WO2023120844A1 - Composition for mass proliferating natural killer cells, comprising novel peptide, and method for mass proliferating natural killer cells by using same - Google Patents

Abstract

The present invention relates to a novel peptide for mass proliferating natural killer cells, a composition for mass proliferating natural killer cells, comprising same, and a method for mass proliferating natural killer cells by using same. The novel peptide replaces IL-2 so as to be used in the differentiation and mass proliferation of NK cells, and thus can reduce side effects caused by IL-2. In addition, the natural killer cells obtained by the method exhibit high differentiation, activity, and cytotoxicity, and thus can be used as a cell therapeutic agent.

Description

Natural killer cell mass propagation composition containing novel peptide and natural killer cell mass propagation method using the same

It relates to a composition for mass proliferation of natural killer cells and a method for mass proliferation of natural killer cells using the same.

Natural killer cells (NK cells) are important cells responsible for innate immunity. Natural killer cells can recognize cancer cell-specific antigens and immediately eliminate cancer cells without any other activation process, directly suppress proliferation and metastasis of cancer cells, and effectively eliminate cancer stem cells, which are important for cancer recurrence. Therefore, it has many advantages in terms of developing anti-cancer immunotherapeutic agents. Natural killer cells have contact-dependent cytotoxicity and produce interferon gamma (IFN-γ) and tumor-necrosis factor-α (TNF-α), which are cytokines related to immune regulation. It induces an immune response of other immune cells and induces apoptosis by secreting Perforin and Granzyme B (GzmB) to eliminate target cells.

Natural killer cells are present in less than 15% of lymphocytes in the blood of healthy adults. On the other hand, in cancer patients, the number, differentiation, and function of natural killer cells are often significantly reduced, so it is necessary to secure a large amount of natural killer cells through activation and proliferation of natural killer cells. However, it is not easy to induce differentiation of natural killer cells at a high rate by in vitro culture from the patient's peripheral blood, and there are limitations in mass-producing natural killer cells, such as a long culture period.

When peripheral blood mononuclear cells are cultured in vitro by adding IL-2 to a commercially available culture medium, natural killer cells with cytotoxicity capable of killing tumor cells can be obtained. Currently, a commercially available culture medium, RPMI1640 or KBM502 (KOHJIN BIO, JAPAN) is used as a culture medium for culturing natural killer cells. RPMI1640 is a cell culture medium designed for culturing floating lymphocytes, and KBM502 is a cell culture medium designed for culturing NK cells. When immune cells are stimulated with IL-2 and various active stimulating substances in such a commercial medium, natural killer cells in a resting state are also stimulated to differentiate and proliferate into natural killer cells, and mass production is possible. do. IL-2 is the most important cytokine for culturing and activating natural killer cells. It is continuously used at a concentration of 1000 U/ml or more during whole blood treatment and proliferation of natural killer cells, and at a concentration of 1250 U/ml or higher or much higher during activation. becomes

However, IL-2 causes serious side effects such as capillary leak syndrome when excessively introduced into the human body, and the price of IL-2 itself is high.

Therefore, for the induction of differentiation and mass proliferation of natural killer cells, it is necessary to use IL-2 at a minimum dose or to develop a substance that can replace IL-2.

Provides novel peptides for mass proliferation of natural killer cells.

A composition for mass proliferation of natural killer cells comprising a first peptide and a second peptide is provided.

Provided is a method for mass propagation of natural killer cells using the composition for mass propagation of natural killer cells.

Provided are natural killer cells or cell populations obtained by the method for mass propagation of natural killer cells.

Provided is a pharmaceutical composition comprising natural killer cells or cell populations obtained by the method for mass propagation of natural killer cells.

One aspect provides a novel peptide for mass proliferation of natural killer cells.

Throughout this specification, conventional one-letter and three-letter codes for amino acids are used. Amino acids referred to by abbreviations herein have been described according to the IUPAC-IUB nomenclature: Alanine, Ala, A; Arginine, Arg, R; Asparagine, Asn, N; aspartic acid, Asp, D; Cysteine, Cys, C; glutamic acid, Glu, E; Glutamine, Gln, Q; Glycine, Gly, G; Histidine, His, H; Isoleucine, Ile, I; Leucine, Leu, L; Lysine, Lys, K; methionine, Met, M; phenylalanine, Phe, F; Proline, Pro, P; serine, Ser, S; threonine, Thr, T; Tryptophan, Trp, W; Tyrosine, Tyr, Y; Valin, Val, V.

The “peptide” refers to a polymer composed of 50 or less amino acid residues linked by amide bonds (or peptide bonds). Peptides can be classified according to the number of amino acid residues, those with two residues are dipeptides, those with three residues are called tripeptides, those with 2 to 10 residues are called oligopeptides, and those with about 10 to 50 residues. It is called a polypeptide. A peptide according to one aspect may be an oligopeptide or polypeptide consisting of 4 to 25 amino acids. The COOH end of the peptide molecule is called the C-terminus, and the NH ₂ end is called the N-terminus.

The peptide molecule can be synthesized according to a conventional method known in the art. For example, the peptide molecule may be synthesized using genetic recombination and a protein expression system, or may be synthesized in vitro through a peptide synthesizer or the like.

The peptide may be non-naturally occurring. The peptide may be an isolated peptide.

In one embodiment, the peptide may be a first peptide comprising the amino acid sequence of SEQ ID NO: 188.

The peptide may be a first peptide represented by the following general formula I:

[Formula I]

X1-His-Gly-His-Gly-X2

In the above general formula I,

X1 is Gly-Val-Gly-His-Gly (SEQ ID NO: 190), Val-Ser-Gly, Ser-Gly, Pro-Ser-Leu-Thr-Gly (SEQ ID NO: 191), Phe-Ile-Val-Ser -Ala (SEQ ID NO: 192), Thr, Leu-Ala-Ser-Leu (SEQ ID NO: 193), Cys-Val-Val-Thr-Gly (SEQ ID NO: 194), Ile-Ser-Gly, Cys-Gly, Ile- Val-Ala-Arg-Ile (SEQ ID NO: 195), Phe-Gly, His-Gly-Asp-Ser-Gly (SEQ ID NO: 196), Ser-Gly, and Tyr-Ala-Met-Ser-Gly (SEQ ID NO: 197 ) consisting of any one amino acid sequence, or absent,

X2 is any one of His-Gly, Val-His, Tyr-Asp, Phe-Val, Val-Pro, Gln-His-Gly, Leu-Ala, Val-Asp, Pro-Leu, Met, and Phe-Ile It consists of the amino acid sequence of, or is absent.

In Formula I, His-Gly-His-Gly (SEQ ID NO: 188) may be a core sequence of the first peptide. The core sequence may be an essential sequence necessary for the first peptide to be used for mass propagation of natural killer cells. Therefore, the first peptide may be one in which X1 and X2 excluding the core sequence do not exist.

The X1 may consist of a sequence of 1 to 5 amino acids.

The X2 may consist of 1 to 3 amino acid sequences.

The first peptide may include, consist essentially of, or consist of any one amino acid sequence of SEQ ID NOs: 1 to 180. Each of SEQ ID NOs: 1 to 180 may include, consist essentially of, or consist of 5 to 12 amino acid sequences.

In another embodiment, the peptide may be a first peptide comprising the amino acid sequence of SEQ ID NO: 189.

The peptide may be a second peptide represented by the following general formula II:

[Formula II]

X3-Arg-Ala-Arg-Arg-Pro-Gly-Leu-Leu-Gly-X4

In the above general formula II,

X3 is Glu, Ser-Val-Leu-Asn-Tyr-Glu (SEQ ID NO: 198), Tyr-Glu, Gly-Leu-Leu-Gly (SEQ ID NO: 199), and Arg-Pro-Gly-Leu-Glu ( SEQ ID NO: 200) consisting of any one amino acid sequence, or absent,

X4 is Ser-Arg-Leu-Arg-Phe-Ile-Pro-Lys (SEQ ID NO: 201), Ala-Ser-Val-Leu-Asp-Asp-Ile-His-Arg-Ala (SEQ ID NO: 202), Ala , Ala-Ser-Val, and Ala-Ser-Val-Leu (SEQ ID NO: 203), or is absent.

In Formula II, Arg-Ala-Arg-Arg-Pro-Gly-Leu-Leu-Gly (SEQ ID NO: 189) may be a core sequence of the second peptide. The core sequence may be an essential sequence necessary for the second peptide to be used for mass propagation of natural killer cells. Accordingly, the second peptide may be one in which X3 and X4 are absent except for the core sequence.

The X3 may consist of a sequence of 1 to 6 amino acids.

The X4 may each consist of 1 to 10 amino acid sequences.

The second peptide may include, consist essentially of, or consist of any one amino acid sequence of SEQ ID NOs: 181 to 187. Each of SEQ ID NOs: 181 to 187 may include, consist essentially of, or consist of 12 to 25 amino acid sequences.

In another embodiment, the peptide may include a derivative thereof. The derivative may be one in which at least one amino acid in the peptide sequence is modified. The modification may be selected from the group consisting of substitution, addition, deletion, modification, and combinations of two or more thereof. The derivative may be a conservative substitution in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids in the peptide, but is not limited thereto.

"Conservative substitution" means the substitution of one amino acid with another amino acid having similar structural and/or chemical properties. Such amino acid substitutions can generally occur based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; Negatively charged (acidic) amino acids include glutamic acid and aspartic acid; Aromatic amino acids include phenylalanine, tryptophan and tyrosine; Hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan. In addition, amino acids can be classified into amino acids with electrically charged side chains and amino acids with uncharged side chains. Amino acids with charged side chains include aspartic acid, glutamic acid, lysine, arginine, and histidine, and amino acids with non-charged side chains can be further classified as nonpolar amino acids or polar amino acids. Non-polar amino acids are glycine, alanine, valine, leucine, and isoleucine. contains methionine, proline; Polar amino acids may include serine, threonine, cysteine, asparagine, and glutamine. Conservative substitutions with amino acids having similar properties as described above can be expected to exhibit the same or similar activity.

