Composition for inducing dendritic cell differentiation, comprising metal-organic framework, and use thereof

Abstract

The present invention relates to a composition for inducing dendritic cell differentiation, comprising a metal-organic framework, and a use thereof, the composition having the effect of not only enabling the increasing of the expression of surface protein markers of dendritic cells, but also enabling the inducing of differentiation into dendritic cells having enhanced viability and migration properties when administered into an individual.

Description

Composition for inducing dendritic cell differentiation comprising a metal-organic framework and use thereof

[0001] The present invention relates to a composition for inducing dendritic cell differentiation comprising a metal-organic framework and its use.

[0002] Dendritic cells (DCs) are immune cells that form part of the mammalian immune system. They function as professional antigen-presenting cells (APCs) that process antigenic substances and present them on their surface so that other cells in the immune system can recognize them.

[0003] Dendritic cells are present in small numbers primarily in tissues in contact with the external environment, such as the skin, nose, lungs, stomach, and intestines, and constitute a heterogeneous population with a phenotype distinct from that of macrophages.

[0004] Specifically, dendritic cells have the ability to induce a primary immune response that can stimulate naive T cells that have never been exposed to an antigen, and are the only immune cells that have the property of inducing immune memory. It is known that the reason dendritic cells can perform such a strong immune response activating function is because, as antigen presenting cells (APCs), they express not only MHC molecules (I / II) on the cell surface, but also costimulatory molecules such as CD80 and CD86, and adhesion molecules such as ICAM-1 in high concentrations, and secrete various cytokines (IFN-alpha, IL-12, IL-18, etc.).

[0005] As mentioned above, dendritic cells are the most powerful antigen-presenting cells, and although they exist in extremely small numbers in the body, they have an excellent function in regulating T-cell immunity, and are therefore actively studied as a treatment for cancer or infectious diseases in clinical studies to induce immunity against specific antigens.

[0006] Specifically, it was confirmed that immature dendritic cells isolated from tissues or blood exhibit immunogenicity when stimulated with antigens in vitro, differentiated into mature dendritic cells, and then reinjected into vivo. If this technology is utilized, it is expected to have great application value as a cell vaccine for inducing immunity against specific antigens of cancer or pathogens.

[0007] Accordingly, there is a need to develop a technology to improve the differentiation characteristics of immature dendritic cells isolated from tissues or blood into mature dendritic cells.

[0008] [Prior Art Literature]

[0009] [Patent Document]

[0010] KR 10-2013-0023797 A

[0011] The purpose of the present invention is to provide a composition for inducing dendritic cell differentiation comprising a metal-organic framework and a use thereof.

[0012] Another object of the present invention is to provide a composition for inducing dendritic cell differentiation, which includes a metal-organic scaffold that can not only increase the expression of surface protein markers of dendritic cells but also induce differentiation into dendritic cells with improved viability and mobility when administered into a subject.

[0013] Another object of the present invention is to provide a method for inducing dendritic cell differentiation using the composition.

[0014] To achieve the above purpose, a composition for inducing dendritic cell differentiation according to one embodiment of the present invention includes a metal-organic framework (MOF).

[0015] The metal-organic framework comprises a metal cluster; and a ligand compound represented by the following chemical formula 1 that coordinately bonds to the metal cluster:

[0016] [Chemical Formula 1]

[0017]

[0018] Here,

[0019] m is an integer from 0 to 4,

[0020] R1 is hydrogen, deuterium, a cyano group, a nitro group, an amino group, a halogen group, a hydroxy group, a substituted or unsubstituted alkylthio group having 1 to 4 carbon atoms, a substituted or unsubstituted alkyl group having 1 to 30 carbon atoms, a substituted or unsubstituted cycloalkyl group having 3 to 20 carbon atoms, a substituted or unsubstituted alkenyl group having 2 to 30 carbon atoms, a substituted or unsubstituted alkynyl group having 2 to 24 carbon atoms, a substituted or unsubstituted aralkyl group having 7 to 30 carbon atoms, a substituted or unsubstituted aryl group having 6 to 30 carbon atoms, a substituted or unsubstituted heteroaryl group having 1 to 60 carbon atoms, a substituted or unsubstituted heteroarylalkyl group having 2 to 30 carbon atoms, a substituted or unsubstituted alkoxy group having 1 to 30 carbon atoms, a substituted or unsubstituted alkylamino group having 1 to 30 carbon atoms, a substituted Or it is selected from the group consisting of an unsubstituted arylamino group having 6 to 30 carbon atoms, a substituted or unsubstituted aralkylamino group having 7 to 30 carbon atoms, a substituted or unsubstituted heteroarylamino group having 1 to 24 carbon atoms, a substituted or unsubstituted alkylsilyl group having 1 to 30 carbon atoms, a substituted or unsubstituted arylsilyl group having 6 to 30 carbon atoms, and a substituted or unsubstituted aryloxy group having 6 to 30 carbon atoms.

[0021] The above R1 is selected from the group consisting of an amino group, a substituted or unsubstituted alkylamino group having 1 to 30 carbon atoms, a substituted or unsubstituted arylamino group having 6 to 30 carbon atoms, a substituted or unsubstituted aralkylamino group having 7 to 30 carbon atoms, and a substituted or unsubstituted heteroarylamino group having 1 to 24 carbon atoms.

