Antibody capable of binding to specific epitope in human IL-4Ralpha and application thereof

Abstract

The invention discloses a human IL-4R antibody or an antigen binding fragment thereof. The antibody or the antigen binding fragment thereof is specifically bound with human IL-4R alpha and is bound with a specific epitope in the human IL-4R alpha. In addition, the invention also provides application of the epitope in screening a binding agent of human IL-4R or a blocking agent of a human IL-4 / IL-13 signal transduction pathway or application of the specific epitope in evaluating the drug effect of the binding agent or the blocking agent.

Description

[0001] Cross-references to related applications

[0002] This patent application claims priority to Chinese Patent Application No. CN202010331685.3, filed on April 24, 2020, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of biopharmaceuticals, and more specifically, to antibodies capable of binding to specific epitopes in human IL-4Rα and their applications. Background Technology

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

[0005] IL-4R is a heterodimer composed of two polypeptide chains. One of the chains, the α chain (IL-4Rα), has a high affinity for IL-4. Furthermore, the IL-4Rα chain forms another form of IL-4R heterodimer with the α chain of the cell surface receptor for IL-13 (IL-13Rα), thus also having a high affinity for IL-13.

[0006] IL-4Rα can produce a soluble form of protein (sIL-4Rα), which can inhibit a range of inflammation-related signaling pathways, such as IL-4-mediated cell proliferation and T cell-mediated IL-5 upregulation. Therefore, blocking antibodies targeting this protein could be helpful in treating and alleviating conditions such as allergic rhinitis, sinusitis, asthma, or eczema. Furthermore, IL-4 and IL-13 are both cytokines with very broad-spectrum biological activity, involved in many inflammation-related responses, and mostly produced by activated T cells, monocytes, mast cells, basophils, and eosinophils. These two interleukins share many common biological functions, with their main biological effects including stimulating the differentiation and proliferation of TH2 cells and stimulating activated B cells to secrete IgE antibodies. Studies have shown that IL-4 and IL-13 play a crucial role in mediating immune responses in autoimmune diseases, allergic diseases, and tumors, and have remained a focus of research.

[0007] Because sIL-4Rα plays a dominant role in IL-4 binding and also involves other cytokines, there is a great deal of research on antibodies targeting sIL-4Rα. Clinically, human monoclonal antibodies targeting this target have been shown to effectively alleviate and treat diseases such as asthma and atopic dermatitis. However, research on the epitopes of sIL-4Rα in immune responses is limited, and its specific epitope characteristics remain unclear. Summary of the Invention

[0008] To address the aforementioned technical problems, the inventors of this invention studied the epitopes of sIL-4Rα. Based on the newly obtained antibody and its species-specific interactions with sIL-4Rα in humans and monkeys, they screened several sites in human sIL-4Rα for mutation and conducted related experiments, identifying several key epitope binding amino acids. These epitope results are of great significance for further research on sIL-4Rα and related antibodies.

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

[0010] Based on the antibody or antigen-binding fragment thereof provided by the present invention, another object of the present invention is to provide a nucleic acid molecule comprising a nucleotide sequence encoding a key domain of the antibody or antigen-binding fragment thereof of the present invention; to provide a vector containing the nucleic acid molecule; to provide a host cell containing the vector; to provide a method for preparing the antibody or antigen-binding fragment thereof; to provide a pharmaceutical composition containing the antibody or antigen-binding fragment thereof; and to provide pharmaceutical use of the antibody or antigen-binding fragment thereof or the pharmaceutical composition.

[0011] Based on the specific epitope in the α chain of human IL-4R provided by the present invention, the present invention also provides the use of the epitope in the preparation of human IL-4R binders or blockers of the human IL-4 / IL-13 signaling pathway, or the use of the epitope in evaluating the efficacy of the binders or blockers.

[0012] The specific solution of the present invention is as follows:

[0013] On one hand, the present invention provides an antibody or antigen-binding fragment thereof that binds to human interleukin-4 receptor (IL-4R), wherein the epitope bound by the antibody or antigen-binding fragment is located on the α chain (IL-4Rα) of human IL-4R, and the epitope includes one or more amino acid residues selected from 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 includes one or more of the amino acid residues D92, V94, and D97 in the amino acid sequence shown in SEQ ID NO:1.

[0015] According to a specific embodiment of the present invention, the epitope includes the following amino acid residues in the amino acid sequence shown 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 binding epitope of the antibody or its antigen-binding fragment provided by the present invention to human IL-4Rα includes the following amino acid residues in the amino acid sequence shown in SEQ ID NO:1:

[0023] (i)Q63 and L64.

[0024] Furthermore, the antibody or its antigen-binding fragment provided by the present invention does not have species cross-binding activity with monkey IL-4R; preferably, it does not bind to monkey IL-4R; more preferably, it does not bind to monkey IL-4Rα; and even more preferably, it does not bind to the amino acid sequence shown in SEQ ID NO:2.

[0025] Regarding the aforementioned cross-binding activity, the present invention provides an antibody or antigen-binding fragment thereof that binds to human interleukin-4 receptor (IL-4R), wherein the epitope bound by the antibody or antigen-binding fragment is located on the α chain of human IL-4R (IL-4Rα), and the epitope includes one or more of amino acid residues L67, L68 and A96 in the amino acid sequence shown in SEQ ID NO:1.

[0026] Preferably, the epitope includes the following amino acid residues in the amino acid sequence shown in SEQ ID NO:1:

[0027] ①L67 and L68;

[0028] ②L67, L68, and A96; or

[0029] ③A96.

[0030] According to a specific embodiment of the present invention, the epitope includes the following amino acid residues in the amino acid sequence shown in 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 epitopes of the present invention include the following amino acid residues in the amino acid sequence shown in 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 invention to the antigen IL-4Rα was detected, and it was found that the EC50 of the binding activity of the antibody of the present invention to sIL-4Rα did not exceed 50 ng / ml, 30 ng / ml, 20 ng / ml, or 10 ng / ml. Furthermore, it was found that the antibody of the present invention did not show binding activity with 100 μl × 0.5 μg / ml of sIL-4Rα-97 coated at 100 μl × 2500 ng / ml, wherein sIL-4Rα-97 is obtained by mutating amino acid residue D at position 97 of sIL-4Rα to A. It was further found that the antibody of the present invention did not show binding activity or showed a significant decrease in binding activity with at least one mutant antigen selected from sIL-4Rα-QSMDH and sIL-4Rα-63 / 64 coated at 100 μl × 2500 ng / ml. 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 invention.

