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WO2022039455A1 - Anticorps ou fragment de liaison à l'antigène de celui-ci qui se lie de manière spécifique à il-33 - Google Patents

Anticorps ou fragment de liaison à l'antigène de celui-ci qui se lie de manière spécifique à il-33 Download PDF

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WO2022039455A1
WO2022039455A1 PCT/KR2021/010812 KR2021010812W WO2022039455A1 WO 2022039455 A1 WO2022039455 A1 WO 2022039455A1 KR 2021010812 W KR2021010812 W KR 2021010812W WO 2022039455 A1 WO2022039455 A1 WO 2022039455A1
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antibody
fragment
amino acid
immunological activity
seq
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Korean (ko)
Inventor
이상호
박수빈
최철용
김선직
르한트룩치
황다현
조상우
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Sungkyunkwan University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/107—Crystal induced conditions; Gout
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/108—Osteoporosis

Definitions

  • the present invention has been made under the support of the Ministry of Science and ICT of the Republic of Korea under the project number 1711136261 and the project number 2021R1F1A1050449. ”, the title of the research project is “Structure-based mechanism of action study of site-specific ubiquitinated Rab7”, organized by Sungkyunkwan University, and the research period is 2021.06.01-2022.05.31.
  • the present invention was made by the project specific number 1711131074 and the project number 2017R1A5A1014560 under the support of the Ministry of Science and ICT of the Republic of Korea, the research management specialized institution of the above project is the National Research Foundation of Korea, the research project name is "Group Research Support (R & D)", The name of the research project is “Non-Lymphatic Organ Immunization Research Center”, the host institution is Sungkyunkwan University, and the research period is 2021.03.01-2022.02.28.
  • the present invention was made by the project specific number 1711134324 and the project number 2021M3A9I4022936 under the support of the Ministry of Science and ICT of the Republic of Korea, and the research management institution for the above project is the National Research Foundation of Korea, and the research project name is "Bio. Medical Technology Development (R & D)" ”, the research project name is “Bio-imaging-based control material discovery and innovation technology development”, the host institution is Sungkyunkwan University, and the research period is 2021.04.01-2021.12.31.
  • the present invention was made by the project specific number 1711127544 and the project number 2020M3A9E4039217 under the support of the Ministry of Science and ICT of the Republic of Korea, and the research management specialized institution of the above project is the National Research Foundation of Korea, and the research project name is "Bio.Medical Technology Development (R&D)" ”, the title of the research project is “Development of nanoprobes based on antibody/aptamer/molecular substances for drug detection and development of popular drug detection kits for the general public”, the lead institution is Sungkyunkwan University, and the research period is 2021.03.01-2021.12.31.
  • the present invention was made by the project number 1395067919 and project number PJ015672012021 under the support of the Rural Development Administration of the Republic of Korea, and the research and management agency of the above project is the Rural Development Administration, and the research project name is "Bio-Green Linked Agricultural Life Innovation Technology Development (R&D)" ,
  • the research project is titled “Development of new material manufacturing technology for environmental stress control using protein design based on agro-biosystem synthesis”, the lead institution is Sungkyunkwan University Industry-Academic Cooperation Foundation, and the research period is 2021.01.01-2021.12.31.
  • the present invention relates to an antibody or fragment having immunological activity that specifically binds to IL-33.
  • Interleukin-33 is a cytokine belonging to the Interleukin-1 family that plays a role in inflammatory conditions. IL-33 is always expressed in the nucleus of epithelial cells or vascular endothelial cells, and is released along with cell destruction due to tissue damage caused by infection or physical and chemical stress, and acts as an alarm. In addition, it is thought that there is a mechanism for IL-33 expression to be increased and secreted by stimulation such as lipopolysaccharide. IL-33 released out of the cell can bind to the IL-33 receptor expressed on the cell and activate an intracellular signal.
  • IL-33 receptor is expressed in various immune cells or epithelial cells, etc., and IL-33 induced intracellular signal transduction occurs.
  • IL-33 is expressed on the surface of immune cells such as Th2 cells, mast cells, eosinophils, basophils, natural killer (NK) T cells, and group 2 innate lymphocytes among the cells of the immune system expressing the IL-33 receptor. It works by binding to the existing receptor ST2, and ST2 combined with IL-33 forms a heterodimer with the accessory receptor IL-1RAcP and then MAPK and NF- ⁇ B pathways as a downstream signaling pathway. Activate it.
  • Th2 cytokines IL-4, IL-5, IL-6, IL-13, etc.
  • IL-1 ⁇ , IL-6 and TNF (tumor necrosis factor)- ⁇ is induced by IL-33 stimulation in mast cells and macrophages among immune system cells expressing IL-33 receptors, It is suggested that this is involved in the pathogenesis of autoantibody-induced arthritis (a model of rheumatoid arthritis) (Damo Xu et al., Journal of Immunology, 2010, Vol. 184, p2620).
  • IL-33 In humans, increased expression of IL-33 is associated with various inflammatory diseases (rheumatoid arthritis, asthma, systemic sclerosis, liver fibrosis, fibrosis such as pulmonary fibrosis, psoriasis, ulcerative colitis, Crohn's disease, multiple sclerosis, ankylosing spondylitis, etc.). It is recognized that IL-33 is involved in the onset and maintenance of various diseases (Yasushi Matsuyama et al., Journal of Rheumatology, 2010, Vol. 37, p18; David Pre ⁇ et al., Journal of Allergy and Clinical Immunology, 2010, Vol.
