Serum-free NK cell culture medium

Abstract

Disclosed herein are activators and expanders for NK cell culture, coating agents for NK cell culture vessels, serum-free media for activation and expansion steps of NK cell culture, and methods of NK cell culture.

Description

Technical Field

[0001] This application relates to the fields of biotechnology and medical technology, specifically to serum-free culture medium for human NK cells and its culture protocol. Background Technology

[0002] NK (natural killer) cells are innate immune cells of the human body's innate immune system, derived from hematopoietic stem cells. The development of NK cells from hematopoietic stem cells depends on the bone marrow and thymus microenvironment. Mature NK cells are mainly distributed in the bone marrow, peripheral blood, liver, spleen, lungs, and lymph nodes. Compared to T cells, allogeneic NK cells have not caused graft-versus-host disease (GVHD) and cytokine release syndrome (CRS) after clinical infusion, and play a broader role in cell immunotherapy. NK cells are not restricted by MHC molecules and can non-specifically recognize and kill target cells. Their main killing pathways are as follows:

[0003] 1) Perforin / Granzyme pathway: NK cells release cytotoxic particles such as perforin and granzyme through exocytosis, activating the caspase pathway to induce target cell necrosis or apoptosis.

[0004] 2) Cytokine pathway: Activated NK cells can synthesize and secrete a variety of cytokines, such as IFN-γ, TNF-α, IL-10, IL-5, IL-8, and GM-CSF, which induce apoptosis in target cells;

[0005] 3) Death receptor pathway: NK cells express Fas (CD95) ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) molecules, which induce apoptosis in CD95+ target cells and TRAIL receptor-positive target cells through a cascade of endogenous enzyme reactions.

[0006] 4) ADCC pathway: Fc receptors (CD16, FcRIIIγ) on the surface of NK cells can bind to antibodies, thereby mediating antibody-dependent cell-mediated cytotoxicity (ADCC).

[0007] 5) Immune checkpoint pathway: Activate NK cells using immune checkpoint blocking antibodies.

[0008] NK cells are mainly divided into two subtypes, including CD3. - CD16 - CD56 + NK Bright and CD3 - CD16+ CD56 + NKDim cells. The former mainly secretes cytokines to kill target cells, while the latter not only highly expresses perforin and granzymes with cytotoxic capabilities, which can directly kill target cells, but also highly expresses CD16, thereby mediating ADCC to enhance the killing ability against target cells. Therefore, NK DIM cells generally have a stronger killing ability.

[0009] NK cells account for only 5-15% of lymphocytes in the blood; therefore, expanding and obtaining sufficient quantities of NK cells in vitro is a prerequisite for immunotherapy. It is worth noting that current human NK cell cultures generally require the addition of human serum to the culture medium, while some require serum-free media with the addition of autologous human plasma, and others use culture methods containing feeder cells. There is an urgent need in this field for improved or alternative methods for NK cell culture. Summary of the Invention

[0010] In one aspect, this application provides an activator for NK cell culture, comprising interleukin-type NK cell activating factors, fat-soluble vitamins, trace elements, and serum substitutes.

[0011] In some implementations, the activator is used for serum-free culture of NK cells.

[0012] In some embodiments, the interleukin-type NK cell activating factor is selected from any combination of IL2, IL7, IL15, IL12, IL18, IL21, or more.

[0013] In some embodiments, the activator also comprises nicotinamide.

[0014] In some embodiments, the activator further comprises dimercaptoethanol.

[0015] In some embodiments, the activator also comprises sodium heparin.

[0016] In some embodiments, the activator further comprises one or more of reduced glutathione, human transferrin, and human serum albumin.

[0017] In some embodiments, the activator further comprises quercetin.

[0018] In some implementations, the NK cells are derived from peripheral blood mononuclear cells (PBMCs).

[0019] In some implementations, the NK cells are derived from umbilical cord blood NK cells.

[0020] In some implementations, the NK cells are obtained from in vitro culture of NK cell lines.

[0021] In some embodiments, the NK cells are obtained from in vitro induction and culture of induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (MSCs).

[0022] In some embodiments, the interleukin-like NK cell activating factors are IL2 and IL15.

[0023] In some embodiments, the interleukin-like NK cell activating factors are IL2, IL15, and IL21.

[0024] In some embodiments, the fat-soluble vitamin is selected from one or more of the following: vitamin A, vitamin E, and vitamin K.

[0025] In some implementations, the vitamin K group is vitamin K1.

[0026] In some embodiments, the fat-soluble vitamins include vitamin A, vitamin E, and vitamin K1.

[0027] In some embodiments, the trace element is provided in the form of a compound selected from those containing one or more of the following elements: selenium, iron, zinc, copper, nickel, cobalt, chromium, silver, aluminum, barium, iodine, vanadium, rubidium, germanium, manganese, tin, and molybdenum.

[0028] In some embodiments, the trace element is provided in the form of one or more compounds selected from the following: sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride, and stannous chloride.

[0029] In some embodiments, the compounds providing the trace elements include sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride, and stannous chloride.

[0030] In some embodiments, the serum substitute is platelet lysate.

[0031] In some embodiments, the activator comprises IL2, IL15, vitamin A, vitamin E, vitamin K1, sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride and stannous chloride, platelet lysate, nicotinamide, dimercaptoethanol, sodium heparin, reduced glutathione, human transferrin and human serum albumin.

[0032] In some embodiments, the activator comprises IL2, IL15, IL21, vitamin A, vitamin E, vitamin K1, sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride and stannous chloride, platelet lysate, nicotinamide, dimercaptoethanol, sodium heparin, reduced glutathione, human transferrin and human serum albumin.

[0033] Secondly, this application provides an amplification agent for NK cell culture, which comprises interleukin-type NK cell amplification factors and serum substitutes.

[0034] In some implementations, the amplifying agent also contains a fat-soluble vitamin.

[0035] In some implementations, the amplifying agent also contains trace elements.

[0036] In some implementations, the amplifying agent is used for serum-free culture of NK cells.

[0037] In some embodiments, the interleukin-type NK cell amplification factor is selected from any combination of IL2, IL15, IL12, IL18, IL21, or more.

[0038] In some embodiments, the amplifying agent does not contain nicotinamide and / or dimercaprol.

[0039] In some implementations, the amplifying agent also comprises sodium heparin.

[0040] In some embodiments, the amplifying agent further comprises one or more of reduced glutathione, human transferrin, and human serum albumin.

[0041] In some implementations, the NK cells are derived from peripheral blood mononuclear cells (PBMCs).

[0042] In some implementations, the NK cells are derived from umbilical cord blood NK cells.

[0043] In some implementations, the NK cells are obtained from in vitro culture of NK cell lines.

[0044] In some embodiments, the NK cells are obtained from in vitro induction and culture of induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (MSCs).

[0045] In some implementations, the interleukin-like NK cell amplification factors are IL2 and IL15.

[0046] In some embodiments, the fat-soluble vitamin is selected from one or more of the following: vitamin A, vitamin E, and vitamin K.

[0047] In some implementations, the vitamin K group is vitamin K1.

[0048] In some embodiments, the fat-soluble vitamins include vitamin A, vitamin E, and vitamin K1.

[0049] In some embodiments, the trace element is provided in the form of a compound selected from those containing one or more of the following elements: selenium, iron, zinc, copper, nickel, cobalt, chromium, silver, aluminum, barium, iodine, vanadium, rubidium, germanium, manganese, tin, and molybdenum.

[0050] In some embodiments, the trace element is provided in the form of one or more compounds selected from the following: sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride, and stannous chloride.

[0051] In some embodiments, the compounds providing the trace elements include sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride, and stannous chloride.

[0052] In some embodiments, the serum substitute is platelet lysate.

[0053] In some embodiments, the amplifying agent comprises IL2, IL15, vitamin A, vitamin E, vitamin K1, sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride and stannous chloride, platelet lysate, sodium heparin, reduced glutathione, human transferrin and human serum albumin.

[0054] Thirdly, this application provides a coating agent for an NK cell culture vessel, comprising an anti-CD38 antibody and an antibody or combination of antibodies targeting one or more of the following antigens: CD3, CD25, CD33, CD52, CD4, CD8, 4-1BB, CD19, CLL1, and CD16.

[0055] In some embodiments, the concentration of the anti-CD38 antibody is 1000 μg / L.

[0056] In some implementations, the anti-CD38 antibody is diluted with physiological saline or phosphate buffer.

[0057] In some embodiments, the coating agent further comprises an anti-CD52 antibody.

[0058] In some embodiments, the concentration of the anti-CD52 antibody is 10,000 μg / L.

[0059] In some implementations, the anti-CD52 antibody is diluted with physiological saline or phosphate buffer.

[0060] In some embodiments, the coating agent further comprises an anti-CD33-CLL1 bispecific antibody.

[0061] In some embodiments, the concentration of the anti-CD33-CLL1 bispecific antibody is 1000 μg / L.

[0062] In some embodiments, the anti-CD33-CLL1 bispecific antibody is diluted with physiological saline or phosphate buffer.

[0063] In some implementations, the NK cells are derived from peripheral blood mononuclear cells (PBMCs).

[0064] In some implementations, the NK cells are derived from umbilical cord blood NK cells.

[0065] In some implementations, the NK cells are obtained from in vitro culture of NK cell lines.

[0066] In some embodiments, the NK cells are obtained from in vitro induction and culture of induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (MSCs).

[0067] In some embodiments, the coating agent comprises anti-CD52 antibody and anti-CD38 antibody.

[0068] In some embodiments, the coating agent comprises an anti-CD38 antibody, an anti-CD52 antibody, and an anti-CD33-CLL1 bispecific antibody.

[0069] Fourthly, this application provides a serum-free culture medium for the activation step of NK cell culture, comprising: a basal culture medium; and the activator described in the first aspect.

[0070] In some embodiments, the serum-free culture medium contains 100-2100 IU / mL of interleukin-type NK cell activating factor, 1-2250 μg / L of fat-soluble vitamins, and 0.05-10 v / v% of serum substitute, based on the total volume of the serum-free culture medium.

[0071] In some embodiments, the serum-free culture medium contains 1000 IU / mL of IL2 and 10 ng / mL of IL15, based on the total volume of the serum-free culture medium.

[0072] In some embodiments, the serum-free culture medium contains 1000 IU / mL IL2, 10 ng / mL IL15 and 10 IU / mL IL21, based on the total volume of the serum-free culture medium.

[0073] In some embodiments, the serum-free culture medium contains 35 μg / L of vitamin A, 1000 μg / L of vitamin E and 100 μg / L of vitamin K1, based on the total volume of the serum-free culture medium.

[0074] In some embodiments, the serum-free culture medium comprises, by total volume, 6.7 μg / L sodium selenite, 20.2 μg / L ferrous chloride, 19.2 μg / L zinc chloride, 16.9 μg / L copper chloride, 0.073 μg / L nickel chloride, 0.044 μg / L cobalt chloride hexahydrate, 0.123 μg / L manganese chloride tetrahydrate, 0.073 μg / L chromium chloride, 0.133 μg / L silver chloride, 13.07 μg / L potassium iodide, 0.012193 μg / L sodium metavanadate, 0.107 μg / L ammonium molybdate tetrahydrate, 11.32 μg / L rubidium chloride, 5.874 μg / L germanium tetrachloride, and 0.3194 μg / L stannous chloride.

[0075] In some embodiments, the serum-free culture medium contains 0.25 v / v% platelet lysis buffer based on the total volume of the serum-free culture medium.

[0076] In some embodiments, the serum-free culture medium contains 1-10 mM of nicotinamide, based on the total volume of the serum-free culture medium.

[0077] In some embodiments, the serum-free culture medium contains 2.5 mM of nicotinamide based on the total volume of the serum-free culture medium.

[0078] In some embodiments, the serum-free culture medium contains 0.001-0.055 mM of dimercaptoethanol, based on the total volume of the serum-free culture medium.

[0079] In some embodiments, the serum-free culture medium contains 0.011 mM of dimercaptoethanol, based on the total volume of the serum-free culture medium.

[0080] In some embodiments, the serum-free culture medium contains 1-10 U / mL of heparin sodium, based on the total volume of the serum-free culture medium.

[0081] In some embodiments, the serum-free culture medium contains 2 U / mL of heparin sodium based on the total volume of the serum-free culture medium.

[0082] In some embodiments, the serum-free culture medium contains 10-1000 μg / L of reduced glutathione, 1000-10000 μg / L of human transferrin, and 1-10 g / L of human serum albumin, based on the total volume of the serum-free culture medium.

[0083] In some embodiments, the serum-free culture medium contains 768.3 μg / L of reduced glutathione, 10,000 μg / L of human transferrin, and 5 g / L of human serum albumin, based on the total volume of the serum-free culture medium.

[0084] In some embodiments, the serum-free culture medium contains, by total volume, 1000 IU / mL IL2, 10 ng / mL IL15, 10 IU / mL IL21, 35 μg / L vitamin A, 1000 μg / L vitamin E, and 100 Vitamin K1 (μg / L), sodium selenite (6.7μg / L), ferrous chloride (20.2μg / L), zinc chloride (19.2μg / L), copper chloride (16.9μg / L), nickel chloride (0.073μg / L), cobalt chloride hexahydrate (0.044μg / L), manganese chloride tetrahydrate (0.123μg / L), chromium chloride (0.073μg / L), silver chloride (0.133μg / L), potassium iodide (13.07μg / L), sodium metavanadate (0.012193μg / L), ammonium molybdate tetrahydrate (0.107μg / L), rubidium chloride (11.32μg / L), germanium tetrachloride (5.874μg / L), stannous chloride (0.3194μg / L), platelet lysis buffer (0.25 v / v %), 2.5 mM nicotinamide, 0.011 mM dimercaptoethanol, 2 U / mL heparin sodium, 768.3 μg / L reduced glutathione, 10000 μg / L human transferrin and 5 g / L human serum albumin.

[0085] In some embodiments, the serum-free culture medium contains, by total volume, 1000 IU / mL IL2, 10 ng / mL IL15, 35 μg / L vitamin A, 1000 μg / L vitamin E, and 100 Vitamin K1 (μg / L), sodium selenite (6.7μg / L), ferrous chloride (20.2μg / L), zinc chloride (19.2μg / L), copper chloride (16.9μg / L), nickel chloride (0.073μg / L), cobalt chloride hexahydrate (0.044μg / L), manganese chloride tetrahydrate (0.123μg / L), chromium chloride (0.073μg / L), silver chloride (0.133μg / L), potassium iodide (13.07μg / L), sodium metavanadate (0.012193μg / L), ammonium molybdate tetrahydrate (0.107μg / L), rubidium chloride (11.32μg / L), germanium tetrachloride (5.874μg / L), stannous chloride (0.3194μg / L), platelet lysis buffer (0.25 v / v %), 2.5 mM nicotinamide, 0.011 mM dimercaptoethanol, 2 U / mL heparin sodium, 768.3 μg / L reduced glutathione, 10000 μg / L human transferrin and 5 g / L human serum albumin.

[0086] In some embodiments, the basal medium is RPMI-1640 basal medium.

[0087] Fifthly, this application provides a serum-free culture medium for the amplification step of NK cell culture, comprising: a basal culture medium; and the amplification agent described in the second aspect.

[0088] In some embodiments, the serum-free culture medium contains 100-2050 IU / mL of interleukin-type NK cell amplification factor and 0.05-1 v / v % serum substitute, based on the total volume of the serum-free culture medium.

[0089] In some embodiments, the serum-free culture medium contains 1-2250 μg / L of fat-soluble vitamins, based on the total volume of the serum-free culture medium.

[0090] In some embodiments, the serum-free culture medium contains 1000 IU / mL of IL2 and 10 ng / mL of IL15, based on the total volume of the serum-free culture medium.

[0091] In some embodiments, the serum-free culture medium contains 35 μg / L of vitamin A, 1000 μg / L of vitamin E and 100 μg / L of vitamin K1, based on the total volume of the serum-free culture medium.

[0092] In some embodiments, the serum-free culture medium comprises, by total volume, 6.7 μg / L sodium selenite, 20.2 μg / L ferrous chloride, 19.2 μg / L zinc chloride, 16.9 μg / L copper chloride, 0.073 μg / L nickel chloride, 0.044 μg / L cobalt chloride hexahydrate, 0.123 μg / L manganese chloride tetrahydrate, 0.073 μg / L chromium chloride, 0.133 μg / L silver chloride, 13.07 μg / L potassium iodide, 0.012193 μg / L sodium metavanadate, 0.107 μg / L ammonium molybdate tetrahydrate, 11.32 μg / L rubidium chloride, 5.874 μg / L germanium tetrachloride, and 0.3194 μg / L stannous chloride.

