Cationic polysaccharide copolymer adjuvant, and vaccine

Abstract

PURPOSE: To obtain a latex polymerization product useful as an adjuvant material for a vaccine by polymerizing an olefin compound in water using a polysaccharide cationic derivative to formulate a surfactant-free latex solution, thereby obtaining a non-viral vector for delivering deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) into cells.CONSTITUTION: Provided is a latex solution comprising a polymer obtained by polymerizing an olefin compound in water in the presence of a polysaccharide cationic derivative. Provided is also a complex obtained by reacting the latex solution with various inactivated viruses, deoxyribonucleic acid (DNA), or ribonucleic acid (RNA).EFFECT: The use of such a non-viral vector enables safe application, in contrast to a vector derived from hazardous viruses, and ensures stable utilization because it is an artificial product. Cell membrane selectivity plays an essential role in improving transformation efficiency as a vector. To further improve transformation efficiency, it is essential to have hydrophobic-hydrophilic domains. Specifically, to improve the efficacy of an adjuvant material for a vaccine, it is essential to form a latex composed of a copolymer material of cationic polysaccharides and vinyl monomers so as to enhance endocytosis and potentiate TLR7 agonists.SELECTED DRAWING: None

Description

Technical Field

[0001] The monomer graft copolymer having a cationic derivative of a water-soluble linear polysaccharide of the present invention - olefin is obtained by graft polymerizing a monomer having an underwater olefin to a cationic derivative of a water-soluble linear polysaccharide having a hydroxyl group to produce a latex polymerization product useful as an adjuvant material for vaccines. The present invention suggests that any cationic derivative of a linear polysaccharide having a hydroxyl group and being water-soluble can be graft polymerized with a monomer having an underwater olefin to produce a latex polymerization product useful as an adjuvant material for vaccines. An adjuvant is an auxiliary agent for the main agent and is used in combination for the purpose of assisting, enhancing, or improving the original action of the active ingredient of the main agent. In the field of immunology, an adjuvant is also called an antigenicity enhancer and is a substance that is injected together with an antigen and used to enhance its antigenicity. The present invention improves vaccines by including the TLR7 agonist possessed by the cationic derivative of the water-soluble linear polysaccharide of the present invention - olefin in the vaccines.

Background Art

[0002] The current development of novel coronavirus vaccines in the world is mainly focused on viral vector vaccines and mRNA vaccines. The institutions that have started clinical trials are the University of Oxford / AstraZeneca in the UK, Pfizer and BioNTech in the US, and Moderna in the US. The vectors are an adeno-associated virus vector (AAV vector) and a liposome vector of lipid nanoparticles (lipofection) respectively. Generally, viral vectors are suspected of being pathogenic and neutralizing antibodies are produced due to their immunogenicity, so repeated administration is contraindicated. Transfection using lipid liposomes has many unstable factors such as limited micelles and is difficult to use in vivo. Also, the stability in vivo becomes a problem for the entire non-viral vector. Most viral vectors are integrated into the host chromosome, and mRNA is stably expressed in the introduced cell line, but there is a question of causing excessive acquired immunity in sensitization by repeated ingestion. Generally, a vaccine is a biological preparation that has an effective role in preventing infectious diseases when inoculated. By inactivating the toxicity and injecting weakened pathogens into the body, antibodies are produced, making it less likely to contract the target infectious disease. Vaccines are broadly classified into live vaccines, inactivated vaccines, and toxoids. Recently, vaccines using messenger RNA and DNA of pathogens have been developed. The problems with vaccines as a whole have remained the same as before. The factors considered in vaccine design include form, manufacturing method, adjuvant, and administration method. The inoculation methods include subcutaneous injection, oral administration, intradermal injection, etc., and are determined for each vaccine. In principle, simultaneous inoculation of multiple vaccines is prohibited for both live vaccines and inactivated vaccines. Since the inoculation interval is also specified when inoculating multiple vaccines more than 4 weeks apart for live vaccines and more than 1 week apart for inactivated vaccines, there are issues that require careful consideration (worldwide, there is a trend towards recognizing simultaneous inoculation of multiple vaccines). In current viral infectious diseases (such as COVID-19) and RNA and viral vaccines, the problem is that it is difficult to manufacture vaccines with high titers, and there are few that are satisfactory in terms of their effectiveness. Therefore, the development of adjuvants to increase the titer must be considered. In drug delivery systems (DDS), it is possible to increase the particle size based on the balance of water solubility and lipid solubility, etc., and the molecular weight, enabling the enhanced permeability and retention (EPR) effect and the reticuloendothelial system (RES) suppression effect. In the EPR effect, particles with a diameter of several tens to 200 nm penetrate the newly formed blood vessels and the drug accumulates in the tumor tissue. In RES suppression, particles with a diameter of 400 nm or more are phagocytosed and eliminated as foreign substances by Kupffer cells in the liver and macrophages in the adrenal glands. Since drugs are decomposed by drug metabolism in the liver, those with a size of 5 nm or less are excreted by filtration in the glomeruli of the kidneys. By achieving such an optimal particle size, it is possible to maintain the drug concentration in the body for a long time. Such a method is called prodrug formation and improves the convenience of the drug. However, in order to be more efficiently taken up by cancer cells, there are problems such as the need to be positively charged with respect to the negatively charged cancer cell surface and the possibility of heat sterilization of DDS for safety reasons. Although latex polymerization products have been conventionally produced as immunoassay materials, most of the production methods involve emulsion polymerization in an aqueous solution in the presence of a surfactant, and soapless products without the presence of a surfactant are desired. This is because the surfactant present in the aqueous solution affects the action of the latex diagnostic agent. As a means to solve this problem, a monomer graft copolymer having a cationic derivative of a water-soluble linear polysaccharide - olefin is produced by graft polymerizing a monomer having an olefin in water with a cationic derivative of a water-soluble linear polysaccharide having a hydroxyl group using a redox initiator or the like, and is produced as a soapless latex polymerization product useful as an immunoassay material. Patents for this soapless cationic derivative of water-soluble linear polysaccharide - monomer graft copolymer latex and latex diagnostic agents have already been established. This is used in antibody adsorption latex diagnostic agents. Emulsion polymerization is a polymerization method in which an olefin monomer is suspended in an aqueous solution and usually emulsified using a surfactant or the like. Specifically, the monomer or growing chain interacts at the water-solvent interface by hydrogen bonding, Coulomb force, charge transfer interaction, van der Waals force, etc., and the polymer chain grows by polymerization to form fine particles in the aqueous solution. Usually, the polymerization product exists as a mixture of the polymerized monomer and the surfactant. The surfactant, which is considered an impurity, sometimes interferes when used in latex diagnostic agents and has been a problem. This time, unexpectedly, using this technique for producing soapless latex, it has been found that a supramolecular compound having substrate selectivity as an artificial enzyme can be formed, and a latex polymerization product useful as a drug delivery (DDS) material for nucleic acids and drugs can be produced.

