Classifications

• • 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
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39591—Stabilisation, fragmentation
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/02—Inorganic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
• • 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
  • A61P11/02—Nasal agents, e.g. decongestants
• • A—HUMAN NECESSITIES
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  • 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
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • 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
• • 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
  • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
• • 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
• • 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
• • 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
  • C07K2317/30—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
  • C07K2317/56—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
  • C07K2317/76—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
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

• the present disclosure belongs to the field of formulations, in particular, the present disclosure relates to stable pharmaceutical compositions comprising anti-IL4R antibodies or antigen-binding fragments thereof, and the pharmaceutical compositions are used for treating and/or preventing excessive IL4 and/or IL13 signaling related disease use.
• Type 2 inflammation-related allergic diseases such as atopic dermatitis, anaphylaxis, allergic rhinitis, and allergic asthma, afflict more than 3 billion people worldwide, and the incidence continues to rise. According to the hygiene hypothesis, the high incidence is partly due to increased living standards and reduced exposure to infectious agents, making the immune system more sensitive to some otherwise harmless allergens (Stephen J. Galli et al., (2008 ) Nature 454(7203):445-454).
• Interleukin 4 (IL4 or IL-4) and IL-13 are two key factors in type 2 immunity that are required to drive most of the key markers associated with type 2 inflammation, for example, immunoglobulin E production and innate Recruitment of cells at sites of inflammation (Gruning G et al., (1998) Science 282:2261-2263; Rankin JA et al., (1996) Proc Natl Acad Sci USA 93:7821-7825; Wills-Karp M et al. , (1998) Science 282:2258-2261).
• IL-4 and IL-13 regulate cell function and activate transcriptional machinery by binding to cell surface receptors, specifically, IL-4 first binds to the IL-4R ⁇ chain with picomolar affinity and then recruits IL-2R ⁇ ( ⁇ c) Form a type I receptor complex, or recruit IL-13R ⁇ 1 to form a type II receptor complex.
• IL-2R ⁇ IL-2R ⁇
• IL-13R ⁇ 1 IL-13R ⁇ 1
• the type II receptor complex can also be formed through the binding of IL-13 to the IL-13R ⁇ 1 chain (binding with nanomolar affinity) and the further recruitment of the IL-4R ⁇ chain.
• IL-13 can also bind IL-13R ⁇ 2 with picomolar affinity to initiate the formation of decoy receptors (Irina G. Luzina et al., (2012) J Leukoc Biol 92(4):753-764). Once the IL-4 receptor complex is formed, intracellular signaling molecules are activated, with STAT6 and IRS signaling activated in response to type I IL-4 receptors, whereas type II IL-4 receptors are not significantly activated IRS (Heller NM et al., (2008) Sci Signal 1(51):ra17-ra17).
• STAT6 signaling is very important for T H 2 cell differentiation and IL-4 production, while IRS can activate PI3K and mTOR signaling pathways (Gadani SP et al., (2012) J Immunol 189:4213-4219). Studies have shown that excessive IL-4 and/or IL-13 signaling may cause allergic diseases. Another study found that STAT6 inhibitors can inhibit the growth of prostate cancer cells (Nappo G et al., (2017) Oncogenesis 6(5):e342). Therefore, some therapeutic antibodies that modulate IL-4 and/or IL-13-mediated signaling have been developed, for example, the antibody Dupilumab against IL4R.
• Antibody drugs for human subjects should maintain biological activity and stability during storage and subsequent use, but antibody drugs have large molecular weights, complex structures, and are prone to degradation, aggregation, or undesired chemical modifications. become unstable. Therefore, there is a need in the art to develop a stable antibody pharmaceutical composition (such as an anti-IL4R antibody pharmaceutical composition) to overcome the above problems, so that the antibody pharmaceutical has a longer shelf life and is suitable for production and administration to patients.
• a stable antibody pharmaceutical composition such as an anti-IL4R antibody pharmaceutical composition
• the present disclosure provides pharmaceutical compositions comprising high concentrations of anti-IL4R ⁇ antibodies or antigen-binding fragments thereof and having an acceptable level of viscosity, in addition, the pharmaceutical compositions of the present disclosure do not result in high levels of aggregation.
• the pharmaceutical composition provided by the present disclosure comprises: (a) an anti-IL4R ⁇ antibody or an antigen-binding fragment thereof, (b) a buffer, (c) a surfactant, and (d) a stabilizer.
• the concentration of the anti-IL4R ⁇ antibody or antigen-binding fragment thereof in the aforementioned pharmaceutical composition is 30 mg/mL to 300 mg/mL, 50 mg/mL to 250 mg/mL, 70 mg/mL to 200 mg/mL, 100 mg/mL mL to 180 mg/mL, 120 mg/mL to 180 mg/mL, 120 mg/mL to 150 mg/mL, or 150 mg/mL to 180 mg/mL. In some embodiments, the concentration of the anti-IL4R ⁇ antibody or antigen-binding fragment thereof in the aforementioned pharmaceutical composition is about 150 mg/mL.
• the concentration of the anti-IL4R ⁇ antibody or antigen-binding fragment thereof in the aforementioned pharmaceutical composition is about 175 mg/mL.
• examples of the concentration of the anti-IL4R ⁇ antibody or antigen-binding fragment thereof in the aforementioned pharmaceutical composition include but are not limited to: about 70 mg/mL, about 80 mg/mL, about 90 mg/mL, about 100 mg/mL, about 110mg/mL, about 120mg/mL, about 125mg/mL, about 130mg/mL, about 135mg/mL, about 140mg/mL, about 145mg/mL, about 150mg/mL, about 155mg/mL, about 160mg/mL, About 165 mg/mL, about 170 mg/mL, about 175 mg/mL, about 180 mg/mL, about 190 mg/mL, or about 200 mg/mL.
• examples of the buffer in the aforementioned pharmaceutical composition include phosphate buffer, acetate buffer or histidine buffer.
• examples of the buffer in the aforementioned pharmaceutical composition include a histidine salt buffer.
• examples of the phosphate buffer include sodium phosphate buffer or potassium phosphate buffer
• examples of the acetate buffer include sodium acetate buffer, potassium acetate buffer, or sodium acetate-acetic acid Buffering agent
• examples of the histidine salt buffer include histidine-hydrochloric acid buffer, histidine-acetic acid buffer, histidine-histidine hydrochloric acid buffer or histidine hydrochloric acid buffer.
• the sodium phosphate buffer comprises disodium hydrogen phosphate and sodium dihydrogen phosphate.
• the sodium acetate-acetic acid buffer comprises sodium acetate and acetic acid.
• the histidine-histidine HCl buffer comprises histidine and histidine HCl.
• the concentration of the buffer in the aforementioned pharmaceutical composition is 1 mM to 100 mM, 2 mM to 80 mM, 4 mM to 60 mM, 8 mM to 40 mM, 10 mM to 30 mM, 10 mM to 20 mM, or 20 mM to 30 mM. In some embodiments, the concentration of the buffering agent in the aforementioned pharmaceutical composition is about 20 mM.
• examples of the concentration of the buffering agent in the aforementioned pharmaceutical composition include, but are not limited to: about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM , about 17mM, about 18mM, about 19mM, about 20mM, about 21mM, about 22mM, about 23mM, about 24mM, about 25mM, about 26mM, about 27mM, about 28mM, about 30mM, about 32mM, about 34mM, about 36mM, about 38mM, or about 40mM.
• the pH of the buffer in the aforementioned pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3. In some embodiments, the pH of the buffer in the aforementioned pharmaceutical composition is about 5.8. In some embodiments, the pH of the buffer in the aforementioned pharmaceutical composition is about 6. In some embodiments, the pH of the buffer in the aforementioned pharmaceutical composition is about 5.
• examples of the pH of the buffer in the aforementioned pharmaceutical composition include, but are not limited to: about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8 , about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.
• the surfactant in the aforementioned pharmaceutical composition is selected from polysorbates (such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate ester 81 or polysorbate 85), poloxamer (such as poloxamer 181, poloxamer 188 or poloxamer 407), polyethylene glycol or polyhydroxy, etc.
• the surfactant in the aforementioned pharmaceutical composition is polysorbate 80.
• the surfactant in the aforementioned pharmaceutical composition is polysorbate 20.
• the concentration of the surfactant in the aforementioned pharmaceutical composition is 0.01 mg/mL to 2 mg/mL, 0.05 mg/mL to 1 mg/mL, 0.1 mg/mL to 0.8 mg/mL, 0.2 mg/mL mL to 0.6mg/mL, or 0.2mg/mL to 0.4mg/mL. In some embodiments, the concentration of the surfactant in the aforementioned pharmaceutical composition is about 0.4 mg/mL.
• examples of the concentration of the surfactant in the aforementioned pharmaceutical composition include, but are not limited to: about 0.05 mg/mL, about 0.06 mg/mL, about 0.07 mg/mL, about 0.08 mg/mL, about 0.09mg/mL, about 0.1mg/mL, about 0.2mg/mL, about 0.3mg/mL, about 0.4mg/mL, about 0.5mg/mL, about 0.6mg/mL, about 0.7mg/mL, about 0.8mg /mL, about 0.9mg/mL, or about 1mg/mL.
