PVR combination to obtain high-purity NK cells to meet the needs of producing and preparing NK products.

[0158] Example 4: Effect verification of the culture scheme of the present invention

[0159] Different batches of iPSC-derived iNK cells were used to verify the effect of the combination of Nectin-2 + PVR on activating NK cells. The concentration of Nectin-2 (1.5 μg / ml) + PVR (1.5 μg / ml) was selected for culture plate coating, and the other culture steps were the same as those in Scheme 1 in Materials and Methods. The control group was coated with mAb-CD16 (3 μg / ml), which is Scheme 4 in Materials and Methods.

[0160] (1) Cell expansion multiple

[0161] After 11 days of activation and expansion, the cell expansion times of each group were detected. The results are as follows Figure 5 The results showed that Nectin-2 (1.5 μg / ml) + PVR (1.5 μg / ml) had a superior activation effect, which could promote the expansion of NK cells by nearly 400 times. Compared with CD16 monoclonal antibody, it showed a better effect in promoting NK activation and expansion, which was about 1.8 times that of CD16 monoclonal antibody.

[0162] (2) Cell killing ability

[0163] After 11 days of culture, NK cells were collected and co-cultured with tumor cells K562 cells at a certain ratio, and the ratio of NK cells to K562 cells (ie, E:T) was 0.3:1, 1:1, and 3:1, respectively. The method for detecting cell killing ability was the same as in Example 2.

[0164] The results are as follows Figure 6 As shown, NK activated by Nectin-2 (1.5μg / ml) + PVR (1.5μg / ml) has high killing activity against K562 tumor cells. When E:T=1:1, the killing efficiency can reach more than 90%, and the killing effect is better than the control group CD16 monoclonal antibody under the condition of low efficiency target ratio.

[0165] (3) Cell purity

[0166] The scheme of Example 3 was used to detect the expression of NK cell marker proteins by flow cytometry. The results are as follows: Figure 7 The results showed that the purity of NK cells activated by Nectin-2 (1.5μg / ml) + PVR (1.5μg / ml) was 99.8%.

[0167] (4) Cell surface receptor expression

[0168] Flow cytometry was used to detect the expression of activating and inhibitory receptors on the surface of NK cells activated by mAb-CD16 and Nectin-2 (1.5 μg / ml) + PVR (1.5 μg / ml) on day 11, including NKp46, NKG2D, NKp30 and NKp44.

[0169] The results are as follows Figure 8As shown, the results showed that different activation reagents had no significant effect on the expression of NK activation receptors after expansion. The expression rate of activation receptor NKp46 was about 50%, the expression rate of NKG2D was 100%, the expression rate of NKp30 was about 70%, and the expression rate of NKp44 was about 80%. For inhibitory receptors NKG2A and CD94 that inhibit NK cell activation and killing function, the expression ratio of NKG2A and CD94 in iPSC-NK activated by mAb-CD16 was higher than that in iPSC-NK activated by Nectin-2 (1.5μg / ml) + PVR (1.5μg / ml).

[0170] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A serum-free, trophoblast-free in vitro method for expanding natural killer (NK) cells, characterized in that: Includes steps: (a) providing a cell culture container, wherein the solid surface of the cell culture container is coated with recombinant proteins Nectin-2 and PVR after being treated with a coating solution; the concentration of Nectin-2 in the coating solution is 0.1-10 μg / ml, and the concentration of PVR is 0.1-10 μg / ml; and the ratio of Nectin-2 to PVR in the coating solution is (1:5) to (5:1); (b) Cultivating NK cells using the cell culture container in step (a).

2. The method according to claim 1, characterized in that The content of Nectin-2 successfully coated on the surface of the solid phase carrier is 0.03-3 μg / cm 2 , the content of PVR is 0.03-3μg / cm 2 .

3. The method according to claim 1, characterized in that The concentration of Nectin-2 in the coating solution is 0.5-5 μg / ml; the concentration of PVR in the coating solution is 0.5-5 μg / ml.

4. The method according to claim 1, characterized in that The ratio of Nectin-2 to PVR in the coating solution is (1:2) to (2:1).

5. The method according to claim 1, characterized in that The step (b) further comprises: (b1) After the cell culture container in step (a) is coated, the coating solution is removed, and culture medium and NK cells to be cultured are immediately added to perform activation culture to obtain activated NK cells; (b2) The NK cells activated in step (b1) are transferred to a culture container for dynamic expansion culture, and the expanded NK cells are harvested.

6. The method according to claim 5, characterized in that Cytokines are also added during the culture process, and the cytokines are selected from the following group: IL-2, IL-12, IL-15, IL-18, IL-21, or a combination thereof.

7. The method according to claim 1, characterized in that The NK cells are iNK cells differentiated from induced pluripotent stem cells (iPSCs).

8. The method according to claim 1, characterized in that The NK cells cultured by the method have one or more of the following characteristics: (1) Amplification factor ≥ 150 times; (2) Purity ≥ 97%; (3) When the effector-target ratio (E:T) is between (1:1) and (3:1), the cytotoxicity to target cells is ≥70%.

9. A use of a recombinant protein combination, characterized in that: The recombinant protein combination is a combination of Nectin-2 and PVR, which is used to prepare an activation reagent. The activation reagent is coated into a solid phase carrier and is used for the activation and expansion of NK cells.

10. A recombinant protein-coated solid phase carrier, characterized in that: The surface of the solid phase carrier is coated with recombinant protein Nectin-2 and recombinant protein PVR, and the solid phase carrier is used for activating and cultivating NK cells.

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