Polymer nanoparticle composition for immune induction and method for producing same

Abstract

The present invention aims to provide an immunity-inducing composition that can induce immunity in an individual from an active ingredient such as a nucleic acid, a polypeptide, or a combination thereof, and that can effectively induce immunity even when the active ingredient is difficult to recognize as an antigen, and a method for producing the same. [Solution] The present invention relates to a composition for inducing immunity, which comprises a nucleic acid, a polypeptide or a combination thereof, a cationic compound, an amphiphilic block copolymer and a polylactate as an active ingredient, and the active ingredient is encapsulated inside a nanoparticle structure formed by the amphiphilic block copolymer and the polylactate, and a method for producing the same.

Description

[Technical field]

[0001] The present invention relates to a polymeric nanoparticle composition capable of inducing immunity and a method for producing the same, and more particularly to a therapeutic or prophylactic vaccine composition that effectively induces T cell immunity against an antigen. [Background technology]

[0002] Safe and efficient drug delivery techniques have been studied for a long time for the treatment with anionic drugs, including nucleic acids, and various delivery methods and delivery techniques have been developed. Delivery methods are broadly divided into viral delivery methods using adenoviruses or retroviruses, etc., and non-viral delivery methods using cationic lipids and cationic polymers, etc. It is known that techniques using viral delivery methods are exposed to risks such as non-specific immune reactions and have many problems in commercialization due to the complex production process. Therefore, recent research has been conducted on non-viral delivery methods to overcome these problems. Compared with viral delivery methods, non-viral delivery methods have the advantages of fewer side effects in terms of in vivo safety and lower production costs in terms of economy.

[0003] Meanwhile, cancer antigen vaccines that induce antigen-specific T cell responses as anticancer therapeutic agents are being actively developed (Non-Patent Document 1). In order to induce an effective anticancer immune response, it is necessary to effectively stimulate cytotoxic T cells by delivering cancer antigens to antigen-presenting cells and increasing the presentation rate of tumor-specific antigen-derived epitopes. For efficient antigen delivery, methods are used in which tumor cells themselves are used as antigens, or peptides, nucleic acids, viruses, or dendritic cells are used as antigens to induce antigen-specific immune responses (Non-Patent Document 2). In particular, nucleic acid mRNA is a non-infectious and non-insertion platform, has relatively few risks such as mutated genes, and has the advantage that it can be produced in large quantities, quickly, and economically by in vitro transcription reactions. Therefore, mRNA has attracted attention as a platform for personalized anticancer vaccines against various types of cancers as well as infectious diseases (Non-Patent Document 3).

[0004] Patent Document 1 discloses an anionic drug delivery composition and a method for producing the same, which comprises an anionic drug as active ingredients, a cationic compound, an amphiphilic block copolymer, and a polylactate, and the anionic drug forms a complex with the cationic compound through electrostatic interaction, and the complex is encapsulated inside a nanoparticle structure formed by the amphiphilic block copolymer and the polylactate. However, the specification only discloses the use of the anionic drug as a delivery method, and does not disclose the effect on the immunity of the subject. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Korean Patent Publication No. 10-2017-0032858 [Non-patent literature]

[0006] [Non-Patent Document 1] npj Vaccines vol.4,Article number:7(2019) [Non-Patent Document 2] Advances in Cancer Research Vol.119,2013,421-475 [Non-Patent Document 3] Nat Rev Drug Discov.2018 Apr;17(4):261-279 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention aims to provide a composition for inducing immunity, which can induce immunity in an individual from an active ingredient such as a nucleic acid, a polypeptide, or a combination thereof, and can effectively induce immunity even when the active ingredient is difficult to recognize as an antigen, and a method for producing the same. [Means for solving the problem]

[0008] One aspect of the present invention provides a composition for inducing immunity, comprising as an active ingredient a nucleic acid, a polypeptide or a combination thereof, a cationic compound, an amphiphilic block copolymer, and a polylactate, wherein the active ingredient is encapsulated within a nanoparticle structure formed by the amphiphilic block copolymer and the polylactate.

[0009] Another aspect of the present invention provides a method for producing a composition for inducing immunity, comprising the steps of: (a) mixing an aqueous solution containing a nucleic acid, a polypeptide or a combination thereof as an active ingredient with a solution in which a cationic compound is dissolved in a water-miscible organic solvent; (b) mixing an aqueous solution of an amphiphilic block copolymer and a polylactate with the mixture of step (a); (c) adding an aqueous solvent to the mixture of step (b) and mixing; and (d) removing the solvent from the mixture of step (c). Effect of the Invention

[0010] The composition for inducing immunity of the present invention has an advantage that it can induce immunity in an individual from an active ingredient such as a nucleic acid, a polypeptide, or a combination thereof, and can effectively induce immunity even when the active ingredient is difficult to recognize as an antigen. By using this, the composition for inducing immunity can be used as an autologous cancer vaccine, and ultimately can be used to prevent or treat cancer.

[0011] In one embodiment, the composition for inducing immunity of the present invention can induce immunity by effectively delivering an active ingredient to the spleen. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a diagram showing the schematic structure of a composition for inducing immunity according to one embodiment of the present invention. [Diagram 2] FIG. 2 is a schematic diagram of the animal experiment method of Example 2. [Diagram 3] FIG. 3 is an image showing the expression level of IFN-γ in splenocytes treated with a composition for inducing immunity according to one embodiment of the present invention. [Figure 4] FIG. 4 is an ELISPOT graph showing the amount of IFN-γ expressed in splenocytes using a composition for inducing immunity according to one embodiment of the present invention. [Diagram 5] FIG. 5 is an image showing organ-specific expression of an immunity-inducing composition according to one embodiment of the present invention. [Figure 6] FIG. 6 is a graph showing the effect of cancer treatment using a composition for inducing immunity according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention will be described in detail below. Among the components of the composition of the present invention, the active ingredient, a nucleic acid, a polypeptide or a combination thereof, may be associated with an antigen capable of inducing an immune response. For example, the nucleic acid may express an antigen, and in another example, the polypeptide may be recognized as an antigen.

