WO2020239134A1 - 抗体白介素4受体的抗体及其应用 - Google Patents

抗体白介素4受体的抗体及其应用 Download PDF

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WO2020239134A1
WO2020239134A1 PCT/CN2020/097686 CN2020097686W WO2020239134A1 WO 2020239134 A1 WO2020239134 A1 WO 2020239134A1 CN 2020097686 W CN2020097686 W CN 2020097686W WO 2020239134 A1 WO2020239134 A1 WO 2020239134A1
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amino acid
seq
acid sequence
antibody
antigen
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宋德勇
董创创
韩静
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Shandong Boan Biotechnology Co Ltd
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Shandong Boan Biotechnology Co Ltd
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Priority to JP2021560459A priority Critical patent/JP7236562B2/ja
Priority to EP20814564.9A priority patent/EP3978528A4/en
Priority to US17/601,837 priority patent/US12338288B2/en
Publication of WO2020239134A1 publication Critical patent/WO2020239134A1/zh
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IG], 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
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • 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
    • C07K2317/70—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the field of biomedicine or biopharmaceutical technology, and more specifically to human anti-IL-4R antibody, its coding sequence, preparation method, composition and application.
  • Interleukin-4 is a cytokine with a variety of biological functions produced by activated type 2 T helper cells (Th2). It is a hydrophobic globular protein composed of 129 amino acid residues. . IL-4 has many target cells: T cells, B cells, hematopoietic cells, fibroblasts and various tumor cells all have IL-4 receptors. There are two types of interleukin-4 receptor (IL-4R): Type I is mainly composed of IL-4R ⁇ chain and ⁇ c chain, and is mainly expressed on the surface of hematopoietic cells.
  • Type II receptors are composed of IL-4R ⁇ chains and IL-13R ⁇ 1 chains, and are mainly expressed on the surface of non-hematopoietic cells and tumor cells (La Porte SL, Juo ZS, Vaclavikova J, et al. Cell, 2008, 132(2): 259-272).
  • IL-4R ⁇ is a key component of type I and type II receptors in the type 2 inflammatory pathway. Blocking IL-4R ⁇ can simultaneously block two powerful regulators of type 2 immune response IL-4 and IL-13 . In the type II inflammation pathway, Th2 cells (T helper 2, belonging to CD4+ T cells) play a key role.
  • Type II immunity is a special immune response that includes innate immunity and adaptive immunity and promotes the formation of an immune barrier on the mucosal surface to eliminate pathogens.
  • type 2 inflammatory pathways play an important role. Therefore, IL-4R ⁇ is one of the key targets for the targeted treatment of allergic diseases (including atopic dermatitis, asthma, idiopathic urticaria, chronic nasal polyp sinusitis, food allergy, etc.).
  • IL-4R anti-interleukin-4 receptor
  • the antibody is an interleukin 4 receptor (IL-4R) antagonist and inhibits IL-4 and IL-13 signaling.
  • IL-4R interleukin 4 receptor
  • the antibody injection is currently used for the treatment of atopic dermatitis and asthma indications.
  • the present invention provides antibodies or antigen-binding fragments thereof having a novel amino acid sequence.
  • the antibodies or antigen-binding fragments thereof include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, Fab, Fab', and F (ab')2, Fv, scFv or dsFv fragments, etc.
  • the antibody or antigen-binding fragment thereof provided by the present invention includes:
  • LCDR1 amino acid sequence is shown in SEQ ID NO: 3
  • LCDR2 amino acid sequence is shown in SEQ ID NO: 4
  • LCDR3 amino acid sequence is shown in SEQ ID NO: 5;
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO: 6, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 7, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 8
  • the light chain of the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL), and the light chain variable region includes the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 9. Any amino acid sequence or an amino acid sequence that has at least 80%, 85% or 90% sequence identity with the above-mentioned light chain variable region amino acid sequence; the antibody or its antigen-binding fragment heavy chain comprises a heavy chain variable region (VH ), the heavy chain variable region includes any amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 10 or includes at least 80%, 85%, or 90% of the amino acid sequence of the above heavy chain variable region Amino acid sequence of identity.
  • the antibody or antigen-binding fragment thereof comprises the following light and heavy chain variable region sequences comprising:
  • the antibody or antigen-binding fragment thereof comprises a heavy chain constant region, wherein the heavy chain constant region comprises a ⁇ -1, ⁇ -2, ⁇ -3 or ⁇ -4 human heavy chain constant region or the human variant of the heavy chain constant region, preferably having the sequence ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY
  • the antibody further comprises a light chain constant region, wherein the light chain constant region comprises a lambda or kappa human light chain constant region, preferably, the sequence of which is RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQCSSPVTKSFTLSKADYEKHKVYACEVTHQCSSPVTKSF
  • the binding antigen of the aforementioned antibody or antigen-binding fragment thereof is IL-4R, preferably, the IL-4R is human IL-4R.
  • the present invention also provides a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof that binds human IL-4R.
  • the nucleic acid encodes an antibody or an antigen-binding fragment thereof containing the following light chain and heavy chain complementarity determining region amino acid sequences:
  • LCDR1 amino acid sequence is shown in SEQ ID NO: 11
  • LCDR2 amino acid sequence is shown in SEQ ID NO: 12
  • LCDR3 amino acid sequence is shown in SEQ ID NO: 13
  • HCDR1 amino acid sequence is shown in SEQ ID NO: 14
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO: 15, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 16. or
  • LCDR1 amino acid sequence is shown in SEQ ID NO: 3
  • LCDR2 amino acid sequence is shown in SEQ ID NO: 4
  • LCDR3 amino acid sequence is shown in SEQ ID NO: 5
  • HCDR1 amino acid sequence is shown in SEQ ID NO: 6
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO: 7
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO: 8
  • the present invention relates to nucleic acids that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to nucleic acids that can hybridize to the nucleic acid of the present invention under stringent conditions.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Fortunately, hybridization occurs when more than 95%. Moreover, the polypeptide encoded by the hybridizable nucleic acid has the same biological function and activity as the mature polypeptide.
  • the recombination method can be used to obtain the relevant sequence in large quantities. This usually involves cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • the biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules that exist in an isolated form.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
  • the present invention also provides a vector containing a nucleotide sequence encoding an antibody that binds IL-4R or an antigen-binding fragment thereof.
  • the vector is an expression vector.
  • the vector of the present invention includes, but is not limited to, a viral vector, such as Adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, etc.; non-viral vectors, such as plasmids, transposon vectors, etc., among which the plasmid vector is preferably a pCDNA3.4 (Life Technology) vector. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • the present invention also provides a host cell for expressing an antibody or antigen-binding fragment thereof that binds to human IL-4R.
  • the host cell contains an expression vector that encodes an antibody that binds to human IL-4R or an antigen-binding fragment thereof or that encodes an antibody that binds to human IL-4R. 4R antibody or nucleic acid of antigen-binding fragment thereof.
  • the present invention also provides a cell for expressing an antibody that binds IL-4R or an antigen-binding fragment thereof, which cell contains an expression vector encoding an antibody that binds IL-4R or an antigen-binding fragment thereof, or an antibody that encodes IL-4R or an antibody
  • the cell is a host cell containing the above-mentioned expression vector.
