Composition for enhancing the activity of natural killer cells

Abstract

The present invention relates to a composition for improving the activity of natural killer cells, and provides a composition for improving the activity of natural killer cells, comprising as an active ingredient a peptide having the amino acid sequence of SEQ ID NO: 1. The peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention not only increases the cell killing ability of natural killer cells, but also reduces the homozygous killing increased by TGF-β, and further improves the sensitivity of natural killer cells to cancer cells, and therefore the peptide having the amino acid sequence of SEQ ID NO: 1 can be usefully used as an active ingredient of anticancer agents or anticancer adjuvants.

Description

[Technical Field]

[0001] The present invention relates to a composition for improving the activity of natural killer cells, and more specifically to a composition for restoring the activity of natural killer cells that has been reduced by TGF-β. [Background technology]

[0002] Natural killer cells (NK cells) are lymphocytes present in bone marrow, lymph nodes, and peripheral blood, accounting for approximately 10–15% of all lymphocytes. Phenotypically, they are CD3-negative and CD56-positive. They play an important role in the innate immune response as immune cells capable of killing cancer cells or virus-infected cells. Natural killer cell function is regulated by the interaction of cell surface receptors with their corresponding target cell ligands, without the need for specific antigen stimulation. These receptors are broadly classified as activating receptors and inhibitory receptors. Activating receptors that transmit activation signals to NK cells and regulate their function include natural cytotoxicity receptors (NCRs), such as NKp30, NKp44, and NKp46, as well as NKG2D. Activated NK cells synthesize and secrete various granules to destroy target cells. Among the major proteins found in granules, perforin and granzymes play key roles in destroying target cells. Perforin aggregates on the target cell membrane to form a complex, opening pores in the membrane and causing cell lysis. Granzymes enter the cell through the pores and activate caspases, inducing cell death through various mechanisms. Natural killer cells also induce apoptosis by binding to death receptors on cancer cells using death ligands such as FasL and TRAIL. This receptor binding activates caspase-8 and caspase-10 within the cancer cell, which in turn activates caspase-3 and caspase-7, ultimately resulting in apoptosis.

[0003] Due to these characteristics, natural killer cells have been used in anti-cancer therapy. However, most successful treatments have been limited to blood cancers, and their effectiveness in solid tumors has been minimal. Treatment of solid tumors is difficult because natural killer cells must migrate and infiltrate into tumor tissues. Even if they do infiltrate tumor tissues, they are unable to exert an adequate anti-cancer effect due to the immunosuppressive microenvironment of tumor tissues.

[0004] On the other hand, TGF-β is a cytokine with immunosuppressive activity that inhibits the anti-cancer activity of T cells and natural killer cells. In particular, TGF-β is produced and secreted in solid tumors, suppressing immune function in the tumor microenvironment and contributing to the growth of cancer tissue. TGF-β secretion in metastatic cancers is closely related to poor cancer prognosis. In fact, inhibiting TGF-β production and signaling enhances the anti-cancer activity of natural killer cells. TGF-β inhibits the production of IFN-γ by natural killer cells and inhibits their killing ability via CD16.

[0005] Therefore, there is a need to research and develop components that can restore the activity of natural killer cells suppressed by TGF-β and improve anti-cancer immune activity against solid cancers or metastatic cancers. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a composition capable of enhancing the activity of natural killer cells. [Means for solving the problem]

[0007] To achieve the above object, the present invention provides a peptide having the amino acid sequence of SEQ ID NO: 1 or code The present invention provides a composition for enhancing the activity of natural killer cells, which comprises, as an active ingredient, a polynucleotide that inhibits the activity of natural killer cells.

[0008] The present invention also provides a peptide having the amino acid sequence of SEQ ID NO: 1 or a peptide having the same. code The present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises, as an active ingredient, a polynucleotide that inhibits the cancer.

[0009] The present invention also provides a peptide having the amino acid sequence of SEQ ID NO: 1 or a peptide having the same. code The present invention provides a cellular therapeutic agent for preventing or treating cancer, which comprises natural killer cells treated with a polynucleotide that targets the cellular therapeutic agent.

[0010] The present invention also provides a peptide having the amino acid sequence of SEQ ID NO: 1 or a peptide having the same. code The present invention provides a method for improving the activity of natural killer cells, which comprises the step of treating natural killer cells with a polynucleotide that inhibits the activity of natural killer cells.

[0011] The present invention also provides a peptide having the amino acid sequence of SEQ ID NO: 1 or a peptide having the same. code and a method for treating cancer, comprising administering to a subject in need of treatment a pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition comprising a polynucleotide that inhibits the cancer.

