Pharmaceutical composition of il2 mutant-antibody fc block fusion protein and use thereof

Abstract

Provided are a pharmaceutical composition of an IL2 mutant-antibody Fc block fusion protein and a preparation method therefor, a lyophilized preparation and a preparation or reconstitution method therefor, an obtained reconstitution solution, a corresponding product, a treatment method for an autoimmune disease and a proliferative disease, and corresponding pharmaceutical use. The pharmaceutical composition comprises: a fusion protein containing an IL2 mutant and an antibody Fc block, a buffer, an osmotic pressure adjuster, and a surfactant, and has good stability.

Description

Pharmaceutical composition of IL2 mutant-antibody Fc block fusion protein and its application Technical Field

[0001] The present invention relates to the field of pharmaceutical preparations, and in particular to a pharmaceutical composition of an IL2 mutant-antibody Fc block fusion protein and applications thereof. Background Art

[0002] Interleukin-2 (IL2) was originally identified as T cell growth factor (TCGF). Studies have found that IL-2 can bind to its receptor to activate the proliferation and activation of immune cells such as T cells and NK cells. IL-2 receptors include IL-2Rα subunit (CD25), IL-2Rβ subunit (CD122) and IL-2Rγ subunit (CD132). Different subunits can form receptor complexes with different affinities, including high-affinity receptor IL-2Rαβγ, intermediate-affinity receptor IL-2Rβγ, and low-affinity receptor IL-2Rα or IL-2Rαβ. Different cells express different types of IL-2R subunits. For example, traditional T cells under resting conditions express CD4 + T, CD8 + T cells generally express IL-2 receptor β (IL-2Rβ, CD122) and IL-2 receptor γ (IL-2Rγ, CD132) on their surface, and rarely express IL-2 receptor α (IL-2Rα, CD25). In addition to expressing IL-2Rβ and IL-2Rγ, regulatory T cells (Tregs) constitutively highly express IL-2Rα.

[0003] Currently, researchers are using IL2 or its mutants to activate immune cells or specific subsets of immune cells to treat tumors or autoimmune diseases. For example, high-dose IL2 is approved for the treatment of malignant melanoma or metastatic renal cell carcinoma, and the IL2-PEG-conjugated drug NKTR-358 has been approved for clinical trials for autoimmune diseases. Further development of new IL2 mutants to improve their stability, yield, and / or alter their binding ability to certain receptor complexes is of great significance for the development of IL-2 drugs. At the same time, IL2 is a large molecular protein that is prone to stability issues such as particles and aggregation in solution. Therefore, after obtaining a new IL2 mutant, it is important to develop a formulation system suitable for the new IL2 mutant and ensure its stability in solution.

[0004] Summary of the Invention

[0005] The present invention provides a pharmaceutical composition and a preparation method thereof, a lyophilized preparation and a preparation or reconstitution method thereof, a reconstituted solution obtained according to the reconstitution method, corresponding pharmaceutical uses, a method for treating autoimmune diseases, and a product.

[0006] In a first aspect, the present invention provides a pharmaceutical composition comprising an antibody Fc block fusion protein comprising an IL2 mutant, a buffer, an osmotic pressure regulator, and a surfactant;

[0007] Preferably, the IL2 mutant comprises at least Y31V, A73L and H79Q mutations compared to wild-type IL2;

[0008] Preferably, the IL2 mutant further comprises one or more mutations selected from the group consisting of a V91R mutation, an H16E mutation, and a D20A mutation, for example, further comprises an H16E mutation and a V91R mutation;

[0009] More preferably, the IL2 mutant has an amino acid sequence that is at least 80% identical to the sequence shown in any one of SEQ ID NOs: 3, 8 to 11, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical.

[0010] The amino acid sequence of the wild-type IL2 is shown in SEQ ID NO: 2.

[0011] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, the following:

[0012] (1) IL2 mutant, linker and antibody Fc block, or

[0013] (2) Antibody Fc block, linker, and IL2 mutant;

[0014] Preferably, the fusion protein forms a homodimer through dimerization of the antibody Fc block;

[0015] Preferably, the linker has (G4S) n An amino acid sequence of, wherein n is selected from 1, 2, 3, 4, 5 or 6;

[0016] Preferably, the linker has an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 12, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical;

[0017] Preferably, the antibody Fc block comprises an N297G mutation;

[0018] Preferably, the antibody Fc block has an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 13, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical;

[0019] More preferably, the fusion protein has an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 14-18, for example, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical.

[0020] In some specific embodiments, the wild-type IL2 has the amino acid sequence shown in SEQ ID NO: 1 or 2.

[0021] In some embodiments, the concentration of the fusion protein is about 1 to 50 mg / mL;

[0022] For example, the concentration of the fusion protein is about 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, 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, 10~20mg / mL, 10~15mg / mL, 20~50mg / mL, 20~40mg / mL, 20~30mg / mL, 30~50mg / mL, 30~40mg / mL, 40~50mg / mL;

[0023] For example, the fusion protein may be present at a concentration of 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, L, 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 or 50 mg / mL; or a concentration range between the above concentration values.

[0024] Preferably, the concentration of the fusion protein is about 2 mg / mL or 5 mg / mL.

[0025] In some 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,

[0026] The concentration of the buffer solution is about 1 to 100 mM;

[0027] For example, the concentration of the buffer is about 1-90 mM, 1-80 mM, 1-70 mM, 1-60 mM, 1-50 mM, 1-40 mM, 1-30 mM, 1-20 mM, 1-10 mM, 10-90 mM, 10-80 mM, 10-70 mM, 10-60 mM, 10-50 mM, 10-40 mM, 10-30 mM, 10-20 mM, 20-90 mM, 20-80 mM, 20-70 mM, 20-60 mM, 20-50 mM, 20-40 mM, 20-30 mM, 30-90 mM, 30-80 mM, 30-70 mM, 30-60 mM, 30-50 mM, 30-40 mM, 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;

[0028] 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 concentration range thereof.

[0029] Preferably, the concentration of the buffer is about 20 mM; and / or,

[0030] The pH value of the buffer solution is about 4.5 to 8.0;

[0031] For example, the pH value of the buffer is about 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;

[0032] For example, the pH value of the buffer solution is about 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0, or a concentration range between the above pH values;

[0033] Preferably, the pH of the buffer is about 5.5.

[0034] In some embodiments, the osmotic pressure regulator is selected from salt, amino acid, sugar or 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;

[0035] Preferably, the osmotic pressure regulator is selected from sodium chloride, glycine, arginine hydrochloride, sucrose, trehalose, mannitol or sorbitol, more preferably sucrose or arginine hydrochloride;

[0036] 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 / v, 5% w / v, 5.5% w / v, 6% w / v, 7% w / v, 8% w / v, 9% w / v, 10% w / v, 11% w / v, 12% w / v, 13% w / v, 14% w / v, 15% w / v, 16% w / v, 17% w / v, 18% w / v, 19% w / v, 20% w / v, 21% w / v, 22% w / v, 23% w / v, 24% w / v, 25% w / v, 26% w / v, 27% w / v, 28% w / v, 29% w / v, 30% w / v, 31% w / v, 32% w / v, 33% w / v, 34% w / v, 35% w / v, 36% w / v, 37% w / v, 38% w / v, 39% w / v, 40% w / v, 41% w / v, 42% w / v, 43% w / v, 44% w / v, 45% w / v, 46% w / v, 47% w / v, 48 % 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 therebetween, preferably 4.5% w / v or 8% w / v.

[0037] More preferably, the concentration of the amino acid is about 1-15% w / v, for example, the concentration of the amino acid is about 1-10% w / v, 1-5% w / v or 5-10% 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 a concentration range therebetween, preferably 2% w / v.

[0038] More preferably, the salt concentration is about 50-200 mM; for example, the salt concentration is about 50-150 mM, 50-100 mM, or 100-150 mM; for example, the salt concentration 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 a concentration range therebetween, preferably 140 mM.

[0039] More preferably, the osmotic pressure regulator is sucrose or arginine hydrochloride.

[0040] In some embodiments, the surfactant is polysorbate 80 (PS-80), polysorbate 20 (PS-20) or poloxamer; and / or,

[0041] 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, such as 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 therebetween, preferably about 0.04% w / v.

[0042] In some embodiments, the pharmaceutical composition comprises the fusion protein, acetic acid-sodium acetate buffer, sucrose, and polysorbate 80; preferably, the pharmaceutical composition comprises about 1 to 50 mg / mL of the fusion protein, about 1 to 100 mM acetic acid-sodium acetate buffer, about 1 to 15% w / v sucrose, and about 0.01 to 0.1% w / v polysorbate 80, with a pH of about 4.5 to 6.0;

[0043] More preferably, the pharmaceutical composition comprises 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 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 sucrose and about 0.01-0.1% w / v, 0.02-0.06% w / v polysorbate 80, and a pH of about 4.5-6.0 or 4.5-5.5;

[0044] More preferably, the pharmaceutical composition comprises 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, with a pH of about 4.5 to 6.0;

[0045] 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, at a pH of about 5.5.