Patent Document 1

Disclosure of the Invention

Problems to be Solved by the Invention

[0003] Most of the currently commercialized drug delivery systems (DDS) are difficult to control the balance such as water solubility and lipid solubility and the molecular weight, and there are problems with safety such as heat sterilization. Furthermore, when using the drug delivery system (DDS), in order to be efficiently taken up by cancer cells, there are problems such as the need to be positively charged with respect to the negatively charged cancer cell surface and the possibility of autoclave sterilization of the DDS for safety reasons. In the invention of Patent No. 4650605, the safety and the possibility of autoclave sterilization are clarified. It not only suppresses side effects by a mere drug delivery system, expands the convenience of drugs, and enhances the drug efficacy, but also has the possibility of having substrate selectivity as an artificial enzyme of a supramolecular anticancer agent complex, and further, by fixing a cancer target substance, an anticancer drug resistance overcoming agent or a chemotherapy enhancer to the anticancer agent by a novel immobilization method, it has been clarified that the anticancer effect is enhanced. Furthermore, in recent technology, a copolymer (DDMC) obtained by graft-polymerizing methyl methacrylate (MMA) onto DEAE-dextran has not only improved transfection efficiency, but also reduced cytotoxicity compared to DEAE-dextran, and the in-vivo residence time has been improved from 1 day to 25 days. This means that DEAE-dextran can be further improved and is effective as an adjuvant for inactivated virus vaccines that have been studied.

