Polymer compound for selectively killing CD44-expressing cancer cells and enhancing killing efficiency, and method for preparing same

Abstract

A polymer compound is provided. The polymer compound comprises a hydrophobic moiety that binds to natural killer (NK) cells, a cancer-cell recognition moiety, and a linker. In the polymer compound, 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, thereby enabling recognition of both NK cells and cancer cells. The length of the linker and the content of the hydrophobic moiety may be controlled to enhance the killing efficiency of CD44-expressing solid cancer cells through the NK cells.

Description

A polymer compound for selectively killing cancer cells containing CD44 and improving killing efficiency, and a method for producing the same

[0001] The present invention relates to a polymer compound that can selectively recognize and kill CD44 cancer cells 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 with a controlled ring length.

[0009] Another technical problem to be solved by the present invention is to provide a polymer compound having a controlled content of hydrophobic moieties.

[0010] Another technical problem that the present invention seeks to solve is to provide a polymer compound capable of recognizing target cancer cells.

[0011] Another technical problem to be solved by the present invention is to provide a polymer compound capable of selectively recognizing cancer cells containing CD44.

[0012] Another technical problem to be solved by the present invention is to provide a polymer compound having improved killing efficiency against cancer cells overexpressing CD44.

[0013] The technical problems to be solved by the present invention are not limited to those described above.

[0014] To solve the above-described technical problems, the present invention provides a polymer compound.

[0015] 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, and wherein the length of the linker and the content of the hydrophobic moiety are controlled, thereby improving the killing efficiency of solid cancer cells including CD44 through the natural killer cells.

[0016] In one embodiment, the linker may comprise polyethylene glycol (PEG).

[0017] In one embodiment, the linker may have a length greater than 0.6 k and less than 5 k.

[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 degree of substitution (DS), defined as the amount of the hydrophobic moiety linked to the cancer cell recognition moiety calculated through NMR analysis, may include greater than 7% and less than 26%.

[0020] In one embodiment, the hydrophobic moiety may comprise one that binds to the surface of a natural killer cell through a lipid-mediated hydrophobic interaction.

[0021] In one embodiment, the cancer cell recognition moiety may comprise hyaluronic acid.

[0022] In one embodiment, the cancer cell recognition moiety may comprise one that selectively recognizes a solid cancer cell comprising CD44.

[0023] 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.

[0024] In one embodiment, the cancer cell recognition moiety may comprise one that recognizes pancreatic cancer cells (MIA PaCa-2), triple-negative breast cancer cells (MDA-MB-231), and colon cancer cells (HCT-116).

[0025] In one embodiment, the polymer compound can inhibit the metastasis of solid cancer cells containing CD44 to other organs.

[0026]

[0027] To solve the technical problems described above, the present invention provides a method for producing a polymer compound.

[0028] According to one embodiment, the method for preparing the polymer compound includes the steps of preparing hyaluronic acid, adding and mixing the hyaluronic acid, EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide), and NHS (N-hydroxy succinimide) into a solvent to prepare a base solution, and adding and stirring a compound having a hydrophobic moiety bonded to one terminal of a linker and an amine group bonded to the other terminal to the base solution, thereby preparing a polymer compound having the hydrophobic moiety bonded to one terminal of the linker and the hyaluronic acid bonded to the other terminal, wherein the method may include controlling the length of the linker and the content of the hydrophobic moiety in the polymer compound to improve the killing efficiency of solid cancer cells including CD44 through the natural killer cells.

[0029] In one embodiment, the linker may comprise polyethylene glycol (PEG) having a length greater than 0.6 k and less than 5 k.

[0030] In one embodiment, the degree of substitution (DS), defined as the amount of the hydrophobic moiety linked to the cancer cell recognition moiety calculated through NMR analysis, may include greater than 7% and less than 26%.

[0031] A polymer compound in which a hydrophobic moiety (DSPE) is bound to one end of a linker (PEG) and a cancer cell recognition moiety (hyaluronic acid) is bound to the other end of the linker can selectively recognize CD44-overexpressing solid cancer cells (e.g., pancreatic cancer cells, triple-negative breast cancer cells, colon cancer cells, etc.) among various cancer cells. In addition, by controlling the length of the linker (more than 0.6 k and less than 5 k) and the content of the hydrophobic moiety (the degree of lipid substitution defined as the value calculated by the amount of the hydrophobic moiety linked to the cancer cell recognition moiety through NMR analysis, of more than 7% and less than 26%), the killing efficiency of solid cancer cells including CD44 through the natural killer cells can be improved.

