Polymeric compound for surface modification of natural killer cells to inhibit metastasis of pancreatic cancer to other organs

Abstract

Provided is a polymer compound capable of selectively recognizing pancreatic cancer cells and inhibiting the metastasis of pancreatic cancer cells to other organs. The polymer compound may comprise: a hydrophobic moiety binding to natural killer cells; a cancer cell-recognition moiety selectively recognizing pancreatic cancer cells; and a linker connecting the hydrophobic moiety and the cancer cell-recognition moiety.

Description

Polymer compounds for surface modification of natural killer cells to inhibit the metastasis of pancreatic cancer to other organs

[0001] The present invention relates to a polymer compound that can bind to the surface of natural killer cells and enhance the anticancer function of the cells, and by injecting the polymer compound into the body, selectively recognizes and kills pancreatic cancer cells, and also suppresses the metastasis of pancreatic cancer cells to other organs.

[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 the cell membrane of natural killer cells.

[0008] Another technical problem to be solved by the present invention is to provide a polymer compound capable of selectively recognizing pancreatic cancer cells on the surface of natural killer cells.

[0009] Another technical challenge that the present invention seeks to solve is to provide a polymer compound capable of inhibiting the metastasis of pancreatic cancer cells to other organs to the surface of natural killer cells.

[0010] Another technical problem that the present invention seeks to solve is to provide a polymer compound capable of modifying the surface of natural killer cells without genetic manipulation.

[0011] Another technical challenge to be solved by the present invention is to provide a polymer compound that can be naturally removed from natural killer cells without external physical or chemical intervention.

[0012] The technical problems to be solved by the present invention are not limited to those described above.

[0013] To solve the above-described technical problems, the present invention provides a polymer compound.

[0014] 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 pancreatic cancer cells, and a cell to which the natural killer cells and the polymer compound are bound can suppress metastasis of pancreatic cancer cells to other organs.

[0015] In one embodiment, natural killer cells and cells combined with the polymer compound can inhibit the metastasis of pancreatic cancer cells to the lungs.

[0016] In one embodiment, the cancer cell recognition moiety may comprise hyaluronic acid.

[0017] 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.

[0018] In one embodiment, the hydrophobic moiety may comprise a lipid and be bound to the surface of the natural killer cell through a hydrophobic interaction mediated by the lipid.

[0019] 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).

[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] In one embodiment, the polymer compound bound to natural killer cells may be removed from the natural killer cells within 36 hours.

[0023] In one embodiment, the polymer compound bound to natural killer cells may be naturally removed from the natural killer cells without external physical and chemical intervention.

[0024] In one embodiment, the cancer cell recognition moiety can recognize a solid cancer cell comprising CD44.

[0025]

[0026] To solve the technical problems described above, the present invention provides a method for producing a polymer compound.

[0027] According to one embodiment, the method for preparing the polymer compound includes the steps of preparing hyaluronic acid, thiolating the hyaluronic acid, and reacting the thiolated hyaluronic acid with a compound having a hydrophobic moiety bonded to one terminal of a linker and a maleimide bonded to the other terminal, 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 recognizing pancreatic cancer cells by the hyaluronic acid and recognizing natural killer cells by the hydrophobic moiety.

[0028] According to one embodiment, the polymer compound may be prepared by a Michael reaction of the thiolated hyaluronic acid and a compound having a hydrophobic moiety attached to one terminal and a maleimide attached to the other terminal.

[0029] According to one embodiment, the step of thiolating the hyaluronic acid may include the step of conjugating the hyaluronic acid with PDPH (3-(2-pyridyldithio)propionyl hydrazide), and the step of adding 2-mercaptoethanol to the hyaluronic acid to which the PDPH is conjugated.

[0030] A polymer compound comprising a hydrophobic moiety (lipid), a cancer cell recognition moiety (hyaluronic 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) and selectively recognize pancreatic cancer cells among various cancer cells. In addition, cells in which natural killer cells and the polymer compound are combined can suppress the metastasis of pancreatic cancer cells to other organs (lungs).

[0031] 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.

[0032] FIG. 2 and FIG. 3 are drawings for explaining the cancer killing process through natural killer cells combined with a polymer compound according to an embodiment of the present invention.

[0033] FIG. 4 is a drawing for explaining a process in which a polymer compound according to an embodiment of the present invention is removed from natural killer cells after binding to the cells.

[0034] FIG. 5 and FIG. 6 are drawings for explaining a method for manufacturing a polymer compound according to an embodiment of the present invention.

[0035] Figure 7 is a diagram comparing HANK cells combined with AF-HA-SH and HANK cells combined with AF-HA-PEG-Lipid.

[0036] Figures 8 and 9 are drawings illustrating the effect of HA-PEG-Lipid on natural killer cells.

