Pharmaceutical compositions of IL2 mutant-antibody Fc block fusion proteins and uses thereof

Abstract

A pharmaceutical composition of an IL2 variant-antibody Fc block fusion protein and its manufacturing method, a lyophilized formulation and its manufacturing or reconstitution method, as well as the resulting reconstituted solution, corresponding products, methods for treating autoimmune diseases and proliferative diseases, and corresponding pharmaceutical uses, wherein the pharmaceutical composition comprises a fusion protein comprising an IL2 variant and an antibody Fc block, a buffer, an osmotic regulator and a surfactant, and has good stability.

Description

[Technical field]

[0001] The present invention relates to the field of drug formulations, specifically to pharmaceutical compositions of IL2 variant-antibody Fc block fusion proteins and uses thereof. [Background technology]

[0002] Interleukin-2 (IL2) was first identified as a T cell growth factor (TCGF), and research has found that IL-2 can bind to its receptor and activate the proliferation and activation of immune cells such as T cells and NK cells. The IL-2 receptor includes the IL-2Rα subunit (CD25), the IL-2Rβ subunit (CD122) and the IL-2Rγ subunit (CD132), and among different subunits, receptor complexes with different affinities can be formed, including the high affinity receptor IL-2Rαβγ, the intermediate affinity receptor IL-2Rβγ, and the low affinity receptor IL-2Rα or IL-2Rαβ. The types of IL-2R subunits expressed in different cells are different, for example, conventional T cell CD4 in resting conditions. + T, CD8 + T cells generally express IL-2 receptor β (IL-2Rβ, CD122) and IL-2 receptor γ (IL-2Rγ, CD132) but rarely express IL-2 receptor α (IL-2Rα, CD25), whereas regulatory T cells (Treg) constitutively express high levels of IL-2Rα in addition to expressing IL-2Rβ and IL-2Rγ. Summary of the Invention [Problem to be solved by the invention]

[0003] Currently, researchers use IL2 or its variants to activate immune cells or certain subpopulations of immune cells to treat tumors or autoimmune diseases. For example, high-dose IL2 has been approved for the treatment of malignant melanoma or metastatic renal cell carcinoma, and IL2 polyethylene glycol drug conjugate NKTR-358 has been approved for clinical development of autoimmune diseases. Further developing new IL2 variants and improving their stability, productivity, and / or modifying their binding ability to certain types of receptor complexes is of great significance to the development of IL-2 drugs. At the same time, IL2 belongs to a high molecular weight protein and is prone to stability problems such as particles and aggregation in solution. Therefore, after obtaining a new IL2 variant, it is of great significance to develop a formulation system suitable for the new IL2 variant and ensure the stability of the new IL2 variant in solution.

[0004] The present invention provides pharmaceutical compositions and methods for their preparation, freeze-dried formulations and methods for their preparation or reconstitution, as well as reconstituted solutions obtained by the above reconstitution methods, corresponding pharmaceutical uses, methods and products for treating autoimmune diseases. [Means for solving the problem]

[0005] In a first aspect, the present invention provides a pharmaceutical composition comprising a fusion protein comprising an IL2 variant and an antibody Fc block, a buffer, an osmotic agent and a surfactant, Preferably, said IL2 variant comprises, compared to wild-type IL2, at least the Y31V, A73L and H79Q mutations, Preferably, the IL2 mutant further comprises one or more of the following mutations: V91R, H16E and D20A, e.g., H16E and V91R, More preferably, the pharmaceutical composition provides an IL2 variant having an amino acid sequence that has at least 80% identity, for example at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, compared to the sequence shown in any one of SEQ ID NOs: 3, 8 to 11.

[0006] The amino acid sequence of the wild-type IL2 is shown in SEQ ID NO:2.

[0007] In some specific embodiments, the fusion protein comprises, from N-terminus to C-terminus: (1) an IL2 variant, a linker and an antibody Fc block, or (2) an antibody comprising an Fc block, a linker, and an IL2 variant; Preferably, the fusion protein forms a homodimer by dimerization of the antibody Fc block, Preferably, the linker is (G4S) n wherein n is selected from 1, 2, 3, 4, 5, or 6; Preferably, the linker has an amino acid sequence having at least 80% identity, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, compared to the sequence shown in SEQ ID NO: 12, Preferably, said antibody Fc block comprises a N297G mutation, Preferably, the antibody Fc block has an amino acid sequence having at least 80% identity, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, compared to the sequence shown in SEQ ID NO: 13, More preferably, the fusion protein has an amino acid sequence having at least 80% identity, for example at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, compared to the sequences shown in SEQ ID NOs: 14 to 18.

[0008] In some specific embodiments, the wild-type IL2 has the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

[0009] In some specific embodiments, the concentration of the fusion protein is about 1 to 50 mg / mL; For example, the concentration of the fusion protein is about 1 to 40 mg / mL, 1 to 30 mg / mL, 1 to 20 mg / mL, 1 to 15 mg / mL, 1 to 10 mg / mL, 5 to 50 mg / mL, 5 to 40 mg / mL, 5 to 30 mg / mL, 5 to 20 mg / mL, 5 to 19 mg / mL, 5 to 18 mg / mL, 5 to 17 mg / mL, 5 to 16 mg / mL, 5 to 15 mg / mL, 5 to 14 mg / mL, 5 to 13 mg / mL, 5 to 16 mg / mL, 5 to 15 mg / mL, 5 to 14 mg / mL, 5 to 16 ... ~12mg / mL, 5~11mg / mL, 5~10mg / mL, 5~9mg / mL, 5~8mg / mL, 5~6mg / mL, 10~50mg / mL, 10~40mg / mL, 10~30mg / mL, 1 0~20mg / mL, 10~15mg / mL, 20~50mg / mL, 20~40mg / mL, 20~30mg / mL, 30~50mg / mL, 30~40mg / mL, 40~50mg / mL, For example, the concentration of the fusion protein may be about 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 31 mg / mL, 32 mg / mL, 33 mg / mL, 34 mg / mL, 35 mg / mL, 36 mg / mL, 37 mg / mL, 38 mg / mL, 39 mg / mL, 40 mg / mL, 41 mg / mL, 42 mg / mL, 43 mg / mL, 44 mg / mL, 45 mg / mL, 46 mg / mL, 47 mg / mL, 48 mg / mL, 49 mg / mL, 50 mg / mL, 51 mg / mL, 52 mg / mL, 53 mg / mL, 54 mg / mL, 55 mg / mL, 56 mg / mL, 57 mg / mL, 58 mg / mL, 59 mg / mL, 60 mg / mL, 61 mg / mL, 62 mg / mL, 63 mg / mL, 64 mg / mL, 65 mg / mL, 66 mg / mL, 67 mg / mL, 68 mg / mL, 69 mg / mL, 70 mg / mL, 71 mg / mL, 72 mg / mL, 73 50 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 31 mg / mL, 32 mg / mL, 33 mg / mL, 34 mg / mL, 35 mg / mL, 36 mg / mL, 37 mg / mL, 38 mg / mL, 39 mg / mL, 40 mg / mL, 41 mg / mL, 42 mg / mL, 43 mg / mL, 44 mg / mL, 45 mg / mL, 46 mg / mL, 47 mg / mL, 48 mg / mL, 49 mg / mL or 50 mg / mL, or a range of concentrations comprised between the above concentration values.

[0010] Preferably, the concentration of the fusion protein is about 2 mg / mL or 5 mg / mL.

[0011] In some specific embodiments, the buffer is selected from acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, histidine-histidine hydrochloride, succinic acid-sodium succinate, or disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, preferably acetic acid-sodium acetate buffer, and / or The concentration of the buffer solution is about 1 to 100 mM. For example, the concentration of the buffer solution may be about 1 to 90 mM, 1 to 80 mM, 1 to 70 mM, 1 to 60 mM, 1 to 50 mM, 1 to 40 mM, 1 to 30 mM, 1 to 20 mM, 1 to 10 mM, 10 to 90 mM, 10 to 80 mM, 10 to 70 mM, 10 to 60 mM, 10 to 50 mM, 10 to 40 mM, 10 to 30 mM, 10 to 20 mM, 20 to 90 mM, 20 to 80 mM, 20 to 70 mM, 20 to 60 mM, 20 to 50 mM. , 20-40mM, 20-30mM, 30-90mM, 30-80mM, 30-70mM, 30-60mM, 30-50mM, 30-40mM, 40-90mM, 40-80mM, 40-70mM, 40-60mM, 40-50mM, 50-90mM, 50-80mM, 50-70mM, 50-60mM, 60-90mM, 60-80mM, 60-70mM, 70-90mM, 80-90mM, For example, the concentration of the buffer is about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM, or a range of concentrations comprised between the above concentration values; Preferably, the concentration of the buffer is about 20 mM; and / or The pH value of the buffer solution is about 4.5 to 8.0. For example, the pH value of the buffer solution is about 4.5 to 7.5, 4.5 to 7.0, 4.5 to 6.5, 4.5 to 6.0, 4.5 to 5.5, 4.5 to 5.0, 5.0 to 8.0, 5.0 to 7.5, 5.0 to 7.0, 5.0 to 6.5, 5.0 to 6.0, 5.0 to 5.5, 5.5 to 8.0, 6 to 7.5, 6.5 to 7.5, 7.0 to 7.5, or 7.5 to 8.0; For example, the pH value of the buffer is about 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0, or a pH range comprised between the above pH values; Preferably, the pH value of the buffer is about 5.5.

[0012] In some specific embodiments, the tonicity adjusting agent is selected from a salt, an amino acid, a sugar or a sugar alcohol, or a combination thereof, preferably the salt is selected from sodium chloride, potassium chloride, or arginine hydrochloride, preferably the amino acid is selected from glycine, arginine, histidine, glutamic acid, or methionine, preferably the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol, or sorbitol; Preferably, the osmotic pressure adjusting agent is selected from sodium chloride, glycine, arginine hydrochloride, sucrose, trehalose, mannitol or sorbitol, more preferably sucrose or arginine hydrochloride; More preferably, the concentration of the sugar or sugar alcohol is about 1 to 15% w / v, and the concentration of the salt is about 50 to 200 mM. For example, the concentration of the sugar or sugar alcohol is about 1 to 10% w / v, 1 to 5% w / v, or 5 to 10% w / v. For example, the concentration of the sugar or sugar alcohol is about 1% w / v, 1.5% w / v, 2% w / v, 2.5% w / v, 3% w / v, 3.5% w / v, 4% w / v, 4.5% w / v. 5% w / v, 5.5% w / v, 6% w / v, 6.5% w / v, 7% w / v, 7.5% w / v, 8% w / v, 8.5% w / v, 9% w / v, 9.5% w / v, 10% w / v, 10.5% w / v, 11% w / v, 11.5% w / v, 12% w / v, 12.5% ​​w / v, 13% w / v, 13.5% w / v, 14% w / v, 14.5% w / v, 15% w / v, or a range of concentrations comprised between the above concentration values, preferably 4.5% w / v or 8% w / v.