  • host cells expressing IL-4R-binding antibodies or antigen-binding fragments thereof include, but are not limited to, mammalian cells, insect cells, plant cells, fungal cells, and prokaryotic cells.
  • Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, and 293 cells.
  • the host cell provided by the present invention for expressing an antibody or antigen-binding fragment thereof that binds IL-4R is HEK293.
  • Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as Escherichia coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Another method is to use MgCl 2 . If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the antibody encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • the recombinant antibody in the above method can be expressed in the cell, on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic cleavage, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.
  • compositions comprising the antibody or antigen-binding fragment thereof that binds to human IL-4R, and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier includes one or more of the following: pharmaceutically acceptable solvents, dispersants, additives, plasticizers, and pharmaceutical excipients. Generally, these substances are non-toxic, inert and pharmaceutically acceptable carrier media.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration (such as injection administration).
  • the present invention also relates to a kit, which comprises any of the antibodies or antibody fragments or nucleic acids that bind to the IL-4R antigen described above.
  • the kit includes an antibody or antigen-binding fragment thereof of any one of the following CDR amino acid sequences:
  • LCDR1 amino acid sequence is shown in SEQ ID NO: 11
  • LCDR2 amino acid sequence is shown in SEQ ID NO: 12
  • LCDR3 amino acid sequence is shown in SEQ ID NO: 13
  • HCDR1 amino acid sequence is shown in SEQ ID NO: 14
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO: 15, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 16; or
  • LCDR1 amino acid sequence is shown in SEQ ID NO: 3
  • LCDR2 amino acid sequence is shown in SEQ ID NO: 4
  • LCDR3 amino acid sequence is shown in SEQ ID NO: 5
  • HCDR1 amino acid sequence is shown in SEQ ID NO: 6
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO: 7, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 8.
  • the kit further includes a detection reagent, a negative control, and a positive control for detecting the IL-4R antigen-antibody reaction.
  • the present invention relates to the use of the antibody or antigen-binding fragment, nucleic acid, vector or cell of any one of the foregoing aspects in the preparation of a pharmaceutical composition for treating or preventing diseases.
  • the present invention relates to the application of the antibody or its antigen-binding fragment or nucleic acid of any one of the foregoing aspects in the preparation of diagnostic and detection kits.
  • a method for treating or preventing diseases comprises administering the antibody or antigen-binding fragment, nucleic acid, vector, cell, or pharmaceutical composition of the present invention to a subject in need.
  • a method of diagnosis and detection which includes administering the antibody or antigen-binding fragment, nucleic acid or kit of the present invention to a subject or sample in need.
  • the use of the antibody or antigen-binding fragment, nucleic acid, carrier, cell or pharmaceutical composition of any one of the foregoing aspects for the treatment and prevention of diseases is provided.
  • the use of the antibody or antigen-binding fragment, nucleic acid, or kit of any one of the foregoing aspects for detection and diagnosis is provided.
  • the disease is preferably an IL-4R-related disease, and further preferably, the IL-4R-related disease is an inflammatory or allergic disease, including asthma, atopic dermatitis, pruritus, and hypertrophic diseases.
  • the inflammatory or allergic disease is asthma;
  • the treatment and/prevention of asthma includes Reduce the incidence of asthma exacerbations, improve one or more asthma parameters, and improve the dependence of patients on inhaled corticosteroids and/or long-acting beta agonists.
  • the inventors successfully obtained a class of anti-IL-4R antibodies.
  • Experimental results show that the IL-4R antibodies obtained in the present invention can effectively block the interaction between IL-4R and its ligands.
  • the obtained candidate antibody has different epitope binding properties with dupilumab, low immunogenicity, long in vivo half-life, and significant in vivo effects in animal models of diseases such as asthma.
  • the present invention has been completed on this basis.
  • Figure 1 shows the serum titer of transgenic mice diluted 5000 times.
  • Figure 2 shows a comparison of candidate antibodies for blocking IL4/IL4R protein binding activity.
  • Figure 3 shows the comparison of BA167, BA173, BA030, Dupilumab and IL4R protein binding activity.
  • Figure 4 shows the comparison of the protein binding activity of BA167, BA173, BA030, and Dupilumab in blocking IL4/IL4R.
  • Figure 5 shows the comparison of the protein binding activity of BA167, BA173, BA030, Dupilumab in blocking IL13+IL13RA1/IL4R.
  • Figure 6 shows the difference in binding of BA167, BA173, BA030 and Dupilimab to Human/Rhesus/Mouse IL4R.
  • Figure 7 shows the IL4-induced TF-1 cell proliferation inhibition experiment.
  • Figure 8 shows the IL13-induced TF-1 cell proliferation inhibition experiment.
  • Figure 9 shows the proliferation inhibitory effect of candidate antibodies on PBMC cells.
  • Figure 10 shows the average concentration-time curve of cynomolgus monkeys after subcutaneous injection of 2.5 mg/kg prescription BA167, BA173, and Dupilumab.
  • FIG 11 shows the changes in serum IgE levels in B-hIL4/hIL4R ⁇ humanized asthma model mice after administration.
  • Figure 12 shows the changes in the BALF eosinophil level of B-hIL4/hIL4R ⁇ humanized asthma model mice after administration.
  • Figures 13A-13C show the immunogenicity detection of BA173, BA167 and Dupilumab antibodies, respectively.
  • Coat protein IL4R (10402-H08H, Yiqiao Shenzhou) with a concentration of 1 ⁇ g/ml with CBS coating solution (pH 9.6 carbonic acid solution), 100ul/hole at 4 degrees overnight; use 3% skimmed milk powder at 37°C for 1h; PBST will dilute the serum to 200X, 1000X, 5000X, 25000X, and add 100ul per well. Incubate at 37°C for 1h; then add HRP-goat anti-human H+L (474-1006, KPL), incubate at 37°C for 1h, after 10min of color development, stop with 2M concentrated sulfuric acid, and read OD450 on the microplate reader. The detection of serum titer is shown in Figure 1.
  • the establishment of the phage library was carried out according to the method described in Carlos F. Barbas III, Phage display: A laboratory manual.
  • the phage library IL4R Q14 was established with the mouse numbered IL4R Q14, with a storage volume of 9.28 ⁇ 10 8 ; the number was IL4R Q6 establishment of mouse phage library of IL4R Q6, capacity 2.8x 10 8; No. IL4R Q29 to establish a mouse phage library IL4R Q29, capacity 6.4x 10 8.
  • IL4R-His protein (Yiqiao Shenzhou, 10402-H08H) coated the plate at 1 ⁇ g/well, placed overnight at 4°C, and blocked the plate with 2% BSA the next day for 1 hour, and added the phage library (2x 10 12 ) Incubate for 2h, wash 4-10 times and elute IL4R-bound phage with Elution Buffer (pH 2.2).
  • IL4RQ14 represents the 14th wild-type mouse immunized
  • BA represents magnetic bead screening
  • the library that was positive by phage enzyme-linked immunoassay (Elisa) method was coated on a plate, and the plate was picked to directly induce expression of the autonomous medium, the supernatant was tested for binding activity, and the selected ones were continued to be detected by ELISA to block the expression of IL4.
  • the ones with IL4 blocking activity were selected, and an ELISA test was performed again to select positive clones that can block IL13/IL13RA1. Molecular construction and production of these positive clones.