  • antibody drugs are growing the fastest in the biopharmaceutical field due to their high therapeutic effect and targeted therapeutic properties.
  • the global market for antibody drugs is growing at an average annual rate of 10-15%, and it is known to have formed a market of $44.7 billion in 2010.
  • Most of the diseases targeted by antibody drugs are concentrated on specific disease indications, such as intractable cancer (49%) and immune diseases (35%), and competition among products for similar indications is fierce. Nevertheless, as there are subdivided therapeutic targets within diseases, the field of anticancer antibody therapy development is expected to continue to grow in the future.
  • An object of the present invention is to provide a novel antibody specific for IL-33 or a fragment having immunological activity thereof.
  • the present invention provides an antibody specific for IL-33 or a fragment having immunological activity thereof.
  • the present invention provides a nucleic acid molecule encoding an antibody or a fragment having immunological activity thereof, a vector comprising the same, and a host cell transformed with the vector.
  • the present invention provides a method for producing an antibody specific for IL-33 or a fragment having immunological activity thereof.
  • the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases.
  • the present invention provides a method for providing information for the diagnosis of inflammatory diseases.
  • the monoclonal antibody or fragments having immunological activity that bind to a specific epitope of IL-33 according to the present invention have high antigen specificity, bind to IL-33 with high affinity and interfere with the binding of IL-33 to ST2. Since it acts as a neutralizing antibody that inhibits MAPK and NF- ⁇ B signaling induced by , IL-33, it can be usefully applied to the prevention or treatment of various diseases related to the release of IL-33, in particular, inflammatory diseases.
  • 1 is a diagram comparing the amino acid sequences of the top 6 single-chain antibody variable fragment (scFv) clones C1_1E1, C2_1D5, C2_2A10, C2_2E1, C2_2E12 and C2_2H5 showing a high binding signal to IL-33.
  • scFv single-chain antibody variable fragment
  • Figures 2a to 2c are diagrams showing the selection of antigen-specific scFv from the scFv-phage antibody pool obtained through panning:
  • C SDA-PAGE analysis result of purified recombinant C2_2E12 scFv antibody (M: size marker; S: antibody secreted medium; P: cell pellet; FT: Ni-NTA agarose binding solution; W: wash solution; and E: C2_2E12 eluate)
  • 3 is a view confirming the binding force of C1_1E1, C2_1D5, C2_2A10, C2_2E1, C2_2E12 and C2_2H5 to IL-33.
  • 5 is a diagram confirming the antigen binding sites of C1_1E1, C2_1D5, C2_2A10, C2_2E1 and C2_2H5.
  • 6a to 6d are diagrams confirming the antigen binding site of C2_2E12:
  • 8A and 8B are diagrams confirming the neutralizing antibody function of C2_2E12.
  • 9A to 9C are diagrams confirming the effect of C2_2E12 on MAPK and NF- ⁇ B signaling induced by IL-33 in order to confirm the role of the neutralizing antibody.
  • FIG. 10 is a diagram showing comparison of amino acid sequences of human-derived IL-33 protein and mouse-derived IL-33 protein.
  • FIG. 11 is a diagram confirming whether the antibody binds to the human-derived IL-33 protein and the mouse-derived IL-33 protein in order to confirm the cross-reactivity of C2_2E12.
  • FIG. 12 is a diagram showing the structure of a diabody in a protein crystal prepared to analyze the structure of the antibody C2_2E12 of the present invention.
  • FIG. 13 is a diagram illustrating a comparison between the crystal structure of C2_2E12 and the modeled molecular structure of itepekimab to confirm that the epitope structures of the C2_2E12 antibody and itepekimab, which are IL-33 specific antibodies, are different.
  • the present invention provides an antibody specific for IL-33 or a fragment having immunological activity thereof.
  • the antibody or fragment having immunological activity thereof is an epitope DLKKDEKKDK (SEQ ID NO: 46) consisting of residues 149 to 158 of the precursor form IL-33 consisting of the amino acid of SEQ ID NO: 1 It can bind to, and can bind to the 150th residue (leucine, L) or the 151st residue (lysine, K) of IL-33 consisting of the amino acid of SEQ ID NO: 1.
  • sequence starting from the 112th amino acid residue of the precursor form of IL-33 may be the mature form of IL-33.
  • the IL-33 may be of human origin.
  • the antibody or fragment having immunological activity thereof may be a monoclonal antibody, and is a neutralizing antibody that inhibits IL-33 and ST2 complex formation and inhibits activation of MAPK or NF- ⁇ B signaling pathway can
  • the fragment having immunological activity of the antibody is Fab, Fd, Fab', dAb, F(ab'), F(ab') 2 , scFv, Fv, single chain antibody, Fv dimer, complementarity It may be any one selected from the group consisting of a crystal region fragment, a humanized antibody, a chimeric antibody, and a diabody, and most preferably an scFv.
  • the antibody or fragment having immunological activity thereof comprises a complementarity determining regions heavy chain (CDRH)1 comprising the amino acid sequence of SEQ ID NO: 8, CDRH2 comprising the amino acid sequence of SEQ ID NO: 9, and SEQ ID NO: 10 to a VH domain comprising a CDRH3 comprising any one selected from the group consisting of the amino acid sequence of 15.
