Anti-il-13 antibody and use thereof

Abstract

The present disclosure relates to an anti-interleukin 13 (IL-13) antibody and the use thereof, and specifically relates to an antibody or antigen-binding fragment thereof that specifically binds to human IL-13, a multispecific antigen-binding molecule, an isolated nucleic acid molecule, a vector, a cell, a pharmaceutical composition, the pharmaceutical use, a method for treating a disease, etc. The anti-IL-13 antibody of the present disclosure can significantly inhibit IL-13-induced proliferation of TF-1 cells, can specifically bind to human IL-13 proteins and has relatively high affinity.

Description

Anti-IL-13 antibodies and their applications

[0001] This disclosure claims priority to Chinese Patent Application No. 202411477367.2, filed with the Chinese Patent Office on October 22, 2024, entitled "Anti-IL-13 Antibody and Its Application", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure relates to the field of biomedicine, specifically to the field of antibodies, and particularly to an anti-IL-13 antibody and its applications. Background Technology

[0003] Interleukin-13 (IL-13) is a type I cytokine with a quadruple helix core linked by two disulfide bonds. It belongs to the T helper cell subtype (Th2) cytokine family, along with IL-3, IL-4, IL-5, and IL-9. In humans, the gene encoding IL-13 is located in the Th2-related cytokine gene cluster on chromosome 5q31. IL-13 is primarily secreted by activated Th2 cells, but can also be produced by activated ILC2 cells, mast cells, NK cells, basophils, and eosinophils. IL-13 has important immunomodulatory activities and affects various immune cells. In T cells, IL-13 inhibits Th1 immune responses and the immunosuppressive activity of Treg cells, while enhancing Th2 immune responses. IL-13 also interferes with the apoptosis pathway of CD4+ T cells, inhibits Th17 cell transformation, and affects the secretion of IL-17A and IL-21. In B cells, IL-13 induces the expression of MHC class II molecules and the low-affinity IgE receptor CD23, promoting B cell proliferation and class switching to IgE. IL-13 can activate macrophages to produce IL-10 and promote the expression of TGF-β, VEGF, etc., contributing to angiogenesis and extracellular matrix protein deposition. IL-13 enhances the activation, aggregation, and survival of eosinophils and promotes mast cell proliferation. IL-13 also stimulates eosinophil migration from peripheral blood to sites of inflammation by inducing the production of IL-5 and eosinophil chemokines. IL-13 also plays an important role in non-immune cells; for example, IL-13 is a strong inducer of VCAM-1 in endothelial cells and can increase the expression of β1 integrin and VCAM-1 on human lung fibroblasts.

[0004] Atopic dermatitis (AD) is a common inflammatory skin disease characterized by recurrent, chronic, eczematous rashes accompanied by significant dryness and itching. It is common in young children but can occur at any age. The complex interactions between the environment and the immune system through the epidermal barrier lead to high heterogeneity in the clinical phenotype of AD, with type 2 (Th2) immune responses considered a key factor in inducing the disease. IL-4 and IL-13 are key cytokines involved in the development of AD, with IL-13 considered a key Th2 cytokine driving peripheral inflammation.

[0005] According to Frost & Sullivan data, the number of people with atopic dermatitis (AD) worldwide reached 649 million in 2019 and is projected to increase to 755 million by 2030. The penetration rate of biological therapies for moderate to severe atopic dermatitis remains low both domestically and internationally, indicating a significant unmet clinical need. There is an urgent need to develop more effective IL-13 antibodies to improve clinical benefits for patients. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this disclosure provides anti-IL-13 antibodies and their uses, specifically providing antibodies that specifically bind to human IL-13 or their antigen-binding fragments, multispecific antigen-binding molecules, isolated nucleic acid molecules, vectors, cells, pharmaceutical compositions, pharmaceutical uses, and methods of treating diseases.

[0007] One aspect of this disclosure provides an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and / or a light chain variable region, the heavy chain variable region comprising HCDR1-3, the light chain variable region comprising LCDR1-3, wherein the HCDR1-3 and / or the LCDR1-3 are selected from those CDRs shown in Table 3 or Table 4; the antibody or the antigen-binding fragment thereof specifically binds to human interleukin-13 (IL-13).

[0008] Another aspect of this disclosure provides an antibody or antigen-binding fragment thereof comprising a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises HCDR1-3 and the light chain variable region comprises LCDR1-3, and the antibody or antigen-binding fragment thereof comprises:

[0009] (a) HCDR1, HCDR2, and HCDR3 of the heavy chain shown in any one of SEQ ID NO: 2, 5, or 9; and / or,

[0010] (b) LCDR1, LCDR2 and LCDR3 of the light chains shown in any one of SEQ ID NO: 3, 4, 6, 7 or 8;

[0011] The antibody or its binding fragment specifically binds to human interleukin-13 (IL-13).

[0012] In some embodiments, the HCDR1 comprises the sequence shown in any one of SEQ ID NO: 16, 23, or a sequence having at least 70% identity or at most 3 amino acid mutations or differences compared to it.

[0013] In some embodiments, the HCDR2 comprises the sequence shown in any one of SEQ ID NO: 17, 19, 20, 24, or a sequence having at least 70% identity or at most 3 amino acid mutations or differences compared to it.

[0014] In some embodiments, the HCDR3 comprises the sequence shown in any one of SEQ ID NO: 18, 25, or a sequence having at least 70% identity or at most 3 amino acid mutations or differences compared to it.

[0015] In some embodiments, the LCDR1 comprises the sequence shown in any one of SEQ ID NO: 10, 13, 21, or a sequence having at least 70% identity or at most 3 amino acid mutations or differences compared to it.

[0016] In some embodiments, the LCDR2 comprises the sequence shown in any one of SEQ ID NO: 11, 14, 15, 22, or a sequence having at least 70% identity or at most 3 amino acid mutations or differences compared to it.

[0017] In some embodiments, the LCDR3 comprises the sequence shown in SEQ ID NO: 12, or a sequence having at least 70% identity or at most 3 amino acid mutations or differences compared to it.

[0018] In some implementations, the HCDR1-3 and / or the LCDR1-3 are determined according to the Kabat or IMGT method.

[0019] In some embodiments, the HCDR1-3 and / or the LCDR1-3 are determined according to the Kabat method, and the sequence of HCDR1 is as shown in SEQ ID NO: 16, the sequence of HCDR2 is as shown in any one of SEQ ID NO: 17, 19 and 20, and the sequence of HCDR3 is as shown in SEQ ID NO: 18; and the sequence of LCDR1 is as shown in SEQ ID NO: 10 or 13, the sequence of LCDR2 is as shown in any one of SEQ ID NO: 11, 14 and 15, and the sequence of LCDR3 is as shown in SEQ ID NO: 12.

[0020] In some embodiments, HCDR1-3 and LCDR1-3 are determined according to the IMGT method, and the sequence of HCDR1 is shown as SEQ ID NO: 23, the sequence of HCDR2 is shown as SEQ ID NO: 24, and the sequence of HCDR3 is shown as SEQ ID NO: 25; and the sequence of LCDR1 is shown as SEQ ID NO: 21, the sequence of LCDR2 is shown as SEQ ID NO: 22, and the sequence of LCDR3 is shown as SEQ ID NO: 12.

