KR20240153888A - Polymeric compounds for surface modification of natural killer cells to inhibit metastasis of triple-negative breast cancer to other organs - Google Patents

Abstract

The present invention relates to a multifunctional polymer for surface modification of natural killer cells and an immuno-cancer technology (particularly for triple-negative breast cancer) utilizing the same to enhance the cancer cell recognition and cancer cell killing functions of natural killer cells.

Description

{Polymeric compounds for surface modification of natural killer cells to inhibit metastasis of triple-negative breast cancer to other organs}

The present invention relates to a polymer compound that can enhance the anticancer function of natural killer cells by attaching to the surface of the cells, by synthesizing and manufacturing the polymer compound, and by injecting the polymer compound into the body, thereby enhancing the recognition and killing effect of cancer cells, and thereby dramatically improving the effect of preventing or treating cancer.

Conventional methods of enhancing immune responses and inducing effector cell activation through administration of cytokine protein preparations such as interleukin or interferon have the potential to cause systemic immunotoxicity side effects.

In addition, it may be difficult to apply existing methods of enhancing immune response and inducing effector cell activation to solid tumors that are difficult for T cells to reach due to immunosuppressive microenvironmental factors, including T cell activation-reducing factors secreted from cancer tissues.

Cell coating technology can be introduced as a means to enhance the immune response and enhance the activity of effector cells as described above, but it has the following limitations.

Layer-by-layer self-assembly can be utilized as the above cell coating technology. Layer-by-layer self-assembly is a simple and versatile deposition process that has been widely applied to solve many problems such as biomolecule deposition, concentration, bioactivity, coating thickness, and release rate.

However, in order to utilize the layer-by-layer method, surface modification of cells is required, and there is a difficulty in the process for surface modification of cells because cationic or anionic substrate materials must be alternately reacted and laminated. In addition, due to the coating material covering the entire surface of the cell, there is a possibility of functional degradation of surface membrane proteins involved in signal transmission and cancer cell recognition. In addition, in cases where cell surface coating is utilized for a single purpose such as preventing cell aggregation, there are many cases where there is a lack of a strategy for multifunctionality that can expect an appropriate immune anticancer treatment effect.

On the other hand, triple-negative breast cancer is not only difficult to suppress in the primary tumor, but also easily metastasizes to other organs, so the treatment effect with existing technologies may be very limited. Since the tumor microenvironment changes and the cancer cells themselves show resistance to various types and forms of drugs/cells administered into the body, the treatment of solid tumors is limited. In the case of existing cell therapy agents, the targeting efficiency for the tumor where the primary tumor is located is low, and there are limits to suppressing the tumor in question, and furthermore, there may be limitations as a combined treatment technology for metastatic cancer.

The present inventors have completed the present invention by synthesizing a polymer compound that has an anticancer effect while preventing functional decline of surface membrane proteins involved in signal transmission and cancer cell recognition even when coating the surface of natural killer cells.

The technical problem to be achieved by the present invention is to provide a multifunctional polymer capable of enhancing the anticancer functionality of natural killer cells through a single surface attachment process, preventing a decrease in the function of a cell signaling membrane protein through partial attachment to a local area on the surface of natural killer cells in the surface attachment process, and maintaining the original function of natural killer cells and reducing side effects through a responsive unmasking process according to the reducing conditions of the cancer cell or cancer tissue environment.

Another technical problem to be achieved by the present invention is to provide a multifunctional polymer capable of effectively treating triple-negative breast cancer by maximizing recognition and killing functions for cancer cells.

Another technical challenge of the present invention is to provide a multifunctional polymer capable of inhibiting tumor metastasis to other organs.

As one embodiment of the present invention for solving the above problem, a polymer compound includes a hydrophobic moiety that binds to natural killer cells, a cancer cell recognition moiety, and a linker, wherein 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, and the linker can have multiple peaks confirmed in the range of 2.09 to 4.34 ppm and the range of 7.33 to 8.19 ppm in the ¹ H-NMR spectra, and a single peak of 6.28 ppm can be confirmed in the range of 6 to 7 ppm.

As one embodiment of the present invention, the polymer compound can inhibit the metastasis of triple-negative breast cancer cells to other organs.

As one embodiment of the present invention, the hydrophobic moiety can bind to natural killer cells, and the cancer cell recognition moiety can recognize cancer cells and promote the death of cancer cells.

As one embodiment of the present invention, the hydrophobic moiety binding to the natural killer cell may be at least one selected from the group consisting of a phospholipid having an alkyl chain having 12 to 24 carbon atoms; a sterol lipid having 10 to 30 carbon atoms; 1,2-bis(diphenylphosphino)ethane (DPPE); and 1,2-bis(dimethylphosphino)ethane (DMPE).

As one embodiment of the present invention, the hydrophobic moiety binding to the natural killer cell can be confirmed to have peaks at 1.27 ppm and 0.84 ppm in the ¹ H-NMR spectra.

As one embodiment of the present invention, the cancer cell recognition moiety may be at least one selected from the group consisting of folic acid, biotin, phenylboronic acid, and phenylboronic acid.

As one embodiment of the present invention, the cancer cell recognition moiety can be confirmed to have multiple peaks in the range of 2.31 to 4.49 ppm and the range of 6.65 to 8.65 ppm in the ¹ H-NMR spectra, and a single peak can be confirmed in the range of 11 to 12 ppm and the range of 12 to 13 ppm, respectively.

As one embodiment of the present invention, the linker may include a structure of <Chemical Formula 1>.

<Chemical Formula 1>

Here, n is an integer from 30 to 50.

As one embodiment of the present invention, the polymer compound may be combined with at least one of a compound for preventing cell internalization, a cationic amino acid, and a fluorescent dye compound to the linker.

As one embodiment of the present invention, the compound for preventing cell internalization may be at least one selected from the group consisting of polyethylene glycol (PEG); polyethylene oxide (PEO); polyvinyl alcohol (PVA); and copolymers thereof.

As one embodiment of the present invention, the cationic amino acid may be selected from the group consisting of arginine, lysine, and histidine.

In another embodiment of the present invention, the polymer compound is a linker in which multiple peaks are confirmed in the range of 2.09 to 4.34 ppm and 7.33 to 8.19 ppm in the ¹ H-NMR spectra, and a single peak of 6.28 ppm is confirmed in the range of 6 to 7 ppm; and

A hydrophobic moiety is included, which is bound to one end of the linker and has peaks confirmed at 1.27 ppm and 0.84 ppm in a ¹ H-NMR spectra, wherein the hydrophobic moiety is bound to a target and can modify the surface of the target.

In another embodiment of the present invention, the target may include a natural killer cell or an exosome.

In another embodiment of the present invention, the polymer compound is intended to be coupled with a cancer cell recognition moiety that recognizes cancer cells and promotes the death of cancer cells, and the cancer cell recognition moiety can be coupled to the other terminal of the linker.

The present invention relates to a polymer compound having a form containing various functional groups, which comprises a hydrophobic moiety for binding to the surface of natural killer cells and a cancer cell recognition moiety, and wherein natural killer cells having their surface modified with the polymer compound have an increased cancer cell recognition ability and can significantly increase cancer cell death by releasing lytic granules and cytokines.

In addition, the cancer cell recognition moiety is cleaved by glutathione released from cancer cells killed by the surface-modified natural killer cells, and the cancer cell recognition moiety continues to bind to the surface of the cancer cell, so that the cancer cell killing effect works synergistically, and the overall cancer cell killing efficacy can be significantly improved.

FIG. 1 is a drawing for explaining a method for enhancing the immune anticancer function of natural killer cells by attaching a polymer compound for modifying the surface of natural killer cells according to one embodiment of the present invention.
Figure 2 is a photograph of natural killer cells coated with a solution containing the polymer compound, labeled with a fluorescent labeling substance, and observed under a fluorescence microscope.
Figure 3 is a graph showing the ratio of fluorescence intensity of the actual coated coating material compared to natural killer cells.
Figure 4 is a graph confirming through flow cytometry that the biopolymer attached to the cell membrane surface of natural killer cells can maintain the attachment function for up to 48 hours. The peak range on the far right indicates the case where no time has passed (0 hr) after the biopolymer was attached to the natural killer cell membrane, the second peak range from the far right indicates the case where 6 hours have passed (6 hr), the third peak range from the far right indicates the case where 12 hours have passed (12 hr), the fourth peak range from the far right indicates the case where 24 hours have passed (24 hr), and the fifth peak range from the far right indicates the case where 48 hours have passed (48 hr) and the peak range of uncoated natural killer cells (no coating). Here, the peak range means including the ranges on the left and right of the part where the peak value appears on the graph.
The left side of Figure 5 is a graph showing the viability of natural killer cells according to the concentration of the coating material, and the right side is a graph showing the viability of natural killer cells according to the passage of time.
Figure 6 is a graph showing that after surface attachment of a biopolymer, signaling receptors originally present on the cell membrane surface of natural killer cells maintain their original characteristics and normally bind and interact with the corresponding ligand substance.
Figure 7 is a drawing showing that after the polymer is attached to the surface, the chemical bond of the ligand for enhancing cancer cell recognition is broken under cancer cell/cancer tissue microenvironment conditions, thereby killing cancer cells.
Figure 8 is a drawing showing the chemical structures of Fmoc-protected aspartate (Fmoc-Asp), cystamine dihydrochloride, and Fmoc-PEDS intermediates confirmed by NMR spectra.
In Figure 9, (A) is the chemical structure of Fmoc-protected aspartate (Fmoc-Asp), (B) is the chemical structure of cystamine, dihydrochloride, and (C) is the chemical structure of the synthesized Fmoc-PEDS intermediate, which were confirmed by FTIR spectra.
In Figure 10, (A) is the chemical structure of the Fmoc-PEDS intermediate, (B) is the chemical structure of the DSPE lipid, and (C) is the chemical structure of the DSPE-Fmoc-PEDS conjugate, which were confirmed by NMR spectra.
In Figure 11, (A) is a picture confirming the chemical structure of the Fmoc-PEDS intermediate, (B) is a picture confirming the chemical structure of the DSPE lipid, and (C) is a picture confirming the chemical structure of the DSPE-Fmoc-PEDS conjugate using FTIR spectra.
In Figure 12, (A) is the chemical structure of succinoyl-DSPE-Fmoc-PEDS, (B) is the chemical structure of folic acid (FA), and (C) is the chemical structure of DSPE-PEDS-FA confirmed through NMR spectra.
In Figure 13, (A) is the chemical structure of succinoyl-DSPE-Fmoc-PEDS, (B) is the chemical structure of folic acid (FA), and (C) is the chemical structure of DSPE-PEDS-FA confirmed through FTIR spectra.
In Figure 14, (A) is PEG1k-COOH, (B) is Fmoc-arginine, (C) is 5CFL dye, and (D) is the chemical structure of the synthesized multifunctional lipid-PEDS5CFL-Arg-PEG1k-FA, which were confirmed by NMR spectra.
In Figure 15, (A) is DSPE-PEDS-FA, (B) is PEG1k-COOH, (C) is Fmoc-arginine, (D) is 5CFL dye, and (E) is the chemical structure of the synthesized multifunctional lipid-PEDS5CFL-Arg-PEG1k-FA, which was confirmed by FT-IR spectra.
Figure 16 shows the chemical structures of (a) DSPE-PEDS-Biotin, (b) 5-CFL, (c) Fmoc-Arg, (d) PEG1k, and (e) DSPE-PEDS5CFL/Arg/PEG1k-Biotin confirmed by NMR spectra.
Figure 17 shows the chemical structures of (a) DSPE-PEDS-Biotin, (b) 5-CFL, (c) Fmoc-Arg, (d) PEG1k, and (e) DSPE-PEDS5CFL/Arg/PEG1k-Biotin confirmed by FT-IR spectra.
Figure 18 is a graph showing the ratio of effector cells attached to target cells (remaining E:T ratio) when the ratio of effector cells (NK-92mi cells or coated NK-92mi cells) to target cells is 10:1.
Figure 19 shows the results of quantifying the amount of DNA in the target cells.
Figure 20 is a graph showing the results of specific cell lysis through NK-92mi cells cultured according to the method of Example 1. The x-axis represents the effector cell and target cell ratio, and the y-axis represents the cell lysis and death ratio.
Figure 21 is a graph showing the results of specific cell lysis through NK-92mi cells cultured according to the method of Example 2. The x-axis represents the effector cell and target cell ratio, and the y-axis represents the cell lysis and death ratio.
Figure 22 is a graph showing the concentration of glutathione released from each target cell (MCF-7 breast cancer cells, MDA-MB-231 breast cancer cells, MIA PaCa-2 pancreatic cancer cells, or human fibroblasts).
Figure 23 is a schematic diagram showing the experimental process of manufacturing a structure that mimics coated natural killer cells to which a natural killer cell coating material is anchored through hydrophobic interactions and using the same to confirm the cancer cell killing effect mediated by antifolates.
Figure 24 is a schematic diagram showing the experimental process for confirming the cancer cell killing effect of coated natural killer cells in the body.
Figure 25A is a hematoxylin and eosin (H&E) stained photograph of tumor tissue harvested from an MDA MB-231 xenograft mouse, where T represents the tumor tissue area and N represents the necrotic area. Here, the white arrow indicates an eosinophilic cell expansion morphology, the white triangle indicates a cell morphology with condensed nuclei, and the black arrow indicates a cell morphology with lysed nuclei. B is a graph quantifying the necrotic area in the hematoxylin and eosin (H&E) stained image of the entire tumor mass, and the ratio of the necrotic area was significantly higher than that of other groups, with a maximum of 36% in the coated NK cells. C is a photograph showing the expression level of cleaved caspase3 as a cell death marker, and the aggregated form in the square indicates the cell portion where cleaved caspase3 was expressed. D is a graph quantifying cleaved caspase3-positive cells in tumor tissue, and the ratio of cleaved caspase3-positive cells in the coated NK cell group was significantly higher than that of all other groups, with a maximum of 26%. E is a photograph confirming the proliferated tumor cell portion with the proliferation marker Ki67, and the aggregated form in the square indicates the portion where the tumor cells proliferated. F is a graph quantifying the tumor cell portion confirmed with the proliferation marker Ki67. The proportion of proliferating tumor cells in the coated NK cell group was about 1/5 of the control group, G is a photograph showing CD56 (human-specific NK cell marker)-positive cells, and the aggregated form shown in the square represents CD56 (human-specific NK cell marker)-positive cells, and H is a graph quantifying the biodistribution of NK cells or coated NK cells in major organs and tumors based on the proportion of CD56-positive cells, showing a high intratumoral distribution of coated NK cells compared to the heart, kidney, liver, lung, and spleen, and the proportion of coated NK cells distributed in tumors was found to be higher than that of the NK cell group.
Figure 26 is a diagram illustrating changes in the tumor microenvironment that occur when coated natural killer cells are administered to a triple-negative breast cancer animal model.
Figure 27 is a diagram illustrating the inhibition of lung metastasis by coated natural killer cells administered to a triple-negative breast cancer animal model.
Figure 28 is a diagram illustrating the inhibition of liver metastasis by coated natural killer cells administered to a triple-negative breast cancer animal model.

