Composition for NK cell proliferation promotion or activation, and use thereof

Abstract

The present invention provides a composition for promoting NK cell proliferation or improving cell lysis activity of NK cells, comprising, as an active ingredient, feeder cells in which membrane bound interleukin-13 (mbIL-13) or membrane bound interleukin-2 (mbIL-2) is expressed on a cell membrane. The composition of the present invention expresses mbIL-13 or mbIL-2 on the cell membrane of the feeder cells, thereby having excellent effects of promoting the NK cell proliferation and inducing improvement of cell lysis activity of NK cells.

Description

Composition for promoting or activating the proliferation of NK cells and its use

[0001] [Cross-reference with related applications]

[0002] This application claims the benefit of priority to Korean Patent Application No. 10-2023-0075200, filed June 12, 2023, the entire contents of which are incorporated herein by reference.

[0003] The present invention relates to a composition for promoting NK cell proliferation or enhancing NK cell cytolytic activity.

[0004]

[0005] Natural killer cells (NK cells) are a type of cytotoxic lymphocyte that plays a crucial role in innate immunity. NK cells respond to virus-infected or cancerous cells, and these responses are determined by signals emitted by NK cell activating and inhibitory receptors that interact with their respective ligands on the target cell. Typically, if the activating signal is stronger than the inhibitory signal, NK cells will attack the target cell. However, if the inhibitory signal is stronger, NK cells will not attack. Therefore, normal cells are not attacked by NK cells because they exhibit inhibitory signals due to the presence of inhibitory receptor ligands (major histocompatibility complex, MHC) on NK cells.

[0006] Some cancer cells can be reduced due to abnormalities in MHC molecules on their surface. In these cases, NK cells lack inhibitory signals, allowing them to attack target cells. They secrete perforin, which punctures the cell membranes of infected or cancerous cells, and release granzymes, which then kill these cells, resulting in cytotoxicity. Many cancer patients, including those with B-cell lymphoma, have been found to have defects in NK cell numbers and anticancer activity, and NK cell dysfunction is known to be closely related to the development and treatment of these cancers.

[0007] Therefore, cancer treatment methods that utilize the anticancer properties of NK cells are emerging, and the development of expansion or culture methods to secure large numbers of high-performance NK cells is crucial. Furthermore, individuals with impaired or abnormal NK cell function are known to be associated with cancer and various immune-related diseases, highlighting the need for research aimed at promoting NK cell proliferation or enhancing cytolytic activity.

[0008]

[0009] The present invention aims to provide genetically engineered support cells for amplification or activation of NK cells.

[0010]

[0011] One aspect of the present invention provides a composition for promoting NK cell proliferation or enhancing NK cell cytolytic activity, which comprises feeder cells expressing membrane bound interleukin-13 (mbIL-13) on the cell membrane.

[0012] Another aspect of the present invention provides a composition for promoting NK cell proliferation or enhancing NK cell cytolytic activity, which comprises feeder cells expressing membrane bound interleukin-2 (mbIL-2) on the cell membrane.

[0013] Another aspect of the present invention provides a composition for promoting NK cell proliferation or enhancing NK cell cytolytic activity, comprising feeder cells expressing membrane bound interleukin-13 (mbIL-13) on the cell membrane and feeder cells expressing membrane bound interleukin-2 (mbIL-2) on the cell membrane.

[0014] Another aspect of the present invention provides a method for promoting the proliferation of NK cells, comprising the step of culturing a sample containing NK cells in a medium containing the composition.

[0015] Another aspect of the present invention provides a method for enhancing the cytolytic activity of NK cells, comprising the step of culturing a sample containing NK cells in a medium containing the composition.

[0016]

[0017] The support cells of the present invention can increase the proliferation of NK cells by at least two to several times compared to cells that are not genetically modified, and improve the cytolytic activity of NK cells. Therefore, a composition including the support cells of the present invention can be used to proliferate NK cells and be used as an immunotherapeutic agent.

[0018]

[0019] Figure 1 is a schematic diagram showing genes that produce mbIL-13 and mbIL-2 for manufacturing support cells of the present invention.

[0020] Figure 2 is a graph confirming the expression characteristics of mbIL-13 and mbIL-2 in support cells according to the present invention.

[0021] Figures 3a and 3b are graphs showing the number of NK cells when NK cells were co-cultured with mbIL-13 and mbIL-2 support cells according to the present invention.

