Vaccine composition or kit for reducing the size or volume of target tissue, containing genetic material encoding an exogenous antigen.

Abstract

The present invention provides a vaccine composition for reducing the size or volume of a target tissue, comprising genetic material encoding a foreign antigen, and preferably, the composition can be provided as a vaccine composition for treating or preventing obesity. The vaccine composition can also be provided as a composition for removing subcutaneous adipocytes.

Description

Technical Field

[0001] This application claims priority based on Korean Patent Application No. 10-2020-0138615 filed on October 23, 2020, and all the content disclosed in the specification and drawings of the application is incorporated into this application.

[0002] The present invention relates to a pharmaceutical composition for inducing an immune response by containing a genetic substance encoding a foreign antigen to reduce the size or volume of a target tissue, and more particularly, to a therapeutic vaccine composition for preventing and / or treating obesity. More specifically, the present invention relates to a composition for inducing cell death of cells constituting a target site by immunotherapy with an antigen gene.

Background Art

[0003] “Obesity” does not simply mean having a high body weight, but rather refers to “a state in which an excessive amount of body fat is accumulated in the body”, and is regarded as a risk factor that increases the incidence of various chronic diseases such as diabetes, cardiovascular diseases, and cancer. Obesity, which is defined as an excessive accumulation of fat, is accompanied by a loss of metabolic, endocrine, and immune functions of adipose tissue, and such pathological remodeling of adipose tissue has attracted attention as a major pathophysiological factor of metabolic diseases.

[0004] For drug treatment for obesity, “Orlistat (generic name)” which is a lipase inhibitor is used. This plays a role in discharging a part of the fat in the body outside the body, but as side effects, it causes diarrhea and fatty stools. In the case of drugs for treating obesity, there are many side effects, and in the female body, it may also decompose fat in undesired sites. At present, there is no appetite suppressant that can be safely used, and in the case of severely obese patients with complications, obesity surgery on the gastrointestinal tract may be helpful.

Summary of the Invention

Problems to be Solved by the Invention

[0005] To solve the problems described above, the present invention aims to provide a novel method or pharmaceutical composition that can be administered to a target tissue to reduce its size or volume. More specifically, the method or pharmaceutical composition can be administered locally to a desired site to kill mast cells. Another embodiment aims to provide a novel obesity treatment or therapeutic vaccine. The vaccine composition can be applied after at least one or more pre-vaccines have been applied to the individual.

[0006] The problem that this invention aims to solve is to provide a novel form of obesity treatment agent or therapeutic vaccine that can eliminate localized obesity and create a supple and smooth body line.

[0007] The problem that this invention aims to solve is to provide a novel adipocyte reduction composition in which the immune system attacks adipocytes that express antigen proteins, thereby promoting the death of adipocytes, and causing the death of adipocytes without affecting adipocytes in undesirable locations. [Means for solving the problem]

[0008] One aspect of the present invention provides a novel concept of a pharmaceutical composition (preferably a vaccine composition) for reducing the size or volume of tissue, in which a genetic material encoding an antigen derived from outside the human body is administered to a target tissue, and when the antigen gene is expressed in the cells constituting the target tissue due to the administered genetic material, the existing immune system acts against it to kill the cells. More specifically, the aim is to provide a vaccine for the prevention or treatment of obesity or a composition for removing subcutaneous fat cells.

[0009] One aspect of the present invention provides a novel method for reducing the size or volume of tissue, in which a genetic material encoding an antigen derived from outside the human body is administered to a target tissue, and when the antigen gene is expressed in the cells constituting the target tissue due to the administered genetic material, the existing immune system acts against it to kill those cells.

[0010] The inventors of this invention discovered that by using an immune response, it is possible to kill the cells constituting a target site and reduce the size of the tissue composed of those cells, thus completing the present invention. In particular, through experiments, it has been confirmed that the present invention can achieve effective removal and / or reduction of fat cells, thereby enabling body shape correction and obesity treatment.

[0011] One embodiment of the present invention is a pharmaceutical composition for administration to a target tissue to reduce the size of the tissue, wherein the composition comprises genetic material encoding a foreign antigen. One embodiment of the present invention provides a target cell death and target tissue reduction application of the genetic material encoding the foreign antigen, and more specifically, a local adipose tissue reduction application of the genetic material. Preferably, the genetic material encoding the foreign antigen comprises one or more viruses selected from the group consisting of yellow fever virus, varicella-zoster virus, and rubella virus, or epitopes thereof. One embodiment may provide a target cell death or target tissue reduction application of the virus or these epitopes. The tissue is not particularly limited and may include, for example, a tissue consisting of one or more cells selected from the group consisting of adipocytes, muscle cells, osteocytes, chondrocytes, skin cells, secretory cells, and hematopoiesis, and preferably the tissue may be adipose tissue or muscle tissue, and more preferably subcutaneous adipose tissue. The composition can induce a reduction or death of adipocytes, in particular adipocytes distributed in subcutaneous fat.