• the stabilizing agent in the aforementioned pharmaceutical composition is selected from one or more of trehalose, mannitol, sucrose, arginine or a pharmaceutically acceptable salt thereof, proline or sodium chloride. kind.
• the stabilizer in the aforementioned pharmaceutical composition comprises sucrose.
• the stabilizer in the aforementioned pharmaceutical composition comprises arginine or a pharmaceutically acceptable salt thereof.
• the stabilizer in the aforementioned pharmaceutical composition comprises proline.
• the stabilizer in the aforementioned pharmaceutical composition comprises sodium chloride.
• the pharmaceutically acceptable salt of arginine include arginine hydrochloride or arginine acetate.
• the stabilizer in the aforementioned pharmaceutical composition comprises sucrose and arginine hydrochloride. In some specific embodiments, the stabilizer in the aforementioned pharmaceutical composition comprises proline and sodium chloride. In some specific embodiments, the stabilizer in the aforementioned pharmaceutical composition comprises sucrose, proline and sodium chloride.
• the concentration of sucrose in the aforementioned pharmaceutical composition is 10 mg/mL to 100 mg/mL, 20 mg/mL to 80 mg/mL, 30 mg/mL to 60 mg/mL, 40 mg/mL to 50 mg/mL, or 50mg/mL to 60mg/mL. In some embodiments, the concentration of sucrose in the aforementioned pharmaceutical composition is about 50 mg/mL.
• examples of the concentration of sucrose in the aforementioned pharmaceutical composition include, but are not limited to: about 20 mg/mL, about 22 mg/mL, about 24 mg/mL, about 26 mg/mL, about 28 mg/mL, about 30 mg /mL, about 32mg/mL, about 34mg/mL, about 36mg/mL, about 38mg/mL, about 40mg/mL, about 42mg/mL, about 44mg/mL, about 46mg/mL, about 48mg/mL, about 50mg /mL, about 52mg/mL, about 54mg/mL, about 56mg/mL, about 58mg/mL, about 60mg/mL, about 62mg/mL, about 64mg/mL, about 66mg/mL, about 68mg/mL, about 70mg /mL, about 72mg/mL, about 74mg/mL, about
• the concentration of arginine or a pharmaceutically acceptable salt thereof (for example, arginine hydrochloride) in the aforementioned pharmaceutical composition is 10 mM to 100 mM, 20 mM to 80 mM, 30 mM to 60 mM, 40 mM to 50 mM , or 50mM to 60mM. In some embodiments, the concentration of arginine or a pharmaceutically acceptable salt thereof (eg, arginine hydrochloride) in the aforementioned pharmaceutical composition is about 50 mM.
• examples of the concentration of arginine or a pharmaceutically acceptable salt thereof (for example, arginine hydrochloride) in the aforementioned pharmaceutical composition include but are not limited to: about 20 mM, about 22 mM, about 24 mM, About 26mM, about 28mM, about 30mM, about 32mM, about 34mM, about 36mM, about 38mM, about 40mM, about 42mM, about 44mM, about 46mM, about 48mM, about 50mM, about 52mM, about 54mM, about 56mM, about 58mM , about 60 mM, about 62 mM, about 64 mM, about 66 mM, about 68 mM, about 70 mM, about 72 mM, about 74 mM, about 76 mM, about 78 mM, or about 80 mM.
• the concentration of proline in the aforementioned pharmaceutical composition is 60mM to 600mM, 80mM to 500mM, 100mM to 450mM, 120mM to 400mM, 150mM to 350mM, 200mM to 350mM, or 250mM to 350mM. In some embodiments, the concentration of proline in the aforementioned pharmaceutical composition is about 250 mM. In some embodiments, the concentration of proline in the aforementioned pharmaceutical composition is about 350 mM.
• examples of the concentration of proline in the aforementioned pharmaceutical composition include but are not limited to: about 120mM, about 130mM, about 140mM, about 150mM, about 160mM, about 170mM, about 180mM, about 190mM, about 200mM, about 210mM, about 220mM, about 230mM, about 240mM, about 250mM, about 260mM, about 270mM, about 280mM, about 290mM, about 300mM, about 310mM, about 320mM, about 330mM, about 340mM, about 350mM, about 360mM, About 370 mM, about 380 mM, about 390 mM, or about 400 mM.
• the concentration of sodium chloride in the aforementioned pharmaceutical composition is 1 mM to 80 mM, 2 mM to 60 mM, 4 mM to 50 mM, 8 mM to 40 mM, 10 mM to 30 mM, or 10 mM to 20 mM. In some embodiments, the concentration of sodium chloride in the aforementioned pharmaceutical composition is about 20 mM.
• examples of the concentration of sodium chloride in the foregoing pharmaceutical composition include, but are not limited to: about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16mM, about 17mM, about 18mM, about 19mM, about 20mM, about 21mM, about 22mM, about 23mM, about 24mM, about 25mM, about 26mM, about 27mM, about 28mM, about 29mM, about 30mM, about 32mM, about 34mM, About 36 mM, about 38 mM, or about 40 mM.
• the pH of the aforementioned pharmaceutical composition is a pH of 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.
• the aforementioned pharmaceutical composition has a pH of about 5.8.
• the aforementioned pharmaceutical composition has a pH of about 6.
• the pH of the aforementioned pharmaceutical composition is about 5.
• examples of the pH of the aforementioned pharmaceutical composition include, but are not limited to: about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.
• the aforementioned pharmaceutical composition comprises: (a) anti-IL4R ⁇ antibody or antigen-binding fragment thereof, (b) phosphate buffer, acetate buffer and/or histidine buffer, (c) Polysorbate 80 or Polysorbate 20, and (d) trehalose, mannitol, sucrose, arginine or a pharmaceutically acceptable salt thereof, proline and/or sodium chloride.
• the aforementioned pharmaceutical composition comprises: (a) an anti-IL4R ⁇ antibody or an antigen-binding fragment thereof, (b) a phosphate buffer or a histidine buffer, (c) polysorbate 80, and (d) ) sucrose, arginine or a pharmaceutically acceptable salt thereof, proline and/or sodium chloride.
• the aforementioned pharmaceutical composition comprises: (a) an anti-IL4R ⁇ antibody or an antigen-binding fragment thereof, (b) a histidine salt buffer, (c) polysorbate 80, and (d) sucrose and arginine acid or a pharmaceutically acceptable salt thereof.
• the aforementioned pharmaceutical composition comprises: (a) anti-IL4R ⁇ antibody or antigen-binding fragment thereof, (b) histidine salt buffer, (c) polysorbate 80, and (d) sucrose, proline acid and sodium chloride.
• the aforementioned pharmaceutical composition comprises: (a) an anti-IL4R ⁇ antibody or an antigen-binding fragment thereof, (b) a histidine salt buffer, (c) polysorbate 80, and (d) proline and Sodium chloride.
• the aforementioned pharmaceutical composition comprises: (a) an anti-IL4R ⁇ antibody or an antigen-binding fragment thereof, (b) a histidine salt buffer, (c) polysorbate 80, and (d) proline.
• the aforementioned pharmaceutical composition comprises: (a) 30 mg/mL to 300 mg/mL of anti-IL4R ⁇ antibody or antigen-binding fragment thereof, (b) 1 mM to 100 mM buffer, (c) 0.01 mg/mL to 2 mg/mL of surfactant, and (d) a stabilizer comprising trehalose, mannitol, sucrose, arginine or a pharmaceutically acceptable salt thereof, proline and/or sodium chloride; And the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.
• the aforementioned pharmaceutical composition comprises: (a) 50 mg/mL to 250 mg/mL of anti-IL4R ⁇ antibody or antigen-binding fragment thereof, (b) 2 mM to 80 mM buffer, (c) 0.05 mg/mL to A surfactant of 1 mg/mL, and (d) a stabilizer comprising trehalose, mannitol, sucrose, arginine or a pharmaceutically acceptable salt thereof, proline and/or sodium chloride; And the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.
• the aforementioned pharmaceutical composition comprises: (a) 70 mg/mL to 200 mg/mL of anti-IL4R ⁇ antibody or antigen-binding fragment thereof, (b) 4 mM to 60 mM buffer, (c) 0.1 mg/mL to 0.8 mg/mL of surfactant, and (d) a stabilizer comprising trehalose, mannitol, sucrose, arginine or a pharmaceutically acceptable salt thereof, proline and/or sodium chloride and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.
• the aforementioned pharmaceutical composition comprises: (a) 100 mg/mL to 180 mg/mL or 120 mg/mL to 180 mg/mL of an anti-IL4R ⁇ antibody or an antigen-binding fragment thereof, (b) 8 mM to 40 mM or 10 mM to 30 mM buffer, (c) 0.2mg/mL to 0.6mg/mL surfactant, and (d) stabilizer, said stabilizer comprises trehalose, mannitol, sucrose, arginine or pharmaceutically acceptable salt, proline and/or sodium chloride; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3 .
