Antibodies against human IL-4RA and their use

Abstract

The present invention relates to an antibody against human interleukin-4 receptor A, a pharmaceutical composition or kit containing the same, and their use in the treatment of eosinophilic esophagitis.

Description

[Technical Field]

[0001] The present invention belongs to the field of treatment of allergic diseases and molecular immunology, and relates to anti-human interleukin-4 receptor A antibodies, pharmaceutical compositions or kits containing same, and their use in the treatment of, for example, eosinophilic esophagitis. [Background technology]

[0002] The interleukin-4 receptor (IL-4R) is a transmembrane receptor that exists in two distinct forms: type I IL-4R, which consists of a high-affinity IL-4Rα subunit (i.e., IL-4RA in the present invention) and a medium-affinity γC subunit. It binds to interleukin-4 (IL-4) and mediates IL-4-induced biological functions such as cell proliferation and activation. Type II IL-4R, which consists of a high-affinity IL-4Rα subunit (IL-4RA) and an interleukin-13 receptorα subunit (IL-13Rα), also functions as a homologous functional receptor capable of binding to interleukin-13 (IL-13) (Wang and Secombes, Cytokine. 2015, Vol. 75, No. 1, pp. 8-13 (2015)). IL-4R is expressed in T, B, and hematopoietic stem cells, as well as endothelial and epithelial cells, muscle cells, fibroblasts, hepatocytes, and brain tissue. After IL-4 binds to its receptor, IL-4R activates various non-receptor protein tyrosine kinases in the cytoplasm in cooperation with the γC subunit or the IL-13Rα subunit, initiating downstream signaling pathways (Nelms et al., Annu Rev Immunol, 17:701-738 (1999); LaPorte et al., Cell, 132(2):259-272 (2008)).

[0003] IL-4RA can bind to IL-4 and IL-13 with high affinity and is the major functional subunit of the above-mentioned type I and type II IL-4Rs, and inhibition of IL-4RA can effectively block the related biological functions mediated by IL-4 and IL-13 (Gessner et al., Immunobiology, 201, 285 (2000)).

[0004] Eosinophilic esophagitis (EoE) is a chronic esophageal disease caused by eosinophil infiltration into the esophagus. It commonly occurs in adolescents and children and manifests as a variety of symptoms, including heartburn / reflux, vomiting, difficulty swallowing, food impact, and even abdominal pain (Gonsalves Nirmala P, Aceves Seema S, Diagnosis and treatment of eosinophilic esophagitis. [J]. J Allergy Clin Immunol, 2020, 145:1-7). EoE patients require dietary restrictions and may require repeated hospital visits, resulting in a poor quality of life.

[0005] EOE is often associated with allergies, with 50% to 60% of EoE patients having a history of allergic conditions, such as rhinitis, bronchial asthma, or atopic dermatitis, which can lead to specific reactive diseases and symptoms. Gastroesophageal reflux disease (GERD), allergens (mainly food allergens), alterations in the epithelial barrier, and potential microbial binding can all contribute to EOE. Food allergens penetrate the epithelium and activate receptors and inflammatory cells, such as eosinophils, which secrete cytokines and induce chronic inflammation, tissue damage, and fibrosis (Vinit C, Dieme A, Courbage S, et al. Eosinophilic esophagitis: Pathophysiology, diagnosis, and management [J]. Archives de Pediatrie, 2019, 26(3):182-190).

[0006] EOE is an immune inflammatory response mediated by T helper cell type 2 (Th2). IL-4 induces early T cells to become Th2 cells, which can secrete IL-5, which enhances eosinophil recruitment by promoting the release of eosinophil chemoactacsin and eosinophil survival, thereby exacerbating the inflammatory response (Joshua B. Wechsler, Ikuo Hirano, Kashi Xiaoyin, Qian Jiaming. Biological Treatment of Eosinophilic Gastroenteritis [J]. Chinese Journal of Clinical Immunology and Allergy, 2018, 12(04):437-444). Part B of a Phase 2 / 3 clinical trial of the anti-human IL-4RA mab dupilumab evaluated its investigational use in patients aged 12 years and older with eosinophilic esophagitis. Results showed that patients receiving dupilumab 300 mg weekly experienced significant improvements in clinical and histological disease indicators and a 64% reduction in EoE symptoms compared with 41% in the placebo group. Summary of the Invention [Means for solving the problem]

[0007] As a result of in-depth research and creative efforts, the present inventors have expressed recombinant human IL-4RA as an antigen using a mammalian cell expression system, immunized mice, and fused the mouse spleen cells with myeloma cells to obtain hybridoma cells. After screening a large number of samples, the present inventors obtained the following hybridoma cell lines:

[0008] The inventors have discovered the following: The hybridoma cell line 13E5 can secrete and produce a specific antibody (designated 13E5) that specifically binds to human IL-4RA, and the antibody can very effectively block the binding of human IL-4RA to IL-4.

[0009] Furthermore, the present inventors have creatively prepared humanized anti-human IL-4RA antibodies (designated 13E5H1L1, 13E5H2L2, 13E5H3L3, 13E5H4L4, and 13E5H4L2, respectively).

[0010] The antibody can effectively bind to human IL-4RA, block the binding of human IL-4RA to its ligands, IL-4 or IL-13, and inhibit the activation of the downstream signaling pathway of human IL-4RA, and may be useful in preparing drugs for the prevention and treatment of eosinophilic esophagitis.

[0011] This provides the following invention: [1] An antibody or an antigen-binding fragment thereof, particularly, the antibody or antigen-binding fragment thereof binds to IL-4RA, preferably human IL-4RA, and is used to treat eosinophilic esophagitis, wherein the antibody is identified as: comprising HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 2, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO: 4; Preferably, based on the IMGT numbering system, the antibody is HCDR1 comprising or consisting of the sequence shown in SEQ ID NO:5 or a variant thereof; an HCDR2 comprising or consisting of the sequence shown in SEQ ID NO:6 or a variant thereof; and an HCDR3 comprising or consisting of the sequence shown in SEQ ID NO:7 or a variant thereof, and the antibody is LCDR1 comprising or consisting of the sequence shown in SEQ ID NO:8 or a variant thereof; an LCDR2 comprising or consisting of the sequence shown in SEQ ID NO:9 or a variant thereof; an LCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 10 or a variant thereof; An antibody or antigen-binding fragment thereof, wherein the sequence of the variant has at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the corresponding sequence.

[0012] [2] The antibody of [1], wherein the antibody comprises: (1)(i) a heavy chain variable region comprising or consisting of the following sequence: The amino acid sequence shown in SEQ ID NO:2 or a variant thereof; (ii) a light chain variable region comprising or consisting of the following sequence: The amino acid sequence shown in SEQ ID NO:4 or a variant thereof; (3)(i) a heavy chain variable region comprising or consisting of the following sequence: The amino acid sequence shown in SEQ ID NO: 12 or a variant thereof; (ii) a light chain variable region comprising or consisting of the following sequence: The amino acid sequence shown in SEQ ID NO: 14 or a variant thereof; (4)(i) a heavy chain variable region comprising or consisting of the following sequence: The amino acid sequence shown in SEQ ID NO: 16 or a variant thereof; (ii) a light chain variable region comprising or consisting of the following sequence: The amino acid sequence shown in SEQ ID NO: 18 or a variant thereof; (5)(i) a heavy chain variable region comprising or consisting of the following sequence: The amino acid sequence shown in SEQ ID NO: 20 or a variant thereof; (ii) a light chain variable region comprising or consisting of the following sequence: The amino acid sequence shown in SEQ ID NO: 22 or a variant thereof; (6) (i) a heavy chain variable region comprising or consisting of the following sequence: The amino acid sequence shown in SEQ ID NO: 24 or a variant thereof; (ii) a light chain variable region comprising or consisting of the following sequence: The amino acid sequence shown in SEQ ID NO: 26 or a variant thereof; (7) (i) a heavy chain variable region comprising or consisting of the following sequence: The amino acid sequence shown in SEQ ID NO: 24 or a variant thereof; (ii) a light chain variable region comprising or consisting of the following sequence: The amino acid sequence set forth in SEQ ID NO: 18 or a variant thereof; or The variant sequence has at least 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the corresponding sequence of an antibody.

[0013] [3] The antibody or antigen-binding fragment thereof according to any one of [1] to [2], wherein the antibody further comprises framework regions FR-H1, FR-H2, FR-H3, and FR-H4 of a heavy chain variable region, and framework regions FR-L1, FR-L2, FR-L3, and FR-L4 of a light chain variable region; (1) FR-H1 comprises or consists of the amino acid sequence of SEQ ID NO: 27 or a variant thereof; FR-H2 comprises or consists of the amino acid sequence of SEQ ID NO:28 or a variant thereof; FR-H3 comprises or consists of the amino acid sequence of SEQ ID NO:29 or a variant thereof; FR-H4 comprises or consists of the amino acid sequence of SEQ ID NO: 30 or a variant thereof; FR-L1 comprises or consists of the amino acid sequence of SEQ ID NO: 31 or a variant thereof; FR-L2 comprises or consists of the amino acid sequence of SEQ ID NO: 32 or a variant thereof; FR-L3 comprises or consists of the amino acid sequence of SEQ ID NO: 33 or a variant thereof; FR-L4 comprises or consists of the amino acid sequence of SEQ ID NO: 34 or a variant thereof; (2) FR-H1 comprises or consists of the amino acid sequence of SEQ ID NO: 35 or a variant thereof; FR-H2 comprises or consists of the amino acid sequence of SEQ ID NO: 36 or a variant thereof; FR-H3 comprises or consists of the amino acid sequence of SEQ ID NO: 37 or a variant thereof; FR-H4 comprises or consists of the amino acid sequence of SEQ ID NO: 38 or a variant thereof; FR-L1 comprises or consists of the amino acid sequence of SEQ ID NO: 39 or a variant thereof; FR-L2 comprises or consists of the amino acid sequence of SEQ ID NO: 40 or a variant thereof; FR-L3 comprises or consists of the amino acid sequence of SEQ ID NO: 41 or a variant thereof; FR-L4 comprises or consists of the amino acid sequence of SEQ ID NO: 42 or a variant thereof; (3) FR-H1 comprises or consists of the amino acid sequence of SEQ ID NO: 43 or a variant thereof; FR-H2 comprises or consists of the amino acid sequence of SEQ ID NO:44 or a variant thereof; FR-H3 comprises or consists of the amino acid sequence of SEQ ID NO: 45 or a variant thereof; FR-H4 comprises or consists of the amino acid sequence of SEQ ID NO: 46 or a variant thereof; FR-L1 comprises or consists of the amino acid sequence of SEQ ID NO: 47 or a variant thereof; FR-L2 comprises or consists of the amino acid sequence of SEQ ID NO: 48 or a variant thereof; FR-L3 comprises or consists of the amino acid sequence of SEQ ID NO: 49 or a variant thereof; FR-L4 comprises or consists of the amino acid sequence of SEQ ID NO: 50 or a variant thereof; (4) FR-H1 comprises or consists of the amino acid sequence of SEQ ID NO: 51 or a variant thereof; FR-H2 comprises or consists of the amino acid sequence of SEQ ID NO: 52 or a variant thereof; FR-H3 comprises or consists of the amino acid sequence of SEQ ID NO: 53 or a variant thereof; FR-H4 comprises or consists of the amino acid sequence of SEQ ID NO: 54 or a variant thereof; FR-L1 comprises or consists of the amino acid sequence of SEQ ID NO: 55 or a variant thereof; FR-L2 comprises or consists of the amino acid sequence of SEQ ID NO: 56 or a variant thereof; FR-L3 comprises or consists of the amino acid sequence of SEQ ID NO: 57 or a variant thereof; FR-L4 comprises or consists of the amino acid sequence of SEQ ID NO: 58 or a variant thereof; (5) FR-H1 comprises or consists of the amino acid sequence of SEQ ID NO: 59 or a variant thereof; FR-H2 comprises or consists of the amino acid sequence of SEQ ID NO: 60 or a variant thereof; FR-H3 comprises or consists of the amino acid sequence of SEQ ID NO: 61 or a variant thereof; FR-H4 comprises or consists of the amino acid sequence of SEQ ID NO: 62 or a variant thereof; FR-L1 comprises or consists of the amino acid sequence of SEQ ID NO: 63 or a variant thereof; FR-L2 comprises or consists of the amino acid sequence of SEQ ID NO:64 or a variant thereof; FR-L3 comprises or consists of the amino acid sequence of SEQ ID NO: 65 or a variant thereof; FR-L4 comprises or consists of the amino acid sequence of SEQ ID NO: 66 or a variant thereof; The sequence of the variant has at least 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the corresponding sequence of an antibody or antigen-binding fragment thereof.

[0014] [4] The antibody or antigen-binding fragment thereof according to any one of [1] to [3], wherein the antibody is a humanized antibody, a chimeric antibody, or a multispecific antibody (e.g., a bispecific antibody).

[0015] [5] The antibody or antigen-binding fragment thereof according to [4], wherein the constant region of the antibody is humanized, preferably derived from human IgG, more preferably derived from IgG4.

[0016] [6] The antibody or antigen-binding fragment thereof according to [5], wherein the heavy chain constant region of the antibody employs an Ig gamma-4 chain C region, more preferably the Ig gamma-4 chain C region of GenBank Accession No. ACCESSION: P01861.1, and the light chain constant region employs an Ig kappa chain C region, more preferably the Ig kappa chain C region of GenBank Accession No. ACCESSION: P01834.

[0017] [7] The antibody or antigen-binding fragment thereof according to any one of [1] to [6], wherein the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, Fab / c, a complementarity-determining region (CDR) fragment, a single-chain antibody (e.g., scFv), a bivalent antibody, or a domain antibody.

