Antibodies that bind to specific epitopes in human il-4rα and uses thereof

Abstract

Disclosed is a human IL-4R antibody or antigen-binding fragment thereof that specifically binds to human IL-4Rα and binds to a specific epitope in human IL-4Rα. Also disclosed are uses of the specific epitope in screening for a binding agent for human IL-4R or a blocker of the human IL-4 / IL-13 signaling pathway or in evaluating the efficacy of the binding agent or blocker using the specific epitope.

Description

[0001] Cross Reference to Related Applications

[0002] This patent application claims the priority benefit of Chinese patent application No. CN202010331685.3, filed on April 24, 2020, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application relates to the field of biopharmaceuticals, and in particular, the present application relates to antibodies capable of binding to specific epitopes in human IL-4Rα and uses thereof. BACKGROUND

[0004] Human interleukin 4 receptor (IL-4R) is a type I transmembrane protein, which is widely expressed in various tissues and organs throughout the body, especially in immune organs, such as tonsils, appendix, lymph nodes, spleen and bone marrow, etc. IL-4R can exert various immunomodulatory effects after binding to ligands such as interleukin 4 (IL-4) and interleukin 13 (IL-13), such as promoting the differentiation of Th2 cells, regulating B cell secretion of IgE antibodies, and stimulating alternative activation of macrophages, etc.

[0005] IL-4R is a heterodimer composed of two polypeptide chains, one of which (IL-4Rα) has a high affinity for IL-4; and the IL-4Rα chain can form another form of IL-4R heterodimer with the cell surface receptor α chain of IL-13 (IL-13Rα), thereby also having a high affinity for IL-13.

[0006] IL-4Rα can produce a soluble form of protein (sIL-4Rα), which can inhibit a series of inflammation-related signaling, such as IL-4-mediated cell proliferation and T cell-mediated IL-5 upregulation, etc. Therefore, blocking antibodies targeting this protein are helpful for the treatment and relief of allergic rhinitis, sinusitis, asthma or eczema, etc. In addition, IL-4 and IL-13 are both cytokines with very broad spectrum of biological activities, which are involved in many kinds of inflammation-related responses, most of which are produced by activated T cells, monocytes, mast cells, basophils and eosinophils. These two interleukins have many commonalities in biological functions, and their main biological effects include stimulating the differentiation and proliferation of TH2 cells, stimulating activated B cells to secrete IgE antibodies, etc. Studies have shown that IL-4 and IL-13 play a very important role in mediating the immune response of autoimmune diseases, allergic diseases, tumors, etc., and have been one of the research hotspots that people have been concerned about.

[0007] Since sIL-4Rα plays a leading role in binding IL-4, and it is also involved in other cytokines, there are many studies on antibodies related to the target sIL-4Rα at present, and it has been proved in clinic that human monoclonal antibodies against the target can effectively relieve and treat diseases such as asthma and atopic dermatitis. However, there are few studies on the epitopes of sIL-4Rα in immune effects at present, and people are still not clear about the specific epitopes. SUMMARY

[0008] In order to solve the above technical problems, the inventors of the present application have studied the epitopes of sIL-4Rα, screened some sites in human sIL-4Rα for mutation according to the newly obtained antibodies and the species specificity of human and monkey existing when the antibodies act on sIL-4Rα, and carried out related experiments to find several key epitope binding amino acids. These epitope results have great significance for further research on sIL-4Rα and related antibodies.

[0009] Therefore, one object of the present application is to provide an antibody or an antigen-binding fragment thereof that binds to human interleukin 4 receptor (IL-4R), wherein the antibody or the antigen-binding fragment thereof binds to a specific epitope in the alpha chain of the human IL-4R (IL-4Rα).

[0010] Based on the antibody or the antigen-binding fragment thereof provided by the present application, other objects of the present application are to provide a nucleic acid molecule comprising a nucleotide sequence encoding a key domain in the antibody or the antigen-binding fragment thereof of the present application; to provide a vector containing the nucleic acid molecule; to provide a host cell containing the vector; to provide a preparation method of the antibody or the antigen-binding fragment thereof; to provide a pharmaceutical composition containing the antibody or the antigen-binding fragment thereof; and to provide a pharmaceutical use of the antibody or the antigen-binding fragment thereof or the pharmaceutical composition.

[0011] Based on the specific epitope in the alpha chain of the human IL-4R provided by the present application, the present application further provides a use of the epitope in preparing a binding agent for the human IL-4R or a blocker for the human IL-4 / IL-13 signaling pathway, or a use of the epitope in evaluating the drug efficacy of the binding agent or the blocker.

[0012] Specific solutions of the present application are as follows:

[0013] In one aspect, the present application provides an antibody or an antigen-binding fragment thereof that binds to human interleukin 4 receptor (IL-4R), wherein the antibody or the antigen-binding fragment thereof binds to a specific epitope in the alpha chain of the human IL-4R (IL-4Rα), and the epitope comprises one or more of 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.

[0014] Preferably, the epitope comprises one or more of amino acid residues D92, V94 and D97 in the amino acid sequence set forth in SEQ ID NO: 1.

[0015] According to the detailed description of the present application, the epitope comprises the following amino acid residues in the amino acid sequence set forth in SEQ ID NO: 1:

[0016] (1) D92;

[0017] (2) D97;

[0018] (3) D92, V94 and D97;

[0019] (4) D92 and V94;

[0020] (5) D92 and D97; or

[0021] (6) V94 and D97.

[0022] And / or, the antibody or antigen-binding fragment thereof provided by the present application has a binding epitope to human IL-4Rα comprising the following amino acid residues in the amino acid sequence set forth in SEQ ID NO: 1:

[0023] (i) Q63 and L64.

[0024] Also, the antibody or antigen-binding fragment thereof provided by the present application does not have species cross-binding activity to monkey IL-4R; preferably, does not bind to monkey IL-4R; more preferably, does not bind to monkey IL-4Rα; further preferably, does not bind to the amino acid sequence set forth in SEQ ID NO: 2.

[0025] In terms of the above cross-binding activity, the present application provides an antibody or antigen-binding fragment thereof binding to human interleukin 4 receptor (IL-4R), wherein the antibody or antigen-binding fragment thereof binds to an epitope located in the α chain (IL-4Rα) of human IL-4R, and the epitope comprises one or more of amino acid residues L67, L68 and A96 in the amino acid sequence set forth in SEQ ID NO: 1.

[0026] Preferably, the epitope comprises the following amino acid residues in the amino acid sequence set forth in SEQ ID NO: 1:

[0027] ① L67 and L68;

[0028] ② L67, L68 and A96; or

[0029] ③ A96.

[0030] According to the specific embodiments of the present application, the epitope includes the following amino acid residues in the amino acid sequence represented by SEQ ID NO: 1:

[0031] (A) L67, L68, A96, H156, and C207;

[0032] (B) A96, H156, and C207;

[0033] (C) L67, L68, H156, and C207;

[0034] (D) L67, L68, A96, and C207; or

[0035] (E) L67, L68, A96, and H156.

[0036] Particularly preferably, the epitope of the present application includes the following amino acid residues in the amino acid sequence represented by SEQ ID NO: 1:

[0037] (P-1) D97, L67, and L68;

[0038] (P-2) D97, L67, L68, A96, and D92;

[0039] (P-3) D97, L67, L68, and D92;

[0040] (P-4) D97, Q63, and L64;

[0041] (P-5) D97, Q63, L64, and D92;

[0042] (P-6) D97, Q63, L64, D92, and A96; or

[0043] (P-7) D97, Q63, L64, L67, and L68.

[0044] The binding of the antibody of the present application to the antigen IL-4Rα was detected, and it was found that the antibody of the present application had a binding activity EC50 of not more than 50 ng / ml, 30 ng / ml, 20 ng / ml or 10 ng / ml to sIL-4Rα. Also, it was found that the antibody of the present application did not show binding activity at 100 ul x 2500 ng / ml to 100 ul x 0.5 ug / ml of sIL-4Rα-97 coated, which was obtained by mutating the 97th amino acid residue D of sIL-4Rα to A. Further, it was found that the antibody of the present application did not show binding activity or showed significantly decreased binding activity at 100 ul x 2500 ng / ml to 100 ul x 0.5 ug / ml of at least one mutant antigen selected from the group consisting of sIL-4Rα-QSMDH and sIL-4Rα-63 / 64 coated. The binding activity was measured by the procedure described in the "Best Mode for Carrying Out the Invention" section "(i) Binding activity detection method" of the present application.

[0045] In addition, the antibody of the present application can block the binding of IL-4 or IL-13 to IL-4R, and thus can be used as a blocking agent for the binding of IL-4 or IL-13 to IL-4R, or as a blocking agent for the IL-4 / IL-13 signaling pathway.

