Pharmaceutical composition containing anti-TSLP antibody

Abstract

Pharmaceutical compositions comprising anti-TSLP antibodies and uses thereof are provided. Specifically, provided is a pharmaceutical composition comprising (a) an anti-TSLP antibody, (b) a buffer agent, (c) a surfactant, and one or more (d) stabilizers, and a use of the pharmaceutical composition in the preparation of drugs for treating and preventing TSLP-related diseases.

Description

[0001] This application is a divisional application of the invention application with an application date of March 2, 2022, Chinese application number 202280017436.4, and invention name “Pharmaceutical composition containing anti-TSLP antibodies”.

[0002] All documents cited or referenced in this application (including but not limited to documents, patents, patent applications) and all documents cited or referenced in documents cited in this application, as well as any manufacturer's instructions, descriptions, product specifications and product pages of any products mentioned in this application or in any document incorporated by reference in this application, are incorporated by reference and can be used in the practice of the present invention. More specifically, all references cited in this application are incorporated by reference to the same extent as if each document was specifically and individually indicated to be incorporated by reference in this application. Moreover, any reference to these publications in this application does not constitute an admission that the publications have become common knowledge in the art in any country. Technical Field

[0003] The present disclosure belongs to the field of pharmaceutical preparations, and in particular, the present disclosure relates to a stable pharmaceutical composition containing an anti-TSLP antibody, or a stable pharmaceutical composition containing a high concentration of an anti-TSLP antibody. Background Art

[0004] Thymic stromal lymphopoietin (TSLP) is a cytokine produced by epithelial cells. It is closely related to IL-7 and binds to TSLPR (a heterodimer of the IL-7 receptor α chain and the TSLP receptor chain). TSLP induces DC cell polarization during the induction phase of the immune response, promotes the differentiation of helper T cells (Th) 2 and the production of Th2 cytokines. In addition, TSLP can directly promote T cell expansion and increase the secretion of Th2 cytokines. Therefore, TSLP is considered to be the main regulator of Th2-driven inflammation, and the upregulation of TSLP is related to the pathogenesis of Th2-type cell-related diseases such as atopic dermatitis and asthma (Rui He et al., (2010) ibid; Ito T et al., (2005) The Journal of Experimental Medicine 202 (9): 1213-1223; He R et al., (2008) Proc Natl Acad Sci USA 105 (33): 11875-11880). On the other hand, TSLP mediates several immune homeostasis in the intestine and thymus. For example, upon bacterial stimulation, TSLP is upregulated in an intestinal epithelial cell line in a strain-dependent manner, synergizing with transforming growth factor-β to promote Treg cell differentiation. TSLP can also be produced by human primary intestinal epithelial cells to regulate CD103+ DC cells become tolerogenic phenotype (Katerina Tsilingiri et al., (2017) Cellular and Molecular Gastroenterology and Hepatology 3(2):174-182; Zeuthen LH et al., (2008) Immunology 123:197–208; IlievID et al., (2009) Gut 58:1481–1489).

[0005] The dual effects of TSLP on the immune system led to the discovery of its two isoforms, the long form and the short form, of which the short form consists of the terminal 63 amino acid residues of the long TSLP. These two isoforms are controlled by different promoters and are expressed depending on the environment, tissue, and stimulus (Harada M et al., (2011) American Journal of Respiratory Cell and Molecular Biology 44:787–793). After infection with highly immunogenic microbial strains, the expression of long TSLP in human intestinal epithelial cells is upregulated and the expression of short TSLP is downregulated; while after infection with commensal Escherichia coli, the opposite expression pattern was observed. The expression pattern of TSLP isoforms has also been studied in some TSLP-related diseases. For example, overexpression of long TSLP has been observed in asthma, ulcerative colitis, atopic dermatitis, and psoriasis, while decreased expression of long TSLP has been found in gluten-induced enteropathy. The expression of the short form of TSLP is downregulated in Crohn's disease, gluten-dependent enteropathy, and atopic dermatitis (Katerina Tsilingiri et al., (2017) supra; Fornasa G et al., (2015) J Allergy Clin Immunol 136:413–422).

[0006] TSLP is associated with a variety of diseases and has become an important clinical target. More and more anti-TSLP antibodies will be developed and used. Therefore, there is a need in the art to develop a pharmaceutical composition comprising an anti-TSLP antibody, so that the anti-TSLP antibody is suitable for production and administration to patients, and can maintain biological activity and stability during storage and subsequent use. Summary of the invention

[0007] The present disclosure provides a pharmaceutical composition comprising an anti-TSLP antibody, which can achieve a stable effect and is suitable for administration in a subject.

[0008] Specifically, the present disclosure provides pharmaceutical compositions containing anti-TSLP antibodies, such as: (i) pharmaceutical compositions capable of inhibiting the generation of high molecular weight impurities; (ii) pharmaceutical compositions capable of inhibiting the generation of charge variants; (iii) pharmaceutical compositions capable of maintaining the biological activity of antibodies; and / or (iv) pharmaceutical compositions capable of reducing the viscosity that increases when the antibody concentration is high.

[0009] The present disclosure provides a pharmaceutical composition comprising: (a) an anti-TSLP antibody, (b) a buffer, (c) a surfactant, and one or more (d) stabilizers.

[0010] In some embodiments, the concentration of the anti-TSLP antibody in the aforementioned pharmaceutical composition is 30 mg / mL to 300 mg / mL, preferably 50 mg / mL to 250 mg / mL, preferably 70 mg / mL to 200 mg / mL, and more preferably 90 mg / mL to 150 mg / mL. In some embodiments, the concentration of the anti-TSLP antibody in the aforementioned pharmaceutical composition is about 120 mg / mL. In some embodiments, the concentration of the anti-TSLP antibody in the aforementioned pharmaceutical composition is about 130 mg / mL. In some embodiments, the concentration of the anti-TSLP antibody in the aforementioned pharmaceutical composition is about 140 mg / mL. In some embodiments, the concentration of the anti-TSLP antibody in the aforementioned pharmaceutical composition is about 150 mg / mL. In some embodiments, the concentration of the anti-TSLP antibody in the aforementioned pharmaceutical composition includes but is not limited to: about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 115 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, or about 150 mg / mL.

[0011] In some embodiments, the buffer in the aforementioned pharmaceutical composition comprises a phosphate buffer, a histidine buffer, an acetate buffer, or a citrate buffer, etc. In some embodiments, the buffer in the aforementioned pharmaceutical composition comprises a phosphate buffer. In some embodiments, the buffer in the aforementioned pharmaceutical composition comprises an acetate buffer. Preferably, the buffer in the aforementioned pharmaceutical composition comprises a histidine buffer.

[0012] In some specific embodiments, the phosphate buffer includes a sodium phosphate buffer or a potassium phosphate buffer, the acetate buffer includes a sodium acetate buffer, a potassium acetate buffer or an ammonium acetate buffer, and the citrate buffer includes a sodium citrate buffer, a potassium citrate buffer or a calcium citrate buffer. In some embodiments, the buffer in the aforementioned pharmaceutical composition comprises a sodium phosphate buffer, such as a sodium phosphate buffer composed of disodium hydrogen phosphate and sodium dihydrogen phosphate. In some embodiments, the buffer in the aforementioned pharmaceutical composition comprises a sodium acetate buffer, such as a sodium acetate buffer composed of sodium acetate and acetic acid. In some embodiments, the buffer in the aforementioned pharmaceutical composition comprises a sodium citrate buffer, such as a sodium citrate buffer composed of citric acid and sodium citrate.

[0013] In some embodiments, the concentration of the buffer in the aforementioned pharmaceutical composition is 1mM to 100mM, preferably 2mM to 80mM, preferably 5mM to 60mM, and more preferably 10mM to 40mM. In some embodiments, the concentration of the buffer in the aforementioned pharmaceutical composition is about 10mM. In some embodiments, the concentration of the buffer in the aforementioned pharmaceutical composition is about 18mM. In some embodiments, the concentration of the buffer in the aforementioned pharmaceutical composition is about 20mM. In some embodiments, the concentration of the buffer in the aforementioned pharmaceutical composition is about 22mM. In some embodiments, the concentration of the buffer in the aforementioned pharmaceutical composition includes but is not limited to: about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, about 20mM, about 21mM, about 22mM, about 23mM, about 24mM, about 25mM, about 26mM, about 27mM, about 28mM, about 30mM, about 32mM, about 34mM, about 36mM, about 38mM, or about 40mM.

[0014] In some embodiments, the pH of the buffer in the aforementioned pharmaceutical composition is 5 to 7, preferably 5.5 to 7, and more preferably 5.5 to 6.5. In some embodiments, the pH of the buffer in the aforementioned pharmaceutical composition is about 5.6. 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 includes but is 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, or about 6.5.

[0015] 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, polysorbate 85), poloxamers (such as poloxamer 181, poloxamer 188, 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.

[0016] In some embodiments, the concentration of the surfactant in the aforementioned pharmaceutical composition is 0.001% to 0.1% (w / v), preferably 0.004% to 0.08% (w / v), preferably 0.006% to 0.06% (w / v), and more preferably 0.008% to 0.04% (w / v). In some embodiments, the concentration of the surfactant in the aforementioned pharmaceutical composition is about 0.01% (w / v). In some embodiments, the concentration of the surfactant in the aforementioned pharmaceutical composition is about 0.02% (w / v). In some embodiments, the concentration of the surfactant in the aforementioned pharmaceutical composition is about 0.04% (w / v). In some embodiments, the concentration of the surfactant in the aforementioned pharmaceutical composition includes but is not limited to: about 0.008% (w / v), about 0.009% (w / v), about 0.01% (w / v), about 0.02% (w / v), about 0.03% (w / v), about 0.04% (w / v), about 0.05% (w / v), or about 0.06% (w / v).

[0017] In some embodiments, the stabilizer in the aforementioned pharmaceutical composition comprises trehalose, mannitol, sucrose, arginine or a pharmaceutically acceptable salt thereof, glycine or proline. In some embodiments, the stabilizer in the aforementioned pharmaceutical composition comprises mannitol. In some embodiments, the stabilizer in the aforementioned pharmaceutical composition comprises arginine. In some embodiments, the stabilizer in the aforementioned pharmaceutical composition comprises proline. In some embodiments, the aforementioned pharmaceutical composition comprises two stabilizers, such as sucrose and proline. The arginine or a pharmaceutically acceptable salt thereof includes arginine, arginine hydrochloride or arginine acetate, etc.

[0018] In some embodiments, the concentration of the stabilizer in the aforementioned pharmaceutical composition is 100mM to 1000mM, 120mM to 800mM, preferably 150mM to 700mM, and more preferably 150mM to 600mM. In some embodiments, the concentration of the stabilizer in the aforementioned pharmaceutical composition is about 200mM. In some embodiments, the concentration of the stabilizer in the aforementioned pharmaceutical composition is about 250mM. In some embodiments, the concentration of the stabilizer in the aforementioned pharmaceutical composition is about 400mM. In some embodiments, the concentration of the stabilizer in the aforementioned pharmaceutical composition is about 500mM. In some embodiments, the concentration of the stabilizer in the aforementioned pharmaceutical composition includes but is not limited to: about 120mM, about 150mM, about 200mM, about 210mM, about 230mM, about 250mM, about 270mM, about 290mM, about 300mM, about 310mM, about 330mM, about 350mM, about 370mM, about 380mM, about 390mM, about 400mM, about 410mM, about 420mM, about 430mM, about 450mM, about 470mM, about 490mM, about 500mM, about 510mM, about 530mM, about 550mM, about 570mM, or about 600mM.

[0019] In some embodiments, the pH of the aforementioned pharmaceutical composition is 5 to 7, preferably 5.5 to 7, and more preferably 5.5 to 6.5. In some embodiments, the pH of the aforementioned pharmaceutical composition is about 5.6. 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 includes but is 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, or about 6.5.

[0020] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 30 mg / mL to about 300 mg / mL of an anti-TSLP antibody, (b) about 1 mM to about 100 mM of a buffer, (c) about 0.001% to about 0.1% (w / v) of a surfactant, and one or more (d) about 100 mM to about 1000 mM of a stabilizer; wherein the pH of the pharmaceutical composition is about 5 to about 7, preferably about 5.5 to about 7, and more preferably about 5.5 to about 6.5.