In this specification, even if it is described as a 'peptide consisting of a specific sequence number', if it has the same or corresponding activity as the peptide consisting of the amino acid sequence of the sequence number, the addition of meaningless sequences before and after the amino acid sequence of the sequence number or natural Mutations that may occur in, or silent mutations thereof are not excluded, and it is obvious that even in the case of having such sequence additions or mutations, they fall within the scope of the present invention. That is, even if there is a difference in some sequences, as long as they show sequence identity of a certain level or higher and exhibit natural killer cell differentiation or mass proliferation activity, they may fall within the scope of the present invention. Specifically, the peptide is 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82% of any one amino acid sequence of SEQ ID NOs: 1 to 187 , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or It may include an amino acid sequence having 99% or more identity, but is not limited thereto.

"Homology" or "identity" means the degree to which two given amino acid sequences or base sequences are related to each other and can be expressed as a percentage. Whether any two peptide sequences have homology, similarity or identity can be determined using known computer algorithms such as, for example, the "FASTA" program. Alternatively, homology, similarity or identity can be determined using, for example, BLAST, or ClustalW of the National Center for Biotechnology Information Database.

The peptide may have its N-terminus and/or C-terminus unmodified, but its N-terminus and/or C-terminus may be chemically modified or It can be protected with an organic group or modified by adding an amino acid to the end of a peptide. As the protecting group for the N-terminus and/or C-terminus, any known in the art may be used. When the C-terminus is not modified, the end of the peptide has a free carboxyl group, but is not particularly limited thereto.

In particular, in the case of chemically synthesized peptides, since the N- and C-termini are charged, the N-terminus and/or C-terminus may be modified to remove these charges. For example, the N-terminus may be acetylated (acetylation) and/or the C-terminus may be amidated (amidation), but is not particularly limited thereto.

The peptide includes all forms of the peptide itself, its salt (eg, pharmaceutically acceptable salt of the peptide), or its solvate.

The type of salt is not particularly limited. However, it is preferably in a form that is safe and effective for an individual, such as a mammal, but is not particularly limited thereto.

In addition, the peptide may be in any form that is pharmaceutically acceptable.

The term "pharmaceutically acceptable" means an amount sufficient to exhibit a therapeutic effect and not causing side effects, and includes the type of disease, age, weight, health, sex of the patient, sensitivity to the drug, and route of administration. , It can be easily determined by a person skilled in the art according to factors well known in the medical field, such as an administration method, the number of administrations, a treatment period, combination or drugs used simultaneously.

The peptide may be in the form of a pharmaceutically acceptable salt thereof. The salt may be a conventional acid addition salt used in the pharmaceutical field, for example, cell therapy field, for example, a salt derived from an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid or nitric acid; and acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, citric acid, maleic acid, malonic acid, methanesulfonic acid, tartaric acid, malic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, oxalic acid or and salts derived from organic acids such as trifluoroacetic acid. In addition, the salt may be a base addition salt such as ammonium, dimethylamine, monomethylamine, monoethylamine, or diethylamine. In addition, the salt includes a common metal salt form, for example, a salt derived from a metal such as lithium, sodium, potassium, magnesium, or calcium. The acid addition salt, base addition salt or metal salt may be prepared according to a conventional method. Pharmaceutically acceptable salts and general methodologies for their preparation are well known in the art.

The peptide may be in the form of a solvate thereof. "Solvate" means that the peptide or its salt forms a complex with solvent molecules.

The peptide may be used for culturing natural killer cells or for mass proliferation of natural killer cells.

“Natural Killer cell (NK cell)” is a type of white blood cell in the blood that is in charge of innate immunity, and is an immune cell that directly destroys virus-infected cells or cancer cells.

“Natural killer cell culture” may mean culturing natural killer cells in vitro .

"Cell proliferation" means that cells undergo a series of cell division steps to increase the number of cells in culture or to differentiate cells. "Natural killer cell proliferation" may include both an increase in the number of NK cells in a culture through a series of cell division steps or an increase in the number of NK cells by differentiation from immature blood cells into NK cells.

“Natural Killer Cell Mass Proliferation” may be used interchangeably with “Natural Killer Cell Mass Culture”, and may mean mass proliferation of the number of natural killer cells.

When peripheral blood mononuclear cells are cultured using the peptide according to one aspect, natural killer cells can be massively proliferated, and natural killer cells obtained therefrom can exhibit excellent activity and cytotoxicity.

Another aspect provides a composition for mass proliferation of natural killer cells comprising the peptide according to one aspect.

The composition may include a first peptide, a second peptide, or a combination thereof.

In one embodiment, the composition may include the first peptide.

In another embodiment, the composition may include a second peptide.

In another embodiment, the composition may include a first peptide and a second peptide.

The natural killer cells may be isolated from an individual. The natural killer cells may be isolated from a subject according to a conventional method. The natural killer cells may be isolated from an individual's blood sample. The subject may be a mammal, such as a human. In one embodiment, the natural killer cells may be derived from peripheral blood mononuclear cells.

“Peripheral blood mononuclear cells (PBMCs)” are peripheral blood cells with round nuclei. These cells are composed of lymphocytes and monocytes, including T cells, B cells, and NK cells. The peripheral blood mononuclear cells can be isolated from the blood of an individual according to a conventional method. For example, the PBMC can be isolated from peripheral blood using a specific gravity centrifugation method using Ficoll.

The composition can selectively proliferate natural killer cells among peripheral blood mononuclear cells.

Since the composition can be used for the purpose of culturing natural killer cells for mass proliferation of natural killer cells, it may be "for culturing natural killer cells".

Based on the total composition, the first peptide is about 0.01 to about 1000 mM, about 0.01 to about 500 mM, about 0.01 to about 100 mM, about 0.1 to about 1000 mM, about 0.1 to about 100 mM, about 0.1 to about 0.1 mM About 50 mM, about 0.1 to about 30 mM, about 0.1 to about 20 mM, about 0.1 to about 10 mM, about 1 to about 100 mM, about 1 to about 50 mM, about 1 to about 30 mM, about 1 to about 20 mM, about 1 to about 10 mM, or about 10 mM.

Based on the total composition, the first peptide is about 0.01 to about 100 μg / mL, about 0.1 to about 100 μg / mL, about 0.1 to about 50 μg / mL, about 0.1 to about 30 μg / mL, about 0.1 to about 20 μg/mL, about 0.1 to about 10 μg/mL, or about 1 μg/mL.

Based on the total composition, the second peptide is about 0.01 to about 1000 mM, about 0.01 to about 500 mM, about 0.01 to about 100 mM, about 0.1 to about 1000 mM, about 0.1 to about 100 mM, about 0.1 to about 0.1 mM About 50 mM, about 0.1 to about 30 mM, about 0.1 to about 20 mM, about 0.1 to about 10 mM, about 1 to about 100 mM, about 1 to about 50 mM, about 1 to about 30 mM, about 1 to about 20 mM, about 1 to about 10 mM, or about 10 mM.

Based on the total composition, the first peptide is about 0.01 to about 100 μg / mL, about 0.1 to about 100 μg / mL, about 0.1 to about 50 μg / mL, about 0.1 to about 30 μg / mL, about 0.1 to about 20 μg/mL, about 0.1 to about 10 μg/mL, or about 0.5 μg/mL.

When the composition includes both the first peptide and the second peptide, the first peptide and the second peptide are each from about 0.01 to about 1000 mM, from about 0.01 to about 500 mM, from about 0.01 to about 0.01 mM, based on the total composition. About 100 mM, about 0.1 to about 1000 mM, about 0.1 to about 100 mM, about 0.1 to about 50 mM, about 0.1 to about 30 mM, about 0.1 to about 20 mM, about 0.1 to about 10 mM, about 1 to about 100 mM, about 1 to about 50 mM, about 1 to about 30 mM, about 1 to about 20 mM, about 1 to about 10 mM, or about 10 mM.

If the concentration of the peptide is out of the range, it may be difficult to obtain the effect of mass proliferation of natural killer cells. Specifically, when the concentration of the peptide is out of the above concentration range, mass proliferation may be rather suppressed due to excessive stimulation signals, and the cost of obtaining the same amount of cells may increase due to excessive use of the peptide.

The composition may further include additional components necessary or helpful for the mass proliferation of natural killer cells. An additional component necessary or helpful for the mass proliferation of natural killer cells may be a natural killer cell activity stimulating substance. The natural killer cell activation stimulating material can differentiate and proliferate natural killer cells in a dormant state into natural killer cells. For example, the composition may further include an antibody, serum, medium, or a combination of two or more thereof. The composition may include all of antibodies, serum and media.

Antibodies that may be included in the composition may be antibodies that are helpful for the mass proliferation of natural killer cells. For example, the antibody may include an anti-CD16 antibody, an anti-CD45 antibody, an anti-NKp44 antibody, an anti-NKp46 antibody, or a combination of two or more thereof. In one embodiment, the antibody may be an anti-CD16 antibody, an anti-CD45 antibody, or a combination thereof. In another embodiment, the antibody may be an anti-CD16 antibody and an anti-CD45 antibody. The concentration of the antibody can be appropriately selected by those skilled in the art within a range showing the effect of mass proliferation of natural killer cells. For example, the concentration of the antibody may be selected in the range of 0.1 to 1.0 ug/ml, but is not limited thereto.

The "serum" refers to the remainder after fibrinogen (fibrinogen) is subtracted from plasma. Serum that may be included in the composition may be of autologous origin, allogeneic origin, or xenogeneic origin. “Autologous” means derived from the same individual. “Allogeneic” means derived from the same species but genetically different individuals. “xenogenic” means derived from another species. The heterologous serum may include, for example, fetal bovine serum (FBS), but is not limited thereto. In one embodiment, the serum may be autologous serum, that is, autologous serum. The serum may be serum separated from peripheral blood from which peripheral blood mononuclear cells are derived. The content of the serum can be appropriately selected by those skilled in the art within a range conducive to the mass proliferation of natural killer cells. For example, the serum content may be selected in the range of 5 to 30% (v/v), but is not limited thereto.