[0046] In terms of the amino acid sequences of the included domains, the antibody or antigen-binding fragment thereof provided by the present application comprises the following combination of heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3 and light chain complementarity determining regions LCDR1, LCDR2, LCDR3:

[0047] (1) HCDR1, HCDR2, HCDR3 shown in SEQ ID NO: 35, 36, 37, respectively; and, LCDR1, LCDR2, LCDR3 shown in SEQ ID NO: 53, 54, 55, respectively;

[0048] (2) HCDR1, HCDR2, HCDR3 shown in SEQ ID NO: 41, 42, 43, respectively; and, LCDR1, LCDR2, LCDR3 shown in SEQ ID NO: 59, 60, 61, respectively;

[0049] (3) HCDR1, HCDR2, HCDR3 shown in SEQ ID NO: 44, 45, 46, respectively; and, LCDR1, LCDR2, LCDR3 shown in SEQ ID NO: 56, 57, 62, respectively;

[0050] (4) HCDR1, HCDR2, HCDR3, shown in SEQ ID NO: 44, 47, 46, respectively; and LCDR1, LCDR2, LCDR3, shown in SEQ ID NO: 63, 64, 65, respectively;

[0051] (5) HCDR1, HCDR2, HCDR3, shown in SEQ ID NO: 48, 49, 46, respectively; and LCDR1, LCDR2, LCDR3, shown in SEQ ID NO: 66, 67, 68, respectively;

[0052] (6) HCDR1, HCDR2, HCDR3, shown in SEQ ID NO: 44, 50, 51, respectively; and LCDR1, LCDR2, LCDR3, shown in SEQ ID NO: 56, 57, 69, respectively; or

[0053] (7) HCDR1, HCDR2, HCDR3, shown in SEQ ID NO: 52, 36, 37, respectively; and LCDR1, LCDR2, LCDR3, shown in SEQ ID NO: 53, 54, 55, respectively.

[0054] The combinations of light and heavy chain CDRs provided herein are from an antibody or fragment thereof of the present application, and can be routinely determined by one of skill in the art based on the variable region amino acid sequences contained in a given antibody or antigen-binding fragment thereof. For example, in accordance with the specific embodiments of the present application, the CDRs in the variable region amino acid sequences are demarcated using the IMGT tool. Combinations of light and heavy chain CDRs demarcated in this manner are also encompassed by the present application.

[0055] In the antibodies or antigen-binding fragments thereof provided herein, preferably, the heavy chain variable region comprises a sequence selected from the group consisting of the amino acid sequence shown in 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,

[0056] Preferably, the light chain variable region comprises an amino acid sequence selected from the group consisting of the amino acid sequence set forth in 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 the amino acid sequence.

[0057] According to the specific embodiments of the present application, the antibody or antigen binding fragment thereof provided by the present application comprises:

[0058] (1) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 3; and, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 4;

[0059] (2) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 7; and, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 8;

[0060] (3) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 9; and, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 10;

[0061] (4) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 11; and, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 12;

[0062] (5) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 15 or an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 15; and, the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 16;

[0063] (6) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 17 or an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 17; and, the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 18 or an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 18;

[0064] (7) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 19 or an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 19; and, the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 20 or an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 20;

[0065] (8) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 27 or an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 27; and, the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 34 or an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 34;

[0066] (9) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 27 or an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 27; and, the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 33 or an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 33;

[0067] (10) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 27 or an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 27; and, the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 28 or an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 28;

[0068] (11) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 23; and, the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 34 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 34;

[0069] (12) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 23; and, the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 33 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 33; or

[0070] (13) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 23; and, the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 28 or an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 28.

[0071] In particular, the antibody or antigen-binding fragment thereof of the present application comprises at least a heavy chain variable region and a light chain variable region, both of which include the above-mentioned CDRs as well as the intervening framework regions (FR), in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Further optionally, the "at least 75% identity" results in up to 25% difference in amino acid sequence that can exist in any of the framework regions in the heavy chain variable region or the light chain variable region, or in any domain or sequence outside of the heavy chain variable region and the light chain variable region in the antibody or antigen-binding fragment thereof of the present application. The difference can result from deletion, addition or substitution of amino acids at any position, wherein the substitution can be conservative substitution or non-conservative substitution.

[0072] Preferably, the antibody or antigen-binding fragment thereof provided by the present application binds to human interleukin 4 receptor (IL-4R), preferably binds to the alpha chain of human IL-4R (IL-4Rα), more preferably binds to human soluble IL-4Rα (sIL-4Rα).

[0073] Preferably, the antibody provided by the present application can be a monoclonal antibody or a single chain antibody. Preferably, the antibody is a murine antibody, a chimeric antibody or a fully or partially humanized antibody. Preferably, the antigen binding fragment is a fragment of the antibody capable of specifically binding to a target. For example, the antigen binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment or a Fv fragment (such as scFv) of the antibody.

[0074] Preferably, the antibody or antigen binding fragment thereof further comprises a constant region of human or murine origin, preferably a heavy chain constant region (CH) and / or a light chain constant region (CL) of human or murine origin. Further preferably, the antibody molecule or antigen binding fragment thereof comprises a heavy chain and a light chain. For example, the antibody is an immunoglobulin, in particular an IgA, IgD, IgE, IgG or IgM, for example a human subtype of IgA, IgD, IgE, IgG or IgM, more preferably a human IgG1, IgG2, IgG3 or IgG4 subtype.

[0075] Preferably, the antibody or antigen binding fragment thereof provided by the present application comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a kappa or lambda type light chain constant region, for example the heavy chain constant region is of IgG (such as IgG1 or IgG4) type and the light chain constant region is of kappa type. Further 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.

[0076] In the context of the present application, "at least 75% identity" is any percentage number of identity between 75% and 100%, for example 75%, 80%, 85%, 90%, even 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0077] In another aspect, the present application also provides a nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain variable region, the light chain variable region, the heavy chain and / or the light chain of the antibody or antigen binding fragment thereof of the present application.

[0078] The nucleic acid molecule of the present application can be cloned into a vector, which in turn transforms or transfects a host cell. Thus, in a further aspect, the present application also provides a vector comprising the nucleic acid molecule of the present application. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, etc.

[0079] The vector or nucleic acid molecule of the present application can be used to transform or transfect a host cell for the purpose of preservation or antibody expression, etc. Thus, in a further aspect, the present application provides a host cell comprising or transformed or transfected with the nucleic acid molecule and / or vector of the present application. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial or an insect, fungal, plant or animal cell.

[0080] The antibody or antigen-binding fragment thereof of the present application can be obtained using any method known in the art. For example, the heavy chain variable region and / or light chain variable region of the antibody, or the heavy chain and / or light chain of the antibody can be obtained from the nucleic acid molecule of the present application, and then assembled into the antibody with optional other domains of the antibody; or, the host cell of the present application is cultured under conditions allowing the expression of the heavy chain variable region and / or light chain variable region of the antibody, or the heavy chain and / or light chain of the antibody for assembly into the antibody. Optionally, the method further comprises a step of recovering the produced antibody.

[0081] The antibody or antigen-binding fragment thereof, nucleic acid molecule, vector and / or host cell of the present application can be included in a composition, more particularly a pharmaceutical composition, e.g. a pharmaceutical preparation, for various purposes as required. Thus, in a further aspect, the present application also provides a composition, preferably a pharmaceutical composition, comprising the antibody or fragment thereof, nucleic acid molecule, vector and / or host cell of the present application, and optionally a pharmaceutically acceptable excipient.

[0082] The above pharmaceutical composition can be formulated into certain dosage forms, preferably for oral administration and parenteral administration (including subcutaneous, intramuscular and intravenous), including but not limited to solid dosage forms, liquid dosage forms, semi-liquid dosage forms, aerosol dosage forms or suppositories, etc. For example, dosage forms suitable for oral administration include tablets, capsules, granules, powders, pills, powders, lozenges, syrups or suspensions; dosage forms suitable for parenteral administration include aqueous or non-aqueous solutions or emulsions, such as subcutaneous injections.

[0083] Preferably, the pharmaceutical composition according to the present application can be administered via parenteral or non-parenteral (enteral) routes that are therapeutically effective for antibody drug treatment. For example, the pharmaceutical composition of the present application is formed into a preparation containing a pharmaceutically acceptable excipient (e.g. a solvent), and is administered systemically or locally.

[0084] In yet another aspect, the present application also provides use of the antibody or antigen binding fragment thereof, nucleic acid molecule, vector, host cell and / or composition of the present application in the manufacture of a medicament. Preferably, the medicament is for preventing, treating or ameliorating a disease or disorder associated with human interleukin 4 receptor (IL-4R) or a disease or disorder associated with human interleukin 4 (IL-4) or human interleukin 13 (IL-13) signaling pathway.

