Pharmaceutical components containing antibodies against IL-4R and their uses.

Abstract

Provided are a pharmaceutical composition containing an anti-IL-4R antibody and the use thereof. The pharmaceutical composition contains an anti-IL-4R antibody or an antigen-binding fragment thereof in a histidine-acetic acid buffer agent, a viscosity modifier, a surfactant, and a stabilizer.

Description

Anti-il-4r antibody pharmaceutical compositions and uses thereof TECHNICAL FIELD

[0001] The present disclosure belongs to the field of pharmaceutical preparations, and in particular relates to a pharmaceutical composition comprising an anti-IL-4R antibody or antigen binding fragment thereof, and uses thereof as a drug for treating immune diseases. BACKGROUND

[0002] The statements herein are provided only to complement the present disclosure and do not necessarily constitute the prior art.

[0003] Allergic diseases are serious medical conditions, including non-life-threatening allergic reactions, and life-threatening allergic diseases. Current treatments for allergy include avoidance of allergens, pharmacological treatment for symptoms, and prevention with allergen-specific immunotherapy.

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

[0005] The biological activity of IL-4 is mediated by a specific cell surface IL-4 receptor (IL-4R). IL-4 receptor (IL-4R) consists of 802 amino acid residues, and IL-4R is expressed on the surface of T cells, B cells, thymocytes, myeloid cells, macrophages, and mast cells. The alpha chain of IL-4R is also a component of IL-13 receptor (IL-13R), so IL-4R can also mediate the biological activity of IL-13. As a new treatment method, a drug containing an IL-4R antagonist and its composition can be administered before, during or after the subject is exposed to an allergen or the onset of an allergic reaction.

[0006] Formulations with high protein concentrations pose challenges to the physical and chemical stability of the protein, and cause difficulties in the preparation, storage and delivery of the protein formulation. One problem is the tendency of the protein to form particles during handling and / or storage, making handling during further processing difficult.

[0007] Currently, there are existing patent applications related to anti-IL-4R antibodies and their formulations, including: WO2010053751, WO2001092340, WO2008054606, WO2014031610, CN106604744A, etc.

[0008] SUMMARY

[0009] Increased viscosity of protein formulations has negative effects on the manufacturing, storage, and administration of the drug from the preparation of the formulation to the delivery of the drug to the patient. Therefore, there is a need to develop relatively high concentration protein formulations with a suitably low viscosity that is suitable for the manufacturing, storage, and administration of the protein formulation.

[0010] The present disclosure provides a pharmaceutical composition comprising an anti-IL-4R antibody or antigen binding fragment thereof and a buffer selected from a histidine-acetate buffer, the pharmaceutical composition having a pH of 4.5-6.0, preferably about 4.5-5.5, most preferably about 5.0.

[0011] In some embodiments, the foregoing pharmaceutical composition, wherein the pH of the buffer is 4.8-5.5, preferably about 5.0.

[0012] In some embodiments, the pH of the buffer in the pharmaceutical composition is 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, about 5.5.

[0013] In some embodiments, the concentration of the histidine-acetate buffer in the pharmaceutical composition is 10 mM to 60 mM, non-limiting examples include 10 mM to 30 mM, 10 mM to 40 mM, 20 mM to 50 mM.

[0014] In some embodiments, the concentration of the buffer in the pharmaceutical composition is about 10 mM to 50 mM, preferably about 10 mM to 30 mM, non-limiting examples include 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM, 30 mM, 32 mM, 34 mM, 36 mM, 38 mM, 40 mM, 42 mM, 44 mM, 46 mM, 48 mM, and 50 mM, most preferably about 20 mM or 50 mM.

[0015] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof in the pharmaceutical composition is at a concentration of 100 mg / mL to 200 mg / mL, 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 point values.

[0016] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof in the pharmaceutical composition is at a concentration of 100 mg / mL to 180 mg / mL.

[0017] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof in the pharmaceutical composition is at a concentration of about 150 mg / mL.

[0018] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof in the pharmaceutical composition is at a concentration of 100 mg / mL or more, preferably 100 mg / mL to 150 mg / mL, most preferably 120 mg / mL.

[0019] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof in the pharmaceutical composition is at a concentration of 100 mg / mL to 140 mg / mL.

[0020] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof in the pharmaceutical composition is at a concentration of 100 mg / mL to 120 mg / mL, 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, 120 mg / mL.

[0021] In some embodiments, the pharmaceutical composition further comprises a viscosity modifier, wherein the viscosity modifier is selected from the group consisting of MgCl2, CaCl2, NaF, NaSCN, KCl, CH3COONa, Na2SO4, Nal, Arg-HCl, arginine, histidine, and lysine, preferably selected from the group consisting of MgCl2, histidine, and arginine hydrochloride (Arg-HCl).

[0022] 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-form amino acid, such as L-arginine, L-lysine, or L-histidine. In some embodiments, the viscosity modifier is selected from the group consisting of MgCl2, CaCl2, NaF, NaSCN, KCl, CH3COONa, Na2SO4, Nal, Arg-HCl, histidine, and lysine. In some embodiments, the viscosity modifier is selected from the group consisting of MgCl2, histidine, and Arg-HCl.

[0023] In some embodiments, the concentration of the viscosity modifier is 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, as well as any range between these point values.

[0024] In some embodiments, the concentration of the viscosity modifier is about 10 mM to about 220 mM. In some embodiments, the concentration of the viscosity modifier is about 50 mM to about 180 mM.

[0025] In some embodiments, the concentration of the viscosity modifier is about 5 mM to about 148 mM.

[0026] In some embodiments, the concentration of the viscosity modifier is about 50 mM to about 120 mM.

[0027] In some embodiments, the concentration of the viscosity modifier is about 5 mM to about 50 mM. In some embodiments, the concentration of the viscosity modifier is about 10 mM to about 40 mM.

[0028] In some embodiments, the viscosity of the pharmaceutical composition is less than 40 mPa.s, less than 30 mPa.s.

[0029] In some embodiments, the viscosity of the pharmaceutical composition is less than 20 mPa.s.

[0030] In some embodiments, the concentration of the viscosity modifier in the pharmaceutical composition is 50 mM to 148 mM, preferably 85 mM to 120 mM.

[0031] In some embodiments, the viscosity modifying agent is:

[0032] i) 5 mM to 220 mM arginine hydrochloride;

[0033] ii) 5 mM to 100 mM histidine; or

[0034] iii) 5 mM to 90 mM MgCl2.

[0035] In some embodiments, the viscosity modifying agent is:

[0036] i) 10 mM to 220 mM arginine hydrochloride;

[0037] ii) 10 mM to 100 mM histidine; or

[0038] iii) 10 mM to 90 mM MgCl2.

[0039] In some embodiments, the viscosity modifying agent is 5 mM to 220 mM arginine hydrochloride, 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.

[0040] In some embodiments, the viscosity modifying agent is 5 mM to 100 mM histidine, non-limiting examples 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 point values.

[0041] In some embodiments, the viscosity modifying agent is 10 mM to 90 mM MgCl2, non-limiting examples 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, or 90 mM, and any range between these point values.

[0042] In some embodiments, the anti-IL-4R antibody or antigen binding fragment thereof in the pharmaceutical composition has a concentration of 100 mg / mL to 200 mg / mL and the viscosity modifying agent is selected from the group consisting of MgCl2, histidine, and arginine hydrochloride.

[0043] In some embodiments, when the concentration of the anti-IL-4R antibody or antigen binding fragment thereof described in the pharmaceutical composition is 100 mg / mL to 200 mg / mL; the viscosity modifier is selected from the group consisting of 50 mM to 200 mM MgCl2, histidine, and arginine hydrochloride.

[0044] In some embodiments, when the concentration of the anti-IL-4R antibody or antigen binding fragment thereof described in the pharmaceutical composition is 100 mg / mL to 200 mg / mL; 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, preferably 50 mM to 180 mM arginine hydrochloride.

[0045] In some embodiments, the concentration of the anti-IL-4R antibody or antigen binding fragment thereof described in the pharmaceutical composition is 120 mg / mL to 150 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 50 mM to 120 mM arginine hydrochloride.

[0046] In some embodiments, the concentration of the anti-IL-4R antibody or antigen binding fragment thereof described in the pharmaceutical composition 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, preferably 10 mM to 40 mM histidine, arginine hydrochloride, or MgCl2.

[0047] In some embodiments, the concentration of the anti-IL-4R antibody or antigen binding fragment thereof described in the pharmaceutical composition is 100 mg / mL to 140 mg / mL; and the viscosity modifier is 30 mM histidine, arginine hydrochloride, or MgCl2.

[0048] In some embodiments, the concentration of the anti-IL-4R antibody or antigen binding fragment thereof described in the pharmaceutical composition is 100 mg / mL to 120 mg / mL; and the viscosity modifier is selected from the group consisting of 5 mM to 50 mM histidine, arginine hydrochloride, or MgCl2, preferably 10 mM to 40 mM histidine, arginine hydrochloride, or MgCl2.

[0049] In some embodiments, the viscosity modifier described in the pharmaceutical composition is selected from the group consisting of 50 mM to 90 mM MgCl2, 85 mM to 100 mM histidine, and 90 mM to 120 mM arginine hydrochloride.

[0050] In some embodiments, the viscosity modifier in the pharmaceutical composition is selected from the group consisting of 5 mM to 50 mM histidine, arginine hydrochloride, or MgCl2, preferably 10 mM to 40 mM histidine, arginine hydrochloride, or MgCl2.

[0051] In some embodiments, the pharmaceutical composition further comprises a surfactant, preferably polysorbate 80.

[0052] In some embodiments, the concentration of the surfactant in the pharmaceutical composition is 0.1 mg / mL to 1.2 mg / mL, preferably 0.8 mg / mL.

[0053] In some embodiments, the concentration of the surfactant in the pharmaceutical composition is about 0.1 mg / mL to 1.0 mg / mL, preferably 0.2 mg / mL to 0.8 mg / mL, more preferably 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 point values, most preferably 0.8 mg / mL.

[0054] In some embodiments, the foregoing pharmaceutical composition, comprising:

[0055] (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 histidine-acetate buffer, pH 4.5-6.0; (c) 50 mM to 220 mM viscosity modifier; and (d) 0.4 mg / mL to 1.2 mg / mL 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; preferably, wherein the viscosity modifier is selected from the group consisting of MgCl2, histidine, and arginine hydrochloride.

[0056] In some embodiments, the pharmaceutical composition further comprises a stabilizer, wherein the stabilizer is preferably selected from the group consisting of trehalose or sucrose, preferably sucrose.

[0057] In some embodiments, the concentration of the stabilizer in the pharmaceutical composition is 20 mg / mL to 70 mg / mL, preferably 40 mg / mL to 60 mg / mL, most preferably 58 mg / mL.

[0058] In some embodiments, the concentration of the stabilizer described in the pharmaceutical composition is 40 mg / mL to 70 mg / mL.

[0059] In some embodiments, the concentration of the sugar in the foregoing pharmaceutical composition is about 50 mg / mL to about 60 mg / mL, preferably 55 mg / mL to 60 mg / mL, non-limiting examples include 50 mg / mL, 51 mg / mL, 52 mg / mL, 53 mg / mL, 54 mg / mL, 55 mg / mL, 56 mg / mL, 57 mg / mL, 58 mg / mL, 59 mg / mL, and 60 mg / mL.

[0060] In some embodiments, the pharmaceutical composition comprises:

[0061] (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, 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.

[0062] In some embodiments, the pharmaceutical composition comprises:

[0063] (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, 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.

[0064] In some embodiments, the pharmaceutical composition comprises:

[0065] (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) 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.

[0066] In some embodiments, the pharmaceutical composition comprises:

[0067] (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;

[0068] or

[0069] (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 histidine-acetate buffer, pH 4.5-5.5; (c) 40 mM to 220 mM viscosity modifier; and (d) 0.4 mg / mL to 1.2 mg / mL polysorbate 80;

[0070] 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.

[0071] In some embodiments, the pharmaceutical composition comprises:

[0072] (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

[0073] (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

[0074] (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

[0075] (a) 100 mg / mL to 180 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof; (b) 10 mM to 30 mM of a histidine-acetate buffer, 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.

[0076] In some embodiments, the pharmaceutical composition comprises:

[0077] (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 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.

[0078] In some embodiments, the pharmaceutical composition comprises:

[0079] (a) 120 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof; (b) 20 mM of a histidine-acetate buffer, pH 4.5-5.5; (c) 30 mM of histidine; (d) 0.8 mg / mL of polysorbate 80; and (e) 58 mg / mL of sucrose.

