Transformed immune cells induce chemotaxis toward heterologous immune cells

Abstract

The present invention relates to immune cells expressing IL-7 (interleukin-7), CCL19 (CC Motif Chemokine Ligand 19), or a combination thereof, and compositions containing the same as active ingredients for the prevention or treatment of cancer or infectious diseases. The present invention provides a multifaceted and synergistic therapeutic effect through the complementary immune response between the patient's endogenous T cells and the infused natural killer cells by injecting only a therapeutically effective amount of immune cells other than T cells. Furthermore, the present invention also provides a significantly improved therapeutic effect when administering T cells and immune cells other than T cells, specifically natural killer cells, by allowing these different cell groups to act more intensively at the lesion site.

Description

[Technical Field]

[0001] The present invention relates to immune cells, particularly immune cells other than T cells, that express specific chemotactic factors to induce migration of T cells to lesion sites, particularly natural killer cells. [Background technology]

[0002] Cancer is a disease fundamentally associated with abnormal tissue growth regulation. Compared to normal cells, cancer cells grow and proliferate excessively, invading neighboring tissues and metastasizing to distant tissues. Anticancer treatments involve physically removing cancerous tissue or using radiation and anticancer drugs to inhibit the expression of genes necessary for cancer cell survival, such as oncogenes, telomerase, growth factor receptors, and signaling molecules, or to induce cell death. However, because these treatments can damage not only cancer cells but also normal cells, immunotherapy has attracted attention as a way to specifically eliminate tumor tissue. In particular, in recent decades, active research has been conducted on immune cell-based anticancer therapies, which use immune cells such as dendritic cells, natural killer cells, and T cells to activate the patient's internal immune response and eliminate tumor tissue.

[0003] Natural killer cells (NKCs) are lymphoid cells that account for approximately 15% of peripheral blood lymphocytes and play an important role in the innate immune response. Specifically, they activate dendritic cells and induce cytotoxic T lymphocytes (CTLs) to specifically target tumors and eliminate tumor cells. NKCs directly kill malignant tumors such as sarcoma, myeloma, lymphoma, and leukemia. The therapeutic effect of ex vivo activated NKCs administered to patients with hematological cancers such as leukemia after allogeneic bone marrow transplantation has been confirmed (Blood Cells Molecules & Disease, 33: 261-266, 2004). However, because most NKCs exist in an inactivated state in normal individuals, active research is underway to improve the therapeutic efficacy of ex vivo activation of NKCs isolated from blood.

[0004] Meanwhile, T cells, the other axis of immune cells, are lymphocytes that are produced in the bone marrow and mature in the thymus. They have memory capacity in the immune system and provide information to B cells to induce antibody production. After undergoing an immune tolerance test in the thymus, T cells differentiate into four types of cells: cytotoxic T cells, helper T cells, regulatory T cells, and memory T cells. Of these, cytotoxic T cells, known as CD8+ T cells, bind to type I MHC to identify cancer cells or infected cells and, like natural killer cells, primarily target diseased cells.

[0005] Although the combined administration of natural killer cells and cytotoxic T cells can maximize the therapeutic effect, there is a need to develop a new cell therapy that can overcome the inefficiency of injecting two cells, each with a therapeutically effective dose.

[0006] Numerous papers and patent documents are referenced throughout this specification and citations are provided, the disclosures of which are incorporated herein by reference in their entirety to more clearly describe the state of the art to which this invention pertains and the content of the present invention. Summary of the Invention [Problem to be solved by the invention]

[0007] The present inventors have conducted extensive research to develop an excellent cell therapy agent that can more efficiently eliminate diseased cells or tissues through a complex immune response mediated by a combination of different immune cells. As a result, they have discovered that when immune cells expressing IL-7, CCL19, or a combination thereof, specifically immune cells other than T cells, most specifically natural killer cells, are injected into a subject, endogenous T cells proliferated and homed by IL-7 and CCL19 and the injected natural killer cells act simultaneously at the lesion site, inducing a multifaceted and synergistic immune response, which led to the completion of the present invention.

[0008] Therefore, an object of the present invention is to provide immune cells that express IL-7, CCL19, or a combination thereof, and compositions for preventing or treating cancer or infectious diseases, comprising the same as an active ingredient.

[0009] Another object of the present invention is to provide a composition for inducing proliferation or homing of heterologous immune cells, comprising as an active ingredient a nucleic acid molecule encoding IL-7, CCL19, or a combination thereof.