The composition may contain plasma instead of serum. The "plasma" refers to liquid components other than blood cells in blood. The plasma may be plasma separated from peripheral blood from which peripheral blood mononuclear cells are derived.

The “medium” may be a medium for culturing natural killer cells or a medium for mass-producing natural killer cells. As the medium, any commercially available medium for culturing natural killer cells, a known medium, or a medium prepared to contain components necessary for culturing natural killer cells may be used. Commercially available media such as KBM502 medium and RPMI1640 medium may be used as the medium.

The medium may contain nutrients, pH adjusting agents, or a combination thereof. The nutritional component may include an amino acid, a vitamin source, a mineral source, and the like. The medium may include, for example, amino acids, vitamins, inorganic compounds, proteins, or a combination of two or more thereof.

Amino acids that may be included in the medium include L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, and L-lysine. , L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and salts or derivatives thereof, but may be at least one selected from the group consisting of, Not limited.

The vitamin that may be included in the medium is at least one selected from the group consisting of para-aminobenzoic acid, D-biotin, choline chloride, cyanocobalamin, folic acid, myoinositol, niacinamide, D-pentothenic acid, pyridoxine HCl, riboflavin, and thiamine HCl It may, but is not limited thereto.

The inorganic compound that may be included in the medium may be at least one selected from the group consisting of Ca(NO ₃ ) ₂ 4H ₂ O, MgSO ₄ , KCl, NaCl, NaHCO ₃ and Na ₂ HPO ₄ , but is not limited thereto.

The protein that may be included in the medium may be albumin, insulin, transferrin, interleukin-2, or a combination of two or more thereof, specifically, daily albumin, recombinant human insulin, human transferrin, recombinant human IL-2 , Or may be a combination of two or more thereof, but is not limited thereto.

The medium may further include glutathione, phenol red, or a combination thereof.

The composition may further include cytokines. The cytokines are IL-2, IL-12, IL-15, IL-18, IL-21, SCF (stem cell factor), FL (fit ligand), and GM-CSF (granulocyte/macrophage colony-stimulation factor). It may be one or more selected from the group consisting of, but is not limited thereto. The composition may contain cytokines at a concentration of 1000 U/ml or less. The composition may contain trace amounts of cytokines. The "trace amount" may mean a small amount that does not cause side effects to the human body.

The composition may not contain cytokines. For example, the composition may not contain IL-2. Since the composition does not contain IL-2, side effects such as capillary leak syndrome due to IL-2 may not be caused. Even if the composition does not contain IL-2, mass proliferation of natural killer cells is possible. Therefore, the peptide according to one aspect included in the composition can be used as a substitute for IL-2.

The composition can mass-proliferate natural killer cells from PBMCs by including the peptide according to one aspect. In addition, natural killer cells that have been massively proliferated using the composition. Compared to natural killer cells proliferated using IL-2. It has excellent differentiation, activity and cytotoxicity to cancer cells. In particular, when a composition containing both the first peptide and the second peptide is used, a synergistic effect can be obtained in the degree of differentiation, activity, and cytotoxicity of natural killer cells.

Another aspect provides a method for mass proliferation of natural killer cells, comprising culturing isolated peripheral blood mononuclear cells (PBMC) in the composition for mass proliferation of natural killer cells according to one aspect.

The culturing may be performed according to a conventional culture method for mass-proliferating natural killer cells from peripheral blood mononuclear cells.

The method may further include isolating peripheral blood mononuclear cells from blood prior to culturing. As the separation method, a conventional method may be used. The blood may be the blood of a mammal, for example, a human. The peripheral blood mononuclear cells may be autologous peripheral blood mononuclear cells. When the peripheral blood mononuclear cells are autologous peripheral blood mononuclear cells, even if some T cells are present in the proliferated NK cell population, there is an advantage in that T cells do not need to be removed because all cells are derived from the patient himself.

The isolated peripheral blood mononuclear cells may be used immediately after isolation, or may be used in a thawed state after cryopreservation.

In one embodiment, the culturing step,

first culturing the isolated peripheral blood mononuclear cells in a first composition for mass proliferation of natural killer cells in a first culture vessel;

secondly culturing the cells and culture medium obtained from the first culture in a second culture vessel in a composition for mass propagation of second natural killer cells;

Thirdly culturing the cells and the culture medium obtained from the second culture in a third culture vessel in a third composition for mass propagation of natural killer cells; and

A fourth culturing of the cells and culture medium obtained from the third culture in a fourth culture vessel may be included.

The first culture vessel, the second culture vessel, the third culture vessel, and the fourth culture vessel may include commercially available dishes, flasks, plates, multi-well plates, culture bags, and the like.

The culture area of the second culture vessel is greater than that of the first culture vessel, the culture area of the third culture vessel is greater than that of the second culture vessel, and the culture area of the fourth culture vessel is It may be larger than the culture area of the third culture vessel. For example, when the culture vessel is a flask, the first culture vessel may be a 25T flask, the second culture vessel may be a 75T flask, the third culture vessel may be a 175T flask, and the fourth culture vessel may be a cell culture bag. , but not limited thereto.

In order to maximally proliferate NK cells, each culture step may have the same or different culture solution components, culture container, and culture period, and finally, NK cell proliferation can be achieved by finding the optimal culture period for each step. For example, during the culture period, NK cell culture can be effectively achieved by adding and culturing the composition (or culture medium) for mass proliferation of NK cells every 2 to 3 days in the flask.

Components and concentrations of the first composition for mass propagation of natural killer cells, the composition for mass propagation of second natural killer cells, and the composition for mass propagation of third natural killer cells may be different from each other. The first composition for mass proliferation of natural killer cells, the composition for mass proliferation of second natural killer cells, and the composition for mass proliferation of third natural killer cells may be peptides according to one aspect (e.g., the first peptide, the second peptide, or any of them). combinations) may be included.

In the fourth culturing step, a cell suspension prepared by adding serum or plasma to the cells obtained from the third culture and the culture medium may be cultured. The serum or plasma may be of autologous origin, allogeneic origin, or heterologous origin. For example, the serum or plasma may be of autologous origin. The serum or plasma content is about 5 to about 30% (v/v), about 5 to about 20% (v/v), about 10 to about 30% (v/v), or about 10 to about 20% It can be selected from the range of (v / v), but is not limited thereto.

The first culture, the second culture, the third culture, and the fourth culture may be suspension culture. The term "suspension culture" refers to a culture method in which cells are suspended in a culture medium.

The culture time of the first culture, the second culture, and the third culture may be a sufficient time to increase the number of cells. For example, the first culture, the second culture, and the third culture may be performed for 2 to 3 days, respectively, but are not limited thereto, and within the range of the number of culture days to obtain the desired number of NK cells. Alternatively, it can be set appropriately outside the range.

As the culture time of the fourth culture, a time sufficient to increase the number of cells may be selected. For example, the fourth culture is about 3 days to about 14 days, about 3 days to about 10 days, about 3 days to about 8 days, about 3 days to about 7 days, about 4 days to about 14 days, about It may be performed for 4 to about 10 days, about 4 to about 8 days, or about 4 to about 7 days, but is not limited thereto, within the range of the number of culture days to obtain the desired number of NK cells, or It can be set appropriately outside the range.

Other culture conditions (eg, culture temperature, culture humidity, etc.) of the first culture, second culture, third culture, and fourth culture can be appropriately selected by those skilled in the art for mass proliferation of natural killer cells.

The method may further include recovering and/or washing natural killer cells after the fourth culturing step. As the method for recovering or washing the natural killer cells, a conventional method for recovering or washing cells may be used.

According to the above method, natural killer cells can be obtained at a high rate. In one embodiment, as a result of culturing PBMCs using the peptide according to one aspect, it was confirmed that the ratio of natural killer cells was 70% or more, specifically, 75% or more. In particular, when both the first peptide and the second peptide were used, it was confirmed that the ratio of natural killer cells was as high as 80% or more.

Another aspect provides natural killer cells or cell populations obtained by the method for mass propagation of natural killer cells according to one aspect.

Natural killer cells obtained by the method according to the above aspect may have a high amount of IFN-4 secretion. In one embodiment, it was confirmed that natural killer cells obtained using the peptide according to one aspect exhibit an IFN-r secretion amount of about 3 pg/mL. This is a higher amount than natural killer cells obtained using IL-2. Therefore, the natural killer cells obtained by the method according to one aspect have excellent activity, and may have increased differentiation ability and high cytotoxicity.

The natural killer cells may have surface markers of CD3-, CD16+CD56+.

Another aspect provides a pharmaceutical composition comprising, as an active ingredient, natural killer cells or cell populations obtained by the method for mass propagation of natural killer cells according to one aspect. The pharmaceutical composition may be a cell therapy agent, for example, an NK cell therapy agent.

“Cell therapy” is a treatment in which living cells are directly injected into a patient.

“Cell therapeutics” refers to living cells used for cell therapy, and drugs manufactured by physically, chemically, or biologically manipulating living autologous, allogeneic, or xenogeneic cells in vitro, such as culturing, proliferating, or selecting them. means

The pharmaceutical composition may be for preventing or treating a disease that can be prevented or treated by administration of natural killer cells.

The term "prevention" refers to any action that suppresses or delays the onset of a disease by administering the composition. The term "treatment" refers to any action that improves or benefits the symptoms of a disease by administration of the composition.

Diseases that can be prevented or treated by administration of the natural killer cells may include cancer, immune-related diseases, and the like, but are not limited thereto.

The pharmaceutical composition may further include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments and flavors for oral administration, and buffers, preservatives, and painless agents for injections. A topical agent, a solubilizing agent, an isotonic agent, and a stabilizer may be mixed and used, and in the case of topical administration, a base, an excipient, a lubricant, and a preservative may be used.

In one embodiment, the pharmaceutical composition may further include a pharmaceutically acceptable excipient.

Formulations of the pharmaceutical composition may be variously prepared by mixing with a pharmaceutically acceptable carrier as described above. For example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be prepared in the form of unit dose ampoules or multiple doses. In addition, it can be formulated into solutions, suspensions, tablets, pills, capsules, and sustained-release preparations.

