Antibodies binding to human il-4r, antigen-binding fragments thereof, and medical uses thereof

Abstract

The present disclosure relates to antibodies, antigen-binding fragments thereof, which bind to human IL-4R, and medical uses thereof. The present disclosure relates to a chimeric antibody, humanized antibody comprising CDR regions from the antibodies, antigen-binding fragments thereof, which bind to human IL-4R, and also to pharmaceutical compositions comprising the antibodies, antigen-binding fragments thereof, which bind to human IL-4R, and uses thereof as medicaments for treating allergic diseases. The present disclosure relates to a humanized antibody, which binds to human IL-4R, for use in the manufacture of a medicament for treating an IL-4R-mediated disease or disorder.

Description

[0001] This application is a divisional application of Chinese patent application No. 201980050263.4, filed on August 23, 2019, entitled "Antibody binding to human IL-4R, its antigen-binding fragment and its pharmaceutical use therein".

[0002] This application claims priority to Chinese applications CN201810971269.2, filed August 24, 2018; CN201811472752.2, filed December 4, 2018; CN201910221311.3, filed March 22, 2019; and CN201910401923.0, filed May 15, 2019. Each of these applications is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure relates to an antibody that binds to human IL-4R, and its antigen-binding fragment. It also relates to a chimeric or humanized antibody comprising a CDR region derived from said antibody, and pharmaceutical compositions comprising an antibody that binds to human IL-4R and its antigen-binding fragment, and their use as a medicament for treating autoimmune diseases. Background Technology

[0004] Allergic diseases are serious medical conditions, including: non-life-threatening allergic reactions that can be cured over time; and life-threatening allergic diseases.

[0005] Current methods for treating allergies include avoiding allergens, pharmacological treatment targeting symptoms, and prevention using allergen-specific immunotherapy.

[0006] Interleukin-4 (IL-4, also known as B cell stimulating factor or BSF-1) has been characterized to stimulate B cell proliferation in response to low concentrations of anti-surface immunoglobulin antibodies. IL-4 has been shown to have broad biological activities, including growth stimulation of T cells, mast cells, granulocytes, megakaryocytes, and erythrocytes. IL-4 induces the expression of MHC-II in resting B cells and enhances the secretion of immunoglobulins IgE and IgG1 by activated B cells.

[0007] The biological activity of IL-4 is mediated by specific cell surface IL-4 receptors (IL-4R). IL-4 receptors (IL-4R) consist of 802 amino acid residues and are expressed on the surface of T cells, B cells, thymocytes, bone marrow cells, macrophages, and mast cells. The α chain of IL-4R is also a component of the IL-13 receptor (IL-13R), therefore IL-4R can also mediate the biological activity of IL-13. Drugs and combinations thereof containing IL-4R antagonists can be administered as a novel treatment method before, during, or after exposure to allergens or the onset of allergic symptoms.

[0008] Currently, numerous pharmaceutical companies worldwide are developing monoclonal antibodies targeting IL-4R for the treatment of various allergic diseases associated with it. Related patent applications include WO2010053751, WO2001092340, WO2008054606, and WO2014031610. Among these, Sanofi's Regeneron's asthma drug dupilumab has been approved for marketing in the treatment of dermatitis, and for asthma, it has completed Phase III clinical trials and entered the market approval stage.

[0009] Current treatment strategies are either ineffective or carry significant risks. Therefore, there is a need in the field to provide highly selective and bioactive antibodies that bind to human IL-4R with high affinity and can neutralize the bioactivity of IL-4R (including blocking binding to IL-4R), thereby providing drugs and compositions thereof, as well as treatment methods, for treating IL-4R-mediated allergic diseases. Summary of the Invention

[0010] In some embodiments described herein, antibodies that specifically bind to IL-4R (also known as antibodies that bind to human IL-4R or anti-human IL-4R antibodies) or antigen-binding fragments thereof are provided, wherein the heavy chain variable region comprises:

[0011] (I) HCDR variants as shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively, or HCDR variants differing from HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 3, 4 and 5 by 3, 2 or 1 amino acid, respectively; or

[0012] (II) HCDR variants that are HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 respectively, or that have 3, 2 or 1 amino acid differences from HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 11, 12 and 13 respectively;

[0013] and / or light chain variable regions, containing:

[0014] (I) LCDR variants as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, or LCDR variants differing from LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 6, 7 and 8 by 3, 2 or 1 amino acid, respectively; or

[0015] (II) LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively, or LCDR variants having 3, 2, or 1 amino acid differences from LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 14, 15, and 16, respectively; or

[0016] (III) LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 38, SEQ ID NO: 7, and SEQ ID NO: 40, respectively, or LCDR variants that differ from LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 38, 7, and 40 by 3, 2, or 1 amino acid, respectively; or

[0017] (IV) LCDR variants as shown in SEQ ID NO: 42, SEQ ID NO: 39 and SEQ ID NO: 8, respectively, or LCDR variants that differ from LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 42, 39 and 8 by 3, 2 or 1 amino acid, respectively.

[0018] In some embodiments, the antibody binding to human IL-4R or its antigen-binding fragment comprises any one of the following (I) to (IV):

[0019] (I) Heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; and

[0020] The light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 respectively;

[0021] (II) Heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; and

[0022] The light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 respectively;

[0023] (III) Heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; and

[0024] The light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 38, SEQ ID NO: 7 and SEQ ID NO: 40 respectively;

[0025] (IV) Heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; and

[0026] The light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 42, SEQ ID NO: 39 and SEQ ID NO: 8, respectively.

[0027] In some implementations, an antibody or its antigen-binding fragment that binds to human IL-4R is used, wherein:

[0028] Heavy chain variable region, containing:

[0029] (I) A sequence as shown in SEQ ID NO: 1 or having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 1; or

[0030] (II) A sequence as shown in SEQ ID NO: 9 or having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 9; or

[0031] (III) A sequence as shown in SEQ ID NO: 43 or having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 43; or

[0032] and / or light chain variable regions, containing:

[0033] (I) A sequence as shown in SEQ ID NO: 2 or having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 2; or

[0034] (II) A sequence as shown in SEQ ID NO: 10 or having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 10; or

[0035] (III) A sequence as shown in SEQ ID NO: 37 or having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 37; or

[0036] (IV) A sequence as shown in SEQ ID NO: 41 or having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 41.

[0037] In at least one embodiment, the antibody or antigen-binding fragment that binds to human IL-4R, wherein:

[0038] The variable region of the heavy chain is shown in sequence SEQ ID NO: 1, and the variable region of the light chain is shown in sequence SEQ ID NO: 2; or

[0039] The variable region of the heavy chain is shown in sequence SEQ ID NO: 9, and the variable region of the light chain is shown in sequence SEQ ID NO: 10; or

[0040] The variable region of the heavy chain is shown in sequence SEQ ID NO: 43, and the variable region of the light chain is shown in sequence SEQ ID NO: 37; or

[0041] The variable region of the heavy chain is shown in sequence SEQ ID NO: 43, and the variable region of the light chain is shown in sequence SEQ ID NO: 41.

[0042] In some implementations, an antibody or antigen-binding fragment thereof binds to human IL-4R, wherein:

[0043] Heavy chain variable region, containing:

[0044] (I) A sequence as shown in one of SEQ ID NO: 25-27 or having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with one of SEQ ID NO: 25-27; or

[0045] (II) A sequence as shown in one of SEQ ID NO: 31-33 or having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with one of SEQ ID NO: 31-33;

[0046] and / or light chain variable regions, containing:

[0047] (I) A sequence as shown in one of SEQ ID NO: 28-30 or having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with one of SEQ ID NO: 28-30; or

[0048] (II) A sequence as shown in one of SEQ ID NO: 34-36 or having at least 70%, 80%, 90%, 95%, 98% or 99% identity with one of SEQ ID NO: 34-36.

[0049] In some specific implementations, the heavy chain variable region is as shown in one of the sequences SEQ ID NO: 25-27, and the light chain variable region is as shown in one of the sequences SEQ ID NO: 28-30.

[0050] In some other specific embodiments, the heavy chain variable region is shown as one of the sequences SEQ ID NO: 31-33, and the light chain variable region is shown as one of the sequences SEQ ID NO: 34-36.

[0051] In some embodiments, the heavy chain of an antibody or its antigen-binding fragment that binds to human IL-4R comprises:

[0052] (I) A sequence as shown in SEQ ID NO: 17 or having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 17; or

[0053] (II) A sequence as shown in SEQ ID NO: 19 or having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 19; or

[0054] (III) A sequence as shown in SEQ ID NO: 44 or having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 44.

[0055] In some embodiments, the light chain of an antibody or its antigen-binding fragment that binds to human IL-4R comprises:

[0056] (I) A sequence as shown in SEQ ID NO: 18 or having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 18; or

[0057] (II) A sequence as shown in SEQ ID NO: 20 or having at least 70%, 80%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 20; or

[0058] (III) A sequence as shown in SEQ ID NO: 45 or having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 45; or

[0059] (IV) A sequence as shown in SEQ ID NO: 46 or having at least 90%, 95%, 98% or 99% identity with SEQ ID NO: 46.