[0032] FIG. 1 is a drawing for explaining a polymer compound according to an embodiment of the present invention.

[0033] FIG. 2 is a drawing for explaining a method for manufacturing a polymer compound according to an embodiment of the present invention.

[0034] Figures 3 and 4 are drawings showing the results of FT-IR analysis of polymer compounds according to experimental examples of the present invention.

[0035] Figure 5 is a drawing showing the results of hydrophobicity analysis of polymer compounds according to experimental examples of the present invention.

[0036] Figure 6 is a fluorescence intensity measurement image and fluorescence intensity measurement graph of cells in which polymer compounds and NK cells are combined according to experimental examples of the present invention.

[0037] Figure 7 is a graph quantitatively comparing the NK cell surface modification efficiency of polymer compounds according to experimental examples of the present invention.

[0038] Figure 8 is a graph summarizing the NK cell surface modification efficiency of polymer compounds according to experimental examples of the present invention in terms of Log P values.

[0039] Figure 9 is a graph showing the coating maintenance ability and cell proliferation ability of polymer compounds according to experimental examples of the present invention combined with NK cells.

[0040] Figure 10 is a graph illustrating the effect of polymer compounds according to experimental examples of the present invention on ligands of NK cells.

[0041] Figure 11 is a graph illustrating the effect of polymer compounds according to experimental examples of the present invention on cytokine secretion of NK cells.

[0042] Figure 12 is a drawing for confirming the targeting ability of NK cells bound with polymer compounds according to experimental examples of the present invention against triple-negative breast cancer cells.

[0043] Figure 13 is a graph quantifying the E:T cluster ratio measured in Figure 12.

[0044] Figure 14 is a drawing for confirming the targeting ability of NK cells to which polymer compounds are bound according to experimental examples of the present invention toward liver cancer cells.

[0045] Figure 15 is a graph quantifying the E:T cluster ratio measured in Figure 14.

[0046] Figure 16 is a drawing for confirming the targeting ability of NK cells to which polymer compounds are bound according to experimental examples of the present invention toward fibroblasts.

[0047] Figure 17 is a graph quantifying the E:T cluster ratio measured in Figure 16.

[0048] Figure 18 is a drawing for explaining the killing ability of NK cells bound with polymer compounds according to experimental examples of the present invention against triple-negative breast cancer cells.

[0049] Figure 19 is a drawing for explaining the killing ability of NK cells to which polymer compounds are combined against liver cancer cells according to experimental examples of the present invention.

[0050] Figure 20 is a drawing for explaining the killing ability of NK cells to which polymer compounds are bound according to experimental examples of the present invention against fibroblasts.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056]

[0057] Polymer compound according to an embodiment

[0058] FIG. 1 is a drawing for explaining a polymer compound according to an embodiment of the present invention.

[0059] Referring to FIG. 1, 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 bound to one end of the linker and the cancer cell recognition moiety is bound to the other end.

[0060] 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.

[0061] 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).

[0062] The cancer cell recognition moiety recognizes and binds to a cancer cell, and may include hyaluronic acid. As described above, when the cancer cell recognition moiety includes hyaluronic acid, the cancer cell recognition moiety can selectively recognize a solid cancer cell that includes CD44. More specifically, the cancer cell recognition moiety that includes hyaluronic acid can selectively recognize a cancer cell that overexpresses CD44. For example, the cancer cells that overexpress CD44 may include pancreatic cancer cells (MIA PaCa-2), triple-negative breast cancer cells (MDA-MB-231), and colon cancer cells (HCT-116). That is, the cancer cell recognition moiety containing hyaluronic acid can selectively recognize pancreatic cancer cells (MIA PaCa-2), triple-negative breast cancer cells (MDA-MB-231), and colon cancer cells (HCT-116) among various cancer cells. Accordingly, the natural killer cells bound to the hydrophobic moiety can selectively kill pancreatic cancer cells (MIA PaCa-2), triple-negative breast cancer cells (MDA-MB-231), and colon cancer cells (HCT-116) among various cancer cells by the cancer cell recognition moiety.

[0063] The linker, which connects the hydrophobic moiety and the cancer cell recognition moiety, may include polyethylene glycol (PEG). Furthermore, the linker may prevent the polymer compound bound to natural killer cells from being endocytosed into the natural killer cells. In other words, the linker may function as a penetration blocker.