[0010] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, the claims and the drawings. [Means for solving the problem]

[0011] According to one aspect, the present invention provides an immune cell that expresses a nucleic acid molecule encoding IL-7 (interleukin-7) or a functional portion thereof; a nucleic acid molecule encoding CCL19 (CC Motif Chemokine Ligand 19) or a functional portion thereof; or a combination thereof.

[0012] The present inventors have conducted extensive research to develop an excellent cell therapy agent that can more efficiently eliminate diseased cells or tissues through a complex immune response using a combination of different immune cells. As a result, they have found that when immune cells expressing IL-7, CCL19, or a combination thereof, specifically immune cells other than T cells, most specifically natural killer cells, are infused into a subject, endogenous T cells proliferated and homed by the IL-7 and CCL19, and the infused natural killer cells act simultaneously at the lesion site, thereby inducing a multifaceted and synergistic immune response.

[0013] As used herein, the term "immune cell" refers to any cell involved in the initiation or promotion of an immune response, and more specifically, refers to an immune effector cell. Examples of immune cells include, but are not limited to, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and dendritic cells. More specifically, the immune cell is an immune cell other than a T cell, and most specifically, a natural killer cell.

[0014] As used herein, the term "T cells" refers to a group of cells within the same scope as understood in the art, specifically including, but not limited to, CD8+ or CD4+ T cells that provide effector or helper functions that directly lyse target cells or induce the death of target cells, and includes any cells classified as "T cells" in the technical field to which the present invention pertains.

[0015] As used herein, the term "functional portion" refers to a fragment of a full-length protein in which some amino acid residues have been deleted, and which is an analog of the full-length protein that maintains its original biological activity and function.

[0016] As used herein, the term "nucleic acid molecule" is intended to encompass DNA (gDNA and cDNA) and RNA molecules, and nucleotides, the basic building blocks of nucleic acid molecules, include not only natural nucleotides but also analogues in which the sugar or base moiety has been modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

[0017] Those skilled in the art will recognize that the nucleic acid molecules of the present invention are not limited to the nucleotide sequences in the attached sequence listing. Nucleotide mutations may not result in changes in protein structure due to codon degeneracy or the presence of different codons encoding biologically equivalent amino acids. Taking into account such mutations with biologically equivalent activity, the nucleic acid molecules used in the present invention are also understood to include sequences that exhibit substantial identity to the sequences set forth in the sequence listing. The term "substantial identity" refers to sequences that exhibit at least 80% homology, more specifically 85% homology, even more specifically 90% homology, and most specifically 95% homology, when the sequences of the present invention are aligned with any other sequence for maximum correspondence and the aligned sequences are analyzed using algorithms commonly used in the art.

[0018] As used herein, the term "express" includes artificial expression of an exogenous gene not expressed by the immune cells of the present invention by introducing it via a gene carrier, natural expression of an endogenous gene by an endogenous expression system, or overexpression of an endogenous gene using a gene carrier to increase the natural expression level. Therefore, the immune cells of the present invention encompass cells that endogenously express IL-7 and CCL19, cells that endogenously express IL-7 and artificially express CCL19, cells that endogenously express CCL19 and artificially express IL-7, and cells that artificially express IL-7 and CCL19.

[0019] As used herein, the term "express" refers to artificially making a gene replicable in a target cell as an extrachromosomal element or by chromosomal integration using a gene carrier to express a foreign gene or overexpress an endogenous gene in a target cell. Therefore, the term "express" has the same meaning as "transformation," "transfection," or "transduction."

[0020] As used herein, the term "gene delivery system" refers to any means for transporting a gene into a cell, and gene delivery has the same meaning as intracellular gene transduction. At the tissue level, the term "gene delivery" has the same meaning as gene spread. Therefore, the gene delivery system of the present invention is described as a gene penetration system and a gene spread system.

[0021] The nucleotide sequences of the IL-7 and CCL19 genes are applicable to all gene delivery systems commonly used for gene transfer, such as plasmids, adenoviruses, adeno-associated viruses, retroviruses, lentiviruses, herpes simplex viruses, vaccinia viruses, liposomes, or niosomes.