[0060] In at least one embodiment, the heavy chain is as shown in sequence SEQ ID NO: 17, and the light chain is as shown in sequence SEQ ID NO: 18.

[0061] In another embodiment, the heavy chain is shown as in sequence SEQ ID NO: 19, and the light chain is shown as in sequence SEQ ID NO: 20.

[0062] In another embodiment, the heavy chain is shown as in sequence SEQ ID NO: 44, and the light chain is shown as in sequence SEQ ID NO: 45.

[0063] In another embodiment, the heavy chain is shown as in sequence SEQ ID NO: 44, and the light chain is shown as in sequence SEQ ID NO: 46.

[0064] In some embodiments, the antibody or antigen-binding fragment against IL-4R is a murine antibody, a chimeric antibody, a human antibody, a humanized antibody, or a fragment thereof.

[0065] In some specific implementations, the antibody or antigen-binding fragment against IL-4R is humanized.

[0066] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment comprises fragments selected from or in combination with the following:

[0067] - FR region sequence derived from hominid light chain IGKV3-11*01;

[0068] - A reversion mutation sequence derived from the FR region of the human light chain IGKV3-11*01, possessing at least 95% identity. In some specific embodiments, the reversion mutation is selected from one or a combination of L46P, L47W, and F71Y.

[0069] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment comprises fragments selected from or in combination with the following:

[0070] - FR region sequence derived from hominid heavy chain IGHV3-48*01;

[0071] - A reversion mutation sequence derived from the FR region of the human germline heavy chain IGHV3-48*01, possessing at least 95% identity. In some specific embodiments, the reversion mutation is selected from one or a combination of S49A, F67S, and A93T.

[0072] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment comprises fragments selected from or in combination with the following:

[0073] - FR region sequence derived from hominid light chain IGKV2D-29*01;

[0074] - A reversion mutation sequence derived from the FR region of the human light chain IGKV2D-29*01, possessing at least 95% identity. In some specific embodiments, the reversion mutation is selected from M4L and / or V58I.

[0075] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment further comprises fragments selected from or in combination with the following:

[0076] - FR region sequence derived from hominid heavy chain IGHV1-2*02;

[0077] - A reversion mutation sequence derived from the FR region of the human germline heavy chain IGHV1-2*02, possessing at least 95% identity. In some specific embodiments, the reversion mutation is selected from one or a combination of M69L, R71I, T73K, and R94K.

[0078] In some embodiments, the heavy chain variable region of the anti-IL-4R antibody or its antigen-binding fragment comprises a heavy chain framework region of human IgG1, IgG2, IgG3, or IgG4 or variants thereof. In some specific embodiments, the heavy chain variable region comprises a heavy chain framework region of human IgG1 or variants thereof; for example, SEQ ID NO: 43 shows a heavy chain variable region or a heavy chain variable region variant having at least 85% sequence identity with it.

[0079] In some embodiments, the antibody against IL-4R or its antigen-binding fragment comprises a constant region of a human κ, λ chain or a variant thereof, such as the light chain variable region shown in SEQ ID NO: 44 or a light chain variable region variant having at least 85% sequence identity with it.

[0080] In some embodiments, an IL-4R humanized antibody or a fragment thereof as described above further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof.

[0081] In at least one embodiment, the antibody comprises a heavy chain constant region of human IgG2 or IgG4. IgG2 or IgG4 has no ADCC toxicity. Alternatively, IgG1 with amino acid mutations that eliminate ADCC (antibody-dependent cell-mediated cytotoxicity) can be used.

[0082] In at least one embodiment, the variant comprises a heavy chain constant region mutation selected from those that reduce or eliminate ADCC function, such as, but not limited to, N297A, L234A, and L235A of IgG1.

[0083] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment, wherein the antibody is a humanized antibody, the heavy chain sequence is as shown in SEQ ID NO: 17 or has at least 85% sequence identity therewith; the light chain sequence is as shown in SEQ ID NO: 18 or has at least 85% sequence identity therewith.

[0084] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment, wherein the antibody is a humanized antibody, the heavy chain sequence is as shown in SEQ ID NO: 19 or has at least 85% sequence identity therewith; the light chain sequence is as shown in SEQ ID NO: 20 or has at least 85% sequence identity therewith.

[0085] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment, wherein the antibody is a humanized antibody, the heavy chain sequence is as shown in SEQ ID NO: 44 or has at least 85% sequence identity therewith; the light chain sequence is as shown in SEQ ID NO: 45 or has at least 85% sequence identity therewith.

[0086] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment, wherein the antibody is a humanized antibody, the heavy chain sequence is as shown in SEQ ID NO: 44 or has at least 85% sequence identity therewith; the light chain sequence is as shown in SEQ ID NO: 46 or has at least 85% sequence identity therewith.

[0087] In some embodiments, an isolated anti-IL-4R antibody or its antigen-binding fragment is provided, characterized in that it competes with any of the antibodies or antigen-binding fragments that bind human IL-4R as described above for binding human IL-4R.

[0088] 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 to human IL-4R according to the present application.

[0089] In other embodiments, a single-chain antibody is provided that contains a light chain variable region and / or a heavy chain variable region of an antibody or antigen-binding fragment of human IL-4R according to the present application.

[0090] In some embodiments, a polynucleotide is provided that encodes an antibody or antigen-binding fragment thereof that binds to human IL-4R according to this application.

[0091] In other embodiments, a polynucleotide is provided that encodes an antibody that competitively binds to IL-4R or its epitope, in accordance with the present application, against IL-4R or its antigen-binding fragment.

[0092] In other embodiments, a polynucleotide is provided that encodes the aforementioned bispecific antibody, multispecific antibody, or single-chain antibody.

[0093] In some embodiments, the polynucleotide according to this application is DNA or RNA.

[0094] In some embodiments, a vector containing the polynucleotides described above is provided, which is a eukaryotic expression vector, a prokaryotic expression vector, or a viral vector.

[0095] In some embodiments, a host cell is provided, which is transformed with the vector described above, wherein the host cell is selected from prokaryotic cells or eukaryotic cells.

[0096] In at least one embodiment, the prokaryotic cells are selected from bacteria, such as Escherichia coli.

[0097] In at least one embodiment, the eukaryotic cells are selected from yeast or mammalian cells, such as Pichia pastoris or CHO cells.

[0098] In some embodiments, a method for detecting or determining IL-4R is provided, the method comprising the step of contacting the above-described anti-IL-4R antibody or its antigen-binding fragment with a sample.

[0099] In some embodiments, a reagent for detecting or measuring human IL-4R is provided, the reagent comprising an anti-IL-4R antibody or an antigen-binding fragment thereof as described above.

[0100] In some embodiments, a reagent for detecting or measuring human IL-4R is provided, the reagent comprising an antibody that competitively binds to IL-4R or its epitope according to the present application.

[0101] In some embodiments, a reagent for detecting or measuring human IL-4R is provided, the reagent comprising the above-mentioned bispecific antibody, multispecific antibody, or single-chain antibody.

[0102] In some embodiments, a diagnostic agent is provided for diagnosing diseases associated with human IL-4R positive cells, the diagnostic agent comprising an anti-IL-4R antibody or an antigen-binding fragment thereof as described above. In other embodiments, the diagnostic agent comprises an antibody that competitively binds to IL-4R or an epitope thereof, in conjunction with an antibody or antigen-binding fragment thereof that binds to human IL-4R according to this application.

[0103] In some embodiments, a pharmaceutical composition is provided comprising:

[0104] -An antibody or antigen-binding fragment thereof that binds to human IL-4R according to this application, and

[0105] - Pharmaceutical excipients, diluents, or carriers.

[0106] In some specific embodiments, a unit dose of the pharmaceutical composition contains 1 mg to 1000 mg of the IL-4R antibody or its antigen-binding fragment according to this application.

[0107] In some specific embodiments, the concentration of the IL-4R antibody or its antigen-binding fragment in the pharmaceutical composition is from 1 mg / L to 1000 mg / L.

[0108] In some specific embodiments, the pharmaceutical composition contains a buffer in a concentration ranging from 1 mM to 1000 mM.

[0109] In some embodiments, the use of the antibody or antigen-binding fragment thereof that binds to human IL-4R as described above is provided in the preparation of a medicament for treating or preventing IL-4R-mediated diseases or conditions.

[0110] In some embodiments, the use of the pharmaceutical composition described above is provided in the preparation of a medicament for treating or preventing IL-4R-mediated diseases or conditions.

[0111] In some embodiments, the use of an antibody or antigen-binding fragment thereof that binds to human IL-4R as described above is provided for the treatment or prevention of a disease or condition.

[0112] In some embodiments, the pharmaceutical composition described above is provided for use in treating or preventing diseases or conditions.

[0113] In the context of this application, the condition or disease may be an immune disease or ailment.

[0114] In some implementations, the diseases or conditions mentioned are selected from: asthma, nasal polyps, chronic sinusitis, allergic dermatitis, 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 kidney disease.

[0115] In at least one implementation, the disease or condition is asthma.

[0116] In other embodiments, the disease or condition is an allergic skin disease.