[0021] In some embodiments, HCDR1-3 and LCDR1-3 are determined according to the Kabat method, and their amino acid sequences are as follows:

[0022] SEQ ID NO:16-18, 13, 14, 12; or

[0023] SEQ ID NO: 16, 19, 18, 13, 11, 12; or

[0024] SEQ ID NO: 16, 19, 18, 10, 15, 12; or

[0025] SEQ ID NO: 16, 19, 18, 13, 15, 12; or

[0026] SEQ ID NO: 16, 19, 18, 10, 14, 12; or

[0027] SEQ ID NO: 16, 20, 18, 13, 15, 12; or

[0028] SEQ ID NO: 16, 20, 18, 10, 14, 12; or

[0029] SEQ ID NO: 16, 20, 18, 13, 14, 12.

[0030] In some embodiments, HCDR1-3 and LCDR1-3 are determined according to the IMGT method, and their amino acid sequences are as follows:

[0031] SEQ ID NO: 23, 24, 25, 21, 22, 12.

[0032] In some embodiments, the heavy chain of the antibody comprises the sequence shown in any one of SEQ ID NO: 2, 5, 9, or a sequence having at least 70% identity or up to 15 amino acid mutations or differences compared to it.

[0033] In some embodiments, the light chain of the antibody comprises the sequence shown in any one of SEQ ID NO: 1, 3-4, 6-8, or a sequence having at least 70% identity or up to 15 amino acid mutations or differences compared to it.

[0034] In some embodiments, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 3.

[0035] In some embodiments, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 4.

[0036] In some embodiments, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 6.

[0037] In some embodiments, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 7.

[0038] In some embodiments, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 8.

[0039] In some embodiments, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 7.

[0040] In some embodiments, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 8.

[0041] In some embodiments, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 3.

[0042] Another aspect of this disclosure provides a mutated antibody or antigen-binding fragment thereof that specifically binds to human IL-13, wherein the mutated antibody contains a CDR region mutation in the heavy chain variable region (VH) and / or the light chain variable region (VL) of the parent, wherein the CDR region mutation in the heavy chain variable region contains S61T and / or A62S, and / or the CDR region mutation in the light chain variable region contains any one or a combination thereof selected from R24K, E59Q, S60T; the mutated antibody or antigen-binding fragment thereof specifically binds to human IL-13.

[0043] In some embodiments, the VH of the parent of the antibody or its antigen-binding fragment contains the sequence shown in SEQ ID NO: 27, or a sequence having at least 70% identity or up to 15 amino acid mutations or differences compared to it.

[0044] In some embodiments, the VL of the parent of the antibody or its antigen-binding fragment contains the sequence shown in SEQ ID NO: 26, or a sequence having at least 70% identity or up to 15 amino acid mutations or differences compared to it.

[0045] In some embodiments, the heavy chain constant region (CH) of the parent of the antibody or its antigen-binding fragment contains the sequence shown in SEQ ID NO: 29, or a sequence having at least 70% identity or up to 15 amino acid mutations or differences compared to it.

[0046] In some embodiments, the light chain constant region (CL) of the parent of the antibody or its antigen-binding fragment contains the sequence shown in SEQ ID NO: 28, or a sequence having at least 70% identity or up to 15 amino acid mutations or differences compared to it.

[0047] In some implementations, the at least 70% identity is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity.

[0048] In some implementations, the maximum three mutations or differences are at most three, two, one, or zero mutations or differences.

[0049] In some implementations, the maximum 15 mutations or differences can be up to 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 mutations or differences.

[0050] In some implementations, the mutation is an insertion, deletion, or substitution.

[0051] In some implementations, the substitution is a conserved amino acid substitution.

[0052] In some implementations, the mutation is a reversion mutation or a hotspot mutation.

[0053] In some embodiments, the antibody or its antigen-binding fragment is selected from: monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, Fab, Fab', Fab'-SH, F(ab')2, Fd, Fv, scFv, bispecific antibodies, and single-domain antibodies.

[0054] In some embodiments, the antibody or its antigen-binding fragment is conjugated to a therapeutic agent or tracer. In some embodiments, the therapeutic agent is selected from radioisotopes, chemotherapeutic agents, and immunomodulators. In some embodiments, the tracer is selected from radiographic contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents, and photosensitizers.

[0055] Another aspect of this disclosure provides a multispecific antigen-binding molecule comprising a first antigen-binding module and a second antigen-binding module, wherein the first antigen-binding module comprises the antibody or antigen-binding fragment, and the second antigen-binding module specifically binds to antigens other than IL-13 or binds to IL-13 antigenic epitopes different from those of the first antigen-binding module.

[0056] In a preferred embodiment, the multispecific antigen-binding molecule is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0057] Another aspect of this disclosure provides an isolated nucleic acid molecule that encodes the antibody or antigen-binding fragment thereof that specifically binds to human IL-13, or the mutated antibody or antigen-binding fragment thereof that specifically binds to human IL-13, or the multispecific antigen-binding molecule.

[0058] Another aspect of this disclosure provides a nucleic acid construct comprising the isolated nucleic acid molecules.

[0059] Another aspect of this disclosure provides a vector comprising the isolated nucleic acid molecule or the nucleic acid construct. Preferably, the vector is an expression vector.

[0060] Another aspect of this disclosure provides a host cell comprising the isolated nucleic acid molecule, the nucleic acid construct, or the vector. Preferably, the host cell is derived from prokaryotic or eukaryotic cells. More preferably, the host cell is derived from mammalian cells, yeast cells, insect cells, *Escherichia coli*, and / or *Bacillus subtilis*; most preferably, the host cell is selected from Expi293 or CHO cells.

[0061] Another aspect of this disclosure provides a method for preparing the antibody that specifically binds to human IL-13 or its antigen-binding fragment, or the mutated antibody that specifically binds to human IL-13 or its antigen-binding fragment, or the multispecific antigen-binding molecule, comprising culturing the host cells under appropriate conditions and isolating the antibody or antigen-binding fragment or multispecific antigen-binding molecule.

[0062] Another aspect of this disclosure provides a pharmaceutical composition comprising the antibody specifically binding to human IL-13 or an antigen-binding fragment thereof, the mutated antibody specifically binding to human IL-13 or an antigen-binding fragment thereof, the multispecific antigen-binding molecule, the isolated nucleic acid molecule, the nucleic acid construct, the vector, the host cell, or a product prepared according to the preparation method described. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, diluent, or adjuvant. In some embodiments, the pharmaceutical composition further comprises additional medicaments for treating and / or preventing diseases associated with aberrant expression and / or activity of IL-13.

[0063] Another aspect of this disclosure provides a kit comprising the aforementioned antibody specifically binding to human IL-13 or its antigen-binding fragment, the aforementioned mutated antibody specifically binding to human IL-13 or its antigen-binding fragment, the aforementioned multispecific antigen-binding molecule, the aforementioned isolated nucleic acid molecule, the aforementioned nucleic acid construct, the aforementioned vector, the aforementioned host cell, or a product prepared according to the aforementioned preparation method, or the aforementioned pharmaceutical composition. In some embodiments, the kit further includes instructions for use.

