WO2012169735A2 - Complex comprising water-soluble fragments of fcεri and composition comprising same for treating allergic diseases mediated by ige - Google Patents

Complex comprising water-soluble fragments of fcεri and composition comprising same for treating allergic diseases mediated by ige Download PDF

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WO2012169735A2
WO2012169735A2 PCT/KR2012/004075 KR2012004075W WO2012169735A2 WO 2012169735 A2 WO2012169735 A2 WO 2012169735A2 KR 2012004075 W KR2012004075 W KR 2012004075W WO 2012169735 A2 WO2012169735 A2 WO 2012169735A2
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fcεri
npb301
cells
complex
water
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PCT/KR2012/004075
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French (fr)
Korean (ko)
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WO2012169735A3 (en
WO2012169735A9 (en
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박병덕
윤선주
김욱동
배종환
김민희
강현정
박은혜
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(주)네오팜
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Priority claimed from KR1020120054613A external-priority patent/KR20120135865A/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy

Definitions

  • the present invention relates to a complex in which a soluble fragment of Fc ⁇ RI, a receptor having high affinity, and a biocompatibility material are conjugated to the Fc region of immunoglobulin E (IgE).
  • the complex conjugated with the water-soluble fragment and the biocompatible material has the effect of treating and preventing allergic diseases by inhibiting the secretion of allergic cytokines by inhibiting the binding of IgE and Fg ⁇ RI, an IgE receptor.
  • Particularly preferred form of the complex according to the present invention is a fusion protein in which the water-soluble fragment of Fc ⁇ RI and the Fc of a human antibody is fused.
  • NPB301 a fusion protein in which the water-soluble fragment of Fc ⁇ RI and the Fc of a human antibody, is provided. It inhibits the binding of the IgE receptor Fc ⁇ RI, inhibits the signaling induced by IgE and Fc ⁇ RI, and thus inhibits the secretion of allergic cytokines, thereby causing atopic dermatitis, allergic rhinitis, asthma, urticaria and contact dermatitis. It has an excellent therapeutic effect in the treatment of allergic diseases.
  • IgE immunoglobulin E
  • Most allergic reactions are caused by the binding of IgE to the surface of the allergic receptor, one of the receptors for the Fc portion (Fc ⁇ ) of IgE is Fc ⁇ RI.
  • IgE binds to Fc ⁇ RI, an IgE receptor expressed on the surface of mast cells or basophilic cells, resulting in allergic signaling.
  • Human Fc ⁇ RI has three different subunits: alpha chain, beta chain (signal amplification factor) and gamma chain (signal transfer factor).
  • Tetramer Fc ⁇ RI composed of one alpha chain and one beta chain and two gamma chains, respectively, is mainly expressed on the cell membranes of tissue cells and basophils and plays an important role in type I allergic responses for cell activation. Only the alpha chain of Fc ⁇ RI binds directly to IgE, and the binding site for IgE spans the extracellular region of the alpha chain (Nature. 2000, 406, 259-266).
  • Fc ⁇ RI to which IgE binds has an ⁇ -subunit (Fc ⁇ RI ⁇ ), and Fc ⁇ RI ⁇ has a size of about 60 kDa, and is composed of a hydrophobic domain existing in the cell membrane and a hydrophilic domain existing outside the cell membrane.
  • Blood IgE is produced by B lymphocytes and is involved in the primary defense against pathogens. IgE phosphorylates Syk protein, a downstream signaling material by binding to specific allergens and binding of mast cell Fc ⁇ RI, and activates mast cells by inducing downstream cell signaling mechanisms. As a result, TNF is secreted out of mast cells, and histamine, chondroitin sulfate, heparin, and protease tryptase and chymase, which are stored in cytoplasmic granules, are fast. It is degranulated in and secreted out of the cell.
  • mast cells and basophils These substances are secreted from mast cells and basophils, and various physiological allergies such as itching, tingling, pruritus, skin disorders, abdominal pain, diarrhea, bronchial contraction, wheezing, dyspnea, bronchial asthma, runny nose, and sneezing Cause disease.
  • the cause of allergic diseases has not been elucidated yet, but the imbalance of human immune mechanisms has been suggested as the cause.
  • increased IgE in blood has been suggested as a major cause of allergic diseases.
  • the concentration of IgE (100-500 IU / ml) in the blood of asthma patients is reported to be significantly increased compared to the normal (20-40 IU / ml), and the concentration of (1000 IU / ml) in atopic patients Much higher is the role of IgE in allergic diseases.
  • allergic rhinitis, asthma and atopic dermatitis have increased expression of Fc ⁇ RI in the blood of patients, the binding between IgE and Fc ⁇ RI receptors in allergic diseases may be very important for the development of the disease (J. Allergy Clin). Immunol. 1980, 66, 305-313).
  • allergen avoidance therapy, antiallergic drug use, and immunotherapy for certain allergens have been used for the treatment of allergic diseases (Clin. Immunol. 1997, 100 (2), 171-176, Allergy 1998, 53, 821-). 832, Allergy Clin. Immunol. 1998, 102, 631-636, Clin.Exp. Allergy. 1999, 29 (11), 1563-1571, Allergy Clin. Immunol. 2002, 110, 154-159).
  • the most widely used treatments for allergic diseases include antihistamines and drugs of the corticosteroid family.
  • beta-2-adrenergic (beta-2 adrenergic) receptors and phosphodiesterase-3 (phosphodiesterase-3) inhibitors and the like are used as therapeutic agents for allergic diseases.
  • Many allergens have been developed over the past few years, but only alleviate the symptoms, and there are no treatments that can cure the root cause of allergies.
  • the efficacy of the drug is insufficient or has a number of disadvantages due to side effects. Therefore, the demand for a new concept of allergy treatment is increasing.
  • Omalizumab (Xolair) is used as a treatment for asthma patients.
  • Omalizumap binds to IgE and inhibits the release of chemicals, a major cause of asthma and allergies.
  • Omalizumab has side effects such as angioedema, anaphylactic reactions and other allergic conditions, and allergic bronchial spasms, and allergic granulomatous vasculitis and idiopathic severe thrombocytopenia have been reported through postmarketing results.
  • the present invention is to provide a therapeutic agent that can effectively and fundamentally treat allergic diseases without side effects, a material having a biocompatibility with a water-soluble fragment of Fc ⁇ RI, a receptor having a high affinity for the Fc region of IgE It is an object to provide a conjugated complex and a method of making such a complex.
  • Particularly preferred form of the complex provided in the present invention is NPB301, which is a fusion protein having the amino acid sequence of SEQ ID NO: 2 where the water-soluble fragment of Fc ⁇ RI and the Fc of a human antibody are fused.
  • the present invention prepared a complex in which a water-soluble fragment of the Fc ⁇ RI receptor is combined with a biocompatible material.
  • 'soluble fragment of Fc ⁇ RI' refers to a fragment in which Fg ⁇ RI lacks an intracellular domain, and thus no signal transduction occurs in cells even though IgE is bound. It is preferably composed of part or all of the extracellular domain having the ability to bind to, and may additionally include a transmembrane domain.
  • Particularly preferred water-soluble fragments of Fc ⁇ RI according to the present invention preferably have the amino acid sequence set forth in SEQ ID NO: 1.
  • the biocompatible material according to the present invention functions to increase the stability of the water-soluble fragment of Fc ⁇ RI and can be used as long as it has a property that does not cause an immune response in the body, polyethylene glycol (PEG), Diacylglycerol (DAG), a fragment crystallizable (Fc) region of a human antibody, and the like are preferred, but are not limited thereto.
  • the Fc region of an antibody refers to a fragment of the constant region portion that is cleaved by treating the antibody with papain enzymes, without limitation if it is derived from IgA, IgD, IgE, IgG and IgM antibodies Particularly preferred are Fc regions derived from IgG1, IgG2, IgG3 and IgG4 antibodies of the IgG antibodies, which are available for the preparation of the complex according to the invention.
  • Preferred complexes according to the invention are fusion proteins in the form of a conjugate of a water-soluble fragment of Fc ⁇ RI and an Fc region of a human antibody.
  • the water-soluble fragment of Fc ⁇ RI and the Fc region of a human antibody can be directly conjugated, and can also be linked using a linker.
  • the linker can be used without limitation as long as it frees the movement of the water-soluble fragment of Fc ⁇ RI in three dimensions without affecting the binding of the water-soluble fragment of Fc ⁇ RI to IgE.
  • Preferred linkers are, but are not limited to, peptide linkers.
  • a preferred fusion protein provided in the present invention is a form in which the water-soluble fragment of Fc ⁇ RI according to SEQ ID NO: 1 and the Fc region of a human antibody are fused to each other by a peptide linker, and in particular, NPB301, a fusion protein having an amino acid sequence such as SEQ ID NO: 2, is desirable.
  • fusion proteins in which mutations in the Fc ⁇ RI soluble fragment, Fc region or linker structure of a human antibody have occurred are also included in the scope of the present invention.
  • One such example is the case where conservative substitution of amino acids in the water-soluble fragment of Fc ⁇ RI occurs.
  • Conservative substitutions mean substitutions with other amino acid residues that have properties similar to those of the original amino acid sequence. For example, lysine, arginine, and histidine have similar properties because they have a base side chain. Aspartic acid and glutamic acid It has similar characteristics in that it has a mountain side chain.
  • glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar characteristics in that they have a non-charged polar side chain, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine are nonpolar.
  • Tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have side chains. Therefore, it will be apparent to those skilled in the art that amino acid substitutions within groups having similar characteristics as described above will not show any change in characteristics.
  • the present invention provides a pharmaceutical composition comprising the complex containing a water-soluble fragment of Fc ⁇ RI as an active ingredient as described above.
  • the pharmaceutical composition may further include conventional pharmaceutically acceptable carriers and excipients.
  • the pharmaceutical composition may be applied to allergic diseases mediated by IgE. Representative diseases include, but are not limited to, allergic rhinitis, asthma, atopic dermatitis, urticaria and contact dermatitis.
  • the present invention provides a method for preventing and treating allergic diseases, wherein the fragment protein is administered to a therapeutic target.
  • the present invention also provides a polynucleotide sequence encoding a fusion protein in which a water-soluble fragment of Fc ⁇ RI or a water-soluble fragment of Fc ⁇ RI and an Fc region of a human antibody are conjugated.
  • a polynucleotide sequence of sequence number 3 which codes NPB301 which is a fusion protein which has the amino acid sequence of sequence number 2 is preferable.
  • the present invention provides a recombinant vector comprising the polynucleotide sequence and a host cell comprising the same, and a method for producing a conjugate comprising the water-soluble fragment of Fc ⁇ RI according to the present invention using the recombinant vector or host cell.
  • a complex in which the water-soluble fragment of Fc ⁇ RI is conjugated with a material other than a protein such as PEG only the water-soluble fragment of Fc ⁇ RI may be produced, followed by conjugation to PEG and the like to prepare the complex.
  • the protein in the case of a complex in the form of a fusion protein of a water-soluble fragment of Fc ⁇ RI and a human Fc region, the protein is linked to each other by an amino bond by connecting a polynucleotide sequence encoding the water-soluble fragment of Fc ⁇ RI and a human Fc region through genetic recombination. It is preferable to prepare by being expressed in the form of one protein.
  • a "recombinant vector” refers to a gene construct that is an expression vector capable of expressing a protein of interest in a suitable host cell, and which contains essential regulatory elements operably linked to express the gene insert.
  • "operably linked” means that the nucleic acid expression control sequence and the nucleic acid sequence encoding the protein of interest is functionally linked to perform a general function. Operative linkage with recombinant vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes generally known in the art. It can be performed easily.
  • Suitable expression vectors may include signal sequences for membrane targeting or secretion in addition to expression control elements such as promoters, initiation codons, termination codons, polyadenylation signals and enhancers. Initiation and termination codons are generally considered to be part of the nucleotide sequence encoding the immunogenic target protein and must be functional in the subject and be in frame with the coding sequence when the gene construct is administered. Generic promoters can be either constitutive or inducible. Prokaryotic cells include, but are not limited to, lac, tac, T3 and T7 promoters.
  • Eukaryotic cells include monkey virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV), for example the long terminal repeat (LTR) promoter of HIV, moronivirus, cytomegalovirus (CMV) ), Epstein Barr virus (EBV), Loose sacoma virus (RSV) promoters, as well as promoters derived from ⁇ -actin promoter, human heroglobin, human muscle creatine, human metallothionein, but are not limited thereto.
  • the expression vector may comprise a selectable marker for selecting a host cell containing the vector.
  • the selection marker is for selecting cells transformed with the vector, and markers conferring a selectable phenotype such as drug resistance, nutritional requirements, resistance to cytotoxic agents or expression of surface proteins can be used. Since only cells expressing a selection marker survive in an environment treated with a selective agent, transformed cells can be selected.
  • the vector when the vector is a replicable expression vector, the vector may include a replication origin, which is a specific nucleic acid sequence from which replication is initiated.
  • various types of vectors such as plasmids, viruses, and cosmids can be used.
  • the type of recombinant vector is not particularly limited as long as it functions to express a desired gene and to produce a desired protein in various host cells of prokaryotic and eukaryotic cells, but has a promoter with strong activity and strong expression, similar to natural state. Vectors that can produce large amounts of foreign protein in form are preferred.
  • Suitable expression vectors for eukaryotic hosts may include, but are not limited to, expression control sequences derived from SV40, bovine papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus and retrovirus. no.
  • Expression vectors that can be used in bacterial hosts include broader hosts, such as bacterial plasmids obtained from Escherichia coli , such as pET, pRSET, pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9, and derivatives thereof.
  • Range plasmids, phage DNA that can be exemplified by a wide variety of phage lambda derivatives such as ⁇ gt10 and ⁇ gt11, NM989, and other DNA phages such as M13 and filamentary single-stranded DNA phages.
  • Useful expression vectors for yeast cells are 2 ° C. plasmids and derivatives thereof.
  • a useful vector for insect cells is pVL941.
  • the recombinant vector is inserted into a host cell to form a transformant.
  • Suitable host cells are Escherichia coli, Bacillus subtilis , Streptomyces sp., Pseudomonas sp., Proteus Prokaryotic cells such as Proteus mirabilis or Staphylococcus sp.
  • fungi such as Aspergillus sp., Pichia pastoris , Saccharomyces cerevisiae , Schizosaccharomyces sp.
  • Eukaryotic cells such as yeast, such as Spora crassa , other lower eukaryotic cells, and cells of higher eukaryotes, such as cells from insects.
  • the host cell is preferably derived from plants, mammals, monkey kidney cells (COS7) cells, NSO cells, SP2 / 0, Chinese hamster ovary (CHO: Chinese hamster ovary) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells and HEK293 cells and the like are available, but are not limited to these. Particularly preferably CHO cells.
  • Transformation into a host cell in the present invention includes any method of introducing a nucleic acid into an organism, cell, tissue or organ and can be carried out by selecting a suitable standard technique according to the host cell as is known in the art. These methods include electroporation, protoplast fusion, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, agitation with silicon carbide fibers, agro bacterial mediated transformation, PEG, dextran sulfate, Lipofectamine and dry / inhibited mediated transformation methods and the like.
  • a soluble protein of the Fc ⁇ RI receptor and a fusion protein of the human Fc region according to the present invention can be produced in a large amount, and the medium and culture conditions are appropriately tolerated by the host cell. Optional is available. Conditions such as temperature, pH of the medium and incubation time can be appropriately adjusted to be suitable for the growth of cells and the mass production of proteins during the culture.
  • the recombinantly produced antibody or antibody fragment as described above can be recovered from the medium or cell digest, and can be isolated and purified by conventional biochemical separation techniques (Sambrook et al., Molecular Cloning: A laborarory Manual, 2nd).