[0022] Figure 4 is a graph comparing the amount of membrane receptors expressed in NK cells when NK cells were co-cultured with mbIL-13 and mbIL-2 support cells according to the present invention and when NK cells were co-cultured with K562 cells.

[0023] Figure 5 is a graph showing the survival rate of cancer cells when co-cultured with mbIL-13 and mbIL-2 support cells and NK cells according to the present invention and when co-cultured with K562 cells and NK cells.

[0024]

[0025] Hereinafter, the present invention will be described in more detail to help understand the present invention.

[0026] The terms and words used in this specification and claims should not be interpreted as limited to their usual or dictionary meanings, but should be interpreted as meanings and concepts that conform to the technical idea of ​​the present invention, based on the principle that the inventor can appropriately define the concept of the term to explain his or her own invention in the best way.

[0027]

[0028] 1. Support cell of the present invention

[0029] One aspect of the present invention provides feeder cells in which membrane bound interleukin-13 (mbIL-13) is expressed on the cell membrane.

[0030] Another aspect of the present invention provides a feeder cell expressing membrane bound interleukin-2 (mbIL-2) on the cell membrane. The term 'feeder cell' of the present invention may refer to a cell that does not have the ability to divide and proliferate by radiation exposure but has metabolic activity and thus produces various metabolites to help the proliferation of target NK cells. The culture feeder cell that can be used in the present specification is an animal cell line into which a gene has been introduced, such as a human chronic myelogenous leukemia cell line (e.g., K562 cells), RPMI8866, EBV_LCL, 721.221, HFWT, etc., and in particular, may be a K562 cell.

[0031] The support cells of the present invention can be used as cultured feeder cells that promote the proliferation of NK cells. Generally, the K562 cell line, a representative feeder cell used for NK cell proliferation, is only used for the proliferation of NK cells, and therefore the K562 cell line itself should not proliferate. Therefore, before culturing with NK cells, the K562 cell line is pretreated with strong radiation so that the K562 cell line itself does not proliferate at all, but only promotes the proliferation of NK cells. Meanwhile, for the efficient proliferation of NK cells, most cytokines such as IL-2 and IL-13 must be continuously exposed to NK cells. Therefore, when cancer cell line-based feeder cells are genetically engineered to express cytokines such as IL-13 or IL-2, the cancer cell line itself that has been pretreated with radiation, etc., cannot survive for a long time during the culture process, and thus there is a problem that the selective proliferation efficiency of NK cells is reduced. However, support cells expressing mbIL-13 or mbIL-2 according to the present invention on the cell membrane can promote the proliferation of NK cells.

[0032] Specifically, the support cells expressing mbIL-13 or mbIL-2 on the cell membrane may be created through genetic engineering.

[0033] The above genetic manipulation "genetic manipulation" or "genetically engineered" means an act of introducing one or more genetic modifications into a cell or a cell produced thereby. It may include an exogenous gene encoding the above-mentioned gene. The above "exogenous" means that the referenced molecule or the referenced activity has been introduced into the host cell. The molecule may be introduced into the host genetic material as an encoding nucleic acid, such as by insertion into the host chromosome, or as a non-chromosomal genetic material such as a plasmid. In relation to the expression of the encoding nucleic acid, the term "exogenous" indicates that the encoding nucleic acid has been introduced into the organism in a form that is expressible. In relation to the biosynthetic activity, the above-mentioned exogenous indicates an activity introduced into the host parent cell. The source may be, for example, a homologous or It may be a heterologous coding nucleic acid. Therefore, "endogenous" refers to the molecule or activity present in the host cell. Similarly, with respect to the expression of a coding nucleic acid, "endogenous" refers to the expression of the coding nucleic acid contained within the organism. The heterologous refers to a molecule or activity from a source other than the species mentioned, and the homologous refers to a molecule or activity from the host parent cell. Therefore, exogenous expression of a coding nucleic acid may utilize either or both heterologous or homologous coding nucleic acids.

