WO2019045529A1 - Self-assembling particles and method for preparing same - Google Patents

Abstract

The present invention relates to immuno-directed particles in which an antigenic protein-amphipathic macromolecule polymer is self-assembled, wherein when using the immuno-directed particles of the present invention, it is possible to induce more immune responses even with a smaller amount of vaccine as compared with a conventional vaccine which simply contains an antigenic protein in a carrier, and to provide an efficient vaccine platform in which side effects such as infections of viral diseases can be eliminated.

Description

Self-assembling particles and method for manufacturing the same

The present invention relates to a self-assembled vaccine particle comprising a protein-amphipathic polymer and a method for producing the self-assembled vaccine particle.

The vaccine is injected with inactivated inactivated pathogenic agent or attenuated pathogenic agent which is weakly pathogenic in the human body prior to infection of the pathogenic organism, thereby activating the immune system of the human body, thereby allowing the immune cell of the human body to form an antibody, Even if they are infected, can prevent the disease caused by the pathogen or minimize the damage to prevent disease.

However, vaccines using inactivated or attenuated pathogens can cause hostility such as pain, redness, fever, chills, etc., and also cause infectious diseases in immunocompromised individuals. Alternatively, purified pathogen components such as hemagglutinin (HA), a specific antigen of influenza virus, and surface antigen vaccine, purified from neuraminidase (NA), may be used , There is a problem that it is difficult to expect the effect of the vaccine due to the immune response with low titer.

In addition, since there is a problem that the vaccine antigen continuously changes in the antigen urine (antigenic drift) of the influenza virus continuously, it is necessary to prepare the vaccine promptly at the early stage of the viral infection, Vaccines containing individuals have difficulties in preventing the emergence of infectious diseases due to the difficulty of rapid production.

Various studies have been made to overcome the disadvantages of such a vaccine. WO2011-151723 discloses a method of increasing the concentration of a vaccine antigen in a liquid composition. However, when a protein is present at a high concentration in a liquid composition state, There still exists a problem that the effect of the vaccine is inhibited by coagulation, degradation and the like.

Therefore, there is a continuing need for improved vaccine preparations which have an effect of inducing an immune response in the body, but which do not cause such a disease as inducing the disease in a subject.

Disclosure of Invention Technical Problem [6] The present invention is directed to solve the problem of low vaccine efficiency of a conventional virus vaccine, and it relates to an immunity inducing particle and a vaccine composition containing the immunity inducing particle which can efficiently induce an immune response by improving such efficiency.

The present invention also relates to a method for producing said immunostimulatory particles.

The present invention relates to an immunoinductive particle comprising a complex of an antigenic protein and an amphipathic polymer, which is formed by self-bonding of the antigenic protein-amphipathic polymer complex and comprises a hydrophobic polymer layer, a hydrophilic polymer layer, Inducing particle having a structure of an antigen protein layer.

The present invention also provides a vaccine composition comprising said immunostimulatory particles.

The present invention also provides a method for producing said immunostimulatory particles.

Also provided is a method for inducing an immune response by administering the immunostimulatory particles of the present invention to an individual.

Although the immunogenic particles according to the present invention do not contain pathogens and contain only antigenic proteins, the density of the antigenic protein presented on the surface per particle is high and the immunity inducing effect is excellent and accompanied by side effects of the vaccine by the attenuated pathogen Moreover, it is possible to produce a simple and rapid production, making it possible to control an infectious disease such as a virus at an early stage.

1A is a schematic view showing a process of synthesizing an amphipathic polymer according to an embodiment of the present invention.

FIG. 1B is a schematic view showing a vaccine particle by an antigen protein-amphipathic polymer self-assembly according to an embodiment of the present invention and particles bound with an antigenic protein on the surface of an amphipathic polymer.

Figures 2a, 2b and 2c are the results of confirming NMR peaks of synthesized materials synthesized according to one embodiment of the present invention.

FIG. 3A shows the results of confirming the peak change of the amphipathic polymer prepared according to an embodiment of the present invention with an FTIR analyzer.

FIG. 3B is a photomicrograph of nanoparticles prepared by varying the content of the hydrophobic polymer in the total mass of the amphipathic polymer according to an embodiment of the present invention. FIG.

FIGS. 4A and 4B are electron micrographs of immunoinducing particles prepared according to an embodiment of the present invention, and graphs illustrating the distribution of the average particle sizes of the immunoglobulin particles. FIG.

FIG. 5 is a microphotograph of immunostimulatory particles formed by self assembly of HA protein-amphipathic polymer complex prepared according to an embodiment of the present invention.

6a and 6b are graphs showing the average particle diameter distribution of the immunity inducing particles according to the degree of modification produced according to an embodiment of the present invention: 6a the particle size according to the degree of modification of the albumin, 6b the hemagglutinin modification degree Size of self-assembled particles according to.

FIGS. 7A and 7B show the results of BCA analysis using the standard curve and the immunity inducing particle used in the BCA analysis using the immunity inducing particles according to an embodiment of the present invention. FIG.

FIG. 8 shows the result of confirming the immunity inducing ability in the mouse using the immunity inducing particles prepared according to one embodiment of the present invention.

Figure 9 shows the standard curve used for BCA analysis using immunostimulatory particles prepared according to one embodiment of the present invention.

FIG. 10 is a graph showing an antibody-induced response using OVA-immunoreactive particles prepared according to an embodiment of the present invention.

FIG. 11 shows the results of confirming the immune response inducing ability of the HA-immunoreactive particles prepared according to one embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, unless further departing from the scope of the invention as defined by the following claims. It is not. It is to be understood that the scope of the present invention includes all modifications, equivalents, and alternatives falling within the scope and spirit of the present invention.