[0064] Natural killer cells (NK cells) combined with the polymer compound can recognize cancer cells containing CD44 through the cancer cell recognition moiety (hyaluronic acid, HA) of the polymer compound, and then combine with the cancer cells through the cancer cell recognition moiety (hyaluronic acid, HA). Thereafter, an activation process in which cytotoxic granules and cytokines are secreted from the natural killer cells occurs, and cancer cells can be killed by the cytotoxic granules and cytokines secreted from the natural killer cells. According to one embodiment, the polymer compound can enhance the cancer cell killing efficiency by promoting the secretion of cytotoxic granules and cytokines from natural killer cells during the activation process.

[0065] According to one embodiment, the polymer compound can control the length of the linker (PEG) to improve the killing efficiency of solid cancer cells including CD44 through the natural killer cells. More specifically, the linker (PEG) may have a length of more than 0.6 k and less than 5 k. In contrast, when the length of the linker (PEG) is 0.6 k or less or 5 k or more, the surface modification efficiency of the natural killer cells (content of the polymer compound bound to the natural killer cells) through the polymer compound may be significantly reduced. That is, when the length of the linker (PEG) is 0.6 k or less or 5 k or more, a problem may occur in which the polymer compound is not bound to the surface of the natural killer cells. As a result, the cancer cell killing efficiency using the natural killer cells bound to the polymer compound may be significantly reduced.

[0066] In addition, according to one embodiment, the polymer compound can control the content of the hydrophobic moiety (e.g., DSPE) to enhance the killing efficiency of solid cancer cells including CD44 through the natural killer cells. More specifically, the polymer compound may have a degree of substitution (DS), which is defined as a value calculated by the amount of the hydrophobic moiety (DSPE) linked to the cancer cell recognition moiety (hyaluronic acid) through NMR analysis, of more than 7% and less than 26%. In contrast, when the degree of lipid substitution is 7% or less or 26% or more, the surface modification efficiency of the natural killer cells through the polymer compound (the content of the polymer compound bound to the natural killer cells) may be significantly reduced. That is, when the degree of lipid substitution is 7% or less or 26% or more, a problem may occur in which the polymer compound is not bound to the surface of the natural killer cells. Due to this, the efficiency of killing cancer cells using natural killer cells combined with the polymer compound may be significantly reduced.

[0067] As a result, the polymer compound in which the hydrophobic moiety (DSPE) is bound to one end of the linker (PEG) and the cancer cell recognition moiety (hyaluronic acid) is bound to the other end of the linker can selectively recognize CD44-overexpressing solid cancer cells (e.g., pancreatic cancer cells, triple-negative breast cancer cells, colon cancer cells, etc.) among various cancer cells. In addition, by controlling the length of the linker (more than 0.6 k and less than 5 k) and the content of the hydrophobic moiety (the degree of lipid substitution defined as the value calculated by the amount of the hydrophobic moiety linked to the cancer cell recognition moiety through NMR analysis of more than 7% and less than 26%), the killing efficiency of solid cancer cells including CD44 through the natural killer cells can be improved.

[0068]

[0069] Method for producing a polymer compound according to an embodiment

[0070] FIG. 2 is a drawing for explaining a method for manufacturing a polymer compound according to an embodiment of the present invention.

[0071] Referring to FIG. 2, a method for producing a polymer compound according to an embodiment of the present invention may include a step of preparing hyaluronic acid (S10), a step of preparing a base solution by mixing the hyaluronic acid with EDC and NHS (S20), and a step of producing the polymer compound (S30). Each step is described in detail below.

[0072] In the above step S10, hyaluronic acid (HA) can be prepared. Thereafter, in step S20, the hyaluronic acid, EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide), and NHS (N-hydroxy succinimide) can be added to a solvent (di-water) and mixed to prepare a base solution. Finally, in step S30, a compound having a hydrophobic moiety (DSPE) bound to one terminal of a linker (PEG) and an amine group (NH2) bound to the other terminal can be added to the base solution and stirred to cause an EDC / NHS coupling reaction between the hyaluronic acid and the compound. Accordingly, the polymer compound (HA-PEG-DSPE) having the hydrophobic moiety (DSPE) bound to one terminal of the linker (PEG) and the hyaluronic acid (HA) bound to the other terminal can be prepared.

[0073]

[0074] Hereinafter, polymer compounds according to embodiments of the present invention are described in more detail through specific experimental examples.

[0075] Preparation of polymer compounds (HA-PEG-DSPE) according to experimental examples

[0076] A base solution was prepared by dissolving 100 mg of hyaluronic acid (Mw 60k, 10 mg / mL, 1 equivalent), 0.075 mmol of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and 0.075 mmol of N-hydroxy succinimide (NHS) in 8 mL of DI water and stirring at room temperature for 6 hours.

