Anti-IL-4R antibody pharmaceutical composition and use thereof

Abstract

The present disclosure relates to an anti-IL-4R antibody pharmaceutical composition and uses thereof. Specifically, the present disclosure relates to a pharmaceutical composition comprising an anti-IL-4R antibody or an antigen-binding fragment thereof in a buffer solution. In addition, the pharmaceutical composition further comprises a sugar, a non-ionic surfactant, and a viscosity modifier. The pharmaceutical composition of the present disclosure has good stability.

Description

[Technical Field]

[0001] Technical Field The present disclosure belongs to the field of drug formulations, and specifically relates to pharmaceutical compositions comprising anti-IL-4R antibodies or antigen-binding fragments thereof, and their use as medicaments for treating immune-mediated diseases. [Background technology]

[0002] Background technology Nothing herein necessarily constitutes prior art, but rather merely provides background information relevant to the present disclosure.

[0003] Allergic diseases are serious medical conditions, including non-life-threatening allergic reactions and life-threatening allergic diseases. Current methods for treating allergies include allergen avoidance, pharmacological treatment of symptoms, and prevention through allergen-specific immunotherapy.

[0004] Interleukin-4 (IL-4, also known as B cell stimulating factor or BSF-1) is characterized by its ability to stimulate B cell proliferation in response to low concentrations of anti-surface immunoglobulin antibodies. IL-4 has been shown to have a wide range of biological activities, including stimulating the proliferation of T cells, mast cells, granulocytes, megakaryocytes, and erythrocytes. IL-4 induces MHC-II expression on resting B cells and enhances the secretion of immunoglobulins IgE and IgG1 by activated B cells.

[0005] The biological activity of IL-4 is mediated by a specific cell surface IL-4 receptor (IL-4R). The IL-4 receptor (IL-4R) consists of 802 amino acid residues and is expressed on the surface of T cells, B cells, thymocytes, myeloid cells, macrophages, and mast cells. The α chain of IL-4R is also a component of the IL-13 receptor (IL-13R), so IL-4R can also mediate the biological activity of IL-13. Drugs and compositions containing IL-4R antagonists can be administered as novel therapies before, during, or after a subject's contact with an allergen or the onset of allergic symptoms.

[0006] High protein concentration formulations pose challenges to the physical and chemical stability of proteins and pose challenges in the preparation, storage, and delivery of protein formulations. One problem is that proteins tend to form particles during handling and / or storage, making them difficult to handle during further processing.

[0007] Currently, existing patent applications related to anti-IL-4R antibodies and their formulations include WO2010053751, WO2001092340, WO2008054606, WO2014031610, CN106604744A, etc. Summary of the Invention

[0008] Summary of the Invention Considering that increased viscosity of protein formulations has adverse effects on everything from preparation of the formulation to delivery of the drug to patients, there is a need to develop relatively high concentration protein formulations with an appropriately low viscosity suitable for preparation, storage, and administration of protein formulations.

[0009] The present disclosure provides a pharmaceutical composition comprising an anti-IL-4R antibody or an antigen-binding fragment thereof and a buffer, wherein the buffer is selected from histidine-acetate buffers, and the pH of the pharmaceutical composition is 4.5 to 6.0, preferably about 4.5 to 5.5, and most preferably about 5.0.

[0010] In some embodiments, the pharmaceutical composition has a pH of the buffer of 4.8 to 5.5, preferably about 5.0.

[0011] In some embodiments, the pharmaceutical composition has a pH value of about 4.5 to 6.0, preferably 4.5 to 5.5, preferably about 4.6 to 5.5, preferably about 4.7 to 5.5, preferably about 4.8 to 5.5, 4.9 to 5.5, preferably about 5.0 to 5.5, preferably about 5.2 to 5.5, or preferably about 5.3 to 5.5, most preferably 5.0; non-limiting examples include about 4.5, about 4.6, about 4.7, about 4.8, about 4.0, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, and about 5.5.

[0012] In some embodiments, the pharmaceutical composition has a concentration of the histidine-acetate buffer between 10 mM and 60 mM, non-limiting examples include 10 mM to 30 mM, 10 mM to 40 mM, and 20 mM to 50 mM.

[0013] In some embodiments, the pharmaceutical composition has a buffer concentration of about 10 mM to 50 mM, preferably about 10 mM to 30 mM, non-limiting examples include 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM, 30 mM, 32 mM, 34 mM, 36 mM, 38 mM, 40 mM, 42 mM, 44 mM, 46 mM, 48 mM, and 50 mM, most preferably about 20 mM or 50 mM.

[0014] In some embodiments, the pharmaceutical composition contains the anti-IL-4R antibody or antigen-binding fragment thereof at a concentration of 100 mg / mL to 200 mg / mL, non-limiting examples of which include 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, 200 mg / mL, and any range between these values.

[0015] In some embodiments, the pharmaceutical composition contains the anti-IL-4R antibody or antigen-binding fragment thereof at a concentration of 100 mg / mL to 180 mg / mL.

[0016] In some embodiments, the pharmaceutical composition has a concentration of the anti-IL-4R antibody or antigen-binding fragment thereof of about 150 mg / mL.

[0017] In some embodiments, the pharmaceutical composition contains the anti-IL-4R antibody or antigen-binding fragment thereof at a concentration of 100 mg / mL or more, preferably 100 mg / mL to 150 mg / mL, and most preferably 120 mg / mL.

[0018] In some embodiments, the pharmaceutical composition contains the anti-IL-4R antibody or antigen-binding fragment thereof at a concentration of 100 mg / mL to 140 mg / mL.

[0019] In some embodiments, the pharmaceutical composition contains the anti-IL-4R antibody or antigen-binding fragment thereof at a concentration of 100 mg / mL to 120 mg / mL, non-limiting examples of which include 102 mg / mL, 104 mg / mL, 106 mg / mL, 108 mg / mL, 110 mg / mL, 112 mg / mL, 114 mg / mL, 116 mg / mL, 118 mg / mL, and 120 mg / mL.

[0020] In some embodiments, the pharmaceutical composition further comprises a viscosity modifier selected from MgCl, CaCl, NaF, NaSCN, KCl, CHCOONa, NaSO, NaI, Arg-HCl, arginine, histidine, and lysine, preferably MgCl, histidine, and arginine hydrochloride (Arg-HCl).

[0021] In some embodiments, the pharmaceutical composition includes a viscosity modifier. In some cases, the antibody formulation has a high viscosity due to the high antibody concentration. In some embodiments, the viscosity modifier is lysine, arginine, or histidine. In some embodiments, the viscosity modifier is arginine. In some embodiments, the viscosity modifier includes a salt form, such as a salt of arginine, lysine, or histidine. In some embodiments, the viscosity modifier is an amino acid, such as an L-amino acid, such as L-arginine, L-lysine, or L-histidine. In some embodiments, the viscosity modifier is selected from MgCl2, CaCl2, NaF, NaSCN, KCl, CH3COONa, Na2SO4, NaI, Arg-HCl, histidine, and lysine. In some embodiments, the viscosity modifier is selected from MgCl2, histidine, and Arg-HCl.

[0022] In some embodiments, the concentration of the viscosity modifier is from about 5 mM to about 220 mM. Non-limiting examples include, but are not limited to, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, or 220 mM, and any range between these values.

[0023] In some embodiments, the concentration of the viscosity modifier is from about 10 mM to about 220 mM, hi some embodiments, the concentration of the viscosity modifier is from about 50 mM to about 180 mM.

[0024] In some embodiments, the concentration of the viscosity modifier is from about 5 mM to about 148 mM.

[0025] In some embodiments, the concentration of the viscosity modifier is from about 50 mM to about 120 mM.

[0026] In some embodiments, the concentration of the viscosity modifier is from about 5 mM to about 50 mM, hi some embodiments, the concentration of the viscosity modifier is from about 10 mM to about 40 mM.

[0027] In some embodiments, the viscosity of the pharmaceutical composition is less than 40 mPa.s, or less than 30 mPa.s.

[0028] In some embodiments, the viscosity of the pharmaceutical composition is less than 20 mPa.s.

[0029] In some embodiments, the pharmaceutical composition contains the viscosity adjusting agent at a concentration of 50 mM to 148 mM, preferably 85 mM to 120 mM.

[0030] In some embodiments, the viscosity modifier is i) 5 mM to 220 mM arginine hydrochloride; ii) 5 mM to 100 mM histidine, or iii) 5 mM to 90 mM MgCl2.

[0031] In some embodiments, the viscosity modifier is i) 10 mM to 220 mM arginine hydrochloride; ii) 10 mM to 100 mM histidine, or iii) 10 mM to 90 mM MgCl2.

[0032] In some embodiments, the viscosity modifier is arginine hydrochloride at 5 mM to 220 mM, non-limiting examples include, but are not limited to, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, or 220 mM, and any range between these values.

[0033] In some embodiments, the viscosity modifier is histidine at 5 mM to 100 mM, non-limiting examples being 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM, and any range between these values.

[0034] In some embodiments, the viscosity modifier is 10 mM to 90 mM MgCl, non-limiting examples being 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, or 90 mM, and any range between these values.

[0035] In some embodiments, the pharmaceutical composition contains the anti-IL-4R antibody or antigen-binding fragment thereof at a concentration of 100 mg / mL to 200 mg / mL, and the viscosity adjusting agent is selected from MgCl2, histidine, and arginine hydrochloride.

[0036] In some embodiments, when the concentration of the anti-IL-4R antibody or antigen-binding fragment thereof is 100 mg / mL to 200 mg / mL, the viscosity adjusting agent in the pharmaceutical composition is selected from 50 mM to 200 mM of MgCl, histidine, and arginine hydrochloride.

[0037] In some embodiments, when the concentration of the anti-IL-4R antibody or antigen-binding fragment thereof is 100 mg / mL to 200 mg / mL, the viscosity adjusting agent in the pharmaceutical composition is selected from 50 mM to 90 mM MgCl2, 50 mM to 100 mM histidine, and 10 mM to 200 mM, preferably 50 mM to 180 mM, arginine hydrochloride.

[0038] In some embodiments, the pharmaceutical composition contains the anti-IL-4R antibody or antigen-binding fragment thereof at a concentration of 120 mg / mL to 150 mg / mL, and the viscosity adjusting agent is selected from 50 mM to 90 mM MgCl, 50 mM to 100 mM histidine, and 50 mM to 120 mM arginine hydrochloride.

[0039] In some embodiments, the pharmaceutical composition contains the anti-IL-4R antibody or antigen-binding fragment thereof at a concentration of 100 mg / mL to 140 mg / mL, and the viscosity adjusting agent is selected from 5 mM to 50 mM of histidine, arginine hydrochloride, or MgCl2, preferably 10 mM to 40 mM of histidine, arginine hydrochloride, or MgCl2.

[0040] In some embodiments, the pharmaceutical composition contains the anti-IL-4R antibody or antigen-binding fragment thereof at a concentration of 100 mg / mL to 140 mg / mL, and the viscosity adjusting agent is 30 mM histidine, arginine hydrochloride, or MgCl2.

[0041] In some embodiments, the pharmaceutical composition contains the anti-IL-4R antibody or antigen-binding fragment thereof at a concentration of 100 mg / mL to 120 mg / mL, and the viscosity adjusting agent is selected from 5 mM to 50 mM of histidine, arginine hydrochloride, or MgCl2, preferably 10 mM to 40 mM of histidine, arginine hydrochloride, or MgCl2.

[0042] In some embodiments, the pharmaceutical composition comprises a viscosity adjusting agent selected from 50 mM to 90 mM MgCl2, 85 mM to 100 mM histidine, and 90 mM to 120 mM arginine hydrochloride.

[0043] In some embodiments, the viscosity adjusting agent in the pharmaceutical composition is selected from 5 mM to 50 mM of histidine, arginine hydrochloride, or MgCl2, preferably 10 mM to 40 mM of histidine, arginine hydrochloride, or MgCl2.

[0044] In some embodiments, the pharmaceutical composition further comprises a surfactant, preferably polysorbate 80.

[0045] In some embodiments, the pharmaceutical composition has a surfactant concentration of 0.1 mg / mL to 1.2 mg / mL, preferably 0.8 mg / mL.

[0046] In some embodiments, the pharmaceutical composition has a surfactant concentration of about 0.1 mg / mL to 1.0 mg / mL, preferably 0.2 mg / mL to 0.8 mg / mL, and more preferably 0.4 mg / mL to 0.8 mg / mL, with non-limiting examples including but not limited to 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.55 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, and any range between these values, with 0.8 mg / mL being most preferred.

[0047] In some embodiments, the pharmaceutical composition comprises: (a) 100 mg / mL to 200 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM of a histidine-acetate buffer having a pH of 4.5 to 6.0, (c) 50 mM to 220 mM of a viscosity modifier, and (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, wherein the viscosity modifier is selected from MgCl2, CaCl2, NaF, NaSCN, KCl, CH3COONa, Na2SO4, NaI, arginine, arginine hydrochloride, histidine, and lysine, and preferably the viscosity modifier is selected from MgCl2, histidine, and arginine hydrochloride.

[0048] In some embodiments, the pharmaceutical composition further comprises a stabilizer, preferably selected from trehalose or sucrose, with sucrose being preferred.

[0049] In some embodiments, the pharmaceutical composition has a stabilizer concentration of 20 mg / mL to 70 mg / mL, preferably 40 mg / mL to 60 mg / mL, and most preferably 58 mg / mL.

[0050] In some embodiments, the pharmaceutical composition has a concentration of the stabilizer of 40 mg / mL to 70 mg / mL.

[0051] In some embodiments, the pharmaceutical composition has a sugar concentration of about 50 mg / mL to about 60 mg / mL, preferably 55 mg / mL to 60 mg / mL, non-limiting examples include 50 mg / mL, 51 mg / mL, 52 mg / mL, 53 mg / mL, 54 mg / mL, 55 mg / mL, 56 mg / mL, 57 mg / mL, 58 mg / mL, 59 mg / mL, and 60 mg / mL.

[0052] In some embodiments, the pharmaceutical composition comprises: The pharmaceutical composition comprises (a) 100 mg / mL to 150 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM of a histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 80 mM to 148 mM or 5 mM to 50 mM of a viscosity adjuster, (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, and (e) 40 mg / mL to 60 mg / mL of sucrose, and the viscosity of the pharmaceutical composition is 20 mPa s or less.

[0053] In some embodiments, the pharmaceutical composition comprises: (a) 100 mg / mL to 150 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM of a histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 80 mM to 148 mM of a viscosity modifier, and (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, Includes.

[0054] In some embodiments, the pharmaceutical composition comprises: (a) 100 mg / mL to 120 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 20 mM histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 10 mM to 40 mM histidine, (d) 0.4 mg / mL to 1.0 mg / mL of polysorbate 80, and (e) 50 mg / mL to 60 mg / mL of sucrose, Includes.

[0055] In some embodiments, the pharmaceutical composition comprises: (a) 100 mg / mL to 140 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM of a histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 10 mM to 40 mM of a viscosity modifier, (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, and (e) 40 mg / mL to 70 mg / mL of sucrose, Or, (a) 100 mg / mL to 200 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM of a histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 40 mM to 220 mM of a viscosity modifier, and (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, Including, Among them, the viscosity modifier is selected from MgCl2, CaCl2, NaF, NaSCN, KCl, CH3COONa, Na2SO4, NaI, arginine, arginine hydrochloride, histidine and lysine.

[0056] In some embodiments, the pharmaceutical composition comprises: (a) 100 mg / mL to 140 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM of a histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 10 mM to 40 mM of a viscosity modifier, (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, and (e) 40 mg / mL to 70 mg / mL of sucrose (wherein the viscosity modifier is histidine, arginine hydrochloride, or MgCl2), or (a) 100 mg / mL to 180 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 40 mM to 90 mM MgCl2, and (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, or (a) 100 mg / mL to 180 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 50 mM to 100 mM histidine, and (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, or (a) 100 mg / mL to 180 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM of a histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 50 mM to 200 mM of arginine hydrochloride, and (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, Includes.

[0057] In some embodiments, the pharmaceutical composition comprises: (a) 100 mg / mL to 140 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 10 mM to 40 mM histidine, (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, and (e) 40 mg / mL to 70 mg / mL of sucrose, Includes.

[0058] In some embodiments, the pharmaceutical composition comprises: (a) 120 mg / mL of an anti-IL-4R antibody or an antigen-binding fragment thereof, (b) 20 mM histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 30 mM histidine, (d) 0.8 mg / mL of polysorbate 80, and (e) 58 mg / mL of sucrose. Includes.

[0059] In some embodiments, the pharmaceutical composition comprises: (a) 120 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 20 mM histidine-acetate buffer having a pH of about 5.0, (c) 30 mM histidine, (d) 0.8 mg / mL of polysorbate 80, and (e) 58 mg / mL of sucrose; Includes.