The present invention comprises a complex of an antigenic protein and an amphipathic polymer, wherein said complex provides self-assembled immunostimulatory particles.

&Quot; Immunogen inducing particle " in the present invention means a particle capable of inducing a humoral immune response or a cellular immune response by stimulating the immune system of an animal or a human within the host. The immunostimulatory particles of the present invention induce an immune response in the host, thereby enabling rapid defense against the same antigen, preventing disease by the antigen or lowering the severity of symptoms. In this sense, the immunostimulatory particles of the present invention can be used as a vaccine and can be used interchangeably with vaccine particles.

The immunopotentiating particle of the present invention is formed by self-assembly of an antigen protein-amphipathic polymer complex, and the hydrophobic polymer layer, the hydrophilic polymer layer and the antigen Protein structure.

Particularly, in the immunostimulatory particle of the present invention, the antigen protein is formed at the outermost part of the particle. Since the antigen protein is contained in the immunoparticle in a state of being bound to the amphipathic polymer forming the self-assembled particle, a larger amount of the antigen protein And has an advantage in that the stability of the vaccine can be enhanced when the antigen protein is stably bound to the particles and is contained in a vaccine preparation or the like.

Conventionally, when an attenuated or inactivated pathogen is used, there is a problem that infection of disease occurs in some individuals. However, since the immunostimulatory particle of the present invention contains only a specific antigen protein, the possibility of such side effect can be effectively solved. However, in the case of a vaccine containing only the purified antigen protein, the effect of the vaccine itself is too small. However, in the case of the present invention, the amount of antigen protein present on the surface of one immunoparticle can be significantly increased, An excellent effect can be recognized in that an excellent immune response can be induced in the administered subject. In one embodiment of the present invention, a simple purified antigen protein and an immunostimulatory particle of the present invention were respectively injected into a mouse to induce an immune response, and the amount of the produced IgM antibody was confirmed. As a result, It was confirmed that it exhibited more excellent antibody forming effect when used.

The immunostimulatory particles of the present invention may further comprise a linker for linking the antigenic protein and the amphipathic polymer. When the antibody further comprises the linker, it may further comprise a linker layer between the hydrophilic polymer layer and the antigen protein layer. The linker layer allows a physically separated space between the antigenic protein and the amphipathic polymer to be secured in the immunity inducing particle, and the immunostimulating particle of the present invention has better particle stability due to the physical space.

In the present invention, the linker means a molecule having a reactive site at both ends so that two substances can be chemically synthesized and connected. The linker of the present invention may be an amine linker molecule having an amine group or a linker molecule having a carboxyl group . The amine linker molecule includes all diamine linker molecules having two amine groups (NH ₂ ). Preferably a diamine-based molecule having one amine group at each end of the molecule. The diamine linker molecule is not limited to the type of the diamine linker molecule in that one end thereof reacts with the carboxyl group of the antigen protein and the other end thereof reacts with the amphipathic polymer to form an antigenic protein complex with the amphipathic polymer, . Further, the linker molecule having a carboxyl group may have two carboxyl groups. When a linker molecule having a carboxy group is used, a linker-antigen protein bond can be formed by reacting with an amine group of an antigen protein.

For example, the linker of the present invention can be selected from the group consisting of hexamethylenediamine, 1,4-diaminobutane, 1,8-diaminooctane, ethylenediamine, 1,6-hexanediamine 1,6-hexanediamine, phenylenediamine, 1,3-propanediamine, 1,13-tridecanediamine, 1,2-ethanediamine, 1,2-ethanediamine or 1,5-pentanediamine, but is not limited thereto. Preferably, the linker of the present invention may be hexamethylenediamine.

When the immunostimulatory particle of the present invention further comprises a linker, the immunogen inducing ability of the immunostimulatory particle of the present invention can be regulated by controlling the degree to which the antigen protein modifies the surface moiety with the linker. Such degree of modification affects the antigenicity and stability of the immunostimulatory particles of the present invention, and when the conditions of the following formula 1 are satisfied, particles having a better immunostimulatory ability can be obtained.

 [Formula 1]

(G) x A of the linker (g / mol) / (unit molar mass of the linker molecule (g / mol)

(Where A is 517.5 kDA / antigen protein molecular weight (kDa)).

The amount of the linker and the amount of the antigen protein in the formula 1 may be the amount of the linker and the antigen protein added to the reaction when the linker and the antigen protein are reacted to prepare the linker-antigen protein conjugate.

The immunostimulatory particles of the present invention may have a degree of modification according to the above formula 1 of less than 3, or less than 2. If the degree of modification is 3 or more, the antigenicity of the antigen protein bound to the particles may be lowered, and the immunity inducing effect may be lowered.

More preferably, in the present invention, the degree of modification of the antigen protein in the immunostimulatory particle is 1/1000 or more to less than 2, or more than 1/500 to 1.8 or less than 1/100 or more to 1.7 or less, 1/80 or more to 1.6 or less , From 1/60 or more to 1.5 or less, from 1/40 or more to 1.4 or less, or from 1/20 or more to 1.3 or less, but is not limited thereto. When the above range is satisfied, the antigenicity of the immunity inducing particle is excellent, and thus it has an excellent immune response inducing effect in the administration individual.

In addition, the antigen protein of the present invention may have a degree of modification of 1/1000 or more according to the above formula (1). When the degree of modification is less than 1/10, the binding between the amphipathic polymer and the antigen protein is reduced, and the hydrophobic interaction for self-assembly is reduced, which may lower the production efficiency of the immunity inducing particles. In addition, there is a problem that the stability of the immunity inducing particle is lowered by the decrease of the hydrophobic interaction.