[0018] [8] A biological material selected from an antibody conjugate, a multispecific antibody (preferably a bispecific antibody), a fusion protein, or a pharmaceutical composition, used to treat eosinophilic esophagitis, the biological material comprising the antibody or antigen-binding fragment thereof according to any one of [1] to [7]; Preferably, the antibody conjugate further comprises a conjugate moiety conjugated to the antibody or antigen-binding fragment thereof, wherein the conjugate moiety is selected from a purification tag (e.g., a His tag), a cytotoxic agent, a detectable label, a radioisotope, a luminescent substance, a colored substance, an enzyme, or polyethylene glycol; Preferably, the multispecific antibody further comprises antibodies or antigen-binding fragments against other antigens and / or other antigen epitopes, Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition is used alone or in combination with one or more drugs, Preferably, the pharmaceutical composition is in a dosage form suitable for oral administration to the gastrointestinal tract (GI), preferably the dosage form is selected from tablets, capsules, pills, powders, granules, emulsions, microemulsions, solutions, suspensions, syrups and elixirs, or the drug is in a dosage form suitable for subcutaneous, intradermal, intravenous, intramuscular, intralesional injection, biological material.

[0019] [9] Use of the antibody or antigen-binding fragment thereof according to any one of [1] to [7], or the biological material according to [8], in the preparation of a drug or kit for treating eosinophilic esophagitis, wherein the kit preferably further comprises an additional therapeutic agent selected from one or more of the following: an IL-4 / IL-13 pathway inhibitor, an IL-1β inhibitor, an IL-5 inhibitor, an IL-8 inhibitor, an IL-9 inhibitor, an IL-13 inhibitor, an IL-17 inhibitor, an IL-25 inhibitor, a JAK inhibitor, a STAT6 inhibitor, a TNF inhibitor, an eosinophil chemokine-3 inhibitor, an IgE inhibitor, a prostaglandin D2 inhibitor, an immunosuppressant, a corticosteroid, a globulin inhibitor, a steroid ... glucocorticoids, long acting beta2-agonists (e.g. salmeterol or formoterol), proton pump inhibitors, decongestants, antihistamines and nonsteroidal anti-inflammatory drugs (NSAIDs), NSAIDs (nonsteroidal anti-inflammatory drugs), antibiotics, antibacterial agents, antiviral agents, antifungal agents, drugs to treat tumors (preferably chemotherapeutic agents or growth inhibitory agents, targeted therapeutic agents, antibody drug conjugates, T cells expressing chimeric antigen receptors, antibodies or antigen-binding fragments thereof, angiogenesis inhibitors, antitumor agents, cancer vaccines, adjuvants and combinations thereof, alkylating agents, antimetabolites, antibiotics, botanical and / or hormonal drugs), The IL-4 / IL-13 pathway inhibitor is not the antibody or antigen-binding fragment thereof described in any one of [1] to [7], Preferably, the IL-4 / IL-13 pathway inhibitor is selected from an anti-IL-4 antibody, an anti-IL-13 antibody, an anti-IL-4 / IL-13 antibody, an IL-4 receptor (IL-4R) inhibitor (e.g., Dupilumab antibody or its biological equivalent, AMG317 or MEDI9314), a JAK inhibitor, or a STAT6 inhibitor.

[0020]

[10] A method for treating eosinophilic esophagitis, comprising administering to a subject in need thereof the antibody or antigen-binding fragment thereof according to any one of [1] to [7] or the biological material according to [8], wherein the subject is preferably selected from mammals (rodents, felines, canines, primates, etc.), preferably a human, and more preferably an infant aged 0 to 1 year, a child aged 1 to 5 years, a juvenile aged 6 to 12 years, an adolescent aged 12 to 18 years, or an adult aged 18 years or older.

[0021]

[11] The method of

[10] , further comprising administering to the subject an additional therapeutic agent or combination therapy selected from one or more of the following: an IL-4 / IL-13 pathway inhibitor, an IL-1β inhibitor, an IL-5 inhibitor, an IL-8 inhibitor, an IL-9 inhibitor, an IL-13 inhibitor, an IL-17 inhibitor, an IL-25 inhibitor, a JAK inhibitor, a STAT6 inhibitor, a TNF inhibitor, an eosinophil chemokine-3 (CCL26) inhibitor, an IgE inhibitor, a prostaglandin D2 inhibitor, an immunosuppressant, a corticosteroid, a glucocorticoid, or a long-acting β2-agonist (e.g., salmeterol or formoterol). ), proton pump inhibitors, decongestants, antihistamines and nonsteroidal anti-inflammatory drugs (NSAIDs), NSAIDs (nonsteroidal anti-inflammatory drugs), antibiotics, antibacterial agents, antiviral agents, antifungal agents, drugs for treating tumors (preferably chemotherapeutic agents or growth inhibitory agents, targeted therapeutic agents, antibody drug conjugates, T cells expressing chimeric antigen receptors, antibodies or antigen-binding fragments thereof, angiogenesis inhibitors, antitumor agents, cancer vaccines, adjuvants and combinations thereof, alkylating agents, antimetabolites, antibiotics, botanical drugs and / or hormone-based drugs), or combinations thereof, said combination therapy comprising dietary therapy and esophageal dilation; The IL-4 / IL-13 pathway inhibitor is not the antibody or antigen-binding fragment thereof described in any one of [1] to [7], Preferably, the IL-4 / IL-13 pathway inhibitor is selected from an anti-IL-4 antibody, an anti-IL-13 antibody, an anti-IL-4 / IL-13 antibody, an IL-4 receptor (IL-4R) inhibitor (e.g., Dupilumab antibody or a biological equivalent thereof, AMG317, or MEDI9314), a JAK inhibitor, or a STAT6 inhibitor; More preferably, the method is one in which the additional therapeutic agent is administered simultaneously or sequentially with the antibody or antigen-binding fragment thereof according to any one of [1] to [7].

[0022]

[12] A hybridoma cell line having the accession number CCTCC NO: C2018131 or a monoclonal antibody secreted by the hybridoma cell line for treating eosinophilic esophagitis.

[0023] In some embodiments of the present invention, there is provided the use of an IL-4 / IL-13 pathway inhibitor in the preparation of a medicament for treating or inhibiting or preventing eosinophilic esophagitis in an individual.

[0024] In some embodiments of the invention, there is provided the use of an antibody or antigen-binding fragment thereof that binds IL-4RA in the preparation of a medicament for treating, inhibiting or preventing eosinophilic esophagitis in an individual.

[0025] In the present invention, the treatment of EoE includes alleviating at least one symptom or sign, including, but not limited to, esophageal eosinophil infiltration, esophageal wall thickening, esophagitis, the appearance of tracheal rings or protrusions in the esophagus, chest and abdominal pain, anorexia, vomiting, difficulty swallowing, and food impaction.

[0026] In some embodiments of the present invention, the term "eosinophil infiltration" refers to the presence of eosinophils in a patient's organs or tissues (including blood, esophagus, stomach, duodenum, and ileum) with 15 or more eosinophils in at least one high-power field in a biopsy.

[0027] In some embodiments of the present invention, the "patients" include subgroups that may exhibit elevated levels of EoE-associated biomarkers, including, but not limited to, esophageal eosinophils, eosinophil chemokine-3 (CCL26), serum eosinophil peroxidase, chemokine ligand 17 (CCL17), IgE, periostin, IL-5, IL-13, thymic stromal lymphopoietin (TSLP), and eosinophil-derived neurotoxin (EDN).

[0028] As used herein, an "allergen" includes any substance, compound, particle, or composition that can elicit an allergic reaction in a susceptible individual, including foods such as dairy products, eggs, wheat, legumes, seafood, nuts, and shellfish.

[0029] In some embodiments of the invention, the patient of the invention is selected from infants aged 0-1 years, children aged 1-5 years, boys aged 6-12 years, adolescents aged 12-18 years, and adults aged 18 years or older.

[0030] In some embodiments of the invention, an IL-4 / IL-13 pathway inhibitor is used in combination with a second therapeutic agent or therapy.

[0031] IL-4 / IL-13 pathway inhibitors according to the present invention include, but are not limited to, anti-IL-4 antibodies, anti-IL-13 antibodies, bispecific anti-IL-4 / IL-13 antibodies, IL-4 receptor (IL-4R) inhibitors, JAK inhibitors, and STAT6 inhibitors. In some embodiments, the IL-4 / IL-13 pathway inhibitor is an IL-4R inhibitor, e.g., an anti-IL-4R antibody, more preferably an anti-IL-4RA antibody.

[0032] In some embodiments of the invention, the methods comprise simultaneously or sequentially administering to the subject or patient a second therapeutic agent, preferably the second therapeutic agent is an IL-1β inhibitor, an IL-5 inhibitor, an IL-8 inhibitor, an IL-9 inhibitor, an IL-13 inhibitor, an IL-17 inhibitor, an IL-25 inhibitor, a JAK inhibitor, a STAT6 inhibitor, a TNF inhibitor, an eosinophil chemokine-3 (CCL26) inhibitor, an IgE inhibitor, a prostaglandin D2 inhibitor, an immunosuppressant, a corticosteroid, a glucocorticoid, a long-acting β2-agonist, a proton pump inhibitor, In some embodiments, the therapeutic agent is selected from the group consisting of anti-inflammatory drugs, decongestants, antihistamines and nonsteroidal anti-inflammatory drugs (NSAIDs), NSAIDs (nonsteroidal anti-inflammatory drugs), antibiotics, antibacterial agents, antiviral agents, antifungal agents, drugs to treat tumors (preferably chemotherapeutic agents or growth inhibitory agents, targeted therapeutic agents, antibody drug conjugates, T cells expressing chimeric antigen receptors, antibody or antigen binding fragments, angiogenesis inhibitors, antitumor agents, cancer vaccines, adjuvants and combinations thereof, alkylating agents, antimetabolites, antibiotics, botanicals and / or hormone-based drugs), or combinations thereof.

[0033] In some embodiments of the present invention, the combination therapy includes, but is not limited to, dietary therapy and esophageal dilation.

[0034] In some embodiments of the present invention, the IL-4R inhibitor is the Dupilumab antibody or a biological equivalent thereof.

[0035] In some embodiments of the invention, the IL-4R inhibitor is AMG317 or MEDI9314.

[0036] In some embodiments of the invention, the long-acting β2-agonist is salmeterol or formoterol.

[0037] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the laboratory operation steps of cell culture, molecular genetics, nucleic acid chemistry, and immunology used in the present invention are all common steps widely used in the corresponding fields. In addition, in order to better understand the present invention, the following provides definitions and interpretations of related terms.

[0038] As used herein, the term "antigen-binding region" refers to a protein or a portion of a protein that specifically binds to a particular antigen. For example, the portion of an antibody that contains amino acid residues that interact with an antigen and confer specificity and affinity to the antibody for the antigen is called the "antigen-binding region." An antigen-binding region typically contains one or more "complementarity-determining regions" ("CDRs"). Some antigen-binding regions further contain one or more "framework" regions ("FRs"). CDRs are amino acid sequences that contribute to antigen-binding specificity and affinity.

[0039] As used herein, the term "antibody" refers to a complete immunoglobulin of any isotype or an antigen-binding fragment thereof capable of specifically binding to a target antigen in competition with a complete antibody, including, for example, chimeric antibodies, humanized antibodies, fully humanized antibodies, and bispecific antibodies or antigen-binding fragments thereof. Such "antibodies" are a type of antigen-binding protein. Complete antibodies typically contain at least two full-length heavy chains and two full-length light chains, but in some cases may contain fewer chains, such as naturally occurring antibodies in camelids, which may contain only heavy chains. An antibody or antigen-binding fragment thereof may be derived from only a single source or may be "chimeric," i.e., different portions of the antibody may be derived from two different sources, as further described below. An antibody or antigen-binding fragment thereof may be produced in a hybridoma by recombinant DNA technology, or by enzymatic or chemical cleavage of a complete antibody. Unless otherwise specified, the term "antibody" includes antibodies containing two full-length heavy chains and two full-length light chains, as well as derivatives, variants, and fragments thereof.

[0040] As used herein, the term "antigen-binding fragment" (or simply "fragment") of an "antibody" or "immunoglobulin chain (heavy or light)" includes a portion of an antibody (regardless of how obtained or synthesized) that lacks at least some amino acids present in the full-length antibody chain but is still capable of specifically binding to an antigen. Such fragments are biologically active because they can specifically bind to a target antigen and compete with other antibodies or antigen-binding fragments thereof for specific binding to a given epitope. In one embodiment, such fragments retain at least one CDR present in the full-length light or heavy chain of the antibody, and in some embodiments, include a single heavy and / or light chain or portion thereof. These biologically active fragments may be produced by recombinant DNA techniques or by, for example, enzymatic or chemical cleavage of intact antibodies. Immunofunctional immunoglobulin fragments include, but are not limited to, Fab, Fab', F(ab'), Fv, domain antibodies, and single-chain antibodies, and may be derived from any mammal, including, but not limited to, human, mouse, rat, camelid, or rabbit. It is also envisioned that functional portions of the antibodies disclosed herein, such as one or more CDRs, may be covalently linked to a second protein or small molecule to generate therapeutic agents directed to specific targets in the body, thereby having bifunctional therapeutic properties or extended serum half-life, such as fusion proteins.

[0041] As used herein, the terms "full antibody chain," "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody or having a heavy chain of the Fc region as defined herein.