On the other hand, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifiers and preservatives may be further included.

The pharmaceutical composition may further include one or more other agents for treating diseases that can be prevented or treated by administration of natural killer cells. Specifically, the other agent may be an anticancer agent or an immune enhancer, but is not limited thereto.

The dosage and frequency of the pharmaceutical composition are determined according to the type of drug as an active ingredient, together with various related factors such as the disease to be treated, the route of administration, the patient's age, sex and weight, and the severity of the disease.

Another aspect provides a method for preventing or treating a disease that can be prevented or treated by administration of natural killer cells, comprising administering an effective amount of natural killer cells according to one aspect to a subject in need thereof.

"Effective amount" or "pharmaceutically effective amount" refers to an amount or dose of an active ingredient that, when administered to a patient in single or multiple doses, provides a desired effect in a patient under diagnosis or treatment. The effective amount can be readily determined by the attending diagnostician as a person skilled in the art by using known techniques or by observing results obtained under similar circumstances. When determining an effective amount for a patient, the mammalian species; his size, age and general health; the specific disease or disorder involved; degree or severity of involvement of the disease or disorder; individual patient response; the specific compound being administered; administration mode; bioavailability characteristics of the administered agent; the selected dosing regimen; use of concomitant medication; A number of factors are taken into account by the attending physician diagnostician, including but not limited to, and other relevant circumstances.

"Individual" means a subject in need of treatment for a disease, and more specifically, means a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow. .

“Administering” means introducing a substance into a patient by any suitable method. The route of administration may be any general route capable of reaching a target in vivo in a patient. The administration may be, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, or intrarectal administration, but is not limited thereto.

The dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, medical condition, food, administration time, administration route, excretion rate and reaction sensitivity, and those skilled in the art can determine these factors Dosage can be appropriately adjusted in consideration of these factors. The number of administrations can be once a day or two or more times within the range of clinically acceptable side effects, and administration can be performed at one or two or more sites, daily or at intervals of 2 to 5 days. The number of administration days may be administered from 1 day to 30 days per treatment. If necessary, the same treatment can be repeated after a titration period. For non-human animals, the same dosage per kg as for humans is used, or the above dosage is converted by the volume ratio (eg, average value) of the organ (heart, etc.) between the target animal and the human. A single dose can be administered.

In the method, an effective amount of natural killer cells can be administered simultaneously, separately, or sequentially with an effective amount of one or more other active ingredients.

Another aspect provides a cell therapy agent containing the peptide and natural killer cells according to one aspect.

The natural killer cells may be self-derived natural killer cells cultured in vitro. The natural killer cells may be obtained by a natural killer cell mass propagation method according to an aspect.

Since the peptide has an activity-stimulating effect on natural killer cells, the effect of a cell therapy agent can be maximized when co-administered to a patient together with self-derived in vitro cultured natural killer cells.

The cell therapy product may further include additional components commonly used in cell therapy products. For example, the cell therapy product may further contain components necessary for injecting cells into a patient.

Each description and embodiment disclosed herein may also apply to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be said that the scope of the present invention is limited by the specific description described below.

The peptide according to one aspect can be used for mass proliferation of natural killer cells.

Since the composition for mass proliferation of natural killer cells according to one aspect can proliferate natural killer cells in large quantities by including a novel peptide, the peptide can replace IL-2, which can cause side effects in the human body.

In the natural killer cell mass propagation method according to one aspect, natural killer cells can be massively propagated by using a novel peptide. Since the natural killer cells obtained by the above method exhibit high differentiation, activity, and cytotoxicity, they can be used as cell therapy agents.

1 is a FACS analysis result showing the NK cell ratio of PBMC before culture.

2 is a FACS analysis result showing the NK cell ratio of the IL-2 treated group (positive control group).

3 is a FACS analysis result showing the NK cell ratio of the first peptide treatment group.

Figure 4 is a FACS analysis result showing the NK cell ratio of the first peptide and the second peptide co-treatment group.

5 is a result showing the NK cell ratio (%) after combination treatment of the first peptide and the second peptide.

6 is a graph showing the amount of IFN-4 secretion (pg/mL) on days 1, 3, and 5 of cell culture. Control: IL-2 treatment group, Peptide: first peptide (SEQ ID NO: 27) treatment group.

7 is a result of calculating cytotoxicity (%) of cells cultured in Example 3 to cancer cells. Pep.1: first peptide (SEQ ID NO: 27) treatment group, Pep.2: second peptide (SEQ ID NO: 217) treatment group, Pep1 + 2: first peptide (SEQ ID NO: 27) and second peptide (SEQ ID NO: 217 ) co-treatment group.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrating the present invention by way of example, and the scope of the present invention is not limited to these examples.

Example 1: Synthesis of Peptides

(1.1) Synthesis of first peptide

A first peptide represented by X1-core sequence-X2 was synthesized.

Table 1 below is a description of X1, core sequence, and X2 of the first peptide.

Table 2 below shows 180 first peptides.

X1:
Any one selected from the following 15 sequences core sequence X2:
Any one selected from the following 11 sequences, or absent Gly-Val-Gly-His-Gly (SEQ ID NO: 190)
His-Gly-His-Gly (SEQ ID NO: 188) His-Gly
Val-Ser-Gly Val-His Ser-Gly Tyr-Asp Pro-Ser-Leu-Thr-Gly (SEQ ID NO: 191) Phe-Val Phe-Ile-Val-Ser-Ala (SEQ ID NO: 192) Val-Pro Thr Gln-His-Gly Leu-Ala-Ser-Leu (SEQ ID NO: 193) Leu-Ala Cys-Val-Val-Thr-Gly (SEQ ID NO: 194) Val-Asp Ile-Ser-Gly Pro-Leu Cys-Gly Met Ile-Val-Ala-Arg-Ile (SEQ ID NO: 195) Phe-Ile Phe-Gly His-Gly-Asp-Ser-Gly (SEQ ID NO: 196) Ser-Gly Tyr-Ala-Met-Ser-Gly (SEQ ID NO: 197)