[0060] In some embodiments, the pharmaceutical composition comprises: (a) 120 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 50 mM histidine-acetate buffer having a pH of about 5.0, (c) 0.8 mg / mL of polysorbate 80, and (d) 58 mg / mL of sucrose; Includes.

[0061] In some embodiments, the pharmaceutical composition comprises: (a) about 100 mg / mL to 180 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) about 10 mM to 30 mM histidine-acetate buffer having a pH of about 4.5 to 5.5, (c) about 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, and (d) about 90 mM to 200 mM of arginine hydrochloride, Includes.

[0062] In some embodiments, the pharmaceutical composition comprises: (a) about 150 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) about 20 mM histidine-acetate buffer having a pH of about 5.0, (c) about 0.8 mg / mL of polysorbate 80, and (d) about 120 mM arginine hydrochloride; Includes.

[0063] In some embodiments, the pharmaceutical composition comprises: (a) 100 mg / mL to 140 mg / mL of an anti-IL-4R antibody or an antigen-binding fragment thereof, (b) 20 mM to 60 mM histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, and (d) 40 mg / mL to 70 mg / mL of sucrose, Includes.

[0064] In some embodiments, the pharmaceutical composition comprises: (a) 100 mg / mL to 140 mg / mL of an anti-IL-4R antibody or an antigen-binding fragment thereof, (b) a 50 mM histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, and (d) 40 mg / mL to 70 mg / mL of sucrose, Includes.

[0065] In some embodiments, the pharmaceutical composition comprises: (a) 100 mg / mL to 120 mg / mL of an anti-IL-4R antibody or an antigen-binding fragment thereof, (b) 20 mM to 60 mM histidine-acetate buffer having a pH of 5.0 to 5.5, (c) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, and (d) 50 mg / mL to 60 mg / mL of sucrose, and preferably (a) 120 mg / mL of an anti-IL-4R antibody or an antigen-binding fragment thereof, (b) 50 mM histidine-acetate buffer having a pH of 5.0 to 5.5, (c) 0.8 mg / mL of polysorbate 80, and (d) 58 mg / mL of sucrose; Includes.

[0066] In some embodiments, the pharmaceutical composition comprises: (a) 120 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 50 mM histidine-acetate buffer having a pH of about 5.0, (c) 0.8 mg / mL of polysorbate 80, and (d) 58 mg / mL of sucrose; Includes.

[0067] In some embodiments, the pharmaceutical composition comprises the anti-IL-4R antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region as set forth below: (i) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively; (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively; (iii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO:38, SEQ ID NO:7, and SEQ ID NO:40, respectively; or (iv) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; and A light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:42, SEQ ID NO:39 and SEQ ID NO:8, respectively. In some embodiments, the pharmaceutical composition comprises the anti-IL-4R antibody or antigen-binding fragment thereof: a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:38, SEQ ID NO:7 and SEQ ID NO:40, respectively; Includes.

[0068] In some embodiments, the pharmaceutical composition comprises the anti-IL-4R antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region as set forth below: (v) a heavy chain variable region sequence set forth in SEQ ID NO:1 or having at least 90% identity thereto, and a light chain variable region sequence set forth in SEQ ID NO:2 or having at least 90% identity thereto; (vi) the heavy chain variable region sequence is set forth in SEQ ID NO:9 or has at least 90% identity to SEQ ID NO:9, and the light chain variable region sequence is set forth in SEQ ID NO:10 or has at least 90% identity to SEQ ID NO:10; (vii) a heavy chain variable region sequence set forth in, or having at least 90% identity to, SEQ ID NO:25, 26, 27, 43, or 47, and a light chain variable region sequence set forth in, or having at least 90% identity to, SEQ ID NO:28, 29, 30, 37, or 41; or (viii) a heavy chain variable region sequence set forth in SEQ ID NO: 31, 32, or 33, or which has at least 90% identity to SEQ ID NO: 31, 32, or 33, and a light chain variable region sequence set forth in SEQ ID NO: 34, 35, or 36, or which has at least 90% identity to SEQ ID NO: 34, 35, or 36; Preferably, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region shown as follows: (IX) the heavy chain variable region sequence is set forth in SEQ ID NO:43 or has at least 90% identity to SEQ ID NO:43, and the light chain variable region sequence is set forth in SEQ ID NO:37 or has at least 90% identity to SEQ ID NO:37; or (X) the heavy chain variable region sequence is set forth in SEQ ID NO:43 or has at least 90% identity to SEQ ID NO:43, and the light chain variable region sequence is set forth in SEQ ID NO:41 or has at least 90% identity to SEQ ID NO:41; or (XI) the heavy chain variable region sequence is set forth in SEQ ID NO:47 or has at least 90% identity to SEQ ID NO:47, and the light chain variable region sequence is set forth in SEQ ID NO:41 or has at least 90% identity to SEQ ID NO:41.

[0069] In some embodiments, the pharmaceutical composition comprises an anti-IL-4R antibody or antigen-binding fragment thereof, wherein the heavy chain variable region sequence is set forth in SEQ ID NO:43 and the light chain variable region sequence is set forth in SEQ ID NO:37.

[0070] In some embodiments, the pharmaceutical composition comprises an anti-IL-4R antibody or antigen-binding fragment thereof comprising a constant region. In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises the constant region of a human κ or λ chain or a variant thereof, and further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 or a variant thereof, for example, IgG4-S228P or IgG4-234A / 235A mutant.

[0071] In some embodiments, the pharmaceutical composition comprises the anti-IL-4R antibody or antigen-binding fragment thereof: a heavy chain as set forth in SEQ ID NO:17 and a light chain as set forth in SEQ ID NO:18, or a heavy chain as set forth in SEQ ID NO:19 and a light chain as set forth in SEQ ID NO:20, or a heavy chain as set forth in SEQ ID NO:44 and a light chain as set forth in SEQ ID NO:45; or a heavy chain as set forth in SEQ ID NO:44 and a light chain as set forth in SEQ ID NO:46, or a heavy chain as shown in SEQ ID NO: 48 and a light chain as shown in SEQ ID NO: 46; Includes.

[0072] In some embodiments, the pharmaceutical composition comprises the anti-IL-4R antibody or antigen-binding fragment thereof comprising a heavy chain set forth in SEQ ID NO:44 and a light chain set forth in SEQ ID NO:45. The present application further relates to an anti-IL-4R antibody having a heavy chain variable region sequence set forth in SEQ ID NO:47 or having at least 90% identity to SEQ ID NO:47 and a light chain variable region sequence set forth in SEQ ID NO:41 or having at least 90% identity to SEQ ID NO:41.

[0073] The present application further relates to an anti-IL-4R antibody comprising a heavy chain shown in SEQ ID NO:48 and a light chain shown in SEQ ID NO:46.

[0074] In one embodiment, the pharmaceutical composition comprises (a) 100 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 4.5 to 6.0, and (c) 0.1 mg / mL of polysorbate 80.

[0075] In one embodiment, the pharmaceutical composition comprises (a) 100 mg / mL of hu25G7-A antibody, and (b) 20 mM histidine-acetate buffer having a pH of 4.5.

[0076] In one embodiment, the pharmaceutical composition comprises: (a) 100 mg / mL of hu25G7-A antibody; (b) 20 mM histidine-acetate buffer having a pH of 4.5; and (c) 0.1 mg / mL of polysorbate 80.

[0077] In one embodiment, the pharmaceutical composition comprises (a) 100 mg / mL of hu25G7-A antibody, and (b) 20 mM histidine-acetate buffer having a pH of 5.0.

[0078] In one embodiment, the pharmaceutical composition comprises: (a) 100 mg / mL of hu25G7-A antibody; (b) 20 mM histidine-acetate buffer having a pH of 5.0; and (c) 0.1 mg / mL of polysorbate 80.

[0079] In one embodiment, the pharmaceutical composition comprises: (a) 100 mg / mL of hu25G7-A antibody; (b) 20 mM histidine-acetate buffer having a pH of 6.0; and (c) 0.1 mg / mL of polysorbate 80.

[0080] In one embodiment, the pharmaceutical composition comprises (a) 100 mg / mL of hu25G7-A antibody, and (b) 20 mM histidine-acetate buffer having a pH of 5.5.

[0081] In one embodiment, the pharmaceutical composition comprises: (a) 100 mg / mL of hu25G7-A antibody; (b) 20 mM histidine-acetate buffer having a pH of 5.5; and (c) 0.1 mg / mL of polysorbate 80.

[0082] In one embodiment, the pharmaceutical composition comprises (a) 100 to 150 mg / mL of the hu25G7-A antibody, and (b) a 20 mM histidine-acetate buffer having a pH of 5.0 to 5.5.

[0083] In one embodiment, the pharmaceutical composition comprises (a) 120 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, and (c) 122 mM NaCl.

[0084] In one embodiment, the pharmaceutical composition comprises (a) 120 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, and (c) 85 mM MgCl 2 .

[0085] In one embodiment, the pharmaceutical composition comprises (a) 120 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, and (c) 148 mM CaCl 2 .

[0086] In one embodiment, the pharmaceutical composition comprises (a) 120 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, and (c) 124 mM KCl.

[0087] In one embodiment, the pharmaceutical composition comprises (a) 120 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, and (c) 86 mM CH3COONa.

[0088] In one embodiment, the pharmaceutical composition comprises (a) 120 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, and (c) 96 mM Na2SO4.

[0089] In one embodiment, the pharmaceutical composition comprises (a) 120 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, and (c) 113 mM NaI.

[0090] In one embodiment, the pharmaceutical composition comprises (a) 120 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, and (c) 74 mM NaF.

[0091] In one embodiment, the pharmaceutical composition comprises (a) 120 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, and (c) 112 mM NaSCN.

[0092] In one embodiment, the pharmaceutical composition comprises (a) 120 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, and (c) 120 mM Arg-HCl.

[0093] In one embodiment, the pharmaceutical composition comprises (a) 120 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, and (c) 118 mM lysine.

[0094] In one embodiment, the pharmaceutical composition comprises (a) 120 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, and (c) 93 mM histidine.

[0095] In one embodiment, the pharmaceutical composition comprises (a) 120 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, and (c) 207 mM proline.

[0096] In one embodiment, the pharmaceutical composition comprises (a) 150 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, and (c) 85 mM MgCl 2 .

[0097] In one embodiment, the pharmaceutical composition comprises (a) 150 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, and (c) 148 mM CaCl 2 .

[0098] In one embodiment, the pharmaceutical composition comprises (a) 150 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, and (c) 93 mM histidine.

[0099] In one embodiment, the pharmaceutical composition comprises (a) 150 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, and (c) 50 mM MgCl 2 .

[0100] In one embodiment, the pharmaceutical composition comprises (a) 150 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, and (c) 90 mM MgCl 2 .

[0101] In one embodiment, the pharmaceutical composition comprises (a) 150 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, and (c) 90 mM CaCl 2 .

[0102] In one embodiment, the pharmaceutical composition comprises (a) 150 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, and (c) 148 mM CaCl 2 .

[0103] In one embodiment, the pharmaceutical composition comprises (a) 150 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, and (c) 90 mM histidine.

[0104] In one embodiment, the pharmaceutical composition comprises (a) 150 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, and (c) 120 mM Arg-HCl.

[0105] In one embodiment, the pharmaceutical composition comprises (a) 150 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, (c) 90 mM MgCl2, and (d) 0.1 mg / mL of polysorbate 80.

[0106] In one embodiment, the pharmaceutical composition comprises (a) 150 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, (c) 90 mM MgCl2, and (d) 1 mg / mL of polysorbate 80.

[0107] In one embodiment, the pharmaceutical composition comprises (a) 120 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, (c) 122 mM NaCl, and (d) 0.1 mg / mL of polysorbate 80.

[0108] In one embodiment, the pharmaceutical composition comprises (a) 120 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, (c) 122 mM NaCl, and (d) 1 mg / mL of polysorbate 80.

[0109] In one embodiment, the pharmaceutical composition comprises (a) 150 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, (c) 90 mM histidine, and (d) 0.1 mg / mL of polysorbate 80.

[0110] In one embodiment, the pharmaceutical composition comprises (a) 150 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, (c) 90 mM histidine, and (d) 1 mg / mL of polysorbate 80.

[0111] In one embodiment, the pharmaceutical composition comprises (a) 150 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, (c) 120 mM Arg-HCl, and (d) 0.1 mg / mL of polysorbate 80.

[0112] In one embodiment, the pharmaceutical composition comprises (a) 150 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, (c) 120 mM Arg-HCl, and (d) 1 mg / mL of polysorbate 80.

[0113] In one embodiment, the pharmaceutical composition comprises: (a) about 165 mg / mL of hu25G7-A antibody; (b) about 20 mM histidine-acetate buffer having a pH of 5.0; and (c) about 50 mM to 90 mM MgCl2.

[0114] In one embodiment, the pharmaceutical composition comprises: (a) about 165 mg / mL of hu25G7-A antibody; (b) about 20 mM histidine-acetate buffer having a pH of 5.0; and (c) about 50 mM to 90 mM histidine.

[0115] In one embodiment, the pharmaceutical composition comprises: (a) about 165 mg / mL of hu25G7-A antibody; (b) about 20 mM histidine-acetate buffer having a pH of 5.0; and (c) about 90 mM to 200 mM Arg-HCl.

[0116] In one embodiment, the pharmaceutical composition comprises (a) 150 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, (c) 120 mM Arg-HCl, and (d) 0.8 mg / mL of polysorbate 80.

[0117] In one embodiment, the pharmaceutical composition comprises (a) 150 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 4.8, (c) 87 mM histidine, and (d) 0.8 mg / mL of polysorbate 80.

[0118] In one embodiment, the pharmaceutical composition comprises (a) 150 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, (c) 100 mM histidine, and (d) 0.8 mg / mL of polysorbate 80.

[0119] In one embodiment, the pharmaceutical composition comprises (a) 150 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, (c) 30 mM histidine, and (d) 0.8 mg / mL of polysorbate 80, and (e) 41.8 mg / mL of sucrose.

[0120] In one embodiment, the pharmaceutical composition comprises (a) 120 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, (c) 30 mM histidine, and (d) 0.8 mg / mL of polysorbate 80, and (e) 58 mg / mL of sucrose.

[0121] In one embodiment, the pharmaceutical composition comprises (a) 120 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.2, (c) 30 mM histidine, and (d) 0.4 mg / mL of polysorbate 80, and (e) 50 mg / mL of sucrose.

[0122] In one embodiment, the pharmaceutical composition comprises (a) 120 mg / mL of hu25G7-A antibody, (b) 50 mM histidine-acetate buffer having a pH of about 5.0, (c) 0.8 mg / mL of polysorbate 80, and (d) 58 mg / mL of sucrose.

[0123] In one embodiment, the pharmaceutical composition comprises: (a) 120 mg / mL to 150 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, (c) 30 mM to 100 mM histidine, (d) 0.8 mg / mL of polysorbate 80, and (e) 41.8 mg / mL to 58 mg / mL of sucrose.

[0124] In one embodiment, the pharmaceutical composition comprises (a) 150 mg / mL of hu25G7-A antibody, (b) 20 mM histidine-acetate buffer having a pH of 5.0, (c) 120 mM arginine hydrochloride, and (d) 0.8 mg / mL of polysorbate 80.

[0125] In one embodiment, the pharmaceutical composition comprises: (a) 100 mg / mL to 140 mg / mL of hu25G7-A antibody, (b) 50 mM histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, and (d) approximately 58 mg / mL of sucrose.

[0126] In one embodiment, the pharmaceutical composition comprises: (a) 100 mg / mL to 140 mg / mL of hu25G7-A antibody, (b) 50 mM histidine-acetate buffer having a pH of 4.8 to 5.5, (c) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, and (d) approximately 58 mg / mL of sucrose.

[0127] In one embodiment, the pharmaceutical composition comprises: (a) 100 mg / mL to 120 mg / mL of hu25G7-A antibody, (b) 50 mM histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 0.4 mg / mL to 0.8 mg / mL of polysorbate 80, and (d) approximately 58 mg / mL of sucrose.

[0128] In one embodiment, the pharmaceutical composition comprises (a) about 132 mg / mL of hu25G7-A antibody, (b) 50 mM histidine-acetate buffer having a pH of about 5.5, (c) about 0.4 mg / mL of polysorbate 80, and (d) about 58 mg / mL of sucrose.

[0129] In one embodiment, the pharmaceutical composition comprises (a) about 100 mg / mL of hu25G7-A antibody, (b) 50 mM histidine-acetate buffer having a pH of about 5.0, (c) about 0.8 mg / mL of polysorbate 80, and (d) about 58 mg / mL of sucrose.