[0042] The term "light chain" includes full-length light chains and fragments thereof containing sufficient variable region sequence to confer binding specificity. A full-length light chain contains a variable region domain, VL, and a constant region domain, CL. The variable region domain of a light chain is at the amino-terminus of the polypeptide. Light chains include kappa chains and lambda chains.

[0043] The term "heavy chain" includes full-length heavy chains and fragments thereof containing sufficient variable region sequence to confer binding specificity. A full-length heavy chain contains a variable region domain, VH, and three constant region domains, CH1, CH2, and CH3. The VH domain is at the amino-terminus of the polypeptide, and the CH domain is at the carboxy-terminus, with CH3 being closest to the carboxy-terminus of the polypeptide. The heavy chain can have any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0044] As used herein, the term "Fab fragment" refers to a fragment consisting of one light chain and one heavy chain variable region. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0045] As used herein, the term "Fc" region includes two heavy chain fragments containing the CH1 and CH2 domains of an antibody, which are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domain.

[0046] As used herein, the term "Fab' fragment" comprises one light chain and a portion of one heavy chain (including the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains) such that an interchain disulfide bond can form between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[0047] As used herein, the term "F(ab')2 fragment" refers to two light chains and two heavy chains containing portions of the constant region between the CH1 and CH2 domains, such that interchain disulfide bonds are formed between the two heavy chains. The F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

[0048] As used herein, the term "Fv region" comprises the variable regions from the heavy and light chains, but lacks the constant regions.

[0049] As used herein, the term "Fd" fragment refers to an antibody fragment consisting of the VH and CH1 domains (Ward et al., Nature 341:544-546 (1989)).

[0050] As used herein, a "dAb" fragment (Ward et al., Nature 341:544-546 (1989)) consists of a VH domain.

[0051] As used herein, the term "Fab'-SH" is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.

[0052] As used in the present invention, "Fab / c" fragments are intermediate antibody degradation products formed by pepsin digestion of immunoglobulins. They possess the advantages of the Fab and Fc regions, namely, strong diffusion ability and slow metabolic elimination from the body, while maintaining high affinity (Liu Jianjun, Journal of Cellular and Molecular Immunology, 1989(4):29-29).

[0053] As used herein, the term "single-chain antibody" refers to an Fv molecule in which the variable regions of the heavy and light chains are linked via a flexible linker to form a single polypeptide chain that forms the antigen-binding region (see, e.g., Bird et al., Science. 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA. 90:5879-5883 (1988)). Single-chain antibodies are described in detail in International Patent Application Publication No. WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203 (the disclosures of which are incorporated by reference).

[0054] As used herein, the term "domain antibody" refers to an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some cases, two or more VH regions are covalently linked via a peptide linker to generate multivalent domain antibodies (particularly bivalent domain antibodies). The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0055] In the present invention, the term "bivalent antigen-binding protein" or "bivalent antibody" comprises two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. A bivalent antibody may be bispecific.

[0056] As used herein, the term "multispecific antigen-binding protein" or "multispecific antibody" is an antigen-binding protein or antibody that targets multiple antigens or epitopes.

[0057] As used herein, the terms "bispecific," "dual-specific," or "bifunctional" antigen-binding proteins or antibodies refer to hybrid antigen-binding proteins or antibodies, each having two different antigen-binding sites. Bispecific antibodies are multispecific antigen-binding proteins or antibodies and can be produced by a variety of methods, including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, for example, Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321; Kostelny et al., 1992, J. Immunol. 148:1547-1553. The two binding sites of a bispecific antigen-binding protein or antibody bind to two different epitopes present on the same or different protein targets.

[0058] As used herein, the terms "mab" and "monoclonal antibody" refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of completely identical antibody molecules, excluding natural mutations that may occur spontaneously. A mab has high specificity for a single epitope on an antigen. In contrast to a monoclonal antibody, a polyclonal antibody typically contains at least two or more different antibodies that recognize different epitopes on the antigen. Monoclonal antibodies are typically obtained using the hybridoma technology first described by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA technology (see, e.g., US Pat. No. 4,816,567).

[0059] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment in which all or part of the CDR regions of a human-derived immunoglobulin (acceptor antibody) have been substituted with the CDR regions of a non-human-derived antibody (donor antibody). These donor antibodies may be non-human (e.g., mouse, rat, or rabbit) antibodies with the desired specificity, affinity, or reactivity. Some amino acid residues in the framework region (FR) of the acceptor antibody can be substituted with corresponding amino acid residues of a non-human antibody or with amino acid residues of another antibody to further improve or optimize the performance of the antibody. For more details on humanized antibodies, see, for example, Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); Presta, Curr. Op. Struct. Biol., 2:593-596 (1992) and Clark, Immunol. Today 21:397-402 (2000).

[0060] As used herein, the term "epitope" refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitope" is also referred to in the art as "antigenic determinant." Epitopes or antigenic determinants are usually composed of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics. For example, an epitope usually contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique conformation and may be "linear" or "conformational." See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996). In a linear epitope, all points of interaction between the protein and the interacting molecule (e.g., an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across separate amino acid residues of the protein.

[0061] As used herein, the terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residues are analogs or mimetics of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms also include amino acid polymers that have been modified or phosphorylated, for example, by the addition of sugar residues to form glycoproteins. Polypeptides and proteins can be produced by naturally occurring cells and non-recombinant cells, or by genetic engineering or recombinant cells, and include molecules having the amino acid sequence of a native protein or molecules with one or more amino acid deletions, additions, and / or substitutions of the native sequence.

[0062] The terms "polypeptide" and "protein" specifically include antibodies, such as anti-human IL-4RA antibodies (also called IL-4RA antibodies), IL-4RA binding proteins, and antibodies or sequences having one or more amino acid deletions, additions, and / or substitutions of antigen-binding proteins.

[0063] The term "polypeptide fragment" refers to a polypeptide having an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion compared to the full-length protein. Such fragments may further contain modified amino acids compared to the full-length protein. In some embodiments, the length of a fragment is about 5 to 500 amino acids. For example, the length of a fragment may be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids. Useful polypeptide fragments include immunologically functional fragments of antibodies containing the binding domain. In the case of human IL-4RA antibodies, useful fragments include, but are not limited to, CDR regions, heavy or light chain variable domains, portions of antibody chains, or variable domains containing just two CDRs.

[0064] The terms "human IL-4RA," "hIL-4RA," "human IL-4 receptor A," and "human IL-4 receptor α subunit" are used interchangeably to refer to the human interleukin-4 receptor α subunit. Human IL-4RA refers to its mature peptide (Genbank ID: NP_001244336.1). IL-4 and IL-13 are the major endogenous agonists of IL-4RA. "IL-4RA" means human IL-4RA unless otherwise specified or clearly understood from the context in which the term is used.

[0065] A "derivative" of a polypeptide is in a manner distinct from an insertion, deletion, or substitution variant, e.g., a polypeptide (e.g., an antigen-binding protein or antibody) that has been chemically modified by the conjugation of other chemical moieties, such as a polypeptide conjugated to PEG.

[0066] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) is one in which the antibody binds to a target antigen within about 10 -5 M, for example, about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 Affinity (K D ) binds to the antigen.

[0067] As used herein, "K" D The term "dissociation equilibrium constant" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction and is used to describe the binding affinity between an antibody and an antigen. Among several parameters measured in molecular binding kinetics, K D The value is the dissociation equilibrium constant, which in antibody drug research is a parameter that specifies the strength of the affinity between the antibody being measured and the target antigen molecule. The calculation method is K D=kdis / kon: The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and antigen. kon: binding rate constant = the rate at which the antigen-antibody complex is formed; the smaller kon, the faster the binding rate between the antibody and antigen. kdis: dissociation rate constant = the rate at which the antibody dissociates from the antigen-antibody complex; the smaller kdis, the slower the rate at which the antibody leaves the antigen and the stronger the binding between the antibody and antigen. Generally, antibodies form at a rate of about 10 -5 Less than M, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 The dissociation equilibrium constant (K D ) binds to an antigen (e.g., L1 protein) and is measured, for example, using surface plasmon resonance (SPR) with a BIACORE meter or Fortebio molecular interaction meter.

[0068] In the present invention, amino acids are typically represented by one-letter and three-letter abbreviations known in the art, for example, alanine may be represented by A or Ala.

[0069] As used herein, the terms "hybridoma" and "hybridoma cell line" are used interchangeably and references to the terms "hybridoma" and "hybridoma cell line" include subclones and progeny of hybridomas.

[0070] As used herein, the term "percentage sequence identity" is used interchangeably with "percentage sequence homology."

[0071] As used herein, the terms "similarity," "sequence similarity," and "identity" refer to the relationship between the sequences of two or more protein or polypeptide molecules, as measured by aligning and comparing the sequences. "Percentage identity" refers to the percentage of identical residues between amino acids in the compared molecules and can be calculated based on the minimum size of the compared molecules. To perform these calculations, alignment gaps (if any) must be resolved using a specific mathematical model or computer program (i.e., "algorithm"). When applied to polypeptides, the term "substantially identical" refers to two peptide sequences that share at least 70%, 75%, or 80% sequence identity, at least 90% or 95% sequence identity, and at least 97%, 98%, or 99% sequence identity when optimally aligned using, for example, the programs GAP or BESTFIT with the default gap weights provided by the programs. In some cases, residues that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted with another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophilicity). Generally, a conservative amino acid substitution does not substantially change the functional properties of a protein. When two or more of the amino acid sequences differ from each other by a conservative substitution, the conservative nature of the substitution can be corrected by adjusting the percentage sequence identity upward. Methods for making such adjustments are well known to those skilled in the art. See, for example, Pearson, Methods Mol. Biol. 243:307-31 (1994). Examples of amino acid groups with side chains with similar chemical properties include: 1) aliphatic hydroxyl side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine.Conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.

[0072] Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al., Science 256:1443-45 (1992), which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0073] Sequence identity of polypeptides is typically measured using sequence analysis software. Protein analysis software matches sequences using measures of similarity assigned to different substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG includes programs such as "Gap" and "Bestfit" that can be used to measure sequence homology or sequence identity between closely related polypeptides (e.g., homologous polypeptides from different species) or between a wild-type protein and its mutant protein (using default parameters specified by the program). See, for example, GCG Version 6.1 (University of Wisconsin, WI). Polypeptide sequences can also be compared using FASTA with default or recommended parameters, see GCG Version 6.10 FASTA (e.g., FASTA2 and FASTA3), which provides alignment and percentage sequence identity of the optimal overlap region between the challenge and search sequences (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000)). When comparing sequences with a database containing multiple sequences from different organisms, another preferred algorithm is the computer program BLAST, particularly blastp or tblastn (using the program's default parameters). See, for example, Altschul et al., Mol. Biol. 215:403-410 (1990); Altschul et al., Nucleic Acids Res. 25:3389-402 (1997).

[0074] As used herein, the term "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, a desired effect. For example, a prophylactically effective amount for a disease (e.g., a disease associated with the binding of IL-4RA to IL-4, e.g., eosinophilic esophagitis) refers to an amount sufficient to prevent, inhibit, or delay the onset of the disease (e.g., a disease associated with the binding of IL-4RA to IL-4, e.g., eosinophilic esophagitis), and a therapeutically effective amount for a disease refers to an amount sufficient to cure or at least partially inhibit the disease and its complications in a patient suffering from the disease. Determining such effective amounts is within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition, such as age, weight, and sex, the dosage form of the drug, and other treatments administered simultaneously.

[0075] Administration method The following content does not limit the application forms of the antibody or antigen-binding fragment thereof, the antibody conjugate, the fusion protein, the drug, or the multi(bi)specific antibody of the present invention.

[0076] In one embodiment, the antibody or antigen-binding fragment thereof, said antibody conjugate, said fusion protein, said drug or said multi(bi)specific antibody of the present invention can be formulated as a pharmaceutical composition suitable for single or multiple administration.

[0077] The antibodies or antigen-binding fragments thereof, antibody conjugates thereof, fusion proteins, drugs or the multi(bi)specific antibodies of the present invention are administered by a variety of suitable routes, including, but not limited to, oral or parenteral (intravenous, intramuscular, topical, subcutaneous, intraperitoneal, spinal or other non-gastrointestinal routes of administration, e.g., by injection or infusion). In some embodiments, the antibodies or antigen-binding fragments thereof, antibody conjugates thereof, fusion proteins, drugs or the multi(bi)specific antibodies of the present invention are administered orally or by injection, e.g., intravenously or intraperitoneally.

[0078] As used herein, the phrase "parenteral administration" refers to patterns of administration other than enteral and topical administration by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracystic, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcystic, subarachnoid, intraspinal, epidural, intrasternal injection and infusion, and in vivo electroporation.

[0079] In some embodiments, the related factor inhibitor (e.g., an anti-human IL-4R antibody) is administered by a non-gastrointestinal route, and in some embodiments, orally. Other non-gastrointestinal routes include topical, epidermal, or mucosal administration routes, such as intranasal, intravaginal, rectal, sublingual, or local administration routes. Administration may be, for example, single, multiple, and / or over one or more extended periods. Suitable dosage forms of the antibody or antigen-binding fragment thereof, antibody conjugate, fusion protein, drug, or multi(bi)specific antibody of the present invention include, but are not limited to, tablets, buccal tablets, pills, capsules (e.g., hard capsules, soft capsules, enteric-coated capsules, microcapsules), elixirs, granules, syrups, injections (intramuscular, intravenous, intraperitoneal), granules, emulsions, suspensions, solutions, dispersions, sustained-release formulations for oral or parenteral administration, and the like.

[0080] The medicament of the present invention comprises a pharmaceutically acceptable carrier and / or excipient.

