WO2021006603A1 - Novel pharmaceutical composition for treating allergic diseases - Google Patents

Novel pharmaceutical composition for treating allergic diseases Download PDF

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WO2021006603A1
WO2021006603A1 PCT/KR2020/008869 KR2020008869W WO2021006603A1 WO 2021006603 A1 WO2021006603 A1 WO 2021006603A1 KR 2020008869 W KR2020008869 W KR 2020008869W WO 2021006603 A1 WO2021006603 A1 WO 2021006603A1
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protein
fcεriα
fusion protein
amino acid
seq
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Korean (ko)
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진현탁
남은주
연제인
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주식회사 프로젠
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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
    • 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
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • 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
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2066—IL-10
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/14—Decongestants or antiallergics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5428—IL-10
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to a pharmaceutical composition, and more particularly, to a pharmaceutical composition for the treatment of novel allergic diseases.
  • Allergy is an irritable disease of the immune system.It refers to a disease in which the body reacts excessively with antigen-antibodies to allergens (usually safe environmental substances such as food, general medicine, dust, pollen, etc.), resulting in various inflammatory reactions in the body. Symptoms start from just feeling bad, itchy, or something on the skin, severe breathing difficulties occur, and usually the skin swells in many cases, especially when the sensitive area, the mucous membrane, swells. Often, allergic asthma or allergic conjunctivitis can be exemplified, and other symptoms such as atopy or allergic rhinitis may appear, and there are many cases where there is only one symptom, but there are also many cases in which several are combined.
  • allergies can be classified into four types. The majority of allergies we talk about are type 1 allergy, which induces anaphylaxis and is called acute hypersensitivity reaction, type 2 allergy due to antibody-mediated hypersensitivity reaction, type 3 due to immune complex, and type 4 due to acquired immunity. .
  • IgE and mast cells cause symptoms.
  • progenitor cells are still unclear, but basophils among leukocytes are the dominant.
  • IgE bound to mast cells like a chemical substance, is a single antigen, but for various reasons, even if the complex binds to an antigen that is not recognized as an antigen, it cannot perform immunity, but in the case of a polyantigen of a specific sequence, it stimulates mast cells. It causes intracellular degranulation. This releases several signaling chemicals such as histamine, leukotriene, prostaglandin and IL-4. Histamine increases the permeability of blood vessels, which in turn causes inflammation as fluid is released from the blood vessels.
  • hypersensitivity shock an acute hypersensitivity reaction, occurs due to vasodilation and airway obstruction due to airway mucosal edema.
  • This irritable shock is a life-threatening symptom and requires immediate epinephrine injection and CPR.
  • antihistamines antileukotrienes, beta-blockers, and steroids have been used in the treatment of allergic diseases, but generally antihistamines and antileukotrienes are combined or nasal spray steroids are often used alone.
  • Omalizumab an anti-IgE antibody, is sometimes used.
  • Dupilumab a monoclonal antibody that binds to IL-4, has also been used.
  • interleukin 10 is an anti-inflammatory cytokine expressed as a non-covalently bound homodimer of about 37 kDa called a cytokine synthesis inhibitory factor (CSIF). .
  • IL-10 plays an important role in the induction and maintenance of immune tolerance, and these dominant anti-inflammatory properties have been known for a long time.
  • IL-10 inhibits the secretion of pro-inflammatory cytokines such as TNF ⁇ , IL-1, IL-6, and IL-12 as well as Th1 cytokines such as IL-2 and IFN ⁇ , and Regulate the differentiation and proliferation of phagocytes, B cells and T cells (Glocker et al ., Ann.
  • the present invention is to solve various problems, including the above-described problems, and an object thereof is to provide a more efficient and safe pharmaceutical composition for treating allergies.
  • the scope of protection of the present invention is not limited to the above purpose.
  • a) Fc ⁇ RI ⁇ protein and IL-10 protein and/or b) there is provided a pharmaceutical composition for treating allergic diseases comprising a fusion protein comprising an Fc ⁇ RI ⁇ protein and an IL-10 protein as an active ingredient.
  • Fc ⁇ RI ⁇ , the Fc region of the antibody and the IL-10 protein are sequentially linked, and optionally between the Fc ⁇ RI ⁇ and the Fc region of the antibody and/or between the Fc region of the antibody and the IL-10 protein
  • a fusion protein comprising a linker peptide is provided.
  • a pharmaceutical composition for treating allergic diseases comprising the fusion protein as an active ingredient is provided.
  • a) Fc ⁇ RI ⁇ protein and IL-10 protein a) Fc ⁇ RI ⁇ protein and IL-10 protein; And/or b) administering a fusion protein including an Fc ⁇ RI ⁇ protein and an IL-10 protein to the individual.
  • the pharmaceutical composition according to an embodiment of the present invention is a drug that can effectively block IgE generated by an allergic reaction, as well as very low the likelihood of hypersensitivity reactions such as anaphylaxis, which is a disadvantage of existing IgE-specific antibody treatments. , It can be used as an allergy treatment with improved safety.
  • FIG. 1 is a schematic diagram showing the overall structure of a fusion protein according to an embodiment of the present invention.
  • Figure 2a is a result of performing PAGE analysis under reduced (left) and non-reduced (right) conditions after expressing the fusion protein (PG110-1) prepared according to an embodiment of the present invention by transient transfection.
  • 2B is a series of chromatograms showing the results of measuring purity by performing SE-HPLC analysis on the fractions (A10, A11, and A12) obtained in the process of purifying the PG110-1
  • 2C is a result of performing PAGE analysis under reduced (left) and non-reduced (right) conditions after expressing the fusion protein (PG110-2) prepared according to an embodiment of the present invention by transient transfection.
  • 2D is a series of chromatograms showing the results of measuring the purity by performing SE-HPLC analysis on the fractions (A11 and A12) obtained in the process of purifying the PG110-2.
  • Figure 3a is a non-reduction for confirming the level of protein expression according to the purification process of the fusion protein (PG110-1) from a stable cell line prepared to produce a fusion protein according to an embodiment of the present invention Photo showing the results of SDS-PAGE analysis under conditions (left) and reducing conditions (right), and FIG. 3B is a result of SEC-HPLC analysis to confirm the purity of the fusion protein (PG110-1) finally purified from the stabilizing cell line. It is a chromatogram representing
  • FIG. 4A is a sensogram showing the result of analyzing the affinity of the Fc ⁇ RI ⁇ -Fc fusion protein with mouse IgE by BLI analysis as a comparative example.
  • FIG. 4B is a mouse of the fusion protein PG110-1 (bottom) according to an embodiment of the present invention It is a sensogram showing the result of analyzing the affinity with IgE by BLI analysis
  • FIG. 4C is a sensogram showing the result of analyzing the affinity of PG110-1 with human IgE according to an embodiment of the present invention by BLI analysis.
  • FIG. 5 is an overview of flow cytometry experiments for analyzing the targeting of U266B1, a membrane IgE-expressing B cell, of the fusion protein PG110 according to an embodiment of the present invention (A), by the fusion protein PG110 according to an embodiment of the present invention
  • Figure 6 shows the affinity of the fusion protein PG110-1 (bottom) and the comparative examples NTIG-IL-10V (top) and NTIG-IL-10Vm proteins (stop) with IL-10R1 according to an embodiment of the present invention. It is a series of graphs showing the results of analysis.
  • FIG. 7 is a result of analyzing the ability of the fusion protein PG110 and the monomeric IL-10 variant protein (IL-10M-1; NTIG-IL-10Vm) to inhibit TNF- ⁇ secretion in mast cells according to an embodiment of the present invention. This is the graph shown.
  • FIG. 8 is a graph showing the results of analyzing the ability to inhibit the secretion of TNF- ⁇ in macrophages according to the treatment concentration of the fusion protein PG110-1 according to an embodiment of the present invention.
  • FIG. 9 is a beta-hexosami that inhibits IgE blocking and mast cell activation when treating mouse bone marrow-derived mast cells with fusion protein PG110-1 and a comparative example of IgE TRAP (Fc ⁇ RI ⁇ -Fc fusion protein) according to an embodiment of the present invention.
  • This is a graph showing the results of analysis by measuring nidase activity.
  • 10 is a fusion protein remaining in the serum for up to 330 hours after administration of the fusion protein according to an embodiment of the present invention to an experimental animal (rat) by various routes (intravenous injection, intraperitoneal injection, intramuscular injection, and subcutaneous injection) It is a graph showing the results of pharmacokinetic analysis by quantifying the amount of.
  • FIG. 11 is a blood toxicity analysis result of investigating whether a change in the number of leukocytes (A), red blood cells (B), and platelets (C) appears when the fusion protein PG110-1 according to an embodiment of the present invention is administered to an experimental animal. It is a series of graphs shown.
  • FIG. 12 is an investigation of whether the fusion protein PG110-1 according to an embodiment of the present invention reduces allergic symptoms in real animals, and the present invention after causing food allergy by oral administration of OVA to OVA-sensitized mice.
  • IgE TRAP Fc ⁇ RI ⁇ -Fc fusion protein
  • B free IgE concentration
  • It is a series of graphs showing the results of measuring the concentration (C) and the concentration (D) of the degranulation enzyme (MCPT-1, mast cell protease-1) in the blood as an allergenic factor.
  • a) Fc ⁇ RI ⁇ protein and IL-10 protein and/or b) there is provided a pharmaceutical composition for treating allergic diseases comprising a fusion protein comprising an Fc ⁇ RI ⁇ protein and an IL-10 protein as an active ingredient.
  • the Fc ⁇ RI ⁇ may be composed of an amino acid sequence described in SEQ ID NO: 2, and the IL-10 protein includes an amino acid sequence 19-178 from which a signal sequence has been removed from the amino acid sequence described in UniProtKB P22301. It may be a mature protein, and may be an IL-10 variant protein in which isoleucine, which is the 87th amino acid of the mature protein, is substituted with alanine, and between the 116th amino acid asparagine and the 117th amino acid lysine.
  • the IL-10 protein may be a monomeric IL-10 variant protein into which a linker peptide having a length of to 12 aa is inserted, and in one embodiment of the present invention, the IL-10 protein is an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 13 It can be composed of.
  • the Fc ⁇ RI ⁇ protein and IL-10 protein can be used as long as they are derived from mammals, but more preferably they are human-derived proteins.
  • fusion protein used in the present invention refers to a recombinant protein (recombinant protein) in which two or more proteins or domains responsible for a specific function in the protein are linked so that each protein or domain is responsible for its natural function.
  • a linker peptide having a generally flexible structure may be inserted between the two or more proteins or domains.
  • the linker peptide is GGGGSGGGGSGGGGSEPKSSDKTHTCPPCP (SEQ ID NO: 3), AAGSGGGGGSGGGGSGGGGS (SEQ ID NO: 8), GGSGG (SEQ ID NO: 9), GGSGGSGGS (SEQ ID NO: 14), GGGSGG (SEQ ID NO: 15), (G 4 S) n (unit: SEQ ID NO: 16, n is an integer of 1 to 10), (GGS) n (n is an integer of 1 to 10), (GS) n (n is an integer of 1 to 10), (GSSGGS) n (unit: SEQ ID NO: 17, n is an integer of 1 to 10), KESGSVSSEQLAQFRSLD (SEQ ID NO: 18), EGKSSGSGSESKST (SEQ ID NO: 19), GSAGSAAGSGEF (SEQ ID NO: 20), (EAAAK) n (unit: SEQ ID NO: 21, n is an integer of 1 to 10), CRRRRRREAEAC (S
  • EPKSCDKTHTCPPCP SEQ ID NO: 40
  • THTCPPCP SEQ ID NO: 41
  • GGGGSGGGGSGGGGSAKNTTAPATTRNTTRGGEEKKKEKEKEEQEERTH TCPPCP SEQ ID NO: 42
  • the Fc ⁇ RI ⁇ protein and the IL-10 protein when included, the Fc ⁇ RI ⁇ protein and the IL-10 protein may be included together in the composition, or formulated separately and administered simultaneously or at different times.
  • the interval between administration of both substances may vary depending on conditions such as the patient's condition, age, symptoms, and the presence or absence of other diseases.
  • the number of administration may be determined according to the type of symptoms. For example, in the case of acute allergic symptoms, it may be desirable to intensively administer for a short period of time, and in the case of chronic allergic symptoms, long-term administration may be necessary.
  • Fc ⁇ RI ⁇ , the Fc region of the antibody and the IL-10 protein are sequentially linked, and optionally between the Fc ⁇ RI ⁇ and the Fc region of the antibody and/or between the Fc region of the antibody and the IL-10 protein
  • a fusion protein comprising a linker peptide is provided.
  • the Fc ⁇ RI ⁇ protein is an IgE Fc receptor, also called an Fc ⁇ receptor, and is a receptor having high affinity for IgE Fc.
  • Fc ⁇ RI is expressed in mast cells and basophils.
  • IgE antibodies bound to Fc ⁇ RI are crosslinked by a multivalent antigen, degranulation occurs in mast cells or basophils, releasing various chemical transport substances including histamine. This release leads to an immediate allergic reaction.
  • the Fc ⁇ RI is a membrane protein composed of one ⁇ -chain, one ⁇ -chain, and two ⁇ -chains bonded by disulfide bonds.
  • the part to which IgE binds is an ⁇ chain (Fc ⁇ RI ⁇ )
  • Fc ⁇ RI ⁇ has a size of about 60 kDa, and is composed of a hydrophobic domain present in the cell membrane and a hydrophilic domain present outside the cell membrane.
  • IgE binds to the extracellular domain of the ⁇ chain.
  • the alpha subunit of the IgE Fc receptor may have the amino acid sequence described in NP_001992.1.
  • the extracellular domain (Fc ⁇ RIa-ECD) of the alpha subunit of the IgE Fc receptor may have the amino acid sequence of SEQ ID NO: 2.
  • the extracellular domain of the alpha subunit of the IgE Fc receptor may be a fragment or variant of the extracellular domain of the alpha subunit of the IgE Fc receptor as long as it can bind to IgE.
  • the variant may be performed through a method of substituting, deleting or adding one or more proteins in the wild-type Fc ⁇ RIa-ECD (Extracellular domain), as long as the function of the ⁇ chain of Fc ⁇ RI is not modified.
  • These various proteins or peptides may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to the amino acid sequence of SEQ ID NO: 2.
  • the Fc region of the antibody may be a hybrid Fc in which all or part of the Fc region of IgA, IgG, IgM, IgD or IgE or domains thereof such as hinge, CH2, CH3 is mixed, and the IgG is IgG1, IgG2, IgG3 , Or IgG4, in particular, so that the affinity for the Fc gamma receptor (Fc ⁇ Rc) and complement (C1a) is low, the effector function of the Fc antibody-dependent cytotoxicity (antibody-dependent cell-mediated cytotoxicity, ADCC) ) Or complement-dependent cytotoxicity (complement-dependent cytotoxicity (CDC)) in which the mutation is induced in the part of the functional group responsible for the half-life of the blood due to high selective affinity for the Fc and/or neonatal Fc receptor (FcRn).
  • Fc ⁇ Rc Fc gamma receptor
  • C1a complement
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent
  • the hybrid Fc may be those described in Korean Patent Nos. 897938, 1380732, 1380729, etc., and the variant Fc is a modified immunoglobulin Fc protein (NTIG) described in International Patent Application PCT/KR2020/006346 I can.
  • the Fc region may be composed of an amino acid sequence of any one of SEQ ID NOs: 4 to 7.
  • the Fc region may be derived from human.
  • one or more linker peptides may be inserted between fusion partners.
  • the linker peptide is as described above.
  • the linker peptide connecting the Fc ⁇ RI ⁇ protein and the Fc region of the antibody may be a hinge region derived from an antibody, a synthetic linker peptide, or a mixture thereof.
  • NTIG' as used in this document means that the 18th and 196th amino acids of the modified Fc domain protein selected from the group consisting of SEQ ID NOs: 4 and 5 are mutated to other amino acids, and there is no effector function such as ADCC and CDC. It refers to a modified Fc domain protein with improved half-life in blood by increasing selective affinity for FcRn.
  • the NTIG may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 7.
  • the IL-10 protein may be a mature form including an amino acid sequence 19-178 from which a signal sequence has been removed from the amino acid sequence described in UniProtKB P22301, based on the mature form.
  • the 87th amino acid isoleucine may be an IL-10 variant protein substituted with alanine, and a linker peptide having a length of 6 to 12 aa is inserted between the 116th amino acid asparagine and the 117th amino acid lysine. It may be a variant protein, and the IL-10 protein may be composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 13.
  • mature form means that a signal sequence used for secretion from a protein produced by translation is cleaved, or an inactive precursor protein such as a prepeptide or propeptide is a protein. It means an active protein produced by cleavage by a cleavage enzyme or the like.
  • the Fc ⁇ RI ⁇ protein and IL-10 protein can be used as long as they are derived from mammals, and any variant can be used as long as they can perform the same or similar function, and more preferably human-derived proteins. to be.
  • a polynucleotide encoding the fusion protein is provided.
  • a recombinant vector comprising the polynucleotide is provided.
  • the polynucleotide may be included in the form of a gene construct operably linked to a regulatory sequence.
  • operably linked to is regulated in such a way that the target nucleic acid sequence (eg, in an in vitro transcription/translation system or in a host cell) can be expressed. It means that it is linked to a sequence.
  • regulatory sequence is meant to include promoters, enhancers and other regulatory elements (eg, polyadenylation signals).
  • the regulatory sequences are those that direct the expression of the nucleic acid of interest in many host cells, and that the nucleic acid of interest can be expressed only in specific tissue cells (e.g., tissue-specific regulatory sequences), and It includes directing expression to be induced by a specific signal (eg, an inducible regulatory sequence).
  • tissue-specific regulatory sequences e.g., tissue-specific regulatory sequences
  • a specific signal eg, an inducible regulatory sequence
  • regulatory sequences that enable expression in eukaryotic and prokaryotic cells are well known to those skilled in the art. As mentioned above, these usually contain regulatory sequences responsible for initiation of transcription and, optionally, a poly-A signal responsible for transcription termination and stabilization of the transcript. Additional regulatory sequences may include, in addition to transcriptional regulators, translation enhancers and/or naturally-combined or heterologous promoter regions.
  • possible regulatory sequences that allow expression in mammalian host cells include CMV-HSV thymidine kinase promoter, SV40, RSV-promoter (Rous sarcoma virus), human kidney element 1 ⁇ -promoter, glucocorticoid-inducible MMTV- Promoter (Moloni mouse tumor virus), a metallothionein-inducible or tetracycline-inducible promoter, or an amplifying agent such as a CMV amplifying agent or an SV40-amplifying agent.
  • neurofibrillation-promoter For expression in neurons, it is contemplated that neurofibrillation-promoter, PGDF-promoter, NSE-promoter, PrP-promoter or thy-1-promoter can be used.
  • Such promoters are known in the art and are described in Charron, J. Biol. Chem. 270: 25739-25745, 1995.
  • a number of promoters have been disclosed including the lac-promoter, the tac-promoter or the trp promoter.
  • the regulatory sequences include transcription termination signals such as SV40-poly-A site or TK-poly-A site downstream of the polynucleotide according to an embodiment of the present invention.
  • suitable expression vectors are known in the art, examples of which are Okayama-Berg cDNA expression vector pcDV1 (Parmacia), pRc/CMV, pcDNA1, pcDNA3 (Invitrogene), pSPORT1 (GIBCO BRL), pGX-27 (Patent No.
  • the vector may further comprise a polynucleotide encoding a secretion signal.
  • the secretion signals are well known to those skilled in the art.
  • a leader sequence that can lead the fusion protein to the cell compartment is combined with the coding sequence of the polynucleotide according to an embodiment of the present invention, and preferably the translated protein or its It is a leader sequence that can secrete proteins directly into the periplasm or extracellular media.
  • the signal sequence of tPA represented by SEQ ID NO: 1 was used, but is not limited thereto, and since the signal sequence is cut and removed during the production of a protein, according to an embodiment of the present invention It is not a component of the fusion protein itself.
  • the vector of the present invention may be prepared by, for example, standard recombinant DNA techniques, and standard recombinant DNA techniques include, for example, ligation of blunt ends and adhesive ends, treatment with restriction enzymes to provide appropriate ends, and inappropriate In order to prevent binding, phosphate group removal by alkaline postage treatment and enzymatic linkage by T4 DNA ligase are included.
  • the vector of the present invention by recombining the DNA encoding the signal peptide obtained by chemical synthesis or gene recombination technology, the mutant IL-10 protein of the present invention, or the DNA encoding the fusion protein containing the same into a vector containing an appropriate regulatory sequence.
  • a vector containing the control sequence can be purchased or manufactured commercially, and in an embodiment of the present invention, a pBispecific vector was used as a backbone.
  • the expression vector may further include a polynucleotide encoding a secretion signal sequence, and the secretion signal sequence induces secretion of a recombinant protein expressed in the cell out of the cell, and a tissue plasminogen activator (tPA) signal sequence ( SEQ ID NO: 1), HSV gDs (herpes simplex virus glycoprotein Ds) signal sequence or growth hormone signal sequence.
  • tPA tissue plasminogen activator
  • the expression vector according to an embodiment of the present invention may be an expression vector capable of expressing the protein in a host cell, and the expression vector is a plasmid vector, a virus vector, a cosmid vector, a phagemid vector, an artificial human chromosome It can be any form such as.
  • a pharmaceutical composition for the treatment of allergic diseases comprising the fusion protein as an active ingredient.
  • the pharmaceutical composition may further contain or be used in combination with a known allergy treatment component.
  • known allergy treatments may include antihistamines, antileukotrienes, and steroids.
