Pharmaceutical composition containing Anti-il-4r antibody and use thereof

Abstract

An anti-IL-4R antibody pharmaceutical composition and its use are provided, the pharmaceutical composition comprising an anti-IL-4R antibody or its antigen-binding fragment in a histidine-acetate buffer, as well as a viscosity modifier, a surfactant and a stabilizer.

Description

Technical Field

[0001] This disclosure pertains to the field of pharmaceutical formulations, specifically relating to a pharmaceutical composition comprising an anti-IL-4R antibody or an antigen-binding fragment thereof, and its use as a treatment for immune diseases. Background Technology

[0002] The statements herein are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

[0003] Allergic diseases are serious medical conditions, including non-life-threatening allergic reactions and life-threatening allergic diseases. Current treatments for allergies include allergen avoidance, symptomatic pharmacological therapy, and prevention using allergen-specific immunotherapy.

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

[0005] The biological activity of IL-4 is mediated by specific cell surface IL-4 receptors (IL-4R). IL-4 receptors (IL-4R) consist of 802 amino acid residues and are expressed on the surface of T cells, B cells, thymocytes, bone marrow cells, macrophages, and mast cells. The α chain of IL-4R is also a component of the IL-13 receptor (IL-13R), therefore IL-4R can also mediate the biological activity of IL-13. Drugs and combinations thereof containing IL-4R antagonists can be administered as a novel treatment method before, during, or after exposure to allergens or the onset of allergic symptoms.

[0006] High protein concentrations pose challenges to the physical and chemical stability of proteins, leading to difficulties in their preparation, storage, and delivery. One issue is the tendency for proteins to form particles during handling and / or storage, which complicates 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] Considering that the increased viscosity of protein formulations has negative effects from formulation preparation to drug delivery to patients, there is a need to develop relatively high-concentration protein formulations with suitable low viscosity, which is suitable for the preparation, storage and administration of protein formulations.

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

[0010] In some embodiments, the aforementioned pharmaceutical composition contains a buffer with a pH of 4.8-5.5, preferably about 5.0.

[0011] In some embodiments, the buffer in the pharmaceutical composition has a pH 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 concentration of the histidine-acetic acid buffer in the pharmaceutical composition is from 10 mM to 60 mM, and 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 concentration of the buffer in the pharmaceutical composition is from about 10 mM to 50 mM, preferably from 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, with most preferably about 20 mM or 50 mM.

[0014] In some embodiments, the concentration of the anti-IL-4R antibody or its antigen-binding fragment in the pharmaceutical composition is from 100 mg / mL to 200 mg / mL, and non-limiting examples 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 concentration of the anti-IL-4R antibody or its antigen-binding fragment in the pharmaceutical composition is from 100 mg / mL to 180 mg / mL.

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

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

[0018] In some embodiments, the concentration of the anti-IL-4R antibody or its antigen-binding fragment in the pharmaceutical composition is from 100 mg / mL to 140 mg / mL.

[0019] In some embodiments, the concentration of the anti-IL-4R antibody or its antigen-binding fragment in the pharmaceutical composition is from 100 mg / mL to 120 mg / mL, and non-limiting examples 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 includes a viscosity modifier selected from MgCl2, CaCl2, NaF, NaSCN, KCl, CH3COONa, Na2SO4, NaI, Arg-HCl, arginine, histidine, and lysine, preferably selected from MgCl2, histidine, and arginine hydrochloride (Arg-HCl).

[0021] In some embodiments, the pharmaceutical composition comprises a viscosity modifier. In some cases, the antibody formulation has a high viscosity due to the high concentration of the antibody. 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 comprises 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-type 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 embodiments 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 point values.

[0023] In some embodiments, the concentration of the viscosity modifier is from about 10 mM to about 220 mM. In 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 viscosity modifier concentration 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. In 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 concentration of the viscosity modifier in the pharmaceutical composition is from 50 mM to 148 mM, preferably from 85 mM to 120 mM.

[0030] In some embodiments, the viscosity modifier is:

[0031] i) Arginine hydrochloride ranging from 5 mM to 220 mM;

[0032] ii) 5 mM to 100 mM histidine; or

[0033] iii) MgCl2 from 5 mM to 90 mM.

[0034] In some embodiments, the viscosity modifier is:

[0035] i) Arginine hydrochloride ranging from 10 mM to 220 mM;

[0036] ii) 10 mM to 100 mM histidine; or

[0037] iii) 10 mM to 90 mM MgCl2.

[0038] In some embodiments, the viscosity modifier is 5 mM to 220 mM arginine hydrochloride, and 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 point values.

[0039] In some embodiments, the viscosity modifier is 5 mM to 100 mM histidine, with non-limiting examples of 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.

[0040] In some embodiments, the viscosity modifier is 10 mM to 90 mM MgCl2, with non-limiting examples of 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.

[0041] In some embodiments, the concentration of the anti-IL-4R antibody or its antigen-binding fragment in the pharmaceutical composition is from 100 mg / mL to 200 mg / mL, and the viscosity modifier is selected from MgCl2, histidine, and arginine hydrochloride.

[0042] In some embodiments, when the concentration of the anti-IL-4R antibody or its antigen-binding fragment in the pharmaceutical composition is 100 mg / mL to 200 mg / mL, the viscosity modifier is selected from 50 mM to 200 mM MgCl2, histidine, and arginine hydrochloride.

[0043] In some embodiments, when the concentration of the anti-IL-4R antibody or its antigen-binding fragment in the pharmaceutical composition is 100 mg / mL to 200 mg / mL; the viscosity modifier 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.

[0044] In some embodiments, the concentration of the anti-IL-4R antibody or its antigen-binding fragment in the pharmaceutical composition is from 120 mg / mL to 150 mg / mL; and the viscosity modifier is selected from 50 mM to 90 mM MgCl2, 50 mM to 100 mM histidine and 50 mM to 120 mM arginine hydrochloride.

[0045] In some embodiments, the concentration of the anti-IL-4R antibody or its antigen-binding fragment in the pharmaceutical composition is from 100 mg / mL to 140 mg / mL; and the viscosity modifier is selected from histidine, arginine hydrochloride or MgCl2 from 5 mM to 50 mM, preferably from 10 mM to 40 mM of histidine, arginine hydrochloride or MgCl2.

[0046] In some embodiments, the concentration of the anti-IL-4R antibody or its antigen-binding fragment in the pharmaceutical composition is from 100 mg / mL to 140 mg / mL; and the viscosity modifier is 30 mM histidine, arginine hydrochloride, or MgCl2.

[0047] In some embodiments, the concentration of the anti-IL-4R antibody or its antigen-binding fragment in the pharmaceutical composition is from 100 mg / mL to 120 mg / mL; and the viscosity modifier is selected from histidine, arginine hydrochloride or MgCl2 from 5 mM to 50 mM, preferably from 10 mM to 40 mM of histidine, arginine hydrochloride or MgCl2.

[0048] In some embodiments, the viscosity modifier in the pharmaceutical composition is selected from 50 mM to 90 mM MgCl2, 85 mM to 100 mM histidine, and 90 mM to 120 mM arginine hydrochloride.

[0049] In some embodiments, the viscosity modifier in the pharmaceutical composition is selected from histidine, arginine hydrochloride or MgCl2 at 5 mM to 50 mM, preferably from 10 mM to 40 mM.

[0050] In some embodiments, the pharmaceutical composition further comprises a surfactant, preferably polysorbate 80.

[0051] In some embodiments, the concentration of the surfactant in the pharmaceutical composition is from 0.1 mg / mL to 1.2 mg / mL, preferably 0.8 mg / mL.

[0052] In some embodiments, the concentration of the surfactant in the pharmaceutical composition is from about 0.1 mg / mL to 1.0 mg / mL, preferably from 0.2 mg / mL to 0.8 mg / mL, more preferably from 0.4 mg / mL to 0.8 mg / mL. Non-limiting examples include, but are 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 the most preferred.

[0053] In some embodiments, the aforementioned pharmaceutical composition comprises:

[0054] (a) 100 mg / mL to 200 mg / mL of anti-IL-4R antibody or its antigen-binding fragment; (b) 10 mM to 30 mM of histidine-acetic acid buffer, pH 4.5-6.0; (c) 50 mM to 220 mM of 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; preferably, wherein the viscosity modifier is selected from MgCl2, histidine, and arginine hydrochloride.

[0055] In some embodiments, the pharmaceutical composition further comprises a stabilizer, wherein the stabilizer is preferably selected from trehalose or sucrose, and more preferably sucrose.

[0056] In some embodiments, the concentration of the stabilizer in the pharmaceutical composition is from 20 mg / mL to 70 mg / mL, preferably from 40 mg / mL to 60 mg / mL, and most preferably 58 mg / mL.

[0057] In some embodiments, the concentration of the stabilizer in the pharmaceutical composition is from 40 mg / mL to 70 mg / mL.

[0058] In some embodiments, the concentration of sugar in the aforementioned pharmaceutical composition is from about 50 mg / mL to about 60 mg / mL, preferably from 55 mg / mL to 60 mg / mL, and 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.

[0059] In some embodiments, the pharmaceutical composition comprises:

[0060] (a) 100 mg / mL to 150 mg / mL of an anti-IL-4R antibody or its antigen-binding fragment; (b) 10 mM to 30 mM of histidine-acetic acid buffer, pH 4.5-5.5; (c) 80 mM to 148 mM, or 5 mM to 50 mM of a viscosity modifier; (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80; and (e) 40 mg / mL to 60 mg / mL of sucrose, wherein the viscosity of the pharmaceutical composition is less than 20 mPa·s.

[0061] In some embodiments, the pharmaceutical composition comprises:

[0062] (a) 100 mg / mL to 150 mg / mL of anti-IL-4R antibody or its antigen-binding fragment; (b) 10 mM to 30 mM of histidine-acetic acid buffer, pH 4.5–5.5; (c) 80 mM to 148 mM of viscosity modifier; and (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80.

[0063] In some embodiments, the pharmaceutical composition comprises:

[0064] (a) 100 mg / mL to 120 mg / mL of anti-IL-4R antibody or its antigen-binding fragment; (b) 10 mM to 20 mM of histidine-acetate buffer, pH 4.5–5.5; (c) 10 mM to 40 mM of 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.

[0065] In some embodiments, the pharmaceutical composition comprises:

[0066] (a) 100 mg / mL to 140 mg / mL of anti-IL-4R antibody or its antigen-binding fragment; (b) 10 mM to 30 mM of histidine-acetate buffer, pH 4.5–5.5; (c) 10 mM to 40 mM of 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;

[0067] or

[0068] (a) 100 mg / mL to 200 mg / mL of anti-IL-4R antibody or its antigen-binding fragment; (b) 10 mM to 30 mM of histidine-acetic acid buffer, pH 4.5–5.5; (c) 40 mM to 220 mM of viscosity modifier; and (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80;

[0069] The viscosity modifiers mentioned therein are selected from: MgCl2, CaCl2, NaF, NaSCN, KCl, CH3COONa, Na2SO4, NaI, arginine, arginine hydrochloride, histidine, and lysine.

[0070] In some embodiments, the pharmaceutical composition comprises:

[0071] (a) 100 mg / mL to 140 mg / mL of anti-IL-4R antibody or its antigen-binding fragment; (b) 10 mM to 30 mM of histidine-acetate buffer, pH 4.5–5.5; (c) 10 mM to 40 mM of 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

[0072] (a) 100 mg / mL to 180 mg / mL of anti-IL-4R antibody or its antigen-binding fragment; (b) 10 mM to 30 mM of histidine-acetate buffer, pH 4.5–5.5; (c) 40 mM to 90 mM of MgCl2; and (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80; or

[0073] (a) 100 mg / mL to 180 mg / mL of anti-IL-4R antibody or its antigen-binding fragment; (b) 10 mM to 30 mM of histidine-acetate buffer, pH 4.5–5.5; (c) 50 mM to 100 mM of histidine; and (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80; or

[0074] (a) 100 mg / mL to 180 mg / mL of anti-IL-4R antibody or its antigen-binding fragment; (b) 10 mM to 30 mM of histidine-acetate buffer, pH 4.5–5.5; (c) 50 mM to 200 mM of arginine hydrochloride; and (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80.

[0075] In some embodiments, the pharmaceutical composition comprises:

[0076] (a) 100 mg / mL to 140 mg / mL of anti-IL-4R antibody or its antigen-binding fragment; (b) 10 mM to 30 mM of histidine-acetate buffer, pH 4.5–5.5; (c) 10 mM to 40 mM of 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.

[0077] In some embodiments, the pharmaceutical composition comprises:

[0078] (a) 120 mg / mL of anti-IL-4R antibody or its antigen-binding fragment; (b) 20 mM histidine-acetate buffer, pH 4.5–5.5; (c) 30 mM histidine; (d) 0.8 mg / mL polysorbate 80 and (e) 58 mg / mL sucrose.

[0079] In some embodiments, the pharmaceutical composition comprises:

[0080] (a) 120 mg / mL of anti-IL-4R antibody or its antigen-binding fragment; (b) 20 mM histidine-acetate buffer, pH approximately 5.0; (c) 30 mM histidine; (d) 0.8 mg / mL polysorbate 80 and (e) 58 mg / mL sucrose.

[0081] In some embodiments, the pharmaceutical composition comprises:

[0082] (a) 120 mg / mL of anti-IL-4R antibody or its antigen-binding fragment; (b) 50 mM histidine-acetate buffer, pH approximately 5.0; (c) 0.8 mg / mL of polysorbate 80; and (d) 58 mg / mL of sucrose.

[0083] In some embodiments, the pharmaceutical composition comprises:

[0084] (a) about 100 mg / mL to 180 mg / mL of anti-IL-4R antibody or its antigen-binding fragment; (b) about 10 mM to 30 mM of histidine-acetate buffer, pH 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.

[0085] In some embodiments, the pharmaceutical composition comprises:

[0086] (a) approximately 150 mg / mL of anti-IL-4R antibody or its antigen-binding fragment; (b) approximately 20 mM of histidine-acetate buffer, pH approximately 5.0; (c) approximately 0.8 mg / mL of polysorbate 80; and (d) approximately 120 mM of arginine hydrochloride.

[0087] In some embodiments, the pharmaceutical composition comprises:

[0088] (a) 100 mg / mL to 140 mg / mL of anti-IL-4R antibody or its antigen-binding fragment; (b) 20 mM to 60 mM of histidine-acetate buffer, pH 4.5–5.5; (c) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80; and (d) 40 mg / mL to 70 mg / mL of sucrose.

[0089] In some embodiments, the pharmaceutical composition comprises:

[0090] (a) 100 mg / mL to 140 mg / mL of anti-IL-4R antibody or its antigen-binding fragment; (b) 50 mM histidine-acetate buffer, pH 4.5–5.5; (c) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80; and (d) 40 mg / mL to 70 mg / mL of sucrose.

[0091] In some embodiments, the pharmaceutical composition comprises:

[0092] (a) 100 mg / mL to 120 mg / mL of anti-IL-4R antibody or its antigen-binding fragment; (b) 20 mM to 60 mM of histidine-acetate buffer, pH 5.0-5.5; (c) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80; and (d) 50 mg / mL to 60 mg / mL of sucrose; preferably, comprising:

[0093] (a) 120 mg / mL of anti-IL-4R antibody or its antigen-binding fragment; (b) 50 mM histidine-acetate buffer, pH 5.0-5.5; (c) 0.8 mg / mL of polysorbate 80 and (d) 58 mg / mL of sucrose.

[0094] In some embodiments, the pharmaceutical composition comprises:

[0095] (a) 120 mg / mL of anti-IL-4R antibody or its antigen-binding fragment; (b) 50 mM histidine-acetate buffer, pH approximately 5.0; (c) 0.8 mg / mL of polysorbate 80; and (d) 58 mg / mL of sucrose.

[0096] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment in the pharmaceutical composition comprises a heavy chain variable region and a light chain variable region as shown below:

[0097] (i) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; and

[0098] The light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 respectively;

[0099] (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; and

[0100] The light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 respectively;

[0101] (iii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; and

[0102] The light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 38, SEQ ID NO: 7, and SEQ ID NO: 40, respectively; or

[0103] (iv) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; and

[0104] The light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 42, SEQ ID NO: 39 and SEQ ID NO: 8, respectively.

[0105] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment in the pharmaceutical composition comprises:

[0106] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; and

[0107] The light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 38, SEQ ID NO: 7 and SEQ ID NO: 40, respectively.