[0081] Compared with the prior art, the present invention has the following advantages, for example: The anti-human IL-4RA antibodies of the present invention can bind to human IL-4RA with high affinity and, by blocking the binding of human IL-4RA to human IL-4 and human IL-13, can inhibit the biological effects of related cells mediated by human IL-4 and human IL-13, such as cell proliferation and IL-4- and IL-13-induced increases in CD23 expression levels. Having advantages such as high activity and immunity to species differences, the antibodies can be used to prepare drugs that block the binding of human IL-4 and IL-13 to human IL-4RA, such as drugs for the treatment or prevention of eosinophilic esophagitis, and have good future application potential and market value. [Brief explanation of the drawings]

[0082] [Figure 1] Binding activity of 13E5 and Dupilumab to the antigen human IL-4RA-hFc or human IL-4RA-mFc. [Figure 2] Binding activity of 13E5H1L1, 13E5H2L2, 13E5H3L3, and Dupilumab to the antigen IL-4RA-mFc. [Figure 3] Binding activity of 13E5H4L2, 13E5H4L4, and Dupilumab to the antigen IL-4RA-mFc. [Figure 4] The activity of 13E5 and Dupilumab competing with human IL4-N-His for binding to human IL-4RA-hFc. [Figure 5] The activity of 13E5H1L1, 13E5H2L2, 13E5H3L3, and Dupilumab to compete with human IL-4-N-His for binding to human IL-4RA-hFc. [Figure 6] The activity of 13E5H4L2, 13E5H4L4, and Dupilumab to compete with human IL-4-N-His for binding to human IL-4RA-hFc. [Figure 7] This shows the results of detecting the ability of 13E5H4L4 and Dupilumab to compete with human IL-4-N-His for binding to human IL-4RA-hFc. [Figure 8]1 shows the results of determining the affinity constant between 13E5H4L4 and human IL-4RA. The antibody concentrations added for each pair of curves from top to bottom in the figure are 25 nM, 6.25 nM, 3.13 nM, 1.56 nM, 0.78 nM, and 0.39 nM, respectively. [Figure 9] 1 shows the results of determining the affinity constant between dupilumab and human IL-4RA. The antibody concentrations added for each pair of curves from top to bottom in the figure are 12.5 nM, 6.25 nM, 3.13 nM, 1.56 nM, 0.78 nM, and 0.39 nM, respectively. [Figure 10] This shows the results of determining the affinity constant between 13E5H1L1 and human IL-4RA. The antibody concentrations added for each pair of curves from top to bottom in the figure are 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, 1.56 nM, 0.78 nM, and 0.39 nM, respectively. [Figure 11] This shows the results of determining the affinity constant between 13E5H2L2 and human IL-4RA. The antibody concentrations added for each pair of curves from top to bottom in the figure are 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, 1.56 nM, 0.78 nM, and 0.39 nM, respectively. [Figure 12] This shows the results of determining the affinity constant between 13E5H3L3 and human IL-4RA. The antibody concentrations added for each pair of curves from top to bottom in the figure are 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, 1.56 nM, and 0.39 nM, respectively. [Figure 13] This shows the results of determining the affinity constant between 13E5H4L2 and human IL-4RA. The antibody concentrations added for each pair of curves from top to bottom in the figure are 12.5 nM, 6.25 nM, 3.13 nM, 1.56 nM, 0.78 nM, and 0.39 nM, respectively. [Figure 14] The results of determining the affinity constant between dupilumab and human IL-4RA. The antibody concentrations added for each pair of curves from top to bottom in the figure are 6.25 nM, 3.13 nM, 1.56 nM, 0.78 nM, and 0.39 nM, respectively. [Figure 15] 13E5H1L1, 13E5H2L2 and Dupilumab inhibit IL-4-induced TF-1 cell proliferation. [Figure 16] 13E5H1L1, 13E5H2L2 and Dupilumab inhibit IL-13-induced TF-1 cell proliferation. [Figure 17] 13E5H4L2, 13E5H4L4 and Dupilumab inhibit IL-4-induced TF-1 cell proliferation. [Figure 18] 13E5H4L2, 13E5H4L4 and Dupilumab inhibit IL-13-induced TF-1 cell proliferation. [Figure 19] 13E5H4L4 and Dupilumab inhibit the IL-4-induced increase in monocyte CD23 expression levels. [Figure 20] 13E5H4L4 and Dupilumab inhibit the IL-13-induced increase in monocyte CD23 expression levels. [Figure 21] 13E5H4L4 inhibits eosinophil chemotaxis to IL-4 after GM-CSF treatment. [Figure 22] 13E5H4L4 inhibits IL-4-induced CCL26 secretion in HUVEC cells. [Figure 23] 13E5H4L4 inhibits IL-4-induced CCL26 secretion in A549 cells. [Figure 24] 13E5H4L4 and Dupilumab inhibit eosinophil chemotaxis into HUVEC cell supernatants after IL-4 treatment. DETAILED DESCRIPTION OF THE INVENTION

[0083] The following provides a detailed description of the present invention, with reference to examples. Those skilled in the art will appreciate that the following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention. Unless specific techniques or conditions are specified in the examples, they are carried out in accordance with the techniques or conditions described in technical literature (see, for example, "Molecular Cloning Experimental Guidelines," 3rd Edition, by J. Sambrook et al., translated by Huang Pedang et al., Science Press) or the product instructions. Reagents or equipment used without a manufacturer's designation are commercially available, commonly available products.

[0084] The BALB / C mice used in the following examples of the present invention were purchased from Guangdong Provincial Medical Experimental Animal Center.

[0085] The positive control antibody Dupilumab VAB 16F3-1 (hereinafter referred to as Dupilumab) used in the following Examples 1 to 8 of the present invention was manufactured by Zhongshan Kangfang Biopharmaceutical Co., Ltd., and its sequence refers to the antibody VAB 16F3-1 heavy chain variable region (the sequence number is shown in SEQ ID NO: 70, the constant region is the Ig gamma-1 chain C region, ACCESSION: P01857) and antibody VAB 16F3-1 light chain variable region (the coding sequence is shown in SEQ ID NO: 71, the constant region is the Ig kappa chain C region, ACCESSION: P01834) described in patent application PCT / US2007 / 021210 of Regeneron Pharmaceuticals, Inc.

[0086] The heavy chain sequence of the isotype control antibody hIgG (also written as hIgG4) in the present invention is SEQ ID NO:68, and the light chain sequence is SEQ ID NO:69.

[0087] Example 1 Preparation of anti-human IL-4RA antibody 13E5 1. Preparation of hybridoma cell line 13E5 The antigen used to prepare anti-IL-4RA antibodies, IL-4RA-mFc, was a fusion protein (synthesized by Zhongshan Kangfang Biomedical Co., Ltd.) of human IL-4RA mature peptide (Genbank ID: NP_001244336.1) and an mFc tag (SEQ ID NO: 67). BALB / C mice (purchased from the Guangdong Provincial Medical Laboratory Animal Center) were immunized with this antigen. Hybridoma cells were generated by cell fusion using spleen cells from immunized BALB / C mice (purchased from the Guangdong Provincial Medical Laboratory Animal Center) and mouse myeloma cells, according to existing technical methods (e.g., Stewart, SJ, "Monoclonal Antibody Production," in Basic Methods in Antibody Production and Characterization, eds. G.C. Howard and D.R. Bethell, Boca Raton: CRC Press, 2000). IL4RA-hFc protein (IL-4RA is the same as above; hFc is a human IgG Fc purification tag, specifically, the Ig gamma-1 chain C region, GenBank number: P01857, positions 114-330) was used as an antigen to coat a microplate. Screening was performed by indirect ELISA to obtain hybridoma cells secreting antibodies that specifically bind to IL-4RA-hFc. The hybridoma cells obtained by indirect ELISA screening were screened by competitive ELISA to find hybridoma cell lines secreting monoclonal antibodies that compete with the ligand IL4-N-his (IL4 NCBI Gene ID: AAH70123.1) for binding to IL-4RA-hFc. Two hybridoma cell lines that stably secrete anti-human IL-4RA antibodies were obtained by limiting dilution. These hybridoma cell lines were named hybridoma cell line LT008, and the monoclonal antibodies they secreted were named 13E5.

[0088] The hybridoma cell line LT008 (IL-4RA-13E5) was deposited at the China Center for Typical Culture Collection (CCTCC) on June 21, 2018, with the deposit number CTCCC NO: C2018131, and was deposited at Wuhan University, Wuhan, China, postal code: 430072.

[0089] 2. Preparation of anti-human IL-4RA antibody 13E5 The LT008 cell lines prepared above were cultured in Chemically Defined Medium (CD medium) containing 1% penicillin-streptomycin in a 5% CO2, 37°C cell incubator. After 7 days, the cell culture supernatant was collected and purified using high-speed centrifugation, microfiltration, and a HiTrap protein A HP column to prepare antibody 13E5.

[0090] Example 2: Sequence analysis of anti-human IL-4RA antibody 13E5 From the LT008 cell line cultured in Example 1, mRNA was extracted according to the method of the cultured cell bacterial total RNA extraction kit (Tiangen, product number DP430).

[0091] cDNA was synthesized and PCR amplified according to the instructions of the Invitrogen SuperScript® III First-Strand Synthesis System for RT-PCR kit.

[0092] The PCR amplification product was directly subjected to TA cloning, and the specific procedure was carried out by referring to the instructions of the pEASY-T1 Cloning Kit (Transgen CT101).

[0093] The product of TA cloning was directly sequenced, and the sequencing results were as follows:

[0094] The nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the fragment length is 348 bp. The encoded amino acid sequence is shown in SEQ ID NO: 2, and the length is 116 amino acids. The sequences of the heavy chain CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.

[0095] The nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:3 and is 321 bp in length. The encoded amino acid sequence is shown in SEQ ID NO:4 and is 107 amino acids in length. The sequences of the light chain CDR1, CDR2, and CDR3 are shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively.

[0096] Example 3: Design and preparation of anti-human IL-4RA humanized antibodies 13E5H1L1, 13E5H2L2, 13E5H3L3, 13E5H4L2, and 13E5H4L4 1. Design of the light and heavy chain sequences of anti-IL-4RA humanized antibodies 13E5H1L1, 13E5H2L2, 13E5H3L3, 13E5H4L2, and 13E5H4L4 An antibody model was simulated on a computer based on the three-dimensional crystal structure of the human IL-4RA protein (Hage T, Reinemer P, Sebald W. Crystals of a 1:1 complex between human interleukin-4 and the extracellular domain of its receptor alpha chain. Eur J Biochem. 1998; 258(2):831-6.) and the sequence of antibody 13E5 obtained in Example 2. Mutations were designed according to the model, and the variable region sequences of antibodies 13E5H1L1, 13E5H2L2, 13E5H3L3, 13E5H4L2, and 13E5H4L4 were obtained (antibody constant region sequence, Ig gamma-4 chain C region, ACCESSION: P01861.1, from the NCBI database; light chain constant region, Ig kappa chain C region, ACCESSION: P01834).

[0097] The designed variable region sequences were as follows: (1) Heavy and light chain sequences of humanized monoclonal antibody 13E5H1L1 The nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:11 and is 348 bp in length. The encoded amino acid sequence is shown in SEQ ID NO:12 and is 116 aa in length. The sequences of the heavy chain CDR1, CDR2, and CDR3 are shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively.

[0098] The nucleic acid sequence of the light chain variable region is shown in SEQ ID NO: 13 and is 321 bp in length. The encoded amino acid sequence is shown in SEQ ID NO: 14 and is 107 aa in length. The sequences of the light chain CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively.

[0099] (2) Heavy and light chain sequences of humanized monoclonal antibody 13E5H2L2 The nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15 and is 348 bp in length. The encoded amino acid sequence is shown in SEQ ID NO: 16 and is 116 aa in length. The sequences of the heavy chain CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.

[0100] The nucleic acid sequence of the light chain variable region is shown in SEQ ID NO: 17 and is 321 bp in length. The encoded amino acid sequence is shown in SEQ ID NO: 18 and is 107 aa in length. The sequences of the light chain CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively.

[0101] (3) Heavy and light chain sequences of humanized monoclonal antibody 13E5H3L3 The nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO: 19 and is 348 bp in length. The encoded amino acid sequence is shown in SEQ ID NO: 20 and is 116 aa in length. The sequences of the heavy chain CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.

[0102] The nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:21 and is 321 bp in length. The encoded amino acid sequence is shown in SEQ ID NO:22 and is 107 aa in length. The sequences of the light chain CDR1, CDR2, and CDR3 are shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively.

[0103] (4) Heavy and light chain sequences of humanized monoclonal antibody 13E5H4L4 The nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:23 and is 348 bp in length. The encoded amino acid sequence is shown in SEQ ID NO:24 and is 116 aa in length. The sequences of the heavy chain CDR1, CDR2, and CDR3 are shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively.

[0104] The nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:25 and is 321 bp in length. The encoded amino acid sequence is shown in SEQ ID NO:26 and is 107 aa in length. The sequences of the light chain CDR1, CDR2, and CDR3 are shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively.

[0105] (5) Heavy and light chain sequences of humanized monoclonal antibody 13E5H4L2 The nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:23, Its encoded amino acid sequence is shown in SEQ ID NO:24, The nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:17, The encoded amino acid sequence is shown in SEQ ID NO:18.

[0106] 2. Preparation of humanized antibodies 13E5H1L1, 13E5H2L2, 13E5H3L3, 13E5H4L2, and 13E5H4L4 The heavy chain constant region used in both cases was the Ig gamma-4 chain C region, ACCESSION: P01861.1, and the light chain constant region used in both cases was the Ig kappa chain C region, ACCESSION: P01834.

