Anti-IL-33 antibodies or antigen-binding fragments thereof and uses thereof

Abstract

The present disclosure relates to the field of biomedicine technology, and provides an anti-IL-33 antibody or an antigen-binding fragment thereof and its use, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the CDR combination of the heavy chain variable region and the light chain variable region is: a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 respectively composed of the amino acid sequences SEQ ID NO: 1, 15, and 3, and a light chain CDR1, a light chain CDR2, and a light chain CDR3 respectively composed of the amino acid sequences SEQ ID NO: 6, 7, and 8. The antibody has a blocking effect on the IL-33-activated NKκB signaling pathway and an inhibitory effect on the IL-33-induced D10.G4.1 cell proliferation at the cellular level, and has potential significance in the treatment of chronic inflammation and autoimmune diseases.

Description

Technical Field

[0001] The present disclosure relates to an anti-IL-33 antibody or an antigen-binding fragment thereof and uses thereof, and in particular to an anti-IL-33 humanized monoclonal antibody with super strong neutralizing activity, which has potential significance in the treatment of chronic inflammatory and autoimmune diseases. Background Art

[0002] Interleukin 33 (IL-33), also known as IL-1F11, is a member of the IL-1 family and is produced by Th2 cells, mast cells and innate lymphocytes. Human and mouse IL-33 cDNA encode polypeptides of 270 and 266 amino acids, respectively, and the molecular weights of the corresponding full-length proteins are 30kDa and 29.9kDa, respectively. IL-33 can cause and regulate a variety of inflammatory responses and may play a role in chronic inflammation and autoimmune diseases. IL-33 is mainly involved in Th2 cell-mediated immune responses, regulates mast cell functions, and also acts as an intracellular nuclear factor that can regulate gene transcription characteristics.

[0003] IL-33 is a specific ligand of ST2. The ST2 gene is expressed in several cells such as fibroblasts and mast cells. It is also specifically expressed in mouse and human Th2 cells. IL-33 signal transduction depends on the expression of its receptor ST2. IL-33 binds to ST2 and activates NFκB and MAPK through downstream signaling molecules myeloid differentiation factor 88 (MyD88), IL-1-related protein kinase (IRAK) and tumor necrosis factor receptor-associated factor 6 (TRAF6), thereby regulating gene transcription, leading to the production of Th2 cytokines IL-4, IL-5 and IL-13 and subsequent biological functions. IL-33 is a powerful chemoattractant for Th2 cells in vitro and in vivo. In vitro, recombinant IL-33 can increase the proportion of Th2 cells, induce morphological changes in Th2 cells in an IL-33 concentration-dependent manner, and cause them to migrate into collagen matrices; in vivo, it attracts adoptively transferred ST2(+) Th2 cells to the site of IL-33 injection. Therefore, IL-33 plays a key role in maintaining chronic inflammation by actively recruiting Th2 cells to sites of inflammation.

[0004] ST2 is selectively expressed on Th2 cells but not on Th1 cells. ST2 is strongly associated with inflammatory responses. Soluble ST2 expression increases in the synovial fluid of patients with Th17 cell-mediated autoimmune diseases such as rheumatoid arthritis, and IL-33 mRNA and protein levels in serum, synovial fluid or inflammatory sites also increase. The level of soluble ST2 in the serum of patients with systemic lupus erythematosus is increased. Therefore, blocking IL-33 and its receptor signaling pathway may provide a new therapeutic strategy for the treatment of arthritis and systemic lupus erythematosus. Studies have shown that administration of IL-33 can stimulate the production of IL-5, inhibit Th1 cell activity, and thus improve atherosclerosis in mice lacking apolipoprotein E. It can be seen that IL-33 also plays a protective role in the development of the disease. In a mouse model of Pseudomonas aeruginosa keratitis, ST2 can reduce corneal infection and inflammatory response by downregulating Th1 cytokines and upregulating the expression of Th2 cytokines, especially IL-10. IL-33 can also reduce the inflammatory response of Pseudomonas aeruginosa keratitis through anti-inflammatory mediators produced by polarized macrophages in the cornea.

[0005] IL-33 can regulate the development and function of mast cells. In vitro, IL-33 promotes the maturation of human CD34+ mast cell precursors and induces mast cells to produce pro-inflammatory cytokines and chemokines. IL-33 can induce mouse bone marrow-derived cultured mast cells (BMCMC) to produce IL-13 and IL-6 in a non-IgE manner. IL-33 promotes the production of these cytokines in a MyD88-dependent manner, but IL-33 does not induce and enhance the degranulation of these mast cells. Under IgE-free conditions, high concentrations of IL-33 can prolong the survival of BMCMC. IL-33 can stimulate the secretion of IL-6 by mouse mast cell line P815 cells and BMCM in a dose- and time-dependent manner, and increase the expression of IL-6 and IL-1b mRNA. IL-33 also induces BMCMC to produce IL-1β, tumor necrosis factor (TNFα), monocyte chemoattractant protein (MCP)-1 and prostaglandin D (PGD)2. IL-33 induces the release of pro-inflammatory cytokines from BMCMCs, indicating that IL-33 has similar pro-inflammatory effects as IL-1 and IL-18. In addition, IL-33 has Th2-type acquired immune response activity that is different from that of IL-1 and IL-18 described above. IL-33 can promote the survival and adhesion of human umbilical cord blood-derived mast cells (HUCBMCs) to fibronectin, and induce the production of IL-8 and IL-13 in HUCBMCs. Therefore, IL-33 may enhance the function of mast cells in allergic reactions and other diseases. The expression of ST2 and IL-33 mRNA and protein in serum and tissues of asthma patients is increased. In the serum or lung tissue of the ovalbumin-induced mouse airway inflammation asthma model, the expression of soluble ST2 and IL-33 mRNA was significantly increased. In the skin of 2,4-dinitrofluorobenzene-induced mouse contact dermatitis, ST2 mRNA expression was increased. In addition, IL-33 expression was significantly increased in allergic conjunctivitis, rhinitis, anaphylactic shock, and atopic dermatitis. These results indicate that IL-33 and its receptor are closely related to the pathogenesis of the above-mentioned allergic diseases.