[0012] The inventors of the present invention provide a composition or kit that has fewer side effects compared to existing fat removal surgeries and drugs, and that can be expected to have the effect of correcting body shape or body form by targeting only a local target area (e.g., adipose tissue in a specific area). The present invention provides a pharmaceutical composition that acts locally on a target area and reduces the size or volume of target tissue using immunotherapy. Preferably, the present invention provides a pharmaceutical composition, vaccine composition, or therapeutic vaccine composition that induces the death of adipocytes, particularly subcutaneous adipocytes.

[0013] As used herein, “target tissue or target site” means a population of cells having the same structure and function for reducing size or volume. “Body shape correction or body shape correction” means that the effect is to reduce the size or volume of tissue (e.g., adipose tissue) accumulated in an undesirable area by killing or reducing the cells that make up the tissue, thereby making the body appear slimmer. As used herein, “working locally” means that the effect of cell death or reduction of tissue size or volume occurs within radii of approximately 10 cm, 9 cm, 8 cm, 7 cm, 6 cm, 5 cm, 4 cm, 3 cm, 2 cm, and 1 cm from the injection site, and preferably within a range of approximately 3 cm.

[0014] When an individual is exposed to an antigen, immune cells functionally perform phagocytosis and antigen presenting. In one embodiment, the pharmaceutical composition of the present invention may be administered after an immune environment has been created by intentionally or unintentionally introducing foreign substances, such as protein antigens or their epitopes, into the body so as to induce an immune response. Alternatively, the pharmaceutical composition may be repeatedly administered into the body through another embodiment. In yet another embodiment, the genetic material contained in the pharmaceutical composition of the present invention may be administered to an individual to induce existing immunity, and then, through repeated administration of the composition, the adipocytes to which the antigen has been presented may die due to substances secreted by the immune response in the human body, such as monocytes, neutrophils, natural killer cells (a type of lymphocyte), cytotoxic T cells, immune cells, bioactive substances, antibodies, and complement, thereby reducing the size or volume of adipose tissue. The composition can be used for body shape correction or body form correction, and can be used for localized body shape correction. Furthermore, the composition can be administered to muscle tissue for wrinkle improvement effects similar to Botox, and for body shape correction through muscle contraction. In addition, it can be used for the removal of positive or malignant tumors, or for the removal of skin warts.