In the present invention, the amphipathic polymer means a particle having a region showing hydrophilicity and a region showing hydrophobicity. The amphipathic polymer can be used without limitation as long as it is a particle having a hydrophilic region and a hydrophobic region. The amphipathic polymer includes a hydrophilic polymer, a hydrophobic polymer, and a combination thereof. The amphipathic polymer may be in the form of a polymer of a hydrophilic polymer and a hydrophobic polymer.

The amphiphilic polymer of the present invention may be in the form of (hydrophilic polymer) m- (hydrophobic polymer) n. Preferably, in the formula 2, K may be an amphipathic polymer satisfying 0.1 to 0.8, or 0.2 to 0.6, or 0.3 to 0.5.

[Formula 2]

K = weight of hydrophilic polymer / weight of amphipathic polymer

More preferably, according to one embodiment of the present invention, the stability of the immunity inducing particles is improved when the weight ratio is 0.1 to 0.6. Particularly, when the amount of the hydrophobic polymer is too small, the hydrophobic interaction in the self-assembled particles may be weakened, and it is important that the above range is satisfied.

In one embodiment, the hydrophilic polymer is a polyalkylene glycol (PAG), a polyacrylic acid (PAA), a polyacrylonitrile (PAN), a polyethylene oxide (PEO), a polyvinyl acetate (PVAc), a polyvinyl alcohol ), Polyvinylpyrrolidone, polyacrylamide, and hydrophilic polyamino acids, and derivatives thereof. Such derivatives include, for example, (mono) methoxypolyethylene glycol, (mono) acetoxypolyethylene glycol, polyethylene glycol, copolymers of polyethylene and propylene glycol, polyvinylpyrrolidone, polyglutamine, polyglutamic acid, polythreonine, , Polyarginine, and polyserine. In a preferred embodiment, the hydrophilic polymer may be methoxypolyethylene glycol (mPEG).

In one embodiment, the hydrophobic polymer can be used without limitation as long as it is a substance capable of forming an amphipathic polymer together with a hydrophilic polymer. For example, the hydrophobic polymer may be at least one selected from the group consisting of polyesters, polyanhydrides, hydrophobic polyamino acids, polyorthoesters, and polyphosphazines. Wherein the hydrophobic polyamino acid is selected from the group consisting of polylysine, polyisoleucine, polyvaline, polyphenylalanine, polyproline, polyglycine, polytryptophan, polyalanine, polylactide, polyglycolide, polycaprolactone, and polymethionine Or more. The hydrophobic polymer includes a derivative thereof.

The amphipathic polymer of the present invention is a hydrophilic polymer comprising mPEG as a hydrophobic polymer and mPEG containing lactide (3,6-Dimethyl-1,4-dioxane-2,5-dione) and a polylactide copolymer (mPEG ) m- (b-PLA) n. In this case, m may be 100 to 200, and n may be 30 to 100.

In the present invention, " antigen protein " means a protein capable of inducing or promoting an immune response to an antigen, and includes fragments including an epitope region of the protein. Examples include influenza viruses such as the hemagglutinin (HA), the neuraminidase (NA), the nucleoprotein (NP), the M1 protein, the M2 protein, the NS1 protein, the NS2 protein : Nuclear export protein), PA protein, PB1 protein (polymerase basic 1 protein), PB1-F2 protein and PB2 protein;

In the case of rabies virus, nuclear protein (N), phosphorus protein (P), substrate protein (M), glycoprotein (G), and viral RNA polymerase (L);

Hepatitis B virus surface antigen (HBsAg), hepatitis B virus core antigen (HbcAg), hepatitis B virus DNA polymerase, HBx protein, preS2 middle surface protein, , Large S protein, viral protein VP1, viral protein VP2, viral protein VP3, and viral protein VP4;

≪ / RTI >

When the immunoinducing particle of the present invention induces an immune response in the form of nanoparticles bound to an amphipathic polymer, it is possible to induce a better immune response at a lower concentration than in the case where only an antigen protein is administered to induce an immune response. Therefore, the immunostimulatory particles of the present invention can be applied to any antigenic protein including a carboxyl group and an antigenic determinant site (epitope) capable of binding to the amphipathic polymer regardless of the type thereof, and thus the present invention is not limited to the kind of antigen protein .

The immunostimulatory particles of the present invention are self-assembled by an amphipathic polymer which is a constituent of the complex, that is, granulated by self-binding of an amphipathic polymer. Accordingly, the particles formed by the self-bonding of the present invention may include a polymer region (hydrophilic polymer layer, hydrophobic polymer layer) and an antigen protein region.

The self-assembled immunostimulatory particles of the present invention may have a membrane structure.

&Quot; Membrane structure " in the present invention means a structure in which the inside is surrounded by a membrane or a shell. The membrane structure may include a fluid or a separate configuration therein. The membrane includes both a single membrane or a multi membrane having two or more single membranes. As an example, when a particle having a single membrane is again surrounded by a single membrane, it can be a particle having multiple membranes. The single membrane and the multiple membrane are concepts that are distinguished from a single layer and a double layer. Particularly, a particle containing only one film of a bilayer structure has a single film, and a particle including a film of a monolayer structure again surrounds a film of a single layer structure means a particle of a multi-film structure .

The nanoparticles having a membrane structure in the present invention may be vesicles, micelles, polymersome, droplets or colloidsome, but are not limited to, But are not limited to, rods, spheres, rings, plates, cylinders, ellipses, spheres, and the like.