Hereinafter, the present invention will be described in detail.

Natural killer cells (NK cells) are a subset of lymphocytes involved in non-conventional immunity. Natural killer cells can be obtained by a variety of techniques known in the art, such as blood sampling, cytopheroplasty, collection, etc.

Characteristics and biological properties of natural killer cells include: expression of surface antigens including CD16, CD56, and/or CD57; the absence of alpha/beta or gamma/delta TCR complexes on the cell surface; the ability to bind to and kill cells that fail to express "self" MHC/HLA antigens by activation of specific cytolytic enzymes; the ability to kill tumor cells or other diseased cells expressing NK activating receptor-ligands; the ability to release cytokines that stimulate or inhibit immune responses; and the ability to undergo multiple cycles of cell division and produce daughter cells that have biological properties similar to the parent cell.

In the present invention, specific details related to the technical characteristics and effects of the polymer compound may be applied identically or similarly to a method for producing the polymer compound, a pharmaceutical composition containing or utilizing the polymer compound, a method for preventing or treating cancer, etc., as long as they do not conflict with the essential aspects of the present invention.

The present invention provides a polymeric compound comprising a hydrophobic moiety that binds to an immune cell; a cancer cell recognition moiety; and a linker comprising a structure represented by the following chemical formula 1.

According to one embodiment of the present invention, the immune cell may be a T cell, a B cell or a natural killer cell, and preferably may be a natural killer cell, but is not limited thereto.

Specifically, the polymer compound of the present invention can provide a polymer compound comprising a hydrophobic moiety that binds to natural killer cells; a cancer cell recognition moiety; and a linker comprising a structure represented by the following chemical formula 1.

In the present invention, “surface modification” may mean binding of a hydrophobic moiety to the surface of a natural killer cell or the surface of an exosome.

The hydrophobic moiety may be bound to one terminal of the chemical formula 1, and the cancer cell recognition moiety may be bound to the other terminal of the chemical formula 1, wherein n may be an integer of 30 to 50.

One terminal of the chemical formula 1 may be linked or bound to the hydrophobic moiety via an amide bond, and the other terminal of the chemical formula 1 may also be linked or bound to the cancer cell recognition moiety via an amide bond.

The above connection may mean that one terminal of chemical formula 1 is connected to a hydrophobic moiety, which may mean that one terminal of chemical formula 1 is directly connected to a hydrophobic moiety, or that one terminal of chemical formula 1 is indirectly connected to a hydrophobic moiety, and in the case of the indirect connection, it may mean that one terminal of chemical formula 1 and the hydrophobic moiety are connected via a linker or the like, and the above bond may mean a chemical bond.

<Chemical Formula 1>

If the above n is less than 30, there may be a problem in which the compound for preventing cell internalization, cationic amino acid, or fluorescent dye compound described below is not sufficiently bound within the structural unit, and if it exceeds 50, the length of the chain becomes excessively long, resulting in too many disulfide bonds within the structural unit, which may result in a problem in which natural killer cells and cancer cells are not properly connected.

The above disulfide bond cleavage can occur randomly within the n repeating units of the chemical formula 1 to form fragments of various lengths. However, it was confirmed that this has no effect on the efficacy of the cancer cell recognition moiety bound to the other terminal of the chemical formula 1 to recognize and kill cancer cells, and that cytotoxicity is not induced as a result.

The hydrophobic moiety can bind to natural killer cells or exosomes, and the cancer cell recognition moiety can recognize cancer cells and promote the death of cancer cells.

In one embodiment of the present invention, one terminal of the chemical formula 1 may be a CH ₃ terminal of a structural unit comprising n disulfide bond structures and repeating, and the other terminal may be a CH ₃ terminal of a structure other than the structural unit comprising n disulfide bond structures and repeating. Here, a hydrophobic moiety may be bound to the one terminal, and a cancer cell recognition moiety may be bound to the other terminal.

In one embodiment of the present invention, a polymer compound including a hydrophobic moiety binding to natural killer cells; a cancer cell recognition moiety; and a linker having the structure of the chemical formula 1 was prepared (Preparation Example 1, Preparation Example 2), and the prepared polymer compound was coated on the surface of natural killer cells (Experimental Example 1, Example 2), and the enhancement of the cancer cell recognition ability of the coated natural killer cells (Experimental Examples 3 to 6), and the cancer cell killing effect of the coated natural killer cells (Experimental Example 10, Experimental Example 11) were confirmed.

Natural killer cells were cultured in a composition containing the DSPE-PEDS _{5CFL-Arg-PEG1k} -FA conjugate, which is an example of the polymer compound manufactured above, to prepare natural killer cells having the DSPE-PEDS _{5CFL-Arg-PEG1k} -FA conjugate bound to their surface.

As a result of analyzing the cancer cell recognition ability of natural killer cells coated with the above DSPE-PEDS 5CFL _-Arg-PEG1k -FA conjugate according to the method of Experimental Example 2, it was shown that the cancer cell recognition ability of natural killer cells coated with the DSPE-PEDS _{5CFL-Arg-PEG1k} -FA conjugate of Preparation Example 1 was significantly improved by about 1.4 times compared to uncoated natural killer cells.

As a result of analyzing the cancer cell killing effect of natural killer cells coated with the DSPE-PEDS _{5CFL-Arg-PEG1k} -FA conjugate according to the method of Experimental Example 3, when natural killer cells coated with the DSPE-PEDS _{5CFL-Arg-PEG1k} -FA conjugate of Preparation Example 1 were co-cultured with cancer cell lines, there was a significant increase in the cell killing effect of the cancer cell lines compared to when uncoated natural killer cells were co-cultured with cancer cell lines. In addition, as the number of natural killer cells coated with the DSPE-PEDS _{5CFL-Arg-PEG1k} -FA conjugate of Preparation Example 1 of the present invention was gradually increased and cultured with cancer cell lines, it was confirmed that the effect of promoting the cell killing effect of the cancer cell lines increased more dramatically.

As a result of analyzing the cancer cell killing effect on natural killer cells coated with the above DSPE-PEDS _{5CFL-Arg-PEG1k} -FA complex according to the method of Experimental Example 5, it was confirmed that natural killer cells coated with the DSPE-PEDS _{5CFL-Arg-PEG1k} -FA complex induced more cancer cell killing and also increased glutathione release.

As a result of analyzing the cancer cell killing effect on natural killer cells coated with the above DSPE-PEDS _{5CFL-Arg-PEG1k} -FA conjugate according to the method of Experimental Example 6, it was confirmed that the disulfide bond of the DSPE-PEDS _{5CFL-Arg-PEG1k} -FA conjugate of Preparation Example 1 was cleaved, dissociating folic acid (FA), and after dissociation, folic acid, which is a cancer cell recognition moiety, was converted into antifolate, thereby additionally inducing cancer cell death.

As a result of analyzing the in vivo cancer cell killing effect of natural killer cells coated with the above DSPE-PEDS _{5CFL-Arg-PEG1k} -FA complex according to the method of Experimental Example 10, it was confirmed that the tumor volume over time in the experimental group injected with the coated natural killer cells was relatively smaller than that in other control or experimental groups, the tumor growth inhibition rate was relatively high, and the survival probability was also high.

The effect of enhancing the cancer suppression ability of natural killer cells coated with the above DSPE-PEDS _{5CFL-Arg-PEG1k} -FA complex was analyzed by histological analysis according to the method of Experimental Example 11. As a result, it was confirmed that natural killer cells coated with the DSPE-PEDS _{5CFL-Arg-PEG1k} -FA complex had enhanced cancer cell infiltration properties, thereby exhibiting excellent cancer cell killing effects.

Natural killer cells were cultured in a composition containing the DSPE-PEDS _{5CFL-Arg-PEG1k} -Biotin conjugate, which is an example of the polymer compound manufactured above, to prepare natural killer cells having the DSPE-PEDS _{5CFL-Arg-PEG1k} -Biotin conjugate bound to their surface.