  • NPB301 which is a soluble protein of the preferred Fc ⁇ RI and human Fc region in the present invention, is preferably produced by culturing and using animal cells as host cells using a polynucleotide sequence according to SEQ ID NO: 3.
  • the complex containing the water-soluble fragment of Fc ⁇ RI according to the present invention effectively inhibits the binding between IgE and Fc ⁇ RI at low concentrations, and thus exhibits an excellent therapeutic effect on various allergic diseases mediated by IgE, and specifically binds only to Fc ⁇ RI. It has no side effects and does not cause an immune response when administered in vivo.
  • Complexes comprising Fc ⁇ RI receptor fragments according to the present invention may inhibit or treat IgE mediated allergic diseases by inhibiting binding between IgE and Fc ⁇ RI.
  • IgE IgE mediated allergic diseases
  • it can be usefully used for the treatment of patients with atopic dermatitis, allergic rhinitis, asthma, urticaria and contact dermatitis.
  • FIG. 1 is a diagram showing a DNA sequence encoding NPB301, a fusion protein according to the present invention.
  • Figure 2 shows the amino acid sequence of NPB301, a fusion protein according to the present invention.
  • FIG. 3 is a view showing the effect of the fusion protein NPB301 according to the present invention on the secretion of beta-hexos aminidayes.
  • Figure 4 is a view showing the histamine secretion inhibitory effect of the fusion protein NPB301 according to the present invention.
  • FIG. 5 is a view showing the PCA inhibitory effect of NPB301 fusion protein according to the present invention.
  • Fig. 6 shows the results of analysis of tissue changes in lung tissue by NPB301 in OVA-induced Fc ⁇ RI ⁇ transgenic mice (Tg mice) asthma model.
  • FIG. 7 shows changes in total cell number, macrophages, eosinophils and lymphocytes by NPB301 in an OVA induced Fc ⁇ RI ⁇ transgenic mouse asthma model.
  • FIG. 8 shows the amount changes of cytokines (IL-4, IL-5, Il-13) by NPB301 in OVA induced Fc ⁇ RI ⁇ transgenic mouse asthma model.
  • FIG. 9 is a view showing the change of the thickness of the ear by NPB301 in contact dermatitis animal model.
  • FIG. 10 shows ear thickness change by NPB301 in TPA induced dermatitis animal model.
  • FIG. 11 shows changes in immunoglobulin E by NPB301 in a peanut allergen animal model.
  • FIG. 12 is a view showing the rhinitis improvement effect by NPB301 in a rhinitis animal model.
  • FIG. 13 is a graph showing changes in percutaneous water loss, intrahydration, and skin thickness after NPB301 intravenous injection in an atopy animal model.
  • FIG. 14 is a graph showing changes in percutaneous water loss, intrahydration, and skin thickness after NPB301 intraperitoneal injection in an atopy animal model.
  • 15 is a view showing the change in skin thickness according to the NPB301 concentration after NPB301 intraperitoneal injection of various concentrations in the OVA atopic animal model.
  • 16 is a diagram showing the effect of atopy improvement by NPB301 in the OVA atopy animal model.
  • 17 is a diagram showing the change in the thickness of the skin after applying NPB301 formulated in the OVA atopic animal model to the affected area.
  • Figure 18 shows the total cell number change by NPB301 in OVA asthma animal model.
  • FIG. 19 shows changes in immunoglobulin E by NPB301 in a TMA dermatitis animal model.
  • cDNA (Catalog No. sc122614) encoding a water soluble fragment of human Fc ⁇ RI receptor was purchased from Origin Technologies (USA). This was recombined with a TT5 vector expressing the Fc of human IgG, and transiently transformed into 293-6E cells, followed by culturing in FreeStyle 293 medium (GIBCO, 12338) to prepare a fusion protein NPB301 according to SEQ ID NO: 2.
  • RBL-SX38 cells were suspended in 3% FBS (Feral Bovine Serum) and DMEM medium (Dulbecco's Modified Eagle's Medium) in 96-well plates for measuring beta hexosminidayes secretion, and then 2 ⁇ 10 4 cells in 96-plate Incubated for 48 hours at 37 °C, 5% CO 2 incubator at / 100 ul.
  • FBS Feal Bovine Serum
  • DMEM medium Dulbecco's Modified Eagle's Medium
  • each cell was washed once with PBS + a buffer (PBS, 0.4 mM MgCl 2 , 1 mM CaCl 2 , 5.6 mM glucose, 0.1% BSA) and then nitropheny-IgE NP-IgE) (0.1 ⁇ g / ml) was dispensed and reacted at 37 ° C., 5% CO 2 incubator for 30 minutes, and then treated with concentration of NPB301 in a 37 ° C., 5% CO 2 incubator for 150 minutes.
  • PBS + a buffer PBS, 0.4 mM MgCl 2 , 1 mM CaCl 2 , 5.6 mM glucose, 0.1% BSA
  • nitropheny-IgE NP-IgE 0.1 ⁇ g / ml
  • NP-BSA (0.1 ug / ml) was then added and reacted for 30 minutes in a 37 ° C., 5% CO 2 incubator and secreted out of the cells to 30 ⁇ l of supernatant containing beta hexoseminidayes, and 0.1% Triton X- 100 ⁇ l of cells were obtained by lysis to 100 ⁇ l of cells, and 30 ⁇ l of the solution contained therein was transferred to a 96-well plate and the substrate buffer (4-p-nitrophenyl-N-acetyl- ⁇ -D- 30 ⁇ l of glucosamine (4-p-nitrophenyl-N-acetyl- ⁇ -D-glucosaminide) 1 mM, sodium citrate, 0.1 M, pH 4.5) was incubated at 37 ° C. for 1 hour, followed by reaction stopping solution ( 50 mM glycine, pH 10.7) was added to terminate the reaction and the absorbance was measured at 405 nm.
  • substrate buffer
  • RBL-SX38 cells were first suspended in 10-well FBS and DMEM medium in 24-well plates, followed by 37 ° C. and 5% CO 2 incubator at 2 ⁇ 10 5 cells / 500 ul in 24-well plates. Incubated for 48 hours. After removing the culture medium, each cell was loaded with Tyrode's buffer, 125 mM NaCl, 5 mM KCl, 0.4 mM MgCl 2 , 1 mM CaCl 2 , 5.6 mM glucose, 0.1% BSA, 10 mM Hepes, pH7.
  • NP-IgE NP-IgE
  • a culture medium at 37 ° C. and 5% CO 2 incubator for 24 hours.
  • NPB301 was treated together.
  • 0.1 ug / ml of NP-BSA was added to react for 30 minutes at 37 °C, 5% CO 2 incubator.
  • 500 ⁇ l of the supernatant was centrifuged, and then 100 ⁇ l of the supernatant was transferred to a 96-well plate and acetylated histamine. Then, the histamine content was measured using the HTRF Kit (CISBIO International, France).
  • NPB301 As a result, as shown in Figure 4 NPB301 was confirmed that the efficient suppression of histamine secretion at low concentrations.
  • NPB301 according to the present invention significantly inhibited PCA.
  • the left ear was normally induced hypersensitivity reaction and turned blue, but the right ear with the hypersensitivity reaction suppressed remained unchanged (see FIG. 5A), and the dye was separated after separating the ear. After the absorbance was also obtained a significant result from the measurement results (see Fig. 5B). From these results, it was confirmed that NPB301 according to the present invention inhibits the binding of IgE and Fc ⁇ RI.
  • OVA ovalbuproliferin
  • Alum 100 ug of OVA (ovalbumin) and 2 mg of Alum were included in a 200 ⁇ l solution.
  • the mixed OVA-alum was continuously mixed at 37 ° C. for about 1 hour.
  • the mixed OVA-alum was divided into 200 ⁇ l into a 1.5 ml tube and put into a 1 ml syringe.
  • 6-week-old females transformed to express human Fc ⁇ RI were intraperitoneally injected 200 ⁇ l over 0, 14 days and 2 times. At 28, 29 and 30 days, 30 ⁇ g of 1% OVA was inhaled through the nasal mucosa.
  • Selected antibody NPB301 was injected at 26, 27, 28, 29 days at 10 mg / kg. After 24 hours, the lung tissue was removed and biopsied, and the number of cells in the bronchoalveolar lavage fluid was counted.
  • the bronchial tubes of anesthetized mice were incised using zoletil, a tube was inserted, a 22-gauge needle was inserted into the trachea, and bronchoalveolar lavage was performed. The samples were collected by injecting 0.9 mL of PBS twice per time. . The collected bronchial alveolar lavage fluid was centrifuged at 4 ° C. and 5000 rpm for 5 minutes, and the supernatant was stored at ⁇ 70 ° C.
  • cytospin 3 Thermo, USA
  • Diff-Quik Diff-Quik Staining was performed. A total of 200 cells were counted to differentially calculate alveolar macrophages, eosinophils, lymphocytes and neutrophils.
  • cytokines such as IL-4, IL-5 and IL-13, which are related to the inflammatory response, were measured.
  • the lung tissue of the asthma model treated with OVA had a thick bronchial wall, but the bronchial tissue of the mouse model injected with NPB301 intravenously recovered to normal (see FIG. 6).
  • the total number of cells, macrophages, eosinophils and lymphocytes in the bronchial alveolar lavage fluid of the asthma model transformed to express OVA-induced Fc ⁇ RI ⁇ was increased compared to the PBS group, but when treated with NPB301, the total bronchoalveolar lavage fluid was treated. It was confirmed that the number of cells, macrophages, eosinophils and lymphocytes significantly decreased (see FIG. 7).
  • TG mice females of 4 weeks old were used. Push the hairs of the pear and apply 100 ⁇ l of 5% Oxazoline (OXA) to sensitize, apply 10 ⁇ l of 0.1% Oxazoline (OXA) to both ears on Day 5, Day 7, Day 9, and Day 11, and apply 5 MPK on Day 9.
  • NPB301 was injected intravenously. Thereafter, the thickness of the ears was measured on Day 13.
  • TG mice 6 week old females were used.
  • 0.05 g (20 ⁇ l) of TPA was applied to the ears on Day 1 and Day 2.
  • 0.5 g (20 ⁇ l) of TPA was applied to the ears on Day 3 and Day 4.
  • NPB301 was intravenously injected at 5 MPK, 10 MPK and 20 MPK on Day 2 and Day 4, and ear thickness was measured on Day 5.
  • TG mice 6 week old females are used. Peanut and cholera toxin were orally administered on an empty stomach once daily for 3 days, and 2 weeks later, peanut and cholera toxin were orally administered on an empty stomach once daily for 3 days. Two weeks later, peanuts were fed at 30-minute intervals, then intraperitoneally administered 10 MPK of NPB301 once a day or once every two days, and total immunoglobulin E in blood was measured using a quantitative kit.
  • mice 6 week old females were used.
  • OVA and Alum were mixed on day 0 and day 7 and sensitized by intraperitoneal injection, then daily inhalation of 150 ⁇ g / 10 ⁇ l of OVA from day 14 to day 20.
  • NPB301 was injected 5 MPK, 10 MPK, 30 minutes before inhaling OVA. Inhaled at a concentration of 20 MPK.
  • OVA was inhaled on Day 21 and immediately after 10 minutes, the number of nose rubs (see FIG. 12A) and the number of sneezes (see FIG. 12B) were measured.
  • the hair of C57BL / 6 mouse was pushed and stimulated 5 times with 3M tape, and 100 ug of OVA was applied to sterile gauze of 1 ⁇ 1 cm length and wrapped with Tegaderm for a week. Changes in skin thickness were measured by exposure to OVA three times every three weeks (Day 1 ⁇ 7, Day 22 ⁇ 28, Day 43 ⁇ 49) and intraperitoneally injected NPB-301 at 0.3, 1, 5 MPK once every two days. .
  • TG mice The hair of TG mice was pushed and stimulated 5 times with 3M tape, and 100 ⁇ g of OVA was applied to sterile gauze of 1 ⁇ 1 cm length and wrapped with Tegaderm for a week. Changes in skin thickness were measured by exposure to OVA three times every three weeks (Day 1-7, Day 22-28, Day 43-49) and intraperitoneally injected NPB-301 at 5 MPK once every two days (see FIG. 16A). ). And total immunoglobulin E in blood was measured using a quantitative kit (see FIG. 16B). As a result of treatment with 5 MPK NPB301 in TG mice, skin thickness gradually decreased when measured at two-day intervals, and IgE in whole blood was also decreased. From the above results, it was confirmed that atopic dermatitis was alleviated (see FIG. 16).
  • the hair of C57BL / 6 mouse was pushed, and stimulated 5 times with 3M tape, and 100 ug of OVA was applied to sterile gauze of 1 ⁇ 1 cm length and wrapped with Tegaderm for a week.
  • Three times every three weeks (Day 1-7, Day 22-28, Day 43-49) was exposed to OVA, 2% NPB301 formulation was applied to the affected area for two days and the change in skin thickness was measured.
  • OVA ovalbuproliferin
  • Alum 25 ug of OVA (ovalbumin) and 1 mg of Alum were included in a 200 ⁇ l solution.
  • the mixed OVA-alum was continuously mixed for 10 minutes using a homogenizer.
  • the mixed OVA-alum was divided into 200 ⁇ l in a 1.5 ml tube and put into a 1 ml syringe. Then, 6-week-old females of balb / c mice were injected intraperitoneally with 200 ⁇ l over 0, 6, 13, 20 days and 4 times. On 26 and 30 days, 50 ⁇ g of 1% OVA was inhaled through the nasal mucosa.
  • NPB301 was injected at 5 mg / kg, 4 days before, 3 days before, 2 days before, and 1 day before inhalation of 50 ⁇ g of 1% OVA.
  • the lung tissue was removed and biopsied, and the number of cells in the bronchoalveolar lavage fluid was counted.
  • the tube was inserted, a 22 gauge needle was inserted into the trachea, and the bronchoalveolar lavage was performed.
  • the samples were collected by injecting 0.9 mL of PBS twice per time. It was.
  • the collected bronchial alveolar lavage fluid was centrifuged at 4 ° C. and 5000 rpm for 5 minutes, and the supernatant was stored at ⁇ 70 ° C. until the next experiment. Cells obtained by centrifugation were observed by using a hemocytometer.
  • the number of bronchial alveolar cells was decreased in the NPB301 group injected 4, 3, 2, 1 days before compared to the control, and among them, the number of bronchial alveolar cells was most decreased the day before OVA inhalation. As a result, it was confirmed that asthma disease is alleviated (see FIG. 18).
  • TMA was applied to TG mice to induce skin inflammation.
  • the hair on the back of the TG mice was pushed and 50 ⁇ l of 5% TMA (in acetone and isoprophylmyristate, 4: 1) was applied.
  • 10 ⁇ l of 5% TMA was applied to both ears.
  • 10 ⁇ l of 2% TMA was applied to both ears once daily from 6 to 15 days.
  • 5 MPK and 10 MPK NPB301 were administered intravenously (twice a week), and on day 17, total immunoglobulin E in blood was measured using a quantitative kit.
  • SEQ ID NO: 1 shows the amino acid sequence of the water soluble fragment of Fc ⁇ RI.
  • SEQ ID NO: 2 shows the amino acid sequence of the complex (NPB301) in which the water-soluble fragment of Fc ⁇ RI and the Fc region of a human antibody are linked by peptide links.
  • SEQ ID NO: 3 shows a nucleotide sequence encoding a complex (NPB301) in which a water-soluble fragment of Fc ⁇ RI and an Fc region of a human antibody are linked by a peptide link.