[0022] The above metal-organic framework comprises one or more metals or metal ions selected from the group consisting of aluminum (Al), iron (Fe), and zirconium (Zr).

[0023] A method for inducing dendritic cell differentiation according to another embodiment of the present invention includes a step of treating immature dendritic cells with the composition and then culturing them.

[0024] The above immature dendritic cells are cells obtained by treating bone marrow cells with granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0025] According to another embodiment of the present invention, dendritic cells are differentiated by the differentiation induction method.

[0026] The above dendritic cells have increased expression of one or more surface protein markers selected from the group consisting of CD40, CD80, CD86, MHC class I, and MHC class II.

[0027] In the present invention, “hydrogen” is hydrogen, light hydrogen, deuterium or tritium, unless specifically limited.

[0028] In the present invention, “halogen group” is fluorine, chlorine, bromine or iodine.

[0029] In the present invention, “alkyl” refers to a monovalent substituent derived from a straight or branched saturated hydrocarbon having 1 to 40 carbon atoms. Examples thereof include, but are not limited to, methyl, ethyl, propyl, isobutyl, sec-butyl, pentyl, iso-amyl, and hexyl.

[0030] In the present invention, “alkenyl” refers to a monovalent substituent derived from a straight or branched unsaturated hydrocarbon having 2 to 40 carbon atoms and at least one carbon-carbon double bond. Examples thereof include, but are not limited to, vinyl, allyl, isopropenyl, and 2-butenyl.

[0031] In the present invention, “alkynyl” refers to a monovalent substituent derived from an unsaturated hydrocarbon having 2 to 40 carbon atoms and a straight or branched chain having at least one carbon-carbon triple bond. Examples thereof include, but are not limited to, ethynyl and 2-propynyl.

[0032] In the present invention, “alkylthio” means the above-described alkyl group bonded via a sulfur linkage (-S-).

[0033] In the present invention, “aryl” refers to a monovalent substituent derived from an aromatic hydrocarbon having 6 to 60 carbon atoms, which is a single ring or a combination of two or more rings. In addition, it may also include a form in which two or more rings are simply attached to each other (pendant) or condensed, and specifically, it may be a naphthyl group, anthracenyl group, phenanthryl group, triphenyl group, pyrenyl group, phenalenyl group, perylenyl group, chrysenyl group, fluorenyl group, etc., but is not limited thereto. The fluorenyl group may be substituted, and adjacent groups may be bonded to each other to form a ring.

[0034] In the present invention, “heteroaryl” refers to a monovalent substituent derived from a monoheterocyclic or polyheterocyclic aromatic hydrocarbon having 6 to 30 carbon atoms. At this time, at least one carbon atom in the ring, preferably 1 to 3 carbon atom(s), is substituted with a heteroatom such as N, O, S, or Se. In addition, a form in which two or more rings are simply attached to each other (pendant) or condensed may be included, and a form condensed with an aryl group may also be included. Examples of such heteroaryls include, but are not limited to, 6-membered monocyclic rings such as pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, and triazinyl; polycyclic rings such as phenoxathienyl, indolizinyl, indolyl, purinyl, quinolyl, benzothiazole, and carbazolyl; and 2-furanyl, N-imidazolyl, 2-isoxazolyl, 2-pyridinyl, and 2-pyrimidinyl.

[0035] In the present invention, “aryloxy” is a monovalent substituent represented by RO-, wherein R represents aryl having 6 to 60 carbon atoms. Examples of such aryloxy include, but are not limited to, phenyloxy, naphthyloxy, and diphenyloxy.

[0036] In the present invention, “alkyloxy” is a monovalent substituent represented by R'O-, wherein R' means alkyl having 1 to 40 carbon atoms, and may include a linear, branched, or cyclic structure. Examples of alkyloxy include, but are not limited to, methoxy, ethoxy, n-propoxy, 1-propoxy, t-butoxy, n-butoxy, and pentoxy.

[0037] In the present invention, “alkoxy” may be straight chain, branched chain, or cyclic chain. The carbon number of the alkoxy is not particularly limited, but is preferably 1 to 20 carbon atoms. Specifically, it may be methoxy, ethoxy, n-propoxy, isopropoxy, i-propyloxy, n-butoxy, isobutoxy, tert-butoxy, sec-butoxy, n-pentyloxy, neopentyloxy, isopentyloxy, n-hexyloxy, 3,3-dimethylbutyloxy, 2-ethylbutyloxy, n-octyloxy, n-nonyloxy, n-decyloxy, benzyloxy, p-methylbenzyloxy, etc., but is not limited thereto.

[0038] As used herein, "aralkyl" refers to an aryl-alkyl group, where aryl and alkyl are as defined above. Preferred aralkyl groups include lower alkyl groups. Non-limiting examples of suitable aralkyl groups include benzyl, 2-phenethyl, and naphthalenylmethyl. Bonding to the parent moiety is via the alkyl group.