As a result of analyzing the cancer cell killing effect of natural killer cells coated with the DSPE-PEDS _{5CFL-Arg-PEG1k} -Biotin conjugate according to the method of Experimental Example 4, when natural killer cells coated with the DSPE-PEDS _{5CFL-Arg-PEG1k} -Biotin conjugate of Preparation Example 2 were co-cultured with cancer cell lines, there was a significant increase in the cell killing effect of the cancer cell lines compared to when uncoated natural killer cells were co-cultured with cancer cell lines. In addition, as the number of natural killer cells coated with the PEDS _{5CFL-Arg-PEG1k} -Biotin conjugate of Preparation Example 2 of the present invention was gradually increased and cultured with cancer cell lines, it was confirmed that the effect of promoting the cell killing effect of the cancer cell lines increased more dramatically.

As a result of analyzing the cancer cell killing effect on natural killer cells coated with the above DSPE-PED _{S5CFL-Arg-PEG1k} -Biotin complex according to the method of Experimental Example 5, it was confirmed that natural killer cells coated with the above DSPE-PEDS _{5CFL-Arg-PEG1k} -Biotin complex induced more cancer cell killing and also increased glutathione release.

In the present invention, “hydrophobic” means a property of having low affinity for water, and this property may mean that it can have high affinity for lipids of biomolecules, and “moiety” may mean a portion within a molecule that has functionality.

In one embodiment of the present invention, the hydrophobic moiety, which is a component of the polymer compound, may mean a portion that has the hydrophobicity described above and specifically binds to a biomolecule, and may mean a portion that has a high affinity for the lipid of the biomolecule and thus firmly attaches (anchores) to the surface of a natural killer cell.

In the present invention, the hydrophobic moiety may be characterized by having hydrophobic properties and thus being capable of binding or attaching to a double membrane of a cell, and the hydrophobic moiety may be composed of a low-molecular-weight substance that can be administered into a living body, such as a lipid, antibody, hormone, or drug, but is not necessarily limited thereto.

Specifically, the hydrophobic moiety herein can bind or attach to the lipid bilayer of an immune cell, and specifically can bind or attach to the lipid bilayer of a natural killer cell.

In one embodiment of the present invention, the polymer compound that recognizes natural killer cells and cancer cells may include a hydrophobic moiety that binds to the natural killer cells, and the hydrophobic moiety that binds to the natural killer cells may be 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-bis(diphenylphosphino)ethane (DPPE); and 1,2-bis(dimethylphosphino)ethane (DMPE), and may be a lipid molecule having a structure similar to the lipid bilayer of the cell membrane of natural killer cells that are the target of surface coating.

As the phospholipid having an alkyl chain of 12 to 24 carbon atoms, preferably, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine (DMPC), dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-distearoyl-3-trimethylammonium-propane chloride (DSTAP) can be used, but the present invention is not limited thereto, and any lipid-type biomolecule that can be attached to and fixed to the surface of natural killer cells and is soluble in a nonpolar solvent can be used without limitation.

As the sterol lipid having 10 to 30 carbon atoms, preferably cholesterol, cholesterol hexasuccinate, 3β-dimethylaminoethane)carbamoyl]cholesterol, ergosterol, stigmasterol, or lanosterol can be used, but any lipid-type biomolecule that can be attached and fixed to the surface of natural killer cells and is soluble in a nonpolar solvent can be used without limitation.

As can be seen in Example 1 of the present invention, the DSPE-PEDS _{5CFL-Arg-PEG1k} -Biotin conjugate is a compound in which the 1,2-Distearoyl-snglycero-3-phosphoethanolamine (DSPE) is partially bound, and the DSPE is a compound in which CH ₂ is continuously bound in the form of a hydrophobic chain that can be anchored to the surface of actual natural killer cells.

The hydrophobic moiety constituting the polymer compound for modifying the surface of natural killer cells of the present invention is attached to the surface of natural killer cells, and the cancer cell recognition moiety recognizes a receptor on the surface of cancer cells, thereby enhancing the ability of the surface-coated natural killer cells to decompose or kill cancer cells.

The above-mentioned enhancement of cancer cell recognition ability is due to the mechanism in which the cell membrane structure of cancer cells is destroyed by the enzymatic action of natural killer cells, and glutathione (GSH) inside the cancer cells flows out of the cells and cleaves the disulfide bonds of high molecular compounds. This can be called a sensitive unmasking process according to the reducing conditions of the cancer tissue environment.

In one embodiment of the present invention, even if the disulfide bond of the polymer compound is cleaved, the cancer cell recognition moiety attached to the end of the polymer compound can bind to a receptor on the surface of the cancer cell and induce the death of the cancer cell (FIG. 7(a), FIG. 7(b)).

That is, the polymer compound of the present invention that recognizes natural killer cells and cancer cells can coat the cell surface by hydrophobic bonding with the cell membrane of natural killer cells through a hydrophobic moiety, and can specifically recognize cancer cells through a cancer cell recognition moiety, thereby increasing the ability of natural killer cells to kill cancer cells.

In addition, due to the death of cancer cells, glutathione present in cancer cells is leaked, and the disulfide bond of the polymer compound of the present invention is broken, converting the cancer cell recognition moiety into a form such as antifolate, thereby inhibiting DNA replication of cancer cells and inducing additional cancer cell death.

Natural killer cells whose surface is modified with the polymer compound of the present invention can significantly increase cancer cell death by increasing cancer cell recognition ability and releasing lytic granules and cytokines, and further enhance cancer cell death by cleaving the cancer cell recognition moiety through the release of glutathione inside the cancer cell.

The polymer compound of the present invention that recognizes natural killer cells and cancer cells may include a cancer cell recognition moiety, and the cancer cell recognition moiety may be at least one selected from the group consisting of folic acid, biotin, phenylboronic acid, and phenylboronic acid, and preferably folic acid or biotin.

The polymer compound of the present invention that recognizes natural killer cells and cancer cells may be combined with at least one of a compound for preventing cell internalization, a cationic amino acid, and a fluorescent dye compound to a linker having the structure of the chemical formula 1.

The compound for preventing cell internalization may be at least one selected from the group consisting of polyethylene glycol (PEG); polyethylene oxide (PEO); polyvinyl alcohol (PVA); and copolymers thereof.

The above-mentioned compound for preventing cell internalization can have the function of preventing a polymer compound that recognizes natural killer cells and cancer cells from being incorporated into the interior of natural killer cells and maintaining attachment to the cell membrane surface.

In the polymer compound of the present invention that recognizes natural killer cells and cancer cells, the cationic amino acid bound to the linker comprising the structure of the chemical formula 1 may be at least one selected from the group consisting of arginine, lysine, and histidine.

The above cationic amino acids can function to induce access to natural killer cells through electrical interactions.

In the polymer compound of the present invention that recognizes natural killer cells and cancer cells, the fluorescent dye compound bound to the linker containing the structure of the chemical formula 1 can enable diagnosis or detection of target cells or cancer through fluorescence imaging when natural killer cells or cancer cells are recognized.

The above fluorescent dye compound may be a fluorescent dye having rhodamine, coumarin, EvoBlue, oxazine, carbopyronine, naphthalene, biphenyl, anthracene, phenanthrene, pyrene or carbazole as a basic skeleton, or a derivative of the above fluorescent dye, and any dye compound exhibiting fluorescent properties may be used without limitation.

The above polymer compound may be characterized in that it does not inhibit the viability and proliferation of natural killer cells even when bound to the surface of natural killer cells. Specifically, it was confirmed that coating the surface of natural killer cells with the polymer compound manufactured in one embodiment of the present invention had no effect on the viability of natural killer cells, and even when the surface of natural killer cells was coated with the polymer compound manufactured in one embodiment of the present invention at a concentration of 2 mg/mL, it was confirmed that there was no effect on the viability of natural killer cells, and it was confirmed that natural killer cells grew well for up to 48 hours after coating at the above concentration (Fig. 5).

In addition, even if a polymer compound binds to the surface of a natural killer cell, the signaling receptors originally present on the surface of the cell membrane of the natural killer cell maintain their original characteristics and can normally bind and interact with the corresponding ligand substance.

This could mean that even if a polymer compound is attached to the surface of natural killer cells, it does not have any physical or biological effect on the activity of various receptors originally present on the surface of natural killer cells, and the cell signaling mechanism through this operates normally.

It is known that lipopolysaccharide (LPS) binds to toll-like receptors on the surface of natural killer cells and causes interferon gamma (IFN-γ) to be released through an intracellular signaling mechanism.

In one embodiment of the present invention, it was confirmed that interferon-γ (IFN-γ) release was normally achieved even when the polymer compound was attached to the surface of natural killer cells, which means that the signal transmission mechanism operates normally. This can be confirmed through the experimental results of Fig. 6.

The method for producing a polymer compound that recognizes natural killer cells and cancer cells of the present invention preferably includes, but is not limited to, the steps of (a) linking a compound represented by the chemical formula 2, which is a hydrophobic moiety, to one terminal of a compound represented by the chemical formula 1 below to provide a compound represented by the chemical formula 3; and (b) linking a cancer cell recognition moiety to the other terminal of the compound represented by the chemical formula 3.

<Chemical Formula 1>

<Chemical Formula 2>

<Chemical Formula 3>

Here, n may be an integer of 30 to 50, X may be one selected from acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ), and 9-fluorenylmethyleneoxycarbonyl (Fmoc), p is a natural number of 12 to 20, and q is a natural number of 12 to 20.

If the above p or q is a natural number less than 12, the length of the lipid attached to the natural killer cells of the polymer compound may become short, which may cause a problem in that the ability to attach to the natural killer cells may decrease. If it is a natural number greater than 20, steric hindrance may be induced or micelles may be formed before coating, which may cause a problem in that the coating efficiency may be reduced.

As an embodiment of the present invention, a method for producing a polymer compound that recognizes natural killer cells and cancer cells may further include a step of replacing X in the chemical formula 1 with any one of a compound for preventing cell internalization, a cationic amino acid, and a fluorescent dye compound.

As an embodiment of the present invention, in the method for producing a polymer compound that recognizes natural killer cells and cancer cells, the cancer cell recognition moiety bound to the other terminal of the chemical formula 3 may be at least one selected from the group consisting of folic acid, biotin, lactobionic acid, and phenylboronic acid.

As an embodiment of the present invention, in the method for producing a polymer compound that recognizes natural killer cells and cancer cells, the compound for preventing cell internalization that can be bound to X of the chemical formula 1 may be at least one selected from the group consisting of polyethylene glycol (PEG); polyethylene oxide (PEO); polyvinyl alcohol (PVA); and copolymers thereof.

As one embodiment of the present invention, in the method for producing a polymer compound that recognizes natural killer cells and cancer cells, the cationic amino acid that can be bound to X of the chemical formula 1 may be at least one selected from the group consisting of arginine, lysine, and histidine.

As one embodiment of the present invention, the pharmaceutical composition for preventing or treating cancer of the present invention may include a polymer compound that recognizes natural killer cells and cancer cells as an active ingredient, the polymer compound including a hydrophobic moiety that binds to natural killer cells; a cancer cell recognition moiety; and a linker having a structure of the chemical formula 1, wherein the hydrophobic moiety is bound to one terminal of the chemical formula 1 and the cancer cell recognition moiety is bound to the other terminal of the chemical formula 1.

<Chemical Formula 1>

Here, n is an integer from 30 to 50.

