WO2021006603A1 - Nouvelle composition pharmaceutique pour le traitement de maladies allergiques - Google Patents

Nouvelle composition pharmaceutique pour le traitement de maladies allergiques Download PDF

Info

Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
protein
fcεriα
fusion protein
amino acid
seq
Prior art date
Application number
PCT/KR2020/008869
Other languages
English (en)
Korean (ko)
Inventor
진현탁
남은주
연제인
Original Assignee
주식회사 프로젠
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 주식회사 프로젠 filed Critical 주식회사 프로젠
Publication of WO2021006603A1 publication Critical patent/WO2021006603A1/fr

Links

Images

Classifications

    • 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.

Abstract

La présente invention concerne une nouvelle composition pharmaceutique pour le traitement de maladies allergiques et, plus particulièrement, une composition pharmaceutique pour le traitement de maladies allergiques, comprenant en tant que principes actifs : a) une protéine FcεRIα et une protéine IL-10 ; et/ou b) une protéine de fusion comprenant une protéine FcεRIα et une protéine IL-10.
PCT/KR2020/008869 2019-07-08 2020-07-07 Nouvelle composition pharmaceutique pour le traitement de maladies allergiques WO2021006603A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR20190082150 2019-07-08
KR10-2019-0082150 2019-07-08

Publications (1)

Publication Number Publication Date
WO2021006603A1 true WO2021006603A1 (fr) 2021-01-14

Family

ID=74113951

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2020/008869 WO2021006603A1 (fr) 2019-07-08 2020-07-07 Nouvelle composition pharmaceutique pour le traitement de maladies allergiques

Country Status (2)

Country Link
KR (1) KR20210006300A (fr)
WO (1) WO2021006603A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116063570A (zh) * 2021-11-02 2023-05-05 广东菲鹏制药股份有限公司 Il10单体融合蛋白及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20080094781A (ko) * 2007-05-30 2008-10-24 주식회사 제넥신 면역글로불린 융합 단백질
WO2012045334A1 (fr) * 2010-10-05 2012-04-12 Synthon Bv Protéines de fusion de l'il‑10 biologiquement actives
KR20120135865A (ko) * 2011-06-07 2012-12-17 (주)네오팜 ＦｃεＲＩ의 수용성 단편을 포함하는 복합체 및 이를 포함하는 ＩｇＥ 매개 알레르기성 질환 치료용 조성물
KR20150038012A (ko) * 2012-08-08 2015-04-08 로슈 글리카트 아게 인터루킨-10 융합 단백질 및 그의 용도
KR20170120579A (ko) * 2015-02-20 2017-10-31 키세이 야쿠힌 고교 가부시키가이샤 Fc-융합 고친화성 IgE 수용체 알파 사슬

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20080094781A (ko) * 2007-05-30 2008-10-24 주식회사 제넥신 면역글로불린 융합 단백질
WO2012045334A1 (fr) * 2010-10-05 2012-04-12 Synthon Bv Protéines de fusion de l'il‑10 biologiquement actives
KR20120135865A (ko) * 2011-06-07 2012-12-17 (주)네오팜 ＦｃεＲＩ의 수용성 단편을 포함하는 복합체 및 이를 포함하는 ＩｇＥ 매개 알레르기성 질환 치료용 조성물
KR20150038012A (ko) * 2012-08-08 2015-04-08 로슈 글리카트 아게 인터루킨-10 융합 단백질 및 그의 용도
KR20170120579A (ko) * 2015-02-20 2017-10-31 키세이 야쿠힌 고교 가부시키가이샤 Fc-융합 고친화성 IgE 수용체 알파 사슬

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116063570A (zh) * 2021-11-02 2023-05-05 广东菲鹏制药股份有限公司 Il10单体融合蛋白及其应用

Also Published As

Publication number Publication date
KR20210006300A (ko) 2021-01-18

Similar Documents

Publication Publication Date Title
Miossec The Th1/Th2 cytokine balance in arthritis
Lecoanet-Henchoz et al. CD23 regulates monocyte activation through a novel interaction with the adhesion molecules CD11b-CD18 and CD11c-CD18
JP3703103B2 (ja) Ｉｌ−４受容体を含有するアレルギー性、ウイルス性、寄生虫性および細菌性疾患ならびにかび感染症の治療、予防または診断剤
AU710369B2 (en) Binding agents for treatment of inflammatory, autoimmune or allergic diseases
US20190119377A1 (en) Recombinant igg fc multimers
JP5557765B2 (ja) 疾患の治療または予防のための可溶型ｃｄ８３タンパク質およびそれをコードする核酸の使用
Zhang et al. Interleukin 10 aggravates experimental autoimmune myasthenia gravis through inducing Th2 and B cell responses to AChR
JP2008500812A (ja) 免疫抑制サイトカイン
Wallner et al. Immunotherapy with T-cell-reactive peptides derived from allergens.
EP3738977A1 (fr) Domaine extracellulaire de sous-unité alpha du récepteur fc ige, composition pharmaceutique le comprenant et son procédé de production
AU755067B2 (en) Immunoregulator
Martin et al. Soluble forms of intercellular adhesion molecule-1 inhibit insulitis and onset of autoimmune diabetes
WO2021006603A1 (fr) Nouvelle composition pharmaceutique pour le traitement de maladies allergiques
WO2021006605A1 (fr) Nouvelle protéine de variant d'il-10 et utilisation de celle-ci
WO2021006604A1 (fr) Nouvelle protéine de fusion et son utilisation
CN112752766A (zh) Cd24用于预防和治疗白血病复发的使用方法
JP2023500066A (ja) ＴＧＦ－βポリペプチド
US20040138113A1 (en) Use of corticotroph-derived glycoprotein hormone to treat inflammation and potentiate glucocorticoid action
JP2011503036A (ja) 再発性多発性硬化症治療のためのｔａｃｉ−免疫グロブリン融合タンパク質
US20220401476A1 (en) Car-t-cell therapy
WO2022019652A1 (fr) Protéine de fusion comprenant un récepteur fc d'ige et son utilisation pour le traitement d'une maladie allergique canine
WO2022015049A1 (fr) Protéine de fusion comprenant un domaine extracellulaire de sous-unité alpha du récepteur fc d'ige et un anticorps anti-il-4r, et utilisation associée
WO2021230705A1 (fr) Nouvelle protéine de fusion recombinante et utilisation associée
WO2024071544A1 (fr) Nouveau récepteur fc recombinant et cellules le comprenant
JP2023548311A (ja) 疾患の治療のための融合タンパク質

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20836881

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 20836881

Country of ref document: EP

Kind code of ref document: A1