[0107] The heavy chain variable region cDNA and light chain variable region cDNA of 13E5H1L1, the heavy chain variable region cDNA and light chain variable region cDNA of 13E5H2L2, the heavy chain variable region cDNA and light chain variable region cDNA of 13E5H3L3, the heavy chain variable region cDNA and light chain variable region cDNA of 13E5H4L2, and the heavy chain variable region cDNA and light chain variable region cDNA of 13E5H4L4 were transformed into pUC57 They were cloned into simple (provided by GenScript Biotechnology Co., Ltd.) vectors, respectively, to obtain pUC57simple-13E5H1, pUC57simple-13E5L1, pUC57simple-13E5H2, pUC57simple-13E5L2, pUC57simple-13E5H3, pUC57simple-13E5L3, pUC57simple-13E5H4, and pUC57simple-13E5L4, respectively. Variable region fragments were obtained by enzymatic digestion according to the standard techniques described in the "Guidelines for Experimental Molecular Cloning (2nd Edition)." These fragments were then subcloned into the pcDNA 3.1 vector containing the corresponding heavy chain constant region fragment and the corresponding light chain constant region fragment (the heavy chain constant region fragment was cloned into the vector pcDNA 3.1 by enzymatic digestion with restriction enzymes (HindIII and EcoRI) and the light chain constant region fragment was cloned into the vector pcDNA3.1 by enzymatic digestion with restriction enzymes (HindIII and EcoRI)). pcDNA3.1-13E5H1, pcDNA3.1-13E5L1, pcDNA3.1-13E5H2, pcDNA3.1-13E5L2, pcDNA3.1-13E5H3, pcDNA3.1-13E5L3, pcDNA3.1-13E5H4, and pcDNA3.1-13E5L4 were obtained. Then, the corresponding light chain and heavy chain recombinant plasmids (pcDNA3.1-13E5H1 and pcDNA3.1-13E5L1, pcDNA3.1-13E5H2 and pcDNA3.1-13E5L2, pcDNA3.1-13E5H3 and pcDNA3.1-13E5L3, pcDNA3.1-13E5H4 and pcDNA3.1-13E5L4, and pcDNA3.1-13E5H4 and pcDNA3.1-13E5L2) were transfected into 293F cells, respectively, and the culture medium was collected and purified.After verifying accuracy by sequencing, endotoxin-free expression plasmids were prepared and transiently transfected into HEK293 cells for antibody expression. After 7 days of culture, the cell culture medium was harvested and affinity purified using a Protein A column to obtain humanized antibodies 13E5H1L1, 13E5H2L2, 13E5H3L3, 13E5H4L2, and 13E5H4L4.

[0108] Example 4. Detection of antibody binding activity to antigen by ELISA method 1. Binding activity of mouse antibody 13E5 and humanized antibodies 13E5H1L1, 13E5H2L2, 13E5H3L3, 13E5H4L2, and 13E5H4L4 to the antigen human IL-4RA-hFc or human IL-4RA-mFc 1.1 Indirect ELISA was used to detect the binding activity of antibodies 13E5, 13E5H1L1, 13E5H2L2, 13E5H3L3, 13E5H4L2, and 13E5H4L4 to the antigen human IL-4RA-hFc or human IL-4RA-mFc.

[0109] Experimental steps: Human IL-4RA-hFc or human IL-4RA-mFc was coated onto a microplate and incubated. After closure, the target antibody was added. After incubation and washing, goat anti-mouse IgG (H+L) and HRP (Jackson ImmunoResearch Inc., product number: 109-035-062) or goat anti-human IgG (H+L) and HRP (Jackson ImmunoResearch Inc., product number: 109-035-088) was added. After incubation and washing, a color reaction was performed with TMB (Neogen, product number 308177). After color development, the absorbance at 450 nm was measured using a microplate reader. Data analysis was performed using SoftMax Pro 6.2.1 software.

[0110] The 450 nm readings indicated that the antibodies 13E5, 13E5H1L1, 13E5H2L2, 13E5H3L3, 13E5H4L2, and 13E5H4L4 all effectively bound to the antigens human IL-4RA-hFc or human IL-4RA-mFc, and the binding efficiency was dose-dependent. Graphical analysis of four-parameter fitting curves, with antibody concentration as the abscissa and absorbance as the ordinate, showed that the antibodies 13E5, 13E5H1L1, 13E5H2L2, 13E5H3L3, 13E5H4L2, and 13E5H4L4 all effectively bound to the antigens human IL-4RA-hFc or human IL-4RA-mFc, with binding activity comparable to that of the positive control antibody dupilumab (Figures 1, 2, and 3).

[0111] [Table 1]

[0112] [Table 2]

[0113] [Table 3]

[0114] 1.2 Competitive ELISA to detect the ability of antibodies 13E5, 13E5H1L1, 13E5H2L2, 13E5H3L3, 13E5H4L2, and 13E5H4L4 to block the binding of IL-4-N-his to the antigen IL-4RA-hFc The half-effective concentrations (EC50) of 13E5, 13E5H1L1, 13E5H2L2, 13E5H3L3, 13E5H4L2, and 13E5H4L4 at which they compete with human IL-4RA-hFc, the ligand of the target antigen, for binding to human IL-4-N-His were measured by ELISA, and the ability of 13E5, 13E5H1L1, 13E5H2L2, 13E5H3L3, 13E5H4L2, and 13E5H4L4 to block the binding of the ligand of the target antigen to human IL-4RA-hFc was examined.

[0115] After coating and sealing a microplate with human IL-4RA-hFc, the target antibody was added, followed by an equal volume of human IL-4-N-His (synthesized by Zhongshan Kangfang Biopharmaceutical Co., Ltd.), and the mixture was incubated. After washing the plate, mouse anti-His, HRP (purchased from Kangwei Century Biotechnology Co., Ltd., product number: CW0285A) was added and incubated. After washing the plate, the mixture was developed with TMB (Neogen, product number 308177). The microplate was immediately placed in a microplate reader, and the OD value of each well was read at a wavelength of 450 nm. The data were analyzed using SoftMax Pro 6.2.1 software. Specific detection results are shown in Tables 4, 5, and 6.

[0116] A four-parameter fitting curve was constructed using antibody concentration as the abscissa and absorbance as the ordinate, and the blocking EC50 values corresponding to the detected antibodies were obtained. As can be seen from Figures 4, 5, and 6, 13E5, 13E5H1L1, 13E5H2L2, 13E5H4L2, and 13E5H4L4 could all effectively block the binding of the ligand human IL4-N-His to the antigen human IL-4RA-hFc, and the blocking efficiency showed a dose-dependent relationship. Here, the ability of 13E5H1L1, 13E5H2L2, 13E5H4L2, and 13E5H4L4 to compete with human IL4-N-His for binding to human IL-4RA-hFc was superior to that of the positive control antibody Dupilumab, which targets the same target.

[0117] [Table 4]

[0118] [Table 5]

[0119] [Table 6]

[0120] 2. Binding activity of humanized antibody 13E5H4L4 to the antigen IL-4RA-hFc 2.1 Competitive ELISA to detect the ability of antibody 13E5H4L4 to block the binding of IL-4-N-his to the antigen IL-4RA-hFc The EC50 (half effective concentration) at which 13E5H4L4 competes with human IL-4-N-His, the ligand of the target antigen, for binding to human IL-4RA-hFc was measured by ELISA, and the activity of 13E5H4L4 to block the binding of the ligand of the target antigen to human IL-4RA-hFc was examined.

[0121] After coating and sealing a microplate with human IL-4RA-hFc, the target antibody was added, followed by an equal volume of human IL-4-N-His (synthesized by Zhongshan Kangfang Biopharmaceutical Co., Ltd.), and the mixture was incubated. After washing the plate, mouse anti-His, HRP (purchased from Kangwei Century Biotechnology Co., Ltd., product number: CW0285A) was added and incubated. After washing the plate, the mixture was developed with TMB (Neogen, product number 308177). The microplate was immediately placed in a microplate reader, and the OD value of each well was read at a wavelength of 450 nm. The data was analyzed using SoftMax Pro 6.2.1 software. Specific detection results are shown in Table 7.

[0122] A four-parameter fitting curve was constructed using antibody concentration as the abscissa and absorbance value as the ordinate, and the blocking EC50 values corresponding to the detected antibodies were obtained. As can be seen from Figure 7, 13E5H4L4 effectively blocked the binding of the ligand human IL4-N-His to the antigen human IL-4RA-hFc, and the blocking efficiency showed a dose-dependent relationship. Here, the ability of 13E5H4L4 to compete with human IL4-N-His for binding to human IL-4RA-hFc was superior to that of the positive control antibody Dupilumab, which targets the same target.

[0123] [Table 7]

[0124] Example 5. Measurement of affinity constants of humanized antibodies 13E5H1L1, 13E5H2L2, 13E5H3L3, 13E5H4L2, and 13E5H4L4 to human IL-4RA The affinity constants of the humanized antibodies 13E5H1L1, 13E5H2L2, 13E5H3L3, 13E5H4L2, 13E5H4L4, and Dupilumab for human IL-4RA were measured using a Fortebio molecular interaction meter.

[0125] 5 μg / mL antigen was immobilized on the AMC sensor for 60 s, the sensor was equilibrated in PBST for 300 s, and the antigen immobilized on the sensor was bound to antibody concentrations of 0.39 to 25 nM (2-fold gradient dilution) for 120 s. The antigen-antibody was then dissociated in PBST for 600 s. The sensor was regenerated with 10 mM glycine at pH 1.7. The data were analyzed using a 1:1 model fitting to obtain affinity constants.

[0126] The results of measuring the affinity constants of humanized antibodies 13E5H4L4 and Dupilumab (as a positive control) with human IL-4RA are shown in Table 8, and the detection results are shown in Figures 8 and 9.

[0127] [Table 8] K Dis the affinity constant, K D =kdis / kon.

[0128] As a result, it was shown that both the humanized antibody 13E5H4L4 and human IL-4RA have strong binding ability, which is equivalent to that of the positive control antibody Dupilumab targeting the same target.

[0129] The results of measuring the affinity constants of the humanized antibodies 13E5H1L1, 13E5H2L2, 13E5H3L3, 13E5H4L2 and Dupilumab (as a positive control drug) to human IL-4RA are shown in Table 9, and the detection results are shown in Figures 10 to 14.

[0130] [Table 9] K D is the affinity constant, K D =kdis / kon.

[0131] The results showed that the antigen affinity of the humanized antibody 13E5H1L1 was superior to that of the positive control antibody Dupilumab, and the dissociation rate constants kdis of 13E5H1L1 and 13E5H2L2 with human IL-4RA were smaller than those of the positive control antibody Dupilumab, suggesting that 13E5H2L2 binds more tightly to human IL-4RA.The binding rate constants of 13E5H1L1 and 13E5H4L2 to the antigen human IL-4RA were greater than those of the positive control antibody Dupilumab, suggesting that the binding rate of 13E5H1L1 and 13E5H4L2 to the antigen human IL-4RA was faster than that of Dupilumab.

[0132] Example 6. Analysis of cell biological activity The cell proliferation assay demonstrated that 13E5H1L1, 13E5H2L2, 13E5H4L2, and 13E5H4L4 have the cell biological activity of blocking the stimulation of TF-1 cell proliferation by human IL-4 and human IL-13. Specifically, the following results were obtained: TF-1 cells (purchased from the American Type Culture Collection, product number CRL-2003) were cultured in normal culture medium (RPMI 1640 + 10% FBS + 2.5 g / L glucose + 2 ng / mL GM-CSF). On the day of the experiment, TF-1 cells were harvested by centrifugation, resuspended in medium without GM-CSF, counted, and seeded at 20,000 cells per well into a 96-well plate. Treatment was performed according to the experimental design: final antibody concentrations were 0.05, 0.5, and 5 nM; IL-4 concentrations were set at three levels: 0.041, 0.41, and 4.1 nM. IL-4 was used in the antibody group at 0.41 nM. IL-13 concentrations were set at three levels: 1.58, 15.8, and 79 nM. IL-13 was used at 15.8 nM. After administration, the 96-well plates were incubated for 72 hours in a 37°C, 5% CO2 incubator. After 72 hours, the CCK8 reagent was added according to the instructions in the CCK8 kit (purchased from Nippon Dojin Co., Ltd., product number: CK04). The mixture was mixed thoroughly and incubated for 3-4 hours in a 37°C, 5% CO2 incubator. The OD (450 nm) was then measured. A cell number-OD curve was also plotted. The TF-1 cells were inoculated into a 96-well plate at a gradient dilution rate, added with CCK8 reagent, and incubated at 37°C in a 5% CO2 incubator for 3-4 hours. The OD (450 nm) was then measured. The cell number for each experimental group was calculated based on the OD values and plotted using GraphPad Prism 5.

[0133] The isotype control antibody used in this study was human anti-Hen Egg Lysozyme IgG (anti-HEL, or human IgG, abbreviated as hIgG). Its sequence was derived from the variable region sequence of Fab F10.6.6 in the Fv domain of anti-protein antibodies described by Acierno et al. as "affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies" (Acierno et al. J Mol Biol. 2007; 374(1):130-46). It was designed as an isotype control antibody in this study and prepared in the laboratory of Zhongshan Kangfang Biopharmaceutical Co., Ltd.