[0085] In yet another aspect, the present application also provides a method of preventing, treating or ameliorating a disease or disorder, which comprises administering (e.g., a therapeutically effective amount of) the antibody or antigen binding fragment thereof, nucleic acid molecule, vector, host cell and / or composition of the present application to a subject in need thereof. Preferably, as mentioned above, the disease or disorder is associated with human interleukin 4 receptor (IL-4R) or associated with human interleukin 4 (IL-4) or human interleukin 13 (IL-13) signaling pathway.

[0086] In the above use or method provided by the present application, the disease or disorder includes an autoimmune disease, an allergic disease, a tumor or a cancer.

[0087] Preferably, the disease or disorder includes an autoimmune disease or an allergic disease, 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 rhinosinusitis with nasal polyps), conjunctivitis, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), inflammatory neuropathy, arthritis (e.g., rheumatoid arthritis), multiple sclerosis, lupus erythematosus, psoriasis, or insulin and non-insulin dependent diabetes mellitus, and other immune or allergy related diseases.

[0088] Alternatively, the disease or disorder includes a tumor or a cancer, 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's or non-Hodgkin's lymphoma, prostate cancer, prostate tumor, lymphoma, and pancreatic cancer, etc.

[0089] Preferably, the medicament is for preventing, treating or ameliorating one or more of the above-mentioned disease types.

[0090] Preferably, the subject is a mammal, preferably a primate; more preferably, the subject is a human.

[0091] Based on the specific epitope in the alpha chain of human IL-4R (IL-4Ra) provided by the present application, the present application also provides the use of the specific epitope in screening a binding agent for human IL-4R or a blocker of human IL-4 / IL-13 signaling pathway or the use of the specific epitope in evaluating the drug efficacy of the binding agent or the blocker. As described above, the epitope includes one or more of the 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.

[0092] Preferably, the epitope includes one or more of the amino acid residues D92, V94 and D97 in the amino acid sequence shown in SEQ ID NO: 1.

[0093] According to the specific embodiments of the present application, the epitope includes the following amino acid residues in the amino acid sequence shown in SEQ ID NO: 1:

[0094] (1) D92;

[0095] (2) D97;

[0096] (3) D92, V94 and D97;

[0097] (4) D92 and V94;

[0098] (5) D92 and D97; or

[0099] (6) V94 and D97.

[0100] And / or, the epitope includes the following amino acid residues (i) Q63 and L64 in the amino acid sequence shown in SEQ ID NO: 1.

[0101] And / or, the epitope includes one or more of the amino acid residues L67, L68 and A96 in the amino acid sequence shown in SEQ ID NO: 1.

[0102] Preferably, the epitope includes the following amino acid residues in the amino acid sequence shown in SEQ ID NO: 1:

[0103] ① L67 and L68;

[0104] ② L67, L68 and A96; or

[0105] ③ A96.

[0106] According to the specific embodiments of the present application, the epitope includes the following amino acid residues in the amino acid sequence shown in SEQ ID NO: 1:

[0107] (A) L67, L68, A96, H156, and C207;

[0108] (B) A96, H156, and C207;

[0109] (C) L67, L68, H156, and C207;

[0110] (D) L67, L68, A96, and C207; or

[0111] (E) L67, L68, A96, and H156.

[0112] Particularly preferably, the epitope of the present application includes the following amino acid residues in the amino acid sequence represented by SEQ ID NO: 1:

[0113] (P-1) D97, L67, and L68;

[0114] (P-2) D97, L67, L68, A96, and D92;

[0115] (P-3) D97, L67, L68, and D92;

[0116] (P-4) D97, Q63, and L64;

[0117] (P-5) D97, Q63, L64, and D92;

[0118] (P-6) D97, Q63, L64, D92, and A96; or

[0119] (P-7) D97, Q63, L64, L67, and L68.

[0120] In connection with the use of the above specific epitopes, the present application provides a screening method for a binding agent that specifically binds to human interleukin 4 receptor (IL-4R) or a blocking agent that specifically blocks the signal transduction pathway of human interleukin 4 (IL-4) or human interleukin 13 (IL-13), the screening method comprising the steps of: contacting a test agent to be screened with human IL-4R or a portion thereof, and then detecting whether the test agent to be screened binds to the epitope described in the present application. Upon detection of the test agent to be screened binding to the epitope described in the present application, it is confirmed to be the binding agent or the blocking agent.

[0121] Alternatively, the present application also provides a method for evaluating the efficacy of an agent, which comprises the following steps: contacting a test agent with human interleukin 4 receptor (IL-4R) or a portion thereof, and then detecting whether the test agent binds to the epitope of the present application. When the test agent is detected to bind to the epitope of the present application, it is confirmed to be a binding agent that specifically binds to human interleukin 4 receptor (IL-4R) or a blocking agent that specifically blocks the signaling pathway of human interleukin 4 (IL-4) or human interleukin 13 (IL-13), thereby having the effect of preventing, treating or improving the diseases or disorders of the present application.

[0122] In the screening method or the method for evaluating the efficacy provided by the present application, preferably, the test agent is 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 portion thereof as an immunogen.

[0123] In the screening method or the method for evaluating the efficacy, preferably, the detection of whether the test agent binds to the epitope of the present application can be performed by mutating one or more amino acid residues included in the epitope provided by the present application by site-directed mutagenesis of human IL-4R or a portion thereof, and comparing the binding activity or affinity of the test agent to human IL-4R or a portion thereof before and after mutation, when the binding activity or affinity to human IL-4R or a portion thereof after mutation is significantly decreased or lost, it is determined that the test agent binds to the epitope of the present application.

[0124] Specifically, the screening method or the method for evaluating the efficacy provided by the present application can comprise the following steps:

[0125] 1) Mutating one or more amino acid residues included in the epitope provided by the present application by site-directed mutagenesis of human interleukin 4 receptor (IL-4R) or a portion thereof, to obtain a mutant of human IL-4R or a portion thereof;

[0126] 2) Contacting a test agent with human IL-4R or a portion thereof and the mutant of human IL-4R or a portion thereof, respectively, and detecting the binding activity or affinity thereof, respectively;

[0127] 3) When the binding activity or affinity of the test agent to the mutant of human IL-4R or a portion thereof is significantly decreased or lost compared to the binding activity or affinity to human IL-4R or a portion thereof, it is determined that the test agent binds to the epitope provided by the present application.

[0128] The skilled person can routinely determine whether the binding activity or affinity has been significantly decreased or lost. For example, when the binding activity or affinity of the agent to be screened to the mutant of human IL-4R or the portion thereof is 1 / 10 of the binding activity or affinity before the mutation, it can be determined that the binding activity or affinity has been significantly decreased.

[0129] Preferably, in step 1), one or more amino acid residues comprised in the epitope are mutated to alanine or to the amino acid residue at the corresponding site of the monkey interleukin 4 receptor to obtain the mutant of human IL-4R or the portion thereof.

[0130] Preferably, the portion of human IL-4R employed in the method of the present application is the alpha chain of human IL-4R, further human sIL-4Rα. Thus preferably, the method is performed employing human IL-4Rα and obtaining a mutant of human IL-4Rα. Further, the method can be performed employing human sIL-4Rα and one or more of the human sIL-4Rα muteins listed in Table 3 below as the mutant in step 1).

[0131] In the present application, human IL-4Rα refers to NCBI Reference Sequence NP_000409.1; the amino acid sequence of human sIL-4Rα is shown in SEQ ID NO: 1; the amino acid sequence of monkey sIL-4Rα is shown in SEQ ID NO: 2.

[0132] Experiments have shown that the antibody or antigen-binding fragment thereof provided by the present application binds to an epitope of human interleukin 4 receptor (IL-4R) located in the alpha chain of human IL-4R (IL-4Rα), and the epitope comprises one or more of the 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. Prior to this, there have been few studies on the epitope of sIL-4Rα in immune effect, and the present application first identifies the key sites of the binding of anti-IL-4R antibody to IL-4R.

[0133] Therefore, the present application provides an antibody against human IL-4R, particularly IL-4Rα, with a brand new binding epitope of IL-4R, which can serve as a novel and effective antibody against human IL-4R, particularly IL-4Rα. Moreover, by elucidating the key sites of the binding of the antibody of the present application to human IL-4Rα, the present application provides another approach for developing potential antibodies or drugs against IL-4R, particularly human IL-4Rα. BRIEF DESCRIPTION OF DRAWINGS

[0134] Hereinafter, embodiments of the present application will be described in detail with reference to the accompanying drawings, in which:

[0135] Figure 1 Sequence alignment of human IL-4Ra (SEQ ID NO: 1) and monkey IL-4Ra (SEQ ID NO: 2) is shown.

[0136] Figure 2 Stability of the antibodies of the present application is shown, wherein Figure 2 A is the result after 2 weeks at 40°C, Figure 2 B is the result after 4 weeks at 40°C.

[0137] Best Mode for Carrying Out the Invention

[0138] In the present application, the term "epitope" refers to the amino acid residues of the antigen corresponding to the binding site when the antibody or fragment thereof binds to the antigen and their corresponding positions in the antigen sequence; and the amino acid residues in the position of the epitope are mutated, which will result in a decrease of more than 10 2 ng / ml or loss of binding activity, as measured by the operation process described below in "(i) Binding activity detection method".

