anti-IL-17A antibody pharmaceutical composition and application thereof

文档序号：1944222 发布日期：2021-12-10 浏览：12次中文

阅读说明：本技术 抗il-17a抗体药物组合物及其用途 (anti-IL-17A antibody pharmaceutical composition and application thereof ) 是由刘洪川刘沛想张静王静刘辉李园园于 2020-06-10 设计创作，主要内容包括：本发明提供一种稳定的抗IL-17A抗体的药物组合物及其在医药上的应用。该药物组合物含有抗IL-17A抗体或其抗原结合片段、缓冲液,还可以含有至少一种稳定剂,任选的还可以含有表面活性剂。所述抗IL-17A抗体如文中所述。(The invention provides a stable anti-IL-17A antibody pharmaceutical composition and application thereof in medicine. The pharmaceutical composition comprises an anti-IL-17A antibody or antigen-binding fragment thereof, a buffer, and may further comprise at least one stabilizer, and optionally may further comprise a surfactant. The anti-IL-17A antibodies are as described herein.)

1. A pharmaceutical composition comprising:

(1) a buffer solution; and

(2) an anti-IL-17A antibody or antigen-binding fragment thereof;

wherein the anti-IL-17A antibody or antigen-binding fragment thereof comprises any one selected from the group consisting of:

(I) HCDR1, HCDR2 and HCDR3 having amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and LCDR1, LCDR2 and LCDR3 having amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; or

(II) HCDR1, HCDR2 and HCDR3 having amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively, and LCDR1, LCDR2 and LCDR3 having amino acid sequences shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively; or

(III) HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, respectively.

2. The pharmaceutical composition of claim 1, wherein the buffer is selected from the group consisting of an acetate buffer, a citrate buffer, a succinate buffer, and a histidine buffer; preferably, the buffer is a histidine buffer; preferably, the buffer is a histidine-hydrochloride buffer; preferably, the buffer has a concentration of about 10 to 30 mM; preferably, the pH of the buffer is about 5.0 to about 6.5, more preferably about 5.0 to about 6.0.

3. The pharmaceutical composition of claim 1 or 2, wherein the pharmaceutical composition further comprises a stabilizer selected from one or more of arginine hydrochloride, proline, glycine, sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol, and trehalose; preferably, the stabilizer comprises at least arginine hydrochloride.

4. The pharmaceutical composition of claim 3, wherein the stabilizing agent is sodium chloride at a concentration of about 30-200 mM; or the stabilizer is mannitol at a concentration of about 100 and 300 mM; or the stabilizer is sorbitol at a concentration of about 100 and 300 mM; or the stabilizer is sucrose at a concentration of about 100 and 300 mM; or the stabilizer is trehalose at a concentration of about 100 and 300 mM; or the stabilizer is arginine hydrochloride at a concentration of about 30-200 mM; or the stabilizer is proline at a concentration of about 100 and 300 mM; or the stabilizer is glycine at a concentration of about 100 and 300 mM;

or the stabilizer is a combination of about 30-200mM sodium chloride and about 30-200mM mannitol; or the stabilizer is a combination of about 30-200mM sodium chloride and about 30-200mM sucrose; or the stabilizer is a combination of about 30-200mM arginine hydrochloride and about 30-200mM mannitol; or the stabilizer is a combination of about 30-200mM arginine hydrochloride and about 30-200mM sucrose; preferably, the stabilizer is a combination of about 30-200mM arginine hydrochloride and about 30-200mM sucrose; more preferably, the stabilizer is a combination of about 60-120mM arginine hydrochloride and about 40-80mM sucrose.

5. The pharmaceutical composition of any one of claims 1-4, wherein the pharmaceutical composition further comprises a surfactant selected from polysorbate 80, polysorbate 20, or poloxamer 188, preferably the surfactant is polysorbate 20; preferably, the surfactant concentration is about 0.01% to about 0.1%, more preferably, the surfactant is about 0.01% to about 0.1% polysorbate 20.

6. The pharmaceutical composition of any one of claims 1-5, wherein the anti-IL-17A antibody comprises any one selected from the group consisting of (I) to (VI):

(I) a heavy chain variable region having an amino acid sequence shown in SEQ ID NO:19 and a light chain variable region having an amino acid sequence shown in SEQ ID NO: 20; or

(II) a heavy chain variable region having an amino acid sequence shown in SEQ ID NO:21 and a light chain variable region having an amino acid sequence shown in SEQ ID NO: 20; or

(III) a heavy chain variable region with an amino acid sequence shown as SEQ ID NO:21 and a light chain variable region with an amino acid sequence shown as SEQ ID NO: 22; or

(IV) heavy chain variable region with amino acid sequence shown as SEQ ID NO. 23 and light chain variable region with amino acid sequence shown as SEQ ID NO. 24; or

(V) a heavy chain variable region having an amino acid sequence shown in SEQ ID NO:23 and a light chain variable region having an amino acid sequence shown in SEQ ID NO: 25; or

(VI) a heavy chain variable region having an amino acid sequence shown in SEQ ID NO:26 and a light chain variable region having an amino acid sequence shown in SEQ ID NO: 27.