[0015] The pharmaceutical composition comprises genetic material encoding at least one antigen, wherein the genetic material includes not only DNA and / or RNA, but also antisense nucleotides, mRNA (messenger RNA), ssRNA (single-stranded RNA), cDNA (complementary DNA), etc., associated with the genetic material. The antigen is a substance that causes an immune response to produce an antibody, and may include tumor antigens, viruses, bacteria, proteins, or antigenic epitopes. The antigen may be any antigen as long as it induces immunity, and may be one or more selected from the group consisting of non-human antigens, viral antigens, bacterial antigens, fungal antigens, protozoan antigens, avian antigens, parasitic antigens, mycoplasma antigens, and plant antigens, which are already known in the art, and the antigen may be expressed as a peptide, a complete protein, or as part thereof. Specifically, non-restrictive examples of viruses include: Retroviridae (e.g., human immunodeficiency virus, e.g., HIV-1); Picornaviridae (e.g., poliovirus, hepatitis A virus; enterovirus, human coxsackievirus, rhinovirus, echovirus); Caliciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviridae (e.g., dengue virus, encephalitis virus, yellow fever virus) Rus); Coronaviridae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus); Bunyaviridae (e.g.,Hantavirus, Bunyavirus, Phlebovirus, and Nylovirus; Arenaviridae (Hemorrhagic fever viruses); Reoviridae (e.g., Reovirus, Orbivirus, and Rotavirus); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae (Parvovirus); Papovaviridae (Papillomavirus, Polyomavirus); Adenoviridae (Many adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpesviruses); Poxviridae (bariola virus, vaccinia virus, poxvirus); and Iridoviridae (e.g., African swine fever virus). Non-restrictive examples of bacterial antigens include Pasteurella, Staphylococci, Streptococcus, and Escherichia coli. This includes Helicobacter pylori, Pseudomonas species, and Salmonella species. Non-restrictive examples of infectious bacteria include Helicobacter pylori, Borelia burgdorferi, Legionella pneumophila, Mycobacteria spp (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansasii, M. gordone), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitides,Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (Billidance group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (Anaerobic species), Streptococcus pneumonia, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus antracis, Corynebacterium diphtheria diphtheria), Corynebacterium genus, Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidum Antigens derived from *Treponema pallidum*, *Treponema pertenue*, *Leptospira*, *Rickettsia*, and *Actinomyces israelli* may be included without limitation. Non-limiting examples of bacterial pathogens used in the present invention include *Streptococcus pneumoniae*, *Moraxella catarrhalis*,This includes Mycoplasma pneumoniae, Klebsiella pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Chlamydia pneumoniae, Legionella pneumophila, or Bordetella partis, or any bacteria that are pathogenic in the lungs. Non-restrictive examples of fungal antigens include those derived from Aspergillus fumigatus, Blastomyces, Coccidioides imitis, Coccidioides posadasi, Cryptococcus neoformans, Cryptococcus gattii, Fusarium, Histoplasma capsulatum, Paesilomyces, Paracoccidioides brasiliensis, Penicillium marnefei, Pneumocystis irovechii, Pseudoalescheria boidii, Skedosporium apiospermum, Rhizopus, Mucor, Abscisia, Kuningamera, Skedosporium prolificans, Stachybotrys cartalum, Trichoderma longibrachiatum, and Trichosporon. It may contain antigenic components derived from parasites, examples of which include: Cestodes (tapeworms), Taenia saginata, Taenia solium, Diphyllobothrium, Hymenolepis nana, Hymenolepis diminuta, Dipyridium caninum, Nematodes (roundworms), A. ascaris rumbricoides, Strongyroides stercolalis, Necatolus americanus, Ancillostoma duodenale, Ancillostoma caninum, Tritulis tritula, Capillaria philippinensis, Tricostrillius species, Tricinella Antigens derived from species such as Necator americanus, Anisakis and subspecies, Angioslongirus costalicensis, Enterobius vermicularis, Trematodes (trematodes), Fasciolopsis buschi, Heterofies species, Echinostomia species, Cronortis sinensis, Opistolchis species, Fasciola species, Metagonimus yokogawai, Cystosoma mansoni, Cystosoma japonicum, Cystosoma mekongii, Cystosoma intercolorum, Echinostomia species and Paragonimus species are preferred, taking into consideration the aspect of tissue size reduction, yellow fever virus, varicella-zoster virus, rubella virus, influenza virus, mumps virus,It may contain one or more viruses selected from the group consisting of and measles viruses, preferably measles viruses. Preferably, the genetic material encoding the antigen may include a nucleic acid molecule having the nucleic acid sequence shown in SEQ ID NO: 1. Alternatively, the genetic material may include a nucleic acid molecule encoding a peptide having SEQ ID NO: 5 or 6.

[0016] Furthermore, the genetic material may include a polynucleotide having a nucleic acid sequence that has at least 85% sequence homology to Sequence ID No. 1 and can reduce the size or volume of the target tissue (i.e., achieve the objective of the present invention). The sequence homology may include sequences of 85% or more, 90% or more, 95% or more, or 99% or more. Alternatively, it may include a polynucleotide having a nucleic acid sequence that has at least 85% sequence homology to the nucleic acid molecule encoding the peptide of Sequence ID No. 5 or 6 and can reduce the size or volume of the target tissue (i.e., achieve the objective of the present invention). The sequence homology may include sequences of 85% or more, 90% or more, 95% or more, or 99% or more.

[0017] The composition may further contain a genetic material encoding one or more cytokines selected from the group consisting of IL-12, IL-2, IL-4, IL-5, IFN-γ, IL-10, IL-1, IL-6, INF-α, INF-β, TNF-α, and TNF-β, and it is preferable to include IL-12 in order to further maximize the immunostimulatory effect of the antigen expressed by the genetic material.

[0018] The composition may be provided as part of a vector for transmitting the genetic material in animal cells. The nucleic acid may be provided in an expression vector, such as a plasmid, and preferably the nucleic acid may contain transcription elements suitable for expression in mammalian cells, such as human cells. As used herein, a “vector” is capable of transporting the genetic material to be administered into a cell. The vector is usable for the transmission of genetic material. Preferably, such a vector may contain a coding sequence operably linked to an expression regulatory sequence.