[0037] Figures 10 and 11 are drawings for confirming the target recognition ability of HANK cells.

[0038] Figure 12 is a graph comparing the amount of cytotoxic granules and cytokines secreted by NK cells and HANK cells against target cells.

[0039] Figure 13 is a graph comparing the lytic ability of NK cells and HANK cells against cancer cells.

[0040] Figures 14 and 15 are diagrams comparing the anticancer efficacy of NK cells and HANK cells against tumor spheroids.

[0041] Figure 16 is a diagram for confirming the retention time of HA-PEG-Lipid bound to NK cells.

[0042] Figure 17 is a diagram comparing the cytokine secretion ability of NK cells and restored NK cells.

[0043] Figure 18 is a diagram comparing the cancer cell lysis ability of NK cells and restored NK cells.

[0044] Figure 19 is a diagram showing the experimental process to confirm the anticancer efficacy of HANK cells against pancreatic cancer.

[0045] Figures 20 and 21 are drawings showing changes in tumor volume, weight, and size as the pancreatic cancer experiment progresses.

[0046] Figure 22 is a diagram comparing the tumor infiltration capabilities of NK cells and HANK cells.

[0047] Figure 23 is a diagram for confirming the biodistribution of NK cells and HANK cells injected into experimental mice.

[0048] Figures 24 and 25 are diagrams comparing the cytotoxic granule diffusion capabilities of NK cells and HANK cells.

[0049] Figures 26 and 27 are diagrams comparing the cytokine diffusion capabilities of NK cells and HANK cells.

[0050] Figures 28 and 29 are diagrams comparing the tumor necrosis abilities of PBS, NK cells, Gemcitabine, and HANK cells.

[0051] Figures 30 and 31 are diagrams comparing the apoptotic areas of tumor masses through PBS, NK cells, Gemcitabine, and HANK cells.

[0052] Figures 32 and 33 are diagrams comparing the cell proliferation inhibition abilities of PBS, NK cells, Gemcitabine, and HANK cells.

[0053] Figure 34 is a diagram showing the experimental process for evaluating the anti-metastatic effect of HANK cells.

[0054] Figure 35 is a drawing confirming the formation of metastatic colonies in lung tissue.

[0055] Figure 36 is a drawing for confirming the alveolar structure in lung tissue.

[0056] Figure 37 is a drawing illustrating the results of introducing human leukocyte antigen into lung tissue.

[0057] Figure 38 is a diagram illustrating the results of introducing an angiogenesis marker into lung tissue.

[0058] Figure 39 is a diagram quantifying the number of metastatic foci in the lungs of experimental mice injected with PBS, NK cells, Gemcitabine, and HANK cells.

[0059] Figure 40 is a drawing quantifying the area of ​​hLA-positive cells confirmed in the lungs of experimental mice injected with PBS, NK cells, Gemcitabine, and HANK cells.

[0060] Figure 41 is a diagram quantifying the area of ​​vWF-positive cells identified in the lungs of experimental mice injected with PBS, NK cells, Gemcitabine, and HANK cells.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066]

[0067] Polymer compound according to an embodiment

[0068] 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, FIGS. 2 and 3 are drawings for explaining a cancer killing process through natural killer cells bound to a polymer compound according to an embodiment of the present invention, and FIG. 4 is a drawing for explaining a process in which a polymer compound according to an embodiment of the present invention is removed from natural killer cells after binding to the natural killer cells.

[0069] 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.

[0070] 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.

[0071] 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).

[0072] 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.

[0073] 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.

[0074] Referring to FIGS. 2 and 3, the process in which the polymer compound bound to natural killer cells via the hydrophobic moiety kills cancer cells is illustrated. In FIGS. 2 and 3, the cell in which the polymer compound and natural killer cells are bound is expressed as a HANK cell. In addition, (a) of FIGS. 2 and 3 illustrates the recognition step of the HANK cell, (b) illustrates the activation step, and (c) illustrates the cancer cell killing step.

[0075] Specifically, the HANK cell can recognize a cancer cell containing CD44 through the cancer cell recognition moiety (hyaluronic acid, HA) of the polymer compound, and then bind to the cancer cell through the cancer cell recognition moiety (hyaluronic acid, HA). Thereafter, an activation process in which cytotoxic granules and cytokines are secreted from 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.

[0076] Referring to FIG. 4, the polymer compound bound to natural killer cells can be naturally removed from the natural killer cells within 36 hours without external physical or chemical intervention. Contrary to the above, if the polymer compound bound to natural killer cells is not removed, various problems such as cytokine release syndrome, neurotoxicity, off-tumor effects, and acute respiratory distress syndrome may occur.

