WO2022039455A1 - Antibody or antigen-binding fragment thereof that specifically binds to il-33 - Google Patents

Abstract

The present invention relates to an antibody that specifically binds to IL-33, or a fragment thereof having immunological activity. A novel monoclonal antibody that binds to a specific epitope of IL-33, or a fragment thereof having immunological activity, of the present invention, may have high antigen specificity and bind to IL-33 with high affinity to interfere with binding of IL-33 and ST2, thereby acting as a neutralizing antibody which inhibits IL-33-induced MAPK and NF-κB signaling, and which can be usefully applied to the prevention or treatment of various diseases related to the release of IL-33, in particular, inflammatory diseases.

Description

Antibody or antigen-binding fragment thereof that specifically binds to IL-33

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.

In addition, 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.

In addition, 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.

In addition, 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.

In addition, 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.

This patent application claims priority to Korean Patent Application No. 10-2020-0103135 filed with the Korean Intellectual Property Office on August 18, 2020, the disclosure of which is incorporated herein by reference.

The present invention relates to an antibody or fragment having immunological activity that specifically binds to IL-33.

Interleukin-33 (IL-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-κB pathways as a downstream signaling pathway. Activate it. Their activation is thought to induce allergic inflammation (asthma, atopic dermatitis, hay fever, anaphylaxis, etc.) by inducing the production of Th2 cytokines (IL-4, IL-5, IL-6, IL-13, etc.) (Tatsukuni Ohno et al., Allergy, 2012, Vol. 67, p1203). In addition, the production of IL-1β, IL-6 and TNF (tumor necrosis factor)-α 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). 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. 125, p752; Koichi Yanaba et al., Clinical Rheumatology, 2011, Vol. 30, p825; AL Rankin et al., Journal of Immunology, 2010, Vol. 184, p1526; Tamar Mchedlidze et al., Immunity, 2013, Vol. 39, p357; Liang-An Hu et al., Asian Pacific Journal of Cancer Prevention, 2013, Vol. 14, p2563; Luca Pastorelli et al., Proceedings of the National Academy of Sciences of the United States of America, 2010, vol. 107, p8017). As the involvement of IL-33 in various diseases, particularly inflammatory diseases, is known, development of IL-33 agonists and antagonists is being made. Polyclonal antibodies are known.

On the other hand, 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.

In order to achieve the above object, the present invention provides an antibody specific for IL-33 or a fragment having immunological activity thereof.

In addition, 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.

In addition, the present invention provides a method for producing an antibody specific for IL-33 or a fragment having immunological activity thereof.

In addition, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases.

In addition, 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-κB 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.

Figures 2a to 2c are diagrams showing the selection of antigen-specific scFv from the scFv-phage antibody pool obtained through panning:

A: 10 scFvs with high ELISA assay values;

B: Comparison of amino acid sequences of C1_1E1, C2_1D5, C2_2A10, C2_2E1, C2_2E12 and C2_2H5; and

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.

4 is a view confirming the binding force of C2_2E12 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:

A: epitope mapping of IL-33;

B: epitope mapping among residues 149-158 of IL-33;

C: stick model of residues in the epitope region of IL-33 recognized by C2_2E12; and

D: Electrostatic interaction between C2_2E12 and IL-33.

7 is a diagram confirming the antigen specificity 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-κB signaling induced by IL-33 in order to confirm the role of the neutralizing antibody.

10 is a diagram showing comparison of amino acid sequences of human-derived IL-33 protein and mouse-derived IL-33 protein.

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.

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.

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.

Hereinafter, the present invention will be described in detail by way of embodiments of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and when it is determined that detailed descriptions of well-known techniques or configurations known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted, and , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of equivalents interpreted therefrom and the description of the claims to be described later.

