WO2012144716A1 - Nanoparticle-based vaccine delivery system having double functions of imaging and delivery - Google Patents
Nanoparticle-based vaccine delivery system having double functions of imaging and delivery Download PDFInfo
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- WO2012144716A1 WO2012144716A1 PCT/KR2011/009553 KR2011009553W WO2012144716A1 WO 2012144716 A1 WO2012144716 A1 WO 2012144716A1 KR 2011009553 W KR2011009553 W KR 2011009553W WO 2012144716 A1 WO2012144716 A1 WO 2012144716A1
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- antigen
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Definitions
- the present invention relates to nanoparticle-based vaccine carriers capable of imaging and delivery.
- Vaccines are the most cost-effective medicines that can prevent and reduce the incidence of up to 99% of diseases, as well as therapeutic effects.
- the use of vaccines is not limited to infectious diseases, but has been widened to various intractable diseases including cancer and autoimmune diseases, and vaccine development is recognized as very important as therapeutic vaccines are introduced.
- cancer vaccines are a novel therapeutic vaccine that destroys cancer cells by inducing a powerful immune response by artificially activating an immune mechanism that specifically acts on cancer cells (eg, introducing cells into antigens).
- APCs antigen-presenting cells
- DC dendritic cells
- the vaccine is useful for immunizing an individual against a target antigen, such as a pathogen antigen or an antigen associated with a cell involved in human disease.
- a target antigen such as a pathogen antigen or an antigen associated with a cell involved in human disease.
- Cell-associated antigens involved in human disease include cancer-associated tumor antigens and antigens associated with autoimmune disease-related cells.
- vaccines that produce target antigens in the cells of vaccinated individuals are effective in inducing cellular arms of the immune system.
- attenuated live vaccines, recombinant vaccines using non-toxic vectors, and DNA vaccines both induce antigen production in cells of vaccinated individuals, leading to cellular mineralization of the immune system.
- subunit vaccines containing only proteins and dead or inactivated vaccines, which induce humoral responses do not induce a good cellular immune response.
- the inventors have sought to develop a vaccine carrier capable of performing both imaging and vaccine delivery.
- a vaccine carrier capable of performing both imaging and vaccine delivery.
- antigens capable of inducing an immune response to gold nanoparticles or magnetic nanoparticles they show excellent performance as vaccine carriers, enable selective transport to lymph nodes, and also provide computed tomography (CT) imaging.
- CT computed tomography
- MRI magnetic resonance imaging
- the present invention has been completed by finding that vaccines based on gold nanoparticles or magnetic nanoparticles successfully achieve immune activation by antigen in lymph nodes and therapeutic effects by immune activity.
- Another object of the present invention is to provide a vaccine pharmaceutical composition for anticancer.
- the present invention provides a composition comprising: (a) gold nanoparticles or magnetic nanoparticles as imaging-carrying bifunctional particles having an imaging and transporting action; And (b) a vaccine delivery system comprising an antigen bound to the surface of the imaging-carrying bifunctional particle, wherein the vaccine delivery system is capable of delivering the antigen and tracing the antigen delivery. It provides a vaccine delivery, characterized in that to enable.
- the inventors have sought to develop a vaccine carrier capable of performing both imaging and vaccine delivery.
- a vaccine carrier capable of performing both imaging and vaccine delivery.
- antigens capable of inducing an immune response to gold nanoparticles or magnetic nanoparticles
- they show excellent performance as vaccine carriers, enable selective transport to lymph nodes, and also provide computed tomography (CT) imaging.
- CT computed tomography
- MRI magnetic resonance imaging
- vaccines based on gold nanoparticles or magnetic nanoparticles successfully achieve immune activation by antigen in lymph nodes and therapeutic effects by immune activity.
- the present invention is to induce as a vaccine delivery system that combines the antigen on the surface of the gold nanoparticles, or magnetic nanoparticles, in vitro (in virto) as well, not just in vivo (in vivo) highly effective immune response in a (e. G., Antibody production) Vaccine carriers.
- vaccine used to describe a vaccine carrier in the present invention means a substance used for automatically immunizing a human or animal, and typically, there is a dead virus vaccine, an attenuated vaccine, an autologous vaccine and a multivalent vaccine.
- nanoparticle means a particle having a size of 1-800 nm, preferably 1-100 nm.
- the nanoparticles used in the present invention have a diameter in nano units, preferably 5-100 nm, more preferably 5-50 nm, and most preferably 12-14 nm.
- Eggplant means nanoparticles.
- This small size facilitates the penetration of the nanoparticles of the invention into cells of interest (eg, immune cells) or immune cell tissue, allowing the penetration of vaccine carriers into cells.
- the present invention comprising such a configuration contributes to the accurate delivery of antigen and induction of immune activation (eg, antibody production) upon antigen delivery in a vaccine delivery vehicle.
- Nanoparticles used in the present invention are gold nanoparticles or magnetic nanoparticles.
- Gold nanoparticles are easy to manufacture in the form of stable particles, are easy to adjust in size, and unlike other heavy metals such as manganese, aluminum, cadmium, lead, mercury, cobalt, nickel and beryllium, they are highly biocompatible. .
- the gold nanoparticles used in the present invention can be prepared, for example, as follows: HAuCl 4 is used as a gold source, and sodium citrate is used as a reducing agent to reduce HAuCl 4 to prepare gold nanoparticles.
- the size of the gold nanoparticles can be adjusted by varying the citrate added. That is, the size of the gold nanoparticles decreases as the amount of citrate increases, so that nucleation increases.
- the gold nanoparticles are larger than 100 nm in diameter, their properties as nanoparticles are greatly reduced, and the binding of functional groups such as the thiol group and the gold surface without the nanomaterial properties is weak. This bound particle is difficult to manufacture.
- Magnetic nanoparticles used in the present invention include any magnetic nanoparticles known in the art.
- the magnetic nanoparticles usable in the present invention are paramagnetic nanoparticles or superparamagnetic nanoparticles, most preferably superparamagnetic signal generating cores.
- Exemplary paramagnetic nanoparticles suitable for the present invention include stable free radicals (eg, stable nitroxides), transition elements, lanthanides, and actinides. Preferred elements are Gd (III), Mn (II), Cu (II), Cr (III), Fe (II), Fe (III), Co (II), Er (II), Ni (II), Eu (III) and Dy (III).
- Exemplary supercrystalline nanoparticles suitable for the present invention include ferro- or ferrimagnetic compounds, such as pure iron, magnetic iron oxides (eg magnetite, Fe 3 O 4 ), ⁇ -Fe 2 O 3 , Manganese ferrite, cobalt ferrite and nickel ferrite.
- ferro- or ferrimagnetic compounds such as pure iron, magnetic iron oxides (eg magnetite, Fe 3 O 4 ), ⁇ -Fe 2 O 3 , Manganese ferrite, cobalt ferrite and nickel ferrite.
- antigens There are many different types of antigens, and if they are foreign to the individual, in principle everyone recognizes them as antigens. Proteins, polysaccharides, nucleic acids, lipids, and complexes thereof are called natural antigens.
- hapten as hapten (adhesive) or phosphorus airport agent. Molecules vary in size, ranging from peptide chains of amino acids to hundreds of thousands of molecules, and viruses, bacteria, and animal cells.
- the antigen may be covalently or non-covalently bound to the nanoparticle surface, preferably covalently bound.
- the antigen can be bound directly or indirectly (eg, via a linker) to the nanoparticles.
- the antigen included in the vaccine carrier of the present invention comprises a microorganism-derived antigen, a virus-derived antigen, a parasite-derived antigen, a plant-derived antigen, an animal-derived antigen, an endogenous antigen and a synthetic antigen.
- a microorganism-derived antigen e.g., a virus-derived antigen, a parasite-derived antigen, a plant-derived antigen, an animal-derived antigen, an endogenous antigen and a synthetic antigen.
- One or more antigens selected from the group a microorganism-derived antigen, a virus-derived antigen, a parasite-derived antigen, a plant-derived antigen, an animal-derived antigen, an endogenous antigen and a synthetic antigen.
- the present invention not only facilitates the production and secretion of antibodies and cytokines involved in the immune response from lymphocytes when the antigen is bound to the surface of an imaging-carrying bifunctional particle and administered to a subject (eg, mammalian oil). It is a vaccine carrier that can exert excellent anticancer efficacy by significantly reducing the size of cancer cells.
- the antigen bound to the imaging-carrying bifunctional particle in the vaccine delivery vehicle of the present invention is a microbe-derived antigen, it preferably comprises a bacterial bacteria-derived antigen, a fungi-derived antigen or a mold-derived antigen.
- the antigen bound to the imaging-carrying bifunctional particle in the vaccine delivery system of the present invention is a virus-derived antigen, an antigen derived from human immunodeficiency virus (HIV), an antigen derived from human Papilloma viruses (HPV), an influenza virus derived Antigens, herpes virus derived antigens, hepatitis virus derived antigens or encephalitis virus derived antigens.
- HIV human immunodeficiency virus
- HPV human Papilloma viruses
- influenza virus derived Antigens derived from human immunodeficiency virus
- herpes virus derived antigens herpes virus derived antigens
- hepatitis virus derived antigens or encephalitis virus derived antigens.
- the antigen bound to the imaging-carrying bifunctional particle in the vaccine delivery vehicle of the present invention is a plant-derived antigen or an animal-derived antigen, it preferably includes an antigen that causes allergy.
- the antigen bound to the imaging-carrying bifunctional particle in the vaccine delivery agent of the present invention is an endogenous antigen, it preferably includes a cancer cell antigen, a cancer causing antigen or an autoimmune disease causing antigen.
- the antigen bound to the imaging-carrying bifunctional particle in the vaccine delivery agent of the present invention is a synthetic (artificial) antigen, it preferably includes a drug antigen.
- the antigen bound to the surface of the imaging-carrying bifunctional particle in the vaccine carrier of the present invention may be bound to molecules of various forms and properties.
- the antigen bound to the surface of the imaging-carrying bifunctional particle in the vaccine carrier of the invention is a single stranded oligonucleotide or polynucleotide, a double stranded oligonucleotide or At least one antigen selected from the group consisting of polynucleotides, proteins, polypeptides, oligopeptides, lipids, lipoproteins, glycolipids, glycoproteins, proteoglycans, polysaccharides and lipopolysaccharides, more preferably polypeptides, One or more antigens selected from the group consisting of oligopeptides, proteins, lipoproteins and glycoproteins, even more preferably polypeptides or proteins.
- adjuvant used in expressing a vaccine carrier in the present invention refers to a substance called an adjuvant or an adjuvant, and more specifically, a lot of antibodies can be generated by the immune system by increasing the response to the vaccine. It means a substance to make.
- the adjuvant is primarily involved in innate immune, but dendritic cells and macrophages that are involved in endogenous immunity secrete chemokines and only cells involved in endogenous immunity. Rather, even the cells involved in adaptive immune are called into the infected area, so the adjuvant also engages in acquired immunity, leading to memory immunity. In addition, by adding an adjuvant to recognize that the immune system is infected with bacteria, dendritic cells, lymphocytes and macrophages recognize the components of the bacteria and are activated to activate the innate immunity.
- the present invention may further comprise an adjuvant or an immunomodulator to effectively enhance immune activity.
- the present invention further comprises an adjuvants.
- the adjuvant is preferably bound to the surface of the antigen or imaging-carrying bifunctional particle, more preferably, is covalently bound to the surface of the antigen or imaging-carrying bifunctional particle, even more preferred. Preferably it is covalently bonded to the surface of the imaging-carrying bifunctional particle.
- the immunoadjuvant additionally included in the vaccine carrier of the present invention in the present invention may be combined molecules of various forms and properties.
- the adjuvant in the present invention CpG oligodeoxynucleotide, unmethylated cystein-phosphate-guanine DNA (DCP), double-stranded RNA, microorganism-derived DNA or RNA, nucleic acid derivatives, Lipids, lipopolysaccharides, lipoproteins, lipopeptides, glycolipids, peptidoglycans, glycopeptides, proteins, recombinant proteins, flagellin, virosomes, Ribi (monophosphoryl-lipid A / trehalose dicorynomycolate) And one or more immunoadjuvant selected from the group consisting of saponins and squalene squalene, nucleic acid derivatives, aluminum salts, calcium salts, complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA), more preferably Preferably CpG oligodeoxynucleotides, unmethylated cystein-phosphate-guanine
- the vaccine carrier of the present invention selectively delivers antigen to lymph nodes.
- Lymph nodes are a major defense against infection and also act as a pathway in the metastasis of malignant tumors. Therefore, the selective delivery of the antigen to the lymph nodes by the vaccine carrier of the present invention shows that the vaccine carrier of the present invention can induce an immune response very effectively in vivo.
- the delivery vehicle of the vaccine of the present invention can be traced by CT imaging or MRI imaging.
- CT imaging or MRI imaging is performed after a predetermined time, it is possible to confirm whether the vaccine delivery agent of the present invention is properly transferred to the lymph nodes. This feature also greatly increases the usefulness of the vaccine carrier of the present invention.
- the invention provides a composition comprising: (a) gold nanoparticles or magnetic nanoparticles as imaging-carrying bifunctional particles for imaging and conveying action; And (b) provides a vaccine pharmaceutical composition for cancer comprising a cancer antigen bound to the surface of the imaging-carrying bifunctional particles.
- the vaccine pharmaceutical composition for anticancer in the present invention is a composition comprising the vaccine carrier, the common content between the two is omitted in order to avoid excessive complexity of the present specification.
- Cancer antigens used in the pharmaceutical compositions of the present invention are P91A, p53, p21 ras , P210, BTA, P198, P1A, gp100, TAG-72, PSMA, G250, Her-2 / neu, CTKA-4, hTERT, VEGF, VEGF-A, MART-1-4, BAGE 1-3, melan-A (MART-1 (Melanoma Antigen Recognized by T cells)), SSX-2, SSX-4, mucin, MAGE-1, MAGE-2, MAGE-3, NY-ESO-1, LAGE, carcinoembryonic antigen (CEA), PRAME, mesothelin, PLK1, GP100 (PMel17), GAGE-1, PSA, PSCA, SAGE and SCP-1 It is not.
- the pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier.
- Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen.
- the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.
- a lubricant e.g., talc, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, a kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mann
- the pharmaceutical composition of the present invention may be administered orally or parenterally, and is preferably applied by parenteral administration.
- Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. Typical dosages of the pharmaceutical compositions of the invention are in the range of 0.001-100 mg / kg on an adult basis.
- compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container.
- the formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.
- the vaccine carrier of the present invention effectively induces an immune response because it selectively transports antigens to the lymph nodes, and moreover, since the lymph nodes play a role in the metastasis of malignant tumors, the anticancer vaccine composition of the present invention is a cancer Very effective in the treatment of
- the present invention provides a vaccine carrier comprising a gold nanoparticle and an antigen, and a vaccine pharmaceutical composition for anticancer.
- the present invention shows little toxicity and side effects on in vivo and can induce high antibody production even when used with antigens with low antigenicity.
- the present invention can significantly induce and enhance humoral mediated immune responses (Th1) and cell mediated immune responses (Th2) in vivo by effectively delivering and presenting antigens to immune cells and enhancing these effects. Through this, it is possible to prevent and treat immunity to antigens causing various diseases and diseases.
- the vaccine carrier of the present invention selectively delivers antigen to lymph nodes. Due to this property, the vaccine carrier of the present invention can induce an immune response very effectively in vivo.
- Figure 1a-1c is a result of measuring the size of the GNP, GNP-RFP and GNP-CpG-RFP in the present invention using an ELS 8000 device.
- the size of nanoparticles after GNP, GNP-RFP, and GNP-CpG-RFP administration was confirmed to be 7.3 nm, 13.7 nm, and 24.3 nm, respectively.
- Figure 2 is a result of measuring the content of gold nanoparticles in the local leaf nodes after administration of GNP-RFP prepared in the present invention to C57BL / 6 mice. Over time, it was found that the content of GNP-RFP increased in local lymph nodes (superficial groin lymph nodes and popliteal lymph nodes).
- Figures 3a-3b was confirmed by silver staining after the administration of GNP-RFP to C57BL / 6 mice by silver staining and also by immunostaining which immune cells capture gold nanoparticles.
- cytokines are markedly expressed in C57BL / 6 mouse immune cells mixed with GNP-RFP or GNP-CpG-RFP.
- Figure 6 in the present invention administered negative control (PBS), RFP, positive control (Alum-RFP), GNP-RFP and GNP-CpG-RFP three times a week intervals (12 ⁇ g antigen / mouse) to C57BL / 6 mice
- the expression pattern of RFP-specific antibody in mouse serum was confirmed by ELISA. It was confirmed that RFP-specific antibodies were highly generated in the mouse serum administered with GNP-RFP and GNP-CpG-RFP.
- Figure 7 is a negative control group (PBS), RFP, GNP-RFP and GNP-CpG-RFP in the present invention administered to C57BL / 6 mice three times a week intervals (12 ⁇ g antigen / mouse), the immune cells in local lymph nodes (CD8 T-cell memory) were isolated and stimulated to measure the amount of interferon gamma (IFN- ⁇ ) expressed from immune cells.
- PBS negative control group
- RFP RFP
- GNP-RFP GNP-RFP
- GNP-CpG-RFP GNP-CpG-RFP in the present invention administered to C57BL / 6 mice three times a week intervals (12 ⁇ g antigen / mouse), the immune cells in local lymph nodes (CD8 T-cell memory) were isolated and stimulated to measure the amount of interferon gamma (IFN- ⁇ ) expressed from immune cells.