[0077] To address this issue, traditional methods have involved introducing and removing suicide genes. However, this necessitates additional drug therapy to activate the suicide gene, which presents additional challenges. However, the polymer compound can be naturally eliminated from natural killer cells without external physical or chemical intervention, making it possible to readily resolve the aforementioned issues.

[0078] Additionally, the binding and removal of the polymer compound to natural killer cells may not impair the inherent function of the natural killer cells. That is, even after the polymer compound is removed from the natural killer cells, the natural killer cells can maintain their inherent function.

[0079] As a result, the polymer compound composed of a hydrophobic moiety (lipid), a cancer cell recognition moiety (hyaluronic 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 of the hydrophobic moiety (lipid) without genetic manipulation, and can selectively recognize solid cancer cells (e.g., pancreatic cancer cells, triple-negative breast cancer cells, colon cancer cells, etc.) in which CD44 is overexpressed among various cancer cells. In addition, since the polymer compound can be naturally removed without external physical and chemical intervention within a predetermined period of time (within 36 hours), problems (e.g., cytokine release syndrome, neurotoxicity, extratumoral effects, acute respiratory distress syndrome, etc.) caused by long-term surface modification of natural killer cells can be easily solved.

[0080]

[0081] Method for producing a polymer compound according to an embodiment

[0082] FIG. 5 and FIG. 6 are drawings for explaining a method for manufacturing a polymer compound according to an embodiment of the present invention.

[0083] Referring to FIGS. 5 and 6, 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 thiolating the hyaluronic acid (S21, S22), and a step of producing the polymer compound using the thiolated hyaluronic acid (S30). Each step is described in detail below.

[0084] In the above step S10, hyaluronic acid (HA) may be prepared. According to one embodiment, in the above step S10, carboxyl groups may be activated by adding EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) and NHS (N-hydroxy succinimide) to a solution in which a hyaluronic acid solution and phosphate-buffered saline (PBS) are mixed.

[0085] In the step S21, the hyaluronic acid can be conjugated with PDPH (3-(2-pyridyldithio)propionyl hydrazide). According to one embodiment, in the step S21, the hyaluronic acid prepared in the step S10 can be conjugated with PDPH by mixing it with PDPH (3-(2-pyridyldithio)propionyl hydrazide), DMAP (4-dimethyl amino pyridine), and DMF (dimethyl formamide). The compound in which the hyaluronic acid is conjugated with PDPH is defined as HA-PDPH.

[0086] In the above step S22, thiolated hyaluronic acid (Thiolated-HA, HA-SA) can be manufactured by adding 2-mercaptoethanol to the hyaluronic acid (HA-PDPH) to which the PDPH is conjugated.

[0087] Finally, in the above step S30, the thiolated hyaluronic acid (HA-SH) can undergo a Michael reaction with a compound in which a hydrophobic moiety (Lipid) is bound to one end of a linker (PEG) and a maleimide is bound to the other end. Accordingly, the polymer compound (HA-PEG-Lipid) in which the hydrophobic moiety (Lipid) is bound to one end of the linker (PEG) and the hyaluronic acid (HA) is bound to the other end can be produced.

[0088]

[0089] Hereinafter, polymer compounds according to embodiments of the present invention are described in more detail through specific experimental examples.

[0090] Preparation of polymer compound (HA-PEG-Lipid) according to experimental example

[0091] After stirring a hyaluronic acid (HA) solution (Mw 60k, 10 mg / mL, 1 equivalent, LifeCore Biomedical) and phosphate-buffered saline (PBS, pH 7.4) for 30 minutes, excess EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, Sigma-Aldrich) and NHS (N-hydroxy succinimide, Sigma-Aldrich) were added to the stirred solution and reacted at room temperature for 3 hours to activate the carboxyl group.

[0092] Afterwards, 30 equivalents of PDPH (3-(2-pyridyldithio)propionyl hydrazide, Sigma-Aldrich), 2 mg of DMAP (4-dimethyl amino pyridine, Sigma-Aldrich), and dimethyl formamide (DMF, Sigma-Aldrich) were further added and reacted at room temperature for 72 hours to obtain an HA-PDPH product in which PDPH was conjugated to HA. In addition, to remove unconjugated PDPH and EDC / NHS from the obtained HA-PDPH product, it was dialyzed against distilled water for 3 days (MWCO 2 kDa) and lyophilized.

[0093] After dissolving 30 mg of HA-PDPH in 10 mL of PBS, 2-mercaptoethanol was added at 0.2 wt% of the initial concentration and stirred at room temperature for 12 h. The mixture produced through stirring was dialyzed against distilled water (MWCO 2 kDa) for 3 days and then lyophilized to produce thiolated HA (HA-SH).