Means for Solving the Problems

[0004] In innate immunity, Toll-like receptor (TLR) is a pattern recognition receptor that recognizes the components of viruses and bacteria, and transmits a signal for infection defense to lymphocytes through the induction of type I interferon (IFN) and inflammatory cytokine production and the maturation of dendritic cells. There are 10 types of these infection defense signals in humans, but the signals induced by TLR activation are defined by the combination of each TLR and downstream adapter molecules, so the final cellular responses are different. In order to induce the desired lymphocyte response, it is necessary to select an optimal TLR ligand, which is a strategy for adjuvant development. In recent years, it has become clear that TLR recognizes not only components derived from microorganisms but also endogenous molecules, and its involvement in autoimmune diseases and inflammatory diseases has attracted attention. In the present invention, the function of TLR that links innate immunity and acquired immunity was considered. Both DEAE-D and the improved DDMC, compounds having an α1-6 sugar chain are commonly TLR7 agonists and are considered to lead to the activation of dendritic cells. Furthermore, DDMC undergoes endocytosis, and TLR7 agonists become larger. Also, due to the RES effect, the antigen presentation period extends to a long term of 25 days (half a day for DEAE-D). In vaccine development, adjuvant development is considered decisive. The main factor is that it is a TLR7 agonist that specifically activates TLR7 possessed by the cationic derivative - olefin of the water-soluble linear polysaccharide of the present invention. Currently, formulations in which paclitaxel is encapsulated in cationic liposomes (Patent Document 2 JP, 2006-517594, A) and drug delivery using drug-encapsulated polymer micelles in which drugs such as anticancer agents are encapsulated in polymer micelles composed of block copolymers having a hydrophilic region and a hydrophobic region are known (Patent Documents 3 to 5). [Patent Document 2] Japanese Patent Application Laid-Open No. 2006-517594 [Patent Document 3] Japanese Patent Application Laid-Open No. 2001-226294. [Patent Document 4] Japanese Patent Application Laid-Open No. 2005-336402 [Patent Document 5] JP 2007-023023 A These target endothelial cells of tumor blood vessels that carry a negative charge, inhibit angiogenesis, and exhibit an antitumor effect, but the reduction of side effects is not complete. In genetic engineering, when transplanting a certain gene into another organism, a so-called carrier is needed to carry the gene, and this is called a vector. Currently, the vectors being used are various viruses, plasmids that parasitize bacteria, and phages that infect bacteria. If these are infected with a gene attached to them, the gene can be introduced into the bacteria. If a complex of a cationic polymer and nucleic acid (DNA, RNA) is used instead of this plasmid or phage, the nucleic acid of the complex can be directly introduced into cells prepared in advance. When using this non-viral vector, unlike vectors of dangerous viruses, it is safe and an artificial substance, so it can be stably used. In genetic engineering, when transplanting a certain gene into another organism, a so-called carrier is needed to carry the gene, and this is called a vector. If taxane is used instead of this nucleic acid and a complex with a cationic polymer is used, the taxane of the complex can be directly introduced into cells prepared in advance. Cationic polysaccharides are promising as cationic polymers because the complex needs to penetrate the cell membrane, and this possibility depends on the reaction between the positive charge of the complex caused by the cationic polysaccharide and the negative charge on the cell membrane surface, and the interaction between the polysaccharide on the cell membrane surface and the complex. Polymer biocompatibility is important regarding the permeation selectivity of the cell membrane as a drug delivery (DDS) material. Furthermore, to impart biocompatibility, the drug delivery (DDS) material of the cationic polymer used needs to have a hydrophobic-hydrophilic domain. Specifically, it is important to form a latex composed of a copolymer of a cationic polysaccharide such as DEAE-dextran and a vinyl monomer, and to have both a hydrophobic part due to the polymerized part of the vinyl monomer and a hydrophilic part due to the cationic polysaccharide.That is, the biocompatibility of the composite latex having the resulting hydrophobic and hydrophilic domains is important. Unexpectedly, by further copolymerizing a cationic polysaccharide and a vinyl monomer, the supramolecular reaction with nucleic acids and taxanes is enhanced, and it has been discovered that the low introduction rate and drug efficacy of nucleic acids and taxanes into cells of the drug delivery (DDS) material of the cationic polysaccharide can be improved.

Effect of the Invention

[0005] Currently, the development of the novel coronavirus vaccine in the world is mainly based on viral vectors and mRNA vaccines. The first institutions to start clinical trials are the University of Oxford and Moderna in the United States. The vectors are the adeno-associated virus vector (AAV vector) and the LNP liposome vector (lipofection) respectively. Generally, viral vectors are suspected of pathogenicity and neutralizing antibodies are generated from their immunogenicity, so repeated administration is contraindicated. Transfection using lipid liposomes has many unstable factors such as limited micelles and is difficult to use in vivo. Also, the stability in vivo becomes a problem for the whole non-viral vectors. Most viral vectors are integrated into the host chromosome, and mRNA is stably expressed in the introduced cell line, but it causes acquired immunity by sensitization and is not suitable for in vivo use. The copolymer (DDMC) obtained by graft-polymerizing methyl methacrylate (MMA) onto DEAE-dextran not only has higher transfection efficiency, but also has reduced cytotoxicity compared to DEAE-dextran, and its in vivo residence time has been improved from 1 day to 25 days. This indicates that DEAE-dextran can be further improved and is effective as an adjuvant for the inactivated virus vaccine being studied. In the study with DEAE-dextran, for example, Figure 1 shows the administration of the Venezuelan equine encephalitis (VEE) inactivated virus vaccine (IVEE) to rhesus monkeys to examine the presence or absence of DEAE-dextran addition. The antibody titer was determined by the plaque reduction neutralization test, and the geometric mean titer (y-axis) was maintained at 400 times when DEAE-dextran was added. That is, in the case of 5 mg / DEAE-D, the maximum peak was maintained for 72 days with a single prophylactic vaccination (10 days without DEAE-D addition). When the DEAE-dextran-MMA copolymer (DDMC) was used, it was maintained at about 4 times higher (Table 1).