[0014] The active ingredient may form a complex with the cationic compound by electrostatic interactions, and the complex may be encapsulated in the nanoparticle structure formed by the amphiphilic block copolymer and polylactate, an exemplary structure of which is shown diagrammatically in Figure 1.

[0015] In the case of nanoparticles, in an aqueous environment, the hydrophilic portion of the amphiphilic block copolymer forms the outer wall of the nanoparticle, while the hydrophobic portion of the amphiphilic block copolymer and the polylactate contained as another component of the amphiphilic block copolymer form the inner wall of the nanoparticle, and the complex of the active ingredient and the cationic compound can be encapsulated inside the nanoparticle formed. Such a nanoparticle structure improves the stability of the active ingredient in blood or body fluids.

[0016] The "nucleic acid" may be, for example, but is not limited to, DNA, RNA, siRNA, shRNA, miRNA, mRNA, aptamer, antisense oligonucleotide, or a combination thereof. The nucleic acid may express an antigen through a series of processes in the body and may code for the antigen. For example, the nucleic acid may be DNA, RNA, or mRNA that includes the base sequence of an antigen or codes for such a polypeptide sequence.

[0017] The "polypeptide" may be capable of being recognized as an antigen. Polypeptide refers to a protein that can be recognized as an antigen through a series of processes in the body, such as the polypeptide sequence of an antigen, an analogue or a precursor thereof.

[0018] The immune-inducing composition according to one embodiment effectively delivers an antigen into the body and has the effect of treating or preventing a disease associated with the antigen. For example, when the disease is a tumor, the nucleic acid may express a tumor antigen or the polypeptide may be a tumor antigen. Examples of tumor antigens include, but are not limited to, Trp2 (tyrosinase-related protein 2), gp100 (glycoprotein 100), tyrosinase, PSA (prostate-specific antigen), WT1 (Wilms' tumor 1), MAGE-1 (melanoma-associated antigen 1), NY-ESO-1 (testicular cancer antigen), MUC-1 (mucin 1), etc.

[0019] The immune-inducing composition of the present invention exhibits efficient and excellent vaccine effects by successfully generating antigens that are difficult to induce immune induction in even when using conventional administration methods or carriers, and as a result, can achieve disease treatment effects through immune induction.

[0020] In another embodiment, the polypeptide may be a viral envelope, a glycoprotein, a bacterium, a cancer cell, or a fragment thereof. In this case, the composition for inducing immunity of the present invention may be for treating or preventing a disease caused by a virus, a bacterium, or a cancer cell.

[0021] The disease includes, but is not limited to, cancer, viral infection, cellular infection, allergy, etc. The composition for inducing immunity can be used as a vaccine for the prevention of a disease.

[0022] The nucleic acid or polypeptide as an active ingredient has chemically modified amino acids, sugars or base backbones, or modified termini, to increase stability in blood or enhance immune-inducing effects.

[0023] In one embodiment, the particle size of the nanoparticles can be defined as the Z average value, and may be, for example, 800 nm or less, 600 nm or less, 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, or 150 nm or less, or may be 10 nm or more. In one embodiment, the particle size of the nanoparticles defined as the Z average value may be, for example, 10 to 800 nm, 10 to 600 nm, 10 to 500 nm, 10 to 400 nm, 10 to 300 nm, 10 to 200 nm, or 10 to 150 nm.

[0024] The "Z-average" refers to the average hydrodynamic diameter of a particle distribution measured using dynamic light scattering (DSL). The nanoparticles may have a monodisperse particle distribution, with a polydispersity index of, for example, 0.01-0.30, 0.05-0.25, or 0.1-0.2.

[0025] In one embodiment, the surface charge of the nanoparticles may be, for example, -40 mV or more, -30 mV or more, -20 mV or more, or -10 mV or more, or 40 mV or less, 30 mV or less, 20 mV or less, or 10 mV or less. In one embodiment, the surface charge of the nanoparticles may be, for example, -40 to 40 mV, -30 to 30 mV, -20 to 20 mV, or -10 to 10 mV. The surface charge can be measured in an environment close to a biological environment, for example, 10 mM HEPES buffer (pH 7.2).

[0026] It is preferable that the particle size and surface charge of the nanoparticles are maintained at the above levels in terms of the stability of the nanoparticle structure, the content of the constituent components, the degree of absorption in the body, and the ease of sterilization as a pharmaceutical composition. For example, when the active ingredient is a nucleic acid, one or more ends of the nucleic acid can be modified with one or more selected from the group consisting of cholesterol, tocopherol, and fatty acids having 10 to 24 carbon atoms. The cholesterol, tocopherol, and fatty acids having 10 to 24 carbon atoms include analogs, derivatives, and metabolites of cholesterol, tocopherol, and fatty acids.

[0027] The content of the active ingredient may be, for example, 30% by weight or less, 25% by weight or less, 20% by weight or less, 15% by weight or less, 10% by weight or less, or 5% by weight or less, or 0.05% by weight or more, 0.1% by weight or more, 0.25% by weight or more, 0.5% by weight or more, or 1% by weight or more, based on the total composition weight. In one embodiment, the content of the active ingredient may be, for example, 0.05-30% by weight, 0.1-25% by weight, 0.25-20% by weight, 0.5-15% by weight, 1-10% by weight, or 1-5% by weight, based on the total composition weight. If the content of the active ingredient is less than the above range based on the weight of the total composition, the amount of the delivery product used compared to the drug is too large, and side effects due to the delivery product may occur. If the content exceeds the above range, the nanoparticle diameter may become too large, the stability of the nanoparticles may decrease, and the loss rate may increase during filter sterilization.

