WO2020038454A1 - 结合人il-4r的抗体、其抗原结合片段及其医药用途 - Google Patents

结合人il-4r的抗体、其抗原结合片段及其医药用途 Download PDF

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WO2020038454A1
WO2020038454A1 PCT/CN2019/102169 CN2019102169W WO2020038454A1 WO 2020038454 A1 WO2020038454 A1 WO 2020038454A1 CN 2019102169 W CN2019102169 W CN 2019102169W WO 2020038454 A1 WO2020038454 A1 WO 2020038454A1
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seq
antibody
antigen
sequence
heavy chain
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PCT/CN2019/102169
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English (en)
French (fr)
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廖成
徐祖朋
蒋家骅
张连山
钱雪明
滕菲
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to KR1020217004544A priority Critical patent/KR20210049792A/ko
Priority to CN201980050263.4A priority patent/CN112513090B/zh
Priority to EP19852040.5A priority patent/EP3808774A4/en
Priority to MX2021001143A priority patent/MX2021001143A/es
Priority to AU2019324403A priority patent/AU2019324403A1/en
Priority to CN202310069400.7A priority patent/CN115991781A/zh
Priority to JP2021508304A priority patent/JP7405831B2/ja
Priority to BR112021002989-3A priority patent/BR112021002989A2/pt
Priority to CA3105527A priority patent/CA3105527A1/en
Priority to US17/269,449 priority patent/US20210238294A1/en
Publication of WO2020038454A1 publication Critical patent/WO2020038454A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G01N33/6857—Antibody fragments
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present disclosure relates to an antibody that binds human IL-4R, and an antigen-binding fragment thereof.
  • the present disclosure also relates to a chimeric antibody or a humanized antibody comprising a CDR region derived from the antibody, and comprising binding human IL- Pharmaceutical composition of 4R antibody and antigen-binding fragment thereof, and use thereof as medicine for treating autoimmune disease.
  • Allergic diseases are serious medical conditions that include: non-life-threatening allergic reactions that can be cured over time; and, life-threatening allergic diseases.
  • Interleukin-4 also known as B-cell stimulating factor or BSF-1
  • BSF-1 B-cell stimulating factor
  • IL-4 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 stimulating growth of T cells, mast cells, granulocytes, megakaryocytes, and red blood cells.
  • IL-4 induces the expression of MHC-II in resting B cells and enhances the secretion of immunoglobulins IgE and IgG1 through activated B cells.
  • IL-4R specific cell surface IL-4 receptors
  • the IL-4 receptor (IL-4R) is composed of 802 amino acid residues, and it 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), so IL-4R can also mediate the biological activity of IL-13.
  • a drug containing an IL-4R antagonist and a composition thereof can be administered.
  • an antibody that specifically binds IL-4R also referred to as an antibody that binds human IL-4R, an antibody against human IL-4R) or an antigen-binding fragment thereof is provided, wherein the heavy chain variable region comprises:
  • the antibody or antigen-binding fragment thereof that binds human IL-4R comprises any one selected from the following (I) to (IV):
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 38, SEQ ID NO: 7 and SEQ ID NO: 40, respectively;
  • the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 42, SEQ ID ID NO: 39 and SEQ ID ID NO: 8, respectively.
  • an antibody or antigen-binding fragment thereof that binds human IL-4R wherein:
  • Heavy chain variable region containing:
  • the antibody or antigen-binding fragment that binds human IL-4R wherein:
  • variable region of the heavy chain is shown in the sequence SEQ ID NO: 1 and the variable region of the light chain is shown in the sequence SEQ ID NO: 2; or
  • variable region of the heavy chain is shown in the sequence SEQ ID NO: 9 and the variable region of the light chain is shown in the sequence SEQ ID NO: 10; or
  • variable region of the heavy chain is shown in the sequence SEQ ID NO: 43 and the variable region of the light chain is shown in the sequence SEQ ID NO: 37; or
  • variable region of the heavy chain is shown in the sequence SEQ ID NO: 43, and the variable region of the light chain is shown in the sequence SEQ ID NO: 41.
  • an antibody or antigen-binding fragment thereof that binds human IL-4R wherein:
  • Heavy chain variable region containing:
  • the heavy chain variable region is shown in one of the sequences SEQ ID NO: 25-27, and the light chain variable region is shown in one of the sequences SEQ ID NO: 28-30.
  • variable region of the heavy chain is shown as one of the sequences SEQ ID NO: 31-33, and the variable region of the light chain is shown as one of the sequences SEQ ID NO: 34-36.
  • the heavy chain of an antibody or antigen-binding fragment thereof that binds human IL-4R comprises:
  • the light chain of a human IL-4R-binding antibody or antigen-binding fragment thereof comprises:
  • the heavy chain is represented by the sequence SEQ ID NO: 17
  • the light chain is represented by the sequence SEQ ID NO: 18.
  • the heavy chain is represented by the sequence SEQ ID NO: 19 and the light chain is represented by the sequence SEQ ID NO: 20.
  • the heavy chain is represented by the sequence SEQ ID NO: 44 and the light chain is represented by the sequence SEQ ID NO: 45.
  • the heavy chain is represented by the sequence SEQ ID NO: 44 and the light chain is represented by the sequence SEQ ID NO: 46.
  • 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.
  • the anti-IL-4R antibody or antigen-binding fragment is humanized.
  • the anti-IL-4R antibody or antigen-binding fragment thereof comprises a fragment selected from the following or a combination thereof:
  • the back mutation is selected from one or a combination of L46P, L47W, and F71Y.
  • the anti-IL-4R antibody or antigen-binding fragment thereof comprises a fragment selected from the following or a combination thereof:
  • the back mutation is selected from one or a combination of S49A, F67S, and A93T.
  • the anti-IL-4R antibody or antigen-binding fragment thereof comprises a fragment selected from the following or a combination thereof:
  • the back mutation is selected from M4L and / or V58I.
  • the anti-IL-4R antibody or antigen-binding fragment thereof further comprises a fragment selected from the following or a combination thereof:
  • the back mutation is selected from one or a combination of M69L, R71I, T73K, R94K.
  • the heavy chain variable region of the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain framework region of human IgG1, IgG2, IgG3, or IgG4 or a variant thereof.
  • the heavy chain variable region comprises the heavy chain framework region of human IgG1 or a variant thereof, for example, SEQ ID NO: 43 shows the heavy chain variable region or has at least 85% sequence identity sexually is a heavy chain variable region variant.
  • the anti-IL-4R antibody or antigen-binding fragment thereof comprises a constant region of a human kappa, lambda chain, or a variant thereof, for example, the light chain variable shown in SEQ ID NO: 44 Region or a light chain variable region variant having at least 85% sequence identity thereto.
  • an IL-4R humanized antibody or fragment thereof as described above further comprising a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 or a variant thereof.
  • the antibody comprises a heavy chain constant region of human IgG2 or IgG4.
  • IgG2 or IgG4 is not ADCC toxic.
  • IgG1 without ADCC (antibody-dependent cell-mediated cytotoxicity) after amino acid mutation is used.
  • the variant comprises a heavy chain constant region mutation selected from the group consisting of a reduction in ADCC function or a loss of ADCC function, such as, but not limited to, N297A, L234A, L235A of IgG1.
  • the anti-IL-4R antibody or antigen-binding fragment thereof wherein the antibody is a humanized antibody, and the heavy chain sequence is as shown in SEQ ID NO: 17 or has at least 85% sequence identity with it ;
  • the light chain sequence is shown in SEQ ID NO: 18 or has at least 85% sequence identity therewith.
  • the anti-IL-4R antibody or antigen-binding fragment thereof wherein the antibody is a humanized antibody, and the heavy chain sequence is as shown in SEQ ID NO: 19 or has at least 85% sequence identity with it ;
  • the light chain sequence is shown in SEQ ID NO: 20 or has at least 85% sequence identity with it.
  • the anti-IL-4R antibody or antigen-binding fragment thereof wherein the antibody is a humanized antibody, and the heavy chain sequence is as shown in SEQ ID NO: 44 or has at least 85% sequence identity with it ;
  • the light chain sequence is shown in SEQ ID NO: 45 or has at least 85% sequence identity therewith.
  • the anti-IL-4R antibody or antigen-binding fragment thereof wherein the antibody is a humanized antibody, and the heavy chain sequence is as shown in SEQ ID NO: 44 or has at least 85% sequence identity with it ;
  • the light chain sequence is shown in SEQ ID NO: 46 or has at least 85% sequence identity therewith.
  • an isolated anti-IL-4R antibody or antigen-binding fragment thereof is provided, which is characterized by competing with any human IL-4R-binding antibody or antigen-binding fragment thereof for binding to human IL-4R as described above. .
  • a bispecific antibody or a multispecific antibody comprising a light chain variable region and / or a heavy chain variable of a human IL-4R-binding antibody or antigen-binding fragment thereof according to the present application. Area.
  • a single chain antibody that contains 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.
  • a polynucleotide that encodes an antibody or antigen-binding fragment thereof that binds human IL-4R according to the present application.
  • a polynucleotide that encodes an antibody that competes against IL-4R or an epitope thereof with an antibody or antigen-binding fragment thereof that binds human IL-4R according to the present application.
  • a polynucleotide that encodes the aforementioned bispecific antibody, multispecific antibody, or single chain antibody.
  • the polynucleotide according to the present application is DNA or RNA.
  • a vector comprising a polynucleotide as described above is provided, which is a eukaryotic expression vector, a prokaryotic expression vector, or a viral vector.
  • a host cell transformed with a vector as described above, the host cell being selected from a prokaryotic cell or a eukaryotic cell.
  • the prokaryotic cell is selected from a bacterium, such as E. coli.
  • the eukaryotic cells are selected from yeast or mammalian cells, such as Pichia or CHO cells.
  • a method for detecting or measuring IL-4R comprising the step of contacting the above-mentioned anti-IL-4R antibody or antigen-binding fragment thereof with a sample.
  • a reagent for detecting or measuring human IL-4R comprising an anti-IL-4R antibody or an antigen-binding fragment thereof as described above.
  • a reagent for detecting or measuring human IL-4R comprising an antibody that targets human IL-4R, or an antigen-binding fragment thereof, that binds human IL-4R according to the present application. -4R or its epitope for competitive binding.
  • a reagent for detecting or measuring human IL-4R comprising the above-mentioned bispecific antibody, multispecific antibody, single chain antibody.
  • a diagnostic agent for diagnosing a disease associated with human IL-4R positive cells comprising an anti-IL-4R antibody or an antigen-binding fragment thereof as described above.
  • the diagnostic agent comprises an antibody that competes with IL-4R or an epitope thereof for binding to human IL-4R or an antigen-binding fragment thereof according to the present application.
  • a pharmaceutical composition which contains:
  • the unit dose of the pharmaceutical composition contains 1 mg to 1000 mg of an IL-4R antibody or an antigen-binding fragment thereof according to the present application.
  • the concentration of the IL-4R antibody or antigen-binding fragment thereof in the pharmaceutical composition is 1 mg / L to 1000 mg / L.
  • the pharmaceutical composition contains a buffering agent, and its content may be 1 mM to 1000 mM.
  • 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 is provided.
  • a pharmaceutical composition as described above for the manufacture of a medicament for the treatment or prevention of an IL-4R-mediated disease or disorder is provided.
  • 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.
  • the use of a pharmaceutical composition as described above for treating or preventing a disease or disorder is provided.
  • the disorder or disease may be an immune disease or disorder.
  • the disease or condition is selected from the group consisting of: asthma, nasal polyps, chronic sinusitis, allergic skin disease, 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.
  • the disease or disorder is asthma.
  • the disease or condition is an allergic skin disease.
  • an antibody or an antigen-binding fragment thereof that binds human IL-4R is provided, wherein the antigen-binding fragment is Fab, Fv, sFv, F (ab ') 2 .
  • a method for treating and / or preventing an IL-4R-mediated disease or disorder comprising: administering to a patient (or subject) in need thereof a therapeutically effective amount (or a prophylactically effective amount) ) As described above binds to a human IL-4R antibody or an antigen-binding fragment thereof.
  • a method for treating and / or preventing an IL-4R-mediated disease or disorder comprising: administering to a patient (or subject) in need thereof a therapeutically effective amount (or a prophylactically effective amount) ) A pharmaceutical composition as described above.
  • a method of treating and / or preventing an immune disease comprising administering to a patient (or subject) in need thereof a therapeutically effective amount (a prophylactically effective amount) of a human IL-binding agent as described above. 4R antibody or antigen-binding fragment thereof or pharmaceutical composition thereof.
  • Human IL-4R (hIL-4R) means a human cytokine receptor that specifically binds to interleukin-4 (IL-4) and IL-4R ⁇ .
  • Antibody refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. According to this, immunoglobulins can be divided into five categories, or isotypes called immunoglobulins, that is, IgM, IgD, IgG, IgA, and IgE. The corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains , Alpha and epsilon chains.
  • Igs of the same class can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds.
  • the light chain is divided into a kappa chain or a lambda chain by different constant regions.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the antibody light chain may further comprise a light chain constant region comprising a human or murine ⁇ , ⁇ chain, or a variant thereof.
  • the antibody heavy chain may further comprise a heavy chain constant region comprising human or murine IgG1, IgG2, IgG3, IgG4 or a variant thereof.
  • variable region The sequence of about 110 amino acids near the N-terminus of the heavy and light chains of the antibody varies greatly and is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region).
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). Three hypervariable regions determine the specificity of an antibody, also known as complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) are composed of three CDR regions and four FR regions.
