KR101831483B1 - MHC-ferritin conjugates, a nanoparticle comprising the same and the use thereof - Google Patents

Abstract

The present invention relates to MHC (Major Histocompatibility Complex) peptides and conjugates of ferritin, nanoparticles comprising the same, and uses thereof.

Description

MHC-ferritin conjugates, nanoparticles containing the same, and uses thereof [0002] MHC-ferritin conjugates, a nanoparticle comprising the same and the use thereof,

The present invention relates to MHC (Major Histocompatibility Complex) peptides and conjugates of ferritin, nanoparticles comprising the same, and uses thereof.

CD8 ⁺ T cells play a major cellular immune response in adaptive immunity and are the main defense immune system to protect the host against viruses and intracellular bacterial infections as well as various cancers. In addition, CD8 ⁺ T cells specifically recognize foreign substances, and antigen recognition of these T cells is caused by T cell receptors (TCR) expressed on the cell surface. However, an antigen presenting cell that not only recognizes a foreign substance that has invaded a living body, that is, an antigen itself, but which presents antigenic peptide fragments together with MHC molecules by digesting the antigen and treating the antigen, for example, dendritic cells dendritic cell), and activated by the TCR stimulation.

Therefore, in order to prevent or treat disease, antigen-specific CD8 ⁺ T cells were appropriately activated and amplified. Up to now, a method of using an antigen-presenting antigen-derived cell to amplify antigen-specific T cells has been mainly used, There were many limitations. Under these circumstances, development of an artificial antigen presenting cell or an artificial antigen presenting cell or complex (aAPC) capable of effectively activating and amplifying antigen-specific CD8 ⁺ T cells is required.

Ferritin is a protein that stores iron and is widely found in prokaryotes and eukaryotes. The molecular weight of ferritin is about 500,000 Da, which is composed of heavy chain and light chain. It has self-assembly ability and shows unique characteristics to form spherical particles. Ferritin is a protein in which 24 monomers (a single monomer or a monomer composed of one of the heavy chain or light chain) are assembled to form a huge spherical tertiary structure. In the case of human ferritin, the outer diameter is about 12 nm and the inner diameter is about 8 nm. Ferritin may be dispersed into monomers depending on pH conditions and may form nanoparticles with 24 monomers combined, which can capture various substances in ferritin.

For example, ferritin was synthesized using silver-binding peptides (Kramer, RM, Li, C .; Carter, DC; Stone, MO; and Naik, RR, Gd-HPDOTA (gadolinium- [10- (2-hydroxypropyl) -1,4,7,10-tetraazacyclododecane-1,4, 7-triacetic acid] has also been reported (Aime, S .; Frullano, L .; and Crich, SG, (2002) Angew. Chem. Int.

As described above, ferritin conjugated with a metal has been used, but there has been no report on the use of it as a synthetic antigen-presenting cell and a vaccine by fusion with a MHC (Major Histocompatibility Complex) peptide.

Accordingly, the present inventors have made intensive efforts to produce an artificial antigen presenting complex that is efficient and safe. As a result, it has been found that a nanoparticle prepared using a MHC (Major Histocompatibility Complex) -peritin conjugate and an antigen peptide Specific T cell can be effectively stimulated and amplified. Thus, it was confirmed that the homozygous aggregate can be used as a therapeutic agent for a disease that requires amplification of T cell-based vaccine and T cell, such as cancer , Thereby completing the present invention.

An object of the present invention is to provide nanoparticles containing 24 combinations of MHC (Major histocompatibility complex) receptor protein and ferritin protein.

Another object of the present invention is to provide a method for promoting T cell proliferation in vitro, comprising the step of reacting the isolated T cells with the nanoparticles.

It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating T cell related diseases selected from the group consisting of cancer, acquired immunodeficiency syndrome and hepatitis C, which comprises the nanoparticle.

It is another object of the present invention to provide a combination of an MHC receptor protein and a ferritin protein.

Yet another object of the present invention is to provide a polynucleotide encoding said conjugate.

It is still another object of the present invention to include a recombinant expression vector comprising the polynucleotide.

It is still another object of the present invention to provide a transformed cell comprising the polynucleotide or the recombinant expression vector.

Yet another object of the present invention is to provide a method for producing nanoparticles comprising 24 complexes of an MHC receptor protein and a ferritin protein, comprising the step of mixing said complex and said antigen peptide.

To achieve the above object, the present invention provides nanoparticles containing 24 combinations of MHC (Major histocompatibility complex) receptor protein and ferritin protein.

In the present invention, nanoparticles prepared by assembling 24 assemblies of MHC receptor protein and ferritin protein were prepared, and it was confirmed that the nanoparticles thus prepared could function as an artificial antigen presenting cell. Specifically, in the present invention, when an antigen peptide is bound to MHC of nanoparticles formed of 24 MHC protein and ferritin protein complexes and reacted with T cells, the amplification of T cells specific to the antigen peptide presented on the nanoparticle is effectively induced . Therefore, the nanoparticles of the present invention can be used for the purpose of reacting T cells separated from a patient with the nanoparticles and then amplifying the T cells and administering them again to the patient. However, the antigen-specific T Direct administration to an individual in need of cell amplification can result in effective proliferation of antigen-specific T cells. Therefore, compared with the conventional dendritic cell vaccine treatment method in which the dendritic cells are isolated and amplified, and then the amplified dendritic cells are again administered to the individual to amplify the T cells, The present invention can be advantageous in that the amplification effect of the antigen-specific T cells can be easily achieved only by the administration of the antigen-specific T cells.

In the present invention, the term "a combination of an MHC receptor protein and a ferritin protein" refers to a combination of an MHC receptor protein and a ferritin protein by a chemical or recombinant method. Such a conjugate is preferably in a connected form so that the antigen presenting ability of the MHC receptor protein and the self-assembly ability of the ferritin protein are not inhibited. The binding of the MHC receptor protein and the ferritin protein may be chemically linked, or two or more polypeptides may be linked by an enzymatic action, or two or more different polypeptides may be expressed in a single polypeptide through manipulation of the gene, I. E., In the form of a fusion protein, but are not limited thereto.

For example, the conjugate may be prepared by techniques well known in the art, for example, peptide synthesis or genetic engineering, and may be particularly efficiently produced by genetic engineering methods. A genetic engineering method is a method of expressing a desired protein in a large amount in a host cell such as E. coli by gene manipulation, and the technology related thereto is described in detail in a well-known document (Molecular Biotechnology: Principle and Application of Recombinant DNA; ASM Press: 1994, J. Chem. Technol. Biotechnol., 1993, 56, 3-13). In the present invention, a polynucleotide sequence encoding an MHC receptor protein and a conjugate of a ferritin protein is inserted into an appropriate expression vector, transformed into an appropriate host cell, and then the host cell is cultured By weight.

Specifically, the binding body of the MHC receptor protein and the ferritin protein of the present invention may be a polynucleotide sequence of SEQ ID NO: 6 or an amino acid sequence of SEQ ID NO: 7, but is not limited thereto.

In the present invention, the term " MHC (Major Histocompatibility Complex) receptor protein " refers to a main histocompatibility complex and plays a role in presenting a part of a protein, e.g., an epitope, in a cell. Since the function of these MHCs can discriminate whether or not they are their own cells through the presented antigen, the antigen bound to the main histocompatibility complex can be recognized by T cells. T cells are antigen-specific immune cells that act in response to specific antigen signals. Unlike B cells, they are characterized by recognizing MHC-bound antigens rather than reacting with free or soluble forms of the antigen.

It plays an important role in determining whether a particular cell is its own or an external cell. T cells are antigen-specific immune cells that act in response to specific antigen signals. B cells are also antigen-specific, but unlike B cells, T cells do not react with antigens in free or soluble form and recognize antigens bound to the cast matrix complex (MHC).

There are three types of MHC, I, II and III, among which MHC classes (class) I and II are mainly involved in antigen presentation. CD4 ⁺ T cells interact with MHC class II mainly to have a helper phenotype, and CD8 ⁺ T cells interact with MHC class I to have cytotoxic T cell characteristics.