[0028] In certain embodiments, the cationic compound may be a cationic lipid or a cationic polymer, more specifically a cationic lipid.

[0029] In one embodiment, the cationic lipid is N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTAP), N,N-dimethyl-(2,3-dioleoyloxy)propylamine (DODMA), N,N,N-trimethyl-(2,3-dioleoyloxy)propylamine (DOTMA), 1,2-diacyl-3-trimethylammonium-propane (TAP), 1,2-diacyl-3-dimethylammonium-propane (DAP), 3β-[N-(N′,N′,N′-trimethylaminoethoxy)propylamine], 1,2-diacyl-3-dimethyl ... The present invention may be one or a combination of two or more selected from the group consisting of 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (TC-cholesterol), 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-cholesterol), 3β-[N-(N'-monomethylaminoethane)carbamoyl]cholesterol (MC-cholesterol), 3β-[N-(aminoethane)carbamoyl]cholesterol (AC-cholesterol), cholesteryloxypropan-1-amine (COPA), N-(N'-aminoethane)carbamoylpropanoic acid tocopherol (AC-tocopherol) and N-(N'-methylaminoethane)carbamoylpropanoic acid tocopherol (MC-tocopherol).

[0030] When using such cationic lipids, it may be preferable to use fewer polycationic lipids with high charge density in order to reduce toxicity of the cationic lipids, and more specifically, it is preferable to use cationic lipids with one functional group capable of exhibiting a positive charge per molecule in aqueous solution.

[0031] Thus, in a preferred embodiment, the cationic lipid may be at least one selected from the group consisting of 3β-[N-(N',N',N'-trimethylaminoethane)carbamoyl]cholesterol (TC-cholesterol), 3β[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-cholesterol), 3β[N-(N'-monomethylaminoethane)carbamoyl]cholesterol (MC-cholesterol), 3β-[N-(aminoethane)carbamoyl]cholesterol (AC-cholesterol), N-(1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTAP), N,N-dimethyl-(2,3-dioleoyloxy)propylamine (DODMA), and N,N,N-trimethyl-(2,3-dioleoyloxy)propylamine (DOTMA).

[0032] On the other hand, in one embodiment, the cationic polymer may be at least one selected from the group consisting of chitosan, glycol chitosan, protamine, polylysine, polyarginine, polyamidoamine (PAMAM), polyethyleneimine, dextran, hyaluronic acid, albumin, polymer polyethyleneimine (PEI), polyamine, and polyvinylamine (PVAm), and more specifically, may be at least one selected from the group consisting of polyethyleneimine (PEI), polyamine, and polyvinylamine (PVAm).

[0033] In one embodiment, the cationic lipid may be a cationic lipid represented by the following formula (1):

[0034] [ka]

[0035] (In the formula, n and m are each 0 to 12, provided that 2≦n+m≦12; a and b are each an integer of 1 to 6; R1 and R2 are each independently selected from the group consisting of saturated and unsaturated hydrocarbon groups having 11 to 25 carbon atoms.

[0036] More specifically, in the formula (1), n ​​and m are each independently 1 to 9, and may be 2≦n+m≦10.

[0037] More specifically, in the formula (1), a and b may each independently be 2 to 4.

[0038] More specifically, in the formula (1), R1 and R2 may each independently be selected from the group consisting of lauryl, myristyl, palmityl, stearyl, arachidyl, behenyl, lignoceryl, celloyl, myristoleyl, palmitoleyl, sapienyl, oleyl, linoleyl, arachidonyl, eicosapentaenyl, erucyl, docosahexaenyl, and celloyl.

[0039] In one embodiment, the cationic lipid is 1,6-dioleoyltriethylenetetramide (N,N'-((ethane-1,2-diylbis(azanediyl))bis(ethane-2,1-diyl))dioleamide), 1,8-dilinoleoyltetraethylenepentamide ((9Z,9'Z,12Z,12'Z)-N,N'-(((azanediylbis(ethane-2,1-diyl))bis(azanediyl))bis(ethane-2,1-diyl))bis(octadeca-9,12-dienamide)), 1,4-dimyristoleoyl and 1,10-dioleoylpentaethylenehexamide (N,N'-(3,6,9,12-tetraazatetradecane-1,14-diyl)dioleamide).

[0040] The content of the cationic compound in the composition for inducing immunity of the present invention may be, for example, 25 parts by weight or less, 20 parts by weight or less, 18 parts by weight or less, 15 parts by weight or less, 12 parts by weight or less, 10 parts by weight or less, or 8 parts by weight or less, or 1 part by weight or more, 1.5 parts by weight or more, 2 parts by weight or more, 2.5 parts by weight or more, or 3 parts by weight or more, relative to 1 part by weight of the active ingredient. In one embodiment, the content of the cationic compound in the composition may be 1 to 25 parts by weight, 1.5 to 10 parts by weight, 2 to 15 parts by weight, 2.5 to 10 parts by weight, or 3 to 8 parts by weight, relative to 1 part by weight of the active ingredient. If the content of the cationic compound in the composition is less than the above range, it may not be possible to form a stable complex with the active ingredient. If the content of the cationic compound exceeds the above range, the nanoparticle size may become too large, the stability may decrease, and the loss rate during filter sterilization may increase.

[0041] When the active ingredient is a nucleic acid, the cationic compound and the nucleic acid are bound to each other by electrostatic interaction to form a complex. In one embodiment, the charge ratio of the nucleic acid (P) and the cationic compound (N) (N / P; the ratio of the cationic charge of the cationic compound to the anionic charge of the nucleic acid) may be 0.5 or more, 1 or more, 2 or more, or 3 or more, and may be 100 or less, 50 or less, 20 or less, or 10 or less, for example, 0.5 to 100, 1 to 50, 2 to 20, 2 to 15, or 3 to 10. When the ratio (N / P) is less than 0.5, it may be difficult to form a complex containing a sufficient amount of nucleic acid, and when the ratio (N / P) is more than 100, toxicity may occur. Furthermore, the N / P value may play an important role in the specific expression of the active ingredient in the spleen.