[0013] More preferably, the concentration of the amino acid is about 1 to 15% w / v, for example, about 1 to 10% w / v, 1 to 5% w / v, or 5 to 10% w / v, for example, about 1% w / v, 1.5% w / v, 2% w / v, 2.5% w / v, 3% w / v, 3.5% w / v, 4% w / v, 4.5% w / v, 5% w / v, 5.5% w / v, 6% w / v, 5% w / v, 6.5% w / v, 7% w / v, 7.5% w / v, 8% w / v, 8.5% w / v, 9% w / v, 9.5% w / v, 10% w / v, 10.5% w / v, 11% w / v, 11.5% w / v, 12% w / v, 12.5% ​​w / v, 13% w / v, 13.5% w / v, 14% w / v, 14.5% w / v, 15% w / v, or a range of concentrations comprised between the above concentration values, preferably 2% w / v.

[0014] More preferably, the concentration of the salt is about 50 to 200 mM, for example, about 50 to 150 mM, 50 to 100 mM, or 100 to 150 mM, for example, about 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM, or a concentration range value comprised between the above concentration values, preferably 140 mM.

[0015] More preferably, the osmolarity adjusting agent is sucrose or arginine hydrochloride.

[0016] In some specific embodiments, the surfactant is polysorbate-80 (PS-80), polysorbate 20 (PS-20) or a poloxamer; and / or The concentration of the surfactant is about 0.01 to 0.1% w / v, for example, 0.01% w / v to 0.05% w / v, 0.05% w / v to 0.1% w / v, 0.02% w / v to 0.08% w / v, for example, about 0.01% w / v, 0.02% w / v, 0.03% w / v, 0.04% w / v, 0.05% w / v, 0.06% w / v, 0.07% w / v, 0.08% w / v, 0.09% w / v, or 1% w / v, or a concentration range value comprised between the above concentration values. Preferably, it is about 0.04% w / v.

[0017] In some specific embodiments, the pharmaceutical composition comprises the fusion protein, acetic acid-sodium acetate buffer, sucrose and polysorbate-80, preferably the pharmaceutical composition comprises about 1-50 mg / mL of the fusion protein, about 1-100 mM acetic acid-sodium acetate buffer, about 1-15% w / v sucrose and about 0.01-0.1% w / v polysorbate-80, and has a pH value of about 4.5-6.0; More preferably, the pharmaceutical composition contains about 1 to 30 mg / mL, 5 to 30 mg / mL, 10 to 30 mg / mL, 10 to 25 mg / mL, 10 to 20 mg / mL, 10 to 15 mg / mL, 15 to 20 mg / mL, 15 to 30 mg / mL or 20 to 30 mg / mL of the fusion protein, about 10 to 50 mM or 10 to 30 mM acetic acid-sodium acetate buffer, about 1 to 15% w / v, 1 to 10% w / v, 5 to 15% w / v, 5 to 10% w / v or 10 to 15% w / v of sucrose, and about 0.01 to 0.1% w / v or 0.02 to 0.06% w / v of polysorbate-80, and has a pH value of about 4.5 to 6.0 or 4.5 to 5.5, More preferably, the pharmaceutical composition contains about 1 to 10 mg / mL of the fusion protein, about 10 to 30 mM acetic acid-sodium acetate buffer, about 5 to 10% w / v sucrose, and about 0.01 to 0.1% w / v polysorbate-80, and has a pH value of about 4.5 to 6.0; More preferably, the pharmaceutical composition comprises about 2 mg / mL or 5 mg / mL of the fusion protein, about 20 mM acetic acid-sodium acetate buffer, about 8% w / v sucrose and about 0.04% w / v polysorbate-80, and has a pH of about 5.5.

[0018] In some specific embodiments, the pharmaceutical composition comprises the fusion protein, acetic acid-sodium acetate buffer, arginine hydrochloride, and polysorbate-80, and preferably comprises about 1-50 mg / mL of the fusion protein, about 1-100 mM acetic acid-sodium acetate buffer, about 50-200 mM arginine hydrochloride, and about 0.01-0.1% w / v polysorbate-80, and has a pH value of about 4.5-6.0; More preferably, the pharmaceutical composition contains about 1 to 30 mg / mL, 5 to 30 mg / mL, 10 to 30 mg / mL, 10 to 25 mg / mL, 10 to 20 mg / mL, 10 to 15 mg / mL, 15 to 20 mg / mL, 15 to 30 mg / mL or 20 to 30 mg / mL of the fusion protein, about 10 to 50 mM or 10 to 30 mM acetic acid-sodium acetate buffer, about 50 to 200 mM, 50 to 150 mM, 100 to 150 mM arginine hydrochloride, and about 0.01 to 0.1% w / v, 0.02 to 0.06% w / v polysorbate-80, and has a pH value of about 4.5 to 6.0 or 4.5 to 6.0, More preferably, the pharmaceutical composition contains about 1 to 10 mg / mL of the fusion protein, about 10 to 30 mM acetic acid-sodium acetate buffer, about 120 to 150 mM arginine hydrochloride, and about 0.01 to 0.1% w / v polysorbate-80, and has a pH value of about 4.5 to 6.0.

[0019] More preferably, the pharmaceutical composition comprises about 2 mg / mL or 5 mg / mL of the fusion protein, about 20 mM acetic acid-sodium acetate buffer, about 140 mM arginine hydrochloride, and about 0.04% w / v or 0.06% w / v polysorbate-80, with a pH value of about 5.5.

[0020] In some specific embodiments, the pharmaceutical composition is an intravenous, intramuscular or subcutaneous injection, preferably a subcutaneous injection.

[0021] In a second aspect, the present invention further provides a method for producing the pharmaceutical composition, the method comprising the step of preparing the fusion protein by mixing with the buffer, an osmotic pressure adjusting agent and a surfactant, preferably the method comprising the step of replacing a stock solution of the fusion protein with the buffer by ultrafiltration concentration.

[0022] In a third aspect, the present invention further provides a lyophilized formulation formed after lyophilizing the pharmaceutical composition described above.

[0023] In a fourth aspect, the present invention further provides a method of producing the lyophilized formulation, comprising the step of lyophilizing the pharmaceutical composition.

[0024] In a fifth aspect, the present invention further provides a method for producing a reconstituted solution of an antibody Fc block fusion protein comprising an IL2 variant, comprising reconstituting said lyophilized formulation with a solvent, preferably said solvent being water for injection.

[0025] In a sixth aspect, the present invention further provides a reconstituted solution of an antibody Fc block fusion protein comprising an IL2 variant produced by the above method.

[0026] In a seventh aspect, the present invention relates to the use of the above-mentioned pharmaceutical composition, lyophilized preparation or reconstituted solution in the manufacture of a medicament for treating an autoimmune disease or a proliferative disease, comprising: Preferably, the autoimmune disease is selected from rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjogren's syndrome, polymyositis, dermatomyositis, scleroderma, psoriasis, plaque psoriasis, alopecia areata, multiple sclerosis, amyotrophic lateral sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, graft-versus-host disease, organ transplant rejection, autoimmune hepatitis, type I diabetes, autoimmune vasculitis, eczema, or asthma; Preferably, said proliferative disease is selected from neoplasms, solid tumors, hematological tumors, malignant ascites or malignant pleural effusion, wherein said solid tumors may be benign or malignant, primary or metastatic, said malignant solid tumors may be carcinomas or sarcomas, such as epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, teratoma, lung tumor, papilloma virus-induced carcinoma, adenocarcinoma, carcinoma, melanoma, hemangiosarcoma, neuroblastoma, metastatic lung cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, Merkel cell carcinoma, ovarian cancer, renal cell carcinoma, metastatic kidney cancer, head and neck cancer, bladder cancer, non-muscle invasive bladder cancer, and said hematological tumors may be selected from leukemias, lymphomas, multiple myelomas, such as B cell lymphoma, T cell lymphoma, cutaneous T cell lymphoma, T cell large granular lymphocyte leukemia, Further uses are provided.

[0027] In an eighth aspect, the present invention relates to the above pharmaceutical composition, lyophilized preparation or reconstituted solution for treating an autoimmune disease or a proliferative disease, Preferably, the autoimmune disease is selected from rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjogren's syndrome, polymyositis, dermatomyositis, scleroderma, psoriasis, plaque psoriasis, alopecia areata, multiple sclerosis, amyotrophic lateral sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, graft-versus-host disease, organ transplant rejection, autoimmune hepatitis, type I diabetes, autoimmune vasculitis, eczema, or asthma; Preferably, said proliferative disease is selected from neoplasms, solid tumors, hematological tumors, malignant ascites or malignant pleural effusion, wherein said solid tumors may be benign or malignant, primary or metastatic, said malignant solid tumors may be carcinomas or sarcomas, such as epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, teratoma, lung tumor, papilloma virus-induced carcinoma, adenocarcinoma, carcinoma, melanoma, hemangiosarcoma, neuroblastoma, metastatic lung cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, Merkel cell carcinoma, ovarian cancer, renal cell carcinoma, metastatic kidney cancer, head and neck cancer, bladder cancer, non-muscle invasive bladder cancer, and said hematological tumors may be selected from leukemias, lymphomas, multiple myelomas, such as B cell lymphoma, T cell lymphoma, cutaneous T cell lymphoma, T cell large granular lymphocyte leukemia, Further provided is a pharmaceutical composition, lyophilized formulation or reconstituted solution as described above.

[0028] In a ninth aspect, the present invention relates to a method for treating an autoimmune disease, the method comprising administering an effective amount of the pharmaceutical composition or reconstituted solution to a subject, preferably, the autoimmune disease is selected from rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjogren's syndrome, polymyositis, dermatomyositis, scleroderma, psoriasis, plaque psoriasis, alopecia areata, multiple sclerosis, amyotrophic lateral sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, graft versus host disease, organ transplant rejection, autoimmune hepatitis, type I diabetes, autoimmune vasculitis, eczema or asthma, A method is further provided.

