WO2014126277A1 - Composition d'un nouveau récepteur de l'interleukine-33 et d'une protéine de liaison et son utilisation - Google Patents

Composition d'un nouveau récepteur de l'interleukine-33 et d'une protéine de liaison et son utilisation Download PDF

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WO2014126277A1
WO2014126277A1 PCT/KR2013/001157 KR2013001157W WO2014126277A1 WO 2014126277 A1 WO2014126277 A1 WO 2014126277A1 KR 2013001157 W KR2013001157 W KR 2013001157W WO 2014126277 A1 WO2014126277 A1 WO 2014126277A1
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disease
autoimmune
receptor
hepatitis
interleukin
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Korean (ko)
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김수현
최지다
유소윤
배수영
이시영
홍재우
곽아름
임유정
전현정
김은솜
조승현
홍광원
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건국대학교 산학협력단
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Priority to PCT/KR2013/001157 priority Critical patent/WO2014126277A1/fr
Priority to KR1020157021498A priority patent/KR101780575B1/ko
Publication of WO2014126277A1 publication Critical patent/WO2014126277A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to novel interleukin-33 receptors and binding protein compositions and uses thereof, which will help to investigate a broad spectrum of IL-33 activity in host defense against pathogens and inflammatory diseases.
  • Interleukin (hereinafter referred to as 'IL')-33 is one of the IL-1 cytokine families and has been identified as a nuclear factor derived from high endothelial venules (Baekkevold, ES, et al. Am). J Pathol 163, 69-79 (2003)). IL-33 is synthesized and predominantly located intracellularly as a precursor IL-33 molecule. Precursor IL-33 does not have a secreted signal peptide; it is a mature protein extracellularly Secreted.
  • IL-33 is a damage related molecular pattern (DAMP) or alarmins secreted from necrotic cells in response to prototypic RNA / DNA virus replication in mice.
  • DAMP damage related molecular pattern
  • ST2 reported as the first rare receptor (also known as IL-1RL1, DER4, Fit-1 or T1) (Bergers, G., Reikerstorfer, A., Braselmann, S., Graninger, P. & Busslinger, M EMBO J 13, 1176-1188 (1994); Klemenz, R., Hoffmann, S. & Werenskiold, AK Proc Natl Acad Sci USA 86, 5708-5712 (1989); Tominaga, S. FEBS letters 258, 301-304. (1989) is a ligand binding chain of the IL-33 receptor (Schmitz, J., et al. Immunity 23, 479-490 (2005)).
  • the IL-33 receptor complex for signaling consists of ligand binding chain ST2 (IL-1R4) and signaling chain IL-1 receptor accessory protein (IL-1RAcP; IL-1R3). After IL-33 binding, the receptor complex is directed to downstream signal transduction molecules such as NF-kB and AP-1 via IL-1 receptor-associated kinase (IRAK), TNF receptor associated factor 6 (TRAF6) and / or MAPKs. Activate it.
  • ST2 is a selection marker for Th2 cells in mice and humans (Trajkovic, V., Sweet, MJ & Xu, D. Cytokine Growth Factor Rev 15, 87-95 (2004); Lohning, M., et al.
  • the present invention has been made in view of the above necessity, and an object of the present invention is to identify a novel interleukin (IL) -33 receptor.
  • IL interleukin
  • Another object of the present invention is to provide a novel composition for preventing or treating autoimmune diseases.
  • the present invention provides a receptor and binding to interleukin (IL) -33 comprising IL-1 receptor accessory protein 2 (IL-1R9) and IL-18 binding protein (IL-18BP) as an active ingredient.
  • IL interleukin
  • IL-1R9 IL-1 receptor accessory protein 2
  • IL-18BP IL-18 binding protein
  • the IL-1 receptor accessory protein 2 (IL-1R9) is preferably composed of one of the amino acid sequences set forth in SEQ ID NO: 1 to 2, but is not limited thereto.
  • the IL-18 binding protein is preferably made of one of the amino acid sequences described in SEQ ID NOs: 3 to 6, but is not limited thereto.
  • the interleukin (IL) -33 is preferably composed of the amino acid sequence of SEQ ID NO: 7 (precursor IL-33) or 8 (Mature IL-33), but is not limited thereto.
  • the present invention is an interleukin (IL) -33-related autoimmune comprising at least one protein selected from the group consisting of IL-1 receptor accessory protein 2 (IL-1R9) and IL-18 binding protein (IL-18BP) as an active ingredient. It provides a composition for preventing and treating diseases.