[0039] In the present invention, “arylamino group” means an amine substituted with an aryl group having 6 to 30 carbon atoms.

[0040] In the present invention, “alkylamino group” means an amine substituted with an alkyl group having 1 to 30 carbon atoms.

[0041] In the present invention, “aralkylamino group” means an amine substituted with an aryl-alkyl group having 6 to 30 carbon atoms.

[0042] In the present invention, “heteroarylamino group” means an amine group substituted with an aryl group and a heterocyclic group having 6 to 30 carbon atoms.

[0043] In the present invention, “heteroaralkyl group” means an aryl-alkyl group substituted with a heterocyclic group.

[0044] In the present invention, “cycloalkyl” refers to a monovalent substituent derived from a monocyclic or polycyclic non-aromatic hydrocarbon having 3 to 40 carbon atoms. Examples of such cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and adamantine.

[0045] In the present invention, “heterocycloalkyl” means a monovalent substituent derived from a non-aromatic hydrocarbon having 3 to 40 carbon atoms, wherein at least one carbon atom in the ring, preferably 1 to 3 carbon atom(s), is substituted with a heteroatom such as N, O, S or Se. Examples of such heterocycloalkyl include, but are not limited to, morpholine and piperazine.

[0046] In the present invention, “alkylsilyl” means silyl substituted with alkyl having 1 to 40 carbon atoms, and “arylsilyl” means silyl substituted with aryl having 6 to 60 carbon atoms.

[0047] In the present invention, “fused ring” means a fused aliphatic ring, a fused aromatic ring, a fused heteroaliphatic ring, a fused heteroaromatic ring, or a combination thereof.

[0048] In the present invention, “forming a ring by bonding with adjacent groups” means forming a substituted or unsubstituted aliphatic hydrocarbon ring; a substituted or unsubstituted aromatic hydrocarbon ring; a substituted or unsubstituted aliphatic heterocycle; a substituted or unsubstituted aromatic heterocycle; or a condensed ring thereof by bonding with adjacent groups.

[0049] Examples of the “aromatic hydrocarbon ring” in the present invention include, but are not limited to, a phenyl group, a naphthyl group, an anthracenyl group, etc.

[0050] In the present invention, “aliphatic heterocycle” means an aliphatic ring containing at least one heteroatom.

[0051] In the present invention, “aromatic heterocycle” means an aromatic ring containing at least one heteroatom.

[0052] In the present invention, "substitution" means that a hydrogen atom bonded to a carbon atom of a compound is replaced with another substituent, and the position of substitution is not limited as long as it is a position where a hydrogen atom is replaced, i.e., a position where a substituent can be substituted, and when two or more are substituted, the two or more substituents may be the same or different from each other. The above substituent is hydrogen, a cyano group, a nitro group, a halogen group, a hydroxy group, an alkyl group having 1 to 30 carbon atoms, an alkenyl group having 2 to 30 carbon atoms, an alkynyl group having 2 to 24 carbon atoms, a heteroalkyl group having 2 to 30 carbon atoms, an aralkyl group having 6 to 30 carbon atoms, an aryl group having 5 to 30 carbon atoms, a heteroaryl group having 2 to 30 carbon atoms, a heteroarylalkyl group having 3 to 30 carbon atoms, an alkoxy group having 1 to 30 carbon atoms, an alkylamino group having 1 to 30 carbon atoms, an arylamino group having 6 to 30 carbon atoms, an aralkylamino group having 6 to 30 carbon atoms, a heteroarylamino group having 2 to 24 carbon atoms, a substituted or unsubstituted alkylsilyl group having 1 to 30 carbon atoms, a substituted or unsubstituted alkyl group having 6 to It may be substituted with one or more substituents selected from the group consisting of 30 arylsilyl groups and substituted or unsubstituted aryloxy groups having 6 to 30 carbon atoms, but is not limited to the above examples.

[0053] In the present invention, “induction of differentiation” includes both inducing differentiation from precursor cells such as monocytes or hematopoietic stem cells into dendritic cells at a phenotypic and functionally immature stage, and inducing these immature dendritic cells into phenotypic and functionally mature dendritic cells suitable for their intended use.

[0054] In the present invention, "immature dendritic cells" are found in the early stage of maturation, and, like mature dendritic cells, do not express cell surface markers such as CD14, and express HLA-DR, CD86, CD80, CD83, or CD40 at low levels, and express CD1a and CCR1, CCR2, CCR5, and CXCR1 at normal levels. Differentiation of immature dendritic cells is initiated by receiving various signals, and this differentiation leads to complete differentiation or partial differentiation depending on the combination of signals received. Immature dendritic cells cannot activate T cells even when they come into contact with T cells because the level of inflammatory cytokines expressed is low.

[0055] In the present invention, "mature dendritic cell" means a cell formed by maturation of immature dendritic cell, and means a cell that expresses cell surface markers involved in B cell and T cell activity, such as MHC class I or MHC class II (HLA-DR), cell adhesion factors (CD54, CD18, CD11), and co-stimulatory factors (e.g., CD86, CD80, CD83, or CD40), at a high level or relatively increased level compared to immature dendritic cells. Typically, mature dendritic cells express CCR7 and CXCR4 at high levels.