[0080] In some embodiments, the pharmaceutical composition comprises:

[0081] (a) 120 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof; (b) 20 mM of a histidine-acetate buffer, pH about 5.0; (c) 30 mM of histidine; (d) 0.8 mg / mL of polysorbate 80; and (e) 58 mg / mL of sucrose.

[0082] In some embodiments, the pharmaceutical composition comprises:

[0083] (a) 120 mg / mL of an anti-IL-4R antibody or antigen-binding fragment thereof; (b) 50 mM of a histidine-acetate buffer, pH about 5.0; (c) 0.8 mg / mL of polysorbate 80; and (d) 58 mg / mL of sucrose.

[0084] In some embodiments, the pharmaceutical composition comprises:

[0085] (a) about 100 mg / mL to 180 mg / mL of an anti-IL-4R antibody, or antigen binding fragment thereof; (b) about 10 mM to 30 mM histidine-acetate buffer, pH about 4.5 to 5.5; (c) about 0.4 mg / mL to 1.2 mg / mL polysorbate 80 and (d) about 90 mM to 200 mM arginine hydrochloride.

[0086] In some embodiments, the pharmaceutical composition comprises:

[0087] (a) about 150 mg / mL of an anti-IL-4R antibody, or antigen binding fragment thereof; (b) about 20 mM histidine-acetate buffer, pH about 5.0; (c) about 0.8 mg / mL polysorbate 80 and (d) about 120 mM arginine hydrochloride.

[0088] In some embodiments, the pharmaceutical composition comprises:

[0089] (a) 100 mg / mL to 140 mg / mL of an anti-IL-4R antibody, or antigen binding fragment thereof; (b) 20 mM to 60 mM histidine-acetate buffer, pH 4.5-5.5; (c) 0.4 mg / mL to 1.2 mg / mL polysorbate 80 and (d) 40 mg / mL to 70 mg / mL sucrose.

[0090] In some embodiments, the pharmaceutical composition comprises:

[0091] (a) 100 mg / mL to 140 mg / mL of an anti-IL-4R antibody, or antigen binding fragment thereof; (b) 50 mM histidine-acetate buffer, pH 4.5-5.5; (c) 0.4 mg / mL to 1.2 mg / mL polysorbate 80 and (d) 40 mg / mL to 70 mg / mL sucrose.

[0092] In some embodiments, the pharmaceutical composition comprises:

[0093] (a) 100 mg / mL to 120 mg / mL of an anti-IL-4R antibody, or antigen binding fragment thereof; (b) 20 mM to 60 mM histidine-acetate buffer, pH 5.0-5.5; (c) 0.4 mg / mL to 1.2 mg / mL polysorbate 80 and (d) 50 mg / mL to 60 mg / mL sucrose; preferably, comprising:

[0094] (a) 120 mg / mL of an anti-IL-4R antibody, or antigen binding fragment thereof; (b) 50 mM histidine-acetate buffer, pH 5.0-5.5; (c) 0.8 mg / mL polysorbate 80 and (d) 58 mg / mL sucrose.

[0095] In some embodiments, the pharmaceutical composition comprises:

[0096] (a) 120 mg / mL of an anti-IL-4R antibody, or antigen-binding fragment thereof; (b) 50 mM histidine-acetate buffer, pH about 5.0; (c) 0.8 mg / mL polysorbate 80; and (d) 58 mg / mL sucrose.

[0097] In some embodiments, the anti-IL-4R antibody, or antigen-binding fragment thereof, in the pharmaceutical composition comprises a heavy chain variable region and a light chain variable region as set forth below:

[0098] (i) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; and

[0099] 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;

[0100] (ii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; and

[0101] a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively;

[0102] (iii) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; and

[0103] a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 38, SEQ ID NO: 7, and SEQ ID NO: 40, respectively; or

[0104] (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

[0105] 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

[0106] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof in the pharmaceutical composition comprises:

[0107] 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

[0108] 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.

[0109] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof in the pharmaceutical composition comprises a heavy chain variable region and a light chain variable region as set forth below:

[0110] (v) a heavy chain variable region sequence as set forth in SEQ ID NO: 1 or at least 90% identity to SEQ ID NO: 1 and a light chain variable region sequence as set forth in SEQ ID NO: 2 or at least 90% identity to SEQ ID NO: 2;

[0111] (vi) a heavy chain variable region sequence as set forth in SEQ ID NO: 9 or at least 90% identity to SEQ ID NO: 9 and a light chain variable region sequence as set forth in SEQ ID NO: 10 or at least 90% identity to SEQ ID NO: 10;

[0112] (vii) a heavy chain variable region sequence as set forth in SEQ ID NO: 25, 26, 27, 43, or 47, or at least 90% identity to SEQ ID NO: 25, 26, 27, 43, or 47, and a light chain variable region sequence as set forth in SEQ ID NO: 28, 29, 30, 37, or 41, or at least 90% identity to SEQ ID NO: 28, 29, 30, 37, or 41; or

[0113] (viii) a heavy chain variable region sequence as set forth in SEQ ID NO: 31, 32, or 33, or at least 90% identity to SEQ ID NO: 31, 32, or 33, and a light chain variable region sequence as set forth in SEQ ID NO: 34, 35, or 36, or at least 90% identity to SEQ ID NO: 34, 35, or 36;

[0114] Preferably, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region as set forth below:

[0115] (IX) a heavy chain variable region sequence as set forth in SEQ ID NO: 43 or at least 90% identity to SEQ ID NO: 43, and a light chain variable region sequence as set forth in SEQ ID NO: 37 or at least 90% identity to SEQ ID NO: 37; or

[0116] (X) a heavy chain variable region sequence as set forth in SEQ ID NO: 43 or at least 90% identity to SEQ ID NO: 43, and a light chain variable region sequence as set forth in SEQ ID NO: 41 or at least 90% identity to SEQ ID NO: 41 ; or

[0117] (XI) a heavy chain variable region sequence as set forth in SEQ ID NO: 47 or at least 90% identity to SEQ ID NO: 47, and a light chain variable region sequence as set forth in SEQ ID NO: 41 or at least 90% identity to SEQ ID NO: 41.

[0118] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof in the pharmaceutical composition comprises 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.

[0119] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof in the pharmaceutical composition comprises a constant region. In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a constant region of a human kappa, lambda chain, or variant thereof; further comprising a heavy chain constant region of a human IgGl, IgG2, IgG3, or IgG4, or variant thereof, such as IgG4-S228P or IgG4-234A / 235A mutant.

[0120] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof in the pharmaceutical composition comprises a heavy chain and a light chain as set forth below:

[0121] a heavy chain as set forth in SEQ ID NO: 17 and a light chain as set forth in SEQ ID NO: 18; or

[0122] a heavy chain as set forth in SEQ ID NO: 19 and a light chain as set forth in SEQ ID NO: 20; or

[0123] a heavy chain as set forth in SEQ ID NO: 44 and a light chain as set forth in SEQ ID NO: 45; or

[0124] a heavy chain as set forth in SEQ ID NO: 44, and a light chain as set forth in SEQ ID NO: 46; or

[0125] a heavy chain as set forth in SEQ ID NO: 48, and a light chain as set forth in SEQ ID NO: 46.

[0126] In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof in the pharmaceutical composition comprises a heavy chain as set forth in SEQ ID NO: 44, and a light chain as set forth in SEQ ID NO: 45.

[0127] The present application also relates to an anti-IL-4R antibody having a heavy chain variable region sequence as set forth in SEQ ID NO: 47 or at least 90% identity to SEQ ID NO: 47, and a light chain variable region sequence as set forth in SEQ ID NO: 41 or at least 90% identity to SEQ ID NO: 41.

[0128] The present application also relates to an anti-IL-4R antibody comprising a heavy chain as set forth in SEQ ID NO: 48, and a light chain as set forth in SEQ ID NO: 46.

[0129] 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.

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

[0131] 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.

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

[0133] 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.

[0134] 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.

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

[0136] 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.

[0137] 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.

[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) 122 mM NaCl.

[0139] 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.

[0140] 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) 148 mM CaCl2.

[0141] 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) 124 mM KCl.

[0142] 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) 86 mM CH3COONa.

[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) 96 mM Na2SO4.

[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) 113 mM NaI.

[0145] 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.

[0146] 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) 112 mM NaSCN.

[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) 120 mM Arg-HCl.

[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) 118 mM lysine.

[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) 93 mM histidine.

[0150] 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.

[0151] 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) 85 mM MgCl2.

[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) 148 mM CaCl2.

[0153] 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.

[0154] 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) 50 mM MgCl2.

[0155] 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 MgCl2.

[0156] 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 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) 148 mM CaCl2.

[0158] 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.

[0159] 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) 120 mM Arg-HCl.

[0160] 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 MgCl2; and (d) 0.1 mg / mL polysorbate 80.

[0161] 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 MgCl2; and (d) 1 mg / mL polysorbate 80.

[0162] 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.

[0163] 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) 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) 0.1 mg / mL polysorbate 80.

[0165] 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.

[0166] 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 Arg-HCl; and (d) 0.1 mg / mL polysorbate 80.

[0167] 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 Arg-HCl; and (d) 1 mg / mL polysorbate 80.

[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 MgCl2.

[0169] 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.

[0170] 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 90 mM to 200 mM Arg-HCl.

[0171] 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 Arg-HCl; 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 4.8; (c) 87 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) 100 mM histidine; and (d) 0.8 mg / mL polysorbate 80.

[0174] 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; (e) 41.8 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 5.0; (c) 30 mM histidine; and (d) 0.8 mg / mL polysorbate 80; (e) 58 mg / mL sucrose.

[0176] 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; (e) 50 mg / mL sucrose.

[0177] 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.

[0178] In one embodiment, the pharmaceutical composition comprises: (a) 120 mg / mL-150 mg / mL hu25G7-A antibody; (b) 20 mM histidine-acetate buffer, pH 5.0; (c) 30 mM-100 mM histidine; (d) 0.8 mg / mL polysorbate 80; and (e) 41.8 mg / mL-58 mg / mL sucrose.

[0179] 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.

[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, 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.

[0181] In one embodiment, the pharmaceutical composition comprises: (a) 100 mg / mL to 140 mg / mL hu25G7-A antibody; (b) 50 mM histidine-acetate buffer, 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.

[0182] In one embodiment, the pharmaceutical composition comprises: (a) 100 mg / mL to 120 mg / mL hu25G7-A antibody; (b) 50 mM histidine-acetate buffer, 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.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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.

[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; (c) about 1.2 mg / mL polysorbate 80; and (d) about 58 mg / mL sucrose.

[0189] 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.

[0190] In one embodiment, the pharmaceutical composition comprises: (a) about 120 mg / mL hu25G7-A antibody; (b) 50 mM histidine-acetate buffer, pH about 5.5; (c) about 1.2 mg / mL polysorbate 80; and (d) about 58 mg / mL sucrose.

[0191] 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.

[0192] The present disclosure also provides a method of preparing the pharmaceutical composition comprising the step of buffer exchange of an anti-IL-4R antibody or antigen binding fragment thereof stock solution.

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

[0194] The present disclosure also provides a lyophilized formulation comprising an anti-IL-4R antibody or antigen binding fragment thereof, wherein the lyophilized formulation is obtained by lyophilizing the aforementioned pharmaceutical composition after dilution.

[0195] The present disclosure also provides a lyophilized formulation comprising an anti-IL-4R antibody or antigen binding fragment thereof, wherein the lyophilized formulation is obtained by lyophilizing the aforementioned pharmaceutical composition after dilution of 1-fold, 2-fold or 3-fold.

[0196] The present disclosure also provides a reconstituted solution comprising an anti-IL-4R antibody or antigen binding fragment thereof, wherein the reconstituted solution is prepared by reconstituting the aforementioned lyophilized formulation.

[0197] In some embodiments, the reconstituted solution comprises the following ingredients:

[0198] (a) 120 mg / mL of an anti-IL-4R antibody or antigen binding fragment thereof; (b) 50 mM histidine-acetate buffer, pH about 5.0; (c) 0.4 mg / mL polysorbate 80 and (e) 50 mg / mL sucrose.

[0199] In one embodiment, the reconstituted 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.

[0200] In one embodiment, the reconstituted 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.

[0201] This disclosure also provides an article of manufacture comprising a container containing the aforementioned pharmaceutical composition, lyophilized formulation, or reconstituted solution.

[0202] 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.

[0203] 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.

[0204] In some implementations, the immune disease or condition is asthma.