[0064] Another aspect of this disclosure provides the use of the antibody specifically binding to human IL-13 or its antigen-binding fragment, the mutated antibody specifically binding to human IL-13 or its antigen-binding fragment, the multispecific antigen-binding molecule, the isolated nucleic acid molecule, the vector, the host cell, or a product prepared according to the preparation method, the pharmaceutical composition, or the kit in the preparation of a medicament for the prevention and / or treatment of diseases associated with abnormal expression and / or activity of IL13. Preferably, the diseases associated with abnormal expression and / or activity of IL13 include atopic dermatitis, allergies, asthma, immune-mediated skin diseases, autoimmune diseases, or other IL13-related diseases.

[0065] In some implementations, allergic diseases include allergic rhinitis, allergic dermatitis, allergic conjunctivitis, atopic dermatitis, food allergies, and urticaria.

[0066] In some implementations, immune-mediated skin diseases include dermatitis, erythema multiforme, and contact dermatitis.

[0067] In some implementation schemes, autoimmune diseases include psoriasis, rheumatoid arthritis, juvenile chronic arthritis, chronic sinusitis with nasal polyps, and inflammatory bowel disease (i.e., ulcerative colitis, Crohn's disease).

[0068] In some implementations, other IL13-related diseases include idiopathic interstitial pneumonia, goblet cell metaplasia, inflammatory and fibrotic lung diseases such as cystic fibrosis, gluten-sensitive enteropathy, Whipple's disease, eosinophilic pneumonia, eosinophilic esophagitis, eosinophilic gastritis, idiopathic pulmonary fibrosis, allergic pneumonia, chronic obstructive pulmonary disease, RSV infection, uveitis, scleroderma, osteoporosis, bronchiectasis, hidradenitis suppurativa, and pemphigoid. Most preferably, the diseases associated with abnormal expression and / or activity of IL13 include atopic dermatitis, allergic asthma, non-allergic asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, eczema, urticaria, food allergy, chronic obstructive pulmonary disease, ulcerative colitis, chronic sinusitis with nasal polyps, RSV infection, uveitis, scleroderma, or osteoporosis.

[0069] Another aspect of this disclosure provides a method for preventing and / or treating diseases associated with abnormal expression and / or activity of IL13, comprising administering to a patient in need an effective amount of the said antibody specifically binding to human IL-13 or its antigen-binding fragment, the mutated antibody specifically binding to human IL-13 or its antigen-binding fragment, the multispecific antigen-binding molecule, the isolated nucleic acid molecule, the vector, the host cell, or a product prepared according to the said preparation method, the pharmaceutical composition, or the kit. Preferably, the diseases associated with abnormal expression and / or activity of IL13 include atopic dermatitis, allergies, asthma, immune-mediated skin diseases, autoimmune diseases, or other IL13-related diseases.

[0070] In some implementations, allergic diseases include allergic rhinitis, allergic dermatitis, allergic conjunctivitis, atopic dermatitis, food allergies, and urticaria.

[0071] In some implementations, immune-mediated skin diseases include dermatitis, erythema multiforme, and contact dermatitis.

[0072] In some implementation schemes, autoimmune diseases include psoriasis, rheumatoid arthritis, juvenile chronic arthritis, chronic sinusitis with nasal polyps, and inflammatory bowel disease (i.e., ulcerative colitis, Crohn's disease).

[0073] In some implementations, other IL13-related diseases include idiopathic interstitial pneumonia, goblet cell metaplasia, inflammatory and fibrotic lung diseases such as cystic fibrosis, gluten-sensitive enteropathy, Whipple's disease, eosinophilic pneumonia, eosinophilic esophagitis, eosinophilic gastritis, idiopathic pulmonary fibrosis, allergic pneumonia, chronic obstructive pulmonary disease, RSV infection, uveitis, scleroderma, osteoporosis, bronchiectasis, hidradenitis suppurativa, and pemphigoid. Most preferably, the diseases associated with abnormal expression and / or activity of IL13 include atopic dermatitis, allergic asthma, non-allergic asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, eczema, urticaria, food allergy, chronic obstructive pulmonary disease, ulcerative colitis, chronic sinusitis with nasal polyps, RSV infection, uveitis, scleroderma, or osteoporosis.

[0074] The disclosed anti-IL-13 antibody has one or more of the following superior technical effects:

[0075] 1. All anti-IL-13 antibodies disclosed herein maintain good binding activity with human IL-13 antigen.

[0076] 2. The anti-IL-13 antibody disclosed herein can significantly inhibit IL-13-induced TF-1 cell proliferation.

[0077] 3. The anti-IL-13 antibody disclosed herein has a specific binding to human IL13 protein and a high affinity level.

[0078] Terminology Definitions and Explanations

[0079] Unless otherwise stated, the terms used in this disclosure have the following meanings: the definitions of groups and terms recorded in this disclosure, including their definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, definitions of specific compounds in the examples, etc., can be arbitrarily combined and combined with each other. A particular term should not be considered uncertain or unclear unless specifically defined, but should be understood in accordance with its ordinary meaning in the art. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient.

[0080] The term "IL-13" refers to Interleukin-13, also known as interleukin-13, which is a type I cytokine with a quadruplex core linked by two disulfide bonds. It belongs to the T helper cell subtype (Th2) cytokine family along with IL-3, IL-4, IL-5, and IL-9.

[0081] The term “antibody”, used in its broadest sense, refers to a polypeptide or combination of polypeptides containing sufficient sequences from the variable regions of the immunoglobulin heavy chain and / or from the variable regions of the immunoglobulin light chain to specifically bind to an antigen. The term “antibody” as used herein encompasses a wide range of forms and structures, provided they exhibit the desired antigen-binding activity. The term “antibody” as used herein includes alternative protein scaffolds or artificial scaffolds having transplanted complementarity-determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (containing mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds containing, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004); the contents of which are incorporated herein by reference. Such scaffolds may also include non-antibody-derived scaffolds, such as scaffold proteins known in the art for use in transplanting CDRs, including but not limited to tendinins, fibronectins, peptide aptamers, etc.

[0082] The term "antibody" in this article includes a typical "quadruple-chain antibody," which belongs to the immunoglobulin family composed of two heavy chains (HC) and two light chains (LC). The heavy chain refers to a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-to-C-terminal direction. Optionally, when the full-length antibody is an IgE isotype, it also includes a heavy chain constant region CH4 domain. The light chain is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-to-C-terminal direction. Heavy chains are linked to each other and to each other with disulfide bonds, forming a "Y"-shaped structure. Because the amino acid composition and sequence of the immunoglobulin heavy chain constant region differ, their antigenicity also differs. Based on this, the "immunoglobulins" in this article can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain can further lead to different subclasses. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA into IgA1 and IgA2. Light chains are classified as κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can possess either a κ or λ chain.

[0083] The “antibody” in this article can be derived from any animal, including but not limited to humans and non-human animals. The non-human animals can be selected from primates, mammals, rodents and vertebrates, such as camels, llamas, guanacos, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g., sharks).