Specifically, the MHC receptor protein may be any one of MHC class I or II, but belongs to MHC class I Peptide, more specifically MHC class IH-2K ^{b &lt}; RTI ID = 0.0 ^> Peptide, and most specifically, it may be composed of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

In the present invention, the term "ferritin protein" refers to a protein that can be used as a constituent component of a biologically-derived nanostructure, and that 24 monomers can self-assemble. In the vertebrate animal, the ferritin monomer includes a light type having a molecular weight of about 19 kDa and a heavy type having a molecular weight of about 21 kDa. In the present invention, the type of the ferritin monomer is not particularly limited, L-type ferritin monomers, H-type ferritin monomers, or both may be used to produce nanoparticles. When both the L-type ferritin monomer and the H-type ferritin monomer are used, all of the nanoparticles mainly composed of the N-type ferritin monomer or the H-type ferritin monomer can be produced. The sequence of the ferritin protein can be easily obtained from a known database such as the NCBI GenBank, and includes, for example, the amino acid sequence of SEQ ID NO: 1, but is not particularly limited thereto.

In addition, in the example of the present invention, the MHC class I H-2K ^b gene was ligated to the 5 'end of the ferritin gene. However, if the ferritin monomer is not limited to self assembly and aggregation, have.

According to one embodiment of the present invention, the nanoparticle comprising the binding substance may be in the form of binding an antigen peptide to an MHC receptor protein of the binding substance constituting the binding substance.

In the present invention, the term "antigenic peptide" refers to a substance capable of stimulating an animal's immune system to induce the production of an antibody. For the purpose of the present invention, the antigenic peptide refers to a substance capable of binding to MHC of the nanoparticle according to the present invention and inducing an antibody thereto. The antigenic peptide may be an MHC receptor May be designed with size and / or sequence capable of binding to the protein.

In addition, the antigenic peptide may be an epitope of an antigenic protein, and can bind to MHC class I or II molecules with moderate efficiency, and may be an amino acid capable of stimulating T cells or binding to T cells in complex with MHC class I or II Sequence. ≪ / RTI > In addition, it plays a role in providing a signal required for B cells to differentiate into antibody producing cells, and can induce lytic cells of target cells by inducing cytotoxic T cells. In particular, the antigen peptides in the present invention are not limited to specific ones that stimulate T cells and enhance the immune response, and it is possible to use proteins, peptides, and epitopes suitable for the purpose. For example, the hepatitis B surface antigen helper T cell epitope, Chlamydia tachycardia trachomitis) major outer membrane protein helper T cell epitopes, plastic modium eight Shifa standing room keomseu prisoners ZH (Plasmodium falciparum circumsporozoite ) helper T cell epitope, Escherichia coli) TraT helper T cell epitopes, Te tanuseu toxoid (Tetanus toxoid) helper T cell epitopes, diphtheria toxoid (diphtheria toxoid) helper T cell epitope, SH testosterone soma only Sony (Schistosoma mansoni ) triose phosphate isomerase helper T cell epitope, measles virus F protein helper T cell epitope, pertussis vaccine, BCG (Bacile Calmette-Guerin), polio vaccine, mumps vaccine including epitopes derived from mumps vaccines, rubella vaccines, rabies vaccines, purified protein derivatives of tuberculin, keyhole limpet hemocyanin, fragments or combinations thereof, and the like. can do.

The present invention also relates to various melanoma-related antigen peptides, ovarian cancer-related antigen peptides, breast cancer-related antigen peptides, lung cancer-related antigen peptides, leukemia-related antigen peptides, multiple myeloma-associated peptides, etc. disclosed in the art as antigen peptides capable of binding antigen- An antigen peptide, a lymphoma-related antigen peptide, and a prostate cancer-associated antigen peptide.

Specifically, MART-1, tyrosinase, gp100, NY-ESO-1, MUC-1, CA-125, Her-2, survivin, telomerase, CAMEL, CEA, livin, 1, SSX-2, PRAME, C-Lectin, Pec60, AES, MAGE-3, G250, FBP, SSX-4, SP17, hTRT, MUC-16, MAGE-1, Topoisomerase II II, Integrin 8 subunit precursor, MUC-1, MAGE-B2, STAT 1, γ-catenin or H-RYK.

(SEQ ID NO: 10), ALALAALLVV (SEQ ID NO: 12), ALLVVDREV (SEQ ID NO: 13), YMNGTMSQV (SEQ ID NO: 14), YMDGTMSQV (SEQ ID NO: 15), ITDQVPFSV (SEQ ID NO: 16), KMASRSMRL ), YLEPGPVTA (SEQ ID NO: 17), AAGIGILTV (SEQ ID NO: 18), ELAGIGILTV (SEQ ID NO: 19), CLTSTVQLV (SEQ ID NO: 20), HLYQGCQVV (SEQ ID NO: 21), KIFGSLAFL SEQ ID NO: 24), VLVKSPNHV (SEQ ID NO: 26), ELVSEFSRM (SEQ ID NO: 27), YLSGANLNL (SEQ ID NO: 28), GPLTPLPV (SEQ ID NO: 29), SLLMWITQC (SEQ ID NO: 32), GLQSPKSPL (SEQ ID NO: 33), VLLKLRRPV (SEQ ID NO: 34), ELYIPSVDL (SEQ ID NO: 35), SLLMWITQV (SEQ ID NO: 36), ILAKFLHWL (SEQ ID NO: 37), STAPPVHNV (SEQ ID NO: 38), FLWGPRALV (SEQ ID NO: 39), FMWGNLTLA (SEQ ID NO: 40), RLVDDFLLV (SEQ ID NO: 41), HLSTAFARV (SEQ ID NO: 42), QLSLLMWIT (SEQ ID NO: 43), ELWTHSYKV (SEQ ID NO: 44), KVAELVHFL (SEQ ID NO: 45), YIFATCLGL (SEQ ID NO: 46), HLYIFATCL (SEQ ID NO: 47), MLMAQEALAFL (SEQ ID NO: 48), STLEKINKT 50, SLLMWITQCFL, ELTLGEFLKL, LTLGEFLKL, SLLEKREKT, TLGEDDPWL, KLGLKPLEV, YLWTSAKNT, SEQ ID NO: ), STAPPAHGV (SEQ ID NO: 58), GMGSEELRL (SEQ ID NO: 59), SLGSPVLGL (SEQ ID NO: 60), YLFFYRKSV (SEQ ID NO: 61), CQQEETFLL (SEQ ID NO: 62), TLAKFSPYL (SEQ ID NO: 63), NLTHVLYPV (SEQ ID NO: 65), SLDQHLIGL (SEQ ID NO: 66), FLDQRVFFV (SEQ ID NO: 67), FLDQRVFVV (SEQ ID NO: 68), FLDQVAFVV (SEQ ID NO: 69), GLDREQLYL (SEQ ID NO: 70), VMQHLLSPL SEQ ID NO: 72), LQPLSGPGL (SEQ ID NO: 73), TLDRDSLYV (SEQ ID NO: 74), QLYLELSQL (SEQ ID NO: 75), KVLEYVIKV (SEQ ID NO: 76), KVADLVGFL ), KTWGQYWQV (SEQ ID NO: 78) and VLDGLDVLL (SEQ ID NO: 79).

In one embodiment of the present invention, a peptide of egg albumin (OVA) protein is used as a typical antigen peptide. The type of antigen peptide bound to the nanoparticle of the present invention according to the antigen-specific T cell to be amplified can be determined by a person skilled in the art And can be selected or devised appropriately. It is not particularly limited to the above example.

The MHC receptor protein and the ferritin protein may be directly connected to each other through a peptide bond or a disulfide bond, or may be connected to each other through a linker in the binding body constituting the nanoparticle.