[0205] In other embodiments, the immune disease or condition is an allergic skin disease.

[0206] 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.

[0207] 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.

[0208] 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.

[0209] In the present application, histidine can act as a buffer and also as a viscosity modifier, therefore, in the pharmaceutical composition, the final content of histidine is the sum of the content of histidine in the buffer and the content of histidine added continuously to reduce the viscosity. For example: in a 20 mM histidine-acetic acid buffer at pH 5.0, 90 mM of histidine is continuously added to increase its effect on reducing viscosity, then the final concentration of histidine in the pharmaceutical composition is 110 mM, and the pH of the pharmaceutical composition is about 5.0. BRIEF DESCRIPTION OF DRAWINGS

[0210] Figure 1: Anti-IL-4R antibody effect on mouse dermatitis experiment results. In the mouse dermatitis model, after sensitization by acetone, humanized antibodies hu25G7-A, hu25G7-B and positive reference antibody dupilumab were administered subcutaneously: twice a week, and the mouse ear thickness was detected on day 27. The results show that hu25G7-A, hu25G7-B and dupilumab can effectively reduce the ear thickness of mice compared with the control group, and hu25G7-B shows a better effect than dupilumab.

[0211] Figure 2: Formulation prescription fitting results.

[0212] Figure 3: Formulation stability change contour plot, where the gray area represents out-of-limit, and the white area represents in-limit. DETAILED DESCRIPTION

[0213] TERMS

[0214] To facilitate the understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise apparent from the context, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0215] The present disclosure incorporates by reference the entire contents of application PCT / CN2019 / 102169.

[0216] “Buffer” refers to a buffer that tolerates pH changes through the action of its acid-base conjugate components. Examples of buffers that control pH in an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0217] A "histidine salt buffer" is a buffer comprising a histidine ion. Examples of histidine salt buffers include histidine-hydrochloric acid, histidine-acetic acid, histidine-phosphoric acid, histidine-sulfuric acid, and the like, preferably a histidine-acetic acid buffer, which is a buffer prepared from histidine and acetic acid, also referred to as a histidine-acetic acid (His-AA) buffer.

[0218] A "citrate buffer" is a buffer comprising a citrate ion. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like. A preferred citrate buffer is a citric acid-sodium citrate buffer.

[0219] A "succinate buffer" is a buffer comprising a succinate ion. Examples of succinate buffers include succinic acid-sodium succinate, succinic acid-potassium succinate, succinic acid-calcium succinate, and the like. A preferred succinate buffer is a succinic acid-sodium succinate buffer.

[0220] A "phosphate buffer" is a buffer comprising a phosphate ion. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, and the like. A preferred phosphate buffer is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

[0221] An "acetate buffer" is a buffer comprising an acetate ion. Examples of acetate buffers include acetic acid-sodium acetate, acetic acid-histidine salt, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, and the like. A preferred acetate buffer is an acetic acid-sodium acetate buffer.

[0222] A "pharmaceutical composition" means a mixture of one or more antibodies or antigen-binding fragments thereof described herein with other chemical components, such as physiologically / pharmaceutically-acceptable carriers and excipients. The purpose of a pharmaceutical composition is to maintain stability of the antibody active ingredient, facilitate administration to an organism, and facilitate absorption of the active ingredient to exert a biological activity.

[0223] Herein, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0224] The solution form of the pharmaceutical composition described in the present disclosure is, unless otherwise specified, an aqueous solution.

[0225] "Substitution" means substitution of a solvent system in which an antibody protein is dissolved, for example, substitution of a high-salt or high-osmotic solvent system containing an antibody protein with a buffer system of a stable formulation by physical manipulation, thereby allowing the antibody protein to exist in a stable formulation. The physical manipulation includes, but is not limited to, ultrafiltration, dialysis, or resuspension after centrifugation.

[0226] “Freeze-dried formulation” means a formulation or pharmaceutical composition obtained after a liquid or solution formulation is subjected to a vacuum freeze-drying step.

[0227] “Sugar” of the present disclosure includes conventional compositions (CH2O) n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like. Examples include glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, xylitol, sorbitol, mannitol, melibiose, melezitose, raffinose, maltotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltulose, and the like. A preferred sugar is a non-reducing disaccharide, more preferably trehalose or sucrose, and most preferably sucrose.

[0228] A surfactant of the present disclosure can be selected from polysorbate 20, polysorbate 80, polyoxyl, Triton, sodium lauryl sulfate, sodium lauryl sulfate, sodium octylglycoside, lauryl / myristyl / linoleyl / stearyl-sulfobetaine, lauryl / myristyl / linoleyl / stearyl-sarcosine, linoleyl / myristyl / cetyl-sarcosine, laurylamidopropyl / karakamidopropyl / linoleamidopropyl / myristylamidopropyl / palmitoylamidopropyl / isostearylamidopropyl-betaine, myristylamidopropyl / palmitoylamidopropyl / isostearylamidopropyl-dimethylamine, sodium methylcocoate, sodium methyloleyltaurate, polyethylene glycol, polypropylene glycol, copolymer of ethylene and propylene glycol, and the like. A preferred surfactant is polysorbate 80 or polysorbate 20, more preferably polysorbate 80.

[0229] The term “viscosity” can be “kinematic viscosity” or “absolute viscosity.” “Kinematic viscosity” is a measure of a fluid’s resistance to 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 more 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, and another fluid takes 400 seconds, the second fluid has a kinematic viscosity that is twice that of the first. “Absolute viscosity,” sometimes also called dynamic or simply viscosity, is the result of kinematic viscosity and fluid density (absolute viscosity = kinematic viscosity x density). The unit of kinematic viscosity is L 2 / T, where L is length and T is time. Typically, kinematic viscosity is expressed in centistokes (cSt). The SI unit of kinematic viscosity is mm 2 / s, which is 1 cSt. Absolute viscosity is expressed in centipoise (cP). The SI unit of absolute viscosity is millipascal-seconds (mPa-s), where 1 cP = 1 mPa-s.

[0230] As used herein, the terms "about," "approximately," or "substantially" mean an acceptable limit of error for a particular value, which varies depending on how the value is measured or determined (i.e., the limitations of the measurement system). For example, "about" can mean within one standard deviation or more than one standard deviation in each performance of the art. Alternatively, "about" or "substantially" can mean a range of ± 20%, ± 15%, ± 10%, or ± 5% of the particular numerical value shown thereafter. Further, particularly for biological systems or processes, the terms can mean up to one order of magnitude or up to 5 times the value. Unless otherwise stated, the meaning of "about" or "substantially" should be assumed to be within an acceptable limit of error for the particular value when that particular value appears in the application and claims.

[0231] The pharmaceutical compositions described herein are capable of achieving a stable effect in which the antibody substantially retains its physical stability, and / or chemical stability, and / or biological activity upon storage. Preferably, the pharmaceutical composition substantially retains its physical and chemical stability as well as its biological activity upon storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. There are a variety of analytical techniques to measure protein stability, which can measure stability upon storage at a selected temperature for a selected period of time.

[0232] A stable pharmaceutical antibody formulation is one in which no significant changes are observed upon storage 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. In addition, a stable liquid formulation includes a liquid formulation that exhibits desirable characteristics upon storage at temperatures including 25°C for periods including 1 month, 3 months, 6 months. Typical acceptable criteria for stability are that generally no more than about 10%, preferably no more than about 5% of the antibody monomer is degraded as measured by SEC-HPLC. The pharmaceutical antibody formulation is a pale yellow to nearly colorless clear liquid or colorless, or clear to slightly opalescent by visual analysis. The concentration, pH, and osmolality of the formulation have no more than a ±10% change. Generally no more than about 10%, preferably no more than about 5% reduction is observed. Generally no more than about 10%, preferably no more than about 5% aggregation is formed.

[0233] An antibody "retains its physical stability" in a pharmaceutical formulation if the antibody shows no significant increase in aggregation, precipitation, and / or denaturation upon visual inspection of color and / or clarity, or as measured by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation can be evaluated by fluorescence spectroscopy, which determines the tertiary structure of the protein, and by FTIR spectroscopy, which determines the secondary structure of the protein.

[0234] An antibody "retains its chemical stability" in a pharmaceutical formulation if it does not show significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often 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 in conjunction with mass spectrometry or MALDI / TOF / MS), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate measurement), and isomerization (evaluated by measuring isoaspartate content, peptide mapping, etc.).

[0235] An antibody "retains its biological activity" in a pharmaceutical formulation if its biological activity at a given time is within a predetermined range of the biological activity exhibited at the time of manufacture of the pharmaceutical formulation. The biological activity of an antibody can be determined, for example, by an antigen binding assay.

[0236] "Human IL-4R" (hIL-4R) means a human cytokine receptor that specifically binds to interleukin-4 (IL-4), IL-4R alpha.

[0237] The three letter and one letter codes for amino acids used herein are as set forth in J. Biol. Chem, 243, p 3558 (1968).

[0238] The term "antibody" (Ab) encompasses any antigen binding molecule or molecular complex that comprises at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (or epitope thereof, e.g., an IL-4R antigen or epitope thereof). The term "antibody" encompasses immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region (CH). The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region (CL). The light chain constant region comprises one domain (CL). The VHand VLregions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VHand VLis composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0239] The FR of an anti-IL-4R antibody (or antigen-binding fragment thereof) can be identical to the human germline sequence, or can be naturally or artificially modified. The antibody can be of a different subclass, e.g., IgG (e.g., IgGl, IgG2, IgG3, or IgG4 subclass), IgAl, IgA2, IgD, IgE, or IgM antibody.

[0240] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR), such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as, for example, a

[0241] An antigen-binding fragment of an antibody typically will comprise at least one variable region. The variable region can be a region of any size or amino acid composition and typically will comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH region and a VL region, the VHand VL regions can be situated relative to one another in any suitable arrangement. For example, the variable region can be dimeric and contain VH-VL or VL-VH dimers.

[0242] In certain embodiments, an antigen-binding fragment can have any configuration of variable and constant regions, which can be either directly linked to one another or can be linked by a full or partial hinge or linker region. The hinge region can be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, such that it creates a flexible and semi-flexible linkage between adjacent variable and / or constant regions in a single polypeptide molecule. Furthermore, an antigen-binding fragment of the present disclosure can comprise a homo- or hetero-dimer (or other multimer) of variable and constant regions that are non-covalently associated with one another and / or with one or more monomeric VHor VL regions (e.g., via disulfide bonds).

[0243] A "murine antibody" is a monoclonal antibody derived from a mouse or rat according to the knowledge and skill in the art. It is prepared by injecting a test subject with an antigen, then isolating a hybridoma that expresses an antibody with the desired sequence or functional properties, the resulting antibody is a mouse-derived antibody when the test subject injected is a mouse, and a rat-derived antibody when the test subject injected is a rat.

[0244] A "chimeric antibody" is an antibody that fuses the variable region of an antibody of a first species (e.g., murine) with the constant region of an antibody of a second species (e.g., human). To create a chimeric antibody, a hybridoma that secretes a monoclonal antibody of the first species is first created, then the variable region gene is cloned from the hybridoma cell, and the constant region gene of the antibody of the second species is cloned as needed, the first species variable region gene and the second species constant region gene are ligated into a chimeric gene, which is then inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system. In a preferred embodiment of the present disclosure, the antibody light chain of the chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or variant thereof. The antibody heavy chain of the chimeric antibody further comprises a heavy chain constant region of a human IgGl, IgG2, IgG3, IgG4 or variant thereof, preferably a human IgGl, IgG2 or IgG4 heavy chain constant region, or a variant of the IgGl, IgG2 or IgG4 heavy chain constant region using amino acid mutations (such as YTE mutations, back mutations, L234A and / or L235A mutations, or S228P mutations).

[0245] The term "humanized antibody" includes CDR-grafted antibody, which refers to an antibody in which the CDR sequences of an animal-derived antibody, such as a murine antibody, are grafted into the variable region framework of a human antibody. Humanized antibodies can overcome the heterogeneity reaction induced by chimeric antibodies due to the presence of a large amount of heterologous protein components. Such framework sequences can be obtained from public DNA databases or published references, including germline antibody gene sequences. The germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at http: / / www.vbase2.org / ), and in Kabat, E. A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in immunogenicity causing a decrease in activity, a small number of back mutations can be made to the human antibody variable region framework sequence to maintain activity. The humanized antibodies of the present disclosure also include humanized antibodies that are further affinity matured by phage display of the CDRs.