  • CDRH complementarity determining regions heavy chain
  • the antibody or fragment having immunological activity thereof is CDRL1 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 16 to 20, selected from the group consisting of the amino acid sequence of SEQ ID NOs: 21 to 26 It may include a VL domain comprising a CDRL2 comprising any one of
  • the antibody or fragment having immunological activity thereof comprises (i) CDRH (complementarity determining regions heavy chain)1 comprising the amino acid sequence of SEQ ID NO: 8, CDRH2 comprising the amino acid sequence of SEQ ID NO: 9, and a VH domain comprising a CDRH3 comprising any one selected from the group consisting of the amino acid sequences of SEQ ID NOs: 10 to 15; and/or (ii) a CDRL1 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 16 to 20, a CDRL2 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 21 to 26, and a sequence It may include a V domain comprising a VL domain comprising CDRL3 comprising any one selected from the group consisting of No. 27 to 31 amino acid sequence.
  • the antibody is not only in the form of a whole antibody, but also includes functional fragments of antibody molecules.
  • the whole antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond.
  • a functional fragment of an antibody molecule refers to a fragment having antigen-binding function
  • antibody fragments include (i) a light chain variable region (VL) and a heavy chain variable region (VH) and a light chain constant region (CL) and a Fab fragment consisting of the first constant region of the heavy chain (CH1); (ii) an Fd fragment consisting of the VH and CH1 domains; (iii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of the VH domain (Ward ES et al., Nature, 1989, Vol.
  • the antibody or fragment having immunological activity thereof may include any one or more selected from the group consisting of amino acid sequences of SEQ ID NOs: 2 to 45 in the variable variable region (V domain).
  • the antibody or fragment having immunological activity thereof is a VH comprising any one selected from the amino acid sequences of SEQ ID NOs: 8 to 15 and 32 to 39, and/or SEQ ID NOs: 16 to 31 and 40 to 45 It may include a VL comprising any one selected from the amino acid sequence of
  • the antibody or fragment having immunological activity thereof may include any one of the amino acid sequences of SEQ ID NOs: 2 to 7.
  • the antibody or immunologically active fragment thereof may include complementarity determining regions CDRH1, CDRH2 and CDRH3 in the heavy chain (VH domain) of the variable region, wherein CDRH1 comprises the amino acid sequence of SEQ ID NO: 8 and CDRH2 may include the amino acid sequence of SEQ ID NO: 9, and CDRH3 may include any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 10 to 15.
  • the antibody or fragment having immunological activity thereof may include complementarity determining regions CDRL1, CDRL2 and CDRL3 in the light chain (VL domain) of the variable region, wherein CDRL1 is the amino acid sequence of SEQ ID NOs: 16 to 20 It may include any one selected from the group consisting of, CDRL2 may include any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 21 to 26, and CDRL3 is amino acids of SEQ ID NOs: 27 to 31 It may include any one selected from the group consisting of sequences.
  • the antibody or fragment having immunological activity is provided.
  • FR1 comprising the amino acid sequence of SEQ ID NO: 32, CDRH1 comprising the amino acid sequence of SEQ ID NO: 8, FR2 comprising the amino acid sequence of SEQ ID NO: 33 or 34, CDRH2 comprising the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: FR3 comprising any one of the amino acid sequences of 35 to 37, CDRH3 comprising any one of the amino acid sequences of SEQ ID NOs: 10 to 15, and FR4 comprising the amino acid sequence of SEQ ID NOs: 38 or 39 a VH domain; and/or
  • FR1 comprising the amino acid sequence of SEQ ID NO: 40
  • CDRL1 comprising any one of the amino acid sequences of SEQ ID NOs: 16 to 20
  • FR2 comprising the amino acid sequence of SEQ ID NO: 41, the amino acid sequence of SEQ ID NO: 27 or 28 CDRL2 comprising
  • FR3 comprising any one of the amino acid sequences of SEQ ID NOs: 42 to 44
  • CDRL3 comprising any one selected from the amino acid sequences of SEQ ID NOs: 27 to 31, and the amino acid sequence of SEQ ID NO: 45 and a V domain comprising a VL domain comprising FR4.
  • the antibody or fragment having immunological activity thereof may be selected from the group consisting of an animal-derived antibody, a chimeric antibody, a humanized antibody, a human antibody, and a fragment having immunological activity thereof.
  • the antibody may be recombinantly or synthetically produced.
  • An animal-derived antibody produced by immunizing an animal to be immunized with a desired antigen may cause an immune rejection reaction when administered to a human for therapeutic purposes.
  • a chimeric antibody is one obtained by substituting a constant region of an animal-derived antibody that causes an anti-isotype reaction with that of a human antibody using a genetic engineering method. Chimeric antibodies have significantly improved anti-isotype response compared to animal-derived antibodies, but still have animal-derived amino acids in the variable region, potentially resulting in anti-idiotypic side effects. are doing A humanized antibody was developed to improve these side effects. This is produced by grafting complementarity determining regions (CDR) regions that play an important role in antigen binding among the variable regions of a chimeric antibody into a human antibody framework.