[0022] When the gene carrier of the present invention is a naked recombinant DNA molecule or a plasmid, various methods can be used for the gene transfer, including microinjection (Capecchi, MR, Cell, 22:479 (1980)), calcium phosphate precipitation (Graham, Flet et al., Virology, 52:456 (1973)), electroporation (Tur-Kaspa et al., Mol. Cell Biol., 6:716-718 (1986)), liposome-mediated transfection (Biochim. Biophys. Acta, 721:185-190 (1982)), DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5:1188-1190 (1985)), and gene bombardment (Yang et al. The gene can be transferred into cells by the method (see, for example, J. Immunol., 1990, 87:9568-9572 (2002)). Specifically, electroporation can be used.

[0023] According to a specific embodiment of the present invention, the IL-7 comprises an amino acid sequence having 85% or more sequence identity, more specifically 90% or more sequence identity, and most specifically 95% or more sequence identity with the amino acid sequence of the first sequence in the sequence listing.

[0024] According to a specific embodiment of the present invention, the CCL19 comprises an amino acid sequence having 85% or more sequence identity, more specifically 90% or more sequence identity, and most specifically 95% or more sequence identity with the amino acid sequence of the second sequence in the sequence listing.

[0025] According to a specific embodiment of the present invention, the nucleic acid molecule encoding IL-7 or a functional part thereof comprises the nucleotide sequence of the third sequence in the sequence listing.

[0026] According to a specific embodiment of the present invention, the nucleic acid molecule encoding CCL19 or a functional part thereof comprises the nucleotide sequence of the fourth sequence in the sequence listing.

[0027] According to the present invention, the third and fourth sequences in the sequence listing are nucleotide sequences in which the base sequences of the IL-7 and CCL19 genes have been codon-optimized so that they are efficiently expressed in natural killer cells.

[0028] In another aspect, the present invention provides a composition for preventing or treating cancer or infectious diseases, which comprises the above-mentioned immune cells of the present invention as an active ingredient.

[0029] When the immune cells of the present invention are used as cell therapy agents, they can be used to treat a variety of tumors and infectious diseases. The immune cells of the present invention, specifically natural killer cells, can be used to treat all types of tumors, including solid cancers and blood cancers, including, but not limited to, gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumors, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma. Infectious diseases that can be prevented or treated using the immune cells of the present invention, specifically natural killer cells, are diseases caused by infection with viruses or pathogenic bacteria, and include all diseases that can be transmitted through the respiratory tract, blood, or skin contact. Such infectious diseases include, but are not limited to, hepatitis B and C, human papilloma virus (HPV) infection, cytomegalovirus infection, viral respiratory diseases, and influenza.

[0030] As used herein, the term "prevention" means inhibiting the occurrence of a disease or condition in a subject who has not been diagnosed as having the disease or condition but is susceptible to such disease or condition.

[0031] As used herein, the term "treatment" refers to (a) the inhibition of the progression of a disease, disorder, or symptom; (b) the alleviation of a disease, disorder, or symptom; or (c) the elimination of a disease, disorder, or symptom. When the immune cells of the present invention are administered to a subject, a complex immune response is initiated by natural killer cells and endogenous T cells induced thereby at the lesion site, or exogenously injected autologous or allogeneic T cells, leading to the death of cancer cells, infected cells, or pathogenic bacteria, thereby inhibiting the progression, eliminating, or alleviating symptoms caused by tumors or infectious diseases. Therefore, the composition of the present invention may be used as a cell therapy composition for a disease by itself, or may be administered together with other pharmacological components, such as therapeutic T cells or other known anticancer agents, to be used as a therapeutic adjunct for the disease. Therefore, as used herein, the terms "treatment" or "therapeutic agent" encompass the meaning of "therapeutic adjunct" or "therapeutic adjunct."

[0032] As used herein, the term "administration" or "administering" refers to administering a therapeutically effective amount of a composition of the present invention directly to a subject, thereby allowing the same amount to be formed in the subject's body.

[0033] In the present invention, the term "therapeutically effective amount" means the content of the composition of the present invention contained in an amount sufficient to provide a therapeutic or prophylactic effect to an individual to whom the composition of the present invention is to be administered, and thus includes a "prophylactically effective amount."

[0034] As used herein, the term "subject" includes, without limitation, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus monkey. Specifically, the subject of the present invention is a human.

[0035] In yet another aspect, the present invention provides a composition for inducing the proliferation or homing of heterologous immune cells, comprising as an active ingredient a nucleic acid molecule encoding IL-7 (interleukin-7) or a functional portion thereof; a nucleic acid molecule encoding CCL19 (CC Motif Chemokine Ligand 19) or a functional portion thereof; or a combination thereof.

