Antibody binding to specific epitopes in human IL-4Rα and application of antibodies

Abstract

A human IL-4R antibody or antigen-binding fragment thereof is disclosed. The antibody or antigen-binding fragment thereof specifically binds to human IL-4Rα and binds to a specific epitope on human IL-4Rα. The present invention also provides the use of the epitope in screening for human IL-4R binding or blocking agents for the human IL-4 / IL-13 signaling pathway, or in assessing the efficacy of such binding or blocking agents.

Description

Technical Field

[0001] [Cross - Reference to Related Applications] This application claims the benefit of priority of Chinese Patent Application No. 202010331685.3, filed on April 24, 2020, the entire disclosure of which is incorporated herein by reference.

[0002] The present disclosure relates to the field of biopharmaceuticals, and particularly to antibodies capable of binding to specific epitopes of human IL - 4Rα and their applications.

Background Art

[0003] Human interleukin 4 receptor (IL - 4R) is a type I transmembrane protein that is widely expressed in various tissues and organs throughout the body, particularly in immune organs such as tonsils, appendix, lymph nodes, spleen, and bone marrow. When IL - 4R binds to ligands such as interleukin 4 (IL - 4) and interleukin 13 (IL - 13), it can exert various immunomodulatory effects, such as promoting the differentiation of Th2 cells, regulating the secretion of IgE antibodies by B cells, and stimulating the alternative activation of macrophages.

[0004] IL - 4R is a heterodimer composed of two polypeptide chains. One of its α - chains (IL - 4Rα) has a high affinity for IL - 4. Also, since IL - 4Rα forms another form of IL - 4R heterodimer with the α - chain of the cell - surface receptor of IL - 13 (IL - 13Rα), it also has a high affinity for IL - 13.

[0005] A soluble protein (sIL-4Rα) can be produced from IL-4Rα. This soluble protein can inhibit various inflammation-related signaling pathways, such as IL-4-mediated cell proliferation and T cell-mediated IL-5 upregulation. Therefore, blocking antibodies targeting this protein are beneficial for the treatment and alleviation of allergic rhinitis, sinusitis, asthma, eczema, and other diseases. Furthermore, both IL-4 and IL-13 are cytokines with very broad biological activity, most abundantly produced by activated T cells, monocytes, mast cells, basophils, and eosinophils, and involved in various inflammatory responses. These two interleukins share many common biological functions, including stimulating the differentiation and proliferation of TH2 cells and stimulating IgE antibody secretion by activated B cells. Research has revealed that IL-4 and IL-13 play crucial roles in mediating immune responses in autoimmune diseases, allergic diseases, tumors, and other diseases, making them a constant source of research interest.

[0006] Because sIL-4Rα controls the binding to IL-4 and is associated with other cytokines, there is currently a lot of research being done on antibodies targeting sIL-4Rα. Human monoclonal antibodies against this target have been clinically demonstrated to be effective in alleviating and treating diseases such as asthma and atopic dermatitis. However, the epitopes of sIL-4Rα in immune responses have been hardly studied, and specific epitopes are still not clearly understood. [Overview of the project] [Problems that the invention aims to solve]

[0007] In response to the above technical challenges, the inventors investigated the epitopes of sIL-4Rα. Based on the species specificity exhibited by the newly obtained antibodies when they reacted with human sIL-4Rα and cynomolgus monkey sIL-4Rα, the inventors selected several positions on sIL-4Rα and performed related experiments by mutating these positions, thereby identifying several important amino acids of the binding epitope. These results regarding the epitope are crucial for further research on sIL-4Rα and related antibodies.

[0008] Therefore, the object of this disclosure is to provide an antibody or antigen-binding fragment thereof that binds to the human interleukin-4 receptor (IL-4R), the antibody or antigen-binding fragment thereof specifically binds to a specific epitope of the alpha chain (IL-4Rα) of human IL-4R.

[0009] Based on the antibodies or antigen-binding fragments provided by this disclosure, another object of this disclosure is to provide nucleic acid molecules comprising nucleotide sequences encoding key domains (which may be more than one) in the antibodies or antigen-binding fragments provided by this disclosure; to provide vectors comprising nucleic acid molecules; to provide host cells comprising such vectors; to provide methods for producing antibodies or antigen-binding fragments; to provide pharmaceutical compositions comprising antibodies or antigen-binding fragments; and to provide pharmaceutical uses of antibodies or antigen-binding fragments or pharmaceutical compositions.

[0010] Based on the specific epitope of the alpha chain of human IL-4R provided by this disclosure, a further object of this disclosure is to provide the use of said epitope in the preparation of human IL-4R binders or human IL-4 / IL-13 signaling pathway blockers, or in the evaluation of the efficacy of such binders or blockers.

[0011] This disclosure provides the following technical solutions.

[0012] In one embodiment, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to the human interleukin-4 receptor (IL-4R), and which binds to an epitope located on the alpha chain of human IL-4R (IL-4Rα) and comprising one or more amino acid residues D92, V94, D97, L67, L68, A96, H156, C207, Q63, and L64 in the amino acid sequence shown in SEQ ID NO: 1.

[0013] Preferably, the epitope comprises one or more amino acid residues D92, V94, and D97 in the amino acid sequence shown in SEQ ID NO: 1.

[0014] According to a particular embodiment of this disclosure, the epitope is the following amino acid residue in the amino acid sequence shown in SEQ ID NO: 1: (1) D92, (2) D97, (3) D92, V94 and D97, (4) D92 and V94, (5) D92 and D97, or (6) V94 and D97, Includes.

[0015] and / or, the antibody or antigen-binding fragment thereof according to this disclosure binds to a human IL-4Rα epitope containing the following amino acid residues in the amino acid sequence shown in SEQ ID NO: 1: (i)Q63 and L64.

[0016] Furthermore, the antibodies or antigen-binding fragments according to this disclosure do not have interspecies binding activity to cynomolgus monkey IL-4R. Preferably, the antibodies or antigen-binding fragments do not bind to cynomolgus monkey IL-4R, more preferably not to cynomolgus monkey IL-4Rα, and even more preferably not to the amino acid sequence shown in SEQ ID NO: 2.

[0017] Regarding the above-mentioned cross-species binding activity, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to the human interleukin-4 receptor (IL-4R), and the antibody or the antigen-binding fragment thereof is located on the alpha chain (IL-4Rα) of human IL-4R and binds to an epitope containing one or more amino acid residues L67, L68, and A96 in the amino acid sequence shown in SEQ ID NO: 1.

[0018] Preferably, the epitope is the following amino acid residues in the amino acid sequence shown in SEQ ID NO: 1: 1. L67 and L68, 2. L67, L68, and A96, or, 3. A96, and includes.

[0019] According to a specific embodiment of the present disclosure, the epitope is the following amino acid residues in the amino acid sequence shown in SEQ ID NO: 1: (A) L67, L68, A96, H156, and C207, (B) A96, H156, and C207, (C) L67, L68, H156, and C207, (D) L67, L68, A96, and C207, or, (E) L67, L68, A96, and H156, and includes.

[0020] Particularly preferably, the epitope according to the present disclosure is the following amino acid residues in the amino acid sequence shown in SEQ ID NO: 1: (P-1) D97, L67, and L68, (P-2) D97, L67, L68, A96, and D92, (P-3) D97, L67, L68, and D92, (P-4) D97, Q63, and L​​​​​​​​​​ When the binding of the antibodies provided in this disclosure to the antigen IL-4Rα was detected, it was found that the antibodies in this disclosure have binding activity to sIL-4Rα with EC50 values ​​of 50 ng / ml, 30 ng / ml, 20 ng / ml, or 10 ng / ml or less. Furthermore, the antibody in this disclosure at a concentration of 100 μl × 2500 ng / ml showed no binding activity to sIL-4Rα-97 coated with 100 μl × 0.5 μg / ml, indicating that sIL-4Rα-97 is obtained by a mutation from amino acid residue D to A at position 97 of sIL-4Rα. In addition, it was found that the antibody in this disclosure at a concentration of 100 μl × 2500 ng / ml showed no binding activity or significantly low binding activity to at least one mutant antigen selected from sIL-4Rα-QSMDH and sIL-4Rα-63 / 64 coated with 100 μl × 0.5 μg / ml. The binding activity was measured according to the procedure described in the section "(I) Method for detecting binding activity" under the subtitle "Modes for Carrying Out the Invention" of this disclosure.