[0012] The present invention also provides (a) a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or code and (b) administering the natural killer cells of step (a) to a subject.

[0013] The present invention also provides a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or a peptide having the same. code and a pharmaceutical composition for preventing or treating cancer, which comprises as an active ingredient a polynucleotide that inhibits the growth of cancer cells; and a method for treating cancer, which comprises administering natural killer cells to a subject. [Effects of the Invention]

[0014] The peptide of the present invention having the amino acid sequence of SEQ ID NO: 1 not only increases the cell killing ability of natural killer cells, but also reduces the increased cell killing caused by TGF-β, and even improves the sensitivity of natural killer cells to cancer cells. Therefore, the peptide of the present invention can be useful as an active ingredient of an anticancer agent or anticancer adjuvant. [Brief explanation of the drawings]

[0015] [Figure 1] 1 shows the results confirming that the cell-killing ability of NK92 cells, which was reduced by TGF-β, was restored by treatment with a peptide consisting of the amino acid sequence of SEQ ID NO: 1. A, B, and C are diagrams outlining the principle by which the peptide exerts its effect on natural killer cells. B, C, and D show the results of an experiment measuring the cell-killing ability of NK92 cells against the lung cancer cell line H460, the lung cancer cell line H3122, and the blood cancer cell line K562, respectively. [Figure 2] 1 shows the results confirming that the cell-killing ability of primary NK cells, which was reduced by TGF-β, was restored by treatment with the peptide consisting of the amino acid sequence of SEQ ID NO: 1. Panels A, B, and C show the experimental results measuring the cell-killing ability of primary NK cells against lung cancer cell line H460, lung cancer cell line H3122, and blood cancer cell line K562, respectively. [Figure 3] 1 shows the reduction in NK92 killing ability against the lung cancer cell line H460 treated with TGF-β and the restoration of the reduced sensitivity by a peptide having the amino acid sequence of SEQ ID NO: 1. (A) is an explanatory diagram showing a schematic diagram of the test of NK92 cell killing ability after treating the lung cancer cell line H460 cells with TGF-β. (B) shows that treatment of H460 cells with TGF-β reduces the killing ability of NK92 by more than half, and that the reduced killing ability is restored by the peptide. [Figure 4]1 shows the results of confirming that TGF-β increases fratricide killing among natural killer cells and that a peptide having the amino acid sequence of SEQ ID NO: 1 reduces fratricide killing. (A) Schematic illustration of the effect of reducing fratricide killing by natural killer cells using NK92 cells reacted with TGF-β as target cells. (B) and (C) show the enhancement of fratricide killing by NK92 and primary NK cells against NK-92 cells reacted with TGF-β, and the reduction of fratricide killing by the peptide, respectively. [Figure 5] 1 shows that the expression of TGF-β in lung cancer cells is suppressed by a peptide having the amino acid sequence of SEQ ID NO: 1. (A) illustrates the effect of the peptide in reducing the expression of TGF-β in the lung cancer cell line H460 cells. (B) shows that the peptide reduces the expression of TGF-β in H460 cells. [Figure 6] The peptide was shown to inhibit lung metastasis of B16F10 melanoma in an animal model. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention will be described in detail below.

[0017] One aspect of the present invention provides a composition for enhancing the activity of natural killer cells.

[0018] The composition for enhancing the activity of natural killer cells of the present invention comprises a peptide having the amino acid sequence of SEQ ID NO: 1 or a functional equivalent thereof, or code It contains a polynucleotide that acts as an active ingredient.