  • CDR complementarity determining regions
  • the most important thing in the CDR grafting technology for producing a humanized antibody is to select an optimized human antibody that can best accept the CDR region of an animal-derived antibody. structure analysis, molecular modeling technology, etc. are used. However, even when the CDR regions of an animal-derived antibody are transplanted into the optimized human antibody framework, there are cases in which amino acids that affect antigen binding are present while being located in the framework of the animal-derived antibody, so that antigen-binding ability is not preserved in many cases. Therefore, it can be said that the application of additional antibody engineering technology to restore antigen binding force is essential.
  • the antibody or fragment having immunological activity thereof may be isolated from a living body (not present in the living body) or may be non-naturally occurring, for example, synthetically or recombinantly produced.
  • the term "antibody” refers to a substance produced by stimulation of an antigen in the immune system, the type is not particularly limited, and can be obtained naturally or non-naturally (eg, synthetically or recombinantly).
  • Antibodies are advantageous for mass expression and production because they are very stable and have a long half-life not only in vitro but also in vivo.
  • the adhesion is very high.
  • a complete antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond.
  • the constant region of an antibody is divided into a heavy chain constant region and a light chain constant region, and the heavy chain constant region has gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ) and epsilon ( ⁇ ) types, subclasses gamma 1 ( ⁇ 1), gamma 2 ( ⁇ 2), gamma 3 ( ⁇ 3), gamma 4 ( ⁇ 4), alpha 1 ( ⁇ 1) and alpha 2 ( ⁇ 2).
  • the constant region of the light chain has kappa ( ⁇ ) and lambda ( ⁇ ) types.
  • the term “heavy chain” refers to a variable region domain V H and three constant region domains C H1 , C H2 and C H3 comprising an amino acid sequence having a variable region sequence sufficient to confer specificity to an antigen. and a full-length heavy chain including a hinge and a fragment thereof.
  • the term “light chain” refers to both full-length light chains and fragments thereof comprising a variable region domain VL and a constant region domain CL comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. interpreted as including
  • variable region or variable domain refers to a portion of an antibody molecule that exhibits many variations in sequence while performing a function of specifically binding to an antigen, and the variable region has complementarity There are determining regions CDR1, CDR2 and CDR3. Between the CDRs, a framework region (FR) portion exists to support the CDR ring.
  • FR framework region
  • the "complementarity determining region” is a ring-shaped region involved in antigen recognition, and the specificity of the antibody for the antigen is determined as the sequence of this region changes.
  • scFv single chain fragment variable
  • scFv single chain fragment variable
  • CDR complementarity determining region
  • the terms “specifically binding” or “specifically recognized” have the same meaning as commonly known to those skilled in the art, and mean that an antigen and an antibody specifically interact to conduct an immunological reaction. .
  • the term "immunologically active fragment” or "antigen binding fragments" of an antibody refers to a fragment of the entire immunoglobulin structure, comprising a portion capable of binding an antigen. means some For example, it may be scFv, (scFv)2, scFv-Fc, Fab, Fab' or F(ab')2, but is not limited thereto.
  • Fab has a structure having a light chain and heavy chain variable regions, a light chain constant region and a heavy chain first constant region (C H1 ), and has one antigen-binding site.
  • Fab' differs from Fab in that it has a hinge region comprising one or more cysteine residues at the C-terminus of the heavy chain C H1 domain.
  • the F(ab') 2 antibody is produced by forming a disulfide bond with a cysteine residue in the hinge region of Fab'.
  • Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region, and a recombinant technique for generating an Fv fragment is well known in the art.
  • the heavy chain variable region and the light chain variable region are linked by a non-covalent bond
  • single-chain Fv single-chain Fv
  • the linker may be a peptide linker consisting of any amino acids from 1 to 100 or 2 to 50 amino acids, and an appropriate sequence is known in the art.
  • the antigen-binding fragment can be obtained using a proteolytic enzyme (for example, Fab can be obtained by restriction digestion of the entire antibody with papain, and F(ab') 2 fragment can be obtained by digestion with pepsin), It can be produced through genetic recombination technology.
  • a proteolytic enzyme for example, Fab can be obtained by restriction digestion of the entire antibody with papain, and F(ab') 2 fragment can be obtained by digestion with pepsin
  • the term "hinge region” refers to a region included in the heavy chain of an antibody, which exists between the C H1 and C H2 regions, and functions to provide flexibility of the antigen-binding site in the antibody. means area.
  • the hinge may be derived from a human antibody, and specifically, may be derived from IgA, IgE, or IgG, such as IgG1, IgG2, IgG3, or IgG4.
  • the present invention provides a nucleic acid molecule encoding an antibody or immunologically active fragment thereof of the present invention, a vector comprising the same, and a host cell transformed with the vector.
  • the nucleic acid molecules of the present invention may be isolated or recombinant, and include single-stranded and double-stranded forms of DNA and RNA as well as corresponding complementary sequences.
  • the nucleic acid is a nucleic acid that has been separated from surrounding genetic sequences present in the genome of the individual from which it was isolated.
  • a nucleic acid synthesized enzymatically or chemically from a template such as a PCR product, a cDNA molecule, or an oligonucleotide
  • a nucleic acid resulting from such a procedure can be understood as an isolated nucleic acid molecule.
  • An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of separate fragments or as a component of a larger nucleic acid construct.
  • a nucleic acid is operably linked when placed into a functional relationship with another nucleic acid sequence.
