HK40045558A - Human il-4r binding antibody, antigen binding fragment thereof, and medical use thereof - Google Patents

Description

Antibodies that bind human IL-4R, antigen-binding fragments thereof, and medical uses thereof

The application claims the priority of Chinese application CN201810971269.2 submitted in 24/08/2018, Chinese application CN201811472752.2 submitted in 04/12/2018, Chinese application CN201910221311.3 submitted in 22/03/2019 and Chinese application CN201910401923.0 submitted in 15/05/2019. Each of the foregoing applications is hereby incorporated by reference in its entirety.

Technical Field

The present disclosure relates to antibodies that bind to human IL-4R, and antigen-binding fragments thereof, and to chimeric or humanized antibodies comprising CDR regions from the antibodies, as well as pharmaceutical compositions comprising antibodies that bind to human IL-4R and antigen-binding fragments thereof, and their use as medicaments for treating autoimmune diseases.

Background

Allergic diseases are serious medical conditions, including: non-life threatening allergic reactions that can heal over a period of time; and, allergic diseases that can be life threatening.

Current methods of treating allergy include allergen avoidance, pharmacological treatment for symptoms, and prophylaxis with allergen-specific immunotherapy.

Interleukin-4 (IL-4, also known as B cell stimulating factor or BSF-1), is characterized by its ability to stimulate B cell proliferation in response to low concentrations of anti-surface immunoglobulin antibodies. IL-4 has been shown to have a wide range of biological activities, including growth stimulation of T cells, mast cells, granulocytes, megakaryocytes, and erythrocytes, among others. IL-4 induces MHC-II expression in resting B cells and enhances secretion of immunoglobulins IgE and IgG1 by activated B cells.

The biological activity of IL-4 is mediated by specific cell surface IL-4 receptors (IL-4R). The IL-4 receptor (IL-4R) consists of 802 amino acid residues, and IL-4R is expressed on the surface of T cells, B cells, thymocytes, bone marrow cells, macrophages, and mast cells. The alpha chain of IL-4R is also a component of the IL-13 receptor (IL-13R), and thus IL-4R may also mediate the biological activity of IL-13. Medicaments containing IL-4R antagonists and compositions thereof may be administered as a novel treatment before, during or after exposure of a subject to an allergen or the appearance of an allergic condition.

Currently, various pharmaceutical companies in various countries are developing monoclonal antibodies against IL-4R for treating various allergic diseases related to the monoclonal antibodies. Related patent applications are WO2010053751, WO2001092340, WO2008054606, WO2014031610, etc. Among them, Dupilumab (Dupilumab), an asthma drug developed by Regeneron, is already marketed for dermatitis indications, and for asthma indications, phase III clinical trials have been completed and the procedure is entered into the marketing approval stage.

The existing treatment strategies have poor curative effect or have larger risks. Accordingly, there remains a need in the art to provide highly selective, highly bioactive antibodies that bind to human IL-4R with high affinity and are capable of neutralizing the biological activity of IL-4R (including blocking binding to IL-4R), thereby providing medicaments and compositions thereof, as well as methods of treatment, that are capable of treating IL-4R-mediated allergic diseases.

Disclosure of Invention

In some embodiments herein, antibodies that specifically bind to IL-4R (also known as antibodies that bind to human IL-4R, anti-human IL-4R) or antigen-binding fragments thereof are provided, wherein the heavy chain variable region comprises:

(I) respectively shown in SEQ ID NO: 3. SEQ ID NO: 4 and SEQ ID NO: 5, HCDR1, HCDR2 and HCDR3 or a variant of SEQ ID NO: 3. HCDR1, HCDR2, HCDR3 variants as shown in 4, 5 having 3, 2 or 1 amino acid differences, respectively; or

(II) are shown as SEQ ID NO: 11. SEQ ID NO: 12 and SEQ ID NO: 13 HCDR1, HCDR2 and HCDR3 or a variant of SEQ ID NO: 11. HCDR1, HCDR2, HCDR3 variants as shown in 12, 13 having 3, 2 or 1 amino acid differences, respectively;

and/or a light chain variable region comprising:

(I) respectively shown in SEQ ID NO: 6. SEQ ID NO: 7 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6. LCDR1, LCDR2, LCDR3 variants with 3, 2 or 1 amino acid differences, respectively, as shown in 7, 8; or

(II) are shown as SEQ ID NO: 14. SEQ ID NO: 15 and SEQ ID NO: 16, or LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 14. LCDR1, LCDR2, LCDR3 variants as shown in 15, 16 having 3, 2 or 1 amino acid differences, respectively; or

(III) as shown in SEQ ID NO: 38. SEQ ID NO: 7 and SEQ ID NO: 40 or LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 38. LCDR1, LCDR2, LCDR3 variants with 3, 2 or 1 amino acid differences, respectively, as shown in 7, 40; or

(IV) are shown as SEQ ID NO: 42. SEQ ID NO: 39 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 42. LCDR1, LCDR2, LCDR3 variants with 3, 2 or 1 amino acid differences, respectively, as shown in 39, 8.

In some embodiments, the antibody or antigen-binding fragment thereof that binds human IL-4R comprises any one selected from the group consisting of (I) to (IV):

(I) a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 3. SEQ ID NO: 4 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in fig. 5; and

a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 6. SEQ ID NO: 7 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown in fig. 8;

(II) a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 11. SEQ ID NO: 12 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown at 13; and

a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 14. SEQ ID NO: 15 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown at 16;

(III) a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 3. SEQ ID NO: 4 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in fig. 5; and

a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 38. SEQ ID NO: 7 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown at 40;

(IV) a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 3. SEQ ID NO: 4 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in fig. 5; and

a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 42. SEQ ID NO: 39 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown in fig. 8.

In some embodiments, an antibody or antigen-binding fragment thereof that binds human IL-4R, wherein:

a heavy chain variable region comprising:

(I) as shown in SEQ ID NO: 1 or a sequence corresponding to SEQ ID NO: 1, sequences having at least 70%, 80%, 90%, 95%, 98%, 99% identity; or

(II) as shown in SEQ ID NO: 9 or a sequence corresponding to SEQ ID NO: 9, sequences having at least 70%, 80%, 90%, 95%, 98%, 99% identity; or

(III) as shown in SEQ ID NO: 43 or a sequence identical to SEQ ID NO: 43 sequences having at least 70%, 80%, 90%, 95%, 98%, 99% identity; or

And/or a light chain variable region comprising:

(I) as shown in SEQ ID NO: 2 or a sequence similar to SEQ ID NO: 2, sequences having at least 70%, 80%, 90%, 95%, 98%, 99% identity; or

(II) as shown in SEQ ID NO: 10 or a sequence identical to SEQ ID NO: 10, sequences having at least 70%, 80%, 90%, 95%, 98%, 99% identity; or

(III) as shown in SEQ ID NO: 37 or a sequence identical to SEQ ID NO: 37, sequences having at least 70%, 80%, 90%, 95%, 98%, 99% identity; or

(IV) as shown in SEQ ID NO: 41 or a sequence corresponding to SEQ ID NO: 41 are sequences having at least 70%, 80%, 90%, 95%, 98%, 99% identity.

In at least one embodiment, the antibody or antigen-binding fragment that binds human IL-4R, wherein:

the heavy chain variable region is shown as a sequence SEQ ID NO: 1, and the light chain variable region is shown as a sequence SEQ ID NO: 2 is shown in the specification; or

The heavy chain variable region is shown as a sequence SEQ ID NO: 9, the light chain variable region is shown as a sequence SEQ ID NO: 10 is shown in the figure; or

The heavy chain variable region is shown as a sequence SEQ ID NO: 43, the light chain variable region is shown as sequence SEQ ID NO: 37 is shown in the figure; or

The heavy chain variable region is shown as a sequence SEQ ID NO: 43, the light chain variable region is shown as sequence SEQ ID NO: shown at 41.

In some embodiments, an antibody or antigen-binding fragment thereof that binds human IL-4R, wherein:

a heavy chain variable region comprising:

(I) as shown in SEQ ID NO: 25-27 or a sequence as set forth in one of SEQ ID NOs: 25-27, or a sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical; or

(II) as shown in SEQ ID NO: 31-33 or a sequence as set forth in one of SEQ ID NOs: 31-33, or a sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical;

and/or a light chain variable region comprising:

(I) as shown in SEQ ID NO: 28-30 or a sequence as set forth in any one of SEQ ID NOs: 28-30, or a sequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical; or

(II) as shown in SEQ ID NO: 34-36 or a sequence as set forth in any one of SEQ ID NOs: 34-36 having at least 70%, 80%, 90%, 95%, 98%, or 99% identity.

In some specific embodiments, the heavy chain variable region is as set forth in SEQ ID NO: 25-27 and the light chain variable region is as shown in SEQ ID NO: shown in one of 28-30.