[0117] In some embodiments, an antibody or antigen-binding fragment thereof that binds to human IL-4R is provided, wherein the antigen-binding fragment is Fab, Fv, sFv, or F(ab')2.

[0118] In some embodiments, a method for treating and / or preventing IL-4R-mediated diseases or conditions is provided, the method comprising: administering to a patient (or subject) in need a therapeutically effective amount (or a preventatively effective amount) of the human IL-4R-binding antibody or its antigen-binding fragment as described above.

[0119] In some embodiments, a method for treating and / or preventing IL-4R-mediated diseases or conditions is provided, the method comprising administering to a patient (or subject) in need a therapeutically effective amount (or a preventatively effective amount) of the pharmaceutical composition as described above. In some embodiments, a method for treating and / or preventing immune diseases is provided, comprising administering to a patient (or subject) in need a therapeutically effective amount (a preventatively effective amount) of an antibody or antigen-binding fragment thereof that binds to human IL-4R as described above, or a pharmaceutical composition thereof.

[0120] the term

[0121] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0122] "Human IL-4R" (hIL-4R) refers to the human cytokine receptor that specifically binds to interleukin-4 (IL-4) and IL-4Rα.

[0123] The amino acid three-letter codes and single-letter codes used in this article are as described in J. Biol. Chem, 243, p3558 (1968).

[0124] Antibodies refer to immunoglobulins, which are tetrapeptide chains composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain can further divide them into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ or λ chains based on differences in the constant region. Each of the five classes of Ig can have either a κ chain or a λ chain.

[0125] The antibody light chain may further include a light chain constant region, which comprises a human or mouse κ, λ chain or a variant thereof.

[0126] The antibody heavy chain may further include a heavy chain constant region, which contains human or mouse IgG1, IgG2, IgG3, IgG4 or variants thereof.

[0127] The sequence of approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains varies considerably and is known as the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are known as the constant region (C region). The variable region includes three hypervariable regions (HVRs) and four relatively conserved backbone regions (FRs). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of a light chain are light chain complementarity determination region 1 (LCDR1), light chain complementarity determination region 2 (LCDR2), and light chain complementarity determination region 3 (LCDR3); the three CDR regions of a heavy chain are heavy chain complementarity determination region 1 (HCDR1), heavy chain complementarity determination region 2 (HCDR2), and heavy chain complementarity determination region 3 (HCDR3).

[0128] Antibodies include murine antibodies, chimeric antibodies, humanized antibodies, and human antibodies, which can be obtained through recombinant synthesis, such as recombinant human antibodies obtained through affinity maturation.

[0129] "Recombinant human antibodies" include human antibodies prepared, expressed, created, or isolated by recombinant methods, the techniques and methods of which are well known in the art, such as (1) antibodies isolated from transgenic, transchromosomal animals (e.g., mice) or hybridomas prepared therefrom of human immunoglobulin genes; (2) antibodies isolated from host cells transformed to express antibodies, such as transgenic tumors; (3) antibodies isolated from recombinant human antibody libraries; and (4) antibodies prepared, expressed, created, or isolated by methods such as splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies contain variable and constant regions that utilize specific germline gene-encoded human germline immunoglobulin sequences, but also include subsequent rearrangements and mutations that occur during antibody maturation.

[0130] "Mouse antibody" herein refers to a monoclonal antibody against human IL-4R prepared in accordance with the knowledge and skills of the art. Preparation involves injecting the test subject with the IL-4R antigen or a polypeptide containing its epitopes, followed by isolation of hybridomas expressing antibodies with the desired sequence or functional characteristics. In some embodiments, the mouse antibody binding to human IL-4R or its antigen-binding fragment may further comprise a light chain constant region of a mouse κ, λ chain or a variant thereof, or further comprise a heavy chain constant region of a mouse IgG1, IgG2, IgG3, or IgG4 or a variant thereof.

[0131] "Human antibody" includes antibodies containing variable and constant regions of human germline immunoglobulin sequences. Human antibodies as described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or through somatic mutations in vivo). However, the term "human antibody" does not include antibodies in which a CDR sequence derived from another mammalian species (such as a mouse) has been grafted onto a human backbone sequence (i.e., "humanized antibody").

[0132] Humanized antibodies, also known as CDR-grafted antibodies, are antibodies created by grafting a non-human CDR sequence into the variable region framework of a human antibody. This overcomes the strong immune response induced by chimeric antibodies that carry a large amount of foreign protein components. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse mutations can be performed on the variable region of the human antibody to maintain its activity.

[0133] Chimeric antibodies are antibodies created by fusing the variable region of a murine antibody with the constant region of a human antibody. They can reduce the immune response induced by murine antibodies. To develop chimeric antibodies, hybridomas secreting murine-specific monoclonal antibodies are selected. The variable region gene is then cloned from mouse hybridoma cells, followed by the desired human antibody constant region gene. The mouse variable region gene and the human constant region gene are linked to form a chimeric gene, which is then inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic industrial system. The constant region of the human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 or their variants, preferably containing the heavy chain constant region of human IgG2 or IgG4. Alternatively, IgG1 with amino acid mutations that eliminates ADCC (antibody-dependent cell-mediated cytotoxicity) can be used.

[0134] "Antigen-binding fragment" refers to Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments and scFv fragments that bind to human IL-4R, and polypeptides or proteins containing the above fragments. The "antigen-binding fragment" contains one or more CDR regions of the antibody described herein. The Fv fragment contains variable regions of the antibody heavy chain and light chain, but no constant regions, and is the smallest antibody fragment with all antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding. Two antibody variable regions can also be linked into a single polypeptide chain using different linkers, called a single-chain antibody or single-chain Fv (scFv).

[0135] "Binding with IL-4R" refers to the ability to interact with human IL-4R. The term "antigen binding site" in this article refers to a three-dimensional spatial site recognized by the antibody or antigen-binding fragment described in this article.

[0136] An epitope is a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed from adjacent amino acids or from non-adjacent amino acids arranged side-by-side through the ternary folding of a protein. Epitopes formed from adjacent amino acids are generally retained after exposure to denaturing solvents, while epitopes formed through ternary folding are generally lost after treatment with denaturing solvents. Epitopes typically comprise at least 3-15 amino acids in a unique spatial conformation. Methods for determining which epitopes bind to a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of epitopes include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0137] "Specific binding" or "selective binding" refers to the binding of an antibody to a predetermined epitope on an antigen. Typically, when recombinant human IL-4R is used as the analyte and an antibody as the ligand, the antibody binds at approximately less than 10^6 mmol / L when measured in an instrument using surface plasmon resonance (SPR) technology. -7 M or even smaller equilibrium dissociation constant (K) D An antibody that recognizes a predetermined antigen binds to it, and its affinity for the predetermined antigen is at least twice that for a nonspecific antigen other than the predetermined antigen or closely related antigens (such as BSA). The term "antibody that recognizes an antigen" may be used interchangeably with the term "antibody that specifically binds."

[0138] “Cross-reactivity” refers to the ability of the antibody described herein to bind to IL-4R from different species. For example, the antibody described herein that binds to human IL-4R may also bind to IL-4R from another species. Cross-reactivity is measured by detecting specific reactivity with purified antigens, or binding or functional interactions with physiologically IL-4R-expressing cells, in binding assays such as SPR and ELISA. Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.

[0139] "Neutralizing" or "blocking" antibodies refer to antibodies whose binding to hIL-4R results in inhibition of the biological activity of hIL-4 and / or hIL-13. This inhibition of hIL-4 and / or IL-13 biological activity can be assessed by measuring one or more well-known indicators of hIL-4 / or hIL-13 biological activity, such as hIL-4 / or hIL-13-induced cell activation and the binding of hIL-4 to hIL-4R, for example, see CN103739711A. "Inhibition of growth" (e.g., involving cells) is intended to include any measurable reduction in cell growth.

[0140] "Inducing an immune response" and "enhancing an immune response" are used interchangeably and refer to the stimulation of an immune response by a specific antigen (i.e., passive or adaptive). In the context of inducing CDC or ADCC, "inducing" refers to stimulating a specific direct cell-killing mechanism.

[0141] "ADCC (antibody-dependent cell-mediated cytotoxicity)" refers to the direct killing of antibody-coated target cells by cells expressing Fc receptors through recognition of the Fc fragment of antibodies. The ADCC effector function of antibodies can be reduced or eliminated by modifying the Fc fragment of IgG. This modification refers to mutations in the constant region of the antibody's heavy chain, such as those selected from N297A, L234A, and L235A of IgG1; IgG2 / 4 chimeras; F235E of IgG4; or L234A / E235A mutations.

[0142] A fusion protein is a protein product obtained through DNA recombination and co-expression of two genes. The recombinant IL-4R extracellular region Fc fusion protein is a fusion protein obtained through DNA recombination and co-expression of the IL-4R extracellular region and a human antibody Fc fragment. The IL-4R extracellular region refers to the portion of the IL-4R protein expressed outside the cell membrane.