  • the DNA of the presequence or secretion leader is operably linked to the DNA of the polypeptide when it is expressed as a preprotein in the form before the polypeptide is secreted
  • a promoter or enhancer ( enhancer) is operably linked to a coding sequence when it affects transcription of the polypeptide sequence
  • a ribosome binding site is operably linked to a coding sequence when positioned to facilitate translation.
  • operably linked means that the DNA sequences to be linked are located contiguously, and in the case of a secretory leader, they are contiguous and in the same reading frame. However, enhancers need not be located adjacent to each other. Linkage is achieved by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional methods.
  • the nucleic acid molecule encoding the antibody or immunologically active fragment of the present invention is an antibody expressed from the coding region due to codon degeneracy or considering codons preferred in the organism in which the antibody is to be expressed.
  • Various modifications can be made to the coding region within a range that does not change the amino acid sequence of It will be appreciated by those skilled in the art that it is included within the scope of the present invention. That is, as long as the nucleic acid molecule of the present invention encodes a protein having an equivalent activity, one or more nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof, and these are also included in the scope of the present invention.
  • the sequence of such a nucleic acid molecule may be single-stranded or double-stranded, and may be a DNA molecule or an RNA (mRNA) molecule.
  • the nucleic acid molecule encoding the antibody or fragment having immunological activity of the present invention according to the present invention may be inserted into an expression vector for protein expression.
  • Expression vectors usually contain a protein operably linked, ie, placed in a functional relationship, with regulatory or regulatory sequences, selectable markers, optional fusion partners, and/or additional elements.
  • the present invention by culturing a host cell transformed with a nucleic acid, preferably an expression vector containing a nucleic acid molecule encoding an antibody according to the present invention or an immunologically active fragment thereof, to induce protein expression.
  • Antibodies or fragments having immunological activity thereof according to the present invention can be produced.
  • host cells can be used, including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. Methods for introducing an exogenous nucleic acid into a host cell are known in the art and will vary depending on the host cell used.
  • E. coli which has a high industrial use value due to low production cost, can be produced as a host cell.
  • the vector of the present invention includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector.
  • Suitable vectors include a signal sequence or leader sequence for membrane targeting or secretion in addition to expression control elements such as promoter, operator, start codon, stop codon, polyadenylation signal and enhancer, and may be prepared in various ways depending on the purpose.
  • the promoter of the vector may be constitutive or inducible.
  • the signal sequence includes a PhoA signal sequence and an OmpA signal sequence when the host is Escherichia sp., and an ⁇ -amylase signal sequence and a subtilisin signal when the host is a Bacillus sp.
  • the vector may also contain a selection marker for selecting a host cell containing the vector, and in the case of a replicable expression vector, an origin of replication.
  • vector refers to a carrier capable of inserting a nucleic acid sequence for introduction into a cell capable of replicating the nucleic acid sequence.
  • Nucleic acid sequences may be exogenous or heterologous.
  • Vectors include, but are not limited to, plasmids, cosmids, and viruses (eg, bacteriophages).
  • viruses eg, bacteriophages.
  • One of skill in the art can construct vectors by standard recombinant techniques (Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988; and Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, NY, 1994 et al.).
  • expression control sequences such as promoter, terminator, enhancer, etc., membrane targeting or secretion according to the type of host cell in which the antibody is to be produced. Sequences and the like can be appropriately selected and combined in various ways depending on the purpose.
  • expression vector refers to a vector comprising a nucleic acid sequence encoding at least a portion of a transcribed gene product. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide.
  • the expression vector may include various regulatory sequences. In addition to regulatory sequences that control transcription and translation, vectors and expression vectors may contain nucleic acid sequences that also serve other functions.
  • the term "host cell” includes eukaryotes and prokaryotes, and refers to any transformable organism capable of replicating the vector or expressing a gene encoded by the vector.
  • a host cell may be transfected or transformed by the vector, which refers to a process in which an exogenous nucleic acid molecule is delivered or introduced into a host cell.
  • the host cell may be a bacterium or an animal cell
  • the animal cell line may be a CHO cell, a HEK cell or an NSO cell
  • the bacterium may be E. coli.
  • the present invention provides the steps of culturing a host cell comprising a vector containing a nucleic acid molecule encoding the aforementioned IL-33 specific antibody or fragment having immunological activity, and culturing the host cell It provides a method for producing an antibody or fragment having immunological activity specific for IL-33, comprising the step of recovering the antibody or fragment having immunological activity thereof from water.
  • the method may further include purifying the antibody or the immunologically active fragment.
  • the antibody or fragment having immunological activity of the present invention may be isolated or purified by various methods known in the art. Standard purification methods include chromatography techniques, electrophoresis, immunization, sedimentation, dialysis, filtration, concentration, and chromatofocusing techniques. As is known in the art, various native proteins, such as, for example, bacterial proteins A, G, and L, bind antibodies and these proteins can be used for purification. Often, purification by a specific fusion partner may be possible.
  • the antibody according to the present invention or a fragment having immunological activity thereof can be mass-produced there is.
  • Appropriate culture methods and medium conditions according to the type of host cells can be easily selected by those skilled in the art from known techniques.
  • the host cell may be a prokaryotic organism such as E. coli or Bacillus subtilis . It may also be a eukaryotic cell derived from yeast, such as Saccharomyces cerevisiae , an insect cell, a plant cell, or an animal cell. More preferably, the animal cells may be autologous or allogeneic animal cells.