The " micelle " means a particle having a hydrophobic core and a hydrophilic shell. In one embodiment, the micelle may be a polymer self-assembled micelle. In the case of self-assembled micelles, various forms can be adopted depending on the type of the polymer forming the copolymer, and the shape of the polymer is not limited thereto. The micelle also includes a multi-layered form comprising two or more monolayers.

The term " polymersome " means a nanoparticle having a bilayer membrane structure having a " hydrophilic region-hydrophobic region-hydrophobic region-hydrophilic region ". The polymer jars include both a single membrane of a bilayer membrane structure and multiple membranes containing two or more of the single membranes.

The " colloidal mule " means a colloidal particle having a size of 1 nm to 1000 nm or a structure in which the granularity is packed tightly. Depending on the type of polymer in the hydrophilic and hydrophobic regions forming the membrane, it may comprise a single layer and a bilayer.

The " droplet " means that the shape of the droplet in the film structure particles is represented by a droplet shape. Includes both single and double layers, and includes both single and multiple layers.

The self-binding form of the immunoadaginized particle of the present invention is more stable in the body than the vaccine of the single antigen protein type and can exhibit the antigen protein on the surface at a higher density than the conventional vaccine in which the antigen protein is bound to the nanoparticle Therefore, it is possible to induce an excellent immune response with an appropriate amount.

The present invention provides a vaccine composition comprising said immunostimulatory particles. The present invention also provides a vaccine preparation comprising the immunostimulatory particles.

The vaccine composition of the present invention may further comprise at least one of a solvent, an adjuvant or an excipient in addition to the immunity inducing particle. The immune enhancer may be Freund ' s incomplete or complete adjuvant, aluminum hydroxide gel, vegetable and mineral oil, etc. Examples of the excipient include aluminum phosphate, aluminum But are not limited to, aluminum hydroxide or aluminum potassium sulfate (alum), and may further include any of the known materials used in the manufacture of vaccines well known to those skilled in the art.

The vaccine composition of the present invention may be prepared as an oral or parenteral preparation and may be prepared as an injectable solution in the form of a parenteral preparation and may be administered orally in the form of intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, But it is not limited to the mode of administration of the agent.

The present invention provides a method for producing said immunostimulatory particles.

The method for producing immunostimulatory particles of the present invention comprises mixing an amphipathic polymer and an antigen protein to form a complex; And self-binding and granulating the complex.

Preferably, an antigenic protein-amphipathic polymer mixed solution to which an amphipathic polymer in a hydrophobic solvent is added to a protein solution containing an antigen protein in a hydrophilic solvent is reacted for 20 to 28 hours with shaking to form an amphipathic polymer-antigen protein complex To do; And a method in which the mixed solution is granulated by self-assembly of an amphipathic polymer-antigen protein complex when the hydrophobic solvent is a colloid phase in which the hydrophobic solvent exists in a droplet state in a hydrophilic solvent containing an antigenic protein.

The protein solution may further comprise glycerol. In this case, it is very effective to prevent the antigen proteins in the composition from aggregating with each other and allow the protein to participate in the reaction more stably in the process of forming the antigen protein-amphipathic polymer complex.

The present invention relates to a method for preparing a protein solution, which comprises adding an amphipathic polymer in a hydrophobic solvent to a protein solution containing an antigen protein in a hydrophilic solvent and then shaking the mixture for a predetermined period of time or longer so that the hydrophobic solvent is dispersed in a hydrophilic solvent, , And the protein is characterized by being able to form particles formed in the outermost layers. The hydrophobic polymer and the antigenic protein in the mixed solvent on the colloid phase may be in an interfacial state on different layers.

The production method of the present invention may further comprise modifying the antigen protein to a linker before the antigen protein-amphipathic polymer complex formation. When the complex is formed by modifying the protein with the linker, the possibility of contact between the moiety of the polymer and the moiety of the linker is increased, thereby increasing the efficiency of forming the antigen protein-amphipathic polymer complex. The degree of modification can be calculated according to the formula 1 described in the description of the immunoinducing particle of the present invention, and the above contents are also applied to the present manufacturing method.

INDUSTRIAL APPLICABILITY The immunostimulatory particles of the present invention can exhibit a strong immune response in a small amount by presenting an antigen protein in the form of nanoparticles irrespective of the kind of the antigenic protein, and thus have an excellent effect as a vaccine. In addition, according to the method of the present invention, immune-inducing particles having an excellent immune reaction inducing effect can be produced without being limited to the kinds of antigen proteins, and thus they can be used variously in the field of vaccine production.

In this aspect, the invention provides a method of inducing an immune response in an individual comprising administering said immunity-inducing particle to a subject. The method of inducing the immune response may be to prevent disease or infection in that it is intended to lower or prevent the possibility of infection of an individual with a disease. The administration can be carried out more than once.

The method for inducing an immune response in an individual may be carried out by administering to a subject an amount of a pharmaceutically effective amount of a pharmaceutically effective amount to induce an immune response in the subject, thereby inducing humoral or cellular immunity response of the individual, Can reduce the likelihood of infection to the disease.

The pharmaceutically effective amount for inducing the immune response may be determined in consideration of physical conditions such as the specific form of the immunity inducing particle, the kind of the antigen protein, the age of the individual, and the body weight.

In one embodiment of the present invention, the immunoreactive particles of the present invention were administered to mice, and the blood of the mice was analyzed. As a result, it was experimentally confirmed that the production of IgM antibody against the antigen protein was effectively induced. Furthermore, it has been confirmed that a larger amount of antibody is produced compared to a particle in which antigen is simply bound to the surface of the purified antigen protein or nanoparticle, and thus it can be used as a vaccine platform having a very high efficacy Respectively.