• the aforementioned pharmaceutical composition comprises: (a) 120 mg/mL to 150 mg/mL or 150 mg/mL to 180 mg/mL of an anti-IL4R ⁇ antibody or an antigen-binding fragment thereof, (b) 10 mM to 20 mM or 20 mM to 30 mM buffering agent, (c) 0.2mg/mL to 0.4mg/mL surfactant, and (d) stabilizing agent comprising trehalose, mannitol, sucrose, arginine or pharmaceutically acceptable salt, proline and/or sodium chloride; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3 .
• the aforementioned pharmaceutical composition comprises: (a) 30 mg/mL to 300 mg/mL of an anti-IL4R ⁇ antibody or an antigen-binding fragment thereof, (b) 1 mM to 100 mM of phosphate buffer, acetate buffer, or Histidine buffer, (c) 0.01 mg/mL to 2 mg/mL polysorbate 80 or polysorbate 20, and (d) 10 mg/mL to 100 mg/mL sucrose, 10 mM to 100 mM arginine or a pharmaceutically acceptable salt thereof, 60mM to 600mM proline, and/or 1mM to 80mM sodium chloride; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.
• the aforementioned pharmaceutical composition comprises: (a) 50 mg/mL to 250 mg/mL of an anti-IL4R ⁇ antibody or an antigen-binding fragment thereof, (b) 2 mM to 80 mM of phosphate buffer, acetate buffer, or Histidine buffer, (c) 0.05 mg/mL to 1 mg/mL polysorbate 80 or polysorbate 20, and (d) 20 mg/mL to 80 mg/mL sucrose, 20 mM to 80 mM arginine or a pharmaceutically acceptable salt thereof, proline from 80mM to 500mM, and/or sodium chloride from 2mM to 60mM; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.
• the aforementioned pharmaceutical composition comprises: (a) 70 mg/mL to 200 mg/mL of an anti-IL4R ⁇ antibody or an antigen-binding fragment thereof, (b) 4 mM to 60 mM of phosphate buffer, acetate buffer, or Histidine buffer, (c) 0.1 mg/mL to 0.8 mg/mL polysorbate 80 or polysorbate 20, and (d) 30 mg/mL to 60 mg/mL sucrose, 30 mM to 60 mM arginine acid or a pharmaceutically acceptable salt thereof, 100mM to 450mM or 120mM to 400mM proline, and/or 4mM to 50mM sodium chloride; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.
• the aforementioned pharmaceutical composition comprises: (a) 100 mg/mL to 180 mg/mL or 120 mg/mL to 180 mg/mL of an anti-IL4R ⁇ antibody or an antigen-binding fragment thereof, (b) 8 mM to 40 mM or 10 mM to 30 mM phosphate buffer, acetate buffer or histidine buffer, (c) 0.2mg/mL to 0.6mg/mL polysorbate 80 or polysorbate 20, and (d) 40mg/mL to 50mg/mL of sucrose, 40mM to 50mM of arginine or a pharmaceutically acceptable salt thereof, 150mM to 350mM of proline, and/or 8mM to 40mM of sodium chloride; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.
• the aforementioned pharmaceutical composition comprises: (a) 120 mg/mL to 150 mg/mL or 150 mg/mL to 180 mg/mL of an anti-IL4R ⁇ antibody or an antigen-binding fragment thereof, (b) 10 mM to 20 mM or 20 mM to 30 mM phosphate buffer, acetate buffer or histidine buffer, (c) 0.2mg/mL to 0.4mg/mL polysorbate 80 or polysorbate 20, and (d) 50mg/mL to 60 mg/mL sucrose, 50 mM to 60 mM arginine or a pharmaceutically acceptable salt thereof, 200 mM to 350 mM or 250 mM to 350 mM proline, and/or 10 mM to 30 mM or 10 mM to 20 mM sodium chloride; and
• the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.
• the aforementioned pharmaceutical composition comprises: (a) 30 mg/mL to 300 mg/mL of anti-IL4R ⁇ antibody or antigen-binding fragment thereof, (b) 1 mM to 100 mM of phosphate buffer or histidine buffer , (c) 0.01mg/mL to 2mg/mL polysorbate 80, and (d) 60mM to 600mM proline and 1mM to 80mM sodium chloride, optionally, also including 10mg/mL to 100mg/ mL of sucrose; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.
• the aforementioned pharmaceutical composition comprises: (a) 50 mg/mL to 250 mg/mL of an anti-IL4R ⁇ antibody or an antigen-binding fragment thereof, (b) 2 mM to 80 mM of phosphate buffer or histidine buffer , (c) 0.05mg/mL to 1mg/mL polysorbate 80, and (d) 80mM to 500mM proline and 2mM to 60mM sodium chloride, optionally, also including 20mg/mL to 80mg/ mL of sucrose; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.
• the aforementioned pharmaceutical composition comprises: (a) 70 mg/mL to 200 mg/mL of an anti-IL4R ⁇ antibody or an antigen-binding fragment thereof, (b) 4 mM to 60 mM of phosphate buffer or histidine buffer , (c) 0.1 mg/mL to 0.8 mg/mL polysorbate 80, and (d) 100 mM to 450 mM or 120 mM to 400 mM proline, and 4 mM to 50 mM sodium chloride, optionally, further comprising 30 mg/mL to 60 mg/mL of sucrose; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.
• the aforementioned pharmaceutical composition comprises: (a) 100 mg/mL to 180 mg/mL or 120 mg/mL to 180 mg/mL of an anti-IL4R ⁇ antibody or an antigen-binding fragment thereof, (b) 8 mM to 40 mM or 10 mM to 30 mM phosphate buffer or histidine buffer, (c) 0.2 mg/mL to 0.6 mg/mL polysorbate 80, and (d) 150 mM to 350 mM proline and 8 mM to 40 mM sodium chloride , optionally, further comprising 40 mg/mL to 50 mg/mL of sucrose; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.
• the aforementioned pharmaceutical composition comprises: (a) 120 mg/mL to 150 mg/mL or 150 mg/mL to 180 mg/mL of an anti-IL4R ⁇ antibody or an antigen-binding fragment thereof, (b) 10 mM to 20 mM or 20 mM to 30 mM phosphate buffer or histidine buffer, (c) 0.2mg/mL to 0.4mg/mL polysorbate 80, and (d) 200mM to 350mM or 250mM to 350mM proline, and 10mM to 30mM or 10mM to 20mM sodium chloride, optionally further comprising 50mg/mL to 60mg/mL sucrose; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5 , 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.
• the aforementioned pharmaceutical composition comprises: (a) 150 mg/mL anti-IL4R ⁇ antibody or antigen-binding fragment thereof, (b) 20 mM histidine-histidine hydrochloride buffer, (c) 0.4 mg/mL mL of polysorbate 80, and (d) 50 mg/mL sucrose and 50 mM arginine hydrochloride; and the pH of the pharmaceutical composition is 5.8.
• the aforementioned pharmaceutical composition comprises: (a) 150 mg/mL anti-IL4R ⁇ antibody or antigen-binding fragment thereof, (b) 20 mM histidine-histidine hydrochloride buffer, (c) 0.4 mg/mL polysorbate 80 in mL, and (d) 250 mM proline and 20 mM sodium chloride; and the pH of the pharmaceutical composition is 5.8.
• the aforementioned pharmaceutical composition comprises: (a) 150 mg/mL anti-IL4R ⁇ antibody or antigen-binding fragment thereof, (b) 20 mM histidine-histidine hydrochloride buffer, (c) 0.4 mg/mL mL of polysorbate 80, and (d) 350 mM of proline; and the pH of the pharmaceutical composition is 5.8.
• the aforementioned pharmaceutical composition comprises: (a) 150 mg/mL anti-IL4R ⁇ antibody or antigen-binding fragment thereof, (b) 20 mM histidine-histidine hydrochloride buffer, (c) 0.4 mg/mL mL of polysorbate 80, and (d) 50 mg/mL of sucrose, 250 mM of proline, and 20 mM of sodium chloride; and the pH of the pharmaceutical composition is 5.8.
• the anti-IL4R ⁇ antibody or antigen-binding fragment thereof described in the aforementioned pharmaceutical composition comprises a heavy chain variable region
• the heavy chain variable region comprises a heavy chain variable region CDR1, a heavy chain variable region, Region CDR2 and heavy chain variable region CDR3, wherein heavy chain variable region CDR1, heavy chain variable region CDR2 and heavy chain variable region CDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 1, 2 and 3 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence.
• the anti-IL4R ⁇ antibody or antigen-binding fragment thereof described in the aforementioned pharmaceutical composition comprises a heavy chain variable region comprising the same expression as SEQ ID NOs: 7, 8 or 9.
• the amino acid sequence shown has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or amino acid sequences with 100% identity.
• the amino acid sequence of SEQ ID NO:8 is:
• the anti-IL4R ⁇ antibody or antigen-binding fragment thereof described in the aforementioned pharmaceutical composition comprises a light chain variable region
• the light chain variable region comprises a light chain variable region CDR1, a light chain variable region, Region CDR2 and light chain variable region CDR3, wherein the light chain variable region CDR1, light chain variable region CDR2 and light chain variable region CDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 4, 5 and 6 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence.
