Polymer compound that binds to natural killer cells so as to selectively kill cancer cells containing sialic acid, and preparation method therefor

Abstract

A polymer compound is provided. The polymer compound comprising a hydrophobic moiety binding to natural killer cells, a cancer cell recognition moiety, and a linker may comprise: the hydrophobic moiety being bound to one end of the linker and the cancer cell recognition moiety being bound to the other end of the linker so that natural killer cells and cancer cells are recognized; and the cancer cell recognition moiety selectively recognizing solid cancer cells containing sialic acid.

Description

A polymer compound that selectively kills cancer cells containing sialic acid by binding to natural killer cells and a method for producing the same

[0001] The present invention relates to a polymer compound that can selectively recognize and kill cancer cells containing sialic acid by synthesizing and manufacturing a polymer compound that can bind to the surface of natural killer cells and enhance the anticancer function of the cells, and injecting the polymer compound into the body, thereby dramatically improving the effect of preventing or treating cancer.

[0002] Immunotherapy is one of the most successful and innovative approaches to treating various cancers. Natural killer (NK) cells, which possess the unique ability to identify and eliminate tumor cells without the drawbacks of T cells, such as autologous T cell deficiency, off-target toxicity, and cytokine release syndrome, are recently attracting attention as a potential replacement for T cells.

[0003] However, there is a problem that the efficacy of immunotherapy is reduced due to the lack of the ability to selectively target tumor cells due to the immunosuppressive tumor microenvironment (TME) and the lack of specific cancer-targeting ligands of NK cells.

[0004] Chimeric antigen receptor (CAR)-based genetic engineering is one technology that can overcome the targeting limitations of NK cells and improve therapeutic performance, and clinical trials are underway worldwide. As interest in and demand for CAR-NK cells grows, challenges related to CAR-based engineering are also emerging.

[0005] Specifically, the manufacturing process for genetically engineered CAR-NK cells suffers from low infection efficiency, and unpredictable mutations can compromise the inherent biological activity of NK cells against tumors. Consequently, the development of new technologies that do not rely on genetic modification is urgently needed.

[0006] To overcome the limitations associated with CAR-NK cell production, NK cell surface engineering technology is being studied. A related technology, glycoengineering, has been developed to target CD22 by modifying the surface of NK cells. However, the expression of targeting ligands through glycoengineering relies entirely on intracellular metabolism, making precise control difficult.

[0007] The technical problem to be solved by the present invention is to provide a polymer compound that can bind to natural killer cells.

[0008] Another technical problem to be solved by the present invention is to provide a polymer compound capable of recognizing target cancer cells.

[0009] Another technical problem to be solved by the present invention is to provide a polymer compound capable of selectively recognizing cancer cells containing sialic acid.

[0010] Another technical problem to be solved by the present invention is to provide a polymer compound having improved killing efficiency against cancer cells overexpressing sialic acid.

[0011] The technical problems to be solved by the present invention are not limited to those described above.

[0012] To solve the above-described technical problems, the present invention provides a polymer compound.

[0013] According to one embodiment, a polymer compound comprising a hydrophobic moiety that binds to natural killer cells, a cancer cell recognition moiety, and a linker, wherein the polymer compound recognizes natural killer cells and cancer cells by having the hydrophobic moiety bound to one end of the linker and the cancer cell recognition moiety bound to the other end of the linker, wherein the cancer cell recognition moiety selectively recognizes a solid cancer cell that contains sialic acid.

[0014] In one embodiment, the cancer cell recognition moiety may comprise phenylboronic acid.

[0015] In one embodiment, the cancer cell recognition moiety may comprise binding to the sialic acid of the solid cancer cell and forming a boron-ester complex.

[0016] According to one embodiment, the polymer compound bound to natural killer cells through the hydrophobic moiety may include binding to cancer cells through the cancer cell recognition moiety, and then promoting the secretion of cytotoxic granules and cytokines from natural killer cells, thereby killing cancer cells.

[0017] In one embodiment, the hydrophobic moiety may comprise a lipid and be bound to the surface of a natural killer cell through a hydrophobic interaction mediated by the lipid.

[0018] In one embodiment, the hydrophobic moiety may include any one of a phospholipid having an alkyl chain having 12 to 24 carbon atoms, a sterol lipid having 10 to 30 carbon atoms, 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE), 1,2-bis(diphenylphosphino)ethane (DPPE), and 1,2-bis(dimethylphosphino)ethane (DMPE).

[0019] In one embodiment, the hydrophobic moiety may further comprise a sub-linker, wherein the sub-linker is connected to one end of the lipid, and the hydrophobic moiety is bound to the linker through the sub-linker.

[0020] In one embodiment, the linker may comprise a polymer compound bound to a natural killer cell that prevents endocytosis into the natural killer cell.

[0021] In one embodiment, the linker may comprise polyethylene glycol (PEG).

[0022] According to one embodiment, the polymer compound may include one represented by the following <Chemical Formula 1>.

[0023] <Chemical Formula 1>

[0024]

[0025] (n: integer greater than or equal to 0)

[0026] In one embodiment, the cancer cell recognition moiety may comprise one that recognizes colon cancer cells (HCT-116), triple-negative breast cancer cells (MDA-MB-231), and liver cancer cells (HepG2).

[0027]

[0028] To solve the technical problems described above, the present invention provides a method for producing a polymer compound.