As an embodiment of the present invention, a pharmaceutical composition for cancer prevention or treatment, which comprises a polymer compound that recognizes natural killer cells and cancer cells as an effective ingredient, is effective for the treatment of prostate cancer, thyroid cancer, stomach cancer, colon cancer, lung cancer, breast cancer (particularly, triple-negative breast cancer), liver cancer, pancreatic cancer, testicular cancer, oral cancer, basal cell carcinoma, brain tumor, gallbladder cancer, bile duct cancer, laryngeal cancer, retinoblastoma, ampulla of Vater, bladder cancer, peritoneal cancer, adrenal cancer, non-small cell lung cancer, tongue cancer, small cell lung cancer, small intestine cancer, meningioma, esophageal cancer, renal pelvic ureteral cancer, kidney cancer, malignant bone tumor, malignant soft tissue tumor, malignant lymphoma, malignant melanoma, eye tumor, urethral cancer, stomach cancer, cyst, pharyngeal cancer, cervical cancer, endometrial cancer, uterine sarcoma, metastatic brain tumor, rectal cancer, vaginal cancer, spinal tumor, salivary gland cancer, tonsil cancer, squamous cell carcinoma, and blood cancer. and can prevent or treat any one or more cancer diseases selected from the group consisting of anal cancer.

As one embodiment of the present invention, a pharmaceutical composition for preventing or treating cancer, which comprises a polymer compound that recognizes natural killer cells and cancer cells as an active ingredient, may additionally contain a pharmaceutically acceptable carrier, excipient or diluent.

The term "pharmaceutically acceptable" of the invention means that the composition exhibits a property of not being toxic to cells or humans exposed to the composition. The composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral dosage forms. When formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are commonly used. The above carrier, excipient and diluent may be at least one selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, saline, methyl hydroxybenzoate, propyl hydroxy benzoate, talc, magnesium stearate, and mineral oil, dextrin, calcium carbonate, propylene glycol and liquid paraffin, but is not limited thereto, and all conventional carriers, excipients or diluents can be used. The above components may be added independently or in combination to the polymer compound which is the active ingredient.

The above pharmaceutical composition may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories.

The base material of the above suppository may include witepsol, macrogol, Tween 60, cocoa butter, laurin butter, glycerogelatin, etc.

The above pharmaceutical composition may have any one dosage form selected from the group consisting of powder, granule, tablet, capsule and liquid forms.

In addition, the present invention provides a method for treating cancer, comprising administering to a subject a pharmaceutical composition comprising a polymer compound comprising a hydrophobic moiety that binds to natural killer cells; a cancer cell recognition moiety; and a linker comprising a structure of chemical formula 1, wherein the hydrophobic moiety is bound to one terminal of the linker and the cancer cell recognition moiety is bound to the other terminal of the linker, and the polymer compound that recognizes natural killer cells and cancer cells.

<Chemical Formula 1>

Here, n is an integer from 30 to 50.

The pharmaceutical composition containing the polymer compound may be administered orally, and may be a solid preparation or a liquid preparation. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing the composition with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. There is no particular restriction on the administration dosage, and it can vary depending on the absorption rate in the body, body weight, patient's age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. The pharmaceutical composition of the present invention is manufactured in consideration of the effective dosage range, and the unit dosage form prepared in this way can be administered several times at regular time intervals using a specialized administration method according to the judgment of a specialist who monitors or observes the administration of the drug and the individual's needs, as needed. The administration can preferably be administered once a day or divided into several times.

The above entity refers to a subject in need of treatment for a disease, and more specifically refers to a mammal (including, for example, a dog, cat, horse, rabbit, zoo animals, non-human animals such as cows, pigs, sheep, and non-human primates). In a specific embodiment, the entity of the present invention is a human.

Hereinafter, the present invention will be described in detail through examples and experimental examples.

However, the following examples and experimental examples are only intended to illustrate the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

Manufacturing Example 1: Manufacturing of a polymer compound for surface modification to enhance the immune anticancer function of natural killer cells

The components used to prepare the polymer compound of the present invention are listed in Table 1.

ingredient manufacturing company 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (N-(3-Dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride, EDC) Sigma-Aldrich, 03449 N-hydroxysuccinimide (NHS) Sigma-Aldrich, 130672 Cystamine dihydrochloride (Cys) Sigma-Aldrich, 30050 4-Dimethylaminopyridine (DMAP) Sigma-Aldrich, 39405 Anhydrous dimethylformamide (N,N-Dimethylformamide, DMF) Sigma-Aldrich, 227056 folic acid (FA) Sigma-Aldrich, F7876 5-carboxyfluorescein (5CFL) Sigma-Aldrich, 86826 triethylamine Sigma-Aldrich, 471283 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine DSPE TCI, 850745P PEG1k-NHS Biopharma PEG Scientific Inc.,HE023017 Fmoc-aspartic acid(Fmoc-Asp) BOC Science, 119062-05-04 Fmoc-arginine(Fmoc-Arg) BOC Science, 91000-69-0 succinic anhydride Daejung, 7673-4405 diethyl ether Duksan, D558 Piperidine Sigma-Aldrich, 104094 Biotin Sigma-Aldrich, B4501 Dimethyl sulfoxide Sigma-Aldrich, 276855

1-1. Synthesis of Fmoc-PEDS intermediate

A reaction mixture was prepared by mixing 1 mmol of Fmoc-ASP, 2 mmol of EDC, and 3 mmol of NHS in 5 mL of anhydrous dimethylformamide (DMF) and stirring at 25 °C for 3 h.

To the above reaction mixture, a solution of cystamine dihydrochloride dissolved in 5 mL of anhydrous dimethylformamide (the concentration of cystamine dihydrochloride was 1 mmol) and 5 mL of 4-dimethylaminopyridine were added.

The reaction mixture containing the above cystamine dihydrochloride and 4-dimethylaminopyridine was left to stand for 15 hours.

1 mmol of EDC, 1 mmol of NHS and 2 mg of 4-dimethylaminopyridine were dissolved in 1 mL of anhydrous dimethylformamide, and further added to the reaction mixture left standing, and stirred at 600 rpm at 60°C for 60 hours in a stirring device (Corning, pC-620D).

After 60 hours, when the reaction was completed, the obtained product was precipitated in 100 mL of diethyl ether at -20°C using centrifugation (LABOGENE, 1580R) at 12,000 rpm for 10 minutes, thereby synthesizing 334 mg of Fmoc-PEDS (Fmoc-poly(ethylene aspartamido disulfide) intermediate.

The molecular weight of the above-mentioned synthesized 334 mg Fmoc-PEDS intermediate was measured using a GPC instrument (Agilent GPC system) equipped with a GPC column (Styragel) using poly(methyl methacrylate) as a GPC (gel permeation chromatography) standard.

The chemical structure of the Fmoc-PEDS intermediate was also confirmed by NMR (500 MHz FT-NMR spectrometer, Bruker, Germany) and FTIR (Perkin Elmer FTIR Spectrum Two, PerkinElmer, USA) spectroscopy.

Figure 8 shows the chemical structures of Fmoc-protected aspartate (Fmoc-Asp), cystamine dihydrochloride, and Fmoc-PEDS intermediates confirmed by NMR spectra.

In Figure 9, (A) shows the chemical structure of Fmoc-protected aspartate (Fmoc-Asp), (B) shows cystamine, dihydrochloride, and (C) shows the chemical structure of the synthesized Fmoc-PEDS intermediate, as confirmed by FTIR spectra.

The degree of polymerization (DP) was calculated using the following formula.

1-2. Formation of DSPE-Fmoc-PEDS complex

1 mmol of EDC, 1.5 mmol of NHS, and 100 mg of the Fmoc-PEDS intermediate synthesized in Preparation Example 1-1 (containing 4.90 μmol of terminal carboxyl group) were dissolved in 5 mL of anhydrous dimethylformamide, and the mixture was stirred at 600 rpm for 3 hours at 25°C using a stirrer (Corning, pC-620D).

After this, a solution of 20 μmoL DSPE dissolved in 5 mL of 4-dimethylaminopyridine was added to the mixture after stirring was completed, and the mixture was maintained for 48 hours under nitrogen (N2) conditions at 25°C to form a reaction mixture.

The above reaction mixture was precipitated into 100 mL of diethyl ether at -20°C and dried under vacuum at -80°C to synthesize 100 mg of DSPE-Fmoc-PEDS conjugate (containing 4.90 μmol of terminal amine group).

The chemical structure of the DSPE-Fmoc-PEDS conjugate was also confirmed by NMR (500 MHz FT-NMR spectrometer, Bruker, Germany) and FTIR (Perkin Elmer FTIR Spectrum Two, PerkinElmer, USA) spectroscopy.

In Figure 10, (A) is the chemical structure of the Fmoc-PEDS intermediate, (B) is the chemical structure of the DSPE lipid, and (C) is the chemical structure of the DSPE-Fmoc-PEDS conjugate, as confirmed by NMR spectra.

As can be confirmed in (A) of Fig. 10, the Fmoc-PEDS intermediate shows multiple peaks in the ranges of 2.09 to 4.34 ppm and 7.33 to 8.19 ppm in the ¹ H-NMR spectra, and a single peak at 6.28 ppm is confirmed in the range of 6 to 7 ppm.

Additionally, as can be confirmed in (B) of Fig. 10, DSPE lipids have peaks confirmed at 1.27 ppm and 0.84 ppm in the ¹ H-NMR spectra.

In Figure 11, (A) shows the chemical structure of the Fmoc-PEDS intermediate, (B) the DSPE lipid, and (C) the DSPE-Fmoc-PEDS conjugate, confirmed by FTIR spectra.

1-3. Synthesis of DSPE-PEDS-FA complex

A reaction mixture was prepared by dissolving 100 mg of DSPE-Fmoc-PEDS and 4.9 mg of succinic anhydride (succinic acid concentration: 49 μmol) synthesized in Manufacturing Example 1-2 in 5 mL of anhydrous dimethylformamide.

The reaction mixture was prepared by stirring the above reaction mixture using a stirring device (Corning, pC-620D) at 25°C for 24 hours at a speed of 600 rpm.

The stirred reaction mixture was precipitated into 100 mL of diethyl ether at -20°C, 10 mL of distilled water (DW) was added, and centrifuged at 10,000 rpm for 10 minutes (LABOGENE, 1580R), and the supernatant containing unreacted succinic anhydride (C ₄ H ₄ O ₃ ) was removed to prepare a reaction mixture.

The reaction mixture from which the above succinic anhydride (C ₄ H ₄ O ₃ ) was removed was dialyzed (molecular weight cutoff: 2 kD) and then lyophilized at 0°C to produce 100 mg of succinoyl-DSPE-Fmoc-PEDS conjugate (equivalent terminal carboxyl group concentration: 4.90 μmol).

For deprotection and FA conjugation of the Fmoc moiety of the succinoyl-DSPE-Fmoc-PEDS conjugate, 100 mg of EDC, NHS, and the succinoyl-DSPE-Fmoc-PEDS conjugate prepared above (equivalent terminal carboxyl group concentration: 4.90 μmol) were dissolved in 5 mL of anhydrous dimethylformamide, wherein the concentration of EDC was adjusted to 1 mmol and the concentration of NHS was adjusted to 1.5 mmol, and the solution was stirred at 600 rpm for 1 hour at 25°C using a stirrer (Corning, pC-620D) to prepare a reaction mixture.