In the present invention, the term "linker" basically refers to two different fusion partners (for example, biological polymers, etc.) with hydrogen bonding, electrostatic interactions, Van der Waals forces, disulfide bonds, Hydrophobic interaction, covalent bonding, or the like. Can have at least one cysteine capable of participating in at least one disulfide bond, preferably under physiological conditions or other standard peptide conditions (e. G., Peptide purification conditions, peptide storage conditions) In addition to the role of hinge, it is also possible to perform a role of providing a gap of a certain size between the fusion partners or as a hinge which provides flexibility or rigidity to the combination. The linker may be a non-peptide linker or a peptide linker, but is not limited thereto.

The term "non-peptide linker" in the present invention refers to a linker that is not in the form of a peptide, specifically a biocompatible linker in which two or more repeating units are bonded. The repeating units may be connected to each other through any covalent bond, not a peptide bond.

Non-peptide linkers that can be used in the present invention include polyethyleneglycol (PEG) homopolymers, polypropylene glycol homopolymers, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohols, polysaccharides, Polyvinyl ethyl ether, biodegradable polymers, lipid polymers, chitins, hyaluronic acid, or a combination thereof. Derivatives thereof which are already known in the art and derivatives which can be easily prepared in the state of the art are included in the scope of the present invention.

In the present invention, the term "peptide linker" refers to a peptide that serves to link two substances together. The peptide linker may be a peptide that is inserted between a protein and a protein such that the structural flexibility of the protein can be increased or the activity of each protein can be enhanced. The peptide linker is not limited in length and / or sequence so far as it does not inhibit the activity of each protein to be bound and does not cause an unnecessary immune response, but specifically includes 1 to 20 amino acids, more specifically 1 to 5 amino acids But it is not limited thereto.

In the present invention, a GGGGS (G ₄ S) linker known to have flexibility and resistance to protease was used. The G ₄ S linker is a linker in which GGGGS is a repeating unit, for example, GGGGS may be repeated three times, but is not limited thereto.

More specifically, the nanoparticles according to the present invention are characterized in that they can function as an artificial antigen presenting complex.

In general, antigen presenting cells (APCs) express MHC-expressing antigens on T cells, and T cells recognize MHC-presenting antigens.

Specifically, TCR stimulation is transmitted to T cells through interaction between CD8 ⁺ T cells and antigen presenting cells, which are antigen-presenting cells that present antigen-presenting peptide fragments together with MHC molecules, . That is, in order to stimulate and proliferate T cells, it is necessary to present antigens bound to MHC. In the present invention, it was confirmed that a nanoparticle can be prepared using a binding substance in which an MHC receptor protein and a ferritin protein are bound so that an antigen peptide binds to MHC, and can be provided as an artificial antigen-presenting complex for inducing a T cell response.

The nanoparticles according to the present invention can exhibit an efficient and safe effect as compared with conventional self-derived antigen-presenting cells.

In addition, if the nanoparticle is formed, the binding site of the ferritin protein and the MHC receptor protein is not particularly limited. However, the MHC receptor protein may be linked to the N- or C-terminal of the ferritin protein through a linker or not. Specifically, the MHC receptor protein may be connected to the N-terminus of the ferritin protein, and the MHC receptor protein may be connected through the linker. However, it is not limited to the above-mentioned position, and may be inserted anywhere inside the ferritin and connected to the outside if the self-assembly of the ferritin is not hindered.

In another embodiment, the invention provides a method of promoting T cell proliferation in vitro, comprising the step of reacting the isolated T cells with the nanoparticles.

In the example of the present invention, the MHC peptide was fused with ferritin to prepare a fusion protein, and MHC / antigen peptide-ferritin nanoparticles were prepared by self-assembly while mixing antigen peptides. The nanoparticles stimulated antigen-specific T cells (Fig. 6). Accordingly, the nanoparticle of the present invention can induce the proliferation of antigen-specific T cells by stimulating antigen-specific T cells and can be used as an artificial antigen-presenting complex, so that it reacts with isolated T cells both in the body and in vitro T cell proliferation can be promoted.

In another embodiment, the present invention provides a pharmaceutical composition for enhancing immunity comprising the nanoparticle. Specifically, the composition may be in the form of a vaccine.

In particular, the nanoparticle according to the present invention can be used as a T cell-based vaccine because it promotes proliferation of T cells.

In addition, by including the nanoparticle, the present invention provides a pharmaceutical composition for immune enhancement that solves all of the problems of toxicity of a live bacterial vaccine and the problem of a dead bacteria vaccine having low antigenicity, .

The pharmaceutical composition for enhancing immunity of the present invention may further comprise a pharmaceutically acceptable carrier, suitable adjuvant and other conventional substances, and may be administered in an immunological effective amount. The term "amount of immunological effect" as used herein means an amount sufficient to exhibit an immunopotentiating effect and an amount not causing side effects or serious or excessive immune response, and the precise dosage level depends on the specific immunogen to be administered And can be determined by a person skilled in the art using known methods in order to examine the occurrence of an immune response. It may also be varied depending on the mode and route of administration, the age, health and weight of the recipient, the nature and severity of symptoms, the type of current therapy, and the number of treatments.

The carrier may contain stabilizers, diluents, and buffers, which are well known in the art. Suitable stabilizers include carbohydrates such as sorbitol, lactose, mannitol, starch, sugar, dextran and glucose; Proteins such as albumin or casein, and the like. Suitable diluents may include salts, Hanks balanced salts, Ringer's solution, and the like. Suitable buffer solutions include alkali metal phosphates, alkali metal carbonates, alkaline earth metal carbonates and the like. Vaccines may also include one or more immunoadjuvants to improve or enhance the immune response. Examples of suitable adjuvants for the immunoadjuvant may include aluminum hydroxide, fraud complete or incomplete adjuvant, DEAE dextran, levamisole, PCG and poly I: C or poly A: U. The vaccine composition of the present invention can be administered through a known administration route. Such methods include, but are not limited to, oral, transdermal, muscular, peritoneal, intravenous, subcutaneous, and nasal passages, and may be administered by any device capable of migrating the active agent to the target cell.

In the example of the present invention, the growth of cancer cells was markedly delayed by promoting proliferation of T cells when MHC / OVA _{257 -264} -ferritin nanoparticle was administered (FIG. 8), and it was observed that the survival rate was high 9). From the experimental results, it was confirmed that the nanoparticles can be used for the prevention or treatment of diseases requiring T cell proliferation, particularly AIDS and hepatitis C virus.

Accordingly, another embodiment of the present invention relates to a pharmaceutical composition for preventing or treating a T cell-related disease selected from the group consisting of cancer, acquired immunodeficiency syndrome and hepatitis C, comprising the nanoparticle.

Specifically, the cancer diseases include cancer of the liver, brain, kidney, pancreas, testicular cancer, ovarian cancer, lung cancer, head and neck cancer, bladder cancer, stomach cancer, uterine cancer, cervical cancer, breast cancer, thyroid cancer, Gallbladder cancer, skin cancer, oral cancer, or prostate cancer. However, the present invention shows the effect of preventing or treating cancer due to immune enhancement. The present invention is not limited to the above cancer diseases.

Specifically, acquired immune deficiency syndrome (AIDS) refers to a disease in which the CD4 ⁺ T cells are destroyed by infection with human immunodeficiency virus (HIV), and the immune system is weakened. The nanoparticles of the present invention induce the proliferation of antigen-specific T cells, and can be used for the prophylactic or therapeutic use of acquired immunodeficiency syndrome, in which the immunity is weakened by deficiency of T cells.

Specifically, hepatitis C refers to a disease in which inflammation of the liver occurs due to the immune response of the body in response to infection with hepatitis C virus (HCV). Unlike hepatitis B, the vaccine is developed And immunoglobulin is also absent. The nanoparticle of the present invention can be used to prevent or treat hepatitis C by directly stimulating T cells to amplify the immune response.

The pharmaceutical composition may be applied to an experimental animal such as a mouse, a rabbit, a rat, a guinea pig, or a hamster or a primate including a human, but is not limited thereto, and is preferably applied to a primate including a human, .