[0019] In a preferred embodiment, the composition comprises a plasmid (e.g., gWiz) that enables protein expression of a gene corresponding to the structural protein of the yellow fever virus (17D) gene in animal cells. TM The vector may include the yellow fever virus. The yellow fever virus may be used without limitation; for example, the yellow fever virus may include, but is not limited to, the 17D strain. The plasmid may include a nucleic acid molecule or a fragment, variant, or analogue thereof that encodes the virus. For example, it may include SEQ ID NO: 1 or a fragment, variant, or analogue thereof.

[0020] In a preferred embodiment, the composition comprises a plasmid (e.g., gWiz) that enables protein expression of a gene corresponding to the structural protein of the rubella virus gene in animal cells. TM The vector may include the rubella virus. The rubella virus may be used without limitation; for example, the rubella virus may include, but is not limited to, the RA27 / 3 strain. The gene encoding the virus may include the gene encoding spike glycoproteins. Examples of the spike glycoprotein may include the E2-E1 heterozygote. For example, the nucleic acid molecule or fragment thereof, variant or analogue may be included that encodes the protein of Sequence ID No. 5.

[0021] In a preferred embodiment, the composition comprises a plasmid (e.g., gWiz) that enables protein expression in animal cells of a gene corresponding to the structural protein of the varicella zoster virus gene. TMThe vector may include the varicella-zoster virus. The varicella-zoster virus may be used without limitation, for example, the varicella-zoster virus may include, but is not limited to, the Oka strain. The nucleic acid molecule that encodes the virus may include the nucleic acid molecule that encodes the surface protein, the gE protein. For example, it may include the nucleic acid molecule that encodes the protein of SEQ ID NO: 6, or a fragment, variant, or analog thereof.

[0022] Here, “this fragment, variant, or analog” can be understood to mean a part of a protein, peptide, nucleic acid, nucleic acid molecule, or gene that achieves the objectives that the protein, peptide, nucleic acid, nucleic acid molecule, or gene of the present invention may have sequence homology of 85% or more, 90% or more, 95% or more, or 99% or more. For example, “including the fragment, variant, or analog” can be understood to mean that as long as the nucleic acid molecule of SEQ ID NO: 1, the nucleic acid molecule encoding SEQ ID NO: 5, or the nucleic acid molecule encoding SEQ ID NO: 6, or the protein encoded by the nucleic acid molecule, achieves the objectives that can be achieved, it is not excluded from the scope of the present invention.

[0023] The exogenous genetic material of the present invention may preferably include genetic material derived from a virus, and the virus may preferably include yellow fever virus, rubella virus, and / or varicella-zoster virus. The virus may be excellent in killing target tissue, preferably adipocytes, and reducing the size of the tissue. Moreover, the effect appears quickly and has few side effects. Furthermore, there is little risk of deterioration even when stored for a long period of time as an injectable preparation.

[0024] In another embodiment, the composition may further enhance the effect of adipocyte death or target tissue reduction by including an immunostimulant capable of enhancing cellular immune responses. The composition may further include a genetic material encoding an immunostimulant capable of improving the immune-enhancing effect, such as an auxiliary genetic material encoding interleukin-12 (IL-12). The auxiliary genetic material may be provided, for example, by inserting a coding region into a pSF-CMV-CMV-Sbfl vector.

[0025] The composition of the present invention can be used in combination with not only the cytokine, the gene encoding the same, or other immunoadjuvants, but also pharmaceutical carriers or excipients widely used in the art. The composition can be formulated for human or veterinary use and administered by various routes. As the administration route, oral, transdermal, intramuscular, peritoneal, intravenous, subcutaneous, nasal routes can be used, but are not limited thereto. More preferably, transdermal, intramuscular, peritoneal, subcutaneous routes can be used. Further, the composition can be administered by any device or route through which the active substance can move to the target cells, and the administration method is not particularly limited as long as the composition of the present invention or the genetic material contained in the composition can be transmitted to the target tissue or the cells constituting the tissue. For example, it can be formulated and provided in the form of an injection, a microneedle patch, etc. For example, when used for injection, the injection can be produced using an aqueous solvent such as physiological saline or Ringer's solution, a vegetable oil, a higher fatty acid ester (e.g., ethyl oleate, etc.), a non-aqueous solvent such as alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.) within a range that does not inhibit the effect of the genetic material, and a stabilizer for preventing deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, butylhydroxyanisole (BHA), tocopherol, ethylenediaminetetraacetic acid (EDTA), etc.), an emulsifier, a buffer for pH adjustment, a preservative for preventing the growth of microorganisms (e.g., phenylmercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.), and other pharmaceutical carriers. When used as a microneedle patch, the fine needles cover insoluble and soluble fine needles.