  • IL-1R9 IL-1 receptor accessory protein 2
  • IL-18BP IL-18 binding protein
  • the autoimmune disease is rheumatoid arthritis, osteoarthritis, pediatric chronic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis and septic arthritis, spondyloarthritis, systemic lupus erythematosus, Crohn's disease, Ulcerative colitis, inflammatory bowel disease, insulin dependent diabetes mellitus, thyroiditis, asthma, allergies, psoriasis, dermatitis, sclerosis, graft-versus-host disease, organ transplant rejection, acute or chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis , Sowing intravascular coagulation, Kawasaki disease, Graves' disease, kidney syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schonlein purpurea, microscopic vasculitis of the kidney, chronic active hepatitis, uveitis, septic shock, Toxin shock syndrome, sepsis syndrome, cache
  • the present invention is to prevent and treat autoimmune diseases comprising an antibody against one or more proteins selected from the group consisting of IL-1 receptor accessory protein 2 (IL-1R9) and IL-18 binding protein (IL-18BP) as an active ingredient It provides a composition for.
  • IL-1 receptor accessory protein 2 IL-1R9
  • IL-18BP IL-18 binding protein
  • Antibodies of the invention can be prepared by any of a variety of techniques known in the art. Particularly preferred methods for preparing the antibodies of the invention include methods using XENOMOUSE transgenic mice, hybridomas and SLAM cell transplantation techniques known in the art for antibody production (Abgenix, Inc., Fremont, CA). Method and the like.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a protein of the present invention and an antibody or antigen-binding portion thereof and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises one or more other additional therapeutic agents for treating diseases in which IL-33 activity is detrimental.
  • Antibodies and antibody portions of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject.
  • the pharmaceutical composition comprises an antibody or antibody portion of the invention and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • pharmaceutically acceptable carriers include one or more or a combination of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like.
  • compositions include isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride.
  • Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances, such as wetting agents or emulsifiers, preservatives or buffers, which enhance the useful life or effectiveness of the antibody or portion of the antibody.
  • Antibodies and antibody portions of the invention can be incorporated into pharmaceutical compositions suitable for parenteral administration.
  • the antibody or portion of the antibody is preferably prepared in an injection solution containing 0.1 to 250 mg / ml of the antibody.
  • Injectable solutions may consist of liquid or lyophilized dosage forms in flint or amber vials, ampoules or prefilled syringes.
  • the buffer may be L-histidine (1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0 (optimally pH 6.0).
  • Other suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate.
  • Sodium chloride can be used to modify the toxicity of the solution at a concentration of 0 to 300 mM (optimally 150 mM in liquid dosage form). Lyophilized dosage forms may contain an antifreeze agent, usually 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose.
  • the lyophilized dosage form can be added with an expandable drug, mainly 1-10% mannitol (optimally 2-4%).
  • Stabilizers mainly 1-50 mM L-methionine (optimally 5-10 mM), can be added to the liquid and lyophilized dosage forms.
  • Other suitable expandable drug mainly 1-10% mannitol (optimally 2-4%).
  • Elongated drugs include glycine and arginine, which may be included as 0-0.05% polysorbate-80 (optimally 0.005-0.01%).
  • Still other surfactants include, but are not limited to, polysorbate 20 and BRIJ surfactants.
  • composition of the present invention may be in various forms.
  • Such forms include, for example, liquid, semisolid and solid dosage forms such as liquid solutions (eg, injection solutions and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories and the like.
  • Preferred forms vary depending on the intended mode of administration and therapeutic use.
  • Generally preferred compositions are compositions similar to those used to passively immunize humans with injection or infusion solutions, such as other antibodies.
  • Preferred modes of administration are parenteral (eg, intravenous, subcutaneous, intraperitoneal, intramuscular).
  • the antibody is administered by intravenous infusion or injection.
  • the antibody is administered by intramuscular or subcutaneous injection.
  • compositions can generally be sterile and must be stable under the conditions of manufacture and storage. Such compositions may be formulated in solution, microemulsions, dispersions, liposomes or other custom structures suitable for high drug concentrations.
  • Sterile injectable solutions can be prepared by mixing the required amount of the active compound (ie, antibody or antibody portion) in a suitable solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by the addition of the active compound to a sterile medium which comprises a basic dispersion medium and the other ingredients described above, as desired.
  • sterile lyophilized powders useful for the preparation of sterile injectable solutions
  • preferred methods of preparation are vacuum drying and spray drying providing the powder of the active ingredient and any desired additional ingredients from the sterile filtration solution described above.