Peptide No. X1 core sequence X2 sequence number peptide 1 Gly-Val-Gly-His-Gly- His-Gly-His-Gly His-Gly One peptide 2 Val-Ser-Gly- His-Gly-His-Gly His-Gly 2 peptide 3 Ser-Gly- His-Gly-His-Gly His-Gly 3 peptide 4 Pro-Ser-Leu-Thr-Gly- His-Gly-His-Gly His-Gly 4 peptide 5 Phe-Ile-Val-Ser-Ala- His-Gly-His-Gly His-Gly 5 peptide 6 Thr- His-Gly-His-Gly His-Gly 6 peptide 7 Leu-Ala-Ser-Leu- His-Gly-His-Gly His-Gly 7 peptide 8 Cys-Val-Val-Thr-Gly- His-Gly-His-Gly His-Gly 8 peptide 9 Ile-Ser-Gly- His-Gly-His-Gly His-Gly 9 peptide 10 Cys-Gly- His-Gly-His-Gly His-Gly 10 peptide 11 Ile-Val-Ala-Arg-Ile- His-Gly-His-Gly His-Gly 11 peptide 12 Phe-Gly- His-Gly-His-Gly His-Gly 12 peptide 13 His-Gly-Asp-Ser-Gly- His-Gly-His-Gly His-Gly 13 peptide 14 Ser-Gly- His-Gly-His-Gly His-Gly 14 peptide 15 Tyr-Ala-Met-Ser-Gly- His-Gly-His-Gly His-Gly 15 peptide 16 Gly-Val-Gly-His-Gly- His-Gly-His-Gly Val-His 16 peptide 17 Val-Ser-Gly- His-Gly-His-Gly Val-His 17 peptide 18 Ser-Gly- His-Gly-His-Gly Val-His 18 peptide 19 Pro-Ser-Leu-Thr-Gly- His-Gly-His-Gly Val-His 19 peptide 20 Phe-Ile-Val-Ser-Ala- His-Gly-His-Gly Val-His 20 peptide 21 Thr- His-Gly-His-Gly Val-His 21 peptide 22 Leu-Ala-Ser-Leu- His-Gly-His-Gly Val-His 22 peptide 23 Cys-Val-Val-Thr-Gly- His-Gly-His-Gly Val-His 23 peptide 24 Ile-Ser-Gly- His-Gly-His-Gly Val-His 24 peptide 25 Cys-Gly- His-Gly-His-Gly Val-His 25 peptide 26 Ile-Val-Ala-Arg-Ile- His-Gly-His-Gly Val-His 26 peptide 27 Phe-Gly- His-Gly-His-Gly Val-His 27 peptide 28 His-Gly-Asp-Ser-Gly- His-Gly-His-Gly Val-His 28 peptide 29 Ser-Gly- His-Gly-His-Gly Val-His 29 Peptide 30 Tyr-Ala-Met-Ser-Gly- His-Gly-His-Gly Val-His 30 peptide 31 Gly-Val-Gly-His-Gly- His-Gly-His-Gly Tyr-Asp 31 peptide 32 Val-Ser-Gly- His-Gly-His-Gly Tyr-Asp 32 peptide 33 Ser-Gly- His-Gly-His-Gly Tyr-Asp 33 Peptide 34 Pro-Ser-Leu-Thr-Gly- His-Gly-His-Gly Tyr-Asp 34 peptide 35 Phe-Ile-Val-Ser-Ala- His-Gly-His-Gly Tyr-Asp 35 Peptide 36 Thr- His-Gly-His-Gly Tyr-Asp 36 peptide 37 Leu-Ala-Ser-Leu- His-Gly-His-Gly Tyr-Asp 37 peptide 38 Cys-Val-Val-Thr-Gly- His-Gly-His-Gly Tyr-Asp 38 peptide 39 Ile-Ser-Gly- His-Gly-His-Gly Tyr-Asp 39 peptide 40 Cys-Gly- His-Gly-His-Gly Tyr-Asp 40 peptide 41 Ile-Val-Ala-Arg-Ile- His-Gly-His-Gly Tyr-Asp 41 peptide 42 Phe-Gly- His-Gly-His-Gly Tyr-Asp 42 peptide 43 His-Gly-Asp-Ser-Gly- His-Gly-His-Gly Tyr-Asp 43 peptide 44 Ser-Gly- His-Gly-His-Gly Tyr-Asp 44 peptide 45 Tyr-Ala-Met-Ser-Gly- His-Gly-His-Gly Tyr-Asp 45 peptide 46 Gly-Val-Gly-His-Gly- His-Gly-His-Gly Phe-Val 46 peptide 47 Val-Ser-Gly- His-Gly-His-Gly Phe-Val 47 peptide 48 Ser-Gly- His-Gly-His-Gly Phe-Val 48 peptide 49 Pro-Ser-Leu-Thr-Gly- His-Gly-His-Gly Phe-Val 49 peptide 50 Phe-Ile-Val-Ser-Ala- His-Gly-His-Gly Phe-Val 50 peptide 51 Thr- His-Gly-His-Gly Phe-Val 51 peptide 52 Leu-Ala-Ser-Leu- His-Gly-His-Gly Phe-Val 52 peptide 53 Cys-Val-Val-Thr-Gly- His-Gly-His-Gly Phe-Val 53 peptide 54 Ile-Ser-Gly- His-Gly-His-Gly Phe-Val 54 peptide 55 Cys-Gly- His-Gly-His-Gly Phe-Val 55 peptide 56 Ile-Val-Ala-Arg-Ile- His-Gly-His-Gly Phe-Val 56 peptide 57 Phe-Gly- His-Gly-His-Gly Phe-Val 57 peptide 58 His-Gly-Asp-Ser-Gly- His-Gly-His-Gly Phe-Val 58 peptide 59 Ser-Gly- His-Gly-His-Gly Phe-Val 59 peptide 60 Tyr-Ala-Met-Ser-Gly- His-Gly-His-Gly Phe-Val 60 peptide 61 Gly-Val-Gly-His-Gly- His-Gly-His-Gly Val-Pro 61 peptide 62 Val-Ser-Gly- His-Gly-His-Gly Val-Pro 62 peptide 63 Ser-Gly- His-Gly-His-Gly Val-Pro 63 peptide 64 Pro-Ser-Leu-Thr-Gly- His-Gly-His-Gly Val-Pro 64 peptide 65 Phe-Ile-Val-Ser-Ala- His-Gly-His-Gly Val-Pro 65 peptide 66 Thr- His-Gly-His-Gly Val-Pro 66 peptide 67 Leu-Ala-Ser-Leu- His-Gly-His-Gly Val-Pro 67 peptide 68 Cys-Val-Val-Thr-Gly- His-Gly-His-Gly Val-Pro 68 peptide 69 Ile-Ser-Gly- His-Gly-His-Gly Val-Pro 69 peptide 70 Cys-Gly- His-Gly-His-Gly Val-Pro 70 peptide 71 Ile-Val-Ala-Arg-Ile- His-Gly-His-Gly Val-Pro 71 peptide 72 Phe-Gly- His-Gly-His-Gly Val-Pro 72 peptide 73 His-Gly-Asp-Ser-Gly- His-Gly-His-Gly Val-Pro 73 peptide 74 Ser-Gly- His-Gly-His-Gly Val-Pro 74 peptide 75 Tyr-Ala-Met-Ser-Gly- His-Gly-His-Gly Val-Pro 75 peptide 76 Gly-Val-Gly-His-Gly- His-Gly-His-Gly Gln-His-Gly 76 peptide 77 Val-Ser-Gly- His-Gly-His-Gly Gln-His-Gly 77 peptide 78 Ser-Gly- His-Gly-His-Gly Gln-His-Gly 78 peptide 79 Pro-Ser-Leu-Thr-Gly- His-Gly-His-Gly Gln-His-Gly 79 peptide 80 Phe-Ile-Val-Ser-Ala- His-Gly-His-Gly Gln-His-Gly 80 peptide 81 Thr- His-Gly-His-Gly Gln-His-Gly 81 peptide 82 Leu-Ala-Ser-Leu- His-Gly-His-Gly Gln-His-Gly 82 peptide 83 Cys-Val-Val-Thr-Gly- His-Gly-His-Gly Gln-His-Gly 83 peptide 84 Ile-Ser-Gly- His-Gly-His-Gly Gln-His-Gly 84 peptide 85 Cys-Gly- His-Gly-His-Gly Gln-His-Gly 85 peptide 86 Ile-Val-Ala-Arg-Ile- His-Gly-His-Gly Gln-His-Gly 86 peptide 87 Phe-Gly- His-Gly-His-Gly Gln-His-Gly 87 peptide 88 His-Gly-Asp-Ser-Gly- His-Gly-His-Gly Gln-His-Gly 88 peptide 89 Ser-Gly- His-Gly-His-Gly Gln-His-Gly 89 peptide 90 Tyr-Ala-Met-Ser-Gly- His-Gly-His-Gly Gln-His-Gly 90 peptide 91 Gly-Val-Gly-His-Gly- His-Gly-His-Gly Leu-Ala 91 peptide 92 Val-Ser-Gly- His-Gly-His-Gly Leu-Ala 92 peptide 93 Ser-Gly- His-Gly-His-Gly Leu-Ala 93 peptide 94 Pro-Ser-Leu-Thr-Gly- His-Gly-His-Gly Leu-Ala 94 peptide 95 Phe-Ile-Val-Ser-Ala- His-Gly-His-Gly Leu-Ala 95 peptide 96 Thr- His-Gly-His-Gly Leu-Ala 96 peptide 97 Leu-Ala-Ser-Leu- His-Gly-His-Gly Leu-Ala 97 peptide 98 Cys-Val-Val-Thr-Gly- His-Gly-His-Gly Leu-Ala 98 peptide 99 Ile-Ser-Gly- His-Gly-His-Gly Leu-Ala 99 Peptide 100 Cys-Gly- His-Gly-His-Gly Leu-Ala 100 peptide 101 Ile-Val-Ala-Arg-Ile- His-Gly-His-Gly Leu-Ala 101 peptide 102 Phe-Gly- His-Gly-His-Gly Leu-Ala 102 peptide 103 His-Gly-Asp-Ser-Gly- His-Gly-His-Gly Leu-Ala 103 peptide 104 Ser-Gly- His-Gly-His-Gly Leu-Ala 104 peptide 105 Tyr-Ala-Met-Ser-Gly- His-Gly-His-Gly Leu-Ala 105 peptide 106 Gly-Val-Gly-His-Gly- His-Gly-His-Gly Val-Asp 106 peptide 107 Val-Ser-Gly- His-Gly-His-Gly Val-Asp 107 peptide 108 Ser-Gly- His-Gly-His-Gly Val-Asp 108 peptide 109 Pro-Ser-Leu-Thr-Gly- His-Gly-His-Gly Val-Asp 109 peptide 110 Phe-Ile-Val-Ser-Ala- His-Gly-His-Gly Val-Asp 110 peptide 111 Thr- His-Gly-His-Gly Val-Asp 111 peptide 112 Leu-Ala-Ser-Leu- His-Gly-His-Gly Val-Asp 112 peptide 113 Cys-Val-Val-Thr-Gly- His-Gly-His-Gly Val-Asp 113 peptide 114 Ile-Ser-Gly- His-Gly-His-Gly Val-Asp 114 peptide 115 Cys-Gly- His-Gly-His-Gly Val-Asp 115 peptide 116 Ile-Val-Ala-Arg-Ile- His-Gly-His-Gly Val-Asp 116 peptide 117 Phe-Gly- His-Gly-His-Gly Val-Asp 117 peptide 118 His-Gly-Asp-Ser-Gly- His-Gly-His-Gly Val-Asp 118 peptide 119 Ser-Gly- His-Gly-His-Gly Val-Asp 119 Peptide 120 Tyr-Ala-Met-Ser-Gly- His-Gly-His-Gly Val-Asp 120 peptide 121 Gly-Val-Gly-His-Gly- His-Gly-His-Gly Pro-Leu 121 peptide 122 Val-Ser-Gly- His-Gly-His-Gly Pro-Leu 122 peptide 123 Ser-Gly- His-Gly-His-Gly Pro-Leu 123 peptide 124 Pro-Ser-Leu-Thr-Gly- His-Gly-His-Gly Pro-Leu 124 peptide 125 Phe-Ile-Val-Ser-Ala- His-Gly-His-Gly Pro-Leu 125 peptide 126 Thr- His-Gly-His-Gly Pro-Leu 126 peptide 127 Leu-Ala-Ser-Leu- His-Gly-His-Gly Pro-Leu 127 peptide 128 Cys-Val-Val-Thr-Gly- His-Gly-His-Gly Pro-Leu 128 peptide 129 Ile-Ser-Gly- His-Gly-His-Gly Pro-Leu 129 Peptide 130 Cys-Gly- His-Gly-His-Gly Pro-Leu 130 peptide 131 Ile-Val-Ala-Arg-Ile- His-Gly-His-Gly Pro-Leu 131 peptide 132 Phe-Gly- His-Gly-His-Gly Pro-Leu 132 peptide 133 His-Gly-Asp-Ser-Gly- His-Gly-His-Gly Pro-Leu 133 peptide 134 Ser-Gly- His-Gly-His-Gly Pro-Leu 134 peptide 135 Tyr-Ala-Met-Ser-Gly- His-Gly-His-Gly Pro-Leu 135 peptide 136 Gly-Val-Gly-His-Gly- His-Gly-His-Gly Met 136 peptide 137 Val-Ser-Gly- His-Gly-His-Gly Met 137 peptide 138 Ser-Gly- His-Gly-His-Gly Met 138 peptide 139 Pro-Ser-Leu-Thr-Gly- His-Gly-His-Gly Met 139 Peptide 140 Phe-Ile-Val-Ser-Ala- His-Gly-His-Gly Met 140 peptide 141 Thr- His-Gly-His-Gly Met 141 peptide 142 Leu-Ala-Ser-Leu- His-Gly-His-Gly Met 142 peptide 143 Cys-Val-Val-Thr-Gly- His-Gly-His-Gly Met 143 peptide 144 Ile-Ser-Gly- His-Gly-His-Gly Met 144 peptide 145 Cys-Gly- His-Gly-His-Gly Met 145 peptide 146 Ile-Val-Ala-Arg-Ile- His-Gly-His-Gly Met 146 peptide 147 Phe-Gly- His-Gly-His-Gly Met 147 peptide 148 His-Gly-Asp-Ser-Gly- His-Gly-His-Gly Met 148 peptide 149 Ser-Gly- His-Gly-His-Gly Met 149 Peptide 150 Tyr-Ala-Met-Ser-Gly- His-Gly-His-Gly Met 150 peptide 151 Gly-Val-Gly-His-Gly- His-Gly-His-Gly Phe-Ile 151 peptide 152 Val-Ser-Gly- His-Gly-His-Gly Phe-Ile 152 peptide 153 Ser-Gly- His-Gly-His-Gly Phe-Ile 153 peptide 154 Pro-Ser-Leu-Thr-Gly- His-Gly-His-Gly Phe-Ile 154 peptide 155 Phe-Ile-Val-Ser-Ala- His-Gly-His-Gly Phe-Ile 155 peptide 156 Thr- His-Gly-His-Gly Phe-Ile 156 peptide 157 Leu-Ala-Ser-Leu- His-Gly-His-Gly Phe-Ile 157 peptide 158 Cys-Val-Val-Thr-Gly- His-Gly-His-Gly Phe-Ile 158 peptide 159 Ile-Ser-Gly- His-Gly-His-Gly Phe-Ile 159 Peptide 160 Cys-Gly- His-Gly-His-Gly Phe-Ile 160 peptide 161 Ile-Val-Ala-Arg-Ile- His-Gly-His-Gly Phe-Ile 161 peptide 162 Phe-Gly- His-Gly-His-Gly Phe-Ile 162 peptide 163 His-Gly-Asp-Ser-Gly- His-Gly-His-Gly Phe-Ile 163 peptide 164 Ser-Gly- His-Gly-His-Gly Phe-Ile 164 peptide 165 Tyr-Ala-Met-Ser-Gly- His-Gly-His-Gly Phe-Ile 165 peptide 166 Gly-Val-Gly-His-Gly- His-Gly-His-Gly 166 peptide 167 Val-Ser-Gly- His-Gly-His-Gly 167 peptide 168 Ser-Gly- His-Gly-His-Gly 168 peptide 169 Pro-Ser-Leu-Thr-Gly- His-Gly-His-Gly 169 Peptide 170 Phe-Ile-Val-Ser-Ala- His-Gly-His-Gly 170 peptide 171 Thr- His-Gly-His-Gly 171 peptide 172 Leu-Ala-Ser-Leu- His-Gly-His-Gly 172 peptide 173 Cys-Val-Val-Thr-Gly- His-Gly-His-Gly 173 peptide 174 Ile-Ser-Gly- His-Gly-His-Gly 174 peptide 175 Cys-Gly- His-Gly-His-Gly 175 peptide 176 Ile-Val-Ala-Arg-Ile- His-Gly-His-Gly 176 peptide 177 Phe-Gly- His-Gly-His-Gly 177 peptide 178 His-Gly-Asp-Ser-Gly- His-Gly-His-Gly 178 peptide 179 Ser-Gly- His-Gly-His-Gly 179 Peptide 180 Tyr-Ala-Met-Ser-Gly- His-Gly-His-Gly 180