[0084] The term "antibody" in this article includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, complete antibodies, fragments of complete antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0085] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that, apart from possible variants (e.g., containing naturally occurring mutations or generated during the manufacturing process of the formulation, such variants are typically present in small amounts), the individual antibodies comprising this group are identical and / or bind to the same epitopes. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. The modifier "monoclonal" herein should not be construed as requiring the production of the antibody or antigen-binding molecule by any particular method. For example, monoclonal antibodies can be prepared using a variety of techniques, including (but not limited to) hybridoma techniques, recombinant DNA methods, phage library display techniques, methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, and other methods known in the art.

[0086] The term "natural antibody" refers to antibodies produced and paired by the immune system of multicellular organisms. The term "engineered antibody" in this article refers to non-natural antibodies obtained through techniques such as genetic engineering and antibody engineering. For example, "engineered antibodies" include humanized antibodies, small molecule antibodies (e.g., scFv), bispecific antibodies, and so on.

[0087] The term "monospecific" refers to having one or more binding sites, where each binding site binds to the same epitope of the same antigen.

[0088] The term "multispecific antibody" refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen. Therefore, terms such as "bispecific," "trispecific," and "quadrispecific" refer to the number of different epitopes that an antibody / antigen binding molecule can bind to.

[0089] The term "valence" indicates the presence of a specified number of binding sites in an antibody / antigen binding molecule. Therefore, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" indicate the presence of one, two, four, and six binding sites in an antibody / antigen binding molecule, respectively.

[0090] In this article, "full-length antibody," "intact antibody," and "complete antibody" are used interchangeably, referring to antibodies with structures that are substantially similar to those of natural antibodies.

[0091] The terms "antigen-binding fragment" and "antibody fragment" in this article include, but are not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies. The terms "antigen-binding fragment" and "antibody fragment" in this article can be understood as local or local variants of intact antibodies having the same antigenic determinant (CDR), monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0092] Papain digestion of the intact antibody produces two identical antigen-binding fragments, called "Fab" fragments, each containing variable domains for both the heavy and light chains, as well as a constant domain for the light chain and a first constant domain (CH1) for the heavy chain. Thus, the term "Fab fragment" in this paper refers to the light chain fragment containing the VL domain and constant domain (CL) of the light chain, and the antibody fragment containing the VH domain and first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment by the addition of a few residues at the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is the Fab' fragment in which the cysteine ​​residues in the constant domain carry a free thiol group. Pepsin treatment produces the F(ab')2 fragment, which has two antigen-binding sites (two Fab fragments) and a portion of the Fc region.

[0093] The “Fv fragment” is the smallest fragment produced by IgG and IgM, containing a complete antigen binding site. The Fv fragment has the same binding properties as Fab and similar three-dimensional binding properties. The VH and VL chains of the Fv fragment are linked together through non-covalent interactions.

[0094] The term "scFv" (single-chain variable fragment) refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994); the contents of which are incorporated herein by reference). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS (SEQ ID NO:30) amino acid sequence or a variant thereof. For example, a adapter having the amino acid sequence (GGGGS)4 (SEQ ID NO:31) may be used, but variants thereof may also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448, the contents of which are incorporated herein by reference). Other adapters that may be used in this disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol. (the contents of which are incorporated herein by reference). In some cases, disulfide bonds can also exist between VH and VL of scFv, forming a disulfide-bonded Fv (dsFv).

[0095] The term "diabody" refers to a polypeptide chain in which the VH and VL domains are expressed on a single polypeptide chain, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with the complementary domain of another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994), the contents of which are incorporated herein by reference).

[0096] The term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain portion is derived from one antibody (which may be derived from a specific species or belong to a specific antibody class or subclass), and another portion of the light chain and / or heavy chain portion is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which retains its binding activity to the target antigen (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)). For example, the term "chimeric antibody" can include antibodies (e.g., human-mouse chimeric antibodies) in which the variable regions of the heavy and light chains of the antibody are derived from a first antibody (e.g., a mouse antibody), while the constant regions of the heavy and light chains of the antibody are derived from a second antibody (e.g., a human antibody).

[0097] The term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase its homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), while all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain or partially retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, the ability to enhance immune cell activity, and the ability to strengthen the immune response.

[0098] The term "fully human antibody" refers to an antibody having variable regions in which both the FR and CDR are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, that constant region is also derived from a human germline immunoglobulin sequence. Fully human antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or through somatic mutations in vivo). However, "fully human antibody" as described herein does not include antibodies in which a CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human frame sequence.

[0099] The term "naked antibody" as used herein refers to an antibody that is not linked, fused to, or conjugated with another agent or molecule (e.g., a label or drug), peptide, or polypeptide. In specific embodiments, naked antibodies expressed by mammalian host cells may be glycosylated by the host cell's glycosylation machinery (e.g., glycosylation enzymes). In some embodiments, naked antibodies are not glycosylated when expressed by host cells that do not possess their own glycosylation machinery (e.g., glycosylation enzymes). In some embodiments, naked antibodies are intact antibodies, while in other embodiments, naked antibodies are antigen-binding fragments of intact antibodies, such as Fab antibody fragments.

[0100] The term "variable region" refers to a region in the antibody heavy or light chain involved in enabling the antibody to bind to an antigen. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies generally have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p.91 (2007). A single VH or VL domain is sufficient to confer antigen-binding specificity. The terms "complementarity-determining region" and "CDR" are used interchangeably in this article. They typically refer to the hypervariable region (HVR) of the heavy chain variable region (VH) or light chain variable region (VL). This region is called the complementarity-determining region because it can form a precise complementarity with the antigen epitope in its spatial structure. The heavy chain variable region CDR can be abbreviated as HCDR, and the light chain variable region CDR can be abbreviated as LCDR. The terms "framework region" and "FR region" are used interchangeably, referring to the amino acid residues in the antibody heavy chain variable region or light chain variable region other than the CDR. A typical antibody variable region consists of four FR regions and three CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0101] The term "CDR" in this paper may be labeled and defined in a manner known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system. The tools and websites used include, but are not limited to, the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abysis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.IMGT.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). The CDR in this paper includes overlaps and subsets of amino acid residues defined in different ways.

[0102] The term "heavy chain constant region" in this document refers to the carboxyl-terminal portion of the antibody heavy chain, which does not directly participate in antibody-antigen binding but exhibits effector functions, such as interaction with the Fc receptor. It has a more conserved amino acid sequence compared to the variable domains of the antibody. A "heavy chain constant region" contains at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof. "Heavy chain constant region" includes a "full-length heavy chain constant region" and a "heavy chain constant region fragment," the former having a structure substantially similar to the natural antibody constant region, while the latter includes only a portion of the full-length heavy chain constant region. Exemplarily, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include the CH1 domain. Exemplarily, a typical "heavy chain constant region fragment" may be selected from the CH1, Fc, or CH3 domains.

[0103] The term "light chain constant region" in this article refers to the carboxyl terminus of the antibody light chain, which does not directly participate in the binding of the antibody to the antigen. The light chain constant region can be selected from the constant κ domain or the constant λ domain.

[0104] The term "Fc" in this document refers to the carboxyl-terminal portion of an antibody obtained by papain hydrolysis of an intact antibody, typically containing the CH3 and CH2 domains of the antibody. Fc regions include, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary slightly, the Fc region of the human IgG heavy chain is generally defined as extending from the amino acid residue at Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the Kabat numbering system) can be removed, for example, during antibody production or purification, or through recombinant engineering of the nucleic acid encoding the antibody heavy chain; therefore, the Fc region may or may not include Lys447.