  • the amino acid sequence of each clone is shown in Table 1:
  • variable region gene amplification by conventional molecular biology techniques (2*EasyPfu PCR SuperMix manufacturer: Transgen, article number: AS211, lot number: #L11228), signal peptide and variable region overlap extension, will carry heavy chain and signal peptide genes
  • the variable region homologous recombination (ClonExpress II One Step Cloning Kit manufacturer: Vazyme Item Number: C112-01 Lot Number: TE222B8) is connected to the vector pCDNA3.4 (Life Technology) with the antibody heavy chain constant region (IgG4) sequence, and Homologous recombination of the variable region with light chain and signal peptide genes (ClonExpress II One Step Cloning Kit Manufacturer: Vazyme Item Number: C112-01 Lot Number: TE222B8) is connected to the vector pCDNA3.4( Life Technology, the sequence is as follows.
  • the transient expression supernatant was purified by Protein A affinity chromatography to obtain IL4R antibody, and combined with UV280 for extinction The coefficient determines the antibody concentration.
  • Dupilumab an IL4R antibody against regenerant IL4R
  • IMGT data The amino acid sequence of Dupilumab was determined by IMGT data and patent US2008160035A1. After the complete gene synthesis, it was inserted into the vector pCDNA3.4 and expressed by HEK293 cells.
  • the amino acid sequence of Dupilumab is as follows:
  • Coated IL4 (11846-HANE, Yiqiao Shenzhou) with a concentration of 0.2 ⁇ g/ml, 100ul/hole 4 degrees overnight; blocked with 3% skimmed milk powder for 1h; meanwhile IL4R-Fc-biotin (0.4 ⁇ g/ml) 50ul and different Concentrations (16 ⁇ g/mL, 4 ⁇ g/mL, 1 ⁇ g/mL, 0.25 ⁇ g/mL, 0.0625 ⁇ g/mL, 0.015625 ⁇ g/mL, 0.00390625 ⁇ g/mL)
  • IL4R-BA167-IgG4 hereinafter or referred to as BA167
  • BA173-IgG4 hereinafter or referred to as BA173
  • BA030 IL4R-BA030-IgG4
  • Coated IL4 (11846-HANE, Yiqiao Shenzhou) with a concentration of 0.2 ⁇ g/ml, 100ul/hole 4 degrees overnight; blocked with 3% skimmed milk powder for 1h; at the same time IL4R-Fc-biotin (0.4 ⁇ g/ml) and different concentrations (30 ⁇ g/mL, 7.5 ⁇ g/mL, 1.875 ⁇ g/mL, 0.46875 ⁇ g/mL, 0.1171875 ⁇ g/mL, 0 ⁇ g/mL)
  • Candidate antibodies were incubated at 37°C for 1h, and then added to the blocked ELISA plate at 37°C.
  • the Elisa method was used to detect the binding of BA167, BA173, BA030 and Dupilumab to human, mouse and cynomolgus IL4R respectively.
  • TF-1 cells, IL4 (Sinobiological, catalog number: 11846-HNAE) and antibodies were all diluted with complete medium (90% RPMI1640, 10% FBS), TF-1 cells were diluted to 4x10 5 cells/mL, and inoculated in In a white 96-well plate, 50 ⁇ L/well, that is, 20000cells/well. Dilute IL4 to 2ng/mL. The antibody was diluted to 5 ⁇ g/mL, and then diluted 4 times in sequence, a total of 6 concentrations. Mix the diluted antibody and IL4 in equal volumes and add them to the cell wells, 50 ⁇ L/well. 1ng/mL IL4 was added to the cell well as a negative control.
  • complete medium 90% RPMI1640, 10% FBS
  • TF-1 cells were diluted to 4x10 5 cells/mL, and inoculated in In a white 96-well plate, 50 ⁇ L/well, that is, 20000cells/well. Dilute
  • TF cells, IL13 (Sinobiological, catalog number: 10369-HNAC) and antibodies were all diluted with complete medium (90% RPMI1640, 10% FBS), and TF-1 cells were diluted to 4x10 5 cells/mL and seeded in white 96 In the well plate, 50 ⁇ L/well, that is, 20000cells/well. Dilute IL13 to 4ng/mL. The antibody was diluted to 7.5 ⁇ g/mL, and then diluted 8 times in sequence, a total of 6 concentrations. Mix the diluted antibody and IL13 in an equal volume and add it to the cell well, 50 ⁇ L/well. Add 2ng/mL IL13 to the cell well as a negative control.
  • IL4 can promote the proliferation of PBMC cells. After adding the antibody, the antibody binds to the receptor of IL4 and inhibits the proliferation of PBMC cells.
  • Cells, IL4 (Sinobiological, catalog number: 11846-HNAE) and antibodies were all diluted with complete medium (90% RPMI 1640, 10% FBS). Resuscitate PBMC, add PHA (Thermo, catalog number: 10576015) at a ratio of 1:100, and cultivate for 4 days. Dilute the antibody to 160 ⁇ g/mL and dilute 10 times in sequence, a total of 9 concentrations. Dilute IL4 to 400ng/mL. After mixing an equal volume of antibody and IL4, add it to a 96-well plate, 50 ⁇ L/well.
  • TF-1 is a human blood leukemia cell.
  • IL4 and IL13 can induce the growth of TF-1 cells by binding to cell surface receptors. After antibody is added, the antibody binds to IL4/IL13 receptors, thereby inhibiting the growth of TF-1 cells. proliferation.
  • BA173 antibody the inhibitory effect of BA173 antibody on IL-4 induced TF-1 cell proliferation is better than other candidate antibodies, and BA173 has the same effect as Dupilumab Similar blocking activity.
  • BA173 and BA167 have a better effect than Dupilumab, indicating that the two antibodies can better bind to IL4 receptors, thereby inhibiting the proliferation of PBMC.
  • Example 4 The study of BA167, BA173 and Dupilumab on the PK of cynomolgus monkeys
  • the dose is 2.5mg/kg, twice administration, the second administration time is 14d (d means day), 0h before administration, 1h after administration, 4h, 10h and 1d, 2d, 3d, 4d, 5d, 7d, 10d, 14d and 1h, 4h, 10h and 1d, 2d, 3d, 4d, 5d, 7d, 10d, 14d, 21d after the second dose
  • the serum was collected to detect the antibody concentration.
  • the method of serum detection was Elisa method. The specific results of the test are shown in Figure 10 and Table 7 below.
  • OVA ovalbuproliferin
  • sensitize by intraperitoneal injection 200 ⁇ L/only.
  • the sensitization time was 0, 7, and 14 days.
  • aerosol inhalation of 2% OVA was used for 30 minutes each time for 5 consecutive days.
  • the test substance was administered on the 20th and 23rd day, and samples were collected for analysis and analysis 24 hours after the last challenge operation. Twenty-four hours after the end of the last nebulization challenge operation, the peripheral blood of the mice was collected, and the serum was tested for OVA-specific IgE antibody; the alveolar lavage fluid was collected for OVA-specific IgE antibody detection.
