Patent Description:
Various treatments such as surgical operation, radiation therapy, and chemotherapy have been developed to treat cancer, but as several side effects have been reported, immunotherapy using immune function of a patient has been recently developed. In particular, immunotherapy using natural killer cells which can undergo large-scale production and freezing has been studied.

Specifically, natural killer cells are a type of lymphocyte that are distributed in bone marrow, spleen, peripheral lymph nodes and peripheral blood of the body. They make up about <NUM>% of peripheral blood lymphocytes, and play an important role in innate immune response (<NPL>)). In addition, natural killer cells are positive for CD56 and CD16, but negative for CD3. Natural killer cells kill cells by release of cytoplasmic granules containing perforin and granzyme. Natural killer cells secrete various cytokines such as IFN-γ, TNF-α, GM-CSF and IL-<NUM>.

In addition, natural killer cells express several receptors on the cell surface, and these receptors are involved in cell adhesion, activation of capacity to kill cells, or inhibition of capacity to kill cells. However, most natural killer cells in the body of a normal subject exist in an inactive state. Therefore, there is a need for activated natural killer cells to eliminate cancer. In addition, for natural killer cells present in the body of a cancer patient, natural killer cells have functional defects due to immune evasion mechanism of cancer cells. Therefore, it is very important to activate natural killer cells to use natural killer cells as a therapeutic agent. Further, it is essential to develop a technique to massively proliferate and freeze natural killer cells in blood from a normal subject or a patient because the number of natural killer cells present in the body is limited.

Meanwhile, IL-<NUM>, also called as T-cell growth factor (TCGF), is a globular glycoprotein that plays a central role in production, survival, and homeostasis of lymphocyte. IL-<NUM> has a size of <NUM> kDa to <NUM> kDa protein and consists of <NUM> amino acids. IL-<NUM> mediates various immune responses by binding to the IL-<NUM> receptor which has three distinct subunits.

In addition, CD80 is known as B7-<NUM> and one of B7 family members among membrane-bound proteins involved in immune regulation by binding to the ligand and thus transmitting costimulatory responses 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.

As such, it is widely known that natural killer cells are important for anticancer treatment, but specific methods that can amplify natural killer cells to use them effectively are still insufficient.

<NPL>) discloses GI101 which comprises the extracellular domain of CD80, which binds CTLA4 and PD-L1, together with a long acting IL2 variant that preferentially binds to IL2Rβ.

<NPL>) discloses fusion protein IL2-B7. <NUM>(IgV+C), which was shown to display strong stimulation of T-cell proliferation.

<NPL>) uses IL2 analogues to identify residues that interact with the α chain of the human IL-<NUM> receptor.

<CIT> discloses "low toxicity" IL-<NUM> analogues and their uses in human immunotherapy and adoptive immunotherapy treatment strategies.

<CIT> discloses CD80 extracellular domain polypeptides and CD80 extracellular domain fusion molecules and their use in the treatment of cancer.

Accordingly, the present inventors prepared natural killer cells by using a fusion protein dimer comprising IL-<NUM> protein and CD80 protein, as a result of researching to prepare activated natural killer cells in a large amount. In addition, the present inventors have identified that natural killer cells thus prepared has increased activity and exhibits excellent anticancer effects, and thus have completed the present invention.

To achieve the above object, in accordance with an exemplary embodiment, provided is use of a composition for culturing natural killer cells including, as an active ingredient, a fusion protein dimer comprising an IL-<NUM> variant and CD80 fragment, as defined in the claims.

In accordance with another exemplary embodiment, provided is a method for culturing natural killer cells including: i) isolating cells that do not express CD3 from peripheral blood mononuclear cells (PBMC); ii) isolating cells that express CD56 from the cells that do not express CD3, isolated in the above step; and iii) culturing the isolated cells in the presence of a fusion protein dimer comprising IL-<NUM> or a variant thereof and CD80 or a fragment thereof, as defined in the claims.

Also described herein is natural killer cells prepared by the method for culturing natural killer cells.

Also described herein is a pharmaceutical composition for use in a method of preventing or treating cancer including the natural killer cells as an active ingredient.

The composition of the present invention for culturing natural killer cells including, as an active ingredient, a fusion protein comprising an IL-<NUM> variant and a CD80 fragment promotes proliferation of natural killer cells, induces expression of CD16 and NKp46, and increases expression and secretion of granzyme B and perforin, and thus may be usefully used in the production of natural killer cells having excellent anticancer immune function.

Exemplary embodiments can be understood in more detail from the following description taken in conjunction with the accompanying drawings, in which:.

An aspect of the present invention provides a composition for culturing a natural killer (NK) cell as defined in the claims including, as an active ingredient, a fusion protein dimer comprising IL-<NUM> or a variant thereof and CD80 or a fragment thereof, wherein the fusion protein is as defined in the claims. In addition, a natural killer cell culture medium including the fusion protein dimer as an active ingredient is provided.

The composition for culturing the natural killer cell may further include any one selected from the group consisting of a medium, a serum, a supplement, and a combination thereof.

The NK cell culture medium may be a medium in which the fusion protein dimer comprising IL-<NUM> protein and CD80 protein is added to a cell culture medium. In this case, 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. As a specific embodiment, the NK cell culture medium may include at least one of components in Table <NUM> to Table <NUM> below.

As used herein, the term "cell culture medium" means a medium used for culturing cells, specifically NK cells, and more specifically CD3-CD56+ cells. This includes components required by cells for cell growth and survival in vitro, or includes components that help 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.

The cell culture medium may include an amino acid component, a vitamin component, an inorganic salt component, other component, and purified water, wherein:.

In addition, the cell culture medium may further include a growth factor or a cytokine. The growth factor may be IGF, bFGF, TGF, HGF, EGF, VEGF, PDGF, or the like alone or at least two thereof, but is not limited thereto. The cytokine may be IL-<NUM>, IL-<NUM>, IL-<NUM>, IFN-γ , IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-<NUM>, or the like alone or at least two thereof, but is not limited thereto.

In addition, the cell culture medium may further include an antibody for activating natural killer cells. The antibody for activating natural killer cells may be an anti-CD3 antibody, an anti-CD2 antibody, an anti-CD335 antibody, or the like alone or at least two thereof, but is not limited thereto. In addition, a bead to which the antibody for activating natural killer cells is bound may be included. Also, a fusion protein including two or more types of antibodies or variable region fragments thereof for activating natural killer cells may be used.

In particular, the NK culture medium may further include any one selected from the group consisting of IL-<NUM>, IL-<NUM>, and a combination thereof.

The IL-<NUM> and IL-<NUM> may be a type of interleukin (IL), and mean proteinaceous bioactive substances produced by immunocompetent cells such as lymphocytes or monocytes and macrophages. The IL-<NUM> and IL-<NUM> may be used when culturing natural killer cells using mononuclear cells as source cells by promoting proliferation of natural killer cells, but there is a problem of low proliferation rate and purity when only these are used alone or in combination (<NPL>).