[0006] IL-33 has other biological functions. Similar to IL-1a, IL-33 is a multifunctional protein. It is not only a pro-inflammatory cytokine, but also an endothelial cell-derived chromatin-associated nuclear factor with transcriptional inhibition properties. IL-33 can significantly inhibit angiotensin-II and phenylephrine-induced cardiomyocyte hypertrophy and fibrosis. It inhibits the phosphorylation of NFκB inhibitor induced by angiotensin-II and phenylephrine and the nuclear binding activity of NFκB, while soluble ST2 can block the inhibitory effect of IL-33. IL-33 can promote angiogenesis by promoting the proliferation, migration and differentiation of endothelial cells, reducing the interaction of intercellular adhesion proteins and increasing vascular permeability.

[0007] In summary, IL-33 is a pro-inflammatory factor with multiple effects. It plays an important role in regulating the innate immune response, especially the function of mast cells. As a pro-inflammatory cytokine, it can cause tissue immunopathological damage. IL-33 can be used to treat autoimmune diseases, allergic disorders, cardiovascular diseases, asthma, gastrointestinal and lung diseases. Summary of the invention

[0008] On the one hand, the present disclosure provides an antibody or an antigen-binding fragment thereof of interleukin 33 (IL-33), which comprises a heavy chain variable region and a light chain variable region, wherein the CDR composition of the heavy chain variable region and the light chain variable region is: a heavy chain CDR1 consisting of the amino acid sequence SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence SEQ ID NO: 15, a heavy chain CDR3 consisting of the amino acid sequence SEQ ID NO: 3, and a light chain CDR1 consisting of the amino acid sequence SEQ ID NO: 6, a light chain CDR2 consisting of the amino acid sequence SEQ ID NO: 7, and a light chain CDR3 consisting of the amino acid sequence SEQ ID NO: 8.

[0009] In some embodiments, the aforementioned antibody or antigen-binding fragment thereof has the following combination of heavy chain variable region and light chain variable region:

[0010] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.16 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.20 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0011] In some embodiments, the aforementioned antibody is an IgG1, IgG2, IgG3 or IgG4 antibody.

[0012] In some preferred embodiments, the heavy chain comprises the amino acid sequence shown in SEQ ID NO. 18 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto;

[0013] In some preferred embodiments, the light chain comprises the amino acid sequence shown in SEQ ID NO.22 or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0014] In another aspect, the present disclosure provides a nucleic acid encoding the aforementioned antibody or antigen-binding fragment thereof.

[0015] In some preferred embodiments, the aforementioned nucleic acid has a nucleotide sequence encoding an antibody heavy chain variable region of SEQ ID NO.17 or any variant thereof, and a nucleotide sequence encoding an antibody light chain variable region of SEQ ID NO.21 or any variant thereof. Wherein, any of the variants preferably have a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO.17 and / or SEQ ID NO.21.

[0016] In some preferred embodiments, the aforementioned nucleic acid has an antibody heavy chain encoding nucleotide sequence of SEQ ID NO.19 or any variant thereof, and an antibody light chain encoding nucleotide sequence of SEQ ID NO.23 or any variant thereof. Wherein, any of the variants preferably have a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO.19 and / or SEQ ID NO.23.

[0017] In another aspect, the present disclosure provides a vector of the aforementioned nucleic acid.

[0018] In another aspect, the present disclosure provides a host cell for the aforementioned nucleic acid vector.

[0019] On the other hand, the present disclosure provides a pharmaceutical composition or pharmaceutical combination comprising the aforementioned antibody or antigen-binding fragment thereof, nucleic acid, vector and / or cell.

[0020] In some embodiments, the composition or pharmaceutical combination further comprises a second therapeutic agent.

[0021] In some preferred embodiments, the second therapeutic agent is selected from beta-2 adrenergic receptor agonists (SABAs), anticholinergics, adrenergic agonists, glucocorticoids, long-acting beta-2 adrenergic receptor agonists (LABAs), leukotriene antagonists, and mast cell stabilizers.

[0022] In another aspect, the present disclosure provides a method for producing the aforementioned antibody or antigen-binding fragment thereof, the method comprising culturing a host cell containing the aforementioned nucleic acid or expression vector.

[0023] On the other hand, the present disclosure provides a use of the aforementioned antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell or pharmaceutical composition or drug combination for preparing a drug or kit for detecting, treating, preventing and / or alleviating autoimmune diseases, allergic disorders, cardiovascular diseases, asthma, gastrointestinal and lung diseases.

[0024] In some preferred embodiments, the autoimmune disease is rheumatoid arthritis.

[0025] The present disclosure provides an anti-IL-33 humanized monoclonal antibody with super strong neutralizing activity. The humanized antibody disclosed in the present invention has a strong blocking effect on the IL-33-activated NKκB signaling pathway at the cellular level, and can strongly inhibit IL-33-induced D10.G4.1 cell proliferation, which has potential significance in the treatment of chronic inflammatory and autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the specification and, together with the description, serve to explain the principles of the specification.

[0027] Figure 1 The SDS-PAGE results of prokaryotically expressed human, cynomolgus monkey, mouse and rat IL-33 proteins are shown.

[0028] Figure 2 The effects of human, cynomolgus monkey, mouse and rat IL-33 proteins on D10.G4.1 cell proliferation are shown.