[0019] The peptide may include the amino acid sequence of SEQ ID NO: 1 or a functional equivalent thereof, or may consist solely of the amino acid sequence of SEQ ID NO: 1 or a functional equivalent thereof. The functional equivalent of the peptide may have the activity of the peptide having the amino acid sequence of SEQ ID NO: 1, in which some or all of the 13 amino acids constituting the amino acid sequence of SEQ ID NO: 1 are substituted with other amino acids, some of the 13 amino acids are deleted, at least one amino acid is added to the 13 amino acids, or some or all of the 13 amino acids are modified. The amino acid substitutions may be conservative substitutions, such as mutual substitutions between aliphatic uncharged amino acids (Gly, Ala, Val, Leu, Ile), mutual substitutions between hydrophilic uncharged amino acids (Ser, Thr, Asn, Gln), mutual substitutions between aromatic amino acids (Phe, Tyr, Trp), mutual substitutions between acidic amino acids (Asp, Glu), mutual substitutions between basic amino acids (His, Lys, Arg), or mutual substitutions between nonpolar uncharged amino acids (Cys, Met, Pro). The deletion or addition of an amino acid may occur, for example, in a portion that does not affect or is not directly involved in the activity of the peptide having the amino acid sequence of SEQ ID NO: 1. In particular, the addition of an amino acid may involve insertion of at least one amino acid into the amino acid sequence of SEQ ID NO: 1, as well as at the N-terminus or C-terminus of the amino acid sequence of SEQ ID NO: 1. Furthermore, the amino acid modification may involve phosphorylation, acetylation, methylation, glycosylation, etc. of some or all of the 13 amino acids constituting the amino acid sequence of SEQ ID NO: 1. In some cases, the functional equivalent may have 60% or more homology, 65% or more, 70% or more, 75% or more, 80% or 85% or more, for example, 90% or more homology with the amino acid sequence of SEQ ID NO: 1.

[0020] The polynucleotide encodes the peptide. codeFor example, it may have the base sequence of SEQ ID NO: 2 or an equivalent base sequence. The equivalent base sequence may be different from the base sequence of SEQ ID NO: 2, but code This means that the sequence of the peptide is the same as the nucleotide sequence of SEQ ID NO: 2. In some cases, the equivalent nucleotide sequence may have 60% or more, 70% or more, 80% or more, or 90% or more, for example, 95% or more, homology to the nucleotide sequence of SEQ ID NO: 2.

[0021] The polynucleotide may be contained in a vector, which is preferably capable of self-replication and more preferably capable of expressing the polynucleotide, and may be, for example, a plasmid, cosmid, or phage.

[0022] Alternatively, a vector containing the polynucleotide may be transformed into a host cell, which is preferably capable of expressing a peptide having the amino acid sequence of SEQ ID NO: 1 or an equivalent thereof from the polynucleotide contained in the vector, and more preferably capable of secreting the expressed peptide extracellularly.

[0023] Natural killer cells are cytotoxic lymphocytes that constitute the main component of the innate immune system. They are defined as large granular lymphocytes (LGLs) and play important roles in both the innate and adaptive immune systems. Natural killer cells include not only mature natural killer cells but also natural killer precursor cells. Furthermore, the natural killer cells may be derived from mammals, such as humans, monkeys, goats, sheep, rats, and mice, and may be derived from humans, monkeys, or mice. The activity of natural killer cells can include the killing ability of target cells such as cancer cells or virus-infected cells, degranulation of natural killer cells, or stimulation of activating receptors or inactivation of inhibitory receptors of natural killer cells, where the activating receptors may be NKG2D, 2B4, DNAM-1, NCRs, etc., and the inhibitory receptors may be PD-1, LAG-3, TIM-3, etc.

[0024] The improvement of natural killer cell activity may refer to an increase in the cytotoxicity of natural killer cells against target cells, a decrease in fratricide killing, or an increase in sensitivity to target cells. The increase in the cytotoxicity of natural killer cells may be due to an increase in the expression or secretion of substances involved in target cell death, such as perforin, granzymes, and interferons, or an increase in the degranulation of granules containing such substances. In particular, the perforin may be perforin-1 or perforin-2, the granzyme may be granzyme A, granzyme B, granzyme H, granzyme K, or granzyme M, and the interferon may be type 1 interferon such as interferon-α, interferon-β, interferon-κ, or interferon-ω, type 2 interferon such as interferon-γ, or type 3 interferon such as interferon-L1. Furthermore, the term "kin killing" refers to the ability of natural killer cells to kill each other, i.e., the phenomenon in which natural killer cells act as both effector cells and target cells. The kin killing between natural killer cells can be increased by TGF-β, and a decrease in the kin killing of natural killer cells means that the degree to which natural killer cells recognize other natural killer cells as target cells decreases. Furthermore, an increase in the sensitivity of natural killer cells to target cells means that natural killer cells can more easily recognize target cells.

[0025] In a specific example of the present invention, it was confirmed that treatment with a peptide consisting of the amino acid sequence of SEQ ID NO: 1 restored the cell-killing ability of NK92 or primary natural killer cells against lung cancer cell lines and blood cancer cell lines, which had been reduced by TGF-β (see Figures 1 and 2), and restored the sensitivity of NK92 or primary natural killer cells against lung cancer cell lines, which had been reduced by TGF-β (see Figure 3), while reducing the homing killing between natural killer cells, which had been increased by TGF-β (see Figure 4). Therefore, the present invention provides a peptide having the amino acid sequence of SEQ ID NO: 1 or a functional equivalent thereof, or said peptide, which has been used in the treatment of lung cancer cells and blood cancer cells, which had been reduced by TGF-β (see Figure 4). code The polynucleotide can be used as an active ingredient for enhancing the activity of natural killer cells.