Depending on the use of the pharmaceutical composition of the present invention and the method of use, the content of the nanoparticle as an active ingredient may be appropriately adjusted according to the choice of a person skilled in the art, and may be used alone or in combination with other pharmacologically acceptable carriers, excipients , A diluent or a subcomponent.

Examples of the pharmaceutically acceptable carrier, excipient or diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, propylhydroxybenzoate, talc, magnesium stearate and mineral oil , Dextrin, calcium carbonate, propylene glycol, liquid paraffin, and physiological saline. However, it is not limited to these, and any conventional carrier, excipient or diluent may be used. In addition, the pharmaceutical composition may further comprise at least one selected from the group consisting of conventional fillers, extenders, binders, disintegrants, anticoagulants, lubricants, humectants, pH adjusters, nutrients, vitamins, electrolytes, alginic acid and its salts, pectic acid and its salts, Glycerin, fragrance, emulsifier or preservative, and the like.

The pharmaceutical composition may be administered either orally or parenterally. For example, the pharmaceutical composition may be administered through various routes including oral, transdermal, subcutaneous, intravenous, or muscular. In addition, the formulations of the compositions may vary depending on the method of use and may be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal . In general, solid dosage forms for oral administration include tablets, pills, soft or hard capsules, pills, powders and granules, which may contain one or more Excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, Sweeteners, fragrances, preservatives, and the like. Forms for parenteral administration include creams, looses, ONITMENTS, PLASTERS, LIQUIDS AND SOULTIONS, AEROSOLS, FRUIDEXTRACTS, Or may be in the form of an isotonic solution (ELIXIR), INFUSIONS, SACHET, PATCH or INJECTIONS, and in case of injectable form, it may preferably be in the form of an isotonic aqueous solution or suspension .

The pharmaceutical composition may further contain other therapeutically useful substances such as sterilizing agents, preservatives, stabilizers, wetting or emulsifying accelerators, adjuvants such as salts and / or buffers for controlling osmotic pressure, and the like, Or coating method, and may be formulated using any other appropriate method known in the art.

In yet another embodiment, the present invention relates to a combination of an MHC receptor protein and a ferritin protein. Specifically, the conjugate may be in the form of a fusion protein. In one embodiment of the present invention, Helicobacter pylori ) ferritin gene at the 5 'end of the MHC class I H-2K ^b Genes were cloned, but the MHC receptor protein and ferritin protein complexes can be linked by chemical means other than recombinant methods, which are genetic engineering means.

In yet another embodiment, the present invention relates to a polynucleotide encoding the above-described conjugate. The polynucleotide may be DNA or RNA, and when the polynucleotide of the present invention is RNA, it can be understood that T (thymine) of DNA is replaced with uracil (U). The polynucleotide can be produced by a known chemical synthesis method.

In another embodiment, the invention relates to a recombinant expression vector comprising said polynucleotide.

&Quot; Expression vector " means a gene construct comprising an essential regulatory element operatively linked to the expression of a gene insert (the polynucleotide), which is capable of expressing a desired protein or RNA of interest in a suitable host cell.

The vector of the present invention includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector. Suitable expression vectors include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoter, operator, initiation codon, termination codon, polyadenylation signal and enhancer, and can be prepared variously according to the purpose. The promoter of the vector may be constitutive or inducible. The expression vector may also include a selection marker for selecting a host cell containing the vector and, if the expression vector is a replicable vector, a replication origin. The signal sequence may include a PhoA signal sequence or an OmpA signal sequence when the host is Escherichia coli, an α-amylase signal sequence or a subtilisin signal sequence when the host is a Bacillus sp. Strain, an MFα signal sequence when the host is yeast, SUC2 signal sequence and the like. When the host is an animal cell, insulin signal sequence, alpha-interferon signal sequence, antibody molecule signal sequence, and the like can be used, but the present invention is not limited thereto.

In another embodiment, the present invention relates to a polynucleotide or a cell transformed with a recombinant vector comprising said polynucleotide.

Transformation includes any method of introducing a polynucleotide, and can be carried out by selecting a suitable standard technique depending on the host cell as is known in the art. Such methods include electroporation, protoplast fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation with silicon carbide fibers, agrobacteria-mediated transformation, PEG (polyethylene glycol) Dextran sulfate, lipofectamine, particle bombardment, and the like.

The transformed bacterium was transformed into Escherichia coli coli), Bacillus subtilis (Bacillus subtilis), Streptomyces (Streptomyces), Pseudomonas (Pseudomonas), Proteus Mira Billy's (Proteus mirabilis), Staphylococcus (Staphylococcus), Agrobacterium Tome Pacific Enschede (Agrobacterium tumefaciens ). < / RTI >

In another embodiment, the present invention relates to a method for producing nanoparticles comprising 24 complexes of MHC (Major histocompatibility complex) receptor protein and ferritin protein, comprising the steps of mixing a complex and an antigen peptide .

Specifically, it may include a step of mixing a complex of an MHC I receptor protein and a ferritin protein, an antigenic peptide, and beta-2-microglobulin. Β2-microglobulin (β2M) is an immune protein that is expressed as a minor component of MHC class I receptor proteins in nucleated cells. The MHC class I receptor protein is a duplex of a-chain and beta microglobulin, and beta-2-microglobulin serves as an immobilized MHC class I receptor protein.

In the role of beta-2-microglobulin as described above, any protein that can stabilize the MHC class I receptor protein or stabilize the structure may be used without limitation.

The fusion protein and nanoparticle of the present invention can be used to stimulate T cell proliferation to enhance the immune response. Therefore, the fusion protein and the nanoparticle can be utilized in the development of a vaccine for immunity enhancement comprising the same, Can be treated.

1 is a schematic diagram of a pET-23a-H-2K ^b -ferritin expression vector cloned into the pET-23a vector system.
FIG. 2 shows the result of SDS-PAGE of the H-2K ^b -ferritin fusion protein present in an inclusion body state.
FIG. 3 is a schematic view showing a process of preparing and purifying nanoparticles by mixing H-2K ^b -ferritin fusion protein and beta-2-microglobulin.
Fig. 4 shows the result of checking the purified nanoparticles.
FIG. 5 shows an electron microscope (TEM) photograph of the nanoparticles produced.
Shows a schematic diagram of experiments to determine the antigen-specific T cells after administration of ferritin nanoparticle-6 is MHC / OVA _{257 -264} as an artificial antigen-presenting complex.
FIG. 7 shows the ratio of antigen-specific T cells on days 7, 9, and 12 after MHC / OVA _{257 -264} -ferritin nanoparticles treated with nothing as a control group and MHC / H60 _{39 -46} -ferritin nanoparticle treatment Lt; / RTI >
FIG. 8 shows the results of _analysis of MHC / OVA _{257 -264} -ferritin nanoparticles and antigen-specific T cell FACS on days 7, 9, and 12 after MHC / H60 _{39 -46} -ferritin nanoparticle treatment as a control will be.
9 is an artificial antigen-presenting complex MHC / OVA as _{257 -264-shows} a schematic diagram of an experiment for comparing the proliferative effect of the ferritin nanoparticle and H-2K ^b / OVA _257-264 specific T cells, the tetramers antigen after each administration will be.
FIG. 10 is a graph comparing the proliferation effects of antigen-specific T cells after administration of 10 μg and 50 μg of MHC / OVA _{257 -264} -ferritin nanoparticles and H-2K ^b / OVA _{257 -264} tetramer, respectively .
FIG. 11 is a graph showing that almost no antigen-specific T cells were observed in the non-administration group and the MHC / H60 _{39 -46} -ferritin nanoparticle-treated group.
FIG. 12 shows the results of confirming the growth of cancer cells in a mouse to which MHC / OVA _{257 -264} -ferritin nanoparticles or H-2K ^b / OVA _{257 -264} tetramer was administered.
13 is a MHC / OVA _{257 -264} - a graph showing the survival rate of ferritin nanoparticle or H-2K ^b / OVA _{257 -264} the tetramer, each administered mice.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  One. MHC - Ferritin Of the fusion protein  Production and refining

Helicobacter the 5 'end of pylori) a ferritin (ferritin derived from bacteria) gene (SEQ ID NO: 1), (GGGGS) ₃ linker (SEQ ID NO: 3) were connected and amplified through PCR in the C57BL / 6 mouse MHC class Ⅰ H-2K was inserted into the BamHI and XhoI sites of the pET-23a vector system, which could be expressed in E. coli , along with the ^b (SEQ ID NO: 2) gene through cloning.