[0045] Furthermore, the antibody of the present invention can block the binding of IL-4 or IL-13 to IL-4R, and therefore can be used as an inhibitor of the binding of IL-4 or IL-13 to IL-4R, or as an inhibitor of the IL-4 / IL-13 signaling pathway.

[0046] Regarding the amino acid sequences of the contained structural domains, the antibody or its antigen-binding fragment provided by the present invention comprises a combination of the following heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 and light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3:

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

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

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

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

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

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

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

[0054] The combination of light and heavy chain CDRs provided by this invention originates from the antibodies or fragments thereof of this invention. Based on the variable region amino acid sequence contained in a given antibody or its antigen-binding fragment, those skilled in the art can conventionally determine the CDRs contained therein. For example, according to a specific embodiment of this invention, the IMGT tool is used to partition the CDRs in the variable region amino acid sequence. Combinations of light and heavy chain CDRs partitioned using methods known in the art are also covered within the scope of this invention.

[0055] In the antibody or antigen-binding fragment provided by the present invention, preferably, the heavy chain variable region comprises a sequence selected from: amino acid sequences 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 with said amino acid sequence; and / or,

[0056] Preferably, the light chain variable region comprises a sequence selected from the following: an amino acid sequence shown 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 with said amino acid sequence.

[0057] According to specific embodiments of the present invention, the antibody or its antigen-binding fragment provided by the present invention contains:

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

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

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

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

[0062] (5) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:15; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:16 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:16.

[0063] (6) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:17 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:17; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:18 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:18.

[0064] (7) The heavy chain variable region comprises an amino acid sequence shown in SEQ ID NO:19 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:19; and the light chain variable region comprises an amino acid sequence shown in SEQ ID NO:20 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:20.

[0065] (8) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:27; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:34 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:34.

[0066] (9) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:27; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:33.

[0067] (10) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:27; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:28 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:28.

[0068] (11) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:23; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:34 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:34.

[0069] (12) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:23; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:33; or

[0070] (13) The heavy chain variable region comprises an amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:23; and the light chain variable region comprises an amino acid sequence shown in SEQ ID NO:28 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:28.

[0071] Specifically, the antibody or its antigen-binding fragment of the present invention comprises at least a heavy chain variable region and a light chain variable region, both of which include the aforementioned CDRs and spaced framework regions (FRs), with the domains arranged as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Further optionally, the maximum 25% difference in amino acid sequence resulting from the "at least 75% identity" can exist in any framework region within the heavy chain variable region or the light chain variable region, or in any domain or sequence other than the heavy chain variable region and the light chain variable region in the antibody or its antigen-binding fragment of the present invention. This difference can be caused by amino acid deletions, additions, or substitutions at any position, wherein substitutions can be conservative or non-conservative.

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

[0073] Preferably, the antibody provided by the present invention 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, Fab' fragment, F(ab')2 fragment, or Fv fragment (such as scFv) of the antibody.

[0074] Preferably, the antibody or its antigen-binding fragment further comprises a human or mouse constant region, more preferably a human or mouse heavy chain constant region (CH) and / or a light chain constant region (CL). More preferably, the antibody molecule or its antigen-binding fragment comprises both a heavy chain and a light chain. For example, the antibody is an immunoglobulin, specifically IgA, IgD, IgE, IgG, or IgM, such as human subtypes of IgA, IgD, IgE, IgG, or IgM, more preferably human IgG1, IgG2, IgG3, or IgG4 subtypes.

[0075] Preferably, the antibody or antigen-binding fragment provided by the present invention comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a κ or λ type light chain constant region, for example, the heavy chain constant region is of type IgG (such as IgG1 or IgG4) and the light chain constant region is of type κ. More preferably, the heavy chain constant region of the monoclonal antibody comprises an amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO:70 or SEQ ID NO:71 or an amino acid sequence having at least 75% identity with the amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO:70 or SEQ ID NO:71; 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 with the amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO:72.

[0076] In the context of this invention, "at least 75% identity" means any percentage of identity between 75% and 100%, such as 75%, 80%, 85%, 90%, or even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0077] On the other hand, the present invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain variable region, a light chain variable region, a heavy chain, and / or a light chain in an antibody of the present invention or an antigen-binding fragment thereof.

[0078] The nucleic acid molecules of this invention can be cloned into a vector, and then transformed or transfected into host cells. Therefore, in another aspect, this invention also provides a vector containing the nucleic acid molecules of this invention. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, or a bacteriophage vector, etc.

[0079] The vectors or nucleic acid molecules of the present invention can be used to transform or transfect host cells for purposes such as preservation or antibody expression. Therefore, in another aspect, the present invention provides a host cell containing the nucleic acid molecules and / or vectors of the present invention, or the host cell being transformed or transfected by the nucleic acid molecules and / or vectors of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as bacterial or insect, fungal, plant, or animal cells.

[0080] The antibodies or antigen-binding fragments thereof provided by this invention 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 can be obtained first from the nucleic acid molecule provided by this invention, or the heavy chain and / or light chain of the antibody can be obtained, and then assembled with optional other structural domains of the antibody to form an antibody; or, the host cells can be cultured while allowing the host cells provided by this invention to express 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 to assemble the antibody. Optionally, the method further includes the step of recovering the generated antibody.

[0081] The antibodies or antigen-binding fragments thereof, nucleic acid molecules, vectors, and / or host cells provided by the present invention can be included in compositions, more particularly in pharmaceutical compositions, such as pharmaceutical formulations, for use in various purposes as needed. Therefore, in another aspect, the present invention also provides a composition, preferably a pharmaceutical composition, comprising the antibodies or fragments thereof described in the present invention, nucleic acid molecules, vectors, and / or host cells, and optionally pharmaceutically acceptable excipients.

[0082] The above-mentioned pharmaceutical compositions can be formulated into specific dosage forms, preferably for oral and parenteral administration (including subcutaneous, intramuscular, and intravenous administration), including but not limited to solid dosage forms, liquid dosage forms, semi-liquid dosage forms, aerosols, or suppositories. 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 invention can be administered via a parenteral or non-parenteral (enteral) route that is effective in treating antibody drugs. For example, the pharmaceutical composition of the invention can be formulated as a preparation containing pharmaceutically acceptable excipients (e.g., solvents) for systemic or local administration.

[0084] In another aspect, the present invention also provides the use of the antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell and / or composition in the preparation of a medicament. Preferably, the medicament is used to prevent, treat or improve diseases or disorders associated with human interleukin-4 receptor (IL-4R) or with human interleukin-4 (IL-4) or human interleukin-13 (IL-13) signaling pathways.