[0139] The following experimental operations or definitions are involved in the present application. It should be noted that the present application can also be implemented using other conventional techniques in the art, and is not limited only to the following experimental operations.

[0140] (i) Binding activity detection method

[0141] 1. Preparation of reagents

[0142] Preparation of antigen: The antigen was prepared into a 100 ug / ml solution using PBS, and stored in a -20°C refrigerator after aliquoting.

[0143] Preparation of the antibody to be tested: The antibody to be tested was diluted to the required concentration of 0, 0.032, 0.16, 0.8, 4, 20, 100, 500, 2500 ng / ml using PBS containing 1% BSA.

[0144] Preparation of secondary antibody working solution: The secondary antibody stock solution (Goat Anti-Human IgG-Fc Secondary Antibody (HRP), sino biological, Cat Number: SSA001) was diluted 16000 times using PBS containing 1% BSA, i.e. the working solution. For example, take 1 ul of the secondary antibody stock solution and add it to 15999 ul of 1% BSA PBS, and mix well by repeatedly inverting. The required volume of secondary antibody working solution can be prepared according to the experimental requirements by scaling up proportionally.

[0145] 2. ELISA detection

[0146] Take 50ul 100ug / ml antigen solution, add to 9.95ml PBS, upside down mix well, that is 0.5ug / ml antigen coating solution. The prepared antigen coating solution is added to the sample groove, using 12 channel pipette (RAININ) to add 96-well enzyme labeled plate (Corning) in each well 100ul. 96-well enzyme labeled plate is wrapped with plastic wrap (or covered) and incubated in 4℃ refrigerator overnight. The next day, take out the 96-well enzyme labeled plate, discard the solution, and gently pat dry on a clean paper towel. Add PBS to the 96-well enzyme labeled plate row by row, 300ul per well. Room temperature for 3min, discard the solution, and gently pat dry on a clean paper towel. Repeat washing 3 times. Add PBS containing 2% BSA to the 96-well enzyme labeled plate row by row, 300ul per well. The enzyme labeled plate is wrapped with plastic wrap (or covered) and incubated in a 37℃ constant temperature incubator for 2h. Take out the 96-well enzyme labeled plate, discard the solution, and gently pat dry on a clean paper towel. Add PBS containing 0.05% Tween-20 (Beyotime, Cat Number: ST825) to the 96-well enzyme labeled plate row by row, 300ul per well. Room temperature for 3min, discard the solution, and gently pat dry on a clean paper towel. Repeat washing 3 times.

[0147] Diluted antibody to be tested was added to the corresponding wells, three replicates were made for each sample, 100ul per well. After wrapping the 96-well plate with plastic wrap (or covering), it was placed in a 37°C incubator for 1h. Take out the 96-well plate, discard the solution, and gently pat dry on a clean paper towel. Add 0.05% Tween-20 in PBS to the 96-well plate row by row, 300ul per well. Stand at room temperature for 3min, discard the solution, and gently pat dry on a clean paper towel. Repeat the washing for 3 times. Add secondary antibody working solution to the 96-well plate row by row, 100ul per well. After wrapping the 96-well plate with plastic wrap (or covering), it was placed in a 37°C incubator for 1h. Take out the 96-well plate, discard the solution, and gently pat dry on a clean paper towel. Add 0.05% Tween-20 in PBS to the 96-well plate row by row, 300ul per well. Stand at room temperature for 3min, discard the solution, and gently pat dry on a clean paper towel. Repeat the washing for 3 times. Add TMB solution (SurModics, Cat Number: TMDS-1000-01) to the 96-well plate row by row, 100ul per well. Place in a 37°C incubator for 5min, immediately add 2M H2SO4 solution to the 96-well plate to stop the reaction. Place the 96-well plate in the flexstation 3 (Molecular Devices) to read the OD450 value, and collect and analyze the data.

[0148] (ii) Cell function activity detection method

[0149] 1. Preparation of reagents

[0150] Preparation of human IL-4 (Invivogen, Cat Number: rhIL-4): Use PBS to prepare a 100ug / ml solution of human IL-4, and store it in the -20°C refrigerator after aliquoting.

[0151] Preparation of human IL-13 (Invivogen, Cat Number: rhIL-13): Use PBS to prepare a 100ug / ml solution of human IL-13, and store it in the -20°C refrigerator after aliquoting.

[0152] Preparation of Quanti-blue (I) solution: Pour a packet of Quanti-blue powder into a sterile 250mL bottle, add 100mL of sterile water, mix gently, incubate at 37°C for 30min, store at 4°C overnight in the dark after complete dissolution, then aliquot 10mL per bottle, store at -20°C in the dark, can be stored for 6 months.

[0153] Preparation of the antibody to be tested: Dilute the antibody to the desired concentration (1000, 200, 40, 8, 1.6, 0.32, 0.064, 0.0128, 0.00256, 0 ng / ml) with DMEM medium (HyClone, Cat No. SH30022.01) containing 10% FBS (Hyclone, Cat No. SV30184.02).

[0154] 2. Cell culture

[0155] Take out the frozen HEK Blue IL-4 / IL13 (Invivogen, Cat No. hkb-il413) cells in liquid nitrogen, and quickly dissolve them in a 37°C water bath with gentle shaking. Transfer the dissolved cell suspension to a 15 ml centrifuge tube, add DMEM medium (containing 10% FBS, 10 μg / ml Blasticidin, 100 μg / ml Zeocin, 100 μg / ml Normocin) to 10 ml, centrifuge at 800 rpm for 5 min, remove the supernatant, retain the cell pellet, repeat the washing once, add 10 ml of DMEM medium, adjust the cell density to 1×10 5 6 6 Transfer 1×10 6 cells into a new T75 cell bottle, and add medium to 10 ml at the same time, continuously pass 2-3 times until the cells are in good condition (cells are transparent, single adherent spindle cells), and then perform the experiment.

[0156] 3. Blocking experiment

[0157] Take the well-grown cells in the T75 cell bottle, discard the supernatant, and gently blow the adherent cells with 10 ml of PBS into a 15 ml centrifuge tube, centrifuge at 800 rpm for 5 min, take the cell pellet, resuspend the cells with 10 ml of PBS again, centrifuge at 800 rpm for 5 min, discard the supernatant, and take the cell pellet. Resuspend the cells with 5 ml of DMEM medium containing 10% FBS, count the cells, add medium, and adjust the cell density to 6.6×10 5The cell suspension was added into 384-well plates at a volume of 30ul per well. The prepared antibodies at different concentrations were added into the 384-well cells (3 repeated wells). Human IL-4 or human IL-13 was diluted to 2.5ng / ml with DMEM medium containing 10% FBS. 2.5ng / ml of human IL-4 or human IL-13 was added into the corresponding cell wells of the 384-well plates at a volume of 10ul per well, so that the final number of cells in each well was 2x104, the final concentration of human IL-4 or human IL-13 was 0.5ng / ml, and the final volume of each well of the 384-well plate was 50ul. The 384-well cell plate was placed in a 37°C, 5% CO2 incubator for incubation.

[0158] 4. Data statistics

[0159] After the 384-well cell plate was incubated in a 5% CO2 incubator for 22h, 45ul of Quanti-blue reagent was added to each well of a new 384-well plate. 5ul of supernatant was taken from each well of the plate previously incubated for 22h and added to the corresponding well of the new 384-well plate. Incubation was carried out at 37°C for 60min. The 384-well plate was placed in a flexstation III, and the OD650 value was read, and the data was collected and analyzed.

[0160] The application will be described below with reference to specific examples. Those skilled in the art will understand that these examples are only used to illustrate the application, and do not limit the scope of the application in any way.

[0161] In the following examples, the experimental methods are conventional methods unless otherwise specified. In the following examples, the medicinal material raw materials, reagent materials, etc. are commercially available products unless otherwise specified. Among them:

[0162] Human sIL-4Rα: NP_000409.1, Met1-His232, see SEQ ID NO: 1;

[0163] Monkey sIL-4Rα: EHH60265.1, Met1-Arg232, see SEQ ID NO: 2.

[0164] Example 1 Preparation of murine, chimeric and humanized antibodies

[0165] Ten mice were immunized with human sIL-4Rα (SEQ ID NO: 1), blood was collected, and the binding activity (i) and cell function activity (ii) were detected as above. Based on the results of the two detections, the two mice with the best results were selected for fusion.

[0166] The fused supernatant of the multiple groups of cells was subjected to the above (1) binding activity and (2) cell function activity detection again, and the results of the two detections were combined to screen the highest activity mother clone for subcloning. After (1) binding activity and (2) cell function activity detection, 14 strains of monoclonal mouse antibodies were finally screened. The 14 monoclonals were sequenced, and 9 pairs of new sequence antibodies were obtained, and the corresponding heavy chain variable region and light chain variable region are as follows; the CDRs divided by IMGT tool are underlined.