  • the sequence from the amino terminal to the carboxy terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to the light chain complementarity determining region 1 (LCDR1), the light chain complementarity determining region 2 (LCDR2), and the light chain complementarity determining region 3 (LCDR3);
  • the three CDR regions of the heavy chain refer to the heavy chain complementarity Determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), and heavy chain complementarity determining region 3 (HCDR3).
  • Antibodies include murine antibodies, chimeric antibodies, humanized antibodies, and human antibodies, which can be obtained recombinantly, for example, recombinant human antibodies obtained by affinity maturation.
  • “Recombinant human antibodies” include human antibodies prepared, expressed, created or isolated by recombinant methods. The techniques and methods involved are well known in the art, such as (1) transgenic, transchromosomal animals from human immunoglobulin genes (E.g., mice) or antibodies isolated from hybridomas; (2) antibodies isolated from host cells transformed to express antibodies, such as transfected tumors; (3) isolated from recombinant combinatorial human antibody libraries Antibodies; and (4) antibodies prepared, expressed, created, or isolated by methods such as splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include rearrangements and mutations that subsequently occur, such as during antibody maturation.
  • a "murine antibody” is herein a monoclonal antibody to human IL-4R prepared according to the knowledge and skill in the art.
  • the test subject is injected with the IL-4R antigen or a polypeptide containing the epitope, and then a hybridoma expressing an antibody having a desired sequence or functional property is isolated.
  • the murine-derived human IL-4R antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine ⁇ , lambda chain or a variant thereof, or further a murine IgG1, The heavy chain constant region of IgG2, IgG3 or IgG4 or a variant thereof.
  • Human antibodies include antibodies having variable and constant regions of human germline immunoglobulin sequences. Human antibodies herein may include amino acid residues that are not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, the term “human antibody” does not include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as a mouse, has been grafted onto a human backbone sequence (ie, a "humanized antibody”) .
  • a "chimeric antibody” is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, and can reduce the immune response response induced by the murine antibody.
  • To establish a chimeric antibody select a hybridoma that secretes a mouse-specific monoclonal antibody, and then clone the variable region gene from the mouse hybridoma cell. Then, clone the constant region gene of the human antibody as needed to change the mouse variable
  • the region gene and the human constant region gene are linked into a chimeric gene and inserted into a human vector. Finally, a chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the constant region of a human antibody may be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably including the human IgG2 or IgG4 heavy chain constant region, or without ADCC (antibody-dependent) cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG1.
  • Antigen-binding fragment refers to a Fab fragment, Fab 'fragment, F (ab') 2 fragment, Fv fragment, scFv fragment that binds to human IL-4R, and a polypeptide or protein containing the above-mentioned fragments, having antigen-binding activity.
  • the "antigen-binding fragment” comprises one or more CDR regions of an antibody described herein.
  • the Fv fragment contains the variable region of the heavy chain and light chain of the antibody, but has no constant region and has the smallest antibody fragment with all antigen-binding sites.
  • Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding.
  • the variable regions of two antibodies can also be linked into a polypeptide chain with different linkers, which are called single chain antibodies 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 an immunoglobulin or antibody.
  • An epitope can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by the tertiary folding of a protein. An epitope formed by adjacent amino acids is usually maintained after exposure to a denaturing solvent, while an epitope formed by tertiary folding is usually lost after treatment with a denaturing solvent.
  • An epitope usually includes at least 3-15 amino acids in a unique spatial conformation. Methods to determine what epitopes are bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation detection analysis. Methods for determining the spatial conformation of an epitope include techniques in the art and techniques described herein, such as X-ray crystal analysis and two-dimensional nuclear magnetic resonance.
  • Specific binding and “selective binding” refer to the binding of an antibody to an epitope on a predetermined antigen.
  • the antibody is measured by surface plasmon resonance (SPR) technology in the instrument, and the antibody is at an equilibrium lower than about 10 -7 M or even less.
  • the dissociation constant (K D ) binds to a predetermined antigen, and its affinity for binding to the predetermined antigen is at least two times its affinity for binding to a non-specific antigen (such as BSA, etc.) other than the predetermined antigen or closely related antigen.
  • K D dissociation constant
  • the term "antibody that recognizes an antigen” is used interchangeably herein with the term “antibody that specifically binds”.
  • Cross-reaction refers to the ability of the antibodies herein to bind to IL-4R from different species.
  • an antibody herein that binds human IL-4R can also bind IL-4R from another species.
  • Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays such as SPR and ELISA, or binding or functional interaction with cells that physiologically express IL-4R. Methods to determine cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.
  • SPR surface plasmon resonance
  • a “neutralizing” or “blocking” antibody means an antibody whose binding to hIL-4R causes inhibition of hIL-4 and / or hIL-13 biological activity. Such inhibition of hIL-4 and / or IL-13 biological activity can be achieved by measuring one or more hIL-4 / or hIL-13 biological activity indicators well known in the art, such as hlL-4 / or hIL-13-induced cells For evaluation, such as activation and binding of hIL-4 to hIL-4R, see, for example, CN103739711A. "Inhibiting growth” (e.g., involving a cell) is intended to include any measurable reduction in cell growth.
  • Induced immune response and “enhanced immune response” are used interchangeably and refer to the stimulation (i.e., passive or adaptive) of a particular antigen by an immune response.
  • the term “induction” with respect to the induction of CDC or ADCC refers to stimulating a specific direct cell killing mechanism.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Fc receptors directly kill target cells coated with antibodies by recognizing the Fc segment of the antibody.
  • Fc segment on IgG ADCC effector function of the antibody can be reduced or eliminated.
  • the modification refers to mutation in the constant region of the heavy chain of the antibody, such as N297A, L234A, L235A selected from IgG1; IgG2 / 4 chimera, F235E of IgG4, or L234A / E235A mutation.
  • a fusion protein is a protein product in which two genes are co-expressed through DNA recombination.
  • Recombinant IL-4R extracellular domain Fc fusion protein Through DNA recombination, a fusion protein that coexpresses the extracellular domain of IL-4R and the human antibody Fc fragment.
  • the IL-4R extracellular region refers to a part of the IL-4R protein expressed outside the cell membrane.
  • 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 also be prepared by conventional methods.
  • the antibodies or antigen-binding fragments described herein are genetically engineered to add one or more human FR regions to a CDR region of non-human origin. Human FR germline sequences can be obtained from the website of ImMunoGeneTics (IMGT) http://imgt.cines.fr, or from the Journal of Immunoglobulins, 2001ISBN012441351.
  • IMGT ImMunoGeneTics
  • 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 a GS expression vector.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • the sequence of the humanized antibody is inserted into the corresponding expression vector by molecular cloning technology, and expressed and produced by the HEK293 cell expression system, then the corresponding humanized antibody can be obtained.
  • mammalian expression systems cause glycosylation of antibodies, especially in the highly conserved N-terminus of the FC region. Stable clones are obtained by expressing antibodies that specifically bind to human-derived antigens.
  • Positive clones were expanded in serum-free medium in the bioreactor to produce antibodies.
  • the culture medium in which the antibody is secreted can be purified and collected by conventional techniques.
  • the antibody can be concentrated by filtration using a conventional method. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange.
  • the resulting product needs to be immediately frozen, such as -70 ° C, or lyophilized.
  • the antibody may be a monoclonal antibody (mAb), which refers to an antibody obtained from a single cloned cell line, and the cell line is not limited to a eukaryotic, prokaryotic, or phage cloned cell line.
  • mAb monoclonal antibody
  • Monoclonal antibodies or antigen-binding fragments can be recombined using techniques such as hybridoma technology, recombinant technology, phage display technology, synthetic technology (such as CDR-grafting), or other existing technologies.
  • Antibodies can be monospecific, bispecific, or multispecific antibodies. Multispecific antibodies can show specificity for different epitopes of the target peptide, or can also include antigen-binding domains that show specificity for more than one target peptide.
  • Human anti-IL-4R antibodies can 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 union, gene fusion, non-covalent binding, or otherwise) to one or more other molecules (such as another antibody or antigen-binding fragment) to produce The second binding specific bispecific or multispecific antibody.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid refer to an exogenous drug, therapeutic agent, diagnostic agent, or composition and animal , Human, subject, cell, tissue, organ, or biological fluid.
  • administering may refer to, for example, treatment, pharmacokinetics, diagnostics, research, and experimental methods.
  • Treatment of a cell includes contact of a reagent with a cell, and contact of a reagent with a fluid, wherein the fluid is in contact with the cell.
  • administering also mean in vitro and ex vivo treatment of a cell, for example, by an agent, diagnostic, binding composition, or by another cell.
  • Treatment when applied to a human, veterinary or research subject refers to therapeutic treatment, preventive or prophylactic measures, research and diagnostic applications.
  • Treatment means the administration to a patient (or subject) of an internal or external therapeutic agent, such as a composition comprising any of the antibodies or antigen fragments thereof, which patient (or subject) has, suspected to have Or may be susceptible to one or more symptoms of the disease, and the therapeutic agent is known to have a therapeutic effect on these symptoms.
  • a therapeutic agent is administered in a treated patient (or subject) or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing the deterioration of such symptoms or inhibiting the development of such symptoms to any clinically measurable Degree.
  • the amount of therapeutic agent (also known as a "therapeutically effective amount") effective to alleviate the symptoms of any particular disease 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 effect on the patient (or (Subject) develops the ability to require efficacy. Evaluate whether the symptoms of the disease have been alleviated by any clinical test method that a doctor or other health care professional usually uses to assess the severity or progression of the symptoms.
  • embodiments of the invention may not be effective in relieving symptoms of a target disease in a single patient (or subject)
  • any statistical test method known in the art such as Student's test, Chi-square test According to the Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test, it should reduce symptoms of the target disease in a statistically significant number of patients (or subjects).
  • Constant modification or “conservative substitution or substitution” refers to the replacement of amino acids in proteins by other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.) Changes are made without altering the biological activity of the protein.
  • Those skilled in the art know that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., P. 224, (4th edition)).
  • structural or functionally similar amino acid substitutions are unlikely to disrupt biological activity.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent the symptoms or conditions 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 can vary depending on factors such as the condition to be treated, the subject's general health, the route and dose of administration, and the severity of the side effects.
  • An effective amount can be the maximum dose or dosage regimen to avoid significant side effects or toxic effects.
  • Exogenous refers to a substance that is produced outside the organism, cell, or human depending on the context.
  • Endogenous refers to a substance that is produced in a cell, organism, or human body by context.
  • “Homology” or “identity” refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit, for example, 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 compared positions x 100%. For example, when the sequences are optimally aligned, if there are 6 matches or homology at 10 positions in the two sequences, then the two sequences are 60% homologous.
  • amino acid sequence having at least 85% sequence identity includes obtained by performing one or more amino acid deletions, insertions or substitution mutations on the parent sequence.
  • the expressions "cell”, “cell line” and “cell culture” are used interchangeably, and all such names include their progeny.
  • the words “transformants” and “transformed cells” include primary test cells and cultures derived therefrom, regardless of the number of metastases. It should also be understood that due to intentional or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Included are mutant offspring that have the same functional or biological activity as those originally screened in the transformed cells.
  • “Pharmaceutical composition” means a mixture containing one or more of the antibodies or antigen-binding fragments described herein, or a physiological / pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiological / pharmacological Pharmaceutically acceptable carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and then exerts the biological activity.
  • Figure 1 In a mouse dermatitis model, after sensitization with acetone, the humanized antibodies hu25G7-A, hu25G7-B and the positive reference antibody dupilizumab are administered subcutaneously: twice a week, day 27 The mouse ear thickness was measured. The results show that compared with the control group, hu25G7-A, hu25G7-B and dupilizumab can effectively reduce the ear thickness of mice, and hu25G7-B shows a better effect than dupilizumab.
  • the following embodiments are used to further describe the present disclosure, but these embodiments do not limit the scope of the present disclosure.
  • the experimental methods without specific conditions in the examples are generally based on conventional conditions, such as the manual of antibody technology experiments and molecular cloning manuals of Cold Spring Harbor; or according to the conditions recommended by raw materials or commercial manufacturers.
  • the reagents without specific sources are conventional reagents purchased on the market.
  • 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 was synthesized by Acrobiosystems, HEK293 expressed and purified.
  • Human IL-4R (h-IL-4R-Fc) recombinant protein with human-derived Fc tag was designed, expressed, and purified.
  • the purified protein can be used in the experiments of the following examples.
  • the CDR amino acid residues of the VL and VH regions of the antibody or antigen-binding fragment conform to the known Kabat numbering rules (LCDR1-3, HCDR2-3), or the Kabat and CHOTHIA (ABM) rules (HCDR1).
  • Anti-human IL4R monoclonal antibodies are produced by immunizing mice. C57BL / 6 mice, female, 6-8 weeks of age (Suzhou Zhaoyan New Drug Research Center Co., Ltd., animal production license number: 201503052).
  • mice SPF level. After the mice were purchased, they were raised in a laboratory environment for 1 week, with a 12/12 hour light / dark cycle adjustment, a temperature of 20-25 ° C, and a humidity of 40-60%. The adapted mice were divided into 3 cages of 5 mice each.
  • the immune antigen was an Fc-tagged human IL-4R recombinant protein (h-IL4R-Fc, 0.73 mg / ml).
  • Freund's adjuvant (sigma, Cat #: F5881) for emulsification: Freund's complete adjuvant (CFA, Pierce, Cat # 77140) was used for the first time, and the rest of the immune-enhancing nucleic acid adjuvant (CpG, Shanghai Shenggong) and aluminum adjuvant Agent (Alum, Thermo Cat # 77161).
  • mice with high antibody titers and plateaus in the serum were selected for spleen cell fusion.
  • IP intraperitoneal
  • ELISA was used to detect the binding characteristics of the IL-4R antibody.