[0021] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 50 mg / mL to about 250 mg / mL of an anti-TSLP antibody, (b) about 2 mM to about 80 mM of a buffer, (c) about 0.004% to about 0.08% (w / v) of a surfactant, and one or more (d) about 120 mM to about 800 mM of a stabilizer; wherein the pH of the pharmaceutical composition is about 5 to about 7, preferably about 5.5 to about 7, and more preferably about 5.5 to about 6.5.

[0022] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 70 mg / mL to about 200 mg / mL of an anti-TSLP antibody, (b) about 5 mM to about 60 mM of a buffer, (c) about 0.006% to about 0.06% (w / v) of a surfactant, and one or more (d) about 150 mM to about 700 mM of a stabilizer; wherein the pH of the pharmaceutical composition is about 5 to about 7, preferably about 5.5 to about 7, and more preferably about 5.5 to about 6.5.

[0023] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 90 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 10 mM to about 40 mM of a buffer, (c) about 0.008% to about 0.04% (w / v) of a surfactant, and one or more (d) about 150 mM to about 600 mM of a stabilizer; wherein the pH of the pharmaceutical composition is about 5 to about 7, preferably about 5.5 to about 7, and more preferably about 5.5 to about 6.5.

[0024] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 10 mM to about 40 mM of a buffer, (c) about 0.008% to about 0.04% (w / v) of a surfactant, and one or more (d) about 150 mM to about 600 mM of a stabilizer; wherein the pH of the pharmaceutical composition is about 5 to about 7, preferably about 5.5 to about 7, and more preferably about 5.5 to about 6.5.

[0025] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 30 mg / mL to about 300 mg / mL of an anti-TSLP antibody, (b) about 1 mM to about 100 mM phosphate buffer, histidine buffer or acetate buffer, (c) about 0.001% to about 0.1% (w / v) polysorbate 20 or polysorbate 80, and (d) about 100 mM to about 1000 mM trehalose, mannitol or sucrose, and / or about 100 mM to about 1000 mM arginine or a pharmaceutically acceptable salt thereof, glycine or proline; wherein the pH of the pharmaceutical composition is about 5 to about 7, preferably about 5.5 to about 7, and more preferably about 5.5 to about 6.5.

[0026] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 50 mg / mL to about 250 mg / mL of an anti-TSLP antibody, (b) about 2 mM to about 80 mM of a phosphate buffer, a histidine buffer or an acetate buffer, (c) about 0.004% to about 0.08% (w / v) of polysorbate 20 or polysorbate 80, and (d) about 120 mM to about 800 mM of trehalose, mannitol or sucrose, and / or about 120 mM to about 800 mM of arginine or a pharmaceutically acceptable salt thereof, glycine or proline; wherein the pH of the pharmaceutical composition is about 5 to about 7, preferably about 5.5 to about 7, and more preferably about 5.5 to about 6.5.

[0027] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 70 mg / mL to about 200 mg / mL of an anti-TSLP antibody, (b) about 5 mM to about 60 mM of a phosphate buffer, a histidine buffer or an acetate buffer, (c) about 0.006% to about 0.06% (w / v) of polysorbate 20 or polysorbate 80, and (d) about 150 mM to about 700 mM of trehalose, mannitol or sucrose, and / or about 150 mM to about 700 mM of arginine or a pharmaceutically acceptable salt thereof, glycine or proline; wherein the pH of the pharmaceutical composition is about 5 to about 7, preferably about 5.5 to about 7, and more preferably about 5.5 to about 6.5.

[0028] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 90 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 10 mM to about 40 mM of a phosphate buffer, a histidine buffer or an acetate buffer, (c) about 0.008% to about 0.04% (w / v) of polysorbate 20 or polysorbate 80, and (d) about 150 mM to about 600 mM of trehalose, mannitol or sucrose, and / or about 150 mM to about 600 mM of arginine or a pharmaceutically acceptable salt thereof, glycine or proline; wherein the pH of the pharmaceutical composition is about 5 to about 7, preferably about 5.5 to about 7, and more preferably about 5.5 to about 6.5.

[0029] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 10 mM to about 40 mM of a phosphate buffer, a histidine buffer or an acetate buffer, (c) about 0.008% to about 0.04% (w / v) of polysorbate 20 or polysorbate 80, and (d) about 150 mM to about 600 mM of trehalose, mannitol or sucrose, and / or about 150 mM to about 600 mM of arginine or a pharmaceutically acceptable salt thereof, glycine or proline; wherein the pH of the pharmaceutical composition is about 5 to about 7, preferably about 5.5 to about 7, and more preferably about 5.5 to about 6.5.

[0030] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 30 mg / mL to about 300 mg / mL of an anti-TSLP antibody, (b) about 1 mM to about 100 mM of a sodium phosphate buffer, a histidine buffer or a sodium acetate buffer, (c) about 0.001% to about 0.1% (w / v) of polysorbate 80, and (d) about 100 mM to about 1000 mM of mannitol, and / or about 100 mM to about 1000 mM of arginine or a pharmaceutically acceptable salt thereof, or proline; wherein the pH of the pharmaceutical composition is about 5 to about 7, preferably about 5.5 to about 7, and more preferably about 5.5 to about 6.5.

[0031] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 50 mg / mL to about 250 mg / mL of an anti-TSLP antibody, (b) about 2 mM to about 80 mM of a sodium phosphate buffer, a histidine buffer or a sodium acetate buffer, (c) about 0.004% to about 0.08% (w / v) of polysorbate 80, and (d) about 120 mM to about 800 mM of mannitol; and / or about 120 mM to about 800 mM of arginine or a pharmaceutically acceptable salt thereof, or proline, wherein the pH of the pharmaceutical composition is about 5 to about 7, preferably about 5.5 to about 7, and more preferably about 5.5 to about 6.5.

[0032] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 70 mg / mL to about 200 mg / mL of an anti-TSLP antibody, (b) about 5 mM to about 60 mM of a sodium phosphate buffer, a histidine buffer or a sodium acetate buffer, (c) about 0.006% to about 0.06% (w / v) of polysorbate 80, and (d) about 150 mM to about 700 mM of mannitol, and / or about 150 mM to about 700 mM of arginine or a pharmaceutically acceptable salt thereof, or proline; wherein the pH of the pharmaceutical composition is about 5 to about 7, preferably about 5.5 to about 7, and more preferably about 5.5 to about 6.5.

[0033] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 90 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 10 mM to about 40 mM of a sodium phosphate buffer, a histidine buffer or a sodium acetate buffer, (c) about 0.008% to about 0.04% (w / v) of polysorbate 80, and (d) about 150 mM to about 600 mM of mannitol, and / or about 150 mM to about 600 mM of arginine or a pharmaceutically acceptable salt thereof, or proline; wherein the pH of the pharmaceutical composition is about 5 to about 7, preferably about 5.5 to about 7, and more preferably about 5.5 to about 6.5.

[0034] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 10 mM to about 40 mM of a sodium phosphate buffer, a histidine buffer or a sodium acetate buffer, (c) about 0.008% to about 0.04% (w / v) of polysorbate 80, and (d) about 150 mM to about 600 mM of mannitol, and / or about 150 mM to about 600 mM of arginine or a pharmaceutically acceptable salt thereof, or proline; wherein the pH of the pharmaceutical composition is about 5 to about 7, preferably about 5.5 to about 7, and more preferably about 5.5 to about 6.5.

[0035] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 10 mM to about 40 mM histidine buffer, (c) about 0.01% (w / v) to about 0.04% (w / v) polysorbate 80 or polysorbate 20, and (d) about 150 mM to about 600 mM proline, mannitol or arginine, wherein the pH of the pharmaceutical composition is about 5.5 to about 6.5, preferably about 5.5 to about 6, for example, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, or about 6.

[0036] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 20 mM histidine buffer, (c) about 0.02% (w / v) polysorbate 80, and (d) about 200 mM mannitol; wherein the pH of the pharmaceutical composition is about 5.5 to about 6.5, preferably about 5.5 to about 6.

[0037] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 20 mM histidine buffer, (c) about 0.02% (w / v) polysorbate 80, and (d) about 150 mM arginine; wherein the pH of the pharmaceutical composition is about 5.5 to about 6.5, preferably about 5.5 to about 6.

[0038] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 20 mM histidine buffer, (c) about 0.02% (w / v) polysorbate 80, and (d) about 400 mM proline; wherein the pH of the pharmaceutical composition is about 5.5 to about 6.5, preferably about 5.5 to about 6.

[0039] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 18 mM histidine buffer, (c) about 0.02% (w / v) polysorbate 80, and (d) about 400 mM proline; wherein the pH of the pharmaceutical composition is about 5.5 to about 6.5, preferably about 5.5 to about 6.

[0040] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 22 mM histidine buffer, (c) about 0.02% (w / v) polysorbate 80, and (d) about 400 mM proline; wherein the pH of the pharmaceutical composition is about 5.5 to about 6.5, preferably about 5.5 to about 6.

[0041] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 18 mM histidine buffer, (c) about 0.02% (w / v) polysorbate 80, and (d) about 500 mM proline; wherein the pH of the pharmaceutical composition is about 5.5 to about 6.5, preferably about 5.5 to about 6.

[0042] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 22 mM histidine buffer, (c) about 0.02% (w / v) polysorbate 80, and (d) about 500 mM proline; wherein the pH of the pharmaceutical composition is about 5.5 to about 6.5, preferably about 5.5 to about 6.

[0043] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 20 mM histidine buffer, (c) about 0.02% (w / v) polysorbate 80, and (d) about 500 mM proline; wherein the pH of the pharmaceutical composition is about 5.5 to about 6.5, preferably about 5.5 to about 6.

[0044] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 18 mM histidine buffer, (c) about 0.02% (w / v) polysorbate 80, and (d) about 450 mM proline; wherein the pH of the pharmaceutical composition is about 5.5 to about 6.5, preferably about 5.5 to about 6.

[0045] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 22 mM histidine buffer, (c) about 0.02% (w / v) polysorbate 80, and (d) about 450 mM proline; wherein the pH of the pharmaceutical composition is about 5.5 to about 6.5, preferably about 5.5 to about 6.

[0046] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 20 mM histidine buffer, (c) about 0.02% (w / v) polysorbate 80, and (d) about 450 mM proline; wherein the pH of the pharmaceutical composition is about 5.5 to about 6.5, preferably about 5.5 to about 6.

[0047] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 18 mM histidine buffer, (c) about 0.02% (w / v) polysorbate 80, and (d) about 350 mM proline; wherein the pH of the pharmaceutical composition is about 5.5 to about 6.5, preferably about 5.5 to about 6.

[0048] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 22 mM histidine buffer, (c) about 0.02% (w / v) polysorbate 80, and (d) about 350 mM proline; wherein the pH of the pharmaceutical composition is about 5.5 to about 6.5, preferably about 5.5 to about 6.

[0049] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 20 mM histidine buffer, (c) about 0.02% (w / v) polysorbate 80, and (d) about 350 mM proline; wherein the pH of the pharmaceutical composition is about 5.5 to about 6.5, preferably about 5.5 to about 6.

[0050] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 18 mM histidine buffer, (c) about 0.02% (w / v) polysorbate 80, and (d) about 250 mM proline; wherein the pH of the pharmaceutical composition is about 5.5 to about 6.5, preferably about 5.5 to about 6.

[0051] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 22 mM histidine buffer, (c) about 0.02% (w / v) polysorbate 80, and (d) about 250 mM proline; wherein the pH of the pharmaceutical composition is about 5.5 to about 6.5, preferably about 5.5 to about 6.

[0052] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 20 mM histidine buffer, (c) about 0.02% (w / v) polysorbate 80, and (d) about 250 mM proline; wherein the pH of the pharmaceutical composition is about 5.5 to about 6.5, preferably about 5.5 to about 6.

[0053] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL of an anti-TSLP antibody, (b) about 20 mM of a histidine buffer, (c) about 0.02% (w / v) of polysorbate 80, and (d) about 400 mM of proline; wherein the pH of the pharmaceutical composition is about 5.8.

