Pharmaceutical composition of IL2 mutant-antibody Fc block fusion protein and use 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] This invention relates to the field of drug formulation, and more specifically to pharmaceutical compositions of IL2 variant-antibody Fc block fusion proteins and their use. [Background technology]

[0002] Interleukin-2 (IL-2) was initially identified as a T cell growth factor (TCGF), and research has shown 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 contains the IL-2Rα subunit (CD25), the IL-2Rβ subunit (CD122), and the IL-2Rγ subunit (CD132), and different subunits can form receptor complexes of different affinities, including the high-affinity receptor IL-2Rαβγ, the intermediate-affinity receptor IL-2Rβγ, and the low-affinity receptor IL-2Rα or IL-2Rαβ. Different types of IL-2R subunits are expressed in different cells; for example, conventional T cells under resting conditions express different types of CD4 + T, CD8 + While T cells generally express IL-2 receptor β (IL-2Rβ, CD122) and IL-2 receptor γ (IL-2Rγ, CD132) and hardly express IL-2 receptor α (IL-2Rα, CD25), regulatory T cells (Tregs) constitutively overexpress IL-2Rα in addition to expressing IL-2Rβ and IL-2Rγ. [Overview of the Initiative] [Problems that the invention aims to solve]

[0003] Currently, researchers are using IL-2 or its variants to activate immune cells or subpopulations of immune cells to treat tumors or autoimmune diseases. For example, high doses of IL-2 are approved for the treatment of malignant melanoma or metastatic renal cell carcinoma, and the IL-2 polyethylene glycol drug conjugate NKTR-358 is approved for clinical trial development for autoimmune diseases. Further development of new IL-2 variants, improving their stability, productivity, and / or altering their ability to bind to specific types of receptor complexes, is of great importance for the development of IL-2-type drugs. At the same time, IL-2 is a high molecular weight protein and is prone to stability problems such as particle formation and aggregation in solution. Therefore, after obtaining a novel IL-2 variant, it is of great importance to develop a formulation system suitable for that novel IL-2 variant and to ensure its stability in solution.

[0004] The present invention provides pharmaceutical compositions and methods for producing the same, lyophilized formulations and methods for producing or redissolving the same, redissolved solutions obtained by the above redissolution method, corresponding pharmaceutical uses, methods for treating autoimmune diseases, and products. [Means for solving the problem]

[0005] In a first embodiment, the present invention relates to a pharmaceutical composition comprising a fusion protein containing an IL2 variant and an antibody Fc block, a buffer, an osmotic regulator, and a surfactant, Preferably, the above IL2 variant contains at least Y31V, A73L, and H79Q mutations compared to wild-type IL2. Preferably, the IL2 variant further comprises one or more mutations from among the V91R mutation, the H16E mutation, and the D20A mutation, for example, further comprising the H16E mutation and the V91R mutation. The present invention provides a pharmaceutical composition 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 Sequence ID No. 2.

[0007] In some specific embodiments, the above fusion protein is arranged sequentially from the N-terminus to the C-terminus. (1) IL2 variant, linker and antibody Fc block, or (2) The antibody comprises an Fc blocker, 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 It has the amino acid sequence, where n is selected from 1, 2, 3, 4, 5 or 6. Preferably, the linker has an amino acid sequence that has at least 80% identity with the sequence shown in Sequence ID No. 12, for example, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. Preferably, the antibody Fc block contains the N297G mutation. Preferably, the antibody Fc block has an amino acid sequence that has at least 80% identity with the sequence shown in SEQ ID NO: 13, for example, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. More preferably, the fusion protein has an amino acid sequence that has at least 80% identity with the sequences shown in SEQ ID NOs. 14-18, for example, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

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

[0009] In some specific embodiments, the concentration of the fusion protein is approximately 1 to 50 mg / mL. For example, the concentrations of the above fusion proteins are approximately 1-40 mg / mL, 1-30 mg / mL, 1-20 mg / mL, 1-15 mg / mL, 1-10 mg / mL, 5-50 mg / mL, 5-40 mg / mL, 5-30 mg / mL, 5-20 mg / mL, 5-19 mg / mL, 5-18 mg / mL, 5-17 mg / mL, 5-16 mg / mL, 5-15 mg / mL, 5-14 mg / mL, 5-13 mg / mL, and 5 ~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 concentrations of the above fusion proteins are approximately 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. The concentration is 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 composed of 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 acetate-sodium acetate buffer, citrate-sodium citrate buffer, histidine-histidine hydrochloride, succinate-sodium succinate, or disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, preferably acetate-sodium acetate buffer, and / or The concentration of the above buffer solution is approximately 1 to 100 mM. For example, buffer concentrations are approximately 1-90mM, 1-80mM, 1-70mM, 1-60mM, 1-50mM, 1-40mM, 1-30mM, 1-20mM, 1-10mM, 10-90mM, 10-80mM, 10-70mM, 10-60mM, 10-50mM, 10-40mM, 10-30mM, 10-20mM, 20-90mM, 20-80mM, 20-70mM, 20-60mM, and 20-50mM. The ranges are 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, and 80-90mM. For example, the concentration of the buffer solution is approximately 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 composed of the above concentration values. Preferably, the concentration of the buffer solution is about 20 mM, and / or The pH value of the above buffer solution is approximately 4.5 to 8.0. For example, the pH values ​​of the above buffers are approximately 4.5-7.5, 4.5-7.0, 4.5-6.5, 4.5-6.0, 4.5-5.5, 4.5-5.0, 5.0-8.0, 5.0-7.5, 5.0-7.0, 5.0-6.5, 5.0-6.0, 5.0-5.5, 5.5-8.0, 6-7.5, 6.5-7.5, 7.0-7.5, or 7.5-8.0. For example, the pH values ​​of the above buffer solution are approximately 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0, or a range of pH values ​​composed of the above pH values. Preferably, the pH value of the buffer solution is approximately 5.5.

[0012] In some specific embodiments, the osmotic pressure regulator is selected from salts, amino acids, sugars or sugar alcohols or combinations 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, and 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, and more preferably sucrose or arginine hydrochloride. More preferably, the concentration of the sugar or sugar alcohol is about 1-15% w / v, and the concentration of the salt is about 50-200 mM, for example, the concentration of the sugar or sugar alcohol is about 1-10% w / v, 1-5% w / v, or 5-10% 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 The values ​​are 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 between the above concentration values. Preferably, it is 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, the concentration of the amino acid is about 1 to 10% w / v, 1 to 5% w / v or 5 to 10% w / v. For example, the concentration of the amino acid 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 is a range value of the concentration composed between the above concentration values. Preferably, it is 2% w / v.

[0014] More preferably, the concentration of the salt is about 50 to 200 mM. For example, the concentration of the salt is about 50 to 150 mM, 50 to 100 mM or 100 to 150 mM. For example, the concentration of the salt is 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 is a range value of the concentration composed between the above concentration values. Preferably, it is 140 mM.

