Composition for culturing natural killer cells and method for preparing natural killer cells by using same

Abstract

The invention relates to: a composition for culturing natural killer cells, comprising, as an active ingredient, a fusion protein comprising an IL-2 protein and a CD80 protein; and a method for preparing natural killer cells by using same. Particularly, a composition for culturing natural killer cells, comprising, as an active ingredient, a fusion protein comprising IL-2 or a variant thereof and CD80 or a fragment thereof, of the present invention, promotes the proliferation of natural killer cells, induces the expression of CD16 and NKp46, and increases the expression and secretion of granzyme B and perforin, thereby being effectively usable in the preparation of natural killer cells having an excellent anticancer immune function.

Description

Composition for culturing natural killer cells and method for producing natural killer cells using the same

[0001] The present invention relates to a composition for culturing natural killer cells and a method for producing natural killer cells using the same.

[0002] Various treatments, including surgery, radiation therapy, and chemotherapy, have been developed to treat cancer. However, due to reports of numerous side effects, immunotherapy methods that utilize the patient's immune system are being developed. In particular, immunotherapy using natural killer cells, which can be mass-produced and frozen, is being studied.

[0003] Specifically, natural killer cells are a type of lymphocyte distributed in the bone marrow, spleen, peripheral lymph nodes, and peripheral blood of the body, accounting for approximately 10% of peripheral blood lymphocytes, and playing an important role in the innate immune response (Ann Rev Immunol., 24: 257-286, 2006). In addition, natural killer cells are positive for CD56 and CD16, but negative for CD3. Natural killer cells kill cells by releasing cytoplasmic granules containing perforin and granzymes. Natural killer cells secrete various cytokines, such as IFN-γ, TNF-α, GM-CSF, and IL-10.

[0004] Furthermore, natural killer cells express several receptors on their cell surface, which are involved in cell adhesion, activation, or inhibition of cell killing ability. However, most natural killer cells present in the body of a normal person are inactive. Therefore, activated natural killer cells are necessary to eradicate cancer. Furthermore, natural killer cells present in the body of cancer patients exhibit functional defects due to the cancer cell's immune evasion mechanism. Therefore, activating natural killer cells is crucial for utilizing natural killer cells as a therapeutic agent. Furthermore, because the number of natural killer cells present in the body is limited, the development of technologies for mass proliferation and freezing of natural killer cells from the blood of a normal person or a patient is essential.

[0005] Meanwhile, IL-2, also known as T-cell growth factor (TCGF), is a globular glycoprotein that plays a central role in lymphocyte production, survival, and homeostasis. IL-2 is a protein weighing 15.5 to 16 kDa and consisting of 133 amino acids. IL-2 mediates various immune responses by binding to the IL-2 receptor, which is composed of three individual subunits.

[0006] CD80, also known as B7-1, is a member of the B7 family of membrane-bound proteins involved in immune regulation by binding to ligands to transduce costimulatory and coinhibitory responses. CD80 is a transmembrane protein expressed on the surface of T cells, B cells, dendritic cells, and monocytes. CD80 is known to bind to CD28, CTLA4 (CD152), and PD-L1.

[0007] Although it is widely known that natural killer cells are important in cancer treatment, there is still a lack of specific methods to amplify them for effective use.

[0008] Accordingly, the present inventors conducted research to mass-produce activated natural killer cells. As a result, they produced natural killer cells using a fusion protein dimer comprising IL-2 and CD80 proteins. Furthermore, they confirmed that the activity of natural killer cells produced in this manner increased and exhibited excellent anticancer effects, thereby completing the present invention.

[0009] To achieve the above purpose, one aspect of the present invention provides a composition for culturing natural killer cells, comprising a fusion protein dimer comprising IL-2 or a variant thereof and CD80 or a fragment thereof as an active ingredient.

[0010] Another aspect of the present invention provides a method for culturing natural killer cells, comprising the steps of: i) isolating cells that do not express CD3 from peripheral blood mononuclear cells (PBMCs); ii) isolating cells that express CD56 from the separated cells that do not express CD3; and iii) culturing the separated cells in the presence of a fusion protein dimer comprising IL-2 or a variant thereof and CD80 or a fragment thereof.

[0011] Another aspect of the present invention provides natural killer cells produced by the above natural killer cell culture method.

[0012] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the natural killer cells as an active ingredient.

[0013] Another aspect of the present invention provides a method for culturing natural killer cells, comprising the steps of: i) isolating cells that do not express CD3 from PBMCs; and ii) culturing the isolated cells in the presence of a fusion protein dimer comprising IL-2 or a variant thereof and CD80 or a fragment thereof.

[0014] Another aspect of the present invention provides natural killer cells produced by the above natural killer cell culture method.

[0015] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the natural killer cells as an active ingredient.

[0016] Another aspect of the present invention provides a method for culturing natural killer cells, comprising the steps of: i) isolating cells expressing CD56 from PBMCs; and ii) culturing the isolated cells in the presence of a fusion protein dimer comprising IL-2 or a variant thereof and CD80 or a fragment thereof.

[0017] Another aspect of the present invention provides natural killer cells produced by the above natural killer cell culture method.

[0018] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the natural killer cells as an active ingredient.

[0019] Another aspect of the present invention provides a method for stimulating the activity of natural killer cells in PBMCs, comprising the step of culturing PBMCs in the presence of a fusion protein dimer comprising IL-2 or a variant thereof and CD80 or a fragment thereof.

[0020] Another aspect of the present invention provides a PBMC manufactured by a method for promoting the activity of natural killer cells in the PBMC.

[0021] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the PBMC as an active ingredient.

[0022] The composition for culturing natural killer cells, which comprises a fusion protein comprising IL-2 or a variant thereof and CD80 or a fragment thereof of the present invention as an active ingredient, promotes the proliferation of natural killer cells, induces the expression of CD16 and NKp46, and increases the expression and secretion of granzyme B and perforin, and can be usefully used for producing natural killer cells with excellent anticancer immune function.

[0023] Figure 1a shows a schematic diagram of the fusion protein dimer used in the present invention.

[0024] Figure 1b shows the obtained fusion protein dimer (GI-101) confirmed by SDS-PAGE.

[0025] Figure 1c shows the content of fusion protein dimer (GI-101) according to absorbance.

[0026] Figure 1d shows the analysis of the obtained fusion protein dimer (GI-101) using size exclusion chromatography (SEC).

[0027] Figure 2a shows the obtained hCD80-Fc fusion protein dimer confirmed by SDS-PAGE.

[0028] Figure 2b shows the analysis of the obtained hCD80-Fc fusion protein dimer using size exclusion chromatography (SEC).

[0029] Figure 3a shows the obtained Fc-IL2v2 fusion protein dimer confirmed by SDS-PAGE.

[0030] Figure 3b shows the analysis of the obtained Fc-IL2v2 fusion protein dimer using size exclusion chromatography (SEC).

[0031] Figure 3c shows the obtained Fc-IL2wt fusion protein dimer confirmed by SDS-PAGE.

[0032] Figure 3d shows the analysis of the obtained Fc-IL2wt fusion protein dimer using size exclusion chromatography (SEC).

[0033] Figure 4a shows the obtained hCD80-Fc-IL2wt fusion protein dimer confirmed by SDS-PAGE.

[0034] Figure 4b shows the analysis of the obtained hCD80-Fc-IL2wt fusion protein dimer using size exclusion chromatography (SEC).

[0035] Figures 5a and 5b show the number of NK cells when cultured under AIM-V (5%SR) conditions.

[0036] Figures 6a and 6b show the survival rate of NK cells when cultured under AIM-V (5%SR) conditions.

[0037] Figures 7a and 7b show the number of NK cells when cultured under AIM-V (5% hABS) conditions.

[0038] Figures 8a and 8b show the survival rate of NK cells when cultured under AIM-V (5% hABS) conditions.

[0039] Figures 9a and 9b show the number of NK cells when cultured under X-vivo (5% hABS) conditions.

[0040] Figures 10a and 10b show the survival rate of NK cells when cultured under X-vivo (5% hABS) conditions.

[0041] Figures 11a and 11b show the number of NK cells when cultured under NK MACS (5% hABS) conditions.

[0042] Figures 12a and 12b show the survival rate of NK cells when cultured under NK MACS (5% hABS) conditions.

[0043] Figure 13 shows the results of FACS analysis confirming the purity of natural killer cells cultured from a composition containing AIM-V (5% SR) medium.

[0044] Figure 14 shows the results of FACS analysis confirming the purity of natural killer cells cultured from a composition containing AIM-V (5% hABS) medium.

[0045] Figure 15 shows the results of FACS analysis confirming the purity of natural killer cells cultured from a composition containing X-vivo15 (5% hABS) medium.

[0046] Figure 16 shows the results of FACS analysis confirming the purity of natural killer cells cultured from a composition containing NK MACS (5% hABS) medium.

[0047] Figure 17 shows the results of analysis on the activity marker of natural killer cells cultured from a composition containing AIM-V (5% SR) medium.

[0048] Figure 18 shows the results of analysis on the inhibition marker of natural killer cells cultured from a composition containing AIM-V (5% SR) medium.

[0049] Figure 19 shows the results of analysis on the activity marker of natural killer cells cultured from a composition containing AIM-V (5% hABS) medium.

[0050] Figure 20 shows the results of analysis on the inhibition marker of natural killer cells cultured from a composition containing AIM-V (5% hABS) medium.

[0051] Figure 21 shows the results of analysis on the activity marker of natural killer cells cultured from a composition containing X-vivo (5% hABS) medium.

[0052] Figure 22 shows the results of analysis on the inhibition marker of natural killer cells cultured from a composition containing X-vivo (5% hABS) medium.

[0053] Figure 23 shows the results of analysis on the activity marker of natural killer cells cultured from a composition containing NK MACS (5% hABS) medium.

[0054] Figure 24 shows the results of analysis on the inhibition marker of natural killer cells cultured from a composition containing NK MACS (5% hABS) medium.

[0055] Figure 25 shows the results of analysis of cytotoxicity markers of natural killer cells cultured from a composition containing AIM-V (5%SR) medium.