[0094] HA-SH (50 mg) was dissolved in 10 mL of PBS to prepare a homogeneous HA-SH solution. Then, a solution of Lipid-PEG-Maleimide (30 eq.) dissolved in 10 mL of DMF was added to the prepared solution, and the mixture was stirred at room temperature for 24 hours. The stirred mixture was dialyzed against distilled water for 3 days (MWCO 12-14 kD) and lyophilized to prepare the final product, HA-PEG-Lipid. More specifically, DSPE (1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine) was used as the lipid. In addition, the above-described manufacturing method is illustrated in Fig. 6.

[0095]

[0096] HANK cell manufacturing according to experimental examples

[0097] Dissolve the polymer compound (HA-PEG-Lipid) according to the above experimental example in MEM alpha, and add 5 x 10 5 NK-92mi natural killer cells were evenly mixed with 100 μL of solution and reacted at room temperature for 30 minutes to produce HANK cells in which the polymer compound (HA-PEG-Lipid) according to the above experimental example was bound to the natural killer cells.

[0098]

[0099] Experimental Example 1: Characterization of HA-PEG-Lipid and HANK cells

[0100] AF-HA-PEG-Lipid was prepared by dissolving 20 mg of HA-PEG-Lipid in 2 mL of PBS and adding 100 nmol of Alex Flour 488 hydrazide (fluorescent dye). In addition, AF-HA-SH was prepared by dissolving 20 mg of HA-SH in 2 mL of PBS and adding 100 nmol of Alex Flour 488 hydrazide (fluorescent dye). Afterwards, AF-HA-PEG-Lipid and AF-HA-SH were combined with natural killer cells to prepare HANK cells.

[0101] Figure 7 is a diagram comparing HANK cells combined with AF-HA-SH and HANK cells combined with AF-HA-PEG-Lipid.

[0102] Referring to Figure 7, optical and fluorescence microscopy images for HANK cells combined with AF-HA-SH and HANK cells combined with AF-HA-PEG-Lipid, respectively, the results of intensity profile analysis of green fluorescence at the red line of the microscopy images, and the mean fluorescence intensity (MFI, x 10) detected by flow cytometry. 4 ) is indicated.

[0103] As can be confirmed by the fluorescence microscope image in Fig. 7, AF-HA-SH was internalized into natural killer cells, whereas AF-HA-PEG-Lipid was uniformly bound to the surface of natural killer cells. In addition, as can be confirmed by the mean fluorescence intensity detected by flow cytometry, in the case of HANK cells bound to AF-HA-PEG-Lipid, the MFI value was saturated at a concentration of 1 mg / mL, whereas in the case of HANK cells bound to AF-HA-SH, it was confirmed that the MFI was low, below 2.5, even at a concentration of 2.5 mg / mL. In other words, in the case of HA-SH, it was confirmed that it was not fixed to the surface of natural killer cells because it did not have a moiety that recognizes natural killer cells, whereas in the case of HA-PEG-Lipid, it was confirmed that it was easily bound to the surface of natural killer cells due to the lipid.

[0104] Figures 8 and 9 are drawings illustrating the effect of HA-PEG-Lipid on natural killer cells.

[0105] Referring to Fig. 8 (a), the survival rate and proliferation rate of HANK cells combined with natural killer cells at 0 to 1 mg / mL of HA-PEG-Lipid are shown. As can be seen in Fig. 8 (a), it can be confirmed that the survival rate and proliferation rate are maintained substantially constant despite the combination of HA-PEG-Lipid with natural killer cells.

[0106] Referring to Fig. 8 (b), the result of applying LPS as a proinflammatory antigen signal to HANK cells is shown. LPS binds to TLR4 of immune cells and induces the secretion of inflammatory cytokines such as IFN-Y. As can be seen in Fig. 8 (b), both NK cells and HANK cells treated with LPS were confirmed to exhibit similar levels of IFN-Y secretion. In other words, Fig. 8 (a) and (b) show that HA-PEG-Lipid bound to the surface of natural killer cells (NK cells) does not interfere with the antigen recognition and subsequent cytokine secretion process after intracellular signal transduction.

[0107] Figure 9 (a) shows the results of analyzing MFI using flow cytometry after detecting TRAIL present on the surface of HANK cells using APC-binding TRAIL antibody, and Figure 9 (b) shows the results of analyzing MFI using flow cytometry after detecting FasL present on the surface of HANK cells using APC-binding FasL antibody.

[0108] FasL and TRAIL are major surface ligands of natural killer cells that can recognize target cancer cells and induce direct apoptosis. As shown in (a) and (b) of Fig. 9, HANK cells were confirmed to exhibit similar MFI to antibody-treated NK cells. In other words, HA-PEG-Lipid does not interfere with the availability of surface ligands of natural killer cells (NK cells).