[0108] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment in the pharmaceutical composition comprises a heavy chain variable region and a light chain variable region as shown below:

[0109] (v) The heavy chain variable region sequence is as shown in SEQ ID NO: 1 or has at least 90% identity with SEQ ID NO: 1, and the light chain variable region sequence is as shown in SEQ ID NO: 2 or has at least 90% identity with SEQ ID NO: 2;

[0110] (vi) The heavy chain variable region sequence is as shown in SEQ ID NO: 9 or has at least 90% identity with SEQ ID NO: 9, and the light chain variable region sequence is as shown in SEQ ID NO: 10 or has at least 90% identity with SEQ ID NO: 10;

[0111] (vii) The heavy chain variable region sequence is as shown in SEQ ID NO: 25, 26, 27, 43 or 47, or has at least 90% identity with SEQ ID NO: 25, 26, 27, 43 or 47; the light chain variable region sequence is as shown in SEQ ID NO: 28, 29, 30, 37 or 41, or has at least 90% identity with SEQ ID NO: 28, 29, 30, 37 or 41; or

[0112] (viii) The heavy chain variable region sequence is as shown in SEQ ID NO: 31, 32 or 33, or has at least 90% identity with SEQ ID NO: 31, 32 or 33, and the light chain variable region sequence is as shown in SEQ ID NO: 34, 35 or 36, or has at least 90% identity with SEQ ID NO: 34, 35 or 36.

[0113] Preferably, the anti-IL-4R antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region as shown below:

[0114] (IX) The heavy chain variable region sequence is as shown in SEQ ID NO: 43 or has at least 90% identity with SEQ ID NO: 43, and the light chain variable region sequence is as shown in SEQ ID NO: 37 or has at least 90% identity with SEQ ID NO: 37; or

[0115] (X) The heavy chain variable region sequence is as shown in SEQ ID NO: 43 or has at least 90% identity with SEQ ID NO: 43, and the light chain variable region sequence is as shown in SEQ ID NO: 41 or has at least 90% identity with SEQ ID NO: 41; or

[0116] (XI) The heavy chain variable region sequence is as shown in SEQ ID NO: 47 or has at least 90% identity with SEQ ID NO: 47, and the light chain variable region sequence is as shown in SEQ ID NO: 41 or has at least 90% identity with SEQ ID NO: 41.

[0117] In some embodiments, the heavy chain variable region sequence of the anti-IL-4R antibody or its antigen-binding fragment in the pharmaceutical composition is shown in SEQ ID NO: 43, and the light chain variable region sequence is shown in SEQ ID NO: 37.

[0118] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment in the pharmaceutical composition includes a constant region. In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment includes a constant region of a human κ, λ chain or a variant thereof; and also includes a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, such as IgG4-S228P or IgG4-234A / 235A mutants.

[0119] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment in the pharmaceutical composition comprises a heavy chain and a light chain as shown below:

[0120] The heavy chain shown in SEQ ID NO: 17 and the light chain shown in SEQ ID NO: 18; or

[0121] The heavy chain shown in SEQ ID NO: 19 and the light chain shown in SEQ ID NO: 20; or

[0122] The heavy chain shown in SEQ ID NO: 44 and the light chain shown in SEQ ID NO: 45; or

[0123] The heavy chain shown in SEQ ID NO: 44 and the light chain shown in SEQ ID NO: 46; or

[0124] The heavy chain shown in SEQ ID NO: 48 and the light chain shown in SEQ ID NO: 46.

[0125] In some embodiments, the anti-IL-4R antibody or its antigen-binding fragment in the pharmaceutical composition comprises the heavy chain shown in SEQ ID NO: 44 and the light chain shown in SEQ ID NO: 45.

[0126] This application also relates to an anti-IL-4R antibody, the heavy chain variable region sequence of which is shown in SEQ ID NO: 47 or has at least 90% identity with SEQ ID NO: 47, and the light chain variable region sequence of which is shown in SEQ ID NO: 41 or has at least 90% identity with SEQ ID NO: 41.

[0127] This application also relates to an anti-IL-4R antibody comprising a heavy chain as shown in SEQ ID NO: 48 and a light chain as shown in SEQ ID NO: 46.

[0128] In one embodiment, the pharmaceutical composition comprises: (a) 100 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetate buffer, pH 4.5-6.0; and (c) 0.1 mg / mL polysorbate 80.

[0129] In one embodiment, the pharmaceutical composition comprises: (a) 100 mg / mL hu25G7-A antibody; and (b) 20 mM histidine-acetate buffer, pH 4.5.

[0130] In one embodiment, the pharmaceutical composition comprises: (a) 100 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetate buffer, pH 4.5; and (c) 0.1 mg / mL polysorbate 80.

[0131] In one embodiment, the pharmaceutical composition comprises: (a) 100 mg / mL hu25G7-A antibody; and (b) 20 mM histidine-acetate buffer, pH 5.0.

[0132] In one embodiment, the pharmaceutical composition comprises: (a) 100 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetate buffer, pH 5.0; and (c) 0.1 mg / mL polysorbate 80.

[0133] In one embodiment, the pharmaceutical composition comprises: (a) 100 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetate buffer, pH 6.0; and (c) 0.1 mg / mL polysorbate 80.

[0134] In one embodiment, the pharmaceutical composition comprises: (a) 100 mg / mL hu25G7-A antibody; and (b) 20 mM histidine-acetate buffer, pH 5.5.

[0135] In one embodiment, the pharmaceutical composition comprises: (a) 100 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetate buffer, pH 5.5; and (c) 0.1 mg / mL polysorbate 80.

[0136] In one embodiment, the pharmaceutical composition comprises: (a) 100-150 mg / mL hu25G7-A antibody; and (b) 20 mM histidine-acetate buffer, pH 5.0-5.5.

[0137] In one embodiment, the pharmaceutical composition comprises: (a) 120 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetic acid buffer, pH 5.0; and (c) 122 mM NaCl.

[0138] In one embodiment, the pharmaceutical composition comprises: (a) 120 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetate buffer, pH 5.0; and (c) 85 mM MgCl2.

[0139] In one embodiment, the pharmaceutical composition comprises: (a) 120 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetic acid buffer, pH 5.0; and (c) 148 mM CaCl2.

[0140] In one embodiment, the pharmaceutical composition comprises: (a) 120 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetic acid buffer, pH 5.0; and (c) 124 mM KCl.

[0141] In one embodiment, the pharmaceutical composition comprises: (a) 120 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetic acid buffer, pH 5.0; and (c) 86 mM CH3COONa.

[0142] In one embodiment, the pharmaceutical composition comprises: (a) 120 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetic acid buffer, pH 5.0; and (c) 96 mM Na2SO4.

[0143] In one embodiment, the pharmaceutical composition comprises: (a) 120 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetate buffer, pH 5.0; and (c) 113 mM NaI.

[0144] In one embodiment, the pharmaceutical composition comprises: (a) 120 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetate buffer, pH 5.0; and (c) 74 mM NaF.

[0145] In one embodiment, the pharmaceutical composition comprises: (a) 120 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetic acid buffer, pH 5.0; and (c) 112 mM NaSCN.

[0146] In one embodiment, the pharmaceutical composition comprises: (a) 120 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetic acid buffer, pH 5.0; and (c) 120 mM Arg-HCl.

[0147] In one embodiment, the pharmaceutical composition comprises: (a) 120 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetate buffer, pH 5.0; and (c) 118 mM lysine.

[0148] In one embodiment, the pharmaceutical composition comprises: (a) 120 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetate buffer, pH 5.0; and (c) 93 mM histidine.

[0149] In one embodiment, the pharmaceutical composition comprises: (a) 120 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetate buffer, pH 5.0; and (c) 207 mM proline.

[0150] In one embodiment, the pharmaceutical composition comprises: (a) 150 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetic acid buffer, pH 5.0; and (c) 85 mM MgCl2.

[0151] In one embodiment, the pharmaceutical composition comprises: (a) 150 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetic acid buffer, pH 5.0; and (c) 148 mM CaCl2.

[0152] In one embodiment, the pharmaceutical composition comprises: (a) 150 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetate buffer, pH 5.0; and (c) 93 mM histidine.

[0153] In one embodiment, the pharmaceutical composition comprises: (a) 150 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetic acid buffer, pH 5.0; and (c) 50 mM MgCl2.

[0154] In one embodiment, the pharmaceutical composition comprises: (a) 150 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetic acid buffer, pH 5.0; and (c) 90 mM MgCl2.

[0155] In one embodiment, the pharmaceutical composition comprises: (a) 150 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetic acid buffer, pH 5.0; and (c) 90 mM CaCl2.

[0156] In one embodiment, the pharmaceutical composition comprises: (a) 150 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetic acid buffer, pH 5.0; and (c) 148 mM CaCl2.

[0157] In one embodiment, the pharmaceutical composition comprises: (a) 150 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetate buffer, pH 5.0; and (c) 90 mM histidine.

[0158] In one embodiment, the pharmaceutical composition comprises: (a) 150 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetic acid buffer, pH 5.0; and (c) 120 mM Arg-HCl.

[0159] In one embodiment, the pharmaceutical composition comprises: (a) 150 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetic acid buffer, pH 5.0; (c) 90 mM MgCl2; and (d) 0.1 mg / mL polysorbate 80.

[0160] In one embodiment, the pharmaceutical composition comprises: (a) 150 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetic acid buffer, pH 5.0; (c) 90 mM MgCl2; and (d) 1 mg / mL polysorbate 80.

[0161] In one embodiment, the pharmaceutical composition comprises: (a) 120 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetate buffer, pH 5.0; (c) 122 mM NaCl; and (d) 0.1 mg / mL polysorbate 80.

[0162] In one embodiment, the pharmaceutical composition comprises: (a) 120 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetic acid buffer, pH 5.0; (c) 122 mM NaCl; and (d) 1 mg / mL polysorbate 80.

[0163] In one embodiment, the pharmaceutical composition comprises: (a) 150 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetate buffer, pH 5.0; (c) 90 mM histidine; and (d) 0.1 mg / mL polysorbate 80.

[0164] In one embodiment, the pharmaceutical composition comprises: (a) 150 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetate buffer, pH 5.0; (c) 90 mM histidine; and (d) 1 mg / mL polysorbate 80.

[0165] In one embodiment, the pharmaceutical composition comprises: (a) 150 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetic acid buffer, pH 5.0; (c) 120 mM Arg-HCl; and (d) 0.1 mg / mL polysorbate 80.

[0166] In one embodiment, the pharmaceutical composition comprises: (a) 150 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetic acid buffer, pH 5.0; (c) 120 mM Arg-HCl; and (d) 1 mg / mL polysorbate 80.

[0167] In one embodiment, the pharmaceutical composition comprises: (a) about 165 mg / mL hu25G7-A antibody; (b) about 20 mM histidine-acetate buffer, pH 5.0; and (c) about 50 mM to 90 mM MgCl2.

[0168] In one embodiment, the pharmaceutical composition comprises: (a) about 165 mg / mL hu25G7-A antibody; (b) about 20 mM histidine-acetate buffer, pH 5.0; and (c) about 50 mM to 90 mM histidine.

[0169] In one embodiment, the pharmaceutical composition comprises: (a) about 165 mg / mL hu25G7-A antibody; (b) about 20 mM histidine-acetic acid buffer, pH 5.0; and (c) about 90 mM to 200 mM Arg-HCl.

[0170] In one embodiment, the pharmaceutical composition comprises: (a) 150 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetic acid buffer, pH 5.0; (c) 120 mM Arg-HCl; and (d) 0.8 mg / mL polysorbate 80.

[0171] In one embodiment, the pharmaceutical composition comprises: (a) 150 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetate buffer, pH 4.8; (c) 87 mM histidine; and (d) 0.8 mg / mL polysorbate 80.

[0172] In one embodiment, the pharmaceutical composition comprises: (a) 150 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetate buffer, pH 5.0; (c) 100 mM histidine; and (d) 0.8 mg / mL polysorbate 80.

[0173] In one embodiment, the pharmaceutical composition comprises: (a) 150 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetate buffer, pH 5.0; (c) 30 mM histidine; and (d) 0.8 mg / mL polysorbate 80; and (e) 41.8 mg / mL sucrose.

[0174] In one embodiment, the pharmaceutical composition comprises: (a) 120 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetate buffer, pH 5.0; (c) 30 mM histidine; and (d) 0.8 mg / mL polysorbate 80; and (e) 58 mg / mL sucrose.

[0175] In one embodiment, the pharmaceutical composition comprises: (a) 120 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetate buffer, pH 5.2; (c) 30 mM histidine; and (d) 0.4 mg / mL polysorbate 80; and (e) 50 mg / mL sucrose.

[0176] In one embodiment, the pharmaceutical composition comprises: (a) 120 mg / mL hu25G7-A antibody; (b) 50 mM histidine-acetate buffer, pH about 5.0; (c) 0.8 mg / mL polysorbate 80; and (d) 58 mg / mL sucrose.

[0177] In one embodiment, the pharmaceutical composition comprises: (a) 120 mg / mL to 150 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetate buffer, pH 5.0; (c) 30 mM to 100 mM histidine; (d) 0.8 mg / mL polysorbate 80; and (e) 41.8 mg / mL to 58 mM sucrose.

[0178] In one embodiment, the pharmaceutical composition comprises: (a) 150 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetate buffer, pH 5.0; (c) 120 mM arginine hydrochloride; and (d) 0.8 mg / mL polysorbate 80.

[0179] In one embodiment, the pharmaceutical composition comprises: (a) 100 mg / mL to 140 mg / mL hu25G7-A antibody; (b) 50 mM histidine-acetate buffer at pH 4.5 to 5.5; (c) 0.4 mg / mL to 1.2 mg / mL polysorbate 80; and (d) about 58 mg / mL sucrose.

[0180] In one embodiment, the pharmaceutical composition comprises: (a) 100 mg / mL to 140 mg / mL hu25G7-A antibody; (b) 50 mM histidine-acetate buffer at pH 4.8 to 5.5; (c) 0.4 mg / mL to 1.2 mg / mL polysorbate 80; and (d) about 58 mg / mL sucrose.

[0181] In one embodiment, the pharmaceutical composition comprises: (a) 100 mg / mL to 120 mg / mL hu25G7-A antibody; (b) 50 mM histidine-acetate buffer at pH 4.5 to 5.5; (c) 0.4 mg / mL to 0.8 mg / mL polysorbate 80; and (d) about 58 mg / mL sucrose.

[0182] In one embodiment, the pharmaceutical composition comprises: (a) about 132 mg / mL hu25G7-A antibody; (b) 50 mM histidine-acetate buffer, pH about 5.5; (c) about 0.4 mg / mL polysorbate 80; and (d) about 58 mg / mL sucrose.

[0183] In one embodiment, the pharmaceutical composition comprises: (a) about 100 mg / mL hu25G7-A antibody; (b) 50 mM histidine-acetate buffer, pH about 5.0; (c) about 0.8 mg / mL polysorbate 80; and (d) about 58 mg / mL sucrose.

[0184] In one embodiment, the pharmaceutical composition comprises: (a) about 100 mg / mL hu25G7-A antibody; (b) 50 mM histidine-acetate buffer, pH about 4.5; (c) about 0.4 mg / mL polysorbate 80; and (d) about 58 mg / mL sucrose.

[0185] In one embodiment, the pharmaceutical composition comprises: (a) about 140 mg / mL hu25G7-A antibody; (b) 50 mM histidine-acetate buffer, pH about 4.5; (c) about 0.8 mg / mL polysorbate 80; and (d) about 58 mg / mL sucrose.

[0186] In one embodiment, the pharmaceutical composition comprises: (a) about 120 mg / mL hu25G7-A antibody; (b) 50 mM histidine-acetate buffer, pH about 4.5; (c) about 1.2 mg / mL polysorbate 80; and (d) about 58 mg / mL sucrose.

[0187] In one embodiment, the pharmaceutical composition comprises: (a) about 100 mg / mL hu25G7-A antibody; (b) 50 mM histidine-acetate buffer, pH about 5; (c) about 1.2 mg / mL polysorbate 80; and (d) about 58 mg / mL sucrose.

[0188] In one embodiment, the pharmaceutical composition comprises: (a) about 100 mg / mL hu25G7-A antibody; (b) 50 mM histidine-acetate buffer, pH about 5.5; (c) about 0.4 mg / mL polysorbate 80; and (d) about 58 mg / mL sucrose.

[0189] In one embodiment, the pharmaceutical composition comprises: (a) about 120 mg / mL hu25G7-A antibody; (b) 50 mM histidine-acetate buffer, pH about 5.5; (c) about 1.2 mg / mL polysorbate 80; and (d) about 58 mg / mL sucrose.