In other specific embodiments, the heavy chain variable region is as set forth in SEQ ID NO: 31-33 and the light chain variable region is as shown in SEQ ID NO: as shown in one of 34-36.

In some embodiments, the heavy chain of an antibody or antigen-binding fragment thereof that binds human IL-4R comprises:

(I) as shown in SEQ ID NO: 17 or a sequence corresponding to SEQ ID NO: 17, a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity; or

(II) as shown in SEQ ID NO: 19 or a sequence corresponding to SEQ ID NO: 19, a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity; or

(III) as shown in SEQ ID NO: 44 or a sequence corresponding to SEQ ID NO: 44 is at least 70%, 80%, 90%, 95%, 98%, or 99% identical.

In some embodiments, a light chain of an antibody or antigen-binding fragment thereof that binds human IL-4R comprises:

(I) as shown in SEQ ID NO: 18 or a sequence corresponding to SEQ ID NO: 18, or a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity; or

(II) as shown in SEQ ID NO: 20 or a sequence corresponding to SEQ ID NO: 20, a sequence having at least 70%, 80%, 90%, 95%, 98%, or 99% identity; or

(III) as shown in SEQ ID NO: 45 or a sequence identical to SEQ ID NO: 45 are at least 90%, 95%, 98%, or 99% identical; or

(IV) as shown in SEQ ID NO: 46 or a sequence corresponding to SEQ ID NO: 46 are at least 90%, 95%, 98% or 99% identical.

In at least one embodiment, the heavy chain is as set forth in sequence SEQ ID NO: 17 and the light chain is as shown in SEQ ID NO: 18, respectively.

In another embodiment, the heavy chain is as set forth in sequence SEQ ID NO: 19 and the light chain is as shown in SEQ ID NO: shown at 20.

In another embodiment, the heavy chain is as set forth in sequence SEQ ID NO: 44 and the light chain is as shown in sequence SEQ ID NO: shown at 45.

In another embodiment, the heavy chain is as set forth in sequence SEQ ID NO: 44 and the light chain is as shown in sequence SEQ ID NO: 46, respectively.

In some embodiments, the anti-IL-4R antibody or antigen binding fragment is a murine antibody, a chimeric antibody, a human antibody, a humanized antibody or a fragment thereof.

In some specific embodiments, the anti-IL-4R antibody or antigen-binding fragment is humanized.

In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a fragment selected from the group consisting of:

-FR region sequences derived from human germline light chain IGKV3-11 x 01;

-a back-mutated sequence derived from an FR region having at least 95% identity to the human germline light chain IGKV3-11 x 01. In some embodiments, the back-mutation is selected from one or a combination of L46P, L47W, F71Y.

In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a fragment selected from the group consisting of:

-FR region sequences derived from human germline heavy chain IGHV3-48 x 01;

-from a back-mutated sequence having at least 95% identity with the FR region of the human germline heavy chain IGHV3-48 x 01. In some embodiments, the back-mutation is selected from one or a combination of S49A, F67S, a 93T.

In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a fragment selected from the group consisting of:

-a FR region sequence derived from human germline light chain IGKV2D-29 x 01;

-a back-mutated sequence derived from an FR region having at least 95% identity to the human germline light chain IGKV2D-29 x 01. In some embodiments, the back-mutation is selected from M4L and/or V58I.

In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof further comprises a fragment selected from the group consisting of:

-FR region sequences derived from human germline heavy chain IGHV1-2 x 02;

-a back-mutated sequence derived from an FR region having at least 95% identity to the human germline heavy chain IGHV1-2 x 02. In some embodiments, the back mutation is selected from one or a combination of M69L, R71I, T73K, R94K.

In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the heavy chain framework regions of human IgG1, IgG2, IgG3, or IgG4, or a variant thereof. In some embodiments, the heavy chain variable region comprises a heavy chain framework region of human IgG1 or a variant thereof, e.g., SEQ ID NO: 43 is a heavy chain variable region or a variant thereof having at least 85% sequence identity thereto.

In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a constant region of a human kappa, lambda chain or variant thereof, e.g., SEQ ID NO: 44 or a light chain variable region variant having at least 85% sequence identity thereto.

In some embodiments, an IL-4R humanized antibody or fragment thereof as described above, further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, or a variant thereof.

In at least one embodiment, the antibody comprises a heavy chain constant region of human IgG2 or IgG 4. Neither IgG2 nor IgG4 had ADCC toxicity. Alternatively, IgG1, which was free of ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation, was used.

In at least one embodiment, the variant comprises a heavy chain constant region mutation selected from the group consisting of: the mutation reduces or eliminates ADCC function, such as, but not limited to, N297A, L234A, L235A of IgG 1.

In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof, wherein the antibody is a humanized antibody having a heavy chain sequence as set forth in SEQ ID NO: 17 or has at least 85% sequence identity thereto; the light chain sequence is shown as SEQ ID NO: 18 or has at least 85% sequence identity thereto.

In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof, wherein the antibody is a humanized antibody having a heavy chain sequence as set forth in SEQ ID NO: 19 or has at least 85% sequence identity thereto; the light chain sequence is shown as SEQ ID NO: 20 or has at least 85% sequence identity thereto.

In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof, wherein the antibody is a humanized antibody having a heavy chain sequence as set forth in SEQ ID NO: 44 or has at least 85% sequence identity thereto; the light chain sequence is shown as SEQ ID NO: 45 or has at least 85% sequence identity thereto.

In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof, wherein the antibody is a humanized antibody having a heavy chain sequence as set forth in SEQ ID NO: 44 or has at least 85% sequence identity thereto; the light chain sequence is shown as SEQ ID NO: 46 or has at least 85% sequence identity thereto.

In some embodiments, an isolated anti-IL-4R antibody or antigen-binding fragment thereof is provided that competes for binding to human IL-4R with any of the antibodies or antigen-binding fragments thereof that bind to human IL-4R as described above.

In some embodiments, a bispecific or multispecific antibody is provided comprising a light chain variable region and/or a heavy chain variable region of an antibody or antigen-binding fragment thereof that binds human IL-4R according to the present application.

In other embodiments, a single chain antibody is provided comprising the light chain variable region and/or the heavy chain variable region of an antibody or antigen-binding fragment thereof that binds human IL-4R according to the present application.

In some embodiments, a polynucleotide is provided that encodes an antibody or antigen-binding fragment thereof that binds human IL-4R according to the present application.

In other embodiments, a polynucleotide is provided that encodes an antibody that competitively binds with an antibody or antigen-binding fragment thereof that binds human IL-4R according to the present application against IL-4R or an epitope thereof.

In other embodiments, a polynucleotide is provided that encodes the aforementioned bispecific antibody, multispecific antibody, or single chain antibody.

In some embodiments, the polynucleotide according to the present application is DNA or RNA.

In some embodiments, there is provided a vector comprising a polynucleotide as described above, which is a eukaryotic expression vector, a prokaryotic expression vector, or a viral vector.

In some embodiments, a host cell transformed with a vector as described above is provided, said host cell being selected from a prokaryotic cell or a eukaryotic cell.

In at least one embodiment, the prokaryotic cell is selected from bacteria, such as E.coli.

In at least one embodiment, the eukaryotic cell is selected from a yeast or a mammalian cell, such as a pichia or CHO cell.

In some embodiments, a method for detecting or determining IL-4R is provided, the method comprising the step of contacting an anti-IL-4R antibody or antigen-binding fragment thereof described above with a sample.

In some embodiments, provided is a reagent for detecting or assaying human IL-4R, the reagent comprising an anti-IL-4R antibody or antigen-binding fragment thereof as described above.

In some embodiments, provided is a reagent for detecting or assaying human IL-4R, the reagent comprising an antibody that competitively binds with an antibody or antigen-binding fragment thereof that binds human IL-4R according to the present application against IL-4R or an epitope thereof.

In some embodiments, provided is an agent for detecting or assaying human IL-4R, said agent comprising a bispecific antibody, a multispecific antibody, a single chain antibody described above.

In some embodiments, a diagnostic agent for diagnosing a disease associated with human IL-4R positive cells is provided, the diagnostic agent comprising an anti-IL-4R antibody or antigen-binding fragment thereof as described above. In other embodiments, the diagnostic agent comprises an antibody that competitively binds with an antibody or antigen-binding fragment thereof that binds human IL-4R according to the present application against IL-4R or an epitope thereof.

In some embodiments, there is provided a pharmaceutical composition comprising:

an antibody or antigen-binding fragment thereof that binds human IL-4R according to the present application, and

-a pharmaceutically acceptable excipient, diluent or carrier.

In some specific embodiments, the unit dose of the pharmaceutical composition contains 1mg to 1000mg of an IL-4R antibody or antigen-binding fragment thereof according to the present application.