[0056] Figure 26 shows the results of analysis of cytotoxicity markers of natural killer cells cultured from a composition containing AIM-V (5% hABS) medium.

[0057] Figure 27 shows the results of analysis of cell killing markers of natural killer cells cultured from a composition containing X-vivo15 (5% hABS) medium.

[0058] Figure 28 shows the results of analysis of cell killing markers of natural killer cells cultured from a composition containing NK MACS (5% hABS) medium.

[0059] Figure 29 shows the results of analyzing the degranulation ability of natural killer cells cultured for 21 days in a composition containing GI-101 (50 nM), GI-101_wt (50 nM), CD80-Fc (50 nM) + Fc-IL2v2 (50 nM), or CD80-Fc (50 nM) + Fc-IL2wt (50 nM) in AIM-V (5% hABS) medium.

[0060] Figure 30 shows the results of analyzing the cancer cell killing effect by natural killer cells cultured for 21 days from a composition containing GI-101 (50 nM), GI-101_wt (50 nM), CD80-Fc (50 nM) + Fc-IL2v2 (50 nM), or CD80-Fc (50 nM) + Fc-IL2wt (50 nM) in AIM-V (5% hABS) medium.

[0061] Figure 31 shows the results of analyzing the degranulation ability of natural killer cells cultured for 21 days in a composition containing GI-101 (50 nM), GI-101_wt (50 nM), CD80-Fc (50 nM) + Fc-IL2v2 (50 nM), or CD80-Fc (50 nM) + Fc-IL2wt (50 nM) in X-vivo15 (5% hABS) medium.

[0062] Figure 32 shows the results of analyzing the cancer cell killing effect by natural killer cells cultured for 21 days from a composition containing GI-101 (50 nM), GI-101_wt (50 nM), CD80-Fc (50 nM) + Fc-IL2v2 (50 nM), or CD80-Fc (50 nM) + Fc-IL2wt (50 nM) in X-vivo15 (5% hABS) medium.

[0063] Figure 33 shows the results of analyzing the degranulation ability of natural killer cells cultured for 21 days in a composition containing GI-101 (50 nM), GI-101_wt (50 nM), CD80-Fc (50 nM) + Fc-IL2v2 (50 nM), or CD80-Fc (50 nM) + Fc-IL2wt (50 nM) in NK MACS (5% hABS) medium.

[0064] Figure 34 shows the results of analyzing the cancer cell killing effect by natural killer cells cultured for 21 days from a composition containing GI-101 (50 nM), GI-101_wt (50 nM), CD80-Fc (50 nM) + Fc-IL2v2 (50 nM), or CD80-Fc (50 nM) + Fc-IL2wt (50 nM) in NK MACS (5% hABS) medium.

[0065] Composition and medium for NK cell proliferation

[0066] One aspect of the present invention provides a composition for culturing natural killer cells, comprising a fusion protein dimer comprising IL-2 or a variant thereof and CD80 or a fragment thereof as an active ingredient. In addition, a medium for culturing natural killer cells, comprising the fusion protein dimer as an active ingredient, is provided.

[0067] The above natural killer cell culture composition may additionally include any one selected from the group consisting of a medium, serum, supplements, and combinations thereof.

[0068] The above NK cell culture medium may be a medium to which a fusion protein dimer including the IL-2 protein and CD80 protein is added to a cell culture medium. At this time, the cell culture medium may include any one selected from the group consisting of amino acids, sugars, inorganic salts, and vitamins. Preferably, the cell culture medium may include all of amino acids, sugars, inorganic salts, and vitamins. In one specific example, the NK cell culture medium may include the components of Tables 1 to 4 below.

[0069] As used herein, the term "cell culture medium" refers to a medium used to culture cells, and more specifically, a medium for culturing NK cells, and more specifically, CD3-CD56+ cells. It contains components required by cells for in vitro cell growth and survival, or components that aid cell growth and survival. Specifically, the components may be vitamins, essential or non-essential amino acids, and trace elements. The medium may be a medium used for culturing cells, preferably eukaryotic cells, and more preferably NK cells.

[0070] The cell culture medium according to the present invention is composed of amino acid components, vitamin components, inorganic salt components, other components, and purified water.

[0071] a) The amino acid component is at least one amino acid or a combination thereof selected from the group consisting of glycine, L-alanine, L-valine, L-leucine, L-isoleucine, L-threonine, L-serine, L-cysteine, L-methionine, L-aspartic acid, L-asparagine, L-glutamic acid, L-glutamine, L-lysine, L-arginine, L-histidine, L-phenylalanine, L-tyrosine, L-tryptophan, L-proline, β-alanine, γ-aminobutyric acid, ornithine, citrulline, homoserine, triiodothyronine, thyroxine, and dihydroxyphenylalanine, and preferably, at least one amino acid or a combination thereof selected from the group consisting of glycine, L-alanine, L-arginine, L-cysteine, L-glutamine, L-histidine, L-lysine, L-methionine, L-proline, L-serine, L-threonine, and L-valine;

[0072] b) The vitamin component is at least one vitamin or a combination thereof selected from the group consisting of biotin, calcium D-pantothenate, folic acid, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, choline chloride, i-inositol, and ascorbic acid, and preferably, at least one or more vitamins or a combination thereof selected from the group consisting of i-inositol, thiamine hydrochloride, niacinamide, and pyridoxine hydrochloride;

[0073] c) The above inorganic salt component is at least one inorganic salt or a combination thereof selected from the group consisting of calcium chloride (CaCl2) (anhydrous), copper sulfate pentahydrate (CuSO4-5H2O), ferric sulfate heptahydrate (FeSO4-7H2O), magnesium chloride (anhydrous), magnesium sulfate (MgSO4) (anhydrous), potassium chloride (KCl), sodium chloride (NaCl), disodium hydrogen phosphate (Na2HPO4), sodium hydrogen phosphate monohydrate (NaH2PO4-H2O), zinc sulfate heptahydrate (ZnSO4-7H2O), ferric nitrate nonahydrate (Fe(NO3)3·9H2O), and sodium bicarbonate (NaHCO3), and preferably, sodium chloride (NaCl), sodium bicarbonate (NaHCO3), potassium chloride (KCl), calcium chloride (CaCl2) (anhydrous), and sodium bicarbonate. At least one inorganic salt selected from the group consisting of monohydrate (NaH2PO4-H2O) or a combination thereof,

[0074] d) The other component is at least one other component or a combination thereof selected from the group consisting of D-glucose (dextrose), sodium pyruvate, hypoxanthine Na, thymidine, linoleic acid, lipoic acid, adenosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'-deoxycytidine HCl and 2'-deoxyguanosine, and preferably, may be sodium pyruvate.

[0075] e) Purified water is used to dissolve the amino acids, vitamins, mineral salts and other components, and may be water obtained through one or more distillations or purified through a filter.

[0076] In addition, the cell culture medium according to the present invention may further include growth factors or cytokines. The growth factors may include IGF, bFGF, TGF, HGF, EGF, VEGF, or PDGF, alone or in combination of two or more, but are not particularly limited thereto. The cytokines may include IL-1, IL-4, IL-6, IFN-γ, IL-10, IL-15, IL-17, or IL-21, alone or in combination of two or more, but are not particularly limited thereto.

[0077] In addition, the cell culture medium according to the present invention may further include an antibody for activating natural killer cells. The activating antibody may be an anti-CD3 antibody, an anti-CD2 antibody, an anti-CD335 antibody, or the like, alone or in combination of two or more, but is not particularly limited thereto. In addition, beads to which the antibody for activating natural killer cells is bound may be included. In addition, a fusion protein comprising two or more of the antibody for activating natural killer cells or variable region fragments may be used.

[0078] In particular, the NK culture medium may additionally contain any one selected from the group consisting of IL-15, IL-21, and combinations thereof.

[0079] The above IL-15 and IL-21 are a type of interleukin (IL), and refer to proteinaceous biologically active substances produced by immune cells such as lymphocytes, monocytes, and macrophages. The above IL-15 and IL-21 promote the proliferation of natural killer cells, so they can be used when culturing natural killer cells using monocytes as source cells. However, when these are used alone or in combination, there is a problem of low proliferation rate and purity (Biossel L. et al., Biology of Blood and Marrow Transplantation, 14, 1031-1038, 2008).

[0080] Specifically, the medium may be a conventional animal cell culture medium such as DMEM (Dulbecco's Modified Eagle's Medium), EDM (Endothelial differentiation medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, a-MEM (a-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), Iscove's Modified Dulbecco's Medium, AIM-V Medium, X-vivo™ 15 Medium, and NK MACS Medium. In one embodiment of the present invention, AIM-V Medium, X-vivo™ 15 Medium, and NK MACS Medium were used as the medium.

[0081] The term "serum" used in the present invention refers to the clear supernatant separated from blood after complete coagulation of the blood. In addition, serum must be added to the synthetic medium for culturing animal cells, and bovine, equine, or human serum is generally used. In the case of bovine serum, fetal bovine serum (FBS), newborn bovine serum, calf serum, or maternal bovine serum can be used depending on the time of blood collection. In the case of human serum, human serum from a donor with blood type AB is used, and human AB serum can be used because it does not have antibodies to blood type A and B antigens, thereby minimizing immune reactivity. In addition, CTS Immune Cell SR or the like can be used as a substitute for the above "serum." In one embodiment of the present invention, human AB serum or CTS Immune Cell SR was used.

[0082] The supplement may use GlutaMAX (Gibco®), an L-Glutamine substitute, to improve stability and cell activity during cell culture. Additionally, the supplement may be NK MACS supplement (Miltenyi Biotec, 130-113-102).

[0083] Fusion protein dimer containing IL-2 protein and CD80 protein

[0084] As used herein, the term "IL-2" or "interleukin-2" means any wild-type IL-2 obtained from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise stated. The IL-2 may be obtained from animal cells, but also includes those obtained from recombinant cells capable of producing IL-2. Furthermore, the IL-2 may be wild-type IL-2 or a variant thereof.