  • a transformant prepared by introducing an autologous or allogeneic animal cell can be administered to an individual and used in cell therapy to treat cancer. Any method known to those skilled in the art may be used for the vector introduction method into the host cell.
  • Other organisms, including transgenic (eg, genetically engineered) mice, or other mammals, may be used to produce antibodies of the invention or fragments with immunological activity thereof (see, eg, US 6,300,129).
  • a mouse engineered by replacing only the variable regions (heavy chain V, D, and J segments, and light chain V and J segments) of a mouse immune gene with the corresponding human variable sequences can generate high-affinity antibodies with human variable sequences. It is known that it can be used for mass production (see, for example, US 6,586,251; US 6,596,541, and US 7,105,348).
  • the present invention provides an antibody specific for IL-33 of the present invention or a fragment having immunological activity thereof; a nucleic acid molecule encoding it; Or it relates to a pharmaceutical composition for preventing or treating inflammatory diseases containing at least one selected from the group consisting of vectors containing the nucleic acid molecule as an active ingredient.
  • the inflammatory disease is toxic shock syndrome and ulcerative colitis, osteoporosis, gout, tendinitis, tendonitis, lupus, fibromyalgia, sinusitis, otitis media, pneumonia, gastritis, enterocolitis, colitis, arthritis, hepatitis, Dermatitis, osteoarthritis, asthma, atopic dermatitis, hay fever, anaphylaxis, rheumatoid arthritis, psoriasis, ulcerative colitis, Crohn's disease, ankylosing spondylitis, inflammatory bowel disease, allergy, allergic rhinitis, allergic conjunctivitis, spring keratoconjunctivitis, seasonal allergy, Pet allergy, food allergy, peanut allergy, atopic dermatitis, chronic rhinosinusitis with nasal polyps (CRSwNP), bronchitis, chronic obstructive pulmonary disease (COPD), viral exacerbation of respiratory diseases,
  • prevention refers to any action that suppresses or delays the occurrence, spread, and recurrence of an inflammatory disease by administration of the composition according to the present invention.
  • treatment refers to any action that improves or advantageously changes symptoms of inflammatory diseases and complications resulting therefrom by administration of the composition according to the present invention.
  • Those of ordinary skill in the art to which the present invention pertains with reference to the data presented by the Korean Medical Association, etc., know the exact standard of the disease for which the composition of the present application is effective, and can determine the degree of improvement, improvement and treatment will be.
  • terapéuticaally effective amount used in combination with an active ingredient in the present invention means an amount effective to prevent or treat an inflammatory disease
  • the therapeutically effective amount of the composition of the present invention depends on several factors, for example, the administration method. , the target site, the patient's condition, etc. Therefore, when used in the human body, the dosage should be determined as an appropriate amount in consideration of both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. These considerations in determining effective amounts are found, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed., 2001, Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed., 1990, Mack Publishing Co.
  • the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not to cause side effects, and the effective dose level is determined by the patient's Health status, type of inflammatory disease, severity of inflammatory disease, drug activity, drug sensitivity, administration method, administration time, administration route and excretion rate, treatment period, factors including drugs used in combination or concurrently, and other medical fields can be determined according to well-known factors in
  • the composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.
  • the pharmaceutical composition of the present invention may include a carrier, diluent, excipient, or a combination of two or more commonly used in biological agents.
  • pharmaceutically acceptable refers to exhibiting properties that are not toxic to cells or humans exposed to the composition.
  • the carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, and for example, Merck Index, 13th ed., Merck & Co. Inc.
  • saline sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components can be mixed and used, and if necessary, other antioxidants, buffers, bacteriostats, etc. Conventional additives may be added.
  • diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets.
  • injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets.
  • it can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18 th ed., 1990).
  • the pharmaceutical composition may be one or more formulations selected from the group including oral formulations, external preparations, suppositories, sterile injection solutions and sprays, and oral or injection formulations are more preferred.
  • the term "administration” means providing a predetermined substance to a subject or patient by any suitable method, and parenteral administration (eg, intravenous, subcutaneous, intraperitoneal) according to a desired method Alternatively, it can be applied locally as an injection formulation) or orally administered, and the dosage varies according to the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease.
  • Liquid formulations for oral administration of the composition of the present invention include suspensions, internal solutions, emulsions, syrups, etc., and various excipients, such as wetting agents, sweetening agents, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. and the like may be included.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.
  • the pharmaceutical composition of the present invention may be administered by any device capable of transporting an active substance to a target cell.
  • Preferred administration methods and formulations are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, and the like.
  • aqueous solvents such as physiological saline and Ringel's solution, vegetable oil, higher fatty acid esters (eg, ethyl oleate), and non-aqueous solvents such as alcohols (eg, ethanol, benzyl alcohol, propylene glycol, glycerin, etc.)
  • Stabilizers for preventing deterioration e.g., ascorbic acid, sodium hydrogen sulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.
  • emulsifiers emulsifiers
  • buffers for pH control to inhibit the growth of microorganisms
  • Pharmaceutical carriers such as preservatives (eg, phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol,
  • the term "individual” refers to monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, bats, and camels including humans that have or may develop the inflammatory disease. , rats, rabbits, or all animals including guinea pigs, and the “sample” may be droplets, sputum, whole blood, plasma, serum, urine or saliva isolated therefrom.
