Micelles composed of amphiphilic peptides and antigen carrier nanoparticles using the same

Abstract

The present invention relates to nanoparticles comprising an amphiphilic peptide that forms a micellar structure by self-assembly and a targeting peptide (preferably a soluble antigen peptide) that electrically binds to the surface of the amphiphilic peptide, and a method for producing the same. The targeting peptide is electrically bound to the surface of the amphiphilic peptide micellar structure to form particles, which can be effectively presented to antigen-presenting cells. By adjusting the weight ratio of the amphiphilic peptide and the targeting peptide, the size of the nanoparticles can be controlled, allowing for intracellular delivery and induction of cytotoxic T cell-mediated immunity. The nanoparticles of the present invention exert their vaccine effect by targeting only antigenic determinants at more precise sites, thereby minimizing side effects. Therefore, they can exhibit excellent antigen-specific antibody and cellular immunotherapy effects and can be used in fields such as the production of various vaccines.

Description

[Technical Field]

[0001] The present invention provides a micellar structure composed of an amphipathic peptide and a nanoparticle having a targeting peptide bound to the surface of the micellar structure. According to the present invention, the nanoparticle is used as an antigen carrier and has a vaccine effect.

[0002] The present invention was developed with the support of the following Korean National Research and Development Programs:

[0003] [Project unique number]1711163105 [Project Number] 2021R1A2C1013022 [Department name] Ministry of Science, ICT and Technology of Korea [Name of issue management (specialized) organization] Korea Research Foundation [Research project name] Individual basic research (Ministry of Science, Technology, Information and Communication) [Research title] Preparation of particulate antigens from soluble incomplete antigens using amphipathic peptides [Name of the organization carrying out the project] Hanyang University [Background technology]

[0004] The process of antibody formation, which plays an important role in the human immune system, can be broadly divided into two stages depending on the type of antigen. Particulate antigens are recognized by pattern recognition receptors present in macrophages and dendritic cells, enter cells by phagocytosis, are degraded by proteasomes, and are then presented on MHC II molecules. Soluble antigens, on the other hand, are recognized by cells with receptors for these antigens, enter cells by receptor-mediated endocytosis, are degraded, and are presented on MHC II molecules.

[0005] It is generally known that antibodies against particulate antigens are frequently produced. Dendritic cells and macrophages, which are uniformly distributed in each tissue for defense, react nonspecifically to particulate antigens and produce antibodies against them. On the other hand, soluble antigens are recognized primarily by B cells and cells differentiated from B cells, which have specific receptors for them. However, B cells are not uniformly distributed in all tissues and are primarily located inside lymph nodes, so a migration process is required (Non-Patent Document 1: Curr Opin Immunol, 65, 1-6, 2020), and a receptor capable of binding to the antigen must be present. Furthermore, because each B cell has a different B cell receptor, antibody production against soluble antigens, as well as the persistence of the generated antibodies, is significantly lower than that against particulate antigens.

[0006] Furthermore, when antibodies are produced in the form of haptens using a portion of the amino acid sequence, or when the extracellularly exposed portion is converted into a recombinant protein using methods such as gene truncation and used as an antigen, problems arise, such as the production of antibodies due to accessibility issues. Furthermore, the formation of covalent bonds between the hapten and the carrier protein can cause structural changes, resulting in the production of antibodies against this portion or antibodies against the large carrier protein, which can cause side effects (Non-Patent Document 2: Sci. Adv. 6, eaaax2285, 2020). To address the issue of the significantly lower in vivo antibody production rate for soluble antigens compared to particulate antigens, attempts have been made to develop various adjuvants, such as nanoparticles. However, due to low efficacy and stability, unacceptable drug resistance, manufacturing difficulties, and toxicity, no successful alternatives have yet been developed (Non-Patent Document 3: Hum. Vacc. Imm, 10:9, 2761-2774, 2014).

[0007] When developing vaccines against nanoparticles, such as bacteria and fungi, antibody production capacity is often not a major consideration. Traditional vaccines that weaken (attenuated vaccines) or inactivate (inactivated vaccines) pathogens have been widely used due to their high antibody production capacity. In these cases, the toxicity and safety of the vaccine itself have been a major concern rather than antibody production capacity.

[0008] In the case of viral vaccines, there are many viruses that cannot be attenuated or inactivated, and in the case of cancer vaccines, attenuation or inactivation is completely impossible because cancer cell-specific antigens and non-specific antigens coexist. To solve these problems, subunit vaccines and recombinant vaccines have been developed as second-generation vaccines, but as mentioned above, the limitations of soluble antigens mean that antibodies are not formed effectively.

[0009] Furthermore, the basic concept of third-generation vaccines is that once a carrier platform is established and the target is determined, the vaccine can be used as a module containing DNA or RNA capable of producing (encoding) the target molecule. This has enabled the development of new vaccines in a very short time. Since then, many researchers have been developing safer and more stable carrier platforms.

[0010] However, mRNA vaccines must produce proteins in the body, and therefore cannot be made shorter than a certain length (if it is too short, the level of protein expression will decrease), making precise targeting difficult.

[0011] The dengue vaccine is an example of a vaccine that requires precise targeting. As the number of people infected with dengue fever continues to increase, and the number of people suffering from pseudomyelitis is also on the rise, the need for vaccine development has also increased. Various research organizations have begun development, and the first approved product was released in 2018. However, after clinical use, significant side effects were observed, leading to restrictions on its use.

[0012] Four types of viruses are currently known to cause dengue fever. Antibodies are primarily produced against the E protein, a surface antigen, and the amino acid sequences of this E protein are extremely similar among the four viruses. More than 52% of the amino acid sequences are identical, with an overall similarity of over 80%. In other words, the sequences of the E proteins, the surface proteins of the four dengue viruses, are over 80% similar. The biggest problem with dengue vaccines is that while they are effective in preventing infection if the correct antibodies are produced and neutralizing antibodies are produced, if they do not bind precisely divalently but only weakly to similar antigens, antibody-dependent enhancement can lead to increased infection.