[0026] Therefore, another aspect of the present invention is a peptide having the amino acid sequence of SEQ ID NO: 1 of the present invention described above or code The present invention provides a method for improving the activity of natural killer cells, which comprises the step of treating natural killer cells with a polynucleotide that inhibits the activity of natural killer cells.

[0027] Meanwhile, the composition for enhancing the activity of natural killer cells can be used as an anti-cancer agent or an anti-cancer adjuvant. Therefore, another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises a peptide having the amino acid sequence of SEQ ID NO: 1 or a functional equivalent thereof, or a compound comprising the peptide. code The polynucleotide may be included as an active ingredient.

[0028] In a specific example of the present invention, when a peptide having the amino acid sequence of SEQ ID NO: 1 was administered to an animal model transplanted with melanoma cells, it was confirmed that the number of cancer nodules metastasizing to the lungs was reduced compared to a negative control group (see FIG. 6). Therefore, the peptide having the amino acid sequence of SEQ ID NO: 1 or a functional equivalent thereof, or the peptide code The polynucleotides can be used as active ingredients for the prevention or treatment of cancer.

[0029] Cancer generally refers to a physiological condition in mammals characterized by uncontrolled cell growth, in which problems arise in the normal control functions of cell division, differentiation, and apoptosis, leading to abnormal overgrowth and infiltration of surrounding tissues and organs, forming masses, and destroying or deforming existing structures. The cancer may be a solid cancer or a metastatic cancer, such as colorectal cancer including colon and rectal cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, brain tumor, head and neck cancer, melanoma, myeloma, leukemia, lymphoma, gastric cancer, lung cancer, pancreatic cancer, liver cancer, esophageal cancer, small intestine cancer, anal cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulva cancer, Hodgkin's disease, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvis cancer, bone cancer, skin cancer, head cancer, neck cancer, cutaneous melanoma, intraocular melanoma, endocrine cell carcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, etc., and particularly, among the above types of cancer, those in which TGF-β is overexpressed or overactivated may be included.

[0030] The prevention means any action that suppresses or delays the occurrence or progression of cancer, and the treatment means any effect that prevents further deterioration of cancer cells, including not only the death of cancer cells but also reduction in the growth of cancer cells, reduction in recurrence, anti-cancer activity of the immune system against cancer cells, formation of immune memory, or reduction in infiltration of cancer cells, but is not limited to these.

[0031] The composition comprises a peptide having the amino acid sequence of SEQ ID NO: 1 or a functional equivalent thereof, or code In addition to the polynucleotide, other active ingredients that exhibit anti-cancer activity may also be included.

[0032] In addition to the active ingredient, the composition may further contain a pharmaceutically acceptable carrier or additive. The term "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not have toxicity beyond the range acceptable for the application (prescription) target. The carrier is defined as a compound that facilitates the addition of the compound into cells or tissues.

[0033] A peptide having the amino acid sequence of SEQ ID NO: 1 or a functional equivalent thereof according to the present invention, or code The polynucleotide can be administered alone or in admixture with any convenient carrier, etc., and such dosage form may be a single dose or multiple dose form. The composition may be a solid or liquid formulation. Solid formulations include, but are not limited to, powders, granules, tablets, capsules, suppositories, etc. Solid formulations may contain, but are not limited to, carriers, flavoring agents, binders, preservatives, disintegrants, lubricants, fillers, etc. Liquid formulations include, but are not limited to, solutions such as water and propylene glycol solutions, suspensions, emulsions, etc., and may be prepared by adding appropriate colorants, flavoring agents, stabilizers, viscosity enhancers, etc. For example, powders may contain the peptide having the amino acid sequence of SEQ ID NO: 1 or a functional equivalent thereof, which is the active ingredient of the present invention, or a compound containing the peptide. code The granules can be prepared by simply mixing the polynucleotide with a suitable pharmaceutically acceptable carrier such as lactose, starch, microcrystalline cellulose, etc. ... codeThe polynucleotide, a pharmaceutically acceptable carrier, and a suitable pharmaceutically acceptable binder such as polyvinylpyrrolidone or hydroxypropyl cellulose are mixed, and then the mixture is prepared by wet granulation using a solvent such as water, ethanol, or isopropanol, or by dry granulation using compression force. Tablets can also be prepared by mixing the granules with a suitable pharmaceutically acceptable lubricant such as magnesium stearate, and then tableting using a tablet press.