[0029] According to one embodiment, the method for preparing the polymer compound may include a step of mixing a hydrophobic moiety and a linker having an amine group at a terminal, thereby forming a first intermediate structure in which the linker is connected to one end of the hydrophobic moiety and the linker has an amine group at a terminal, a step of preparing a second intermediate structure by converting the amine group of the first intermediate structure into a carboxylic acid group, and a step of reacting the second intermediate structure with phenylboronic acid to prepare a polymer compound in which the hydrophobic moiety is connected to one end of the linker and the phenylboronic acid is connected to the other end.

[0030] In one embodiment, the second intermediate structure may include conversion of an amine group to a carboxylic acid group through succinylation of the first intermediate structure.

[0031] In one embodiment, the hydrophobic moiety comprises a compound in which polyethylene glycol (PEG) is bonded to one end of 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE), and the linker may comprise polyethylene glycol (PEG).

[0032] A polymer compound composed of a hydrophobic moiety (lipid), a cancer cell recognition moiety (phenylboronic acid), and a linker (PEG) connecting the hydrophobic moiety and the cancer cell recognition moiety can easily modify the surface of natural killer cells (modify natural killer cells so that they can recognize specific cancer cells) through a hydrophobic action via the hydrophobic moiety (lipid), and can selectively recognize solid cancer cells (e.g., colon cancer cells, triple-negative cancer cells, liver cancer cells, etc.) that overexpress sialic acid among various cancer cells and then effectively kill them.

[0033] FIG. 1 is a drawing for explaining a state in which a polymer compound according to an embodiment of the present invention binds to natural killer cells and a cancer killing process through natural killer cells.

[0034] FIG. 2 is a drawing for more specifically explaining a state in which a polymer compound according to an embodiment of the present invention binds to natural killer cells.

[0035] FIG. 3 is a drawing for explaining the binding of a cancer cell recognition moiety of a polymer compound according to an embodiment of the present invention to sialic acid of cancer cells.

[0036] FIG. 4 is a drawing for explaining step S10 of a method for producing a polymer compound according to an embodiment of the present invention.

[0037] FIG. 5 is a drawing for explaining step S20 of a method for manufacturing a polymer compound according to an embodiment of the present invention.

[0038] FIG. 6 is a drawing for explaining step S30 of a method for manufacturing a polymer compound according to an embodiment of the present invention.

[0039] Figures 7 and 8 show the results of FT-IR analysis of polymer compounds, compounds used in the manufacturing process thereof, and products according to experimental examples of the present invention. 1 This is a diagram showing the results of H-NMR analysis.

[0040] Figure 9 PBA, DSPE PEG -di(PEG), and DSPE PEG This is a diagram illustrating the bonding of -di(PEG-PBA) and sialic acid.

[0041] Figure 10 is a drawing for explaining the binding strength of a polymer compound and various sugars according to an experimental example of the present invention.

[0042] Figure 11 shows a fluorescence intensity measurement image of a cell in which a polymer compound and NK cells are combined according to an experimental example of the present invention.

[0043] Figure 12 is a drawing for explaining NK cell binding efficiency according to the concentration of a polymer compound according to an experimental example of the present invention.

[0044] Figure 13 is a graph showing the viability of PBA-NK cells.

[0045] Figure 14 is a graph illustrating the effect of a polymer compound according to experimental examples of the present invention on cytokine secretion of NK cells.

[0046] Figure 15 is a drawing for explaining the effect of a polymer compound according to an experimental example of the present invention on the ligand of NK cells.

[0047] Figure 16 is a diagram illustrating the cancer cell targeting ability of PBA-NK cells.

[0048] Figures 17 and 18 are drawings illustrating the cytotoxic granule (Granzyme B, Perforin) secretion ability of PBA-NK cells.

[0049] Figure 19 is a diagram illustrating the cytokine (IFN-Y) secretion ability of PBA-NK cells.

[0050] Figure 20 is a drawing illustrating the killing ability of PBA-NK cells against target cells.

[0051] Figure 21 is a fluorescence microscope image of triple-negative breast cancer cell (MDA-MB-231) spheroids treated with NK cells and PBA-NK cells.

[0052] Figure 22 is a graph quantifying the fluorescence intensity measured in Figure 21.

[0053] Figure 23 is a fluorescence microscopy image of triple-negative breast cancer cell (MDA-MB-231) spheroids stained with EthD-1 after treatment with NK cells and PBA-NK cells.

[0054] Figure 24 is a graph quantifying the intensity and spheroid area of ​​EthD-1 measured in Figure 23.

[0055] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the attached drawings. However, the technical concept of the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided to ensure that the disclosed content is thorough and complete and to sufficiently convey the spirit of the present invention to those skilled in the art.

[0056] In this specification, when a component is referred to as being on another component, it means that it can be formed directly on the other component, or a third component may be interposed between them. In addition, in the drawings, the thicknesses of films and regions are exaggerated for the purpose of effectively explaining the technical contents.

[0057] Also, although terms such as first, second, and third have been used to describe various components in various embodiments of this specification, these components should not be limited by these terms. These terms are only used to distinguish one component from another. Thus, what is referred to as a first component in one embodiment may be referred to as a second component in another embodiment. Each embodiment described and illustrated herein also includes its complementary embodiments. Also, the term "and / or" has been used herein to mean including at least one of the components listed before and after.

[0058] In the specification, singular expressions include plural expressions unless the context clearly dictates otherwise. In addition, terms such as "comprise" or "have" are intended to specify the presence of a feature, number, step, component, or combination thereof described in the specification, and should not be construed as excluding the presence or addition of one or more other features, numbers, steps, components, or combinations thereof. In addition, the term "connection" is used in the present specification to mean both indirectly connecting multiple components and directly connecting them.