[0013] Therefore, to create a multivalent vaccine that can minimize side effects, it is necessary to construct a vaccine that contains only antigenic determinants at more precise sites. However, conventional nucleic acid vaccine platforms cannot meet this requirement. Therefore, the present inventors have completed the present invention to provide an antigen that targets only the modified portion of a virus or tumor marker. [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] Curr Opin Immunol,65,1-6,2020 [Non-patent document 2] Sci.Adv.6,eaaax2285,2020 [Non-patent document 3] Hum.Vacc.Imm,10:9,2761-2774,2014 Summary of the Invention [Problem to be solved by the invention]

[0015] One aspect is to provide nanoparticles comprising an amphiphilic peptide that self-assembles to form a micellar structure and a targeting peptide (e.g., a soluble antigenic peptide) that is electrically bound to the surface of the amphiphilic peptide.

[0016] Another aspect is to provide a method for producing nanoparticles, which includes the steps of: mixing an amphipathic peptide in an aqueous solution to form a micellar structure; and mixing a target peptide (e.g., a soluble antigen peptide) and electrically binding it to the surface of the formed micellar structure.

[0017] Another aspect of the present invention is to provide nanoparticles for multivalent vaccines that use only antigenic determinants at more precise sites, thereby minimizing side effects. [Means for solving the problem]

[0018] In order to overcome the limitations of the above-mentioned conventional technology, the present inventors have developed a method in which various amphipathic peptides with small molecular weights of approximately 10 amino acids or less form particulate micelles, and then electrically bind haptenic peptides to the surface of these micelles and present them to antigen-presenting cells.

[0019] The present invention will be described in detail below.

[0020] The terms used in this application are merely used to describe particular embodiments and are not intended to limit the present invention. Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which this disclosure belongs. The present invention provides nanoparticles comprising an amphiphilic peptide that self-assembles to form a micellar structure (see FIG. 1) and a targeting peptide that electrically binds to the surface of the amphiphilic peptide (or a micellar structure composed of the amphiphilic peptide).

[0021] As used herein, the term "amphiphilic peptide" refers to a peptide that contains both polar hydrophilic amino acids and non-polar hydrophobic amino acids. Because of this amino acid composition, amphiphilic peptides can interact with both hydrophilic and hydrophobic molecules.

[0022] In one embodiment of the present invention, the amphipathic peptide may contain 1 to 10 (or 1 to 15) amino acids, and in one embodiment of the present invention, the amphipathic peptide is a peptide represented by the following general formula I:

[0023] [General formula I] CnXm Here, C is a charged amino acid, X is a hydrophobic amino acid which may be the same or different, n is an integer of 1 to 5, and m is an integer of 3 to 10.

[0024] That is, the nanoparticles of the present invention may include micelles formed from amphipathic peptides represented by the general formula I: CnXm(I) (wherein C is a charged amino acid, X is a hydrophobic amino acid which may be the same or different, n is an integer of 1 to 5, and m is an integer of 3 to 10). In one embodiment, the number of charged amino acids may be 2 to 5, and the number of hydrophobic amino acids may be 4 to 8. In one embodiment of the present invention, the amphipathic peptide may include, as hydrophilic amino acids, positively charged amino acids such as arginine (R), histidine (H), and lysine (K), or negatively charged amino acids such as aspartic acid (D), glutamic acid (E), etc. In one embodiment, the hydrophilic amino acids are located on the surface of the micelle, and the surface of the micelle may be positively or negatively charged.

[0025] In one embodiment of the present invention, the amphipathic peptide may contain one or more hydrophobic amino acids selected from the group consisting of valine, phenylalanine, tryptophan, isoleucine, leucine, proline, methionine, and alanine.

[0026] In one embodiment of the present invention, the amphipathic peptide may include arginine (R) as a hydrophilic amino acid and valine (V) as a hydrophobic amino acid.

[0027] In one embodiment of the present invention, the amphipathic peptide may be R3V6 (RRRVVVVVV, SEQ ID NO: 1) consisting of the amino acid sequence of SEQ ID NO: 1, which contains three positively charged hydrophilic amino acids, arginine, and six hydrophobic amino acids, valine; R4V7 (RRRRVVVVVVV, SEQ ID NO: 2), which contains the amino acid sequence of SEQ ID NO: 2, which contains four positively charged hydrophilic amino acids, arginine, and seven hydrophobic amino acids, valine; E3V6 (EEEVVVVVV, SEQ ID NO: 3), which contains the amino acid sequence of SEQ ID NO: 3, which contains three negatively charged hydrophilic amino acids, glutamic acid, and six hydrophobic amino acids, valine; or E4V7 (EEEEVVVVVVV, SEQ ID NO: 4), which contains the amino acid sequence of SEQ ID NO: 4, which contains four negatively charged hydrophilic amino acids, glutamic acid, and seven hydrophobic amino acids, valine.

[0028] In one embodiment of the present invention, the "target peptide" is a peptide that binds (electrically) to the amphipathic peptide. The "target peptide" of the present invention is preferably an "antigen peptide," for example, a "hapten peptide."

[0029] In the present invention, the term "hapten peptide" refers to a low molecular weight substance (e.g., a low molecular weight peptide, a low molecular weight toxin, etc.) that can be electrically bound to the surface of a micelle composed of the amphipathic peptide. Haptens have small molecular weights and sizes and are unable to produce antibodies by themselves through immune reactions in the body. However, in vitro, they can act as specific antigens that react with corresponding antibodies and form precipitates in antigen-antibody reactions. Furthermore, haptens can produce antibodies when bound to a carrier, such as a carrier protein, that is large enough to induce antibodies.

[0030] In one embodiment of the present invention, the "hapten peptide" consists of 6 to 15 amino acids or 8 to 12 amino acids, and preferably consists of a sequence of about 10 amino acids.

[0031] The "targeting peptide" of the present invention preferably has a "positive charge" or "negative charge" as a whole, and has a charge opposite to the surface charge of the micelle structure. The targeting peptide of the present invention has a charge of, for example, +1, +2, +3, -1, -2, or -3.

[0032] In one embodiment of the present invention, the target peptide may be bound to the surface of the amphipathic peptide. Specifically, the target peptide may be electrically bound to the amphipathic peptide with opposite polarity (see FIGS. 2 and 3).

[0033] In a preferred embodiment of the present invention, the "hapten peptide" may consist of the amino acid sequence of SEQ ID NO: 5-11.