[0085] In this specification, IL-2 or its variants are collectively referred to as "IL-2 protein" or "IL-2 polypeptide." IL-2, IL-2 protein, IL-2 polypeptide, and IL-2 variants specifically bind to, for example, the IL-2 receptor. This specific binding can be confirmed by methods known to those skilled in the art.

[0086] A specific example of the IL-2 may have the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36. In addition, the IL-2 may be in a mature form. Specifically, the mature IL-2 may not include a signal sequence and may have the amino acid sequence of SEQ ID NO: 10. In this case, the IL-2 may be used as a concept that includes a fragment in which a portion of the N-terminus or C-terminus of the wild-type IL-2 is deleted (truncated).

[0087] In addition, the fragment of IL-2 may be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids are deleted consecutively from the N-terminus of the protein having the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36. In addition, the fragment of the IL-2 may be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids are deleted consecutively from the C-terminus of the protein having the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36.

[0088] As used herein, the term "IL-2 variant" refers to a form in which a portion of an amino acid sequence of full-length IL-2 or a fragment of the above-mentioned IL-2 is substituted. That is, the IL-2 variant may have an amino acid sequence different from that of wild-type IL-2 or a fragment thereof. However, the IL-2 variant may have an activity equivalent to or similar to that of wild-type IL-2. Here, "IL-2 activity" may mean, for example, specific binding to an IL-2 receptor, and this specific binding can be measured by a method known to those skilled in the art.

[0089] Specifically, the IL-2 variant may be one in which some of the amino acids of wild-type IL-2 are substituted. As a specific example of the IL-2 variant by amino acid substitution, at least one of the 38th, 42nd, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 may be substituted.

[0090] Specifically, the IL-2 variant may be one in which at least one of the 38th, 42nd, 45th, 61st, or 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 is substituted with another amino acid. In addition, when IL-2 is in a form in which a part of the N-terminus of the amino acid sequence of SEQ ID NO: 35 is deleted, the amino acid at the complementarily corresponding position in the amino acid sequence of SEQ ID NO: 10 may be substituted with another amino acid. For example, when IL-2 has the amino acid sequence of SEQ ID NO: 35, the IL-2 variant may be one in which at least one of the 58th, 62nd, 65th, 81st, or 92nd amino acids in the amino acid sequence of SEQ ID NO: 35 is substituted with another amino acid. These correspond to the 38th, 42nd, 45th, 61st, and 72nd amino acid residues of the amino acid sequence of SEQ ID NO: 10, respectively. In one embodiment, one, two, three, four, five, six, seven, eight, nine, or ten amino acids may be substituted, as long as IL-2 activity is maintained. In another embodiment, one to five amino acids may be substituted.

[0091] In one specific example, the IL-2 variant may be a form in which two amino acids are substituted. Specifically, the IL-2 variant may be a form in which the 38th and 42nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Furthermore, in one specific example, the IL-2 variant may be a form in which the 38th and 45th amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Furthermore, in one specific example, the IL-2 variant may be a form in which the 38th and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Furthermore, in one specific example, the IL-2 variant may be a form in which the 38th and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Furthermore, in one specific example, the IL-2 variant may be a form in which the 42nd and 45th amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be one in which the 42nd and 61st amino acids are substituted in the amino acid sequence of SEQ ID NO: 10. In addition, as a specific example, the IL-2 variant may be one in which the 42nd and 72nd amino acids are substituted in the amino acid sequence of SEQ ID NO: 10. In addition, as a specific example, the IL-2 variant may be one in which the 45th and 61st amino acids are substituted in the amino acid sequence of SEQ ID NO: 10. In addition, as a specific example, the IL-2 variant may be one in which the 45th and 72nd amino acids are substituted in the amino acid sequence of SEQ ID NO: 10. In addition, as a specific example, the IL-2 variant may be one in which the 61st and 72nd amino acids are substituted in the amino acid sequence of SEQ ID NO: 10.

[0092] Furthermore, the IL-2 variant may be a form in which three amino acids are substituted. Specifically, the IL-2 variant may be a form in which the 38th, 42nd, and 45th amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 42nd, and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 42nd, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 45th, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be one in which the 38th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be one in which the 42nd, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be one in which the 42nd, 45th, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be one in which the 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted.

[0093] In addition, the IL-2 variant may be a form in which four amino acids are substituted. Specifically, the IL-2 variant may be a form in which the 38th, 42nd, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 42nd, 45th, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 42nd, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Additionally, as a specific example, the IL-2 variant may have amino acids at positions 42, 45, 61, and 72 substituted in the amino acid sequence of SEQ ID NO: 10.

[0094] Furthermore, the IL-2 variant may be a form in which five amino acids are substituted. Specifically, the IL-2 variant may be a form in which the 38th, 42nd, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are all substituted with different amino acids.

[0095] At this time, the “other amino acid” introduced by the substitution may be any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. However, in the amino acid substitution of the IL-2 variant, the 38th position in the amino acid sequence of SEQ ID NO: 10 cannot be substituted with arginine, the 42nd position cannot be substituted with phenylalanine, the 45th position cannot be substituted with tyrosine, the 61st position cannot be substituted with glutamic acid, and the 72nd position cannot be substituted with leucine.

[0096] In the amino acid substitution of the IL-2 variant, arginine, which is the 38th amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with an amino acid other than arginine. Preferably, in the amino acid substitution of the IL-2 variant, arginine, which is the 38th amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with alanine (R38A).

[0097] In the amino acid substitution of the IL-2 variant, phenylalanine, which is the 42nd amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with an amino acid other than phenylalanine. Preferably, in the amino acid substitution of the IL-2 variant, phenylalanine, which is the 42nd amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with alanine (F42A).

[0098] In the amino acid substitution of the IL-2 variant, tyrosine, which is the 45th amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with an amino acid other than tyrosine. Preferably, in the amino acid substitution of the IL-2 variant, tyrosine, which is the 45th amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with alanine (Y45A).

[0099] In the amino acid substitution of the IL-2 variant, glutamic acid, which is the 61st amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with an amino acid other than glutamic acid. Preferably, in the amino acid substitution of the IL-2 variant, glutamic acid, which is the 61st amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with arginine (E61A).

[0100] In the amino acid substitution of the IL-2 variant, leucine, which is the 72nd amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with an amino acid other than leucine. Preferably, in the amino acid substitution of the IL-2 variant, leucine, which is the 72nd amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with glycine (L72G).

[0101] Specifically, the IL-2 variant may have at least one substitution selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G in the amino acid sequence of SEQ ID NO: 10.

[0102] Specifically, the IL-2 variant may have amino acid substitutions at two, three, four or five positions selected from the group consisting of R38A, F42A, Y45A, E61R and L72G.

[0103] In addition, the IL-2 variant may be a form in which two amino acids are substituted. Specifically, the IL-2 variant may be a form in which R38A and F42A are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A and Y45A are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A and E61R are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A and L72G are substituted. In addition, as a specific example, the IL-2 variant may be a form in which F42A and Y45A are substituted. In addition, as a specific example, the IL-2 variant may be a form in which F42A and E61R are substituted. In addition, as a specific example, the IL-2 variant may be a form in which F42A and L72G are substituted. Additionally, as a specific example, the IL-2 variant may have substitutions of E61R and L72G.

[0104] Furthermore, the IL-2 variant may be a form in which three amino acids are substituted. Specifically, the IL-2 variant may be a form in which R38A, F42A, and Y45A are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A, F42A, and E61R are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A, F42A, and L72G are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A, Y45A, and E61R are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A, Y45A, and L72G are substituted. In addition, as a specific example, the IL-2 variant may be a form in which F42A, Y45A, and E61R are substituted. In addition, as a specific example, the IL-2 variant may be one in which substitutions occur at F42A, Y45A, and L72G. In addition, as a specific example, the IL-2 variant may be one in which substitutions occur at F42A, E61R, and L72G. In addition, as a specific example, the IL-2 variant may be one in which substitutions occur at Y45A, E61R, and L72G.

[0105] In addition, the IL-2 variant may be a form in which four amino acids are substituted. Specifically, the IL-2 variant may be a form in which substitutions occur at R38A, F42A, Y45A, and E61R. In addition, as a specific example, the IL-2 variant may be a form in which substitutions occur at R38A, F42A, Y45A, and L72G. In addition, as a specific example, the IL-2 variant may be a form in which substitutions occur at R38A, F42A, E61R, and L72G. In addition, as a specific example, the IL-2 variant may be a form in which substitutions occur at R38A, Y45A, E61R, and L72G. In addition, as a specific example, the IL-2 variant may be a form in which substitutions occur at F42A, Y45A, E61R, and L72G.

[0106] Furthermore, the IL-2 variant may have substitutions such as R38A, F42A, Y45A, E61R, and L72G.

[0107] Preferably, one specific example of the IL-2 variant may be one in which a substitution of any one of the following combinations (a) to (d) has occurred in the amino acid sequence of SEQ ID NO: 10:

[0108] (a) R38A / F42A

[0109] (b) R38A / F42A / Y45A

[0110] (c) R38A / F42A / E61R

[0111] (d) R38A / F42A / L72G

[0112] At this time, if IL-2 has the amino acid sequence of SEQ ID NO: 35, it may have an amino acid substitution at a position complementarily corresponding to SEQ ID NO: 10. In addition, even if IL-2 is a fragment of the amino acid sequence of SEQ ID NO: 35, the amino acid at a position complementarily corresponding to SEQ ID NO: 10 may be substituted.

[0113] Specifically, the variant of IL-2 may have an amino acid sequence of SEQ ID NO: 6, 22, 23 or 24.

[0114] In addition, the IL-2 variant may be characterized by low toxicity in vivo. In this case, the low toxicity in vivo may be a side effect caused by IL-2 binding to the alpha chain (IL-2Rα) of the IL-2 receptor. In order to improve the side effects caused by the binding of IL-2 and IL-2Rα, various IL-2 variants have been developed, and those disclosed in U.S. Patent No. 5,229,109 and Republic of Korea Patent No. 10-1667096 can be used as the IL-2 variant. In particular, the IL-2 variant described in the present application has a low binding affinity to the alpha chain (IL-2Rα) of the IL-2 receptor, and thus has lower in vivo toxicity than the wild-type IL-2.