[0246] Grafting of CDRs can result in a decrease in the affinity of the resulting antibody or antigen-binding fragment thereof for the antigen due to framework residues that contact the antigen. Such interactions can be the result of somatic hypermutation. Thus, it can still be desirable to graft such donor framework amino acids into the framework of the humanized antibody. Amino acid residues from the non-human antibody or antigen-binding fragment thereof that participate in antigen binding can be identified by examining animal monoclonal antibody variable region sequences and structures. Each residue in the CDR donor framework that differs from the germline can be considered relevant. If the closest germline cannot be determined, then the sequence can be compared to a subclass consensus sequence or to a consensus sequence of animal antibody sequences with a high percent similarity. Rare framework residues are considered likely to be the result of somatic hypermutation and thus play an important role in binding.

[0247] In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof can further comprise a light chain constant region of a human or murine kappa, lambda chain or variant thereof, or further comprise a heavy chain constant region of a human or murine IgGl, IgG2, IgG3, IgG4 or variant thereof.

[0248] "Conventional variants" of human antibody heavy chain constant regions and human antibody light chain constant regions refer to variants of heavy chain constant regions or light chain constant regions derived from humans that are known in the art to not alter the structure and function of the antibody variable region, exemplary variants include IgGl, IgG2, IgG3 or IgG4 heavy chain constant region variants that are site directed engineered and amino acid substituted, specific substitutions are known in the art as the YTE mutation, the L234A and / or L235A mutation, or the S228P mutation, or mutations that result in a knob-into-hole structure (such that the antibody heavy chain has a combination of knob-Fc and hole-Fc), these mutations have been shown to result in antibodies with new properties, but do not alter the function of the antibody variable region.

[0249] "Human antibody" and "human-derived antibody" are used interchangeably and can be an antibody derived from a human or an antibody obtained from a transgenic organism engineered to produce specific human antibodies in response to antigenic challenge and can be produced by any method known in the art. In certain technologies, elements of the human heavy and light chain loci are introduced into a cell strain that lacks endogenous immunoglobulin heavy and light chain loci. The transgenic organism can synthesize human antibodies specific for an antigen and the organism can be used to generate human antibody-secreting hybridomas. Human antibodies 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 by genetic or chromosomal transfection methods and phage display techniques, or from in vitro activated B cells, all of which are known in the art.

[0250] "Monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts. Each monoclonal antibody of a monoclonal antibody preparation (preparation) is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure can be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and use of transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

[0251] In addition, while two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be produced as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., 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 encompassed within the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same fashion as are intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulin.

[0252] Antigen-binding fragments can also be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al., 1995 Protein Eng. 8(10): 1057-1062; and U.S. Patent No. 5641870).

[0253] Fab is an antibody fragment having a molecular weight of about 50,000 Da and having antigen binding activity, which is obtained by treating an IgG antibody with a protease, papain, which cleaves the amino acid residue at position 224 of the H chain, in which about half of the N-terminal side of the H chain and the entire L chain are bound together by a disulfide bond.

[0254] F(ab')2 is an antibody fragment having a molecular weight of about 100,000 Da and having antigen binding activity, which is obtained by digesting the lower part of two disulfide bonds in the hinge region of IgG with pepsin, and contains two Fab regions connected at the hinge position.

[0255] Fab' is an antibody fragment having a molecular weight of about 50,000 Da and having antigen binding activity, which is obtained by cleaving the disulfide bond of the hinge region of the above F(ab')2. Fab' can be produced by treating F(ab')2 that specifically recognizes and binds to an antigen with a reducing agent such as dithiothreitol.

[0256] In addition, the Fab' fragment of the antibody can be expressed by inserting DNA encoding the Fab' fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector, and introducing the vector into a prokaryote or a eukaryote to express the Fab'.

[0257] The term "single-chain antibody", "single-chain Fv" or "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules can have the general formula: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example using 1-4 (including 1, 2, 3 or 4) repeats of the variant (Holliger et al. (1993), Proc Natl Acad Sci USA. 90:6444-6448). Other linkers useful in the present 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.

[0258] Diabodies refer to antibody fragments with bivalent antigen-binding activity that are dimerized scFv. In bivalent antigen-binding activity, the two antigens can be the same or different.

[0259] dsFv is obtained by linking polypeptides in which one amino acid residue in each of VH and VL is replaced with a cysteine residue via a disulfide bond between the cysteine residues. The amino acid residue to be replaced with a cysteine residue can be selected based on the three-dimensional structure prediction of an antibody according to a known method (Protein Engineering. 7:697 (1994)).

[0260] Antigen-binding fragments in some embodiments of the present disclosure can be produced by the following steps: obtaining cDNAs encoding VH and / or VL and other domains as required of a monoclonal antibody of the present disclosure that specifically recognizes and binds to an antigen, constructing DNA encoding the antigen-binding fragment, inserting the DNA into a prokaryotic expression vector or eukaryotic expression vector, and then introducing the expression vector into a prokaryote or eukaryote to express the antigen-binding fragment.

[0261] An "Fc region" can be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxy-terminus. The numbering of the residues in the Fc region is that of the EU index as in Kabat. 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 an immunoglobulin generally has two constant regions domains, CH2 and CH3.

[0262] The term "amino acid difference" or "amino acid mutation" refers to the presence of an amino acid change or mutation in a variant protein or polypeptide as compared to a parent protein or polypeptide, including an insertion, deletion, or substitution of one or more amino acids from the parent protein or polypeptide.

[0263] The "variable region" of an antibody refers to the variable region of the antibody light chain (VL) or the variable region of the antibody heavy chain (VH) either alone or in combination. As 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 and contribute to the formation of the antigen binding site of the antibody with CDRs from the other chain. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th Ed. 1991, National Institutes of Health, Bethesda MD)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al. J. Molec. Biol. 273:927-948 (1997)). As used herein, CDRs can refer to CDRs determined by either approach or by a combination of the two approaches.

[0264] The term "antibody framework" or "FR region" refers to the portion of a variable domain VL or VH that acts as a scaffold for the antigen binding loops (CDRs) of the variable domain. In essence, it is the variable domain without the CDRs.

[0265] The terms“complementarity determining region” and“CDR” refer to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding. Typically, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, LCDR3). The boundaries of the amino acid sequences of the CDRs can be determined using any of a variety of well-known schemes, including the“Kabat” numbering convention (see Kabat et al. (1991),“Sequences of Proteins of Immunological Interest,” 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD), the“Chothia” numbering convention Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001, and the ImMunoGenTics (IMGT) numbering convention (Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)), among others. For example, for the canonical format, following the Kabat convention, 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 in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following the Chothia convention, the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and in the VL are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). By combining the CDR definitions of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. Following the IMGT convention, the CDR amino acid residues in the VH are numbered approximately 27-38 (CDR1), 56-65 (CDR2), and 105-117 (CDR3), and in the VL are numbered approximately 27-38 (CDR1), 56-65 (CDR2), and 105-117 (CDR3).Following IMGT rules, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0266] "Antibody constant region domain" refers to a domain derived from the constant region of the light and heavy chains of an antibody, including CL and CHI, CH2, CH3, and CH4 domains derived from different classes of antibodies.

[0267] "Epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes usually consist of chemically distinct groups of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids that together form a particular spatial and polar property that interacts with the antibody. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996).

[0268] The terms "specifically binds," "selectively binds," "selectively binds," and "specifically binds" refer to the binding of an antibody to an epitope on a predetermined antigen.

[0269] 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 regions of the antibody "arms" interact with the antigen at multiple amino acid sites through weak noncovalent forces; the greater the interaction, the stronger the affinity. As used herein, the term "high affinity" for an antibody or antigen-binding fragment thereof (e.g., a Fab fragment) generally refers to an antibody or antigen-binding fragment thereof having a K -9 M or less. D (1E -10 M or less. D , 1E -11 M or less. D , 1E -12 M or less. D , 1E -13 M or less. D , 1E -14 M or less. D , etc.

[0270] The term "KD" or "K D " refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, an antibody binds to an antigen with a K -8 M, e.g., less than about 1E -9 M, 1E -10 M, or 1E -11M or smaller dissociation equilibrium constant (KD) for binding to an antigen, e.g., as determined using surface plasmon resonance (SPR) technology in a BIACORE instrument. The smaller the KD value, the greater the affinity.

[0271] The term "nucleic acid molecule" refers to DNA molecules and RNA molecules. A nucleic acid molecule can be single-stranded or double-stranded, DNA or RNA, e.g., a double-stranded DNA or mRNA. A nucleic acid is "operably linked" when it is functionally connected to another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0272] The term "vector" means a construct capable of delivering one or more genes or sequences of interest and preferably expressing them in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or bacteriophage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

[0273] Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, e.g., Current Protocols in Immunology, John Wiley & Sons, Inc., 5-8 and 15 chapters. For example, a mouse can be immunized with an antigen or fragment thereof, the resulting antibodies can be renatured, purified, and subjected to amino acid sequencing using conventional methods. Antigen-binding fragments can likewise be prepared using conventional methods. The antibodies or antigen-binding fragments described herein have one or more human FR regions added to the CDR regions of non-human origin using genetic engineering methods. Human FR germline sequences can be obtained by alignment of the IMGT human antibody variable region germline gene database and MOE software, or from Immunoglobulin Journal, 2001 ISBN 012441351.

[0274] 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. Bacteria that are readily transformed include members of the enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microbial hosts include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line), HEK293 cells (non-limiting examples of which are HEK293E cells), and NSO cells.

[0275] Engineered antibodies or antigen binding fragments can be produced and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As an alternative art, mammalian expression systems result in glycosylation of the antibody, 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 expanded in serum-free media in a bioreactor to produce the antibody. The culture fluid in which the antibody is secreted can be purified using conventional techniques. For example, purification can be performed using a protein A or protein G Sepharose FF column with a modified buffer. Non-specifically bound components are washed away. The bound antibody is eluted using a pH gradient method, and the antibody fragments are detected using SDS-PAGE and collected. The antibody can be filtered and concentrated using conventional methods. Soluble mixtures and multimers can also be removed using conventional methods, such as molecular sieving, ion exchange. The resulting product is immediately frozen, such as at -70°C, or lyophilized.

[0276] "Administer," "dose," "give" and "treat" when applied to an animal, human, test subject, cell, tissue, organ or biological fluid mean to provide contact of an exogenous drug, therapeutic agent, diagnostic agent, composition or human manipulation (such as "euthanasia" in the examples) to the animal, human, test subject, cell, tissue, organ or biological fluid. "Give" and "treat" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of a cell includes contact of a reagent with a cell, and contact of a reagent with a fluid that is in contact with a cell. "Give" and "treat" also mean by a reagent, diagnostic, binding composition or by another cell in vitro and ex vivo treatment of, for example, a cell. "Treat" when applied to a human, veterinary or research test subject means therapeutic treatment, prophylaxis or preventative measures, research and diagnostic applications.

[0277] "Treat" means to administer to a patient (or subject) having (or suspected of having, or susceptible to, one or more symptoms of a disease, a therapeutic agent known to have a therapeutic effect on such symptoms, e.g., a composition comprising any one of the compounds of the embodiments of the disclosure, which is to be used internally or externally. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in the patient (or subject) or population being treated, to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable extent. The amount of a therapeutic agent effective to alleviate any particular symptom of a disease (also referred to as a "therapeutically effective amount") can vary according to factors such as the disease state, age, and weight of the patient (or subject), and the ability of the drug to elicit a desired effect in the patient (or subject). Whether a disease symptom has been alleviated can be assessed by any clinical detection method typically used by a physician or other professional health care provider to assess the severity or progression of the symptom. Although embodiments of the disclosure (e.g., a method of treatment or article of manufacture) can not be effective in alleviating every symptom of a disease of interest, it is determined that the symptom of interest should be alleviated in a statistically significant number of patients (or subjects) according to any statistical test known in the art, such as the Student t-test, the Chi-square test, the U-test according to Mann and Whitney, the Kruskal-Wallis test (H-test), the Jonckheere-Terpstra test and the Wilcoxon test.

[0278] "Amino acid conservative modifications" or "amino acid conservative substitutions" refer to the substitution of one amino acid for another amino acid in a protein or polypeptide that has similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation, and rigidity, etc.) such that the change can often be made without altering the biological activity or other desired properties of the protein or polypeptide (e.g., antigen affinity and / or specificity). Those skilled in the art recognize that, in general, a single amino acid substitution in a non-essential region of a polypeptide will not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th ed.)). Moreover, substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity.