[0143] Methods for producing and purifying antibodies and antigen-binding fragments are well-known and readily available in existing technologies, such as Cold Spring Harbor's Guide to Antibody Experimentation, Chapters 5-8 and 15. For example, mice can be immunized with human IL-4R or fragments thereof, and the resulting antibodies can be refolded, purified, and sequenced amino acids using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described herein are prepared by adding one or more human FR regions to non-human CDR regions using genetic engineering methods. Human FR germline sequences are available from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr or from the Immunoglobulin Journal, 2001 ISBN012441351.

[0144] Engineered antibodies or antigen-binding fragments can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. Using molecular cloning techniques, the sequences of the humanized antibodies described in this paper are inserted into the corresponding expression vectors, and the corresponding humanized antibodies are produced using the HEK293 cell expression system. As a more recommended existing technique, mammalian expression systems lead to glycosylation of the antibodies, particularly at the highly conserved N-terminus of the FC region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. Cultures secreting antibodies can be purified and collected using conventional techniques. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving and ion exchange. The resulting product should be immediately frozen, such as at -70°C, or lyophilized.

[0145] Antibodies can be monoclonal antibodies (mAbs), which are antibodies obtained from a single clonal cell line. These cell lines are not limited to eukaryotic, prokaryotic, or phage clonal cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained through recombination using techniques such as hybridoma technology, recombinant technology, phage display technology, synthetic techniques (such as CDR-grafting), or other existing technologies.

[0146] Antibodies can be monospecific, bispecific, or multispecific. Multispecific antibodies may exhibit specificity for different epitopes of a target peptide, or may contain antigen-binding domains that exhibit specificity for more than one target peptide. Human anti-IL-4R antibodies may be linked to or co-expressed with another functional molecule (such as another peptide or protein). For example, antibodies or fragments thereof may be functionally (e.g., through chemical coupling, gene fusion, non-covalent binding, or other means) linked to one or more other molecules (such as another antibody or antigen-binding fragment) to produce bispecific or multispecific antibodies with a second binding specificity.

[0147] "Administration," "giving," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "giving," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Administration," "giving," and "treatment" also mean the treatment of, for example, cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. "Treatment," when applied to humans, veterinary, or research subjects, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0148] "Treatment" means administering an oral or topical therapeutic agent, such as a composition containing any of the antibodies or antigen fragments thereof described herein, to a patient (or subject) who has, is suspected of having, or is susceptible to symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient (or subject) or population in an amount that effectively relieves symptoms of one or more diseases, whether by inducing the regression of such symptoms or inhibiting their progression to any clinically measurable extent. The amount of a therapeutic agent that effectively relieves symptoms of any specific disease (also referred to as the "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's (or subject's) disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient (or subject). Whether the symptoms have been relieved can be evaluated using any clinical test that a physician or other healthcare professional typically uses to assess the severity or progression of the symptoms. Although the implementation method described herein (e.g., treatment method or product) may be ineffective in alleviating the symptoms of the target disease in a single patient (or subject), it should reduce the symptoms of the target disease in a statistically significant number of patients (or subjects) according to any statistical test known in the art, such as the Student t test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0149] "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, main chain conformation, and rigidity), allowing for frequent alterations without changing the protein's biological activity. Those skilled in the art will recognize that, in general, the substitution of a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to disrupt biological activity.

[0150] An "effective dose" includes a dose sufficient to improve or prevent the symptoms or condition of a medical condition. An effective dose also means a dose sufficient to allow or facilitate diagnosis. The effective dose for a specific subject or veterinary subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective dose may be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0151] "Exogenous" refers to substances that are produced outside an organism, cell, or human body, depending on the context.

[0152] "Endogenous" refers to substances that are produced in cells, organisms, or the human body in accordance with their context.

[0153] "Homology" or "identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if every position in two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage of 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, multiplied by 100%. For example, at optimal sequence alignment, if six out of ten positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of homology is obtained by aligning the two sequences. The term "at least 85% sequence identity" as used herein refers to a variant sequence having at least 85% homology with the parent sequence. In some schemes, this homology is at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%; in some specific schemes, it is 90%, 95%, or more than 99%; and in other specific schemes, it is at least 95% sequence homology. The amino acid sequence having at least 85% sequence identity includes sequences obtained by performing one or more amino acid deletions, insertions, or substitutions on the parent sequence.

[0154] The terms “cell,” “cell line,” and “cell culture” used herein are used interchangeably, and all such names include their progeny. Therefore, the terms “transformation” and “transformed cell” include primary test cells and cultures derived from them, regardless of the number of transfections. It should also be understood that, due to intentional or unintentional mutations, all progeny cannot be exactly identical in terms of DNA content. This includes mutant progeny with the same function or biological activity as those screened from the original transformed cells.

[0155] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur. For example, "optionally contains 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a particular sequence may, but do not have to, be present.

[0156] "Pharmaceutical composition" means a mixture containing one or more antibodies or antigen-binding fragments described herein, or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity. Attached Figure Description

[0157] Figure 1 In a mouse dermatitis model, after sensitization with acetone, humanized antibodies hu25G7-A and hu25G7-B, and the positive control antibody dupilumab were administered subcutaneously twice a week. Ear thickness was measured on day 27. Results showed that, compared to the control group, hu25G7-A, hu25G7-B, and dupilumab all effectively reduced ear thickness in mice, with hu25G7-B showing a superior effect compared to dupilumab. Detailed Implementation

[0158] The following examples are used to further describe this disclosure, but these examples are not intended to limit the scope of this disclosure. Experimental methods not specifically described in the examples are generally performed under conventional conditions, such as those described in Cold Spring Harbor's Antibody Technology Manual or Molecular Cloning Manual; or under conditions recommended by the raw material or commercial manufacturer. Reagents not specifically named are commercially available, conventional reagents.

[0159] Example 1: Immunization and Detection in Mice

[0160] The his-tagged human IL-4R (h-IL-4R-his) recombinant protein, the his-tagged mouse IL-4R (m-IL-4R-his) recombinant protein, and the his-tagged rhesus monkey IL-4R (rhesus-IL-4R-his) recombinant protein were synthesized, expressed, and purified by Acrobiosystems using HEK293.

[0161] The recombinant human IL-4R (h-IL-4R-Fc) protein with a human Fc tag was designed, expressed, and purified in-house. The purified protein was used in the experiments described in the following examples.

[0162] In this embodiment, the number and position of CDR amino acid residues in the VL and VH regions of the antibody or antigen-binding fragment conform to the known Kabat numbering rules (LCDR1-3, HCDR2-3), or conform to the Kabat and CHOTHIA (ABM) rules (HCDR1).

[0163] Table 1. Immunogen Information

[0164] 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

[0165] Anti-human IL4R monoclonal antibody was produced by immunizing mice. The experimental mice used were female C57BL / 6 mice, 6–8 weeks old (Suzhou Zhaoyan New Drug Research Center Co., Ltd., Animal Production License No.: 201503052).

[0166] Housing environment: SPF grade. Mice were housed in a laboratory environment for one week after purchase, with a 12 / 12-hour light / dark cycle, temperature 20-25℃, and humidity 40-60%. Once acclimatized, the mice were divided into three cages of five mice each. The immunogen was Fc-tagged recombinant human IL-4R protein (h-IL4R-Fc, concentration 0.73 mg / ml). Emulsification was performed using Freund's adjuvant (Sigma, Cat#: F5881): initial immunization used Freund's complete adjuvant (CFA, Pierce, Cat#77140), followed by booster immunizations using nucleic acid adjuvants (CpG, Shanghai Sangon Biotech) and aluminum adjuvants (Alum, Thermo Cat#77161).

[0167] On day 0, mice were injected intraperitoneally (IP) with 70 μg / mouse of emulsified antigen. On days 14, 28, 42, 56, and 77, based on the presence of back masses and abdominal swelling, 0.1 ml of antigen was injected into the back and intraperitoneally respectively. Blood samples were collected on days 21, 35, 49, 63, and 84 for serum analysis. The antibody titer in the mouse serum was determined using the ELISA method described in Test Example 1. After the fourth immunization, mice with high antibody titers that were trending towards a plateau were selected for spleen cell fusion. Three days prior to fusion, a booster immunization was administered via intraperitoneal (IP) injection of 10 μg / mouse of an antigen solution prepared with phosphate-buffered saline. An optimized PEG-mediated fusion procedure was used to fuse spleen lymphocytes with myeloma Sp2 / 0 cells (…). CRL-8287 TM Hybridoma cells are obtained by fusing them together.

[0168] Example 2: ELISA testing and screening of antibodies

[0169] 1. ELISA combined with experiment:

[0170] ELISA assays are used to detect the binding properties of IL-4R antibodies. Microplates directly coated with his-tagged recombinant IL-4R protein are used. After adding antibodies to each well, the antibody-antigen binding activity is detected by adding secondary antibody (HRP-conjugated anti-primary Fc antibody) and HRP substrate TMB.