[0034] In one embodiment of the present invention, the hapten may be a peptide constructed based on a portion of the amino acid sequence of a DDOST protein (dolichyl-diphosphooligosaccharide-protein glycosyltransferase 48 kDa subunit precursor glycosyltransferase, NP_005207). For example, the hapten peptide of the present invention may consist of any one of the sequences of SEQ ID NOS: 5 to 7. SEQ ID NOS: 5 (SDLGQHTLIV) of the present invention exhibits charge-1 in a neutral solution, SEQ ID NOS: 6 (LEDTLSSE) exhibits charge-2 in a neutral solution, and SEQ ID NOS: 7 (PGSQRYSQTGN) exhibits charge+1 in a neutral solution. However, any "hapten" that can be chemically bound to a KLH protein or albumin protein (a compound that carries a charge and can be electrically bound to the surface of a carrier peptide) and then induces the formation of antibodies against the hapten when injected into a living body may be used as the hapten peptide proposed in the present invention.

[0035] In one embodiment of the present invention, the size of the nanoparticles can be controlled by adjusting the weight ratio of the amphipathic peptide acting as a carrier to the targeting peptide, for example, by adjusting the weight ratio (w / w) of the amphipathic peptide to the targeting peptide to 1:5 to 5:1, 1:4 to 4:1, or 1:3 to 3:1.

[0036] In one embodiment of the present invention, the nanoparticles may contain an amphipathic peptide and a hapten in a weight ratio (w / w) of 1:3 to 3:1. Specifically, when the amphipathic peptide and the hapten peptide were mixed at weight ratios of 3:1 and 1:3, particle size analysis using a Zetasizer confirmed that the particle sizes could be adjusted to 60 to 150 nm and 300 to 800 nm, respectively. Such size adjustment of the nanoparticles can induce antibody immunity or cellular immunity.

[0037] In one embodiment of the present invention, the nanoparticles have a size of 50 nm to 150 nm, 50 nm to 300 nm, 50 nm to 700 nm, 150 nm to 400 nm, or 100 nm to 300 nm, and preferably have a size of 150 nm to 350 nm.

[0038] In one embodiment of the present invention, the size of the nanoparticles may be 60-150 nm or 300-800 nm when the amphipathic peptide and hapten peptide are contained in a weight ratio of 1:3 or 3:1. In this case, it was confirmed that the phagocytosis of the nanoparticles by macrophages is increased (see FIG. 1), and they are presented on the surface by antigen presenting cells (APCs), thereby more effectively increasing immunoglobulin production.

[0039] In one embodiment of the present invention, the size of the nanoparticles may be 60-150 nm or 300-800 nm when the amphipathic peptide and hapten peptide are contained in a weight ratio of 3:1 or 1:3. In this case, it was confirmed that the nanoparticles are phagocytosed by macrophages relatively little (see FIG. 1), resulting in a decrease in immunoglobulin formation. Meanwhile, it was confirmed that the number of cytotoxic T lymphocytes specific to the hapten peptide is increased compared to helper T cells and B cells specific to the hapten peptide.

[0040] The present invention also provides a method for producing nanoparticles, which includes the steps of: mixing an amphipathic peptide in an aqueous solution to form a micellar structure; and mixing a target peptide and electrically binding it to the surface of the formed micellar structure.

[0041] In one embodiment of the present invention, the amphiphilic peptide may be a peptide represented by the following general formula I: [General formula I] CnXm Here, C is a charged amino acid, X is a hydrophobic amino acid that is different from each other, n is an integer of 1 to 5, and m is an integer of 3 to 10.

[0042] In one embodiment of the present invention, the step of forming the micellar structure is performed in a glucose solution having a concentration of 5 to 20%. Specifically, mixing the amphipathic peptide and the hapten peptide in a glucose solution having a concentration of 5 to 20% can form a more stabilized micellar structure.

[0043] In one embodiment of the present invention, the step of forming the micelle structure may be performed by further including cholesterol in a weight ratio of 1:5 to 5:1 relative to the amphipathic peptide. Specifically, the amphipathic peptide and cholesterol may be mixed in a weight ratio of 1:5, 1:3, 1:1, etc., and then left at room temperature for 2 hours to form particles with a relatively small size deviation.

[0044] In one embodiment of the present invention, the method for producing nanoparticles is characterized by mixing an amphipathic peptide and a targeting peptide in a weight ratio of 1:5 to 5:1, and forming the targeting peptide into particles.

[0045] In one embodiment of the present invention, in the particle-forming step, the antigen peptide may be a hapten peptide.

[0046] The present invention also provides a vaccine composition comprising the nanoparticles, which can be used in immunotherapy.

[0047] The vaccine composition of the present invention can target, but is not limited to, dengue virus infection, severe fever with thrombocytopenia syndrome, or influenza virus infection.

[0048] In one embodiment of the present invention, the vaccine may be used to prevent or treat diseases that involve immune cells (B cells or T cells).

[0049] As used herein, the term "prevention" refers to any action that inhibits or delays the onset of a disease in an individual by administering a pharmaceutical composition according to one embodiment.

[0050] As used herein, the term "treatment" refers to any action that improves or beneficially alters the symptoms of a disease by administering a pharmaceutical composition according to one embodiment.

[0051] The present invention also provides a pharmaceutical composition for preventing or treating immune diseases, comprising the nanoparticles.

[0052] According to one embodiment of the present invention, the immune disease may be any disease caused by infection with a pathogen, such as a virus or a microorganism, or any disease induced by a pathogenic substance. Specifically, the pathogen itself, a part thereof, or the pathogenic substance may be used as a hapten. The pharmaceutical composition may further include a pharmaceutically acceptable carrier or diluent. The pharmaceutically acceptable carrier or diluent may be any known in the art. The carrier or diluent may be lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water (e.g., saline and sterile water), syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, Ringer's solution, a buffer, maltodextrin solution, glycerol, ethanol, dextran, albumin, or any combination thereof. The pharmaceutical compositions may further comprise lubricating agents, wetting agents, sweetening agents, flavoring agents, emulsifying agents, suspending agents, or preservatives.