[0085] On the other hand, the present invention also provides a method for preventing, treating, or improving a disease or disorder, the method comprising administering (e.g., a therapeutically effective amount) of an antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, and / or composition of the present invention to a subject in need. Preferably, as described above, the disease or disorder is associated with human interleukin-4 receptor (IL-4R) or with the human interleukin-4 (IL-4) or human interleukin-13 (IL-13) signaling pathway.

[0086] In the above-described uses or methods provided by this invention, the disease or disorder includes autoimmune diseases, allergic diseases, tumors, or cancer.

[0087] Preferably, the disease or disorder includes autoimmune or allergic diseases, such as dermatitis (e.g., atopic dermatitis), asthma (e.g., inflammatory asthma, such as type 2 inflammatory asthma), chronic esophagitis (e.g., eosinophilic esophagitis), eczema, rhinitis (e.g., allergic rhinitis), nasal polyps (e.g., chronic sinusitis with nasal polyps), conjunctivitis, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), inflammatory neuropathy, arthritis (e.g., rheumatoid arthritis), multiple sclerosis, lupus erythematosus, psoriasis, or insulin and non-insulin-dependent diabetes mellitus, as well as other immune or allergy-related diseases.

[0088] Alternatively, the disease or disorder may include tumors or cancers such as colorectal 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, HIV-associated Kaposi's sarcoma, Hodgkin's or non-Hodgkin's lymphoma, prostate cancer, prostate tumors, lymphoma, and pancreatic cancer, etc.

[0089] Preferably, the drug is used to prevent, treat, or improve one or more of the above-mentioned disease types.

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

[0091] Based on the specific epitope in the α chain (IL-4Rα) of human IL-4R provided by this invention, this invention also provides the use of said specific epitope in screening human IL-4R binders or blockers of the human IL-4 / IL-13 signaling pathway, or in evaluating the efficacy of said binders or blockers. As described above, said epitope includes one or more amino acid residues selected from the amino acid sequence shown in SEQ ID NO:1, namely D92, V94, D97, L67, L68, A96, H156, C207, Q63, and L64.

[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 a specific embodiment of the present invention, 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 in the amino acid sequence shown in SEQ ID NO:1: (i) Q63 and L64.

[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] ①L67 and L68;

[0104] ②L67, L68, and A96; or

[0105] ③A96.

[0106] According to a specific embodiment of the present invention, 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 epitopes of the present invention include the following amino acid residues in the amino acid sequence shown in 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] Regarding the application of the aforementioned specific epitopes, this invention provides a method for screening binders that specifically bind to human interleukin-4 receptor (IL-4R) or blockers that specifically block the signaling pathways of human interleukin-4 (IL-4) or human interleukin-13 (IL-13). The screening method includes the following steps: contacting the reagent to be screened with human IL-4R or a portion thereof, and then detecting whether the reagent to be screened binds to the epitope described in this invention. When the reagent to be screened is detected to bind to the epitope described in this invention, it is confirmed as the binder or blocker.

[0121] Alternatively, the present invention also provides a method for evaluating the efficacy of a reagent, the method comprising the following steps: contacting the reagent to be screened with a human interleukin-4 receptor (IL-4R) or a portion thereof, and then detecting whether the reagent to be screened binds to the epitope described in the present invention. When the reagent to be screened is detected to bind to the epitope described in the present invention, it is confirmed as a binder that specifically binds to the human interleukin-4 receptor (IL-4R) or a blocker that specifically blocks the signal transduction 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 described in the present invention.

[0122] In the screening method or efficacy evaluation method provided by this invention, preferably, the reagent to be screened is an antibody, such as a polyclonal antibody or a monoclonal antibody. More preferably, the antibody is obtained by immunizing animals with human IL-4R or a portion thereof as an immunogen.

[0123] In the screening method or efficacy evaluation method, preferably, the detection of whether the reagent to be screened binds to the epitope of the present invention can be performed as follows: by performing site-directed mutagenesis on human IL-4R or a portion thereof, causing mutation of one or more amino acid residues included in the epitope provided by the present invention, comparing the binding activity or affinity of the reagent to be screened with human IL-4R or a portion thereof before and after the mutation, and determining that the reagent binds to the epitope of the present invention when the binding activity or affinity with the mutated human IL-4R or a portion thereof decreases or is even lost.

[0124] Specifically, the screening method or efficacy evaluation method provided by the present invention may include the following steps:

[0125] 1) By performing site-directed mutagenesis on human interleukin-4 receptor (IL-4R) or a portion thereof, one or more amino acid residues included in the epitope provided by the present invention are mutated, thereby obtaining a mutant of human IL-4R or a portion thereof;

[0126] 2) The reagent to be screened was brought into contact with human IL-4R or a portion thereof and mutants of human IL-4R or a portion thereof, and the binding activity or affinity with them was detected respectively;

[0127] 3) Compared to the binding activity or affinity with human IL-4R or a portion thereof, when the binding activity or affinity of the reagent to be screened with mutants of human IL-4R or a portion thereof is significantly reduced or lost, the reagent is determined to bind to the epitope provided by the present invention.

[0128] Those skilled in the art can routinely determine whether the binding activity or affinity has significantly decreased or been lost. For example, when the binding activity or affinity of the screening reagent with a mutant of human IL-4R or a 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 significantly decreased.

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

[0130] Preferably, the portion of human IL-4R used in the method of the present invention is the α chain of human IL-4R, and more preferably human sIL-4Rα. Therefore, it is preferable to use human IL-4Rα and obtain a mutant of human IL-4Rα to perform the method. Further, the method can be performed using human sIL-4Rα, with one or more of the human sIL-4Rα mutant proteins listed in Table 3 below used as mutants in step 1).

[0131] In this invention, human IL-4Rα is described in NCBI reference sequence NP_000409.1; the amino acid sequence of human sIL-4Rα is shown in SEQ ID NO:1; and the amino acid sequence of monkey sIL-4Rα is shown in SEQ ID NO:2.

[0132] Experiments have demonstrated that the binding epitope of the antibody or its antigen-binding fragment provided by this invention to the human interleukin-4 receptor (IL-4R) is located on the α chain of human IL-4R (IL-4Rα), and the epitope includes one or more amino acid residues selected from 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 was limited research on the epitopes of sIL-4Rα in immune responses, while this invention is the first to clearly identify the key binding site between the anti-IL-4R antibody and IL-4R.

