Polymeric compound for surface modification of natural killer cells for promoting cell priming

Abstract

The present invention relates to a polymeric compound for surface modification of natural killer cells for promoting cell priming. The polymeric compound for surface modification of natural killer cells, according to an embodiment of the present invention, has the effects of promoting the priming of natural killer cells by inducing the clustering of natural killer cells and increasing the production of interferon gamma (IFN-γ), which is known to more effectively eliminate tumor cells by activating macrophages and T cells, and thus can be effectively used for the prevention or treatment of cancer.

Description

Polymer compounds for natural killer cell surface modification to promote cell priming

[0001] The present invention relates to a polymeric compound for modifying the surface of natural killer cells to promote cell priming.

[0002] Natural killer (NK) cells are lymphocytes that can bind to specific tumor cells and virus-infected cells without antigen stimulation and kill them by inserting granules containing perforin.

[0003] First, NK cells may fail to recognize cancer due to a failure of immune surveillance, and second, they may fail to kill cancer by binding to certain proteins on the surface of cancer cells due to immune evasion.

[0004] One of the traditional NK cell activation techniques is chemical activation using polyethyleneimide for DNA complexation and transformation. Another method involves treating with a cytokine cocktail during the expansion process; for example, interleukin (IL)-2, IL-12, IL-15, IL-18, IL-21, and IFN-αβ are widely used to induce NK cell proliferation or enhance tumor cytotoxicity. Delivering exogenous cytokines to NK cells can provide sufficient effects. Alternatively, tumor-induced cell activation using irradiated tumor cells as stimulants is also used.

[0005] Cytokines administered in bolus form require continuous and repeated administration during the expansion of NK cells due to their short half-life. Furthermore, there is a problem in that NK cell activation is often insufficient because cytokines are frequently inactivated in the culture medium, which can act as a technical obstacle in the mass production of autologous or allogeneic cell therapies. Additionally, overexpression of immune checkpoint receptors has been reported in NK cells activated by the co-administration of cytokines and glucocorticoids. This phenomenon can occur if cytokine stimulation persists for more than 96 hours, and in particular, increased expression of programmed PD-1 can reduce the therapeutic efficacy of NK cells by promoting binding to PD-L1 on the surface of tumor cells.

[0006] The background description of the invention is provided to facilitate a better understanding of the present invention. The matters described in the background description should not be construed as an acknowledgment that they exist as prior art.

[0007] Meanwhile, conventional natural killer cell activation technology, particularly activation of natural killer cells through cytokine treatment, has problems such as cytokine inactivation and increased expression of immune checkpoints during NK cell expansion, so the need for a new natural killer cell priming technology without these side effects was recognized.

[0008] Furthermore, it was noted that one of the morphological characteristics exhibited by NK cells during the initial expansion phase is cluster formation, and while NK cells have a single-cell form when migrating into blood vessels in the body, during the expansion process in vitro / in vitro, floating NK cell populations form clusters through the development of the extracellular matrix (ECM).

[0009] These structural changes often facilitate diffusive paracrine signaling molecule exchange and membrane contact signaling within NK cells. Consequently, these interactions further activate the production of cytokine activators such as IL-2, and the activated NK cells acquire enhanced anticancer efficacy.

[0010] Accordingly, the inventors of the present invention sought to develop a polymeric compound that modifies the surface of natural killer cells through cell membrane fixation so as to induce the formation of NK cell clusters without cytokines or a cocktail of growth factors.

[0011] As a result, the inventors of the present invention confirmed that cell cluster formation is promoted when the surface of natural killer cells is coated using a polymer compound having a structure in which multiple hydrophobic two-tail carbon chains interacting with the cell membrane are connected to an aromatic ring.

[0012] Accordingly, the problem to be solved by the present invention is to provide a polymeric compound for modifying the surface of natural killer cells to promote cell priming, comprising: a hydrophobic moiety that binds to a cell membrane; and an aryl linker.

[0013] In addition, another problem that the present invention aims to solve is to provide a natural killer cell with a surface modified by combining the polymer compound for surface modification of the natural killer cell.

[0014] In addition, another problem that the present invention aims to solve is to provide a method for preparing a polymer compound for surface modification of natural killer cells to promote cell priming.

[0015] In addition, another problem that the present invention aims to solve is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising, as an active ingredient, natural killer cells whose surface is modified with a polymeric compound comprising a hydrophobic moiety that binds to a cell membrane and an aryl linker.

[0016] In addition, another problem that the present invention aims to solve is to provide a cell therapy composition for cancer prevention or treatment comprising natural killer cells whose surface is modified with a polymeric compound comprising a hydrophobic moiety that binds to a cell membrane and an aryl linker as an active ingredient.

[0017] In addition, another problem that the present invention aims to solve is to provide a method for promoting the priming of natural killer cells.

[0018] The problems of the present invention are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0019] To solve the problem described above, a polymer compound for modifying the surface of natural killer cells to promote cell priming is provided, comprising: a hydrophobic moiety that binds to a cell membrane according to one embodiment of the present invention; and an aryl linker.

[0020] According to a feature of the present invention, the hydrophobic moiety may be selected from the group consisting of phospholipids having alkyl chains having 12 to 24 carbon atoms, sterol lipids having 10 to 30 carbon atoms, 1,2-bis(diphenylphosphino)ethane (DPPE), and 1,2-bis(dimethylphosphino)ethane (DMPE).

[0021] According to another feature of the present invention, the hydrophobic moiety may be a compound represented by Formula 1 or a derivative thereof.

[0022] [Chemical Formula 1]

[0023]

[0024] q is an integer greater than 0, and

[0025] p is an integer greater than 0.

[0026] According to another feature of the present invention, the aryl linker may be selected from the group consisting of phenyl linker, naphthalene linker, anthracenyl linker, perylenyl linker, pyrrole linker, furanyl linker, thiophene linker, and oxazolyl linker.

[0027] According to another feature of the present invention, the polymer compound for modifying the surface of natural killer cells may further comprise a cell internalization-preventing moiety.

[0028] According to another feature of the present invention, the cell internalization prevention moiety may be selected from the group consisting of polyethylene glycol (PEG), polyethylene oxide (PEO), and polyvinyl alcohol (PVA).

[0029] According to another feature of the present invention, the cell internalization-preventing moiety may be a compound represented by Formula 2 or a derivative thereof.

[0030] [Chemical Formula 2]

[0031]

[0032] n is an integer greater than 0.

[0033] According to another feature of the present invention, the polymer compound for surface modification of natural killer cells may induce cell clustering of natural killer cells.

[0034] According to another feature of the present invention, the polymer compound for natural killer cell surface modification may increase the production of interferon gamma (IFN-γ).