SEQ ID NO: direction The primer sequence (5'-3 ') 4 Forward 5'-GGA TCC GGC GGC GGC GGC AGC GGC GGC GGC GGC AGC GGC GGC GGC GGC AGC atg tta tca aaa gac atc-3 ' 5 Reverse 5'-CTC GAG TCA TTA AGA TTT CCT-3 '

The plasmid prepared by the cloning was transformed into competent cells and then overexpressed using 1 mM IPTG at 37 ° C for 4 hours.

Bacterial cells were collected and sonicated in solution buffer (50 mM TrisHCl, 25% sucrose, 1 mM NaEDTA, 0.1% NaAzide, 10 mM DTT, pH 8.0) Thereafter, the reaction was performed by adding lysozyme, DNase I, MgCl ₂ , lysis buffer (50 mM TrisHCl, 1% Triton X-100, 0.1% Na deoxycholate, 100 mM NaCl, 0.1% NaAzide, 10 mM DTT, pH 8.0) ) Was centrifuged at 11,000 g for 20 minutes and washed with a washing buffer (50 mM Tris-HCl, 0.5% Triton X-100, 100 mM NaCl, 1 mM NaEDTA, 0.1% NaAzide, 1 mM DTT, pH 8.0) containing Triton X- To release the inclusion body. To remove Triton X-100, the pellet was centrifuged at 11,000 g for 20 minutes to precipitate the pellet. The pellet was washed with a washing buffer (50 mM TrisHCl, 100 mM NaCl, 1 mM NaEDTA, 0.1% NaAzide, 1 mM DTT , pH 8.0). The pellet precipitated by centrifugation at 11,000 g for 20 min was dissolved in 8 M urea (25 mM MES, 8 M Urea, 10 mM NaEDTA, 0.1 mM DTT, pH 8.0), transferred to an ultracentrifugation tube, rotor) for 20 minutes at 48,100 rpm to finally obtain the supernatant as fusion protein. The final purified fusion proteins were identified by 10% SDS-PAGE (FIG. 2) and stored at -80 ° C. in divided portions. The gene sequence and amino acid sequence of the prepared H-2K ^b -ferritin fusion protein are shown in SEQ ID NO: 6 and SEQ ID NO: 7, respectively.

Example  2. MHC / Antigen Peptides - Ferritin 24- mer Nanoparticle ( homopolymer ) And MHC / antigen peptide Tetramer  making

By the over-expression in E. coli obtained fusion protein MHC (H-2K ^b) - ferritin heavy chain (heavy chain) and the beta-2-microglobulin (beta-2- microglobulin) the H-2K present in a high concentration ^b - restricted peptide (H the -2K ^b -restricted peptide) with refolding buffer (refolding buffer, 100mM Tris-HCl , 400mM L-arginine-HCl, 2mM NaEDTA, 0.5mM oxid. glutathione, 5mM red. glutathione) folding (folding in), with MHC / Antigen peptide-ferritin fusion protein nanoparticles were prepared.

OVA _257-264 (SIINFEKL, SEQ ID NO: 8) which is a well-known T cell epitope of ovalbumin with H-2K ^b -restricted peptide was used as a control and H60 _{39 -46} (LTFNYRNL, SEQ ID NO: 9) To prepare nanoparticles. And then purified using gel filtration chromatography (Figures 3 and 4).

H-2K ^b / OVA _{257 -264} monomer (monomer) was also made in the same way, the produced _{^{H-2K b / OVA 257 -264}} - ferritin nanoparticle and H-2K ^b / OVA _{257 -264} method size exclusion purification of a monomer (monomer) (size exclusion purification) of Superdex-75 fast performance liquid chromatography and then purified via (FPLC) was used. The monomer is biotinylated with a biotin-protein ligase BirA, d-biotin and ATP to a biotinylation site at the C-terminus of the heavy chain, After the gel filtration, the purified monomer was reacted with PE-conjugated streptavidin or PE-free streptavidin at a molar ratio of 10: 1 to prepare a tetramer.

Example  3. Manufactured Of nanoparticles  Transmission electron microscope ( Transmission electron  microscope, TEM )

3 μl of the purified MHC / antigen peptide-ferritin fusion protein nanoparticle solution was placed on an EM grid and stained with 3 ul of 2% acetic acid uranyl acetate solution, dried in air and observed with a transmission electron microscope (TEM) , A nanometer-sized (approximately 27 nm) spherical shape considered to be an MHC / antigen peptide-ferritin fusion protein nanoparticle was observed (FIG. 5).

Example  4. MHC / Antigen Peptides - Ferritin Of nanoparticles OVA  Identification of the proliferative effect of specific T cells

MHC / antigen peptide-ferritin nanoparticles as an artificial antigen-presenting complex were tested for the ability to stimulate and proliferate specific T cells against OVA antigen. OVA antigen-specific T cells in the splenocytes of OT1 transgenic mice were administered to the tail vein And one day later, MHC / antigen peptide-ferritin nanoparticles were administered (FIG. 6). Example 2 of the MHC / OVA _{257 -264} produced by a ferritin nanoparticle-the MHC / antigen peptide of ferritin nanoparticles were used.

Specifically, OVA antigen-specific T cells in the splenocytes of OT1 transgenic mice were injected into the tail vein of female C57BL / 6 mice in an amount of ⁵ × 10 ⁵ cells / mouse to increase the number of T cells capable of specifically reacting with OVA in the state after a day MHC / OVA _{257 -264} - ferritin nanoparticle and H-2Kb / OVA _{257 -264} tetramers were administered to the respective tail vein by 100㎍, as a control group not administered with any group MHC / H60 _{39 - 46} - ferritin nanoparticles were used.

OVA-specific T cells in the blood obtained from the Retro-Orbital plexus were measured by tetramer staining on days 7, 9, and 12 after administration, and the results were shown in FIG. 7 Respectively.

On the 7th day after administration, MHC / OVA ₂₅₇ - ₂₆₄ - ferritin nanoparticle group showed more antigen - specific T cells than the other two control groups. On the 9th day and 12th day after the administration, the decrease was observed as compared with the 7th day, and both the 7th, 9th, and 12th days of the control group showed a low value without a large change.

From these results, it was confirmed that MHC / antigen peptide-ferritin nanoparticle stimulates and proliferates T cells specifically to the antigen.

Example  5. OVA  Identification of antigen-specific cytotoxic T cells

The MHC / OVA _{257 -264} prepared in Example 2-ferritin nanoparticle and the control of MHC / H60 _39-46 - after administration of the ferritin nanoparticle or 10ug 50ug the tail vein of female C57BL / 6 mice, mice in 7 day after the blood obtained from the orbital jeongmaekchong washed with FACS buffer (0.5% FBS, 0.09% NaN 3 in PBS), purified rat anti-mouse CD16 / CD32 (BD biosciences, California , USA) and a streptavidin 5㎍ / ㎖ Tabividin (Invitrogen) was reacted on ice for 20 minutes. Washed with FACS buffer and stained by reacting with FITC-conjugated anti-CD44, PE-conjugated OVA257-264 / Kb tetramer, PE-Cy5-conjugated anti-CD8 and antigen presenting cell (APC) -conjugated anti-CD45 . After incubation with FACS lysing solution for 20 min at room temperature, red blood cells were dissolved and fixed with FACS Calibur cytometer (BD Bioscience, SanDiego, Calif.). Then, CD8 ⁺ cells were collected and a dot plot was plotted on the Y-axis as tetramer and the X-axis as CD44, and the double positive cells were regarded as activated OT-1 cells. Flow cytometry was performed using Flowjo software (TreeStar Inc., Ashalend, OR, USA).