[0059] In addition, when describing the present invention below, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description will be omitted.

[0060]

[0061] Polymer compound according to an embodiment

[0062] FIG. 1 is a drawing for explaining a state in which a polymer compound according to an embodiment of the present invention binds to natural killer cells and a cancer killing process through natural killer cells, FIG. 2 is a drawing for explaining in more detail a state in which a polymer compound according to an embodiment of the present invention binds to natural killer cells, and FIG. 3 is a drawing for explaining the binding of a cancer cell recognition moiety of a polymer compound according to an embodiment of the present invention to sialic acid of cancer cells.

[0063] Referring to FIGS. 1 to 3, a polymer compound according to an embodiment of the present invention may include a hydrophobic moiety, a cancer cell recognition moiety, and a linker connecting the hydrophobic moiety and the cancer cell recognition moiety. That is, the polymer compound may have a structure in which the hydrophobic moiety is bonded to one end of the linker and the cancer cell recognition moiety is bonded to the other end.

[0064] The hydrophobic moiety can recognize natural killer cells (NK cells) and bind to the surface of the natural killer cells. Accordingly, the polymer compound can be fixed to the surface of the natural killer cells by the hydrophobic moiety. More specifically, the hydrophobic moiety is configured to include a lipid and can bind to the surface of the natural killer cells through a hydrophobic interaction mediated by the lipid.

[0065] In one embodiment, the hydrophobic moiety may include any one of a phospholipid having an alkyl chain having 12 to 24 carbon atoms, a sterol lipid having 10 to 30 carbon atoms, 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE), 1,2-bis(diphenylphosphino)ethane (DPPE), and 1,2-bis(dimethylphosphino)ethane (DMPE).

[0066] The hydrophobic moiety may further include a sub-linker, and may have a structure in which the sub-moiety is bonded to one end of a lipid, and the hydrophobic moiety may be bonded to the linker through the sub-linker. According to one embodiment, the sub-linker may include polyethylene glycol (PEG). For example, when DSPE is used as the lipid of the hydrophobic moiety and PEG is used as the sub-linker, the hydrophobic moiety may be DSPE. PEG can be expressed as

[0067] The cancer cell recognition moiety recognizes and binds to cancer cells, and may include phenylboronic acid (PBA). As described above, when the cancer cell recognition moiety includes phenylboronic acid, the cancer cell recognition moiety can selectively recognize solid cancer cells that include sialic acid. More specifically, the cancer cell recognition moiety including phenylboronic acid can selectively recognize cancer cells that overexpress sialic acid. For example, the cancer cells that overexpress sialic acid may include colon cancer cells (HCT-116), triple-negative breast cancer cells (MDA-MB-231), and hepatoma cells (HepG2). That is, the cancer cell recognition moiety containing phenylboronic acid can selectively recognize colon cancer cells (HCT-116), triple-negative breast cancer cells (MDA-MB-231), and liver cancer cells (HepG2) among various cancer cells. Accordingly, the natural killer cells bound to the hydrophobic moiety can selectively kill colon cancer cells (HCT-116), triple-negative breast cancer cells (MDA-MB-231), and liver cancer cells (HepG2) among various cancer cells by the cancer cell recognition moiety.

[0068] The linker is intended to connect the hydrophobic moiety and the cancer cell recognition moiety, and may include polyethylene glycol (PEG). In addition, the linker may prevent the polymer compound bound to natural killer cells from being endocytosed into the natural killer cells.

[0069] As illustrated in FIG. 1, the natural killer cells (PBA-NK cells) bound to the polymer compound can recognize cancer cells containing sialic acid through the cancer cell recognition moiety (phenylboronic acid, PBA) of the polymer compound, and then bind to the cancer cells through the cancer cell recognition moiety (phenylboronic acid, PBA). More specifically, as illustrated in FIG. 3, the cancer cell recognition moiety (phenylboronic acid, PBA) can bind to the sialic acid of the solid cancer cells to form a boron-ester complex. Thereafter, an activation process in which cytotoxic granules and cytokines are secreted from the natural killer cells (NK cells) occurs, and cancer cells can be killed by the cytotoxic granules and cytokines secreted from the natural killer cells. In one embodiment, the polymer compound can enhance cancer cell killing efficiency by promoting the secretion of cytotoxic granules and cytokines from natural killer cells during the activation process.

[0070] According to one embodiment, the polymer compound can be expressed by the following <Chemical Formula 1>. In the following <Chemical Formula 1>, the red structural formula is the hydrophobic moiety (DSPE PEG ) means the linker (PEG), and the green structural formula means the cancer cell recognition moiety (PBA). That is, the polymer compound is the hydrophobic moiety (DSPE PEG ) may have a structure in which two linkers (PEG) are bonded to one end of the linker, and the cancer cell recognition moiety (PBA) is bonded to each linker (PEG).

[0071] <Chemical Formula 1>

[0072]

[0073] (n: integer greater than or equal to 0)

[0074] As a result, the polymer compound composed of a hydrophobic moiety (lipid), a cancer cell recognition moiety (phenylboronic acid), and a linker (PEG) connecting the hydrophobic moiety and the cancer cell recognition moiety can easily modify the surface of natural killer cells (modify natural killer cells so that they can recognize specific cancer cells) through the hydrophobic action via the hydrophobic moiety (lipid), and can selectively recognize solid cancer cells (e.g., colon cancer cells, triple-negative cancer cells, liver cancer cells, etc.) that overexpress sialic acid among various cancer cells and then effectively kill them.