[0035] According to another feature of the present invention, the polymer compound for surface modification of natural killer cells may enhance the anticancer effect of natural killer cells.

[0036] In order to solve the problem described above, a natural killer cell with a modified surface is provided by combining a polymer compound for surface modification of the natural killer cell according to another embodiment of the present invention.

[0037] In order to solve the problem described above, a method for preparing a polymer compound for modifying the surface of natural killer cells to promote cell priming is provided, according to another embodiment of the present invention, comprising the steps of: (a) activating the carboxyl groups of an aryl linker comprising a plurality of carboxyl groups; and (b) attaching a polymer compound comprising a hydrophobic moiety, an anti-internalization moiety, and an amine group to each of the activated carboxyl group sites of the aryl linker.

[0038] According to the features of the present invention, a polymeric compound comprising a hydrophobic moiety, a cell internalization prevention moiety, and an amine group may be a compound represented by Formula 3 or a derivative thereof.

[0039] [Chemical Formula 3]

[0040]

[0041] q is an integer greater than 0, and

[0042] p is an integer greater than 0, and

[0043] n is an integer greater than 0.

[0044] According to another feature of the present invention, the polymer compound in which step (b) is completed may be a compound represented by Formula 4 or a derivative thereof.

[0045] [Chemical Formula 4]

[0046]

[0047] q is an integer greater than 0, and

[0048] p is an integer greater than 0, and

[0049] n is an integer greater than 0.

[0050] To solve the problem described above, a pharmaceutical composition for cancer prevention or treatment is provided, comprising a natural killer cell whose surface is modified with a polymeric compound comprising a hydrophobic moiety that binds to a cell membrane according to another embodiment of the present invention and an aryl linker, as an active ingredient.

[0051] According to a feature of the present invention, the polymer compound may further comprise a cell internalization-preventing moiety.

[0052] According to another feature of the present invention, the cancer may be selected from the group consisting of thyroid cancer, stomach cancer, colorectal cancer, lung cancer, breast cancer, liver cancer, pancreatic cancer, testicular cancer, oral cancer, basal cell carcinoma, brain tumor, gallbladder cancer, bile duct cancer, laryngeal cancer, retinoblastoma, ampulla of Vater cancer, bladder cancer, peritoneal cancer, adrenal cancer, non-small cell lung cancer, tongue cancer, small cell lung cancer, small intestine cancer, meningioma, esophageal cancer, renal pelvis and ureter cancer, kidney cancer, malignant bone tumor, malignant soft tissue tumor, malignant lymphoma, malignant melanoma, eye tumor, urethral cancer, stomach cancer, sarcoma, pharyngeal cancer, cervical cancer, endometrial cancer, uterine sarcoma, metastatic brain tumor, rectal cancer, vaginal cancer, spinal cord tumor, salivary gland cancer, tonsil cancer, squamous cell carcinoma, hematological cancer and anal cancer.

[0053] To solve the problem described above, a cell therapy composition for cancer prevention or treatment is provided, comprising a natural killer cell whose surface is modified with a polymeric compound comprising a hydrophobic moiety that binds to a cell membrane according to another embodiment of the present invention and an aryl linker, as an active ingredient.

[0054] According to a feature of the present invention, the polymer compound may further comprise a cell internalization-preventing moiety.

[0055] To solve the problem described above, a method for promoting the priming of natural killer cells is provided, comprising the steps of: immobilizing a polymer compound for modifying the surface of natural killer cells to promote cell priming, comprising a hydrophobic moiety that binds to a cell membrane according to another embodiment of the present invention and an aryl linker, onto the surface of natural killer cells; and inducing cell aggregation by bringing the natural killer cells, on which the polymer compound for modifying the surface of natural killer cells to promote cell priming is immobilized, into contact with other natural killer cells in vivo.

[0056] According to a feature of the present invention, the polymer compound may further comprise a cell internalization-preventing moiety.

[0057] A polymer compound for surface modification of natural killer cells according to one embodiment of the present invention has the effect of inducing the aggregation of natural killer cells to promote the priming of natural killer cells, and increasing the production of interferon gamma (IFN-γ), which is known to activate macrophages and T cells to more effectively eliminate tumor cells.

[0058] Therefore, natural killer cells with surfaces modified with the polymer compound of the present invention have the effect of enhancing the anticancer immune response by promoting the priming of natural killer cells, and thus can be usefully used for the prevention or treatment of cancer.

[0059] The effects according to the present invention are not limited to those exemplified above, and various other effects are included in this specification.

[0060] FIGS. 1a and 1b illustrate, exemplarily, the process of manufacturing a polymer compound for cell surface modification according to one embodiment of the present invention.

[0061] FIG. 2 is according to one embodiment of the present invention (DSPE- PEG This shows the results of the structural characteristic analysis of -NH)3-T.

[0062] FIG. 3 is according to one embodiment of the present invention (DSPE- PEG This shows the results of the cell viability evaluation of natural killer cells treated with -NH)3-T.

[0063] FIG. 4 is according to one embodiment of the present invention (DSPE- PEG This shows the results of the structural analysis of natural killer cell clusters coated with -NH)3-T.

[0064] FIG. 5 is according to one embodiment of the present invention (DSPE- PEG This shows the results of the cytokine release analysis of natural killer cells coated with -NH)3-T.

[0065] The advantages and features of the present invention and the methods for achieving them will become clear by referring to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0066] In the following, terms used within this specification are explained for clarity of explanation.

[0067] In this document, expressions such as "have," "can have," "include," or "can include" refer to the existence of the relevant feature (e.g., numerical values, functions, actions, or components, etc.) and do not exclude the existence of additional features.

[0068] In this document, "or" means "and / or" unless otherwise noted. Expressions such as "A or B," "at least one of A or / and B," or "one or more of A or / and B" may include all possible combinations of items listed together. For example, "A or B," "at least one of A and B," or "at least one of A or B" may refer to cases including (1) at least one A, (2) at least one B, or (3) both at least one A and at least one B.

[0069] The term “priming” as used in the present invention refers to an important process of preparing to respond more quickly and strongly to a specific stimulus, and in the present invention, it refers to a process of preparing natural killer cells to be activated so that they can effectively attack tumor cells or virus-infected cells. In this process, NK cells are activated by specific signals or ligands, which enhances their ability to recognize and attack tumor cells or infected cells.

[0070] The term “surface modification” as used in the present invention may refer to the binding of hydrophobic moiety to the surface of a cell, i.e., the cell membrane.

[0071] The term “Aryl Linker” as used in this invention refers to a molecular structure containing an aryl group, which is a functional group derived from an aromatic ring compound. It is generally used in drug design to enhance molecular stability and impart desired properties in specific chemical reactions.