[0029] Figure 3 The SDS-PAGE results of eukaryotically expressed human and cynomolgus monkey soluble ST-2 proteins are shown.

[0030] Figure 4 The binding of human IL-33 to human ST-2 is shown.

[0031] Figure 5 The results of HEK293-NFκB luc-hST-2-2C3 luciferase reporter gene cell assay are shown.

[0032] Figure 6 The binding of the chimeric antibody 75D11 to IL-33 is shown.

[0033] Figure 7 Blockade of IL-33 / ST-2 binding by the chimeric antibody 75D11 is shown.

[0034] Figure 8 The chimeric antibody 75D11 shows the inhibition of IL-33-induced NFκB signaling pathway.

[0035] Fig. 9 Blockade of IL-33-induced proliferation of D10.G4.1 cells by chimeric antibody 75D11 is shown.

[0036] Fig.10Blockade of IL-5 release from IL-33-stimulated KU812 cells by the chimeric antibody 75D11 is shown.

[0037] Fig.11 The affinity of the chimeric antibody 75D11 to IL-33 is shown.

[0038] Fig.12 The binding of humanized antibody h75D11 H2K1 to IL-33 is shown.

[0039] Fig.13 Blockade of IL-33 / ST-2 binding by humanized antibody h75D11 H2K1 is shown.

[0040] Fig.14 The humanized antibody h75D11 H2K1 shows the inhibition of IL-33-induced NFκB signaling pathway.

[0041] Fig.15 The humanized antibody h75D11 H2K1 shows the inhibition of IL-33-induced proliferation of D10.G4.1 cells.

[0042] Fig.16 The humanized antibody h75D11 H2K1 shows the inhibition of IL-5 release from IL-33-stimulated KU812 cells.

[0043] Fig.17 The affinity of humanized antibody h75D11 H2K1 to IL-33 is shown. DETAILED DESCRIPTION

[0044] I. Definitions

[0045] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

[0046] As used herein and unless otherwise specified, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0047] Sequence "identity" or "identity" has a meaning recognized in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule (see, e.g., Computational Molecular Biology, Lesk, AM, ed., Oxford University Pres, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). Although there are many methods to measure the identity between two polynucleotides or polypeptides, the term "identity" is well known to those of skill in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)).

[0048] With respect to the variable domains of antibodies, the term "variable" refers to certain parts of related molecules that have extensive sequence differences between antibodies and are used for specific recognition and binding of a particular antibody to its specific target. However, the variability is not evenly distributed throughout the variable domain of an antibody. The variability is concentrated in three segments called complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) or hypervariable regions, which are located in the variable domains of light and heavy chains. The more conserved parts of the variable domains are called framework (FR) regions or framework sequences. Each variable domain of a natural heavy chain and light chain includes four FR regions, which mainly adopt a β-pleated configuration, connecting three CDRs, which form loops that connect the β-pleated structure and in some cases form part of the β-pleated structure. The CDRs of each chain are usually linked together in proximity by the FR regions and, with the help of the CDRs from the other chain, contribute to the formation of the antibody target binding site (epitope or determinant) (see Kabat et al. Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD (1987)). As used herein, the numbering of immunoglobulin amino acid residues is based on the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated. A CDR may have the ability to specifically bind to a cognate epitope.

[0049] As used herein, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than the full length, but contains at least a portion of the variable region (e.g., one or more CDRs and / or one or more antibody combining sites) of the antibody that binds to an antigen, and thus retains binding specificity and at least a portion of the specific binding ability of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment was derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives.

[0050] As used herein, "monoclonal antibody" refers to a population of identical antibodies, meaning that each individual antibody molecule in a monoclonal antibody population is identical to other antibody molecules. This property is in contrast to the properties of a polyclonal population of antibodies, which contains antibodies with a variety of different sequences. Monoclonal antibodies can be prepared by many well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared by immortalized B cells, for example, by fusion with myeloma cells to produce hybridoma cell lines or by infecting B cells with viruses such as EBV. Recombinant technology can also be used to prepare antibodies from a clonal population of host cells in vitro by transforming host cells with a plasmid carrying an artificial sequence of nucleotides encoding the antibody.

[0051] As used herein, a full-length antibody is an antibody having two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region, such as antibodies naturally produced by antibody-secreting B cells and antibodies produced synthetically with the same domains.

[0052] As used herein, the term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0053] As used herein, the term "humanized" antibody refers to a non-human (e.g., mouse) antibody form that is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of an antibody) containing minimal sequence derived from a non-human immunoglobulin. Preferably, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues in a complementary determining region (CDR) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) such as a mouse, rat, or rabbit having the desired specificity, affinity, and capacity.

[0054] In addition, in humanization, it is also possible to mutate the amino acid residues in the CDR1, CDR2 and / or CDR3 regions of VH and / or VL, thereby improving one or more binding properties (e.g., affinity) of the antibody. For example, PCR-mediated mutations can be used to introduce mutations, and their effects on antibody binding or other functional properties can be evaluated using in vitro or in vivo tests described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions or deletions. In addition, the mutations in the CDR are generally no more than one or two. Therefore, the humanized antibodies described in the present disclosure also encompass antibodies comprising 1 or 2 amino acid mutations in the CDR.

[0055] As used herein, the term "CDR" refers to a complementarity-determining region, and each heavy chain and light chain of a known antibody molecule has three CDRs. CDRs are also referred to as hypervariable regions and are present in the variable regions of each heavy chain and light chain of an antibody, with very high variability sites in the primary structure of the CDRs. In this specification, the CDRs of the heavy chain are represented by CDR1, CDR2, and CDR3 from the amino terminal of the amino terminal sequence of the heavy chain, and the CDRs of the light chain are represented by CDR1, CDR2, and CDR3 from the amino terminal of the amino terminal sequence of the light chain. These sites are adjacent to each other in the tertiary structure and determine the specificity of the antigen to which the antibody binds.