[0029] In some specific embodiments, the effective amount is 0.001 to 10mpk, for example, 0.001mpk, 0.002mpk, 0.003mpk, 0.004mpk, 0.005mpk, 0.006mpk, 0.007mpk, 0.008mpk, 0.009mpk, 0.01mpk, 0.02mpk, 0.03mpk, 0.04mpk, 0.05mpk, 0.06mpk , 0.07mpk, 0.08mpk, 0.09mpk, 0.1mpk, 0.2mpk, 0.3mpk, 0.4mpk, 0.5mpk, 0.6mpk, 0.7mpk, 0.8mpk, 0.9mpk, 1mpk, 2mpk, 3mpk, 4mpk, 5mpk, 6mpk, 7mpk, 8mpk, 9mpk or 10mpk, or a range value of effective amounts comprised between the above effective amounts.

[0030] In a tenth aspect, the present invention relates to a method for treating a proliferative disease, said method comprising administering to a subject an effective amount of said pharmaceutical composition or reconstituted solution, preferably said proliferative disease is selected from neoplasms, solid tumors, hematological tumors, malignant ascites or malignant pleural effusion, said solid tumors may be benign or malignant, primary or metastatic, said malignant solid tumors are carcinomas or sarcomas, such as epithelial cell carcinomas, endothelial cell carcinomas, squamous cell carcinomas, gli ... the tumor may be selected from the group consisting of bladder cancer, bladder cancer, non-muscle invasive ... A method is further provided.

[0031] In an eleventh aspect, the present invention further provides an article of manufacture comprising a container containing the above-mentioned pharmaceutical composition, lyophilized formulation or reconstitution solution. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Definitions and Explanations of Terms Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings that are understood by those of ordinary skill in the art.

[0033] Unless otherwise stated, the term "IL2" or "IL-2" according to the present invention refers to any natural or recombinant IL-2 derived from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), as well as domestic or agricultural mammals. "IL2" or "IL-2" according to the present invention includes incompletely processed IL-2 (e.g., IL-2 containing an N-terminal signal peptide) and any form that produces fully processed IL-2 within a cell. "IL2" or "IL-2" according to the present invention further includes naturally occurring variants and fragments of IL-2, such as truncated or allelic variants. "IL2" or "IL-2" according to the present invention further includes non-naturally occurring variants, such as IL-2 variants artificially modified by genetic engineering.

[0034] A "wild-type IL-2" is otherwise the same as an IL-2 variant, except that at each mutated amino acid position of the IL-2 variant, the IL-2 form of the wild-type amino acid is retained. Illustratively, if the IL-2 variant is an incompletely processed IL-2, the wild-type form of the variant is an incompletely processed mature IL-2, if the IL-2 variant is a fully processed IL-2, the wild-type form of the variant is a fully processed IL-2, and if the IL-2 variant is a truncated form of IL-2, the wild-type form of the variant is the corresponding truncated form of IL-2 having the wild-type sequence. Illustratively, a "wild-type IL-2" according to the present invention may have the amino acid sequence shown below:

[0035] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF XQSIISTLT (SEQ ID NO: 1), where the amino acid residue "X" at position 125 represents C, S, A or V.

[0036] In the present invention, the term "mutation" includes amino acid substitution, deletion, insertion, or any combination thereof. "Mutation" according to the present invention can be generated using genetic or chemical methods known in the art, including, but not limited to, methods such as site-directed mutagenesis, PCR, gene synthesis, etc.

[0037] The "mutation site" number of an IL-2 mutant according to the present invention is counted from the amino acid residue A at position 1 of "wild-type IL-2" shown in SEQ ID NO: 1. Exemplarily, the "Y31 mutation" of the present invention refers to a mutation of an IL-2 mutant occurring at the amino acid residue (Tyr, Y) at position 31 of wild-type IL-2 shown in SEQ ID NO: 1. Exemplarily, the "Y31V mutation" of the present invention refers to an IL-2 mutant in which the amino acid residue at position 31 of wild-type IL-2 shown in SEQ ID NO: 1 is mutated from Y (Tyr) to V (Val).

[0038] In the present invention, the term " / " used between mutation sites means "and" and indicates that the mutations before and after " / " exist simultaneously in the same IL-2 mutant. For example, "Y31 / A73 / H79" indicates that the same IL-2 mutant has mutations at the Y31 site, the A73 site, and the H79 site simultaneously, and "Y31V / A73L / H79Q" indicates that the same IL-2 mutant has mutations at the Y31 site, the A73L site, and the H79Q site simultaneously.

[0039] In the present invention, the term "fusion protein" refers to a protein product obtained by linking the coding regions of two or more genes by recombinant gene methods, chemical methods or other suitable methods, and expressing the recombinant genes under the control of the same regulatory sequence. In the fusion protein of the present invention, the coding regions of the two or more genes can be fused at one or more positions by a sequence encoding a linker peptide. The linker peptide can be used to construct the fusion protein of the present invention.

[0040] In the present invention, the term "Linker" refers to a peptide used in the present invention to link IL-2 with another protein molecule or protein fragment to ensure correct folding and stability of the protein. Such other molecules include, but are not limited to, antibody Fc blocks. The "Linker" of the present invention is preferably (GGGGS)n, where n can be 0, 1, 2, 3, 4, 5 or 6.

[0041] In the present invention, the term "antibody Fc block" refers to the constant region of an immunoglobulin chain, in particular the carboxy-terminus or a part thereof of the heavy chain constant region of an immunoglobulin, which does not have antigen binding activity and is the site where the antibody molecule interacts with effector molecules or cells. "Fc" according to the present invention may be any Fc or variant thereof from a human or non-human mammal. For example, an immunoglobulin Fc may comprise a combination of two or more domains of the heavy chain CH1, CH2, CH3, CH4 and an immunoglobulin hinge region. Fc may be derived from immunoglobulins of different species, preferably human. Depending on the amino acid sequence of the heavy chain constant region, immunoglobulins can be divided into different types, there are mainly five types of immunoglobulins: IgA, IgD, IgE, IgG and IgM. Some of them can be further divided into subclasses (isotypes), e.g. IgG-1, IgG-2, IgG-3, IgG-4, IgA-1 and IgA-2. "Fc" preferably comprises at least one immunoglobulin hinge region and the CH2 and CH3 domains of IgG. More preferably, it comprises one CH2 domain, one CH3 domain and one immunoglobulin hinge region of IgG1, and the starting amino acid position of the hinge region can vary. Unless otherwise stated, amino acid residues in an Fc, constant region or antibody according to the invention are numbered according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0042] In the present invention, the term "pharmaceutical composition" refers to a mixture containing one or more IL-2 variants, fusion proteins, nucleic acid fragments, vectors or host cells according to the present invention. The mixture further comprises other ingredients, including but not limited to pharma- ceutical acceptable carriers, diluents or adjuvants thereof. The pharmaceutical composition according to the present invention is intended to facilitate administration to the living body and facilitate the absorption of the active ingredient to exert its biological activity.

[0043] In the present invention, the term "treatment" refers to surgical or therapeutic treatment aimed at preventing or alleviating (reducing) the progression of undesirable physiological changes or pathologies in a subject to be treated, such as an autoimmune disease (e.g., systemic lupus erythematosus). Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stable disease state (i.e., not worsening), delay or slowing of disease progression, improvement or mitigation of disease state, and remission (whether partial or total remission), whether detectable or undetectable. Subjects in need of treatment include those already suffering from a disease or disorder, and those susceptible to a disease or disorder, or those in whom a disease or disorder is to be prevented. When referring to terms such as slowing, alleviation, reduction, mitigation, remission, and the like, the meaning also includes situations such as resolution, disappearance, non-occurrence, and the like.

[0044] In the present invention, the term "subject" refers to a living organism undergoing treatment for a particular disease or condition (e.g., an autoimmune disease) according to the present invention. Examples of subjects and patients include mammals undergoing treatment for a disease or condition, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, cows or other members of the bovine family, sheep and horses.

[0045] In the present invention, the term "effective amount" refers to an amount of a therapeutic agent that is effective to prevent or ameliorate the symptoms of a disease or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject. "Effective amount" also refers to an amount of a compound that is sufficient to ameliorate a symptom, e.g., treat, cure, prevent, or ameliorate an associated medical condition, or treat, cure, prevent, or ameliorate an increase in the rate of such a condition. When an active ingredient is administered alone to an individual, the therapeutically effective dose is the amount of that ingredient alone. When a combination is used, the therapeutically effective dose refers to the combined dose of the active ingredients that produces a therapeutic effect, regardless of combined, sequential, or simultaneous administration.

[0046] In the present invention, the term "autoimmune disease" refers to a disease of a subject characterized by damage to cells, tissues and / or organs caused by an immune response of the subject against its own cells, tissues and / or organs. Exemplary autoimmune diseases include, but are not limited to, rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjogren's syndrome, polymyositis, dermatomyositis, scleroderma, psoriasis, plaque psoriasis, alopecia areata, multiple sclerosis, amyotrophic lateral sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, graft-versus-host disease, organ transplant rejection, autoimmune hepatitis, type I diabetes, autoimmune vasculitis, eczema or asthma.

[0047] In the present invention, the term "proliferative disorder" refers to the occurrence of an undesirable medical condition or disease state, which may or may not be cancerous, caused by unregulated and / or abnormal growth of cells or tissues. This includes, but is not limited to, neoplasms, solid tumors, hematological tumors, malignant ascites or malignant pleural effusion. In the present invention, a "solid tumor" may be benign or malignant, primary or metastatic, and a malignant solid tumor may be a carcinoma or a sarcoma. Exemplarily, in the present invention, "solid tumor" includes, but is not limited to, epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, teratoma, lung tumor, papilloma virus-induced cancer, adenocarcinoma, carcinoma, melanoma, angiosarcoma, neuroblastoma, metastatic lung cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, Merkel cell carcinoma, ovarian cancer, renal cell carcinoma, metastatic kidney cancer, head and neck cancer, bladder cancer, and non-muscle layer invasive bladder cancer. Exemplarily, in the present invention, "blood tumor" includes, but is not limited to, leukemia, lymphoma, and multiple myeloma, such as B cell lymphoma, T cell lymphoma, cutaneous T cell lymphoma, and T cell large granular lymphocyte leukemia.