[0130] In one embodiment, the pharmaceutical composition comprises (a) about 100 mg / mL of hu25G7-A antibody, (b) 50 mM histidine-acetate buffer having a pH of about 4.5, (c) about 0.4 mg / mL of polysorbate 80, and (d) about 58 mg / mL of sucrose.

[0131] In one embodiment, the pharmaceutical composition comprises (a) about 140 mg / mL of hu25G7-A antibody, (b) 50 mM histidine-acetate buffer having a pH of about 4.5, (c) about 0.8 mg / mL of polysorbate 80, and (d) about 58 mg / mL of sucrose.

[0132] In one embodiment, the pharmaceutical composition comprises (a) about 120 mg / mL of hu25G7-A antibody, (b) 50 mM histidine-acetate buffer having a pH of about 4.5, (c) about 1.2 mg / mL of polysorbate 80, and (d) about 58 mg / mL of sucrose.

[0133] In one embodiment, the pharmaceutical composition comprises (a) about 100 mg / mL of hu25G7-A antibody, (b) 50 mM histidine-acetate buffer having a pH of about 5, (c) about 1.2 mg / mL of polysorbate 80, and (d) about 58 mg / mL of sucrose.

[0134] In one embodiment, the pharmaceutical composition comprises (a) about 100 mg / mL of hu25G7-A antibody, (b) 50 mM histidine-acetate buffer having a pH of about 5.5, (c) about 0.4 mg / mL of polysorbate 80, and (d) about 58 mg / mL of sucrose.

[0135] In one embodiment, the pharmaceutical composition comprises (a) about 120 mg / mL of hu25G7-A antibody, (b) 50 mM histidine-acetate buffer having a pH of about 5.5, (c) about 1.2 mg / mL of polysorbate 80, and (d) about 58 mg / mL of sucrose.

[0136] In one embodiment, the pharmaceutical composition comprises: (a) about 120 mg / mL of hu25G7-A antibody; (b) 50 mM histidine-acetate buffer having a pH of about 5; (c) about 0.8 mg / mL of polysorbate 80; and (d) about 58 mg / mL of sucrose.

[0137] The present disclosure further provides a method for preparing the above pharmaceutical composition, comprising the step of replacing a stock solution of an anti-IL-4R antibody or antigen-binding fragment thereof with a buffer.

[0138] The present disclosure further provides a lyophilized formulation comprising an anti-IL-4R antibody or an antigen-binding fragment thereof, which is obtainable by lyophilizing the pharmaceutical composition described above.

[0139] The present disclosure further provides a lyophilized preparation comprising an anti-IL-4R antibody or an antigen-binding fragment thereof, which is obtained by diluting the above-mentioned pharmaceutical composition and then lyophilizing it.

[0140] The present disclosure further provides a lyophilized formulation comprising an anti-IL-4R antibody or an antigen-binding fragment thereof, which is obtained by diluting the above-mentioned pharmaceutical composition 1-fold, 2-fold, or 3-fold and then lyophilizing it.

[0141] The present disclosure further provides a reconstituted solution containing an anti-IL-4R antibody or an antigen-binding fragment thereof, which is prepared by reconstituting the above-mentioned lyophilized formulation.

[0142] In some embodiments, the reconstitution solution comprises: (a) 120 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 50 mM histidine-acetate buffer having a pH of about 5.0, (c) 0.4 mg / mL of polysorbate 80, and (e) 50 mg / mL of sucrose.

[0143] In one embodiment, the reconstitution solution comprises (a) about 120 mg / mL of hu25G7-A antibody, (b) about 50 mM histidine-acetate buffer, (c) about 0.8 mg / mL of polysorbate 80, and (d) about 58 mg / mL of sucrose, and the pH of the pharmaceutical composition is about 5.3.

[0144] In one embodiment, the reconstitution solution comprises (a) about 150 mg / mL of hu25G7-A antibody, (b) about 20 mM histidine-acetate buffer, (c) about 0.8 mg / mL of polysorbate 80, and (d) about 120 mM arginine hydrochloride, and the pH of the pharmaceutical composition is about 5.3.

[0145] The present disclosure further provides a product comprising a container containing the pharmaceutical composition, lyophilized formulation, or reconstitution solution.

[0146] The present disclosure further provides a method for treating or preventing an immune disease or condition, comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition, lyophilized formulation, or reconstituted solution, wherein preferably the immune disease is an IL-4R-mediated disease or condition.

[0147] In some embodiments, the immune-mediated disease or condition is selected from asthma, nasal polyps, chronic sinusitis, allergic skin disease, eosinophilic esophagitis, chronic obstructive pulmonary disease, allergic rhinitis, arthritis, inflammatory diseases, allergic reactions, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, and kidney disease; preferably, the disease or condition is asthma or allergic skin disease.

[0148] In some embodiments, the immune-mediated disease or condition is asthma.

[0149] In another embodiment, the immune disease or condition is an allergic skin disease.

[0150] The present disclosure further provides use of the above-mentioned pharmaceutical composition, or lyophilized formulation, or reconstituted solution of the lyophilized formulation, or product in the preparation of a medicament for treating or preventing an immune disease or condition, preferably wherein the immune disease or condition is an IL-4R-mediated disease or condition.

[0151] The pharmaceutical composition, freeze-dried formulation, or reconstituted solution of the freeze-dried formulation, or product according to the present disclosure can be used as a medicament, preferably as a medicament for treating or preventing an immune disease or condition, more preferably as a medicament for treating an IL-4R-mediated disease or condition.

[0152] The pharmaceutical composition, or freeze-dried formulation, or reconstituted solution of the freeze-dried formulation, or product of the present disclosure can be used as a medicament, and is preferably used as a medicament for treating or preventing an immune disease or condition, more preferably the immune disease or condition is selected from asthma, nasal polyps, chronic sinusitis, allergic skin disease, eosinophilic esophagitis, chronic obstructive pulmonary disease, allergic rhinitis, arthritis, inflammatory disease, allergic reaction, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, and kidney disease, and preferably the disease or condition is asthma or allergic skin disease.

[0153] In the present application, histidine can be used as a buffer and also has the effect of a viscosity adjuster, so the final histidine content in a pharmaceutical composition is the sum of the histidine content in the buffer and the content of histidine added to reduce viscosity. For example, if 90 mM of histidine is further added to a 20 mM histidine-acetate buffer having a pH of 5.0 to improve its viscosity-reducing effect, the final histidine concentration in the pharmaceutical composition will be 110 mM, and the pH of the pharmaceutical composition will be approximately 5.0. [Brief explanation of the drawings]

[0154] [Figure 1] This figure shows experimental results demonstrating the effect of anti-IL-4R antibodies on mouse dermatitis. After acetone sensitization in a mouse dermatitis model, humanized antibodies hu25G7-A, hu25G7-B, and the positive control antibody dupilumab were administered subcutaneously twice weekly, and the ear thickness of the mice was measured on day 27. The results clearly show that, compared with the control group, hu25G7-A, hu25G7-B, and dupilumab all reduced the ear thickness of the mice, with hu25G7-B demonstrating a more potent effect than dupilumab. [Figure 2] This is the fitting result of the formulation. [Figure 3] 1 is a contour plot of the change in formulation stability, in which the grey area indicates that the limit has been exceeded and the white area indicates that the limit has not been exceeded. DETAILED DESCRIPTION OF THE INVENTION

[0155] term In order that the present disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless expressly defined elsewhere herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0156] The present disclosure incorporates the entire contents of application PCT / CN2019 / 102169 into this application.

[0157] "Buffer" refers to a buffer that can withstand changes in pH due to the action of its acid-base conjugate components. Examples of buffers that control pH within an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0158] A "histidine salt buffer" is a buffer containing histidine ions. Examples of histidine salt buffers include histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate, etc., and preferably histidine acetate buffer, which is prepared from histidine and acetic acid and is also called histidine-acetic acid (His-AA) buffer.

[0159] A "citrate buffer" is a buffer containing citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate buffers, etc. A preferred citrate buffer is citric acid-sodium citrate buffer.

[0160] A "succinate buffer" is a buffer containing succinate ions. Examples of succinate buffers include succinic acid-sodium succinate, succinic acid-potassium succinate, succinic acid-calcium succinate buffers, etc. A preferred succinate buffer is succinic acid-sodium succinate buffer.

[0161] A "phosphate buffer" is a buffer containing phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citrate buffer, etc. A preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

[0162] An "acetate buffer" is a buffer containing acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, acetic acid histidine, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate buffers, etc. A preferred acetate buffer is acetic acid-sodium acetate buffer.

[0163] A "pharmaceutical composition" refers to a mixture containing one or more antibodies or antigen-binding fragments thereof described herein and other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The pharmaceutical composition is intended to maintain the stability of the antibody active ingredient, facilitate administration to the body, and promote absorption of the active ingredient, thereby enabling it to exert its biological activity.

[0164] As used herein, the terms "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0165] Unless otherwise specified, the solution form of the pharmaceutical composition described in the present disclosure is an aqueous solution.

[0166] "Replacement" refers to the replacement of a solvent system in which an antibody protein is dissolved, such as by physically replacing a high-salt or hypertonic solvent system containing the antibody protein with the buffer system of a stable formulation, resulting in the antibody protein being present in a stable formulation. Such physical manipulations include, but are not limited to, ultrafiltration, dialysis, or centrifugation followed by reconstitution.

[0167] "Lyophilized formulation" refers to a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after the process of vacuum freeze-drying a liquid or solution formulation.

[0168] "Sugars" of the present disclosure are of general composition (CHO) including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. nand derivatives thereof, which may be selected from glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerin, arabinitol, xylitol, sorbitol, mannitol, melibiose, melezitose, raffinose, manninotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, isomaltulose, etc. Preferred sugars are non-reducing disaccharides, more preferably trehalose or sucrose, and most preferably sucrose.

[0169] The surfactant of the present disclosure may be selected from polysorbate 20, polysorbate 80, poloxamer, Triton, sodium dodecyl sulfonate, sodium lauryl sulfonate, sodium octyl glycoside, lauryl / myristyl / linoleyl / stearyl-sulfobetaine, lauryl / myristyl / linoleyl / stearyl-sarcosine, linoleyl / myristyl / cetyl-betaine, lauramidopropyl / cocamidopropyl / linoleylamidopropyl / myristoylamidopropyl / palmitoylamidopropyl / isostearylamidopropyl-betaine, myristoylamidopropyl / palmitoylamidopropyl / isostearylamidopropyl-dimethylamine, sodium methyl cocoyl, sodium methyl oleyl taurate, polyethylene glycol, polypropylene glycol, copolymers of ethylene and propylene glycol, etc. Preferred surfactants are polysorbate 80 or polysorbate 20, more preferably polysorbate 80.

[0170] The term "viscosity" may be "kinematic viscosity" or "absolute viscosity." "Kinematic viscosity" is a metric of a fluid's resistance to flow under the influence of gravity. When equal volumes of two fluids are placed in the same capillary viscometer and allowed to flow by gravity, the more viscous fluid will take longer to flow through the capillary than the less viscous fluid. For example, if one fluid requires 200 seconds to complete its flow and the other requires 400 seconds, the latter fluid has a kinematic viscosity metric that is twice as long as the first. "Absolute viscosity," sometimes called dynamic or simple viscosity, is the product of kinematic viscosity and fluid density (absolute viscosity = kinematic viscosity × density). The units of kinematic viscosity are L 2 / T, where L is length and T is time. Kinematic viscosity is usually expressed in centistokes (cSt). The SI unit of kinematic viscosity is mm 2 / s, which is 1 cSt. Absolute viscosity is expressed in centipoise (cP). The SI unit of absolute viscosity is millipascal-second (mPa·s), of which 1 cP = 1 mPa·s.

[0171] As used herein, the terms "about" and "approximately" refer to a numerical value that is within an acceptable error range of a specific value as determined by one of ordinary skill in the art, where the numerical value depends on how it is measured or determined (i.e., the limitations of the measurement system). For example, in the respective practice of the art, "about" may mean within or more than 1 standard deviation. Alternatively, "about" or "substantially comprising" may mean a range of ±20%, ±15%, ±10%, or ±5% of the specific numerical value thereafter. However, particularly for biological systems or processes, the term may mean at most one order of magnitude or up to five times the numerical value. Unless otherwise indicated, when specific values ​​appear in the present application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range of the specific value.

[0172] The pharmaceutical compositions described herein can achieve a stable effect in that the antibodies therein substantially retain their physical stability, chemical stability, and / or biological activity after storage. Preferably, the pharmaceutical composition substantially retains its physical and chemical stability and biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, various analytical techniques are available to measure protein stability after storage at a selected temperature for a selected period of time.

[0173] A stable drug-antibody formulation is one that shows no significant change when stored at refrigerated temperatures (2-8°C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably 2 years. A stable liquid formulation includes a liquid formulation that exhibits desired characteristics after storage at temperatures including 25°C for periods including 1 month, 3 months, and 6 months. A typical acceptable standard for stability is that the antibody monomer decomposes, as measured by SEC-HPLC, typically no more than about 10%, preferably no more than about 5%. Visual analysis reveals that the drug-antibody formulation is a pale yellow, almost colorless, transparent liquid or colorless, or clear to slightly opalescent. The concentration, pH, and osmolality of the formulation vary by no more than ±10%. A decrease of no more than about 10%, preferably no more than about 5%, is typically observed. Aggregation typically forms at no more than about 10%, preferably no more than about 5%.

[0174] An antibody "retains its physical stability" in a drug formulation if it does not exhibit significant increased aggregation, precipitation, and / or denaturation after visual inspection of color and / or clarity, or as measured by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines protein tertiary structure) and FTIR spectroscopy (which determines protein secondary structure).

[0175] If the antibody does not undergo significant chemical changes, the antibody "retains its chemical stability" in the drug formulation. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that constantly alter the chemical structure of proteins include hydrolysis or truncation (assessed by methods such as size-exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide mapping coupled with mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion-exchange chromatography, capillary isoelectric focusing, peptide mapping, and measurement of isoaspartic acid), and isomerization (assessed by measurement of isoaspartic acid content, peptide mapping, etc.).

[0176] An antibody "retains its biological activity" in a drug formulation if the antibody's biological activity over a given period of time is within a given range of the biological activity exhibited at the time of preparation of the drug formulation. Antibody biological activity can be determined, for example, by antigen binding assays.

[0177] "Human IL-4R" (hIL-4R) refers to a human cytokine receptor that specifically binds to interleukin-4 (IL-4), IL-4Rα.

[0178] The three-letter and one-letter codes for amino acids used herein are as described in J. Biol. Chem, 243, p. 3558 (1968).

[0179] The term "antibody (Ab)" includes any antigen-binding molecule or molecular complex containing at least one complementarity-determining region (CDR) that specifically binds to or interacts with a specific antigen (or epitope thereof, e.g., IL-4R antigen or epitope thereof). The term "antibody" includes immunoglobulin molecules and multimers thereof (e.g., IgM) that contain four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region (CH). This heavy chain constant region contains three regions (domains): CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region (CL). The light chain constant region contains one region (domain, CL). The VH and VL regions can be further divided into hypervariable regions called complementarity-determining regions (CDRs) and relatively conserved regions called framework regions (FRs) scattered between the hypervariable regions. Each VH and VL region is composed of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxy terminus.

[0180] The FRs of an anti-IL-4R antibody (or antigen-binding fragment thereof) may be identical to the human germline sequence or may be naturally or artificially modified. The antibody may be of a different subclass, for example, an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subclass), an IgA1, an IgA2, an IgD, an IgE, or an IgM antibody.

[0181] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed by the term "antigen-binding fragment" as used herein. Antigen-binding fragments of antibodies typically contain at least one variable region. The variable region may be any size or amino acid region and generally contains CDRs adjacent to or within one or more framework sequences. In antigen-binding fragments containing a VH region and a VL region, the VH and VL regions may be positioned opposite each other in any suitable arrangement. For example, the variable region may be a dimer, comprising a VH-VL or VL-VH dimer.

[0182] In some embodiments, in any variable and constant region configuration, the antigen-binding fragment may have variable and constant regions directly connected to each other or connected by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, such that a flexible or semi-flexible linkage is generated between adjacent variable and / or constant regions in a single polypeptide molecule. Additionally, antigen-binding fragments of the present disclosure may include homodimers or heterodimers (or other multimers) having variable and constant regions non-covalently linked to each other and / or linked (e.g., by disulfide bonds) to one or more monomeric VH or VL regions.

[0183] In the present disclosure, a "mouse antibody" refers to a monoclonal antibody derived from a mouse or rat, prepared using knowledge and techniques in this field. During preparation, an antigen is injected into a test subject, and then hybridomas expressing antibodies with the desired sequence or functional properties are isolated. When the test subject injected is a mouse, the antibody produced is a mouse-derived antibody, and when the test subject injected is a rat, the antibody produced is a rat-derived antibody.