[0279] "Binding to IL-4R" means the ability to interact with human IL-4R. The term "antigen binding site" herein refers to the three-dimensional space recognized by an antibody or antigen binding fragment herein.

[0280] "Cross-reactivity" refers to the ability of an antibody herein to bind to IL-4R from a different species. For example, an antibody herein that binds to human IL-4R can also bind to IL-4R of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigen, or binding to or functional interaction with cells physiologically expressing IL-4R in a binding assay (e.g., SPR and ELISA). Methods to determine cross-reactivity include standard binding assays as described herein, e.g., surface plasmon resonance (SPR) analysis, or flow cytometry.

[0281] "Neutralizing" or "blocking" antibody means an antibody whose binding to hIL-4R results in inhibition of hIL-4 and / or hIL-13 biological activity. Such inhibition of hIL-4 and / or IL-13 biological activity can be assessed by measuring one or more indicators of hIL-4 and / or hIL-13 biological activity well known in the art, such as hIL-4 and / or hIL-13 induced cell activation and hIL-4 binding to hIL-4R, etc., as described in CN103739711A. "Inhibiting growth" (e.g., with respect to a cell) is intended to include any measurable decrease in cell growth.

[0282] "Inducing an immune response" and "enhancing an immune response" are used interchangeably and refer to the stimulation (i.e., either passively or adaptively) of an immune response to a particular antigen. The term "inducing" with respect to inducing CDC or ADCC refers to the stimulation of a particular direct cell killing mechanism.

[0283] "ADCC (antibody-dependent cell-mediated cytotoxicity)" refers to the lysis of a target cell by a cell expressing Fc receptors, which recognizes the Fc portion of the antibody bound to the target cell. The ADCC effect of an antibody can be reduced or eliminated by modification of the Fc portion of IgG, such as mutations in the constant region of the heavy chain, such as N297A, L234A, L235A of IgGl, IgG2 / 4 chimera, F235E, or L234A / E235A mutations of IgG4.

[0284] Engineered antibodies or antigen binding fragments can be produced and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. The sequences of the humanized antibodies herein can be inserted into the corresponding expression vectors using molecular cloning techniques, and expressed using the HEK293 cell expression system, to produce the corresponding humanized antibodies. As a more preferred prior art, mammalian expression systems result in 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 expanded in serum-free media in bioreactors to produce antibodies. The antibody-secreting culture fluid can be purified, collected using conventional techniques. The antibodies can be filtered and concentrated using conventional methods. Soluble aggregates and multimers can also be removed using conventional methods, such as molecular sieves, ion exchange. The resulting product is immediately frozen, such as at -70°C, or lyophilized.

[0285] "Effective amount" or "effective dose" means the amount of a drug, compound, or pharmaceutical composition necessary to effect any one or more of the beneficial or desired results. For prophylactic use, beneficial or desired results include eliminating or reducing risk, lessening severity, or delaying onset of a disease or disorder, including biochemical, histological, and / or behavioral symptoms of the disorder, its complications, and intermediate pathological phenotypes presenting during development of the disorder. For therapeutic use, beneficial or desired results include clinical results, such as reducing incidence or improving one or more symptoms of various disease states associated with the target antigens of the present disclosure, reducing the dosage of other medications required to treat a disease state, enhancing the effect of another medication, and / or delaying the progression of a disease state associated with the target antigens of the present disclosure in a patient (or subject).

[0286] "Exogenous" refers to a substance produced in or outside of a cell, organism, or human body, as the case can be.

[0287] "Endogenous" refers to a substance produced in or inside of a cell, organism, or human body, as the case can be.

[0288] "Isolated" refers to the state of purification and in this context means that the specified molecule is essentially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates or other materials, such as cellular debris and growth media. Generally, the term "isolated" is not intended to mean completely free from these materials or the absence of water, buffers, or salts unless they are present in amounts that significantly interfere with experimental or therapeutic uses of the compounds as described herein.

[0289] "Homology" or "identity" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, such as an adenine or an isoleucine, then the molecules are homologous at this position. A 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 x 100%. For example, if 6 of 10 positions in two sequences are matched or homologous, then the two sequences are 60% homologous. In general, comparisons are made at the amino acid level. The "at least 85% sequence identity" described herein means that the variant has at least 85% homology to the parent sequence, in some aspects, it has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology; in some specific aspects, it has 90%, 95%, or 99% or more; in other specific aspects, it has at least 95% sequence homology. The amino acid sequence having at least 85% sequence identity includes one or more amino acid deletion, insertion, or substitution mutations from the parent sequence.

[0290] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include their progeny. The terms "transformant" and "transformed cell" therefore include the primary subject cell and cultures derived from it, regardless of the number of transfers. It is also understood that all progeny can not be identical to the parental cell in DNA content, as a result of deliberate or inadvertent mutation. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.

[0291] "Optional" or "optionally" means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of the specified sequence can or can not be present.

[0292] Exemplary antibody pharmaceutical composition (formulation) preparation process:

[0293] First step: take a certain amount of purified anti-IL-4R antibody solution, solvent exchange with buffer without antibody (preferably ultrafiltration), at least 6 times volume replacement through ultrafiltration membrane, the antibody is concentrated to a certain concentration. Add a certain volume of other auxiliary material mother liquor, and dilute with buffer to make the antibody and each auxiliary material reach the required concentration, mix well. The stock solution is filtered and the control sample is detected for sterility. The stock solution is passed through a 0.22 μm PVDF filter cartridge, and the filtrate is collected.

[0294] Second step: adjust the volume to 2.15 mL, fill the filtrate into a 2 mL vial, add a plug, and take samples for control detection of volume difference at the beginning, middle and end of filling.

[0295] Third step: open the capping machine, add aluminum cap, and perform capping.

[0296] Fourth step: visual inspection to confirm that the product has no volume inaccuracy and other defects. Print and paste the vial label; print the carton label, fold the carton, pack the carton, and paste the carton label.

[0297] Examples

[0298] The following examples are used to further illustrate the present disclosure, but these examples do not limit the scope of the present disclosure. The experimental methods in the examples, unless otherwise specified, are generally carried out according to conventional conditions, such as the Antibody Techniques Laboratory Manual of Cold Spring Harbor, Molecular Cloning Manual; or according to the conditions recommended by the manufacturer of the raw materials or products. Reagents not specified by source are conventional reagents purchased on the market.

[0299] Antibody preparation examples

[0300] Example 1: Mouse immunization and detection

[0301] His-tagged human IL-4R (h-IL-4R-his) recombinant protein, his-tagged mouse IL-4R (m-IL-4R-his) recombinant protein, and his-tagged rhesus IL-4R (rhesus-IL-4R-his) recombinant protein were synthesized by Acrobiosystems company, and purified after expression in HEK293.

[0302] His-tagged human IL-4R (h-IL-4R-his) recombinant protein, his-tagged mouse IL-4R (m-IL-4R-his) recombinant protein, and his-tagged rhesus IL-4R (rhesus-IL-4R-his) recombinant protein were synthesized by Acrobiosystems company, and purified after expression in HEK293.

[0303] The CDR amino acid residues of the VL and VH regions of the antibody or antigen binding fragment in this example meet the known Kabat numbering rules (LCDR1-3, HCDR2-3) and AbM rules (HCDR1) in number and position.

[0304] Table 1. Immunogen information

[0305] Name Amino acid sequence Start End Database accession number / commercial designation 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

[0306] Anti-human IL-4R monoclonal antibodies were produced by immunizing mice. C57BL / 6 mice, female, 6-8 weeks old (Suzhou Zhaoyan New Drug Research Center Co., Ltd., Animal Production License No: 201503052) were used in the experiment.

[0307] Housing environment: SPF level. After the mice were purchased, they were raised in the laboratory environment for 1 week, with 12 / 12 hour light / dark cycle regulation, temperature 20-25℃; humidity 40-60%. The mice that had adapted to the environment were divided into 3 cages, 5 mice per cage. The immunogen was human IL-4R recombinant protein with Fc tag (h-IL4R-Fc, concentration 0.73 mg / mL). Emulsified with Freund's adjuvant (sigma, Cat#: F5881): first with Freund's complete adjuvant (CFA, Pierce, Cat#: 77140), and the rest with nucleic acid adjuvant (CpG, Shanghai Sangon) and aluminum adjuvant (Alum, Thermo Cat#: 77161).

[0308] On day 0, 70 μg / mouse of the emulsified antigen was injected intraperitoneally (IP). On days 14, 28, 42, 56, 77, the antigen was injected intraperitoneally (0.1 mL each) according to the back lump and abdominal swelling. Blood was collected on days 21, 35, 49, 63, 84 for blood test, and the mouse serum was detected by the ELISA method of Example 2 to determine the antibody titer in the mouse serum. After the fourth immunization, the mice with high antibody titer and titer tending to plateau in the serum were selected for spleen cell fusion, and 10 μg / mouse of the antigen solution prepared with phosphate buffer was injected intraperitoneally (IP) 3 days before fusion for boosting. The spleen lymphocytes were fused with myeloma cells Sp2 / 0 cells (ATCC, CRL-8287) using an optimized PEG-mediated fusion procedure to obtain hybridoma cells. CRL-8287 TM ).

[0309] Example 2: ELISA test and screening of antibodies

[0310] 1. ELISA binding experiment:

[0311] ELISA experiment was used to detect the binding property of anti-IL-4R antibody. The enzyme-labeled plate was coated with his-tagged IL-4R recombinant protein, and after the antibody was added to each well, the activity of the antibody binding to the antigen was detected by adding secondary antibody (HRP-conjugated anti-Fc antibody) and HRP substrate TMB.

[0312] Human or Rhesus IL-4R-his protein coated 96-well enzyme-labeled plate, 100 μL per well at a concentration of 0.5 μg / mL, 4°C overnight. Wash three times with 250 μL per well. Add 200 μL / well blocking solution and incubate at room temperature for 2 hours. Wash three times with 250 μL per well. Add 100 μL of anti-IL-4R antibody diluted with diluent to each well. Incubate at room temperature for 1 hour. Wash three times with 250 μL per well. Add 100 μL of HRP-labeled goat anti-human IgG secondary antibody diluted 1:20,000 with diluent to each well. Incubate at room temperature for 1 hour. Wash three times with 250 μL per well. Add 100 μL TMB to each well and avoid light for 15 minutes. Add 50 μL of 0.16 M / L sulfuric acid per well. Read the OD value at 450 nm with Thermo MultiSkanFc enzyme-labeled instrument, and calculate the binding EC 50 value of anti-IL-4R antibody to IL-4R.

[0313] 2. ELISA blocking experiment:

[0314] This experiment detects the blocking of the selected anti-human IL-4R antibody to human IL-4R and human IL-4 by in vitro blocking experiment. The specific method is to coat the Fc-tagged IL-4R recombinant protein on the 96-well enzyme-labeled plate, add the antibody that binds to human IL-4R to fully bind and occupy the epitope, then add IL-4 (Biolegend, Cat# 574004), and through biotin-conjugated anti-IL-4 antibody and Neutravidin-HRP (Pierce, Cat# 31001), detect whether IL-4 can still bind to IL-4R, and calculate the IC 50 value of IL-4R antibody to IL-4 / IL-4R binding.

[0315] Human IL-4R-Fc protein coated 96-well ELISA plate, 0.5 μg / mL concentration per 100 μL per well, 4°C incubate overnight. Wash three times with 250 μL per well. Add 200 μL / well blocking solution and incubate at room temperature for 2 hours. Wash three times with 250 μL per well. Add 100 μL per well of anti-IL-4R test antibody diluted with diluent, incubate at room temperature for 1 hour. Wash three times with 250 μL per well. Add 100 μL per well of diluted IL-4, incubate at room temperature for 1 hour, and wash three times. Add 100 μL per well of diluted biotin-conjugated anti-IL-4 antibody, incubate at room temperature for 1 hour, and wash three times. Dilute HRP-labeled Neutravidin 1:5000 with diluent, incubate at room temperature for 1 hour. Wash three times with 250 μL per well. Add 100 μL TMB per well, avoid light for 15 minutes. Add 50 μL 0.16 M / L sulfuric acid per well. Thermo MultiSkan FC microplate reader reads OD value at 450 nm, calculates the IC 50 value of IL-4R antibody blocking IL-4R and IL-4 binding.

[0316] Example 3: Reporter cell activity experiment of antibodies binding to human IL-4R

[0317] HEK-Blue IL-4 cells were purchased from Invivogen (Cat# hkb-stat6), which were stably transfected with human IL-4R gene and STAT6-mediated SEAP gene group, and the activation level of IL-4R signaling pathway could be characterized by detecting the secreted SEAP in the supernatant of SEAP substrate QUANTI-Blue.