5.90 μmol folic acid (FA) was dissolved in a solvent containing 5 mL of 4-dimethylaminopyridine and 5 mL of dimethyl sulfoxide (DMSO), and the mixture was mixed with the stirred reaction mixture, followed by stirring at 25°C under a nitrogen (N2) atmosphere for 48 hours.

After completion of the 48-hour stirring, 3 mL of piperidine solution was added for deprotection of the Fmoc moiety and FA conjugation, and the DSPE-PEDS-FA conjugate was synthesized by stirring at 600 rpm for 30 minutes using a stirring device.

The above-mentioned synthesized lipid (DSPE)-PEDS-FA conjugate (lipid-PEDS-FA) was precipitated in 100 mL of diethyl ether at -20°C, centrifuged at 10,000 rpm for 15 minutes, dried under vacuum at -80°C, dialyzed (cutoff molecular weight 2 kD) against distilled water (DW) to remove impurities, and lyophilized.

The chemical structure of the synthesized lipid-PEDS-FA conjugate was confirmed by NMR (500 MHz FT-NMR spectrometer, Bruker, Germany) and FT-IR (Perkin Elmer FTIR Spectrum Two, PerkinElmer, USA) spectroscopy.

In Figure 12, (A) is the chemical structure of succinoyl-lipid (DSPE)-Fmoc-PEDS, (B) is folic acid (FA), and (C) is the chemical structure of DSPE-lipid (PEDS)-FA confirmed through NMR spectra.

As can be confirmed in (B) of Fig. 12, the ¹ H-NMR spectra of folic acid (FA) show multiple peaks in the ranges of 2.31 to 4.49 ppm and 6.65 to 8.65 ppm, and a single peak in the ranges of 11 to 12 ppm and 12 to 13 ppm, respectively.

In Figure 13, (A) is the chemical structure of succinoyl-lipid(PEDS)-Fmoc-PEDS, (B) is folic acid (FA), and (C) is the chemical structure of lipid(PEDS)-PEDS-FA confirmed through FT-IR spectra.

1-4. Synthesis of DSPE-PEDS5CFL-Arg-PEG1k-FA conjugate

21.23 μmoL 5CFL, 106.15 μmoL Fmoc-Arg, EDC, and NHS were dissolved in 5 mL of anhydrous dimethylformamide solution, and a solution adjusted to 1 mmol of EDC and 1.5 mmol of NHS was stirred at 25°C for 1 hour to prepare a reaction mixture.

Thereafter, the DSPE-PEDS-FA conjugate of Manufacturing Example 1-3 (the concentration of grafted amine groups in the polymer was 212 μmol) and 20 μL of triethylamine were added to the reaction mixture in which the 5CFL solution, Fmoc-Arg solution, EDC, and NHS were dissolved in anhydrous dimethylformamide solution and stirred, and the reaction mixture was prepared by stirring at 25°C for 48 hours.

Thereafter, the reaction mixture was precipitated in 100 mL of diethyl ether at -20°C for 48 hours, dialyzed (cutoff molecular weight 2 kD), and lyophilized to synthesize 100 mg of 5CFL, PEG1k-NHS, and a compound in which Arginine and DSPE-PEDS-FA are bound (DSPE-PEDS _{5CFL-Arg-PEG1k} -FA conjugate).

The chemical structure of the functional moiety of the above-formed DSPE-PEDS _{5CFL-Arg-PEG1k} -FA conjugate was confirmed by NMR (500 MHz FTNMR spectrometer, Bruker, Germany) and FT-IR (Perkin Elmer FTIR Spectrum Two, PerkinElmer, USA) spectroscopy.

In Figure 14, (A) is PEG1k-COOH, (B) is Fmoc-arginine, (C) is 5CFL dye, and (D) is the chemical structure of the synthesized multifunctional lipid-PEDS _{5CFL-Arg-PEG1k} -FA, confirmed by NMR spectra.

In Figure 15, (A) is DSPE-PEDS-FA, (B) is PEG1k-COOH, (C) is Fmoc-arginine, (D) is 5CFL dye, and (E) is the chemical structure of the synthesized multifunctional lipid-PEDS _{5CFL-Arg-PEG1k} -FA, which were confirmed by FT-IR spectra.

Manufacturing Example 2: Manufacturing of DSPE-PEDS5CFL-Arg-PEG1k-Biotin

2-1. Synthesis of DSPE-PEDS-Biotin complex

A DSPE-Fmoc-PEDS-Biotin conjugate was prepared in the same manner as in Manufacturing Example 1-3 using 100 mg of the DSPE-Fmoc-PEDS conjugate synthesized in Manufacturing Example 1-2, except that 5 mL of 5.90 μmol folic acid in the reaction mixture stirred with the DSPE-Fmoc-PEDS conjugate was replaced with 5 mL of 5.90 μmol biotin.

The chemical structure of the formed succinoyl-DSPE-Fmoc-PEDS-Biotin conjugate was confirmed by NMR (500 MHz FT-NMR spectrometer, Bruker, Germany) and FT-IR (Perkin Elmer FTIR Spectrum Two, PerkinElmer, USA) spectroscopy.

In Fig. 16, (a) shows the chemical structures of DSPE-PEDS-Biotin, (b) 5-CFL, (c) Fmoc-Arg, (d) PEG1k, and (e) DSPE-PEDS _{5CFL/Arg/PEG1k} -Biotin, which were confirmed by NMR spectra.

In Fig. 17, (a) shows the chemical structures of DSPE-PEDS-Biotin, (b) 5-CFL, (c) Fmoc-Arg, (d) PEG1k, and (e) DSPE-PEDS _{5CFL/Arg/PEG1k} -Biotin, which were confirmed by FT-IR spectra.

2-2. DSPE-PEDS _{5CFL-Arg-PEG1k} -Synthesis of biotin complexes

Using the same method as in Manufacturing Example 1-4, the DSPE-Fmoc-PEDS-Biotin conjugate synthesized in Manufacturing Example 2-1 was used to synthesize a compound in which 5CFL, PEG1k-NHS, and Arginine and lipid (DSPE)-PEDS-FA were bound (DSPE-PEDS _{5CFL-Arg-PEG1k} -Biotin conjugate).

The chemical structure of the functional moiety of the above-formed DSPE-PEDS _{5CFL-Arg-PEG1k} -Biotin conjugate was confirmed by NMR (500 MHz FT-NMR spectrometer, Bruker, Germany) and FT-IR (Perkin Elmer FTIR Spectrum Two, PerkinElmer, USA) spectroscopy.

Figure 16 shows the chemical structures of (a) DSPE-PEDS-Biotin, (b) 5-CFL, (c) Fmoc-Arg, (d) PEG1k, and (e) DSPE-PEDS _{5CFL/Arg/PEG1k} -Biotin confirmed by NMR spectra.

Figure 17 shows the chemical structures of (a) DSPE-PEDS-Biotin, (b) 5-CFL, (c) Fmoc-Arg, (d) PEG1k, and (e) DSPE-PEDS _{5CFL/Arg/PEG1k} -Biotin confirmed by FT-IR spectra.

Manufacturing Example 3: Cell culture

NK-92mi cells, natural killer cells that bind to the surface of cancer cells, were obtained from ATCC (American Type Culture Collection, USA).

The above NK-92mi cells were seeded at a concentration of 1 x 10 ⁵ cells/mL in a T25 culture flask and cultured in 10 mL MEMα (Minimum Essential Medium Alpha, Gibco, USA) containing 12.5% fetal bovine serum (FBS, Gibco), 12.5% horse serum (Gibco), 1% penicillin-streptomycin solution (Corning, USA), 0.2 mM inositol (Sigma-Aldrich, USA), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), and 0.02 mM folic acid (Sigma-Aldrich). Cell culture was performed at 37°C, 5% CO, and 95% humidity for 48 hours.

Breast cancer cell line MCF-7 (ATCC), breast cancer cell line MDA-MB-231 (ATCC), pancreatic cancer cell line MIA PaCa-2 (ATCC), and normal cell line human dermal Fibroblast (Lonza, USA) were also provided by ATCC. The cells were cultured in a culture medium containing 89% Dulbecco's modified Eagle's medium (DMEM, Corning), 1% penicillin-streptomycin solution, and 10% FBS (Corning) at 37°C, 5% CO, and 95% humidity for 24 hours.

The breast cancer cell line MCF-7 (ATCC) was seeded at a concentration of 5 x 10 ⁴ cells/mL in a T25 culture flask, the breast cancer cell line MDA-MB-231 (ATCC) was seeded at a concentration of 5 x 10 ⁴ cells/mL in a T25 culture flask, the pancreatic cancer cell line MIA PaCa-2 (ATCC) was seeded at a concentration of 5 x 10 ⁴ cells/mL in a T25 culture flask, and the normal cell line human dermal Fibroblast (Lonza, USA) was seeded at a concentration of 5 x 10 ⁴ cells/mL in a T25 culture flask and used in the experiment.

Example 1: DSPE-PEDS _{5CFL-Arg-PEG1k} - Natural killer cells cultured under a coating composition containing FA complex

10 mL αMEM medium containing 5 x 10 ⁵ cells of NK-92mi cells cultured by the method of Manufacturing Example 3 was mixed with 100 μL of a coating solution having a concentration of 2 mg/mL, prepared by dissolving powdered DSPE-PEDS _{5CFL-Arg-PEG1k} -FA prepared by the method of Manufacturing Example 1 in αMEM medium, and the NK-92mi cells were cultured at 25°C for 30 minutes to prepare NK-92mi cells whose surfaces were coated with the DSPE-PEDS _{5CFL-Arg-PEG1k} -FA compound.

The surface-coated NK-92mi cells were washed twice with 1 mL of DPBS (dulbecco's phosphate buffered saline, Sigma-Aldrich).

Example 2: DSPE-PEDS _{5CFL-Arg-PEG1k} - Natural killer cells cultured under a coating composition containing a biotin complex

1 mg of DSPE-PEDS _{5CFL-Arg-PEG1k} -Biotin, manufactured by the method of Manufacturing Example 2, was dissolved in 0.5 mL of αMEM at 100% concentration to prepare 0.5 mL of a coating solution (2 mg/mL).

NK-92mi cells (5 x 10 ⁵ cells) were centrifuged to form a cell pellet, 0.1 mL of the coating solution (2 mg/mL) was added to the cell pellet, and the pellet was incubated at 25°C for 30 minutes to prepare NK-92mi cells whose surface was coated with the DSPE-PEDS _{5CFL-Arg-PEG1k} -Biotin compound.

The surface-coated NK-92mi cells were washed twice with 1 mL of DPBS (dulbecco's phosphate buffered saline, Sigma-Aldrich).

Comparative Example 1. Uncoated NK-92mi cells

Culture was performed in the same manner as in Examples 1 and 2, except that DSPE-PEDS _{5CFL-Arg-PEG1k} -FA and DSPE-PEDS _{5CFL-Arg-PEG1k} -Biotin were not added to the cell culture medium.

Experimental Example 1. Confirmation of coating form, coating efficiency, and coating maintenance ability on the surface of NK-92mi cells

1-1: Confirmation of coating form

To visualize the coating morphology, the surface-coated NK-92mi cells were labeled with a fluorescent substance, 5-carboxyfluorescein, and observed under a fluorescence microscope (Ti-E System, Nikon, Japan).

As a result of the above, Fig. 2 confirmed that the coating component including the DSPE-PEDS _{5CFL-Arg-PEG1k} -FA conjugate was well coated on the surface of NK-92mi cells.