Specifically, the medium may be a conventional medium for culturing animal cells, such as DMEM (Dulbecco's Modified Eagle's Medium), EDM (Endothelial differentiation medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI <NUM>, F-<NUM>, F-<NUM>, a-MEM (a-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), Iscove's Modified Dulbecco's Medium, AIM-V Medium, X-VIVO™ <NUM> Medium, NK MACS Medium. In an embodiment of the present invention, AIM-V Medium, X-VIVO™ <NUM> Medium and NK MACS Medium were used as a medium.

The term "serum" as used in the present invention means clear supernatant separated from blood after the blood has been completely clotted. In addition, it is required to add serum to a synthetic medium for culturing animal cells, and it is common to use bovine, horse, or human serum. For bovine-derived serum, fetal bovine serum (FBS), newborn bovine serum, calf serum, bovine serum, or the like may be used depending on the timing of blood collection. For human-derived serum, human serum from a donor whose blood type is AB is used, and human AB serum which is free of antibodies to A and B blood type antigens so that can minimize immune reactivity may be used. In addition, CTS Immune Cell SR, or the like may be used as an alternative to the "serum. " In an embodiment of the present invention, human AB serum or CTS Immune Cell SR was used.

GLUTAMAX (GIBCO®), a L-Glutamine alternative, may be used as the supplement to improve stability and cell activity during cell culture. In addition, the supplement may be NK MACS supplement (Miltenyi Biotec, <NUM>-<NUM>-<NUM>).

As used herein, the term "IL-<NUM>" or "interleukin-<NUM>", unless otherwise stated, refers to any wild-type IL-<NUM> obtained from any vertebrate source, including mammals, for example, primates (such as humans) and rodents (such as mice and rats). IL-<NUM> may be obtained from animal cells, and also includes one obtained from recombinant cells capable of producing IL-<NUM>. In addition, IL-<NUM> may be wild-type IL-<NUM> or a variant thereof.

In the present specification, IL-<NUM> or a variant thereof may be collectively expressed by the term "IL-<NUM> protein" or "IL-<NUM> polypeptide". IL-<NUM>, an IL-<NUM> protein, an IL-<NUM> polypeptide, and an IL-<NUM> variant specifically bind to, for example, an IL-<NUM> receptor. This specific binding may be identified by methods known to those skilled in the art.

IL-<NUM> may have the amino acid sequence of SEQ ID NO: <NUM> or SEQ ID NO: <NUM>. Here, IL-<NUM> may also be in a mature form. Specifically, the mature IL-<NUM> may not comprise a signal sequence, and may have the amino acid sequence of SEQ ID NO: <NUM>. Here, IL-<NUM> may be used under a concept encompassing a fragment of wild-type IL-<NUM> in which a portion of N-terminus or C-terminus of the wild-type IL-<NUM> is truncated.

In addition, the fragment of IL-<NUM> may be in a form in which <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> contiguous amino acids are truncated from N-terminus of a protein having the amino acid sequence of SEQ ID NO: <NUM> or SEQ ID NO: <NUM>. In addition, the fragment of IL-<NUM> may be in a form in which <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> contiguous amino acids are truncated from C-terminus of a protein having the amino acid sequence of SEQ ID NO: <NUM> or SEQ ID NO: <NUM>.

As used herein, the term "IL-<NUM> variant" refers to a form in which a portion of amino acids in the full-length IL-<NUM> or the above-described fragment of IL-<NUM> is substituted. That is, an IL-<NUM> variant may have an amino acid sequence different from wild-type IL-<NUM> or a fragment thereof. However, an IL-<NUM> variant may have activity equivalent or similar to the wild-type IL-<NUM>. Here, "IL-<NUM> activity" may, for example, refer to specific binding to an IL-<NUM> receptor, which specific binding can be measured by methods known to those skilled in the art.

Specifically, the IL-<NUM> variant to be used in the invention is obtained by substitution of at least the <NUM>th and <NUM>nd amino acids in the amino acid sequence of SEQ ID NO: <NUM>.

Specifically, the IL-<NUM> variant to be used in the invention is obtained by substitution of at least the <NUM>th and <NUM>nd amino acid in the amino acid sequence of SEQ ID NO: <NUM> with another amino acid. In addition, when IL-<NUM> is in a form in which a portion of N-terminus in the amino acid sequence of SEQ ID NO: <NUM> is truncated, the amino acid at a position complementarily corresponding to that in the amino acid sequence of SEQ ID NO: <NUM> may be substituted with another amino acid. For example, when IL-<NUM> has the amino acid sequence of SEQ ID NO: <NUM>, its IL-<NUM> variant may be obtained by substitution of at least one of <NUM>th, <NUM>nd, <NUM>th, <NUM>st, or <NUM>nd amino acid in the amino acid sequence of SEQ ID NO: <NUM> with another amino acid. These amino acid residues correspond to the respective <NUM>th, <NUM>nd, <NUM>th, <NUM>st, and <NUM>nd amino acid residues in the amino acid sequence of SEQ ID NO: <NUM>. According to an embodiment, two, three, four, five, six, seven, eight, nine, or ten amino acids may be substituted as long as such IL-<NUM> variant maintains IL-<NUM> activity. According to another embodiment, two to five amino acids may be substituted.

In an embodiment, an IL-<NUM> variant may be in a form in which two amino acids are substituted. Specifically, the IL-<NUM> variant is obtained by substitution of the <NUM>th and <NUM>nd amino acids in the amino acid sequence of SEQ ID NO: <NUM>.

Furthermore, an IL-<NUM> variant may be in a form in which three amino acids are substituted. Specifically, the IL-<NUM> variant may be obtained by substitution of the <NUM>th, <NUM>nd, and <NUM>th amino acids in the amino acid sequence of SEQ ID NO: <NUM>. In addition, in an embodiment, the IL-<NUM> variant may be obtained by substitution of the <NUM>th, <NUM>nd, and <NUM>st amino acids in the amino acid sequence of SEQ ID NO: <NUM>. In addition, in an embodiment, the IL-<NUM> variant may be obtained by substitution of the <NUM>th, <NUM>nd, and <NUM>nd amino acids in the amino acid sequence of SEQ ID NO: <NUM>.

In addition, an IL-<NUM> variant may be in a form in which four amino acids are substituted. Specifically, the IL-<NUM> variant may be obtained by substitution of the <NUM>th, <NUM>nd, <NUM>th, and <NUM>st amino acids in the amino acid sequence of SEQ ID NO: <NUM>. In addition, in an embodiment, the IL-<NUM> variant may be obtained by substitution of the <NUM>th, <NUM>nd, <NUM>th, and <NUM>nd amino acids in the amino acid sequence of SEQ ID NO: <NUM>. In addition, in an embodiment, the IL-<NUM> variant may be obtained by substitution of the <NUM>th, <NUM>nd, <NUM>st, and <NUM>nd amino acids in the amino acid sequence of SEQ ID NO: <NUM>.