[0042] In one embodiment, the amphiphilic block copolymer may be an AB type block copolymer comprising a hydrophilic A block and a hydrophobic B block, which forms a core-shell type polymer nanoparticle in an aqueous solution, in which the hydrophobic B block forms the core (inner wall) and the hydrophilic A block forms the shell (outer wall).

[0043] In one embodiment, the hydrophilic A block may be at least one selected from the group consisting of polyalkylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, and derivatives thereof.

[0044] More specifically, the hydrophilic A block may be at least one selected from the group consisting of monomethoxypolyethylene glycol, monoacetoxypolyethylene glycol, polyethylene glycol, a copolymer of polyethylene and propylene glycol, and polyvinylpyrrolidone.

[0045] In one embodiment, the hydrophilic A block may have a number average molecular weight of 200 to 50,000 Daltons, more specifically, 1,000 to 20,000 Daltons, and more specifically, 1,000 to 5,000 Daltons.

[0046] If necessary, a functional group capable of reaching a specific tissue or cell, a ligand, or a functional group capable of promoting intracellular delivery can be chemically bound to the end of the hydrophilic A block to regulate the biodistribution of the polymer nanoparticle delivery body formed of the amphiphilic block copolymer and polylactate, or to enhance the delivery efficiency of the nanoparticle delivery body to cells. In one embodiment, the functional group or ligand may be at least one selected from the group consisting of monosaccharides, polysaccharides, vitamins, peptides, proteins, and antibodies against cell surface receptors. More specifically, the functional group or ligand may be at least one selected from the group consisting of anisamide, vitamin B9 (folic acid), vitamin B12, vitamin A, galactose, lactose, mannose, hyaluronic acid, RGD peptide, NGR peptide, transferrin, and antibodies against transferrin receptors.

[0047] The hydrophobic B block is a biocompatible and biodegradable polymer, which in one embodiment may be at least one selected from the group consisting of polyesters, polyanhydrides, polyamino acids, polyorthoesters, and polyphosphazines.

[0048] More specifically, the hydrophobic B block may be at least one selected from the group consisting of polylactic acid, polyglycolide, polycaprolactone, polydioxane-2-one, a copolymer of polylactic acid and glycolide, a copolymer of polylactic acid and polydioxane-2-one, a copolymer of polylactic acid and polycaprolactone, and a copolymer of polyglycolide and polycaprolactone.

[0049] In one embodiment, the hydrophobic B block may have a number average molecular weight of 50 to 50,000 Daltons, more specifically, 200 to 20,000 Daltons, and even more specifically, 1,000 to 5,000 Daltons.

[0050] In one embodiment, in order to increase the hydrophobicity of the hydrophobic B block and improve the stability of the nanoparticles, the hydrophobic B block can be modified by chemically bonding tocopherol, cholesterol, or a fatty acid having 10 to 24 carbon atoms to the hydroxy group at the end of the hydrophobic B block.

[0051] In one embodiment, the content of the amphiphilic block copolymer containing the hydrophilic block (A) and the hydrophobic block (B) in the composition may be, for example, 150 parts by weight or less, 100 parts by weight or less, 70 parts by weight or less, 50 parts by weight or less, or 30 parts by weight or less, or 1 part by weight or more, 10 parts by weight or more, or 15 parts by weight or more, relative to 1 part by weight of the active ingredient. For example, the content of the amphiphilic block copolymer in the composition may be 1 to 150 parts by weight, 10 to 100 parts by weight, 10 to 70 parts by weight, 15 to 50 parts by weight, or 15 to 30 parts by weight, relative to 1 part by weight of the active ingredient. If the content of the amphiphilic block copolymer in the composition is less than the above range, the nanoparticle diameter may become too large, the stability of the nanoparticles may decrease, and the loss rate during filter sterilization may increase. If the content of the amphiphilic block copolymer exceeds the above range, the content of the active ingredient to be taken in may be too small.

[0052] In one embodiment, the ratio of the hydrophilic block (A) and the hydrophobic block (B) in the amphiphilic block copolymer may be 40 to 70% by weight, more specifically 50 to 60% by weight, based on the total weight of the copolymer. When the ratio of the hydrophilic block (A) is less than 40% by weight based on the total weight of the copolymer, the solubility of the polymer in water is low, and it may be difficult to form nanoparticles. Therefore, in order to give the copolymer sufficient water solubility to form nanoparticles, the ratio of the hydrophilic block (A) is preferably 40% by weight or more. On the other hand, when the ratio of the hydrophilic block (A) is more than 70% by weight based on the total weight of the copolymer, the hydrophilicity is too high, the stability of the polymer nanoparticles is reduced, and it may be difficult to use it as a solubilized composition of an active ingredient / cationic compound complex. Therefore, in consideration of nanoparticle stability, the ratio of the hydrophilic block (A) is preferably 70% by weight or less.

[0053] In one embodiment, the amphiphilic block copolymer allows the complex of the active ingredient and the cationic compound in the aqueous solution to be encapsulated inside the nanoparticle structure, and the ratio of the weight (a) of the active ingredient and the cationic compound complex to the weight (b) of the amphiphilic block copolymer [a / b×100; (weight of active ingredient+weight of cationic compound) / weight of amphiphilic block copolymer×100] may be 90% or less, 80% or less, 60% or less, 50% or less, or 45% or less, or 1% or more, 2% or more, 5% or more, 10% or more, 15% or more, 20% or more, 21% or more, or 25% or more, for example, 1 to 90%, 2 to 50%, 10 to 50%, 15 to 50%, or 21% to 45%. When the weight ratio (a / b×100) is less than 1%, the content of the complex of the active ingredient and the cationic compound becomes too small, making it difficult to meet the effective content at which the active ingredient can act effectively. When the weight ratio (a / b×100) exceeds 90%, nanoparticle structures of appropriate size may not be formed due to the molecular weight of the amphiphilic block copolymer or the amount of the complex of the active ingredient and the cationic compound.