  • Proper fluidity of the solution can be maintained, for example, through the use of a coating such as lecithin, the maintenance of the required particle size in the case of dispersions and the use of surfactants.
  • Sustained absorption of the injectable composition can be provided by the addition of an absorption retardant such as a monostearate salt and gelatin to the composition.
  • the antibodies and portions of antibodies of the invention can be administered by a variety of methods known in the art, but the preferred route / administration of route for various therapeutic uses is subcutaneous injection, intravenous injection or infusion. As will be appreciated by those skilled in the art, the route and / or mode of administration will depend upon the desired result.
  • the active compound may be formulated with a carrier that prevents the compound from being rapidly released, including controlled release formulations including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used here, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for preparing such formulations are patented or generally known to those skilled in the art.
  • an antibody or antibody portion of the invention can be administered orally, such as with an inert diluent or an assimilable edible carrier.
  • an inert diluent or an assimilable edible carrier Such compounds (and other ingredients, if necessary) may also be filled into hard or soft skin gelatin capsules, compressed into tablets, or added directly to the subject diet.
  • the compound may be mixed with excipients to form intake tablets, buccal tablets, troches, capsules; It can be used in the form of elixirs, suspensions, syrups, wafers and the like.
  • the pharmaceutical composition of the invention may comprise a "therapeutically effective amount” or “prophylactically effective amount” of an antibody or portion of an antibody of the invention.
  • therapeutically effective amount means the dosage and the amount effective for the required period of time to obtain the desired therapeutic result.
  • a therapeutically effective amount of the antibody or portion of the antibody can be determined by one skilled in the art and will vary depending on factors such as the disease state, age, sex and body weight of the individual and the ability of the antibody or portion of the antibody to elicit a desired response in the individual. Can be.
  • a therapeutically effective amount is an amount in which a therapeutically beneficial effect exceeds any toxic or detrimental effect of the antibody or portion of the antibody.
  • prophylactically effective amount is meant a dosage that is effective to obtain the desired prophylactic result and an effective amount for the required period of time. In general, the prophylactically effective amount will be less than the therapeutically effective amount because the prophylactic dose is used in subjects prior to or during the early stages of the disease.
  • the dosage regimen may be adjusted to provide the desired optimal response (eg, a therapeutic or prophylactic response).
  • a therapeutic or prophylactic response e.g. one bolus tablet may be administered, several divided doses may be administered over time, or doses may be reduced or increased in proportion to the exigencies of the therapeutic situation.
  • parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subject to be treated. Thus, each unit comprises a predetermined amount of active compound calculated to provide the desired therapeutic effect in association with the required pharmaceutical carrier.
  • dosage unit form of the present invention are specific to (a) the unique characteristics of the active compound and the specific therapeutic or prophylactic effect to be obtained, and (b) the combination of the active compound in the treatment of sensitivity of the subject. It depends on the restrictions and depends directly on them.
  • Exemplary ranges of non-limiting amounts to a therapeutically or prophylactically effective amount of an antibody or antibody portion of the invention are 0.1 to 20 mg / kg, more preferably 1 to 10 mg / kg.
  • the dosage value is determined by the practitioner of the person who manages or supervises the individual's needs and composition dosage
  • IL-33 can be used for Th1 and Th2-related autoimmune diseases by regulating both Th1 and Th2 cytokine.
  • IL-18BP is used for both Th1 (rheumatoid arthritis, colitis, psoriasis) and Th2 (Asthma and atopic dermatitis).
  • Th1 rheumatoid arthritis, colitis, psoriasis
  • Th2 Thma and atopic dermatitis.
  • IL-33 when acting alone, induces a Th2 immune response if Th1 acts in concert with IL-12, IL-2, IFNalpha.
  • IL-18BP so far focuses only on IL-18-induced IFN gamma inhibition and has been registered as a Th1 immunosuppressive therapeutic agent and the inventor of the present invention is related.
  • IL-1R9 has been known to have no ligand as an orphan receptor until now, but IL-33 is a ligand of IL-1R9 and Fc-IL-1R9 does not directly bind to IL-33. This is the first report that acts as a co-receptor and plays an important role in the Th1 immune response induced by IL-33, or IFN gamma induction.
  • a neutralizing antibody that inhibits the function of IL-1R9 may be prepared and used to specifically and selectively inhibit the Th1 immune response of IL-33.
  • Th1-related autoimmune diseases include autoimmune hepatitis, chronic rheumatoid arthritis, insulin-dependent diabetes, ulcerative colitis, Crohn's disease, multiple sclerosis, autoimmune myocarditis, psoriasis, scleroderma, myasthenia gravis, multiple myositis / skin myositis, Hashimoto's disease, autoimmune cytopenia (e.g., erythrocytopenia, aplastic anemia, etc.), Sjogren's syndrome, vasculitis syndrome, systemic lupus erythematosus, etc. are preferable.