[0077] Afterwards, a solution of 0.05 mmol of DSPE-PEG-NH2 dissolved in 2 mL of DMF (dimethylformamide) and DMAP (4-dimethylaminopyridine) were added to the above-described base solution, and the mixture was stirred at room temperature for 48 hours.

[0078] Finally, the stirred reaction mixture was dialyzed against DI water (MWCO 12-14 kDa) for 3 days to produce a HA-PEG-DSPE polymer compound. The above-described manufacturing method is also illustrated in Fig. 2.

[0079] In addition, polymer compounds were prepared using PEGs of different lengths (0 k, 0.6 k, 2 k, 5 k), and the prepared polymer compounds are expressed as PEG 0, PEG 600, PEG 2000, and PEG 5000 in explaining the experimental examples below.

[0080] In addition, for the case where PEG with a length of 2 k was used, the degree of substitution (DS), defined as the amount of DSPE linked to the hyaluronic acid calculated through NMR analysis, was manufactured differently as 7%, 18%, and 26%, and the manufactured polymer compounds are expressed as PEG 2000 7%, PEG 2000 18%, and PEG 2000 26% in explaining the experimental examples below. Specifically, HA-PEG-DSPE having different degrees of lipid substitution was manufactured by varying the content of DSPE-PEG-NH2 used in the above-described manufacturing process. HA-PEG-DSPE with a lipid substitution degree of 7% was prepared using 70 μg of DSPE-PEG-NH2, HA-PEG-DSPE with a lipid substitution degree of 18% was prepared using 140 μg of DSPE-PEG-NH2, and HA-PEG-DSPE with a lipid substitution degree of 26% was prepared using 280 μg of DSPE-PEG-NH2.

[0081]

[0082] Experimental Example 1: Characterization of Polymer Compounds

[0083] Figures 3 and 4 are drawings showing the results of FT-IR analysis of polymer compounds according to experimental examples of the present invention.

[0084] Referring to (a) of Fig. 3, the FT-IR analysis results for HA (hyaluronic acid), DSPE-PEG-NH2, HA-PEG(2000, 7%)-DSPE(DS=7%), HA-PEG(2000, 18%)-DSPE(DS=18%), and HA-PEG(2000, 26%)-DSPE(DS=26%) are shown, respectively; and referring to (b) of Fig. 3, the FT-IR analysis results for HA (hyaluronic acid), DSPE-PEG-NH2, and HA-PEG-DSPE are shown, respectively; and referring to (a) of Fig. 4, the FT-IR analysis results for HA (hyaluronic acid), DSPE-PEG 0.6k -NH2, and HA-PEG 0.6k-The FT-IR analysis results for each DSPE are shown, and with reference to Fig. 4 (b), HA (hyaluronic acid), DSPE-PEG 2k -NH2, and HA-PEG 2k -The FT-IR analysis results for each DSPE are shown, and with reference to Fig. 4 (c), HA (hyaluronic acid), DSPE-PEG 5k -NH2, and HA-PEG 5k -The FT-IR analysis results for each DSPE are shown. As can be seen in Figures 3 and 4, it can be confirmed that DSPE and HA are easily bound to PEGs with different lengths (0.6 k, 2 k, 5 k).

[0085] Figure 5 is a drawing showing the results of hydrophobicity analysis of polymer compounds according to experimental examples of the present invention.

[0086] Referring to (a) of Fig. 5, the results of measuring the Log P value to confirm the hydrophobicity for each of HA-PEG0-DSPE, HA-PEG600-DSPE, HA-PEG2000-DSPE, HA-PEG5000-DSPE, and HA (hyaluronic acid) are shown, and referring to (b) of Fig. 5, the results of measuring the Log P value to confirm the hydrophobicity for each of HA-PEG2000, 7%-DSPE, HA-PEG2000, 18%-DSPE, HA-PEG2000, 26%, and HA (hyaluronic acid) are shown. The Log P value was calculated through the following <Mathematical Formula 1>.

[0087] <Mathematical Formula 1>

[0088] Log P = (solute)actanol / (solute)water

[0089] As can be seen in (a) of Fig. 5, as the length of PEG increases (0 -> 600 -> 2000 -> 5000), the Log P value increases, confirming that the hydrophobicity decreases. In addition, as can be seen in (b) of Fig. 5, as the lipid substitution degree value increases (7% -> 18% -> 26%), the Log P value decreases. In other words, it can be seen that the hydrophobicity decreases as the lipid content decreases (the lower the lipid substitution degree value).