[0133] Therefore, this invention provides antibodies against human IL-4R, particularly IL-4Rα, possessing novel IL-4R binding epitopes, which can serve as novel and effective antibodies against human IL-4R, particularly IL-4Rα. Furthermore, by elucidating the key binding sites of the antibodies against human IL-4Rα, this invention provides another avenue for developing potential antibodies or drugs against IL-4R, particularly human IL-4Rα. Attached Figure Description

[0134] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0135] Figure 1 The sequence alignment results of human IL-4Rα (sequence 1; SEQ ID NO:1) and monkey IL-4Rα (sequence 0; SEQ ID NO:2) are shown.

[0136] Figure 2 The stability of the antibody of the present invention is shown, wherein Figure 2 A represents the result after being placed at 40℃ for 2 weeks. Figure 2 B represents the results after being placed at 40℃ for 4 weeks.

[0137] The best way to implement an invention

[0138] In this invention, the term "epitope" refers to the antigenic amino acid residue at the binding site when an antibody or fragment binds to an antigen and its corresponding position in the antigen sequence; and, as measured by the procedure described in "(I) Binding Activity Detection Method" below, mutation of the amino acid residue at the epitope position will result in a decrease in binding activity greater than 10%. 2 ng / ml or loss of binding activity.

[0139] The present invention relates to the following experimental procedures or definitions. It should be noted that the present invention can also be implemented using other conventional techniques in the art, and is not limited to the following experimental procedures.

[0140] (I) Combining activity detection methods

[0141] 1. Preparation of reagents

[0142] Antigen preparation: The antigen was prepared into a 100ug / ml solution using PBS, aliquoted, and stored at -20℃.

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

[0144] Preparation of secondary antibody working solution: Dilute the secondary antibody stock solution (Goat Anti-Human IgG-Fc Secondary Antibody (HRP), sino biological, Cat Number: SSA001) 16000 times with PBS containing 1% BSA to obtain the working solution. For example, add 1 μL of the secondary antibody stock solution to 15999 μL of PBS containing 1% BSA and mix thoroughly by repeatedly inverting the container. The solution can be scaled up proportionally according to experimental needs to prepare the required volume of secondary antibody working solution.

[0145] 2. ELISA testing

[0146] Take 50 μL of 100 μg / ml antigen solution and add it to 9.95 ml of PBS. Mix thoroughly by inverting the container to obtain the 0.5 μg / ml antigen coating solution. Add the prepared antigen coating solution to the sample well and use a 12-channel pipette (RAININ) to add 100 μL to each well of a 96-well microplate (Corning). Wrap the 96-well microplate with plastic wrap (or cover it) and incubate overnight at 4°C. The next day, remove the 96-well microplate, discard the solution, and gently pat dry on a clean paper towel. Add 300 μL of PBS to each well of the 96-well microplate, row by row. Incubate at room temperature for 3 minutes, discard the solution, and gently pat dry on a clean paper towel. Repeat the washing process 3 times. Add 300 μL of PBS containing 2% BSA to each well of the 96-well microplate, row by row. Wrap the ELISA plate with plastic wrap (or cover it) and incubate at 37°C for 2 hours. Remove the 96-well ELISA plate, discard the solution, and gently pat dry on a clean paper towel. Add 300 μL of PBS containing 0.05% Tween-20 (Beyotime, Cat Number: ST825) to each well of the 96-well ELISA plate, row by row. Incubate at room temperature for 3 minutes, discard the solution, and gently pat dry on a clean paper towel. Repeat the washing process 3 times.

[0147] Add the diluted antibody to the corresponding wells sequentially, with three replicates per sample, 100 μL per well. Wrap the ELISA plate with plastic wrap (or cover it) and incubate at 37°C for 1 hour. Remove the 96-well ELISA plate, discard the solution, and gently pat dry on a clean paper towel. Add 300 μL of PBS containing 0.05% Tween-20 to each well of the 96-well ELISA plate, row by row. Incubate at room temperature for 3 minutes, discard the solution, and gently pat dry on a clean paper towel. Repeat the washing process three times. Add 100 μL of secondary antibody working solution to each well of the 96-well ELISA plate, row by row. Wrap the ELISA plate with plastic wrap (or cover it) and incubate at 37°C for 1 hour. Remove the 96-well ELISA plate, discard the solution, and gently pat dry on a clean paper towel. Add 300 μL of PBS containing 0.05% Tween-20 to each well of a 96-well microplate, row by row. Incubate at room temperature for 3 minutes, discard the solution, and gently pat dry on a clean paper towel. Repeat washing 3 times. Add 100 μL of TMB solution (SurModics, Cat Number: TMDS-1000-01) to each well of a 96-well microplate, row by row. Incubate at 37°C for 5 minutes, then immediately add 2M H2SO4 solution to the 96-well microplate to stop the reaction. Place the 96-well microplate in a FlexStation 3 (Molecular Devices) and read the OD450 value. Collect, calculate, and analyze the data.

[0148] (II) Methods for Detecting Cell Functional Activity

[0149] 1. Preparation of reagents

[0150] Preparation of human IL-4 (Invivogen, catalog number: rhIL-4): Prepare a 100ug / ml solution of human IL-4 using PBS, aliquot and store at -20°C.

[0151] Preparation of human IL-13 (Invivogen, catalog number: rhIL-13): Prepare a 100ug / ml solution of human IL-13 using PBS, aliquot and store at -20°C.

[0152] Preparation of Quanti-blue(I) solution: Pour one packet of Quanti-blue powder into a sterile 250mL bottle, add 100mL of sterile water, mix gently, incubate at 37℃ for 30min, and after complete dissolution, place in a 4℃ refrigerator protected from light overnight. Then dispense into 10mL vials and store at -20℃ protected from light. It can be stored for 6 months.

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

[0154] 2. Cell Culture

[0155] Retrieve frozen HEK Blue IL-4 / IL13 (Invivogen, Cat No. hkb-il413) cells from liquid nitrogen and gently vortex continuously in a 37°C water bath to rapidly thaw them. Transfer the thawed cell suspension to a 15ml centrifuge tube, add DMEM medium (containing 10% FBS, 10μg / ml Blasticidin, 100μg / ml Zeocin, 100μg / ml Normocin) to a final volume of 10ml, centrifuge at 800rpm for 5min, aspirate the supernatant, retain the cell pellet, wash once more, add 10ml of DMEM medium, and adjust the cell density to 1×10⁶ cells / mL. 5 -1×10 6 Cells were cultured at a density of 10 cells / ml and transferred to T75 cell culture flasks (Nunc). The flasks were then incubated at 37°C in a 5% CO2 incubator (Thermo). Every 2-3 days, the cell suspension was collected, centrifuged at 800 rpm for 5 min, resuspended in 10 ml of culture medium, and the cells were counted at 1 × 10⁻⁶ cells / ml. 6 Each cell was transferred into a new T75 cell culture flask, and culture medium was added to 10 ml. The cells were passaged 2-3 times until they were in good condition (clear cells, single adherent spindle-shaped cells) before experiments could be performed.