[0075]

[0076] Method for producing a polymer compound according to an embodiment

[0077] FIG. 4 is a drawing for explaining step S10 of a method for producing a polymer compound according to an embodiment of the present invention, FIG. 5 is a drawing for explaining step S20 of a method for producing a polymer compound according to an embodiment of the present invention, and FIG. 6 is a drawing for explaining step S30 of a method for producing a polymer compound according to an embodiment of the present invention.

[0078] Referring to FIGS. 4 to 6, a method for manufacturing a polymer compound according to an embodiment of the present invention may include a step (S10) of forming a first intermediate structure (MS1), a step of forming a second intermediate structure (MS2), and a step (S30) of manufacturing a polymer compound (PC). Each step is described in detail below.

[0079] In the above step S10, a first intermediate structure (MS1) can be formed by mixing tricarboxylic benzoic acid, a hydrophobic moiety, and a linker. According to one embodiment, the hydrophobic moiety is a compound (DSPE) in which a sub-linker (PEG) is bound to one end of a lipid (DSPE), and an amine group (NH2) is present at the end of the sub-linker (PEG). PEG -NH2) may be included. According to one embodiment, the linker may include a compound (Bis-PEG Amine) having an amine group (NH2) at the end of PEG. According to one embodiment, the first intermediate structure (MS1) may include a compound (DSPE) having two linkers connected to one end of the hydrophobic moiety and an amine group at the end. PEG -di(PEG)) may be included. The structural formula of the compound used as the hydrophobic moiety is DSPE as shown in Figure 4. PEG -NH2, and the structural formula of the compound used as the linker is the same as BisPEG shown in Figure 4, and the structural formula of the compound expressed as the first intermediate structure (MS1) is DSPE shown in Figure 4. PEG -Same as -di(PEG).

[0080] In the above step S20, the second intermediate structure (MS2) can be formed by converting the amine group at the end of the first intermediate structure (MS1) into a carboxylic acid group through succinylation of the first intermediate structure (MS1). The structural formula of the compound represented by the second intermediate structure (MS2) is Succinoyl DSPE shown in FIG. 5. PEG -Same as -di(PEG).

[0081] In the above S30 step, the second intermediate structure (MS2) and phenylboronic acid (PBA) are reacted to form the hydrophobic moiety (DSPE) at one end of the linker (PEG). PEG ) can be combined and a polymer compound (PC) having phenylboronic acid (PBA) combined at the other end can be prepared. The structural formula of the polymer compound (PC) is DSPE shown in Figure 6. PEG -It is the same as di(PEG-PBA).

[0082]

[0083] Hereinafter, polymer compounds according to embodiments of the present invention are described in more detail through specific experimental examples.

[0084] Preparation of polymer compound (DSPEPEG-di(PEG-PBA)) according to experimental example

[0085] A first base solution was prepared by dissolving 1 mmol of tricarboxylic benzoic acid, 3 mmol of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and 5 mmol of NHS (N-hydroxy succinimide, Sigma-Aldrich) in 5 mL of dimethyl formamide (DMF) and stirring for 6 hours under a N2 atmosphere at room temperature (RT).

[0086] 2 mmol of Bis-PEG Amine, 1 mmol of DSPE were added to the prepared first base solution. PEG -NH2, and 5 mg of DMAP (4-dimethyl amino pyridine) were added and stirred for 48 hours under N2 atmosphere and room temperature (RT). The mixture obtained through stirring was transferred to a dialysis tube (MWCO 6 kDa) and dialyzed against distilled water (DW) for 3 days to remove unreacted substances, and then freeze-dried to obtain the first intermediate structure (DSPE PEG -di(PEG)) was prepared.

[0087] First intermediate structure (DSPE) PEG -di(PEG)), excess succinic anhydride, and DMAP were dissolved in anhydrous DMF and stirred for 24 hours at room temperature (RT) under an N2 atmosphere. The mixture obtained through stirring was transferred to a dialysis tube (MWCO 2 kDa) and dialyzed against distilled water (DW) for 3 days to remove unreacted substances, and then freeze-dried to obtain the second intermediate structure (Succinoyl DSPE PEG -di(PEG)) was prepared.

[0088] 1 mmol of the second intermediate structure (Succinoyl DSPE) PEG -di(PEG)), an excess of EDC, and an excess of NHS were dissolved in 5 mL of anhydrous DMF, and stirred at room temperature (RT) for 48 hours to prepare a second base solution.

[0089] 5 mmol of PBA (Phenylboronic Acid) and 5 mg of DMAP were added to the second base solution and stirred for 48 hours at room temperature (RT) under an N2 atmosphere. The mixture obtained through stirring was transferred to a dialysis tube (MWCO 2 kDa) and dialyzed against distilled water (DW) for 3 days to remove unreacted substances, and then freeze-dried to obtain DSPE. PEG -di(PEG-PBA) polymer compound was prepared. The above-described preparation method is illustrated in FIGS. 4 to 6.

[0090]

[0091] Experimental Example 1: Characterization of Polymer Compounds

[0092] Figures 7 and 8 show the results of FT-IR analysis of polymer compounds, compounds used in the manufacturing process thereof, and products according to experimental examples of the present invention. 1 This is a diagram showing the results of H-NMR analysis.

