Polymer compound for cell surface modification for improving drug delivery efficiency

Abstract

The present invention relates to a polymer compound for cell surface modification for improving drug delivery efficiency. A polymer compound for natural killer cell surface modification according to an embodiment of the present invention includes a DSPE lipid having high hydrophobicity, and thus has the effect of increasing cell membrane immobilization efficiency and binding retention time. The polymer compound includes a prodrug at one end, and thus can improve the efficacy of immunotherapy through a target drug delivery system mediated by immune cells such as natural killer cells.

Description

Polymeric compounds for cell surface modification to improve drug delivery efficiency

[0001] The present invention relates to a polymer compound for cell surface modification to improve drug delivery efficiency.

[0002] Adoptive cell therapy involves collecting lymphocytes or natural killer cells from the body, isolating and proliferating them, and then re-injecting them. CAR-T therapy, which has evolved from the previous method of isolating and proliferating tumor-infiltrating lymphocytes to inject them, involves genetically engineering T lymphocytes to attach chimeric antigen receptors and injecting them. This therapy is gaining attention and is currently being used for blood cancers. However, despite the initial success of natural killer cell-based adoptive cell therapy for blood cancers, the therapeutic effect on solid tumors remains minimal.

[0003] Solid tumors form a dense extracellular matrix that acts as a physical barrier and secrete immunosuppressive cytokines, inducing the exaustion of peripheral immune cells, particularly through the formation of the tumor microenvironment (TME), and inhibiting the infiltration and cytotoxic function of natural killer cells, thereby promoting the growth and metastasis of tumor cells.

[0004] Meanwhile, layer-by-layer self-assembly can be utilized as a cell coating technology. The aforementioned layer-by-layer method is a simple and versatile deposition process that is widely applied to address many issues, such as biomolecular deposition, concentration, bioactivity, coating thickness, and release rate.

[0005] However, utilizing the above-mentioned layer-by-layer method requires surface modification of the cells, and this surface modification presents process difficulties as it involves alternating reactions and stacking of cationic or anionic substrate materials. Furthermore, the covering of the entire cell surface with a coating material may lead to a potential degradation of the function of surface membrane proteins involved in signal transduction and cancer cell recognition. In addition, when cell surface coatings are used for a single purpose, such as preventing cell aggregation, there is often a lack of strategies for multifunctionality that can be expected to provide appropriate immuno-oncology therapeutic effects.

[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, although natural killer (NK) cell-based adoptive cell therapy has garnered attention as a novel anticancer treatment due to its ability to target and eliminate cancer cells, the need for a new adoptive cell therapy applicable to solid tumors was recognized, as it is difficult to apply this therapy to solid tumors that form a tumor microenvironment and induce exhaustion of peripheral immune cells.

[0008] Furthermore, it was noted that pre-sensitizing cancer cells by administering a first-line anticancer drug before the anticancer chemotherapy stage can improve antitumor efficacy while minimizing drug resistance and side effects by regulating the tumor microenvironment.

[0009] Accordingly, the inventors of the present invention aimed to develop a drug delivery platform capable of providing natural killer cells and a prodrug together.

[0010] As a result, the inventors of the present invention devised a technology in which a primary anticancer drug is dissociated from a polymer compound by glutathione (GSH) released from cancer cells dissolved by natural killer cells, thereby delivering the anticancer drug to surrounding cancer cells.

[0011] Accordingly, the problem to be solved by the present invention is to provide a polymeric compound for cell surface modification to improve drug delivery efficiency, comprising a hydrophobic moiety that binds to a cell membrane, a moiety that prevents cell internalization, a disulfide linker, and an anticancer prodrug moiety.

[0012] In addition, another problem that the present invention aims to solve is to provide natural killer cells whose surfaces are modified with the polymer compound.

[0013] In addition, another problem that the present invention aims to solve is to provide a drug delivery system whose surface is modified with the above-mentioned polymer compound.

[0014] In addition, another problem that the present invention aims to solve is to provide a method for manufacturing a polymeric compound comprising a hydrophobic moiety that binds to a cell membrane, a moiety that prevents cell internalization, a disulfide linker, and an anticancer prodrug moiety.

[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 a polymeric compound comprising a hydrophobic moiety that binds to a cell membrane; a moiety that prevents cell internalization; a disulfide linker; and an anticancer prodrug moiety.

[0016] In addition, another problem that the present invention aims to solve is to provide a method for preventing or treating cancer, comprising the step of administering the above-mentioned pharmaceutical composition for cancer prevention or treatment to an individual.

[0017] 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.

[0018] To solve the problem described above, a polymer compound for modifying a cell surface to improve drug delivery efficiency is provided, comprising a hydrophobic moiety that binds to a cell membrane, a cell internalization prevention moiety, a disulfide linker, and an anticancer prodrug moiety according to one embodiment of the present invention.

[0019] 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).

[0020] According to another feature of the present invention, the hydrophobic moiety may be a compound represented by Formula 1 or a derivative thereof.

[0021] [Chemical Formula 1]

[0022]

[0023] q can be an integer greater than 0, and

[0024] p can be an integer greater than 0.

[0025] 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.

[0026] [Chemical Formula 2]

[0027]

[0028] n can be an integer greater than 0.

[0029] 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).

[0030] According to another feature of the present invention, the disulfide linker may be a compound represented by Chemical Formula 3 or a derivative thereof.

[0031] [Chemical Formula 3]

[0032]

[0033] R and R' may each be independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclil, and substituted heterocyclil.

[0034] According to another feature of the present invention, the alkyl group may be a methylene having 1 to 30 repeating structural units.

[0035] According to another feature of the present invention, the cancer may be selected from the group consisting of prostate cancer, 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.

[0036] According to another feature of the present invention, the prodrug may be selected from the group consisting of gemcitabine, fludarabine, clofarabine, cladribine, azacitidine, decitabine, cytarabine, nelarabine, nimustine, temozolomide, dacarbazine, thioguanine, carboquone, doxorubicin, entinostat, pemetrexed, and methotrexate.

[0037] According to another feature of the present invention, the anticancer prodrug moiety may be covalently bonded to a disulfide linker.

[0038] According to another feature of the present invention, the cell membrane may be the cell membrane of an immune cell or an artificial cell membrane.

[0039] According to another feature of the present invention, the immune cell may be a natural killer cell.

[0040] According to another feature of the present invention, the polymer compound may deliver an anticancer prodrug, which is dissociated as the disulfide bond is cleaved by glutathione released from cancer cells, to surrounding cancer cells.

[0041] To solve the problem described above, a natural killer cell with a surface modified by combining the polymer compound according to another embodiment of the present invention is provided.

[0042] To solve the problem described above, a drug delivery system having a surface modified with the polymer compound according to another embodiment of the present invention is provided.

[0043] In order to solve the problem as described above, according to another embodiment of the present invention

[0044] (a) A step of activating the carboxyl groups of a disulfide containing carboxyl groups at both ends;

[0045] (b) a step of attaching a polymeric compound comprising a hydrophobic moiety, an anti-internalization moiety, and an amine group to one end of a disulfide and

[0046] (c) A method for preparing a polymer compound comprising the step of binding a prodrug to the other end of the disulfide of the polymer compound in which step (b) is completed.

[0047] According to a feature 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 4 or a derivative thereof.

[0048] [Chemical Formula 4]

[0049]

[0050] q can be an integer greater than 0, and

[0051] p can be an integer greater than 0.