Administration, and after 7 days, MHC / OVA _{257 -264} compared to the control - was found to be significantly plenty of OT-1 cell activation when administered to ferritin nanoparticle (FIG. 8), through a result, MHC / antigen Peptide-ferritin nanoparticles stimulated and proliferated T cells specifically for the antigen.

Example 6. Comparison of the effect of MHC / antigen peptide-ferritin nanoparticles and MHC / antigen peptide tetramer on the proliferation of antigen-specific T cells

In order to compare the T cell stimulation efficiency of MHC / antigen peptide-ferritin nanoparticles and MHC / antigen peptide tetramer as an artificial antigen-presenting complex, MHC / antigen peptide-ferritin nanoparticle and MHC / antigen peptide fusion protein tetramer MHC / OVA _257-264 -ferritin nanoparticles and MHC / OVA _257-264 tetramer prepared in Example 2 were used.

Specifically, OVA antigen-specific T cells in the splenocytes of OT1 transgenic mice were injected into the tail vein of female C57BL / 6 mice in an amount of ⁵ × 10 ⁵ cells / mouse to increase the number of T cells capable of specifically reacting with OVA in the state after a day MHC / OVA _{257 -264} - ferritin nanoparticle and H-2Kb / OVA _{257 -264} the tetramer into the tail vein 10㎍, was administered by 50㎍, the control group is the group with MHC / H60 not administered anything _{39 -46} -ferritin nanoparticle (Fig. 9).

On the seventh day after the administration, OVA-specific T cells present in the blood obtained from the Retro-Orbital plexus were measured using tetramer staining and compared. The results are shown in FIGS. 9 and 10 .

As a result, there was no difference when 10 μg of each of MHC / OVA _{257 -264} -ferritin nanoparticle and H-2K ^b / OVA _{257 -264} tetramer was administered, but when 50 μg was administered, MHC / OVA _{257 -264} -ferritin nanoparticle, it was confirmed that more OVA antigen-specific T cells were present (FIG. 10). In the control group, there was almost no OVA antigen-specific T cells (FIG. 11).

These results indicate that MHC / antigen peptide-ferritin nanoparticles can stimulate and proliferate antigen-specific T cells more effectively than MHC tetramers when administered over 50 μg. From these results, it can be seen that the nanoparticles formed from the MHC-ferritin conjugate of the present invention can be used as an artificial antigen-presenting complex.

Example  7. MHC / Antigen Peptides - Ferritin Of nanoparticles  Confirming Cancer Cell Growth Inhibitory Effect

Example 2 a MHC / antigen peptide prepared in-ferritin increase of OVA antigen specific T cells due to a nanoparticle in order to ensure that the cytotoxicity that can inhibit the growth of cancer cells present, MHC / OVA _{257 -264} - ferritin nano particles, HC / H60 _{39 -46} - ferritin nanoparticle and was administered into the tail vein of H-2K ^b / OVA _{257 -264} tetramers in an amount of each 10㎍ or 50㎍, to generate solid tumors for 19 days after the second dose 1 x ¹⁰⁶ cells of EG7 tumor cells were subcutaneously administered. As a result, MHC / OVA _{257 -264} - For the treated group, the ferritin nanoparticle, HC / H60 _39-46 - ferritin case of administration of the nanoparticle, or administration of a H-2K ^b / OVA _{257 -264} tetramers, nothing It was confirmed that the growth of cancer cells was significantly delayed as compared with the mice not administered (Fig. 12). In addition, the cancer cell sizes in the mouse was determined to have died, MHC / OVA _{257 -264} when more than 200mm - showed ferritin nanoparticle or H-2K ^b / OVA mice administered _257-264 tetramers are high viability (Fig. 13).

These results MHC / OVA _{257 -264} - will show that ferritin was administered when the nano-particles, this by the OVA antigen specific T cell growth The growth of the cancer cells by the cytotoxic according suppressed can exhibit a protective effect on cancer .

In summary, the above results show that the MHC / antigen peptide-ferritin nanoparticles according to the present invention can be used as an artificial antigen-presenting complex for stimulating antigen-specific T cells in vivo, Such as cancer, acquired immunodeficiency syndrome (HIV), or hepatitis C virus (HCV).