[0190] In one embodiment, the pharmaceutical composition comprises: (a) about 120 mg / mL hu25G7-A antibody; (b) 50 mM histidine-acetate buffer, pH about 5; (c) about 0.8 mg / mL polysorbate 80; and (d) about 58 mg / mL sucrose.

[0191] This disclosure also provides a method for preparing the pharmaceutical composition, comprising the step of replacing the stock solution of an anti-IL-4R antibody or its antigen-binding fragment with a buffer.

[0192] This disclosure also provides a lyophilized formulation containing an anti-IL-4R antibody or an antigen-binding fragment thereof, wherein the lyophilized formulation is obtained by freeze-drying the aforementioned pharmaceutical composition.

[0193] This disclosure also provides a lyophilized formulation containing an anti-IL-4R antibody or an antigen-binding fragment thereof, wherein the lyophilized formulation is obtained by lyophilizing the aforementioned pharmaceutical composition after dilution.

[0194] This disclosure also provides a lyophilized formulation containing an anti-IL-4R antibody or its antigen-binding fragment, wherein the lyophilized formulation is obtained by lyophilizing the aforementioned pharmaceutical composition after diluting it by 1, 2, or 3 times.

[0195] This disclosure also provides a reconstituted solution containing an anti-IL-4R antibody or its antigen-binding fragment, characterized in that the reconstituted solution is prepared by reconstituted the aforementioned lyophilized formulation.

[0196] In some embodiments, the reconstituted solution comprises the following components:

[0197] (a) 120 mg / mL of anti-IL-4R antibody or its antigen-binding fragment; (b) 50 mM histidine-acetate buffer, pH approximately 5.0; (c) 0.4 mg / mL of polysorbate 80; and (e) 50 mg / mL of sucrose.

[0198] In one embodiment, the reconstitution solution comprises: (a) about 120 mg / mL hu25G7-A antibody; (b) about 50 mM histidine-acetate buffer; (c) about 0.8 mg / mL polysorbate 80; and (d) about 58 mg / mL sucrose, and the pH of the pharmaceutical composition is about 5.3.

[0199] In one embodiment, the reconstitution solution comprises: (a) about 150 mg / mL hu25G7-A antibody; (b) about 20 mM histidine-acetate buffer; (c) about 0.8 mg / mL polysorbate 80; and (d) about 120 mM arginine hydrochloride, and the pH of the pharmaceutical composition is about 5.3.

[0200] This disclosure also provides an article of manufacture comprising a container containing the aforementioned pharmaceutical composition, lyophilized formulation, or reconstituted solution.

[0201] This disclosure also provides a method for treating or preventing an immune disease or condition, the method comprising administering to a subject a therapeutically effective amount of the aforementioned pharmaceutical composition or lyophilized preparation or reconstituted solution, preferably wherein the immune disease is an IL-4R-mediated disease or condition.

[0202] In some embodiments, the immune disease or condition is selected from: asthma, nasal polyps, chronic sinusitis, allergic dermatitis, eosinophilic esophagitis, chronic obstructive pulmonary disease, allergic rhinitis, arthritis, inflammatory diseases, allergic reactions, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, and nephropathy; preferably, the disease or condition is asthma or allergic dermatitis.

[0203] In some implementations, the immune disease or condition is asthma.

[0204] In other embodiments, the immune disease or condition is an allergic skin disease.

[0205] This disclosure also provides the use of the aforementioned pharmaceutical compositions, or lyophilized formulations or reconstituted solutions of lyophilized formulations, or articles thereof in the preparation of medicaments for the treatment or prevention of immune diseases or conditions, preferably wherein said immune diseases or conditions are IL-4R-mediated diseases or conditions.

[0206] The pharmaceutical compositions, or lyophilized preparations or reconstituted solutions of lyophilized preparations, or articles thereof disclosed herein may be used as pharmaceuticals, preferably as pharmaceuticals for the treatment or prevention of immune diseases or conditions, and more preferably as pharmaceuticals for the treatment of IL-4R-mediated diseases or conditions.

[0207] The pharmaceutical compositions, lyophilized preparations, or reconstituted solutions of lyophilized preparations, or articles thereof disclosed herein may be used as medicines, preferably as medicines for treating or preventing immune diseases or conditions, more preferably, wherein the immune diseases or conditions are selected from: asthma, nasal polyps, chronic sinusitis, allergic dermatitis, eosinophilic esophagitis, chronic obstructive pulmonary disease, allergic rhinitis, arthritis, inflammatory diseases, allergic reactions, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, and nephropathy; preferably, the disease or condition is asthma or allergic dermatitis.

[0208] In this application, histidine acts as both a buffer and a viscosity modifier. Therefore, the final histidine content in the pharmaceutical composition is the sum of the histidine content in the buffer and the histidine content added to further reduce viscosity. For example, in a 20mM histidine-acetic acid buffer at pH 5.0, if 90mM of histidine is added to enhance its viscosity-reducing effect, the final histidine concentration in the pharmaceutical composition will be 110mM, and the pH of the pharmaceutical composition will be approximately 5.0. Attached Figure Description

[0209] Figure 1Figure 1 shows the experimental results of the effect of anti-IL-4R antibody on mouse dermatitis. In a mouse dermatitis model, after sensitization with acetone, humanized antibodies hu25G7-A and hu25G7-B, and the positive control antibody dupilumab were administered subcutaneously twice a week. Ear thickness was measured on day 27. The results showed that, compared with the control group, hu25G7-A, hu25G7-B, and dupilumab all effectively reduced ear thickness in mice, with hu25G7-B showing a superior effect compared to dupilumab.

[0210] Figure 2 : Results of fitting the formulation.

[0211] Figure 3 Contour plot of formulation stability changes, where gray areas indicate limits exceeded and white areas indicate limits not exceeded. Detailed Implementation

[0212] the term

[0213] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0214] This disclosure incorporates the entire contents of application PCT / CN2019 / 102169 into this application.

[0215] "Buffer" refers to a buffer that tolerates pH changes through the action of its acid-base conjugate components. Examples of buffers that maintain pH within an appropriate range include acetates, succinates, gluconates, histidines, oxalates, lactates, phosphates, citrates, tartrates, fumarates, glycylglycine, and other organic acid buffers.

[0216] "Histidine buffer" is a buffer containing histidine ions. Examples of histidine buffers include histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate, etc., with histidine-acetate buffer being preferred. Histidine-acetate buffer is prepared by reacting histidine with acetic acid and is also called histidine-acetic acid (His-AA) buffer.

[0217] "Citrate buffer" is a buffer that includes citrate ions. Examples of citrate buffers include sodium citrate, potassium citrate, calcium citrate, and magnesium citrate buffers. A preferred citrate buffer is sodium citrate buffer.

[0218] "Succinate buffer" is a buffer that includes succinate ions. Examples of succinate buffers include sodium succinate-sodium succinate, potassium succinate-potassium succinate, and calcium succinate-calcium succinate buffers. A preferred succinate buffer is sodium succinate-sodium succinate buffer.

[0219] A "phosphate buffer" is a buffer that contains phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid buffer, etc. A preferred phosphate buffer is the disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

[0220] "Acetate buffer" is a buffer that includes acetate ions. Examples of acetate buffers include sodium acetate, histidine acetate, potassium acetate, calcium acetate, magnesium acetate, etc. Preferred acetate buffer is sodium acetate buffer.

[0221] "Pharmaceutical composition" refers to a mixture containing one or more antibodies or antigen-binding fragments described herein, along with other chemical components, such as physiological / pharmaceutical-grade carriers and excipients. The purpose of a pharmaceutical composition is to maintain the stability of the antibody's active ingredient, facilitate administration to the organism, and enhance the absorption of the active ingredient to exert its biological activity.

[0222] In this article, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0223] Unless otherwise specified, the solvents in the solution forms of the pharmaceutical compositions described in this disclosure are all aqueous solutions.

[0224] "Displacement" refers to the replacement of the solvent system in which the antibody protein is dissolved. For example, using a buffer system of a stabilizing formulation, a high-salt or hypertonic solvent system containing the antibody protein is replaced by a physical manipulation, thereby ensuring the antibody protein remains in the stabilizing formulation. The physical manipulation methods include, but are not limited to, ultrafiltration, dialysis, or reconstitution after centrifugation.

[0225] "Lyophilized formulation" refers to a pharmaceutical composition or formulation obtained by a vacuum freeze-drying step after the liquid or solution form has been processed.

[0226] The "sugar" disclosed herein comprises a conventional composition (CH2O). nAnd its derivatives, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. Selectable sugars include glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabinitol, xylitol, sorbitol, mannitol, melitriose, maltotriose, stachyose, maltose, lactulose, maltitol, maltitol, lactitol, isomaltulose, etc. Preferred sugars are non-reducing disaccharides, more preferably trehalose or sucrose, and most preferably sucrose.

[0227] The surfactants disclosed herein may be selected from polysorbate 20, polysorbate 80, polyhydroxyene, Triton, sodium dodecyl sulfonate, sodium lauryl sulfonate, sodium octyl glycoside, lauryl / myristyl / linoleyl / stearyl-sulfobetaine, lauryl / myristyl / linoleyl / stearyl-sarcosine, linoleyl / myristyl / cetyl-betaine, lauramide propyl / cocarbamate propyl / linoleamide propyl / myristamidopropyl / palmitamidopropyl / isostearamidopropyl-betaine, myristamidopropyl / palmitamidopropyl / isostearamidopropyl-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.

[0228] The term "viscosity" can be either "kinematic viscosity" or "absolute viscosity." Kinematic viscosity is a measure of how much a fluid resists flow under the influence of gravity. When two equal volumes of fluid are placed in the same capillary viscometer and allowed to flow by gravity, the viscous fluid takes longer to flow through the capillary than the less viscous fluid. For example, if one fluid takes 200 seconds to complete its flow while the other takes 400 seconds, the kinematic viscosity of the second fluid is twice that of the first. Absolute viscosity, sometimes called kinetic or simple viscosity, is the result of kinematic viscosity and fluid density (absolute viscosity = kinematic viscosity × density). The unit of kinematic viscosity is liters (L). 2 / T, where L is length and T is time. Kinematic viscosity is typically expressed in centiliters (cSt). The SI unit for 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), where 1 cP = 1 mPa-s.

[0229] As used herein, the terms “about” and “approximately” mean a numerical value within the acceptable error range of a specific value determined by a person skilled in the art, the numerical value depending in part on how it is measured or determined (i.e., the limits of the measurement system). For example, in every practice in the art, “about” may mean within or above 1 standard deviation. Alternatively, “about” or “substantially includes” may mean a range of ±20%, ±15%, ±10%, or ±5% of the specific numerical value shown thereafter. Furthermore, particularly for biological systems or processes, the term may mean at most one order of magnitude or at most five times the numerical value. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of “about” or “substantially includes” should be assumed to be within the acceptable error range of that specific value.

[0230] The pharmaceutical compositions disclosed herein achieve a stable effect: the antibodies therein substantially retain their physical stability, and / or chemical stability, and / or biological activity after storage. Preferably, the pharmaceutical compositions substantially retain their physical and chemical stability, as well as their 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 for measuring protein stability, which can measure stability after storage at a selected temperature for a selected period of time.

[0231] Stable drug antibody formulations are those in which no significant changes are observed when stored at refrigerated temperatures (2-8°C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. Additionally, stable liquid formulations include those that exhibit the desired characteristics after storage at temperatures including 25°C for periods of 1 month, 3 months, and 6 months. Typical acceptable criteria for stability are as follows: Degradation of antibody monomers typically not exceeding about 10%, preferably not exceeding about 5%, as determined by SEC-HPLC. Visually, the drug antibody formulation is a pale yellow, nearly colorless, clear liquid or colorless, or clear to slightly milky white. The concentration, pH, and osmotic pressure of the formulation exhibit variations not exceeding ±10%. A reduction of not more than about 10%, preferably not more than about 5%, is typically observed. Aggregation typically forms at a rate of not more than about 10%, preferably not more than about 5%.

[0232] If, after visual inspection of color and / or clarity, or by means of UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS), the antibody does not show significant increase in aggregation, precipitation, and / or denaturation, then the antibody “retains its physical stability” in the pharmaceutical formulation. Changes in protein conformation can be evaluated by fluorescence spectroscopy (which determines the tertiary structure of the protein) and by FTIR spectroscopy (which determines the secondary structure of the protein).

[0233] If an antibody does not show significant chemical changes, then the antibody "retains its chemical stability" in the pharmaceutical formulation. Chemical stability can be assessed by detecting and quantifying the chemically altered form of the protein. Degradation processes that frequently alter the chemical structure of a protein include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and SDS-PAGE), oxidation (evaluated by methods such as peptide mapping combined with mass spectrometry or MALDI / TOF / MS), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, and isofpartate measurement), and isomerization (evaluated by measuring isofpartate content, peptide mapping, etc.).

[0234] If the antibody's biological activity at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared, then the antibody "retains its biological activity" in the pharmaceutical formulation. The biological activity of an antibody can be determined, for example, by an antigen-binding assay.

[0235] "Human IL-4R" (hIL-4R) refers to the human cytokine receptor that specifically binds to interleukin-4 (IL-4) and IL-4Rα.

[0236] The amino acid three-letter codes and single-letter codes used in this article are as described in J. Biol. Chem, 243, p3558 (1968).

[0237] The term "antibody (Ab)" includes any antigen-binding molecule or molecular complex comprising at least one complementarity-determining region (CDR) that specifically binds to or interacts with a specific antigen (or its epitope, such as the IL-4R antigen or its epitope). The term "antibody" includes: an immunoglobulin molecule comprising four polypeptide chains interconnected by disulfide bonds, two heavy (H) chains and two light (L) chains, and its polymers (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated 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 comprises a light chain variable region (abbreviated 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 subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0238] The FR of an anti-IL-4R antibody (or its antigen-binding fragment) may be identical to that of a human lineage or may be naturally or artificially modified. The antibody may be a different subclass, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subclasses), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0239] 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) the smallest recognition unit consisting of amino acid residues mimicking the hypervariable region of an antibody (e.g., a separated complementarity-determining region (CDR), such as a CDR3 peptide) or a restricted FR3-CDR3-FR4 peptide. Other engineered molecules, such as region-specific antibodies, single-domain antibodies, region-deleted antibodies, chimeric antibodies, CDR-implanted antibodies, biantibodies, triantibodies, tetraantibodies, microantibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and squalane-variable IgNAR regions, are also included in the term "antigen-binding fragment" as used herein.

[0240] Antigen-binding fragments of antibodies typically contain at least one variable region. The variable region can be of any size or amino acid composition and generally contains a CDR adjacent to or within one or more frame sequences. In antigen-binding fragments having both VH and VL regions, the VH and VL regions can be arranged opposite each other in any suitable configuration. For example, the variable region can be dimerized and contain VH-VL or VL-VH dimers.

[0241] In some embodiments, in any configuration of variable and constant regions, the antigen-binding fragment may have the variable and constant regions directly linked to each other or linked by complete or partial hinge or linker regions. The hinge region may consist of at least two amino acids (e.g., 5, 10, 15, 20, 40, 60, or more), such that it creates flexible and semi-flexible linkages between adjacent variable and / or constant regions within a single polypeptide molecule. Furthermore, the antigen-binding fragment disclosed herein may comprise a homodimer or heterodimer (or other multimer) 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.

[0242] In this disclosure, "mouse-derived antibody" refers to a monoclonal antibody derived from a mouse or rat and prepared in accordance with the knowledge and skills in the art. Preparation involves injecting an antigen into the test subject, followed by isolating a hybridoma expressing an antibody with the desired sequence or functional characteristics. When the injected test subject is a mouse, the resulting antibody is a mouse-derived antibody; when the injected test subject is a rat, the resulting antibody is a rat-derived antibody.

[0243] A chimeric antibody is an antibody formed by fusing the variable region of a first-species (e.g., mouse) antibody with the constant region of a second-species (e.g., human) antibody. To create a chimeric antibody, a hybridoma secreting a first-species monoclonal antibody is first established. Then, the variable region gene is cloned from the hybridoma cells. Next, the constant region gene of the second-species antibody is cloned as needed. The first-species variable region gene and the second-species constant region gene are linked to form 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 a preferred embodiment disclosed herein, the antibody light chain of the chimeric antibody further comprises a light chain constant region of a human κ, λ chain, or a variant thereof. The heavy chain of the chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, preferably comprising a heavy chain constant region of human IgG1, IgG2 or IgG4, or using a variant of the heavy chain constant region of IgG1, IgG2 or IgG4 with amino acid mutations (such as YTE mutation, reversion mutation, L234A and / or L235A mutation, or S228P mutation).