Abstract

The present invention relates to a complex in which water-soluble fragments of FcεRI, which is a receptor having a high affinity to the Fc region of immunoglobulin E (IgE), are bound with a material having biocompatibility. The complex in which the water-soluble fragments of FcεRI are bound with the material having biocompatibility according to the present invention inhibits the coupling of IgE and the FcεRI, which is a receptor of the IgE, so as to suppress the secretion of allergic cytokine. Thus, the complex has the effect of treating and preventing allergic diseases.

Description

FCεRI의 수용성 단편을 포함하는 복합체 및 이를 포함하는 IGE 매개 알레르기성 질환 치료용 조성물Complex containing a water-soluble fragment of FCεRI and composition for the treatment of IGE mediated allergic disease comprising the same
본 발명은 면역글로불린 E(IgE)의 Fc 부위에 고친화성을 갖는 수용체인 FcεRI의 수용성(soluble) 단편과 생체 적합성(biocompatibility)을 가지는 물질이 접합된 복합체에 대한 발명으로, 본 발명에 따른 FcεRI의 수용성 단편과 생체 적합성을 가지는 물질이 접합된 복합체는 IgE와 IgE 수용체인 FcεRI의 결합을 저해하여 알레르기성 사이토카인의 분비를 억제시킴으로써 알레르기 질환을 치료 및 예방하는 효과가 있다.The present invention relates to a complex in which a soluble fragment of FcεRI, a receptor having high affinity, and a biocompatibility material are conjugated to the Fc region of immunoglobulin E (IgE). The complex conjugated with the water-soluble fragment and the biocompatible material has the effect of treating and preventing allergic diseases by inhibiting the secretion of allergic cytokines by inhibiting the binding of IgE and FgεRI, an IgE receptor.
특히 본 발명에 따른 바람직한 복합체의 형태는 FcεRI의 수용성 단편과 인간 항체의 Fc가 융합된 융합 단백질로, 본 발명에서 제공되는 FcεRI의 수용성 단편과 인간 항체의 Fc가 융합된 융합 단백질인 NPB301은 IgE와 IgE 수용체인 FcεRI의 결합을 억제하여, IgE와 FcεRI에 의해 유도되는 신호전달을 저해하며, 이에 따라 알레르기성 사이토카인의 분비를 억제시킴으로써 아토피성 피부염, 알레르기성 비염, 천식, 두드러기 및 접촉성 피부염 등의 알레르기성 질환의 치료에 탁월한 치료효능을 나타낸다. Particularly preferred form of the complex according to the present invention is a fusion protein in which the water-soluble fragment of FcεRI and the Fc of a human antibody is fused. NPB301, a fusion protein in which the water-soluble fragment of FcεRI and the Fc of a human antibody, is provided. It inhibits the binding of the IgE receptor FcεRI, inhibits the signaling induced by IgE and FcεRI, and thus inhibits the secretion of allergic cytokines, thereby causing atopic dermatitis, allergic rhinitis, asthma, urticaria and contact dermatitis. It has an excellent therapeutic effect in the treatment of allergic diseases.
특정한 음식이나 약에 과민반응하는 알레르기 반응으로서 체내 고민감성 즉각적 반응 형태를 나타내는 천식, 알레르기성 비염 등의 질환이 있다. 알레르기 반응에서의 즉각적인 반응은 이뮤노글로블린 E(IgE)에 의해 매개되어진다. 대부분의 알레르기 반응은 IgE가 알레르기 수용체의 표면에 결합함으로써 유발되어지는데, IgE의 Fc 부분(Fcε)에 대한 수용체중 대표적인 것이 FcεRI이다. IgE는 비만 세포(mast cell) 또는 호염기(basophilia)성 세포 표면에서 발현되어지는 IgE 수용체인 FcεRI와 결합을 하여 알레르기를 일으키는 신호전달을 하게 된다. As an allergic reaction to hypersensitivity to certain foods or drugs, there are diseases such as asthma and allergic rhinitis, which have a form of an immediate response in the body. Immediate response in allergic reactions is mediated by immunoglobulin E (IgE). Most allergic reactions are caused by the binding of IgE to the surface of the allergic receptor, one of the receptors for the Fc portion (Fcε) of IgE is FcεRI. IgE binds to FcεRI, an IgE receptor expressed on the surface of mast cells or basophilic cells, resulting in allergic signaling.
인간 FcεRI은 알파쇄, 베타쇄(시그날 증폭인자), 감마쇄(시그날 전달인자)의 3개의 서로 다른 서브유닛을 가지고 있다. 각각 한 개의 알파쇄 및 베타쇄 및 두 개의 감마쇄로 구성된 테트라머 FcεRI는 주로 조직세포 및 호염기구의 세포막상에서 발현되고, 세포의 활성화를 위한 I형 알레르기 반응에서 중요한 역할을 한다. FcεRI 중 알파쇄만이 IgE와 직접 결합하며 IgE에 대한 결합 부위는 알파쇄의 세포외 영역 전체에 걸쳐 있다(Nature. 2000, 406, 259-266). 이 중 IgE가 결합하는 FcεRI은 α-서브유닛을 가지고 있는데(FcεRIα), FcεRIα는 60 kDa 정도의 크기를 가지며, 세포막 안에 존재하는 소수성 도메인과 세포막 외부에 존재하는 친수성 도메인으로 구성된다.Human FcεRI has three different subunits: alpha chain, beta chain (signal amplification factor) and gamma chain (signal transfer factor). Tetramer FcεRI, composed of one alpha chain and one beta chain and two gamma chains, respectively, is mainly expressed on the cell membranes of tissue cells and basophils and plays an important role in type I allergic responses for cell activation. Only the alpha chain of FcεRI binds directly to IgE, and the binding site for IgE spans the extracellular region of the alpha chain (Nature. 2000, 406, 259-266). Among them, FcεRI to which IgE binds has an α-subunit (FcεRIα), and FcεRIα has a size of about 60 kDa, and is composed of a hydrophobic domain existing in the cell membrane and a hydrophilic domain existing outside the cell membrane.
혈중 IgE는 B 림포사이트에 의해 생산되어지는데, 병원체(pathogens)에 대한 일차적 방어기작에 포함되어 진다. IgE는 특정 알레르겐에 결합 및 비만세포 FcεRI의 결합에 의하여 하위 신호전달물질인 Syk 단백질을 인산화시키고, 하위 세포신호전달 기작이 유도되어 비만세포가 활성화된다. 그 결과로 인해 비만 세포 밖으로 TNF가 분비되고, 세포질의 과립에 저장되어 있던 히스타민, 콘드로이틴 설페이트(chondroitin sulfate), 헤파린 및 단백질 분해효소인 트립테이즈(tryptase)와 카이메이즈(chymase)등이 빠른 시간 내에 탈과립되어 세포 밖으로 분비된다. 비만세포와 호염기성 세포에서 분비되는 이러한 물질들은 각각 가려움증, 담마진(두드러기), 소양증, 피부이상, 복통, 설사, 기관지 수축, 천명, 호흡곤란, 기관지 천식증상, 콧물, 재채기 등의 다양한 생리학적 알레르기 질환을 일으킨다. Blood IgE is produced by B lymphocytes and is involved in the primary defense against pathogens. IgE phosphorylates Syk protein, a downstream signaling material by binding to specific allergens and binding of mast cell FcεRI, and activates mast cells by inducing downstream cell signaling mechanisms. As a result, TNF is secreted out of mast cells, and histamine, chondroitin sulfate, heparin, and protease tryptase and chymase, which are stored in cytoplasmic granules, are fast. It is degranulated in and secreted out of the cell. These substances are secreted from mast cells and basophils, and various physiological allergies such as itching, tingling, pruritus, skin disorders, abdominal pain, diarrhea, bronchial contraction, wheezing, dyspnea, bronchial asthma, runny nose, and sneezing Cause disease.
알레르기성 질환의 원인은 아직 명확하게 규명되고 있지는 않지만, 인체 면역 기전의 불균형 등이 원인으로 제시되고 있다. 또한 혈중 내 IgE의 증가 또한 주요한 알레르기성 질환의 원인으로 제시되고 있다. 특히 천식 환자의 혈중 내 IgE의 농도 (100~500 IU/ml)가 정상인(20~40 IU/ml)에 비하여 매우 증가되어 있는 것으로 보고되어 있으며, 아토피 환자의 경우 그 농도가 (1000 IU/ml) 훨씬 높아 알레르기성 질환에 있어 IgE의 역할이 강조되고 있다. 이외에도 알레르기성 비염, 천식 및 아토피 피부염에서 환자의 혈액 중 FcεRI의 발현은 증가되어져 있기 때문에, 알레르기 질환에서 IgE와 FcεRI 수용체 사이의 결합은 질환의 발병에 있어 매우 중요하다 할 수 있다(J. Allergy Clin. Immunol. 1980, 66, 305-313).The cause of allergic diseases has not been elucidated yet, but the imbalance of human immune mechanisms has been suggested as the cause. In addition, increased IgE in blood has been suggested as a major cause of allergic diseases. In particular, the concentration of IgE (100-500 IU / ml) in the blood of asthma patients is reported to be significantly increased compared to the normal (20-40 IU / ml), and the concentration of (1000 IU / ml) in atopic patients Much higher is the role of IgE in allergic diseases. In addition, since allergic rhinitis, asthma and atopic dermatitis have increased expression of FcεRI in the blood of patients, the binding between IgE and FcεRI receptors in allergic diseases may be very important for the development of the disease (J. Allergy Clin). Immunol. 1980, 66, 305-313).
현재 알레르기 질환의 치료를 위해서 알레르겐 회피요법, 항 알레르기 약물사용, 특정 알레르겐에 대한 면역 치료 요법 등이 사용되고 있다(Clin. Immunol. 1997, 100(2), 171-176, Allergy 1998, 53, 821-832, Allergy Clin. Immunol. 1998, 102, 631-636, Clin. Exp. Allergy. 1999, 29 (11), 1563-1571, Allergy Clin. immunol. 2002, 110, 154-159). 가장 널리 사용되는 알레르기 질환 치료제로는 항히스타민 약품과 코르티코스테로이드(corticosteroid) 계통의 약품이 있다. 이외에도 베타-2-아드레네직(beta-2 adrenergic) 수용체의 촉매제나 포스포다이에스테라제-3(phosphodiesterase-3) 저해제 등이 알레르기성 질환의 치료제로 사용되고 있다. 지난 수년간 다수의 알레르기 치료용 약품이 개발 되었으나 증상을 완화하는 데 그칠 뿐, 실제로 알레르기의 근본 원인을 치료할 수 있는 치료제는 없는 실정이다. 또한 약제의 효능이 불충분하거나 부작용으로 인한 많은 단점을 가지고 있다. 따라서 새로운 개념의 알레르기 치료제에 대한 수요가 증가하고 있는 현실이다. Currently, allergen avoidance therapy, antiallergic drug use, and immunotherapy for certain allergens have been used for the treatment of allergic diseases (Clin. Immunol. 1997, 100 (2), 171-176, Allergy 1998, 53, 821-). 832, Allergy Clin. Immunol. 1998, 102, 631-636, Clin.Exp. Allergy. 1999, 29 (11), 1563-1571, Allergy Clin. Immunol. 2002, 110, 154-159). The most widely used treatments for allergic diseases include antihistamines and drugs of the corticosteroid family. In addition, beta-2-adrenergic (beta-2 adrenergic) receptors and phosphodiesterase-3 (phosphodiesterase-3) inhibitors and the like are used as therapeutic agents for allergic diseases. Many allergens have been developed over the past few years, but only alleviate the symptoms, and there are no treatments that can cure the root cause of allergies. In addition, the efficacy of the drug is insufficient or has a number of disadvantages due to side effects. Therefore, the demand for a new concept of allergy treatment is increasing.
전통적인 면역 요법은 알레르겐을 장기간에 걸쳐 그 양을 증가시키면서 알레르기 환자에 투입하는 방법이다. 이 방법은 알레르기성 비염의 치료에 효과를 보였고 알레르기성 천식에도 일정 부분 효과를 보였다. 이러한 면역요법은 T 세포 반응을 Th2 유형에서 Th1으로 변경함으로써 알레르기 환자의 증상을 완화시키게 하고 알레르겐 특이적 IgG 반응을 유도한다. 하지만 이러한 면역 요법 역시 환자의 증상을 완화시키는 작용을 할 뿐, 근본적인 치료는 되지 못한다는 단점을 갖고 있다. Traditional immunotherapy is a method of injecting allergens into allergic patients with increasing amounts over time. This method has been shown to be effective in the treatment of allergic rhinitis and in part to allergic asthma. This immunotherapy alters the T cell response from Th2 type to Th1 to alleviate the symptoms of allergic patients and induce allergen specific IgG responses. However, these immunotherapy also works to alleviate the symptoms of the patient, and has the disadvantage of not being a fundamental treatment.
또한 현재 IgE를 타겟으로 하는 항체 제품으로는 오말리주맵(Omalizumab, 상품명 : 졸레어(Xolair))이 천식환자 치료제로 사용되고 있다. 오말리주맵은 IgE에 결합함으로써 천식과 알레르기의 주된 원인인 화학물질 방출을 억제하는 역할을 한다. 하지만 오말리주맵은 혈관부종, 아나필락시스 반응 및 다른 알레르기성 상태, 알레르기성 기관지 경련 등의 부작용이 있으며, 시판 후 결과를 통해 심각한 이상인 알레르기성 육아종 혈관염, 특발성 중증 혈소판감소증이 보고되고 있다. In addition, as an antibody product targeting IgE, Omalizumab (Xolair) is used as a treatment for asthma patients. Omalizumap binds to IgE and inhibits the release of chemicals, a major cause of asthma and allergies. However, Omalizumab has side effects such as angioedema, anaphylactic reactions and other allergic conditions, and allergic bronchial spasms, and allergic granulomatous vasculitis and idiopathic severe thrombocytopenia have been reported through postmarketing results.
따라서, 부작용 없이 알레르기 질환을 효율적이고 근본적으로 치료할 수 있는 치료제 개발에 대한 수요는 지속적으로 증가하고 있는 실정이다. Therefore, the demand for the development of a therapeutic agent capable of effectively and fundamentally treating allergic diseases without side effects is continuously increasing.
상기와 같은 문제를 해결하고자 본 발명은 부작용 없이 알레르기 질환을 효율적이고 근본적으로 치료할 수 있는 치료제를 제공하기 위하여, IgE의 Fc 부위에 고친화성을 갖는 수용체인 FcεRI의 수용성 단편과 생체 적합성을 가지는 물질이 접합된 복합체 및 그러한 복합체의 제조방법을 제공하는 것을 목적으로 한다. 특히 본 발명에서 제공되는 바람직한 복합체의 형태는 FcεRI의 수용성 단편과 인간 항체의 Fc가 융합된 서열번호 2의 아미노산 서열을 가지는 융합 단백질인 NPB301이다. In order to solve the above problems, the present invention is to provide a therapeutic agent that can effectively and fundamentally treat allergic diseases without side effects, a material having a biocompatibility with a water-soluble fragment of FcεRI, a receptor having a high affinity for the Fc region of IgE It is an object to provide a conjugated complex and a method of making such a complex. Particularly preferred form of the complex provided in the present invention is NPB301, which is a fusion protein having the amino acid sequence of SEQ ID NO: 2 where the water-soluble fragment of FcεRI and the Fc of a human antibody are fused.
본 발명의 또 다른 목적은 상기의 복합체를 함유하는 알레르기성 비염, 천식, 아토피 피부 질환, 두드러기 및 접촉성 피부염을 치료하기 위한 약학 조성물을 제공하는 것이다. It is another object of the present invention to provide a pharmaceutical composition for treating allergic rhinitis, asthma, atopic skin disease, urticaria and contact dermatitis containing the complex.