[0093] In some embodiments, the serum-free culture medium contains 0.1 v / v % platelet lysis buffer, based on the total volume of the serum-free culture medium.

[0094] In some embodiments, the serum-free culture medium contains 1-10 U / mL of heparin sodium, based on the total volume of the serum-free culture medium.

[0095] In some embodiments, the serum-free culture medium contains 2 U / mL of heparin sodium based on the total volume of the serum-free culture medium.

[0096] In some embodiments, the serum-free culture medium contains 10-1000 μg / L of reduced glutathione, 0.1-10000 μg / L of human transferrin, and 1-10 g / L of human serum albumin, based on the total volume of the serum-free culture medium.

[0097] In some embodiments, the serum-free culture medium contains 768.3 μg / L of reduced glutathione, 10,000 μg / L of human transferrin, and 5 g / L of human serum albumin, based on the total volume of the serum-free culture medium.

[0098] In some embodiments, the serum-free culture medium contains, by total volume, 1000 IU / mL IL2, 10 ng / mL IL15, 35 μg / L vitamin A, 1000 μg / L vitamin E, and 100 Vitamin K1 (μg / L), sodium selenite (6.7μg / L), ferrous chloride (20.2μg / L), zinc chloride (19.2μg / L), copper chloride (16.9μg / L), nickel chloride (0.073μg / L), cobalt chloride hexahydrate (0.044μg / L), manganese chloride tetrahydrate (0.123μg / L), chromium chloride (0.073μg / L), silver chloride (0.133μg / L), potassium iodide (13.07μg / L), sodium metavanadate (0.012193μg / L), ammonium molybdate tetrahydrate (0.107μg / L), rubidium chloride (11.32μg / L), germanium tetrachloride (5.874μg / L), stannous chloride (0.3194μg / L), platelet lysis buffer (0.1 v / v %), 2.5 μg / L mM nicotinamide, 0.011 mM dimercaptoethanol, 2 U / mL heparin sodium, 768.3 μg / L reduced glutathione, 10000 μg / L human transferrin and 5 g / L human serum albumin.

[0099] In some embodiments, the basal medium is RPMI-1640 basal medium.

[0100] Sixthly, this application provides an NK cell culture kit comprising one, any two, or all three of (i) to (iii):

[0101] (i) the coating agent described in the third aspect,

[0102] (ii) the activator described in the first aspect, and

[0103] (iii) The amplifying agent described in the second aspect.

[0104] Seventhly, this application provides an NK cell culture kit comprising one, any two, or all three of (i) to (iii):

[0105] (i) the coating agent described in the third aspect,

[0106] (ii) The serum-free culture medium for the activation step of NK cell culture as described in the fourth aspect, and

[0107] (iii) The serum-free culture medium for the amplification step of NK cell culture as described in the fifth aspect.

[0108] Eighthly, this application provides one, any combination of, or all of the following uses in NK cell culture: (i) to (v)

[0109] (i) the coating agent described in the third aspect,

[0110] (ii) The activator described in the first aspect,

[0111] (iii) The amplifying agent described in the second aspect,

[0112] (iv) The serum-free culture medium for the activation step of NK cell culture as described in aspect four, and

[0113] (v) Serum-free culture medium for the amplification step of NK cell culture as described in the fifth aspect.

[0114] Ninthly, this application provides a method for culturing NK cells, comprising,

[0115] (1) The culture container is coated with the coating agent described in the third aspect, and

[0116] (2) Add a cell population containing NK cells to the culture container after coating.

[0117] In some implementations, the cell population is a PBMC cell population.

[0118] In a tenth aspect, this application provides a method for culturing NK cells, comprising,

[0119] The cell population containing NK cells is brought into contact with the activator described in the first aspect or the serum-free culture medium for the activation step of NK cell culture described in the fourth aspect.

[0120] In some implementations, the cell population is a PBMC cell population.

[0121] In its eleventh aspect, this application provides a method for culturing NK cells, comprising,

[0122] The cell population containing NK cells is brought into contact with the amplification agent described in the second aspect or the serum-free culture medium for the amplification step of NK cell culture described in the fifth aspect.

[0123] In some implementations, the NK cells are activated prior to contact.

[0124] In some implementations, the cell population is a PBMC cell population.

[0125] As a non-limiting example, this application provides the following implementation scheme:

[0126] 1. An activator for NK cell culture, comprising interleukin-type NK cell activating factors, fat-soluble vitamins, trace elements, and serum substitutes.

[0127] 2. The activator as described in embodiment 1, wherein the activator is used for serum-free culture of NK cells.

[0128] 3. The activator as described in Embodiment 1, wherein the interleukin-type NK cell activating factor is selected from any combination of IL2, IL7, IL15, IL12, IL18, IL21 or above.

[0129] 4. The activator as described in embodiment 1 further comprises nicotinamide.

[0130] 5. The activator as described in embodiment 1 further comprises dimercaptoethanol.

[0131] 6. The activator as described in embodiment 1 further comprises sodium heparin or quercetin.

[0132] 7. The activator as described in Embodiment 1 further comprises one or more of reduced glutathione, human transferrin, and human serum albumin.

[0133] 8. The activator as described in embodiment 1, wherein,

[0134] The NK cells were obtained from peripheral blood mononuclear cells (PBMCs); or

[0135] The NK cells are obtained from NK cells derived from umbilical cord blood; or

[0136] The NK cells were obtained from in vitro culture of NK cell lines; or

[0137] The NK cells were obtained from in vitro induction and culture of induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (MSCs).

[0138] 9. The activator as described in Embodiment 1, wherein the interleukin-like NK cell activating factors are IL2 and IL15.

[0139] 10. The activator as described in Embodiment 1, wherein the interleukin-like NK cell activating factors are IL2, IL15, and IL21.

[0140] 11. The activator as described in embodiment 1, wherein the fat-soluble vitamin is selected from one or more of the following: vitamin A, vitamin E and vitamin K.

[0141] 12. The activator as described in embodiment 1, wherein the vitamin K group is vitamin K1.

[0142] 13. The activator as described in embodiment 1, wherein the fat-soluble vitamins include vitamin A, vitamin E and vitamin K1.

[0143] 14. The activator as described in embodiment 1, wherein the trace element is provided in the form of a compound comprising one or more of the following elements: selenium, iron, zinc, copper, nickel, cobalt, chromium, silver, aluminum, barium, iodine, vanadium, rubidium, germanium, manganese, tin, and molybdenum.

[0144] 15. The activator as described in embodiment 1, wherein the trace element is provided in the form of one or more compounds selected from the following: sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride, and stannous chloride.

[0145] 16. The activator as described in embodiment 1, wherein the compound providing the trace element comprises sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride, and stannous chloride.

[0146] 17. The activator as described in embodiment 1, wherein the serum substitute is platelet lysis buffer.

[0147] 18. The activator as described in Embodiment 1, comprising IL2, IL15, vitamin A, vitamin E, vitamin K1, sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride and stannous chloride, platelet lysate, nicotinamide, dimercaptoethanol, sodium heparin, reduced glutathione, human transferrin and human serum albumin.

[0148] 19. The activator as described in Embodiment 1, comprising IL2, IL15, IL21, vitamin A, vitamin E, vitamin K1, sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride and stannous chloride, platelet lysate, nicotinamide, dimercaptoethanol, sodium heparin, reduced glutathione, human transferrin and human serum albumin.

[0149] 20. An amplification agent for NK cell culture, comprising interleukin-type NK cell amplification factors and serum substitutes.

[0150] 21. The amplifying agent as described in embodiment 20 further comprises a fat-soluble vitamin.

[0151] 22. The amplifying agent as described in embodiment 20 further comprises trace elements.

[0152] 23. The amplification agent as described in embodiment 20, wherein the amplification agent is used for serum-free culture of NK cells.

[0153] 24. The amplification agent as described in embodiment 20, wherein the interleukin-type NK cell amplification factor is selected from any combination of IL2, IL15, IL12, IL18, IL21 or above.

[0154] 25. The amplification agent as described in embodiment 20 does not contain nicotinamide and / or dimercaprol.

[0155] 26. The amplifying agent as described in embodiment 20, wherein,

[0156] The NK cells were obtained from peripheral blood mononuclear cells (PBMCs); or

[0157] The NK cells are obtained from NK cells derived from umbilical cord blood; or

[0158] The NK cells were obtained from in vitro culture of NK cell lines; or

[0159] The NK cells were obtained from in vitro induction and culture of induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (MSCs).

[0160] 27. The amplifying agent as described in embodiment 20 further comprises one or more of sodium heparin, reduced glutathione, human transferrin, and human serum albumin.

[0161] 28. The amplification agent as described in embodiment 20, wherein the interleukin-type NK cell amplification factors are IL2 and IL15.

[0162] 29. The amplifying agent as described in embodiment 21, wherein the fat-soluble vitamin is selected from one or more of the following: vitamin A, vitamin E, and vitamin K.

[0163] 30. The amplifying agent as described in embodiment 29, wherein the vitamin K group is vitamin K1.

[0164] 31. The amplifying agent as described in embodiment 21, wherein the fat-soluble vitamins include vitamin A, vitamin E and vitamin K1.

[0165] 32. The amplifying agent as described in embodiment 22, wherein the trace element is provided in a compound selected from those comprising one or more of the following elements: selenium, iron, zinc, copper, nickel, cobalt, chromium, silver, aluminum, barium, iodine, vanadium, rubidium, germanium, manganese, tin, and molybdenum.

[0166] 33. The amplifying agent as described in embodiment 22, wherein the trace element is provided in the form of one or more compounds selected from the following: sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride, and stannous chloride.

[0167] 34. The amplifying agent as described in embodiment 22, wherein the compound providing the trace element includes sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride, and stannous chloride.

[0168] 35. The amplification agent as described in embodiment 20, wherein the serum substitute is platelet lysis buffer.

[0169] 36. The amplification agent as described in embodiment 20, comprising IL2, IL15, vitamin A, vitamin E, vitamin K1, sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride and stannous chloride, platelet lysate, sodium heparin, reduced glutathione, human transferrin and human serum albumin.

[0170] 37. A coating agent for NK cell culture containers comprising an anti-CD38 antibody and an antibody or combination of antibodies targeting one or more of the following antigens: CD3, CD25, CD33, CD52, CD4, CD8, 4-1BB, CD19, CLL1, and CD16.

[0171] 38. The coating agent as described in embodiment 37, wherein the concentration of the anti-CD38 antibody is 1000 μg / L.

[0172] 39. The coating agent as described in embodiment 38, wherein the anti-CD38 antibody is diluted with physiological saline or phosphate buffer.

[0173] 40. The coating agent as described in embodiment 37 further comprises an anti-CD52 antibody.

[0174] 41. The coating agent as described in embodiment 40, wherein the concentration of the anti-CD52 antibody is 10000 μg / L.

[0175] 42. The coating agent as described in embodiment 41, wherein the anti-CD52 antibody is diluted with physiological saline or phosphate buffer.

[0176] 43. The coating agent as described in embodiment 37 further comprises an anti-CD33-CLL1 bispecific antibody.

[0177] 44. The coating agent as described in embodiment 43, wherein the concentration of the anti-CD33-CLL1 bispecific antibody is 1000 μg / L.

[0178] 45. The coating agent as described in embodiment 44, wherein,

[0179] The NK cells were obtained from peripheral blood mononuclear cells (PBMCs); or

[0180] The NK cells are obtained from NK cells derived from umbilical cord blood; or

[0181] The NK cells were obtained from in vitro culture of NK cell lines; or

[0182] The NK cells were obtained from in vitro induction and culture of induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (MSCs).

[0183] 46. ​​The coating agent as described in embodiment 37, wherein the coating agent comprises an anti-CD52 antibody and an anti-CD38 antibody.

[0184] 47. The coating agent as described in embodiment 37, wherein the coating agent comprises an anti-CD38 antibody, an anti-CD52 antibody, and an anti-CD33-CLL1 bispecific antibody.

[0185] 48. A serum-free culture medium for the activation step of NK cell culture, comprising:

[0186] Basic culture medium; and

[0187] The activator as described in any one of Implementation Schemes 1-19.

[0188] 49. The serum-free culture medium as described in embodiment 48, wherein the serum-free culture medium comprises 100-2100 IU / mL of interleukin-type NK cell activating factor, 1-2250 μg / L of fat-soluble vitamins and 0.05-10 v / v% of serum substitute, based on the total volume of the serum-free culture medium.

[0189] 50. The serum-free culture medium as described in embodiment 48, wherein the serum-free culture medium contains 1000 IU / mL of IL2 and 10 ng / mL of IL15 based on the total volume of the serum-free culture medium.

[0190] 51. The serum-free culture medium as described in embodiment 48, wherein the serum-free culture medium comprises 1000 IU / mL IL2, 10 ng / mL IL15 and 10 IU / mL IL21 based on the total volume of the serum-free culture medium.

[0191] 52. The serum-free culture medium as described in embodiment 48, wherein the serum-free culture medium comprises 35 μg / L of vitamin A, 1000 μg / L of vitamin E and 100 μg / L of vitamin K1, based on the total volume of the serum-free culture medium.

[0192] 53. The serum-free culture medium as described in embodiment 48, wherein, based on the total volume of the serum-free culture medium, the serum-free culture medium comprises 6.7 μg / L sodium selenite, 20.2 μg / L ferrous chloride, 19.2 μg / L zinc chloride, 16.9 μg / L copper chloride, 0.073 μg / L nickel chloride, 0.044 μg / L cobalt chloride hexahydrate, 0.123 μg / L manganese chloride tetrahydrate, 0.073 μg / L chromium chloride, 0.133 μg / L silver chloride, 13.07 μg / L potassium iodide, 0.012193 μg / L sodium metavanadate, 0.107 μg / L ammonium molybdate tetrahydrate, 11.32 μg / L rubidium chloride, 5.874 μg / L germanium tetrachloride, and 0.3194 μg / L stannous chloride.

[0193] 54. The serum-free culture medium as described in embodiment 48, wherein the serum-free culture medium contains 0.25 v / v% platelet lysis buffer based on the total volume of the serum-free culture medium.

[0194] 55. The serum-free culture medium as described in embodiment 48, wherein the serum-free culture medium contains 1-10 mM nicotinamide based on the total volume of the serum-free culture medium.

[0195] 56. The serum-free culture medium as described in embodiment 48, wherein the serum-free culture medium contains 2.5 mM nicotinamide based on the total volume of the serum-free culture medium.

[0196] 57. The serum-free culture medium as described in embodiment 48, wherein the serum-free culture medium contains 0.001-0.055 mM of dimercaptoethanol based on the total volume of the serum-free culture medium.

[0197] 58. The serum-free culture medium as described in embodiment 48, wherein the serum-free culture medium contains 0.011 mM dimercaptoethanol based on the total volume of the serum-free culture medium.

[0198] 59. The serum-free culture medium as described in embodiment 48, wherein the serum-free culture medium contains 1-10 U / mL of heparin sodium based on the total volume of the serum-free culture medium.

[0199] 60. The serum-free culture medium as described in embodiment 48, wherein the serum-free culture medium contains 2 U / mL of heparin sodium based on the total volume of the serum-free culture medium.

[0200] 61. The serum-free culture medium as described in embodiment 48, wherein the serum-free culture medium comprises 10-1000 μg / L of reduced glutathione, 1000-10000 μg / L of human transferrin and 1-10 g / L of human serum albumin, based on the total volume of the serum-free culture medium.

[0201] 62. The serum-free culture medium as described in embodiment 48, wherein the serum-free culture medium comprises 768.3 μg / L of reduced glutathione, 10000 μg / L of human transferrin and 5 g / L of human serum albumin, based on the total volume of the serum-free culture medium.

[0202] 63. The serum-free culture medium as described in embodiment 48, wherein, based on the total volume of the serum-free culture medium, the serum-free culture medium comprises 1000 IU / mL IL2, 10 ng / mL IL15, 10 IU / mL IL21, 35 μg / L vitamin A, 1000 μg / L vitamin E, and 100 Vitamin K1 (μg / L), sodium selenite (6.7μg / L), ferrous chloride (20.2μg / L), zinc chloride (19.2μg / L), copper chloride (16.9μg / L), nickel chloride (0.073μg / L), cobalt chloride hexahydrate (0.044μg / L), manganese chloride tetrahydrate (0.123μg / L), chromium chloride (0.073μg / L), silver chloride (0.133μg / L), potassium iodide (13.07μg / L), sodium metavanadate (0.012193μg / L), ammonium molybdate tetrahydrate (0.107μg / L), rubidium chloride (11.32μg / L), germanium tetrachloride (5.874μg / L), stannous chloride (0.3194μg / L), platelet lysis buffer (0.25 v / v %), 2.5 mM nicotinamide, 0.011 mM dimercaptoethanol, 2 U / mL heparin sodium, 768.3 μg / L reduced glutathione, 10000 μg / L human transferrin and 5 g / L human serum albumin.