[0048] In the present invention, the term "IL-2 receptor α subunit" (IL-2Rα), also referred to as "CD25," refers to any naturally occurring IL-2 receptor α subunit or variants thereof from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), and includes the "full-length" unprocessed IL-2 receptor α subunit and any form of intracellularly processed IL-2 receptor α subunit, further including naturally occurring IL-2 receptor α subunit variants such as truncated variants or allelic variants, and further including variants that have been artificially engineered based on the naturally occurring IL-2 receptor α subunit.

[0049] In the present invention, the term "IL-2 receptor β subunit" (IL-2Rβ), also known as "CD122," refers to any naturally occurring IL-2 receptor β subunit or variants thereof from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), and includes the "full-length" unprocessed IL-2 receptor β subunit and any form of intracellularly processed IL-2 receptor β subunit, further including naturally occurring IL-2 receptor β subunit variants such as truncated variants or allelic variants, and further including variants that have been artificially engineered based on the naturally occurring IL-2 receptor β subunit.

[0050] In the present invention, the term "IL-2 receptor gamma subunit" (IL-2Rγ), also referred to as "CD132", refers to any naturally occurring IL-2 receptor gamma subunit or variants thereof from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), and includes the "full-length" unprocessed IL-2 receptor gamma subunit and any form of intracellularly processed IL-2 receptor gamma subunit, further including naturally occurring IL-2 receptor gamma subunit variants such as truncated variants or allelic variants, and further including variants artificially engineered based on the naturally occurring IL-2 receptor gamma subunit.

[0051] In the present invention, the term "Treg" refers to "regulatory T cells" or "T 制御細胞 These specific CD4 T cells, also called CD4+ T cells, can block other T cell responses. + Refers to a type of T cell. Tregs are characterized by expressing the IL-2 receptor α subunit (CD25) and the transcription factor Forkhead box protein P3 (FOXP3), and play an important role in the induction and maintenance of peripheral self-tolerance to antigens. Tregs require IL-2 for their function and development, as well as for the induction of their inhibitory properties.

[0052] As used herein, the terms "percent (%) sequence identity" and "percent (%) sequence identity" are interchangeable and refer to the percent of amino acid (or nucleotide) residues of a candidate sequence that are identical to the amino acid (or nucleotide) residues of a reference sequence after aligning the sequences to achieve maximum percent sequence identity and introducing gaps (if necessary) (e.g., for optimal alignment, gaps can be introduced in one or both of the candidate and reference sequences, and non-homologous sequences can be ignored for purposes of comparison). For purposes of determining percent sequence identity, alignment can be achieved in a variety of ways well known to those of skill in the art, for example using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAIi) software. Those of skill in the art can determine appropriate parameters to be used for measuring alignment, including any algorithm required to achieve maximum alignment within the full length of the sequences being compared. For example, a reference sequence aligned for comparison with a candidate sequence can show that the candidate sequence exhibits 50% to 100% sequence identity over the entire length of the candidate sequence or over a selected portion of consecutive amino acid (or nucleotide) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purposes may, for example, be at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence. When a position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence, then the molecules are identical at that position. [Brief description of the drawings]

[0053] [Figure 1] Mouse body weight change curves: Body weight change curves after administration of CsA, AMG592 and IL2-1-2 injections to DTH model mice, 10 mice per group. Data points represent the average body weight of animals in a group, and error bars represent standard error of the mean (SEM). [Diagram 2]Mouse weight change curves: Weight change rates after administration of CsA, AMG592 and IL2-1-2 injections to DTH model mice. There were 10 mice in each group. Data points represent the average weight change rate of animals in a group, and error bars represent standard error of the mean (SEM). [Diagram 3] DTH model mouse ear thickness change curve: Ear thickness change curve after CsA, AMG592 and IL2-1-2 injection in DTH model mice. There were 10 mice in each group. Data points represent the mean ear thickness change within the group, and error bars represent standard error of the mean (SEM). ****P<0.0001: compared with vehicle control group. [Figure 4] The curves show the change rate of ear thickness after injection of CsA, AMG592 and IL2-1-2 in DTH model mice. There were 10 mice in each group. Data points represent the mean ear thickness change within the group, and error bars represent the standard error of the mean (SEM). ****P<0.0001: compared with the vehicle control group. EXAMPLES

[0054] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become more apparent with the description. In the examples, conditions not specified are general conditions or conditions recommended by the manufacturer. Reagents or equipment used without a specified manufacturer are all general products available on the market.

[0055] The embodiments of the present invention are merely illustrative and do not limit the scope of the present invention. Without departing from the spirit and scope of the present invention, the details and forms of the technical solutions of the present invention may be modified or replaced, but it should be understood by those skilled in the art that all such modifications and replacements are within the protection scope of the present invention.

[0056] Regarding the explanation of Examples 1 to 5 1. Replacing the Ultrafiltration Concentrate Unless otherwise stated, the replacement of ultrafiltration concentrate in the following examples means adding a protein stock solution to the corresponding ultrafiltration tube, using a centrifuge to carry out ultrafiltration concentration, the protein is blocked, the buffer solution of the protein stock solution passes through the ultrafiltration membrane to achieve the purpose of protein concentration, then adding the target solution to be replaced, the buffer solution of the original protein stock solution is diluted, ultrafiltration concentration is continued, and after concentration is completed, the target solution to be replaced is continued to be added, ultrafiltration concentration is continued, and the operation is repeated until the replacement of the liquid is completed.

[0057] 2. English abbreviations and their meanings Unless otherwise stated, the following English abbreviations have the meanings set out in the table below.

[0058] [Table 1]

[0059] 3. Method for detecting the binding activity of IL2 mutant fusion protein according to Example 5 (1) Coating: Select a carbonate buffer solution of pH 9.6 (50 mM Na2CO3-NaHCO3, pH 9.6) as the coating solution, dilute human IL-2Ralpha protein (Acro, ILA-H52H9) to 0.5 μg / mL in the coating solution, add 100 μL / well to the microplate reader, and coat overnight (>16 h) at 2-8 °C. (2) Blocking: After washing the plate, PBS containing 3% BSA was added at 300 μL / well, and blocking was carried out at 37° C. for 1 hour, after which the plate was washed. (3) Sample dilution: The IL2 mutant fusion protein was diluted to a starting concentration of 200 ng / mL (details are shown in Table 2).

[0060] [Table 2]

[0061] (4) Diluted samples of IL2 mutant fusion proteins were added at 100 μL / well and incubated at 37° C. for 40 to 80 minutes. (5) After washing, mouse anti-human IgG Fc γ-HRP (Jackson Immuno, 209-035-098) diluted at 1:10,000 (v / v) was added at 100 μL / well and incubated at 37° C. for 40 to 80 minutes. (6) After washing, add 100 μL / well of the color-developing solution TMB (Thermo, 34029) to the microplate reader and react for 5 to 9 minutes in the dark at room temperature. Then, add 100 μL / well of 1 M sulfuric acid to stop the reaction. Read the plate using a microplate reader within 2 minutes. The measurement wavelength was 450 nm and the reference wavelength was 630 nm. (7) Using the software SoftMax Pro or GraphPad Prism provided with the Molecular Devices microplate reader or a homogenous analysis software, the EC50 of the test sample and the control sample were calculated by four-parameter regression. The relative activity of the test sample was calculated by the following formula: Relative binding activity of the test sample (%) = Control EC50 / Test sample EC50 × 100. If the relative activity of the test sample was in the range of 70% to 130%, it was judged to be a pass.

[0062] Example 1 Production and purification of IL2 mutant fusion proteins 1. Design of IL2 mutant fusion proteins (1) Using multiple algorithms, we designed the mutation sites Y31V / A73L / H79Q to improve the thermostability of IL2. (2) Using a variety of algorithms, functional mutation sites V91R, H16E, D20A, or H16E / V91R were designed to reduce the interaction between IL2 and the IL2 receptor βγ subunit complex. (3) The above thermostable and functional mutations were combined to obtain the following IL2 mutants containing the following combination mutations:

[0063] Y31V / A73L / H79Q / V91R, Y31V / A73L / H79Q / H16E, Y31V / A73L / H79Q / D20A, Y31V / A73L / H79Q / H16E / V91R.

[0064] To extend the half-life, the IL2 thermostable mutant or combination mutant was further linked to Fc via a linker to form a fusion protein (hereinafter referred to as IL2 mutant fusion protein), the individual elements and their overall sequences are shown in Table 3.

[0065] Compared to the wild-type IL2 fusion protein (SEQ ID NO: 19), the IL2 mutant fusion proteins according to the invention have the following features:

[0066] (1) When analyzed by the DSF method, the Tm values ​​of all IL2 mutant fusion proteins containing thermostable mutations were increased compared to the wild-type IL2 fusion protein. The Tm value of IL2 mutant 1-linker-Fc was increased by about 8-9°C, the Tm value of IL2 mutant 1-2-linker-Fc was increased by more than 12°C, the Tm value of IL2 mutant 1-3-linker-Fc was increased by 8-9°C, the Tm value of IL2 mutant 1-4-linker-Fc was increased by about 9-10°C, and the Tm value of IL2 mutant 1-5-linker-Fc was increased by about 11-12°C. (2) Compared with the wild-type IL2 fusion protein, the binding activity of the IL2 mutant fusion protein containing the functional mutation to the human IL2 receptor βγ dimer on the cell surface was inhibited, and the CD4 + CD25 - FoxP3 - T cells or CD8 + Activation of STAT5 phosphorylation levels in T cells was significantly reduced, the effect on Treg cell proliferation was similar to that of the wild-type IL2 fusion protein, and proliferation levels of NK cells were significantly reduced. (3) Compared with the wild-type IL2 fusion protein or Fc-linker-IL2 variant 6, IL2 variant 1-2-linker-Fc showed better therapeutic effects in efficacy (DTH model mouse and GVHD mouse model), higher exposure and bioavailability in pharmacokinetics, and longer half-life, with more obvious subcutaneous administration. (4) The IL2 variant 1-2-linker-Fc (Y31V / A73L / H79Q / V91R, SEQ ID NO: 15) was selected for further process development.

[0067] [Table 3-1]

[0068] [Table 3-2]

[0069] 2. Expression of IL2 mutant fusion proteins The IL2 mutant fusion protein was expressed in CHO as the host cell and cultured in OPM-CHO CD063 medium (manufacturer: OPM, catalog number: C483260), the culture period did not exceed 14 days, and the reactor control parameters were pH 6.8-7.2, dissolved oxygen (DO) 20%-80%, rotation speed 75RPM-80RPM, initial culture temperature 36.0°C-37.0°C, and the culture temperature was reduced to 34.0±0.5°C until harvest when cultured until the 6th day.