[0015] More preferably, the osmotic pressure regulator is sucrose or arginine hydrochloride.

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

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

[0018] In some specific embodiments, the pharmaceutical composition comprises a fusion protein, an acetate-sodium acetate buffer, arginine hydrochloride, and polysorbate-80, preferably comprising about 1 to 50 mg / mL of the fusion protein, about 1 to 100 mM of the acetate-sodium acetate buffer, about 50 to 200 mM of arginine hydrochloride, and about 0.01 to 0.1% w / v of polysorbate-80, with a pH of about 4.5 to 6.0. More preferably, the pharmaceutical composition comprises the above fusion protein in an amount of about 1-30 mg / mL, 5-30 mg / mL, 10-30 mg / mL, 10-25 mg / mL, 10-20 mg / mL, 10-15 mg / mL, 15-20 mg / mL, 15-30 mg / mL, or 20-30 mg / mL, about 10-50 mM or 10-30 mM of acetate-sodium acetate buffer, about 50-200 mM, 50-150 mM, or 100-150 mM of arginine hydrochloride, and about 0.01-0.1% w / v or 0.02-0.06% w / v of polysorbate-80, with a pH value of about 4.5-6.0 or 4.5-6.0. More preferably, the pharmaceutical composition comprises about 1 to 10 mg / mL of the fusion protein, about 10 to 30 mM of acetate-sodium acetate buffer, about 120 to 150 mM of arginine hydrochloride, and about 0.01 to 0.1% w / v of polysorbate-80, with a pH 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 of acetate-sodium acetate buffer, about 140 mM of arginine hydrochloride, and about 0.04% w / v or 0.06% w / v of polysorbate-80, with a pH of about 5.5.

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

[0021] In a second aspect, the present invention further provides a method for producing the above-mentioned pharmaceutical composition, wherein the method comprises the step of preparing the fusion protein by mixing it with the buffer, an osmotic pressure regulator, and a surfactant, and preferably the method comprises the step of replacing the 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 above-mentioned pharmaceutical composition.

[0023] In a fourth aspect, the present invention further provides a method for producing the above-mentioned 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 redissolution solution of an antibody Fc block fusion protein containing an IL2 variant, comprising redissolving the lyophilized formulation in a solvent, preferably the solvent being water for injection.

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

[0026] In a seventh aspect, the present invention relates to the use of the above-mentioned pharmaceutical composition, lyophilized formulation, or redissolving solution in the manufacture of a drug for treating an autoimmune disease or a proliferative disorder, Preferably, the above autoimmune disease is selected from rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjögren's syndrome, polymyositis, dermatomyositis, scleroderma, psoriasis, psoriasis vulgaris, 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 1 diabetes mellitus, autoimmune vasculitis, eczema, or asthma. Preferably, the proliferative disorder is selected from neoplasms, solid tumors, hematological malignancies, malignant ascites, or malignant pleural effusions, where the solid tumor may be benign or malignant, primary or metastatic, the malignant solid tumor may be carcinoma or sarcoma, such as epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, teratoma, lung tumor, papillomavirus-induced carcinoma, 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, or non-muscle-invasive bladder cancer, and the hematological malignancy may be selected from leukemia, lymphoma, multiple myeloma, such as B-cell lymphoma, T-cell lymphoma, cutaneous T-cell lymphoma, or T-cell macrogranular lymphocyte leukemia. To provide further use.

[0027] In the eighth aspect, the present invention relates to the above-mentioned pharmaceutical composition, lyophilized preparation, or redissolving solution for treating an autoimmune disease or a proliferative disorder, Preferably, the above autoimmune disease is selected from rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjögren's syndrome, polymyositis, dermatomyositis, scleroderma, psoriasis, psoriasis vulgaris, 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 1 diabetes mellitus, autoimmune vasculitis, eczema, or asthma. Preferably, the proliferative disorder is selected from neoplasms, solid tumors, hematological malignancies, malignant ascites, or malignant pleural effusions, where the solid tumor may be benign or malignant, primary or metastatic, the malignant solid tumor may be carcinoma or sarcoma, such as epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, teratoma, lung tumor, papillomavirus-induced carcinoma, 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, or non-muscle-invasive bladder cancer, and the hematological malignancy may be selected from leukemia, lymphoma, multiple myeloma, such as B-cell lymphoma, T-cell lymphoma, cutaneous T-cell lymphoma, or T-cell macrogranular lymphocyte leukemia. The above-mentioned pharmaceutical composition, lyophilized preparation, or re-dissolving solution is further provided.

[0028] In a ninth aspect, the present invention relates to a method for treating an autoimmune disease, wherein the method comprises administering an effective amount of the pharmaceutical composition or redissolving solution to a subject, preferably the autoimmune disease being selected from rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjögren's syndrome, polymyositis, dermatomyositis, scleroderma, psoriasis, psoriasis vulgaris, 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 1 diabetes mellitus, autoimmune vasculitis, eczema, or asthma. Further methods will be provided.

[0029] In some specific embodiments, the effective amount is 0.001 to 10 mpk, for example, 0.001 mpk, 0.002 mpk, 0.003 mpk, 0.004 mpk, 0.005 mpk, 0.006 mpk, 0.007 mpk, 0.008 mpk, 0.009 mpk, 0.01 mpk, 0.02 mpk, 0.03 mpk, 0.04 mpk, 0.05 mpk, 0.06 mpk. , 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 an effective amount configured between the above effective amounts.

[0030] In a tenth aspect, the present invention relates to a method for treating a proliferative disorder, wherein the method comprises administering an effective amount of the pharmaceutical composition or redissolving solution to a subject, preferably the proliferative disorder being selected from neoplasms, solid tumors, hematological malignancies, malignant ascites, or malignant pleural effusion, wherein the solid tumor may be benign or malignant, primary or metastatic, and the malignant solid tumor may be carcinoma or sarcoma, such as epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, or This may include hematomas, lung tumors, papillomavirus-induced cancers, adenocarcinomas, carcinomas, melanomas, angiosarcomas, neuroblastomas, metastatic lung cancers, non-small cell lung cancers, small cell lung cancers, breast cancers, Merkel cell carcinomas, ovarian cancers, renal cell carcinomas, metastatic kidney cancers, head and neck cancers, bladder cancers, and non-muscle-invasive bladder cancers. The above hematological malignancies may be selected from leukemias, lymphomas, multiple myelomas, such as B-cell lymphomas, T-cell lymphomas, cutaneous T-cell lymphomas, and T-cell macrogranule lymphocyte leukemias. Further methods will be provided.

[0031] In an eleventh aspect, the present invention further provides a product comprising a container containing the above-mentioned pharmaceutical composition, lyophilized preparation, or redissolving solution. [Modes for carrying out the invention]

[0032] Definitions and explanations of terms Unless otherwise defined, the scientific and technical terms relating to the present invention shall have meanings as understood by those skilled in the art.