[0203] 64. The serum-free culture medium as described in embodiment 48, wherein, based on the total volume of the serum-free culture medium, the serum-free culture medium comprises 1000 IU / mL IL2, 10 ng / mL IL15, 35 μg / L vitamin A, 1000 μg / L vitamin E, and 100 Vitamin K1 (μg / L), sodium selenite (6.7μg / L), ferrous chloride (20.2μg / L), zinc chloride (19.2μg / L), copper chloride (16.9μg / L), nickel chloride (0.073μg / L), cobalt chloride hexahydrate (0.044μg / L), manganese chloride tetrahydrate (0.123μg / L), chromium chloride (0.073μg / L), silver chloride (0.133μg / L), potassium iodide (13.07μg / L), sodium metavanadate (0.012193μg / L), ammonium molybdate tetrahydrate (0.107μg / L), rubidium chloride (11.32μg / L), germanium tetrachloride (5.874μg / L), stannous chloride (0.3194μg / L), platelet lysis buffer (0.25 v / v %), 2.5 mM nicotinamide, 0.011 mM dimercaptoethanol, 2 U / mL heparin sodium, 768.3 μg / L reduced glutathione, 10000 μg / L human transferrin and 5 g / L human serum albumin.

[0204] 65. The serum-free culture medium as described in embodiment 48, wherein the basal culture medium is RPMI-1640 basal culture medium.

[0205] 66. A serum-free culture medium for the expansion step of NK cell culture, comprising,

[0206] Basic culture medium; and

[0207] The amplifying agent as described in any one of Implementation Schemes 20-36.

[0208] 67. The serum-free culture medium as described in embodiment 66, wherein the serum-free culture medium contains 100-2050 IU / mL of interleukin-type NK cell amplification factor and 0.05-1 v / v % serum substitute, based on the total volume of the serum-free culture medium.

[0209] 68. The serum-free culture medium as described in embodiment 66, wherein the serum-free culture medium contains 1-2250 μg / L of fat-soluble vitamins based on the total volume of the serum-free culture medium.

[0210] 69. The serum-free culture medium as described in embodiment 66, wherein the serum-free culture medium contains 1000 IU / mL of IL2 and 10 ng / mL of IL15 based on the total volume of the serum-free culture medium.

[0211] 70. The serum-free culture medium as described in embodiment 66, wherein the serum-free culture medium comprises 35 μg / L of vitamin A, 1000 μg / L of vitamin E and 100 μg / L of vitamin K1, based on the total volume of the serum-free culture medium.

[0212] 71. The serum-free culture medium as described in embodiment 66, wherein, based on the total volume of the serum-free culture medium, the serum-free culture medium comprises 6.7 μg / L sodium selenite, 20.2 μg / L ferrous chloride, 19.2 μg / L zinc chloride, 16.9 μg / L copper chloride, 0.073 μg / L nickel chloride, 0.044 μg / L cobalt chloride hexahydrate, 0.123 μg / L manganese chloride tetrahydrate, 0.073 μg / L chromium chloride, 0.133 μg / L silver chloride, 13.07 μg / L potassium iodide, 0.012193 μg / L sodium metavanadate, 0.107 μg / L ammonium molybdate tetrahydrate, 11.32 μg / L rubidium chloride, 5.874 μg / L germanium tetrachloride, and 0.3194 μg / L stannous chloride.

[0213] 72. The serum-free culture medium as described in embodiment 66, wherein the serum-free culture medium contains 0.1 v / v % platelet lysis buffer based on the total volume of the serum-free culture medium.

[0214] 73. The serum-free culture medium as described in embodiment 66, wherein the serum-free culture medium contains 1-10 U / mL of heparin sodium based on the total volume of the serum-free culture medium.

[0215] 74. The serum-free culture medium as described in embodiment 66, wherein the serum-free culture medium contains 2 U / mL of heparin sodium based on the total volume of the serum-free culture medium.

[0216] 75. The serum-free culture medium as described in embodiment 66, wherein the serum-free culture medium comprises 10-1000 μg / L of reduced glutathione, 1000-10000 μg / L of human transferrin and 1-10 g / L of human serum albumin, based on the total volume of the serum-free culture medium.

[0217] 76. The serum-free culture medium as described in embodiment 66, wherein the serum-free culture medium comprises 768.3 μg / L of reduced glutathione, 10000 μg / L of human transferrin and 5 g / L of human serum albumin, based on the total volume of the serum-free culture medium.

[0218] 77. The serum-free culture medium as described in embodiment 66, wherein, based on the total volume of the serum-free culture medium, the serum-free culture medium comprises 1000 IU / mL IL2, 10 ng / mL IL15, 35 μg / L vitamin A, 1000 μg / L vitamin E, and 100 Vitamin K1 (μg / L), sodium selenite (6.7μg / L), ferrous chloride (20.2μg / L), zinc chloride (19.2μg / L), copper chloride (16.9μg / L), nickel chloride (0.073μg / L), cobalt chloride hexahydrate (0.044μg / L), manganese chloride tetrahydrate (0.123μg / L), chromium chloride (0.073μg / L), silver chloride (0.133μg / L), potassium iodide (13.07μg / L), sodium metavanadate (0.012193μg / L), ammonium molybdate tetrahydrate (0.107μg / L), rubidium chloride (11.32μg / L), germanium tetrachloride (5.874μg / L), stannous chloride (0.3194μg / L), platelet lysis buffer (0.1 v / v %), 2.5 μg / L mM nicotinamide, 0.011 mM dimercaptoethanol, 2 U / mL heparin sodium, 768.3 μg / L reduced glutathione, 10000 μg / L human transferrin and 5 g / L human serum albumin.

[0219] 78. The serum-free culture medium as described in embodiment 66, wherein the basal culture medium is RPMI-1640 basal culture medium.

[0220] 79. An NK cell culture kit comprising one, any two, or all three of (i) through (iii):

[0221] (i) The coating agent according to any one of embodiments 37-47,

[0222] (ii) The activator according to any one of embodiments 1-19, and

[0223] (iii) The amplifying agent according to any one of embodiments 20-36.

[0224] 80. An NK cell culture kit comprising one, any two, or all three of (i) to (iii):

[0225] (i) The coating agent according to any one of embodiments 37-47,

[0226] (ii) The serum-free culture medium for the activation step of NK cell culture according to any one of embodiments 48-65, and

[0227] (iii) Serum-free culture medium for the amplification step of NK cell culture as described in any one of embodiments 66-78.

[0228] 81. Use of one, any, or all of (i) to (v) in NK cell culture:

[0229] (i) The coating agent according to any one of embodiments 37-47,

[0230] (ii) The activator according to any one of embodiments 1-19,

[0231] (iii) The amplifying agent according to any one of embodiments 20-36,

[0232] (iv) The serum-free culture medium for the activation step of NK cell culture according to any one of embodiments 48-65, and

[0233] (v) Serum-free culture medium for the amplification step of NK cell culture as described in any one of embodiments 66-78.

[0234] 82. NK cell culture methods, including,

[0235] (1) Coating the culture container with the coating agent described in any one of embodiments 37-47, and

[0236] (2) Add a cell population containing NK cells to the culture container after coating.

[0237] 83. The NK cell culture method as described in embodiment 82, wherein the cell population is a PBMC cell population and optionally undergoes an NK cell enrichment step.

[0238] 84. NK cell culture methods, including,

[0239] The cell population containing NK cells is brought into contact with the activator of any one of embodiments 1-19 or the serum-free culture medium for the activation step of NK cell culture of any one of embodiments 48-65.

[0240] 85. The NK cell culture method as described in embodiment 84, wherein the cell population is a PBMC cell population and optionally undergoes an NK cell enrichment step.

[0241] 86. NK cell culture methods, including,

[0242] Contact the cell population containing NK cells with the amplification agent of any one of embodiments 20-36 or the serum-free culture medium for the amplification step of NK cell culture of any one of embodiments 66-78.

[0243] 87. The NK cell culture method as described in embodiment 86, wherein the NK cells have been activated prior to contact.

[0244] 88. The NK cell culture method as described in embodiment 86, wherein the cell population is a PBMC cell population and optionally undergoes an NK cell enrichment step. Attached Figure Description

[0245] In order to more intuitively demonstrate the technical effects of the embodiments of this application and further understand the technical solution, accompanying drawings are provided to explain this application. It should be understood that the following drawings only show part of the content of this application and do not constitute a limitation on this application.

[0246] Figure 1 These are the PBMC cell flow cytometry results of each volunteer after NK cell enrichment in Example 1.

[0247] Figure 2 This is the cell proliferation curve corresponding to the test for volunteer A cells.

[0248] Figure 3 This is the cell proliferation curve of the volunteer's B cells in the corresponding test.

[0249] Figure 4 This is the cell proliferation curve of the volunteer's C cells in the corresponding test.

[0250] Figure 5 This is a comparison of the terminal expansion fold of cells in volunteers D and E, respectively.

[0251] Figures 6A to 6D These are the flow cytometry results of terminal NK cells tested for each culture protocol. Detailed Implementation

[0252] Currently, China mainly relies on imported human serum, which is costly and subject to restrictions from foreign manufacturers. Furthermore, existing technologies for analyzing human serum only scratch the surface, leaving room for potential risks from unknown viruses and components. Separating autologous plasma not only complicates NK cell preparation but also leads to inconsistent culture stability due to individual differences. While using genetically engineered feeder cells yields the highest NK cell expansion efficiency, the inactivated feeder cells cannot be completely removed after culture, and there is a risk of NK cell mutation. Therefore, the field of NK cell therapy urgently needs a high-performance culture medium that is free of allogeneic components, serum, and feeder layers, and does not require the addition of autologous plasma to achieve low-cost, efficient, safe, and stable expansion of high-purity NK cells.

[0253] In view of the current technical deficiencies in this field, this application provides a serum-free culture protocol for human NK cells, a method for preparing the culture medium, and its uses. The purpose of this application is to achieve low-cost, simpler, more efficient, safer, and more stable expansion and harvesting of high-purity NK cells that meet clinical treatment needs.

[0254] The serum-free culture medium of this application exhibits excellent performance, contains no heterologous components, requires no feeder cell support, is low in cost, and has good versatility. The serum-free culture medium consists of a basal medium and additives. The basal medium is, for example, RPMI-1640. The additives may include fat-soluble vitamins, trace elements, nicotinamide, reduced glutathione, dimercaptoethanol, human transferrin, human serum albumin, activating factors, and / or serum substitutes to achieve efficient NK cell expansion, ensuring that the viability and quantity of NK cells meet clinical needs, and has broad application prospects in the biomedical field. This application also provides an NK cell culture kit containing serum-free culture medium.

[0255] The serum-free NK cell culture protocol used in this application avoids contamination from exogenous feeder cells and can culture NK cells efficiently without the need for additional human serum or autologous plasma. The final cell purity can reach over 95%, and it is compatible with NK cell culture from more different donors, improving the universality, stability and safety of the culture and reducing production costs.

[0256] CD16 + CD56 + NK cells have a stronger ADCC effect and are mainly used in the field of anti-tumor therapy, followed by anti-aging, autoimmune diseases, and antiviral therapy.

[0257] In one aspect, this application provides an activator for NK cell culture, comprising interleukin-type NK cell activating factors, fat-soluble vitamins, trace elements, and serum substitutes.

[0258] In some implementations, the activator is used for serum-free culture of NK cells.

[0259] In some embodiments, the interleukin-type NK cell activating factor is selected from any combination of IL2, IL7, IL15, IL12, IL18, IL21, or more.

[0260] In some embodiments, the activator further comprises nicotinamide. In some embodiments, the activator further comprises dimercaprol. In some embodiments, the activator further comprises sodium heparin.

[0261] In some embodiments, the activator further comprises one or more of reduced glutathione, human transferrin, and human serum albumin.

[0262] In some embodiments, the activator further comprises quercetin.

[0263] In some embodiments, the interleukin-like NK cell activating factors are IL2 and IL15.

[0264] In some embodiments, the interleukin-like NK cell activating factors are IL2, IL15, and IL21.

[0265] In some embodiments, the fat-soluble vitamin is selected from one or more of the following: vitamin A, vitamin E, and vitamin K.

[0266] In some implementations, the vitamin K group is vitamin K1.

[0267] In some embodiments, the vitamin A is vitamin A1 or vitamin A2.

[0268] In some embodiments, the vitamin E is α, β, γ, or δ-tocopherol.

[0269] In some embodiments, the fat-soluble vitamins include vitamin A, vitamin E, and vitamin K1.

[0270] In some embodiments, the trace element is provided in the form of a compound selected from those containing one or more of the following elements: selenium, iron, zinc, copper, nickel, cobalt, chromium, silver, aluminum, barium, iodine, vanadium, rubidium, germanium, manganese, tin, and molybdenum.

[0271] In some embodiments, the trace element is provided in the form of one or more compounds selected from the following: sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride, and stannous chloride.

[0272] In some embodiments, the compounds providing the trace elements include sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride, and stannous chloride.

[0273] In some embodiments, the serum substitute is platelet lysate.

[0274] In some embodiments, the activator comprises IL2, IL15, vitamin A, vitamin E, vitamin K1, sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride and stannous chloride, platelet lysate, nicotinamide, dimercaptoethanol, sodium heparin, reduced glutathione, human transferrin and human serum albumin.

[0275] In some embodiments, the activator comprises IL2, IL15, IL21, vitamin A, vitamin E, vitamin K1, sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride and stannous chloride, platelet lysate, nicotinamide, dimercaptoethanol, sodium heparin, reduced glutathione, human transferrin and human serum albumin.

[0276] Secondly, this application provides an amplification agent for NK cell culture, which comprises interleukin-type NK cell amplification factors and serum substitutes.

[0277] In some embodiments, the amplifying agent further comprises a fat-soluble vitamin. In some embodiments, the amplifying agent further comprises trace elements.

[0278] In some implementations, the amplifying agent is used for serum-free culture of NK cells.

[0279] In some embodiments, the interleukin-type NK cell amplification factor is selected from any combination of IL2, IL15, IL12, IL18, IL21, or more.

[0280] In some embodiments, the amplifying agent does not contain nicotinamide and / or dimercaprol. In some embodiments, the amplifying agent also contains sodium heparin.

[0281] In some embodiments, the amplifying agent further comprises one or more of reduced glutathione, human transferrin, and human serum albumin.

[0282] In some implementations, the interleukin-like NK cell amplification factors are IL2 and IL15.

[0283] In some embodiments, the fat-soluble vitamin is selected from one or more of the following: vitamin A, vitamin E, and vitamin K.

[0284] In some embodiments, the vitamin K group is vitamin K1. In some embodiments, the vitamin A is vitamin A1 or vitamin A2. In some embodiments, the vitamin E is α, β, γ, or δ-tocopherol.

[0285] In some embodiments, the fat-soluble vitamins include vitamin A, vitamin E, and vitamin K1.

[0286] In some embodiments, the trace element is provided in the form of a compound selected from those containing one or more of the following elements: selenium, iron, zinc, copper, nickel, cobalt, chromium, silver, aluminum, barium, iodine, vanadium, rubidium, germanium, manganese, tin, and molybdenum.

[0287] In some embodiments, the trace element is provided in the form of one or more compounds selected from the following: sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride, and stannous chloride.

[0288] In some embodiments, the compounds providing the trace elements include sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride, and stannous chloride.

[0289] In some embodiments, the serum substitute is platelet lysate.

[0290] In some embodiments, the amplifying agent comprises IL2, IL15, vitamin A, vitamin E, vitamin K1, sodium selenite, ferrous chloride, zinc chloride, copper chloride, nickel chloride, cobalt chloride hexahydrate, manganese chloride tetrahydrate, chromium chloride, silver chloride, potassium iodide, sodium metavanadate, ammonium molybdate tetrahydrate, rubidium chloride, germanium tetrachloride and stannous chloride, platelet lysate, sodium heparin, reduced glutathione, human transferrin and human serum albumin.

[0291] Thirdly, this application provides a coating agent for an NK cell culture vessel, comprising an anti-CD38 antibody and an antibody or combination of antibodies targeting one or more of the following antigens: CD3, CD25, CD33, CD52, CD4, CD8, 4-1BB, CD19, CLL1, and CD16.

[0292] In some embodiments, the concentration of the anti-CD38 antibody is 1000 μg / L.

[0293] In some implementations, the anti-CD38 antibody is diluted with physiological saline or phosphate buffer.

[0294] In some embodiments, the coating agent further comprises an anti-CD52 antibody.