Furthermore, an IL-<NUM> variant may be in a form in which five amino acids are substituted. Specifically, the IL-<NUM> variant may be obtained by substitution of each of the <NUM>th, <NUM>nd, <NUM>th, <NUM>st, and <NUM>nd amino acids in the amino acid sequence of SEQ ID NO: <NUM> with another amino acid.

Here, the "another 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, regarding amino acid substitution for the IL-<NUM> variant, in the amino acid sequence of SEQ ID NO: <NUM>, the <NUM>th amino acid cannot be substituted with arginine, the <NUM>nd amino acid cannot be substituted with phenylalanine, the <NUM>th amino acid cannot be substituted with tyrosine, the <NUM>st amino acid cannot be substituted with glutamic acid, and the <NUM>nd amino acid cannot be substituted with leucine.

Regarding amino acid substitution for an IL-<NUM> variant, in the amino acid sequence of SEQ ID NO: <NUM>, the <NUM>th amino acid, arginine, may be substituted with an amino acid other than arginine. Preferably, regarding amino acid substitution for an IL-<NUM> variant, in the amino acid sequence of SEQ ID NO: <NUM>, the <NUM>th amino acid, arginine, may be substituted with alanine (R38A).

Regarding amino acid substitution for an IL-<NUM> variant, in the amino acid sequence of SEQ ID NO: <NUM>, the <NUM>nd amino acid, phenylalanine, may be substituted with an amino acid other than phenylalanine. Preferably, regarding amino acid substitution for an IL-<NUM> variant, in the amino acid sequence of SEQ ID NO: <NUM>, the <NUM>nd amino acid, phenylalanine, may be substituted with alanine (F42A).

Regarding amino acid substitution for an IL-<NUM> variant, in the amino acid sequence of SEQ ID NO: <NUM>, the <NUM>th amino acid, tyrosine, may be substituted with an amino acid other than tyrosine. Preferably, regarding amino acid substitution for an IL-<NUM> variant, in the amino acid sequence of SEQ ID NO: <NUM>, the <NUM>th amino acid, tyrosine, may be substituted with alanine (Y45A).

Regarding amino acid substitution for an IL-<NUM> variant, in the amino acid sequence of SEQ ID NO: <NUM>, the <NUM>st amino acid, glutamic acid, may be substituted with an amino acid other than glutamic acid. Preferably, regarding amino acid substitution for an IL-<NUM> variant, in the amino acid sequence of SEQ ID NO: <NUM>, the <NUM>st amino acid, glutamic acid, may be substituted with arginine (E61R).

Regarding amino acid substitution for an IL-<NUM> variant, in the amino acid sequence of SEQ ID NO: <NUM>, the <NUM>nd amino acid, leucine, may be substituted with an amino acid other than leucine. Preferably, regarding amino acid substitution for an IL-<NUM> variant, in the amino acid sequence of SEQ ID NO: <NUM>, the <NUM>nd amino acid, leucine, may be substituted with glycine (L72G).

Specifically, an IL-<NUM> variant may be obtained by 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: <NUM>.

Specifically, an IL-<NUM> variant may be obtained by amino acid substitutions at two, three, four, or five positions among the positions selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G.

In addition, an IL-<NUM> variant may be in a form in which two amino acids are substituted. Specifically, an IL-<NUM> variant may be obtained by the substitutions, R38A and F42A.

Furthermore, an IL-<NUM> variant may be in a form in which three amino acids are substituted. Specifically, an IL-<NUM> variant may be obtained by the substitutions, R38A, F42A, and Y45A. In addition, in an embodiment, an IL-<NUM> variant may be obtained by the substitutions, R38A, F42A, and E61R. In addition, in an embodiment, an IL-<NUM> variant may be obtained by the substitutions, R38A, F42A, and L72G.

In addition, an IL-<NUM> variant may be in a form in which four amino acids are substituted. Specifically, an IL-<NUM> variant may be obtained by the substitutions, R38A, F42A, Y45A, and E61R. In addition, in an embodiment, an IL-<NUM> variant may be obtained by the substitutions, R38A, F42A, Y45A, and L72G. In addition, in an embodiment, an IL-<NUM> variant may be obtained by the substitutions, R38A, F42A, E61R, and L72G.

Furthermore, an IL-<NUM> variant may be obtained by the substitutions, R38A, F42A, Y45A, E61R, and L72G.

Preferably, an embodiment of the IL-<NUM> variant may comprise which are any one selected from the following substitution combinations (a) to (d) in the amino acid sequence of SEQ ID NO: <NUM>:.

Here, when IL-<NUM> has the amino acid sequence of SEQ ID NO: <NUM>, an amino acid substitution may be present at a position complementarily corresponding to that in the amino acid sequence of SEQ ID NO: <NUM>. In addition, even when IL-<NUM> is a fragment of the amino acid sequence of SEQ ID NO: <NUM>, an amino acid substitution may be present at a position complementarily corresponding to that in the amino acid sequence of SEQ ID NO: <NUM>.

Specifically, an IL-<NUM> variant may have the amino acid sequence of SEQ ID NO: <NUM>, <NUM>, <NUM>, or <NUM>.

In addition, an IL-<NUM> variant may be characterized by having low in vivo toxicity. Here, the low in vivo toxicity may be a side effect caused by binding of IL-<NUM> to the IL-<NUM> receptor alpha chain (IL-2Rα). Various IL-<NUM> variants have been developed to ameliorate the side effect caused by binding of IL-<NUM> to IL-2Rα, and such IL-<NUM> variants may be those disclosed in <CIT> and <CIT>. In particular, IL-<NUM> variants described in the present application have low binding ability for the IL-<NUM> receptor alpha chain (IL-2Rα) and thus have lower in vivo toxicity than the wild-type IL-<NUM>.

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

As used herein, the term "CD80 fragment" refers to a truncated form of CD80. In addition, the CD80 fragment to be used in the invention comprises an extracellular domain of CD80. An embodiment of the CD80 fragment may be obtained by elimination of the <NUM>st to <NUM>th amino acids from N-terminus which are a signal sequence of CD80. Specifically, an embodiment of the CD80 fragment may be a protein consisting of the <NUM>th to <NUM>th amino acids in SEQ ID NO: <NUM>. In addition, an embodiment of the CD80 fragment may be a protein consisting of the <NUM>th to <NUM>nd amino acids in SEQ ID NO: <NUM>. In addition, an embodiment of the CD80 fragment may be a protein consisting of the <NUM>th to <NUM>nd amino acids in SEQ ID NO: <NUM>. In addition, an embodiment of the CD80 fragment may be a protein consisting of the <NUM>th to <NUM>th amino acids in SEQ ID NO: <NUM>. In addition, an embodiment of the CD80 fragment may be a protein consisting of the <NUM>nd to <NUM>nd amino acids in SEQ ID NO: <NUM>. In an embodiment, a CD80 fragment may have the amino acid sequence of SEQ ID NO: <NUM>.