[0072] The term “phenyl linker” used in the present invention refers to a molecular structure containing a phenyl group, which is an aromatic ring compound derived from a benzene ring.

[0073] The term "cell therapy" as used in this invention refers to a pharmaceutical product used for the purposes of treatment, diagnosis, and prevention by performing a series of actions, such as proliferating or selecting living autologous, allogenic, or xenogenic cells in vitro or altering the biological characteristics of cells by other methods, in order to restore the function of cells and tissues. The United States has managed cell therapy products as pharmaceutical products since 1993, and Korea since 2002. Such cell therapy products can be broadly classified into two categories: the first is stem cell therapy products for tissue regeneration or the recovery of organ function, and the second is immunocell therapy products for regulating immune responses, such as suppressing or enhancing immune responses within the body. In this invention, the term refers to immunocell therapy products.

[0074] In one aspect, the present invention relates to a polymeric compound for modifying the surface of natural killer cells to promote cell priming, comprising: a hydrophobic moiety that binds to a cell membrane; and an aryl linker.

[0075] In one embodiment of the present invention, the hydrophobic moiety may be selected from the group consisting of phospholipids having alkyl chains having 12 to 24 carbon atoms, sterol lipids having 10 to 30 carbon atoms, 1,2-bis(diphenylphosphino)ethane (DPPE), and 1,2-bis(dimethylphosphino)ethane (DMPE), but is not limited thereto.

[0076] In one embodiment of the present invention, the phospholipid having an alkyl chain having 12 to 24 carbon atoms may preferably be 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine (DMPC), dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-distearoyl-3-trimethylammonium-propane chloride (DSTAP), but is not limited thereto, and any lipid-type biomolecule that can be attached to and immobilized on the surface of a cell and is soluble in a non-polar solvent may be used without limitation.

[0077] In one embodiment of the present invention, the sterol lipid having 10 to 30 carbon atoms may preferably be cholesterol, cholesterol hexasuccinate, 3βN'-dimethylaminoethane)carbamoyl cholesterol, ergosterol, stigmasterol, or lanosterol, but any biomolecule in the form of lipids that can be attached to and fixed to the surface of a cell and is soluble in a non-polar solvent may be used without limitation.

[0078] Furthermore, the cell surface coating ability of conventional lipid-based biomaterials is significantly influenced by hydrogenation (carbon chain length) and structural similarity to phospholipids within the cell membrane (two-tail lipid structure). For example, DMPE lipids (two-tailed C14 carbon chain) had a problem of poor cell surface coating efficiency due to their lower hydrophobicity compared to DSPE lipids.

[0079] In one embodiment of the present invention, the hydrophobic moiety may be a compound represented by Formula 1 or a derivative thereof, but is not limited thereto.

[0080] [Chemical Formula 1]

[0081]

[0082] p may be an integer greater than 0, preferably an integer from 1 to 30, but is not limited thereto.

[0083] q may be an integer greater than 0, preferably an integer from 1 to 30, but is not limited thereto.

[0084] In one embodiment of the present invention, the aryl linker may be selected from the group consisting of phenyl linker, naphthalene linker, anthracenyl linker, perylenyl linker, pyrrole linker, furanyl linker, thiophene linker, and oxazolyl linker, preferably may be a phenyl linker, and more preferably may be a trimesic acid having a structure in which a carboxyl group (-COOH) is attached to the 1st, 3rd, and 5th positions of a benzene ring, but is not limited thereto.

[0085] In one embodiment of the present invention, the polymer compound for modifying the surface of natural killer cells may further comprise a cell internalization-preventing moiety, but is not limited thereto.

[0086] In one embodiment of the present invention, the cell internalization prevention moiety may be selected from the group consisting of polyethylene glycol (PEG), polyethylene oxide (PEO), and polyvinyl alcohol (PVA), but is not limited thereto.

[0087] In one embodiment of the present invention, the cell internalization-preventing moiety may be a compound represented by Formula 2 or a derivative thereof, but is not limited thereto.

[0088] [Chemical Formula 2]

[0089]

[0090] n may be an integer greater than 0, preferably an integer from 1 to 50, but is not limited thereto.

[0091] In one embodiment of the present invention, the cell internalization prevention moiety can function to maintain attachment to the cell membrane surface without the cell and the polymer compound being incorporated into the natural killer cell.

[0092] In one embodiment of the present invention, the polymer compound for surface modification of natural killer cells may induce cell clustering of natural killer cells, but is not limited thereto.

[0093] In one embodiment of the present invention, the polymer compound for modifying the surface of natural killer cells may increase the production of interferon gamma (IFN-γ), but is not limited thereto.

[0094] In one embodiment of the present invention, the polymer compound for surface modification of natural killer cells may enhance the anticancer effect of natural killer cells, but is not limited thereto.

[0095] In one aspect, the present invention relates to a natural killer cell whose surface is modified by combining a polymer compound for surface modification of the natural killer cell.

[0096] In one aspect, the present invention relates to a method for preparing a polymer compound for modifying the surface of natural killer cells to promote cell priming.

[0097] Hereinafter, with reference to FIG. 3, a method for preparing a polymer compound for surface modification of natural killer cells to promote priming according to one embodiment of the present invention will be described. For convenience of explanation, the description will be made with reference to FIG. 1a.

[0098] Referring to FIG. 1a, the method includes (a) a step of activating the carboxyl groups of an aryl linker comprising a plurality of carboxyl groups (S110) and (b) a step of attaching a polymer compound comprising a hydrophobic moiety, an anti-internalization moiety, and an amine group to each activated carboxyl group site of the aryl linker (S120).

[0099] According to one embodiment of the present invention, (a) a step (S110) of activating the carboxyl groups of an aryl linker comprising a plurality of carboxyl groups comprises a step (S210) of preparing a first reaction mixture by dissolving an aryl linker comprising a plurality of carboxyl groups, EDC (3-ethylcarbodiimide hydrochloride), and NHS (N-hydroxysuccinimide) in an inorganic dimethylformamide (anhydrous DMF), and a step (S220) of activating the carboxyl groups by stirring the first reaction mixture.

[0100] In one embodiment of the present invention, an aryl linker comprising a plurality of carboxyl groups, EDC (3-ethylcarbodiimide hydrochloride), and NHS (N-hydroxysuccinimide) may be mixed in a molar ratio of 1:29:48, but is not limited thereto.

[0101] In one embodiment of the present invention, the stirring may be performed at room temperature for 1 to 5 hours, preferably 2.5 to 3.5 hours.