[0052] According to a feature of the present invention, the polymer compound in which step (b) is completed may be a compound represented by Formula 5 or a derivative thereof.

[0053] [Chemical Formula 5]

[0054]

[0055] q can be an integer greater than 0, and

[0056] p can be an integer greater than 0, and

[0057] n can be an integer greater than 0, and

[0058] R and R' may each be independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclil, and substituted heterocyclil.

[0059] According to a feature of the present invention, a polymeric compound comprising a hydrophobic moiety that binds to a cell membrane, a cell internalization prevention moiety, a disulfide linker, and an anticancer prodrug moiety may be a compound represented by Formula 6 or a derivative thereof.

[0060] [Chemical Formula 6]

[0061]

[0062] q can be an integer greater than 0, and

[0063] p can be an integer greater than 0, and

[0064] n can be an integer greater than 0, and

[0065] R and R' may each be independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclil, and substituted heterocyclil, and

[0066] A may be selected from gemcitabine, temozolomide, irinotecan, ifosfamide, fludarabine, and clofarabine.

[0067] To solve the problem described above, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising as an active ingredient a polymeric compound comprising a hydrophobic moiety that binds to a cell membrane according to another embodiment of the present invention; a moiety that prevents cell internalization; a disulfide linker; and an anticancer prodrug moiety.

[0068] To solve the problem described above, a method for preventing or treating cancer is provided, comprising the step of administering the pharmaceutical composition for preventing or treating cancer according to another embodiment of the present invention to an individual.

[0069] A polymer compound for surface modification of natural killer cells according to one embodiment of the present invention includes a DSPE lipid having high hydrophobicity, which has the effect of increasing cell membrane immobilization efficiency and binding maintenance period, and the polymer compound includes a prodrug at one end, which can enhance the efficacy of immunotherapy through a targeted drug delivery system mediated by immune cells such as natural killer cells.

[0070] The effects according to the present invention are not limited to those exemplified above, and various other effects are included in this specification.

[0071] 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.

[0072] FIG. 2 is a DSPE- according to an embodiment of the present invention PEG This shows the results of the structural characteristic analysis of -ss-Gem.

[0073] Figure 3 illustrates the results of evaluating the cell viability and proliferation ability of natural killer cells by Gem alone treatment according to one embodiment of the present invention.

[0074] FIGS. 4a and 4b illustrate various DSPEs according to an embodiment of the present invention. PEGThis shows the results of evaluating the cell viability and proliferation ability of surface-modified natural killer cells according to -ss-Gem concentration.

[0075] FIGS. 4C and 4D illustrate the results of evaluating the anticancer function of GCNK cells coated with a lipid-gemcitabine conjugate at a concentration of 3.3 μg / mL or 33 μg / mL according to one embodiment of the present invention.

[0076] FIG. 5 is a DSPE- according to an embodiment of the present invention PEG This shows the results of the evaluation of the availability of cell membrane components in natural killer cells whose surfaces were modified with -ss-Gem.

[0077] FIG. 6 is a DSPE- according to an embodiment of the present invention. PEG This shows the results of measuring the zeta potential of the surface of natural killer cells modified with -ss-Gem.

[0078] FIG. 7a is a DSPE- according to an embodiment of the present invention PEG This shows the results of confirming the gemcitabine release profile by GSH from -ss-Gem.

[0079] FIGS. 7b and 7c illustrate the results of evaluating cell viability of PANC-1 and MIA PaCa-2 exposed to released gemcitabine according to one embodiment of the present invention.

[0080] FIG. 8 illustrates the results of E / T cluster quantification for effector cells (NK or GCNK cells) and target cells (PANC-1, MIA PaCa-2, and normal fibroblasts) according to one embodiment of the present invention.

[0081] FIGS. 9a to 9c illustrate the results of an analysis of calcein release from NK and GCNK cells to pancreatic cancer cells and normal fibroblasts according to one embodiment of the present invention.

[0082] FIGS. 10a and 10b illustrate the results of evaluating MICA / B expression levels in PANC-1 and MIA PaCa-2 exposed to released gemcitabine according to one embodiment of the present invention.

[0083] FIGS. 11a and 11b are DSPE- according to an embodiment of the present invention PEG This shows the results of the analysis of calcein release from PANC-1 and MIA PaCa-2 following treatment of natural killer cells surface-modified with -ss-Gem.

[0084] 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.

[0085] In the following, terms used within this specification are explained for clarity of explanation.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] The term “prodrug” as used in this invention refers to a drug that is converted into an active form through metabolic processes within the body, and in this invention, it refers to a scope that includes both prodrugs and their derivatives.

[0090] The term “disulfide linker” used in the present invention refers to a compound having a structure in which two sulfur atoms are bonded, and said disulfide linkers play an important role in biochemistry and molecular biology, and are particularly used to stabilize the secondary and tertiary structures of proteins.

[0091] The term “artificial cell membrane” as used in this invention refers to a synthetic structure created by mimicking the structure and function of a natural cell membrane, having a double membrane form composed mainly of phospholipids, and possessing selective permeability and protective functions similar to those of a cell membrane.

[0092] The term “Drug Delivery System” as used in this invention refers to a system designed to efficiently deliver a drug to a specific part of the body, minimize side effects, and maximize drug efficacy.

[0093] The inventors recognized the problems with the adoptive cell therapy for solid tumors described above, and to solve these problems, they developed a polymeric compound in which a phospholipid acting as an anchor for fixing the cell membrane, PEG (Polyethylene Glycol) which improves solubility and prevents intracellular invasion, a disulfide linker that is cleaved by glutathione (GSH) released from cancer cells, and an anticancer prodrug are sequentially combined.

[0094] In one aspect, the present invention relates to a polymeric compound for modifying a cell surface to improve drug delivery efficiency, comprising a hydrophobic moiety that binds to a cell membrane, a moiety that prevents cell internalization, a disulfide linker, and an anticancer prodrug moiety.

[0095] 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.

[0096] In one embodiment of the present invention, the phospholipid having an alkyl chain having 12 to 24 carbon atoms is preferably 1 , 2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) , 1 , 2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) , 1 , 2-Distearoyl-sn-glycero-3-phosphocholine(DSPC) , 1 , 2-Dioleoyl-sn-glycero-3-phosphocholine(DOPC) , 1 ,2-Dipalmitoyl-sn-glycero-3-phosphocholine(DPPC) , 1 , 2-Dimyristoyl-sn-glycero-3-phosphocholine(DMPC) , 1 , 2-Dioleoyl-3-trimethylammonium-propane(chloride salt)(DOTAP) , 1 , 2-Distearoyl-3-trimethylammonium-propane (chloride salt) (DSTAP) may be used, but is not limited thereto. Any lipid-type biomolecule 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.

[0097] In one embodiment of the present invention, the sterol lipid having 10 to 30 carbon atoms is preferably cholesterol. , Cholesterol hexasuccinate , 3β , N'-Dimethylaminoethane)Carbamoyl]Cholesterol , ergosterol , Stigmasterol or lanosterol, etc., may be used, but any lipid-type biomolecule 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.

[0098] 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.

[0099] Therefore, to improve coating retention on the cell surface, it is desirable to use a highly hydrophobic DSPE lipid (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine) as a cell membrane anchor.

[0100] In one embodiment of the present invention, the hydrophobic moiety may be a compound represented by Formula 1 or a derivative thereof.

[0101] [Chemical Formula 1]

[0102]

[0103] q can be an integer greater than 0, preferably an integer from 1 to 30, and

[0104] p may be an integer greater than 0, preferably an integer from 1 to 30, but is not limited thereto.