  • the antihistamines are chlorpheniramine, triprolidine, piprinhydrinate, hydroxyzine, mequitazine, diphenhydramine, dimenhydrin Dimenhydrinate, meclizine, cetirizine, loratadine, levocetirizine, ebastine, ketotifen, fexofenadine, azela May be azelastine, desloratadine, bepotastine, olopatadine, emedastine, epinastine, or levocabastine .
  • the anti-leukotriene agent may be montelukast, pranlukast, or zafirlukast.
  • the steroid preparation may be cortisol, glucocorticoid, prednisone, prednisolone, methylprednisolone, triamcinolone, dexamethasone, desonide, or corticosterone.
  • the allergic disease may be allergic rhinitis, allergic bronchial asthma, atopic dermatitis, allergic conjunctivitis, chronic idiopathic urticaria, drug allergy, food allergy or anaphylaxis.
  • the allergic disease may be IgE mediated.
  • the composition may include a badly acceptable carrier, and may additionally include a pharmaceutically acceptable adjuvant, excipient, or diluent in addition to the carrier.
  • pharmaceutically acceptable refers to a composition that is physiologically acceptable and, when administered to a human, does not usually cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions.
  • the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.
  • fillers, anti-aggregating agents, lubricants, wetting agents, flavoring agents, emulsifying agents and preservatives may additionally be included.
  • compositions according to an embodiment of the present invention may be formulated using a method known in the art to enable rapid release, or sustained or delayed release of the active ingredient when administered to a mammal.
  • Formulations include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, and sterile powder forms.
  • composition according to an embodiment of the present invention may be administered by various routes, for example, oral, parenteral, for example, suppository, transdermal, intravenous, intraperitoneal, intramuscular, intralesional, nasal, intrathecal administration It can be administered, and can also be administered using an implanted device for sustained release or continuous or repeated release.
  • the frequency of administration may be administered once a day or divided into several times a day within a desired range, and the administration period is not particularly limited.
  • composition according to an embodiment of the present invention may be formulated in a suitable form together with a generally used pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include, for example, carriers for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycol, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
  • composition according to the present invention is a suspension agent, a solubilizer, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, a painless agent, a buffer agent, Antioxidants and the like may be appropriately included.
  • Pharmaceutically acceptable carriers and formulations suitable for the present invention including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, the latest edition.
  • the dosage of the composition to a patient depends on many factors, including the patient's height, body surface area, age, the specific compound administered, sex, time and route of administration, general health, and other drugs administered simultaneously.
  • the pharmaceutically active protein may be administered in an amount of 100 ng/body weight (kg)-10 mg/body weight (kg), more preferably 1 to 500 ⁇ g/kg (body weight). Preferably, it may be administered at 5 to 50 ⁇ g/kg (body weight), and the dosage may be adjusted in consideration of the above factors.
  • the individual may be a human or non-human mammal, and the non-human mammal may be an animal belonging to a primate, a carnivorous tree, a right tree, a rodent tree, or a base tree.
  • the term "therapeutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type and severity of the subject, Age, sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including concurrent drugs and other factors well known in the medical field.
  • the therapeutically effective amount of the composition of the present invention may be 0.1 mg/kg to 1 g/kg, more preferably 1 mg/kg to 500 mg/kg, but the effective dosage may be depending on the age, sex and condition of the patient. Can be adjusted appropriately.
  • the present inventors used Fc ⁇ RI ⁇ , a receptor that specifically binds to IgE, and a dimeric IL-10 variant protein (IL-10V or IL-10M) or a monomeric IL-10 variant protein (IL-10Vm) with a modified Fc region, respectively.
  • a fusion protein linked to both sides with an Fc region interposed was designed.
  • the present inventors prepared a polynucleotide encoding a fusion protein having the configuration shown in Table 1 below using oligonucleotide synthesis and PCR, and then inserted it into a pBispecific expression vector (Genexin, Korea), and then ExpiCHO After transfecting the expression vector into ExpiCHO-S cells using a kit (Thermo Fisher, USA), incubating for 1 day in an 8% CO 2 , 37°C incubator, lowering the temperature to 32°C and further incubating for 7 days I did.
  • Example 2 Example 3 Signal sequence SEQ ID NO: 1 SEQ ID NO: 1 SEQ ID NO: 1 Fc ⁇ RI ⁇ SEQ ID NO: 2 SEQ ID NO: 2 SEQ ID NO: 2 Linker 1 SEQ ID NO: 3 SEQ ID NO: 3 NTIG SEQ ID NO: 6 SEQ ID NO: 6 Linker 2 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 9 IL-10Vm SEQ ID NO: 10 SEQ ID NO: 11 SEQ ID NO: 13
  • the modified Fc used in Table 1 is an Fc in which the hinge portion of IgG1 and CH2 and CH3 of IgD and IgG4 are mixed, and the present inventors named it "NTIG", including The fusion protein, Fc ⁇ RI ⁇ -NTIG-IL-10Vm, was named "PG110".
  • Examples 1 and 2 differ only in the linker 2 (SEQ ID NOs: 8 and 9) connecting NTIG and IL-10Vm and the linker (spacer) peptide portion inserted into the interior of IL-10Vm (SEQ ID NOs: 10 and 11).
  • IL-10Vm used in Example 2 is the same as the previously reported monomeric IL-10 variant protein (Josephson et al ., J.
  • Example 1 Biol. Chem . 275(18): 13552-13557, 2000). Accordingly, in the case of Example 1, it was named "PG110-1", and in the case of Example 2, it was named "PG110-2".
  • T threonine
  • M methionine
  • L leucine
  • PG-110-1 of Example 1 achieved a high purity of 93.9% in fraction 10. After formulating this, it was used for the analysis of IL-10 activity in the future, and fractions 11 (A11) and 12 (A12) showing somewhat lower purity were combined to perform secondary purification.
  • Example 4 Establishment of a stable cell line expressing Fc ⁇ RI ⁇ -NTIG-IL-10Vm
  • the present inventors then constructed a stable cell line capable of stably expressing PG110-1 of Example 1 above.
  • the expression vector pAD15 expression vector (WO2015/009052A) containing the gene construct encoding the Fc ⁇ RI ⁇ -NTIG-IL-10Vm fusion protein constructed above was converted into CHO DG44 (from Dr. Chasin, Columbia University, USA). transfected using the transfection system Neon ⁇ the cells were injected.
  • 10% dFBS Gibco, USA, 30067-334) without HT (5-hydroxytrypamine), MEM ⁇ (Gibco, 12561, USA, Cat No. 12561-049), HT+ (Gibco, USA, 11067- 030) medium was used to perform HT selection.
  • methotrexate (MTX) amplification was performed using HT-selected clones.
  • MTX amplification was performed in the form of a mini-pool on a plate, and after selecting one type of amplification confirmed, LDC (Limiting Dilution Cloning; 96 wells, 30 plates) was performed to obtain the final cell line. Secured.
  • the finally obtained cell line was subjected to 700 ml batch culture in hyCellCHO medium, and Protein A purification was performed on the culture medium obtained on the 5th day of culture to confirm the purified protein, and the purified protein was obtained through SDS-PAGE and SE-HPLC. And the purity was analyzed.
  • the fusion protein according to an embodiment of the present invention was normally expressed from the established stable cell line (purity 95.3%).
  • the present inventors determined the affinity of the control group Fc ⁇ RI ⁇ -Fc fusion protein (Korean Patent Laid-Open Publication 10-2019-0084885 disclosed) and the fusion protein (Fc ⁇ RI ⁇ -NTIG-IL-10Vm) of an embodiment of the present invention with mouse IgE and human IgE in the biolayer. It was analyzed through interferometric analysis (BLI assay).
  • the present inventors first used Dip and Read TM Amine Reactive 2 nd Generation (AR2G) Reagent Kit (forteBio, Cat No. 18-5092) to a 96 plate as a control Fc ⁇ RI ⁇ -Fc fusion protein and the fusion protein of Example 1 was attached. Specifically, after dispensing 200 ⁇ l of DW into a 96-well plate, amine biosensor included in the kit was inserted and hydrated for 10 minutes. Subsequently, 200 ⁇ l of DW was additionally dispensed, EDC:NHS was mixed at 1:1 with 1/20 of the required sample amount, diluted in DW, and 200 ⁇ l each was dispensed into a 96-well plate.
  • AR2G Amine Reactive 2 nd Generation
  • the Fc ⁇ RI ⁇ -Fc fusion protein and the Fc ⁇ RI ⁇ -NTIG-IL-10Vm fusion protein were diluted to 10 ⁇ g/ml in 10 mM acetate pH 5.0 solution, and 200 ⁇ l each was added to the 96-well plate. Then, 200 ⁇ l of 1 M ethanolamine was added to the 96-well plate, and then the Biosensor plate and the sample plate were added to Octet. It was put into a K2 instrument and measured. After the measurement was completed, 200 ⁇ l of 1x Kinetics buffer was added to the sample plate, and a baseline was determined.
  • anti-DNP mouse IgE (Sigma) was diluted in 1x Kinetic buffer at various concentrations (50 pM to 3.125 nM), and then dispensed to 200 ⁇ l on a sample plate, and then Octet The degree of binding was measured by performing BLI analysis with a K2 instrument.
  • Fc ⁇ RI ⁇ -Fc PG110-1 (Fc ⁇ RI ⁇ -NTIG-IL-10Vm) Primary Secondary 3rd Primary Secondary 3rd Average K D (nM) 0.364 0.478 0.940 0.224 0.282 0.346 Average: 0.594 Average: 0.284 Average K on (1/Ms) 1.23x10 5 1.49x 10 5 1.66x10 5 1.52x10 5 1.44x10 5 1.39x10 5 Average K dis (1/s) 4.48x10 -5 7.10x10 -5 1.56x10 -4 3.40x10 -5 4.06x10 -5 4.82x10 -5 Average R 2 0.996 0.999 0.997 0.999 0.999 0.999 0.999
  • the K D value of the fusion protein (Fc ⁇ RI ⁇ -NTIG-IL-10Vm, PG110-1) according to the embodiment of the present invention is 0.284 nM, which is the control Fc ⁇ RI ⁇ -Fc fusion. It was found to be somewhat lower than that of the protein, but since the difference in this degree was not a significant difference, it was confirmed that the binding ability with IgE did not decrease by the addition of the IL-10 protein.
  • the present inventors investigated whether the fusion protein of the present invention can target cells expressing IgE on the membrane, such as IgE B cells, from the results of Experimental Example 1 above.
  • the present inventors cultured the U266B1 cell line known to express membrane IgE, followed by performing Live-dead staining for 20 minutes, removing residual staining reagents through washing, and fusion protein according to an embodiment of the present invention.
  • (PG110-1) was reacted at various concentrations (0, 50, 200 and 800 ⁇ g/ml) for 30 minutes, and then unreacted protein was removed by washing, and then PE-binding IgG4 antibody was treated for 20 minutes to be stained.
  • PG110-1 was reacted at various concentrations (0, 50, 200 and 800 ⁇ g/ml) for 30 minutes, and then unreacted protein was removed by washing, and then PE-binding IgG4 antibody was treated for 20 minutes to be stained.
  • FIGS. 5A and 5B was performed (FIGS. 5A and 5B ).
  • the fusion protein according to an embodiment of the present invention was able to target cells expressing IgE on the cell membrane surface. This suggests that the fusion protein according to an embodiment of the present invention targets cells expressing membrane IgE, so that IL-10 can selectively exhibit an effect on the cells.
  • the present inventors analyzed the affinity of the fusion protein (PG110-1) with the IL-10 receptor 1 (IL-10R1) according to an embodiment of the present invention similar to Experimental Example 1 described above (BLI assay) It was analyzed through. At this time, as a control, Fc ⁇ RI ⁇ is not linked, and isoleucine, the 87th amino acid of wild-type human IL-10 mature protein, is substituted with alanine in the modified Fc region, thereby suppressing the immune activation function.
  • NTIG-IL-10V fusion protein between the fusion protein (NTIG-IL-10V) and the fusion protein of NTIG-IL-10V, asparagine (N), the 116th amino acid of the IL-10 variant protein, and lysine (K), the 117th amino acid A fusion protein (NTIG-IL-10Vm) to which a monomeric IL-10 variant protein (IL-10Vm) into which a linker (spacer) peptide (GGSGGSGGS, SEQ ID NO: 14) is inserted was used was used.
  • the NTIG-IL-10Vm is the same as PG110-1 according to an embodiment of the present invention, except that Fc ⁇ RI ⁇ is not connected.
  • the present inventors first attached the IL-10R His-tag protein to 96 plates using Dip and Read TM Amine Reactive 2 nd Generation (AR2G) Reagent Kit (forteBio, Cat No. 18-5092). Specifically, after dispensing 200 ⁇ l of DW into a 96-well plate, amine biosensor included in the kit was inserted and hydrated for 10 minutes. Subsequently, 200 ⁇ l of DW was additionally dispensed, EDC:NHS was mixed at 1:1 with 1/20 of the required sample amount, diluted in DW, and 200 ⁇ l each was dispensed into a 96-well plate.
  • AR2G Amine Reactive 2 nd Generation
  • IL-10R His-tag protein was diluted to 10 ⁇ g/ml in 10 mM acetate pH 5.0 solution, and then 200 ⁇ l was added to the 96-well plate. Thereafter, 200 ⁇ l of 1 M ethanolamine was added to the 96-well plate, and then the Biosensor plate and the sample plate were added to Octet. It was put into a K2 instrument and measured. After the measurement was completed, 200 ⁇ l of 1x Kinetics buffer was added to the sample plate, and a baseline was determined.
  • the fusion protein (Fc ⁇ RI ⁇ -NTIG-IL-10Vm) prepared according to an embodiment of the present invention, and the NTIG-IL-10V and NTIG-IL-10Vm fusion proteins used as controls were respectively added to various concentrations (0, 62.5 , 125, 250, 500 and 1,000 nM) in 1x Kinetic buffer and then dispensed to 200 ⁇ l on a sample plate, and then Octet BLI analysis was performed with a K2 instrument to measure binding force.
  • the fusion protein (NTIG-IL-10Vm) according to an embodiment of the present invention has a Kd value of 29.2 nM, and the monomeric IL-10 fusion protein (NTIG-IL-10V ) was about three times the Kd value (11.1 nM). Therefore, it was found that the affinity with IL-10R was slightly lower than that of the monomeric IL-10 fusion protein. Conventional research results are also predictable because monomeric IL-10 has a lower affinity for IL-10R compared to dimeric IL-10 protein.
  • the fusion protein (Fc ⁇ RI ⁇ -NTIG-IL-10Vm, PG110-1) of Example 1 of the present invention had a KD value of less than 0.001 nM, indicating a higher affinity with IL-10R. This is a result showing that the monomeric IL-10 variant has sufficient binding capacity to IL-10R, despite being linked to the API Fc ⁇ RI ⁇ .
  • Experimental Example 4 Immune Suppression Activity Assay
  • bone marrow-derived mast cells were 1x10 4 cells/in a 96-well plate containing RPMI medium containing 10% FBS, 1% antibiotics, rmSCF 20 ng/ml and rmIL-3 10 ng/ml. Dispense so as to be 50 ⁇ l/well, dilute the fusion protein (NTIG-IL-10Vm) containing the monomeric IL-10 variant protein and the fusion protein (PG110-1) of the example according to the concentration, and then anti-DNP IgE was diluted to 1 ⁇ g/mL, 50 ⁇ l each was added to a 96-well plate, and incubated for 24 hours at 37° C. and 5% CO 2 .
  • NTIG-IL-10Vm the fusion protein
  • PG110-1 monomeric IL-10 variant protein
  • anti-DNP IgE was diluted to 1 ⁇ g/mL, 50 ⁇ l each was added to a 96-well plate, and incubated for 24 hours at 37° C.
  • DNP-BSA Antigen
  • DNP-BSA Antigen
  • both the PG110-1 of the present invention and the monomeric IL-10 variant protein (NTIG-IL-10Vm) used as a comparative example have concentration-dependent TNF- ⁇ secretion inhibitory ability.
  • the fusion protein (PG-11O) according to an embodiment of the present invention exhibited very excellent TNF- ⁇ secretion inhibitory ability even at a lower concentration, and effective blocking of IgE was possible.
  • the present inventors confirmed the ability of the PG110-1 protein to inhibit the secretion of TNF- ⁇ in mast cells according to an embodiment of the present invention, followed by TNF- ⁇ in macrophages, which are immune cells closely related to the TNF- ⁇ -mediated inflammatory response. It was investigated whether it can suppress secretion.
  • RAW264.7 cells were prepared at a concentration of 5 ⁇ 10 5 cells/mL, and then 100 ⁇ L was dispensed into a 96-well plate, followed by incubation in a 37°C, 5% CO 2 incubator for 12 hours. Then, the PG110-1 according to an embodiment of the present invention was sequentially diluted and then treated on the RAW264.7 cells in the culture. At this time, TNF- ⁇ expression was induced by treating 100 ⁇ L of LPS (400 ng/mL) in a 96 well plate.
  • LPS 400 ng/mL
  • the PG110-1 fusion protein according to an embodiment of the present invention inhibited the expression of TNF- ⁇ in macrophages in a concentration-dependent manner.
  • the present inventors performed pharmacokinetic analysis to determine how stable the fusion protein of the present invention is when administered in vivo.
  • the fusion protein (PG110-1) targets 3 SD rats per group by intravenous injection, intraperitoneal injection, intramuscular injection, and subcutaneous injection at a dose of 1 mg/Kg, respectively.
  • intravenous injection is 0 minutes, 5 minutes, 1 hour, 5 hours, 10 hours, 24 hours, 48 hours , 72 hours, 120 hours, 168 hours, 240 hours and 336 hours, and for other administration methods (intraperitoneal injection, intramuscular injection, and subcutaneous injection), serum at the same time as the intravenous injection, except for measurement after 5 minutes.
  • intravenous injection is 0 minutes, 5 minutes, 1 hour, 5 hours, 10 hours, 24 hours, 48 hours , 72 hours, 120 hours, 168 hours, 240 hours and 336 hours, and for other administration methods (intraperitoneal injection, intramuscular injection, and subcutaneous injection), serum at the same time as the intravenous injection, except for measurement after 5 minutes.
  • the fusion protein was quantified using human IgG4 Fc ELISA.
  • the fusion protein according to an embodiment of the present invention exhibited a modest decrease over time regardless of the route of administration, which is a fusion protein according to an embodiment of the present invention. This shows that it remains stable for a considerable period of time in the body.
  • the present inventors consisted of 3 mice per group targeting magnetic ICR mice, and administered PG110-1 at 0, 50, 150 and 300 mg/kg doses for each group, and blood was collected on the 11th day after drug administration. Then, a white blood cell differential count, a total RBC, and a total platelet were analyzed using a hemocytometer (XN-V, SYSMEX, JAPAN). As a result, as shown in FIG. 11, when PG110-1 was administered, the number of blood cells and the like was not affected at all.
  • the present inventors have conducted oral administration of OVA to an experimental animal sensitized with egg white albumin (OVA), a cause of allergies. We investigated whether diarrhea, which is an allergic symptom, was improved.
  • OVA egg white albumin
  • OVA ovalbuproliferin
  • Alum a solution of 50 ⁇ g of OVA (ovalbumin) and 1 mg of Alum was administered intraperitoneally at 14 days intervals to Balb/c mice (Coretech) to sensitize. Subsequently, on days 28, 30, 32, 34, and 36, OVA 50 mg was orally administered at intervals of 2 days over a total of 5 times to induce food allergy in the intestine.
  • drugs constituting each group were administered on the 31st day.
  • IgE TRAP 5 mg/kg
  • PG110-1 7.3 mg/kg
  • OVA was administered orally over a total of 5 times to check whether diarrhea occurred.At 37 days, mice were autopsied and the number of mast cells in the small intestine, IgE concentration in the blood, and the concentration of degranulation enzymes in the blood mast cells (MCPT -1, Mast cell protease-1) was analyzed. As a result, as can be seen in Figure 12, compared to the control and IgE TRAP- administered mice in the mice belonging to the group administered with PG110-1 according to an embodiment of the present invention it was confirmed the effect of reducing food allergy.
  • the fusion protein according to an embodiment of the present invention blocks IgE and thereby inhibits the activation of mast cells. This action seems to be due to the inactivation of anti-Fc ⁇ RI ⁇ antibody and thereby inhibition of the activation of mast cells.
  • the fusion protein according to an embodiment of the present invention may block free IgE as well as target B cells expressing IgE presented on the membrane.
  • regulatory T cells regulatory T cells, T reg ) (Hsu et al ., J. Immunol . 2015 Oct 15;195(8):3665). -3674, 2015)).
  • the fusion protein according to an embodiment of the present invention contains the IL-10 protein, it is expected to target various immune cells expressing the IL-10 receptor and inhibit their function.
  • immune cells include mast cells that secrete anti-inflammatory cytokines, monocytes and macrophages responsible for the function of secreting anti-inflammatory cytokines and presenting antigens, and anti-inflammatory cytokines that also have antigen-presenting ability.
  • the fusion protein according to an embodiment of the present invention inhibits the activation of the immune cells and their functions, especially the secretion of anti-inflammatory cytokines, the ability to present antigens, etc., thereby preventing various immune-related diseases caused by overactivated immune functions.
  • it can be used for the treatment of allergic diseases such as atopic diseases, food allergies, chronic spontaneous urticaria, and asthma.
  • the fusion protein according to an embodiment of the present invention is a very useful drug candidate that can improve the administration convenience of allergic patients.
  • the fusion protein according to an embodiment of the present invention may be usefully used in the development of an allergy treatment.