본 발명의 또 다른 목적은 상기의 복합체를 이용한 알레르기 질환, 특히 알레르기성 비염, 천식, 아토피 피부 질환, 두드러기 및 접촉성 피부염 등을 억제하는 방법을 제공하는 것이다.It is another object of the present invention to provide a method for inhibiting allergic diseases, in particular allergic rhinitis, asthma, atopic skin disease, urticaria and contact dermatitis, etc. using the complex.
상기 목적을 달성하기 위하여, 본 발명은 FcεRI 수용체의 수용성 단편을 생체 적합성 물질과 결합시킨 복합체를 제조하였다. In order to achieve the above object, the present invention prepared a complex in which a water-soluble fragment of the FcεRI receptor is combined with a biocompatible material.
본 발명에서의 ‘FcεRI의 수용성 단편(soluble fragment)’은 FcεRI 중에서 세포내 도메인(intracellular domain)이 결여되어 있어, IgE가 결합되더라도 세포 내로 신호전달(signal transduction)이 일어나지 않는 단편을 의미하며, IgE과 결합할 수 있는 능력을 가지는 세포외 도메인(extracellular domain)의 일부 또는 전부로 구성됨이 바람직하며, 추가적으로 세포막 도메인(transmembrane domain)이 포함될 수 있다. 특히 본 발명에 따른 바람직한 FcεRI의 수용성 단편은 서열번호 1에 기재된 아미노산 서열을 가지는 것이 바람직하다. In the present invention, 'soluble fragment of FcεRI' refers to a fragment in which FgεRI lacks an intracellular domain, and thus no signal transduction occurs in cells even though IgE is bound. It is preferably composed of part or all of the extracellular domain having the ability to bind to, and may additionally include a transmembrane domain. Particularly preferred water-soluble fragments of FcεRI according to the present invention preferably have the amino acid sequence set forth in SEQ ID NO: 1.
본 발명에 따른 생체 적합성 물질은 FcεRI의 수용성 단편의 체내 안정성(stability)을 증가시키는 기능을 하며 체내에서 면역반응 등을 유발하지 않는 특성을 가진 것이라면 어떤 것이라도 사용가능하며, 폴리에틸렌글리콜(PEG), 디아실글리세롤(DAG), 인간 항체의 Fc(fragment crystallizable) 영역 등이 바람직하지만 이에 한정되는 것은 아니다. The biocompatible material according to the present invention functions to increase the stability of the water-soluble fragment of FcεRI and can be used as long as it has a property that does not cause an immune response in the body, polyethylene glycol (PEG), Diacylglycerol (DAG), a fragment crystallizable (Fc) region of a human antibody, and the like are preferred, but are not limited thereto.
항체의 Fc 영역은 항체를 파파인(papain) 효소로 처리해서, 절단되는 불변영역(constant region) 부분의 단편을 의미하며, IgA, IgD, IgE, IgG 및 IgM 항체에서 유래된 것이라면 어떤 것이라도 제한 없이 본 발명에 따른 복합체 제조에 이용가능하며, 특히 그 중에서 IgG 항체의 IgG1, IgG2, IgG3 및 IgG4 항체 유래의 Fc 영역이 특히 바람직하다. The Fc region of an antibody refers to a fragment of the constant region portion that is cleaved by treating the antibody with papain enzymes, without limitation if it is derived from IgA, IgD, IgE, IgG and IgM antibodies Particularly preferred are Fc regions derived from IgG1, IgG2, IgG3 and IgG4 antibodies of the IgG antibodies, which are available for the preparation of the complex according to the invention.
본 발명에 따른 바람직한 복합체는 FcεRI의 수용성 단편과 인간 항체의 Fc 영역이 접합된 형태의 융합 단백질(fusion protein)이다. FcεRI의 수용성 단편과 인간 항체의 Fc 영역은 직접 접합하는 것도 가능하며, 링커(linker)를 사용하여 연결하는 것도 가능하다. 링커는 FcεRI의 수용성 단편과 IgE의 결합에 영향을 미치지 않으면서, 3차원적으로 FcεRI의 수용성 단편의 움직임을 자유롭게 하는 것이라면 본 발명이 속하는 기술분야에서 통상적으로 사용될 수 있는 어떤 것이라도 제한 없이 사용가능하며, 바람직한 링커는 펩타이드 링커(peptide linker)이지만, 이에 한정되는 것은 아니다. Preferred complexes according to the invention are fusion proteins in the form of a conjugate of a water-soluble fragment of FcεRI and an Fc region of a human antibody. The water-soluble fragment of FcεRI and the Fc region of a human antibody can be directly conjugated, and can also be linked using a linker. The linker can be used without limitation as long as it frees the movement of the water-soluble fragment of FcεRI in three dimensions without affecting the binding of the water-soluble fragment of FcεRI to IgE. Preferred linkers are, but are not limited to, peptide linkers.
본 발명에서 제공되는 바람직한 융합 단백질은 서열번호 1에 따른 FcεRI의 수용성 단편과 인간 항체의 Fc 영역이 펩타이드 링커에 의해 서로 융합된 형태이며, 특히 서열번호 2와 같은 아미노산 서열을 가지는 융합단백질인 NPB301이 바람직하다. A preferred fusion protein provided in the present invention is a form in which the water-soluble fragment of FcεRI according to SEQ ID NO: 1 and the Fc region of a human antibody are fused to each other by a peptide linker, and in particular, NPB301, a fusion protein having an amino acid sequence such as SEQ ID NO: 2, is desirable.
또한 본 발명의 융합 단백질의 특성이 유지되는 한, FcεRI 수용성 단편, 인간 항체의 Fc 영역 또는 링커 구조에서의 변이가 일어난 융합 단백질도 본 발명의 권리범위에 포함된다. 그러한 예로서 FcεRI의 수용성 단편 내에서의 아미노산의 보존적 치환(conservative substitution)이 일어난 경우를 들 수 있다. 보존적 치환은 원래의 아미노산 서열과 유사한 특성을 가지는 다른 아미노산 잔기로의 치환을 의미하는데, 예를 들어 라이신, 아르기닌, 히스티딘은 염기 곁사슬을 가지고 있어 유사한 특성을 가지고, 아스파라틱산과 글루타믹산은 산 곁사슬을 가진다는 점에서 유사한 특성을 가진다. 또한, 글라이신, 아스파라긴, 글루타민, 세린, 트레오닌, 티로신, 시스테인, 트립토판은 비전하 극성 곁사슬을 가진다는 점에서 특성이 유사하며, 알라닌, 발린, 루신, 트레오닌, 아이소류신, 프롤린, 페닐알라닌, 메티오닌은 비극성 곁사슬을 가지고 있다는 점에서, 티로신, 페닐알라닌, 트립토판, 히스티딘은 방향족 곁사슬을 가지고 있다는 점에서 유사한 특성을 가진다. 따라서, 상기와 같이 유사한 특성을 가지는 그룹 내에서의 아미노산 치환이 일어나더라도 별다른 특성 변화를 보이지 않을 것이라는 점은 통상의 기술자에게는 자명한 것이다.As long as the characteristics of the fusion protein of the present invention are maintained, fusion proteins in which mutations in the FcεRI soluble fragment, Fc region or linker structure of a human antibody have occurred are also included in the scope of the present invention. One such example is the case where conservative substitution of amino acids in the water-soluble fragment of FcεRI occurs. Conservative substitutions mean substitutions with other amino acid residues that have properties similar to those of the original amino acid sequence. For example, lysine, arginine, and histidine have similar properties because they have a base side chain. Aspartic acid and glutamic acid It has similar characteristics in that it has a mountain side chain. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar characteristics in that they have a non-charged polar side chain, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine are nonpolar. Tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have side chains. Therefore, it will be apparent to those skilled in the art that amino acid substitutions within groups having similar characteristics as described above will not show any change in characteristics.
본 발명에서는 상기와 같이 FcεRI의 수용성 단편을 포함하는 복합체를 유효성분으로 하는 약제학적 조성물을 제공한다. 상기 약제학적 조성물에는 약학적으로 허용가능한 통상적인 담체 및 부형제 등이 추가적으로 포함될 수 있다. 상기 약제학적 조성물은 IgE에 의해 매개되는 모든 알레르기 질환에 적용될 수 있는데, 대표적인 질환으로는 알레르기성 비염, 천식, 아토피 피부 질환, 두드러기 및 접촉성 피부염 등이 있지만, 이에 한정되는 것은 아니다. 또한, 본 발명은 상기 단편 단백질은 치료대상에게 투여하는 것을 특징으로 하는 알레르기 질환의 예방 및 치료방법을 제공한다. The present invention provides a pharmaceutical composition comprising the complex containing a water-soluble fragment of FcεRI as an active ingredient as described above. The pharmaceutical composition may further include conventional pharmaceutically acceptable carriers and excipients. The pharmaceutical composition may be applied to allergic diseases mediated by IgE. Representative diseases include, but are not limited to, allergic rhinitis, asthma, atopic dermatitis, urticaria and contact dermatitis. In addition, the present invention provides a method for preventing and treating allergic diseases, wherein the fragment protein is administered to a therapeutic target.
또한 본 발명에서는 FcεRI의 수용성 단편 또는 FcεRI의 수용성 단편과 인간 항체의 Fc 영역이 접합된 융합 단백질을 인코딩하는 폴리뉴클레오티드 서열을 제공한다. 특히 서열번호 2에 기재된 아미노산 서열을 가지는 융합 단백질인 NPB301을 인코딩하는 서열번호 3에 기재된 폴리뉴클레오티드 서열이 바람직하다. The present invention also provides a polynucleotide sequence encoding a fusion protein in which a water-soluble fragment of FcεRI or a water-soluble fragment of FcεRI and an Fc region of a human antibody are conjugated. Especially the polynucleotide sequence of sequence number 3 which codes NPB301 which is a fusion protein which has the amino acid sequence of sequence number 2 is preferable.
본 발명은 상기 폴리뉴클레오티드 서열을 포함하는 재조합 벡터 및 이를 포함하는 숙주세포, 그리고 상기 재조합 벡터 또는 숙주세포를 이용하여 본 발명에 따른 FcεRI의 수용성 단편을 포함하는 접합체를 제조하는 방법을 제공한다. FcεRI의 수용성 단편이 PEG 등의 단백질이 아닌 물질과 접합된 복합체의 경우에는 FcεRI의 수용성 단편만을 생산한 후, PEG 등과 접합시키는 단계를 추가적으로 수행하여 복합체를 제조할 수 있다. 또한 FcεRI의 수용성 단편과 인간 Fc 영역의 융합 단백질 형태의 복합체의 경우에는 유전자 재조합 방법을 통해 FcεRI의 수용성 단편과 인간 Fc 영역을 인코딩하는 폴리뉴클레오티드 서열을 연결시켜, 상기 단백질이 서로 아미노 결합으로 연결되어 하나의 단백질의 형태로 발현되도록 하여 제조하는 것이 바람직하다. The present invention provides a recombinant vector comprising the polynucleotide sequence and a host cell comprising the same, and a method for producing a conjugate comprising the water-soluble fragment of FcεRI according to the present invention using the recombinant vector or host cell. In the case of a complex in which the water-soluble fragment of FcεRI is conjugated with a material other than a protein such as PEG, only the water-soluble fragment of FcεRI may be produced, followed by conjugation to PEG and the like to prepare the complex. In addition, in the case of a complex in the form of a fusion protein of a water-soluble fragment of FcεRI and a human Fc region, the protein is linked to each other by an amino bond by connecting a polynucleotide sequence encoding the water-soluble fragment of FcεRI and a human Fc region through genetic recombination. It is preferable to prepare by being expressed in the form of one protein.
본 발명에서 "재조합 벡터"란 적당한 숙주세포에서 목적 단백질을 발현할 수 있는 발현 벡터로서, 유전자 삽입물이 발현되도록 작동가능하게 연결된 필수적인 조절 요소를 포함하는 유전자 작제물을 말한다. 본 발명에서 "작동가능하게 연결된 (operably linked)"는 일반적 기능을 수행하도록 핵산 발현조절 서열과 목적하는 단백질을 코딩하는 핵산 서열이 기능적으로 연결되어 있는 것을 말한다. 재조합 벡터와의 작동적 연결은 본 발명이 속하는 기술분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 본 발명이 속하는 기술 분야에서 일반적으로 알려진 효소 등을 사용하여 용이하게 수행할 수 있다.As used herein, a "recombinant vector" refers to a gene construct that is an expression vector capable of expressing a protein of interest in a suitable host cell, and which contains essential regulatory elements operably linked to express the gene insert. In the present invention, "operably linked" means that the nucleic acid expression control sequence and the nucleic acid sequence encoding the protein of interest is functionally linked to perform a general function. Operative linkage with recombinant vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes generally known in the art. It can be performed easily.
본 발명에서 사용될 수 있는 적합한 발현 벡터는 프로모터, 개시코돈, 종결코돈, 폴리아데닐화 시그널 및 인핸서 같은 발현 조절 엘리먼트 외에도 막 표적화 또는 분비를 위한 시그널 서열을 포함할 수 있다. 개시 코돈 및 종결 코돈은 일반적으로 면역원성 표적 단백질을 코딩하는 뉴클레오타이드 서열의 일부로 간주되며, 유전자 작제물이 투여되었을 때 개체에서 반드시 작용을 나타내야 하며 코딩 서열과 인프레임(in frame)에 있어야 한다. 일반 프로모터는 구성적 또는 유도성일 수 있다. 원핵 세포에는 lac, tac, T3 및 T7 프로모터가 있으나 이로 제한되지는 않는다. 진핵세포에는 원숭이 바이러스 40(SV40), 마우스 유방 종양 바이러스(MMTV) 프로모터, 사람 면역 결핍 바이러스(HIV), 예를 들면 HIV의 긴 말단 반복부(LTR) 프로모터, 몰로니 바이러스, 시토메갈로바이러스(CMV), 엡스타인 바 바이러스(EBV), 로우스 사코마 바이러스(RSV)프로모터 뿐만 아니라, β-액틴 프로모터, 사람 헤로글로빈, 사람 근육 크레아틴, 사람 메탈로티오네인 유래의 프로모터가 있으나 이것으로 제한되지는 않는다. 상기 발현 벡터는 벡터를 함유하는 숙주 세포를 선택하기 위한 선택성 마커를 포함할 수 있다. 선택마커는 벡터로 형질전환된 세포를 선별하기 위한 것으로, 약물 내성, 영양 요구성, 세포 독성제에 대한 내성 또는 표면 단백질의 발현과 같은 선택가능 표현형을 부여하는 마커들이 사용될 수 있다. 선택제 (selective agent)가 처리된 환경에서 선별 마커를 발현하는 세포만 생존하므로 형질전환된 세포가 선별 가능하다. 또한, 벡터는 복제가능한 발현벡터인 경우, 복제가 개시되는 특정 핵산 서열인 복제원점 (replication origin)을 포함할 수 있다. 재조합 발현 벡터로는 플라스미드, 바이러스, 코즈미드 등 다양한 형태의 벡터를 사용할 수 있다. 재조합 벡터의 종류는 원핵세포 및 진핵세포의 각종 숙주세포에서 원하는 유전자를 발현하고 원하는 단백질을 생산하는 기능을 하는 한 특별히 한정되지 않지만, 강력한 활성을 나타내는 프로모터와 강한 발현력을 보유하면서 자연 상태와 유사한 형태의 외래 단백질을 대량으로 생산할 수 있는 벡터가 바람직하다. Suitable expression vectors that may be used in the present invention may include signal sequences for membrane targeting or secretion in addition to expression control elements such as promoters, initiation codons, termination codons, polyadenylation signals and enhancers. Initiation and termination codons are generally considered to be part of the nucleotide sequence encoding the immunogenic target protein and must be functional in the subject and be in frame with the coding sequence when the gene construct is administered. Generic promoters can be either constitutive or inducible. Prokaryotic cells include, but are not limited to, lac, tac, T3 and T7 promoters. Eukaryotic cells include monkey virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV), for example the long terminal repeat (LTR) promoter of HIV, moronivirus, cytomegalovirus (CMV) ), Epstein Barr virus (EBV), Loose sacoma virus (RSV) promoters, as well as promoters derived from β-actin promoter, human heroglobin, human muscle creatine, human metallothionein, but are not limited thereto. . The expression vector may comprise a selectable marker for selecting a host cell containing the vector. The selection marker is for selecting cells transformed with the vector, and markers conferring a selectable phenotype such as drug resistance, nutritional requirements, resistance to cytotoxic agents or expression of surface proteins can be used. Since only cells expressing a selection marker survive in an environment treated with a selective agent, transformed cells can be selected. In addition, when the vector is a replicable expression vector, the vector may include a replication origin, which is a specific nucleic acid sequence from which replication is initiated. As the recombinant expression vector, various types of vectors such as plasmids, viruses, and cosmids can be used. The type of recombinant vector is not particularly limited as long as it functions to express a desired gene and to produce a desired protein in various host cells of prokaryotic and eukaryotic cells, but has a promoter with strong activity and strong expression, similar to natural state. Vectors that can produce large amounts of foreign protein in form are preferred.