  • autoimmune cytopenia e.g., erythrocytopenia, aplastic anemia, etc.
  • Sjogren's syndrome vasculitis syndrome
  • systemic lupus erythematosus etc.
  • the Th2-related autoimmune diseases include allergic asthma, perennial allergic rhinitis, seasonal allergic rhinitis, atopic dermatitis, contact hypersensitivity (including contact dermatitis), conjunctivitis, especially allergic conjunctivitis, eosinophilic bronchitis, food allergy, eosinophilic gastroenteritis, Inflammatory bowel disease, ulcerative colitis, Crohn's disease, mastocytosis and autoimmune diseases such as hyper IgE syndrom and systemic lupus erythematosus, psoriasis, acne, multiple sclerosis, transplant rejection, reperfusion injury, chronic obstructive pulmonary Diseases, rheumatoid arthritis, psoriatic arthritis and osteoarthritis, and the like.
  • IL-33 affinity chromatography isolated new IL-33 interacting proteins, IL-1R9 (IL-1RAcPL2; IL-1R9) and IL-18BP (IL-18 binding protein).
  • IL-33 interacts with IL-18BP but not IL-1R9 .
  • IL-18BP inhibits IFN ⁇ and IL-8, while anti-IL-1R9 selectively inhibits IFN ⁇ .
  • mature IL-33 induces IFN ⁇ , but precursor IL-33 fails to induce IFN ⁇ , which will help to investigate a broad spectrum of IL-33 activity in host defense against pathogens and inflammatory diseases.
  • the newly identified novel IL-33 receptor related molecules, IL-1R9 (IL-1RAcPL2) and IL-18BP are selectively involved in the biological activity of IL-33 in several cell types.
  • IL-1R4 and IL-1R3 are expressed in HMC-1 cells while human PBMCs express IL-1R4 and IL-1R9, which contributes to IFN ⁇ production. This result was demonstrated as anti-IL-1R9 antibody that specifically neutralizes IL-33-induced IFN ⁇ in human PBMC.
  • Water soluble IL-18BP and IL-1R4 specifically bound IL-33 (FIG. 2) and inhibited IL-33-induced cytokines.
  • anti-IL-1R9 could not block IL-8 induction in HMC-1 cells (FIG. 5A), but specifically inhibited IL-33-induced IFN ⁇ in PBMCs (FIG. 5B).
  • This result suggests a unique configuration of the IL-33 receptor in other cell types. That is, in HMC-1 cells the IL-33 receptor consists of IL-1R4 and IL-1R3, and in PBMC the IL-33 receptor consists of IL-1R4 and IL-1R9 (FIG. 5C).
  • IL of the second chain IL-1R3 or IL-1R9, a -33 receptor has a binding affinity to ligand after IL-33 / IL-1R4 precomplex formation (FIG. 5C).
  • Events of IL-33 / receptor complexes on cell membranes that induce downstream cytokines are hampered by soluble IL-33 receptors and binding proteins (FIG. 5C, right panel)
  • IFN ⁇ producing cell population was significantly increased in both human PBMC and mouse splenocytes / lymph node cells (FIG. 7).
  • the synergistic effect of the IL-12-induced IFN ⁇ producing cell population was more effective than that of LPS.
  • the results of the present invention will be an important experimental tool for analyzing the precise role of IL-33 in the immune response.
  • IL-33 protein 5 mg was produced to prepare IL-33 ligand-affinity chromatography as described in Immunity 10, 127-136 (1999). Concentrated human urine was applied to an IL-33 ligand-affinity column to isolate the water-soluble IL-33 binding protein. After washing, the acidic solution-eluted IL-33 binding protein was examined by Western blot. Prior to western blot, we used the IL-1R family to select candidate receptors that cause IL-1R9 (IL-1RAcPL2) to have high homology (35.3% identity) with IL-1R3 (IL-1RAcP). In comparison, the homology of the IL-33 receptor was analyzed.
  • IL-1RAcPL2 candidate receptors that cause IL-1R9
  • IL-1RAcP IL-1R3
  • Urine water soluble IL-1R3 (IL-1RAcP) and IL-1R4 (ST2) of the predicted molecular weight were detected (FIG. 1C / d) and the results were consistent with previous reports (Smith, DE, et al. Immunity 18, 87 -96 (2003); Yanagisawa, K., et al. J Biochem 121, 95-103 (1997)).