[0105] The term "single-domain antibody" in this article refers to a single-domain antibody consisting only of the heavy chain variable region obtained from the variable region of a naturally occurring heavy chain antibody that lacks the light chain in a cloned camel.

[0106] The term "identity" in this document can be calculated as follows: To determine the percentage of "identity" between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position. Taking into account the number of vacancies that need to be introduced for optimal alignment of the two sequences and the length of each vacancy, the percentage of identity between the two sequences varies with the common positions of the sequences.

[0107] The term "nucleic acid" in this document includes any compound and / or substance comprising a polymer containing nucleotides. Each nucleotide consists of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, nucleic acid molecules are described by the sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented as 5′ to 3′. In this document, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers containing mixtures of two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derived sugar or phosphate backbones or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as carriers for the direct expression of the antibodies disclosed herein in vitro and / or in vivo, such as in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) carriers can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA carrier and / or the expression of the encoded molecule, thereby allowing the mRNA to be injected into a subject to generate antibodies in vivo (see, for example, Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038 / nm.4356 or EP 2 101 823 B1).

[0108] As used herein, the term "vector" includes nucleic acid vectors, such as DNA vectors (e.g., plasmids), RNA vectors, viruses, or other suitable replicons (e.g., viral vectors). Various vectors have been developed for delivering polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells. The expression vectors of this disclosure contain polynucleotide sequences and additional sequence elements, for example, for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. Some vectors that can be used to express the antibodies and antibody fragments of this disclosure include plasmids containing regulatory sequences (e.g., promoter and enhancer regions) that guide gene transcription. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to guide efficient transcription of the gene carried on the expression vector. The expression vectors of this disclosure may also contain polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or norsocrine.

[0109] The term "host cell" in this article refers to a cell in which foreign nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their progeny, regardless of the number of passages. Progeny may not be identical to parental cells in their nucleic acid contents and may contain mutations. This article includes mutant progeny with the same function or biological activity as those screened or selected in the initially transformed cells.

[0110] The term "treatment" refers to surgical or therapeutic treatment aimed at preventing, slowing (reducing) undesirable physiological changes or lesions in the treated individual, such as the progression of cancer, autoimmune diseases, and viral infections. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, disease severity reduction, disease stability (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of disease status, and remission (whether partial or complete), whether detectable or undetectable. Individuals requiring treatment include those already suffering from the condition or disease, those susceptible to the condition or disease, or those intending to prevent the condition or disease. When terms such as slowing, reducing, weakening, mitigating, or remission are used, they also imply elimination, disappearance, or non-occurrence.

[0111] The term "effective dose" refers to the amount of a therapeutic agent, administered alone or in combination with another therapeutic agent to cells, tissues, or subjects, that is effective in preventing or alleviating symptoms of a disease or the progression of that disease. "Effective dose" also refers to the amount of a compound sufficient to relieve symptoms, such as treating, curing, preventing, or alleviating an associated medical condition, or increasing the rate at which such symptoms are treated, cured, prevented, or alleviated. When an active ingredient is administered to an individual alone, the therapeutically effective dose refers to that ingredient alone. When a combination is used, the therapeutically effective dose refers to the combined amount of active ingredients that produce the therapeutic effect, regardless of whether they are administered in combination, consecutively, or simultaneously.

[0112] The term "subject" refers to an organism that receives treatment for a specific disease or condition as described in this disclosure. Examples of subjects and patients include mammals, such as humans, primates (e.g., monkeys), or non-primate mammals that receive treatment for a disease or condition.

[0113] The amount of the disclosed compound constituting a “therapeutic effective amount” varies depending on the compound, the disease state and its severity, the route of administration, and the age of the mammal to be treated, but may routinely be determined by a person skilled in the art based on their own knowledge and the content of this disclosure.

[0114] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0115] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present disclosure or salts thereof with pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate the administration of the disclosed compounds to an organism.

[0116] The term "pharmaceuticalally acceptable excipient" refers to excipients that do not cause significant irritation to the organism and do not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

[0117] The word “comprise” or “include” and its English variants such as comprises or comprising can be understood as having an open, non-exclusive meaning, that is, “including but not limited to”.

[0118] The pharmaceutical compositions disclosed herein are suitable for parenteral administration, such as in suitable unit dosage forms as sterile solutions, suspensions, or lyophilized products. For example, the pharmaceutical compositions disclosed herein may be in the form of sterile aqueous solutions for intramuscular or subcutaneous administration. The pharmaceutical compositions disclosed herein may accept other solvents or media, such as water, Ringer's solution, or isotonic sodium chloride solution, during use.

[0119] The antibodies disclosed herein can be prepared by a variety of methods known to those skilled in the art, including prokaryotic expression, eukaryotic expression, artificial synthesis, and other equivalent substitutions known to those skilled in the art. Attached Figure Description

[0120] Unless otherwise defined in this disclosure, scientific and technical terms used in connection with this disclosure shall have the meanings understood by one of ordinary skill in the art.

[0121] Figures 1A-1B show the binding activity of the anti-IL13 antibody to the human IL13 protein.

[0122] Figures 2A-2B show the inhibitory activity of anti-IL13 antibody on human IL13-induced HEK293-IL4R&IL13R-STAT6 reporter gene signaling.

[0123] Figures 3A-3B show the inhibitory activity of anti-IL13 antibody on human IL13-induced TF-1 cell proliferation. Detailed Implementation

[0124] The present disclosure will be further described below with reference to specific embodiments, and the advantages and features of the present disclosure will become clearer with the description. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available products.

[0125] The embodiments disclosed herein are merely exemplary and do not constitute any limitation on the scope of this disclosure. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions disclosed herein without departing from the spirit and scope of this disclosure, but all such modifications and substitutions fall within the protection scope of this disclosure.

[0126] Example 1: Design of Lebrikizumab antibody CDR mutation and preparation of corresponding mutant antibodies

[0127] Based on the crystal structure of the Lebrikizumab antibody and IL13 complex (PDB ID: 4I77), the amino acid interactions at the three-dimensional structural interface were analyzed, and antibodies maintaining activity were obtained by mutating CDR sites using various algorithms. The Lebrikizumab antibody CDR mutation design is shown in Table 1. The specific sequence information of the Lebrikizumab mutant antibody is shown in Table 2. Based on the specific sequences in Table 2, the anti-IL3 antibody disclosed in this paper was prepared, including the parental Lebrikizumab and mutants Lebrikizumab H0L8, Lebrikizumab H5L1, Lebrikizumab H5L2, Lebrikizumab H5L6, Lebrikizumab H5L7, Lebrikizumab H1L6, Lebrikizumab H1L7, and Lebrikizumab H1L8.Among them, the heavy chain of Lebrikizumab H0L8 is the parental Lebrikizumab heavy chain, and the light chain is based on the parental Lebrikizumab light chain, containing the mutations R24K, E59Q, and S60T; the heavy chain of Lebrikizumab H5L1 is based on the parental Lebrikizumab heavy chain, containing the mutations S61T and A62S, and the light chain is based on the parental Lebrikizumab light chain, containing the mutation R24K; the heavy chain of Lebrikizumab H5L2 is based on the parental Lebrikizumab heavy chain, containing the mutations S61T and A62S, and the light chain is based on the parental Lebrikizumab light chain, containing the mutation E59Q; Lebrikizumab The heavy chain of Lebrikizumab H5L6 is based on the parental Lebrikizumab heavy chain, containing the mutations S61T and A62S; the light chain is based on the parental Lebrikizumab light chain, containing the mutations R24K and E59Q. The heavy chain of Lebrikizumab H5L7 is based on the parental Lebrikizumab heavy chain, containing the mutations S61T and A62S; the light chain is based on the parental Lebrikizumab light chain, containing the mutations E59Q and S60T. The heavy chain of Lebrikizumab H1L6 is based on the parental Lebrikizumab heavy chain, containing the mutation S61T; the light chain is based on the parental Lebrikizumab light chain, containing the mutations R24K and E59Q. The H1L7 heavy chain is based on the parental Lebrikizumab heavy chain and contains the S61T mutation; the light chain is based on the parental Lebrikizumab light chain and contains the E59Q and S60T mutations. The Lebrikizumab H1L8 heavy chain is based on the parental Lebrikizumab heavy chain and contains the S61T mutation; the light chain is based on the parental Lebrikizumab light chain and contains the R24K, E59Q, and S60T mutations.