[0056] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually comprise chemically active surface patterns of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics.

[0057] As used herein, "specific binding" or "immunospecifically binding" with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with a cognate antigen through non-covalent interactions between the antibody combining sites of the antibody and the antigen. The antigen may be an isolated antigen or present in a tumor cell. Typically, an antibody that immunospecifically binds (or specifically binds) to an antigen is present in an amount of about or 1×10 7 M -1 or 1×10 8 M -1 or greater affinity constant Ka (or 1×10 -7 M or 1×10 -8M or lower dissociation constant (Kd)) binds to the antigen. Affinity constants can be determined by standard kinetic methods of antibody reactions, e.g., immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art (see, e.g., Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); also see U.S. Pat. No. 7,229,619 describing exemplary SPR and ITC methods for calculating the binding affinity of antibodies). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (see, BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335).

[0058] As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), usually linked together by a phosphodiester bond. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA.

[0059] As used herein, an isolated nucleic acid molecule is a nucleic acid molecule isolated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. "Isolated" nucleic acid molecules such as cDNA molecules can be substantially free of other cellular materials or culture media when prepared by recombinant technology, or substantially free of chemical precursors or other chemical compositions when chemosynthesized. The exemplary isolated nucleic acid molecules provided herein include the isolated nucleic acid molecules of the antibody or Fab provided by encoding.

[0060] As used herein, "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, usually by restriction digestion and ligation. Vectors also include those containing nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells, for amplification of nucleic acids or for expression / display of polypeptides encoded by nucleic acids. Vectors are usually kept free, but can be designed to integrate genes or parts thereof into chromosomes of the genome. Artificial chromosome vectors are also contemplated, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles are well known to those skilled in the art.

[0061] As used herein, vectors also include “viral vectors” or “viral vectors.” Viral vectors are engineered viruses that are operably linked to exogenous genes to transfer (as a vehicle or shuttle) the exogenous genes into cells.

[0062] As used herein, "expression vector" includes vectors capable of expressing DNA, which is operably linked to regulatory sequences such as promoter regions that can affect the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more replication origins, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, expression vectors refer to recombinant DNA or RNA constructs, such as plasmids, phages, recombinant viruses or other vectors, which, when introduced into appropriate host cells, result in the expression of cloned DNA. Suitable expression vectors are well known to those skilled in the art, and include expression vectors that are replicable in eukaryotic cells and / or prokaryotic cells and expression vectors that remain free or are integrated into the host cell genome.

[0063] As used herein, the term "affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can usually be expressed in terms of an equilibrium dissociation constant (KD). The equilibrium dissociation constant is the ratio of the dissociation rate constant to the association rate constant (kdis and kon, respectively). The smaller the KD, the smaller the dissociation, representing the stronger the affinity between the antibody and the antigen. Affinity can be measured by conventional methods known in the art, for example, using a KD determined in a BIACORE instrument using surface plasmon resonance (SPR). Typically, an antibody dissociates from an antigen with an equilibrium dissociation constant (KD) of no more than 1×10-5M, for example, less than about 1×10-6M, 1×10-7M, 1×10-8M, 1×10-9M, or 1×10-10M or less.

[0064] II. Detailed description of specific implementation scheme

[0065] On the one hand, the present disclosure provides an antibody or an antigen-binding fragment thereof of interleukin 33 (IL-33), which comprises a heavy chain variable region and a light chain variable region, wherein the CDR combination of the heavy chain variable region and the light chain variable region is: a heavy chain CDR1 consisting of the amino acid sequence SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence SEQ ID NO: 15, a heavy chain CDR3 consisting of the amino acid sequence SEQ ID NO: 3, and a light chain CDR1 consisting of the amino acid sequence SEQ ID NO: 6, a light chain CDR2 consisting of the amino acid sequence SEQ ID NO: 7, and a light chain CDR3 consisting of the amino acid sequence SEQ ID NO: 8.

[0066] In some embodiments, the aforementioned antibody or antigen-binding fragment thereof has the following combination of heavy chain variable region and light chain variable region:

[0067] The heavy chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO.16, and the light chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO.20.

[0068] In some embodiments, the aforementioned antibody is an IgG1, IgG2, IgG3 or IgG4 antibody.

[0069] In some preferred embodiments, the aforementioned heavy chain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO.18.

[0070] In some preferred embodiments, the aforementioned light chain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO.22.

[0071] In another aspect, the present disclosure provides a nucleic acid encoding the aforementioned antibody or antigen-binding fragment thereof.

[0072] In some preferred embodiments, the aforementioned nucleic acid has a nucleotide sequence encoding an antibody heavy chain variable region of SEQ ID NO.17 or any variant thereof, and a nucleotide sequence encoding an antibody light chain variable region of SEQ ID NO.21 or any variant thereof. Wherein, any of the variants preferably have a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO.17 and / or SEQ ID NO.21.

[0073] In some preferred embodiments, the aforementioned nucleic acid has a nucleotide sequence encoding an antibody heavy chain of SEQ ID NO.19 or any variant thereof, and a nucleotide sequence encoding an antibody light chain of SEQ ID NO.23 or any variant thereof. Wherein, any of the variants preferably have a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO.19 and / or SEQ ID NO.23.

[0074] In another aspect, the present disclosure provides a vector of the aforementioned nucleic acid.

[0075] In another aspect, the present disclosure provides a host cell for the aforementioned nucleic acid vector.

[0076] On the other hand, the present disclosure provides a pharmaceutical composition or pharmaceutical combination comprising the aforementioned antibody or antigen-binding fragment thereof, nucleic acid, vector and / or cell.