In addition, the IL-<NUM> variant and the CD80 fragment to be used in the invention are attached to each other via a linker or a carrier. In the present description, the linker and the carrier may be used interchangeably.

The linker links two proteins. An embodiment of the linker may include <NUM> to <NUM> amino acids, albumin or a fragment thereof, an Fc domain of an immunoglobulin, or the like. Here, the Fc domain of immunoglobulin refers to a protein that comprises heavy chain constant region <NUM> (CH2) and heavy chain constant region <NUM> (CH3) of an immunoglobulin, and does not comprise heavy and light chain variable regions and light chain constant region <NUM> (CH1) of an immunoglobulin. The immunoglobulin may be IgG, IgA, IgE, IgD, or IgM, and may preferably be IgG4. Here, Fc domain of wild-type immunoglobulin G4 may have the amino acid sequence of SEQ ID NO: <NUM>.

In addition, the Fc domain of an immunoglobulin may be an Fc domain variant as well as wild-type Fc domain. In addition, as used herein, the term "Fc domain variant" may refer to a form which is different from the wild-type Fc domain in terms of glycosylation pattern, has a high glycosylation as compared with the wild-type Fc domain, or has a low glycosylation as compared with the wild-type Fc domain, or a deglycosylated form. In addition, an aglycosylated Fc domain is included therein. The Fc domain or a variant thereof may be adapted to have an adjusted number of sialic acids, fucosylations, or glycosylations, through culture conditions or genetic manipulation of a host.

In addition, glycosylation of the Fc domain of an immunoglobulin may 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 in a mixed form of respective Fc regions of immunoglobulins, IgG, IgA, IgE, IgD, and IgM. In addition, the Fc domain variant may be in a form in which some amino acids of the Fc domain are substituted with other amino acids. An embodiment of the Fc domain variant may have the amino acid sequence of SEQ ID NO: <NUM>.

The fusion protein to be used in the invention has the following structural formula (I) or (II):.

N'-X-[linker (<NUM>)]n-Fc domain-[linker (<NUM>)]m-Y-C'     (I).

N'-Y-[linker (<NUM>)]n-Fc domain-[linker (<NUM>)]m-X-C'     (II).

Here, in the structural formulas (I) and (II),.

Preferably, the fusion protein to be used in the invention may consist of the structural formula (I). The IL-<NUM> variant is as described in the claims. In addition, the CD80 protein is as described in the claims. The CD80 fragment may be a fragment obtained by truncation of up to about <NUM> contiguous amino acid residues from the N-terminus or C-terminus of the wild-type CD80. Alternatively, the CD80 fragment may be an extracellular immunoglobulin-like domain having the activity of binding to the T cell surface receptors CTLA-<NUM> and CD28.

Specifically, the fusion protein may have the amino acid sequence of SEQ ID NO: <NUM>, <NUM>, <NUM>, or <NUM>. According to another embodiment, the fusion protein includes a polypeptide having a sequence identity of <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% to the amino acid sequence of SEQ ID NO: <NUM>, <NUM>, <NUM>, or <NUM>. Here, the identity is, for example, percent homology, and may be determined through homology comparison software such as BlastN software of the National Center of Biotechnology Information (NCBI).

The peptide linker (<NUM>) to be used in the invention is included between the CD80 fragment and the Fc domain. The peptide linker (<NUM>) may consist of <NUM> to <NUM> contiguous amino acids, <NUM> to <NUM> contiguous amino acids, <NUM> to <NUM> contiguous amino acids, or <NUM> to <NUM> contiguous amino acids. In an embodiment, the peptide linker (<NUM>) may consist of <NUM> amino acids. In addition, the peptide linker (<NUM>) may comprise at least one cysteine. Specifically, the peptide linker (<NUM>) may comprise one, two, or three cysteines. In addition, the peptide linker (<NUM>) may be derived from the hinge of an immunoglobulin. In an embodiment, the peptide linker (<NUM>) may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: <NUM>.

The peptide linker (<NUM>) may consist of <NUM> to <NUM> contiguous amino acids, <NUM> to <NUM> contiguous amino acids, or <NUM> to <NUM> contiguous amino acids. In an embodiment, the peptide linker (<NUM>) may be (G4S)n (where n is an integer of <NUM> to <NUM>). Here, in (G4S)n, n may be <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>. In an embodiment, the peptide linker (<NUM>) may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: <NUM>.

Also described herein is a dimer obtained by binding of two fusion proteins, each of which comprises an IL-<NUM> protein and a CD80 protein. The fusion protein comprising IL-<NUM> or a variant thereof and CD80 or a fragment thereof is as described above.

Here, the binding between the fusion proteins constituting the dimer may be achieved by, but is not limited to, a disulfide bond formed by cysteines present in the linker. The fusion proteins constituting the dimer may be the same or different fusion proteins from each other. Preferably, the dimer may be a homodimer. An embodiment of the fusion protein constituting the dimer may be a protein having the amino acid sequence of SEQ ID NO: <NUM>.

Another aspect of the present invention provides a method for culturing a natural killer cell, including: i) isolating a cell that do not express CD3 from peripheral blood mononuclear cells (PBMC); ii) isolating a cell that express CD56 from the cell that do not express CD3 isolated in the above step; and iii) culturing the isolated cells in the presence of a fusion protein dimer comprising IL-<NUM> or a variant thereof and CD80 or a fragment thereof, as defined by the claims.

The term "PBMC" as used in the present invention means a peripheral blood mononuclear cell. The PBMC is composed of lymphocytes (T cells, B cells, natural killer cells) and monocytes, and can be isolated from whole blood by Ficoll and centrifugation. The PBMC may be isolated from whole blood obtained from an individual.

The fusion protein dimer is as described in detail for a composition for culturing natural killer cells. The fusion protein dimer may be treated at a concentration of <NUM> to <NUM>. Specifically, the fusion protein dimer may be treated at a concentration of <NUM> to <NUM>, <NUM> to <NUM>, or <NUM> to <NUM>. In an embodiment of the present invention, the fusion protein dimer was treated at a concentration of <NUM> or <NUM>.

A method for culturing the isolated cells may be performed using a method widely known in the art. Specifically, the culture temperature in the step of culturing the isolated cells may be <NUM> to <NUM>, or <NUM> to <NUM>. In an embodiment of the present invention, culture was performed at a temperature of <NUM>. In addition, in the step of culturing the isolated cells, CO<NUM> concentration condition during culture may be <NUM>% to <NUM>%, and preferably, it may be cultured in a <NUM>% CO<NUM> condition.