[0046] In some embodiments, the pharmaceutical composition comprises a fusion protein, an acetic acid-sodium acetate buffer, arginine hydrochloride, and polysorbate 80; preferably, the pharmaceutical composition comprises about 1 to 50 mg / mL of the fusion protein, about 1 to 100 mM acetic acid-sodium acetate buffer, about 50 to 200 mM arginine hydrochloride, and about 0.01 to 0.1% w / v polysorbate 80, with a pH of about 4.5 to 6.0;

[0047] More preferably, the pharmaceutical composition comprises 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 acetic acid-sodium acetate buffer, about 50-200 mM, 50-150 mM, 100-150 mM arginine hydrochloride and about 0.01-0.1% w / v, 0.02-0.06% w / v polysorbate 80, and a pH of about 4.5-6.0 or 4.5-6.0;

[0048] More preferably, the pharmaceutical composition comprises about 1-10 mg / mL of the fusion protein, about 10-30 mM acetic acid-sodium acetate buffer, about 120-150 mM arginine hydrochloride and about 0.01-0.1% w / v polysorbate 80, with a pH of about 4.5-6.0.

[0049] 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 of about 5.5.

[0050] In some embodiments, the pharmaceutical composition is administered intravenously, intramuscularly or subcutaneously, preferably subcutaneously.

[0051] In a second aspect, the present invention also provides a method for preparing the aforementioned pharmaceutical composition, which comprises the step of mixing the fusion protein with the buffer, osmotic pressure regulator and surfactant; preferably, the method comprises the step of replacing the stock solution of the fusion protein into the buffer by ultrafiltration concentration.

[0052] In a third aspect, the present invention further provides a lyophilized preparation, wherein the lyophilized preparation is formed by lyophilizing the aforementioned pharmaceutical composition.

[0053] In a fourth aspect, the present invention further provides a method for preparing the aforementioned lyophilized preparation, wherein the method comprises the step of freeze-drying the aforementioned pharmaceutical composition.

[0054] In a fifth aspect, the present invention further provides a method for preparing a reconstituted solution of an antibody Fc block fusion protein containing an IL2 mutant, comprising reconstituted the aforementioned lyophilized preparation with a solvent, preferably, the solvent is water for injection.

[0055] In a sixth aspect, the present invention further provides a reconstituted solution of the antibody Fc block fusion protein containing the IL2 mutant prepared according to the aforementioned method.

[0056] In a seventh aspect, the present invention also provides use of the aforementioned pharmaceutical composition, lyophilized preparation or reconstituted solution in the preparation of a medicament for treating an autoimmune disease or a proliferative disease;

[0057] Preferably, the 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, 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;

[0058] Preferably, the proliferative disease is selected from neoplasms, solid tumors, hematologic tumors, 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 a carcinoma or sarcoma, for example, 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 renal carcinoma, head and neck cancer, bladder cancer, non-muscle invasive bladder cancer; the hematologic tumor may be selected from leukemia, lymphoma, multiple myeloma, for example, B cell lymphoma, T cell lymphoma, cutaneous T cell lymphoma, T cell large granular lymphocytic leukemia.

[0059] In an eighth aspect, the present invention further provides the aforementioned pharmaceutical composition, lyophilized preparation or reconstituted solution for use in treating autoimmune diseases or proliferative diseases;

[0060] Preferably, the 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, 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;

[0061] Preferably, the proliferative disease is selected from neoplasms, solid tumors, hematologic tumors, 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 a carcinoma or sarcoma, for example, 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 renal carcinoma, head and neck cancer, bladder cancer, non-muscle invasive bladder cancer; the hematologic tumor may be selected from leukemia, lymphoma, multiple myeloma, for example, B cell lymphoma, T cell lymphoma, cutaneous T cell lymphoma, T cell large granular lymphocytic leukemia.

[0062] In a ninth aspect, the present invention further provides a method for treating an autoimmune disease, wherein the method comprises administering to a subject an effective amount of the aforementioned pharmaceutical composition or reconstituted solution; preferably, the 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, 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.

[0063] 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 k, 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 an effective amount range consisting of the above effective amounts.

[0064] In a tenth aspect, the present invention further provides a method for treating a proliferative disease, wherein the method comprises administering to a subject an effective amount of the aforementioned pharmaceutical composition or reconstituted solution; preferably, the proliferative disease is selected from a neoplasm, a solid tumor, a hematologic tumor, malignant ascites, or a malignant pleural effusion; wherein the solid tumor may be benign or malignant, primary or metastatic, and the malignant solid tumor may be a carcinoma or a sarcoma, for example, 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 renal carcinoma, head and neck cancer, bladder cancer, or non-muscle invasive bladder cancer; the hematologic tumor may be selected from a leukemia, a lymphoma, or multiple myeloma, for example, a B-cell lymphoma, a T-cell lymphoma, a cutaneous T-cell lymphoma, or a T-cell large granular lymphocytic leukemia.

[0065] In an eleventh aspect, the present invention further provides a product comprising a container containing the aforementioned pharmaceutical composition, lyophilized preparation or reconstituted solution.

[0066] Definitions and Explanations of Terms

[0067] 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.

[0068] Unless otherwise indicated, the term "IL2" or "IL-2" as used herein refers to any natural or recombinant IL-2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) and domesticated or agricultural mammals. "IL2" or "IL-2" as used herein includes unprocessed mature IL-2 (e.g., IL-2 comprising an N-terminal signal peptide) and any form of IL-2 produced and processed in cells. "IL2" or "IL-2" as used herein also includes natural variants and fragments of IL-2, such as splice variants or allelic variants. "IL2" or "IL-2" as used herein also includes non-naturally occurring mutants, such as IL-2 mutants artificially modified by genetic engineering.

[0069] "Wild-type IL-2" is a form of IL-2 that is otherwise identical to an IL-2 mutant, except that the wild-type amino acid is retained at each mutated amino acid position in the IL-2 mutant. For example, if the IL-2 mutant is unprocessed mature IL-2, then the wild-type form of the mutant is unprocessed mature IL-2; if the IL-2 mutant is processed mature IL-2, then the wild-type form of the mutant is processed mature IL-2; if the IL-2 mutant is a truncated form of IL-2, then the wild-type form of the mutant is the corresponding truncated form of IL-2 having the wild-type sequence. For example, the "wild-type IL-2" of the present invention may have the following amino acid sequence: The 125th amino acid residue "X" represents C, S, A or V.

[0070] The term "mutation" in the present invention includes amino acid substitution, deletion, insertion or any combination thereof. The "mutation" in the present invention can be generated by genetic or chemical methods known in the art to generate amino acid mutations, including but not limited to site-directed mutagenesis, PCR, gene synthesis and the like.

[0071] The "mutation site" numbering of the IL-2 mutants described herein is counted starting from the amino acid residue A at position 1 of the "wild-type IL-2" set forth in SEQ ID NO: 1. For example, the "Y31 mutation" of the present invention refers to an IL-2 mutant in which the amino acid residue at position 31 (Tyr, Y) of the wild-type IL-2 set forth in SEQ ID NO: 1 is mutated. For example, the "Y31V mutation" of the present invention refers to an IL-2 mutant in which the amino acid residue at position 31 of the wild-type IL-2 set forth in SEQ ID NO: 1 is mutated from Y (Tyr) to V (Val).

[0072] The term " / " used herein between mutation sites represents "and," meaning that the mutations before and after the " / " are present simultaneously in the same IL-2 mutant. For example, "Y31 / A73 / H79" refers to mutations at Y31, A73, and H79 in the same IL-2 mutant, while "Y31V / A73L / H79Q" refers to the presence of Y31V, A73L, and H79Q mutations in the same IL-2 mutant.

[0073] The term "fusion protein" herein refers to a protein product produced by linking the coding regions of two or more genes through genetic recombination, chemical methods, or other appropriate methods, with expression under the control of the same regulatory sequences. In the fusion proteins of the present invention, the coding regions of two or more genes can be fused at one or more locations via a sequence encoding a linker peptide. Linkers can be used to construct the fusion proteins of the present invention.

[0074] The term "linker" herein refers to a peptide used to connect IL-2 to another protein molecule or protein fragment to ensure proper protein folding and stability. The other molecule includes, but is not limited to, an antibody Fc block. The "linker" herein is preferably (GGGGS)n, where n can be 0, 1, 2, 3, 4, 5, or 6.