[0244] The term "humanized antibody," including CDR-grafted antibodies, refers to antibodies produced by grafting the CDR sequence of an animal-derived antibody, such as a murine antibody, into the variable region (or framework region) of a human antibody. Humanized antibodies can overcome the heterologous response induced by chimeric antibodies due to the large amount of heterologous protein components they carry. Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences or from publicly available references. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the VBase human germline sequence database (available at http: / / www.vbase2.org / ) and in Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in activity due to reduced immunogenicity, a small amount of reversion mutation can be performed on the aforementioned human antibody variable region framework sequence to maintain activity. The humanized antibodies disclosed herein also include humanized antibodies that have undergone affinity maturation of CDR by phage display.

[0245] Due to contact residues with the antigen, CDR transplantation can lead to a decrease in the affinity of the resulting antibody or its antigen-binding fragment for the antigen due to the framework residues in contact with the antigen. Such interactions may be a result of high somatic mutations. Therefore, it may still be necessary to transplant such donor framework amino acids into the framework of humanized antibodies. The amino acid residues involved in antigen binding from non-human antibodies or their antigen-binding fragments can be identified by examining the variable region sequence and structure of animal monoclonal antibodies. Residues in the CDR donor framework that differ from the species can be considered relevant. If the closest species cannot be determined, the sequence can be compared with a subclass common sequence or a common sequence of animal antibody sequences with a high percentage of similarity. Rare framework residues are thought to be a result of high somatic mutations, thus playing an important role in binding.

[0246] In one embodiment of this disclosure, the antibody or its antigen-binding fragment may further comprise a light chain constant region of a human or mouse κ, λ chain or a variant thereof, or further comprise a heavy chain constant region of a human or mouse IgG1, IgG2, IgG3, IgG4 or a variant thereof.

[0247] "Conventional variants" of the human antibody heavy chain constant region and human antibody light chain constant region refer to variants of the heavy chain constant region or light chain constant region derived from humans that do not alter the structure and function of the antibody variable region, as disclosed in the prior art. Exemplary variants include IgG1, IgG2, IgG3, or IgG4 heavy chain constant region variants that involve site-specific modifications and amino acid substitutions in the heavy chain constant region. Specific substitutions include YTE mutations, L234A and / or L235A mutations, or S228P mutations known in the prior art, or mutations that obtain a knock-in-hole structure (resulting in the antibody heavy chain having a knock-Fc and hole-Fc combination). These mutations have been shown to give antibodies new properties without altering the function of the antibody variable region.

[0248] "Human antibody" and "human-derived antibody" are used interchangeably. It can be an antibody derived from a human being or obtained from a transgenic organism "modified" 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 in which endogenous heavy and light chain loci are targeted and disrupted. The transgenic organism can synthesize antigen-specific human antibodies and can be used to produce human antibody-secreting hybridomas. A human antibody can also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources. Fully human antibodies can also be constructed using gene or chromosome transfection methods and phage display technology, or from in vitro activated B cells, all of which are known in the art.

[0249] "Monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, meaning that, apart from possible variant antibodies (e.g., those containing naturally occurring mutations or mutations generated during the manufacture of a monoclonal antibody formulation, which are typically present in small amounts), the individual antibodies constituting said population recognize and / or bind to the same epitopes. Each monoclonal antibody in a monoclonal antibody preparation (formulation) targets a single determinant cluster on an antigen. Therefore, the modifier "monoclonal" indicates the characteristics of an antibody as obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the manufacture of the antibody by any particular method. For example, monoclonal antibodies used according to this 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 part of the human immunoglobulin loci, as well as other exemplary methods for preparing monoclonal antibodies, which are described herein.

[0250] Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked by synthetic linkers using recombinant methods, thereby enabling the production of a single protein chain in which the VL and VH regions pair to form a monovalent molecule (referred to as 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). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and fragments are screened for functionality in the same manner as for intact antibodies. The antigen-binding portion can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.

[0251] The antigen-binding fragment can also be incorporated into a single-chain molecule containing a pair of tandem Fv fragments (VH-CH1-VH-CH1), which together with the complementary light chain polypeptide form a pair of antigen-binding regions (Zapata et al., 1995 ProteinEng. 8(10): 1057-1062; and US Patent US5641870).

[0252] Fab is an antibody fragment with a molecular weight of approximately 50,000 Da and antigen-binding activity obtained by treating IgG antibodies with the protease papain (which cleaves the amino acid residue at position 224 of the H chain). Approximately half of the N-terminal side of the H chain and the entire L chain are linked together by disulfide bonds.

[0253] F(ab')2 is an antibody fragment with a molecular weight of approximately 100,000 Da and antigen-binding activity, containing two Fab regions connected at the hinge position, obtained by digesting the portion below the two disulfide bonds in the hinge region of IgG with pepsin.

[0254] Fab' is an antibody fragment with a molecular weight of approximately 50,000 Da and antigen-binding activity obtained by cleaving the disulfide bonds in the hinge region of the aforementioned F(ab')2. Fab' can be produced by treating F(ab')2 that specifically recognizes and binds to antigens with a reducing agent such as dithiothreitol.

[0255] In addition, Fab' can be expressed by inserting DNA encoding the Fab' fragment of an antibody into a prokaryotic or eukaryotic expression vector and then introducing the vector into a prokaryote or eukaryote.

[0256] The terms “single-chain antibody,” “single-chain Fv,” or “scFv” refer to molecules comprising a variable domain (or region; VH) of the antibody heavy chain and a variable domain (or region; VL) of the antibody light chain linked by a linker. Such scFv molecules may have the general formula: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof, for example, using variants with 1–4 repeats (including 1, 2, 3, or 4) (Holliger et al. (1993), Proc Natl Acad Sci USA. 90:6444-6448). Other connectors available for use in this disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur J Immuno. 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 Immunol Immunother. 50:51-59.

[0257] A diabody is an antibody fragment in which scFv is dimerized; it is an antibody fragment with bivalent antigen-binding activity. In bivalent antigen-binding activity, the two antigens can be the same or different.

[0258] dsFv is obtained by linking polypeptides in which one amino acid residue in each VH and VL is replaced by a cysteine ​​residue via disulfide bonds between cysteine ​​residues. The amino acid residues to be replaced by cysteine ​​residues can be selected based on the prediction of the antibody's three-dimensional structure using known methods (Protein Engineering. 7:697 (1994)).

[0259] In some embodiments of this disclosure, the antigen-binding fragment can be produced by the following steps: obtaining cDNA encoding the VH and / or VL of the monoclonal antibody that specifically recognizes and binds to the antigen disclosed herein, and other desired domains; 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.

[0260] The "Fc region" can be a native sequence Fc region or a variant Fc region. While the boundaries of the Fc region in the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. Residues in the Fc region are numbered using the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD., 1991. The Fc region of immunoglobulins typically has two constant region domains, CH2 and CH3.

[0261] The term "amino acid difference" or "amino acid mutation" refers to an alteration or mutation of amino acids in a variant protein or polypeptide compared to the original protein or polypeptide, including the insertion, deletion, or substitution of one or more amino acids based on the original protein or polypeptide.

[0262] The “variable region” of an antibody refers to the variable region (VL) of the antibody light chain alone or in combination, or the variable region (VH) of the antibody heavy chain. As is known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs) (also known as hypervariable regions). The CDRs in each chain are held together tightly by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th edition, 1991, National Institutes of Health, Bethesda MD)); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al., J. Molec. Biol. 273:927-948 (1997)). As used herein, a CDR may refer to a CDR determined by either method or a combination of both methods.

[0263] The term "antibody framework" or "FR region" refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain. Essentially, it is a variable domain without a CDR.

[0264] The terms “complementarity-determining region” and “CDR” refer to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable domain and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable domain. The amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the “Kabat” numbering rule (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the “Chothia” numbering rule (Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001), and the ImMunoGenTics (IMGT) numbering rule (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003)), etc. For example, in the classic format, following Kabat rules, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following Chothia rules, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Combining the CDR definitions from Kabat and Chothia, the CDR is composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) from human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) from human VL. Following the IMGT rules, the CDR amino acid residues in VH are approximately numbered 27-38 (CDR1), 56-65 (CDR2), and 105-117 (CDR3), while those in VL are approximately numbered 27-38 (CDR1), 56-65 (CDR2), and 105-117 (CDR3).Following the IMGT rules, the CDR region of an antibody can be determined using the IMGT / DomainGapAlign procedure.

[0265] "Antibody constant region domains" refer to the domains derived from the constant regions of the light and heavy chains of antibodies, including CL and CH1, CH2, CH3, and CH4 domains derived from different classes of antibodies.

[0266] An epitope, or antigenic determinant, is a site on an antigen where an immunoglobulin or antibody specifically binds. Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed (1996).

[0267] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to an epitope on a pre-defined antigen.

[0268] The term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single epitope. Within each antigenic site, the variable region of the antibody "arm" interacts with the antigen at multiple amino acid sites via weak non-covalent forces; the greater the interaction, the stronger the affinity. As used herein, the term "high affinity" for an antibody or its antigen-binding fragment (e.g., the Fab fragment) generally refers to an antibody with a 1E affinity. -9 M or smaller K D (e.g., 1E) -10 M or smaller K D 1E -11 M or smaller K D 1E -12 M or smaller K D 1E -13 M or smaller K D 1E -14 M or smaller K D Antibody or antigen-binding fragments (such as antibodies or antigens).

[0269] The term "KD" or "K" D "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Typically, antibodies dissociate at a rate less than approximately 1E. -8 M, for example, less than approximately 1E -9 M, 1E -10 M or 1E -11An antigen is bound by a dissociation equilibrium constant (KD) of M or smaller, for example, as determined in a BIACORE instrument using surface plasmon resonance (SPR) technology. The smaller the KD value, the greater the affinity.

[0270] The term "nucleic acid molecule" refers to both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded DNA or RNA molecules, such as double-stranded DNA or mRNA. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0271] The term "vector" refers to a construct capable of delivering one or more target genes or sequences and preferably expressing them in host cells. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as production cells.

[0272] Methods for producing and purifying antibodies and antigen-binding fragments are well-known in the prior art, such as those described in Cold Spring Harbor's Guide to Antibody Laboratory Techniques, Chapters 5-8 and 15. For example, mice can be immunized with antigens or fragments thereof, and the resulting antibodies can be renatured, purified, and sequenced using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in this disclosure are prepared by adding one or more human FR regions to non-human CDR regions using genetic engineering methods. Human FR germline sequences can be obtained by alignment with the IMGT Human Antibody Variable Region Germline Gene Database and MOE software, or from the Immunoglobulin Journal, 2001 ISBN012441351.

[0273] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; members of the Bacillaceae family, 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-restrictive implementations such as HEK293E cells), and NSO cells.

[0274] Engineered antibodies or antigen-binding fragments can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As an alternative existing technology, mammalian expression systems lead to glycosylation of antibodies, particularly at the highly conserved N-terminal site in the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to the antigen. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. The culture medium secreting antibodies can be purified using conventional techniques, such as using a Sepharose FF column with adjusted buffer for Protein A or Protein G. Non-specifically bound components are washed away. The bound antibodies are then eluted using a pH gradient, and antibody fragments are detected by SDS-PAGE and collected. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, such as at -70°C, or lyophilized.

[0275] "Administration," "dosage," "giving," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, composition, or human intervention to an animal, human, subject, cell, tissue, organ, or biological fluid. "Giving" and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and a cell, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cell. "Giving" and "treatment" also mean, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo, such as cells. "Treatment," when applied to humans, veterinary, or research subjects, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0276] "Treatment" means administering an oral or topical therapeutic agent, such as a composition comprising any of the compounds disclosed herein, to a patient (or subject) who has (or is suspected of having, or is susceptible to) symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient (or subject) or population in an amount that effectively relieves symptoms of one or more diseases to induce the regression of such symptoms or inhibit the progression of such symptoms to any clinically measurable degree. The amount of therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's (or subject's) disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient (or subject). Whether the disease symptoms have been relieved can be evaluated using any clinical testing methods commonly used by a physician or other healthcare professional to assess the severity or progression of the symptoms. Although the embodiments disclosed herein (e.g., treatment methods or products) may not be effective in alleviating symptoms of every target disease, they should reduce symptoms of the target disease in a statistically significant number of patients (or subjects) according to any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0277] "Conserved amino acid modification" or "conserved amino acid substitution" refers to the substitution of an amino acid in a protein or polypeptide with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity), thereby allowing for frequent alterations without changing the protein or polypeptide's biological activity or other desired properties (e.g., antigen affinity and / or specificity). Those skilled in the art will recognize that, generally, the substitution of a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al., (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th edition)). Furthermore, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.

[0278] "Binding with IL-4R" refers to the ability to interact with human IL-4R. The term "antigen binding site" in this article refers to a three-dimensional spatial site recognized by the antibody or antigen-binding fragment described in this article.

[0279] “Cross-reactivity” refers to the ability of the antibody described herein to bind to IL-4R from different species. For example, the antibody described herein that binds to human IL-4R may also bind to IL-4R from another species. Cross-reactivity is measured by detecting specific reactivity with purified antigens, or binding or functional interactions with physiologically IL-4R-expressing cells, in binding assays such as SPR and ELISA. Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.

[0280] "Neutralizing" or "blocking" antibodies refer to antibodies whose binding to hIL-4R results in inhibition of the biological activity of hIL-4 and / or hIL-13. This inhibition of hIL-4 and / or IL-13 biological activity can be assessed by measuring one or more well-known indicators of hIL-4 / or hIL-13 biological activity, such as hIL-4 / or hIL-13-induced cell activation and the binding of hIL-4 to hIL-4R, for example, see CN103739711A. "Inhibition of growth" (e.g., involving cells) is intended to include any measurable reduction in cell growth.

[0281] "Inducing an immune response" and "enhancing an immune response" are used interchangeably and refer to the stimulation of an immune response by a specific antigen (i.e., passive or adaptive). In the context of inducing CDC or ADCC, "inducing" refers to stimulating a specific direct cell-killing mechanism.

[0282] "ADCC (antibody-dependent cell-mediated cytotoxicity)" refers to the direct killing of antibody-coated target cells by cells expressing Fc receptors through recognition of the Fc fragment of antibodies. The ADCC effector function of antibodies can be reduced or eliminated by modifying the Fc fragment of IgG. This modification refers to mutations in the constant region of the antibody's heavy chain, such as those selected from N297A, L234A, and L235A of IgG1; IgG2 / 4 chimeras; F235E of IgG4; or L234A / E235A mutations.

[0283] Engineered antibodies or antigen-binding fragments can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. Using molecular cloning techniques, the sequences of the humanized antibodies described in this paper are inserted into the corresponding expression vectors, and the corresponding humanized antibodies are produced using the HEK293 cell expression system. As a more recommended existing technique, mammalian expression systems lead to glycosylation of the antibodies, particularly at the highly conserved N-terminus of the FC region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. Cultures secreting antibodies can be purified and collected using conventional techniques. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving and ion exchange. The resulting product should be immediately frozen, such as at -70°C, or lyophilized.

[0284] "Effective amount" or "effective dose" means the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic outcome. For prophylactic use, beneficial or desired outcomes include eliminating or reducing risk, mitigating severity, or delaying the onset of a condition, including the condition itself, its complications, and the biochemical, histological, and / or behavioral symptoms of intermediate pathological phenotypes present during the development of the condition. For therapeutic use, beneficial or desired outcomes include clinical outcomes such as reducing the incidence of various target antigen-related conditions disclosed herein or improving one or more symptoms of said conditions, reducing the dosage of other agents required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of the target antigen-related condition disclosed in a patient (or subject).

[0285] "Exogenous" refers to substances that are produced outside of an organism, cell, or human body, depending on the circumstances.

[0286] "Endogenous" refers to substances that are produced in cells, organisms, or the human body, depending on the circumstances.

[0287] "Separated" refers to a purified state, and in this context means that the specified molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth media. Generally, the term "separated" is not intended to mean the complete absence of these materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compound as described herein.

[0288] "Homology" or "identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if every position in two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, multiplied by 100%. For example, at optimal sequence alignment, if six out of ten positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of homology is obtained by aligning the two sequences. The term "at least 85% sequence identity" as used herein refers to a variant sequence having at least 85% homology with the parent sequence. In some schemes, this homology is at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%; in some specific schemes, it is 90%, 95%, or more than 99%; and in other specific schemes, it is at least 95% sequence homology. The amino acid sequence having at least 85% sequence identity includes sequences obtained by performing one or more amino acid deletions, insertions, or substitutions on the parent sequence.