- IFN- ⁇ interferon gamma
- FIG. 8 shows immune cells (CD8 T-cell memory) in local lymph nodes after administration of C57BL / 6 mice (12 ⁇ g antigen / mouse) of negative control (PBS), RFP, GNP-RFP and GNP-CpG-RFP in the present invention. ) Were isolated and stimulated with RFP to immune cells, and IFN- ⁇ -expressing immune cells were examined by FACS. In the GNP-RFP-administered group, 22.66% and 28.92% in the GNP-CpG-RFP-administered group were identified as immune cells expressing IFN- ⁇ .
- naive T cells by separating immune cells from local lymph nodes after administration of C57BL / 6 mice (12 ⁇ g antigen / mouse) of negative control group (PBS), RFP, GNP-RFP and GNP-CpG-RFP in the present invention. This is the result of the FACS investigation. GNP-RFP and GNP-CpG-RFP administration group showed lower naive T cell count (distribution) than the negative control group.
- Figure 10 after the administration of the negative control group (PBS), RFP, GNP-RFP and GNP-CpG-RFP in C57BL / 6 mice, isolating immune cells from local lymph nodes regulatory T cells (Treg) This is the result of checking the number of. As a result of confirming the number of regulatory T cells (Treg) expressing Foxp3 using the Foxp3 antibody, the number of regulatory T cells in the GNP-RFP and GNP-CpG-RFP-administered groups was compared with those of the negative control group. Little difference was found.
- FIG. 11 is a negative control group (PBS), GNP-RFP and GNP-CpG-RFP in the present invention, after administration of C57BL / 6 mice, isolating immune cells from local lymph nodes, separating only T cells and stimulating with RFP. This is the result of confirming the proliferation of cells. Unlike the negative control group in the GNP-RFP and GNP-CpG-RFP administration group, it was confirmed that T cells proliferate depending on the RFP concentration.
- FIG. 12 shows B16F10-RFP cancer after one week (0 day) after immunizing negative control group (PBS), GNP, RFP, GNP-RFP, and GNP-CpG-RFP three times a week at C57BL / 6 mice The result of observing the growth of cancer after administration of the cells. In the group immunized with GNP-RFP, cancer growth was delayed for about a month.
- PBS negative control group
- FIG. 13 shows B16F10-RFP cancer after one week (0 day) after immunizing negative control group (PBS), GNP, RFP, GNP-RFP and GNP-CpG-RFP to C57BL / 6 mice three times a week apart This is a result of comparing the survival rate of mice after administration of the cells.
- the group that immunized with GNP-RFP was found to have a 60-day longer survival compared to the negative control group.
- Figure 14 in the present invention after immunizing the control group (PBS), GNP, RFP, GNP-RFP and GNP-CpG-RFP three times a week at C57BL / 6 mice, do not express RFP after a week (0 day) Cancer growth after administration of B16F10 cancer cells. As a result, it was confirmed that the size of the cancer grows similarly in all experimental groups. These results confirm that GNP conjugates induce RFP specific immune responses.
- Figure 15 shows the negative control group (PBS), GNP, RFP, GNP-RFP and GNP-CpG-RFP when the size of the cancer after administration of B16F10-RFP cancer cells to C57BL / 6 mice in the present invention
- PBS negative control group
- FIG. 16 is a mucin peptide specific in mouse serum after immunizing negative control group (PBS), CGG Mucin peptide, CAL Mucin peptide, GNP- CGG MUC and GNP- CAL MUC three times a week at C57BL / 6 mice This is a comparison result confirming the expression pattern of the antibody by ELISA. As a result, it was confirmed that many mucin-specific antibodies covalently bound to mucin peptides were produced.
- PBS positive control group
- Figure 17 is a negative control group (PBS), CGG Mucin peptide, CAL Mucin peptide, GNP- CGG MUC and GNP- CAL MUC immunized three times a week intervals in C57BL / 6 mice, a week later (0 day)
- B16F1-mucin cancer cells expressing mucin protein were subcutaneously administered to the animals of each experimental group to observe the growth of cancer.
- the experimental group administered gold nanoparticles significantly inhibited cancer growth compared to the negative control.
- Figure 18 is a negative control group (PBS), CAL Mucin peptideGNP, and GNP- CAL MUC immunized three times a week to C57BL / 6 mice in the present invention, B16F1-mucin cancer cells were administered subcutaneously of experimental animals It is a result of comparing survival rate.
- the group immunized with GNP- CAL MUC was found to have a longer survival rate compared to the negative control group.
- 20A-20C show the results of measuring the size of ELS 8000 after the preparation of RFP-magnetic nanoparticles.
- Figure 21 shows the antibody induced analysis by RFP-magnetic nanoparticles.
- % used to refer to the concentration of a particular substance is (weight / weight)% solids / solid, (weight / volume)%, and liquid / Liquid is (volume / volume)%.
- Gold nanoparticles were synthesized as follows: Milli-Q water (150 mL) solution containing 2.2 mM sodium citrate was placed in a 250 mL round-bottom flask and heated. The mixture was heated to reflux for 15 minutes with vigorous stirring using mantel. After boiling, 1 ml of HAuCl 4 at a concentration of 23.4 mM was injected. The prepared particles were stabilized with negatively charged citrate ions and the stabilized particles were suspended.
- the gene encoding the RFP was amplified using a polymerase chain reaction (PCR) from a pDSRed2_C1 vector (clontech, Palo Alto, USA) containing all DNA sequences of DsRed. All molecular biology experiments were conducted following standard protocols (Molecular Cloning, Cold Spring Harbor, New York). Forward primer 5-ATAGAAA to bind two cysteines (marked codons in bold in reverse primer) at C-terminus CATATG GCCTCCTCCGAGAAC-3 and Reverse Primer 5-ATA CTCGAG TTA ACAACA CAGGAACAGGTG-3 was designed (Genotech, South Korea).
- PCR polymerase chain reaction
- PCR products were purified using a DNA purification kit (GeneAll, South Korea), and the restriction enzyme having an underlined site in the primer Nde I (NEB, Ipswich, MA) Xho Cuts were made using I (NEB, Ipswich, Mass.).
- the cleaved DNA fragments were linked to the pET28b (Novagen, Northumberland, UK) vector. Plasmids prepared for expressing proteins Escherichia coli Strain (DE3; Novagen, Northumberland, UK) was transformed. 1 mM IPTG (isopropyl ⁇ -D-thiogalactopyranoside; Sigma, St. Louis, MO) was treated at 37 ° C. for 6 hours to induce transformants by the strain.
- lysis buffer 50 mM sodium phosphate containing pH 8.0, 300 mM NaCl and 5 mM imidazole
- Cell lysates were centrifuged at 1,550 g at 4 ° C. for 1 hour.
- Suspension was placed in a gravity flow column (BioRad, Hercules, CA) filled with Ni-NTA affinity resin (Peptron, Daejeon, Korea) pre-equilibrated with Lysis buffer (3 ml bad volume per liter of medium). .
- RFP was extracted using a 50 mM sodium phosphate (pH 8.0) solution containing 300 mM NaCl and 300 mM imidazole. Fractions containing RFP were collected using a Superdex 200 column (Amersham Pharmacia, Bucks, UK) pre-equilibrated with PBS (phosphate-buffered saline; pH 7.4) solution and the proteins were further purified.
- PBS phosphate-buffered saline; pH 7.4
- Gold particles stabilized with 10 nm citrate were synthesized as described above.
- the synthesized gold particles were then modified with two cysteine modified RFP proteins or thiol modified A10-CpG1668 sequences. That is, in the case of RFP-GNP, a water-soluble protein solution (200 mL; 200 mg / mL) was added to a final concentration of 4 ⁇ M gold nanoparticle solution, followed by stirring at room temperature for 24 hours. In the case of RFP-CpG-GNP, a water-soluble protein solution (200 mL; 200 mg / mL) was added to a final concentration of 4 ⁇ M gold nanoparticle solution, followed by stirring at room temperature for 1 hour.
- thiol modified A10-CpG1668 sequence (5 nmole) was added to the RFP-GNP solution and stirred for 23 hours at room temperature. Centrifugation (20,000 x g, 30 minutes) was used to remove excess protein and DNA oligos from the nanoparticles.
- the hydrodynamic particle size of GNP dispersed in distilled water was measured using ELS 8000 (Otsuka Electronics Korea, Seoul, Korea).
- the morphology and dispersibility of GNP was measured by transmission electron microscopy (TEM) using Philips TECNAI F20 (Philips Electronic Instrument Corp., Mahwah, NJ) operating at 200 kV.
- Plasmon uptake of GNP was measured by UV-vis spectrophotometry using NEOSYS-2000 (Sinco, Daejeon, Korea).
- the size of the nanoparticles after GNP conjugation was found to be 7.3 nm for GNP, 13.7 nm for GNP-RFP, and 24.3 nm for GNP-RFP-CpG (FIGS. 1A-1C).
- PET / SPECT / CT system (Inveon TM; Siemens Preclinical Solutions, Knoxville, TN) was used. Images were taken at 60 kVp X-ray voltage, 500 ⁇ A anodic current, and 500 millisecond exposure time for each 360 rotational step. One bed position was scanned for 7 minutes to get the whole body of C57BL / 6 mice. The second order slice of each bed position was reconstructed using the modified Feldkamp algorithm with a ramp filter.
- the image was reconstructed on a 512 x 512 pixel grid with a 50 x 50 ⁇ m pixel size.
- the system was calculated using 50-mL polypropylene tubes containing water as described in the Inveon TM Instruction Manual.
- the resolution of the reconstructed image was 111 ⁇ m.
- CT data for the target area were analyzed using HUs.
- LPS Lipopolysaccharides; 10 ng / ml
- a 10 CpG 1668 10 ⁇ M
- GNP 8 nM
- GNP-RFP 8 nM
- GNP-CpG-RFP 8 nM
- RNAs were extracted using Welprep TM (Jeil Biotechservices Inc., Daegu, Korea). 1 ⁇ g of total RNA was reverse transcribed with UmProm-II reverse transcriptase (Promega) and amplified with MJ Mini TM PCR system (Bio-Rad, Hercules, CA).
- IL-6 5-TTCCTCTCTGCAAGAGACT-3, 5-TGTATCTCTCTGAAGGACT-3; Actin, 5'-TCATGAAGTGTGACGTTGACATCCGT-'3,5'-TTGCGGTGCACGATGGAGGGGCCGGA-'3; IL-12p40, 5'-GAAGTTCAACATCAAGAGCAGTAG-'3, 5'-AGGGAGAAGTAGGAATGGGG-30; IL-1 ⁇ , 5'-CCTGTGGCCTTGGGCCTCAA-'3, 5'-GAGGTGCTGATGTACCAGTTGG-'3; TNF- ⁇ , 5'-AAAATTCGAGTGACAAGCCTGTAG-'3, 5'-CCCTTGAAGAGAACCTGGGAGTAG-'3; iNOS2, 5'-GATGTTGAACTATGTCCTATCTCC-'3, 5'-AACACCACTTTCACCAAGAC-'3.
- mice C57BL / 6 mice were immunized with boosters (PBS, RFP, GNP-RFP, and GNP-RFP-CpG) on the soles of boosters on days 1, 8 and 15.
- boosters PBS, RFP, GNP-RFP, and GNP-RFP-CpG
- T-cells were isolated from draininig lymph nodes and na ⁇ ve T-cell counts were examined using FACs analysis (using naive T-cell markers CD62L and CD45RB).
- the GNP conjugate administration group showed less naT cell distribution compared with the negative control group (FIG. 9).
- mice C57BL / 6 mice were immunized with boosters (PBS, RFP, GNP-RFP, and GNP-RFP-CpG) on the soles of boosters on days 1, 8 and 15.
- boosters PBS, RFP, GNP-RFP, and GNP-RFP-CpG
- T-cells were isolated from draining lymph nodes and exposed to various concentrations of trypsinized RFP, followed by heat-inactivated T-cell medium containing 10% (vol / vol) FBS (HyClone). T-cells were incubated for 5672 hours in 96-well plates with flat bottoms. After 5672 hours of T-cell incubation, 0.5 ⁇ Ci of NEN ([H 3 ] -thymidine) was added to each well and further incubated for 16 hours. After the cultured cells were recovered, [H 3 ] -thymidine uptake was measured using liquid scintillation counting.
- C57BL / 6 mice were immunized by injecting PBS, RFP, GNP-RFP and GNP-RFP-CpG into the soles on days 1, 8 and 15.
- PBS PBS
- RFP GNP-RFP
- GNP-RFP-CpG GNP-RFP-CpG
- B16F10 cells 5 ⁇ 10 5 cells / mouse
- wild type B16F10 cells 5 ⁇ 10 5 cells / mouse
- GNP conjugates were immunized three times a week at mice and cancer growth was observed after administration of B16F10 cancer cells that did not express RFP. As a result, it was confirmed that the growth of cancer in all the experimental groups similarly (Fig. 14). From these results, it was concluded that GNP conjugates induce RFP specific immune responses.
- mice 6-week-old female C57BL / 6 mice were injected subcutaneously with 5 ⁇ 10 5 transformed B16F10 cells delivered to the dorsal flank on day 0. On day 10, mice were randomly divided into groups when tumors became more than 30 mm 3 and PBS, RFP, GNP-RFP, and GNP-RFP-CpG at various concentrations in the dorsal region at 10, 13, 16, 21 and 26 days. Tumor size was observed after injection.
- the group administered with the GNP conjugate showed a more delayed growth of cancer compared to the negative control (FIG. 15).
- CAL-mucin CALNN PDTRPAPGSTAPPAHGVTSA PDTRPAPGST; CGG-mucin: CGGGG PDTRPAPGSTAPPAHGVTSA PDTRPAPGST. (Anigen, South Korea)
- mucin antigen peptides were dissolved in distilled water at a concentration of 10 mM and mixed with 6 ⁇ M gold nanoparticles (GNP). Then, the mixture was covalently bonded to the gold nanoparticles with mucin peptides by reaction covalent bonding at room temperature for 24 hours, and then confirmed whether the covalent bonds with the gold nanoparticles by UV and visible spectroscopy. Mucin peptides were quantified.
- mice were administered to the soles of mice three times at weekly intervals, and 7 days later, B16F1-mucin cancer cells expressing mucin protein to the animals of each experimental group were administered subcutaneously to the experimental animals. Observed.
- GNP conjugates were immunized three times a week at mice, followed by cancer growth after administration of B16F1 cancer cells that did not express mucin. As a result, it was confirmed that the growth of cancer in all the experimental groups similarly (Fig. 19). These results led to the conclusion that GNP conjugates induce mucin specific immune responses.
- FIG. 20a is for G1 of Table 5 below and 34.2 ⁇ 7.5 nm particle size
- FIG. 20b is for G2 of Table 5 below and 32.4 ⁇ 6.4 nm particle size
- FIG. 20c is for G3 of Table 5 below, 34 ⁇ 6.7 nm particle size.
- Serum samples were obtained from C57BL / 6 mice immunized three times each at three weekly intervals using physiological saline, RFP (12 ⁇ g / mouse) and magnetic nanoparticle-RFP (12 ⁇ g / mouse).
- Anti-RFP antibody in serum was analyzed using ELISA (anti-mouse IgG-HRP, San Cruz Biotechnology, Inc.). As a result, it was confirmed that high antibody was produced in the serum of the group treated with magnetic nanoparticle-RFP. (FIG. 21).
Abstract
The present invention relates to a gold nanoparticle or a magnetic nanoparticle as a particle having a double function of imaging-delivering for imaging and delivery effects, an antigen-containing vaccine delivery system, an anticancer vaccine, and a pharmaceutical anticancer vaccine composition. The present invention rarely shows any in vivo toxicity and side effects, and can induce a high antigen generation rate even when used with an antigen having a low antigenicity. Also, the present invention effectively delivers and presents the antigen to an immunocyte, thus significantly inducing and enhancing an in vivo humoral (Th1) immune response and a cellular (Th2) immune response, and through such effects, enables immunoprophylaxis and immunotherapy against antigens causing various illnesses and diseases.
Description
본 발명은 이미징 및 운반이 가능한 나노입자-기반 백신 전달체에 관한 것이다.The present invention relates to nanoparticle-based vaccine carriers capable of imaging and delivery.
백신은 치료효과 뿐만 아니라 예방효과가 있어 질병의 99%까지 발병률을 줄일 수 있는 비용 대비 효과가 가장 큰 의약품이다. 특히 최근에는 백신의 용도가 감염성 질병에만 국한되는 것이 아니라, 암 및 자가면역질환을 포함한 각종 난치성 질환으로 넓혀지고 있고, 치료 백신이 등장함에 따라 백신 개발이 매우 중요하게 인식되고 있다.Vaccines are the most cost-effective medicines that can prevent and reduce the incidence of up to 99% of diseases, as well as therapeutic effects. In particular, in recent years, the use of vaccines is not limited to infectious diseases, but has been widened to various intractable diseases including cancer and autoimmune diseases, and vaccine development is recognized as very important as therapeutic vaccines are introduced.
인체에서는 항원 제시 세포에 의하여 종양세포의 항원이 효율적으로 제시되지 않기 때문에 이에 대한 면역반응이 효과적으로 유도되지 않는다. 암 치료백신은 암세포에 특이적으로 작용하는 면역기전을 인위적으로 활성화시켜(항원제시 세포 도입 등) 강력한 면역반응을 유발하여 암세포를 파괴하는 새로운 개념의 치료용 백신이다. 이용 가능한 백신의 접근방법으로 사용되는 것 중에서 수지상 세포(dendritic cells, DC)와 같은 항원제시 세포(antigen-presenting cell, APC)를 사용한 세포백신은 효과적인 T 세포 면역을 생성시키는데 확실한 방법으로 알려져 있다(Rosenberg, S. A. et al., Nat. Med., 10, 909-915, 2004).In the human body, antigen-presenting cells are not efficiently presented by antigen-presenting cells, so that immune responses to them are not effectively induced. Cancer vaccines are a novel therapeutic vaccine that destroys cancer cells by inducing a powerful immune response by artificially activating an immune mechanism that specifically acts on cancer cells (eg, introducing cells into antigens). Among the vaccine approaches available, cell vaccines using antigen-presenting cells (APCs), such as dendritic cells (DC), are known as a reliable method for generating effective T cell immunity. Rosenberg, SA et al., Nat. Med ., 10, 909-915, 2004).