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Abstract

The present invention relates to a novel pharmaceutical composition for treating allergic diseases and, more particularly, to a pharmaceutical composition for treating allergic diseases, comprising as active ingredients: a) an FcεRIα protein and an IL-10 protein; and/or b) a fusion protein including an FcεRIα protein and an IL-10 protein.

Description

신규 알러지 질환 치료용 약학적 조성물Pharmaceutical composition for the treatment of new allergic diseases
본 발명은 약학적 조성물에 관한 것으로서, 보다 구체적으로는 신규 알러지 질환 치료용 약학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition, and more particularly, to a pharmaceutical composition for the treatment of novel allergic diseases.
알러지(allergy)는 면역체계의 과민성 질환으로 신체가 알레르겐(음식, 일반 의약품, 먼지, 꽃가루 등 보통 안전한 환경 물질)에 지나친 항원항체 반응이 일어나 결국 신체 내에 다양한 염증성 반응이 유발되는 질환을 의미하며, 증상은 그저 기분이 나빠지거나 가렵거나 피부에 뭔가 나는 정도부터 시작해서, 심하게는 호흡곤란이 발생되는 경우도 있으며, 보통 피부가 부풀어 오르는 경우가 많은데, 특히 예민한 부위인 점막이 부풀어 오르는 경우가 많다. 흔히 알레르기성 천식이나 알러지성 결막염을 예로 들 수 있으며, 그 외에 아토피나 알러지성 비염 등의 증상으로 나타나기도 하며, 증상이 한 가지만 있는 경우도 많지만 여러 가지가 복합적으로 나타나는 경우 역시 많다.Allergy is an irritable disease of the immune system.It refers to a disease in which the body reacts excessively with antigen-antibodies to allergens (usually safe environmental substances such as food, general medicine, dust, pollen, etc.), resulting in various inflammatory reactions in the body. Symptoms start from just feeling bad, itchy, or something on the skin, severe breathing difficulties occur, and usually the skin swells in many cases, especially when the sensitive area, the mucous membrane, swells. Often, allergic asthma or allergic conjunctivitis can be exemplified, and other symptoms such as atopy or allergic rhinitis may appear, and there are many cases where there is only one symptom, but there are also many cases in which several are combined.
Gell & Cooms의 분류법을 이용하면 알러지를 총 4형으로 분류할 수 있다. 우리가 흔히 말하는 알러지의 대다수이자 아나필락시스를 유발하고 급성 과민반응으로 불리고 있는 제1형 알러지, 항체 매개 과민반응의 제2형 알러지, 면역복합체에 의한 제3형 그리고 후천 면역에 의한 제4형이 있다.Using the Gell & Cooms classification method, allergies can be classified into four types. The majority of allergies we talk about are type 1 allergy, which induces anaphylaxis and is called acute hypersensitivity reaction, type 2 allergy due to antibody-mediated hypersensitivity reaction, type 3 due to immune complex, and type 4 due to acquired immunity. .
제1형, 즉 일반적인 의미의 알러지에서는 항체 중 IgE와 비만세포(mast cell)가 증상을 유발한다. 비만세포는 전구세포가 아직 불확실하나 백혈구 중 호염구(Basophils)가 유력하다. 비만세포와 결합한 IgE는, 화학물질처럼 단일항원이나, 여러 이유로 항원이지만 항원으로 인식하지 못하는 항원에 대해서는 복합체가 결합하더라도 면역 작용을 수행하지 못하나, 특정 서열의 다가항원인 경우에는 비만세포를 자극하여 세포내 탈과립화(Degranulation)를 유발하게 된다. 이로 인해 히스타민(histamine), 류코트리엔(leukotriene), 프로스타글란딘(prostaglandin) 및 IL-4 등 여러 신호적 화학물질을 방출한다. 히스타민은 혈관의 투과성을 증대시키는데, 결국 혈관 내 체액이 혈관 밖으로 방출됨에 따라 염증이 발생한다. 아울러, 체액의 방출에 따라 혈압은 감소하게 되며, 급성 과민 반응인 과민성 쇼크는 혈관 확장과 기도 점막 부종에 따른 기도 폐쇄로 인해 발생한다. 이러한 과민성 쇼크는 생명을 위협하는 증상으로 즉시 에피네프린 주사와 심폐소생술을 해야 한다. In type 1, that is, allergy in the general sense, among antibodies, IgE and mast cells cause symptoms. In mast cells, progenitor cells are still unclear, but basophils among leukocytes are the dominant. IgE bound to mast cells, like a chemical substance, is a single antigen, but for various reasons, even if the complex binds to an antigen that is not recognized as an antigen, it cannot perform immunity, but in the case of a polyantigen of a specific sequence, it stimulates mast cells. It causes intracellular degranulation. This releases several signaling chemicals such as histamine, leukotriene, prostaglandin and IL-4. Histamine increases the permeability of blood vessels, which in turn causes inflammation as fluid is released from the blood vessels. In addition, blood pressure decreases as body fluids are released, and hypersensitivity shock, an acute hypersensitivity reaction, occurs due to vasodilation and airway obstruction due to airway mucosal edema. This irritable shock is a life-threatening symptom and requires immediate epinephrine injection and CPR.
현재까지, 알러지 질환의 치료에는 항히스타민제, 항류코트리엔제, 베타차단제, 및 스테로이드제 등을 사용하는데, 일반적으로 항히스타민제와 항류코트리엔제를 조합하거나 비강 분무 스테로이드제를 단독으로 많이 사용한다. 아울러, 높은 IgE 수치가 기록되는 알러지의 경우에는 항-IgE 항체인 오말리주맙(Omalizumab)이 사용되기도 한다. 최근에는 IL-4에 결합하는 단클론 항체인 듀필루맙(Dupilumab)이 사용되기도 한다. 그러나, 오말리주맙의 경우 IgE에 대한 낮은 친화도(KD = 1.49x10-8) 때문에 IgE 기저값의 상한선에서의 사용에 있어서 제한의 문제나 항-FcεRI 자가-항체를 가지고 있는 환자에 대하여는 사용이 불가능한데, 만성 두드러기 환자의 35~40%는 항-FcεRI 자가-항체를 보유하고 있는 것으로 보고되고 있다. 가장 큰 문제점은 아나필락시스에 대한 잠재적 위험성이 있다는 것이다.Until now, antihistamines, antileukotrienes, beta-blockers, and steroids have been used in the treatment of allergic diseases, but generally antihistamines and antileukotrienes are combined or nasal spray steroids are often used alone. In addition, in the case of allergies in which high IgE levels are recorded, Omalizumab, an anti-IgE antibody, is sometimes used. Recently, Dupilumab, a monoclonal antibody that binds to IL-4, has also been used. However, in the case of omalizumab, due to its low affinity for IgE (K D = 1.49x10 -8 ), its use at the upper limit of the baseline IgE value is limited, or it is not used for patients with anti-FcεRI auto-antibodies. It is not possible, but it is reported that 35-40% of patients with chronic urticaria have anti-FcεRI auto-antibodies. The biggest problem is that there is a potential risk for anaphylaxis.
이러한 문제점 때문에, 항-IgE 항체 대신에 FcεRIα와 같은 IgE 수용체 단백질의 IgE 결합 도메인을 사용하고자 하는 연구가 이루어지고 있다(US4962035, US5639660, US6602983 등). 그러나, FcεRIα을 유효성분으로 하는 알러지 치료제는 동물의약용으로만 상용화되었을 뿐, 인체를 대상으로 한 약물로 아직 임상 승인된 약물은 없는 실정이다.Because of this problem, studies to use the IgE binding domain of an IgE receptor protein such as FcεRIα instead of an anti-IgE antibody are being conducted (US4962035, US5639660, US6602983, etc.). However, allergy treatments using FcεRIα as an active ingredient have been commercialized only for animal medicine, and there is no clinically approved drug for the human body yet.
한편, 인터류킨 10(이하, "IL-10"라 지칭함)은 사이토카인 합성 저해 인자(cytokine synthesis inhibitory factor, CSIF)라고 불리우는 약 37 kDa의 비공유 결합된 동종이량체로 발현되는 항-염증성 사이토카인이다. IL-10은 면역관용의 유도와 유지에 중요한 역할을 하며, 이러한 우세한 항-염증 특성은 오랜 기간 동안 알려져 왔다. IL-10은 TNFα, IL-1, IL-6, 및 IL-12 뿐만 아니라 IL-2 및 IFNγ와 같은 Th1 사이토카인과 같은 염증-촉진성 사이토카인(pro-inflammatory cytokine)의 분비를 억제하고 대식세포, B 세포 및 T 세포의 분화 및 증식을 조절한다(Glocker et al., Ann. NY Acad. Sci. 1246: 102-107, 2011; Moore et al., Annu. Rev. Immunol.19, 683-765, 2001); Waal Malefyt et al., J. Exp. Med. 174: 915-924, 1991; Williams et al., Immunol. 113: 281-292, 2004). 또한, 이는 항원 제시의 강력한 억제제로서, MHC II 발현뿐만 아니라 공동자극 인자 CD80 및 CD86의 상향조절을 억제한다(Mosser & Yhang, Immunol. Rev. 226: 205-218, 2008). 이러한 특성 때문에 IL-10은 염증성 장질환이나 건선과 같은 자가면역 질환 등의 치료제로 사용하려는 연구가 진행되어 왔다.On the other hand, interleukin 10 (hereinafter referred to as "IL-10") is an anti-inflammatory cytokine expressed as a non-covalently bound homodimer of about 37 kDa called a cytokine synthesis inhibitory factor (CSIF). . IL-10 plays an important role in the induction and maintenance of immune tolerance, and these dominant anti-inflammatory properties have been known for a long time. IL-10 inhibits the secretion of pro-inflammatory cytokines such as TNFα, IL-1, IL-6, and IL-12 as well as Th1 cytokines such as IL-2 and IFNγ, and Regulate the differentiation and proliferation of phagocytes, B cells and T cells (Glocker et al ., Ann. NY Acad. Sci. 1246: 102-107, 2011; Moore et al ., Annu. Rev. Immunol . 19, 683- 765, 2001); Waal Malefyt et al ., J. Exp. Med . 174: 915-924, 1991; Williams et al ., Immunol . 113: 281-292, 2004). In addition, it is a potent inhibitor of antigen presentation, inhibiting MHC II expression as well as upregulation of costimulatory factors CD80 and CD86 (Mosser & Yhang, Immunol. Rev. 226: 205-218, 2008). Because of these characteristics, studies have been conducted to use IL-10 as a therapeutic agent for inflammatory bowel disease or autoimmune diseases such as psoriasis.
본 발명은 상술한 문제점을 포함하여 여러 가지 문제점을 해결하기 위한 것으로서, 보다 효율적이고 안전한 알러지 치료용 약학적 조성물을 제공하는 것을 목적으로 한다. 그러나, 본 발명의 보호범위가 상기 목적으로 제한되는 것은 아니다.The present invention is to solve various problems, including the above-described problems, and an object thereof is to provide a more efficient and safe pharmaceutical composition for treating allergies. However, the scope of protection of the present invention is not limited to the above purpose.
본 발명의 일 관점에 따르면, a) FcεRIα 단백질 및 IL-10 단백질; 및/또는 b) FcεRIα 단백질 및 IL-10 단백질을 포함하는 융합단백질을 유효성분으로 포함하는 알러지 질환 치료용 약학적 조성물이 제공된다.According to one aspect of the present invention, a) FcεRIα protein and IL-10 protein; And/or b) there is provided a pharmaceutical composition for treating allergic diseases comprising a fusion protein comprising an FcεRIα protein and an IL-10 protein as an active ingredient.
본 발명의 다른 일 관점에 따르면, FcεRIα, 항체의 Fc 영역 및 IL-10 단백질이 순차적으로 연결되고, 선택적으로 상기 FcεRIα 및 항체의 Fc 영역 사이 및/또는 상기 항체의 Fc 영역 및 IL-10 단백질 사이에 링커 펩타이드를 포함하는, 융합단백질이 제공된다.According to another aspect of the present invention, FcεRIα, the Fc region of the antibody and the IL-10 protein are sequentially linked, and optionally between the FcεRIα and the Fc region of the antibody and/or between the Fc region of the antibody and the IL-10 protein A fusion protein comprising a linker peptide is provided.
본 발명의 다른 일 관점에 따르면, 상기 융합단백질을 유효성분으로 포함하는 알러지 질환 치료용 약학적 조성물이 제공된다.According to another aspect of the present invention, a pharmaceutical composition for treating allergic diseases comprising the fusion protein as an active ingredient is provided.
본 발명의 다른 일 관점에 따르면, a) FcεRIα 단백질 및 IL-10 단백질; 및/또는 b) FcεRIα 단백질 및 IL-10 단백질을 포함하는 융합단백질을 개체에 투여하는 단계를 포함하는 상기 개체의 알러지 치료방법이 제공된다.According to another aspect of the present invention, a) FcεRIα protein and IL-10 protein; And/or b) administering a fusion protein including an FcεRIα protein and an IL-10 protein to the individual.
본 발명의 일 실시예에 따른 약학적 조성물은 알러지 반응에 의해 생성되는 IgE를 효과적으로 차단할 수 있을 뿐만 아니라, 기존 IgE 특이적 항체치료제의 단점인 아낙필라시스와 같은 과민반응의 가능성을 매우 낮춘 약물로서, 안전성이 개선된 알러지 치료제로 사용될 수 있다. The pharmaceutical composition according to an embodiment of the present invention is a drug that can effectively block IgE generated by an allergic reaction, as well as very low the likelihood of hypersensitivity reactions such as anaphylaxis, which is a disadvantage of existing IgE-specific antibody treatments. , It can be used as an allergy treatment with improved safety.
도 1은 본 발명의 일 실시예에 따른 융합단백질의 전반적인 구조를 나타내는 개요도이다.1 is a schematic diagram showing the overall structure of a fusion protein according to an embodiment of the present invention.
도 2a는 본 발명의 일 실시예에 따라 제조된 융합단백질(PG110-1)을 일시적 형질감염에 의해 발현시킨 후 정제 단계별로 환원(좌측) 및 비환원(우측) 조건에서 PAGE 분석을 수행한 결과를 나타내는 사진이고, 도 2b는 상기 PG110-1을 정제하는 과정에서 수득된 분획(A10, A11, 및 A12)에 대하여 SE-HPLC 분석을 수행하여 순도를 측정한 결과를 나타내는 일련의 크로마토그램이며, 도 2c는 본 발명의 일 실시예에 따라 제조된 융합단백질(PG110-2)을 일시적 형질감염에 의해 발현시킨 후 정제 단계별로 환원(좌측) 및 비환원(우측) 조건에서 PAGE 분석을 수행한 결과를 나타내는 사진이고, 도 2d는 상기 PG110-2를 정제하는 과정에서 수득된 분획(A11 및 A12)에 대하여 SE-HPLC 분석을 수행하여 순도를 측정한 결과를 나타내는 일련의 크로마토그램이다.Figure 2a is a result of performing PAGE analysis under reduced (left) and non-reduced (right) conditions after expressing the fusion protein (PG110-1) prepared according to an embodiment of the present invention by transient transfection. 2B is a series of chromatograms showing the results of measuring purity by performing SE-HPLC analysis on the fractions (A10, A11, and A12) obtained in the process of purifying the PG110-1, 2C is a result of performing PAGE analysis under reduced (left) and non-reduced (right) conditions after expressing the fusion protein (PG110-2) prepared according to an embodiment of the present invention by transient transfection. 2D is a series of chromatograms showing the results of measuring the purity by performing SE-HPLC analysis on the fractions (A11 and A12) obtained in the process of purifying the PG110-2.
도 3a는 본 발명의 일 실시예에 따른 융합단백질을 생산하도록 제조된 안정 세포주(stable cell line)로부터 상기 융합단백질(PG110-1)의 정제공정에 따른 단백질의 발현 정도를 확인하기 위한 비-환원조건(좌측) 및 환원조건(우측)에서의 SDS-PAGE 분석 결과를 나타내는 사진이고, 도 3b는 상기 안정화 세포주에서 최종 정제된 융합단백질(PG110-1)의 순도를 확인하기 위한 SEC-HPLC 분석결과를 나타내는 크로마토그램이다. Figure 3a is a non-reduction for confirming the level of protein expression according to the purification process of the fusion protein (PG110-1) from a stable cell line prepared to produce a fusion protein according to an embodiment of the present invention Photo showing the results of SDS-PAGE analysis under conditions (left) and reducing conditions (right), and FIG. 3B is a result of SEC-HPLC analysis to confirm the purity of the fusion protein (PG110-1) finally purified from the stabilizing cell line. It is a chromatogram representing
도 4a는 비교예로 FcεRIα-Fc 융합단백질의 마우스 IgE와의 친화도를 BLI 분석으로 분석한 결과를 나타내는 센소그램이고 도 4b는 본 발명의 일 실시예에 따른 융합단백질 PG110-1(하단)의 마우스 IgE와의 친화도를 BLI 분석으로 분석한 결과를 나타내는 센소그램이며, 도 4c는 본 발명의 일 실시예에 따른 PG110-1의 인간 IgE와의 친화도를 BLI 분석으로 분석한 결과를 나타내는 센소그램이다.4A is a sensogram showing the result of analyzing the affinity of the FcεRIα-Fc fusion protein with mouse IgE by BLI analysis as a comparative example. FIG. 4B is a mouse of the fusion protein PG110-1 (bottom) according to an embodiment of the present invention It is a sensogram showing the result of analyzing the affinity with IgE by BLI analysis, and FIG. 4C is a sensogram showing the result of analyzing the affinity of PG110-1 with human IgE according to an embodiment of the present invention by BLI analysis.
도 5는 본 발명의 일 실시예에 따른 융합단백질 PG110의 막 IgE 발현 B 세포인 U266B1의 표적화 여부를 분석하기 위한 유세포분석 실험 개요(A), 본 발명의 일 실시예에 따른 융합단백질 PG110에 의한 막 IgE 발현 B 세포의 검출과정을 개략적으로 도시한 개요도(B) 및 실제 본 발명의 융합단백질 PG110을 이용하여 U266B1 B 세포를 표적화한 유세포 분석 결과를 나타내는 일련의 히스토그램(C)이다. 5 is an overview of flow cytometry experiments for analyzing the targeting of U266B1, a membrane IgE-expressing B cell, of the fusion protein PG110 according to an embodiment of the present invention (A), by the fusion protein PG110 according to an embodiment of the present invention A schematic diagram (B) schematically showing the detection process of membrane IgE-expressing B cells, and a series of histograms (C) showing the results of flow cytometry targeting U266B1 B cells using the actual fusion protein PG110 of the present invention.
도 6은 본 발명의 일 실시예에 따른 융합단백질 PG110-1(하단)과 비교예인 NTIG-IL-10V(상단) 및 NTIG-IL-10Vm 단백질(중단)의 IL-10R1과의 친화도를 BLI 분석으로 분석한 결과를 나타내는 일련의 그래프이다.Figure 6 shows the affinity of the fusion protein PG110-1 (bottom) and the comparative examples NTIG-IL-10V (top) and NTIG-IL-10Vm proteins (stop) with IL-10R1 according to an embodiment of the present invention. It is a series of graphs showing the results of analysis.
도 7은 본 발명의 일 실시예에 따른 융합단백질 PG110 및 단량체성 IL-10 변이체 단백질(IL-10M-1; NTIG-IL-10Vm)의 비만세포에서의 TNF-α 분비 억제능을 분석한 결과를 나타낸 그래프이다.7 is a result of analyzing the ability of the fusion protein PG110 and the monomeric IL-10 variant protein (IL-10M-1; NTIG-IL-10Vm) to inhibit TNF-α secretion in mast cells according to an embodiment of the present invention. This is the graph shown.
도 8은 본 발명의 일 실시예에 따른 융합단백질 PG110-1의 처리농도에 따른 대식세포에서의 TNF-α 분비 억제능을 분석한 결과를 나타낸 그래프이다.8 is a graph showing the results of analyzing the ability to inhibit the secretion of TNF-α in macrophages according to the treatment concentration of the fusion protein PG110-1 according to an embodiment of the present invention.
도 9는 본 발명의 일 실시예에 따른 융합단백질 PG110-1 및 비교예로서 IgE TRAP(FcεRIα-Fc 융합단백질)을 마우스 골수유래 비만세포에 처리 시 IgE 차단 및 비만세포 활성화 억제능을 베타-헥소사미니다아제 활성을 측정함으로써 분석한 결과를 나타내는 그래프이다.9 is a beta-hexosami that inhibits IgE blocking and mast cell activation when treating mouse bone marrow-derived mast cells with fusion protein PG110-1 and a comparative example of IgE TRAP (FcεRIα-Fc fusion protein) according to an embodiment of the present invention. This is a graph showing the results of analysis by measuring nidase activity.
도 10은 본 발명의 일 실시예에 따른 융합단백질을 실험동물(랫트)에 다양한 경로(정맥주사, 복강주사, 근육주사, 및 피하주사)로 투여한 이후 최대 330시간 까지 혈청 내 남아 있는 융합단백질의 양을 정량함으로써 약물 동태학 분석을 수행한 결과를 나타내는 그래프이다.10 is a fusion protein remaining in the serum for up to 330 hours after administration of the fusion protein according to an embodiment of the present invention to an experimental animal (rat) by various routes (intravenous injection, intraperitoneal injection, intramuscular injection, and subcutaneous injection) It is a graph showing the results of pharmacokinetic analysis by quantifying the amount of.