(1.2) Synthesis of second peptide A second peptide represented by X3-core sequence-X4 was synthesized.

Table 3 below is a description of X3, core sequence, and X4 of the second peptide.

Table 4 below shows seven kinds of the second peptide.

X3:
Any one selected from the following 5 sequences, or absent core sequence X4:
Any one selected from the following 5 sequences, or absent Glu Arg-Ala-Arg-Arg-Pro-Gly-Leu-Leu-Gly (SEQ ID NO: 189) Ser-Arg-Leu-Arg-Phe-Ile-Pro-Lys (SEQ ID NO: 201)
Ser-Val-Leu-Asn-Tyr-Glu (SEQ ID NO: 198) Ala-Ser-Val-Leu-Asp-Asp-Ile-His-Arg-Ala (SEQ ID NO: 202) Tyr-Glu Ala Gly-Leu-Leu-Gly (SEQ ID NO: 199) Ala-Ser-Val Arg-Pro-Gly-Leu-Glu (SEQ ID NO: 200) Ala-Ser-Val-Leu (SEQ ID NO: 203)

Peptide No. X3 core sequence X4 sequence number peptide 181 Glu Arg-Ala-Arg-Arg-Pro-Gly-Leu-Leu-Gly Ser-Arg-Leu-Arg-Phe-Ile-Pro-Lys 181 peptide 182 Ser-Val-Leu-Asn-Tyr-Glu Arg-Ala-Arg-Arg-Pro-Gly-Leu-Leu-Gly 182 peptide 183 Ser-Val-Leu-Asn-Tyr-Glu Arg-Ala-Arg-Arg-Pro-Gly-Leu-Leu-Gly Ala-Ser-Val-Leu-Asp-Asp-Ile-His-Arg-Ala 183 peptide 184 Tyr-Glu Arg-Ala-Arg-Arg-Pro-Gly-Leu-Leu-Gly Ala 184 peptide 185 Arg-Ala-Arg-Arg-Pro-Gly-Leu-Leu-Gly Ala-Ser-Val 185 peptide 186 Gly-Leu-Leu-Gly Arg-Ala-Arg-Arg-Pro-Gly-Leu-Leu-Gly Ala 186 peptide 187 Arg-Pro-Gly-Leu-Glu Arg-Ala-Arg-Arg-Pro-Gly-Leu-Leu-Gly Ala-Ser-Val-Leu 187

Example 2: Isolation of blood and peripheral blood mononuclear cells (PBMC) Blood was collected according to the following steps, and peripheral blood mononuclear cells were isolated therefrom.

1. A total of 60 ml of blood was collected and collected in a tube containing heparin.

2. 30 ml each of blood was slowly added to two 50 ml tubes containing Ficoll-Paque Plus (GE Healthcare) along the tube walls to avoid mixing with Ficoll and blood.

3. Centrifuged at 2000 rpm for 25 minutes.

4. After separating the buffy coat layer containing lymphocytes among the four layers (from the upper layer: plasma layer-buffy layer-Ficoll layer-red blood cell layer) separated by the density gradient, a 50 ml conical tube ( conical tube).

5. The separated buffy coat layer was adjusted to a volume of 50 ml with Dulbecco's phosphate buffered saline (DPBS, Thermo Fisher Scientific), mixed, and then centrifuged at 1300 rpm for 10 minutes to discard the supernatant and remove lymphocytes. The washing process for separation was repeated 2-3 times in total.

6. Plasma from the separation process was placed in a 50 ml tube, inactivated in a 56°C constant temperature water bath for 30 minutes, centrifuged at 2300 rpm for 10 minutes, and only the supernatant was transferred to a new tube and stored for cell culture Autologous serum was used.

Example 3: NK cell differentiation and proliferation

NK cells were massively proliferated from PBMCs according to the following steps.

1. The PBMC prepared in Example 2 was well dissolved in KBM502 (KOHJIN BIO, JAPAN), and the first peptide 1 ul / mL (10 mM), the second peptide 0.5 ul / mL (10 mM), or together with both Anti-CD16 antibody, anti-CD56 antibody, enzyme mix, and 20% (v/v) autologous serum were added and cultured in a 25T flask for 2-3 days.

2. After culturing in the 25T flask, as the number of cells increased, the bottom of the flask was scraped well with a scraper, and all the culture medium and cells were transferred to the T75. 1 ul/mL (10 mM) of the first peptide, 0.5 ul/mL (10 mM) of the second peptide, CD16, CD56, enzyme mix, and 20% autologous serum were added and cultured in a 75T flask for 2 to 3 days.

3. After culturing in the 75T flask, as the number of cells increased, the bottom of the flask was scraped well with a scraper, and all the culture medium and cells were transferred to the T175. 1 ul/mL (10 mM) of the first peptide, 0.5 ul/mL (10 mM) of the second peptide, CD16, CD56, enzyme mix, and 20% autologous serum were added and cultured in a 175T flask for 2 to 3 days.

4. After scraping the bottom of the T175 flask during final culture with a scraper, cell suspension was made by adding 10 to 15 ml of the remaining autologous plasma or FBS (or similar) thereto. Next, after adding the cell suspension and 1L of the culture medium to a cell culture bag with 1L carbon dioxide permeability, mix well, put in a 37 ℃, 5% CO₂ incubator and further culture for about 4 to 7 days to proliferate the cells in large quantities made it

5. Divide the cultured final culture into two 500 ml centrifuge tubes, centrifuge at 2500 rpm for 20 minutes, discard the supernatant, mix the remaining cells with DPBS, and centrifuge again 2-3 times. repeated.

Experimental Example 1: Confirmation of the number of cells differentiated into NK cells

The cells obtained in Example 3 were analyzed according to the following steps, and the number of cells differentiated into NK cells was confirmed.

1. Using peptide 1 (SEQ ID NO: 27) as the first peptide and peptide 181 (SEQ ID NO: 217) as the second peptide, cells were obtained by performing the same method as in Example 3. Cells obtained by performing the same method as in Example 3 using IL-2 instead of the first and second peptides were used as a positive control.

2. After washing and centrifuging 1x10 ⁶ cells with FACS buffer (eBioscience, 00-4222-26), the supernatant is discarded, and only the cells are mixed well with FACS buffer again. ) was busy.

3. CD3-FITC (eBioscience), an antibody capable of identifying T cells, CD16-APC (eBioscience), and CD56-PE (eBioscience), antibodies capable of identifying T cells, were added to the FACS tube in which the cells were dispensed, respectively. Then, it was reacted at 4°C for 30 minutes.