[0077] In another aspect, the present disclosure provides a combination product comprising the aforementioned antibody or antigen-binding fragment thereof, nucleic acid, vector and / or cell and a second therapeutic agent.

[0078] The anti-IL-33 antibodies or antigen-binding fragments thereof disclosed herein can be used in combination with any known treatment for inflammatory diseases, including any agent or combination of agents known to be useful for treating inflammatory diseases, or agents that have been used or are currently being used to treat inflammatory diseases. For example. Agents used to treat asthma are divided into two general categories: rapid relief drugs for treating acute symptoms; and long-term control drugs for preventing further exacerbations. Rapid onset treatments include, for example, short-acting beta-2 adrenergic receptor agonists (SABAs) (e.g., salbutamol); anticholinergics (e.g., ipratropium bromide), adrenergic agonists (e.g., epinephrine). Long-term control treatments include, for example, glucocorticoids (e.g., fluticasone propionate); long-acting beta-2 adrenergic receptor agonists (LABAs); leukotriene antagonists (e.g., zafirlukast); and mast cell stabilizers (e.g., sodium cromoglycate).

[0079] In another aspect, the present disclosure provides a method for producing the aforementioned antibody or antigen-binding fragment thereof, the method comprising culturing a host cell containing the aforementioned nucleic acid or expression vector.

[0080] On the other hand, the present disclosure provides a use of the aforementioned antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell or pharmaceutical composition or drug combination for preparing a drug or kit for detecting, treating, preventing and / or alleviating autoimmune diseases, allergic disorders, cardiovascular diseases, asthma, gastrointestinal and lung diseases.

[0081] In some preferred embodiments, the autoimmune disease is rheumatoid arthritis.

[0082] For purposes of clarity and concise description, features are described herein as part of the same or separate embodiments, however, it will be understood that the scope of the present disclosure may include embodiments having a combination of all or some of the described features.

[0083] Example

[0084] Example 1: Material preparation

[0085] (1) Antigen preparation

[0086] Human IL-33 (SEQ ID NO: 11), cynomolgus monkey IL-33 (SEQ ID NO: 12), mouse IL-33 (SEQ ID NO: 13) and rat IL-33 (SEQ ID NO: 14) were synthesized. The sequences are shown in Table 1. The full-length fragments were cloned into a prokaryotic expression plasmid, and the plasmid was used to transform BL21 E. coli. Since the protein itself contains 4 free sulfhydryl groups (-SH), the protein needs to be purified under reducing conditions and stored in 1mM EDTA and 2mM DTT solution. Human, cynomolgus monkey, mouse and rat IL-33 proteins were purified, and the SDS-PAGE results are as follows: Figure 1 The effect of purified IL-33 on cell proliferation was detected using D10.G4.1 cells. Figure 2 As shown, human, cynomolgus monkey, mouse and rat IL-33 proteins all have biological activity.

[0087] Table 1 Sequences of human IL-33, cynomolgus monkey IL-33, mouse IL-33 and rat IL-33

[0088]

[0089]

[0090] (2) Soluble ST-2 protein

[0091] The human and cynomolgus ST-2 sequences were cloned into a self-constructed eukaryotic expression plasmid system. HEK293.6E cells were transfected with this plasmid, and the cell supernatant was collected and purified by Protein A affinity chromatography to obtain human and cynomolgus ST-2 soluble proteins, such as Figure 3 As shown. Enzyme-linked immunosorbent assay (ELISA) detected that human IL-33 could bind to human ST-2 protein, as shown Figure 4 shown.

[0092] (3) DNA plasmid

[0093] Human IL-33 and cynomolgus monkey IL-33 were constructed into piCAG vectors to construct piCAG-hIL-33 (PADRE), piCAG-cynoIL-33 (PADRE) and piCAG-hIL-33-NTT plasmids, which were used for gene gun immunization of mice.

[0094] (4) HEK293-NFκB luc-hST-2 reporter gene cell line

[0095] 10 μg of piCAG-NFκB luc hygro plasmid was mixed with 5 × 10 6 The HEK293 cells were mixed and electroporated, with the voltage set to 220V and the capacitance to 950μF; 200μg / mL hygromycin B solution was added 24-48 hours after transfection, and the screening medium was replaced every 2-4 days. After a period of pressurized screening, the stable cells were stimulated with different concentrations of IL-1β for 24 hours and the fluorescence signal was detected. After that, monoclonal screening was performed, and the HEK293-NFκB luc luciferase reporter gene monoclonal stable cell line was successfully constructed. Using lentiviral infection technology, the human ST-2 gene was introduced into the HEK293-NFκB luc luciferase reporter gene stable cell line, and 1μg / mL puromycin solution was added, and the screening medium was replaced every 2-4 days. After a period of pressurized screening, the stable cells were stimulated with different concentrations of IL-33 for 24 hours and the fluorescence signal was detected. The results are as follows Figure 5 As shown, the fluorescence signal gradually increased with the increase of IL-33 concentration, indicating that IL-33 can activate the NFκB signaling pathway of cells.

[0096] Example 2: Generation of anti-IL33 antibodies

[0097] (1) Mouse immunization

[0098] Female Balb / C mice aged 4-6 weeks were selected and immunized by antigen, gene gun or combination. The gene gun immunization dose was 20 μg IL-33 plasmid per mouse per immunization, with a one-week interval between each immunization. The antigen immunization dose was 50 μg IL-33 protein per mouse per immunization. The antigen was mixed with TiterMax adjuvant (Sigma-Aldrich) in equal volumes and then immunized subcutaneously and in the foot pad, with a two-week interval between each immunization. The serum was screened after it had blocking activity for human IL-33 / ST-2 binding at the antigen level.