[0026] In addition, the genetic material and / or auxiliary genetic material contained in the composition can be transmitted into the body according to the ordinary gene transfer methods in the art. As non-limiting examples, physical transfer methods such as electroporation (electrical perforation method), a gene gun, etc., which are gene transfer methods, chemical transfer methods such as lipid-gene conjugates (Lipid-DNA complex: Lipoplex), polymer-gene conjugates (Polymer-DNA complex: Polyplex), liposomes, dendrimers, nanoparticles, or other suitable transfer vectors, etc. can all be used.

[0027] The composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient for the composition to exhibit a therapeutic, ameliorating, or preventive effect, or a vaccine effect, and also means an amount that does not cause side effects or serious or excessive immune reactions. Considering the characteristic of the composition of the present invention aiming to induce an immune reaction and induce cell death, the genetic material can be administered, and it is preferably administered 4 to 8 times, preferably 5 times, every 2 to 3 days. When administered as described above, a tissue reduction or shrinkage effect can be obtained by cell death at the target site. The composition can show a difference in the cell death effect the more it is administered in a large amount or multiple times, and can be used by adjusting an appropriate dosage for each site.

[0028] Another aspect of the present invention provides a method for reducing or decreasing the size or volume of tissue, comprising the step of administering a genetic material encoding a protein antigen, a vector containing the genetic material, or a composition containing the same to a target tissue of an individual. The method can preferably kill cells constituting subcutaneous adipose tissue. The i) method or ii) the genetic material encoding the protein antigen, a vector containing the genetic material, or a composition containing the same, or a kit containing the same, can be used for applications to improve or treat obesity in an individual, for wrinkle improvement, for muscle reduction, for removal or reduction of positive or malignant tumors, for wart removal, etc., and can be used for cosmetic purposes to remove fat bags around the eyes, thereby for use in reducing dark circles under the eyes.

[0029] The method may further include a step of creating an immune environment in the body before administering the genetic material, a vector containing the genetic material, or a composition containing these to a target tissue. The step of creating an immune environment in the body may include a step of administering an exogenous substance capable of inducing an immune response. The exogenous substance may be any substance that uses a protein as an antigen, without limitation.

[0030] Certain aspects of the present invention may be provided not only for the treatment, improvement, or prevention of various indications, but also for cosmetic purposes to improve appearance.

[0031] Another embodiment of the present invention provides a kit comprising (a) a vaccine composition for reducing the size or volume of a target tissue, comprising genetic material encoding an exogenous antigen, and (b) an administration guidebook for administering the vaccine. The kit may further comprise (c) a composition comprising a protein antigen or the epitope for creating an immune environment before administration of the composition in (a). Here, (c) can be understood as at least one pre-vaccine or composition for inducing an immune response.

[0032] The “pre-vaccine” as used in this disclosure may be understood as a vaccine administered before the administration of the vaccine composition for reducing the size or volume of the target tissue of the present invention, and may be understood as a vaccine composition administered before the administration of the vaccine composition for reducing the size or volume of the target tissue of the present invention to obtain existing immunity. The pre-vaccine may contain the same antigenic substance as the vaccine composition for reducing the size or volume of the target tissue of the present invention. For example, (a) a vaccine composition for reducing the size or volume of the target tissue, comprising genetic material encoding an exogenous antigen, may include yellow fever virus, rubella virus, and / or varicella-zoster virus, and (c) may also include yellow fever virus, rubella virus, and / or varicella-zoster virus.

[0033] The vaccine composition (a) may further contain genetic material encoding cytokines, and any substances that can be used to enhance the immune effect of the composition (a) may be included without limitation. The cytokines may include one or more selected from the group consisting of IL-12, IL-2, IL-4, IL-5, IFN-γ, IL-10, IL-1, IL-6, INF-α, INF-β, TNF-α, and TNF-β.

[0034] The kit of the present invention may include at least one additional formulation as defined herein, in relation to pharmaceutical compositions, antimicrobial agents, DNase inhibitors, RNase inhibitors, solubilizers, and the like. The kit may consist of, for example, one or more kit components (e.g., containers). For example, each container may be a syringe, pre-filled syringe, vial, bottle, jar, sealing sleeve, envelope or pouch, tube or blister pack or any other preferred form in which the container is configured to prevent premature mixing of the components. Each of the different components may be provided individually, or some of the different components may be provided together (i.e., in the same container). The kit may also include an administration guide having information on the administration, method of administration and dosage of any component.