[0184] A "chimeric antibody" is an antibody in which the variable region of an antibody of a first species (e.g., mouse) is fused with the constant region of an antibody of a second species (e.g., human). To produce a chimeric antibody, first, hybridomas secreting a monoclonal antibody of the first species are prepared, and the variable region genes are cloned from the hybridoma cells. If necessary, the constant region genes of an antibody of a second species are also cloned. The variable region genes of the first species and the constant region genes of the second species are ligated into a chimeric gene, which is then inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system. In one preferred embodiment of the present disclosure, the antibody light chain of the chimeric antibody further comprises a light chain constant region of a human κ or λ chain or a variant thereof. The antibody heavy chain of the chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, or a variant thereof, and preferably comprises a heavy chain constant region of human IgG1, IgG2, or IgG4, or a variant of the heavy chain constant region of IgG1, IgG2, or IgG4 using amino acid mutations (e.g., YTE mutation, back mutation, L234A and / or L235A mutation, or S228P mutation).

[0185] The term "humanized antibody," including CDR-grafted antibodies, refers to antibodies produced by grafting CDR sequences from an animal-derived antibody, such as a mouse antibody, onto the framework region of a human antibody variable region. Humanized antibodies can overcome the heterologous reactions induced by chimeric antibodies carrying large amounts of heterologous proteins. Such framework sequences can be obtained from consensus DNA databases containing germline antibody gene sequences or from published references. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at http: / / www.vbase2.org / ) and Kabat, EA et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. To avoid reduced activity due to reduced immunogenicity, minor backmutations of the framework sequences of the human antibody variable region can be used to maintain activity. The humanized antibodies of the present disclosure also include humanized antibodies that have been affinity-matured against their CDRs by phage display.

[0186] Due to antigen contact residues, CDR grafting can reduce the affinity of the resulting antibody or its antigen-binding fragment to the antigen due to framework residues that contact the antigen. Such interactions may be the result of extensive somatic mutation. Therefore, it may still be necessary to graft such donor framework amino acids into the framework of a humanized antibody. Amino acid residues involved in antigen binding from non-human antibodies or their antigen-binding fragments can be identified by examining the sequence and structure of animal monoclonal antibody variable regions. Each residue in the CDR donor framework that differs from the germline is considered relevant. If the closest germline cannot be determined, the sequence can be compared to a subclass consensus sequence or a consensus sequence of animal antibody sequences with a high percentage of similarity. Rare framework residues may be the result of extensive somatic mutation and therefore play an important role in binding.

[0187] In one embodiment of the present disclosure, the antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a human or mouse κ or λ chain or a variant thereof, or may further comprise a heavy chain constant region of a human or mouse IgG1, IgG2, IgG3, IgG4 or a variant thereof.

[0188] "Conventional variants" of human antibody heavy chain constant regions and human antibody light chain constant regions refer to variants of human-derived heavy chain constant regions that do not change the structure and function of the antibody variable regions disclosed in the prior art. Exemplary variants include variants of IgG1, IgG2, IgG3, or IgG4 heavy chain constant regions in which the heavy chain constant region has been modified at specific points and subjected to amino acid substitutions. Specific substitutions include, for example, the YTE mutation, the L234A and / or L235A mutation, or the S228P mutation known in the prior art, or a mutation resulting in a knob-into-hole structure (so that the antibody heavy chain has a combination of knob-Fc and hole-Fc). These mutations have been confirmed to confer new properties to antibodies without changing the function of the antibody variable region.

[0189] The terms "human antibody" and "humanized antibody" are used interchangeably and may refer to antibodies of human origin or antibodies obtained from transgenic organisms that have been "engineered" to produce specific human antibodies in response to antigenic stimulation, and can be produced by any method known in the art. In some techniques, elements of human heavy and light chain loci are introduced into cell lines, and endogenous heavy and light chain loci in these cell lines are targetedly disrupted. Transgenic organisms are capable of synthesizing human antibodies specific to human antigens, and such organisms can be used to produce human antibody-secreting hybridomas. A human antibody may be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from the DNA of one or more humans. Fully human antibodies may be constructed by genetic or chromosomal transfection methods and phage display technology, or by in vitro activated B cells, all of which are known in the art.

[0190] A "monoclonal antibody" refers to an antibody obtained from a group of substantially homogeneous antibodies; i.e., except for possible variant antibodies (e.g., including naturally occurring mutations or mutations that arise during the preparation of a monoclonal antibody preparation, which are typically present in minor amounts), the individual antibodies comprising the group recognize and / or bind to the same epitope. Each monoclonal antibody of a monoclonal antibody preparation (preparation) is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a group of substantially homogeneous antibodies and should not be construed as requiring the antibody to be prepared by any particular method. For example, monoclonal antibodies used in accordance with the present disclosure can be prepared by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or portions of the human immunoglobulin loci; such methods, as well as other exemplary methods for preparing monoclonal antibodies, are described herein.

[0191] Although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker using recombinant techniques to produce a single protein chain (called a 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) that pairs with the VL and VH domains to form a monovalent molecule. Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. Such antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0192] The antigen-binding fragment may be assembled into a single-chain molecule comprising a pair of tandem Fv fragments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen-binding regions (Zapata et al., 1995 Protein Eng. 8(10): 1057-1062, and U.S. Pat. No. 5,641,870).

[0193] Fab is an antibody fragment with a molecular weight of approximately 50,000 Da that is obtained by treating an IgG antibody with the protease papain (which cleaves the 224th amino acid residue of the H chain) and has antigen-binding activity. Approximately half of the N-terminal H chain is connected to the entire L chain via a disulfide bond.

[0194] F(ab')2 is an antibody fragment containing two Fab regions linked at the hinge position, with a molecular weight of approximately 100,000 Da, and possessing antigen-binding activity, obtained by digesting the lower portion of the two disulfide bonds in the hinge region of IgG with pepsin.

[0195] Fab' is an antibody fragment with a molecular weight of approximately 50,000 Da that has antigen-binding activity and is obtained by cleaving the disulfide bond in the hinge region of the F(ab')2. Fab' can be produced by treating F(ab')2, which specifically recognizes and binds to an antigen, with a reducing agent such as dithiothreitol.

[0196] Furthermore, Fab' can be expressed by inserting DNA encoding the Fab' fragment of an antibody into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote.

[0197] The terms "single-chain antibody," "single-chain Fv," or "scFv" are intended to include molecules of an antibody heavy chain variable domain (or region, VH) and an antibody light chain variable domain (or region, VL) connected by a linker. Such scFv molecules can have the general formula NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, using, for example, variants of one to four (including one, two, three, or four) repeats (Holliger et al. (1993), Proc Natl Acad Sci USA. 90: 6444-6448). Other linkers that can be used in the present disclosure are described in Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur JI mmuno. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J Mol Biol. 293: 41-56, and Roovers et al. (2001), Cancer I mmunol I mmunother. 50: 51-59.

[0198] A diabody is an antibody fragment formed by dimerization of scFvs and has bivalent antigen-binding activity, in which the two antigens may be homologous or different.

[0199] A dsFv can be obtained by connecting polypeptides in which one amino acid residue in each of the VH and VL domains is substituted with a cysteine ​​residue via a disulfide bond between the cysteine ​​residues. The amino acid residue to be substituted with a cysteine ​​residue can be selected based on prediction of the three-dimensional structure of an antibody by a known method (Protein Engineering, 7: 697 (1994)).

[0200] In some embodiments of the present disclosure, antigen-binding fragments can be produced by the steps of obtaining cDNA encoding the VH and / or VL and any other desired domains of a monoclonal antibody that specifically recognizes and binds to an antigen of the present disclosure, constructing DNA encoding the antigen-binding fragment, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into a prokaryote or eukaryote to express the antigen-binding fragment.

[0201] The "Fc region" may be a native-sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxy-terminus thereof. The numbering of residues in the Fc region is, for example, that of the EU index in Kabat. Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin usually has two constant region domains, CH2 and CH3. The term "amino acid difference" or "amino acid mutation" means that a variant protein or polypeptide has an amino acid change or mutation compared to an original protein or polypeptide, including an insertion, deletion, or substitution of one or more amino acids based on the original protein or polypeptide.

[0202] The "variable region" of an antibody refers to the variable region of the antibody light chain (VL) or the variable region of the antibody heavy chain (VH), either alone or in combination. As is known in the art, the heavy and light chain variable regions each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs) (also called hypervariable regions). The CDRs in each chain are tightly held together by the FRs and, together with the CDRs from another chain, promote the formation of the antibody antigen-binding site. There are at least two techniques for determining CDRs: (1) a method based on interspecies sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th edition, 1991, National Institutes of Health, Bethesda, MD)), and (2) a method based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al., J. Molec. Biol. 273:927-948 (1997)). As used herein, CDRs can refer to CDRs determined by any one method or a combination of two methods. The term "antibody framework" or "FR region" refers to the part of a variable domain VL or VH that serves as a support for the antigen binding rings (CDRs) of that variable domain. Essentially, it is a variable domain without the CDRs.

[0203] The terms "complementarity-determining region" and "CDR" refer to one of the six hypervariable regions in an antibody variable domain that primarily mediates antigen binding. Typically, each heavy chain variable region has three CDRs (HCDR1, HCDR2, and HCDR3), and each light chain variable region has three CDRs (LCDR1, LCDR2, and LCDR3). The amino acid sequence boundaries of CDRs can be determined by any one of a variety of known methods, including the "Kabat" numbering convention (Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention (Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains[J]. 2001), and the ImMunoGenTics (IMGT) numbering convention (see Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77(2003)). For example, in a typical format, according to the Kabat rules, the CDR amino acid residues in the heavy chain variable region (VH) are numbered 31 to 35 (HCDR1), 50 to 65 (HCDR2), and 95 to 102 (HCDR3), and the CDR amino acid residues in the light chain variable region (VL) are numbered 24 to 34 (LCDR1), 50 to 56 (LCDR2), and 89 to 97 (LCDR3). According to the Chothia rules, the CDR amino acid residues in the VH are numbered 26 to 32 (HCDR1), 52 to 56 (HCDR2), and 95 to 102 (HCDR3), and the amino acid residues in the VL are numbered 24 to 34 (LCDR1), 50 to 56 (LCDR2), and 89 to 97 (LCDR3).According to the combined Kabat and Chothia CDR definition, the CDRs are composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. According to the IMGT rules, the CDR amino acid residue numbers in VH are approximately 27-38 (CDR1), 56-65 (CDR2), and 105-117 (CDR3), and those in VL are approximately 27-38 (CDR1), 56-65 (CDR2), and 105-117 (CDR3). According to the IMGT rules, the CDR regions of an antibody can be determined using the program IMGT / DomainGapAlign.

[0204] "Antibody constant region domain" refers to domains from the constant regions of the light and heavy chains of an antibody, including CL and CH1, CH2, CH3 and CH4 domains from antibodies of different classes.

[0205] "Epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope usually includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique spatial conformation. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

[0206] The terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen.

[0207] The term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single epitope. At each antigen site, the variable region of an antibody "arm" interacts with the antigen at multiple amino acid sites through weak non-covalent forces; the greater the interaction, the stronger the affinity. As used herein, the term "high affinity" for an antibody or antigen-binding fragment thereof (e.g., a Fab fragment) generally refers to an antibody with high affinity at a single epitope. -9 KD below M (e.g., 1E -10 KD below M, 1E -11 KD below M, 1E -12 KD below M, 1E -13 KD below M, 1E -14 K below M D The term "antibody" refers to an antibody or antigen-binding fragment having a nucleotide sequence such as nucleotides.

[0208] The term "KD" or "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, antibodies have a dissociation equilibrium constant of less than about 1E-8 M, e.g., about 1E -9 M, 1E -10 M or 1E -11 It binds to the antigen with a dissociation equilibrium constant (KD) smaller than or equal to M, as measured, for example, in a BIACORE instrument using surface plasmon resonance (SPR) technology. The smaller the KD value, the greater the affinity.

[0209] The term "nucleic acid molecule" refers to a DNA molecule or an RNA molecule. A nucleic acid molecule may be a single-stranded or double-stranded DNA or RNA molecule, e.g., double-stranded DNA or mRNA. A nucleic acid is "operatively linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operatively linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.

[0210] The term "vector" refers to a construct that can deliver and suitably express one or more target genes or sequences in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, or phage vectors, DNA or RNA expression vectors associated with cationic flocculants, DNA or RNA expression vectors encapsulated in liposomes, and some eukaryotic cells, such as producer cells.

[0211] Methods for producing and purifying antibodies and antigen-binding fragments well known in the art are described, for example, in Chapters 5-8 and 15 of Reisenko's Antibody Laboratory Techniques Manual. For example, mice can be immunized with an antigen or its fragment, and the resulting antibodies can be renatured, purified, and amino acid sequenced using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described herein incorporate one or more human-derived FR regions into non-human CDR regions via genetic engineering techniques. Human FR germline sequences can be obtained by aligning the IMGT human antibody variable region germline gene database and software MOE, or from the Immunoglobulin Journal, 2001, ISBN 012441351.

[0212] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant, or animal cells. Bacteria amenable to transformation include members of the Enterobacteriaceae family, such as strains of Escherichia coli and Salmonella, Bacillaceae, such as Bacillus subtilis, Pneumococcus, Streptococcus, and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line), HEK293 cells (non-limiting examples include HEK293E cells), and NS0 cells.

[0213] Engineered antibodies or antigen-binding fragments can be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As an alternative, mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminal site of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to antigens. Positive clones are expanded in serum-free medium in bioreactors to produce antibodies. The culture medium secreting the antibodies can be purified by conventional techniques, for example, through a protein A or protein G Sepharose FF column containing a regulated buffer. Nonspecifically bound components are washed away. The bound antibodies are then eluted using a pH gradient, and the antibody fragments are detected and collected by SDS-PAGE. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and multimers may be removed by conventional methods, such as molecular sieving or ion exchange. The resulting product must be immediately frozen, such as at -70°C, or lyophilized.

[0214] "Giving," "administration," "delivering," and "treating," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to providing an exogenous agent, therapeutic agent, diagnostic agent, composition, or artificial manipulation (e.g., "euthanasia" in the examples) to contact an animal, human, subject, cell, tissue, organ, or biological fluid. "Delivering" and "treating" can refer, for example, to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes contact of reagents with cells and contact of reagents with fluids, where the fluid contacts the cells. "Delivering" and "treating" also refer to ex vivo and in vitro treatment, e.g., of cells, with a reagent, diagnostic, binding composition, or through another cell. When applied to a human, veterinary, or research subject, "treatment" refers to therapeutic treatment, preventative or prophylactic measures, and research and diagnostic applications.

[0215] "Treatment" refers to the administration of an internal or external therapeutic agent, including, for example, a composition of any one of the disclosed embodiments, to a patient (or subject) who has (or is suspected of having, or is susceptible to) one or more disease symptoms, and for whom the therapeutic agent is known to have a therapeutic effect against those symptoms. Typically, the patient (or subject) or group being treated is administered an amount of therapeutic agent that effectively alleviates one or more disease symptoms, thereby inducing the resolution of those symptoms or inhibiting their progression to any clinically measurable extent. The amount of therapeutic agent that effectively alleviates any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on various factors, such as the patient's (or subject's) disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Alleviation of disease symptoms can be assessed by any clinical detection method commonly used by physicians or other professional health care providers to assess the severity or progression of the condition. Although an embodiment (e.g., a method of treatment or product) of the present disclosure may not effectively alleviate all of the respective target disease symptoms, it should alleviate the target disease symptoms in a statistically significant number of patients (or subjects) as determined by any statistical testing method known in the art, such as Student's t-test, chi-square test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0216] "Conservative amino acid modification" or "conservative amino acid substitution" means that an amino acid in a protein or polypeptide can be routinely substituted with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main-chain conformation, and rigidity) without altering the biological activity or other desired properties (e.g., antigen affinity and / or specificity) of the protein or polypeptide. Those skilled in the art will appreciate that single amino acid substitutions in non-essential regions of a polypeptide typically do not substantially alter biological activity (see, e.g., Watson et al., (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th edition)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to destroy biological activity.

[0217] "Binding to IL-4R" means being able to interact with human IL-4R. The term "antigen-binding site" herein refers to the three-dimensional spatial site recognized by an antibody or antigen-binding fragment herein.

[0218] "Cross-reactivity" refers to the ability of an antibody herein to bind to IL-4R from a different species. For example, an antibody herein that binds to human IL-4R may also bind to IL-4R from another species. Cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays (e.g., SPR and ELISA) or binding or functional interaction with cells that physiologically express IL-4R. Methods for determining cross-reactivity include standard binding assays described herein, such as, for example, surface plasmon resonance (SPR) analysis or flow cytometry.