[0099] (2) Phage screening

[0100] Mouse lymph nodes and spleen cells were taken, and the RNA of the cells was extracted by TRNzol lysis method, and then reverse transcribed to synthesize single-stranded cDNA, which was used as a template to amplify the variable region sequence of the antibody. The IL-33 immune library was constructed on the independently established Fab antibody fragment phage display platform. Human IL-33 protein or cynomolgus monkey IL-33 protein was added to the solid phase immunotube (Maxisorp immunotube) and liquid phase (SAbeads) to screen the IL-33 antibody library. Fab that can specifically recognize human and cynomolgus monkey IL-33 was obtained, and Fab positive clones that block the binding of IL-33 to ST-2 were further screened. After screening the Fab phage library, 14 clones that bound to human IL-33 were obtained, namely 70D5, 75D11, 77B1, 78F1, 79F4, 80A12, 80F1, 80H3, 81A12, 81D11, 82A4, 82G5, 82H10, and 84D1, as shown in Table 2.

[0101] Table 2 Phage display library screening Fab activity

[0102]

[0103]

[0104] Based on the comprehensive results of Fab binding to human IL-33 at the antigen level, Fab blocking human IL-33 / ST-2 at the antigen level, Fab blocking human IL-33 / ST-2 at the cell level and Fab affinity detection by Octect instrument, 75D11 Fab has good biological activity.

[0105] (3) Sequence acquisition of positive clones

[0106] According to the results of phage screening, the variable region sequence of the positive clone (75D11) was amplified. The amplified product was sequenced to obtain the heavy chain and light chain variable region sequences of the candidate antibody. The variable region sequences of the antibody light and heavy chains were cloned into eukaryotic expression vectors by PCR, and the full-length IgG antibody was expressed in CHO-18 cells, and the activity of the full-length antibody was analyzed. The 75D11 sequence is shown in Table 3.

[0107] Table 3 75D11 sequence

[0108]

[0109]

[0110] Example 3: Chimeric Antibody Activity Detection

[0111] (1) Binding of chimeric antibodies to human IL-33

[0112] 1 μg / mL human IL-33 protein was coated on a 96-well ELISA plate with a coating solution of pH 7.4 in PBS solution at 4°C overnight. Wash three times with PBS, and add 200 μl of PBS containing 3% BSA to each well for blocking for 1 hour. After washing once with PBS, add 100 μl of anti-IL-33 antibody to each well and incubate at room temperature for 60 minutes; after washing three times with PBST and PBS respectively, add 100 μl of HRP-labeled anti-human IgGFc secondary antibody to each well and incubate at room temperature for 60 minutes. After washing three times with PBST and PBS respectively, add 100 μl of TMB substrate to each well and develop at 37°C for 10 minutes, then terminate the reaction with 50 μl of 2M sulfuric acid solution per well, and read the absorbance at a wavelength of 450 nm. The results show that the chimeric antibody 75D11 can bind to human IL-33 protein, such as Figure 6 shown.

[0113] (2) Chimeric antibodies block human IL-33 / ST-2 binding

[0114] 2μg / mL human ST-2 recombinant protein was coated on a 96-well ELISA plate with a coating solution of pH 7.4 in PBS solution at 4°C overnight. Wash three times with PBS, and add 200μl of PBS containing 3% BSA to each well for blocking for 1 hour. Incubate the chimeric antibody and biotin-labeled human IL-33 protein in a blocked U-shaped 96-well plate at room temperature for 30 minutes. Wash the 96-well ELISA plate three times with PBS, add 100μl of the incubated mixture to each well, and incubate at room temperature for 1 hour. After washing three times with PBST and PBS respectively, add 100μl SA-HRP to each well and incubate at room temperature in the dark for 30 minutes. After washing three times with PBST and PBS respectively, add 100μl TMB substrate to each well and develop at 37°C for 10 minutes, terminate the reaction with 50μl 2M sulfuric acid solution per well, and read the absorbance at a wavelength of 450nm. The results show that the chimeric antibody 75D11 can block the binding of IL33 / ST-2, such as Figure 7 shown.

[0115] (3) Blockade of IL-33-activated NFκB signaling by chimeric antibodies

[0116] HEK293-NFκB luc-hST-2 cells were added to a 96-well plate at 2 × 10 cells per well. 4 The cells were incubated overnight at 37°C and 5% CO2 in a cell culture incubator. The next day, different concentrations of chimeric antibodies were incubated with human IL-33 protein at 37°C for 30 minutes and then added to the cells. The cells were incubated at 37°C and 5% CO2 for 24 hours. Luciferase substrate was added and the chemiluminescent signal was detected by an enzyme reader. The luciferase reporter gene method was used to determine whether the chimeric antibody had a blocking effect at the cellular level. The results showed that the chimeric antibody 75D11 had good blocking activity and IC 50 is 1.79nM, such as Figure 8 shown.

[0117] (4) Chimeric antibody blocks IL-33-induced D10.G4.1 cell proliferation

[0118] D10.G4.1 cells were added to a 96-well plate at 2 × 10 cells per well. 4 The cells were starved overnight in a 37°C, 5% CO2 cell culture incubator. The next day, different concentrations of antibodies were incubated with human IL-33 protein at 37°C for 30 minutes and then added to the cells. The cells were incubated in a 37°C, 5% CO2 cell culture incubator for 72 hours. Cell titer Glo detection reagent (Promega, catalog number G7570) was added and the chemiluminescent signal was detected by an enzyme reader. The results showed that the chimeric antibody 75D11 had blocking activity, IC 50 is 4.99nM, such as Fig. 9 shown.