[0034] A peptide having the amino acid sequence of SEQ ID NO: 1 or a functional equivalent thereof according to the present invention, or code Depending on the disease to be treated and the condition of the individual, the polynucleotide may be administered orally, by injection (e.g., intramuscular injection, intraperitoneal injection, intravenous injection, infusion, subcutaneous injection, implant), inhalant, nasal administration, vaginal administration, rectal administration, sublingual administration, transdermal administration, topical administration, etc., but is not limited to these. It may be formulated into an appropriate dosage unit dosage form containing a non-toxic, pharmaceutically acceptable carrier, additive, or vehicle that is commonly used depending on the administration route.

[0035] The composition can be administered daily at a dose of about 0.0001 mg / kg to about 10 g / kg, or about 0.001 mg / kg to about 1 g / kg. However, the dosage can vary depending on the tablet size of the mixture, the patient's condition (age, sex, weight, etc.), the severity of the condition being treated, etc. If necessary, the total daily dosage can be administered in several divided doses throughout the day for convenience.

[0036] Another aspect of the present invention is a peptide having the amino acid sequence of SEQ ID NO: 1 or code The present invention provides a cellular therapeutic agent for preventing or treating cancer, which comprises natural killer cells treated with a polynucleotide that targets the cellular therapeutic agent.

[0037] The term "cellular therapeutic agent" refers to a pharmaceutical product (as defined by the U.S. FDA) that is used for therapeutic, diagnostic, and preventive purposes using cells and tissues isolated from an individual, cultured, and specially processed, and that is used for therapeutic, diagnostic, and preventive purposes through a series of actions such as the ex vivo expansion and selection of living autologous, allogenic, or xenogenic cells to restore the function of cells or tissues, or by changing the biological characteristics of cells in other ways.

[0038] In the present invention, the therapeutic agent is intended to be used for the treatment of cancer, and includes a peptide having the amino acid sequence of SEQ ID NO: 1 or a peptide having the same. code When natural killer cells with increased killing power against cancer cells are treated with a polynucleotide corresponding to the target gene, the cells can be used as a cell therapy agent for the treatment and prevention of cancer.

[0039] Another aspect of the present invention is a peptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention or a peptide comprising the same. code and a method for treating cancer, comprising administering to a subject a pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition comprising, as an active ingredient, a polynucleotide that targets the cancer.

[0040] Yet another aspect of the present invention provides a method for treating cancer, comprising the steps of: (a) the peptide of the present invention having the amino acid sequence of SEQ ID NO: 1 or code administering the polynucleotide to natural killer cells; and (b) administering the natural killer cells of step (a) to a subject.

[0041] The method of the present invention comprises administering to a subject a peptide of the present invention or code Since natural killer cells are treated with a polynucleotide corresponding to the target gene and have increased killing power against cancer cells, the description of the overlapping content will be omitted to avoid excessive complexity of this specification. [Example]

[0042] The present invention will be described in detail below with reference to examples. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

[0043] [Example 1] [1-1]NK92 cell culture NK92 cells were cultured according to ATCC culture conditions in α-MEM (Welgene) medium supplemented with 12.5% ​​FBS (Fetal Bovine Serum, Corning, United States Origin, 35-015-CV) and 12.5% ​​horse serum (Gibco), supplemented with 0.2 mM myo-inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 1% antibiotic-antimycotic (Gibco), and IL-2 cytokine (Peprotech) at a concentration of 20 ng / ml. The cells were cultured in a water-jacketed incubator (Thermo Electron Corporation) at 37°C with 5% CO2. Cells: 1 x 10 5 The cells were cultured in a T-75 flask (Thermo) at a concentration of 20–30 ml at 1000 rpm for 5 minutes every 2–3 days, and the supernatant was removed and the culture medium was replaced.