[0075] The term "antibody Fc block" herein refers to the constant region of an immunoglobulin chain, particularly the carboxyl terminus or a portion thereof, which lacks antigen-binding activity and is the site of interaction between the antibody molecule and effector molecules or cells. The "Fc" herein can be any Fc or variant thereof, derived from human or non-human mammals. For example, an immunoglobulin Fc can comprise a combination of two or more of the heavy chain CH1, CH2, CH3, and CH4 domains, and an immunoglobulin hinge region. Fc can be derived from immunoglobulins of different species, preferably human. Based on the amino acid sequence of the heavy chain constant region, immunoglobulins can be divided into five main classes: IgA, IgD, IgE, IgG, and IgM. Some of these can be further divided into subclasses (isotypes), such as 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, it comprises a CH2 domain, a CH3 domain and an immunoglobulin hinge region of IgG1, and the starting amino acid position of the hinge region can vary. Unless otherwise indicated, the amino acid residues in the Fc, constant region or antibody of the present invention are numbered according to the EU numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991, also known as the EU index.

[0076] The term "pharmaceutical composition" herein refers to a mixture containing one or more IL-2 mutants, fusion proteins, nucleic acid fragments, vectors, or host cells described herein, and other components, including, but not limited to, pharmaceutically acceptable carriers, diluents, or adjuvants. The purpose of the pharmaceutical composition described herein is to facilitate administration to a living organism, promote absorption of the active ingredient, and thereby exert its biological activity.

[0077] The term "treatment" of the present invention refers to surgical or pharmaceutical treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or pathological changes in the treatment subject, such as the progression of autoimmune diseases (such as systemic lupus erythematosus). Useful or desired clinical results include, but are not limited to, alleviation of symptoms, weakening of the degree of disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Objects in need of treatment include objects already suffering from a disease or disease, objects susceptible to a disease or disease, or objects intended to prevent a disease or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, and alleviating, their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0078] The term "subject" as used herein refers to an organism that is being treated for a particular disease or condition (e.g., an autoimmune disease) as described herein. Examples of subjects and patients include mammals such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, cattle or other members of the bovine family, sheep, and horses, among others, being treated for a disease or condition.

[0079] As used herein, the term "effective amount" refers to an 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 ameliorating a disease symptom or the progression of that disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0080] The term "autoimmune disease" herein refers to a condition of a subject characterized by damage to cells, tissues, and / or organs caused by an immune response against its own cells, tissues, and / or organs. Exemplarily, 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, 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.

[0081] The term "proliferative disease" of the present invention refers to a condition in which the growth of cells or tissues is unregulated and / or abnormal, which can lead to the development of an unwanted condition or disease, and may or may not be cancerous, including but not limited to neoplasms, solid tumors, hematological tumors, malignant ascites or malignant pleural effusions. "Solid tumors" of the present invention may be benign or malignant, primary or metastatic; malignant solid tumors may be carcinomas or sarcomas. Exemplarily, "solid tumors" of 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 renal cancer, head and neck cancer, bladder cancer, and non-muscle invasive bladder cancer. Illustratively, the "hematologic malignancy" of the present invention 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 lymphocytic leukemia.

[0082] The term "IL-2 receptor α subunit" (IL-2Rα), also known as "CD25" in the present invention, refers to any natural IL-2 receptor α subunit or its mutants from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), including "full-length" unprocessed IL-2 receptor α subunit and any form of IL-2 receptor α subunit derived from processing in cells, as well as naturally occurring IL-2 receptor α subunit variants, such as splice variants or allelic variants, and also includes mutants artificially modified based on the natural IL-2 receptor α subunit.

[0083] The term "IL-2 receptor β subunit" (IL-2Rβ), also known as "CD122" in the present invention, refers to any natural IL-2 receptor β subunit or its mutants from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), including "full-length" unprocessed IL-2 receptor β subunit and any form of IL-2 receptor β subunit derived from processing in cells, as well as naturally occurring IL-2 receptor β subunit variants, such as splice variants or allelic variants, and also includes mutants artificially modified based on the natural IL-2 receptor β subunit.

[0084] The term "IL-2 receptor γ subunit" (IL-2Rγ), also known as "CD132" in the present invention, refers to any natural IL-2 receptor γ subunit or its mutants from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), including "full-length" unprocessed IL-2 receptor γ subunit and any form of IL-2 receptor γ subunit derived from processing in cells, as well as naturally occurring IL-2 receptor γ subunit variants, such as splice variants or allelic variants, and also includes mutants artificially modified based on the natural IL-2 receptor γ subunit.

[0085] The term "Treg" in the present invention is also called "regulatory T cells" or "T 调节细胞 ", refers to a specialized CD4 T cell that can suppress the response of other T cells + T cell type. Tregs 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 key role in inducing and maintaining peripheral self-tolerance to antigens. Tregs require IL-2 for their function and development, as well as for the induction of their suppressive characteristics.

[0086] As used herein, the terms "percent (%) sequence identity" and "percent (%) sequence identity" are interchangeable and refer to the percentage 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 sequences and introducing gaps (if necessary) to achieve maximum percent sequence identity (e.g., for optimal alignment, gaps can be introduced into one or both of the candidate and reference sequences, and for the purpose of comparison, non-homologous sequences can be ignored). For the purpose of determining percent sequence identity, alignment can be achieved in a variety of ways well known to those skilled in the art, such as using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAIi) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm that requires achieving maximum alignment over the full length of the compared sequences. For example, a reference sequence aligned for comparison with a candidate sequence can show that the candidate sequence exhibits from 50% to 100% sequence identity over the full length of the candidate sequence or a selected portion of the continuous amino acid (or nucleotide) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purposes can be, for example, 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

[0087] Figure 1. Body weight changes in DTH model mice after administration of CsA, AMG592, and IL2-1-2. Ten mice per group. Data points represent the mean body weight within each group, and error bars represent the standard error (SEM).

[0088] Figure 2 shows the weight change rate of DTH model mice after administration of CsA, AMG592, and IL2-1-2. Each group consisted of 10 mice. Data points represent the mean weight change rate of animals within each group, and error bars represent the standard deviation (SEM).

[0089] Figure 3. Changes in ear thickness in DTH model mice after administration of CsA, AMG592, and IL2-1-2. Ten mice per group. Data points represent the mean change in ear thickness within each group, and error bars represent the standard error of the mean (SEM). ****P < 0.0001, compared with the vehicle control group.

[0090] Figure 4. Changes in ear thickness in DTH model mice after administration of CsA, AMG592, and IL2-1-2. Ten mice per group. Data points represent the mean change in ear thickness within each group, and error bars represent the standard error of the mean (SEM). ****P < 0.0001, compared with the vehicle control group. DETAILED DESCRIPTION

[0091] 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 as the description proceeds. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer were used. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0092] The embodiments of the present invention are merely exemplary and do not limit the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements shall fall within the scope of protection of the present invention.

[0093] Description of Examples 1-5

[0094] 1. Ultrafiltration and concentration

[0095] Unless otherwise specified, the ultrafiltration concentration and liquid exchange in the following embodiments refers to: adding the protein stock solution to the corresponding ultrafiltration tube, using a centrifuge for ultrafiltration concentration, the protein is retained, and the buffer of the protein stock solution flows through the ultrafiltration membrane to achieve the purpose of protein concentration, and then adding the target solution to be replaced, the buffer of the original protein stock solution is diluted, and ultrafiltration concentration is continued. After concentration is completed, the target solution to be replaced is continued to be added, and ultrafiltration concentration is continued. The operation is repeated until the liquid exchange is completed.

[0096] 2. English abbreviations and their meanings

[0097] Unless otherwise specified, the following English abbreviations have the meanings shown in the following table:

[0098] Table 1 Abbreviations

[0099] 3. Binding activity detection method of IL2 mutant fusion protein involved in Example 5

[0100] (1) Coating: Select carbonate buffer (50 mM Na2CO3-NaHCO3, pH 9.6) at pH 9.6 as the coating solution. Dilute human IL-2R alpha protein (Acro, ILA-H52H9) to 0.5 μg / mL with the coating solution. Add 100 μl / well to the ELISA plate and coat overnight (>16 h) at 2-8°C.

[0101] (2) Blocking: Wash the plate, then add PBS containing 3% BSA at 300 μl / well, block at 37°C for 1 hour, and then wash the plate.

[0102] (3) Sample dilution: The IL2 mutant fusion protein was diluted with a starting concentration of 200 ng / mL (see Table 2 for details).

[0103] Table 2 IL2 mutant fusion protein dilution method

[0104] Note: Take 120 μL of prepared sample 1, add 280 μL of sample diluent, and mix to obtain sample 2; take 120 μL of prepared sample 2, add 280 μL of sample diluent, and mix to obtain sample 3. The dilution of samples 4 to 8 can be done in the same way.