[0115] As used herein, the term "CD80," also known as "B7-1," is a membrane protein present on dendritic cells, activated B cells, and monocytes. CD80 provides costimulatory signals essential for the activation and survival of T cells. CD80 is known to be a ligand for two different proteins present on the surface of T cells, CD28 and CTLA-4. CD80 consists of 288 amino acids, and may specifically have the amino acid sequence of SEQ ID NO: 11. In addition, as used herein, "CD80 protein" refers to full-length CD80 or a CD80 fragment.

[0116] As used herein, the term "CD80 fragment" refers to a truncated form of CD80. In addition, the CD80 fragment may be an extracellular domain of CD80. A specific example of the CD80 fragment may be one in which the 1st to 34th amino acids from the N-terminus, which is the signal sequence of CD80, are removed. Specifically, a specific example of the CD80 fragment may be a protein consisting of amino acids 35th to 288th of SEQ ID NO: 11. In addition, a specific example of the CD80 fragment may be a protein consisting of amino acids 35th to 242nd of SEQ ID NO: 11. In addition, a specific example of the CD80 fragment may be a protein consisting of amino acids 35th to 232nd of SEQ ID NO: 11. In addition, a specific example of the CD80 fragment may be a protein consisting of amino acids 35th to 139th of SEQ ID NO: 11. Additionally, one specific example of the CD80 fragment may be a protein consisting of amino acids 142 to 242 of SEQ ID NO: 11. In one embodiment, the CD80 fragment may have an amino acid sequence of SEQ ID NO: 2.

[0117] Additionally, the IL-2 protein and the CD80 protein may be linked by a linker or carrier. Specifically, the IL-2 or a variant thereof and the CD80 (B7-1) or a fragment thereof may be linked by a linker or carrier. In the present specification, the terms linker and carrier are used interchangeably.

[0118] The linker connects two proteins. A specific example of the linker may include 1 to 50 amino acids, albumin or a fragment thereof, or an Fc domain of an immunoglobulin. In this case, the Fc domain of the immunoglobulin refers to a protein that includes the heavy chain constant region 2 (CH2) and the heavy chain constant region 3 (CH3) of the immunoglobulin, and does not include the variable region of the heavy and light chains of the immunoglobulin and the light chain constant region 1 (CH1). The immunoglobulin may be IgG, IgA, IgE, IgD, or IgM, and preferably IgG4. In this case, the Fc domain of the wild-type immunoglobulin G4 may have the amino acid sequence of SEQ ID NO: 4.

[0119] In addition, the Fc domain of the immunoglobulin may be a wild-type Fc domain as well as an Fc domain variant. In addition, the term "Fc domain variant" as used herein may have a different glycosylation pattern from that of the wild-type Fc domain, or may have increased glycosylation compared to the wild-type Fc domain, decreased glycosylation compared to the wild-type Fc domain, or a deglycosylated form. In addition, an aglycosylated Fc domain is also included. The Fc domain or variant may have sialic acid, fucosylation, or glycosylation content controlled through culture conditions or genetic manipulation of the host.

[0120] In addition, the sugar chain of the Fc domain of the immunoglobulin can be modified by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. In addition, the Fc domain variant may be a form in which the Fc region of the immunoglobulin is mixed with that of IgG, IgA, IgE, IgD, or IgM. In addition, the Fc domain variant may be a form in which some amino acids of the Fc domain are substituted with other amino acids. A specific example of the Fc domain variant may have the amino acid sequence of SEQ ID NO: 12.

[0121] The fusion protein may have a structure in which CD80 and IL-2 proteins are linked to the N-terminus and C-terminus, respectively, of the Fc domain as a linker (or carrier), or IL-2 and CD80 are linked to each other (Fig. 1a). The linkage of the N-terminus or C-terminus of the Fc domain to CD-80 or IL-2 may be arbitrarily achieved by a linker peptide.

[0122] Specifically, the fusion protein may be composed of the following structural formula (I) or (II):

[0123] N'-X-[linker (1)]n-Fc domain-[linker (2)]mYC' (I)

[0124] N'-Y-[linker (1)]n-Fc domain-[linker (2)]mXC' (II)

[0125] At this time, in the structural formulas (I) and (II),

[0126] The above N' is the N-terminus of the fusion protein,

[0127] The above C' is the C-terminus of the fusion protein,

[0128] The above X is CD80 protein,

[0129] The above Y is IL-2 protein,

[0130] The above linker (1) and linker (2) are peptide linkers,

[0131] The above n and m are each independently O or 1.

[0132] Preferably, the fusion protein may be composed of structural formula (I). The IL-2 protein is as described above. In addition, the CD80 protein is as described above. According to one specific example, the IL-2 protein may be an IL-2 variant in which one to five amino acids are substituted compared to wild-type IL-2. The CD80 protein may be a fragment in which up to about 34 amino acid residues are consecutively deleted (truncated) from the N-terminus or C-terminus of wild-type CD80. Alternatively, the CD80 protein may be an extracellular immunoglobulin-like domain having binding activity to T cell surface receptors CTLA-4 and CD28.

[0133] Specifically, the fusion protein may have an amino acid sequence of SEQ ID NO: 9, 26, 28 or 30. In another specific embodiment, the fusion protein comprises a polypeptide having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9, 26, 28 or 30. Here, the identity can be determined, for example, by percent homology, homology comparison software such as BlastN software of the National Center of Biotechnology Information (NCBI).

[0134] A peptide linker (1) may be included between the CD80 protein and the Fc domain. The peptide linker (1) may be composed of 5 to 80 consecutive amino acids, 20 to 60 consecutive amino acids, 25 to 50 consecutive amino acids, or 30 to 40 amino acids. In one specific example, the peptide linker (1) may be composed of 30 amino acids. In addition, the peptide linker (1) may include at least one cysteine. Specifically, it may include one, two, or three cysteines. In addition, the peptide linker (1) may be derived from the hinge of an immunoglobulin. In one specific example, the peptide linker (1) may be a peptide linker composed of the amino acid sequence of SEQ ID NO: 3.

[0135] The above peptide linker (2) may be composed of 1 to 50 consecutive amino acids, or 3 to 30 consecutive amino acids, or 5 to 15 amino acids. In one specific example, the peptide linker (2) may be (G4S)n (wherein n is an integer from 1 to 10). In this case, n in (G4S)n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the peptide linker (2) may be a peptide linker composed of an amino acid sequence of SEQ ID NO: 5.

[0136] Another aspect of the present invention provides a dimer comprising two fusion proteins comprising the IL-2 protein and the CD80 protein. The fusion protein comprising the IL-2 or a variant thereof and the CD80 or a fragment thereof is as described above.

[0137] At this time, the bond between the fusion proteins constituting the dimer may be formed by a disulfide bond via a cysteine ​​present in the linker, but is not limited thereto. The fusion proteins constituting the dimer may be the same, or may be different fusion proteins. Preferably, the dimer may be a homodimer. One example of the fusion protein constituting the dimer may be a protein having the amino acid sequence of SEQ ID NO: 9.

[0138] NK cell culture method 1

[0139] Another aspect of the present invention provides a method for culturing natural killer cells, comprising the steps of: i) isolating cells that do not express CD3 from peripheral blood mononuclear cells (PBMCs); ii) isolating cells that express CD56 from the cells that do not express CD3 separated in the step; and iii) culturing the separated cells in the presence of a fusion protein dimer comprising IL-2 or a variant thereof and CD80 or a fragment thereof.

[0140] The term "PBMC" used in the present invention refers to peripheral blood mononuclear cells. PBMCs are composed of lymphocytes (T cells, B cells, natural killer cells) and monocytes, and can be separated from whole blood using Ficoll and centrifugation. The PBMCs may be isolated from whole blood obtained from an individual.

[0141] The above fusion protein dimer is the same as that described above in the composition for culturing natural killer cells. The fusion protein dimer can be treated at a concentration of 1 nM to 500 nM. Specifically, the fusion protein dimer can be treated at a concentration of 1 nM to 500 nM, 5 nM to 300 nM, or 10 nM to 150 nM. In one embodiment of the present invention, the fusion protein dimer was treated at a concentration of 1.6 nM and 50 nM.

[0142] The method for culturing the above-mentioned isolated cells can be performed using methods widely known in the art. Specifically, the culture temperature in the step of culturing the above-mentioned isolated cells may be 27°C to 40°C or 30°C to 37°C. In one embodiment of the present invention, the culture was performed at 37°C. In addition, the CO2 concentration condition during the culturing step of culturing the above-mentioned isolated cells may be 1% to 10%, and preferably, the culture may be performed under 5% CO2 conditions.

[0143] In the step of culturing the above-mentioned separated cells, the culturing period may be 5 to 25 days, 6 to 23 days, or 7 to 21 days. In one embodiment of the present invention, the culturing period was 20 days, and a significant difference in proliferation was observed from the 5th day.

[0144] Obtained NK cells and their uses

[0145] Another aspect of the present invention provides natural killer cells produced by the above natural killer cell culture method.

[0146] The above natural killer cells may have increased expression of CD16 and NKp46. The above natural killer cells may have increased expression of granzyme B and perforin. Natural killer cells cultured according to the above natural killer cell culture method can be frozen, and their function is not impaired even when thawed again.

[0147] The above natural killer cells exhibit high expression of activating receptors such as CD16 and NKp46, which increases their killing capacity against cancer cell lines and their secretion of granzyme B and perforin, suggesting excellent anticancer effects. Therefore, using large quantities of activated natural killer cells suitable for clinical application, effective therapeutic agents for cancer treatment can be developed. Furthermore, the above natural killer cells may exhibit high expression of NKp30 and DNAM1.

[0148] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the natural killer cells as an active ingredient.

[0149] In addition, the natural killer cells produced by the above natural killer cell culture method may be included in an amount of 10 to 95 wt% based on the total weight of the pharmaceutical composition. In addition, the pharmaceutical composition may additionally include one or more effective ingredients exhibiting the same or similar function in addition to the above effective ingredient.