Hereinafter, the present invention will be described in detail with reference to Production Examples and Experimental Examples. The following Examples and Experiments are illustrative of the present invention and are not intended to limit the scope of the present invention.

[ Manufacturing example  One] Amphipathic  Synthesis of polymer

The amphipathic polymer to be bound to the antigen protein of the present invention can be prepared by reacting biocompatible polymer mPEG (Mw: 2,000) and lactide (3,6-dimethyl -1,4-dioxane-2,5-dione, Sigma Aldrich), and then an amphipathic polymer, mPEG-b-PLA, was synthesized through a ring opening reaction.

Specifically, 1 g of mPEG, a small amount of Sn (Oct) 2 (Tin (Ⅱ) 2-ethylhexanoate, Sigma Aldrich), lactide and 50 mL of toluene (anhydrous, Sigma Aldrich) were added to a 3-neck rounded flask, , And vacuum and N2 purge were repeatedly treated. After reaching 120 deg. C, N2 purge was stopped and stirred. The solution was clarified and allowed to react for 24 hours. It was then concentrated under reduced pressure and heated to the product gel state before adding 10 mL of DCM (Dichloromethane, Sigma Aldrich). The product was slowly dropped into diethyl ether and stirred for about 30 minutes while precipitating, and the precipitate was decompressed and separated. The step of separation by settling and decompression was repeated three times, and the separated material was dried for one day.

[Reaction Scheme 1]

Then, mPEC-b-PLA-COOH was prepared by replacing the OH group of mPEG-b-PLA with the COOH group in order to bind the above-mentioned copolymer and the antigen protein. Specifically, 500 mg of mPEG-b-PLA synthesized above, 80 mg of succinic anhydride (Sigma Aldrich) and 35.7 mg of DMAP (Sigma Aldrich) were placed in 10 mL of dichloromethane (DCM, Sigma Aldrich) ) Was added dropwise. After reaction at ambient temperature for 24 hours, the resultant was precipitated with cold diethyl ether and vacuum dried to obtain mPEG-b-PLA-COOH.

MPEG-b-PLA-NHS was synthesized using NHS-sulfo as the mPEG-b-PLA-COOH synthesized above. Specifically, 325 mg of mPEG-b-COOH synthesized above, 17 mg of NHS (Thermo Scientific) and 21 mg of DCC (Sigma Aldrich) were placed in 10 mL of dichloromethane (DCM, Sigma Aldrich), and 20 μL of triethylamine Drop method. The reaction was allowed to proceed at ambient temperature for 24 hours. The resultant was precipitated with cold diethyl ether and vacuum dried to obtain mPEG-b-PLA-NHS.

NMR was used to confirm the synthesis of each step of the copolymer synthesized by NMR. As shown in FIGS. 2A, 2B, and 2C, peaks of PLA and PEG are common and a peak of each functional group is observed.

Further, (mPEG) m- (b-PLA) n was synthesized by varying polymerization ratios of mPEG and lactide (3,6-Dimethyl-1,4-dioxane-2,5-dione) . The amounts of mPEG and lactide (3,6-Dimethyl-1,4-dioxane-2,5-dione) were adjusted as shown in Table 1 below.

mPEG (g)	DL-Lactide (g)	Mw (g / mol)	f mPEG (weight%)	m	n
One	4.5	8262.967	0.242044	181.8	86.98565
One	2.9	4928.322	0.405818	181.8	40.6711409395973
One	3.1	5596.044	0.357395	181.8	49.94505
One	3.3	5566.649	0.359283	181.8	49.53678

As shown in FIG. 3A, the FTIR analysis results confirmed that the peak of the C-H moiety decreased as the moiety of mPEG decreased in the copolymer. As the degree of polymerization of lactic acid was increased, the ratio of PEG in the polymer was decreased and the C-H bond peak was relatively decreased compared to the C = O bond peak. (MPEG) m- (b-PLA) was obtained by varying the polymerization ratios of mPEG and lactide (3,6-Dimethyl-1,4-dioxane-2,5- ) n were synthesized and the morphological characteristics of the self - assembled particles were confirmed according to the proportion of the hydrophilic polymer.

mPEG (g)	DL-Lactide (g)	Mw (g / mol)	f mPEG (% by weight)	m	n
One	2.9	4928.322	0.405818	181.8	40.6711409395973
One	4.5	8262.967	0.242044	181.8	86.98565
0.5	9	39398.86	0.057	181.8	519.42857

As shown in FIG. 3B, it was confirmed that the shape of the self-assembled particles produced varies depending on the weight ratio of the hydrophilic polymer in the amphipathic polymer. Particularly, when the ratio (f) of the hydrophilic polymer is 0.05% by weight, there is a problem that the shape of the rod is formed but the stability of the shape of the particle is unstable.

 [Preparation Example 2] Polymer-protein complex formation and granulation

The immunoinducing particle of the present invention is characterized in that it is a particle formed by self-assembly of an antigen protein-amphipathic polymer complex. In order to have immunity-inducing ability, the antigen protein must be located at the outermost position of self-assembly. Thus, the self-assembled particles having the antigen protein of the present invention at the outermost periphery were prepared by the following method. As antigenic proteins, ovalbumin (Thermo Scientific) and hemagglutinin (A / California / 04/2009 (H1N1), SEQ ID NO: 1) were used.