[0289] The terms “cell,” “cell line,” and “cell culture” used herein are used interchangeably, and all such names include their progeny. Therefore, the terms “transformant” and “transformed cell” include primary test cells and cultures derived therefrom, regardless of the number of transfections. It should also be understood that, due to intentional or unintentional mutations, all progeny cannot be exactly identical in terms of DNA content. This includes mutant progeny with the same function or biological activity as those screened from the original transformed cells.

[0290] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur. For example, "optionally contains 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a particular sequence may, but do not have to, be present.

[0291] Exemplary antibody drug composition (formulation) preparation process:

[0292] Step 1: Take a certain amount of purified anti-IL-4R antibody solution and perform solvent replacement (preferably ultrafiltration) with an antibody-free buffer. After at least 6 volume replacements via ultrafiltration, concentrate the antibody to a certain concentration. Add a certain volume of other excipient stock solution and dilute with buffer to achieve the required concentrations of antibody and excipients, then mix thoroughly. After filtration, take a sample from the control room for sterility testing. Pass the stock solution through a 0.22μm PVDF filter and collect the filtrate.

[0293] Step 2: Adjust the volume to 2.15 mL, fill the filtrate into 2 mL vials, stopper them, and take samples at the beginning, middle and end of filling to check the volume difference.

[0294] Step 3: Turn on the capping machine, add the aluminum cap, and perform capping.

[0295] Step 4: Visual inspection to confirm the product is free of defects such as inaccurate filling. Print and affix vial labels; print carton labels, fold cartons, pack, and affix carton labels.

[0296] Example

[0297] The following examples are used to further describe this disclosure, but these examples are not intended to limit the scope of this disclosure. Experimental methods not specifically described in the examples are generally performed under conventional conditions, such as those described in Cold Spring Harbor's Antibody Technology Manual or Molecular Cloning Manual; or under conditions recommended by the raw material or commercial manufacturer. Reagents not specifically named are commercially available, conventional reagents.

[0298] Antibody preparation examples

[0299] Example 1: Immunization and Detection in Mice

[0300] The his-tagged human IL-4R (h-IL-4R-his) recombinant protein, the his-tagged mouse IL-4R (m-IL-4R-his) recombinant protein, and the his-tagged rhesus monkey IL-4R (rhesus-IL-4R-his) recombinant protein were synthesized by Acrobiosystems, expressed on HEK293, and then purified.

[0301] The recombinant human IL-4R (h-IL-4R-Fc) protein with a human Fc tag was designed, expressed, and purified in-house. The purified protein was used in the experiments described in the following examples.

[0302] In this embodiment, the number and position of CDR amino acid residues in the VL and VH regions of the antibody or antigen-binding fragment conform to the known Kabat numbering rules (LCDR1-3, HCDR2-3) and AbM rules (HCDR1).

[0303] Table 1. Immunogen Information

[0304] name amino acid sequence start and end Database ID / Product ID h-IL-4R-his Met26-His232 NP_000409.1 m-IL-4R-his Ile26-Arg233 NP_001008700 rhesus-IL-4R-his Met26-Arg232 G7Q0S7 h-IL-4R-Fc Met1-His232 NP_000409.1

[0305] Anti-human IL-4R monoclonal antibody was produced by immunizing mice. The experimental mice were female C57BL / 6 mice, 6–8 weeks old (Suzhou Zhaoyan New Drug Research Center Co., Ltd., Animal Production License No.: 201503052).

[0306] Housing environment: SPF grade. Mice were housed in a laboratory environment for one week after purchase, with a 12 / 12-hour light / dark cycle, temperature 20-25℃, and humidity 40-60%. Once acclimatized, the mice were divided into three cages of five mice each. The immunogen was Fc-tagged recombinant human IL-4R protein (h-IL4R-Fc, concentration 0.73 mg / mL). Emulsification was performed using Freund's adjuvant (Sigma, Cat#: F5881): initial immunization used Freund's complete adjuvant (CFA, Pierce, Cat#77140), followed by booster immunizations using nucleic acid adjuvants (CpG, Shanghai Sangon Biotech) and aluminum adjuvants (Alum, Thermo Cat#77161).

[0307] On day 0, mice were injected intraperitoneally (IP) with 70 μg / mouse of emulsified antigen. On days 14, 28, 42, 56, and 77, based on the presence of back masses and abdominal swelling, 0.1 mL of antigen was injected into the back and intraperitoneally respectively. Blood samples were collected on days 21, 35, 49, 63, and 84 for serum analysis. The antibody titer in mouse serum was determined using the ELISA method described in Example 2. After the fourth immunization, mice with high antibody titers in their serum that were trending towards a plateau were selected for spleen cell fusion. Three days prior to fusion, a booster immunization was administered via intraperitoneal (IP) injection of 10 μg / mouse of an antigen solution prepared with phosphate buffer. An optimized PEG-mediated fusion procedure was used to fuse spleen lymphocytes with myeloma Sp2 / 0 cells (…). CRL-8287 TM Hybridoma cells are obtained by fusing them together.

[0308] Example 2: ELISA testing and screening of antibodies

[0309] 1. ELISA combined with experiment:

[0310] ELISA assays are used to detect the binding properties of anti-IL-4R antibodies. Microplates coated with his-tagged recombinant IL-4R protein are used. After adding antibodies to each well, the antibody-antigen binding activity is detected by adding secondary antibody (HRP-conjugated anti-primary Fc antibody) and HRP substrate TMB.

[0311] Coat 96-well microplates with human or rhesus monkey IL-4R-his protein at a concentration of 0.5 μg / mL (100 μL per well) and incubate overnight at 4°C. Wash three times with 250 μL of washing buffer per well. Add 200 μL / well blocking buffer and incubate at room temperature for 2 hours. Wash three times with 250 μL of washing buffer per well. Add 100 μL of diluted anti-IL-4R antibody to each well. Incubate at room temperature for 1 hour. Wash three times with 250 μL of washing buffer per well. Add 100 μL of HRP-labeled goat anti-human IgG secondary antibody diluted 1:20000 with diluent to each well. Incubate at room temperature for 1 hour. Wash three times with 250 μL of washing buffer per well. Add 100 μL of TMB to each well and react in the dark for 15 minutes. Add 50 μL of 0.16 M / L sulfuric acid per well. The Thermo MultiSkanFc microplate reader reads the 450nm OD value and calculates the EC50 of the anti-IL-4R antibody binding to IL-4R. 50 value.

[0312] 2. ELISA blocking assay:

[0313] This experiment used an in vitro blocking assay to detect the blocking effect of screened anti-human IL-4R antibodies on the binding of human IL-4R and human IL-4. Specifically, Fc-tagged recombinant IL-4R protein was coated onto a 96-well microplate. After the antibody binding to human IL-4R fully occupied the epitope, IL-4 (Biolegend, Cat#574004) was added. The binding of IL-4 to IL-4R was then detected using a biotin-conjugated anti-IL-4 antibody and Neutravidin-HRP (Pierce, Cat#31001). The IC50 of the IL-4R antibody on the blocking effect of IL-4R binding was calculated. 50 value.

[0314] Human IL-4R-Fc protein was coated onto 96-well microplates at a concentration of 0.5 μg / mL (100 μL per well) and incubated overnight at 4°C. Wash three times with 250 μL of washing buffer per well. Add 200 μL / well blocking buffer and incubate at room temperature for 2 hours. Wash three times with 250 μL of washing buffer per well. Add 100 μL of diluted anti-IL-4R antibody to each well and incubate at room temperature for 1 hour. Wash three times with 250 μL of washing buffer per well. Add 100 μL of diluted IL-4 to each well and incubate at room temperature for 1 hour. Wash three times with washing buffer. Add 100 μL of diluted biotin-conjugated anti-IL-4 antibody to each well and incubate at room temperature for 1 hour. Wash three times with washing buffer. Incubate with HRP-labeled Neutravidin diluted 1:5000 at room temperature for 1 hour. Wash three times with 250 μL of washing buffer per well. Add 100 μL of TMB to each well and incubate in the dark for 15 minutes. Add 50 μL of 0.16 M / L sulfuric acid to each well. Use a Thermo MultiSkanFc microplate reader to read the OD value at 450 nm and calculate the IC50 of the IL-4R antibody blocking the binding of IL-4R and IL-4. 50 value.

[0315] Example 3: Reporter gene activity assay using antibodies binding to human IL-4R

[0316] HEK-Blue IL-4 cells were purchased from Invivogen (Cat#hkb-stat6). These cells were stably transfected with the human IL-4R gene and the STAT6-mediated SEAP genome. The activation level of the IL-4R signaling pathway can be characterized by detecting the secreted SEAP in the supernatant using the SEAP substrate QUANTI-Blue.

[0317] This experiment detected the activation of HEK-Blue IL-4 in cells, based on IC50. 50 The size was used to evaluate the in vitro cell activity of the IL-4R antibody.

[0318] HEK-Blue IL-4 cells were cultured in DMEM medium containing 10% FBS, 100 μg / mL Zeocin (Invivogen, Cat#ant-zn-05), and 10 μg / mL Blastidin (Invivogen, Cat#ant-bl-05). Cells were passaged 2–3 times per week at a ratio of 1:5 or 1:10. Before passage, the medium was aspirated, the cell layer was washed with 5 mL of 0.25% trypsin, the trypsin was then aspirated, and the cells were digested in an incubator for 3–5 minutes. The cells were then resuspended in fresh medium. 100 μL of the cell suspension was added to each 96-well cell culture plate at a density of 5 × 10⁻⁶ cells / well. 5Cells / mL, culture medium was DMEM containing 10% FBS, 100 μg / mL Zeocin and 30 μg / mL Blasticidin, with only 100 μl of sterile water added to the periphery of the 96-well plate. The plate was incubated for 24 hours (37℃, 5% CO2). After cell attachment, 100 μl of serially diluted test antibody was added to each well. The plate was incubated for 20–24 hours (37℃, 5% CO2). 20 μl of cell supernatant was transferred from each well to a new 96-well plate, along with 180 μl of QUANTI-Blue substrate solution. The plate was incubated in the dark for 1–3 hours. The absorbance at 620 nm was measured using a Thermo MultiSkan FPGA.

[0319] Example 4: Experiment on the inhibition of TF-1 cell proliferation by antibody binding to human IL-4R

[0320] TF-1 cells (ATCC CRL-2003) are lymphoma cells that express IL-4R and are sensitive to cytokines such as IL-4 / IL-13. IL-4 can stimulate the proliferation of TF-1 cells in the absence of GM-CSF. This experiment investigated the neutralizing activity of different anti-IL-4R antibodies by adding neutralizing antibodies to block the IL-4 pathway and inhibit TF-1 cell proliferation.

[0321] TF-1 cells were cultured in RPMI 1640 medium containing 10% FBS and 2 ng / mL GM-CSF (R&D, Cat#215-GM-010); passaged 2–3 times per week at a ratio of 1:10. 100 μL of cell suspension was added to each 96-well cell culture plate at a density of 2 × 10⁻⁶ cells / well. 5 Cells / mL, cultured in 10% FBS RPMI 1640 medium, with only 100 μL of sterile water added to the periphery of the 96-well plate. 50 μL of serially diluted test antibody and 50 μL of IL-4 (R&D, Cat#204-IL-050) at a final concentration of 0.7 ng / mL were added to each well. The plate was incubated for 72 hours (37°C, 5% CO2). After culture, cell proliferation was detected using a CTG kit (Promega, Cat#G7572).

[0322] Example 5: In vitro binding affinity and kinetics experiment

[0323] The affinity of the antibody against human IL-4R was determined using a Biacore GE instrument.

[0324] Following the instructions for the Human Anti-Capture Kit (Cat.#BR-1008-39, GE), the human anti-capture antibody was covalently coupled to the CM5 biosensor chip of a Biacore instrument (Biacore X100, GE) to affinity capture a certain amount of the test antibody. Then, a series of concentration gradients of IL-4R antigen (all IL-4R antigens were purchased from Acrobiosystems, Cat#ILR-H5221) were passed through the chip surface. The reaction signal was monitored in real time using the Biacore instrument (Biacore X100, GE) to obtain binding and dissociation curves. After each cycle of dissociation, the biochip was regenerated by washing with the regeneration solution provided in the human anti-capture kit. The amino-coupling kit used in the experiment was purchased from GE (Cat.#BR-1000-50, GE), and the buffer was HBS-EP + 10× buffer solution (Cat.#BR-1006-69, GE) diluted to 1× (pH 7.4) with DIWater.

[0325] The experimental data were fitted using BiacoreX100 evaluation software 2.0 and GE software in a (1:1) combination model to obtain the affinity values.

[0326] Example 6: Antibody Sequence and Preparation

[0327] Through the ELISA binding assay (ELISA binding of human IL-4R-his) and ELISA blocking assay (ELISA blocking of human IL-4 / IL-4R) in Example 2, the experiment in Example 3 inhibiting the activation of HEK293-Blue IL-4 cells under IL-4 stimulation, and the experiment in Example 4 inhibiting the proliferation of TF-1 cells under IL-4 stimulation, the two monoclonal hybridoma cell lines with the best in vitro activity were selected. Their activity detection results are shown in Table 2.

[0328] Table 2. Results of hybridoma cell line activity assay

[0329]

[0330] Monoclonal hybridoma cell lines 25G7 and 7B10 were selected, and antibody sequences were cloned from them. The process of cloning sequences from hybridomas is as follows:

[0331] Log-phase hybridoma cells were collected, and RNA was extracted using Trizol (Invitrogen, 15596-018) (following the kit instructions). Reverse transcription (PrimeScript) was then performed. TMReverse Transcriptase (Takara, cat#2680A). The cDNA obtained by reverse transcription was amplified by PCR using a mouse Ig-Primer Set (Novagen, TB326Rev.B 0503) and then sent to a sequencing company for sequencing. The resulting antibody sequence was then analyzed.

[0332] The heavy and light chain variable region sequences of mouse monoclonal antibody 25G7 are as follows:

[0333] 25G7HCVR

[0334]

[0335] 25G7LCVR

[0336]

[0337] The CDR sequences it contains are shown in Table 3.

[0338] Table 3. CDR sequence of monoclonal antibody 25G7

[0339] name sequence serial number HCDR1 GFTFSDYGMH SEQ ID NO: 3 HCDR2 FISSGSSIIYYADIVKG SEQ ID NO: 4 HCDR3 GNKRGFFDY SEQ ID NO: 5 LCDR1 NASSSVSYMY SEQ ID NO: 6 LCDR2 LTSNLAS SEQ ID NO: 7 LCDR3 QQWRSNPPMLT SEQ ID NO: 8

[0340] The heavy and light chain variable region sequences of mouse monoclonal antibody 7B10 are as follows:

[0341] 7B10 HCVR

[0342]

[0343] 7B10 LCVR

[0344]

[0345] The CDR sequences it contains are shown in Table 4.

[0346] Table 4. CDR sequence of monoclonal antibody 7B10

[0347] name sequence serial number HCDR1 GYTFTSYWMH SEQ ID NO: 11 HCDR2 LIHPNSDTTKFSENFKT SEQ ID NO: 12 HCDR3 SKIITTIVARHWYFDV SEQ ID NO: 13 LCDR1 KASQSVDYGGDSYMN SEQ ID NO: 14

[0348] LCDR2 AASNLES SEQ ID NO: 15 LCDR3 QHSNENPPT SEQ ID NO: 16

[0349] The obtained variable region sequence was ligated into the human constant region sequence to obtain the human-mouse chimeric antibody sequence. Using molecular cloning technology, the chimeric antibody sequence was inserted into the corresponding expression vector. Using the HEK293 cell expression system, the human-mouse chimeric antibodies 25G7-C and 7B10-C were obtained.

[0350] The purified chimeric antibody was subjected to in vitro activity assays using the methods described in Examples 2-5 above, and the data are shown in Table 5. The results showed that the 25G7-C antibody significantly outperformed the reference antibody dupilumab in blocking IL-4 binding and inhibiting cell proliferation (as described in WHO Drug Information, Vol. 26, No. 4, 2012).

[0351] Table 5. In vitro activity assay

[0352]

[0353] Example 7: Mouse Antibody Humanization Experiment

[0354] The obtained murine antibodies 25G7 and 7B10 were humanized. Based on the typical structure of the obtained murine antibody VH / VLCDR, the variable region sequences of the heavy and light chains were compared with the human antibody Germline database to obtain highly homologous human germline templates. The human germline light chain framework region was derived from the human κ light chain gene, with the preferred human germline light chain templates IGKV3-11*01 (SEQ ID NO: 22, for antibody 25G7) and IGKV2D-29*01 (SEQ ID NO: 24, for antibody 7B10). The human germline heavy chain framework region was derived from the human heavy chain, with the preferred human germline heavy chain templates IGHV3-48*01 (SEQ ID NO: 21, for antibody 25G7) and IGHV1-2*02 (SEQ ID NO: 23, for antibody 7B10).