  • An enzyme-labeled plate coated with a his-tagged IL-4R recombinant protein was directly coated. After the antibody was added to each well, the secondary antibody (HRP-conjugated antibody against primary antibody Fc) and HRP substrate TMB were used to detect the binding of the antibody to the antigen Of activity.
  • Human or rhesus IL-4R-his protein was coated on a 96-well microtiter plate, 100 ⁇ l per well at a concentration of 0.5 ⁇ g / ml, and incubated overnight at 4 ° C. Wash the washing solution three times, 250 ⁇ l per well. Shake each wash for 10 seconds to ensure adequate washing. Add 200 ⁇ l / well blocking solution and incubate at room temperature for 2 hours. Wash the washing solution three times, 250 ⁇ l per well. Shake each wash for 10 seconds to ensure adequate washing. Add 100 ⁇ l of anti-IL-4R test antibody diluted with the dilution solution to each well. Incubate for 1 hour at room temperature. Wash the washing solution three times, 250 ⁇ l per well.
  • Human IL-4R-Fc protein was coated on a 96-well microtiter plate, 100 ⁇ l per well at a concentration of 0.5 ⁇ g / ml, and incubated overnight at 4 ° C. Wash the washing solution three times, 250 ⁇ l per well. Shake each wash for 10 seconds to ensure adequate washing. Add 200 ⁇ l / well blocking solution and incubate at room temperature for 2 hours. Wash the washing solution three times, 250 ⁇ l per well. Shake each wash for 10 seconds to ensure adequate washing. Add 100 ⁇ l of anti-IL-4R test antibody diluted with the dilution solution to each well, and incubate for 1 hour at room temperature. Wash the washing solution three times, 250 ⁇ l per well.
  • HEK-Blue IL-4 cells were purchased from Invivogen (Cat # hkb-stat6). The cells were stably transfected with human IL-4R gene and STAT6-mediated SEAP genome. The secretion of the supernatant can be detected by the SEAP substrate QUANTI-Blue SEAP to characterize the level of activation of the IL-4R signaling pathway.
  • HEK-Blue IL-4 cells were cultured in DMEM medium containing 10% FBS, 100 ⁇ g / ml Zeocin (Invivogen, Cat # ant-zn-05) and 10 ⁇ g / ml Blasticidin (Invivogen, Cat # ant-bl-05). ; Passage 2 to 3 times a week, pass down 1: 5 or 1:10. During passaging, aspirate the medium, rinse the cell layer with 5ml 0.25% trypsin, and then remove the trypsin.
  • the plates were incubated in an incubator for 20-24 hours (37 °C, 5% CO 2 ). Take 20 ⁇ l of cell supernatant from each well into a new 96-well flat bottom plate, add 180 ⁇ l of QUANTI-Blue substrate solution, and incubate the culture plate in the incubator in the dark for 1-3 hours. The absorbance at 620 nm was measured using a microplate reader (Thermo MultiSkanFc).
  • TF-1 cells are lymphoma cells that express IL-4R and are sensitive to cytokines such as IL-4 / IL-13. IL-4 can stimulate the proliferation of TF-1 cells without GM-CSF.
  • IL-4R antibodies were compared by adding neutralizing antibodies to block the pathway of IL-4 and inhibit the proliferation of TF-1 cells.
  • Cell TF-1 was cultured in RPMI1640 medium containing 10% FBS, 2ng / ml GM-CSF (R & D, Cat # 215-GM-010); passaged 2 to 3 times a week, and the passage ratio was 1:10.
  • the affinity of the humanized human IL-4R antibody and human IL-4R to be tested was determined using a Biacore, GE instrument.
  • the human anti-capture antibody was covalently coupled to the biosensor chip CM5 of Biacore instrument (Biacore X100, GE) according to the method of the human anti-capture kit (Cat. # BR-1008-39, GE), so as to kiss And capture a certain amount of test antibody; then the IL-4R antigen (IL-4R antigen was purchased from Acrobiosystems, Cat # ILR-H5221) under a series of concentration gradients was flowed on the surface of the chip, using a Biacore instrument (Biacore X100, GE ) Real-time detection of reaction signals to obtain binding and dissociation curves.
  • Biacore instrument Biacore X100, GE
  • the biochip is washed and regenerated with the regeneration solution configured 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) Dilute to 1X (pH 7.4) with DIWater.
  • Example 2 Inhibition of HEK293-Blue IL- in Example 3 by the ELISA binding experiment (human IL-4R-his ELISA binding) and ELISA blocking experiment (human IL-4 / IL-4R ELISA blocking) in Example 2 above.
  • ELISA binding experiment human IL-4R-his ELISA binding
  • ELISA blocking experiment human IL-4 / IL-4R ELISA blocking
  • Monoclonal hybridoma cell lines 25G7 and 7B10 with the best activity in vitro were selected and the monoclonal antibody sequences were cloned.
  • the process of cloning sequences from hybridomas is as follows. Logarithmic growth phase hybridoma cells were collected, and RNA was extracted using Trizol (Invitrogen, 15596-018) (in accordance with the kit instructions) and reverse transcription (PrimeScript TM Reverse Transcriptase, Takara, cat # 2680A). The reverse-transcribed cDNA was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and then sent to a sequencing company for sequencing, and the obtained antibody sequence was analyzed.
  • variable region of the heavy and light chains of the mouse monoclonal antibody 25G7 is as follows:
  • variable region of the heavy and light chains of murine mAb 7B10 is as follows:
  • variable region sequence was connected to a human constant region sequence to obtain a human-mouse chimeric antibody sequence.
  • sequence of the chimeric antibody was inserted into the corresponding expression vector.
  • human-mouse chimeric antibodies 25G7-C and 7B10-C can be obtained.
  • the two strains (25G7 and 7B10) with the highest functional activity among the obtained murine antibodies were humanized.
  • the heavy and light chain variable region sequences were compared with the antibody Germline database to obtain a human germline template with high homology.
  • the human germline light chain framework region is derived from the human kappa light chain gene, and the human germline light chain template IGKV3-11 * 01 (SEQ ID NO: 22 for antibody 25G7) and IGKV2D-29 * 01 (SEQ ID ID NO : 24 for antibody 7B10).
  • the human germline heavy chain framework region is derived from the human heavy chain, preferably the human germline heavy chain template IGHV3-48 * 01 (SEQ ID NO: 21 for antibody 25G7) and IGHV1-2 * 02 (SEQ ID NO: 23, used To antibody 7B10).
  • the human germline template sequence is shown below.
  • the CDR region of the murine antibody was transplanted onto the selected humanized template, the humanized variable region was replaced, and then recombined with the IgG constant region. Then, based on the three-dimensional structure of the mouse-derived antibody, back-mutation was performed on the embedded residues, the residues that directly interact with the CDR regions, and the residues that have an important influence on the conformation of VL and VH to obtain a series of human Source molecule.
  • Hu7B10-VH-a, hu7B10-VH-b, and hu7B10-VH-c were modified by drugs to change the first position of the heavy chain human germline template from Q to E.
  • Hu25G7 is also made into a medicinal modification.
  • 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, respectively. , Shown in SEQ ID NO: 34-36.
  • the final humanized antibody hu25G7 (using the VH-c heavy chain and VL-a light chain) and the hu7B10 antibody molecule (using the VH-b heavy chain and VL-b light chain), and their respective complete light and heavy chain sequences are shown in SEQ ID NO: 17-20.
  • the humanized antibodies hu25G7 and hu7B10 were tested for in vitro activity as described in Examples 2-5.
  • the test results are shown in Table 8.
  • the results showed that both hu25G7 and hu7B10 only bind to human IL-4R, but not to rhesus IL-4R, indicating that both antibodies bind to epitopes of different origins from humans and rhesus monkeys and can specifically bind Human IL-4R.
  • Both antibodies can block IL-4 / IL-4R binding and intracellular signaling pathways, resulting in neutralization of IL-4 activation and inhibition of TF-1 cell proliferation.
  • the blocking and inhibitory activity of hu25G7 remains It is significantly better than the reference antibody dupilizumab, but the affinity K D value is relatively low.
  • 25G7 antibody was affinity matured by yeast display platform technology. Based on the hu25G7 antibody, an affinity matured yeast library for 6 CDRs was designed and degenerate primers were designed. The designed mutant amino acids were introduced into the hu25G7-scFv antibody library by PCR and homologous recombination. The size of each library was about 10 9. The constructed yeast library was verified by the second-generation sequencing (GENEWIZ) method. Sex.
  • Biotinylated human IL-4R was used to screen high-affinity antibodies from the hu25G7-scFv yeast library. After two rounds of MACS screening (streptomycin magnetic beads, Invitrogen) and two rounds of FACS screening (BD FACSAriaTM FUSION), the yeast was selected Monoclonal culture and inducible expression. FACS (BD FACSCanto II) was used to detect the binding of yeast monoclonal to human IL-4R. Yeast monoclonal sequencing with higher affinity than the wild-type 25G7 antibody was selected and verified. The sequencing clones were compared and analyzed. After removing the redundant sequences, the non-redundant sequences are converted into full-length human antibody molecules for mammalian cell expression.
  • MACS screening streptomycin magnetic beads, Invitrogen
  • FACS screening BD FACSAriaTM FUSION
  • the affinity-purified full-length antibody was subjected to affinity measurement using BIAcoreTM X-100 (GE Life Sciences).
  • the candidate antibody molecules with higher affinity for human IL-4R were selected as follows. The affinity of these antibody molecules for human IL-4R is higher than that of wild-type hu25G7 antibody.
  • the affinity of hu25G7-A antibody molecule is comparable to that of dupilizumab, while the affinity of hu25G7-B molecule is significantly better than that of dupilizumab.
  • EIVLTQSPATLSLSPGERATLSCRASSSVPYMYWYQQKPGQAPRLLIYLTSNLASGIPARFSGSGSDFDFTLTISSLEPEDFAVYYCQQWRAYPPMLTFGGGTKVEIK (SEQ ID ID: 37) contains the CDR sequences shown in Table 9.
  • the sequence of the light chain variable region of antibody hu25G7-B is as follows:
  • 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 and hu25G7 light chain constant region are recombined to obtain the hu25G7-B antibody chain.
  • the heavy chain variable region can be recombined with the hu25G7 heavy chain constant region to obtain the hu25G7-A / hu25G7-B antibody heavy chain.
  • Example 3 and Example 4 detect hu25G7-A and hu25G7-B antibodies; both hu25G7-A and hu25G7-B antibodies can block IL-4 / IL-4R binding, and intracellular signaling pathways cause The activation of IL-4 and IL-13 was neutralized, and the proliferation of TF-1 cells was inhibited.
  • Various activity data are shown in Table 11.
  • both hu25G7-A and hu25G7-B showed beneficial effects.
  • the effect was repeatedly verified, and the results showed that under the same conditions, hu25G7-A inhibited the IL-13 proliferation (IC 50 ) value of 11.68 in TF-1 cells.
  • hu25G7 than dopilizumab has a significantly improved effect in blocking the binding of IL-4, IL-13 and IL-4R, and in cell proliferation caused by the binding.
  • Example 11 Effect of humanized antibodies on dermatitis in mice
  • mice purchased from Saiye Model Biological Research Center (Taicang) Co., Ltd.
  • D0 Day0
  • 20 ⁇ L of a 1% OXZ acetone olive oil solution was evenly spread on both ears (both sides) of the mice for challenge, and the challenge was performed every 72 hours.
  • This experiment consists of a normal control group (only acetone and olive oil solution is applied during sensitization and challenge), a model control group, hu25G7-A, hu25G7-B, and dupilizumab group, a total of 5 groups, each group of 3 to 5 mice
  • the administration dose was 50 mg / kg
  • the administration mode was subcutaneous administration, which was administered twice a week (for details, see Table 12).
  • the thickness of the ear was measured with a vernier caliper, and the results are shown in FIG. 1.
  • Model control group 5 (3 females + 2 males) S.C. - 2 times a week hu25G7-A 5 (3 females + 2 males) S.C. 50mg / kg 2 times a week hu25G7-B 3 (2 female + 1 male) S.C. 50mg / kg 2 times a week
  • Dupilizumab 4 (2 female + 2 male) S.C. 50mg / kg 2 times a week
  • hu25G7-A, hu25G7-B, and dupilizumab mice was significantly lower than that of the model control group. That is, hu25G7-A, hu25G7-B, and dupilizumab can be used to treat dermatitis, and the effect of hu25G7-B is better than that of dupilizumab.