[0109]

[0110] Experimental Example 2: In vitro anticancer effect mediated by immune synapses

[0111] Figures 10 and 11 are drawings for confirming the target recognition ability of HANK cells.

[0112] Referring to Figures 10 and 11, the target recognition ability of NK cells for target cells (NK cell + target cell), CD44 of HANK cells Block Target recognition ability for target cells (HANK cell + CD44 Block Target cell), and the target recognition ability of HANK cells for the target cell (HANK cell + Target cell) are measured and displayed. Specifically, CD44 positive cancer cell lines (MIA PaCa-2, MDA-MB-231, and HCT-116) were used as target cells, and CD44 Block Target cells were used as CD44-blocked cells pretreated with 1 mg / mL of hyaluronic acid (HA) for 2 hours as described above. In addition, target recognition ability was evaluated by quantification of effector / target (E / T) clusters.

[0113] As can be seen in Figures 10 and 11, the target recognition ability of NK cells for target cells (NK cell + target cell) and CD44 of HANK cells Block Target recognition ability for target cells (HANK cell + CD44 Block The target recognition ability of HANK cells for the target cell (HANK cell + Target cell) was 4.77% and 4.15%, respectively, while the target recognition ability of HANK cells for the target cell (HANK cell + Target cell) was 19.79%, which was significantly high. In other words, it can be seen that HANK cells have a high target recognition ability for CD44.

[0114] Figure 12 is a graph comparing the amount of cytotoxic granules and cytokines secreted by NK cells and HANK cells against target cells.

[0115] Referring to Figure 12, the secretion amounts of cytotoxic granules (Granzyme B, Perforin) and cytokines (IFN-Y, TNF-α) released from NK cells after co-culturing target cells and NK cells are measured and shown. In addition, the secretion amounts of cytotoxic granules (Granzyme B, Perforin) and cytokines (IFN-Y, TNF-α) released from HANK cells after co-culturing target cells and HANK cells are measured and shown. MIA PaCa-2 was used as the target cell.

[0116] As can be seen in Figure 12, HANCK cells can be confirmed to actively secrete cytotoxic granules (Granzyme B, Perforin) and cytokines (IFN-Υ, TNF-α) compared to NK cells. That is, it can be seen that the secretion of cytotoxic granules (Granzyme B, Perforin) and cytokines (IFN-Υ, TNF-α) of NK cells is promoted by HA-PEG-Lipid.

[0117] Figure 13 is a graph comparing the lytic ability of NK cells and HANK cells against cancer cells.

[0118] Referring to Fig. 13, NK cells and HANK cells are cultured with cancer cells (MIA PaCa-2, MDA-MB-231, HCT-116, and Fibroblast), respectively, and the cancer cell lysis rate (specific cell lysis, %) is measured and displayed. The E:T Ratio shown in Fig. 13 refers to the culture ratio of NK cells or HANK cells:cancer cells.

[0119] As shown in Fig. 13, HANK cells showed significantly higher lysis rates for CD44-overexpressing MIA PaCa-2, MDA-MB-231, and HCT-116 compared to NK cells. In other words, it can be seen that the target recognition ability of NK cells for MIA PaCa-2, MDA-MB-231, and HCT-116 was enhanced by HA-PEG-Lipid. In addition, it was confirmed that HANK cells could not lyse fibroblasts lacking CD44. In other words, it can be seen that HANK cells selectively recognize cancer cells overexpressing CD44 among various cancer cells.

[0120] Figures 14 and 15 are diagrams comparing the anticancer efficacy of NK cells and HANK cells against tumor spheroids.

[0121] Referring to Figures 14 and 15, tumor spheroids were cultured with NK cells and HANK cells, respectively, and then the anticancer efficacy was measured by measuring fluorescence intensity. The Control shown in Figures 14 and 15 refers to tumor spheroids.

[0122] As can be seen in Fig. 14, the tumor spheroids cultured with HANK cells had significantly deformed morphology, and as can be seen in Fig. 15, the tumor spheroids cultured with HANK cells had significantly reduced fluorescence intensity compared to the tumor spheroids cultured with NK cells.

[0123]

[0124] Experimental Example 3: Recovery of HANK cells into NK cells

[0125] Figure 16 is a diagram for confirming the retention time of HA-PEG-Lipid bound to NK cells.

[0126] Referring to Figure 16, the retention time of HA-PEG-Lipid bound to NK cells is confirmed and shown through fluorescence intensity analysis. As can be confirmed in Figure 16, HA-PEG-Lipid bound to NK cells can be confirmed to be removed from NK cells within 36 hours without external physical or chemical intervention.

[0127] Figure 17 is a diagram comparing the cytokine secretion ability of NK cells and restored NK cells.