[0105] (4) Add 100 μl / well of diluted IL2 mutant fusion protein sample and incubate at 37°C for 40-80 min.

[0106] (5) After washing, add mouse anti-human IgG Fcγ-HRP (Jackson Immuno, 209-035-098) diluted at 1:10000 (v / v) at 100 μl / well and incubate at 37°C for 40-80 min.

[0107] (6) After washing, add 100 μl / well of TMB (Thermo, 34029) to the ELISA plate. After reacting at room temperature in the dark for 5-9 min, add 100 μl / well of 1 M sulfuric acid to terminate the reaction. Read the plate within 2 min using a microplate reader. The measurement wavelength is 450 nm and the reference wavelength is 630 nm.

[0108] (7) Calculate the EC50 values ​​of the test and control samples using SoftMax Pro or GraphPad Prism software (available with a Molecular Devices microplate reader) or equivalent analysis software using four-parameter regression. Calculate the relative activity of the test sample using the following formula: Relative binding activity of the test sample (%) = EC50 of the control sample / EC50 of the test sample × 100. The test sample is considered qualified when its relative activity is between 70% and 130%.

[0109] Example 1 Preparation and purification of IL2 mutant fusion protein

[0110] 1. Design of IL2 mutant fusion protein

[0111] (1) Using multiple algorithms, we designed mutation sites Y31V / A73L / H79Q to improve the thermal stability of IL2.

[0112] (2) Using multiple algorithms, functional mutation sites V91R, H16E, D20A, or H16E / V91R were designed to weaken the interaction between IL2 and the IL2 receptor βγ subunit complex.

[0113] (3) Combining the above-mentioned thermostability mutations and functional mutations to obtain IL2 mutants containing the following combined mutations:

[0114] Y31V / A73L / H79Q / V91R;

[0115] Y31V / A73L / H79Q / H16E;

[0116] Y31V / A73L / H79Q / D20A;

[0117] Y31V / A73L / H79Q / H16E / V91R.

[0118] To extend the half-life, the IL2 thermostable mutant or combined mutant was further linked to Fc via a linker to form a fusion protein (hereinafter referred to as IL2 mutant fusion protein). The elements and their overall sequences are shown in Table 3.

[0119] Compared with the wild-type IL2 fusion protein (SEQ ID NO: 19), the IL2 mutant fusion protein of the present invention has the following characteristics:

[0120] (1) DSF analysis showed that compared with the wild-type IL2 fusion protein, the Tm values ​​of the IL2 mutant fusion proteins containing thermostability mutations were all increased: the Tm value of IL2 mutant 1-linker-Fc increased by about 8 to 9°C, the Tm value of IL2 mutant 1-2-linker-Fc increased by more than 12°C, the Tm value of IL2 mutant 1-3-linker-Fc increased by 8 to 9°C, the Tm value of IL2 mutant 1-4-linker-Fc increased by about 9 to 10°C, and the Tm value of IL2 mutant 1-5-linker-Fc increased by about 11 to 12°C.

[0121] (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 CD4 + CD25 - FoxP3 - T cells or CD8 + The activation of STAT5 phosphorylation level in T cells was significantly reduced, and the effect on Treg cell proliferation was similar to that of wild-type IL2 fusion protein, and the proliferation level of NK cells was significantly reduced.

[0122] (3) Compared with wild-type IL2 fusion protein or Fc-linker-IL2 mutant 6, IL2 mutant 1-2-linker-Fc showed better efficacy in terms of pharmacodynamics (DTH model mice and GVHD mouse model), higher exposure and bioavailability, and longer half-life in terms of pharmacokinetic, among which subcutaneous administration was more obvious.

[0123] (4) The IL2 mutant 1-2-linker-Fc (Y31V / A73L / H79Q / V91R, SEQ ID NO: 15) was selected for subsequent process development.

[0124] Table 3 Sequence information of IL2 mutant fusion proteins

[0125] 2. Expression of IL2 mutant fusion protein

[0126] The IL2 mutant fusion protein was expressed using CHO as a host cell and cultured in OPM-CHO CD063 medium (manufacturer: OPM, catalog number: C483260). The culture period did not exceed 14 days. The reactor control parameters were 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. When the culture reached the 6th day, the culture temperature was reduced to 34.0±0.5°C until harvest.

[0127] 3. Purification of IL2 mutant fusion protein

[0128] The IL2 mutant fusion protein was purified using a multi-step chromatography, concentration, and filtration process. The fermentation harvest supernatant was captured by Protein A affinity chromatography (AT Protein A Diamond Plus). The captured IL2 mutant fusion protein solution was incubated at low pH to inactivate potential viruses. After neutralization, the IL2 mutant fusion protein solution was deep filtered to remove precipitates. Cation exchange chromatography (Diamond S) was then performed 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. Filtration was then performed using a nanofiltration membrane to remove potential endogenous viruses. The IL2 mutant fusion protein solution was then concentrated and buffer exchanged using an ultrafiltration membrane. Finally, excipients were added and the concentration was adjusted, followed by filtration to obtain a stock solution of the IL2 mutant fusion protein. In subsequent examples, the stock solution was exchanged for the corresponding formulation system using ultrafiltration and concentration.

[0129] Examples 2 to 5 Develop formulations for maintaining the stability of the IL2 mutant 1-2-linker-Fc (V91R / Y31V / A73L / H79Q, SEQ ID NO: 15) in Example 1

[0130] Example 2 Screening of buffer system

[0131] 1. Screening plan

[0132] (1) Design 12 different buffer formulation systems F1-F12, the specific formulation systems are shown in Table 4. (2) The IL2 mutant fusion protein stock solution obtained in Example 1 was replaced into F1-F12 by ultrafiltration concentration and liquid replacement treatment, and the protein concentration was adjusted to 2 mg / mL. (3) Through 25°C accelerated stability test and 40°C accelerated stability test, the stability of IL2 mutant fusion protein in 12 buffer systems was comprehensively investigated, and the optimal buffer system was screened for subsequent screening of excipients and surfactants. The investigation indicators include appearance, pH value, protein concentration, purity (SEC-HPLC), CE-SDS (NR), CE-SDS (R), and dynamic light scattering (DLS). The investigation plan is detailed in Table 4.

[0133] Table 4 Preparation buffer system and screening plan

[0134] Note: X = appearance, pH, protein concentration, DLS, SEC-HPLC, CE-SDS (NR&R).

[0135] 2. Buffer system screening results

[0136] The main results of the buffer system screening are summarized as follows:

[0137] Table 5 Appearance test results of the formulation in the accelerated stability test at 25°C or 40°C

[0138] Table 6 pH test results of the formulation in the accelerated stability test at 25℃ or 40℃

[0139] Table 7 Concentration (mg / mL) test results of the formulation in the accelerated stability test at 25°C or 40°C

[0140] Table 8 DLS (radius, nm) test results of the formulation in the accelerated stability test at 25°C or 40°C

[0141] Table 9 SEC-HPLC test results of the formulation in the 25°C accelerated stability test

[0142] Table 10 SEC-HPLC test results of the formulation in the 40°C accelerated stability test

[0143] Table 11 CE-SDS (NR) test results of the formulation in the accelerated stability test at 25°C

[0144] Table 12 CE-SDS (NR) test results of the formulation in the accelerated stability test at 40°C

[0145] Table 13 CE-SDS(R) test results of the formulation in the accelerated stability test at 25°C

[0146] Table 14 CE-SDS(R) test results of the formulation in the accelerated stability test at 40°C

[0147] 3. Inspection results

[0148] (1) Appearance ranking: F1, F3 > F2, F7, F8, F9, F11 > F10, F12 > F4, F5, F6.

[0149] (2) pH value: The pH values ​​of the 12 buffer systems were stable and showed no significant changes.

[0150] (3) Ranking of protein concentration: F1, F2, F3, F6, F7, F8, F9, F11, F12 > F5 > F4, F10.

[0151] (4) DLS ranking: F1, F5, F9>F6, F7, F8>F3, F4, F10, F11, F12>F2.

[0152] (5) SEC-HPLC ranking: F2, F3, F5, F6, F7, F8, F9>F4, F10>F1>F11, F12.

[0153] (6) CE-SDS (NR) ranking: F2, F3, F6 > F1, F5, F7, F8, F9 > F4, F10 > F11, F12.

[0154] (7) CE-SDS(R) ranking: F8, F9>F3, F6>F2, F7, F10>F1, F4, F5, F11, F12.

[0155] Summary: After comprehensive consideration of 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) outperformed the other formulations and showed better stability. Therefore, F3 was selected for the next step of development.