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Abstract

结合人IL-4R的抗体、其抗原结合片段及其医药用途。一种嵌合抗体、人源化抗体，其包含来自结合人IL-4R的抗体及其抗原结合片段的CDR区，以及包含结合人IL-4R的抗体及其抗原结合片段的药物组合物，及其作为治疗过敏性疾病药物的用途。一种人源化的结合人IL-4R的抗体，在制备用于治疗IL-4R介导的疾病或病症的药物中的用途。

Description

结合人IL-4R的抗体、其抗原结合片段及其医药用途
本申请要求2018年08月24日提交的中国申请CN201810971269.2、2018年12月04日提交的中国申请CN201811472752.2、2019年03月22日提交的中国申请CN201910221311.3、2019年05月15日提交的中国申请CN201910401923.0的优先权。前述申请各自以全文引用的方式并入在此。
技术领域
本公开涉及一种结合人IL-4R的抗体，以及其抗原结合片段，本公开还涉及一种嵌合抗体或人源化抗体，其包含来自所述抗体的CDR区，以及包含结合人IL-4R的抗体及其抗原结合片段的药物组合物，以及其作为治疗自身免疫疾病药物的用途。
背景技术
过敏性疾病是严重的医学病症，包括：非危及生命的过敏反应，其经过一段时间可治愈；以及，可危及生命的过敏性疾病。
目前治疗过敏的方法包括避免过敏原、针对症状的药理学治疗、以及用过敏原特异性的免疫治疗进行预防。
白细胞介素-4(IL-4，又称为B细胞刺激因子或BSF-1)，其响应低浓度的抗表面免疫球蛋白抗体，刺激B细胞增殖的能力得到表征。IL-4己被证明具有广泛的生物活性，包括对T细胞、肥大细胞、粒细胞、巨核细胞和红细胞等起到生长刺激。IL-4诱导MHC-II在静息B细胞中的表达，并通过激活的B细胞增强免疫球蛋白IgE和IgG1的分泌。
IL-4的生物活性是由特定的细胞表面IL-4受体(IL-4R)介导的。IL-4受体(IL-4R)由802个氨基酸残基组成，在T细胞、B细胞、胸腺细胞、骨髓细胞、巨噬细胞和肥大细胞表面都有IL-4R表达。IL-4R的α链同时也是IL-13受体(IL-13R)的组成部分，因此IL-4R也可介导IL-13的生物活性。在受试者接触过敏原或过敏症状出现之前、期间或之后，作为新的治疗方法，可施用含有IL-4R拮抗剂的药物及其组合物。
目前各国已有多家制药公司正在研发针对IL-4R的单克隆抗体，用于治疗与其相关的各类过敏性疾病。相关专利申请如WO2010053751、WO2001092340、WO2008054606、WO2014031610等。其中赛诺菲旗下再生元公司(Regeneron)研发的哮喘药物度匹鲁单抗(dupilumab)已获批上市用于皮炎适应症，对于哮喘适应症，也已完成III期临床试验，并进入上市报批阶段。
目前已有的治疗策略，疗效不佳，或具有较大风险。因此，本领域仍需要提供高选择性、高生物活性的结合人IL-4R的抗体，其以高亲和性与人IL-4R结合并 能够中和IL-4R的生物活性(包括阻断与IL-4R的结合)，从而提供能够治疗IL-4R介导的过敏性疾病的药物及其组合物、以及治疗方法。
发明内容
本文的一些实施方案中，提供特异性结合IL-4R的抗体(也作结合人IL-4R的抗体、抗人IL-4R的抗体)或其抗原结合片段，其中重链可变区，包含：
(I)分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3或与SEQ ID NO：3、4、5所示的HCDR1、HCDR2、HCDR3分别具有3、2或1个氨基酸差异的HCDR变体；或
(II)分别如SEQ ID NO：11、SEQ ID NO：12和SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3或与SEQ ID NO：11、12、13所示的HCDR1、HCDR2、HCDR3分别具有3、2或1个氨基酸差异的HCDR变体；
和/或轻链可变区，包含：
(I)分别如SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3或与SEQ ID NO：6、7、8所示的LCDR1、LCDR2、LCDR3分别具有3、2或1个氨基酸差异的LCDR变体；或
(II)分别如SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示的LCDR1、LCDR2和LCDR3或与SEQ ID NO：14、15、16所示的LCDR1、LCDR2、LCDR3分别具有3、2或1个氨基酸差异的LCDR变体；或
(III)分别如SEQ ID NO：38、SEQ ID NO：7和SEQ ID NO：40所示的LCDR1、LCDR2和LCDR3或与SEQ ID NO：38、7、40所示的LCDR1、LCDR2、LCDR3分别具有3、2或1个氨基酸差异的LCDR变体；或
(IV)分别如SEQ ID NO：42、SEQ ID NO：39和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3或与SEQ ID NO：42、39、8所示的LCDR1、LCDR2、LCDR3分别具有3、2或1个氨基酸差异的LCDR变体。
在一些实施方案中，结合人IL-4R的抗体或其抗原结合片段包含选自以下(I)至(IV)中的任一项：
(I)重链可变区，其包含分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3；
(II)重链可变区，其包含分别如SEQ ID NO：11、SEQ ID NO：12和SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示的LCDR1、LCDR2和LCDR3；
(III)重链可变区，其包含分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：38、SEQ ID NO：7和SEQ ID NO：40所示的LCDR1、LCDR2和LCDR3；
(IV)重链可变区，其包含分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：42、SEQ ID NO：39和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3。
一些实施方案中，结合人IL-4R的抗体或其抗原结合片段，其中：
重链可变区，包含：
(I)如SEQ ID NO：1所示或与SEQ ID NO：1具有至少70％、80％、90％、95％、98％、99％同一性的序列；或
(II)如SEQ ID NO：9所示或与SEQ ID NO：9具有至少70％、80％、90％、95％、98％、99％同一性的序列；或
(III)如SEQ ID NO：43所示或与SEQ ID NO：43具有至少70％、80％、90％、95％、98％、99％同一性的序列；或
和/或轻链可变区，包含：
(I)如SEQ ID NO：2所示或与SEQ ID NO：2具有至少70％、80％、90％、95％、98％、99％同一性的序列；或
(II)如SEQ ID NO：10所示或与SEQ ID NO：10具有至少70％、80％、90％、95％、98％、99％同一性的序列；或
(III)如SEQ ID NO：37所示或与SEQ ID NO：37具有至少70％、80％、90％、95％、98％、99％同一性的序列；或
(IV)如SEQ ID NO：41所示或与SEQ ID NO：41具有至少70％、80％、90％、95％、98％、99％同一性的序列。
在至少一个实施方案中，所述结合人IL-4R的抗体或抗原结合片段，其中：
重链可变区如序列SEQ ID NO：1所示，轻链可变区如序列SEQ ID NO：2所示；或
重链可变区如序列SEQ ID NO：9所示，轻链可变区如序列SEQ ID NO：10所示；或
重链可变区如序列SEQ ID NO：43所示，轻链可变区如序列SEQ ID NO：37所示；或
重链可变区如序列SEQ ID NO：43所示，轻链可变区如序列SEQ ID NO：41所示。
在一些实施方案中，结合人IL-4R的抗体或其抗原结合片段，其中：
重链可变区，包含：
(I)如SEQ ID NO：25-27之一所示或与SEQ ID NO：25-27之一具有至少70％、80％、90％、95％、98％或99％同一性的序列；或
(II)如SEQ ID NO：31-33之一所示或与SEQ ID NO：31-33之一具有至少70％、80％、90％、95％、98％、或99％同一性的序列；
和/或轻链可变区，包含：
(I)如SEQ ID NO：28-30之一所示或与SEQ ID NO：28-30之一具有至少70％、80％、90％、95％、98％或99％同一性的序列；或
(II)如SEQ ID NO：34-36之一所示或与SEQ ID NO：34-36之一具有至少70％、80％、90％、95％、98％或99％同一性的序列。
在一些具体的实施方案中，重链可变区如序列SEQ ID NO：25-27之一所示，并且轻链可变区如序列SEQ ID NO：28-30之一所示。
在另一些具体的实施方案中，重链可变区如序列SEQ ID NO：31-33之一所示，并且轻链可变区如序列SEQ ID NO：34-36之一所示。
在一些实施方案中，结合人IL-4R的抗体或其抗原结合片段的重链，包含：
(I)如SEQ ID NO：17所示或与SEQ ID NO：17具有至少70％、80％、90％、95％、98％或99％同一性的序列；或
(II)如SEQ ID NO：19所示或与SEQ ID NO：19具有至少70％、80％、90％、95％、98％、或99％同一性的序列；或
(III)如SEQ ID NO：44所示或与SEQ ID NO：44具有至少70％、80％、90％、95％、98％、或99％同一性的序列。
在一些实施方案中，结合人IL-4R的抗体或其抗原结合片段的轻链，包含：
(I)如SEQ ID NO：18所示或与SEQ ID NO：18具有至少70％、80％、90％、95％、98％或99％同一性的序列；或
(II)如SEQ ID NO：20所示或与SEQ ID NO：20具有至少70％、80％、90％、95％、98％或99％同一性的序列；或
(III)如SEQ ID NO：45所示或与SEQ ID NO：45具有至少90％、95％、98％或99％同一性的序列；或
(IV)如SEQ ID NO：46所示或与SEQ ID NO：46具有至少90％、95％、98％或99％同一性的序列。
在至少一个实施方案中，重链如序列SEQ ID NO：17所示，和轻链如序列SEQ ID NO：18所示。
在另一个实施方案中，重链如序列SEQ ID NO：19所示，和轻链如序列SEQ ID NO：20所示。
在另一个实施方案中，重链如序列SEQ ID NO：44所示，和轻链如序列SEQ  ID NO：45所示。
在另一个实施方案中，重链如序列SEQ ID NO：44所示，和轻链如序列SEQ ID NO：46所示。
在一些实施方案中，所述的抗IL-4R的抗体或抗原结合片段是鼠源抗体、嵌合抗体、人抗体、人源化抗体或其片段。
在一些具体的实施方案中，所述抗IL-4R的抗体或抗原结合片段是人源化的。
在一些实施方案中，所述的抗IL-4R的抗体或其抗原结合片段，包含选自以下的片段或其组合：
-源自人种系轻链IGKV3-11*01的FR区序列；
-源自与人种系轻链IGKV3-11*01的FR区具有至少95％同一性的回复突变序列。在一些具体实施方案中，所述的回复突变选自L46P、L47W、F71Y中的一个或组合。
在一些实施方案中，所述的抗IL-4R的抗体或其抗原结合片段，包含选自以下的片段或其组合：
-源自人种系重链IGHV3-48*01的FR区序列；
-源自与人种系重链IGHV3-48*01的FR区具有至少95％同一性的回复突变序列。在一些具体实施方案中，所述的回复突变选自S49A、F67S、A93T中的一个或组合。
在一些实施方案中，所述的抗IL-4R的抗体或其抗原结合片段，包含选自以下的片段或其组合：
-源自人种系轻链IGKV2D-29*01的FR区序列；
-源自与人种系轻链IGKV2D-29*01的FR区具有至少95％同一性的回复突变序列。在一些具体实施方案中，所述的回复突变选自M4L和/或V58I。
在一些实施方案中，所述的抗IL-4R抗体或其抗原结合片段进一步包含选自以下的片段或其组合：
-源自人种系重链IGHV1-2*02的FR区序列；
-源自与人种系重链IGHV1-2*02的FR区具有至少95％同一性的回复突变序列。在一些具体实施方案中，所述的回复突变选自M69L、R71I、T73K、R94K中的一个或组合。
在一些实施方案中，所述的抗IL-4R的抗体或其抗原结合片段中，其重链可变区包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链框架区。在一些具体的方案中，重链可变区包含人源IgG1或其变体的重链框架区，例如，SEQ ID NO：43所示的是重链可变区或与其具有至少85％序列同一性的是重链可变区变体。
在一些实施方案中，所述的抗IL-4R的抗体或其抗原结合片段包含人源κ、λ链或其变体的恒定区，例如，SEQ ID NO：44所示的的轻链可变区或与其具有至 少85％序列同一性的的轻链可变区变体。
在一些实施方案中，一种如上所述的IL-4R人源化抗体或其片段，还包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区。
在至少一个实施方案中，所述抗体包含人源IgG2或IgG4的重链恒定区。IgG2或IgG4没有ADCC毒性。或者，使用氨基酸突变后无ADCC(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG1。
在至少一个实施方案中，所述变体包含选自以下的重链恒定区突变：所述突变使得ADCC功能降低、或使得ADCC功能缺失，例如但不限于IgG1的N297A、L234A、L235A。
在一些实施方案中，所述的抗IL-4R的抗体或其抗原结合片段，其中所述抗体为人源化抗体，重链序列如SEQ ID NO：17所示或与其具有至少85％序列同一性；轻链序列如SEQ ID NO：18所示或与其具有至少85％序列同一性。
在一些实施方案中，所述的抗IL-4R的抗体或其抗原结合片段，其中所述抗体为人源化抗体，重链序列如SEQ ID NO：19所示或与其具有至少85％序列同一性；轻链序列如SEQ ID NO：20所示或与其具有至少85％序列同一性。
在一些实施方案中，所述的抗IL-4R的抗体或其抗原结合片段，其中所述抗体为人源化抗体，重链序列如SEQ ID NO：44所示或与其具有至少85％序列同一性；轻链序列如SEQ ID NO：45所示或与其具有至少85％序列同一性。
在一些实施方案中，所述的抗IL-4R的抗体或其抗原结合片段，其中所述抗体为人源化抗体，重链序列如SEQ ID NO：44所示或与其具有至少85％序列同一性；轻链序列如SEQ ID NO：46所示或与其具有至少85％序列同一性。
在一些实施方案中，提供一种分离的抗IL-4R抗体或其抗原结合片段，其特征在于与如上所述的任一结合人IL-4R的抗体或其抗原结合片段竞争结合人IL-4R。
在一些实施方案中，提供一种双特异性抗体或多特异性抗体，其含有根据本申请的结合人IL-4R的抗体或其抗原结合片段的轻链可变区和/或重链可变区。
在另一些实施方案中，提供一种单链抗体，其含有根据本申请的结合人IL-4R的抗体或其抗原结合片段的轻链可变区和/或重链可变区。
在一些实施方案中，提供一种多核苷酸，其编码根据本申请的结合人IL-4R的抗体或其抗原结合片段。
在另一些实施方案中，提供一种多核苷酸，其编码这样的抗体，其与根据本申请的结合人IL-4R的抗体或其抗原结合片段，针对IL-4R或其表位进行竞争性结合。
在另一些实施方案中，提供一种多核苷酸，其编码前述双特异性抗体、多特异性抗体、或单链抗体。
在一些实施方案中，根据本申请的多核苷酸是DNA或RNA。
在一些实施方案中，提供一种含有如上所述多核苷酸的载体，其为真核表达载体、原核表达载体或病毒载体。
在一些实施方案中，提供一种宿主细胞，其转化有如上所述的载体，所述宿主细胞选自原核细胞或真核细胞。
在至少一个实施方案中，原核细胞选自细菌，例如大肠杆菌。
在至少一个实施方案中，真核细胞选自酵母菌或哺乳动物细胞，例如毕赤酵母或CHO细胞。