[0022] Furthermore, since the antibodies provided in this disclosure can block the binding of IL-4 or IL-13 to IL-4R, they can be used as inhibitors of the binding of IL-4 or IL-13 to IL-4R, or as inhibitors of the IL-4 / IL-13 signaling pathway.

[0023] Therefore, with respect to the amino acid sequence of the included domains, the antibody or its antigen-binding fragment according to this disclosure is a combination of the following heavy chain complementarity-determining regions HCDR1, HCDR2, HCDR3 and light chain complementarity-determining regions LCDR1, LCDR2, LCDR3: (1) HCDR1, HCDR2 and HCDR3 shown in sequence numbers 35, 36 and 37, and LCDR1, LCDR2 and LCDR3 shown in sequence numbers 53, 54 and 55, (2) HCDR1, HCDR2 and HCDR3 shown in Sequence ID No. 41, Sequence ID No. 42 and Sequence ID No. 43, and LCDR1, LCDR2 and LCDR3 shown in Sequence ID No. 59, Sequence ID No. 60 and Sequence ID No. 61, (3) HCDR1, HCDR2 and HCDR3 shown in Sequence ID No. 44, Sequence ID No. 45 and Sequence ID No. 46, and LCDR1, LCDR2 and LCDR3 shown in Sequence ID No. 56, Sequence ID No. 57 and Sequence ID No. 62, (4) HCDR1, HCDR2, and HCDR3 shown in Sequence ID No. 44, Sequence ID No. 47, and Sequence ID No. 46, and LCDR1, LCDR2, and LCDR3 shown in Sequence ID No. 63, Sequence ID No. 64, and Sequence ID No. 65, (5) HCDR1, HCDR2 and HCDR3 shown in Sequence ID No. 48, Sequence ID No. 49 and Sequence ID No. 46, and LCDR1, LCDR2 and LCDR3 shown in Sequence ID No. 66, Sequence ID No. 67 and Sequence ID No. 68, (6) HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs. 44, 50 and 51, and LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs. 56, 57 and 69, or (7) HCDR1, HCDR2 and HCDR3 shown in Sequence ID No. 52, Sequence ID No. 36 and Sequence ID No. 37, and LCDR1, LCDR2 and LCDR3 shown in Sequence ID No. 53, Sequence ID No. 54 and Sequence ID No. 55, Includes.

[0024] The light-chain and heavy-chain CDR combinations provided herein originate from specific antibodies or fragments thereof provided in this disclosure. The CDRs contained in the variable region amino acid sequence of a given antibody or its antigen-binding fragment can be routinely determined by those skilled in the art. For example, according to certain embodiments of this disclosure, the IMGT tool was used to define the CDRs in the variable region amino acid sequence. All light-chain and heavy-chain CDR combinations defined by methods known in the art are encompassed within the scope of this disclosure.

[0025] In the antibody or antigen-binding fragment thereof according to the present disclosure, preferably, the heavy chain variable region includes the amino acid sequence shown in the following sequence: SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 23, or SEQ ID NO: 27, or an amino acid sequence having at least 75% identity to said amino acid sequence, and / or preferably, the light chain variable region includes the amino acid sequence shown in the following sequence: SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 33, or SEQ ID NO: 34, or an amino acid sequence having at least 75% identity to said amino acid sequence.

[0026] According to certain embodiments of this disclosure, in an antibody or antigen-binding fragment thereof, (1) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 4. (2) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 8. (3) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 10. (4) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 11, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 12. (5) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 15, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 16. (6) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 17, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 18 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 18. (7) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 19 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 19, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 20 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 20. (8) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 34 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 34. (9) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 33 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 33. (10) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 28 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 28. (11) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 23 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 23, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 34 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 34. (12) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 23 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 23, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 33 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 33, or (13) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 23 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 23, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 28 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 28.

[0027] In particular, the antibody or its antigen-binding fragment according to this disclosure comprises at least a heavy chain variable region and a light chain variable region, both of which include the CDR and framework region (FR) in a configuration such as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Alternatively, differences of up to 25% in the amino acid sequence, based on "at least 75% identity," may exist in any framework region of the heavy chain variable region or light chain variable region, or in any domain or sequence of the antibody or its antigen-binding fragment according to this disclosure other than the heavy chain variable region and light chain variable region. These differences may result from the deletion, addition, or substitution of amino acids at any position, and such substitutions may be conserved or non-conservative.

[0028] Preferably, the antibody or antigen-binding fragment according to this disclosure binds to the human interleukin-4 receptor (IL-4R), preferably to the alpha chain of human IL-4R (IL-4Rα), and more preferably to human soluble IL-4Rα (sIL-4Rα).

[0029] Preferably, the antibodies according to this disclosure may be monoclonal antibodies or single-chain antibodies. Preferably, the antibodies are mouse antibodies, chimeric antibodies, or fully or partially humanized antibodies. Preferably, the antigen-binding fragment is a fragment of the antibody that can specifically bind to a target. For example, the antigen-binding fragment is a Fab fragment, Fab' fragment, F(ab')2 fragment, or Fv fragment (e.g., scFv) of the antibody.

[0030] Preferably, the antibody or its antigen-binding fragment further comprises a human or mouse constant region, preferably a human or mouse heavy chain constant region (CH) and / or a light chain constant region (CL). More preferably, the antibody or its antigen-binding fragment comprises a heavy chain and a light chain. For example, the antibody is an immunoglobulin, particularly IgA, IgD, IgE, IgG, or IgM, for example, human IgA, IgD, IgE, IgG, or IgM, more preferably human IgG1, IgG2, IgG3, or IgG4 subtype.

[0031] Preferably, the antibody or antigen-binding fragment according to this disclosure comprises a heavy chain constant region of IgG, IgA, IgM, IgD, or IgE, and / or a kappa-type or lambda-type light chain constant region. For example, the heavy chain constant region is of the IgG type (e.g., IgG1 or IgG4), and the light chain constant region is of the kappa type. More preferably, the heavy chain constant region of the monoclonal antibody comprises an amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 70 or SEQ ID NO: 71, or an amino acid sequence having at least 75% identity to the amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 70 or SEQ ID NO: 71, and the light chain constant region of the monoclonal antibody comprises an amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 72, or an amino acid sequence having at least 75% identity to the amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 72.

[0032] In the context of this disclosure, “at least 75% identity” means any percentage identity between 75% and 100%, such as 75%, 80%, 85%, 90%, and even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0033] In another embodiment, the Disclosure also provides nucleic acid molecules comprising a heavy chain variable region, a light chain variable region, a heavy chain, and / or a nucleotide sequence encoding a light chain of an antibody or its antigen-binding fragment according to the Disclosure.

[0034] The nucleic acid molecules according to this disclosure can be cloned into a vector, which can then be transformed or transfected into host cells. Therefore, in a further embodiment, this disclosure also provides a vector comprising the nucleic acid molecules according to this disclosure. The vector may be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, etc.

[0035] The vectors or nucleic acid molecules according to this disclosure may be used to transform or transfect host cells for storage or antibody expression, etc. Accordingly, in a further embodiment, this disclosure provides host cells comprising the nucleic acid molecules and / or vectors according to this disclosure, or host cells transformed or transfected with the nucleic acid molecules and / or vectors according to this disclosure. The host cells may be any prokaryotic or eukaryotic cells, such as bacterial or insect, fungal, plant, or animal cells.