[0134] For human IgG, we commissioned Nanjing King's Biological Sciences to perform codon optimization and gene synthesis for antibody heavy and light chain (full sequence or variable region) genes. Following the standard techniques outlined in the "Guidelines for Molecular Cloning (Second Edition)," we subcloned the heavy and light chain genes into mammalian antibody heavy and light chain expression vectors (both pcDNA3.1 vectors) using standard molecular cloning techniques, including enzyme digestion, DNA gel recovery, ligation, colony PCR, and enzyme digestion analysis. Furthermore, we sequenced the heavy and light chain genes in the recombinant expression vectors (heavy chain sequence: SEQ ID NO: 68, light chain sequence: SEQ ID NO: 69). After verifying accuracy by sequencing, we produced large quantities of endotoxin-free expression plasmids and transiently transfected the heavy and light chain expression plasmids into HEK293 cells for recombinant antibody expression. After 7 days of culture, the cell culture medium was harvested and affinity purified using rProtein A column (GE). The harvested antibody samples were then analyzed for quality by SDS-PAGE and SEC-HPLC standard analytical techniques.

[0135] The detection results are shown in Figures 15 to 18. As can be seen from Figures 15 to 18, both human IL-4 and human IL-13 effectively promoted the proliferation of TF-1 cells in a dose-dependent manner, and compared to the isotype control antibody (human IgG), Dupilumab, 13E5H1L1, 13E5H2L2, 13E5H4L2, and 13E5H4L4 all specifically inhibited the proliferation of TF-1 cells induced by IL-4 and IL-13 in a dose-dependent manner.

[0136] The results showed that 13E5H1L1, 13E5H2L2, 13E5H4L2, and 13E5H4L4 could specifically inhibit the proliferation of human IL-4 and human IL-13-induced TF-1 cells, and the activity of 13E5H1L1 and 13E5H4L4 was equivalent to that of the positive control drug Dupilumab.

[0137] Example 7. Inhibition of increased PBMC cell CD23 expression levels by 13E5H4L4 The purpose of this example was to detect the neutralizing biological activity of 13E5H4L4 against the increase in cell surface CD23 expression level of human PBMCs induced by human IL-4 and human IL-13 by flow cytometry. Specifically, the following was performed: Fresh human PBMCs were isolated from normal human peripheral blood (heparin anticoagulated) by Ficoll density gradient centrifugation, washed three times by centrifugation, and then counted to a cell density of 2.5 × 10 6The solution was adjusted to 1 mL / mL and inoculated into a low-adsorption 96-well plate. 200 μL of PBMCs were added per well (i.e., 500,000 per well). 25 μL of the antibodies Dupilumab and 13E5H4L4 (final concentrations of 30, 3, and 0.3 nM, respectively) were added per well, and a blank control and isotype control human IgG (final concentration of 30 nM) were also added. The mixture was then incubated at room temperature for 30 minutes. After 30 min, 25 μL of human IL-4 (final concentration 100 pM) or 25 μL of human IL-13 (final concentration 300 ng / mL) was added and the cells were incubated in an incubator for 2.5 days. After 2.5 days, PBMCs from each group were collected into 1.5 mL EP tubes. 500 μL of 1% PBSA was added to each tube, and the cells were centrifuged at 1000 x g for 5 min. The supernatant was removed. 50 μL of CD23-PE antibody (50:1 diluted in 1% PBSA) was added and the cells were incubated on ice for 40 min. After 40 min, 1 mL of 1% PBSA was added, the cells were centrifuged at 1000 x g for 5 min, and the supernatant was removed. 200 μL of resuspended cells in 1% PBSA were added, and the cells were transferred to a flow cytometer for detection.

[0138] The detection results are shown in Figures 19 and 20.

[0139] As a result, it was shown that human IL-4 and human IL-13 increased the expression level of CD23 on the surface of mononuclear cells in human PBMCs, and that 13E5H4L4 specifically bound to human IL-4RA and effectively inhibited the increase in CD23 expression level caused by IL-4 and IL-13.

[0140] Example 8. Effect of anti-IL-4RA antibody on IL-4-induced human eosinophil chemotaxis After peripheral blood from healthy volunteers turned red, it was centrifuged at 1200 rpm for 5 min. Cells were washed twice with 15 mL HBSS (Hank's Balanced Salt Solution, manufactured by Zhongshan Kangfang Biopharmaceutical Co., Ltd.) and once with complete medium (1640 + 10% FBS). Cells were resuspended in complete medium, counted, and cultured overnight with 50 pM granulocyte-macrophage colony-stimulating factor (GM-CSF) (Peprotech, product number 300-03). The next day, a transwell chamber (Thermo, product number 140629) was placed in a 24-well plate, and 100 μL of complete medium was added to the upper chamber. The plate was then equilibrated at 37°C for 1 h. Cells were harvested and incubated with complete medium, 13E5H4L4 antibody (Zhongshan Kangfang Biopharmaceuticals Co., Ltd., lot number 201811), or isotype control antibody hIgG4 (Zhongshan Kangfang Biopharmaceuticals Co., Ltd., lot number 20190910) at 37°C for 1 h. After 1 h, the complete medium in the upper chamber was discarded, and the cell and complete medium or antibody preincubation solution was added to the upper chamber. 400 μL of complete medium containing 1 nM IL-4 (Peprotech, part number 200-04) was added to the lower chamber. The cells were then placed in a 37°C incubator for 2.5 h of chemotaxis. After 2.5 hours, the upper chamber was removed, and the cells in the lower chamber were collected and centrifuged. FITC-labeled anti-human CD66b antibody (Biolegend, part number 305104) and APC-labeled anti-human CD16 antibody (Biolegend, part number 302012) were added and incubated on ice for 30 minutes, protected from light. After centrifuging, the cells were resuspended in 200 μL of 1% PBSA (PBS + 1% BSA (Sigma, part number V900933-1KG)) and the eosinophil concentration in the lower chamber was measured by flow cytometry. Chemotaxis inhibition rate (%) = (eosinophil concentration in the control group - eosinophil concentration in the experimental group) / eosinophil concentration in the control group * 100%.

[0141] The results are shown in FIG. 21 and demonstrate that, compared to the isotype control, the anti-IL4-RA antibody 13E5H4L4 was able to effectively inhibit the chemotaxis of eosinophils to IL-4 after GM-CSF treatment.

[0142] Example 9. Inhibition of IL-4-stimulated CCL26 secretion by anti-IL-4RA antibody Cytokines (e.g., IL-1β, TNF-α, IL-4, and IL-13) can stimulate endothelial cells to produce large amounts of chemokines. IL-4 and IL-13, which are overexpressed in EoE, stimulate esophageal squamous epithelial cells to produce the chemokine eotaxin-3 (CCL26), which is an effective eosinophil attractant that induces activated eosinophils to chemotaxis into the esophagus, degranulates them, and releases toxic products, resulting in esophageal damage and remodeling. It has also been shown that CCL26 is highly expressed in eosinophilic esophagitis lesions (Rothenberg ME. Biology and Treatment of Eosinophilic Esophagitis [J]. Gastroenterology, 2009, 137(4):1238-1249).

[0143] Human non-small cell lung cancer cell line alveolar basal epithelial cells A549 (purchased from the Chinese Academy of Sciences) were collected and resuspended in DMEM with 10% FBS to give 5 × 10 5 500 μL / well was seeded into a 24-well plate and cultured at 37°C in a 5% carbon dioxide incubator. Human umbilical vein endothelial cells (HUVECs) (purchased from Aucelles) were harvested and resuspended in endothelial cell medium. 5 × 10 5 500 μL of the solution was seeded into a 24-well plate and cultured at 37°C in a 5% CO2 incubator. After 2 hours, the cells adhered to the plate, and then IL-4 (purchased from Peprotech, product number 200-04) and antibodies were added for stimulation for 3 days. The supernatant was collected and the CCL26 content was detected using a kit (R&D, product number DCC260B).

[0144] The results are shown in Figures 22 and 23. As a result, it was shown that the anti-IL-4 RA antibody 13E5H4L4 reduced IL-4-stimulated CCL26 secretion in HUVEC cells and A549 cell lines, and had a relatively good concentration-dependent effect, with ****p<0.001 compared to the IL-4 group.

[0145] Example 10. Inhibition of IL-4-stimulated eosinophil chemotaxis by anti-IL-4RA antibody Harvest HUVEC cells and resuspend them in DMEM or endothelial cell medium with 10% FBS at 5 × 10 5Each well was seeded into a separate 24-well plate and cultured for 2 hours in a 37°C, 5% CO2 incubator. After the cells adhered to the plate, IL-4 and antibodies were added and treated for 3 days. The cell supernatant was collected as conditioned medium. Fresh blood (from a healthy donor) was collected using heparin sodium anticoagulant. Fresh blood was mixed with HBSS at a 1:3 ratio. Ficoll was added to a 50 mL centrifuge tube, and the diluted blood was slowly added to the upper layer at a 3:4 ratio. The tube was centrifuged at 25°C, 420 x g, and 0°C for 30 minutes. The upper layer was aspirated, and the lower red blood cell layer was collected. Add 1X red blood cell lysis buffer at a 1:5 volume ratio, swirl gently, and lyse for 15-20 min. Centrifuge at 1200 x g for 5 min to collect granulocytes. Add 10 mL of HBSS to resuspend the cells, then centrifuge at 1200 x g for 5 min at 25°C and wash twice, once with 1640 medium. Count and viability were determined using AOPI. Eosinophil sorting kit (Miltenyi, product number: 130-092-010) was used to separate eosinophils (EOS). A transwell chamber was placed in a 24-well plate, and 100 μL of 1640 medium was added to the upper chamber. The plate was then equilibrated at 37°C for 1 h. Eosinophils (EOS) were added to the upper chamber at 100 μL / well (200,000 cells), and 400 μL of conditioned medium (i.e., the cell supernatant) was added to each lower chamber. Chemotaxis was then performed for 2.5 hours in an incubator. The upper chamber was removed, and the lower chamber cells were collected and centrifuged at 250 x g for 5 minutes to remove the supernatant. FITC anti-human CD66b antibody (1 μL / 100 μL) (Biolegend, Part Number 305104) and PE anti-human CD16 antibody (1 μL / 100 μL) (Biolegend, Part Number 360704) were added and incubated. After washing, the cells were resuspended in 200 μL of 1% PBSA. 50 μL of cells were collected and analyzed for EOS cell count using a flow cytometer. Inhibition rate %=(IL-4 group-antibody group) / (IL-4 group)×100%, calculated after subtracting the blank control group from each treatment group.

[0146] The results are shown in Figure 24. As a result, in the supernatant of IL-4-treated HUVEC cells, the inhibitory effect of EOS chemotaxis increased with increasing anti-IL-4RA antibody concentration, indicating that anti-IL-4RA antibodies can gradually inhibit IL-4-stimulated eosinophil chemotaxis. Furthermore, in the HUVEC cell line, the chemotaxis inhibitory rate of 13E5H4L4 against EOS was higher than that of the positive control dupilumab (purchased from Regeneron, lot number: 7L615F), indicating that 13E5H4L4 had an even better inhibitory effect against IL-4-stimulated EOS chemotaxis.

[0147] Compared with the IL-4 group, *p<0.05, **p<0.01, ***p<0.001.

[0148] Although the above has specifically described preferred embodiments of the present invention, the creation of the present invention is not limited to the above examples. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are included in the scope defined by the claims of this application.