[0054] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 10 mM to about 40 mM phosphate or acetate buffer, (c) about 0.01% (w / v) to about 0.04% (w / v) polysorbate 80 or polysorbate 20, and (d) about 200 mM to about 600 mM proline, arginine or mannitol; wherein the pH of the pharmaceutical composition is about 5 to about 7, for example, 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 6.5, or about 7.

[0055] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 20 mM phosphate or acetate buffer, (c) about 0.01% (w / v) to about 0.04% (w / v) polysorbate 80 or polysorbate 20, and (d) about 200 mM mannitol; wherein the pH of the pharmaceutical composition is about 5 to about 7.

[0056] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 120 mg / mL to about 150 mg / mL of an anti-TSLP antibody, (b) about 20 mM sodium phosphate or sodium acetate buffer, (c) about 0.02% (w / v) to about 0.04% (w / v) polysorbate 80 or polysorbate 20, and (d) about 200 mM mannitol; wherein the pH of the pharmaceutical composition is about 5 to about 7.

[0057] In the aforementioned pharmaceutical composition, an isotonic agent or preservative may be appropriately added as desired, and the isotonic agent or preservative may be used appropriately and in an amount within the range of the amount that can achieve the desired effect. Among them, the isotonic agent includes sodium chloride, potassium chloride, calcium chloride, etc.; the preservative includes methyl parahydroxybenzoate, ethyl parahydroxybenzoate, sorbic acid, phenol, cresol, chlorocresol, benzyl alcohol, etc.

[0058] In an optional embodiment, the anti-TSLP antibody in the aforementioned pharmaceutical composition comprises a heavy chain variable region, wherein the heavy chain variable region comprises a VH-CDR1 region, a VH-CDR2 region and a VH-CDR3 region, wherein the VH-CDR1 region, the VH-CDR2 region and the VH-CDR3 region respectively comprise: the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3.

[0059] In an optional embodiment, the anti-TSLP antibody in the aforementioned pharmaceutical composition comprises a heavy chain variable region, and the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NOs: 7, 8 or 9 (X1 = R, X2 = V, X3 = R; X1 = R, X2 = V, X3 = V; X1 = R, X2 = A, X3 = R; X1 = K, X2 = A, X3 = R; or X1 = K, X2 = A, X3 = V). The amino acid sequence as shown in SEQ ID NO: 7 can be encoded by the nucleotide sequence as shown in SEQ ID NOs: 17 or 18, and the amino acid sequence as shown in SEQ ID NO: 9 (X1 = R, X2 = V, X3 = R) can be encoded by the nucleotide sequence as shown in SEQ ID NO: 19.

[0060] In an optional embodiment, the anti-TSLP antibody in the aforementioned pharmaceutical composition comprises a light chain variable region, which comprises a VL-CDR1 region, a VL-CDR2 region and a VL-CDR3 region, wherein the VL-CDR1 region, the VL-CDR2 region and the VL-CDR3 region may respectively comprise: the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6.

[0061] In an optional embodiment, the anti-TSLP antibody in the aforementioned pharmaceutical composition comprises a light chain variable region, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NOs: 10 or 11 (X1=S, X2=V; X1=A, X2=I; or X1=S, X2=I). The amino acid sequence as shown in SEQ ID NOs: 10 can be encoded by a nucleotide sequence as shown in SEQ ID NOs: 20 or 21, and the amino acid sequence as shown in SEQ ID NO: 11 (X1=A, X2=I) can be encoded by a nucleotide sequence as shown in SEQ ID NO: 22.

[0062] In an optional embodiment, the anti-TSLP antibody in the aforementioned pharmaceutical composition comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises a VH-CDR1 region, a VH-CDR2 region and a VH-CDR3 region, and the light chain variable region comprises a VL-CDR1 region, a VL-CDR2 region and a VL-CDR3 region, wherein the VH-CDR1 region, the VH-CDR2 region, the VH-CDR3 region, the VL-CDR1 region, the VL-CDR2 region and the VL-CDR3 region respectively comprise: the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4, 5 and 6.

[0063] In an optional embodiment, the anti-TSLP antibody in the aforementioned pharmaceutical composition comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region may comprise an amino acid sequence selected from the following: (1) as shown in SEQ ID NOs: 7 and 10, respectively; (2) as shown in SEQ ID NOs: 8 and 11 (X1=S, X2=V), respectively; (3) as shown in SEQ ID NOs: 9 (X1=R, X2=V, X3=R) and 11 (X1=S, X2=V), respectively; (4) as shown in SEQ ID NOs: 9 (X1=R, X2=V, X3=V) and 11 (X1=S, X2=V), respectively; (5) as shown in SEQ ID NOs: 9 (X1=R, X2=A, X3=R) and 11 (X1=S, X2=V), respectively; (6) as shown in SEQ ID NOs: NOs: 9 (X1 = K, X2 = A, X3 = R) and 11 (X1 = S, X2 = V); (7) as shown in SEQ ID NOs: 9 (X1 = K, X2 = A, X3 = V) and 11 (X1 = S, X2 = V); (8) as shown in SEQ ID NOs: 8 and 11 (X1 = A, X2 = I); (9) as shown in SEQ ID NOs: 9 (X1 = R, X2 = V, X3 = R) and 11 (X1 = A, X2 = I); (10) as shown in SEQ ID NOs: 9 (X1 = R, X2 = V, X3 = V) and 11 (X1 = A, X2 = I); (11) as shown in SEQ ID NOs: 9 (X1 = R, X2 = A, X3 = R) and 11 (X1 = A, X2 = I); (12) as shown in SEQ ID NOs: NOs: 9 (X1 = K, X2 = A, X3 = R) and 11 (X1 = A, X2 = I); (13) as shown in SEQ ID NOs: 9 (X1 = K, X2 = A, X3 = V) and 11 (X1 = A, X2 = I); (14) as shown in SEQ ID NOs: 8 and 11 (X1 = S, X2 = I); (15) as shown in SEQ ID NOs: 9 (X1 = R, X2 = V, X3 = R) and 11 (X1 = S, X2 = I); (16) as shown in SEQ ID NOs: 9 (X1 = R, X2 = V, X3 = V) and 11 (X1 = S, X2 = I); (17) as shown in SEQ ID NOs: NOs: 9 (X1 = R, X2 = A, X3 = R) and 11 (X1 = S, X2 = I); (18) are respectively shown in SEQ ID NOs: 9 (X1 = K, X2 = A, X3 = R) and 11 (X1 = S, X2 = I); or (19) are respectively shown in SEQ ID NOs: 9 (X1 = K, X2 = A, X3 = V) and 11 (X1 = S, X2 = I).

[0064] In an optional embodiment, the anti-TSLP antibody in the aforementioned pharmaceutical composition comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region and a heavy chain constant region, and the light chain comprises a light chain variable region and a light chain constant region; the heavy chain constant region may comprise a human IgG1 heavy chain constant region having an amino acid sequence as shown in SEQ ID NOs: 12 or 37, or a human IgG4 heavy chain constant region having an amino acid sequence as shown in SEQ ID NO: 13, or a fragment of these heavy chain constant regions; the light chain constant region may comprise a human κ light chain constant region having an amino acid sequence as shown in SEQ ID NO: 14 or a fragment thereof; the heavy chain constant region may also be a mouse IgG1 heavy chain constant region having an amino acid sequence as shown in SEQ ID NO: 15, and the light chain constant region may be a mouse κ light chain constant region having an amino acid sequence as shown in SEQ ID NO: 16. The amino acid sequences shown in SEQ ID NOs: 12-16 and 37 may be encoded by the nucleotide sequences shown in SEQ ID NOs: 23-27 and 38, respectively.

[0065] In an optional embodiment, the anti-TSLP antibody in the aforementioned pharmaceutical composition can be a full-length antibody, such as a full-length antibody of IgG1, IgG2 or IgG4 isotype. In other embodiments, the anti-TSLP antibody in the aforementioned pharmaceutical composition can be a single-chain variable region (scFv) antibody, or an antibody fragment, such as a Fab or F(ab')2 fragment.

[0066] In an optional embodiment, the anti-TSLP antibody in the aforementioned pharmaceutical composition is the anti-TSLP antibody in PCT / CN2020 / 113289.

[0067] The present disclosure also provides a method for preparing the aforementioned pharmaceutical composition, which includes the step of contacting the aforementioned anti-TSLP antibody with a buffer, such as replacing the anti-TSLP antibody into the buffer, preferably, the buffer is a phosphate buffer, a histidine buffer or an acetate buffer, the concentration of the buffer is 1mM to 100mM, 2mM to 80mM, preferably 5mM to 60mM, more preferably 10mM to 40mM, and the pH of the buffer is 5 to 7, preferably 5.5 to 7, more preferably 5.5 to 6.5. The method for preparing the aforementioned pharmaceutical composition further includes adding one or more stabilizers and surfactants, regardless of the order, wherein the stabilizer comprises trehalose, mannitol, sucrose, arginine or a pharmaceutically acceptable salt thereof, glycine or proline, etc., and the surfactant comprises polysorbate 20 or polysorbate 80, etc.

[0068] The present disclosure also provides a method for preparing a lyophilized formulation comprising an anti-TSLP antibody, comprising 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. The skilled person understands that any method of removing water from the pharmaceutical composition of the present disclosure is applicable to the present disclosure.

[0069] The present disclosure also provides a lyophilized preparation comprising an anti-TSLP antibody, which is prepared by the aforementioned method for preparing a lyophilized preparation.

[0070] The present disclosure also provides a lyophilized preparation comprising an anti-TSLP antibody, wherein the lyophilized preparation can form the aforementioned pharmaceutical composition after being reconstituted.

[0071] The present disclosure also provides a product, which includes a container containing the aforementioned pharmaceutical composition or the aforementioned lyophilized preparation.

[0072] The pharmaceutical compositions or lyophilized formulations of the present disclosure can be administered according to known methods, such as injection or infusion over a period of time in an appropriate manner, for example, by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, topical application, inhalation or by sustained release or delayed release.

[0073] The pharmaceutical composition of the present disclosure may also contain one or more other active compounds, preferably those that do not adversely affect each other and have complementary activities, as long as it is necessary for the specific symptoms to be treated. In addition or alternatively, the pharmaceutical composition may include an anti-TSLP antibody and another disease-specific protein, such as TSLPR, IgE, IL-13 or IL-5, such molecules being appropriately present in the pharmaceutical composition in an amount effective for the intended target.

[0074] The present disclosure also provides the use of the pharmaceutical composition, lyophilized preparation or product of the present disclosure in the preparation of a drug for treating and preventing TSLP-related diseases. In some embodiments, the TSLP-related diseases include asthma, ulcerative colitis, atopic dermatitis and psoriasis.

[0075] The present disclosure also provides a method for treating and preventing TSLP-related diseases, which comprises administering the pharmaceutical composition, lyophilized formulation or product of the present disclosure to a subject. In some embodiments, the TSLP-related diseases include asthma, ulcerative colitis, atopic dermatitis and psoriasis.

[0076] In summary, the present disclosure relates to the following:

[0077] 1. A pharmaceutical composition comprising: (a) an anti-TSLP antibody, (b) a buffer, (c) a surfactant, and one or more (d) stabilizers.

[0078] 2. The pharmaceutical composition described in item 1, wherein the anti-TSLP antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises VH-CDR1, VH-CDR2 and VH-CDR3, and the light chain variable region comprises VL-CDR1, VL-CDR2 and VL-CDR3, wherein the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4, 5 and 6, respectively.