도 11은 본 발명의 일 실시예에 따른 융합단백질 PG110-1을 실험동물에 투여 시 백혈구(A), 적혈구(B) 및 혈소판(C)의 수의 변화가 나타나는지 여부를 조사한 혈액독성 분석 결과를 나타내는 일련의 그래프이다.FIG. 11 is a blood toxicity analysis result of investigating whether a change in the number of leukocytes (A), red blood cells (B), and platelets (C) appears when the fusion protein PG110-1 according to an embodiment of the present invention is administered to an experimental animal. It is a series of graphs shown.
도 12는 본 발명의 일 실시예에 따른 융합단백질 PG110-1이 실제 동물에서 알러지 증상을 감소시킬 지 여부를 조사한 것으로서, OVA로 감작된 마우스에 대하여 OVA 경구 투여로 음식물 알러지를 유발시킨 후 본 발명의 일 실시예에 따른 PG110-1 및 비교예로 IgE TRAP(FcεRIα-Fc 융합단백질) 투여 시 설사증상의 변화(A), 실험 종료 후 희생된 마우스로부터 측정된 유리 IgE 농도(B), 총 IgE 농도(C) 및 알러지 인자로서 혈중 비만세포 내의 탈과립 효소(MCPT-1, mast cell protease-1)의 농도(D)를 측정한 결과를 나타내는 일련의 그래프이다.FIG. 12 is an investigation of whether the fusion protein PG110-1 according to an embodiment of the present invention reduces allergic symptoms in real animals, and the present invention after causing food allergy by oral administration of OVA to OVA-sensitized mice. Changes in diarrhea symptoms when PG110-1 according to an embodiment of PG110-1 and a comparative example of IgE TRAP (FcεRIα-Fc fusion protein) administration (A), free IgE concentration (B) measured from the sacrificed mice after the end of the experiment, and total IgE It is a series of graphs showing the results of measuring the concentration (C) and the concentration (D) of the degranulation enzyme (MCPT-1, mast cell protease-1) in the blood as an allergenic factor.
본 발명의 일 관점에 따르면, a) FcεRIα 단백질 및 IL-10 단백질; 및/또는 b) FcεRIα 단백질 및 IL-10 단백질을 포함하는 융합단백질을 유효성분으로 포함하는 알러지 질환 치료용 약학적 조성물이 제공된다.According to one aspect of the present invention, a) FcεRIα protein and IL-10 protein; And/or b) there is provided a pharmaceutical composition for treating allergic diseases comprising a fusion protein comprising an FcεRIα protein and an IL-10 protein as an active ingredient.
상기 약학적 조성물에 있어서, 상기 FcεRIα는 서열번호 2로 기재되는 아미노산 서열로 구성될 수 있고, 상기 IL-10 단백질은 UniProtKB P22301에 기재된 아미노산 서열에서 신호서열이 제거된 19-178번째 아미노산 서열을 포함하는 성숙형(mature form) 단백질일 수 있고, 상기 성숙형 단백질의 87번째 아미노산인 이소류신이 알라닌으로 치환된 IL-10 변이체 단백질일 수 있으며, 116번째 아미노산인 아스파라긴 및 117번째 아미노산인 라이신 사이에 6 내지 12 a.a.의 길이를 갖는 링커 펩타이드가 삽입된 단량체성 IL-10 변이체 단백질일 수 있으며, 본 발명의 일 구체예에서 상기 IL-10 단백질은 서열번호 10 내지 13으로 구성되는 군으로부터 선택되는 아미노산 서열로 구성될 수 있다.In the pharmaceutical composition, the FcεRIα may be composed of an amino acid sequence described in SEQ ID NO: 2, and the IL-10 protein includes an amino acid sequence 19-178 from which a signal sequence has been removed from the amino acid sequence described in UniProtKB P22301. It may be a mature protein, and may be an IL-10 variant protein in which isoleucine, which is the 87th amino acid of the mature protein, is substituted with alanine, and between the 116th amino acid asparagine and the 117th amino acid lysine. It may be a monomeric IL-10 variant protein into which a linker peptide having a length of to 12 aa is inserted, and in one embodiment of the present invention, the IL-10 protein is an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 13 It can be composed of.
상기 약학적 조성물에 있어서, 상기 FcεRIα 단백질 및 IL-10 단백질은 포유동물 유래의 것이라면 어느 것이라도 사용 가능하나, 더욱 바람직하게는 인간 유래의 단백질이다.In the pharmaceutical composition, the FcεRIα protein and IL-10 protein can be used as long as they are derived from mammals, but more preferably they are human-derived proteins.
본 발명에서 사용되는 용어 "융합단백질"은 둘 이상의 단백질 또는 단백질 내 특정 기능을 담당하는 도메인이 각각의 단백질 또는 도메인이 본연의 기능을 담당하도록 연결된 재조합 단백질(recombinant protein)을 의미한다. 상기 둘 이상의 단백질 또는 도메인 사이에는 통상적으로 유연한 구조를 갖는 링커 펩타이드(linker peptide)가 삽입될 수 있다. 상기 링커 펩타이드는 GGGGSGGGGSGGGGSEPKSSDKTHTCPPCP(서열번호 3), AAGSGGGGGSGGGGSGGGGS(서열번호 8), GGSGG(서열번호 9), GGSGGSGGS(서열번호 14), GGGSGG(서열번호 15), (G4S)n(단위체: 서열번호 16, n은 1 내지 10의 정수), (GGS)n(n은 1 내지 10의 정수), (GS)n(n은 1 내지 10의 정수), (GSSGGS)n(단위체: 서열번호 17, n은 1 내지 10의 정수), KESGSVSSEQLAQFRSLD(서열번호 18), EGKSSGSGSESKST(서열번호 19), GSAGSAAGSGEF(서열번호 20), (EAAAK)n(단위체: 서열번호 21, n은 1 내지 10의 정수), CRRRRRREAEAC(서열번호 22), A(EAAAK)4ALEA(EAAAK)4A(서열번호 23), GGGGGGGG(서열번호 24), GGGGGG(서열번호 25), AEAAAKEAAAAKA(서열번호 26), PAPAP(서열번호 27), (Ala-Pro)n(n은 1 내지 10의 정수), VSQTSKLTRAETVFPDV(서열번호 28), PLGLWA(서열번호 29), TRHRQPRGWE(서열번호 30), AGNRVRRSVG(서열번호 31), RRRRRRRR(서열번호 32), GFLG(서열번호 33), GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE(서열번호 34), GGGGSGGGGSGGGGSEKEKE EQEERTHTCPPCP(서열번호 35), RNTGRGGEEKKGSKEKEEQEERETKTPECP(서열번호 36), GGGGSGGGGSGGGGSEPKSCDKTHTCPPCP(서열번호 37), GSGGGSGTLVTVSSESKYGPPCPPCP(서열번호 38), EPKSSDKTHTCPPCP(서열번호 39), EPKSCDKTHTCPPCP(서열번호 40), THTCPPCP(서열번호 41), GGGGSGGGGSGGGGSAKNTTAPATTRNTTRGGEEKKKEKEKEEQEERTHTCPPCP(서열번호 42) 등이 포함될 수 있다. The term "fusion protein" used in the present invention refers to a recombinant protein (recombinant protein) in which two or more proteins or domains responsible for a specific function in the protein are linked so that each protein or domain is responsible for its natural function. A linker peptide having a generally flexible structure may be inserted between the two or more proteins or domains. The linker peptide is GGGGSGGGGSGGGGSEPKSSDKTHTCPPCP (SEQ ID NO: 3), AAGSGGGGGSGGGGSGGGGS (SEQ ID NO: 8), GGSGG (SEQ ID NO: 9), GGSGGSGGS (SEQ ID NO: 14), GGGSGG (SEQ ID NO: 15), (G 4 S) n (unit: SEQ ID NO: 16, n is an integer of 1 to 10), (GGS) n (n is an integer of 1 to 10), (GS) n (n is an integer of 1 to 10), (GSSGGS) n (unit: SEQ ID NO: 17, n is an integer of 1 to 10), KESGSVSSEQLAQFRSLD (SEQ ID NO: 18), EGKSSGSGSESKST (SEQ ID NO: 19), GSAGSAAGSGEF (SEQ ID NO: 20), (EAAAK) n (unit: SEQ ID NO: 21, n is an integer of 1 to 10), CRRRRRREAEAC (SEQ ID NO: 22), A (EAAAK) 4 ALEA (EAAAK) 4 A (SEQ ID NO: 23), GGGGGGGG (SEQ ID NO: 24), GGGGGG (SEQ ID NO: 25), AEAAAKEAAAAKA (SEQ ID NO: 26), PAPAP (SEQ ID NO: 27) ), (Ala-Pro)n (n is an integer from 1 to 10), VSQTSKLTRAETVFPDV (SEQ ID NO: 28), PLGLWA (SEQ ID NO: 29), TRHRQPRGWE (SEQ ID NO: 30), AGNRVRRSVG (SEQ ID NO: 31), RRRRRRRR (SEQ ID NO: 32), GFLG (SEQ ID NO: 33), GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (SEQ ID NO: 34), GGGGSGGGGSGGGGSEKEKE EQEERTHTCPPCP (SEQ ID NO: 35), RNTGRGGEETCGGKGSKEKEEQEERETKTPECP (SEQ ID NO: 36), GGGCPGS (SEQ ID NO. 39), EPKSCDKTHTCPPCP (SEQ ID NO: 40), THTCPPCP (SEQ ID NO: 41), GGGGSGGGGSGGGGSAKNTTAPATTRNTTRGGEEKKKEKEKEEQEERTH TCPPCP (SEQ ID NO: 42) and the like may be included.
상기 약학적 조성물에 있어서, FcεRIα 단백질 및 IL-10 단백질을 포함하는 경우, 상기 FcεRIα 단백질 및 IL-10 단백질은 조성물에 함께 포함될 수도 있고, 개별적으로 제형화되어 동시 또는 시차를 두고 투여될 수 있다. 시차를 두고 투여될 경우, 양 물질의 투여 간격은 환자의 상태, 나이, 증상, 다른 질환의 유무 등의 조건에 따라서 달라질 수 있다. 아울러, 투여 횟수의 경우 증상의 종류에 따라서 결정될 수 있다. 예컨대, 급성 알러지 증상일 경우 단기간 동안 집중적으로 투여를 하는 것이 바람직할 수 있고, 만성 알러지 증상일 경우에는 장기간 투여가 필요할 수 있다. In the pharmaceutical composition, when the FcεRIα protein and the IL-10 protein are included, the FcεRIα protein and the IL-10 protein may be included together in the composition, or formulated separately and administered simultaneously or at different times. When administered at a time difference, the interval between administration of both substances may vary depending on conditions such as the patient's condition, age, symptoms, and the presence or absence of other diseases. In addition, the number of administration may be determined according to the type of symptoms. For example, in the case of acute allergic symptoms, it may be desirable to intensively administer for a short period of time, and in the case of chronic allergic symptoms, long-term administration may be necessary.
본 발명의 다른 일 관점에 따르면, FcεRIα, 항체의 Fc 영역 및 IL-10 단백질이 순차적으로 연결되고, 선택적으로 상기 FcεRIα 및 항체의 Fc 영역 사이 및/또는 상기 항체의 Fc 영역 및 IL-10 단백질 사이에 링커 펩타이드를 포함하는, 융합단백질이 제공된다.According to another aspect of the present invention, FcεRIα, the Fc region of the antibody and the IL-10 protein are sequentially linked, and optionally between the FcεRIα and the Fc region of the antibody and/or between the Fc region of the antibody and the IL-10 protein A fusion protein comprising a linker peptide is provided.
상기 융합단백질에 있어서, 상기 FcεRIα 단백질은 Fcε 수용체라고도 불리우는 IgE Fc 수용체로서, IgE Fc에 대해 고친화성인 수용체이다. FcεRI은 비만세포, 호염기구에서 발현된다. FcεRI에 결합한 IgE 항체가 다가 항원에 의해 가교되면, 비만세포 또는 호염 기구에서 탈과립이 생겨 히스타민을 비롯한 여러 가지 화학 전달 물질을 방출한다. 이러한 방출에 의해 즉시형 알러지 반응이 나타난다.In the fusion protein, the FcεRIα protein is an IgE Fc receptor, also called an Fcε receptor, and is a receptor having high affinity for IgE Fc. FcεRI is expressed in mast cells and basophils. When IgE antibodies bound to FcεRI are crosslinked by a multivalent antigen, degranulation occurs in mast cells or basophils, releasing various chemical transport substances including histamine. This release leads to an immediate allergic reaction.
상기 FcεRI는 하나의 α사슬과 한 개의 β사슬 및 2황화 결합한 2개의 γ사슬로 구성된 막단백질이다. 이 중 IgE가 결합하는 부분은 α사슬(FcεRIα)로서, FcεRIα는 60 kDa 정도의 크기를 가지며, 세포막 안에 존재하는 소수성 도메인과 세포막 외부에 존재하는 친수성 도메인으로 구성된다. 특히, α사슬의 세포외 도메인에 IgE가 결합하게 된다.The FcεRI is a membrane protein composed of one α-chain, one β-chain, and two γ-chains bonded by disulfide bonds. Of these, the part to which IgE binds is an α chain (FcεRIα), and FcεRIα has a size of about 60 kDa, and is composed of a hydrophobic domain present in the cell membrane and a hydrophilic domain present outside the cell membrane. In particular, IgE binds to the extracellular domain of the α chain.
구체적으로, 상기 IgE Fc 수용체의 알파 서브유닛은 NP_001992.1에 기재된 아미노산 서열을 가질 수 있다. 또한, IgE Fc 수용체의 알파 서브유닛의 세포외 도메인(FcεRIa-ECD)은 서열번호 2의 아미노산 서열을 가질 수 있다. 본 명세서에서 IgE Fc 수용체의 알파 서브유닛의 세포외 도메인은 IgE와 결합을 할 수 있는 한, IgE Fc 수용체의 알파 서브유닛의 세포외 도메인의 단편 또는 변이체일 수 있다.Specifically, the alpha subunit of the IgE Fc receptor may have the amino acid sequence described in NP_001992.1. In addition, the extracellular domain (FcεRIa-ECD) of the alpha subunit of the IgE Fc receptor may have the amino acid sequence of SEQ ID NO: 2. In the present specification, the extracellular domain of the alpha subunit of the IgE Fc receptor may be a fragment or variant of the extracellular domain of the alpha subunit of the IgE Fc receptor as long as it can bind to IgE.
상기 변이체는 FcεRⅠ의 α사슬의 기능을 변형시키지 않는 한, 야생형 FcεRIa-ECD(Extracellular domain)에서 하나 이상의 단백질을 치환, 결실 또는 추가하는 방법을 통하여 수행될 수 있다. 이러한 다양한 단백질 또는 펩타이드는 서열번호 2의 아미노산 서열과 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 또는 99% 이상 동일한 것일 수 있다.The variant may be performed through a method of substituting, deleting or adding one or more proteins in the wild-type FcεRIa-ECD (Extracellular domain), as long as the function of the α chain of FcεRI is not modified. These various proteins or peptides may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to the amino acid sequence of SEQ ID NO: 2.
상기 항체의 Fc 영역은 IgA, IgG, IgM, IgD 또는 IgE의 Fc 영역 또는 이들의 도메인들 예컨대 힌지, CH2, CH3의 전부 또는 일부가 혼합된 하이브리드 Fc일 수 있고, 상기 IgG는 IgG1, IgG2, IgG3, 또는 IgG4일 수 있으며, 특히, Fc 감마 수용체(FcγRc) 및 보체(C1a)에 대한 친화도가 낮도록, Fc의 효과기(effector) 기능인 항체-의존성 세포독성(antibody-dependent cell-mediated cytotoxicity, ADCC) 또는 보체-의존성 세포독성(complement-dependent cytotoxicity, CDC)을 담당하는 기능기 부분에 변이가 유도된 변이체 Fc 및/또는 신생아 Fc 수용체(neonatal Fc receptor, FcRn)에 대한 선택적 친화도가 높아서 혈중반감기가 증가될 수 있도록 변이된 변이체 Fc일 수 있다. 이 중, 상기 하이브리드 Fc는 대한민국 특허 제897938호, 제1380732호, 제1380729호 등에 기재된 것을 사용할 수 있고, 상기 변이체 Fc는 국제특허출원 PCT/KR2020/006346에 기재된 변형 면역글로불린 Fc 단백질(NTIG)일 수 있다. 더욱 구체적으로 상기 Fc 영역은 서열번호 4 내지 7 중 어느 하나의 아미노산 서열로 구성될 수 있다. 아울러, 상기 Fc 영역은 인간으로부터 유래한 것일 수 있다. 아울러, 상기 융합단백질은 융합 파트너 사이에 하나 이상의 링커 펩타이드가 삽입될 수 있다. 상기 링커 펩타이드는 상술한 바와 같다. 특히, 상기 FcεRIα 단백질 및 항체의 Fc 영역 사이를 연결하는 링커 펩타이드는 항체 유래의 힌지 부위, 합성 링커 펩타이드 또는 이들이 혼용된 것일 수 있다. The Fc region of the antibody may be a hybrid Fc in which all or part of the Fc region of IgA, IgG, IgM, IgD or IgE or domains thereof such as hinge, CH2, CH3 is mixed, and the IgG is IgG1, IgG2, IgG3 , Or IgG4, in particular, so that the affinity for the Fc gamma receptor (FcγRc) and complement (C1a) is low, the effector function of the Fc antibody-dependent cytotoxicity (antibody-dependent cell-mediated cytotoxicity, ADCC) ) Or complement-dependent cytotoxicity (complement-dependent cytotoxicity (CDC)) in which the mutation is induced in the part of the functional group responsible for the half-life of the blood due to high selective affinity for the Fc and/or neonatal Fc receptor (FcRn). It may be a mutant Fc that has been mutated so that is increased. Among these, the hybrid Fc may be those described in Korean Patent Nos. 897938, 1380732, 1380729, etc., and the variant Fc is a modified immunoglobulin Fc protein (NTIG) described in International Patent Application PCT/KR2020/006346 I can. More specifically, the Fc region may be composed of an amino acid sequence of any one of SEQ ID NOs: 4 to 7. In addition, the Fc region may be derived from human. In addition, in the fusion protein, one or more linker peptides may be inserted between fusion partners. The linker peptide is as described above. In particular, the linker peptide connecting the FcεRIα protein and the Fc region of the antibody may be a hinge region derived from an antibody, a synthetic linker peptide, or a mixture thereof.
본 문서에서 사용되는 용어 'NTIG'는 서열번호 4 및 5로 구성되는 군으로부터 선택되는 변형 Fc 도메인 단백질의 18번째 및 196번째 아미노산이 다른 아미노산으로 변이가 되어 ADCC 및 CDC와 같은 효과기 기능이 없으면서도 FcRn에 대한 선택적 친화도가 증가하여 혈중 반감기가 개선된 변형 Fc 도메인 단백질을 의미한다. 이러한, 상기 NTIG는 서열번호 6 및 7로 구성되는 군으로부터 선택되는 아미노산 서열을 포함할 수 있다.The term'NTIG' as used in this document means that the 18th and 196th amino acids of the modified Fc domain protein selected from the group consisting of SEQ ID NOs: 4 and 5 are mutated to other amino acids, and there is no effector function such as ADCC and CDC. It refers to a modified Fc domain protein with improved half-life in blood by increasing selective affinity for FcRn. Such, the NTIG may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 7.
상기 융합단백질에 있어서, 상기 IL-10 단백질은 UniProtKB P22301에 기재된 아미노산 서열에서 신호서열이 제거된 19-178번째 아미노산 서열을 포함하는 성숙형(mature form)일 수 있고, 상기 성숙형을 기준으로의 87번째 아미노산인 이소류신이 알라닌으로 치환된 IL-10 변이체 단백질일 수 있으며, 116번째 아미노산인 아스파라긴 및 117번째 아미노산인 라이신 사이에 6 내지 12 a.a.의 길이를 갖는 링커 펩타이드가 삽입된 단량체성 IL-10 변이체 단백질일 수 있으며, 상기 IL-10 단백질은 서열번호 10 내지 13으로 구성되는 군으로부터 선택되는 아미노산 서열로 구성될 수 있다.In the fusion protein, the IL-10 protein may be a mature form including an amino acid sequence 19-178 from which a signal sequence has been removed from the amino acid sequence described in UniProtKB P22301, based on the mature form. The 87th amino acid isoleucine may be an IL-10 variant protein substituted with alanine, and a linker peptide having a length of 6 to 12 aa is inserted between the 116th amino acid asparagine and the 117th amino acid lysine. It may be a variant protein, and the IL-10 protein may be composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 13.
본 문서에서 사용되는 용어 "성숙형(mature form)"은 번역에 의해 생산된 단백질에서 분비에 사용되는 신호서열이 절단되거나, 프리펩타이드(prepeptide) 또는 프로펩타이드(propeptide)와 같이 불활성 전구체 단백질이 단백질 절단효소 등에 의해 절단됨으로써 생성되는 활성형 단백질을 의미한다.As used herein, the term "mature form" means that a signal sequence used for secretion from a protein produced by translation is cleaved, or an inactive precursor protein such as a prepeptide or propeptide is a protein. It means an active protein produced by cleavage by a cleavage enzyme or the like.
상기 융합단백질에 있어서, 상기 FcεRIα 단백질 및 IL-10 단백질은 포유동물 유래의 것이라면 어느 것이라도 사용 가능하고, 동일 또는 유사한 기능을 할 수 있다면 어떠한 변이체라도 사용 가능하며, 더욱 바람직하게는 인간 유래의 단백질이다.In the fusion protein, the FcεRIα protein and IL-10 protein can be used as long as they are derived from mammals, and any variant can be used as long as they can perform the same or similar function, and more preferably human-derived proteins. to be.