[0318] This experiment detects the activation of HEK-Blue IL-4 cells, and evaluates the in vitro cell activity of IL-4R antibodies according to the IC 50 value.

[0319] HEK-Blue IL-4 cells were cultured in DMEM medium containing 10% FBS, 100 μg / mL Zeocin (Invivogen, Cat# ant-zn-05) and 10 μg / mL Blasticidin (Invivogen, Cat# ant-bl-05). Subcultured 2-3 times a week, with a subculture ratio of 1:5 or 1:10. When subcultured, aspirate the culture medium, rinse the cell layer with 5 mL of 0.25% trypsin, then aspirate the trypsin and place the cells in the incubator for 3-5 minutes for digestion, add fresh culture medium to resuspend the cells. Add 100 μL of cell suspension to a 96-well cell culture plate, with a density of 5 x 10 5Cells were seeded at 2 x 104cells / mL in 10% FBS, 100 μg / mL Zeocin and 30 μg / mL Blasticidin in DMEM, 100 μL sterile water was added to the periphery of the 96 well plate. The plate was incubated in the incubator for 24 hours (37°C, 5% CO2). After the cells adhered, 100 μL of the antibody to be tested was added to each well. The plate was incubated in the incubator for 20-24 hours (37°C, 5% CO2). 20 μL of the cell supernatant was taken from each well to a new 96 well flat bottom plate, 180 μL of QUANTI-Blue substrate solution was added, and the plate was incubated in the incubator for 1-3 hours in the dark. The absorbance at 620 nm was measured using a microplate reader (Thermo MultiSkan FC).

[0320] Example 4: Antibodies binding to human IL-4R inhibit TF-1 cell proliferation assay

[0321] TF-1 cells (ATCC CRL-2003) are lymphocytic leukemia cells that express IL-4R and are sensitive to IL-4 / IL-13 and other cytokines. IL-4 can stimulate the proliferation of TF-1 cells in the absence of GM-CSF. In this experiment, the neutralizing activity of different anti-IL-4R antibodies was compared by adding the antibodies to block the IL-4 pathway and inhibit the proliferation of TF-1 cells.

[0322] Cells were cultured in RPMI1640 medium containing 10% FBS, 2 ng / mL GM-CSF (R&D, Cat# 215-GM-010); passaged 2-3 times a week, with a 1:10 split ratio. 100 μL of cell suspension was added to each well of a 96-well cell culture plate, at a density of 2 x 104cells / mL; 100 μL sterile water was added to the periphery of the 96-well plate. 50 μL of the antibody to be tested was added to each well, and 50 μL of IL-4 (R&D, Cat# 204-IL-050) at a final concentration of 0.7 ng / mL was added to each well. The plate was incubated in the incubator for 72 hours (37°C, 5% CO2). After the incubation, the cell proliferation was detected using the CTG kit (Promega, Cat# G7572). 5 Cells were cultured in RPMI1640 medium containing 10% FBS, 2 ng / mL GM-CSF (R&D, Cat# 215-GM-010); passaged 2-3 times a week, with a 1:10 split ratio. 100 μL of cell suspension was added to each well of a 96-well cell culture plate, at a density of 2 x 104cells / mL; 100 μL sterile water was added to the periphery of the 96-well plate. 50 μL of the antibody to be tested was added to each well, and 50 μL of IL-4 (R&D, Cat# 204-IL-050) at a final concentration of 0.7 ng / mL was added to each well. The plate was incubated in the incubator for 72 hours (37°C, 5% CO2). After the incubation, the cell proliferation was detected using the CTG kit (Promega, Cat# G7572).

[0323] Example 5: In vitro binding affinity and kinetics assay

[0324] The affinity of the humanized IL-4R antibody to be tested was determined using Biacore, a GE instrument.

[0325] Human anti-capture antibody was covalently coupled to a Biacore instrument (Biacore X100, GE) biosensor chip CM5 according to the method in the human anti-capture kit (Cat. # BR-1008-39, GE) instructions to affinity capture a certain amount of antibody to be tested; then a series of concentration gradients of IL-4R antigen (all IL-4R antigens were purchased from Acrobiosystems, Cat# ILR-H5221) were flowed through the chip surface, and the reaction signal was detected in real time by the Biacore instrument (Biacore X100, GE) to obtain the binding and dissociation curves. After each cycle of dissociation was completed, the biosensor chip was washed and regenerated with the regeneration solution provided in the human anti-capture kit. The amino coupling kit used in the experiment was purchased from GE Company (Cat. # BR-1000-50, GE), and the buffer was HBS-EP+10x buffer solution (Cat. # BR-1006-69, GE) diluted with D.I. Water to 1x (pH 7.4).

[0326] The data obtained from the experiment were fitted with the Biacore X100 evaluation software 2.0 GE software using a (1:1) binding model to obtain the affinity value.

[0327] Example 6: Sequences and preparation of antibodies

[0328] The two monoclonal hybridoma cell strains with the best in vitro activity were selected by the ELISA binding experiment (human IL-4R-his ELISA binding) and the ELISA blocking experiment (human IL-4 / IL-4R ELISA blocking) of Example 2 above, the experiment of inhibiting the activation of HEK293-Blue IL-4 cells under IL-4 stimulation in Example 3, and the experiment of inhibiting the proliferation of TF-1 cells under IL-4 stimulation in Example 4. The activity detection results of the two monoclonal hybridoma cell strains are shown in Table 2.

[0329] Table 2. Activity detection results of hybridoma cell strains

[0330]

[0331] The monoclonal hybridoma cell strains 25G7 and 7B10 were selected, and the antibody sequences thereof were cloned. The sequence cloning process from the hybridoma was as follows:

[0332] The logarithmically growing hybridoma cells were collected, and the RNA was extracted with Trizol (Invitrogen, 15596-018) (according to the kit instructions), reverse transcribed (PrimeScript TMReverse Transcriptase, Takara, cat#2680A). The cDNA obtained by reverse transcription was subjected to PCR amplification using mouse Ig-Primer Set (Novagen, TB326Rev.B 0503) and then sent to a sequencing company for sequencing, and the obtained antibody sequence was analyzed.

[0333] The heavy chain and light chain variable region sequences of the murine monoclonal antibody 25G7 are as follows:

[0334] 25G7 HCVR

[0335]

[0336] 25G7 LCVR

[0337]

[0338] The CDR sequences contained therein are shown in Table 3.

[0339] Table 3. CDR sequences of monoclonal antibody 25G7

[0340] Name SEQ ID NO 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

[0341] The heavy chain and light chain variable region sequences of the murine monoclonal antibody 7B10 are as follows:

[0342] 7B10 HCVR

[0343]

[0344] 7B10 LCVR

[0345]

[0346] The CDR sequences contained therein are shown in Table 4.

[0347] Table 4. CDR sequences of monoclonal antibody 7B10

[0348] Name Sequence No. HCDR1 GYTFTSYWMH SEQ ID NO: 11 HCDR2 LIHPNSDTTKFSENFKT SEQ ID NO: 12 HCDR3 SKIITTIVARHWYFDV SEQ ID NO: 13 LCDR1 KASQSVDYGGDSYMN SEQ ID NO: 14

[0349] LCDR2 AASNLES SEQ ID NO: 15 LCDR3 QHSNENPPT SEQ ID NO: 16

[0350] The obtained variable region sequences are connected to human constant region sequences to obtain human-mouse chimeric antibody sequences. The chimeric antibody sequences are inserted into corresponding expression vectors by molecular cloning technology. The human-mouse chimeric antibodies 25G7-C and 7B10-C are obtained by using the HEK293 cell expression system.

[0351] The purified chimeric antibodies are subjected to corresponding in vitro activity detection by the methods of the foregoing Examples 2-5, and the data are shown in Table 5. The results show that the blocking effect of the 25G7-C antibody on IL-4 binding and the inhibitory effect on cell proliferation are significantly better than the reference antibody dupilumab (reference WHO Drug Information, Vol. 26, No. 4, 2012 synthesis).

[0352] Table 5. In vitro activity detection

[0353]

[0354] Example 7: Humanization experiment of mouse antibody

[0355] The obtained mouse-derived antibodies 25G7 and 7B10 are humanized. On the basis of the obtained mouse-derived antibody VH / VLCDR typical structure, the heavy and light chain variable region sequences are compared with the human antibody Germline database to obtain human germline templates with high homology. Among them, the human germline light chain framework region is from human kappa light chain gene, and the human germline light chain templates IGKV3-11*01 (SEQ ID NO: 22, used for antibody 25G7) and IGKV2D-29*01 (SEQ ID NO: 24, used for antibody 7B10) are preferred. The human germline heavy chain framework region is from human heavy chain, and the human germline heavy chain templates IGHV3-48*01 (SEQ ID NO: 21, used for antibody 25G7) and IGHV1-2*02 (SEQ ID NO: 23, used for antibody 7B10) are preferred.

[0356] The human germline template sequences are shown as follows.

[0357] Human germline heavy chain template IGHV3-48*01:

[0358]

[0359] Human germline light chain template IGKV3-11*01:

[0360]

[0361] Human germline heavy chain template IGHV1-2*02:

[0362]

[0363]

[0364] Human germline light chain template IGKV2D-29*01:

[0365]

[0366] The CDR regions of the mouse-derived antibody were grafted onto the selected humanized templates, then recombined with the IgG constant region, and subjected to back mutation to obtain a series of humanized molecules.

[0367] Among them, the hu7B10-VH-a, hu7B10-VH-b, hu7B10-VH-c were made into drug modification, the first position of the heavy chain human germline template was changed from Q to E. The heavy chain variable region sequences of the two antibodies after humanization are shown in SEQ ID NO: 25-27, SEQ ID NO: 31-33, respectively; the light chain variable region sequences are shown in SEQ ID NO: 28-30, SEQ ID NO: 34-36, respectively.

[0368] hu25G7-VH-a:

[0369]

[0370] hu25G7-VH-b:

[0371]

[0372] hu25G7-VH-c:

[0373]

[0374] hu25G7-VL-a:

[0375]

[0376] hu25G7-VL-b:

[0377]

[0378] hu25G7-VL-c:

[0379]

[0380] hu7B10-VH-a:

[0381]

[0382]

[0383] hu7B10-VH-b:

[0384]

[0385] hu7B10-VH-c:

[0386]

[0387] hu7B10-VL-a:

[0388]

[0389] hu7B10-VL-b:

[0390]

[0391] hu7B10-VL-c:

[0392]

[0393] Backmutation design for hybridoma clone 25G7 is shown in Table 6.

[0394] Table 6. Template selection and backmutation design

[0395]

[0396] Note: Amino acid positions for backmutations are determined using Kabat numbering convention.

[0397] Backmutation design for hybridoma clone 7B10 is shown in Table 7 below:

[0398] Table 7. Template selection and backmutation design

[0399]

[0400] Note: Amino acid positions for backmutations are determined using Kabat numbering convention.

[0401] 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 each are shown in SEQ ID NOs: 17-20.

[0402] hu25G7 HC

[0403]

[0404] hu25G7 LC

[0405]

[0406] hu7B10 HC

[0407]

[0408] hu7B10 LC

[0409]

[0410] Using molecular cloning techniques, the sequences of the humanized antibodies are inserted into the corresponding expression vectors, and using the HEK293 cell expression system, the corresponding humanized antibodies can be obtained.

[0411] Example 8: Humanized antibody activity data

[0412] The humanized antibodies hu25G7 and hu7B10 were subjected to the in vitro activity detection described in Examples 2-5, and the test results are shown in Table 8.

[0413] The results show that hu25G7 and hu7B10 both bind to human IL-4R, but not to rhesus IL-4R, indicating that both antibodies bind to epitopes that are not homologous between humans and rhesus monkeys, and can specifically bind to human IL-4R. Both antibodies can block IL-4 / IL-4R binding and intracellular signaling pathways, resulting in neutralization of the activation effect of IL-4 and inhibition of the proliferation of TF-1 cells, and the blocking and inhibitory activity of hu25G7 is still significantly better than that of the reference antibody dupilumab.

[0414] Table 8. In vitro activity detection

[0415]

[0416] Example 9: Affinity maturation experiment of humanized antibody hu25G7

[0417] The hu25G7 antibody was subjected to affinity maturation by a yeast display platform technology. Affinity-matured yeast libraries for 6 CDRs were designed and prepared based on the hu25G7 antibody. Degenerate primers were designed, and the designed mutant amino acids were introduced into the hu25G7-scFv antibody library by PCR and homologous recombination. The size of each library was about 10 9 The constructed yeast library was verified for library diversity by a second-generation sequencing method (GENEWIZ).