[0295] In some embodiments, the concentration of the anti-CD52 antibody is 10,000 μg / L.

[0296] In some implementations, the anti-CD52 antibody is diluted with physiological saline or phosphate buffer.

[0297] In some embodiments, the coating agent further comprises an anti-CD33-CLL1 bispecific antibody.

[0298] In some embodiments, the concentration of the anti-CD33-CLL1 bispecific antibody is 1000 μg / L.

[0299] In some embodiments, the anti-CD33-CLL1 bispecific antibody is diluted with physiological saline or phosphate buffer.

[0300] In some embodiments, the coating agent comprises anti-CD52 antibody and anti-CD38 antibody.

[0301] In some embodiments, the coating agent comprises an anti-CD38 antibody, an anti-CD52 antibody, and an anti-CD33-CLL1 bispecific antibody.

[0302] Fourthly, this application provides a serum-free culture medium for the activation step of NK cell culture, comprising: a basal culture medium; and the activator described in the first aspect.

[0303] In some embodiments, the serum-free culture medium contains 100-2100 IU / mL of interleukin-type NK cell activating factor, 1-2250 μg / L of fat-soluble vitamins, and 0.05-10 v / v% of serum substitute, based on the total volume of the serum-free culture medium.

[0304] In some embodiments, the serum-free culture medium contains 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or 2100 IU / mL or any of the above values ​​as a range of interleukin-type NK cell activating factors, based on the total volume of the serum-free culture medium.

[0305] In some embodiments, the serum-free culture medium comprises, based on the total volume of the serum-free culture medium, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 85 Fat-soluble vitamins in the range of 0, 900, 950, 1000, 1050, 1100, 1135, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200 or 2250 μg / L or any value between the above.

[0306] In some embodiments, the serum-free culture medium comprises, by total volume, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 v / v% or any range of the above values.

[0307] In some embodiments, the serum-free culture medium contains 1000 IU / mL of IL2 and 10 ng / mL of IL15, based on the total volume of the serum-free culture medium.

[0308] In some embodiments, the serum-free culture medium contains 1000 IU / mL IL2, 10 ng / mL IL15 and 10 IU / mL IL21, based on the total volume of the serum-free culture medium.

[0309] In some embodiments, the serum-free culture medium contains 35 μg / L of vitamin A, 1000 μg / L of vitamin E and 100 μg / L of vitamin K1, based on the total volume of the serum-free culture medium.

[0310] In some embodiments, the serum-free culture medium comprises, by total volume, 6.7 μg / L sodium selenite, 20.2 μg / L ferrous chloride, 19.2 μg / L zinc chloride, 16.9 μg / L copper chloride, 0.073 μg / L nickel chloride, 0.044 μg / L cobalt chloride hexahydrate, 0.123 μg / L manganese chloride tetrahydrate, 0.073 μg / L chromium chloride, 0.133 μg / L silver chloride, 13.07 μg / L potassium iodide, 0.012193 μg / L sodium metavanadate, 0.107 μg / L ammonium molybdate tetrahydrate, 11.32 μg / L rubidium chloride, 5.874 μg / L germanium tetrachloride, and 0.3194 μg / L stannous chloride.

[0311] In some embodiments, the serum-free culture medium contains 0.1-20 μg / L, preferably 0.5-10 μg / L, and more preferably 6.7 μg / L, sodium selenite, based on the total volume of the serum-free culture medium.

[0312] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 20.2 μg / L, of ferrous chloride, based on the total volume of the serum-free culture medium.

[0313] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 19.2 μg / L, of zinc chloride, based on the total volume of the serum-free culture medium.

[0314] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 16.9 μg / L, of copper chloride, based on the total volume of the serum-free culture medium.

[0315] In some embodiments, the serum-free culture medium contains 0.0001-0.1 μg / L, preferably 0.073 μg / L, of nickel chloride, based on the total volume of the serum-free culture medium.

[0316] In some embodiments, the serum-free culture medium contains 0.0001-0.1 μg / L, preferably 0.044 μg / L, of cobalt chloride hexahydrate, based on the total volume of the serum-free culture medium.

[0317] In some embodiments, the serum-free culture medium contains 0.0001-0.5 μg / L, preferably 0.123 μg / L, of manganese chloride tetrahydrate, based on the total volume of the serum-free culture medium.

[0318] In some embodiments, the serum-free culture medium contains 0.0001-0.1 μg / L, preferably 0.073 μg / L, chromium chloride, based on the total volume of the serum-free culture medium.

[0319] In some embodiments, the serum-free culture medium contains 0.0001-0.5 μg / L, preferably 0.133 μg / L, of silver chloride, based on the total volume of the serum-free culture medium.

[0320] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 13.07 μg / L, of potassium iodide, based on the total volume of the serum-free culture medium.

[0321] In some embodiments, the serum-free culture medium contains 0.0001-0.1 μg / L, preferably 0.012193 μg / L, sodium metavanadate, based on the total volume of the serum-free culture medium.

[0322] In some embodiments, the serum-free culture medium contains 0.0001-0.5 μg / L, preferably 0.107 μg / L, of ammonium molybdate tetrahydrate, based on the total volume of the serum-free culture medium.

[0323] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 11.32 μg / L, of rubidium chloride, based on the total volume of the serum-free culture medium.

[0324] In some embodiments, the serum-free culture medium contains 0.1-10 μg / L, preferably 5.874 μg / L, germanium tetrachloride, based on the total volume of the serum-free culture medium.

[0325] In some embodiments, the serum-free culture medium contains 0.0001-0.5 μg / L, preferably 0.3194 μg / L stannous chloride, based on the total volume of the serum-free culture medium.

[0326] In some embodiments, the serum-free culture medium contains 0.25 v / v% platelet lysis buffer based on the total volume of the serum-free culture medium.

[0327] In some embodiments, the serum-free culture medium contains 1-10 mM of nicotinamide, based on the total volume of the serum-free culture medium.

[0328] In some embodiments, the serum-free culture medium contains nicotinamide in the range of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mM or any of the above values, based on the total volume of the serum-free culture medium.

[0329] In some embodiments, the serum-free culture medium contains 2.5 mM of nicotinamide based on the total volume of the serum-free culture medium.

[0330] In some embodiments, the serum-free culture medium contains 0.001-0.055 mM of dimercaptoethanol, based on the total volume of the serum-free culture medium.

[0331] In some embodiments, the serum-free culture medium comprises 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.02, 0.021, 0.022, 0.023, 0.024, 0.0 25, 0.026, 0.027, 0.028, 0.029, 0.03, 0.031, 0.032, 0.033, 0.034, 0.035, 0.036, 0.037, 0.038, 0.039, 0.04, 0.041, 0.042, 0.043, 0.044, 0.045, 0.046, 0.047, 0.048, 0.049, 0.05, 0.051, 0.052, 0.053, 0.054 or 0.055 mM or any value between the above ranges of dimercaprol.

[0332] In some embodiments, the serum-free culture medium contains 0.011 mM of dimercaptoethanol, based on the total volume of the serum-free culture medium.

[0333] In some embodiments, the serum-free culture medium contains 1-10 U / mL of heparin sodium, based on the total volume of the serum-free culture medium.

[0334] In some embodiments, the serum-free culture medium contains heparin sodium at concentrations of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 U / mL, or any of the above values, based on the total volume of the serum-free culture medium.

[0335] In some embodiments, the serum-free culture medium contains 2 U / mL of heparin sodium based on the total volume of the serum-free culture medium.

[0336] In some embodiments, the serum-free culture medium contains 10-1000 μg / L of reduced glutathione, 1000-10000 μg / L of human transferrin, and 1-10 g / L of human serum albumin, based on the total volume of the serum-free culture medium.

[0337] In some embodiments, the serum-free culture medium contains 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 768.3, 800, 850, 900, 950, or 1000 μg / L or any of the above values, based on the total volume of the serum-free culture medium.

[0338] In some embodiments, the serum-free culture medium comprises, based on the total volume of the serum-free culture medium, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, ​​3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650 3700, 3750, 3800, 3850, 3900, 3950, 4000, 4050, 4100, 4150, 4200, 4250, 4300, 4350, 4400, 4450, 4500, 4550, 4600, 4650, 4700, 4750, 4800, 4850, 4900 4950, 5000, 5050, 5100, 5150, 5200, 5250, 5300, 5350, 5400, 5450, 5500, 5550, 5600, 5650, 5700, 5750, 5800, 5850, 5900, 5950, 6000, 6050, 6100, 6150 6200, 6250, 6300, 6350, 6400, 6450, 6500, 6550, 6600, 6650, 6700, 6750, 6800, 6850, 6900, 6950, 7000, 7050, 7100, 7150, 7200, 7250, 7300, 7350, 7400 7450, 7500, 7550, 7600, 7650, 7700, 7750, 7800, 7850, 7900, 7950, 8000, 8050, 8100, 8150, 8200, 8250, 8300, 8350, 8400, 8450, 8500, 8550, 8600, 8650Human transferrin in concentrations of 8700, 8750, 8800, 8850, 8900, 8950, 9000, 9050, 9100, 9150, 9200, 9250, 9300, 9350, 9400, 9450, 9500, 9550, 9600, 9650, 9700, 9750, 9800, 9850, 9900, 9950, or 10000 μg / L, or any value within the range of the above.

[0339] In some embodiments, the serum-free culture medium contains 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 g / L or any of the above values, based on the total volume of the serum-free culture medium.

[0340] In some embodiments, the serum-free culture medium contains 768.3 μg / L of reduced glutathione, 10,000 μg / L of human transferrin, and 5 g / L of human serum albumin, based on the total volume of the serum-free culture medium.

[0341] In some embodiments, the serum-free culture medium contains, by total volume, 1000 IU / mL IL2, 10 ng / mL IL15, 10 IU / mL IL21, 35 μg / L vitamin A, 1000 μg / L vitamin E, and 100 Vitamin K1 (μg / L), sodium selenite (6.7μg / L), ferrous chloride (20.2μg / L), zinc chloride (19.2μg / L), copper chloride (16.9μg / L), nickel chloride (0.073μg / L), cobalt chloride hexahydrate (0.044μg / L), manganese chloride tetrahydrate (0.123μg / L), chromium chloride (0.073μg / L), silver chloride (0.133μg / L), potassium iodide (13.07μg / L), sodium metavanadate (0.012193μg / L), ammonium molybdate tetrahydrate (0.107μg / L), rubidium chloride (11.32μg / L), germanium tetrachloride (5.874μg / L), stannous chloride (0.3194μg / L), platelet lysis buffer (0.25 v / v %), 2.5 mM nicotinamide, 0.011 mM dimercaptoethanol, 2 U / mL heparin sodium, 768.3 μg / L reduced glutathione, 10000 μg / L human transferrin and 5 g / L human serum albumin.

[0342] In some embodiments, the serum-free culture medium contains, by total volume, 1000 IU / mL IL2, 10 ng / mL IL15, 35 μg / L vitamin A, 1000 μg / L vitamin E, and 100 Vitamin K1 (μg / L), sodium selenite (6.7μg / L), ferrous chloride (20.2μg / L), zinc chloride (19.2μg / L), copper chloride (16.9μg / L), nickel chloride (0.073μg / L), cobalt chloride hexahydrate (0.044μg / L), manganese chloride tetrahydrate (0.123μg / L), chromium chloride (0.073μg / L), silver chloride (0.133μg / L), potassium iodide (13.07μg / L), sodium metavanadate (0.012193μg / L), ammonium molybdate tetrahydrate (0.107μg / L), rubidium chloride (11.32μg / L), germanium tetrachloride (5.874μg / L), stannous chloride (0.3194μg / L), platelet lysis buffer (0.25 v / v %), 2.5 mM nicotinamide, 0.011 mM dimercaptoethanol, 2 U / mL heparin sodium, 768.3 μg / L reduced glutathione, 10000 μg / L human transferrin and 5 g / L human serum albumin.

[0343] In some embodiments, the serum-free culture medium contains 0.1-20 μg / L, preferably 0.5-10 μg / L, and more preferably 6.7 μg / L, sodium selenite, based on the total volume of the serum-free culture medium.

[0344] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 20.2 μg / L, of ferrous chloride, based on the total volume of the serum-free culture medium.

[0345] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 19.2 μg / L, of zinc chloride, based on the total volume of the serum-free culture medium.

[0346] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 16.9 μg / L, of copper chloride, based on the total volume of the serum-free culture medium.

[0347] In some embodiments, the serum-free culture medium contains 0.0001-0.1 μg / L, preferably 0.073 μg / L, of nickel chloride, based on the total volume of the serum-free culture medium.

[0348] In some embodiments, the serum-free culture medium contains 0.0001-0.1 μg / L, preferably 0.044 μg / L, of cobalt chloride hexahydrate, based on the total volume of the serum-free culture medium.

[0349] In some embodiments, the serum-free culture medium contains 0.0001-0.5 μg / L, preferably 0.123 μg / L, of manganese chloride tetrahydrate, based on the total volume of the serum-free culture medium.

[0350] In some embodiments, the serum-free culture medium contains 0.0001-0.1 μg / L, preferably 0.073 μg / L, chromium chloride, based on the total volume of the serum-free culture medium.

[0351] In some embodiments, the serum-free culture medium contains 0.0001-0.5 μg / L, preferably 0.133 μg / L, of silver chloride, based on the total volume of the serum-free culture medium.

[0352] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 13.07 μg / L, of potassium iodide, based on the total volume of the serum-free culture medium.

[0353] In some embodiments, the serum-free culture medium contains 0.0001-0.1 μg / L, preferably 0.012193 μg / L, sodium metavanadate, based on the total volume of the serum-free culture medium.

[0354] In some embodiments, the serum-free culture medium contains 0.0001-0.5 μg / L, preferably 0.107 μg / L, of ammonium molybdate tetrahydrate, based on the total volume of the serum-free culture medium.

[0355] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 11.32 μg / L, of rubidium chloride, based on the total volume of the serum-free culture medium.

[0356] In some embodiments, the serum-free culture medium contains 0.1-10 μg / L, preferably 5.874 μg / L, germanium tetrachloride, based on the total volume of the serum-free culture medium.

[0357] In some embodiments, the serum-free culture medium contains 0.0001-0.5 μg / L, preferably 0.3194 μg / L stannous chloride, based on the total volume of the serum-free culture medium.

[0358] In some embodiments, the basal medium is RPMI-1640 basal medium.

[0359] The basal culture medium is selected from serum-free cell culture media or other cell culture media supplemented with serum or serum substitutes. The cell culture media include, but are not limited to, serum-free cell culture media or other cell culture media supplemented with serum or serum substitutes. An example of a basal culture medium is the commercially available RPMI-1640 medium.

[0360] Fifthly, this application provides a serum-free culture medium for the amplification step of NK cell culture, comprising: a basal culture medium; and the amplification agent described in the second aspect.

[0361] In some embodiments, the serum-free culture medium contains 100-2050 IU / mL of interleukin-type NK cell amplification factor and 0.05-1 v / v % serum substitute, based on the total volume of the serum-free culture medium.

[0362] In some embodiments, the serum-free culture medium contains 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or 2050 IU / mL or any of the above values, based on the total volume of the serum-free culture medium.

[0363] In some embodiments, the serum-free culture medium comprises, by total volume, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 v / v % or any of the above values ​​as a serum substitute.

[0364] In some embodiments, the serum-free culture medium contains 1-2250 μg / L of fat-soluble vitamins, based on the total volume of the serum-free culture medium.

[0365] In some embodiments, the serum-free culture medium comprises, based on the total volume of the serum-free culture medium, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 85 Fat-soluble vitamins of 0, 900, 950, 1000, 1050, 1100, 1135, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200 or 2250 μg / L or any of the above values.

[0366] In some embodiments, the serum-free culture medium contains 1000 IU / mL of IL2 and 10 ng / mL of IL15, based on the total volume of the serum-free culture medium.

[0367] In some embodiments, the serum-free culture medium contains 35 μg / L of vitamin A, 1000 μg / L of vitamin E and 100 μg / L of vitamin K1, based on the total volume of the serum-free culture medium.

[0368] In some embodiments, the vitamin A is vitamin A1 or vitamin A2.

[0369] In some embodiments, the vitamin E is α, β, γ, or δ-tocopherol.

[0370] In some embodiments, the serum-free culture medium comprises, by total volume, 6.7 μg / L sodium selenite, 20.2 μg / L ferrous chloride, 19.2 μg / L zinc chloride, 16.9 μg / L copper chloride, 0.073 μg / L nickel chloride, 0.044 μg / L cobalt chloride hexahydrate, 0.123 μg / L manganese chloride tetrahydrate, 0.073 μg / L chromium chloride, 0.133 μg / L silver chloride, 13.07 μg / L potassium iodide, 0.012193 μg / L sodium metavanadate, 0.107 μg / L ammonium molybdate tetrahydrate, 11.32 μg / L rubidium chloride, 5.874 μg / L germanium tetrachloride, and 0.3194 μg / L stannous chloride.