[0035] Another embodiment of the present invention provides a syringe filled with a vaccine composition for reducing the size or volume of a target tissue, the composition comprising genetic material encoding an exogenous antigen. The syringe may further comprise genetic material encoding a cytokine. The cytokine may comprise one or more selected from the group consisting of IL-12, IL-2, IL-4, IL-5, IFN-γ, IL-10, IL-1, IL-6, INF-α, INF-β, TNF-α, and TNF-β. [Effects of the Invention]

[0036] The present invention provides a novel composition for improving or treating obesity that can kill cells in a specific targeted area, thereby reducing or decreasing the size or volume of the tissue. The composition can be administered to obtain a body shape correction or body shape improvement effect.

[0037] The present invention can provide a novel form of obesity treatment agent that can eliminate localized obesity and create a supple and smooth body line.

[0038] The obesity treatment agent of the present invention is effective for selective local reduction, as it can selectively reduce only the tissue in the desired area without affecting the tissue or the cells that make up the tissue in the undesired area.

[0039] Furthermore, the composition of the present invention can also be used for cosmetic purposes to improve external appearance. [Brief explanation of the drawing]

[0040] [Figure 1]This shows the results of inducing pre-existing immunity by administering the yellow fever vaccine strain 17D virus. To confirm the creation of an immune environment, serum was isolated from the blood two weeks after administration of the vaccine strain, and the IgG antibody titer against the yellow fever virus was measured. NC indicates normal mice that received no administration, ND (Normal diet) indicates mice fed a normal diet, and HFD (High fat diet) indicates obesity-induced mice fed a high-calorie diet. [Figure 2] This is a result of measuring the change in body weight before and after administration of the therapeutic vaccine, using the body weight immediately before administration as the baseline. [Figure 3] This result shows the degree of change in adipose tissue (AT) weight after administration of a therapeutic vaccine. [Figure 4] The results of the statistical analysis of the rate of change in AT (Adipose tissue) weight are shown. The statistical analysis revealed that the P-VALUE for MOCK (fake control group) / therapeutic agent (therapeutic vaccine) was 0.03007, and the P-VALUE for MOCK / therapeutic agent + IL-12 was 0.0005. Statistically, significant differences were observed in both the therapeutic agent alone treatment and the therapeutic agent + IL-12 treatment compared to MOCK. This indicates that significant differences were observed in both the therapeutic agent alone treatment and the simultaneous treatment with the therapeutic agent and IL-12. [Figure 5] This shows a vector map of the yellow fever virus (17D) genome (GenBank:X03700.1) with the genetic material (SEQ ID NO: 1) encoding structural proteins (capsid protein, prM protein, envelope protein, 119-2452 bp (2334 bp)) inserted into a gWizTM vector. A tga (stop codon) was added to the end. [Figure 6]This vector map shows the insertion of sequence numbers 2 and 3, which are nucleic acid sequences corresponding to the coding regions (p35: 127-774 bp, p40: 35-1042 bp) of IL-12 p35 (GenBank: M86672.1) and p40 (GenBank: M86671.1), into the pSF-CMV-CMV-Sbfl vector along with the Kozak sequence, respectively. IL-12 p35 was inserted into the restriction enzyme sites EcoRI and XhoI at MCS site I, and IL-12p40 was inserted into the restriction enzyme sites SalI and SpeI at MCS site II. [Modes for carrying out the invention]

[0041] The present invention will be described in more detail below with reference to examples to aid in understanding the invention. However, the examples of the present invention can be modified into various other forms, and the scope of the invention should not be construed as being limited to the following examples. The examples of the present invention are provided to give a more complete explanation of the invention to a person of average knowledge in the art.

[0042] [1. Manufacturing of vaccines for the breakdown of fat cells] For the experiment, genetic material for administration to existing immune-induced individuals and an immunostimulant that can be administered together with the genetic material were manufactured as described below.

[0043] (1) Preparation of foreign antigens 1) Preparation of the genetic material of the yellow fever virus First, we prepared the genetic material (SEQ ID NO: 1) encoding structural proteins (capsid protein, prM protein, envelope protein, 119-2452 bp (2334 bp)) from the yellow fever virus (17D) genome (GenBank: X03700.1).

[0044] 2) Preparation of the genetic material of the rubella virus We prepared genetic material encoding the E1-E2 proteins of rubella virus strain RA27 / 3, which will be used to encrypt sequence number 5.