[0090]

[0091] Experimental Example 2: Analysis of the NK cell surface modification efficiency of polymer compounds

[0092] Dissolve the polymer compound (HA-PEG-DSPE) in MEM alpha at a concentration of 1.0 mg / mL, and add 5 x 10 5 Canine NK92-mi natural killer (NK) cells were evenly mixed in 100 μL of solution. The NK cell surface was modified (by binding HA-PEG-DSPE to NK cells) for 30 minutes at room temperature, and then washed twice with MEM alpha. The cells were then lysed in 250 μL of RIPA buffer and stored at 4°C for 30 minutes. Finally, 250 μL of distilled water was added to dilute the solution, and the solution was transferred to a 96-well plate. The fluorescence intensity was measured at 480 / 535 nm.

[0093] FIG. 6 is a fluorescence intensity measurement image and fluorescence intensity measurement graph of cells in which polymer compounds and NK cells are combined according to experimental examples of the present invention, FIG. 7 is a graph quantitatively comparing the NK cell surface modification efficiency of polymer compounds according to experimental examples of the present invention, and FIG. 8 is a graph summarizing the NK cell surface modification efficiency of polymer compounds according to experimental examples of the present invention in terms of Log P values.

[0094] As can be seen in Fig. 6, it can be confirmed that the fluorescence intensity is not practically measured for the polymer compounds using PEG600 and PEG5000. On the other hand, it can be confirmed that the polymer compound using PEG2000 measures strong fluorescence intensity on the surface of NK cells. That is, the polymer compound using PEG2000 is well fixed to the surface of NK cells, whereas the polymer compounds using PEG600 and PEG5000 are not fixed to the surface of NK cells. Accordingly, in order to improve the cancer cell killing efficiency of NK cells through the HA-PEG-DSPE polymer compound binding to NK cells, it can be seen that the length of PEG should be controlled to be greater than 0.6 k and less than 5 k.

[0095] As can be seen in Figure 7, the polymer compound using PEG2000 showed a higher NK cell coating efficiency (cell coating efficacy, ng / 10) compared to the polymer compounds using PEG600 and PEG5000. 5 It can be confirmed that the coating efficiency of NK cells is high. In particular, when the degree of lipid substitution increases from 7% to 18% in the polymer compound using PEG2000, the coating efficiency of NK cells increases, but when it increases from 18% to 26%, the coating efficiency of NK cells is significantly reduced. Consequently, it can be seen that in order to improve the cancer cell killing efficiency of NK cells through the HA-PEG-DSPE polymer compound binding to NK cells, the degree of lipid substitution must be controlled to be greater than 7% and less than 26%.

[0096] Referring to Fig. 8, the NK cell coating efficiency according to Log P is summarized for HA-PEG-DSPE having various PEG lengths and various lipid substitution degrees. The Log P value was calculated using the above-described <Mathematical Formula 1>. As can be seen in Fig. 8, HA-PEG-DSPE having a PEG length of 2 k and a lipid substitution degree of 18% has a significantly higher NK cell coating efficiency compared to other HA-PEG-DSPEs.

[0097]

[0098] Experimental Example 3: Analysis of the Effects of Polymer Compounds on NK Cells

[0099] Figure 9 is a graph showing the coating maintenance ability and cell proliferation ability of polymer compounds according to experimental examples of the present invention combined with NK cells.

[0100] Referring to Fig. 9 (a), the coating sustainability of HA-PEG-DSPE is shown in cells bound to NK cells with various HA-PEG-DSPEs. Specifically, the coating sustainability was measured using HA-PEG-DSPE containing PEG 600, PEG 2000 (7%), PEG 2000 (18%), PEG 2000 (26%), and PEG 5000, and the coating sustainability was confirmed through the change in MFI value according to the incubation time (min). As can be seen in Fig. 9 (a), it can be confirmed that all different HA-PEG-DSPEs have similar coating sustainability.

[0101] Referring to Fig. 9 (b), the cell proliferation ability (fold change) of cells bound to various HA-PEG-DSPEs is shown. Specifically, the measurement was performed using HA-PEG-DSPE containing PEG 600, PEG 2000 (7%), PEG 2000 (18%), PEG 2000 (26%), and PEG 5000. In addition, the cell proliferation ability of NK cells not bound to HA-PEG-DSPE was also measured as a control. As can be seen in Fig. 9 (b), it can be confirmed that all different HA-PEG-DSPEs have similar cell proliferation abilities.

[0102] That is, when binding HA-PEG-DSPE to NK cells, it can be seen that changes in PEG length and lipid content do not substantially affect the coating retention ability and cell proliferation ability.