[0171] Coat 96-well microplates with human or rhesus monkey IL-4R-his protein at a concentration of 0.5 μg / ml (100 μl per well) and incubate overnight at 4°C. Wash three times with 250 μl of washing buffer per well, vortexing for 10 seconds each time to ensure thorough washing. Add 200 μl / well blocking buffer and incubate at room temperature for 2 hours. Wash three times with 250 μl of washing buffer per well, vortexing for 10 seconds each time to ensure thorough washing. Add 100 μl of the anti-IL-4R antibody diluted with diluent to each well. Incubate at room temperature for 1 hour. Wash three times with 250 μl of washing buffer per well. Add 100 μl of HRP-labeled goat anti-human IgG secondary antibody diluted 1:20000 with diluent to each well. Incubate at room temperature for 1 hour. Wash three times with 250 μl of washing buffer per well. Add 100 μl of TMB to each well and incubate in the dark for 15 minutes. Add 50 μl of 0.16 M / L sulfuric acid to each well. Use a ThermoMultiSkanFc microplate reader to read the OD value at 450 nm and calculate the EC50 of the IL-4R antibody binding to IL-4R. 50 value.

[0172] 2. ELISA blocking assay:

[0173] This experiment used an in vitro blocking assay to detect the blocking effect of screened anti-human IL-4R antibodies on the binding of human IL-4R and human IL-4. Specifically, Fc-tagged recombinant IL-4R protein was coated onto a 96-well microplate. After the antibody binding to human IL-4R fully occupied the epitope, IL-4 (Biolegend, Cat#574004) was added. The binding of IL-4 to IL-4R was then detected using a biotin-conjugated anti-IL-4 antibody and Neutravidin-HRP (Pierce, Cat#31001). The IC50 of the IL-4R antibody on the blocking effect of IL-4R binding was calculated. 50 value.

[0174] Human IL-4R-Fc protein was coated onto 96-well microplates at a concentration of 0.5 μg / ml (100 μl per well) and incubated overnight at 4°C. Wash three times with 250 μl of washing buffer per well, vortexing for 10 seconds each time to ensure thorough washing. Add 200 μl / well blocking buffer and incubate at room temperature for 2 hours. Wash three times with 250 μl of washing buffer per well, vortexing for 10 seconds each time to ensure thorough washing. Add 100 μl of diluted anti-IL-4R antibody to each well and incubate at room temperature for 1 hour. Wash three times with 250 μl of washing buffer per well. Add 100 μl of diluted IL-4 to each well and incubate at room temperature for 1 hour, then wash three times with washing buffer. Add 100 μl of diluted biotin-conjugated anti-IL-4 antibody to each well and incubate at room temperature for 1 hour, then wash three times with washing buffer. Incubate with HRP-labeled Neutravidin diluted 1:5000 using dilution buffer for 1 hour at room temperature. Wash three times with washing buffer, 250 μl per well. Add 100 μl of TMB to each well and incubate in the dark for 15 minutes. Add 50 μl of 0.16 M / L sulfuric acid to each well. Read the OD value at 450 nm using a Thermo MultiSkanFc microplate reader and calculate the IC50 of the IL-4R antibody blocking the binding of IL-4R and IL-4. 50 value.

[0175] Example 3: Reporter gene activity assay using antibodies binding to human IL-4R

[0176] HEK-Blue IL-4 cells were purchased from Invivogen (Cat#hkb-stat6). These cells were stably transfected with the human IL-4R gene and the STAT6-mediated SEAP genome. The activation level of the IL-4R signaling pathway can be characterized by detecting the secreted SEAP in the supernatant using the SEAP substrate QUANTI-Blue.

[0177] This experiment detected the activation of HEK-Blue IL-4 in cells, based on IC50. 50The in vitro cellular viability of IL-4R antibodies was evaluated by size. HEK-Blue IL-4 cells were cultured in DMEM medium containing 10% FBS, 100 μg / ml Zeocin (Invivogen, Cat#ant-zn-05), and 10 μg / ml Blastidin (Invivogen, Cat#ant-bl-05); passaged 2–3 times per week at a passage ratio of 1:5 or 1:10. At passage, the medium was aspirated, the cell layer was washed with 5 ml of 0.25% trypsin, the trypsin was then aspirated, and the cells were digested in an incubator for 3–5 minutes. The cells were then resuspended in fresh medium. 100 μL of the cell suspension was added to each 96-well cell culture plate at a density of 5 × 10⁻⁶ cells / well. 5 Cells / ml, culture medium was DMEM containing 10% FBS, 100ug / ml Zeocin and 30ug / ml Blasticidin, with only 100μl of sterile water added to the periphery of the 96-well plate. The plate was incubated for 24 hours (37℃, 5% CO2). After cell attachment, 100μl of serially diluted test antibody was added to each well. The plate was incubated for 20-24 hours (37℃, 5% CO2). 20μl of cell supernatant was transferred from each well to a new 96-well plate, along with 180μl of QUANTI-Blue substrate solution. The plate was incubated in the dark for 1-3 hours. The absorbance at 620nm was measured using a Thermo MultiSkan FPGA.

[0178] Example 4: Experiment on the inhibition of TF-1 cell proliferation by antibody binding to human IL-4R

[0179] TF-1 cells (ATCC CRL-2003) are lymphoma cells expressing IL-4R and sensitive to cytokines such as IL-4 / IL-13. IL-4 can stimulate the proliferation of TF-1 cells in the absence of GM-CSF. This experiment aimed to inhibit TF-1 cell proliferation by adding neutralizing antibodies to block the IL-4 pathway and compare the neutralizing activity of different IL-4R antibodies. TF-1 cells were cultured in RPMI 1640 medium containing 10% FBS and 2 ng / ml GM-CSF (R&D, Cat#215-GM-010); passaged 2-3 times per week at a passage ratio of 1:10. 100 μL of cell suspension was added to each 96-well cell culture plate at a density of 2 × 10⁻⁶ cells / well. 5Cells / ml, cultured in 10% FBS RPMI 1640 medium, with only 100 μl of sterile water added to the periphery of the 96-well plate. 50 μl of serially diluted test antibody and 50 μl of IL-4 (R&D, Cat#204-IL-050) at a final concentration of 0.7 ng / ml were added to each well. The plate was incubated for 72 hours (37°C, 5% CO2). After incubation, cell proliferation was detected using a CTG kit (Promega, Cat#G7572).

[0180] Example 5: In vitro binding affinity and kinetics experiment

[0181] The affinity of the humanized antibody to human IL-4R for human IL-4R was determined using a Biacore GE instrument.

[0182] Following the instructions for the Human Anti-Capture Kit (Cat.#BR-1008-39, GE), the human anti-capture antibody was covalently coupled to the CM5 biosensor chip of a Biacore instrument (Biacore X100, GE) to affinity capture a certain amount of the test antibody. Then, a series of concentration gradients of IL-4R antigen (all IL-4R antigens were purchased from Acrobiosystems, Cat#ILR-H5221) were passed through the chip surface. The reaction signal was monitored in real time using the Biacore instrument (Biacore X100, GE) to obtain binding and dissociation curves. After each cycle of dissociation, the biochip was regenerated by washing with the regeneration solution provided in the human anti-capture kit. The amino-coupling kit used in the experiment was purchased from GE (Cat.#BR-1000-50, GE), and the buffer solution was HBS-EP + 10× buffer solution (Cat.#BR-1006-69, GE) diluted to 1X (pH 7.4) with DIWater.

[0183] The experimental data were fitted using the BiacoreX100 evaluation software 2.0 GE software with a (1:1) Binding model to obtain the affinity values.

[0184] Example 6: Antibody Sequence and Preparation

[0185] Through the ELISA binding assay (ELISA binding of human IL-4R-his) and ELISA blocking assay (ELISA blocking of human IL-4 / IL-4R) in Example 2, the experiment in Example 3 inhibiting the activation of HEK293-Blue IL-4 cells under IL-4 stimulation, and the experiment in Example 4 inhibiting the proliferation of TF-1 cells under IL-4 stimulation, the two monoclonal hybridoma cell lines with the best in vitro activity were selected. Their activity detection results are shown in Table 2.

[0186] Table 2. Selection of hybridoma cell lines with the best in vitro activity

[0187]

[0188] The monoclonal hybridoma cell lines 25G7 and 7B10, which exhibited the best in vitro activity, were selected, and their monoclonal antibody sequences were cloned. The sequence cloning process from the hybridomas is as follows: Hybridoma cells in the logarithmic growth phase were collected, and RNA was extracted using Trizol (Invitrogen, 15596-018) (following the kit instructions). Reverse transcription (PrimeScript) was then performed. TM Reverse Transcriptase (Takara, cat#2680A). The cDNA obtained by reverse transcription was amplified by PCR using a mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503) and then sent to a sequencing company for sequencing. The resulting antibody sequence was then analyzed.

[0189] The heavy and light chain variable region sequences of mouse monoclonal antibody 25G7 are as follows:

[0190] 25G7 HCVR

[0191] EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYGMHWVRQAPEKGLEWVAFISSGSSIIYYADIVKGRSTISRDNAKNTLFLQMTSLRSEDTAMYYCTRGNKRGFFDYWGQGTILTVSS(SEQ ID NO: 1)

[0192] 25G7 LCVR

[0193] QIVLTQSPALMSASPGEKVTMTCNASSSVSYMYWYQRKPRSSPKPWIYLTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWRSNPPMLTFGSGTKLEVK(SEQ ID NO: 2)

[0194] The CDR sequences it contains are shown in Table 3.