[0079] 3. The pharmaceutical composition of item 1 or 2, wherein the anti-TSLP antibody comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region may comprise an amino acid sequence selected from the following: (1) as shown in SEQ ID NOs: 7 and 10, respectively; (2) as shown in SEQ ID NOs: 8 and 11 (X1=S, X2=V), respectively; (3) as shown in SEQ ID NOs: 9 (X1=R, X2=V, X3=R) and 11 (X1=S, X2=V), respectively; (4) as shown in SEQ ID NOs: 9 (X1=R, X2=V, X3=V) and 11 (X1=S, X2=V), respectively; (5) as shown in SEQ ID NOs: 9 (X1=R, X2=A, X3=R) and 11 (X1=S, X2=V), respectively; (6) as shown in SEQ ID NOs: NOs: 9 (X1 = K, X2 = A, X3 = R) and 11 (X1 = S, X2 = V); (7) as shown in SEQ ID NOs: 9 (X1 = K, X2 = A, X3 = V) and 11 (X1 = S, X2 = V); (8) as shown in SEQ ID NOs: 8 and 11 (X1 = A, X2 = I); (9) as shown in SEQ ID NOs: 9 (X1 = R, X2 = V, X3 = R) and 11 (X1 = A, X2 = I); (10) as shown in SEQ ID NOs: 9 (X1 = R, X2 = V, X3 = V) and 11 (X1 = A, X2 = I); (11) as shown in SEQ ID NOs: 9 (X1 = R, X2 = A, X3 = R) and 11 (X1 = A, X2 = I); (12) as shown in SEQ ID NOs: ID NOs: 9 (X1 = K, X2 = A, X3 = R) and 11 (X1 = A, X2 = I); (13) as shown in SEQ ID NOs: 9 (X1 = K, X2 = A, X3 = V) and 11 (X1 = A, X2 = I); (14) as shown in SEQ ID NOs: 8 and 11 (X1 = S, X2 = I); (15) as shown in SEQ ID NOs: 9 (X1 = R, X2 = V, X3 = R) and 11 (X1 = S, X2 = I); (16) as shown in SEQ ID NOs: 9 (X1 = R, X2 = V, X3 = V) and 11 (X1 = S, X2 = I); (17) as shown in SEQ ID NOs: NOs: 9 (X1 = R, X2 = A, X3 = R) and 11 (X1 = S, X2 = I); (18) are respectively shown in SEQ ID NOs: 9 (X1 = K, X2 = A, X3 = R) and 11 (X1 = S, X2 = I); or (19) are respectively shown in SEQ ID NOs: 9 (X1 = K, X2 = A, X3 = V) and 11 (X1 = S, X2 = I).

[0080] 4. The pharmaceutical composition according to any one of items 1-3, wherein the concentration of the anti-TSLP antibody is 30 mg / ml to 300 mg / ml, preferably 50 mg / ml to 250 mg / ml, preferably 70 mg / ml to 200 mg / ml, more preferably 90 mg / ml to 150 mg / ml, and most preferably 120 mg / ml to 150 mg / ml.

[0081] 5. The pharmaceutical composition according to any one of items 1 to 4, wherein the buffer comprises a phosphate buffer, a histidine buffer, an acetate buffer or a citrate buffer.

[0082] 6. The pharmaceutical composition described in item 5, wherein the phosphate buffer is a sodium phosphate buffer, the acetate buffer is a sodium acetate buffer, and the citrate buffer is a sodium citrate buffer.

[0083] 7. The pharmaceutical composition according to any one of items 1-6, wherein the concentration of the buffer is 1 mM to 100 mM, preferably 2 mM to 80 mM, more preferably 5 mM to 60 mM, and most preferably 10 mM to 40 mM.

[0084] 8. The pharmaceutical composition described in any one of items 1-7, wherein the surfactant comprises polysorbate 80 or polysorbate 20.

[0085] 9. The pharmaceutical composition according to any one of items 1 to 8, wherein the concentration of the surfactant is 0.001% to 0.1% (w / v), preferably 0.004% to 0.08% (w / v), more preferably 0.006% to 0.06% (w / v), and most preferably 0.008% to 0.04% (w / v).

[0086] 10. The pharmaceutical composition according to any one of items 1 to 9, wherein the stabilizer comprises trehalose, mannitol, sucrose, arginine or a pharmaceutically acceptable salt thereof, glycine or proline.

[0087] 11. The pharmaceutical composition according to any one of items 1 to 10, wherein the concentration of the stabilizer is 100 mM to 1000 mM, preferably 120 mM to 800 mM, more preferably 150 mM to 700 mM, and most preferably 150 mM to 600 mM.

[0088] 12. The pharmaceutical composition according to any one of items 1 to 11, wherein the pH of the pharmaceutical composition is 5 to 7, preferably 5.5 to 7, and more preferably 5.5 to 6.5.

[0089] 13. The pharmaceutical composition of any one of items 1 to 12, wherein the pharmaceutical composition comprises:

[0090] (a) 30 mg / ml to 300 mg / ml of anti-TSLP antibody,

[0091] (b) 1 mM to 100 mM buffer,

[0092] (c) 0.001% to 0.1% (w / v) of a surfactant, and

[0093] one or more (d) 100 mM to 1000 mM of a stabilizer;

[0094] The pH of the pharmaceutical composition is 5 to 7, preferably 5.5 to 7, more preferably 5.5 to 6.5.

[0095] 14. The pharmaceutical composition of any one of items 1 to 13, wherein the pharmaceutical composition comprises:

[0096] (a) 50 mg / ml to 250 mg / ml of anti-TSLP antibody,

[0097] (b) 2 mM to 80 mM buffer,

[0098] (c) 0.004% to 0.08% (w / v) of a surfactant, and

[0099] one or more (d) 120 mM to 800 mM of a stabilizer;

[0100] The pH of the pharmaceutical composition is 5 to 7, preferably 5.5 to 7, more preferably 5.5 to 6.5.

[0101] 15. The pharmaceutical composition of any one of items 1 to 14, wherein the pharmaceutical composition comprises:

[0102] (a) 70 mg / ml to 200 mg / ml of anti-TSLP antibody,

[0103] (b) 5 mM to 60 mM buffer,

[0104] (c) 0.006% to 0.06% (w / v) of a surfactant, and

[0105] one or more (d) 150 mM to 700 mM stabilizers;

[0106] The pH of the pharmaceutical composition is 5 to 7, preferably 5.5 to 7, more preferably 5.5 to 6.5.

[0107] 16. The pharmaceutical composition of any one of items 1 to 15, wherein the pharmaceutical composition comprises:

[0108] (a) 90 mg / ml to 150 mg / ml of anti-TSLP antibody,

[0109] (b) 10 mM to 40 mM buffer,

[0110] (c) 0.008% to 0.04% (w / v) of a surfactant, and

[0111] one or more (d) 150 mM to 600 mM stabilizers;

[0112] The pH of the pharmaceutical composition is 5 to 7, preferably 5.5 to 7, more preferably 5.5 to 6.5.

[0113] 17. The pharmaceutical composition of any one of items 1 to 16, wherein the pharmaceutical composition comprises:

[0114] (a) 120 mg / ml to 150 mg / ml of anti-TSLP antibody,

[0115] (b) 10 mM to 40 mM buffer,

[0116] (c) 0.008% to 0.04% (w / v) of a surfactant, and

[0117] one or more (d) 150 mM to 600 mM stabilizers;

[0118] The pH of the pharmaceutical composition is 5 to 7, preferably 5.5 to 7, more preferably 5.5 to 6.5.

[0119] 18. A lyophilized preparation containing an anti-TSLP antibody, wherein the lyophilized preparation is obtained by freeze-drying the pharmaceutical composition described in any one of items 1 to 17.

[0120] 19. A lyophilized preparation containing an anti-TSLP antibody, which can form the pharmaceutical composition described in any one of items 1 to 17 after reconstitution.

[0121] 20. A product comprising a container containing the pharmaceutical composition according to any one of items 1 to 17, or the lyophilized preparation according to any one of items 18 to 19.

[0122] 21. A method for treating and preventing TSLP-related diseases, comprising administering a therapeutically effective amount of the pharmaceutical composition described in any one of items 1 to 17, the lyophilized preparation described in any one of items 18 to 19, or the product described in item 20 to a patient in need thereof.

[0123] 22. The method of claim 21, wherein the TSLP-related diseases include asthma, ulcerative colitis, atopic dermatitis and psoriasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] Figure 1 : Shows the ability of humanized antibodies hu1C5F12E9-V8 and hu1C5F12E9-V14 to bind to human TSLP in capture ELISA.

[0125] Figure 2 : Shows the binding ability of humanized antibodies hu1C5F12E9-V8 and hu1C5F12E9-V14 to cynomolgus monkey TSLP in indirect ELISA.

[0126] Figure 3 : Shows the blocking ability of humanized antibodies hu1C5F12E9-V8 and hu1C5F12E9-V14 on the binding of human TSLP to TSLPR / IL7R in competition ELISA.

[0127] Figure 4 : Shows the ability of humanized antibodies hu1C5F12E9-V8 and hu1C5F12E9-V14 to block the binding of reference product Tezepelumab to human TSLP in competition ELISA.

[0128] Figure 5 : Shown is the ability of humanized antibodies hu1C5F12E9-V8 and hu1C5F12E9-V14 to block the binding of human TSLP to engineered BAF3 cells expressing human TSLPR and IL7R in a cell-based ligand blocking FACS assay.

[0129] Figure 6A-6B : Shown are the inhibitory effects of humanized antibodies hu1C5F12E9-V8 (A) and hu1C5F12E9-V14 (B) on BAF3 cell survival and proliferation in cell-based functional assays.

[0130] Figure 7 : Shows the ability of humanized antibodies hu1C5F12E9-V8 (IgG1), hu1C5F12E9-V8 (IgG4), hu1C5F12E9-V14 (IgG1), and hu1C5F12E9-V14 (IgG4) to bind to human TSLP in capture ELISA.

[0131] Figure 8 : Shows the blocking ability of humanized antibodies hu1C5F12E9-V8 (IgG1), hu1C5F12E9-V8 (IgG4), hu1C5F12E9-V14 (IgG1) and hu1C5F12E9-V14 (IgG4) on the binding of human TSLP to TSLPR / IL7R in competition ELISA.

[0132] Fig. 9: Shows the ability of humanized antibodies hu1C5F12E9-V8 (IgG1), hu1C5F12E9-V8 (IgG4), hu1C5F12E9-V14 (IgG1), and hu1C5F12E9-V14 (IgG4) to block the binding of the reference article to human TSLP in competition ELISA.

[0133] Fig.10 : Shown is the ability of humanized antibodies hu1C5F12E9-V8(IgG1), hu1C5F12E9-V8(IgG4), hu1C5F12E9-V14(IgG1), and hu1C5F12E9-V14(IgG4) to block the binding of human TSLP to engineered BAF3 cells expressing human TSLPR and IL7R in a cell-based ligand blocking FACS assay.

[0134] Figures 11A-11B : Shown are the inhibitory effects of humanized antibodies hu1C5F12E9-V8(IgG1), hu1C5F12E9-V8(IgG4) (A), hu1C5F12E9-V14(IgG1), and hu1C5F12E9-V14(IgG4) (B) on BAF3 cell survival and proliferation in cell-based functional assays. Figures 12A-12B : Shown is the ability of humanized antibodies hu1C5F12E9-V8(IgG1), hu1C5F12E9-V8(IgG4) (A), hu1C5F12E9-V14(IgG1) and hu1C5F12E9-V14(IgG4) (B) to block the interaction of human TSLP with engineered HEK293T cells in a cell-based reporter gene assay.

[0135] Fig.13 : Shows the results of protein thermal shift assays of hu1C5F12E9-V8(IgG1), hu1C5F12E9-V8(IgG4), hu1C5F12E9-V14(IgG1), and hu1C5F12E9-V14(IgG4) antibodies. DETAILED DESCRIPTION

[0136] The following description of the present disclosure is merely for the purpose of illustrating various embodiments of the present disclosure.

[0137] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, 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 disclosure belongs.

[0138] "Phosphate buffer" is a buffer containing phosphate ions. Examples of phosphate buffers include sodium phosphate buffer, potassium phosphate buffer, etc., and a preferred phosphate buffer is sodium phosphate buffer.

[0139] "Acetate buffer" is a buffer including acetate ions. Examples of acetate buffers include potassium acetate buffer, ammonium acetate buffer, sodium acetate buffer, and the like, and a preferred acetate buffer is sodium acetate buffer.

[0140] "Citrate buffer" is a buffer including citrate ions. Examples of citrate buffers include sodium citrate buffer, potassium citrate buffer, calcium citrate buffer, and the like, and a preferred citrate buffer is sodium citrate buffer.