[0119] (5) Chimeric antibody blocks IL-5 release from IL-33-stimulated KU812 cells

[0120] KU812 cells were added to a 96-well plate at 2 × 10 4 The cells were cultured overnight at 37°C and 5% CO2 in a cell culture incubator. The next day, different concentrations of antibodies were incubated with human IL-33 protein at 37°C for 30 minutes and then added to the cells. The cells were incubated at 37°C and 5% CO2 for 48 hours. The supernatant was collected and the IL-5 cytokine secretion was detected using a kit. The results showed that the chimeric antibody 75D11 could block the IL-5 cytokine released by KU812 cells induced by IL-33. 50 is 6.47nM, such as Fig.10 shown.

[0121] (6) Chimeric antibody affinity

[0122] The kinetic binding activity of anti-IL-33 antibodies to human IL-33 was measured by surface plasmon resonance (SPR) using the BIAcore T200 system. 2 μg / ml of anti-IL-33 antibody was immobilized on the sensor chip Protein A, and 1×HBS-EP buffer was used as the running buffer at a flow rate of 10 μl / min for 40 seconds. Different concentrations of human IL-33 of 0-21.7 nM were injected onto the immobilized anti-IL-33 antibody surface at a flow rate of 30 μl / min. After each injection cycle, the Protein A chip surface was regenerated at a flow rate of 30 μl / min with a regeneration buffer of 10 mM glycine (pH 2.0). The binding rate constant (ka) and dissociation rate constant (kd) and the equilibrium dissociation constant KD were analyzed using background subtraction binding sensorgrams. The resulting data set was fitted to the Langmuir 1:1 kinetics model using BIAevaluation software. The results showed that the chimeric antibody 75D11 antibody binds to human IL-33 with high affinity. Fig.11 shown.

[0123] In summary, the chimeric antibody 75D11 has good binding activity to IL-33 and blocking activity on the binding of IL-33 to ST-2, as shown in Table 4.

[0124] Table 4 Affinity and activity of chimeric antibody 75D11

[0125]

[0126] Example 4: Humanization of anti-IL-33 chimeric antibodies

[0127] Humanization was performed based on the chimeric antibody 75D11. The light and heavy chain derivatives were fully synthesized and cloned into a vector containing the antibody κ chain constant region Cκ or the human IgG4 constant region CH1-CH3. The light and heavy chain derivative plasmids were transfected into CHO cells and expressed for 7-9 days. The supernatant was collected and purified by Protein A column to obtain the humanized molecule h75D11 H2K1. The sequence of h75D11H2K1 is shown in Table 5.

[0128] Table 5h75D11 H2K1 sequence

[0129]

[0130]

[0131]

[0132] Example 5: Humanized Antibody Activity Detection

[0133] (1) Binding of humanized antibodies to human IL-33

[0134] 1 μg / mL human IL-33 protein was coated on a 96-well ELISA plate with a coating solution of pH 7.4 in PBS solution at 4°C overnight. Wash three times with PBS, and add 200 μl of PBS containing 3% BSA to each well for blocking for 1 hour. After washing once with PBS, add 100 μl of anti-IL-33 antibody to each well and incubate at room temperature for 60 minutes; after washing three times with PBST and PBS, add 100 μl of HRP-labeled anti-human IgGFc secondary antibody to each well and incubate at room temperature for 60 minutes. After washing three times with PBST and PBS, add 100 μl of TMB substrate to each well and develop at 37°C for 10 minutes. After that, stop the reaction with 50 μl of 2M sulfuric acid solution per well, and read the absorbance at a wavelength of 450 nm. The results showed that the humanized antibody h75D11 H2K1 can bind to IL-33 at the antigen level, EC 50 is 0.02nM, such as Fig.12 shown.

[0135] (2) Humanized antibodies block IL-33 / ST-2 binding

[0136] 2μg / mL human ST-2 recombinant protein was coated on a 96-well ELISA plate, and the coating solution was a PBS solution with pH 7.4, overnight at 4°C. Wash three times with PBS, and add 200μl of PBS containing 3% BSA to each well for 1 hour. Incubate the humanized antibody and biotin-labeled human IL-33 protein in a blocked U-shaped 96-well plate at room temperature for 30 minutes. Wash the 96-well ELISA plate three times with PBS, add 100μl of the incubated mixture to each well, and incubate at room temperature for 1 hour. After washing three times with PBST and PBS, add 100μl SA-HRP to each well and incubate at room temperature in the dark for 30 minutes. After washing three times with PBST and PBS, add 100μl TMB substrate to each well and color at 37°C for 10 minutes, stop the reaction with 50μl 2M sulfuric acid solution per well, and read the absorbance at a wavelength of 450nm. The results showed that the humanized antibody h75D11 H2K1 blocked the binding of IL-33 to ST-2 at the antigen level. 50 is 1.53nM, such as Fig.13 shown.

[0137] (3) Inhibition of IL-33-induced NFκB signaling pathway by humanized antibodies

[0138] HEK293-NFκB luc-hST-2 cells were added to a 96-well plate at 2 × 10 cells per well. 4 Cells were incubated overnight in a 37°C, 5% CO2 cell culture incubator. The next day, different concentrations of humanized antibodies were incubated with human IL-33 protein at 37°C for 30 minutes and then added to the cells. The cells were incubated in a 37°C, 5% CO2 cell culture incubator for 24 hours. Luciferase substrate was added and the chemiluminescent signal was detected by an enzyme reader. The luciferase reporter gene method was used to determine whether the humanized antibody had a blocking effect at the cellular level. The results showed that the humanized antibody h75D11 H2K1 could block the IL-33-activated NKκB signaling pathway at the cellular level. 50 is 0.37nM, such as Fig.14 shown.