[0056] In the present invention, “subject” includes, without limitation, mammals including dogs, cats, rats, livestock, humans, birds, reptiles, farmed fish, etc.

[0057] The present invention relates to a composition for inducing dendritic cell differentiation comprising a metal-organic framework and a use thereof. The composition not only increases the expression of a surface protein marker of dendritic cells, but also has the effect of inducing differentiation into dendritic cells with improved viability and mobility when administered to a subject.

[0058] FIG. 1 illustrates a frontal structure of a metal-organic framework according to one embodiment of the present invention.

[0059] FIG. 2 illustrates a side structure of a metal-organic framework according to one embodiment of the present invention.

[0060] Figure 3 shows the results of scanning electron microscopy measurements on a metal-organic framework according to one embodiment of the present invention.

[0061] FIG. 4 shows the results of X-ray diffraction analysis for a metal-organic framework according to one embodiment of the present invention.

[0062] FIG. 5 shows the N2 adsorption / desorption isotherm, specific surface area, and pore measurement results for a metal-organic framework according to one embodiment of the present invention.

[0063] Figure 6 shows the results of FT-IR measurements for a metal-organic framework according to one embodiment of the present invention.

[0064] Figure 7 shows the results of a cytotoxicity test for a metal-organic framework according to one embodiment of the present invention.

[0065] Figure 8 shows the results of verifying the ability of a metal-organic framework according to one embodiment of the present invention to induce dendritic cell differentiation and the activation of differentiated dendritic cells.

[0066] The present invention relates to a composition for inducing dendritic cell differentiation comprising a metal-organic framework (MOF).

[0067] Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement them. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

[0068] The term "metal-organic framework (MOF)" used herein refers to a porous material in which metal clusters and organic linkers (or organic bridging ligands) are linked by coordination bonds to form a three-dimensional structure, and various MOFs can be created depending on the selection of metal ions and organic ligands. The MOF is characterized by porosity with empty spaces within the structure, and the pore size, porosity, three-dimensional structure, surface area, etc. can be designed in various ways depending on the type and bonding method of the metal ions and organic ligands that make up the MOF. Due to this porosity, the MOF not only has a very large surface area but also has an open pore structure, so it can transport a large amount of molecules or solvents compared to other porous materials known in the art, and when used as a catalyst or gas storage, it has the advantage of having many active sites, which can maximize efficiency. In addition, the MOF is not easily deformed at high temperatures and has a rigid framework, so it has excellent chemical and thermal stability.

[0069] Accordingly, the metal-organic framework (MOF) is known to perform various functions such as gas storage, catalyst, drug delivery, and chemical sensor, as it has the property of transporting large quantities of molecules or solvents, etc., and storing catalysts or gases by utilizing its porous structural characteristics.

[0070] Meanwhile, as previously mentioned, dendritic cells (DDCs) are the most potent antigen-presenting cells (professional antigen-presenting cells; APCs) and play a crucial role in immune induction and immune regulation. In particular, they are comprised of a heterogeneous population with a distinct phenotype from macrophages, and unlike B cells and macrophages, which have relatively weak antigen-presenting capabilities, they are characterized by being potent antigen-presenting cells.

[0071] Since dendritic cells present in the peripheral blood mononuclear cells (PBMCs) of an average person account for only about 1%, methods for differentiating dendritic cells from precursor cells have been studied to obtain a number of cells that can be applied clinically.

[0072] Specifically, the differentiation process of dendritic cells is carried out through ex vivo culture using cytokines from peripheral blood-derived monocytes or hematopoietic stem cells from peripheral blood, umbilical cord blood, and bone marrow, and is largely divided into two stages. In stage 1, differentiation is induced into dendritic cells that are phenotypic and functionally immature from precursor cells such as monocytes and hematopoietic stem cells, and stage 2 is the stage of differentiating these immature dendritic cells into mature dendritic cells using differentiation-inducing substances that can phenotypic and functionally mature them to suit their intended use.

[0073] In particular, the characteristics and functions of dendritic cells differentiated in the above two steps are clearly distinguished as immune enhancement or immune suppression depending on the characteristics of the differentiation-inducing substance. Therefore, it is necessary to appropriately control the differentiation of dendritic cells in vitro to provide optimal conditions suited to the symptoms of various immune diseases.

[0074] Specifically, cytokine cocktails, which are a combination of various cytokines, pathogen-associated molecular patterns (PAMPs) commonly possessed by infectious agents or toll-like receptor ligands for these, sterilized bacteria with their toxins removed, and cancer cells that have induced necrosis, are being developed and utilized as substances that induce dendritic cell differentiation. However, while in vitro analysis of the differentiation-inducing substances confirms an increase in immunological activity, clinical studies on humans have not shown a sufficient level of increase in immunological activity. This is analyzed to be due to the fact that dendritic cells manufactured in vitro have a very low production of IL-12, which is essential for inducing cellular immunity, have low survival rates and mobility when administered in the body, and lose function of dendritic cells due to a systemic immunosuppressive environment.

[0075] Accordingly, the present invention aims to provide a composition for inducing differentiation and maturation of dendritic cells, which can overcome the above-mentioned problems and improve the immune activity, viability, and mobility of dendritic cells.