[0128] Referring to Figure 17, the cytokine (IFN-Y, pg / mL) secretion ability of NK cells and restored NK cells is compared and shown. Restored NK cells refer to a state in which HA-PEG-Lipid is removed from NK cells after HA-PEG-Lipid is bound to NK cells to form HANK cells. In addition, Control in Figure 17 represents a normal state, and LPS represents the result of applying LPS as a proinflammatory antigen signal. LPS binds to TLR4 of immune cells to induce the secretion of inflammatory cytokines such as IFN-Y.

[0129] As can be seen in Figure 17, there was no substantial difference in the cytokine secretion ability of NK cells and restored NK cells. In other words, it can be seen that the binding and removal of HA-PEG-Lipid did not affect the cytokine secretion ability of NK cells.

[0130] Figure 18 is a diagram comparing the cancer cell lysis ability of NK cells and restored NK cells.

[0131] Referring to Figure 18, the cancer cell lytic ability of NK cells and restored NK cells is compared and shown. MIA PaCa-2, MDA-MB-231, HCT-116 with overexpression of CD44 and Fibroblasts without CD44 were used as cancer cells.

[0132] As shown in Figure 18, NK cells and restored NK cells showed no substantial difference in their lytic ability against MIA PaCa-2, MDA-MB-231, and HCT-116 cancer cells. This indicates that binding and removal of HA-PEG-Lipid did not affect the cancer cell lytic ability of NK cells. In addition, restored NK cells still lacked the lytic ability against CD44-deficient Fibroblasts.

[0133]

[0134] Experimental Example 4: Evaluation of the anticancer efficacy of HANK cells against pancreatic cancer.

[0135] Figure 19 is a diagram showing the experimental process to confirm the anticancer efficacy of HANK cells against pancreatic cancer.

[0136] Referring to Figure 19, after forming a tumor by injecting MIA PaCa-2, a pancreatic cancer cell, into experimental mice, PBS (250 μL), NK cells (10 7 The anticancer efficacy of each was evaluated by administering Gemcitabine (120 mg / kg), HANK cells (107 cells), and PBS as a control. Gemcitabine, a widely known drug for the treatment of pancreatic cancer, was used to confirm the efficacy of HANK cells.

[0137] Figures 20 and 21 are drawings showing changes in tumor volume, weight, and size as the pancreatic cancer experiment progresses.

[0138] Referring to Figures 20 and 21, the volume, weight, and size changes of tumors of experimental mice administered PBS, NK cells, Gemcitabine, and HANK cells are shown. As can be seen in Figures 20 and 21, the tumors of experimental mice administered HANK cells are confirmed to be significantly reduced in volume, weight, and size.

[0139] Figure 22 is a diagram comparing the tumor infiltration capabilities of NK cells and HANK cells.

[0140] Referring to Figure 22, the results using human-specific anti-CD56 are shown to confirm the tumor-infiltrating ability of NK cells and HANK cells. As can be seen in Figure 22, NK cells are mainly accumulated at the tumor margin, whereas HANK cells are distributed throughout the entire tumor area. In other words, HANK cells have a significantly higher tumor-infiltrating ability compared to NK cells.

[0141] Figure 23 is a diagram for confirming the biodistribution of NK cells and HANK cells injected into experimental mice.

[0142] Referring to Figure 23, the results of confirming biodistribution after injecting NK cells and HANK cells into experimental mice are shown. To confirm biodistribution, human specific anti-CD56 was used.

[0143] As shown in Figure 23, only trace amounts of NK cells and HANK cells were detected in the heart, kidney, and liver, whereas large amounts of NK cells and HANK cells were detected in the lungs and tumors. Accordingly, it can be seen that the metastasis of pancreatic cancer to the lungs can be effectively prevented.

[0144] Figures 24 and 25 are diagrams comparing the cytotoxic granule diffusion capabilities of NK cells and HANK cells.

[0145] Referring to Figures 24 and 25, the results of confirming the intratumoral distribution of Granzyme B, a cytotoxic granule secreted from NK cells and HANK cells, are shown. To confirm the intratumoral distribution of Granzyme B, human specific anti-CD56 was used.

[0146] As can be seen in Fig. 24, Granzyme B secreted from NK cells was mainly distributed in the peripheral area of ​​the tumor, whereas Granzyme B secreted from HANK cells was distributed throughout the entire tumor area. In addition, as can be seen in Fig. 25, the Granzyme B+ / CD56+ cell area ratio (are %) was significantly higher in HANK cells compared to NK cells. Therefore, it can be seen from Figs. 24 and 25 that HA-PEG-Lipid promotes the secretion of cytotoxic granules by NK cells.

[0147] Figures 26 and 27 are diagrams comparing the cytokine diffusion capabilities of NK cells and HANK cells.