Table 1

Best Mode for Carrying Out the Invention

[0006] Hereinafter, the cationic derivative of the linear polysaccharide of the present invention - a monomer graft copolymer having an olefin will be described in detail. The cationic derivative of the linear polysaccharide - a monomer graft copolymer having an olefin is obtained through the following step (2). It has been found that this polymer forms a complex with an anticancer agent through step (3) and is taken into cancer cells to cause cell death of the cancer cells. (1) Preparation of cationic derivative of linear polysaccharide When it exists in a solid state, the formula of the unit of the cationic derivative of the linear polysaccharide is

Chemical formula

Chemical formula

Example

[0007] Example 1 2 g of DEAE (diethylaminoethyl)-dextran hydrochloride with a nitrogen content of 5% based on dextran with an average molecular weight Mw of 500,000 was dissolved in 50 ml of water. Then, 8 ml of methyl methacrylate (MMA) was added. After thoroughly replacing the air in the reaction solution and the reaction vessel with nitrogen gas, while stirring well, 100 mg of cerium (IV) ammonium nitrate nitrate dissolved in 15 ml of 0.1 N nitric acid whose dissolved air was replaced with nitrogen gas was added to start the reaction. The reaction was carried out at 30 °C for 2 hours to form latex. At the end of the reaction, 3 ml of a 1% solution of hydroquinone was used as a terminator. Then, the reaction solution was poured into three times the amount of methanol to obtain a precipitate. This precipitate was thoroughly washed with hot water, centrifuged, and dried under reduced pressure at 50 °C. Then, the dried product was placed in a Soxhlet extractor and extracted with acetone for 24 hours to obtain 1.5 g of the hydrochloride of DEAE (diethylaminoethyl)-dextran-MMA copolymer. Nitrogen content 1.7%, grafting rate 200% Yield of 25% relative to DEAE-dextran This does not dissolve in water, which is a good solvent for DEAE-dextran hydrochloride, nor in acetone, which is a good solvent for polymethyl methacrylate. Looking at the infrared absorption spectrum of this substance, the absorption of the carbonyl group not seen in DEAE-dextran hydrochloride is at a wave number of 1730 cm -1It can be found nearby. Example 2 After performing the same reaction as in Example 1, the reaction-terminated solution of the latex was not injected into methanol, but dialysis was carried out in water to remove unreacted substances and initiators, and a DEAE-dextran-MMA copolymer latex was obtained. This is useful as a recombinant vector and shows test results. The test method was carried out according to the procedure of (4) - Protocol A in the column of (Best Mode for Carrying Out the Invention). After incubating the transformed cells, COS-1 cells, at 37°C for 50 hours, the expression efficiency was examined. That is, the expression efficiency of transformation for observing the effect of the vector depended on the luciferase activity expressed in the COS-1 cells incorporated. When comparing the sample of Example 2 with the value of DEAE-dextran hydrochloride having a nitrogen content of 5% using dextran with an average molecular weight Mw of 500,000 as the matrix being 1, a luciferase activity 5 times higher was obtained. Example 3 The latex of the DEAE (diethylaminoethyl)-dextran-MMA copolymer obtained in Example 2 was adjusted to a solution of 10 mg / ml in terms of DEAE (diethylaminoethyl)-dextran. When 2 ml of this solution was added to 1 ml of a DNA solution (20 mg / ml) derived from salmon sperm, it completely precipitated in 0.4 hours, and a complex of 20 mg of DEAE (diethylaminoethyl)-dextran-MMA copolymer and DNA was obtained. When the same operation was carried out with the hydrochloride of the raw material DEAE (diethylaminoethyl)-dextran, it took 96 hours to completely precipitate. Figure 1 is the infrared absorption spectrum of the product. Wavenumber 1000 cm -1 to 1100 cm -1 Absorption of the pyranose ring derived from DEAE (diethylaminoethyl)-dextran is observed, and absorption due to the stretching vibration of P-O derived from DNA is observed near 1220 cm -1 and absorption due to the carbonyl group C=O derived from MMA is observed near 1730 cm -1 Example 4 ​The latex of the DEAE (diethylaminoethyl)-dextran-MMA copolymer obtained in Example 2 is adjusted to a solution of 10 mg / ml in terms of DEAE (diethylaminoethyl)-dextran. When 2 ml of this solution was added to 1 ml of a yeast-derived RNA solution (20 mg / ml), it completely precipitated in 4 hours, and a complex of 10 mg of DEAE (diethylaminoethyl)-dextran-MMA copolymer and RNA was obtained. When the same operation was carried out with the hydrochloride salt of the raw material DEAE (diethylaminoethyl)-dextran, it took 144 hours to completely precipitate. Figure 2 is the infrared absorption spectrum of the product. From a wavenumber of 1000 cm -1 to 1100 cm -1 the absorption of the pyranose ring derived from DEAE (diethylaminoethyl)-dextran is observed. Near 1230 cm -1 the absorption due to the stretching vibration of P-O derived from RNA is observed, and near 1730 cm -1 the absorption of the carbonyl group C=O derived from MMA is observed. Example 5 4 g of DEAE (diethylaminoethyl)-pullulan hydrochloride with a nitrogen content of 4% based on pullulan with an average molecular weight Mw of 200,000 was dissolved in 80 ml of water. Then, 10 ml of methanol and 35 ml of styrene monomer were added. After thoroughly replacing the air in the reaction solution and the reaction vessel with nitrogen gas and stirring well, 200 mg of ceric ammonium nitrate nitrate dissolved in 30 ml of 0.1 N nitric acid whose dissolved air had been replaced with nitrogen gas was added to start the reaction. The reaction was carried out at room temperature for 1 hour to form a latex. 