在一些实施方案中，提供一种用于检测或测定IL-4R的方法，所述方法包括使上述抗IL-4R抗体或其抗原结合片段与样本接触的步骤。
在一些实施方案中，提供一种用于检测或测定人IL-4R的试剂，所述试剂包含如上所述的抗IL-4R抗体或其抗原结合片段。
在一些实施方案中，提供一种用于检测或测定人IL-4R的试剂，所述试剂包含这样的抗体，其与根据本申请的结合人IL-4R的抗体或其抗原结合片段，针对IL-4R或其表位进行竞争性结合。
在一些实施方案中，提供一种用于检测或测定人IL-4R的试剂，所述试剂包含上述双特异性抗体、多特异性抗体、单链抗体。
在一些实施方案中，提供一种用于诊断与人IL-4R阳性细胞相关的疾病的诊断剂，所述诊断剂包含如上所述的抗IL-4R抗体或其抗原结合片段。在另一些实施方案中，所述诊断剂包含这样的抗体，其与根据本申请的结合人IL-4R的抗体或其抗原结合片段，针对IL-4R或其表位进行竞争性结合。
在一些实施方案中，提供一种药物组合物，其含有：
-根据本申请的结合人IL-4R的抗体或其抗原结合片段，和
-可药用的赋形剂、稀释剂或载体。
在一些具体的实施方案中，所述药物组合物的单位剂量含有1mg至1000mg根据本申请的IL-4R抗体或其抗原结合片段。
在一些具体的实施方案中，所述药物组合物中的IL-4R抗体或其抗原结合片段的浓度为1mg/L至1000mg/L。
在一些具体的实施方案中，所述药物组合物含有缓冲剂，其含量可以为1mM至1000mM。
在一些实施方案中，提供如上所述的结合人IL-4R的抗体或其抗原结合片段在制备用于治疗或预防IL-4R介导的疾病或病症的药物中的用途。
在一些实施方案中，提供如上所述的药物组合物在制备用于治疗或预防IL-4R介导的疾病或病症的药物中的用途。
在一些实施方案中，提供一种如上所述的结合人IL-4R的抗体或其抗原结合 片段在治疗或预防疾病或病症中的用途。
在一些实施方案中，提供如上所述的药物组合物在治疗或预防疾病或病症中的用途。
在本申请上下文中，所述病症或疾病可以是免疫性疾病或病症。
在一些实施方案中，所述的疾病或病症选自：哮喘、鼻息肉、慢性鼻窦炎、过敏性皮肤病、嗜酸细胞性食管炎、慢性阻塞性肺病、过敏性鼻炎、关节炎、炎症性疾病、变应性反应、自体免疫淋巴组织增生性综合征、自体免疫性溶血性贫血、巴雷特食管、自体免疫葡萄膜炎、结核病和肾病。
在至少一个实施方案中，所述疾病或病症是哮喘。
在另一些实施方案中，所述疾病或病症是过敏性皮肤病。
在一些实施方案中，提供一种结合人IL-4R的抗体或其抗原结合片段，其中所述的抗原结合片段为Fab、Fv、sFv、F(ab’) 2。
在一些实施方案中，提供一种治疗和/或预防IL-4R介导的疾病或病症的方法，该方法包括：向有需求的患者(或受试者)给予治疗有效量(或预防有效量)的如上所述的结合人IL-4R抗体或其抗原结合片段。
在一些实施方案中，提供一种治疗和/或预防IL-4R介导的疾病或病症的方法，该方法包括：向有需求的患者(或受试者)给予治疗有效量(或预防有效量)的如上所述的药物组合物。在一些实施方案中，提供一种治疗和/或预防免疫性疾病的方法，包括向有需求的患者(或受试者)给予治疗有效量(预防有效量)的如上所述的结合人IL-4R的抗体或其抗原结合片段或其药物组合物。
术语
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除显而易见在本文中的它处另有明确定义，否则本文使用的所有其它技术和科学术语都具有所属领域的一般技术人员通常理解的含义。
“人IL-4R”(hIL-4R)意指与白细胞介素-4(IL-4)、IL-4Rα特异性结合的人细胞因子受体。
本文中所用的氨基酸三字母代码和单字母代码如J.Biol.Chem，243，p3558(1968)中所述。
“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
抗体轻链可进一步包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体。
抗体重链可进一步包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1、IgG2、IgG3、IgG4或其变体。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。轻链的3个CDR区指轻链互补决定区1(LCDR1)、轻链互补决定区2(LCDR2)、和轻链互补决定区3(LCDR3)；重链的3个CDR区指重链互补决定区1(HCDR1)、重链互补决定区2(HCDR2)和重链互补决定区3(HCDR3)。
抗体包括鼠源抗体，嵌合抗体，人源化抗体，人抗体，其可以是重组获得的，例如可以是亲和力成熟得到的重组人抗体。
“重组人抗体”包括通过重组方法制备、表达、创建或分离的人抗体，所涉及的技术和方法在本领域中是熟知的，诸如(1)从人免疫球蛋白基因的转基因、转染色体动物(例如小鼠)或由其制备的杂交瘤中分离的抗体；(2)从经转化以表达抗体的宿主细胞如转染瘤中分离的抗体；(3)从重组组合人抗体文库中分离的抗体；以及(4)通过将人免疫球蛋白基因序列剪接到其他DNA序列等方法制备、表达、创建或分离的抗体。此类重组人抗体包含可变区和恒定区，这些区域利用特定的由种系基因编码的人种系免疫球蛋白序列，但也包括随后诸如在抗体成熟过程中发生的重排和突变。
“鼠源抗体”在本文中为根据本领域知识和技能制备的对人IL-4R的单克隆抗体。制备时用IL-4R抗原或包含其表位的多肽注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在一些实施方案中，所述的鼠源结合人IL-4R的抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1，IgG2，IgG3或IgG4或其变体的重链恒定区。
“人抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本文的人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(如通过体外随机或位点特异性诱变或通过体内体细胞突变所引入的突变)。然而，术语“人抗体”不包括这样的抗体，即其中已将衍生自另一种哺乳动物物种(诸如小鼠)种系的CDR序列移植到人骨架序列上(即“人源化抗体”)。
“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将非人物种的CDR序列移植到人的抗体可变区框架中产生的抗 体。可以克服嵌合抗体由于携带大量异种蛋白成分，从而诱导的强烈的免疫应答反应。为避免在免疫原性下降的同时引起活性的下降，可对所述的人抗体可变区可进行最少反向突变，以保持活性。
“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要选建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再要据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入人载体中，最后在真核工业系统或原核工业系统中表达嵌合抗体分子。人抗体的恒定区可选自人源IgG1，IgG2，IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG2或IgG4重链恒定区，或者使用氨基酸突变后无ADCC(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG1。
“抗原结合片段”指具有抗原结合活性的Fab片段，Fab’片段，F(ab’)2片段，与人IL-4R结合的Fv片段、scFv片段，以及，包含上述片段的多肽或蛋白。所述“抗原结合片段”包含本文所述抗体的一个或多个CDR区。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体(single chain antibody)或单链Fv(scFv)。
“与IL-4R结合”，指能与人IL-4R相互作用。本文的术语“抗原结合位点”指由本文抗体或抗原结合片段识别的三维空间位点。
“表位”是指抗原上与免疫球蛋白或抗体特异性结合的位点。表位可以由相邻的氨基酸、或通过蛋白质的三级折叠而并列的不相邻的氨基酸形成。由相邻的氨基酸形成的表位通常在暴露于变性溶剂后保持，而通过三级折叠形成的表位通常在变性溶剂处理后丧失。表位通常以独特的空间构象包括至少3-15个氨基酸。确定什么表位由给定的抗体结合的方法在本领域中是熟知的，包括免疫印迹和免疫沉淀检测分析等。确定表位的空间构象的方法包括本领域中的技术和本文所述的技术，例如X射线晶体分析法和二维核磁共振等。
“特异性结合”、“选择性结合”是指抗体与预定的抗原上的表位结合。通常，当使用重组人IL-4R作为分析物，并使用抗体作为配体，在仪器中通过表面等离子体共振(SPR)技术测定时，抗体以大约低于10 -7M或甚至更小的平衡解离常数(K D)与预定的抗原结合，并且其与预定抗原结合的亲和力是其与预定抗原或紧密相关的抗原之外的非特异性抗原(如BSA等)结合的亲和力的至少两倍。术语“识别抗原的抗体”在本文中可以与术语“特异性结合的抗体”互换使用。
“交叉反应”是指本文的抗体与来自不同物种的IL-4R结合的能力。例如， 结合人IL-4R的本文的抗体也可以结合另一物种的IL-4R。交叉反应性是通过在结合测定(例如SPR和ELISA)中检测与纯化抗原的特异性反应性，或与生理表达IL-4R的细胞的结合或功能性相互作用来测量。确定交叉反应性的方法包括如本文所述的标准结合测定，例如表面等离子体共振(SPR)分析，或流式细胞术。
“中和”或“阻断”抗体意指其与hIL-4R的结合造成对hIL-4和/或hIL-13生物活性抑制的抗体。这种hIL-4和/或IL-13生物活性的抑制可通过测量本领域众所周知的一个或多个hIL-4/或hIL-13生物活性指标，如hlL-4/或hIL-13诱导的细胞活化和hIL-4与hIL-4R的结合等指标来评估，例如参见CN103739711A中的。“抑制生长”(例如涉及细胞)旨在包括细胞生长任何可测量的降低。
“诱导免疫应答”和“增强免疫应答”可互换使用，并指免疫应答对特定抗原的刺激(即，被动或适应性的)。针对诱导CDC或ADCC的术语，“诱导”是指刺激特定的直接细胞杀伤机制。
“ADCC(antibody-dependent cell-mediated cytotoxicity)”，抗体依赖的细胞介导的细胞毒作用，是指表达Fc受体的细胞通过识别抗体的Fc段直接杀伤被抗体包被的靶细胞。可通过对IgG上Fc段的修饰，降低或消除抗体的ADCC效应功能。所述的修饰指在抗体的重链恒定区进行突变，如选自IgG1的N297A，L234A，L235A；IgG2/4嵌合体，IgG4的F235E，或L234A/E235A突变。
融合蛋白是一种通过DNA重组得到的两个基因共表达的蛋白产物。重组IL-4R胞外区Fc融合蛋白通过DNA重组，把IL-4R胞外区和人抗体Fc片段共表达的融合蛋白。所述的IL-4R胞外区，是指IL-4R蛋白表达在细胞膜以外的部分。
生产和纯化抗体和抗原结合片段的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南，5-8章和15章。如，小鼠可以用人IL-4R或其片段免疫，所得到的抗体能被复性，纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。本文所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人FR区。人FR种系序列可以从ImMunoGeneTics(IMGT)的网站http：//imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。利用分子克隆技术将本文的人源化抗体的序列插入到相应的表达载体中，利用HEK293细胞表达系统表达生产，即可获得相应的人源化抗体。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在FC区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。 可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛，离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
抗体可以是单克隆抗体(mAb)，是指由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的，原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术，合成技术(如CDR-grafting)，或其它现有技术进行重组得到。
抗体可以是单特异、双特异或多特异抗体。多特异性抗体可对靶标肽的不同表位显示特异性，或也可包含对一个以上靶标肽显示特异性的抗原结合域。人抗IL-4R抗体可被连接到另一个功能分子(如另一个肽或蛋白质)上，或与之共表达。例如，抗体或其片段可功能性的(如通过化学欧联、基因融合、非共价结合或其他方式)与一个或多个其他分子(如另一个抗体或抗原结合片段)连接，以产生具有第二结合特异性的双特异或多特异抗体。
“施用”、“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“施用”、“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“施用”、“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者(或受试者)内用或外用治疗剂，诸如包含本文的任一种抗体或其抗原片段的组合物，所述患者(或受试者)已经具有、疑似具有或易感于一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者(或受试者)或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床可测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者(或受试者)的疾病状态、年龄和体重，以及药物在患者(或受试者)产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本本的实施方案(例如治疗方法或制品)在缓解单个患者(或受试者)的目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者(或受试者)中应当减轻目标疾病症状。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大 小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene，The Benjamin/Cummings Pub.Co.，第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。
“有效量”包含足以改善或预防医字病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定受试者或兽医学受试者的有效量可依据以下因素而变化：如待治疗的病症、受试者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“外源性”指根据背景在生物、细胞或人体外产生的物质。
“内源性”指根据背景在细胞、生物或人体内产生的物质。
“同源性”或“同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100％的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。本文所述“至少85％序列同一性”是指变体与亲本序列相比，两序列具有至少85％同源，在一些方案中，其具有至少86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％序列同源；在一些具体的方案中，其具有90％、95％或99％以上；在另一些具体的方案中，其具有至少95％序列同源。所述具有至少85％序列同一性的氨基酸序列包括通过对亲本序列进行一个或者多个氨基酸缺失、插入或替换突变获得。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括其后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“药物组合物”表示含有一种或多种本文所述抗体或抗原结合片段或其生理 学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