[0045] 3) Preparation of genetic material for the varicella zoster virus We prepared the genetic material that encodes the gE protein of the Oka strain of varicella-zoster virus, which encodes sequence number 6.

[0046] (2) Preparation of vectors to be expressed in animal cells gWiz, a protein expressionable in animal cells. TM The genetic material of the yellow fever virus was inserted into the vector, and the death of adipocytes was observed as a representative result. The vector map is shown in Figure 5.

[0047] (3) Preparation of accessory genetic material Next, an immunostimulant was prepared as an auxiliary genetic material. Sequence IDs 2 and 3, which are nucleic acid sequences corresponding to the coding regions of IL-12 p35 (GenBank: M86672.1) and p40 (GenBank: M86671.1) (p35: 127~774 bp, p40: 35~1042 bp), were inserted into the pSF-CMV-CMV-Sbfl vector along with the Kozak sequence, respectively. The vector map is shown in Figure 6.

[0048] Furthermore, a Kossack sequence is inserted at the beginning during the synthesis of each IL-12 molecule.

[0049] The cloned YF antigen plasmid and mRNA12 plasmid were transformed into DH5a competent cells. These cells were cultured in large quantities, and the plasmids were recovered using a plasmid prep kit.

[0050] [2. Confirmation of fat cell death] (1) Experimental method 1) Production of diet-induced obese (DIO) mice Three-week-old female C57BL / 6J mice were fed either a standard diet or a 60% high-fat diet. Their body weight was measured every other week, and after 10-15 weeks of rearing, mice that gained more than 25% of the body weight of normal mice were considered obesity-inducing mice and used in experiments.

[0051] 2) Induction of existing immunity and measurement of antibody titers The formation of pre-existing immunity was induced using a weakened live virus, which is a vaccine strain virus. The 17D virus was used as the vaccine strain virus.

[0052] 17D virus (attenuated live virus) was injected into the left hind leg muscle of a mouse in a 2x10⁻¹⁰ 5 Three injections were administered at a concentration of pfu / injection, two weeks apart. Blood was collected two weeks after the last injection, and serum was separated. Antibodies specific to yellow fever virus (YFV) were detected in the separated serum by ELISA analysis. For ELISA analysis, 5 x 10⁻¹⁵ units of the 17D virus used in the injections were used. 4 Plates were coated with pfu / well concentration, and the analytical sample (serum) was diluted and reacted for 2 hours. After washing, the plate was treated with anti-mouse IgG-HRP secondary antibody diluted to 1 / 4000, and reacted for 2 hours. After washing, the chromogenic solution was added and reacted for 10 minutes, and the OD value was measured.

[0053] Two weeks after administration of the yellow fever vaccine strain 17D virus, serum was isolated from the blood and the IgG antibody titer against YFV was measured, indicating that normal immune induction (antibodies) had occurred.

[0054] The results are shown in Figure 3. To confirm antibody production in the mice, YFV-specific total IgG antibody titers were preferentially analyzed. The degree of antibody titer formation was indicated by absorbance (OD value) using ELISA analysis. Compared to the NC group, the ND and HFD groups showed an OD value of approximately 1, confirming antibody production. Therefore, it can be confirmed that immunity induction occurred.

[0055] 3) Administration of therapeutic vaccines and measurement of fat weight Normal mice and obese mice were divided into two groups: one receiving the yellow fever vaccine and the other not. All mice were fed a normal diet starting three days before the therapeutic vaccine was administered. The therapeutic vaccine was injected into two sites: the inguinal adipose tissue and the interscapular adipose tissue on one side of each mouse, divided into left and right halves. Five doses were administered every 2-3 days, with a total dose of 150 μg / site (100 μg / site of antigen DNA and 50 μg / site of IL-12 DNA). An empty vector was used to match the total amount of antigen DNA administered. Body weight was measured before and after the therapeutic vaccine administration. Seven days after the last therapeutic vaccine administration, the mice were dissected, and the inguinal adipose tissue and interscapular adipose tissue were separated and weighed.