[0054] The nanoparticle structure in the composition of the present invention includes polylactate (e.g., polylactate sodium salt (PLANa)). The polylactate is distributed in the core (inner wall) of the nanoparticle to increase the hydrophobicity of the core and stabilize the nanoparticle, and at the same time helps to effectively avoid the reticuloendothelial system (RES) in the body. That is, the anion of the carboxylic acid in the polylactate effectively binds with the cationic complex to reduce the surface charge of the polymer nanoparticle. As a result, the surface positive charge of the polymer nanoparticle is less than that of the polymer nanoparticle without polylactate, so that it can be efficiently delivered to the target site (e.g., cancer cells, inflammatory cells, etc.) without being captured by the reticuloendothelial system.

[0055] In one embodiment, the polylactate, which is a component independent of the amphiphilic block copolymer, is a component of the inner wall of the nanoparticle, and the number average molecular weight of the polylactate may be 500 to 50,000 Daltons, more specifically 1,000 to 50,000 Daltons. When the number average molecular weight of the polylactate is less than 500 Daltons, the hydrophobicity is too low, so the polylactate may not easily exist in the core (inner wall) of the nanoparticle, and when the number average molecular weight of the polylactate is more than 50,000 Daltons, the particles of the polymer nanoparticles may be too large.

[0056] In one embodiment, the content of the polylactate in the composition may be 80 parts by weight or less, 60 parts by weight or less, 50 parts by weight or less, 40 parts by weight or less, or 30 parts by weight or less, or 1 part by weight or more, 5 parts by weight or more, 10 parts by weight or more, 15 parts by weight or more, or 20 parts by weight or more, relative to 1 part by weight of the active ingredient. For example, the content of the polylactate in the composition may be 1 to 80 parts by weight, 1 to 50 parts by weight, 1 to 40 parts by weight, 5 to 30 parts by weight, or 20 to 60 parts by weight, relative to 1 part by weight of the active ingredient. If the content of the polylactate in the composition is less than the above range, the delivery efficiency of the active ingredient may decrease. If the content of the polylactate exceeds the above range, the nanoparticles may become too large, making filtration through a sterilizing membrane difficult or reducing stability.

[0057] In one embodiment, among the terminals of the polylactate (e.g., sodium polylactate), the terminal opposite the metal carboxylate (e.g., sodium carboxylate) may be substituted with a group selected from the group consisting of hydroxy, acetoxy, benzoyloxy, decanoyloxy, palmitoyloxy, and alkoxy having 1 to 2 carbon atoms.

[0058] In one embodiment, the polylactate may be selected from the group consisting of compounds of the following formulas 2 to 7:

[0059] [ka]

[0060] (Wherein, A is -COO-CHZ-, B is -COO-CHY-, -COO-CH2CH2CH2CH2CH2- or -COO-CH2CH2OCH2; R is a hydrogen atom, an acetyl group, a benzoyl group, a decanoyl group, a palmitoyl group, a methyl group, or an ethyl group; Z and Y are each a hydrogen atom, a methyl group, or a phenyl group; M is Na, K, or Li; n is an integer from 1 to 30, m is an integer from 0 to 20.

[0061] [ka]

[0062] (wherein X is a methyl group; Y' is a hydrogen atom or a phenyl group; p is an integer from 0 to 25; q is an integer from 0 to 25, with the proviso that p+q is an integer from 5 to 25; R is a hydrogen atom, an acetyl group, a benzoyl group, a decanoyl group, a palmitoyl group, a methyl group, or an ethyl group; M is Na, K, or Li; Z is a hydrogen atom, a methyl group, or a phenyl group.

[0063] [ka]

[0064] (Wherein, W-M' is [ka] or [ka] and The PAD is selected from the group consisting of D,L-polylactic acid, D-polylactic acid, polymandelic acid, copolymers of D,L-lactic acid and glycolic acid, copolymers of D,L-lactic acid and mandelic acid, copolymers of D,L-lactic acid and caprolactone, and copolymers of D,L-lactic acid and 1,4-dioxane-2-one; R is a hydrogen atom, an acetyl group, a benzoyl group, a decanoyl group, a palmitoyl group, a methyl group, or an ethyl group; M is independently Na, K, or Li.

[0065] [ka]

[0066] [In the formula, S is [ka]

[0067] (L is -NR1-(R1 is a hydrogen atom or C 1-10 alkyl) or -O-, a is an integer of 0 to 4, b is an integer of 1 to 10, and M is Na, K, or Li; Q is CH3, CH2CH3, CH2CH2CH3, CH2CH2CH2CH3, or CH2C6H5; The PAD is at least one selected from the group consisting of D,L-polylactic acid, D-polylactic acid, polymandelic acid, a copolymer of D,L-lactic acid and glycolic acid, a copolymer of D,L-lactic acid and mandelic acid, a copolymer of D,L-lactic acid and caprolactone, and a copolymer of D,L-lactic acid and 1,4-dioxane-2-one.

[0068] [ka]

[0069] [wherein R' is -PAD-OC(O)-CH2CH2-C(O)-OM (PAD is selected from the group consisting of D,L-polylactic acid, D-polylactic acid, polymandelic acid, copolymers of D,L-lactic acid and glycolic acid, copolymers of D,L-lactic acid and mandelic acid, copolymers of D,L-lactic acid and caprolactone, and copolymers of D,L-lactic acid and 1,4-dioxane-2-one, and M is Na, K, or Li), a is an integer from 1 to 4.