[0141] "Histidine buffer" is a buffer comprising histidine ions. Examples of histidine buffers include histidine-acetic acid buffer, histidine-hydrochloric acid buffer, histidine-histidine hydrochloride buffer, preferably, the histidine buffer is prepared by histidine (L-Histidine), and the pH is further adjusted with acetic acid or hydrochloric acid.

[0142] "Buffer" refers to a pharmaceutically acceptable agent capable of maintaining the pH of a pharmaceutical composition within a desired pH range. Suitable buffers for use in the present disclosure include phosphate buffers, acetate buffers, citrate buffers or histidine buffers. In a preferred embodiment, the buffer suitable for use in the present disclosure is a histidine buffer, which is prepared by histidine (L-Histidine) and further adjusted in pH with acetic acid or hydrochloric acid.

[0143] "Stabilizer" refers to a pharmaceutically acceptable agent used to maintain the stability of the active ingredient in the pharmaceutical composition. In the present disclosure, the stabilizer also has the function of an anti-adhesive agent and / or an isotonic agent.

[0144] "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 antibody suitable for production and administration to a patient, 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.

[0145] 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 period of time. 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) and differential scanning calorimetry (DSC), then the active ingredient "retains its physical stability" in the pharmaceutical composition. Preferably, when using the pharmaceutical composition of the present disclosure, 5% or less, 4% or less, preferably 3% or less of the active ingredient forms aggregates (also referred to as high molecular weight impurities), such as by SEC-UPLC 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.). If the biological activity of the active ingredient (e.g., antibody) at a given time is within a predetermined range of biological activity exhibited when the pharmaceutical composition is prepared, then the active ingredient "retains its biological activity" in the pharmaceutical composition for a given time, such as determined by an antigen binding assay. Other methods for evaluating the stability of pharmaceutical compositions are also described in the following examples, such as using a HVROC-S viscometer to measure viscosity. Preferably, when the pharmaceutical composition of the present disclosure exhibits a viscosity of about 20 mPa·s, about 19 mPa·s, about 18 mPa·s, about 15 mPa·s or less, it is considered to have a low viscosity.

[0146] "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).

[0147] "Charge variant" refers to a variant 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).

[0148] 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.

[0149] "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 in the present disclosure, "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).

[0150] 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, include fractions thereof (such as tenths and hundredths of integers), unless otherwise indicated.

[0151] Throughout this disclosure, unless the context dictates otherwise, the words "comprise," "include," and "contain" will be understood to mean including the stated step or element or set of steps or elements, but not excluding any other step or element or set 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. "Consisting essentially 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 this disclosure. Thus, the phrase "consisting essentially 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.

[0152] The term "TSLP" refers to thymic stromal lymphopoietin. The term "TSLP" includes variants, isoforms, homologs, orthologs, and paralogs. For example, in some cases, antibodies specific for human TSLP protein may cross-react with TSLP proteins from species other than humans (such as monkeys). In other embodiments, antibodies specific for human TSLP protein may be completely specific for human TSLP protein and may not cross-react with proteins from other species or other types, or may cross-react with TSLP derived from certain other species but not all other species.

[0153] The term "human TSLP" refers to a TSLP protein having a human amino acid sequence, such as the amino acid sequence of human TSLP with Genbank Accession No. NP_149024.1. The terms "monkey or rhesus TSLP" and "mouse TSLP" refer to monkey and mouse TSLP sequences, respectively, such as the amino acid sequences with Genbank Accession Nos. NP_001100503.1 and NP_067342.1, respectively.

[0154] "Antibody" is intended to include full-length antibodies and any antigen-binding fragments (i.e., antigen-binding portions) or single chains thereof. Conventional full-length antibodies are glycoproteins comprising two heavy (H) chains and two light (L) chains, the heavy 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, which 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 a light chain constant region, which 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.

[0155] The "antigen-binding portion" of an antibody (or simply "antibody portion") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., TSLP 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 portion" 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 comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) 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 Fc domains are paired to form monovalent molecules (referred to as single-chain Fc (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). These single-chain antibodies are also encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments can be obtained by conventional techniques known to those skilled in the art, and can be functionally screened in the same manner as full-length antibodies.

[0156] An "isolated antibody" is an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to a TSLP protein is substantially free of antibodies that specifically bind to antigens other than the TSLP protein). However, an isolated antibody that specifically binds to a human TSLP protein may have cross-binding properties to other antigens, such as TSLP proteins of other species. In addition, an isolated antibody is substantially free of other cellular materials and / or chemicals.

[0157] "Mouse antibody" refers to an antibody including an antibody 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 of the present disclosure 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" as used herein does not include antibodies in which CDR sequences from other mammalian species are inserted into mouse framework region sequences.

[0158] "Chimeric antibodies" refer to antibodies that are obtained by combining non-human genetic material with human genetic material. Or more generally, chimeric antibodies refer to antibodies that combine genetic material from one species with genetic material from another species.

[0159] "Humanized antibodies" are antibodies that are derived from non-human species but whose protein sequences have been modified to increase their similarity to naturally occurring antibodies in humans.

[0160] "Isotype" refers to the antibody class (eg, IgM or IgGl) encoded by the heavy chain constant region genes.

[0161] "An antibody that recognizes an antigen" and "antibody specific for an antigen / antibody having specificity for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen."

[0162] An antibody that "specifically binds to human TSLP" refers to an antibody that binds to human TSLP protein (and possibly TSLP protein of one or more non-human species) but does not substantially bind to non-TSLP proteins. Preferably, the antibody binds to human TSLP protein with "high affinity", i.e., K D The value is 5.0×10 -8 M or less, preferably 1.0×10 -8 M or less, more preferably 7.0×10 -9 M or smaller.

[0163] "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., the K of the binding protein or cell is D The value is 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 -3 M or more, more preferably 1.0×10 -2 M or larger.

[0164] For IgG antibodies, "high affinity" refers to the KD The value is 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 less. However, for other antibody isotypes, “high affinity” binding may be different. For example, “high affinity” binding for an IgM isotype refers to a K of D Value is 10 -6 M or less, preferably 10 -7 M or less, more preferably 10 -8 M or smaller.

[0165] The term "K assoc ” or “K a ” refers to the association rate of a specific antibody-antigen interaction, and the term “K dis ” or “K d ” refers to the dissociation rate of a specific antibody-antigen interaction. The term “K D ” refers to the dissociation constant, which is obtained from K d With K a The ratio (K d / K a ) and expressed as molar concentration (M). D The K value can be determined using methods well known in the art to determine the antibody K 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.

[0166] The term “EC 50 ”, also known as the half-maximal effect concentration, refers to the antibody concentration that induces a response halfway between baseline and maximum after a specific exposure time.

[0167] The term "IC50", also known as half maximal inhibitory concentration, refers to the concentration of an antibody that inhibits a specific biological or biochemical function by 50% relative to the absence of the antibody.

[0168] "Subject" includes any human or non-human animal. "Non-human animals" include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians and reptiles; although mammals, such as non-human primates, sheep, dogs, cats, cows and horses are preferred.

[0169] The term "therapeutically effective amount" refers to an amount sufficient to prevent or improve symptoms associated with a disease or condition (e.g., a TSLP-related disease), 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 determine the actual effective amount.

[0170] Anti-TSLP Antibodies in the Pharmaceutical Compositions of the Disclosure

[0171] The anti-TSLP antibody or antigen-binding portion thereof in the pharmaceutical composition of the present disclosure may be an antibody with structural and chemical characteristics as described below and in the Examples. The amino acid SEQ ID NOs of the heavy / light chain variable regions of the antibodies are summarized in Table 1 below, and some antibodies have the same VH or VL. The heavy chain constant region of the antibody may be a human IgG1 heavy chain constant region having an amino acid sequence as shown in SEQ ID NOs: 12 or 37, or 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 light chain constant region having an amino acid sequence as shown in SEQ ID NO: 14. These antibodies may also contain a mouse IgG1 or IgG2 heavy chain constant region, and / or a mouse κ light chain constant region. The antibody may be composed of two heavy chains and two light chains connected by a disulfide bond, the C-terminus of the heavy chain variable region being connected to the N-terminus of the heavy chain constant region, and the C-terminus of the light chain variable region being connected to the N-terminus of the light chain constant region.

[0172] 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.

[0173] The VH and VL sequences (or CDR sequences) of other anti-TSLP antibodies that bind to human TSLP can be "mixed and paired" with the VH and VL sequences (or CDR sequences) of the anti-TSLP antibodies in the pharmaceutical compositions of the present disclosure. Preferably, when the VH and VL chains (or CDRs in these chains) are mixed and paired, the VH sequence from a particular VH / VL pair is replaced by a structurally similar VH sequence. Similarly, it is preferred that the VL sequence from a particular VH / VL pair is replaced with a structurally similar VL sequence.

[0174] Thus, in one embodiment, the anti-TSLP antibody or antigen-binding portion thereof in the pharmaceutical composition of the present disclosure comprises:

[0175] (a) a heavy chain variable region comprising the amino acid sequence listed in Table 1; and

[0176] (b) a light chain variable region comprising the amino acid sequence listed in Table 1, or VL of another anti-TSLP antibody, wherein the antibody specifically binds to human TSLP.

[0177]

[0178] In another embodiment, the anti-TSLP antibody or antigen-binding portion thereof in the pharmaceutical composition of the present disclosure comprises:

[0179] (a) CDR1, CDR2 and CDR3 of the heavy chain variable region listed in Table 1; and

[0180] (b) CDR1, CDR2 and CDR3 of the light chain variable region listed in Table 1, or CDRs of another anti-TSLP antibody, wherein the antibody specifically binds to human TSLP.

[0181] In another embodiment, the anti-TSLP antibody or antigen-binding portion thereof in the pharmaceutical composition of the present disclosure includes the heavy chain variable region CDR2 of the anti-TSLP antibody and the CDRs of other antibodies that bind to human TSLP, 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-TSLP antibody.

[0182] It is well known in the art that the CDR3 domain can alone 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 a common CDR3 sequence.

[0183] In another embodiment, the anti-TSLP antibody in the pharmaceutical composition of the present disclosure comprises CDR2 of the heavy chain variable region of the anti-TSLP antibody and CDR3 of at least the heavy chain variable region and / or light chain variable region of the anti-TSLP antibody, or CDR3 of the heavy chain variable region and / or light chain variable region of another anti-TSLP antibody, wherein the antibody can specifically bind to human TSLP. These antibodies preferably compete with the anti-TSLP antibody in the pharmaceutical composition of the present disclosure for binding to TSLP; (b) retain functional properties; (c) bind to the same epitope; and / or (d) have similar binding affinity. In another embodiment, the anti-TSLP antibody in the pharmaceutical composition of the present disclosure may also comprise CDR2 of the light chain variable region of the anti-TSLP antibody, or CDR2 of the light chain variable region of another anti-TSLP antibody, wherein the antibody specifically binds to human TSLP. In another embodiment, the anti-TSLP antibody in the pharmaceutical composition of the present disclosure may further include the heavy chain variable region and / or light chain variable region CDR1 of an anti-TSLP antibody, or the heavy chain variable region and / or light chain variable region CDR1 of another anti-TSLP antibody, wherein the antibody specifically binds to human TSLP.

[0184] Conservative modification

[0185] In another embodiment, the anti-TSLP antibody in the pharmaceutical composition of the present disclosure comprises a CDR1, CDR2, and CDR3 sequence that is different from the heavy chain variable region and / or light chain variable region of the anti-TSLP antibody in the pharmaceutical composition of the present disclosure, and the difference is due to one or more conservative modifications. It should be understood in the art that some 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; Hall et al. 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.

[0186] Therefore, in one embodiment, the anti-TSLP antibody in the pharmaceutical composition 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:

[0187] (a) the heavy chain variable region CDR1 comprises the sequence listed in Table 1, and / or conservative modifications thereof; and / or

[0188] (b) the heavy chain variable region CDR2 comprises the sequence listed in Table 1, and / or conservative modifications thereof; and / or

[0189] (c) the heavy chain variable region CDR3 comprises the sequence listed in Table 1, and / or conservative modifications thereof; and / or

[0190] (d) the light chain variable region CDR1, and / or CDR2, and / or CDR3 comprises the sequence listed in Table 1, and / or conservative modifications thereof; and

[0191] (e) The antibody specifically binds to human TSLP.