4. Subsequently, the cells were washed with FACS buffer and analyzed for surface antigen characteristics of CD3- and CD16+CD56+ representing NK cells using FACS (Fluorescenceactivated cell sorting, BD FACSCalibur™).

Table 5 below shows the NK cell ratio according to the culture method of Example 3.

group Proportion of NK cells PBMCs before culture 17.31% IL-2 treatment group (positive control group) 67.31% First peptide treatment group 75.95% Second peptide treatment group 71.43% 1st peptide + 2nd peptide treatment group 83.76%

1 is a FACS analysis result showing the NK cell ratio of PBMC before culture. FIG. 2 is a FACS analysis result showing the NK cell ratio of the IL-2 treated group (positive control group).

3 is a FACS analysis result showing the NK cell ratio of the first peptide treatment group.

Figure 4 is a FACS analysis result showing the NK cell ratio of the first peptide and the second peptide co-treatment group.

As shown in Table 5 and FIGS. 1 to 4, the ratio of NK cells was higher in the first peptide and/or second peptide treated group than in the IL-2 treated group. In particular, the NK cell ratio was the highest in the group treated with the first peptide and the second peptide simultaneously.

Additionally, in the same manner as described above, the NK cell ratio by single treatment or combined treatment of the 187 peptides of Example 1 was analyzed.

The analysis results of the NK cell ratio by single treatment of each of the 180 first peptides are shown in Table 6 below.

Proportion of NK cells (%)
(Mean, N=3 (for each sample)) STD
(Standard Deviation) IL-2 treatment group (positive control group) 67.31 0.78 peptide 1 75.95 0.15 peptide 2 72.33 0.13 peptide 3 73.24 0.32 peptide 4 75.1 0.00 peptide 5 74.15 0.47 peptide 6 72.31 0.66 peptide 7 71.54 0.30 peptide 8 69.84 0.14 peptide 9 70.33 0.96 peptide 10 73.11 0.38 peptide 11 72.42 0.41 peptide 12 71.23 0.19 peptide 13 69.88 0.12 peptide 14 70.54 0.55 peptide 15 70.13 0.10 peptide 16 69.98 0.74 peptide 17 71.33 0.55 peptide 18 71.85 0.56 peptide 19 72.15 0.96 peptide 20 73.32 0.67 peptide 21 74.22 0.29 peptide 22 73.12 0.57 peptide 23 69.64 0.69 peptide 24 70.34 0.85 peptide 25 70.65 0.98 peptide 26 70.95 0.61 peptide 27 71.34 0.84 peptide 28 74.12 0.80 peptide 29 73.43 0.94 Peptide 30 72.54 0.06 peptide 31 72.87 0.50 peptide 32 71.98 0.73 peptide 33 71.54 0.55 Peptide 34 73.76 0.53 peptide 35 70.33 0.72 Peptide 36 75.12 0.42 peptide 37 74.98 0.75 peptide 38 73.77 0.27 peptide 39 73.54 0.51 peptide 40 72.44 0.92 peptide 41 71.53 0.98 peptide 42 72.67 0.43 peptide 43 73.51 0.85 peptide 44 71.23 0.22 peptide 45 71.33 0.90 peptide 46 72.76 0.05 peptide 47 71.55 0.53 peptide 48 72.87 0.23 peptide 49 73.98 0.64 peptide 50 74.12 0.72 peptide 51 70.34 0.43 peptide 52 71.54 0.99 peptide 53 70.55 0.36 peptide 54 71.85 0.23 peptide 55 70.56 0.44 peptide 56 73.18 0.64 peptide 57 74.10 0.50 peptide 58 76.07 0.37 peptide 59 74.87 0.30 peptide 60 73.03 0.56 peptide 61 71.56 0.65 peptide 62 70.13 0.14 peptide 63 70.62 0.57 peptide 64 73.90 0.54 peptide 65 72.87 0.79 peptide 66 72.03 0.03 peptide 67 70.08 0.88 peptide 68 70.85 0.12 peptide 69 70.92 0.94 peptide 70 70.41 0.81 peptide 71 71.81 0.01 peptide 72 72.18 0.22 peptide 73 73.03 0.67 peptide 74 73.88 0.31 peptide 75 75.16 0.77 peptide 76 73.84 0.66 peptide 77 70.09 0.65 peptide 78 70.97 0.11 peptide 79 71.60 0.70 peptide 80 71.93 0.12 peptide 81 71.78 0.67 peptide 82 75.07 0.24 peptide 83 73.53 0.45 peptide 84 73.42 0.90 peptide 85 73.54 0.80 peptide 86 72.39 0.60 peptide 87 71.65 0.62 peptide 88 73.93 0.09 peptide 89 70.74 0.29 peptide 90 75.89 0.26 peptide 91 75.00 0.89 peptide 92 73.87 0.39 peptide 93 74.21 0.67 peptide 94 73.04 0.51 peptide 95 71.70 0.97 peptide 96 73.07 0.19 peptide 97 73.92 0.36 peptide 98 71.35 0.58 peptide 99 71.83 0.88 Peptide 100 73.63 0.53 peptide 101 71.91 0.71 peptide 102 73.06 0.46 peptide 103 74.62 0.34 peptide 104 74.34 0.64 peptide 105 70.51 0.77 peptide 106 72.05 0.16 peptide 107 71.23 0.58 peptide 108 72.32 0.10 peptide 109 72.50 0.33 peptide 110 73.14 0.67 peptide 111 73.35 0.73 peptide 112 75.70 0.97 peptide 113 74.96 0.99 peptide 114 72.49 0.46 peptide 115 71.89 0.94 peptide 116 70.27 0.22 peptide 117 70.90 0.45 peptide 118 73.16 0.15 peptide 119 72.56 0.77 Peptide 120 72.04 0.28 peptide 121 70.53 0.62 peptide 122 70.75 0.20 peptide 123 70.27 0.93 peptide 124 70.28 0.76 peptide 125 71.53 0.22 peptide 126 72.54 0.20 peptide 127 73.00 0.31 peptide 128 73.66 0.98 peptide 129 74.69 0.40 Peptide 130 73.22 0.15 peptide 131 70.02 0.10 peptide 132 71.21 0.32 peptide 133 71.03 0.20 peptide 134 71.16 0.63 peptide 135 71.39 0.96 peptide 136 74.34 0.82 peptide 137 74.02 0.81 peptide 138 72.83 0.03 peptide 139 73.04 0.15 Peptide 140 72.17 0.26 peptide 141 72.37 0.98 peptide 142 73.90 0.52 peptide 143 71.09 0.72 peptide 144 76.10 0.16 peptide 145 75.13 0.20 peptide 146 74.70 0.73 peptide 147 74.22 0.17 peptide 148 73.25 0.34 peptide 149 72.46 0.60 Peptide 150 73.54 0.64 peptide 151 74.50 0.87 peptide 152 71.65 0.52 peptide 153 72.18 0.86 peptide 154 73.26 0.07 peptide 155 72.29 0.34 peptide 156 73.16 0.03 peptide 157 74.77 0.61 peptide 158 74.76 0.50 peptide 159 70.93 0.94 Peptide 160 72.26 0.75 peptide 161 71.10 0.40 peptide 162 72.57 0.04 peptide 163 73.64 0.29 peptide 164 75.99 0.21 peptide 165 74.86 0.43 peptide 166 72.38 0.16 peptide 167 72.00 0.05 peptide 168 70.79 0.10 peptide 169 71.00 0.86 Peptide 170 73.16 0.92 peptide 171 73.02 0.11 peptide 172 71.28 0.63 peptide 173 70.65 0.40 peptide 174 71.08 0.17 peptide 175 70.21 0.37 peptide 176 70.14 0.23 peptide 177 71.52 0.01 peptide 178 72.31 0.74 peptide 179 72.89 0.03 Peptide 180 74.24 0.04

The analysis results of the NK cell ratio by single treatment of each of the 7 second peptides are shown in Table 7 below.

Proportion of NK cells (%)
(Mean, N=3 (for each sample)) STD
(Standard Deviation) IL-2 treatment group (positive control group) 67.31 0.78 peptide 181 71.43 0.88 peptide 182 69.33 0.53 peptide 183 70.24 0.71 peptide 184 71.11 0.46 peptide 185 70.67 0.34 peptide 186 68.99 0.64 peptide 187 69.98 0.77

The analysis results of the NK cell ratio by the combination treatment of the first peptide and the second peptide are shown in Table 8 and FIG. 5 below.

Proportion of NK cells (%) STD IL-2 treatment group 67.31 0.78 combination 1 peptide 1 peptide 181 83.76 0.17 peptide 182 81.78 0.65 peptide 183 82.32 0.78 peptide 184 80.45 0.13 peptide 185 81.88 0.59 peptide 186 83.21 0.25 peptide 187 81.11 0.44 Combination 2 peptide 16 peptide 181 82.18 0.80 peptide 182 81.97 0.79 peptide 183 82.85 0.04 peptide 184 81.17 0.20 peptide 185 82.77 0.69 peptide 186 82.10 0.73 peptide 187 81.60 0.98 combination 3 peptide 31 peptide 181 81.83 0.98 peptide 182 82.55 0.88 peptide 183 81.37 0.87 peptide 184 82.84 0.36 peptide 185 83.47 0.03 peptide 186 81.69 0.74 peptide 187 81.88 0.25 combination 4 peptide 46 peptide 181 82.89 0.32 peptide 182 82.84 0.67 peptide 183 83.29 0.20 peptide 184 83.08 0.01 peptide 185 81.31 0.67 peptide 186 82.99 0.24 peptide 187 84.19 0.81 Combination 5 peptide 61 peptide 181 81.91 0.74 peptide 182 83.01 0.61 peptide 183 82.61 0.54 peptide 184 80.87 0.83 peptide 185 83.83 0.99 peptide 186 82.96 0.32 peptide 187 81.61 0.43 Combination 6 peptide 76 peptide 181 82.20 0.88 peptide 182 83.07 0.59 peptide 183 81.94 0.54 peptide 184 82.60 0.62 peptide 185 83.17 0.44 peptide 186 81.03 0.90 peptide 187 82.00 0.44 Combination 7 peptide 91 peptide 181 84.00 0.58 peptide 182 81.40 0.43 peptide 183 82.59 0.36 peptide 184 83.04 0.43 peptide 185 81.02 0.54 peptide 186 82.71 0.40 peptide 187 83.75 0.01 Combination 8 peptide 106 peptide 181 81.35 0.07 peptide 182 81.92 0.12 peptide 183 83.11 0.07 peptide 184 83.04 0.27 peptide 185 82.88 0.13 peptide 186 82.64 0.76 peptide 187 81.00 0.37 combination 9 peptide 121 peptide 181 82.72 0.28 peptide 182 84.26 0.46 peptide 183 82.52 0.80 peptide 184 83.03 0.41 peptide 185 82.62 0.25 peptide 186 81.22 0.11 peptide 187 83.34 0.79 Combination 10 peptide 136 peptide 181 84.05 0.54 peptide 182 81.82 0.61 peptide 183 82.33 0.67 peptide 184 82.96 0.04 peptide 185 82.03 0.90 peptide 186 82.40 0.37 peptide 187 82.57 0.03 Combination 11 peptide 151 peptide 181 80.86 0.01 peptide 182 82.40 0.73 peptide 183 83.58 0.56 peptide 184 81.97 0.94 peptide 185 82.18 0.14 peptide 186 82.65 0.25 peptide 187 80.74 0.45 Combination 12 peptide 166 peptide 181 82.83 0.01 peptide 182 83.59 0.80 peptide 183 81.58 0.38 peptide 184 82.71 0.96 peptide 185 82.54 0.38 peptide 186 83.54 0.37 peptide 187 83.02 0.46