[0093] Referring to Fig. 7, DSPE according to the above experimental example PEG Tricarboxylic benzoic acid (TBA), polyethylene glycol (PEG), and DSPE used in the manufacturing process of the -di(PEG-PBA) polymer compound PEG -NH2 and DSPE, the first intermediate structure produced during the manufacturing process PEG FT-IR analysis results for each of -di(PEG) (a) and 1 The results of H-NMR analysis (b) are shown. As can be seen in Fig. 7, tricarboxylic benzoic acid (TBA), polyethylene glycol (PEG), and DSPE PEG -DSPE, the first intermediate structure, is obtained through the use of NH2 PEG -It can be confirmed that di(PEG) has been synthesized.

[0094] Referring to Fig. 8, DSPE according to the above experimental example PEG Phenylboronic Acid (PBA) used in the manufacturing process of the -di(PEG-PBA) polymer compound, and DSPE, the first intermediate structure generated in the manufacturing process. PEG -di(PEG), DSPE, the second intermediate structure PEG -di(PEG)-COOH, and the final product, DSPE PEG FT-IR analysis results for each of -di(PEG-PBA) (a) and 1 The results of H-NMR analysis (b) are shown. As can be seen in Fig. 8, the second intermediate structure, DSPE PEG -di(PEG)-COOH is combined with phenylboronic acid (PBA) to form the final product, DSPE PEG -It can be confirmed that di(PEG-PBA) has been synthesized.

[0095]

[0096] Experimental Example 2: Analysis of the binding between polymer compounds and sugars

[0097] Figure 9 PBA, DSPE PEG -di(PEG), and DSPE PEG This is a diagram illustrating the bonding of -di(PEG-PBA) and sialic acid.

[0098] Referring to Figure 9, PBA, DSPE PEG -di(PEG), and DSPE PEG -di(PEG-PBA) is mixed with a pH 7.4 buffer solution and sialic acid, and the fluorescence intensity (au) measured from the mixture is expressed. Since PBA exhibits a fluorescent photo-induced electron transfer (PET) effect when bound to sugar, the binding between the above-mentioned compounds and sialic acid can be determined through fluorescence intensity measurement.

[0099] As can be seen in Figure 9, DSPE without PBA PEG -Di(PEG) no fluorescence intensity was measured, whereas PBA and DSPE PEG -For di(PEG-PBA), it can be confirmed that the fluorescence intensity is measured. That is, DSPE without PBA PEG -di(PEG) does not bind to sialic acid, whereas PBA and DSPE PEG -di(PEG-PBA) can be seen to bind to sialic acid.

[0100] Figure 10 is a drawing for explaining the binding strength of a polymer compound and various sugars according to an experimental example of the present invention.

[0101] Referring to Fig. 10, DSPE PEG-di(PEG-PBA) was mixed with a buffer solution and various sugars, and the results of analyzing the fluorescence intensity measured from the mixture to determine the binding affinity are shown. More specifically, sialic acid, glucose, arabinose, and sucrose were used as sugars, and the binding affinity was determined by measuring the initial fluorescence intensity (I O ) The value of the final fluorescence intensity (I) compared to O / I) was confirmed. In addition, Fig. 10 (a) shows the result when a buffer solution of pH 6.5 was used as a buffer solution, and Fig. 10 (b) shows the result when a buffer solution of pH 7.4 was used as a buffer solution.

[0102] As can be seen in Fig. 10, DSPE, a polymer compound according to the above experimental example PEG -di(PEG-PBA) was confirmed to have a significantly higher binding affinity to sialic acid compared to other sugars (glucose, arabinose, sucrose).

[0103]

[0104] Experimental Example 3: Fluorescence intensity analysis of NK cells bound to polymer compounds

[0105] Polymeric compounds (DSPE) in MEM alpha at a concentration of 1.0 mg / mL PEG -di(PEG-5CFL)) was dissolved and 5 x 10 5 Dog NK92-mi natural killer cells (NK cells) were evenly mixed in 100 μL of solution. The surface of NK cells was modified (DSPE on NK cells) for 30 minutes at room temperature. PEGAfter binding to -di(PEG-5CFL), the cells were washed twice with MEM alpha. Afterwards, the cells were lysed with 250 μL of RIPA buffer and stored at 4°C for 30 minutes. Finally, 250 μL of distilled water was added to dilute the cells and transferred to a 96-well plate, and the fluorescence intensity was measured at 480 / 535 nm. 5CFL (5-carboxyfluorescein) is a fluorescent dye and was used instead of PBA to measure the fluorescence intensity.

[0106] Figure 11 shows a fluorescence intensity measurement image of a cell in which a polymer compound and NK cells are combined according to an experimental example of the present invention.

[0107] Referring to Figure 11, DSPE is applied to NK cells as described in Experimental Example 3. PEG -di(PEG-5CFL) is combined with the image measuring the fluorescence intensity of the cells. As can be seen in Figure 11, DSPE is present on the surface of NK cells. PEG -It can be confirmed that di(PEG-5CFL) is uniformly coated.

[0108] Figure 12 is a drawing for explaining NK cell binding efficiency according to the concentration of a polymer compound according to an experimental example of the present invention.

[0109] Referring to Figure 12, the results of measuring the fluorescence intensity by the method described in Experimental Example 3 for each of NK cells, Coated NK cells (0.75 mg / mL), and Coated NK cells (1 mg / mL) are quantitatively shown. More specifically, NK cells are DSPE PEG -di(PEG-5CFL) represents the unbound state, and Coated NK cells (0.75 mg / mL) represent the state in which NK cells are coated with 0.75 mg / mL of DSPE PEG -di(PEG-5CFL) is combined, and Coated NK cell (1 mg / mL) is NK cell coated with 1 mg / mL of DSPEPEG -Indicates a state in which di(PEG-5CFL) is bound.