[0192] The anti-TSLP antibodies in the pharmaceutical compositions of the present disclosure have one or more of the following functional properties, such as high affinity for human TSLP.

[0193] In various embodiments, the anti-TSLP antibodies in the pharmaceutical compositions of the present disclosure can be, for example, mouse, human, chimeric, or humanized antibodies.

[0194] The term "conservative sequence modification" as used herein refers to an amino acid modification that does not significantly affect or change the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the anti-TSLP antibodies in the pharmaceutical compositions of the present disclosure by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions are amino acid residues that are replaced with amino acid residues having similar side chains. Groups of amino acid residues having similar side chains are known in the art. Therefore, one or more amino acid residues in the CDR region of the anti-TSLP antibodies in the pharmaceutical compositions of the present disclosure can be replaced with other amino acid residues from the same side chain group, and the resulting antibodies can be functionally tested using the functional assays described herein.

[0195] The preparation and purification methods of anti-TSLP antibodies in the present disclosure have been recorded in the patent document with application number PCT / CN2020 / 113289, and the entire contents of the aforementioned application document are introduced into the present disclosure.

[0196] For the sake of clarity, the present disclosure is further illustrated by examples, but the examples do not limit the scope of the present disclosure. The anti-TSLP antibody 1C5F12E9 in the examples is the antibody 1C5F12E9 in PCT / CN2020 / 113289, wherein the mouse anti-TSLP antibody 1C5F12E9 is produced by hybridoma technology according to the method described in PCT / CN2020 / 113289 and obtained after in vitro functional screening.

[0197] Example 1 Humanization of anti-TSLP mouse monoclonal antibody

[0198] The mouse anti-TSLP antibody 1C5F12E9 was humanized. The humanization of the mouse antibody was performed using an established CDR transplantation method, as described below. In order to select the acceptor framework for the humanization of the mouse antibody 1C5F12E9, the sequences of the antibody light chain variable region and the heavy chain variable region were BLASTed with the human immunoglobulin gene database. The human germline antibody with the highest homology to the mouse antibody 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 mutated to obtain more candidate heavy / light chain variable regions. A total of 18 exemplary humanized 1C5F12E9 antibodies, i.e., hu1C5F12E9-V1 to hu1C5F12E9-V18, were obtained, and their heavy / light chain variable region sequences are shown in Table 1.

[0199] A vector encoding a humanized 1C5F12E9 heavy chain variable region and a human IgG4 heavy chain constant region (SEQ ID NO: 13) and a vector encoding a humanized 1C5F12E9 light chain variable region and a human κ light chain constant region (SEQ ID NO: 14) were constructed and transiently transfected into 50 mL 293F suspension cells using 1 mg / mL PEI at a ratio of 60%:40% light chain construct: heavy chain construct. After six days of culture in a shake flask, the cell supernatant containing the humanized antibodies was collected, the cells in the supernatant were precipitated by centrifugation, and then the above 18 antibodies were purified from the cell supernatant.

[0200] Example 2 Characterization of Exemplary Humanized Anti-TSLP Monoclonal Antibodies

[0201] The binding affinity and binding kinetics of purified exemplary humanized 1C5F12E9 antibodies to human TSLP were evaluated by the Biacore T200 system (GE Healthcare, Pittsburgh, PA, USA).

[0202] Briefly, in-house synthesized recombinant human TSLP-his (SEQ ID NO: 28) or cynomolgus monkey TSLP-his protein (SEQ ID NO: 29) was dissolved in CH3COONa buffer (provided by Biocore) at a final concentration of 10 μg / mL and then covalently linked to a CM5 chip (carboxymethylated dextran coated chip, GE Healthcare #BR100530) 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, gradient dilutions of purified humanized 1C5F12E9 antibody (2-fold gradient dilutions in HBS-EP+ buffer with a starting concentration of 100 nM) and reference Tezepelumab (also known as TSLP-BM, prepared in-house using the heavy and light chains shown in SEQ ID NOs: 35 and 36, 2-fold gradient dilutions in HBS-EP+ buffer with a starting concentration of 100 nM) were flowed through the chip at a flow rate of 50 μL / min. The antigen-antibody binding kinetics were tracked for 4 minutes, and the dissociation kinetics were tracked for 13 minutes. The binding and dissociation curves were fitted to a 1:1 Langmuir binding model using BIAcore evaluation software, and the KD, Ka, and Kd values ​​were determined. The results are shown in Table 2 below.

[0203] Table 2. Binding affinity of humanized 1C5F12E9 monoclonal antibody

[0204]

[0205] The lower limit of the Kd value measured by Biacore is 1.00E-05, and the Kd value below 1.00E-05 can be roughly calculated from the corresponding sensorgram. The results show that the binding affinity of all humanized 1C5F12E9 antibodies to human TSLP is higher than that of Tezepelumab.

[0206] Example 3 Characterization of humanized anti-TSLP antibodies hu1C5F12E9-V8 and hu1C5F12E9-V14

[0207] Humanized antibodies hu1C5F12E9-V8 and hu1C5F12E9-V14 were selected for further characterization. Specifically, their binding affinity / capacity for human and cynomolgus monkey TSLP and other functions were determined by Biacore, capture ELISA, indirect ELISA, competitive ELISA, cell-based ligand blocking FACS and cell-based functional assays according to the methods in Example 2 and below. The results are shown in Tables 3, 4 and 5. Figure 1-5 and 6A-6B.

[0208] For capture ELISA, 2 μg / mL affinity purified goat anti-human IgG antibody (Jackson ImmunoResearch, 109-005-098) dissolved in PBS was coated in 96-well plates, 100 μL / well, and incubated overnight at 4°C. The plates were washed 4 times with washing buffer (PBS+0.05% Tween-20, PBST), and then 200 μL of blocking buffer (PBST containing 5% w / v skim milk) was added to each well and blocked for 2 h at 37°C. The plates were washed again, and 100 μL / well of serial dilutions (starting at 66.7 nM, 5-fold serial dilutions in PBST containing 2.5% skim milk) of the humanized anti-TSLP antibody, Tezepelumab or hIgG (Hualan Biological Engineering Inc.) of the present disclosure were added, incubated at 37°C for 40 min, and then the plates were washed 4 times again. 100 μL / well of biotinylated human TSLP-his protein (SEQ ID NO: 28, prepared in-house, dissolved in PBST containing 2.5% skim milk, final concentration 0.23 nM) was added to the 96-well plate with capture antibody, incubated at 37°C for 40 min, washed 4 times, and then 100 μL of HRP-labeled streptavidin (diluted 1:10000 in PBST, Jackson ImmunoResearch, 016-030-084) was added to each well and incubated at 37°C for 40 min. After the last wash, 100 μL / well of TMB (Innoreagents) was added to the plate for incubation. After 15 min, 50 μL / well of 1M H2SO4 was added at room temperature to terminate the reaction, and the absorbance of each well was read on a microplate reader using dual wavelength mode (TMB detection wavelength 450 nm, reference wavelength 630 nm). The OD (450-630) values ​​and the corresponding antibody concentrations were plotted. The data were analyzed using Graphpad Prism software and the EC50 values ​​were obtained. Figure 1 shown.

[0209] For indirect ELISA, 2 μg / mL cynomolgus monkey TSLP-his protein (SEQ ID NO: 29, prepared in-house) dissolved in carbonate / bicarbonate buffer (pH 9.6) was coated in a 96-well plate, 100 μL / well, and incubated overnight at 4°C. The plate was washed 4 times with washing buffer (PBS+0.05% Tween-20, PBST), and then 200 μL of blocking buffer (PBST containing 5% w / v skim milk) was added to each well and blocked at 37°C for 2 h. The plate was washed again, and 100 μL / well of serial dilutions (starting at 66.7 nM, 5-fold serial dilutions in PBST containing 2.5% skim milk) of the humanized anti-TSLP antibody, Tezepelumab or hIgG of the present disclosure was added and incubated at 37°C for 40 min. Wash the plate again 4 times, and add 100 μL / well of peroxidase-labeled affinity-purified F(ab')2 fragmented goat anti-human IgG antibody (Jackson Immunoresearch, 109-036-098) and incubate at 37°C for 40 min. After the last wash, add 100 μL / well of TMB (Innoreagents) for incubation. After 15 min, add 50 μL / well of 1M H2SO4 at room temperature to terminate the reaction, and read the absorbance of each well on a microplate reader using dual wavelength mode (TMB detection wavelength 450 nm, reference wavelength 630 nm). Plot the OD (450-630) value and the corresponding antibody concentration. Use Graphpad Prism software to analyze the data and obtain the EC50 value. The results of some antibodies are shown in Figure 2. Figure 2 shown.

[0210] The ability of humanized anti-TSLP antibodies to block the binding of TSLP to TSLPR / IL7R was determined using a competitive ELISA. Briefly, TSLPR-Fc protein (SEQ ID NO: 30, prepared in-house) was dissolved in PBS at a final concentration of 1 μg / mL, and IL7Ra-Fc protein (SEQ ID NO: 31, prepared in-house) was dissolved in PBS at a final concentration of 1 μg / mL. The two solutions (100 μL each) were coated in a 96-well plate and incubated overnight at 4°C. The next day, the plate was washed with wash buffer (PBS + 0.05% Tween-20, PBST), and PBST containing 5% w / v skim milk was added and blocked at 37°C for 2 h. Then, the plate was washed with wash buffer. Humanized anti-TSLP antibody or control was diluted with biotin-labeled human TSLP-Fc (SEQ ID NO: 32, prepared in-house, dissolved in PBST containing 2.5% skim milk, final concentration 0.29 nM), starting at 66.7 nM, and diluted 3-fold, and incubated at room temperature for 40 min. Then 100 μL / well of the antibody / TSLP-Fc mixture was added to the plate coated with TSLPR / IL7R. After incubation at 37°C for 40 min, the plate was washed 4 times with wash buffer. HRP-labeled streptavidin was then added and incubated at 37°C for 40 min to detect biotin-labeled human TSLP-Fc bound to TSLPR / IL7R. The plate was washed again with wash buffer. Finally, TMB was added and the reaction was terminated with 1M H2SO4. The absorbance of each well was read on a microplate reader using dual wavelength mode (TMB detection wavelength 450nm, reference wavelength 630nm), and then the OD (450-630) value and the corresponding antibody concentration were plotted. The data were analyzed using GraphpadPrism software and the IC50 value was obtained. The results for some antibodies are shown in Figure 2. Figure 3 shown.

[0211] The ability of humanized anti-TSLP antibodies to block the binding of a reference substance (Tezepelumab) to human TSLP was determined using a competitive ELISA. Briefly, 2 μg / mL Tezepelumab dissolved in PBS 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% Tween-20, PBST), and PBST containing 5% w / v skim milk was added and blocked at 37°C for 2 h. At the same time, anti-TSLP antibodies or reference substances were diluted with biotin-labeled human TSLP-Fc (SEQ ID NO: 32, dissolved in PBST containing 2.5% skim milk, final concentration 0.047 nM), and 4-fold serial dilutions were made starting at 40 nM, and incubated at room temperature for 40 min. Then, 100 μL / well of the antibody / TSLP-Fc-biotin mixture was added to the 96-well plate coated with the reference substance. After incubation at 37°C for 40 min, wash the plate 4 times with wash buffer. Then add HRP-labeled streptavidin and incubate at 37°C for 40 min to detect biotin-labeled human TSLP-Fc bound to the reference. Finally, wash the plate with wash buffer. Add TMB and terminate the reaction with 1M H2SO4. Read the absorbance of each well on a microplate reader using dual wavelength mode (TMB detection wavelength 450nm, reference wavelength 630nm), and then plot the OD (450-630) value and the corresponding antibody concentration. Analyze the data using Graphpad Prism software and obtain the IC50 value. The results for some antibodies are shown in Figure 2. Figure 4 shown.