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> Ewha university-Industry Collaboration Foundation <120> MHC-ferritin conjugates, a nanoparticle comprising the same and          the use thereof <130> KPA140383-KR <160> 79 <170> Kopatentin 2.0 <210> 1 <211> 507 <212> DNA <213> Helicobacter pylori <220> <221> gene &Lt; 222 > (1) <223> ferritin <400> 1 atgttatcaa aagacatcat taagttgcta aacgaacaag tgaataagga aatgaactct 60 tccaacttgt atatgagcat gagttcatgg tgctataccc atagcttaga tggcgcgggg 120 cttttcttgt ttgaccatgc ggctgaagaa tacgagcatg ctaaaaagct tattatcttc 180 ttgaatgaaa acaatgtgcc tgtgcaattg accagcatca gcgcgcctga gcataagttt 240 gaaggtttga ctcaaatttt ccaaaaagcc tatgaacatg agcaacacat cagcgagtct 300 attaacaata tcgtagatca cgccataaaa agcaaagatc atgcgacttt caatttcttg 360 caatggtatg tggctgaaca gcatgaagaa gaagtgcttt tcaaggatat tttggataaa 420 attgagttga ttggtaatga aaaccatggc ttgtatttag ccgatcagta tgtcaaaggg 480 atcgctaaaa gcaggaaatc ttaatga 507 <210> 2 <211> 280 <212> PRT <213> Artificial Sequence <220> <223> H-2Kb peptide <400> 2 Met Gly Pro His Ser Leu Arg Tyr Phe Val Thr Ala Val Ser Arg Pro   1 5 10 15 Gly Leu Gly Glu Pro Arg Tyr Met Glu Val Gly Tyr Val Asp Asp Thr              20 25 30 Glu Phe Val Arg Phe Asp Ser Asp Ala Glu Asn Pro Arg Tyr Glu Pro          35 40 45 Arg Ala Arg Trp Met Glu Gln Glu Gly Pro Glu Tyr Trp Glu Arg Glu      50 55 60 Thr Gln Lys Ala Lys Gly Asn Glu Gln Ser Phe Arg Val Asp Leu Arg  65 70 75 80 Thr Leu Leu Gly Tyr Tyr Asn Gln Ser Lys Gly Gly Ser His Thr Ile                  85 90 95 Gln Val Ile Ser Gly Cys Glu Val Gly Ser Asp Gly Arg Leu Leu Arg             100 105 110 Gly Tyr Gln Gln Tyr Ala Tyr Asp Gly Cys Asp Tyr Ile Ala Leu Asn         115 120 125 Glu Asp Leu Lys Thr Trp Thr Ala Ala Asp Met Ala Ala Leu Ile Thr     130 135 140 Lys His Lys Trp Glu Gln Ala Gly Glu Ala Glu Arg Leu Arg Ala Tyr 145 150 155 160 Leu Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Lys Asn Gly                 165 170 175 Asn Ala Thr Leu Leu Arg Thr Asp Ser Pro Lys Ala His Val Thr His             180 185 190 His Ser Arg Pro Glu Asp Lys Val Thr Leu Arg Cys Trp Ala Leu Gly         195 200 205 Phe Tyr Pro Ala Asp Ile Thr Leu Thr Trp Gln Leu Asn Gly Glu Glu     210 215 220 Leu Ile Gln Asp Met Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly 225 230 235 240 Thr Phe Gln Lys Trp Ala Ser Val Val Val Pro Leu Gly Lys Glu Gln                 245 250 255 Tyr Tyr Thr Cys His Val Tyr His Gln Gly Leu Pro Glu Pro Leu Thr             260 265 270 Leu Arg Trp Glu Pro Pro Ser Ser         275 280 <210> 3 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> (GGGGS) 3 linker <400> 3 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser   1 5 10 15 <210> 4 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 4 ggatccggcg gcggcggcag cggcggcggc ggcagcggcg gcggcggcag catgttatca 60 aaagacatc 69 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 5 ctcgagtcat taagatttcc t 21 <210> 6 <211> 1398 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide sequence of H-2Kb-ferritin fusion protein <400> 6 atgggtccac actcgctgag gtatttcgtc accgccgtgt cccggcccgg cctcggggag 60 ccccggtaca tggaagtcgg ctacgtggac gacacggagt tcgtgcgctt cgacagcgac 120 gcggagaatc cgagatatga gccgcgggcg cggtggatgg agcaggaggg gcccgagtat 180 tgggagcggg agacacagaa agccaagggc aatgagcaga gtttccgagt ggacctgagg 240 accctgctcg gctactacaa ccagagcaag ggcggctctc acactattca ggtgatctct 300 ggctgtgaag tggggtccga cgggcgactc ctccgcgggt accagcagta cgcctacgac 360 ggctgcgatt acatcgccct gaacgaagac ctgaaaacgt ggacggcggc ggacatggcg 420 gcgctgatca ccaaacacaa gtgggagcag gctggtgaag cagagagact cagggcctac 480 ctggagggca cgtgcgtgga gtggctccgc agatacctga agaacgggaa cgcgacgctg 540 ctgcgcacag attccccaaa ggcccatgtg acccatcaca gcagacctga agataaagtc 600 accctgaggt gctgggccct gggcttctac cctgctgaca tcaccctgac ctggcagttg 660 aatggggagg agctgatcca ggacatggag cttgtggaga ccaggcctgc aggggatgga 720 accttccaga agtgggcatc tgtggtggtg cctcttggga aggagcagta ttacacatgc 780 cacgtgtacc atcaggggct gcctgagccc ctcaccctga gatgggagcc tcctccatcc 840 ggatccggcg gcggcggcag cggcggcggc ggcagcggcg gcggcggcag catgttatca 900 aaagacatca ttaagttgct aaacgaacaa gtgaataagg aaatgaactc ttccaacttg 960 tatatgagca tgagttcatg gtgctatacc catagcttag atggcgcggg gcttttcttg 1020 tttgaccatg cggctgaaga atacgagcat gctaaaaagc ttattatctt cttgaatgaa 1080 aacaatgtgc ctgtgcaatt gaccagcatc agcgcgcctg agcataagtt tgaaggtttg 1140 actcaaattt tccaaaaagc ctatgaacat gagcaacaca tcagcgagtc tattaacaat 1200 atcgtagatc acgccataaa aagcaaagat catgcgactt tcaatttctt gcaatggtat 1260 gtggctgaac agcatgaaga agaagtgctt ttcaaggata ttttggataa aattgagttg 1320 attggtaatg aaaaccatgg cttgtattta gccgatcagt atgtcaaagg gatcgctaaa 1380 agcaggaaat cttaatga 1398 <210> 7 <211> 464 <212> PRT <213> Artificial Sequence <220> <223> H-2Kb-ferritin fusion protein <400> 7 Met Gly Pro His Ser Leu Arg Tyr Phe Val Thr Ala Val Ser Arg Pro   1 5 10 15 Gly Leu Gly Glu Pro Arg Tyr Met Glu Val Gly Tyr Val Asp Asp Thr              20 25 30 Glu Phe Val Arg Phe Asp Ser Asp Ala Glu Asn Pro Arg Tyr Glu Pro          35 40 45 Arg Ala Arg Trp Met Glu Gln Glu Gly Pro Glu Tyr Trp Glu Arg Glu      50 55 60 Thr Gln Lys Ala Lys Gly Asn Glu Gln Ser Phe Arg Val Asp Leu Arg  65 70 75 80 Thr Leu Leu Gly Tyr Tyr Asn Gln Ser Lys Gly Gly Ser His Thr Ile                  85 90 95 Gln Val Ile Ser Gly Cys Glu Val Gly Ser Asp Gly Arg Leu Leu Arg             100 105 110 Gly Tyr Gln Gln Tyr Ala Tyr Asp Gly Cys Asp Tyr Ile Ala Leu Asn         115 120 125 Glu Asp Leu Lys Thr Trp Thr Ala Ala Asp Met Ala Ala Leu Ile Thr     130 135 140 Lys His Lys Trp Glu Gln Ala Gly Glu Ala Glu Arg Leu Arg Ala Tyr 145 150 155 160 Leu Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Lys Asn Gly                 165 170 175 Asn Ala Thr Leu Leu Arg Thr Asp Ser Pro Lys Ala His Val Thr His             180 185 190 His Ser Arg Pro Glu Asp Lys Val Thr Leu Arg Cys Trp Ala Leu Gly         195 200 205 Phe Tyr Pro Ala Asp Ile Thr Leu Thr Trp Gln Leu Asn Gly Glu Glu     210 215 220 Leu Ile Gln Asp Met Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly 225 230 235 240 Thr Phe Gln Lys Trp Ala Ser Val Val Val Pro Leu Gly Lys Glu Gln                 245 250 255 Tyr Tyr Thr Cys His Val Tyr His Gln Gly Leu Pro Glu Pro Leu Thr             260 265 270 Leu Arg Trp Glu Pro Pro Ser Gly Ser Gly Gly Gly Gly Ser Gly         275 280 285 Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Leu Ser Lys Asp Ile Ile     290 295 300 Lys Leu Leu Asn Glu Gln Val Asn Lys Glu Met Asn Ser Ser Asn Leu 305 310 315 320 Tyr Met Ser Ser Ser Trp Cys Tyr Thr His Ser Leu Asp Gly Ala                 325 330 335 Gly Leu Phe Leu Phe Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys             340 345 350 Lys Leu Ile Ile Phe Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr         355 360 365 Ser Ile Ser Ala Pro Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe     370 375 380 Gln Lys Ala Tyr Glu His Glu Gln His Ile Ser Glu Ser Ile Asn Asn 385 390 395 400 Ile Val Asp His Ala Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe                 405 410 415 Leu Gln Trp Tyr Val Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys             420 425 430 Asp Ile Leu Asp Lys Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu         435 440 445 Tyr Leu Ala Asp Gln Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser     450 455 460 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> OVA257-264 peptide <400> 8 Ser Ile Ile Asn Phe Glu Lys Leu   1 5 <210> 9 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> H60 39-46 peptide <400> 9 Leu Thr Phe Asn Tyr Arg Asn Leu   1 5 <210> 10 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 10 Ser Ile Leu Ser Leu Lys Glu Ala Ser Thr   1 5 10 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 11 Lys Met Ala Ser Arg Ser Met Arg Leu   1 5 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 12 Ala Leu Ala Leu Ala Ala Leu   1 5 10 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 13 Ala Leu Leu Val Val Asp Arg Glu Val   1 5 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 14 Tyr Met Asn Gly Thr Met Ser Gln Val   1 5 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 15 Tyr Met Asp Gly Thr Met Ser Gln Val   1 5 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 16 Ile Thr Asp Gln Val Pro Phe Ser Val   1 5 <210> 17 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 17 Tyr Leu Glu Pro Gly Pro Val Thr Ala   1 5 <210> 18 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 18 Ala Ala Gly Ile Gly Ile Leu Thr Val   1 5 <210> 19 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 19 Glu Leu Ala Gly Ile Gly Ile Leu Thr Val   1 5 10 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 20 Cys Leu Thr Ser Thr Val Gln Leu Val   1 5 <210> 21 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 21 His Leu Tyr Gln Gly Cys Gln Val Val   1 5 <210> 22 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 22 Lys Ile Phe Gly Ser Leu Ala Phe Leu   1 5 <210> 23 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 23 Ile Ile Ser Ala Val Val   1 5 <210> 24 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 24 Pro Leu Thr Ser Ile Ile Ser Ala Val   1 5 <210> 25 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 25 Val Met Ala Gly Val Gly Ser Pro Tyr Val   1 5 10 <210> 26 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 26 Val Leu Val Lys Ser Pro Asn His Val   1 5 <210> 27 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 27 Glu Leu Val Ser Glu Phe Ser Arg Met   1 5 <210> 28 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 28 Tyr Leu Ser Gly Ala Asn Leu Asn Leu   1 5 <210> 29 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 29 Gly Pro Leu Thr Pro Leu Pro Val   1 5 <210> 30 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 30 Ser Leu Leu Met Trp Ile Thr Gln Cys   1 5 <210> 31 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 31 Lys Ala Leu Phe Ala Gly Pro Pro Val   1 5 <210> 32 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 32 Tyr Leu Glu Thr Phe Arg Glu Gln Val   1 5 <210> 33 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 33 Gly Leu Gln Ser Pro Lys Ser Pro Leu   1 5 <210> 34 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 34 Val Leu Leu Lys Leu Arg Arg Pro Val   1 5 <210> 35 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 35 Glu Leu Tyr Ile Pro Ser Val Asp Leu   1 5 <210> 36 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 36 Ser Leu Leu Met Trp Ile Thr Gln Val   1 5 <210> 37 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 37 Ile Leu Ala Lys Phe Leu His Trp Leu   1 5 <210> 38 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 38 Ser Thr Ala Pro Pro Val His Asn Val   1 5 <210> 39 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 39 Phe Leu Trp Gly Pro Arg Ala Leu Val   1 5 <210> 40 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 40 Phe Met Trp Gly Asn Leu Thr Leu Ala   1 5 <210> 41 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 41 Arg Leu Val Asp Asp Phe Leu Leu Val   1 5 <210> 42 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 42 His Leu Ser Thr Ala Phe Ala Arg Val   1 5 <210> 43 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 43 Gln Leu Ser Leu Leu Met Trp Ile Thr   1 5 <210> 44 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 44 Glu Leu Trp Thr His Ser Tyr Lys Val   1 5 <210> 45 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 45 Lys Val Ala Glu Leu Val His Phe Leu   1 5 <210> 46 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 46 Tyr Ile Phe Ala Thr Cys Leu Gly Leu   1 5 <210> 47 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 47 His Leu Tyr Ile Phe Ala Thr Cys Leu   1 5 <210> 48 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 48 Met Leu Met Ala Gln Glu Ala Leu Ala Phe Leu   1 5 10 <210> 49 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 49 Ser Thr Leu Glu Lys Ile Asn Lys Thr   1 5 <210> 50 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 50 Lys Ala Ser Glu Lys Ile Phe Tyr Val   1 5 <210> 51 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 51 Ser Leu Leu Met Trp Ile Thr Gln Cys Phe Leu   1 5 10 <210> 52 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 52 Glu Leu Thr Leu Gly Glu Phe Leu Lys Leu   1 5 10 <210> 53 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 53 Leu Thr Leu Gly   1 5 <210> 54 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 54 Ser Leu Leu Glu Lys Arg Glu Lys Thr   1 5 <210> 55 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 55 Thr Leu Gly Asp Asp Pro Trp Leu   1 5 <210> 56 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 56 Lys Leu Gly Leu Lys Pro Leu Glu Val   1 5 <210> 57 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 57 Tyr Leu Trp Thr Ser Ala Lys Asn Thr   1 5 <210> 58 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 58 Ser Thr Ala Pro Pro Ala His Gly Val   1 5 <210> 59 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 59 Gly Met Gly Ser Glu Glu Leu Arg Leu   1 5 <210> 60 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 60 Ser Leu Gly Ser Pro Val Leu Gly Leu   1 5 <210> 61 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 61 Tyr Leu Phe Phe Tyr Arg Lys Ser Val   1 5 <210> 62 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 62 Cys Gln Gln Glu Glu Thr Phe Leu Leu   1 5 <210> 63 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 63 Thr Leu Ala Lys Phe Ser Pro Tyr Leu   1 5 <210> 64 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 64 Asn Leu Thr His Val Leu Tyr Pro Val   1 5 <210> 65 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 65 Ser Thr Phe Lys Asn Trp Pro Phe Leu   1 5 <210> 66 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 66 Ser Leu Leu Gln His Leu Ile Gly Leu   1 5 <210> 67 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 67 Phe Leu Asp Gln Arg Val Phe Phe Val   1 5 <210> 68 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 68 Phe Leu Asp Gln Arg Val Phe Val Val   1 5 <210> 69 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 69 Phe Leu Asp Gln Val Ala Phe Val Val   1 5 <210> 70 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 70 Gly Leu Asp Arg Glu Gln Leu Tyr Leu   1 5 <210> 71 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 71 Val Met Gln His Leu Leu Ser Pro Leu   1 5 <210> 72 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 72 Gln Gln Thr His Gly Ile Thr Arg Leu   1 5 <210> 73 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 73 Leu Gln Pro Leu Ser Gly Pro Gly Leu   1 5 <210> 74 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 74 Thr Leu Asp Arg Asp Ser Leu Tyr Val   1 5 <210> 75 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 75 Gln Leu Tyr Leu Glu Leu Ser Gln Leu   1 5 <210> 76 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 76 Lys Val Leu Glu Tyr Val Ile Lys Val   1 5 <210> 77 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 77 Lys Val Ala Asp Leu Val Gly Phe Leu   1 5 <210> 78 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 78 Lys Thr Trp Gly Gln Tyr Trp Gln Val   1 5 <210> 79 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antigen peptide <400> 79 Val Leu Asp Gly Leu Asp Val Leu Leu   1 5