[0036] The antibodies or antigen-binding fragments thereof according to this disclosure can be obtained by any method known in the art. For example, the heavy chain variable region and / or light chain variable region of an antibody, or the heavy chain and / or light chain of an antibody, can be obtained from the nucleic acid molecule according to this disclosure, and then assembled with any other domain of the antibody to obtain the antibody. Alternatively, the host cells according to this disclosure can be cultured under conditions that allow the host cells to express the heavy chain variable region and / or light chain variable region of an antibody, or the heavy chain and / or light chain of an antibody, and to assemble them into the antibody. Optionally, the above method may further include a step of recovering the produced antibody.

[0037] The antibodies or their antigen-binding fragments, nucleic acid molecules, vectors and / or host cells according to this disclosure may be included in compositions, more particularly pharmaceutical compositions such as pharmaceutical preparations, for a variety of purposes actually required. Accordingly, in a further embodiment, this disclosure also provides compositions, preferably pharmaceutical compositions, comprising the antibodies or their fragments, nucleic acid molecules, vectors and / or host cells according to this disclosure and optionally pharmaceutically acceptable excipients.

[0038] Pharmaceutical compositions can be prepared in certain dosage forms, including but not limited to solid, liquid, semi-liquid, aerosol, and suppository forms, preferably for oral and parenteral administration (including subcutaneous, intramuscular, and intravenous administration). For example, dosage forms suitable for oral administration include tablets, capsules, granules, powders, pills, lozenges, syrups, and suspensions, while dosage forms suitable for parenteral administration include aqueous or non-aqueous solutions and emulsions, such as subcutaneous injections.

[0039] Preferably, the pharmaceutical compositions according to the present disclosure can be administered via a therapeutically effective parenteral or non-parenteral (non-enteral) route for antibody drugs. For example, the pharmaceutical compositions according to the present disclosure can be formed into formulations comprising pharmaceutically acceptable excipients (e.g., vehicles) for systemic or topical administration.

[0040] In further embodiments, the disclosure also provides the use of antibodies or their antigen-binding fragments, nucleic acid molecules, vectors, host cells and / or compositions in the manufacture of pharmaceuticals. Preferably, the pharmaceuticals are for the prevention, treatment or improvement of diseases or disorders related to the human interleukin-4 receptor (IL-4R) or diseases or disorders related to the signaling pathways of human interleukin-4 (IL-4) or human interleukin-13 (IL-13).

[0041] In another embodiment, the Disclosure also provides a method for preventing, treating, or improving a disease or disorder, the method comprising administering (e.g., a therapeutically effective amount) an antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, and / or composition according to the Disclosure to a subject in need thereof. Preferably, the disease or disorder is associated with the human interleukin-4 receptor (IL-4R), or the signaling pathway of human interleukin-4 (IL-4) or human interleukin-13 (IL-13), as described above.

[0042] In the uses or methods provided by this disclosure, disease or disorder includes autoimmune diseases, allergic diseases, tumors, or cancers.

[0043] Preferably, the disease or disorder includes autoimmune or allergic diseases, such as dermatitis (e.g., atopic dermatitis), asthma (e.g., inflammatory asthma such as type 2 inflammatory asthma), chronic esophagitis (e.g., eosinophilic esophagitis), eczema, rhinitis (e.g., allergic rhinitis), nasal polyps (e.g., chronic sinusitis with nasal polyps), conjunctivitis, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), inflammatory neuropathy, arthritis (e.g., rheumatoid arthritis), multiple sclerosis, lupus erythematosus, psoriasis, or insulin- and non-insulin-dependent diabetes mellitus, as well as other immune or allergy-related disorders.

[0044] Alternatively, disease or disorder includes tumors or cancers such as intestinal cancer, melanoma, skin cancer, breast cancer, uterine cancer, cervical cancer, endometrial cancer, ovarian cancer, testicular cancer, mesothelioma, prostate cancer, bladder cancer, anal cancer, glioblastoma, astrocytoma, liver cancer, kidney cancer, esophageal cancer, stomach cancer, lung cancer, head and neck cancer, myeloma, bone cancer, AIDS-related Kaposi's sarcoma, Hodgkin lymphoma or non-Hodgkin lymphoma, prostate cancer, prostate tumor, lymphoma, and pancreatic cancer.

[0045] Preferably, the pharmaceutical product is for the prevention, treatment, or improvement of one or more of the above-mentioned diseases or disorders.

[0046] Preferably, the subject is a mammal, preferably a primate, and more preferably a human.

[0047] Based on the specific epitope of the human IL-4R alpha chain (IL-4Rα) provided by this disclosure, this disclosure also provides the use of the specific epitope in screening for human IL-4R binders or human IL-4 / IL-13 signaling pathway blockers, or in evaluating the efficacy of binders or blockers. As described above, the epitope comprises one or more amino acid residues D92, V94, D97, L67, L68, A96, H156, C207, Q63, and L64 in the amino acid sequence shown in SEQ ID NO: 1.

[0048] Preferably, the epitope comprises one or more amino acid residues D92, V94, and D97 in the amino acid sequence shown in SEQ ID NO: 1.

[0049] According to a particular embodiment of this disclosure, the epitope is the following amino acid residue in the amino acid sequence shown in SEQ ID NO: 1: (1) D92, (2) D97, (3) D92, V94 and D97, (4) D92 and V94, (5) D92 and D97, or (6) V94 and D97, Includes.

[0050] And / or, the epitope includes the following amino acid residues in the amino acid sequence shown in SEQ ID NO: 1: (i) Q63 and L64.

[0051] and / or, the epitope comprises one or more amino acid residues L67, L68 and A96 in the amino acid sequence shown in SEQ ID NO: 1.

[0052] Preferably, the epitope is the following amino acid residue in the amino acid sequence shown in SEQ ID NO: 1: 1. L67 and L68, 2. L67, L68 and A96, or 3.A96, Includes.

[0053] According to a particular embodiment of this disclosure, the epitope is the following amino acid residue in the amino acid sequence shown in SEQ ID NO: 1: (A) L67, L68, A96, H156 and C207, (B) A96, H156 and C207, (C)L67, L68, H156 and C207, (D)L67, L68, A96 and C207, or (E)L67, L68, A96 and H156, Includes.

[0054] Particularly preferred is the epitope according to this disclosure, which is the following amino acid residue in the amino acid sequence shown in SEQ ID NO: 1: (P-1) D97, L67 and L68, (P-2) D97, L67, L68, A96 and D92, (P-3) D97, L67, L68 and D92, (P-4) D97, Q63 and L64, (P-5) D97, Q63, L64 and D92, (P-6) D97, Q63, L64, D92 and A96, or, (P-7) D97, Q63, L64, L67 and L68, Includes.

[0055] Based on the use of specific epitopes as described above, the Disclosure provides a method for screening for conjugates that specifically bind to the human interleukin-4 receptor (IL-4R) or blockers that specifically block the signaling pathway of human interleukin-4 (IL-4) or human interleukin-13 (IL-13), comprising the steps of contacting a drug to be screened with human IL-4R or a portion thereof, and then detecting whether the drug is capable of binding to the epitope according to the Disclosure. If a drug capable of binding to the epitope according to the Disclosure is detected, the drug is identified as a conjugate or blocker.