[0128] Table 1. Mutation numbers and corresponding sequence numbers for the Lebrikizumab antibody CDR region mutation design.

[0129] Table 2. Parental and mutant amino acid sequences of Lebrikizumab antibody (light and heavy chains).

[0130] Note: Bold and underlined amino acids indicate amino acids that have mutated relative to the parent.

[0131] According to the Kabat numbering system, the Kabat analysis results of the CDR regions of the Lebrikizumab parental and mutant antibody sequences are shown in Table 3.

[0132] Table 3. Kabat analysis results of the CDR region of the amino acid sequences of Lebrikizumab parental and mutant antibodies.

[0133] According to the IMGT numbering system, the IMGT analysis results of the CDR region of the above-mentioned Lebrikizumab parental and mutant antibody sequences are shown in Table 4.

[0134] Table 4. IMGT analysis results of the CDR region of the amino acid sequences of Lebrikizumab parental and mutant antibodies.

[0135] Example 2: Identification of the activity of the disclosed anti-human IL-13 antibody

[0136] 2.1 ELISA method for detecting the binding activity of anti-human IL-13 antibody to human IL-13-his-Biotin protein

[0137] Human IL-13-his-Biotin protein (Acro, #IL3-H82E5) was diluted with 50 ng / well in PBS (Meilunbio, #PWL050) buffer at pH 7.4. This was added to 96-well microplates (Corning, #9018) pre-coated with 250 ng / well of Streptavidin (Sigma, #S4762) and incubated overnight at 4°C. The next day, before the experiment, the plates were washed three times with PBST buffer (pH 7.4 PBS containing 0.1% Tween-20), and then 100 μL / well of different concentrations of the antibody to be tested, diluted with sample diluent, was added. The plates were incubated at 37°C for 1 hour. After incubation, the plates were washed three times with PBST, and then 100 μL / well of HRP-labeled goat anti-human IgG full-length secondary antibody (Jackson Immuno, #109-035-098), diluted with sample diluent, was added. The plates were incubated at 37°C for 1 hour. After incubation, the plates were washed five times with PBST, and 50 μL / well of TMB chromogenic substrate (KPL, catalog number #5120-0077) was added. The plates were incubated at room temperature for 5–15 min, and then 50 μL / well of 1M hydrochloric acid was added to terminate the reaction. The absorbance was read at 450 nm using a Biotek PowerWave microplate reader, and the EC50 value of the anti-human IL-13 antibody binding to human IL-13-his-Biotin protein was calculated. The isotype was anti-FTIC-hIgG4. The results are shown in Figures 1A and 1B and Table 5-1. All antibodies, after amino acid mutation, maintained good binding activity to the human IL-13 antigen.

[0138] Table 5-1-1 Binding activity of anti-human IL-13 antibodies

[0139] 2.2 Detection of the inhibitory activity of anti-human IL13 antibody against human IL13-induced HEK293-IL4R&IL13R-STAT6 reporter gene signaling.

[0140] HEK293T-IL-4Rα & IL-13Rα1-STAT6 cells were cultured in T-175 cell culture flasks to achieve 90% confluence. The culture medium was aspirated, and the cells were washed once with PBS buffer, then digested with trypsin. After stopping the digestion, the cells were washed twice with PBS buffer. The cell pellet was resuspended in DMEM medium containing 2% (w / w) fetal bovine serum to a final volume of 4 × 10⁻⁶. 5Cells per milliliter. 50 μL / well was added to a white translucent reaction plate (purchased from Cabernet Biotechnology, catalog number: 62096), followed by 25 μL of DMEM medium diluted with 2% (w / w) fetal bovine serum to a final concentration of 1 ng / mL human IL13. Then, 25 μL / well of serially diluted test antibody in DMEM medium containing 2% (w / w) fetal bovine serum was added. The plates were incubated at 37°C and 5% (v / v) CO2 for five hours. Detection was performed using the Bio-Glo luciferase reporter assay kit (purchased from Meilun Biotechnology, catalog number: PWL110). 50 μL / well of reagent was added, and results were read using a PE EnSight microplate reader. Data were analyzed using software (GraphPad Prism10), and IC50 values ​​were calculated, with the isotype set to anti-FTIC-hIgG4. The results are shown in Figures 2A and 2B and Table 5-2. The antibody with amino acid mutation can significantly inhibit the HEK293-IL4R&IL13R-STAT6 reporter gene signal induced by human IL13.

[0141] Table 5-2 Blocking activity of anti-human IL-13 antibodies

[0142] 2.3 Detection of the inhibitory activity of anti-human IL13 antibody against IL13-induced TF-1 cell proliferation

[0143] TF-1-luciferase suspension cells were cultured in T-75 cell culture flasks until the logarithmic growth phase. After centrifugation, the supernatant was discarded, and the cell pellet was washed twice with PBS. The cell density was adjusted to 8 × 10⁶ cells / year using 1640 medium containing 2% (w / w) fetal bovine serum. 5 Add 50 μL per well to a white translucent reaction plate (purchased from Cabernet Biotechnology, catalog number: 62096) and starve the cells at 37°C for 24 hours. Add 25 μL of human IL13 diluted to a final concentration of 2 ng / mL in 1640 medium with 2% (w / w) fetal bovine serum, followed by 25 μL / well of serially diluted test antibody in DMEM medium containing 2% (w / w) fetal bovine serum. Incubate at 37°C and 5% (v / v) CO2 for 72 hours. Detect using the Bio-Glo luciferase reporter assay kit (purchased from Meilun Biotechnology, catalog number: PWL110), adding 50 μL of reagent per well and reading the results using a PE EnSight microplate reader. Analyze the data using software (GraphPad Prism10), perform data fitting, and calculate the IC50 value, where the isotype is anti-FTIC-hIgG4. The results are shown in Figures 3A and 3B and Table 5-3. The antibody with amino acid mutation can significantly inhibit IL13-induced TF-1 cell proliferation.