[0070] [ka]

[0071] (In the formula, X and X' are independently hydrogen, alkyl having 1 to 10 carbon atoms, or aryl having 6 to 20 carbon atoms, Y and Z are independently Na, K, or Li; m and n are independently an integer of 0 to 95, provided that <m+n<100であり、 a and b are independently an integer from 1 to 6; R is -(CH2) k - (k is an integer of 0 to 10), a divalent alkenyl having 2 to 10 carbon atoms, a divalent aryl having 6 to 20 carbon atoms, or a combination thereof.)

[0072] In one embodiment, the polylactate may be a compound of formula (2) or formula (3).

[0073] In one embodiment, the composition of the present invention may further contain 0.01 to 50% by weight, more specifically 0.1 to 10% by weight, of a fusogenic lipid relative to the total weight of the entire composition in order to increase the efficiency of delivery of mRNA to cells.

[0074] When the complex of mRNA and cationic lipid is mixed, the fusogenic lipid binds to the complex by hydrophobic interaction to form a complex of mRNA, cationic lipid and fusogenic lipid. The complex containing the fusogenic lipid is encapsulated inside the nanoparticle structure of the amphiphilic block copolymer.

[0075] In one embodiment, the fusogenic lipid may be one or a combination of two or more selected from the group consisting of phospholipids, cholesterol, and tocopherol.

[0076] More specifically, the phospholipid may be at least one selected from the group consisting of phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidic acid. The phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidic acid may each have one or two C 10-24 It may be in a form bound to a fatty acid. The cholesterol and tocopherol mentioned above include analogues, derivatives and metabolites of cholesterol and tocopherol, respectively.

[0077] More specifically, the fusogenic lipid may be dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, dilinoleoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, 1,2-diphytanoyl-3-sn-phosphatidylethanolamine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine. The phosphatidylcholine may be one or a combination of two or more selected from the group consisting of dilauroylphosphatidylcholine, dilinoleoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine, 1,2-diphytanoyl-3-sn-phosphatidylcholine, dilauroylphosphatidic acid, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, distearoylphosphatidic acid, dioleoylphosphatidic acid, dilinoleoylphosphatidic acid, 1-palmitoyl-2-oleoylphosphatidic acid, 1,2-diphytanoyl-3-sn-phosphatidic acid, cholesterol, and tocopherol.

[0078] More specifically, the fusogenic lipid may be at least one selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), dipalmitoleoylphosphocholine (1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine; DPPC), dioleoylphosphocholine (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC) and dipalmitoleoylphosphoethanolamine (1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine; DPPE).

[0079] In one embodiment, the compositions of the present invention may further comprise divalent or trivalent metal ions.

[0080] The divalent or trivalent metal ion is preferably calcium (Ca2+ ), Magnesium (Mg 2+ ), Barium (Ba 2+ ), Chromium (Cr 3+ ), iron (Fe 3+ ), Manganese (Mn 2+ ), Nickel (Ni 2+ ), Copper (Cu 2+ ), Zinc (Zn 2+ ) or aluminum (Al 3+ ) etc. can be selected.

[0081] The metal ions can be added to the polymer nanoparticle composition in the form of sulfates, hydrochlorides, carbonates, phosphates and hydroxides. The metal ions can be added preferably in the form of calcium chloride (CaCl2), magnesium chloride (MgCl2), zinc chloride (ZnCl2), aluminum chloride (AlCl3), iron chloride (FeCl3), calcium carbonate (CaCO3), magnesium carbonate (MgCO3), calcium phosphate (Ca3(PO4)2), magnesium phosphate (Mg3(PO4)2), aluminum phosphate (AlPO4), magnesium sulfate (MgSO4), calcium hydroxide (Ca(OH)2), magnesium hydroxide (Mg(OH)2), aluminum hydroxide (Al(OH)3), zinc hydroxide (Zn(OH)2) or mixtures thereof.

[0082] By adjusting the equivalent of the divalent or trivalent metal ion, the release rate of the drug encapsulated inside the polymer nanoparticle can be adjusted. Specifically, when the divalent or trivalent metal ion is contained in the polymer nanoparticle composition in an amount of 1 equivalent or less relative to the equivalent of the carboxylic acid group of the polylactate, the number of ions bound to the carboxyl end group of the polylactate is small, and the release rate of the drug is fast. When the divalent or trivalent metal ion is contained in an amount of 1 equivalent or more relative to the equivalent of the carboxylic acid group of the polylactate, the number of ions bound to the carboxyl end group of the polylactate is large, and the release rate of the drug is sustained. Therefore, in order to increase the release rate of the drug in the blood, a small equivalent of the metal ion can be used, and in order to sustain the release rate of the drug, a large equivalent of the metal ion can be used.

[0083] The divalent or trivalent metal ion may be contained in an amount of 0.01 to 10 equivalents, 0.1 to 5 equivalents, or 0.2 to 2 equivalents relative to the equivalent of the carboxyl terminal group of the polylactate.

[0084] In a particular embodiment, the composition of the present invention, which includes an active ingredient-cationic compound complex encapsulated in a nanoparticle structure of an amphiphilic block copolymer and polylactate, can be administered via routes such as blood vessels, muscles, subcutaneous, oral, bone, transdermal or local tissue, and can be formulated into various oral or parenteral administration preparations. Examples of the oral administration preparations include tablets, capsules, powders and liquids, and examples of the parenteral administration preparations include various preparations such as eye drops and injections. In a preferred embodiment, the composition may be an injectable preparation. For example, when the composition of the present invention is lyophilized, it can be redissolved in distilled water for injection, 0.9% saline, 5% dextrose aqueous solution, or the like to prepare an injectable preparation.

[0085] In one embodiment, the immune-inducing composition can be used to treat or prevent cancer. The immune-inducing composition can efficiently deliver an active ingredient to the spleen. In this process, even if the composition is an autoantigen that is difficult to recognize as an antigen by a single administration, the composition induces immunity against the antigen, so that the same effect as a vaccine can be expected. Therefore, in one embodiment, the immune-inducing composition may target the spleen. These effects can be obtained not only by local administration but also by systemic administration.