본 발명의 다른 일 관점에 따르면, 상기 융합단백질을 암호화하는 폴리뉴클레오타이드가 제공된다.According to another aspect of the present invention, a polynucleotide encoding the fusion protein is provided.
본 발명의 다른 일 관점에 따르면, 상기 폴리뉴클레오타이드를 포함하는 재조합 벡터가 제공된다.According to another aspect of the present invention, a recombinant vector comprising the polynucleotide is provided.
상기 재조합 벡터에 있어서, 상기 폴리뉴클레오타이드는 조절서열에 작동가능하게 연결된 유전자 컨스트럭트 형태로 포함될 수 있다.In the recombinant vector, the polynucleotide may be included in the form of a gene construct operably linked to a regulatory sequence.
본 문서에서 사용되는 용어 "작동 가능하게 연결된(operably linked to)"이란 목적으로 하는 핵산서열(예컨대, 시험관내 전사/번역 시스템에서 또는 숙주세포에서)이 그의 발현이 이루어질 수 있도록 하는 방식으로 상기 조절서열에 연결되어 있다는 것을 의미한다.As used herein, the term "operably linked to" is regulated in such a way that the target nucleic acid sequence (eg, in an in vitro transcription/translation system or in a host cell) can be expressed. It means that it is linked to a sequence.
상기 "조절서열"이란 용어는 프로모터, 인핸서 및 다른 조절 요소(예, 폴리아데닐화 신호)를 포함하는 의미이다. 조절서열에는 많은 숙주세포에서 목적으로 하는 핵산이 항상적으로 발현될 수 있도록 지시하는 것, 특정한 조직세포에서만 목적으로 하는 핵산이 발현될 수 있도록 지시하는 것(예, 조직특이적 조절서열), 그리고 특정 신호에 의해 발현이 유도되도록 지시하는 것(예, 유도성 조절서열)이 포함된다. 발현벡터의 설계는 형질전환될 숙주세포의 선택 및 원하는 단백질 발현의 수준 등과 같은 인자에 따라 달라질 수 있다는 것은 당업자라면 이해할 수 있다. 본 발명의 발현벡터는 숙주 세포에 도입되어 상기 융합 단백질을 발현할 수 있다. 상기 진핵세포 및 원핵세포에서 발현을 가능하게 하는 조절서열들은 당업자에게 잘 알려져 있다. 상술한 바와 같이, 이들은 보통 전사개시를 담당하는 조절서열들 및, 선택적으로 전사물의 전사종결 및 안정화를 담당하는 폴리-A 신호를 포함한다. 추가적인 조절서열들은 전사조절인자 외에도 번역 증진인자 및/또는 천연-조합 또는 이종성 프로모터 영역을 포함할 수 있다. 예를 들어 포유류 숙주 세포에서 발현을 가능하게 하는 가능한 조절서열들은 CMV-HSV 티미딘 키나아제 프로모터, SV40, RSV-프로모터(로우스 육종 바이러스), 인간 신장 요소 1α-프로모터, 글루코코르티코이드-유도성 MMTV-프로모터(몰로니 마우스 종양 바이러스), 메탈로티오네인-유도성 또는 테트라사이클린-유도성 프로모터 또는, CMV 증폭제 또는 SV40-증폭제와 같은 증폭제를 포함한다. 신경 세포 내 발현을 위해, 신경미세섬유-프로모터(neurofilament-promoter), PGDF-프로모터, NSE-프로모터, PrP-프로모터 또는 thy-1-프로모터들이 사용될 수 있다는 것이 고려되고 있다. 상기 프로모터들은 당 분야에 알려져 있으며, 문헌(Charron, J. Biol. Chem. 270: 25739-25745, 1995)에 기술되어 있다. 원핵세포내 발현을 위해, lac-프로모터, tac-프로모터 또는 trp 프로모터를 포함하는 다수의 프로모터들이 개시되어 있다. 전사를 개시할 수 있는 인자들 외에, 상기 조절서열들은 본 발명의 일 실시예에 따른 폴리뉴클레오타이드의 하류(downstream)에 SV40-폴리-A 부위 또는 TK-폴리-A 부위와 같은 전사 종결 신호를 포함할 수도 있다. 본 문서에서, 적당한 발현 벡터들은 당 분야에 알려져 있으며, 그 예로는 오카야마-베르그(Okayama-Berg) cDNA 발현 벡터 pcDV1(Parmacia), pRc/CMV, pcDNA1, pcDNA3(Invitrogene), pSPORT1(GIBCO BRL), pGX-27(특허 제1442254호), pX(Pagano et al., Science 255: 1144-1147, 1992), 효모 2-혼성(two-hybrid) 벡터, 가령 pEG202 및 dpJG4-5(Gyuris et al., Cell 75: 791-803, 1995) 또는 원핵 발현 벡터, 가령 람다 gt11 또는 pGEX(Amersham Pharmacia)가 있다. 본 발명의 핵산 분자들 외에, 벡터는 분비 신호를 암호화하는 폴리뉴클레오타이드를 추가로 포함할 수 있다. 상기 분비신호들은 당업자에게 잘 알려져 있다. 그리고, 사용된 발현 시스템에 따라, 융합단백질을 세포 구획으로 이끌 수 있는 리더서열(leader sequence)이 본 발명의 일 실시예에 따른 폴리뉴클레오타이드의 코딩 서열에 조합되며, 바람직하게는 해독된 단백질 또는 이의 단백질을 세포질 주변 또는 세포외 매질로 직접 분비할 수 있는 리더 서열이다. 본 발명의 일 실시예에서는 서열번호 1로 기재되는 tPA의 신호서열이 사용되었으나, 이로 제한되는 것은 아니며, 상기 신호서열은 단백질의 생산 과정에서 절단되어 제거되기 때문에, 본 발명의 일 실시예에 따른 융합단백질 자체의 구성요소는 아니다.The term "regulatory sequence" is meant to include promoters, enhancers and other regulatory elements (eg, polyadenylation signals). The regulatory sequences are those that direct the expression of the nucleic acid of interest in many host cells, and that the nucleic acid of interest can be expressed only in specific tissue cells (e.g., tissue-specific regulatory sequences), and It includes directing expression to be induced by a specific signal (eg, an inducible regulatory sequence). It can be understood by those skilled in the art that the design of the expression vector may vary depending on factors such as the selection of host cells to be transformed and the level of desired protein expression. The expression vector of the present invention can be introduced into a host cell to express the fusion protein. Regulatory sequences that enable expression in eukaryotic and prokaryotic cells are well known to those skilled in the art. As mentioned above, these usually contain regulatory sequences responsible for initiation of transcription and, optionally, a poly-A signal responsible for transcription termination and stabilization of the transcript. Additional regulatory sequences may include, in addition to transcriptional regulators, translation enhancers and/or naturally-combined or heterologous promoter regions. For example, possible regulatory sequences that allow expression in mammalian host cells include CMV-HSV thymidine kinase promoter, SV40, RSV-promoter (Rous sarcoma virus), human kidney element 1α-promoter, glucocorticoid-inducible MMTV- Promoter (Moloni mouse tumor virus), a metallothionein-inducible or tetracycline-inducible promoter, or an amplifying agent such as a CMV amplifying agent or an SV40-amplifying agent. For expression in neurons, it is contemplated that neurofibrillation-promoter, PGDF-promoter, NSE-promoter, PrP-promoter or thy-1-promoter can be used. Such promoters are known in the art and are described in Charron, J. Biol. Chem. 270: 25739-25745, 1995. For expression in prokaryotic cells, a number of promoters have been disclosed including the lac-promoter, the tac-promoter or the trp promoter. In addition to factors capable of initiating transcription, the regulatory sequences include transcription termination signals such as SV40-poly-A site or TK-poly-A site downstream of the polynucleotide according to an embodiment of the present invention. You may. In this document, suitable expression vectors are known in the art, examples of which are Okayama-Berg cDNA expression vector pcDV1 (Parmacia), pRc/CMV, pcDNA1, pcDNA3 (Invitrogene), pSPORT1 (GIBCO BRL), pGX-27 (Patent No. 1442254), pX (Pagano et al ., Science 255: 1144-1147, 1992), yeast two-hybrid vectors, such as pEG202 and dpJG4-5 (Gyuris et al ., Cell 75: 791-803, 1995) or a prokaryotic expression vector such as lambda gt11 or pGEX (Amersham Pharmacia). In addition to the nucleic acid molecules of the present invention, the vector may further comprise a polynucleotide encoding a secretion signal. The secretion signals are well known to those skilled in the art. And, depending on the expression system used, a leader sequence that can lead the fusion protein to the cell compartment is combined with the coding sequence of the polynucleotide according to an embodiment of the present invention, and preferably the translated protein or its It is a leader sequence that can secrete proteins directly into the periplasm or extracellular media. In one embodiment of the present invention, the signal sequence of tPA represented by SEQ ID NO: 1 was used, but is not limited thereto, and since the signal sequence is cut and removed during the production of a protein, according to an embodiment of the present invention It is not a component of the fusion protein itself.
또한, 본 발명의 벡터는 예를 들면, 표준 재조합 DNA 기술에 의하여 제조될 수 있으며, 표준 재조합 DNA 기술에는 예를 들면, 평활말단 및 접착말단 라이게이션, 적절한 말단을 제공하기 위한 제한 효소 처리, 부적합한 결합을 방지하기 위하여 알칼리 포스테이즈 처리에 의한 인산기 제거 및 T4 DNA 라이게이즈에 의한 효소적 연결 등이 포함된다. 화학적 합성 또는 유전자 재조합 기술에 의하여 얻어진 신호 펩타이드를 코딩하는 DNA, 본 발명의 변이체 IL-10 단백질 또는 이를 포함하는 융합단백질을 암호화하는 DNA를 적절한 조절서열이 포함되어 있는 벡터에 재조합함으로써 본 발명의 벡터가 제조될 수 있다. 상기 조절 서열이 포함되어 있는 벡터는 상업적으로 구입 또는 제조할 수 있으며, 본 발명의 일 실시예에서는 pBispecific 벡터를 백본으로 사용하였다.In addition, the vector of the present invention may be prepared by, for example, standard recombinant DNA techniques, and standard recombinant DNA techniques include, for example, ligation of blunt ends and adhesive ends, treatment with restriction enzymes to provide appropriate ends, and inappropriate In order to prevent binding, phosphate group removal by alkaline postage treatment and enzymatic linkage by T4 DNA ligase are included. The vector of the present invention by recombining the DNA encoding the signal peptide obtained by chemical synthesis or gene recombination technology, the mutant IL-10 protein of the present invention, or the DNA encoding the fusion protein containing the same into a vector containing an appropriate regulatory sequence. Can be prepared. A vector containing the control sequence can be purchased or manufactured commercially, and in an embodiment of the present invention, a pBispecific vector was used as a backbone.
상기 발현벡터는 분비 신호서열을 암호화하는 폴리뉴클레오타이드를 추가로 포함할 수 있으며, 상기 분비 신호서열은 세포내에서 발현되는 재조합 단백질의 세포 밖으로의 분비를 유도하며, tPA(tissue plasminogen activator) 신호서열(서열번호 1), HSV gDs(단순포진 바이러스 당단백질 Ds) 신호서열 또는 성장호르몬 신호서열일 수 있다.The expression vector may further include a polynucleotide encoding a secretion signal sequence, and the secretion signal sequence induces secretion of a recombinant protein expressed in the cell out of the cell, and a tissue plasminogen activator (tPA) signal sequence ( SEQ ID NO: 1), HSV gDs (herpes simplex virus glycoprotein Ds) signal sequence or growth hormone signal sequence.
본 발명의 일 실시예에 따른 상기 발현벡터는 숙주세포에서 상기 단백질을 발현하도록 할 수 있는 발현벡터일 수 있으며, 상기 발현벡터는 플라스미드 벡터, 바이러스 벡터, 코스미드 벡터, 파지미드 벡터, 인공 인간 염색체 등 그 어떠한 형태를 나타내더라도 무방하다.The expression vector according to an embodiment of the present invention may be an expression vector capable of expressing the protein in a host cell, and the expression vector is a plasmid vector, a virus vector, a cosmid vector, a phagemid vector, an artificial human chromosome It can be any form such as.
본 발명의 다른 일 관점에 따르면, 상기 융합단백질을 유효성분으로 포함하는 알러지성 질환 치료용 약학적 조성물이 제공된다.According to another aspect of the present invention, there is provided a pharmaceutical composition for the treatment of allergic diseases comprising the fusion protein as an active ingredient.
상기 약학적 조성물은, 공지의 알러지 치료제 성분을 추가로 포함하거나 병용하여 사용될 수 있다. 이러한 공지의 알러지 치료제로는 항히스타민제, 항류코트리엔제, 및 스테로이드제가 포함될 수 있다. 상기 항히스타민제는 클로르페니라민(chlorpheniramine), 트리프롤리딘(triprolidine), 피프린히드리네이트(piprinhydrinate), 히드록시진(hydroxyzine), 메퀴타진(mequitazine), 디펜히드라민(diphenhydramine), 디멘히드리네이트(dimenhydrinate), 메클리진(meclizine), 세티리진(cetirizine), 로라타딘(loratadine), 레보세티리진(levocetirizine), 에바스틴(ebastine), 케토티펜(ketotifen), 펙소페나딘(fexofenadine), 아젤라스틴(azelastine), 데스로라타딘(desloratadine), 베포타스틴(bepotastine), 올로파타딘(olopatadine), 에메다스틴(emedastine), 에피나스틴(epinastine), 또는 레보카바스틴(levocabastine)일 수 있다. 상기 항류코트리엔제는 몬테루카스트(motelukast), 프란루카스트(pranlukast), 또는 자피르무카스트(zafirlukast)일 수 있다. 상기 스테로이드제제는 코르티솔, 클루코코르티코이드, 프레드니손, 프레드니솔론, 메틸프레드니솔론, 트리암시놀론, 덱사메타손, 데소니드, 또는 코르티코스테론일 수 있다.The pharmaceutical composition may further contain or be used in combination with a known allergy treatment component. Such known allergy treatments may include antihistamines, antileukotrienes, and steroids. The antihistamines are chlorpheniramine, triprolidine, piprinhydrinate, hydroxyzine, mequitazine, diphenhydramine, dimenhydrin Dimenhydrinate, meclizine, cetirizine, loratadine, levocetirizine, ebastine, ketotifen, fexofenadine, azela May be azelastine, desloratadine, bepotastine, olopatadine, emedastine, epinastine, or levocabastine . The anti-leukotriene agent may be montelukast, pranlukast, or zafirlukast. The steroid preparation may be cortisol, glucocorticoid, prednisone, prednisolone, methylprednisolone, triamcinolone, dexamethasone, desonide, or corticosterone.
상기 약학적 조성물에 있어서, 상기 알러지 질환은 알러지성 비염, 알러지성 기관지 천식, 아토피성 피부염, 알러지성 결막염, 만성 특발성 두드러기, 약물 알러지, 식품 알러지 또는 아나필락시스일 수 있다. 특히, 상기 알러지 질환은 IgE 매개성일 수 있다.In the pharmaceutical composition, the allergic disease may be allergic rhinitis, allergic bronchial asthma, atopic dermatitis, allergic conjunctivitis, chronic idiopathic urticaria, drug allergy, food allergy or anaphylaxis. In particular, the allergic disease may be IgE mediated.
상기 조성물은 악학적으로 허용 가능한 담체를 포함할 수 있고, 상기 담체 외에 약학적으로 허용가능한 보조제, 부형제 또는 희석제를 추가적으로 포함할 수 있다.The composition may include a badly acceptable carrier, and may additionally include a pharmaceutically acceptable adjuvant, excipient, or diluent in addition to the carrier.
본 문서에서 사용되는 용어 "약학적으로 허용 가능한"이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 위장 장애, 현기증과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 조성물을 말한다. 상기 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 또한, 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다. The term "pharmaceutically acceptable" as used herein refers to a composition that is physiologically acceptable and, when administered to a human, does not usually cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils. In addition, fillers, anti-aggregating agents, lubricants, wetting agents, flavoring agents, emulsifying agents and preservatives may additionally be included.
또한, 본 발명의 일 실시예에 따른 약학적 조성물은 포유동물에 투여 시, 활성 성분의 신속한 방출, 또는 지속 또는 지연된 방출이 가능하도록 당업계에 공지된 방법을 사용하여 제형화될 수 있다. 제형은 분말, 과립, 정제, 에멀젼, 시럽, 에어로졸, 연질 또는 경질 젤라틴 캅셀, 멸균 주사용액, 멸균 분말 형태를 포함한다. In addition, the pharmaceutical composition according to an embodiment of the present invention may be formulated using a method known in the art to enable rapid release, or sustained or delayed release of the active ingredient when administered to a mammal. Formulations include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, and sterile powder forms.
본 발명의 일 실시예에 따른 조성물은 다양한 경로로 투여될 수 있으며, 예를 들면, 경구, 비경구, 예를 들면 좌제, 경피, 정맥, 복강, 근육내, 병변내, 비강, 척추관내 투여로 투여될 수 있으며, 또한 서방형 또는 연속적 또는 반복적 방출을 위한 이식 장치를 사용하여 투여될 수 있다. 투여횟수는 원하는 범위 내에서 하루에 1회, 또는 수회로 나누어 투여할 수 있으며, 투여 기간도 특별히 한정되지 않는다. The composition according to an embodiment of the present invention may be administered by various routes, for example, oral, parenteral, for example, suppository, transdermal, intravenous, intraperitoneal, intramuscular, intralesional, nasal, intrathecal administration It can be administered, and can also be administered using an implanted device for sustained release or continuous or repeated release. The frequency of administration may be administered once a day or divided into several times a day within a desired range, and the administration period is not particularly limited.
본 발명의 일 실시예에 따른 조성물은 일반적으로 사용되는 약학적으로 허용가능한 담체와 함께 적합한 형태로 제형화될 수 있다. 약학적으로 허용되는 담체로는 예를 들면, 물, 적합한 오일, 식염수, 수성 글루코스 및 글리콜 등과 같은 비경구 투여용 담체 등이 있으며 안정화제 및 보존제를 추가로 포함할 수 있다. 적합한 안정화제로는 아황산수소나트륨, 아황산나트륨 또는 아스코르브산과 같은 항산화제가 있다. 적합한 보존제로는 벤즈알코늄 클로라이드, 메틸- 또는 프로필-파라벤 및 클로로부탄올이 있다. 또한 본 발명에 따른 조성물은 그 투여방법이나 제형에 따라 필요한 경우, 현탁제, 용해보조제, 안정화제, 등장화제, 보존제, 흡착방지제, 계면활성화제, 희석제, 부형제, pH 조정제, 무통화제, 완충제, 산화방지제 등을 적절히 포함할 수 있다. 상기에 예시된 것들을 비롯하여 본 발명에 적합한 약학적으로 허용되는 담체 및 제제는 문헌[Remington's Pharmaceutical Sciences, 최신판]에 상세히 기재되어 있다. The composition according to an embodiment of the present invention may be formulated in a suitable form together with a generally used pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, carriers for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycol, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. In addition, the composition according to the present invention is a suspension agent, a solubilizer, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, a painless agent, a buffer agent, Antioxidants and the like may be appropriately included. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, the latest edition.
상기 조성물의 환자에 대한 투여량은 환자의 신장, 체표면적, 연령, 투여되는 특정 화합물, 성별, 투여 시간 및 경로, 일반적인 건강, 및 동시에 투여되는 다른 약물들을 포함하는 많은 요소들에 따라 다르다. 약학적으로 활성인 단백질은 100 ng/체중(kg) - 10 ㎎/체중(㎏)의 양으로 투여될 수 있고, 더 바람직하게는 1 내지 500 ㎍/kg(체중)으로 투여될 수 있으며, 가장 바람직하게는 5 내지 50 ㎍/kg(체중)으로 투여될 수 있는데, 상기 요소들을 고려하여 투여량이 조절될 수 있다.The dosage of the composition to a patient depends on many factors, including the patient's height, body surface area, age, the specific compound administered, sex, time and route of administration, general health, and other drugs administered simultaneously. The pharmaceutically active protein may be administered in an amount of 100 ng/body weight (kg)-10 mg/body weight (kg), more preferably 1 to 500 μg/kg (body weight). Preferably, it may be administered at 5 to 50 μg/kg (body weight), and the dosage may be adjusted in consideration of the above factors.
본 발명의 다른 일 관점에 따르면, 치료적으로 유효한 양의 a) FcεRIα 단백질 및 IL-10 단백질; 및/또는 b) FcεRIα 단백질 및 IL-10 단백질을 포함하는 융합단백질을 개체에 투여하는 단계를 포함하는 상기 개체의 알러지 치료방법이 제공된다.According to another aspect of the present invention, a therapeutically effective amount of a) FcεRIα protein and IL-10 protein; And/or b) administering a fusion protein including an FcεRIα protein and an IL-10 protein to the individual.
상기 치료방법에 있어서, 상기 개체는 인간 또는 비인간 포유동물일 수 있으며, 상기 비인간 포유동물은 영장류, 식육목, 우제목, 설치목, 또는 기제목에 속하는 동물일 수 있다.In the treatment method, the individual may be a human or non-human mammal, and the non-human mammal may be an animal belonging to a primate, a carnivorous tree, a right tree, a rodent tree, or a base tree.
