Pharmaceutical compositions of anti-IL4R antibodies and uses thereof

Abstract

The present invention provides an anti-IL4R antibody pharmaceutical composition and uses thereof, the pharmaceutical composition comprises an anti-IL4R antibody or an antigen binding fragment thereof and a buffer agent, and the pharmaceutical composition can further comprise a surfactant and a stabilizer.

Description

[0001] This application is a divisional application of the invention application with an application date of August 25, 2022, Chinese application number 202280046126.5, and invention name “Pharmaceutical compositions of anti-IL4R antibodies and their uses”. Technical Field

[0002] The present disclosure belongs to the field of preparations, and in particular, the present disclosure relates to a stable pharmaceutical composition comprising an anti-IL4R antibody or an antigen-binding fragment thereof, and use of the pharmaceutical composition for treating and / or preventing diseases associated with excessive IL4 and / or IL13 signaling. Background Art

[0003] Allergic diseases associated with type 2 inflammation, such as atopic dermatitis, anaphylaxis, allergic rhinitis and allergic asthma, affect more than 3 billion people worldwide, and the incidence continues to rise. According to the hygiene hypothesis, the high incidence is partly due to the fact that with the improvement of living standards, the chance of contact with infectious sources has decreased, making the immune system more sensitive to some originally harmless allergens (Stephen J. Galli et al., (2008) Nature 454 (7203): 445-454). Interleukin 4 (IL4 or IL-4) and IL-13 are two key factors in type 2 immunity that are required to drive most of the key hallmarks associated with type 2 inflammation, such as the production of immunoglobulin E and the recruitment of innate cells to sites of inflammation (Gruning G et al., (1998) Science 282:2261-2263; Rankin JA et al., (1996) Proc Natl Acad Sci USA 93:7821-7825; Wills-Karp M et al., (1998) Science 282:2258-2261).

[0004] IL-4 and IL-13 regulate cell function and activate transcriptional mechanisms by binding to cell surface receptors. Specifically, IL-4 first binds to the IL-4Rα chain with picomolar affinity, then recruits IL-2Rγ (γc) to form a type I receptor complex, or recruits IL-13Rα1 to form a type II receptor complex. Studies have found that non-hematopoietic cells do not express or low-express γc, but highly express IL-13Rα1, while lymphocytes do the opposite. Bone marrow cells are between these two types of cells. The type II receptor complex can also be formed by IL-13 binding to the IL-13Rα1 chain (binding with nanomolar affinity) and further recruiting the IL-4Rα chain. In addition, IL-13 can also bind to IL-13Rα2 with picomolar affinity to initiate the formation of decoy receptors (Irina G. Luzina et al., (2012) J Leukoc Biol 92 (4): 753-764). Once the IL-4 receptor complex is formed, intracellular signaling molecules will be activated, among which STAT6 and IRS signaling will be activated in response to type I IL-4 receptor, while type II IL-4 receptor cannot significantly activate IRS (Heller NM et al., (2008) Sci Signal 1(51):ra17-ra17). H 2 cell differentiation and IL-4 production are very important, and IRS can activate PI3K and mTOR signaling pathways (Gadani SP et al., (2012) J Immunol 189: 4213-4219). Studies have shown that excessive IL-4 and / or IL-13 signaling may cause allergic diseases. Other studies have found that STAT6 inhibitors can inhibit the growth of prostate cancer cells (Nappo G et al., (2017) Oncogenesis 6 (5): e342). Therefore, some therapeutic antibodies that regulate IL-4 and / or IL-13-mediated signaling have been developed, for example, Dupilumab, an antibody targeting IL4R.

[0005] Antibody drugs for human subjects should maintain biological activity and stability during storage and subsequent use, but antibody drugs have large molecular weights and complex structures, and are easily degraded, polymerized, or undergo undesirable chemical modifications, etc., and become unstable. Therefore, there is a need in the art to develop stable antibody drug compositions (e.g., anti-IL4R antibody drug compositions) to overcome the above problems, so that antibody drugs have a longer shelf life and are suitable for production and administration to patients. However, when antibodies are present at high concentrations, it is also necessary to overcome problems such as high levels of aggregation, osmotic pressures higher than physiological, and low injectability due to increased viscosity. SUMMARY OF THE INVENTION

[0007] The present disclosure provides a pharmaceutical composition comprising an anti-IL4Rα antibody or an antigen-binding fragment thereof at a high concentration and having an acceptable viscosity level. In addition, the pharmaceutical composition of the present disclosure does not cause a high level of aggregation.

[0008] The present disclosure provides a pharmaceutical composition comprising: (a) an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) a buffer, (c) a surfactant, and (d) a stabilizer.

[0009] In some embodiments, the concentration of the anti-IL4Rα antibody or its antigen-binding fragment in the aforementioned pharmaceutical composition is 30 mg / mL to 300 mg / mL, 50 mg / mL to 250 mg / mL, 70 mg / mL to 200 mg / mL, 100 mg / mL to 180 mg / mL, 120 mg / mL to 180 mg / mL, 120 mg / mL to 150 mg / mL, or 150 mg / mL to 180 mg / mL. In some embodiments, the concentration of the anti-IL4Rα antibody or its antigen-binding fragment in the aforementioned pharmaceutical composition is about 150 mg / mL. In some embodiments, the concentration of the anti-IL4Rα antibody or its antigen-binding fragment in the aforementioned pharmaceutical composition is about 175 mg / mL. In some embodiments, examples of the concentration of the anti-IL4Rα antibody or its antigen-binding fragment in the aforementioned pharmaceutical composition include, but are not limited to, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 125 mg / mL, about 130 mg / mL, about 135 mg / mL, about 140 mg / mL, about 145 mg / mL, about 150 mg / mL, about 155 mg / mL, about 160 mg / mL, about 165 mg / mL, about 170 mg / mL, about 175 mg / mL, about 180 mg / mL, about 190 mg / mL, or about 200 mg / mL.

[0010] In some embodiments, examples of the buffer in the aforementioned pharmaceutical composition include phosphate buffer, acetate buffer or histidine salt buffer. Preferably, examples of the buffer in the aforementioned pharmaceutical composition include histidine salt buffer. In some specific embodiments, examples of the phosphate buffer include sodium phosphate buffer or potassium phosphate buffer, examples of the acetate buffer include sodium acetate buffer, potassium acetate buffer or sodium acetate-acetate buffer, and examples of the histidine salt buffer include histidine-hydrochloric acid buffer, histidine-acetate buffer, histidine-histidine hydrochloride buffer or histidine hydrochloride buffer.

[0011] In some specific embodiments, the sodium phosphate buffer comprises disodium hydrogen phosphate and sodium dihydrogen phosphate. In some specific embodiments, the sodium acetate-acetic acid buffer comprises sodium acetate and acetic acid. In some specific embodiments, the histidine-histidine hydrochloride buffer comprises histidine and histidine hydrochloride.

[0012] In some embodiments, the concentration of the buffer in the aforementioned pharmaceutical composition is 1mM to 100mM, 2mM to 80mM, 4mM to 60mM, 8mM to 40mM, 10mM to 30mM, 10mM to 20mM, or 20mM to 30mM. In some embodiments, the concentration of the buffer in the aforementioned pharmaceutical composition is about 20mM. In some embodiments, examples of the concentration of the buffer in the aforementioned pharmaceutical compositions include, but are not limited to, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 30 mM, about 32 mM, about 34 mM, about 36 mM, about 38 mM, or about 40 mM.

[0013] In some embodiments, the pH of the buffer in the aforementioned pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3. In some embodiments, the pH of the buffer in the aforementioned pharmaceutical composition is about 5.8. In some embodiments, the pH of the buffer in the aforementioned pharmaceutical composition is about 6. In some embodiments, the pH of the buffer in the aforementioned pharmaceutical composition is about 5. In some embodiments, examples of the pH of the buffer in the aforementioned pharmaceutical composition include, but are not limited to, about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.

[0014] In some embodiments, the surfactant in the aforementioned pharmaceutical composition is selected from polysorbates (such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81 or polysorbate 85), poloxamers (such as poloxamer 181, poloxamer 188 or poloxamer 407), polyethylene glycol or polyhydroxyl bodies, etc. In some embodiments, the surfactant in the aforementioned pharmaceutical composition is polysorbate 80. In some embodiments, the surfactant in the aforementioned pharmaceutical composition is polysorbate 20.

[0015] In some embodiments, the concentration of the surfactant in the aforementioned pharmaceutical composition is 0.01 mg / mL to 2 mg / mL, 0.05 mg / mL to 1 mg / mL, 0.1 mg / mL to 0.8 mg / mL, 0.2 mg / mL to 0.6 mg / mL, or 0.2 mg / mL to 0.4 mg / mL. In some embodiments, the concentration of the surfactant in the aforementioned pharmaceutical composition is about 0.4 mg / mL. In some embodiments, examples of the concentration of the surfactant in the aforementioned pharmaceutical composition include but are not limited to: about 0.05 mg / mL, about 0.06 mg / mL, about 0.07 mg / mL, about 0.08 mg / mL, about 0.09 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, or about 1 mg / mL.

[0016] In some embodiments, the stabilizer in the aforementioned pharmaceutical composition is selected from one or more of trehalose, mannitol, sucrose, arginine or a pharmaceutically acceptable salt thereof, proline or sodium chloride. In some embodiments, the stabilizer in the aforementioned pharmaceutical composition comprises sucrose. In some embodiments, the stabilizer in the aforementioned pharmaceutical composition comprises arginine or a pharmaceutically acceptable salt thereof. In some embodiments, the stabilizer in the aforementioned pharmaceutical composition comprises proline. In some embodiments, the stabilizer in the aforementioned pharmaceutical composition comprises sodium chloride. Examples of pharmaceutically acceptable salts of arginine include arginine hydrochloride or arginine acetate. In some specific embodiments, the stabilizer in the aforementioned pharmaceutical composition comprises sucrose and arginine hydrochloride. In some specific embodiments, the stabilizer in the aforementioned pharmaceutical composition comprises proline and sodium chloride. In some specific embodiments, the stabilizer in the aforementioned pharmaceutical composition comprises sucrose, proline and sodium chloride.

[0017] In some embodiments, the concentration of sucrose in the aforementioned pharmaceutical composition is 10 mg / mL to 100 mg / mL, 20 mg / mL to 80 mg / mL, 30 mg / mL to 60 mg / mL, 40 mg / mL to 50 mg / mL, or 50 mg / mL to 60 mg / mL. In some embodiments, the concentration of sucrose in the aforementioned pharmaceutical composition is about 50 mg / mL. In some embodiments, examples of the concentration of sucrose in the aforementioned pharmaceutical compositions include, but are not limited to, about 20 mg / mL, about 22 mg / mL, about 24 mg / mL, about 26 mg / mL, about 28 mg / mL, about 30 mg / mL, about 32 mg / mL, about 34 mg / mL, about 36 mg / mL, about 38 mg / mL, about 40 mg / mL, about 42 mg / mL, about 44 mg / mL, about 46 mg / mL, about 48 mg / mL, about 50 mg / mL, about 52 mg / mL, about 54 mg / mL, about 56 mg / mL, about 58 mg / mL, about 60 mg / mL, about 62 mg / mL, about 64 mg / mL, about 66 mg / mL, about 68 mg / mL, about 70 mg / mL, about 72 mg / mL, about 74 mg / mL, about 76 mg / mL, about 78 mg / mL, or about 80 mg / mL.