[0056] Alternatively, the Disclosure also provides a method for evaluating the efficacy of a drug, comprising the steps of contacting the drug to be evaluated with a human interleukin-4 receptor (IL-4R) or a portion thereof, and then detecting whether the drug is capable of binding to the epitope according to the Disclosure. If a drug capable of binding to the epitope according to the Disclosure is detected, the drug is identified as a conjugate that specifically binds to the human interleukin-4 receptor (IL-4R), or as an antagonist that specifically blocks the signaling pathway of human interleukin-4 (IL-4) or human interleukin-13 (IL-13), and therefore has efficacy for preventing, treating, or improving the diseases or disorders described in the Disclosure.

[0057] In the methods for screening binders or blockers, or for evaluating the efficacy of a drug, according to this disclosure, the drug to be screened or evaluated is preferably an antibody, such as a polyclonal antibody or a monoclonal antibody. More preferably, the antibody is obtained by immunizing an animal with human IL-4R or a part thereof as an immunogen.

[0058] In a method for screening binders or blockers, or for evaluating the efficacy of a drug, preferably, whether a drug can bind to the epitope according to this disclosure can be detected by mutating one or more amino acid residues contained in the epitope according to this disclosure by site-directed mutagenesis of human IL-4R or a part thereof, comparing the binding activity or binding affinity of the drug to the mutated human IL-4R or a part thereof with that of the pre-mutation drug, and identifying the drug as being able to bind to the epitope according to this disclosure if the binding activity or binding affinity of the drug to the mutated human IL-4R or a part thereof is significantly low or even lost.

[0059] In particular, the method for screening binders or blockers, or for evaluating the efficacy of a drug, as disclosed herein, 1) A step of obtaining a variant of human IL-4R or a part thereof by introducing site-directed mutagenesis into human interleukin-4 receptor (IL-4R) or a part thereof, thereby mutating one or more amino acid residues contained in the epitope according to this disclosure, 2) A step of contacting the drug to be screened or evaluated with human IL-4R or a part thereof, and a variant of human IL-4R or a part thereof, and detecting the binding activity or binding affinity to them, respectively. 3) If the binding activity or affinity of the drug to a mutated human IL-4R or a part thereof is significantly lower or lost compared to the binding activity or affinity of the drug to a mutated human IL-4R or a part thereof, the step of identifying that the drug is capable of binding to the epitope according to this disclosure, It may include.

[0060] A person skilled in the art can routinely determine whether binding activity or binding affinity is significantly low or lost. For example, if the binding activity or binding affinity of a drug to a variant or part thereof of human IL-4R is one-tenth of the binding activity or binding affinity of the pre-mutation variant, it can be determined that the binding activity or binding affinity is significantly low.

[0061] Preferably, in step 1), one or more amino acid residues contained by the epitope are mutated to alanine or the amino acid residue of the cynomolgus monkey interleukin 4 receptor at the corresponding position to obtain a variant of human IL-4R or a part thereof.

[0062] Preferably, the portion of human IL-4R used in the method according to this disclosure is the alpha chain of human IL-4R, and more preferably human sIL-4R-alpha. Therefore, preferably, the above method is carried out using human IL-4Rα to obtain a variant of human IL-4Rα. Furthermore, the above method can be carried out using human sIL-4Rα, and one or more variants of human sIL-4Rα listed in Table 3 below are used as the variant in step 1).

[0063] In this disclosure, human IL-4Rα refers to the sequence with NCBI accession number NP_000409.1, the amino acid sequence of human sIL-4Rα is shown in SEQ ID NO: 1, and the amino acid sequence of cynomolgus monkey sIL-4Rα is shown in SEQ ID NO: 2.

[0064] Experiments demonstrate that the antibodies or antigen-binding fragments according to this disclosure bind to an epitope located on the alpha chain (IL-4Rα) of the human interleukin-4 receptor (IL-4R), and that this epitope contains one or more amino acid residues D92, V94, D97, L67, L68, A96, H156, C207, Q63, and L64 in the amino acid sequence shown in SEQ ID NO: 1. Although the sIL-4Rα epitope in immunoreactions has not been extensively studied, this disclosure defines for the first time a crucial position for the binding of anti-IL-4R antibodies to IL-4R.

[0065] Accordingly, this disclosure provides an antibody against human IL-4R, particularly IL-4Rα, that binds to a novel epitope of IL-4R. This antibody may be a novel, useful, and effective antibody against human IL-4R, particularly IL-4Rα. Furthermore, this disclosure provides an alternative approach to the development of potential antibodies or drugs against IL-4R, particularly human IL-4Rα, by elucidating the key position in the binding of the antibody provided herein to human IL-4Rα.

[0066] Embodiments of this disclosure will be described in detail below with reference to the accompanying drawings. [Brief explanation of the drawing]

[0067] [Figure 1] This figure shows the alignment of human IL-4Rα (sequence_1; SEQ ID NO: 1) and cynomolgus monkey IL-4Rα (sequence_2; SEQ ID NO: 2). [Figure 2] This figure shows the stability of the antibodies of this disclosure; Panel 2A shows the results after storage at 40°C for 2 weeks, and Panel 2B shows the results after storage at 40°C for 4 weeks. [Modes for carrying out the invention]

[0068] In this disclosure, the term "epitope" refers to the amino acid residue(s) of the antigen corresponding to the binding site when an antibody or fragment thereof binds to the antigen, and the corresponding position(s) of the amino acid residue(s) in the sequence of the antigen. Furthermore, a mutation in the amino acid residue(s) of an epitope, when measured according to the procedure described in "(I) Method for detecting binding activity" below, results in a binding activity of 10 2 The level drops below ng / ml, or the binding activity is lost.

[0069] This disclosure includes the following experimental procedures or definitions. It should be noted that this disclosure can also be carried out by other prior art in the art and is not limited to the following experimental procedures.

[0070] (I) Method for detecting binding activity 1. Preparation of reagents Antigen preparation: The antigen was prepared as a 100 μg / ml solution in PBS, divided into smaller portions, and stored in a refrigerator at -20°C.

[0071] Preparation of detectable antibodies: Antibodies to be detected were diluted in PBS containing 1% BSA to desired concentrations of 0 ng / ml, 0.032 ng / ml, 0.16 ng / ml, 0.8 ng / ml, 4 ng / ml, 20 ng / ml, 100 ng / ml, 500 ng / ml, and 2500 ng / ml.

[0072] Preparation of secondary antibody working solution: The secondary antibody stock solution (goat anti-human IgG-Fc secondary antibody (HRP), sino biological, catalog number SSA001) was diluted 16,000 times with PBS containing 1% BSA to obtain the working solution. For example, 1 μl of the secondary antibody stock solution was added to 15,999 μl of PBS containing 1% BSA, and the resulting mixture was thoroughly mixed by repeatedly inverting the mixture. The desired volume of secondary antibody working solution can be prepared by scaling up according to experimental requirements.

[0073] 2. ELISA detection 50 μl of 100 μg / ml antigen solution was added to 9.95 ml of PBS, and the resulting mixture was thoroughly mixed by inverting the mixture to obtain a 0.5 μg / ml antigen coating solution. The prepared antigen coating solution was pipetted using a 12-channel pipette (RAININ) and added to a 96-well plate (Corning) at a rate of 100 μl per well. The 96-well plate was wrapped (or covered) with antiseptic film and incubated overnight in a refrigerator at 4°C. The next day, the 96-well plate was removed, the solution inside was discarded, and the plate was gently tapped on a clean paper towel to dry. 300 μl of PBS was added to each well of the 96-well plate. After leaving the plate at room temperature for 3 minutes, the solution inside was discarded, and the plate was gently tapped on a clean paper towel to dry. The plate was then washed three times. 300 μl of PBS containing 2% BSA was added to each well of the 96-well plate. The 96-well plate was wrapped (or covered) with a preservative film and incubated in a 37°C incubator for 2 hours. The 96-well plate was removed, the solution was discarded, and the plate was gently tapped on a clean paper towel to dry. PBS containing 0.05% Tween-20 (Beyotime, catalog number ST825) was added to the 96-well plate at a rate of 300 μl per well. After the plate was left at room temperature for 3 minutes, the solution was discarded, and the plate was gently tapped on a clean paper towel to dry. The plate was then washed three times.