[0219] A "neutralizing" or "inhibitory" antibody refers to an antibody that inhibits the biological activity of hIL-4 and / or hIL-13 upon its binding to hIL-4R. Such inhibition of the biological activity of hIL-4 and / or IL-13 can be assessed by one or more indicators of hIL-4 and / or hIL-13 biological activity known in the art, such as hIL-4 and / or hIL-13-induced cell activation and hIL-4 binding to hIL-4R; see, for example, the description in CN103739711A. "Inhibition of proliferation" (e.g., with respect to cells) is intended to include any measurable decrease in cell proliferation.

[0220] The terms "induction of an immune response" and "enhancement of an immune response" may be used interchangeably and refer to the stimulation of an immune response (i.e., passive or adaptive) to a specific antigen. With respect to the term induction of CDC or ADCC, "induction" refers to the stimulation of a specific direct cell-killing mechanism.

[0221] "ADCC (antibody-dependent cell-mediated cytotoxicity)" refers to the direct killing of antibody-coated target cells by cells expressing Fc receptors through recognition of the Fc portion of the antibody. The ADCC effector function of an antibody can be reduced or eliminated by modifying the Fc portion of an IgG. The modification refers to a mutation in the heavy chain constant region of the antibody, and is selected from, for example, N297A, L234A, L235A in IgG1, IgG2 / 4 chimera, F235E in IgG4, or the L234A / E235A mutation.

[0222] Engineered antibodies or antigen-binding fragments can be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. The humanized antibody sequences described herein can be inserted into corresponding expression vectors using molecular cloning techniques and then expressed in a HEK293 cell expression system to obtain the corresponding humanized antibody. As a preferred conventional technique, mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminal site of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are cultured in serum-free medium in a bioreactor to produce antibodies. The culture medium secreting the antibodies can be purified and collected by conventional techniques. The antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and multimers can be removed by conventional methods, such as molecular sieving and ion exchange. The resulting product must be immediately frozen, such as at -70°C, or lyophilized.

[0223] An "effective amount" or "effective dosage" refers to the amount of a drug, compound, or pharmaceutical composition required to achieve any one or more beneficial or desired therapeutic results. For prophylactic uses, beneficial or desired results include eliminating or reducing the risk, reducing the severity, or delaying the onset of a disease, including the disease, its complications, and the biochemical, histological, and / or behavioral manifestations of intermediate pathological phenotypes manifested during the progression of the disease. For therapeutic applications, beneficial or desired results include clinical results such as reducing the incidence of or ameliorating one or more symptoms of various target antigen-associated diseases of the present disclosure, reducing the dosage of other drugs required to treat the disease, improving the therapeutic efficacy of other drugs, and / or delaying the progression of a target antigen-associated disease of the present disclosure in a patient (or subject).

[0224] "Exogenous" means a substance that originates outside the organism, cell, or human body, as the case may be.

[0225] "Endogenous" means a substance that occurs within a cell, organism, or human, as the case may be.

[0226] "Isolated" refers to a purified state, in this case meaning that the designated molecule is substantially free from other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cellular debris, growth medium, etc. Generally, the term "isolated" is not intended to imply the total absence of these materials, or the absence of water, buffers, or salts, unless they are present in amounts that would clearly interfere with experimental or therapeutic uses of the compounds described herein.

[0227] "Homology" or "identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit, for example, when each position in two DNA molecules is occupied by adenine, the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences, divided by the number of positions compared, multiplied by 100%. For example, if the sequences are optimally aligned, and 6 of 10 positions in the two sequences are matched or homologous, the two sequences are 60% homologous. Typically, two sequences are compared when aligned to obtain the maximum percentage of homology. As used herein, "at least 85% sequence identity" means that when comparing a variant and a parent sequence, the two sequences have at least 85% sequence identity, and in some embodiments, at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity; in some specific embodiments, 90%, 95%, or 99% or more; and in other specific embodiments, at least 95% sequence identity. The amino acid sequence having at least 85% sequence identity includes those obtained by deleting, inserting, or substituting one or more amino acids from the parent sequence.

[0228] As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably, and all such designations include their progeny. Thus, the terms "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard for the number of transfers. It should also be understood that all progeny may not be precisely identical in DNA content, due to intentional or unintentional mutations. Mutant progeny that have the same function or biological activity as screened for from the originally transformed cell are included.

[0229] "Optionally" or "optionally" means that the subsequently described event or circumstance may occur, but is not necessarily so, and the phrase includes cases where the event or circumstance occurs and cases where it does not. For example, "optionally comprising 1 to 3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of a particular sequence may be present, but are not necessarily present.

[0230] An exemplary process for preparing an antibody pharmaceutical composition (formulation) is as follows: Step 1: The solvent of a certain amount of purified anti-IL-4R antibody solution is replaced (preferably by ultrafiltration) with antibody-free buffer, and the solution is passed through an ultrafiltration membrane at least six times its volume to concentrate the antibody to a certain concentration. A certain volume of the mother solution of other auxiliary materials is added and diluted with buffer to achieve the desired concentration of antibody and each auxiliary material, and then the mixture is mixed uniformly. After filtering the stock solution, it is sampled using the center console to test for sterility. The stock solution is passed through a 0.22 μm PVDF filter, and the filtrate is collected. Step 2: Adjust the loading volume to 2.15 mL, fill the filtrate into a 2 mL vial, stopper it, and sample it at the start, during, and end of filling to detect any difference in the loading volume using the center console. Step 3: Turn on the capping machine, apply the aluminum cap, and crimp the cap. Step 4: Visually inspect the product to ensure there are no defects such as incorrect filling. Print and attach labels to the vials, print labels for the cartons, fold the cartons, place the products in the cartons, and attach the carton labels. [Example]

[0231] The present disclosure will be further described below with reference to examples, but these examples do not limit the scope of the present disclosure. Experimental methods for which specific conditions are not specified in the examples generally follow standard conditions, such as those in the Reisenko Antibody Technology Experimental Manual and Molecular Cloning Manual, or conditions suggested by raw material or product manufacturers. Reagents for which specific sources are not specified are standard commercially available reagents.

[0232] Example of antibody preparation Example 1: Mouse immunization and detection His-tagged human IL-4R (h-IL-4R-his) recombinant protein, his-tagged mouse IL-4R (m-IL-4R-his) recombinant protein, and his-tagged rhesus IL-4R (rhesus-IL-4R-his) recombinant protein were synthesized by Acrobiosystems, expressed in HEK293 cells, and purified.

[0233] A human Fc-tagged human IL-4R (h-IL-4R-Fc) recombinant protein was designed, expressed, and purified, and the purified protein was applicable to the experiments in each of the following examples.

[0234] The numbers and positions of CDR amino acid residues in the VL and VH regions of the antibody or antigen-binding fragment of this example conform to the known Kabat numbering rules (LCDR1-3, HCDR2-3) and AbM rules (HCDR1).

[0235] [Table 1]

[0236] Anti-human IL-4R monoclonal antibodies were produced by immunizing mice. Experiments were performed using 6- to 8-week-old female C57BL / 6 mice (Suzhou Zhaoyan New Drug Research Center Co., Ltd., Animal Production License Number: 201503052).

[0237] Housing environment: SPF. After purchase, the mice were housed in a laboratory environment with a 12 / 12-hour light / dark cycle, a temperature of 20-25°C, and humidity of 40-60% for one week. After adapting to the environment, the mice were divided into three cages, with five mice per cage. The immunizing antigen was Fc-tagged human IL-4R recombinant protein (h-IL4R-Fc, concentration: 0.73 mg / mL). The antigen was emulsified with Freund's adjuvant (Sigma, Cat#: F5881): complete Freund's adjuvant (CFA, Pierce, Cat# 77140) was used for the first immunization, and nucleic acid adjuvant (CpG, Shanghai Zhonggong) and aluminum adjuvant (Alum, Thermo, Cat# 77161) were used for the remaining booster immunizations.

[0238] On day 0, mice were injected intraperitoneally (IP) with 70 μg of emulsified antigen. On days 14, 28, 42, 56, and 77, the dorsal and abdominal cavities were selected for antigen injection (0.1 mL each) based on the dorsal mass and abdominal swelling. Blood samples were collected on days 21, 35, 49, 63, and 84, and the mouse sera were analyzed using the ELISA method described in Example 2 to determine antibody titers in the mouse sera. After the fourth immunization, mice with high serum antibody titers that tended to plateau were selected for spleen cell fusion. Three days before fusion, the mice were boosted and intraperitoneally injected with 10 μg of antigen solution prepared in phosphate buffer solution. Hybridoma cells were obtained by fusing spleen lymphocytes with myeloma Sp2 / 0 cells (ATCC® CRL-8287™) using an optimized PEG-mediated fusion process.

[0239] Example 2: ELISA testing and screening of antibodies 1. ELISA binding experiment: ELISA experiments were used to detect the binding properties of anti-IL-4R antibodies. Using a microplate coated with His-tagged IL-4R recombinant protein, the antibody was added to each well, followed by the addition of a secondary antibody (an anti-primary Fc antibody coupled with HRP) and the HRP substrate TMB to detect the antibody's binding activity to the antigen.

[0240] A 96-well microplate was coated with human or rhesus monkey IL-4R-his protein at a concentration of 0.5 μg / mL, 100 μL per well, and incubated overnight at 4°C. After washing three times with washing buffer, 250 μL per well was added. 200 μL of blocking solution was added per well and incubated at room temperature for 2 hours. After washing three times with washing buffer, 250 μL per well was added. 100 μL of anti-IL-4R test antibody diluted with diluent was added per well and incubated at room temperature for 1 hour. After washing three times with washing buffer, 250 μL per well was added. 100 μL of HRP-conjugated goat anti-human IgG secondary antibody diluted 1:20,000 in diluent was added per well and incubated at room temperature for 1 hour. After washing three times with washing buffer, 250 μL per well was added. 100 μL of TMB was added per well and incubated in the dark for 15 minutes. 50 μL of 0.16 M / L sulfuric acid was added to each well. The OD value at 450 nm was read using a Thermo MultiSkanFc microplate reader, and the EC binding value of the anti-IL-4R antibody to IL-4R was calculated. 50 The value was calculated.

[0241] 2. ELISA inhibition experiment: In this experiment, we performed an in vitro inhibition experiment to detect the inhibition of human IL-4 binding to human IL-4R by the screened anti-human IL-4R antibodies. Specifically, a 96-well microplate was coated with Fc-tagged IL-4R recombinant protein, and an antibody that binds to human IL-4R was added to sufficiently bind to the epitope. IL-4 (Biolegend, Cat. #574004) was then added. The ability of IL-4 to bind to IL-4R was then detected using a biotin-coupled anti-IL-4 antibody and Neutravidin-HRP (Pierce, Cat. #31001). The IC value of the IL-4R antibody's inhibition of IL-4 / IL-4R binding was then calculated. 50 The value was calculated.

[0242] A 96-well microplate was coated with human IL-4R-Fc protein at a concentration of 0.5 μg / mL, 100 μL per well, and incubated overnight at 4°C. The plate was washed three times with washing buffer, followed by 250 μL per well. 200 μL of blocking solution was added and incubated at room temperature for 2 hours. The plate was washed three times with washing buffer, followed by 250 μL per well. 100 μL of anti-IL-4R test antibody diluted in diluent was added per well and incubated at room temperature for 1 hour. The plate was washed three times with washing buffer, followed by 250 μL per well. 100 μL of diluted IL-4 was added per well and incubated at room temperature for 1 hour. The plate was washed three times with washing buffer, followed by 250 μL per well. 100 μL of diluted biotin-coupled anti-IL-4 antibody was added per well and incubated at room temperature for 1 hour. The plate was washed three times with washing buffer. HRP-labeled Neutravidin diluted 1:5000 with diluent was added and incubated at room temperature for 1 hour. After washing three times with washing buffer, 250 μL of the solution was added per well. 100 μL of TMB was added per well and incubated in the dark for 15 minutes. 50 μL of 0.16 M / L sulfuric acid was added per well. OD values ​​at 450 nm were read using a Thermo MultiSkanFc microplate reader, and the IC value of inhibition of IL-4 binding to IL-4R by IL-4R antibodies was calculated. 50 The value was calculated.

[0243] Example 3: Reporter gene cellular activity experiments of antibodies that bind to human IL-4R HEK-Blue IL-4 cells were purchased from Invivogen (Cat#hkb-stat6) and stably transfected with the human IL-4R gene and the STAT6-mediated SEAP genome. The activation level of the IL-4R signaling pathway can be characterized by detecting SEAP secreted into the supernatant using the SEAP substrate QUANTI-Blue.

[0244] This experiment was conducted to detect the activation of HEK-Blue cells by IL-4. 50 The in vitro cellular activity of the IL-4R antibody was evaluated based on the size of the antibody.

[0245] HEK-Blue IL-4 cells were cultured in DMEM medium containing 10% FBS, 100 μg / mL Zeocin (Invivogen, Cat. #ant-zn-05), and 10 μg / mL Blasticidin (Invivogen, Cat. #ant-bl-05). They were passaged 2-3 times a week at a passage ratio of 1:5 or 1:10. At the time of passage, the medium was aspirated, the cell layer was rinsed with 5 mL of 0.25% trypsin, the trypsin was aspirated, and the cells were placed in an incubator for 3-5 minutes to digest. Fresh medium was then added and the cells were resuspended. 100 μL of the cell suspension was added to a 96-well cell culture plate, and the cells were cultured at a density of 5 × 10 5The cells were cultured at 100 μL / mL in DMEM containing 10% FBS, 100 μg / mL Zeocin, and 30 μg / mL Blasticidin. 100 μL of sterile water was added to the periphery of the 96-well plate. The culture plate was incubated in an incubator for 24 hours (37°C, 5% CO2). After the cells had contacted the wall, 100 μL of gradient-diluted test antibody was added to each well. The culture plate was incubated in an incubator for 20–24 hours (37°C, 5% CO2). 20 μL of cell supernatant was placed in a new 96-well flat plate, 180 μL of QUANTI-Blue substrate solution was added, and the culture plate was incubated in the dark for 1–3 hours. The absorbance at 620 nm was measured using a Thermo MultiSkanFc microplate reader.

[0246] Example 4: Experiment on suppression of TF-1 cell proliferation by antibodies that bind to human IL-4R TF-1 cells (ATCC CRL-2003) are lymphoma cells that express IL-4R and are sensitive to cytokines such as IL-4 and IL-13. IL-4 can stimulate the proliferation of TF-1 cells in the absence of GM-CSF. In this experiment, we compared the neutralizing activity of different anti-IL-4R antibodies by adding neutralizing antibodies to inhibit the IL-4 pathway and suppress TF-1 cell proliferation.

[0247] TF-1 cells were cultured in RPMI 1640 medium containing 10% FBS and 2 ng / mL GM-CSF (R&D, Cat. #215-GM-010) and passaged at a 1:10 ratio two to three times per week. 100 μL of the cell suspension was added to a 96-well cell culture plate at a density of 2 × 10 cells / mL in RPMI 1640 medium containing 10% FBS. 100 μL of sterile water was added to the periphery of the 96-well plate. 50 μL of a gradient-diluted test antibody and 50 μL of IL-4 (R&D, Cat. #204-IL-050) at a final concentration of 0.7 ng / mL were added per well, and the culture plate was incubated in an incubator for 72 hours (37°C, 5% CO2). After incubation, cell proliferation was detected using a CTG kit (Promega, Cat. #G7572).

[0248] Example 5: In vitro binding affinity and kinetic experiments The affinity of the test humanized IL-4R antibodies to human IL-4R was measured using a Biacore, GE instrument. Following the instructions in the instructions for the human anti-capture kit (Cat. # BR-1008-39, GE), a fixed amount of test antibody was affinity-captured by covalently binding the human anti-capture antibody to a biosensor chip CM5 of a Biacore instrument (Biacore X100, GE). A gradient series of IL-4R antigens (all IL-4R antigens were purchased from Acrobiosystems, Cat. # ILR-H5221) was then passed over the chip surface. The response signal was detected in real time by the Biacore instrument (Biacore X100, GE), yielding binding and dissociation curves. After each dissociation cycle, the biochip was regenerated by washing with the regeneration solution provided in the human anti-capture kit. The amine coupling kit used in the experiments was purchased from GE (Cat. # BR-1000-50, GE), and the buffer was HBS-EP + 10x buffer solution (Cat. # BR-1006-69, GE) diluted with DI water to 1x (pH 7.4).

[0249] The data obtained in the experiment was fitted with a (1:1) binding model using BiacoreX100 evaluation software 2.0 GE software to obtain affinity values.