• the anti-IL4R ⁇ antibody or antigen-binding fragment thereof described in the aforementioned pharmaceutical composition comprises a light chain variable region comprising the same expression as SEQ ID NOs: 10, 11 or 12.
• the amino acid sequence shown has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or amino acid sequences with 100% identity.
• the amino acid sequence of SEQ ID NO: 11 is:
• the anti-IL4R ⁇ antibody or antigen-binding fragment thereof described in the aforementioned pharmaceutical composition comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region Contains CDR1, CDR2 and CDR3 respectively, of which heavy chain variable region CDR1, heavy chain variable region CDR2, heavy chain variable region CDR3, light chain variable region CDR1, light chain variable region CDR2 and light chain variable region CDR3 Comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 Amino acid sequences that are %, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.
• amino acid sequence of SEQ ID NO:8 is:
• amino acid sequence of SEQ ID NO: 11 is:
• the anti-IL4R ⁇ antibody or antigen-binding fragment thereof described in the aforementioned pharmaceutical composition comprises a heavy chain and a light chain, and the heavy chain comprises a heavy chain variable region and a heavy chain constant region, and the light chain comprises a heavy chain variable region and a heavy chain constant region.
• the chain comprises a light chain variable region and a light chain constant region, wherein the C-terminal of the heavy chain variable region is linked to the N-terminal of the heavy chain constant region, and the C-terminal of the light chain variable region is connected to the N-terminal of the light chain constant region,
• the heavy chain variable region and the light chain variable region comprise the above-mentioned amino acid sequences
• the heavy chain constant region has the human IgG4 constant region of the amino acid sequence shown in SEQ ID NO: 13
• the light chain constant region has the human IgG4 constant region such as SEQ ID NO: 14 Human kappa constant region of amino acid sequence shown.
• the anti-IL4R ⁇ antibody or antigen-binding fragment thereof described in the aforementioned pharmaceutical composition may be a full-length antibody, such as an IgG1, IgG2 or IgG4 isotype full-length antibody, preferably an IgG4 isotype full-length antibody. long antibody.
• the anti-IL4R ⁇ antibody or antigen-binding fragment thereof in the aforementioned pharmaceutical composition may be a single-chain variable region (scFv) antibody, or an antibody fragment, such as a Fab or F(ab') 2 fragment.
• the anti-IL4R ⁇ antibody or antigen-binding fragment thereof described in the aforementioned pharmaceutical composition is the C2C1A1A1 antibody described in the patent document with application number PCT/CN2021077784.
• the present disclosure also provides a method for preparing the aforementioned pharmaceutical composition, which includes the step of contacting the above-mentioned anti-IL4R ⁇ antibody or its antigen-binding fragment with a buffer, for example, displacing the anti-IL4R ⁇ antibody or its antigen-binding fragment into the buffer, so that Said buffering agent comprises phosphate buffering agent, acetate buffering agent or histidine salt buffering agent etc., further includes addition of surfactant and stabilizing agent, without distinguishing the sequence, wherein said stabilizing agent comprises trehalose, nectar alcohol, sucrose, arginine or a pharmaceutically acceptable salt thereof, proline and/or sodium chloride, and the surfactant comprises polysorbate, poloxamer, polyethylene glycol or polyhydroxy.
• a buffer for example, displacing the anti-IL4R ⁇ antibody or its antigen-binding fragment into the buffer
• Said buffering agent comprises phosphate buffering agent, acetate buffering agent or histidine salt buffering agent etc
• the present disclosure also provides a method for preparing a freeze-dried preparation comprising an anti-IL4R ⁇ antibody or an antigen-binding fragment thereof, which includes the step of freeze-drying the aforementioned pharmaceutical composition.
• the freeze-drying is performed according to methods known in the art, including but not limited to the steps of pre-freezing, primary drying and secondary drying. Those skilled in the art understand that any method of removing water from the pharmaceutical compositions of the present disclosure is suitable for use in the present disclosure.
• the present disclosure also provides a lyophilized preparation comprising an anti-IL4R ⁇ antibody or an antigen-binding fragment thereof, which is prepared by the aforementioned method for preparing a lyophilized preparation.
• the present disclosure also provides a lyophilized preparation comprising an anti-IL4R ⁇ antibody or an antigen-binding fragment thereof, and the lyophilized preparation can form the aforementioned pharmaceutical composition after reconstitution.
• the present disclosure also provides a product, which includes a container filled with the foregoing pharmaceutical composition or the foregoing freeze-dried preparation.
• the pharmaceutical composition or lyophilized formulation of the present disclosure can be administered according to known methods, such as injection or infusion over a period of time in a suitable manner, for example, subcutaneously, intravenously, intraperitoneally, intramuscularly, arterially intralesional or intraarticular routes, topically, by inhalation or by sustained or delayed release.
• a pharmaceutical composition or lyophilized formulation of the present disclosure may be diluted with a suitable diluent to a suitable concentration prior to administration to provide the optimum desired response (eg, a therapeutic response).
• the present disclosure provides a method of reducing IL4 and/or IL13 signaling in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition, lyophilized formulation or preparation of the present disclosure.
• IL4 signals through receptors comprising IL-4R ⁇ and ⁇ c
• IL13 signals through receptors comprising IL-4R ⁇ and IL13R ⁇ 1.
• IL4 and/or IL13 signaling is manifested in the activation and/or proliferation of B cells, eosinophils, macrophages, fibroblasts, and smooth muscle cells (eg, airway smooth muscle cell proliferation, etc.).
• the present disclosure provides a method of reducing a Type 2 immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition, lyophilized formulation or preparation of the present disclosure.
• the present disclosure provides a method of treating a disease associated with excessive IL4 and/or IL13 signaling in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition, a lyophilized formulation of the present disclosure or products.
• the diseases associated with excessive IL4 and/or IL13 signaling are allergic diseases, non-limiting examples include atopic dermatitis, anaphylaxis, allergic rhinitis or allergic asthma.
• the present disclosure provides a method of treating a tumor associated with increased STAT6 activation in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition, lyophilized formulation or preparation of the present disclosure.
• the tumor comprises a solid tumor or a non-solid tumor.
• examples of the tumor include, but are not limited to, melanoma, lung cancer, kidney cancer, prostate cancer, cervical cancer, colorectal cancer, gastric cancer, pancreatic cancer, ovarian cancer, and urothelial cancer.
• Buffering agent refers to a pharmaceutically acceptable substance or mixture of substances capable of maintaining the pH of a pharmaceutical composition to a desired pH range.
• buffers suitable for use in the pharmaceutical compositions of the present disclosure include phosphate buffer, acetate buffer or histidine buffer.
• a “phosphate buffer” is a buffer comprising phosphate ions, eg, a buffer comprising phosphoric acid and/or phosphate salts, wherein phosphoric acid includes phosphoric acid and/or hydrates thereof, and phosphate includes phosphate salts and/or hydrates thereof.
• phosphate buffer include but are not limited to sodium phosphate buffer or potassium phosphate buffer, the preferred phosphate buffer is sodium phosphate buffer.
• the sodium phosphate buffer includes sodium dihydrogen phosphate monohydrate (NaH 2 PO 4 ⁇ H 2 O) and disodium hydrogen phosphate dodecahydrate (Na 2 HPO 4 ⁇ 12H 2 O).
• Acetate buffer is a buffer comprising acetate ions, for example comprising acetic acid and/or acetate salts, wherein acetic acid comprises acetic acid and/or hydrates thereof, and acetate comprises acetate and/or its hydrate.
• acetate buffers include but are not limited to potassium acetate buffer, ammonium acetate buffer or sodium acetate buffer, sodium acetate-acetate buffer, the preferred acetate buffer is sodium acetate-acetate buffer.
• the sodium acetate-acetic acid buffer comprises acetic acid and sodium acetate, wherein the acetic acid comprises acetic acid and/or its hydrate, the sodium acetate comprises sodium acetate and/or its hydrate, in one specific example, the sodium acetate-acetic acid buffer comprises acetic acid And sodium acetate trihydrate (CH 3 COONa ⁇ 3H 2 O).
• a "histidine salt buffer” is a buffer comprising histidine ions, for example comprising histidine and/or a histidine salt, wherein histidine comprises histidine and/or a hydrate thereof, histidine Amino acid salts include histidine salts and/or hydrates thereof.
• histidine salt buffers include histidine-hydrochloric acid buffers, histidine-acetic acid buffers, histidine-histidine hydrochloride or histidine hydrochloride buffers, and preferred histidine salt buffers are Histidine-Histidine HCl buffer.
• Histidine-histidine hydrochloride includes histidine and histidine hydrochloride, wherein histidine includes histidine and/or its hydrate, histidine hydrochloride includes histidine hydrochloride and/or its hydrate, in
• the histidine-histidine hydrochloride buffer comprises histidine and histidine hydrochloride monohydrate (C 6 H 9 N 3 O 2 ⁇ HCl ⁇ H 2 O).
• Stabilizer refers to a pharmaceutically acceptable substance or mixture of substances used to maintain the stability of the active ingredient in the pharmaceutical composition.
• a stabilizer may also serve as a viscosity reducer and/or an isotonic agent, etc.