[0034] The term "vector" in the present invention refers to a vector capable of expressing a target protein in a suitable host cell, and a genetic construct including regulatory elements operably linked to express a gene insert. The vector may include expression regulatory elements such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, or an enhancer, and the promoter of the vector may be constitutive or inducible. In addition, the vector may be an expression vector capable of stably expressing the target protein in a host cell. The expression vector may be a conventional one used in the art to express foreign proteins in plants, animals, or microorganisms. The recombinant vector may be constructed by various methods known in the art. For example, the vector may include a selectable marker for selecting a host cell containing the vector, and, if it is a replicable vector, may include an origin of replication. Additionally, the vector may be self-replicating or may be introduced into the host DNA, and the vector may be selected from the group consisting of a plasmid, a lentivirus, an adenovirus, an adeno-associated virus, a retrovirus, a herpes simplex virus, and a bathysitis virus.

[0035] Additionally, in the above vector, the polynucleotide sequence encoding the aforementioned target protein may be operably linked to a promoter. As used herein, the term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, whereby the control sequence controls the transcription or translation of the other nucleic acid sequence.

[0036] Therefore, the support cells are transformed with a vector containing a nucleic acid encoding mbIL-13 or mbIL-2 protein.

[0037] The term "membrane bound interleukin" of the present invention refers to an interleukin bound to a cell membrane, and may be distinct from an interleukin secreted extracellularly. mbIL-13 may have a sequence homology of about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more to the nucleic acid sequence of SEQ ID NO: 1 or its amino acid sequence. mbIL-2 may have a sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% to the nucleic acid sequence of SEQ ID NO: 2 or its amino acid sequence.

[0038] The term homology of the present invention means that one oligonucleotide strand (sense or antisense strand) of the activating RNA or DNA has similarity to the coding strand or template strand within the region of the target gene promoter sequence.

[0039] The support cell in which mbIL-13 of the present invention is expressed on the cell membrane may contain a nucleic acid encoding mbIL-13 (SEQ ID NO: 1).

[0040] The support cell in which the mbIL-2 of the present invention is expressed on the cell membrane may contain a nucleic acid encoding mbIL-2 (SEQ ID NO: 2).

[0041] The support cell of the present invention, in which mbIL-13 and mbIL-2 are expressed on the cell membrane, may include a nucleic acid encoding mbIL-13 (SEQ ID NO: 1) and a nucleic acid encoding mbIL-2 (SEQ ID NO: 2).

[0042] In addition, the support cell of the present invention, in which mbIL-13 or mbIL-2 is expressed on the cell membrane, may further include a CD8a TM (transmembrane) gene (SEQ ID NO: 3) at one end of the nucleic acid encoding the mbIL-13 or mbIL-2.

[0043] In addition, the support cell of the present invention, in which mbIL-13 or mbIL-2 is expressed on the cell membrane, may further include a CD8a SP (signal peptide) gene (SEQ ID NO: 4) at the other end of the nucleic acid encoding mbIL-13 or mbIL-2.

[0044] In addition, the support cell of the present invention, in which mbIL-13 or mbIL-2 is expressed on the cell membrane, may further include a CD8 α hinge gene (SEQ ID NO: 5) between the nucleic acid encoding the mbIL-13 or mbIL-2 and the TM (transmembrane) gene of the CD8a.

[0045] In addition, the support cells of the present invention, in which mbIL-13 or mbIL-2 is expressed on the cell membrane, may further include a marker gene (Flag tag) in order to visually confirm whether mbIL-13 or mbIL-2 is expressed. The marker may be one commonly used in the art to identify specific cells, and an example thereof may include a gene encoding a fluorescent protein. Specifically, the gene encoding the fluorescent protein may be used without limitation as long as it can express a protein that generates fluorescence on its own, and the gene encoding the fluorescent protein may be a gene of GFP, GFPUV, or RFP, and preferably may include a gene represented by SEQ ID NO: 6.

[0046] Accordingly, the support cell may comprise a nucleic acid encoding mbIL-13 or mbIL-2, a CD8a SP (signal peptide) gene and a CD8a TM (transmembrane) gene at the end of the nucleic acid.

[0047] Additionally, the support cell may include a nucleic acid encoding mbIL-13 or mbIL-2, a CD8a SP (signal peptide) gene at the end of the nucleic acid, a CD8a TM (transmembrane) gene, and a flag tag gene.

[0048] In a specific embodiment of the present invention, the support cell was transformed with a vector comprising a nucleic acid encoding mbIL-13, an SP gene of CD8a, a TM gene of CD8a, a CD8 α hinge gene, and a flag tag gene at the end of the nucleic acid.