[0167] Y0188-1

[0168] Heavy chain variable region (SEQ ID NO: 3; CDRs are SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 in turn)

[0169]

[0170] Light chain variable region (SEQ ID NO: 4; CDRs are SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55 in turn)

[0171]

[0172] Y0188-2

[0173] Heavy chain variable region (SEQ ID NO: 5; CDRs are SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 in turn)

[0174]

[0175] Light chain variable region (SEQ ID NO: 6; CDRs are SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58 in turn)

[0176]

[0177] Y0188-3

[0178] Heavy chain variable region (SEQ ID NO: 7; CDRs are SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 in turn)

[0179]

[0180] Light chain variable region (SEQ ID NO: 8; CDRs are SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61 in turn)

[0181]

[0182] Y0188-4

[0183] Heavy chain variable region (SEQ ID NO: 9; CDRs in order: SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46)

[0184]

[0185] Light chain variable region (SEQ ID NO: 10; CDRs in order: SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 62)

[0186]

[0187] Y0188-6

[0188] Heavy chain variable region (SEQ ID NO: 11; CDRs in order: SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 46)

[0189]

[0190] Light chain variable region (SEQ ID NO: 12; CDRs in order: SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65)

[0191]

[0192] Y0188-8

[0193] Heavy chain variable region (SEQ ID NO: 13; CDRs in order: SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40)

[0194]

[0195] Light chain variable region (SEQ ID NO: 14; CDRs in order: SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58)

[0196]

[0197] Y0188-9

[0198] Heavy chain variable region (SEQ ID NO: 15; CDRs in order: SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 46)

[0199]

[0200] Light chain variable region (SEQ ID NO: 16; CDRs in order: SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68)

[0201]

[0202] Y0188-10

[0203] Heavy chain variable region (SEQ ID NO: 17; CDRs in order: SEQ ID NO: 44, SEQ ID NO: 50, SEQ ID NO: 51)

[0204]

[0205] Light chain variable region (SEQ ID NO: 18; CDRs in order: SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 69)

[0206]

[0207]

[0208] Y0188-14

[0209] Heavy chain variable region (SEQ ID NO: 19; CDRs in order: SEQ ID NO: 52, SEQ ID NO: 36, SEQ ID NO: 37)

[0210]

[0211] Light chain variable region (SEQ ID NO: 20; CDRs in order: SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55)

[0212]

[0213] According to the above variable region sequence, 9 chimeric antibodies were further constructed, using the sequence shown in SEQ ID NO: 71 as the heavy chain constant region coding sequence, and the sequence shown in SEQ ID NO: 72 as the light chain constant region coding sequence, the 9 chimeric antibodies were expressed and purified, named with the mouse anti-name plus Q, the IC50 of the chimeric antibodies was detected using the operation process described above in "(ii) Cell function activity detection method", and the affinity KD of the chimeric antibodies to human sIL-4Rα was detected by Biacore, and the results are shown in Table 1 below.

[0214] Table 1. Cell function activity and affinity detection results of chimeric antibodies

[0215]

[0216] The chimeric antibody of clone No. 14 (Y0188-14Q) was selected for humanization, and 7 humanized heavy chain variable regions and 7 humanized light chain variable regions were obtained. The humanized heavy chain variable region and light chain variable region are shown below; the CDRs divided by IMGT tool are shown in underlined.

[0217] Heavy chain variable region:

[0218] >HV3-15-14H (SEQ ID NO: 21; CDRs are SEQ ID NO: 52, SEQ ID NO: 36, SEQ ID NO: 37 in turn)

[0219]

[0220] >HV3-48-14H (SEQ ID NO: 22; CDRs are SEQ ID NO: 52, SEQ ID NO: 36, SEQ ID NO: 37 in turn)

[0221]

[0222] >HV3-73*2-14H (SEQ ID NO: 23; CDRs are SEQ ID NO: 52, SEQ ID NO: 36, SEQ ID NO: 37 in turn)

[0223]

[0224] >HV3-72-14H (SEQ ID NO: 24; CDRs are SEQ ID NO: 52, SEQ ID NO: 36, SEQ ID NO: 37 in turn)

[0225]

[0226] Y01-14H (SEQ ID NO: 25; CDRs in order: SEQ ID NO: 52, SEQ ID NO: 36, SEQ ID NO: 37)

[0227]

[0228] 162-14H (SEQ ID NO: 26; CDRs in order: SEQ ID NO: 52, SEQ ID NO: 36, SEQ ID NO: 37)

[0229]

[0230] VH73-14H (SEQ ID NO: 27; CDRs in order: SEQ ID NO: 52, SEQ ID NO: 36, SEQ ID NO: 37)

[0231]

[0232] Light chain variable region:

[0233] Y01-14L (SEQ ID NO: 28; CDRs in order: SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55)

[0234]

[0235] 164-14L (SEQ ID NO: 29; CDRs in order: SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55)

[0236]

[0237] KV4-14L (SEQ ID NO: 30; CDRs in order: SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55)

[0238]

[0239] KV1-27-14L (SEQ ID NO: 31; CDRs in order: SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55)

[0240]

[0241] KV1-9-14L (SEQ ID NO: 32; CDRs in order: SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55)

[0242]

[0243] KV1-NL1-14L (SEQ ID NO: 33; CDRs in order: SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55)

[0244]

[0245] KV1D-43-14L (SEQ ID NO: 34; CDRs in order: SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55)

[0246]

[0247] The above-mentioned light and heavy chain humanized sequences were paired with each other, and the sequence shown in SEQ ID NO: 71 was used as the heavy chain constant region encoding sequence, and the sequence shown in SEQ ID NO: 72 was used as the light chain constant region encoding sequence, to express, and then the IC50 of the 49 combination antibodies obtained was detected by the same operation process described in the above "(ii) Cell function activity detection method", and further verified the cell function activity (KD) by Biacore, and finally 6 combinations were preferred, as shown in Table 2.

[0248] Table 2. Cell function activity detection results of humanized antibodies

[0249]

[0250] Example 2 Epitope binding research

[0251] 2.1 Species difference of antibody binding to human and monkey IL-4Rα

[0252] Using the monkey sIL-4Rα (EHH60265.1, Met1-Arg232; SEQ ID NO: 2) as an antigen, the binding activity of the antibody to the antigen was detected by the operation described in the above "(i) Binding activity detection method".

[0253] 2.2 Mutation site selection in antigen sequence

[0254] The sequences of human and monkey IL-4Rα were aligned, see Figure 1It is speculated that the different sites of IL-4Rα sequence of human and monkey are the key sites for the binding of the antibody to IL-4Rα, so that the antibody binds to the different sequence of human but not to the different sequence of monkey. Therefore, the sites of sIL-4Rα of human which are different from those of monkey are subjected to single-point mutation or combined mutation into the amino acids at the corresponding sites of sIL-4Rα of monkey, and the non-alanine near the different sites of human and monkey sIL-4Rα is selectively subjected to single-point or combined mutation into alanine, and then the human sIL-4Rα mutants with different mutation combinations are constructed (see Table 3 below), and the cell function activity experiment is used for verification.

[0255] Table 3. Human sIL-4Rα mutant proteins

[0256]

[0257] 2.3 Detection of binding activity

[0258] The human sIL-4Rα protein and the different sIL-4Rα mutant proteins shown in Table 3 are used as antigens, and 6 human-derived high-activity antibodies are used as primary antibodies, and the binding activity of the antibodies to the antigens is detected by using the operation described in "(i) Binding activity detection method" above. The results are shown in Table 4.

[0259] Similarly, the human sIL-4Rα protein and the different sIL-4Rα mutant proteins shown in Table 3 are used as antigens, and the 9 chimeric antibodies screened are used as primary antibodies, and the binding activity of the antibodies to the antigens is also detected. The results are shown in Table 5.

[0260] Through screening, it is found that the binding activity of the antibodies to the sIL-4Rα mutant proteins of the multiple human-monkey different sites and the sites near them changes or even reverses.

[0261]

[0262]

[0263] As can be seen from the data in Table 4, as for the five sites of L67, L68, A96, H156 and C207, when the amino acid residues at any one of them are mutated into the amino acids at the corresponding sites of monkey IL-4Rα, the binding activity of the humanized antibodies of the present application significantly decreases when the mutation site combination contains L67 and L68; and at the same time, when only L67 and L68 are individually mutated, the humanized antibodies of the present application maintain the original binding activity.

[0264] As for the three sites of D92, V94 and D97, when the amino acid residues at any one of them are mutated into alanine, the binding activity of the humanized antibodies of the present application significantly decreases when the mutation site combination contains D97 or only D97 is mutated.