[0070] 3. Purification of IL2 Mutant Fusion Proteins The IL2 mutant fusion protein was purified in sequence using multi-step chromatography, concentration and filtration unit operations. The supernatant of the fermentation harvest liquid was captured by Protein A affinity chromatography (AT ProteinA Diamond Plus). The captured IL2 mutant fusion protein solution was treated with low pH incubation to inactivate potential viruses. After neutralization of the IL2 mutant fusion protein solution, the precipitate was removed by deep filtration. Next, cation exchange chromatography (Diamond S) was performed in sequence to remove impurities such as HCP and aggregates, and anion exchange chromatography (POROS 50HQ) was performed to remove impurities such as HCD, HCP and shed Protein A. The solution was filtered by a nanofiltration membrane pack to remove potential endogenous and exogenous viruses. Next, the IL2 mutant fusion protein solution was concentrated in an ultrafiltration membrane pack and replaced with a buffer solution. Finally, additives were added to adjust the concentration and filtered to obtain the IL2 mutant fusion protein stock solution. In the subsequent examples, the stock solutions were replaced with the corresponding formulation systems by an exchange procedure of the ultrafiltration concentrate.

[0071] Examples 2 to 5 Development of a formulation that maintains the stability of the IL2 mutant 1-2-linker-Fc (V91R / Y31V / A73L / H79Q, SEQ ID NO: 15) in Example 1 Example 2 Screening of buffer systems 1. Screening Method (1) Twelve different buffer formulation systems F1-F12 were designed, and the specific formulation systems are shown in Table 4. (2) The IL2 mutant fusion protein stock solution obtained in Example 1 was replaced with F1-F12 by exchanging the ultrafiltration concentrate, and the protein concentration was adjusted to 2 mg / mL. (3) The stability of the IL2 mutant fusion protein in 12 buffer systems was comprehensively considered through 25°C accelerated stability tests and 40°C accelerated stability tests, and the optimal buffer system was screened for subsequent additive and surfactant screening. The consideration indexes included appearance, pH value, protein concentration, purity (SEC-HPLC), CE-SDS (NR), CE-SDS (R), and dynamic light scattering (DLS). The details of the consideration method were shown in Table 4.

[0072] [Table 4]

[0073] 2. Screening results of buffer systems The main results of the buffer system screening are summarized below.

[0074] [Table 5]

[0075] [Table 6]

[0076] [Table 7]

[0077] [Table 8]

[0078] [Table 9]

[0079] [Table 10]

[0080] [Table 11]

[0081] [Table 12]

[0082] [Table 13]

[0083] [Table 14]

[0084] 3.Results of consideration (1) Appearance superiority ranking: F1, F3 > F2, F7, F8, F9, F11 > F10, F12 > F4, F5, F6. (2) pH value: The pH values ​​of the 12 buffer systems were stable and did not change significantly. (3) Protein concentration ranking: F1, F2, F3, F6, F7, F8, F9, F11, F12>F5>F4, F10. (4) DLS superiority / inferiority order: F1, F5, F9>F6, F7, F8>F3, F4, F10, F11, F12>F2. (5) SEC-HPLC superiority order: F2, F3, F5, F6, F7, F8, F9>F4, F10>F1>F11, F12. (6)CE-SDS(NR) Superiority: F2, F3, F6>F1, F5, F7, F8, F9>F4, F10>F11, F12. (7) CE-SDS(R) Superiority: F8, F9>F3, F6>F2, F7, F10>F1, F4, F5, F11, F12.

[0085] Summary: Taking into account the various indicators detected in the 25°C accelerated stability test and the 40°C accelerated stability test, formulation F3 (20 mM acetic acid-sodium acetate, pH 5.5) was superior to the other formulations and showed better stability, so F3 was selected as the next development.

[0086] Example 3 Additive screening 1. Screening Method Based on the optimal buffer system (20 mM acetic acid-sodium acetate, pH 5.5) screened in Example 2, seven formulations were designed, and the stability of the IL2 mutant fusion protein (IL2 mutant fusion protein concentration is 2 mg / mL) after the addition of sodium chloride, glycine, arginine hydrochloride, sucrose, trehalose, mannitol or sorbitol was independently examined, and the optimal formulation was screened for subsequent surfactant screening. The examination indexes included appearance, pH value, concentration, SEC-HPLC, DLS, CE-SDS (NR&R), and the specific formulation composition and examination method are shown in Table 15.

[0087] [Table 15]

[0088] 2. Screening results of additives The main results of the additive screening are summarized below.

[0089] [Table 16]

[0090] [Table 17]

[0091] [Table 18]

[0092] [Table 19-1]

[0093] [Table 19-2]

[0094] [Table 20-1]

[0095] [Table 20-2]

[0096] [Table 21-1]

[0097] [Table 21-2]

[0098] 3.Results of consideration (1) Appearance superiority ranking: F3-2>F3-1>F3-3>F3-5>F3-4, F3-6>F3-7. (2) pH value ranking: There was no significant difference in the data between the groups. (3) Concentration ranking: There was no significant difference in the data between the groups. (4) DLS superiority / inferiority order: F3-2>F3-1>F3-3, F3-4, F3-5, F3-6>F3-7. (5) SEC-HPLC superiority ranking: F3-1, F3-3, F3-4, F3-5, F3-6>F3-7>F3-2. (6)CE-SDS(R) Superiority order: F3-1, F3-3, F3-4, F3-5, F3-6>F3-7>F3-2. (7)CE-SDS(NR) Superiority order: F3-2, F3-3>F3-4>F3-5>F3-7>F3-6>F3-1.

[0099] Summary: Based on the appearance, groups F3-4, F3-5, F3-6 and F3-7, which showed particles in appearance at long-term stability 2-8℃, were excluded. The content of oligomers detected by CE-SDS(NR) in the F3-1 formulation containing glycine was higher than that of F3-2 and F3-3 at 40℃-4W, indicating that IL-2 protein is more easily decomposed in the F3-1 formulation. The SEC-HPLC detection index of F3-2 was not significantly different from the other groups, and the proportion of other peaks was relatively high at 40℃-4W in CE-SDS(R), but there was no significant difference from the other groups at long-term stability 2-8℃ and 25℃. Therefore, considering the stability data comprehensively, the F3-2 and F3-3 formulations showed better stability than the other formulations. Formulations F3-2 (containing 140 mM arginine hydrochloride) and F3-3 (containing 8% w / v sucrose) were selected to further explore the stability of the formulations after the addition of surfactants.

[0100] Example 4 Surfactant Considerations 1. Method of consideration Based on the F3-2 and F3-3 formulations, the stability of the formulations after adding the surfactant PS-80 was examined. Details of the formulation conditions are shown in Table 22. Details of the examination method are shown in Table 23. The examination indexes include appearance, pH, protein concentration, DLS, SEC-HPLC, CE-SDS (NR&R), and insoluble particulate matter (MFI). Based on the examination results, an appropriate formulation was selected for confirmation of the formulation stability.

[0101] [Table 22]

[0102] [Table 23]

[0103] 2. Screening Results The main results are summarized below.

[0104]

Table 24

[0105]

Table 25

[0106]

Table 26

[0107]

Table 27

[0108]

Table 28

[0109]

Table 29

[0110]

Table 30

[0111]

Table 31

[0112] 3. Survey results In this example, the protective effect of polysorbate 80 (PS80), a surfactant, on proteins in a pH 5.5 acetic acid system was examined. As shown in the results, the protein has good resistance to high temperatures, repeated freezing and thawing, and shaking at concentrations of 2 mg / mL and 5 mg / mL under conditions of 20 mM acetic acid-sodium acetate, 140 mM arginine hydrochloride, 0.04% or 0.06% PS80 (w / v), and the protein also has good resistance to high temperatures, repeated freezing and thawing, and shaking at a protein concentration of 2 mg / mL under conditions of 20 mM acetic acid-sodium acetate, 8% sucrose (w / v), and 0.04% PS80 (w / v). There were no significant differences in appearance, pH, concentration, protein particle size (DLS), SEC-HPLC, and CE (NR) between the formulations. Formula F3-3-1 had better CE-SDS(R) results in protein purity at 40℃-4W compared with other formulations using arginine hydrochloride and 8% sucrose as an additive, and the ratio in other peaks was 2.68%, which was 4.25%-5.05% lower than other formulations. From the above, PS80 can effectively protect proteins from shear forces in the environment and manufacturing process and prevent protein aggregation.

[0113] 4. Drug discovery research The stability of 5 mg / mL of IL-2 mutant fusion protein was examined in 20 mM acetic acid-sodium acetate, pH 5.2, 8% sucrose (w / v), 0.02% PS80 (w / v) system. The results showed that 5 mg / mL of IL2 mutant fusion protein had good druggability in the druggability study formulation, and the details were shown in Table 32.

[0114] [Table 32]

[0115] 5. Conclusion Considering that, when screening surfactants, formulation F3-3-1 showed better results in CE-SDS(R) compared with other formulations, and at the same time, 2 mg / mL and 5 mg / mL both belong to low concentration protein, and 5 mg / mL of IL2 mutant fusion protein showed good stability in drug discovery studies of formulations similar to F3-3-1, 5 mg / mL of IL2 mutant fusion protein, 20 mM acetic acid-acetate, pH 5.5, 8% sucrose (w / v), 0.04% PS80 (w / v) was selected as the target formulation for the next formulation confirmation stability study.

[0116] Example 5 Formulation confirmation stability 1. Formulation confirmation stability method The target formulation (5 mg / mL IL2 mutant fusion protein, 20 mM acetic acid-sodium acetate, pH 5.5, 8% sucrose (w / v), 0.04% PS80 (w / v)) was selected and formulation confirmation stability study was performed. 1 mL of the liquid formulation was dispensed into a 2 mL vial, and a closed packaging system was assembled using a rubber stopper and an aluminum plastic cap, and its stability was examined by placing it upright and inverted, respectively, and the examination method is shown in Table 33.

[0117] [Table 33]

[0118] 2. Formulation confirmation stability results The main formulation-validated stability results are summarized in the table below.

[0119] [Table 34-1]

[0120] [Table 34-2]

[0121] 3. Formulation confirmation stability study conclusion

[0122] After 3 months at low temperatures of 2-8°C, and 3 months of accelerated stability at 25°C, the normal and inverted samples showed no significant changes in the conventional detection indicators, insoluble particulates, purity, charge isomers, activity, etc., and there was no significant difference between the normal and inverted samples. After 4 weeks of accelerated stability at 40°C, the main peak of the charge variants decreased by 9.2% and tended to approach the acidic peak value, and the purity and charge variants changed significantly.