[0049] In addition, in a specific embodiment of the present invention, the support cell was transformed with a vector comprising a nucleic acid encoding mbIL-2, an SP gene of CD8a, a TM gene of CD8a, a CD8 α hinge gene, and a flag tag gene at the end of the nucleic acid.

[0050]

[0051] 2. Composition containing the above support cells and its use

[0052] Another aspect of the present invention provides a composition for promoting NK cell proliferation or enhancing NK cell cytolytic activity.

[0053] The composition for promoting NK cell proliferation or enhancing cytolytic activity of NK cells of the present invention comprises the feeder cells described above in the above "1. Feeder cells of the present invention." Therefore, the composition for promoting NK cell proliferation or enhancing cytolytic activity of NK cells of the present invention may comprise at least one selected from the group consisting of i) feeder cells expressing membrane-bound interleukin-13 (mbIL-13) on the cell membrane; and ii) a mixture of feeder cells expressing mbIL-13 on the cell membrane and feeder cells expressing membrane-bound interleukin-2 (mbIL-2) on the cell membrane.

[0054] In the present invention, "NK cells" are cytotoxic lymphocytes that constitute a major component of the innate immune system, and are defined as large granular lymphocytes (LGL) and constitute a third cell differentiated from B and T lymphocytes that produce common lymphoid progenitors (CLPs). The NK cells include NK cells without additional modification derived from any tissue source, and may include not only mature NK cells but also natural killer progenitor cells. The NK cells are activated in response to interferon or macrophage-derived cytokines, and NK cells contain two types of surface receptors that control the cytolytic activity of cells, which are labeled as "activating receptors" and "inhibitory receptors." In addition, the NK cells may be generated from hematopoietic cells, such as hematopoietic stem or progenitors, from any source, such as placental tissue, placental perfusate, umbilical cord blood, placental blood, peripheral blood, spleen, liver, etc.

[0055] The composition of the present invention can be used to promote the proliferation of NK cells.

[0056] The promotion of the above proliferation can be achieved by co-culturing the composition of the present invention with NK cells.

[0057] Specifically, the promotion of proliferation may mean that the number of NK cells increases by about 2 times or more, specifically, 2.5 times or more, 3 times or more, 3.5 times or more, 4 times or more, 4.5 times or more, 5 times or more, 5.5 times or more, or 6 times or more, particularly 6.5 times or more, after co-culturing NK cells with the composition of the present invention for 1 day or more, 4 days or more, 7 days or more, 10 days or more, specifically 11 days or more, 12 days or more, 13 days or more, 14 days or more, 15 days or more, 16 days or more, 17 days or more, 18 days or more, 19 days or more, or 20 days or more, particularly 21 days or more, compared to the case where NK cells are co-cultivated with non-genetically modified K562 feeder cells.

[0058] According to a specific embodiment of the present invention, when the composition for promoting NK cell proliferation including the K562-mbIL13 supporter cells of the present invention is co-cultured with NK cells, the proliferation of NK cells is promoted, and the number of NK cells is increased by about 6.7 times compared to when co-cultured with general K562 supporter cells, and when the composition for promoting NK cell proliferation including the K562-mbIL2 supporter cells of the present invention is co-cultured with NK cells, the proliferation of NK cells is promoted, and the number of NK cells is increased by about 14.5 times compared to when co-cultured with general K562 supporter cells.

[0059] Additionally, the composition of the present invention can be used to enhance the cytolytic activity of NK cells.

[0060] The enhancement of the cytolytic activity of the above NK cells can be achieved by co-culturing the composition of the present invention with NK cells.

[0061] Specifically, the enhancement of the cytolytic activity of the NK cells may mean that the cytolytic activity or the intrinsic immunomodulatory ability of the NK cells is activated compared to parent cells, for example, hematopoietic cells, or natural killer precursor cells. The population of NK cells with enhanced cytolytic activity can be assessed by detecting one or more functionally related markers, for example, CD16, CD56, CD57, CD69, CD94, CD161, CD158a, CD158b, NKp30, NKp44, NKp46, DNAM-1, 2B4, NKp46, CD94, KIR (e.g., KIR2DL1, KIR2DL2 / 3, KIR3DL1), or the NKG2 family of activating receptors (e.g., NKG2A, NKG2C, NKG2D), specifically CD56, NKp30 or NKG2D. Specifically, after co-culturing NK cells with the composition of the present invention, it can be evaluated whether the expression level of CD56, NKp30 or NKG2D is greater than when co-culturing NK cells with general K562 support cells that have not been genetically modified, and the comparison of the expression levels can be made by evaluating whether the expression of CD56, NKp30 or NKG2D is 1.05 times or more, for example, 1.1 times or more, 1.15 times or more, specifically, 1.2 times or more, 1.4 times or more, 1.6 times or more, 1.8 times or more, 2 times or more, 2.2 times or more, 2.4 times or more, 2.6 times or more, 2.8 times or more, 3 times or more, 3.2 times or more, and particularly, 3.4 times or more, compared to when co-culturing NK cells with general K562 support cells.