[0018] In some embodiments, the concentration of arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride) in the aforementioned pharmaceutical composition is 10mM to 100mM, 20mM to 80mM, 30mM to 60mM, 40mM to 50mM, or 50mM to 60mM. In some embodiments, the concentration of arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride) in the aforementioned pharmaceutical composition is about 50mM. In some embodiments, examples of the concentration of arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride) in the aforementioned pharmaceutical compositions include, but are not limited to, about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, about 30 mM, about 32 mM, about 34 mM, about 36 mM, about 38 mM, about 40 mM, about 42 mM, about 44 mM, about 46 mM, about 48 mM, about 50 mM, about 52 mM, about 54 mM, about 56 mM, about 58 mM, about 60 mM, about 62 mM, about 64 mM, about 66 mM, about 68 mM, about 70 mM, about 72 mM, about 74 mM, about 76 mM, about 78 mM, or about 80 mM.

[0019] In some embodiments, the concentration of proline in the aforementioned pharmaceutical composition is 60mM to 600mM, 80mM to 500mM, 100mM to 450mM, 120mM to 400mM, 150mM to 350mM, 200mM to 350mM, or 250mM to 350mM. In some embodiments, the concentration of proline in the aforementioned pharmaceutical composition is about 250mM. In some embodiments, the concentration of proline in the aforementioned pharmaceutical composition is about 350mM. In some embodiments, examples of the concentration of proline in the aforementioned pharmaceutical compositions include, but are not limited to, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM, about 310 mM, about 320 mM, about 330 mM, about 340 mM, about 350 mM, about 360 mM, about 370 mM, about 380 mM, about 390 mM, or about 400 mM.

[0020] In some embodiments, the concentration of sodium chloride in the aforementioned pharmaceutical composition is 1mM to 80mM, 2mM to 60mM, 4mM to 50mM, 8mM to 40mM, 10mM to 30mM, or 10mM to 20mM. In some embodiments, the concentration of sodium chloride in the aforementioned pharmaceutical composition is about 20mM. In some embodiments, examples of the concentration of sodium chloride in the aforementioned pharmaceutical compositions include, but are not limited to, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 32 mM, about 34 mM, about 36 mM, about 38 mM, or about 40 mM.

[0021] In some embodiments, the pH of the aforementioned pharmaceutical composition is a pH of 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3. In some embodiments, the pH of the aforementioned pharmaceutical composition is about 5.8. In some embodiments, the pH of the aforementioned pharmaceutical composition is about 6. In some embodiments, the pH of the aforementioned pharmaceutical composition is about 5. In some embodiments, examples of the pH of the aforementioned pharmaceutical composition include, but are not limited to, about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.

[0022] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) a phosphate buffer, an acetate buffer and / or a histidine buffer, (c) polysorbate 80 or polysorbate 20, and (d) trehalose, mannitol, sucrose, arginine or a pharmaceutically acceptable salt thereof, proline and / or sodium chloride.

[0023] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) a phosphate buffer or a histidine salt buffer, (c) polysorbate 80, and (d) sucrose, arginine or a pharmaceutically acceptable salt thereof, proline and / or sodium chloride.

[0024] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) a histidine salt buffer, (c) polysorbate 80, and (d) sucrose and arginine or a pharmaceutically acceptable salt thereof.

[0025] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) a histidine salt buffer, (c) polysorbate 80, and (d) sucrose, proline, and sodium chloride.

[0026] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) a histidine salt buffer, (c) polysorbate 80, and (d) proline and sodium chloride.

[0027] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) a histidine salt buffer, (c) polysorbate 80, and (d) proline.

[0028] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) 30 mg / mL to 300 mg / mL of an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) 1 mM to 100 mM of a buffer, (c) 0.01 mg / mL to 2 mg / mL of a surfactant, and (d) a stabilizer comprising trehalose, mannitol, sucrose, arginine or a pharmaceutically acceptable salt thereof, proline and / or sodium chloride; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.

[0029] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) 50 mg / mL to 250 mg / mL of an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) 2 mM to 80 mM of a buffer, (c) 0.05 mg / mL to 1 mg / mL of a surfactant, and (d) a stabilizer comprising trehalose, mannitol, sucrose, arginine or a pharmaceutically acceptable salt thereof, proline and / or sodium chloride; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.

[0030] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) 70 mg / mL to 200 mg / mL of an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) 4 mM to 60 mM of a buffer, (c) 0.1 mg / mL to 0.8 mg / mL of a surfactant, and (d) a stabilizer comprising trehalose, mannitol, sucrose, arginine or a pharmaceutically acceptable salt thereof, proline and / or sodium chloride; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.

[0031] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) 100 mg / mL to 180 mg / mL or 120 mg / mL to 180 mg / mL of an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) 8 mM to 40 mM or 10 mM to 30 mM of a buffer, (c) 0.2 mg / mL to 0.6 mg / mL of a surfactant, and (d) a stabilizer comprising trehalose, mannitol, sucrose, arginine or a pharmaceutically acceptable salt thereof, proline and / or sodium chloride; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.

[0032] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) 120 mg / mL to 150 mg / mL or 150 mg / mL to 180 mg / mL of an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) 10 mM to 20 mM or 20 mM to 30 mM of a buffer, (c) 0.2 mg / mL to 0.4 mg / mL of a surfactant, and (d) a stabilizer comprising trehalose, mannitol, sucrose, arginine or a pharmaceutically acceptable salt thereof, proline and / or sodium chloride; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.

[0033] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) 30 mg / mL to 300 mg / mL of an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) 1 mM to 100 mM phosphate buffer, acetate buffer or histidine buffer, (c) 0.01 mg / mL to 2 mg / mL polysorbate 80 or polysorbate 20, and (d) 10 mg / mL to 100 mg / mL sucrose, 10 mM to 100 mM arginine or a pharmaceutically acceptable salt thereof, 60 mM to 600 mM proline, and / or 1 mM to 80 mM sodium chloride; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.

[0034] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) 50 mg / mL to 250 mg / mL of an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) 2 mM to 80 mM phosphate buffer, acetate buffer or histidine buffer, (c) 0.05 mg / mL to 1 mg / mL polysorbate 80 or polysorbate 20, and (d) 20 mg / mL to 80 mg / mL sucrose, 20 mM to 80 mM arginine or a pharmaceutically acceptable salt thereof, 80 mM to 500 mM proline, and / or 2 mM to 60 mM sodium chloride; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.

[0035] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) 70 mg / mL to 200 mg / mL of an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) 4 mM to 60 mM phosphate buffer, acetate buffer or histidine buffer, (c) 0.1 mg / mL to 0.8 mg / mL polysorbate 80 or polysorbate 20, and (d) 30 mg / mL to 60 mg / mL sucrose, 30 mM to 60 mM arginine or a pharmaceutically acceptable salt thereof, 100 mM to 450 mM or 120 mM to 400 mM proline, and / or 4 mM to 50 mM sodium chloride; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.

[0036] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) 100 mg / mL to 180 mg / mL or 120 mg / mL to 180 mg / mL of an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) 8 mM to 40 mM or 10 mM to 30 mM phosphate buffer, acetate buffer or histidine buffer, (c) 0.2 mg / mL to 0.6 mg / mL polysorbate 80 or polysorbate 20, and (d) 40 mg / mL to 50 mg / mL sucrose, 40 mM to 50 mM arginine or a pharmaceutically acceptable salt thereof, 150 mM to 350 mM proline, and / or 8 mM to 40 mM sodium chloride; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.

[0037] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) 120 mg / mL to 150 mg / mL or 150 mg / mL to 180 mg / mL of an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) 10 mM to 20 mM or 20 mM to 30 mM of a phosphate buffer, an acetate buffer or a histidine buffer, (c) 0.2 mg / mL to 0.4 mg / mL of polysorbate 80 or polysorbate 20, and (d) ) 50 mg / mL to 60 mg / mL sucrose, 50 mM to 60 mM arginine or a pharmaceutically acceptable salt thereof, 200 mM to 350 mM or 250 mM to 350 mM proline, and / or 10 mM to 30 mM or 10 mM to 20 mM sodium chloride; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.

[0038] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) 30 mg / mL to 300 mg / mL of an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) 1 mM to 100 mM phosphate buffer or histidine buffer, (c) 0.01 mg / mL to 2 mg / mL polysorbate 80, and (d) 60 mM to 600 mM proline and 1 mM to 80 mM sodium chloride, optionally, further comprising 10 mg / mL to 100 mg / mL sucrose; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.

[0039] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) 50 mg / mL to 250 mg / mL of an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) 2 mM to 80 mM phosphate buffer or histidine buffer, (c) 0.05 mg / mL to 1 mg / mL polysorbate 80, and (d) 80 mM to 500 mM proline and 2 mM to 60 mM sodium chloride, optionally, also comprising 20 mg / mL to 80 mg / mL sucrose; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.

[0040] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) 70 mg / mL to 200 mg / mL of an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) 4 mM to 60 mM phosphate buffer or histidine buffer, (c) 0.1 mg / mL to 0.8 mg / mL polysorbate 80, and (d) 100 mM to 450 mM or 120 mM to 400 mM proline, and 4 mM to 50 mM sodium chloride, optionally, further comprising 30 mg / mL to 60 mg / mL sucrose; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.

[0041] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) 100 mg / mL to 180 mg / mL or 120 mg / mL to 180 mg / mL of an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) 8 mM to 40 mM or 10 mM to 30 mM phosphate buffer or histidine buffer, (c) 0.2 mg / mL to 0.6 mg / mL polysorbate 80, and (d) 150 mM to 350 mM proline and 8 mM to 40 mM sodium chloride, optionally, further comprising 40 mg / mL to 50 mg / mL sucrose; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.

[0042] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) 120 mg / mL to 150 mg / mL or 150 mg / mL to 180 mg / mL of an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) 10 mM to 20 mM or 20 mM to 30 mM phosphate buffer or histidine buffer, (c) 0.2 mg / mL to 0.4 mg / mL polysorbate 80, and (d) 200 mM to 350 mM or 250 mM to 350 mM proline, and 10 mM to 30 mM or 10 mM to 20 mM sodium chloride, optionally, further comprising 50 mg / mL to 60 mg / mL sucrose; and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3.

[0043] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) 150 mg / mL of an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) 20 mM histidine-histidine hydrochloride buffer, (c) 0.4 mg / mL polysorbate 80, and (d) 50 mg / mL sucrose and 50 mM arginine hydrochloride; and the pH of the pharmaceutical composition is 5.8.

[0044] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) 150 mg / mL of an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) 20 mM histidine-histidine hydrochloride buffer, (c) 0.4 mg / mL polysorbate 80, and (d) 250 mM proline and 20 mM sodium chloride; and the pH of the pharmaceutical composition is 5.8.

[0045] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) 150 mg / mL of an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) 20 mM histidine-histidine hydrochloride buffer, (c) 0.4 mg / mL polysorbate 80, and (d) 350 mM proline; and the pH of the pharmaceutical composition is 5.8.

[0046] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) 150 mg / mL of an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) 20 mM histidine-histidine hydrochloride buffer, (c) 0.4 mg / mL polysorbate 80, and (d) 50 mg / mL sucrose, 250 mM proline and 20 mM sodium chloride; and the pH of the pharmaceutical composition is 5.8.

[0047] In an optional embodiment, the anti-IL4Rα antibody or antigen-binding fragment thereof in the aforementioned pharmaceutical composition comprises a heavy chain variable region, wherein the heavy chain variable region comprises a heavy chain variable region CDR1, a heavy chain variable region CDR2 and a heavy chain variable region CDR3, wherein the heavy chain variable region CDR1, the heavy chain variable region CDR2 and the heavy chain variable region CDR3 respectively comprise an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NOs: 1, 2 and 3.

[0048] In an optional embodiment, the anti-IL4Rα antibody or antigen-binding fragment thereof in the aforementioned pharmaceutical composition comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NOs: 7, 8 or 9. Wherein, the amino acid sequence of SEQ ID NO: 8 is:

[0049] EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYGMSWVRQAPGKGLVX1VX2TINSNGGSTSYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARFFRFRNAMDYWGQGTLVTVSS, where, X 1＝ W and X2=S, X1=L and X2=A, or X1=W and X2=A.