[0144] Table 5-3 Blocking activity of anti-human IL-13 antibodies

[0145] 2.4 Biacore assay for the affinity between anti-human IL13 antibody and human IL13 protein

[0146] The specific binding between the tested anti-IL13 antibodies and human IL13 protein was detected using a BIAcore 8K. The experiment employed a ProteinA chip (purchased from Cytiva, catalog number #29-1275-56) to capture the antibodies. Human IL13 protein was serially diluted, and the affinity between the antibody and antigen was determined using multi-cycle kinetics. In each cycle, after capturing the antibody, a gradient concentration of TSLP protein was injected, and the binding and dissociation processes of the antigen and antibody were recorded. After each cycle, the ProteinA chip was regenerated using Glycine pH 1.5 (purchased from Cytiva, catalog number #BR-1003-54) to remove protein from the chip. Finally, a 1:1 binding model was applied to analyze the data and fit the antibody-antigen binding kinetic parameters, including the binding rate constant ka, dissociation rate constant kd, equilibrium dissociation constant KD, and maximum binding signal Rmax. Table 6 shows that all antibodies with amino acid mutations exhibited specific binding with human IL13 protein and showed high affinity levels.

[0147] Table 6. Affinity of anti-human IL13 antibody to human IL13 protein

[0148] Various changes and equivalent substitutions may be made to the embodiments of this disclosure without departing from the spirit and scope of this disclosure. Unless otherwise stated in the context, any feature, step, or embodiment of the embodiments of this disclosure may be combined with any other feature or embodiment.

Claims

1. An antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment comprises a heavy chain variable region and / or a light chain variable region, the heavy chain variable region comprising HCDR1-3, the light chain variable region comprising LCDR1-3, wherein HCDR1-3 and / or the LCDR1-3 are selected from the CDRs shown in Table 3 or Table 4; or, The antibody or its antigen-binding fragment comprises: (a) HCDR1, HCDR2, and HCDR3 of the heavy chain shown in any one of SEQ ID NO: 2, 5, or 9; and / or, (b) LCDR1, LCDR2 and LCDR3 of the light chains shown in any one of SEQ ID NO: 3, 4, 6, 7 or 8; The antibody or its antigen-binding fragment specifically binds to human interleukin-13 (IL-13).

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the HCDR1 comprises the sequence shown in any one of SEQ ID NO: 16, 23, or a sequence having at least 70% identity or at most 3 amino acid mutations or differences compared to it; and The HCDR2 comprises the sequence shown in any one of SEQ ID NO: 17, 19, 20, 24, or a sequence having at least 70% identity or at most 3 amino acid mutations or differences compared to it; and The HCDR3 comprises the sequence shown in any one of SEQ ID NO: 18 or 25, or a sequence having at least 70% identity or at most 3 amino acid mutations or differences compared to it; and / or The LCDR1 comprises the sequence shown in any one of SEQ ID NO: 10, 13, 21, or a sequence having at least 70% identity or at most 3 amino acid mutations or differences compared to it; and The LCDR2 comprises the sequence shown in any one of SEQ ID NO: 11, 14, 15, 22, or a sequence having at least 70% identity or at most 3 amino acid mutations or differences compared to it; and The LCDR3 comprises the sequence shown in SEQ ID NO: 12, or a sequence having at least 70% identity or at most 3 amino acid mutations or differences compared to it.

3. The antibody or its antigen-binding fragment according to claim 1 or 2, wherein HCDR1-3 and / or LCDR1-3 are determined according to the Kabat or IMGT method; Preferably, HCDR1-3 and LCDR1-3 are determined according to the Kabat method, and the sequence of HCDR1 is as shown in SEQ ID NO: 16, the sequence of HCDR2 is as shown in any one of SEQ ID NO: 17, 19 and 20, and the sequence of HCDR3 is as shown in SEQ ID NO: 18; and the sequence of LCDR1 is as shown in SEQ ID NO: 10 or 13, the sequence of LCDR2 is as shown in any one of SEQ ID NO: 11, 14 and 15, and the sequence of LCDR3 is as shown in SEQ ID NO: 12; or Preferably, HCDR1-3 and LCDR1-3 are determined according to the IMGT method, and the sequence of HCDR1 is as shown in SEQ ID NO: 23, the sequence of HCDR2 is as shown in SEQ ID NO: 24, and the sequence of HCDR3 is as shown in SEQ ID NO: 25; and the sequence of LCDR1 is as shown in SEQ ID NO: 21, the sequence of LCDR2 is as shown in SEQ ID NO: 22, and the sequence of LCDR3 is as shown in SEQ ID NO: 12; More preferably, HCDR1-3 and LCDR1-3 are determined according to the Kabat method, and their amino acid sequences are as follows: SEQ ID NO:16-18, 13, 14, 12; or SEQ ID NO: 16, 19, 18, 13, 11, 12; or SEQ ID NO: 16, 19, 18, 10, 15, 12; or SEQ ID NO: 16, 19, 18, 13, 15, 12; or SEQ ID NO: 16, 19, 18, 10, 14, 12; or SEQ ID NO: 16, 20, 18, 13, 15, 12; or SEQ ID NO: 16, 20, 18, 10, 14, 12; or SEQ ID NO: 16, 20, 18, 13, 14, 12; or More preferably, HCDR1-3 and LCDR1-3 are determined according to the IMGT method, and their amino acid sequences are as follows: SEQ ID NO: 23, 24, 25, 21, 22, 12.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the heavy chain of the antibody comprises the sequence shown in any one of SEQ ID NO: 2, 5, 9, or a sequence having at least 70% identity or at most 15 amino acid mutations or differences compared to it.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the light chain of the antibody comprises the sequence shown in any one of SEQ ID NO: 1, 3-4, 6-8, or a sequence having at least 70% identity or at most 15 amino acid mutations or differences compared to it.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the amino acid sequence of the heavy chain of the antibody is as shown in SEQ ID NO: 2, and the amino acid sequence of the light chain of the antibody is as shown in SEQ ID NO: 3; or The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 4; or The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 6; or The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 7; or The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 8; or The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 7; or The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 8; or The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO:

3.

7. A mutated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof contains a CDR region mutation in the heavy chain variable region (VH) and / or the light chain variable region (VL) of the parent, the CDR region mutation in the heavy chain variable region containing S61T and / or A62S, and / or the CDR region mutation in the light chain variable region containing any one or a combination of R24K, E59Q, and S60T; the mutated antibody or antigen-binding fragment thereof specifically binds to human IL-13.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein the parental VH of the antibody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 27, or a sequence having at least 70% identity or at most 15 amino acid mutations or differences compared to it.

9. The antibody or antigen-binding fragment thereof according to claim 7 or 8, wherein the parental VL of the antibody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 26, or a sequence having at least 70% identity or at most 15 amino acid mutations or differences compared to it.

10. The antibody or antigen-binding fragment thereof according to any one of claims 7-9, wherein the heavy chain constant region (CH) of the parent of the antibody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 29, or a sequence having at least 70% identity or at most 15 amino acid mutations or differences compared to it.