  • the average serum IgE level in the G1 control group was 28.85ng/mL, and the IgE levels in the Dupilumab, BA173 and BA167 groups were 2.0ng/mL, 3.4ng/mL, 2.1ng/mL, respectively; eosinophil count control group average 384.50X 10 2 (number / mL), Dupilumab, BA173 and BA167 group group eosinophils cells were 31.1X 10 2 (two /mL),12.8X 10 2 (two /mL),14.0X 10 2 (pcs/mL).
  • the BA167 and BA173 groups can significantly reduce the IgE level in the serum of asthma model mice and the number of eosinophils in the alveolar lavage fluid at a dose of 25mg/kg. Effectively alleviate asthma, help reduce the incidence of asthma exacerbations, improve asthma-related evaluation parameters, and improve patients' dependence on inhaled corticosteroids and/or long-acting beta agonists.
  • Candidate antibodies BA173 and BA167 both showed similar effects in the above animal models to those in asthma models, exhibiting resistance to atopic dermatitis, pruritus, neutropenia, allergic reactions, nasal polyps, eosinophilic esophagitis, Skin infections and chronic sinusitis have good relief or treatment effects.

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Abstract

提供特异性结合IL-4R抗原的抗体或其抗原结合片段、其制备方法、组合物和应用。该IL-4R抗体可用于制备于治疗和/或预防炎症或过敏症得药物，还可用于免疫学检测IL-4R抗原。

Description

抗体白介素4受体的抗体及其应用 技术领域
本发明涉及生物医学或生物制药技术领域，更具体地涉及人抗IL-4R的抗体，及其编码序列、制备方法、组合物和应用。
背景技术
白细胞介素-4(interleukin-4；IL-4)是由活化的2型T辅助细胞(Th2)产生的具有多种生物学功能的细胞因子，由129个氨基酸残基组成疏水性的球形蛋白质。IL-4有很多靶细胞：T细胞、B细胞、造血细胞、成纤维细胞和各种肿瘤细胞等都具有IL-4受体。白细胞介素-4受体(IL-4R)有两种类型：Ⅰ型主要由IL-4Rα链和γc链组成,主要在造血细胞表面表达。II型受体由IL-4Rα链和IL-13Rα1链构成，主要在非造血细胞和肿瘤细胞表面常表达(La Porte SL,Juo ZS,Vaclavikova J,et al.Cell,2008,132(2):259-272)。IL-4Rα是2型炎症通路中I型受体和II型受体的关键组成部分，阻断IL-4Rα可同时阻断两个2型免疫反应的强效调节因子IL-4和IL-13。在II型炎症通路中，Th2细胞(T helper 2，属于CD4+T细胞)扮演关键角色。II型免疫是一种包含先天性免疫和适应性免疫并促使在黏膜表面形成免疫屏障清除病原体的特殊免疫反应。在过敏性疾病发生过程中，2型炎症通路起到重要作用。因此，IL-4Rα是针对性治疗过敏性疾病(包括特应性皮炎、哮喘、特发性荨麻疹、慢性鼻息肉性鼻窦炎以及食物过敏等)的关键靶点之一。
目前，赛诺菲公司的靶向白介素4受体(IL-4R)的抗体药物dupilumab已获得美国FDA批准上市，商品名
Figure PCTCN2020097686-appb-000001
该抗体是白介素4受体(IL-4R)拮抗剂，抑制IL-4及IL-13信号。目前该抗体注射剂用于特应性皮炎及哮喘适应症的治疗。
然而，面对患者对于疾病治疗的药物需求，尤其是抗体药物的需求，仍然亟待有免疫原性更低、半衰期更长，药物效果更优的IL-4R抗体药物。
发明内容
本发明提供了具有新的氨基酸序列的抗体或其抗原结合片段，所述抗体或其抗原结合片段为包括单克隆抗体、多克隆抗体、嵌合抗体、人源化抗体、Fab、Fab’、F(ab’)2、Fv、scFv或dsFv片段等。
本发明所提供的抗体或其抗原结合片段，包括：
1)3个轻链互补决定区，其中，LCDR1氨基酸序列如SEQ ID NO:11所示，LCDR2氨基酸序列如SEQ ID NO:12所示，LCDR3氨基酸序列如SEQ ID NO:13所示；和3个重链互补决定区，其中，HCDR1氨基酸序列如SEQ ID NO:14所示，HCDR2氨基酸序列如SEQ ID NO:15所示，HCDR3氨基酸序列如SEQ ID NO:16。或
2)3个轻链互补决定区，其中，LCDR1氨基酸序列如SEQ ID NO:3所示，LCDR2氨基酸序列如SEQ ID NO:4所示，LCDR3氨基酸序列如SEQ ID NO:5所示；和
3个重链互补决定区，其中，HCDR1氨基酸序列如SEQ ID NO:6所示，HCDR2氨基酸序列如SEQ ID NO:7所示，HCDR3氨基酸序列如SEQ ID NO:8
在本发明的一个方面中，所述抗体或其抗原结合片段轻链包含轻链可变区(VL)，所述轻链可变区包括SEQ ID NO:1或SEQ ID NO:9所示的任一氨基酸序列或包括与上述轻链可变区氨基酸序列具有至少80％、85％或90％序列同一性的氨基酸序列；所述抗体或其抗原结合片段重链包含重链可变区(VH)，所述重链可变区包括SEQ ID NO:2或SEQ ID NO:10所示的 任一氨基酸序列或包括与上述重链可变区氨基酸序列具有至少80％、85％或90％序列同一性的氨基酸序列。优选地，所述抗体或其抗原结合片段包含以下轻、重链可变区序列包含：
1)SEQ ID NO:1所示的氨基酸序列轻链可变区和/或包含与SEQ ID NO:2所示的氨基酸序列的重链可变区，或者
2)SEQ ID NO:9所示的氨基酸序列的轻链可变区和/或SEQ ID NO:10所示的氨基酸序列的重链可变区。
在一个方面，所述抗体或其抗原结合片段包含重链恒定区，其中所述重链恒定区包含γ-1、γ-2、γ-3或γ-4人重链恒定区或所述人重链恒定区的变体，优选地，其序列为ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。在一个方面，所述抗体还包含轻链恒定区，其中所述轻链恒定区包含λ或κ人轻链恒定区，优选地，其序列为RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
在本发明的一个方面，上述抗体或其抗原结合片段的结合抗原是IL-4R，优选地，所述IL-4R是人IL-4R。
本发明还提供了编码含有结合人IL-4R的抗体或其抗原结合片段的核酸序列。
在本发明的一个方面中，所述核酸编码含有以下轻链和重链互补决定区氨基酸序列的抗体或其抗原结合片段：
1)LCDR1氨基酸序列如SEQ ID NO:11所示，LCDR2氨基酸序列如SEQ ID NO:12所示，LCDR3氨基酸序列如SEQ ID NO:13所示；和HCDR1氨基酸序列如SEQ ID NO:14所示，HCDR2氨基酸序列如SEQ ID NO:15所示，HCDR3氨基酸序列如SEQ ID NO:16。或
2)LCDR1氨基酸序列如SEQ ID NO:3所示，LCDR2氨基酸序列如SEQ ID NO:4所示，LCDR3氨基酸序列如SEQ ID NO:5所示；和HCDR1氨基酸序列如SEQ ID NO:6所示，HCDR2氨基酸序列如SEQ ID NO:7所示，HCDR3氨基酸序列如SEQ ID NO:8