현재 다양한 종양성 질환 및 감염 질환에 대한 새로운 백신들이 지속적으로 개발이 되고 있다. 약독화된 병원균 또는 복제를 하지 않는 비활성화된 병원균을 이용했던 예전의 백신과는 대조적으로, 요즘의 백신은 합성, 재조합, 또는 정제된 소단위 항원으로 이루어진다.Currently, new vaccines against various neoplastic and infectious diseases are constantly being developed. In contrast to previous vaccines that used attenuated pathogens or inactivated pathogens that did not replicate, modern vaccines consist of synthetic, recombinant, or purified subunit antigens.
또한, 백신은 병원체 항원 또는 사람 질병에 연루된 세포와 연관된 항원과 같은 표적 항원에 대해 개체를 면역화하는데 유용하다. 사람 질병에 연루된 세포 관련 항원에는 암 관련 종양 항원 및 자가면역 질환 관련 세포와 연관된 항원이 포함된다. 이러한 백신 디자인에서, 백신 접종한 개체의 세포 내에서 표적 항원을생산하는 백신이 면역계의 세포 무기화(cellular arm)를 유도하는데 효과적임은 공지되어 있다. 구체적으로, 약독화된 생백신, 비독성 벡터를 사용하는 재조합 백신 및 DNA 백신은 모두 백신접종된 개체의 세포 내에서 항원 생성을 유도하여 면역계의 세포 무기화를 유도한다. 반면에, 체액 반응을 유도하는, 단백질 및 죽은 또는 불활성화된 백신만을 함유하는 서브 유닛 백신은, 양호한 세포 면역반응을 유도하지 못한다.In addition, the vaccine is useful for immunizing an individual against a target antigen, such as a pathogen antigen or an antigen associated with a cell involved in human disease. Cell-associated antigens involved in human disease include cancer-associated tumor antigens and antigens associated with autoimmune disease-related cells. In such vaccine designs, it is known that vaccines that produce target antigens in the cells of vaccinated individuals are effective in inducing cellular arms of the immune system. Specifically, attenuated live vaccines, recombinant vaccines using non-toxic vectors, and DNA vaccines both induce antigen production in cells of vaccinated individuals, leading to cellular mineralization of the immune system. On the other hand, subunit vaccines containing only proteins and dead or inactivated vaccines, which induce humoral responses, do not induce a good cellular immune response.
따라서, 일반적인 화학요법의 비특이적 특성과 표적치료제의 암의 저항성 증가는 종양을 치료하는데 있어서 새로운 면역요법을 이용한 치료방법이 대두되고 있으며, 이러한 면역요법에 있어서 현재 존재하는 면역요법의 가장 큰 단점인 항원 전달 시 항원전달세포(antigen-presenting cell, APC)로의 정확한 전달 및 활성화를 개선할 수 있는 새로운 방법이 필요한 실정이다.Therefore, the nonspecific characteristics of general chemotherapy and the increased resistance of cancer to the targeted therapeutics have emerged as therapeutic methods using new immunotherapy to treat tumors, and the antigen, which is the biggest disadvantage of immunotherapy currently existing in such immunotherapy, is emerging. There is a need for new methods to improve the accurate delivery and activation of antigen-presenting cells (APCs) during delivery.
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.
본 발명자들은 이미징 및 백신 운반을 모두 수행할 수 있는 백신 전달체를 개발하고자 노력하였다. 그 결과, 금 나노입자 또는 자성 나노입자에 면역반응을 유발시킬 수 있는 항원을 결합시키는 경우에는 백신 전달체로서 우수한 퍼포먼스(performance)를 나타내며 림프절로의 선택적 운반을 가능하게 하며 또한 CT(computed tomography) 조영 또는 MRI(Magnetic resonance imaging) 조영을 통한 운반 경로의 추적도 가능하다는 것을 발견하였다. 더불어, 금 나노입자 또는 자성 나노입자를 기반으로 하는 백신은 림프절에서의 항원에 의한 면역 활성화 및 면역 활성에 의한 치료 효과를 성공적으로 달성함을 규명함으로써, 본 발명을 완성하게 되었다.The inventors have sought to develop a vaccine carrier capable of performing both imaging and vaccine delivery. As a result, when binding antigens capable of inducing an immune response to gold nanoparticles or magnetic nanoparticles, they show excellent performance as vaccine carriers, enable selective transport to lymph nodes, and also provide computed tomography (CT) imaging. Alternatively, it has been found that traceability of the delivery route through magnetic resonance imaging (MRI) imaging is possible. In addition, the present invention has been completed by finding that vaccines based on gold nanoparticles or magnetic nanoparticles successfully achieve immune activation by antigen in lymph nodes and therapeutic effects by immune activity.
따라서, 본 발명의 목적은 백신 전달체를 제공하는데 목적이 있다.Accordingly, it is an object of the present invention to provide a vaccine delivery vehicle.
본 발명의 다른 목적은 항암용 백신 약제학적 조성물을 제공하는데 있다.Another object of the present invention is to provide a vaccine pharmaceutical composition for anticancer.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.
본 발명의 일 양태에 따르면, 본 발명은 (a) 이미징 및 운반 작용을 하는 이미징-운반 이중기능 입자로서의 금 나노입자 또는 자성 나노입자; 및 (b) 상기 이미징-운반 이중기능 입자의 표면에 결합된 항원(antigen)을 포함하는 백신 전달체(delivery system)로서, 상기 백신 전달체는 상기 항원의 전달 및 상기 항원 전달의 추적(tracing)을 할 수 있도록 하는 것을 특징으로 하는 백신 전달체를 제공한다.According to one aspect of the present invention, the present invention provides a composition comprising: (a) gold nanoparticles or magnetic nanoparticles as imaging-carrying bifunctional particles having an imaging and transporting action; And (b) a vaccine delivery system comprising an antigen bound to the surface of the imaging-carrying bifunctional particle, wherein the vaccine delivery system is capable of delivering the antigen and tracing the antigen delivery. It provides a vaccine delivery, characterized in that to enable.
본 발명자들은 이미징 및 백신 운반을 모두 수행할 수 있는 백신 전달체를 개발하고자 노력하였다. 그 결과, 금 나노입자 또는 자성 나노입자에 면역반응을 유발시킬 수 있는 항원을 결합시키는 경우에는 백신 전달체로서 우수한 퍼포먼스(performance)를 나타내며 림프절로의 선택적 운반을 가능하게 하며 또한 CT(computed tomography) 조영 또는 MRI(Magnetic resonance imaging) 조영을 통한 운반 경로의 추적도 가능하다는 것을 발견하였다. 더불어, 금 나노입자 또는 자성 나노입자를 기반으로 하는 백신은 림프절에서의 항원에 의한 면역 활성화 및 면역 활성에 의한 치료 효과를 성공적으로 달성함을 규명하였다.The inventors have sought to develop a vaccine carrier capable of performing both imaging and vaccine delivery. As a result, when binding antigens capable of inducing an immune response to gold nanoparticles or magnetic nanoparticles, they show excellent performance as vaccine carriers, enable selective transport to lymph nodes, and also provide computed tomography (CT) imaging. Alternatively, it has been found that traceability of the delivery route through magnetic resonance imaging (MRI) imaging is possible. In addition, it has been found that vaccines based on gold nanoparticles or magnetic nanoparticles successfully achieve immune activation by antigen in lymph nodes and therapeutic effects by immune activity.
본 발명은 금 나노입자 또는 자성 나노입자의 표면에 항원을 결합시킨 백신 전달체로서, 인 비트로(in virto) 뿐 만 아니라 인 비보(in vivo)에서 매우 효과적으로 면역 반응(예컨대, 항체 생산)을 유도시킬 수 있는 백신 전달체이다.The present invention is to induce as a vaccine delivery system that combines the antigen on the surface of the gold nanoparticles, or magnetic nanoparticles, in vitro (in virto) as well, not just in vivo (in vivo) highly effective immune response in a (e. G., Antibody production) Vaccine carriers.
본 발명에서 백신 전달체를 설명하면 사용하는 용어 "백신"은 사람이나 동물을 자동적으로 면역하기 위하여 사용되는 물질을 의미하며, 대표적으로 사균백신, 약독화백신, 자가백신 및 다가백신이 있다.The term "vaccine" used to describe a vaccine carrier in the present invention means a substance used for automatically immunizing a human or animal, and typically, there is a dead virus vaccine, an attenuated vaccine, an autologous vaccine and a multivalent vaccine.
본 발명의 백신 전달체의 한 구성성분인 "나노입자"는 1-800 nm 바람직하게는 1-100 nm 크기를 가지는 입자를 의미한다.One component of the vaccine carrier of the present invention, "nanoparticle", means a particle having a size of 1-800 nm, preferably 1-100 nm.
본 발명의 바람직한 구현예에 따르면, 본 발명에서 이용되는 나노입자는 나노 단위의 직경, 바람직하게는 5-100 nm, 보다 바람직하게는 5-50 nm, 가장 바람직하게는 12-14 nm의 직경을 가지는 나노입자를 의미한다.According to a preferred embodiment of the present invention, the nanoparticles used in the present invention have a diameter in nano units, preferably 5-100 nm, more preferably 5-50 nm, and most preferably 12-14 nm. Eggplant means nanoparticles.
이러한 작은 크기는 본 발명의 나노입자가 목적의 세포(예컨대, 면역 세포) 또는 면역 세포 조직으로 침투하는 것을 용이하게 하여 세포내에 백신 전달체의 침투를 가능하게 한다. 이와 같은 구성을 포함하는 본 발명은 백신 전달체에서의 항원 전달시 항원을 정확하게 전달 및 면역 활성화(예컨대, 항체 생산)를 유도할 수 있는데 기여한다.This small size facilitates the penetration of the nanoparticles of the invention into cells of interest (eg, immune cells) or immune cell tissue, allowing the penetration of vaccine carriers into cells. The present invention comprising such a configuration contributes to the accurate delivery of antigen and induction of immune activation (eg, antibody production) upon antigen delivery in a vaccine delivery vehicle.
본 발명에서 사용되는 나노입자는 금 나노입자 또는 자성 나노입자이다.Nanoparticles used in the present invention are gold nanoparticles or magnetic nanoparticles.
금 나노입자는 안정된 입자의 형태로 제조가 쉬우며, 크기 조절이 용이하고, 망간, 알루미늄, 카드늄, 납, 수은, 코발트, 니켈 및 베릴륨 등의 중금속과 달리 인체에 무해하여 높은 생체친화성을 가진다.Gold nanoparticles are easy to manufacture in the form of stable particles, are easy to adjust in size, and unlike other heavy metals such as manganese, aluminum, cadmium, lead, mercury, cobalt, nickel and beryllium, they are highly biocompatible. .
본 발명에서 이용되는 금 나노입자는 예컨대, 다음과 같이 제조될 수 있다: HAuCl4를 금 공급원으로 하고, 소듐 시트레이트를 환원제로 하여 HAuCl4를 환원시켜 금 나노입자를 제조한다. 이 경우, 금 나노입자의 크기는 첨가하는 시트레이트를 달리해 줌으로써 조절이 가능한다. 즉, 시트레이트의 첨가량을 증가시킬수록 핵형성(nucleation)이 많이 되기 때문에 금 나노입자의 크기는 감소한다.The gold nanoparticles used in the present invention can be prepared, for example, as follows: HAuCl 4 is used as a gold source, and sodium citrate is used as a reducing agent to reduce HAuCl 4 to prepare gold nanoparticles. In this case, the size of the gold nanoparticles can be adjusted by varying the citrate added. That is, the size of the gold nanoparticles decreases as the amount of citrate increases, so that nucleation increases.
금 나노입자는 직경이 100 nm 이상으로 커질 경우 나노입자로서의 특성이 크게 감소될 뿐 아니라, 나노 물질의 특성이 없는 금 표면과 티올기 등의 작용기와의 결합은 약하기 때문에 금 입자를 매개로 하여 항원이 결합된 입자는 제조하기 어렵다.When the gold nanoparticles are larger than 100 nm in diameter, their properties as nanoparticles are greatly reduced, and the binding of functional groups such as the thiol group and the gold surface without the nanomaterial properties is weak. This bound particle is difficult to manufacture.
본 발명에서 이용되는 자성 나노입자는 당업계에 공지된 어떠한 자성 나노입자도 포함한다. 바람직하게는, 본 발명에서 이용 가능한 자성 나노입자는 상자성(paramagnetic) 나노입자 또는 초상자성(superparamagnetic) 나노입자이며, 가장 바람직하게는 초상자성 신호 발생 코어이다. 본 발명에 적합한 예시적인 상자성 나노입자는 안정된 자유 라디칼(예컨대, 안정된 니트록사이드(nitroxides)), 전이원소, 란탄족 원소 및 악티늄족 원소를 포함한다. 바람직한 원소는, Gd(Ⅲ), Mn(Ⅱ), Cu(Ⅱ), Cr(Ⅲ), Fe(Ⅱ), Fe(Ⅲ), Co(Ⅱ), Er(Ⅱ), Ni(Ⅱ), Eu(Ⅲ) 및 Dy(Ⅲ)를 포함한다. 본 발명에 적합한 예시적인 초상자정 나노입자는 페로-(ferro-) 또는 페리마그네틱 화합물(ferrimagnetic compounds), 예컨대, 순수 철, 자성 산화철(예컨대, 마그네타이트, Fe3O4), γ-Fe2O3, 망간 페라이트, 코발트 페라이트 및 니켈 페라이트를 포함한다. Magnetic nanoparticles used in the present invention include any magnetic nanoparticles known in the art. Preferably, the magnetic nanoparticles usable in the present invention are paramagnetic nanoparticles or superparamagnetic nanoparticles, most preferably superparamagnetic signal generating cores. Exemplary paramagnetic nanoparticles suitable for the present invention include stable free radicals (eg, stable nitroxides), transition elements, lanthanides, and actinides. Preferred elements are Gd (III), Mn (II), Cu (II), Cr (III), Fe (II), Fe (III), Co (II), Er (II), Ni (II), Eu (III) and Dy (III). Exemplary supercrystalline nanoparticles suitable for the present invention include ferro- or ferrimagnetic compounds, such as pure iron, magnetic iron oxides (eg magnetite, Fe 3 O 4 ), γ-Fe 2 O 3 , Manganese ferrite, cobalt ferrite and nickel ferrite.
본 발명에서 백신 전달체를 표현하면 사용하는 용어 "항원(antigen)"은 면역계를 자극하여 생체에 특이적 면역반응을 유도하는 물질을 의미하며, 본 발명에서 용어 "면역원(immunogen)"과 혼용되어 사용될 수 있다. 항원물질의 종류는 대단히 다양하며 그 개체에 대해 이물질이라면 원칙적으로 모두가 항원으로 인식한다. 단백질, 다당질, 핵산, 지질 및 이들의 복합체등을 천연항원이라 한다. 또한 화학적으로 합성된 물질이라도 단백질(담체)등과 결합하면 면역계를 자극시킬 수 있다. 이들을 합텐(부착제) 또는 인공항원이라 한다. 분자의 크기는 여러가지인데 아미노산 여러 개의 펩티드사슬로부터 분자량이 수십만의 물질까지, 또 바이러스, 세균, 동물세포 등도 항원이 될 수 있다.The term "antigen" used to express a vaccine carrier in the present invention means a substance that stimulates the immune system to induce a specific immune response in the living body, and is used in the present invention in combination with the term "immunogen". Can be. There are many different types of antigens, and if they are foreign to the individual, in principle everyone recognizes them as antigens. Proteins, polysaccharides, nucleic acids, lipids, and complexes thereof are called natural antigens. In addition, even chemically synthesized substances, when combined with proteins (carriers), can stimulate the immune system. These are called hapten (adhesive) or phosphorus airport agent. Molecules vary in size, ranging from peptide chains of amino acids to hundreds of thousands of molecules, and viruses, bacteria, and animal cells.
본 발명에서 항원은 나노입자 표면에 공유 또는 비공유 결합될 수 있으며, 바람직하게는 공유결합 된다. 공유결합 되는 경우, 항원은 나노입자에 직접 또는 간접적(예컨대, 링커를 통하여)으로 결합될 수 있다.In the present invention, the antigen may be covalently or non-covalently bound to the nanoparticle surface, preferably covalently bound. When covalently bound, the antigen can be bound directly or indirectly (eg, via a linker) to the nanoparticles.
본 발명의 바람직한 구현예에 따르면, 본 발명의 백신 전달체에 포함된 항원은 미생물 유래 항원, 바이러스 유래 항원, 기생충 유래 항원, 식물 유래 항원, 동물 유래 항원, 내재성(endogenous) 항원 및 합성 항원으로 이루어진 군으로부터 선택된 하나 이상의 항원을 포함한다.According to a preferred embodiment of the present invention, the antigen included in the vaccine carrier of the present invention comprises a microorganism-derived antigen, a virus-derived antigen, a parasite-derived antigen, a plant-derived antigen, an animal-derived antigen, an endogenous antigen and a synthetic antigen. One or more antigens selected from the group.