  • the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive includes starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate. , lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, Calcium stearate, sucrose, dextrose, sorbitol and talc and the like may be used.
  • the pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.
  • the present invention comprises the steps of obtaining a sample separated from the body from the subject; treating the sample with the antibody or fragment having immunological activity of the present invention; And it provides a method of providing information for diagnosing an inflammatory disease comprising the step of determining whether the level of IL-33 contained in the sample of the subject is higher than the level of IL-33 contained in the sample of the normal group.
  • the method may further include determining that the subject has an inflammatory disease.
  • the present invention provides an antibody according to an aspect of the present invention or a fragment having immunological activity thereof; Or it provides a method for preventing or treating an inflammatory disease comprising administering the pharmaceutical composition according to an aspect of the present invention to a subject in need of treatment.
  • the subject is a mammal or a human.
  • the mammal includes, but is not limited to, dogs, cats, cattle, horses, pigs, sheep, and non-human primates.
  • the method for preventing or treating an inflammatory disease of the present invention includes an antibody or a fragment having immunological activity thereof according to an aspect of the present invention; Or, since it includes the step of administering the above-described pharmaceutical composition according to an aspect of the present invention to a subject in need of treatment, descriptions of common contents therebetween are omitted in order to avoid excessive complexity of the present specification.
  • Condition 1 Round Antigen concentration( ⁇ g ml -1 ) Tier of input(cfu a ) Tier of output(cfu a ) Tier of output(cfu a )
  • Condition 2 Round Antigen concentration( ⁇ g ml -1 ) Tier of input(cfu a ) Tier of output(cfu a ) Tier of output(cfu a )
  • scFv phage ELISA was performed. Specifically, the scFv obtained through the fifth round of panning in Example 1-1 was spread and grown on an LB (Luria-Bertani) plate, and then 384 scFv clones were selected. They were cultured in 96-well cell culture plates, and ELISA was performed by inoculating the GST-tagged IL-33-coated plates. Here, a plate containing only GST protein, not IL-33 antigen, containing scFv was used as a negative control for ELISA.
  • the K d value was obtained by ELISA analysis while the concentration of the antigen was fixed and the concentration of the antibody was changed (Fig. 4 and Table 2). .
  • a stop codon (stop codon) was inserted to construct recombinant GST-IL-33, and the binding affinity of C1_1E1, C2_1D5, C2_2A10, C2_2E1 or C2_2H5 to each of them was confirmed.
  • amino acid residues 149 to 158 of IL-33 are C1_1E1, C2_1D5 , it was confirmed that it is important for binding to C2_2A10, C2_2E1 or C2_2H5 ( FIG. 5 ).
  • alanine (A) for amino acid residues 149 to 158 of IL-33, binding affinity to C2_2E12 was confirmed, and amino acid residues 150 and 151 of IL-33 were confirmed to be important for binding to C2_2E12 ( 6B). Accordingly, HADDOCK (High Ambiguity-Driven biomolecular DOCKing)-derived molecular docking of C2_2E12 to IL-33 was confirmed and shown in Table 3 below. Residues in the epitope region of IL-33 recognized by C2_2E12 were identified as a stick model. And the electrostatic interaction of C2_2E12 and IL-33 was confirmed and represented by PyMOL (Schrodinger) (FIGS. 6C and D).
  • Molecule C2_2E12 HADDOCK score (AU a ) -97.8 ⁇ 11.4 Cluster size 5 R.m.s.d. from the overall lowest-energy structure ( ⁇ ) 0.5 ⁇ 0.3 van der Waals energy (kcal mol -1 ) -40.0 ⁇ 2.9 Electrostatic energy (kcal mol -1 ) -261.1 ⁇ 60.1 Desolvation energy (kcal mol -1 ) -6.8 ⁇ 5.6 Restraints violation energy (kcal mol -1 ) 12.2 ⁇ 14.47 Buried surface area ( ⁇ 2 ) 1350.8 ⁇ 90.0 Z-score -1.3
  • a cross-reactivity experiment was performed to confirm the antigen specificity of C2_2E12 among the IL-33-specific antibodies obtained through the panning. Specifically, using C2_2E12 as a primary antibody (0.5 mg/ml, 1:100 dilution) and anti-HA-HRP as a secondary antibody (0.2 mg/ml, 1:5000 dilution), IL- among interleukin proteins As a result of immunoblot analysis of IL-1b, which is statistically closest to 33, IL-6, and GST, BSA and IlvC proteins that are not related to interleukin, C2_2E12 specifically binds only to IL-33. was confirmed (FIG. 7).
  • IL-33-specific antibody C2_2E12 can function as a neutralizing antibody capable of inhibiting the binding between IL-33 and ST2.
  • HMC-1 cells that internally express ST2 and IL-1RAcP and those that do not express ST2 and IL-1RAcP HeLa cells were stimulated with human IL-33 (1 ng/ml, 8 min), treated with increasing amounts of C2_2E12, and then each cell was lysed to release ERK, JNK and I ⁇ B ⁇ in the MAPK and NF- ⁇ B signaling pathways. The pattern of phosphorylation band change was confirmed by immunoblot analysis.