3 ml of a 1% solution of hydroquinone was used as a terminator at the end of the reaction. The subsequent purification and drying steps were carried out in the same manner as in Example 1 to obtain 7 g of the hydrochloride salt of DEAE (diethylaminoethyl)-pullulan-styrene copolymer. Nitrogen content 0.92%, grafting rate 350% Yield based on DEAE-pullulan 38% Example 6 After performing the same reaction as in Example 5, instead of injecting the reaction-terminated solution of the latex into methanol, dialysis was carried out in water to remove unreacted substances and the initiator, and a DEAE-pullulan-styrene copolymer latex was obtained. This was useful as a recombinant vector. According to the same procedure as in Example 2, the luciferase expression activity of the latex solution of this was 1.5 times the value of DEAE-dextran hydrochloride in Example 2 with the value of Example 2 being set to 1, and luciferase expression activity was obtained. Example 7 Adjust the latex of the DEAE (diethylaminoethyl)-pullulan-styrene copolymer obtained in Example 6 to a solution of 10 mg / ml in terms of DEAE (diethylaminoethyl)-pullulan. When 2 ml of this solution was added to 1 ml of a DNA solution (20 mg / ml) derived from salmon sperm, it completely precipitated in 2.5 hours, and a complex of 12 mg of DEAE (diethylaminoethyl)-pullulan-styrene copolymer and DNA was obtained. Example 8 Adjust the latex of the DEAE (diethylaminoethyl)-pullulan-styrene copolymer obtained in Example 6 to a solution of 10 mg / ml in terms of DEAE (diethylaminoethyl)-pullulan. When 2 ml of this solution was added to 1 ml of a RNA solution (20 mg / ml) derived from yeast, it completely precipitated in 5 hours, and a complex of 9 mg of DEAE (diethylaminoethyl)-pullulan-styrene copolymer and RNA was obtained. Example 9 4 g of AE (aminoethyl)-dextran hydrochloride with a nitrogen content of 5% based on dextran with an average molecular weight Mw of 40,000 was dissolved in 90 ml of water. Then, 5 ml of methanol and 20 ml of butyl methacrylate were added. After thoroughly replacing the air in the reaction solution and the reaction vessel with nitrogen gas, while stirring well, 50 mg of cerium (IV) ammonium nitrate dissolved in 15 ml of 0.1 N nitric acid whose dissolved air had been replaced with nitrogen gas was added to start the reaction. The reaction was carried out at room temperature for 30 minutes to form a latex. At the end of the reaction, 3 ml of a 1% hydroquinone solution was used as a terminator. The subsequent purification and drying steps were carried out in the same manner as in Example 1 to obtain 6 g of the hydrochloride of AE (aminoethyl)-dextran-butyl methacrylate copolymer. Nitrogen content 1.3%, grafting ratio 300%, yield based on AE-dextran 38% This product is insoluble in water, which is a good solvent for AE-dextran hydrochloride, and also insoluble in acetone, which is a good solvent for polybutyl methacrylate. Example 10 After carrying out the same reaction as in Example 9, the reaction-terminated solution of the latex was not poured into methanol but was dialyzed in water to remove unreacted substances and initiators, obtaining an AE (aminoethyl)-dextran-butyl methacrylate copolymer latex. This was useful as a recombinant vector. Following the same procedure as in Example 2, the luciferase expression activity of the latex solution of this product was 1.5 times the value of the DEAE-dextran hydrochloride in Example 2 when the value of the DEAE-dextran hydrochloride in Example 2 was taken as 1. Example 11 The latex of the AE (aminoethyl)-dextran-butyl methacrylate copolymer obtained in Example 10 was adjusted to a solution of 10 mg / ml in terms of AE (aminoethyl)-dextran. When 2 ml of this solution was added to 1 ml of a DNA solution (20 mg / ml) derived from salmon sperm, it completely precipitated in 3 hours, obtaining a complex of 12 mg of AE (aminoethyl)-dextran-butyl methacrylate copolymer and DNA. Example 12 The latex of the AE (aminoethyl)-dextran-butyl methacrylate copolymer obtained in Example 10 is adjusted to a solution of 10 mg / ml in terms of AE (aminoethyl)-dextran. When 2 ml of this solution was added to 1 ml of a yeast-derived RNA solution (20 mg / ml), it completely precipitated in 5 hours, and a complex of 10 mg of the AE (aminoethyl)-dextran-butyl methacrylate copolymer and RNA was obtained. Example 13 4 g of HPTMA (2-hydroxypropyltrimethylammonium)-pullulan hydrochloride with a nitrogen content of 3% based on pullulan with an average molecular weight Mw of 30,000 was dissolved in 100 ml of water, and then 30 ml of methyl acrylate monomer was added. After thoroughly replacing the air in the reaction solution and the reaction vessel with nitrogen gas, while stirring well, 200 mg of ceric ammonium nitrate nitrate dissolved in 20 ml of 0.1 N nitric acid whose dissolved air had been replaced with nitrogen gas was added to start the reaction. The reaction was carried out at room temperature for 1 hour to form a latex. At the end of the reaction, 4 ml of