[0156] 3. Blocking test

[0157] Collect healthy T75 cells from a cell culture flask, discard the supernatant, gently pipette the adherent cells off the flask with 10 ml of PBS, transfer to a 15 ml centrifuge tube, centrifuge at 800 rpm for 5 min, collect the cell pellet, resuspend the cells in 10 ml of PBS, centrifuge at 800 rpm for 5 min, discard the supernatant, and collect the cell pellet. Resuspend the cells in 5 ml of DMEM medium containing 10% FBS, count the cells, add culture medium, and adjust the cell density to 6.6 × 10⁶ cells / mL. 5Cells / ml. Add 30 μL of cell suspension to each well of a 384-well plate. Add 10 μL of prepared antibody at different concentrations to each well of the 384-well plate (3 replicates). Dilute human IL-4 or human IL-13 to 2.5 ng / ml with DMEM medium containing 10% FBS. Add 10 μL of 2.5 ng / ml human IL-4 or human IL-13 to each well of the corresponding cell well in the 384-well plate, so that the final cell count in each well is 2 × 10⁶ cells / ml. 4 The final concentration of human IL-4 or human IL-13 was 0.5 ng / ml, and the final volume of each well in the 384-well plate was 50 μl. The 384-well cell culture plates were placed in a 37°C, 5% CO2 incubator for static culture.

[0158] 4. Data Statistics

[0159] After incubating the 384-well cell culture plates statically in a 5% CO2 incubator for 22 hours, 45 μL of Quanti-Blue reagent was added to each well of a new 384-well plate. 5 μL of supernatant from each well of the previously incubated 22-hour plate was added to the corresponding wells of the new 384-well plate. The plates were incubated at 37°C for 60 minutes. The 384-well plates were then placed in a FlexStation III incubator, and the OD650 values ​​were read. Data were collected, calculated, and analyzed.

[0160] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0161] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the medicinal materials and reagents used in the following examples are commercially available products. 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 mouse anti-antibody, chimeric antibody, and humanized antibody

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

[0166] The supernatants from the fusion cells were subjected to the same binding activity and cellular functional activity tests as described in (I) and (II) above. Based on the combined results of these two tests, the parent clone with the highest activity was selected for subcloning. After further screening using (I) binding activity and (II) cellular functional activity tests, 14 monoclonal mouse antibodies were finally obtained. Sequencing of these 14 monoclonal antibodies revealed 9 pairs of novel sequences. The corresponding heavy chain variable regions and light chain variable regions are shown below; the CDRs defined using the 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)

[0169] EVQLVESGGGLVQPKGSLKLSCAASGFTFN TYGMH WVRQAPGKGLEWVA HIRSKSSNYATYYADSVKD RFTISRDDSQSMLYLQMNNLKTEDTAMYYCVR WFRAMDY WGQGTSVTVSS

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

[0171] DIVMTQSHKFMSTSVGDRVSITC KASQDVSTAVA WYQEKPGQSPKLLIY WASTRHT GVPDRFTGSGSGTDYTLTISSVQAEDLALYYC QQHYSTPLT FGAGTKLELKY0188-2

[0172] Heavy chain variable region (SEQ ID NO:5; CDRs are: SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40)

[0173] EVQLIESGGGLVQPKGSLKLSCAASGFTFN MYAMD WVRQAPGKGLEWVA RIRSKGSNFETNYADSVKD RFTISRDDSQSMVYLQMINLKTEDTAMYYCVR HRGGAWFAY WGQGTLVSVSA

[0174] Light chain variable region (SEQ ID NO:6; CDRs are: SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58)

[0175] DIVVTQSPASLAVSLGQRATISC RASKSVSTSGYSYMH WYQQKPGQPPKLLIY LASNLES GVPARFSGSGSGTDFTLNIHPVEEEDVAIYYC QHSRELPLT FGAGTKLELK Y0188-3

[0176] Heavy chain variable region (SEQ ID NO:7; CDRs are: SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43)

[0177] QVQLVETGGGLVRPGNSLKLSCVTSGFTFS NYRMH WLRQPPGKRLEWIA VITVKSNNYGANYAESVKG RFAISRDDSKSSVYLEMNRLREEDTATYFCSR ERAYGNPFDY WGQGTTLTVSS

[0178] Light chain variable region (SEQ ID NO:8; CDRs are: SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61)

[0179] DIQMTQSPSSLSASLGERVSLTC RASQEISGYLS WLQQKPDGTIKRLIY AASTLD SGVPKRFSGSRSGSDYSLTISSLESEDFADYYC LQYGSYPYT FGGGTKLEIKY0188-4

[0180] Heavy chain variable region (SEQ ID NO:9; CDRs are: SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46)

[0181] EVQLVESGGGLVQPKGSLKLSCAASGFTFN MYAMN WVRQAPGQGLEWVA RIRSKSNNYATYYADSVKD RFIISRDDSESMVYLQMSNLRAADTAMYYCVR HLRAMDY WGQGTSVTVSS

[0182] Light chain variable region (SEQ ID NO:10; CDRs are: SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:62)

[0183] DIVLTQSPASLTVSLGQRATISC RASKSVSTSGYSYMH WYQQKPGQPPKLLIY LASNLES GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHSRELPIT FGSGTKLEIK Y0188-6

[0184] Heavy chain variable region (SEQ ID NO:11; CDRs are: SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:46)

[0185] EVQLVESGGGLVQPKGSLKLSCAASGFSFN MYAMN WVRQAPGKGLEWVA RIRTKSNHYSTYYADSVKD RFTISRDDSASMFYLQMNNLKTEDTAMYFCVR HLRAMDY WGQGTSVTVSS

[0186] Light chain variable regions (SEQ ID NO:12; CDRs are: SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65)

[0187] DIVLTQSPASLVVSLGQRATISC RASQSVSTSGYSYMH WYQQKPGQPPKLLIY LASNVQS GVPARFSGSGSGTDFTLNIHPVEEEDVATYYC HHNRDLPFT FGSGTKLEIK

[0188] Y0188-8

[0189] Heavy chain variable region (SEQ ID NO:13; CDRs are: SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40)