[0086] Another aspect of the present invention provides a method for producing a composition for inducing immunity, comprising the steps of: (a) mixing a nucleic acid, a polypeptide or a combination thereof, a cationic compound, an amphiphilic block copolymer and a polylactate as active ingredients with a solvent which is an aqueous solution, a water-miscible organic solvent or a combination thereof; and (b) adding an aqueous solvent to the mixture of step (a) and mixing the mixture.

[0087] The step (a) can include (a-1) a step of mixing an aqueous solution containing a nucleic acid, a polypeptide or a combination thereof as an active ingredient with a solution in which a cationic compound is dissolved in a water-miscible organic solvent, and (a-2) a step of mixing the mixture of the step (a-1) with an aqueous solution of an amphiphilic block copolymer and a polylactate.

[0088] Moreover, the step (a) may further include the steps of (a-1) mixing an aqueous solution containing a nucleic acid, a polypeptide, or a combination thereof as an active ingredient with an aqueous solution containing a polylactate, and (a-2) mixing the mixture of the step (a-1) with an aqueous solution containing a water-miscible organic solvent containing a cationic compound and an amphiphilic block copolymer.

[0089] The method may further comprise the step of removing the solvent from the mixture of step (b).

[0090] The "aqueous solution" refers to, for example, water, sterile purified water, a buffer, an injection solution, etc., and may be a buffer further containing an organic acid. The aqueous solution may be, for example, a citrate buffer, a PBS buffer, etc., but is not limited thereto.

[0091] The "water-miscible organic solvent" may be, but is not limited to, alcohol, acetone, acetonitrile, water mixtures thereof, or mixtures thereof.

[0092] According to another embodiment, the production method may further comprise a step of adding a freeze-drying auxiliary and freeze-drying after the step of removing the solvent.

[0093] According to another embodiment, the method of manufacture may further comprise the step of sterilizing the aqueous polymeric nanoparticle solution through a sterile filter prior to said lyophilization.

[0094] The freeze-drying aid used in the present invention is added to help the freeze-dried composition maintain the cake form or to help the amphiphilic block copolymer composition dissolve quickly and uniformly during the redissolution process after freeze-drying. Specifically, the freeze-drying aid may be at least one selected from the group consisting of lactose, mannitol, sorbitol and sucrose. The content of the freeze-drying aid may be 1-90% by weight, more specifically 10-60% by weight, based on the total dry weight of the freeze-dried composition.

[0095] The nanoparticles in the composition produced by the production method of the present invention are stable in blood, as a complex of an active ingredient and a cationic compound is encapsulated in an amphiphilic block copolymer and polylactate nanoparticle structure, and their size may be specifically 10 to 200 nm, more specifically 10 to 150 nm.

[0096] The present invention will now be described in detail with reference to the following examples, which are intended only to illustrate the present invention and are not intended to limit the scope of the present invention in any manner.

[0097] [Examples 1 and 2] Preparation of a composition for inducing immunity containing tumor antigen-expressing mRNA as an active ingredient 100 μL of Trp2 mRNA (TriLink, cat ♯ L7008, 10 mM citrate buffer, 1 mg / mL) and 76.4 μL of PLANa aqueous solution (10 mg / mL) were prepared and mixed. Next, 30 μL of cationic lipid 2OT (N,N'-((ethane-1,2-diylbis(azanediyl))bis(ethane-2,1-diyl))dioleamide) solution (20 mM sodium acetate buffer, 20 mg / mL) and 166 μL of mPEG-PLA aqueous solution (10 mg / mL) were mixed and added to the prepared Trp2 mRNA and PLANa mixed solution to form a complex. After that, PBS was filled up to an mRNA concentration of 10 μg / 200 μL, and the mixture was filtered through a 0.22 μm PVDF filter to eliminate the formulation containing large particles.

[0098] [Table 1]

[0099] mPEG-PLA: monomethoxypolyethylene glycol-polylactide block copolymer PLANa: Polylactic acid sodium salt

[0100] [Experimental Example 2] Confirmation of protein expression after intravenous injection of mouse immunity-inducing composition The immune-inducing compositions of Examples 1 and 2 were administered to the hind legs of mice using PBS buffer as a carrier. After administration three times on days 0, 7, and 14, the mice were sacrificed on day 20, the spleens were harvested, and splenocytes were isolated. Splenocytes were removed from the spleen, the tissue was placed in a 40 μm cell strainer, and homogenized using the tip of a syringe plunger. 5 mL of serum-free medium was added and centrifuged (1,200 rpm, 5 min, 4° C.). After removing the supernatant, 1 mL of red blood cell lysis solution was added. After reacting at room temperature for 3 minutes, 1 mL of PBS was added for neutralization. After centrifugation (1,200 rpm, 5 min, 4° C.), the cells were suspended in 1 mL of medium and stored on ice. After staining with 0.4% trypan blue solution, live splenocytes were counted using a hemocytometer.

[0101] To confirm whether IFN-γ was expressed in each test group and control group, the results were graphed using ELISPOT. 5 ~1×10 6 The splenocytes were placed in an ELISPOT plate and OVA peptide (10 μg / mL) was added as a stimulant. After 36 hours of reaction in a humidified incubator at 37°C under 5% CO2 conditions, IFN-γ expression was measured using a detection antibody against IFN-γ and streptavidin-ALP, and the results were shown in a graph. In splenocytes, IFN-γ secretion is thought to be mainly the result of activation of T cells that recognize antigenic epitopes.