3)SEQ ID NO:1所示的氨基酸序列轻链可变区和/或包含与SEQ ID NO:2所示的氨基酸序列的重链可变区，或者
4)SEQ ID NO:9所示的氨基酸序列的轻链可变区和/或SEQ ID NO:10所示的氨基酸序列的重链可变区。
本发明涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的核酸。本发明特别涉及在严格条件下与本发明所述核酸可杂交的核酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC,0.1％SDS,60℃；或(2)杂交时加有变性剂,如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上,更好是95％以上时才发生杂交。并且,可杂交的核酸编码的多肽与成熟多肽有相同的生物学功能和活性。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明 所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还提供了含有编码结合IL-4R的抗体或其抗原结合片段的核苷酸序列的载体，优选地，所述载体是表达载体，本发明所述载体包括但不限于，病毒载体，如腺病毒载体、逆转录病毒载体、腺相关病毒载体等；非病毒载体，如质粒、转座子载体等，其中质粒载体优选为pCDNA3.4(Life Technology)载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
本发明还提供了用于表达结合人IL-4R的抗体或其抗原结合片段的宿主细胞，该宿主细胞含有编码结合人IL-4R的抗体或其抗原结合片段的表达载体或编码结合人IL-4R的抗体或其抗原结合片段的核酸。
本发明还提供了用于表达结合IL-4R的抗体或其抗原结合片段的细胞，该细胞含有编码结合IL-4R的抗体或其抗原结合片段的表达载体或编码结合IL-4R的抗体或其抗原结合片段的核酸，优选地，所述细胞是含有上述表达载体的宿主细胞。在本发明的一个方面中，表达结合IL-4R的抗体或其抗原结合片段的宿主细胞包括但不限于哺乳动物细胞、昆虫细胞、植物细胞、真菌细胞、原核细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。优选地，本发明提供用于表达结合IL-4R的抗体或其抗原结合片段的宿主细胞是HEK293。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔，脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的抗体。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组抗体可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
根据本发明的另一方面，还提供一种组合物，其包含上文所述结合人IL-4R的抗体或其抗原结合片段、以及药学上可接受的载体。所述药学上可接受的载体包括以下中的一种或多种：药学上可接受的溶剂、分散剂、附加剂、塑形剂、药物辅料。通常，这些物质是无毒的、惰性的和药学上可接受的载体介质。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或如局部给药(如注射给药)。
本发明还涉及一种试剂盒，其包含上文任一所述结合IL-4R抗原的抗体或其抗体片段、核酸。在本发明的一个方面中,所述试剂盒包含以下任一组CDR氨基酸序列的抗体或其抗原 结合片段：
1)LCDR1氨基酸序列如SEQ ID NO:11所示，LCDR2氨基酸序列如SEQ ID NO:12所示，LCDR3氨基酸序列如SEQ ID NO:13所示；和HCDR1氨基酸序列如SEQ ID NO:14所示，HCDR2氨基酸序列如SEQ ID NO:15所示，HCDR3氨基酸序列如SEQ ID NO:16；或
2)LCDR1氨基酸序列如SEQ ID NO:3所示，LCDR2氨基酸序列如SEQ ID NO:4所示，LCDR3氨基酸序列如SEQ ID NO:5所示；和HCDR1氨基酸序列如SEQ ID NO:6所示，HCDR2氨基酸序列如SEQ ID NO:7所示，HCDR3氨基酸序列如SEQ ID NO:8。
在本发明的一个方面中,所述试剂盒还包括用于对IL-4R抗原抗体反应进行检测的检测试剂、阴性对照、阳性对照。
在另一方面，本发明涉及前述任一方面所述的抗体或其抗原结合片段、核酸、载体或细胞在制备治疗或预防疾病的药物组合物中的应用。
在另一方面，本发明涉及前述任一方面所述的抗体或其抗原结合片段、核酸在制备诊断、检测试剂盒中的应用。
在另一方面，提供一种治疗或预防疾病的方法，包括将本发明的抗体或抗原结合片段、核酸、载体、细胞或药物组合物给予有需要的受试者。
在另一方面，提供一种诊断、检测的方法，包括将本发明的抗体或抗原结合片段、核酸或试剂盒给予有需要的受试者或样本。
在另一方面，提供前述任一方面所述的抗体或其抗原结合片段、核酸、载体、细胞或药物组合物用于治疗、预防疾病的用途。
在另一方面，提供前述任一方面所述的抗体或其抗原结合片段、核酸、或试剂盒用于检测、诊断的用途。
根据本发明的另一方面，所述疾病优选是IL-4R相关疾病，进一步优选地，所述IL-4R相关病症是炎症或过敏性疾病，包括哮喘、特应性皮炎、瘙痒症、嗜中性白血球减少症、过敏反应、鼻息肉、嗜酸性食管炎、皮肤感染、慢性鼻窦炎等；优选地，所述炎症或过敏性疾病是哮喘；进一步优选地，所述哮喘的治疗和/预防包括减少哮喘加重发生率，改善1至多种哮喘参数，改善患者对吸入皮质类固醇和/或长效β激动剂的依赖性等。
本发明人通过广泛而深入的研究，经过大量的筛选，成功获得一类抗IL-4R抗体，实验结果表明，本发明获得的IL-4R抗体能够有效阻断IL-4R与其配体之间的相互作用，令人意外的是，经鉴定，获得候选抗体具有与dupilumab不同表位结合特性，并且免疫原性低，体内半衰期长，在哮喘等疾病动物模型中体内效果显著。在此基础上完成了本发明。
附图说明
图1示出了血清稀释5000倍的转基因小鼠血清滴度。
图2示出了候选抗体阻断IL4/IL4R的蛋白结合活性的比较。
图3示出了BA167、BA173、BA030、Dupilumab与IL4R蛋白结合活性的比较。
图4示出了BA167、BA173、BA030、Dupilumab阻断IL4/IL4R的蛋白结合活性比较。
图5示出了BA167、BA173、BA030、Dupilumab阻断IL13+IL13RA1/IL4R的蛋白结合活性比较。
图6示出了BA167、BA173、BA030和Dupilimab与Human/Rhesus/Mouse IL4R的结合 差异。
图7示出了IL4诱导的TF-1细胞增殖抑制实验。
图8示出了IL13诱导的TF-1细胞增殖抑制实验。
图9示出了候选抗体对PBMC细胞的增殖抑制作用。
图10示出了食蟹猴皮下注射2.5mg/kg处方BA167、BA173、Dupilumab后体体内平均浓度-时间曲线。
图11示出了给药后B-hIL4/hIL4Rα双人源化哮喘模型小鼠血清中IgE水平变化。
图12示出了给药后B-hIL4/hIL4Rα双人源化哮喘模型小鼠BALF嗜酸细胞水平的变化。
图13A-13C依次分别示出了对BA173、BA167和Dupilumab抗体免疫原性检测。
具体实施方式
参照以下实施例可以更好地理解本发明。但是，应理解，以下实施例仅用于举例说明目的，而不应被理解为以任何方式限制本发明的保护范围。
实施例1.抗IL4R抗体的产生
1.1免疫
用IL4R(Sinobiological，目录号10402-H08H)与弗氏佐剂乳化后免疫山东博安全人抗体转基因小鼠BoAn-hMab。首免使用弗氏完全佐剂，二免至三免使用弗氏不完全佐剂，本次共免疫9只小鼠。选取血清滴度较高的小鼠加强免疫，3天后处死小鼠取出脾脏用于后续实验。检测血清滴度主要采用ELISA法。以CBS包被液(pH9.6碳酸溶液)包被浓度为1μg/ml的蛋白IL4R(10402-H08H，义翘神州)，100ul/孔4度过夜；用3％脱脂奶粉37℃封闭1h；用PBST将血清稀释至200X、1000X、5000X、25000X，每孔加100ul。37度孵育1h；然后加入HRP-山羊抗人H+L(474-1006，KPL)，37℃孵育1h，显色10min后，用2M浓硫酸终止，酶标仪上读取OD450。检测血清滴度如图1所示。