[0105] In one embodiment of the present invention, the cell internalization-preventing moiety may be a compound represented by Formula 2 or a derivative thereof.

[0106] [Chemical Formula 2]

[0107]

[0108] n may be an integer greater than 0, preferably an integer from 30 to 70, but is not limited thereto.

[0109] 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).

[0110] The above-mentioned moiety for preventing cell internalization can function to maintain attachment to the cell membrane surface without preventing cells and macromolecular compounds from incorporating into the interior of natural killer cells.

[0111] In one embodiment of the present invention, the disulfide linker may be a compound represented by Chemical Formula 3 or a derivative thereof.

[0112] [Chemical Formula 3]

[0113]

[0114] R and R' may each be independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclil, and substituted heterocyclil, but are not limited thereto.

[0115] In one embodiment of the present invention, the alkyl may be methylene (CH2) having 1 to 30 repeating units.

[0116] In one embodiment of the present invention, the cancer may be selected from the group consisting of prostate cancer, 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.

[0117] In one embodiment of the present invention, the prodrug may be included without limitation as long as it is a compound known to those skilled in the art as a drug that is converted into an active form through a metabolic process in the body, preferably a prodrug containing an amine group capable of chemically bonding with other polymers through an EDC / NHS coupling reaction, or a prodrug derivative in which an amine group is introduced, and more preferably may be selected from the group consisting of gemcitabine, fludarabine, clofarabine, cladribine, azacitidine, decitabine, cytarabine, and nelarabine.

[0118] Representative prodrugs containing amine groups include gemcitabine, fludarabine, clofarabine, cladribine, azacitidine, decitabine, cytarabine, nelarabine, nimustine, temozolomide, dacarbazine, thioguanine, carboquone, doxorubicin, entinostat, pemetrexed, and methotrexate.

[0119] In one embodiment of the present invention, the anticancer prodrug moiety may be covalently bonded to a disulfide linker.

[0120] In one embodiment of the present invention, the cell membrane may be included without limitation as long as it is a cell having a cell membrane, and preferably may be an immune cell or a cell membrane of a drug delivery system coated with an artificial cell membrane.

[0121] In one embodiment of the present invention, the immune cell may be selected from T cells, B cells, natural killer cells, neutrophils, eosinophils, basophils, monocytes, macrophages, and dendritic cells, preferably may be a T cell or a natural killer cell, and more preferably may be a natural killer cell.

[0122] In one embodiment of the present invention, the polymer compound may deliver an anticancer prodrug, which is dissociated as the disulfide bond is cleaved by glutathione released from cancer cells, to surrounding cancer cells, but is not limited thereto.

[0123] In one aspect, the present invention relates to natural killer cells whose surface is modified with the above-mentioned polymer compound.

[0124] In the surface-modified natural killer cells according to the present invention, the description of the polymer compound is as described above.

[0125] In one aspect, the present invention relates to a drug delivery system whose surface is modified with the above-mentioned polymer compound.

[0126] In the drug delivery system according to the present invention, the description of the polymer compound is as described above.

[0127] In one embodiment of the present invention, the drug delivery system may have a structure in which a target substance (e.g., a drug) to be delivered is supported inside an artificial cell membrane, but is not limited thereto.

[0128] In one aspect, the present invention

[0129] (a) A step of activating the carboxyl groups of a disulfide containing carboxyl groups at both ends (S310);

[0130] (b) a step (S320) of attaching a polymer compound comprising a hydrophobic moiety, a cell internalization prevention moiety, and an amine group to one end of a disulfide and

[0131] (c) A method for manufacturing a polymer compound comprising the step (S330) of binding a prodrug to the other end of the disulfide of the polymer compound in which the above step (b) is completed.

[0132] In one embodiment of the present invention, the polymer compound comprising the hydrophobic moiety; the cell internalization prevention moiety; and the amine group may be a compound represented by Formula 4 or a derivative thereof.

[0133] [Chemical Formula 4]

[0134]

[0135] q can be an integer greater than 0, preferably an integer from 1 to 30, and

[0136] p may be an integer greater than 0, preferably an integer from 1 to 30, but is not limited thereto.

[0137] In one embodiment of the present invention, the polymer compound in which step (b) is completed may be a compound represented by Formula 5 or a derivative thereof.

[0138] [Chemical Formula 5]

[0139]

[0140] q can be an integer greater than 0, and

[0141] p can be an integer greater than 0, preferably an integer from 1 to 30, and

[0142] n can be an integer greater than 0, preferably an integer from 30 to 70, and

[0143] R and R' may each be independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclil, and substituted heterocyclil, but are not limited thereto.

[0144] In one embodiment of the present invention, the polymeric compound comprising a hydrophobic moiety that binds to a cell membrane, a cell internalization prevention moiety, a disulfide linker, and an anticancer prodrug moiety may be a compound represented by Formula 6 or a derivative thereof.

[0145] [Chemical Formula 6]

[0146]

[0147] q can be an integer greater than 0, preferably an integer from 1 to 30, and

[0148] p can be an integer greater than 0, preferably an integer from 1 to 30, and

[0149] n can be an integer greater than 0, preferably an integer from 30 to 70, and

[0150] R and R' may each be independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclil, and substituted heterocyclil, but are not limited thereto.

[0151] A may be selected from gemcitabine, fludarabine, clofarabine, cladribine, azacitidine, decitabine, cytarabine, nelarabine, nimustine, temozolomide, dacarbazine, thioguanine, carboquone, doxorubicin, entinostat, pemetrexed, and methotrexate, but is not limited thereto.

[0152] In one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of cancer, comprising as an active ingredient a polymeric compound comprising a hydrophobic moiety that binds to a cell membrane; a moiety that prevents cell internalization; a disulfide linker; and an anticancer prodrug moiety.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] The composition according to the present invention may comprise a pharmaceutically effective amount of an active substance alone or 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 disorders or dizziness.

[0158] 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 to the active substance, which is the active ingredient, either independently or in combination.

[0159] 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.

[0160] The active ingredient of the present invention can be administered in various oral and parenteral formulations during clinical administration, and when formulated, it is manufactured using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0161] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, troches, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin, with one or more active substances of the present invention. In addition, lubricants such as magnesium stirate talc are also used in addition to simple excipients. Liquid dosage forms 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.

[0162] 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.

[0163] The pharmaceutical composition of the present invention may be administered by any device capable of moving the active substance to target cells. Preferred modes of administration and formulations include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, etc. The injection may be prepared using aqueous solvents such as physiological saline solution or Ringer's solution, non-aqueous solvents such as vegetable oils, higher fatty acid esters (e.g., ethyl oleate), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.), and may include a pharmaceutical carrier such as a stabilizer to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), an emulsifier, a buffer to adjust pH, and a preservative to inhibit microbial growth (e.g., phenylmercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

[0164] The pharmaceutical composition of the present invention can also be provided in the form of an external preparation containing an active substance as an active ingredient.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] In addition, the effective dosage of the active substance of the present invention to the human body 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.

[0169] 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.

[0170] In one aspect, the present invention relates to a method for preventing or treating cancer, comprising the step of administering the pharmaceutical composition for preventing or treating cancer to an individual.

[0171] In the present invention, the term "individual" refers to a subject requiring a method for the prevention, control, or treatment of a disease, and may be used without limitation and may include humans, dogs, monkeys, cats, rodents, e.g., mice, genetically modified mice, etc. More specifically, it refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cattle.