7. The pharmaceutical composition of any one of claims 1-5, wherein the anti-IL-17A antibody comprises any one selected from the group consisting of (I) to (VI):

(I) a heavy chain amino acid sequence as shown in SEQ ID NO:28 and a light chain amino acid sequence as shown in SEQ ID NO: 29; or

(II) a heavy chain amino acid sequence as shown in SEQ ID NO:30 and a light chain amino acid sequence as shown in SEQ ID NO: 29; or

(III) a heavy chain amino acid sequence as shown in SEQ ID NO:30 and a light chain amino acid sequence as shown in SEQ ID NO: 31; or

(IV) a heavy chain amino acid sequence as shown in SEQ ID NO:32 and a light chain amino acid sequence as shown in SEQ ID NO: 33; or

(V) the heavy chain amino acid sequence shown as SEQ ID NO:32 and the light chain amino acid sequence shown as SEQ ID NO: 34; or

(VI) a heavy chain amino acid sequence as shown in SEQ ID NO:35 and a light chain amino acid sequence as shown in SEQ ID NO: 36.

8. The pharmaceutical composition of any one of claims 1-7, wherein the anti-IL-17A antibody or antigen-binding fragment thereof is present at a concentration of about 1-300mg/mL, preferably about 20-200mg/mL, more preferably about 60-180mg/mL, most preferably about 80-150 mg/mL.

9. The pharmaceutical composition according to any one of claims 1 to 7, which comprises a component represented by any one of the following (1) to (12):

(1) (a) about 60mg/mL to 180mg/mL of an anti-IL-17A antibody or antigen-binding fragment thereof; (b) about 10-30mM histidine buffer, pH about 5.0-6.0; (c) about 100mM sorbitol; (d) and about 0.01% -0.1% polysorbate 20; or

(2) (a) about 60mg/mL to 180mg/mL of an anti-IL-17A antibody or antigen-binding fragment thereof; (b) about 10-30mM histidine buffer, pH about 5.0-6.0; (c) about 30mM to about 200mM arginine hydrochloride; (d) and about 0.01% -0.1% polysorbate 20; or

(3) (a) about 60mg/mL to 180mg/mL of an anti-IL-17A antibody or antigen-binding fragment thereof; (b) about 10-30mM histidine buffer, pH about 5.0-6.0; (c) a combination of about 30mM to about 200mM sodium chloride and about 30-200mM mannitol; (d) and about 0.01% -0.1% polysorbate 20; or

(4) (a) about 60mg/mL to 180mg/mL of an anti-IL-17A antibody or antigen-binding fragment thereof; (b) about 10-30mM histidine buffer, pH about 5.0-6.0; (c) about 30mM to about 200mM sodium chloride in combination with about 30-200mM sucrose; (d) and about 0.01% -0.1% polysorbate 20; or

(5) (a) about 60mg/mL to 180mg/mL of an anti-IL-17A antibody or antigen-binding fragment thereof; (b) about 10-30mM histidine buffer, pH about 5.0-6.0; (c) (ii) arginine hydrochloride in a combination of about 30mM to about 200mM and mannitol in a combination of about 30-200 mM; (d) and about 0.01% -0.1% polysorbate 20; or

(6) (a) about 60mg/mL to 180mg/mL of an anti-IL-17A antibody or antigen-binding fragment thereof; (b) about 10-30mM histidine buffer, pH about 5.0-6.0; (c) (ii) arginine hydrochloride in a combination of about 30mM to about 200mM and sucrose in a combination of about 30-200 mM; (d) and about 0.01% -0.1% polysorbate 20; or

(7) (a) about 80mg/mL of an anti-IL-17A antibody or antigen-binding fragment thereof; (b) about 20mM histidine buffer, pH about 5.5; (c) a combination of about 50mM sodium chloride and about 140mM mannitol; (d) and about 0.02% polysorbate 20; or

(8) (a) about 150mg/mL of an anti-IL-17A antibody or antigen-binding fragment thereof; (b) about 30mM histidine buffer, pH about 5.5; (c) about 135mM arginine hydrochloride; (d) and about 0.02% polysorbate 20; or

(9) (a) about 150mg/mL of an anti-IL-17A antibody or antigen-binding fragment thereof; (b) about 30mM histidine buffer, pH about 5.5; (c) a combination of about 50mM sodium chloride and about 120mM sucrose; (d) and about 0.02% polysorbate 20;

