WO2012169735A2 - Complexe comprenant des fragments hydrosolubles de fcεri et composition le contenant pour traiter les maladies allergiques médiées par l'ige - Google Patents

Complexe comprenant des fragments hydrosolubles de fcεri et composition le contenant pour traiter les maladies allergiques médiées par l'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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WO2012169735A3 (fr
WO2012169735A9 (fr
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박병덕
윤선주
김욱동
배종환
김민희
강현정
박은혜
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Neopharm Co Ltd
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Neopharm Co Ltd
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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.

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Abstract

Cette invention concerne un complexe contenant des fragments hydrosolubles de FceRI, qui est un récepteur ayant une forte affinité pour la région Fc de l'immunoglobuline E (IgE), liés à un matériau doué de biocompatibilité. Le complexe contenant des fragments hydrosolubles de FceRI liés à un matériau doué de biocompatibilité selon cette invention inhibe le couplage de l'IgE et du FceRI, qui est un récepteur de l'IgE, de façon à supprimer la sécrétion d'une cytokine allergique. Le complexe selon l'invention a, par conséquent, pour effet de traiter et de prévenir les maladies allergiques.
PCT/KR2012/004075 2011-06-07 2012-05-23 Complexe comprenant des fragments hydrosolubles de fcεri et composition le contenant pour traiter les maladies allergiques médiées par l'ige Ceased WO2012169735A2 (fr)

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KR1020120054613A KR20120135865A (ko) 2011-06-07 2012-05-23 ＦｃεＲＩ의 수용성 단편을 포함하는 복합체 및 이를 포함하는 ＩｇＥ 매개 알레르기성 질환 치료용 조성물

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107207623A (zh) * 2015-02-20 2017-09-26 橘生药品工业株式会社 Fc融合高亲和力IgE受体α链
US20220347236A1 (en) * 2018-01-12 2022-11-03 Gl INNOVATION, INC. Composition comprising probiotics and polypeptide having binding affinity for ige and use thereof
US12409203B2 (en) 2019-07-08 2025-09-09 Gi Innovation, Inc. Polypeptide dimer with high sialic acid content, comprising extracellular domain of alpha subunit of IGE FC receptor, and pharmaceutical composition comprising same

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL116436A (en) * 1995-12-18 2006-12-31 Yissum Res Dev Co Fc?Á-PE CHIMERIC PROTEIN FOR TARGETED TREATMENT OF ALLERGY RESPONSES AND
AR008077A1 (es) * 1996-07-26 1999-12-09 Talarico Salinas Laura Beatriz Un polipeptido de fusion o una sal del mismo, su uso, un proceso para prepararlos, una composicion farmaceutica que los comprende, y un vector.
WO2003008584A1 (fr) * 2001-07-19 2003-01-30 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute Anticorps de recepteur ige antihumain de type humain et fragment d'anticorps

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107207623A (zh) * 2015-02-20 2017-09-26 橘生药品工业株式会社 Fc融合高亲和力IgE受体α链
KR20170120579A (ko) 2015-02-20 2017-10-31 키세이 야쿠힌 고교 가부시키가이샤 Fc-융합 고친화성 IgE 수용체 알파 사슬
US10077297B2 (en) 2015-02-20 2018-09-18 Kissei Pharmaceutical Co., Ltd. Fc fusion high affinity IgE receptor α-chain
KR20230132610A (ko) 2015-02-20 2023-09-15 센주 세이야꾸 가부시키가이샤 Fc-융합 고친화성 IgE 수용체 알파 사슬
US20220347236A1 (en) * 2018-01-12 2022-11-03 Gl INNOVATION, INC. Composition comprising probiotics and polypeptide having binding affinity for ige and use thereof
US12128076B2 (en) * 2018-01-12 2024-10-29 Gi Innovation, Inc. Composition comprising probiotics and polypeptide having binding affinity for IgE and use thereof
US12409203B2 (en) 2019-07-08 2025-09-09 Gi Innovation, Inc. Polypeptide dimer with high sialic acid content, comprising extracellular domain of alpha subunit of IGE FC receptor, and pharmaceutical composition comprising same

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