• “Pharmaceutical composition” is intended to encompass a product comprising a specified active ingredient, such as an antibody, optionally in a specified amount, as well as any product produced directly or indirectly by combining the specified active ingredient, optionally in a specified amount.
• the purpose of a pharmaceutical composition is to make the active ingredient suitable for manufacture and administration to a patient, and to maintain biological activity and/or stability during storage and subsequent use.
• the pharmaceutical composition is a water-soluble injection, and the water-soluble injection includes but is not limited to a water-soluble preparation that has not been lyophilized or a water-soluble preparation reconstituted from a lyophilized powder.
• the pharmaceutical composition is a lyophilized formulation.
• “pharmaceutical composition” and “formulation” are not mutually exclusive.
• a “stable” or “stabilized” pharmaceutical composition refers to a pharmaceutical composition in which the active ingredient (eg, antibody) substantially retains its physical and/or chemical stability and/or biological activity when preserved therein.
• active ingredient eg, antibody
• Various analytical techniques for measuring the stability of active ingredients are known in the art, for example in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) Reviewed in Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at selected temperatures and other storage conditions for selected periods of time.
• an active ingredient "retains its physical stability" in a pharmaceutical composition if it does not exhibit a significant increase in aggregation, precipitation and/or denaturation.
• an active ingredient when using a pharmaceutical composition of the present disclosure, 10% or less, 5% or less, 4% or less active ingredient, such as by SEC or any other suitable method for measuring aggregate formation Aggregates (also known as macromolecular impurities) are formed.
• An active ingredient eg, an antibody "retains its chemical stability" in a pharmaceutical composition if it does not exhibit a significant chemical change. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the antibody. Processes that often alter protein chemical structure include hydrolysis or truncation (evaluated by methods such as size-exclusion chromatography and SDS-PAGE), oxidation (by methods such as peptide mapping coupled with mass spectrometry or MALDI/TOF/MS), to evaluate), deamidation (evaluated by methods such as ion-exchange chromatography, capillary isoelectrophoresis, peptide mapping, isoaspartic acid measurement, etc.) and isomerization (by measuring isoaspartic acid content, Peptide mapping, etc.
• An active ingredient "retains its biological activity” in a pharmaceutical composition for a given period of time if the biological activity of the active ingredient (such as an antibody) for the given period of time is within a predetermined range of the biological activity exhibited when the pharmaceutical composition was prepared.
• biological activity as determined, for example, by an antigen binding assay.
• Macromolecular impurities or “aggregates” refer to the general term of impurities with a molecular weight larger than that of the target active ingredient (eg, antibody).
• Charge variant refers to a variant that directly or indirectly causes the charge of the antibody molecule to change through glycosylation, deamidation, oxidation and/or isomerization, etc.
• Concentrated electrophoresis (CIEF) and cation exchange chromatography (CEX-HPLC) were used for detection.
• a pharmaceutical composition refers to one pharmaceutical composition or more than one pharmaceutical composition.
• “About” or “approximately” means that a value is within an acceptable error range for the particular value as determined by one of ordinary skill in the art, depending in part on how it is measured or determined (ie, the limits of the measurement system). For example, “about” or “approximately” can mean within 1 or more than 1 standard deviation in the art. Alternatively “about” or “approximately” means a range of plus or minus 15%, 10%, 5% or 1% of the stated value. Furthermore, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to 5 times a value.
• any percentage range, ratio range or integer range should be understood to include the value of any integer within the recited range and, where appropriate, fractions thereof (such as tenths and hundredths of integers). ), unless otherwise noted.
• IL4R ⁇ refers to the interleukin 4 receptor alpha subunit.
• IL4R ⁇ includes variants, isoforms, homologs, orthologs and paralogs.
• an antibody specific for human IL4R ⁇ protein may cross-react with IL4R ⁇ protein of a species other than human (eg, monkey).
• antibodies specific for human IL4R ⁇ protein may be fully specific for human IL4R ⁇ protein and may not cross-react with other species or types of proteins, or may be specific for human IL4R ⁇ protein derived from certain but not all other species. Species cross-react with IL4R ⁇ .
• human IL4R ⁇ refers to an IL4R ⁇ protein having a human amino acid sequence, such as the amino acid sequence of human IL4R ⁇ with Genbank accession number NP_001244335.1.
• cynomolgus IL4R ⁇ and “marmoset IL4R ⁇ ” refer to IL4R ⁇ sequences, which have, for example, the amino acid sequences of Genbank accession numbers EHH60265.1 and NP_001244161.1, respectively.
• antibody herein includes full-length antibodies and any antigen-binding fragments thereof or single chains thereof.
• Full-length antibodies are glycoproteins comprising two heavy (H) chains and two light (L) chains linked by disulfide bonds.
• Each heavy chain is composed of a heavy chain variable region (abbreviated as VH ) and a heavy chain constant region.
• the heavy chain constant region consists of three domains, CH1 , CH2 and CH3 .
• Each light chain is composed of a light chain variable region (abbreviated as V L ) and a light chain constant region.
• the light chain constant region consists of one domain, CL .
• the VH and VL regions can also be divided into hypervariable regions called complementarity determining regions (CDRs), which are separated by more conserved framework region (FR) regions.
• CDRs complementarity determining regions
• FR conserved framework region
• Each VH and VL consists of three CDRs and four FRs, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminal to the carboxyl terminal.
• the variable regions of the heavy and light chains contain the binding domains that interact with the antigen.
• the constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various immune system cells (eg, effector cells) and the first component (Clq) of the classical complement system.
• an "antigen-binding fragment” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (eg, IL4R ⁇ protein). It has been demonstrated that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies.
• binding fragments encompassed by "antigen-binding fragments" of antibodies include: (i) Fab fragments, monovalent fragments composed of VL, VH , CL and CH1 domains; ( ii) F(ab') 2 fragments, including bivalent fragments of two Fab fragments connected by disulfide bonds at the hinge region; (iii) Fd fragment composed of V H and CH1 ; (iv) Fd fragment composed of antibody single arm V L and V H Fv fragments; (v) dAb fragments composed of VH (Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity determining regions (CDRs); and (vii) Nanobodies, namely A heavy chain variable region comprising a single variable domain and two constant domains.
• the two structural domains VL and VH of the Fv fragment are encoded by different genes, they can be connected by a synthetic linker to form a single protein chain through recombination, in which the VL and VH regions are paired to form a monovalent Molecules (termed single-chain Fv (scFv); see for example Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc.Natl.Acad.Sci.USA 85:5879- 5883).
• Such single chain antibodies are also encompassed within the term "antigen-binding fragment" of an antibody.
• These antibody fragments can be obtained by common techniques known to those skilled in the art, and can be functionally screened in the same manner as full-length antibodies.
• isolated antibody refers to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds IL4R ⁇ protein is substantially free of antibodies that specifically bind to antigens other than IL4R ⁇ protein) .
• An isolated antibody that specifically binds human IL4R ⁇ protein may, however, have cross-reactivity to other antigens, such as IL4R ⁇ proteins from other species.
• an isolated antibody is substantially free of other cellular material and/or chemicals.
• mouse antibody refers to an antibody in which both the framework and CDR regions of the variable regions are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region also is derived from mouse germline immunoglobulin sequences.
• the mouse antibodies herein may comprise amino acid residues not encoded by mouse germline immunoglobulin sequences (eg, mutations introduced by random or point mutations in vitro, or by somatic mutation in vivo). However, the term “mouse antibody” as used herein does not include antibodies in which CDR sequences from other mammalian species are inserted into the sequence of the mouse framework region.
• chimeric antibody refers to an antibody obtained by combining genetic material of non-human origin and genetic material of human origin. Or more generally, chimeric antibodies are antibodies that combine genetic material from one species with genetic material from another species.
• humanized antibody refers to an antibody derived from a non-human species but whose protein sequence has been modified to increase its similarity to natural human antibodies.
• a “humanized antibody” contains complementarity determining regions (CDRs) derived from non-human antibodies and framework regions (FRs) and constant regions derived from human antibodies.
• isotype refers to the antibody class (eg, IgM or IgGl) encoded by the heavy chain constant region genes.
• antibody that recognizes an antigen and “antibody specific/antibody specific for an antigen” are used interchangeably herein with the term “antibody that specifically binds an antigen”.
• an antibody that "specifically binds human IL4R ⁇ ” refers to an antibody that binds human IL4R ⁇ protein (and possibly IL4R ⁇ protein of one or more non-human species) but does not substantially bind non-IL4R ⁇ protein.
• the antibody binds human IL4R ⁇ protein with "high affinity", i.e., with 5.0 ⁇ -8 M or less, preferably 1.0 ⁇ 10-8 M or less, more preferably 7.0 ⁇ 10-9 M or less KD binds human IL4R ⁇ protein.
• the term "substantially does not bind" proteins or cells means does not bind to proteins or cells, or does not bind to them with high affinity, i.e., at 1.0 x 10 -6 M or greater, preferably 1.0 x 10 -5 M or greater, more preferably 1.0 ⁇ 10 -4 M or greater, more preferably 1.0 ⁇ 10 -3 M or greater, more preferably 1.0 ⁇ 10 -2 M or greater KD- binding protein or cell.