[0102] In one embodiment of the present invention, the room temperature may be 1 ℃ to 35 ℃, preferably 21 ℃ to 25 ℃, but is not limited thereto.

[0103] According to one embodiment of the present invention, the step (S120) of combining a polymer compound comprising a hydrophobic moiety, an anti-cell internalization moiety, and an amine group to each activated carboxyl group site of an aryl linker comprises the step (S310) of preparing a second reaction mixture by dissolving the polymer compound comprising the carboxyl-activated aryl linker, the anti-cell internalization moiety, and the amine group in inorganic dimethylformamide (anhydrous DMF), and the step (S320) of combining the amine group of the polymer compound with the carboxyl group of the aryl linker by stirring the second reaction mixture.

[0104] Additionally, (b) the step of attaching a polymeric compound comprising a hydrophobic moiety, an anti-internalization moiety, and an amine group to each activated carboxyl group site of the aryl linker (S120) may further include the step (S330) of washing the second reaction mixture after the reaction is complete to remove the unreacted aryl linker and polymeric compound and the unreacted carboxyl group remaining.

[0105] In one embodiment of the present invention, the polymeric compound comprising a hydrophobic moiety, a cell internalization prevention moiety, and an amine group may be a compound represented by Formula 3 or a derivative thereof, but is not limited thereto.

[0106] [Chemical Formula 3]

[0107]

[0108] p may be an integer greater than 0, preferably an integer from 1 to 30, but is not limited thereto.

[0109] q may be an integer greater than 0, preferably an integer from 1 to 30, but is not limited thereto.

[0110] n may be an integer greater than 0, preferably an integer from 1 to 50, but is not limited thereto.

[0111] In one embodiment of the present invention, the stirring may be performed at room temperature for 36 to 60 hours, preferably 42 to 54 hours.

[0112] In one embodiment of the present invention, the room temperature may be 1 ℃ to 35 ℃, preferably 21 ℃ to 25 ℃, but is not limited thereto.

[0113] In one embodiment of the present invention, the polymer compound in which step (b) is completed may be a compound represented by Formula 4 or a derivative thereof, but is not limited thereto.

[0114] [Chemical Formula 4]

[0115]

[0116] p may be an integer greater than 0, preferably an integer from 1 to 30, but is not limited thereto.

[0117] q may be an integer greater than 0, preferably an integer from 1 to 30, but is not limited thereto.

[0118] n may be an integer greater than 0, preferably an integer from 1 to 50, but is not limited thereto.

[0119] Meanwhile, the method for manufacturing polymer compounds is not limited to the chemical synthesis method through EDC / NHS coupling shown in FIG. 1a, and can be produced through methods known in the art, such as chemical synthesis methods, enzymatic conversion methods, and photochemical methods.

[0120] In one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of cancer comprising natural killer cells whose surface is modified with a polymeric compound comprising a hydrophobic moiety that binds to a cell membrane and an aryl linker as an active ingredient.

[0121] In one embodiment of the present invention, the polymer compound may further comprise a cell internalization-preventing moiety, but is not limited thereto.

[0122] In one embodiment of the present invention, the cancer may be selected from the group consisting of thyroid cancer, stomach cancer, colorectal cancer, lung cancer, breast cancer, liver cancer, pancreatic cancer, testicular cancer, oral cancer, basal cell carcinoma, brain tumor, gallbladder cancer, bile duct cancer, laryngeal cancer, retinoblastoma, ampulla of Vater cancer, bladder cancer, peritoneal cancer, adrenal cancer, non-small cell lung cancer, tongue cancer, small cell lung cancer, small intestine cancer, meningioma, esophageal cancer, renal pelvis and ureter cancer, kidney cancer, malignant bone tumor, malignant soft tissue tumor, malignant lymphoma, malignant melanoma, eye tumor, urethral cancer, stomach cancer, sarcoma, pharyngeal cancer, cervical cancer, endometrial cancer, uterine sarcoma, metastatic brain tumor, rectal cancer, vaginal cancer, spinal cord tumor, salivary gland cancer, tonsil cancer, squamous cell carcinoma, hematological cancer and anal cancer, but is not limited thereto.

[0123] The term "prevention" above refers to any act that reduces the frequency or severity of pathological phenomena. Prevention may be complete or partial. In this case, it may refer to a phenomenon in which symptoms of cancer within an individual are reduced compared to the case where the composition is not used.

[0124] The above "treatment" refers to any act of clinical intervention intended to alter the natural processes of the target or cell to be treated, and may be performed during the progression of a clinical pathological condition or to prevent it. The intended therapeutic effects may include preventing the onset or recurrence of the disease, alleviating symptoms, reducing all direct or indirect pathological consequences associated with the disease, preventing metastasis, slowing the rate of disease progression, alleviating or temporarily resolving the disease state, or improving the prognosis.

[0125] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used in the present invention, the term "pharmaceuticalally effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause adverse effects. The effective dose level may be determined based on factors including the patient's health status, type and severity of the disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. Considering all of the above factors, it is important to administer an amount that obtains maximum effect with a minimum amount without adverse effects, and this can be easily determined by a person skilled in the art.

[0126] In the present invention, the term "individual" refers to an individual intended for the prevention or treatment of cancer, and is not particularly limited to animals including humans, for example, non-primates (e.g., cattle, pigs, horses, cats, dogs, rats, and mice) and mammals including primates (e.g., monkeys, e.g., cynomolgous monkeys and chimpanzees). In some cases, it may be an individual excluding humans.

[0127] The composition according to the present invention may comprise, alone, natural killer cells whose surface is modified with a polymeric compound comprising a hydrophobic moiety that binds to a cell membrane in a pharmaceutically effective amount; and an aryl linker, or may comprise one or more pharmaceutically acceptable carriers, excipients, or diluents. In the above, "pharmaceutically effective amount" refers to an amount sufficient to prevent, improve, and treat symptoms of cancer. In the above, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and, when administered to humans, does not typically cause allergic reactions or similar reactions such as gastrointestinal disturbances or dizziness.

[0128] Additionally, a composition comprising a pharmaceutically acceptable carrier may be one or more formulations selected from the group comprising oral formulations, topical preparations, suppositories, sterile injectable solutions, and sprays, such as various formulations including capsules, liquids, injections, soft capsules, granules, or tablets. When formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. The above carrier, excipient, and diluent may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, physiological saline, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol and liquid paraffin, but are not limited thereto, and any conventional carrier, excipient, or diluent may be used. The above components may be added independently or in combination to natural killer cells whose surface has been modified with a polymeric compound comprising a hydrophobic moiety that binds to the cell membrane, which is the above active ingredient; and an aryl linker.