[0371] In some embodiments, the serum-free culture medium contains 0.1-20 μg / L, preferably 0.5-10 μg / L, and more preferably 6.7 μg / L, sodium selenite, based on the total volume of the serum-free culture medium.

[0372] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 20.2 μg / L, of ferrous chloride, based on the total volume of the serum-free culture medium.

[0373] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 19.2 μg / L, of zinc chloride, based on the total volume of the serum-free culture medium.

[0374] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 16.9 μg / L, of copper chloride, based on the total volume of the serum-free culture medium.

[0375] In some embodiments, the serum-free culture medium contains 0.0001-0.1 μg / L, preferably 0.073 μg / L, of nickel chloride, based on the total volume of the serum-free culture medium.

[0376] In some embodiments, the serum-free culture medium contains 0.0001-0.1 μg / L, preferably 0.044 μg / L, of cobalt chloride hexahydrate, based on the total volume of the serum-free culture medium.

[0377] In some embodiments, the serum-free culture medium contains 0.0001-0.5 μg / L, preferably 0.123 μg / L, of manganese chloride tetrahydrate, based on the total volume of the serum-free culture medium.

[0378] In some embodiments, the serum-free culture medium contains 0.0001-0.1 μg / L, preferably 0.073 μg / L, chromium chloride, based on the total volume of the serum-free culture medium.

[0379] In some embodiments, the serum-free culture medium contains 0.0001-0.5 μg / L, preferably 0.133 μg / L, of silver chloride, based on the total volume of the serum-free culture medium.

[0380] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 13.07 μg / L, of potassium iodide, based on the total volume of the serum-free culture medium.

[0381] In some embodiments, the serum-free culture medium contains 0.0001-0.1 μg / L, preferably 0.012193 μg / L, sodium metavanadate, based on the total volume of the serum-free culture medium.

[0382] In some embodiments, the serum-free culture medium contains 0.0001-0.5 μg / L, preferably 0.107 μg / L, of ammonium molybdate tetrahydrate, based on the total volume of the serum-free culture medium.

[0383] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 11.32 μg / L, of rubidium chloride, based on the total volume of the serum-free culture medium.

[0384] In some embodiments, the serum-free culture medium contains 0.1-10 μg / L, preferably 5.874 μg / L, germanium tetrachloride, based on the total volume of the serum-free culture medium.

[0385] In some embodiments, the serum-free culture medium contains 0.0001-0.5 μg / L, preferably 0.3194 μg / L stannous chloride, based on the total volume of the serum-free culture medium.

[0386] In some embodiments, the serum-free culture medium contains 0.1 v / v % platelet lysis buffer, based on the total volume of the serum-free culture medium.

[0387] In some embodiments, the serum-free culture medium contains 1-10 U / mL of heparin sodium, based on the total volume of the serum-free culture medium.

[0388] In some embodiments, the serum-free culture medium contains heparin sodium at concentrations of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 U / mL, or any of the above values, based on the total volume of the serum-free culture medium.

[0389] In some embodiments, the serum-free culture medium contains 2 U / mL of heparin sodium, based on the total volume of the serum-free culture medium.

[0390] In some embodiments, the serum-free culture medium contains 10-1000 μg / L of reduced glutathione, 1000-10000 μg / L of human transferrin, and 1-10 g / L of human serum albumin, based on the total volume of the serum-free culture medium.

[0391] In some embodiments, the serum-free culture medium contains 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 768.3, 800, 850, 900, 950, or 1000 μg / L or any of the above values, based on the total volume of the serum-free culture medium.

[0392] In some embodiments, the serum-free culture medium comprises, based on the total volume of the serum-free culture medium, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, ​​3000, 30 50, 3100, 3150, 3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3800, 3850, 3900, 3950, 4000, 4050, 4100, 4150, 4200, 4250, 4300, 4350, 4400, 4450, 4500, 4550, 4600, 4650, 4700, 4750, 4800, 4850, 4900, 4950, 5000, 5050, 5100, 5150, 5200, 5250, 5300, 5350, 5400, 54 50, 5500, 5550, 5600, 5650, 5700, 5750, 5800, 5850, 5900, 5950, 6000, 6050, 6100, 6150, 6200, 6250, 6300, 6350, 6400, 6450, 6500, 6550, 6600, 6650, 6700, 6750, 6800, 6850, 6900, 6950, 7000, 7050, 7100, 7150, 7200, 7250, 7300, 7350, 7400, 7450, 7500, 7550, 7600, 7650, 7700, 7750, 7800, 78 Human transferrin in the range of 50, 7900, 7950, 8000, 8050, 8100, 8150, 8200, 8250, 8300, 8350, 8400, 8450, 8500, 8550, 8600, 8650, 8700, 8750, 8800, 8850, 8900, 8950, 9000, 9050, 9100, 9150, 9200, 9250, 9300, 9350, 9400, 9450, 9500, 9550, 9600, 9650, 9700, 9750, 9800, 9850, 9900, 9950 or 10000 μg / L or any value between the above.

[0393] In some embodiments, the serum-free culture medium contains 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 g / L or any of the above values, based on the total volume of the serum-free culture medium.

[0394] In some embodiments, the serum-free culture medium contains 768.3 μg / L of reduced glutathione, 10,000 μg / L of human transferrin, and 5 g / L of human serum albumin, based on the total volume of the serum-free culture medium.

[0395] In some embodiments, the serum-free culture medium contains, by total volume, 1000 IU / mL IL2, 10 ng / mL IL15, 35 μg / L vitamin A, 1000 μg / L vitamin E, and 100 Vitamin K1 (μg / L), sodium selenite (6.7μg / L), ferrous chloride (20.2μg / L), zinc chloride (19.2μg / L), copper chloride (16.9μg / L), nickel chloride (0.073μg / L), cobalt chloride hexahydrate (0.044μg / L), manganese chloride tetrahydrate (0.123μg / L), chromium chloride (0.073μg / L), silver chloride (0.133μg / L), potassium iodide (13.07μg / L), sodium metavanadate (0.012193μg / L), ammonium molybdate tetrahydrate (0.107μg / L), rubidium chloride (11.32μg / L), germanium tetrachloride (5.874μg / L), stannous chloride (0.3194μg / L), platelet lysis buffer (0.1 v / v %), 2.5 μg / L mM nicotinamide, 0.011 mM dimercaptoethanol, 2 U / mL heparin sodium, 768.3 μg / L reduced glutathione, 10000 μg / L human transferrin and 5 g / L human serum albumin.

[0396] In some embodiments, the serum-free culture medium contains 0.1-20 μg / L, preferably 0.5-10 μg / L, and more preferably 6.7 μg / L, sodium selenite, based on the total volume of the serum-free culture medium.

[0397] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 20.2 μg / L, of ferrous chloride, based on the total volume of the serum-free culture medium.

[0398] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 19.2 μg / L, of zinc chloride, based on the total volume of the serum-free culture medium.

[0399] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 16.9 μg / L, of copper chloride, based on the total volume of the serum-free culture medium.

[0400] In some embodiments, the serum-free culture medium contains 0.0001-0.1 μg / L, preferably 0.073 μg / L, of nickel chloride, based on the total volume of the serum-free culture medium.

[0401] In some embodiments, the serum-free culture medium contains 0.0001-0.1 μg / L, preferably 0.044 μg / L, of cobalt chloride hexahydrate, based on the total volume of the serum-free culture medium.

[0402] In some embodiments, the serum-free culture medium contains 0.0001-0.5 μg / L, preferably 0.123 μg / L, of manganese chloride tetrahydrate, based on the total volume of the serum-free culture medium.

[0403] In some embodiments, the serum-free culture medium contains 0.0001-0.1 μg / L, preferably 0.073 μg / L, chromium chloride, based on the total volume of the serum-free culture medium.

[0404] In some embodiments, the serum-free culture medium contains 0.0001-0.5 μg / L, preferably 0.133 μg / L, of silver chloride, based on the total volume of the serum-free culture medium.

[0405] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 13.07 μg / L, of potassium iodide, based on the total volume of the serum-free culture medium.

[0406] In some embodiments, the serum-free culture medium contains 0.0001-0.1 μg / L, preferably 0.012193 μg / L, sodium metavanadate, based on the total volume of the serum-free culture medium.

[0407] In some embodiments, the serum-free culture medium contains 0.0001-0.5 μg / L, preferably 0.107 μg / L, of ammonium molybdate tetrahydrate, based on the total volume of the serum-free culture medium.

[0408] In some embodiments, the serum-free culture medium contains 1-50 μg / L, preferably 11.32 μg / L, of rubidium chloride, based on the total volume of the serum-free culture medium.

[0409] In some embodiments, the serum-free culture medium contains 0.1-10 μg / L, preferably 5.874 μg / L, germanium tetrachloride, based on the total volume of the serum-free culture medium.

[0410] In some embodiments, the serum-free culture medium contains 0.0001-0.5 μg / L, preferably 0.3194 μg / L stannous chloride, based on the total volume of the serum-free culture medium.

[0411] In some embodiments, the basal medium is RPMI-1640 basal medium.

[0412] The basal culture medium is selected from serum-free cell culture media or other cell culture media supplemented with serum or serum substitutes. The cell culture media include, but are not limited to, serum-free cell culture media or other cell culture media supplemented with serum or serum substitutes. An example of a basal culture medium is the commercially available RPMI-1640 medium.

[0413] Sixthly, this application provides an NK cell culture kit comprising one, any two, or all three of (i) to (iii):

[0414] (i) the coating agent described in the third aspect,

[0415] (ii) the activator described in the first aspect, and

[0416] (iii) The amplifying agent described in the second aspect.

[0417] In some implementations, the NK cell culture kit includes (i), (ii) and (iii); or (i) and (ii); or (i) and (iii); or (ii) and (iii); or (i); or (ii); or (iii).

[0418] Seventhly, this application provides an NK cell culture kit comprising one, any two, or all three of (i) to (iii):

[0419] (i) the coating agent described in the third aspect,

[0420] (ii) The serum-free culture medium for the activation step of NK cell culture as described in the fourth aspect, and

[0421] (iii) The serum-free culture medium for the amplification step of NK cell culture as described in the fifth aspect.

[0422] In some implementations, the NK culture kit includes (i), (ii) and (iii); or (i) and (ii); or (i) and (iii); or (ii) and (iii); or (i); or (ii); or (iii).

[0423] Eighthly, this application provides one, any combination of, or all of the following uses in NK cell culture: (i) to (v)

[0424] (i) the coating agent described in the third aspect,

[0425] (ii) The activator described in the first aspect,

[0426] (iii) The amplifying agent described in the second aspect,

[0427] (iv) The serum-free culture medium for the activation step of NK cell culture as described in aspect four, and

[0428] (v) Serum-free culture medium for the amplification step of NK cell culture as described in the fifth aspect.

[0429] In some embodiments, this application provides the use of (i) above in NK cell culture.

[0430] In some embodiments, this application provides the use of (ii) above in NK cell culture.

[0431] In some embodiments, this application provides the use of (iii) above in NK cell culture.

[0432] In some embodiments, this application provides the use of (iv) above in NK cell culture.

[0433] In some embodiments, this application provides the use of (v) above in NK cell culture.

[0434] In some embodiments, this application provides the use of (i) and (ii) above in NK cell culture.

[0435] In some embodiments, this application provides the use of (i) and (iii) above in NK cell culture.

[0436] In some embodiments, this application provides the use of (i) and (iv) above in NK cell culture.

[0437] In some embodiments, this application provides the use of (i) and (v) above in NK cell culture.

[0438] In some embodiments, this application provides the use of (ii) and (iii) above in NK cell culture.

[0439] In some embodiments, this application provides the use of (ii) and (iv) above in NK cell culture.

[0440] In some embodiments, this application provides the use of (ii) and (v) above in NK cell culture.

[0441] In some embodiments, this application provides the use of (iii) and (iv) above in NK cell culture.

[0442] In some embodiments, this application provides the use of (iii) and (v) above in NK cell culture.

[0443] In some embodiments, this application provides the use of (iv) and (v) above in NK cell culture.

[0444] In some embodiments, this application provides the use of (i), (ii) and (iii) above in NK cell culture.

[0445] In some embodiments, this application provides the use of (i), (ii) and (iv) above in NK cell culture.

[0446] In some embodiments, this application provides the use of (i), (ii) and (v) above in NK cell culture.

[0447] In some embodiments, this application provides the use of (i), (iii) and (iv) above in NK cell culture.

[0448] In some embodiments, this application provides the use of (i), (iii) and (v) above in NK cell culture.

[0449] In some embodiments, this application provides the use of (i), (iv) and (v) above in NK cell culture.

[0450] In some embodiments, this application provides the use of (ii), (iii) and (iv) above in NK cell culture.

[0451] In some embodiments, this application provides the use of (ii), (iii) and (v) above in NK cell culture.

[0452] In some embodiments, this application provides the use of (ii), (iv) and (v) above in NK cell culture.

[0453] In some embodiments, this application provides the use of (iii), (iv) and (v) above in NK cell culture.

[0454] In some embodiments, this application provides the uses of (i), (ii), (iii), and (iv) above in NK cell culture. In some embodiments, this application provides the uses of (i), (ii), (iii), and (v) above in NK cell culture. In some embodiments, this application provides the uses of (i), (ii), (iv), and (v) above in NK cell culture. In some embodiments, this application provides the uses of (i), (iii), (iv), and (v) above in NK cell culture. In some embodiments, this application provides the uses of (ii), (iii), (iv), and (v) above in NK cell culture. In some embodiments, this application provides the uses of (i), (ii), (iii), (iv), and (v) above in NK cell culture.

[0455] Ninthly, this application provides a method for culturing NK cells, comprising,

[0456] (1) The culture container is coated with the coating agent described in the third aspect, and

[0457] (2) Add a cell population containing NK cells to the culture container after coating.

[0458] In some implementations, the cell population is a PBMC cell population.

[0459] In a tenth aspect, this application provides a method for culturing NK cells, comprising,

[0460] The cell population containing NK cells is brought into contact with the activator described in the first aspect or the serum-free culture medium for the activation step of NK cell culture described in the fourth aspect.

[0461] In some implementations, the cell population is a PBMC cell population.

[0462] In its eleventh aspect, this application provides a method for culturing NK cells, comprising,

[0463] The cell population containing NK cells is brought into contact with the amplification agent described in the second aspect or the serum-free culture medium for the amplification step of NK cell culture described in the fifth aspect.

[0464] In some implementations, the NK cells are activated prior to contact.

[0465] In some implementations, the cell population is a PBMC cell population.

[0466] The basal culture medium is selected from serum-free cell culture media or other cell culture media supplemented with serum or serum substitutes. The cell culture media include, but are not limited to, serum-free cell culture media or other cell culture media supplemented with serum or serum substitutes. An example of a basal culture medium is the commercially available RPMI-1640 medium.

[0467] In addition, this application also provides a method for in vitro culture of NK cells, the brief operation steps of which are as follows:

[0468] 1) NK cells are enriched from peripheral blood or umbilical cord blood using NK cell enrichment reagents. However, NK cell lines such as NK92 can skip this step directly;

[0469] 2) Incubate the cells with the coating agent on a culture dish. After incubation, remove excess coating agent, add activation medium, and inoculate the enriched NK or PBMC cells. Activate and culture for 5-9 days.

[0470] 3) After activation culture, continue culturing for 6-10 days using amplification medium, and then harvest the cells.

[0471] The NK cell enrichment reagent can be any reagent capable of enriching NK cells, including but not limited to enrichment reagents independently developed by the applicant, as well as any reagent or combination thereof capable of removing non-NK cells such as B cells, T cells, and granulocytes. Preferably, the enriched NK cells account for 40% or more of the PBMCs, and double-negative T cells (CD4+) account for a certain percentage. - CD8 - CD3 + (less than 0.1%)

[0472] This invention also provides a method for in vitro culture of NK cells, comprising three stages: a coating stage, an activation stage, and an expansion stage. Other culture conditions or external environments comply with industry standards in the field, including but not limited to culture temperature, humidity, carbon dioxide concentration, and culture medium pH.