  • IL-18BPa inhibits precursor IL-33-induced IL-8 production in HMC-1 cells.
  • IL-33-induced IL-8 in human HMC-1 cells was inhibited by FcIL-18BPa (FIG. 3A), but IL-8 inhibition was not obtained by FcIL-18BPc (FIG. 3B).
  • FcIL-18BPa and c sufficiently inhibit dose-dependent mature IL-33-induced IL-8 (FIG. 3C / d). Inhibition of IL-33-induced IL-8 production by FcIL-18BPa More effective than that of FcIL-18BPc.
  • FcIL-1R4 and FcIL-18BP interact with mature IL-33, we have combined in mature IL-33-induced IL-8 production in HMC-1 cells.
  • FcIL-1R4 + FcIL-18BPa or FcIL-1R4 + FcIL-18BPc was tested.
  • IL-8 was effectively inhibited by the combined inhibitors and the dose response of the combined inhibitors was more linear than IL-18BP alone (FIG. 3E). / f).
  • precursor IL-33 (100 ng / ml) stimulated IL-8 production in HMC-1 cells showed that the biological activity of precursor IL-33 was 10 / compared with that of mature IL-33 (10 ng / ml). 20 times less active.
  • precursor and mature IL-33-induced IFN ⁇ in the presence of IL-12.
  • Human peripheral blood mononuclear cells (PBMC) and mouse splenocytes were stimulated with precursor and mature IL-33 in the presence of IL-12. Contrary to the results of FIG.
  • IL -One R9 Is PBMC In IL -33 is a component of the receptor HMC This is not the case for -1 cells
  • IL-33 ligand affinity chromatography isolated the rare receptor IL-1R9 (IL-1RAcPL2) (FIG. 1A), but recombinant FcIL-1R9 was unable to interact with both precursor and mature IL-33. It suggests that 1R9 acts as a signal transduction chain that has no direct binding affinity with the ligand. Therefore, we performed IL-33 analysis with anti-IL-1R9 and FcIL-1R4 to understand how IL-1R9 is involved in IL-33 biological activity. Mature IL-33 significantly increased IL-8 production in human HMC-1 (FIG. 5A).
  • the IL-33 inhibitory assay shows that HMC-1 is IL-1R4 (ST2) and IL- PBMCs express IL-1R4 (ST2) and IL-1R9 (IL-1RAcPL2) while expressing 1R3 (IL-1RAcP).
  • IL-33 / IL-1R4 precomplex recruits IL-1R3 in HMC-1 or IL-1R9 in PBMC (FIG. 5C, left to right panel).
  • IL-33 / IL-18BP and IL-33 / IL-33 inhibitory complexes such as sIL-1R4 are present in the circulation and inhibit IL-33 access to their receptors on cell membranes (FIG. 5C, right panel).
  • FcIL-18BPa and c as well as FcIL-1R4 specifically inhibited IL-18- and IL-33-induced IFN ⁇ while FcIL-1R4 was directed to IL-18-induced IFN ⁇ (indicated by the “#” symbol). There was no effect and this result was consistent with the immunoprecipitation assay (FIG. 2C / d). Similar inhibition was obtained with inhibitors combined in two or three and the inhibition was significant in FcIL-1R4 in combination with FcIL18BPa or FcIL18BPc (FIG. 6C).
  • IFN ⁇ producing cells were measured in human PBMC and mouse splenocytes after stimulation of IL-12, IL-33, and LPS.
  • IL-4 and IFN ⁇ producing cells were examined by FACS analysis 24 hours after stimulation as shown in FIG. 7.
  • IL-12 + IL-33 significantly increased the number of IFN ⁇ producing cells but no IL-4 producing cells were detected (FIG. 7A).
  • the increase of IFN ⁇ producing cells by IL-33 + LPS was IL It was weaker than that of -33 + IL-12.
  • the same experiment was repeated with mouse spleen cells (FIG. 7B) and lymph node cells (FIG. 7C). The results were very similar to human PBMCs.
  • T-helper (Th) T-cell subtype immune responses are regulated by specific cytokines that regulate unique subset differentiation and proliferation. It is important to understand the regulation of cytokines because unbalanced cytokine production leads to several immune diseases.
  • the present invention suggests that the merging edge of the Th1 / Th2 immune response is tightly regulated by IL-1 family cytokines and receptor related molecules.
  • the novel IL-33 receptors and binding proteins comprising the biological activity of mature IL-33 in the IFN ⁇ production of the present invention will be useful for investigating important immune regulation associated with Th1 / Th2 immune responses.