[0033] Unless otherwise stated, the terms “IL2” or “IL-2” as used in this invention refer 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 livestock or agricultural mammals. The terms “IL2” or “IL-2” as used in this invention include incompletely processed IL-2 (e.g., IL-2 containing an N-terminal signal peptide) and any form that produces fully processed IL-2 in a cell. The terms “IL2” or “IL-2” as used in this invention further include natural variants and fragments of IL-2, such as shear mutants or allele mutants. The terms “IL2” or “IL-2” as used in this invention further include unnaturally occurring variants, such as IL-2 mutants artificially modified by genetic engineering.

[0034] "Wild-type IL-2" is identical to the IL-2 mutant in all other respects, except that the IL-2 form of the wild-type amino acids is retained at each mutant amino acid position of the IL-2 mutant. For example, if the IL-2 mutant is incompletely processed IL-2, the wild-type form of the mutant is incompletely processed mature IL-2; if the IL-2 mutant is fully processed IL-2, the wild-type form of the mutant is fully processed IL-2; and if the IL-2 mutant is a cleavage form of IL-2, the wild-type form of the mutant is the corresponding IL-2 cleavage form having the wild-type sequence. For example, the "wild-type IL-2" described in the present invention may have the amino acid sequence shown below.

[0035] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF XQSIISTLT (Sequence ID 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 substitutions, deletions, insertions, or any combination thereof. The "mutations" described in 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, and gene synthesis.

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

[0038] In this invention, the term " / " used between mutation sites means "and," indicating that the mutations before and after the " / " are simultaneously present in the same IL-2 mutant. For example, "Y31 / A73 / H79" means that the same IL-2 mutant is simultaneously mutated at the Y31, A73, and H79 sites, and "Y31V / A73L / H79Q" means that the same IL-2 mutant is simultaneously mutated with the Y31V, A73L, and H79Q mutations.

[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 a genetic recombination method, a chemical method, or other suitable method, and expressing the genetic recombination under the control of the same regulatory sequence. In the fusion protein of the present invention, the coding regions of 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 to link IL-2 to another protein molecule or protein fragment to ensure the correct folding and stability of the protein. The other molecules include, but are not limited to, antibody Fc blocks. The "linker" of the present invention is preferably (GGGGS)n, where n may be 0, 1, 2, 3, 4, 5, or 6.

[0041] In the present invention, the term "antibody Fc block" refers to the carboxyl terminus or a portion thereof of the constant region of the immunoglobulin chain, particularly the heavy chain constant region of immunoglobulin, which does not have antigen-binding activity and is the site where the antibody molecule interacts with effector molecules or cells. The "Fc" described in the present invention may be any Fc or its variants derived from human or non-human mammals. For example, immunoglobulin Fc may include a combination of two or more heavy chain domains CH1, CH2, CH3, CH4 and an immunoglobulin hinge region. Fc may be derived from different species, preferably human immunoglobulins. Based on the amino acid sequence of the heavy chain constant region, immunoglobulins can be classified into different types, mainly five types: IgA, IgD, IgE, IgG, and IgM. Some of these can be further divided into subclasses (isotypes), for example, IgG-1, IgG-2, IgG-3, IgG-4, IgA-1, and IgA-2. "Fc" preferably comprises at least one immunoglobulin hinge region, as well as the CH2 and CH3 domains of IgG. More preferably comprises one CH2 domain, one CH3 domain, and one immunoglobulin hinge region of IgG1, wherein the starting amino acid position of the hinge region can be varied. Unless otherwise stated, the Fc, constant region, or amino acid residues in the antibody described in the present invention are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interests, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0042] In the present invention, the term "pharmaceutical composition" refers to a mixture comprising one or more IL-2 variants, fusion proteins, nucleic acid fragments, vectors, or host cells described in the present invention. The mixture further comprises other components, including but not limited to its pharmaceutically acceptable carrier, diluent, or adjuvant. The pharmaceutical compositions described in the present invention are intended to facilitate administration to a living organism and aid in the absorption of the active ingredient to exert biological activity.

[0043] In this invention, the term "treatment" refers to surgical or therapeutic treatment aimed at preventing or mitigating (reducing) undesirable physiological changes or disease progression in a target of treatment, such as autoimmune diseases (e.g., systemic lupus erythematosus). Beneficial or desirable clinical outcomes include, but are not limited to, symptom reduction, disease severity, stable disease state (i.e., no worsening), delayed or slowed disease progression, improvement or mitigation of disease state, and remission (whether partial or total remission), whether detectable or undetectable. Targets requiring treatment include those already suffering from the disease, those susceptible to the disease, or those for whom prevention of the disease is necessary. When referring to terms such as slowing, reduction, decrease, mitigation, and remission, the meaning also includes situations such as resolution, disappearance, and non-occurrence.

[0044] In the present invention, the term "subject" refers to an organism undergoing treatment for a specific disease or condition described in the present invention (e.g., an autoimmune disease). 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, cattle or other members of the Bovidae family, sheep, and horses.

[0045] In the present invention, the term “effective dose” refers to the amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or relieving the symptoms of a disease or the progression of said disease. “Effective dose” also refers to the amount of a compound sufficient to relieve symptoms, for example, to treat, cure, prevent or relieve a related medical condition, or to treat, cure, prevent or relieve the rate of progression of such a condition. When an active ingredient is administered alone to an individual, the therapeutic effective dose is only the amount of that ingredient. When a combination is used, the therapeutic effective dose refers to the combined dose of the active ingredients that produces a therapeutic effect, regardless of whether it is administered in combination, sequentially, or simultaneously.

[0046] In the present invention, the term "autoimmune disease" refers to a disease of an object characterized by damage to cells, tissues and / or organs caused by the object initiating an immune response against its own cells, tissues and / or organs. Exemplary examples of autoimmune diseases include, but are not limited to, rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjögren's syndrome, polymyositis, dermatomyositis, scleroderma, psoriasis, psoriasis vulgaris, 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 1 diabetes mellitus, autoimmune vasculitis, eczema, or asthma.

[0047] In the present invention, the term “proliferative disorder” refers to the development of an undesirable medical condition or disease, whether cancerous or not, caused by unregulated and / or abnormal growth of cells or tissues. This includes, but is not limited to, neoplasms, solid tumors, hematological malignancies, malignant ascites, or malignant pleural effusions. In the present invention, “solid tumors” may be benign or malignant, primary or metastatic, and malignant solid tumors may be carcinomas or sarcomas. Exemplary examples in the present invention include, but are not limited to, epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, teratoma, lung tumor, papillomavirus-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-invasive bladder cancer. Exemplary examples in the present invention include, but are not limited to, leukemia, lymphoma, multiple myeloma, such as B-cell lymphoma, T-cell lymphoma, cutaneous T-cell lymphoma, and T-cell large granule lymphocyte leukemia.