  • IL-33 treatment increased JNK expression, increased JNK and ERK phosphorylation, and decreased JNK and ERK phosphorylation in a concentration-dependent manner of C2_2E12.
  • I ⁇ B ⁇ was decreased due to IL-33 treatment, and then increased in a concentration-dependent manner of C2_2E12 ( FIG. 9 ). That is, it was confirmed that C2_2E12 inhibits IL-33-induced MAPK and NF- ⁇ B signaling in a concentration-dependent manner, and this shows that C2_2E12 also acts as a neutralizing antibody in cells.
  • the peptide sequences of human-derived IL-33 and mouse-derived IL-33 are shown in SEQ ID NOs: 1 and 47, and the protein sequences of human-derived and mouse-derived IL-33 were aligned and compared using ClustalW. The results of comparing the peptide sequences of human-derived IL-33 and mouse-derived IL-33 are shown in FIG. 10 .
  • the mature form of IL-33 corresponds to amino acid residues 112 to 270 in human-derived proteins and amino acid residues 102 to 266 in mouse-derived proteins.
  • a specific protease acts to cut between amino acid residues 111 and 112 in the case of human-derived IL-33 and between amino acid residues 101 and 102 in the case of mouse-derived IL-33 (“cleavage site”).
  • the amino acid sequence portion of human-derived IL-33 recognized by the C2_2E12 antibody is indicated by a black line ( FIG. 10 ). Among these, positions with a large sequence difference between human-derived IL-33 and mouse-derived IL-33 are indicated by black triangles.
  • Human-derived IL-33 (112-270) and mouse-derived IL-33 (102-266) were expressed in Escherichia coli (BL21(DE3) strain) in the form of pGST2 vector encoded in the form of GST-fusion protein, glutathione Sepharose 4B It was purified using resin (GE Healthcare).
  • C2_2E12 scFv was cloned into pComb3x vector and expressed in ER2537 E. coli cells and used in the experiment in the form of lysate.
  • the C2_2E12 antibody of the present invention that recognizes human-derived IL-33 did not recognize mouse-derived antigen (mouse IL-33, abbreviation mIL-33) unlike general antibodies, and human-derived IL-33 was specifically bound to
  • the sequence difference between human-derived IL-33 and mouse-derived IL-33 shown in FIG. 10 is considered to be a major cause of not showing cross-reactivity.
  • the purified C2_2E12 diabody was concentrated to 10 mg/ml and crystallized through vapor diffusion method.
  • the C2_2E12 antibody was expressed and purified by converting it into a diabody form.
  • the linker sequence corresponding to ⁇ GGGGS> 2 was deleted and R87T/E89C mutation was introduced.
  • the cloned C2_2E12 diabody was expressed by introducing (transfection) into human-derived Expi293 cells. After expression at 37°C for 5 days, the supernatant was added to Ni-NTA resin and purified. The final purification was performed using size exclusion chromatography using a Superdex 200 column.
  • Crystals were formed after incubation at 25% PEG3350, 0.2 M NaCl, 0.1 M sodium citrate pH 5.5, 22°C for about 2 months, and diffraction data were collected at the Pohang Accelerator Laboratory. Molecular replacement was performed using the PDB ID:6QB3 structure as a search model. Finally, the three-dimensional molecular structure of the C2_2E12 diabody was identified with a resolution of 2.75 ⁇ . The three-dimensional molecular structure is shown in FIG. 12, and the analysis results are shown in Table 4.
  • two modified scFvs made a polymeric diabody. In the crystal structure, it was observed that all 12 diabodies were present in the asymmetric unit of the protein crystal.
  • Example 8 Human-derived IL-33 complex structural modeling and comparison of the structure of C2_2E12 antibody and Itepekimab
  • the three-dimensional molecular structure of Itepekimab was modeled using SwissModel (Waterhouse et al., Nucleic Acids Res., 2018, Vol. 46(W1), pW296).
  • the complex structure between C2_2E12 and IL-33 was calculated using the HADDOCK server (van Zundert et al., J. Mol. Biol., 2015, Vol. 428, p720). The results are shown in FIG. 13 .
  • the C2_2E12 crystal structure was very similar to the C2_2E12 structure predicted through modeling.
  • the modeled itepekimab structure was overlaid on the complex structure model derived through molecular docking calculation and analyzed.
  • the structure of the CDR3 (HCDR3) portion of the heavy chain was significantly different.

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Abstract

La présente invention concerne un anticorps qui se lie de manière spécifique à IL-33, ou un fragment de celui-ci ayant une activité immunologique. L'invention concerne un nouvel anticorps monoclonal qui se lie à un épitope spécifique de l'IL-33, ou un fragment de celui-ci ayant une activité immunologique, selon la présente invention, peut avoir une spécificité à l'antigène élevée et se lie à IL-33 avec une affinité élevée pour interférer avec la liaison de l'IL-33 et de ST2, ce qui agit comme un anticorps neutralisant qui inhibe la signalisation MAPK et NF-κB induite par IL-33, et qui peut être utilement appliqué à la prévention ou au traitement de diverses maladies liées à la libération d'IL-33, en particulier des maladies inflammatoires.
PCT/KR2021/010812 2020-08-18 2021-08-13 Anticorps ou fragment de liaison à l'antigène de celui-ci qui se lie de manière spécifique à il-33 Ceased WO2022039455A1 (fr)

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