[0062] In addition, NK cells with enhanced cytolytic activity may lower the survival rate of cancer cells or kill all cancer cells. Specifically, when NK cells are co-cultured with the composition of the present invention, it may mean that the survival rate of cancer cells is lowered or all cancer cells are killed compared to when NK cells are co-cultured with general K562 support cells. The lowering of the survival rate may mean that the survival rate (%) of cancer cells is lowered to 75% or less, for example, 65% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, and particularly 25% or less compared to when NK cells are co-cultured with K562 support cells.

[0063] According to a specific embodiment of the present invention, when a composition for enhancing the cytolytic activity of NK cells including K562-mbIL13 support cells of the present invention was co-cultured with NK cells, the expression of NKG2D of NK cells increased compared to NK cells proliferated by K562, and the cytolytic activity of NK cells against cancer cells (NALM6) was enhanced, so that the survival rate of cancer cells was confirmed to be lower than that of NK cells proliferated by K562.

[0064] In addition, according to a specific embodiment of the present invention, when a composition for enhancing the cytolytic activity of NK cells including the K562-mbIL2 support cells of the present invention was co-cultured with NK cells, the expression of CD56, NKG2D and NKp30 of NK cells increased compared to NK cells proliferated by K562, and the cytolytic activity of NK cells against cancer cells (NALM6) was enhanced, and it was confirmed that the survival rate of cancer cells was lower than that of NK cells proliferated by K562.

[0065] The composition of the present invention includes the support cells, and may include the support cells in an amount of 0.005 to 100.0 wt%, or 0.01 to 90.0 wt%, or 0.01 to 80 wt%, or 0.01 to 70 wt%, or 0.1 to 60 wt%, or 0.5 to 50 wt% based on the total weight of the composition, but is not limited thereto. When the effective content of the support cells is less than 0.005 wt%, the effect of promoting proliferation of NK cells or enhancing cytolytic activity may be reduced.

[0066] In addition to the support cells of the present invention, the composition of the present invention may further contain other components that can synergistically enhance the effects of promoting NK cell proliferation or promoting cytolytic activity, within a range that does not impair the effects of the present invention. For example, the composition may further contain cytokines.

[0067] The above cytokine can refer to a protein (~5-20 kDa) that plays a role in cell signaling. Cytokines are released by cells and affect the behavior of the cells that release the cytokines and / or other cells. Non-limiting examples of cytokines include chemokines, interferons, interleukins, lymphokines, tumor necrosis factor, monokines, and colony-stimulating factors. Cytokines can be produced by a wide range of cells, including but not limited to immune cells such as macrophages, B lymphocytes, T lymphocytes, mast cells, and monocytes, endothelial cells, fibroblasts, and stromal cells. Cytokines are interleukins or their mutants. Many interleukins are synthesized by helper CD4 T lymphocytes, as well as monocytes, macrophages, and endothelial cells. Interleukins can promote the development and differentiation of T and B lymphocytes and hematopoietic cells. Non-limiting examples of interleukins include IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8 (CXCL8), IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, IL36 or mutants thereof.

[0068]

[0069] Another aspect of the present invention provides a pharmaceutical composition comprising the support cells for preventing or treating cancer or diseases related to immunodeficiency.

[0070] The above disease may refer to a single pathological condition, in particular cancer, infectious disease, inflammatory disease, metabolic disease, autoimmune disorder, degenerative disease, apoptosis-related disease and graft rejection.