[0139] (4) Inhibition of IL-33-stimulated D10.G4.1 cell proliferation by humanized antibodies

[0140] D10.G4.1 cells were added to a 96-well plate at 2 × 10 cells per well. 4The cells were starved overnight in a 37°C, 5% CO2 cell culture incubator. The next day, different concentrations of humanized antibodies were incubated with human IL-33 protein at 37°C for 30 minutes and then added to the cells. The cells were incubated in a 37°C, 5% CO2 cell culture incubator for 72 hours. Cell titer Glo detection reagent (Promega, Cat. No. G7570) was added and the chemiluminescent signal was detected with an enzyme reader. The results showed that the humanized antibody h75D11 H2K1 had an inhibitory effect on IL-33-induced D10.G4.1 cell proliferation at the cellular level, IC 50 is 23.16nM, such as Fig.15 shown.

[0141] (5) Inhibition of IL-5 release from KU812 cells stimulated with IL-33 by humanized antibodies

[0142] KU812 cells were added to a 96-well plate at 2 × 10 4 The cells were cultured overnight at 37°C and 5% CO2 in a cell culture incubator. The next day, different concentrations of humanized antibodies and human IL-33 protein were incubated at 37°C for 30 minutes and then added to the cells. The cells were incubated at 37°C and 5% CO2 for 48 hours. The supernatant was collected and the IL-5 cytokine secretion was detected using a kit. The results showed that the humanized antibody h75D11 H2K1 had inhibitory activity on the release of IL-5 by KU812 cells stimulated by IL-33 at the cellular level, and IC 50 is 6.55nM, such as Fig.16 shown.

[0143] (6) Humanized antibody affinity determination

[0144] The kinetic binding activity of anti-IL-33 antibodies to human IL-33 was measured by surface plasmon resonance (SPR) using the BIAcore T200 system. 2 μg / ml of anti-IL-33 antibody was immobilized on the sensor chip Protein A, and 1×HBS-EP buffer was used as the running buffer at a flow rate of 10 μl / min for 40 seconds. Different concentrations of human IL-33 of 0-21.7 nM were injected onto the immobilized anti-IL-33 antibody surface at a flow rate of 30 μl / min. After each injection cycle, the Protein A chip surface was regenerated at a flow rate of 30 μl / min with a regeneration buffer of 10 mM glycine (pH 2.0). The binding rate constant (ka) and dissociation rate constant (kd) and the equilibrium dissociation constant KD were analyzed using background subtraction binding sensorgrams. The resulting data set was fitted to the Langmuir 1:1 kinetics model using BIAevaluation software. The results showed that the humanized antibody h75D11H2K1 had a high affinity for binding to IL-33, with a KD value of 0.23 pM. Fig.17 shown.

[0145] In summary, the results of H2K1 activity of humanized antibody h75D11 are summarized in Table 6.

[0146] Table 6 Affinity and activity of humanized antibody h75D11 H2K1

[0147]

Claims

1. An antibody or antigen-binding fragment thereof to interleukin 33 (IL-33), comprising a heavy chain variable region and a light chain variable region, wherein the CDR combination of the heavy chain variable region and the light chain variable region is: a heavy chain CDR1 consisting of the amino acid sequence SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence SEQ ID NO: 15, a heavy chain CDR3 consisting of the amino acid sequence SEQ ID NO: 3, and a light chain CDR1 consisting of the amino acid sequence SEQ ID NO: 6, a light chain CDR2 consisting of the amino acid sequence SEQ ID NO: 7, and a light chain CDR3 consisting of the amino acid sequence SEQ ID NO:

8.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 16, and the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:

20.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

20. 4 . The antibody or antigen-binding fragment thereof according to claim 1 , wherein the antibody is an IgG1, IgG2, IgG3 or IgG4 type antibody.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the heavy chain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 18, and the light chain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:

22.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 18, and the light chain comprises the amino acid sequence shown in SEQ ID NO:

22.

7. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein The nucleic acid has an antibody heavy chain variable region encoding nucleotide sequence that is at least 80% identical to SEQ ID NO: 17, and an antibody light chain variable region encoding nucleotide sequence that is at least 80% identical to SEQ ID NO:

21.

8. The nucleic acid according to claim 7, wherein the nucleic acid has a nucleotide sequence encoding an antibody heavy chain variable region as shown in SEQ ID NO: 17, and a nucleotide sequence encoding an antibody light chain variable region as shown in SEQ ID NO:

21.

9. The nucleic acid of claim 7 or 8, wherein the nucleic acid has an antibody heavy chain encoding nucleotide sequence that is at least 80% identical to SEQ ID NO: 19, and an antibody light chain encoding nucleotide sequence that is at least 80% identical to SEQ ID NO:

23.

10. The nucleic acid according to claim 7 or 8, wherein the nucleic acid has a nucleotide sequence encoding an antibody heavy chain as shown in SEQ ID NO: 19, and a nucleotide sequence encoding an antibody light chain as shown in SEQ ID NO:

23.

11. A vector comprising the nucleic acid of any one of claims 7-10.

12. A host cell comprising the nucleic acid of any one of claims 7 to 10 or the vector of claim 11.

13. A pharmaceutical composition or drug combination comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

14. The pharmaceutical composition or pharmaceutical combination according to claim 13, wherein the composition or pharmaceutical combination further comprises a second therapeutic agent.

15. The pharmaceutical composition or pharmaceutical combination according to claim 14, wherein the second therapeutic agent is selected from beta-2 adrenergic receptor agonists (SABA), anticholinergics, adrenergic agonists, glucocorticoids, long-acting beta-2 adrenergic receptor agonists (LABA), leukotriene antagonists and mast cell stabilizers.

16. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the method comprising culturing the host cell according to claim 12.

17. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 or the pharmaceutical composition or pharmaceutical combination according to any one of claims 13 to 15 in the preparation of a drug or kit for detecting and / or treating asthma.

18. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 or the pharmaceutical composition or pharmaceutical combination according to any one of claims 13 to 15 for preparing a medicament for alleviating asthma.

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