In some specific embodiments, the concentration of the IL-4R antibody or antigen-binding fragment thereof in the pharmaceutical composition is from 1mg/L to 1000 mg/L.

In some specific embodiments, the pharmaceutical composition contains a buffer, which may be present in an amount of 1mM to 1000 mM.

In some embodiments, there is provided the use of an antibody or antigen-binding fragment thereof that binds human IL-4R as described above in the manufacture of a medicament for the treatment or prevention of an IL-4R-mediated disease or disorder.

In some embodiments, there is provided a use of a pharmaceutical composition as described above in the manufacture of a medicament for treating or preventing an IL-4R-mediated disease or disorder.

In some embodiments, there is provided a use of an antibody or antigen-binding fragment thereof that binds human IL-4R as described above in the treatment or prevention of a disease or disorder.

In some embodiments, there is provided a use of a pharmaceutical composition as described above in the treatment or prevention of a disease or disorder.

In the context of the present application, the disorder or disease may be an immunological disease or disorder.

In some embodiments, the disease or disorder is selected from: asthma, nasal polyps, chronic sinusitis, allergic skin diseases, eosinophilic esophagitis, chronic obstructive pulmonary disease, allergic rhinitis, arthritis, inflammatory diseases, allergic reactions, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, barrett's esophagus, autoimmune uveitis, tuberculosis, and renal diseases.

In at least one embodiment, the disease or disorder is asthma.

In other embodiments, the disease or disorder is an allergic skin disease.

In some embodiments, an antibody or antigen-binding fragment thereof that binds human IL-4R is provided, wherein the antigen-binding fragment is Fab, Fv, sFv, F (ab')₂。

In some embodiments, there is provided a method of treating and/or preventing an IL-4R mediated disease or disorder, the method comprising: administering to a patient (or subject) in need thereof a therapeutically effective amount (or a prophylactically effective amount) of an antibody that binds human IL-4R or an antigen-binding fragment thereof, as described above.

In some embodiments, there is provided a method of treating and/or preventing an IL-4R mediated disease or disorder, the method comprising: administering to a patient (or subject) in need thereof a therapeutically effective amount (or a prophylactically effective amount) of a pharmaceutical composition as described above. In some embodiments, there is provided a method of treating and/or preventing an immune disease, comprising administering to a patient (or subject) in need thereof a therapeutically effective amount (prophylactically effective amount) of an antibody or antigen-binding fragment thereof that binds human IL-4R, or a pharmaceutical composition thereof, as described above.

Term(s) for

In order that the disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

"human IL-4R" (hIL-4R) means a human cytokine receptor that specifically binds to interleukin-4 (IL-4), IL-4R α.

Amino acid three letter codes and one letter codes as used herein are as described in j.biol.chem, 243, p3558 (1968).

"antibody" refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus, their antigenicity. Accordingly, immunoglobulins can be classified into five classes, otherwise known as the isotype of immunoglobulins, i.e., IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being the μ, δ, γ, α, and ε chains, respectively. The same class of igs can be divided into different subclasses according to differences in amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, and for example, iggs can be classified into IgG1, IgG2, IgG3 and IgG 4. Light chains are classified as either kappa or lambda chains by differences in the constant regions. Each of the five classes of Ig may have either a kappa chain or a lambda chain.

The antibody light chain may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or variant thereof.

The antibody heavy chain may further comprise a heavy chain constant region comprising human or murine IgG1, IgG2, IgG3, IgG4, or variants thereof.

The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, being variable regions (V-regions); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable regions include 3 hypervariable regions (HVRs) and 4 Framework Regions (FRs) which are relatively sequence conserved. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) is composed of 3 CDR regions and 4 FR regions, arranged sequentially from amino terminus to carboxy terminus in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The 3 CDR regions of the light chain refer to light chain complementarity determining region 1(LCDR1), light chain complementarity determining region 2(LCDR2), and light chain complementarity determining region 3(LCDR 3); the 3 CDR regions of the heavy chain refer to heavy chain complementarity determining region 1(HCDR1), heavy chain complementarity determining region 2(HCDR2), and heavy chain complementarity determining region 3(HCDR 3).

Antibodies include murine antibodies, chimeric antibodies, humanized antibodies, human antibodies, which may be recombinantly obtained, e.g., may be affinity matured recombinant human antibodies.

"recombinant human antibodies" include human antibodies made, expressed, created or isolated by recombinant methods, involving techniques and methods well known in the art, such as (1) antibodies isolated from transgenes of human immunoglobulin genes, transchromosomal animals (e.g., mice), or hybridomas made therefrom; (2) antibodies isolated from host cells transformed to express the antibodies, such as transfectomas; (3) antibodies isolated from a library of recombinant combinatorial human antibodies; and (4) antibodies prepared, expressed, created or isolated by splicing human immunoglobulin gene sequences to other DNA sequences, and the like. Such recombinant human antibodies comprise variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations such as occur during antibody maturation.

"murine antibody" is herein a monoclonal antibody to human IL-4R prepared according to the knowledge and skill in the art. Preparation is accomplished by injecting a subject with the IL-4R antigen or a polypeptide comprising an epitope thereof, and then isolating hybridomas that express antibodies having the desired sequence or functional properties. In some embodiments, the murine antibody that binds human IL-4R, or an antigen binding fragment thereof, may further comprise a light chain constant region of a murine kappa, lambda chain, or variant thereof, or further comprise a heavy chain constant region of a murine IgG1, IgG2, IgG3, or IgG4, or variant thereof.

"human antibodies" include antibodies having variable and constant regions of human germline immunoglobulin sequences. The human antibodies herein can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e., "humanized antibodies").

"humanized antibody", also known as CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting CDR sequences of a non-human species into the framework of the variable region of a human antibody. Can overcome the strong immune response induced by the chimeric antibody due to carrying a large amount of heterologous protein components. To avoid a decrease in activity associated with a decrease in immunogenicity, the human antibody variable regions may be subjected to minimal back-mutation to maintain activity.

"chimeric antibody" is an antibody obtained by fusing a variable region of a murine antibody with a constant region of a human antibody, and can reduce an immune response induced by the murine antibody. Establishing a chimeric antibody, selecting and establishing a hybridoma secreting a mouse-derived specific monoclonal antibody, cloning a variable region gene from a mouse hybridoma cell, cloning a constant region gene of a human antibody according to needs, connecting the mouse variable region gene and the human constant region gene into a chimeric gene, inserting the chimeric gene into a human vector, and finally expressing a chimeric antibody molecule in a eukaryotic industrial system or a prokaryotic industrial system. The constant region of the human antibody may be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising human IgG2 or IgG4 heavy chain constant region, or IgG1 which has no ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.

"antigen-binding fragment" refers to Fab fragments, Fab 'fragments, F (ab') 2 fragments, Fv fragments that bind to human IL-4R, scFv fragments, and polypeptides or proteins comprising the foregoing fragments, which have antigen-binding activity. The "antigen-binding fragment" comprises one or more CDR regions of an antibody described herein. The Fv fragment contains the variable regions of the antibody heavy and light chains, but lacks the constant region, and has the smallest antibody fragment with the entire antigen-binding site. Generally, Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding. Two antibody variable regions can also be joined into a single polypeptide chain using different linkers, called single chain antibodies (scFv) or single chain fv (scFv).

"binding to IL-4R" refers to the ability to interact with human IL-4R. The term "antigen binding site" herein refers to a three-dimensional spatial site recognized by an antibody or antigen binding fragment herein.

An "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed from contiguous amino acids, or non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are typically retained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost after denaturing solvent treatment. Epitopes typically comprise at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitope is bound by a given antibody are well known in the art and include immunoblot and immunoprecipitation detection assays, and the like. Methods of determining the spatial conformation of an epitope include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, among others.

"specific binding", "selective binding" refers to binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody is less than about 10 when measured in an instrument by Surface Plasmon Resonance (SPR) techniques using recombinant human IL-4R as the analyte and an antibody as the ligand^-7M or even smaller equilibrium dissociation constant(K _D) Binds to a predetermined antigen and binds to the predetermined antigen with at least twice the affinity as it binds to a non-specific antigen other than the predetermined antigen or closely related antigens (e.g., BSA, etc.). The term "antibody recognizing an antigen" is used interchangeably herein with the term "specifically binding antibody".

"Cross-reactive" refers to the ability of an antibody herein to bind to IL-4R from a different species. For example, an antibody herein that binds human IL-4R may also bind IL-4R of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigens, or binding or functional interactions with cells that physiologically express IL-4R in binding assays (e.g., SPR and ELISA). Methods of determining cross-reactivity include standard binding assays as described herein, such as Surface Plasmon Resonance (SPR) analysis, or flow cytometry.