본 문서에서 사용되는 용어 "치료적으로 유효한 양(therapeutically effective amount)"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물의 치료적으로 유효한 양은 0.1 mg/kg 내지 1 g/kg, 더 바람직하게는 1 mg/kg 내지 500 mg/kg일 수 있으나, 유효 투여량은 환자의 나이, 성별 및 상태에 따라 적절히 조절될 수 있다.As used herein, the term "therapeutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type and severity of the subject, Age, sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including concurrent drugs and other factors well known in the medical field. The therapeutically effective amount of the composition of the present invention may be 0.1 mg/kg to 1 g/kg, more preferably 1 mg/kg to 500 mg/kg, but the effective dosage may be depending on the age, sex and condition of the patient. Can be adjusted appropriately.
이하, 실시예 및 실험예를 통하여 본 발명을 더 상세히 설명한다. 그러나 본 발명은 이하에서 개시되는 실시예 및 실험예에 한정되는 것이 아니라 서로 다른 다양한 형태로 구현될 수 있는 것으로, 이하의 실시예 및 실험예는 본 발명의 개시가 완전하도록 하며, 본 발명이 속한 기술분야의 통상의 지식을 가진 자에게 발명의 범주를 완전하게 알려주기 위해 제공되는 것이다. Hereinafter, the present invention will be described in more detail through examples and experimental examples. However, the present invention is not limited to the embodiments and experimental examples disclosed below, but may be implemented in various different forms, and the following examples and experimental examples are intended to complete the disclosure of the present invention, and to which the present invention belongs. It is provided to fully inform the scope of the invention to those of ordinary skill in the art.
실시예 1 내지 3: FcεRIα-NTIG-IL-10Vm의 제조Examples 1 to 3: Preparation of FcεRIα-NTIG-IL-10Vm
본 발명자들은 IgE와 특이적으로 결합하는 수용체인 FcεRIα와 이량체성 IL-10 변이체 단백질(IL-10V or IL-10M) 또는 단량체성 IL-10 변이체 단백질(IL-10Vm)을 변형된 Fc 영역과 각각 또는 Fc 영역을 사이에 두고 양쪽으로 연결된 융합단백질을 고안하였다.The present inventors used FcεRIα, a receptor that specifically binds to IgE, and a dimeric IL-10 variant protein (IL-10V or IL-10M) or a monomeric IL-10 variant protein (IL-10Vm) with a modified Fc region, respectively. Alternatively, a fusion protein linked to both sides with an Fc region interposed was designed.
구체적으로, 본 발명자들은 하기 표 1과 같은 구성을 갖는 융합단백질을 암호화하는 폴리뉴클레오타이드를 올리고뉴클레오타이드 합성 및 PCR을 이용하여 제조한 후, 이를 pBispecific 발현벡터(제넥신, 대한민국)에 삽입한 후, ExpiCHO kit(Thermo Fisher, USA)를 이용하여 ExpiCHO-S 세포에 상기 발현벡터를 형질감염시킨 후 8% CO2, 37℃ incubator 에서 1일 동안 배양한 후, 온도를 32℃로 낮추어 7일 동안 추가 배양하였다. 그런 다음, Protein A 친화성 크로마토그래피를 이용한 정제를 수행하였고, SDS-FAGE를 통해 후보물질이 정제되었는지 확인하였으며, 후보물질의 pI 값에 맞게 제형화 완충액을 제조하여 제형화하였다. 제형화가 완료된 물질을 Nanodrop을 이용하여 정량하고 SE-HPLC를 이용하여 순도를 확인하였다.Specifically, the present inventors prepared a polynucleotide encoding a fusion protein having the configuration shown in Table 1 below using oligonucleotide synthesis and PCR, and then inserted it into a pBispecific expression vector (Genexin, Korea), and then ExpiCHO After transfecting the expression vector into ExpiCHO-S cells using a kit (Thermo Fisher, USA), incubating for 1 day in an 8% CO 2 , 37°C incubator, lowering the temperature to 32°C and further incubating for 7 days I did. Then, purification using Protein A affinity chromatography was performed, it was confirmed that the candidate material was purified through SDS-FAGE, and a formulation buffer was prepared and formulated according to the pI value of the candidate material. The formulated material was quantified using Nanodrop, and purity was confirmed using SE-HPLC.
구성요소Component 실시예 1Example 1 실시예 2Example 2 실시예 3Example 3
신호서열Signal sequence 서열번호 1SEQ ID NO: 1 서열번호 1SEQ ID NO: 1 서열번호 1SEQ ID NO: 1
FcεRIαFcεRIα 서열번호 2SEQ ID NO: 2 서열번호 2SEQ ID NO: 2 서열번호 2SEQ ID NO: 2
링커 1 Linker 1 서열번호 3SEQ ID NO: 3 서열번호 3SEQ ID NO: 3 서열번호 3SEQ ID NO: 3
NTIGNTIG 서열번호 6SEQ ID NO: 6 서열번호 6SEQ ID NO: 6 서열번호 6SEQ ID NO: 6
링커 2 Linker 2 서열번호 8SEQ ID NO: 8 서열번호 9SEQ ID NO: 9 서열번호 9SEQ ID NO: 9
IL-10VmIL-10Vm 서열번호 10SEQ ID NO: 10 서열번호 11SEQ ID NO: 11 서열번호 13SEQ ID NO: 13
상기 표 1에서 사용된 변형된 Fc는 도 1에 도시된 바와 같이, IgG1의 힌지부분과 IgD 및 IgG4의 CH2 및 CH3가 혼합된 형태의 Fc로서 본 발명자들은 이를 "NTIG"로 명명하였고, 이를 포함하는 융합단백질인 FcεRIα-NTIG-IL-10Vm을 "PG110"로 명명하였다. 상기 실시예 1과 2는 NTIG와 IL-10Vm을 연결하는 링커 2(서열번호 8 및 9)와 IL-10Vm의 내부에 삽입된 링커(스페이서) 펩타이드 부분에서만 차이가 난다(서열번호 10 및 11). 실시예 2에서 사용된 IL-10Vm은 종래 보고된 단량체성 IL-10 변이체 단백질과 동일한 것이다(Josephson et al., J. Biol. Chem. 275(18): 13552-13557, 2000). 이에, 실시예 1의 경우 "PG110-1", 실시예 2의 경우 "PG110-2"로 명명하였다. 상기 NTIG는 서열번호 4로 기재되는 아미노산 서열을 갖는 변이 Fc 도메인단백질의 18번째 아미노산인 트레오닌(T)이 글루타민(Q)으로 변이되고, 196번째 아미노산인 메티오닌(M)이 류신(L)으로 변이된 서열번호 6으로 표시되는 아미노산 서열을 갖는 것을 사용하였다.그 결과, 도 2a 및 2b에서 확인되는 바와 같이, 실시예 1의 PG-110-1의 경우 10번 분획에서 93.9%라는 고순도를 달성하여 이를 제형화한 후 향후 IL-10 활성 분석에 사용하였고, 그보다 다소 낮은 순도를 나타낸 11번 분획(A11) 및 12번 분획(A12)는 병합하여 2차 정제를 진행하였다.As shown in Figure 1, the modified Fc used in Table 1 is an Fc in which the hinge portion of IgG1 and CH2 and CH3 of IgD and IgG4 are mixed, and the present inventors named it "NTIG", including The fusion protein, FcεRIα-NTIG-IL-10Vm, was named "PG110". Examples 1 and 2 differ only in the linker 2 (SEQ ID NOs: 8 and 9) connecting NTIG and IL-10Vm and the linker (spacer) peptide portion inserted into the interior of IL-10Vm (SEQ ID NOs: 10 and 11). . IL-10Vm used in Example 2 is the same as the previously reported monomeric IL-10 variant protein (Josephson et al ., J. Biol. Chem . 275(18): 13552-13557, 2000). Accordingly, in the case of Example 1, it was named "PG110-1", and in the case of Example 2, it was named "PG110-2". In the NTIG, threonine (T), the 18th amino acid of the mutant Fc domain protein having the amino acid sequence shown in SEQ ID NO: 4, is mutated to glutamine (Q), and methionine (M), which is the 196th amino acid, is mutated to leucine (L). As a result, as shown in Figs. 2a and 2b, PG-110-1 of Example 1 achieved a high purity of 93.9% in fraction 10. After formulating this, it was used for the analysis of IL-10 activity in the future, and fractions 11 (A11) and 12 (A12) showing somewhat lower purity were combined to perform secondary purification.
아울러, 실시예 2의 PG-110-2의 경우, 도 2c 및 2d에서 확인되는 바와 같이, 실시예 1보다 생산되는 양이 적을 뿐 아니라 다소 낮은 순도인 65 내지 75%의 순도를 나타냈다.In addition, in the case of PG-110-2 of Example 2, as shown in FIGS. 2C and 2D, the amount produced was less than that of Example 1, and a purity of 65 to 75% was exhibited, which is a somewhat lower purity.
실시예 4: FcεRIα-NTIG-IL-10Vm 발현 안정적 세포주(stable cell line) 확립Example 4: Establishment of a stable cell line expressing FcεRIα-NTIG-IL-10Vm
본 발명자들은 이어 상기 실시예 1의 PG110-1을 안정적으로 발현할 수 있는 안정적 세포주(stable cell line)을 구축하였다.The present inventors then constructed a stable cell line capable of stably expressing PG110-1 of Example 1 above.
구체적으로, 상기에서 구축된 FcεRIα-NTIG-IL-10Vm 융합단백질을 암호화하는 유전자 컨스트럭트를 포함하는 발현벡터 pAD15 발현벡터(WO2015/009052A)를 CHO DG44(from Dr. Chasin, Columbia University, USA)세포에 Neonㄾ transfection system을 이용하여 형질주입하였다. 1차 스크리닝 과정으로 HT(5-hydroxytrypamine)가 없는 10% dFBS(Gibco, USA, 30067-334), MEMα(Gibco, 12561, USA, Cat No. 12561-049), HT+(Gibco, USA, 11067-030) 배지를 사용하여 HT 선별을 수행하였다. 이후, DHFR(dihydrofolate reductase)-시스템을 이용하여 생산성을 증폭시키기 위해, HT 선별된 클론들을 이용하여 메토트렉세이트(MTX) 증폭을 수행하였다. 높은 생산성의 클론을 확보하기 위해 플레이트 상에서 미니 풀의 형태로 MTX 증폭을 수행하였으며 증폭이 확인된 1종에 대해 선별한 후, LDC(Limiting Dilution Cloning; 96 wells, 30 plates)를 진행하여 최종 세포주를 확보하였다.Specifically, the expression vector pAD15 expression vector (WO2015/009052A) containing the gene construct encoding the FcεRIα-NTIG-IL-10Vm fusion protein constructed above was converted into CHO DG44 (from Dr. Chasin, Columbia University, USA). transfected using the transfection system Neon ㄾ the cells were injected. As a first screening process, 10% dFBS (Gibco, USA, 30067-334) without HT (5-hydroxytrypamine), MEMα (Gibco, 12561, USA, Cat No. 12561-049), HT+ (Gibco, USA, 11067- 030) medium was used to perform HT selection. Thereafter, in order to amplify productivity using the DHFR (dihydrofolate reductase)-system, methotrexate (MTX) amplification was performed using HT-selected clones. In order to secure high-productivity clones, MTX amplification was performed in the form of a mini-pool on a plate, and after selecting one type of amplification confirmed, LDC (Limiting Dilution Cloning; 96 wells, 30 plates) was performed to obtain the final cell line. Secured.
최종 확보된 세포주를 hyCellCHO 배지에서 700 ml 배치 배양 수행하였으며 배양 5일차에 수득된 배양액에 대해 Protein A 정제를 수행하여 정제된 단백질을 확인하였으며, 정제된 단백질은 SDS-PAGE 및 SE-HPLC를 통해 양 및 순도를 분석하였다. The finally obtained cell line was subjected to 700 ml batch culture in hyCellCHO medium, and Protein A purification was performed on the culture medium obtained on the 5th day of culture to confirm the purified protein, and the purified protein was obtained through SDS-PAGE and SE-HPLC. And the purity was analyzed.
그 결과, 도 3a 및 3b에서 확인되는 바와 같이, 확립된 안정적 세포주로부터 본 발명의 일 실시예에 따른 융합 단백질이 정상적으로 발현이 됨을 알 수 있었다(순도 95.3%). As a result, as shown in FIGS. 3a and 3b, it was found that the fusion protein according to an embodiment of the present invention was normally expressed from the established stable cell line (purity 95.3%).
실험예 1: IgE 결합능 분석Experimental Example 1: IgE binding ability analysis
본 발명자들은 대조군인 FcεRIα-Fc 융합단백질(대한민국 공개특허 10-2019-0084885 개시)과 본 발명의 실시예의 융합단백질(FcεRIα-NTIG-IL-10Vm)의 마우스 IgE 및 인간 IgE와의 친화도를 생물층 간섭계 분석(BLI assay)을 통해서 분석하였다. The present inventors determined the affinity of the control group FcεRIα-Fc fusion protein (Korean Patent Laid-Open Publication 10-2019-0084885 disclosed) and the fusion protein (FcεRIα-NTIG-IL-10Vm) of an embodiment of the present invention with mouse IgE and human IgE in the biolayer. It was analyzed through interferometric analysis (BLI assay).
이를 위해 우선 본 발명자들은 Dip and ReadTM Amine Reactive 2nd Generation(AR2G) Reagent Kit(forteBio, Cat No. 18-5092)를 이용하여 96 플레이트에 대조군으로 FcεRIα-Fc 융합단백질 및 실시예 1의 융합단백질을 부착시켰다. 구체적으로, 96웰 플레이트에 D.W. 200 μl를 분주한 후, 상기 키트에 포함된 amine biosensor를 꽂아 10분 동안 수화시켰다. 이어, D.W. 200 μl를 추가로 분주한 후, 필요한 시료 양의 1/20로 EDC:NHS를 1:1로 혼합한 후, D.W.에 희석하여 96웰 플레이트에 200 μl 씩 분주하였다. 이어, FcεRIα-Fc 융합단백질 및 FcεRIα-NTIG-IL-10Vm 융합단백질을 10 mM acetate pH 5.0 용액에 10 μg/ml이 되도록 희석한 후 상기 96웰 플레이트에 200 μl씩 첨가하였다. 이후, 1 M 에탄올아민을 200 μl 씩 96웰 플레이트에 첨가한 후, Biosensor 플레이트와 샘플 플레이트를 Octet　 K2 기기에 넣고 측정하였다. 측정이 끝난 후 샘플 플레이트에 1x Kinetics 완충액을 200 μl를 첨가한 후, 기저값(baseline)을 정하였다. 이후, 상기 실시예에서 항-DNP 마우스 IgE(Sigma)를 다양한 농도(50 pM 내지 3.125 nM)로 1x Kinetic 완충액에 희석한 후 샘플 플레이트에 200 μl로 분주한 후, Octet　 K2 기기로 BLI 분석을 수행함으로써 결합 정도를 측정하였다. To this end, the present inventors first used Dip and Read TM Amine Reactive 2 nd Generation (AR2G) Reagent Kit (forteBio, Cat No. 18-5092) to a 96 plate as a control FcεRIα-Fc fusion protein and the fusion protein of Example 1 Was attached. Specifically, after dispensing 200 μl of DW into a 96-well plate, amine biosensor included in the kit was inserted and hydrated for 10 minutes. Subsequently, 200 μl of DW was additionally dispensed, EDC:NHS was mixed at 1:1 with 1/20 of the required sample amount, diluted in DW, and 200 μl each was dispensed into a 96-well plate. Subsequently, the FcεRIα-Fc fusion protein and the FcεRIα-NTIG-IL-10Vm fusion protein were diluted to 10 μg/ml in 10 mM acetate pH 5.0 solution, and 200 μl each was added to the 96-well plate. Then, 200 μl of 1 M ethanolamine was added to the 96-well plate, and then the Biosensor plate and the sample plate were added to Octet.　It was put into a K2 instrument and measured. After the measurement was completed, 200 μl of 1x Kinetics buffer was added to the sample plate, and a baseline was determined. Thereafter, in the above example, anti-DNP mouse IgE (Sigma) was diluted in 1x Kinetic buffer at various concentrations (50 pM to 3.125 nM), and then dispensed to 200 μl on a sample plate, and then Octet　 The degree of binding was measured by performing BLI analysis with a K2 instrument.
FcεRIα-FcFcεRIα-Fc PG110-1 (FcεRIα-NTIG-IL-10Vm)PG110-1 (FcεRIα-NTIG-IL-10Vm)
1차Primary 2차Secondary 3차3rd 1차Primary 2차Secondary 3차3rd
평균 KD (nM) Average K D (nM) 0.3640.364 0.4780.478 0.9400.940 0.2240.224 0.2820.282 0.3460.346
평균: 0.594Average: 0.594 평균: 0.284Average: 0.284
평균 Kon (1/Ms)Average K on (1/Ms) 1.23x105 1.23x10 5 1.49x 105 1.49x 10 5 1.66x105 1.66x10 5 1.52x105 1.52x10 5 1.44x105 1.44x10 5 1.39x105 1.39x10 5
평균 Kdis (1/s)Average K dis (1/s) 4.48x10-5 4.48x10 -5 7.10x10-5 7.10x10 -5 1.56x10-4 1.56x10 -4 3.40x10-5 3.40x10 -5 4.06x10-5 4.06x10 -5 4.82x10-5 4.82x10 -5
평균 R2 Average R 2 0.9960.996 0.9990.999 0.9970.997 0.9990.999 0.9990.999 0.9990.999
그 결과, 도 4a, 4b 및표 2에서 나타난 바와 같이, 본 발명의 실시예에 따른 융합단백질(FcεRIα-NTIG-IL-10Vm, PG110-1)의 KD 값은 0.284 nM로 대조군인 FcεRIα-Fc 융합단백질보다 다소 낮은 것으로 나타났는데, 이 정도의 차이는 유의한 차이는 아니므로, IL-10 단백질의 부가에 의해 IgE와의 결합능이 떨어지지 않음을 확인할 수 있었다.As a result, as shown in Figures 4a, 4b and Table 2, the K D value of the fusion protein (FcεRIα-NTIG-IL-10Vm, PG110-1) according to the embodiment of the present invention is 0.284 nM, which is the control FcεRIα-Fc fusion. It was found to be somewhat lower than that of the protein, but since the difference in this degree was not a significant difference, it was confirmed that the binding ability with IgE did not decrease by the addition of the IL-10 protein.
상기 결과와 함께, 본 발명에서 사용한 인간 FcεRIα가 인간의 IgE와 결합하는 지 여부를 확인하기 위하여, 본 발명자들은 실시예의 융합단백질(PG110-1)의 인간 IgE와의 결합 친화도를 상술한 방법을 이용하여 분석하였다. In addition to the above results, in order to confirm whether human FcεRIα used in the present invention binds to human IgE, the present inventors used the method described above to determine the binding affinity of the fusion protein (PG110-1) to human IgE of the embodiment. And analyzed.
PG110-1 (FcεRIα-NTIG-IL-10Vm)PG110-1 (FcεRIα-NTIG-IL-10Vm)
평균 KD (nM) Average K D (nM) 0.3460.346
평균 Kon (1/Ms) Average K on (1/Ms) 2.19x105 2.19x10 5
평균 Kdis (1/s)Average K dis (1/s) 7.59x10-5 7.59x10 -5
평균 R2 Average R 2 0.99550.9955
그 결과, 도 4c 및 표 3에서 나타난 바와 같이, 본 발명의 일 실시예에 따른 융합단백질은 인간 IgE와도 상기 마우스 IgE와 유사한 정도의 친화도롤 나타냈다. 실험예 2: 막 IgE를 발현하는 세포(IgE B cell)에 대한 표적화 여부 확인 As a result, as shown in Fig. 4c and Table 3, the fusion protein according to an embodiment of the present invention showed similar degree of affinity to human IgE as well as the mouse IgE. Experimental Example 2: Confirmation of targeting to membrane IgE-expressing cells (IgE B cells)
본 발명자들은 상기 실험예 1의 결과로부터 본 발명의 융합단백질이 막에 IgE를 발현하는 세포, 예컨대 IgE B cell과 같은 세포를 표적할 수 있는지 여부를 조사하였다.The present inventors investigated whether the fusion protein of the present invention can target cells expressing IgE on the membrane, such as IgE B cells, from the results of Experimental Example 1 above.
이를 위해 구체적으로 본 발명자들은 막 IgE를 발현하는 것으로 알려진 U266B1 세포주를 배양한 뒤 Live-dead 염색을 20분간 수행하고, 세척을 통해 잔류 염색 시약을 제거하고, 본 발명의 일 실시예에 따른 융합단백질(PG110-1)을 다양한 농도(0, 50, 200 및 800 μg/ml)로 30분간 처리하여 반응시킨 후, 마찬가지로 미반응 단백질을 세척으로 제거한 다음, PE-결합 IgG4 항체를 20분간 처리하여 염색을 수행하였다(도 5A 및 5B). Specifically for this, the present inventors cultured the U266B1 cell line known to express membrane IgE, followed by performing Live-dead staining for 20 minutes, removing residual staining reagents through washing, and fusion protein according to an embodiment of the present invention. (PG110-1) was reacted at various concentrations (0, 50, 200 and 800 μg/ml) for 30 minutes, and then unreacted protein was removed by washing, and then PE-binding IgG4 antibody was treated for 20 minutes to be stained. Was performed (FIGS. 5A and 5B ).