[0071] The pharmaceutical composition of the present invention comprises the support cells, and may comprise the support cells in an amount of 0.005 to 100.0 wt%, or 0.01 to 90.0 wt%, or 0.01 to 80 wt%, or 0.01 to 70 wt%, or 0.1 to 60 wt%, or 0.5 to 50 wt% based on the total weight of the composition, but is not limited thereto. When the effective content of the support cells is less than 0.005 wt%, the effect of promoting proliferation of NK cells or enhancing cytolytic activity may be reduced.

[0072] The pharmaceutical composition of the present invention may additionally contain, in addition to the support cells, other components that can synergistically enhance the effects of the support cells, as long as they do not impair the effects of the present invention. For example, the composition may further contain cytokines.

[0073] The route of administration of the pharmaceutical composition above includes oral, intravenous, intramuscular, intraarterial, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal, and may be applied by topical application, for example. The parenteral route includes subcutaneous, intradermal, intravenous, intramuscular, or intralesional injection or infusion techniques.

[0074] The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount.

[0075] In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined according to factors including the type and severity of the individual, age, sex, type of disease, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered singly or in multiple doses. It is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects by taking all of the above factors into consideration, and it can be easily determined by a person skilled in the art.

[0076] The pharmaceutical composition of the present invention is not particularly limited and can be applied to any subject that aims to enhance immunity by promoting the proliferation of NK cells or enhancing cytolytic activity. For example, the pharmaceutical composition of the present invention can be used not only for humans but also for non-human animals such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cows, sheep, pigs, goats, birds, and fish.

[0077] The above pharmaceutical composition may contain one or more active ingredients exhibiting the same or similar function as the above supporting cells. The above composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, external preparations, suppositories, and sterile injectable solutions, each according to a conventional method.

[0078] Solid preparations for oral administration include powders, granules, tablets, capsules, soft capsules, and pills. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included.

[0079] As preparations for parenteral administration, they can be formulated and used in the form of external preparations such as powders, granules, tablets, capsules, sterile aqueous solutions, liquids, non-aqueous solvents, suspensions, emulsions, syrups, suppositories, aerosols, etc. and sterile injection preparations according to conventional methods, and preferably, external pharmaceutical compositions for skin such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasms can be prepared and used, but are not limited thereto. Non-aqueous solvents and suspensions can be used, such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can be used, such as witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin.

[0080] The above composition may additionally contain auxiliary agents such as preservatives, stabilizers, wetting agents or emulsifying promoters, salts for osmotic pressure control and / or buffers, and other therapeutically useful substances, and may be formulated according to conventional mixing, granulation or coating methods.

[0081] The dosage of the above pharmaceutical composition may vary depending on several factors including the age, body weight, general health, sex, time of administration, route of administration, excretion rate, drug combination, and severity of the specific disease of the subject.

[0082] In addition, the pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers. In addition, the pharmaceutical composition can be administered at a dosage within the range of 0.001 to 200 mg / kg for adults, and when the pharmaceutical composition is an external preparation, it is recommended to apply 1 to 5 times a day in an amount of 1.0 to 3.0 ml for adults and continue for more than 1 month, but the dosage does not limit the scope of the present invention.

[0083] When the above pharmaceutical composition is formulated in a unit dosage form, it is preferable that the support cells of the present invention as an active ingredient are contained in a unit dosage of about 0.01 to 1,500 mg, and the dosage required for adult treatment is usually in the range of about 1 to 500 mg per day depending on the frequency and intensity of administration, but is not limited thereto, and for some patients, a higher daily dosage may be preferable.

[0084] The above pharmaceutical composition may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, anticancer therapy, immunotherapy, radiotherapy, and biological response modifiers.

[0085]

[0086] Another aspect of the present invention provides a method for promoting the proliferation of NK cells, comprising the step of culturing a sample containing NK cells in a medium containing the composition.

[0087] Another aspect of the present invention provides a method for enhancing the cytolytic activity of NK cells, comprising the step of culturing a sample containing NK cells in a medium containing the composition.

[0088] The sample may be a biological sample derived from an individual, for example, a mammal including a human. Furthermore, the biological sample may be isolated from the individual and may be blood, whole blood, serum, plasma, lymph, urine, feces, tissue, cells, organs, bone marrow, saliva, sputum, cerebrospinal fluid, or a combination thereof. In particular, the biological sample may include PBMCs, purified NK cells, or primary resting cells (i.e., isolated directly from blood).

[0089] The above culturing step may be performed in the presence of cytokines.