[0036] The nucleic acid molecules used in the present invention have already been described in detail, so their description will be omitted to avoid excessive repetition.

[0037] As used herein, the term "heterogeneous immune cells" refers to immune cells of a different type from the subject cells into which the nucleic acid molecules of the present invention are introduced. Specifically, the heterogeneous immune cells are T cells or dendritic cells. According to the present invention, immune cells that simultaneously express IL-7 and CCL19, specifically immune cells other than T cells, most specifically natural killer cells, can be injected into a subject to induce T cell proliferation and chemotaxis, thereby allowing a large number of endogenous T cells to migrate to the lesion site. The term "chemotaxis" refers to the positive (toward the stimulus) or negative (away from the stimulus) migration of migratory cells induced by a chemical stimulus, specifically positive migration. According to the present invention, T cells are proliferated and differentiated by IL-7 and activated by the corresponding chemotactic factor, CCL19, to migrate to natural killer cells expressing IL-7 and CCL19, thereby forming a heterogeneous cell population at the lesion site composed of mutually complementary immune cells, namely, T cells and natural killer cells. Therefore, by introducing the composition of the present invention into natural killer cells, a therapeutically effective dose of a single cell can serve as a therapeutic adjuvant that achieves the effects of combined administration of different cell groups.

[0038] According to a specific embodiment of the present invention, the nucleic acid molecules may be contained in a composition either inserted together in one gene carrier or inserted separately in two gene carriers.

[0039] According to yet another aspect of the present invention, there is provided a method for preventing or treating cancer or an infectious disease, comprising administering the immune cells of the present invention to a subject. The immune cells used in the present invention and the cancers and infectious diseases that can be prevented or treated thereby have already been described in detail, so a detailed description thereof will be omitted to avoid excessive repetition.

[0040] According to yet another aspect, the present invention provides a method for inducing the proliferation or homing of heterologous immune cells, comprising the step of introducing into immune cells a nucleic acid molecule encoding interleukin-7 (IL-7) or a functional portion thereof; a nucleic acid molecule encoding CC Motif Chemokine Ligand 19 (CCL19) or a functional portion thereof; or a combination thereof. The nucleic acid molecule and heterologous immune cells used in the present invention have already been described in detail, so their description will be omitted to avoid excessive repetition. [Effects of the Invention]

[0041] The features and advantages of the present invention can be summarized as follows: (a) The present invention provides immune cells that express IL-7, CCL19, or a combination thereof, and compositions for preventing or treating cancer or infectious diseases, comprising the same as an active ingredient. (b) The present invention can achieve multifaceted and synergistic therapeutic effects by injecting only a therapeutically effective amount of immune cells other than T cells, specifically natural killer cells, through the mutually complementary immune responses between the patient's endogenous T cells and the infused natural killer cells. (c) The present invention also enables the combined administration of T cells and immune cells other than T cells, specifically natural killer cells, to achieve significantly improved therapeutic effects by allowing these different cell groups to act more intensively on the lesion site. [Brief explanation of the drawings]

[0042] [Figure 1] FIG. 1 is a schematic diagram showing the structure of the pEF1-IRES empty vector used as a negative control vector.

[0043] [Figure 2] FIG. 1 is a schematic diagram showing the structure of the pEF1-IRES vector into which the IL-7 and CCL19 genes have been inserted.

[0044] [Figure 3] FIG. 1 is a schematic diagram showing the structure of the pEF1-IRES vector into which the CCL19 and IL-7 genes have been inserted. DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention will be described in more detail with reference to the following examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of explaining the present invention in more detail, and that the scope of the present invention is not limited to these examples according to the gist of the present invention. [Example]

[0046] Example 1: Construction of IL-7 and CCL19 expression vectors To generate human NK cells expressing IL-7 and CCL19, we used the pEF1α-IRES bicistronic mammalian expression vector (Takara, CAT#631970) under the control of the human elongation factor 1 alpha (EF1α) promoter.

[0047] The ORF sequences of the genes to be inserted into the vector, IL-7 and / or CCL19, were codon-optimized and synthesized (Sequences 3 and 4 in the Sequence Listing, respectively). IL-7 or CCL19 was cloned into the multiple cloning site A (MCS A) of the pEF1a-IRES vector, and CCL19 or IL-7 was cloned into MCS B. The pEF1a-IRES vector contained an internal ribosome entry site (IRES) between the two MCSs, allowing for simultaneous expression of the two genes. The structure of the negative control vector, the empty vector, is shown in Figure 1, the IL-7 / CCL19 expression vector in Figure 2, and the CCL19 / IL7 expression vector in Figure 3.