附图说明
图1：小鼠皮炎模型中，经丙酮致敏后，人源化抗体hu25G7-A、hu25G7-B和阳性参照抗体度匹鲁单抗以皮下给药方式：一周给药两次，第27天检测小鼠耳朵厚度。结果显示，和对照组相比，hu25G7-A、hu25G7-B与度匹鲁单抗均能有效降低小鼠的耳朵厚度，且hu25G7-B显示出比度匹鲁单抗更优的效果。
具体实施方式
以下结合实施例用于进一步描述本公开，但这些实施例并非限制着本公开的范围。实施例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1：小鼠免疫接种及检测
带his标签的人IL-4R(h-IL-4R-his)重组蛋白、带his标签的小鼠IL-4R(m-IL-4R-his)重组蛋白和带his标签的恒河猴IL-4R(rhesus-IL-4R-his)重组蛋白由Acrobiosystems公司合成、HEK293表达以及纯化。
带人源Fc标签的人IL-4R(h-IL-4R-Fc)重组蛋白经自行设计、表达以及纯化。纯化的蛋白可用于下述各实施例实验中。
本实施例中抗体或抗原结合片段的VL区和VH区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDR2-3)，或符合Kabat和CHOTHIA(ABM)规则(HCDR1)。
表1.免疫原信息
名称 氨基酸序列起止 数据库编号/商品号
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
抗人IL4R单克隆抗体通过免疫小鼠产生。实验用C57BL/6小鼠，雌性，6～8周龄(苏州昭衍新药研究中心有限公司，动物生产许可证号：201503052)。
饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时的光/暗周期调节，温度20-25℃；湿度40-60％。将已适应环境的小鼠分成3笼，每笼5只。免疫抗原为带Fc标签的人IL-4R重组蛋白(h-IL4R-Fc，浓度为0.73mg/ml)。用 弗氏佐剂(sigma，Cat#：F5881)乳化：首次用弗氏完全佐剂(CFA，Pierce，Cat#77140)，其余加强免疫用核酸类佐剂(CpG，上海生工)和铝佐剂(Alum，Thermo Cat#77161)。
第0天腹膜内(IP)注射70μg/只的乳化后抗原。第14、28、42、56、77天，根据背部结块和腹部肿胀情况，选择背部和腹膜内注射抗原(各注射0.1ml)。于第21、35、49、63、84天采血进行血检，用测试例1的ELISA方法检测小鼠血清，确定小鼠血清中的抗体滴度。在第4次免疫以后，选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合，融合前3天加强免疫，腹膜内(IP)注射10ug/只的磷酸盐缓冲液配制的抗原溶液。采用优化的PEG介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(
Figure PCTCN2019102169-appb-000001
CRL-8287 TM)进行融合得到杂交瘤细胞。
实施例2：抗体的ELISA测试及筛选
1、ELISA结合实验：
ELISA实验被用来检测IL-4R抗体的结合特性。用直接包被带his标签的IL-4R重组蛋白的酶标板，抗体加入各孔后，通过加入二抗(HRP偶联的抗一抗Fc的抗体)和HRP底物TMB检测抗体与抗原结合的活性。
人或恒河猴IL-4R-his蛋白包被96孔酶标板，按0.5μg/ml浓度每孔100μl，4℃孵育过夜。洗液洗三遍，每孔250μl。每次洗涤震荡10秒以保证充分清洗。加入200μl/孔封闭液室温孵育2小时。洗液洗三遍，每孔250μl。每次洗涤震荡10秒以保证充分清洗。每孔加100μl用稀释液稀释好的抗IL-4R待测抗体。室温孵育1小时。洗液洗三遍，每孔250μl。每孔加入100μl用稀释液按1：20000倍稀释的HRP标记的山羊抗人IgG二抗。室温孵育1小时。洗液洗三遍，每孔250μl。每孔加入100μl TMB，避光反应15分钟。加入50μl每孔的0.16M/L硫酸。Thermo MultiSkanFc酶标仪读取450nm OD值，计算IL-4R抗体对IL-4R的结合EC 50值。
2、ELISA阻断实验：
本实验通过体外阻断实验，检测所筛选出来的抗人IL-4R抗体阻断人IL-4R和人IL-4结合。具体方法是，将带Fc标签的IL-4R重组蛋白包被到96孔酶标板上，加入结合人IL-4R的抗体充分结合占据表位后，再加入IL-4(Biolegend，Cat#574004)，通过生物素偶联的抗IL-4抗体以及Neutravidin-HRP(Pierce，Cat#31001)，检测IL-4是否还能够结合到IL-4R上，计算IL-4R抗体对IL-4/IL-4R结合的阻断的IC 50值。
人IL-4R-Fc蛋白包被96孔酶标板，按0.5μg/ml浓度每孔100μl，4℃孵育过夜。洗液洗三遍，每孔250μl。每次洗涤震荡10秒以保证充分清洗。加入200μl/孔封闭液室温孵育2小时。洗液洗三遍，每孔250μl。每次洗涤震荡10秒以保证充分清洗。每孔加100μl用稀释液稀释好的抗IL-4R待测抗体，室温孵育1小时。 洗液洗三遍，每孔250μl。每孔加入100μl稀释好的IL-4，室温孵育1小时，洗液洗三遍。每孔加入100μl稀释好的生物素偶联的抗IL-4抗体，室温孵育1小时，洗液洗三遍。用稀释液按1：5000倍稀释的HRP标记的Neutravidin，室温孵育1小时。洗液洗三遍，每孔250μl。每孔加入100μl TMB，避光反应15分钟。加入50μl每孔的0.16M/L硫酸。Thermo MultiSkanFc酶标仪读取450nm OD值，计算IL-4R抗体对IL-4R与IL-4结合阻断的IC 50值。
实施例3：结合人IL-4R的抗体的报告基因细胞活性实验
HEK-Blue IL-4细胞购自Invivogen(Cat#hkb-stat6)，该细胞稳定转染了人IL-4R基因和STAT6介导的SEAP基因组，可以通过SEAP底物QUANTI-Blue检测上清中分泌的SEAP来表征IL-4R信号通路的活化水平。
本实验通过检测细胞HEK-Blue IL-4的活化，根据IC 50大小评价IL-4R抗体的体外细胞活性。在含10％FBS、100μg/ml Zeocin(Invivogen，Cat#ant-zn-05)和10μg/ml Blasticidin(Invivogen，Cat#ant-bl-05)的DMEM培养基中培养HEK-Blue IL-4细胞；一周传代2～3次，传代比列1：5或1：10。传代时，吸掉培养基，用5ml 0.25％的胰酶冲洗细胞层，然后吸掉胰酶，将细胞放在培养箱中消化3～5分钟，加入新鲜培养基重悬细胞。在96孔细胞培养板中加入100μL的细胞悬液，密度为5×10 5细胞/ml，培养基为10％FBS，100ug/ml Zeocin和30ug/ml Blasticidin的DMEM，96孔板外围只加入100μl无菌水。将培养板在培养箱培养24小时(37℃，5％CO 2)。细胞贴壁后，每孔加入100μl梯度稀释的待测抗体。将培养板在培养箱孵育20-24小时(37℃，5％CO 2)。每孔取20μl细胞上清到一个新的96孔平底板中，加入180μl QUANTI-Blue底物溶液，将培养板在培养箱内避光孵育1-3小时。用酶标仪(Thermo MultiSkanFc)测定在620nm处的吸光度。
实施例4：结合人IL-4R的抗体抑制TF-1细胞增殖实验
TF-1细胞(ATCC CRL-2003)是表达IL-4R，对IL-4/IL-13等细胞因子敏感的淋巴癌细胞。IL-4可以刺激TF-1细胞在无GM-CSF的情况下增殖。本实验通过加入中和性抗体，阻断IL-4的作用通路，抑制TF-1细胞增殖，来比较不同IL-4R抗体的中和活性。细胞TF-1培养在含10％FBS，2ng/ml GM-CSF(R&D，Cat#215-GM-010)的RPMI1640培养基中；一周传代2～3次，传代比列1：10。在96孔细胞培养板中加入100μL的细胞悬液，密度为2×10 5细胞/ml，培养基为10％FBS RPMI1640培养基，96孔板外围只加入100μl无菌水。每孔加入50μl梯度稀释的待测抗体和50μl终浓度0.7ng/ml的IL-4(R&D，Cat#204-IL-050)，将培养板在培养箱孵育72小时(37℃，5％CO 2)。培养结束后，用CTG试剂盒(Promega，Cat#G7572)检测细胞增殖。
实施例5：体外结合亲和力和动力学实验
用Biacore，GE仪器测定待测人源化结合人IL-4R的抗体和人IL-4R亲和力。
按照人抗捕获试剂盒(Cat.#BR-1008-39，GE)说明书中的方法将人抗捕获抗体共价偶联于Biacore仪器(Biacore X100，GE)的生物传感芯片CM5上，从而亲和捕获一定量的待测抗体；然后于芯片表面流经一系列浓度梯度下的IL-4R抗原(IL-4R抗原均购自Acrobiosystems，Cat#ILR-H5221)，利用Biacore仪器(Biacore X100，GE)实时检测反应信号从而获得结合和解离曲线。在每个循环解离完成后，用人抗捕获试剂盒里配置的再生溶液将生物芯片洗净再生。实验中用到的氨基偶联试剂盒购自GE公司(Cat.#BR-1000-50，GE)，缓冲液为HBS-EP+10×缓冲溶液(Cat.#BR-1006-69，GE)用D.I.Water稀释至1X(pH 7.4)。
实验得到的数据用BiacoreX100evaluation software2.0GE软件以(1：1)Binding模型进行拟合，得出亲和力数值。
实施例6：抗体的序列与制备
通过如上实施例2的ELISA结合实验(人IL-4R-his的ELISA结合)和ELISA阻断实验(人IL-4/IL-4R的ELISA阻断)、实施例3中抑制HEK293-Blue IL-4细胞在IL-4刺激下活化的实验，以及实施例4中抑制TF-1细胞在IL-4刺激下增殖的实验，选出体外活性最好的两株单克隆杂交瘤细胞株。其活性检测结果见表2。
表2.选出体外活性最好的杂交瘤细胞株
Figure PCTCN2019102169-appb-000002
挑选出体外活性最好的单克隆杂交瘤细胞株25G7和7B10，克隆其中的单克隆抗体序列。从杂交瘤中克隆序列过程如下。收集对数生长期杂交瘤细胞，用Trizol(Invitrogen，15596-018)提取RNA(按照试剂盒说明书步骤)，反转录(PrimeScript TMReverse Transcriptase，Takara，cat#2680A)。将反转录得到的cDNA采用mouse Ig-Primer Set(Novagen，TB326 Rev.B 0503)进行PCR扩增后送测序公司测序，并分析所得抗体序列。
鼠单抗25G7的重链和轻链可变区序列如下：
25G7 HCVR
Figure PCTCN2019102169-appb-000003
Figure PCTCN2019102169-appb-000004
25G7 LCVR
Figure PCTCN2019102169-appb-000005
其含有的CDR序列如表3所示。
表3.单抗25G7的CDR序列
名称 序列 编号
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
鼠单抗7B10的重链和轻链可变区序列如下：
7B10 HCVR
Figure PCTCN2019102169-appb-000006
7B10 LCVR
Figure PCTCN2019102169-appb-000007
其含有的CDR序列如表4所示。
表4.单抗7B10的CDR序列
名称 序列 编号
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
将获得的可变区序列接上人的恒定区序列，得到人-鼠嵌合的抗体序列。利用分子克隆技术，把嵌合抗体的序列插入到相应的表达载体中。利用HEK293细胞表达系统，即可获得人-鼠嵌合抗体25G7-C和7B10-C。
经前述实施例2-5的方法，对纯化的嵌合抗体进行相应的体外活性检测，数据见表5。结果显示，25G7-C抗体对IL-4结合的阻断效果，以及对细胞增殖的抑制 效果均显著优于参照抗体度匹鲁单抗(参考WHO Drug Information，Vol.26，No.4，2012合成)。
表5.体外活性检测
Figure PCTCN2019102169-appb-000008
实施例7：小鼠抗体人源化实验
对所得鼠源抗体中功能活性最强的两株(25G7和7B10)进行人源化。在所获得的鼠源抗体VH/VLCDR典型结构的基础上，将重、轻链可变区序列与抗体Germline数据库比较，获得同源性高的人种系模板。其中，人类种系轻链框架区来自人κ轻链基因，优选人种系轻链模版IGKV3-11*01(SEQ ID NO：22，用于抗体25G7)和IGKV2D-29*01(SEQ ID NO：24，用于抗体7B10)。人类种系重链框架区来自人重链，优选人种系重链模版IGHV3-48*01(SEQ ID NO：21，用于抗体25G7)和IGHV1-2*02(SEQ ID NO：23，用于抗体7B10)。
人种系模板序列如下所示。
人种系重链模版IGHV3-48*01：
Figure PCTCN2019102169-appb-000009
人种系轻链模板IGKV3-11*01：
Figure PCTCN2019102169-appb-000010
人种系重链模版IGHV1-2*02：
Figure PCTCN2019102169-appb-000011
人种系轻链模板IGKV2D-29*01：
Figure PCTCN2019102169-appb-000012
将鼠源抗体的CDR区移植到选择的人源化模板上，替换人源化可变区，再与IgG恒定区重组。然后，以鼠源抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对VL和VH的构象有重要影响的残基进行回复突变，得到一系列人源化分子。
其中，对Hu7B10-VH-a、hu7B10-VH-b、hu7B10-VH-c做成药性修饰，将重链人种系模板的第一位由Q变为E。对hu25G7也做成药性修饰。两株抗体的人源化后的重链可变区序列分别如SEQ ID NO：25-27，SEQ ID NO：31-33所示；轻链可变区序列分别如SEQ ID NO：28-30，SEQ ID NO：34-36所示。
hu25G7-VH-a：
Figure PCTCN2019102169-appb-000013
hu25G7-VH-b：
Figure PCTCN2019102169-appb-000014
hu25G7-VH-c：
Figure PCTCN2019102169-appb-000015
hu25G7-VL-a：
Figure PCTCN2019102169-appb-000016
hu25G7-VL-b：
Figure PCTCN2019102169-appb-000017
hu25G7-VL-c：
Figure PCTCN2019102169-appb-000018
hu7B10-VH-a：
Figure PCTCN2019102169-appb-000019
hu7B10-VH-b：
Figure PCTCN2019102169-appb-000020
hu7B10-VH-c：
Figure PCTCN2019102169-appb-000021
hu7B10-VL-a：
Figure PCTCN2019102169-appb-000022
hu7B10-VL-b：
Figure PCTCN2019102169-appb-000023
hu7B10-VL-c：
Figure PCTCN2019102169-appb-000024
杂交瘤克隆25G7的模板选择和回复突变设计见表6。
表6.模板选择和回复突变设计
Figure PCTCN2019102169-appb-000025