[0250] Example 6: Antibody Sequence and Preparation Two monoclonal hybridoma cell lines with the highest in vitro activity were selected based on the ELISA binding experiment (ELISA binding of human IL-4R-his) and ELISA inhibition experiment (ELISA inhibition of human IL-4 / IL-4R) in Example 2, the experiment of inhibiting the activation of HEK293-Blue IL-4 cells under IL-4 stimulation in Example 3, and the experiment of inhibiting the proliferation of TF-1 cells under IL-4 stimulation in Example 4. The activity detection results are shown in Table 2.

[0251] [Table 2]

[0252] The monoclonal hybridoma cell lines 25G7 and 7B10 were selected and the antibody sequences therein were cloned. The process for cloning the sequences from the hybridomas is as follows:

[0253] Hybridoma cells in the logarithmic growth phase were harvested, and RNA was extracted using Trizol (Invitrogen, 15596-018) (according to the kit's instructions) and reverse-transcribed (PrimeScript™ Reverse Transcriptase, Takara, cat # 2680A). The reverse-transcribed cDNA was subjected to PCR amplification using a mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503), and then sent to a sequencing company for analysis of the resulting antibody sequences.

[0254] The heavy and light chain variable region sequences of the murine monoclonal antibody 25G7 are as follows: 25G7 HCVR EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYGMHWVRQAPEKGLEWVAFISSGSSIIYYADIVKGRSTISRDNAKNTLFLQMTSLRSEDTAMYYCTRGNKRGFFDYWGQGTILTVSS(SEQ ID NO:1) 25G7 LCVR QIVLTQSPALMSASPGEKVTMTCNASSSVSYMYWYQRKPRSSPKPWIYLTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWRSNPPMLTFGSGTKLEVK(SEQ ID NO:2)

[0255] The CDR sequences contained therein are as shown in Table 3. [Table 3]

[0256] The heavy and light chain variable region sequences of the murine monoclonal antibody 7B10 are as follows: 7B10 HCVR QVQLQQPGTELLKPGASVSLSCKASGYTFTSYWMHWVKQRPGQGLEWIGLIHPNSDTTKFSENFKTRATLTIDKSSSTAYMKLSSLTSEDSAVYYCAKSKIITTIVARHWYFDVWGTGTTVTVSS(SEQID NO:9) 7B10 LCVR DIVLTQSPPSLAVSLGQRATISCKASQSVDYGGDSYMNWYQQKLGQPPKVLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDVATYYCQHSNENPPTFGGGTKLEIK(SEQID NO:10)

[0257] The CDR sequences contained therein are as shown in Table 4. [Table 4]

[0258] The resulting variable region sequences were then ligated to human constant region sequences to obtain human-mouse chimeric antibody sequences. The chimeric antibody sequences were then inserted into the corresponding expression vectors using molecular cloning techniques. Using the HEK293 cell expression system, the human-mouse chimeric antibodies 25G7-C and 7B10-C were obtained.

[0259] The purified chimeric antibodies were subjected to in vitro activity detection using the methods described in Examples 2 to 5 above, and the data are shown in Table 5. As is clear from the results, the inhibitory effect of the 25G7-C antibody on IL-4 binding and the inhibitory effect on cell proliferation are both significantly superior to those of the reference antibody dupilumab (synthesized with reference to WHO Drug Information, Vol. 26, No. 4, 2012).

[0260] [Table 5]

[0261] Example 7: Humanization experiment of mouse antibody The resulting mouse antibodies 25G7 and 7B10 were humanized. Based on typical VH / VLCDR structures of the resulting mouse antibodies, the heavy and light chain variable region sequences were compared with the Germline database of human antibodies to obtain highly homologous human germline templates. The human germline light chain framework regions were derived from the human kappa light chain gene, preferably the human germline light chain templates IGKV3-11*01 (SEQ ID NO: 22, used for antibody 25G7) and IGKV2D-29*01 (SEQ ID NO: 24, used for antibody 7B10). The human germline heavy chain framework regions were derived from the human heavy chain, preferably the human germline heavy chain templates IGHV3-48*01 (SEQ ID NO: 21, used for antibody 25G7) and IGHV1-2*02 (SEQ ID NO: 23, used for antibody 7B10).

[0262] The human germline template sequence is shown below:

[0263] Human germline heavy chain template IGHV3-48*01: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR(SEQ ID NO:21) Human germline light chain template IGKV3-11*01: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWP(SEQ ID NO:22) Human germline heavy chain template IGHV1-2*02: QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR(SEQ ID NO:23) Human germline light chain template IGKV2D-29*01: DIVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTYLYWYLQKPGQPPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSIQLP(SEQ ID NO:24)

[0264] The CDR regions of the murine antibodies were grafted onto the selected humanized templates, further recombined with IgG constant regions, and backmutated to obtain a series of humanized molecules. Here, hu7B10-VH-a, hu7B10-VH-b, and hu7B10-VH-c were pharmacologically modified by changing the first position of the heavy chain human germline template from Q to E. The humanized heavy chain variable region sequences of the two antibody strains are shown in SEQ ID NOs: 25-27 and 31-33, respectively, and the light chain variable region sequences are shown in SEQ ID NOs: 28-30 and 34-36, respectively.

[0265] [ka] [ka]

[0266] The back mutations of hybridoma clone 25G7 were designed as shown in Table 6. [Table 6]

[0267] The back mutations of hybridoma clone 7B10 were designed as shown in Table 7 below. [Table 7]

[0268] Exemplary humanized antibody molecules hu25G7 (using a VH-c heavy chain and a VL-a light chain) and hu7B10 (using a VH-b heavy chain and a VL-b light chain) have their respective complete light and heavy chain sequences shown in SEQ ID NOs: 17-20.

[0269] hu25G7 HC EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAFISSGSSIIYYADIVKGRSTISRDNAKNSLYLQMNSLRAEDTAVYYCTRGNKRGFFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQ KSLSLSLGK(SEQ ID NO:17) hu25G7 LC EIVLTQSPATLSLSPGERATLSCNASSSVSYMYWYQQKPGQAPRLLIYLTSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWRSNPPMLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:18) hu7B10 HC EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGLIHPNSDTTKFSENFKTRVTMTIDTSISTAYMELSRLRSDDTAVYYCAKSKIITTIVARHWYFDV WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:19) hu7B10 LC DIVLTQTPLSLSVTPGQPASISCKASQSVDYGGDSYMNWYLQKPGQPPQLLIYAASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQHSNENPPTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:20)

[0270] By using molecular cloning technology, the sequence of a humanized antibody can be inserted into a corresponding expression vector and produced by expression in a HEK293 cell expression system to obtain the corresponding humanized antibody.

[0271] Example 8: Humanized Antibody Activity Data The humanized antibodies hu25G7 and hu7B10 were subjected to in vitro activity detection as described in Examples 2 to 5, and the test results are shown in Table 8.

[0272] The results clearly show that both hu25G7 and hu7B10 bind only to the human IL-4R, not to the rhesus monkey IL-4R, but both antibodies bind to epitopes that are non-homologous between humans and rhesus monkeys, demonstrating their ability to specifically bind to the human IL-4R. Both antibodies can inhibit IL-4 / IL-4R binding and the intracellular signaling pathway, thereby neutralizing the activation of IL-4 and suppressing the proliferation of TF-1 cells. The inhibitory and suppressive activities of hu25G7 are significantly superior to those of the reference antibody dupilumab.

[0273] [Table 8]

[0274] Example 9: Affinity maturation experiments of humanized antibody hu25G7 Affinity maturation of the hu25G7 antibody was performed using yeast display platform technology. Yeast libraries for affinity maturation of six CDRs based on the hu25G7 antibody were designed and constructed. Degenerate primers were designed, and the designed mutant amino acids were introduced into the hu25G7-scFv antibody library using PCR and homologous recombination methods. The size of each library was approximately 109. The diversity of the constructed yeast libraries was verified using second-generation sequencing (GENEWIZ) methods.

[0275] High-affinity antibodies were screened from the hu25G7-scFv yeast library using biotin-labeled human IL-4R. After two rounds of MACS screening (streptomycin magnetic beads, Invitrogen) and two rounds of FACS screening (BD FACSAria™ FUSION), yeast monoclonals were selected and cultured for expression. Binding of the yeast monoclonals to human IL-4R was detected using a BD FACSCanto II. Yeast monoclonals with higher affinity than the wild-type 25G7 antibody were selected and verified by sequencing. Sequenced clones were subjected to alignment analysis, and redundant sequences were removed. Non-redundant sequences were converted into full-length human antibody molecules and expressed in mammalian cells. After affinity purification, the full-length antibodies were subjected to affinity testing using a BIAcore™ X-100 (GE Life Sciences) to select candidate antibody molecules with high affinity for human IL-4R. These antibody molecules have higher affinity for human IL-4R than the wild-type hu25G7 antibody, where the affinity of the hu25G7-A antibody molecule is comparable to that of dupilumab, while the affinity of the hu25G7-B molecule is clearly superior to that of dupilumab.

[0276] The final light chain variable region sequence of antibody hu25G7-A obtained after affinity maturation is as follows: hu25G7-A LCVR EIVLTQSPATLSLSPGERATLSCRASSSVPYMYWYQQKPGQAPRLLIYLTSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWRAYPPMLTFGGGTKVEIK(SEQ ID NO:37)

[0277] The CDR sequences contained therein are as shown in Table 9. [Table 9]

[0278] The light chain variable region sequence of antibody hu25G7-B is as follows: hu25G7-B LCVR EIVLTQSPATLSLSPGERATLSCRASPGVPPLAWYQQKPGQAPRLLIYLASSRPSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWRSNPPMLTFGGGTKVEIK(SEQ ID NO:41)

[0279] The CDR sequences contained therein are as shown in Table 10. [Table 10]

[0280] The light chain variable region hu25G7-A LCVR was recombined with the hu25G7 light chain constant region to obtain the hu25G7-A antibody light chain, and the light chain variable region hu25G7-B LCVR was recombined with the hu25G7 light chain constant region to obtain the hu25G7-B antibody light chain.

[0281] The amino acid residues of hu25G7-VH-c were optimized to obtain the heavy chain variable regions hu25G7-A / B VH and hu25G7-C VH. hu25G7-A / B VH: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAFISSGSSIIYYADIVKGRSTISRDNAKNTLYLQMNSLRAEDTAVYYCTRGNKRGFFDYWGQGTLVTVSS(SEQ ID NO:43) hu25G7-C VH: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAFISSGSSIIYYADIVKGRSTISRDNAKNTLYLQMSSLRAEDTAVYYCTRGNKRGFFDYWGQGTLVTVSS (SEQ ID NO:47)

[0282] The heavy chain variable regions can be recombined with the hu25G7 heavy chain constant region to obtain hu25G7-A / hu25G7-B and hu25G7-C antibody heavy chains.

[0283] The complete heavy chain sequences of hu25G7-A and hu25G7-B are shown in SEQ ID NO:44. hu25G7 HC (i.e., hu25G7-A / hu25G7-B antibody heavy chain) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAFISSGSSIIYYADIVKGRSTISRDNAKNTLYLQMNSLRAEDTAVYYCTRGNKRGFFDYWGQGT LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:44) hu25G7-C antibody heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAFISSGSSIIYYADIVKGRSTISRDNAKNTLYLQMSSLRAEDTAVYYCTRGNKRGFFDYWGQGT LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:48)

[0284] The complete light chain sequences of each are shown in SEQ ID NOs: 45-46. hu25G7-A LC EIVLTQSPATLSLSPGERATLSCRASSSVPYMYWYQQKPGQAPRLLIYLTSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWRAYPPMLTFGGGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:45) hu25G7-B / hu25G7-C LC EIVLTQSPATLSLSPGERATLSCRASPGVPPLAWYQQKPGQAPRLLIYLASSRPSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWRSNPPMLTFGGGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:46)

[0285] Example 10: Affinity Maturation Activity Data of Humanized Antibodies According to Examples 3 and 4, two antibodies, hu25G7-A and hu25G7-B, were detected. Both antibodies, hu25G7-A and hu25G7-B, can inhibit IL-4 / IL-4R binding and intracellular signaling pathways, thereby neutralizing the activation effects of IL-4 and IL-13 and suppressing the proliferation of TF-1 cells. The activity data for each antibody are shown in Table 11.

[0286] [Table 11]

[0287] In experiments to inhibit IL-13-stimulated TF-1 cell proliferation, both hu25G7-A and hu25G7-B demonstrated beneficial effects. Compared to dupilumab, hu25G7-A and hu25G7-B exhibited significantly improved effects on the binding of IL-4 and IL-13 to IL-4R and the inhibition of cell proliferation through this binding.

[0288] Example 11: Study of the effect of humanized antibodies on mouse dermatitis Construction of a mouse dermatitis model: IL-4 / IL-4Rα transgenic mice (purchased from Scitech Model Biological Research Center (Taicang) Co., Ltd.) were selected. Each mouse had an abdominal area of ​​approximately 3 cm x 3 cm. 100 μL of 1.5% OXZ acetone olive oil solution (acetone:olive oil = 4:1) was uniformly applied to the area. The day of sensitization was designated Day 0 (D0). On Day 7 (Day 7), 20 μL of 1% OXZ acetone olive oil solution was uniformly applied to both ears (both sides) of the mice for challenge. The mice were challenged once every 72 hours.

[0289] In this experiment, five groups were established: a normal control group (applied with only acetone-olive oil solution during sensitization and challenge), a model control group, and hu25G7-A, hu25G7-B, and dupilumab groups, with 3-5 mice per group. The dose of the treatment group was 50 mg / kg, administered subcutaneously twice weekly (specific information is shown in Table 12). Ear thickness was measured using a vernier caliper on day 27, and the results are shown in Figure 1.

[0290] [Table 12]

[0291] The results show that mice in the model control group showed obvious pathological damage to their ears, with ear thickness significantly higher than that of the normal control group. On day 27, mice in the hu25G7-A, hu25G7-B, and dupilumab groups had ear thickness significantly lower than that of the model control group. This means that hu25G7-A, hu25G7-B, and dupilumab are all suitable for treating dermatitis, and the efficacy of hu25G7-B is superior to that of dupilumab.

[0292] Example of formulation preparation The equipment used during the preparation and the calculation method of the results are as follows:

[0293] SEC size exclusion chromatography: This is an analytical method for separating solutes based on the correlation between the pore size of gel pores and the coil dimensions of polymer sample molecules. SEC monomer content percentage=Amonomer / Atotal*100% (Amonomer is the peak area of ​​the main peak monomer in the sample, and Atotal is the sum of all peak areas). SEC measurement equipment: Agilent 1260, column: water, XBrige BEH200Å SEC (300 × 7.8 mm 3.5 μm).

[0294] CE Capillary Gel Electrophoresis: This is a method of electrophoresis in which gel is transferred to a capillary as a support medium, and samples are separated based on their molecular weight at a constant voltage. Percent purity of non-reduced CE = A Main / A Total * 100% (A Main is the peak area of ​​the main peak in the sample, and A Total is the sum of all peak areas). CE measuring equipment: Beckman, model number plus800

[0295] iCIEF Imaging Capillary Isoelectric Focusing: This is a technique for separating proteins based on their isoelectric points (pI). iCIEF neutral peak content percentage = neutral peak area / total area*100% (total area is the sum of the areas of the acidic peak, neutral peak and basic peak). The manufacturer of the equipment used for iCIEF measurement is Simple Protein, and the model number is Muarice.

[0296] Measurement of viscosity: The viscosity was measured using a rheometer (manufacturer: Anton Paar, model number: MCR xx2), the measurement temperature was 25°C, and the sample was placed directly on the measurement plate.

[0297] Osmotic pressure: Osmotic pressure was measured using the freezing point method. Based on the direct proportional relationship between the freezing point depression value and the molar concentration of the solution, a highly sensitive thermosensor was used to measure the freezing point of the solution and convert it into osmotic pressure using an electrical charge. The device manufacturer is Loser, and the model number is OM815.

[0298] Example 1. Screening of buffer systems Formulations containing 100 mg / mL hu25G7-A antibody and 0.1 mg / mL polysorbate 80 (PS80) were prepared in different buffer systems ranging from pH 4.5 to 6.5 to examine the stability of the antibody in the different buffer systems. The specific formulations were designed as shown in the table below.

[0299] [Table 13]

[0300] The samples were sterilized and filtered into bottles, and the appearance and SEC of the samples were examined under high temperature conditions of 40°C. The data are shown in the table below.

[0301] [Table 14]

[0302] As the results show, the His-AA buffer system was selected because the SA / CA / AA buffer system had poor appearance. The His-AA system had a relatively good appearance at pH 4.5 to 5.5. Except for His-AA at pH 6.5, there was no significant difference in SEC between the buffer systems, with pH 5.0 and 6.0 being slightly better. Therefore, His-AA at pH 4.5 to 6.0 was selected as the buffer system for this antibody.