1.2噬菌体库的建立
取免疫小鼠的脾脏细胞，加入Trizol(Thermo Scientific，目录货号15596-026)，待裂解充分后加入1/5体积的氯仿，充分混匀，室温放置20min后4℃ 12000rpm离心20min，取上层水溶液，并加入等体积的异丙醇，室温放置20min，4℃ 12000rpm离心20min，弃去上清水溶液，加入75％乙醇洗涤两次，4℃ 12000rpm离心5min，弃去水溶液，保留沉淀，室温风干后加入DEPC水重悬沉淀获得RNA，获得的RNA使用罗氏反转录试剂盒Transcriptor First Strand cDNA Synthesis Kit(Roche Applied Science，目录货号4897030001)按照其说明书将RNA反转录成cDNA。噬菌体库的建立步骤参照Carlos F.Barbas III，Phage display：A laboratory manual中记载的方法进行，以编号为IL4R Q14的小鼠建立的噬菌体库IL4R Q14，库容9.28x 10 8；以编号为IL4R Q6的小鼠建立的噬菌体库IL4R Q6，库容2.8x 10 8；以编号为IL4R Q29的小鼠建立的噬菌体库IL4R Q29，库容6.4x 10 8。
1.3筛选
1.平板筛选用IL4R-His蛋白(义翘神州，10402-H08H)以1μg/孔包被平板，4℃放置过夜，第二天通过2％BSA封闭平板1h，加入噬菌体库(2x 10 12)孵育2h，洗涤4-10次后用Elution Buffer(pH 2.2)洗脱IL4R结合的噬菌体。
2.磁珠筛选，将IL4R-Fc蛋白(义翘神州，10402-H02H)按照常规步骤进行生物素化(投入的IL4R蛋白与生物素摩尔比1:2)，再与Thermo的磁珠(Invitrogen Dynabeads M-280 Streptavidin，00355871)结合后与噬菌体库孵育，洗涤4-10次后用Elution Buffer(pH 2.2) 洗脱IL4R特异性结合的噬菌体。
平板筛选获得克隆IL4RQ14-BA030\BA034、IL4RQ6-BA167\BA173、IL4RQ24-BA420、IL4RQ29-BA1301。其中，IL4RQ14代表免疫的第14只野生型小鼠，BA代表磁珠筛选。
将通过噬菌体酶联免疫(Elisa)法检测阳性的库涂布平板，挑取直接进行自主培养基的诱导表达，上清检测结合活性，挑选出的继续利用ELISA检测阻断IL4的表达。将具有IL4阻断活性的挑选出来，再进行一次ELISA检测，挑选出能阻断IL13/IL13RA1的阳性克隆。将这些阳性克隆进行分子构建与生产。
实施例2.阻断抗体的分子构建与生产
将克隆IL4RQ14-BA030、BA034(本发明中简称BA030、BA034)、IL4RQ6-BA167、BA173(本发明中简称BA167、BA173)、IL4RQ24-BA420(本发明中简称BA1301)、IL4RQ29-BA1301(本发明中简称BA1301)送Invitrogen生物技术有限公司测序。各克隆氨基酸序列如下表1：
表1 具有阻断活性克隆的氨基酸序列
Figure PCTCN2020097686-appb-000002
Figure PCTCN2020097686-appb-000003
通过常规的分子生物学技术进行可变区基因扩增(2*EasyPfu PCR SuperMix厂家：Transgen货号：AS211批号：#L11228)、信号肽与可变区重叠延伸，将带有重链和信号肽基因的可变区同源重组(ClonExpress Ⅱ One Step Cloning Kit厂家：Vazyme货号：C112-01批号：TE222B8)连接入带有抗体重链恒定区(IgG4)序列的载体pCDNA3.4(Life Technology)，将带有轻链和信号肽基因的可变区同源重组(ClonExpress Ⅱ One Step Cloning Kit厂家：Vazyme 货号：C112-01批号：TE222B8)连接入带有抗体轻链恒定区列的载体pCDNA3.4(Life Technology,序列如下所示。
重链恒定区(IgG4)序列：
Figure PCTCN2020097686-appb-000004
轻链恒定区序列：
Figure PCTCN2020097686-appb-000005
然后共转染进入HEK293细胞，在37℃\8％CO 2\125rpm摇床中培养，6-7天后的瞬时表达上清通过Protein A亲和层析，纯化获得IL4R抗体，并通过UV280结合消光系数确定抗体浓度。
对照抗体生产：通过IMGT数据及专利US2008160035A1确定再生元IL4R抗体Dupilumab的氨基酸序列，全基因合成后插入载体pCDNA3.4通过HEK293细胞表达。Dupilumab氨基酸序列如下所示：
Dupilumab轻链氨基酸序列：
Figure PCTCN2020097686-appb-000006
Dupilumab重链氨基酸序列：
Figure PCTCN2020097686-appb-000007
Figure PCTCN2020097686-appb-000008
实施例3 BA030、BA167、BA173与Dupilumab的比较
3.1筛选抗体与IL4R蛋白的阻断活性的比较
包被浓度为0.2μg/ml的IL4(11846-HANE，义翘神州)，100ul/孔4度过夜；用3％脱脂奶粉封闭1h；同时IL4R-Fc-biotin(0.4μg/ml)50ul和不同浓度(16μg/mL、4μg/mL、1μg/mL、0.25μg/mL、0.0625μg/mL、0.015625μg/mL、0.00390625μg/mL)候选抗体50ul共100ul，加入到封闭过的ELISA板上37℃共孵育1h；PBST洗三次后，加入链霉素/HRP(R&D，目录号：890803)，37℃孵育1h；每孔加入100ul TMB显色液(北京梅科万德，目录号：1001)显色10min后，加入50ul 2M的浓硫酸终止，酶标仪上读取OD450。结果见图2及表2所示。
表2 候选抗体阻断IL4/IL4R的蛋白结合活性
Figure PCTCN2020097686-appb-000009
综合分析抗体序列、阻断数据等，我们选择IL4R-BA167-IgG4(以下或简称为BA167)、IL4R-BA173-IgG4(以下或简称为BA173)、IL4R-BA030-IgG4(以下或简称为BA030)与Dupilumab抗体进行比较实验。
3.2 BA167、BA173、BA030、Dupilumab与IL4R蛋白的结合活性的比较
以CBS包被液(pH9.6碳酸溶液)包被不同浓度(0.4μg/ml、0.2μg/ml、0.1μg/ml、0.05μg/ml、0.025μg/ml、0.0125μg/ml、0.00625μg/ml、0μg/ml)的蛋白IL4R(10402-H08H，义翘神州)，100ul/孔4度过夜；用3％脱脂奶粉37℃封闭1h；每孔加入2μg/ml候选抗体各100ul，37度孵育1h；然后加入山羊抗人IgG(H+L)/HRP(KPL，目录号：474-1006)，37℃孵育1h，显色10min后，每孔加入50μL 2M H 2SO 4终止显色酶标仪上读取OD450。结果见图3及表3所示，3株抗体与对照抗体相比，具有对IL4R蛋白接近的结合灵敏度。
表3.对IL4R蛋白的结合灵敏度
Figure PCTCN2020097686-appb-000010
3.3候选抗体阻断IL4/IL4R的蛋白结合
包被浓度为0.2μg/ml的IL4(11846-HANE，义翘神州)，100ul/孔4度过夜；用3％脱脂奶粉封闭1h；同时IL4R-Fc-biotin(0.4μg/ml)和不同浓度(30μg/mL、7.5μg/mL、1.875μg/mL、0.46875μg/mL、0.1171875μg/mL、0μg/mL)候选抗体37℃共孵育1h，然后加入到封闭过的ELISA板上，37℃再孵育1h；继而加入链霉素/HRP(R&D，目录号：890803)，37℃孵育1h；显色10min后，每孔加入50μL 2M H 2SO 4终止。酶标仪上读取OD450。结果见图4及表 4，与对照相比，3株抗体都能够有效阻断IL4与IL4R的结合，且具有接近的阻断活性。
表4.