[0150] The dosage of the above pharmaceutical composition may be adjusted according to various factors including the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients included in the composition, the type of formulation, and the patient's age, weight, general health, sex, and diet, the time of administration, the route of administration, and the excretion rate of the composition, the treatment period, and concurrently used drugs.

[0151] However, for a desirable effect, the dosage of the pharmaceutical composition is 1Х10 based on the active ingredient, natural killer cells. 2 cells / ㎏ to 1.0Х10 13 cells / ㎏, 1Х10 7 cells / ㎏ to 1.5Х10 11 It can be cells / kg. At this time, administration can be done once a day or divided into several times.

[0152] Additionally, the pharmaceutical composition can be administered to a subject using various methods known in the art. The route of administration can be appropriately selected by a person skilled in the art, taking into account the administration method, volume of body fluid, viscosity, etc.

[0153] The above cancer may be any one selected from the group consisting of stomach cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

[0154] Treatment method using the obtained NK cells

[0155] In another aspect of the present invention, a method for treating cancer is provided, comprising administering the NK cells to a subject suffering from cancer. The NK cells and the cancer are as described above. In another aspect of the present invention, the use of the NK cells for treating cancer is provided.

[0156] NK cell culture method 2

[0157] Another aspect of the present invention provides a method for culturing natural killer cells, comprising the steps of: i) isolating cells that do not express CD3 from PBMCs; and ii) culturing the isolated cells in the presence of a fusion protein dimer comprising IL-2 or a variant thereof and CD80 or a fragment thereof.

[0158] Another aspect of the present invention provides natural killer cells produced using the above natural killer cell culture method. Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the natural killer cells as an active ingredient. Another aspect of the present invention provides a method for treating cancer, comprising administering the natural killer cells to a subject suffering from cancer. In this case, the NK cells and the cancer are as described above. Another aspect of the present invention provides the use of the NK cells for treating cancer.

[0159] The above natural killer cells exhibit high expression of activating receptors such as CD16 and NKp46, which increases their killing capacity against cancer cell lines and their secretion of granzyme B and perforin, suggesting excellent anticancer effects. Therefore, using large quantities of activated natural killer cells suitable for clinical application, effective therapeutic agents for cancer treatment can be developed. Furthermore, the above natural killer cells may exhibit high expression of NKp30 and DNAM1.

[0160] The above cancer may be any one selected from the group consisting of stomach cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

[0161] NK cell culture method 3

[0162] Another aspect of the present invention provides a method for culturing natural killer cells, comprising the steps of: i) isolating cells expressing CD56 from PBMCs; and ii) culturing the isolated cells in the presence of a fusion protein dimer comprising IL-2 or a variant thereof and CD80 or a fragment thereof.

[0163] Another aspect of the present invention provides natural killer cells produced using the above natural killer cell culture method. These natural killer cells exhibit high expression of activating receptors, such as CD16 and NKp46, leading to increased killing capacity against cancer cell lines and increased secretion of granzyme B and perforin, and thus are expected to exhibit excellent anticancer effects. Therefore, a large quantity of activated natural killer cells suitable for clinical application can be used to produce an effective therapeutic agent for cancer treatment. Furthermore, these natural killer cells may exhibit high expression of NKp30 and DNAM1.

[0164] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the natural killer cells as an active ingredient. Another aspect of the present invention provides a method for treating cancer, comprising administering the NK cells to a subject suffering from cancer. The NK cells and the cancer are as described above. Another aspect of the present invention provides a use of the NK cells for treating cancer.

[0165] The above cancer may be any one selected from the group consisting of stomach cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

[0166] NK cell culture method 4

[0167] Another aspect of the present invention provides a method for stimulating the activity of natural killer cells in PBMCs, comprising the step of culturing PBMCs in the presence of a fusion protein dimer comprising IL-2 or a variant thereof and CD80 or a fragment thereof.

[0168] Another aspect of the present invention provides PBMCs manufactured by a method for stimulating the activity of natural killer cells within the PBMCs. Natural killer cells within the PBMCs exhibit high expression of activating receptors, such as CD16 and NKp46, which increases their ability to kill cancer cell lines and their secretion of granzyme B and perforin, and thus exhibit excellent anticancer effects. Therefore, a therapeutic agent effective for cancer treatment can be manufactured using PBMCs containing a large quantity of activated natural killer cells that can be clinically applied. Furthermore, the natural killer cells within the PBMCs may exhibit high expression of NKp30 and DNAM1.

[0169] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the PBMC as an active ingredient. Another aspect of the present invention provides a method for treating cancer, comprising administering the NK cells to a subject suffering from cancer. The NK cells and the cancer are as described above. Another aspect of the present invention provides a use of the NK cells for treating cancer.

[0170] The above cancer may be any one selected from the group consisting of stomach cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

[0171] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are intended only to illustrate the present invention, and the present invention is not limited to the following examples.

[0172] I. GI-101, Preparation of Natural Killer Cells and Natural Killer Cell Culture Composition

[0173] Manufacturing Example 1. Manufacturing of hCD80-Fc-IL-2 variant (2M): GI-101

[0174] To produce a fusion protein comprising a human CD80 fragment, an Fc domain, and an IL-2 variant, a polynucleotide comprising a base sequence (SEQ ID NO: 8) encoding a fusion protein comprising a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge linked to a linker (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 variant (2M) (R38A, F42A) (SEQ ID NO: 6) with two amino acids substituted in this order from the N-terminus was synthesized using the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was cultured in CHO cells (Expi-CHO) TM ) was introduced to express the fusion protein of sequence number 9. After introducing the vector, the culture was cultured for 7 days in an environment of 37°C, 125 RPM, and CO28% concentration, and the culture solution was collected and the fusion protein was purified. The purified fusion protein dimer was named "GI-101".

[0175] The fusion protein was purified using chromatography using MabSelect SuRe protein A resin. The fusion protein was bound in 25 mM Tris, 25 mM NaCl, pH 7.4. The solution was eluted with 100 mM NaCl, 100 mM acetic acid (pH 3). The fusion protein was collected by adding 20% ​​1 M Tris-HCl (pH 9) to the collection tube. The collected fusion protein was dialyzed against PBS buffer for 16 h.

[0176] Afterwards, the absorbance at a wavelength of 280 nm was measured over time using size exclusion chromatography using a TSKgel G3000SWXL column (TOSOH Bioscience) to secure a high concentration of the fusion protein. At this time, the separated and purified fusion protein was subjected to SDS-PAGE under reducing (R) or non-reducing (NR) conditions, and its purity was confirmed by staining with Coomassie blue (Fig. 1b). When detected using NanoDrop, it was confirmed that the fusion protein was contained at a concentration of 2.78 mg / ml (Fig. 1c). In addition, the results of the analysis using size exclusion chromatography are as shown in Fig. 1d.

[0177] Manufacturing Example 2. Manufacturing of Fc-IL-2 variant (2M) dimer: Fc-IL-2v2

[0178] To produce a fusion protein comprising an Fc domain and an IL-2 variant, a polynucleotide comprising the base sequence (SEQ ID NO: 45) encoding a fusion protein comprising a signal peptide (SEQ ID NO: 1), an Ig hinge (SEQ ID NO: 38), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 variant (2M) (R38A, F42A) (SEQ ID NO: 6) with two amino acids substituted in this order from the N-terminus was synthesized as a polynucleotide using the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was introduced into CHO cells (Expi-CHO™) to express the fusion protein of SEQ ID NO: 44. After introduction of the vector, the culture medium was collected after 7 days of culture in an environment of 37°C, 125 RPM, and CO28%, and the fusion protein dimer was purified. The purified fusion protein dimer was named “Fc-IL2v2”.

[0179] The purification and collection of the fusion protein were performed using the same method as in Manufacturing Example 1. The purified fusion protein was separated and purified by SDS-PAGE under reducing (R) or non-reducing (NR) conditions, and its purity was confirmed by staining with Coomassie blue (Fig. 3a). As a result, it was confirmed that the fusion protein formed a dimer. In addition, the results of the analysis using size exclusion chromatography are as shown in Fig. 3b.

[0180] Manufacturing Example 3. Manufacturing of Fc-IL-2 dimer: Fc-IL-2wt

[0181] To produce a fusion protein comprising an Fc domain and wild-type IL-2, a polynucleotide comprising the base sequence (SEQ ID NO: 43) encoding a fusion protein comprising a signal peptide (SEQ ID NO: 1), an Ig hinge (SEQ ID NO: 38), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and wild-type IL-2 (SEQ ID NO: 10) in this order from the N-terminus was synthesized using the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was introduced into CHO cells (Expi-CHO™) to express the fusion protein of SEQ ID NO: 42. After introduction of the vector, the culture medium was harvested and the fusion protein dimer was purified after culturing for 7 days in an environment of 37°C, 125 RPM, and CO28%. The purified fusion protein dimer was named "Fc-IL2wt".

[0182] The purification and collection of the fusion protein were performed using the same method as in Manufacturing Example 1. The purified fusion protein was separated and purified by SDS-PAGE under reducing (R) or non-reducing (NR) conditions, and its purity was confirmed by staining with Coomassie blue (Fig. 3c). As a result, it was confirmed that the fusion protein formed a dimer. In addition, the results of the analysis using size exclusion chromatography are as shown in Fig. 3d.

[0183] Manufacturing Example 4. Manufacturing of hCD80-Fc-IL-2 wild-type dimer: hCD80-Fc-IL-2wt

[0184] To produce a fusion protein comprising a human CD80 fragment, an Fc domain, and an IL-2 wild-type protein, a polynucleotide comprising a base sequence (SEQ ID NO: 41) encoding a fusion protein comprising a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge linked to a linker (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 wild-type protein (SEQ ID NO: 10) in this order from the N-terminus was synthesized using the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was cultured in CHO cells (Expi-CHO) TM ) was introduced to express the fusion protein of sequence number 46. After introducing the vector, the culture was cultured for 7 days in an environment of 37°C, 125 RPM, and CO28% concentration, and the culture medium was collected to purify the fusion protein dimer. The purified fusion protein dimer was named "hCD80-Fc-IL2wt".