본 발명에 따른 FcεRI의 수용성 단편, 또는 FcεRI의 수용성 단편과 인간 Fc 영역의 융합 단백질을 발현시키기 위해 다양한 발현 숙주/벡터 조합이 이용될 수 있다. 진핵숙주에 적합한 발현 벡터로는 SV40, 소 유두종바이러스, 아네노바이러스, 아데노-연관 바이러스(adeno-associated virus), 시토메갈로바이러스 및 레트로바이러스로부터 유래된 발현 조절 서열 등이 사용될 수 있지만 이에 한정되는 것은 아니다. 세균 숙주에 사용할 수 있는 발현 벡터에는 pET, pRSET, pBluescript, pGEX2T, pUC벡터, col E1, pCR1, pBR322, pMB9 및 이들의 유도체와 같이 대장균(Escherichia coli)에서 얻어지는 세균성 플라스미드, RP4와 같이 보다 넓은 숙주 범위를 갖는 플라스미드, λgt10과 λgt11, NM989와 같은 매우 다양한 파지 람다(phage lambda) 유도체로 예시될 수 있는 파지 DNA, 및 M13과 필라멘트성 단일가닥의 DNA 파지와 같은 기타 다른 DNA 파지가 포함된다. 효모 세포에 유용한 발현 벡터는 2℃ 플라스미드 및 그의 유도체이다. 곤충 세포에 유용한 벡터는 pVL941이다. Various expression host / vector combinations can be used to express the water-soluble fragment of FcεRI, or the fusion protein of the human Fc region, with the water-soluble fragment of FcεRI according to the present invention. Suitable expression vectors for eukaryotic hosts may include, but are not limited to, expression control sequences derived from SV40, bovine papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus and retrovirus. no. Expression vectors that can be used in bacterial hosts include broader hosts, such as bacterial plasmids obtained from Escherichia coli , such as pET, pRSET, pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9, and derivatives thereof. Range plasmids, phage DNA that can be exemplified by a wide variety of phage lambda derivatives such as λgt10 and λgt11, NM989, and other DNA phages such as M13 and filamentary single-stranded DNA phages. Useful expression vectors for yeast cells are 2 ° C. plasmids and derivatives thereof. A useful vector for insect cells is pVL941.
상기 재조합 벡터는 숙주세포에 삽입되어 형질전환체를 형성하는데, 적합한 숙주세포는 대장균, 바실러스 서브틸리스(Bacillus subtilis), 스트렙토마이세스 속 (Streptomyces sp.), 슈도모나스 속(Pseudomonas sp.), 프로테우스 미라빌리스(Proteus mirabilis) 또는 스타필로코쿠스 속(Staphylococcus sp.)과 같은 원핵 세포일 수 있다. 또한, 아스페르길러스 속(Aspergillus sp.)과 같은 진균, 피치아 파스토리스(Pichia pastoris), 사카로마이세스 세레비지애(Saccharomyces cerevisiae), 쉬조사카로마세스 속(Schizosaccharomyces sp.) 및 뉴로스포라 크라사(Neurospora crassa)와 같은 효모, 그 밖의 하등진핵 세포, 및 곤충으로부터의 세포와 같은 고등 진핵생물의 세포와 같은 진핵 세포일 수 있다. The recombinant vector is inserted into a host cell to form a transformant. Suitable host cells are Escherichia coli, Bacillus subtilis , Streptomyces sp., Pseudomonas sp., Proteus Prokaryotic cells such as Proteus mirabilis or Staphylococcus sp. Also, fungi such as Aspergillus sp., Pichia pastoris , Saccharomyces cerevisiae , Schizosaccharomyces sp. Eukaryotic cells such as yeast, such as Spora crassa , other lower eukaryotic cells, and cells of higher eukaryotes, such as cells from insects.
또한 상기 숙주세포는 바람직하게는 식물, 포유동물로부터 유래할 수 있는데, 원숭이 신장 세포7(COS7 : monkey kidney cells) 세포, NSO 세포, SP2/0, 차이니즈 햄스터 난소(CHO : chinese hamster ovary) 세포, W138, 어린 햄스터 신장(BHK : baby hamster kidney)세포, MDCK, 골수종 세포주, HuT 78 세포 및 HEK293 세포 등이 이용가능하며, 이에 한정되지 않는다. 특히 바람직하게는 CHO 세포이다. In addition, the host cell is preferably derived from plants, mammals, monkey kidney cells (COS7) cells, NSO cells, SP2 / 0, Chinese hamster ovary (CHO: Chinese hamster ovary) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells and HEK293 cells and the like are available, but are not limited to these. Particularly preferably CHO cells.
본 발명에서 숙주세포로의형질 전환"은 핵산을 유기체, 세포, 조직 또는 기관에 도입하는 어떤 방법도 포함되며 당 분야에서 공지된 바와 같이 숙주 세포에 따라 적합한 표준 기술을 선택하여 수행할 수 있다. 이런 방법에는 전기충격유전자전달법(electroporation), 원형질 융합, 인산 칼슘(CaPO4) 침전, 염화 칼슘(CaCl2) 침전, 실리콘 카바이드 섬유 이용한 교반, 아그로 박테리아 매개된 형질전환, PEG, 덱스트란 설페이트, 리포펙타민 및 건조/억제 매개된 형질전환 방법 등이 포함되나 이에 제한되지 않는다. Transformation into a host cell in the present invention includes any method of introducing a nucleic acid into an organism, cell, tissue or organ and can be carried out by selecting a suitable standard technique according to the host cell as is known in the art. These methods include electroporation, protoplast fusion, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, agitation with silicon carbide fibers, agro bacterial mediated transformation, PEG, dextran sulfate, Lipofectamine and dry / inhibited mediated transformation methods and the like.
재조합 벡터가 발현되는 형질전환체를 영양배지에서 배양함으로써 본 발명에 따른 FcεRI 수용체의 수용성 단편과 인간 Fc 영역의 융합 단백질을 대량으로 생산할 수 있으며, 배지와 배양조건은 숙주 세포에 따라 관용되는 것을 적당히 선택 이용할 수 있다. 배양시 세포의 생육과 단백질의 대량 생산에 적합하도록 온도, 배지의 pH 및 배양시간 등의 조건들을 적절하게 조절할 수 있다. 상기와 같이 재조합적으로 생산된 항체 또는 항체 단편은 배지 또는 세포 분해물로부터 회수될 수 있으며, 통상적인 생화학 분리 기술에 의해서 분리, 정제가 가능하다(Sambrook et al., Molecular Cloning: A laborarory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press(1989); Deuscher, M., Guide to Protein Purification Methods Enzymology, Vol. 182. Academic Press. Inc., San Diego, CA(1990)). 이에는 전기영동, 원심분리, 겔여과, 침전, 투석, 크로마토그래피(이온교환 크로마토그래피, 친화성 크로마토그래피, 면역흡착 크로마토그래피, 크기 배제 크로마토그래피 등), 등전점 포커싱 및 이의 다양한 변화 및 복합 방법 등이 이용가능하지만 이에 한정되지 않으며, 특히 프로테인 A(Protein A)를 이용하여 분리, 정제하는 것이 바람직하다.By culturing the transformant expressing the recombinant vector in a nutrient medium, a soluble protein of the FcεRI receptor and a fusion protein of the human Fc region according to the present invention can be produced in a large amount, and the medium and culture conditions are appropriately tolerated by the host cell. Optional is available. Conditions such as temperature, pH of the medium and incubation time can be appropriately adjusted to be suitable for the growth of cells and the mass production of proteins during the culture. The recombinantly produced antibody or antibody fragment as described above can be recovered from the medium or cell digest, and can be isolated and purified by conventional biochemical separation techniques (Sambrook et al., Molecular Cloning: A laborarory Manual, 2nd). Ed., Cold Spring Harbor Laboratory Press (1989); Deuscher, M., Guide to Protein Purification Methods Enzymology, Vol. 182. Academic Press. Inc., San Diego, CA (1990)). These include electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (ion exchange chromatography, affinity chromatography, immunosorbent chromatography, size exclusion chromatography, etc.), isoelectric focusing, and various variations and complex methods thereof. These may be used but are not limited thereto, and in particular, it is preferable to separate and purify using Protein A.
특히 본 발명에서의 바람직한 FcεRI의 수용성 단편과 인간 Fc 영역의 융합 단백질인 NPB301은 서열번호 3에 따른 폴리뉴클레오티드 서열을 이용하여 동물세포를 숙주세포로 이용, 배양함으로써 생산하는 것이 바람직하다. In particular, NPB301, which is a soluble protein of the preferred FcεRI and human Fc region in the present invention, is preferably produced by culturing and using animal cells as host cells using a polynucleotide sequence according to SEQ ID NO: 3.
본 발명에 따른 FcεRI의 수용성 단편을 포함하는 복합체는 IgE와 FcεRI 간의 결합을 낮은 농도로도 효율적으로 억제하여, IgE에 의해 매개되는 다양한 알레르기성 질환에 우수한 치료효과를 나타내며, FcεRI에만 특이적으로 결합, 작용하므로 부작용이 거의 없으며, 체내 투여시 면역반응을 유발하지 않는다.The complex containing the water-soluble fragment of FcεRI according to the present invention effectively inhibits the binding between IgE and FcεRI at low concentrations, and thus exhibits an excellent therapeutic effect on various allergic diseases mediated by IgE, and specifically binds only to FcεRI. It has no side effects and does not cause an immune response when administered in vivo.
본 발명에 따른 FcεRI 수용체 단편을 포함하는 복합체는 IgE와 FcεRI 간의 결합을 억제하여 IgE 매개된 알레르기 질환을 예방 또는 치료할 수 있다. 특히 아토피성 피부염, 알레르기성 비염, 천식, 두드러기 및 접촉성 피부염 환자의 치료용도로 유용하게 사용될 수 있다.Complexes comprising FcεRI receptor fragments according to the present invention may inhibit or treat IgE mediated allergic diseases by inhibiting binding between IgE and FcεRI. In particular, it can be usefully used for the treatment of patients with atopic dermatitis, allergic rhinitis, asthma, urticaria and contact dermatitis.
도 1은 본 발명에 따른 융합단백질인 NPB301을 인코딩하는 DNA 서열을 나타낸 도면.1 is a diagram showing a DNA sequence encoding NPB301, a fusion protein according to the present invention.
도 2는 본 발명에 따른 융합단백질인 NPB301의 아미노산 서열을 나타낸 도면.Figure 2 shows the amino acid sequence of NPB301, a fusion protein according to the present invention.
도 3은 본 발명에 따른 융합단백질인 NPB301이 베타-헥소스아미니데이즈 분비에 미치는 영향을 나타내는 도면.3 is a view showing the effect of the fusion protein NPB301 according to the present invention on the secretion of beta-hexos aminidayes.
도 4는 본 발명에 따른 융합단백질인 NPB301의 히스타민 분비 억제효과를 나타내는 도면.Figure 4 is a view showing the histamine secretion inhibitory effect of the fusion protein NPB301 according to the present invention.
도 5는 본 발명에 따른 융합단백질인 NPB301의 PCA 저해효과를 나타내는 도면.5 is a view showing the PCA inhibitory effect of NPB301 fusion protein according to the present invention.
(A) 형질전환 마우스를 이용한 귀에서의 과민 반응 육안 관찰 결과(A) Visual observation of hypersensitivity reactions in the ears using transgenic mice
(B) 형질전환 마우스를 이용한 귀 색 변화의 흡광도 측정 결과(B) Absorbance Measurement of Ear Color Change Using Transgenic Mouse
도 6은 OVA 유도된 FcεRIα 트랜스제닉 마우스(Tg mice) 천식 모델에서의 NPB301에 의한 폐 조직의 변화를 조직 생검으로 분석한 결과를 나타내는 도면.Fig. 6 shows the results of analysis of tissue changes in lung tissue by NPB301 in OVA-induced FcεRIα transgenic mice (Tg mice) asthma model.
도 7은 OVA 유도된 FcεRIα 트랜스제닉 마우스 천식 모델에서의 NPB301에 의한 전체 세포수, 대식구, 호산구 및 림프구 수 변화를 나타내는 도면.FIG. 7 shows changes in total cell number, macrophages, eosinophils and lymphocytes by NPB301 in an OVA induced FcεRIα transgenic mouse asthma model.
도 8은 OVA 유도된 FcεRIα 트랜스제닉 마우스 천식 모델에서의 NPB301에 의한 사이토카인(IL-4, IL-5, Il-13)의 양 변화를 나타내는 도면.FIG. 8 shows the amount changes of cytokines (IL-4, IL-5, Il-13) by NPB301 in OVA induced FcεRIα transgenic mouse asthma model.
도 9는 접촉성피부염 동물모델에서의 NPB301에 의한 귀의 두께 변화를 나타내는 도면.9 is a view showing the change of the thickness of the ear by NPB301 in contact dermatitis animal model.
도 10은 TPA 유도된 피부염증 동물 모델에서의 NPB301에 의한 귀의 두께 변화를 나타내는 도면.FIG. 10 shows ear thickness change by NPB301 in TPA induced dermatitis animal model.
도 11은 땅콩 알레르기 유발 동물모델에서의 NPB301에 의한 면역글로불린 E의 변화를 나타내는 도면.FIG. 11 shows changes in immunoglobulin E by NPB301 in a peanut allergen animal model. FIG.
도 12는 비염 동물모델에서의 NPB301에 의한 비염 개선효과를 나타내는 도면.12 is a view showing the rhinitis improvement effect by NPB301 in a rhinitis animal model.
(A) 코 문지르기 횟수를 측정한 결과(A) Results of measuring the number of nose rubs
(B) 재채기 횟수를 측정한 결과(B) measuring the number of sneezes
도 13은 아토피 동물모델에서 NPB301 정맥주사 후 경피 수분손실도, 피부 내 수화도, 및 피부두께의 변화를 나타내는 도면.FIG. 13 is a graph showing changes in percutaneous water loss, intrahydration, and skin thickness after NPB301 intravenous injection in an atopy animal model. FIG.
도 14는 아토피 동물모델에서 NPB301 복강주사 후 경피 수분손실도, 피부 내 수화도, 및 피부두께의 변화를 나타내는 도면.FIG. 14 is a graph showing changes in percutaneous water loss, intrahydration, and skin thickness after NPB301 intraperitoneal injection in an atopy animal model. FIG.