In addition, the terms used in this specification are terms used to properly express the preferred embodiment of the present invention, which may vary depending on the intention of a user or operator or customs in the field to which the present invention belongs. Accordingly, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

Throughout this specification, common one-letter and three-letter codes for naturally occurring amino acids are used as well as generally accepted for other amino acids such as Aib (alpha-aminoisobutyric acid), Sar (N-methylglycine), etc. A three-character code is used. Amino acids referred to by abbreviation in the present invention are also described according to the IUPAC-IUB nomenclature as follows:

Alanine: A, Arginine: R, Asparagine: N, Aspartic Acid: D, Cysteine: C, Glutamic Acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine : M, phenylalanine: F, proline: P, serine: S, threonine: T, tryptophan: W, tyrosine: Y and valine: V.

According to one aspect of the present invention, the present invention provides an antibody specific for IL-33 or a fragment having immunological activity thereof.

In one embodiment, 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.

In one embodiment, the sequence starting from the 112th amino acid residue of the precursor form of IL-33 may be the mature form of IL-33.

In one embodiment, the IL-33 may be of human origin.

In one embodiment, 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-κB signaling pathway can

In one embodiment, 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.

In one embodiment, 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.

In one embodiment, 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

In one embodiment, 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, and examples of 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. 341, p544); (v) an isolated CDR region; (vi) a F(ab')2 fragment, which is a bivalent fragment comprising two linked Fab fragments; (vii) a single chain Fv molecule (scFv) joined by a peptide linker that joins the VH domain and the VL domain to form an antigen binding site; (viii) a bispecific single-chain Fv dimer (PCT/US92/09965) and (ix) a multivalent or multispecific fragment produced by gene fusion, diabody WO94/13804), and the like.

In one embodiment, 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).

In one embodiment, 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

In one embodiment, the antibody or fragment having immunological activity thereof may include any one of the amino acid sequences of SEQ ID NOs: 2 to 7.

In one embodiment, 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.

In one embodiment, 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.

In one embodiment, the antibody or fragment having immunological activity

i) 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

ii) 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.

In the present invention, 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.

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. can

In the present invention, 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). can 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. In addition, since the antibody essentially has a dimer structure, the adhesion (avidity) 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 (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.

As used herein, 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. In addition, 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

As used herein, the term “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. 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.

As used herein, the term "scFv (single chain fragment variable)" refers to a single-chain antibody made by expressing only the variable region of an antibody through genetic recombination, and a single-chain form in which the VH region and the VL region of the antibody are connected by a short peptide chain. refers to the antibody of The term “scFv” is intended to include scFv fragments, including antigen-binding fragments, unless otherwise indicated or understood otherwise by context. This is obvious to those skilled in the art.

As used herein, the term "complementarity determining region (CDR)" refers to the amino acid sequence of the hypervariable region of the heavy chain and light chain of an immunoglobulin. The heavy and light chains may each comprise three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs may provide key contact residues for the binding of an antibody to an antigen or epitope.

In the present invention, 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. .

As used herein, 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. Among the antigen-binding fragments, 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. In a double-chain Fv (two-chain Fv), the heavy chain variable region and the light chain variable region are linked by a non-covalent bond, and in single-chain Fv (single-chain Fv), the heavy chain variable region and the single chain variable region are generally shared through a peptide linker. Since they are linked by a bond or are linked directly at the C-terminus, they can form a dimer-like structure like a double-stranded 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.

As used herein, 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. For example, 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.

According to another aspect of the present invention, 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. In the case of a nucleic acid isolated from a naturally occurring source, 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. In the case of 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. For example, 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, and a promoter or enhancer ( enhancer) is operably linked to a coding sequence when it affects transcription of the polypeptide sequence, or a ribosome binding site is operably linked to a coding sequence when positioned to facilitate translation. In general, 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. In an appropriate state, 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. A variety of suitable 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. Preferably, 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 α-amylase signal sequence and a subtilisin signal when the host is a Bacillus sp. When the host is yeast, the MFα signal sequence, SUC2 signal sequence, etc., and when the host is an animal cell, an insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, etc. can be used, It is not limited thereto. 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.

As used herein, the term "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). 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.).

In one embodiment, when constructing the vector, 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.

As used herein, the term "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.

In the present invention, 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.

In one embodiment, 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, and the bacterium may be E. coli.

According to another aspect of the present invention, 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.

In one embodiment, after recovering the antibody or the immunologically active fragment from the host cell culture, 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.