By "neutralizing" or "blocking" antibody is meant an antibody whose binding to hIL-4R results in inhibition of hIL-4 and/or hIL-13 biological activity. Such inhibition of hIL-4 and/or IL-13 bioactivity can be assessed by measuring one or more hIL-4 and/or hIL-13 bioactivity indicators well known in the art, such as hlL-4 and/or hIL-13-induced cell activation and hIL-4 binding to hIL-4R, for example, in CN 103739711A. "inhibiting growth" (e.g., reference to a cell) is intended to include any measurable decrease in cell growth.

"inducing an immune response" and "enhancing an immune response" are used interchangeably and refer to stimulation (i.e., passive or adaptive) of an immune response to a particular antigen. The term "induce" with respect to induction of CDC or ADCC refers to stimulation of a specific direct cell killing mechanism.

"ADCC (antibody-dependent cell-mediated cytotoxicity)", antibody-dependent cell-mediated cytotoxicity (Fc-mediated cytotoxicity), refers to the direct killing of Fc receptor-expressing cells by recognition of the Fc fragment of an antibody against antibody-coated target cells. The ADCC effector function of an antibody may be reduced or eliminated by modification of the Fc-fragment of the IgG. The modification refers to mutation in the heavy chain constant region of an antibody, such as N297A, L234A, L235A selected from IgG 1; IgG2/4 chimeras, F235E for IgG4, or L234A/E235A mutations.

The fusion protein is a protein product obtained by co-expression of two genes obtained through DNA recombination. Recombinant IL-4R extracellular region Fc fusion protein is a fusion protein which is obtained by coexpressing an IL-4R extracellular region and a human antibody Fc fragment through DNA recombination. The IL-4R extracellular region refers to a part of the IL-4R protein which is expressed outside a cell membrane.

Methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as the antibody test technical guide of cold spring harbor, chapters 5-8 and 15. For example, mice can be immunized with human IL-4R or fragments thereof, and the resulting antibodies can be renatured, purified, and amino acid sequenced using conventional methods. Antigen-binding fragments can likewise be prepared by conventional methods. The antibodies or antigen-binding fragments described herein are genetically engineered to incorporate one or more human FR regions in CDR regions of non-human origin. The human FR germline sequence can be obtained from the website http of imminogenetics (imgt): i/imgt. cines. fr or from the journal of immunoglobulins, 2001ISBN 012441351.

The engineered antibody or antigen-binding fragment can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains may be cloned and recombined into a GS expression vector. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. The sequence of the humanized antibody is inserted into a corresponding expression vector by using a molecular cloning technology, and the corresponding humanized antibody can be obtained by expression and production by using an HEK293 cell expression system. As a more recommended prior art, mammalian expression systems lead to glycosylation of antibodies, particularly at the highly conserved N-terminus of the FC region. Stable clones were obtained by expressing antibodies that specifically bind to antigens of human origin. Positive clones were expanded in bioreactor serum-free medium to produce antibodies. The antibody-secreting culture medium can be purified and collected by conventional techniques. The antibody can be concentrated by filtration by a conventional method. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is either immediately frozen, e.g., -70 ℃, or lyophilized.

The antibody may be a monoclonal antibody (mAb) which refers to an antibody obtained from a single clonal cell line, not limited to eukaryotic, prokaryotic, or phage clonal cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombinant technology, phage display technology, synthetic techniques (e.g., CDR-grafting), or other known techniques.

The antibody may be a monospecific, bispecific or multispecific antibody. Multispecific antibodies may exhibit specificity for different epitopes of a target peptide, or may also comprise antigen binding domains that exhibit specificity for more than one target peptide. The human anti-IL-4R antibody may be linked to, or co-expressed with, another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical ligation, genetic fusion, non-covalent binding, or other means) to one or more other molecules (e.g., another antibody or antigen-binding fragment) to produce a bispecific or multispecific antibody having a second binding specificity.

"administration," "administering," and "treating," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration," "administering," and "treating" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells and contacting the reagent with a fluid, wherein the fluid is in contact with the cells. "administering", "administering" and "treating" also mean treating, for example, a cell in vitro and ex vivo by an agent, a diagnostic, a binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

By "treating" is meant administering a therapeutic agent, such as a composition comprising any of the antibodies or antigenic fragments thereof herein, either internally or externally to a patient (or subject) who has, is suspected of having, or is susceptible to one or more disease symptoms for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in the patient (or subject) or population being treated in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable degree. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the disease state, age, and weight of the patient (or subject), and the ability of the drug to produce a desired therapeutic effect in the patient (or subject). Whether a disease symptom has been reduced can be assessed by any clinical test commonly used by physicians or other health professional to assess the severity or progression of the symptom. Although embodiments of the present invention (e.g., methods of treatment or articles of manufacture) may be ineffective in alleviating symptoms of a target disease in a single patient (or subject), they should alleviate symptoms of the target disease in a statistically significant number of patients (or subjects), as determined by any statistical test method known in the art, such as Student's t-test, chi-square test, U-test by Mann and Whitney, Kruskal-Wallis test (H-test), Jonckhere-Terpstra test, and Wilcoxon test.

"conservative modification" or "conservative substitution" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, and rigidity, etc.) so that changes can be made frequently without changing the biological activity of the protein. It is known to The person skilled in The art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter The biological activity (see, for example, Watson et al (1987) Molecular Biology of The Gene, The Benjamin/Cummings pub. Co., p. 224, (4 th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to abolish biological activity.

An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular subject or veterinary subject may vary depending on the following factors: such as the condition to be treated, the general health of the subject, the method and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

"exogenous" refers to a substance produced outside an organism, cell, or human body depending on the background.

"endogenous" refers to a substance produced in a cell, organism, or human body by background.

"homology" or "identity" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100%. For example, two sequences are 60% homologous if there are 6 matches or homologies at 10 positions in the two sequences when the sequences are optimally aligned. In general, comparisons are made when aligning two sequences to obtain the greatest percentage of homology. As used herein, "at least 85% sequence identity" means that the variant has at least 85% homology, in some embodiments at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology to the parent sequence; in some specific embodiments, it has 90%, 95%, or 99% or more; in other embodiments, it has at least 95% sequence homology. The amino acid sequence having at least 85% sequence identity comprises a sequence obtained by one or more amino acid deletion, insertion or substitution mutations in a parent sequence.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny thereof. Thus, the words "transformant" and "transformed cell" include the primary test cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.

"optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of a particular sequence may, but need not, be present.

"pharmaceutical composition" means a mixture comprising one or more antibodies or antigen-binding fragments described herein, or physiologically/pharmaceutically acceptable salts or prodrugs thereof, in admixture with other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

Drawings

FIG. 1: in a mouse dermatitis model, after acetone sensitization, a humanized antibody hu25G7-A, hu25G7-B and a positive reference antibody doluzumab are administered in a subcutaneous mode: the administration was performed twice a week, and the ear thickness of the mice was measured on day 27. The results show that both hu25G7-A, hu25G7-B and dulucizumab were effective in reducing the thickness of the ears in mice compared to the control group, and hu25G7-B showed superior effects compared to dulucizumab.

Detailed Description

The present disclosure is further described below with reference to examples, but these examples do not limit the scope of the present disclosure. The experimental methods in the examples, in which specific conditions are not specified, are generally carried out according to conventional conditions such as antibody technical laboratory manual of cold spring harbor, molecular cloning manual; or according to the conditions recommended by the manufacturer of the raw material or the goods. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

Example 1: mouse immunization and detection

His-tagged human IL-4R (h-IL-4R-his) recombinant protein, his-tagged mouse IL-4R (m-IL-4R-his) recombinant protein, and his-tagged rhesus IL-4R (rhesus-IL-4R-his) recombinant protein were synthesized by Acrobiosystems, HEK293 expression, and purification.

The human IL-4R (h-IL-4R-Fc) recombinant protein with the human Fc tag is self-designed, expressed and purified. The purified protein was used in the experiments described in the following examples.

The CDR amino acid residues in the VL and VH regions of the antibody or antigen-binding fragment of this example are in number and position in accordance with the known Kabat numbering convention (LCDR1-3, HCDR2-3), or in accordance with the Kabat and CHOTHIA (ABM) convention (HCDR 1).

TABLE 1 Immunogen information

Name (R)	Start and stop of amino acid sequence	Database numbering/commodity number
h-IL-4R-his	Met26-His232	NP_000409.1
m-IL-4R-his	Ile26-Arg233	NP_001008700
rhesus-IL-4R-his	Met26-Arg232	G7Q0S7
h-IL-4R-Fc	Met1-His232	NP_000409.1

Anti-human IL4R monoclonal antibodies were generated by immunizing mice. C57BL/6 mice for experiments, female, 6-8 weeks old (Suzhou Zhao-derived new drug research center, Inc., animal production license number: 201503052).