[0053] The pharmaceutical composition may be formulated using pharmaceutically acceptable carriers and / or excipients by methods known to those skilled in the art and prepared in unit dose form or in multi-dose containers. The dosage form may be a solution, suspension, syrup, or emulsion in an oil or aqueous solvent, or an extract, powder, powder, granules, tablet, or capsule, and may further contain a dispersant or stabilizer. The aqueous solvent may contain physiological saline or PBS. The pharmaceutical composition according to one embodiment may be formulated for oral or parenteral administration, preferably a parenteral administration. For intramuscular, intraperitoneal, subcutaneous, and intravenous administration, a sterile solution of the active ingredient is typically prepared, and a buffer may be added to appropriately adjust the pH of the solution. For intravenous administration, an isotonicity agent may be added to impart isotonicity to the formulation.

[0054] The dosage (effective amount) of a pharmaceutical composition according to one embodiment can be varied depending on factors such as formulation method, administration method, patient age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate, and reaction sensitivity. Those skilled in the art can appropriately adjust the dosage taking these factors into account. The administration frequency can be once daily or two or more times per day within the range of clinically acceptable side effects. The administration site can be one or more sites, daily or every 2 to 5 days, for a total administration period of 1 to 30 days. If necessary, the same treatment can be repeated at an appropriate time. For non-human animals, the same dosage per kg as for humans can be administered, or the dosage can be calculated based on, for example, the volume ratio (e.g., average value) of organs (e.g., heart) between the target animal and humans. Possible administration routes include parenteral (e.g., subcutaneous, intramuscular, intra-arterial, intraperitoneal, intradural, or intravenous), topical (including transdermal), injection, or insertion of an implantable device or substance. Animals that may be treated in accordance with one embodiment include humans and other mammals of interest, including, for example, humans, monkeys, mice, rats, rabbits, sheep, cattle, dogs, horses, pigs, and the like.

[0055] The present invention also provides a health functional food for preventing or improving immune diseases, which contains the nanoparticles as an active ingredient.

[0056] The nanoparticles, immune diseases and prevention are as described above.

[0057] As used herein, "amelioration" refers to any action that suppresses or delays the onset of an immune disorder in an individual by administering a composition according to one embodiment.

[0058] The health functional food defined in the present invention may be a health functional food whose functionality and safety for the human body have been fully established as newly defined through the Health Functional Food Act revised in 2008, and which is listed in the regulations regarding the certification of functional ingredients for health functional foods as stipulated in Notification No. 2008-72 of the Ministry of Food and Drug Safety of Korea.

[0059] When the composition of the present invention is used in a functional health food, it can be added directly or in combination with other functional health foods or functional health food ingredients, and can be used appropriately in a conventional manner. The amount of active ingredient to be added can be determined appropriately depending on the intended use. Generally, the active ingredient constituting the composition of the present invention may be contained in an amount of 0.01 to 15 wt.%, preferably 0.2 to 10 wt.%, of the total weight of the food. When prepared as a beverage, it may contain 0.1 to 30 g, preferably 0.2 to 5 g, per 100 mL. The entire beverage may be composed of natural ingredients. However, in the case of long-term intake for health regulation and hygiene purposes, the amount may be less than the above range, and since there are no safety issues, the active ingredient may also be used in an amount greater than the above range.

[0060] The composition for a health functional food according to the present invention can be formulated into a conventional health functional food dosage form known in the art. The health functional food can be prepared, for example, in the form of powder, granules, tablets, pills, capsules, suspension, emulsion, syrup, tablet, liquid, extract, gum, tea, jelly, or beverage, and is preferably formulated in the form of a beverage. The food-scientifically acceptable carrier or additive can be any carrier or additive known in the art to be usable in the preparation of the desired dosage form. Foods used for animal feed may also be used.

[0061] The functional health food may contain, depending on the purpose or preference, nutrients, vitamins, electrolytes, flavorings, colorants, pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, etc. In addition, it may contain fruit pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages. Furthermore, the functional health food composition may further contain food additives, and the suitability of a food additive as a "food additive" may be determined in accordance with the specifications and standards for that item, such as the general provisions and general test methods for food additives approved by the Korea Food and Drug Administration, unless otherwise specified.

[0062] The present invention also provides a use of the nanoparticles for the manufacture of a medicament for the prevention or treatment of an immune disease. [Effects of the Invention]

[0063] According to the present invention, when an amphipathic peptide that forms a micellar structure by self-assembly and a target peptide (e.g., a soluble antigen peptide) that electrically binds to the surface of the amphipathic peptide are mixed at a certain weight ratio, nanoparticles of adjustable size can be formed. Depending on the size, the nanoparticles can be presented to auxiliary T lymphocytes to induce antibody production or activate cytotoxic T lymphocytes and cellular immunity, thereby demonstrating a target peptide-specific immunotherapeutic effect.

[0064] In the present invention, the nanoparticles can induce immune responses of antigen-presenting cells, B cells, and auxiliary T cells without chemical modification, thereby producing antibodies in the body, and can induce the production of target peptide-specific cytotoxic T lymphocytes. Therefore, they can be used in fields such as vaccine production, as well as in cellular immunity, which has not yet been put to practical use, and can be used in the prevention and treatment of various diseases such as various viral diseases and cancer.

[0065] The nanoparticles of the present invention use hapten peptides as target peptides, allowing for the construction of antigenic determinants at more precise sites than conventional methods, and can therefore be used as multivalent vaccines with minimized side effects. In other words, the nanoparticles (vaccine platform) of the present invention can provide antigens that target only the modified portions of viruses or tumor markers. [Brief explanation of the drawings]