[0044] [1-2] Primary NK cell culture Umbilical cord blood donated by a hospital (IRB approval number P01-201610-31-002) was treated with 2 μl of Rosettesep Human CD3 depletion cocktail (STEMCELL technologies, catalog #15621) per 1 ml of blood and incubated for 20 minutes. The blood was then gradually added in 35 ml increments to 15 ml of Ficoll (GE Healthcare, Ficoll-paque premium) increments. Care was taken to avoid mixing the blood and Ficoll layers. To separate the cell layer, the blood was centrifuged (Beckman Coulter) at 2000 rpm with Decel 0 for 25 minutes to separate red blood cells and CD3 cells. The CD3-separated cells were incubated with 10 ml of ACK buffer for 10 minutes to remove remaining red blood cells, and then washed twice with phosphate-buffered saline (PBS) supplemented with 2% FBS. The cells were washed by centrifuging at 1500 rpm for 5 minutes in a centrifuge (Beckman Coulter) and removing the supernatant. The cells thus obtained were cultured with natural killer cells. The culture conditions for natural killer cells were alpha-MEM (Welgene) supplemented with 10% FBS (Corning, United States Origin, 35-015-CV) and 10 ng / ml IL-15 (Peprotech), 10 ng / ml IL-21 (Peprotech), 10 -6 The cells were cultured in a medium containing 1% hydrocortisone (Peprotech) and 1% antibiotic-antimycotic (Gibco). The cell culture density was 2 x 10 6 The cells were cultured in a 6-well plate at a concentration of 100 cells / ml and centrifuged at 1200 rpm for 5 minutes in a centrifuge (Beckman Coulter) every 2 to 3 days. The supernatant was removed and the culture medium was replaced. The degree of NK cell differentiation was confirmed by FACSCanto II (BD Biosciences) flow cytometry every 3 days. FACS flow cytometry analysis was performed by placing 2 × 10 cells in a FACS tube (Falcon). 4 The cells were aliquoted and washed in FBS-supplemented PBS (FACS buffer) at 1500 rpm for 3 minutes in a centrifuge (Beckman Coulter). The supernatant was removed, and 100 μL of FACS buffer was added. 1 μL each of CD56 APC (BD) antibody and CD3 PE (BD) antibody was added and stained for 20 minutes at 4°C. Then, 500 μL of FACS buffer was added and washed by centrifugation at 1500 rpm for 3 minutes in a centrifuge (Beckman Coulter). After removing the supernatant, 200 μL of FACS buffer was added, and analysis was performed using a FACSCanto II (BD Biosciences). On days 7-9 of culture, the CD56 differentiation rate was confirmed to be approximately 90%, and experiments were carried out using primary natural killer (NK) cells whose differentiation was confirmed.

[0045] [1-3] Lung cancer cell line H460, H3122 cell culture The target cell lines for the killing assay were the lung cancer cell lines H460 and H3122. H460 and H3122 cells were cultured in RPMI (Welgene) medium supplemented with 10% FBS (Corning, United States Origin, 35-015-CV) and 1% antibiotic-antimycotic (Gibco) according to ATCC culture conditions. They were cultured in a water-jacketed CO2 incubator (Thermo Electron Corporation) at 37°C with 5% CO2. The cells were cultured at a density of 2 x 10 5The cells were cultured in 100mm x 20mm cell culture dishes (corning) at a concentration of 100 cells / ml in a volume of approximately 10ml. Cells were observed and the culture medium was changed every 2-3 days. When changing the culture medium for H460 and H3122, the supernatant was removed and the cells were incubated with 2ml of 0.025% Trysin-EDTA (Gibco) for 3 minutes at 37°C. The adherent cells were then detached and centrifuged at 1000 rpm for 5 minutes in a centrifuge (Beckman Coulter), and the culture medium was replaced with new medium.

[0046] [1-4] Blood cancer cell line K562 cell culture In the killing assay, the blood cancer cell line K562 was cultured and used as the target cell line. K562 cells were cultured in RPMI (Welgene) medium supplemented with 10% FBS (Fetal Bovine Serum, Corning, United States Origin, 35-015-CV) and 1% antibiotic-antimycotic (Gibco) according to ATCC culture conditions, in a 37°C CO2 incubator (water-jacketed CO2 incubator, Thermo Electron Corporation) with 5% CO2. The cells were cultured at 2 x 10 5 The cells were cultured in a 75T flask (corning) at a concentration of 100 cells / ml in a volume of approximately 20 ml. The culture medium was changed every 2 to 3 days.

[0047] [1-5] Preparation of peptide having the amino acid sequence of SEQ ID NO: 1 Additionally, a peptide consisting of 13 amino acids of SEQ ID NO: 1 was synthesized by Peptron Co., Ltd. (Daejeon, Korea) and used in the following experiments.

[0048] [Example 2] Restoration of TGF-induced decreased cytotoxicity of natural killer cells The effect of the peptides prepared in the above Examples [1-5] on the cell killing ability of natural killer cells, which had been reduced by TGF-β, was examined.