[0149] Sequence (Note: Underlined regions indicate CDR regions) 13E5 heavy chain variable region: GAGGTGCAGCTGCAGCAGAGCGGACCAGAGCTGGTGAAGCCTGGCGCCTCTGTGAAGATCAGCTGTAAGACCTCCGGCTACACCTTCACAGAGTATACAATCCACTGGGTGAAGCAGAACCACGGCAAGAGCCTGGAGTGGATCGGCGGCATCAATCCTAACAATGGCGGCACC GTGTACAACCAGAACTTCAAGGGCAAGGCCACCCTGACAGTGGACAAGAGCTCCTCTACCGCCTATATGGAGCTGAGGTCTCTGACAAGCGAGGACTCCGCCGTGTACTATTGCGCCAGAGTGCGGAGAGGCATGGATTACTGGGGCCAGGGCACCTCCGTGACAGTGAGCTCC (SEQ ID NO:1) EVQLQQSGPELVKPGASVKISCKTS GYTFTEYT IHWVKQNHGKSLEWIGG INPNNGGT VYNQNFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYC ARVRRGMDY WGQGTSVTVSS (SEQ ID NO:2) 13E5 Empty Railroad GACATCGTGATGACCCAGTCCCACAAGTTTATGTCCACATCTGTGGGCGAGGTGTCCATCACCTGTAAGGCCTCTCAGGATGTGACCACAGCCGTGGCCTGGTACCAGCAGAAGCCAGGCCAGTCTCCCAAGCTGCTGATCTATAGCGCCTCCTACCGG TATACAGGCGTGCCCGACAGATTCACCGGCTCTGGCAGCGGCACAGATTTCACCTTTACAATCAGCTCCGTGCAGGCAGAGGACCTGGCCGTGTACTATTGCCAGCAGCACTACTCTGCCCCTTGGACCTTCGGCGGAGGAAAACCTGGAGATCAAG(SEQ ID NO:3) DIVMTQSHKFMSTSVGDRVSITCKAS QDVTTA VAWYQQKPGQSPKLLIY SAS YRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYC QQHYSAPWT FGGGTNLEIK (SEQ ID NO:4) 13E5 CDR HCDR1:GYFTEYTE (SEQ ID NO:5) HCDR2:INPNNGGT (SEQ ID NO:6) HCDR3:ARVRRGMDY (SEQ ID NO:7) LCDR1:QDVTTA (SEQ ID NO:8) LCDR2:SAS (SEQ ID NO:9) LCDR3:QQHYSAPWT (SEQ ID NO:10) 13E5H1 Switchboard CAGGTGCAGCTGCAGCAGTCCGGAGCAGAGGTGGTGAAGCCAGGAGCCAGCGTGAAGATCTCCTGTAAGACCTCTGGCTACACCTTCACAGAGTATACAATCCACTGGGTGAAGCAGGCACACGGACAGAGCCTGGAGTGGATCGGCGGCATCAACCCTAACAATGGCGGCACCGTGTACAATCAGAAGTTTCAGGGCAAGGCCACCCTGACAGTGGACAAGTCTACCAGCACAGCCTATATGGAGCTGAGGTCCCTGACCTCTGAGGACACAGCCGTGTACTATTGCGCCCGGGTGCGGAGAGGCATGGATTACTGGGGCCAGGGCACCTCCGTGACAGTGAGCTCC (SEQ ID NO:11) QVQLQQSGAEVVKPGASVKISCKTS GYTFTEYT IHWVKQAHGQSLEWIGG INPNNGGT VYNQKFQGKATLTVDKSTSTAYMELRSLTSEDTAVYYC ARVRRGMDY WGQGTSVTVSS (SEQ ID NO:12) Variable region of light chain of 13E5L1 GACATCCAGATGACCCAGTCTCCTAAGTCTCTGAGCACATCCGTGGGCGACCGGGTGACCATCACATGTAGAGCCAGCCAGGATGTGACCACAGCAGTGGCATGGTACCAGCAGAAGCCTGGCAAGTCCCCTAAGCTGCTGATCTATTCTGCCAGCTACAGGTATACCGGAGTGCCATCTCGGTTCTCCGGCTCTGGCAGCGGCACAGACTTCACCTTTACAATCAGCTCCGTGCAGCCAGAGGATCTGGCCACCTACTATTGCCAGCAGCACTACAGCGCCCCATGGACCTTTGGCGGAGGAACAAACCTGGAGATCAAG(SEQ ID NO:13) DIQMTQSPKSLSTSVGDRVTITCRAS QDVTTA VAWYQQKPGKSPKLLIY SASYRYTGVPSRFSGSGSGTDFTFTISSVQPEDLATYYC QQHYSAPWT FGGGTNLEIK (SEQ ID NO:14) Variable region of the heavy chain of 13E5H2 CAGGTGCAGCTGGTGCAGTCCGGAGCAGAGGTGGTGAAGCCAGGAGCCAGCGTGAAGGTGTCCTGTAAGACCTCTGGCTACACCTTCACAGAGTATACAATCCACTGGGTGCGGCAGGCACCAGGACAGTCTCTGGAGTGGATCGGCGGCATCAACCCTAACAATGGCGGCACCAACTACAATCAGAAGTTTCAGGGCAAGGTGACCCTGACAGTGGACAAGTCCACCTCTACAGCCTATATGGAGCTGAGCTCCCTGAGGTCTGAGGACACAGCCGTGTACTATTGCGCCCGCGTGCGGAGAGGCATGGATTACTGGGGCCAGGGCACCAGCGTGACAGTGTCTAGC (SEQ ID NO:15) QVQLVQSGAEVVKPGASVKVSCKTS GYTFTEYT IHWVRQAPGQSLEWIGG INPNNGGT NYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYYC ARVRRGMDY WGQGTSVTVSS (SEQ ID NO:16) Variable region of the light chain of 13E5L2 GACATCCAGATGACCCAGAGCCCTAGCTCCCTGAGCGCCTCCGTGGGCGACAGGGTGACCATCACATGTAGAGCCTCCCAGGATGTGACCACAGCAGTGGCATGGTACCAGCAGAAGCCAGGCAAGGCCCCTAAGCTGCTGATCTACTCCGCCTCTAGCAGGTATACCGGAGTGCCATCTCGGTTCTCTGGCAGCGGCTCCGGCACAGACTTTACCCTGACAATCTCCTCTGTGCAGCCAGAGGATCTGGCCACATACTATTGCCAGCAGCACTATTCTGCCCCCTGGACCTTTGGCGGCGGCACAAACCTGGAGATCAAG(SEQ ID NO:17) DIQMTQSPSSLSASVGDRVTITCRAS QDVTTA VAWYQQKPGKAPKLLIY SAS SRYTGVPSRFSGSGSGTDFTLTISSVQPEDLATYYC QQHYSAPWT FGGGTNLEIK(SEQ ID NO:18) Variable region of the heavy chain of 13E5H3 CAGGTGCAGCTGGTGCAGTCCGGAGCAGAGGTGGTGAAGCCAGGAGCCAGCGTGAAGGTGTCCTGTAAGGCCTCTGGCTACACCTTCACAGAGTATACCATCCACTGGGTGCGGCAGGCACCAGGACAGGGACTGGAGTGGATCGGCGGCATCAACCCTAACAATGGCGGCACAAATTACGCCCAGAAGTTTCAGGGCAGGGTGACCATCACAGTGGACAAGTCCACCTCTACAGCCTATATGGAGCTGAGCTCCCTGAGGTCTGAGGACACCGCCGTGTACTATTGCGCCCGCGTGCGGAGAGGCATGGATTACTGGGGCCAGGGCACCAGCGTGACAGTGTCTAGC (SEQ ID NO:19) QVQLVQSGAEVVKPGASVKVSCKAS GYTFTEYT IHWVRQAPGQGLEWIGG INPNNGGTNYAQKFQGRVTITVDKSTSTAYMELSSLRSEDTAVYYC ARVRRGMDY WGQGTSVTVSS (SEQ ID NO:20) Variable region of light chain of 13E5L3 GACATCCAGATGACCCAGAGCCCTAGCTCCCTGAGCGCCTCCGTGGGCGACAGGGTGACCATCACATGTAGAGCCTCCCAGGATGTGACCACAGCCCTGGCATGGTACCAGCAGAAGCCAGGCAAGGCCCCTAAGCTGCTGATCTACTCCGCCTCTAGCCTGTATACCGGAGTGCCATCTCGGTTCTCTGGCAGCGGCTCCGGCACAGACTTTACCCTGACAATCTCCTCTCTGCAGCCAGAGGATTTCGCCACATACTATTGCCAGCAGCACTATTCTGCCCCCTGGACCTTTGGCGGCGGCACAAACCTGGAGATCAAG (SEQ ID NO:21) DIQMTQSPSSLSASVGDRVTITCRAS QDVTTA LAWYQQKPGKAPKLLIY SAS SLYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQHYSAPWT FGGGTNLEIK (SEQ ID NO:22) Variable region of heavy chain of 13E5H4 CAGGTGCAGCTGGTGCAGTCCGGAGCAGAGGTGGTGAAGCCAGGAGCCAGCGTGAAGGTGTCCTGTAAGACCTCTGGCTACACCTTCACAGAGTATACAATCCACTGGGTGCGGCAGGCACCAGGACAGTCTCTGGAGTGGATCGGCGGCATCAACCCTAACAATGGCGGCACCgtcTACAATCAGAAGTTTCAGGGCAAGGTGACCCTGACAGTGGACAAGTCCACCTCTACAGCCTATATGGAGCTGAGCTCCCTGAGGTCTGAGGACACAGCCGTGTACTATTGCGCCCGCGTGCGGAGAGGCATGGATTACTGGGGCCAGGGCACCAGCGTGACAGTGTCTAGC (SEQ ID NO:23) QVQLVQSGAEVVKPGASVKVSCKTS GYTFTEYT IHWVRQAPGQSLEWIGG INPNNGGT VYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYYC ARVRRGMDY WGQGTSVTVSS (SEQ ID NO:24) 13E5L4: Light chain variable region GACATCCAGATGACCCAGAGCCCTAGCTCCCTGAGCGCCTCCGTGGGCGACAGGGTGACCATCACATGTAGAGCCTCCCAGGATGTGACCACAGCAGTGGCATGGTACCAGCAGAAGCCAGGCAAGGCCCCTAAGCTGCTGATCTACTCCGCCTCTTAcAGGTATACCGGAGTGCCATCTCGGTTCTCTGGCAGCGGCTCCGGCACAGACTTTACCCTGACAATCTCCTCTGTGCAGCCAGAGGATCTGGCCACATACTATTGCCAGCAGCACTATTCTGCCCCCTGGACCTTTGGCGGCGGCACAAACCTGGAGATCAAG (SEQ ID NO:25) DIQMTQSPSSLSASVGDRVTITCRAS QDVTTA VAWYQQKPGKAPKLLIY SASYRYTGVPSRFSGSGSGTDFTLTISSVQPEDLATYYC QQHYSAPWT FGGGTNLEIK (SEQ ID NO:26) 13E5 Railroad Railway FR-H1:EVQLQQSGPELVKPGASVKISCKTS(SEQ ID NO:27) FR-H2:IHWVKQNHGKSLEWIGG(SEQ ID NO:28) FR-H3:VYNQNFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYC(SEQ ID NO:29) FR-H4:WGQGTSVTVSS(SEQ ID NO:30) : : : : : : : : : : : : : : : : : : : : : : 13E5 snowflake snowflakes FR-L1:DIVMTQSHKFMSTSVGDRVSITCKAS(SEQ ID NO:31) FR-L2:VAWYQQKPGQSPKLLIY(SEQ ID NO:32) FR-L3:YRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYC(SEQ ID NO:33) FR-L4:FGGGTNLEIK(SEQ ID NO:34) 13E5H1 Liquid Range: FR-H1:QVQLQQSGAEVVKPGASVKISCKTS(SEQ ID NO:35) FR-H2:IHWVKQAHGQSLEWIGG(SEQ ID NO:36) FR-H3:VYNQKFQGKATLTVDKSTSTAYMELRSLTSEDTAVYYC(SEQ ID NO:37) FR-H4:WGQGTSVTVSS (SEQ ID NO:38) 13E5L1 Leader Line: FR-L1:DIQMTQSPKSLSTSVGDRVTITCRAS(SEQ ID NO:39) FR-L2:VAWYQQKPGKSPKLLIY(SEQ ID NO:40) FR-L3: YRYTGVPSRFSGSGSGTDFTFTISSVQPEDLATYYC (SEQ ID NO:41) FR-L4: FGGGTNLEIK (SEQ ID NO:42) 13E5H2 framework region: FR-H1: QVQLVQSGAEVVKPGASVKVSCKTS (SEQ ID NO:43) FR-H2: IHWVRQAPGQSLEWIGG (SEQ ID NO:44) FR-H3: NYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYYC (SEQ ID NO:45) FR-H4: WGQGTSVTVSS (SEQ ID NO:46)<000078l>13E5L2 framework region: FR-L1: DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO:47) FR-L2: VAWYQQKPGKAPKLLIY (SEQ ID NO:48) FR-L3: SRYTGVPSRFSGSGSGTDFTLTISSVQPEDLATYYC (SEQ ID NO:49) FR-L4: FGGGTNLEIK (SEQ ID NO:50) 13E5H3 framework region: FR-H1: QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO:51) FR-H2: IHWVRQAPGQGLEWIGG (SEQ ID NO:52) FR-H3: NYAQKFQGRVTITVDKSTSTAYMELSSLRSEDTAVYYC (SEQ ID NO:53) FR-H4: WGQGTSVTVSS (SEQ ID NO:54) 13E5L3 framework region: FR-L1: DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO:55) FR-L2: LAWYQQKPGKAPKLLIY (SEQ ID NO:56) FR-L3:SLYTGVPSRFSGSGSGTFTLTISSLQPEDFATYYC(SEQ ID NO:57) FR-L4:FGGGTNLEIK (SEQ ID NO:58) 13E5H4: Framework Area: FR-H1:QVQLVQSGAEVVKPGASVKVSCKTS(SEQ ID NO:59) FR-H2:IHWVRQAPGQSLEWIGG(SEQ ID NO:60) FR-H3:VYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYYC(SEQ ID NO:61) FR-H4:WGQGTSVTVSS (SEQ ID NO:62) 13E5L4 Framework Area: FR-L1:DIQMTQSPSSLSASVGDRVTITCRAS(SEQ ID NO:63) FR-L2:VAWYQQKPGKAPKLLIY(SEQ ID NO:64) FR-L3:YRYTGVPSRFSGSGSGTFTLTISSVQPEDLATYYC(SEQ ID NO:65) FR-L4:FGGGTNLEIK (SEQ ID NO:66) mFc tag sequence: PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIE RTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:67) hIgG heavy chain sequence EVQLEQSGAELMKPGASVKISCKATGYTFTTYWIEWIKQRPGHSLEWIGEILPGDSDSTYYNEKVKGKVTFTADASSNTAYMQLSLSLTSEDSAVYCARGDGFYVYWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:68) sequence of hIgG DIELTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKYTSQSMSGIPSRFSGSGSGTDFTLSINSVETEDFGVYFCQQSGSWPRTFGGGTKLDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:69) VAB 16F3-1 VH-2 EVQLVESGGGLVQPGRSLRLSCEAS GFTFDDYA MHWVRQAPGKGLEWVSG LSRTSVSI GYADSVKGRFTISRDNAKNSLYLEMNSLRPEDTALYC AKWGTRGYFDY WGQGTLVTVSS(SEQ ID NO:70) 16F3-1 VL DIQMTQSPSSVSASVGDRVTITCRAS QDISIW LAWYQQSPGKAPKLLIN VAS RLQSGVPSRFSGSGSGTDFTLTINSLQPEDFVTYYC QQANSFPIT FGQGTRLATK(SEQ ID NO:71)

Claims

1. An antibody or an antigen-binding fragment thereof, in particular, the antibody or an antigen-binding fragment thereof binds to IL-4RA, preferably human IL-4RA, is used for treating eosinophilic esophagitis, and based on the Kabat, IMGT, Chothia or AbM numbering system, the antibody comprises HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 2, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO: 4, preferably, based on the IMGT numbering system, the antibody comprises HCDR1 comprising the sequence shown in SEQ ID NO: 5 or a variant thereof, or consisting of the same, HCDR2 comprising the sequence shown in SEQ ID NO: 6 or a variant thereof, or consisting of the same, HCDR3 comprising the sequence shown in SEQ ID NO: 7 or a variant thereof, or consisting of the same, and the antibody comprises LCDR1 comprising the sequence shown in SEQ ID NO: 8 or a variant thereof, or consisting of the same, LCDR2 comprising the sequence shown in SEQ ID NO: 9 or a variant thereof, or consisting of the same, LCDR3 comprising the sequence shown in SEQ ID NO: 10 or a variant thereof, or consisting of the same, wherein the sequence of the variant has at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the corresponding sequence, an antibody or an antigen-binding fragment thereof.