[0156] Example 3 Screening of auxiliary materials

[0157] 1. Screening plan

[0158] Based on the optimal buffer system (20 mM acetic acid-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 was 2 mg / mL) after the addition of sodium chloride, glycine, arginine hydrochloride, sucrose, trehalose, mannitol, or sorbitol was independently evaluated. One optimal formulation was selected for subsequent surfactant screening. Evaluation criteria included appearance, pH, concentration, SEC-HPLC, DLS, and CE-SDS (NR&R). The specific formulation composition and evaluation plan are shown in Table 15.

[0159] Table 15 Excipient screening plan

[0160] Note: X = appearance, pH value, concentration, DLS, SEC-HPLC, CE-SDS (NR&R).

[0161] 2. Screening results of excipients

[0162] The main results of excipient screening are summarized below.

[0163] Table 16 Appearance test results of the formulation at different temperatures / time points

[0164] Table 17 pH value or protein concentration test results of the formulation at different temperatures / time points

[0165] Table 18 DLS test results (diameter, nm) of the formulation at different temperatures / time points

[0166] Table 19-1 SEC-HPLC test results of preparations at different temperatures / time points

[0167] Table 19-2 SEC-HPLC test results of the preparation at different temperatures / time points

[0168] Table 20-1 CE-SDS(R) test results of the preparation at different temperatures / time points

[0169] Table 20-2 CE-SDS(R) test results of the formulation at different temperatures / time points

[0170] Table 21-1 CE-SDS (NR) test results of the preparation at different temperatures / time points

[0171] Table 21-2 CE-SDS (NR) test results of the preparation at different temperatures / time points

[0172] 3. Inspection results

[0173] (1) Appearance ranking: F3-2>F3-1>F3-3>F3-5>F3-4, F3-6>F3-7.

[0174] (2) Ranking of pH values: There was no significant difference in the data among the groups.

[0175] (3) Ranking of concentrations: There was no significant difference in the data among the groups.

[0176] (4) DLS ranking: F3-2>F3-1>F3-3, F3-4, F3-5, F3-6>F3-7.

[0177] (5) SEC-HPLC ranking: F3-1, F3-3, F3-4, F3-5, F3-6>F3-7>F3-2.

[0178] (6) CE-SDS(R) ranking: F3-1, F3-3, F3-4, F3-5, F3-6 > F3-7 > F3-2.

[0179] (7) CE-SDS (NR) ranking: F3-2, F3-3 > F3-4 > F3-5 > F3-7 > F3-6 > F3-1.

[0180] Summary: Based on their appearance, formulations F3-4, F3-5, F3-6, and F3-7, which exhibited particle formation during long-term stability at 2-8°C, were initially excluded. Formulation F3-1, containing glycine, showed higher oligomer content detected by CE-SDS(NR) at 40°C-4 weeks than formulations F3-2 and F3-3, indicating that IL-2 protein is more susceptible to degradation in formulation F3-1. SEC-HPLC parameters for formulation F3-2 were similar to those of the other formulations. CE-SDS(R) showed a higher proportion of other peaks at 40°C-4 weeks, but no significant differences were observed between the two formulations during long-term stability at 2-8°C and 25°C. Therefore, considering the comprehensive stability data, formulations F3-2 and F3-3 outperformed the other formulations and demonstrated excellent stability. Formulations F3-2 (containing 140 mM arginine hydrochloride) and F3-3 (containing 8% w / v sucrose) were selected for further stability evaluation after the addition of surfactants.

[0181] Example 4 Investigation of surfactants

[0182] 1. Inspection plan

[0183] Based on formulations F3-2 and F3-3, the stability of the formulations after adding the surfactant PS-80 was investigated. The formulation details are shown in Table 22. The investigation plan is detailed in Table 23. The investigation criteria included appearance, pH, protein concentration, DLS, SEC-HPLC, CE-SDS (NR&R), and particulate matter insoluble fraction (MFI). Based on the investigation results, an appropriate formulation was selected for formulation stability confirmation.

[0184] Table 22 Preparation prescription

[0185] Table 23 Inspection plan

[0186] Note:

[0187] X = appearance, pH, protein concentration, DLS, SEC, CE-SDS (NR&R), particulate matter insoluble (MFI).

[0188] Shaking test (300 rpm, 25°C): 25°C, 300 rpm, continuous shaking.

[0189] Freeze-thaw test (-80~25℃): Multiple rounds of freeze-thaw. In each round of freeze-thaw, the sample is placed at -80℃ until frozen, and then placed at 25℃ until completely thawed, forming one round of freeze-thaw.

[0190] 2. Filter results

[0191] The main results are summarized as follows:

[0192] Table 24 Appearance test results at different temperatures / time points

[0193] Table 25 pH test results at different temperatures / time points

[0194] Table 26 Protein concentration (mg / mL) test results at different temperatures / time points

[0195] Table 27 DLS test results (diameter, nm) at different temperatures / time points

[0196] Table 28 Insoluble particles (MFI, particles / mL) detection results at different temperatures / time points

[0197] Table 29 SEC-HPLC test results at different temperatures / time points

[0198] Table 30 CE-SDS (NR) test results at different temperatures / time points

[0199] Table 31 CE-SDS(R) test results at different temperatures / time points

[0200] 3. Inspection results

[0201] This example examines the protective effect of the surfactant polysorbate 80 (PS80) on proteins in an acetic acid system at pH 5.5. The results show that the protein exhibits good tolerance to high temperatures, freeze-thaw cycles, and shaking at concentrations of 2 mg / mL and 5 mg / mL in the presence of 20 mM acetic acid-sodium acetate, 140 mM arginine hydrochloride, and 0.04% or 0.06% PS80 (w / v). The protein also exhibits good tolerance to high temperatures, freeze-thaw cycles, and shaking at a concentration of 2 mg / mL in the presence of 20 mM acetic acid-sodium acetate, 8% sucrose (w / v), and 0.04% PS80 (w / v). No significant differences were observed between the formulations in appearance, pH, concentration, protein particle size (DLS), SEC-HPLC, or CE (NR). Formulation F3-3-1, which uses 8% sucrose as an excipient, exhibited superior protein purity by CE-SDS (R) at 40°C-4W compared to other formulations using arginine hydrochloride. The proportion of other peaks was 2.68%, lower than the 4.25%-5.05% observed in other formulations. In summary, PS80 effectively protects proteins from environmental and production shear stresses, preventing aggregation.

[0202] 4. Drugability Research

[0203] The stability of 5 mg / mL IL-2 mutant fusion protein in 20 mM acetic acid-sodium acetate, pH 5.2, 8% sucrose (w / v), 0.02% PS80 (w / v) system was investigated. The results showed that 5 mg / mL IL2 mutant fusion protein exhibited good drugability in the formulation of drugability study, as shown in Table 32 for details.

[0204] Table 32 Drugability study results

[0205] 5. Conclusion

[0206] Considering that formulation F3-3-1 showed better results in CE-SDS(R) compared with other formulations when screening surfactants, and considering that 2 mg / mL and 5 mg / mL are both low-concentration proteins, and 5 mg / mL IL2 mutant fusion protein showed good stability in the drugability study of a formulation similar to F3-3-1, 5 mg / mL, 20 mM acetic acid-acetate, pH 5.5, 8% sucrose (w / v), and 0.04% PS80 (w / v) were selected as the target formulation for the next step of formulation confirmation stability study.

[0207] Example 5 Prescription Confirmation Stability

[0208] 1. Prescription confirmation stability plan

[0209] 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 for formulation confirmation stability study: 1 mL of the liquid preparation was dispensed into 2 mL vials, and a closed packaging system consisting of a rubber stopper and an aluminum-plastic cap was used. The vials were placed upright and inverted to investigate their stability. The investigation plan is shown in Table 33.

[0210] Table 33 Prescription confirmation stability plan

[0211] Note: X = appearance, pH, protein concentration, icIEF, SEC-HPLC, CE-SDS (NR), CE-SDS (R), insoluble particles (MFI), binding activity assay.

[0212] 2. Prescription confirmation stability results

[0213] The main results of the formulation confirmation stability are summarized in the table below.

[0214] Table 34-1 Prescription confirmation stability results

[0215] Table 34-2 Prescription confirmation stability results

[0216] 3 Conclusions of the prescription confirmation stability study

[0217] After three months of low-temperature storage at 2-8°C and three months of accelerated stability testing at 25°C, no significant changes were observed in conventional test parameters, insoluble particulates, purity, charge variants, or activity for both upright and inverted samples. No significant differences were observed between upright and inverted samples. During four weeks of accelerated stability testing at 40°C, the main peak of charge variants decreased by 9.2%, primarily trending toward an acidic peak, and significant changes were observed in purity and charge variants.

[0218] In summary, during the low-temperature (2-8°C) real-time stability test and the 25°C accelerated stability test, the sample's conventional test parameters, insoluble particulates, purity, charge variants, and activity remained unchanged, remaining within the specified range. This indicates that the IL2 mutant fusion protein exhibits good stability in this formulation. In the 40°C accelerated stability test, purity and charge variants showed a downward trend, indicating that the formulation of this invention is sensitive to high temperatures.