[0148] Referring to Figures 26 and 27, the results of confirming the intratumoral distribution of TNF-α, a cytokine secreted from NK cells and HANK cells, are shown. To confirm the intratumoral distribution of TNF-α, human specific anti-CD56 was used.

[0149] As can be seen in Fig. 26, TNF-α secreted from NK cells was mainly distributed in the peripheral area of ​​the tumor, whereas Granzyme B secreted from HANK cells was distributed throughout the entire tumor area. In addition, as can be seen in Fig. 27, the TNF-α / CD56+ cell area ratio (are %) was significantly higher in HANK cells compared to NK cells. Therefore, it can be seen from Figs. 26 and 27 that HA-PEG-Lipid promotes cytokine secretion of NK cells.

[0150] Figures 28 and 29 are diagrams comparing the tumor necrosis abilities of PBS, NK cells, Gemcitabine, and HANK cells.

[0151] Referring to Figure 28, the degree of tumor necrosis is shown by staining the subcutaneous tumors of experimental mice injected with PBS (a), NK cells (b), Gemcitabine (c), and HANK cells (d) with hematoxylin and eosin (H&E), and referring to Figure 29, the subcutaneous tumor necrosis area (Necrosis area, %) of experimental mice injected with PBS, NK cells, Gemcitabine, and HANK cells is shown by quantification.

[0152] As can be seen in Figures 28 and 29, HANK cells exhibit significantly improved tumor necrosis ability compared to NK cells and Gemcitabine.

[0153] Figures 30 and 31 are diagrams comparing the apoptotic areas of tumor masses through PBS, NK cells, Gemcitabine, and HANK cells.

[0154] Referring to Figure 30, the results are shown in which the apoptotic area of ​​the tumor mass is indicated by cleaved caspase3 in experimental mice injected with PBS (a), NK cells (b), Gemcitabine (c), and HANK cells (d), and referencing Figure 31, the results show the quantification of the positive area of ​​cleaved caspase3 in experimental mice injected with PBS (a), NK cells (b), Gemcitabine (c), and HANK cells (d).

[0155] As shown in Figure 30, HANK cells show a strong expression of cleaved caspase3 compared to NK cells and Gemcitabine. Furthermore, as shown in Figure 31, HANK cells show a significantly higher positive area ratio for cleaved caspase3 compared to NK cells and Gemcitabine.

[0156] Figures 32 and 33 are diagrams comparing the cell proliferation inhibition abilities of PBS, NK cells, Gemcitabine, and HANK cells.

[0157] Referring to Figure 32, the results of staining Ki67 in subcutaneous tumors of experimental mice injected with PBS (a), NK cells (b), Gemcitabine (c), and HANK cells (d) to determine the degree of cell proliferation are shown, and referring to Figure 33, the results of quantifying the proportion of Ki67 positive cells in experimental mice injected with PBS (a), NK cells (b), Gemcitabine (c), and HANK cells (d) are shown.

[0158] As shown in Figure 32, HANK cells exhibit significantly lower Ki67 expression compared to NK cells and Gemcitabine. Furthermore, as shown in Figure 33, HANK cells exhibit significantly lower cell proliferation areas compared to NK cells and Gemcitabine. In other words, HANK cells exhibit significantly higher cell proliferation inhibition capabilities compared to NK cells and Gemcitabine.

[0159]

[0160] Experimental Example 5: Evaluation of the Anti-Metastatic Effect of HANK Cells

[0161] Figure 34 is a diagram showing the experimental process for evaluating the anti-metastatic effect of HANK cells.

[0162] Referring to Figure 34, after forming a tumor by injecting MIA PaCa-2, a pancreatic cancer cell, into experimental mice, PBS (250 μL), NK cells (10 7 The anti-metastatic effects of HANK cells (107 cells) and Gemcitabine (120 mg / kg) were evaluated. PBS was used as a control, and Gemcitabine, a widely known drug for the treatment of pancreatic cancer, was used to confirm the efficacy of HANK cells.

[0163] Figure 35 is a drawing confirming the formation of metastatic colonies in lung tissue.

[0164] Referring to Figure 35, the results of confirming the formation of metastatic colonies in the lung tissue of experimental mice injected with PBS (a), NK cells (b), Gemcitabine (c), and HANK cells (d) are shown. As can be seen in Figure 35, metastatic colonies were formed in the injection of PBS (a), NK cells (b), and Gemcitabine (c), but no metastatic colonies were formed in the injection of HANK cells (d).

[0165] Figure 36 is a drawing for confirming the alveolar structure in lung tissue.