[0303] Example 2. Antibody concentration screening Different concentrations of hu25G7-A formulations were prepared in a 20 mM His-AA buffer system at pH 5.0 and 5.5, and the formulation components were as follows: 1) 20 mM His-AA pH 5.0, 100 mg / mL hu25G7-A 2) 20 mM His-AA pH 5.5, 100 mg / mL hu25G7-A 3) 20 mM His-AA pH 5.0, 150 mg / mL hu25G7-A 4) 20 mM His-AA pH 5.5, 150 mg / mL hu25G7-A 5) 20 mM His-AA pH 5.0, 139 mg / mL hu25G7-A 6) 20 mM His-AA pH 5.0, 127 mg / mL hu25G7-A 7) 20 mM His-AA pH 5.0, 120 mg / mL hu25G7-A 8) 20 mM His-AA pH 5.5, 138 mg / mL hu25G7-A 9) 20 mM His-AA pH 5.5, 122 mg / mL hu25G7-A

[0304] After the formulation was completed, a sample was taken to measure the viscosity of the formulation. The sample was then sterile filtered and bottled, and the appearance, SEC, non-reduced CE-SDS, and iCIEF of the sample were examined at a high temperature of 40°C to determine the effect of different antibody concentrations on the viscosity and stability of the hu25G7-A formulation. The results are shown in the table below.

[0305] [Table 15]

[0306] [Table 16]

[0307] As can be seen from the results, there were no significant differences in appearance, SEC, CE, or iCIEF between different antibody concentrations. That is, in His-AA buffer solutions at pH 5.0 or pH 5.5, the antibody concentration did not have a significant effect on formulation stability, but the higher the antibody concentration, the greater the viscosity of the formulation. In the pH 5.0 buffer solution, the viscosity was approximately 14 mPa·s when the antibody concentration was 100 mg / mL, and approximately 22 mPa·s when the antibody concentration was 120 mg / mL.

[0308] Example 3. Screening of viscosity modifiers hu25G7-A formulations containing different viscosity modifiers were prepared in 20 mM His-AA (pH 5.0) buffer at an antibody concentration of 120 mg / mL. The effects of different viscosity modifiers on the viscosity of the formulation were investigated using viscosity as the evaluation index. To meet the isotonicity requirements of subcutaneous formulations, each viscosity modifier was added to the formulation at an osmolality of 270 mosm / kg of the blank viscosity modifier, and its effect was examined. Specific results are shown in the table below.

[0309] [Table 17]

[0310] The results show that, apart from proline, NaCl, MgCl2, CaCl2, KCl, CH3COONa, Na2SO4, NaI, NaF, NaSCN, Arg-HCl, Lys (lysine), and His (histidine) all significantly reduce the viscosity of the formulation at their maximum concentrations, with Arg-HCl, MgCl2, CaCl2, NaF, NaSCN, and histidine showing better effects, and Arg-HCl, MgCl2, CaCl2, and histidine being preferred viscosity modifiers.

[0311] Example 4. Effect of viscosity modifiers on the viscosity of highly concentrated formulations To test the effect of viscosity modifiers, a Hu25G7-A formulation was prepared in 20 mM His-AA buffer at pH 5.0 with a hu25G7-A antibody concentration of 150 mg / mL, and the specific components were as follows: 1) No viscosity modifiers (N / A) 2) 85 mM MgCl 3) 148 mM CaCl 4) 93 mM histidine

[0312] Using viscosity as an evaluation index, the effects of different auxiliary materials on the viscosity of high-concentration formulations were investigated, and the results are shown in the table below. [Table 18]

[0313] As is clear from the results, when the antibody concentration is 150 mg / mL, MgCl2, CaCl2 and histidine can reduce the viscosity of the formulation to 20 mPa.s or less, and have a good viscosity-reducing effect.

[0314] Example 5. Effect of viscosity modifiers on formulation stability Formulations containing different concentrations of supplementary materials were prepared at a Hu25G7-A antibody concentration of 150 mg / mL in a 20 mM His-AA buffer system at pH 5.0, and the effects of different concentrations of supplementary materials on the thermal stability of the formulations (left at 40°C for 17 days) were investigated. The experimental design and results are shown in the table below.

[0315] [Table 19] [Table 20]

[0316] As is clear from the results, MgCl2, Arg-HCl, NaCl, and histidine have a relatively small effect on formulation stability. Among them, the stability of formulations containing MgCl2, Arg-HCl, NaCl, and histidine is superior to that of formulations containing CaCl2. The CaCl2 group showed a greater decrease in CE under high temperature conditions than the other formulation samples. The stability of formulations containing MgCl2, Arg-HCl, and histidine is also good, so MgCl2, Arg-HCl, and histidine are preferred.

[0317] Example 6. Comprehensive evaluation of the effect of viscosity modifiers on formulation stability A formulation containing 0.1 mg / mL PS80, 150 mg / mL Hu25G7-A, and a viscosity modifier was prepared in a 20 mM His-AA buffer solution at pH 5.0, where the PS80 concentration in the sample was 1 mg / mL after shaking for 15 days. The effects of different auxiliary materials on the viscosity and stability of the Hu25G7-A formulation were investigated. The experimental design was as follows: 1) N / A (does not contain viscosity modifiers) 2) 90 mM MgCl2 3) 120 mM Arg-HCl 4) 90 mM histidine

[0318] After the formulation was completed, a sample was taken to measure the viscosity of the formulation. The sample was then sterile filtered and bottled. The appearance, SEC, and non-reduced CE were examined under high temperature (40°C), repeated freeze-thawing (-35°C to 4°C), and shaking (25°C, 300 rpm) conditions to evaluate the effect of different auxiliary ingredients on the viscosity and stability of the formulation. The results are shown in the table below.

[0319] [Table 21] [Table 22]

[0320] As is clear from the results, After adding the viscosity modifier, the appearance became opalescent, but there was no difference between the various additives. When the formulation contained 0.1 mg / mL of PS80, the group containing the viscosity modifier showed particles after 1 day of shaking. However, when the PS80 was 1 mg / mL, the appearance remained clear after 15 days of shaking.

[0321] The SEC results at 40°C show that the SEC monomer content decreased slightly after the addition of the viscosity modifier, but there was no significant difference between the samples in each group, with the Arg-HCl sample group and the histidine sample group performing relatively well, and the histidine sample having the best stability.The CE results at 40°C show that the CE decreased slightly after the addition of the viscosity modifier, but there was no significant difference between the various auxiliary materials.

[0322] Example 7. Viscosity modifier concentration screening Formulations containing different Hu25G7-A antibody concentrations and viscosity modifiers were prepared in a 20 mM His-AA buffer system at pH 5.0, and the viscosity of the formulations was investigated. The experimental design and results are shown in the table below.

[0323] [Table 23]

[0324] As is clear from the results, increasing the concentrations of the viscosity modifiers His, MgCl2 and Arg-HCl can clearly reduce the viscosity of the formulation.

[0325] Example 8. Stability testing of formulations In order to improve the appearance of the formulation, the amount of basic amino acid (viscosity modifier) ​​added in the formulation was reduced, and the following formulation samples were prepared: 1. 20 mM His-AA, 30 mM histidine, 0.8 mg / mL PS80, 41.8 mg / mL sucrose, 120 mg / mL antibody Hu25G7-A at pH 5.0, the final content of histidine in the sample being 50 mM. 2. 20 mM His-AA, 87 mM histidine, 0.8 mg / mL PS80, 150 mg / mL antibody Hu25G7-A, pH 4.8. 3. 20 mM His-AA, 100 mM histidine, 0.8 mg / mL PS80, 150 mg / mL antibody Hu25G7-A, pH 5.0. 4. 20 mM His-AA, 120 mM arginine hydrochloride, 0.8 mg / mL PS80, 150 mg / mL antibody Hu25G7-A, pH 5.0.

[0326] After the formulation was completed, some formulation samples were taken to measure the viscosity of the formulation. The samples were then sterile filtered and bottled, and their stability was tested at 4°C and 25°C. The sample appearance, osmolality, pH, SEC, non-reduced CE, and iCIEF were measured to evaluate the viscosity and stability of the different formulation samples. The results are shown in the table below.

[0327] [Table 24]

[0328] [Table 25]

[0329] As can be seen from the results, formulation samples 1 to 4 all had a pH drift within 0.1, relatively good buffering capacity, an osmotic pressure within the isotonic range, and a viscosity within 20 mPa·s. After storage at 25°C for 3 months, sample 1 showed a slight decrease in CE / iCIEF, but after storage at 4°C for 3 months, the appearance remained transparent, and there was no significant change in chemical stability. After storage at 4°C for 11 months, samples 1 to 4 were transparent in appearance, and the SEC, iCIEF, and CE-SDS detection results were all stable, indicating that formulations 1 to 4 are relatively stable.

[0330] Example 9. Sugar concentration screening To develop a subcutaneous formulation and reduce irritation upon injection, it is most desirable to control the osmotic pressure to isotonicity. Therefore, the sugar concentration was screened to determine the isotonic sugar concentration. A formulation containing 50 mM His-AA, 58 mg / mL sucrose, 0.8 mg / mL PS80, and 120 mg / mL hu25G7-A antibody at pH 5.0 was prepared, and the osmotic pressure was measured in triplicate by freezing point analysis.

[0331] [Table 26]

[0332] As can be seen from the results, when the sugar concentration is 58 mg / mL, the osmolality is about 297 mosm / kg, which is isotonic.

[0333] Example 10. Freeze-drying process screening A formulation of 25 mM His-AA, 60 mg / mL hu25G7-A, 25 mg / mL sucrose, and 0.2 mg / mL PS80 at pH 4.9 was prepared, sterile filtered, filled at 3.4 mL per vial, and lyophilized according to the procedure in the table below.

[0334] [Table 27]

[0335] After lyophilization, the sample was removed from the box and appeared as a white cake, bulging without any pitting. It was then reconstituted in approximately 1.5 mL of water for injection. The reconstituted sample contained 50 mM His-AA, 120 mg / mL hu25G7-A, 50 mg / mL sucrose, and 0.4 mg / mL PS80 at a pH of 5.3. The reconstituted sample was analyzed for each parameter. The results clearly show that after dilution, freezing, and concentration, the reconstituted solution exhibited excellent performance.

[0336] [Table 28]

[0337] Example 11. Optimization of formulation A DOE experiment was designed using the pH of the 50 mM His-AA buffer, the concentration of Hu25G7-A antibody, and the concentration of PS80 as variables. The variables were pH 4.5-5.5, PS80 0.4-1.2 mg / mL, and Hu25G7-A antibody protein concentration 100-140 mg / mL. Using JMP software, a series of hu25G7-A formulations were obtained, each containing 58 mg / mL of sucrose. The details are shown in Table 29. The formulation stability was assessed by forced degradation, including high temperature (40°C), shaking (300 rpm), and freeze-thawing (-35°C to 4°C). Appearance, viscosity, SEC, non-reduced CE, and iCIEF were used as evaluation indices. The results are shown in Table 30. Statistical analysis of the results was performed using the least squares method.

[0338] [Table 29]

[0339] [Table 30]

[0340] As can be seen from the results, after five freeze-thaw cycles, each formulation remains clear in appearance.

[0341] The forced degradation data were fitted, and the fit of the viscosity, 40°C CE, and iCIEF degradation difference values ​​was relatively good, demonstrating the validity of the model. The results are shown in Figure 2. When the PS concentration was 0.8 mg / mL, a contour map was plotted using the antibody concentration and pH as the horizontal and vertical coordinates, and the viscosity and 40°C CE / iCIEF degradation difference values ​​as indicators. A contour map of the formulation stability change was plotted with the limits of viscosity < 30 cP, CE reduction < 3%, and iCIEF reduction < 30%. The results are shown in Figure 3.

[0342] The results clearly show that the lower the protein concentration, the lower the pH, and the lower the viscosity of the formulation. The CE results are optimal at pH 5.0, and the neutral peak of iCIEF is optimal at pH 5.1. Combining the contour plot and viscosity results, stable formulations are those with antibody concentrations of 100-140 mg / mL, PS80 values ​​of 0.4-1.2 mg / mL, and pH values ​​of 4.5-5.5. Furthermore, formulations are more stable when the antibody concentration is 110-130 mg / mL and the pH is 4.85-5.45.

[0343] Example 12. Formulation stability detection A formulation containing 50 mM His-AA, 120 mg / mL hu25G7-A antibody, 58 mg / mL sucrose, and 0.8 mg / mL PS80 at pH 5.0 was prepared, sterile filtered, and bottled. A stability study was then performed at 4°C, and the results are shown in Table 31.

[0344] [Table 31]