[0190] EVQLIESGGGLVQPKGSLKLSCAASGFTFN MYAMD WVRQAPGKGLEWVA RIRSKGSNFETNYADSVKD RFTISRDDSQSMVYLQMNNLKTEDTAMYYCVR HRGGAWFAY WGQGTLVTVSA

[0191] Light chain variable region (SEQ ID NO:14; CDRs are: SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58)

[0192] DIVVTQSPASLAVSLGQRATISC RASKSVSTSGYSYMH WYQQKPGQPPKLLIY LASNLES GVPARFSGSGSGTDFTLNIHPVEEEDVAIYYC QHSRELPLT FGAGTKLELK

[0193] Y0188-9

[0194] Heavy chain variable region (SEQ ID NO:15; CDRs are: SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:46)

[0195] EVQLVESGGGLVRPKGSLKLSCAASGFSFN TYAMN WVRQAPGKGLEWIV WIRSKSHNYATYYADSVKD RFTISRDDSESMLYLQMNNLKTEDTAMYYCVR HLRAMDY WGQGTSVTVSS

[0196] Light chain variable region (SEQ ID NO:16; CDRs are: SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68)

[0197] DIVLTQSPASLAVSLGQRATISC RASKSVSASGYSYMH WYQQKPGQPPKLLIY LASNLQS GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHSRELPPT FGGGTKLEIK

[0198] Y0188-10

[0199] Heavy chain variable region (SEQ ID NO:17; CDRs are: SEQ ID NO:44, SEQ ID NO:50, SEQ ID NO:51)

[0200] EVRLVESGGGLVQPKGSLKLSCEASGFSFN MYAMN WVRQAPGKGLEWIT HIRSKSNNYATYYADSVKD RFIISRDDSESMVYLQMNNLKTEDTAMYYCVR LLRALDY WGQGTSVTVSS

[0201] Light chain variable region (SEQ ID NO:18; CDRs are: SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:69)

[0202] DIVLTQSPASLAVFLGQRATISC RASKSVSTSGYSYMH WYQQKAGQPPKLLIY LASNLES GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC HHSRELPIT FGSGTKLEMK

[0203] Y0188-14

[0204] Heavy chain variable region (SEQ ID NO:19; CDRs are: SEQ ID NO:52, SEQ ID NO:36, SEQ ID NO:37)

[0205] EVQLVESGGGLVQPKGSLKLSCAASGFTFN MYGMH WVRQAPGKGLEWVA HIRSKSSNYATYYADSVKD RLTISRDDSQSMLYLQMNNLKTEDTAMYYCVR WFRAMDY WGQGTSVTVSS

[0206] Light chain variable regions (SEQ ID NO:20; CDRs are: SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55)

[0207] DIVMTQSHKFMSTSVGDRVSITC KASQDVSTAVA WYQEKPGQSPKLLIY WASTRHT GVPDRFTGSGSGTDYTLTISSVQAEDLALYYC QQHYSTPLT FGAGTKLELK

[0208] Nine chimeric antibodies were further constructed based on the above variable region sequence. The sequence shown in SEQ ID NO:71 was used as the coding sequence for the heavy chain constant region, and the sequence shown in SEQ ID NO:72 was used as the coding sequence for the light chain constant region. These nine chimeric antibodies were expressed and purified, and named with the mouse antibody name plus Q. The IC50 of the chimeric antibodies was detected using the operation procedure described in "(II) Cellular Functional Activity Detection Method" above. The affinity KD between the chimeric antibodies and human sIL-4Rα was detected using Biacore. The results are shown in Table 1 below.

[0209] Table 1. Results of cellular functional activity and affinity assays for chimeric antibodies.

[0210]

[0211]

[0212] The chimeric antibody of clone 14 (Y0188-14Q) was selected for humanization, resulting in 7 humanized heavy chain variable regions and 7 humanized light chain variable regions. The humanized heavy chain and light chain variable regions are shown below; the CDRs defined using the IMGT tool are shown underlined.

[0213] Heavy chain variable region:

[0214] >HV3-15-14H (SEQ ID NO:21; CDRs are: SEQ ID NO:52, SEQ ID NO:36, SEQ ID NO:37)

[0215] EVQLVESGGGLVKPGGSLRLSCAASGFTFS MYGMH WVRQAPGKGLEWVG HIRSKSSNYATYYADSVKD RFTISRDDSKNTLYLQMNSLKTEDTAVYYCTT WFRAMDY WGQGTLVTVSS

[0216] >HV3-48-14H (SEQ ID NO:22; CDRs are: SEQ ID NO:52, SEQ ID NO:36, SEQ ID NO:37)

[0217] EVQLVESGGGLVQPGGSLRLSCAASGFTFS MYGMH WVRQAPGKGLEWVS HIRSKSSNYATYYADSVKD RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR WFRAMDY WGQGTLVTVSS

[0218] >HV3-73*2-14H (SEQ ID NO:23; CDRs are: SEQ ID NO:52, SEQ ID NO:36, SEQ ID NO:37)

[0219] EVQLVESGGGLVQPGGSLKLSCAASGFTFS MYGMH WVRQASGKGLEWVG HIRSKSSNYATYYADSVKD RFTISRDDSKNTAYLQMNSLKTEDTAVYYCTR WFRAMDY WGQGTLVTVSS

[0220] >HV3-72-14H (SEQ ID NO:24; CDRs are: SEQ ID NO:52, SEQ ID NO:36, SEQ ID NO:37)

[0221] EVQLVESGGGLVQPGGSLRLSCAASGFTFS MYGMH WVRQAPGKGLEWVG HIRSKSSNYATYYADSVKD RFTISRDDSKNSLYLQMNSLKTEDTAVYYCAR WFRAMDY WGQGTLVTVSS

[0222] >Y01-14H (SEQ ID NO:25; CDRs are: SEQ ID NO:52, SEQ ID NO:36, SEQ ID NO:37)

[0223] EVQLVESGGGLVQPGGSLRLSCAASGFTFS MYGMH WVRQAPGKGLEWVS HIRSKSSNYATYYADSVKD RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR WFRAMDY WGQGTLVTVSS

[0224] >162-14H (SEQ ID NO:26; CDRs are: SEQ ID NO:52, SEQ ID NO:36, SEQ ID NO:37)

[0225] EVQLVESGGGLEQPGGSLRLSCAGSGFTFR MYGMH WVRQAPGKGLEWVS HIRSKSSNYATYYADSVKD RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK WFRAMDY WGQGTTVTVSS