1-2: Checking the coating efficiency

The surface of uncoated NK-92mi cells was treated with coating solutions containing the DSPE-PEDS _{5CFL-Arg-PEG1k} -FA conjugate at concentrations of 0.5 mg/mL, 1 mg/mL, and 2 mg/mL, respectively, and the cell coating efficiency was confirmed using a cell analyzer (flow cytometry, (Beckman Coulter, USA)) to determine the optimal coating concentration of 2 mg/mL.

Figure 3 shows the results above. On the left, the coating efficiency of NK-92mi cells changes as the concentration of the coating solution changes to 0.5 mg/mL, 1 mg/mL, and 2 mg/mL, respectively. On the right, the coating efficiency of NK-92mi cells is analyzed using a fluorescent label, and the fluorescence intensity of the supernatant at each concentration is measured using microplate spectrophotometry (Ex/Em = 485/535 nm wavelength).

Here, when the concentration of the coating solution was 2 mg/mL, the fluorescence intensity was measured to be the highest, confirming that the coating efficiency was the best in the coating solution having the concentration of 2 mg/mL.

1-3: Check coating retention ability

NK-92mi cells coated with the above surface were incubated in complete growth medium at 37°C.

Cells were collected at 0 h, 6 h, 12 h, 24 h, and 48 h, and the fluorescence signals were measured using a cell analyzer (flow cytometry, (Beckman Coulter, USA)) and compared with the fluorescence signals of uncoated NK-92mi cells to analyze how long the DSPE-PEDS _{5CFL-Arg-PEG1k} -FA conjugate remained.

Figure 4 shows that the results are consistent with the fluorescence signal of uncoated NK-92mi cells after 48 hours, confirming that the DSPE-PEDS _{5CFL-Arg-PEG1k} -FA conjugate exists on the surface of NK-92mi cells for up to 48 hours after coating.

Experimental Example 2: Confirmation of improved cancer cell recognition ability

MCF-7 breast cancer cells, MDA-MB-231 breast cancer cells, MIA PaCa-2 pancreatic cancer cells, and human fibroblasts (hFibroblasts) as a normal control were seeded at 10,000 cells per well in 96-well plates and incubated at 37°C, 5% CO2, and 95°C for 24 hours.

To sufficiently saturate the folic acid receptor (FAR) on the surface of the breast cancer and pancreatic cancer cells, 0.1 mL of folic acid (1 mM) was added to the cell medium at 37°C for 2 hours.

NK-92mi cells were coated in the same manner as in Example 1 or Example 2, labeled with 10 μM CellTracker™Blue CMAC Dye (Invitrogen, USA) at 37°C for 30 minutes, and washed twice with DPBS. As a control, NK-92mi cells of Comparative Example 1 were labeled with 10 μM CellTracker™Blue CMAC Dye (Invitrogen, USA) at 37°C for 30 minutes, and washed twice with DPBS.

The ratio of the effector cells (NK-92mi cells (Comparative Example 1) or coated NK-92mi cells (Example 1 or 2)) was set to 10, and the ratio of target cells (MCF-7 breast cancer cells, MDA-MB-231 breast cancer cells, MIA PaCa-2 pancreatic cancer cells, and human fibroblasts, respectively) was set to 1, so that the effector cells to target cells (E:T) were 10:1, and co-cultured at 37°C for 30 minutes, and unbound effector cells were collected. Thereafter, the unbound effector cells were quantified using a standard curve based on the fluorescence intensity of the labeled effector cells to calculate the remaining effector cell to target cell (E:T) ratio.

Figure 18 is a graph showing the ratio of effector cells recognizing target cells to target cells.

The results above confirmed that the coated NK-92mi cells: showed the highest cancer cell recognition level in the target cell (MCF-7 breast cancer cells, MDA-MB-231 breast cancer cells, or MIA PaCa-2 pancreatic cancer cells) group, and this showed that the cancer cell recognition ability of the coated natural killer cells was significantly improved by about 1.4 times compared to that of the uncoated natural cells.

Experimental Example 3: Confirmation of enhanced cancer cell killing ability of coated natural killer cells - FA

An experiment was performed to verify the degradation or death of MCF-7 breast cancer cells, MDAMB-231 breast cancer cells, or MIA PaCa-2 pancreatic cancer cells by incubating the effector cells (NK-92mi cells (Comparative Example 1) or coated NK-92mi cells (Example 1 or 2)) to target cells (MCF-7 breast cancer cells, MDA-MB-231 breast cancer cells, MIA PaCa-2 pancreatic cancer cells, and human fibroblasts, respectively) at a ratio of 1:1, 2:1, 4:1, and 10:1 for 24 hours under conditions of 37°C, 5% CO2, and 95% humidity. Here, the coated NK-92mi cells used cells cultured by the method of Example 1, and the uncoated NK-92mi cells used cells cultured by the method of Comparative Example 1.

The lysis/death of cells by natural killer cells was confirmed using the calcein-AM release assay. The numerical ratio of cell lysis/death was calculated using the known calcein-AM release assay.

Figure 20 shows the results of confirming the killing effect of NK-92mi cells on cancer cell lines cultured according to the methods of Example 1 and Comparative Example 1.

As a result, as can be confirmed in FIG. 20, when natural killer cells coated with the DSPE-PEDS _{5CFL-Arg-PEG1k} -FA conjugate of Manufacturing Example 1 of the present invention were cultured together with cancer cell lines, there was a significant increase in the cell killing effect of the cancer cell lines compared to when uncoated natural killer cells were cultured together with cancer cell lines. In addition, as the number of natural killer cells coated with the DSPE-PEDS _{5CFL-Arg-PEG1k} -FA conjugate of Manufacturing Example 1 of the present invention was gradually increased and cultured with cancer cell lines, the effect of promoting the cell killing effect of the cancer cell lines was confirmed to increase even more dramatically.

However, when natural killer cells were cultured together with human fibroblasts, which were not cancer cell lines used as a control, it was confirmed that cell death was not induced even if the natural killer cells coated with the DSPE-PEDS _{5CFL-Arg-PEG1k} -FA complex of Manufacturing Example 1 of the present invention were cultured in large quantities.

Experimental Example 4: Confirmation of enhanced cancer cell killing ability of coated natural killer cells - Biotin

An experiment was performed to verify the degradation or death of MCF-7 breast cancer cells, MDA-MB-231 breast cancer cells, or MIA PaCa-2 pancreatic cancer cells by incubating the effector cells (NK-92mi cells (Comparative Example 1) or coated NK-92mi cells (Example 1 or 2)) to target cells (MCF-7 breast cancer cells, MDA-MB-231 breast cancer cells, MIA PaCa-2 pancreatic cancer cells, and human fibroblasts, respectively) at a ratio of 1:1, 2: ₁ , 4:1, and 10:1 under conditions of 37°C, 5% CO2, and 95% humidity for 24 hours. Here, the coated NK-92mi cells used cells cultured by the method of Example 2, and the uncoated NK-92mi cells used cells cultured by the method of Comparative Example 1.

The lysis/death of cells by natural killer cells was confirmed using the calcein-AM release assay. The numerical ratio of cell lysis/death was calculated using the known calcein-AM release assay.

Figure 21 shows the results of confirming the killing effect of NK-92mi cells on cancer cell lines cultured according to the methods of Example 2 and Comparative Example 1.

As a result, as can be confirmed in FIG. 21, when natural killer cells coated with the DSPE-PEDS _{5CFL-Arg-PEG1k} -Biotin conjugate of Manufacturing Example 1 of the present invention were cultured together with cancer cell lines, there was a significant increase in the cell killing effect of the cancer cell lines compared to when uncoated natural killer cells were cultured together with cancer cell lines. In addition, as the number of natural killer cells coated with the DSPE-PEDS _{5CFL-Arg-PEG1k} -Biotin conjugate of Manufacturing Example 2 of the present invention was gradually increased and cultured with cancer cell lines, the effect of promoting the cell killing effect of the cancer cell lines was confirmed to increase even more dramatically.

However, when natural killer cells were cultured together with human fibroblasts, which were not cancer cell lines used as a control, it was confirmed that cell death was not induced even if the natural killer cells coated with the DSPE-PEDS _{5CFL-Arg-PEG1k} -Biotin complex of Manufacturing Example 2 of the present invention were cultured in large quantities.

Experimental Example 5: Confirmation of cancer cell death effect through confirmation of glutathione release

In the same manner as in Experimental Example 3, the ratio of effector cells (NK-92mi cells (Comparative Example 1) or coated NK-92mi cells (Example 1 or 2)) to target cells (MCF-7 breast cancer cells, MDA-MB-231 breast cancer cells, MIA PaCa-2 pancreatic cancer cells, and human fibroblasts, respectively) was set to 10:1, and incubated for 4 hours under the conditions of 37°C, 5% CO2, and 95% humidity, and the degree of glutathione (GSH) release from MCF-7 breast cancer cells, MDA-MB-231 breast cancer cells, or MIA PaCa-2 pancreatic cancer cells was measured. Here, Triton X-100 is a positive control that artificially destroys cancer cells to release glutathione (GSH) inside the cancer cells.

Measurement of glutathione released from destroyed cancer cells was performed using the EZ-glutathione assay kit (DoGenBio) according to the manufacturer's protocol.

Figure 22 is a graph showing the results of confirming the concentration of glutathione released from each target cell (MCF-7 breast cancer cells, MDA-MB-231 breast cancer cells, MIA PaCa-2 pancreatic cancer cells, or human fibroblasts).

As a result, as can be confirmed in FIG. 22, when NK-92mi cells coated with the DSPE-PEDS _{5CFL-Arg-PEG1k} -Biotin conjugate of Manufacturing Example 1 of the present invention were co-cultured with a cancer cell line, the glutathione release effect was significantly improved compared to when uncoated NK-92mi cells were co-cultured with a cancer cell line. Through this, it was confirmed that NK-92mi cells coated with the DSPE-PEDS _{5CFL-Arg-PEG1k} -Biotin conjugate of Manufacturing Example 1 of the present invention were significantly excellent in destroying cancer cells.

When natural killer cells and cancer cells were co-cultured, the natural killer cells destroyed the cancer cell line, releasing glutathione (GSH). This effect was confirmed to be due to the fact that more cancer cell death was induced in the coated NK cell group, which also increased glutathione release.

As a result, as shown in Figure 22, in the general cell line triton-x100 treatment group, it was confirmed that glutathione (GSH) was released even when the general cell line was killed.

However, when natural killer cells and normal cells were co-cultured, the natural killer cells or coated natural killer cells did not destroy the normal cells, so no release of glutathione (GSH) occurred.

Experimental Example 6: Cancer cell death effect through antifolate mediation

In order to confirm the cancer cell killing effect through anti-folate-mediated, a structure was fabricated by immobilizing the DSPE-PEDS _{5CFL-Arg-PEG1k} -FA conjugate of Manufacturing Example 1 of the present invention on a gelatin-coated solution, and the following experiment was performed by simulating the glutathione environment released from cancer cells.

10 μL of 2% (w/v) gelatin solution (derived from porcine skin, Sigma-Aldrich, G1890) was dropped onto the bottom surface of a polycarbonate transwell (0.4 μm pore, Corning, CLS3428-24EA), and the gelatin-treated transwell was dried at room temperature for 2 h.

Afterwards, 1 mL of the prepared 5 mM 1,2-distearoyl-sn-glycero 3-phosphoethanolamine-N-[succinimidyl (PEG)] (DSPE-PEG-NHS, Nanosoft Polymers, USA) solution was added to the gelatin-coated transwell and stirred for 1 hour to conjugate the amino group of gelatin with the NHS moiety of 1,2-distearoyl-sn-glycero 3-phosphoethanolamine-N-[succinimidyl (PEG)] and expose the lipid portion (DSPE) to the outside.