[0355] The racial template sequence is shown below.

[0356] Ethnographic heavy chain template IGHV3-48*01:

[0357]

[0358] Ethnic Light Link Template IGKV3-11*01:

[0359]

[0360] Ethnographic heavy chain template IGHV1-2*02:

[0361]

[0362]

[0363] Humanoid Light Chain Template IGKV2D-29*01:

[0364]

[0365] The CDR region of the murine antibody was transplanted onto a selected humanized template, then recombined with the constant region of IgG, and reversed through mutation to obtain a series of humanized molecules.

[0366] Specifically, hu7B10-VH-a, hu7B10-VH-b, and hu7B10-VH-c underwent pharmacodynamic modifications by changing the first digit of the humanized heavy chain template from Q to E. The humanized heavy chain variable region sequences of the two antibodies are shown in SEQ ID NO: 25-27 and SEQ ID NO: 31-33, respectively; the light chain variable region sequences are shown in SEQ ID NO: 28-30 and SEQ ID NO: 34-36, respectively.

[0367] hu25G7-VH-a:

[0368]

[0369] hu25G7-VH-b:

[0370]

[0371] hu25G7-VH-c:

[0372]

[0373] hu25G7-VL-a:

[0374]

[0375] hu25G7-VL-b:

[0376]

[0377] hu25G7-VL-c:

[0378]

[0379] hu7B10-VH-a:

[0380]

[0381]

[0382] hu7B10-VH-b:

[0383]

[0384] hu7B10-VH-c:

[0385]

[0386] hu7B10-VL-a:

[0387]

[0388] hu7B10-VL-b:

[0389]

[0390] hu7B10-VL-c:

[0391]

[0392] The reverse mutation design of hybridoma clone 25G7 is shown in Table 6.

[0393] Table 6. Template Selection and Reverse Mutation Design

[0394]

[0395] Note: The amino acid positions of the reversion mutations are determined using the Kabat numbering rules.

[0396] Hybridoma clone 7B10 reverse mutation design is shown in Table 7 below:

[0397] Table 7. Template Selection and Reverse Mutation Design

[0398]

[0399] Note: The amino acid positions of the reversion mutations are determined using the Kabat numbering rules.

[0400] Exemplary humanized antibody hu25G7 (using VH-c heavy chain and VL-a light chain) and hu7B10 antibody molecule (using VH-b heavy chain and VL-b light chain), the complete light and heavy chain sequences of which are shown in SEQ ID NO: 17-20.

[0401] hu25G7 HC

[0402]

[0403] hu25G7 LC

[0404]

[0405] hu7B10 HC

[0406]

[0407] hu7B10 LC

[0408]

[0409] By using molecular cloning technology, the sequence of a humanized antibody is inserted into a corresponding expression vector, and then expressed and produced using the HEK293 cell expression system, the corresponding humanized antibody can be obtained.

[0410] Example 8: Humanized Antibody Activity Data

[0411] The in vitro activity of humanized antibodies hu25G7 and hu7B10 was tested as described in Examples 2-5, and the results are shown in Table 8.

[0412] The results showed that both hu25G7 and hu7B10 bound only to human IL-4R and not to rhesus monkey IL-4R, indicating that both antibodies bound to epitopes different from those in humans and rhesus monkeys, and could specifically bind to human IL-4R. Both antibodies blocked IL-4 / IL-4R binding and intracellular signaling pathways, leading to the neutralization of IL-4 activation and inhibition of TF-1 cell proliferation. Among them, the blocking and inhibitory activities of hu25G7 were significantly better than those of the reference antibody dupilumab.

[0413] Table 8. In vitro activity assay

[0414]

[0415] Example 9: Affinity maturation experiment of humanized antibody hu25G7

[0416] Affinity maturation of the hu25G7 antibody was performed using yeast display platform technology. Based on the hu25G7 antibody, affinity-matured yeast libraries targeting six CDRs were designed and prepared. Degenerate primers were designed, and designed mutant amino acids were introduced into the hu25G7-scFv antibody library via PCR and homologous recombination. The size of each library was less than 10 μL. 9 The constructed yeast library was then validated for diversity using next-generation sequencing (GENEWIZ).

[0417] High-affinity antibodies against human IL-4R were screened from the hu25G7-scFv yeast library using biotin-labeled human IL-4R. After two rounds of MACS screening (streptomycin magnetic beads, Invitrogen) and two rounds of FACS screening (BD FACSAria™ FUSION), yeast clones were selected, cultured, and induced to express the antibodies. The binding of yeast clones to human IL-4R was detected using FACS (BD FACSCanto II), and yeast clones with higher affinity than wild-type 25G7 antibodies were selected for sequencing verification. After alignment analysis of the sequenced clones and removal of redundant sequences, the non-redundant sequences were converted into full-length human antibody molecules for expression in mammalian cells. The affinity-purified full-length antibodies were then subjected to affinity assays using a BIAcore™ X-100 (GE Healthcare) to select candidate antibody molecules with high affinity for human IL-4R. These antibody molecules have a higher affinity for human IL-4R than wild-type hu25G7 antibodies. Among them, the affinity of hu25G7-A antibody molecules is comparable to that of dupilumab, while the affinity of hu25G7-B molecules is significantly better than that of dupilumab.

[0418] The light chain variable region sequence of antibody hu25G7-A, obtained after affinity maturation, is as follows:

[0419] hu25G7-A LCVR

[0420]

[0421] The CDR sequences it contains are shown in Table 9.

[0422] Table 9. LCDR sequence of hu25G7-A

[0423] name sequence serial number LCDR1 RASSSVPYMY SEQ ID NO: 38 LCDR2 LTSNLAS SEQ ID NO: 7 LCDR3 QQWRAYPPMLT SEQ ID NO: 40

[0424] The light chain variable region sequence of antibody hu25G7-B is as follows:

[0425] hu25G7-B LCVR

[0426]

[0427] The CDR sequences it contains are shown in Table 10.

[0428] Table 10. CDR Sequences

[0429] name sequence serial number LCDR1 RASPGVPPLA SEQ ID NO: 42 LCDR2 LASSRPS SEQ ID NO: 39 LCDR3 QQWRSNPPMLT SEQ ID NO: 8

[0430] The above-mentioned light chain variable region hu25G7-A LCVR recombines with the hu25G7 light chain constant region to obtain the hu25G7-A antibody light chain; the above-mentioned light chain variable region hu25G7-B LCVR recombines with the hu25G7 light chain constant region to obtain the hu25G7-B antibody light chain.

[0431] Optimization of the amino acid residues in hu25G7-VH-c yielded the heavy chain variable regions hu25G7-A / B VH and hu25G7-C VH.

[0432] hu25G7-A / B VH:

[0433]

[0434] hu25G7-C VH:

[0435]

[0436] The aforementioned variable region of the heavy chain can recombine with the constant region of the hu25G7 heavy chain to obtain hu25G7-A / hu25G7-B and hu25G7-C antibody heavy chains.

[0437] The complete heavy chain sequences of hu25G7-A and hu25G7-B are shown in SEQ ID NO: 44.

[0438] hu25G7HC (i.e., hu25G7-A / hu25G7-B antibody heavy chain)

[0439]

[0440]

[0441] hu25G7-C antibody heavy chain:

[0442]

[0443] The complete light chain sequences are shown in SEQ ID NO: 45-46.

[0444] hu25G7-A LC

[0445]

[0446] hu25G7-B / hu25G7-C LC

[0447]

[0448] Example 10: Humanized Antibody Affinity Maturation Activity Data

[0449] The two antibodies, hu25G7-A and hu25G7-B, were tested according to Examples 3 and 4. Both antibodies could block the binding of IL-4 / IL-4R and neutralize the activation of IL-4 and IL-13 caused by intracellular signaling pathways, thus inhibiting the proliferation of TF-1 cells. The activity data are shown in Table 11.

[0450] Table 11. Comparison of Activity Data

[0451]

[0452]

[0453] In experiments inhibiting IL-13-induced TF-1 cell proliferation, both hu25G7-A and hu25G7-B showed beneficial effects. Both hu25G7-A and hu25G7-B significantly enhanced the effects of dupilumab in blocking the binding of IL-4, IL-13, and IL-4R, and in promoting cell proliferation induced by these bindings.

[0454] Example 11: Study on the effect of humanized antibodies on mouse dermatitis

[0455] Establishment of a mouse dermatitis model: IL-4 / IL-4Rα transgenic mice (purchased from Cyagen Model Organisms Research Center (Taicang) Co., Ltd.) were used. Each mouse had an abdominal area of ​​approximately 3cm × 3cm. 100μL of a 1.5% OXZ acetone-olive oil solution (acetone:olive oil = 4:1) was applied evenly to sensitize the mice, and the day of sensitization was recorded as Day 0. Starting on Day 7, 20μL of a 1% OXZ acetone-olive oil solution was applied evenly to both ears (both sides) for challenge, with challenges occurring every 72 hours.

[0456] This experiment included a normal control group (treated with only acetone-olive oil solution during sensitization and challenge), a model control group, and hu25G7-A, hu25G7-B, and dupilumab groups, for a total of 5 groups, with 3-5 mice in each group. The dosage for the treatment groups was 50 mg / kg, administered subcutaneously twice a week (details are shown in Table 12). Ear thickness was measured using calipers on day 27, and the results are as follows. Figure 1 As shown.

[0457] Table 12. Application plans for each group

[0458] Group Animal number Administration method Dosage Dosage frequency* normal control group 3 (male) SC - Twice a week Model control group 5(3 females + 2 males) SC - Twice a week hu25G7-A 5(3 females + 2 males) SC 50mg / kg Twice a week hu25G7-B 3(2 females + 1 male) SC 50mg / kg Twice a week Dupilumab 4(2 females + 2 males) SC 50mg / kg Twice a week

[0459] The results showed that the ears of mice in the model control group had significant pathological damage, and the ear thickness was significantly higher than that of the normal control group. On day 27, the ear thickness of mice in the hu25G7-A, hu25G7-B, and dupilumab groups was significantly lower than that of the model control group. This indicates that hu25G7-A, hu25G7-B, and dupilumab can all be used to treat dermatitis, and hu25G7-B is more effective than dupilumab.

[0460] Formulation preparation examples

[0461] The equipment used in the preparation process and the calculation methods for the results are as follows:

[0462] SEC size exclusion chromatography:

[0463] An analytical method for separating solutes based on the relative relationship between the pore size of the gel and the coil size of the polymer sample molecules.

[0464] SEC monomer content percentage = A monomer / A total * 100% (A monomer is the peak area of ​​the main peak monomer in the sample, and A total is the sum of the peak areas of all peaks).

[0465] Instruments used for SEC analysis: Agilent 1260; Column: Waters, XBrige SEC (300×7.8mm3.5μm).

[0466] CE capillary gel electrophoresis:

[0467] An electrophoresis method in which a gel is transferred into a capillary as a supporting medium and separated according to the molecular weight of the sample under a certain voltage.

[0468] Non-reduced CE purity percentage = A main peak / A total * 100% (A main peak is the peak area of ​​the main peak in the sample, and A total is the sum of the areas of all peaks).

[0469] CE testing instrument: Beckman Plus 800

[0470] iCIEF Imaging Capillary Isofocusing Electrophoresis:

[0471] A technique for separating proteins based on their different isoelectric points (pI).

[0472] iCIEF neutral peak content percentage = neutral peak area / total area * 100% (total area is the sum of the areas of acidic peak, neutral peak and basic peak).

[0473] The instrument used for iCIEF measurement is manufactured by Simple Protein, and its model is Muarice.

[0474] Viscosity determination: Viscosity was determined using a rheometer (manufacturer Anton Paar, model MCR xx2) at a temperature of 25 degrees Celsius. The sample was placed directly on the measuring plate for testing.

[0475] Osmotic pressure: Osmotic pressure is determined by the freezing point method. Based on the direct proportionality between freezing point depression and the molar concentration of the solution, a highly sensitive temperature sensing element is used to determine the freezing point of the solution, and the result is converted into osmotic pressure via electrical charge. Instrument manufacturer: Loser, model OM815.

[0476] Example 1. Screening of Buffer Systems

[0477] 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, and the stability of the antibody in different buffer systems was investigated. Specific formulation designs are shown in the table below.

[0478] Table 13. Composition Table of Pharmaceutical Samples

[0479] Sample number Buffer system 1 20mM histidine-acetic acid (His-AA) pH 4.5 2 20mM His-AA pH5.0 3 20mM Acetic Acid-Sodium Acetate (AA) pH 5.5 4 20mM succinic acid-sodium succinate (SA) pH 5.5 5 20mM citric acid - sodium citrate (CA) pH 5.5 6 20mM His-AA pH 5.5 7 20mM His-AA pH 6.0 8 20mM His-AA pH 6.5

[0480] The samples were aseptically filtered and filled, and their appearance and SEC were examined under a high temperature of 40℃. The data are shown in the table below.

[0481] Table 14. Stability results of formulation samples

[0482]

[0483] Note: D15 indicates testing on day 15, M indicates month, T0 indicates testing immediately after sample preparation, and N / A indicates no testing.

[0484] The results showed that the SA / CA / AA buffer had a poor appearance, therefore His-AA was chosen as the buffer system. In the His-AA system, the appearance was better at pH 4.5-5.5; except for His-AA at pH 6.5, there was no significant difference in SEC among the buffer systems, with pH 5.0 and 6.0 being slightly better. Therefore, His-AA, pH 4.5-6.0, was chosen as the buffer system for this antibody.

[0485] Example 2. Antibody concentration screening

[0486] Different concentrations of hu25G7-A formulations were prepared in 20 mM His-AA buffer systems at pH 5.0 and 5.5. The formulation components are as follows:

[0487] 1)20mM His-AA pH5.0, 100mg / mL hu25G7-A

[0488] 2)20mM His-AA pH5.5, 100mg / mL hu25G7-A

[0489] 3) 20mM His-AA pH5.0, 150mg / mL hu25G7-A

[0490] 4) 20mM His-AA pH5.5, 150mg / mL hu25G7-A

[0491] 5) 20mM His-AA pH5.0, 139mg / mL hu25G7-A

[0492] 6) 20mM His-AA pH5.0, 127mg / mL hu25G7-A

[0493] 7) 20mM His-AA pH5.0, 120mg / mL hu25G7-A

[0494] 8) 20mM His-AA pH5.5, 138mg / mL hu25G7-A

[0495] 9) 20mM His-AA pH5.5, 122mg / mL hu25G7-A

[0496] After the formulation was prepared, a portion of the formulation sample was first taken to test its viscosity. Then, the sample was aseptically filtered and filled into vials. The appearance, SEC, non-reduced CE-SDS, and iCIEF of the samples were examined under a high temperature of 40℃. The effects of different antibody concentrations on the viscosity and stability of the hu25G7-A formulation were also investigated. The results are shown in the table below:

[0497] Table 15. Results of viscosity determination of formulation

[0498] sample Viscosity (mPa·s) 1 13.7 2 25.5 3 90.0 4 140.5 5 34.6 6 26.5 7 21.8 8 93.2 9 52.6

[0499] Table 16. Results of formulation stability assay

[0500]

[0501] The results showed that there were no significant differences in appearance, SEC, CE, and iCIEF among different antibody concentrations, indicating that antibody concentration had little effect on the stability of the formulation in His-AA buffer at pH 5.0 or pH 5.5. However, the higher the antibody concentration, the greater the viscosity of the formulation. In the buffer at pH 5.0, 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.

[0502] Example 3. Screening of viscosity modifiers

[0503] A hu25G7-A formulation with an antibody concentration of 120 mg / mL and containing different viscosity modifiers was prepared in 20 mM His-AA (pH 5.0) buffer. Viscosity was used as the evaluation index to investigate the effect of different viscosity modifiers on the formulation viscosity. To meet the isotonic requirement of the subcutaneous formulation, each viscosity modifier was added to the formulation at an osmotic pressure of 270 mosm / kg (the blank viscosity modifier) ​​to evaluate its effect. Specific results are shown in the table below.