[0226] >VH73-14H (SEQ ID NO:27; CDRs are: SEQ ID NO:52, SEQ ID NO:36, SEQ ID NO:37)

[0227] EVQLVESGGGLVQPGGSLKLSCAASGFTFS MYGMH WVRQASGKGLEWVG HIRSKSSNYATYYADSVKD RFTISRDDSKNTAYLQMNSLKTEDTAVYYCTR WFRAMDY WGQGTTVTVSS

[0228] Light chain variable region:

[0229] >Y01-14L (SEQ ID NO:28; CDRs are: SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55)

[0230] EIVLTQSPGTLSLSPGERATLSC KASQDVSTAVA WYQQKPGQAPRLLIY WASTRHT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQHYSTPLT FGQGTKVEIK

[0231] >164-14L (SEQ ID NO:29; CDRs are: SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55)

[0232] DIVMTQSPLSLPVTPGEPASISC KASQDVSTAVA WYLQKSGQSPQLLIY WASTRHT GVPDRFSGSGSGTDFTLKISRVEAEDVGFYYC QQHYSTPLT FGQGTKLEIK

[0233] >KV4-14L (SEQ ID NO:30; CDRs are: SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55)

[0234] DIVMTQSPDSLAVSLGERATINC KASQDVSTAVA WYQQKPGQPPKLLIY WASTRHT GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQHYSTPLT FGGGTKVEIK

[0235] >KV1-27-14L (SEQ ID NO:31; CDRs are: SEQ ID NO:53, SEQ ID NO:54, SEQ IDNO:55)

[0236] DIQMTQSPSSLSASVGDRVTITC KASQDVSTAVA WYQQKPGKVPKLLIY WASTRHT GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QQHYSTPLT FGGGTKVEIK

[0237] >KV1-9-14L (SEQ ID NO:32; CDRs are: SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55)

[0238] DIQLTQSPSFLSASVGDRVTITC KASQDVSTAVA WYQQKPGKAPKLLIY WASTRHT GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC QQHYSTPLT FGGGTKVEIK

[0239] >KV1-NL1-14L (SEQ ID NO:33; CDRs are: SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55)

[0240] DIQMTQSPSSLSASVGDRVTITC KASQDVSTAVA WYQQKPGKAPKLLLY WASTRHT GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQHYSTPLT FGGGTKVEIK

[0241] >KV1D-43-14L (SEQ ID NO:34; CDRs are: SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55)

[0242] AIRMTQSPFSLSASVGDRVTITC KASQDVSTAVA WYQQKPAKAPKLFIY WASTRHT GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQHYSTPLT FGGGTKVEIK

[0243] The above-mentioned humanized light and heavy chain sequences were paired together, and the sequence shown in SEQ ID NO:71 was used as the coding sequence for the heavy chain constant region, and the sequence shown in SEQ ID NO:72 was used as the coding sequence for the light chain constant region. The IC50 of the 49 combined antibodies were then detected using the same procedure described in "(II) Cell Functional Activity Detection Method" above. Cell functional activity (KD) was further verified using Biacore, and finally 6 combinations were selected, as shown in Table 2.

[0244] Table 2. Results of cellular functional activity assays for humanized antibodies

[0245]

[0246] Example 2 Combined with epitope research

[0247] 2.1 Species differences in antibody binding to human and monkey IL-4Rα

[0248] Using monkey sIL-4Rα (EHH60265.1, Met1-Arg232; SEQ ID NO:2) as the antigen, the binding activity between the antibody and the antigen was detected using the procedure described in "(I) Binding Activity Detection Method" above.

[0249] 2.2 Selection of mutation sites in antigen sequences

[0250] Sequence alignment of human and monkey IL-4Rα was performed, see [link / reference]. Figure 1 It is hypothesized that the differential sites in the human-monkey IL-4Rα sequence may be key sites for antibody binding to IL-4Rα, allowing it to bind to the differential human sequence but not the differential monkey sequence. Therefore, single-point mutations or combination mutations were performed on the differential sites in human sIL-4Rα to the corresponding amino acids in monkey sIL-4Rα. Furthermore, non-alanine amino acids near the differential sites in human-monkey sIL-4Rα were selectively mutated to alanine using single-point mutations or combination mutations. Human sIL-4Rα mutants with different mutation combinations were then constructed (see Table 3 below) and verified using cellular functional activity experiments.

[0251] Table 3. Human sIL-4Rα mutant protein

[0252]

[0253] 2.3 Combining activity detection

[0254] Using human sIL-4Rα protein and different sIL-4Rα mutant proteins shown in Table 3 as antigens, and six humanized highly active antibodies as primary antibodies, the binding activity between the antibodies and antigens was detected using the procedures described in "(I) Binding Activity Detection Method" above. The results are shown in Table 4.

[0255] Similarly, using human sIL-4Rα protein and the different sIL-4Rα mutant proteins shown in Table 3 as antigens, and the nine chimeric antibodies obtained through screening as primary antibodies, the binding activity of the antibodies to the antigens was also tested. The results are shown in Table 5.

[0256] Through screening, it was found that the binding activity of the antibody to the sIL-4Rα mutant protein at multiple human-monkey differential sites and nearby sites changed or even reversed.

[0257]

[0258]

[0259] As shown in Table 4, regarding the five sites L67, L68, A96, H156, and C207, after mutating any one of the amino acid residues to the corresponding amino acid in monkey IL-4Rα, when the combination of mutation sites includes L67 and L68, the binding activity of the humanized antibody of the present invention is significantly reduced; while when only L67 and L68 are mutated individually, the humanized antibody of the present invention maintains its original binding activity.

[0260] Regarding the three sites D92, V94, and D97, after mutating any of the amino acid residues to alanine, the binding activity of the humanized antibody of the present invention is significantly reduced when the combination of mutation sites includes D97 or only mutates D97.

[0261]

[0262]

[0263] As shown in Table 5, regarding the D92, V94, and D97 sites, mutating any one of these amino acid residues to alanine significantly reduced the binding activity of chimeric antibodies, except for Y0188-2Q and Y0188-8Q, when the combination of mutation sites included D97 or only D97 was mutated. This is consistent with the binding results obtained from humanized antibodies mentioned above.

[0264] Regarding the five sites L67, L68, A96, H156, and C207, mutating any one of these amino acid residues to the corresponding amino acid in monkey IL-4Rα significantly reduced the binding activity of chimeric antibodies Y0188-1Q, Y0188-3Q, and Y0188-10Q when the mutation site combination included L67 and L68 or L67, L68, and A96. However, when only L67 and L68 were mutated, all chimeric antibodies except Y0188-2Q and Y0188-8Q maintained their original binding activity. Furthermore, regarding A96, the binding activity of chimeric antibody Y0188-3Q was significantly reduced when the mutation site combination included A96.