First, the antigen protein and hexamethylenediamine (linker) were reacted to modify the antigen protein with the linker. To reduce aggregation of the antigen protein or albumin, 5 vol% glycerol was added to the OVA (1 mg / 1 mL) solution. To the 300 μl of the OVA (1 mg / 1 ml) solution was added 40 μl of EDAC / Sulfo-NHS solution (20 mg each of 100 μl of DW), and 80 μl of hexamethylenediamine (104 mg / DW 2 ml) was added to the mixture. After vortexing the mixed solution for 1 hour at room temperature, the excess reagent was removed by centrifugation using a 30k Memphane filter (12300 rcf, 10 min, repeated 3 times). OVA-NH ₂ in which hexamethylenediamine was bonded to COOH (glutamic acid) group of ovalbumin was obtained.

Next, 20 μl of mPEG-b-PLA-NHS (4 mg in 100 μl of chloroform) was added to the solution containing OVA-NH ₂ (glycerol (5 v / v%)) to form an amphipathic polymer- The DPBS, the OVA content 1 mg / mL), and vortexing at room temperature overnight to produce the mPEG-b-PLA-HA complex while granulating the complex. By vortexing, chloroform is present in the form of droplets in the hydrophilic solvent of the OVA-NH ₂ solution. In this case, the amphipathic polymer - antigen protein complex was interfaced with the hydrophilic solvent between the chloroform droplet and the hydrophilic solvent and self - assembled and granulated. Specifically, the hydrophobic polymer in the chloroform droplet forms the inside of the particle, and the hydrophilic polymer is formed on the hydrophobic polymer layer by the interaction of the hydrophobic polymer, and the antigen protein is formed on the outermost layer of the particle by the linker. Through this, self-assembled particles (proteSome) of mPEG-b-PLA-OVA complex were obtained. The mPEB-b-PLA-HA complex was formed and granulated in the same manner as above to obtain self-assembled particles of mPEB-b-PLA-HA complex.

The granulation degree of the mPEG-b-PLA-OVA complex and the mPEB-b-PLA-HA complex was confirmed using DLS and electron microscope, and the particle size was measured.

As shown in FIGS. 4A and 4B, it was confirmed that the OVA-amphipathic polymer composite formed particles well by self-assembly, and that the average size of the OVA-polymer particles modified by 1/2 was 148 nm . Further, as shown in Fig. 5, it was confirmed that the hemagglutinin-amphipathic polymer complex formed particles well by self-assembly.

[Preparation Example 3] Confirmation of optimal conditions for protein antigen modification

Since the degree of surface modification of the protein antigen is related to the antigenicity of the protein and the stability of the vaccine, experiments were carried out to confirm the antigenicity by varying the degree of protein modification by the linker hexamethylenediamine (Hexa.). The OVA protein (45 kDa) was used as a stock solution (OVA 1 μg / μl) and the amphipathic polymer mPEG-b-PLA-NHS (molecular weight 6 kDa) synthesized in Preparation Example 1 was used.

Modification ratio, when said amount (mg) a modified constant (k) of the linker required to modify the antigenic proteins 1mg, represents a (x k amount of antigen) the value of the amount of linker /. In this experiment, the amount of hexamethylenediamine (unit molar mass: 116.21 g / mol) required to modify 1 mg of OVA protein was 11.5 mg. In addition, the amount of hexamethylenediamine required to modify 1 mg of hemaglutinin was 6.088 mg.

Experimental group	Modification	OVA (占 퐂)	Polymer (㎍)	Hexa. (占 퐂)
Example 1	One	200	400	2300
Example 2	1/2	200	400	1150
Example 3	1/4	200	400	575
Example 4	1/8	200	400	287.5
Example 5	2	200	400	143.75

For each of the OVA protein and hemagglutinin (HA, 85 kDA) protein, the degree of protein modification by hexamethylenediamine was changed to 1, 1/10 or 1/100. The particle sizes of Examples 6-8 and Examples 9-10 were analyzed via DLS.

Experimental group	Modification (approx.)	OVA (占 퐂)	Polymer (㎍)	Hexa. (占 퐂)
Example 6	One	300	800	3420
Example 7	1/10	300	800	342
Example 8	1/100	300	800	34.2
Experimental group	Modification (approx.)	HA (占 퐂)	Polymer (㎍)	Hexa. (占 퐂)
Example 9	One	300	800	1710
Example 10	1/10	300	800	171
Example 11	1/100	300	800	17.1

As shown in Fig. 6, the hydrodynamic diameter was measured to be about 500 nm, and a tendency was shown that the size variation occurred as the modification ratio was lowered.

[Preparation Example 4] Preparation of surface-bound particles of protein

In order to confirm the difference in effect as a vaccine for particles in which the antigen protein is bound to the surface of the self-assembled particles of the antigenic protein-amphipathic polymer complex of the present invention and the polymer, the surface of the nanoparticles .

The antigen protein (OVA, HA) was modified to 1/10 of the degree of modification and then granulated in the self-assembled form using the amphipathic polymer mPEG-b-PLA-NHS (molecular weight 6 kDa). Then, the EDC / Sulfo-NHS stock was added and antigen protein OVA (45 kDA) was added to prepare OVA-surface-bound nanoparticles (Comparative Example 2) by mixing the self-assembled nanoparticles with the OVA protein. The HA protein was also treated in the same manner as above to prepare HA-surface-bound nanoparticles (Comparative Example 3). The amount of the antigen protein used and the amount of the reactant were prepared in the same manner as in the case of the modification degree 1/10 in Table 4.

[Experimental Example 1] Determination of the degree of antibody formation by vaccine particles

Using the immunosuppressive particles (ProteSome) prepared according to the method of Preparation Example 2, the antibody formation effect was confirmed as follows.