a 1% solution of hydroquinone was used as a terminator. The subsequent purification and drying steps were carried out in the same manner as in Example 1 to obtain 2 g of the hydrochloride of the HPTMA (2-hydroxypropyltrimethylammonium)-pullulan-methyl acrylate copolymer. Nitrogen content 1.2%, grafting rate 150% Yield of HPTMA-pullulan 20% Example 14 After carrying out the same reaction as in Example 13, dialysis was carried out in water to remove unreacted substances and initiators, and an HPTMA (2-hydroxypropyltrimethylammonium)-pullulan-methyl acrylate copolymer latex was obtained. This was useful as a recombinant vector. According to the same procedure as in Example 2, the luciferase expression activity of the latex solution of this product was 1.1 times the value of the DEAE-dextran hydrochloride in Example 2 when the value of the latter was set to 1. Example 15 The latex of the HPTMA (2-hydroxypropyltrimethylammonium)-pullulan-methyl acrylate copolymer obtained in Example 14 is adjusted to a solution of 10 mg / ml in terms of HPTMA (2-hydroxypropyltrimethylammonium)-pullulan. When 2 ml of this solution was added to 1 ml of a DNA solution (20 mg / ml) derived from salmon sperm, it completely precipitated in 5 hours, and a complex of 10 mg of HPTMA (2-hydroxypropyltrimethylammonium)-pullulan-methyl acrylate copolymer and DNA was obtained. Example 16 The latex of the HPTMA (2-hydroxypropyltrimethylammonium)-pullulan-methyl acrylate copolymer obtained in Example 14 is adjusted to a solution of 10 mg / ml in terms of HPTMA (2-hydroxypropyltrimethylammonium)-pullulan. When 2 ml of this solution was added to 1 ml of a RNA solution (20 mg / ml) derived from yeast, it completely precipitated in 6 hours, and a complex of 9 mg of HPTMA (2-hydroxypropyltrimethylammonium)-pullulan-methyl acrylate copolymer and RNA was obtained. Example 17 2 g of TEAE (triethylaminoethyl)-dextran hydrochloride with a nitrogen content of 2% based on dextran with an average molecular weight Mw of 300,000 was dissolved in 50 ml of water, 15 ml of methyl acrylate (MA) was added, and after thoroughly replacing the air in the reaction solution and the reaction vessel with nitrogen gas, while stirring well, 250 mg of ceric ammonium nitrate nitrate dissolved in 15 ml of 0.1 N nitric acid whose dissolved air was replaced with nitrogen gas was added to start the reaction. The reaction was carried out at 30 °C for 2 hours to form a latex. For the end of the reaction, 3 ml of a 1% solution of hydroquinone was used as a terminator. Then, the reaction solution was poured into three times the amount of methanol to obtain a precipitate. This precipitate was thoroughly washed with hot water, centrifuged, and dried under reduced pressure at 50 °C. Then, the dried product was placed in a Soxhlet extractor and extracted with acetone for 24 hours to obtain 2 g of the hydrochloride of TEAE (triethylaminoethyl)-dextran-MA copolymer. Nitrogen content 0.7%, grafting rate 185% Yield of TEAE-dextran 35% This substance is insoluble in water, which is a good solvent for TEAE-dextran hydrochloride, and also insoluble in acetone, which is a good solvent for methyl acrylate. Example 18 After carrying out the same reaction as in Example 17, the reaction-terminated solution of the latex was not poured into methanol, but dialysis was carried out in water to remove unreacted substances and initiators, and a TEAE-dextran-MMA copolymer latex was obtained. This is useful as a recombinant vector and shows test results. The test method was carried out according to the procedure in (4) of the column "(Best Mode for Carrying Out the Invention)". That is, the expression efficiency of transformation for observing the effect of the vector was based on the luciferase activity incorporated into COS-1 cells. When the sample of Example 18 was compared with the value of DEAE-dextran hydrochloride having a nitrogen content of 5% using dextran with an average molecular weight Mw of 500,000 as the matrix being 1, a luciferase activity three times as high was obtained. Example 19 The latex of the TEAE (triethylaminoethyl)-dextran-MA copolymer obtained in Example 18 was adjusted to a solution of 10 mg / ml in terms of TEAE (diethylaminoethyl)-dextran. When 2 ml of this solution was added to 1 ml of a DNA solution (20 mg / ml) derived from salmon sperm, it completely precipitated in 3 hours, and a complex of 15 mg of TEAE (triethylaminoethyl)-dextran-MA copolymer and DNA was obtained. Example 20 The latex of the TEAE (triethylaminoethyl)-dextran-MA copolymer obtained in Example 18 was adjusted to a solution of 10 mg / ml in terms of TEAE (triethylaminoethyl)-dextran. When 2 ml of this solution was added to 1 ml of a yeast-derived RNA solution (20 mg / ml), it completely precipitated in 5 hours, and a complex of 8 mg of TEAE (triethylaminoethyl)-dextran-MA copolymer and RNA was obtained. Example 21 The latex of the DEAE (diethylaminoethyl)-dextran-MMA copolymer obtained in Example 2 is adjusted to a solution of 10 mg / ml in terms of DEAE (diethylaminoethyl)-dextran. This solution was administered to rhesus monkeys with inactivated Venezuelan equine encephalitis (VEE) virus vaccine (IVEE) under the same conditions as described in Fig. 1 at 5 mg / kg in terms of DEAE-D, and the effectiveness was examined under the same conditions as the addition of DEAE-dextran described in Fig. 1, and the effectiveness shown in Table 1 was obtained.