  • IL-33 can be used for Th1 and Th2-related autoimmune diseases by regulating both Th1 and Th2 cytokine.
  • the Th1-related autoimmune diseases include autoimmune hepatitis, chronic rheumatoid arthritis, insulin dependent diabetes mellitus, ulcerative colitis, Crohn's disease, multiple sclerosis, autoimmune myocarditis, psoriasis, scleroderma, myasthenia gravis, multiple myositis / skin myositis, Hashimoto Disease, autoimmune cytopenia (e.g., erythrocytopenia, aplastic anemia, etc.), Sjogren's syndrome, vasculitis syndrome, systemic lupus erythematosus, preferably
  • the Th2-related autoimmune diseases include allergic asthma, perennial allergic rhinitis, seasonal allergic rhinitis, atopic dermatitis, contact hypersensitivity (including contact dermatitis), conjunctivitis, especially allergic conjunctivitis, eosinophilic bronchitis, food allergy, eosinophilic gastroenteritis, Inflammatory bowel disease, ulcerative colitis, Crohn's disease, mastocytosis and autoimmune diseases such as hyper IgE syndrom and systemic lupus erythematosus, psoriasis, acne, multiple sclerosis, transplant rejection, reperfusion injury, chronic obstructive pulmonary Diseases, rheumatoid arthritis, psoriatic arthritis, and osteoarthritis.
  • FIG. 1 shows Western blot of water soluble IL-33 binding protein isolated from IL-33 ligand affinity chromatography.
  • Human IL-33 ligand-affinity chromatography isolated urine IL-33 interacting proteins and detected by Western blot. Four fractions were eluted from the IL-33 ligand affinity column.
  • Anti-IL-1R9 antibody recognizes urine soluble IL-1R9. A band with a molecular weight of about 53 kDa was detected in the eluted fractions. The theoretical molecular weight of mature IL-1R9 extracellular domain (340 polypeptide) is 39 kDa.
  • the predicted four N-linked glycosylation sites (triplet sequence Asn-X-Ser or Asn-X-Thr, X may be unspecified amino acids) increase the negligible molecular weight of urine IL-1R9.
  • Western blots with specific antibodies recognize urine (b) IL-18BP, (c) IL-1R4 or (d) IL-1R3. Data represent one of three independent experiments.
  • FIG. 2 is a diagram showing the specific interaction of the precursor and mature IL-33 with Fc receptor and binding protein.
  • precursor IL-33 were immunoprecipitated with recombinant FcIL-18BPa and c isoform, respectively.
  • recombinant FcIL-1R4 was used for immunoprecipitation of (c) Mature IL-18, (d) precursor IL-18, (e) Mature IL-33, or (f) precursor IL-33.
  • Each ligand 100 ng
  • Fc fusion protein 500 ng
  • a complex of ligand and Fc fusion protein was precipitated with protein A beads (10 ml). The mixed components are shown at the top. Data represent one of four independent experiments.
  • FIG. 3 shows inhibition of precursor or mature IL-33-induced IL-18 in HMC-1 cells.
  • (e and f) FcIL-1R4 in combination with FcIL-18BPa or FcIL-18BPc inhibited mature IL-33-induced IL-18 production and the dose reactivity of the combined inhibitors was more linear compared to IL-18BP alone.
  • the concentration of Fc fusion inhibitor is shown at the bottom of the graph and calculated in molar ratio. Data represent one of three independent experiments.
  • IL-33 receptor components are dependent on cell type.
  • Human HMC-1 cells were stimulated with mature IL-33 (20 ng / ml) alone or IL-33 mixed with FcIL-1R4 (0.5 mg / ml). For antibody neutralization, mature IL-33 was added 1 hour after pretreatment of anti-IL-1R9 (1 mg / ml) and control anti-IL-1R8.
  • human PBMCs were stimulated with mature IL-33 in the presence of IL-12. In contrast to HMC-1 cell assays, mature IL-33-induced IFN ⁇ production was completely inhibited by anti-IL-1R9.
  • FIG. 6 shows the additional effects of IFN ⁇ induction by IL-18 + IL-33 and IFN ⁇ inhibition by Fc-fused IL-33R inhibitory proteins.
  • FIG. (a) IL-12 + IL-18 or IL-12 + IL-33 increased the induction of IFN ⁇ whereas IL-12 + IL-18 in the presence of IL-33 had an additional effect on IFN ⁇ induction.