[0049] [2-1] Preparation of effector cells For this, first 1x10 5 20 μM of the peptide from Example [1-5] or 5 μM of the TGF-β signaling inhibitor SB431542 were added to 100 cells / ml of NK92 cells or primary NK cells, and the cells were cultured at 37°C for 2 hours. After that, 10 ng / ml of TGF-β was added and the cells were cultured again under the same conditions for 24 hours. 20 μM of the peptide from Example [1-5] or 5 μM of SB431542 were then added and the cells were cultured at 37°C for 2 hours. After that, 10 ng / ml of TGF-β was added and the cells were cultured again under the same conditions for 24 hours. The NK92 cells and primary NK cells cultured as described above were used as effector cells for the killing assay.

[0050] [2-2] Preparation of target cells On the other hand, lung cancer cell lines H460 and H3122 cells and blood cancer cell line K562 were used as target cells to evaluate cell killing activity. 6 H460, H3122, and K562 cell lines (cells / ml each) were stained by culturing in culture medium containing 5 μl of calcein-AM (Invitrogen) at 37°C for 1 hour, and then washed by centrifugation at 1000 rpm for 5 minutes.

[0051] [2-3] Evaluation of cell killing ability Each target cell stained with calcein-AM in Example [2-2] above was dispensed into a 96-well plate and treated with the effector cells prepared in Example [2-1] above. When NK92 cells were used as effector cells, they were treated so that the effector cell to target cell ratio (E:T ratio) was 20:1. When primary NK cells were used as effector cells, they were treated so that the E:T ratio was 10:1. The cells were then cultured at 37°C for 4 hours. The cultures were centrifuged at 100 rcf for 5 minutes to separate the supernatant, which was then dispensed in 100 μl aliquots into 96-well black plates and subjected to ELISA assays (fluorescence 480 nm / 530 nm, SpectraMax i3x Molecular Device). As a result, as shown in Figure 1, it was confirmed that TGF-β reduced the cell-killing ability of NK92 cells against all three types of target cells, but that the cell-killing ability of NK92 cells was restored by the peptides of Examples [1-5] and SB431542. Furthermore, the effect of the peptides of Examples [1-5] in restoring the cell-killing ability of natural killer cells was confirmed not only in NK92 cells, but also in primary NK cells, as shown in Figure 2.

[0052] [Example 3] Restoration of TGF-decreased sensitivity of natural killer cells to cancer cells The effect of the peptides prepared in the above Examples [1-5] on the sensitivity of natural killer cells to cancer cells, which had been reduced by TGF-β, was examined. For this, first 1x10 520μM of the peptide from Example [1-5] or 5μM of the TGF-β signal inhibitor SB431542 were added to the H460 lung cancer cell line at 1x10 cells / ml, and the cells were cultured at 37°C for 2 hours. Then, 10ng / ml of TGF-β was added and the cells were cultured again under the same conditions for 24 hours. Next, 20μM of the peptide from Example [1-5] or 5μM of SB431542 were added and the cells were cultured at 37°C for 2 hours. Then, 10ng / ml of TGF-β was added and the cells were cultured again under the same conditions for 24 hours. The H460 cells cultured as described above were cultured at 1x10 cells / ml. 6 The cells / ml were stained by culturing in culture medium containing 5 μl of calcein-AM (Invitrogen) at 37°C for 1 hour, then washed by centrifugation at 1000 rpm for 5 minutes to prepare the "target" cells. As described above, each target cell stained with calcein-AM was dispensed into a 96-well plate, and untreated (fresh) NK92 cells were used as effector cells at an E:T ratio of 20:1 and cultured for 4 hours at 37°C. The cultured cells were centrifuged at 100 rcf for 5 minutes to separate the supernatant, which was then dispensed in 100 μl aliquots into a 96-well black plate and subjected to an ELISA assay (fluorescence 480 nm / 530 nm, SpectraMax i3x Molecular Device). As a result, as shown in Figure 3, natural killer cells did not exhibit sufficient cell-killing ability against H460 cells treated with TGF-β, but it was confirmed that the cell-killing ability of natural killer cells was restored against H460 cells treated with the peptide of Example [1-5] or SB431542.