[0103] Figure 10 is a graph illustrating the effect of polymer compounds according to experimental examples of the present invention on ligands of NK cells.

[0104] Referring to Fig. 10, in order to evaluate whether TRAIL and FasL, two representative cell membrane ligands required for NK cell recognition of cancer cells, function normally, cells bound to various HA-PEG-DSPEs were treated with TRAIL antibodies and FasL antibodies, and then TRAIL and FasL present on the surface of each cell were detected and the MFI was analyzed using flow cytometry. Specifically, the measurement was performed using HA-PEG-DSPE with PEG 600, PEG 2000 (7%), PEG 2000 (18%), PEG 2000 (26%), and PEG 5000. In addition, NK cells (Non coated) to which HA-PEG-DSPE was not bound were also measured as a control. In addition, Fig. 10 (a) shows the results for FasL, and Fig. 10 (b) shows the results for TRAIL.

[0105] As can be seen in Fig. 10, it can be confirmed that all NK cells bound to different HA-PEG-DSPEs function normally with TRAIL and FasL ligands. In other words, when HA-PEG-DSPE is bound to NK cells, it can be confirmed that changes in PEG length and lipid content do not affect TRAIL and FasL ligands, which are ligands unique to NK cells.

[0106] Figure 11 is a graph illustrating the effect of polymer compounds according to experimental examples of the present invention on cytokine secretion of NK cells.

[0107] Referring to Fig. 11, in order to evaluate whether the secretion of cytokine (IFN-Y), a representative substance of NK cells for killing cancer cells, functions normally, the amount (pg / mL) of cytokine (IFN-Y) secreted from cells bound to NK cells with various HA-PEG-DSPEs is quantified and shown. Specifically, the measurement was performed using HA-PEG-DSPE with PEG 600, PEG 2000 (7%), PEG 2000 (18%), PEG 2000 (26%), and PEG 5000. In addition, NK cells (Non coated) to which HA-PEG-DSPE was not bound were also measured as a control. In addition, Fig. 11 (a) shows a state in which LPS, a substance that promotes cytokine secretion, is not treated, and Fig. 11 (b) shows a state in which LPS is treated.

[0108] As can be seen in Figure 11, it can be confirmed that all NK cells bound to different HA-PEG-DSPEs function normally in secretion of cytokines (IFN-Y). In other words, when HA-PEG-DSPE is bound to NK cells, it can be confirmed that changes in PEG length and lipid content do not affect the secretion of cytokines (IFN-Y), which is one of the unique functions of NK cells.

[0109]

[0110] Experimental Example 4: Analysis of the Cancer Cell Targeting Ability of NK Cells Conjugated with Polymer Compounds

[0111] NK cells were stained with calcein AM (green reagent) and target cells with cell tracker red (red reagent), and then conjugated with various HA-PEG-DSPE conjugates. The conjugated cells were then co-cultured with target cells at a 1:1 ratio for 30 minutes, and the effector cell-to-target cell cluster ratio (E:T cluster), which is detected in both FITC and APC regions, was detected by flow cytometry.

[0112] FIG. 12 is a diagram for confirming the targeting ability of NK cells bound with polymer compounds according to experimental examples of the present invention against triple-negative breast cancer cells, and FIG. 13 is a graph quantifying the E:T cluster ratio measured in FIG. 12.

[0113] Referring to FIGS. 12 and 13, the E:T cluster was detected using the method according to Experimental Example 4 described above, but triple-negative breast cancer cells (MDA-MB-231) with overexpressed CD44 were used as target cells. More specifically, (a) of FIG. 12 shows the results for NK cells not bound to HA-PEG-DSPE, (b) of FIG. 12 shows the results for NK cells bound to HA-PEG-DSPE using PEG 600, (c) of FIG. 12 shows the results for NK cells bound to HA-PEG-DSPE using PEG 2000 (7%), (d) of FIG. 12 shows the results for NK cells bound to HA-PEG-DSPE using PEG 2000 (18%), (e) of FIG. 12 shows the results for NK cells bound to HA-PEG-DSPE using PEG 2000 (26%), and (f) of FIG. 12 shows the results for NK cells bound to HA-PEG-DSPE using PEG 5000.

[0114] As can be seen in Figures 12 and 13, it can be confirmed that the targeting ability for triple-negative breast cancer cells (MDA-MB-231) is significantly higher in the case of HA-PEG-DSPE using PEG 2000 (18%) compared to other HA-PEG-DSPEs.