도 15는 OVA 아토피 동물모델에서 다양한 농도의 NPB301 복강주사 후, NPB301 농도에 따른 피부두께의 변화를 나타내는 도면.15 is a view showing the change in skin thickness according to the NPB301 concentration after NPB301 intraperitoneal injection of various concentrations in the OVA atopic animal model.
도 16은 OVA 아토피 동물모델에서의 NPB301에 의한 아토피 개선효과를 나타내는 도면.16 is a diagram showing the effect of atopy improvement by NPB301 in the OVA atopy animal model.
(A) 5 MPK 복강주사 후 피부두께의 변화를 측정한 결과(A) Measurement of skin thickness change after 5 MPK intraperitoneal injection
(B) 5 MPK 복강주사 후 면역글로불린 E의 변화를 측정한 결과(B) Measurement of changes in immunoglobulin E after 5 MPK intraperitoneal injection
도 17은 OVA 아토피 동물모델에서 제형화 된 NPB301을 환부에 바른 후 피부의 두께 변화를 나타내는 도면.17 is a diagram showing the change in the thickness of the skin after applying NPB301 formulated in the OVA atopic animal model to the affected area.
도 18은 OVA 천식 동물모델에서의 NPB301에 의한 전체 세포수 변화를 나타내는 도면.Figure 18 shows the total cell number change by NPB301 in OVA asthma animal model.
도 19는 TMA 피부염증 동물모델에서의 NPB301에 의한 면역글로불린 E의 변화를 나타내는 도면.FIG. 19 shows changes in immunoglobulin E by NPB301 in a TMA dermatitis animal model.
이하 본 발명을 실시예와 첨부된 도면을 참조하여 상세히 설명한다. 그러나 이들은 본 발명을 보다 상세하게 설명하기 위한 것으로, 본 발명의 권리범위가 하기의 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to embodiments and the accompanying drawings. However, these are intended to explain the present invention in more detail, and the scope of the present invention is not limited by the following examples.
[실시예 1] FcεRI 수용체 단편을 포함하는 융합 단백질의 제조Example 1 Preparation of Fusion Protein Containing FcεRI Receptor Fragments
FcεRI 수용체 단편을 포함하는 융합 단백질을 제조하기 위하여, 오리진 테크놀로지스(Origene Technologies, 미국)에서 인간 FcεRI 수용체의 수용성 단편을 코딩하는 cDNA(카탈로그 No. sc122614)를 구입하였다. 이를 인간 IgG의 Fc를 발현하는 TT5 벡터에 재조합시키고, 293-6E 세포로 일시적인 형질전환을 한 후 FreeStyle 293 배지(GIBCO, 12338)에서 배양하여 서열번호 2에 따른 융합 단백질 NPB301을 제조하였다. To prepare fusion proteins comprising FcεRI receptor fragments, cDNA (Catalog No. sc122614) encoding a water soluble fragment of human FcεRI receptor was purchased from Origin Technologies (USA). This was recombined with a TT5 vector expressing the Fc of human IgG, and transiently transformed into 293-6E cells, followed by culturing in FreeStyle 293 medium (GIBCO, 12338) to prepare a fusion protein NPB301 according to SEQ ID NO: 2.
[실시예 2] 선별된 항체의 탈과립 억제 효과Example 2 Degranulation Inhibitory Effect of Selected Antibodies
베타 헥소스아미니데이즈 분비량 측정을 위해 96-웰 플레이트에 RBL-SX38 세포를 3% FBS(Feral Bovine Serum)와 DMEM 배지(Dulbecco's Modified Eagle's Medium)에 현탁시킨 후 96-플레이트에 2 × 104 cells/100 ul로 37℃, 5% CO2 배양기에서 48시간 배양하였다. 배양 배지를 제거한 후 각 세포들을 PBS+a 버퍼(buffer) (PBS, 0.4 mM MgCl2, 1 mM CaCl2, 5.6 mM 포도당, 0.1% BSA)로 1회 세척한 다음 니트로페닐-IgE(nitropheny-IgE; NP-IgE) (0.1 ㎍/㎖)를 분주한 뒤 37℃, 5% CO2 배양기에서 30분간 반응 후, NPB301을 농도별로 처리하여 37℃, 5% CO2배양기에서 150분 동안 반응시켰다. 이후 NP-BSA(0.1 ug/ml) 을 가하여 37℃, 5% CO2 배양기에서 30분 동안 반응시키고 세포 밖으로 분비되는 베타 헥소스아미니데이즈를 포함하는 상층액 30 ㎕과, 0.1% 트리톤 X-100 60 ㎕으로 용해(lysis)시켜 세포 내의 베타 헥소스아미니데이즈가 수득되어 포함된 용액 30 ㎕를 96-웰 플레이트에 옮기고 기질버퍼 (4-p-니트로페닐-N-아세틸-β-D-글루코사미니드(4-p-nitrophenyl-N-acetyl-β-D-glucosaminide) 1 mM, 소듐 시트레이트, 0.1 M, pH 4.5) 30 ㎕를 넣고 37℃에서 1시간 동안 배양시킨 후 반응정지 용액(50 mM 글리신, pH 10.7)을 가하여 반응을 종결시키고 405 nm에서 흡광도를 측정하였다.RBL-SX38 cells were suspended in 3% FBS (Feral Bovine Serum) and DMEM medium (Dulbecco's Modified Eagle's Medium) in 96-well plates for measuring beta hexosminidayes secretion, and then 2 × 10 4 cells in 96-plate Incubated for 48 hours at 37 ℃, 5% CO 2 incubator at / 100 ul. After removing the culture medium, each cell was washed once with PBS + a buffer (PBS, 0.4 mM MgCl 2 , 1 mM CaCl 2 , 5.6 mM glucose, 0.1% BSA) and then nitropheny-IgE NP-IgE) (0.1 μg / ml) was dispensed and reacted at 37 ° C., 5% CO 2 incubator for 30 minutes, and then treated with concentration of NPB301 in a 37 ° C., 5% CO 2 incubator for 150 minutes. NP-BSA (0.1 ug / ml) was then added and reacted for 30 minutes in a 37 ° C., 5% CO 2 incubator and secreted out of the cells to 30 μl of supernatant containing beta hexoseminidayes, and 0.1% Triton X- 100 μl of cells were obtained by lysis to 100 μl of cells, and 30 μl of the solution contained therein was transferred to a 96-well plate and the substrate buffer (4-p-nitrophenyl-N-acetyl-β-D- 30 μl of glucosamine (4-p-nitrophenyl-N-acetyl-β-D-glucosaminide) 1 mM, sodium citrate, 0.1 M, pH 4.5) was incubated at 37 ° C. for 1 hour, followed by reaction stopping solution ( 50 mM glycine, pH 10.7) was added to terminate the reaction and the absorbance was measured at 405 nm.
그 결과, NPB301을 처리한 경우 베타 헥소스아미니데이즈 분비량이 감소됨을 확인할 수 있었으며, NPB301의 농도가 증가됨에 따라 분비량 감소가 더욱 강력하게 일어나, 탈과립 현상을 억제함을 확인할 수 있었다(도 3 참조).As a result, it was confirmed that the beta hexose minidayes secretion decreases when treated with NPB301, and as the concentration of NPB301 is increased, the secretion decreases more strongly, thereby inhibiting the degranulation phenomenon (see FIG. 3). ).
[실시예 3] 히스타민(histamine) 분비 억제 효능Example 3 Histamine Secretion Inhibitory Effect
히스타민의 분비량 측정을 위해 먼저 24-웰 플레이트에 RBL-SX38 세포를 10% FBS와 DMEM 배지에 현탁시킨 후, 24-웰 플레이트에 2 × 105 cells/500 ul로 37℃, 5% CO2 배양기에서 48시간 배양하였다. 배양 배지를 제거한 후 각 세포들을 티로드 버퍼(Tyrode’s buffer, 125 mM NaCl, 5 mM KCl, 0.4 mM MgCl2, 1 mM CaCl2, 5.6 mM 포도당, 0.1% BSA, 10 mM 허피스(Hepes), pH7.2)로 2회 세척한 다음 NP-IgE 0.5 ㎍/㎖를 분주한 뒤 배양배지로 37℃, 5% CO2배양기에서 24시간 반응 하였다. 이 때 NPB301을 함께 처리하였다. 24시간 후, 티로드 버퍼로 3회 세척한 다음, NP-BSA 0.1 ug/㎖를 가하여 37℃, 5% CO2배양기에서 30분 동안 반응시켰다. 이후, 상층액 500 ㎕를 원심분리하여 다시 상층액 100 ㎕를 96- 웰 플레이트에 옮기고 히스타민을 아세틸화 시킨 후, 히스타민 함량을 HTRF Kit(CISBIO International, 프랑스)를 이용하여 측정하였다. In order to measure the amount of histamine secreted, RBL-SX38 cells were first suspended in 10-well FBS and DMEM medium in 24-well plates, followed by 37 ° C. and 5% CO 2 incubator at 2 × 10 5 cells / 500 ul in 24-well plates. Incubated for 48 hours. After removing the culture medium, each cell was loaded with Tyrode's buffer, 125 mM NaCl, 5 mM KCl, 0.4 mM MgCl 2 , 1 mM CaCl 2 , 5.6 mM glucose, 0.1% BSA, 10 mM Hepes, pH7. After washing twice with 2), 0.5 μg / ml of NP-IgE was dispensed and reacted in a culture medium at 37 ° C. and 5% CO 2 incubator for 24 hours. At this time, NPB301 was treated together. After 24 hours, three washes with T-load buffer, and then 0.1 ug / ㎖ of NP-BSA was added to react for 30 minutes at 37 ℃, 5% CO 2 incubator. Thereafter, 500 μl of the supernatant was centrifuged, and then 100 μl of the supernatant was transferred to a 96-well plate and acetylated histamine. Then, the histamine content was measured using the HTRF Kit (CISBIO International, France).
그 결과 도 4에 나타난 바와 같이 NPB301은 히스타민의 분비를 낮은 농도에서도 효율적으로 억제하는 효능이 있음을 확인할 수 있었다.As a result, as shown in Figure 4 NPB301 was confirmed that the efficient suppression of histamine secretion at low concentrations.
[실시예 4] PCA(Passive Cutaneous Anaphylaxis) 동물실험을 통한 NPB301의 IgE와 FcεRI의 결합 억제 효능 확인Example 4 Confirmation of the Inhibition Effect of IgE and FcεRI of NPB301 by PCA (Passive Cutaneous Anaphylaxis) Animal Experiment
인간 FcεRI을 발현하도록 형질전환된 마우스 6주령 암컷을 사용하여, 왼쪽 귀에는 NP-IgE(0.1 ug/20 ㎕)를 피하 주사하고, 동시에 오른쪽 귀에는 NP-IgE(0.1 ug/20 ㎕) 와 NPB301(0.08 ug/10㎕)를 피하 주사하였다. 24시간 경과 후, 1% 에반스 블루(EVANS Blue) 파랑 염색약이 용해된 NP-BSA 1 mg를 꼬리에 정맥주사하였다. 90분 경과 후 귀를 절단하고, 포름알데히드를 첨가한 후, 55℃에서 24시간 반응시키고, 620 nm에서 흡광도를 측정하였다.Six-week-old females transformed to express human FcεRI were injected subcutaneously with NP-IgE (0.1 ug / 20 μl) in the left ear, while NP-IgE (0.1 ug / 20 μl) and NPB301 in the right ear. (0.08 ug / 10 μl) was injected subcutaneously. After 24 hours, 1 mg of EV-BSA with 1% Evans Blue blue dye was intravenously injected into the tail. After 90 minutes, the ear was cut off, formaldehyde was added, the reaction was carried out at 55 ° C. for 24 hours, and the absorbance was measured at 620 nm.
그 결과 도 5에 도시된 바와 같이, 본 발명에 따른 NPB301은 PCA를 유의성 있게 저해함을 확인할 수 있었다. 구체적으로 육안으로 확인한 결과, 왼쪽 귀에서는 정상적으로 과민증 반응이 유도되어 파란색으로 변하였지만, 과민증 반응이 억제된 오른쪽 귀는 색깔이 변하지 않고 그대로 유지되었으며(도 5A 참조), 귀를 분리한 후 염색약을 분리한 후 흡광도를 측정결과에서도 유의성 있는 결과를 얻게 되었다(도 5B 참조). 이러한 결과로부터 본 발명에 따른 NPB301이 IgE와 FcεRI의 결합을 억제함을 확인할 수 있었다. As a result, as shown in Figure 5, it could be confirmed that NPB301 according to the present invention significantly inhibited PCA. Specifically, as a result of visual inspection, the left ear was normally induced hypersensitivity reaction and turned blue, but the right ear with the hypersensitivity reaction suppressed remained unchanged (see FIG. 5A), and the dye was separated after separating the ear. After the absorbance was also obtained a significant result from the measurement results (see Fig. 5B). From these results, it was confirmed that NPB301 according to the present invention inhibits the binding of IgE and FcεRI.
[실시예 5] 천식동물 모델을 이용한 NPB301의 과민반응 세포 수의 변화에 미치는 영향 확인Example 5 Confirmation of the Effect of NPB301 on the Change of Hypersensitivity Cell Number Using Asthma Model
OVA(ovalbumin) 100 ug과 알럼(Alum) 2 mg을 200 μl 용액에 포함되도록 하였다. 혼합된 OVA-알럼을 37℃에서 1시간 정도 계속 혼합하였다. 혼합이 끝난 OVA-알럼은 1.5 ml 튜브에 200 μl 씩 분주하고 1 ml 주사기에 하나씩 넣어주었다. 이후, 인간 FcεRI을 발현하도록 형질전환된 마우스 6주령 암컷에 0, 14 일, 2차에 걸쳐 200 μl씩 복강주사하였다. 28, 29, 30일에 코 점막을 통하여 1% OVA 30 μg을 흡입하도록 하였다. 100 ug of OVA (ovalbumin) and 2 mg of Alum were included in a 200 μl solution. The mixed OVA-alum was continuously mixed at 37 ° C. for about 1 hour. The mixed OVA-alum was divided into 200 μl into a 1.5 ml tube and put into a 1 ml syringe. Subsequently, 6-week-old females transformed to express human FcεRI were intraperitoneally injected 200 μl over 0, 14 days and 2 times. At 28, 29 and 30 days, 30 μg of 1% OVA was inhaled through the nasal mucosa.
선별된 항체 NPB301은 10 mg/kg로 26, 27, 28, 29일에 주사하였다. 24시간 후 폐조직을 떼어내어 생검하고, 기관지 폐포 세척액에서 세포 수를 계수하였다. 졸레틸(Zoletil)을 이용해 마취한 마우스의 기관지를 절개한 후 관을 삽입하여 22게이지 주사바늘을 기관에 삽입한 뒤, 기관지 폐포 세척을 시행하였는데 1회당 0.9 mL의 PBS를 2회 주입하여 수거하였다. 수거된 기관지 폐포 세척액은 4℃, 5000 rpm에서 5분간 원심분리하고, 상층액은 다음 실험시까지 -70℃에 보관하였다. 원심분리로 얻어 낸 세포를 혈구 계선반을 이용하여 총 세포수를 세었으며, 사이토스핀(cytospin) 3(Thermo 사, 미국)를 이용하여 유리 슬라이드에 세포를 도말하고, 디프-퀵(Diff-Quik) 염색을 실시하였다. 총 200개의 세포를 세어 폐포 대식세포, 호산구, 림프구, 호중구를 감별 계산하였다. 또한 염증반응과 관련 있는 IL-4, IL-5 및 IL-13 등의 사이토카인의 농도를 측정하였다. Selected antibody NPB301 was injected at 26, 27, 28, 29 days at 10 mg / kg. After 24 hours, the lung tissue was removed and biopsied, and the number of cells in the bronchoalveolar lavage fluid was counted. The bronchial tubes of anesthetized mice were incised using zoletil, a tube was inserted, a 22-gauge needle was inserted into the trachea, and bronchoalveolar lavage was performed. The samples were collected by injecting 0.9 mL of PBS twice per time. . The collected bronchial alveolar lavage fluid was centrifuged at 4 ° C. and 5000 rpm for 5 minutes, and the supernatant was stored at −70 ° C. until the next experiment. Cells obtained by centrifugation were counted using a hemocytometer and plated on glass slides using cytospin 3 (Thermo, USA), and Diff-Quik ) Staining was performed. A total of 200 cells were counted to differentially calculate alveolar macrophages, eosinophils, lymphocytes and neutrophils. In addition, the levels of cytokines such as IL-4, IL-5 and IL-13, which are related to the inflammatory response, were measured.