[0110] As can be seen in Figure 12, DSPE PEG -DSPE compared to NK cells not conjugated with -di(PEG-5CFL) PEG -The fluorescence intensity of NK cells bound to di(PEG-5CFL) was high, and 0.75 mg / mL of DSPE PEG -1 mg / mL of DSPE than di(PEG-5CFL) PEG It can be confirmed that the fluorescence intensity of NK cells bound with -di(PEG-5CFL) is high. That is, 1 mg / mL of DSPE is used for surface modification of NK cells. PEG -di(PEG-PBA) can be found to be suitable.

[0111]

[0112] Experimental Example 4: Analysis of the Effects of Polymer Compounds on NK Cells

[0113] Polymeric compounds (DSPE) in MEM alpha at a concentration of 1.0 mg / mL PEG -di(PEG-PBA)) was dissolved and 5 x 10 5 Dog NK92-mi natural killer cells (NK cells) were evenly mixed in 100 μL of solution. The surface of NK cells was modified (DSPE on NK cells) for 30 minutes at room temperature. PEG After binding to -di(PEG-PBA), the cells were washed twice with MEM alpha. Afterwards, the cells were lysed with 250 μL of RIPA buffer and stored at 4°C for 30 minutes. DSPE was added to NK cells. PEG Cells bound with -di(PEG-PBA) are expressed as PBA-NK cells.

[0114] Figure 13 is a graph showing the viability of PBA-NK cells.

[0115] Referring to Figure 13, various concentrations of DSPE PEG-di(PEG-PBA)-bound NK cell (PBA-NK cell) viability (Cell viability, Fold change). More specifically, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, and 1 mg / mL of DSPE PEG -di(PEG-PBA) was used. In addition, DSPE was used as a control. PEG -di(PEG-PBA) also shows the viability of NK cells not bound to it.

[0116] As can be seen in Figure 13, DSPE PEG NK cells (PBA-NK cells) combined with -di(PEG-PBA) are DSPE PEG - It can be confirmed that there is no substantial difference in viability between NK cells that are not bound to di(PEG-PBA). That is, DSPE PEG -di(PEG-PBA) was found to have no significant effect on the viability of NK cells. In addition, DSPE PEG It was also confirmed that the concentration of -di(PEG-PBA) did not affect viability.

[0117] Figure 14 is a graph illustrating the effect of a polymer compound according to experimental examples of the present invention on cytokine secretion of NK cells.

[0118] Referring to Figure 14, the amount (pg / mL) of cytokine (IFN-Y) secreted from NK cells and PBA-NK cells is quantified and shown to evaluate whether the secretion of cytokine (IFN-Y), a representative substance of NK cells for killing cancer cells, functions normally. Figure 14 (a) shows a state in which LPS, a substance that promotes cytokine secretion, has not been treated, and Figure 14 (b) shows a state in which LPS has been treated.

[0119] As can be seen in Figure 14, the amount of cytokine (IFN-Y) secreted by NK cells and PBA-NK cells is similar. That is, DSPE PEG When -di(PEG-PBA) is bound to NK cells, it can be confirmed that it does not affect the secretion of cytokines (IFN-Y), which is one of the unique functions of NK cells.

[0120] Figure 15 is a drawing for explaining the effect of a polymer compound according to an experimental example of the present invention on the ligand of NK cells.

[0121] Referring to Fig. 15, in order to evaluate whether TRAIL and FasL, two representative cell membrane ligands required for NK cell recognition of cancer cells, function normally, NK cells and PBA-NK cells were treated with TRAIL antibody and FasL antibody, and then TRAIL and FasL present on the surface of each cell were detected and the MFI was analyzed using flow cytometry. Fig. 15 (a) shows the results for TRAIL, and Fig. 15 (b) shows the results for FasL.

[0122] As can be seen in Figure 15, both NK cells and PBA-NK cells can be confirmed to function normally with TRAIL and FasL ligands. That is, DSPE PEG When -di(PEG-PBA) is bound to NK cells, it can be confirmed that it does not affect TRAIL and FasL ligands, which are unique ligands of NK cells.

[0123]

[0124] Experimental Example 5: Analysis of PBA-NK cell targeting ability against cancer cells

[0125] NK cells were stained with calcein AM (green reagent) and target cells with cell tracker red (red reagent), and then subjected to DSPE. PEG-di(PEG-PBA) was combined. Afterwards, the combined cells were co-cultured with target cells at a 1:1 ratio for 30 minutes, and then effector cell clusters and target cell clusters (E:T clusters) captured in both FITC and APC regions were detected by flow cytometry.

[0126] Figure 16 is a diagram illustrating the cancer cell targeting ability of PBA-NK cells.

[0127] Referring to Fig. 16, the E:T cluster was detected by the method according to the experimental example 5 described above, and sialic acid-overexpressing colon cancer cells (HCT-116), triple-negative breast cancer cells (MDA-MB-231), hepatoma cells (HepG2), and normal fibroblast cells were used as target cells. In addition, (1) shown in (a) to (d) of Fig. 16 is DSPE PEG -di(PEG-PBA) shows the results for the target cells of NK cells not bound, and (2) is DSPE PEG SA of NK cells not bound to -di(PEG-PBA) block The results for the target cell are shown, and (3) is DSPE PEG -di(PEG-PBA) shows the results for Tager cells of PBA-NK cells combined with NK cells, and (4) is DSPE PEG SA of PBA-NK cells bound to -di(PEG-PBA) NK cells block Shows the results for the target cell. Also, SA block Target cell refers to a target cell in which sialic acid is blocked by excessive treatment with PBA.