[0048] Example 2: Confirmation of IL-7 and CCL19 expression The NK92 cell line was purchased from ATCC (CAT#CRL-2407) and cultured in Alpha Minimum Essential Medium (α-MEM) containing 12.5% ​​horse serum (Sigma, CAT#H1270), 12.5% ​​fetal bovine serum (FBS, Gibco, CAT#10099141), and interleukin-2 (IL-2; Peprotech, CAT#200-02). To generate the NK92 cell line expressing IL-7 and CCL19 (hereafter referred to as the "transformed NK92 cell line"), 1 × 10 cells were cultured. 6 Transformed NK92 cell lines were generated by electroporation at a density of NK92 cells / 0.1 mL.

[0049] Electroporation is Neon TMTransfection was performed using a Transfection System (ThermoFisher, CAT#MPK5000) under the following conditions: voltage of 1800-1850V, pulse length of 10 ms, one time, and 10-20 μg of DNA (negative control vector, IL-7 / CCL19 expression vector, or CCL19 / IL-7 expression vector prepared in Example 1). After culturing for 1 day in 2 mL of RPMI1640 medium (Gibco #11875168) containing 10% FBS, the expression levels of IL-7 or CCL19 were measured, and a Transwell assay was performed.

[0050] To measure the expression levels of IL-7 and CCL19 in the transformed NK92 cell line, the culture medium was collected in a 6-well plate and centrifuged at 800 g for 5 minutes to separate the supernatant. 0.1 mL of the supernatant per well was appropriately diluted and used in an enzyme-linked immunosorbent assay (ELISA) to measure the expression levels of IL-7 or CCL19 (IL-7, Komabiotech, CAT#k0331215; CCL19, Abcam, CAT#ab100601).

[0051] The immunosorbent assay experiments for IL-7 and CCL19 were performed according to the standard experimental methods of each manufacturer, and the final values ​​were measured at a wavelength of 450 nm and are shown in Table 1. [Table 1] *ND: Not detected

[0052] The results in Table 1 confirmed that the IL-7 / CCL19 NK92 cell line and the CCL19 / IL-7 NK92 cell line secreted IL-7 and CCL19. In contrast, IL-7 and CCL19 were not detected or were detected at insufficient levels in the negative control cells injected with the empty vector.

[0053] Example 3: Analysis of T cell chemotaxis and proliferation by IL-7 and CCL19-expressing NK cells To confirm the chemotactic and proliferative effects of IL-7 and CCL19 secreted by NK cells on T cells, a 12-well transwell (Corning, CAT#CLS3421) was used, and the chamber filter was a 5 μm polycarbonate filter.

[0054] Comparison of IL-7 / CCL19 NK92 cells and negative control groups with the HuT78 T cell line HuT78 T cells (ATCC, CAT#TIB-161) 1 × 10 7 The cells were cultured for 24 hours in IMDM (Gibco, CAT#12440053) medium supplemented with 1% PS and no FBS. 6 100 μl of HuT78 cells at a density of 100 cells / mL were dispensed into the upper chamber using the same medium as above, and the culture medium of the IL-7 / CCL19 NK92 cell line prepared in Example 2, which had been cultured for 2-3 days, was dispensed into the lower chamber.

[0055] After culturing for one day in a cell incubator at 37°C and 5% CO2, the upper chamber was removed and the cells were further cultured for three days. To measure the number of cells in the lower chamber, 400 μl of medium per well was centrifuged at 800 g for 5 minutes, and the supernatant was removed to obtain a concentrated sample. 10 μl of the concentrated sample was mixed with 10 μl of Trypan blue at a 1:1 ratio and used for Countess. TM The results were calculated as the total number of viable cells in 400 μl of the culture medium, and are shown in Table 2 below. [Table 2]

[0056] Comparison of IL-7 / CCL19 NK92 cells, CCL19 / IL-7 NK92 cells, and negative control groups using PBMC-derived T cells To confirm the chemotaxis of T cells isolated from peripheral blood mononuclear cells (PBMC), T cells were isolated from PBMC using EasySep TM Isolate 2 x 10 cells using the Human T cell Isolation Kit (STEMCELL #17951). 6 T cells at a concentration of 100 cells / mL were diluted with RPMI1640 medium containing 2% FBS, and 100 μL of the diluted solution was dispensed into the upper chamber. The lower chamber contained the culture medium obtained by culturing the IL-7 / CCL19 and CCL19 / IL-7 transformed NK92 cell lines prepared in Example 2 for one day.