[0074] The antibodies to be detected, diluted sequentially as described above, were added to the corresponding wells at a rate of 100 μl per well, and each sample was replicated in 3 wells. The 96-well plate was wrapped (or covered) with antiseptic film and incubated in a 37°C incubator for 1 hour. The 96-well plate was removed, the solution inside was discarded, and the plate was gently tapped on a clean paper towel to dry. PBS containing 0.05% Tween-20 was added to the 96-well plate at a rate of 300 μl per well. After leaving the plate at room temperature for 3 minutes, the solution inside was discarded, and the plate was gently tapped on a clean paper towel to dry. The plate was then washed three times. The secondary antibody working solution was added to the 96-well plate at a rate of 100 μl per well. The 96-well plate was wrapped (or covered) with antiseptic film and incubated in a 37°C incubator for 1 hour. Next, the 96-well plate was removed, the solution inside was discarded, and the plate was gently tapped on a clean paper towel to dry. PBS containing 0.05% Tween-20 was added to the 96-well plate at a rate of 300 μl per well. After leaving the plate at room temperature for 3 minutes, the solution inside was discarded, and the plate was gently tapped on a clean paper towel to dry. The plate was then washed three times. TMB substrate solution (SurModics, catalog number TMDS-1000-01) was added to the 96-well plate at a rate of 100 μl per well. The plate was then incubated in a 37°C incubator for 5 minutes, and immediately afterward, 2M H2SO4 solution was added to the 96-well plate to stop the reaction. The 96-well plate was placed in a flexstation 3 (Molecular Devices), the OD450 value was read, and data for calculation and analysis was collected.

[0075] (II) Methods for detecting cellular functional activity 1. Preparation of reagents Preparation of human IL-4 (Invivogen, catalog number rhIL-4): Human IL-4 was prepared as a 100 μg / ml solution in PBS, divided into smaller portions, and stored in a refrigerator at -20°C.

[0076] Preparation of human IL-13 (Invivogen, catalog number rhIL-13): Human IL-13 was prepared as a 100 μg / ml solution in PBS, divided into smaller portions, and stored in a refrigerator at -20°C.

[0077] Preparation of Quanti-blue(I) solution: Pour one pack of Quanti-blue(trademark) powder into a sterile 250 mL bottle, then add 100 mL of sterile water. Gently mix the resulting mixture, and place the bottle in a 37°C bath for 30 minutes. Once the powder is completely dissolved, place the bottle in a 4°C refrigerator and leave it in the dark overnight. Afterwards, divide the solution into smaller portions, 10 mL per tube, and store in the dark at -20°C. This solution can be stored for 6 months.

[0078] Preparation of detectable antibodies: The detectable antibodies were diluted to desired concentrations (1000 ng / ml, 200 ng / ml, 40 ng / ml, 8 ng / ml, 1.6 ng / ml, 0.32 ng / ml, 0.064 ng / ml, 0.0128 ng / ml, 0.00256 ng / ml, and 0 ng / ml) in DMEM medium (HyClone, catalog number SH30022.01) containing 10% FBS (Hyclone, catalog number SV30184.02).

[0079] 2.Cell culture Frozen HEK-Blue™ IL-4 / IL-13 cells (Invivogen, catalog number hkb-il413) were removed from liquid nitrogen and rapidly thawed by gentle shaking in a 37°C water bath. The thawed cell suspension was transferred to a 15 ml centrifuge tube, and 10 ml of DMEM medium (containing 10% FBS, 10 μg / ml blasticidin, 100 μg / ml zeocin, and 100 μg / ml normocin) was added to the tube. The tube was then centrifuged at 800 rpm for 5 minutes. The supernatant was aspirated, and the remaining cell pellet was washed once. 10 ml of DMEM medium was added to the cells, and the cell density was increased to 1 × 10⁶. 5 cells / ml~1×10 6The cell concentration was adjusted to cells / ml. Next, the cell suspension was transferred to a T75 cell culture bottle (Nunc) and placed in a 37°C, 5% CO2 incubator (Thermo) for steady-state culture. Every 2-3 days, the cell suspension was collected, centrifuged at 800 rpm for 5 minutes, and the resulting cells were resuspended in 10 ml of culture medium. From there, 1 × 10⁶ cells were extracted. 6 The cells were counted and transferred to a new T75 cell culture bottle, and at the same time, culture medium was added up to 10 ml. The cells were subculturised two to three times, and once the cell proliferation was good (the cells were clear, spindle-shaped, and adherent), the following experiments could be performed.

[0080] 3. Blocking experiment A T75 cell bottle containing cells in good growth condition was taken, the supernatant was discarded, and the attached cells were gently detached in 10 ml of PBS. The detached cells were transferred to a 15 ml centrifuge tube, then centrifuged at 800 rpm for 5 minutes, and the cell pellet was resuspended in 10 ml of PBS. The cells were centrifuged again at 800 rpm for 5 minutes, the supernatant was discarded, and the remaining cell pellet was resuspended in 5 ml of DMEM medium containing 10% FBS. After counting the number of cells, the medium was replenished to achieve a cell density of 6.6 × 10⁶. 5 The cell suspension was adjusted to cells / ml. 30 μl of the cell suspension was added to each well of a 384-well plate. 10 μl of the detectable antibody, prepared as described above, was added to the cells in the wells of the 384-well plate (each sample was replicated in 3 wells). Human IL-4 or human IL-13 was diluted to 2.5 ng / ml in DMEM medium containing 10% FBS, and the final cell count in each well was 2 × 10⁶. 4 To achieve a final concentration of 0.5 ng / ml of human IL-4 or human IL-13 and a final volume of 50 μl in each well of a 384-well plate, 10 μl of 2.5 ng / ml of human IL-4 or human IL-13 was added to each corresponding well of the 384-well plate. The 384-well plate was placed in a 37°C, 5% CO2 incubator for steady cell culture.

[0081] 4. Data Statistics After incubating a 384-well plate in a 5% CO2 incubator for 22 hours, 45 μl of Quanti-blue solution was added to each well of a new 384-well plate. The supernatant from each well of the plate incubated for 22 hours was pipetteed and added to the corresponding wells of a new 384-well plate, 5 μl per well, and then incubated at 37°C for 60 minutes. The 384-well plates were placed in a flexstation 3, the OD650 values ​​were read, and data for calculation and analysis were collected.

[0082] The present disclosure will be described below with reference to specific examples. Those skilled in the art will understand that these examples are merely illustrative and do not limit the scope of the present disclosure in any way.

[0083] The experimental procedures in the following examples are all conventional unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0084] Human sIL-4Rα:NP_000409.1, Met1-His232, shown in Sequence ID No. 1. The results for the cynomolgus monkey sIL-4Rα:EHH60265.1, Met1-Arg232, are shown in Sequence ID No. 2. [Examples]

[0085] Example 1: Preparation of mouse antibodies, chimeric antibodies, and humanized antibodies Ten mice were immunized with human sIL-4Rα (SEQ ID NO: 1). Blood samples were collected from the mice and detected according to the procedures described in "(I) Method for detecting binding activity" and "(II) Method for detecting cellular function activity". Based on a comprehensive analysis of the two detection results, the two mice showing the best results were selected for fusion.

[0086] The supernatants obtained from different fusions were again detected according to the procedures described in "(I) Method for detecting binding activity" and "(II) Method for detecting cell function activity". Based on a comprehensive analysis of the two detection results, the parent clone showing the highest activity was selected for subcloning. Detection was performed according to the procedures described in "(I) Method for detecting binding activity" and "(II) Method for detecting cell function activity", and finally 14 types of mouse monoclonal antibodies were screened. The base sequences of the 14 types of monoclonal antibodies were determined, and 9 sets of antibodies with new sequences were obtained. The amino acid sequences of the heavy chain variable region and light chain variable region of the antibodies are shown below, and the CDR defined by the IMGT tool is underlined.