[0265] Regarding the three sites Q63 and L64, mutating both to alanine significantly reduced or even eliminated the binding activity of chimeric antibodies, except for Y0188-1Q, Y0188-2Q, and Y0188-8Q.

[0266] Example 3 antibody stability

[0267] Antibodies E5 and B5 were prepared at concentrations of 0.59 mg / ml, 0.54 mg / ml, and 6.44 mg / ml, respectively, in 10 mM Histidine, 150 mM NaCl, and pH 6. After incubation at 40℃ for 2 weeks and 4 weeks, the assay was performed using the method described in "(II) Cell Functional Activity Detection Method" above. The relationship between antibody concentration and OD650 was obtained as shown in the figure. Figure 2 Then the IC50 (ng / mL) was calculated, as shown in Table 6 below.

[0268] Table 6. Antibody stability results

[0269]

[0270] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to human interleukin-4 receptor (IL-4R), wherein the epitope to which the antibody or antigen-binding fragment binds is located on the α chain of human IL-4R (IL-4Rα), and the epitope comprises one or more amino acid residues selected from the amino acid sequence shown in SEQ ID NO:1, namely D92, V94, D97, L67, L68, A96, H156, C207, Q63, and L64.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, 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; more preferably, the epitope includes the following amino acid residues in the amino acid sequence shown in SEQ ID NO:1: (1)D92； (2)D97； (3) D92, V94 and D97; (4) D92 and V94; (5) D92 and D97; or (6) V94 and D97; Preferably, the binding epitope of the antibody or its antigen-binding fragment to human IL-4Rα includes the following amino acid residues in the amino acid sequence shown in SEQ ID NO:1: (i) Q63 and L64.

3. The antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The antibody or its antigen-binding fragment does not have species cross-binding activity with monkey IL-4R; preferably, it does not bind to monkey IL-4R; more preferably, it does not bind to monkey IL-4Rα; and even more preferably, it does not bind to the amino acid sequence shown in SEQ ID NO:

2. Preferably, the epitope includes one or more of amino acid residues L67, L68, and A96 in the amino acid sequence shown in SEQ ID NO:1; preferably, it includes: ①L67 and L68; ②L67, L68, and A96; or ③A96； More preferably, the epitope includes the following amino acid residues in the amino acid sequence shown in SEQ ID NO:1: (A)L67, L68, A96, H156 and C207; (B)A96, H156 and C207; (C)L67, L68, H156 and C207; (D)L67, L68, A96, and C207; or (E)L67, L68, A96 and H156.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The epitope includes the following amino acid residues in the amino acid sequence shown in SEQ ID NO:1: (P-1)D97, L67 and L68; (P-2)D97, L67, L68, A96 and D92; (P-3)D97, L67, L68 and D92; (P-4)D97, Q63 and L64; (P-5)D97, Q63, L64 and D92; (P-6)D97, Q63, L64, D92, and A96; or (P-7)D97, Q63, L64, L67 and L68.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that, The antibody or its antigen-binding fragment includes 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: (1) HCDR1, HCDR2, HCDR3 shown in SEQ ID NO:35, 36, 37 in sequence; and LCDR1, LCDR2, LCDR3 shown in SEQ ID NO:53, 54, 55 in sequence; (2) HCDR1, HCDR2, and HCDR3, which are shown in SEQ ID NO:41, 42, and 43 respectively; and LCDR1, LCDR2, and LCDR3, which are shown in SEQ ID NO:59, 60, and 61 respectively; (3) HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO:44, 45, and 46 respectively; and LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO:56, 57, and 62 respectively; (4) HCDR1, HCDR2, HCDR3 shown in SEQ ID NO:44, 47, 46 in sequence; and LCDR1, LCDR2, LCDR3 shown in SEQ ID NO:63, 64, 65 in sequence; (5) HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO:48, 49, and 46 respectively; and LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO:66, 67, and 68 respectively; (6) HCDR1, HCDR2, and HCDR3, sequentially shown in SEQ ID NO:44, 50, and 51; and LCDR1, LCDR2, and LCDR3, sequentially shown in SEQ ID NO:56, 57, and 69; or (7) HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO:52, 36, and 37 respectively; and LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO:53, 54, and 55 respectively; Preferably, the heavy chain variable region comprises a sequence selected from: 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 with said amino acid sequence; and / or, the light chain variable region comprises a sequence selected from: Amino acid sequences shown 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 amino acid sequences having at least 75% identity with said amino acid sequences.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, characterized in that, In the antibody or its antigen-binding fragment: (1) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:3; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:

4. (2) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:7; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:

8. (3) The heavy chain variable region comprises an amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:9; and the light chain variable region comprises an amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:

10. (4) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:11; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:12 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:

12. (5) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:15; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:16 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:

16. (6) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:17 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:17; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:18 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:

18. (7) The heavy chain variable region comprises an amino acid sequence shown in SEQ ID NO:19 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:19; and the light chain variable region comprises an amino acid sequence shown in SEQ ID NO:20 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:

20. (8) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:27; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:34 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:

34. (9) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:27; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:

33. (10) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:27; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:28 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:

28. (11) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:23; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:34 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:

34. (12) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:23; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:33; or (13) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:23; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:28 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:

28.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that, The antibody or its antigen-binding fragment binds to human interleukin-4 receptor (IL-4R), preferably to the α chain of human IL-4R (IL-4Rα), and more preferably to soluble human IL-4Rα. Preferably, the antibody is 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 that can specifically bind to IL-4R or IL-4Rα.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, characterized in that, The antibody or its antigen-binding fragment further comprises a human or mouse constant region, preferably a human or mouse heavy chain constant region (CH) and / or a light chain constant region (CL). Preferably, the antibody molecule or its antigen-binding fragment comprises a heavy chain and a light chain; Preferably, the antibody is an immunoglobulin, specifically IgA, IgD, IgE, IgG or IgM, such as human subtypes of IgA, IgD, IgE, IgG or IgM, more preferably human IgG1, IgG2, IgG3 or IgG4 subtypes; More preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a κ or λ type light chain constant region, for example, the heavy chain constant region is of the IgG type (such as IgG1 or IgG4) and the light chain constant region is of the κ type.

9. A nucleic acid molecule comprising a nucleotide sequence encoding VH, VL, CH and / or CL in an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

10. A vector comprising the nucleic acid molecule of claim 9.

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