농도 (μg/ml)Concentration (μg/ml) 800800 200200 5050 00
몰농도 (μM)Molar concentration (μM) 5.865.86 1.41.4 0.370.37 00
표적화 비율 (%) Targeting rate (%) 71.471.4 43.143.1 11.211.2 1.211.21
그 결과, 도 5C 및 상기 표 4에 나타난 바와 같이, 본 발명의 일 실시예에 따른 융합단백질은 IgE를 세포막 표면에 발현하는 세포를 표적화할 수 있었다. 이는 본 발명의 일 실시예에 따른 융합단백질이 막 IgE를 발현하는 세포를 표적으로 하여 해당 세포에 선택적으로 IL-10이 효과를 나타낼 수 있음을 시사하는 것이다. As a result, as shown in FIG. 5C and Table 4, the fusion protein according to an embodiment of the present invention was able to target cells expressing IgE on the cell membrane surface. This suggests that the fusion protein according to an embodiment of the present invention targets cells expressing membrane IgE, so that IL-10 can selectively exhibit an effect on the cells.
실험예 3: IL-10 수용체와의 친화도 분석Experimental Example 3: Analysis of affinity with IL-10 receptor
본 발명자들은 본 발명의 일 실시예에 따른 융합단백질(PG110-1)의 IL-10 수용체 1(IL-10R1)과의 친화도를 상술한 실험예 1과 유사하게 생물층 간섭계 분석(BLI assay)을 통해서 분석하였다. 이때 대조군으로는 FcεRIα가 연결되지 않고, 변형된 Fc 영역에 야생형 인간 IL-10 성숙형 단백질의 87번째 아미노산인 이소류신이 알라닌으로 치환되어, 면역 활성화 기능이 억제된 이량체성 IL-10 변이체 단백질(IL-10V)이 연결된 융합단백질(NTIG-IL-10V) 및 상기 NTIG-IL-10V의 융합단백질에서 IL-10 변이체 단백질의 116번째 아미노산인 아스파라긴(N)과 117번째 아미노산인 라이신(K) 사이에 링커(스페이서) 펩타이드(GGSGGSGGS, 서열번호 14)가 삽입된 단량체성 IL-10 변이체 단백질(IL-10Vm)이 연결된 융합단백질(NTIG-IL-10Vm)이 사용되었다. 상기 NTIG-IL-10Vm은 FcεRIα이 연결되지 않은 점을 제외하면 본 발명의 일 실시예에 따른 PG110-1과 동일하다. The present inventors analyzed the affinity of the fusion protein (PG110-1) with the IL-10 receptor 1 (IL-10R1) according to an embodiment of the present invention similar to Experimental Example 1 described above (BLI assay) It was analyzed through. At this time, as a control, FcεRIα is not linked, and isoleucine, the 87th amino acid of wild-type human IL-10 mature protein, is substituted with alanine in the modified Fc region, thereby suppressing the immune activation function. -10V) between the fusion protein (NTIG-IL-10V) and the fusion protein of NTIG-IL-10V, asparagine (N), the 116th amino acid of the IL-10 variant protein, and lysine (K), the 117th amino acid A fusion protein (NTIG-IL-10Vm) to which a monomeric IL-10 variant protein (IL-10Vm) into which a linker (spacer) peptide (GGSGGSGGS, SEQ ID NO: 14) is inserted was used was used. The NTIG-IL-10Vm is the same as PG110-1 according to an embodiment of the present invention, except that FcεRIα is not connected.
이를 위해 우선 본 발명자들은 Dip and ReadTM Amine Reactive 2nd Generation(AR2G) Reagent Kit(forteBio, Cat No. 18-5092)를 이용하여 96 플레이트에 IL-10R His-tag 단백질을 부착시켰다. 구체적으로, 96웰 플레이트에 D.W. 200 μl를 분주한 후, 상기 키트에 포함된 amine biosensor를 꽂아 10분 동안 수화시켰다. 이어, D.W. 200 μl를 추가로 분주한 후, 필요한 시료 양의 1/20로 EDC:NHS를 1:1로 혼합한 후, D.W.에 희석하여 96웰 플레이트에 200 μl 씩 분주하였다. 이어, IL-10R His-tag 단백질을 10 mM acetate pH 5.0 용액에 10 μg/ml이 되도록 희석한 후 상기 96웰 플레이트에 200 μl씩 첨가하였다. 이후, 1 M 에탄올아민을 200 μl 씩 96웰 플레이트에 첨가한 후, Biosensor 플레이트와 샘플 플레이트를 Octet　 K2 기기에 넣고 측정하였다. 측정이 끝난 후 샘플 플레이트에 1x Kinetics 완충액을 200 μl를 첨가한 후, 기저값(baseline)을 정하였다. 이후, 본 발명의 일 실시예에 따라 제조된 융합단백질(FcεRIα-NTIG-IL-10Vm), 그리고 대조군으로 사용된 NTIG-IL-10V 및 NTIG-IL-10Vm 융합단백질을 각각 다양한 농도(0, 62.5, 125, 250, 500 및 1,000 nM)로 1x Kinetic 완충액에 희석한 후 샘플 플레이트에 200 μl로 분주한 후, Octet　K2 기기로 BLI 분석을 수행하여 결합력을 측정하였다. To this end, the present inventors first attached the IL-10R His-tag protein to 96 plates using Dip and Read TM Amine Reactive 2 nd Generation (AR2G) Reagent Kit (forteBio, Cat No. 18-5092). Specifically, after dispensing 200 μl of DW into a 96-well plate, amine biosensor included in the kit was inserted and hydrated for 10 minutes. Subsequently, 200 μl of DW was additionally dispensed, EDC:NHS was mixed at 1:1 with 1/20 of the required sample amount, diluted in DW, and 200 μl each was dispensed into a 96-well plate. Subsequently, IL-10R His-tag protein was diluted to 10 μg/ml in 10 mM acetate pH 5.0 solution, and then 200 μl was added to the 96-well plate. Thereafter, 200 μl of 1 M ethanolamine was added to the 96-well plate, and then the Biosensor plate and the sample plate were added to Octet.　It was put into a K2 instrument and measured. After the measurement was completed, 200 μl of 1x Kinetics buffer was added to the sample plate, and a baseline was determined. Thereafter, the fusion protein (FcεRIα-NTIG-IL-10Vm) prepared according to an embodiment of the present invention, and the NTIG-IL-10V and NTIG-IL-10Vm fusion proteins used as controls were respectively added to various concentrations (0, 62.5 , 125, 250, 500 and 1,000 nM) in 1x Kinetic buffer and then dispensed to 200 μl on a sample plate, and then Octet　BLI analysis was performed with a K2 instrument to measure binding force.
Sample IDSample ID NTIG-IL-10VNTIG-IL-10V NTIG-IL-10VmNTIG-IL-10Vm FcεRIα-NTIG-IL-10VmFcεRIα-NTIG-IL-10Vm
KD (nM)K D (nM) 11.1±0.911.1±0.9 29.2±8.8529.2±8.85 < 0.001<0.001
Kon(1/Ms)K on (1/Ms) 71,100±6,67071,100±6,670 20,550±2,31220,550±2,312 11,500±98011,500±980
Kdis(1/s)K dis (1/s) 0.00080.0008 0.00060.0006 < 1.0E-07<1.0E-07
R2 R 2 0.970.97 0.970.97 0.980.98
그 결과, 도 6 및 표 5에서 나타난 바와 같이, 본 발명의 일 실시예에 따른 융합단백질(NTIG-IL-10Vm)은 Kd 값은 29.2 nM로 단량체성 IL-10 융합단백질(NTIG-IL-10V)의 Kd 값(11.1 nM)의 약 세 배 정도로 나타났다. 따라서, IL-10R과의 친화력은 단량체성 IL-10 융합단백질에 비해 다소 떨어지는 것으로 나타났다. 종래 연구결과도 단량체성 IL-10의 경우 이량체성 IL-10 단백질에 비해 IL-10R과의 친화성이 떨어지는 것으로 나타났기 때문에, 충분히 예상할 수 있는 바이다. 반면, 본원발명의 실시예 1의 융합단백질(FcεRIα-NTIG-IL-10Vm, PG110-1)은 KD 값이 0.001 nM 미만으로 나타나, IL-10R과의 친화도가 더 높은 것으로 나타났다. 이는 API인 FcεRIα와 연결되었음에도 불구하고 단량체성 IL-10 변이체가 IL-10R에 충분한 결합능이 있음을 보여주는 결과이다. 실험예 4: 면역 억제 활성 분석 As a result, as shown in Figure 6 and Table 5, the fusion protein (NTIG-IL-10Vm) according to an embodiment of the present invention has a Kd value of 29.2 nM, and the monomeric IL-10 fusion protein (NTIG-IL-10V ) Was about three times the Kd value (11.1 nM). Therefore, it was found that the affinity with IL-10R was slightly lower than that of the monomeric IL-10 fusion protein. Conventional research results are also predictable because monomeric IL-10 has a lower affinity for IL-10R compared to dimeric IL-10 protein. On the other hand, the fusion protein (FcεRIα-NTIG-IL-10Vm, PG110-1) of Example 1 of the present invention had a KD value of less than 0.001 nM, indicating a higher affinity with IL-10R. This is a result showing that the monomeric IL-10 variant has sufficient binding capacity to IL-10R, despite being linked to the API FcεRIα. Experimental Example 4: Immune Suppression Activity Assay
4-1: 비만세포에서의 TNF-α 분비 억제능 분석4-1: Analysis of the ability to inhibit TNF-α secretion in mast cells
상기 비만세포 증식능 분석에 이어서, IL-10 융합단백질의 비만세포에서의 TNF-α 분비능 감소에 미치는 영향 즉, 면역 억제 활성을 조사하였다.Following the mast cell proliferation assay, the effect of the IL-10 fusion protein on the reduction of TNF-α secretion ability in mast cells, that is, the immunosuppressive activity, was investigated.
이를 위해, 구체적으로, 10% FBS, 1% 항생제, rmSCF 20 ng/ml 및 rmIL-3 10 ng/ml을 포함하는 RPMI 배지가 담겨진 96웰 플레이트에 골수유래 비만세포(BMMC)를 1x104 cells/50 μl/well이 되도록 분주하고, 단량체성 IL-10 변이체 단백질을 포함하는 융합단백질(NTIG-IL-10Vm)와 실시예의 융합단백질(PG110-1)을 농도에 맞게 희석한 다음, 항-DNP IgE를 1 μg/mL로 희석한 후 96웰 플레이트에 50 μl씩 첨가하고, 37℃ 및 5% CO2 조건에서 24시간 동안 배양하였다. 이어, DNP-BSA(Antigen)을 100 ng/mL이 되도록 희석하여 96웰 플레이트에 50 μl 씩 첨가한 후, 37℃ 및 5% CO2 조건에서 밤새 반응시켰다. 그런 다음 4℃에서 1,500 rpm의 속도로 5분간 원심분리를 한 후, 새로운 96웰 플레이트에 회수된 상등액 150 μl를 분주한 후 TNF-α ELISA 키트(BioLegend, USA)를 이용하여 TNF-α의 농도를 측정하였다. To this end, specifically, bone marrow-derived mast cells (BMMC) were 1x10 4 cells/in a 96-well plate containing RPMI medium containing 10% FBS, 1% antibiotics, rmSCF 20 ng/ml and rmIL-3 10 ng/ml. Dispense so as to be 50 μl/well, dilute the fusion protein (NTIG-IL-10Vm) containing the monomeric IL-10 variant protein and the fusion protein (PG110-1) of the example according to the concentration, and then anti-DNP IgE Was diluted to 1 μg/mL, 50 μl each was added to a 96-well plate, and incubated for 24 hours at 37° C. and 5% CO 2 . Then, DNP-BSA (Antigen) was diluted to 100 ng/mL, added to a 96-well plate in 50 μl each, and reacted overnight at 37° C. and 5% CO 2 . Then, after centrifuging for 5 minutes at a speed of 1,500 rpm at 4°C, 150 μl of the recovered supernatant was dispensed into a new 96-well plate, and the concentration of TNF-α using a TNF-α ELISA kit (BioLegend, USA). Was measured.
본 발명의 일 실시예에 따른 융합단백질의 비만세포에서의 TNF-α 분비 억제율Inhibition rate of TNF-α secretion in mast cells of a fusion protein according to an embodiment of the present invention
농도(nM)Concentration (nM) 00 1.561.56 66 2525 100100
NTIG-IL-10VmNTIG-IL-10Vm 0%0% 6.3%6.3% 27%27% 43%43% 45%45%
PG110PG110 0%0% 58%58% 65%65% 80%80% 83%83%
그 결과, 상기 표 6 및 도 7에서 확인되는 바와 같이, 본원발명의 PG110-1 및 비교예로 사용된 단량체성 IL-10 변이체 단백질(NTIG-IL-10Vm) 모두 농도의존적인 TNF-α 분비 억제능을 나타냈으나, 본 발명의 일 실시예에 따른 융합단백질(PG-11O)이 더 낮은 농도에서도 매우 우수한 TNF-α 분비 억제능을 나타내, IgE의 효과적인 차단이 가능함을 확인할 수 있었다.As a result, as shown in Table 6 and FIG. 7, both the PG110-1 of the present invention and the monomeric IL-10 variant protein (NTIG-IL-10Vm) used as a comparative example have concentration-dependent TNF-α secretion inhibitory ability. However, it was confirmed that the fusion protein (PG-11O) according to an embodiment of the present invention exhibited very excellent TNF-α secretion inhibitory ability even at a lower concentration, and effective blocking of IgE was possible.
4-2: 대식세포에서의 LPS 유도 TNF-α 분비 억제능 분석4-2: Analysis of LPS-induced TNF-α secretion inhibition ability in macrophages
본 발명자들은 본 발명의 일 실시예에 따른 PG110-1 단백질의 비만세포에서의 TNF-α 분비능 억제능을 확인한데 이어, TNF-α 매개 염증반응에 밀접하게 관련된 면역세포인 대식세포에서도 역시 TNF-α 분비를 억제할 수 있는지 조사하였다.The present inventors confirmed the ability of the PG110-1 protein to inhibit the secretion of TNF-α in mast cells according to an embodiment of the present invention, followed by TNF-α in macrophages, which are immune cells closely related to the TNF-α-mediated inflammatory response. It was investigated whether it can suppress secretion.
이를 위해 구체적으로, RAW264.7 세포를 5x105 cells/mL 농도로 준비한 후 96 웰 플레이트에 100 μL씩 분주한 후, 37 ℃, 5% CO2 배양기에서 12시간 동안 배양하였다. 그런 다음 본 발명의 일 실시예에 따른 PG110-1을 순차적으로 희석한 후 상기 배양중인 RAW264.7 세포에 처리하였다. 이 때, LPS(400 ng/mL)를 96 웰 플레이트에 100 μL씩 처리함으로써 TNF-α 발현을 유도하였다. 그런 다음, 37 ℃, 5% CO2 배양기에서 12시간 동안 배양하고, 상층액 150 μL를 분리한 후 상층액에 포함된 TNF-α 발현량을 상기 실험예 4-1과 동일한 방법으로 측정하였다(표 7 및 도 8).For this purpose, RAW264.7 cells were prepared at a concentration of 5 ×10 5 cells/mL, and then 100 μL was dispensed into a 96-well plate, followed by incubation in a 37°C, 5% CO 2 incubator for 12 hours. Then, the PG110-1 according to an embodiment of the present invention was sequentially diluted and then treated on the RAW264.7 cells in the culture. At this time, TNF-α expression was induced by treating 100 μL of LPS (400 ng/mL) in a 96 well plate. Then, after incubation for 12 hours at 37° C., 5% CO 2 incubator, 150 μL of the supernatant was separated, and the expression level of TNF-α contained in the supernatant was measured in the same manner as in Experimental Example 4-1 ( Table 7 and Figure 8).
본 발명의 일 실시예에 따른 융합단백질(PG110-1)의 대식세포에서의 TNF-α 분비 억제율Inhibition rate of TNF-α secretion in macrophages of fusion protein (PG110-1) according to an embodiment of the present invention
농도 (nM)Concentration (nM) IC50(nM)IC 50 (nM)
0.0190.019 0.0960.096 0.480.48 2.42.4 1212 6060 100100 300300 15071507
TNF-α 분비 저해율 (%)TNF-α secretion inhibition rate (%) 7%7% 13%13% 10%10% 14%14% 22%22% 31%31% 38%38% 40%40% 42%42% 10.4210.42
그 결과, 표 7 및 도 8에서 확인되는 바와 같이, 본 발명의 일 실시예에 따른 PG110-1 융합단백질은 농도의존적으로 대식세포에서의 TNF-α의 발현을 억제하였다.As a result, as shown in Tables 7 and 8, the PG110-1 fusion protein according to an embodiment of the present invention inhibited the expression of TNF-α in macrophages in a concentration-dependent manner.
실험예 5: IgE 차단 및 비만세포 활성화 억제 분석Experimental Example 5: IgE blocking and mast cell activation inhibition assay
본 발명자들은 본 발명의 일 실시예에 따른 PG110 단백질이 실제 IgE와 결합하여 그 기능을 차단하고 IgE 자극시 탈과립화에 의해 히스타민 등의 알러지 반응 매개물질을 분비함으로써 알러지 반응을 유발하는 비만 세포(mast cells)의 활성화를 억제할 수 있는지 여부를 조사하였다.The present inventors believe that the PG110 protein according to an embodiment of the present invention binds to actual IgE, blocks its function, and secretes allergic reaction mediators such as histamine by degranulation upon stimulation of IgE, thereby causing an allergic reaction. cells) could be inhibited.
이를 위해 구체적으로, 대조군인 IgE TRaP(FcεRIα-Fc) 및 본 발명의 일 실시예에 따른 융합단백질 PG110-1(FcεRIα-NTIG-IL10Vm)을 농도별로 준비한 후 항-DNP 마우스 IgE(1 ug/mL)와 혼합한 후 상온에서 30분간 인큐베이션 하였다. 이어, 마우스의 골수로부터 분화된 비만세포를 준비하고, 이를 HBSS 버퍼로 세척해준 후 5x105 cells/mL 농도로 맞춰준 후 96-웰 플레이트에 60 μL씩 분주하였다. 그런 다음, IgE 및 약물을 처리했던 시료 20 μL를 사전에 준비된 비만세포에 첨가한 후 37 ℃, 5% CO2 배양기에서 30분 동안 배양하였다. 이후 DNP BSA(500 ng/mL)를 20 μL 첨가한 후 다시 37 ℃, 5% CO2 배양기에서 30분 동안 배양하였고, 이어, 1500 rpm, 4℃의 조건으로 원심분리한 후 상층액 30 μL를 분리하였다. 분리된 상층액 30 μL와 기질(4-Nitrophenyl N-acetyl-β-D-glucosaminide, 5.84 mM) 30 μL를 혼합한 후 37 ℃의 온도의 조건으로 5% CO2 배양기에서 25분 동안 배양하였다. 그런 다음, 0.1 M 소디움 카보네이트 버퍼(sodium carbonate buffer, pH 10) 140 μL를 혼합하여 반응을 종결시켰다. 이 후, 405 nm에서 흡광도를 측정하여 분비된 베타-헥소사미니다아제(β-hexosaminidase)의 상대량을 비교하여 각 약물의 농도에 따른 비만세포 억제 효과를 확인하였다(도 9 및 표 8). 상기 베타-헥소사미니다아제는 비만세포에서 탈과립화를 유발하는 알러지 반응 지표 분자이다. 그 결과, 도 9 및 표 8에서 확인되는 바와 같이, 본 발명의 일 실시예에 따른 PG110은 대조군인 IgETRAP 보다 소폭이기는 하나 좀 더 우수한 IgE에 의한 비만세포 활성화 억제능을 나타냈다. To this end, specifically, after preparing a control group IgE TRaP (FcεRIα-Fc) and the fusion protein PG110-1 (FcεRIα-NTIG-IL10Vm) according to an embodiment of the present invention by concentration, anti-DNP mouse IgE (1 ug/mL ) And incubated at room temperature for 30 minutes. Subsequently, mast cells differentiated from the bone marrow of the mouse were prepared, washed with HBSS buffer, adjusted to a concentration of 5 ×10 5 cells/mL, and then dispensed into 96-well plates by 60 μL. Then, 20 μL of a sample treated with IgE and drug was added to the mast cells prepared in advance, and then incubated for 30 minutes in a 37°C, 5% CO 2 incubator. Thereafter, 20 μL of DNP BSA (500 ng/mL) was added, and then incubated for 30 minutes in a 37°C, 5% CO 2 incubator, followed by centrifugation under conditions of 1500 rpm and 4°C, and 30 μL of the supernatant was added. Separated. 30 μL of the separated supernatant and 30 μL of the substrate (4-Nitrophenyl N-acetyl-β-D-glucosaminide, 5.84 mM) were mixed and incubated for 25 minutes in a 5% CO 2 incubator at a temperature of 37°C. Then, 140 μL of 0.1 M sodium carbonate buffer (pH 10) was mixed to terminate the reaction. Thereafter, absorbance was measured at 405 nm and the relative amount of secreted beta-hexosaminidase was compared to confirm the mast cell inhibitory effect according to the concentration of each drug (FIG. 9 and Table 8). The beta-hexosaminidase is an allergic reaction indicator molecule that causes degranulation in mast cells. As a result, as can be seen in Figure 9 and Table 8, PG110 according to an embodiment of the present invention exhibited a slightly better inhibitory ability to inhibit mast cell activation by IgE than the control IgE TRAP .
본 발명의 일 실시예에 따른 융합단백질의 IgE에 의한 비만세포 활성화 억제 정도 분석 결과Analysis of the degree of inhibition of mast cell activation by IgE of a fusion protein according to an embodiment of the present invention
1차 시험1st test 2차 시험2nd test 3차 시험3rd test 평균Average
IC50 of IgETRAP (nM)IC 50 of IgE TRAP (nM) 19.5819.58 17.8817.88 22.3122.31 19.9219.92
IC50 of PG110-1 (nM)IC 50 of PG110-1 (nM) 21.6421.64 14.7814.78 14.4214.42 16.94716.947
실험예 6: 약물 동태학(pharmacokinetics) 분석Experimental Example 6: pharmacokinetics analysis
본 발명자들은 본 발명의 융합단백질이 체내 투여시 안정성이 얼마나 되는지 확인하기 위해, 약물 동태학 분석을 수행하였다.The present inventors performed pharmacokinetic analysis to determine how stable the fusion protein of the present invention is when administered in vivo.