[0185] The fusion protein was purified using chromatography using MabSelect SuRe protein A resin. The fusion protein was bound in 25 mM Tris, 25 mM NaCl, pH 7.4. The solution was eluted with 100 mM NaCl, 100 mM acetic acid (pH 3). The fusion protein was collected by adding 20% ​​1 M Tris-HCl (pH 9) to the collection tube. The collected fusion protein was dialyzed against PBS buffer for 16 h.

[0186] Afterwards, using a TSKgel G3000SWXL column (TOSOH Bioscience), size exclusion chromatography was performed to measure the absorbance at a wavelength of 280 nm over time to secure a high concentration of fusion protein. At this time, the separated and purified fusion protein was subjected to SDS-PAGE under reducing (R) or non-reducing (NR) conditions, and its purity was confirmed by staining with Coomassie blue (Fig. 4a). As a result, it was confirmed that the fusion protein formed a dimer. In addition, the results of the analysis using size exclusion chromatography are as shown in Fig. 4b.

[0187] Manufacturing Example 5. Manufacturing of hCD80-Fc dimer: hCD80-Fc

[0188] To produce a fusion protein comprising a human CD80 fragment and an Fc domain, a polynucleotide (SEQ ID NO: 39) comprising a base sequence encoding a fusion protein comprising a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge linked to a linker (SEQ ID NO: 3), and an Fc domain (SEQ ID NO: 4) in this order from the N-terminus was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was cultured in CHO cells (Expi-CHO TM ) was introduced to express the fusion protein of sequence number 40. After introducing the vector, the culture was cultured for 7 days in an environment of 37°C, 125 RPM, and CO28% concentration, and the culture medium was collected to purify the fusion protein dimer. The purified fusion protein dimer was named "hCD80-Fc".

[0189] The fusion protein was purified using chromatography using MabSelect SuRe protein A resin. The fusion protein was bound in 25 mM Tris, 25 mM NaCl, pH 7.4. The solution was eluted with 100 mM NaCl, 100 mM acetic acid (pH 3). The fusion protein was collected by adding 20% ​​1 M Tris-HCl (pH 9) to the collection tube. The collected fusion protein was dialyzed against PBS buffer for 16 h.

[0190] Afterwards, using a TSKgel G3000SWXL column (TOSOH Bioscience), size exclusion chromatography was performed to measure the absorbance at a wavelength of 280 nm over time to secure a high concentration of fusion protein. At this time, the separated and purified fusion protein was subjected to SDS-PAGE under reducing (R) or non-reducing (NR) conditions, and its purity was confirmed by staining with Coomassie blue (Fig. 2a). As a result, it was confirmed that the fusion protein formed a dimer. In addition, the results of the analysis using size exclusion chromatography are as shown in Fig. 2b.

[0191] Preparation Example 1. Preparation of natural killer cell culture medium

[0192] A natural killer cell culture medium composition was prepared by adding substances corresponding to addition conditions 1 to 4 of Table 5 below to each basic culture medium having the composition of Tables 1 to 4 below.

[0193] Basic culture medium 1 ingredient name Manufacturer Cat. # Capacity Final concentration CTS TMImmune Cell SR (Serum replacement)ThermoA25961020.5ml5%GlutaMAX 100XGibcoA12860-010.5ml1XrhIL-15R&D systems247-ILB / CFAdd immediately before use66.7IUrhiL-21R&D systems8879-IL / CFAdd immediately before use0.341IUanti-CD2 / CD335 BeadsMiltenyi Biotec130-094-483OKT3Biolegend31732610ng / mLAIM-V mediumGibco0870112-DKto 50ml

[0194] Basic culture medium 2-ingredient name Manufacturer Cat. # Volume Final concentration Human AB serum Sigma H45 22 2.5 ml 5% GlutaMAX 100 X Gibco A 12860-01 0.5 ml 1 X rhIL-15 R&D systems 247-ILB / CF Add immediately before use 66.7 IU rhIL-2 1 R&D systems 8879-IL / CF Add immediately before use 0.341 IU anti-CD2 / CD3 35 Beads Miltenyi Biotec 130-094-483 OKT 3 Biolegend 31732 610 ng / m LAIM-V medium Gibco 0870112-DKto 50 ml

[0195] Basic culture medium 3-ingredient name Manufacturer Cat. # Volume Final concentration Human AB serum Sigma H45 22 2.5 ml 5% GlutaMAX 100 X Gibco A 12 860-0 1 0.5 ml 1 X rhIL-1 5 R&D systems 2 47-ILB / CF Add immediately before use 66.7 IU rhIL-2 1 R&D systems 8 879-IL / CF Add immediately before use 0.341 IU anti-CD2 / CD3 35 Beads Miltenyi Biotec 130-094-483 OKT 3 Biolegend 3 17 32 6 10 ng / mL X-vivo TM 15 MediumLonza04-418Qto 50ml

[0196] Basic culture medium 4 ingredients Manufacturer Cat. # Volume Final concentration Human AB serum Sigma H45 22 2.5ml 5% NK MACS supplement Miltenyi Biotec 130-113-10 2 500 ㎕ 1% anti-CD2 / CD335 beads Miltenyi Biotec 130-094-483 OKT3 Biolegend 317 32 6 10 ng / mL NK MACS medium 130-112-96 8 to 50ml

[0197] Additive Substance Classification Ingredient Name Manufacturer Dose Final Concentration Addition Conditions 1GI-101GI-Innovation Add immediately before use 1.6nM / 50nM Addition Conditions 2GI-101WTGI-Cell Add immediately before use 1.6nM / 50nM Addition Conditions 3CD80-Fc+Fc-IL2WTGI-Cell Add immediately before use CD80-Fc(1.6nM)+ Fc-IL2WT(1.6nM) / CD80-Fc(50nM)+ Fc-IL2WT(50nM) Addition Conditions 4CD80-Fc+Fc-IL2v2GI-Cell Add immediately before use CD80-Fc(1.6nM)+ Fc-IL2v2(1.6nM) / CD80-Fc(50nM)+ Fc-IL2v2(50nM)

[0198] Example 1. Preparation of CD3(-)CD56(+) natural killer cells derived from peripheral blood mononuclear cells (PBMCs).

[0199] To obtain CD3(-) cells, peripheral blood mononuclear cells (PBMCs, Zen-Bio. Inc, NC 27709, USA, Cat#: SER-PBMC-200-F) were counted using an ADAM-MC2 automated cell counter (NanoEnTek, Cosmogenetech Co., Ltd.). The PBMCs were transferred to a new tube and centrifuged at 300×g and 4°C for 5 minutes. MACS buffer (pH 7.2) containing 0.5% (v / v) bovine serum albumin (BSA) and 2 mM EDTA in PBS was prepared. After centrifugation, 1×10 7The cell pellet was suspended by treating it with 80 μl of MACs buffer and 20 μl of CD3 magnetic beads (Miltenyi biotech, 130-050-101) per cell number, and incubated at 4°C for 15 minutes. For washing, 10 ml of MACs buffer was added, centrifuged at 300×g and 4°C for 10 minutes, and the cell pellet was resuspended in 0.5 ml of MACs buffer.

[0200] 2 ml of MACs buffer was first flowed through an LD column (Miltenyi Biotec, Bergisch Gladbach, Germany, Cat#: 130-042-901), and then the cell suspension was flowed. Then, CD3(-) cells passing through the LD column were collected. At this time, 2 ml of MACs buffer was flowed three times to obtain CD3(-) cells so that the cells remaining in the LD column could be sufficiently separated. The obtained CD3(-) cells were counted using a cell counter, placed in a new tube, and centrifuged at 300×g and 4℃ for 5 minutes. After that, the supernatant was removed, and 1×10 7 80 μl of MACs buffer and 20 μl of CD56 magnetic beads (Miltenyi biotech, Cat#: 130-050-401) were added per cell number and reacted at 4°C for 15 minutes. For washing, 10 ml of MACs buffer was added, centrifuged at 300×g, 4°C for 10 minutes, and the cell pellet was resuspended in 0.5 ml of MACs buffer.

[0201] 3 ml of MACs buffer was first flowed through an LS column (Miltenyi Biotec, Bergisch Gladbach, Germany, Cat#: 130-042-901), followed by the above cell suspension. At this time, 2 ml of MACs buffer was flowed three times to ensure that the cells remaining in the LS column could be sufficiently separated. After that, the LS column was separated from the magnet stand, 5 ml of MACs buffer was added, and pressure was applied with a piston to obtain CD3(-)CD56(+) natural killer cells. The obtained CD3(-)CD56(+) natural killer cells were transferred to a new tube and centrifuged at 300×g, 4℃ for 5 minutes. After removing the supernatant, the cells were suspended in the basic culture medium listed in Tables 1 to 4 considering the culture conditions. The cell number of the suspended cells was measured using a cell counter.

[0202] Example 2. Culture of CD3(-)CD56(+) natural killer cells derived from peripheral blood mononuclear cells (PBMCs).

[0203] 100 μL of CD335 (NKp46)-Biotin and 100 μL of CD2-Biotin included in the NK Cell Activation / Expansion Kit (Cat#: 130-112-968) (Miltenyi Biotec, Bergisch Gladbach, Germany) were added to a 1.5 mL microtube and mixed, then 500 μL of Anti-Biotin MACSiBead Particles were added and mixed. Afterwards, 300 μL of MACs buffer was added and mixed at 2°C to 8°C for 2 hours using a Microtube Rotator. Considering the cell number, 1x10 6NK activation beads (5 μL per cell) were transferred to a new tube. 1 mL of PBS was added and centrifuged at 300 × g for 5 min. After removing the supernatant, NK MACs medium (Cat#: 130-094-483) (Miltenyi Biotec, Bergisch Gladbach, Germany) was added to 10 6 After adding 5 μL per NK cell and releasing the beads, they were inoculated into the CD3(-)CD56(+) natural killer cells isolated in Example 1.