[0048] In the present invention, the term "IL-2 receptor α subunit" (IL-2Rα) is also referred to as "CD25" and refers to any natural IL-2 receptor α subunit or its variant derived from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), and includes "full-length" unprocessed IL-2 receptor α subunits and any form of IL-2 receptor α subunit processed intracellularly, further including naturally occurring IL-2 receptor α subunit variants such as shear variants or allele variants, and further including variants artificially modified based on the natural IL-2 receptor α subunit.

[0049] In the present invention, the term "IL-2 receptor β subunit" (IL-2Rβ) is also referred to as "CD122" and refers to any natural IL-2 receptor β subunit or its variant derived from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), and includes "full-length" unprocessed IL-2 receptor β subunits and any form of IL-2 receptor β subunit processed intracellularly, further including naturally occurring IL-2 receptor β subunit variants such as shear variants or allele variants, and further including variants artificially modified based on the natural IL-2 receptor β subunit.

[0050] In the present invention, the term "IL-2 receptor γ subunit" (IL-2Rγ) is also referred to as "CD132" and refers to any natural IL-2 receptor γ subunit or its variant derived from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), and includes "full-length" unprocessed IL-2 receptor γ subunits and any form of IL-2 receptor γ subunit processed intracellularly, further including naturally occurring IL-2 receptor γ subunit variants such as shear variants or allele variants, and further including variants artificially modified based on the natural IL-2 receptor γ subunit.

[0051] In this invention, the term "Treg" refers to "regulatory T cell" or "T 制御細胞 Also known as , it is a specific CD4 that can inhibit the response of other T cells. + This refers to a type of T cell. Treg cells are characterized by the expression of the IL-2 receptor α subunit (CD25) and the transcription factor forkhead box protein P3 (FOXP3), and play a crucial role in inducing and maintaining peripheral self-tolerance to antigens. Treg cells require IL-2 to achieve their function, development, and the induction of their inhibitory properties.

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

[0053] [Figure 1] Mouse weight change curves: These are weight change curves for DTH model mice after administration of CsA, AMG592, and IL2-1-2 injection, with 10 mice in each group. Data points represent the average weight of the animals within each group, and error lines represent the standard error (SEM). [Figure 2]Mouse weight change rate curves: These curves show the weight change rate of DTH model mice after administration of CsA, AMG592, and IL2-1-2 injection. Each group consists of 10 mice. Data points represent the average weight change rate of animals within a group, and error lines represent the standard error (SEM). [Figure 3] Curves of change in ear thickness in DTH model mice: These are curves of change in ear thickness after administration of CsA, AMG592, and IL2-1-2 injection to DTH model mice. Each group consists of 10 mice. Data points represent the mean change in ear thickness within the group, and the error line represents the standard error (SEM). ****P<0.0001: Comparison with the vehicle control group. [Figure 4] These are the rate of change curves of earlobe thickness after administration of CsA, AMG592, and IL2-1-2 injection to DTH model mice. Each group consisted of 10 mice. Data points represent the mean change in earlobe thickness within the group, and the error line represents the standard error (SEM). ****P<0.0001: Comparison 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 clearer with further explanation. Unless specific conditions are stated in the examples, general conditions or conditions recommended by the manufacturer shall be followed. Unless the manufacturer of the reagents or equipment used is specified, they are all common products available on the market.

[0055] The embodiments of the present invention are merely illustrative and do not limit the scope of the invention. Details and forms of the technical proposals of the present invention can be modified or substituted without departing from the spirit and scope of the invention, and those skilled in the art will understand that all such modifications and substitutions fall within the scope of the protection of the invention.

[0056] Explanation of Examples 1-5 1. Replacement of ultrafiltration concentrate Unless otherwise specified, the replacement of the ultrafiltration concentrate in the following examples means adding the protein stock solution to the corresponding ultrafiltration tube, performing ultrafiltration concentration using a centrifuge until the protein is blocked and 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, diluting the buffer solution of the original protein stock solution, continuing the ultrafiltration concentration, continuing to add the target solution to be replaced after concentration is complete, and continuing the ultrafiltration concentration until the liquid replacement is complete.

[0057] 2. English abbreviations and their meanings Unless otherwise specified, the following English abbreviations have the meanings shown in the table below.

[0058] [Table 1]

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

[0060] [Table 2]

[0061] (4) Add the diluted IL2 mutant fusion protein sample to 100 μL / well and incubate at 37°C for 40-80 minutes. (5) After washing, 100 μL / well of mouse anti-human IgG Fc γ-HRP (Jackson Immuno, 209-035-098) diluted 1:10000 (v / v) was added and incubated at 37°C for 40-80 minutes. (6) After washing, 100 μL / well of color-developing solution TMB (Thermo, 34029) was added to the microplate reader, and the reaction was allowed to proceed at room temperature in the dark for 5 to 9 minutes. Then, 100 μL / well of 1 M sulfuric acid was added to stop the reaction, and the reading was taken using the microplate reader within 2 minutes. The measurement wavelength was set to 450 nm and the reference wavelength to 630 nm. (7) The EC50 of the sample and control were calculated using four-parameter regression with the SoftMax Pro or GraphPad Prism software included with the Molecular Devices microplate reader, or with isomorphic analysis software. The relative activity of the sample was calculated using the following formula: Relative binding activity of the sample (%) = Control EC50 / Sample EC50 × 100. A sample was judged to be acceptable if its relative activity was between 70% and 130%.

[0062] Example 1: Production and purification of IL2 mutant fusion protein 1. Design of IL2 mutant fusion proteins (1) Using various algorithms, we designed mutation sites Y31V / A73L / H79Q to improve the thermal stability of IL2. (2) Using various 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) By combining the above-mentioned thermal stability mutations and functional mutations, IL2 mutants containing the following combination mutations were obtained.

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

[0064] To extend the half-life, an IL2 thermostable variant or a combinatorial variant is further linked to Fc via a linker to constitute a fusion protein (hereinafter referred to as an IL2 variant fusion protein), and each element and its overall sequence are shown in Table 3.

[0065] Compared with the wild-type IL2 fusion protein (SEQ ID NO: 19), the IL2 variant fusion proteins described in the present invention have the following characteristics.

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

[0067] [Table 3-1]

[0068] [Table 3-2]

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

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

[0071] Examples 2-5: 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 formulations F1 to F12 were designed, and the specific formulations are shown in Table 4. (2) The IL2 mutant fusion protein stock solution obtained in Example 1 was replaced with F1-F12 by an ultrafiltration concentrate exchange process, and the protein concentration was adjusted to 2 mg / mL. (3) The stability of the IL2 mutant fusion protein in the twelve buffer systems was comprehensively examined by 25°C and 40°C accelerated stability tests, and the optimal buffer system was screened for subsequent additive and surfactant screening. The evaluation indicators included appearance, pH value, protein concentration, purity (SEC-HPLC), CE-SDS(NR), CE-SDS(R), and dynamic light scattering (DLS). Details of the evaluation method are shown in Table 4.