[0087] Y0188-1 Heavy chain variable region (SEQ ID NO: 3; CDRs are sequential: SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37) EVQLVESGGGLVQPKGSLKLSCAASGFTFN TYGMH WVRQAPGKGLEWVA HIRSKSSNYATYYADSVKD RFTISRDDSQSMLYLQMNNLKTEDTAMYYCVR WFRAMDY WGQGTSVTVSS Light chain variable region (SEQ ID NO: 4; CDRs are sequential: SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55) DIVMTQSHKFMSTSVGDRVSITC KASQDVSTAVA WYQEKPGQSPKLLIY WASTRHT GVPDRFTGSGSGTDYTLTISSVQAEDLALYYC QQHYSTPLT FGAGTKLELK Y0188-2 Heavy chain variable region (SEQ ID NO: 5; CDRs are sequential: SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40) EVQLIESGGGLVQPKGSLKLSCAASGFTFN MYAMD WVRQAPGKGLEWVA RIRSKGSNFETNYADSVKD RFTISRDDSQSMVYLQMINLKTEDTAMYYCVR HRGGAWFAY WGQGTLVSVSA Light chain variable region (SEQ ID NO: 6; CDRs are sequential: SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58) DIVVTQSPASLAVSLGQRATISC RASKSVSTSGYSYMH WYQQKPGQPPKLLIY LASNLES GVPARFSGSGSGTDFTLNIHPVEEEDVAIYYC QHSRELPLT FGAGTKLELK Y0188-3 Heavy chain variable region (SEQ ID NO: 7; CDRs are sequential: SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43) QVQLVETGGGLVRPGNSLKLSCVTSGFTFS NYRMH WLRQPPGKRLEWIA VITVKSNNYGANYAESVKG RFAISRDDSKSSVYLEMNRLREEDTATYFCSR ERAYGNPFDY WGQGTTLTVSS Light chain variable region (SEQ ID NO: 8; CDRs are sequential: SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61) DIQMTQSPSSLSASLGERVSLTC RASQEISGYLS WLQQKPDGTIKRLIY AASTLD SGVPKRFSGSRSGSDYSLTISSLESEDFADYYC LQYGSYPYT FGGGTKLEIK Y0188-4 Heavy chain variable region (SEQ ID NO: 9; CDRs are sequential: SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46) EVQLVESGGGLVQPKGSLKLSCAASGFTFN MYAMN WVRQAPGQGLEWVA RIRSKSNNYATYYADSVKD RFIISRDDSESMVYLQMSNLRAADTAMYYCVR HLRAMDY WGQGTSVTVSS Light chain variable region (SEQ ID NO: 10; CDRs are sequential: SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 62) DIVLTQSPASLTVSLGQRATISC RASKSVSTSGYSYMH WYQQKPGQPPKLLIY LASNLES GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHSRELPIT FGSGTKLEIK Y0188-6 Heavy chain variable region (SEQ ID NO: 11; CDRs are sequential: SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 46) EVQLVESGGGLVQPKGSLKLSCAASGFSFN MYAMN WVRQAPGKGLEWVA RIRTKSNHYSTYYADSVKD RFTISRDDSASMFYLQMNNLKTEDTAMYFCVR HLRAMDY WGQGTSVTVSS Light chain variable region (SEQ ID NO: 12; CDRs are sequential: SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65) DIVLTQSPASLVVSLGQRATISC RASQSVSTSGYSYMH WYQQKPGQPPKLLIY LASNVQS GVPARFSGSGSGTDFTLNIHPVEEEDVATYYC HHNRDLPFT FGSGTKLEIK Y0188-8 Heavy chain variable region (SEQ ID NO: 13; CDRs are sequential: SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40) EVQLIESGGGLVQPKGSLKLSCAASGFTFN MYAMD WVRQAPGKGLEWVA RIRSKGSNFETNYADSVKD RFTISRDDSQSMVYLQMNNLKTEDTAMYYCVR HRGGAWFAY WGQGTLVTVSA Light chain variable region (SEQ ID NO: 14; CDRs are sequential: SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58) DIVVTQSPASLAVSLGQRATISC RASKSVSTSGYSYMH WYQQKPGQPPKLLIY LASNLES GVPARFSGSGSGTDFTLNIHPVEEEDVAIYYC QHSRELPLT FGAGTKLELK Y0188-9 Heavy chain variable region (SEQ ID NO: 15; CDRs are sequential: SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 46) EVQLVESGGGLVRPKGSLKLSCAASGFSFN TYAMN WVRQAPGKGLEWIV WIRSKSHNYATYYADSVKD RFTISRDDSESMLYLQMNNLKTEDTAMYYCVR HLRAMDY WGQGTSVTVSS Light chain variable region (SEQ ID NO: 16; CDRs are sequential: SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68) DIVLTQSPASLAVSLGQRATISC RASKSVSASGYSYMH WYQQKPGQPPKLLIY LASNLQS GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHSRELPPT FGGGTKLEIK Y0188-10 Heavy chain variable region (SEQ ID NO: 17; CDRs are sequential: SEQ ID NO: 44, SEQ ID NO: 50, SEQ ID NO: 51) EVRLVESGGGLVQPKGSLKLSCEASGFSFN MYAMN WVRQAPGKGLEWIT HIRSKSNNYATYYADSVKD RFIISRDDSESMVYLQMNNLKTEDTAMYYCVR LLRAL mark WGQGTSVTVSS Light chain variable region (SEQ ID NO: 18; CDRs are sequential: SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 69) DIVLTQSPASLAVFLGQRATISC RASKSVSTSGYSYMH WYQQKAGQPPKLLIY LASNLES GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC HHSRELPIT FGSGTKLEMK Y0188-14 Heavy chain variable region (SEQ ID NO: 19; CDRs are sequential: SEQ ID NO: 52, SEQ ID NO: 36, SEQ ID NO: 37) EVQLVESGGGLVQPKGSLKLSCAASGFTFN MYGMH WVRQAPGKGLEWVA HIRSKSSNYATYYADSVKD RLTISRDDSQSMLYLQMNNLKTEDTAMYYCVR WFRAMDY WGQGTSVTVSS Light chain variable region (SEQ ID NO: 20; CDRs are sequential: SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55) DIVMTQSHKFMSTSVGDRVSITC KASQDVSTAVA WYQEKPGQSPKLLIY WASTRHT GVPDRFTGSGSGTDYTLTISSVQAEDLALYYC QQHYSTPLT FGAGTKLELK

[0088] Using the variable region sequences described above, nine chimeric antibodies were constructed using the sequence shown in SEQ ID NO: 71 as the heavy chain constant region and the sequence shown in SEQ ID NO: 72 as the light chain constant region. The nine chimeric antibodies were expressed and purified, and named mouse antibody name plus Q. The IC50 values ​​of the chimeric antibodies were detected according to the procedure described in "(II) Method for detecting cellular functional activity," and the affinity (KD) of the chimeric antibodies against human sIL-4Rα was measured using a BIACORE instrument. The results are shown in Table 1 below.

[0089] [Table 1]

[0090] The chimeric antibody (Y0188-14Q) of clone number 14 was selected and humanized to obtain seven humanized sequences from the heavy chain variable region and seven humanized sequences from the light chain variable region. The humanized amino acid sequences of the heavy chain variable region and light chain variable region are shown below, with the CDR defined by the IMGT tool underlined.