[0090] The meaning of the above proliferation promotion, the meaning of the enhancement of cell lysis activity, and cytokines are the same as those described above, so their description is omitted.

[0091] Additionally, the culturing step may be performed for 24 hours to 30 days. For example, the culturing step may be performed for a period of 24 hours or more, 2 days or more, 4 days or more, 6 days or more, 8 days or more, 10 days or more, specifically 11 days or more, 12 days or more, 13 days or more, 14 days or more, 15 days or more, 16 days or more, 17 days or more, 18 days or more, 19 days or more, or 20 days or more, particularly 21 days or more.

[0092]

[0093] Hereinafter, the present invention will be described in detail through manufacturing examples and working examples.

[0094] However, the following manufacturing examples, experimental examples, and examples are only for illustrating the present invention, and the content of the present invention is not limited to the following manufacturing examples and examples.

[0095]

[0096] [Example]

[0097] Example 1. Production of genetically engineered feeder cells

[0098] Feeder cells expressing membrane bound interleukin-13 (mbIL-13) and feeder cells expressing membrane bound interleukin-2 (mbIL-2) were prepared.

[0099] Specifically, the transmembrane (TM) of CD8a (SEQ ID NO: 3) was linked to one end of the human mbIL-13 gene (SEQ ID NO: 1) or the human mbIL-2 gene (SEQ ID NO: 2), and the signal peptide (SP) of CD8a (SEQ ID NO: 4) was linked to the other end. In addition, the CD8 α hinge gene (SEQ ID NO: 5) and the Flag tag gene (SEQ ID NO: 6) were inserted and linked between the mbIL-13 gene and the CD8a TM gene (Fig. 1). The combination of genes as above was cloned into the pLVX-puro vector to produce a vector for recombinant production. Thereafter, the recombinant genes produced above were transfected into 293T cells together with the packaging vector for virus production. After a period of time, the medium was replaced with fresh medium, and the cells were cultured for 48 hours, and then the medium containing the virus was collected. The recovered medium was centrifuged at 500 X g for 10 minutes, and only the pure medium containing the virus was separated using a 0.45 ㎛ filter to produce mbIL-13 or mbIL-2 expressing viruses, respectively.

[0100] Afterwards, the mbIL-13 or mbIL-2 expressing virus was dissolved in a medium containing K562 cells and added together with polybrene (8 ㎍ / ml), and cell culture was performed for 48 hours.

[0101] Next, in order to select only cells infected with the above virus, puromycin antibiotic was treated. After puromycin selection, the expression of mbIL-13 was analyzed using a cell analyzer (FACS). As a result, as shown in Figures 2a and 2b, a K562 cell line expressing mbIL-13 (hereinafter, "K562-mbIL13") and a K562 cell line expressing mbIL-2 (hereinafter, "K562-mbIL2") were produced, and it was confirmed that the above marker was expressed in the K562-mbIL13 cell line and the K562-mbIL2 cell line.

[0102]

[0103] Experimental Example 1. NK cell proliferation promotion effect of the support cells of the present invention

[0104] In order to confirm whether the support cells of the present invention manufactured in Example 1 promote the proliferation of NK cells, each support cell was co-cultured with NK cells isolated from peripheral blood mononuclear cells (PBMC), and the number of NK cells was measured after a certain period of time.

[0105] Specifically, for the isolation of peripheral blood mononuclear cells (PBMCs), whole blood from a normal donor was diluted with PBS in a 1:1 ratio (15 ml whole blood: 15 ml PBS) and overlaid on 15 ml Lymphoprep. Next, centrifugation was performed without brakes at 1200 X g for 25 minutes at room temperature (acceleration 1, deceleration 0), cells were harvested from the buffy coat layer, and washed three times with PBS at 400 X g for 7 minutes. Through this, PBMCs were obtained. Through the above process, five types of PBMCs were obtained from five donors and used in this experiment. To isolate NK cells from PBMCs, a human NK cell isolation kit (EasySep™) was used. RPMI1640+10% FBS or NK macs+5% human serum was used as culture medium.

[0106] As a result, as shown in FIGS. 3A and 3B, when co-cultured with the K562-mbIL2 or K562-mbIL13 cells of the present invention, the number of NK cells significantly increased compared to when co-cultured with general K562 cells. The same test was repeatedly performed using the above five types of PBMC, and in all cases, the support cells developed by our research team showed a superior NK cell proliferation promotion effect than general K562 cells.