[0219] Example 6 Pharmacodynamic Study of IL2 Mutant Fusion Protein Injection in DTH Model Mice

[0220] In this example, the single-agent efficacy of an IL2 mutant fusion protein injection was evaluated in a KLH-induced delayed-type hypersensitivity (DTH) model mouse model. The IL2 mutant fusion protein was 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. A positive control was Fc-linker-IL2 mutant 6 (V91K), i.e., AMG592, with the sequence shown in SEQ ID NO: 20. The injection formulations for both IL2-1-2 and AMG592 were 5 mg / mL IL2-1-2 or AMG592, 20 mM acetic acid-sodium acetate, pH 5.5, 8% sucrose (w / v), and 0.04% PS80 (w / v).

[0221] 6.1 Animal grouping

[0222] BALB / c mice aged 6-8 weeks were used, and each mouse was weighed and randomly divided into 8 experimental groups (G0-G7) based on weight, with 10 mice in each group. Dosing began on the day of grouping, recorded as Day 0. IL2-1-2 and AMG592 were administered subcutaneously every three days in each dose group, and CsA was administered intraperitoneally once a day until the end of the experiment.

[0223] 6.2 Animal Model Construction Plan

[0224] 6.2.1 Reagent preparation

[0225] (1) 3 mg / ml KLH: Weigh the lyophilized powder and prepare a 3 mg / ml KLH solution with PBS.

[0226] (2) 1 mg / ml KLH emulsion: Mix KLH (3 mg / ml): IFA: CFA in a volume ratio of 1:1:1 and emulsify the antigen using a connecting tube syringe method to fully emulsify the antigen to form a viscous emulsion.

[0227] (3) 1 mg / ml KLH solution: 3 mg / ml KLH was diluted 3 times with PBS to 1 mg / ml.

[0228] 6.2.2 DTH modeling method

[0229] BALB / c mice were sensitized on day 0 by injection of 100 μl of a 1 mg / ml KLH emulsion (KLH, IFA, and CFA in a 1:1:1 volume ratio) into two points on the right side of the back. A control group received an equal volume of the emulsion without KLH. On day 5, each mouse was stimulated with an intradermal injection of 10 μl of a 1 mg / ml KLH solution into the right ear, producing symptoms of delayed-type hypersensitivity in the local skin tissue of the model mice. The thickness of the right ear was measured with a micrometer before and 24, 48, 72, and 96 hours after local KLH injection to assess the extent of local delayed-type hypersensitivity in the different treatment groups.

[0230] 6.3 Grouped dosing regimen is shown in Table 35

[0231] Table 35 Dosage regimen of IL2 mutant fusion protein injection in DTH model mice

[0232] 6.4 Detection indicators

[0233] 6.4.1 Body weight measurement: Weigh the animals before grouping and every three days after grouping. The weight is in grams and is rounded to one decimal place.

[0234] 6.4.2 Measurement of mouse ear thickness:

[0235] (1) Before stimulation, the thickness of the right ear of each mouse was measured with a digital micrometer as the background value.

[0236] (2) After stimulation, the thickness of the mouse ear pieces was measured at 24h, 48h, 72h, and 96h.

[0237] 6.4.3 General clinical observation: During the adaptive feeding period and the experimental period, the animals should be observed at least twice a week. The observation contents should include but not be limited to the animal's mental state, diet, etc. Any unexpected situation must be recorded in the experimental record book (paper).

[0238] 6.5 Experiment Termination

[0239] 6.5.1 The animal's health should be carefully observed between dosing intervals. If any one or more of the following conditions occur, dosing should be suspended until the animal returns to normal:

[0240] (1) When the animal's weight is less than 81% of the weight at the start of drug treatment, stop the drug administration. When the animal's weight recovers to 90% of the weight at the start of drug treatment, continue the drug administration.

[0241] (2) After administration, the animal's movements become slow or abnormal, or acute stress symptoms occur;

[0242] 6.5.2 Humane endpoints for experimental animals: During the experiment, when an animal's condition becomes abnormal, the animal's health status will be assessed to decide whether to provide treatment, whether the experiment can continue, or whether to perform euthanasia.

[0243] 6.5.3 Euthanasia: When the experiment is terminated 96 hours after the stimulation of mice or when a humane endpoint is implemented, the animals are euthanized by asphyxiation with an overdose of CO2.

[0244] 6.6 Statistical Analysis Methods

[0245] Raw data from measurements and observations must be recorded. Analyses were performed based on the raw data, and the results were expressed as mean ± SEM. Statistical analysis was performed on the data. Two-way ANOVA was used to analyze changes in ear thickness, and one-way ANOVA was used to analyze body weight on day 10. P < 0.05 was considered statistically significant.

[0246] 6.7 Results and Discussion

[0247] 6.7.1 Effect of IL2-1-2 Injection on Mouse Body Weight in the DTH Model

[0248] The body weight of experimental animals serves as an indirect indicator for assessing drug toxicity. The body weights and rates of weight change of mice following administration of CsA, AMG592, and various doses of IL2-1-2 are shown in Figures 1 and 2 and Table 36. DTH model mice were administered each therapeutic drug subcutaneously every three days. During the experimental period, the body weights of the mice fluctuated. Except for the hormone-administered CsA group, which saw a decrease in body weight, but no mice lost more than 10% of their body weight, the body weights of the mice in the other treatment groups showed an upward trend. All mice were healthy, with no other cases of morbidity or mortality. Treatment was well tolerated at doses of 10 mg / kg for CsA, 1 mg / kg for AMG592, and 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, and 1 mg / kg for IL2 mutant 1-2.

[0249] Table 36 Effects of Mutant 1-2 Injection on Body Weight of DTH Model Mice

[0250] aMean ± standard error of the mean (SEM), **P < 0.005.

[0251] 6.7.2 Effect of IL2-1-2 Injection on Ear Thickness in DTH Model Mice

[0252] The changes in ear thickness of DTH model mice in each treatment group are shown in Figures 3-4. The onset of DTH model reached its peak 48 hours after KLH stimulation. The change in ear thickness of model group mice at the peak of onset was 15.7 mm × 10 -2 The ear thickness of mice increased by 74.1% compared with the initial thickness of the mice. The model was successfully established. The ear thickness of mice in the 1, 0.3, 0.1 and 0.03 mg / kg IL2-1-2 injection treatment groups increased by 2.45, 4.01, 9.09 and 12.26 mm×10 -2 , changes of 10.2%, 22.0%, 41.3%, and 53.6% in the mouse ear pieces compared to before KLH stimulation. The positive drugs CsA 10mg / kg and AMG592 1mg / kg also had a significant therapeutic effect on this model, showing a certain therapeutic effect compared with the vehicle control group (P<0.0001), but the therapeutic effects of both were inferior to those of 0.3 and 1mg / kg IL2-1-2. In summary, IL2-1-2 has a significant therapeutic effect in KLH-induced DTH model mice.

[0253] Example 7 Pharmacodynamic Study of IL2 Mutant Fusion Protein Injection in BALB / c Mice

[0254] In this example, the pharmacodynamic properties of an IL2 mutant fusion protein injection were evaluated in BALB / c mice. The IL2 mutant fusion protein was 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. A positive control was Fc-linker-IL2 mutant 6 (V91K), i.e., AMG592, with the sequence shown in SEQ ID NO: 20. The injection formulations for both IL2-1-2 and AMG592 were 5 mg / mL IL2-1-2 or AMG592, 20 mM acetic acid-sodium acetate, pH 5.5, 8% sucrose (w / v), and 0.04% PS80 (w / v).

[0255] 7.1 Animal grouping

[0256] After 6-8 week old BALB / c mice were adapted to feeding, each mouse was weighed and randomly assigned to five experimental groups (G1-G5) based on their weight using EXCEL, with 5 mice in each group. Dosing began on the day of grouping, which was recorded as Day 1.

[0257] 7.2 Grouped dosing regimen, as shown in Table 37.

[0258] Table 37 Dosage regimen of IL2 mutant fusion protein injection in BALB / c mice

[0259] 7.3 Detection indicators

[0260] 7.3.1 Body weight measurement: Weigh the animals before grouping and before blood collection after grouping, in grams.