[0050] In an optional embodiment, the anti-IL4Rα antibody or antigen-binding fragment thereof in the aforementioned pharmaceutical composition comprises a light chain variable region, wherein the light chain variable region comprises a light chain variable region CDR1, a light chain variable region CDR2 and a light chain variable region CDR3, wherein the light chain variable region CDR1, the light chain variable region CDR2 and the light chain variable region CDR3 respectively comprise an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NOs: 4, 5 and 6.

[0051] In an optional embodiment, the anti-IL4Rα antibody or antigen-binding fragment thereof in the aforementioned pharmaceutical composition comprises a light chain variable region, wherein the light chain variable region comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NOs: 10, 11 or 12. Wherein, the amino acid sequence of SEQ ID NO: 11 is:

[0052] DIQMTQSPSSLSASVGDRVTITCRTSENIYSYLAWYQQKPGKAPKX1LX2YNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYGPPTWTFGQGTKVEIK, where X1=L and X2=I, X1=F and X2=V, or X1=F and X2=I.

[0053] In an optional embodiment, the anti-IL4Rα antibody or antigen-binding fragment thereof in the aforementioned pharmaceutical composition comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region respectively comprise CDR1, CDR2 and CDR3, wherein the heavy chain variable region CDR1, the heavy chain variable region CDR2, the heavy chain variable region CDR3, the light chain variable region CDR1, the light chain variable region CDR2 and the light chain variable region CDR3 respectively comprise an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NOs: 1, 2, 3, 4, 5 and 6.

[0054] In an optional embodiment, the anti-IL4Rα antibody or antigen-binding fragment thereof in the aforementioned pharmaceutical composition comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise: (1) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NOs: 7 and 10, respectively; (2) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NOs: 8 and 11, respectively, wherein X1=W and X2=S in SEQ ID NO: 8; (3) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NOs: 8 and 11, respectively, wherein X1=W and X2=S in SEQ ID NO: 8; NOs:8 and 12 have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs:8 and 12, wherein X1=W and X2=S in SEQ ID NO:8; (4) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs:9 and 11, respectively; (5) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs:9 and 11, respectively; NOs:9 and 12 have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs:9 and 12; (6) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs:8 and 11, respectively, wherein X1 in SEQ ID NO:8 is L and X2 is A; (7) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs:8 and 11, respectively, wherein X1 in SEQ ID NO:8 is L and X2 is A; (8) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs: The amino acid sequences shown in NOs:8 and 12 have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, wherein X1 in SEQ ID NO:8 = L and X2 = A;(8) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs: 8 and 11, respectively, wherein X1 in SEQ ID NO: 8 = W and X2 = A; or (9) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences set forth in SEQ ID NOs: 8 and 12, respectively, wherein X1 in SEQ ID NO: 8 = W and X2 = A.;

[0055] Wherein, the amino acid sequence of SEQ ID NO:8 is:

[0056] EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYGMSWVRQAPGKGLVX1VX2TINSNGGSTSYPDSVKGRFTISSRDNAKNTLYLQMNSLRAEDTAVYYCARFFRFRNAMDYWGQGTLVTVSS;

[0057] Wherein, the amino acid sequence of SEQ ID NO:11 is:

[0058] DIQMTQSPSSLSASVGDRVTITCRTSENIYSYLAWYQQKPGKAPKX1LX2YNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYGPPTWTFGQGTKVEIK, where X1=L and X2=I, X1=F and X2=V, or X1=F and X2=I.

[0059] In an optional embodiment, the anti-IL4Rα antibody or antigen-binding fragment thereof described in the aforementioned pharmaceutical composition comprises a heavy chain and a light chain, the heavy chain comprises a heavy chain variable region and a heavy chain constant region, the light chain comprises a light chain variable region and a light chain constant region, wherein the C-terminus of the heavy chain variable region is connected to the N-terminus of the heavy chain constant region, and the C-terminus of the light chain variable region is connected to the N-terminus of the light chain constant region, wherein the heavy chain variable region and the light chain variable region comprise the above-mentioned amino acid sequence, the heavy chain constant region has a human IgG4 constant region with an amino acid sequence as shown in SEQ ID NO: 13, and the light chain constant region has a human κ constant region with an amino acid sequence as shown in SEQ ID NO: 14.

[0060] In an optional embodiment, the anti-IL4Rα antibody or antigen-binding fragment thereof described in the aforementioned pharmaceutical composition may be a full-length antibody, such as a full-length antibody of the IgG1, IgG2 or IgG4 isotype, preferably a full-length antibody of the IgG4 isotype. In other embodiments, the anti-IL4Rα antibody or antigen-binding fragment thereof described in the aforementioned pharmaceutical composition may be a single-chain variable region (scFv) antibody, or an antibody fragment, such as a Fab or F(ab')2 fragment.

[0061] In an optional embodiment, the anti-IL4Rα antibody or antigen-binding fragment thereof described in the aforementioned pharmaceutical composition is the C2C1A1A1 antibody described in the patent document with application number PCT / CN2021077784.

[0062] The present disclosure also provides a method for preparing the aforementioned pharmaceutical composition, comprising the step of contacting the aforementioned anti-IL4Rα antibody or its antigen-binding fragment with a buffer, for example, replacing the anti-IL4Rα antibody or its antigen-binding fragment into a buffer, wherein the buffer comprises a phosphate buffer, an acetate buffer or a histidine salt buffer, etc., and further comprising adding a surfactant and a stabilizer, regardless of the order of addition, wherein the stabilizer comprises trehalose, mannitol, sucrose, arginine or a pharmaceutically acceptable salt thereof, proline and / or sodium chloride, and the surfactant comprises polysorbate, poloxamer, polyethylene glycol or polyhydroxymer.

[0063] The present disclosure also provides a method for preparing a lyophilized preparation comprising an anti-IL4Rα antibody or an antigen-binding fragment thereof, which comprises the step of freeze-drying the aforementioned pharmaceutical composition. In some embodiments, the freeze-drying is performed according to methods known in the art, including but not limited to the steps of pre-freezing, primary drying and secondary drying. It is understood by those skilled in the art that any method for removing water from the pharmaceutical composition of the present disclosure is applicable to the present disclosure.

[0064] The present disclosure also provides a lyophilized preparation comprising an anti-IL4Rα antibody or an antigen-binding fragment thereof, which is prepared by the aforementioned method for preparing a lyophilized preparation.

[0065] The present disclosure also provides a lyophilized preparation comprising an anti-IL4Rα antibody or an antigen-binding fragment thereof, wherein the lyophilized preparation can form the aforementioned pharmaceutical composition after being reconstituted.

[0066] The present disclosure also provides a product, which includes a container containing the aforementioned pharmaceutical composition or the aforementioned lyophilized preparation.

[0067] The pharmaceutical composition or lyophilized preparation of the present disclosure can be administered according to known methods, such as injection or infusion in a suitable manner over a period of time, for example, by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, topical administration, inhalation or by sustained release or delayed release. The pharmaceutical composition or lyophilized preparation of the present disclosure can be diluted to a suitable concentration with a suitable diluent before administration to provide the best desired response (e.g., therapeutic response).

[0068] The present disclosure provides a method for reducing IL4 and / or IL13 signaling in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition, lyophilized formulation or product of the present disclosure. IL4 signals through receptors comprising IL-4Rα and γc, while IL13 signals through receptors comprising IL-4Rα and IL13 Rα1. IL4 and / or IL13 signaling is manifested as activation and / or proliferation of B cells, eosinophils, macrophages, proliferation of fibroblasts, and proliferation of smooth muscle cells (e.g., proliferation of airway smooth muscle cells, etc.).

[0069] The present disclosure provides a method of reducing a type 2 immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition, lyophilized formulation or preparation of the present disclosure.

[0070] The present disclosure provides a method for treating a disease associated with excessive IL4 and / or IL13 signaling in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition, lyophilized formulation or product of the present disclosure. The disease associated with excessive IL4 and / or IL13 signaling is an allergic disease, non-limiting examples of which include atopic dermatitis, allergic reactions, allergic rhinitis or allergic asthma.

[0071] The present disclosure provides a method for treating a tumor associated with increased STAT6 activation in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition, a lyophilized formulation or a product of the present disclosure. In some embodiments, the tumor comprises a solid tumor or a non-solid tumor. In some embodiments, examples of the tumor include, but are not limited to, melanoma, lung cancer, kidney cancer, prostate cancer, cervical cancer, colorectal cancer, gastric cancer, pancreatic cancer, ovarian cancer and urothelial cancer.

[0072] Based on the following specific description and examples, other features and advantages of the present invention will become clear, and specific description and examples should not be interpreted as limiting. The contents of all documents, Genbank records, patents, and published patent applications cited in this application are expressly incorporated herein by reference. DETAILED DESCRIPTION OF THE INVENTION

[0074] In order to better understand the present disclosure, some terms are first defined. Other definitions are listed throughout the detailed description. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0075] "Buffer" refers to a pharmaceutically acceptable substance or mixture of substances capable of maintaining the pH of a pharmaceutical composition within a desired pH range. Examples of buffers suitable for use in the pharmaceutical compositions of the present disclosure include phosphate buffers, acetate buffers, or histidine buffers.

[0076] "Phosphate buffer" is a buffer containing phosphate ions, such as a buffer containing phosphoric acid and / or a phosphate salt, wherein phosphoric acid includes phosphoric acid and / or its hydrate, and phosphate includes phosphate and / or its hydrate. Examples of phosphate buffers include, but are not limited to, sodium phosphate buffers or potassium phosphate buffers, and a preferred phosphate buffer is a sodium phosphate buffer. In a specific example, the sodium phosphate buffer includes sodium dihydrogen phosphate monohydrate (NaH2PO4·H2O) and disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O).

[0077] "Acetate buffer" is a buffer including acetate ions, such as a buffer including acetic acid and / or acetate, wherein acetic acid includes acetic acid and / or its hydrate, and acetate includes acetate and / or its hydrate. Examples of acetate buffers include, but are not limited to, potassium acetate buffer, ammonium acetate buffer or sodium acetate buffer, sodium acetate-acetic acid buffer, and a preferred acetate buffer is sodium acetate-acetic acid buffer. Sodium acetate-acetic acid buffer contains acetic acid and sodium acetate, wherein acetic acid includes acetic acid and / or its hydrate, and sodium acetate includes sodium acetate and / or its hydrate. In a specific example, sodium acetate-acetic acid buffer includes acetic acid and sodium acetate trihydrate (CH3COONa·3H2O).

[0078] "Histidine salt buffer" is a buffer including histidine ions, such as a buffer containing histidine and / or histidine salts, wherein histidine includes histidine and / or its hydrates, and histidine salts include histidine salts and / or its hydrates. Examples of histidine salt buffers include histidine-hydrochloride buffer, histidine-acetate buffer, histidine-histidine hydrochloride or histidine hydrochloride buffer, and a preferred histidine salt buffer is histidine-histidine hydrochloride buffer. Histidine-histidine hydrochloride includes histidine and histidine hydrochloride, wherein histidine includes histidine and / or its hydrates, and histidine hydrochloride includes histidine hydrochloride and / or its hydrates. In a specific example, the histidine-histidine hydrochloride buffer includes histidine and histidine hydrochloride monohydrate (C6H9N3O2·HCl·H2O).

[0079] "Stabilizer" refers to a pharmaceutically acceptable substance or substance mixture used to maintain the stability of the active ingredient in the pharmaceutical composition. In the present disclosure, the stabilizer can also be used as a viscosity reducer and / or an isotonic agent, etc.