[0057] After culturing for one day in a cell incubator at 37°C and 5% CO2, the upper chamber was removed and the cells were further cultured for three days. To measure the number of cells in the lower chamber, 500 μl of medium per well was centrifuged at 800 g for 5 minutes, and the supernatant was removed to obtain a concentrated sample. 10 μl of the concentrated sample was mixed with 10 μl of Trypan blue in a 1:1 ratio and used for Countess. TM The results were calculated as the total cell number and are shown in Table 3 below. [Table 3]

[0058] As shown in Tables 2 and 3, the number of T cells that migrated to the lower chamber was significantly higher in the IL-7 / CCL19 NK92 cell line and CCL19 / IL-7 NK92 cell line treatment groups than in the negative control group (empty vector-transfected NK92 cell line). This confirmed the chemotaxis of T cells and the proliferation-inducing effect of NK92 cell lines transformed to secrete IL-7 and CCL19.

[0059] Example 4: Measurement of the cancer cell-killing effect of transformed NK92 cell line and T cells transferred by it, and secreted factors related to this 2 x 10 HepG2 liver cancer cell line (ATCC #HB-8065) 5 The cells were diluted in RPMI1640 medium containing 10% FBS at a concentration of 100 cells / mL, then 0.1 mL was dispensed into each well of a 96-well plate and cultured for 1 day. 0.5 mL of RPMI1640 medium containing the transformed NK92 cell line, which had been cultured for 1 day by electroporation in the same manner as in Example 2, was transferred to the lower chamber of a transwell chamber in the same manner as in Example 3, and 2 × 10 T cells isolated from PBMCs were placed in the upper chamber. 5 T cell migration was induced for 1 day by transferring 1 × 10 cells / 0.1 mL to the lower chamber. 5 CD3 / CD28 Dynabeads and 100 U IL-2 were added to activate the migrated T cells. After one day of culture, the upper chamber was removed and 0.1 mL of medium was transferred per well to a 96-well plate containing HepG2 liver cancer cell lines. The cells were then cultured for an additional three days. After washing three times with PBS (Phosphate Buffer Saline), the medium was replaced with 0.1 mL of 10% FBS-RPMI 1640 medium containing 1% CCK-8 solution and cultured for an additional two hours. The absorbance at 450 nm was measured to determine the cell viability of the HepG2 liver cancer cell line. The cell viability was calculated as follows: The results are shown in Table 4.

number

[0060] In addition, using the medium collected from the above-mentioned killing assay, the secretion levels of granzyme B (Grz-B), interferon-γ (IFN-γ), and tumor necrosis factor (TNF)-α from activated T cells and activated NK cells were measured by enzyme-linked immunosorbent assay (ELISA; Grz-B, Abcam #ab235635; IFN-γ, Komabiotech #K0331121; TNF-α, Komabiotech #K0331131). The results are shown in Table 5. Each value in Tables 4 and 5 is expressed as the mean ± standard deviation. [Table 4] [Table 5] *ND: Not detected

[0061] As shown in Table 4, the experimental groups containing the IL-7 / CCL19 NK92 cell line and the CCL19 / IL-7 NK92 cell line demonstrated significantly higher killing effects on HepG2 cells than the negative control group, which was an NK92 cell line transfected with an empty vector. As shown in Table 5, factors related to the killing effects on HepG2 cells were also secreted at higher levels in the IL-7 / CCL19 NK92 cell line and CCL19 / IL-7 NK92 cell line test groups compared to the negative control group. These findings confirm the potential use of both the IL-7 / CCL19 NK cell line and the CCL19 / IL-7 NK cell line as effective anti-cancer therapeutic agents.

[0062] Although certain parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A natural killer cell transformed with a nucleic acid molecule encoding IL-7 (interleukin-7) or a functional portion thereof; and a nucleic acid molecule encoding CCL19 (CC Motif Chemokine Ligand 19) or a functional portion thereof.

2. A composition for preventing or treating cancer or infectious diseases, comprising the natural killer cells of claim 1 as an active ingredient.

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