[0195] Table 3. CDR sequence of monoclonal antibody 25G7

[0196]

[0197]

[0198] The heavy and light chain variable region sequences of mouse monoclonal antibody 7B10 are as follows:

[0199] 7B10 HCVR

[0200] QVQLQQPGTELLKPGASVSLSCKASGYTFTSYWMHWVKQRPGQGLEWIGLIHPNSDTTKFSENFKTRATLTIDKSSSTAYMKLSSSLTSEDSAVYYCAKSKIITTIVARHWYFDVWGTGTTVTVSS (SEQ ID NO: 9)

[0201] 7B10 LCVR

[0202] DIVLTQSPPSLAVSLGQRATISCKASQSVDYGGDSYMNWYQQKLGQPPKVLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDVATYYCQHSNENPPTFGGGTKLEIK(SEQ ID NO: 10)

[0203] The CDR sequences it contains are shown in Table 4.

[0204] Table 4. CDR sequence of monoclonal antibody 7B10

[0205] 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

[0206] The obtained variable region sequence was ligated into the human constant region sequence to obtain the human-mouse chimeric antibody sequence. Using molecular cloning technology, the chimeric antibody sequence was inserted into the corresponding expression vector. Using the HEK293 cell expression system, the human-mouse chimeric antibodies 25G7-C and 7B10-C were obtained.

[0207] The purified chimeric antibody was subjected to in vitro activity assays using the methods described in Examples 2-5 above, and the data are shown in Table 5. The results showed that the 25G7-C antibody significantly outperformed the reference antibody dupilumab in blocking IL-4 binding and inhibiting cell proliferation (as described in WHO Drug Information, Vol. 26, No. 4, 2012).

[0208] Table 5. In vitro activity assay

[0209]

[0210]

[0211] Example 7: Mouse Antibody Humanization Experiment

[0212] The two most functionally active murine antibodies (25G7 and 7B10) were humanized. Based on the typical VH / VLCDR structures of the obtained murine antibodies, the variable region sequences of the heavy and light chains were compared with the antibody Germline database to obtain highly homologous human germline templates. The human germline light chain framework region was derived from the human κ light chain gene, with the preferred human germline light chain templates IGKV3-11*01 (SEQ ID NO: 22, used for antibody 25G7) and IGKV2D-29*01 (SEQ ID NO: 24, used for antibody 7B10). The human germline heavy chain framework region was derived from the human heavy chain, with the preferred human germline heavy chain templates IGHV3-48*01 (SEQ ID NO: 21, used for antibody 25G7) and IGHV1-2*02 (SEQ ID NO: 23, used for antibody 7B10).

[0213] The racial template sequence is shown below.

[0214] Ethnographic heavy chain template IGHV3-48*01:

[0215] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVS YISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ IDNO: 21)

[0216] Ethnic Light Link Template IGKV3-11*01:

[0217] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDAS NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWP (SEQ ID NO: 22)

[0218] Ethnographic heavy chain template IGHV1-2*02:

[0219] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEW MGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR(SEQ ID NO: 23)

[0220] Humanoid Light Chain Template IGKV2D-29*01:

[0221] DIVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTYLYWYLQKPGQPPQL LIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSIQLP (SEQ IDNO: 24)

[0222] The CDR region of the murine antibody was transplanted onto a selected humanized template, replacing the humanized variable region, and then recombined with the IgG constant region. Then, based on the three-dimensional structure of the murine antibody, reverse mutations were performed on embedded residues, residues that directly interact with the CDR region, and residues that significantly affect the conformation of VL and VH, resulting in a series of humanized molecules.

[0223] Specifically, Hu7B10-VH-a, hu7B10-VH-b, and hu7B10-VH-c underwent pharmacodynamic modifications, changing the first digit of the human heavy chain template from Q to E. Hu25G7 also underwent pharmacodynamic modifications. The humanized heavy chain variable region sequences of the two antibodies are shown in SEQ ID NO: 25-27 and SEQ ID NO: 31-33, respectively; the light chain variable region sequences are shown in SEQ ID NO: 28-30 and SEQ ID NO: 34-36, respectively.

[0224] hu25G7-VH-a:

[0225] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQ A PGKGLEWVAFISSGSSIIYYADIVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGNKRGFFDYWGQGTLVTVSS(SEQ ID NO: 25)

[0226] hu25G7-VH-b:

[0227] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQ A PGKGLEWVAFISSGSSSIIYYADIVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC T RGNKRGFFDYWGQGTLVTVSS(SEQ ID NO: 26)

[0228] hu25G7-VH-c:

[0229] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQA PGKGLEWVAFISSGSSIIYYADIVKGR S TISRDNAKNSLYLQMNSLRAEDTAVYYC T RGNKRGFFDYWGQGTLVTVSS(SEQ ID NO:27)

[0230] hu25G7-VL-a:

[0231] EIVLTQSPATLSLSPGERATLSCNASSSVSYMYWYQQKPGQAPRLLIYLTSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWRSNPPMLTFGGGTKVEIK(SEQ ID NO:28)

[0232] hu25G7-VL-b:

[0233] EIVLTQSPATLSLSPGERATLSCNASSSVSYMYWYQQKPGQAPRLLIYLTSNLASGIPARFSGSGSGTD Y TLTISSLEPEDFAVYYCQQWRSNPPMLTFGGGTKVEIK(SEQ ID NO:29)

[0234] hu25G7-VL-c:

[0235] EIVLTQSPATLSLSPGERATLSCNASSSVSYMYWYQQKPGQAPR PW IYLTSNLASGIPARFSGSGSGTD Y TLTISSLEPEDFAVYYCQQWRSNPPMLTFGGGTKVEIK(SEQ ID NO:30)

[0236] hu7B10-VH-a:

[0237] EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGLIHPNSDTTKFSENFKTRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSKIITTIVARHWYFDVWGQGTTVTVSS(SEQ ID NO:31)

[0238] hu7B10-VH-b:

[0239] EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGLIHPNSDTTKFSENFKTRVTMTIDTSISTAYMELSRLRSDDTAVYYCAKSKIITTIVARHWYFDVWGQGTTVTVSS(SEQ ID NO: 32)

[0240] hu7B10-VH-c:

[0241] EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGLIHPNSDTTKFSENFKTRVTLTIDKSISTAYMELSRLRSDDTAVYYCAKSKIITTIVARHWYFDVWGQGTTVTVSS(SEQ ID NO: 33)

[0242] hu7B10-VL-a:

[0243] DIVMTQTPLSLSVTPGQPASISCKASQSVDYGGDSYMNWYLQKPGQPPQLLIYAASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQHSNENPPTFGG GTKVEIK(SEQ ID NO: 34)

[0244] hu7B10-VL-b:

[0245] DIVLTQTPLSLSVTPGQPASISCKASQSVDYGGDSYMNWYLQKPGQPPQLL IYAASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQHSNENPPTFGGG TKVEIK(SEQ ID NO: 35)

[0246] hu7B10-VL-c:

[0247] DIVMTQTPLSLSVTPGQPASISCKASQSVDYGGDSYMNWYLQKPGQPPQL LIYAASNLESGIPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQHSNENPPTFGGG TKVEIK(SEQ ID NO: 36)

[0248] The template selection and back mutation design for hybridoma clone 25G7 are shown in Table 6.

[0249] Table 6. Template Selection and Reverse Mutation Design

[0250]

[0251] Template selection and reverse mutation design for hybridoma clone 7B10 are shown in Table 7 below:

[0252] Table 7. Template Selection and Reverse Mutation Design

[0253]

[0254] Based on the small-scale expression tests and comparisons of the number of reversion mutations of the antibodies in each of the above light and heavy chain combinations, the final humanized antibody hu25G7 (using VH-c heavy chain and VL-a light chain) and hu7B10 antibody molecule (using VH-b heavy chain and VL-b light chain) were selected through comprehensive evaluation. Their complete light and heavy chain sequences are shown in SEQ ID NO: 17-20.

[0255] hu25G7 HC

[0256] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAFISSGSSIIYYADIVKGRSTISRDNAKNSLYLQMNSLRAEDTAVYYCTRGNKRGFFDYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLP SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK (SEQ ID NO: 17)

[0257] hu25G7 LC

[0258] EIVLTQSPATLSLSPGERATLSCNASSSVSYMYWYQQKPGQAPRLLIYLTSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWRSNPPMLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQID NO:18)

[0259] hu7B10 HC

[0260] EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGLIHPNSDTTKFSENFKTRVTMTIDTSISTAYMELSRLRSDDTAVYYCAKSKIITTIVARHWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:19)

[0261] hu7B10 LC

[0262] DIVLTQTPLSLSVTPGQPASISCKASQSVDYGGDSYMNWYLQKPGQPPQLLIYAASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQHSNENPPTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:20)

[0263] By using molecular cloning technology, the sequence of a humanized antibody is inserted into a corresponding expression vector, and then expressed and produced using the HEK293 cell expression system, the corresponding humanized antibody can be obtained.