In the step of culturing the isolated cells, culture period may be <NUM> days to <NUM> days, <NUM> days to <NUM> days, or <NUM> days to <NUM> days. In an embodiment of the present invention, culture period was <NUM> days, and a significant difference in proliferation appeared from the 5th day.

Also described herein are natural killer cells prepared by the method for culturing natural killer cells.

The natural killer cells may have increased expression of CD16 and NKp46. The natural killer cells may have increased expression of granzyme B and perforin. The natural killer cells cultured according to the method for culturing natural killer cells may be frozen and the function of cells is not impaired even when thawed again.

Due to high expression of activating receptors such as CD16 and NKp46, the natural killer cells exhibit increased killing capacity against a cancer cell line and increased secretion of granzyme B and perforin, and thus an excellent anticancer effect may be expected. Therefore, a therapeutic agent effective for treating cancer may be prepared, using a large amount of activated natural killer cells which are clinically applicable. In addition, the natural killer cells may have high expression of NKp30 or DNAM1.

In addition, natural killer cells prepared by the method for culturing natural killer cells may be included in amount of <NUM> to <NUM>% by weight based on the total weight of the pharmaceutical composition. Further, the pharmaceutical composition may further include, in addition to the active ingredient, at least one active ingredient that exhibits the same or similar functions.

A dosage of the pharmaceutical composition may be adjusted according to various factors including type of disease, severity of disease, kinds and content of active ingredients and other ingredients included in the composition, kinds of formulation, and age, weight, general health condition, gender, and diet of a patient, time of administration, route of administration, and secretion rate of a composition, duration of treatment, and simultaneously used drugs.

However, for a desirable effect, a dosage of the pharmaceutical composition may be <NUM>× <NUM><NUM> cells/kg to <NUM>× <NUM><NUM> cells/kg, and <NUM>× <NUM><NUM> cells/kg to <NUM>× <NUM><NUM> cells/kg based on the natural killer cells, which is an active ingredient. In this case, the dose may be administered once a day, or may be divided in several times.

In addition, the pharmaceutical composition may be administered to an individual by various methods known in the art. The route of administration may be appropriately selected by a person skilled in the art in consideration of the method of administration, volume of body fluid, viscosity, or the like.

The cancer may be any one selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, larynx cancer, acute lymphoblastic leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary cancer, and lymphoma.

Also described herein is a method for treating cancer including administering the NK cell to an individual having cancer. In this case, the NK cells and cancer are as described above. Still another aspect of the present invention provides use of the NK cell to treat cancer.

Also described herein is a method for culturing a natural killer cell, including: i) isolating a cell that do not express CD3 from PBMCs; and ii) culturing the isolated cell in the presence of a fusion protein dimer comprising IL-<NUM> or a variant thereof and CD80 or a fragment thereof.

Also described herein are natural killer cells prepared by the method for culturing natural killer cell Also described herein is a pharmaceutical composition for preventing or treating cancer, including the natural killer cell as an active ingredient. Also described herein is a method for treating cancer including administering the NK cell to an individual having cancer. In this case, the NK cell and cancer are as described above. Also described herein is use of the NK cell to treat cancer.

Also described herein is a method for culturing natural killer cells, including: i) isolating a cell that express CD56 from PBMCs; and ii) culturing the isolated cell in the presence of a fusion protein dimer comprising IL-<NUM> or a variant thereof and CD80 or a fragment thereof.

Also described herein is a natural killer cell prepared by the method for culturing natural killer cell. Due to high expression of activating receptors such as CD16 and NKp46, the natural killer cells exhibit increased killing capacity against a cancer cell line and increased secretion of granzyme B and perforin, and thus an excellent anticancer effect may be expected. Therefore, a therapeutic agent effective for treating cancer may be prepared, using a large amount of activated natural killer cells which are clinically applicable. In addition, the natural killer cells may have high expression of NKp30 or DNAM1.

Also described herein is a pharmaceutical composition for preventing or treating cancer, including the natural killer cell as an active ingredient. Also described herein is a method for treating cancer including administering the NK cell to an individual having cancer. In this case, NK cells and cancer are as described above. Also described herein is use of the NK cells to treat cancer.

Also described herein is a method for promoting the activity of natural killer cells in PBMCs, including culturing PBMCs in the presence of a fusion protein dimer comprising IL-<NUM> or a variant thereof and CD80 or a fragment thereof.

Also described herein is PBMCs prepared by the method for promoting the activity of natural killer cells in the PBMCs. The natural killer cells in PBMC have high expression of an activating receptor such as CD16 and NKp46 so that increase the cell killing capacity against cancer cell lines and secretion of granzyme B and perforin, and thus excellent anticancer effects can be expected. Therefore, a therapeutic agent effective for treating cancer may be prepared using PBMCs including a large amount of activated natural killer cells which are clinically applicable. In addition, the natural killer cells in PBMCs may have high expression of NKp30 or DNAM1.

Also described herein is a pharmaceutical composition for preventing or treating cancer including the PBMCs as an active ingredient. Also described herein is a method for treating cancer including administering the NK cells to an individual having cancer. In this case, NK cells and cancer are as described above. Also described herein is use of the NK cells to treat cancer.

Hereinafter, the present disclosure will be described in more detail by way of the following examples. However, the following examples are only for illustrating the present disclosure, and the scope of the present disclosure is not limited thereto.

In order to produce a fusion protein including a human CD80 fragment, a Fc domain, and an IL-<NUM> variant, a polynucleotide including a nucleotide sequence (SEQ ID NO: <NUM>) encoding a fusion protein comprising a signal peptide (SEQ ID NO: <NUM>), a CD80 fragment (SEQ ID NO: <NUM>), a linker-conjugated Ig hinge (SEQ ID NO: <NUM>), a Fc domain (SEQ ID NO: <NUM>), a linker (SEQ ID NO: <NUM>), and an IL-<NUM> variant (<NUM>) in which two amino acids are substituted (R38A, F42A) (SEQ ID NO: <NUM>) in this order from N-terminus was synthesized through Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific Inc. , and cloned into a pcDNA3_4 vector. In addition, the vector was introduced into CHO cells (EXPI-CHO™) to express a fusion protein of SEQ ID NO: <NUM>. After introducing the vector, cells were cultured in an environment of <NUM>, <NUM> RPM, and <NUM>% CO<NUM> for <NUM> days, and then collected to purify a fusion protein. The purified fusion protein dimer was named as "GI-<NUM>.

Purification was performed using chromatography including MabSelect SuRe protein A resin. The fusion protein was bound under the condition of <NUM> Tris, <NUM> NaCl, and pH <NUM>. Then, it was eluted with <NUM> NaCl and <NUM> acetic acid at pH <NUM>. After putting <NUM>% of <NUM> Tris-HCl at pH <NUM> into a collection tube, the fusion protein was collected. The collected fusion protein was dialyzed into PBS buffer for <NUM> hours to change.