[0115] FIG. 14 is a drawing for confirming the targeting ability of NK cells bound with polymer compounds according to experimental examples of the present invention toward liver cancer cells, and FIG. 15 is a graph quantifying the E:T cluster ratio measured in FIG. 14.

[0116] Referring to FIGS. 14 and 15, E:T clusters were detected using the method according to Experimental Example 4 described above, but hepatoma cells (HepG2) with little CD44 expression were used as target cells. More specifically, (a) of FIG. 14 shows the results for NK cells not bound to HA-PEG-DSPE, (b) of FIG. 14 shows the results for NK cells bound to HA-PEG-DSPE using PEG 600, (c) of FIG. 14 shows the results for NK cells bound to HA-PEG-DSPE using PEG 2000 (7%), (d) of FIG. 14 shows the results for NK cells bound to HA-PEG-DSPE using PEG 2000 (18%), (e) of FIG. 14 shows the results for NK cells bound to HA-PEG-DSPE using PEG 2000 (26%), and (f) of FIG. 14 shows the results for NK cells bound to HA-PEG-DSPE using PEG 5000.

[0117] As can be seen in Figures 14 and 15, all of the different HA-PEG-DSPEs have virtually no targeting ability against liver cancer cells. In other words, HA-PEG-DSPE selectively recognizes cancer cells overexpressing CD44.

[0118] FIG. 16 is a drawing for confirming the targeting ability of NK cells to which polymer compounds are bound according to experimental examples of the present invention toward fibroblasts, and FIG. 17 is a graph quantifying the E:T cluster ratio measured in FIG. 16.

[0119] Referring to FIGS. 16 and 17, the E:T cluster was detected using the method according to Experimental Example 4 described above, but fibroblasts, which are normal cells, were used as target cells. More specifically, (a) of FIG. 16 shows the results for NK cells not bound to HA-PEG-DSPE, (b) of FIG. 16 shows the results for NK cells bound to HA-PEG-DSPE using PEG 600, (c) of FIG. 16 shows the results for NK cells bound to HA-PEG-DSPE using PEG 2000 (7%), (d) of FIG. 16 shows the results for NK cells bound to HA-PEG-DSPE using PEG 2000 (18%), (e) of FIG. 16 shows the results for NK cells bound to HA-PEG-DSPE using PEG 2000 (26%), and (f) of FIG. 16 shows the results for NK cells bound to HA-PEG-DSPE using PEG 5000.

[0120] As can be seen in Figures 16 and 17, all of the different HA-PEG-DSPEs have virtually no targeting ability toward fibroblasts. This again demonstrates that HA-PEG-DSPE selectively recognizes CD44-overexpressing cancer cells.

[0121]

[0122] Experimental Example 5: Analysis of the Cancer Cell Killing Ability of NK Cells Combined with Polymer Compounds

[0123] After dissolving the HA-PEG-DSPE polymer compound in MEM alpha at a concentration of 1.0 mg / mL, 6 x 10 5Canine 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.

[0124] Target cell 6 x 10 4 Dogs were stained with 60 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 a ratio of 10:1 and cultured at 37°C for 4 hours. The supernatant was obtained and the fluorescence intensity was measured at 480 / 535 mm.

[0125] Figure 18 is a drawing for explaining the killing ability of NK cells bound with polymer compounds according to experimental examples of the present invention against triple-negative breast cancer cells.

[0126] Referring to Fig. 18, the lysis ability (Specific cell lysis, %) of target cells was measured by the method according to Experimental Example 5 described above, and triple-negative breast cancer cells (MDA-MB-231) with overexpressed CD44 were used as target cells. In addition, HA-PEG-DSPE with various polymer compounds such as PEG 600, PEG 2000 (7%), PEG 2000 (18%), PEG 2000 (26%), and PEG 5000 was used, and the lysis ability of NK cells not bound to HA-PEG-DSPE was also measured. Fig. 18 (a) shows a schematic diagram of the process in which NK cells bound to polymer compounds kill triple-negative breast cancer cells (Breast cancer), and Fig. 18 (b) quantitatively shows the solubility for triple-negative breast cancer cells (MDA-MB-231).

[0127] As can be seen in Fig. 18, NK cells bound to HA-PEG-DSPE show improved solubility in triple-negative breast cancer cells (Breast cancer) compared to NK cells not bound to HA-PEG-DSPE. In particular, in the case of HA-PEG-DSPE using PEG 2000 (18%), it can be confirmed that the solubility for triple-negative breast cancer cells (MDA-MB-231) is significantly improved.

[0128] Figure 19 is a drawing for explaining the killing ability of NK cells to which polymer compounds are combined against liver cancer cells according to experimental examples of the present invention.