[0418] The biotin-labeled human IL-4R was used to screen high-affinity antibodies from the hu25G7-scFv yeast library. After two rounds of MACS screening (streptavidin magnetic beads, Invitrogen) and two rounds of FACS screening (BD FACSAriaTM FUSION), the yeast monoclonal was selected for culture and expression induction. The binding of the yeast monoclonal to human IL-4R was detected by FACS (BD FACSCanto II), and the yeast monoclonal with higher affinity than the wild-type 25G7 antibody was selected for sequencing verification. After comparison and analysis of the sequencing clones, the non-redundant sequences were converted into full-length human antibody molecules, which were expressed in mammalian cells. The full-length antibodies after affinity purification were subjected to affinity determination by BIAcoreTM X-100 (GE Life Sciences), and the candidate antibody molecules with higher affinity to human IL-4R were selected. The affinity of these antibody molecules to human IL-4R was higher than that of the wild-type hu25G7 antibody. Among them, the affinity of the hu25G7-A antibody molecule was comparable to that of dupilumab, and the affinity of the hu25G7-B molecule was significantly better than that of dupilumab.

[0419] The variable region sequence of the light chain of the antibody hu25G7-A obtained by affinity maturation is as follows:

[0420] hu25G7-A LCVR

[0421]

[0422] The CDR sequences contained therein are shown in Table 9.

[0423] Table 9. LCDR sequences of hu25G7-A

[0424] Name Sequence No. LCDR1 RASSSVPYMY SEQ ID NO: 38 LCDR2 LTSNLAS SEQ ID NO: 7 LCDR3 QQWRAYPPMLT SEQ ID NO: 40

[0425] The variable region sequence of the light chain of the antibody hu25G7-B obtained by affinity maturation is as follows:

[0426] hu25G7-B LCVR

[0427]

[0428] The CDR sequences contained therein are shown in Table 10.

[0429] Table 10. CDR sequences

[0430] Name Sequence No. LCDR1 RASPGVPPLA SEQ ID NO: 42 LCDR2 LASSRPS SEQ ID NO: 39 LCDR3 QQWRSNPPMLT SEQ ID NO: 8

[0431] The above light chain variable region hu25G7-A LCVR is recombined with hu25G7 light chain constant region to obtain hu25G7-A antibody light chain; the above light chain variable region hu25G7-B LCVR is recombined with hu25G7 light chain constant region to obtain hu25G7-B antibody light chain.

[0432] The above light chain variable region hu25G7-A LCVR is recombined with hu25G7 light chain constant region to obtain hu25G7-A antibody light chain; the above light chain variable region hu25G7-B LCVR is recombined with hu25G7 light chain constant region to obtain hu25G7-B antibody light chain.

[0433] hu25G7-A / B VH:

[0434]

[0435] hu25G7-C VH:

[0436]

[0437] The above light chain variable region hu25G7-A LCVR is recombined with hu25G7 light chain constant region to obtain hu25G7-A antibody light chain; the above light chain variable region hu25G7-B LCVR is recombined with hu25G7 light chain constant region to obtain hu25G7-B antibody light chain.

[0438] The complete heavy chain sequence of hu25G7-A and hu25G7-B is shown in SEQ ID NO: 44.

[0439] hu25G7 HC (i.e. hu25G7-A / hu25G7-B antibody heavy chain)

[0440]

[0441]

[0442] hu25G7-C antibody heavy chain:

[0443]

[0444] The respective complete light chain sequences are shown as SEQ ID NOs: 45-46.

[0445] hu25G7-A LC

[0446]

[0447] hu25G7-B / hu25G7-C LC

[0448]

[0449] Example 10: Activity data of affinity maturation of humanized antibodies

[0450] The two antibodies hu25G7-A and hu25G7-B were tested according to Example 3 and Example 4; both hu25G7-A and hu25G7-B can block the binding of IL-4 / IL-4R, and the activation of IL-4 and IL-13 caused by the intracellular signaling pathway is neutralized, and the proliferation of TF-1 cells is inhibited, and the activity data are shown in Table 11.

[0451] Table 11. Comparison of activity data

[0452]

[0453]

[0454] In the experiment of inhibiting the proliferation of TF-1 cells caused by IL-13 stimulation, hu25G7-A and hu25G7-B both showed beneficial effects. hu25G7-A and hu25G7-B had significantly improved effects in blocking the binding of IL-4, IL-13 and IL-4R, and the cell proliferation caused by the binding, compared with dupilumab.

[0455] Example 11: Study on the effect of humanized antibodies on mouse dermatitis

[0456] Establishment of mouse dermatitis model: IL-4 / IL-4Rα transgenic mice (purchased from Sino Model Animal Technology Co., Ltd. (Taicang)) were selected, and each mouse had an abdominal area of about 3 cm x 3 cm, which was uniformly coated with 1.5% OXZ acetone olive oil solution (acetone: olive oil = 4: 1) 100 μL for sensitization, and the sensitization day was counted as D0 (Day 0). Starting from the seventh day (Day 7), 1% OXZ acetone olive oil solution 20 μL was uniformly coated on both ears (two sides) of the mouse for attack, and the attack was performed every 72 hours.

[0457] The experiment set normal control group (only smearing acetone olive oil solution when sensitizing and exciting), model control group, hu25G7-A, hu25G7-B and dupilumab group, a total of 5 groups, 3-5 mice in each group, the dose of the drug administration group is 50mg / kg, the administration mode is subcutaneous administration, twice a week (see Table 12 for specific information). On the 27th day, the ear thickness was detected by vernier caliper, and the results are shown in Figure 1.

[0458] Table 12. Administration scheme of each group

[0459] Group Animal number Administration mode Administration dose Administration frequency* Normal control group 3 (male) S.C. - twice a week Model control group 5 (3 female + 2 male) S.C. - twice a week hu25G7-A 5 (3 female + 2 male) S.C. 50mg / kg twice a week hu25G7-B 3 (2 female + 1 male) S.C. 50mg / kg twice a week Dupilumab 4 (2 female + 2 male) S.C. 50mg / kg twice a week

[0460] The results show that the mice in the model control group have obvious pathological damage to the ears, and the ear thickness is significantly higher than that of the normal control group. The ear thickness of the mice in the hu25G7-A, hu25G7-B and dupilumab groups on the 27th day is significantly lower than that of the model control group. That is, hu25G7-A, hu25G7-B and dupilumab can be used to treat dermatitis, and the effect of hu25G7-B is more excellent than that of dupilumab.

[0461] Preparation of the preparation

[0462] The equipment used in the preparation process and the calculation method of the results are as follows:

[0463] SEC molecular exclusion chromatography:

[0464] An analytical method for separating solutes according to the relative relationship between the pore size of the gel pores and the size of the polymer sample molecules.

[0465] SEC monomer content percentage = Amonomer / Atotal*100% (Amonomer is the peak area of the main monomer peak in the sample, and Atotal is the sum of all peak areas).

[0466] SEC instrument for determination: Agilent 1260; column: waters, XBrige SEC (300x7.8mm 3.5μm).

[0467] CE capillary gel electrophoresis:

[0468] A method of electrophoresis in which the gel is moved into a capillary as a supporting medium, and the sample is separated according to the size of the molecular weight under a certain voltage.

[0469] 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, A total is the sum of all peak areas).

[0470] Instrument for CE determination: Beckman model plus800

[0471] iCIEF imaging capillary isoelectric focusing electrophoresis:

[0472] Technology for separation according to different protein isoelectric points pI.

[0473] iCIEF neutral peak content percentage = neutral peak area / total area * 100% (total area is the sum of acidic peak, neutral peak and basic peak areas).

[0474] Instrument for iCIEF determination: simple protein, model muarice.

[0475] Viscosity determination: rheometer (manufacturer Anton Paar, model MCR xx2) was used to determine the viscosity, the determination temperature was 25 degrees, the sample was directly placed on the test plate for testing.

[0476] Osmotic pressure: osmotic pressure was determined by freezing point method, based on the proportional relationship between freezing point drop value and molar concentration of the solution, high sensitivity temperature sensing element was used to determine the freezing point of the solution, and the osmotic pressure was converted by electric quantity. Instrument manufacturer Loser, model OM815.

[0477] Example 1. Screening of buffer system

[0478] In different buffer systems at pH 4.5-6.5, 100 mg / mL hu25G7-A antibody containing 0.1 mg / mL polysorbate 80 (PS80) formulations were prepared, and the stability of the antibody in different buffer systems was investigated. The specific prescription design is shown in the following table.

[0479] Table 13. Formulation sample ingredient table

[0480] Sample No. Buffer system 120 mM histidine-acetic acid (His-AA) pH 4.5 220 mM His-AA pH 5.0 320 mM acetic acid-sodium acetate (AA) pH 5.5 320 mM succinic acid-sodium succinate (SA) pH 5.5 320 mM citric acid-sodium citrate (CA) pH 5.5 320 mM His-AA pH 5.5 320 mM His-AA pH 6.0 320 mM His-AA pH 6.5

[0481] The samples were sterile filtered and filled, and the appearance and SEC of the samples under high temperature conditions at 40°C were investigated, respectively, and the data are shown in the following table.

[0482] Table 14. Formulation sample stability results

[0483]

[0484] Note: D15 indicates day 15 test, M indicates month, T0 indicates direct test after sample configuration, N / A indicates not tested.

[0485] The results show that the appearance of the SA / CA / AA buffer is poor, so the His-AA buffer system is selected. In the His-AA system, the appearance is better at pH 4.5-5.5; there is no significant difference between the SEC of each buffer system except His-AA pH 6.5, and the SEC of pH 5.0 and 6.0 is slightly better. Therefore, the buffer system of this antibody is selected as His-AA, and the pH is 4.5-6.0.

[0486] Example 2. Antibody concentration screening

[0487] In 20 mM His-AA pH 5.0, 5.5 buffer system, different concentrations of hu25G7-A formulation were prepared, and the formulation components were as follows:

[0488] 1) 20 mM His-AA pH 5.0, 100 mg / mL hu25G7-A

[0489] 2) 20 mM His-AA pH 5.5, 100 mg / mL hu25G7-A

[0490] 3) 20 mM His-AA pH 5.0, 150 mg / mL hu25G7-A

[0491] 4) 20 mM His-AA pH 5.5, 150 mg / mL hu25G7-A

[0492] 5) 20 mM His-AA pH 5.0, 139 mg / mL hu25G7-A

[0493] 6) 20 mM His-AA pH 5.0, 127 mg / mL hu25G7-A

[0494] 7) 20 mM His-AA pH 5.0, 120 mg / mL hu25G7-A

[0495] 8) 20 mM His-AA pH 5.5, 138 mg / mL hu25G7-A

[0496] 9) 20 mM His-AA pH 5.5, 122 mg / mL hu25G7-A

[0497] After the preparation of the formulation, the viscosity of the formulation was first determined by taking a portion of the sample. Then the sample was sterile filtered and filled, and the appearance, SEC, non-reduced CE-SDS, and iCIEF of the sample were investigated under high temperature conditions at 40°C. The effect of different antibody concentrations on the viscosity and stability of the hu25G7-A formulation was investigated. The results are shown in the following table:

[0498] Table 15. Results of determination of the viscosity of the formulation

[0499] Sample viscosity mPa.s 113.7 225.5 390.0 41 0.55 34.6 62 6.5 21.8 93.2 95 2.6

[0500] Table 16. Results of determination of the stability of the formulation

[0501]

[0502] The results show that there is no significant difference in appearance, SEC, CE, and iCIEF among different antibody concentrations, i.e., the antibody concentration has 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, when the antibody concentration is 100 mg / mL, the viscosity is about 14 mPa.s, and when the antibody concentration is 120 mg / mL, the viscosity is about 22 mPa.s.

[0503] Example 3. Screening of viscosity regulators

[0504] In 20 mM His-AA (pH 5.0) buffer, hu25G7-A formulations with an antibody concentration of 120 mg / mL and different viscosity regulators were prepared. The effect of different viscosity regulators on the viscosity of the formulation was investigated by taking the viscosity as an evaluation index. In order to meet the need of isotonicity of the subcutaneous formulation, the osmotic pressure of each viscosity regulator was 270 mosm / kg, and the effect was investigated. The specific results are shown in the following table.