[0473] The coating stage refers to pre-incubation with a coating agent on a culture dish, incubating at room temperature or 37 degrees Celsius for two hours or overnight in a 4-degree Celsius refrigerator before use, preferably incubating at 37 degrees Celsius for two hours; the culture dish can be a conventional commercially available T25, T75, T175, or T225 culture flask or other culture plates with the same materials, preferably untreated TC culture flasks; the coating solution is phosphate-buffered saline (PBS) or physiological saline solution containing the coating agent, preferably physiological saline solution containing the coating agent; the coating agent is... Single or multiple monoclonal antibody combinations, or single or multiple bispecific antibody combinations, are available. Antibodies include anti-CD3, CD25, CD33, CD52, CD4, CD8, 4-1BB, CD19, CLL1, CD38, and CD16 antibodies. Anti-CD25, CD33, CD52, CLL1, and CD38 monoclonal antibodies are preferred, anti-CD33, CLL1, CD38, and CD52 monoclonal antibodies are more preferred, and anti-CD38 monoclonal antibody, CD33-CLL1 bispecific antibody, and CD52 monoclonal antibody are most preferred.

[0474] In one embodiment, the coating agent contains a final concentration of 0.1-20 μg / mL, preferably 0.5-10 μg / mL, most preferably 1 μg / mL of anti-CD33-CLL1 bispecific antibody and a final concentration of 0.5-20 μg / mL, preferably 1-15 μg / mL, most preferably 10 μg / mL of anti-CD52 monoclonal antibody;

[0475] In a more preferred embodiment, the coating agent contains an anti-CD38 monoclonal antibody at a final concentration of 0.1-20 μg / mL, preferably 0.5-10 μg / mL, most preferably 1 μg / mL, and an anti-CD52 monoclonal antibody at a final concentration of 0.5-20 μg / mL, preferably 1-15 μg / mL, most preferably 10 μg / mL.

[0476] In the most preferred embodiment, the coating agent contains an anti-CD38 monoclonal antibody at a final concentration of 0.1-20 μg / mL, preferably 0.5-10 μg / mL, most preferably 1 μg / mL; an anti-CD52 monoclonal antibody at a final concentration of 0.5-20 μg / mL, preferably 1-15 μg / mL, most preferably 10 μg / mL; and an anti-CD33-CLL1 bispecific antibody at a final concentration of 0.1-20 μg / mL, preferably 0.5-10 μg / mL, most preferably 1 μg / mL.

[0477] In a specific implementation, the coating agent contains an anti-CD52 monoclonal antibody at a final concentration of about 10 μg / mL, an anti-CD38 monoclonal antibody at a final concentration of about 1 μg / mL, and / or an anti-CD33-CLL1 bispecific antibody at a final concentration of 1 μg / mL.

[0478] The activation phase refers to adding activation medium to a culture dish that has been incubated and had excess coating agent removed, followed by inoculation and culture of enriched PBMC cells. The culture period for the activation phase can be 5-9 days, preferably 6-8 days, and more preferably 7 days; after the activation phase, CD3... - CD56 + The proportion of NK cells can reach 70% or more, preferably 80% or more, and more preferably 90% or more; the seeding density of the PBMC cells is preferably 1.0 × 10⁻⁶. 6 ~2.5×10 6 cells / mL, more preferably 1.5 × 10⁻⁶ cells / mL. 6 ~2.0×10 6 cells / mL;

[0479] The activation medium comprises a basal medium and an activator; the basal medium is a conventional commercial RPMI-1640; the activator comprises, but is not limited to, interleukin-2 (IL2), interleukin-7 (IL7), interleukin-15 (IL15), interleukin-12 (IL12), interleukin-18 (IL18), and interleukin-21 (IL21), preferably a combination of IL2 and IL15, and more preferably a combination of IL2, IL15, and IL21;

[0480] The activator also includes, but is not limited to, fat-soluble vitamins, trace elements, serum substitutes, heparin sodium, ascorbic acid, metformin, taurine, polyethylene glycol (PEG), nicotinamide, reduced glutathione, quercetin, 2-mercaptoethanol, rh-transferrin, and / or human serum albumin.

[0481] The fat-soluble vitamins include, but are not limited to, vitamin A, vitamin E, and vitamin K; the trace elements include, but are not limited to, selenium (Se), iron (Fe), zinc (Zn), copper (Cu), nickel (Ni), cobalt (Co), chromium (Cr), silver (Ag), aluminum (Al), barium (Ba), iodine (I), vanadium (V), rubidium (Rb), germanium (Ge), manganese (Mn), tin (Sn), and molybdenum (Mo); the serum substitutes include, but are not limited to, platelet lysate.

[0482] In one embodiment, the activation culture medium comprises RPMI-1640, 100-2000 IU / mL, preferably 1000 IU / mL IL2, 5-50 ng / mL, preferably 10 ng / mL IL15, 1-10 U / mL, preferably 2 U / mL heparin sodium, 1-10 g / L, preferably 5 g / L human serum albumin, 1-100 μg / mL, preferably 1-20 μg / mL, more preferably 10 μg / mL human transferrin, 1-50 μg / L, preferably 2-10 μg / L, more preferably 3.5 μg / L vitamin A, 10 Vitamin E at concentrations of -2000 μg / L, preferably 50-1000 μg / L, more preferably 100 μg / L; Vitamin K1 at concentrations of 1-200 μg / L, preferably 10 μg / L; Reduced glutathione at concentrations of 10-1000 μg / L, preferably 50-100 μg / L, more preferably 76.83 μg / L; Sodium selenite at concentrations of 0.1-20 μg / L, preferably 0.5-10 μg / L, more preferably 0.67 μg / L; Ferrous chloride at concentrations of 1-50 μg / L, preferably 2.02 μg / L; Zinc chloride at concentrations of 1-50 μg / L, preferably 1.92 μg / L; 1-5 μg / L of other vitamins. 0 μg / L, preferably 1.69 μg / L, copper chloride; 0.0001-0.1 μg / L, preferably 0.0073 μg / L, nickel chloride; 0.0001-0.1 μg / L, preferably 0.0044 μg / L, cobalt chloride hexahydrate; 0.0001-0.5 μg / L, preferably 0.0123 μg / L, manganese chloride tetrahydrate; 0.0001-0.1 μg / L, preferably 0.0073 μg / L, chromium chloride; 0.0001-0.5 μg / L, preferably 0.0133 μg / L, silver chloride; 1-50 μg / L, preferably 1. Potassium iodide at 307 μg / L, sodium metavanadate at 0.0001-0.1 μg / L (preferably 0.0012193 μg / L), ammonium molybdate tetrahydrate at 0.0001-0.5 μg / L (preferably 0.0107 μg / L), rubidium chloride at 1-50 μg / L (preferably 1.132 μg / L), germanium tetrachloride at 0.1-10 μg / L (preferably 0.5874 μg / L), stannous chloride at 0.0001-0.5 μg / L (preferably 0.03194 μg / L), and platelet lysis buffer at 0.05-10% (preferably 0.1-5%), more preferably 2.5%.

[0483] In another preferred embodiment, the activation culture medium comprises RPMI-1640, preferably 1000 IU / mL IL2, preferably 10 ng / mL IL15, preferably 2 U / mL sodium heparin, preferably 5 g / L human serum albumin, preferably 10 μg / mL human transferrin, preferably 35 μg / L vitamin A, preferably 1000 μg / L vitamin E, preferably 100 μg / L vitamin K1, preferably 768.3 μg / L reduced glutathione, preferably 6.7 μg / L sodium selenite, preferably 20.2 μg / L ferrous chloride, preferably 19.2 μg / L zinc chloride, preferably 16.9 μg / L copper chloride, preferably 0.073 μg / L nickel chloride, preferably... The following are preferred: 0.044 μg / L cobalt chloride hexahydrate, preferably 0.123 μg / L manganese chloride tetrahydrate, preferably 0.073 μg / L chromium chloride, preferably 0.133 μg / L silver chloride, preferably 13.07 μg / L potassium iodide, preferably 0.012193 μg / L sodium metavanadate, preferably 0.107 μg / L ammonium molybdate tetrahydrate, preferably 11.32 μg / L rubidium chloride, preferably 5.874 μg / L germanium tetrachloride, preferably 0.3194 μg / L stannous chloride, and preferably 0.25% platelet lysis buffer.

[0484] In another embodiment, the activation culture medium comprises RPMI-1640, preferably 1000 IU / mL IL2, preferably 10 ng / mL IL15, preferably 2 U / mL sodium heparin, preferably 5 g / L human serum albumin, preferably 5 μg / mL human transferrin, preferably 17.5 μg / L vitamin A, preferably 500 μg / L vitamin E, preferably 50 μg / L vitamin K1, preferably 384.15 μg / L reduced glutathione, preferably 3.35 μg / L sodium selenite, preferably 10.1 μg / L ferrous chloride, preferably 9.6 μg / L zinc chloride, preferably 8.45 μg / L copper chloride, preferably 0.0365 μg / L nickel chloride, preferably... 0.022 μg / L cobalt chloride hexahydrate, preferably 0.0615 μg / L manganese chloride tetrahydrate, preferably 0.0365 μg / L chromium chloride, preferably 0.0665 μg / L silver chloride, preferably 6.535 μg / L potassium iodide, preferably 0.0060965 μg / L sodium metavanadate, preferably 0.0535 μg / L ammonium molybdate tetrahydrate, preferably 5.66 μg / L rubidium chloride, preferably 2.937 μg / L germanium tetrachloride, preferably 0.1597 μg / L stannous chloride, preferably 1.25% platelet lysis buffer;

[0485] In yet another preferred embodiment, the activation culture medium comprises RPMI-1640, preferably 1000 IU / mL IL2, preferably 10 ng / mL IL15, 1-100 ng / mL, preferably 10 ng / mL IL21, preferably 2 U / mL heparin sodium, preferably 5 g / L human serum albumin, 1-10 mM, preferably 2.5 mM nicotinamide, 0.001-0.055 mM, preferably 0.011 mM dimercaptoethanol, preferably 10 μg / mL human transferrin, preferably 35 μg / L vitamin A, preferably 1000 μg / L vitamin E, preferably 100 μg / L vitamin K1, preferably 768.3 μg / L reduced glutathione, preferably 6.7 μg / L sodium selenite, preferably 20.2 μg / L ferrous chloride, preferably 19.2 μg / L sodium selenite, preferably 19.2 μg / L sodium selenite, preferably 10 ... The following are preferred concentrations: zinc chloride (L), copper chloride (16.9 μg / L), nickel chloride (0.073 μg / L), cobalt chloride hexahydrate (0.044 μg / L), manganese chloride tetrahydrate (0.123 μg / L), chromium chloride (0.073 μg / L), silver chloride (0.133 μg / L), potassium iodide (13.07 μg / L), sodium metavanadate (0.012193 μg / L), ammonium molybdate tetrahydrate (0.107 μg / L), rubidium chloride (11.32 μg / L), germanium tetrachloride (5.874 μg / L), stannous chloride (0.3194 μg / L), and platelet lysis buffer (0.25%).

[0486] The expansion phase refers to further culturing the activated NK cells in an expansion medium for approximately 15 days, yielding NK cells with the CD3 phenotype. - CD56 + CD16 + CD56 + It can be above 90%, with a preferred ratio of above 95%.

[0487] The amplification medium comprises a basal medium and an amplification agent; the basal medium is a conventional commercial RPMI-1640; the amplification agent comprises, but is not limited to, interleukin-2 (IL2), interleukin-15 (IL15), interleukin-12 (IL12), interleukin-18 (IL18), and interleukin-21 (IL21), preferably a combination of IL2 and IL15; the amplification agent also comprises, but is not limited to, fat-soluble vitamins, trace elements, serum substitutes, heparin sodium, ascorbic acid, metformin, taurine, polyethylene glycol (PEG), nicotinamide, reduced glutathione, quercetin, 2-mercaptoethanol, rh-transferrin, and / or human serum albumin.

[0488] The fat-soluble vitamins include, but are not limited to, vitamin A, vitamin E, and vitamin K; the trace elements include, but are not limited to, selenium (Se), iron (Fe), zinc (Zn), copper (Cu), nickel (Ni), cobalt (Co), chromium (Cr), silver (Ag), aluminum (Al), barium (Ba), iodine (I), vanadium (V), rubidium (Rb), germanium (Ge), manganese (Mn), tin (Sn), and molybdenum (Mo); the serum substitutes include, but are not limited to, platelet lysate.

[0489] In one embodiment, the amplification medium comprises RPMI-1640, 100-2000 IU / mL, preferably 1000 IU / mL IL2, 5-50 ng / mL, preferably 10 ng / mL IL15, 1-10 U / mL, preferably 2 U / mL heparin sodium, 1-10 g / L, preferably 5 g / L human serum albumin, and 0.05-1%, preferably 0.1% platelet lysis buffer;

[0490] In another preferred embodiment, the amplification medium comprises RPMI-1640, preferably 1000 IU / mL IL2, preferably 10 ng / mL IL15, preferably 2 U / mL heparin sodium, preferably 5 g / L human serum albumin, 1-100 μg / mL, preferably 1-20 μg / mL, more preferably 10 μg / mL human transferrin, 1-50 μg / L, preferably 35 μg / L vitamin A, and 10-2000 μg / L, preferably 1000 μg / L of... Vitamin E, 1-200 μg / L, preferably 100 μg / L; Vitamin K1, 10-1000 μg / L, preferably 768.3 μg / L; Reduced glutathione, 0.1-20 μg / L, preferably 6.7 μg / L; Sodium selenite, 1-50 μg / L, preferably 20.2 μg / L; Ferrous chloride, 1-50 μg / L, preferably 19.2 μg / L; Zinc chloride, 1-50 μg / L, preferably 16.9 μg / L; Copper chloride, 0.0001-0.1 μg / L. The concentrations are as follows: nickel chloride (0.073 μg / L, preferably 0.0001-0.1 μg / L, preferably 0.044 μg / L, cobalt chloride hexahydrate (0.0001-0.5 μg / L, preferably 0.123 μg / L, manganese chloride tetrahydrate (0.0001-0.1 μg / L, preferably 0.073 μg / L, preferably 0.0001-0.5 μg / L, preferably 0.133 μg / L, silver chloride (1-50 μg / L, preferably 13.07 μg / L)). Potassium iodide, 0.0001-0.1 μg / L, preferably 0.012193 μg / L; sodium metavanadate, 0.0001-0.5 μg / L, preferably 0.107 μg / L; ammonium molybdate tetrahydrate, 1-50 μg / L, preferably 11.32 μg / L; rubidium chloride, 0.1-10 μg / L, preferably 5.874 μg / L; germanium tetrachloride, 0.0001-0.5 μg / L, preferably 0.3194 μg / L; and platelet lysis buffer, preferably 0.1%.

[0491] The serum-free culture medium described in this application contains fat-soluble vitamins, which overcomes the deficiency of conventional culture media that only contain water-soluble vitamins. This plays an important role in maintaining the integrity and stability of cell membranes and the survival of NK cells. The addition of trace elements not only promotes cell proliferation but also, to some extent, substitutes for protein factors in performing biological functions. Reducing trace elements, antioxidants, and vitamins work together synergistically to exert antioxidant and cell viability-maintaining effects, thereby significantly reducing the aging process of NK cells and enabling them to proliferate in vitro and maintain high cell viability. Platelet lysis buffer, a common serum substitute, is prepared by lysing platelets and sterile filtration. It has been widely used in the culture of umbilical cord blood mesenchymal stem cells. Due to its high concentration of cell growth factors, its appropriate addition to the NK cell growth medium can enhance NK cell activation and proliferation.

[0492] The NK cells harvested from the expanded culture described in this invention can be further concentrated, replaced, washed, and formulated before being stored at room temperature, refrigerated, or frozen. They can also be used for patient reinfusion directly or after further processing, or for other research purposes.

[0493] The present invention also provides an NK culture kit comprising the above-mentioned coating agent, activation medium and amplification medium.

[0494] The coating agent of this application can be a single or multiple monoclonal antibody combination, or a single or multiple bispecific antibody combination. Optional antibodies include anti-CD3, CD25, CD33, CD52, CD4, CD8, 4-1BB, CD19, CLL1, CD38 and CD16 antibodies.

[0495] The activators in this application include, but are not limited to, interleukin-2 (IL2), interleukin-7 (IL7), interleukin-15 (IL15), interleukin-12 (IL12), interleukin-18 (IL18), and interleukin-21 (IL21).

[0496] The activators in this application also include, but are not limited to, fat-soluble vitamins, trace elements, serum substitutes, heparin sodium, ascorbic acid, metformin, taurine, polyethylene glycol (PEG), nicotinamide, reduced glutathione, quercetin, 2-mercaptoethanol, rh-transferrin, and / or human serum albumin.

[0497] The fat-soluble vitamins include, but are not limited to, vitamin A, vitamin E, and vitamin K; the trace elements include, but are not limited to, selenium (Se), iron (Fe), zinc (Zn), copper (Cu), nickel (Ni), cobalt (Co), chromium (Cr), silver (Ag), aluminum (Al), barium (Ba), iodine (I), vanadium (V), rubidium (Rb), germanium (Ge), manganese (Mn), tin (Sn), and molybdenum (Mo); the serum substitutes include, but are not limited to, platelet lysate.