[0053] [Example 4] Inhibition of natural killer cell homing (fratricide) We investigated how TGF-β affects fratricide between natural killer cells, and further investigated how the peptides prepared in Examples [1-5] above affect fratricide between natural killer cells. For this purpose, 1x10 cells were cultured in the same manner as in Example [2-1]. 6 NK92 cells (1000 cells / ml) were stained by culturing in culture medium supplemented with 5 μL of calcein-AM (Invitrogen) at 37°C for 1 hour, then centrifuged at 1000 rpm for 5 minutes and washed to prepare "target" cells. Each target cell stained with calcein-AM was dispensed into a 96-well plate and treated with untreated (fresh) NK92 cells or primary NK cells as effector cells at a ratio of 20:1 or 10:1 as in Examples 2-3 above. The cultures were centrifuged at 100 rcf for 5 minutes to separate the supernatant, which was then dispensed in 100 μL aliquots into a 96-well black plate and subjected to an ELISA assay (fluorescence 480 nm / 530 nm, SpectraMax i3x Molecular Devices). As a result, as shown in Figure 4, it was confirmed that the natural killer cells had very high activity of killing their own relatives against NK92 cells treated with TGF-β, but that the peptides of Examples [1-5] and SB431542 significantly reduced the natural killer cells' activity of killing their own relatives.

[0054] [Example 5] Suppression of TGF-β expression in cancer cells The effect of the peptides prepared in the above Examples [1-5] on the expression of TGF-β in cancer cells was examined. For this, 2x10 5Lung cancer cell line H460 cells / ml were cultured for 2 hours to stabilize, then treated with the peptides from Examples [1-5] at concentrations of 5 μM, 10 μM, and 20 μM, or SB431542 at 5 μM, and cultured at 37°C for 24 hours. Then, the cells were again treated with the peptides from Examples [1-5] or SB431542 at the same concentrations and cultured at 37°C for 24 hours. The cultures were centrifuged at 100 rcf for 5 minutes to separate the supernatant and pellet. The supernatant was dispensed in 100 μl aliquots into 96-well black plates, and TGF-β secretion was measured by ELISA (absorbance 450 nm, SpectraMax i3x Molecular Device). As a result, as shown in FIG. 5, it was confirmed that the amount of TGF-β secreted from H460 cells was reduced in a concentration-dependent manner when the peptide of Example [1-5] was treated.

[0055] [Example 6] Anti-cancer effects in animal models To confirm the anti-cancer effect of the peptide prepared in Example [1-5] in vivo, 25 mg / kg of the peptide prepared in Example [1-5] was intraperitoneally administered to 24-month-old C57BL6J mice, four times every other week for four weeks. 5 B16F10 melanoma cells were administered into the tail vein, and the number of cancer nodules that had metastasized to the lungs was counted to confirm the anti-cancer effect. As a result, as shown in FIG. 6, it was confirmed that the number of nodules in the lungs of mice administered with the peptide of Example [1-5] was significantly reduced compared to the negative control group. Although the preferred embodiments of the present invention have been described above as examples, the scope of the present invention is not limited to the specific embodiments described above, and a person having ordinary knowledge in the art would be able to make appropriate modifications within the scope of the claims of the present invention.

Claims

1. A composition for enhancing the activity of natural killer cells, comprising as an active ingredient a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or a polynucleotide encoding the peptide.

2. The composition for enhancing natural killer cell activity according to claim 1, wherein the peptide suppresses the expression or activity of TGF-β.

3. The composition for enhancing the activity of natural killer cells according to claim 2, wherein the peptide enhances the cell-killing ability of natural killer cells.

4. The composition for enhancing natural killer cell activity according to claim 2, wherein the peptide reduces the fratricide activity of natural killer cells.

5. The composition for enhancing natural killer cell activity according to claim 1, wherein the composition is an anti-cancer agent or an anti-cancer adjuvant.

6. The composition for enhancing natural killer cell activity according to claim 5 , wherein the cancer is a solid cancer or a metastatic cancer.

7. A pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or a polynucleotide encoding the peptide.

8. 8. The pharmaceutical composition for preventing or treating cancer according to claim 7, wherein the peptide suppresses the expression or activity of TGF-β in the cancer.

9. The pharmaceutical composition for preventing or treating cancer according to claim 7, wherein the peptide enhances the susceptibility of natural killer cells to cancer cells of the cancer.

10. The pharmaceutical composition for preventing or treating cancer according to claim 7, wherein the cancer is one in which TGF-β is overexpressed or overactivated.

11. A cell therapy agent for preventing or treating cancer, comprising natural killer cells treated with a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or a polynucleotide encoding the same.

12. A method for enhancing the in vitro or ex vivo activity of natural killer cells, comprising the step of treating natural killer cells with a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or a polynucleotide encoding the same.

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