[0172] The pharmaceutical composition of the present invention may be administered in a therapeutically effective amount or a pharmaceutically effective amount.

[0173] In the present invention, the term "therapeutically effective amount" refers to a pharmaceutically acceptable amount of salt of a composition effective for preventing or treating a target disease, and the therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the method of administration, the target site, the patient's condition, etc. Therefore, when used in humans, the dosage should be determined as an appropriate amount by considering both safety and efficacy. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. Such matters to be considered when determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and EW Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

[0174] In the present invention, the term “pharmacologically 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 side 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. Taking all of the above factors into consideration, it is important to administer an amount that obtains maximum effect with a minimum amount without side effects, and this can be easily determined by a person skilled in the art.

[0175] 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.

[0176] Example 1: Preparation of a polymer compound for natural killer cell surface modification

[0177] 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.

[0178] Referring to FIG. 1b, the present invention designs and synthesizes a lipid-prodrug conjugate that modifies the surface of natural killer cells through cell membrane fixation. The lipid-prodrug conjugate is designed with a functional moiety consisting of a hydrophobic two-tail carbon chain that binds to the surface of natural killer cells and a prodrug that is released upon cancer cell lysis to enhance natural killer cell-mediated anticancer efficacy.

[0179] The first step is DSPE- corresponding to the hydrophobic moiety PEG - A step of reacting NH2 with 3,3-dithiodipropionic acid (DTPA) to produce a terminal carboxyl group having a dithiol linker moiety.

[0180] Specifically, DTPA (0.036 mmol), EDC (3-ethylcarbodiimide hydrochloride, 0.073 mmol), and NHS (N-hydroxysuccinimide, 0.091 mmol) were dissolved in 2 mL of inorganic dimethylformamide (anhydrous DMF), and the reaction mixture was stirred at room temperature for 30 minutes to activate the carboxyl groups. In another flask, DSPE- PEG -NH2 (0.036 mmol) and 2 mg DMAP (4-dimethylaminopyridine) were dissolved in 2 mL of inorganic dimethylformamide (anhydrous DMF). Subsequently, the carboxyl-activated DTPA solution was DSPE- PEG It was slowly added to the -NH2 solution. The reaction mixture was stirred at room temperature for 24 hours under N2 protection. After the reaction was complete, the mixture was dialyzed (MWCO 2kD) and freeze-dried to obtain a white solid powder (DSPE-) with a yield of 84.2%. PEG -ss-COOH) was obtained. All reactions were carried out under N2 protection.

[0181] The second step is the prodrug gemcitabine (Gem) DSPE- PEG This is the step of binding with -ss-COOH. Specifically, 50 mg of DSPE- PEG-ss-COOH (0.016 mmol), 3.1 mg of EDC (0.016 mmol), and 2.8 mg of NHS (0.025 mmol) were dissolved in 2 mL of inorganic dimethylformamide (anhydrous DMF) and stirred at room temperature for 30 minutes under N2 protection. Subsequently, an excess of 8.66 mg of gemcitabine (0.033 mmol) and 2 mg of DMAP were added to DSPE- PEG It was added to a -ss-COOH solution. The reaction mixture was continuously stirred at room temperature for 24 hours under N2 protection. After the reaction was complete, the mixture was dialyzed (MWCO 2kD) and freeze-dried to obtain a white solid powder (DSPE-) with a yield of 77.2%. PEG -ss-Gem) was obtained.

[0182] Example 2: Characterization of DSPE-PEG-SS-Gem

[0183] DSPE-, the final product PEG To analyze the structural characteristics of the -ss-Gem, proton nuclear magnetic resonance (1H-NMR) (500 MHz FT-NMR spectrometer, Bruker, Germany) was performed on the compounds generated at each step.

[0184] As a result, as shown in Fig. 2, DSPE- PEG -NH2 showed NMR signals corresponding to amide bond protons (δ 7.78 and 7.38 ppm), phosphate hydroxyl protons (δ 5.14 ppm), ethylene oxide repeating units of PEG (δ 3.51 ppm), -CH2- moiety of lipid chains (δ 1.18–1.23 ppm), and terminal methyl group protons of lipid chains (δ 0.85 ppm). DSPE- PEG-ss-Gem showed NMR signals corresponding to the proton of the newly formed amide bond (δ 7.57 ppm), the hydroxyl proton corresponding to DSPE-PEG (δ 5.05 ppm), the ethylene oxide unit of the PEG chain (δ 3.51 ppm), the -CH2- proton of the newly formed terminal dithiol-based molecule (δ 2.94, 2.88, 2.64, and 2.60 ppm), the -CH2- molecule of the lipid chain (δ 2.26, 1.49, and 1.23 ppm), and the terminal methyl proton (δ 0.85) (Fig. 2a). It was confirmed that DSPE-PEG-ss-COOH was successfully synthesized through the ratio of the proton signal area of ​​the lipid terminal methyl proton and the PEG repeating unit relative to the -CH2- proton of the newly formed terminal dithiol-based moiety (δ 2.94 - 2.60 ppm, 8 H).

[0185] DSPE-, the final product PEG NMR analysis of the structure of the -ss-Gem confirmed a signal corresponding to a DPSE-PEG intermediate containing gemcitabine and dithiol (Fig. 2b). DSPE- PEGThe NMR analysis results of -ss-Gem are as follows: alkene ring protons and paranox ring CH2 protons corresponding to gemcitabine (δ 8.12, 8.11 ppm), amide bond protons of DPSE-PEG and newly formed amide (δ 7.78 - 7.37 ppm), hydroxyl protons corresponding to the paranox ring CH2 protons of DSPE-PEG and gemcitabine (δ 6.63 - 5.77 ppm), ethylene oxide units of the PEG chain (δ 3.50 ppm), -CH2- protons of the dithiol moiety (δ 2.98 - 2.64 ppm), and -CH2- and terminal methyl moiety of the lipid chain (δ 2.33 - 1.23 and 0.85 ppm). The integration ratio of the terminal methyl proton (δ 0.85 ppm, 6H), PEG repeat unit (δ 3.50 ppm, 181 H), and alkene ring proton belongs to Gem (δ 8.12 ppm, 0.95 H), thereby further confirming the successful binding of gemcitabine, with a conversion rate exceeding 95% and DSPE- PEG The average molecular weight of -ss-Gem was found to be approximately 3227 g / mol.

[0186] Example 3: Optimization of DSPE-PEG-SS-Gem Concentration for Natural Killer Cell Surface Coating

[0187] First, to evaluate the proliferative capacity of NK cells treated with soluble gemcitabine, NK cells were treated with gemcitabine at concentrations of 0 to 100 μM, and then cell viability was evaluated using the EZ-CYTOX kit (DoGenBio, South Korea) with the WST-1 assay.

[0188] Specifically, to evaluate the proliferative capacity of GCNK cells, 5 x 10 4NK cells were seeded into U-bottom 96-well plates at a volume of 200 μL and treated with dissolved gemcitabine at concentrations of 0–100 μM. The plates were then incubated for 0, 24, and 48 hours at 37°C in a 5% CO2 environment. After each time point, 20 μL of EZ-CYTOX solution was added to each well, and the plates were incubated for an additional 3 hours. The cultured plates were centrifuged at 400 g for 5 minutes, and 100 μL of the supernatant was transferred to a new 96-well flat-bottom culture plate. The absorbance of the supernatant was measured at 450 nm using a SpectraMax iD3 (Molecular Devices, USA).