[0129] Referring to Fig. 19, the lysis ability (Specific cell lysis, %) of target cells was measured by the method according to Experimental Example 5 described above, and liver cancer cells (HepG2) with little CD44 expression were used as target cells. In addition, HA-PEG-DSPE with various polymer compounds such as PEG 600, PEG 2000 (7%), PEG 2000 (18%), PEG 2000 (26%), and PEG 5000 was used, and the lysis ability of NK cells not bound to HA-PEG-DSPE was also measured. Fig. 19 (a) shows a schematic diagram of the process in which NK cells bound to polymer compounds kill liver cancer cells, and Fig. 19 (b) quantitatively shows the solubility for liver cancer cells (HepG2).

[0130] As can be seen in Figure 19, NK cells bound to HA-PEG-DSPE show virtually no difference in killing ability against liver cancer cells compared to NK cells not bound to HA-PEG-DSPE. In other words, NK cells bound to HA-PEG-DSPE selectively kill cancer cells overexpressing CD44.

[0131] Figure 20 is a drawing for explaining the killing ability of NK cells to which polymer compounds are bound according to experimental examples of the present invention against fibroblasts.

[0132] Referring to Fig. 20, the lysis ability (Specific cell lysis, %) of target cells was measured by the method according to Experimental Example 5 described above, but normal fibroblast cells were used as target cells. In addition, HA-PEG-DSPE containing various polymer compounds such as PEG 600, PEG 2000 (7%), PEG 2000 (18%), PEG 2000 (26%), and PEG 5000 was used, and the lysis ability of NK cells not bound to HA-PEG-DSPE was also measured. Fig. 20 (a) shows a schematic diagram of the process in which NK cells bound to polymer compounds react with fibroblast cells (Normal cells), and Fig. 20 (b) quantitatively shows the solubility for fibroblasts.

[0133] As can be seen in Figure 20, both NK cells bound to HA-PEG-DSPE and NK cells not bound to HA-PEG-DSPE do not lyse normal fibroblast cells. In other words, it can be seen once again that NK cells bound to HA-PEG-DSPE selectively kill cancer cells with overexpressed CD44.

[0134]

[0135] 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.

[0136] 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. A polymer compound comprising a polymer that improves the killing efficiency of solid cancer cells containing CD44 through natural killer cells by controlling the length of the linker and the content of the hydrophobic moiety.

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

3. In paragraph 2, The above linker is a polymer compound having a length of more than 0.6 k and less than 5 k.

4. In paragraph 1, 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).

5. In paragraph 4, A polymer compound comprising a degree of substitution (DS), defined as a value calculated by NMR analysis as the amount of the hydrophobic moiety linked to the cancer cell recognition moiety, of more than 7% and less than 26%.

6. In paragraph 4, A polymer compound comprising the hydrophobic moiety that is bound to the surface of a natural killer cell through a lipid-mediated hydrophobic action.

7. In paragraph 1, The cancer cell recognition moiety is a polymer compound containing hyaluronic acid.

8. In paragraph 7, The cancer cell recognition moiety is a polymer compound that selectively recognizes solid cancer cells containing CD44.

9. 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.

10. In paragraph 1, A polymer compound comprising the cancer cell recognition moiety that recognizes pancreatic cancer cells (MIA PaCa-2), triple-negative breast cancer cells (MDA-MB-231), and colon cancer cells (HCT-116).

11. In paragraph 1, A polymer compound that inhibits the metastasis of solid cancer cells containing CD44 to other organs.

12. Step of preparing hyaluronic acid; and A step of preparing a base solution by adding and mixing the hyaluronic acid, EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide), and NHS (N-hydroxy succinimide) into a solvent; and A step of adding a compound having a hydrophobic moiety bonded to one end of a linker and an amine group bonded to the other end into the base solution and stirring the compound, thereby producing a polymer compound having the hydrophobic moiety bonded to one end of the linker and the hyaluronic acid bonded to the other end, A method for producing a polymer compound, comprising controlling the length of the linker and the content of the hydrophobic moiety in the polymer compound, thereby improving the killing efficiency of solid cancer cells including CD44 through the natural killer cells.

13. In paragraph 12, A method for producing a polymer compound comprising polyethylene glycol (PEG) having a length of more than 0.6 k and less than 5 k, wherein the linker is 14. In paragraph 12, A method for producing a polymer compound, wherein the degree of substitution (DS), defined as a value calculated by NMR analysis of the amount of the hydrophobic moiety linked to the cancer cell recognition moiety, is greater than 7% and less than 26%.

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