Industrial Applicability

[0008] Most of the currently commercialized drug delivery materials have poor cationicity, are difficult to autoclave, and have problems with targeting and safety. When using a drug delivery material such as the cationic polysaccharide copolymer of the present invention, autoclaving and the like are easy, so it can be stably used. The cationic polysaccharide copolymer of the present invention easily binds to the hydrophobic part of inactivated virus or nucleic acid by hydrophobic binding force to form a supramolecular cationic polysaccharide copolymer-nucleic acid complex. This supramolecular complex formation is an important first step in the inactivated virus or nucleic acid taxane delivery system. Since it has a hydrophobic-hydrophilic domain, it enhances the supramolecular reaction with inactivated virus or nucleic acid, and since it is cationic, it is easily introduced into cells by endocytosis, increases the probability of being taken up by endosomes (transport vesicles), and exhibits the property of being a TLR7 agonist that specifically activates TLR7 possessed by endocytosis. Above all, the cationic polysaccharide copolymer of the present invention is chemically stable. For example, its solution can withstand autoclaving at 120 °C for 15 minutes. To enhance the drug delivery material to the industrial level, excellent reproducibility, low cost, and especially chemical stability are important. In addition, formalin-inactivation of viral vaccines considerably reduces the toxicity to the host in relation to the administration of live attenuated vaccines, but unfortunately this process often also reduces the antigenicity and protective effect of the vaccine. Various methods of compensating for toxicity and effects, including the use of adjuvants, are used to enhance immunogenicity. The availability of new adjuvants that are safe and effective is valuable for improving the immunogenicity of weak but potentially useful antigens. DEAE-dextran of anion exchange resin has been established as an effective adjuvant for inactivated foot-and-mouth disease virus for immunization of guinea pigs and pigs. DDMC is considered particularly useful as an adjuvant and carrier for COVID-19 vaccines, especially mRNA vaccines, due to its stable DDS function, intracellular endocytosis, and long residence time in vivo.

Brief Description of the Drawings

[0009]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 3

Claims

1. General formula 【Chemical 1】 wherein the formula of the unit of the water-soluble linear polysaccharide cationic derivative is 【Chemical 2】 represented by, and the formula of the unit of the polymer starting from the double bond of the olefin compound in the formula is 【Chemical Formula 3】 represented by, using a water-soluble linear polysaccharide cationic derivative as the main polymer, and a compound having an olefin as the graft chain, with a grafting rate in the range of 2% to 5000%, consisting of (Chemical Formula 2) and this (Chemical Formula 3), the above (Chemical Formula 1) represents a cationic partial substitution product of a polysaccharide based on a water-soluble linear polysaccharide, and is composed of a copolymer obtained by graft polymerization of a monomer having an olefin onto the cationic partial substitution product of the polysaccharide with a water-soluble linear polysaccharide as the matrix, an adjuvant material for vaccines.