[0076] Specifically, in the present invention, a metal-organic framework (MOF) is used as a differentiation-inducing material capable of differentiating immature dendritic cells into mature dendritic cells, thereby overcoming the aforementioned problems in the differentiation process, and not only increasing the expression of surface protein markers of differentiated dendritic cells, but also inducing dendritic cells with improved viability and mobility when administered into a subject, thereby specifying this.

[0077] A composition for inducing dendritic cell differentiation according to one embodiment of the present invention comprises a metal-organic framework (MOF).

[0078] The metal-organic framework comprises a metal cluster; and a ligand compound represented by the following chemical formula 1 that coordinately bonds to the metal cluster:

[0079] [Chemical Formula 1]

[0080]

[0081] Here,

[0082] m is an integer from 0 to 4,

[0083] R1 is hydrogen, deuterium, a cyano group, a nitro group, an amino group, a halogen group, a hydroxy group, a substituted or unsubstituted alkylthio group having 1 to 4 carbon atoms, a substituted or unsubstituted alkyl group having 1 to 30 carbon atoms, a substituted or unsubstituted cycloalkyl group having 3 to 20 carbon atoms, a substituted or unsubstituted alkenyl group having 2 to 30 carbon atoms, a substituted or unsubstituted alkynyl group having 2 to 24 carbon atoms, a substituted or unsubstituted aralkyl group having 7 to 30 carbon atoms, a substituted or unsubstituted aryl group having 6 to 30 carbon atoms, a substituted or unsubstituted heteroaryl group having 1 to 60 carbon atoms, a substituted or unsubstituted heteroarylalkyl group having 2 to 30 carbon atoms, a substituted or unsubstituted alkoxy group having 1 to 30 carbon atoms, a substituted or unsubstituted alkylamino group having 1 to 30 carbon atoms, a substituted Or it is selected from the group consisting of an unsubstituted arylamino group having 6 to 30 carbon atoms, a substituted or unsubstituted aralkylamino group having 7 to 30 carbon atoms, a substituted or unsubstituted heteroarylamino group having 1 to 24 carbon atoms, a substituted or unsubstituted alkylsilyl group having 1 to 30 carbon atoms, a substituted or unsubstituted arylsilyl group having 6 to 30 carbon atoms, and a substituted or unsubstituted aryloxy group having 6 to 30 carbon atoms.

[0084] Meanwhile, the R1 is selected from the group consisting of an amino group, a substituted or unsubstituted alkylamino group having 1 to 30 carbon atoms, a substituted or unsubstituted arylamino group having 6 to 30 carbon atoms, a substituted or unsubstituted aralkylamino group having 7 to 30 carbon atoms, and a substituted or unsubstituted heteroarylamino group having 1 to 24 carbon atoms.

[0085] Specifically, R1 of the ligand may be a compound that is an amino group, and more specifically, may be a compound represented by the following chemical formula 2:

[0086] [Chemical Formula 2]

[0087]

[0088] -COO of the ligand compound represented by the above chemical formula 2 - The metal can form coordination bonds with metal clusters to form metal-organic frameworks.

[0089] As described above, the metal-organic framework of the present invention can induce differentiation of immature dendritic cells into mature dendritic cells, thereby increasing the expression rate of surface protein markers of the differentiated dendritic cells. Through this, the immune-inducing activity of dendritic cells can be maximized.

[0090] The above metal-organic framework comprises one or more metals or metal ions selected from the group consisting of aluminum (Al), iron (Fe), and zirconium (Zr).

[0091] Specifically, the metal-organic framework may be synthesized using the selected metal or metal ion, and may specifically include the metal or metal ion and an organic ligand coordinated thereto. Specifically, the metal part of the metal-organic framework may be aluminum (Al, Al 3+ ), iron (Fe, Fe 3+ ) and zirconium (Zr, Zr 4+) may be composed of one or more metals or metal ions thereof selected from the group consisting of:

[0092] In one embodiment, the metal-organic framework can be an aluminum-based MOF, an iron-based MOF, and / or a zirconium-based MOF.

[0093] In one embodiment, the aluminum-based MOF may include, but is not limited to, one or more selected from the group consisting of Al-MIL-53, Al-MIL-53-NH2, Al-MIL-88, Al-MIL-88-NH2, Al-MIL-100, Al-MIL-100-NH2, Al-MIL-101, Al-MIL-101-NH2, Al-MIL-125, and Al-MIL-125-NH2.

[0094] In one embodiment, the iron-based MOF may include, but is not limited to, one or more selected from the group consisting of Fe-MIL-53, Fe-MIL-53-NH2, Fe-MIL-88, Fe-MIL-88-NH2, Fe-MIL-100, Fe-MIL-100-NH2, Fe-MIL-101, Fe-MIL-101-NH2Fe-MIL-125 and Fe-MIL-125-NH2.