본 발명은 이미징-운반 이중기능 입자의 표면에 항원을 결합하여 대상체(예컨대, 포류유)에 투여하는 경우 림프구로부터 면역반응에 관여하는 항체 및 사이토카인을 매우 효과적으로 생산 및 분비를 촉진시킬 뿐 만 아니라 암 세포의 크기를 현저히 줄임으로써 우수한 항암 효능을 발휘할 수 있는 백신 전달체이다.The present invention not only facilitates the production and secretion of antibodies and cytokines involved in the immune response from lymphocytes when the antigen is bound to the surface of an imaging-carrying bifunctional particle and administered to a subject (eg, mammalian oil). It is a vaccine carrier that can exert excellent anticancer efficacy by significantly reducing the size of cancer cells.
본 발명에서의 백신 전달체에서 이미징-운반 이중기능 입자에 결합된 항원이 미생물 유래 항원인 경우, 바람직하게는 박테리아균 유래 항원, 진균(fungi) 유래 항원 또는 사상균(mold) 유래 항원을 포함한다.When the antigen bound to the imaging-carrying bifunctional particle in the vaccine delivery vehicle of the present invention is a microbe-derived antigen, it preferably comprises a bacterial bacteria-derived antigen, a fungi-derived antigen or a mold-derived antigen.
또한, 본 발명에서의 백신 전달체에서 이미징-운반 이중기능 입자에 결합된 항원이 바이러스 유래 항원인 경우, 바람직하게는 HIV(human immunodeficiency virus) 유래 항원, HPV(human Papilloma viruses) 유래 항원, 인플루엔자 바이러스 유래 항원, 헤르페스 바이러스 유래 항원, 헤파티티스 바이러스 유래 항원 또는 뇌염 바이러스 유래 항원를 포함한다.In addition, when the antigen bound to the imaging-carrying bifunctional particle in the vaccine delivery system of the present invention is a virus-derived antigen, an antigen derived from human immunodeficiency virus (HIV), an antigen derived from human Papilloma viruses (HPV), an influenza virus derived Antigens, herpes virus derived antigens, hepatitis virus derived antigens or encephalitis virus derived antigens.
또한, 본 발명에서의 백신 전달체에서 이미징-운반 이중기능 입자에 결합된 항원이 식물 유래 항원 또는 동물 유래 항원인 경우, 바람직하게는 알러지(alergy)를 유발하는 항원을 포함한다.In addition, when the antigen bound to the imaging-carrying bifunctional particle in the vaccine delivery vehicle of the present invention is a plant-derived antigen or an animal-derived antigen, it preferably includes an antigen that causes allergy.
또한, 본 발명에서의 백신 전달체에서 이미징-운반 이중기능 입자에 결합된 항원이 내재성 항원인 경우, 바람직하게는 암 세포 항원, 암 유발 항원 또는 자가면역질환 유발 항원을 포함한다.In addition, when the antigen bound to the imaging-carrying bifunctional particle in the vaccine delivery agent of the present invention is an endogenous antigen, it preferably includes a cancer cell antigen, a cancer causing antigen or an autoimmune disease causing antigen.
또한, 본 발명에서의 백신 전달체에서 이미징-운반 이중기능 입자에 결합된 항원이 합성(인공) 항원인 경우, 바람직하게는 약물(drug) 항원을 포함한다.In addition, when the antigen bound to the imaging-carrying bifunctional particle in the vaccine delivery agent of the present invention is a synthetic (artificial) antigen, it preferably includes a drug antigen.
본 발명에서의 본 발명에서의 백신 전달체에서 이미징-운반 이중기능 입자의 표면에 결합된 항원은 다양한 형태 및 성질의 분자들(molecules)이 결합될 수 있다.In the present invention, the antigen bound to the surface of the imaging-carrying bifunctional particle in the vaccine carrier of the present invention may be bound to molecules of various forms and properties.
본 발명의 바람직한 구현예에 따르면, 본 발명에서의 백신 전달체에서 이미징-운반 이중기능 입자의 표면에 결합된 항원은 단일쇄(single strand) 올리고뉴클레오타이드 또는 폴리뉴클레오타이드, 이중쇄(double strand) 올리고뉴클레오타이드 또는 폴리뉴클레오타이드, 단백질, 폴리펩타이드, 올리고펩타이드, 지질, 지질단백질, 당지질, 당당백질, 프로테오글리칸, 폴리사카라이드 및 리포폴리사카라이드로 이루어진 군으로부터 선택된 하나 이상의 항원을 포함하며, 보다 바람직하게는 폴리펩타이드, 올리고펩타이드, 단백질, 지질단백질 및 당당백질로 이루어진 군으로부터 선택된 하나 이상의 항원을 포함하고, 보다 더 바람직하게는 폴리펩타이드 또는 단백질을 포함한다.According to a preferred embodiment of the invention, the antigen bound to the surface of the imaging-carrying bifunctional particle in the vaccine carrier of the invention is a single stranded oligonucleotide or polynucleotide, a double stranded oligonucleotide or At least one antigen selected from the group consisting of polynucleotides, proteins, polypeptides, oligopeptides, lipids, lipoproteins, glycolipids, glycoproteins, proteoglycans, polysaccharides and lipopolysaccharides, more preferably polypeptides, One or more antigens selected from the group consisting of oligopeptides, proteins, lipoproteins and glycoproteins, even more preferably polypeptides or proteins.
본 발명에서 백신 전달체를 표현하면서 사용하는 용어 "면역보조제(adjuvant)"는 면역 보강제 또는 항원 보강제로 불리는 물질을 의미하며, 보다 구체적으로 백신에 대한 반응을 높여 면역시스템에 의해 항체가 많이 생성 될 수 있도록 하는 물질을 의미한다. The term "adjuvant" used in expressing a vaccine carrier in the present invention refers to a substance called an adjuvant or an adjuvant, and more specifically, a lot of antibodies can be generated by the immune system by increasing the response to the vaccine. It means a substance to make.
면역보조제는 일차적으로 내재 면역(innate immune)에 관여하나, 내재 면역에 관여하는 수상돌기세포(dendritic cell)와 대식세포(macrophage)는 케모카인(chemokine) 등을 분비하여 내재 면역에 관여하는 세포뿐 만 아니라, 후천성 면역(adaptive immune)에 관여하는 세포까지 감염된 곳으로 불려 들이기 때문에 결국 면역보조제도 후천성 면역에 관여함으로써 기억면역이 생기도록 한다. 또한, 면역 시스템에서 세균에 감염되었다는 것을 인지하도록 하기 위해서 면역보조제를 넣어줌으로서 수상돌기세포, 림프구 및 대식세포가 세균의 성분을 인식하게 되고 활성화 되어 내재 면역을 활성화 시킨다.The adjuvant is primarily involved in innate immune, but dendritic cells and macrophages that are involved in endogenous immunity secrete chemokines and only cells involved in endogenous immunity. Rather, even the cells involved in adaptive immune are called into the infected area, so the adjuvant also engages in acquired immunity, leading to memory immunity. In addition, by adding an adjuvant to recognize that the immune system is infected with bacteria, dendritic cells, lymphocytes and macrophages recognize the components of the bacteria and are activated to activate the innate immunity.
본 발명은 면역 활성을 효과적으로 향상시키기 위하여 면역보조제 또는 면역조절제를 추가적으로 포함할 수 있다.The present invention may further comprise an adjuvant or an immunomodulator to effectively enhance immune activity.
본 발명의 바람직한 구현예에 따르면, 본 발명은 면역보조제(adjuvants)를 추가적으로 포함한다.According to a preferred embodiment of the present invention, the present invention further comprises an adjuvants.
본 발명에서 면역보조제는 항원 또는 이미징-운반 이중기능 입자의 표면에 결합되어 있는 것이 바람직하며, 보다 바람직하게는 항원 또는 이미징-운반 이중기능 입자의 표면에 공유 결합되어 있는 것이 바람직하고, 보다 더 바람직하게는 이미징-운반 이중기능 입자의 표면에 공유 결합되어 있는 것이 바람직하다.In the present invention, the adjuvant is preferably bound to the surface of the antigen or imaging-carrying bifunctional particle, more preferably, is covalently bound to the surface of the antigen or imaging-carrying bifunctional particle, even more preferred. Preferably it is covalently bonded to the surface of the imaging-carrying bifunctional particle.
본 발명에서의 본 발명에서의 백신 전달체에서 추가적으로 포함하는 면역보조제는 다양한 형태 및 성질의 분자들이 결합될 수 있다.The immunoadjuvant additionally included in the vaccine carrier of the present invention in the present invention may be combined molecules of various forms and properties.
본 발명의 바람직한 구현예에 따르면, 본 발명에서 면역보조제는 CpG 올리고데옥시뉴클레오타이드, 언메틸레티드 CpG DNA(unmethylated cystein-phosphate-guanine DNA), 이중쇄 RNA, 미생물유래 DNA 또는 RNA, 핵산 유도체, 지질, 리포폴리사카라이드, 리포프로테인, 리포펩타이드, 당지질, 펩티도글리칸, 글리코펩타이드, 단백질, 재조합 단백질, 플라젤린(flagellin), 비로좀(virosome), Ribi(monophosphoryl-lipid A/trehalose dicorynomycolate), 사포닌 및 스쿠알렌(squalene) 스쿠랄렌(squalene), 핵산 유도체, 알루미늄염, 칼슘염, CFA(complete Freund's adjuvant) 및 IFA(incomplete Freund's adjuvant)으로 이루어진 군으로부터 선택된 하나 이상의 면역보조제를 포함하며, 보다 바람직하게는 CpG 올리고데옥시뉴클레오타이드, 언메틸레티드 CpG DNA(unmethylated cystein-phosphate-guanine DNA), 이중쇄 RNA, 미생물유래 DNA 또는 RNA 및 핵산 유도체로 이루어진 군으로부터 선택된 하나 이상의 면역보조제를 포함하고, 보다 더 바람직하게는 CpG 올리고데옥시뉴클레오타이드 또는 언메틸레티드 CpG DNA(unmethylated cystein-phosphate-guanine DNA)를 포함하며, 가장 바람직하게는 CpG 올리고데옥시뉴클레오타이드이다. According to a preferred embodiment of the present invention, the adjuvant in the present invention CpG oligodeoxynucleotide, unmethylated cystein-phosphate-guanine DNA (DCP), double-stranded RNA, microorganism-derived DNA or RNA, nucleic acid derivatives, Lipids, lipopolysaccharides, lipoproteins, lipopeptides, glycolipids, peptidoglycans, glycopeptides, proteins, recombinant proteins, flagellin, virosomes, Ribi (monophosphoryl-lipid A / trehalose dicorynomycolate) And one or more immunoadjuvant selected from the group consisting of saponins and squalene squalene, nucleic acid derivatives, aluminum salts, calcium salts, complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA), more preferably Preferably CpG oligodeoxynucleotides, unmethylated cystein-phosphate-guanine DNA (DCP), double stranded RNA, microbial DNA or RNA At least one immunoadjuvant selected from the group consisting of nucleic acid derivatives, more preferably CpG oligodeoxynucleotides or unmethylated cystein-phosphate-guanine DNA (CpG DNA), and most preferably CpG Oligodeoxynucleotides.
본 발명의 바람직한 구현예에 따르면, 본 발명의 백신 전달체는 림프절로 선택적으로 항원을 운반한다. 림프절은 감염에 대한 주요한 방어 기전이며 또한 악성 종양의 전이에서 통로 역할을 한다. 따라서, 본 발명의 백신 전달체가 림프절로 선택적으로 항원을 운반하는 것은, 본 발명의 백신 전달체가 생체 내에서 매우 유효하게 면역반응을 유도할 수 있음을 보여주는 것이다.According to a preferred embodiment of the present invention, the vaccine carrier of the present invention selectively delivers antigen to lymph nodes. Lymph nodes are a major defense against infection and also act as a pathway in the metastasis of malignant tumors. Therefore, the selective delivery of the antigen to the lymph nodes by the vaccine carrier of the present invention shows that the vaccine carrier of the present invention can induce an immune response very effectively in vivo.
본 발명의 바람직한 구현예에 따르면, 본 발명의 백신 전달체는 CT 이미징 또는 MRI 이미징으로 운반 경로가 추적될 수 있다. 본 발명의 백신 전달체를 인체 내에 투여하고 일정시간 후 CT 이미징 또는 MRI 이미징을 하면, 본 발명의 백신 전달체가 림프절로 제대로 이동하였는 지 여부를 확인할 수 있다. 이러한 특징도, 본 발명의 백신 전달체의 유용성을 크게 높이는 것이다. According to a preferred embodiment of the present invention, the delivery vehicle of the vaccine of the present invention can be traced by CT imaging or MRI imaging. When the vaccine delivery agent of the present invention is administered in a human body and CT imaging or MRI imaging is performed after a predetermined time, it is possible to confirm whether the vaccine delivery agent of the present invention is properly transferred to the lymph nodes. This feature also greatly increases the usefulness of the vaccine carrier of the present invention.
본 발명의 다른 양태에 따르면, 본 발명은 (a) 이미징 및 운반 작용을 하는 이미징-운반 이중기능 입자로서의 금 나노입자 또는 자성 나노입자; 및 (b) 상기 이미징-운반 이중기능 입자의 표면에 결합된 암 항원을 포함하는 항암용 백신 약제학적 조성물을 제공한다.According to another aspect of the invention, the invention provides a composition comprising: (a) gold nanoparticles or magnetic nanoparticles as imaging-carrying bifunctional particles for imaging and conveying action; And (b) provides a vaccine pharmaceutical composition for cancer comprising a cancer antigen bound to the surface of the imaging-carrying bifunctional particles.
본 발명에서의 항암용 백신 약제학적 조성물은 상기 백신 전달체를 포함하는 조성물이므로, 이 둘 사이에 공통된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여 그 기재를 생략한다.Since the vaccine pharmaceutical composition for anticancer in the present invention is a composition comprising the vaccine carrier, the common content between the two is omitted in order to avoid excessive complexity of the present specification.
본 발명의 약제학 조성물에 이용되는 암 항원은 P91A, p53, p21ras, P210, BTA, P198, P1A, gp100, TAG-72, PSMA, G250, Her-2/neu, CTKA-4, hTERT, VEGF, VEGF-A, MART-1-4, BAGE 1-3, melan-A (MART-1 (Melanoma Antigen Recognized by T cells)), SSX-2, SSX-4, 뮤신, MAGE-1, MAGE-2, MAGE-3, NY-ESO-1, LAGE, CEA(carcinoembryonic antigen), PRAME, 메소텔린, PLK1, GP100 (PMel17), GAGE-1, PSA, PSCA, SAGE 및 SCP-1를 포함하나, 이에 한정되는 것은 아니다.Cancer antigens used in the pharmaceutical compositions of the present invention are P91A, p53, p21 ras , P210, BTA, P198, P1A, gp100, TAG-72, PSMA, G250, Her-2 / neu, CTKA-4, hTERT, VEGF, VEGF-A, MART-1-4, BAGE 1-3, melan-A (MART-1 (Melanoma Antigen Recognized by T cells)), SSX-2, SSX-4, mucin, MAGE-1, MAGE-2, MAGE-3, NY-ESO-1, LAGE, carcinoembryonic antigen (CEA), PRAME, mesothelin, PLK1, GP100 (PMel17), GAGE-1, PSA, PSCA, SAGE and SCP-1 It is not.
본 발명의 약제학적 조성물은 약제학적으로 허용되는 담체를 포함할 수 있다. 본 발명의 약제학적 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약제학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences (19th ed., 1995)에 상세히 기재되어 있다.The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
본 발명의 약제학적 조성물은 경구 또는 비경구 투여할 수 있으며, 바람직하게는 비경구 투여 방식으로 적용된다.The pharmaceutical composition of the present invention may be administered orally or parenterally, and is preferably applied by parenteral administration.
본 발명의 약제학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. 본 발명의 약제학적 조성물의 일반적인 투여량은 성인 기준으로 0.001-100 ㎎/kg 범위 내이다.Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. Typical dosages of the pharmaceutical compositions of the invention are in the range of 0.001-100 mg / kg on an adult basis.
본 발명의 약제학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액, 시럽제 또는 유화액 형태이거나 엑스제, 산제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.
상술한 바와 같이, 본 발명의 백신 전달체는 림프절로 선택적으로 항원을 운반하기 때문에 효과적으로 면역반응을 유도하며 더욱이, 림프절은 악성 종양의 전이에서 통로 역할을 하기 때문에, 본 발명의 항암용 백신 조성물은 암의 치료에 매우 유효하다.As described above, the vaccine carrier of the present invention effectively induces an immune response because it selectively transports antigens to the lymph nodes, and moreover, since the lymph nodes play a role in the metastasis of malignant tumors, the anticancer vaccine composition of the present invention is a cancer Very effective in the treatment of
본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:
(a) 본 발명은 금 나노입자와 항원을 포함하는 백신 전달체 및 항암용 백신 약제학적 조성물을 제공한다.(a) The present invention provides a vaccine carrier comprising a gold nanoparticle and an antigen, and a vaccine pharmaceutical composition for anticancer.
(b) 본 발명은 인 비보상에서 독성 및 부작용을 거의 나타내지 않으며, 항원성이 낮은 항원과 함께 사용하는 경우에도 높은 항체 생성을 유도할 수 있다.(b) The present invention shows little toxicity and side effects on in vivo and can induce high antibody production even when used with antigens with low antigenicity.
(c) 또한, 본 발명은 항원을 면역 세포로 효과적으로 전달 및 제시함으로써, 인 비보에서 체액매개 면역반응(Th1) 및 세포매개 면역 반응(Th2)을 유의적으로 유도 및 향상시킬 수 있으며 이러한 효과를 통하여 다양한 질환 및 질병을 유발하는 항원에 대한 면역 예방 및 치료가 가능하다.(c) In addition, the present invention can significantly induce and enhance humoral mediated immune responses (Th1) and cell mediated immune responses (Th2) in vivo by effectively delivering and presenting antigens to immune cells and enhancing these effects. Through this, it is possible to prevent and treat immunity to antigens causing various diseases and diseases.