[0212] Cell-based ligand blocking FACS, using the cell line BAF3-3E6 expressing human TSLPR (amino acid residues 1-371 of Uniprot No. Q9HC73.1, SEQ ID NO: 33) and human IL7R (amino acid residues 1-459 of Uniprot No. P16871.1, SEQ ID NO: 34) on the cell surface, flow cytometry (FACS) was used to evaluate the activity of humanized anti-TSLP antibodies in blocking the binding of TSLP-Fc protein to cell surface TSLPR / IL7R. BAF3-3E6 cell line was prepared by transfecting BAF3 cells (iCell Bioscience Inc., MIMCL-021) with recombinant plasmid pCMV-TP (TSLPR coding sequence inserted between EcoRI and Xbal sites) and recombinant plasmid pCMV3-SP (IL7R coding sequence inserted between HindIII and Xbal) according to the instructions of lipofectamine 3000 transfection reagent (Thermo Fisher). Briefly, the anti-TSLP antibodies of the present disclosure, references or negative control hIgG (human immunoglobulin for intravenous injection (pH 4) (Hualan Biological Engineering Inc.) were diluted with human TSLP-Fc solution (SEQ ID NO: 32, prepared in-house, dissolved in FACS buffer, final concentration of 0.38 nM) and serially diluted 2-fold starting at 30 nM and incubated at room temperature for 40 min. BAF3-3E6 cells were harvested from cell culture flasks, washed twice, and then resuspended in phosphate-buffered saline (PBS) containing 2% v / v fetal bovine serum (FACS buffer). In a solution containing 1×10 5 100 μL / well of antibody / TSLP-Fc-biotin mixture was added to 96-well plates with 10 cells / well and incubated at 4°C for 40 min. The cells were washed twice with FACS buffer, and then 100 μL / well of R-phycoerythrin-labeled streptavidin (diluted 1:1000 in FACS buffer, Jackson Immunoresearch, 016-110-084) was added and incubated at 4°C in the dark for 40 min. The cells were washed twice and resuspended in FACS buffer. Fluorescence values ​​were measured using a Becton Dickinson FACS Canto II-HTS. Data were analyzed using Graphpad Prism software and IC values ​​were obtained. 50 The results for some antibodies are as follows Figure 5 shown.

[0213] The proliferation and survival of BAF3 cells are normally dependent on IL-3. However, when these cells are engineered to express human TSLPR and human IL7R, and TSLP is added to the cell culture medium, they can survive in the absence of IL-3. The inhibitory activity of humanized anti-TSLP antibodies on the proliferation of BAF3-3E6 cells expressing TSLPR (SEQ ID NO: 33) / IL7R (SEQ ID NO: 34) was further tested by a cell-based functional assay. Briefly, 100 μL of 8×10 3 RPMI1640 medium (Gibco, A10491-01) of BAF3-3E6 cells in the logarithmic growth phase was inoculated into a 96-well plate, wherein the RPMI1640 medium contained 10% FBS (Gibco, A10099-141). Then, 50 μL of human TSLP-his protein (SEQ ID NO: 28, prepared in-house, dissolved in RPMI-1640, final concentration of 6.4 ng / mL) was mixed with 50 μL of humanized anti-TSLP antibody or control (5-fold gradient dilution in the medium with a starting concentration of 40 μg / mL), and the mixture was incubated at room temperature for 30 minutes. Then, 100 μL / well of the antibody / TSLP-his mixture was added to the 96-well plate containing BAF3-3E6 cells and cultured in a 37°C, CO2-containing incubator for 72 hours. Thereafter, the 96-well plate containing cells was placed in a Cell 100 well plate. The cells were incubated at 37°C for 10 min using a luminescent cell viability assay kit (Promega, G7572, 50 μL / well). 200Pro was used to measure the chemiluminescence value. Graphpad Prism software was used to analyze the data and obtain the IC 50 The results for some antibodies are as follows Figure 6A-6B shown.

[0214] The data demonstrate that hu1C5F12E9-V8 and hu1C5F12E9-V14 display in vitro activity comparable to that of the parental mouse and chimeric antibodies.

[0215] As shown in Table 3, Figure 1 and Figure 2 As shown, compared with the reference, the humanized antibodies hu1C5F12E9-V8 and hu1C5F12E9-V14 have higher binding affinity / activity to human TSLP and are comparable to the binding affinity / ability to cynomolgus monkey TSLP.

[0216] Figure 3 and 5It was shown that humanized antibodies hu1C5F12E9-V8 and hu1C5F12E9-V14 were able to block the binding of human TSLP to human TSLPR / IL7R.

[0217] Figure 6A-6B The results showed that humanized antibodies hu1C5F12E9-V8 and hu1C5F12E9-V14 were able to block the interaction between TSLP and TSLPR / IL7R, leading to blockade of the TSLP pathway and death of BAF3-3E6 cells at low antibody concentrations, while the reference product required high antibody levels to exert such efficacy.

[0218] Table 3. Binding affinities of monoclonal antibodies hu1C5F12E9-V8 and hu1C5F12E9-V14

[0219]

[0220]

[0221] Then, according to the methods described in Examples 2-3 and the following methods, the humanized antibodies hu1C5F12E9-V8 and hu1C5F12E9-V14 having a human IgG1 heavy chain constant region (SEQ ID NO: 12) and a human κ light chain constant region (SEQ ID NO: 14) were compared with hu1C5F12E9-V8 and hu1C5F12E9-V14 having a human IgG4 heavy chain constant region (SEQ ID NO: 13) and a human κ light chain constant region (SEQ ID NO: 14) for binding affinity / capacity to human and cynomolgus monkey TSLP and other functions by Biacore, capture ELISA, competition ELISA, cell-based ligand blocking FACS, cell-based functional assay, cell-based reporter gene assay and protein thermal shift assay. The results are shown in Figure 2-3. Figure 7-10 , 11A-11B, 12A-12B and 13.

[0222] To determine the thermal stability of four anti-TSLP humanized antibodies, GloMelt TM Thermal Shift Protein Stability Kit (Biotium, 33022-T) was used to determine the Tm (melting temperature) of proteins by thermal shift assay. TMThe 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, LightCycler 480II), and the parameters of the Melt Curve program in the thermal cycler are shown in Table 4.

[0223] Table 4. Parameters of the Melt Curve program

[0224] step temperature Heating rate Duration Initial hold 25℃ NA 30s Melt curve 25-99℃ 0.1℃ / s NA

[0225] In the cell-based reporter gene assay, the reporter gene cell line HEK293T-TSLPR / IL7R / STAT5-Luc expressing human TSLPR (SEQ ID NO: 33) and human IL7R (SEQ ID NO: 34) on the cell surface was used. According to the instructions of lipofectamine 3000 transfection reagent (Thermo Fisher), HEK293T cells were transfected with recombinant plasmid pCMV-TP (TSLPR coding sequence inserted between EcoRI and Xbal sites), recombinant plasmid pCMV3-SP (IL7R coding sequence inserted between HindIII and Xbal) and pGL4.52 [luc2P / STAT5RE / Hygro] (Promega) to obtain the target gene. CRL-11268), HEK293T-TSLPR / IL7R / STAT5-Luc cells were prepared in-house. Briefly, HEK293T-TSLPR / IL7R / STAT5-Luc cells were harvested from cell culture flasks. Then, 100 μL of 5×10 4DMEM medium (Gibco, 10566-016) containing 10% FBS (Gibco, 10099-141) of 10 cells was inoculated into a 96-well cell culture plate (Corning, 30218026). At the same time, 50 μL of human TSLP-his (SEQ ID NO: 28, dissolved in DMEM medium containing 10% FBS, with a final concentration of 160 ng / mL) was mixed with 50 μL of serially diluted anti-TSLP antibodies hu1C5F12E9-V8 (IgG1), hu1C5F12E9-V8 (IgG4), hu1C5F12E9-V14 (IgG1), hu1C5F12E9-V14 (IgG4) and Tezepelumab (5-fold serial dilution in DMEM medium containing 10% FBS with a starting concentration of 200 μg / mL) and incubated at room temperature for 30 min. Then, 100 μL / well of the anti-TSLP antibody / TSLP-his mixture was added to a 96-well cell culture plate and cultured in a 37°C, CO2-containing incubator for 16-18 hours. 100 μL of supernatant was discarded from each well, and then 50 μL / well of luciferase detection reagent (Promega, E6120) was added. After 10 minutes, the plate was analyzed using a Tecan Infinite 200Pro plate reader. The data of the luminescence signal was analyzed using Graphpad prism software and the IC50 value was obtained.

[0226] like Figure 7 , 8 , 10, 11A-11B and 12A-12B, hu1C5F12E9-V8(IgG1), hu1C5F12E9-V8(IgG4), hu1C5F12E9-V14(IgG1) and hu1C5F12E9-V14(IgG4) have comparable or better in vitro activities than Tezepelumab. In particular, Figures 11A-11B As shown in Figures 12A-12B, the humanized antibody was able to block the interaction between TSLP and TSLPR / IL7R at an antibody concentration much lower than that of the reference, thereby leading to the blockade of the TSLP pathway and the death of BAF3-3E6 cells.

[0227] like Fig.13 As shown, the melting temperatures (T1, T2) of hu1C5F12E9-V8(IgG1), hu1C5F12E9-V8(IgG4), hu1C5F12E9-V14(IgG1) and hu1C5F12E9-V14(IgG4) are (69.5°C, 80°C), (66.5°C, 76°C), (69.5°C, 80°C) and (66.5°C, 76°C), respectively.

[0228] Example 4 Screening of pharmaceutical compositions

[0229] Size exclusion chromatography (SEC-UPLC): used to determine the purity of antibodies in samples, using Thermo Vanquish F high performance liquid chromatography, Waters ACQUITY UPLC Protein BEH SEC Column Gel chromatography column, Waters ACQUITY UPLC Protein BEH SEC Guard Column The pre-column was prepared and eluted with 50mmol / L phosphate buffer-200mmol / L sodium chloride solution (pH 7.0) as the mobile phase, and the detection wavelength was 280nm. The percentage of high molecular impurities and immunoglobulin monomers was calculated by area normalization method.

[0230] Differential scanning calorimetry (DSC): MicroCal VP-Capillary DSC (Malvern) was used to analyze the unfolding temperature (Tm) of the samples. The samples were diluted to a concentration of 1 mg / mL with the following program: the scan start temperature was 20 °C, the scan end temperature was 110 °C, and the heating rate was 60 °C / h.

[0231] The aggregation temperature (Tagg) of the sample was detected using Dynaproplate ReaderⅢ (Wyatt). The sample was added to a 384-well sample plate, sealed with a sealing film, and then the 384-well sample plate was centrifuged to remove bubbles. The test was then performed using the following procedure: the temperature was raised from 35°C to 85°C, and the scanning time was 5 seconds.

[0232] The viscosity of the sample was analyzed using a HVROC-S viscometer (Rheosense), the Auto mode was selected, and the test results were recorded.

[0233] Capillary isoelectric focusing (CIEF): used to determine the charge variants of the sample, using a ProteinSimple CE3 imaging capillary isoelectric focusing electrophoresis instrument, Maurice fast fully automatic protein characterization analysis system, ProteinSimple coated quartz capillaries, and a UV detector, with detection at a UV detection wavelength of 280 nm. Set the injection time to 60 seconds, pre-focus at 1500V for 1 minute, and focus at 3000V for 5 minutes. Calculate the peak area percentage of the charge variant by peak area normalization method.

[0234] CE-SDS reduction electrophoresis: Beckman Coulter Pa 800Plus biopharmaceutical analysis system, non-coated fused silica capillary with an inner diameter of 50 μm, a total length of 31 cm, and an effective length of 21 cm, PDA detector, and detection at a detection wavelength of 220 nm. The corrected peak area percentages of light chain, heavy chain, and non-glycosylated heavy chain were calculated by area normalization. CE-SDS non-reducing electrophoresis: Beckman Coulter Pa800Plus biopharmaceutical analysis system, non-coated fused silica capillary with an inner diameter of 50 μm, a total length of 31 cm, and an effective length of 21 cm, PDA detector, and detection at a detection wavelength of 220 nm. The corrected peak area percentages of the main peak were calculated by area normalization.