[0095] In one embodiment, the zirconium-based MOF may include at least one selected from the group consisting of UIO-66, UIO-66-NH2, UIO-67, UIO-67-NH2, PCN-128, PCN-222, PCN-223, PCN-224, MOF-525, MOF-545, MOF-801, MOF-808, and MOF-867, but is not limited thereto. Specifically, according to one embodiment, it was confirmed that UIO-66-NH2, as a representative example of the zirconium-based MOF, can effectively induce differentiation of immature dendritic cells into mature dendritic cells.

[0096] A pharmaceutical composition according to another embodiment of the present invention may include the composition.

[0097] The pharmaceutical composition may include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" may refer to a carrier or diluent that does not stimulate a living organism and does not inhibit the biological activity or properties of the injected compound. Here, "pharmaceutically acceptable" means that the carrier does not inhibit the activity of the active ingredient and does not exhibit toxicity exceeding the adaptability of the subject of application (prescription). Any carrier commonly used in the art and pharmaceutically acceptable for the pharmaceutical composition may be used. Non-limiting examples of the above carriers include lactose, dextrose, maltodextrin, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, glycerol, ethanol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. These may be used alone or in combination of two or more. The pharmaceutical composition may be prepared as an oral formulation or a parenteral formulation according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. The above pharmaceutical composition can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions, each according to a conventional method.

[0098] When formulating the above pharmaceutical composition, it may be prepared using a diluent or excipient such as a generally used filler, bulking agent, binder, wetting agent, disintegrant, or surfactant, but may not be limited thereto.

[0099] When the above pharmaceutical composition is manufactured into an oral dosage form, it can be manufactured into a dosage form such as powder, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers, etc., using a suitable carrier according to a method known in the art. At this time, examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, xylitol, etc.; starches such as corn starch, potato starch, and wheat starch; celluloses such as cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyols, and vegetable oils. In case of formulation, the formulation may include diluents and / or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants, as needed.

[0100] When the above pharmaceutical composition is prepared as a parenteral dosage form, it can be formulated in the form of injections, transdermal administration, nasal inhalation, and suppositories using a suitable carrier according to a method known in the art. When formulated as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, and preferably, Ringer's solution, phosphate-buffered saline (PBS) containing triethanolamine, sterile water for injection, and isotonic solutions such as 5% dextrose can be used. When formulated as a transdermal dosage form, it can be formulated in the form of ointments, creams, lotions, gels, external solutions, pastes, liniments, aerosols, etc. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, or carbon dioxide, and in the case of formulating it as a suppository, the base can be witepsol, tween 61, polyethylene glycol, cacao butter, laurin butter, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene stearate, sorbitan fatty acid ester, etc.

[0101] The pharmaceutical composition may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" refers to an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention, and the effective dosage level may be determined based on the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and the excretion rate, the treatment period, the drug used in combination or simultaneously with the composition of the present invention used, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered alone or in combination with a component known to exhibit a therapeutic effect on known inflammation, allergic diseases, atopic dermatitis, or asthma. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects.

[0102] The dosage of the pharmaceutical composition may be determined by a person skilled in the art in consideration of the intended use, the degree of toxicity of the disease, the patient's age, weight, sex, medical history, or the type of substance used as the active ingredient. For example, the pharmaceutical composition of the present invention may be administered at about 0.1 ng to about 1,000 mg / kg, preferably 1 ng to about 100 mg / kg, per adult, and the frequency of administration of the composition of the present disclosure is not particularly limited thereto, but may be administered once a day or administered in divided doses several times. The dosage or frequency of administration does not limit the scope of the present disclosure in any way.

[0103] Meanwhile, one embodiment of the present invention provides a method for inducing differentiation of dendritic cells in a subject, comprising a step of administering the composition for inducing dendritic cell differentiation to the subject, and the same portion as described above can be applied to the method.

[0104] A method for inducing dendritic cell differentiation according to another embodiment of the present invention includes a step of treating immature dendritic cells with the composition and then culturing them.

[0105] The above immature dendritic cells are cells obtained by treating bone marrow cells with granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0106] However, the immature dendritic cells are not limited to cells obtained by the above method, and the type of cells is not limited as long as they are capable of differentiating into mature dendritic cells.

[0107] Meanwhile, the bone marrow cells may be bone marrow-derived hematopoietic stem cells, but are not limited thereto.

[0108] According to another embodiment of the present invention, dendritic cells are differentiated by the differentiation induction method.

[0109] The above dendritic cells have increased expression of one or more surface protein markers selected from the group consisting of CD40, CD80, CD86, MHC class I, and MHC class II.

[0110] Specifically, dendritic cells induced to differentiate by treating with the composition for inducing dendritic cell differentiation of the present invention have the characteristic of an improved expression rate of surface protein markers such as MHC-I, MHC-II, CD40, CD80, CD83, and CD86, and through this, have the characteristic of being able to induce immune activation such as inducing strong T cell proliferation and promoting the production of IFN-γ and IL-2 from T cells.

[0111] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0112]

[0113] Manufacturing example

[0114] Synthesis of UIO-66-NH2

[0115] 100 ml of N,N-Dimethylformamide (DMF) and 3.8 g of Benzoic acid were added to a 100 ml container and stirred. After all of the Benzoic acid had dissolved, 120 mg of ZrCl4 was additionally added to the container and stirred. After all of the ZrCl4 had dissolved, 2-Aminoterephthalic Acid was additionally added to the container and stirred to dissolve all of the reagents. The container was sealed with Teflon tape and reacted at 120°C for 24 hours.