  • (c) Two or three Fc-fused IL-33R inhibitory proteins sufficiently inhibited IFN ⁇ production compared to a single inhibitor. Mean ⁇ SEM human IFN ⁇ ; *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001 (N 3 / group). Data represent one of three independent experiments.
  • Figure 7 shows the increase in IFN ⁇ producing cell population by costimulatory factors.
  • Flow cells after stimulating (a) human PBMCs, (b) mouse splenocytes and (c) lymph node cells with IL-33, LPS, IL-12, IL-12 + IL-33, or LPS + IL-33
  • the analysis was performed. Intracellular staining of IL-4 and IFN ⁇ indicates that IL-12 + IL-33 increases the population of IFN ⁇ producing cells but not IL-4.
  • the increased IFN ⁇ producing cell population obtained by LPS was less pronounced than that of IL-12 in both human and mouse cells. Data represent one of three independent experiments.
  • IL-33R related inhibitory molecules that inhibit IFN ⁇ induced by several costimulatory factors.
  • mouse splenocytes with (a) IL-2 + IL-33, IL-12 + IL-33, IFNa + IL-33, or TNF ⁇ + IL-33, the stimulator The cells were incubated with FcIL-1R4 + FcIL-18BPa for 30 minutes at room temperature. The most significant induction of IFN ⁇ was observed in IL-12 + IL-33. IL-2 and IFNa also increased IFN ⁇ production less markedly but TNF ⁇ failed to produce IFN ⁇ .
  • FcIL-1R4 + FcIL-18BPa effectively inhibited IL-33-induced IFN ⁇ production.
  • Mean ⁇ SEM human IFN ⁇ ; *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001 (N 4 per group). Data represent one of three independent experiments.
  • FIG. 9 shows a Western blot of IL-12 + IL-33 activated signal molecule.
  • PhosphSTAT4, NF-kB, p38MAPK, and p44 / 42MAPK were detected by stimulation of IL-2, IL-12 + IL-33, or IL-12 + IL-18 at various time points. Phosphorylation of STAT4 was observed 4 hours after stimulation and lasted up to 36 hours. Increased phosphSTAT4 was sustained by IL-33 compared to IL-12 alone. PhosphNF-kB activation was detected 30 minutes after IL-12 stimulation and its activation was maintained by IL-33 and IL-18.
  • Phosphp38MAPK and p44 / 42MAPK activation were not correlated with IFN ⁇ production.
  • the same signaling molecules were examined with several IL-33R related inhibitory molecules.
  • Human mature IL-33 (5 mg) was immobilized by coupling to Affigel-15 beads (1 ml volume) according to manufacturer's instructions (Bio-Rad laboratories, Hercules, Calif.). One batch of 250 ml crude urine protein, concentrated 500 times, was subjected to its ligand-affinity chromatography at 4 ° C. overnight. The IL-33 ligand-affinity column was washed with 500 ml of phosphate buffer containing 0.5 M sodium chloride, pH8. Binding proteins were eluted at a volume of 1 ml containing 25 mM citric acid, pH 2.2, and the eluted fractions were immediately neutralized with 2M glycine.
  • IL-33 receptor related molecules For detection of derived IL-33 receptor related molecules from IL-33 ligand-affinity purified water soluble protein, 20 ml of eluted fractions were loaded in 10% SDS-PAGE.
  • Anti-IL-18BP (YbdYbiotech, Seoul Korea), anti-IL-1R3, IL-1R4, and anti-IL-1R9 antibodies (R & D Systems, Minneapolis MN) were purchased and used for western blot.
  • Peroxidase-attached secondary antibodies Jackson Immuno Research Laboratories, West Grove, PA
  • Supex Neuroonex, Seoul Korea
  • LAS-4000 imaging device Flujifilm, Japan
  • western blots were performed with anti-IL-33 and IL-18 antibodies (YbdYbiotech).
  • human PBMC cells were stimulated with several combined cytokine stimulators at various time points in the presence or absence of IL-33 inhibitors.
  • Cells were disrupted with kinase disruption buffer (Cell Signaling Technology, Beverly, MA) and then subjected to 10% SDS-PAGE. Transfer the samples to a nitrocellulose membrane. The membranes were blocked in 3% BSA / TBST (Santa Cruz Biotechnology, Santa Cruz, CA).
  • the membranes were first probed with anti-phosph-NF-kB, anti-phosph-p38MAPK, or anti-phosph-p44 / 42MAPK (Cell Signaling Technology) and finally standardized with anti-GAPDH (Santa Cruz Biotechnology).
  • Recombinant precursor and mature IL-33 proteins were expressed in E. coli , rosetta cells and purified by TALON affinity chromatography (Invitrogen, Carlsbad, Calif.) Using His6-tag at the N-terminus of the recombinant protein.