• high affinity for an IgG antibody means that the antibody has 1.0 ⁇ 10 -6 M or less, preferably 5.0 ⁇ 10 -8 M or less, more preferably 1.0 ⁇ 10 -8 M or less, for an antigen. Smaller, more preferably a K D of 7.0 ⁇ 10 -9 M or less, more preferably 1.0 ⁇ 10 -9 M or less. But for other antibody isotypes, "high affinity" binding may be different. For example, for the IgM isotype, "high affinity” means that the antibody has 1.0 x 10 -6 M or less, preferably 1.0 x 10 -7 M or less, more preferably 1.0 x 10 -8 M or less KD .
• K assoc or "K a” refers to the association rate of a specific antibody-antigen interaction
• K dis or “K d” refers to the solution of a specific antibody-antigen interaction. off speed.
• KD refers to the dissociation constant, which is the ratio of Kd to Ka (ie, Kd / Ka ) and is expressed in molarity (M).
• the KD value of an antibody can be determined using methods well known in the art. A preferred method of determining the KD value of an antibody is using surface plasmon resonance, preferably using a biosensor system, such as the Biacore TM system.
• EC 50 also known as the half-maximal effect concentration
• IC 50 also known as the half maximal inhibitory concentration
• IC 50 refers to the concentration of an antibody that inhibits a specific biological or biochemical function by 50% relative to the absence of the antibody.
• subject includes any human or non-human animal.
• non-human animal includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians and reptiles; preferably mammals, such as Non-human primates, sheep, dogs, cats, cows and horses.
• therapeutically effective amount refers to an amount of an antibody of the invention sufficient to prevent or ameliorate symptoms associated with a disease or disorder (eg, cancer), and/or lessen the severity of the disease or disorder. It should be understood that the therapeutically effective amount is related to the disease being treated, wherein the actual effective amount can be readily determined by those skilled in the art.
• identity refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. Sequence comparisons and percent identities between two sequences can be determined by the default settings of the BLASTN/BLASTP algorithm on the website of the National Center for Biotechnology Information.
• Xn and Xaa are equivalent and refer to unspecified amino acids (Unspecified Amino Acid), and the scope covered by them is specified by the subsequent definitions in the relevant expressions.
• anti-IL4R ⁇ antibodies or antigen-binding fragments thereof of the present disclosure specifically bind human IL4R ⁇ with comparable (if not better) binding affinity/capacity than reported anti-IL4R ⁇ antibodies such as Dupilumab.
• the anti-IL4R ⁇ antibody or antigen-binding fragment thereof in the present disclosure can block the binding of IL4R ⁇ to IL4 or IL13-IL13R ⁇ 1, thereby blocking the corresponding intracellular signal transduction.
• IL4R ⁇ antibody such as Dupilumab
• the anti-IL4R ⁇ antibody of the present invention is a humanized monoclonal antibody.
• the anti-IL4R ⁇ antibodies of the invention can be, for example, mouse, chimeric or human monoclonal antibodies.
• Anti-IL4R ⁇ antibodies in the present disclosure are monoclonal antibodies with structural and chemical properties as described below and in the Examples.
• the amino acid sequence IDs of the heavy/light chain variable regions of the antibodies are summarized in Table 1 below, some antibodies have the same VH or VL .
• the heavy chain constant region of the antibody may be a human IgG4 heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 13, and the light chain constant region of the antibody may be human ⁇ having the amino acid sequence shown in SEQ ID NO: 14 constant region.
• the heavy chain variable region CDRs and light chain variable region CDRs in Table 1 have been defined by the Kabat numbering system. However, CDR regions may also be determined by other numbering systems such as Chothia, IMGT, AbM or Contact numbering systems/methods based on heavy/light chain variable region sequences, as is well known in the art. Where specific CDR sequences are used to define an antibody or antigen-binding fragment thereof, the scope of said antibody encompasses an antibody or antigen-binding fragment thereof defined by a CDR sequence defined by any numbering system in the art (e.g. Chothia, IMGT, AbM or Contact numbering systems) .
• VH and VL sequences (or CDR sequences) of other anti-IL4R ⁇ antibodies that bind human IL4R ⁇ can be “mixed and paired” with the VH and VL sequences (or CDR sequences) of the anti-IL4R ⁇ antibodies of the present disclosure.
• VH and VL chains (or CDRs in these chains) are mixed and paired, the VH sequence from a particular VH / VL pair is replaced by a structurally similar VH sequence.
• an anti-IL4R ⁇ antibody or antigen-binding fragment thereof of the present disclosure comprises:
• the anti-IL4R ⁇ antibody or antigen-binding fragment thereof of the present disclosure comprises:
• an anti-IL4R ⁇ antibody of the disclosure or an antigen-binding fragment thereof comprises CDR2 of the heavy chain variable region of an anti-IL4R ⁇ antibody of the disclosure and the CDRs of other antibodies that bind human IL4R ⁇ , such as from another anti-IL4R ⁇ CDR1 and/or CDR3 of the heavy chain variable region of an antibody, and/or CDR1, CDR2 and/or CDR3 of the light chain variable region.
• the CDR3 domain alone is capable of determining the binding specificity of an antibody for a cognate antigen, and based on the common CDR3 sequence can predictively generate antibodies with the same binding A variety of specific antibodies.
• the anti-IL4R ⁇ antibody of the present disclosure or antigen-binding fragment thereof comprises the CDR2 of the heavy chain variable region of the anti-IL4R ⁇ antibody of the disclosure and at least the heavy chain variable region of the anti-IL4R ⁇ antibody of the disclosure and/or the CDR3 of the light chain variable region, or the CDR3 of the heavy chain variable region and/or the light chain variable region of another anti-IL4R ⁇ antibody, wherein the antibody specifically binds human IL4R ⁇ .
• These antibodies preferably (a) compete for IL4R ⁇ binding with the anti-IL4R ⁇ antibodies of the disclosure; (b) retain functional properties; (c) bind the same epitope; and/or (d) have similar binding affinities.
• the anti-IL4R ⁇ antibody of the present disclosure may further comprise the CDR2 of the light chain variable region of the anti-IL4R ⁇ antibody of the present disclosure, or the CDR2 of the light chain variable region of another anti-IL4R ⁇ antibody, wherein the antibody Specifically binds human IL4R ⁇ .
• the anti-IL4R ⁇ antibody of the present disclosure can also comprise the heavy chain variable region and/or the CDR1 of the light chain variable region of the anti-IL4R ⁇ antibody of the present disclosure, or the heavy chain of another anti-IL4R ⁇ antibody can be CDR1 of the variable region and/or light chain variable region, wherein the antibody specifically binds human IL4R ⁇ .
• an anti-IL4R ⁇ antibody or antigen-binding fragment thereof of the present disclosure comprises a heavy chain variable region and/or a light chain variable region, each of which comprises a CDR1 , CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 sequences are different from the CDR1, CDR2 and CDR3 sequences of the anti-IL4R ⁇ antibody in the present disclosure, and the difference is derived from one or more conservative modifications. It is understood in the art that certain conservative sequence modifications do not abolish antigen binding. See, e.g., Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng.
• an anti-IL4R ⁇ antibody of the present disclosure or an antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region comprising, respectively, CDR1, CDR2 and CDR3, wherein:
• CDR1 of the heavy chain variable region comprises the sequences listed in Table 1, and/or conservative modifications thereof;
• CDR2 of the heavy chain variable region comprises the sequences listed in Table 1, and/or conservative modifications thereof; and/or
• CDR3 of the heavy chain variable region comprises the sequences listed in Table 1, and/or conservative modifications thereof; and/or
• CDR1, and/or CDR2, and/or CDR3 of the light chain variable region comprise the sequences listed in Table 1, and/or conservative modifications thereof;
• conservative sequence modification refers to amino acid modifications that do not significantly affect or alter the binding properties of an antibody. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the present disclosure by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions refer to the substitution of amino acid residues for amino acid residues with similar side chains. Groups of amino acid residues having similar side chains are known in the art.
• amino acid residues include those with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., For example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (for example, alanine, valine, leucine acid, isoleucine, proline, phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains ( For example, amino acids of tyrosine, phenylalanine, tryptophan, histidine).
• basic side chains e.g., lysine, arginine, histidine
• acidic side chains e.g., aspartic acid, glutamic acid
• one or more amino acid residues in the CDR regions of the anti-IL4R ⁇ antibodies of the present disclosure can be replaced with other amino acid residues from the same side chain group, and the resulting antibodies can be tested using the functional assays described in the present disclosure. It is tested for retained function (ie, the function described above).
• compositions, lyophilized formulations or preparations of the present disclosure have a variety of in vitro and in vivo uses involving, for example, the treatment of diseases associated with excessive IL4 and/or IL13 signaling.
• anti-IL4R ⁇ antibodies or antigen-binding fragments thereof can block the binding of IL4R ⁇ to IL4 or IL13-IL13R ⁇ 1 to reduce type 2 immunity.