[0066] [Figure 1] Figure 1 is a schematic diagram showing the micelle formation conditions confirmed for all possible amphipathic peptides to secure the nanoparticles (vaccine platforms) of the present invention. The nanoparticles (peptide platforms) of the present invention refer to amphipathic peptides that can form micelles through self-assembly in aqueous solution. Furthermore, the formed micelles are disassembled within antigen-presenting cells and presented on MHC class 2 molecules, which may generate antibodies against the micelle-forming peptides. To minimize the formation of antibodies against these peptides, the peptides were composed of only two types of bipolar amino acids. [Figure 2] Figure 2 is a schematic diagram showing the steps of preparing nanoparticles (modular antigens in Figure 2). According to the present invention, nanoparticles are formed by binding a target peptide (hapten peptide) to the surface of a micelle formed from an amphiphilic peptide. [Figure 3] 3 is a schematic diagram showing the formation of nanoparticles (peptide vaccine platform) according to the present invention. The nanoparticles of the present invention are formed by adding a target peptide (preferably a hapten peptide) to an amphiphilic peptide and then granulating it. [Figure 4] Figure 4 shows an image (left) and an electron microscope image (right) of the size measurements of particles formed by amphiphilic peptide (R3V6) and R3V6 (or R3L6)-hapten peptide according to one embodiment of the present invention. As shown in Figure 4, micelles composed only of amphiphilic peptide form spherical particles with a size of 100 to 300 nm, and when the hapten peptide is electrically attached to the amphiphilic peptide, particles with a size of 150 to 350 nm are formed. [Figure 5] Figure 5 shows images of nanoparticles internalized by macrophages taken with a confocal fluorescence microscope according to size. It can be seen that the degree of uptake by macrophage cell lines differs depending on the size of the nanoparticles produced, and that modular vaccines with an average size of around 200-300 nm are well absorbed. [Figure 6] 6 is a graph showing the expression of target protein-specific IgG and IgM antibodies in mouse blood following administration of nanoparticles prepared according to the present invention. Treatment with nanoparticles of 200-300 nm size (●) showed greater immunogenicity than administration of nanoparticles of 30-50 nm size (■). [Figure 7] Figure 7 shows an image comparing antibodies formed by the nanoparticles of the present invention and the KLH conjugate method. To compare the amount of antibody produced, we also compared the amount of antibody produced by conjugating the nanoparticles with keyhole limpet hemocyanin, which is the most commonly used soluble incomplete antigen for antibody induction. When soluble incomplete antigens alone were injected, no antibodies were produced (lane 1 in the top panel of Figure 7), but antibodies were produced when the nanoparticles were conjugated with keyhole limpet hemocyanin (lane 2 in the top panel of Figure 7). It was also confirmed that antibodies could be induced in larger amounts using the nanoparticles of the present invention than with the commonly used keyhole limpet hemocyanin method (lanes 3 and 4 in the top panel of Figure 7). [Figure 8] Figure 8 shows images of nanoparticles internalized by macrophages taken with a confocal fluorescence microscope. Macrophages were incubated with nanoparticles for 2 hours (nanoparticle concentration: 40 μg / ml). The hapten was displayed with red fluorescence. Nuclei were counterstained with blue. (A) Amphipathic peptide:hapten peptide ratio: 3:1 (B) Amphipathic peptide:hapten peptide ratio: 1:3 [Figure 9]Figure 9 shows the titration curve for the hapten peptide, confirming the specific IgG response. BALB / c mice were immunized on days 0 and 21 with nanoparticles containing a carrier protein, hapten, and adjuvant in a 3:1 (w / w) or 1:3 (w / w) ratio. Blood samples were collected twice a week and analyzed by ELISA. (In Figure 9, the "microparticles" refer to the amphiphilic peptide.) [Figure 10] Figure 10 shows the effect of nanoparticles on antigen-specific IgG responses. BALB / c mice were immunized with emulsified 3:1 (w / w) or 1:3 (w / w) nanoparticles on days 0 and 21. Mice were sacrificed on day 35, and whole blood was collected. Serum was analyzed by dot blot for whole proteins containing haptens as moieties. (In Figure 10, "microparticles" refers to amphipathic peptides.) [Figure 11] Figure 11 shows histogram images of splenocyte B cells that reacted with Alexa 647-labeled DDOST protein according to one embodiment of the present invention. In this experiment, flow cytometry was performed on splenocytes harvested from immunized mice. The Alexa 647-positive channel was used to detect hapten-specific B cells, and the hapten-specific B cells were the lower gate. Figure 11A shows the results for the amphipathic peptide (carrier), with the lower gated group representing 15.1%. Figure 11B shows the results for the hapten peptide, with the lower gated group representing 14.3%. Figure 11C shows the results for the (amphipathic peptide:hapten peptide) 3:1 (w / w) nanoparticles, with the lower gated group representing 33.6%. Figure 11D shows the results for the 1:3 (w / w) nanoparticles, with the lower gated group representing 23.5%. [Figure 12]Figure 12 shows the results of flow cytometry of hapten-specific cytotoxic T cell populations. Spleen cells harvested from immunized mice were analyzed using flow cytometry. The Alexa 647-positive channel was used to detect hapten-specific cytotoxic T cells. Hapten- and CD8-specific T cells were sub-gated. Figure 12A shows the results of carrier analysis, with the sub-gated group showing 0.78%. Figure 12B shows the results of hapten analysis, with the sub-gated group showing 0.67%. Figure 12C shows the results of 3:1 (w / w) nanoparticle analysis, with the sub-gated group showing 1%. Figure 12D shows the results of 1:3 (w / w) nanoparticle analysis, with the sub-gated group showing 2.1%. DETAILED DESCRIPTION OF THE INVENTION

[0067] The present invention will be described in more detail below with reference to examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples and experimental examples.

[0068] Examples and Experiments Experimental Example 1. Materials and Methods 1.1 Production of nanoparticles (soluble haptens) using amphiphilic peptides Recombinant amphipathic peptides and hapten peptides were produced and provided by Anizen, Gwangju, South Korea. To prepare nanoparticles (particulate soluble haptens) using amphipathic peptides, amphipathic peptides (peptides SEQ ID NOS: 1-4) were dissolved in distilled water at a concentration of 4 mg / ml. Hapten peptides (peptides SEQ ID NOS: 5-11) were dissolved in distilled water at a concentration of 0.5 mg / ml. A fixed amount of hapten (50 μg) was then mixed with two different amounts of amphipathic peptides at a ratio of 3:1 and 1:3 (w / w) and incubated at room temperature (25°C) for 30 minutes.