[0265]

[0266]

[0267] From the data in Table 5, it can be seen that, as for the three sites D92, V94 and D97, when the amino acid residue at any one of them is mutated into alanine, the binding activity of the chimeric antibodies is significantly decreased, except for Y0188-2Q and Y0188-8Q, when the mutation site combination contains D97 or only D97 is mutated. This is consistent with the binding results of the humanized antibodies described above.

[0268] As for the five sites L67, L68, A96, H156 and C207, when the amino acid residue at any one of them is mutated into the amino acid at the corresponding site in monkey IL-4Rα, the binding activity of the chimeric antibodies Y0188-1Q, Y0188-3Q and Y0188-10Q is significantly decreased when the mutation site combination contains L67 and L68 or L67, L68 and A96; while when only L67 and L68 are individually mutated, the chimeric antibodies, except for Y0188-2Q and Y0188-8Q, maintain the original binding activity. In addition, as for A96, when the mutation site combination contains A96, the binding activity of the chimeric antibody Y0188-3Q is significantly decreased.

[0269] As for the three sites Q63 and L64, when both of them are simultaneously mutated into alanine, the binding activity of the chimeric antibodies, except for Y0188-1Q, Y0188-2Q and Y0188-8Q, is significantly decreased or even lost.

[0270] Example 3 Antibody stability

[0271] The antibodies ES and B5 were prepared in 10 mM Histidine, 150 mM NaCl, pH 6 at concentrations of 0.59 mg / ml, 0.54 mg / ml and 6.44 mg / ml, respectively, and then placed at 40°C for 2 weeks and 4 weeks, respectively. The relationship between the antibody concentration and OD650 was determined by the method described above in "(ii) Cell function activity detection method", and the results are shown in Figures 6A and 6B. Figure 2 Then, the IC50 (ng / mL) was calculated, as shown in Table 6 below.

[0272] Table 6. Antibody stability results

[0273]