[0129] In addition, the pharmaceutical composition of the present invention may have any one formulation selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid formulations, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Witepsol, macrogol, Tween 61, cacao oil, laurin oil, glycerol, gelatin, etc. may be used as the base for suppositories.

[0130] Natural killer cells whose surface is modified with a polymeric compound comprising a hydrophobic moiety that binds to the cell membrane of the present invention and an aryl linker can be administered in various oral and parenteral formulations during clinical administration, and when formulated, they are prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0131] Solid formulations for oral administration include tablets, pills, powders, granules, capsules, troches, etc., and these solid formulations are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin, with natural killer cells whose surface is modified with a polymeric compound comprising one or more hydrophobic moiety that binds to the cell membrane of the present invention; and an aryl linker. In addition, lubricants such as magnesium styrate talc are also used in addition to simple excipients. Liquid formulations for oral administration include suspensions, liquid formulations, emulsions, or syrups, and may include various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.

[0132] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, witepsol, macrogol, tween 61, cocoa paste, laurin paste, glycerol, gelatin, etc. may be used.

[0133] The pharmaceutical composition of the present invention may be administered by any device capable of moving natural killer cells, whose surface is modified with a polymeric compound comprising a hydrophobic moiety that binds to a cell membrane and an aryl linker, to target cells. Preferred modes of administration and formulations include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, etc. Injectable preparations can be prepared using aqueous solvents such as physiological saline solution and Ringer's gel solution, vegetable oils, higher fatty acid esters (e.g., ethyl oleate), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin), etc., and may include pharmaceutical carriers such as stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers to adjust pH, and preservatives to inhibit microbial growth (e.g., phenylmercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

[0134] The pharmaceutical composition of the present invention can also be provided as a formulation of a topical agent comprising natural killer cells whose surface is modified with a polymeric compound comprising a hydrophobic moiety that binds to a cell membrane; and an aryl linker.

[0135] When the pharmaceutical composition of the present invention is used as a topical application for the skin, it may additionally contain adjuvants commonly used in the field of dermatology, such as fatty substances, organic solvents, solvents, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic emulsifiers, nonionic emulsifiers, fillers, metal ion chelating agents, chelating agents, preservatives, vitamins, blockers, humectants, essential oils, dyes, pigments, hydrophilic activators, lipophilic activators, or lipid vesicles, and any other ingredients commonly used in topical applications for the skin. Furthermore, said ingredients may be introduced in amounts commonly used in the field of dermatology. When the pharmaceutical composition of the present invention is provided as a topical application for the skin, it may be in the form of an ointment, patch, gel, cream, or spray, but is not limited thereto.

[0136] The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, malt syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight with respect to the composition, but is not limited thereto.

[0137] The term "administration" as used in the present invention means providing a specific composition of the present invention to an individual by any appropriate method.

[0138] In addition, the effective dosage to the human body of natural killer cells whose surface is modified with a polymeric compound comprising a hydrophobic moiety that binds to the cell membrane of the present invention and an aryl linker may vary depending on the patient's age, body weight, gender, form of administration, health condition, and degree of disease, and is generally about 0.001-100 mg / kg / day, preferably 0.01-35 mg / kg / day. Based on an adult patient weighing 70 kg, it is generally 0.07-7000 mg / day, preferably 0.7-2500 mg / day, and may be administered in divided doses once or several times a day at regular intervals according to the judgment of a doctor or pharmacist.

[0139] The pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, or biological response modifiers.

[0140] In one aspect, the present invention relates to a cell therapy composition comprising a natural killer cell whose surface is modified with a polymeric compound comprising a hydrophobic moiety that binds to a cell membrane and an aryl linker as an active ingredient.

[0141] The administration route of the cell therapy composition of the present invention may be administered through any general route as long as it can reach the target tissue. It may be administered in a conventional manner via rectal, intravenous (intravenous therapy, iv), intra-arterial, intraperitoneal, intramuscular, intrasternal, transdermal, local, intraocular, or intradermal routes, but is not limited thereto.

[0142] The compositions of the present invention may be formulated in a suitable form with pharmaceutical carriers commonly used in cell therapy. "Pharmaceuticalally acceptable" means a composition that is physiologically acceptable and, when administered to humans, does not typically cause allergic reactions or similar reactions such as gastrointestinal disturbances or dizziness. Pharmaceutically acceptable carriers include, for example, parenteral delivery carriers such as water, suitable oils, saline solution, aqueous glucose, and glycol, and may additionally contain stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Other pharmaceutically acceptable carriers may be referenced in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

[0143] In addition, the composition of the present invention may be administered by any device capable of moving the cell therapeutic agent to target cells.

[0144] In addition, the cell therapy composition of the present invention may include a therapeutically effective amount of cell therapy for the treatment of a disease.

[0145] The above “therapeutically effective amount” refers to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal, or human as conceived by a researcher, veterinarian, physician, or other clinician, and includes an amount that induces the alleviation of symptoms of the disease or disorder being treated. It is obvious to those skilled in the art that the cell therapy agent included in the composition of the present invention will vary according to the desired effect. Therefore, the optimal cell therapy agent content can be easily determined by those skilled in the art and may be adjusted according to various factors including the type of disease, the severity of the disease, the content of other ingredients contained in the composition, the type of formulation, and the patient's age, weight, general health status, gender, diet, time of administration, route of administration and secretion rate of the composition, duration of treatment, and concurrently used drugs. It is important to include an amount that can obtain maximum effect with a minimum amount without side effects, taking all of the above factors into consideration.

[0146] When the cell therapy composition of the present invention is administered once or several times a day, the cell therapy agent included in the composition is 1 × 10 per kg of body weight. 8 Up to 1×10 9 It is desirable to include the number of cells.

[0147] In one aspect, the present invention relates to a method for promoting the priming of natural killer cells, comprising the steps of: immobilizing a polymer compound for modifying the surface of natural killer cells to promote cell priming, comprising a hydrophobic moiety that binds to a cell membrane; and an aryl linker, onto the surface of natural killer cells; and inducing cell aggregation by bringing the natural killer cells, on which the polymer compound for modifying the surface of natural killer cells to promote cell priming is immobilized, into contact with other natural killer cells in vivo.

[0148] In one embodiment of the present invention, the polymer compound may further comprise a cell internalization-preventing moiety, but is not limited thereto.

[0149] In one embodiment of the present invention, the priming of natural killer cells may occur in vitro and / or in vivo.

[0150] The present invention will be explained in more detail below through examples. However, since these examples are merely illustrative of the present invention, the scope of the present invention should not be interpreted as being limited by these examples.