[0116] Afterwards, the particles synthesized through the above reaction were washed three times each using DMF and EtOH, and then dried at 80°C to obtain a powder.

[0117] Experimental example

[0118] Analysis of synthetic results

[0119] For scanning electron microscope (SEM) measurements, the powder sample was placed in a 2 mg to 3 mg Eppen tube, 100 uL of ethanol was added, and bath sonication was performed. 10 uL was then spotted onto a silicon wafer and dried in an oven at 80°C. The dried silicon wafer was then attached to an SEM mount using carbon tape and moved to an SEM device to capture images (Company name: Zeiss / Model name: ULTRA PLUS).

[0120] Meanwhile, for X-ray diffraction (XRD) measurement, the powder sample was carefully placed on the XRD sample holder, and the sample was compressed by pressing it with an appropriate force using a glass slide glass. After that, all powder scattered around the sample holder was carefully removed, and the sample holder was moved to the equipment, set up, and measurements were performed in the range of 2°C to 30°C (Company name: Bruker / Model name: D2 phaser).

[0121] Meanwhile, for N2 adsorption isotherm and BET specific surface area-pore measurement, about 40 mg of completely dried sample was prepared and carefully placed in a glass sampler using a glass funnel, and all powder adhering to the glass tube except for the lower sample container was removed. Afterwards, the upper filter cap was attached, and degas was performed by vacuum and heat treatment at 120℃ for 12 hours in a pretreatment device. The weight of the pretreated sample was measured using a microbalance, and the weight of the empty cell that was weighed in advance was subtracted to calculate the weight of the intact sample. The glass sampler was attached to the N2 adsorption-desorption device and the measurement was performed (Company name: MICROTAAC / Model name: BELSORP MINI X).

[0122] The structural images for UIO-66-NH2 particles are shown in Figs. 1 and 2.

[0123] Figure 1 is a front view of the structure of UIO-66-NH2, and Figure 2 shows an image of the side structure.

[0124] The scanning electron microscope observation results for UIO-66-NH2 are as shown in Fig. 3, the X-ray diffraction analysis results are as shown in Fig. 4, and the FT-IR measurement results are as shown in Fig. 6. Referring to this, it can be confirmed that the manufactured particles exhibit the crystal structure of UIO-66-NH2 and were synthesized.

[0125] Meanwhile, Fig. 5 shows the adsorption / desorption isotherm, specific surface area, and pore measurement results for N2. As a result of BET (Brunauer-Emmett-Teller) analysis through N2 adsorption, the specific surface area of ​​UIO-66-NH2 particles is 792.02 m 2 / g, and the pore diameter was confirmed to be 1.71 nm.

[0126] Cytotoxicity assessment

[0127] Mouse fibroblasts (L929) were placed in a 96-well plate at 9,000 cells per well and incubated at 37°C for 24 hours. Thereafter, the existing medium in each well was removed, and the UIO-66-NH2 was suspended in the medium at concentrations of 100 ug / mL, 50 ug / mL, 10 ug / mL, 5 ug / mL, and 1 ug / mL, respectively, to prepare MOF suspensions (UIO-66-NH2) representing each concentration, and 100 uL of this was added to each well. Thereafter, after incubating at 37°C for 24 hours, the cells were washed three times with PBS to remove the medium and particles attached to the plate and cell surface. Afterwards, 10% Ez-Cytox was added to each well, and after reacting at 37°C for 1 hour, the absorbance was measured at 450 nm using a microplate reader (reference wavelength 600 nm to 650 nm).

[0128] The results of evaluating cytotoxicity using the above method are as shown in Fig. 7, and referring to Fig. 7, it was confirmed that there was no cytotoxicity up to a MOF suspension with a concentration of 100 ug / mL.

[0129] Verification of the ability to induce differentiation into dendritic cells and the activation of differentiated dendritic cells

[0130] The ability to induce differentiation of immature dendritic cells into mature dendritic cells of the present invention through the above MOF treatment and the immune activation of differentiated dendritic cells were verified using the following methods.

[0131] GM-CSF / recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) was purchased from Biolegend (CA, USA), and RPMI 1640 medium (Gibco, Cat no. 22400), fetal bovine serum (FBS; Gibco, Cat no. 16000044), and penicillin / streptomycin (Gibco, Cat no. 15140163) were purchased from Thermo Fisher Scientific (MA, USA).

[0132] Specifically, bone marrow cells were treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) (Biolegend (CA, USA)) at a concentration of 20 ng / mL for 6 days to obtain immature bone marrow dendritic cells differentiated from bone marrow cells. Thereafter, the immature bone marrow dendritic cells were cultured at 1 x 10 5 The immature bone marrow dendritic cells were seeded in a 96-well plate at a density of 10 cells / well and cultured in RPMI medium (RPMI 1640, (Gibco, Cat no. 22400)). Subsequently, a suspension of UIO-66-NH2 prepared at concentrations of 25, 50, and 100 μg / mL was added to the immature bone marrow dendritic cells and co-cultured at 37°C for 24 hours.