[0345] As can be seen from the results, the above formulation remains stable after being stored at 4°C for 4 months. Further aspects of the present invention are described below: [Section 1] A pharmaceutical composition comprising an anti-IL-4R antibody or an antigen-binding fragment thereof and a buffer, wherein the buffer is a histidine-acetate buffer, and the pH of the buffer is 4.5 to 6.0, preferably 4.5 to 5.5. [Section 2] Item 2. The pharmaceutical composition according to Item 1, wherein the concentration of the histidine-acetate buffer is 10 mM to 60 mM, preferably 10 mM to 30 mM. [Section 3] Item 3. The pharmaceutical composition according to Item 1 or 2, wherein the concentration of the anti-IL-4R antibody or antigen-binding fragment thereof is 100 mg / mL or more, preferably 100 mg / mL to 200 mg / mL, preferably 100 mg / mL to 180 mg / mL, and more preferably 100 mg / mL to 150 mg / mL. [Section 4] Item 4. The pharmaceutical composition according to any one of Items 1 to 3, further comprising a viscosity modifier selected from MgCl, CaCl, NaF, NaSCN, KCl, CHCOONa, NaSO, NaI, arginine, arginine hydrochloride, histidine, and lysine, preferably MgCl, histidine, and arginine hydrochloride. [Section 5] The concentration of the viscosity modifier is 5 mM to 220 mM, preferably 5 mM to 148 mM, and more preferably, the viscosity modifier is i) 5 mM to 220 mM arginine hydrochloride; ii) 5 mM to 100 mM histidine, or iii) 5 mM to 90 mM MgCl2; Item 5. The pharmaceutical composition according to Item 4. [Section 6] Item 6. The pharmaceutical composition according to Item 5, wherein the concentration of the anti-IL-4R antibody or antigen-binding fragment thereof is 100 mg / mL to 200 mg / mL, preferably 120 mg / mL to 150 mg / mL, and the viscosity modifier is selected from 50 mM to 90 mM MgCl, 50 mM to 100 mM histidine, and 10 mM to 200 mM, preferably 50 mM to 180 mM, arginine hydrochloride. [Section 7] Item 6. The pharmaceutical composition according to Item 5, wherein the concentration of the anti-IL-4R antibody or antigen-binding fragment thereof is 100 mg / mL to 140 mg / mL, preferably 100 mg / mL to 120 mg / mL, and the viscosity modifier is selected from 5 mM to 50 mM histidine, arginine hydrochloride, or MgCl2, preferably 10 mM to 40 mM histidine, arginine hydrochloride, or MgCl2, and most preferably 30 mM histidine, arginine hydrochloride, or MgCl2. [Section 8] Item 8. The pharmaceutical composition according to any one of Items 1 to 7, further comprising a surfactant, preferably a polysorbate, and most preferably polysorbate 80 or polysorbate 20. [Section 9] Item 9. The pharmaceutical composition according to Item 8, wherein the concentration of the surfactant is 0.1 mg / mL to 1.2 mg / mL, preferably 0.4 mg / mL to 1.0 mg / mL, and most preferably 0.8 mg / mL. [Section 10] Item 10. The pharmaceutical composition according to any one of Items 1 to 5 and 7 to 9, further comprising a stabilizer, preferably trehalose or sucrose, more preferably sucrose. [Section 11] Item 11. The pharmaceutical composition according to Item 10, wherein the concentration of the stabilizer is 20 mg / mL to 70 mg / mL, preferably 40 mg / mL to 60 mg / mL, and most preferably 58 mg / mL. [Section 12] (a) 100 mg / mL to 150 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM of a histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 80 mM to 148 mM of a viscosity modifier, and (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, or (a) 100 mg / mL to 120 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 20 mM histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 5 mM to 50 mM histidine, (d) 0.4 mg / mL to 1.0 mg / mL of polysorbate 80, and (e) 50 mg / mL to 60 mg / mL of sucrose, 10. A pharmaceutical composition comprising: [Section 13] (a) 100 mg / mL to 140 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM of a histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 10 mM to 40 mM of a viscosity modifier, (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, and (e) 40 mg / mL to 70 mg / mL of sucrose, Or, (a) 100 mg / mL to 200 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM of a histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 40 mM to 220 mM of a viscosity modifier, and (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, Including, Wherein, the viscosity modifier is selected from MgCl2, CaCl2, NaF, NaSCN, KCl, CH3COONa, Na2SO4, NaI, arginine, arginine hydrochloride, histidine and lysine; Preferably, (a) 100 mg / mL to 140 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM of a histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 10 mM to 40 mM of a viscosity modifier, (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, and (e) 40 mg / mL to 70 mg / mL of sucrose (wherein the viscosity modifier is histidine, arginine hydrochloride, or MgCl2), or (a) 100 mg / mL to 180 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 40 mM to 90 mM MgCl2, and (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, or (a) 100 mg / mL to 180 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 50 mM to 100 mM histidine, and (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, or (a) 100 mg / mL to 180 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM of a histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 50 mM to 200 mM of arginine hydrochloride, and (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, 10. A pharmaceutical composition comprising: [Section 14] (a) 100 mg / mL to 120 mg / mL of an anti-IL-4R antibody or an antigen-binding fragment thereof, (b) 20 mM to 60 mM histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, and (d) 40 mg / mL to 70 mg / mL of sucrose, and preferably (a) 120 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 50 mM histidine-acetate buffer having a pH of 5.0, (c) 0.8 mg / mL of polysorbate 80, and (d) 58 mg / mL of sucrose; 10. A pharmaceutical composition comprising: [Section 15] (a) about 150 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) about 20 mM histidine-acetate buffer having a pH of about 5.0, (c) about 0.8 mg / mL of polysorbate 80, and (d) about 120 mM arginine hydrochloride; 10. A pharmaceutical composition comprising: [Section 16] The anti-IL-4R antibody or antigen-binding fragment thereof is (i) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:38, SEQ ID NO:7 and SEQ ID NO:40, respectively; (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively; (iii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively; or (iv) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:42, SEQ ID NO:39 and SEQ ID NO:8, respectively; and preferably a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:38, SEQ ID NO:7 and SEQ ID NO:40, respectively; Including, More preferably, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region as set forth in any one of the following: (v) a heavy chain variable region sequence set forth in SEQ ID NO:1 or having at least 90% identity thereto, and a light chain variable region sequence set forth in SEQ ID NO:2 or having at least 90% identity thereto; (vi) the heavy chain variable region sequence is set forth in SEQ ID NO:9 or has at least 90% identity to SEQ ID NO:9, and the light chain variable region sequence is set forth in SEQ ID NO:10 or has at least 90% identity to SEQ ID NO:10; (vii) a heavy chain variable region sequence set forth in, or having at least 90% identity to, SEQ ID NO:25, 26, 27, 43, or 47, and a light chain variable region sequence set forth in, or having at least 90% identity to, SEQ ID NO:28, 29, 30, 37, or 41; or (viii) a heavy chain variable region sequence set forth in SEQ ID NO: 31, 32, or 33, or which has at least 90% identity to SEQ ID NO: 31, 32, or 33, and a light chain variable region sequence set forth in SEQ ID NO: 34, 35, or 36, or which has at least 90% identity to SEQ ID NO: 34, 35, or 36; Most preferably, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region as set forth in any one of the following: (IX) a heavy chain variable region sequence set forth in SEQ ID NO: 43 or having at least 90% identity to SEQ ID NO: 43, and a light chain variable region sequence set forth in SEQ ID NO: 37 or having at least 90% identity to SEQ ID NO: 37; (X) the heavy chain variable region sequence is set forth in SEQ ID NO:43 or has at least 90% identity to SEQ ID NO:43, and the light chain variable region sequence is set forth in SEQ ID NO:41 or has at least 90% identity to SEQ ID NO:41; or (XI) the heavy chain variable region sequence is set forth in SEQ ID NO:47 or has at least 90% identity to SEQ ID NO:47, and the light chain variable region sequence is set forth in SEQ ID NO:41 or has at least 90% identity to SEQ ID NO:41; Item 16. The pharmaceutical composition according to any one of Items 1 to 15. [Section 17] The anti-IL-4R antibody or antigen-binding fragment thereof comprises a constant region, and preferably the anti-IL-4R antibody comprises: a heavy chain as set forth in SEQ ID NO:17 and a light chain as set forth in SEQ ID NO:18, or a heavy chain as set forth in SEQ ID NO:19 and a light chain as set forth in SEQ ID NO:20, or a heavy chain as set forth in SEQ ID NO:44 and a light chain as set forth in SEQ ID NO:45; or a heavy chain as set forth in SEQ ID NO:44 and a light chain as set forth in SEQ ID NO:46, or a heavy chain as shown in SEQ ID NO: 48 and a light chain as shown in SEQ ID NO: 46; Including, More preferably, The anti-IL-4R antibody comprises a heavy chain shown in SEQ ID NO: 44 and a light chain shown in SEQ ID NO: 45. Item 17. The pharmaceutical composition according to Item 16. [Section 18] Item 18. A method for preparing the pharmaceutical composition according to any one of Items 1 to 17, comprising replacing a stock solution of an anti-IL-4R antibody or an antigen-binding fragment thereof with a buffer. [Section 19] A lyophilized preparation comprising an anti-IL-4R antibody or an antigen-binding fragment thereof, which is obtained by lyophilizing the pharmaceutical composition according to any one of Items 1 to 17. [Section 20] A reconstituted solution containing an anti-IL-4R antibody or an antigen-binding fragment thereof, which is obtained by reconstituting the lyophilized formulation according to Item 19, and preferably comprises: (a) 120 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof; (b) 50 mM histidine-acetate buffer; (c) 0.4 mg / mL of polysorbate 80; and (d) 50 mg / mL of sucrose, wherein the pH of the pharmaceutical composition is about 5.3; or (a) 120 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof; (b) 50 mM histidine-acetate buffer; (c) 0.8 mg / mL of polysorbate 80; and (d) 50 mg / mL of sucrose, wherein the pH of the pharmaceutical composition is about 5.3; or (a) 120 mg / mL of an anti-IL-4R antibody or an antigen-binding fragment thereof, (b) 20 mM of a histidine-acetate buffer, (c) 0.8 mg / mL of polysorbate 80, and (d) 120 mM of arginine hydrochloride, wherein the pH of the pharmaceutical composition is about 5.3. A reconstitution solution characterized by: [Section 21] A product comprising a container containing the pharmaceutical composition according to any one of Items 1 to 17, the lyophilized preparation according to Item 19, or the reconstitution solution according to Item 20. [Section 22] A method for treating or preventing an immune disease or condition, comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition according to any one of Items 1 to 17, the lyophilized preparation according to Item 19, or the reconstituted solution according to Item 20. Preferably, the immune disease or condition is an IL-4R-mediated disease or condition. More preferably, the immune disease or condition is selected from asthma, nasal polyps, chronic sinusitis, allergic skin disease, eosinophilic esophagitis, chronic obstructive pulmonary disease, allergic rhinitis, arthritis, inflammatory disease, allergic reaction, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, and kidney disease. Most preferably, the immune disease or condition is asthma or an allergic skin disease.

Claims

1. A pharmaceutical composition comprising an anti-IL-4R antibody or an antigen-binding fragment thereof and a buffer, wherein the buffer is a histidine-acetate buffer and the pH of the buffer is 4.5 to 6.0; The anti-IL-4R antibody or antigen-binding fragment thereof is (i) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 38, SEQ ID NO: 7 and SEQ ID NO: 40, respectively; (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively; (iii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively; or (iv) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 42, SEQ ID NO: 39, and SEQ ID NO: 8, respectively; Includes.

2. The pharmaceutical composition described in claim 1, wherein the pH of the buffer is 4.5 to 5.

5.

3. 2. The pharmaceutical composition of claim 1, wherein the concentration of the histidine-acetate buffer is 10 mM to 60 mM.

4. The pharmaceutical composition described in claim 3, wherein the concentration of the histidine-acetate buffer is 10 mM to 30 mM.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the concentration of the anti-IL-4R antibody or antigen-binding fragment thereof is 100 mg / mL or more.

6. The pharmaceutical composition of claim 5, wherein the concentration of the anti-IL-4R antibody or its antigen-binding fragment is 100 mg / mL to 200 mg / mL.

7. The pharmaceutical composition of claim 5, wherein the concentration of the anti-IL-4R antibody or its antigen-binding fragment is 100 mg / mL to 180 mg / mL.

8. The pharmaceutical composition of claim 5, wherein the concentration of the anti-IL-4R antibody or its antigen-binding fragment is 100 mg / mL to 150 mg / mL.

9. Further comprising a viscosity modifier, the viscosity modifier being MgCl 2 , CaCl 2 , NaF, NaSCN, KCl, CH 3 COONa, Na 2 SO 4 9. The pharmaceutical composition according to claim 1, wherein the hydroxybenzoate is selected from the group consisting of arginine, arginine hydrochloride, histidine and lysine.

10. 10. The pharmaceutical composition of claim 9, wherein the viscosity modifier is present in a concentration of 5 mM to 220 mM.

11. The pharmaceutical composition described in claim 9, wherein the concentration of the viscosity adjuster is 5 mM to 148 mM.

12. The concentration of the anti-IL-4R antibody or antigen-binding fragment thereof is 100 mg / mL to 200 mg / mL, and the viscosity adjusting agent is 50 mM to 90 mM MgCl 2 12. The pharmaceutical composition of claim 1, wherein the soluble ...

13. The pharmaceutical composition of claim 12, wherein the concentration of the anti-IL-4R antibody or its antigen-binding fragment is 120 mg / mL to 150 mg / mL, and the viscosity adjuster is 50 mM to 180 mM arginine hydrochloride.

14. 14. The pharmaceutical composition of claim 1, further comprising a surfactant, wherein the surfactant is polysorbate 80 or polysorbate 20.

15. 15. The pharmaceutical composition of claim 14, wherein the concentration of the surfactant is 0.1 mg / mL to 1.2 mg / mL.

16. The pharmaceutical composition described in claim 14, wherein the concentration of the surfactant is 0.4 mg / mL to 1.0 mg / mL.

17. The pharmaceutical composition of claim 14, wherein the concentration of the surfactant is 0.8 mg / mL.

18. 18. The pharmaceutical composition of claim 1, further comprising a stabilizer, wherein the stabilizer is trehalose or sucrose, and the concentration of the stabilizer is from 20 mg / mL to 70 mg / mL.

19. (a) 100 mg / mL to 150 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof; (b) 10 mM to 30 mM of a histidine-acetate buffer having a pH of 4.5 to 5.5; (c) 80 mM to 148 mM of a viscosity modifier; and (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80; or (a) 100 mg / mL to 120 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 20 mM histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 5 mM to 50 mM histidine, (d) 0.4 mg / mL to 1.0 mg / mL of polysorbate 80, and (e) 50 mg / mL to 60 mg / mL of sucrose. or (a) 100 mg / mL to 140 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 10 mM to 40 mM of a viscosity modifier, (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, and (e) 40 mg / mL to 70 mg / mL of sucrose. or (a) 100 mg / mL to 200 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof; (b) 10 mM to 30 mM of a histidine-acetate buffer having a pH of 4.5 to 5.5; (c) 40 mM to 220 mM of a viscosity modifier; and (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80; A pharmaceutical composition comprising: wherein the viscosity modifier is selected from MgCl2, CaCl2, NaF, NaSCN, KCl, CH3COONa, Na2SO4, NaI, arginine, arginine hydrochloride, histidine and lysine; The anti-IL-4R antibody or antigen-binding fragment thereof is (i) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 38, SEQ ID NO: 7 and SEQ ID NO: 40, respectively; (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively; (iii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively; or (iv) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 42, SEQ ID NO: 39 and SEQ ID NO: 8, respectively; Includes:

20. (a) 100 mg / mL to 140 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM of histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 10 mM to 40 mM of a viscosity modifier, (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, and (e) 40 mg / mL to 70 mg / mL of sucrose (wherein the viscosity modifier is histidine, arginine hydrochloride, or MgCl2), or (a) 100 mg / mL to 180 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 40 mM to 90 mM MgCl 2 , and (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, or (a) 100 mg / mL to 180 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 50 mM to 100 mM histidine, and (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, or (a) 100 mg / mL to 180 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 10 mM to 30 mM histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 50 mM to 200 mM arginine hydrochloride, and (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, or (a) 100 mg / mL to 120 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 20 mM to 60 mM histidine-acetate buffer having a pH of 4.5 to 5.5, (c) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80, and (d) 40 mg / mL to 70 mg / mL of sucrose, or (a) 120 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) 50 mM histidine-acetate buffer having a pH of 5.0, (c) 0.8 mg / mL of polysorbate 80, and (d) 58 mg / mL of sucrose, or (a) about 150 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof, (b) about 20 mM histidine-acetate buffer having a pH of about 5.0, (c) about 0.8 mg / mL of polysorbate 80, and (d) about 120 mM arginine hydrochloride; A pharmaceutical composition comprising: wherein the anti-IL-4R antibody or antigen-binding fragment thereof is (i) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 38, SEQ ID NO: 7 and SEQ ID NO: 40, respectively; (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively; (iii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively; or (iv) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 42, SEQ ID NO: 39 and SEQ ID NO: 8, respectively; Includes:

21. The pharmaceutical composition of any one of claims 1 to 20, wherein the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region shown in any one of the following: (v) a heavy chain variable region sequence set forth in SEQ ID NO: 1 or having at least 90% identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 2 or having at least 90% identity thereto; (vi) the heavy chain variable region sequence is set forth in SEQ ID NO: 9 or has at least 90% identity to SEQ ID NO: 9, and the light chain variable region sequence is set forth in SEQ ID NO: 10 or has at least 90% identity to SEQ ID NO: 10; (vii) a heavy chain variable region sequence set forth in, or having at least 90% identity to, SEQ ID NO: 25, 26, 27, 43, or 47, and a light chain variable region sequence set forth in, or having at least 90% identity to, SEQ ID NO: 28, 29, 30, 37, or 41; or (viii) the heavy chain variable region sequence is set forth in SEQ ID NO: 31, 32, or 33, or has at least 90% identity to SEQ ID NO: 31, 32, or 33, and the light chain variable region sequence is set forth in SEQ ID NO: 34, 35, or 36, or has at least 90% identity to SEQ ID NO: 34, 35, or 36.

22. The pharmaceutical composition of claim 21, wherein the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region shown in any one of the following: (IX) the heavy chain variable region sequence is set forth in SEQ ID NO: 43 or has at least 90% identity to SEQ ID NO: 43, and the light chain variable region sequence is set forth in SEQ ID NO: 37 or has at least 90% identity to SEQ ID NO: 37; (X) the heavy chain variable region sequence is set forth in SEQ ID NO: 43 or has at least 90% identity to SEQ ID NO: 43, and the light chain variable region sequence is set forth in SEQ ID NO: 41 or has at least 90% identity to SEQ ID NO: 41; or (XI) the heavy chain variable region sequence is set forth in SEQ ID NO: 47 or has at least 90% identity to SEQ ID NO: 47, and the light chain variable region sequence is set forth in SEQ ID NO: 41 or has at least 90% identity to SEQ ID NO:

41.

23. The anti-IL-4R antibody or antigen-binding fragment thereof comprises a constant region, and the anti-IL-4R antibody comprises: a heavy chain as set forth in SEQ ID NO: 17 and a light chain as set forth in SEQ ID NO: 18, or a heavy chain as set forth in SEQ ID NO: 19 and a light chain as set forth in SEQ ID NO: 20, or a heavy chain as set forth in SEQ ID NO: 44 and a light chain as set forth in SEQ ID NO: 45, or a heavy chain as set forth in SEQ ID NO: 44 and a light chain as set forth in SEQ ID NO: 46, or a heavy chain as shown in SEQ ID NO: 48 and a light chain as shown in SEQ ID NO: 46; 22. The pharmaceutical composition of claim 21, comprising:

24. A method for preparing the pharmaceutical composition according to any one of claims 1 to 23, comprising the step of replacing a stock solution of the anti-IL-4R antibody or antigen-binding fragment thereof with a buffer.

25. A lyophilized preparation comprising an anti-IL-4R antibody or an antigen-binding fragment thereof, the lyophilized preparation being obtainable by lyophilizing the pharmaceutical composition according to any one of claims 1 to 23.

26. A reconstituted solution comprising an anti-IL-4R antibody or an antigen-binding fragment thereof, the reconstituted solution being obtainable by reconstituting the lyophilized formulation of claim 25.

27. A product comprising a container containing the pharmaceutical composition of any one of claims 1 to 23, the lyophilized formulation of claim 25, or the reconstitution solution of claim 26.

28. 24. The pharmaceutical composition according to any one of claims 1 to 23, for treating or preventing an immune disease or condition, wherein the immune disease or condition is selected from asthma, nasal polyps, chronic sinusitis, allergic skin disease, eosinophilic esophagitis, chronic obstructive pulmonary disease, allergic rhinitis, arthritis, inflammatory diseases, allergic reactions, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, and kidney disease.

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