(d) 본 발명의 백신 전달체는 림프절로 선택적으로 항원을 운반한다. 이러한 특성에 의해, 본 발명의 백신 전달체는 생체 내에서 매우 유효하게 면역반응을 유도할 수 있다.(d) The vaccine carrier of the present invention selectively delivers antigen to lymph nodes. Due to this property, the vaccine carrier of the present invention can induce an immune response very effectively in vivo.
도 1a-1c는 본 발명에서 GNP, GNP-RFP 및 GNP-CpG-RFP를 ELS 8000기를 이용하여 크기를 측정한 결과이다. GNP, GNP-RFP 및 GNP-CpG-RFP 투여 후의 나노파티클의 크기는 각각 7.3 ㎚, 13.7 ㎚, 및 24.3 ㎚으로 확인되었다.Figure 1a-1c is a result of measuring the size of the GNP, GNP-RFP and GNP-CpG-RFP in the present invention using an ELS 8000 device. The size of nanoparticles after GNP, GNP-RFP, and GNP-CpG-RFP administration was confirmed to be 7.3 nm, 13.7 nm, and 24.3 nm, respectively.
도 2는 본 발명에서 제조된 GNP-RFP를 C57BL/6 마우스에 투여한 후 국부 리프절에서의 금 나노입자의 함량을 측정한 결과이다. 시간이 지남에 따라서, 국부 림프절(표재성 서혜부 림프절 및 오금 림프절)에서 GNP-RFP의 함량이 증가하는 것으로 확인되었다.Figure 2 is a result of measuring the content of gold nanoparticles in the local leaf nodes after administration of GNP-RFP prepared in the present invention to C57BL / 6 mice. Over time, it was found that the content of GNP-RFP increased in local lymph nodes (superficial groin lymph nodes and popliteal lymph nodes).
도 3a-3b는 GNP-RFP를 C57BL/6 마우스에 투여한 후 오금 림프절에서 존재 하는지를 은 염색으로 확인하고 또한 어떤 면역세포가 금 나노입자을 포집하는지를 면역염색을 통하여 확인하였다.Figures 3a-3b was confirmed by silver staining after the administration of GNP-RFP to C57BL / 6 mice by silver staining and also by immunostaining which immune cells capture gold nanoparticles.
도 4a-4b는 GNP-RFP 또는 GNP-CpG-RFP를 C57BL/6 마우스에 투여한 후 CT 이미징을 이용하여 관찰될수 있는지를 분석하였다. 그 결과, 오금 림프절에서 시간이 지남(0, 24시간 및 48시간)에 따라 GNP가 축적되는 것을 확인할 수 있었다.4A-4B analyzed whether CT or GNP-RFP or GNP-CpG-RFP could be observed using CT imaging after administration to C57BL / 6 mice. As a result, it was confirmed that GNP accumulates over time (0, 24 and 48 hours) in the popliteal lymph nodes.
도 5는 본 발명에서 제조된 GNP 컨쥬게이트를 C57BL/6 마우스 면역세포(마우스 대식세포, RAW264.7)와 혼합하였을 때, 면역세포에서의 사이토카인의 발현 양상을 조사한 결과이다. 그 결과, GNP-RFP 또는 GNP-CpG-RFP이 혼합된 C57BL/6 마우스 면역세포에서 사이토카인이 현저히 발현되는 것으로 확인되었다.5 is a result of examining the expression of cytokines in immune cells when the GNP conjugate prepared in the present invention is mixed with C57BL / 6 mouse immune cells (mouse macrophage, RAW264.7). As a result, it was confirmed that cytokines are markedly expressed in C57BL / 6 mouse immune cells mixed with GNP-RFP or GNP-CpG-RFP.
도 6은 본 발명에서 음성대조군(PBS), RFP, 양성대조군(Alum-RFP), GNP-RFP 및 GNP-CpG-RFP를 C57BL/6 마우스에 일주일 간격으로 3회 투여(12 ㎍ 항원/마우스)한 후 마우스 혈청에서 RFP 특이적인 항체의 발현양상을 ELISA로 확인한 비교 결과이다. GNP-RFP 및 GNP-CpG-RFP를 투여한 마우스 혈청에서 RFP 특이적인 항체가 높게 생성 되는 것을 확인하였다.Figure 6 in the present invention administered negative control (PBS), RFP, positive control (Alum-RFP), GNP-RFP and GNP-CpG-RFP three times a week intervals (12 μg antigen / mouse) to C57BL / 6 mice The expression pattern of RFP-specific antibody in mouse serum was confirmed by ELISA. It was confirmed that RFP-specific antibodies were highly generated in the mouse serum administered with GNP-RFP and GNP-CpG-RFP.
도 7은 본 발명에서 음성대조군(PBS), RFP, GNP-RFP 및 GNP-CpG-RFP를 C57BL/6 마우스에 일주일 간격으로 3회 투여(12 ㎍ 항원/마우스)한 후, 국소 림프절에서 면역세포(CD8 T-세포 메모리)들을 분리하여 자극을 주어 면역세포로부터 발현되는 인터페론 감마(IFN-γ)의 양을 측정한 비교 결과이다.Figure 7 is a negative control group (PBS), RFP, GNP-RFP and GNP-CpG-RFP in the present invention administered to C57BL / 6 mice three times a week intervals (12 μg antigen / mouse), the immune cells in local lymph nodes (CD8 T-cell memory) were isolated and stimulated to measure the amount of interferon gamma (IFN-γ) expressed from immune cells.
도 8은 본 발명에서 음성대조군(PBS), RFP, GNP-RFP 및 GNP-CpG-RFP를 C57BL/6 마우스 투여(12 ㎍ 항원/마우스)한 후, 국소 림프절에서 면역세포(CD8 T-세포 메모리)들을 분리하여 면역세포에 RFP로 자극한 후 IFN-γ를 발현하는 면역세포를 FACS로 조사한 결과이다. GNP-RFP 투여군에서는 22.66%, GNP-CpG-RFP 투여군에서는 28.92%가 IFN-γ를 발현하는 면역세포로 확인되었다.FIG. 8 shows immune cells (CD8 T-cell memory) in local lymph nodes after administration of C57BL / 6 mice (12 μg antigen / mouse) of negative control (PBS), RFP, GNP-RFP and GNP-CpG-RFP in the present invention. ) Were isolated and stimulated with RFP to immune cells, and IFN-γ-expressing immune cells were examined by FACS. In the GNP-RFP-administered group, 22.66% and 28.92% in the GNP-CpG-RFP-administered group were identified as immune cells expressing IFN-γ.
도 9는 본 발명에서 음성대조군(PBS), RFP, GNP-RFP 및 GNP-CpG-RFP를 C57BL/6 마우스 투여(12 ㎍ 항원/마우스)한 후, 국소 림프절에서 면역세포들을 분리하여 naive T 세포의 수를 FACS로 조사한 결과이다. GNP-RFP 및 GNP-CpG-RFP 투여군이 음성대조군에 비하여 적은 naive T 세포 수(분포도)를 나타내었다.9 is naive T cells by separating immune cells from local lymph nodes after administration of C57BL / 6 mice (12 μg antigen / mouse) of negative control group (PBS), RFP, GNP-RFP and GNP-CpG-RFP in the present invention. This is the result of the FACS investigation. GNP-RFP and GNP-CpG-RFP administration group showed lower naive T cell count (distribution) than the negative control group.
도 10은 본 발명에서 음성대조군(PBS), RFP, GNP-RFP 및 GNP-CpG-RFP를 C57BL/6 마우스 투여한 후, 국소 림프절에서 면역세포들을 분리하여 조절 T 세포(regulatory T cells, Treg)의 수를 확인한 결과이다. Foxp3 항체를 이용하여 Foxp3를 발현하는 조절 T 세포(regulatory T cells, Treg)의 수를 확인한 결과, GNP-RFP 및 GNP-CpG-RFP 투여군에서의 조절 T 세포 수는 음성대조군에서의 조절 T 세포와의 거의 차이가 없는 것으로 확인되었다.Figure 10 after the administration of the negative control group (PBS), RFP, GNP-RFP and GNP-CpG-RFP in C57BL / 6 mice, isolating immune cells from local lymph nodes regulatory T cells (Treg) This is the result of checking the number of. As a result of confirming the number of regulatory T cells (Treg) expressing Foxp3 using the Foxp3 antibody, the number of regulatory T cells in the GNP-RFP and GNP-CpG-RFP-administered groups was compared with those of the negative control group. Little difference was found.
도 11은 본 발명에서 음성대조군(PBS), GNP-RFP 및 GNP-CpG-RFP를 C57BL/6 마우스 투여한 후, 국소 림프절에서 면역세포들을 분리하여 T 세포만 분리한 후 RFP로 자극을 주어 T 세포의 증식여부를 확인한 결과이다. GNP-RFP 및 GNP-CpG-RFP 투여군에서 음성대조군과는 달리 RFP 농도에 따라 의존적으로 T 세포가 증식하는 것으로 확인되었다.FIG. 11 is a negative control group (PBS), GNP-RFP and GNP-CpG-RFP in the present invention, after administration of C57BL / 6 mice, isolating immune cells from local lymph nodes, separating only T cells and stimulating with RFP. This is the result of confirming the proliferation of cells. Unlike the negative control group in the GNP-RFP and GNP-CpG-RFP administration group, it was confirmed that T cells proliferate depending on the RFP concentration.
도 12는 본 발명에서 음성대조군(PBS), GNP, RFP, GNP-RFP 및 GNP-CpG-RFP를 C57BL/6 마우스에 일주일 간격으로 3회 면역시킨 후, 일주일 후(0 day) B16F10-RFP 암 세포를 투여한 이후 암의 성장 여부를 관찰한 결과이다. GNP-RFP를 면역시킨 군에서 암의 성장이 한 달 정도 더 지연되는 결과를 나타내었다.FIG. 12 shows B16F10-RFP cancer after one week (0 day) after immunizing negative control group (PBS), GNP, RFP, GNP-RFP, and GNP-CpG-RFP three times a week at C57BL / 6 mice The result of observing the growth of cancer after administration of the cells. In the group immunized with GNP-RFP, cancer growth was delayed for about a month.
도 13은 본 발명에서 음성대조군(PBS), GNP, RFP, GNP-RFP 및 GNP-CpG-RFP를 C57BL/6 마우스에 일주일 간격으로 3회 면역시킨 후, 일주일 후(0 day) B16F10-RFP 암 세포를 투여한 이후 마우스의 생존율을 비교한 결과이다. GNP-RFP를 면역한 군이 음성대조군과 비교하여 개체의 생존율이 60일정도 더 연장되는 것으로 확인되었다.FIG. 13 shows B16F10-RFP cancer after one week (0 day) after immunizing negative control group (PBS), GNP, RFP, GNP-RFP and GNP-CpG-RFP to C57BL / 6 mice three times a week apart This is a result of comparing the survival rate of mice after administration of the cells. The group that immunized with GNP-RFP was found to have a 60-day longer survival compared to the negative control group.
도 14는 본 발명에서 음성대조군(PBS), GNP, RFP, GNP-RFP 및 GNP-CpG-RFP를 C57BL/6 마우스에 일주일 간격으로 3회 면역시킨 후, 일주일 후(0 day) RFP를 발현하지 않은 B16F10 암 세포를 투여한 이후의 암 성장을 비교한 결과이다. 그 결과, 모든 실험군에서 암의 크기가 비슷하게 성장하는 결과를 확인하였다. 이러한 결과를 통하여, GNP 컨쥬게이트들이 RFP 특이적인 면역반응을 유도한다는 것을 확인할 수 있었다.Figure 14 in the present invention, after immunizing the control group (PBS), GNP, RFP, GNP-RFP and GNP-CpG-RFP three times a week at C57BL / 6 mice, do not express RFP after a week (0 day) Cancer growth after administration of B16F10 cancer cells. As a result, it was confirmed that the size of the cancer grows similarly in all experimental groups. These results confirm that GNP conjugates induce RFP specific immune responses.
도 15는 본 발명에서 B16F10-RFP 암 세포를 C57BL/6 마우스에 투여한 후 암의 크기가 70 ㎣이 되었을 때, 음성대조군(PBS), GNP, RFP, GNP-RFP 및 GNP-CpG-RFP를 암 조직 근처 마우스 등 부위에 각각 투여한 후 10, 13, 16, 21 및 26 일째에 암의 성장(크기)을 비교한 결과이다. 그 결과, GNP 컨쥬게이트를 투여한 군이 음성 대조군에 비해서 암의 성장이 더 지연되는 결과를 나타내었다.Figure 15 shows the negative control group (PBS), GNP, RFP, GNP-RFP and GNP-CpG-RFP when the size of the cancer after administration of B16F10-RFP cancer cells to C57BL / 6 mice in the present invention This is a result of comparing the growth (size) of the cancer at 10, 13, 16, 21 and 26 days after administration to the dorsal region near the cancer tissue, respectively. As a result, the group to which the GNP conjugate was administered showed a delay of cancer growth more than the negative control group.
도 16은 본 발명에서 음성대조군(PBS), CGGMucin peptide, CALMucin peptide, GNP-CGGMUC 및 GNP-CALMUC를 C57BL/6 마우스에 일주일 간격으로 3회 면역시킨 후, 마우스 혈청에서 뮤신 펩타이드 특이적인 항체의 발현양상을 ELISA로 확인한 비교 결과이다. 그 결과, 뮤신 펩타이드를 공유 결합한 뮤신 특이적인 항체가 많이 생산되는 것으로 확인되었다. FIG. 16 is a mucin peptide specific in mouse serum after immunizing negative control group (PBS), CGG Mucin peptide, CAL Mucin peptide, GNP- CGG MUC and GNP- CAL MUC three times a week at C57BL / 6 mice This is a comparison result confirming the expression pattern of the antibody by ELISA. As a result, it was confirmed that many mucin-specific antibodies covalently bound to mucin peptides were produced.
도 17은 본 발명에서 음성대조군(PBS), CGGMucin peptide, CALMucin peptide, GNP-CGGMUC 및 GNP-CALMUC를 C57BL/6 마우스에 일주일 간격으로 3회 면역시킨 후, 일주일 후(0 day)에 각 실험군의 동물에 뮤신 단백질을 발현하는 B16F1-뮤신 암세포를 실험동물의 피하로 투여하여 암의 성장여부를 관찰한 결과이다. 그 결과, 음성 대조군에 비해서 금나노 입자를 투여한 실험 군이 유의적으로 암 성장을 억제하는 것으로 관찰 되었다.Figure 17 is a negative control group (PBS), CGG Mucin peptide, CAL Mucin peptide, GNP- CGG MUC and GNP- CAL MUC immunized three times a week intervals in C57BL / 6 mice, a week later (0 day) In the experimental group, B16F1-mucin cancer cells expressing mucin protein were subcutaneously administered to the animals of each experimental group to observe the growth of cancer. As a result, it was observed that the experimental group administered gold nanoparticles significantly inhibited cancer growth compared to the negative control.
도 18은 본 발명에서 음성대조군(PBS), CALMucin peptideGNP, 및 GNP-CALMUC를 C57BL/6 마우스에 일주일 간격으로 3회 면역시킨 후, B16F1-뮤신 암세포를 실험동물의 피하로 투여하여 마우스의 생존율을 비교한 결과이다. GNP-CALMUC를 면역한 군이 음성대조군과 비교하여 개체의 생존율이 더 연장되는 것으로 확인되었다.Figure 18 is a negative control group (PBS), CAL Mucin peptideGNP, and GNP- CAL MUC immunized three times a week to C57BL / 6 mice in the present invention, B16F1-mucin cancer cells were administered subcutaneously of experimental animals It is a result of comparing survival rate. The group immunized with GNP- CAL MUC was found to have a longer survival rate compared to the negative control group.
도 19는 본 발명에서 음성대조군(PBS), CGGMucin peptide, CALMucin peptide, GNP-CGGMUC 및 GNP-CALMUC를 C57BL/6 마우스에 일주일 간격으로 3회 면역시킨 후, 7일 후에 각 실험군의 동물에 뮤신 단백질을 발현하지 않는 B16F1 암세포를 실험동물의 피하로 투여하여 암의 성장여부를 관찰한 결과이다. 그 결과, 모든 실험군에서 암의 크기가 비슷하게 성장하는 결과를 확인하였다. 이러한 결과를 통하여, GNP 컨쥬게이트들이 뮤신 특이적인 면역반응을 유도한다는 것을 확인할 수 있었다.19 is immunized three times at weekly intervals to C57BL / 6 mice with negative control group (PBS), CGG Mucin peptide, CAL Mucin peptide, GNP- CGG MUC and GNP- CAL MUC in the present invention, after 7 days of each experimental group B16F1 cancer cells that do not express mucin protein in animals were administered subcutaneously in experimental animals to observe the growth of cancer. As a result, it was confirmed that the size of the cancer grows similarly in all experimental groups. Through these results, it was confirmed that GNP conjugates induce mucin-specific immune response.
도 20a-20c는 RFP-자성나노입자의 제조 후 ELS 8000기를 이용하여 크기를 측정한 결과이다. 20A-20C show the results of measuring the size of ELS 8000 after the preparation of RFP-magnetic nanoparticles.
도 21은 RFP-자성나노입자에 의한 항체 유발 분석 결과이다.Figure 21 shows the antibody induced analysis by RFP-magnetic nanoparticles.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .
실시예Example
본 명세서 전체에 걸쳐, 특정 물질의 농도를 나타내기 위하여 사용되는 %는 별도의 언급이 없는 경우, 고체/고체는 (중량/중량) %, 고체/액체는 (중량/부피) %, 그리고 액체/액체는 (부피/부피) %이다.Throughout this specification,% used to refer to the concentration of a particular substance is (weight / weight)% solids / solid, (weight / volume)%, and liquid / Liquid is (volume / volume)%.
화학 제품Chemical products
실험에 사용된 모든 제품은 고순도의 화학제품이며, Merck사, Sigma-Aldrich사, Fluka사 및 Ambion사로부터 구입하였다. ELISA 키트는 R&D Systems사로부터 구입하였으며, 하이드로겐 테트라클로아레이트(Ⅲ) 트리하이드레이트(Hydrogen tetrachloroaurate(Ⅲ) trihydrate; 99.9+%, Aldrich)는 보편적인 것으로 사용하였다. 모든 유리제품을 아세톤으로 세척하고 탈이온수로 린스하였으며, 사용 전에 100℃에 보관하였다. 모든 시약들은 제조사의 추천 방법에 따라 제조하였다.All products used in the experiments are high purity chemicals and were purchased from Merck, Sigma-Aldrich, Fluka and Ambion. ELISA kits were purchased from R & D Systems and Hydrogen tetrachloroaurate (III) trihydrate (99.9 +%, Aldrich) was used universally. All glassware was washed with acetone and rinsed with deionized water and stored at 100 ° C. before use. All reagents were prepared according to the manufacturer's recommendations.
실시예 1: GNPs(Gold nanoparticles) 합성Example 1 Synthesis of Gold Nanoparticles (GNPs)
다음과 같은 방법으로 GNPs(Gold nanoparticles)를 합성하였다: 2.2 mM 소듐 시트레이트(sodium citrate)가 포함된 Milli-Q 수(150 ㎖) 용액을 250 ㎖ 3구 환저(round-bottom) 플라스크에 담아 열 맨텔(mantel)를 이용하여 강하게 휘저으면서 15분 동안 가열하여 환류시켰다. 끓기 시작한 후, 23.4 mM 농도의 HAuCl4 1 ㎖를 주입하였다. 제조된 파티클을 음성적으로 전하를 가진 시트레이트 이온으로 안정화시켰으며 안정화된 파티클을 부유시켰다.Gold nanoparticles (GNPs) were synthesized as follows: Milli-Q water (150 mL) solution containing 2.2 mM sodium citrate was placed in a 250 mL round-bottom flask and heated. The mixture was heated to reflux for 15 minutes with vigorous stirring using mantel. After boiling, 1 ml of HAuCl 4 at a concentration of 23.4 mM was injected. The prepared particles were stabilized with negatively charged citrate ions and the stabilized particles were suspended.
실시예 2: RFP(red fluorescent proteins)의 유전자 엔지니어링, 발현 및 정제Example 2: Gene Engineering, Expression and Purification of Red Fluorescent Proteins (RFP)
DsRed의 모든 DNA 서열을 포함하고 있는 pDSRed2_C1 벡터(clontech, Palo Alto, USA)로부터 PCR(polymerase chain reaction)을 이용하여 RFP를 코딩하는 유전자를 증폭하였다. 모든 분자생물학 실험들은 표준 프로토콜을 따라 실시되었다(Molecular Cloning, Cold Spring Harbor, New York). C-말단에 2개의 시스테인(역방향 프라이머에서 볼드체로된 표기된 코돈)이 결합되도록 정방향 프라이머 5- ATAGAAACATATGGCCTCCTCCGAGAAC-3와 역방향 프라이머 5-ATACTCGAGTTAACAACACAGGAACAGGTG-3를 디자인하였다(Genotech, 대한민국). DNA 정제 키트(GeneAll, 대한민국)를 이용하여 PCR 결과물을 정제하였으며, 상기 프라이머에 밑줄로 표시된 부위를 가진 제한 효소 NdeI(NEB, Ipswich, MA)와 XhoI(NEB, Ipswich, MA)를 이용하여 절단하였다. 절단된 DNA 단편들을 pET28b(Novagen, Northumberland, UK) 벡터에 연결하였다. 단백질을 발현시키기 위하여 제조된 플라스미드를 Escherichia coli 균주(DE3; Novagen, Northumberland, UK)에 형질전환시켰다. 37℃에서 6시간 동안 1 mM IPTG(isopropyl β-D-thiogalactopyranoside; Sigma, St. Louis, MO)를 처리하여 균주에 의하여 형질전환체(transformant)를 유도시켰다. 그 다음, 균주를 모은 후 모아진 균주를 라이시스(lysis) 버퍼(pH 8.0, 300 mM NaCl과 5 mM 이미다졸을 포함하고 있는 50 mM 소듐 포스페이트)에 부유하였으며, 초음파로 라이시스시켰다. 세포 용해물을 4℃에서 1시간 동안 1,550 g로 원심분리하였다. 라이시스 버퍼(배지 리터당 3 ㎖ 배드 볼륨)로 전-평형화시킨 Ni-NTA 친화성 레진(Peptron, Daejeon, Korea)으로 채워진 중력-유동(gravity flow) 컬럼(BioRad, Hercules, CA)에 부유물을 넣었다. 컬럼과 라이시스 버퍼의 10 비드 볼륨을 포함하는 매트릭스를 세척한 후, 300 mM NaCl과 300 mM 이미다졸을 포함하는 50 mM 소듐 포스페이트(pH 8.0) 용액을 이용하여 RFP를 추출하였다. PBS(phosphate-buffered saline; pH 7.4) 용액으로 전-평형화시킨 Superdex 200 컬럼(Amersham Pharmacia, Bucks, UK)을 이용하여 RFP를 포함하는 분획물을 모으고, 단백질을 추가적으로 정제하였다.The gene encoding the RFP was amplified using a polymerase chain reaction (PCR) from a pDSRed2_C1 vector (clontech, Palo Alto, USA) containing all DNA sequences of DsRed. All molecular biology experiments were conducted following standard protocols (Molecular Cloning, Cold Spring Harbor, New York). Forward primer 5-ATAGAAA to bind two cysteines (marked codons in bold in reverse primer) at C-terminusCATATGGCCTCCTCCGAGAAC-3 and Reverse Primer 5-ATACTCGAGTTAACAACACAGGAACAGGTG-3 was designed (Genotech, South Korea). PCR products were purified using a DNA purification kit (GeneAll, South Korea), and the restriction enzyme having an underlined site in the primerNdeI (NEB, Ipswich, MA)XhoCuts were made using I (NEB, Ipswich, Mass.). The cleaved DNA fragments were linked to the pET28b (Novagen, Northumberland, UK) vector. Plasmids prepared for expressing proteinsEscherichia coli Strain (DE3; Novagen, Northumberland, UK) was transformed. 1 mM IPTG (isopropyl β-D-thiogalactopyranoside; Sigma, St. Louis, MO) was treated at 37 ° C. for 6 hours to induce transformants by the strain. The collected strains were then suspended in lysis buffer (50 mM sodium phosphate containing pH 8.0, 300 mM NaCl and 5 mM imidazole) and sonicated by ultrasound. Cell lysates were centrifuged at 1,550 g at 4 ° C. for 1 hour. Suspension was placed in a gravity flow column (BioRad, Hercules, CA) filled with Ni-NTA affinity resin (Peptron, Daejeon, Korea) pre-equilibrated with Lysis buffer (3 ml bad volume per liter of medium). . After washing the column and the 10 bead volume of the Lysis buffer, RFP was extracted using a 50 mM sodium phosphate (pH 8.0) solution containing 300 mM NaCl and 300 mM imidazole. Fractions containing RFP were collected using a Superdex 200 column (Amersham Pharmacia, Bucks, UK) pre-equilibrated with PBS (phosphate-buffered saline; pH 7.4) solution and the proteins were further purified.
실시예 3: 금 나노입자 콘쥬게이션(conjugation)Example 3: Gold Nanoparticle Conjugation
상기 기술된 바에 따라 10 nm 시트레이트로 안정화된 금 파티클을 합성하였다. 그 다음, 합성된 금 파티클들을 2개의 시스테인으로 변형된 RFP 단백질 또는 티올 변형된 A10-CpG1668 시퀀스로 변형시켰다. 즉, RFP-GNP의 경우 최종 농도 4 μM 금 나노입자 용액에 수용성 단백질 용액(200 ㎖; 200 ㎎/㎖)을 첨가한 후 실온에서 24시간 동안 저어주었다. RFP-CpG-GNP의 경우, 최종 농도 4 μM 금 나노입자 용액에 수용성 단백질 용액(200 ㎖; 200 ㎎/㎖)을 첨가한 후 실온에서 1시간 동안 저어주었다. 이에 따라, 티올 변형 A10-CpG1668 시퀀스(5 nmole)는 RFP-GNP 용액에 첨가한 후 실온에서 23시간 저어주었다. 원심분리(20,000 x g, 30 분)를 이용하여 나노파티클로부터 잉여의 단백질 및 DNA 올리고를 제거하였다. Gold particles stabilized with 10 nm citrate were synthesized as described above. The synthesized gold particles were then modified with two cysteine modified RFP proteins or thiol modified A10-CpG1668 sequences. That is, in the case of RFP-GNP, a water-soluble protein solution (200 mL; 200 mg / mL) was added to a final concentration of 4 μM gold nanoparticle solution, followed by stirring at room temperature for 24 hours. In the case of RFP-CpG-GNP, a water-soluble protein solution (200 mL; 200 mg / mL) was added to a final concentration of 4 μM gold nanoparticle solution, followed by stirring at room temperature for 1 hour. Accordingly, thiol modified A10-CpG1668 sequence (5 nmole) was added to the RFP-GNP solution and stirred for 23 hours at room temperature. Centrifugation (20,000 x g, 30 minutes) was used to remove excess protein and DNA oligos from the nanoparticles.
실시예 4: 금 나노입자의 크기 측정Example 4 Measurement of Size of Gold Nanoparticles
ELS 8000(Otsuka Electronics Korea, Seoul, Korea)을 이용하여 증류수에 흩어진 GNP의 유체역학적 파티클 크기를 측정하였다. 200 kV에서 작동하는 Philips TECNAI F20(Philips Electronic Instrument Corp., Mahwah, NJ)을 이용하여 TEM(transmission electron microscopy)으로 GNP의 형태학 및 분산성을 측정하였다. NEOSYS-2000(Sinco, Daejeon, Korea)를 이용하여 자외선 및 가시광선 분광 분석법(UV-vis spectrophotometry)으로 GNP의 플라즈몬(plasmonic) 흡수도를 측정하였다. The hydrodynamic particle size of GNP dispersed in distilled water was measured using ELS 8000 (Otsuka Electronics Korea, Seoul, Korea). The morphology and dispersibility of GNP was measured by transmission electron microscopy (TEM) using Philips TECNAI F20 (Philips Electronic Instrument Corp., Mahwah, NJ) operating at 200 kV. Plasmon uptake of GNP was measured by UV-vis spectrophotometry using NEOSYS-2000 (Sinco, Daejeon, Korea).
그 결과, GNP 컨쥬게이션 후의 나노파티클의 크기는 GNP가 7.3 ㎚, GNP-RFP가 13.7 ㎚, 그리고 GNP-RFP-CpG가 24.3 ㎚으로 확인되었다(도 1a-1c).As a result, the size of the nanoparticles after GNP conjugation was found to be 7.3 nm for GNP, 13.7 nm for GNP-RFP, and 24.3 nm for GNP-RFP-CpG (FIGS. 1A-1C).
실시예 5: 국부 림프절에서 금 함량 평가Example 5 Evaluation of Gold Content in Local Lymph Nodes
발바닥(foot pad)를 통하여 C57BL/6 마우스 (오리엔트 바이오, 대한민국)에 GNP-RFP를 주입시킨 후 마우스 국부 림프절에서의 금의 양을 특정하기 위하여, 각 2, 5 및 8일 마다 국부 림프절을 모았으며, 오토샘플러(AS-93 plus, Perkin Elmer instruments, U.S.A.)가 장착된 고해상 섹터 필드 ICP-OES(inductively coupled plasma atomic emission spectroscopy; Optima 4300 DV, Perkin Elmer instruments, U.S.A.)에 의하여 측정된 세포내 철 함량을 이용하여 금을 정량화하였다. 샘플 인트로덕션(introduction)을 위하여 미라 미스트 네불라아저(Perkin Elmer)가 장착된 스코트 타입 스프레이 챔버를 이용하였다.Local lymph nodes were collected every 2, 5 and 8 days to determine the amount of gold in mouse local lymph nodes after GNP-RFP was injected into C57BL / 6 mice (Oriental Bio, South Korea) through the foot pad. Intracellular iron measured by high resolution sector field ICP-OES (inductively coupled plasma atomic emission spectroscopy; Optima 4300 DV, Perkin Elmer instruments, USA) equipped with an autosampler (AS-93 plus, Perkin Elmer instruments, USA). Gold was quantified using the content. A sample type spray chamber equipped with a Mira Mist Nebulazer (Perkin Elmer) was used for sample introduction.
그 결과, GNP-RFP를 C57BL/6 마우스 마우스에 투여한 후 마우스 국소 림프절에서의 금(Au)의 함량은 시간이 지남에 따라 증가하는 것을 확인되었다(도 2).As a result, after administration of GNP-RFP to C57BL / 6 mouse mice, the content of gold (Au) in the mouse regional lymph nodes was confirmed to increase over time (Fig. 2).
실시예 6: 마우스 국부 림프절에서 은(silver) 염색 및 면역조직화학분석Example 6 Silver Staining and Immunohistochemical Analysis in Mouse Local Lymph Nodes
실버 인헨스먼트 키트(SE-100, sigma)를 이용하여 5분간 국부 림프절을 염색하였고, PBS로 3회 이상 세척하였다. 현미경으로 실버로 염색된 부분을 분석하여 금 나노입자을 확인하였다 (도 3a). 또한, 면역조직화학분석을 위하여 분리한 국부 림프절을 다양한 항체(BD Bioscience)와 결합시켰다 (도 3b).Local lymph nodes were stained for 5 minutes using a silver enhancement kit (SE-100, sigma) and washed three more times with PBS. The silver stained portion was analyzed under a microscope to identify the gold nanoparticles (Fig. 3a). In addition, isolated local lymph nodes were combined with various antibodies (BD Bioscience) for immunohistochemical analysis (FIG. 3B).
GNP-RFP를 C57BL/6 마우스에 투여한 후 오금 림프절에서 어떤 면역세포가 금 나노입자을 포집하는지를 은 염색과 면역염색을 통하여 확인하였다. 그 결과, 대식세포, 랑게르한스 세포와 pDCs(plasmacytoid dendritic cells) 면역세포에서의 금 나노입자가 확인되었다.After administration of GNP-RFP to C57BL / 6 mice, it was confirmed by silver staining and immunostaining which immune cells capture gold nanoparticles in popliteal lymph nodes. As a result, gold nanoparticles were identified in macrophages, Langerhans cells and pDCs (plasmacytoid dendritic cells).
실시예 7: CT 이미징(imaging)Example 7: CT Imaging
로-에너지 X-레이 튜브(X-ray energy, 35-80 kVp), 하이-레졸류션 포스포 스크린 및 2,048 x 3,072 픽셀(55 x 84 mm)을 가진 차지-커플-디바이스 디텍터로 구성된 작은 동물용 PET/SPECT/CT 시스템(InveonTM; Siemens Preclinical Solutions, Knoxville, TN)을 이용하였다. 60 kVp X-레이 전압, 500 μA 양극전류, 그리고 각각의 360 회전 스텝에 대한 500 밀리세컨드 노출시간에서 이미지를 얻었다. C57BL/6 마우스의 전체 몸을 얻기 위하여 하나의 베드 포지션을 7분 동안 스캐닝하였다. 램프 필터와 함께 변형된 Feldkamp 알고리즘을 이용하여 각 베드 포지션의 2차 스라이스를 복원하였다. 50 x 50 ㎛ 픽셀 크기를 가진 512 x 512 픽셀 그리드상에 이미지를 복원하였다. HU(Hounsfield units)에 복원하기 위하여, InveonTM 지침 매뉴얼에 기재된 바에 따라 물이 들어있는 50-㎖ 폴리프로필렌 튜브를 이용하여 시스템을 연산하였다. 복원된 이미지의 해상도는 111 ㎛이었다. 목적영역에 대한 CT 데이터는 HUs을 이용하여 분석하였다.For small animals consisting of a charge-couple-device detector with X-ray energy (35-80 kVp), high-resolution phosphor screen and 2,048 x 3,072 pixels (55 x 84 mm) PET / SPECT / CT system (Inveon ™; Siemens Preclinical Solutions, Knoxville, TN) was used. Images were taken at 60 kVp X-ray voltage, 500 μA anodic current, and 500 millisecond exposure time for each 360 rotational step. One bed position was scanned for 7 minutes to get the whole body of C57BL / 6 mice. The second order slice of each bed position was reconstructed using the modified Feldkamp algorithm with a ramp filter. The image was reconstructed on a 512 x 512 pixel grid with a 50 x 50 μm pixel size. To restore to Hounsfield units (HU), the system was calculated using 50-mL polypropylene tubes containing water as described in the Inveon ™ Instruction Manual. The resolution of the reconstructed image was 111 μm. CT data for the target area were analyzed using HUs.
그 결과, 오금 림프절에서 시간이 지남(0, 24시간 및 48시간)에 따라 GNP가 축적되는 것을 확인할 수 있었다(도 4a 및 4b).As a result, it was confirmed that GNP accumulates over time (0, 24 hours and 48 hours) in the popliteal lymph nodes (FIGS. 4A and 4B).