[0204] Next, the total number of CD3(-)CD56(+) natural killer cells prepared above was 2.5x10 5 The cells were suspended in a culture medium composition containing the additives prepared in Preparation Example 1 and seeded in a 48-well plate, and cultured under conditions of 37°C and 5% CO2. Thereafter, the cell count was checked every two days, and when the cells occupied more than 80% of the culture vessel (confluency), the cells were subcultured in the following order: 48-well plate, 24-well plate, 12-well plate, 6-well plate, and 25T flask, and finally, all cells were harvested on the 21st day.

[0205] Example 3. Cell count measurement and cell viability comparison

[0206] The total cell number and viability of cultured natural killer cells were measured using a cell counter (ADAM-MC2) on days 5, 9, 11, 13, 15, 17, and 21. Since cell proliferation rates vary depending on the treated substance and culture medium type, the cell number was measured on the above dates based on the speed at which 80% of the passage criterion was filled.

[0207] The results of comparing the total cell number and viability of CD3-CD56+ cells cultured under the conditions of the culture medium composition prepared in Preparation Example 1 are shown in Tables 6 to 13 and Figures 5a to 12b.

[0208] As a result, it was confirmed that the culture medium composition containing GI-101 manufactured by Manufacturing Example 1 had a greater total number of natural killer cells than the control group (CD80-Fc+Fc-IL2v2, CD80-Fc+Fc-IL2WT added) regardless of the treatment concentration under the four basic culture medium conditions (Tables 1 to 4) (Figs. 5a, 5b, 7a, 7b, 9a, 9b, 11a, and 11b).

[0209] In addition, in terms of cell viability, when GI-101 was added, high viability was observed regardless of the basic culture medium and concentration (Fig. 6a, Fig. 6b, Fig. 8a, Fig. 8b, Fig. 10a, Fig. 10b, Fig. 12a and Fig. 12b).

[0210] The above results confirmed that GI-101 plays an important role in improving the proliferation ability and survival rate of natural killer cells compared to the control group (CD80-Fc+Fc-IL2v2, CD80-Fc+Fc-IL2WT added), regardless of the basic culture medium and concentration.

[0211] TOTAL CELL NUMBER ( X10 5 )Culture conditionsConcentrationTreatment materialCD3-CD56+ cellsDAY0(seeding)DAY5DAY9DAY11DAY13DAY15DAY17DAY21Table 150nMGI-1012.503.178.56-23.84-27.3823.76GI-101WT2.502.366.40---15.4513.33CD80-Fc+Fc-IL2v22.502.626.61-16.16-21.9313.19CD80-Fc+Fc-IL2WT2.501.87- 11.0413.92- 17.8613.331.6nMGI-1012.502.276.79-13.98-15.6211.19GI-101WT2.501.92 4.65----3.58CD80-Fc+Fc-IL2v22.500.23------CD80-Fc+Fc-IL2WT2.501.76- -6.96- 6.644.46

[0212] Cell Viability ( % )배양가자농도세리래의CD3-CD56+ cellsDAY0(seeding)DAY5DAY9DAY11DAY13DAY15DAY17DAY21표 150nMGI-101-76.5882.41-90.94-86.6884.19GI-101WT-75.9883.42---89.2789.53CD 80-Fc+Fc-IL2v2-78.1881.73-90.57-88.7689.64CD80-Fc+Fc-IL2WT-83.64-86.5092.1 9-89.6282.361.6nMGI-101-75.0882.94-87.85-90.3488.22GI-101WT-77.5582.32--- -87.01CD80-Fc+Fc-IL2v2-49.77------CD80-Fc+Fc-IL2WT-74.51--86.47-88.9093.29

[0213] TOTAL CELL NUMBER ( X10 5 )배양가자농도세리래의CD3-CD56+ cellsDAY0(seeding)DAY5DAY9DAY11DAY13DAY15DAY17DAY21표 250nMGI-1012.511.7138.81-214.88415.25-694.59GI-101WT2.58.5730.5862.3196.42--152. 16CD80-Fc+Fc-IL2v22.58.12--90.54122.27-153.14CD80-Fc+Fc-IL2WT2.57.8029.97-92.3211 8.56-152.231.6nMGI-1012.55.6028.69-55.39--94.22GI-101WT2.56.5814.92-35.18--37.81C D80-Fc+Fc-IL2v22.56.0219.85-56.26--75.83CD80-Fc+Fc-IL2WT2.56.0418.39-46.50--55.51

[0214] CELL VIABILITY (%) 250nMGI-101-93.3195.64-96.4796.58-93.21GI-101WT-94.3895.4596.0694.96--92.14C D80-Fc+Fc-IL2v2-93.32--97.0195.96-91.73CD80-Fc+Fc-IL2WT-91.5196.49-96.1995.23 -91.811.6nMGI-101-91.8193.75-95.14--85.22GI-101WT-93.5693.56-94.81--88.63CD8 0-Fc+Fc-IL2v2-93.5794.00-94.36--89.53CD80-Fc+Fc-IL2WT-90.8794.04-95.01--87.36

[0215] TOTAL CELL NUMBER ( X10 5 )배양가자농도세리래의CD3-CD56+ cellsDAY0(seeding)DAY5DAY9DAY11DAY13DAY15DAY17DAY21표 350nMGI-1012.506.0724.64-46.1361.25-99.60GI-101WT2.506.9824.90-49.7564.15-6 7.71CD80-Fc+Fc-IL2v22.504.11-21.60---56.02CD80-Fc+Fc-IL2WT2.505.7323.52-51.1 766.32-76.191.6nMGI-1012.506.4725.14-54.1369.59-86.68GI-101WT2.503.34-----1 0.91CD80-Fc+Fc-IL2v22.501.07------CD80-Fc+Fc-IL2WT2.504.6113.16-16.27--13.16

[0216] CELL VIABILITY (%) 350nMGI-101-89.7290.65-90.5988.11-82.08GI-101WT-90.7492.95-93.2090.52-8 7.38CD80-Fc+Fc-IL2v2-87.57-88.80---87.63CD80-Fc+Fc-IL2WT-88.6093.44-95.1 394.99-91.381.6nMGI-101-90.0792.11-92.6490.80-85.62GI-101WT-85.76-----7 5.98CD80-Fc+Fc-IL2v2-66.33------CD80-Fc+Fc-IL2WT-88.0590.17-89.08--79.81

[0217] TOTAL CELL NUMBER ( X10 5 )배양가자농도세리래의CD3-CD56+ cellsDAY0(seeding)DAY5DAY9DAY11DAY13DAY15DAY17DAY21표 450nMGI-1012.506.3137.3658.21-146.76-467.75GI-101WT2.505.7430.2452.00-126.99-347.5 9CD80-Fc+Fc-IL2v22.506.9631.2051.52-130.65-285.63CD80-Fc+Fc-IL2WT2.506.3035.5250.08 -147.54-350.281.6nMGI-1012.506.7537.2863.68-166.42-419.02GI-101WT2.502.46-8.089.84 --8.93CD80-Fc+Fc-IL2v22.500.73------CD80-Fc+Fc-IL2WT2.503.8618.85-48.9067.86-134.21

[0218] CELL VIABILITY (%) Culture Conditions Concentration Treatment Substance CD3-CD56+ cells DAY0 (seeding) DAY5 DAY9 DAY11 DAY13 DAY15 DAY17 DAY21 Table 450 nMGI-101-94.7 296.9 397.24-96.20-96.0 4GI-101WT-94.9 096.7 996.11-63.11-64.73 CD80-Fc+Fc-IL2v2-95.3 397.8 396.70-95.73-96.43 CD80-Fc+Fc-IL2WT-95.9 997.8 697. 52-97.09-96.391.6nMGI-101-94.7597.7296.89-94.67-95.00GI-101WT-89.00-88.06- --88.22CD80-Fc+Fc-IL2v2-75.94------CD80-Fc+Fc-IL2WT-91.4096.61--96.49-95.99

[0219] II. Characterization of Natural Killer Cells Using Natural Killer Cell Culture Compositions

[0220] Example 4. Confirmation of natural killer cell purity

[0221] The CD3-CD56+ natural killer cells obtained in Example 2 were centrifuged at 300×g for 5 minutes, the supernatant was removed, and 1 ml of FACS buffer was added to loosen the pellet. Then, the cell pellet was resuspended in 1 ml of FACS buffer prepared by adding 3% (v / v) FBS, 10 mM EDTA, 20 mM HEPES, 10 μg / ml polymyxin B, 100 U / ml penicillin, 100 μg / ml streptomycin, and 1 mM sodium pyruvate to PBS. After that, 2×10 6 Diluted with FACS buffer to obtain 10 cells / ml.

[0222] 100 μl of the diluted cell solution was added to each 5 ml FACS tube, and 100 μl of FACS buffer was additionally added. The tubes were treated with PerCP-labeled anti-human CD3 antibody (PerCP human anti-CD3 (Clone UCHT1)) and PE / cy7-labeled anti-human CD56 antibody (PE / cy7 human anti-CD56 (Clone B159)). After incubation at 4°C for 20 minutes, 200 μl of FACS buffer was added, and centrifugation was performed at 1,500 rpm for 3 minutes. The supernatant was removed, and 200 μl of FACS buffer was added to resuspend the cells, and the cell phenotype was confirmed using a flow cytometer (Cytek® Aurora, Cytek, Fremont, CA, USA).

[0223] Information on antibodies used in the above experiment is shown in Table 14. In addition, the purity of CD3-CD56+ natural killer cells cultured for 21 days under the conditions of the culture medium composition prepared in Preparation Example 1 was measured and shown in Figures 13 to 16.

[0224] TargetColorCloneProducerCat#NK markerCD3PerCPUCHT1BioLegend300428CD56APC / cy75.1H11BioLegend362512

[0225] Example 5. Identification of Activation and Inhibition Markers of Natural Killer Cells

[0226] The CD3-CD56+ natural killer cells obtained in Example 2 were centrifuged at 300×g for 5 minutes, the supernatant was removed, and 1 ml of FACS buffer was added to dissolve the pellet.