Figure PCTCN2020097686-appb-000011
3.4候选抗体阻断IL13+IL13RA1/IL4R的蛋白结合
包被浓度为0.8μg/ml的IL13RA1(10943-H08H，义翘神州)，100ul/孔4度过夜；用3％脱脂奶粉封闭1h；同时IL4R-Fc-biotin(0.24μg/ml)、IL13(1μg/ml，10369-HANE，义翘神州)和不同浓度(30μg/mL、7.5μg/mL、1.875μg/mL、0.46875μg/mL、0.1171875μg/mL、0μg/mL)候选抗体37℃共孵育1h，然后加入到封闭过的ELISA板上，37℃再孵育1h；PBST清洗三次后，加入链霉素/HRP(R&D，目录号：890803)，37℃孵育1h；PBST清洗四次后，TMB显色10min，每孔加入50μL 2M H 2SO 4终止，酶标仪上读取OD450。结果见图5及表5，与对照抗体相比，3株抗体都能够有效阻断IL13+IL13RA1与IL4R的结合，且具有相似的阻断活性。
表5.阻断IL13+IL13RA1/IL4R的蛋白结合活性
Figure PCTCN2020097686-appb-000012
3.5 BA167、BA173、BA030和Dupilumab在不同种属上的结合
通过Elisa法检测BA167、BA173、BA030和Dupilumab分别与人、小鼠、食蟹猴IL4R的结合。
以CBS包被液(pH9.6碳酸溶液)包被不同浓度(5μg/ml、1.25μg/ml、0.3125μg/ml、0.078125μg/ml)的huamn IL4R(10402-H08H，义翘神州)、Rhesus IL4R(ILR-C52H8,ACRO)、Mouse IL4R(ILR-M52H1,ACRO)100ul/孔4度过夜；用3％脱脂奶粉37℃封闭1h；每孔加入5μg/ml候选抗体各100ul，37度孵育1h；然后加入山羊抗人IgG/HRP(KPL，目录号：474-1006)，37℃孵育1h。PBST清洗四次后，TMB显色10min，每孔加入50μL 2M H 2SO 4终止，酶标仪上读取OD450。
结果显示4组抗体与人IL-4R蛋白具有相似的结合，而在食蟹猴IL4R蛋白上具有不一样的结合表现，见图6，该结果抗体预示BA167、BA173、BA030抗体与Dupilumab在IL4R上具有不同的表位。
3.6 BA030、BA167、BA173与Dupilumab抗体在体外细胞功能上的比较
3.6.1 IL4诱导的TF-1细胞增殖抑制实验
TF-1细胞、IL4(Sinobiological，目录号：11846-HNAE)和抗体均用完全培养基(90％RPMI1640,10％FBS)进行稀释，把TF-1细胞稀释至4x10 5cells/mL，接种于白色96孔板中，50μL/孔，即20000cells/孔。把IL4稀释至2ng/mL。把抗体稀释至5μg/mL，然后依次4倍稀释，共6个浓度。把稀释的抗体和IL4等体积混合后加入到细胞孔中，50μL/孔。把1ng/mL的IL4加入细胞孔作为阴性对照。把完全培养基加入细胞孔作为阳性对照。所有样品设置复孔。培养96h后，使用CellTiter-Glo(Promega，目录号：G7572)试剂盒检测细胞量。实验结果见图7和表6所示。
3.6.2 IL13诱导的TF-1细胞增殖抑制实验
TF细胞、IL13(Sinobiological，目录号：10369-HNAC)和抗体均用完全培养基(90％RPMI1640,10％FBS)进行稀释，把TF-1细胞稀释至4x10 5cells/mL，接种于白色96孔板中，50μL/孔，即20000cells/孔。把IL13稀释至4ng/mL。把抗体稀释至7.5μg/mL，然后依次8倍稀释，共6个浓度。把稀释的抗体和IL13等体积混合后加入到细胞孔中，50μL/孔。把2ng/mL的IL13加入细胞孔作为阴性对照。把完全培养基加入细胞孔作为阳性对照。所有样品设置复孔。培养96h后，使用CellTiter-Glo(Promega，目录号：G7572)试剂盒检测细胞量。实验结果见图8和表6所示。
3.6.3对PBMC细胞的增殖抑制作用
IL4可以促进PBMC细胞的增殖，加入抗体后，抗体与IL4的受体结合，抑制了PBMC细胞的增殖。细胞、IL4(Sinobiological，目录号：11846-HNAE)和抗体均用完全培养基(90％RPMI 1640,10％FBS)进行稀释。复苏PBMC，按1:100比例加入PHA(Thermo，目录号：10576015)，培养4天。把抗体稀释至160μg/mL，在依次10倍稀释，共9个浓度。把IL4稀释至400ng/mL。把抗体和IL4等体积混合后，加入96孔板中，50μL/孔。加入PHA处理的PBMC，50μL/孔。每孔加入10μL CCK8(Dojindo，目录号：CK08)，显色4h后读取OD450nm。实验结果见图9和表6所示。
表6：细胞活性IC50数据统计
Figure PCTCN2020097686-appb-000013
TF-1是人血液白血病细胞，IL4、IL13可以通过结合细胞表面受体从而诱导TF-1细胞的生长，加入抗体后，抗体与IL4/IL13的受体结合，从而抑制了TF-1细胞的增殖。从上述3.6.1-3.6.2实验可以发现，在TF-1的细胞增殖抑制实验中，BA173抗体的抑制IL-4诱导的TF-1细胞增殖效果优于其他候选抗体，且BA173具有与Dupilumab相似的阻断活性。在3.6.3实验中，BA173和BA167比Dupilumab的作用效果更好，说明两个抗体能更好的与IL4的受体结合，从而抑制PBMC的增殖。
实施例4 BA167、BA173与Dupilumab在食蟹猴PK上的研究
每个抗体选3只食蟹猴皮下注射给药，剂量为2.5mg/kg，两次给药，第二次给药时间为14d(d表示天)，给药前0h、给药后1h、4h、10h及1d、2d、3d、4d、5d、7d、10d、14d及第二次给药后1h、4h、10h及1d、2d、3d、4d、5d、7d、10d、14d、21d和28d采血清检测抗体浓度，检测血清方法为Elisa法，检测具体结果见下图10及表7
表7 食蟹猴皮下注射2.5mg/kg处方BA167、BA173、Dupilumab后体内的药代参数