[0189] As a result, as shown in Figure 3, all concentrations of gemcitabine reduced the viability of NK cells, and NK cells treated with 0.01 μM gemcitabine showed moderate viability (1.17 times) after 24 hours of culture, but when the concentration of gemcitabine was increased to 100 μM, cell viability was found to decrease significantly at both 24 and 48 hours.

[0190] These results suggest that pre-sensitization of cancer cells through gemcitabine treatment may limit the survival rate of natural killer cells.

[0191] Next, the optimal DSPE- PEG To determine the cell membrane immobilization concentration of -ss-Gem, DSPE- at various concentrations (0 μg / mL to 333 μg / mL) was used. PEG The surface of NK cells was coated with -ss-Gem. Afterward, the surface-coated NK cells were cultured for 48 hours, and the viability and proliferation ability of NK-92mi cells were evaluated.

[0192] To culture NK-92 mi cells (American Type Culture Collection, ATCC, USA), a complete culture medium was prepared using Minimum Essential Medium Alpha (MEMα, Gibco, USA). This medium consists of 12.5% ​​fetal bovine serum (FBS, Gibco), 12.5% ​​horse serum (Gibco), 1% penicillin-streptomycin (P / S) solution (Corning, USA), 0.2 mM inositol (Sigma-Aldrich, USA), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), and 0.02 mM folic acid (Sigma-Aldrich). PANC-1 (ATCC), MIA PaCa-2 (ATCC), and human fibroblasts (Lonza) were cultured in high-glucose Dulbecco's Modified Eagle's Medium (Corning) supplemented with 10% FBS (Corning) and 1% P / S solution. The culture environment of all cells was maintained at 37°C in a humid environment containing 5% CO2.

[0193] DSPE- on the cell surface of natural killer cells PEG To immobilize the -ss-Gem, the NK cell surface is conditioned with DSPE-, a synthetic lipid-gem conjugate. PEG It was coated with -ss-Gem. Simply put, 1 × 10 6 Canine natural killer cells in 200 μL of coating solution (DSPE- in powder form) PEG -ss-Gem dissolved in MEM-α) was cultured at concentrations of 0 to 333 μg / mL. After incubating at room temperature for 30 minutes, DSPE- PEGTo remove the material not coated with -ss-Gem, the cells were washed twice with 1 mL of MEM-α. Subsequently, uncoated natural killer cells (NK cells) and natural killer cells coated with lipid-gemcitabine conjugate (GCNK cells) were used in the experiment.

[0194] Cell viability was assessed using the same method as previously mentioned, and DSPE- in the range of 0 to 333 μg / mL PEG 5 x 10 surface-coated with -ss-Gem 4 GCNK cells (natural killer cells coated with lipid-gemcitabine conjugates) were inoculated into U-bottom 96-well plates. After culturing for 0, 24, and 48 hours, the supernatant was measured using the same method described above.

[0195] As a result, as shown in Figures 4a and 4b, 0-3.3 μg / mL of DSPE- PEG GCNK cells treated with -ss-Gem were shown to proliferate normally during the culture period, but higher concentrations (> 33 μg / mL) were found to inhibit cell proliferation. Therefore, lipid-gemcitabine conjugates (DSPE-) for natural killer cell surface coating. PEG The optimal concentration of -ss-Gem was determined to be 3.3 μg / mL, which does not inhibit the proliferation of natural killer cells.

[0196] Next, the anticancer function of GCNK cells at the above optimal and high concentrations was investigated.

[0197] Specifically, NK cells (1 x 10 6 ) is DSPE- in the same way as the method described above PEG They were coated with -ss-Gem (3.3 or 33 μg / mL). Target cancer cells (PANC-1 and MIA PaCa-2) were prepared by staining with 15 μM Calcein-AM. Then, DSPE- PEGThe anticancer function of GCNK cells was evaluated by co-culturing GCNK cells coated with -ss-Gem (3.3 or 33 μg / mL) with Calcein-AM stained cancer cells (PANC-1 and MIA PaCa-2) for 24 hours.

[0198] As a result, GCNK cells coated with 33 μg / mL of lipid-gemcitabine conjugate showed a 46% decrease in anticancer function against PACN-1 cancer cells compared to GCNK cells coated with 3.3 μg / mL (Fig. 4c), while no significant difference was observed in MIA PaCa-2 cancer cells (Fig. 4d). These results suggest that high concentrations of lipid-gem can induce NK cell toxicity and reduce anticancer function.

[0199] Therefore, 3.3 μg / mL of DSPE- PEG It was confirmed that -ss-Gem is the optimal cell coating concentration that enhances anticancer function while providing excellent NK cell compatibility.

[0200] In addition, to analyze the amount of lipid-prodrug conjugates on the surface of NK cells and the density of prodrugs immobilized on the cell surface, a lipid-fluorescent dye conjugate (DSPE-) conjugated with a fluorescent amine (FL) instead of gemcitabine was used. PEG -ss-FL) was synthesized, and the specific method for preparing the lipid-fluorescent dye conjugate is as follows.

[0201] DSPE-PEG-ss-COOH (1 mmol), EDC (1 mmol), and NHS (1.5 mmol) were dissolved in anhydrous DMF (1 mL) and stirred at room temperature for 3 hours. Then, FL (1.5 mmol) and DMAP (2 mg) were dissolved in anhydrous DMF (1 mL) and added to the above solution. The reaction mixture was stirred under dark conditions at room temperature for 24 hours. The reaction product (DSPE- PEG -ss-FL) was precipitated in cold diethyl ether, dialyzed in DW (MWCO 2 kDa), and then freeze-dried.

[0202] The amount of lipid-prodrug conjugates on the surface of NK cells and the density of prodrugs immobilized on the cell surface were investigated by the following methods.

[0203] Specifically, NK cells (1 x 10 6 ) is DSPE- in the same way as the method described above PEG It was coated with DSPE-ss-FL (333 μg / mL). PEG NK cells coated with -ss-FL were lysed using 250 μL of Radioimmunoprecipitation Assay (RIPA) buffer (ELPIS-BIOTECH) at 4°C for 30 minutes. To calculate the amount of coating material immobilized on the NK cells, the fluorescence intensity of the cell lysates was measured using a SpectraMax iD3 (Ex / Em = 485 / 535 nm). To account for interference from the cell lysates, the fluorescence intensity of uncoated NK cell lysates was also measured. Then, various concentrations of DSPE- PEG The amount of material coated on NK cells was calculated using a standard curve generated by -ss-FL.

[0204] As a result, approximately 175.79 ng of lipid-fluorescent dye conjugates were found in NK cells (10 5 It was fixed to cells). Based on these results, when cells were coated with 3.3 μg / mL of lipid-gemcitabine conjugate, approximately 1.75 ng of the conjugate was fixed to NK cells (10 5 It can be estimated that it is fixed to cells. Since 1 mg of lipid-gemcitabine conjugate contains 81.5 μg of gemcitabine, GCNK cells with 1.75 ng of fixed material contain approximately 0.14 ng (0.53 pmol) of gemcitabine. NK cells coated with 3.3 μg / mL of lipid-Gem (10 5 The cells) maintained the viability of NK cells by containing about 0.53 pmol of gemcitabine.

[0205] Example 4: Confirmation of maintenance of cell membrane solubleness in GCNK cells

[0206] The presence of recognition ligands and activating receptors on the cell membrane of NK cells is essential for identifying cancer cells and performing key functions. FasL and TRAIL are major surface ligands of NK cells that recognize target cancer cells and directly induce apoptosis. Additionally, membrane components such as the activation marker CD69 play a crucial role in the initial activation of NK cells and promote cytokine release. Therefore, it is necessary to evaluate whether the surface soluble compartments are maintained after the NK cell coating process.