2. General formula 【Chemical 1】 wherein the formula of the unit of the water-soluble linear polysaccharide cationic derivative is 【Chemical 2】 represented by, and the formula of the unit of the polymer starting from the double bond of the olefin compound in the formula is [Chemical Formula 3] represented by, using a water-soluble linear polysaccharide cationic derivative as the main polymer, and a compound having an olefin as the graft chain, with a grafting rate in the range of 2% to 5000%, consisting of (Chemical Formula 2) and this (Chemical Formula 3), the above (Chemical Formula 1) represents a cationic partial substitution product of a polysaccharide based on a water-soluble linear polysaccharide, and is a method for producing an adjuvant material for vaccines composed of a copolymer obtained by graft polymerization of a monomer having an olefin onto the cationic partial substitution product of the polysaccharide with a water-soluble linear polysaccharide as the matrix.

3. General formula 【Chemical 1】 wherein the formula of the unit of the water-soluble linear polysaccharide cationic derivative is 【Chemical Formula 2】 represented by, and the formula of the unit of the polymer starting from the double bond of the olefin compound in the formula is 【Chemical Formula 3】 represented by, using a water-soluble linear polysaccharide cationic derivative as the main polymer, and a compound having an olefin as the graft chain, with a grafting rate in the range of 2% to 5000%, consisting of (Chemical Formula 2) and this (Chemical Formula 3), the above (Chemical Formula 1) represents a cationic partial substitution product of a polysaccharide based on a linear polysaccharide, and is a vaccine composed of a copolymer adjuvant material obtained by graft polymerization of a monomer having an olefin onto the cationic partial substitution product of the polysaccharide with a linear polysaccharide as the matrix and a virus antigen.

4. General formula 【Chemical 1】 wherein the formula of the unit of the water-soluble linear polysaccharide cationic derivative is 【Chemical Formula 2】 represented by, and the formula of the unit of the polymer starting from the double bond of the olefin compound in the formula is [Chemical Formula 3] It is represented by the following formula, using a cationic derivative of a water-soluble linear polysaccharide as the main polymer, and a compound having an olefin as the graft chain, and having a grafting ratio in the range of 2% to 5000%. A copolymer obtained by graft polymerization of a monomer having an olefin onto a cationic partial substituent of a polysaccharide having a linear polysaccharide as a parent, which consists of (Chemical Formula 2) and this (Chemical Formula 3) as represented by the above (Chemical Formula 1). 【Chemical Formula 5】 A vaccine comprising a complex of a copolymer obtained by grafting an olefin monomer and deoxyribonucleic acid (DNA), which is formed by adding a deoxyribonucleotide represented by the following formula as a repeating unit to an aqueous solution of a cationic partial substituent of a polysaccharide having a linear polysaccharide as a parent.

5. General formula 【Chemical Formula 1】 In this formula, the formula of the unit of the water-soluble linear polysaccharide cationic derivative is [Chemical Formula 2] represented by the following formula, and the formula of the unit of the polymer starting from the double bond of the olefin compound in the formula is 【Chemical Formula 3】 represented by the following formula, using a cationic derivative of a water-soluble linear polysaccharide as the main polymer, and a compound having an olefin as the graft chain, and having a grafting ratio in the range of 2% to 5000%. A copolymer obtained by graft polymerization of a monomer having an olefin onto a cationic partial substituent of a polysaccharide having a linear polysaccharide as a parent, which consists of (Chemical Formula 2) and this (Chemical Formula 3) as represented by the above (Chemical Formula 1). [Chemical Formula 6] A vaccine comprising a complex of a copolymer obtained by grafting an olefin monomer and ribonucleic acid (RNA), which is formed by adding a ribonucleotide represented by the following formula as a repeating unit to an aqueous solution of a cationic partial substituent of a polysaccharide having a linear polysaccharide as a parent.

6. A vaccine delivery system (DDS) characterized in that the formation of the complex of the copolymer obtained by graft polymerization of a monomer having an olefin onto a cationic partial substituent of a polysaccharide having a linear polysaccharide as a parent, which consists of (Chemical Formula 2) and this (Chemical Formula 3) as represented by the above (Chemical Formula 1), using the cationic derivative of the water-soluble linear polysaccharide in Claim 1 as the main polymer and having a grafting ratio in the range of 2% to 5000%, and a nucleic acid as the first step.

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