[0123] In summary, in the process of examining the real-time stability at low temperature (2-8°C) and the accelerated test at 25°C, there were no significant changes in the conventional detection indicators of the sample, insoluble particles, purity, charge isomers, activity, etc., and all the indicators were within the range of the quality standards, suggesting that the IL2 mutant fusion protein has relatively good stability in this formulation. In the examination experiment of the accelerated stability at high temperature 40°C, there was a tendency for the purity and charge isomers to decrease, suggesting that the formulation shown in the present invention is sensitive to high temperatures.

[0124] Example 6 Study of IL2 mutant fusion protein injections in DTH model mice single agent pharmacodynamics This example evaluates the efficacy of single agent injection of IL2 mutant fusion protein in KLH-induced delayed type hypersensitivity (DTH) model mice, where the IL2 mutant fusion protein is IL2 mutant 1-2-linker-Fc (V91R / Y31V / A73L / H79Q), hereinafter referred to as IL2-1-2, the sequence of which is shown in SEQ ID NO: 15. The positive control is Fc-linker-IL2 mutant 6 (V91K), i.e., AMG592, the sequence of which is shown in SEQ ID NO: 20. The injection formulations of the above IL2-1-2 and AMG592 were both 5 mg / mL IL2-1-2 or AMG592, 20 mM acetic acid-sodium acetate, pH 5.5, 8% sucrose (w / v), 0.04% PS80 (w / v).

[0125] 6.1. Animal Grouping BALB / c mice aged 6 to 8 weeks were used, and each mouse was weighed and randomly divided into 8 experimental groups, G0 to G7, with 10 mice in each group, according to their body weight. Administration began on the day of grouping, which was recorded as Day 0. IL2-1-2 and AMG592 were administered by subcutaneous injection once every 3 days for each dose group, and CsA was administered by intraperitoneal injection once a day, and administration was continued until the end of the experiment.

[0126] 6.2. How to construct an animal model 6.2.1. Preparation of Reagents (1) 3 mg / mL KLH: The lyophilized powder was weighed out and a 3 mg / mL KLH solution was prepared in PBS. (2) The volume ratio of 1 mg / mL KLH emulsion:KLH (3 mg / mL):IFA:CFA is 1:1:1, and the antigen is emulsified by the communicating tube syringe method, so that the antigen can be sufficiently emulsified to form a viscous emulsion. (3) 1 mg / mL KLH solution: 3 mg / mL KLH was diluted 3-fold with PBS to 1 mg / mL.

[0127] 6.2.2. Modeling method for DTH model BALB / c mice were sensitized on day 0 by injecting a total of 100μL of 1mg / mL KLH emulsion (emulsified with a volume ratio of 1:1:1 between KLH, IFA, and CFA) into two sites on the right side of the back. The normal control group was injected with an equal volume of emulsion without KLH. On day 5, 10μL of 1mg / mL KLH solution was injected into the right ear skin of each mouse to stimulate the mice, causing delayed-type hypersensitivity symptoms in the local skin tissue of the model mice. The thickness of the right ear of the mice was measured using a micrometer before and 24h, 48h, 72h, and 96h after the local KLH injection into the right ear to evaluate the degree of local delayed-type hypersensitivity in the mice of different treatment groups.

[0128] 6.3. Group administration method is shown in Table 35

[0129] [Table 35]

[0130] 6.4. Detection Indicators 6.4.1. Body weight detection: The animals were weighed before grouping, and after the animals were grouped, the mice were weighed once every 3 days, with the unit being g and the weight being kept to one decimal place.

[0131] 6.4.2. Mouse ear thickness measurement: (1) Before stimulation, the thickness of the right ear of each mouse was measured using a digital outer micrometer and used as the background value. (2) After stimulation, the thickness of the mouse ear pieces was measured 24 h, 48 h, 72 h, and 96 h later.

[0132] 6.4.3. General clinical observations: During the adaptation and experimental periods, observations should be conducted at least twice a week. Observations should include, but are not limited to, the animals' mental state, feeding habits, etc. Any unexpected observations should be recorded in the experimental record notebook (paper).

[0133] 6.5. End of experiment 6.5.1. During the dosing interval, attention should be paid to observing the health status of the animals. If any one or more of the following situations occur, dosing should be suspended until the animal has returned to normal: (1) Treatment was stopped when the animal's body weight fell below 81% of its body weight at the start of drug treatment, and treatment was continued when the animal's body weight returned to 90% of its body weight at the start of drug treatment. (2) After administration, the animals' behavior was delayed or abnormal, or they experienced acute stress phenomena.

[0134] 6.5.2. Humane End Point of Experimental Animals: If the animal's condition is abnormal during the experiment, the animal's health status will be evaluated to determine whether treatment should be administered, whether the experiment can be continued, or whether euthanasia should be performed.

[0135] 6.5.3. Euthanasia: 96 h after mouse stimulation, if the experiment was to be terminated or a humane endpoint was to be implemented, the animals were euthanized by asphyxiation with excess CO2.

[0136] 6.6.Statistical analysis methods The original data of measurements and observations should be recorded. Analysis processing was carried out based on the original data, and the results were expressed as the mean number and standard error (Mean ± SEM). At the same time, statistical analysis was carried out on the data, and the changes in the thickness of the mouse ear pieces were analyzed by Two-way ANOVA, and the body weight of the mice on the 10th day was analyzed by One-way ANOVA, and P<0.05 was considered to be statistically significant.

[0137] 6.7. Results and Discussion 6.7.1. Effect of IL2-1-2 injections on mouse body weight in the DTH model The body weight of the experimental animals was used as a reference index to indirectly measure drug toxicity. The body weight and the body weight change rate of the mice after administration of CsA, AMG592 and different doses of IL2-1-2 are shown in Figures 1, 2 and Table 36. Each therapeutic drug was administered subcutaneously to DTH model mice once every 3 days, and during the observation period, the body weight of the mice fluctuated, and except for the decrease in body weight of the mice in the hormone-administered CsA group, no mice lost more than 10% of their body weight, and all mice in the other administration groups showed a tendency to increase in body weight. All the mice were in good condition, and there were no other symptoms or deaths. It was found that the treatment with 10 mg / kg CsA, 1 mg / kg AMG592, and 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg and 1 mg / kg doses of IL2 variant 1-2 were all tolerable.

[0138] [Table 36]

[0139] 6.7.2. Effect of IL2-1-2 injection on changes in ear thickness in DTH model mice The changes in ear thickness of the DTH model mice in each treatment group are shown in Figures 3-4. The onset of the DTH model peaked 48 hours after KLH stimulation, and the change in ear thickness of the model mice at the peak of onset was 15.7 mm × 10 -2The ear thickness of the mice increased by 74.1% compared to the initial ear thickness. The model was successfully established, and the IL2-1-2 injection solution increased the ear thickness of the mice by 2.45, 4.01, 9.09 and 12.26 mm × 10 in the 1, 0.3, 0.1 and 0.03 mg / kg treatment groups, respectively. -2 The increase in the number of mice with KLH stimulation was 10.2%, 22.0%, 41.3% and 53.6% compared to the mice with ears before KLH stimulation. The positive drugs CsA 10mg / kg and AMG592 1mg / kg also had a clear therapeutic effect on this model, and showed a certain therapeutic effect compared to the solvent control group (P<0.0001), but the therapeutic effect of both was lower than that of IL2-1-2 at 0.3 and 1mg / kg. From the above, IL2-1-2 had a clear therapeutic effect in the KLH-induced DTH model mice.

[0140] Example 7 Pharmacokinetic Study of IL2 Mutant Fusion Protein Injections in BALB / c Mice This example evaluates the pharmacokinetic properties of IL2 variant fusion protein injections in BALB / c mice, where the IL2 variant fusion protein is IL2 variant 1-2-linker-Fc (V91R / Y31V / A73L / H79Q), hereafter referred to as IL2-1-2, with the sequence set forth in SEQ ID NO: 15. The positive control used Fc-linker-IL2 variant 6 (V91K), i.e., AMG592, with the sequence set forth in SEQ ID NO: 20. The above IL2-1-2 and AMG592 injection formulations were both 5 mg / mL IL2-1-2 or AMG592, 20 mM acetic acid-sodium acetate, pH 5.5, 8% sucrose (w / v), 0.04% PS80 (w / v).

[0141] 7.1. Animal Grouping After 6-8 week old BALB / c mice were properly raised, the weight of each mouse was weighed, and based on the weight of the mice calculated using random numbers using Excel, the mice were randomly divided into 5 experimental groups, G1-G5, with 5 mice in each group. The administration was started on the day of grouping, which was recorded as Day 1.

[0142] 7.2. Group administration method is shown in Table 37.

[0143] [Table 37]

[0144] 7.3.Detection Indicators 7.3.1. Body weight determination: The animals were weighed before grouping, and after grouping the animals were weighed before blood sampling, and the weight was expressed in g.

[0145] 7.3.2. Flow cytometric classification of immune cells in mouse peripheral blood Four days after administration, the mice were euthanized and the heart was punctured to collect peripheral blood. Immune cell surface markers (CD3+, CD4+, CD25+) and nuclear factors (Foxp3+, Ki-67+) were labeled with fluorescent tag antibodies. Immune cell counting and grouping analysis were performed using the following FACS method. (1) After euthanizing the mice at the end of the experiment, the heart was punctured and mouse peripheral blood was collected in an EDTA-2K anticoagulant tube. The anticoagulant tube was gently inverted to thoroughly mix the blood with the anticoagulant to prevent clotting. (2) 300 μL of uncoagulated whole blood was transferred to a flow tube. An antibody mixture of cell surface markers (CD3+, CD4+, CD25+) was added to the flow tube and incubated at room temperature in the dark for 20 minutes. (3) 1 mL of lysis solution was added to the flow tube to lyse the red blood cells and incubated at room temperature in the dark for 5 minutes. Centrifuged at 400 g / rcf for 6 minutes at 20°C and the supernatant was discarded. (4) Step (3) was repeated to re-lyse the red blood cells. (5) The cells were resuspended in 4 mL of washing solution (PBS). The cell suspension was filtered through a filter membrane and then transferred to a new flow tube. (6) 500 μL of fixative was added dropwise to the flow tube while shaking, and incubated overnight at 4°C in the dark. (7) 2 mL of transmembrane nuclei solution was added to the flow tube and shaken, the flow tube was centrifuged at 500 g / rcf at 4°C for 5 min, and the supernatant was discarded. (8) Step (7) was repeated, and the cells were treated again with 3 mL of transmembrane nuclei solution. (9) Nuclear factor (Foxp3+, Ki-67+) antibody mixture was added to the flow tube and incubated at 4°C in the dark for 40 min, shaking the flow tube once every 20 min. (10) 4 mL of wash solution was added to the flow tube while shaking, the flow tube was centrifuged at 500 g / rcf at 4°C for 5 min, the supernatant was discarded, and the cells were resuspended in 200 μL of PBS. (11) Counting microspheres were equilibrated by vortexing for at least 30 s at room temperature. Add 50 μL of counting microspheres to the flow tubes and perform FACS detection on the flow cytometer, and each flow tube should be shaken thoroughly before placing it on the instrument. (12) Based on the FACS results, the immune cell types were analyzed using FlowJo software, and the cell numbers were calculated according to the instructions of the counting microspheres.