[0504] Table 17. Viscosity Results

[0505] Viscosity modifier Concentration (mM) Average viscosity (mPa·s) No blank added N / A 16 NaCl 122 8 <![CDATA[MgCl2]]> 85 5.3 <![CDATA[CaCl2]]> 148 5.6 KCl 124 7.7 <![CDATA[CH3COONa]]> 86 8.8 <![CDATA[Na2SO4]]> 96 9.5 NaI 113 8.3 NaF 74 6.3 NaSCN 112 6.6 Arg-HCl 120 7.2 Lys 118 8.7 His 93 6.6 Pro 207 12.9

[0506] The results showed that, except for proline, NaCl, MgCl2, CaCl2, KCl, CH3COONa, Na2SO4, NaI, NaF, NaSCN, Arg-HCl, Lysine, and Histidine all significantly reduced the viscosity of the formulation at their respective maximum concentrations. Among them, Arg-HCl, MgCl2, CaCl2, NaF, NaSCN, and histidine showed better effects, and Arg-HCl, MgCl2, CaCl2, and histidine were preferred as viscosity modifiers.

[0507] Example 4. Effect of viscosity modifiers on the viscosity of high-concentration formulations

[0508] To test the effect of the viscosity modifier, a Hu25G7-A antibody formulation with a concentration of 150 mg / mL was prepared in 20 mM His-AA, pH 5.0 buffer. The specific components are as follows:

[0509] 1) Contains no viscosity modifier (N / A)

[0510] 2) 85mM MgCl2

[0511] 3) 148mM CaCl2

[0512] 4) 93mM histidine

[0513] Using viscosity as the evaluation index, the effects of different excipients on the viscosity of high-concentration formulations were investigated, and the results are shown in the table below.

[0514] Table 18. Viscosity Results

[0515] sample Viscosity (mPa·s) 1 88.80 2 16.86 3 17.22 4 19.00

[0516] The results showed that when the antibody concentration was 150 mg / mL, MgCl2, CaCl2 and histidine could all reduce the viscosity of the formulation to below 20 mPa·s, and the viscosity-reducing effect was good.

[0517] Example 5. Effect of viscosity modifiers on formulation stability

[0518] In a 20 mM His-AA pH 5.0 buffer system, formulations containing Hu25G7-A antibody at a concentration of 150 mg / mL and different concentrations of excipients were prepared to investigate the effect of different concentrations of excipients on the thermal stability of the formulations (after 17 days at 40°C). The experimental design and results are shown in the table below.

[0519] Table 19. Formulation Samples

[0520] sample Viscosity modifier 1 Blank (No added) 2 50mM MgCl2 3 90mM MgCl2 4 90mM CaCl2 5 148mM CaCl2 6 90mM histidine 7 122mM NaCl 8 120mM Arg-HCl

[0521] Table 20. Effect of excipient dosage on sample stability

[0522]

[0523] The results showed that MgCl2, Arg-HCl, NaCl and histidine had little effect on the stability of the formulation. The formulation containing MgCl2, Arg-HCl, NaCl and histidine had better stability than the formulation containing CaCl2. The CaCl2 group showed a greater decrease in CE under high temperature conditions than other formulation samples. The formulations containing MgCl2, Arg-HCl and histidine all had good stability. Therefore, MgCl2, Arg-HCl and histidine are preferred.

[0524] Example 6. Comprehensive evaluation of the effect of viscosity modifiers on formulation stability

[0525] In a 20 mM His-AA pH 5.0 buffer, a formulation containing 0.1 mg / mL PS80, 150 mg / mL Hu25G7-A, and a viscosity modifier was prepared. The concentration of PS80 in the 15-day sample was 1 mg / mL. The effects of different excipients on the viscosity and stability of the Hu25G7-A formulation were investigated. The experimental design is as follows.

[0526] 1) N / A (without viscosity modifier)

[0527] 2) 90mM MgCl2

[0528] 3) 120mM Arg-HCl

[0529] 4) 90mM histidine

[0530] After the formulation was prepared, a portion of the formulation sample was first taken to test its viscosity. Then, the sample was aseptically filtered and filled into vials. The appearance, SEC (secret chemical index), and non-reduced CE (colour index) of the samples were examined under the following conditions: 40℃ high temperature, repeated freeze-thaw cycles (-35℃ to 4℃), and shaking (25℃, 300 rpm). The effects of different excipients on the viscosity and stability of the formulation were evaluated. The results are shown in the table below.

[0531] Table 21. Viscosity Measurement Results

[0532] sample Viscosity (mPa·s) 1 82.2 2 12.1 3 15.8 4 17.7

[0533] Table 22. Stability Results

[0534]

[0535]

[0536] Note: D1 indicates testing after 1 day of vibration, and D15 indicates testing after 15 days of vibration.

[0537] The results show:

[0538] After adding the viscosity modifier, the appearance became opalescent, but there was no difference between the excipients. When the formulation contained 0.1 mg / mL PS80, the group containing the viscosity modifier showed particles after shaking for one day. However, when the PS80 concentration was 1 mg / mL, the appearance remained clear after shaking for 15 days.

[0539] The 40℃ SEC results showed that the SEC monomer content decreased slightly after the addition of the viscosity modifier, but there was no significant difference between the samples. The Arg-HCl and histidine sample groups showed better results, with the histidine sample exhibiting the best stability. The 40℃ CE results showed that the CE decreased slightly after the addition of the viscosity modifier, but the differences between the excipients were not significant.

[0540] Example 7. Concentration screening of viscosity modifiers

[0541] Formulations containing different concentrations of Hu25G7-A antibody and viscosity modifiers were prepared in a 20 mM His-AA pH 5.0 buffer system, and the viscosity of the formulations was investigated. The experimental design and results are shown in the table below.

[0542] Table 23. Viscosity Measurement Results

[0543]

[0544] The results showed that increasing the concentrations of viscosity modifiers His, MgCl2, and Arg-HCl significantly reduced the viscosity of the formulation.

[0545] Example 8. Formulation stability test

[0546] To improve the appearance of the formulation, the amount of basic amino acids (viscosity modifiers) added was reduced. The following formulation samples were prepared:

[0547] 1. 20mM His-AA pH 5.0, 30mM histidine, 0.8mg / mL PS80, 41.8mg / mL sucrose, antibody Hu25G7-A 120mg / mL, the final histidine content in this sample is 50mM;

[0548] 2. 20mM His-AA pH 4.8, 87mM histidine, 0.8mg / mL PS80, 150mg / mL antibody Hu25G7-A;

[0549] 3. 20mM His-AA pH 5.0, 100mM histidine, 0.8mg / mL PS80, 150mg / mL antibody Hu25G7-A;

[0550] 4. 20mM His-AA pH 5.0, 120mM arginine hydrochloride, 0.8mg / mL PS80, 150mg / mL antibody Hu25G7-A;

[0551] After the formulation was prepared, a portion of the formulation sample was first taken to test its viscosity. Then, the sample was aseptically filtered and filled into vials, and its stability at 4°C and 25°C was examined. The appearance, osmotic pressure, pH, SEC, non-reducing CE, and iCIEF of the samples were measured to evaluate the viscosity and stability of different formulation samples. The results are shown in the table.

[0552] Table 24. Viscosity, pH and Osmotic Pressure Results

[0553]

[0554] Table 25. Stability results at 4℃ and 25℃

[0555]

[0556]

[0557] The results showed that the pH drift of formulations 1 to 4 was within 0.1, indicating good buffering capacity; the osmotic pressure was within the isotonic range, and the viscosity was within 20 mPa·s; after 3 months of storage at 25°C, the CE / iCIEF of formulation 1 decreased slightly; after 3 months of storage at 4°C, the appearance remained clear, and the chemical stability showed no significant change. After 11 months of storage at 4°C, the appearance of formulations 1 to 4 remained clear, and the detection results of SEC, iCIEF, and CE-SDS were all relatively stable. Therefore, formulations 1 to 4 are all relatively stable.

[0558] Example 9. Sugar concentration screening

[0559] To develop a subcutaneous formulation and reduce injection irritation, isotonicity was optimal. Therefore, glucose concentrations were screened to determine the isotonic glucose concentration. An antibody formulation containing 50 mM His-AA at pH 5.0, 58 mg / mL sucrose, 0.8 mg / mL PS80, and 120 mg / mL hu25G7-A was prepared. Osmometry was measured using the freezing point method, and the test was performed three times.

[0560] Table 26. Osmolarity of Formulations (mosm / kg)

[0561] Test 1 Detection 2 Detection 3 average value 290 298 304 297

[0562] The results showed that the osmotic pressure was approximately 297 mosm / kg when the sugar concentration was 58 mg / mL, indicating isotonicity.

[0563] Example 10. Screening of freeze-drying processes

[0564] Prepare a formulation containing 25 mM His-AA (pH 4.9), 60 mg / mL hu25G7-A, 25 mg / mL sucrose, and 0.2 mg / mL PS80. Filter aseptically, fill 3.4 mL vials, and freeze-dry according to the procedure in the table below.

[0565] Table 27. Freeze-drying process

[0566] Process parameters Set temperature (°C) Set time (min) Duration (h) Vacuum level (Pa) Feeding 5 N / A N / A N / A Pre-freezing 5 10 1 N / A Pre-freezing -45 50 2.5 N / A One-time drying -5 60 30 20 Secondary drying 25 60 1 10 Secondary drying 25 1 7.5 1

[0567] After lyophilization, the sample appeared as a white, full-bodied powder without any collapse. It was reconstituted with approximately 1.5 mL of water for injection. The composition of the reconstituted sample was as follows: 50 mM His-AA, 120 mg / mL hu25G7-A, 50 mg / mL sucrose, 0.4 mg / mL PS80, pH 5.3. Various tests were performed on the reconstituted sample. The results showed that the reconstituted solution exhibited good performance in all parameters after the lyophilization process.

[0568] Table 28. Stability results before and after freeze-drying

[0569]

[0570]

[0571] Example 11. Optimization of Formulation

[0572] A design for DOE experiments was conducted using the pH of 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. A series of Hu25G7-A formulations were obtained using JMP software, all containing 58 mg / mL sucrose (see Table 29). The stability of the formulations was assessed through forced degradation, including high-temperature (40°C), shaking (300 rpm), and freeze-thaw tests (-35°C to 4°C). Appearance, viscosity, SEC, non-reducing CE, and iCIEF were used as evaluation indicators. The results are shown in Table 30, and statistical analysis was performed using the least squares method.

[0573] Table 29. DOE Formulation Screening Experiment Preparation Components and Viscosities

[0574]

[0575] Table 30. Results of DOE Prescription Screening Experiment

[0576]

[0577]

[0578] Note: M1.6 represents 1.6 months; D14 represents 14 days; D0 represents the start of the experiment.

[0579] The results showed that after five freeze-thaw cycles, the appearance of each formulation remained clear.

[0580] The forced degradation data were fitted, and the viscosity and the degradation difference at 40°C CE and iCIEF showed a better fit, indicating that the model was effective. Results are shown below. Figure 2 When the PS concentration was 0.8 mg / mL, contour plots were drawn with antibody concentration and pH as the x and y axes, and viscosity and the difference in CE / iCIEF degradation at 40 degrees Celsius as indicators. Contour plots of formulation stability changes were drawn with viscosity <30 cP, CE decrease <3%, and iCIEF decrease <30% as limits. The results are shown in […]. Figure 3 .

[0581] The results showed that the lower the protein concentration and the lower the pH, the lower the viscosity of the formulation. The optimal CE result was observed at pH 5.0, and the optimal iCIEF neutral peak was observed at pH 5.1. Combining contour plots and viscosity results, the following formulations were found to be stable: antibody concentration 100-140 mg / mL, PS80 0.4-1.2 mg / mL, pH 4.5-5.5. Furthermore, the formulation was more stable at antibody concentrations of 110-130 mg / mL and pH 4.85-5.45.

[0582] Example 12. Formulation stability testing

[0583] A formulation containing 50 mM His-AA (pH 5.0), 120 mg / mL hu25G7-A antibody, 58 mg / mL sucrose, and 0.8 mg / mL PS80 was prepared, aseptically filtered, and bottled. A stability test was then conducted at 4°C, and the results are shown in Table 31.

[0584] Table 31. Results of stability test at 4℃

[0585]