2. The antibody according to claim 1, wherein the antibody comprises the following: (1) (i) a heavy chain variable region comprising the following sequence or consisting of the following sequence: the amino acid sequence shown in SEQ ID NO: 2 or a variant thereof, and (ii) a light chain variable region comprising the following sequence or consisting of the following sequence: the amino acid sequence shown in SEQ ID NO: 4 or a variant thereof, (2) (i) a heavy chain variable region comprising the following sequence or consisting of the following sequence: the amino acid sequence shown in SEQ ID NO: 12 or a variant thereof, and (ii) a light chain variable region comprising the following sequence or consisting of the following sequence: the amino acid sequence shown in SEQ ID NO: 14 or a variant thereof, (3) (i) a heavy chain variable region comprising the following sequence or consisting of the following sequence: the amino acid sequence shown in SEQ ID NO: 16 or a variant thereof, and (ii) A light chain variable region comprising the following sequence or consisting of the following sequence: The amino acid sequence shown in SEQ ID NO: 18 or a variant thereof, (4) (i) A heavy chain variable region comprising the following sequence or consisting of the following sequence: The amino acid sequence shown in SEQ ID NO: 20 or a variant thereof, and (ii) A light chain variable region comprising the following sequence or consisting of the following sequence: The amino acid sequence shown in SEQ ID NO: 22 or a variant thereof, (5) (i) A heavy chain variable region comprising the following sequence or consisting of the following sequence: The amino acid sequence shown in SEQ ID NO: 24 or a variant thereof, and (ii) A light chain variable region comprising the following sequence or consisting of the following sequence: The amino acid sequence shown in SEQ ID NO: 26 or a variant thereof, (6) (i) A heavy chain variable region comprising the following sequence or consisting of the following sequence: The amino acid sequence shown in SEQ ID NO: 24 or a variant thereof, and (ii) A light chain variable region comprising the following sequence or consisting of the following sequence: The amino acid sequence shown in SEQ ID NO: 18 or a variant thereof, or The sequence of the variant is a sequence having at least 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the corresponding sequence, an antibody.

3. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 2, wherein the antibody further comprises framework regions FR-H1, FR-H2, FR-H3 and FR-H4 of the heavy chain variable region, and framework regions FR-L1, FR-L2, FR-L3 and FR-L4 of the light chain variable region, (1) FR-H1 comprises or consists of the amino acid sequence of SEQ ID NO: 27 or a variant thereof, FR-H2 comprises or consists of the amino acid sequence of SEQ ID NO: 28 or a variant thereof, FR-H3 comprises or consists of the amino acid sequence of SEQ ID NO: 29 or a variant thereof, FR-H4 comprises or consists of the amino acid sequence of SEQ ID NO: 30 or a variant thereof, FR-L1 comprises or consists of the amino acid sequence of SEQ ID NO: 31 or a variant thereof, FR-L2 comprises or consists of the amino acid sequence of SEQ ID NO: 32 or a variant thereof, FR-L3 comprises or consists of the amino acid sequence of SEQ ID NO: 33 or a variant thereof, FR-L4 comprises or consists of the amino acid sequence of SEQ ID NO: 34 or a variant thereof, (2) FR-H1 comprises or consists of the amino acid sequence of SEQ ID NO: 35 or a variant thereof, FR-H2 comprises or consists of the amino acid sequence of SEQ ID NO: 36 or a variant thereof, FR-H3 comprises or consists of the amino acid sequence of SEQ ID NO: 37 or a variant thereof, FR-H4 comprises or consists of the amino acid sequence of SEQ ID NO: 38 or a variant thereof, FR-L1 comprises or consists of the amino acid sequence of SEQ ID NO: 39 or a variant thereof, FR-L2 comprises or consists of the amino acid sequence of SEQ ID NO: 40 or a variant thereof, FR-L3 comprises or consists of the amino acid sequence of SEQ ID NO: 41 or a variant thereof, FR-L4 comprises or consists of the amino acid sequence of SEQ ID NO: 42 or a variant thereof, (3) FR-H1 comprises or consists of the amino acid sequence of SEQ ID NO: 43 or a variant thereof, FR-H2 comprises or consists of the amino acid sequence of SEQ ID NO: 44 or a variant thereof, FR-H3 comprises or consists of the amino acid sequence of SEQ ID NO: 45 or a variant thereof, FR-H4 comprises or consists of the amino acid sequence of SEQ ID NO: 46 or a variant thereof, FR-L1 comprises or consists of the amino acid sequence of SEQ ID NO: 47 or a variant thereof, FR-L2 comprises or consists of the amino acid sequence of SEQ ID NO: 48 or a variant thereof, FR-L3 comprises or consists of the amino acid sequence of SEQ ID NO: 49 or a variant thereof, FR-L4 comprises or consists of the amino acid sequence of SEQ ID NO: 50 or a variant thereof, (4) FR-H1 comprises or consists of the amino acid sequence of SEQ ID NO: 51 or a variant thereof, FR-H2 comprises or consists of the amino acid sequence of SEQ ID NO: 52 or a variant thereof, FR-H3 comprises or consists of the amino acid sequence of SEQ ID NO: 53 or a variant thereof, FR-H4 comprises, or consists of, the amino acid sequence of SEQ ID NO: 54 or a variant thereof, FR-L1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 55 or a variant thereof, FR-L2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 56 or a variant thereof, FR-L3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 57 or a variant thereof, FR-L4 comprises, or consists of, the amino acid sequence of SEQ ID NO: 58 or a variant thereof, (5) FR-H1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 59 or a variant thereof, FR-H2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 60 or a variant thereof, FR-H3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 61 or a variant thereof, FR-H4 comprises, or consists of, the amino acid sequence of SEQ ID NO: 62 or a variant thereof, FR-L1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 63 or a variant thereof, FR-L2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 64 or a variant thereof, FR-L3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 65 or a variant thereof, FR-L4 comprises, or consists of, the amino acid sequence of SEQ ID NO: 66 or a variant thereof, The sequence of the variant has at least 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the corresponding sequence, an antibody or an antigen-binding fragment thereof.

4. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 2, wherein the antibody is a humanized antibody, a chimeric antibody or a multispecific antibody (e.g., a bispecific antibody), an antibody or an antigen-binding fragment thereof.

5. An antibody or an antigen-binding fragment thereof according to claim 4, wherein the constant region of the antibody is humanized, preferably derived from human IgG, more preferably derived from IgG4, an antibody or an antigen-binding fragment thereof.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein the heavy-chain constant region of the antibody employs an Ig gamma-4 chain C region, more preferably the Ig gamma-4 chain C region of GenBank accession number ACCESSION: P01861.1, and the light-chain constant region employs an Ig kappa chain C region, more preferably the Ig kappa chain C region of GenBank accession number ACCESSION: P01834.

7. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 2, wherein the antigen-binding fragment is selected from Fab, Fab', F(ab') ２ , Fd, Fv, dAb, Fab / c, complementarity-determining region (CDR) fragment, single-chain antibody (e.g., scFv), bivalent antibody or domain antibody.

8. A biological material, wherein the biological material is selected from an antibody conjugate, a multispecific antibody (preferably a bispecific antibody), a fusion protein, or a pharmaceutical composition, is used for treating eosinophilic esophagitis, and the biological material contains the antibody or antigen-binding fragment thereof according to claim 1, Preferably, the antibody conjugate further contains a conjugate moiety conjugated to the antibody or antigen-binding fragment thereof, and the conjugate moiety is selected from a purification tag (e.g., His tag), a cytotoxic agent, a detectable label, a radioisotope, a luminescent substance, a colored substance, an enzyme, or polyethylene glycol. Preferably, the multispecific antibody further contains an antibody or antigen-binding fragment against another antigen and / or another antigen epitope. Preferably, the pharmaceutical composition further contains a pharmaceutically acceptable carrier and / or excipient. Preferably, the pharmaceutical composition is used alone or in combination with one or more drugs. Preferably, the pharmaceutical composition has a dosage form suitable for oral administration to the gastrointestinal tract (GI), and preferably, the dosage form is selected from tablets, capsules, pills, powders, granules, emulsions, microemulsions, solutions, suspensions, syrups, and elixirs, or the drug has a dosage form suitable for subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, or intralesional injection.

9. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 and the biological material according to claim 8 in the preparation of a drug or kit for treating eosinophilic esophagitis. Preferably, the kit further comprises one or more additional therapeutic agents selected from the following: IL-4 / IL-13 pathway inhibitors, IL-1β inhibitors, IL-5 inhibitors, IL-8 inhibitors, IL-9 inhibitors, IL-13 inhibitors, IL-17 inhibitors, IL-25 inhibitors, JAK inhibitors, STAT6 inhibitors, TNF inhibitors, eosinophil chemokine-3 (CCL26) inhibitors, IgE inhibitors, prostaglandin D2 inhibitors, immunosuppressants, corticosteroids, glucocorticoids, long-acting β2-agonists (e.g., salmeterol or formoterol), proton pump inhibitors, decongestants, antihistamines and non-steroidal anti-inflammatory drugs (NSAIDs), NSAIDs (non-steroidal anti-inflammatory drugs), antibiotics, antibacterial agents, antiviral agents, antifungal agents, drugs for treating tumors (preferably chemotherapeutic agents or growth inhibitors, targeted therapeutic agents, antibody-drug conjugates, T cells expressing chimeric antigen receptors, antibodies or antigen-binding fragments thereof, angiogenesis inhibitors, antitumor agents, cancer vaccines, adjuvants and combinations thereof, alkylating agents, antimetabolites, antibiotics, plant-based drugs and / or hormonal drugs), The IL-4 / IL-13 pathway inhibitor is not the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, Preferably, the IL-4 / IL-13 pathway inhibitor is selected from anti-IL-4 antibodies, anti-IL-13 antibodies, anti-IL-4 / IL-13 antibodies, IL-4 receptor (IL-4R) inhibitors (e.g., Dupilumab antibody or its biological equivalent, AMG317 or MEDI9314), JAK inhibitors, STAT6 inhibitors for use.

10. A method for treating eosinophilic esophagitis, comprising administering to a subject in need of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 and the biological material according to claim 8, preferably, the subject is selected from mammals (rodents, felines, canines, primates, etc.), preferably, the subject is a human, more preferably, an infant from 0 to 1 year old, a child from 1 to 5 years old, a juvenile from 6 to 12 years old, an adolescent from 12 to 18 years old and an adult over 18 years old, a method for treating eosinophilic esophagitis.

11. The method according to claim 10, wherein The method further comprises administering to the subject a further therapeutic agent or combination therapy selected from one or more of the following: an IL-4 / IL-13 pathway inhibitor, an IL-1β inhibitor, an IL-5 inhibitor, an IL-8 inhibitor, an IL-9 inhibitor, an IL-13 inhibitor, an IL-17 inhibitor, an IL-25 inhibitor, a JAK inhibitor, a STAT6 inhibitor, a TNF inhibitor, an eosinophil chemokine-3 (CCL26) inhibitor, an IgE inhibitor, a prostaglandin D2 inhibitor, an immunosuppressant, a corticosteroid, a glucocorticoid, a long-acting β2-agonist (such as salmeterol or formoterol), a proton pump inhibitor, a decongestant, an antihistamine and a non-steroidal anti-inflammatory agent (NSAID), an NSAID (non-steroidal anti-inflammatory drug), an antibiotic, an antibacterial agent, an antiviral agent, an antifungal agent, a drug for treating tumors (preferably a chemotherapeutic agent or a growth inhibitor, a targeted therapeutic agent, an antibody-drug conjugate, a T cell expressing a chimeric antigen receptor, an antibody or an antigen-binding fragment thereof, an angiogenesis inhibitor, an antitumor agent, a cancer vaccine, an adjuvant and combinations thereof, an alkylating agent, an antimetabolite, an antibiotic, a plant-based drug and / or a hormonal drug), or a combination thereof, The combination therapy includes diet therapy and esophageal dilation, The IL-4 / IL-13 pathway inhibitor is not the antibody or its antigen-binding fragment according to any one of claims 1 to 2, Preferably, the IL-4 / IL-13 pathway inhibitor is selected from an anti-IL-4 antibody, an anti-IL-13 antibody, an anti-IL-4 / IL-13 antibody, an IL-4 receptor (IL-4R) inhibitor (such as a Dupilumab antibody or its biological equivalent, AMG317 or MEDI9314), a JAK inhibitor, and a STAT6 inhibitor, More preferably, the further therapeutic agent is administered simultaneously or sequentially with the antibody or its antigen-binding fragment according to any one of claims 1 to 2.

12. A hybridoma cell line with deposit number CCTCC NO: C2018131 or a monoclonal antibody secreted by the hybridoma cell line for treating eosinophilic esophagitis.

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