[0080] "Pharmaceutical composition" is intended to cover products comprising specific active ingredients (e.g., antibodies) in optionally specific amounts, as well as any product produced directly or indirectly by combining specific active ingredients in optionally specific amounts. The purpose of a pharmaceutical composition is to make the active ingredients suitable for production and administration to patients, and to maintain biological activity and / or stability during storage and subsequent use. In some embodiments, the pharmaceutical composition is a water-soluble injection, including but not limited to a water-soluble preparation that has not been lyophilized or a water-soluble preparation reconstituted with a lyophilized powder. In other embodiments, the pharmaceutical composition is a lyophilized preparation. In the present disclosure, "pharmaceutical composition" and "preparation" are not mutually exclusive.

[0081] A "stable" or "stabilized" pharmaceutical composition refers to a pharmaceutical composition in which the active ingredient (e.g., an antibody) substantially retains its physical stability and / or chemical stability and / or biological activity when stored therein. A variety of analytical techniques for measuring the stability of active ingredients are known in the art, for example, as reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature and other storage conditions for a selected time period. For example, if the active ingredient does not show a significant increase in aggregation, precipitation and / or denaturation after visual inspection of color and / or clarity, or as measured by UV light scattering, size exclusion chromatography (SEC), differential scanning calorimetry (DSC), or differential scanning fluorimetry (DSF), then the active ingredient "retains its physical stability" in the pharmaceutical composition. Preferably, when using the pharmaceutical composition of the present disclosure, 10% or less, 5% or less, 4% or less of the active ingredient forms aggregates (also referred to as high molecular weight impurities), such as by SEC or any other suitable method for measuring aggregate formation. An active ingredient (e.g., an antibody) "retains its chemical stability" in a pharmaceutical composition if it does not show significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the antibody. Processes that often alter the chemical structure of a protein include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide mapping in combination with mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing electrophoresis, peptide mapping, isoaspartate measurement), and isomerization (assessed by measuring isoaspartate content, peptide mapping, etc.). An active ingredient (e.g., an antibody) "retains its biological activity" over a given period of time in a pharmaceutical composition if the biological activity of the active ingredient over a given period of time is within a predetermined range of the biological activity exhibited when the pharmaceutical composition is prepared, such as determined by an antigen binding assay.

[0082] "High molecular weight impurities" or "aggregates" are a general term for impurities with a molecular weight greater than that of the target active ingredient (eg, antibody).

[0083] "Charge variants" refer to variants in which the charge of the antibody molecule is changed directly or indirectly due to glycosylation, deamidation, oxidation and / or isomerization. These charge variants can be detected by capillary isoelectric focusing (CIEF) and cation exchange chromatography (CEX-HPLC).

[0084] The articles "a", "an", and "the" are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. For example, "a pharmaceutical composition" refers to one pharmaceutical composition or more than one pharmaceutical composition.

[0085] "About" or "approximately" means that the value is within the acceptable error range of the specific value determined by a person of ordinary skill in the art, and the value depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, in the art, "about" or "approximately" can mean a standard deviation within or exceeding 1. Or "about" or "approximately" means a range of plus or minus 15%, 10%, 5%, or 1% of the value. In addition, particularly for biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. Unless otherwise specified, "about XX" or "approximately XX" or "substantially including "XX" means a value within the acceptable error range of the specific value "XX" (including the value "XX" itself, as well as a value within the acceptable error range of the value determined by a person of ordinary skill in the art).

[0086] As described herein, any percentage range, ratio range or integer range should be understood to include the value of any integer within the recited range and, where appropriate, fractions thereof (such as tenths and hundredths of integers), unless otherwise indicated.

[0087] In this article, unless the context otherwise dictates, the words "comprise", "include" and "contain" will be understood to mean including the described step or element or a group of steps or elements, but not excluding any other step or element or a group of steps or elements. "Consisting of" means including and limited to what follows the phrase "consisting of". Thus, the phrase "consisting of" means that the listed elements are required or necessary, and no other elements may be present. "Substantially consisting of" means including any element listed after this phrase, and is limited to other elements that do not interfere with or contribute to the activity or action of the listed elements as described in detail in the present invention. Thus, the phrase "substantially consisting of" means that the listed elements are required or necessary, but other elements are optional and may be present or absent depending on whether they affect the activity or action of the listed elements.

[0088] The term "IL4Rα" refers to the interleukin 4 receptor alpha subunit. The term "IL4Rα" includes variants, subtypes, homologs, orthologs, and paralogs. For example, in some cases, antibodies specific for human IL4Rα protein may cross-react with IL4Rα proteins of species other than humans (e.g., monkeys). In other embodiments, antibodies specific for human IL4Rα protein may be completely specific for human IL4Rα protein and may not cross-react with proteins of other species or other types, or may cross-react with IL4Rα derived from certain other species but not all other species.

[0089] The term "human IL4Rα" refers to an IL4Rα protein having a human amino acid sequence, such as the amino acid sequence of human IL4Rα with Genbank accession number NP_001244335.1. The terms "cynomolgus monkey IL4Rα" and "marmoset IL4Rα" refer to IL4Rα sequences, such as the amino acid sequences with Genbank accession numbers EHH60265.1 and NP_001244161.1, respectively.

[0090] The term "antibody" herein includes full-length antibodies and any antigen-binding fragments thereof or single chains. Full-length antibodies are glycoproteins comprising two heavy (H) chains and two light (L) chains, the heavy chains and light chains being linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as V H ) and a heavy chain constant region. The heavy chain constant region consists of three domains, namely, C H1 , C H2 and C H3 Each light chain consists of a light chain variable region (V L ) and the light chain constant region. The light chain constant region consists of a domain C L Composition. H and V L The V domains can also be divided into regions of hypervariability, termed complementarity determining regions (CDRs), separated by more conserved framework regions (FRs). H and V L It is composed of three CDRs and four FRs, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including a variety of immune system cells (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0091] As used herein, an "antigen-binding fragment" of an antibody (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., IL4Rα protein). It has been demonstrated that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Examples of binding fragments encompassed in the "antigen-binding fragment" of an antibody include: (i) a Fab fragment, which is composed of V L 、V H , C L and C H1 (ii) a F(ab')2 fragment, a bivalent fragment consisting of two Fab fragments linked by a disulfide bond at the hinge region; (iii) a F(ab')2 fragment consisting of a V H and C H1 (iv) an Fd fragment consisting of an antibody single-arm V L and V H Fv fragment composed of V H (vi) a separate complementarity determining region (CDR); and (vii) a nanobody, i.e., a heavy chain variable region comprising a single variable domain and two constant domains. In addition, although the two domains V of the Fv fragment are L and V H Encoded by different genes, they can be recombined to form a single protein chain through a synthetic linker. L and V H The domains are paired to form monovalent molecules (called single-chain Fv (scFv); see, for example, Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single-chain antibodies are also encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments can be obtained by common techniques known to those skilled in the art, and can be functionally screened in the same manner as full-length antibodies.

[0092] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to an IL4Rα protein is substantially free of antibodies that specifically bind to antigens other than the IL4Rα protein). However, an isolated antibody that specifically binds to a human IL4Rα protein may have cross-reactivity to other antigens, such as IL4Rα proteins of other species. In addition, the isolated antibody is substantially free of other cellular materials and / or chemicals.

[0093] As used herein, the term "mouse antibody" refers to antibodies in which the framework region and CDR region in the variable region are both from mouse germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also from a mouse germline immunoglobulin sequence. The mouse antibody herein may comprise amino acid residues not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced by random mutations or point mutations in vitro or by somatic mutations in vivo). However, the term "mouse antibody" used herein does not include antibodies in which CDR sequences from other mammalian species are inserted into mouse framework region sequences.

[0094] The term "chimeric antibody" refers to an antibody obtained by combining non-human genetic material with human genetic material. Or more generally, a chimeric antibody refers to an antibody that combines genetic material from one species with genetic material from another species.

[0095] As used herein, the term "humanized antibody" refers to an antibody derived from a non-human species but whose protein sequence has been modified to increase its similarity to a natural human antibody. In some specific examples, a "humanized antibody" contains a complementary determining region (CDR) derived from a non-human antibody and a framework region (FR) and a constant region derived from a human antibody.

[0096] The term "isotype" refers to the antibody class (eg, IgM or IgGl) encoded by the heavy chain constant region genes.

[0097] The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen / an antibody having specificity for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0098] As used herein, an antibody that "specifically binds to human IL4Rα" refers to an antibody that binds to human IL4Rα protein (and possibly IL4Rα protein of one or more non-human species) but does not substantially bind to non-IL4Rα proteins. Preferably, the antibody binds to human IL4Rα protein with "high affinity", i.e., with an affinity of 5.0× -8 M or less, preferably 1.0×10 -8 M or less, more preferably 7.0×10 -9 M or smaller K D Binds to human IL4Rα protein.

[0099] As used herein, the term "substantially not binding" a protein or cell means not binding to the protein or cell, or not binding to the protein or cell with high affinity, i.e., with an affinity of 1.0×10 -6 M or more, preferably 1.0×10 -5 M or more, more preferably 1.0×10 -4 M or more, more preferably 1.0×10 -3M or more, more preferably 1.0×10 -2 M or larger K D Binding proteins or cells.

[0100] The term "high affinity" for IgG antibodies means that the antibody has a binding affinity of 1.0 × 10 -6 M or less, preferably 5.0×10 -8 M or less, more preferably 1.0×10 -8 M or less, more preferably 7.0×10 -9 M or less, more preferably 1.0×10 -9 M or smaller K D . But for other antibody isotypes, “high affinity” binding may be different. For example, for the IgM isotype, “high affinity” means that the antibody has a 1.0×10 -6 M or less, preferably 1.0×10 -7 M or less, more preferably 1.0×10 -8 M or smaller K D .

[0101] The term "K assoc ” or “K a " refers to the association rate of a specific antibody-antigen interaction, and the term "K" as used herein dis ” or “K d " refers to the dissociation rate of a specific antibody-antigen interaction. The term "K" as used herein D ” refers to the dissociation constant, which is K d With K a The ratio (K d / K a ) and expressed as molar concentration (M). D The value can be determined using methods well known in the art. D A preferred method of quantifying the value is by using surface plasmon resonance, preferably using a biosensor system such as Biacore TM The system is measured.

[0102] The term “EC 50 ”, also known as the half-maximal effect concentration, is the antibody concentration that induces a response halfway between baseline and maximum after a specific exposure time. The term “IC 50 ", also known as the half-maximal inhibitory concentration, refers to the antibody concentration that inhibits a specific biological or biochemical function by 50% relative to the absence of the antibody.

[0103] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians and reptiles; preferably mammals, such as non-human primates, sheep, dogs, cats, cows and horses.

[0104] The term "therapeutically effective amount" refers to an amount of an antibody of the present invention sufficient to prevent or improve symptoms associated with a disease or condition (e.g., cancer), and / or to reduce the severity of a disease or condition. It should be understood that the therapeutically effective amount is related to the disease being treated, wherein those skilled in the art can easily discern the actual effective amount.

[0105] The term "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. The sequence comparison and identity percentage between two sequences can be determined by the default settings of the BLASTN / BLASTP algorithm on the website of the National Center for Biotechnology Information of the United States.

[0106] The term "Xn" is equivalent to "Xaa" and refers to an unspecified amino acid, and its scope is specified by the subsequent definition in the relevant expression.

[0107] Various aspects of the invention are described in more detail below.

[0108] Anti-IL4Rα antibody or antigen-binding fragment thereof

[0109] The anti-IL4Rα antibodies or antigen-binding fragments thereof in the present disclosure specifically bind to human IL4Rα with comparable (if not better) binding affinity / ability than reported anti-IL4Rα antibodies (such as Dupilumab).