[0498] The amplification agents of this application include, but are not limited to, interleukin-2 (IL2), interleukin-15 (IL15), interleukin-12 (IL12), interleukin-18 (IL18), and interleukin-21 (IL21); the amplification agents of this application also include, but are not limited to, fat-soluble vitamins, trace elements, serum substitutes, heparin sodium, ascorbic acid, metformin, taurine, polyethylene glycol (PEG), nicotinamide, reduced glutathione, quercetin, 2-mercaptoethanol, rh-transferrin, and / or human serum albumin.

[0499] The fat-soluble vitamins include, but are not limited to, vitamin A, vitamin E, and vitamin K; the trace elements include, but are not limited to, selenium (Se), iron (Fe), zinc (Zn), copper (Cu), nickel (Ni), cobalt (Co), chromium (Cr), silver (Ag), aluminum (Al), barium (Ba), iodine (I), vanadium (V), rubidium (Rb), germanium (Ge), manganese (Mn), tin (Sn), and molybdenum (Mo); the serum substitutes include, but are not limited to, platelet lysate.

[0500] The serum-free culture medium-based NK cell production process described in this application includes, but is not limited to, NK cell coating, activation, and expansion.

[0501] The culture protocols of this application are applicable to, but not limited to, the subsequent culture of PBMCs derived from fresh or frozen peripheral blood, and the enrichment and subsequent culture of NK cells from PBMCs derived from fresh or frozen peripheral blood. Other NK cell cultures, such as those enriched from umbilical cord blood and NK92, should also be included within the scope of protection of this application.

[0502] Example

[0503] This application will be described in more detail by way of specific examples. The following embodiments are provided for illustrative purposes only and are not intended to limit this application in any way. Those skilled in the art will readily recognize that various non-critical parameters can be changed or modified to produce substantially the same results.

[0504] The basic culture media mentioned in the examples are all commercially available RPMI-1640. You can directly purchase liquid reagents or purchase lyophilized culture medium powder, fully dissolve it with water for injection, and filter it through a 0.22-micron filter membrane before use.

[0505] Table 1.1: Formulation of RPMI-1640 basal medium

[0506]

[0507] Table 1.2: Reagent Sources

[0508]

[0509] The cell counting method, flow cytometry phenotyping analysis, and Calcein AM release assay used in the embodiments of this invention are all conventional detection methods, and will only be briefly described in the following embodiments.

[0510] Example 1: NK cell enrichment

[0511] In this embodiment, NK cells or PBMC cells are derived from peripheral blood apheresis leukocytes (hPB Leukopak) provided by healthy volunteers. NK cells are enriched from hPB Leukopak, and most T cells are removed.

[0512] The following examples will use PBMC cells from five donors: volunteer A, volunteer B, volunteer C, volunteer D, and volunteer E. After enriching NK cells, CD3 - CD56 + The proportion of NK cells in PBMCs is shown in Table 2 below. Figure 1 :

[0513] Table 2: Flow cytometry results of initial NK cells after enrichment

[0514]

[0515] Example 2: Serum-free NK cell culture protocol a

[0516] The serum-free NK cell culture protocol of this embodiment includes a coating agent (for coating the culture container), an activation medium, and an amplification medium. The coating agent is diluted with physiological saline. The activation medium and the amplification medium are based on RPMI-1640 and supplemented with activators and amplifiers.

[0517] Table 3: Components and their concentrations in Example 2

[0518]

[0519] Example 3: Serum-free NK cell culture protocol b

[0520] The serum-free NK cell culture protocol of this embodiment includes a coating agent (for coating the culture solvent), an activation medium, and an amplification medium. The coating agent is diluted with physiological saline. Both the activation medium and the amplification medium use RPMI-1640 as the basal medium and add activators and amplifiers.

[0521] Table 4: Components and their concentrations in Example 3

[0522]

[0523] Example 4: Serum-free NK cell culture protocol c

[0524] The serum-free NK cell culture protocol of this embodiment includes a coating agent (for coating the culture solvent), an activation medium, and an amplification medium. The coating agent is diluted with physiological saline. Both the activation medium and the amplification medium use RPMI-1640 as the basal medium and add activators and amplifiers.

[0525] Table 5: Components and their concentrations in Example 4

[0526]

[0527] Example 5: Serum-free NK cell culture protocol

[0528] The serum-free NK cell culture protocol of this embodiment includes a coating agent (for coating the culture solvent), an activation medium, and an amplification medium. The coating agent is diluted with physiological saline. Both the activation medium and the amplification medium use RPMI-1640 as the basal medium and add activators and amplifiers.

[0529] Table 6: Components and their concentrations in Example 5

[0530]

[0531] Example 6: Serum-free NK cell culture protocol e

[0532] The serum-free NK cell culture protocol of this embodiment includes a coating agent (for coating the culture solvent), an activation medium, and an amplification medium. The coating agent is diluted with physiological saline. Both the activation medium and the amplification medium use RPMI-1640 as the basal medium and add activators and amplifiers.

[0533] Table 7: Components and their concentrations in Example 6

[0534]

[0535] Example 7: Serum-free NK cell culture protocol

[0536] The serum-free NK cell culture protocol of this embodiment includes a coating agent (for coating the culture solvent), an activation medium, and an amplification medium. The coating agent is diluted with physiological saline. Both the activation medium and the amplification medium use RPMI-1640 as the basal medium and add activators and amplifiers.

[0537] Table 8: Components and their concentrations used in Example 7

[0538]

[0539] Example for reference: Commercial serum-free culture medium regimen

[0540] This reference example uses Shanghai Bei'anji Biotechnology HIPP-T009 serum-free lymphocyte culture medium as the basal medium, and is used in conjunction with a coating agent and added activating factors. The specific components are shown in Table 9 below:

[0541] Table 9: Serum-free culture medium components of the reference example

[0542]

[0543] Example 8: Serum-free in vitro culture of NK cells

[0544] The PBMC cells obtained after enriching NK cells in Example 1 were cultured using the following method:

[0545] Coating phase (Day 0): Dilute the corresponding coating agent to the given concentration with physiological saline (as shown in Tables 3 to 8 for coating agents and concentrations), and add T25cm 2 Place in a culture flask and incubate at 37°C for 2 hours;

[0546] Activation phase (Day 0-7): T25cm 2 Remove the culture flask, remove excess coating agent, add activation medium, and inoculate with PBMC cells or enriched initial NK cells, controlling the initial density at 1.0 × 10⁶ cells / year. 6 ~2.5×106 Cells / mL. On the third and fifth days, replenish the culture medium as needed based on the color change, maintaining a cell density greater than 0.4 × 10⁶ cells / mL. 6 cells / mL;

[0547] Expansion phase (Days 7-14~17): Transplant cells to T75cm 2 Add amplification medium to the culture flask and continue culturing. Add fluid every other day to maintain a cell density greater than 0.4 × 10⁻⁶ cells / day. 6 Cells / mL, and depending on the cell density and volume, determine whether to transfer to a larger culture flask or bag. The total culture time is 14-17 days, after which the cells are harvested.

[0548] The NK cells enriched from the five volunteers mentioned in Example 1 were tested in one or more serum-free culture protocols as described in Examples 2-7 and the Reference Example. Specifically,

[0549] NK cells from volunteer A were tested in Examples 2-4, where culture protocol number A1 was used in Example 2, culture protocol number A2 was used in Example 4, and culture protocol number A3 was used in Example 3.

[0550] NK cells from volunteer B were tested in Examples 4 and 5, where culture protocol B1 was used in Example 4 and culture protocol B2 was used in Example 5.

[0551] NK cells from volunteer C were tested in Examples 4-7, wherein culture protocol number C1 was used in Example 4, culture protocol number C2 was used in Example 5, culture protocol number C3 was used in Example 6, and culture protocol number C4 was used in Example 7.

[0552] NK cells from volunteers D and E were tested in Example 6 and the reference example, respectively, where the culture protocols corresponding to Example 6 were numbered D1 and E1, and the culture protocols corresponding to the reference example were numbered D2 and E2.

[0553] NK cell viability and cell proliferation assay

[0554] Cells corresponding to the culture protocols of each embodiment were counted during culture (AOPI staining method), and cell proliferation curves were plotted. After culture, the final cell viability, total number of viable cells, and fold increase were measured. The cell proliferation curve for the culture protocol corresponding to volunteer A cells is shown below. Figure 2 Cell viability, total viable cell count, and fold expansion are shown in Table 10; cell proliferation curves for the corresponding culture protocols of volunteer B cells are shown in Table 10. Figure 3 Cell viability, total viable cells, and fold expansion are shown in Table 11; cell proliferation curves for the culture protocols corresponding to volunteer C cells are shown in Table 11. Figure 4Cell viability, total viable cells, and fold expansion are shown in Table 12; the comparison of the final cell fold expansion at the end of the culture protocol tests for volunteers D and E is shown in Table 12. Figure 5 The cell viability, total number of viable cells, and expansion fold are shown in Table 13. The results show that the exemplary scheme of this application, as shown in Example 6, is significantly superior to the commercial culture scheme shown in the reference example in terms of cell viability, total number of viable cells, and expansion fold.

[0555] Table 10: Cell viability, total number of viable cells, and fold increase of cells for volunteer A.

[0556]

[0557] Table 11: Cell viability, total number of viable cells, and fold expansion of B cells in volunteers.

[0558]

[0559] Table 12: Cell viability, total number of viable cells, and fold expansion of C cells in volunteers.

[0560]

[0561] Table 13: Cell viability, total viable cell count, and fold increase in cell count for volunteers D and E.

[0562]

[0563] NK cell flow cytometry phenotyping

[0564] The CD3, CD16, and CD56 phenotypes of the NK cells obtained in each embodiment were detected using conventional flow cytometry. The flow cytometry results are shown in Table 14. Figures 6A to 6D .

[0565] Table 14: Results of terminal NK cell phenotypic detection

[0566]

[0567] The proportion of NK cells in A1, A3, and A2 cells shows a gradually increasing trend. Without being bound by any specific theory, it is believed that the decrease in platelet lysate concentration in A1, A3, and A2 cells (2.5%, 1.25%, and 0.25%, respectively) is related to this trend. This is because platelet lysate contains a high concentration of TGF-β, which has an inhibitory effect on NK cells. Therefore, controlling the concentration of platelet lysate used should help increase CD3+. - CD56 + NK cells and CD16 + CD56 +NK cell percentage. In addition, nicotinamide and dimercaprol were added in Example 4 (A2), which, without being bound by any particular theory, help promote NK cell proliferation.

[0568] NK cell killing effect detection

[0569] The killing effect of NK cells on tumor cells was detected using the Calcein AM release assay, i.e., the fluorescence value of the final released Calcein was detected by an enzyme-linked immunosorbent assay (ELISA) reader. The killing rate was calculated as (fluorescence value of the experimental group - fluorescence value of the natural minimum group) / (fluorescence value of the maximum group - fluorescence value of the natural minimum group) × 100%. For the killing assay of volunteer A cells, NCI-H292 cells were used with an effector-to-target ratio of 3:1 and a co-incubation time of 4 hours. The results are shown in Table 15 below. For the killing assay of volunteer C cells, U937 cells were used with an effector-to-target ratio of 10:1 and a co-incubation time of 4 hours. The results are shown in Table 16 below.

[0570] Table 15: Percentage of NCI-H292 tumor cells killed by volunteer A cells

[0571]

[0572] Table 16: Percentage of C cells killed by volunteers in the U937 leukemia cell line

[0573]

[0574] The test results above indicate that the culture protocol in this embodiment has a good proliferation effect, CD3 - CD56 + and CD16 + CD56 + The proportion of NK cells is relatively high, reaching about 95%, and it has a high killing effect on both lung cancer cell line NCI-H292 and leukemia cell line U937.

[0575] The above description is merely a preferred embodiment of this application and is for illustrative purposes only, and is not intended to limit this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions provided in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A coating agent for an NK cell culture vessel, the coating agent being (i) an anti-CD38 antibody and an anti-CD52 antibody; or (ii) an anti-CD38 antibody, an anti-CD52 antibody, and an anti-CD33-CLL1 bispecific antibody; wherein the concentration of the anti-CD38 antibody is 0.1-20 pg / mL, the concentration of the anti-CD52 antibody is 0.5-20 pg / mL, and the concentration of the anti-CD33-CLL1 bispecific antibody is 0.1-20 pg / mL.

2. The coating agent of claim 1, wherein the concentration of the anti-CD38 antibody is 0.5-10 pg / mL, the concentration of the anti-CD52 antibody is 1-15 pg / mL, and the concentration of the anti-CD33-CLL1 bispecific antibody is 0.5-10 pg / mL.

3. The coating agent of claim 1 or 2, wherein the concentration of the anti-CD38 antibody is 1000 pg / L.

4. The coating agent of claim 3, wherein the anti-CD38 antibody is diluted with physiological saline or phosphate buffer.

5. The coating agent of claim 1, wherein the concentration of the anti-CD52 antibody is 10000 pg / L.

6. The coating agent of claim 5, wherein the anti-CD52 antibody is diluted with physiological saline or phosphate buffer.

7. The coating agent of claim 1, wherein the concentration of the anti-CD33-CLL1 bispecific antibody is 1000 pg / L.

8. An NK cell culture kit comprising the coating agent of any one of claims 1-7; or comprising the coating agent of any one of claims 1-7 and an activator for NK cell culture and an expander for NK cell culture, wherein the NK cells are obtained from peripheral blood mononuclear cell (PBMC)-derived NK cells; wherein the activator is (i) or (ii): (i) an anti-CD16 antibody, an anti-CD2 antibody, an anti-CD3 antibody, an anti-CD28 antibody, an anti-CD31 antibody, an anti-CD40 antibody, an anti-CD48 antibody, an anti-CD54 antibody, an anti-CD86 antibody, an anti-CD137 antibody, an anti-CD244 antibody, an anti-CD276 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti-CD274 antibody, an anti-CD273 antibody, an anti (i) a combination of 1000 IU / mL of IL2, 10 ng / mL of IL15, 35 pg / L of vitamin A, 1000 pg / L of vitamin E, 100 pg / L of vitamin Kl, 0.1-20 pg / L of sodium selenite, 1-50 pg / L of ferrous chloride, 1-50 pg / L of zinc chloride, 1-50 pg / L of copper chloride, 0.0001-0.1 pg / L of nickel chloride, 0.0001-0.1 pg / L of cobalt chloride hexahydrate, 0.0001-0.5 pg / L of manganese chloride tetrahydrate, 0.0001-0.1 pg / L of chromous chloride, 0.0001-0.5 pg / L of silver chloride, 1-50 pg / L of potassium iodide, 0.0001-0.1 pg / L of sodium metavanadate, 0.0001-0.5 pg / L of ammonium molybdate tetrahydrate, 1-50 pg / L of rubidium chloride, 0.1-10 pg / L of germanium tetrachloride, 0.0001-0.5 pg / L of stannous chloride, 0.25 v / v% of platelet lysate, 1-10 mM of nicotinamide, 0.001-0.055 mM of dithiothreitol, 1-10 U / mL of sodium heparin, 10-1000 pg / L of reduced glutathione, 1-100 pg / mL of human transferrin, and 1-10 g / L of human blood albumin; or (ii) a combination of 1000 IU / mL of IL2, 10 ng / mL of IL15, 10 ng / mL of IL21, 35 pg / L of vitamin A, 1000 pg / L of vitamin E, 100 pg / L of vitamin Kl, 0.1-20 pg / L of sodium selenite, 1-50 pg / L of ferrous chloride, 1-50 pg / L of zinc chloride, 1-50 pg / L of copper chloride, 0.0001-0.1 pg / L of nickel chloride, 0.0001-0.1 pg / L of cobalt chloride hexahydrate, 0.0001-0.5 pg / L of manganese chloride tetrahydrate, 0.0001-0.1 pg / L of chromous chloride, 0.0001-0.5 pg / L of silver chloride, 1-50 pg / L of potassium iodide, 0.0001-0.1 pg / L of sodium metavanadate, 0.0001-0.5 pg / L of ammonium molybdate tetrahydrate, 1-50 pg / L of rubidium chloride, 0.1-10 pg / L of germanium tetrachloride, 0.0001-0.5 pg / L of stannous chloride, 0.25 v / v% of platelet lysate, 1-10 mM of nicotinamide, 0.001-0.055 mM of dithiothreitol, 1-10 U / mL of sodium heparin, 10-1000 pg / L of reduced glutathione, 1-100 pg / mL of human transferrin, and 1-10 g / L of human blood albumin; wherein the expansion agent is (iii) (iii) a combination of 1000 IU / mL of IL2, 10 ng / mL of IL15, 35 pg / L of vitamin A, 1000 pg / L of vitamin E, 100 pg / L of vitamin K1, 0.1-20 pg / L of sodium selenite, 1-50 pg / L of ferrous chloride, 1-50 pg / L of zinc chloride, 1-50 pg / L of copper chloride, 0.0001-0.1 pg / L of nickel chloride, 0.0001-0.1 pg / L of cobalt chloride hexahydrate, 0.0001-0.5 pg / L of manganese chloride tetrahydrate, 0.0001-0.1 pg / L of chromous chloride, 0.0001-0.5 pg / L of silver chloride, 1-50 pg / L of potassium iodide, 0.0001-0.1 pg / L of sodium metavanadate, 0.0001-0.5 pg / L of ammonium molybdate tetrahydrate, 1-50 pg / L of rubidium chloride, 0.1-10 pg / L of germanium tetrachloride, 0.0001-0.5 pg / L of stannous chloride, 0.1 v / v% of platelet lysate, 1-10 U / mL of sodium heparin, 10-1000 pg / L of reduced glutathione, 1-100 pg / mL of human transferrin, and 1-10 g / L of human blood albumin.