Then, absorbance at a wavelength of <NUM> over time was measured by using size exclusion chromatography with TSKgel G3000SWXI, column (TOSOH Bioscience) to obtain a high concentration of fusion protein. At this time, the isolated and purified fusion protein was subjected to SDS-PAGE under the reducing (R) or non-reducing (NR) conditions, and stained with coomassie blue to confirm its purity (<FIG>). It was confirmed that the fusion protein was included at a concentration of <NUM>/ml as detected using NanoDrop (<FIG>). Also, the result analyzed using size exclusion chromatography is as shown in <FIG>.

In order to produce a fusion protein comprising a Fc domain and an IL-<NUM> variant, a polynucleotide including a nucleotide sequence (SEQ ID NO: <NUM>) encoding a fusion protein comprising a signal peptide (SEQ ID NO: <NUM>), an Ig hinge (SEQ ID NO: <NUM>), a Fc domain (SEQ ID NO: <NUM>), a linker (SEQ ID NO: <NUM>), and an IL-<NUM> variant (<NUM>) in which two amino acids are substituted (R38A, F42A) (SEQ ID NO: <NUM>) in this order from N-terminus was synthesized through Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific Inc. , and cloned into a pcDNA3_4 vector. In addition, the vector was introduced into CHO cells (EXPI-CHO™) to express a fusion protein of SEQ ID NO: <NUM>. After introducing the vector, the cells were cultured in an environment of <NUM>, <NUM> RPM, and <NUM>% CO<NUM> for <NUM> days, and then collected to purify a fusion protein dimer. The purified fusion protein dimer was named as "Fc-IL2v2.

The purification and collection of the fusion protein were performed in the same manner as in the Preparatory Example <NUM>. The isolated and purified fusion protein was subjected to SDS-PAGE under the reducing (R) or non-reducing (NR) conditions, and stained with coomassie blue to confirm its purity (<FIG>). As a result, it was confirmed that the fusion protein forms a dimer. Also, the result analyzed using size exclusion chromatography is as shown in <FIG>.

In order to produce a fusion protein comprising a Fc domain and a wild-type IL-<NUM>, a polynucleotide including a nucleotide sequence (SEQ ID NO: <NUM>) encoding a fusion protein comprising a signal peptide (SEQ ID NO: <NUM>), an Ig hinge (SEQ ID NO: <NUM>), a Fc domain (SEQ ID NO: <NUM>), a linker (SEQ ID NO: <NUM>), and a wild-type IL-<NUM> (SEQ ID NO: <NUM>) in this order from N-terminus was synthesized through Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific Inc. , and cloned into a pcDNA3_4 vector. In addition, the vector was introduced into CHO cells (EXPI-CHO™) to express a fusion protein of SEQ ID NO: <NUM>. After introducing the vector, the cells were cultured in an environment of <NUM>, <NUM> RPM, and <NUM>% CO<NUM> for <NUM> days, and then collected to purify a fusion protein dimer. The purified fusion protein dimer was named as "Fc-IL2wt.

In order to produce a fusion protein comprising a human CD80 fragment, a Fc domain, and an IL-<NUM> wile-type protein, a polynucleotide including a nucleotide sequence (SEQ ID NO: <NUM>) encoding a fusion protein comprising a signal peptide (SEQ ID NO: <NUM>), a CD80 fragment (SEQ ID NO: <NUM>), a linker-conjugated Ig hinge (SEQ ID NO: <NUM>), a Fc domain (SEQ ID NO: <NUM>), a linker (SEQ ID NO: <NUM>), and IL-<NUM> wild-type (SEQ ID NO: <NUM>) in this order from N-terminus was synthesized through Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific Inc. , and cloned into a pcDNA3_4 vector. In addition, the vector was introduced into CHO cells (EXPI-CHO™) to express a fusion protein of SEQ ID NO: <NUM>. After introducing the vector, the cells were cultured in an environment of <NUM>, <NUM> RPM, and <NUM>% CO<NUM> concentration for <NUM> days, and then collected to purify a fusion protein dimer. The purified fusion protein dimer was named as "hCD80-Fc-II,2wt.

Then, absorbance at a wavelength of <NUM> over time was measured by using size exclusion chromatography with TSKgel G3000SWXI, column (TOSOH Bioscience) to obtain a high concentration of fusion protein. At this time, the isolated and purified fusion protein was subjected to SDS-PAGE under the reducing (R) or non-reducing (NR) conditions, and stained with coomassie blue to confirm its purity (<FIG>). As a result, it was confirmed that the fusion protein forms a dimer. Also, the result analyzed using size exclusion chromatography is as shown in <FIG>.

In order to produce a fusion protein comprising a human CD80 fragment and a Fc domain, a polynucleotide (SEQ ID NO: <NUM>) including a nucleotide sequence encoding a fusion protein comprising a signal peptide (SEQ ID NO: <NUM>), a CD80 fragment (SEQ ID NO: <NUM>), a linker-conjugated Ig hinge (SEQ ID NO: <NUM>), and a Fc domain (SEQ ID NO: <NUM>) in this order from N-terminus was synthesized through Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific Inc. , and cloned into a pcDNA3_4 vector. In addition, the vector was introduced into CHO cells (EXPI-CHO™) to express a fusion protein of SEQ ID NO: <NUM>. After introducing the vector, the cells were cultured in an environment of <NUM>, <NUM> RPM, and <NUM>% CO<NUM> for <NUM> days, and then collected to purify a fusion protein dimer. The purified fusion protein dimer was named "hCD80-Fc.

Natural killer cell culture media compositions were prepared by respectively adding substances corresponding to the adding conditions <NUM> to <NUM> of Table <NUM> to each basal culture medium having composition of Table <NUM> to Table <NUM> below.

In order to obtain CD3(-) cells, the number of PBMCs (peripheral blood mononuclear cells, Zen-Bio. Inc, NC <NUM>, USA, Cat#: SER-PBMC-<NUM>-F) was counted using an ADAM-MC2 automated cell counter (NanoEnTek, purchased from Cosmo Genetech Co. The PBMCs were transferred to a new tube, and then centrifuged at <NUM>×g for <NUM> minutes at a temperature of <NUM>. <NUM>% (v/v) bovine serum albumin (BSA) and EDTA at a concentration of <NUM> were included in PBS to prepare MACS buffer (pH <NUM>). After centrifugation was completed, a cell pellet was treated with <NUM>µl of MACs buffer and <NUM>µl of CD3 magnetic beads (Miltenyi biotech, <NUM>-<NUM>-<NUM>) per <NUM>×<NUM><NUM> cells to suspend, and then incubated at a temperature of <NUM> for <NUM> minutes. <NUM> of MACs buffer was added for washing and centrifuged at <NUM>×g for <NUM> minutes at a temperature of <NUM>, and then the cell pellet was resuspended in <NUM> of MACs buffer.