杂交瘤克隆7B10的模板选择和回复突变设计，见下表7：
表7.模板选择和回复突变设计
Figure PCTCN2019102169-appb-000026
经由以上各轻重链组合的抗体小规模表达测试和回复突变数量比较，综合评判并选择出最终的人源化抗体hu25G7(使用VH-c重链和VL-a轻链)和hu7B10抗体分子(使用VH-b重链和VL-b轻链)，其各自的完整轻重链序列如SEQ ID NO：17-20所示。
hu25G7 HC
Figure PCTCN2019102169-appb-000027
hu25G7 LC
Figure PCTCN2019102169-appb-000028
hu7B10 HC
Figure PCTCN2019102169-appb-000029
hu7B10 LC
Figure PCTCN2019102169-appb-000030
利用分子克隆技术，将人源化抗体的序列插入到相应的表达载体中，利用HEK293细胞表达系统表达生产，即可获得相应的人源化抗体。
实施例8：人源化抗体活性数据
对人源化抗体hu25G7和hu7B10进行实施例2-5所述的体外活性检测，试验结果见表8。结果显示，hu25G7和hu7B10均只与人IL-4R结合，而不与恒河猴IL-4R 结合，说明两个抗体均结合在人与恒河猴不同源的表位上，能特异性地结合人IL-4R。两个抗体均能阻断IL-4/IL-4R结合以及细胞内信号通路，导致IL-4的激活作用被中和，TF-1细胞的增殖被抑制，其中hu25G7的阻断及抑制活性仍显著优于参照抗体度匹鲁单抗，但亲和力K D值相对较低一些。
表8.体外活性检测
Figure PCTCN2019102169-appb-000031
实施例9：人源化抗体hu25G7的亲和力成熟实验
为获得药效更佳的抗人IL-4R抗体，通过酵母展示平台技术对25G7抗体进行亲和力成熟，在hu25G7抗体的基础上设计并制备针对6个CDR的亲和力成熟的酵母文库，设计简并引物，通过PCR和同源重组的方法将设计的突变氨基酸引入hu25G7-scFv抗体文库中，每个文库的大小在10 9左右，构建好的酵母文库通过二代测序(GENEWIZ)的方法验证文库的多样性。
用生物素标记人的IL-4R从hu25G7-scFv酵母文库中筛选高亲和力的抗体，经过两轮MACS筛选(链霉素磁珠，Invitrogen)和两轮FACS筛选(BD FACSAriaTM FUSION)，再挑选酵母单克隆培养和诱导表达，通过FACS(BD FACSCanto II)检测酵母单克隆对人IL-4R的结合，选出比野生型25G7抗体亲和力高的酵母单克隆测序验证，经过对测序克隆进行比对分析，去除冗余序列之后，将非冗余序列转换成全长人的抗体分子，进行哺乳动物细胞表达。亲和纯化之后的全长抗体采用BIAcoreTM X-100(GE Life Sciences)进行亲合力测定，挑选出对人IL-4R较高亲和力的候选抗体分子如下。这些抗体分子对人IL-4R的亲和力高于野生型hu25G7抗体，其中hu25G7-A抗体分子的亲和力和度匹鲁单抗相当，而hu25G7-B分子的亲和力明显更优于度匹鲁单抗。
亲和力成熟最终得到抗体hu25G7-A的轻链可变区序列如下：
hu25G7-A LCVR
EIVLTQSPATLSLSPGERATLSCRASSSVPYMYWYQQKPGQAPRLLIYLTSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWRAYPPMLTFGGGTKVEIK(SEQ ID NO：37)其含有的CDR序列如表9所示。
表9.CDR序列
名称 序列 编号
LCDR1 RASSSVPYMY SEQ ID NO：38
LCDR2 LTSNLAS SEQ ID NO：7
LCDR3 QQWRAYPPMLT SEQ ID NO：40
抗体hu25G7-B的轻链可变区序列如下：
hu25G7-B LCVR
Figure PCTCN2019102169-appb-000032
其含有的CDR序列如表10所示。
表10.CDR序列
名称 序列 编号
LCDR1 RASPGVPPLA SEQ ID NO：42
LCDR2 LASSRPS SEQ ID NO：39
LCDR3 QQWRSNPPMLT SEQ ID NO：8
上述轻链可变区hu25G7-A LCVR与hu25G7轻链恒定区重组，得到hu25G7-A抗体轻链；上述轻链可变区hu25G7-B LCVR与hu25G7轻链恒定区重组，得到hu25G7-B抗体轻链。
对hu25G7-VH-c不稳定氨基酸残基优化，增强成药性，得到重链可变区hu25G7-VH
Figure PCTCN2019102169-appb-000033
上述重链可变区可与hu25G7重链恒定区重组，得到hu25G7-A/hu25G7-B抗体重链。
hu25G7-A和hu25G7-B完整的重链序列如SEQ ID NO：44所示。
hu25G7HC
Figure PCTCN2019102169-appb-000034
各自完整的轻链序列如SEQ ID NO：45-46所示。
hu25G7-A LC
Figure PCTCN2019102169-appb-000035
hu25G7-B LC
Figure PCTCN2019102169-appb-000036
实施例10：人源化抗体亲和力成熟活性数据
实施例3和实施例4对hu25G7-A及hu25G7-B两个抗体进行检测；hu25G7-A及hu25G7-B两个抗体均能阻断IL-4/IL-4R结合，以及细胞内信号通路导致IL-4及IL-13的激活作用被中和，TF-1细胞的增殖被抑制，各项活性数据见表11。
表11.活性数据比较
Figure PCTCN2019102169-appb-000037
在抑制IL-13刺激引起的TF-1细胞增殖实验中，hu25G7-A和hu25G7-B均表现出有益效果。对效果进行重复验证，结果显示，在相同条件下，hu25G7-A对TF-1细胞中的IL-13增殖抑制(IC 50)值为11.68，对照度匹鲁单抗对TF-1细胞中的IL-13增殖抑制(IC 50)值为22.85。hu25G7比度匹鲁单抗在阻断IL-4、IL-13与IL-4R的结合方面，及所述结合引起的细胞增殖方面具有显著提高的效果。
实施例11：人源化抗体对小鼠皮炎的作用研究
建立小鼠皮炎模型：选用IL-4/IL-4Rα转基因小鼠(购自赛业模式生物研究中心(太仓)有限公司)，每只小鼠腹部范围约为3cm×3cm，均匀涂抹1.5％OXZ丙酮橄榄油溶液(丙酮：橄榄油＝4：1)100μL致敏，致敏当日计为D0(Day 0)。第七天(Day 7)开始将1％OXZ丙酮橄榄油溶液20μL均匀涂抹于小鼠双耳(两 面)进行攻击，每隔72小时攻击一次。
本实验设正常对照组(致敏和激发时仅涂抹丙酮橄榄油溶液)、模型对照组、hu25G7-A、hu25G7-B和度匹鲁单抗组，共5组，每组3～5只小鼠，给药组给药剂量为50mg/kg，给药方式为皮下给药，一周给药两次(具体信息见表12)。第27天用游标卡尺检测耳朵厚度，结果如图1所示。
表12.各组施用方案
组别 动物数 给药方式 给药剂量 给药频率*
正常对照组 3(雄) S.C. - 一周2次
模型对照组 5(3雌+2雄) S.C. - 一周2次
hu25G7-A 5(3雌+2雄) S.C. 50mg/kg 一周2次
hu25G7-B 3(2雌+1雄) S.C. 50mg/kg 一周2次
度匹鲁单抗 4(2雌+2雄) S.C. 50mg/kg 一周2次
结果显示，模型对照组小鼠耳朵出现明显病理性损伤，耳朵厚度显著高于正常对照组。hu25G7-A、hu25G7-B及度匹鲁单抗组小鼠第27天耳朵厚度显著低于模型对照组。即hu25G7-A、hu25G7-B与度匹鲁单抗均可以用于治疗皮炎，且hu25G7-B的效果较之度匹鲁单抗更优异。

Claims (19)

  1. 一种抗IL-4R抗体或其抗原结合片段，其中：
    重链可变区，包含：
    (I)分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；或
    (II)分别如SEQ ID NO：11、SEQ ID NO：12和SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3；
    和/或，轻链可变区，包含：
    (I)分别如SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3；或
    (II)分别如SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示的LCDR1、LCDR2和LCDR3；或
    (III)分别如SEQ ID NO：38、SEQ ID NO：7和SEQ ID NO：40所示的LCDR1、LCDR2和LCDR3；或
    (IV)分别如SEQ ID NO：42、SEQ ID NO：39和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3。
  2. 根据权利要求1所述的抗IL-4R抗体或其抗原结合片段，其包含选自以下(I)至(IV)中的任一项：
    (I)重链可变区，其包含分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；和
    轻链可变区，其包含分别如SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3；
    (II)重链可变区，其包含分别如SEQ ID NO：11、SEQ ID NO：12和SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3；和
    轻链可变区，其包含分别如SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示的LCDR1、LCDR2和LCDR3；
    (III)重链可变区，其包含分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；和
    轻链可变区，其包含分别如SEQ ID NO：38、SEQ ID NO：7和SEQ ID NO：40所示的LCDR1、LCDR2和LCDR3；
    (IV)重链可变区，其包含分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；和
    轻链可变区，其包含分别如SEQ ID NO：42、SEQ ID NO：39和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3。
  3. 根据权利要求1-2任一项所述的抗IL-4R抗体或其抗原结合片段，其进一步包含选自以下的片段或其组合：
    -源自人种系轻链IGKV3-11*01的FR区序列；
    -源自与人种系轻链IGKV3-11*01的FR区具有至少95％同一性的回复突变序列；
    优选地，所述的回复突变选自L46P、L47W、F71Y中的一个或组合；
    和/或，所述的抗IL-4R抗体或其抗原结合片段进一步包含选自以下的片段或其组合：
    -源自人种系重链IGHV3-48*01的FR区序列；
    -源自与人种系重链IGHV3-48*01的FR区具有至少95％同一性的回复突变序列；
    优选地，所述的回复突变选自S49A、F67S、A93T中的一个或组合。
  4. 根据权利要求1-2任一项所述的抗IL-4R抗体或其抗原结合片段，其进一步包含选自以下的片段或其组合：
    -源自人种系轻链IGKV2D-29*01的FR区序列；
    -源自与人种系轻链IGKV2D-29*01的FR区具有至少95％同一性的回复突变序列；
    优选地，所述的回复突变选自M4L和/或V58I；
    和/或，所述的抗IL-4R抗体或其抗原结合片段进一步包含选自以下的片段或其组合：
    -源自人种系重链IGHV1-2*02的FR区序列；
    -源自与人种系重链IGHV1-2*02的FR区具有至少95％同一性的回复突变序列；
    优选地，所述的回复突变选自M69L、R71I、T73K、R94K中的一个或组合。
  5. 根据权利要求1-2任一项所述的抗IL-4R抗体或其抗原结合片段，其中：
    所述抗原结合片段选自：Fab、Fab’-SH、Fv、scFv、(Fab’)2片段；
    所述抗体的重链可变区包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链框架区；优选地，所述抗体的重链可变区包含人源IgG1、IgG2或IgG4的重链框架区；
    更优选地，所述抗体的重链可变区包含人源IgG4的重链框架区。
  6. 根据权利要求1-2任一项所述的抗IL-4R抗体或其抗原结合片段，其中：
    重链可变区，包含：
    (I)如SEQ ID NO：1所示或与SEQ ID NO：1具有至少90％、95％、98％、99％同一性的序列；或
    (II)如SEQ ID NO：9所示或与SEQ ID NO：9具有至少90％、95％、98％、99％同一性的序列；或
    (III)如SEQ ID NO：43所示或与SEQ ID NO：43具有至少90％、95％、98％、99％同一性的序列；
    和/或，轻链可变区，包含：
    (I)如SEQ ID NO：2所示或与SEQ ID NO：2具有至少90％、95％、98％、99％同一性的序列；或
    (II)如SEQ ID NO：10所示或与SEQ ID NO：10具有至少90％、95％、98％、99％同一性的序列；或
    (III)如SEQ ID NO：37所示或与SEQ ID NO：37具有至少90％、95％、98％、99％同一性的序列；或
    (IV)如SEQ ID NO：41所示或与SEQ ID NO：41具有至少90％、95％、98％、99％同一性的序列；
    优选地，所述抗IL-4R抗体或其抗原结合片段包含：
    SEQ ID NO：1所示的重链可变区，SEQ ID NO：2所示的轻链可变区；或
    SEQ ID NO：9所示的重链可变区，SEQ ID NO：10所示的轻链可变区；或
    SEQ ID NO：43所示的重链可变区，SEQ ID NO：37所示的轻链可变区；或
    SEQ ID NO：43所示的重链可变区，SEQ ID NO：41所示的轻链可变区。
  7. 根据权利要求1-2任一项所述的抗IL-4R抗体或其抗原结合片段，其中：
    重链可变区，包含：
    (I)如SEQ ID NO：25-27之一所示或与SEQ ID NO：25-27之一具有至少90％、95％、98％或99％同一性的序列；或
    (II)如SEQ ID NO：31-33之一所示或与SEQ ID NO：31-33之一具有至少90％、95％、98％、或99％同一性的序列；
    和/或轻链可变区，包含：
    (I)如SEQ ID NO：28-30之一所示或与SEQ ID NO：28-30之一具有至少90％、95％、98％或99％同一性的序列；或
    (II)如SEQ ID NO：34-36之一所示或与SEQ ID NO：34-36之一具有至少90％、95％、98％或99％同一性的序列。
  8. 根据权利要求1-2任一项所述的抗IL-4R抗体或其抗原结合片段，其中：
    重链，包含
    (I)如SEQ ID NO：17所示或与SEQ ID NO：17具有至少90％、95％、98％ 或99％同一性的序列；或
    (II)如SEQ ID NO：19所示或与SEQ ID NO：19具有至少90％、95％、98％、或99％同一性的序列；或
    (III)如SEQ ID NO：44所示或与SEQ ID NO：44具有至少90％、95％、98％、或99％同一性的序列；
    和/或轻链，包含
    (I)如SEQ ID NO：18所示或与SEQ ID NO：18具有至少90％、95％、98％或99％同一性的序列；或
    (II)如SEQ ID NO：20所示或与SEQ ID NO：20具有至少90％、95％、98％或99％同一性的序列；或
    (III)如SEQ ID NO：45所示或与SEQ ID NO：45具有至少90％、95％、98％或99％同一性的序列；或
    (IV)如SEQ ID NO：46所示或与SEQ ID NO：46具有至少90％、95％、98％或99％同一性的序列；