[0151] Example 1: Preparation of a polymer compound for natural killer cell surface modification

[0152] FIGS. 1a and 1b illustrate, exemplarily, the process of manufacturing a polymer compound for surface modification of natural killer cells according to the present invention.

[0153] Referring to FIG. 1b, the present invention devised a priming technique that designs a lipid polymer into a triangular amphiphilic structure to induce the clustering of natural killer cells through the insertion of various hydrophobic lipids into the cell membrane and thereby activates the natural killer cells.

[0154] In this experiment, 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (hereinafter DSPE) was used as the hydrophobic moiety (or lipid polymer), polyethylene glycol (PEG) was used as the cell internalization moiety, and trimesic acid (hereinafter TMA linker), which has a structure in which carboxyl groups (-COOH) are attached to the 1st, 3rd, and 5th carbon positions of a benzene ring, was used as the aryl linker.

[0155] DSPE-PEG-NH2 (molecular weight 2850 g / mol, purity 95% by GPC) was purchased from Nanosoft Polymers, Inc. (Winston Salem, NC, USA). 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, purity >98.0%) was purchased from Tokyo Chemical Industry (Tokyo, Japan). N-hydroxy succinimide (NHS, purity 98%), dimethyl formamide (DMF, purity 99.8%, anhydrous), 4-dimethylaminopyridine (DMAP, purity ≥99%), and trimesic acid (TMA) (95%) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0156] The first step is to activate the carboxyl group of the TMA linker.

[0157] First, 0.1 mmol of TMA linker (20 mg) was dissolved in 10 mL of inorganic dimethylformamide (anhydrous DMF) along with 0.29 mmol of EDC (3-ethylcarbodiimide hydrochloride, 54.5 mg) and 0.48 mmol of NHS (N-hydroxysuccinimide, 54.7 mg), and the reaction mixture was stirred at room temperature for 3 hours to activate the carboxyl group.

[0158] The second step is to combine the TMA linker with DSPE-PEG.

[0159] The TMA linker and DSPE-PEG were synthesized using the EDC / NHS reaction method. Specifically, the carboxyl-activated TMA linker was reacted with 0.32 mmol of DSPE-PEG-NH2 (849.9 mg) in anhydrous DMF by adding 0.3 mmol of DMAP (37.2 mg). The reaction mixture was stirred at room temperature for 48 hours under N2 protection. The reaction mixture was washed with distilled water (DW) for 3 days to remove unreacted DSPE-PEG-NH2, TMA, EDC, NHS, and single / double bonds of DSPE-PEG-NH and TMA, leaving only the lipid conjugate in which DSPE-PEG is attached to three substituents of the TMA linker. The washed reaction solution was freeze-dried to obtain a white solid powder with a yield of 64%, and the lipid conjugate (DSPE- PEG It was named -NH)3-T.

[0160] Example 2: Characterization of (DSPE-PEG-NH)3-T

[0161] The final product (DSPE- PEG For the compounds generated at each step to analyze the structural characteristics of -NH)3-T 1 H-NMR (500 MHz FT-NMR, Bruker, Billerica, MA, USA) was performed.

[0162] As a result, as shown in FIGS. 2a and 2b (DSPE- PEG As a result confirmed by NMR analysis of -NH)3-T, 1 H-NMR (500 MHz, DMSO-d6) NMR signals of δ 8.97, 8.87, 8.64, 8.57, 8.56, 8.54, 8.43, 7.93, 7.51, 3.51, 2.25, 1.49, 1.23, 1.17, 0.98, and 0.85 ppm were confirmed.

[0163] The NMR signal of the TMA linker is 1H-NMR (500 MHz, dimethyl sulfoxide (DMSO)-d6) δ 13.59 ppm and 8.66 ppm, DSPE- PEG The NMR signal of -NH2 is 1 H-NMR (500 MHz, DMSO-d6) δ is 7.72 and 7.57, 3.51, 2.37, 2.26, 1.49, 1.23, 1.06, and 0.85 ppm.

[0164] Synthesized (DSPE- PEG -NH)3-T exhibited major NMR signals for terminal methyl groups (δ 0.85 ppm), lipid chain methyl protons (δ 0.98–2.25 ppm), PEG repeating units (δ 3.51 ppm), DSPE-PEG amide protons (δ 7.51–7.93 ppm), and core phenyl ring protons (δ 8.64 ppm). Additionally, signals corresponding to newly formed amide bond protons were observed at δ 8.43–8.57 and δ 8.75–8.97 ppm, thereby (DSPE- PEG It was confirmed that -NH)3-T was successfully synthesized.

[0165] In addition, by comparing the peak integral value at δ 8.64 ppm (3H), the terminal CH3 group peak at δ 0.85 ppm (19H), and the PEG ethylene glycol repeating unit peak at δ 3.51 ppm (628H), it was further confirmed that three DSPE-PEG molecules were successfully conjugated to the benzene ring of the TMA linker (Fig. 2b).

[0166] In addition, it was confirmed that there is no intermediate signal in the NMR spectrum, indicating the presence of (DSPE- in the white solid powder PEG It was confirmed that the purity of -NH)3-T is 95% or higher.

[0167] Example 3: Evaluation of cell viability of natural killer cells surface-modified with (DSPE-PEG-NH)3-T

[0168] The (DSPE- prepared in Example 1 PEGAfter treating natural killer cells (hereinafter referred to as NK cells) with -NH)3-T, cell viability was evaluated using the CellTiter-Blue® kit (Promega Corporation, US) with the WST-1 assay.

[0169] NK-92 mi cells (American Type Culture Collection, ATCC, USA) were prepared with a complete growth medium using Minimum Essential Medium Alpha (MEMα, Gibco, USA). The complete growth medium consisted of 12.5% ​​fetal bovine serum (Gibco), 12.5% ​​horse serum (Gibco), 1% penicillin streptomycin solution (Corning, NY, USA), 0.2 mM inositol (Sigma-Aldrich), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), and 0.02 mM folic acid (Sigma-Aldrich). The culture environment for all cells was maintained at 37°C in a humid environment containing 5% CO2.

[0170] (DSPE- PEG -NH)3-T was dissolved in complete growth medium at concentrations of 0 to 200 μg / mL. 2 × 10⁶ NK cells were dissolved in 200 μL of (DSPE- PEG The cells were suspended in -NH)3-T solution. The suspended cells were dispensed into a U-bottom 96-well cell culture plate (SPL Life Science), cultured at room temperature for 30 minutes, and then cultured at 37°C for 6 hours.

[0171] To evaluate cell viability, 40 μL of CellTiter-Blue® (Promega, Madison, WI, USA) was added to each well. The treated cells were cultured at 37 °C for 3 additional hours, then centrifuged at 400× g for 5 minutes, and the supernatant was transferred to a new flat-bottom 96-well cell culture plate. The fluorescence intensity (FI) of the supernatant was measured using a microplate spectrophotometer (Ex / Em = 560 / 590 nm wavelength). Cell viability was calculated using the following Equation 1.