먼저, 폐조직 생검 결과, OVA를 처리 한 천식 동물 모델의 폐 조직은 기관지벽이 두꺼워져 있지만, NPB301을 정맥 주사한 마우스 모델의 기관지 조직은 정상으로 회복되는 것을 확인할 수 있었다(도 6 참조). First, as a result of lung tissue biopsy, the lung tissue of the asthma model treated with OVA had a thick bronchial wall, but the bronchial tissue of the mouse model injected with NPB301 intravenously recovered to normal (see FIG. 6).
또한, OVA에 의해 유도된 FcεRIα를 발현하도록 형질전환된 천식 모델의 기관지 폐포 세척액에서 전체 세포수, 대식구, 호산구 및 림프구의 수가 PBS 그룹에 비해 증가하지만, NPB301을 처리할 경우, 기관지 폐포 세척액에서 전체 세포수, 대식구, 호산구 및 림프구의 수가 현저하게 감소함을 확인할 수 있었으며(도 7 참조), In addition, the total number of cells, macrophages, eosinophils and lymphocytes in the bronchial alveolar lavage fluid of the asthma model transformed to express OVA-induced FcεRIα was increased compared to the PBS group, but when treated with NPB301, the total bronchoalveolar lavage fluid was treated. It was confirmed that the number of cells, macrophages, eosinophils and lymphocytes significantly decreased (see FIG. 7).
NPB301를 처리한 결과, 기관지 폐포에서 염증반응에 관계된 사이토카인인 IL-4, IL-5, IL-13 등의 양이 현저하게 감소됨을 확인할 수 있었다(도 8 참조).As a result of treatment with NPB301, it was confirmed that the amount of cytokines IL-4, IL-5, IL-13, etc. related to the inflammatory response in the bronchoalveolar was significantly reduced (see FIG. 8).
[실시예 6] 표면 플라즈몬 공명(Biacore)을 이용한 NPB301과 human IgE의 친화도 측정Example 6 Measurement of Affinity between NPB301 and Human IgE Using Surface Plasmon Resonance (Biacore)
GE의 BIAcore3000을 이용하여 CM5 sensor chip에 NPB301을 10 mM sodium acetate(pH 5.0) 조건에서 1030 RU로 고정(immobilization)화 하고, EDC/NHS를 이용하여 표면을 활성화 시킨 후, 1 M 에탄올아민(ethanolamine)방법으로 고정시켰다. 대조 셀에는 NPB301을 넣지 않고 동일 조건으로 고정시켰다. 인간 IgE 또는 마우스 IgE를 HBS-EP buffer에 25 내지 0 nM 로 1/2 희석하여 CM5 chip에 작은 농도에서 높은 농도 순으로 흘려주어 kinetics를 측정하고 10 mM Glycine(pH 2.0) buffer를 이용하여 재생하였다. Biacore(SPR기법)로 NPB301과 인간 IgE 또는 마우스 IgE의 친화도를 측정하였다. 그 결과 하기 표 1에서도 확인할 수 있듯이, KD = 1.43 × 1010(M)로 측정되어 NPB301과 인간 IgE의 친화도(affinity)가 좋은 것으로 측정되었다. Immobilization of NPB301 to 1030 RU under 10 mM sodium acetate (pH 5.0) on a CM5 sensor chip using GE's BIAcore3000, and after surface activation using EDC / NHS, 1 M ethanolamine Method). The control cell was fixed under the same conditions without adding NPB301. Human IgE or mouse IgE was diluted 1/2 to 0 nM in HBS-EP buffer and flowed to the CM5 chip in small to high concentrations to measure kinetics and regenerated using 10 mM Glycine (pH 2.0) buffer. . The affinity between NPB301 and human IgE or mouse IgE was measured by Biacore (SPR). As a result, as can be seen in Table 1, KD = 1.43 × 10 10 (M) was measured, it was determined that the affinity of NPB301 and human IgE (affinity) is good.
Figure PCTKR2012004075-appb-I000001
Figure PCTKR2012004075-appb-I000001
[실시예 7] 접촉성피부염 동물모델을 이용한 NPB301의 효능평가Example 7 Evaluation of Efficacy of NPB301 Using Contact Dermatitis Animal Model
TG 마우스 4주령 암컷을 사용하였다. 배의 털을 밀고 5% Oxazoline(OXA) 100㎕를 도포하여 감작시킨 후, Day 5, Day 7, Day 9, Day 11에 0.1% Oxazoline(OXA) 10 ㎕를 양쪽 귀에 도포하고, Day 9에는 5MPK의 NPB301을 정맥주사 하였다. 이 후, Day 13에 귀의 두께를 측정하였다. TG mice females of 4 weeks old were used. Push the hairs of the pear and apply 100 μl of 5% Oxazoline (OXA) to sensitize, apply 10 μl of 0.1% Oxazoline (OXA) to both ears on Day 5, Day 7, Day 9, and Day 11, and apply 5 MPK on Day 9. NPB301 was injected intravenously. Thereafter, the thickness of the ears was measured on Day 13.
그 결과, OXA 대조군에 비해서 5MPK의 NPB301을 정맥주사한 그룹의 평균값이30% 가량 낮아져 NPB301에 의해 접촉성피부염의 피부 두께가 상당히 감소한 것을 확인할 수 있었다(도 9 참조).As a result, compared to the OXA control group, the average value of the intravenous group injected with 5 MPK NPB301 was lowered by 30%, indicating that the skin thickness of contact dermatitis was significantly reduced by NPB301 (see FIG. 9).
[실시예 8] TPA 유도 피부염증 동물모델을 이용한 NPB301의 효능평가Example 8 Evaluation of NPB301 Efficacy Using TPA Induced Dermatitis Animal Model
TG 마우스 6주령 암컷을 사용하였다. Day 1, Day 2에 0.05 g(20 ㎕)의 TPA를 귀에 도포하였다. Day 3, Day 4에 0.5 g(20 ㎕)의 TPA를 귀에 도포하였다. Day 2, Day 4에 NPB301을 5 MPK, 10 MPK, 20 MPK로 정맥주사하여, Day 5에 귀의 두께를 측정하였다. TG mice 6 week old females were used. 0.05 g (20 μl) of TPA was applied to the ears on Day 1 and Day 2. 0.5 g (20 μl) of TPA was applied to the ears on Day 3 and Day 4. NPB301 was intravenously injected at 5 MPK, 10 MPK and 20 MPK on Day 2 and Day 4, and ear thickness was measured on Day 5.
귀의 두께 측정 결과, TPA에 의해 유도된 부종이 NPB301을 정맥주사 하였을 때 NPB301의 농도에 따라 효과적으로 억제되는 것을 확인할 수 있었다(도 10 참조). As a result of measuring the thickness of the ear, it was confirmed that the edema induced by TPA was effectively suppressed according to the concentration of NPB301 when intravenously injected with NPB301 (see FIG. 10).
[실시예 9] 땅콩 알레르기 유발 동물모델을 이용한 NPB301의 효능평가Example 9 Evaluation of Efficacy of NPB301 Using Peanut Allergy-Induced Animal Model
TG 마우스 6주령 암컷을 사용한다. 땅콩과 콜레라 톡신을 3일 동안 1일 1회 공복에 경구투여하고 2주 후 땅콩과 콜레라 톡신을 3일 동안 1일 1회 공복에 경구투여 반복하였다. 2주 후, 30분 간격으로 땅콩을 먹인 뒤, 하루에 한 번, 또는 이틀에 한 번씩 일주일간 10 MPK의 NPB301을 복강투여하고, 혈액 내 전체 면역글로블린 E를 정량 Kit을 이용하여 측정하였다 . TG mice 6 week old females are used. Peanut and cholera toxin were orally administered on an empty stomach once daily for 3 days, and 2 weeks later, peanut and cholera toxin were orally administered on an empty stomach once daily for 3 days. Two weeks later, peanuts were fed at 30-minute intervals, then intraperitoneally administered 10 MPK of NPB301 once a day or once every two days, and total immunoglobulin E in blood was measured using a quantitative kit.
그 결과, T/G 마우스에 10 MPK NPB301을 처리한 결과 매일 주사하였을 때 뿐 아니라 이틀간격으로 주사하였을 때에도 정상 수준의 면역글로불린 E의 결과를 확인하였고, 상기 결과로부터 NPB301이 땅콩 알레르기 질환을 완화시켜주는 결과를 확인할 수 있었다(도 11 참조).As a result, the results of treatment with 10 MPK NPB301 in T / G mice confirmed the results of normal levels of immunoglobulin E not only when injected daily but also every two days, from which NPB301 alleviated peanut allergy disease. The results were confirmed (see FIG. 11).
[실시예 10] 비염 동물모델을 이용한 NPB301의 효능평가Example 10 Evaluation of Efficacy of NPB301 Using Rhinitis Animal Model
Balb/c 마우스 6주령 암컷을 사용하였다. Day 0과 Day 7에 OVA와 Alum을 섞어 복강주사로 감작시킨 후에 Day 14에서 Day 20까지 매일 OVA 150 ug/10 ㎕를 코로 흡입시키는데 OVA를 코로 흡입하기 30분 전에 NPB301을 5 MPK, 10 MPK, 20 MPK의 농도로 흡입시켜 주었다. Day 21에 OVA를 흡입시키고 직후에 10분간 코 문지르기 횟수(도 12A 참조)와 재채기 횟수(도 12B 참조)를 측정하였다. Balb / c mice 6 week old females were used. OVA and Alum were mixed on day 0 and day 7 and sensitized by intraperitoneal injection, then daily inhalation of 150 μg / 10 μl of OVA from day 14 to day 20. NPB301 was injected 5 MPK, 10 MPK, 30 minutes before inhaling OVA. Inhaled at a concentration of 20 MPK. OVA was inhaled on Day 21 and immediately after 10 minutes, the number of nose rubs (see FIG. 12A) and the number of sneezes (see FIG. 12B) were measured.
도 12에서도 확인할 수 있듯이, 비염 동물모델에 NPB301을 처리한 결과 코 문지르는 횟수와 재채기 하는 횟수가 감소하는 결과를 확인하였고, 상기 결과로부터 NPB301이 비염 질환을 완화시켜주는 결과를 확인할 수 있었다. As can be seen in Figure 12, as a result of the treatment of NPB301 in the rhinitis animal model was confirmed that the number of times rubbing and sneezing is reduced, from the results it can be confirmed that NPB301 alleviates rhinitis diseases.
[실시예 11] 아토피 동물모델을 이용한 NPB301의 효능평가Example 11 Evaluation of Efficacy of NPB301 Using Atopic Animal Model
무모 마우스 7 주령 암컷을 사용하였다. 5% Oxazoline(OXA) 100 ㎕을 도포 하여 감작시킨 7일 경과 후 0.1% OXA 100 ㎕를 마우스의 등에 이틀에 한번 20일간 고루 도포하였다. 아토피 피부염 유발이 종료된 후 TEWL(경피 수분손실도), 피부 내수화도를 측정하기 위해 TEWameter TM 300(Courage & Khazaka, Cologne, Germany)을 사용하고 피부두께를 측정한 후 일정한 그룹을 나눈다. 5, 10, 20 MPK NPB301을 하루에 한번 정맥주사(도 13 참조)복강주사(도 14 참조)후 측정하였다. Hairless mice 7 week old females were used. After 7 days of sensitization by applying 100 μl of 5% Oxazoline (OXA), 100 μl of 0.1% OXA was evenly applied to the back of the mouse once every two days for 20 days. After the induction of atopic dermatitis, TEWameter TM 300 (Courage & Khazaka, Cologne, Germany) is used to measure TEWL (transdermal water loss) and skin hydration, and the skin is divided into groups. 5, 10, 20 MPK NPB301 was measured after intravenous injection (see FIG. 13) once per day (see FIG. 14).
그 결과 경피 수분손실도, 피부 내 수화도, 피부두께 모두 완화되는 결과를 확인 할 수 있었다.As a result, it was confirmed that the water loss, transdermal hydration, and skin thickness were alleviated.
[실시예 12] OVA 아토피 동물모델을 이용한 NPB301의 효능평가Example 12 Evaluation of Efficacy of NPB301 Using OVA Atopic Animal Model
(1) NPB301 처리 농도에 따른 피부두께 변화(1) Change of skin thickness according to NPB301 treatment concentration
C57BL/6 mouse의 털을 밀고, 3M tape으로 5번 자극을 주어 1 × 1 cm 길이의 멸균 거즈에 100 ug의 OVA를 도포하여 Tegaderm으로 일주일 동안 감싸주었다. 3주 간격으로 3회(Day 1~7, Day 22~28, Day 43~49) OVA에 노출시키고 NPB-301을 0.3, 1, 5 MPK로 이틀에 한번 복강주사하여 피부두께의 변화를 측정하였다. The hair of C57BL / 6 mouse was pushed and stimulated 5 times with 3M tape, and 100 ug of OVA was applied to sterile gauze of 1 × 1 cm length and wrapped with Tegaderm for a week. Changes in skin thickness were measured by exposure to OVA three times every three weeks (Day 1 ~ 7, Day 22 ~ 28, Day 43 ~ 49) and intraperitoneally injected NPB-301 at 0.3, 1, 5 MPK once every two days. .
그 결과 NPB301을 0.3, 1.2, 5 MPK 주사한 그룹 모두에서 피부두께가 감소하는 경향을 확인하였고, 상기의 결과로부터 아토피 피부가 호전되는 것을 확인할 수 있었다(도 15 참조).As a result, it was confirmed that the skin thickness decreased in all the groups injected with NPB301 0.3, 1.2, and 5 MPK, and it was confirmed that the atopic skin improved from the above results (see FIG. 15).
(2) NPB301 처리에 따른 혈액 내 전체 면역글로블린 E 변화(2) Total immunoglobulin E changes in blood following NPB301 treatment
TG mice의 털을 밀고, 3M tape으로 5번 자극을 주어 1 × 1 cm 길이의 멸균 거즈에 100 ug의 OVA를 도포하여 Tegaderm으로 일주일 동안 감싸주었다. 3주 간격으로 3회(Day 1~7, Day 22~28, Day 43~49) OVA에 노출시키고 NPB-301을 5 MPK로 이틀에 한번 복강주사하여 피부 두께의 변화를 측정하였다(도 16A 참조). 및 혈액 내 전체 면역글로블린 E를 정량 Kit을 이용하여 측정하였다(도 16B 참조). TG 마우스에 5 MPK NPB301을 처리한 결과 이틀간격으로 측정하였을 때 점차 피부두께가 감소하는 경향을 보았으며, 전체 혈액 내 IgE도 감소하는 결과를 보였다. 상기의 결과로부터 아토피 피부염증이 완화되는 것을 확인할 수 있었다(도 16 참조). The hair of TG mice was pushed and stimulated 5 times with 3M tape, and 100 μg of OVA was applied to sterile gauze of 1 × 1 cm length and wrapped with Tegaderm for a week. Changes in skin thickness were measured by exposure to OVA three times every three weeks (Day 1-7, Day 22-28, Day 43-49) and intraperitoneally injected NPB-301 at 5 MPK once every two days (see FIG. 16A). ). And total immunoglobulin E in blood was measured using a quantitative kit (see FIG. 16B). As a result of treatment with 5 MPK NPB301 in TG mice, skin thickness gradually decreased when measured at two-day intervals, and IgE in whole blood was also decreased. From the above results, it was confirmed that atopic dermatitis was alleviated (see FIG. 16).