Claims (17)

Nanoparticles containing 24 MHC (major histocompatibility complex) receptor proteins, antigen peptides, and ferritin protein complexes,
Wherein the antigenic peptide is bound to an MHC receptor protein and the MHC receptor protein is linked to the N-terminus of ferritin.
delete 2. The nanoparticle of claim 1, wherein the MHC receptor protein and the ferritin protein of the conjugate are linked together by a linker.
The nanoparticle of claim 1, wherein the nanoparticle is capable of functioning as an artificial antigen presenting complex.
delete 2. The nanoparticle of claim 1, wherein the MHC receptor protein is MHC class I.
A method for promoting proliferation of T cells in vitro, comprising the step of reacting isolated T cells with the nanoparticles of any one of claims 1, 3, 4 and 6.
A pharmaceutical composition for enhancing immunity comprising nanoparticles according to any one of claims 1, 3, 4 and 6.
9. The composition of claim 8, wherein the composition is in the form of a vaccine.
A pharmaceutical composition for preventing or treating a T cell related disease selected from the group consisting of cancer, acquired immunodeficiency syndrome and hepatitis C, comprising the nanoparticles of any one of claims 1, 3, 4 and 6 Gt;
As a combination of an MHC receptor protein, an antigenic peptide, and a ferritin protein,
Wherein said antigenic peptide is bound to an MHC receptor protein and said MHC receptor protein is linked to the N-terminus of ferritin.
12. The conjugate of claim 11, wherein the conjugate is in the form of a fusion protein.
A polynucleotide encoding the combination of claim 12.
14. A recombinant expression vector comprising the polynucleotide of claim 13.
A transformed cell comprising the polynucleotide of claim 13 or the recombinant expression vector of claim 14.
A method for producing nanoparticles comprising 24 complexes of MHC (Major histocompatibility complex) receptor protein and ferritin protein, comprising the step of mixing MHC receptor protein, antigen peptide and ferritin protein,
Wherein said antigenic peptide is bound to an MHC receptor protein and said MHC receptor protein is linked to the N-terminus of ferritin.
17. The method of claim 16,
Wherein the method comprises admixing a complex of an MHC I receptor protein and a ferritin protein, an antigenic peptide, and beta-2-microglobulin.

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