[0207] To this end, NK cells and GCNK cells were cultured with FasL, TRAIL, and CD69 conjugated with APC (Allophycocyanin) at 4°C for 30 minutes. Afterward, they were washed twice with cold DPBS, and the solubility of NK cell membrane components was evaluated by flow cytometry.

[0208] As a result, as shown in Figure 5, GCNK cells exhibited an MFI similar to that of NK cells, confirming that the coating of the lipid-gemcitabine conjugate did not affect the solubility of NK surface ligands and activating molecules.

[0209] Additionally, the zeta potentials of NK and GCNK cell surfaces were measured using dynamic light scattering (DLS). GCNK cell surfaces were coated with 3.3 μg / mL of lipid-gemcitabine conjugate. Briefly, 1 × 10⁶ 6 NK cells were cultured with 200 μL of coating solution. The coating process was performed in the same manner as described in Example 3. After washing twice with 1X DPBS, the cells were resuspended in 1 mL of 1X DPBS, and the zeta potential of the GCNK cells was analyzed using a Malvern Zetasizer Pro (Malvern, UK).

[0210] The zeta potential analysis results for NK and GCNK cells showed -12.27 ± 0.54 and -12.75 ± 0.67 mV, respectively (Fig. 6). These results suggest that DSPE-, a lipid-mediated cell surface coating material... PEG This means that -ss-Gem does not inhibit the intrinsic properties of the NK cell membrane.

[0211] Therefore, lipid-gemcitabine conjugates can be utilized as effective membrane immobilization materials that promote targeted prodrug delivery without interfering with the recognition-activation mechanism of NK cells.

[0212] Example 5: Confirmation of Glutathione-Reactive Gemcitabine Release Profile

[0213] In designing a modular lipid-prodrug conjugate, the inventors used a cell membrane anchor (i.e., DSPE lipid) and a glutathione (GSH) reactive disulfide linker for the active release of the integrated prodrug at a specific tumor site. Thus, after cancer cells are destroyed by lysis granules of GCNK cells, local diffusion of GSH released by cancer cell damage can cleave the disulfide bonds of the lipid-gemcitabine conjugate, and the cleavage of the disulfide bonds releases gemcitabine, which can further increase cytotoxicity against cancer cells.

[0214] GSH is a dominant and abundant antioxidant in the body that accumulates intensively in cancer cells, while showing relatively low levels in plasma and normal cells.

[0215] To investigate whether gemcitabine is released from the lipid-gemcitabine conjugate by GSH, the release profiles of gemcitabine from the lipid-gemcitabine conjugate at various time intervals were compared under conditions without GSH and in the presence of GSH.

[0216] First, a lipid-gemcitabine conjugate at a concentration of 1 mg / mL in PBS was prepared and placed in a dialysis bag (MWCO 2KDa). Then, the dialysis bag was placed in 4 mL of PBS or GSH (10 or 100 μM) solution and incubated at 37°C for various time intervals (0, 2, 4, 8, 24, 48 hours). At each selected time interval, the PBS buffer outside the dialysis bag was collected, and an equal amount of fresh buffer was added to the dialysis bag. The amount of released gemcitabine was measured using a UV-visible SpectraMax iD3 (Molecular Devices, USA) at 268 nm and quantified using a standard curve based on the absorbance values ​​of standard gemcitabine.

[0217] As a result, as shown in Fig. 7a, under conditions where 100 μM of GSH is present, about 55% of gemcitabine was released within the first 2 hours, and 99% was released within 8 hours. Complete release (100%) was achieved within 24 hours. These results indicate that NK cells coated with a lipid-gemcitabine conjugate can (1) retain gemcitabine in the bloodstream and (2) selectively release gemcitabine in response to increased GSH levels resulting from NK cell-mediated cancer cell killing.

[0218] In addition, the biological activity of gemcitabine released from the lipid-gemcitabine conjugate was quantified through cytotoxicity against pancreatic cancer cells (PANC-1 and MIA PaCa-2).

[0219] PANC-1 and MIA PaCa-2 cancer cells in 96-well plates, 5 x 10⁶ cells per well 3Cells were inoculated with 100 μL of medium at cell density and incubated for 24 hours at 37°C under 5% CO2 conditions. Then, freshly prepared gemcitabine and released gemcitabine samples (i.e., gemcitabine collected from previous release profile verification experiments) were added to 200 μL of each well at a final concentration of 1 μM and incubated for 24 hours. Subsequently, 20 μL of EZ-CYTOX solution was added to each well, and the plates were incubated for another 3 hours. The absorbance of the solution was measured at 450 nm using a SpectraMax iD3.

[0220] As a result, as shown in Figures 7b and 7c, it was confirmed that there was no significant difference in the survival rate of PANC-1 and MIA PaCa-2 cells exposed to the newly prepared gemcitabine or the released gemcitabine. This means that the released gemcitabine exhibits a level of biological activity equivalent to that of the newly prepared gemcitabine and can induce a therapeutic effect on cancer cells after targeted prodrug delivery through GCNK cells.

[0221] Example 6: Effects of Natural Killer Cell-Mediated Gemcitabine Delivery on Cancer Cells

[0222] The mechanism of the anticancer efficacy of GCNK cells may proceed as follows.

[0223] First, pancreatic cancer cell targeting mediated by the unique cytotoxic efficacy of NK cells through membrane-dependent target recognition, the formation of post-recognition immune synapses between NK cells and cancer cells, cancer lysis by lysis granules secreted from NK cells, subsequent leakage of GSH from the destroyed cancer cell bodies, cleavage of disulfide bonds in response to the leaked GSH, release of Gem and subsequent delivery to cancer cells, increased expression of the stress-induced ligand MICA / B at the cancer cell membrane, and further activation of NK cells are induced.

[0224] Therefore, the inventors aimed to demonstrate that GCNK cells are non-toxic to normal cells while systematically evaluating whether they function against target pancreatic cancer cells. First, a target recognition analysis was performed to evaluate the ability of GCNK cells to specifically recognize pancreatic cancer cells compared to normal control cells (i.e., fibroblasts). For this experiment, two types of pancreatic cancer cell lines (PANC-1 and MIA PaCa-2) and normal control cells (fibroblasts) were co-cultured with effector cells (NK or GCNK cells). The target recognition ability of effector NK cells was evaluated through the quantification of effector-target (E / T) cluster formation.

[0225] Specifically, two types of pancreatic cancer cell lines (PANC-1 and MIA PaCa-2) and a normal control (fibroblast) were stained with 1 μM CellTracker Red CMTPX dye (Invitrogen) and washed twice with FBS-free DMEM. NK cells were stained with 0.1 μM Calcein AM (Invitrogen) at 37°C for 30 minutes and washed twice with FBS-free DMEM. Subsequently, 3.3 μg / mL of lipid-gemcitabine conjugate was coated onto the calcein-stained NK cells using the method described in Example 3. Then, green-stained NK or GCNK cells (0.5 × 10⁶ 6 Cells) red-stained target cells (0.5 × 10⁶ 6 Cells were cultured in 1.5 mL Eppendorf tubes at 37°C for 30 minutes at an effector-target (E:T) ratio of 1:1. Since E / T clusters exhibit fluorescent signals in both effector and target cells, cells emitting both green and red fluorescent signals were selectively gated and quantified as a percentage of total detected cell events. To ensure accuracy, non-fluorescent stained cells were used as a gating control, and each sample set was analyzed in three replicates.