[0586] The results showed that the above-mentioned preparation remained stable after being placed at 4 degrees Celsius for 4 months. sequence list <110> Jiangsu Hengrui Medicine Co., Ltd., Shanghai Hengrui Medicine Co., Ltd. <120> An anti-IL-4R antibody pharmaceutical composition and its uses <130> 721013CPCT <150> CN202010107765.0 <151> 2020-02-21 <160> 48 <170> SIPOSequenceListing 1.0 <210> 1 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(118) <223> Heavy chain variable region of mouse monoclonal antibody 25G7 <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Ser Ser Gly Ser Ser Ile Ile Tyr Tyr Ala Asp Ile Val 50 55 60 Lys Gly Arg Ser Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Thr Arg Gly Asn Lys Arg Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ile Leu Thr Val Ser Ser 115 <210> 2 <211> 108 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(108) <223> Light chain variable region of mouse monoclonal antibody 25G7 <400> 2 Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Asn Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Arg Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Arg Ser Asn Pro Pro Met 85 90 95 Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Val Lys 100 105 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(10) <223> HCDR1 of mouse monoclonal antibody 25G7 <400> 3 Gly Phe Thr Phe Ser Asp Tyr Gly Met His 1 5 10 <210> 4 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(17) <223> HCDR2 of mouse monoclonal antibody 25G7 <400> 4 Phe Ile Ser Ser Gly Ser Ser Ile Ile Tyr Tyr Ala Asp Ile Val Lys 1 5 10 15 Gly <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(9) <223> HCDR3 of mouse monoclonal antibody 25G7 <400> 5 Gly Asn Lys Arg Gly Phe Phe Asp Tyr 1 5 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(10) <223> LCDR1 of mouse monoclonal antibody 25G7 <400> 6 Asn Ala Ser Ser Ser Val Ser Tyr Met Tyr 1 5 10 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(7) <223> LCDR2 of mouse monoclonal antibody 25G7 <400> 7 Leu Thr Ser Asn Leu Ala Ser 1 5 <210> 8 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(11) <223> LCDR3 of mouse monoclonal antibody 25G7 <400> 8 Gln Gln Trp Arg Ser Asn Pro Pro Met Leu Thr 1 5 10 <210> 9 <211> 125 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(125) <223> Heavy chain variable region of mouse monoclonal antibody 7B10 <400> 9 Gln Val Gln Leu Gln Gln Pro Gly Thr Glu Leu Leu Lys Pro Gly Ala 1 5 10 15 Ser Val Ser Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile His Pro Asn Ser Asp Thr Thr Lys Phe Ser Glu Asn Phe 50 55 60 Lys Thr Arg Ala Thr Leu Thr Ile Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Lys Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Lys Ile Ile Thr Thr Ile Val Ala Arg His Trp Tyr Phe 100 105 110 Asp Val Trp Gly Thr Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 10 <211> 111 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(111) <223> Light chain variable region of mouse monoclonal antibody 7B10 <400> 10 Asp Ile Val Leu Thr Gln Ser Pro Pro Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Gly 20 25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Leu Gly Gln Pro Pro 35 40 45 Lys Val Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Val Ala Thr Tyr Tyr Cys Gln His Ser Asn 85 90 95 Glu Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(10) <223> HCDR1 of mouse monoclonal antibody 7B10 <400> 11 Gly Tyr Thr Phe Thr Ser Tyr Trp Met His 1 5 10 <210> 12 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(17) <223> HCDR2 of mouse monoclonal antibody 7B10 <400> 12 Leu Ile His Pro Asn Ser Asp Thr Thr Lys Phe Ser Glu Asn Phe Lys 1 5 10 15 Thr <210> 13 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(16) <223> HCDR3 of mouse monoclonal antibody 7B10 <400> 13 Ser Lys Ile Ile Thr Thr Ile Val Ala Arg His Trp Tyr Phe Asp Val 1 5 10 15 <210> 14 <211> 15 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(14) <223> LCDR1 of mouse monoclonal antibody 7B10 <400> 14 Lys Ala Ser Gln Ser Val Asp Tyr Gly Gly Asp Ser Tyr Met Asn 1 5 10 15 <210> 15 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(7) <223> LCDR2 of mouse monoclonal antibody 7B10 <400> 15 Ala Ala Ser Asn Leu Glu Ser 1 5 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(9) <223> LCDR3 of mouse monoclonal antibody 7B10 <400> 16 Gln His Ser Asn Glu Asn Pro Pro Thr 1 5 <210> 17 <211> 445 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(445) <223> Heavy chain of humanized antibody hu25G7 <400> 17 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Ser Ser Gly Ser Ser Ile Ile Tyr Tyr Ala Asp Ile Val 50 55 60 Lys Gly Arg Ser Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Asn Lys Arg Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 18 <211> 215 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(215) <223> Light chain of humanized antibody hu25G7 <400> 18 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Asn Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Arg Ser Asn Pro Pro Met 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 19 <211> 455 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(455) <223> Heavy chain of humanized antibody hu7B10 <400> 19 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Leu Ile His Pro Asn Ser Asp Thr Thr Lys Phe Ser Glu Asn Phe 50 55 60 Lys Thr Arg Val Thr Met Thr Ile Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Lys Ile Ile Thr Thr Ile Val Ala Arg His Trp Tyr Phe 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 20 <211> 218 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(218) <223> Light chain of humanized antibody hu7B10 <400> 20 Asp Ile Val Leu Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Gly 20 25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Leu Gln Lys Pro Gly Gln Pro Pro 35 40 45 Gln Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser 65 70 75 80 Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln His Ser Asn 85 90 95 Glu Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 21 <211> 98 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(98) <223> Germline Heavy Chain Template IGHV3-48*01 <400> 21 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> 22 <211> 95 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(95) <223> Human germline light chain template IGKV3-11*01 <400> 22 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro 85 90 95 <210> 23 <211> 98 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(98) <223> Human germline heavy chain template IGHV1-2*02 <400> 23 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> twenty four <211> 100 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(100) <223> Humanoid Light Chain Template IGKV2D-29*01 <400> twenty four Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Pro 35 40 45 Pro Gln Leu Leu Ile Tyr Glu Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ser 85 90 95 Ile Gln Leu Pro 100 <210> 25 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(118) <223> hu25G7-VH-a <400> 25 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Ser Ser Gly Ser Ser Ile Ile Tyr Tyr Ala Asp Ile Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asn Lys Arg Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 26 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(118) <223> hu25G7‑VH‑b <400> 26 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Ser Ser Gly Ser Ser Ile Ile Tyr Tyr Ala Asp Ile Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Asn Lys Arg Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 27 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(118) <223> hu25G7-VH-c <400> 27 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Ser Ser Gly Ser Ser Ile Ile Tyr Tyr Ala Asp Ile Val 50 55 60 Lys Gly Arg Ser Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Asn Lys Arg Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 28 <211> 108 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(108) <223> hu25G7-VL-a <400> 28 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Asn Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Arg Ser Asn Pro Pro Met 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 29 <211> 108 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(108) <223> hu25G7-VL-b <400> 29 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Asn Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Arg Ser Asn Pro Pro Met 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 30 <211> 108 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(108) <223> hu25G7-VL-c <400> 30 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Asn Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Pro Trp Ile Tyr 35 40 45 Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Arg Ser Asn Pro Pro Met 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 31 <211> 125 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(125) <223> hu7B10-VH-a <400> 31 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Leu Ile His Pro Asn Ser Asp Thr Thr Lys Phe Ser Glu Asn Phe 50 55 60 Lys Thr Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Lys Ile Ile Thr Thr Ile Val Ala Arg His Trp Tyr Phe 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 32 <211> 125 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(125) <223> hu7B10-VH-b <400> 32 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Leu Ile His Pro Asn Ser Asp Thr Thr Lys Phe Ser Glu Asn Phe 50 55 60 Lys Thr Arg Val Thr Met Thr Ile Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Lys Ile Ile Thr Thr Ile Val Ala Arg His Trp Tyr Phe 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 33 <211> 125 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(125)<0ooo2279><223> hu7B10-VH-c <400> 33 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Leu Ile His Pro Asn Ser Asp Thr Thr Lys Phe Ser Glu Asn Phe 50 55 60 Lys Thr Arg Val Thr Leu Thr Ile Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Lys Ile Ile Thr Thr Ile Val Ala Arg His Trp Tyr Phe 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 34 <211> 111 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(111) <223> hu7B10-VL-a <400> 34 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Gly 20 25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Leu Gln Lys Pro Gly Gln Pro Pro 35 40 45 Gln Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser 65 70 75 80 Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln His Ser Asn 85 90 95 Glu Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 35 <211> 111 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(111) <223> hu7B10-VL-b <400> 35 Asp Ile Val Leu Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Gly 20 25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Leu Gln Lys Pro Gly Gln Pro Pro 35 40 45 Gln Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser 65 70 75 80 Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln His Ser Asn 85 90 95 Glu Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 36 <211> 111 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(111) <223> hu7B10-VL-c <400> 36 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Gly 20 25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Leu Gln Lys Pro Gly Gln Pro Pro 35 40 45 Gln Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser 65 70 75 80 Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln His Ser Asn 85 90 95 Glu Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 37 <211> 108 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(108) <223> Light chain variable region of hu25G7-A <400> 37 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 [[ID=三十九]]Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Pro Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser It should be noted that there is a mislabeling in the original text where "[[ID=三十九]]" is used instead of a proper ID. This has been retained in the translation as it is in the original. Also, the text might be more accurately processed and presented in a more organized format in a proper biological or patent document management system. 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Arg Ala Tyr Pro Pro Met 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 38 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(10) <223> LCDR1 of hu25G7-A <400> 38 Arg Ala Ser Ser Ser Val Pro Tyr Met Tyr 1 5 10 <210> 39 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(7) <223> LCDR2 of hu25G7-B <400> 39 Leu Ala Ser Ser Ser Arg Pro Ser 1 5 <210> 40 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(11) <223> LCDR3 of hu25G7-A <400> 40 Gln Gln Trp Arg Ala Tyr Pro Pro Met Leu Thr 1 5 10 <210> 41 <211> 108 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(108) <223> Light chain variable region of hu25G7-B <400> 41 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Pro Gly Val Pro Pro Leu 20 25 30 Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Leu Ala Ser Ser Arg Pro Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Arg Ser Asn Pro Pro Met 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 42 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(10) <223> LCDR1 of hu25G7-B <400> 42 Arg Ala Ser Pro Gly Val Pro Pro Leu Ala 1 5 10 <210> 43 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(118) <223> hu25G7-A / B VH optimized heavy chain variable region <400> 43 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Ser Ser Gly Ser Ser Ile Ile Tyr Tyr Ala Asp Ile Val 50 55 60 Lys Gly Arg Ser Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Asn Lys Arg Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 44 <211> 445 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(445) <223> Heavy chain of hu25G7-A and hu25G7-B <400> 44 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Ser Ser Gly Ser Ser Ile Ile Tyr Tyr Ala Asp Ile Val 50 55 60 Lys Gly Arg Ser Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Asn Lys Arg Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 45 <211> 215 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(215) <223> Light chain of hu25G7-A <400> 45 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Pro Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Arg Ala Tyr Pro Pro Met 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 46 <211> 215 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <222> (1)..(215) <223> Light chain of hu25G7-B <400> 46 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Pro Gly Val Pro Pro Leu 20 25 30 Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Leu Ala Ser Ser Arg Pro Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Arg Ser Asn Pro Pro Met 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 47 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> hu25G7-C VH <400> 47 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Ser Ser Gly Ser Ser Ile Ile Tyr Tyr Ala Asp Ile Val 50 55 60 Lys Gly Arg Ser Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Asn Lys Arg Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 48 <211> 445 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> hu25G7-C heavy chain[[ID=CI]] <400> 48 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Ser Ser Gly Ser Ser Ile Ile Tyr Tyr Ala Asp Ile Val 50 55 60 Lys Gly Arg Ser Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Asn Lys Arg Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445

Claims

1. A pharmaceutical composition comprising an anti-IL-4R antibody or antigen-binding fragment thereof and a buffer, the buffer being a histidine-acetate buffer, the buffer having a pH of 4.5 to 6.0, wherein the anti-IL-4R antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable region comprising a HCDR1, a HCDR2, and a 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; 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.

2. The pharmaceutical composition according to claim 1, wherein, the buffer having a pH of 4.5 to 5.

5.

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

4. The pharmaceutical composition of claim 1, wherein the concentration of the histidine-acetate buffer is 10 mM to 30 mM.

5. The pharmaceutical composition of claim 1, 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 1, wherein the concentration of the anti-IL-4R antibody or antigen-binding fragment thereof is 100 mg / mL to 200 mg / mL.

7. The pharmaceutical composition of claim 1, wherein the concentration of the anti-IL-4R antibody or antigen-binding fragment thereof is 100 mg / mL to 180 mg / mL.

8. The pharmaceutical composition of claim 1, wherein the concentration of the anti-IL-4R antibody or antigen-binding fragment thereof is 100 mg / mL to 150 mg / mL.

9. The pharmaceutical composition of claim 1, further comprising a viscosity modifier, wherein the viscosity modifier is selected from the group consisting of MgCl2, CaCl2, NaF, NaSCN, KCl, CH3COONa, Na2SO4, Nal, arginine, arginine hydrochloride, histidine, and lysine.

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

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

12. The pharmaceutical composition of claim 9, wherein 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.

13. The pharmaceutical composition of claim 10, wherein the concentration of the anti-IL-4R antibody or antigen-binding fragment thereof is 100 mg / mL to 200 mg / mL and the viscosity modifier is selected from the group consisting of 50 mM to 90 mM MgCl2; 50 mM to 100 mM histidine; and 10 mM to 200 mM arginine hydrochloride.

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

15. The pharmaceutical composition of claim 10, wherein the concentration of the anti-IL-4R antibody or antigen-binding fragment thereof is 100 mg / mL to 140 mg / mL; and the viscosity modifier is selected from the group consisting of 5 mM to 50 mM histidine, arginine hydrochloride, or MgCl2.

16. The pharmaceutical composition of claim 15, wherein the concentration of the anti-IL-4R antibody or antigen-binding fragment thereof is 100 mg / mL to 120 mg / mL; and the viscosity modifier is 10 mM to 40 mM histidine, arginine hydrochloride, or MgCl2.

17. The pharmaceutical composition of claim 1, further comprising a surfactant.

18. The pharmaceutical composition of claim 17, wherein the surfactant is a polysorbate.

19. The pharmaceutical composition of claim 17, wherein the surfactant is polysorbate 80 or polysorbate 20.

20. The pharmaceutical composition of claim 17, wherein the concentration of the surfactant is 0.1 mg / mL to 1.2 mg / mL.

21. The pharmaceutical composition of claim 17, wherein the concentration of the surfactant is 0.4 mg / mL to 1.0 mg / mL.

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

23. The pharmaceutical composition of claim 1, further comprising a stabilizer.

24. The pharmaceutical composition of claim 23, wherein the stabilizer is trehalose or sucrose.

25. The pharmaceutical composition of claim 23, wherein the concentration of the stabilizer is 20 mg / mL to 70 mg / mL.

26. The pharmaceutical composition of claim 25, wherein the concentration of the stabilizer is 40 mg / mL to 60 mg / mL.

27. The pharmaceutical composition of claim 25, wherein the concentration of the stabilizer is 58 mg / mL.

28. A pharmaceutical composition comprising: (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 buffer, pH 4.5-5.5; (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 comprising: (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 of a histidine-acetate buffer, pH 4.5-5.5; (c) 5 mM to 50 mM of 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; wherein the anti-IL-4R antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable region comprising a HCDR1, a HCDR2, and a HCDR3 as set forth in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; and 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; (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 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; 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 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.

29. A pharmaceutical composition comprising: (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, pH 4.5-5.5; (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, pH 4.5-5.5; (c) 40 mM to 220 mM of a viscosity modifier; and (d) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80; wherein the viscosity modifier is selected from the group consisting of: MgCl2, CaCl2, NaF, NaSCN, KCl, CH3COONa, Na2SO4, Nal, arginine, arginine hydrochloride, histidine, and lysine; wherein the anti-IL-4R antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable region comprising a HCDR1, a HCDR2, and a HCDR3 as set forth in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; and 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; (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 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; 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 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.

30. The pharmaceutical composition of claim 29, wherein 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, pH 4.5-5.5; (c) 10 mM to 40 mM viscosity modifier; (d) 0.4 mg / mL to 1.2 mg / mL polysorbate 80; and (e) 40 mg / mL to 70 mg / mL 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, pH 4.5-5.5; (c) 40 mM to 90 mM MgCl2; and (d) 0.4 mg / mL to 1.2 mg / mL 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, pH 4.5-5.5; (c) 50 mM to 100 mM histidine; and (d) 0.4 mg / mL to 1.2 mg / mL 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, pH 4.5-5.5; (c) 50 mM to 200 mM arginine hydrochloride; and (d) 0.4 mg / mL to 1.2 mg / mL polysorbate 80.

31. A pharmaceutical composition comprising: (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, pH 4.5-5.5; (c) 40 mM to 90 mM MgCl2; and (d) 0.4 mg / mL to 1.2 mg / mL 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, pH 4.5-5.5; (c) 50 mM to 100 mM histidine; and (d) 0.4 mg / mL to 1.2 mg / mL 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, pH 4.5-5.5; (c) 50 mM to 200 mM arginine hydrochloride; and (d) 0.4 mg / mL to 1.2 mg / mL polysorbate 80. (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 of a histidine-acetate buffer, pH 4.5-5.5; (c) 0.4 mg / mL to 1.2 mg / mL of polysorbate 80 and (d) 40 mg / mL to 70 mg / mL of sucrose; wherein the anti-IL-4R antibody, or antigen-binding fragment thereof, comprises: (i) a heavy chain variable region comprising a HCDR1, a HCDR2, and a HCDR3 as set forth in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; and 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; (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 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; 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 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.

32. The pharmaceutical composition of claim 31, comprising: (a) 120 mg / mL of an anti-IL-4R antibody, or antigen-binding fragment thereof; (b) 50 mM of a histidine-acetate buffer, pH 5.0; (c) 0.8 mg / mL of polysorbate 80 and (d) 58 mg / mL of sucrose.

33. A pharmaceutical composition comprising: (a) about 150 mg / mL of an anti-IL-4R antibody, or antigen-binding fragment thereof; (b) about 20 mM of a histidine-acetate buffer, pH about 5.0; (c) about 0.8 mg / mL of polysorbate 80 and (d) about 120 mM of arginine hydrochloride; wherein the anti-IL-4R antibody, or antigen-binding fragment thereof, comprises: (i) a heavy chain variable region comprising a HCDR1, a HCDR2, and a HCDR3 as set forth in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; and 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; (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 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; 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; (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.

34. The pharmaceutical composition of any one of claims 1 to 33, wherein 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: (v) a heavy chain variable region sequence as set forth in SEQ ID NO: 1 and a light chain variable region sequence as set forth in SEQ ID NO:

2.

35. The pharmaceutical composition of any one of claims 1 to 33, wherein 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: (IX) a heavy chain variable region sequence as set forth in SEQ ID NO: 43 and a light chain variable region sequence as set forth in SEQ ID NO: 37; or (X) a heavy chain variable region sequence as set forth in SEQ ID NO: 43 and a light chain variable region sequence as set forth in SEQ ID NO:

41.

36. The pharmaceutical composition of claim 1, wherein the anti-IL-4R antibody or antigen-binding fragment thereof comprises a constant region.

37. The pharmaceutical composition of claim 36, wherein the anti-IL-4R antibody comprises a heavy chain and a light chain as set forth in any one of: 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: 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.

38. A method of preparing the pharmaceutical composition of any one of claims 1 to 37, comprising the step of buffer exchange of an anti-IL-4R antibody or antigen-binding fragment thereof stock solution.

39. A lyophilized formulation comprising an anti-IL-4R antibody or antigen-binding fragment thereof, wherein the lyophilized formulation is obtained by lyophilizing the pharmaceutical composition of any one of claims 1 to 38.

40. A reconstituted solution comprising an anti-IL-4R antibody or antigen binding fragment thereof, wherein The reconstituted solution is obtained by reconstituting the lyophilized formulation of claim 39.

41. The reconstituted solution of claim 40, comprising: (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 polysorbate 80; and (d) 50 mg / mL sucrose, and 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) 0.4 mg / mL polysorbate 80; and (d) 50 mg / mL sucrose, and 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 polysorbate 80 and (d) 50 mg / mL sucrose, and 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) 20 mM histidine-acetate buffer; (c) 0.8 mg / mL polysorbate 80 and (d) 120 mM arginine hydrochloride, and the pH of the pharmaceutical composition is about 5.

3.

42. An article of manufacture comprising a container housing a pharmaceutical composition of any one of claims 1 to 37, or a lyophilized formulation of claim 39, or a reconstituted solution of any one of claims 40-41.

43. Use of a pharmaceutical composition of any one of claims 1 to 37, or a lyophilized formulation of claim 39, or a reconstituted solution of any one of claims 40-41, for the manufacture of a medicament for the treatment or prevention of an immune disease or disorder, said immune disease or disorder being asthma or dermatitis.

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