[0274] The above description of the specific embodiments of the present application does not limit the present application, and various changes or modifications can be made to the present application by those skilled in the art without departing from the spirit of the present application, and all such changes or modifications shall fall within the scope of the appended claims of the present application. SEQUENCE LISTING <110> Suzhou Conplast Biomedicals Co., Ltd. <120> Antibodies binding to specific epitopes in human IL-4R alpha and uses thereof <130> LC21210003P-CN <140> CN202180030276.2 <141> 2021-04-19 <150> CN202010331685.3 <151> 2020-04-24 <160> 72 <170> PatentIn version 3.3 <210> 1 <211> 232 <212> PRT <213> Homo sapiens <400> 1 Met Gly Trp Leu Cys Ser Gly Leu Leu Phe Pro Val Ser Cys Leu Val 1 5 10 15 Leu Leu Gln Val Ala Ser Ser Gly Asn Met Lys Val Leu Gln Glu Pro 20 25 30 Thr Cys Val Ser Asp Tyr Met Ser Ile Ser Thr Cys Glu Trp Lys Met 35 40 45 Asn Gly Pro Thr Asn Cys Ser Thr Glu Leu Arg Leu Leu Tyr Gln Leu 50 55 60 Val Phe Leu Leu Ser Glu Ala His Thr Cys Ile Pro Glu Asn Asn Gly 65 70 75 80 Gly Ala Gly Cys Val Cys His Leu Leu Met Asp Asp Val Val Ser Ala Gly Ala Gly Cys Val Cys His Leu Leu Met Asp Asp Val Val Ser Ala85 90 95 Asp Asn Tyr Thr Leu Asp Leu Trp Ala Gly Gin Gin Leu Leu Trp Lys 100 105 110 Gly Ser Phe Lys Pro Ser Glu His Val Lys Pro Arg Ala Pro Gly Asn 115 120 125 Leu Thr Val His Thr Asn Val Ser Asp Thr Leu Leu Leu Thr Trp Ser 130 135 140 Asn Pro Tyr Pro Pro Asp Asn Tyr Leu Tyr Asn His Leu Thr Tyr Ala 145 150 155 160 Val Asn Ile Trp Ser Glu Asn Asp Pro Ala Asp Phe Arg Ile Tyr Asn 165 170 175 Val Thr Tyr Leu Glu Pro Ser Leu Arg Ile Ala Ala Ser Thr Leu Lys 180 185 190 Ser Gly Ile Ser Tyr Arg Ala Arg Val Arg Ala Trp Ala Gin Cys Tyr 195 200 205 Asn Thr Thr Trp Ser Glu Trp Ser Pro Ser Thr Lys Trp His Asn Ser 210 215 220 Tyr Arg Glu Pro Phe Glu Gin His 225 230 <210> 2 <211> 232 <212> PRT <213> Macaca fascicularis <400> 2 Met Gly Trp Leu Cys Ser Gly Leu Leu Phe Pro Val Ser Cys Leu Val 1 5 10 15 Leu Leu Gln Val Ala Ser Ser Gly Ser Met Lys Val Leu Gln Glu Pro 20 25 30 Ala Cys Val Ser Asp Tyr Met Ser Ile Ser Thr Cys Glu Trp Lys Met 35 40 45 Gly Gly Pro Thr Asn Cys Ser Ala Glu Leu Arg Leu Leu Tyr Gln Leu 50 55 60 Val Phe Gln Ser Ser Glu Thr His Thr Cys Val Pro Glu Asn Asn Gly 65 70 75 80 Gly Val Gly Cys Val Cys His Leu Leu Met Asp Asp Val Val Ser Met 85 90 95 Asp Asn Tyr Thr Leu Asp Leu Trp Ala Gly Gln Gln Leu Leu Trp Lys 100 105 110 Gly Ser Phe Lys Pro Ser Glu His Val Lys Pro Arg Ala Pro Gly Asn 115 120 125 Leu Thr Val His Thr Asn Val Ser Asp Thr Val Leu Leu Thr Trp Ser 130 135 140 Asn Pro Tyr Pro Pro Asp Asn Tyr Leu Tyr Asn Asp Leu Thr Tyr Ala 145 150 155 160 Val Asn Ile Trp Ser Glu Asn Asp Pro Ala Tyr Ser Arg Ile His Asn 165 170 175 Val Thr Tyr Leu Lys Pro Thr Leu Arg Ile Pro Ala Ser Thr Leu Lys 180 185 190 Ser Gly Ile Ser Tyr Arg Ala Arg Val Arg Ala Trp Ala Gln His Tyr 195 200 205 Asn Thr Thr Trp Ser Glu Trp Ser Pro Ser Thr Lys Trp Tyr Asn Ser 210 215 220 Tyr Arg Glu Pro Phe Glu Gln Arg 225 230 <210> 3 <211> 118 <212> PRT <213> artificial sequence <220> <223> Y0188-1, VH <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala His Ile Arg Ser Lys Ser Ser Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Met 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg Trp Phe Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 4 <211> 107 <212> PRT <213> artificial sequence <220> <223> Y0188‑1, VL <400> 4 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Glu Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 5 <211> 120 <212> PRT <213> artificial sequence <220> <223> Y0188‑2, VH <400> 5 Glu Val Gln Leu Ile Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Met Tyr 20 25 30 Ala Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Gly Ser Asn Phe Glu Thr Asn Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Met 65 70 75 80 Val Tyr Leu Gln Met Ile Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Arg Gly Gly Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Ser Val Ser Ala 115 120 <210> 6 <211> 111 <212> PRT <213> artificial sequence <220> <223> Y0188-2, VL <400> 6 Asp Ile Val Val Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Val Ala Ile Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 7 <211> 121 <212> PRT <213> artificial sequence <220> <223> Y0188-3, VH <400> 7 Gln Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val Arg Pro Gly Asn 1 5 10 15 Ser Leu Lys Leu Ser Cys Val Thr Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Arg Met His Trp Leu Arg Gln Pro Pro Gly Lys Arg Leu Glu Trp Ile 35 40 45 Ala Val Ile Thr Val Lys Ser Asn Asn Tyr Gly Ala Asn Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Val Tyr Leu Glu Met Asn Arg Leu Arg Glu Glu Asp Thr Ala Thr Tyr 85 90 95 Phe Cys Ser Arg Glu Arg Ala Tyr Gly Asn Pro Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 8 <211> 107 <212> PRT <213> artificial sequence <220> <223> Y0188‑3, VL <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr 20 25 30 Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Gly Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 9 <211> 118 <212> PRT <213> artificial sequence <220> <223> Y0188‑4, VH <400> 9 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Met Tyr 20 25 30 Ala Met Asn Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Val 35 40 45 Ala Arg He Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe He He Ser Arg Asp Asp Ser Glu Ser Met 65 70 75 80 Val Tyr Leu Gin Met Ser Asn Leu Arg Ala Ala Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Leu Arg Ala Met Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 10 <211> 111 <212> PRT <213> artificial sequence <220> <223> Y0188-4, VL <400> 10 Asp He Val Leu Thr Gin Ser Pro Ala Ser Leu Thr Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Ile Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 11 <211> 118 <212> PRT <213> artificial sequence <220> <223> Y0188‑6, VH <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Asn Met Tyr 20 25 30 Ala Met Asn Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg He Arg Thr Lys Ser Asn His Tyr Ser Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr He Ser Arg Asp Asp Ser Ala Ser Met 65 70 75 80 Phe Tyr Leu Gin Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Phe Cys Val Arg His Leu Arg Ala Met Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 12 <211> 111 <212> PRT <213> artificial sequence <220> <223> Y0188-6, VL <400> 12 Asp He Val Leu Thr Gin Ser Pro Ala Ser Leu Val Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr He Ser Cys Arg Ala Ser Gin Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Val Gln Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Val Ala Thr Tyr Tyr Cys His His Asn Arg 85 90 95 Asp Leu Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 13 <211> 120 <212> PRT <213> artificial sequence <220> <223> Y0188‑8, VH <400> 13 Glu Val Gln Leu Ile Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Met Tyr 20 25 30 Ala Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Gly Ser Asn Phe Glu Thr Asn Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gin Ser Met 65 70 75 80 Val Tyr Leu Gin Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Arg Gly Gly Ala Trp Phe Ala Tyr Trp Gly Gin 100 105 110 Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 14 <211> 111 <212> PRT <213> artificial sequence <220> <223> Y0188-8, VL <400> 14 Asp Ile Val Val Thr Gin Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Val Ala Ile Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 15 <211> 118 <212> PRT <213> artificial sequence <220> <223> Y0188‑9, VH <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Arg Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Val Trp Ile Arg Ser Lys Ser His Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Glu Ser Met 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 16 <211> 111 <212> PRT <213> artificial sequence <220> <223> Y0188-9, VL <400> 16 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Ala Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Gln Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 17 <211> 118 <212> PRT <213> artificial sequence <220> <223> Y0188-10, VH <400> 17 Glu Val Arg Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Glu Ala Ser Gly Phe Ser Phe Asn Met Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Thr His Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Ile Ile Ser Arg Asp Asp Ser Glu Ser Met 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg Leu Leu Arg Ala Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 18 <211> 111 <212> PRT <213> artificial sequence <220> <223> Y0188-10, VL <400> 18 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Phe Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys His His Ser Arg 85 90 95 Glu Leu Pro Ile Thr Phe Gly Ser Gly Thr Lys Leu Glu Met Lys 100 105 110 <210> 19 <211> 118 <212> PRT <213> artificial sequence <220> <223> Y0188-14, VH <400> 19 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Met Tyr 20 25 30 Gly Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala His He Arg Ser Lys Ser Ser Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Leu Thr He Ser Arg Asp Asp Ser Gin Ser Met 65 70 75 80 Leu Tyr Leu Gin Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg Trp Phe Arg Ala Met Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 20 <211> 107 <212> PRT <213> artificial sequence <220> <223> Y0188-14, VL <400> 20 Asp He Val Met Thr Gin Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser lie Thr Cys Lys Ala Ser Gin Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu lie 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr lie Ser Ser Val Gin Ala 65 70 75 80 Glu Asp Leu Ala Leu Tyr Tyr Cys Gin Gin His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 21 <211> 118 <212> PRT <213> artificial sequence <220> <223> HV3-15-14H, VH <400> 21 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Met Tyr 20 25 30 Gly Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly His He Arg Ser Lys Ser Ser Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr He Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Thr Trp Phe Arg Ala Met Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 22 <211> 118 <212> PRT <213> artificial sequence <220> <223> HV3-48-14H, VH <400> 22 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Met Tyr 20 25 30 Gly Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Arg Ser Lys Ser Ser Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Trp Phe Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 23 <211> 118 <212> PRT <213> artificial sequence <220> <223> HV3‑73*2‑14H, VH <400> 23 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Met Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Arg Ser Lys Ser Ser Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Arg Trp Phe Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 24 <211> 118 <212> PRT <213> artificial sequence <220> <223> HV3‑72‑14H, VH <400> 24 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Met Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly His Ile Arg Ser Lys Ser Ser Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Trp Phe Arg Ala Met Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 25 <211> 118 <212> PRT <213> artificial sequence <220> <223> 201‑14H, VH <400> 25 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Met Tyr 20 25 30 Gly Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Arg Ser Lys Ser Ser Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Trp Phe Arg Ala Met Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 26 <211> 118 <212> PRT <213> artificial sequence <220> <223> 162‑14H, VH <400> 26 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Glu Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Arg Met Tyr 20 25 30 Gly Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser His Ile Arg Ser Lys Ser Ser Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Lys Trp Phe Arg Ala Met Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 27 <211> 118 <212> PRT <213> artificial sequence <220> <223> VH73-14H, VH <400> 27 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Met Tyr 20 25 30 Gly Met His Trp Val Arg Gin Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly His He Arg Ser Lys Ser Ser Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr He Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Arg Trp Phe Arg Ala Met Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 28 <211> 107 <212> PRT <213> artificial sequence <220> <223> Y01‑14L, VL <400> 28 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 29 <211> 107 <212> PRT <213> artificial sequence <220> <223> 164‑14L, VL <400> 29 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Leu Gln Lys Ser Gly Gln Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala 65 70 75 80 Glu Asp Val Gly Phe Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 30 <211> 107 <212> PRT <213> artificial sequence <220> <223> KV4‑14L, VL <400> 30 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr lie Asn Cys Lys Ala Ser Gin Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu lie 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gin Gin His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu lie Lys 100 105 <210> 31 <211> 107 <212> PRT <213> artificial sequence <220> <223> KV1-27-14L, VL <400> 31 Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr lie Thr Cys Lys Ala Ser Gin Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 32 <211> 107 <212> PRT <213> artificial sequence <220> <223> KV1‑9‑14L, VL <400> 32 Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 33 <211> 107 <212> PRT <213> artificial sequence <220> <223> KV1-NL1-14L, VL <400> 33 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 34 <211> 107 <212> PRT <213> artificial sequence <220> <223> KV1D-43-14L, VL <400> 34 Ala Ile Arg Met Thr Gln Ser Pro Phe Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Ala Lys Ala Pro Lys Leu Phe Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 35 <211> 5 <212> PRT <213> artificial sequence <220> <223> HCDR1 <400> 35 Thr Tyr Gly Met His 1 5 <210> 36 <211> 19 <212> PRT <213> artificial sequence <220> <223> HCDR2 <400> 36 His Ile Arg Ser Lys Ser Ser Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp <210> 37 <211> 7 <212> PRT <213> artificial sequence <220> <223> HCDR3 <400> 37 Trp Phe Arg Ala Met Asp Tyr 1 5 <210> 38 <211> 5 <212> PRT <213> artificial sequence <220> <223> HCDR1 <400> 38 Met Tyr Ala Met Asp 1 5 <210> 39 <211> 19 <212> PRT <213> artificial sequence <220> <223> HCDR2 <400> 39 Arg Ile Arg Ser Lys Gly Ser Asn Phe Glu Thr Asn Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp <210> 40 <211> 9 <212> PRT <213> artificial sequence <220> <223> HCDR3 <400> 40 His Arg Gly Gly Ala Trp Phe Ala Tyr 1 5 <210> 41 <211> 5 <212> PRT <213> artificial sequence <220> <223> HCDR1 <400> 41 Asn Tyr Arg Met His 1 5 <210> 42 <211> 19 <212> PRT <213> artificial sequence <220> <223> HCDR2 <400> 42 Val Ile Thr Val Lys Ser Asn Asn Tyr Gly Ala Asn Tyr Ala Glu Ser 1 5 10 15 Val Lys Gly <210> 43 <211> 10 <212> PRT <213> artificial sequence <220> <223> HCDR3 <400> 43 Glu Arg Ala Tyr Gly Asn Pro Phe Asp Tyr 1 5 10 <210> 44 <211> 5 <212> PRT <213> artificial sequence <220> <223> HCDR1 <400> 44 Met Tyr Ala Met Asn 1 5 <210> 45 <211> 19 <212> PRT <213> artificial sequence <220> <223> HCDR2 <400> 45 Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp <210> 46 <211> 7 <212> PRT <213> artificial sequence <220> <223> HCDR3 <400> 46 His Leu Arg Ala Met Asp Tyr 1 5 <210> 47 <211> 19 <212> PRT <213> artificial sequence <220> <223> HCDR2 <400> 47 Arg Ile Arg Thr Lys Ser Asn His Tyr Ser Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp <210> 48 <211> 5 <212> PRT <213> artificial sequence <220> <223> HCDR1 <400> 48 Thr Tyr Ala Met Asn 1 5 <210> 49 <211> 19 <212> PRT <213> artificial sequence <220> <223> HCDR2 <400> 49 Trp Ile Arg Ser Lys Ser His Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp <210> 50 <211> 19 <212> PRT <213> artificial sequence <220> <223> HCDR2 <400> 50 His Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp <210> 51 <211> 7 <212> PRT <213> artificial sequence <220> <223> HCDR3 <400> 51 Leu Leu Arg Ala Leu Asp Tyr 1 5 <210> 52 <211> 5 <212> PRT <213> artificial sequence <220> <223> HCDR1 <400> 52 Met Tyr Gly Met His 1 5 <210> 53 <211> 11 <212> PRT <213> artificial sequence <220> <223> LCDR1 <400> 53 Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala 1 5 10 <210> 54 <211> 7 <212> PRT <213> artificial sequence <220> <223> LCDR2 <400> 54 Trp Ala Ser Thr Arg His Thr 1 5 <210> 55 <211> 9 <212> PRT <213> artificial sequence <220> <223> LCDR3 <400> 55 Gln Gln His Tyr Ser Thr Pro Leu Thr 1 5 <210> 56 <211> 15 <212> PRT <213> artificial sequence <220> <223> LCDR1 <400> 56 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His 1 5 10 15 <210> 57 <211> 7 <212> PRT <213> artificial sequence <220> <223> LCDR2 <400> 57 Leu Ala Ser Asn Leu Glu Ser 1 5 <210> 58 <211> 9 <212> PRT <213> artificial sequence <220> <223> LCDR3 <400> 58 Gln His Ser Arg Glu Leu Pro Leu Thr 1 5 <210> 59 <211> 11 <212> PRT <213> artificial sequence <220> <223> LCDR1 <400> 59 Arg Ala Ser Gin Glu lie Ser Gly Tyr Leu Ser 1 5 10 <210> 60 <211> 7 <212> PRT <213> artificial sequence <220> <223> LCDR2 <400> 60 Ala Ala Ser Thr Leu Asp Ser 1 5 <210> 61 <211> 9 <212> PRT <213> artificial sequence <220> <223> LCDR3 <400> 61 Leu Gin Tyr Gly Ser Tyr Pro Tyr Thr 1 5 <210> 62 <211> 9 <212> PRT <213> artificial sequence <220> <223> LCDR3 <400> 62 Gln His Ser Arg Glu Leu Pro lie Thr 1 5 <210> 63 <211> 15 <212> PRT <213> artificial sequence <220> <223> LCDR1 <400> 63 Arg Ala Ser Gin Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His 1 5 10 15 <210> 64 <211> 7 <212> PRT <213> artificial sequence <220> <223> LCDR2 <400> 64 Leu Ala Ser Asn Val Gln Ser 1 5 <210> 65 <211> 9 <212> PRT <213> artificial sequence <220> <223> LCDR3 <400> 65 His His Asn Arg Asp Leu Pro Phe Thr 1 5 <210> 66 <211> 15 <212> PRT <213> artificial sequence <220> <223> LCDR1 <400> 66 Arg Ala Ser Lys Ser Val Ser Ala Ser Gly Tyr Ser Tyr Met His 1 5 10 15 <210> 67 <211> 7 <212> PRT <213> artificial sequence <220> <223> LCDR2 <400> 67 Leu Ala Ser Asn Leu Gln Ser 1 5 <210> 68 <211> 9 <212> PRT <213> artificial sequence <220> <223> LCDR3 <400> 68 Gln His Ser Arg Glu Leu Pro Pro Thr 1 5 <210> 69 <211> 9 <212> PRT <213> artificial sequence <220> <223> LCDR3 <400> 69 His His Ser Arg Glu Leu Pro Ile Thr 1 5 <210> 70 <211> 993 <212> DNA <213> Homo sapiens <400> 70 gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720 atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcacgagg ctctgcacaa ccactacacg 960 cagaagagcc tctccctgtc tccgggtaaa tga 993 <210> 71 <211> 995 <212> DNA <213> Homo sapiens <400> 71 GCTAGCACCA AGGGCCCATC GGTCTTCCCC CTGGCGCCCT GCTCCAGGAG CACCTCCGAG 60 AGCACAGCCG CCCTGGGCTG CCTGgtCAAG GACTACTTCC CCgaACCgGT GACGgtGTcG 120 TGGAACtCAG GCgCCCTGA CCAGCggCgTG ACACACCTT CCCGGCTGT CCTACAGTCCT CA 180 GGACTCTACT CCCTCAGCAG CGTGgtGAcc GTGCCCTCA GCAGCTTggG CACGAAGACC 240 TACACCTGCA ACGTAGATCA CAAGCCCAGC AACACCAGG GTGGACAAga GAGTTGAgtC 300 AAATATGGTC CCCATGCCC ATCATGCCCAG CCACCTGAgt TCCTGGGGGG ACCATCAGTC 360 TTCCTGTTCC CCCCAAAGCC CAAGGACACC TCtCATGATC TCCTGGACCC CTGAGGTCAcG 420 TGCgtGgtGG TGGACGTGAG CCAGGAAGAC CCCGAGGTCC AGTTCAACTG GTACGTGGAT 480 GGCgtGGAGG TGCATAATGC CAAGACAAAG CCgCGGAGGA GCAGTTCAAC AGCACGTAC 540 CgtGTGgtCA GCgtCCTCAC CgtCCTGCAC CAGGACTGGC TGAACGGCAA GGAGTACAAG 600 TGCAAGGTCT CCAACAAAGG CCTCCGTCCt CCATCGAGAA ACCATCTCCA AAGCCAAA 660 GGGCAGCCCC GAGAGCCACA GGTGTACACC CTGCCCCCAT CCCAGGAAGA GATGACCAAG 720 aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 780 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 840 gacggctcct tcttcctcta cagcaggctc accgtggaca agagcaggtg gcaggagggg 900 aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 960 ctctccctgt ctccgggtaa atgagttcta gctgg 995 <210> 72 <211> 327 <212> DNA <213> Homo sapiens <400> 72 acggtggctg caccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga 60 actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg 120 aaggtggata acgccctcca atcgggtaac tcccaggaga gtgtcacaga gcaggacagc 180 aaggacagca cctacagcct cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa 240 cacaaagtct acgcctgcga agtcacccat cagggcctga gctcgcccgt cacaaagagc 300 ttcaacaggg gagagtgtta gagggag 327