(3) NPB301의 제형에 따른 피부두께 변화(3) Skin thickness change according to the formulation of NPB301
C57BL/6 mouse의 털을 밀고, 3M tape으로 5번 자극을 주어 1 × 1 cm 길이의 멸균 거즈에 100 ug의 OVA를 도포하여 Tegaderm으로 일주일 동안 감싸주었다. 3주 간격으로 3회(Day 1~7, Day 22~28, Day 43~49) OVA에 노출시키고 2% NPB301 제형을 만들어 이틀간 환부에 발라주고 피부두께의 변화를 측정하였다. The hair of C57BL / 6 mouse was pushed, and stimulated 5 times with 3M tape, and 100 ug of OVA was applied to sterile gauze of 1 × 1 cm length and wrapped with Tegaderm for a week. Three times every three weeks (Day 1-7, Day 22-28, Day 43-49) was exposed to OVA, 2% NPB301 formulation was applied to the affected area for two days and the change in skin thickness was measured.
그 결과, C57BL/6 mouse의 아토피 환부에 2% NPB301을 이틀간 도포하였을 때 피부두께가 감소하는 경향을 확인하였고, 상기의 결과로부터 아토피 피부염증이 완화되는 것을 확인할 수 있었다(도 17 참조). As a result, when the 2% NPB301 was applied to the atopic dermatitis of the C57BL / 6 mouse for two days, the skin thickness tended to decrease, and it was confirmed that atopic dermatitis was alleviated from the above results (see FIG. 17).
[실시예 13] OVA 천식 동물모델을 이용한 NPB301의 효능평가 Example 13 Evaluation of Efficacy of NPB301 Using OVA Asthma Animal Model
OVA(ovalbumin) 25 ug과 알럼(Alum) 1 mg을 200 ㎕ 용액에 포함되도록 하였다. 혼합된 OVA-알럼을 호모게나이저를 이용하여 10분정도 계속 혼합하였다. 혼합이 끝난 OVA-알럼은 1.5 ml 튜브에 200 ㎕씩 분주하고 1 ml 주사기에 하나씩 넣어주었다. 이후, balb/c 마우스 6주령 암컷에 0, 6, 13, 20일, 4차에 걸쳐 200 ㎕씩 복강 주사하였다. 26, 30일에 코 점막을 통하여 1% OVA 50 ㎍을 흡입하도록 하였다. NPB301은 5 mg/kg로 1% OVA 50 ㎍ 흡입 4일 전, 3일 전, 2일 전, 1일 전에 주사하였다. 24시간 후 폐조직을 떼어내어 생검하고, 기관지 폐포 세척액에서 세포 수를 계수하였다. 졸레틸(Zoletil)을 이용해 마취한 마우스의 기관지를 절개한 후, 관을 삽입하고 22 게이지 주사바늘을 기관에 삽입한 뒤, 기관지 폐포 세척을 시행하였는데 1회당 0.9 mL의 PBS를 2회 주입하여 수거하였다. 수거된 기관지 폐포 세척액은 4℃, 5000 rpm 에서 5분간 원심분리하고, 상층액은 다음 실험시까지 -70℃에 보관하였다. 원심분리로 얻어 낸 세포를 혈구 계선반을 이용하여 총 세포수를 관찰하였다. 25 ug of OVA (ovalbumin) and 1 mg of Alum were included in a 200 μl solution. The mixed OVA-alum was continuously mixed for 10 minutes using a homogenizer. The mixed OVA-alum was divided into 200 μl in a 1.5 ml tube and put into a 1 ml syringe. Then, 6-week-old females of balb / c mice were injected intraperitoneally with 200 μl over 0, 6, 13, 20 days and 4 times. On 26 and 30 days, 50 μg of 1% OVA was inhaled through the nasal mucosa. NPB301 was injected at 5 mg / kg, 4 days before, 3 days before, 2 days before, and 1 day before inhalation of 50 μg of 1% OVA. After 24 hours, the lung tissue was removed and biopsied, and the number of cells in the bronchoalveolar lavage fluid was counted. After bronchial anesthesia of the anesthetized mouse using Zoletil, the tube was inserted, a 22 gauge needle was inserted into the trachea, and the bronchoalveolar lavage was performed. The samples were collected by injecting 0.9 mL of PBS twice per time. It was. The collected bronchial alveolar lavage fluid was centrifuged at 4 ° C. and 5000 rpm for 5 minutes, and the supernatant was stored at −70 ° C. until the next experiment. Cells obtained by centrifugation were observed by using a hemocytometer.
그 결과 control 에 비교하여 4, 3, 2, 1일 전 투입한 NPB301 그룹에서 기관지 폐포 세포수가 감소하였고, 그 중 OVA 흡입 하루 전에서 기관지 폐포 세포수가 제일 많이 감소되는 경향이 나타남을 확인하였고, 상기의 결과로부터 천식질환이 완화되는 것을 확인할 수 있었다(도 18 참조). As a result, the number of bronchial alveolar cells was decreased in the NPB301 group injected 4, 3, 2, 1 days before compared to the control, and among them, the number of bronchial alveolar cells was most decreased the day before OVA inhalation. As a result, it was confirmed that asthma disease is alleviated (see FIG. 18).
[실시예 14] TMA 피부염증 동물모델을 이용한 NPB301의 효능평가 Example 14 Evaluation of Efficacy of NPB301 Using TMA Dermatitis Animal Model
TG mice에 TMA를 도포하여 피부염증을 유발시켰다. TG mice에 등에 있는 털을 밀고 5% TMA 50 ㎕(in acetone and isoprophylmyristate, 4:1)을 도포하였다. 5일 경과 후 5% TMA 10 ㎕를 양쪽 귀에 도포하였다. 6일부터 15일까지 매일 1회 2% TMA 10 ㎕를 양쪽 귀에 계속 도포하였다. 9일째와 13일째 되는 날 5MPK, 10MPK의 NPB301을 정맥투여 하고(주2회), 17일에 혈액 내 전체 면역글로블린 E를 정량 Kit을 이용하여 측정하였다. TMA was applied to TG mice to induce skin inflammation. The hair on the back of the TG mice was pushed and 50 μl of 5% TMA (in acetone and isoprophylmyristate, 4: 1) was applied. After 5 days, 10 μl of 5% TMA was applied to both ears. 10 μl of 2% TMA was applied to both ears once daily from 6 to 15 days. On day 9 and day 13, 5 MPK and 10 MPK NPB301 were administered intravenously (twice a week), and on day 17, total immunoglobulin E in blood was measured using a quantitative kit.
TG 마우스에 NPB301을 처리한 결과, 면역글로불린 E의 수치가 정상치에 가깝게 낮아지는 것을 확인하였고, 상기의 결과로부터 NPB301이 염증 질환을 완화시켜주는 것을 확인할 수 있었다(도 19 참조).As a result of treatment with NPB301 in TG mice, it was confirmed that the level of immunoglobulin E was lowered to a normal value, and it was confirmed that NPB301 relieves inflammatory diseases (see FIG. 19).
서열목록 1은 FcεRI의 수용성 단편의 아미노산 서열을 나타낸 것이다.SEQ ID NO: 1 shows the amino acid sequence of the water soluble fragment of FcεRI.
서열목록 2는 FcεRI의 수용성 단편과 인간 항체의 Fc 영역이 펩타이드 링크로 연결된 복합체(NPB301)의 아미노산 서열을 나타낸 것이다. SEQ ID NO: 2 shows the amino acid sequence of the complex (NPB301) in which the water-soluble fragment of FcεRI and the Fc region of a human antibody are linked by peptide links.
서열목록 3은 FcεRI의 수용성 단편과 인간 항체의 Fc 영역이 펩타이드 링크로 연결된 복합체(NPB301)를 인코딩하는 염기서열을 나타낸 것이다.SEQ ID NO: 3 shows a nucleotide sequence encoding a complex (NPB301) in which a water-soluble fragment of FcεRI and an Fc region of a human antibody are linked by a peptide link.

Claims (17)

  1. FcεRI의 수용성(soluble) 단편과 생체 적합성(biocompatibility)을 가지는 물질이 접합된 복합체(NPB301).A complex in which a soluble fragment of FcεRI and a material having biocompatibility are conjugated (NPB301).
  2. 청구항 제1항에 있어서, FcεRI의 수용성 단편은 서열번호 1로 기재된 아미노산 서열을 가지는 것을 특징으로 하는 복합체(NPB301).The complex (NPB301) of claim 1, wherein the water soluble fragment of FcεRI has the amino acid sequence set forth in SEQ ID NO: 1.
  3. 청구항 제1항에 있어서, 생체 적합성을 가지는 물질은 인간 항체 Fc 영역, 폴리에틸렌글리콜(PEG) 또는 디아실글리세롤(DAG) 임을 특징으로 하는 복합체(NPB301).The complex (NPB301) according to claim 1, wherein the biocompatible material is a human antibody Fc region, polyethylene glycol (PEG) or diacylglycerol (DAG).
  4. 청구항 제1항에 있어서, FcεRI의 수용성 단편과 인간 항체 Fc 영역이 접합된 복합체(NPB301).The complex according to claim 1, wherein the water-soluble fragment of FcεRI and a human antibody Fc region are conjugated (NPB301).
  5. 청구항 제4항에 있어서, FcεRI의 수용성 단편과 인간 항체 Fc 영역이 펩타이드 링커로 연결된 것을 특징으로 하는 복합체(NPB301).The complex (NPB301) according to claim 4, wherein the water soluble fragment of FcεRI and the human antibody Fc region are linked by a peptide linker.
  6. 청구항 제5항에 있어서, 서열번호 2의 아미노산 서열을 가지는 것을 특징으로 하는 복합체(NPB301).The complex (NPB301) according to claim 5, which has an amino acid sequence represented by SEQ ID NO: 2.
  7. 청구항 제1항 내지 제6항 중 어느 한 항에 따른 복합체 및 하나 이상의 약제학적으로 허용 가능한 담체를 포함하는 알레르기성 질환 치료용 약제학적 조성물.A pharmaceutical composition for the treatment of allergic diseases comprising a complex according to any one of claims 1 to 6 and at least one pharmaceutically acceptable carrier.
  8. 청구항 제7항에 있어서, 상기 알레르기성 질환은 아토피성 피부염, 알레르기성 비염, 천식, 두드러기 및 접촉성 피부염에서 선택됨을 특징으로 하는 약제학적 조성물.8. The pharmaceutical composition of claim 7, wherein the allergic disease is selected from atopic dermatitis, allergic rhinitis, asthma, urticaria and contact dermatitis.
  9. 청구항 제4항 내지 제6항에 따른 FcεRI의 수용성 단편과 인간 항체 Fc 영역의 복합체(NPB301)를 인코딩하는 폴리뉴클레오티드. A polynucleotide encoding a complex (NPB301) of a water soluble fragment of FcεRI and a human antibody Fc region according to claim 4.
  10. 청구항 제9항에 있어서, 서열번호 3에 기재된 서열을 가지는 것을 특징으로 하는 폴리뉴클레오티드.10. The polynucleotide according to claim 9, which has the sequence set forth in SEQ ID NO: 3.
  11. 청구항 제9항 또는 제10항에 따른 폴리뉴클레오티드를 포함하는 재조합 발현 벡터.Recombinant expression vector comprising a polynucleotide according to claim 9.
  12. 제11항의 재조합 발현 벡터로 형질전환된 숙주세포.A host cell transformed with the recombinant expression vector of claim 11.
  13. 청구항 제12항에 있어서, 상기 숙주세포는 동물세포, 식물세포, 효모, 대장균, 곤충세포에서 선택된 것임을 특징으로 하는 숙주세포.The host cell of claim 12, wherein the host cell is selected from animal cells, plant cells, yeast, E. coli, and insect cells.
  14. 청구항 제13항에 있어서, 상기 형질전환된 숙주 세포는 원숭이 신장 세포7(COS7 : monkey kidney cells) 세포, NSO 세포, SP2/0 세포, 차이니즈 햄스터 난소(CHO : chinese hamster ovary) 세포, W138, 어린 햄스터 신장(BHK : baby hamster kidney) 세포, MDCK, 골수종 세포주, HuT 78 세포 및 HEK293 세포, 대장균, 바실러스 서브틸리스(Bacillus subtilis), 스트렙토마이세스 속(Streptomyces sp.), 슈도모나스 속(Pseudomonas sp.), 프로테우스 미라빌리스(Proteus mirabilis) 또는 스타필로코쿠스 속(Staphylococcus sp.), 아스페르길러스 속(Aspergillus sp.), 피치아 파스토리스(Pichia pastoris), 사카로마이세스 세레비지애(Saccharomyces cerevisiae), 쉬조사카로마세스 속(Schizosaccharomyces sp.) 및 뉴로스포라 크라사(Neurospora crassa)에서 선택됨을 특징으로 하는 숙주세포.The method of claim 13, wherein the transformed host cell is a monkey kidney cells (COS7) cells, NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, young Baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells and HEK293 cells, Escherichia coli, Bacillus subtilis , Streptomyces sp., Pseudomonas sp. ), Proteus mirabilis or Staphylococcus sp., Aspergillus sp., Pichia pastoris , Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), Schizosaccharomyces sp. And Neurospora crassa .
  15. 청구항 제11항 내지 제14항 중 어느 한 항에 따른 벡터 또는 숙주세포를 배양하는 단계를 포함하는 FcεRI의 수용성 단편과 인간 항체 Fc 영역의 복합체를 생산하는 방법.15. A method of producing a complex of a water-soluble fragment of FcεRI and a human antibody Fc region comprising culturing the vector or host cell according to any one of claims 11-14.
  16. (a) FcεRI의 수용성 단편을 인코딩하는 폴리뉴클레오티드를 포함하는 벡터를 이용하여 형질전환된 숙주세포를 배양하여 FcεRI의 수용성 단편을 생산하는 단계; 및(a) culturing the transformed host cell using a vector comprising a polynucleotide encoding a water-soluble fragment of FcεRI to produce a water-soluble fragment of FcεRI; And
    (b) (a) 단계에서 생산된 FcεRI의 수용성 단편에 생체적합성 물질을 접합하는 단계;(b) conjugating the biocompatible material to the water-soluble fragment of FcεRI produced in step (a);
    를 포함하는 FcεRI의 수용성 단편을 포함하는 복합체를 생산하는 방법.Method for producing a complex comprising a water-soluble fragment of FcεRI comprising a.
  17. 청구항 제16항에 있어서, 상기 생체적합성 물질은 폴리에틸렌글리콜(PEG) 또는 디아실글리세롤(DAG) 중에서 선택된 어느 하나 이상임을 특징으로 하는 FcεRI의 수용성 단편을 포함하는 복합체를 생산하는 방법.The method of claim 16, wherein the biocompatible material is at least one selected from polyethylene glycol (PEG) or diacylglycerol (DAG).
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