As shown in Tables 6 to 8 and FIG. 5, 180 first peptides and 7 second peptides had a ratio of NK cells of 70% or more when treated alone, so compared to the IL-2 treated group (positive control group), NK It was confirmed that the percentage of cells was significantly high. In particular, when the first peptide and the second peptide were used in combination, the ratio of NK cells was 80% or more, so it was confirmed that there was a synergistic effect. Therefore, it was found that culturing PBMCs using the peptide according to one embodiment can obtain NK cells at a high rate.

Experimental Example 2: Confirmation of IFN-r Secretion by NK Cells

Cells cultured in Example 3 were collected and an experiment was conducted to confirm the secretion amount of IFN-r, which is an indicator of NK cell activity. IFN-r is an indicator substance indicating the activity of NK cells, and is considered to have high cytotoxicity and increase differentiation capacity as the secretion amount increases.

Specifically, the experiment used a human IFN-r ELISA Kit (BD, 550612). In Example 3, after acquiring 1 ml of the supernatant of the culture solution in each flask on the 1st day (25T), 3rd day (75T), and 5th day (175T) of culture, centrifugation was performed to separate and store only the supernatant for use in experiments. did Put the supernatant stored above in the IFN-r-coated 96-well plate and reacted in a 37° C. incubator for 2 hours. Then, after washing each well several times with the washing buffer provided in the kit, the secondary antibody and the chromogenic substrate were sequentially added and reacted. Finally, it was confirmed at 450 nm using a Bio-Rad Microplate Reader, i-Mark.

6 is a graph showing the amount of IFN-4 secretion (pg/mL) on days 1, 3, and 5 of cell culture.

As shown in FIG. 6 , a higher amount of IFN-r secretion was observed in cells cultured by treatment with the first peptide without using IL-2.

Experimental Example 3: Confirmation of Cytotoxicity of NK Cells to Cancer Cells

In order to evaluate the cytotoxicity of the cultured cells against cancer cells in Example 3, K562 (Korea Cell Line Bank), a blood cancer cell line, was purchased and used. The blood cancer cell line K562 used as a target cell was counted with a hemocytometer and the number of 1.0 × 10 ⁴ cells was used at a ratio of 1, and the differentiated NK cells used as effecter cells were cultured The cells were counted and the cell numbers of 5×10 ⁴ , 10×10 ⁴ , and 20×10 ⁴ were used as ratios. Target cells and effector cells were divided into a 96-well plate with RPMI1640 medium (serum free) at ratios of 1:5, 1:10, and 1:20 (target cells:effect cells) and co-cultured in a 37°C incubator for 4 hours ( co-culture). Then, the amount of LDH secreted from the dead cells was measured using the EZ-LDH (DoGen, DG-LDH500) Kit with a 450 nm filter in a Bio-Rad Microplate Reader and i-Mark to determine cell-mediated cytotoxicity (Cell-LDH500). mediated cytotoxicity) was confirmed. At this time, the calculation method used to obtain the cytotoxicity (%) is as follows.

Cytotoxicity (%) = (A - B - C ) / (D - B) X 100

A: Background control

B: Target cell control

C: Effector cell control

D: Target cell high control

7 is a result of calculating cytotoxicity (%) of cells cultured in Example 3 to cancer cells.

As shown in FIG. 7, the first peptide-treated group showed a similar level of cytotoxicity to that of the IL-2-treated group. The second peptide-treated group and the first and second peptide-treated groups showed higher cytotoxicity than the IL-2-treated group. In particular, the group treated with the first peptide and the second peptide showed the highest cytotoxicity.

In summary, the degree of differentiation and activity into NK cells was higher in the group treated with the peptide according to one embodiment than in the group treated with IL-2 in the cell differentiation step. In particular, it was confirmed that the group treated with the first peptide and the second peptide showed the highest degree of differentiation, activity, and cytotoxicity. This indicates that the peptide according to one embodiment can be used for NK cell-induced differentiation and mass culture by replacing IL-2 in the cell activation step. Furthermore, when the peptide according to one embodiment is co-administered to a patient along with autologous in vitro cultured NK cells, it is expected to be more effective as an NK cell therapeutic agent such as anticancer and antiviral.

The present invention has industrial applicability as an NK cell therapeutic agent, such as anticancer and antiviral agents, when co-administered to a patient.

Sequence Listing of the present invention is attached separately.

Claims (14)

A composition for mass proliferation of natural killer cells comprising a first peptide and a second peptide, The first peptide is represented by the following general formula I, [Formula I] X1-His-Gly-His-Gly-X2 In the above general formula I, X1 is Gly-Val-Gly-His-Gly, Val-Ser-Gly, Ser-Gly, Pro-Ser-Leu-Thr-Gly, Phe-Ile-Val-Ser-Ala, Thr, Leu-Ala-Ser -Leu, Cys-Val-Val-Thr-Gly, Ile-Ser-Gly, Cys-Gly, Ile-Val-Ala-Arg-Ile, Phe-Gly, His-Gly-Asp-Ser-Gly, Ser-Gly , and Tyr-Ala-Met-Ser-Gly consisting of any one amino acid sequence, X2 is any one of His-Gly, Val-His, Tyr-Asp, Phe-Val, Val-Pro, Gln-His-Gly, Leu-Ala, Val-Asp, Pro-Leu, Met, and Phe-Ile consists of the amino acid sequence of, or is absent; The second peptide is represented by the following general formula II, [Formula II] X3-Arg-Ala-Arg-Arg-Pro-Gly-Leu-Leu-Gly-X4 In the above general formula II, X3 consists of any one amino acid sequence of Glu, Ser-Val-Leu-Asn-Tyr-Glu, Tyr-Glu, Gly-Leu-Leu-Gly, and Arg-Pro-Gly-Leu-Glu, or does not exist, X4 is Ser-Arg-Leu-Arg-Phe-Ile-Pro-Lys, Ala-Ser-Val-Leu-Asp-Asp-Ile-His-Arg-Ala, Ala, Ala-Ser-Val, and Ala- A composition for mass proliferation of natural killer cells, which consists of any one amino acid sequence of Ser-Val-Leu or is absent. The composition for mass proliferation of natural killer cells according to claim 1, wherein the first peptide consists of any one amino acid sequence of SEQ ID NOs: 1 to 180. The composition for mass propagation of natural killer cells according to claim 1, wherein the second peptide consists of an amino acid sequence of any one of SEQ ID NOs: 181 to 187. The composition for mass proliferation of natural killer cells according to claim 1, wherein the natural killer cells are derived from peripheral blood mononuclear cells (PBMC). The composition for mass proliferation of natural killer cells according to claim 1, wherein the first peptide and the second peptide are included at a concentration of 0.1 to 100 mM, respectively, based on the entire composition. The composition for mass proliferation of natural killer cells according to claim 1, further comprising an antibody, serum, medium, or a combination of two or more thereof. The composition of claim 6, wherein the antibody is an anti-CD16 antibody, an anti-CD45 antibody, or a combination thereof. A method for mass proliferation of natural killer cells, comprising culturing isolated peripheral blood mononuclear cells (PBMC) in the composition for mass proliferation of natural killer cells according to any one of claims 1 to 7. The method according to claim 8, wherein the culturing step, first culturing the isolated peripheral blood mononuclear cells in a first culture vessel in a composition for mass proliferation of natural killer cells; secondly culturing the cells and culture medium obtained from the first culture in a second culture vessel in a composition for mass propagation of second natural killer cells; Thirdly culturing the cells and the culture medium obtained from the second culture in a third culture vessel in a third composition for mass propagation of natural killer cells; and A method for mass propagation of natural killer cells comprising the step of fourth culturing the cells and culture medium obtained from the third culture in a fourth culture vessel. The method according to claim 9, wherein the culture area of the second culture vessel is greater than the culture area of the first culture vessel, the culture area of the third culture vessel is greater than the culture area of the second culture vessel, and the fourth culture vessel The culture area of is larger than the culture area of the third culture container, a method for mass propagation of natural killer cells. The method of claim 9, wherein in the fourth culturing step, the cell suspension prepared by adding serum to the cells obtained from the third culturing and the culture medium is cultured. The method of claim 9, wherein the first culture, the second culture, and the third culture are performed for 2 to 3 days, respectively. The method of claim 9, wherein the fourth culture is performed for 4 to 8 days. A pharmaceutical composition for preventing or treating cancer comprising natural killer cells obtained by the method of mass propagation of natural killer cells of claim 8.

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