• the present disclosure provides a method of treating type 2 immune-related allergic disease in a subject, the method comprising administering to the subject a pharmaceutical composition, lyophilized formulation or preparation of the present disclosure.
• the allergic disease may be atopic dermatitis, anaphylaxis, allergic rhinitis or allergic asthma.
• STAT6 can be activated due to IL4 or IL13 signaling, and STAT6 inhibitors have been found to inhibit cancer cell growth.
• the present disclosure provides a method of inhibiting tumor cell growth in a subject, the method comprising administering to the subject a pharmaceutical composition, lyophilized formulation or preparation of the present disclosure.
• tumors include, but are not limited to, melanoma, lung cancer, renal cancer, prostate cancer, cervical cancer, colorectal cancer, gastric cancer, pancreatic cancer, ovarian cancer, and urothelial cancer.
• the present disclosure provides a method of reducing or inhibiting cellular activation in response to IL-4 or IL-13 in a subject.
• inhibiting activation includes inhibiting cytokine production or secretion.
• inhibiting activation includes inhibiting proliferation.
• Cells that respond to IL-4 by activating hybrid IL-4R ⁇ / ⁇ c receptors include, but are not limited to, B cells, eosinophils, and macrophages.
• Cells that respond to IL-13 by activating hybrid IL-4R ⁇ /IL-13R ⁇ 1 receptors include, but are not limited to, fibroblasts and smooth muscle cells. Accordingly, in one embodiment, the present disclosure provides methods of inhibiting smooth muscle cell proliferation. In another embodiment, the present disclosure provides methods of inhibiting fibroblast proliferation.
• the binding affinity and binding kinetics of the anti-IL4R ⁇ antibodies (mAbs) of the present disclosure were characterized by Biacore T200 system (GE Healthcare, Pittsburgh, PA, USA).
• recombinant human IL4R ⁇ -his protein (prepared in-house, amino acid sequence shown in SEQ ID NO: 15), cynomolgus monkey IL4R ⁇ -his protein (Sino biological inc., Product catalog 90897-C08H), or marmoset IL4R ⁇ -his protein (customized product of Sino biological inc., also known as cal-IL4R ⁇ -his, amino acid sequence as shown in SEQ ID NO: 16) flowed through at a flow rate of 30 ⁇ L/min chip. Antigen-antibody binding kinetics were followed for 2 minutes and dissociation kinetics were followed for 10 minutes. The binding and dissociation curves were fitted to a 1:1 Langmuir binding model using BIA evaluation software to determine K D , K a and K d values.
• the binding activity of the anti-IL4R ⁇ antibodies of the present disclosure to IL4R ⁇ was determined by capture ELISA, flow cytometry (FACS) and indirect ELISA.
• affinity-purified goat anti-human IgG Fc ⁇ fragment specific, Jackson Immunoresearch, catalog 109-005-098
• PBS PBS+0.05% w/v Tween-20, PBST
• PBST blocking buffer
• the plate was washed again, and 100 ⁇ L of the anti-IL4R ⁇ antibody, reference substance or negative control in the present disclosure was added in a serial dilution (66.7 nM as the initial concentration, 5-fold serial dilution in PBST containing 2.5% w/v skimmed milk) to each well hIgG (human immunoglobulin for intravenous injection (pH 4), Hualan Biological Engineering Inc.), co-incubated at 37°C for 40 minutes, and then washed the plate 4 times.
• hIgG human immunoglobulin for intravenous injection (pH 4), Hualan Biological Engineering Inc.
• the binding activity of the anti-IL4R ⁇ antibody in the present disclosure to IL4R ⁇ expressed on the surface of 293F-IL4R ⁇ cells was detected by flow cytometry (FACS). Briefly, 293F cells (Thermofisher Inc., cat. 11625019) were transfected with a pCMV-TP plasmid construct with 1-825 between EcoRI and XbaI encoding human IL4R ⁇ (uniprot#P24394-1). amino acid residues), and a pool of stable cells (named 293F-IL4R ⁇ ) was selected for subsequent cell-based binding FACS and cell-based ligand-blocking FACS analysis.
• FACS flow cytometry
• 293F-IL4R ⁇ cells were harvested from cell culture flasks, washed twice, and resuspended in FACS buffer (phosphate buffered saline (PBS) containing 2% v/v fetal bovine serum). Then, add 100 ⁇ L/well of anti-IL4R ⁇ antibody or control substance serially diluted in FACS buffer (with 80 nM as the initial concentration, 4-fold serial dilution) in a 96-well plate containing 2 ⁇ 10 5 cells/well, and store on ice Bath for 40 minutes.
• FACS buffer phosphate buffered saline (PBS) containing 2% v/v fetal bovine serum).
• PBS phosphate buffered saline
• fetal bovine serum fetal bovine serum
• Anti-IL4R ⁇ antibodies of the present disclosure were tested for cross-reactivity with cynomolgus monkey IL4R ⁇ protein or cal-IL4R ⁇ -his protein. Briefly, cynomolgus monkey IL4R ⁇ -his protein (Sino biological inc., catalog 90897-C08H) was dissolved at a concentration of 2 ⁇ g/mL in carbonate/bicarbonate buffer (pH 9.6) or dissolved in carbon Salt/bicarbonate buffer (pH 9.6) with a concentration of 0.2 ⁇ g/mL cal-IL4R ⁇ -his protein (Sino biological inc.
• the anti-IL4R ⁇ antibody or reference substance in the present disclosure was gradiently diluted with PBST buffer containing 2.5% w/v skimmed milk (with 80nM as the initial concentration, 4-fold gradient dilution), and the anti-IL4R ⁇ antibody or reference substance of the gradient dilution was Add 100 ⁇ L/well to the plate coated with IL4R ⁇ , and incubate with human IL4R ⁇ -his protein for 40 minutes at 37°C. The plate was washed 4 times with washing buffer, and then 100 ⁇ L of biotin-labeled human IL4 protein (Sino biological inc., product catalog 11846-HNAE) at a concentration of 0.56 nM was added to each well, and incubated at 37° C.
• biotin-labeled human IL4 protein (Sino biological inc., product catalog 11846-HNAE) at a concentration of 0.56 nM was added to each well, and incubated at 37° C.
• the ability of the anti-IL4R ⁇ antibodies of the present disclosure to block the binding of a reference, human IL4R ⁇ was determined by a competition ELISA. Briefly, the reference substance dissolved in PBS at a concentration of 2 ⁇ g/mL was coated in a 96-well plate at 100 ⁇ L/well, and incubated overnight at 4°C. The next day, the plate was washed with washing buffer (PBS+0.05% w/v Tween-20, PBST), and PBST containing 5% w/v skimmed milk was added to block at 37°C for 2 hours.
• washing buffer PBS+0.05% w/v Tween-20, PBST
• biotin-labeled human IL4R ⁇ -his protein prepared in-house, amino acid sequence shown in SEQ ID NO: 15
• PBST containing 2.5% w/v skimmed milk at a concentration of 0.55 nM
• the anti-IL4R ⁇ antibody or control substance in the present disclosure 100 nM as the initial concentration, 4-fold serial dilution
• 100 ⁇ L/well of the antibody/IL4R ⁇ -his mixture was added to the plate coated with the reference. After incubation at 37°C for 40 minutes, the plate was washed with wash buffer.
• 293F-IL4R ⁇ cells were harvested from cell culture flasks, washed twice, and resuspended in FACS buffer (PBS containing 2% v/v fetal bovine serum). Then, add 100 ⁇ L/well of anti-IL4R ⁇ antibody or control substance serially diluted in FACS buffer (with 80 nM as the initial concentration, 4-fold serial dilution) in a 96-well plate containing 1 ⁇ 10 5 cells/well, and store on ice. Bath for 40 minutes.
• FACS buffer PBS containing 2% v/v fetal bovine serum
• IL4 and IL13 are able to bind to IL4R ⁇ and induce STAT6 phosphorylation in HEK293T-IL4R ⁇ -STAT6-STAT6LUC-LB2 cells. STAT6 phosphorylation is critical in the IL4/IL13 signaling pathway.
• pcDNA3.1-Puro YouBio biological inc., product catalog VT9222
• plasmid construct (with the nucleotide encoding human IL4R ⁇ between BamHI and XhoI)
• STAT6 plasmid (Sino biological inc., product catalog HG13190-NH) (with nucleotides encoding human STAT6 between KpnI and XbaI)
• STAT6 luciferase reporter gene plasmid STAT6-Luc stably transfected with native expression of IL13R ⁇ 1 HEK293T cells (ATCC CRL-11268), HEK293T-IL4R ⁇ -STAT6-STAT6LUC-LB2 cells were prepared in-house. The single cell clone LB2 was then selected for all subsequent functional assays.
• HEK293T-IL4R ⁇ -STAT6-STAT6LUC-LB2 cells in logarithmic phase were resuspended in medium (RPMI1640+10% FBS), seeded into 96-well plates at 100 ⁇ L/well, and each well contained 5 ⁇ 10 5 cells. Then, 50 ⁇ L of serially diluted anti-IL4R ⁇ antibody of the present disclosure or control substance (including anti-CD22 antibody prepared in-house) was added to each well (100 nM as the initial concentration, 5-fold serial dilution), and incubated at 37° C. for 30 minutes.