[0235] The biological activity was detected by enzyme-linked immunosorbent assay (ELISA). 2 μg / mL TSLP protein (Sinobiological, 16135-H08H) was coated in a 96-well plate, 100 μL / well, and incubated at 2-8°C overnight. After washing the 96-well plate, 250 μL of blocking solution (PBS solution containing 3% BSA) was added to each well and incubated at 25°C for 2 hours. After washing the 96-well plate, the reference sample and the test sample (initial concentration of 4000 ng / mL, 4-fold gradient dilution, a total of 7 dilutions) were added and incubated at 25°C for 2 hours, wherein the reference sample is a representative pharmaceutical composition sample of the process, and the test sample is a pharmaceutical composition sample to be tested, and the preparation method is as follows. After washing the 96-well plate, HRP-labeled goat anti-human antibody (PekinElmer, NEF802001EA, diluted according to the instructions) was added at 100 μL / well and incubated at 25°C for 1 hour. After washing the 96-well plate, add 100 μL TMB to each well and incubate at 25°C in the dark for 5 minutes. Finally, add 1M H2SO4 to stop the color development. After standing at room temperature for 5 minutes, use an ELISA reader to detect the absorbance at 450nm with 650nm as the reference wavelength and record the measurement results. Test sample biological activity (%) = (reference sample EC50 value / test sample EC50 value) × 100%

[0236] Preparation of buffer: Sodium phosphate buffer is prepared from sodium dihydrogen phosphate monohydrate (NaH2PO4·H2O) and disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), for example, 20mM sodium phosphate buffer (pH6.0) is prepared from about 2.54g / L NaH2PO4·H2O and about 0.573g / L Na2HPO4·12H2O; Sodium acetate buffer is prepared from anhydrous sodium acetate and acetic acid, for example, 20mM sodium acetate buffer (pH6.0) is prepared from about 1.58g / L anhydrous sodium acetate and about 0.116g / L acetic acid; Histidine buffer is prepared from histidine (L-Histidine) and adjusted to the target pH with acetic acid, for example, 20mM histidine buffer (pH6.0) is prepared from about 3.1g / L histidine, and the pH is adjusted to 6 with acetic acid.

[0237] By ultrafiltration centrifugation, the anti-TSLP antibody (hu1C5F12E9-V8 (IgG1) ', having a human IgG1 heavy chain constant region shown in SEQ ID NO: 37 and a human κ light chain constant region shown in SEQ ID NO: 14) was replaced into the buffer selected in Table 5, and concentrated after the replacement was completed. Excipients were added according to the pharmaceutical composition selected in Table 5. After mixing evenly, the mixture was sterilized by filtration through a 0.22 μm filter membrane. The Tm values, Tagg values ​​and SEC-UPLC test results of different pharmaceutical compositions are shown in Table 5.

[0238] Table 5. Tm, Tagg, and SEC-UPLC test results of the screened pharmaceutical compositions

[0239]

[0240]

[0241] Exemplarily, histidine buffer was selected as a buffer for further screening, and the anti-TSLP antibody (hu1C5F12E9-V8 (IgG1) ', having a human IgG1 heavy chain constant region shown in SEQ ID NO: 37 and a human κ light chain constant region shown in SEQ ID NO: 14) was replaced into the histidine buffer screened in Table 6 by ultrafiltration centrifugation, and concentrated after the replacement was completed, and excipients were added according to the screened pharmaceutical composition in Table 6. After mixing evenly, sterilization was performed by filtering through a 0.22 μm filter membrane. The Tm value, Tagg value, viscosity and SEC-UPLC test results of different pharmaceutical compositions are shown in Table 6.

[0242] Table 6. Tm, Tagg, viscosity and SEC-UPLC test results of the screened pharmaceutical compositions

[0243]

[0244] Exemplarily, histidine buffer is selected as a buffer for further screening, and the anti-TSLP antibody (hu1C5F12E9-V8 (IgG1) ', having a human IgG1 heavy chain constant region shown in SEQ ID NO: 37 and a human κ light chain constant region shown in SEQ ID NO: 14) is replaced into the histidine buffer screened in Table 7 by ultrafiltration centrifugation. After the replacement is completed, it is concentrated, and excipients are added according to the pharmaceutical composition screened in Table 7. After mixing evenly, filter and sterilize through a 0.22 μm filter membrane. The appearance, visible foreign matter, biological activity, viscosity and SEC-UPLC test results of different pharmaceutical compositions are shown in Table 7; the stability study results of different pharmaceutical compositions at 2-8°C are shown in Table 8.

[0245] Table 7. Appearance, visible foreign matter, biological activity, viscosity and SEC-UPLC test results of the screened pharmaceutical compositions

[0246]

[0247]

[0248] Table 8 Stability study results of the screened pharmaceutical compositions at 2-8°C

[0249]

[0250] Exemplarily, histidine buffer is selected as a buffer and proline is further screened as a stabilizer. The anti-TSLP antibody (hu1C5F12E9-V8 (IgG1) ', having a human IgG1 heavy chain constant region shown in SEQ ID NO: 37 and a human κ light chain constant region shown in SEQ ID NO: 14) is replaced into the histidine buffer (acetic acid to adjust pH to 5.8) screened in Table 9 by ultrafiltration centrifugation. After the replacement is completed, it is concentrated and the excipients are added according to the pharmaceutical composition screened in Table 9. After mixing evenly, it is sterilized by filtration through a 0.22 μm filter membrane. The viscosity, SEC-UPLC, CIEF and CE-SDS test results of different pharmaceutical compositions are shown in Table 10.

[0251] Table 9. Screened drug compositions

[0252] Element F1 F2 Anti-TSLP Antibodies 150mg / mL (150g / L) 120 mg / mL (120 g / L) Histidine 20mmol / L (3.1g / L) 20mmol / L (3.1g / L) Proline 500mmol / L(57.56g / L) 400mmol / L(46.05g / L) Polysorbate 80 0.02%w / v(0.2g / L) 0.02%w / v(0.2g / L) pH 5.8 5.8

[0253] Table 10. Viscosity, SEC-UPLC, CIEF and CE-SDS test results of the screened pharmaceutical compositions

[0254]

[0255]

[0256] Illustratively, pharmaceutical composition F2 in Table 9 was selected for stability study at 2-8°C. The results of the stability study of pharmaceutical composition F2 at 2-8°C are shown in Table 11.

[0257] Table 11. Results of stability study of the screened pharmaceutical compositions at 2-8°C

[0258]

[0259] Those skilled in the art will further appreciate that the present invention can be implemented in other specific forms without departing from its spirit and core attributes. In the foregoing description, the present invention discloses only exemplary embodiments, and it should be understood that other variations are also included within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments described in detail herein. On the contrary, with respect to the scope and content of the present invention, reference should be made to the appended claims.

Claims

1. A pharmaceutical composition comprising: (a) an anti-TSLP antibody, (b) a buffer, (c) a surfactant, and one or more (d) stabilizers, wherein: The anti-TSLP antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises a VH-CDR1 region, a VH-CDR2 region and a VH-CDR3 region, the light chain variable region comprises a VL-CDR1 region, a VL-CDR2 region and a VL-CDR3 region, wherein the VH-CDR1 region, the VH-CDR2 region, the VH-CDR3 region, the VL-CDR1 region, the VL-CDR2 region and the VL-CDR3 region respectively comprise: the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4, 5 and 6.

2. The pharmaceutical composition according to claim 1, wherein The anti-TSLP antibody comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region may comprise an amino acid sequence selected from the following: (1) as shown in SEQ ID NOs: 7 and 10, respectively; (2) as shown in SEQ ID NOs: 8 and 11 (X1=S, X2=V), respectively; (3) as shown in SEQ ID NOs: 9 (X1=R, X2=V, X3=R) and 11 (X1=S, X2=V), respectively; (4) as shown in SEQ ID NOs: 9 (X1=R, X2=V, X3=V) and 11 (X1=S, X2=V), respectively; (5) as shown in SEQ ID NOs: 9 (X1=R, X2=A, X3=R) and 11 (X1=S, X2=V), respectively; (6) as shown in SEQ ID NOs: NOs: 9 (X1 = K, X2 = A, X3 = R) and 11 (X1 = S, X2 = V); (7) as shown in SEQ ID NOs: 9 (X1 = K, X2 = A, X3 = V) and 11 (X1 = S, X2 = V); (8) as shown in SEQ ID NOs: 8 and 11 (X1 = A, X2 = I); (9) as shown in SEQ ID NOs: 9 (X1 = R, X2 = V, X3 = R) and 11 (X1 = A, X2 = I); (10) as shown in SEQ ID NOs: 9 (X1 = R, X2 = V, X3 = V) and 11 (X1 = A, X2 = I); (11) as shown in SEQ ID NOs: 9 (X1 = R, X2 = A, X3 = R) and 11 (X1 = A, X2 = I); (12) as shown in SEQ ID NOs: NOs: 9 (X1 = K, X2 = A, X3 = R) and 11 (X1 = A, X2 = I); (13) as shown in SEQ ID NOs: 9 (X1 = K, X2 = A, X3 = V) and 11 (X1 = A, X2 = I); (14) as shown in SEQ ID NOs: 8 and 11 (X1 = S, X2 = I); (15) as shown in SEQ ID NOs: 9 (X1 = R, X2 = V, X3 = R) and 11 (X1 = S, X2 = I); (16) as shown in SEQ ID NOs: 9 (X1 = R, X2 = V, X3 = V) and 11 (X1 = S, X2 = I); (17) as shown in SEQ ID NOs: NOs: 9 (X1 = R, X2 = A, X3 = R) and 11 (X1 = S, X2 = I); (18) are respectively shown in SEQ ID NOs: 9 (X1 = K, X2 = A, X3 = R) and 11 (X1 = S, X2 = I); or (19) are respectively shown in SEQ ID NOs: 9 (X1 = K, X2 = A, X3 = V) and 11 (X1 = S, X2 = I).

3. The pharmaceutical composition according to any one of claims 1 to 2, wherein The concentration of the anti-TSLP antibody is 30 mg / ml to 300 mg / ml, preferably 50 mg / ml to 250 mg / ml, preferably 70 mg / ml to 200 mg / ml, more preferably 90 mg / ml to 150 mg / ml, and most preferably 120 mg / ml to 150 mg / ml.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein The buffer includes phosphate buffer, histidine buffer, acetate buffer or citrate buffer.

5. The pharmaceutical composition according to claim 4, wherein The phosphate buffer is a sodium phosphate buffer, the acetate buffer is a sodium acetate buffer, and the citrate buffer is a sodium citrate buffer.

6. The pharmaceutical composition according to any one of claims 1-5, wherein the concentration of the buffer is 1 mM to 100 mM, preferably 2 mM to 80 mM, more preferably 5 mM to 60 mM, most preferably 10 mM to 40 mM.

7. The pharmaceutical composition according to any one of claims 1-6, wherein the surfactant comprises polysorbate 80 or polysorbate 20.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the concentration of the surfactant is 0.001% to 0.1% (w / v), preferably 0.004% to 0.08% (w / v), more preferably 0.006% to 0.06% (w / v), and most preferably 0.008% to 0.04% (w / v).

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the stabilizer comprises trehalose, mannitol, sucrose, arginine or a pharmaceutically acceptable salt thereof, glycine or proline.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the concentration of the stabilizer is 100 mM to 1000 mM, preferably 120 mM to 800 mM, more preferably 150 mM to 700 mM, most preferably 150 mM to 600 mM.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the pH of the pharmaceutical composition is 5 to 7, preferably 5.5 to 7, more preferably 5.5 to 6.

5.

12. A lyophilized preparation containing an anti-TSLP antibody, wherein the lyophilized preparation is obtained by freeze-drying the pharmaceutical composition according to any one of claims 1 to 11.

13. A lyophilized preparation containing an anti-TSLP antibody, wherein the lyophilized preparation can form the pharmaceutical composition according to any one of claims 1 to 11 after being reconstituted.

14. A product comprising a container containing the pharmaceutical composition according to any one of claims 1 to 11 or the lyophilized preparation according to any one of claims 12 to 13.

15. A method for treating and preventing TSLP-related diseases, comprising administering a therapeutically effective amount of the pharmaceutical composition of any one of claims 1 to 11, the lyophilized preparation of any one of claims 12 to 13, or the product of claim 14 to a patient in need thereof.

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