구체적으로, 각 그룹당 3두의 SD 랫트를 대상으로 본 발명의 일 실시예에 따른 융합단백질(PG110-1)을 1 mg/Kg의 투여량으로 정맥 주사, 복강주사, 근육주사 및 피하주사로 각각 1.0 mL/kg, 1.0 mL/kg, 0.4 mL/kg 및 1.0 mL/kg의 부피로 투여한 후, 정맥주사의 경우 0분, 5분, 1시간, 5시간, 10시간, 24시간, 48시간, 72시간, 120시간, 168시간, 240시간 및 336시간 동안 그리고 다른 투여방법(복강주사, 근육주사, 및 피하주사)의 경우에는 5분 후 측정을 제외하고는 상기 정맥주사와 동일한 시간대에 혈청을 채취해서 인간 IgG4 Fc ELISA 를 이용하여 상기 융합단백질을 정량하였다.Specifically, the fusion protein (PG110-1) according to an embodiment of the present invention targets 3 SD rats per group by intravenous injection, intraperitoneal injection, intramuscular injection, and subcutaneous injection at a dose of 1 mg/Kg, respectively. After administration in volumes of 1.0 mL/kg, 1.0 mL/kg, 0.4 mL/kg, and 1.0 mL/kg, intravenous injection is 0 minutes, 5 minutes, 1 hour, 5 hours, 10 hours, 24 hours, 48 hours , 72 hours, 120 hours, 168 hours, 240 hours and 336 hours, and for other administration methods (intraperitoneal injection, intramuscular injection, and subcutaneous injection), serum at the same time as the intravenous injection, except for measurement after 5 minutes. Was collected, and the fusion protein was quantified using human IgG4 Fc ELISA.
그 결과, 도 10에 나타난 바와 같이, 본 발명의 일 실시예에 따른 융합단백질은 투여경로와 무관하게 시간의 경과에 따라서 완만한 감소양상을 나타냈는데, 이는 본 발명의 일 실시예에 따른 융합단백질이 체내에서 상당 기간 안정적으로 유지가 됨을 보여주는 것이다. As a result, as shown in Fig. 10, the fusion protein according to an embodiment of the present invention exhibited a modest decrease over time regardless of the route of administration, which is a fusion protein according to an embodiment of the present invention. This shows that it remains stable for a considerable period of time in the body.
실험예 7: 혈액독성 분석Experimental Example 7: Blood Toxicity Analysis
종래의 재조합 IL-10은 투여농도를 높이거나, 반복 투여시 혈액 내 헤모글로빈 농도가 감소함으로써 빈혈 증상이 나타나고, 혈소판 농도가 감소함에 따라 혈소판 감소증(thromocytopenia)이 나타나는 부작용이 보고된 바 있다(Tilg et al., J. Immunol. 164(4): 2204-2209, 2002; Fedorak et al., Gastroendocrinol. 119(6): 1473-1482, 2000). 이에 본 발명자들은 본 발명의 일 실시예에 따른 융합단백질이 그러한 부작용을 나타낼 지 여부에 대하여 조사하였다.Conventional recombinant IL-10 has been reported to cause anemia symptoms by increasing the administration concentration or decreasing the hemoglobin concentration in the blood when repeated administration, and thromocytopenia as the platelet concentration decreases (Tilg et al. al ., J. Immunol . 164(4): 2204-2209, 2002; Fedorak et al., Gastroendocrinol. 119(6): 1473-1482, 2000). Accordingly, the present inventors investigated whether the fusion protein according to an embodiment of the present invention exhibits such side effects.
이를 위해 구체적으로, 본 발명자들은 자성 ICR 마우스를 대상으로 그룹당 3마리씩 구성 후 각 그룹별로 PG110-1를 0, 50, 150 및 300 mg/kg 용량으로 투여하고, 약물 투여 후 11일차에 혈액을 채취하고, 혈구분석기(XN-V, SYSMEX, JAPAN)를 이용하여 감별 백혈구수(white blood cell differential count), 총적혈구수(total RBC), 및 혈소판수(total platelet)을 분석하였다. 그 결과 도 11에서 나타나는 바와 같이, PG110-1 투여 시 혈구 세포 등의 수에는 영향이 전혀 없었다.Specifically, for this, the present inventors consisted of 3 mice per group targeting magnetic ICR mice, and administered PG110-1 at 0, 50, 150 and 300 mg/kg doses for each group, and blood was collected on the 11th day after drug administration. Then, a white blood cell differential count, a total RBC, and a total platelet were analyzed using a hemocytometer (XN-V, SYSMEX, JAPAN). As a result, as shown in FIG. 11, when PG110-1 was administered, the number of blood cells and the like was not affected at all.
실험예 8: 음식물 알러지에 대한 효능 실험Experimental Example 8: Efficacy experiment on food allergy
본 발명자들은 본 발명의 일 실시예에 따른 융합단백질 PG110이 실제 알러지 질환에 대하여 효과를 나타낼 수 있을지 확인하기 위해, 알러지 유발원인 난백 알부민(OVA)로 감작시킨 실험동물을 대상으로 OVA 경구 투여시 발생하는 알러지 증상인 설사 증상이 얼마나 개선이 되는지 여부를 조사하였다.In order to confirm whether the fusion protein PG110 according to an embodiment of the present invention can exhibit an effect on an actual allergic disease, the present inventors have conducted oral administration of OVA to an experimental animal sensitized with egg white albumin (OVA), a cause of allergies. We investigated whether diarrhea, which is an allergic symptom, was improved.
구체적으로, Balb/c 마우스(코아텍)들에 대하여 OVA(ovalbumin) 50 μg 및 알럼(Alum) 1 mg을 섞은 용액을 14일 간격으로 2회 복강 투여하여 감작(sensitization)시켰다. 이어, 28, 30, 32, 34, 36일째 총 5회에 걸쳐 2일 간격으로 OVA 50 mg을 경구 투여하여 장에 음식물 알러지를 유발하였다. 그 과정에서 31일째에 각 그룹을 구성하는 약물을 투여해주었다. 제1그룹(대조군)에는 인산완충액(PBS)을, 제2그룹에는 IgETRAP(5 mg/kg)을, 제3그룹에는 본 발명의 일 실시예에 따른 PG110-1(7.3 mg/kg)을 각각 몰수를 맞추어 계산한 후 투여하였다. 총 5회에 걸쳐 OVA를 경구 투여하면서 설사 발생 여부를 확인하였으며, 37일째 마우스를 부검하여 각 그룹에 속한 마우스에 대하여 소장 내 비만세포의 수, 혈중 IgE 농도 및 혈중 비만세포의 탈과립 효소 농도(MCPT-1, Mast cell protease-1)를 분석하였다. 그 결과, 도 12에서 확인되는 바와 같이, 본 발명의 일 실시예에 따른 PG110-1을 투여한 군에 속한 마우스에서 대조군 및 IgETRAP 투여군 마우스에 비하여 식품 알러지가 완화되는 효과를 확인할 수 있었다.Specifically, a solution of 50 μg of OVA (ovalbumin) and 1 mg of Alum was administered intraperitoneally at 14 days intervals to Balb/c mice (Coretech) to sensitize. Subsequently, on days 28, 30, 32, 34, and 36, OVA 50 mg was orally administered at intervals of 2 days over a total of 5 times to induce food allergy in the intestine. In the process, drugs constituting each group were administered on the 31st day. Phosphoric acid buffer (PBS) in the first group (control), IgE TRAP (5 mg/kg) in the second group, and PG110-1 (7.3 mg/kg) according to an embodiment of the present invention in the third group. Each was administered after calculating the number of moles. OVA was administered orally over a total of 5 times to check whether diarrhea occurred.At 37 days, mice were autopsied and the number of mast cells in the small intestine, IgE concentration in the blood, and the concentration of degranulation enzymes in the blood mast cells (MCPT -1, Mast cell protease-1) was analyzed. As a result, as can be seen in Figure 12, compared to the control and IgE TRAP- administered mice in the mice belonging to the group administered with PG110-1 according to an embodiment of the present invention it was confirmed the effect of reducing food allergy.
상술한 바와 같이, 본 발명의 일 실시예에 따른 융합단백질은 IgE를 차단하고 그로 인해서 비만세포의 활성화를 억제하는 것으로 확인되었다. 이러한 작용은 항-FcεRIα 항체의 불활성화 및 이에 의한 비만세포의 활성화 억제에 기인하는 것으로 보인다. 더구나, 본 발명의 일 실시예에 따른 융합단백질은 유리 IgE를 차단할 뿐만 아니라 막에 제시된 IgE를 발현하는 B 세포를 표적화할 수 있다. 아울러, 종래 문헌에 따르면, 조절 T 세포(regulatory T cell, Treg)의 활성 및 분화에 기여할 수 있는 것으로 알려지고 있다(Hsu et al., J. Immunol. 2015 Oct 15;195(8):3665-3674, 2015)). 더구나, 본 발명의 일 실시예에 따른 융합단백질은 IL-10 단백질을 포함하고 있기 때문에, IL-10 수용체를 발현하고 있는 다양한 면역 세포들을 대상으로 하여 이들의 기능을 억제할 것으로 예상된다. 이러한 면역세포들로는 향-염증성 사이토카인을 분비하는 비만세포, 향-염증성 사이토카인을 분비하고 항원을 제시하는 기능을 담당하는 단핵구 및 대식세포, 향-염증성 사이토카인을 분비하여 역시 항원 제시능을 가지는 수지상 세포, 사이토카인을 분비하는 호산구(eosinophil), 케모카인 및 향-염증성 사이토카인을 분비하는 호중구(neutrophil), 알러젠 특이적 IgE를 분비하는 B 세포, 및 알러젠 특이적 Th1/Th2 세포가 포함된다. 따라서, 본 발명의 일 실시예에 따른 융합단백질은 상기 면역세포들의 활성화와 그들의 기능 특히 향-염증성 사이토카인의 분비, 항원제시능 등을 억제함으로써, 과활성화된 면역기능에 기인한 다양한 면역관련 질환들 예컨대, 아토피성 질환, 음식 알러지, 만성 자발성 두드러기(chronic spontaneous urticaria), 천식 등의 알러지 질환의 치료에 사용될 수 있다.As described above, it was confirmed that the fusion protein according to an embodiment of the present invention blocks IgE and thereby inhibits the activation of mast cells. This action seems to be due to the inactivation of anti-FcεRIα antibody and thereby inhibition of the activation of mast cells. Moreover, the fusion protein according to an embodiment of the present invention may block free IgE as well as target B cells expressing IgE presented on the membrane. In addition, according to the conventional literature, it is known that it can contribute to the activity and differentiation of regulatory T cells (regulatory T cells, T reg ) (Hsu et al ., J. Immunol . 2015 Oct 15;195(8):3665). -3674, 2015)). Moreover, since the fusion protein according to an embodiment of the present invention contains the IL-10 protein, it is expected to target various immune cells expressing the IL-10 receptor and inhibit their function. These immune cells include mast cells that secrete anti-inflammatory cytokines, monocytes and macrophages responsible for the function of secreting anti-inflammatory cytokines and presenting antigens, and anti-inflammatory cytokines that also have antigen-presenting ability. Dendritic cells, eosinophils secreting cytokines, neutrophils secreting chemokines and anti-inflammatory cytokines, B cells secreting allergen-specific IgE, and allergen-specific Th1/Th2 cells. Therefore, the fusion protein according to an embodiment of the present invention inhibits the activation of the immune cells and their functions, especially the secretion of anti-inflammatory cytokines, the ability to present antigens, etc., thereby preventing various immune-related diseases caused by overactivated immune functions. For example, it can be used for the treatment of allergic diseases such as atopic diseases, food allergies, chronic spontaneous urticaria, and asthma.
따라서, 본 발명의 일 실시예에 따른 융합단백질은 알러지 환자의 투여 편의성을 개선시킬 수 있는 매우 유용한 약물 후보임을 알 수 있다.Therefore, it can be seen that the fusion protein according to an embodiment of the present invention is a very useful drug candidate that can improve the administration convenience of allergic patients.
본 발명은 상술한 실시예 및 실험예를 참고로 설명되었으나 이는 예시적인 것에 불과하며, 당해 기술분야에서 통상의 지식을 가진 자라면 이로부터 다양한 변형 및 균등한 다른 실시예가 가능하다는 점을 이해할 것이다. 따라서 본 발명의 진정한 기술적 보호 범위는 첨부된 특허청구범위의 기술적 사상에 의하여 정해져야 할 것이다.The present invention has been described with reference to the above-described embodiments and experimental examples, but these are merely exemplary, and those of ordinary skill in the art will appreciate that various modifications and equivalent other embodiments are possible therefrom. Therefore, the true technical protection scope of the present invention should be determined by the technical spirit of the appended claims.
본 발명의 일 실시예에 따른 융합단백질은 알러지 치료제의 개발에 유용하게 사용될 수 있다.The fusion protein according to an embodiment of the present invention may be usefully used in the development of an allergy treatment.

Claims (20)

  1. a) FcεRIα 단백질 및 IL-10 단백질; 및/또는 b) FcεRIα 단백질 및 IL-10 단백질을 포함하는 융합단백질을 유효성분으로 포함하는 알러지 질환 치료용 약학적 조성물.a) FcεRIα protein and IL-10 protein; And / or b) a pharmaceutical composition for the treatment of allergic diseases comprising a fusion protein comprising an FcεRIα protein and IL-10 protein as an active ingredient.
  2. 제1항에 있어서,The method of claim 1,
    상기 FcεRIα는 서열번호 2로 기재되는 아미노산 서열로 구성되는, 약학적 조성물.The FcεRIα is composed of the amino acid sequence shown in SEQ ID NO: 2, pharmaceutical composition.
  3. 제1항에 있어서,The method of claim 1,
    상기 IL-10 단백질은 인간 IL-10 단백질의 성숙형(mature form) 기준으로 87번째 아미노산인 이소류신이 알라닌으로 치환된 IL-10 변이체 단백질인, 약학적 조성물.The IL-10 protein is an IL-10 variant protein in which isoleucine, which is the 87th amino acid based on the mature form of human IL-10 protein, is substituted with alanine.
  4. 제3항에 있어서,The method of claim 3,
    상기 IL-10 단백질은 성숙형(mature form)을 기준으로 116번째 아미노산인 아스파라긴 및 117번째 아미노산인 라이신 사이에 6 내지 12 a.a.의 길이를 갖는 링커 펩타이드가 삽입된 단량체성 IL-10 변이체 단백질인, 약학적 조성물.The IL-10 protein is a monomeric IL-10 variant protein in which a linker peptide having a length of 6 to 12 aa is inserted between asparagine as the 116th amino acid and lysine as the 117th amino acid based on the mature form, Pharmaceutical composition.
  5. 제1항에 있어서,The method of claim 1,
    상기 융합단백질은 상기 FcεRIα 단백질 및 IL-10 단백질 사이에 항체의 Fc 영역 및 이량체화 도메인로 구성되는 군으로부터 선택되는 하나 이상의 추가 펩타이드가 삽입이 된 것인, 약학적 조성물.The fusion protein is one or more additional peptides selected from the group consisting of an Fc region and a dimerization domain of an antibody are inserted between the FcεRIα protein and the IL-10 protein.
  6. 제1항에 있어서, The method of claim 1,
    상기 융합단백질은 상기 FcεRIα 단백질 및 IL-10 단백질이 링커 펩타이드에 연결된 것인, 약학적 조성물.The fusion protein is that the FcεRIα protein and IL-10 protein are linked to a linker peptide.
  7. 제5항에 있어서,The method of claim 5,
    상기 융합단백질은, 상기 FcεRIα 단백질 및 추가 펩타이드 및/또는 상기 추가 펩타이드 및 IL-10 단백질 사이에 링커 펩타이드를 포함하는, 약학적 조성물.The fusion protein, the FcεRIα protein and an additional peptide and/or a linker peptide between the additional peptide and the IL-10 protein, a pharmaceutical composition.
  8. 제1항 내지 제7항 중 어느 한 항에 있어서, 상기 알러지 질환은 알러지성 비염, 약물 알러지, 알러지성 기관지 천식, 아토피성 피부염, 알러지성 결막염, 만성 특발성 두드러기,식품 알러지 또는 아나필락시스인, 약학적 조성물.The pharmaceutical according to any one of claims 1 to 7, wherein the allergic disease is allergic rhinitis, drug allergy, allergic bronchial asthma, atopic dermatitis, allergic conjunctivitis, chronic idiopathic urticaria, food allergy or anaphylaxis. Composition.
  9. FcεRIα, 항체의 Fc 영역 및 IL-10 단백질이 순차적으로 연결되고, 선택적으로 상기 FcεRIα 및 항체의 Fc 영역 사이 및/또는 상기 항체의 Fc 영역 및 IL-10 단백질 사이에 링커 펩타이드를 포함하는, 융합단백질.FcεRIα, the Fc region of the antibody and the IL-10 protein are sequentially linked, and optionally, a fusion protein comprising a linker peptide between the FcεRIα and the Fc region of the antibody and/or between the Fc region and the IL-10 protein of the antibody .
  10. 제9항에 있어서,The method of claim 9,
    상기 FcεRIα 단백질은 서열번호 2로 기재되는 아미노산 서열로 구성되는, 융합단백질.The FcεRIα protein is composed of the amino acid sequence described in SEQ ID NO: 2, a fusion protein.
  11. 제9항에 있어서,The method of claim 9,
    상기 항체의 Fc 영역은 IgA, IgG, IgM, IgD 또는 IgE의 Fc 영역 또는 이들의 도메인들 예컨대 힌지, CH2, CH3의 전부 또는 일부가 혼합된 하이브리드 Fc인, 융합단백질.The Fc region of the antibody is a hybrid Fc in which all or part of the Fc region of IgA, IgG, IgM, IgD or IgE or domains thereof such as hinge, CH2, CH3 is mixed, a fusion protein.
  12. 제9항에 있어서, The method of claim 9,
    상기 항체의 Fc 영역은 서열번호 4 내지 7 중 어느 하나의 아미노산 서열로 구성되는, 융합단백질.The Fc region of the antibody is composed of the amino acid sequence of any one of SEQ ID NOs: 4 to 7, a fusion protein.
  13. 제9항에 있어서,The method of claim 9,
    상기 IL-10 단백질은 인간 IL-10 단백질의 성숙형(mature form) 기준으로 87번째 아미노산인 이소류신이 알라닌으로 치환된 IL-10 변이체 단백질인, 융합단백질.The IL-10 protein is an IL-10 variant protein in which isoleucine, which is the 87th amino acid based on the mature form of human IL-10 protein, is substituted with alanine.
  14. 제13항에 있어서,The method of claim 13,
    상기 IL-10 변이체 단백질은 성숙형 형태(mature form)을 기준으로 아미노산 서열의 116번째 아미노산인 아스파라긴 및 117번째 아미노산인 라이신 사이에 6 내지 12 a.a.의 길이를 갖는 링커 펩타이드가 삽입된 단량체성 IL-10 변이체 단백질인, 융합단백질.The IL-10 variant protein is a monomeric IL- in which a linker peptide having a length of 6 to 12 aa is inserted between asparagine, the 116th amino acid of the amino acid sequence, and lysine, the 117th amino acid, based on the mature form. 10 Variant proteins, fusion proteins.
  15. 제9항에 있어서,The method of claim 9,
    상기 IL-10 단백질은, 서열번호 10 내지 13으로 구성되는 군으로부터 선택되는 아미노산 서열로 구성되는, 융합단백질.The IL-10 protein, consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 13, a fusion protein.
  16. 제9항 내지 제15항 중 어느 한 항의 융합단백질을 암호화하는 폴리뉴클레오타이드.A polynucleotide encoding the fusion protein of any one of claims 9 to 15.
  17. 제16항의 폴리뉴클레오타이드를 포함하는 벡터.A vector comprising the polynucleotide of claim 16.
  18. 제9항 내지 제15항 중 어느 한 항의 융합단백질을 유효성분으로 포함하는, 알러지 질환 치료용 약학적 조성물.A pharmaceutical composition for treating allergic diseases, comprising the fusion protein of any one of claims 9 to 15 as an active ingredient.
  19. 제18항에 있어서, The method of claim 18,
    상기 알러지 질환은 알러지성 비염, 약물 알러지, 알러지성 기관지 천식, 아토피성 피부염, 알러지성 결막염, 만성 특발성 두드러기, 식품 알러지 또는 아나필락시스인, 약학적 조성물.The allergic disease is allergic rhinitis, drug allergy, allergic bronchial asthma, atopic dermatitis, allergic conjunctivitis, chronic idiopathic urticaria, food allergy or anaphylaxis, pharmaceutical composition.
  20. 치료적으로 유효한 양의 a) FcεRIα 단백질 및 IL-10 단백질; 및/또는 b) FcεRIα 단백질 및 IL-10 단백질을 포함하는 융합단백질을 개체에 투여하는 단계를 포함하는 상기 개체의 알러지 치료방법.A therapeutically effective amount of a) FcεRIα protein and IL-10 protein; And/or b) administering a fusion protein comprising an FcεRIα protein and an IL-10 protein to the individual.
PCT/KR2020/008869 2019-07-08 2020-07-07 Novel pharmaceutical composition for treating allergic diseases WO2021006603A1 (en)

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