[0261] 7.3.2 Immune cell typing in mouse peripheral blood by flow cytometry

[0262] On day 4 after administration, mice were euthanized and peripheral blood was collected by cardiac puncture. Fluorescent antibodies were used to label immune cell surface markers (CD3+, CD4+, CD25+) and nuclear factors (Foxp3+, Ki-67+). Immune cell counts and grouping analyses were performed using the following FACS method:

[0263] (1) After euthanasia of the mouse at the end point, cardiac puncture was performed and peripheral blood of the mouse was collected into EDTA-2K anticoagulant tubes. The anticoagulant tubes were gently inverted to mix the blood and anticoagulant thoroughly to prevent coagulation. (2) 300 μl of uncoagulated whole blood was transferred to the flow cytometry tube. A mixture of antibodies against cell surface markers (CD3+, CD4+, CD25+) was added to the flow cytometry tube and incubated at room temperature in the dark for 20 minutes. (3) 1 ml of lysis buffer was added to the flow cytometry tube to lyse the red blood cells and incubated at room temperature in the dark for 5 minutes. Centrifuge at 400 g / rcf at 20°C for 6 minutes and discard the supernatant. (4) Repeat step (3) to lyse the red blood cells again. (5) Resuspend the cells with 4 ml of washing solution (PBS). Filter the cell suspension through a filter membrane and transfer it to a new flow cytometry tube. (6) 500 μl of fixative solution was added dropwise to the flow cytometry tube while shaking and incubate at 4°C in the dark overnight. (7) Add 2 ml of transmembrane and transnuclear solution to the flow tube and shake it, centrifuge the flow tube at 4°C, 500 g / rcf for 5 minutes, and discard the supernatant. (8) Repeat step (7) and treat the cells again with 3 ml of transmembrane and transnuclear solution. (9) Add a mixture of antibodies against nuclear factors (Foxp3+, Ki-67+) to the flow tube, incubate at 4°C in the dark for 40 minutes, and shake the flow tube every 20 minutes. (10) Add 4 ml of washing solution to the flow tube and shake it, centrifuge the flow tube at 4°C, 500 g / rcf for 5 minutes, discard the supernatant, and resuspend the cells with 200 μl of PBS. (11)

[0264] Vortex the equilibrated counting microspheres at room temperature for at least 30 seconds. Add 50 μl of counting microspheres to the flow cytometer tube and perform FACS analysis on the flow cytometer. Each flow cytometer tube should be thoroughly shaken before loading. (12) Analyze the immune cell types based on the FACS results using FlowJo software and count the cells according to the instructions on the counting microspheres.

[0265] 7.3.3 General clinical observation: During the adaptive feeding period and the experimental period, the animals should be observed at least twice a week. The observation contents include but are not limited to the animal's mental state, diet, etc. Any unexpected situation must be recorded in the experimental record book (paper).

[0266] 7.4 Experiment Termination

[0267] 7.4.1 The animal's health should be carefully observed between dosing intervals. If any one or more of the following conditions occur, dosing should be suspended until the animal returns to normal:

[0268] ① If the animal's body weight is lower than 81% of the weight at the start of drug treatment, stop the drug administration, and continue the drug administration when the animal's body weight recovers to 90% of the weight at the start of drug treatment;

[0269] ②After administration, the animal moves slowly or abnormally, or experiences acute stress;

[0270] 7.4.2 Humane endpoints for experimental animals: During the experiment, when an animal's condition becomes abnormal, the animal's health status is assessed to decide whether to provide treatment, whether the experiment can continue, or whether to perform euthanasia.

[0271] 7.4.3 Euthanasia: At the end of the experiment on day 26 after sensitization, the animals were euthanized by asphyxiation with an overdose of CO2.

[0272] 7.5 Statistical Analysis Methods

[0273] The raw data of measurement and observation must be recorded. The analysis is based on the raw data and the results are expressed as mean ± SEM.

[0274] 7.6 Results and Discussion

[0275] 7.6.1 Effects of IL2-1-2 Injection on Peripheral Blood Immune Cells in BALB / c Mice

[0276] Table 38 shows the fold change in peripheral blood immune cell counts in BALB / c mice treated with different doses of IL2-1-2 compared to the vehicle control group. Subcutaneous administration of IL2-1-2 significantly stimulated Treg cell proliferation in mice. Three days after administration of IL2-1-2 or AMG592 significantly stimulated Treg cells compared to the vehicle control group. Subcutaneous administration of 0.1, 0.3, and 1 mg / kg of IL2-1-2 resulted in fold changes of 13.73, 35.27, and 12.05 in peripheral blood Treg cell counts, respectively. The increase in Treg cell counts in the 1 mg / kg AMG592 group was greater than in the 1 mg / kg IL2-1-2 group, but less than in the 0.3 mg / kg group. This result suggests that IL2-1-2 can significantly stimulate Treg cell proliferation at relatively low doses. The fold change in the Treg / CD4+ ratio is shown in Table 39. In the experiment, it was observed that CD4+Foxp3 cells or CD3+CD4- cells were not activated or slightly activated.

[0277] Table 38. Changes in peripheral blood immune cell counts

[0278] Table 39 Treg / CD4+ ratio change fold

[0279] 7.7 Conclusion

[0280] The results showed that subcutaneous injection of IL2-1-2 injection could significantly promote the proliferation of Treg cells in the peripheral blood of BALB / c mice, and the mice were obviously tolerant to the dose of the test molecule.

[0281] Example 8 Pharmacodynamic Study of IL2 Mutant Fusion Protein Injection in SD Rats

[0282] In this example, the pharmacodynamic properties of an IL2 mutant fusion protein injection were evaluated in SD rats. The IL2 mutant fusion protein was IL2 mutant 1-2-linker-Fc (V91R / Y31V / A73L / H79Q), hereinafter referred to as IL2-1-2, with the sequence set forth in SEQ ID NO: 15. The IL2-1-2 injection formulation was 5 mg / mL IL2-1-2, 20 mM acetic acid-sodium acetate, pH 5.5, 8% sucrose (w / v), and 0.04% PS80 (w / v).

[0283] 8.1 Animal grouping

[0284] After acclimation, 6-8 week-old SD rats were randomly assigned to six experimental groups, with three males and three females in each group. Dosing began on the day of grouping (designated as Day 1), with IL2-1-2 injection administered four times subcutaneously or via the tail vein on Days 1, 8, 15, and 22.

[0285] 8.2 The grouped dosing regimen is shown in Table 40.

[0286] Table 40 Dosage regimen of IL2 mutant fusion protein injection in SD rats

[0287] Note: sc: subcutaneous injection, iv: intravenous injection

[0288] 8.3 Observation indicators

[0289] 8.3.1 Weight Monitoring

[0290] The animals were weighed after grouping and before each administration and recorded in g.

[0291] 8.3.2 Flow Cytometry Analysis of Immune Cell Phenotypes in Rat Peripheral Blood

[0292] Peripheral blood was 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 fluorescently labeled antibodies. The number of immune cells was calculated using fluorescence-activated cell sorting (FACS) as follows:

[0293] (1) Collect whole blood from the rat jugular vein into an anticoagulant tube containing EDTA-2K. Gently invert the anticoagulant tube to thoroughly mix the blood with the anticoagulant to prevent it from clotting. (2) Transfer 300 μl of unclotted whole blood to a flow cytometer. Add a mixture of antibodies against cell surface markers (CD3+, CD4+, CD25+) to the flow cytometer and incubate at room temperature in the dark for 20 minutes. (3)

[0294] Add 1 ml of lysis buffer to the flow cytometry tube to lyse the red blood cells and incubate at room temperature in the dark for 5 minutes. Centrifuge the flow cytometry tube at 20°C at 400g / rcf and discard the supernatant. (4) Repeat step (3) to lyse the red blood cells again. (5) Resuspend the cells with 4 ml of washing buffer (PBS). Filter the cell suspension through a filter membrane and transfer it to a new flow cytometry tube. (6) Add 500 μl of fixative solution dropwise to the flow cytometry tube while shaking and incubate overnight at 4°C in the dark. (7) Add 2 ml of transmembrane and transnuclear solution to the flow cytometry tube and shake it. Centrifuge the flow cytometry tube at 4°C at 500g / rcf for 5 minutes and discard the supernatant. (8) Repeat step (7) and treat the cells again with 3 ml of transmembrane and transnuclear solution. (9) Add a mixture of antibodies against nuclear factors (Foxp3+, Ki-67+) to the flow cytometry tube and incubate at 4°C in the dark for 40 minutes, shaking the flow cytometry tube every 20 minutes. (10) Add 4 ml of washing solution to the flow cytometer and shake. Centrifuge the flow cytometer at 500 g / rcf at 4°C for 5 minutes, discard the supernatant and resuspend the cells in 200 μl of PBS. (11) Vortex the counting microspheres for at least 30 seconds at room temperature. Add 50 μl of counting microspheres to the flow cytometer and perform FACS analysis on the flow cytometer. Each flow cytometer needs to be shaken thoroughly before loading. (12) Analyze the type of immune cells based on the FACS results using FlowJo software and count the number of cells according to the instructions of the counting microspheres.