실시예 8: Example 8:
in vitroin vitro
면역 반응 Immune response
열 불활성화시킨 10% (v/v) FBS(fetal bovine serum; Invitrogen), 100 유니트/㎖ Penicillin 및 100 ㎎/㎖ 스트렙토마이신(Invitrogen)을 포함하는 DMEM(Dulbeccos modified Eagles medium)에 RAW264.7(a murine monocytic cell line, ATCC) 세포주를 배양하였다.RAW264.7 in DMEM (Dulbeccos modified Eagles medium) containing heat-inactivated 10% (v / v) fetal bovine serum (Invitrogen), 100 units / ml Penicillin and 100 mg / ml streptomycin (Invitrogen) A murine monocytic cell line (ATCC) cell line was cultured.
GNP 콘쥬게이트에 의하여 발생된 사이토카인 mRNA 증가를 확인하기 위하여, 2시간, 4시간 동안 RAW264.7 세포주에 LPS(Lipopolysaccharides; 10 ng/㎖), A10CpG 1668 (10 μM), GNP (8 nM), GNP-RFP (8 nM) 및 GNP-CpG-RFP (8 nM)를 각각 처리하였다(표 1).To confirm cytokine mRNA increase caused by GNP conjugates, LPS (Lipopolysaccharides; 10 ng / ml), A 10 CpG 1668 (10 μM), GNP (8 nM) in RAW264.7 cell lines for 2 and 4 hours. ), GNP-RFP (8 nM) and GNP-CpG-RFP (8 nM), respectively, were treated (Table 1).
표 1
Table 1
| 그룹 | 첨가 물질 | 농도 |
| 1 | LPS | 10 ng/㎖ |
| 2 | A10CpG1668 | 10 μM |
| 3 | GNP | 8 nM |
| 4 | GNP-RFP | 8 nM |
| 5 | GNP-RFP-CpG | 8 nM |
| 6 | 음성대조군(medium treated) | - |
| group | Additive material | density |
| One | | 10 ng / |
| 2 | | 10 μM |
| 3 | | 8 |
| 4 | GNP- | 8 |
| 5 | GNP-RFP- | 8 |
| 6 | Negative treated | - |
그 다음, 모든 세포주들을 모아서 총 RNAs를 WelprepTM(Jeil Biotechservices Inc., Daegu, Korea)을 이용하여 추출하였다. 총 RNA의 1 ㎍을 UmProm-Ⅱ 역전사효소(Promega)로 역전사 시켰으며 MJ MiniTM PCR 시스템(Bio-Rad, Hercules, CA)으로 증폭하였다.Then all cell lines were pooled and total RNAs were extracted using Welprep ™ (Jeil Biotechservices Inc., Daegu, Korea). 1 μg of total RNA was reverse transcribed with UmProm-II reverse transcriptase (Promega) and amplified with MJ Mini ™ PCR system (Bio-Rad, Hercules, CA).
다음과 같은 프라이머를 이용하여 역전사 PCR 분석을 실시하였다: IL-6, 5-TTCCTCTCTGCAAGAGACT-3, 5-TGTATCTCTCTGAAGGACT-3; Actin, 5'-TCATGAAGTGTGACGTTGACATCCGT-'3,5'-TTGCGGTGCACGATGGAGGGGCCGGA-'3; IL-12p40, 5'-GAAGTTCAACATCAAGAGCAGTAG-'3, 5'-AGGGAGAAGTAGGAATGGGG-30; IL-1β, 5'-CCTGTGGCCTTGGGCCTCAA-'3, 5'-GAGGTGCTGATGTACCAGTTGG-'3; TNF-α, 5'-AAAATTCGAGTGACAAGCCTGTAG-'3, 5'-CCCTTGAAGAGAACCTGGGAGTAG-'3; iNOS2, 5'-GATGTTGAACTATGTCCTATCTCC-'3, 5'-AACACCACTTTCACCAAGAC-'3.Reverse transcription PCR analysis was performed using the following primers: IL-6, 5-TTCCTCTCTGCAAGAGACT-3, 5-TGTATCTCTCTGAAGGACT-3; Actin, 5'-TCATGAAGTGTGACGTTGACATCCGT-'3,5'-TTGCGGTGCACGATGGAGGGGCCGGA-'3; IL-12p40, 5'-GAAGTTCAACATCAAGAGCAGTAG-'3, 5'-AGGGAGAAGTAGGAATGGGG-30; IL-1β, 5'-CCTGTGGCCTTGGGCCTCAA-'3, 5'-GAGGTGCTGATGTACCAGTTGG-'3; TNF-α, 5'-AAAATTCGAGTGACAAGCCTGTAG-'3, 5'-CCCTTGAAGAGAACCTGGGAGTAG-'3; iNOS2, 5'-GATGTTGAACTATGTCCTATCTCC-'3, 5'-AACACCACTTTCACCAAGAC-'3.
GNP 컨쥬게이트를 C57BL/6 마우스 면역세포(마우스 대식세포, RAW264.7)와 혼합하였을 때, 면역세포에서의 사이토카인의 발현양상을 조사하였다. 그 결과, GNP-RFP 또는 GNP-CpG-RFP이 혼합된 C57BL/6 마우스 면역세포에서 사이토카인이 현저히 발현되는 것으로 확인되었다(도 5).When GNP conjugate was mixed with C57BL / 6 mouse immune cells (mouse macrophage, RAW264.7), the expression patterns of cytokines in immune cells were examined. As a result, it was confirmed that cytokines are remarkably expressed in C57BL / 6 mouse immune cells mixed with GNP-RFP or GNP-CpG-RFP (FIG. 5).
실시예 9: 혈청에서의 RFP-특이적 IgG 수준 분석Example 9: Analysis of RFP-specific IgG Levels in Serum
PBS, RFP(12 ㎍/마우스), GNP-RFP(12 ㎍/마우스) 및 GNP-RFP-CpG(12 ㎍/마우스)를 이용하여 3차례 주 간격으로 3회 각각 면역화시킨 C57BL/6 마우스로부터 혈청 샘플을 수득하였다. ELISA를 이용하여 혈청에 있는 항-RFP 항체를 분석하였다.(anti-mouse IgG-HRP, San Cruz Biotechnology, Inc.)Serum from C57BL / 6 mice immunized three times each at three weekly intervals using PBS, RFP (12 μg / mouse), GNP-RFP (12 μg / mouse) and GNP-RFP-CpG (12 μg / mouse) Samples were obtained. Anti-RFP antibodies in serum were analyzed using ELISA. (Anti-mouse IgG-HRP, San Cruz Biotechnology, Inc.)
그 결과, GNP-RFP의 투여군 마우스 혈청에서 높은 항체가 생성 되는 것을 확인할 수 있었다(도 6).As a result, it was confirmed that a high antibody is produced in the mouse serum of the GNP-RFP administration group (FIG. 6).
실시예 10: 마우스 CD8 T-세포로부터의 IFN-γ를 측정Example 10: Measuring IFN-γ from Mouse CD8 T-cells
1, 8 및 15일에 부스터(booster)를 이용하여 발바닥로 PBS, RFP, GNP-RFP 및 GNP-RFP-CpG를 각각 투여하여 C57BL/6 마우스에 주입하였다(표 2). 36일째에, 배수(draining) 림프절로부터 림프구를 분리하였으며, 트립신 처리된 RFP에 노출시키고 ELISA 및 FACs을 통하여 IFN-γ를 측정하였다.(Anti-Mouse IFN gamma, eBioscience,Inc.)On day 1, 8 and 15, boosters were used to inject CBSBL / 6 mice to the soles of PBS, RFP, GNP-RFP and GNP-RFP-CpG, respectively (Table 2). On day 36, lymphocytes were isolated from draining lymph nodes, exposed to trypsinized RFPs and IFN-γ measured via ELISA and FACs. (Anti-Mouse IFN gamma, eBioscience, Inc.)
표 2
TABLE 2
| 그룹 | 첨가 물질 | 농도 (㎍/㎖) | 시간 | 마우스 개체 수 |
| 1 | 음성대조군(media) | - | 72 | 3 |
| 2 | 트립신 처리된 RFP | 25 | 72 | 3 |
| group | Additive material | Concentration (μg / ml) | time | Mouse object count |
| One | Voice control | - | 72 | 3 |
| 2 | | 25 | 72 | 3 |
그 결과, GNP-RFP 또는 GNP-RFP-CpG를 투여한 마우스 국소 림프절에서의 면역세포에서 IFN-γ의 발현양이 현저히 증가하는 것을 확인할 수 있었다(도 7).As a result, it was confirmed that the expression level of IFN-γ was significantly increased in immune cells in mouse regional lymph nodes administered with GNP-RFP or GNP-RFP-CpG (FIG. 7).
또한, IFN-γ를 발현하는 면역세포의 분포를 FACS로 조사한 결과, GNP-RFP 투여군에서는 22.66%, GNP-RFP-CpG 투여군에서 28.92%가 IFN-γ를 발현하는 면역세포로 확인되었다(도 8).In addition, when the distribution of immune cells expressing IFN- [gamma] was examined by FACS, 22.66% in the GNP-RFP-administered group and 28.92% in the GNP-RFP-CpG-administered group were identified as immune cells expressing IFN- [gamma] (FIG. 8). ).
실시예 11: GNPs 백신화 후 Example 11: After GNPs Vaccination
인 비보In Vivo
상에서의 T-세포 수T-cell count on the stomach
1, 8 및 15일에 부스터(booster)를 이용하여 발바닥로 PBS, RFP, GNP-RFP 및 GNP-RFP-CpG를 투여하여 C57BL/6 마우스를 면역화시켰다. 60일째에, 드레이닝(drainnig) 림프절로부터 T-세포를 분리하였으며, FACs 분석(나이브(na) T-세포 마커인 CD62L과 CD45RB를 이용)을 이용하여 나이브(na) T-세포수를 조사하였다.C57BL / 6 mice were immunized with boosters (PBS, RFP, GNP-RFP, and GNP-RFP-CpG) on the soles of boosters on days 1, 8 and 15. On day 60, T-cells were isolated from draininig lymph nodes and naïve T-cell counts were examined using FACs analysis (using naive T-cell markers CD62L and CD45RB). .
그 결과, GNP 컨쥬게이트 투여군이 음성대조군과 비교하여 적은 naT 세포 분포도를 나타내었다(도 9). As a result, the GNP conjugate administration group showed less naT cell distribution compared with the negative control group (FIG. 9).
또한, Foxp3 항체를 이용하여 Foxp3를 발현하는 조절 T 세포(regulatory T cells, Treg)의 수를 확인한 결과, GNP-컨쥬게이트 투여군과 음성대조군의 차이가 거의 없는 것으로 확인되었다(도 10).In addition, as a result of confirming the number of regulatory T cells (Treg) expressing Foxp3 using the Foxp3 antibody, it was confirmed that there is almost no difference between the GNP-conjugated group and the negative control group (FIG. 10).
실시예 12: GNPs 백신화시킨 후 Example 12 After GNPs Vaccination
in vivoin vivo
상에서의 T-세포 증식T-cell proliferation in the stomach
1, 8 및 15일에 부스터(booster)를 이용하여 발바닥로 PBS, RFP, GNP-RFP 및 GNP-RFP-CpG를 투여하여 C57BL/6 마우스를 면역화시켰다. 60일째에, 배수 림프절로부터 T-세포를 분리하였으며, 다양한 농도로 트립신처리된 RFP에 노출시킨 다음, 열불활성화시킨 10% (vol/vol) FBS (HyClone)를 포함하는 T-세포 배지를 포함하는 편평한 바닥을 가진 96-웰 플레이트에서 T-세포를 5672시간 동안 배양하였다. T-세포를 5672시간 배양시킨 후, NEN([H3]-thymidine) 0.5 μCi을 각각의 웰에 첨가하였으며 16시간 동안 추가적으로 배양시켰다. 배양된 세포를 회수한 후 리퀴드 신틸레이션 카운팅을 이용하여 [H3]-티미딘 흡수를 측정하였다.C57BL / 6 mice were immunized with boosters (PBS, RFP, GNP-RFP, and GNP-RFP-CpG) on the soles of boosters on days 1, 8 and 15. On day 60, T-cells were isolated from draining lymph nodes and exposed to various concentrations of trypsinized RFP, followed by heat-inactivated T-cell medium containing 10% (vol / vol) FBS (HyClone). T-cells were incubated for 5672 hours in 96-well plates with flat bottoms. After 5672 hours of T-cell incubation, 0.5 μCi of NEN ([H 3 ] -thymidine) was added to each well and further incubated for 16 hours. After the cultured cells were recovered, [H 3 ] -thymidine uptake was measured using liquid scintillation counting.
그 결과, GNP 컨쥬게이트 투여 면역군들에서 음성대조군과는 달리 RFP의 농도에 따라 의존적으로 세포가 증식하는 것으로 나타났다(도 11).As a result, in the GNP conjugate-administered immune groups, the cells proliferated depending on the concentration of RFP, unlike the negative control group (FIG. 11).
실시예 13: Example 13:
in vivoin vivo
에서 암 백신화Vaccination in Cancer
암백신 효율을 평가하기 위하여, 1, 8 및 15일에 발바닥에 PBS, RFP, GNP-RFP 및 GNP-RFP-CpG를 주입하여 C57BL/6 마우스를 면역화시켰다. 21일째에, RFP로 형질전환시킨 B16F10 세포(5 x 105 세포/마우스) 또는 야생형 B16F10 세포(5 x 105 세포/마우스)를 마우스 등에 접종하고 종양 크기를 관찰하였다. 또한, 종양 접종 후 생존률을 모니터링하였다.To assess cancer vaccine efficiency, C57BL / 6 mice were immunized by injecting PBS, RFP, GNP-RFP and GNP-RFP-CpG into the soles on days 1, 8 and 15. On day 21, B16F10 cells (5 × 10 5 cells / mouse) or wild type B16F10 cells (5 × 10 5 cells / mouse) transformed with RFPs were seeded in mice and tumor size was observed. In addition, survival after tumor inoculation was monitored.
그 결과, GNP 컨쥬게이트를 투여한 면역군에서 암의 성장이 한 달 정도 더 지연되는 결과는 관찰하였다(도 12).As a result, the growth of cancer was delayed by one month in the immune group administered with GNP conjugate (FIG. 12).
또한, GNP 컨쥬게이트를 마우스에 일주일 간격으로 3회 면역시킨 후 B16F10-RFP 암 세포를 투여한 이후 마우스의 생존율에 있어서, GNP-RFP를 면역한 군이 음성대조군들에 비해서 개체의 생존율이 60일정도 더 연장되었다(도 13). In addition, in the survival rate of mice after immunizing GNP conjugates to mice three times at weekly intervals, and then administering B16F10-RFP cancer cells, the group that was immunized with GNP-RFP had a 60-day survival rate compared to the negative controls. Is further extended (FIG. 13).
뿐 만 아니라, GNP 컨쥬게이트를 마우스에 일주일 간격으로 3회 면역시킨 후 RFP를 발현하지 않은 B16F10 암 세포를 투여한 이후의 암 성장을 관찰하였다. 그 결과, 모든 실험군에서 암의 크기가 비슷하게 성장하는 결과를 확인하였다(도 14). 이러한 결과를 통하여, GNP 컨쥬게이트들이 RFP 특이적인 면역반응을 유도한다는 결론을 얻게 되었다.In addition, GNP conjugates were immunized three times a week at mice and cancer growth was observed after administration of B16F10 cancer cells that did not express RFP. As a result, it was confirmed that the growth of cancer in all the experimental groups similarly (Fig. 14). From these results, it was concluded that GNP conjugates induce RFP specific immune responses.
실시예 14: 고형 종양(solid tumor)에 대한 Example 14: against solid tumors
인 비보In Vivo
에서의 항암 효율Anticancer efficiency
암 치료율을 평가하기 위하여, 0일째에 6주령 암컷 C57BL/6 마우스에 등옆구리로 전달되는 5 x 105 형질전환된 B16F10 세포를 피하 주입하였다. 10일째에, 종양이 30 ㎣ 이상이 되는 경우 마우스를 그룹으로 임의로 나누었으며 10, 13, 16, 21 및 26일에 PBS, RFP, GNP-RFP 및 GNP-RFP-CpG를 등 부위에 다양한 농도로 주입한 후 종양 크기를 관찰하였다.To assess cancer treatment, 6-week-old female C57BL / 6 mice were injected subcutaneously with 5 × 10 5 transformed B16F10 cells delivered to the dorsal flank on day 0. On day 10, mice were randomly divided into groups when tumors became more than 30 mm 3 and PBS, RFP, GNP-RFP, and GNP-RFP-CpG at various concentrations in the dorsal region at 10, 13, 16, 21 and 26 days. Tumor size was observed after injection.
그 결과, GNP 컨쥬게이트를 투여한 군이 음성 대조군에 비해서 암의 성장이 더 지연되는 결과를 나타내었다(도 15).As a result, the group administered with the GNP conjugate showed a more delayed growth of cancer compared to the negative control (FIG. 15).