• IL4 protein 600 pg/mL, Sino biological inc., catalog 11846-HNAE
• IL13 protein 50 ng/mL, Sino biological inc., catalog 10369-HNAC
• 50 ⁇ L of IL4 protein 600 pg/mL, Sino biological inc., catalog 11846-HNAE
• IL13 protein 50 ng/mL, Sino biological inc., catalog 10369-HNAC
• the cells were washed twice, 200 ⁇ L of permeabilization buffer (BD biosciences inc., catalog number 558050) was added to each well, and the cells were incubated on ice for 30 minutes. Plates were washed 3 times with staining buffer. Then anti-pSTAT6 antibody (pSTAT6 stock solution diluted 20 times, BD biosciences inc., catalog 562079) was added, and left to stand on ice for 60 minutes. Plates were finally washed twice and resuspended in staining buffer. Fluorescence values were measured using a Becton Dickinson FACS Canto II-HTS. Data were analyzed using Graphpad Prism software and IC50 values were obtained.
• permeabilization buffer BD biosciences inc., catalog number 558050
• mice anti-IL4R ⁇ monoclonal antibodies were sequenced, and the sequence IDs are summarized in Table 1.
• the heavy chain variable region and light chain variable region of the mouse anti-IL4R ⁇ monoclonal antibody C2C1A1A1 were cloned into the vector containing the human IgG4 heavy chain constant region (SEQ ID NO: 13) and the vector containing the human ⁇ light chain constant region (SEQ ID NO: 13). ID NO: 14), wherein the C-terminal of the variable region is connected to the N-terminal of the corresponding constant region.
• Cell supernatants were harvested after six days of culture in shake flasks, and cells in the supernatant were pelleted by centrifugation and filtered through a 0.22 ⁇ m filter for immunoglobulin isolation. Chimeric antibodies were purified by protein A affinity chromatography. Briefly, a protein A Sepharose column (bestchrom (Shanghai) Biosciences, catalog AA0273) was washed with 5 to 10 column volumes of PBS buffer. The cell supernatant was passed through a protein A sepharose column, and the column was washed with PBS buffer until the absorbance of the protein reached baseline.
• BIAcore replace goat anti-human IgG with goat anti-mouse IgG (GE healthcare, catalog BR100838, Mouse Antibody Capture Kit), covalently attached to the CM5 chip;
• Mouse anti-IL4R ⁇ antibody C2C1A1A1 was humanized and further characterized. Humanization of mouse antibodies was performed using established CDR grafting methods, as described below.
• the sequences of the light and heavy chain variable regions of the respective mouse antibodies were BLASTed against the human immunoglobulin gene database.
• the human germline antibody with the highest homology was selected as the recipient backbone for humanization. Insert mouse antibody heavy/light chain variable region CDRs into the selected framework, and further back-mutate the residues in the framework to obtain more candidate heavy/light chain variable regions.
• Cell supernatants were harvested after six days of culture in shake flasks, and cells in the supernatant were pelleted by centrifugation and filtered through a 0.22 ⁇ m filter for immunoglobulin isolation.
• Antibodies were purified by protein A affinity chromatography. Briefly, a protein A Sepharose column (bestchrom (Shanghai) Biosciences, catalog AA0273) was washed with 5 to 10 column volumes of PBS buffer. The cell supernatant was passed through a protein A sepharose column, and the column was washed with PBS buffer until the absorbance of the protein reached baseline.
• Dissociation % (End(RU)-Start(RU))/Start(RU)
• Humanized antibodies huC2C1A1A1-V14 and huC2C1A1A1-V15 was further tested.
• Tm melting temperature
• GloMelt TM Thermal Shift Protein Stability Kit Biotium, catalog 33022-T, lot number 181214.
• GloMelt TM dye was thawed to room temperature. Vortex and centrifuge the vial containing the dye. Then, add 5 ⁇ L of 200X dye to 95 ⁇ L of PBS to prepare 10X dye. Add 2 ⁇ L of 10 ⁇ dye and 10 ⁇ g of humanized antibody to the reaction system, and add PBS to a total reaction volume of 20 ⁇ L.
• the centrifuge tubes containing the dye and antibody were centrifuged briefly and placed in a real-time PCR thermal cycler (Roche, LightCycler 480 II) with a Melt Curve program set using the parameters in Table 4.
• the results are shown in Tables 5-1, 5-2 and 5-3.
• the data show that the humanized C2C1A1A1 antibody showed comparable (if not better) binding affinity/activity to human IL4R ⁇ and Blocking capacity of IL4R ⁇ -IL4/IL13.
• Size exclusion chromatography used to determine the purity of the antibody in the sample, using Thermo Vanquish F high performance liquid chromatography, Waters ACQUITY UPLC Protein BEH SEC Column ( 1.7 ⁇ m, 4.6mm ⁇ 300mm) chromatographic column, Waters ACQUITY UPLC Protein BEH SEC Guard Column ( 1.7 ⁇ m, 4.6mm ⁇ 30mm) as the pre-column, with 50mmol/L phosphate buffer + 200mmol/L sodium chloride (pH 7.0) as the mobile phase for elution, and the detection wavelength is 280nm. Calculate the percentage content of macromolecule impurities and immunoglobulin monomers by the area normalization method.
• DSF Differential Scanning Fluorescence
• iCIEF Whole-column imaging capillary isoelectric focusing: used to determine the charge variants of samples, using imaging capillary isoelectric focusing electrophoresis (ProteinSimple iCE3) and fast automatic protein characterization analysis system (Protein Simple, Maurice), Coated quartz capillary (Protein Simple), ultraviolet detector, detection is carried out under the ultraviolet detection wavelength 280nm.
• Imaging capillary isoelectric focusing electrophoresis ProteinSimple iCE3
• fast automatic protein characterization analysis system Protein Simple, Maurice
• Coated quartz capillary Protein Simple
• ultraviolet detector detection is carried out under the ultraviolet detection wavelength 280nm.
• Set the injection time to 60 seconds, 1500 volts to pre-focus for 1 minute, and 300 volts to focus for 10 minutes. Calculate the percent peak area of each charge variant by the peak area normalization method.
• Histidine-histidine hydrochloride buffering agent comprises histidine and histidine hydrochloride
• histidine-histidine hydrochloride buffering agent comprises histidine and histidine hydrochloride monohydrate, e.g., 20 mM histidine-histidine hydrochloride buffer (pH 5.8) consisting of about 1.17 mg/mL histidine and about 2.62 mg/mL histidine hydrochloride monohydrate;
• the sodium phosphate buffer comprises disodium hydrogen phosphate and sodium dihydrogen phosphate
• the sodium phosphate buffer comprises sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate dodecahydrate, for example, 20mM sodium phosphate buffer (pH 6.0) is formulated from about 2.539mg/mL sodium dihydrogen phosphate monohydrate and about 0.573mg/mL disodium hydrogen phosphate dodecahydrate;
• Sodium acetate-acetic acid buffering agent comprises sodium acetate and acetic acid, and in a specific embodiment, sodium acetate-acetic acid buffering agent comprises sodium acetate trihydrate and acetic acid, for example, 20mM sodium acetate-acetic acid buffering agent (pH 6.0 ) is formulated from about 2.605mg/mL sodium acetate trihydrate and about 0.141mg/mL acetic acid.
• the anti-IL4R ⁇ antibody (huC2C1A1A1-V15) was replaced by ultrafiltration into the buffer in Table 6, concentrated after the replacement, and then added with surfactant and stabilizer according to Table 6. After mixing evenly, filter and sterilize through a 0.22 ⁇ m filter membrane, and dispense into vials, stopper and cap.
• the SEC-UPLC detection results of the pharmaceutical compositions F1-F4 are shown in Table 7, wherein the growth of polymer impurities (from 1.36% to 5.66%) in F4 after storage at 40° C. for one month is higher than that in F1-F3.
• 20 mM histidine-histidine hydrochloride is selected as the buffer.
• the anti-IL4R ⁇ antibody (huC2C1A1A1-V15) was substituted into the buffer in Table 8 by ultrafiltration, concentrated after the replacement, and then added surfactant and stabilizer according to Table 8. After mixing evenly, filter and sterilize through a 0.22 ⁇ m filter membrane, and dispense into vials, stopper and cap.
• the Tm and SEC-UPLC detection results of the pharmaceutical compositions F5-F7 are shown in Table 9, among which, F6 has the least growth of polymer impurities after being stored at 40°C for one month.
• 20 mM histidine-histidine hydrochloride is selected as the buffer.
• the anti-IL4R ⁇ antibody (huC2C1A1A1-V15) was substituted into the buffer in Table 10 by ultrafiltration, concentrated after the replacement, and then added surfactant and stabilizer according to Table 10. After mixing evenly, filter and sterilize through a 0.22 ⁇ m filter membrane, and dispense into vials, stopper and cap.
• the Tm, viscosity, SEC-UPLC and iCIEF test results of pharmaceutical compositions F8 and F9 are shown in Table 11, wherein the viscosity of F8 is lower than that of F9.

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