[0166] Referring to Figure 36, the results of staining the lung tissue of experimental mice injected with PBS (a), NK cells (b), Gemcitabine (c), and HANK cells (d) with hematoxylin and eosin (H&E) to confirm the alveolar structure are shown. As can be seen in Figure 36, multiple nonalveolar cell clusters are observed when PBS (a), NK cells (b), and Gemcitabine (c) are injected, whereas typical alveolar structures are observed when HANK cells (d) are injected.

[0167] Figure 37 is a drawing illustrating the results of introducing human leukocyte antigen into lung tissue.

[0168] Referring to Figure 37, the results of introducing human leukocyte antigen (hLA TP3) into the lungs of experimental mice injected with PBS (a), NK cells (b), Gemcitabine (c), and HANK cells (d) to indicate metastatic colonies in the lungs are shown. As can be seen in Figure 37, hLA-positive cell clusters were detected when PBS (a), NK cells (b), and Gemcitabine (c) were injected, whereas hLA-positive cell clusters were not detected when HANK cells (d) were injected.

[0169] Figure 38 is a diagram illustrating the results of introducing an angiogenesis marker into lung tissue.

[0170] Referring to Figure 38, the results of indicating lung metastatic colonies by introducing von Willebrand factor (vWF), an angiogenesis marker of vascularized tumors, into the lungs of experimental mice injected with PBS (a), NK cells (b), Gemcitabine (c), and HANK cells (d) are shown. As can be seen in Figure 38, vWF was strongly expressed when PBS (a), NK cells (b), and Gemcitabine (c) were injected, whereas vWF expression was barely observed when HANK cells (d) were injected.

[0171] Figure 39 is a diagram quantifying the number of metastatic foci in the lungs of experimental mice injected with PBS, NK cells, Gemcitabine, and HANK cells.

[0172] As can be seen in Figure 39, the number of metastatic foci in the lung was significantly lower when HANK cells were injected compared to when PBS, NK cells, and Gemcitabine were injected.

[0173] Figure 40 is a drawing quantifying the area of ​​hLA-positive cells confirmed in the lungs of experimental mice injected with PBS, NK cells, Gemcitabine, and HANK cells.

[0174] As can be seen in Figure 40, the area of ​​hLA-positive cells is significantly lower when HANK cells are injected compared to when PBS, NK cells, and Gemcitabine are injected.

[0175] Figure 41 is a diagram quantifying the area of ​​vWF-positive cells identified in the lungs of experimental mice injected with PBS, NK cells, Gemcitabine, and HANK cells.

[0176] As can be seen in Figure 41, the area of ​​vWF-positive cells is significantly lower when HANK cells are injected compared to when PBS, NK cells, and Gemcitabine are injected.

[0177] As a result, it was found that HANK cells conjugated with HA-PEG-Lipid to natural killer cells (NK cells) can be easily used for the treatment of cancers with overexpression of CD44, and can also significantly reduce metastasis to the lung.

[0178]

[0179] 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.

[0180] 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 above cancer cell recognition moiety selectively recognizes pancreatic cancer cells, A cell in which natural killer cells and the above polymer compound are combined is a polymer compound that inhibits the metastasis of pancreatic cancer cells to other organs.

2. In paragraph 1, A cell in which natural killer cells and the polymer compound are combined is a polymer compound that inhibits the metastasis of pancreatic cancer cells to the lungs.

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

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 the natural killer cell through a hydrophobic interaction 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 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.

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

9. In paragraph 1, A polymer compound comprising a polymer compound bound to natural killer cells, wherein the polymer compound is removed from the natural killer cells within 36 hours.

10. In paragraph 9, A polymer compound comprising a polymer compound bound to natural killer cells, wherein the polymer compound is naturally removed from natural killer cells without external physical and chemical intervention.

11. In paragraph 1, The cancer cell recognition moiety is a polymer compound that recognizes a solid cancer cell containing CD44.

12. Step of preparing hyaluronic acid; A step of thiolating the above hyaluronic acid; and A step of reacting the thiolated hyaluronic acid with a compound having a hydrophobic moiety bonded to one end of a linker and a maleimide bonded to the other end, 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 recognizing pancreatic cancer cells by the hyaluronic acid and recognizing natural killer cells by the hydrophobic moiety.

13. In paragraph 12, A method for producing a polymer compound, comprising producing the polymer compound by a Michael reaction of the thiolated hyaluronic acid and a compound having a hydrophobic moiety bonded to one terminal and a maleimide bonded to the other terminal.

14. In paragraph 12, The step of thiolating the above hyaluronic acid is: A step of conjugating the above hyaluronic acid with PDPH (3-(2-pyridyldithio)propionyl hydrazide); and A method for producing a polymer compound, comprising a step of adding 2-mercaptoethanol to the hyaluronic acid to which the PDPH is conjugated.

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