11. The antibody or antigen-binding fragment thereof according to any one of claims 7-10, wherein the light chain constant region (CL) of the parent of the antibody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 28, or a sequence having at least 70% identity or at most 15 amino acid mutations or differences compared to it.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1-11, wherein the at least 70% identity is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1-12, wherein the most three mutations or differences are at most three, two, one or zero mutations or differences.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1-13, wherein the most 15 mutations or differences are at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 mutations or differences.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1-14, wherein the mutation is an insertion, deletion or substitution; preferably, the substitution is a conserved amino acid substitution; more preferably, the mutation is a reversion mutation or a hotspot mutation.

16. The antibody or antigen-binding fragment thereof according to any one of claims 1-15, wherein the antibody or antigen-binding fragment thereof is selected from: monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, Fab, Fab', Fab'-SH, F(ab')2, Fd, Fv, scFv, bispecific antibodies, and single-domain antibodies.

17. The antibody or antigen-binding fragment thereof according to any one of claims 1-16, wherein the antibody or antigen-binding fragment thereof is conjugated with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from: radioisotopes, chemotherapeutic agents and immunomodulators; and / or the tracer is selected from: radiocontrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers.

18. A multispecific antigen-binding molecule, wherein the multispecific antigen-binding molecule comprises a first antigen-binding module and a second antigen-binding module, the first antigen-binding module comprising an antibody or antigen-binding fragment as described in any one of claims 1-17, and the second antigen-binding module specifically binds to antigens other than IL-13 or binds to IL-13 antigenic epitopes different from those of the first antigen-binding module; preferably, the multispecific antigen-binding molecule is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

19. An isolated nucleic acid molecule, wherein the nucleic acid molecule encodes an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-17, or a multispecific antigen-binding molecule as claimed in claim 18.

20. A nucleic acid construct comprising the isolated nucleic acid molecule of claim 19.

21. A vector comprising the isolated nucleic acid molecule of claim 19 or the nucleic acid construct of claim 20; preferably, the vector is an expression vector.

22. A host cell comprising the isolated nucleic acid molecule of claim 19, the nucleic acid construct of claim 20, or the vector of claim 21; preferably, the host cell is derived from prokaryotic or eukaryotic cells; more preferably, the host cell is derived from mammalian cells, yeast cells, insect cells, Escherichia coli, and / or Bacillus subtilis; most preferably, the host cell is selected from Expi293 or CHO cells.

23. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1-17, or the multispecific antigen-binding molecule according to claim 18, comprising culturing the host cell according to claim 22 under appropriate conditions and isolating the antibody or antigen-binding fragment, or the multispecific antigen-binding molecule.

24. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-17, a multispecific antigen-binding molecule as described in claim 18, an isolated nucleic acid molecule as described in claim 19, a nucleic acid construct as described in claim 20, a carrier as described in claim 21, a host cell as described in claim 22, or a product prepared according to the preparation method described in claim 23; preferably, the composition further comprises a pharmaceutically acceptable carrier, diluent, or adjuvant; and / or preferably, the pharmaceutical composition further comprises additional medicaments for treating and / or preventing diseases associated with abnormal expression and / or activity of IL13.

25. A kit comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-17, a multispecific antigen-binding molecule according to claim 18, an isolated nucleic acid molecule according to claim 19, a nucleic acid construct according to claim 20, a vector according to claim 21, a host cell according to claim 22, or a product prepared according to the preparation method of claim 23, or a pharmaceutical composition according to claim 24; optionally, the kit further comprising instructions for use.

26. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-17, the multispecific antigen-binding molecule according to claim 18, the isolated nucleic acid molecule according to claim 19, the nucleic acid construct according to claim 20, the vector according to claim 21, the host cell according to claim 22, or the product prepared according to the preparation method according to claim 23, or the pharmaceutical composition according to claim 24, or the kit according to claim 25 in the preparation of a medicament for the prevention and / or treatment of diseases associated with abnormal expression and / or activity of IL13; Preferably, the diseases associated with abnormal expression and / or activity of IL13 include atopic dermatitis, allergies, asthma, immune-mediated skin diseases, autoimmune diseases, or other diseases related to IL13; More preferably, allergic diseases include allergic rhinitis, allergic dermatitis, allergic conjunctivitis, atopic dermatitis, food allergies, and urticaria; and / or immune-mediated skin diseases include psoriasis, erythema multiforme, and contact dermatitis; and / or autoimmune diseases include psoriasis, rheumatoid arthritis, juvenile chronic arthritis, chronic sinusitis with nasal polyps, inflammatory bowel disease (i.e., ulcerative colitis, Crohn's disease); and / or other IL13-related diseases include idiopathic interstitial pneumonia, goblet cell metaplasia, inflammatory and fibrotic lung diseases such as cystic fibrosis, gluten-sensitive enteropathy, Whipple's disease, eosinophilic pneumonia, eosinophilic esophagitis, eosinophilic gastritis, idiopathic pulmonary fibrosis, allergic pneumonia, chronic obstructive pulmonary disease, RSV infection, uveitis, scleroderma, osteoporosis, bronchiectasis, hidradenitis suppurativa, and pemphigoid. Most preferably, the diseases associated with abnormal expression and / or activity of IL13 include atopic dermatitis, allergic asthma, non-allergic asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, eczema, urticaria, food allergy, chronic obstructive pulmonary disease, ulcerative colitis, chronic sinusitis with nasal polyps, RSV infection, uveitis, scleroderma, or osteoporosis.

27. A method for preventing and / or treating diseases associated with abnormal expression and / or activity of IL13, comprising administering to a patient in need an effective amount of an antibody or antigen-binding fragment thereof as described in any one of claims 1-17, a multispecific antigen-binding molecule as described in claim 18, an isolated nucleic acid molecule as described in claim 19, a nucleic acid construct as described in claim 20, a vector as described in claim 21, a host cell as described in claim 22, or a product prepared according to the preparation method described in claim 23, or a pharmaceutical composition as described in claim 24, or a kit as described in claim 25; Preferably, the diseases associated with abnormal expression and / or activity of IL13 include atopic dermatitis, allergies, asthma, immune-mediated skin diseases, autoimmune diseases, or other diseases related to IL13; More preferably, allergic diseases include allergic rhinitis, allergic dermatitis, allergic conjunctivitis, atopic dermatitis, food allergies, and urticaria; and / or immune-mediated skin diseases include psoriasis, erythema multiforme, and contact dermatitis; and / or autoimmune diseases include psoriasis, rheumatoid arthritis, juvenile chronic arthritis, chronic sinusitis with nasal polyps, inflammatory bowel disease (i.e., ulcerative colitis, Crohn's disease); and / or other IL13-related diseases include idiopathic interstitial pneumonia, goblet cell metaplasia, inflammatory and fibrotic lung diseases such as cystic fibrosis, gluten-sensitive enteropathy, Whipple's disease, eosinophilic pneumonia, eosinophilic esophagitis, eosinophilic gastritis, idiopathic pulmonary fibrosis, allergic pneumonia, chronic obstructive pulmonary disease, RSV infection, uveitis, scleroderma, osteoporosis, bronchiectasis, hidradenitis suppurativa, and pemphigoid. Most preferably, the diseases associated with abnormal expression and / or activity of IL13 include atopic dermatitis, allergic asthma, non-allergic asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, eczema, urticaria, food allergy, chronic obstructive pulmonary disease, ulcerative colitis, chronic sinusitis with nasal polyps, RSV infection, uveitis, scleroderma, or osteoporosis.

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