실시예 15: 뮤신 항원 펩타이드와 금 나노입자와의 화학적 결합체 제조 및 Example 15 Preparation of Chemical Conjugates of Mucin Antigen Peptides with Gold Nanoparticles and
in vivoin vivo
백신 실험 Vaccine Experiment
다음과 같은 두 가지 형태의 뮤신 항원 펩타이드를 제조하였다: 1. CAL-뮤신: CALNN PDTRPAPGSTAPPAHGVTSA PDTRPAPGST; 2. CGG-뮤신: CGGGG PDTRPAPGSTAPPAHGVTSA PDTRPAPGST.(애니젠, 대한민국)Two forms of mucin antigen peptides were prepared: 1. CAL-mucin: CALNN PDTRPAPGSTAPPAHGVTSA PDTRPAPGST; CGG-mucin: CGGGG PDTRPAPGSTAPPAHGVTSA PDTRPAPGST. (Anigen, South Korea)
상기와 같은 이런 뮤신 항원 펩타이드를 증류수에 10 mM 농도로 녹인 뒤 6 μM 금 나노입자(GNP)와 혼합하였다. 그 다음, 혼합물을 상온에서 24시간동안 반응 공유결합으로 금 나노입자에 뮤신 펩타이드를 공유결합으로 결합 시킨 후, 자외선 및 가시선 분광 분석법을 이용하여 금 나노입자와 공유결합여부를 확인하였으며 HPLC 분석을 통해서 뮤신 펩타이드를 정량하였다.Such mucin antigen peptides were dissolved in distilled water at a concentration of 10 mM and mixed with 6 μM gold nanoparticles (GNP). Then, the mixture was covalently bonded to the gold nanoparticles with mucin peptides by reaction covalent bonding at room temperature for 24 hours, and then confirmed whether the covalent bonds with the gold nanoparticles by UV and visible spectroscopy. Mucin peptides were quantified.
하기 표 3과 같은 실험군으로 마우스의 발바닥에 일주일 간격으로 3번 투여 후 14일 후에 혈액을 취하여 뮤신 펩타이드에 대한 항체 생성여부를 ELISA 방법으로 확인하였다(anti-mouse IgG-HRP, San Cruz Biotechnology, Inc.).In the experimental group as shown in Table 3, blood was taken 14 days after the administration to the sole of the mouse three times at weekly intervals, and the production of antibodies to the mucin peptide was confirmed by ELISA (anti-mouse IgG-HRP, San Cruz Biotechnology, Inc.). .).
표 3
TABLE 3
| 실험군 | 물질 | 농도 (펩타이드) |
| G1 | PBS | 0.03 mL |
| G2 | CGGMucin peptide | 1.5 nmole |
| G3 | CALMucin peptide | 1.5 nmole |
| G4 | GNP-CGGMUC | 1.5 nmole |
| G5 | GNP-CALMUC | 1.5 nmole |
| Experimental group | matter | Concentration (peptide) |
| G1 | PBS | 0.03 mL |
| G2 | CGGMucin peptide | 1.5 nmole |
| G3 | CALMucin peptide | 1.5 nmole |
| G4 | GNP-CGGMUC | 1.5 nmole |
| G5 | GNP-CALMUC | 1.5 nmole |
그 결과, 뮤신 펩타이드와 공유결합 한 금 나노입자를 투여한 실험 군에서 뮤신 특이적인 항체가 많이 생산되는 것으로 확인되었다(도 16).As a result, it was confirmed that a lot of mucin-specific antibodies are produced in the experimental group administered with the gold nanoparticles covalently bonded to the mucin peptide (Fig. 16).
뮤신 항원 펩타이드와 금 나노입자와의 화학적 결합체를 이용한 in vivo 항암 치료실험In vivo chemotherapy using chemical conjugate of mucin antigen peptide and gold nanoparticles
항암 능력 평가를 위해서 아래의 실험군으로 마우스의 발바닥에 일주일 간격으로 3번 투여 후 7일 후에 각 실험군의 동물에 뮤신 단백질을 발현하는 B16F1-뮤신 암세포를 실험동물의 피하로 투여하여 암의 성장여부를 관찰하였다.To evaluate anticancer activity, the following experimental group was administered to the soles of mice three times at weekly intervals, and 7 days later, B16F1-mucin cancer cells expressing mucin protein to the animals of each experimental group were administered subcutaneously to the experimental animals. Observed.
표 4
Table 4
| 실험군 | 물질 | 농도 (펩타이드) |
| G1 | 생리식염수 | 0.03 mL |
| G2 | CGGMucin peptide | 1.5 nmole |
| G3 | CALMucin peptide | 1.5 nmole |
| G4 | GNP-CGGMUC | 1.5 nmole |
| G5 | GNP-CALMUC | 1.5 nmole |
| Experimental group | matter | Concentration (peptide) |
| G1 | Saline solution | 0.03 mL |
| G2 | CGG Mucin peptide | 1.5 nmole |
| G3 | CAL Mucin peptide | 1.5 nmole |
| G4 | GNP- CGG MUC | 1.5 nmole |
| G5 | GNP- CAL MUC | 1.5 nmole |
그 결과, 도 15에 나타난 바와 같이 암 크기의 변화에 있어서 음성 대조군에 비해서 금 나노입자를 투여한 실험군이 유의적으로 암 성장을 억제시키는 것으로 확인되었다.As a result, as shown in FIG. 15, it was confirmed that the experimental group to which gold nanoparticles were administered significantly inhibited cancer growth in the change in cancer size compared to the negative control.
또한, GNP 컨쥬게이트를 마우스에 일주일 간격으로 3회 면역시킨 후 B16F1-뮤신 암 세포를 투여한 이후 마우스의 생존율에 있어서, GNP-CALMUCIN를 면역한 군이 음성대조군들에 비해서 개체의 생존율이 현저히 더 연장되었다(도 18). In addition, in the survival rate of mice after immunizing GNP conjugates to mice three times at weekly intervals and then administering B16F1-mucin cancer cells, the groups that immunized with GNP- CAL MUCIN showed significantly higher survival rates than the negative controls. Further extended (FIG. 18).
뿐 만 아니라, GNP 컨쥬게이트를 마우스에 일주일 간격으로 3회 면역시킨 후 뮤신을 발현하지 않은 B16F1 암 세포를 투여한 이후의 암 성장을 관찰하였다. 그 결과, 모든 실험군에서 암의 크기가 비슷하게 성장하는 결과를 확인하였다(도 19). 이러한 결과를 통하여, GNP 컨쥬게이트들이 뮤신 특이적인 면역반응을 유도한다는 결론을 얻게 되었다.In addition, GNP conjugates were immunized three times a week at mice, followed by cancer growth after administration of B16F1 cancer cells that did not express mucin. As a result, it was confirmed that the growth of cancer in all the experimental groups similarly (Fig. 19). These results led to the conclusion that GNP conjugates induce mucin specific immune responses.
실시예 16: 자성나노입자를 이용한 백신 실험Example 16: Vaccine Experiment with Magnetic Nanoparticles
자성나노입자(TLC-SPION)를 이용한 백신 실험을 진행하기 위해서 2개의 시스테인으로 변형된 RFP(red fluorescence protein) 단백질을 화학적으로 컨쥬게이션을 실시하였다. 즉, RFP-자성나노입자의 경우 최종 농도 15 mg(0.5 mL) 자성나노입자 용액에 수용성 단백질 용액(0.2 ㎖; 117 ㎍/㎖)을 첨가한 후 실온에서 아래의 표 5와 같이 컨쥬게이션을 24시간 동안 실시 한 후, 자성 나노입자를 자성체로 결합시켜 잉여의 단백질을 제거하였다. 도 20a는 하기 표 5의 G1에 대한 것이고 34.2± 7.5 nm 입자크기, 도 20b는 하기 표 5의 G2 대한 것이고 32.4± 6.4 nm 입자크기, 도 20c는 하기 표 5의 G3에 대한 것이며, 34± 6.7 nm 입자크기를 나타낸다.In order to proceed with a vaccine experiment using magnetic nanoparticles (TLC-SPION), two cysteine modified red fluorescence protein (RFP) proteins were chemically conjugated. That is, in the case of RFP-magnetic nanoparticles, a water-soluble protein solution (0.2 ml; 117 µg / ml) was added to a final concentration of 15 mg (0.5 mL) magnetic nanoparticle solution, followed by conjugation at room temperature as shown in Table 5 below. After running for a period of time, the magnetic nanoparticles were bound to the magnetic body to remove excess protein. FIG. 20a is for G1 of Table 5 below and 34.2 ± 7.5 nm particle size, FIG. 20b is for G2 of Table 5 below and 32.4 ± 6.4 nm particle size, FIG. 20c is for G3 of Table 5 below, 34 ± 6.7 nm particle size.
표 5
Table 5
| - | 자성나노입자 | RFP | EDC | NHS | 완충액 | 부피 |
| G1 | 15 mg/0.5mL | 117 μg/ 0.2 mL | 20 μmole100 μL | 20 μmole100 μL | pH 7.4 | 0.9 mL |
| G2 | 15 mg/0.5mL | 117 μg/ 0.2 mL | 20 μmole100 μL | 20 μmole100 μL | pH 8 | 0.9 mL |
| G3 | 15 mg/0.5mL | 117 μg/ 0.2 mL | 20 μmole100 μL | 20 μmole100 μL | pH 9 | 0.9 mL |
| - | Magnetic Nanoparticles | RFP | EDC | NHS | | volume | |
| G1 | |||||||
| 15 mg / 0.5mL | 117 μg / 0.2 | 20 | 20 | pH 7.4 | 0.9 | ||
| G2 | |||||||
| 15 mg / 0.5mL | 117 μg / 0.2 | 20 | 20 | pH | 8 | 0.9 | |
| G3 | |||||||
| 15 mg / 0.5mL | 117 μg / 0.2 | 20 | 20 | pH 9 | 0.9 mL |
혈청에서의 RFP-특이적 항체 수준 분석RFP-specific Antibody Level Analysis in Serum
생리식염수, RFP(12 ㎍/마우스) 및 자성나노입자-RFP(12 ㎍/마우스) 를 이용하여 3차례 주 간격으로 3회 각각 면역화시킨 C57BL/6 마우스로부터 혈청 샘플을 수득하였다. ELISA를 이용하여 혈청에 있는 항-RFP 항체를 분석하였다(anti-mouse IgG-HRP, San Cruz Biotechnology, Inc.) 그 결과, 자성나노입자-RFP의 투여군 마우스 혈청에서 높은 항체가 생성 되는 것을 확인할 수 있었다(도 21).Serum samples were obtained from C57BL / 6 mice immunized three times each at three weekly intervals using physiological saline, RFP (12 μg / mouse) and magnetic nanoparticle-RFP (12 μg / mouse). Anti-RFP antibody in serum was analyzed using ELISA (anti-mouse IgG-HRP, San Cruz Biotechnology, Inc.). As a result, it was confirmed that high antibody was produced in the serum of the group treated with magnetic nanoparticle-RFP. (FIG. 21).
이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
Claims (14)
- (a) 이미징 및 운반 작용을 하는 이미징-운반 이중기능 입자로서의 금 나노입자 또는 자성 나노입자; 및 (b) 상기 이미징-운반 이중기능 입자의 표면에 결합된 항원(antigen)을 포함하는 백신 전달체(delivery system)로서, 상기 백신 전달체는 상기 항원의 전달 및 상기 항원 전달의 추적(tracing)을 할 수 있도록 하는 것을 특징으로 하는 백신 전달체.(a) gold nanoparticles or magnetic nanoparticles as imaging-carrying bifunctional particles for imaging and transporting action; And (b) a vaccine delivery system comprising an antigen bound to the surface of the imaging-carrying bifunctional particle, wherein the vaccine delivery system is capable of delivering the antigen and tracing the antigen delivery. Vaccine delivery, characterized in that.
- 제 1 항에 있어서, 상기 항원은 미생물 유래 항원, 바이러스 유래 항원, 기생충 유래 항원, 식물 유래 항원, 동물 유래 항원, 내재성(endogenous) 항원 및 합성 항원으로 이루어진 군으로부터 선택된 하나 이상의 항원을 포함하는 것을 특징으로 하는 백신 전달체.The method of claim 1, wherein the antigen comprises at least one antigen selected from the group consisting of microbial antigen, virus derived antigen, parasite derived antigen, plant derived antigen, animal derived antigen, endogenous antigen and synthetic antigen. A vaccine carrier characterized by the above.
- 제 2 항에 있어서, 상기 미생물 유래 항원은 박테리아균 유래 항원, 진균(fungi) 유래 항원 또는 사상균(mold) 유래 항원인 것을 특징으로 하는 백신 전달체.The vaccine carrier according to claim 2, wherein the microorganism-derived antigen is a bacterial bacteria-derived antigen, a fungi-derived antigen, or a mold-derived antigen.
- 제 2 항에 있어서, 상기 바이러스 유래 항원은 HIV(human immunodeficiency virus) 유래 항원, HPV(human Papilloma viruses) 유래 항원, 인플루엔자 바이러스 유래 항원, 헤르페스 바이러스 유래 항원, 헤파티티스 바이러스 유래 항원 또는 뇌염 바이러스 유래 항원인 것을 특징으로 하는 전달체.According to claim 2, wherein the virus-derived antigen is HIV (human immunodeficiency virus) antigen, HPV (human Papilloma viruses) antigen, influenza virus-derived antigen, herpes virus-derived antigen, hepatitis virus-derived antigen or encephalitis virus-derived antigen It is a transfer body characterized by the above-mentioned.
- 제 2 항에 있어서, 상기 식물 유래 항원 또는 동물 유래 항원은 알러지를 유발하는 항원인 것을 특징으로 하는 백신 전달체.The vaccine delivery vehicle according to claim 2, wherein the plant-derived antigen or the animal-derived antigen is an allergen.
- 제 2 항에 있어서, 상기 내재성 항원은 암 세포 항원, 암 유발 항원 또는 자가면역질환 유발 항원인 것을 특징으로 하는 백신 전달체.The vaccine delivery vehicle according to claim 2, wherein the endogenous antigen is a cancer cell antigen, a cancer causing antigen or an autoimmune disease causing antigen.
- 제 2 항에 있어서, 상기 합성 항원은 약물(drug) 항원인 것을 특징으로 하는 백신 전달체.3. The vaccine delivery system according to claim 2, wherein the synthetic antigen is a drug antigen.
- 제 1 항에 있어서, 상기 항원은 단일쇄(single strand) 올리고뉴클레오타이드 또는 폴리뉴클레오타이드, 이중쇄(double strand) 올리고뉴클레오타이드 또는 폴리뉴클레오타이드, 단백질, 폴리펩타이드, 올리고펩타이드, 지질, 지질단백질, 당지질, 당당백질, 프로테오글리칸, 폴리사카라이드 및 리포폴리사카라이드로 이루어진 군으로부터 선택된 하나 이상의 항원을 포함하는 것을 특징으로 하는 백신 전달체.The method of claim 1, wherein the antigen is a single strand oligonucleotide or polynucleotide, double strand oligonucleotide or polynucleotide, protein, polypeptide, oligopeptide, lipid, lipoprotein, glycolipid, glycoprotein A vaccine carrier comprising at least one antigen selected from the group consisting of, proteoglycans, polysaccharides and lipopolysaccharides.
- 제 1 항에 있어서, 상기 백신 전달체는 면역보조제(adjuvants)를 추가적으로 포함하는 것을 특징으로 하는 백신 전달체.The vaccine delivery system of claim 1, wherein the vaccine delivery system further comprises an adjuvants.
- 제 9 항에 있어서, 상기 면역보조제는 상기 항원 또는 상기 이미징-운반 이중기능 입자의 표면에 결합되어 있는 것을 특징으로 하는 백신 전달체.10. The vaccine delivery system of claim 9, wherein said adjuvant is bound to the surface of said antigen or said imaging-carrying bifunctional particles.
- 제 9 항에 있어서, 상기 면역보조제는 CpG 올리고데옥시뉴클레오타이드, 언메틸레티드 CpG DNA(unmethylated cystein-phosphate-guanine DNA), 이중쇄 RNA, 미생물유래 DNA 또는 RNA, 핵산 유도체, 지질, 리포폴리사카라이드, 리포프로테인, 리포펩타이드, 당지질, 펩티도글리칸, 글리코펩타이드, 단백질, 재조합 단백질, 플라젤린(flagellin), 비로좀(virosome), Ribi(monophosphoryl-lipid A/trehalose dicorynomycolate), 사포닌 및 스쿠알렌(squalene) 스쿠랄렌(squalene) 및 핵산 유도체로 이루어진 군으로부터 선택된 하나 이상의 면역보조제를 포함하는 것을 특징으로 하는 백신 전달체.10. The method of claim 9, wherein the adjuvant CpG oligodeoxynucleotide, unmethylated cystein-phosphate-guanine DNA (DCP), double-stranded RNA, microorganism-derived DNA or RNA, nucleic acid derivatives, lipids, lipopolysaka Rydes, lipoproteins, lipopeptides, glycolipids, peptidoglycans, glycopeptides, proteins, recombinant proteins, flagellin, virosomes, monophosphoryl-lipid A / trehalose dicorynomycolate, saponins, and squalene squalene) A vaccine carrier comprising at least one adjuvant selected from the group consisting of squalene and nucleic acid derivatives.
- 제 1 항에 있어서, 상기 백신 전달체는 림프절로 선택적으로 상기 항원을 운반하는 것을 특징으로 하는 백신 전달체.2. The vaccine delivery system of claim 1, wherein said vaccine delivery vehicle selectively carries said antigen to lymph nodes.
- 제 1 항에 있어서, 상기 이미징-운반 이중기능 입자는 5-50 nm의 크기를 가지는 것을 특징으로 하는 백신 전달체.The vaccine delivery vehicle of claim 1, wherein the imaging-carrying bifunctional particle has a size of 5-50 nm.
- (a) 이미징 및 운반 작용을 하는 이미징-운반 이중기능 입자로서의 금 나노입자 또는 자성 나노입자; 및 (b) 상기 이미징-운반 이중기능 입자의 표면에 결합된 암 항원을 포함하는 항암용 백신 약제학적 조성물.(a) gold nanoparticles or magnetic nanoparticles as imaging-carrying bifunctional particles for imaging and transporting action; And (b) a cancer antigen bound to the surface of the imaging-carrying bifunctional particle.
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| CN114469895A (en) * | 2022-03-14 | 2022-05-13 | 海南大学 | Preparation method of metal polyphenol nano vaccine for delivering antigen and immune environment regulator and obtained product |
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