[0128] As can be seen in (a) to (c) of Figure 16, PBA-NK cells have significantly higher targeting ability against colon cancer cells (HCT-116), triple-negative breast cancer cells (MDA-MB-231), and liver cancer cells (HepG2) compared to NK cells. In addition, PBA-NK cells have SA block It can be confirmed that the targeting ability for target cells is significantly low. In addition, as can be confirmed in (d) of Figure 16, it can be confirmed that PBA-NK cells have virtually no targeting ability for normal cells, fibroblasts. Accordingly, DSPE PEG -di(PEG-PBA) was found to be able to selectively recognize cancer cells (colon cancer cells, triple-negative breast cancer cells, liver cancer cells) that overexpress sialic acid.

[0129] Figures 17 and 18 are drawings for explaining the cytotoxic granule (Granzyme B, Perforin) secretion ability of PBA-NK cells, and Figure 19 is a drawing for explaining the cytokine (IFN-Y) secretion ability of PBA-NK cells.

[0130] Referring to Fig. 17, the amount of Granzyme B secretion (ng / mL) for the target cells of NK cells and PBA-NK cells is measured and shown, and referring to Fig. 18, the amount of Perforin secretion (ng / mL) for the target cells of NK cells and PBA-NK cells is measured and shown, and referring to Fig. 19, the amount of IFN-Y secretion (ng / mL) for the target cells of NK cells and PBA-NK cells is measured and shown. As target cells, colon cancer cells (HCT-116), triple-negative breast cancer cells (MDA-MB-231), hepatoma cells (HepG2), and normal fibroblast cells were used. In addition, (1) shown in (a) to (d) of Figs. 17 to 19 shows the results for the target cells of NK cells, and (2) shows the SA of NK cells. block (3) shows the results for the Tager cell of PBA-NK cell, and (4) shows the results for the SA of PBA-NK cell. block Shows the results for the target cell. Also, SA block Target cell refers to a target cell in which sialic acid is blocked by excessive treatment with PBA.

[0131] As can be seen in Figures 17 to 19, PBA-NK cells secrete significantly higher amounts of cytotoxic granules (Granzyme B, Perforin) and cytokines (IFN-Y) against target cells compared to NK cells. That is, DSPE PEG -di(PEG-PBA) was found to promote the secretion of cytotoxic granules (Granzyme B, Perforin) and cytokines (IFN-Y) of NK cells. In addition, SA of PBA-NK cells blockIt was confirmed that the amount of cytotoxic granules (Granzyme B, Perforin) and cytokines (IFN-Y) secreted for target cells was significantly low, and that cytotoxic granules (Granzyme B, Perforin) and cytokines (IFN-Y) were practically not secreted for normal cells, fibroblasts. Accordingly, DSPE PEG -di(PEG-PBA) was found to be able to selectively recognize cancer cells (colon cancer cells, triple-negative breast cancer cells, liver cancer cells) that overexpress sialic acid.

[0132]

[0133] Experimental Example 6: Analysis of the Cancer Cell Killing Ability of PBA-NK Cells

[0134] DSPE in MEM alpha at a concentration of 1.0 mg / mL PEG After dissolving the -di(PEG-PBA) polymer compound, 6 x 10 5 Canine NK92-mi natural killer cells (NK cells) were evenly mixed with 120 μL of the composite material solution. The NK cell surface was modified (by binding HA-PEG-DSPE to NK cells) at room temperature for 30 minutes, and then washed twice with MEM alpha.

[0135] Target cell 6 x 10 4 Dogs were stained with 600 μL of 15 μM Calcein-AM solution at 37°C for 30 minutes, then washed twice with HDMEM. Surface-modified NK cells and stained target cells were placed together in a 96-well plate at ratios of 1:1, 5:1, and 10:1, and cultured at 37°C for 4 hours. The supernatant was obtained and the fluorescence intensity was measured at 480 / 535 mm.

[0136] Figure 20 is a drawing illustrating the killing ability of PBA-NK cells against target cells.

[0137] Referring to Fig. 20, the lysis ability (Specific cell lysis, %) of the target cell was measured by the method according to the experimental example 6 described above, and as the target cells, colon cancer cells (HCT-116), triple-negative breast cancer cells (MDA-MB-231), hepatoma cells (HepG2) overexpressing sialic acid, and normal fibroblast cells were used. In addition, the black indicators shown in (a) to (d) of Fig. 20 represent the results for the target cells of NK cells, and the purple indicators represent the SA of NK cells. block The results for the target cell are shown, the green indicator shows the results for the tager cell of the PBA-NK cell, and the red indicator shows the SA of the PBA-NK cell. block Shows the results for the target cell. Also, SA block Target cell refers to a target cell in which sialic acid is blocked by excessive treatment with PBA.

[0138] As can be seen in Figure 20, PBA-NK cells have a significantly higher solubility (specific cell lysis, %) for target cells compared to NK cells. That is, DSPE PEG -di(PEG-PBA) was found to enhance the killing ability of NK cells against colon cancer cells (HCT-116), triple-negative breast cancer cells (MDA-MB-231), and hepatoma cells (HepG2). In addition, PBA-NK cells were found to have no substantial killing ability against normal cells, fibroblasts. Accordingly, DSPE PEG It was found that NK cells conjugated with -di(PEG-PBA) selectively kill cancer cells with overexpressed sialic acid.