[0069] 1.2 Nanoparticle size characterization To characterize the size of the amphiphilic peptides and nanoparticles, the diameters of micelles and nanoparticles formed by the amphiphilic peptide (carrier) were measured using a Zetasizer Nano ZS system (Malvern Instruments, Malvern, UK). After dissolution, the micelle stock solution was placed in a cuvette. After 30 minutes of incubation, the nanoparticles were diluted to a final volume of 1 ml. The average size was then analyzed using Dispersion Technology software 4.3. The nanoparticles were diluted using ultrapure water (refractive index 1.33, viscosity 0.89) and used for analysis at room temperature. Observations are presented as triplicate means + / - standard deviation.

[0070] 1.3 Cellular absorption of nanoparticles Macrophages, RAW264.7, were purchased from ATCC, Virginia, USA. To detect nanoparticle uptake by cells, nanoparticles were labeled with Alexa 647. After labeling, the nanoparticles were incubated with carriers and prepared at 40 μg / ml. Cells were plated in 8-chamber slides, 1×10 4 The cells were seeded in 100 ml of PBS and set up for 1 day. Cells were treated with nanoparticles at two different ratios (1:3 or 3:1) for 2 hours. Cells were fixed in 4% PBS for 10 minutes. Cells were blocked with 1% bovine serum albumin for 30 minutes. DAPI counterstaining was performed for 1 minute without antibody treatment. After mounting a cover glass, the cells were observed under a confocal fluorescence microscope SP5 TCS (Leica Biosystems, USA).

[0071] 1.4 Immunization of mice Female BALB / C mice were purchased from Nara Biotech, Seoul, Korea, and housed under pathogen-free conditions at 6 weeks of age. This study was conducted in strict accordance with the National Institutes of Health's Guidelines for the Care and Use of Laboratory Animals. The protocol was approved by the Animal Care and Use Committee of Hanyang University Animal Hospital. For immunization, nanoparticles were emulsified with aluminum hydroxide (20 mg / 100 μL, Sigma-Aldrich, St. Louis, MO, USA). Mice were injected with 150 μg of carrier dissolved in 150 μl of phosphate-buffered saline (PBS). Mice were injected with 50 μg of hapten dissolved in 150 μl of PBS. Mice were injected with 3:1 (w / w) nanoparticles (150 μg of carrier and 50 μg of hapten) combined with 150 μl of PBS and 40 mg of aluminum hydroxide. Mice were injected with 150 μl of PBS and 1:3 (w / w) nanoparticles (16.67 μg of carrier and 50 μg of hapten) combined with 40 mg of aluminum hydroxide. All groups were injected intraperitoneally and again on day 21. Blood was collected twice weekly via the jugular vein and centrifuged at 2000 g for 20 minutes. Serum was collected from the centrifuged blood and frozen at -20°C until analysis by ELSIA. Mice were sacrificed on day 35. Whole blood was collected and centrifuged at 2000 g for 20 minutes. Serum was collected from the centrifuged blood and frozen at -20°C until analysis by dot blot. Spleens were collected to harvest splenocytes for flow cytometry.

[0072] 1.6 Antibody detection by dot blot To determine nanoparticle (or hapten peptide)-specific antibodies, serially diluted recombinant proteins were seeded onto PVDF membranes at concentrations ranging from 1.2 μg / µL to 12 ng / µL. The membranes were blocked with 2.5% nonfat milk in PBS (blocking buffer) for 2 hours at room temperature. 20 μL of serum was diluted to a final volume of 3 mL with blocking buffer and applied to the membrane for 2 hours at room temperature. After serum treatment, the wells were incubated with horseradish peroxidase-conjugated anti-mouse IgG antibody for 1 hour at room temperature. The membranes were then treated with enhanced chemiluminescence (ECL) solution and analyzed directly on a Chemiscope 3400 (CLINX, China). Spots were visualized within 10 seconds. The membranes were washed three times with wash buffer (PBS with Tween) at all stages.

[0073] 1.7 Harvesting of spleen cells Spleens were collected from sacrificed mice on day 35 and stored in RPMI 1640 (Coring, Kennebunk, ME, USA) with 10% FBS without antibiotics. A 70 μm cell strainer was placed in a 50 mL conical tube and washed with 5 mL of RPMI 1640. The spleen was excised with scissors, placed on the cell strainer, and gently triturated. The strainer was washed again with 5 mL of RPMI 1640. The suspended cells were transferred to a 15 mL conical tube and the sample was stored on ice. The suspended cells were centrifuged at 550 g for 5 minutes, and the supernatant was discarded. 1 mL of ACK lysis buffer (Thermofisher Scientific, Massachusetts, USA) was added to the 15 mL conical tube and incubated at room temperature for 5 minutes. 4 mL of RPMI 1640 was added to the conical tube, and the suspended cells were centrifuged at 550 g for 5 minutes. This step was repeated until the red spots disappeared in the cell pellet. Finally, the cell pellet was resuspended in 5 mL of RPMI 1640 and used directly for flow cytometry.

[0074] 1.8 Live-cell flow cytometry Live-cell flow cytometry was performed to identify nanoparticle (or hapten peptide)-specific lymphocyte populations. To detect hapten-specific B and T lymphocytes, DDOST recombinant protein containing the hapten peptide was labeled with Alexa 647 (ab269823, Abcam, Cambridge, UK). The harvested splenocytes were centrifuged at 550 g for 5 minutes, and the supernatant was discarded. The cell pellet was resuspended in cold DPBS. This step was repeated three times for washing. After washing, the cells were filtered through a 40 μm cell strainer and counted. 2 × 10 6 The cells were transferred to a 1.75 ml E tube and treated with FcR blocking reagent (for mice) at 4°C for 10 minutes.

[0075] To characterize B lymphocytes, Alexa 488-conjugated anti-mouse CD19 antibody (ab270176, Abcam, Cambridge, UK) and Alexa 647-labeled recombinant protein were treated with FcR blocking reagent (130-092-575, Miltenybiotec, Bergisch Gladbach, Germany) for 1 hour at 4°C. The treated cells were washed three times with FACS buffer. After washing, the cells were resuspended in 1 mL of cold DPBS. PI was added to the cells for 10 minutes to detect viable cells, and the cells were analyzed using FACS.