[0072] [Table 4]

[0073] 2. Screening results for the buffer system 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) Ranking of appearance quality: F1, F3 > F2, F7, F8, F9, F11 > F10, F12 > F4, F5, F6. (2) pH values: The pH values ​​of the 12 buffer systems remained stable, and no significant changes were observed. (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: When all the indicators detected in the 25°C and 40°C accelerated stability tests were combined, formulation F3 (20 mM sodium acetate, pH 5.5) showed superior stability compared to the other formulations, and therefore F3 was selected as the next development.

[0086] Example 3: Screening of additives 1. Screening Method Based on the optimal buffer system (20 mM acetate-sodium acetate, pH 5.5) screened in Example 2, seven formulations were designed. The stability of the IL2 mutant fusion protein (IL2 mutant fusion protein concentration of 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 to screen subsequent surfactants. The indicators considered included appearance, pH value, concentration, SEC-HPLC, DLS, and CE-SDS (NR&R). The specific formulation compositions and methods of consideration are shown in Table 15.

[0087] [Table 15]

[0088] 2. Results of additive screening 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) Ranking of appearance quality: F3-2 > F3-1 > F3-3 > F3-5 > F3-4, F3-6 > F3-7. (2) pH value ranking: There were no significant differences in the data among the groups. (3) Concentration ranking: There were no significant differences in the data among the groups. (4) DLS superiority / inferiority order: F3-2>F3-1>F3-3, F3-4, F3-5, F3-6>F3-7. (5) SEC-HPLC ranking: F3-1, F3-3, F3-4, F3-5, F3-6 > F3-7 > F3-2. (6)CE-SDS(R) Superiority: 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 2-8°C, were first excluded. Formula F3-1, which contains glycine, showed a higher oligomer content detected by CE-SDS(NR) than F3-2 and F3-3 at 40°C-4W, indicating that the IL-2 protein is more easily degraded in formula F3-1. The SEC-HPLC detection index for F3-2 did not differ significantly from the other groups, and while the proportion of other peaks was relatively high at 40°C-4W, there were no significant differences from the other groups at 2-8°C and 25°C. Therefore, considering the stability data comprehensively, formulas F3-2 and F3-3 showed superior stability compared to the other formulas. We selected formulations F3-2 (containing 140 mM arginine hydrochloride) and F3-3 (containing 8% w / v sucrose) and further investigated the stability of the formulations after adding surfactants.

[0100] Example 4: Consideration of Surfactants 1. Method of consideration The stability of formulations after adding the surfactant PS-80 was examined based on formulations F3-2 and F3-3. Details of the formulations are shown in Table 22. Details of the examination method are shown in Table 23. Examination indicators included appearance, pH, protein concentration, DLS, SEC-HPLC, CE-SDS (NR&R), and insoluble microparticles (MFI). Based on the examination results, an appropriate formulation was selected to confirm 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. Investigation Results In this example, the protective effect of polysorbate 80 (PS80), a surfactant for proteins in a pH 5.5 acetic acid system, was investigated. As shown in the results, proteins exhibited good resistance to high temperatures, repeated freeze-thaw cycles, and shaking at concentrations of 2 mg / mL and 5 mg / mL under conditions of 20 mM sodium acetate, 140 mM arginine hydrochloride, and 0.04% or 0.06% PS80 (w / v). Furthermore, proteins exhibited good resistance to high temperatures, repeated freeze-thaw cycles, and shaking at a protein concentration of 2 mg / mL under conditions of 20 mM sodium acetate, 8% sucrose (w / v), and 0.04% PS80 (w / v). There were no significant differences between the formulations in appearance, pH, concentration, protein particle size (DLS), SEC-HPLC, and CE(NR). Formulation F3-3-1, with 8% sucrose added, showed superior CE-SDS(R) results in protein purity at 40°C-4W compared to other formulations using arginine hydrochloride, with a peak ratio of 2.68%, which was 4.25% to 5.05% lower than the other formulations. Therefore, PS80 can effectively protect proteins from environmental and manufacturing shear forces and prevent protein aggregation.

[0113] 4. Research on drug discovery The stability of a 5 mg / mL IL-2 mutant fusion protein was investigated in a 20 mM sodium acetate-sodium acetate, pH 5.2, 8% sucrose (w / v), and 0.02% PS80 (w / v) system. The results showed that the 5 mg / mL IL-2 mutant fusion protein had good drug discovery potential in drug discovery studies, details of which are shown in Table 32.

[0114] [Table 32]

[0115] 5. Conclusion When screening surfactants, considering that formulation F3-3-1 showed superior results in CE-SDS(R) compared to other formulations, and that both 2 mg / mL and 5 mg / mL belong to the low-concentration protein category, and that the 5 mg / mL IL2 mutant fusion protein showed good stability in drug discovery studies of formulations similar to F3-3-1, we selected 5 mg / mL IL2 mutant fusion protein, 20 mM acetate-acetate, pH 5.5, 8% sucrose (w / v), and 0.04% PS80 (w / v) as the target formulation for the following formulation confirmation stability study.

[0116] Example 5: Formulation Stability Confirmation 1. Prescription confirmation stability method A target formulation (5 mg / mL IL2 mutant fusion protein, 20 mM sodium acetate, pH 5.5, 8% sucrose (w / v), 0.04% PS80 (w / v)) was selected, and a formulation confirmation stability study was conducted. 1 mL of the liquid formulation was dispensed into a 2 mL vial, and a sealed packaging system was assembled using a rubber stopper and an aluminum-plastic cap. The stability was examined both in the normal and reversed orientations, and the methods of examination are shown in Table 33.

[0117] [Table 33]

[0118] 2. Prescription confirmation stability results The main results regarding prescription confirmation stability are summarized in the table below.

[0119] [Table 34-1]

[0120] [Table 34-2]

[0121] 3. Conclusion of Prescription Verification Stability Study

[0122] After leaving the samples at a low temperature of 2-8°C for 3 months and examining the accelerated stability at 25°C for 3 months, there were no significant changes in conventional detection indicators, insoluble particles, purity, charge isomers, or activity between the upright and reversed samples, and there were no significant differences between the upright and reversed samples. When the accelerated stability at 40°C was examined for 4 weeks, the main peak of the charge variant decreased by 9.2%, tending to approach the acidic peak value, and the purity and charge variants changed significantly.

[0123] In summary, during the real-time stability testing at low temperatures (2-8°C) and the 25°C accelerated stability test, there were no significant changes in the conventional detection indicators, insoluble particles, purity, charge isomers, or activity of the sample. All indicators remained within the quality standards, suggesting that the IL2 mutant fusion protein exhibits relatively good stability in this formulation. In the high-temperature (40°C) accelerated stability test, there was a tendency for purity and charge isomers to decrease, suggesting that the formulation presented in this invention is sensitive to high temperatures.