After transforming the vector according to the present invention into an appropriate host cell, for example, E. coli or yeast cell, and then culturing the transformed host cell, 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). For example, 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).

In one aspect, 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.

In one embodiment, 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, viral infections in children and adults ( Respiratory syncytial virus (RSV), rhinovirus, influenza), urticaria, eosinophilic esophagitis, chronic fibrosis, liver fibrosis, systemic sclerosis, idiopathic pulmonary fibrosis (IPF), scleroderma, liver fibrosis, fibrosis including pulmonary fibrosis, non-alcoholic steatohepatitis (NASH), chronic kidney disease, acute kidney injury, sepsis, pancreatitis, type 1 diabetes, graft-versus-host disease (GVHD), Alzheimer's disease, rheumatoid arthritis, irritable bowel syndrome (IBS), ulcerative colitis , multiple sclerosis, psoriasis, celiac disease, and may be any one or more selected from the group consisting of Raynaud's disease or Raynaud's phenomenon.

In the present invention, the term "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.

As used herein, the term “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.

The term "therapeutically effective amount" used in combination with an active ingredient in the present invention means an amount effective to prevent or treat an inflammatory disease, and 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. As used herein, the term "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. As used herein, the term “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. The compound described in , 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. In addition, 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. Furthermore, 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).

In one embodiment, 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.

As used herein, 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. For injection, 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, buffers for pH control, to inhibit the growth of microorganisms Pharmaceutical carriers such as preservatives (eg, phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.) may be included.

As used herein, 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.

According to another aspect of the present invention, 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.

In one embodiment, when 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.

According to another aspect of the present invention, 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.

In one embodiment, 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.

The present invention will be described in more detail through the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited thereto.

Example 1. IL-33 specific scFv screening

1-1. biopanning

10 μg of antigen large synthetic human scFv library was put into an immunotube, coated at 37° C. for 1 hour, and blocked at room temperature (room temperature, RT) for 1 hour with PBS buffer containing 5% skim milk. One set of scFv library cells with a diversity of 1.0 X 10 ¹² were phage-infected, incubated at 30° C. for 16 hours, concentrated with polyethylene glycol 8000, and suspended in PBS buffer solution. Put the prepared library phage into the blocked immunotube. The reaction was carried out for 1 hour. After washing with 1xPBST and extracting only single-chain antibody-phages that specifically bind to the antigen through the elution solution, positive phage pools were obtained through a panning process in which the phages were again infected with E. coli and amplified. In the first round of panning, only the number of times was increased in the washing step with the amplified phage, and 2, 3, 4, and 5 rounds of panning were performed in the same manner for the rest, and are shown in Table 1 below.

Condition 1
Round	Antigen concentration(μg ml -1 )	Tier of input(cfu a )	Tier of output(cfu a )	Tier of output(cfu a )
One	50	1.6x10 12	2.9x10 8	3.0x10 8
2	10	1.9x10 11	1.6x10 7	3.6x10 7
3	7.5	1.2x10 13	8.8x10 7	8.0x10 7
4	5	1.0x10 11	5.4x10 6	1.1x10 7
5	2.5	1.9x10 11	1.5x10 8	1.2x10 8
Condition 2
Round	Antigen concentration(μg ml -1 )	Tier of input(cfu a )	Tier of output(cfu a )	Tier of output(cfu a )
One	50	1.6x10 12	2.9x10 8	3.0x10 8
2	10	1.9x10 11	3.7x10 7	3.0x10 7
3	7.5	1.2x10 13	6.8x10 7	1.8x10 7
4	5	1.0x10 11	3.4x10 7	2.5x10 7
5	2.5	1.9x10 11	3.4x10 7	2.3x10 8