[0139]

[0140] Experimental Example 7: Analysis of the Tumor Spheroid Destruction Ability of PBA-NK Cells

[0141] Triple-negative breast cancer cell (MDA-MB-231) spheroids were cultured with NK cells and PBA-NK cells, respectively, and the structural destruction ability was measured by measuring fluorescence intensity.

[0142] Figure 21 is a fluorescence microscope image of triple-negative breast cancer cell (MDA-MB-231) spheroids treated with NK cells and PBA-NK cells, and Figure 22 is a graph quantifying the fluorescence intensity measured in Figure 21.

[0143] (a) shown in Figure 21 shows triple-negative breast cancer cell (MDA-MB-231) spheroids as a control, (b) shows triple-negative breast cancer cell (MDA-MB-231) spheroids treated with NK cells, and (c) shows triple-negative breast cancer cell (MDA-MB-231) spheroids treated with PBA-NK cells.

[0144] As can be seen in Figures 21 and 22, PBA-NK cells have a significantly higher ability to destroy triple-negative breast cancer cell (MDA-MB-231) spheroids compared to NK cells.

[0145] Figure 23 is a fluorescence microscopy image of triple-negative breast cancer cell (MDA-MB-231) spheroids stained with EthD-1 after NK cell and PBA-NK cell treatment, and Figure 24 is a graph quantifying the intensity of EthD-1 and spheroid area measured in Figure 23.

[0146] (a) shown in Figure 23 shows triple-negative breast cancer cell (MDA-MB-231) spheroids as a control, (b) shows triple-negative breast cancer cell (MDA-MB-231) spheroids treated with NK cells, and (c) shows triple-negative breast cancer cell (MDA-MB-231) spheroids treated with PBA-NK cells.

[0147] As can be seen in Figures 23 and 24, PBA-NK cells have a significantly higher ability to destroy triple-negative breast cancer cell (MDA-MB-231) spheroids compared to NK cells.

[0148]

[0149] While the present invention has been described in detail using preferred embodiments, the scope of the present invention is not limited to the specific embodiments described above, and should be interpreted in accordance with the appended claims. Furthermore, those skilled in the art will appreciate that numerous modifications and variations are possible without departing from the scope of the present invention.

[0150] The present invention can be used in the medical industry.

Claims

1. A polymer compound comprising a hydrophobic moiety that binds to natural killer cells, a cancer cell recognition moiety, and a linker, The hydrophobic moiety is bound to one end of the linker and the cancer cell recognition moiety is bound to the other end of the linker to recognize natural killer cells and cancer cells. The cancer cell recognition moiety is a polymer compound that selectively recognizes solid cancer cells containing sialic acid.

2. In paragraph 1, The above cancer cell recognition moiety is a polymer compound containing phenylboronic acid.

3. In paragraph 2, A polymer compound comprising the cancer cell recognition moiety that forms a boron-ester complex with the sialic acid of the solid cancer cell.

4. In paragraph 1, The polymer compound, which is bound to natural killer cells through the hydrophobic moiety, binds to cancer cells through the cancer cell recognition moiety, A polymer compound that kills cancer cells by promoting the secretion of cytotoxic granules and cytokines from natural killer cells.

5. In paragraph 1, A polymer compound comprising the hydrophobic moiety, which comprises a lipid and is bound to the surface of a natural killer cell through a hydrophobic action mediated by the lipid.

6. In paragraph 5, The hydrophobic moiety is a polymer compound comprising any one of a phospholipid having an alkyl chain having 12 to 24 carbon atoms, a sterol lipid having 10 to 30 carbon atoms, 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE), 1,2-bis(diphenylphosphino)ethane (DPPE), and 1,2-bis(dimethylphosphino)ethane (DMPE).

7. In paragraph 5, The above hydrophobic moiety further comprises a sub-linker, A polymer compound comprising a sub-linker connected to one end of the lipid, and a hydrophobic moiety bonded to the linker through the sub-linker.

8. In paragraph 1, A polymer compound comprising the linker that prevents the polymer compound bound to the natural killer cell from being endocytosed into the natural killer cell.

9. In paragraph 1, The above linker is a polymer compound containing polyethylene glycol (PEG).

10. In paragraph 1, The above polymer compound is a polymer compound including the one represented by the following <Chemical Formula 1>. <Chemical Formula 1> (n: integer greater than or equal to 0) 11. In paragraph 1, A polymer compound comprising the cancer cell recognition moiety that recognizes colon cancer cells (HCT-116), triple-negative breast cancer cells (MDA-MB-231), and liver cancer cells (HepG2).

12. A step of mixing a hydrophobic moiety and a linker including an amine group at a terminal, thereby forming a first intermediate structure in which the linker is connected to one end of the hydrophobic moiety and the linker includes an amine group at a terminal; A step of manufacturing a second intermediate structure by converting the amine group of the first intermediate structure into a carboxylic acid group; and A method for producing a polymer compound, comprising a step of reacting the second intermediate structure with phenylboronic acid to produce a polymer compound in which the hydrophobic moiety is bonded to one terminal of the linker and the phenylboronic acid is bonded to the other terminal.

13. In paragraph 12, A method for producing a polymer compound, wherein the second intermediate structure comprises converting an amine group into a carboxylic acid group through succinylation of the first intermediate structure.

14. In paragraph 12, The hydrophobic moiety comprises a compound in which polyethylene glycol (PEG) is bound to one end of 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE), The above linker is a method for producing a polymer compound containing polyethylene glycol (PEG).

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