[0227] The cell pellet was resuspended in 1 ml of FACS buffer prepared by adding 3% (v / v) FBS, 10 mM EDTA, 20 mM HEPES, 10 μg / ml polymyxin B, 100 U / ml penicillin, 100 μg / ml streptomycin, and 1 mM sodium pyruvate to PBS. Then, 2 × 10 cells were counted using a cell counter.6 Diluted with FACS buffer to obtain 10 cells / ml. Add 100 ㎕ of the diluted cell solution to a 5 ㎖ FACS tube and analyze using a flow cytometer with Pe-CF594-labeled anti-human CD16 antibody (PE-CF594 human anti-CD16 (Clone 3G8)), APC-labeled anti-human DNAM1 antibody (APC human anti-DNAM1 (Clone 11A8)), BV605-labeled anti-human NKG2C antibody (BV605 human anti-NKG2C (Clone 134591)), BV650-labeled anti-human NKG2D antibody (BV650 human anti-NKG2D (Clone 1D11)), BB515-labeled anti-human NKp46 antibody (BB515 human anti-NKp46 (Clone 9E2)), BV480-labeled anti-human NKp30 antibody (BV480 human Anti-NKp30 (Clone p30-15)), PE-labeled anti-human PD-1 antibody (PE human anti-PD-1 (Clone EH12.2H7)), and APC-labeled anti-human NKG2A antibody (APC anti-human NKG2A (Clone 131411)) were used for detection. After incubation at 4°C for 20 minutes, 100 μl of FACS buffer was added, and centrifugation was performed at 1,500 rpm for 3 minutes.

[0228] The supernatant was removed, 200 μl of FACS buffer was added, and the cells were suspended. The phenotype of the cells was confirmed using a flow cytometer (Cytek® Aurora, Cytek, Fremont, CA, USA). The antibody information used in the above experiment is shown in Table 15. The activity and inhibition markers of CD3-CD56+ natural killer cells cultured for 21 days under the conditions of the culture medium composition prepared in Preparation Example 1 were confirmed and are shown in Figures 17 to 24.

[0229] TargetColorCloneProducerCat#Activation markerCD16PE-CF5943G8BD562293DNAM1APC11A8BioLegend338312NKp46BB5159E2BD564536NKp30BV480p30-15BD746491Inhibition markerPD-1PEEH12.2H7BioLegend329906NKG2AAPC131411R&D systemsFAB1059A-100

[0230] Example 6. Confirmation of granzyme B, perforin, and interferon gamma secretion capacity of natural killer cells.

[0231] To confirm the granzyme and perforin secretion capacity of CD3-CD56+ natural killer cells obtained in Example 2, the expression levels of granzyme B, perforin, and interferon gamma in natural killer cells were measured through intracellular staining. The cultured natural killer cells were centrifuged at 300×g for 5 minutes, and the supernatant was removed. After that, 2x10 6 Each culture composition was diluted to the number of cells / ml.

[0232] The prepared cells were dispensed into each well of a 96-well plate at 200 μl, and 1% (v / v) Stimulation Cocktail (1X) (Thermo Scientific, Waltham, MA, USA) was added and incubated at 37°C, CO2 for 4 hours. Afterwards, the plate was centrifuged at 300×g for 5 minutes, and the supernatant was removed. Then, the cell pellet was resuspended in 100 μl of FACS buffer prepared by adding 3% (v / v) FBS, 10 mM EDTA, 20 mM HEPES, 10 μg / ml polymyxin B, 100 U / ml penicillin, 100 μg / ml streptomycin, and 1 mM sodium pyruvate to PBS. The supernatant was removed, and 100 μl of BD Cytofix / Cytoperm™ buffer (perm / fixation buffer, BD science) was added for fixation and permeation, followed by incubation at 4°C for 30 minutes. An additional 100 μl of FACS buffer was added, and centrifugation was performed at 1,500 rpm for 3 minutes.

[0233] The cells were treated with Pe / cy7-labeled anti-human Granzyme B antibody (PE / cy7 anti-human Granzyme B (Clone NGZB)), APC-labeled anti-human perforin antibody (APC anti-human Perforin (Clone B-D48)), and BV421-labeled anti-human interferon gamma antibody (BV421 anti-human IFN-gamma (Clone B27)). After incubation at 4°C for 20 minutes, 100 μl of FACS buffer was added, and centrifugation was performed at 1,500 rpm for 3 minutes. The supernatant was removed, and 200 μl of FACS buffer (fixation buffer) was added to suspend the cells, and the expression level of the cells was confirmed using a flow cytometer.

[0234] Information on antibodies used in the above experiment is shown in Table 16. Markers of CD3-CD56+ natural killer cells cultured for 21 days under the conditions of the culture medium composition prepared in Preparation Example 1 were confirmed and are shown in Figures 25 to 28.

[0235] TargetColorCloneProducerCat#CytotoxicityPerforinAPCB-D48BioLegend353312IFN-γBV421B27BioLegend506538GranzymeB(GB11)PE / cy7NGZBebioscience25-8898-82

[0236] III. Analysis of the cancer cell killing ability of natural killer cells according to culture composition

[0237] Example 8. Confirmation of the degranulation and apoptotic effects of natural killer cells on cancer cells.

[0238] Specifically, the K562 cancer cell line (American Type Culture Collection, ATCC) was diluted in PBS to the cell number shown in Table 17 below and dispensed into each well.

[0239] E: T1: 11: 51: 10(E) NK cells2.5Х10 5 cells1Х10 5 cells1Х10 5 cells(T) K5622.5Х10 5 cells5Х10 5 cells1Х10 6 cells

[0240] Specifically, the K562 cancer cell line was cultured at 1×10 7 After diluting each culture composition to the number of cells / ml, it was dispensed into each well according to the cell number specified in the table above. After that, natural killer cells were also dispensed at 5×10 6After diluting with each culture composition to the number of cells / ml, the cells were dispensed per well according to the number of cells specified in the table above, and centrifuged at 30×g for 3 minutes. After that, the cells were cultured for 4 hours at 37°C and 5% CO2, and then treated with BV421-labeled anti-human CD3 antibody (BV421 human anti-CD3 (Clone UCHT1)), PE-labeled anti-human CD16 antibody (PE human anti-CD16 (Clone 3G8)), PE / cy7-labeled anti-human CD56 antibody (PE / cy7 human anti-CD56 (Clone B159)), and FITC-labeled anti-human CD107a antibody (Clone H4A3) and reacted on ice for 20 minutes.

[0241] After that, 100 ㎕ of FACS buffer was added and centrifuged at 1,300 rpm, 4℃ for 5 minutes. After removing the supernatant, 7-AAD Viability Staining Solution was treated and incubated at room temperature for 15 minutes under light protection. After that, 100 ㎕ of FACS buffer was added and centrifuged at 1,300 rpm, 4℃ for 5 minutes. Again, 200 ㎕ of FACS buffer was added and centrifuged at 1,300 rpm, 4℃ for 5 minutes. After repeating the above process once more, the supernatant was removed, 400 ㎕ of FACS buffer was added, and analysis was performed using a flow cytometer (Cytek® Aurora, Cytek, Fremont, CA, USA).

[0242] The degranulation ability and apoptotic effect of natural killer cells cultured for 21 days in a composition in which 50 nM of each of the additives in Table 5 was added to the basic culture medium in Tables 1 to 4 through the above experiment are shown in Figures 29 to 34.

Claims

A composition for culturing an isolated natural killer cell without CD3+ or CD56- cells comprising as an active ingredient a fusion protein dimer, wherein the fusion protein comprises the following structural formula (I) or (II): N'-X-[linker (1)]n-Fc domain-[linker (2)]m-Y-C' - formula (I) N'-Y-[linker (1)]n-Fc domain-[linker (2)]m-X-C' - formula (II), wherein, N′ is the N-terminus of the fusion protein, C′ is the C-terminus of the fusion protein, X is the CD80 protein or the fragment thereof, Y is the IL-2 protein variant, the linkers (1) and (2) are peptide linkers, and n and m are each independently 0 or 1, wherein the IL-2 protein variant comprises any one of the following substitution combinations (a) to (d) in the amino acid sequence of SEQ ID NO: 10: (a) R38A / F42A (b) R38A / F42A / Y45A (c) R38A / F42A / E61R, (d) R38A / F42A / L72G,wherein the CD80 protein fragment comprises an extracellular domain of CD80.The composition of claim 1, wherein the IL-2 variant comprises the amino acid sequence of SEQ ID NO: 6, 22, 23 or 24.The composition of claim 1, wherein the fragment of CD80 comprises the 35th to 242nd amino acids in the amino acid sequence of SEQ ID NO: 11. The composition of claim 1, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 9, 26, 28 or 30. A method for in vitro culturing a natural killer cell, which comprises: culturing the isolated NK cells without CD3+ or CD56- cells in the presence of a fusion protein dimer,wherein the fusion protein comprises the following structural formula (I) or (II): N'-X-[linker (1)]n-Fc domain-[linker (2)]m-Y-C' - formula (I) N'-Y-[linker (1)]n-Fc domain-[linker (2)]m-X-C' - formula (II), wherein, N′ is the N-terminus of the fusion protein, C′ is the C-terminus of the fusion protein, X is the CD80 protein or the fragment thereof, Y is the IL-2 protein variant, the linkers (1) and (2) are peptide linkers, and n and m are each independently 0 or 1, wherein the IL-2 protein variant comprises any one of the following substitution combinations (a) to (d) in the amino acid sequence of SEQ ID NO: 10: (a) R38A / F42A (b) R38A / F42A / Y45A (c) R38A / F42A / E61R, (d) R38A / F42A / L72G,wherein the CD80 protein fragment comprises an extracellular domain of CD80. The method of claim 5, wherein the fusion protein dimer is treated at a concentration of 1 nM to 500 nM.The method of claim 5, wherein the culture period in the culturing step is 5 days to 25 days.The method of claim 5, wherein the IL-2 variant comprises the amino acid sequence of SEQ ID NO: 6, 22, 23 or 24.The method of claim 5, wherein the fusion protein comprises of the amino acid sequence of SEQ ID NO: 9, 26, 28 or 30.

Images