Finally, a 2 mg/mL coating solution of Example 1 was applied to the lipid for 30 minutes to produce a structure simulating the surface of natural killer cells (Fig. 23).

As in Experimental Example 1, MCF-7 breast cancer cells, MDA-MB-231 breast cancer cells, MIA PaCa-2 pancreatic cancer cells, and human fibroblasts (hFibroblasts) as a normal control were seeded at 10,000 cells per well in a 24-well plate and incubated for 24 hours under conditions of 37°C, 5% CO2, and 95% humidity.

The cell culture medium was replaced with fresh cell culture medium containing 100 μM glutathione (Sigma-Aldrich, G6013), and the coated transwell was inserted into the plate seeded with cells and cultured, and incubated at 37°C for 24 hours.

Then, cells were separated by trypsin treatment, collected by centrifugation, lysed using a homogenizer, and the amount of DNA in target cells cultured in transwells was quantified according to the manufacturer's protocol using Quant-iT PicoGreen dsDNA Reagent Kit (Thermo Fisher Scientific, USA).

Figure 19 shows the results of verifying whether the dissociated FA, which was formed by breaking the disulfide bond of the DSPE-PEDS _{5CFL-Arg-PEG1k} -FA complex of Manufacturing Example 1 of the present invention by glutathione, promotes cancer cell death by interfering with DNA synthesis of cancer cells.

Through the above results, it was confirmed that in an environment where glutathione (GSH) was treated, compared to when only GSH was treated or when coated with gelatin, the disulfide bond of the DSPE-PEDS _{5CFL-Arg-PEG1k} -FA conjugate of Manufacturing Example 1 of the present invention fixed to a gelatin-coated transwell was broken, dissociating folic acid (FA), and after dissociation, folic acid, which is a cancer cell recognition moiety, was converted into antifolate, thereby additionally inducing cancer cell death.

Experimental Example 7: Measurement of viability of coated natural killer cells

NK-92mi cells cultured by the method of Manufacturing Example 3 were cultured in 10 mL αMEM medium, and 100 μL of a coating solution having a concentration of 2 mg/mL, prepared by dissolving powdered DSPE-PEDS _{5CFL-Arg-PEG1k} -FA prepared by the method of Manufacturing Example 1 in αMEM medium, was mixed, and the NK-92mi cells were cultured at 25°C for 30 minutes to prepare NK-92mi cells whose surfaces were coated with the DSPE-PEDS _{5CFL-Arg-PEG1k} -FA compound.

In the process of preparing NK-92mi cells coated with the above surface, NK-92mi cells were prepared separately by adjusting the concentration of the coating solution to 0.5 mg/mL and 1 mg/mL, respectively.

The surface-coated NK-92mi cells were washed three times with 1 mL of DPBS (dulbecco's phosphate buffered saline, Sigma-Aldrich) at each prepared concentration (0.5 mg/mL, 1 mg/mL, 2 mg/mL).

The viability of the surface-coated NK-92mi cells, adjusted to each concentration of the above washing, was evaluated through the WST-1 assay using EZ-Cytox (DoGenBio, Korea). UV absorbance was measured at 450 nm using an Infinite M200 micro-plate reader (Tecan, Zurich, Switzerland).

The graph on the left in Fig. 5 is the result of measuring the viability of surface-coated NK-92mi cells by adjusting each concentration through the WST-1 assay. Compared to the control group with an uncoated surface, there was almost no change in absorbance, indicating that coating the surface of NK-92mi cells with a solution containing up to 2 mg/mL of the DSPE-PEDS _{5CFL-Arg-PEG1k} -FA compound did not affect the viability.

Experimental Example 8: Confirmation of the proliferation ability of coated natural killer cells

NK-92mi cells cultured by the method of Manufacturing Example 3 were cultured in 10 mL αMEM medium, and 100 μL of a coating solution having a concentration of 2 mg/mL, prepared by dissolving powdered DSPE-PEDS _{5CFL-Arg-PEG1k} -FA prepared by the method of Manufacturing Example 1 in αMEM medium, was mixed, and the NK-92mi cells were cultured at 25°C for 30 minutes to prepare NK-92mi cells whose surfaces were coated with the DSPE-PEDS _{5CFL-Arg-PEG1k} -FA compound.

In the process of preparing NK-92mi cells coated with the above surface, NK-92mi cells were prepared separately by adjusting the concentration of the coating solution to 0.5 mg/mL and 1 mg/mL, respectively.

The surface-coated NK-92mi cells were washed three times with 1 mL of DPBS (dulbecco's phosphate buffered saline, Sigma-Aldrich) at each prepared concentration (0.5 mg/mL, 1 mg/mL, 2 mg/mL).

A comparison of proliferation capacity according to cell culture density was performed during long-term cell culture. The proliferation capacity of each cell was confirmed by calculating the proliferation rate for each passage through the number of cells initially seeded and the number of cells obtained after culture was completed, and the results are shown on the right side of Fig. 6.

Experimental Example 9: Measurement of secretion of immune factors from coated natural killer cells

To confirm the extent of secretion of immune factors through antigen recognition of NK-92mi cells coated by the method of Example 1, the amount of interferon gamma (IFN-γ) secretion dependent on lipopolysaccharide (LPS) was analyzed.

1.5 X 10 ⁵ uncoated NK-92mi cells of Comparative Example 1 and 1.5 X 10 ⁵ uncoated NK-92mi cells of Example 1 were cultured in the medium containing 1 μg/mL of LPS (Escherichia coli O26:B6, Sigma-Aldrich), and the cells were incubated at 37°C for 24 hours, centrifuged, and the medium was collected. Thereafter, 1.5 X 10 ⁵ uncoated NK-92mi cells of Comparative Example 1 and 1.5 X 10 ⁵ uncoated NK-92mi cells of Example 1 were each incubated at 37°C for 24 hours, centrifuged, and the medium was collected, which was used as a control.

As a result, as can be confirmed in Fig. 6, in the control group that was not treated with lipopolysaccharide, the interferon gamma secretion amount of uncoated NK-92mi cells was slightly higher than that of coated NK-92mi cells, but in the experimental group treated with lipopolysaccharide, the interferon gamma secretion amount of coated NK-92mi cells was higher than that of uncoated NK-92mi cells.

Through this, it was confirmed that the interferon gamma secretion amount of NK-92mi cells was at the same level as the interferon gamma secretion amount of coated NK-92mi cells, and in the experimental group treated with lipopolysaccharide, the interferon gamma secretion amount of NK-92mi cells was improved to the same level as the interferon gamma secretion amount of coated NK-92mi cells.

In summary, we were able to confirm that the coating material coated on the surface of NK cells does not affect the series of processes in which antigen substances such as LPS are recognized, a corresponding cell signaling mechanism occurs, and the resulting substance, interferon gamma, is released outside the cell.

Experimental Example 10: Confirmation of the cancer cell killing effect of coated natural killer cells in the body

To confirm the cancer cell killing effect of NK-92mi cells coated by the method of Example 1, an MDA-MB-231 xenograft model was prepared.

The above xenograft model was prepared by subcutaneously inoculating ^1x107 MDA-MB-231 cells into the left flank of 40 8-week-old nude (BALB/c nu/nu) mice (narabio, Korea).

Afterwards, when the tumor volume of the xenografted mice reached 100 mm ³ , the xenografted mice were randomly divided into the control group, the atezolizumab injection group, the uncoated NK-92mi cell injection group, and the coated NK cell injection group (n = 10 mice per group).

The classified control group or each injection group was inoculated with 250 μL of phosphate-buffered saline (PBS).

Afterwards, the uncoated NK-92mi cell injection group was intravenously injected with ^1x107 uncoated NK-92mi cells, and the coated NK cell injection group was intravenously injected with ^1x107 coated NK cells. Meanwhile, the atezolizumab injection group was intraperitoneally injected with 10 mg/kg atezolizumab (BioXCell, USA) every 3 days.

The control group was inoculated with only 250 μL of phosphate-buffered saline (PBS) during the above process and did not receive any other inoculations.

Afterwards, five mice per group (total of 20 mice) were randomly selected and the changes in tumor volume, tumor growth inhibition ratio (TGI), fold change in body weight change, and survival probability were analyzed as in the experimental examples below.

The experimental process for confirming the in vivo cancer cell killing effect of coated natural killer cells is schematically shown in Figure 24A.

10-1: Measurement of fold change in tumor volume

To determine the effect of cancer cell death in the body, tumor growth was monitored for 14 days, and the recorded values were divided by the initial value to calculate the tumor volume using the following formula.

Tumor volume = 0.5 x a x ^{b 2} (where a is the diameter of the longest tumor tissue and b is the diameter of the shortest tumor tissue.)

As a result, as shown in Figure 24E, the tumor volume of each mouse in the coated NK-92mi cell injection group was lower than that of the other control or injection groups.

In addition, as shown in B of Fig. 24, the average tumor volume of the uncoated NK-92mi cell injection group was suppressed compared to the control group, but the tumors continued to grow. On the other hand, the coated NK-92mi cell injection group showed a remarkable decrease in the average tumor volume from day 4. The tumor volume of the coated NK-92mi cell injection group after 14 days was 1/4 compared to the control group, and after 14 days, the tumor volume was 1/2 compared to the atezolizumab injection group, confirming a remarkable cancer cell killing effect.

10-2: Measurement of tumor growth inhibition rate

To determine the effect of cancer cell killing in the body, tumor growth was monitored for 14 days and the tumor growth inhibition rate (TGI) was calculated using the following formula.

(from here is the average tumor volume of the treated group at a given point in time, is the mean tumor volume of the treated group at the initial time point, is the average tumor volume of the control group at a given point in time, is the mean tumor volume of the control group at the initial time point.)

Here, the average tumor volume is the average value calculated by calculating the tumor volume of each mouse using the tumor volume calculation formula of Experimental Example 10-1.

As a result, as shown in C of Figure 24, the tumor growth inhibition rate of the uncoated NK-92mi cell injection group was 10%, and the tumor growth inhibition rate of the coated NK-92mi cell injection group was 80%, confirming that the tumor growth inhibition rate of the coated NK-92mi cell injection group was 8 times greater than that of the uncoated NK-92mi cell injection group, indicating a significant anticancer effect.

Meanwhile, in the case of the atezolizumab injection group, tumor suppression was significantly promoted compared to the control group and the uncoated NK-92mi cell injection group, but compared to the coated NK-92mi cell injection group, the tumor volume was doubled after 14 days, and the tumor growth inhibition rate was 60%, confirming that the anticancer effect was relatively lower compared to the coated NK-92mi cell injection group.

10-3: Measuring weight change

To determine the effect of cancer cell death in the body, the average body weight of mice in the control group and each injection group was monitored for 14 days.

As a result, as shown in Figure 24D, the fold change in body weight of mice classified into each group was almost constant, and thus, it was confirmed that the treatment administered by classifying into each group did not cause systemic toxicity.

10-4: Measuring survival probability

To determine the effect of cancer cell death in the body, the survival rate of mice classified into each group (10 mice per group) was monitored for 100 days.

The survival probability was calculated using the following formula.

(Here, the daily survival population refers to the number of individuals observed and surviving at a certain point in time during the day.)