[0172] [Mathematical Formula 1]

[0173]

[0174] As a result, as shown in Fig. 3 (DSPE- PEG It was confirmed that no cytotoxicity occurred, as no decrease in NK cell viability was observed until the -NH)3-T concentration reached 200 μg / mL.

[0175] Example 4: Structural analysis of natural killer cell clusters coated with (DSPE-PEG-NH)3-T

[0176] Generally, NK cells develop into a spherical shape starting 24 hours after the initial proliferation period. Therefore (DSPE- PEG To evaluate the degree of cluster formation promoted by -NH)3-T, the sphericity of the NK spheres was measured after 6 hours.

[0177] First, (DSPE- PEG To fix -NH)3-T to the surface of NK cells (DSPE- PEG -NH)3-T was dissolved in complete growth medium at a concentration of 1 mg / mL. 5 × 10⁴ NK cells were cultured with 100 μL of (DSPE-PEG-NH)₃-T solution at room temperature for 30 minutes. Subsequently, (DSPE- PEGTo remove NK cells not coated with -NH)3-T, cells were washed twice with 500 μL of DPBS (Dulbecco's phosphate-buffered saline). On the cell surface (DSPE- PEG -NH)3-T coated NK cells (hereinafter TLNK cells) were resuspended in complete growth medium at a concentration of 5 × 10³ cells / 100 μL.

[0178] Agar-coated well plates were prepared by adding 50 μL of 1.5% (w / v) agar (E&S Bioelectronics Company, Daejeon-si, Republic of Korea), dissolved in distilled water using a microwave, to flat-bottom 96-well cell culture plates (SPL Life Science, Pocheon-si, Republic of Korea). Subsequently, 5 × 10³ NK cells or TLNK cells were cultured in the agar-coated well plates. Each plate was cultured for 6, 24, and 48 hours; spheroid size was analyzed after 6 hours of culture, and spheroid volume was analyzed after 24 and 48 hours. Cells were cultured in a humid environment containing 5% CO2 at 37 ℃.

[0179] Spheroid images of NK cells and TLNK cells were taken using an optical microscope (Ti-E System, Nikon, Tokyo, Japan). To evaluate clustering efficiency (i.e., spheroid volume and sphericity), ImageJ software 1.53e was used, spheroid volume was calculated using Equation 2, and sphericity was calculated using Equation 3.

[0180] [Mathematical Formula 2]

[0181]

[0182] [Mathematical Formula 3]

[0183]

[0184] As a result, as shown in Fig. 4, the sphericity of the pure NK spheres formed without material treatment is 0.45 (Fig. 4A), (DSPE- PEG The sphericity of the TLNK spheres formed using -NH)3-T was found to be 0.52 (Fig. 4B). These (DSPE- PEG The increased sphericity due to the -NH)3-T coating indicates that the distance between NK cells within the sphere is relatively close and uniform (Fig. 4C). These results suggest that the exchange of signaling molecules and the induction of membrane contact signals within the NK population can be activated.

[0185] The volumes observed in both NK and TLNK spheres showed similar trends over time. These results (DSPE- PEG This suggests that -NH)3-T contributes to the uniform aggregation of cell aggregates without interfering with the inherent NK cell clustering function. Furthermore, the spherical arrangement of these NK cell aggregates increased the likelihood of individual constituent cells coming into contact with neighboring cells. Although (DSPE- PEG Although -NH)3-T did not directly affect the macroscopic dimension of NK cell clusters, it is expected that cluster formation by the substance could cause changes in intercellular contact, thereby affecting the core structure within the clusters.

[0186] Example 4: Analysis of Cytokine Release from Natural Killer Cells Coated with (DSPE-PEG-NH)3-T

[0187] To evaluate the levels of IFN-γ released from NK cells by inflammatory stimulation, 1 μg / mL of LPS (Escherichia coli O26:B6, Sigma-Aldrich) was applied to NK cell globules or TLNK cell globules and incubated in plates at 37°C for 6 and 24 hours. The IFN-γ secretion response was analyzed using the Enzyme-Linked ImmunoSorbent Assay Kit (PeproTech, Cranbury, NJ, USA) according to the manufacturer's instructions.

[0188] As a result, as shown in Figure 5, compared to naturally formed pure NK spheres, TLNK spheres were found to have improved IFN-γ secretion levels when spheres were formed for 6 and 24 hours.

[0189] These results were obtained from the (DSPE- prepared in Example 1 PEG This suggests that -NH)3-T does not inhibit sequential signaling mechanisms during the priming process of NK cells.

[0190] The above signaling mechanism includes the binding of membrane receptors to external antigen molecules, the initiation of signal transduction into intracellular pathways, stepwise signal regulation for cytokine expression, and the secretion of generated cytokines through the NK cell membrane.

[0191] In addition, the significant increase in the amount of IFN-γ secreted from TLNK globules was (DSPE- PEG -NH)3-T induction of NK cell aggregation and cell priming methods through this process show that it improved membrane-to-membrane contact within NK cell populations and enabled more frequent signal exchange in pure NK globules without natural ECM formation.

[0192] Although embodiments of the present invention have been described in more detail with reference to the attached drawings, the present invention is not necessarily limited to these embodiments and may be modified in various ways within the scope of the technical spirit of the present invention. Accordingly, the embodiments disclosed in the present invention are intended to explain, not limit, the technical spirit of the present invention, and the scope of the technical spirit of the present invention is not limited by these embodiments. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of protection of the present invention shall be interpreted by the claims below, and all technical spirits within an equivalent scope shall be interpreted as being included within the scope of rights of the present invention.