    优选地，所述抗IL-4R抗体或其抗原结合片段包含选自以下的任一项：
    SEQ ID NO：17所示的重链，和SEQ ID NO：18所示的轻链；或
    SEQ ID NO：19所示的重链，和SEQ ID NO：20所示的轻链；或
    SEQ ID NO：44所示的重链，和SEQ ID NO：45所示的轻链；或
    SEQ ID NO：44所示的重链，和SEQ ID NO：46所示的轻链。
  9. 根据权利要求1-2任一项所述的抗IL-4R抗体或其抗原结合片段，其为鼠源抗体、嵌合抗体、人抗体、人源化抗体或其片段。
  10. 一种多核苷酸，其编码权利要求1至9任一项所述的抗IL-4R抗体或其抗原结合片段。
  11. 一种载体，其含有权利要求10所述的多核苷酸，所述载体为真核表达载体、原核表达载体或病毒载体。
  12. 一种宿主细胞，其包含权利要求11所述的载体，
    优选地，所述宿主细胞选自：细菌、酵母、哺乳动物细胞，
    更优选地，所述宿主细胞选自：大肠杆菌、毕赤酵母、中国仓鼠卵巢细胞、人胚肾293细胞。
  13. 一种用于检测或测定IL-4R的方法，所述方法包括：
    使权利要求1至9任一项所述的抗IL-4R抗体或其抗原结合片段与样本接触 的步骤。
  14. 一种试剂，所述试剂包含权利要求1至9任一项所述的抗IL-4R抗体或其抗原结合片段。
  15. 根据权利要求14所述的试剂，所述试剂用于诊断与人IL-4R阳性细胞相关的疾病。
  16. 一种药物组合物，其含有：
    权利要求1至9任一项所述的抗IL-4R抗体或其抗原结合片段；以及
    可药用的赋形剂、稀释剂或载体。
  17. 选自以下的任一项或其组合在制备用于治疗或预防IL-4R介导的疾病或病症、或用于治疗或预防免疫性疾病或病症的药物中的用途：
    权利要求1至9任一项所述的抗IL-4R抗体或其抗原结合片段、权利要求10所述的多核苷酸、权利要求16所述的药物组合物，
    优选地，所述疾病或病症选自：
    哮喘、鼻息肉、慢性鼻窦炎、过敏性皮肤病、嗜酸细胞性食管炎、慢性阻塞性肺病、过敏性鼻炎、关节炎、炎症性疾病、变应性反应、自体免疫淋巴组织增生性综合征、自体免疫性溶血性贫血、巴雷特食管、自体免疫葡萄膜炎、结核病和肾病；
    更优选地，所述疾病或病症为哮喘或过敏性皮肤病。
  18. 一种治疗或预防IL-4R介导的疾病或病症、和/或治疗或预防免疫性疾病或病症的方法，包括：
    向受试者施用权利要求1至9任一项所述的抗IL-4R抗体或其抗原结合片段、权利要求16所述的药物组合物，
    优选地，所述疾病或病症选自：
    哮喘、鼻息肉、慢性鼻窦炎、过敏性皮肤病、嗜酸细胞性食管炎、慢性阻塞性肺病、过敏性鼻炎、关节炎、炎症性疾病、变应性反应、自体免疫淋巴组织增生性综合征、自体免疫性溶血性贫血、巴雷特食管、自体免疫葡萄膜炎、结核病和肾病；更优选地，所述疾病或病症为哮喘或过敏性皮肤病。
  19. 一种用于制备抗IL-4R抗体或其抗原结合片段的方法，包括如下步骤：
    在权利要求12所述的宿主细胞中表达抗IL-4R抗体或其抗原结合片段，以及
    从所述宿主细胞中分离抗IL-4R抗体或其抗原结合片段。
PCT/CN2019/102169 2018-08-24 2019-08-23 结合人il-4r的抗体、其抗原结合片段及其医药用途 WO2020038454A1 (zh)

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EP19852040.5A EP3808774A4 (en) 2018-08-24 2019-08-23 HUMAN IL-4R BINDING ANTIBODIES, ANTIGEN BINDING FRAGMENT AND RELATED MEDICAL USE
MX2021001143A MX2021001143A (es) 2018-08-24 2019-08-23 Anticuerpo de union a il-4r humana, fragmento de union a antigeno y uso medico del mismo.
AU2019324403A AU2019324403A1 (en) 2018-08-24 2019-08-23 Human IL-4R binding antibody, antigen binding fragment thereof, and medical use thereof
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JP2021508304A JP7405831B2 (ja) 2018-08-24 2019-08-23 ヒトｉｌ－４ｒ結合抗体、その抗原結合フラグメント、およびそれらの医学的使用
BR112021002989-3A BR112021002989A2 (pt) 2018-08-24 2019-08-23 anticorpo para ligação de il-4r de humano, fragmento para ligação de antígeno e uso médico do mesmo
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111996172A (zh) * 2020-09-08 2020-11-27 中国食品药品检定研究院 一种用于测定il-4靶向治疗性抗体生物学活性的方法
CN114652853A (zh) * 2020-12-22 2022-06-24 江苏恒瑞医药股份有限公司 抗il-4r抗体-药物偶联物及医药用途
WO2022135441A1 (zh) 2020-12-22 2022-06-30 江苏恒瑞医药股份有限公司 抗il-4r抗体或其抗原结合片段的复合物及医药用途
WO2023143351A1 (zh) 2022-01-29 2023-08-03 上海盛迪医药有限公司 糖皮质激素的药物偶联物

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20240199688A1 (en) 2022-05-02 2024-06-20 Regeneron Pharmaceuticals, Inc. Methods for reducing lipase activity
US20240024472A1 (en) 2022-05-02 2024-01-25 Regeneron Pharmaceuticals, Inc. Anti-Interleukin-4 Receptor (IL-4R) Antibody Formulations
WO2024061279A1 (en) * 2022-09-22 2024-03-28 Biosion Inc. Recombinant bispecific antibodies targeting tslp and il4r
WO2024197119A1 (en) * 2023-03-22 2024-09-26 Sanofi Biotechnology Methods for treating chronic obstructive pulmonary disease (copd) by administering an il-4r antagonist
CN117924491B (zh) * 2023-12-22 2024-10-18 华润生物医药有限公司 抗il-4r抗体及其用途

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001092340A2 (en) 2000-05-26 2001-12-06 Immunex Corporation Use of interleukin-4 antagonists and compositions thereof
WO2008054606A2 (en) 2006-10-02 2008-05-08 Regeneron Pharmaceuticals, Inc. High affinity human antibodies to human il-4 receptor
WO2010053751A1 (en) 2008-10-29 2010-05-14 Regeneron Pharmaceuticals, Inc. High affinity human antibodies to human il-4 receptor
WO2014031610A1 (en) 2012-08-21 2014-02-27 Sanofi Methods for treating or preventing asthma by administering an il-4r antagonist
CN108373505A (zh) * 2018-04-20 2018-08-07 北京智仁美博生物科技有限公司 抗il-4r抗体及其用途
CN108409860A (zh) * 2017-02-10 2018-08-17 上海麦济生物技术有限公司 抗人白细胞介素-4受体α单克隆抗体、其制备方法和应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101620539B1 (ko) * 2007-12-21 2016-05-13 메디뮨 리미티드 인터루킨-4 수용체 알파(ＩＬ-4Ｒα)에 대한 결합 구성원-173
EP3539985A1 (en) * 2012-02-24 2019-09-18 AbbVie Stemcentrx LLC Anti sez6 antibodies and methods of use
EP3188757A1 (en) * 2014-09-03 2017-07-12 Medimmune Limited Stable anti-il-4r-alpha antibody formulation
BR112017020915A2 (pt) * 2015-04-02 2018-07-10 Intervet Int Bv ?anticorpo de mamífero isolado ou fragmento de ligação ao antígeno do mesmo, anticorpo caninizado ou fragmento de ligação ao antígeno caninizado do mesmo, anticorpo monoclonal caninizado ou fragmento de ligação ao antígeno do mesmo, ácido nucleico isolado, vetor de expressão, célula hospedeira, peptídeo isolado, proteína de fusão, composição farmacêutica, e, método para diminuir a atividade de uma célula imune?
CN108339118A (zh) * 2017-01-23 2018-07-31 瑞阳(苏州)生物科技有限公司 治疗或预防阻塞性睡眠呼吸暂停的药物组合物

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001092340A2 (en) 2000-05-26 2001-12-06 Immunex Corporation Use of interleukin-4 antagonists and compositions thereof
WO2008054606A2 (en) 2006-10-02 2008-05-08 Regeneron Pharmaceuticals, Inc. High affinity human antibodies to human il-4 receptor
WO2010053751A1 (en) 2008-10-29 2010-05-14 Regeneron Pharmaceuticals, Inc. High affinity human antibodies to human il-4 receptor
CN103739711A (zh) 2008-10-29 2014-04-23 瑞泽恩制药公司 抗人il-4受体的高亲和性人抗体
EP3064511A1 (en) * 2008-10-29 2016-09-07 Regeneron Pharmaceuticals, Inc. High affinity human antibodies to human il-4 receptor
WO2014031610A1 (en) 2012-08-21 2014-02-27 Sanofi Methods for treating or preventing asthma by administering an il-4r antagonist
CN108409860A (zh) * 2017-02-10 2018-08-17 上海麦济生物技术有限公司 抗人白细胞介素-4受体α单克隆抗体、其制备方法和应用
CN108373505A (zh) * 2018-04-20 2018-08-07 北京智仁美博生物科技有限公司 抗il-4r抗体及其用途

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
"Drug Information", vol. 26, 2012, WHO
"The Immunoglobulin FactsBook", 2001
J. BIOL. CHEM, vol. 243, 1968, pages 3558
See also references of EP3808774A4
WATSON ET AL.: "Molecular Biology of the Gene", 1987, THE BENJAMIN/CUMMINGS PUB. CO., pages: 224
YANG GUANGYONG,GUO HAITAO,LIU XIMING,HE GUANGZHI,TIAN WEIYI,CAI KUN,WANG PING,WANG WENJIA: "Construction and Panning of scFv Phage Display Library Against Recombinant Interleukin 4 Receptor", CHINESE JOURNAL OF CELLULAR AND MOLECULAR IMMUNOLOGY, vol. 32, no. 6, 31 December 2016 (2016-12-31), pages 829 - 833, XP055770247, ISSN: 1007-8738, DOI: 10.13423/j.cnki.cjcmi.007799 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111996172A (zh) * 2020-09-08 2020-11-27 中国食品药品检定研究院 一种用于测定il-4靶向治疗性抗体生物学活性的方法
CN114652853A (zh) * 2020-12-22 2022-06-24 江苏恒瑞医药股份有限公司 抗il-4r抗体-药物偶联物及医药用途
WO2022135441A1 (zh) 2020-12-22 2022-06-30 江苏恒瑞医药股份有限公司 抗il-4r抗体或其抗原结合片段的复合物及医药用途
EP4269447A4 (en) * 2020-12-22 2024-07-24 Jiangsu Hengrui Pharmaceuticals Co Ltd ANTI-IL-4R ANTIBODY COMPLEX OR ANTIGEN-BINDING FRAGMENT THEREOF AND ASSOCIATED MEDICAL USE
CN114652853B (zh) * 2020-12-22 2024-09-13 江苏恒瑞医药股份有限公司 抗il-4r抗体-药物偶联物及医药用途
WO2023143351A1 (zh) 2022-01-29 2023-08-03 上海盛迪医药有限公司 糖皮质激素的药物偶联物

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