[0091] Heavy chain variable region: >HV3-15-14H (Sequence ID 21; CDRs are sequential: Sequence ID 52, Sequence ID 36, Sequence ID 37) EVQLVESGGGLVKPGGSLRLSCAASGFTFS MYGMH WVRQAPGKGLEWVG HIRSKSSNYATYYADSVKD RFTISRDDSKNTLYLQMNSLKTEDTAVYYCTT WFRAMDY WGQGTLVTVSS >HV3-48-14H (Sequence ID 22; CDRs sequentially: Sequence ID 52, Sequence ID 36, Sequence ID 37) EVQLVESGGGLVQPGGSLRLSCAASGFTFS MYGMH WVRQAPGKGLEWVS HIRSKSSNYATYYADSVKD RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR WFRAMDY WGQGTLVTVSS >HV3-73 * 2-14H (Sequence ID 23; CDRs are sequential: Sequence ID 52, Sequence ID 36, Sequence ID 37) EVQLVESGGGLVQPGGSLKLSCAASGFTFS MYGMH WVRQASGKGLEWVG HIRSKSSNYATYYADSVKD RFTISRDDSKNTAYLQMNSLKTEDTAVYYCTR WFRAMDY WGQGTLVTVSS >HV3-72-14H (Sequence ID 24; CDRs sequentially: Sequence ID 52, Sequence ID 36, Sequence ID 37) EVQLVESGGGLVQPGGSLRLSCAASGFTFS MYGMH WVRQAPGKGLEWVG HIRSKSSNYATYYADSVKD RFTISRDDSKNSLYLQMNSLKTEDTAVYYCAR WFRAMDY WGQGTLVTVSS >Y01-14H (Sequence ID 25; CDRs are sequential: Sequence ID 52, Sequence ID 36, Sequence ID 37) EVQLVESGGGLVQPGGSLRLSCAASGFTFS MYGMH WVRQAPGKGLEWVS HIRSKSSNYATYYADSVKD RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR WFRAMDY WGQGTLVTVSS >162-14H (Sequence ID 26; CDRs are sequential: Sequence ID 52, Sequence ID 36, Sequence ID 37) EVQLVESGGGLEQPGGSLRLSCAGSGFTFR MYGMH WVRQAPGKGLEWVS HIRSKSSNYATYYADSVKD RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK WFRAMDY WGQGTTVTVSS >VH73-14H (Sequence ID 27; CDRs sequentially: Sequence ID 52, Sequence ID 36, Sequence ID 37) EVQLVESGGGLVQPGGSLKLSCAASGFTFS MYGMH WVRQASGKGLEWVG HIRSKSSNYATYYADSVKD RFTISRDDSKNTAYLQMNSLKTEDTAVYYCTR WFRAMDY WGQGTTVTVSS Light chain variable region: >Y01-14L (Sequence ID 28; CDRs are sequential: Sequence ID 53, Sequence ID 54, Sequence ID 55) EIVLTQSPGTLSLSPGERATLSC KASQDVSTAVA WYQQKPGQAPRLLIY WASTRHT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQHYSTPLT FGQGTKVEIK >164-14L (Sequence ID 29; CDRs are sequential: Sequence ID 53, Sequence ID 54, Sequence ID 55) DIVMTQSPLSLPVTPGEPASISC KASQDVSTAVA WYLQKSGQSPQLLIY WASTRHT GVPDRFSGSGSGTDFTLKISRVEAEEDVGFYYC QQHYSTPLT FGQGTKLEIK >KV4-14L (Sequence ID 30; CDRs are sequential: Sequence ID 53, Sequence ID 54, Sequence ID 55) DIVMTQSPDSLAVSLGERATINC KASQDVSTAVA WYQQKPGQPPKLLIY WASTRHT GVPDRFSGSGSGTDFLTISSLQAEDVAVYYC QQHYSTPLT FGGGTKVEIK >KV1-27-14L (Sequence ID 31; CDRs are sequential: Sequence ID 53, Sequence ID 54, Sequence ID 55) DIQMTQSPSSLSASVGDRVTITC KASQDVSTAVA WYQQKPGKVPKLLIY WASTRHT GVPSRFSGSGSGTDFLTISSLQPEDVATYYC QQHYSTPLT FGGGTKVEIK >KV1-9-14L (Sequence ID 32; CDRs are sequential: Sequence ID 53, Sequence ID 54, Sequence ID 55) DIQLTQSPSFLSASVGDRVTITC KASQDVSTAVA WYQQKPGKAPKLLIY WASTRHT GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC QQHYSTPLT FGGGTKVEIK >KV1-NL1-14L (Sequence ID 33; CDRs are sequential: Sequence ID 53, Sequence ID 54, Sequence ID 55) DIQMTQSPSSLSASVGDRVTITC KASQDVSTAVA WYQQKPGKAPKLLLY WASTRHT GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQHYSTPLT FGGGTKVEIK >KV1D-43-14L (Sequence ID 34; CDRs are sequential: Sequence ID 53, Sequence ID 54, Sequence ID 55) AIRMTQSPFSLSASVGDRVTITC KASQDVSTAVA WYQQKPAKAPKLFIY WASTRHT GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQHYSTPLT FGGGTKVEIK

[0092] Using humanized sequences of light chain variable regions and heavy chain variable regions, 49 types of humanized antibodies were constructed using the sequence shown in SEQ ID NO: 71 as the sequence encoding the heavy chain constant region and the sequence shown in SEQ ID NO: 72 as the sequence encoding the light chain constant region. The humanized antibodies were expressed, and the IC50 value of the antibodies was detected according to the procedure described in "(II) Method for detecting cellular functional activity," and the cellular functional activity (KD) of the antibodies was confirmed using a BIACORE instrument. Six preferred combinations of variable regions were obtained. The results are shown in Table 2 below.

[0093] [Table 2]

[0094] Example 2: Research on binding epitopes 2.1 Species differences in antibody binding to human IL-4Rα and cynomolgus monkey IL-4Rα Using cynomolgus monkey sIL-4Rα (EHH60265.1, Met1-Arg232; SEQ ID NO: 2) as the antigen, the binding activity of the antibody to the antigen was detected according to the procedure described in "(I) Method for detecting binding activity".

[0095] 2.2 Selection of the site to mutate in the antigen sequence The sequences of human IL-4Rα and cynomolgus monkey IL-4Rα were aligned as shown in Figure 1. Several positions that differ between the human IL-4Rα sequence and the cynomolgus monkey IL-4Rα sequence are important positions responsible for antibody binding to IL-4Rα, and it is presumed that these positions would allow the antibody to bind to the human sequence rather than the cynomolgus monkey sequence. Therefore, in the human IL-4Rα sequence, amino acids (sometimes multiple) at positions (sometimes multiple) that differ from those in the cynomolgus monkey IL-4Rα sequence were subjected to single point mutations or compound mutations to change these amino acids (sometimes multiple) to the amino acids (sometimes multiple) at corresponding positions (sometimes multiple) in the cynomolgus monkey IL-4Rα sequence. Additionally, non-alanine amino acids (sometimes multiple) located near the positions (sometimes multiple) where the different amino acids existed were subjected to single point mutations or compound mutations to change these amino acids (sometimes multiple) to alanine. Next, we constructed mutants of human sIL-4Rα with different combinations of mutations (see Table 3 below), and verified their activity according to the procedure described in "(II) Method for detecting cellular functional activity."

[0096] [Table 3]

[0097] 2.3 Detection of binding activity The binding activity of six types of humanized high-activity antibodies to antigens was detected using human sIL-4Rα protein and various sIL-4Rα variants shown in Table 3 as antigens, and these antibodies as primary antibodies, according to the procedure described in "(I) Method for detecting binding activity". The results are shown in Table 4.

[0098] The binding activity of nine chimeric antibodies to the antigens was similarly detected using human sIL-4Rα protein and various sIL-4Rα variants shown in Table 3 as antigens, and these antibodies as primary antibodies. The results are shown in Table 5.