9. The NK cell culture kit of claim 8, wherein, In the activator: the concentration of sodium selenite is 0.5-10 pg / L; the concentration of reduced glutathione is 50-100 pg / L; and / or the concentration of human transferrin is 1-20 pg / mL.

10. The NK cell culture kit of claim 8, wherein, In the expansion agent: the concentration of sodium selenite is 0.5-10 pg / L; the concentration of reduced glutathione is 50-100 pg / L; and / or the concentration of human transferrin is 1-20 pg / mL.

11. The NK cell culture kit of claim 8, wherein: the activator is a combination of 1000 IU / mL of IL2, 10 pg / L of IL15, 10 pg / L of IL21, 2 U / mL of sodium heparin, 5000000 pg / L of human serum albumin, 0.25 v / v% of platelet lysate, 2.5 mM of nicotinamide, 0.011 mM of dithiothreitol, 10000 pg / L of human transferrin, 35 pg / L of vitamin A, 1000 pg / L of vitamin E, 100 pg / L of vitamin Kl, 768.3 pg / L of reduced glutathione, 6.7 pg / L of sodium selenite, 20.2 pg / L of ferrous chloride, 19.2 pg / L of zinc chloride, 16.9 pg / L of copper chloride, 0.073 pg / L of nickel chloride, 0.044 pg / L of cobalt chloride hexahydrate, 0.123 pg / L of manganese chloride tetrahydrate, 0.073 pg / L of chromous chloride, 0.133 pg / L of silver chloride, 13.07 pg / L of potassium iodide, 0.012193 pg / L of sodium metavanadate, 0.107 pg / L of ammonium molybdate tetrahydrate, 11.32 pg / L of rubidium chloride, 5.874 pg / L of germanium tetrachloride, 0.3194 pg / L of stannous chloride; and the expansion agent is 1000 IU / mL of IL2, 10 pg / L of IL15, 2 U / mL of sodium heparin, 5000000 pg / L of human serum albumin, 0.1 v / v% of platelet lysate, 10000 pg / L of human transferrin, 35 pg / L of vitamin A, 1000 pg / L of vitamin E, 100 pg / L of vitamin Kl, 768.3 pg / L of reduced glutathione, 6.7 pg / L of sodium selenite, 20.2 pg / L of ferrous chloride, 19.2 pg / L of zinc chloride, 16.9 pg / L of copper chloride, 0.073 pg / L of nickel chloride, 0.044 pg / L of cobalt chloride hexahydrate, 0.123 pg / L of manganese chloride tetrahydrate, 0.073 pg / L of chromous chloride, 0.133 pg / L of silver chloride, 13.07 pg / L of potassium iodide, 0.012193 pg / L of sodium metavanadate, 0.107 pg / L of ammonium molybdate tetrahydrate, 11.32 pg / L of rubidium chloride, 5.874 pg / L of germanium tetrachloride, 0.3194 pg / L of stannous chloride.

12. The NK cell culture kit of claim 8, wherein The activator is a combination of 1000 IU / mL of IL2, 10 pg / L of IL15, 2 U / mL of sodium heparin, 5000000 pg / L of human blood albumin, 0.25 v / v% of platelet lysate, 2.5 mM of nicotinamide, 0.011 mM of dithiothreitol, 10000 pg / L of human transferrin, 35 pg / L of vitamin A, 1000 pg / L of vitamin E, 100 pg / L of vitamin K1, 768.3 pg / L of reduced glutathione, 6.7 pg / L of sodium selenite, 20.2 pg / L of ferrous chloride, 19.2 pg / L of zinc chloride, 16.9 pg / L of copper chloride, 0.073 pg / L of nickel chloride, 0.044 pg / L of cobalt chloride hexahydrate, 0.123 pg / L of manganese chloride tetrahydrate, 0.073 pg / L of chromous chloride, 0.133 pg / L of silver chloride, 13.07 pg / L of potassium iodide, 0.012193 pg / L of sodium metavanadate, 0.107 pg / L of ammonium molybdate tetrahydrate, 11.32 pg / L of rubidium chloride, 5.874 pg / L of germanium tetrachloride, 0.3194 pg / L of stannous chloride; and The activator is a combination of 1000 IU / mL of IL2, 10 pg / L of IL15, 2 U / mL of sodium heparin, 5000000 pg / L of human blood albumin, 0.25 v / v% of platelet lysate, 2.5 mM of nicotinamide, 0.011 mM of dithiothreitol, 10000 pg / L of human transferrin, 35 pg / L of vitamin A, 1000 pg / L of vitamin E, 100 pg / L of vitamin K1, 768.3 pg / L of reduced glutathione, 6.7 pg / L of sodium selenite, 20.2 pg / L of ferrous chloride, 19.2 pg / L of zinc chloride, 16.9 pg / L of copper chloride, 0.073 pg / L of nickel chloride, 0.044 pg / L of cobalt chloride hexahydrate, 0.123 pg / L of manganese chloride tetrahydrate, 0.073 pg / L of chromous chloride, 0.133 pg / L of silver chloride, 13.07 pg / L of potassium iodide, 0.012193 pg / L of sodium metavanadate, 0.107 pg / L of ammonium molybdate tetrahydrate, 11.32 pg / L of rubidium chloride, 5.874 pg / L of germanium tetrachloride, 0.3194 pg / L of stannous chloride.

13. An NK cell culture kit comprising (i) the coating agent of any one of claims 1-7; or comprising (i) the coating agent of any one of claims 1-7 and one or both of (ii) and (iii): (ii) a serum-free medium for the activation step of NK cell culture comprising a first basal medium and an activator for NK cell culture, and (iii) a serum-free medium for the expansion step of NK cell culture comprising a second basal medium and an expansion agent for NK cell culture; wherein the NK cells are obtained from peripheral blood mononuclear cell (PBMC)-derived NK cells. The activator is either (iv) or (v): (iv) 1000 IU / mL IL2, 10 ng / mL IL15, 35 μg / L Vitamin A, 1000 μg / L Vitamin E, 100 μg / L Vitamin K1, 0.1-20 μg / L Sodium selenite, 1-50 μg / L Ferrous chloride, 1-50 μg / L Zinc chloride, 1-50 μg / L Copper chloride, 0.0001-0.1 μg / L Nickel chloride, 0.0001-0.1 μg / L Cobalt chloride hexahydrate, 0.0001-0.5 μg / L... Manganese chloride tetrahydrate (0.0001-0.1 μg / L), chromium chloride (0.0001-0.5 μg / L), silver chloride (0.0001-0.5 μg / L), potassium iodide (1-50 μg / L), sodium metavanadate (0.0001-0.1 μg / L), ammonium molybdate tetrahydrate (0.0001-0.5 μg / L), rubidium chloride (1-50 μg / L), germanium tetrachloride (0.1-10 μg / L), stannous chloride (0.0001-0.5 μg / L), platelet lysis buffer (0.25 v / v%), 1-10 μg / L... A combination of mM nicotinamide, 0.001-0.055 mM dimercaptoethanol, 1-10 U / mL heparin sodium, 10-1000 μg / L reduced glutathione, 1-100 μg / mL human transferrin, and 1-10 g / L human serum albumin; or (v) 1000 IU / mL IL2, 10 ng / mL IL15, 10 ng / mL IL21, 35 μg / L Vitamin A, 1000 μg / L Vitamin E, 100 μg / L Vitamin K1, 0.1-20 μg / L Sodium selenite, 1-50 μg / L Ferrous chloride, 1-50 μg / L Zinc chloride, 1-50 μg / L Copper chloride, 0.0001-0.1 μg / L Nickel chloride, 0.0001-0.1 μg / L Cobalt chloride hexahydrate, 0.00 0.01-0.5 μg / L manganese chloride tetrahydrate, 0.0001-0.1 μg / L chromium chloride, 0.0001-0.5 μg / L silver chloride, 1-50 μg / L potassium iodide, 0.0001-0.1 μg / L sodium metavanadate, 0.0001-0.5 μg / L ammonium molybdate tetrahydrate, 1-50 μg / L rubidium chloride, 0.1-10 μg / L germanium tetrachloride, 0.0001-0.5 μg / L stannous chloride, 0.25 v / v% platelet lysis buffer, 1-10 A combination of mM nicotinamide, 0.001-0.055 mM dimercaptoethanol, 1-10 U / mL heparin sodium, 10-1000 μg / L reduced glutathione, 1-100 μg / mL human transferrin and 1-10 g / L human serum albumin. The amplifying agent mentioned above is the following (vi): (vi) a combination of 1000 IU / mL of IL2, 10 ng / mL of IL15, 35 pg / L of vitamin A, 1000 pg / L of vitamin E, 100 pg / L of vitamin Kl, 0.1-20 pg / L of sodium selenite, 1-50 pg / L of ferrous chloride, 1-50 pg / L of zinc chloride, 1-50 pg / L of copper chloride, 0.0001-0.1 pg / L of nickel chloride, 0.0001-0.1 pg / L of cobalt chloride hexahydrate, 0.0001-0.5 pg / L of manganese chloride tetrahydrate, 0.0001-0.1 pg / L of chromous chloride, 0.0001-0.5 pg / L of silver chloride, 1-50 pg / L of potassium iodide, 0.0001-0.1 pg / L of sodium metavanadate, 0.0001-0.5 pg / L of ammonium molybdate tetrahydrate, 1-50 pg / L of rubidium chloride, 0.1-10 pg / L of germanium tetrachloride, 0.0001-0.5 pg / L of stannous chloride, 0.1 v / v% of platelet lysate, 1-10 U / mL of sodium heparin, 10-1000 pg / L of reduced glutathione, 1-100 pg / mL of human transferrin, and 1-10 g / L of human blood albumin.

14. The NK cell culture kit of claim 13, wherein, In the activator: the concentration of sodium selenite is 0.5-10 pg / L; the concentration of reduced glutathione is 50-100 pg / L; and / or the concentration of human transferrin is 1-20 pg / mL.

15. The NK cell culture kit of claim 13, wherein, In the expansion agent: the concentration of sodium selenite is 0.5-10 pg / L; the concentration of reduced glutathione is 50-100 pg / L; and / or the concentration of human transferrin is 1-20 pg / mL.

16. The NK cell culture kit of claim 13, wherein: the activator is a combination of 1000 IU / mL of IL2, 10 pg / L of IL15, 10 pg / L of IL21, 2 U / mL of sodium heparin, 5000000 pg / L of human serum albumin, 0.25 v / v% of platelet lysate, 2.5 mM of nicotinamide, 0.011 mM of dithiothreitol, 10000 pg / L of human transferrin, 35 pg / L of vitamin A, 1000 pg / L of vitamin E, 100 pg / L of vitamin Kl, 768.3 pg / L of reduced glutathione, 6.7 pg / L of sodium selenite, 20.2 pg / L of ferrous chloride, 19.2 pg / L of zinc chloride, 16.9 pg / L of copper chloride, 0.073 pg / L of nickel chloride, 0.044 pg / L of cobalt chloride hexahydrate, 0.123 pg / L of manganese chloride tetrahydrate, 0.073 pg / L of chromous chloride, 0.133 pg / L of silver chloride, 13.07 pg / L of potassium iodide, 0.012193 pg / L of sodium metavanadate, 0.107 pg / L of ammonium molybdate tetrahydrate, 11.32 pg / L of rubidium chloride, 5.874 pg / L of germanium tetrachloride, 0.3194 pg / L of stannous chloride; and the expansion agent is 1000 IU / mL of IL2, 10 pg / L of IL15, 2 U / mL of sodium heparin, 5000000 pg / L of human serum albumin, 0.1 v / v% of platelet lysate, 10000 pg / L of human transferrin, 35 pg / L of vitamin A, 1000 pg / L of vitamin E, 100 pg / L of vitamin Kl, 768.3 pg / L of reduced glutathione, 6.7 pg / L of sodium selenite, 20.2 pg / L of ferrous chloride, 19.2 pg / L of zinc chloride, 16.9 pg / L of copper chloride, 0.073 pg / L of nickel chloride, 0.044 pg / L of cobalt chloride hexahydrate, 0.123 pg / L of manganese chloride tetrahydrate, 0.073 pg / L of chromous chloride, 0.133 pg / L of silver chloride, 13.07 pg / L of potassium iodide, 0.012193 pg / L of sodium metavanadate, 0.107 pg / L of ammonium molybdate tetrahydrate, 11.32 pg / L of rubidium chloride, 5.874 pg / L of germanium tetrachloride, 0.3194 pg / L of stannous chloride.

17. The NK cell culture kit of claim 13, wherein: The activator is a combination of 1000 IU / mL of IL2, 10 pg / L of IL15, 2 U / mL of sodium heparin, 5000000 pg / L of human blood albumin, 0.25 v / v% of platelet lysate, 2.5 mM of nicotinamide, 0.011 mM of dithiothreitol, 10000 pg / L of human transferrin, 35 pg / L of vitamin A, 1000 pg / L of vitamin E, 100 pg / L of vitamin Kl, 768.3 pg / L of reduced glutathione, 6.7 pg / L of sodium selenite, 20.2 pg / L of ferrous chloride, 19.2 pg / L of zinc chloride, 16.9 pg / L of copper chloride, 0.073 pg / L of nickel chloride, 0.044 pg / L of cobalt chloride hexahydrate, 0.123 pg / L of manganese chloride tetrahydrate, 0.073 pg / L of chromous chloride, 0.133 pg / L of silver chloride, 13.07 pg / L of potassium iodide, 0.012193 pg / L of sodium metavanadate, 0.107 pg / L of ammonium molybdate tetrahydrate, 11.32 pg / L of rubidium chloride, 5.874 pg / L of germanium tetrachloride, 0.3194 pg / L of stannous chloride; and The activator is a combination of 1000 IU / mL of IL2, 10 pg / L of IL15, 2 U / mL of sodium heparin, 5000000 pg / L of human blood albumin, 0.25 v / v% of platelet lysate, 2.5 mM of nicotinamide, 0.011 mM of dithiothreitol, 10000 pg / L of human transferrin, 35 pg / L of vitamin A, 1000 pg / L of vitamin E, 100 pg / L of vitamin Kl, 768.3 pg / L of reduced glutathione, 6.7 pg / L of sodium selenite, 20.2 pg / L of ferrous chloride, 19.2 pg / L of zinc chloride, 16.9 pg / L of copper chloride, 0.073 pg / L of nickel chloride, 0.044 pg / L of cobalt chloride hexahydrate, 0.123 pg / L of manganese chloride tetrahydrate, 0.073 pg / L of chromous chloride, 0.133 pg / L of silver chloride, 13.07 pg / L of potassium iodide, 0.012193 pg / L of sodium metavanadate, 0.107 pg / L of ammonium molybdate tetrahydrate, 11.32 pg / L of rubidium chloride, 5.874 pg / L of germanium tetrachloride, 0.3194 pg / L of stannous chloride; and 18. The NK cell culture kit of any one of claims 13-17, wherein the first basal medium is RPMI-1640 basal medium.

19. The NK cell culture kit of any one of claims 13-17, wherein the second basal medium is RPMI-1640 basal medium.

20. Use of the coating agent of any one of claims 1-7 in NK cell culture, wherein the NK cells are obtained from peripheral blood mononuclear cell (PBMC)-derived NK cells.

21. A method of NK cell culture, comprising, (1) coating a culture vessel with the coating agent of any one of claims 1 to 7, and (2) adding a cell population comprising NK cells to the coated culture vessel; wherein the cell population is a PBMC cell population.

22. The NK cell culture method of claim 21, wherein the cell population is subjected to an NK cell enrichment step.

Images