A breeding environment: SPF grade. After the mouse is purchased, the mouse is raised in a laboratory environment for 1 week, and the light/dark period is adjusted for 12/12 hours at the temperature of 20-25 ℃; the humidity is 40-60%. The acclimated mice were divided into 3 cages of 5 mice each. The immunizing antigen is human IL-4R recombinant protein with Fc tag (h-IL4R-Fc, concentration of 0.73 mg/ml). Emulsification with Freund's adjuvant (sigma, Cat #: F5881): freund's complete adjuvant (CFA, Pierce, Cat #77140) was used for the first time, and nucleic acid adjuvant (CpG, Shanghai Biotechnology) and aluminum adjuvant (Alum, Thermo Cat #77161) were used for the rest of booster immunizations.

Day 0 Intraperitoneal (IP) injection of 70. mu.g/pellet of post-emulsification antigen. On days 14, 28, 42, 56, 77, dorsal and intraperitoneal injections (0.1 ml each) were selected based on dorsal clotting and abdominal swelling. Blood was collected on days 21, 35, 49, 63 and 84 for blood test, and mouse serum was tested by the ELISA method of test example 1 to determine the antibody titer in the mouse serum. After the 4 th immunization, mice with high antibody titers in serum and titers that plateau were selected for splenocyte fusion, boosted 3 days prior to fusion, and injected Intraperitoneally (IP) with 10 ug/mouse of antigen solution in phosphate buffer. Spleen lymphocytes and myeloma cells Sp2/0 cells using an optimized PEG-mediated fusion procedure (CRL-8287 ^TM) And carrying out fusion to obtain hybridoma cells.

Example 2: ELISA testing and screening of antibodies

1. ELISA binding experiments:

ELISA experiments were used to detect the binding properties of IL-4R antibodies. After the antibody was added to each well using an elisa plate directly coated with his-labeled IL-4R recombinant protein, the activity of the antibody binding to the antigen was detected by adding a secondary antibody (HRP-conjugated anti-primary anti-Fc antibody) and HRP substrate TMB.

Human or rhesus IL-4R-his protein was coated on a 96-well microplate, 100. mu.l per well at a concentration of 0.5. mu.g/ml, and incubated overnight at 4 ℃. The wash solution was washed three times, 250. mu.l per well. Each wash was shaken for 10 seconds to ensure adequate cleaning. Add 200. mu.l/well blocking solution and incubate at room temperature for 2 hours. The wash solution was washed three times, 250. mu.l per well. Each wash was shaken for 10 seconds to ensure adequate cleaning. Mu.l of the anti-IL-4R test antibody diluted with the diluent was added to each well. Incubate at room temperature for 1 hour. The wash solution was washed three times, 250. mu.l per well. Add 100. mu.l of diluent per well at a rate of 1: 20000-fold diluted anti-human IgG secondary HRP-labeled goat antibody. Incubate at room temperature for 1 hour. The wash solution was washed three times, 250. mu.l per well. Mu.l of TMB was added to each well and the reaction was protected from light for 15 minutes. 50 μ L of 0.16M/L sulfuric acid per well was added. Reading 450nm OD value by Thermo MultiSkanFc plate reader, calculating binding EC of IL-4R antibody on IL-4R₅₀The value is obtained.

2. ELISA blocking experiments:

the experiment detects that the screened anti-human IL-4R antibody blocks the combination of human IL-4R and human IL-4 through an in vitro blocking experiment. The specific method comprises the steps of coating an IL-4R recombinant protein with Fc label on a 96-hole enzyme label plate, adding an antibody which is combined with human IL-4R to fully combine to occupy epitope, then adding IL-4(Biolegend, Cat #574004), detecting whether IL-4 can be combined with IL-4R through biotin-coupled anti-IL-4 antibody and Neutravidin-HRP (Pierce, Cat #31001), and calculating IC of IL-4R antibody for blocking IL-4/IL-4R combination₅₀The value is obtained.

The human IL-4R-Fc protein was coated on a 96-well microplate, 100. mu.l per well at a concentration of 0.5. mu.g/ml, and incubated overnight at 4 ℃. The wash solution was washed three times, 250. mu.l per well. Each wash was shaken for 10 seconds to ensure adequate cleaning. Add 200. mu.l/well blocking solution and incubate at room temperature for 2 hours. The wash solution was washed three times, 250. mu.l per well. Each wash was shaken for 10 seconds to ensure adequate cleaning. Mu.l of the anti-IL-4R test antibody diluted with the diluent was added to each well and incubated at room temperature for 1 hour. The wash solution was washed three times, 250. mu.l per well. Mu.l of diluted IL-4 was added to each well, incubated for 1 hour at room temperature, and washed three times with the wash solution. Diluting with 100. mu.l of diluent per wellThe biotin-conjugated anti-IL-4 antibody of (4) was incubated at room temperature for 1 hour and washed three times with a washing solution. Diluting with diluent according to the proportion of 1: HRP-labeled Neutravidin, diluted 5000-fold, was incubated at room temperature for 1 hour. The wash solution was washed three times, 250. mu.l per well. Mu.l of TMB was added to each well and the reaction was protected from light for 15 minutes. 50 μ L of 0.16M/L sulfuric acid per well was added. Reading the OD value of 450nm by a Thermo MultiSkanFc plate reader, and calculating the IC of the IL-4R antibody for the IL-4R and IL-4 binding blockage₅₀The value is obtained.

Example 3: reporter gene cell activity assay for antibodies that bind human IL-4R

HEK-Blue IL-4 cells were purchased from Invivogen (Cat # hkb-STAT6), stably transfected with the human IL-4R gene and STAT 6-mediated SEAP genome, and the level of activation of the IL-4R signaling pathway was characterized by detecting secreted SEAP in the supernatant using the SEAP substrate QUANTI-Blue.

This experiment was performed by detecting the activation of cell HEK-Blue IL-4, according to IC₅₀Size IL-4R antibody was evaluated for in vitro cellular activity. HEK-Blue IL-4 cells were cultured in DMEM medium containing 10% FBS, 100. mu.g/ml Zeocin (Invivogen, Cat # ant-zn-05) and 10. mu.g/ml Blastidin (Invivogen, Cat # ant-bl-05); and (3) carrying out passage 2-3 times in one week, wherein the passage ratio is 1: 5 or 1: 10. and (3) sucking off the culture medium during passage, flushing a cell layer by using 5ml of 0.25% pancreatin, sucking off the pancreatin, digesting the cells in an incubator for 3-5 minutes, and adding a fresh culture medium to resuspend the cells. Add 100. mu.L of cell suspension to 96 well cell culture plates at a density of 5X 10⁵Cells/ml, DMEM with 10% FBS, 100ug/ml Zeocin and 30ug/ml Blasticidin, 100. mu.l sterile water was added to the periphery of the 96-well plate. The plates were incubated in an incubator for 24 hours (37 ℃, 5% CO)₂). After cell attachment, 100. mu.l of the test antibody was added to each well in a gradient. Incubate the plates in an incubator for 20-24 hours (37 ℃, 5% CO)₂). Mu.l of cell supernatant per well was taken to a new 96-well flat bottom plate, 180. mu.l of QUANTI-Blue substrate solution was added, and the plate was incubated in an incubator for 1-3 hours in the absence of light. The absorbance at 620nm was measured with a microplate reader (Thermo MultiSkanFc).

Example 4: inhibition of TF-1 cell proliferation assay by antibodies that bind to human IL-4R

TF-1 cells (ATCC CRL-2003) are lymphoid cancer cells which express IL-4R and are sensitive to cytokines such as IL-4/IL-13. IL-4 stimulates the proliferation of TF-1 cells in the absence of GM-CSF. In this experiment, neutralizing activity of different IL-4R antibodies was compared by adding neutralizing antibodies, blocking the pathway of action of IL-4, and inhibiting proliferation of TF-1 cells. Cell TF-1 was cultured in GM-CSF (R) containing 10% FBS, 2ng/ml&D, Cat #215-GM-010) in RPMI1640 medium; and (3) carrying out passage 2-3 times in one week, wherein the passage ratio is 1: 10. add 100. mu.L of cell suspension to 96 well cell culture plates at a density of 2X 10⁵Cells/ml, medium 10% FBS RPMI1640 medium, 100. mu.l sterile water only added to the periphery of 96-well plates. Add 50. mu.l of the test antibody diluted in a gradient and 50. mu.l of IL-4 (R) at a final concentration of 0.7ng/ml to each well&D, Cat #204-IL-050), plates were incubated in an incubator for 72 hours (37 ℃, 5% CO)₂). After the culture was completed, cell proliferation was detected by using CTG kit (Promega, Cat # G7572).