[0264] Example 8: Humanized Antibody Activity Data

[0265] The in vitro activity assays of the humanized antibodies hu25G7 and hu7B10, as described in Examples 2-5, were performed, and the results are shown in Table 8. The results showed that both hu25G7 and hu7B10 bound only to human IL-4R and not to rhesus monkey IL-4R, indicating that both antibodies bound to epitopes different from those in humans and rhesus monkeys, and could specifically bind to human IL-4R. Both antibodies blocked IL-4 / IL-4R binding and intracellular signaling pathways, leading to the neutralization of IL-4 activation and inhibition of TF-1 cell proliferation. The blocking and inhibitory activities of hu25G7 were significantly superior to those of the reference antibody dupilumab, but the affinity K... D The value is relatively low.

[0266] Table 8. In vitro activity assay

[0267]

[0268]

[0269] Example 9: Affinity maturation experiment of humanized antibody hu25G7

[0270] To obtain a more effective anti-human IL-4R antibody, affinity maturation of the 25G7 antibody was performed using a yeast display platform. Based on the hu25G7 antibody, affinity-matured yeast libraries targeting six CDRs were designed and prepared. Degenerate primers were designed, and designed mutant amino acids were introduced into the hu25G7-scFv antibody library via PCR and homologous recombination. Each library was approximately 10 μL in size. 9The constructed yeast library was then validated for diversity using next-generation sequencing (GENEWIZ).

[0271] High-affinity antibodies against human IL-4R were screened from the hu25G7-scFv yeast library using biotin-labeled human IL-4R. After two rounds of MACS screening (streptomycin magnetic beads, Invitrogen) and two rounds of FACS screening (BD FACSAria™ FUSION), yeast clones were selected, cultured, and induced to express the antibodies. The binding of yeast clones to human IL-4R was detected using FACS (BD FACSCanto II). Yeast clones with higher affinity than wild-type 25G7 antibodies were selected for sequencing verification. After alignment analysis of the sequenced clones and removal of redundant sequences, the non-redundant sequences were converted into full-length human antibody molecules for expression in mammalian cells. The affinity-purified full-length antibodies were then analyzed using a BIAcore™ X-100 (GE Life Sciences) to determine their affinity, and candidate antibody molecules with high affinity for human IL-4R were selected as follows. These antibody molecules have a higher affinity for human IL-4R than wild-type hu25G7 antibodies. Among them, the affinity of hu25G7-A antibody molecules is comparable to that of dupilumab, while the affinity of hu25G7-B molecules is significantly better than that of dupilumab.

[0272] The light chain variable region sequence of antibody hu25G7-A, obtained after affinity maturation, is as follows:

[0273] hu25G7-A LCVR

[0274] The CDR sequence contained in EIVLTQSPATLSLSPGERATLSCRASSSVPYMYWYQQKPGQAPRLLIYLTSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWRAYPPMLTFGGGTKVEIK (SEQ ID NO: 37) is shown in Table 9.

[0275] Table 9. CDR Sequences

[0276] LCDR1 RASSSVPYMY SEQ ID NO: 38 LCDR2 LTSNLAS SEQ ID NO: 7 LCDR3 QQWRAYPPMLT SEQ ID NO: 40

[0277] The light chain variable region sequence of antibody hu25G7-B is as follows:

[0278] hu25G7-B LCVR

[0279] EIVLTQSPATLSLSPGERATLSCRASPGVPPLAWYQQKPGQAPRLLIYLASSRPSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWRSNPPMLTFGGGTKVEIK(SEQ ID NO: 41)

[0280] The CDR sequences it contains are shown in Table 10.

[0281] Table 10. CDR Sequences

[0282]

[0283]

[0284] The above-mentioned light chain variable region hu25G7-A LCVR recombines with the hu25G7 light chain constant region to obtain the hu25G7-A antibody light chain; the above-mentioned light chain variable region hu25G7-B LCVR recombines with the hu25G7 light chain constant region to obtain the hu25G7-B antibody light chain.

[0285] Optimization of unstable amino acid residues in hu25G7-VH-c enhances drug-like properties, yielding the heavy chain variable region hu25G7-VH.

[0286] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVA FISSGSSSIIYYADIVKGRSTISRDNAKNTLYLQMNSLRAEDTAVYYCTRGNKRGF FDYWGQGTLVTVSS(SEQ ID NO: 43)

[0287] The aforementioned variable region of the heavy chain can recombine with the constant region of the hu25G7 heavy chain to obtain the hu25G7-A / hu25G7-B antibody heavy chain.

[0288] The complete heavy chain sequences of hu25G7-A and hu25G7-B are shown in SEQ ID NO: 44.

[0289] hu25G7 HC

[0290] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAFISSGSSIIYYADIVKGRSTISRDNAKNTLYLQMNSLRAEDTAVYYCTRGNKRGFFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 44)

[0291] The respective complete light chain sequences are shown in SEQ ID NO: 45-46.

[0292] hu25G7-A LC

[0293] EIVLTQSPATLSLSPGERATLSCRASSSVPYMYWYQQKPGQAPRLLIYLTSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWRAYPPMLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 45)

[0294] hu25G7-B LC

[0295] EIVLTQSPATLSLSPGERATLSCRASPGVPPLAWYQQKPGQAPRLLIYLASSRPSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWRSNPPMLTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQID NO:46)

[0296] Example 10: Humanized Antibody Affinity Maturation Activity Data

[0297] Examples 3 and 4 tested the two antibodies hu25G7-A and hu25G7-B. Both antibodies hu25G7-A and hu25G7-B could block the binding of IL-4 / IL-4R and neutralize the activation of IL-4 and IL-13 caused by intracellular signaling pathways, thus inhibiting the proliferation of TF-1 cells. The activity data are shown in Table 11.

[0298] Table 11. Comparison of Activity Data

[0299]

[0300] In the experiment inhibiting IL-13-induced TF-1 cell proliferation, both hu25G7-A and hu25G7-B showed beneficial effects. Repeated verification of these effects showed that, under the same conditions, hu25G7-A significantly inhibited IL-13-induced proliferation in TF-1 cells (IC50-90%). 50 The value was 11.68, indicating that dupilumab inhibited IL-13 proliferation in TF-1 cells (IC50). 50 The value was 22.85. Hu25G7 bidupilumab significantly enhanced the blocking effect of IL-4, IL-13, and IL-4R on cell proliferation induced by these bindings.

[0301] Example 11: Study on the effect of humanized antibodies on mouse dermatitis

[0302] Establishment of a mouse dermatitis model: IL-4 / IL-4Rα transgenic mice (purchased from Cyagen Model Organisms Research Center (Taicang) Co., Ltd.) were used. Each mouse had an abdominal area of ​​approximately 3cm × 3cm. 100μL of a 1.5% OXZ acetone-olive oil solution (acetone:olive oil = 4:1) was applied evenly to sensitize the mice, and the day of sensitization was designated as Day 0. Starting on Day 7, 20μL of a 1% OXZ acetone-olive oil solution was applied evenly to both ears (both sides) for challenge, with challenges occurring every 72 hours.

[0303] This experiment included a normal control group (treated with only acetone-olive oil solution during sensitization and challenge), a model control group, and hu25G7-A, hu25G7-B, and dupilumab groups, for a total of 5 groups, with 3-5 mice in each group. The dosage for the treatment groups was 50 mg / kg, administered subcutaneously twice a week (details are shown in Table 12). Ear thickness was measured using calipers on day 27, and the results are as follows. Figure 1 As shown.

[0304] Table 12. Application plans for each group

[0305] normal control group 3 (male) SC - Twice a week Model control group 5(3 females + 2 males) SC - Twice a week hu25G7-A 5(3 females + 2 males) SC 50mg / kg Twice a week hu25G7-B 3(2 females + 1 male) SC 50mg / kg Twice a week Dupilumab 4(2 females + 2 males) SC 50mg / kg Twice a week

[0306] The results showed that the ears of mice in the model control group had significant pathological damage, and the ear thickness was significantly higher than that of the normal control group. On day 27, the ear thickness of mice in the hu25G7-A, hu25G7-B, and dupilumab groups was significantly lower than that of the model control group. This indicates that hu25G7-A, hu25G7-B, and dupilumab can all be used to treat dermatitis, and hu25G7-B is more effective than dupilumab.

Claims

1. An anti-IL-4R antibody or an antigen-binding fragment thereof, comprising any one of the following (I) to (IV): (I) Heavy chain variable region, which includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 respectively; and The light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 respectively; (II) Heavy chain variable region, which includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 respectively; and The light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 respectively; (III) Heavy chain variable region, which includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 respectively; and The light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 38, SEQ ID NO: 7 and SEQ ID NO: 40 respectively; (IV) Heavy chain variable region, which includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 respectively; and The light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 42, SEQ ID NO: 39 and SEQ ID NO: 8, respectively; Furthermore, the anti-IL-4R antibody or its antigen-binding fragment also contains a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4.