[0505] Table 17. Viscosity results

[0506] Viscosity regulator concentration (mM) Average viscosity mPa.s blank N / A 16 NaCl 122 8 MgCl2 55.3 CaCl2 148 5.6 KCl 124 7.7 CH3COONa 868.8 Na2SO4 96 9.5 NaI 113 8.3 NaF 746.3 NaSCN 112 6.6 Arg-HCl 120 7.2 Lys 118 8.7 His 93 6.6 Pro 207 12.9

[0507] The results show that, in addition to proline, NaCl, MgCl2, CaCl2, KCl, CH3COONa, Na2SO4, Nal, NaF, NaSCN, Arg-HCl, Lys (lysine) and His (histidine) all have a significant effect on reducing the viscosity of the preparation at the corresponding maximum concentration. Among them, Arg-HCl, MgCl2, CaCl2, NaF, NaSCN and histidine have better effects, and Arg-HCl, MgCl2, CaCl2 and histidine are preferably selected as viscosity adjusting agents.

[0508] Example 4. Effect of viscosity adjusting agents on the viscosity of high-concentration preparations

[0509] To test the effect of viscosity adjusting agents, a hu25G7-A antibody preparation with a concentration of 150 mg / mL Hu25G7-A was prepared in a 20 mM His-AA, pH 5.0 buffer, and the specific components were as follows:

[0510] 1) No viscosity adjusting agent (N / A)

[0511] 2) 85 mM MgCl2

[0512] 3) 148 mM CaCl2

[0513] 4) 93 mM histidine

[0514] The effect of different excipients on the viscosity of high-concentration preparations was investigated with viscosity as the evaluation index, and the results are shown in the following table.

[0515] Table 18. Viscosity results

[0516] Sample viscosity mPa.s 188.80 216.86 17.22 19.00

[0517] The results show that when the antibody concentration is 150 mg / mL, MgCl2, CaCl2 and histidine can all reduce the viscosity of the preparation to below 20 mPa.s, and have a good viscosity-reducing effect.

[0518] Example 5. Effect of viscosity adjusting agents on the stability of the preparation

[0519] In a 20 mM His-AA pH 5.0 buffer system, a Hu25G7-A antibody preparation with a concentration of 150 mg / mL was prepared with different concentrations of excipients, and the effect of different concentrations of excipients on the thermal stability (placed at 40°C for 17 days) of the preparation was investigated. The experimental design and results are shown in the following table.

[0520] Table 19. Preparation samples

[0521] Sample Viscosity Modifiers 1 Blank (no addition) 250 mM MgCl2 390 mM MgCl2 490 mM CaCl2 148 mM CaCl2 690 mM Histidine 712 mM NaCl 812 mM Arg-HCl 820

[0522] Table 20. Effect of excipient amount on sample stability

[0523]

[0524] The results show that MgCl2, Arg-HCl, NaCl and histidine have less effect on the stability of the preparation, and the stability of the preparation containing MgCl2, Arg-HCl, NaCl and histidine is better than that of the preparation containing CaCl2. The CE decrease of the CaCl2 group under high temperature conditions is greater than that of other preparation samples. The stability of the preparation containing MgCl2, Arg-HCl and histidine is good, so MgCl2, Arg-HCl and histidine are preferred.

[0525] Example 6. Comprehensive evaluation of the effect of viscosity modifiers on the stability of the preparation

[0526] In 20 mM His-AA pH 5.0 buffer, a preparation containing 0.1 mg / mL PS80, 150 mg / mL Hu25G7-A and viscosity modifiers was prepared, and the concentration of PS80 in the sample shaken for 15 days was 1 mg / mL. The effect of different excipients on the viscosity and stability of Hu25G7-A preparation was investigated. The experimental design is as follows.

[0527] 1) N / A (no viscosity modifier)

[0528] 2) 90 mM MgCl2

[0529] 3) 120 mM Arg-HCl

[0530] 4) 90 mM histidine

[0531] After the preparation was completed, the viscosity of the preparation sample was first detected. Then the sample was sterile filtered and filled, and the appearance, SEC and non-reduced CE of the sample under the conditions of high temperature (40°C), repeated freeze-thaw (-35°C to 4°C), shaking (25°C, 300 rpm) were investigated, respectively, to evaluate the effect of different excipients on the viscosity and stability of the preparation. The results are shown in the following table.

[0532] Table 21. Viscosity determination results

[0533] Sample Viscosity mPa.s 182.2 212.1 15.8 417.7

[0534] Table 22. Stability results

[0535]

[0536]

[0537] Note: Shake D1 means detection after shaking for 1 day, and shake D15 means detection after shaking for 15 days.

[0538] The results show that:

[0539] After adding the viscosity regulator, the appearance showed opalescence, but there was no difference among the adjuvants; when the formulation contained 0.1 mg / mL PS80, the appearance showed particles after shaking for one day with the viscosity regulator. However, when the PS80 was 1 mg / mL, the appearance was still clear after shaking for 15 days.

[0540] The 40°C SEC results showed that after adding the viscosity regulator, the SEC monomer content decreased slightly, but there was no significant difference among the samples, among which the Arg-HCl sample group and the histidine sample group were better, and the histidine sample had the best stability. The 40°C CE results showed that after adding the viscosity regulator, the CE decreased slightly, but there was no significant difference among the adjuvants.

[0541] Example 7. Concentration screening of viscosity regulator

[0542] In a 20 mM His-AA pH 5.0 buffer system, formulations containing different concentrations of Hu25G7-A antibody and viscosity regulators were prepared to investigate the viscosity of the formulations. The experimental design and results are shown in the following table.

[0543] Table 23. Viscosity determination results

[0544]

[0545] The results show that increasing the concentrations of viscosity regulators His, MgCl2 and Arg-HCl can significantly reduce the viscosity of the formulation.

[0546] Example 8. Formulation stability test

[0547] To improve the appearance of the formulation, the amount of basic amino acid (viscosity regulator) added to the formulation was reduced, and the formulation samples were prepared as follows:

[0548] 1. 20 mM His-AA pH 5.0, 30 mM histidine, 0.8 mg / mL PS80, 41.8 mg / mL sucrose, antibody Hu25G7-A 120 mg / mL, the final content of histidine in this sample is 50 mM;

[0549] 2. 20 mM His-AA pH 4.8, 87 mM Histidine, 0.8 mg / mL PS80, 150 mg / mL antibody Hu25G7-A;

[0550] 3. 20 mM His-AA pH 5.0, 100 mM Histidine, 0.8 mg / mL PS80, 150 mg / mL antibody Hu25G7-A;

[0551] 4. 20 mM His-AA pH 5.0, 120 mM Arginine-HCl, 0.8 mg / mL PS80, 150 mg / mL antibody Hu25G7-A;

[0552] After the preparation of the formulations, first, the viscosity of the formulations was detected. Then the samples were sterile filtered and filled, and the stability at 4°C and 25°C was investigated, respectively. The appearance, osmotic pressure, pH, SEC, non-reduced CE, iCIEF of the samples were detected, and the viscosity and stability of different formulation samples were evaluated, and the results are shown in the table.

[0553] Table 24. Viscosity, pH and osmotic pressure results

[0554]

[0555] Table 25. 4°C, 25°C stability results

[0556]

[0557]

[0558] The results show that: the pH drifts of the No. 1 to No. 4 formulation samples are all within 0.1, and the buffer capacity is good; the osmotic pressure is in the isotonic range, and the viscosity is all within 20 mPa.s; the CE / iCIEF of the No. 1 sample decreases slightly after being placed at 25 degrees for 3 months; after being placed at 4 degrees for 3 months, the appearance is still clear, and the chemical stability has no significant change. The appearance of the No. 1 to No. 4 samples is clear after being placed at 4°C for 11 months, and the detection results of SEC, iCIEF and CE-SDS are relatively stable, so the above No. 1 to No. 4 formulations are relatively stable.

[0559] Example 9. Sugar concentration screening

[0560] In order to develop a subcutaneous formulation and reduce the irritation of injection, the osmotic pressure control is isotonic, so the isotonic sugar concentration is determined by screening the sugar concentration. A formulation containing 50 mM His-AA pH 5.0, 58 mg / mL sucrose, 0.8 mg / mL PS80 and 120 mg / mL hu25G7-A antibody was prepared, and the osmotic pressure was determined by freezing point method, and tested for 3 times.

[0561] Table 26. Osmolality of Formulations (mosm / kg)

[0562] Test 1 Test 2 Test 3 Average 290 298 304 297

[0563] The results show that the osmolality is about 297 mosm / kg, isotonic, when the sugar concentration is 58 mg / mL.

[0564] Example 10. Lyophilization Process Screening

[0565] A formulation of 25 mM His-AA pH 4.9, 60 mg / mL hu25G7-A, 25 mg / mL sucrose, 0.2 mg / mL PS80 was prepared, sterile filtered, and filled into 3.4 mL / vial, and lyophilized according to the procedure in the table below.

[0566] Table 27. Lyophilization Procedure

[0567] Process Parameter Set Temperature (°C) Set Time (min) Hold Time (h) Vacuum (Pa) Feed 5 N / A N / A N / A Pre-freeze 5 10 1 N / A Pre-freeze -4 5 0 2.5 N / A Primary Drying -5 6 0 3 0 2 Secondary Drying 2 5 6 0 1 1 0 Secondary Drying 2 5 1 7.5 1

[0568] After lyophilization, the vials were removed from the shelf and the samples appeared as white cake with full appearance and no collapse. About 1.5 mL of water for injection was used to reconstitute the samples, and the reconstituted samples had the following composition: 50 mM His-AA, 120 mg / mL hu25G7-A, 50 mg / mL sucrose, 0.4 mg / mL PS80, pH 5.3. The reconstituted samples were tested for various parameters. The results showed that the reconstituted solution was good in all parameters after dilution of the concentrated solution.

[0569] Table 28. Stability Results Before and After Lyophilization

[0570]

[0571]

[0572] Example 11. Optimization of Formulation

[0573] A series of hu25G7-A formulation recipes were obtained by using JMP software with 50 mM His-AA buffer pH, Hu25G7-A antibody concentration and PS80 concentration as variables: pH 4.5-5.5, PS80 0.4-1.2 mg / mL, Hu25G7-A antibody protein concentration 100-140 mg / mL. The formulations all contained 58 mg / mL sucrose, and the specific formulation recipes are shown in Table 29. The stability of the formulations was detected by forced degradation, including high temperature (40 degrees), shaking (300 rpm), freeze-thaw experiments (-35 degrees C to 4 degrees C). The appearance, viscosity, SEC, non-reducing CE, iCIEF were used as evaluation indexes, and the results are shown in Table 30. The results were statistically analyzed by the least squares method.

[0574] Table 29. Formulation ingredients and viscosity of DOE formulation screening experiments

[0575]

[0576] Table 30. Results of DOE formulation screening experiments

[0577]

[0578]

[0579] Note: M1.6 means 1.6 months; D14 means 14 days, and D0 means the beginning of the experiment.

[0580] The results showed that after 5 freeze-thaw cycles, the appearance of each formulation was still clear.

[0581] The forced degradation data were fitted, and the viscosity and 40-degree CE, iCIEF degradation difference were well fitted, the model was effective, and the results are shown in Figure 2. When the PS concentration was 0.8 mg / mL, the antibody concentration and pH were used as the horizontal and vertical coordinates, the viscosity, 40-degree CE / iCIEF degradation difference were used as the indexes to draw the contour map, and the viscosity <30 cP, CE reduction <3%, iCIEF reduction <30% were used as the limits to draw the contour map of the stability change of the formulation, and the results are shown in Figure 3.

[0582] The results showed that the lower the protein concentration, the lower the pH, and the lower the viscosity of the formulation; when the pH was 5.0, the CE result was optimal, and when the pH was 5.1, the neutral peak in iCIEF was optimal. Combined with the contour map and the viscosity results, the stable formulation was as follows: antibody concentration 100-140 mg / mL, 0.4-1.2 mg / mL PS80, pH 4.5-5.5. Further, when the antibody concentration was 110-130 mg / mL and the pH was 4.85-5.45, the formulation was more stable.

[0583] Example 12. Formulation stability detection

[0584] A formulation of 50 mM His-AA pH 5.0, hu25G7-A antibody 120 mg / mL, 58 mg / ml sucrose, 0.8 mg / mL PS80 was prepared, sterile filtered, and filled into vials. A 4°C stability experiment was then performed and the results are shown in Table 31.

[0585] Table 31. Results of 4°C stability experiment

[0586]

[0587] The results show that the above formulation is stable after 4 months at 4°C.

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