[0099] Screening revealed that the binding activity of antibodies against sIL-4Rα variants with mutant amino acids (sometimes multiple) at or near these positions, which have different amino acids between human and cynomolgus monkey sIL-4Rα, is altered, or even reversed, compared to the binding activity to sIL-4Rα itself.

[0100] [Table 4]

[0101] As is evident from the data in Table 4, for five positions, L67, L68, A96, H156, and C207, one of the amino acid residues was mutated to the amino acid residue at the corresponding position in cynomolgus monkey IL-4Rα. When L67 and L68 were included in the combination of mutation sites, the binding activity of the humanized antibodies provided in this disclosure was significantly lower than their binding activity to sIL-4R-His. On the other hand, when L67 and L68 were mutated individually, the humanized antibodies in this disclosure maintained their original binding activity to sIL-4R-His.

[0102] Regarding the three positions D92, V94, and D97, if any one of these amino acid residues was mutated to alanine, and the combination of mutation sites included D97, or if only D97 was mutated, the binding activity of the humanized antibodies provided in this disclosure was significantly lower compared to their binding activity to sIL-4R-His.

[0103] [Table 5]

[0104] As is evident from the data in Table 5, for the three positions D92, V94, and D97, if any one of the amino acid residues was mutated to alanine, and if D97 was included in the combination of mutation sites, or if only D97 was mutated, the binding activity of the chimeric antibodies provided in this disclosure was significantly lower than their binding activity to sIL-4R-His, with the exception of Y0188-2Q and Y0188-8Q.

[0105] Regarding five positions, L67, L68, A96, H156, and C207, if any one of these amino acid residues was mutated to the amino acid at the corresponding position in cynomolgus monkey IL-4Rα, and L67 and L68, or L67, L68, and A96 were included in the combination of mutation sites, the binding activity of the chimeric antibodies Y0188-1Q, Y0188-3Q, and Y0188-10Q was significantly lower compared to their binding activity to sIL-4R-His. On the other hand, when L67 and L68 were mutated individually, the chimeric antibodies, with the exception of Y0188-2Q and Y0188-8Q, all maintained their original binding activity to sIL-4R-His. Furthermore, regarding A96, if A96 was included in the combination of mutation sites, the binding activity of the chimeric antibody Y0188-3Q was significantly lower compared to its binding activity to sIL-4R-His.

[0106] Regarding the two positions Q63 and L64, when both positions were mutated to alanine, the binding activity of the chimeric antibodies was significantly lower compared to their binding activity to sIL-4R-His, with the exception of Y0188-1Q, Y0188-2Q, and Y0188-8Q.

[0107] Example 3: Antibody Stability Antibodies E5 and B5 were prepared in 10 mM histidine, 150 mM NaCl, and pH 6 at concentrations of 0.59 mg / mL, 0.54 mg / mL, and 6.44 mg / mL, respectively. These were stored at 40°C for 2 weeks and 4 weeks, and detected according to the procedure described in "(II) Method for detecting cellular functional activity." A graph of antibody concentration versus OD650 value was plotted (Figure 2), and the IC50 (ng / mL) was then calculated. The results are shown in Table 6 below.

[0108] [Table 6]

[0109] The foregoing description relating to embodiments of the present invention is not intended to limit the invention, and those skilled in the art can make various changes and modifications to the invention without departing from the spirit of the invention, which should be included in the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to the human interleukin-4 receptor (IL-4R), The antibody or its antigen-binding fragment comprises a heavy chain variable region including heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region including light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3. The HCDR1, HCDR2, and HCDR3 are amino acid sequences represented by SEQ ID NO: 52, SEQ ID NO: 36, and SEQ ID NO: 37, respectively. An antibody or antigen-binding fragment thereof, wherein the LCDR1, LCDR2, and LCDR3 are amino acid sequences represented by SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO: 55, respectively.

2. (1) The heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 19, and the light chain variable region is the amino acid sequence shown in SEQ ID NO:

20. (2) The heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region is the amino acid sequence shown in SEQ ID NO:

33. (3) The antibody or antigen-binding fragment according to claim 1, wherein the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 23, and the light chain variable region is the amino acid sequence shown in SEQ ID NO:

33.

3. The antibody or antigen-binding fragment according to claim 1 or 2, wherein the antibody or antigen-binding fragment binds to the alpha chain (IL-4Rα) of the human interleukin-4 receptor.

4. The antibody or antigen-binding fragment according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof binds to human soluble IL-4Rα (sIL-4Rα).

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody is a monoclonal antibody or a single-chain antibody.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody is a mouse antibody, a chimeric antibody, or a fully or partially humanized antibody.

7. The antibody or antigen-binding fragment according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment further comprises a human or mouse heavy chain constant region (CH) and / or light chain constant region (CL).

8. The antibody or antigen-binding fragment according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment comprises a heavy chain and a light chain.

9. The antibody or antigen-binding fragment according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment thereof is human immunoglobulin IgA, IgD, IgE, IgG, or IgM.

10. The antibody or antigen-binding fragment according to any one of claims 1 to 9, wherein the antibody or antigen-binding fragment thereof is a human immunoglobulin IgG1, IgG2, IgG3, or IgG4 subtype.

11. The antibody or antigen-binding fragment according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment comprises a heavy chain constant region of IgG, IgA, IgM, IgD, or IgE, and / or a kappa-type or lambda-type light chain constant region.

12. The antibody or antigen-binding fragment thereof according to claim 11, wherein the heavy chain constant region is of the IgG type and the light chain constant region is of the kappa type.

13. A nucleic acid molecule comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12.

14. A vector comprising the nucleic acid molecule described in claim 13.

15. A host cell containing the nucleic acid molecule described in claim 13 or the vector described in claim 14, or a host cell transformed or transfected with the nucleic acid molecule described in claim 13 or the vector described in claim 14.

16. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, a nucleic acid molecule according to claim 13, a vector according to claim 14, or a host cell according to claim 15.

17. The composition according to claim 16, wherein the composition is a pharmaceutical composition.

18. The composition according to claim 16, wherein the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

19. The composition according to claim 16, wherein the composition is a pharmaceutical composition prepared in dosage forms for oral and parenteral administration.

20. The use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, a nucleic acid molecule according to claim 13, a vector according to claim 14, a host cell according to claim 15, or a composition according to claim 18 or 19 in the manufacture of a pharmaceutical product for the prevention, treatment, or improvement of a disease or disorder related to the human interleukin-4 receptor (IL-4R), or a disease or disorder related to the signaling pathway of human interleukin-4 (IL-4) or human interleukin-13 (IL-13).

21. The use according to claim 20, wherein the disease or disorder includes an autoimmune disease, an allergic disease, a tumor, or cancer.

22. The use according to claim 20, wherein the disease or disorder includes at least one selected from the group consisting of dermatitis, asthma, chronic esophagitis, eczema, rhinitis, nasal polyps, conjunctivitis, inflammatory bowel disease, inflammatory neuropathy, arthritis, multiple sclerosis, lupus erythematosus, psoriasis, or insulin and non-insulin-dependent diabetes mellitus, and other immune or allergy-related diseases.

23. The use according to claim 20, wherein the disease or disorder is selected from the group consisting of intestinal cancer, melanoma, skin cancer, breast cancer, uterine cancer, cervical cancer, endometrial cancer, ovarian cancer, testicular cancer, mesothelioma, prostate cancer, bladder cancer, anal cancer, glioblastoma, astrocytoma, liver cancer, kidney cancer, esophageal cancer, gastric cancer, lung cancer, head and neck cancer, myeloma, bone cancer, AIDS-related Kaposi's sarcoma, Hodgkin lymphoma or non-Hodgkin lymphoma, prostate tumor, lymphoma, and pancreatic cancer.

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