<NUM> of MACs buffer was first flowed into the LD column (Miltenyi Biotec, Bergisch Gladbach, Germany, Cat#: <NUM>-<NUM>-<NUM>), and then the cell suspension was flowed. Then, CD3(-) cells passing through the LD column were obtained. At this time, CD3(-) cells were obtained by flowing <NUM> of MACs buffer three times so that the cells remaining in the LD column could be sufficiently separated. The obtained CD3(-) cells were counted using a cell counter, and then placed in a new tube and centrifuged at <NUM>×g for <NUM> minutes at a temperature of <NUM>. Then, the supernatant was removed, and then <NUM>µl of MACs buffer and <NUM>µl of CD56 magnetic beads (Miltenyi biotech, Cat#: <NUM>-<NUM>-<NUM>) were added per <NUM>×<NUM><NUM> cells, followed by incubation at a temperature of <NUM> for <NUM> minutes. <NUM> of MACs buffer was added for washing and centrifuged at <NUM>×g for <NUM> minutes at a temperature of <NUM>, and then the cell pellet was resuspended in <NUM> of MACs buffer.

<NUM> of MACs buffer was first flowed into the LS column (Miltenyi Biotec, Bergisch Gladbach, Germany, Cat#: <NUM>-<NUM>-<NUM>), and then the cell suspension was flowed. At this time, <NUM> of MACs buffer was flowed three times so that the cells remaining in the LS column could be sufficiently separated. Then, after the LS column was separated from a magnet stand, <NUM> 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 was placed in a new tube and centrifuged at <NUM>×g for <NUM> minutes at a temperature of <NUM>. After removing the supernatant, the cells were suspended in the basal culture media shown in Table <NUM> to Table <NUM> in consideration of the culture conditions. The number of suspended cells was counted using a cell counter.

<NUM>µL of CD335 (NKp46)-biotin and <NUM>µL of CD2-biotin included in a NK Cell Activation/Expansion Kit (Cat#: <NUM>-<NUM>-<NUM>) (Miltenyi Biotec, Bergisch Gladbach, Germany) were placed in a <NUM> microtube and mixed, and then <NUM>µL of Anti-Biotin MACSiBead Particles was added and mixed. Then, <NUM>µL of MACs buffer was added, and mixed at <NUM> to <NUM> for <NUM> hours using a microtube rotator. Considering the number of cells, <NUM>µL of NK activation beads per <NUM>×<NUM><NUM> cells was transferred to a new tube. <NUM> of PBS was added and centrifuged at <NUM>×g for <NUM> minutes. After removing the supernatant, NK MACs medium (Cat#: <NUM>-<NUM>-<NUM>) (Miltenyi Biotec, Bergisch Gladbach, Germany) to be used was added on the basis of <NUM>µL per <NUM><NUM> NK cells, and released beads, followed by inoculating into the CD3(-)CD56(+) natural killer cells isolated in Example <NUM>.

Next, the prepared CD3(-)CD56(+) natural killer cells were suspended in a culture medium composition containing an additive prepared in Preparation example <NUM> so that the total number of cells was <NUM>×<NUM><NUM>, and seeded in a <NUM>-well plate, followed by culturing under the condition of <NUM> and <NUM>% CO<NUM>. Then, the number of cells was determined every <NUM> days to subculture in the order of a <NUM>-well plate, a <NUM>-well plate, a <NUM>-well plate, a <NUM>-well plate, and a 25T flask when the cells were confluent <NUM>% or more of culture vessel (confluency), and finally all cells were harvested on day <NUM>.

The total number of cells and viability of the cultured natural killer cells were counted using a cell counter (ADAM-MC2) on days <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>. At this time, the number of cells were counted on the dates above as the cells reach <NUM>% confluency which is a criterion for subculture because the proliferation rate of cells varies depending on a treated material and type of culture medium.

The results of comparing the total number of cells and viability of CD3-CD56+ cells cultured under the conditions of the culture medium composition prepared in Preparation example <NUM> are shown in Tabled <NUM> to <NUM>, and <FIG>.

As a result, it was confirmed that all culture media compositions to which GI-<NUM> prepared by Preparatory example <NUM> was added had the total number of natural killer cells greater than the control group (addition of CD80-Fc+Fc-IL2v2 or CD80-Fc+Fc-IL2WT), regardless of the treatment concentration in four basal culture medium conditions (Tables <NUM> to <NUM>) (<FIG>, <FIG>, <FIG>, <FIG>, <FIG>, <FIG>, <FIG> and <FIG>).

In addition, even for cells viability, when GI-<NUM> was added, all culture media compositions exhibited high viability regardless of the basal culture medium and the concentration (<FIG>, <FIG>, <FIG>, <FIG>, <FIG>, <FIG>, <FIG> and <FIG>).

Based on the results, it was confirmed that GI-<NUM> plays an important role in improving proliferation ability and viability of natural killer cells as compared with the control group (addition of CD80-Fc+Fc-II,2v2 or CD80-Fc+Fc-II,2WT), regardless of the basal culture medium and the concentration.

CD3-CD56+ natural killer cells obtained from Example <NUM> were respectively centrifuged at <NUM>×g condition for <NUM> minutes to remove the supernatant, and <NUM> of FACS buffer was added to release the pellet. Then, <NUM>% (v/v) FBS, <NUM> EDTA, <NUM> HEPES, <NUM>µg/ml polymyxin B, <NUM> U/ml penicillin, <NUM>µg/ml streptomycin, and <NUM> sodium pyruvate were added to PBS to prepare FACS buffer, and <NUM> of prepared FACS buffer was added to resuspend the cell pellet. Next, it was diluted with FACS buffer to <NUM>×<NUM><NUM> cells/ml using a cell counter.

<NUM>µl of the diluted cell solution was added to each of a <NUM> FACS tube, and <NUM>µl of FACS buffer was further added thereto, followed by treatment with a PerCP-labeled anti-human CD3 antibody (PerCP human anti-CD3(Clone UCHT1)) and a PE/cy7-labeled anti-human CD56 antibody (PE/cy7 human anti-CD56(Clone B159)). Then, after incubating at <NUM> for <NUM> minutes, <NUM>µl of FACS buffer was added and centrifuged at <NUM>,<NUM> rpm for <NUM> minutes. The supernatant was removed, and <NUM>µl of FACS buffer was added to suspend, and then phenotype of the cells was determined using a flow cytometer (CYTEK® Aurora, Cytek, Fremont, CA, USA).

Information about antibodies used in the experiment is shown in Table <NUM>. In addition, the purities of CD3-CD56+ natural killer cells cultured for <NUM> days under the conditions of the culture media compositions prepared in Preparation example <NUM> were measured and shown in <FIG>.