[0110] The anti-IL4Rα antibodies or antigen-binding fragments thereof in the present disclosure can block the binding of IL4Rα to IL4 or IL13-IL13Rα1, thereby blocking the corresponding intracellular signal transduction, and have comparable or higher blocking activity than reported anti-IL4Rα antibodies (such as Dupilumab).

[0111] Preferably, the anti-IL4Rα antibody of the present invention is a humanized monoclonal antibody. In addition, or alternatively, the anti-IL4Rα antibody of the present invention may be, for example, a mouse, chimeric or human monoclonal antibody.

[0112] The anti-IL4Rα antibodies in the present disclosure are monoclonal antibodies with structural and chemical characteristics as described below and in the Examples. The amino acid sequence IDs of the heavy / light chain variable regions of the antibodies are summarized in Table 1 below. Some antibodies have the same V H or V LThe heavy chain constant region of the antibody may be a human IgG4 heavy chain constant region having an amino acid sequence as shown in SEQ ID NO:13, and the light chain constant region of the antibody may be a human κ constant region having an amino acid sequence as shown in SEQ ID NO:14.

[0113] Table 1. Amino acid sequence IDs of heavy / light chain variable regions

[0114]

[0115]

[0116] The heavy chain variable region CDRs and light chain variable region CDRs in Table 1 have been defined by the Kabat numbering system. However, as is well known in the art, the CDR region can also be determined based on the heavy / light chain variable region sequence by other numbering systems such as Chothia, IMGT, AbM or Contact numbering systems / methods. When referring to antibodies or antigen-binding fragments thereof defined by specific CDR sequences, the scope of the antibodies encompasses antibodies or antigen-binding fragments thereof defined by CDR sequences defined by any numbering system (e.g., Chothia, IMGT, AbM or Contact numbering system) in the art.

[0117] V of other anti-IL4Rα antibodies binding to human IL4Rα H and V L The sequence (or CDR sequence) may be the same as the V sequence of the anti-IL4Rα antibody of the present disclosure. H and V L Sequences (or CDR sequences) are "mixed and paired". Preferably, when V H and V L When chains (or CDRs within those chains) are mixed and paired, H / V L Centered V H The sequence is structurally similar to V H Likewise, it is preferred to replace the specific V H / V L Centered V L Sequence replaced with a structurally similar V L sequence.

[0118] Therefore, in one embodiment, the anti-IL4Rα antibody or antigen-binding fragment thereof of the present disclosure comprises:

[0119] (a) a heavy chain variable region comprising the amino acid sequence listed in Table 1; and

[0120] (b) a light chain variable region comprising the amino acid sequence listed in Table 1, or a V region of another anti-IL4Rα antibody L, wherein the antibody or antigen-binding fragment thereof specifically binds to human IL4Rα.

[0121] In another embodiment, the anti-IL4Rα antibody or antigen-binding fragment thereof of the present disclosure comprises:

[0122] (a) CDR1, CDR2 and CDR3 of the heavy chain variable region listed in Table 1; and

[0123] (b) CDR1, CDR2 and CDR3 of the light chain variable region listed in Table 1, or CDRs of another anti-IL4Rα antibody, wherein the antibody or antigen-binding fragment thereof specifically binds to human IL4Rα.

[0124] In another embodiment, the anti-IL4Rα antibody or antigen-binding fragment thereof of the present disclosure comprises CDR2 of the heavy chain variable region of the anti-IL4Rα antibody of the present disclosure and CDRs of other antibodies that bind to human IL4Rα, such as CDR1 and / or CDR3 of the heavy chain variable region, and / or CDR1, CDR2 and / or CDR3 of the light chain variable region from another anti-IL4Rα antibody.

[0125] Furthermore, it is well known in the art that the CDR3 domain alone can determine the binding specificity of an antibody to a cognate antigen, independent of the CDR1 and / or CDR2 domains, and that multiple antibodies with the same binding specificity can be predictively generated based on the common CDR3 sequence.

[0126] Therefore, in another embodiment, the anti-IL4Rα antibody or antigen-binding fragment thereof in the present disclosure comprises the CDR2 of the heavy chain variable region of the anti-IL4Rα antibody of the present disclosure and at least the CDR3 of the heavy chain variable region and / or the light chain variable region of the anti-IL4Rα antibody of the present disclosure, or the CDR3 of the heavy chain variable region and / or the light chain variable region of another anti-IL4Rα antibody, wherein the antibody specifically binds to human IL4Rα. These antibodies preferably compete with the anti-IL4Rα antibody of the present disclosure for binding to IL4Rα; (b) retain functional properties; (c) bind to the same epitope; and / or (d) have similar binding affinity. In another embodiment, the anti-IL4Rα antibody in the present disclosure may also comprise the CDR2 of the light chain variable region of the anti-IL4Rα antibody of the present disclosure, or the CDR2 of the light chain variable region of another anti-IL4Rα antibody, wherein the antibody specifically binds to human IL4Rα. In another embodiment, the anti-IL4Rα antibody of the present disclosure may further include CDR1 of the heavy chain variable region and / or light chain variable region of the anti-IL4Rα antibody of the present disclosure, or CDR1 of the heavy chain variable region and / or light chain variable region of another anti-IL4Rα antibody, wherein the antibody specifically binds to human IL4Rα.

[0127] In another embodiment, the anti-IL4Rα antibody or antigen-binding fragment thereof in the present disclosure comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region and the light chain variable region respectively comprise CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 sequences are different from the CDR1, CDR2 and CDR3 sequences of the anti-IL4Rα antibody in the present disclosure, and the difference is due to one or more conservative modifications. It should be understood in the art that certain conservative sequence modifications will not eliminate antigen binding. See, for example, Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hallet al., (1992) J. Immunol. 149: 1605-12; Kelley and O'Connell (1993) Biochem. 32: 6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10: 341-6 and Beers et al. al., (2000) Clin. Can. Res. 6:2835-43.

[0128] Therefore, in one embodiment, the anti-IL4Rα antibody or antigen-binding fragment thereof of the present disclosure comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise CDR1, CDR2 and CDR3, respectively, wherein:

[0129] (a) CDR1 of the heavy chain variable region comprises the sequence listed in Table 1, and / or conservative modifications thereof; and / or

[0130] (b) CDR2 of the heavy chain variable region comprises the sequence listed in Table 1, and / or conservative modifications thereof; and / or

[0131] (c) CDR3 of the heavy chain variable region comprises the sequence listed in Table 1, and / or conservative modifications thereof; and / or

[0132] (d) CDR1, and / or CDR2, and / or CDR3 of the light chain variable region comprise the sequences listed in Table 1, and / or conservative modifications thereof; and

[0133] (e) The antibody or antigen-binding fragment thereof specifically binds to human IL4Rα.

[0134] As used herein, the term "conservative sequence modification" refers to amino acid modifications that do not significantly affect or change the binding properties of an antibody. Such conservative modifications include amino acid replacements, additions and deletions. Modifications can be introduced into the antibodies of the present disclosure by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid replacement refers to replacing an amino acid residue with an amino acid residue with a similar side chain. Amino acid residue groups with similar side chains are known in the art. These amino acid residue groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR region of the anti-IL4Rα antibodies of the present disclosure can be replaced with other amino acid residues from the same side chain group, and the resulting antibodies can be tested for retention of function (i.e., the functions described above) using the functional assays described in the present disclosure.

[0135] The pharmaceutical compositions, lyophilized formulations or preparations disclosed herein have a variety of in vitro and in vivo uses, involving, for example, the treatment of diseases associated with excessive IL4 and / or IL13 signaling.

[0136] On the one hand, since anti-IL4Rα antibodies or antigen-binding fragments thereof can block the binding of IL4Rα to IL4 or IL13-IL13Rα1 to reduce type 2 immunity. The present disclosure provides a method for treating type 2 immunity-related allergic diseases in a subject, the method comprising administering a pharmaceutical composition, lyophilized preparation or product of the present disclosure to the subject. The allergic disease may be atopic dermatitis, allergic reaction, allergic rhinitis or allergic asthma.

[0137] On the other hand, since IL4 or IL13 signaling can activate STAT6, and it has been found that STAT6 inhibitors can inhibit cancer cell growth. The present disclosure provides a method for inhibiting tumor cell growth in a subject, the method comprising administering a pharmaceutical composition, lyophilized formulation or product of the present disclosure to the subject. Non-limiting examples of the tumor include, but are not limited to, melanoma, lung cancer, kidney cancer, prostate cancer, cervical cancer, colorectal cancer, gastric cancer, pancreatic cancer, ovarian cancer and urothelial cancer.

[0138] On the other hand, the present disclosure provides a method for reducing or inhibiting cell activation in response to IL-4 or IL-13 in a subject. In some embodiments, inhibiting activation includes inhibiting the production or secretion of cytokines. In some embodiments, inhibiting activation includes inhibiting proliferation. Cells that respond to IL-4 by activating the hybrid IL-4Rα / γc receptor include, but are not limited to, B cells, eosinophils, and macrophages. Cells that respond to IL-13 by activating the hybrid IL-4Rα / IL-13Rα1 receptor include, but are not limited to, fibroblasts and smooth muscle cells. Therefore, in one embodiment, the present disclosure provides a method for inhibiting smooth muscle cell proliferation. In another embodiment, the present disclosure provides a method for inhibiting fibroblast proliferation.

[0139] The present invention is further described by the following examples, which should not be construed as limiting.All references, Genebank sequences, patents and published patent applications cited in this application are expressly incorporated herein by reference. Example

[0140] The preparation and purification methods of the anti-IL4Rα antibodies disclosed in the present invention have been recorded in the patent document with application number PCT / CN2021077784, and the entire contents of the PCT / CN2021077784 application document are introduced into the present disclosure.

[0141] Example 1 Determination of affinity of anti-IL4Rα antibody using BIACORE surface plasmon resonance

[0142] The binding affinity and binding kinetics of the anti-IL4Rα antibodies (mAbs) of the present disclosure were characterized by Biacore T200 system (GE Healthcare, Pittsburgh, PA, USA).

[0143] Briefly, goat anti-human IgG (GE healthcare, catalog BR100839, HumanAntibody Capture Kit) was covalently linked to a CM5 chip (carboxymethylated dextran coated chip) via primary amine groups using a standard amine coupling kit provided by Biacore (GE Healthcare, Pittsburgh, PA, USA). Unreacted portions on the biosensor surface were blocked with ethanolamine. Then, the anti-IL4Rα antibody of the present disclosure at a concentration of 66.67 nM and the anti-IL4Rα antibody reference ( Also called BM) flowed through the chip at a flow rate of 10 μL / min. Then, recombinant human IL4Rα-his protein (prepared in-house, the amino acid sequence is shown in SEQ ID NO: 15), cynomolgus monkey IL4Rα-his protein (Sino biological inc., product catalog 90897-C08H), or marmoset IL4Rα-his protein (Sino biological inc. custom product, also called cal-IL4Rα-his, the amino acid sequence is shown in SEQ ID NO: 16) diluted in HBS EP buffer (provided by Biacore) was flowed through the chip at a flow rate of 30 μL / min. The antigen-antibody binding kinetics were tracked for 2 minutes, and the dissociation kinetics were tracked for 10 minutes. The binding and dissociation curves were fitted to the 1:1 Langmuir binding model using BIA evaluation software to determine K D , K a and K d value.

[0144] Example 2 Binding activity of anti-IL4Rα antibody to IL4Rα

[0145] The binding activity of the anti-IL4Rα antibodies of the present disclosure to IL4Rα was determined by capture ELISA, flow cytometry (FACS) and indirect ELISA.