^a colony forming unit

1-2. IL-33 specific scFv selection

In order to select antigen-specific scFv from the scFv-phage antibody pool obtained through the panning, 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. As a result of ELISA analysis, 10 scFvs with high absorbance values at 450 nm were selected (FIG. 2A), and the top 6 clones C1_1E1, C2_1D5, C2_2A10, C2_2E1, C2_2E12 and C2_2H5 showing a high binding signal to IL-33 were selected. The sequences of the selected C1_1E1 (SEQ ID NO: 3), C2_1D5 (SEQ ID NO: 4), C2_2A10 (SEQ ID NO: 5), C2_2E1 (SEQ ID NO: 6), C2_2E12 (SEQ ID NO: 2) and C2_2H5 (SEQ ID NO: 7) were analyzed (Fig. 1 and 2B), the size (kDa) of C2_2E12 was confirmed by Western blot (FIG. 2C). The affinity K _d values of the clones C1_1E1, C2_1D5, C2_2A10, C2_2E1, C2_2E12 and C2_2H5 for IL-33 were measured by ELISA analysis (FIG. 3 and Table 2).

Anti-IL-33 scFvs	K d (nM)	R 2
C1_1E1	48	0.9878
C2_1D5	36	0.9947
C2_2A10	57	0.9768
C2_2E1	35	0.9888
C2_2E12	28	0.9673
C2_2H5	31	0.9622

Example 2. Confirmation of antigen binding affinity of IL-33 specific scFv

Among the IL-33-specific antibodies obtained through the panning, in order to confirm the binding affinity of C2_2E12 with the antigen, 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). .

Example 3. Identification of antigen binding site of IL-33 specific scFv

3-1. Identification of antigen binding sites of C1_1E1, C2_1D5, C2_2A10, C2_2E1 and C2_2H5

To identify the antigen binding sites of the IL-33-specific antibodies C1_1E1, C2_1D5, C2_2A10, C2_2E1 and C2_2H5 obtained through the panning, 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. As a result, 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 ).

3-2. Identification of the antigen binding site of C2_2E12

In order to confirm the antigen binding site of the IL-33 specific antibody C2_2E12 obtained through the panning, 127, 138, 148, 158, 169, 187, 197, 206, 216, 222, 232, among the amino acid sequences of IL-33, After constructing recombinant GST-IL-33 by inserting a stop codon after amino acid residues 243 and 251, respectively, the binding affinity of C2_2E12 to each of them was confirmed. As a result, amino acid residues 149 to 158 of IL-33 were C2_2E12 It was confirmed that it is important for binding to (FIG. 6A). Accordingly, by substituting 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 (Å)	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 (Å 2 )	1350.8 ± 90.0
Z-score	-1.3

^a AU, arbitrary unit.

Through the above results, it was confirmed that all clones recognized the same epitope.

Example 4. Confirmation of antigen specificity of IL-33 specific scFv

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).

Example 5. Confirmation of neutralizing antibody function of IL-33 specific scFv

5-1. Inhibition of IL-33 and ST2 complex formation of C2_2E12

In vitro pull-down analysis was performed to confirm that the IL-33-specific antibody C2_2E12 can function as a neutralizing antibody capable of inhibiting the binding between IL-33 and ST2. Specifically, GST-tagged IL-33 was fixed/conjugated to glutathione resin (Glutathione Sepharose 4B), reacted with IL-33 by adding ST2-Fc fusion protein, and then increased the amount of C2_2E12 and IL- Whether the binding between 33 and ST2 was inhibited was confirmed by SDS-PAGE and Western blot analysis (protein molar concentration ratios were GST-IL-33: ST2-Fc: C2_2E12 = 1: 1: 0.1, 1:1:1, respectively. 1:1:10 and 1:1:100 (M)). As a result, it was confirmed that C2_2E12 interfered with the binding of IL-33 to ST2 ( FIG. 8 ).

5-2. Confirmation of C2_2E12 Neutralization Efficacy in Human Cells

To confirm whether the IL-33-specific antibody C2_2E12 functions as a neutralizing antibody even in cells, human mast cell (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 IκBα in the MAPK and NF-κB signaling pathways. The pattern of phosphorylation band change was confirmed by immunoblot analysis. As a result, in HMC-1 cells expressing ST2 and IL-1RAcP, 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. (Fig. 9). In addition, IκBα 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-κB signaling in a concentration-dependent manner, and this shows that C2_2E12 also acts as a neutralizing antibody in cells.