  • the TALON affinity-purified proteins were performed by high performance liquid chromatography (GE Healthcare, Waukesha, Wi.).
  • the two-step purified recombinant protein was subjected to LAL method (Cape cod, East Falmouth, MA) according to the manufacturer's instructions. Was used in the experiment after testing the level (below 0.3EU / mg recombinant protein).
  • HMC-1 cells were purchased from the American Type Culture Collection (ATCC) and maintained as directed by ATCC. HMC-1 cells were cultured in Iscove's modified Dulbecco's medium (IMDM) suspended with 10% FBS. Bioassays were performed in 96-well plates. HMC-1 cells (1 10 5 / ml) were seeded into each well before assay. Several other IL-33 receptor related inhibitors and several stimulants were added to the cells. The plate was placed in a cell incubator for 18 hours and cytokines were measured in the supernatant.
  • IMDM Iscove's modified Dulbecco's medium
  • PBMCs Human PBMCs were separated by density centrifugation of blood in Ficoll-Paque TM PLUS (GE healthcare Life Sciences, NJ, USA). PBMCs were washed twice with brine (0.9% Sodium chloride) and resuspended in culture medium (RPMI 1640). Mouse splenocytes were obtained from female C57BL / 6 mice and disrupted with RBC disruption buffer (Sigma, St. Louis, Mo.) to remove red blood cells. The cells were washed twice with culture medium (RPMI 1640) and resuspended in the same medium.
  • PBMC peripheral blood mononuclear cells
  • mouse splenocytes (1 ⁇ 10 6 ) were seeded in 96-well plates at 100 ml volume and then 100 ml of culture medium (control) or 100 ml in the presence or absence of IL-33 receptor related inhibitors Incubated with several stimulants. Costimulatory factors, IL-2, IL-12 (Peprotech, Rocky Hill, NJ), IFNa2 (LG biotech, Seoul Korea), TNF ⁇ (YbdYbiotech) were used to treat primary cells. After 18 hours of stimulation, the cell culture supernatant was used for cytokine determination.
  • Mouse splenocytes and human PBMCs were obtained as above and used for flow cytometry.
  • splenocytes were cultured in 2 x 10 6 cells / well of 24-well plates in 1 ml medium and IL-12 (1 ng / ml), IL-18 (20 ng / ml; YbdYbiotech), IL-33 (20 ng). / ml) and LPS (1 mg / ml; Sigma) alone or in the combinations described.
  • GolgiStop (BD Bioscience, San Jose, Calif.) was added to the cells for cytokine accumulation.
  • PBMCs (2 ⁇ 10 6 ) were seeded in 24-well plates at 500 ml volume and incubated with several stimulants in the same manner as mouse splenocytes. After 24 h incubation, the cells were stained for FACS analysis.
  • IL-8 and IFN ⁇ ELISA kits were purchased from R & D systems. Cytokine levels were measured in culture supernatants (100 ml) by sandwich ELISA according to the manufacturer's instructions.

Abstract

La présente invention concerne une composition d'un nouveau récepteur de l'interleukine-33 et d'une protéine de liaison ainsi que son utilisation. La présente invention contribuera à vérifier un large spectre d'activités de l'IL-33 quant à la défense de l'hôte contre des pathogènes et des maladies inflammatoires.
PCT/KR2013/001157 2013-02-14 2013-02-14 Composition d'un nouveau récepteur de l'interleukine-33 et d'une protéine de liaison et son utilisation WO2014126277A1 (fr)

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KR1020157021498A KR101780575B1 (ko) 2013-02-14 2013-02-14 신규한 인터루킨-33 수용체와 결합 단백질 조성물 및 그 용도

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US11708608B2 (en) 2014-11-10 2023-07-25 Genentech, Inc. Therapeutic and diagnostic methods for IL-33-mediated disorders
US11725050B2 (en) 2014-11-10 2023-08-15 Genentech, Inc. Anti-interleukin-33 antibodies and uses thereof
US11760797B2 (en) 2020-03-13 2023-09-19 Genentech, Inc. Anti-interleukin-33 antibodies and uses thereof
CN113151481A (zh) * 2021-04-13 2021-07-23 东营市人民医院 生物标志物在食管鳞癌分化等级评估中的应用
WO2023178192A1 (fr) 2022-03-15 2023-09-21 Compugen Ltd. Anticorps antagonistes de l'il-18bp et leur utilisation en monothérapie et polythérapie dans le traitement du cancer

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