CD3-CD56+ natural killer cells obtained from Example <NUM> were respectively centrifuged at <NUM>×g condition for <NUM> minutes to remove the supernatant, and <NUM> of FACS buffer was added to release the pellet.

<NUM>% (v/v) FBS, <NUM> EDTA, <NUM> HEPES, <NUM>µg/ml polymyxin B, <NUM> U/ml penicillin, <NUM>µg/ml streptomycin, and <NUM> sodium pyruvate were added to PBS to prepare FACS buffer, and <NUM> of prepared FACS buffer was added to resuspend the cell pellet. Then, it was diluted with FACS buffer to <NUM>×<NUM><NUM> cells/ml using a cell counter. <NUM>µl of the diluted cell solution was added each of a <NUM> FACS tube, and confirmed by using a 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 <NUM>)), 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-<NUM>)), PE-labeled anti-human PD-<NUM> antibody (PE human anti-PD-<NUM> (Clone EH12.2H7)), and APC-labeled anti-human NKG2A antibody (APC anti-human NKG2A (Clone <NUM>)), using a flow cytometer. Then, after incubating at <NUM> for <NUM> minutes, <NUM>µl of FACS buffer was added and centrifuged at <NUM>,<NUM> rpm for <NUM> minutes.

The supernatant was removed, and <NUM>µl of FACS buffer was added to suspend, and then phenotype of the cells was determined using a flow cytometer (CYTEK® Aurora, Cytek, Fremont, CA, USA). Information of antibodies used in the experiment is shown in Table <NUM>. The activation and inhibition markers for CD3-CD56+ natural killer cells cultured for <NUM> days under the conditions of the culture media compositions prepared in Preparation example <NUM> were identified and shown in <FIG>.

In order to determine granzyme and perforin secretory capacity of CD3-CD56+ natural killer cells obtained from Example <NUM>, an amount of expression of granzyme B, perforin, and interferon gamma in the natural killer cells were measured by intracellular staining. The cultured natural killer cells were centrifuged at <NUM>×g condition for <NUM> minutes and the supernatant was removed. Then, it was diluted with each culture composition to <NUM>×<NUM><NUM> cells/ml using a cell counter.

<NUM>µl of the prepared cells were dispensed into each well of a <NUM>-well plate, and then <NUM>% (v/v) Stimulation Cocktail(1X) (Thermo Scientific, Waltham, MA, USA) was added and incubated at <NUM>, CO<NUM> condition for <NUM> hours. Then, the plates were centrifuged at <NUM>×g condition for <NUM> minutes and the supernatant was removed. Next, <NUM>% (v/v) FBS, <NUM> EDTA, <NUM> HEPES, <NUM>µg/ml polymyxin B, <NUM> U/ml penicillin, <NUM>µg/ml streptomycin, and <NUM> sodium pyruvate were added to PBS to prepare FACS buffer, and <NUM>µl of prepared FACS buffer was added to resuspend the cell pellet. The supernatant was removed, and <NUM>µl of BD CYTOFIX/CYTOPERM™ buffer (perm/fixation buffer, BD science) was added for fixation and permeation and then suspended, followed by incubation at <NUM> for <NUM> minutes. <NUM>µl of FACS buffer was further added and centrifuged at <NUM>,<NUM> rpm for <NUM> minutes.

A PE/cy7-labeled anti-human granzyme B antibody (PE/cy7 anti-human Granzyme B (Clone NGZB)), an 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)) were treated. Then, after incubating at <NUM> for <NUM> minutes, <NUM>µl of FACS buffer was added and centrifuged at <NUM>,<NUM> rpm for <NUM> minutes. After supernatant was removed and <NUM>µl of FACS buffer (fixation buffer) was added to suspended, an amount of expression of the cells was determined using a flow cytometer.

Information of antibodies used in the experiment is shown in Table <NUM>. The markers for CD3-CD56+ natural killer cells cultured for <NUM> days under the conditions of the culture media compositions prepared in Preparation example <NUM> were identified and shown in <FIG>.

Specifically, a K562 cancer cell line (American Type Culture Collection, ATCC) was diluted in PBS to number of cells shown in Table <NUM> below, and dispensed into each well.

Specifically, a K562 cancer cell line was diluted with each culture composition to <NUM>×<NUM><NUM> cells/ml, and then dispensed according to the number of cells specified in the above table for each well. Then, natural killer cells were also diluted with each culture composition to <NUM>×<NUM><NUM> cells/ml, and then dispensed according to the number of cells specified in the above table for each well, and centrifuged at <NUM>×g condition for <NUM> minutes. Next, after culture at <NUM>, <NUM>% CO<NUM> condition for <NUM> hours, a BV421-labeled anti-human CD3 antibody (BV421 human anti-CD3 (Clone UCHT1)), a PE-labeled anti-human CD16 antibody (PE human anti-CD16 (Clone 3G8)), a PE/cy7-labeled anti-human CD56 antibody (PE/cy7 human anti-CD56(Clone B159)), and a FITC-labeled anti-human CD107a antibody (FITC anti-human CD107a antibody (Clone H4A3)) were treated and incubated on ice for <NUM> minutes.

Then, <NUM>µL of FACS buffer was added and centrifuged at <NUM>,<NUM> rpm, <NUM> condition for <NUM> minutes. After removing the supernatant, <NUM>-AAD Viability Staining Solution was treated, and light was blocked to react at room temperature for <NUM> minutes. Then, <NUM>µL of FACS buffer was added and centrifuged at <NUM>,<NUM> rpm, <NUM> condition for <NUM> minutes. <NUM>µL of FACS buffer was added again, and centrifuged at <NUM>,<NUM> rpm, <NUM> condition for <NUM> minutes. After repeating the above process once more, the supernatant was removed, and <NUM>µL of FACS buffer was added, followed by analysis using a flow cytometer (CYTEL® Aurora, Cytek, Fremont, CA, USA).

Claim 1:
A use of a composition for in vitro culturing an isolated natural killer cell without CD3+ or CD56- cells, the composition comprising as an active ingredient a fusion protein dimer comprising an IL-<NUM> variant and a CD80 fragment,
wherein the fusion protein comprises the following structural formula (<NUM>) or (II):

        N'-X-[linker (<NUM>)]n-Fc domain-[linker (<NUM>)]m-Y-C' -     formula (<NUM>)

        N'-Y-[linker (<NUM>)]n-Fc domain-[linker (<NUM>)]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 fragment,
Y is the IL-<NUM> variant,
the linkers (<NUM>) and (<NUM>) are peptide linkers, and
n and m are each independently <NUM> or <NUM>,
wherein the IL-<NUM> variant comprises a substitution of the 38th and <NUM>nd amino acids in the amino acid sequence of SEQ ID NO: <NUM>,
wherein the CD80 fragment comprises the extracellular domain of CD80.