2. The anti-IL-4R antibody or its antigen-binding fragment according to claim 1, further comprising fragments selected from or combinations thereof: - FR region sequence derived from hominid light chain IGKV3-11*01; - A reversion mutation sequence derived from the FR region of the human light chain IGKV3-11*01 with at least 95% identity; And / or, the anti-IL-4R antibody or its antigen-binding fragment further comprises fragments selected from or combinations thereof: - FR region sequence derived from hominid heavy chain IGHV3-48*01; - A reversion mutation sequence derived from the FR region of the human heavy chain IGHV3-48*01 with at least 95% identity.

3. The anti-IL-4R antibody or its antigen-binding fragment according to claim 1, comprising fragments selected from or combinations thereof: - FR region sequence derived from hominid light chain IGKV3-11*01; - A reversion mutation sequence derived from the FR region of the human light chain IGKV3-11*01 with at least 95% identity, wherein the reversion mutation is selected from one or a combination of L46P, L47W, and F71Y; And / or, the anti-IL-4R antibody or its antigen-binding fragment further comprises fragments selected from or combinations thereof: - FR region sequence derived from hominid heavy chain IGHV3-48*01; - A reversion mutation sequence derived from the FR region of the human germline heavy chain IGHV3-48*01 with at least 95% identity, wherein the reversion mutation is selected from one or a combination of S49A, F67S, and A93T.

4. The anti-IL-4R antibody or its antigen-binding fragment according to claim 1, further comprising fragments selected from or combinations thereof: - FR region sequence derived from hominid light chain IGKV2D-29*01; - A reversion mutation sequence derived from the FR region of the human light chain IGKV2D-29*01 with at least 95% identity; And / or, the anti-IL-4R antibody or its antigen-binding fragment further comprises fragments selected from or combinations thereof: - FR region sequence derived from hominid heavy chain IGHV1-2*02; - A reversion mutation sequence derived from the FR region of the human germline heavy chain IGHV1-2*02 with at least 95% identity.

5. The anti-IL-4R antibody or its antigen-binding fragment according to claim 1, further comprising fragments selected from or combinations thereof: - FR region sequence derived from hominid light chain IGKV2D-29*01; - A reversion mutation sequence derived from the FR region of the human light chain IGKV2D-29*01 with at least 95% identity, wherein the reversion mutation is selected from M4L and / or V58I; And / or, the anti-IL-4R antibody or its antigen-binding fragment further comprises fragments selected from or combinations thereof: - FR region sequence derived from hominid heavy chain IGHV1-2*02; - A reversion mutation sequence derived from the FR region of the human germline heavy chain IGHV1-2*02 with at least 95% identity, wherein the reversion mutation is selected from one or a combination of M69L, R71I, T73K, and R94K.

6. The anti-IL-4R antibody or its antigen-binding fragment according to claim 1, wherein: The antigen-binding fragment is selected from: Fab, Fab'-SH, Fv, scFv, (Fab')2 fragment; The heavy chain variable region of the antibody includes the heavy chain framework region of human IgG1, IgG2, IgG3 or IgG4.

7. The anti-IL-4R antibody or its antigen-binding fragment according to claim 1, wherein: (I) The heavy chain variable region contains a sequence as shown in SEQ ID NO: 1 or having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 1, and the light chain variable region contains a sequence as shown in SEQ ID NO: 2 or having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 2; or (II) The heavy chain variable region contains a sequence as shown in SEQ ID NO: 9 or having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 9, and the light chain variable region contains a sequence as shown in SEQ ID NO: 10 or having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 10; or (III) The heavy chain variable region contains a sequence as shown in SEQ ID NO: 43 or having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 43, and the light chain variable region contains a sequence as shown in SEQ ID NO: 37 or having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 37; or (IV) The heavy chain variable region contains a sequence as shown in SEQ ID NO: 43 or having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 43, and the light chain variable region contains a sequence as shown in SEQ ID NO: 41 or having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO:

41.

8. The anti-IL-4R antibody or its antigen-binding fragment according to claim 7, wherein, The anti-IL-4R antibody or its antigen-binding fragment comprises: The heavy chain variable region shown in SEQ ID NO: 1, and the light chain variable region shown in SEQ ID NO: 2; or The heavy chain variable region shown in SEQ ID NO: 9, and the light chain variable region shown in SEQ ID NO: 10; or The heavy chain variable region shown in SEQ ID NO: 43, and the light chain variable region shown in SEQ ID NO: 37; or The heavy chain variable region shown in SEQ ID NO: 43, and the light chain variable region shown in SEQ ID NO:

41.

9. The anti-IL-4R antibody or its antigen-binding fragment according to claim 1, wherein: (I) The heavy chain variable region contains a sequence as shown in or having at least 90%, 95%, 98%, or 99% identity with any of SEQ ID NO: 25-27, and the light chain variable region contains a sequence as shown in or having at least 90%, 95%, 98%, or 99% identity with any of SEQ ID NO: 28-30; or (II) The heavy chain variable region contains a sequence as shown in one of SEQ ID NO: 31-33 or having at least 90%, 95%, 98%, or 99% identity with one of SEQ ID NO: 31-33, and the light chain variable region contains a sequence as shown in one of SEQ ID NO: 34-36 or having at least 90%, 95%, 98%, or 99% identity with one of SEQ ID NO: 34-36.

10. The anti-IL-4R antibody or its antigen-binding fragment according to claim 1, wherein: The anti-IL-4R antibody or its antigen-binding fragment contains the heavy chain constant region of human IgG2 or IgG4.

11. The anti-IL-4R antibody or its antigen-binding fragment according to claim 1, wherein: (I) The heavy chain comprises a sequence as shown in SEQ ID NO: 17 or having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 17, and the light chain comprises a sequence as shown in SEQ ID NO: 18 or having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 18; or (II) The heavy chain comprises a sequence as shown in SEQ ID NO: 19 or having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 19, and the light chain comprises a sequence as shown in SEQ ID NO: 20 or having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 20; or (III) The heavy chain comprises a sequence as shown in SEQ ID NO: 44 or having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 44, and the light chain comprises a sequence as shown in SEQ ID NO: 45 or having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 45; or (IV) The heavy chain comprises a sequence as shown in SEQ ID NO: 44 or having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 44, and the light chain comprises a sequence as shown in SEQ ID NO: 46 or having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO:

46.

12. The anti-IL-4R antibody or its antigen-binding fragment according to claim 11, wherein, The anti-IL-4R antibody or its antigen-binding fragment comprises any one of the following: The heavy chain shown in SEQ ID NO: 17 and the light chain shown in SEQ ID NO: 18; or The heavy chain shown in SEQ ID NO: 19 and the light chain shown in SEQ ID NO: 20; or The heavy chain shown in SEQ ID NO: 44 and the light chain shown in SEQ ID NO: 45; or The heavy chain shown in SEQ ID NO: 44 and the light chain shown in SEQ ID NO:

46.

13. The anti-IL-4R antibody or its antigen-binding fragment according to claim 1, wherein it is a chimeric antibody, a humanized antibody or a fragment thereof.

14. A polynucleotide encoding the anti-IL-4R antibody or its antigen-binding fragment as claimed in any one of claims 1 to 13.

15. A vector containing the polynucleotide of claim 14, wherein the vector is a eukaryotic expression vector, a prokaryotic expression vector, or a viral vector.

16. A host cell comprising the vector of claim 15.

17. The host cell according to claim 16, wherein, The host cells are selected from bacteria, yeast, and mammalian cells.

18. The host cell according to claim 16, wherein, The host cells were selected from: Escherichia coli, Pichia pastoris, Chinese hamster ovary cells, and human embryonic kidney 293 cells.

19. Use of the anti-IL-4R antibody or its antigen-binding fragment according to any one of claims 1 to 13 in the preparation of a reagent for detecting or determining IL-4R, said use comprising: The step of contacting the anti-IL-4R antibody or its antigen-binding fragment as described in any one of claims 1 to 13 with the sample.

20. A reagent comprising the anti-IL-4R antibody or its antigen-binding fragment as described in any one of claims 1 to 13.

21. The reagent of claim 20, wherein the reagent is used to diagnose diseases associated with human IL-4R positive cells.

22. A pharmaceutical composition comprising: The anti-IL-4R antibody or its antigen-binding fragment according to any one of claims 1 to 13; and Pharmaceutical excipients, diluents, or carriers.

23. Use of any one or a combination of the following in the preparation of a medicament for the treatment or prevention of IL-4R-mediated diseases or conditions, or for the treatment or prevention of immune diseases or conditions: The anti-IL-4R antibody or its antigen-binding fragment according to any one of claims 1 to 13, the polynucleotide according to claim 14, and the pharmaceutical composition according to claim 22; The disease or condition mentioned is selected from: asthma, chronic sinusitis, allergic dermatitis, eosinophilic esophagitis, chronic obstructive pulmonary disease, or allergic rhinitis.

24. The use according to claim 23, wherein, The disease or condition mentioned is asthma or allergic skin disease.

25. A method for preparing an anti-IL-4R antibody or an antigen-binding fragment thereof, comprising the following steps: Expressing an anti-IL-4R antibody or its antigen-binding fragment in the host cells according to any one of claims 16-18, and Isolate anti-IL-4R antibodies or antigen-binding fragments thereof from the host cells.

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