[0076] To characterize T lymphocytes, FITC-conjugated anti-mouse CD8 antibody (ab237367, Abcam, Cambridge, UK) and Alexa 647-labeled recombinant protein were treated with FcR blocking reagent for 1 hour at 4°C. The treated cells were washed three times with FACS buffer. After washing, the cells were resuspended in 1 mL of cold DPBS. PI was added to the cells to detect viable cells for 10 minutes, and the cells were immediately analyzed using a FACS Conto II (BD Biosciences, San Diego, USA). In all experiments, cell recovery was performed by centrifugation at 300 g for 3 minutes.

[0077] Experimental Example 2. Results 2.1 Nanoparticle size measurement First, in this example, the sizes of the nanoparticles formed by the amphipathic peptide (R3V6) and the amphipathic peptide (R3V6)-hapten peptide were measured and confirmed using an electron microscope (Figure 4). Referring to Figure 4, it can be seen that the amphipathic peptide alone forms micelles (spherical particles) with a size of 50 to 300 nm (preferably 100 to 300 nm), and when the hapten peptide is electrically attached to the micelle structure, particles with a size of 60 to 150 nm or 300 to 800 nm are formed, depending on the ratio. To characterize the carrier and nanoparticle sizes, we also measured the diameters of micelles and nanoparticles formed by the amphiphilic peptide. More specifically, a fixed amount of hapten peptide was mixed with two different amounts of amphiphilic peptide at weight ratios of 3:1 and 1:3. The sizes of the micelles prepared by the present invention were 100-300 nm, the sizes of the 3:1 (w / w) nanoparticles were 60-150 nm, and the sizes of the 1:3 (w / w) nanoparticles were 300-800 nm (Table 1). [Table 1] 2.2 Cellular absorption of nanoparticles Macrophages are one of the antigen-presenting cells, so when the nanoparticles according to the present invention act as antigens, cellular absorption must occur.

[0078] In this example, a hapten peptide was labeled with the fluorescent substance Alexa 647 and mixed with an amphipathic peptide (R3V6) at adjusted ratios. Nanoparticles with average sizes of 30-50 nm and 200-300 nm were then administered to a macrophage cell line. Referring to Figure 5, it can be seen that nanoparticles with a size of 200-300 nm are well absorbed into macrophages, whereas nanoparticles with a size of 30-50 nm are significantly less absorbed. This indicates that the nanoparticles prepared according to the present invention are more effective as a vaccine when formed with a size of 150-350 nm.

[0079] In addition, the hapten peptide was mixed with two different amphiphilic peptides at a weight ratio of 3:1 (left image in Figure 8) and 1:3 (right image in Figure 8), and then the resulting nanoparticles were applied to a macrophage cell line. After incubation for 2 hours, it was confirmed that the nanoparticles were absorbed by the macrophages (Figure 8).

[0080] 2.3 Detection of nanoparticle (or hapten peptide)-specific antibodies 2.3.1 Increase in nanoparticle (or hapten peptide)-specific IgG antibodies To detect an increase in nanoparticle (or hapten peptide)-specific IgG antibodies, blood was collected from the mice twice a week and ELISA was performed. IgM increased only in the group injected with a mixture of amphipathic peptide (micelle structure) and hapten peptide, reaching its highest level around day 7 and then rapidly decreasing. Although the results are complex and not shown, there was no significant increase in either IgM or IgG antibody expression in the groups injected with amphipathic peptide or hapten peptide alone.

[0081] Specifically, mice were injected with 200-300 nm nanoparticles (▲, ● in Figure 6) and 30-50 nm nanoparticles (◆, ■ in Figure 6) produced according to the present invention. 15 days later, the mice were injected again with the same amount of nanoparticles, and the expression of target peptide (hapten peptide)-specific IgG and IgM antibodies in the mouse blood was confirmed (Figure 6). Referring to Figure 6, it can be seen that hapten peptide-specific IgM and IgG were induced in the same manner as a typical vaccine.

[0082] The results of examining the expression of IgG and IgM antibodies depending on the mixing ratio of amphipathic peptide and hapten peptide are shown in Figure 9. Although no significant increase in IgG was observed after the primary inoculation, an increase in optical density (OD) was observed after the secondary booster injection in the group in which amphipathic peptide and hapten peptide were mixed at a ratio of 1:3 (w / w). Furthermore, the group treated with a 3:1 (w / w) mixture of amphipathic peptide and hapten peptide showed a significantly lower increase in OD than the 1:3 group (Figure 9).

[0083] 2.3.2 Comparison of antibodies generated by modular vaccine and KLH conjugate methods Thirty-three days after vaccination, dot blot analysis of the targeted protein was performed for the first time using antisera isolated from the blood. Similar to the ELISA data, good results were observed when nanoparticles of 200-300 nm were administered.

[0084] To compare the amount of antibody production, we also compared the amount of antibody produced by inducing antibodies using soluble incomplete antigens conjugated to keyhole limpet hemocyanin, which is the most commonly used method for inducing antibodies. We found that, while no antibodies were produced when the hapten peptide alone was injected (lane 1 in the upper panel of Figure 7), antibodies were produced when the hapten peptide was conjugated to keyhole limpet hemocyanin (lane 2 in the upper panel of Figure 7). We also found that, when antibodies were induced using the nanoparticles of the present invention, the same or even larger amounts of antibodies were induced compared to the commonly used keyhole limpet hemocyanin method (lanes 3 and 4 in the upper panel of Figure 7).

[0085] Furthermore, when the antibodies confirmed by Western blot were pre-reacted with soluble truncated antigen (lower panel of Figure 7), all bands disappeared, confirming that all the antibodies produced were specifically produced against the soluble truncated antigen. After the experiment, the spleens of the animals were removed, and splenocytes were isolated and analyzed for B and T cells via flow cytometry.

[0086] 2.3.3 Detection of nanoparticle-specific IgG antibodies To confirm nanoparticle (or hapten peptide)-specific IgG antibodies, whole blood was collected from mice sacrificed on day 35 and a dot blot assay was performed. As shown in the ELISA results above, the 1:3 (w / w) nanoparticle dots were confirmed to be the most conclusive dots, which were highly concentrated and diluted as the seeded recombinant protein concentration decreased. However, no concentrated dots were observed in the groups injected with amphiphilic peptide micelles or hapten peptide alone. Furthermore, in the group where the amphiphilic peptide and hapten were mixed at a 3:1 (w / w) ratio, very light but distinct dots were observed (Figure 10). As shown in the ELISA results above, the group where the amphiphilic peptide and hapten were mixed at a 3:1 (w / w) ratio demonstrated a lower antibody production rate than the group where the amphiphilic peptide and hapten were mixed at a 1:3 (w / w) ratio.