[0295] 8.3.3 General clinical observations

[0296] Clinical observations should be conducted at least twice a week during the adaptive feeding period and the experimental period. Observations should include, but are not limited to, the animals' health, performance, daily activities, mental state, diet, etc. Any unexpected situations must be recorded in the experimental record book (paper).

[0297] 8.4 Experiment Termination

[0298] 8.4.1 The health status of the animals should be carefully observed during the experiment and between dosing intervals. If any one or more of the following conditions occur, dosing should be suspended until the animals return to normal:

[0299] (1) When the animal's body weight is less than 81% of its pre-treatment weight, drug administration should be stopped. When the animal's body weight recovers to 90% of its pre-treatment weight, drug administration can be continued.

[0300] (2) After administration, the animal's movements become slow or abnormal, or acute stress symptoms occur.

[0301] 8.4.2 Humane Endpoint

[0302] The animal's health status is assessed to determine whether treatment or experimentation should continue, or whether euthanasia should be performed if any one or more of the following conditions occur:

[0303] (1) The animal moves abnormally or becomes paralyzed;

[0304] (2) The animal's body weight was less than 20% of its body weight before grouping;

[0305] (3) The animal’s body temperature is too low and it is in a dying state, etc.

[0306] 8.4.3 Euthanasia

[0307] At the humane endpoint or at the end of the experiment, animals were euthanized with an overdose of CO2.

[0308] Statistical Analysis

[0309] The results were expressed as mean ± SEM.

[0310] 8.6 Results

[0311] 8.6.1 Effects of IL2-1-2 on the Number and Proportion of Immune Cells in Rat PBMCs

[0312] Table 41 shows the changes in the number and proportion of immune cells in the peripheral blood of SD rats after IL2-1-2 treatment. IL2-1-2 induced Treg cell expansion in SD rats following both subcutaneous and tail vein injection. Compared to the control, low-dose IL2-1-2 (0.005 mg / kg subcutaneously or 0.002 mg / kg via tail vein injection) showed a slight effect on Treg cell expansion from day 4 to day 25 after administration. Following subcutaneous administration of a medium-dose IL2-1-2 (0.05 mg / kg), the number of Treg cells in the rats' peripheral blood increased to 9.55-fold the baseline value on day 4 and remained at 3.16-fold the baseline value on day 25. Following subcutaneous administration of a high-dose IL2-1-2 (1 mg / kg), the number of Treg cells in the rats' peripheral blood peaked on day 11 (13.8-fold the baseline value) and decreased to 3.18-fold the baseline value on day 25. Following tail vein injection of a high-dose of IL2-1-2 (1 mg / kg), the number of Treg cells in the rats' peripheral blood significantly increased on days 4 and 11, reaching 21.25-fold and 30.13-fold of baseline values, respectively. However, similar to the other treatment groups, the number of Treg cells decreased to 3.26-fold of baseline values ​​on day 25. This phenomenon may be due to the production of anti-drug antibodies (ADA) in the rats after multiple drug administration. The expansion pattern of Ki-67+ Treg cells was consistent with the trend of increased Treg cell numbers.

[0313] The changes in the ratio of Treg / CD4+ cells are 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 activation effect on the number of CD4+Foxp3- cells and CD3+CD4- cells in the peripheral blood of rats, 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 expansion of the above cells.

[0314] Table 41. Changes in cell number ratio (based on baseline values)

[0315] Note: The results are expressed as mean ± standard error

[0316] Table 42 Changes in the ratio of Treg / CD4+ cells

[0317] Note: The results are expressed as mean ± standard error

[0318] 8.7 Conclusion

[0319] The above experimental results show that IL2-1-2 can induce the proliferation of Treg cells when injected subcutaneously or through the tail vein into SD rats, and SD rats show good tolerance to all doses of IL2-1-2.

Claims

1. A pharmaceutical composition comprising: A fusion protein comprising an IL2 mutant and an antibody Fc block, a buffer, an osmotic pressure regulator, and a surfactant; The fusion protein comprises, from N-terminus to C-terminus: (1) IL2 mutant, linker and antibody Fc block, or (2) Antibody Fc block, linker, and IL2 mutant; Preferably, the IL2 mutant comprises at least Y31V, A73L and H79Q mutations compared to wild-type IL2; More preferably, the IL2 mutant further comprises one or more mutations selected from the group consisting of a V91R mutation, an H16E mutation, and a D20A mutation, for example, further comprising 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 mutant has an amino acid sequence that is at least 80% identical to the sequence shown in any one of SEQ ID NOs: 3, 8 to 11.

2. The pharmaceutical composition according to claim 1, wherein The linker has an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 12, or the linker has (G4S) n The sequence shown, wherein n is selected from 1, 2, 3, 4, 5 or 6; The antibody Fc block has an N297G mutation, or the antibody Fc block has an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 13; Preferably, the fusion protein has an amino acid sequence that is at least 80% identical to the sequence shown in any one of SEQ ID NOs: 14 to 18; More preferably, the fusion protein forms a homodimer through dimerization of the antibody Fc block.

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

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein The buffer is selected from the group consisting of 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 4.5-8.

0.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein The osmotic pressure regulator is selected from: salt, amino acid, sugar or 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 osmotic pressure regulator is selected from sodium chloride, glycine, arginine hydrochloride, sucrose, trehalose, mannitol or sorbitol, and most preferably is sucrose or arginine hydrochloride; The concentration of the sugar or sugar alcohol is 1-15% w / v, and the concentration of the salt is 50-200 mM.

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

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein 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, with a pH value of 4.5-6.

0.

8. The pharmaceutical composition according to any one of claims 1 to 6, wherein The pharmaceutical composition comprises: a fusion protein, an 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, with a pH value of 4.5-6.

0.

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

10. A method for preparing the pharmaceutical composition according to any one of claims 1 to 9, wherein: The method comprises the step of mixing the fusion protein with the buffer, the osmotic pressure regulator and the surfactant; preferably, the method comprises the step of replacing the stock solution of the fusion protein into the buffer by ultrafiltration concentration.

11. A freeze-dried preparation, wherein The lyophilized preparation is formed by lyophilizing the pharmaceutical composition according to any one of claims 1 to 9.

12. A method for preparing the lyophilized preparation according to claim 11, wherein: The method comprises the step of freeze-drying the pharmaceutical composition according to any one of claims 1 to 9.

13. A method for preparing a reconstituted solution of the lyophilized preparation according to claim 11, comprising reconstituted the lyophilized preparation according to claim 12 with a solvent, preferably, the solvent is water for injection.

14. The reconstituted solution of the Fc fusion protein containing the IL2 mutant prepared by the method according to claim 13.

15. Use of 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 in the preparation of a medicament for treating autoimmune diseases or proliferative diseases; Preferably, the 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, 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, 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; the malignant solid tumor may be a carcinoma or a sarcoma, for example, 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 renal cancer, head and neck cancer, bladder cancer, non-muscle invasive bladder cancer; the hematological tumor may be selected from leukemia, lymphoma, multiple myeloma, for example, B cell lymphoma, T cell lymphoma, cutaneous T cell lymphoma, T cell large granular lymphocytic leukemia.

16. 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, for use in treating autoimmune diseases or proliferative diseases; Preferably, the 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, 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, 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; the malignant solid tumor may be a carcinoma or a sarcoma, for example, 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 renal cancer, head and neck cancer, bladder cancer, non-muscle invasive bladder cancer; the hematological tumor may be selected from leukemia, lymphoma, multiple myeloma, for example, B cell lymphoma, T cell lymphoma, cutaneous T cell lymphoma, T cell large granular lymphocytic leukemia.

17. A method for treating an autoimmune disease, wherein: The method comprises administering to a subject an effective amount of the pharmaceutical composition according to any one of claims 1 to 9 or the reconstituted solution according to claim 14; preferably, the 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, 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 1 diabetes, autoimmune vasculitis, eczema or asthma; preferably, the effective amount is 0.001 to 10 mpk.

18. A method for treating a proliferative disease, comprising administering to a subject an effective amount of the pharmaceutical composition according to any one of claims 1 to 9 or the reconstituted solution according to claim 14; preferably, the proliferative disease is selected from the group consisting of neoplasms, solid tumors, hematologic tumors, malignant ascites, and malignant pleural effusion; wherein: The solid tumor may be benign or malignant, primary or metastatic; the malignant solid tumor may be a carcinoma or a sarcoma, for example, 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 renal cancer, head and neck cancer, bladder cancer, non-muscle invasive bladder cancer; the hematological tumor may be selected from leukemia, lymphoma, multiple myeloma, for example, B cell lymphoma, T cell lymphoma, cutaneous T cell lymphoma, T cell large granular lymphocytic leukemia.

19. A product comprising 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.

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