[0124] Example 6: Study of IL2 mutant fusion protein injection in DTH model mouse monotherapy pharmacodynamics This example evaluated the monotherapy efficacy of an IL2 mutant fusion protein injection in a KLH-induced delayed-type hypersensitivity (DTH) model mouse. The IL2 mutant fusion protein was IL2 mutant 1-2-linker-Fc (V91R / Y31V / A73L / H79Q), hereafter referred to as IL2-1-2, and its sequence is shown in SEQ ID NO: 15. The positive control was Fc-linker-IL2 mutant 6 (V91K), i.e., AMG592, and its sequence is shown in SEQ ID NO: 20. The injection formulations for both IL2-1-2 and AMG592 were 5 mg / mL of IL2-1-2 or AMG592, 20 mM sodium acetate, pH 5.5, 8% sucrose (w / v), and 0.04% PS80 (w / v).

[0125] 6.1. Classification of Animals Using 6-8 week old BALB / c mice, the body weight of each mouse was weighed, and they were randomly divided into eight experimental groups, G0-G7, with 10 mice in each group, according to their body weight. Administration was started on the day of group division and designated as Day 0. Both IL2-1-2 and AMG592 were administered by subcutaneous injection once every three days, while CsA was administered by intraperitoneal injection once daily. Administration continued until the end of the experiment.

[0126] 6.2. Method for constructing animal models 6.2.1. Preparation of Reagents (1) 3 mg / mL of KLH: Lipo-dried powder was weighed and prepared as a 3 mg / mL KLH solution 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 can be emulsified by the connecting tube syringe method, and the antigen can be sufficiently emulsified to form a viscous emulsion. (3) 1 mg / mL KLH solution: 3 mg / mL KLH was diluted three times to 1 mg / mL with PBS.

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

[0128] 6.3. The group division and administration methods are shown in Table 35.

[0129] [Table 35]

[0130] 6.4 Detection Indicators 6.4.1. Body weight detection: The weight of the animals was weighed before grouping, and after grouping, the weight of the mice was weighed once every three days, in grams, and retained to one decimal place.

[0131] 6.4.2. Measurement of mouse ear thickness: (1) Before stimulation, the thickness of the right ear piece of each mouse was measured using a digital outer diameter micrometer and used as the background value. (2) The thickness of mouse ear pieces was measured at 24h, 48h, 72h, and 96h after stimulation.

[0132] 6.4.3. General Clinical Observation: Observe the animals at least twice a week during the adaptation and experimental periods. Observations should include, but are not limited to, the animals' mental state and eating habits. Unexpected observations should be recorded in an experimental logbook (on paper).

[0133] 6.5. Experiment complete 6.5.1. Care should be taken to observe the health condition of the animals during the administration interval, and if any or more of the following situations occur, administration should be temporarily suspended until the animals return to a normal state. (1) Administration was stopped when the animal's body weight fell below 81% of its body weight at the start of drug treatment, and resumed when it returned to 90% of its body weight at the start of drug treatment. (2) After administration, the animal's behavior was delayed or abnormal, or an acute stress phenomenon occurred.

[0134] 6.5.2. The End of Human-Centered Approach to Experimental Animals: If an abnormality occurred in the animal's condition during the experimental process, the animal's health was evaluated, and a decision was made on whether to provide treatment, whether the experiment could be continued, or whether to perform euthanasia.

[0135] 6.5.3. Euthanasia: 96 hours after stimulation of mice, the experiment was terminated, or if an anthropogenic endpoint was implemented, the animals were euthanized by asphyxiation using excess CO2.

[0136] 6.6.Statistical analysis methods The raw data for measurement and observation must be recorded. Analysis was performed based on the raw data, and the results were expressed as the mean number and standard error (Mean ± SEM). Simultaneously, statistical analysis was performed on the data. Changes in mouse ear piece thickness were analyzed using two-way ANOVA, and mouse body weight on day 10 was analyzed using one-way ANOVA. A p-value of < 0.05 was considered statistically significant.

[0137] 6.7. Results and Review 6.7.1. Effect of IL2-1-2 injection on mouse body weight in the DTH model Body weight of experimental animals was used as a reference index to indirectly measure drug toxicity. Body weight and percentage change in body weight of mice after administration of CsA, AMG592, and different doses of IL2-1-2 are shown in Figures 1 and 2 and Table 36. Each therapeutic agent was administered subcutaneously to DTH model mice once every three days. During observations throughout the experiment, mouse body weight fluctuated, and except for the decrease in body weight of mice in the hormone-administered CsA group, no mice experienced a decrease in body weight exceeding 10%, and all other administration groups showed a tendency for increased body weight. All mouse conditions were normal, with no other onset or death, and treatment with doses of 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 IL2 mutant 1-2 was found to be acceptable.

[0138] [Table 36]

[0139] 6.7.2. Effect of IL2-1-2 injection on changes in ear piece thickness in DTH model mice The changes in ear piece thickness in each treatment group of DTH model mice are shown in Figures 3-4. DTH model onset peaked 48 hours after KLH stimulation, and the change in ear piece thickness at the peak of onset in the model mice was 15.7 mm × 10⁶. -2The ear thickness increased by 74.1% compared to the initial ear piece thickness of the mouse, and the model construction was successful. In the IL2-1-2 injection treatment groups of 1, 0.3, 0.1, and 0.03 mg / kg, the ear piece thickness of the mouse was 2.45, 4.01, 9.09, and 12.26 mm × 10⁻¹. -2 The levels increased, changing by 10.2%, 22.0%, 41.3%, and 53.6% compared to mouse ear pieces before KLH stimulation. The positive drugs CsA 10 mg / kg and AMG592 1 mg / kg also had clear therapeutic effects in this model and showed some therapeutic effect compared to the solvent control group (P<0.0001), but their therapeutic effects were lower than those of IL2-1-2 at 0.3 and 1 mg / kg, respectively. From these results, IL2-1-2 had a clear therapeutic effect in KLH-induced DTH model mice.

[0140] Example 7: Pharmacokinetic study of IL2 mutant fusion protein injection in BALB / c mice This example evaluates the pharmacokinetic properties of an IL2 mutant fusion protein injection in BALB / c mice. The IL2 mutant fusion protein was IL2 mutant 1-2-linker-Fc (V91R / Y31V / A73L / H79Q), hereafter referred to as IL2-1-2, and its sequence is shown in SEQ ID NO: 15. The positive control was Fc-linker-IL2 mutant 6 (V91K), i.e., AMG592, and its sequence is shown in SEQ ID NO: 20. The injection formulations for both IL2-1-2 and AMG592 were 5 mg / mL of IL2-1-2 or AMG592, 20 mM sodium acetate, pH 5.5, 8% sucrose (w / v), and 0.04% PS80 (w / v).

[0141] 7.1. Classification of Animals After properly rearing 6-8 week old BALB / c mice, the body weight of each mouse was weighed, and using Excel, the mice were randomly divided into five experimental groups (G1-G5) of 5 mice each based on their body weight. Administration was started on the day of group division and labeled as Day 1.