Example 6. Confirmation of mouse and human IL-33 cross-reactivity of C2_2E12 antibody

The following experiment was performed to confirm whether the IL-33 specific antibody C2_2E12 antibody was cross-reactive with human-derived IL-33 (112-270) and mouse-derived IL-33.

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.

Specifically, 13.5, 6.8, 3.4 pmole for human-derived IL-33 (“GST-hIL-33”) and 54, 27, 13.5, 2.7 for mouse-derived IL-33 (“GST-mIL-33”) , 1.4 pmol was added to each lane, electrophoresed, and immunoblot was performed using C2_2E12-HA as the primary antibody and anti-HA-HRP as the secondary antibody. The results are shown in FIG. 11 .

11, 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.

Example 7. Identification of the three-dimensional crystal structure of the C2_2E12 antibody

To confirm the three-dimensional crystal structure of the IL-33 specific antibody C2_2E12 antibody of the present invention, the purified C2_2E12 diabody was concentrated to 10 mg/ml and crystallized through vapor diffusion method. In order to facilitate crystallization, the C2_2E12 antibody was expressed and purified by converting it into a diabody form.

Specifically, after transferring the C2_2E12 (VH-<GGGGS> ₃ -VL-His ₆ -HA tag: based on protein sequence) gene in the pComb3X vector to the pSF vector, 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.

The conditions required for crystallization were as follows:

Crystals were formed after incubation at 25% PEG3350, 0.2 M NaCl, 0.1 M sodium citrate pH 5.5, 22℃ 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 Å. The three-dimensional molecular structure is shown in FIG. 12, and the analysis results are shown in Table 4.

As shown in Fig. 12, 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.

division	C2_2E12
Wavelength (Å)	0.9794
Resolution range (Å)	48.82 - 2.75 (2.849 - 2.75)
space group		C 1 2 1
unit cell
a , b , c (Å)	184.754, 115.265, 141.521
α , β , γ (°)	90, 96.164, 90
total reflections	529505
Unique reflections	75472 (7012)
Multiplicity	7.0
Completeness (%)	98.18 (91.89)
Mean I / σ ( I )	13.73
Wilson B -factor (Å 2 )	50.14
R merge	0.151
R meas	0.164
R pim	0.061
CC 1/2	0.987
Reflections used in refinement	75368 (7011)
Reflections used for R free	1996 (185)
R work	0.2201 (0.3042)
R free	0.2810 (0.3666)
Number of non-hydrogen atoms	20640
macromolecules	20270
ligands	270
solvent	175
protein residues	2738
RMS (bonds)	0.002
RMS(angles)	0.46
Ramachandran favored (%)	91.80
Ramachandran allowed (%)	7.64
Ramachandran outliers (%)	0.56
Rotamer outliers (%)	5.07
Clash score	27.31
Average B-factor (Å 2 )	61.40
macromolecules	61.45
ligands	68.59
solvent	48.43
Number of TLS groups	57

Statistics for the highest-resolution shell are shown in parentheses.

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 .

As shown in FIG. 13 , the C2_2E12 crystal structure was very similar to the C2_2E12 structure predicted through modeling. Using the previously calculated human-derived IL-33 crystal structure (PDB ID: 4KC3) and the modeled C2_2E12 structure, the modeled itepekimab structure was overlaid on the complex structure model derived through molecular docking calculation and analyzed. As a result, it was analyzed that the structure of the CDR3 (HCDR3) portion of the heavy chain was significantly different. These results suggest that the probability that the epitope recognition mode of the antibody C2_2E12 of the present invention and itepekimab is different is very high.