Example 5: in vitro binding affinity and kinetics experiments

The affinity of the humanized human IL-4R-binding antibody to be tested and human IL-4R was determined using Biacore, GE instrument.

Covalently coupling human anti-capture antibodies to a biosensor chip CM5 of a Biacore instrument (Biacore X100, GE) according to the method of the specification of the human anti-capture kit (Cat. # BR-1008-39, GE) to affinity capture a quantity of the antibody to be detected; the surface of the chip was then flowed over a series of concentration gradients of IL-4R antigen (IL-4R antigen was purchased from Acrobiosystems, Cat # ILR-H5221) and reaction signals were detected in real time using a Biacore instrument (Biacore X100, GE) to obtain binding and dissociation curves. After each cycle of dissociation is completed, the biochip is washed and regenerated with a regeneration solution prepared in a human anti-capture kit. The amino coupling kit used in the experiment was purchased from GE corporation (Cat. # BR-1000-50, GE) and the buffer was HBS-EP +10 Xbuffer solution (Cat. # BR-1006-69, GE) diluted to 1X (pH 7.4) with D.I.Water.

The data obtained from the experiment were fitted to the (1: 1) Binding model using BiacoreX100evaluation software2.0GE software to obtain affinity values.

Example 6: antibody sequences and preparation

Two monoclonal hybridoma cell lines with the best in vitro activity were selected by the ELISA binding assay (ELISA binding of human IL-4R-his) and ELISA blocking assay (ELISA blocking of human IL-4/IL-4R) as described above in example 2, the assay for inhibiting the activation of HEK293-Blue IL-4 cells under IL-4 stimulation in example 3, and the assay for inhibiting the proliferation of TF-1 cells under IL-4 stimulation in example 4. The results of the activity measurements are shown in Table 2.

TABLE 2 selection of hybridoma cell lines with the best in vitro activity

The monoclonal hybridoma cell lines 25G7 and 7B10 with the best in vitro activity were selected and the monoclonal antibody sequences were cloned. The sequence cloning from the hybridoma is as follows. The logarithmic growth phase hybridoma cells were harvested, RNA extracted (according to kit instructions) using Trizol (Invitrogen, 15596-018), and reverse transcribed (PrimeScript)^TMReverse Transcriptase, Takara, cat # 2680A). The cDNA obtained by reverse transcription was subjected to PCR amplification using mouse Ig-Primer Set (Novagen, TB326 Rev. B0503), and then sequenced by a sequencer, and the sequence of the obtained antibody was analyzed.

The variable regions of the heavy and light chains of murine mab 25G7 were as follows:

25G7 HCVR

25G7 LCVR

it contains CDR sequences as shown in Table 3.

TABLE 3 CDR sequences of mAb 25G7

Name (R)	Sequence of	Numbering
HCDR1	GFTFSDYGMH	SEQ ID NO：3
HCDR2	FISSGSSIIYYADIVKG	SEQ ID NO：4
HCDR3	GNKRGFFDY	SEQ ID NO：5
LCDR1	NASSSVSYMY	SEQ ID NO：6
LCDR2	LTSNLAS	SEQ ID NO：7
LCDR3	QQWRSNPPMLT	SEQ ID NO：8

The variable regions of the heavy and light chains of murine mab 7B10 were as follows:

7B10 HCVR

7B10 LCVR

it contains CDR sequences as shown in Table 4.

TABLE 4 CDR sequences of mAb 7B10

Name (R)	Sequence of	Numbering
HCDR1	GYTFTSYWMH	SEQ ID NO：11
HCDR2	LIHPNSDTTKFSENFKT	SEQ ID NO：12
HCDR3	SKIITTIVARHWYFDV	SEQ ID NO：13
LCDR1	KASQSVDYGGDSYMN	SEQ ID NO：14
LCDR2	AASNLES	SEQ ID NO：15
LCDR3	QHSNENPPT	SEQ ID NO：16

The obtained variable region sequence was ligated to a human constant region sequence to obtain a human-mouse chimeric antibody sequence. The sequence of the chimeric antibody is inserted into a corresponding expression vector using molecular cloning techniques. Human-mouse chimeric antibodies 25G7-C and 7B10-C were obtained using the HEK293 cell expression system.

Purified chimeric antibodies were tested for their respective in vitro activities by the methods described in examples 2-5 above, and the data are shown in Table 5. The results show that the 25G7-C antibody has a blocking effect on IL-4 binding and an inhibition effect on cell proliferation which are both significantly better than those of a reference antibody dolitumumab (refer to WHO Drug Information, Vol.26, No.4, 2012 synthesis).

TABLE 5 in vitro Activity assay

Example 7: mouse antibody humanization experiments

The two most functionally active strains of the murine antibody obtained (25G7 and 7B10) were humanized. On the basis of the obtained typical structure of the mouse antibody VH/VLCDR, the heavy chain variable region sequence and the light chain variable region sequence are compared with an antibody Germline database to obtain a human Germline template with high homology. Wherein the human germline light chain framework regions are derived from human kappa light chain genes, preferably human germline light chain templates IGKV3-11 x 01(SEQ ID NO: 22 for antibody 25G7) and IGKV2D-29 x 01(SEQ ID NO: 24 for antibody 7B 10). The human germline heavy chain framework regions are derived from human heavy chains, preferably human germline heavy chain templates IGHV3-48 x 01(SEQ ID NO: 21 for antibody 25G7) and IGHV1-2 x 02(SEQ ID NO: 23 for antibody 7B 10).

The human germline template sequence is shown below.

Human germline heavy chain template IGHV3-48 × 01:

human germline light chain template IGKV3-11 × 01:

human germline heavy chain template IGHV1-2 × 02:

human germline light chain template IGKV2D-29 x 01:

the CDR regions of the murine antibody were grafted onto a selected humanized template, replacing the humanized variable regions, and recombined with IgG constant regions. Then, based on the three-dimensional structure of the murine antibody, the embedded residues, residues that interact directly with the CDR regions, and residues that have significant effects on the conformation of VL and VH are subjected to back-mutation to yield a series of humanized molecules.

Wherein, the Hu7B10-VH-a, the Hu7B10-VH-B and the Hu7B10-VH-c are modified to change the first position of the heavy chain human germline template from Q to E. The hu25G7 is also modified by drug property. The sequences of the humanized heavy chain variable regions of the two antibodies are respectively shown as SEQ ID NO: 25-27, SEQ ID NO: 31-33; the light chain variable region sequences are respectively shown as SEQ ID NO: 28-30, SEQ ID NO: 34-36.

hu25G7-VH-a：

hu25G7-VH-b：

hu25G7-VH-c：

hu25G7-VL-a：

hu25G7-VL-b：

hu25G7-VL-c：

hu7B10-VH-a：

hu7B10-VH-b：

hu7B10-VH-c：

hu7B10-VL-a：

hu7B10-VL-b：

hu7B10-VL-c：

The template selection and back-mutation design of hybridoma clone 25G7 are shown in Table 6.

TABLE 6 template selection and Back-mutation design

Template selection and back-mutation design for hybridoma clone 7B10, see table 7 below:

TABLE 7 template selection and Back-mutation design

Through the above antibody small-scale expression tests and the comparison of the number of back mutations for each light-heavy chain combination, the final humanized antibody hu25G7 (using VH-c heavy chain and VL-a light chain) and hu7B10 antibody molecules (using VH-B heavy chain and VL-B light chain) were synthetically judged and selected, each of which had the complete light-heavy chain sequence as set forth in SEQ ID NO: 17-20.

hu25G7 HC

hu25G7 LC

hu7B10 HC

hu7B10 LC

The sequence of the humanized antibody is inserted into a corresponding expression vector by using a molecular cloning technology, and the corresponding humanized antibody can be obtained by using an HEK293 cell expression system for expression and production.

Example 8: humanized antibody Activity data

The in vitro activity assays described in examples 2-5 were performed on humanized antibodies hu25G7 and hu7B10, and the results are shown in Table 8. The results show that both hu25G7 and hu7B10 bound only to human IL-4R and not to rhesus IL-4R, indicating that both antibodies bind to epitopes that are not homologous to human and rhesus and specifically bind to human IL-4R. Both antibodies can block IL-4/IL-4R binding and intracellular signal paths, so that the activation of IL-4 is neutralized, and the proliferation of TF-1 cells is inhibited, wherein the blocking and inhibiting activities of hu25G7 are still remarkably superior to those of a reference antibody, namely piruzumab, but the affinity K is higher than that of the reference antibody_DThe value is relatively low.