[0146] 2.1 Capture ELISA

[0147] Briefly, affinity purified goat anti-human IgG (Fcγ fragment specific, Jackson Immunoresearch, catalog 109-005-098) at a concentration of 2 μg / mL in PBS was coated in a 96-well plate at 100 μL / well and incubated overnight at 4° C. The plate was washed once with washing buffer (PBS+0.05% w / v Tween-20, PBST), and then 200 μL / well of blocking buffer (PBST containing 5% w / v skim milk) was added and blocked at 37° C. for 2 hours. The plate was washed again, and 100 μL of a gradient dilution (starting at 66.7 nM, 5-fold gradient dilution in PBST containing 2.5% w / v skim milk) of the anti-IL4Rα antibody disclosed herein, a reference substance, or a negative control substance hIgG (human immunoglobulin for intravenous injection (pH 4), Hualan Biological Engineering Inc.) was added to each well, incubated at 37°C for 40 minutes, and then the plate was washed 4 times. 100 μL / well of biotin-labeled human IL4Rα-his protein (prepared in-house, amino acid sequence as shown in SEQ ID NO: 15, dissolved in PBST containing 2.5% w / v skim milk, concentration of 0.14 nM) was added to the 96-well plate captured with anti-IL4Rα antibody, incubated at 37°C for 40 minutes, washed 4 times, and then 100 μL / well of HRP-labeled streptavidin (diluted 1:10000 in PBST, Jackson Immuno Research, product catalog 016-030-084) was added and incubated at 37°C for 40 minutes. After the last wash, 100 μL / well of ELISA substrate TMB (Innoreagents, product catalog TMB-S-002) was added for incubation. After 10 minutes, 50 μL / well of 1M H2SO4 was added at 25°C to terminate the reaction, and the absorbance value was read at 450 nm. The data were analyzed using GraphpadPrism software, and the EC 50 value.

[0148] 2.2 Cell-based binding FACS

[0149] The binding activity of the anti-IL4Rα antibodies of the present disclosure to IL4Rα expressed on the surface of 293F-IL4Rα cells was detected by flow cytometry (FACS). Briefly, 293F cells (Thermofisher Inc., catalog 11625019) were transfected with a pCMV-TP plasmid construct having nucleotides encoding human IL4Rα (amino acid residues 1-825 of uniprot#P24394-1) between EcoRI and XbaI, and a stable cell pool (named 293F-IL4Rα) was selected for subsequent cell-based binding FACS and cell-based ligand blocking FACS analysis. 293F-IL4Rα cells were harvested from cell culture flasks, washed twice, and resuspended in FACS buffer (phosphate buffered saline (PBS) containing 2% v / v fetal bovine serum). Then, in a flask containing 2×10 5 Add 100 μL / well of anti-IL4Rα antibody or control diluted in FACS buffer (starting at 80 nM, 4-fold serial dilution) to a 96-well plate with 10 cells / well and place on ice for 40 minutes. After washing the cells twice with FACS buffer, add 100 μL / well of R-phycoerythrin-labeled affinity-purified goat anti-human IgG (Fcγ fragment specific, Jackson Immunoresearch, product catalog 109-115-098, diluted 1:1000 in FACS buffer). After incubation at 4°C in the dark for 40 minutes, wash the cells 3 times and resuspend them in FACS buffer. Fluorescence values ​​were measured using BectonDickinson FACS Canto II-HTS. Data were analyzed using Graphpad Prism software, and EC values ​​were obtained. 50 value.

[0150] 2.3 Indirect ELISA

[0151] The cross-reaction of the anti-IL4Rα antibody in the present disclosure with the cynomolgus IL4Rα protein or cal-IL4Rα-his protein was detected. Briefly, 2 μg / mL of cynomolgus IL4Rα-his protein (Sino biological inc., product catalog 90897-C08H) dissolved in carbonate / bicarbonate buffer (pH 9.6) or 0.2 μg / mL of cal-IL4Rα-his protein (Sino biological inc. custom product, product catalog BAX2) dissolved in carbonate / bicarbonate buffer (pH 9.6) were coated in 96-well plates at 100 μL / well and incubated at 37°C for 2 hours. The plate was washed once with washing buffer (PBS+0.05% w / v Tween-20, PBST), and then 200 μL / well of blocking buffer (PBST containing 5% w / v skim milk) was added and blocked at 37°C for 2 hours. Wash the plate again, add 100 μL of the anti-IL4Rα antibody or control disclosed in the present disclosure (0.004-66.7 nM, starting at 66.7 nM, 5-fold gradient dilution in PBST containing 2.5% w / v skim milk) to each well, and incubate at 37°C for 40 minutes. Wash the plate 4 times, then add 100 μL / well of peroxidase-labeled affinity-purified F(ab')2 fragmented goat anti-human IgG (Fcγ fragment specific, Jackson Immunoresearch, product catalog 109-036-098, diluted 1:5000 in PBST buffer), and incubate at 37°C for 40 minutes. After the last wash, add 100 μL / well of TMB (Innoreagents) for incubation. After 3-10 minutes, add 50 μL / well of 1M H2SO4 at 25°C to terminate the reaction, and read the absorbance value at 450nm. The data were analyzed using Graphpad Prism software, and the EC 50 value.

[0152] Example 3 Blocking activity of anti-IL4Rα antibodies on IL4Rα-reference or IL4Rα-IL4 interactions

[0153] 3.1 Ligand blocking ELISA

[0154] The ability of the anti-IL4Rα antibodies of the present disclosure to block IL4-IL4Rα interactions was determined by competitive ELISA. Briefly, human IL4Rα-his protein (prepared in-house, amino acid sequence as shown in SEQ ID NO: 15) dissolved in PBS at a concentration of 2 μg / mL was coated in a 96-well plate at 100 μL / well and incubated overnight at 4°C. The next day, the plate was washed with wash buffer (PBS + 0.05% w / v Tween-20, PBST), and PBST containing 5% w / v skim milk was added and blocked at 37°C for 2 hours. Then, the plate was washed again with wash buffer.

[0155] The anti-IL4Rα antibody or control substance in the present disclosure was diluted with PBST buffer containing 2.5% w / v skim milk (with a starting concentration of 80nM and a 4-fold gradient dilution), and the gradient diluted anti-IL4Rα antibody or control substance was added to the plate coated with IL4Rα at 100μL / well, and incubated with human IL4Rα-his protein at 37°C for 40 minutes. The plate was washed 4 times with washing buffer, and then 100μL of biotin-labeled human IL4 protein (Sino biologicalinc., product catalog 11846-HNAE) at a concentration of 0.56nM was added to each well and incubated at 37°C for 40 minutes. The plate was washed again with washing buffer, and then 100μL / well of HRP-labeled streptavidin (diluted at a ratio of 1:10000 in PBST buffer, JacksonImmunoresearch, product catalog 016-030-084) was added and incubated at 37°C for 40 minutes. The plate was washed again with wash buffer. Finally, TMB was added and 1M H2SO4 was used to terminate the reaction, and the absorbance was read at 450 nm. The data were analyzed using GraphpadPrism software and the IC 50 value.

[0156] 3.2 Reference Blocking ELISA

[0157] The ability of the anti-IL4Rα antibodies of the present disclosure to block the binding of the reference substance to human IL4Rα was determined by competitive ELISA. Briefly, the reference substance dissolved in PBS at a concentration of 2 μg / mL was coated in a 96-well plate at 100 μL / well and incubated overnight at 4°C. The next day, the plate was washed with washing buffer (PBS+0.05% w / v Tween-20, PBST), and PBST containing 5% w / v skim milk was added and blocked at 37°C for 2 hours. During the blocking of the 96-well plate, the anti-IL4Rα antibodies of the present disclosure or the reference substance (starting at 100 nM, 4-fold serial dilution) were diluted with biotin-labeled human IL4Rα-his protein (prepared in-house, the amino acid sequence is shown in SEQ ID NO: 15, dissolved in PBST containing 2.5% w / v skim milk, the concentration is 0.55 nM), and incubated at 25°C for 40 minutes. After washing the plate, add 100 μL / well of the antibody / IL4Rα-his mixture to the plate coated with the reference. Incubate at 37°C for 40 minutes and wash the plate with wash buffer. Then add 100 μL / well of HRP-labeled streptavidin to the plate and incubate at 37°C for 40 minutes to detect biotin-labeled human IL4Rα-his bound to the plate. Wash the plate again with wash buffer. Finally, add TMB and stop the reaction with 1M H2SO4, and read the absorbance at 450nm. Analyze the data using GraphpadPrism software and obtain IC 50 value.

[0158] 3.3 Cell-based ligand blocking FACS

[0159] Using the 293F-IL4Rα cells prepared as described above, flow cytometry (FACS) was used to evaluate the activity of anti-IL4Rα antibodies in blocking the binding of IL4 protein to cell surface IL4Rα.

[0160] Briefly, 293F-IL4Rα cells were harvested from cell culture flasks, washed twice, and resuspended in FACS buffer (PBS containing 2% v / v fetal bovine serum). 5100 μL / well of anti-IL4Rα antibody or control diluted in FACS buffer (starting at 80 nM, 4-fold dilution) was added to a 96-well plate with 10 cells / well and placed on ice for 40 minutes. After washing the plate twice with FACS buffer, 100 μL of 1.67 nM biotin-labeled human IL4 protein (Sino biological inc., product catalog 11846-HNAE) was added to each well and incubated at 4°C in the dark for 40 minutes. After washing the plate twice with FACS buffer, 100 μL of R-phycoerythrin-labeled streptavidin (diluted 1:500 with FACS buffer, Jackson Immunoresearch, product catalog 016-110-084) was added to each well and incubated at 4°C in the dark for 40 minutes. After washing the cells twice, resuspend them in FACS buffer. Fluorescence values ​​were measured using Becton Dickinson FACS Canto II-HTS. Data were analyzed using Graphpad Prism software, and IC 50 value.

[0161] Example 4 Cell-based functional assay of anti-IL4Rα antibody

[0162] IL4 and IL13 can bind to IL4Rα and induce STAT6 phosphorylation in HEK293T-IL4Rα-STAT6-STAT6LUC-LB2 cells. STAT6 phosphorylation is crucial in the IL4 / IL13 signaling pathway.

[0163] Briefly, HEK293T-IL4Rα-STAT6-STAT6LUC-LB2 cells were prepared in-house by stably transfecting HEK293T cells (ATCC CRL-11268) naturally expressing IL13Rα1 with pcDNA3.1-Puro (YouBio biological inc., catalog VT9222) plasmid construct (with nucleotides encoding human IL4Rα between BamHI and XhoI), STAT6 plasmid (Sino biologicalinc., catalog HG13190-NH) (with nucleotides encoding human STAT6 between KpnI and XbaI), and STAT6 luciferase reporter plasmid STAT6-Luc (Yeasen biological inc., catalog 11588ES03). Single cell clone LB2 was then selected for all subsequent functional assays.

[0164] The inhibitory effect of the anti-IL4Rα antibody of the present disclosure on IL4- and IL13-induced STAT6 phosphorylation was tested.

[0165] Briefly, HEK293T-IL4Rα-STAT6-STAT6LUC-LB2 cells in the logarithmic phase were resuspended in culture medium (RPMI1640 + 10% FBS) and seeded into 96-well plates at 100 μL / well, with 5 × 10 5 cells. Then, 50 μL of the anti-IL4Rα antibody or control substance (including the anti-CD22 antibody prepared in-house) in the present disclosure was added to each well (with a starting concentration of 100 nM, 5-fold gradient dilution) and incubated at 37° C. for 30 minutes. Then 50 μL of IL4 protein (600 pg / mL, Sino biological inc., product catalog 11846-HNAE) or IL13 protein (50 ng / mL, Sinobiological inc., product catalog 10369-HNAC) was added to each well and incubated at 37° C. for 20 minutes. The plate was centrifuged and washed twice with staining buffer (prepared in-house, DPBS+0.5% w / v BSA+2mM EDTA), and then 50 μL of fixation buffer (BDbiosciences inc., product catalog 5545655) was added to each well and incubated at 4° C. for 30 minutes. The cells were washed twice, 200 μL of permeabilization buffer (BD biosciences inc., catalog number 558050) was added to each well, and ice bathed for 30 minutes. The plate was washed 3 times with staining buffer. Then anti-pSTAT6 antibody (pSTAT6 stock solution diluted 20 times, BD biosciences inc., product catalog number 562079) was added and allowed to stand on ice for 60 minutes. Finally, the plate was washed twice and resuspended in staining buffer. Fluorescence values ​​were measured using BectonDickinson FACS Canto II-HTS. Data were analyzed using Graphpad Prism software and IC 50 value.