The experiment was divided into the group treated with OVA only (positive control group), the group treated with the immunity inducing particles of the present invention (Examples 1, 2, 3 and 4) and the group treated with nothing (negative control) . In the immunostimulatory particle treatment group, modification of the antigen protein by hexamethylenediamine was started from the modification 1 of Example 1, and each of the prepared immunostimulatory particles was reduced by 1/2.

Immunoreactive particles were inoculated by BCA analysis after the quantitative control. Specifically, they were quantitated by a reliable standard curve (Fig. 7a) with R ² ≥ 0.99 and analyzed both before and after the first inoculation and the second inoculation To control the density. However, in the case of the first injection group of Example 2, the first dose was carried out at a low concentration because the production amount of the nanoparticles was small. The contents of the specifically inoculated vaccines are shown in Table 5 below.

	Experimental group	mPEG-b-PLA-NHS	Primary vaccination (/ / 50))	Second inoculation (/ / 50))
Comparative Example 1	Only OVA	X	22	17.73672
Example 1	ProteSome 1	200 μg / 10 μl	22.09521	17.73672
Example 2	ProteSome 1/2	200 μg / 10 μl	9.232413	17.73672
Example 3	ProteSome 1/4	200 μg / 10 μl	21.73613	17.73672
Example 4	ProteSome 1/8	200 μg / 10 μl	22.06306	17.73672
Negative control 1	Negative	X	X	X

 Specifically, for the mice (n = 18, 3 mice for each test group), blood sampling (pre-inoculation blood sampling) was carried out before vaccination and then the first vaccine was inoculated. On the 14th day after the first vaccination day, blood collection was carried out (primary blood collection after inoculation) and the second vaccination was inoculated. On the 28th day after the second inoculation, blood collection (second blood collection after inoculation) was carried out. Antibodies were analyzed by ELISA after dilution by 50, 100, 200 and 400 times with the serum of the blood obtained by the primary blood collection and the secondary blood collection after inoculation. The results of analysis according to the treatment groups are shown in FIG.

As shown in FIG. 8, when the protein modification for the granulation was reduced to 1/2, IgG was expressed to 1/8 times larger, and the IgG value of the Example 4 (ProteSome 1/8) And 4.9 times, 7.6 times, and 11.9 times and 18.7 times, respectively, in 50X, 100X, 200X and 400X compared to the single inoculation with OVA. Therefore, it was confirmed that when the degree of modification of the antigen protein of the present invention was lowered, the immune response inducing effect was further improved.

[Experimental Example 2] Confirmation of antibody formation by vaccine particles

Using the immunosorbent particles (ProteSome) prepared according to the method of Preparation Example 3, the antibody formation effect was confirmed as follows.

Experiments were performed on the OVA antigen protein-treated group (Comparative Example 1; OVA), the immunoinductive particle treated group of the present invention (Examples 5, 6 and 7), the surface-bound nanoparticle group (Comparative Example 1 and Comparative Example 2) (Negative control group 2).

Immunoreactive particles were inoculated after BCA analysis and after controlling the density. Specifically, they were quantitated with a reliable standard curve (Fig. 9) at ² ≥ 0.99 and analyzed both before and after the first inoculation and the second inoculation To control the density. The contents of the specifically inoculated vaccines are shown in Table 6 below.

OVA	mPEG-b-PLA-NHS (占 퐂 / 50 占 퐇)	Primary vaccination (/ / 50))	Second inoculation (/ / 50))
Comparative Example 1 (OVA alone)	X	20	20
Example 6	334	20	20
Example 7	334	20	20
Example 8	334	20	20
Comparative Example 2 (Surface Bonded Particle)	334	20	20
Negative control 2	X	X	X

Specifically, mice (n = 30, 5 mice per each experimental group) were inoculated with a primary vaccine after blood collection (pre-inoculation) was carried out before vaccination. On the 14th day after the first vaccination day, blood collection was carried out (primary blood collection after inoculation) and the second vaccination was inoculated. On the 28th day after the second inoculation, blood collection (second blood collection after inoculation) was carried out.

The degree of antibody (IgG) induction by enzyme immunoassay (ELISA) was determined by measuring the absorbance intensity of the serum of the blood obtained by the primary blood collection and the secondary blood collection after inoculation. The absorption intensity of each experimental group is shown in Table 7. Coated with orbumin (OVA) at 500 ng / well in a 96-well plate. Serum dilution was performed in four steps from 100 times to 800 times, and finally the two samples (N = 3) were finally excluded from the average of the results (N = 5) derived from each example.

Dilution	OVA P (Comparative Example 2)	OVA 1 (Example 6)	OVA 0.1 (Example 7)	OVA 0.01 (Example 8)
1/100	1.178129	1.922218	1.471825	2.780263
1/200	0.92587	1.880971	1.634751	3.156385
1/400	1.187006	1.522782	1.495559	2.788081
1/800	1.273497	1.381065	1.605979	2.765058

In the case of inoculating the self-assembled antigen particles of Examples 6 to 8 of the present invention, a higher antibody production effect was observed compared to the case of inoculation of antigen protein single immunization (positive control 1) and antigen surface binding particles (Comparative Example 1) Respectively. Depending on the degree of modification, the antibody inducing effect was clearly higher at 1/100, which is the lowest level. Therefore, the self-assembled antigen particles of the present invention exhibit high antibody-inducing ability as compared to antigen particles or protein alone bound to the surface of the amphipathic particles, so that they can be used as an effective vaccine platform as compared with conventional vaccine preparations This is possible.