[0193] [National R&D projects that supported this invention]

[0194] [Project ID] 2710006360

[0195] [Assignment No.] 00419061

[0196] [Ministry Name] Ministry of Science and ICT

[0197] [Name of Project Management (Specialized) Agency] Korea Institute for Science and Technology Commercialization

[0198] [Research Project Name] Support Project for Activating Industry-Academic-Research Cooperation

[0199] [Project Title] IP Advancement and Commercialization for the Promotion of Commercialization of Immunotherapy with Enhanced Anticancer Functionality Using Cell Membrane-Bound Polymer Material Technology

[0200] [Name of Project Performing Organization] Dongguk University Industry-Academic Cooperation Foundation

[0201] [Research Period] April 1, 2024 ~ December 31, 2024

[0202]

[0203] [Project ID] 2710003135

[0204] [Assignment No.] 00277800

[0205] [Ministry Name] Ministry of Science and ICT

[0206] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0207] [Research Project Name] Individual Basic Research (Ministry of Science and ICT)

[0208] [Project Title] Development of Immuno-oncology Cell Complexes for Pancreatic Cancer Treatment Using Triple-Function Enhanced Cell Membrane Activation Materials

[0209] [Name of Project Performing Organization] Dongguk University Industry-Academic Cooperation Foundation

[0210] [Research Period] 2024.03.01 ~ 2025.02.28

[0211]

[0212] [Project ID] 2710006048

[0213] [Assignment No.] 00398030

[0214] [Ministry Name] Ministry of Science and ICT

[0215] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0216] [Research Project Name] Group Research Support

[0217] [Project Title] Laboratory for Immunocell Conversion for Anticancer Therapy Based on In Situ Surface Modification through the Advancement of Organic and Inorganic Biomaterials

[0218] [Name of Project Performing Organization] Dongguk University Industry-Academic Cooperation Foundation

[0219] [Research Period] May 1, 2024 – April 30, 2025

Claims

1. Hydrophobic moiety that binds to the cell membrane; and Includes an aryl linker Polymer compound for natural killer cell surface modification to promote cell priming.

2. In Paragraph 1, The above hydrophobic moiety is selected from the group consisting of phospholipids having alkyl chains having 12 to 24 carbon atoms, sterol lipids having 10 to 30 carbon atoms, 1,2-bis(diphenylphosphino)ethane (DPPE), and 1,2-bis(dimethylphosphino)ethane (DMPE). Polymer compound for natural killer cell surface modification to promote cell priming.

3. In Paragraph 1, The above hydrophobic moiety is a polymeric compound for natural killer cell surface modification to promote cell priming, wherein the above hydrophobic moiety is a compound represented by Chemical Formula 1 or a derivative thereof: [Chemical Formula 1] q is an integer greater than 0, and p is an integer greater than 0.

4. In Paragraph 1, The above aryl linker is selected from the group consisting of phenyl linker, naphthalene linker, anthracenyl linker, perylenyl linker, pyrrole linker, furanyl linker, thiophene linker, and oxazolyl linker. Polymer compound for natural killer cell surface modification to promote cell priming.

5. In Paragraph 1, The above polymer compound for surface modification of natural killer cells is, further including a cell internalization-preventing moiety, Polymer compound for natural killer cell surface modification to promote cell priming.

6. In Paragraph 5, The above cell internalization-inhibiting moiety is selected from the group consisting of polyethylene glycol (PEG), polyethylene oxide (PEO), and polyvinyl alcohol (PVA). Polymer compound for natural killer cell surface modification to promote cell priming.

7. In Paragraph 5, The above cell internalization-inhibiting moiety is a compound represented by Chemical Formula 2 or a derivative thereof, Polymeric compounds for natural killer cell surface modification to promote cell priming: [Chemical Formula 2] n is an integer greater than 0.

8. In Paragraph 1, The above polymer compound for surface modification of natural killer cells is, Inducing cell clustering of natural killer cells, Polymer compound for natural killer cell surface modification to promote cell priming.

9. In Paragraph 1, The above polymer compound for surface modification of natural killer cells is, Increasing the production of interferon gamma (IFN-γ), Polymer compound for natural killer cell surface modification to promote cell priming.

10. In Paragraph 1, The above polymer compound for surface modification of natural killer cells is, That which enhances the anticancer effect of natural killer cells, Polymer compound for natural killer cell surface modification to promote cell priming.

11. A natural killer cell whose surface is modified by bonding a polymer compound of any one of claims 1 to 10. 12.(a) A step of activating the carboxyl group of an aryl linker comprising a plurality of carboxyl groups and (b) a step comprising attaching a polymeric compound comprising a hydrophobic moiety, an anti-internalization moiety, and an amine group to each activated carboxyl group site of an aryl linker, Method for preparing a polymer compound for natural killer cell surface modification to promote cell priming.

13. In Paragraph 12, The polymeric compound comprising the above-mentioned hydrophobic moiety, cell internalization prevention moiety, and amine group is, Method for preparing a polymer compound for natural killer cell surface modification to promote cell priming, which is a compound represented by Chemical Formula 3 or a derivative thereof: [Chemical Formula 3] n is an integer greater than 0, and q is an integer greater than 0, and p is an integer greater than 0.

14. In Paragraph 12, The polymer compound after completing step (b) above is, A compound represented by Chemical Formula 4 or a derivative thereof, Method for preparing a polymer compound for natural killer cell surface modification to promote cell priming: [Chemical Formula 4] n is an integer greater than 0, and q is an integer greater than 0, and p is an integer greater than 0.

15. A pharmaceutical composition for the prevention or treatment of cancer comprising, as an active ingredient, natural killer cells whose surface is modified with a polymeric compound comprising a hydrophobic moiety that binds to a cell membrane; and an aryl linker.

16. In Paragraph 15, A pharmaceutical composition for the prevention or treatment of cancer, wherein the above cancer is selected from the group consisting of thyroid cancer, stomach cancer, colorectal cancer, lung cancer, breast cancer, liver cancer, pancreatic cancer, testicular cancer, oral cancer, basal cell carcinoma, brain tumor, gallbladder cancer, bile duct cancer, laryngeal cancer, retinoblastoma, ampullary cancer, bladder cancer, peritoneal cancer, adrenal cancer, non-small cell lung cancer, tongue cancer, small cell lung cancer, small intestine cancer, meningioma, esophageal cancer, renal pelvis and ureter cancer, kidney cancer, malignant bone tumor, malignant soft tissue tumor, malignant lymphoma, malignant melanoma, eye tumor, urethral cancer, stomach cancer, sarcoma, pharyngeal cancer, cervical cancer, endometrial cancer, uterine sarcoma, metastatic brain tumor, rectal cancer, vaginal cancer, spinal cord tumor, salivary gland cancer, tonsil cancer, squamous cell carcinoma, hematological cancer, and anal cancer.

17. A cell therapy composition for preventing or treating cancer, comprising natural killer cells whose surface is modified with a polymeric compound comprising a hydrophobic moiety that binds to a cell membrane; and an aryl linker.

18. A step of immobilizing a polymer compound for modifying the surface of a natural killer cell to promote cell priming, comprising a hydrophobic moiety that binds to a cell membrane; and an aryl linker, onto the surface of a natural killer cell; and A method for promoting the priming of natural killer cells, comprising the step of inducing cell aggregation by bringing natural killer cells, on which a polymer compound for modifying the surface of natural killer cells to promote cell priming is immobilized on the cell surface in vivo, into contact with other natural killer cells.

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