As a result, as shown in F of Fig. 24, the survival rate of the coated NK-92mi cell injection group was 80% at 100 days, the atezolizumab injection group was 60%, and the control group and the uncoated NK-92mi cell injection group were 0%, confirming that the coated NK-92mi cell injection group had a significantly superior anticancer effect compared to the control group or other injection groups.

Experimental Example 11: Enhanced cancer suppression ability and biocompatibility of coated natural killer cells through histological analysis

A xenograft mouse model was prepared using the same method as Experimental Example 10 above, and five mice per group (total of 20 mice) were randomly selected and euthanized for histological analysis.

Tumors and major organs collected from xenograft mice of each group above were fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde for 24 hours and then stored in paraffin.

Afterwards, specimen sections (5-μm thick) were stained with fluorescent dye compounds, and the slides were heated at 98°C for 20 min in 100 mL of 10 mM citric acid buffer (pH 6.0), and cooled to 25°C for 20 min.

After the cooling, the samples were incubated at 4°C for 16 h in a solution containing antibodies each recognizing CD56 (MAB24083; R&D Systems, USA; 1:200), cleaved caspase3 (9661, Cell Signaling Technology, USA; 1:200), Ki67 (ab16667, Abcam; 1:200), HMGB-1 (ab18256, Abcam; 1:100), CD8a (14-0081-82, Invitrogen; 1:100), HLA (Class I ABC, ab70328, Abcam; 1:200), and von Willebrand factor (vWF , AB7356, Sigma-Aldrich; 1:200).

After the above incubation, the samples were incubated with secondary antibody (Invitrogen; 1:200) at 20°C for 2 h and counterstained with TO-PRO-3 (TP3, T3605, Invitrogen, 1 μM).

Fluorescence detection was performed using a confocal laser microscope (DMi8; Leica, Germany). Tumor and central necrotic areas were traced on H&E images and measured using ImageJ software. The number of marker-positive cells was counted and the area positive for specific markers was measured on immunohistochemical images. Five regions of interest (ROIs; 200 × 200 μm) were randomly selected from single-stained slides of each sample (five samples per group) (total of 25 ROIs per group).

Tumor tissues from MDA-MB-231 xenograft mice (five mice per group) were stained with hematoxylin and eosin (H&E). In Fig. 25A , T represents the tumor tissue area and N represents the necrotic area.

As a result of hematoxylin and eosin (H&E) staining of tumor tissues, as shown in Figure 25A, tumor tissues showing necrotic morphology such as eosinophilic cell expansion, nuclear fusion, and nuclear lysis were observed much more in the atezolizumab and coated NK-92mi cell injection groups than in the control and uncoated NK-92mi cell injection groups, confirming that the anticancer effect of the NK-92mi cell injection group was remarkable.

In addition, as shown in Figure 25B, the necrosis area percentage was the highest at 36% in the coated NK-92mi cells, confirming that it induced the most extensive tumor necrosis compared to the other groups.

Cleaved caspase 3 is a protein that induces the death of cancer cells through apoptosis. The higher the expression of cleaved caspase 3, the more active apoptosis is induced, indicating a superior anticancer effect.

As a result of analyzing the expression level of cleaved caspase3, as shown in Figure 25C, the control group and

Since it was observed much more in the atezolizumab and coated NK-92mi cell injection group than in the uncoated NK-92mi cell injection group, it was confirmed that the anticancer effect of the NK-92mi cell injection group was remarkable.

Figures 25E and F show the extent of tumor cell proliferation using the proliferation marker Ki67. Here, extensive expression of Ki67 was observed in the control group, atezolizumab injection group, and uncoated NK-92mi cell injection group, but significantly lower expression was observed in the coated NK-92mi cell injection group.

The relative proportion of tumor or infiltrating cells in the group injected with coated NK-92mi cells and the group injected with uncoated NK-92mi cells was determined using human-specific CD56 (a natural killer cell marker).

As a result, as shown in FIG. 25G, it was confirmed that the number of NK cells expressing human-specific CD56 was significantly increased in the sample of the group injected with coated NK-92mi cells compared to the sample of the group injected with uncoated NK-92mi cells. Through this, it was found that when NK cells are coated using the polymer compound of Example 1 of the present invention, the characteristic of infiltrating cancer cells is improved compared to uncoated NK cells, thereby exhibiting an excellent cancer cell killing effect.

A lower relative proportion of tumor or infiltrating cells was observed in samples from the group injected with coated NK-92mi cells.

In Figure 25H, CD56+cell ratio represents the relative proportion of human-specific CD56 expressed in total cells. Here, the relative proportion of CD56 was observed in the liver, lung, and spleen, but not in the heart and lung.

In conclusion, histological analysis results confirmed that administration of coated natural killer cells effectively inhibited tumor growth in a xenograft model.

Experimental Example 12: Confirmation of changes in the tumor microenvironment when coated natural killer cells are administered to a triple-negative breast cancer animal model

Figure 26 is a diagram illustrating changes in the tumor microenvironment that occur when coated natural killer cells are administered to a triple-negative breast cancer animal model.

NK-92mi cells coated by the method of Example 1 were administered to a triple-negative breast cancer animal model, and changes in the tumor microenvironment were confirmed. Specifically, the methods described in Experimental Examples 10 and 11 were used. In addition, the coated natural injury cells are indicated as SCNK cells in Fig. 26.

In Figure 26A, a cancer with a relatively high response rate is referred to as a hot tumor, and a cancer with a low response rate is referred to as a cold tumor. It can be seen that a change into a tumor with a high response rate can be induced by the coated natural killer cells.

In Figures B and C of Figure 26, it can be confirmed that the expression of HMGB1, a representative antigen marker that can activate innate immunity and a cancer cell death marker, increases.

In Figures D and E of 26, it can be confirmed that CD8+ cytotoxic T cells that have infiltrated into tumor tissue increase.

Figures 26F and G showed an increase in inflammatory cytokines such as IFN- γ and TNF-477 present in the blood.

Through this, it can be seen that the administration of the coated natural killer cells can control the immune response within the tumor microenvironment where triple-negative breast cancer is formed.

Experimental Example 13: Confirmation of inhibition of lung metastasis by coated natural killer cells administered to triple-negative breast cancer animal model

Figure 27 is a diagram illustrating the inhibition of lung metastasis by coated natural killer cells administered to a triple-negative breast cancer animal model.

NK-92mi cells coated by the method of Example 1 were administered to a triple-negative breast cancer animal model, and inhibition of metastasis to the lungs was confirmed. Specifically, the methods described in Experimental Examples 10 and 11 were used. In addition, the coated natural injury cells are indicated as SCNK cells in Fig. 27.

Atezolizumab is a commercialized monoclonal antibody drug for the treatment of triple-negative breast cancer. Human leukocyte antigen (HLA, human major histocompatibility complex) is a marker for colonies of cancer cells that have metastasized to the lungs, and Von Willebrand factor (vWF) is a marker of angiogenesis in tumor tissues with developed blood vessels. The decrease in the two factors shows that the lung metastasis inhibition effect by coated natural killer cells (SCNK cells) is superior to that of unmodified natural killer cells (NK cells) or atezolizumab.

Experimental Example 14: Confirmation of inhibition of liver metastasis by coated natural killer cells administered to triple-negative breast cancer animal model

Figure 28 is a diagram illustrating the inhibition of liver metastasis by coated natural killer cells administered to a triple-negative breast cancer animal model.

NK-92mi cells coated by the method of Example 1 were administered to a triple-negative breast cancer animal model, and inhibition of metastasis to the liver was confirmed. Specifically, the methods described in Experimental Examples 10 and 11 were used. In addition, the coated natural injury cells are indicated as SCNK cells in Fig. 28.

Atezolizumab is a commercialized monoclonal antibody drug for the treatment of triple-negative breast cancer. As shown in Experimental Example 12, the expression of two types of cancer markers (HLA and vWF) was also reduced in liver tissue, demonstrating that the liver metastasis inhibition effect by coated SCNK cells was superior to that of unmodified NK cells or atezolizumab.

Although the embodiments have been described above by way of limited examples and drawings, those skilled in the art can make various modifications and variations from the above description. For example, appropriate results can be achieved even if the described techniques are performed in a different order from the described method, and/or the described components are combined or combined in a different form from the described method, or are replaced or substituted by other components or equivalents. Therefore, other implementations, other embodiments, and equivalents to the claims also fall within the scope of the following claims.

Claims (14)

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 coupled to one end of the linker and the cancer cell recognition moiety is coupled to the other end of the linker to recognize natural killer cells and cancer cells.
The above linker is a polymer compound in which multiple peaks are confirmed in the ranges of 2.09 to 4.34 ppm and 7.33 to 8.19 ppm in the ¹ H-NMR spectra, and a single peak at 6.28 ppm is confirmed in the range of 6 to 7 ppm.
In the first paragraph,
A polymer compound that inhibits the metastasis of triple-negative breast cancer cells to other organs.
In the first paragraph,
A polymer compound characterized in that the hydrophobic moiety binds to natural killer cells, and the cancer cell recognition moiety recognizes cancer cells and promotes the death of cancer cells.
In the first paragraph,
A polymer compound in which the hydrophobic moiety binding to the above natural killer cell is at least one selected from the group consisting of a phospholipid having an alkyl chain having 12 to 24 carbon atoms; a sterol lipid having 10 to 30 carbon atoms; 1,2-bis(diphenylphosphino)ethane (DPPE); and 1,2-bis(dimethylphosphino)ethane (DMPE).
In the first paragraph,
The hydrophobic moiety that binds to the above natural killer cells is a polymer compound whose peaks are confirmed at 1.27 ppm and 0.84 ppm in the ¹ H-NMR spectra.
In the first paragraph,
The cancer cell recognition moiety is a polymer compound comprising at least one selected from the group consisting of folic acid, biotin, lactobionic acid, and phenylboronic acid.
In the first paragraph,
The above cancer cell recognition moiety is a polymer compound in which multiple peaks are confirmed in the ranges of 2.31 to 4.49 ppm and 6.65 to 8.65 ppm in the ¹ H-NMR spectra, and a single peak is confirmed in the ranges of 11 to 12 ppm and 12 to 13 ppm, respectively.
In the first paragraph,
The above linker is a polymer compound comprising a structure of <chemical formula 1>.
<Chemical formula 1>

Here, n is an integer from 30 to 50.
In the first paragraph,
A polymer compound comprising at least one of a compound for preventing cell internalization, a cationic amino acid, and a fluorescent dye compound bound to the linker.
In Article 9,
The compound for preventing cell internalization is a polymer compound selected from the group consisting of polyethylene glycol (PEG); polyethylene oxide (PEO); polyvinyl alcohol (PVA); and copolymers thereof.
In Article 9,
A polymer compound wherein the cationic amino acid is at least one selected from the group consisting of arginine, lysine, and histidine.
¹ H-NMR spectra results show multiple peaks in the range of 2.09 to 4.34 ppm and 7.33 to 8.19 ppm, and a single peak at 6.28 ppm in the range of 6 to 7 ppm; and
A hydrophobic moiety is bonded to one end of the above linker and has peaks at 1.27 ppm and 0.84 ppm in the ¹ H-NMR spectra,
A polymer compound in which the hydrophobic moiety is bonded to a target to modify the surface of the target.
In Article 12,
The above target is a polymer compound containing natural killer cells or exosomes.
In Article 12,
To be combined with a cancer cell recognition moiety that recognizes cancer cells and promotes the death of cancer cells,
A polymer compound in which the cancer cell recognition moiety is coupled to the other terminal of the linker.