[Experimental Example 3] Confirmation of antibody formation by vaccine particles

Using the immunogenic particles (ProteSome) containing the HA protein prepared according to the method of Preparation Example 3, the antibody formation effect was confirmed as follows.

Experiments were conducted on the immunized particles of the present invention (Examples 8, 9 and 10), the surface-bound nanoparticles (Comparative Example 3) and the non-treated group (negative control 3) However, the method of analyzing the experimental conditions and results was the same as in Experiment 2.

HA	mPEG-b-PLA-NHS (占 퐂 / 50 占 퐇)	Primary vaccination (/ / 50))	Second inoculation (/ / 50))
Example 9	334	20	20
Example 10	334	20	20
Example 11	334	20	20
Comparative Example 3 (Surface Bonded Particle)	334	20	20
Negative control 3	-	-	-

The cells were coated with hemagglutinin (600 ng / well) in a 96-well plate and assayed by enzyme immunoassay (ELISA). (N = 5) except for one sample which is out of the average of the result value (N = 6) derived for each embodiment, and finally, the result is shown.

N = 5	HA P (Comparative Example 3)	HA 1 (Example 9)	HA 0.1 (Example 10)	HA 0.01 (Example 11)	NC (negative control group 3)
Average	0.84721	1.28734	2.3174	1.49575	0.0735
STD	0.224198	0.291706	0.331991	0.128784	0.003486

As shown in FIG. 11, in the case of the immunity inducing particle according to the present invention, it was confirmed that the immunoreactivity inducing effect was higher than that of nanoparticles surface-bound to the nanoparticles in all experimental groups. These results are consistent with the OVA protein of Test Example 2. It can be seen that the immunostimulatory particles of the present invention have a very excellent effect as a vaccine preparation having excellent immunoreactivity.

Claims (14)

1. An immunity inducing particle comprising a complex of an antigen protein and an amphipathic polymer,

   Wherein the antigen-protein-amphipathic polymer composite is formed by self-assembly of the antigen protein-amphipathic polymer complex and has a structure of a hydrophobic polymer layer, a hydrophilic polymer layer and an antigen protein layer in order from the inside of the particle.
2. The method according to claim 1,

   Wherein the complex of the antigen protein and the amphipathic polymer further comprises a linker.
3. 3. The method of claim 2,

   Wherein the particle further comprises a linker layer between the hydrophilic polymer layer and the antigen protein layer.
4. 3. The method of claim 2,

   Wherein the immunity inducing particle has a degree of modification of less than 3 in the following formula (1).

   [Formula 1]

   (G) x A of the linker (g / mol) / (unit molar mass of the linker molecule (g / mol)

   (Where A is 517.5 kDA / antigen protein molecular weight (kDa)).
5. The method according to claim 1,

   Wherein the amphipathic polymer comprises a hydrophilic polymer and a hydrophobic polymer.
6. The method according to claim 1,

   Wherein the amphipathic polymer satisfies the following inequality (2): K is 0.1 to 0.8,

   [Formula 2]

   K = weight of hydrophilic polymer / weight of amphipathic polymer
7. The method according to claim 1,

   The hydrophilic polymer may be at least one selected from the group consisting of polyalkylene glycols (PAG), polyacrylic acid (PAA), polyacrylonitrile (PAN), polyethylene oxide (PEO), polyvinyl acetate (PVAc), polyvinyl alcohol And at least one selected from the group consisting of polyvinyl pyrrolidone and polyacrylamide.
8. The method according to claim 1,

   The hydrophobic polymer may be at least one selected from the group consisting of polyesters, polyanhydrides, polyorthoesters, polyphosphazines, polylysines, polyisols, polyvalins, polyphenylalanines, polyprolines, polyglycines, polytryptophanes, polyalanines, polylactides, At least one member selected from the group consisting of polyglycolide, polycaprolactone, and polymethionine.
9. The method according to claim 1,

   The antigen protein may be selected from the group consisting of influenza virus hemagglutinin (HA), the neuraminidase (NA), the nucleoprotein (NP), M1 protein, M2 protein, NS1 protein, NS2 protein NEP protein: nuclear export protein), PA protein, PB1 protein (polymerase basic 1 protein), PB1-F2 protein and PB2 protein;

   In the case of rabies virus, nuclear protein (N), phosphorus protein (P), substrate protein (M), glycoprotein (G), and viral RNA polymerase (L);

   Hepatitis B virus surface antigen (HBsAg), hepatitis B virus core antigen (HbcAg), hepatitis B virus DNA polymerase, HBx protein, preS2 middle surface protein, , Large S protein, viral protein VP1, viral protein VP2, viral protein VP3, and viral protein VP4;

   ≪ / RTI >
10. Reacting an antigen protein-amphipathic polymer mixed solution prepared by adding an amphipathic polymer in a hydrophobic solvent to a protein solution containing an antigen protein in a hydrophilic solvent with shaking for 20 to 28 hours to form an amphipathic polymer-antigen protein complex; And

    Wherein the mixed solution comprises a hydrophilic solvent containing an antigenic protein and the hydrophobic solvent is a colloidal phase present in a droplet state, the hydrophilic solvent is granulated by self-assembly of the amphipathic polymer-antigen protein complex.
11. 11. The method of claim 10,

    Wherein the protein solution further comprises glycerol.
12. A vaccine composition comprising an immunostimulatory particle according to claim 1.
13. 13. The method of claim 12,

    Wherein the composition further comprises an adjuvant.
14. A method for inducing an immune response in an individual comprising administering to the individual an immunostimulatory particle according to claim 1.

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