[0146] 7.3.3. General clinical observations: During the adaptation and experimental periods, observations should be conducted at least twice a week. Observations should include, but are not limited to, the animals' mental state, feeding habits, etc. Any unexpected observations should be recorded in the experimental record notebook (paper).

[0147] 7.4. End of experiment 7.4.1. During the dosing interval, the animal's health condition should be carefully observed. If any one or more of the following situations occur, administration should be suspended until the animal has returned to normal: (1) Treatment was stopped when the animal's body weight fell below 81% of its body weight at the start of drug treatment, and treatment was continued when the animal's body weight returned to 90% of its body weight at the start of drug treatment. (2) After administration, the animals' behavior was delayed or abnormal, or they experienced acute stress phenomena.

[0148] 7.4.2. Humane End Point of Experimental Animals: If any abnormality was found in the animal's condition during the experiment, the animal's health status was evaluated to determine whether treatment should be administered, whether the experiment could be continued, or whether euthanasia should be performed.

[0149] 7.4.3. Euthanasia: At the end of the experiment, 26 days after sensitization, animals were euthanized by asphyxiation with overdose of CO2.

[0150] 7.5.Statistical analysis methods The original data of measurements and observations must be recorded. Analysis was performed based on the original data, and the results were expressed as mean numbers and standard errors (Mean ± SEM).

[0151] 7.6. Results and Discussion 7.6.1.Effect of IL2-1-2 injection on peripheral blood immune cells in BALB / c mice After BALB / c mice were treated with different doses of IL2-1-2, the fold change in peripheral blood immune cell counts was compared with the solvent control group mice, as shown in Table 38. Subcutaneous injection of IL2-1-2 was observed to have a significant promoting effect on mouse Treg cell proliferation. Compared with the solvent control group, 3 days after administration of IL2-1-2 or AMG592, Treg cells were significantly stimulated. After subcutaneous administration of 0.1, 0.3, and 1 mg / kg IL2-1-2, the fold change in mouse peripheral blood Treg cell counts was 13.73, 35.27, and 12.05, respectively. The degree of increase in Treg cell counts in the 1 mg / kg AMG592 group mice was higher than that in the 1 mg / kg IL2-1-2 group mice, but lower than that in the 0.3 mg / kg group mice, indicating that IL2-1-2 has a good promoting effect on Treg cells at a relatively low dose. The fold change in Treg / CD4+ ratio was shown in Table 39. In the experiment, it was observed that CD4+Foxp3 cells or CD3+CD4- cells were not activated or were only slightly activated.

[0152] [Table 38]

[0153] [Table 39]

[0154] 7.7. Conclusion The results showed that subcutaneous injection of IL2-1-2 injection significantly promoted the proliferation of Treg cells in the peripheral blood of BALB / c mice, and the mice were obviously tolerant to the dose of the tested molecule.

[0155] Example 8 Pharmacokinetic study of IL2 mutant fusion protein injection in SD rats This example evaluates the pharmacokinetic properties of IL2 mutant fusion protein injections in SD rats, where the IL2 mutant fusion protein is IL2 mutant 1-2-linker-Fc (V91R / Y31V / A73L / H79Q), hereinafter referred to as IL2-1-2, with the sequence shown in SEQ ID NO: 15. The above IL2-1-2 injection formulations were all 5 mg / mL IL2-1-2, 20 mM acetic acid-sodium acetate, pH 5.5, 8% sucrose (w / v), 0.04% PS80 (w / v).

[0156] 8.1. Animal Grouping Six to eight week old SD rats were properly raised and then randomly allocated to six experimental groups, each with three males and three females. The administration started on the day of grouping (denoted as day 1), and IL2-1-2 injection was administered four times by subcutaneous or tail vein injection on days 1, 8, 15, and 22, respectively.

[0157] 8.2. Grouping and administration methods are shown in Table 40.

[0158] [Table 40]

[0159] 8.3. Observation Indicators 8.3.1. Weight monitoring After grouping and before each administration, the animals were weighed and recorded in grams.

[0160] 8.3.2. Flow cytometry of immune cell phenotypes in rat peripheral blood Peripheral blood samples were collected from rats before administration on day 1, and on days 4, 11, and 25 after administration. Immune cell surface markers (CD3+, CD4+, CD25+) and nuclear factors (Foxp3+, Ki-67+) were labeled with fluorescent tags. The number of immune cells was calculated by the following fluorescence-activated cell sorting (FACS) method. (1) Whole blood was collected from the rat jugular vein into an anticoagulant tube containing EDTA-2K. The anticoagulant tube was gently inverted to thoroughly mix the blood with the anticoagulant to prevent clotting. (2) 300 μL of uncoagulated whole blood was transferred to a flow tube. An antibody mixture of cell surface markers (CD3+, CD4+, CD25+) was added to the flow tube and incubated at room temperature in the dark for 20 minutes. (3) 1 mL of lysis solution was added to the flow tube to lyse the red blood cells and incubated at room temperature in the dark for 5 minutes. The flow tube was centrifuged at 400 g / rcf at 20°C and the supernatant was discarded. (4) Step (3) was repeated to re-lyse the red blood cells. (5) The cells were resuspended in 4 mL of washing solution (PBS). The cell suspension was filtered through a filter membrane and then transferred to a new flow tube. (6) 500 μL of fixative was added drop by drop to the flow tube while shaking and incubated overnight at 4°C in the dark. (7) 2 mL of transmembrane nuclei solution was added to the flow tube and shaken, the flow tube was centrifuged at 500 g / rcf at 4°C for 5 min, and the supernatant was discarded. (8) Step (7) was repeated, and the cells were treated again with 3 mL of transmembrane nuclei solution. (9) Nuclear factor (Foxp3+, Ki-67+) antibody mixture was added to the flow tube and incubated at 4°C in the dark for 40 min, and the flow tube was shaken once every 20 min. (10) 4 mL of washing solution was added to the flow tube and shaken, the flow tube was centrifuged at 500 g / rcf at 4°C for 5 min, the supernatant was discarded, and the cells were resuspended in 200 μL of PBS. (11) Counting microspheres were vortexed at room temperature for at least 30 seconds to equilibrate. 50 μL of counting microspheres were added to the flow tube and FACS detection was performed on the flow cytometer, and each flow tube should be shaken thoroughly before placing it in the instrument. (12) Based on the FACS results, immune cell types were analyzed using FlowJo software, and cell numbers were calculated according to the instructions in the counting microspheres.

[0161] 8.3.3. General Clinical Observations During the adaptation and experimental periods, clinical observations should be made at least twice a week, including but not limited to the animal's health condition, expression, daily activity, mental state, feeding status, etc. Any unexpected observations should be recorded in the experimental record notebook (paper).

[0162] 8.4. End of experiment 8.4.1 During the course of the experiment and between dosing intervals, careful observation of the animal's health status should be made. If any one or more of the following situations occurs, dosing should be suspended until the animal has returned to normal: (1) Treatment was stopped when the animal's body weight fell below 81% of its pre-treatment weight and continued when it returned to 90% of its pre-treatment weight. (2) After administration, the animals' behavior was delayed or abnormal, or they experienced acute stress phenomena.

[0163] 8.4.2. The End of Humanism The health status of the animals was assessed to determine whether to continue with treatment or experiment or to perform euthanasia if any one or more of the following conditions occurred: (1) The animal's behavior is abnormal or paralyzed. (2) the body weight of the animals is less than 20% of the body weight before grouping; (3) The animal’s body temperature is too low and it is in a critical condition.

[0164] Euthanasia At the humane endpoint or termination of the experiment, animals were euthanized using CO2 overdose.

[0165] 8.5. Statistical analysis The results were expressed as mean and standard error (Mean±SEM).

[0166] 8.6.Results 8.6.1. Effect of IL2-1-2 on the number and ratio of immune cells in rat PBMCs The percentage changes in immune cell counts in SD rats in peripheral blood after IL2-1-2 administration are shown in Table 41. After IL2-1-2 was injected into SD rats subcutaneously and via tail vein, it could induce Treg cell expansion. Compared with the control, low dose IL2-1-2 (0.005 mg / kg subcutaneous injection or 0.002 mg / kg tail vein injection) showed a slight promoting effect on Treg cell expansion from day 4 to day 25 after administration. After subcutaneous injection of medium dose IL2-1-2 (0.05 mg / kg), the number of Treg cells in rat peripheral blood increased to 9.55 times the baseline value on day 4 and was maintained at 3.16 times the baseline value on day 25. After subcutaneous injection of high doses of IL2-1-2 (1 mg / kg), the number of Treg cells in the peripheral blood of rats reached a peak value (13.8 times the baseline value) on day 11 and decreased to 3.18 times the baseline value on day 25. After tail vein injection of high doses of IL2-1-2 (1 mg / kg), the number of Treg cells in the peripheral blood of rats increased significantly on days 4 and 11, reaching 21.25 and 30.13 times the baseline value, respectively. However, similar to the other treatment groups, the number of Treg cells also decreased to 3.26 times the baseline value on day 25. This phenomenon may have been due to the production of anti-drug antibodies (ADA) in the body after multiple administrations to rats. The expansion pattern of Ki-67+ cells in Treg cells was also consistent with the increasing trend of Treg cell numbers.