[0142] 7.2. The group division and administration methods are shown in Table 37.

[0143] [Table 37]

[0144] 7.3. Detection Indicators 7.3.1. Detection of body weight: The body weight of the animals was weighed before grouping, and after grouping, before blood sampling, with the unit being grams.

[0145] 7.3.2. Classification of immune cells in mouse peripheral blood obtained by flow cytometry Four days after administration, peripheral blood was collected by euthanizing the mice and then puncturing their hearts. Immune cell surface markers (CD3+, CD4+, CD25+) and nuclear factors (Foxp3+, Ki-67+) were labeled with fluorescently tagged antibodies. Immune cell counting and group analysis were performed using the following FACS method. (1) After euthanizing the mice at the end of the experiment, the hearts were punctured and peripheral blood was collected in an EDTA-2K anticoagulant tube. The anticoagulant tube was gently inverted to thoroughly mix the blood and anticoagulant to prevent coagulation. (2) 300 μL of uncoagulated whole blood was transferred to a flow tube. A mixture of cell surface markers (CD3+, CD4+, CD25+) antibodies was added to the flow tube and incubated at room temperature in the dark for 20 minutes. (3) 1 mL of lysate was added to the flow tube to lyse the red blood cells and incubated at room temperature in the dark for 5 minutes. The cells were centrifuged at 20°C at 400 g / rcf for 6 minutes and the supernatant was discarded. (4) Step (3) was repeated to relyse the red blood cells. (5) The cells were resuspended in 4 mL of washing solution (PBS). The cell suspension was filtered through a filtration membrane and then transferred to a new flow tube. (6) Add 500 μL of fixative drop by drop to the flow tube while shaking, and incubate overnight in the dark at 4°C. (7) Add 2 mL of transmembrane nucleus solution to the flow tube and shake, then centrifuge the flow tube at 500 g / rcf at 4°C for 5 minutes and discard the supernatant. (8) Repeat step (7) and treat the cells again with 3 mL of transmembrane nucleus solution. (9) Add the antibody mixture of nuclear factors (Foxp3+, Ki-67+) to the flow tube and incubate in the dark at 4°C for 40 minutes, shaking the flow tube once every 20 minutes. (10) Add 4 mL of washing solution to the flow tube and shake, then centrifuge the flow tube at 500 g / rcf at 4°C for 5 minutes, discard the supernatant and resuspend the cells in 200 μL of PBS. (11) Equilibrate the counting microspheres by vortexing them at room temperature for at least 30 seconds. 50 μL of counting microspheres were added to the flow tube, and FACS detection was performed using a flow cytometer. Each flow tube should be shaken thoroughly before being placed in the instrument. (12) Based on the FACS results, the types of immune cells were analyzed using FlowJo software, and the cell counts were calculated according to the instructions for counting microspheres.

[0146] 7.3.3. General Clinical Observation: Observe the animals at least twice a week during the adaptation and experimental periods. Observations should include, but are not limited to, the animals' mental state and eating habits. Unexpected observations should be recorded in an experimental logbook (on paper).

[0147] 7.4. Experiment complete 7.4.1. During the administration interval, the health status of the animals must be carefully monitored, and if any one or more of the following situations occur, administration must be temporarily suspended until the animals return to a normal state. (1) Administration was stopped when the animal's body weight fell below 81% of its body weight at the start of drug treatment, and resumed when it returned to 90% of its body weight at the start of drug treatment. (2) After administration, the animal's behavior was delayed or abnormal, or an acute stress phenomenon occurred.

[0148] 7.4.2. The End of Human-Centered Approach to Experimental Animals: If an abnormality occurred in the animal's condition during the experimental process, the animal's health was evaluated, and a decision was made on whether to provide treatment, whether the experiment could be continued, or whether to perform euthanasia.

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

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

[0151] 7.6. Results and Review 7.6.1. Effects of IL2-1-2 injection on peripheral blood immune cells in BALB / c mice Table 38 shows the multiplier of change in peripheral blood immune cell counts compared to solvent control mice after BALB / c mice were treated with different doses of IL2-1-2. Subcutaneous injection of IL2-1-2 showed a significant promoting effect on mouse Treg cell proliferation. Compared to the solvent control group, Treg cells were significantly stimulated 3 days after administration of IL2-1-2 or AMG592. After subcutaneous administration of 0.1, 0.3, and 1 mg / kg of IL2-1-2, the multipliers of change in peripheral blood Treg cell counts in mice were 13.73, 35.27, and 12.05, respectively. The increase in Treg cell count in mice administered 1 mg / kg of AMG592 was higher than that in mice administered 1 mg / kg of IL2-1-2, but lower than that in mice administered 0.3 mg / kg. This result indicates that IL2-1-2 has a good promoting effect on Treg cells at relatively low doses. The multipliers of change in the Treg / CD4+ ratio are shown in Table 39. In the experiment, CD4+ Foxp3 cells or CD3+ CD4- cells were observed to be either unactivated or slightly activated.

[0152] [Table 38]

[0153] [Table 39]

[0154] 7.7. Conclusion The results showed that subcutaneous injection of IL2-1-2 solution significantly promoted Treg cell proliferation in peripheral blood of BALB / c mice, and that the mice clearly possessed tolerance to the dose of the test molecule.

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

[0156] 8.1. Classification of Animals After appropriately rearing 6-8 week old SD rats, they were randomly assigned to six experimental groups of three males and three females each. Administration began on the day of group assignment (marked as day 1), and IL2-1-2 injection solution was administered four times by subcutaneous or tail vein injection on days 1, 8, 15, and 22.

[0157] 8.2. The group division 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 dose, the animals' weight was 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. Immunocyte surface markers (CD3+, CD4+, CD25+) and nuclear factors (Foxp3+, Ki-67+) were labeled with fluorescent tags. The number of immune cells was calculated using 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 and anticoagulant to prevent coagulation. (2) 300 μL of uncoagulated whole blood was transferred to a flow tube. A mixture of cell surface markers (CD3+, CD4+, CD25+) antibodies was added to the flow tube and incubated at room temperature in the dark for 20 minutes. (3) 1 mL of lysate 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 relyse the red blood cells. (5) The cells were resuspended in 4 mL of washing solution (PBS). After filtering the cell suspension through a filtration membrane, it was 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) Add 2 mL of transmembrane sorbent to the flow tube and shake. Centrifuge the flow tube at 500 g / rcf at 4°C for 5 minutes and discard the supernatant. (8) Repeat step (7) and treat the cells again with 3 mL of transmembrane sorbent. (9) Add the antibody mixture of nuclear factors (Foxp3+, Ki-67+) to the flow tube and incubate in the dark at 4°C for 40 minutes, shaking the flow tube once every 20 minutes. (10) Add 4 mL of washing solution to the flow tube and shake. Centrifuge the flow tube at 500 g / rcf at 4°C for 5 minutes and discard the supernatant and resuspend the cells in 200 μL of PBS. (11) Equilibrate the counting microspheres by vortexing them at room temperature for at least 30 seconds. Add 50 μL of counting microspheres to the flow tube and perform FACS detection using a flow cytometer. Each flow tube should be shaken thoroughly before placing it in the instrument. (12) Based on the FACS results, the types of immune cells were analyzed using FlowJo software, and the number of cells was calculated according to the instructions for counting microspheres.