[0226] As a result, as shown in Figure 8, similar levels of E / T cluster formation (approximately 8%) were observed between the NK-PANC-1 group and the GCNK-PANC-1 group. Similarly, when NK cells or GCNK cells were co-cultured with MIA PaCa-2 cells, a similar level of E / T cluster formation (approximately 3%) was observed. On the other hand, in the NK and GCNK cell groups co-cultured with fibroblasts, almost no E / T cluster formation was observed (less than 1%).

[0227] Subsequently, the anticancer efficacy of NK and GCNK cells against pancreatic cancer cells and normal fibroblasts was evaluated through calcein release analysis.

[0228] Specifically, the in vitro anticancer efficacy of GCNK cells coated with a 3.3 μg / mL lipid-Gem conjugate was evaluated using a calcein release assay. Green-stained target cells (PANC-1, MIA PaCa-2, and fibroblasts) and effector cells (NK cells and GCNK cells) were prepared by the method described in Example 3. Subsequently, 1 x 10 4 Target cells were co-cultured with NK or GCNK cells at 37°C for 4 or 24 hours at E / T ratios of 0.5:1, 1:1, 5:1, and 10:1. Two control groups were established to calculate specific lysis rates: a spontaneous release group (containing only target cells) and a maximum release group (target cells 100% lysed by treatment with 5% Triton X-100 for 30 minutes). Specific cell lysis rates were calculated using Equation 1.

[0229] [Mathematical Formula 1]

[0230]

[0231] As a result, as shown in Fig. 9, both NK and GCNK cells exhibited anticancer activity against PANC-1 (28.5% and 28.0%, respectively, at a 10:1 E:T ratio) and MIA PaCa-2 (13.8% and 10.2%, respectively, at a 10:1 E:T ratio) cancer cells (Figs. 9a and 9b), which was consistent with the target recognition trend. On the other hand, no targeted cytotoxic activity was observed against normal fibroblasts (less than 1.5% at a 10:1 E:T ratio) (Fig. 9c). These results indicate that (1) the inherent cancer-targeting ability of NK cells was maintained even after coating the cell surface with the lipid-gemcitabine conjugate, and (2) gemcitabine was selectively delivered to cancer cells through NK-cancer cell interactions, protecting normal cells and reducing non-target effects.

[0232] Gemcitabine-induced DNA damage exerts immunomodulatory effects within the tumor microenvironment (TME), specifically increasing the expression of stress-inducing ligands on the surface of cancer cells. One significant consequence of this DNA damage is the increased expression of MICA / B in pancreatic cancer cells. MICA / B is a stress-inducing ligand that binds to the NKG2D receptor on NK cells, facilitating critical interactions that promote NK cell-mediated cancer cell recognition and destruction. When MICA / B binds to NKG2D, it recruits DNAX-activated protein 10 (DAP10) within NK cells, activating the phosphorylated inositol 3-kinase (PI3K) signaling pathway, which plays a vital role in enhancing NK cell cytotoxicity. Activated NK cells kill cancer cells directly and indirectly by secreting various pro-inflammatory cytokines and cytotoxic granules. Therefore, gemcitabine's ability to promote MICA / B expression in cancer cells implies that pancreatic cancer cells may become more vulnerable to NK cell-mediated lysis. Therefore, the expression levels of MICA / B in two pancreatic cancer cells (PANC-1 and MIA PaCa-2) were evaluated.

[0233] The surface expression levels of MICA / B in pancreatic cancer cells were evaluated using flow cytometry to assess signal transduction by gemcitabine delivered from the conjugate. 1 x 10⁶ 4 Uncoated NK cells or GCNK cells were co-cultured with pancreatic cancer cells at an E / T (Effector / Target) ratio of 1:1 for 24 hours. Then, the remaining pancreatic cancer cells were washed twice with cold 1X DPBS, and the cells were labeled with APC anti-human MICA / MICB antibodies at 4°C for 30 minutes. The cells were washed twice and resuspended in cold 1X DPBS for flow cytometry analysis. To ensure accuracy, unfluorescently stained pancreatic cancer cells were used as a negative control.

[0234] As a result, as shown in Figures 10a and 10b, MICA / B expression was found to be significantly increased in PANC-1 cells co-cultured with GCNK cells compared to when co-cultured with non-coated NK cells (Figure 10a). This increase in MICA / B expression implies that gemcitabine was effectively delivered to the target cancer cells during cancer cell lysis mediated by cancer-targetable GCNK cells. Gemcitabine released from the surface of GCNK cells promoted MICA / B expression in cancer cells, thereby promoting NK cell activation through interaction with NKG2D on the surface of NK cells. However, in MIA PaCa-2, similar levels of MICA / B expression were observed in both NK cells and GCNK cells (Figure 10b), and these results are consistent with a differential response to gemcitabine treatment.

[0235] These results suggest that PANC-1 is more sensitive to gemcitabine than MIA PaCa-2 in terms of MICA / B expression, and that gemcitabine-responsive MICA / B expression in pancreatic cancer cells may be related not only to membrane interactions with NK cells but also to GCNK cell-mediated downstream anticancer cytotoxicity of gemcitabine.

[0236] Example 7: Enhancement of anticancer efficacy of GCNK cells against PANC-1 cancer cells

[0237] To determine whether anticancer efficacy is enhanced by GCNK cell-mediated Gem delivery, the anticancer efficacy of NK cells immobilized with a prodrug was evaluated through calcein release assay.

[0238] Target cancer cells (PANC-1 and MIA PaCa-2) were labeled with 15 μM calcein-AM (Invitrogen) for 30 minutes at 37°C. Prepared GCNK cells (5 x 10⁶ 4 cells) are PANC-1 and MIA PaCa-2 pancreatic cancer cells stained with calcein-AM (1 x 10⁶ 4 The cells were cultured for 24 hours. Then, calcein released from the dead cancer cells was measured using SpectraMax iD3 (Ex / Em = 485 / 535 nm). The specific lysis rate was calculated using Equation 1 above.

[0239] As a result, as shown in Figure 11, when examining the 24-hour culture results, the cytolytic activity of GCNK cells against PANC-1 cells was significantly increased compared to non-coated NK cells at 1:1 and 5:1 E:T ratios (Figure 11a). These results imply that increased MICA / B expression is closely associated with enhanced cytotoxic activity of NK cells, and indicate that GCNK cell-mediated gemcitabine delivery can induce a series of immune responses in PANC-1 cancer cells, including NK cell activation and subsequent tumor cell lysis.

[0240] In addition, it was found that the anticancer efficacy was not sufficiently increased for MIA PaCa-2 (Fig. 11b), which implies that MICA / B expression mediated by GCNK cell-mediated gemcitabine delivery can regulate the level of antitumor activity against various pancreatic cancer cells.

[0241] On the other hand, when the co-culture period was short (i.e., 4 hours), this trend was not observed (Fig. 9a, b), which supports the fact that at least 24 hours are required for the synergistic anticancer efficacy of NK cell-mediated cytotoxicity and prodrug Gem-mediated cancer cell death to manifest. This may be the critical period for the completion of the stepwise mechanism of anticancer efficacy of GCNK cells described above. Accordingly, the lipid-gemcitabine conjugate prepared according to one embodiment of the present invention was successfully immobilized on the surface of NK cells, enabling targeted delivery of the prodrug to PANC-1 cancer cells upon cell lysis.