Claims (18)

1. An antibody specific for IL-33 or a fragment having immunological activity thereof.
2. The antibody or fragment having immunological activity specific for IL-33 according to claim 1, which binds to a peptide fragment consisting of amino acid residues 149 to 158 of IL-33 consisting of the amino acid sequence of SEQ ID NO: 1.
3. The antibody or fragment having immunological activity specific for IL-33 according to claim 1, which binds to the 150th residue or the 151st residue of IL-33 consisting of the amino acid sequence of SEQ ID NO: 1.
4. According to claim 1, wherein the monoclonal antibody, IL-33 specific antibody or fragment having an immunological activity thereof.
5. According to claim 1, 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 selected from the group consisting of the amino acid sequence of SEQ ID NOs: 10 to 15 An antibody or fragment having immunological activity specific for IL-33, comprising a VH domain comprising CDRH3 comprising any one of
6. The method according to claim 1, wherein the CDRL1 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 16 to 20, CDRL2 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 21 to 26, and the sequence An antibody or fragment having immunological activity specific for IL-33, comprising a VL domain comprising CDRL3 comprising any one selected from the group consisting of No. 27 to 31 amino acid sequence.
7. The method of claim 1,

   (i) any one selected from the group consisting of 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 the amino acid sequence of SEQ ID NOs: 10 to 15 a VH domain comprising a CDRH3 comprising; and

   (ii) CDRL1 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 16 to 20, CDRL2 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 21 to 26, and SEQ ID NOs: 27 to 31. An antibody or fragment having immunological activity specific for IL-33, comprising a VL domain comprising CDRL3 comprising any one selected from the group consisting of a 31 amino acid sequence.
8. According to claim 1, wherein the fragment having immunological activity is Fab, Fd, Fab', dAb, F(ab'), F(ab')2, scFv (single chain fragment variable), Fv, single chain antibody, Fv Any one selected from the group consisting of dimers, complementarity determining region fragments, humanized antibodies, chimeric antibodies and diabodies, IL-33 specific antibody or fragment having immunological activity thereof.
9. The IL33-specific antibody or fragment having immunological activity according to claim 1, which is a neutralizing antibody that inhibits the formation of a complex between IL-33 and ST2.
10. The antibody or fragment having immunological activity specific for IL-33 according to claim 1, which inhibits the activation of MAPK or NF-κB signaling pathway.
11. A nucleic acid molecule encoding the antibody of any one of claims 1 to 10 or a fragment having immunological activity thereof.
12. A vector comprising the nucleic acid molecule of claim 11 .
13. A host cell comprising the vector of claim 12 .
14. a) culturing a host cell comprising a vector comprising a nucleic acid molecule encoding the antibody of any one of claims 1 to 10 or a fragment having immunological activity thereof; and

    b) a method for producing an antibody specific for IL-33 or a fragment having immunological activity thereof, comprising the step of recovering the antibody or fragment having immunological activity thereof from the host cell culture.
15. The antibody of any one of claims 1 to 10 or a fragment having immunological activity thereof; a nucleic acid molecule encoding it; And a pharmaceutical composition for the prevention or treatment of inflammatory diseases containing at least one selected from the group consisting of a vector containing the nucleic acid molecule as an active ingredient.
16. The method of claim 15, wherein 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, vernal 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, in children and adults of viral infections (respiratory syncytial virus (RSV), rhinovirus, influenza), urticaria, eosinophilic esophagitis, chronic fibrosis, liver fibrosis, systemic sclerosis, idiopathic pulmonary fibrosis (IPF), scleroderma, liver fibrosis, pulmonary fibrosis fibrosis of the back, non-alcoholic steatohepatitis (NASH), chronic kidney disease, acute kidney injury, sepsis, pancreatitis, type 1 diabetes, graft-versus-host disease (GVHD), Alzheimer's disease, rheumatoid arthritis, irritable bowel syndrome ( IBS), ulcerative colitis, multiple sclerosis, psoriasis, celiac disease, and any one or more selected from the group consisting of Raynaud's disease or Raynaud's phenomenon, a pharmaceutical composition for preventing or treating inflammatory diseases.
17. The method of claim 15, wherein the fragment having immunological activity is Fab, Fd, Fab', dAb, F(ab'), F(ab')2, single chain fragment variable (scFv), Fv, single chain antibody, Fv A pharmaceutical composition for preventing or treating an inflammatory disease, which is any one selected from the group consisting of dimers, complementarity determining region fragments, humanized antibodies, chimeric antibodies, and diabodies.
18. (a) obtaining a sample isolated from the subject in vitro;

    (b) treating the sample with the antibody of any one of claims 1 to 10 or a fragment having immunological activity thereof; and

    (c) 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.

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