[0166] Example 5 Production and characterization of chimeric antibodies

[0167] The heavy chain variable region and light chain variable region of mouse anti-IL4Rα monoclonal antibody were sequenced, and the sequence IDs are summarized in Table 1.

[0168] The heavy chain variable region and light chain variable region of the mouse anti-IL4Rα monoclonal antibody C2C1A1A1 were cloned into a vector containing a human IgG4 heavy chain constant region (SEQ ID NO: 13) and a vector containing a human κ light chain constant region (SEQ ID NO: 14), respectively, wherein the C-terminus of the variable region was connected to the N-terminus of the corresponding constant region.

[0169] A vector comprising nucleotides encoding a heavy chain variable region linked to a human IgG4 heavy chain constant region, and a vector comprising nucleotides encoding a light chain variable region linked to a human kappa light chain constant region, were transiently transfected into 50 mL of 293F suspension cells with 1 mg / mL PEI at a ratio of 60%:40% light chain construct:heavy chain construct.

[0170] After culturing for six days in a shake flask, the cell supernatant was harvested, the cells in the supernatant were centrifuged, and filtered through a 0.22 μm filter to separate immunoglobulins. The chimeric antibody was purified by protein A affinity chromatography. In short, the protein A agarose column (bestchrom (Shanghai) Biosciences, product catalog AA0273) was washed with 5 to 10 column volumes of PBS buffer. The cell supernatant was passed through the protein A agarose column, and then the column was washed with PBS buffer until the absorbance of the protein reached baseline. The column was eluted with elution buffer (0.1 M glycine-HCl, pH 2.7), and the eluate was immediately collected in a 1.5 mL tube and neutralized with neutralization buffer (1 M Tris-HCl, pH 9.0). The immunoglobulin-containing parts were merged and dialyzed overnight at 4 ° C in PBS.

[0171] According to the protocol in the previous examples, the purified antibodies were detected by capture ELISA, competition ELISA, BIAcore affinity assay, cell-based binding FACS assay and cell-based functional assay; for mouse C2C1A1A1, the detection method was slightly modified based on the previous examples as described below:

[0172] For capture ELISA, 2 μg / mL goat anti-mouse IgG (Fcγ fragment specific, Jackson Immunoresearch, catalog 115-005-008) was used instead of goat anti-human IgG, 100 μL / well;

[0173] For BIAcore, goat anti-mouse IgG (GE healthcare, catalog BR100838, MouseAntibody Capture Kit) was used instead of goat anti-human IgG and covalently linked to the CM5 chip;

[0174] For cell-based binding FACS, R-phycoerythrin-labeled affinity purified goat anti-human IgG was replaced with R-phycoerythrin-labeled affinity purified F(ab')2 fragmented goat anti-mouse IgG (Jackson Immunoresearch, catalog 115-116-146) at 100 μL / well.

[0175] The results are shown in Table 2, and the data indicate that the chimeric antibody has similar binding affinity / potency and blocking activity as its parental mouse antibody.

[0176] Table 2. Binding and functional activities of chimeric antibodies

[0177]

[0178] Example 6 Humanization of anti-IL4Rα monoclonal antibody

[0179] The mouse anti-IL4Rα antibody C2C1A1A1 was humanized and further characterized. Humanization of the mouse antibody was performed using an established CDR grafting method as described below.

[0180] In order to select the acceptor framework for the humanization of mouse antibody C2C1A1A1, the sequences of the light chain variable region and the heavy chain variable region of each mouse antibody were BLASTed with the human immunoglobulin gene database. The human germline antibody with the highest homology was selected as the acceptor framework for humanization. The mouse antibody heavy / light chain variable region CDRs were inserted into the selected framework, and the residues in the framework were further back-mutated to obtain more candidate heavy / light chain variable regions. A total of 16 exemplary humanized C2C1A1A1 antibodies, i.e., huC2C1A1A1-V1 to huC2C1A1A1-V16, were obtained, and their heavy / light chain variable region sequence IDs are shown in Table 1.

[0181] A vector comprising nucleotides encoding a humanized heavy chain variable region linked to a human IgG4 heavy chain constant region (SEQ ID NO: 13), and a vector comprising nucleotides encoding a humanized light chain variable region linked to a human kappa light chain constant region (SEQ ID NO: 14) were transiently transfected into 50 mL of 293F suspension cells with 1 mg / mL PEI at a ratio of 60%:40% light chain construct:heavy chain construct.

[0182] After culturing for six days in a shake flask, the cell supernatant was harvested, the cells in the supernatant were centrifuged, and filtered through a 0.22 μm filter to separate immunoglobulins. The antibody was purified by protein A affinity chromatography. In short, the protein A agarose column (bestchrom (Shanghai) Biosciences, product catalog AA0273) was washed with 5 to 10 column volumes of PBS buffer. The cell supernatant was flowed through the protein A agarose column, and then the column was washed with PBS buffer until the absorbance of the protein reached baseline. The column was eluted with elution buffer (0.1 M glycine-HCl, pH 2.7), and the eluate was immediately collected in a 1.5 mL tube and neutralized with neutralization buffer (1 M Tris-HCl, pH 9.0). The immunoglobulin-containing parts were merged and dialyzed overnight at 4 ° C in PBS.

[0183] Example 7 Characterization of humanized antibodies

[0184] According to the protocol in the previous example, the binding affinity of the humanized antibody to human IL4Rα was evaluated by BIAcore technology to determine K a , K d and K D The values ​​are summarized in Table 3.

[0185] Table 3. Binding affinity of humanized C2C1A1A1 monoclonal antibodies

[0186]

[0187] Dissociation % = (End (RU) - Start (RU)) / Start (RU)

[0188] The results showed that the humanized antibodies had similar human IL4Rα binding affinities as the chimeric antibodies, and all humanized huC2C1A1A1 antibodies showed higher human IL4Rα binding affinities compared with the reference.

[0189] Humanized antibodies huC2C1A1A1-V14 and huC2C1A1A1-V15 were further tested by Biacore, capture ELISA, indirect ELISA, cell-based binding FACS, competition ELISA, cell-based ligand blocking FACS and cell-based functional assays according to the protocols in the previous examples.

[0190] The thermal stability of the humanized antibody huC2C1A1A1-V15 was also tested. TMThermal Shift Protein Stability Kit (Biotium, catalog 33022-T, lot number 181214) was used to determine the Tm (melting temperature) of proteins by thermal shift assay. TM The dye was thawed to room temperature. The vial containing the dye was vortexed and centrifuged. Then, 5 μL of 200× dye was added to 95 μL PBS to prepare 10× dye. 2 μL of 10× dye and 10 μg of humanized antibody were added to the reaction system, and PBS was added to a total reaction volume of 20 μL. The centrifuge tube containing the dye and antibody was briefly centrifuged and placed in a real-time PCR thermal cycler (Roche, LightCycler480II), in which the Melt Curve program was set using the parameters in Table 4. The results are shown in Tables 5-1, 5-2 and 5-3, and the data show that the humanized C2C1A1A1 antibody exhibits comparable (if not better) human IL4Rα binding affinity / activity and blocking ability against IL4Rα-IL4 / IL13 compared to the reference.

[0191] Table 4. Parameters of the Melt Curve program

[0192]

[0193]

[0194] Table 5-1. Binding activity and functional activity of humanized monoclonal antibodies

[0195]

[0196] Table 5-2. Binding activity and functional activity of humanized monoclonal antibodies

[0197]

[0198] Table 5-3. Binding activity and functional activity of humanized monoclonal antibodies

[0199]

[0200] “*” indicates not detected.

[0201] Example 8 Preparation and Screening of Pharmaceutical Compositions

[0202] Detection method:

[0203] (1) Size Exclusion Chromatography (SEC-UPLC): used to determine the purity of antibodies in samples, using ThermoVanquish F high performance liquid chromatography, Waters ACQUITY UPLC Protein BEH SEC Column ( 1.7μm, 4.6mm×300mm) chromatographic column, Waters ACQUITY UPLC Protein BEH SEC Guard Column ( 1.7μm, 4.6mm×30mm) as a pre-column, 50mmol / L phosphate buffer + 200mmol / L sodium chloride (pH 7.0) as the mobile phase for elution, and the detection wavelength is 280nm. The percentage of high molecular impurities and immunoglobulin monomers is calculated by area normalization method.

[0204] (2) Differential scanning fluorescence (DSF): A protein stability analyzer (NanoTemper, Prometheus NT.48) was used to analyze the unfolding temperature (Tm) of the sample. 10 μl of the sample was taken into the sample chamber using a capillary tube. The procedure was as follows: the scan start temperature was 25°C, the scan end temperature was 95°C, and the heating rate was 0.3°C / min.

[0205] (3) Use HVROC-S viscometer (Rheosense, μVISC) to analyze the viscosity of the sample, select the Auto mode, and record the test results.

[0206] (4) Whole-column imaging capillary isoelectric focusing (iCIEF): used to determine the charge variants of the sample, using an imaging capillary isoelectric focusing electrophoresis instrument (ProteinSimple iCE3), a fast fully automatic protein characterization analysis system (ProteinSimple, Maurice), coated quartz capillaries (Protein Simple), and a UV detector. The detection was performed at a UV detection wavelength of 280 nm. The injection time was set to 60 seconds, pre-focusing at 1500 volts for 1 minute, and focusing at 300 volts for 10 minutes. The peak area percentage of each charge variant was calculated by the peak area normalization method.

[0207] Preparation of buffer:

[0208] (1) Histidine-histidine hydrochloride buffer comprises histidine and histidine hydrochloride. In a specific embodiment, the histidine-histidine hydrochloride buffer comprises histidine and histidine hydrochloride monohydrate. For example, 20 mM histidine-histidine hydrochloride buffer (pH 5.8) is composed of about 1.17 mg / mL histidine and about 2.62 mg / mL histidine hydrochloride monohydrate.

[0209] (2) The sodium phosphate buffer comprises disodium hydrogen phosphate and sodium dihydrogen phosphate. In a specific embodiment, the sodium phosphate buffer comprises sodium dihydrogen phosphate monohydrate and sodium dihydrogen phosphate dodecahydrate. For example, 20 mM sodium phosphate buffer (pH 6.0) is prepared from about 2.539 mg / mL sodium dihydrogen phosphate monohydrate and about 0.573 mg / mL sodium dihydrogen phosphate dodecahydrate.

[0210] (3) Sodium acetate-acetic acid buffer comprises sodium acetate and acetic acid. In a specific embodiment, the sodium acetate-acetic acid buffer comprises sodium acetate trihydrate and acetic acid. For example, 20 mM sodium acetate-acetic acid buffer (pH 6.0) is prepared by about 2.605 mg / mL sodium acetate trihydrate and about 0.141 mg / mL acetic acid.

[0211] By ultrafiltration, the anti-IL4Rα antibody (huC2C1A1A1-V15) was replaced into the buffer of Table 6, concentrated after the replacement, and then the surfactant and stabilizer were added according to Table 6. After uniform mixing, the mixture was sterilized by filtration through a 0.22 μm filter membrane, and dispensed into vials, stoppered and capped. The SEC-UPLC test results of the pharmaceutical compositions F1-F4 are shown in Table 7, where the growth of high molecular impurities in F4 after storage at 40°C for 1 month (from 1.36% to 5.66%) was higher than that of F1-F3.