[0056] ND: Normal diet, HFD: 60% high fat diet

[0057] [Table 1]

[0058] Figures 2 to 4 show the average percentage change in body weight and the effect of reducing adipose tissue after administration of the therapeutic agent for each group in Table 1 above. Referring to Figure 2, body weight was measured before and after administration of the therapeutic vaccine (therapeutic agent) (labeled "treatment" in the figure), and the change in body weight was measured using the body weight immediately before administration as a baseline. In the group that received no treatment, body weight gradually increased, but the MOCK, therapeutic vaccine 1, and therapeutic vaccine 2 groups all showed a decrease in body weight, with the groups administered therapeutic vaccines 1 and 2 showing an even more significant decrease. Furthermore, the group administered IL-12 together showed the greatest decrease in body weight. Figure 3 shows the results of separating the left and right inguinal adipose tissue (AT) and interscapular adipose tissue (AT) of mice on the 7th day after administration of the therapeutic vaccine five times, measuring the weight of the AT, and comparing the amount of decrease in fat weight with the AT of the same individual that did not receive an empty vector or therapeutic vaccine as a baseline. It can be seen that the weight of adipose tissue decreased when treated with therapeutic vaccines 1 and 2 compared with the Control group (no treatment) and the empty vector group. In particular, the change in adipose tissue weight was significantly greater in the treatment group treated with therapeutic vaccine 2, which was administered together with IL-12. [Industrial applicability]

[0059] The present invention can prevent and / or treat obesity. The present invention provides a method for preventing and / or treating obesity by injection at a local site.

Claims

1. A vaccine composition for reducing the size or volume of a target tissue, comprising genetic material encoding an exogenous antigen, The aforementioned foreign antigen is a structural protein of the yellow fever virus, and A vaccine composition for reducing the size or volume of a target tissue, wherein the target tissue is adipose tissue.

2. The vaccine composition for reducing the size or volume of a target tissue according to claim 1, characterized in that the target tissue is a tissue composed of adipocytes.

3. The composition is a vaccine composition for reducing the size or volume of a target tissue according to claim 1, which induces a reduction or death of adipocytes.

4. The vaccine composition for reducing the size or volume of a target tissue according to claim 1, characterized in that the genetic material is DNA or RNA.

5. The vaccine composition for reducing the size or volume of a target tissue according to claim 1, wherein the genetic material comprises the nucleic acid molecule of Sequence ID No.

1.

6. The vaccine composition for reducing the size or volume of a target tissue according to claim 1, further comprising a genetic material encoding a cytokine.

7. The vaccine composition for reducing the size or volume of a target tissue according to claim 6, characterized in that the cytokine is one or more selected from the group consisting of IL-12, IL-2, IL-4, IL-5, IFN-γ, IL-10, IL-1, IL-6, INF-α, INF-β, TNF-α, and TNF-β.

8. The vaccine composition for reducing the size or volume of a target tissue according to claim 1, characterized in that the foreign antigen induces an intracellular immune response.

9. The aforementioned composition acts locally, The vaccine composition for reducing the size or volume of a target tissue, as described in claim 1, characterized in that the vaccine composition is applied after at least one protein for creating an immune environment has been applied to the individual.

10. The genetic material is administered by oral, transdermal, intramuscular, peritoneal, intravenous, subcutaneous, or nasal route injection, or A vaccine composition for reducing the size or volume of a target tissue, according to claim 1, characterized by being administered to the target tissue via electroporation, a gene gun, liposomes, dendrimers, nanoparticles, or a transfer vector.

11. The vaccine composition for reducing the size or volume of target tissue according to claim 1, characterized in that the amount of genetic material contained in the composition is 0.1 to 1000 μg / site at the time of a single dose.

12. (a) A vaccine composition for reducing the size or volume of a target tissue, comprising genetic material encoding an exogenous antigen, (b) A dosing guide for administering the vaccine composition, Includes, The aforementioned foreign antigen is a structural protein of the yellow fever virus, and The aforementioned target tissue is adipose tissue; this is a kit for reducing the size or volume of adipose tissue.

13. The kit according to claim 12, wherein the kit further comprises (c) a composition comprising a protein antigen for creating an immune environment before administration of the vaccine composition of (a).

14. The kit according to claim 12, wherein the vaccine composition (a) further comprises a genetic material encoding a cytokine.

15. A syringe for reducing the size or volume of adipose tissue, filled with a vaccine composition for reducing the size or volume of target tissue, which contains genetic material encoding an exogenous antigen, The aforementioned foreign antigen is a structural protein of the yellow fever virus, and The target tissue is adipose tissue; a syringe for reducing the size or volume of adipose tissue.

16. The syringe according to claim 15, further comprising genetic material encoding a cytokine.

17. It contains genetic material that codes for at least one foreign antigen, The aforementioned foreign antigen is a structural protein of the yellow fever virus, and the composition is for removing subcutaneous fat cells.

18. The composition for removing subcutaneous fat cells according to claim 17, further comprising a genetic material encoding a cytokine.

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