Claims

1. An antibody or antigen-binding fragment thereof that binds human interleukin 4 receptor (IL-4R), the antibody or antigen-binding fragment thereof comprising the following heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3 and light chain complementarity determining regions LCDR1, LCDR2, LCDR3 in its heavy chain variable region and light chain variable region: HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 52, 36, and 37, respectively; and, LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 53, 54, and 55, respectively. In the antibody or antigen-binding fragment thereof:

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, (1) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 19; and, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 20; (2) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27; and, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 34; (3) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27; and, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 33; (4) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27; and, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28; (5) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23; and, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 34; (6) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23; and, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 33; or (7) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23; and, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

28. The antibody or antigen-binding fragment thereof binds to an alpha chain of human IL-4R (IL-4Ra).

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein, The antibody or antigen-binding fragment thereof binds to soluble human IL-4Ra.

4. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein, The antibody is a monoclonal antibody or a single chain antibody.

5. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein, The antibody is a murine, chimeric, or fully or partially humanized antibody.

6. The antibody or antigen-binding fragment thereof of claim 5, wherein, The antigen-binding fragment is a fragment of the antibody that is capable of specifically binding to IL-4R or IL-4Ra.

7. The antibody or antigen-binding fragment thereof of claim 5, wherein, The antibody or antigen-binding fragment thereof further comprises a constant region of human or murine origin.

8. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein, The antibody or antigen-binding fragment thereof further comprises a heavy chain constant region (CH) and / or a light chain constant region (CL) of human or murine origin.

9. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein, The antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain.

10. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein, The antibody is an immunoglobulin of IgA, IgD, IgE, IgG, or IgM type.

11. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein, The antibody is an immunoglobulin of human subtype IgA, IgD, IgE, IgG, or IgM.

12. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein, The antibody is an immunoglobulin of human subtype IgG1, IgG2, IgG3, or IgG4.

13. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein, ​ 14. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein, The antibody or antigen-binding fragment thereof comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a light chain constant region of κ or λ type.

15. The antibody or antigen-binding fragment thereof of claim 14, wherein, The heavy chain constant region of the antibody is of IgG type and the light chain constant region is of κ type.

16. The antibody or antigen-binding fragment thereof of claim 14, wherein, The heavy chain constant region of the antibody is of IgG1 or IgG4 type and the light chain constant region is of κ type.

17. A nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 16.

18. A vector comprising the nucleic acid molecule of claim 17.

19. A host cell comprising or transformed or transfected with the nucleic acid molecule of claim 17 or the vector of claim 18, wherein, The host cell is not a plant cell.

20. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, the nucleic acid molecule of claim 17, the vector of claim 18 or the host cell of claim 19.

21. The composition of claim 20, wherein, The composition is a pharmaceutical composition.

22. The composition of claim 21, wherein, The pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

23. The composition of claim 21 or 22, wherein, The pharmaceutical composition is in a dosage form for oral or parenteral administration.

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