[0102] As control groups, PBS carrier and free mRNA not processed into nanoparticles were administered. As a comparison group, a commercially available transfection reagent, TransIT (registered trademark), was used, and as a positive control group, Trp2 peptide and I:C (polyinosinic acid:polycytidylic acid) were administered subcutaneously. "I:C" is known to act as an immune adjuvant by stimulating TLR3 (Toll-like receptor) expressed on the cell surface of B cells, macrophages, and dendritic cells. Therefore, when used in combination with the antigenic substance Trp2, immune activity is induced, making it suitable as a positive control group. Table 2 below summarizes the test groups and the dose of antigenic substance (mRNA) administered to each test group.

[0103] [Table 2]

[0104] The results are shown in Figures 3 and 4. As a result of observing IFN-γ expression, strong IFN-γ was detected in the groups administered with the immunity-inducing compositions of Examples 1 and 2 and in the positive control group.

[0105] [Experimental Example 3] Confirmation of mouse splenocyte expression rate of immune-inducing composition The immune-inducing composition was prepared in the same manner as in Example 2, except that instead of the mRNA expressing Trp2, mRNA capable of expressing luciferase (luc: Trilink, cat #L7202) was introduced (Example 3). 10 μg of mRNA from Example 3, Comparative Example 1 with a small N / P (nitrogen to phosphate) ratio, and Comparative Example 2 with a large N / P ratio were administered to the tail vein of mice (BALB / cAnNCrlOri, n=3). Six hours after administration, the mice were sacrificed, and each organ was removed to measure the luminescence expression pattern (L=liver, S=spleen, H=heart, Lu=lung, K=kidney). As a result, as shown in FIG. 5, it was confirmed that luminescence expression was significantly higher in the spleen than in other organs.

[0106] [Experimental Example 4] Confirmation of the cancer treatment effect of the immune-inducing composition The composition for inducing immunity of Example 2 was used. Mice (BALB / cAnNCrlOri, n=3) were inoculated with 2.5×10 melanoma cells B16F10 cells. 5 The mice were intravenously administered with 10 μg of mRNA and 10 μg of PBS as a carrier into the tail vein on the third day, and 10 μg of mRNA was administered for immunization on the third, fifth, seventh, and nineth days. After sacrifice on the 19th day, metastasis to lung nodules was observed. As control groups, mice were administered with only the carrier PBS and with only the mRNA. One-way ANOVA post hoc Tukey's multiple comparisons on the number of lung nodules showed the statistics shown in Figure 6 (***p<0.001, ****p<0.0001 vs.mRNA). As a result, it was observed that the lung metastasis of the immune-inducing composition of the present invention was significantly lower than that of the control group, confirming the effectiveness of the cancer treatment by immune induction.

[0107] All animal experiments were performed in accordance with protocols approved by the Institute for Laboratory Animal Care and Use (IACUC).

Claims

1. A composition for inducing immunity, comprising: Nucleic acids, polypeptides or combinations thereof as active ingredients; 1,6-dioleoyltriethylenetetramine as a cationic compound; As an amphiphilic block copolymer, an A-B type block copolymer consisting of a hydrophilic A block of monomethoxypolyethylene glycol and a hydrophobic B block of polylactic acid, and Polylactate sodium salt (PLANa) as polylactate Including, The content of the cationic compound is 3 to 8 parts by weight per 1 part by weight of the active ingredient. The content of the amphiphilic block copolymer is 15 to 30 parts by weight per 1 part by weight of the active ingredient. The content of the polylactate is 5 to 30 parts by weight per 1 part by weight of the active ingredient, The active ingredient is encapsulated within the nanoparticle structure formed by the amphiphilic block copolymer and the polylactate; The composition for inducing immunity is characterized in that it is intended to efficiently deliver an active ingredient to the spleen and to induce immunity using the active ingredient to treat or prevent cancer.

2. The composition for inducing immunity according to claim 1 , wherein the nucleic acid expresses a tumor antigen.

3. The composition for inducing immunity according to claim 1 , wherein the polypeptide can be recognized as a tumor antigen.

4. The composition for inducing immunity according to claim 1, wherein the surface charge of the nanoparticles is -40 to 40 mV.

5. The immune-inducing composition according to claim 1, wherein the hydrophobic B block is modified by chemically bonding tocopherol, cholesterol, or a fatty acid having 10 to 24 carbon atoms to its terminal hydroxyl group.

6. 2. The composition for inducing immunity according to claim 1, wherein the hydrophilic A block has a number average molecular weight of 200 to 50,000 daltons, and the hydrophobic B block has a number average molecular weight of 50 to 50,000 daltons.

7. An immune-inducing composition as described in claim 2 or 3, which is for treating or preventing cancer associated with a tumor antigen.

8. An immune-inducing composition as described in claim 2 or 3, for use as a vaccine against cancer associated with a tumor antigen.

9. A method for producing a composition for inducing immunity, comprising: (a) mixing a nucleic acid, a polypeptide or a combination thereof as an active ingredient, 1,6-dioleoyltriethylenetetramide as a cationic compound, an A-B type block copolymer consisting of a hydrophilic A block of monomethoxypolyethylene glycol and a hydrophobic B block of polylactic acid as an amphiphilic block copolymer, and polylactic acid sodium salt (PLANa) as a polylactate salt with a solvent which is an aqueous solution, a water-miscible organic solvent or a combination thereof; (b) adding an aqueous solvent to the mixture of step (a) and mixing the mixture; Including, The amount of the cationic compound mixed is 3 to 8 parts by weight per 1 part by weight of the active ingredient. The amount of the amphiphilic block copolymer mixed is 15 to 30 parts by weight per 1 part by weight of the active ingredient, The amount of polylactate mixed is 5 to 30 parts by weight per 1 part by weight of the active ingredient. The active ingredient is encapsulated within the nanoparticle structure formed by the amphiphilic block copolymer and the polylactate; A method for producing a composition for inducing immunity, characterized in that the composition is for efficiently delivering an active ingredient to the spleen and for inducing immunity using the active ingredient to treat or prevent cancer.

Images