[0212] Table 6. Ingredients of the pharmaceutical composition

[0213]

[0214] Table 7. SEC-UPLC test results of pharmaceutical compositions

[0215]

[0216]

[0217] Exemplarily, 20 mM histidine-histidine hydrochloride is selected as a buffer. The anti-IL4Rα antibody (huC2C1A1A1-V15) is replaced into the buffer of Table 8 by ultrafiltration, concentrated after the replacement, and then the surfactant and stabilizer are added according to Table 8. After mixing evenly, sterilize by filtration through a 0.22 μm filter membrane, and dispense into vials, stopper and cap. The Tm and SEC-UPLC test results of the pharmaceutical compositions F5-F7 are shown in Table 9, among which F6 has the least growth of high molecular impurities after storage at 40°C for 1 month.

[0218] Table 8. Ingredients of the pharmaceutical composition

[0219]

[0220] “ / ” means none.

[0221] Table 9. Tm and SEC-UPLC test results of the pharmaceutical composition

[0222]

[0223] “*” indicates not detected.

[0224] Exemplarily, 20mM histidine-histidine hydrochloride is selected as a buffer. The anti-IL4Rα antibody (huC2C1A1A1-V15) is replaced into the buffer of Table 10 by ultrafiltration, concentrated after the replacement, and then the surfactant and stabilizer are added according to Table 10. After mixing evenly, sterilize by filtration through a 0.22μm filter membrane, and dispense into vials, stopper and cap. The Tm, viscosity, SEC-UPLC and iCIEF test results of pharmaceutical compositions F8 and F9 are shown in Table 11, among which the viscosity of F8 is lower than that of F9.

[0225] Table 10. Ingredients of the pharmaceutical composition

[0226]

[0227] “ / ” means none.

[0228] Table 11. Tm, viscosity, SEC-UPLC and iCIEF test results of the pharmaceutical composition

[0229]

[0230] “*” indicates not detected.

[0231] Although the present disclosure has been described in conjunction with one or more embodiments, it should be understood that the present disclosure not only covers these embodiments, but is also intended to cover all alternatives, modifications and equivalents included in the spirit and scope of the appended claims. All documents cited herein are fully incorporated herein by reference.

[0232] The sequence information of the present disclosure is summarized in Table 12 below.

[0233] Table 12. Sequence information

[0234]

[0235]

[0236]

Claims

1. A pharmaceutical composition comprising: (a) an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) a buffer, (c) a surfactant, and (d) a stabilizer.

2. The pharmaceutical composition according to claim 1, wherein the anti-IL4Rα antibody or antigen-binding fragment thereof comprises a heavy chain variable region CDR1, a heavy chain variable region CDR2, a heavy chain variable region CDR3, a light chain variable region CDR1, a light chain variable region CDR2 and a light chain variable region CDR3, and the heavy chain variable region CDR1, the heavy chain variable region CDR2, the heavy chain variable region CDR3, the light chain variable region CDR1, the light chain variable region CDR2 and the light chain variable region CDR3 respectively comprise an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NOs: 1, 2, 3, 4, 5 and 6.

3. The pharmaceutical composition according to claim 1 or 2, wherein the anti-IL4Rα antibody or antigen-binding fragment thereof comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NOs: 7, 8 or 9, wherein The amino acid sequence of SEQ ID NO:8 is: EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYGMSWVRQAPGKGLVX1VX2TINSNGGSTSYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARFFRFRNAMDYWGQGTLVTVSS, wherein X1=W and X2=S, X1=L and X2=A, or X1=W and X2=A in SEQ ID NO:8; and / or The anti-IL4Rα antibody or antigen-binding fragment thereof comprises a light chain variable region, wherein the light chain variable region comprises an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NOs: 10, 11 or 12, wherein the amino acid sequence of SEQ ID NO: 11 is: DIQMTQSPSSLSASVGDRVTITCRTSENIYSYLAWYQQKPGKAPKX1LX2YNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYYGPPTWTFGQGTKVEIK, wherein X1=L and X2=I, X1=F and X2=V, or X1=F and X2=I in SEQ ID NO:

11.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the anti-IL4Rα antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise: (1) an amino acid sequence having at least 85% identity to the amino acid sequence shown in SEQ ID NOs: 7 and 10, respectively; (2) an amino acid sequence having at least 85% identity to the amino acid sequence shown in SEQ ID NOs: 8 and 11, respectively, wherein X1=W and X2=S in SEQ ID NO: 8; (3) an amino acid sequence having at least 85% identity to the amino acid sequence shown in SEQ ID NOs: 8 and 12, respectively, wherein X1=W and X2=S in SEQ ID NO: 8; (4) an amino acid sequence having at least 85% identity to the amino acid sequence shown in SEQ ID NOs: 9 and 11, respectively; (5) an amino acid sequence having at least 85% identity to the amino acid sequence shown in SEQ ID NOs: 9 and 12, respectively; (6) an amino acid sequence having at least 85% identity to the amino acid sequence shown in SEQ ID NOs: 8 and 11, respectively, wherein SEQ ID NO:8, wherein X1=L and X2=A; (7) an amino acid sequence having at least 85% identity to the amino acid sequences set forth in SEQ ID NOs:8 and 12, respectively, wherein X1=L and X2=A in SEQ ID NO:8; (8) an amino acid sequence having at least 85% identity to the amino acid sequences set forth in SEQ ID NOs:8 and 11, respectively, wherein X1=W and X2=A in SEQ ID NO:8; or (9) an amino acid sequence having at least 85% identity to the amino acid sequences set forth in SEQ ID NOs:8 and 12, respectively, wherein X1=W and X2=A in SEQ ID NO:8; The amino acid sequence of SEQ ID NO:8 is: EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYGMSWVRQAPGKGLVX1VX2TINSNGGSTSYPDSVKGRF TISRDNAKNTLYLQMNSLRAEDTAVYYCARFFRFRNAMDYWGQGTLVTVSS; The amino acid sequence of SEQ ID NO: 11 is: DIQMTQSPSSLSASVGDRVTITCRTSENIYSYLAWYQQKPGKAPKX1LX2YNAKTLAEGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQHYYGPPTWTFGQGTKVEIK, wherein, In SEQ ID NO:11, X1=L and X2=I, X1=F and X2=V, or X1=F and X2=I.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the anti-IL4Rα antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 13, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

14.

6. The pharmaceutical composition according to any one of claims 1-5, wherein the concentration of the anti-IL4Rα antibody or its antigen-binding fragment is 30 mg / mL to 300 mg / mL, 50 mg / mL to 250 mg / mL, 70 mg / mL to 200 mg / mL, 100 mg / mL to 180 mg / mL, 120 mg / mL to 180 mg / mL, 120 mg / mL to 150 mg / mL, or 150 mg / mL to 180 mg / mL.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the buffer comprises a phosphate buffer, an acetate buffer or a histidine salt buffer; preferably, the phosphate buffer is a sodium phosphate buffer, the acetate buffer is a sodium acetate-acetate buffer, and / or the histidine salt buffer is a histidine-histidine hydrochloride buffer; Optionally, the concentration of the buffer is 1 mM to 100 mM, 2 mM to 80 mM, 4 mM to 60 mM, 8 mM to 40 mM, 10 mM to 30 mM, 10 mM to 20 mM, or 20 mM to 30 mM.

8. The pharmaceutical composition of any one of claims 1-7, wherein the surfactant comprises polysorbate 80 or polysorbate 20; optionally, wherein the concentration of the surfactant is 0.01 mg / mL to 2 mg / mL, 0.05 mg / mL to 1 mg / mL, 0.1 mg / mL to 0.8 mg / mL, 0.2 mg / mL to 0.6 mg / mL, or 0.2 mg / mL to 0.4 mg / mL.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the stabilizer is selected from one or more of trehalose, mannitol, sucrose, arginine or a pharmaceutically acceptable salt thereof, proline or sodium chloride; Optionally, the concentration of sucrose is 10 mg / mL to 100 mg / mL, 20 mg / mL to 80 mg / mL, 30 mg / mL to 60 mg / mL, 40 mg / mL to 50 mg / mL, or 50 mg / mL to 60 mg / mL; the concentration of arginine or a pharmaceutically acceptable salt thereof is 10 mM to 100 mM, 20 mM to 80 mM, 30 mM to 60 mM, 40 mM to 50 mM, or 50 mM to 60 mM; the concentration of proline is 60mM to 600mM, 80mM to 500mM, 100mM to 450mM, 120mM to 400mM, 150mM to 350mM, 200mM to 350mM, or 250mM to 350mM; and / or the concentration of sodium chloride is 1mM to 80mM, 2mM to 60mM, 4mM to 50mM, 8mM to 40mM, 10mM to 30mM, or 10mM to 20mM.

10. The pharmaceutical composition of any one of claims 1-9, wherein the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.

3.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the pharmaceutical composition comprises: (a) 30 mg / mL to 300 mg / mL of an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) 1 mM to 100 mM buffer, (c) 0.01 mg / mL to 2 mg / mL of a surfactant, and (d) a stabilizer, the stabilizer comprising trehalose, mannitol, sucrose, arginine or a pharmaceutically acceptable salt thereof, proline and / or sodium chloride, and the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.3; optionally, the pharmaceutical composition comprises: (a) 30 mg / mL to 300 mg / mL of an anti-IL4Rα antibody or an antigen-binding fragment thereof, (b) 1 mM to 100 mM phosphate buffer, acetate buffer or histidine buffer, (c) 0.01 mg / mL to 2 mg / mL of polysorbate 80 or polysorbate 20, and (d) 10 mg / mL to 100 mg / mL of sucrose, 10 mM to 100 mM of arginine or a pharmaceutically acceptable salt thereof, 60 mM to 600 mM of proline, and / or 1 mM to 80 mM of sodium chloride, And the pH of the pharmaceutical composition is 4 to 7, 5 to 7, 5 to 6.5, 5.3 to 6.5, 5.3 to 6.3, 5.3 to 5.8, or 5.8 to 6.

3.

12. A lyophilized preparation containing an anti-IL4Rα antibody or an antigen-binding fragment thereof, wherein the lyophilized preparation is obtained by freeze-drying the pharmaceutical composition according to any one of claims 1 to 11; or the lyophilized preparation can form the pharmaceutical composition according to any one of claims 1 to 11 after being reconstituted.

13. A method for treating an allergic disease associated with excessive IL4 and / or IL13 signaling in a subject, the method comprising administering to the subject the pharmaceutical composition of any one of claims 1 to 11 or the lyophilized formulation of claim 12; Preferably, the allergic disease is atopic dermatitis, anaphylaxis, allergic rhinitis or allergic asthma.

14. A method for treating a tumor associated with increased STAT6 activation in a subject, the method comprising administering to the subject the pharmaceutical composition of any one of claims 1 to 11 or the lyophilized formulation of claim 12; Preferably, the tumor is a solid tumor; more preferably, the tumor is melanoma, lung cancer, kidney cancer, prostate cancer, cervical cancer, colorectal cancer, gastric cancer, pancreatic cancer, ovarian cancer and / or urothelial carcinoma.

15. A method for reducing IL4 and / or IL13 signaling in a subject or reducing a type 2 immune response in a subject, the method comprising administering to the subject the pharmaceutical composition of any one of claims 1 to 11 or the lyophilized formulation of claim 12; Preferably, the IL4 and / or IL13 signal transduction is manifested as activation and / or proliferation of B cells, eosinophils, macrophages, proliferation of fibroblasts, and proliferation of smooth muscle; preferably, the proliferation of smooth muscle cells is proliferation of airway smooth muscle cells.

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