[0087] 2.4 Identification of nanoparticle-specific lymphocyte populations 2.4.1 Nanoparticle-specific B cell populations To identify nanoparticle (or hapten peptide)-specific B cell populations, live-cell flow cytometry was performed using recombinant proteins containing the hapten, labeled with Alexa 647. After gating on CD19-positive B cells, histograms were set to the Alexa 647-positive channel and subgated with bars (Figure 11). There was no significant difference in the percentage of cells reacting to the target protein between the group treated with amphipathic peptide alone (Figure 11, A) and the group treated with hapten peptide alone (Figure 11, B). However, in the group treated with a 3:1 ratio of amphipathic peptide:hapten peptide (Figure 11, C), the percentage of cells reacting to the target protein was 8% higher than in the groups treated with amphipathic peptide or hapten peptide. In the group treated with a 1:3 ratio of amphipathic peptide:hapten peptide (Figure 11, D), the percentage of cells reacting to the target protein was approximately 18% higher than in the groups treated with amphipathic peptide or hapten peptide (see Table 2). [Table 2] 2.4.2 Hapten-specific cytotoxic T cell populations To identify hapten-specific T cell populations, we performed live-cell flow cytometry using Alexa 647-labeled recombinant proteins containing the hapten. Dot plots are shown, with each axis representing a target-specific channel (X axis: CD8-specific channel, Y axis: hapten-specific channel). Similar to B cell analysis, there was no significant difference in the percentage of cells reacting to the target protein between the groups treated with amphiphile peptide alone (Figure 12, A) and the hapten peptide alone (Figure 12, B). However, the percentage of cells reacting to the target protein in the amphiphile peptide:hapten peptide = 1:3 group was 0.22% higher than the amphiphile peptide alone group and 0.33% higher than the hapten peptide alone group (Figure 12, A, B, and D). In contrast, in the amphipathic peptide:hapten peptide = 3:1 group, the number of cytotoxic T cells was 1.32% higher than that of the group injected with amphipathic peptide alone and 1.43% higher than that of the group injected with hapten peptide alone (Figures 12A, B, and C), confirming that the group with relatively small particle size is more likely to increase cytotoxic T cells.

[0088] The present invention has been described above with reference to preferred embodiments. Those skilled in the art will recognize that the present invention can be embodied in various modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the claims, not the foregoing description, and all variations within the scope of the claims are to be construed as being within the scope of the present invention.

[0089] Sequence Catalog Free Text SEQ ID NO: 1 Amphiphilic peptide R3V6 RRRVVVVVV SEQ ID NO: 2 Amphiphilic peptide R4V7 RRRRVVVVVVV SEQ ID NO: 3 Amphiphilic peptide E3V6 EEEVVVVVV SEQ ID NO:4 Amphiphilic peptide E4V7 EEEEVVVVVVV SEQ ID NO:5 Happen peptide 1 SDLGQHTLIV SEQ ID NO:6 Happen peptide 2 LEDTLSSE SEQ ID NO:7 Happen peptide 3 PGSQRYSQTGN SEQ ID NO:8 Dengue virus subtype1 hapten peptide APTSEIQLTD SEQ ID NO:9 Dengue virus subtype2 hapten peptide SSITEAELTG SEQ ID NO: 10 Dengue virus subtype3 hapten peptide ASTVEAILPE SEQ ID NO: 11 Dengue virus subtype4 hapten peptide SPSVEVKLPD

Claims

1. A nanoparticle comprising an amphiphilic peptide that self-assembles to form a micellar structure and a targeting peptide that electrically binds to the surface of the amphiphilic peptide.

2. The nanoparticle of claim 1, wherein the amphiphilic peptide is a peptide represented by the following general formula I: [General formula I] CnXm Here, C is a charged amino acid, X is a hydrophobic amino acid, n is an integer from 1 to 5, and m is an integer from 3 to 10.

3. The nanoparticle of claim 1 , wherein the amphipathic peptide comprises arginine (R) as a hydrophilic amino acid and valine (V) as a hydrophobic amino acid.

4. The nanoparticles according to claim 1, wherein the amphiphilic peptide is one or more selected from the group consisting of peptides represented by the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO:

4.

5. The nanoparticle of claim 1 , wherein the targeting peptide has a polarity opposite to the surface polarity of the micellar structure.

6. The nanoparticle of claim 1 , wherein the target peptide is an antigenic peptide.

7. The nanoparticle of claim 6 , wherein the antigen peptide is a hapten peptide of an antigen of interest.

8. The nanoparticle according to claim 1, wherein the target peptide consists of any one of the amino acid sequences of SEQ ID NO: 5 to SEQ ID NO:

11.

9. The nanoparticles according to claim 1, characterized in that the amphipathic peptide and the targeting peptide are contained in a weight ratio of 1:5 to 5:

1.

10. The nanoparticles of claim 1, wherein the size of the nanoparticles is between 50 nm and 1,000 nm.

11. A vaccine composition comprising the nanoparticles of claim 1.

12. The vaccine composition according to claim 11, which is for preventing dengue virus infection, severe fever with thrombocytopenia syndrome, or influenza virus infection.

13. (a) mixing an amphiphilic peptide into an aqueous solution to form a micellar structure; and (b) mixing a target peptide and electrically binding it to the surface of the formed micellar structure.

14. 14. The method for producing nanoparticles according to claim 13, wherein the amphiphilic peptide is a peptide represented by the following general formula I: [General formula I] CnXm Here, C is a charged amino acid, X is a hydrophobic amino acid, n is an integer from 1 to 5, and m is an integer from 3 to 10.

15. The method for producing nanoparticles according to claim 13, wherein the amphipathic peptide and the targeting peptide in step (b) are mixed in a weight ratio of 1:5 to 5:

1.

16. The method for producing nanoparticles according to claim 13 , wherein the target peptide is a hapten peptide.

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