[0133] Meanwhile, a group treated with 50 ng / mL Lipopolysaccharide (LPS) was prepared as a positive control group so that it could be compared with the test group.

[0134] To analyze maturation markers of mature bone marrow dendritic cells differentiated from immature dendritic spheroids through 24-hour culture, cells were harvested after incubation, individually labeled with fluorescently conjugated primary antibodies, and then analyzed using a CytoFLEX flow cytometer (Beckman Coulter, CA, USA).

[0135] The results of verifying the differentiation induction ability and activation of differentiated dendritic cells using the above method are as shown in Fig. 8. Referring to Fig. 8, not only was it confirmed that immature dendritic cells differentiated into mature dendritic cells when treated with a MOF suspension, but also, in the case of differentiated dendritic cells, it was confirmed that the expression of surface protein markers increased in a concentration-dependent manner depending on the treated MOF suspension.

[0136]

[0137] Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims also fall within the scope of the present invention.

[0138] This project (outcome) is the result of a local government-university collaboration-based regional innovation project, supported by the National Research Foundation of Korea and funded by the Ministry of Education in 2024 (2021RIS-001).

[0139] [Project ID] 1345370811

[0140] [Project Number] 2021RIS-001

[0141] [Ministry Name] Ministry of Education (P13)

[0142] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0143] [Research Project Name] Local Government-University Cooperation-Based Regional Innovation Project

[0144] [Research Project Name] Local Government-University Cooperation-Based Regional Innovation Project (Chungbuk Regional Innovation Platform)

[0145] [Contribution rate]

[0146] [Name of Project Performing Organization] (Chungbuk Regional Innovation Platform) Chungbuk National University

[0147] [Research Period] March 1, 2024 - February 28, 2025

[0148] The present invention relates to a composition for inducing dendritic cell differentiation comprising a metal-organic framework and its use.

Claims

1. Containing a metal-organic framework (MOF) A composition for inducing dendritic cell differentiation.

2. In paragraph 1, The metal-organic framework comprises a metal cluster; and A ligand compound represented by the following chemical formula 1 that coordinates to the above metal cluster. Composition for inducing dendritic cell differentiation: [Chemical Formula 1] Here, m is an integer from 0 to 4, R1 is hydrogen, deuterium, a cyano group, a nitro group, an amino group, a halogen group, a hydroxy group, a substituted or unsubstituted alkylthio group having 1 to 4 carbon atoms, a substituted or unsubstituted alkyl group having 1 to 30 carbon atoms, a substituted or unsubstituted cycloalkyl group having 3 to 20 carbon atoms, a substituted or unsubstituted alkenyl group having 2 to 30 carbon atoms, a substituted or unsubstituted alkynyl group having 2 to 24 carbon atoms, a substituted or unsubstituted aralkyl group having 7 to 30 carbon atoms, a substituted or unsubstituted aryl group having 6 to 30 carbon atoms, a substituted or unsubstituted heteroaryl group having 1 to 60 carbon atoms, a substituted or unsubstituted heteroarylalkyl group having 2 to 30 carbon atoms, a substituted or unsubstituted alkoxy group having 1 to 30 carbon atoms, a substituted or unsubstituted alkylamino group having 1 to 30 carbon atoms, a substituted Or it is selected from the group consisting of an unsubstituted arylamino group having 6 to 30 carbon atoms, a substituted or unsubstituted aralkylamino group having 7 to 30 carbon atoms, a substituted or unsubstituted heteroarylamino group having 1 to 24 carbon atoms, a substituted or unsubstituted alkylsilyl group having 1 to 30 carbon atoms, a substituted or unsubstituted arylsilyl group having 6 to 30 carbon atoms, and a substituted or unsubstituted aryloxy group having 6 to 30 carbon atoms.

3. In paragraph 2, The above R1 is selected from the group consisting of an amino group, a substituted or unsubstituted alkylamino group having 1 to 30 carbon atoms, a substituted or unsubstituted arylamino group having 6 to 30 carbon atoms, a substituted or unsubstituted aralkylamino group having 7 to 30 carbon atoms, and a substituted or unsubstituted heteroarylamino group having 1 to 24 carbon atoms. A composition for inducing dendritic cell differentiation.

4. In paragraph 1, The above metal-organic framework comprises one or more metals or metal ions selected from the group consisting of aluminum (Al), iron (Fe), and zirconium (Zr). A composition for inducing dendritic cell differentiation.

5. A step of treating immature dendritic cells with a composition according to any one of claims 1 to 4 and then culturing them. Method for inducing dendritic cell differentiation.

6. In paragraph 5, The above immature dendritic cells are cells obtained by treating bone marrow cells with granulocyte-macrophage colony-stimulating factor (GM-CSF). Method for inducing dendritic cell differentiation.

7. Differentiated by the differentiation induction method according to Article 5 Dendritic cells.

8. In paragraph 7, The above dendritic cells have increased expression of one or more surface protein markers selected from the group consisting of CD40, CD80, CD86, MHC class I and MHC class II. Dendritic cells.

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