[0167] The ratio changes of Treg / CD4+ cell counts were shown in Table 42. It was observed that low dose (0.005 mg / kg subcutaneous injection or 0.002 mg / kg tail vein injection) and medium dose (0.05 mg / kg subcutaneous injection) of IL2-1-2 had no or slight activating effect on CD4+Foxp3- cell and CD3+CD4- cell counts in rat peripheral blood, while high dose (1 mg / kg subcutaneous injection or 1 mg / kg tail vein injection) of IL2-1-2 could slightly activate the expansion of the above cells.

[0168] [Table 41]

[0169] [Table 42]

[0170] 8.7. Conclusion The above experimental results showed that both subcutaneous and tail vein injection of IL2-1-2 into SD rats could induce the proliferation of Treg cells, and SD rats had good tolerance to all doses of IL2-1-2.

Claims

1. 1. A pharmaceutical composition comprising: a fusion protein comprising an IL2 variant and an antibody Fc block, a buffer, an osmotic agent and a surfactant, The fusion protein comprises, in order from the N-terminus to the C-terminus: (1) an IL2 variant, a linker and an antibody Fc block, or (2) comprising an antibody Fc block, a linker, and an IL2 variant; Preferably, said IL2 mutant comprises at least the mutations Y31V, A73L and H79Q compared to wild-type IL2, More preferably, the IL2 mutant further comprises one or more of the following mutations: a V91R mutation, an H16E mutation, and a D20A mutation, e.g. an H16E mutation and a V91R mutation; The amino acid sequence of the wild-type IL2 is shown in SEQ ID NO:2, More preferably, the IL2 variant has an amino acid sequence having at least 80% identity compared to the sequence shown in any one of SEQ ID NOs: 3, 8 to 11. Pharmaceutical compositions.

2. The linker has an amino acid sequence having at least 80% identity compared to the sequence shown in SEQ ID NO: 12, or the linker has ４ S) ｎ wherein n is selected from 1, 2, 3, 4, 5, or 6; the antibody Fc block has a N297G mutation, or the antibody Fc block has an amino acid sequence with at least 80% identity compared to the sequence set forth in SEQ ID NO: 13; Preferably, the fusion protein has an amino acid sequence having at least 80% identity compared to the sequence shown in any one of SEQ ID NOs: 14 to 18, More preferably, the fusion protein forms a homodimer by dimerization of the antibody Fc block. The pharmaceutical composition of claim 1.

3. The concentration of the fusion protein is 1-50 mg / mL. The pharmaceutical composition according to any one of claims 1 to 2.

4. the buffer is selected from acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, histidine-histidine hydrochloride, succinic acid-sodium succinate, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, preferably acetic acid-sodium acetate buffer, The concentration of the buffer solution is 1 to 100 mM; The pH value of the buffer solution is 4.5 to 8.0; The pharmaceutical composition according to any one of claims 1 to 3.

5. The osmolality adjusting agent is selected from a salt, an amino acid, a sugar or a sugar alcohol or a combination thereof, preferably the salt is selected from sodium chloride, potassium chloride or arginine hydrochloride, preferably the amino acid is selected from glycine, arginine, histidine, glutamic acid or methionine, preferably the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol or sorbitol; More preferably, the osmolality adjusting agent is selected from sodium chloride, glycine, arginine hydrochloride, sucrose, trehalose, mannitol or sorbitol, most preferably sucrose or arginine hydrochloride; The sugar or sugar alcohol has a concentration of 1 to 15% w / v, and the salt has a concentration of 50 to 200 mM. The pharmaceutical composition according to any one of claims 1 to 4.

6. The surfactant is selected from polysorbate 80 (PS-80), polysorbate 20 (PS-20) or poloxamer, and the concentration of the surfactant is 0.01-0.1% w / v; The pharmaceutical composition according to any one of claims 1 to 5.

7. The pharmaceutical composition comprises the fusion protein, acetic acid-sodium acetate buffer, sucrose and polysorbate-80, preferably the pharmaceutical composition comprises 1-50 mg / mL of the fusion protein, 1-100 mM acetic acid-sodium acetate buffer, 1-15% w / v sucrose and 0.01-0.1% w / v polysorbate-80, and has a pH value of 4.5-6.

0. The pharmaceutical composition according to any one of claims 1 to 6.

8. The pharmaceutical composition comprises a fusion protein, acetic acid-sodium acetate buffer, arginine hydrochloride and polysorbate-80, preferably the pharmaceutical composition comprises 1-50 mg / mL of the fusion protein, 1-100 mM acetic acid-sodium acetate buffer, 50-200 mM arginine hydrochloride and 0.01-0.1% w / v polysorbate-80, and the pH value is 4.5-6.0; The pharmaceutical composition according to any one of claims 1 to 6.

9. The pharmaceutical composition is an intravenous, intramuscular or subcutaneous injection, preferably a subcutaneous injection; The pharmaceutical composition according to any one of claims 1 to 8.

10. A method for producing the pharmaceutical composition according to any one of claims 1 to 9, comprising the steps of: The method includes a step of preparing the fusion protein by mixing it with the buffer, the osmotic pressure adjusting agent and the surfactant, and preferably, the method includes a step of replacing a stock solution of the fusion protein with the buffer by ultrafiltration concentration.

11. A lyophilized formulation comprising: Formed after lyophilization of the pharmaceutical composition according to any one of claims 1 to 9, Lyophilized formulation.

12. A method for producing the lyophilized formulation of claim 11, comprising the steps of: The method comprises the step of lyophilizing the pharmaceutical composition according to any one of claims 1 to 9. method.

13. A method for producing a reconstituted solution of the lyophilized formulation according to claim 11, comprising the steps of: The method includes reconstituting the lyophilized formulation according to claim 12 with a solvent, preferably the solvent being water for injection. method.

14. A reconstituted solution of an Fc fusion protein containing an IL2 mutant, produced by the method of claim 13.

15. Use of the pharmaceutical composition according to any one of claims 1 to 9, the lyophilized formulation according to claim 11 or the reconstituted solution according to claim 14 in the manufacture of a medicament for treating an autoimmune disease or a proliferative disease, comprising: Preferably, the autoimmune disease is selected from rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjogren's syndrome, polymyositis, dermatomyositis, scleroderma, psoriasis, plaque psoriasis, alopecia areata, multiple sclerosis, amyotrophic lateral sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, graft-versus-host disease, organ transplant rejection, autoimmune hepatitis, type I diabetes, autoimmune vasculitis, eczema, or asthma; Preferably, said proliferative disease is selected from neoplasms, solid tumors, hematological tumors, malignant ascites or malignant pleural effusion, wherein said solid tumors may be benign or malignant, primary or metastatic, said malignant solid tumors may be carcinomas or sarcomas, such as epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, teratoma, lung tumor, papilloma virus induced carcinoma, adenocarcinoma, carcinoma, melanoma, hemangiosarcoma, neuroblastoma, metastatic lung cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, Merkel cell carcinoma, ovarian cancer, renal cell carcinoma, metastatic kidney cancer, head and neck cancer, bladder cancer, non-muscle invasive bladder cancer, and said hematological tumors may be selected from leukemias, lymphomas, multiple myelomas, such as B cell lymphoma, T cell lymphoma, cutaneous T cell lymphoma, T cell large granular lymphocyte leukemia, use.

16. A pharmaceutical composition according to any one of claims 1 to 9, a lyophilized formulation according to claim 11 or a reconstituted solution according to claim 14 for treating an autoimmune disease or a proliferative disease, comprising: Preferably, the autoimmune disease is selected from rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjogren's syndrome, polymyositis, dermatomyositis, scleroderma, psoriasis, plaque psoriasis, alopecia areata, multiple sclerosis, amyotrophic lateral sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, graft-versus-host disease, organ transplant rejection, autoimmune hepatitis, type I diabetes, autoimmune vasculitis, eczema, or asthma; Preferably, said proliferative disease is selected from neoplasms, solid tumors, hematological tumors, malignant ascites or malignant pleural effusion, wherein said solid tumors may be benign or malignant, primary or metastatic, said malignant solid tumors may be carcinomas or sarcomas, such as epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, teratoma, lung tumor, papilloma virus induced carcinoma, adenocarcinoma, carcinoma, melanoma, hemangiosarcoma, neuroblastoma, metastatic lung cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, Merkel cell carcinoma, ovarian cancer, renal cell carcinoma, metastatic kidney cancer, head and neck cancer, bladder cancer, non-muscle invasive bladder cancer, and said hematological tumors may be selected from leukemias, lymphomas, multiple myelomas, such as B cell lymphoma, T cell lymphoma, cutaneous T cell lymphoma, T cell large granular lymphocyte leukemia, A pharmaceutical composition, a lyophilized formulation or a reconstituted solution.

17. 1. A method of treating an autoimmune disease, comprising: The present invention includes administering an effective amount of the pharmaceutical composition according to any one of claims 1 to 9 or the reconstituted solution according to claim 14 to a subject, and preferably, the autoimmune disease is selected from rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjogren's syndrome, polymyositis, dermatomyositis, scleroderma, psoriasis, plaque psoriasis, alopecia areata, multiple sclerosis, amyotrophic lateral sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, graft versus host disease, organ transplant rejection, autoimmune hepatitis, type I diabetes, autoimmune vasculitis, eczema, or asthma, and preferably, the effective amount is 0.001 to 10 mpk. method.

18. 1. A method of treating a proliferative disease, comprising: The method includes administering an effective amount of the pharmaceutical composition according to any one of claims 1 to 9 or the reconstituted solution according to claim 14 to a subject, and preferably, the proliferative disease is selected from neoplasms, solid tumors, hematological tumors, malignant ascites or malignant pleural effusion, wherein the solid tumor may be benign or malignant, primary or metastatic, and the malignant solid tumor is carcinoma or sarcoma, such as epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, teratoma, lung tumor, papilloma, or the like. The tumor may be a roman virus-induced cancer, an adenocarcinoma, a carcinoma, a melanoma, angiosarcoma, a neuroblastoma, a metastatic lung cancer, a non-small cell lung cancer, a small cell lung cancer, a breast cancer, a Merkel cell carcinoma, an ovarian cancer, a renal cell carcinoma, a metastatic kidney cancer, a head and neck cancer, a bladder cancer, a non-muscle invasive bladder cancer, and the hematological tumor may be selected from a leukemia, a lymphoma, a multiple myeloma, e.g., a B cell lymphoma, a T cell lymphoma, a cutaneous T cell lymphoma, a T cell large granular lymphocyte leukemia, method.

19. A container containing the pharmaceutical composition according to any one of claims 1 to 9, the lyophilized preparation according to claim 11, or the reconstituted solution according to claim 14. product.

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