Figure PCTCN2020097686-appb-000014
食蟹猴皮下第二次给药Dupilumab、BA173、BA167后，出现不同程度的蓄积，其Cmax2/Cmax1值接近2，AUC2st/AUC1st值≥2。以第二次给药末端消除相求算T1/2值分别为4.2±3.0d、15.7±1.9d、22.2±8.1d。以上数据可以发现，给药后BA167、BA173在食蟹猴血清中更加持久，相比Dupilumab每周给药，可显著延长给药周期。
实施例5 BA167、BA173与Dupilumab基于B-hIL-4/hIL-4RA双人源化小鼠哮喘模型的药物药效实验
配40μg OVA(卵清蛋白)，腹腔注射致敏，200μL/只。致敏时间为第0、7、14天。在第21-25天，雾化吸入2％OVA激发，每次30min，连续5天。受试品在第20、23天给药，最后一次激发操作结束24小时后采集样本检测分析。末次雾化激发操作结束24小时后，采小鼠外周血，血清进行OVA特异性的IgE抗体检测；采集肺泡灌洗液进行OVA特异性IgE抗体检测。末次雾化激发操作结束24小时后，采集小鼠肺泡灌洗液通过流式细胞术进行嗜酸细胞浸润检测。实验结果如图11和图12所示，其中G1:1X Buffer；G2:Dupilumab；G3:BA173；G4:BA167。
在实验终点，血清中G1对照组平均IgE水平为28.85ng/mL，Dupilumab、BA173组和BA167组IgE水平分别为2.0ng/mL，3.4ng/mL、2.1ng/mL；嗜酸性粒细胞数对照组平均为384.50X 10 2(个/mL)，Dupilumab、BA173组和BA167组嗜酸性粒细胞数分别为31.1X 10 2(个/mL)，12.8X 10 2(个/mL)，14.0X 10 2(个/mL)。在本实验药效模型上，BA167、BA173组在25mg/kg剂量下可显著减少哮喘模型鼠血清中IgE水平以及肺泡灌洗液中嗜酸粒细胞数量。有效缓解哮喘，有助于减少哮喘加重发生率，改善哮喘相关评价参数，改善患者对吸入皮质类固醇和/或长效β激动剂的依赖性。
同时，在特应性皮炎、瘙痒症、嗜中性白血球减少症、过敏反应、鼻息肉、嗜酸性食管炎、皮肤感染、慢性鼻窦炎动物模型中，分别对各组动物给药，结果显示，候选抗体BA173和BA167在以上动物模型中均显示出与哮喘模型中相似的效果，展现出对特应性皮炎、瘙痒症、嗜中性白血球减少症、过敏反应、鼻息肉、嗜酸性食管炎、皮肤感染、慢性鼻窦炎良好的缓解或治疗效果。
实施例6 BA167、BA173与Dupilumab在食蟹猴上的免疫原性
以CBS包被液(pH9.6碳酸溶液)包被BA167、BA173与Dupilumab为0.125μg/ml，100ul/孔4度过夜；用3％脱脂奶粉37℃封闭1h；每孔加入PBST稀释的100X血清100ul，37度孵育1h；PBST洗两次后加入BA167-biotin、BA173-biotin与Dupilumab-biotin 0.125μg/ml，每孔100ul，37℃孵育1h。PBST洗两次后，加入链霉素/HRP，37℃孵育1h；PBST洗四次后TMB显色10min，每孔加入50μL 2M H 2SO4终止，酶标仪上读取OD450。
结果如图13所示(101-303代表猴编号)，Dupilumab实验组有一只猴子在第21天产生了较强的免疫原性，在第28天时OD值接近2。BA173和BA167的OD值均未超过0.3，在食蟹猴上没有产生免疫原性。说明我们的抗体未来在疗效和安全性上会更高，给药后体内副反应少，具有更低肝脏毒性。

Claims (10)

  1. 一种抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包括：
    1)3个轻链互补决定区，其中，LCDR1氨基酸序列如SEQ ID NO:11所示，LCDR2氨基酸序列如SEQ ID NO:12所示，LCDR3氨基酸序列如SEQ ID NO:13所示；和
    3个重链互补决定区，其中，HCDR1氨基酸序列如SEQ ID NO:14所示，HCDR2氨基酸序列如SEQ ID NO:15所示，HCDR3氨基酸序列如SEQ ID NO:16。或
    2)3个轻链互补决定区，其中，LCDR1氨基酸序列如SEQ ID NO:3所示，LCDR2氨基酸序列如SEQ ID NO:4所示，LCDR3氨基酸序列如SEQ ID NO:5所示；和
    3个重链互补决定区，其中，HCDR1氨基酸序列如SEQ ID NO:6所示，HCDR2氨基酸序列如SEQ ID NO:7所示，HCDR3氨基酸序列如SEQ ID NO:8
  2. 根据权利要求1所述的抗体或其抗原结合片段，其特征在于包括氨基酸序列为SEQ ID NO:1的轻链可变区，和氨基酸序列为SEQ ID NO:2的重链可变区；或者包括氨基酸序列为SEQ ID NO:9的轻链可变区，和氨基酸序列为SEQ ID NO:10的重链可变区
  3. 根据权利要求1至2任一所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段结合抗原是IL-4R。
  4. 一种核酸，其编码权利要求1-4任一项所述抗体或其抗原结合片段。
  5. 一种载体，其包含权利要求4的核酸；优选地，所述载体是表达载体。
  6. 一种细胞，其包含权利要求4的核酸或权利要求5的载体。
  7. 一种药物组合物，其特征在于含有权利要求1-3任一项所述抗体或其抗原结合片段，或权利要求4所述的核酸，或权利要求5所述的载体，或权利要求6所述的细胞，及药学可接受的载体。
  8. 一种试剂盒，含有权利要求1-3任一项所述抗体或其抗原结合片段，或权利要求5所述的核酸。
  9. 权利要求1-3任一项所述抗体或其抗原结合片段，或权利要求5所述的核酸用于预防、治疗、检测或诊断与IL-4R相关的疾病的应用，优选地，所述IL-4R相关疾病是炎症或过敏性疾病。
  10. 根据权利要求9所述应用，所述炎症或过敏性疾病包括哮喘、特应性皮炎、瘙痒症、嗜中性白血球减少症、过敏反应、鼻息肉、嗜酸性食管炎、皮肤感染、慢性鼻窦炎；优选地，所述炎症或过敏性疾病是哮喘。
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