[0161] 8.3.3. General Clinical Observations During the adaptation and experimental periods, clinical observations should be conducted at least twice a week. Observations should include, but are not limited to, the animal's health status, expression, daily activities, mental state, and eating habits. Unexpected observations should be recorded in an experimental logbook (on paper).

[0162] 8.4. Experiment complete 8.4.1. Care must be taken to observe the health status of the animals during the experimental process and between doses. If any or more of the following situations occur, administration must be temporarily suspended until the animals return to a normal state. (1) Administration was stopped when the animal's body weight fell below 81% of its body weight before drug treatment, and resumed when it returned to 90% of its body weight before drug treatment. (2) After administration, the animal's behavior was delayed or abnormal, or an acute stress phenomenon occurred.

[0163] 8.4.2. The End of Humanism By evaluating the animals' health status, we determined whether to continue treatment or experiments, or whether to perform euthanasia if any or more of the following conditions occurred: (1) The animal's behavior is abnormal or paralyzed, (2) The weight of the animals is less than 20% of their weight before grouping, (3) The animal's body temperature is too low, and it is in a near-death state.

[0164] 8.4.3. Euthanasia At the end of the human-centered experiment or at the conclusion of the experiment, the animals were euthanized using excess CO2.

[0165] 8.5. Statistical analysis The results were analyzed using the mean number and standard error (Mean ± SEM).

[0166] 8.6.Results 8.6.1. Effects of IL2-1-2 on the number and ratio of immune cells in rat PBMCs Table 41 shows the changes in the ratio of immune cell counts in peripheral blood of SD rats after IL2-1-2 administration. IL2-1-2 can induce Treg cell amplification in SD rats after injection subcutaneously and via the tail vein. Compared to controls, low doses of IL2-1-2 (0.005 mg / kg subcutaneous injection or 0.002 mg / kg tail vein injection) slightly promoted Treg cell amplification from day 4 to day 25 post-administration. After subcutaneous injection of a medium dose of 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 a high dose of IL2-1-2 (1 mg / kg), the number of Treg cells in rat peripheral blood reached a peak value on day 11 (13.8 times the baseline value) and decreased to 3.18 times the baseline value on day 25. After tail vein injection of a high dose of IL2-1-2 (1 mg / kg), the number of Treg cells in rat peripheral blood increased significantly on both days 4 and 11, reaching 21.25 times and 30.13 times the baseline value, respectively. However, similar to the other treatment groups, the number of Treg cells decreased to 3.26 times the baseline value on day 25. This phenomenon may be due to the production of anti-drug antibodies (ADA) in the rats' bodies after multiple administrations. The amplification pattern of Ki-67+ cells in Treg cells also coincided with the increasing trend in the number of Treg cells.

[0167] The change in the ratio of Treg / CD4+ cell numbers is shown in Table 42. Low doses (0.005 mg / kg subcutaneous injection or 0.002 mg / kg tail vein injection) and medium doses (0.05 mg / kg subcutaneous injection) of IL2-1-2 had no or slight activating effect on the number of CD4+Foxp3- cells and CD3+CD4- cells in rat peripheral blood, but high doses (1 mg / kg subcutaneous injection or 1 mg / kg tail vein injection) of IL2-1-2 were observed to slightly activate the amplification of the above cells.

[0168] [Table 41]

[0169] [Table 42]

[0170] 8.7. Conclusion The above experimental results demonstrate that subcutaneous and tail vein injections of IL2-1-2 into SD rats can induce Treg cell proliferation, and that SD rats exhibit good tolerance to all doses of IL2-1-2.

Claims

1. A pharmaceutical composition, The fusion protein includes an IL2 variant and an antibody Fc block, a buffer, an osmotic regulator, and a surfactant. (1) The fusion protein has the amino acid sequence described in Sequence ID No. 15, and forms a homodimer by dimerization of the antibody Fc block. (2) The buffer solution is an acetate-sodium acetate buffer solution, the concentration of the buffer solution is 1 to 100 mM, and the pH value of the buffer solution is 4.5 to 6.

0. (3) The osmotic pressure adjusting agent is sucrose, and the concentration of the sucrose is 1 to 15% w / v. (4) The surfactant is polysorbate-80, the concentration of the surfactant is 0.01 to 0.1% w / v, and the pH value is 4.5 to 6.

0. Pharmaceutical composition.

2. The concentration of the fusion protein is 1 to 50 mg / mL. The pharmaceutical composition according to claim 1.

3. It is a freeze-dried preparation, A pharmaceutical composition according to claim 1 or 2 is formed after freeze-drying. Freeze-dried preparation.

4. The use of the pharmaceutical composition according to claim 1 or 2 in the manufacture of a drug for treating an autoimmune disease or a proliferative disorder, The aforementioned autoimmune diseases are selected from rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjögren's syndrome, polymyositis, dermatomyositis, scleroderma, psoriasis, psoriasis vulgaris, 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 1 diabetes mellitus, autoimmune vasculitis, eczema, or asthma. The proliferative disorder is selected from neoplasms, solid tumors, hematological malignancies, malignant ascites, or malignant pleural effusions, where the solid tumor may be benign or malignant, primary or metastatic, the malignant solid tumor may be carcinoma or sarcoma, such as epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, teratoma, lung tumor, papillomavirus-induced carcinoma, 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, or non-muscle-invasive bladder cancer, and the hematological malignancy may be selected from leukemia, lymphoma, or multiple myeloma. use.

5. A pharmaceutical composition according to claim 1 or 2 for treating an autoimmune disease or a proliferative disorder, The aforementioned autoimmune diseases are selected from rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjögren's syndrome, polymyositis, dermatomyositis, scleroderma, psoriasis, psoriasis vulgaris, 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 1 diabetes mellitus, autoimmune vasculitis, eczema, or asthma. The proliferative disorder is selected from neoplasms, solid tumors, hematological malignancies, malignant ascites, or malignant pleural effusions, where the solid tumor may be benign or malignant, primary or metastatic, the malignant solid tumor may be carcinoma or sarcoma, such as epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, teratoma, lung tumor, papillomavirus-induced carcinoma, 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, or non-muscle-invasive bladder cancer, and the hematological malignancy may be selected from leukemia, lymphoma, or multiple myeloma. Pharmaceutical composition.

6. A container containing the pharmaceutical composition according to claim 1 or 2, product.

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