TABLE 8 in vitro Activity assay

Example 9: affinity maturation assay for humanized antibody hu25G7

In order to obtain anti-human IL-4R antibody with better drug effect, affinity maturation is carried out on 25G7 antibody by yeast display platform technology, affinity maturation yeast libraries aiming at6 CDRs are designed and prepared on the basis of hu25G7 antibody, degenerate primers are designed, designed mutant amino acids are introduced into hu25G7-scFv antibody library by PCR and homologous recombination method, and the size of each library is 10⁹On the other hand, the diversity of the constructed yeast library was verified by the method of second generation sequencing (GENEWIZ).

The method comprises the steps of labeling human IL-4R with biotin to screen high-affinity antibodies from a hu25G7-scFv yeast library, performing two rounds of MACS screening (streptomycin magnetic beads, Invitrogen) and two rounds of FACS screening (BD FACSAria (TM) FUSION), selecting yeast monoclonals, culturing and inducing expression, detecting the binding of the yeast monoclonals to human IL-4R through FACS (BD FACSCAnto II), selecting yeast monoclonals with higher affinity than wild-type 25G7 antibodies, performing sequencing verification, performing alignment analysis on the sequenced clones, removing redundant sequences, converting non-redundant sequences into full-length human antibody molecules, and performing mammalian cell expression. Affinity assay of the full-length antibody after affinity purification was performed using BIAcoreTM X-100(GE Life Sciences), and candidate antibody molecules having higher affinity for human IL-4R were selected as follows. The affinity of the antibody molecules to human IL-4R is higher than that of the wild type hu25G7 antibody, wherein the affinity of the hu25G7-A antibody molecule is equivalent to that of dolugumab, and the affinity of the hu25G7-B molecule is obviously better than that of dolugumab.

The light chain variable region sequence of the antibody hu25G7-A finally obtained by affinity maturation is as follows:

hu25G7-A LCVR

EIVLTQSPATLSLSPGERATLSCRASSSVPYMYWYQQKPGQAPRLLIYLTSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWRAYPPMLTFGGGTKVEIK (SEQ ID NO: 37) having CDR sequences shown in Table 9.

TABLE 9 CDR sequences

Name (R)	Sequence of	Numbering
LCDR1	RASSSVPYMY	SEQ ID NO：38
LCDR2	LTSNLAS	SEQ ID NO：7
LCDR3	QQWRAYPPMLT	SEQ ID NO：40

The light chain variable region sequence of antibody hu25G7-B is as follows:

hu25G7-B LCVR

it contains CDR sequences as shown in Table 10.

TABLE 10 CDR sequences

Name (R)	Sequence of	Numbering
LCDR1	RASPGVPPLA	SEQ ID NO：42
LCDR2	LASSRPS	SEQ ID NO：39
LCDR3	QQWRSNPPMLT	SEQ ID NO：8

The light chain variable region hu25G7-A LCVR is recombined with the hu25G7 light chain constant region to obtain the hu25G7-A antibody light chain; the light chain variable region hu25G7-B LCVR described above was recombined with the hu25G7 light chain constant region to give the hu25G7-B antibody light chain.

Optimizing hu25G7-VH-c unstable amino acid residue, enhancing drug property, and obtaining the heavy chain variable region hu25G7-VH

The heavy chain variable region described above can be recombined with the hu25G7 heavy chain constant region to give the hu25G7-A/hu25G7-B antibody heavy chain.

The complete heavy chain sequences of hu25G7-A and hu25G7-B are shown in SEQ ID NO: as shown at 44.

hu25G7HC

The respective complete light chain sequences are shown in SEQ ID NO: 45-46.

hu25G7-A LC

hu25G7-B LC

Example 10: humanized antibody affinity maturation Activity data

Examples 3 and 4 detected two antibodies, hu25G7-A and hu25G 7-B; both hu25G7-A and hu25G7-B antibodies blocked IL-4/IL-4R binding, and intracellular signaling pathways resulted in neutralization of IL-4 and IL-13 activation, and inhibition of TF-1 cell proliferation, with activity data as shown in Table 11.

TABLE 11 comparison of Activity data

In the experiment of inhibiting the TF-1 cell proliferation caused by IL-13 stimulation, both hu25G7-A and hu25G7-B show beneficial effects. Repeated validation of the effect revealed that hu25G7-A inhibited IL-13 proliferation (IC) in TF-1 cells under the same conditions₅₀) The value was 11.68, inhibition of IL-13 proliferation (IC) in TF-1 cells by control P-mumab₅₀) The value was 22.85. The hu25G7 piruzumab has a significantly improved effect on blocking the binding of IL-4, IL-13 and IL-4R, and on cell proliferation caused by said binding.

Example 11: effect of humanized antibody on mouse dermatitis

Establishing a mouse dermatitis model: IL-4/IL-4 Ra transgenic mice (purchased from the model Bioresearch center of the Seikagaku Co., Ltd.) each having an abdominal range of about 3 cm. times.3 cm were uniformly sensitized with 100. mu.L of a 1.5% solution of OXZ acetone in olive oil (acetone: olive oil: 4: 1), and the sensitization Day was D0(Day 0). On Day seven (Day 7) the challenge was initiated by applying 20 μ L of a 1% OXZ acetone olive oil solution evenly to both ears (both sides) of the mice, once every 72 hours.

In the experiment, a normal control group (only an acetone olive oil solution is smeared during sensitization and excitation), a model control group, hu25G7-A, hu25G7-B and a doluvian monoclonal antibody group are arranged, 5 groups are provided, 3-5 mice in each group are provided, the administration dose of an administration group is 50mg/kg, the administration mode is subcutaneous administration, and the administration is carried out twice a week (the specific information is shown in table 12). The thickness of the ear was measured with a vernier caliper on day 27, and the results are shown in FIG. 1.

TABLE 12 administration regimens for each group

Group of	Number of animals	Mode of administration	Dosage to be administered	Frequency of administration
Normal control group	3 (Male)	S.C.	-	2 times a week
Model control group	5(3 female +2 male)	S.C.	-	2 times a week
hu25G7-A	5(3 female +2 male)	S.C.	50mg/kg	2 times a week
hu25G7-B	3(2 female +1 male)	S.C.	50mg/kg	2 times a week
Dolu monoclonal antibody	4(2 female +2 male)	S.C.	50mg/kg	2 times a week

The result shows that the ears of the mice in the model control group have obvious pathological damage, and the ear thickness is obviously higher than that of the normal control group. The ear thickness of the hu25G7-A, hu25G7-B and DOPILUMAb mice was significantly lower than that of the model control group on day 27. That is, hu25G7-A, hu25G7-B and dolugumab can be used for treating dermatitis, and hu25G7-B has more excellent effect than dolugumab.

Claims (19)

1. An anti-IL-4R antibody or antigen-binding fragment thereof, wherein:

   a heavy chain variable region comprising:

   (I) respectively shown in SEQ ID NO: 3. SEQ ID NO: 4 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in fig. 5; or

   (II) are shown as SEQ ID NO: 11. SEQ ID NO: 12 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown at 13;

   and/or, a light chain variable region comprising:

   (I) respectively shown in SEQ ID NO: 6. SEQ ID NO: 7 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown in fig. 8; or

   (II) are shown as SEQ ID NO: 14. SEQ ID NO: 15 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown at 16; or

   (III) as shown in SEQ ID NO: 38. SEQ ID NO: 7 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown at 40; or

   (IV) are shown as SEQ ID NO: 42. SEQ ID NO: 39 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown in fig. 8.
2. The anti-IL-4R antibody or antigen-binding fragment thereof of claim 1, comprising any one selected from the following (I) to (IV):

   (I) a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 3. SEQ ID NO: 4 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in fig. 5; and

   a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 6. SEQ ID NO: 7 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown in fig. 8;

   (II) a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 11. SEQ ID NO: 12 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown at 13; and

   a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 14. SEQ ID NO: 15 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown at 16;

   (III) a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 3. SEQ ID NO: 4 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in fig. 5; and

   a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 38. SEQ ID NO: 7 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown at 40;

   (IV) a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 3. SEQ ID NO: 4 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in fig. 5; and

   a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 42. SEQ ID NO: 39 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown in fig. 8.
3. The anti-IL-4R antibody or antigen-binding fragment thereof of any one of claims 1-2, further comprising a fragment selected from the group consisting of:

   -FR region sequences derived from human germline light chain IGKV3-11 x 01;

   -a back-mutated sequence derived from an FR region having at least 95% identity to the human germline light chain IGKV3-11 x 01;

   preferably, the back mutation is selected from one or a combination of L46P, L47W and F71Y;

   and/or, the anti-IL-4R antibody or antigen binding fragment thereof further comprises a fragment selected from the group consisting of:

   -FR region sequences derived from human germline heavy chain IGHV3-48 x 01;

   -from a back-mutated sequence having at least 95% identity with the FR region of human germline heavy chain IGHV3-48 x 01;

   preferably, the back mutation is selected from one or a combination of S49A, F67S, a 93T.
4. The anti-IL-4R antibody or antigen-binding fragment thereof of any one of claims 1-2, further comprising a fragment selected from the group consisting of:

   -a FR region sequence derived from human germline light chain IGKV2D-29 x 01;

   -a back-mutated sequence derived from an FR region having at least 95% identity to the human germline light chain IGKV2D-29 x 01;

   preferably, the back-mutations are selected from M4L and/or V58I;

   and/or, the anti-IL-4R antibody or antigen binding fragment thereof further comprises a fragment selected from the group consisting of:

   -FR region sequences derived from human germline heavy chain IGHV1-2 x 02;

   -from a back-mutated sequence having at least 95% identity with the FR region of human germline heavy chain IGHV1-2 x 02;

   preferably, the back mutation is selected from one or a combination of M69L, R71I, T73K and R94K.
5. The anti-IL-4R antibody or antigen-binding fragment thereof of any one of claims 1-2, wherein:

   the antigen binding fragment is selected from the group consisting of: fab, Fab '-SH, Fv, scFv, (Fab') 2 fragments;

   the heavy chain variable region of the antibody comprises a heavy chain framework region of human IgG1, IgG2, IgG3, or IgG4, or a variant thereof; preferably, the heavy chain variable region of the antibody comprises the heavy chain framework region of human IgG1, IgG2, or IgG 4;

   more preferably, the heavy chain variable region of the antibody comprises the heavy chain framework region of human IgG 4.
6. The anti-IL-4R antibody or antigen-binding fragment thereof of any one of claims 1-2, wherein:

   a heavy chain variable region comprising:

   (I) as shown in SEQ ID NO: 1 or a sequence corresponding to SEQ ID NO: 1, sequences having at least 90%, 95%, 98%, 99% identity; or

   (II) as shown in SEQ ID NO: 9 or a sequence corresponding to SEQ ID NO: 9 sequences having at least 90%, 95%, 98%, 99% identity; or

   (III) as shown in SEQ ID NO: 43 or a sequence identical to SEQ ID NO: 43 sequences having at least 90%, 95%, 98%, 99% identity;

   and/or, a light chain variable region comprising:

   (I) as shown in SEQ ID NO: 2 or a sequence similar to SEQ ID NO: 2, sequences having at least 90%, 95%, 98%, 99% identity; or

   (II) as shown in SEQ ID NO: 10 or a sequence identical to SEQ ID NO: 10 sequences having at least 90%, 95%, 98%, 99% identity; or

   (III) as shown in SEQ ID NO: 37 or a sequence identical to SEQ ID NO: 37, sequences having at least 90%, 95%, 98%, 99% identity; or

   (IV) as shown in SEQ ID NO: 41 or a sequence corresponding to SEQ ID NO: 41 are at least 90%, 95%, 98%, 99% identical;

   preferably, the anti-IL-4R antibody or antigen-binding fragment thereof comprises:

   SEQ ID NO: 1, SEQ ID NO: 2; or

   SEQ ID NO: 9, SEQ ID NO: 10; or

   SEQ ID NO: 43, SEQ ID NO: 37; or

   SEQ ID NO: 43, SEQ ID NO: 41, or a light chain variable region.
7. The anti-IL-4R antibody or antigen-binding fragment thereof of any one of claims 1-2, wherein:

   a heavy chain variable region comprising:

   (I) as shown in SEQ ID NO: 25-27 or a sequence as set forth in one of SEQ ID NOs: 25-27, or a sequence having at least 90%, 95%, 98%, or 99% identity; or

   (II) as shown in SEQ ID NO: 31-33 or a sequence as set forth in one of SEQ ID NOs: 31-33, or a sequence having at least 90%, 95%, 98%, or 99% identity;

   and/or a light chain variable region comprising:

   (I) as shown in SEQ ID NO: 28-30 or a sequence as set forth in any one of SEQ ID NOs: 28-30, having at least 90%, 95%, 98%, or 99% identity; or

   (II) as shown in SEQ ID NO: 34-36 or a sequence as set forth in any one of SEQ ID NOs: 34-36 having at least 90%, 95%, 98%, or 99% identity.
8. The anti-IL-4R antibody or antigen-binding fragment thereof of any one of claims 1-2, wherein:

   heavy chain comprising

   (I) As shown in SEQ ID NO: 17 or a sequence corresponding to SEQ ID NO: 17 sequences having at least 90%, 95%, 98% or 99% identity; or

   (II) as shown in SEQ ID NO: 19 or a sequence corresponding to SEQ ID NO: 19, a sequence having at least 90%, 95%, 98%, or 99% identity; or

   (III) as shown in SEQ ID NO: 44 or a sequence corresponding to SEQ ID NO: 44 is at least 90%, 95%, 98%, or 99% identical;

   and/or a light chain comprising

   (I) As shown in SEQ ID NO: 18 or a sequence corresponding to SEQ ID NO: 18 with at least 90%, 95%, 98%, or 99% identity; or

   (II) as shown in SEQ ID NO: 20 or a sequence corresponding to SEQ ID NO: 20 with at least 90%, 95%, 98% or 99% identity; or

   (III) as shown in SEQ ID NO: 45 or a sequence identical to SEQ ID NO: 45 are at least 90%, 95%, 98%, or 99% identical; or

   (IV) as shown in SEQ ID NO: 46 or a sequence corresponding to SEQ ID NO: 46 are at least 90%, 95%, 98%, or 99% identical;

   preferably, the anti-IL-4R antibody or antigen-binding fragment thereof comprises any one selected from the group consisting of:

   SEQ ID NO: 17, and SEQ ID NO: 18, a light chain; or

   SEQ ID NO: 19, and SEQ ID NO: 20, a light chain; or

   SEQ ID NO: 44, and the heavy chain of SEQ ID NO: 45, a light chain; or

   SEQ ID NO: 44, and the heavy chain of SEQ ID NO: 46, or a light chain as shown.
9. The anti-IL-4R antibody or antigen-binding fragment thereof according to any one of claims 1-2, which is a murine antibody, a chimeric antibody, a human antibody, a humanized antibody or a fragment thereof.
10. A polynucleotide encoding the anti-IL-4R antibody or antigen-binding fragment thereof of any one of claims 1 to 9.
11. A vector comprising the polynucleotide of claim 10, wherein the vector is a eukaryotic expression vector, a prokaryotic expression vector, or a viral vector.
12. A host cell comprising the vector of claim 11,

    preferably, the host cell is selected from the group consisting of: bacteria, yeast, mammalian cells,

    more preferably, the host cell is selected from the group consisting of: escherichia coli, Pichia pastoris, Chinese hamster ovary cells, human embryonic kidney 293 cells.
13. A method for detecting or determining IL-4R, the method comprising:

    a step of contacting the anti-IL-4R antibody or antigen-binding fragment thereof of any one of claims 1 to 9 with a sample.
14. An agent comprising an anti-IL-4R antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.
15. The reagent according to claim 14, which is used for diagnosing a disease associated with human IL-4R positive cells.
16. A pharmaceutical composition comprising:

    an anti-IL-4R antibody or antigen-binding fragment thereof according to any one of claims 1 to 9; and

    a pharmaceutically acceptable excipient, diluent or carrier.
17. Use of any one or a combination thereof selected from the group consisting of:

    the anti-IL-4R antibody or antigen-binding fragment thereof of any one of claims 1 to 9, the polynucleotide of claim 10, the pharmaceutical composition of claim 16,

    preferably, the disease or condition is selected from:

    asthma, nasal polyps, chronic sinusitis, allergic skin diseases, eosinophilic esophagitis, chronic obstructive pulmonary disease, allergic rhinitis, arthritis, inflammatory diseases, allergic reactions, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, barrett's esophagus, autoimmune uveitis, tuberculosis, and renal diseases;

    more preferably, the disease or disorder is asthma or an allergic skin disease.
18. A method of treating or preventing an IL-4R mediated disease or disorder, and/or treating or preventing an immune disease or disorder, comprising:

    administering to a subject the anti-IL-4R antibody or antigen-binding fragment thereof of any one of claims 1 to 9, the pharmaceutical composition of claim 16,

    preferably, the disease or condition is selected from:

    asthma, nasal polyps, chronic sinusitis, allergic skin diseases, eosinophilic esophagitis, chronic obstructive pulmonary disease, allergic rhinitis, arthritis, inflammatory diseases, allergic reactions, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, barrett's esophagus, autoimmune uveitis, tuberculosis, and renal diseases; more preferably, the disease or disorder is asthma or an allergic skin disease.
19. A method for making an anti-IL-4R antibody or antigen-binding fragment thereof, comprising the steps of:

    expressing an anti-IL-4R antibody or antigen-binding fragment thereof in the host cell of claim 12, and

    isolating an anti-IL-4R antibody or antigen-binding fragment thereof from the host cell.