[0107]

[0108] Experimental Example 2. Effect of the support cells of the present invention on promoting NK cell lysis activity.

[0109] 2-1. Confirmation of NK cell membrane receptor phenotype characteristics

[0110] Through the process of Experimental Example 1, the membrane receptor phenotype characteristics of NK cells amplified by K562-mbIL13 and K562-mbIL2 cells were confirmed.

[0111] 1 X 10 proliferated NK cells 6 After washing the cells with FACS buffer (PBS containing 1% FBS), the cells were treated with fluorescent dye-tagged anti-human NKG2D, CD3, and CD56 antibodies for 30 minutes. The cells were then washed with FACS buffer, and data were acquired and analyzed using a FACS Calibur. K562 feeder cells were used as a control.

[0112] As a result, as shown in Fig. 4, it was confirmed that the NKG2D membrane receptor was expressed at a higher level in NK cells cultured using the K562-mbIL13 support cells of the present invention compared to the case where the cells were cultured using K562 cells.

[0113] In addition, as shown in Fig. 4, it was confirmed that NK cells cultured using the K562-mbIL2 support cells of the present invention expressed CD56, NKG2D, and NKp30 membrane receptors at higher levels than when cultured using K562 cells. A total of five PBMCs were used for the experiment, and it was confirmed that the support cells developed by the present research team further increased NKG2D expression in NK cells in four of the PBMCs.

[0114] 2-2. Confirmation of NK cell cytolytic activity

[0115] In order to confirm the efficacy of NK cells cultured by the support cells of the present invention as an anticancer therapeutic agent, the cytolytic activity of NK cells against cancer cells was measured by luminescence analysis.

[0116] Specifically, NALM6 (NALM6-luc), a cancer cell expressing luciferase, was mixed with NK cells proliferated by support cells at effector-to-target (E:T) ratios of 0:1, 0.2:1, 1:1, and 5:1. The number of viable NALM6 cells was calculated by measuring the luminescence value after 4 or 24 hours.

[0117] As a result, as illustrated in Fig. 5, it was confirmed that the cytolytic activity of NK cells proliferated with K562-mbIL2 or K562-mbIL13 support cells of the present invention was much superior to that of NK cells proliferated with K562 cells. Therefore, it can be seen that when NK cells are cultured using a composition including the support cells of the present invention, the cytolytic activity of NK cells is enhanced.

Claims

1. A composition for promoting NK cell proliferation or enhancing NK cell cytolytic activity, comprising feeder cells expressing membrane bound interleukin-13 (mbIL-13) on the cell membrane.

2. A composition for promoting NK cell proliferation or enhancing NK cell cytolytic activity, comprising feeder cells expressing membrane bound interleukin-2 (mbIL-2) on the cell membrane.

3. In claim 1, A composition for promoting NK cell proliferation or enhancing NK cell cytolytic activity, further comprising feeder cells expressing membrane bound interleukin-2 (mbIL-2) on the cell membrane.

4. In claim 1 or claim 2, A composition for promoting NK cell proliferation or enhancing NK cell cytolytic activity, wherein the supporting cells are selected from the group consisting of K562, RPMI8866, EBV_LCL, 721.221, HFWT cells, and combinations thereof.

5. In claim 1, A composition for promoting NK cell proliferation or enhancing NK cell cytolytic activity, comprising a nucleic acid encoding the above mbIL-13.

6. In claim 5, A composition for promoting NK cell proliferation or enhancing NK cell cytolytic activity, wherein one end of the nucleic acid encoding the above mbIL-13 further comprises a CD8a TM (transmembrane).

7. In claim 6, A composition for promoting NK cell proliferation or enhancing NK cell cytolytic activity, further comprising a CD8a SP (signal peptide) at the other end of the nucleic acid encoding the above mbIL-13.

8. A pharmaceutical composition for preventing or treating cancer or a disease related to immunodeficiency, comprising the composition of any one of claims 1 to 7.

9. A method for promoting the proliferation of NK cells, comprising the step of culturing a sample containing NK cells in a medium containing one or more compositions of claims 1 to 7.

10. A method for enhancing the cytolytic activity of NK cells, comprising the step of culturing a sample containing NK cells in a medium containing one or more compositions of claims 1 to 7.

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