[0242]

[0243] [National R&D projects that supported this invention]

[0244] [Project ID] 2710006360

[0245] [Assignment No.] 00419061

[0246] [Ministry Name] Ministry of Science and ICT

[0247] [Name of Project Management (Specialized) Agency] Korea Institute for Science and Technology Commercialization

[0248] [Research Project Name] Support Project for Activating Industry-Academic-Research Cooperation

[0249] [Project Title] IP Advancement and Commercialization for the Promotion of Commercialization of Immunotherapy with Enhanced Anticancer Functionality Using Cell Membrane-Bound Polymer Material Technology

[0250] [Name of Project Performing Organization] Dongguk University Industry-Academic Cooperation Foundation

[0251] [Research Period] April 1, 2024 ~ December 31, 2024

[0252]

[0253] [Project ID] 2710003135

[0254] [Assignment No.] 00277800

[0255] [Ministry Name] Ministry of Science and ICT

[0256] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0257] [Research Project Name] Individual Basic Research (Ministry of Science and ICT)

[0258] [Project Title] Development of Immuno-oncology Cell Complexes for Pancreatic Cancer Treatment Using Triple-Function Enhanced Cell Membrane Activation Materials

[0259] [Name of Project Performing Organization] Dongguk University Industry-Academic Cooperation Foundation

[0260] [Research Period] 2024.03.01 ~ 2025.02.28

Claims

1. Hydrophobic moiety that binds to the cell membrane; Cell internalization prevention moiety; Disulfide linker and Polymeric compounds for cell surface modification to improve drug delivery efficiency, comprising anticancer prodrug moiety.

2. In Paragraph 1, A polymer compound for cell surface modification to improve drug delivery efficiency, wherein the 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).

3. In Paragraph 1, The above hydrophobic moiety is a polymeric compound for cell surface modification to enhance drug delivery efficiency, 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-mentioned cell internalization-preventing moiety is a polymeric compound for cell surface modification to enhance drug delivery efficiency, which is a compound represented by Chemical Formula 2 or a derivative thereof: [Chemical Formula 2] n is an integer greater than 0.

5. In Paragraph 4, A polymer compound for modifying the cell surface to improve drug delivery efficiency, wherein the cell internalization-preventing moiety is selected from the group consisting of polyethylene glycol (PEG), polyethylene oxide (PEO), and polyvinyl alcohol (PVA).

6. In Paragraph 1, The above disulfide linker is a polymeric compound for cell surface modification to improve drug delivery efficiency, which is a compound represented by Chemical Formula 3 or a derivative thereof: [Chemical Formula 3] R and R' are each independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclil, and substituted heterocyclil.

7. In Paragraph 6, A polymer compound for cell surface modification to improve drug delivery efficiency, characterized in that the above alkyl is methylene having 1 to 30 repeating units.

8. In Paragraph 1, A polymer compound for cell surface modification to improve drug delivery efficiency, wherein the above cancer is selected from the group consisting of prostate cancer, 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.

9. In Paragraph 1, A polymeric compound for cell surface modification to improve drug delivery efficiency, wherein the above-mentioned prodrug is selected from the group consisting of gemcitabine, fludarabine, clofarabine, cladribine, azacitidine, decitabine, cytarabine, nelarabine, nimustine, temozolomide, dacarbazine, thioguanine, carboquone, doxorubicin, entinostat, pemetrexed, and methotrexate.

10. In Paragraph 1, A polymeric compound for cell surface modification to improve drug delivery efficiency, wherein the above anticancer prodrug moiety is covalently bonded to a disulfide linker.

11. In Paragraph 1, The above cell membrane is a polymer compound for modifying the cell surface to improve drug delivery efficiency, wherein the cell membrane is a cell membrane of an immune cell or a drug delivery vehicle coated with an artificial cell membrane.

12. In Paragraph 11, The above immune cell is a natural killer cell, a polymer compound for cell surface modification to improve drug delivery efficiency.

13. In Paragraph 1, The above polymer compound is a polymer compound for cell surface modification to improve drug delivery efficiency, which delivers an anticancer prodrug dissociated by the cleavage of disulfide bonds by glutathione released from cancer cells to surrounding cancer cells.

14. Natural killer cells whose surface has been modified with a polymer compound of any one of claims 1 to 13.

15. A drug delivery system whose surface is modified with a polymer compound of any one of claims 1 to 13. 16.(a) A step of activating the carboxyl groups of a disulfide containing carboxyl groups at both ends; (b) a step of attaching a polymeric compound comprising a hydrophobic moiety, an anti-internalization moiety, and an amine group to one end of a disulfide and (c) A method for preparing a polymeric compound comprising a hydrophobic moiety that binds to a cell membrane, a cell internalization prevention moiety, a disulfide linker, and an anticancer prodrug moiety, comprising the step of binding a prodrug to the other end of the disulfide of the polymeric compound in which step (b) is completed.

17. In Paragraph 16, A method for preparing a polymeric compound comprising a hydrophobic moiety that binds to a cell membrane, a cell-internalization-preventing moiety, a disulfide linker, and an anticancer prodrug moiety, wherein the polymeric compound comprising the above hydrophobic moiety; the cell-internalization-preventing moiety and an amine group is a compound represented by Chemical Formula 4 or a derivative thereof; [Chemical Formula 4] q is an integer greater than 0, and p is an integer greater than 0.

18. In Paragraph 16, A method for preparing a polymer compound in which the above step (b) is completed, comprising a hydrophobic moiety that binds to a cell membrane, a cell internalization-preventing moiety, a disulfide linker, and an anticancer prodrug moiety, wherein the polymer compound is represented by Chemical Formula 5 or a derivative thereof: [Chemical Formula 5] q is an integer greater than 0, and p is an integer greater than 0, and n is an integer greater than 0, and R and R' are each independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclil, and substituted heterocyclil.

19. In Paragraph 16, A method for preparing a polymeric compound comprising a hydrophobic moiety that binds to the cell membrane, a cell internalization-preventing moiety, a disulfide linker, and an anticancer prodrug moiety, wherein the polymeric compound comprising the above-mentioned hydrophobic moiety, the cell internalization-preventing moiety, the disulfide linker, and the anticancer prodrug moiety is a compound represented by Chemical Formula 6 or a derivative thereof: [Chemical Formula 6] q is an integer greater than 0, and p is an integer greater than 0, and n is an integer greater than 0, and R and R' are each independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclil, and substituted heterocyclil, and A is selected from gemcitabine, fludarabine, clofarabine, cladribine, azacitidine, decitabine, cytarabine, nelarabine, nimustine, temozolomide, dacarbazine, thioguanine, carboquone, doxorubicin, entinostat, pemetrexed, and methotrexate.

20. 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; a moiety that prevents cell internalization; a disulfide linker; and an anticancer prodrug moiety.

21. In Paragraph 20, A pharmaceutical composition for the prevention or treatment of cancer, wherein the above cancer is selected from the group consisting of prostate cancer, 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.

22. In Paragraph 20, A pharmaceutical composition for the prevention or treatment of cancer, wherein the above-mentioned prodrug is selected from the group consisting of gemcitabine, fludarabine, clofarabine, cladribine, azacitidine, decitabine, cytarabine, nelarabine, nimustine, temozolomide, dacarbazine, thioguanine, carboquone, doxorubicin, entinostat, pemetrexed, and methotrexate.

23. A method for preventing or treating cancer, comprising the step of administering the pharmaceutical composition of claim 20 to an individual.

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