(10) (a) about 150mg/mL of an anti-IL-17A antibody or antigen-binding fragment thereof; (b) about 30mM histidine buffer, pH about 5.5; (c) (ii) arginine hydrochloride in a combination of about 90mM and mannitol in a combination of about 50 mM; (d) and about 0.02% polysorbate 20;

(11) (a) about 150mg/mL of an anti-IL-17A antibody or antigen-binding fragment thereof; (b) about 30mM histidine buffer, pH about 5.5; (c) (ii) arginine hydrochloride in a combination of about 90mM and sucrose in a combination of about 50 mM; (d) and about 0.02% polysorbate 20;

(12) (a) about 150mg/mL of an anti-IL-17A antibody or antigen-binding fragment thereof; (b) about 15mM histidine buffer, pH about 5.5; (c) (ii) arginine hydrochloride in a combination of about 90mM and sucrose in a combination of about 50 mM; (d) and about 0.02% polysorbate 20.

10. Use of a pharmaceutical composition according to any one of claims 1-9 in the manufacture of a medicament for the treatment or prevention of an IL-17A mediated disease; preferably, the disease includes inflammatory and autoimmune diseases, such as arthritis, rheumatoid arthritis, ankylosing spondylitis, chronic obstructive pulmonary disease, Systemic Lupus Erythematosus (SLE), lupus nephritis, asthma, multiple sclerosis, cystic fibrosis, psoriasis.

Technical Field

The present invention relates to the field of therapeutic pharmaceutical compositions. In particular, the invention relates to the field of pharmaceutical formulations comprising a humanized antibody that specifically binds to IL-17A.

Background

Interleukin 17 (IL-17), also known as CTLA-8 or IL-17A, plays a key role in the immune system. Six members of the IL-17 family, including IL-17A, IL-17B, IL-17C, IL-17D, IL-17E and IL-17F, all contain 4 highly conserved cysteine residues which are crucial, but the biological effects of the members are greatly different, wherein the homology and biological functions of IL-17A and IL-17F are the closest, and the study is the most intensive at present. IL-17A expressed in vivo has a 23 amino acid N-terminal signal peptide which is cleaved to yield mature IL-17A. Mature IL-17A is linked by disulfide bonds, typically secreted and present as homodimers, and sometimes linked to IL-17F to form heterodimers IL-17 AF. IL-17A or IL-17 generally refers to IL-17A homodimeric protein, which is produced primarily by helper T cells 17(T helper 17, Thl7) and may be synthetically secreted by other immune cells such as γ δ T cells, LTi (lymphoma Tissue indicator cells), ILCs (lnnate lymphoma cells) and NKT (Natural Killer T) cells. The expression regulation of IL-17A is very complex, and researches find that cytokines IL-6, IL-1 beta, TGF beta and the like induce the initial CD4+ T cell to be differentiated into Th17, but at the moment, the Th17 cell has weak stability, secretes a small amount of IL-17A, and has small tissue damage effect; IL-23, when present, causes inflammatory outbreaks and tissue damage by promoting Th17 cell stability and continuing to secrete IL-17A, up-regulating proinflammatory factors (IL-22, CSF-2 and IFN-gamma) and down-regulating the expression of proinflammatory factors (IL-2, IL-27 and IL-12). Therefore, the IL-17A pathway plays a critical role in tissue injury when IL-23 is abnormally expressed in tissues.

IL-17 is generally secreted in a site-specific manner and acts by binding to the IL-17 receptor (IL-17R) on the surface of target cells in local tissues. The IL-17R family includes five members of IL-17RA, IL-17RB, IL-17RC, IL-17RD and IL-17RE, and is widely expressed on various cell membranes. IL-17 acts primarily by binding to the IL-17RA/IL-17RC complex on the surface of cells of nonhematopoietic origin (e.g., epithelial cells, mesenchymal cells), promotes secretion of cytokines (such as IL-6, G-CSF, GM-CSF, IL-10, TGF- β, TNF- α), chemokines (including IL-8, CXCL1, and MCP-1) and prostaglandins (e.g., PGE2) by cells, induces neutrophil and macrophage aggregation, and releases Reactive Oxygen Species (ROS) to damage tissues.

Autoimmune diseases seriously threaten human health, such as psoriasis, rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, multiple sclerosis and the like. The research finds that the dyssecretion of IL-17 is closely related to the occurrence, development and development of the diseases. Antibodies targeting IL-17 can effectively alleviate the symptoms of autoimmune diseases by inhibiting the IL-17-IL-17R signaling pathway. Cosentyx (secukinumab) developed by Nowa is the first IL-17 monoclonal antibody in the world, and the approved indications comprise moderate to severe plaque psoriasis (plaque pseudoasis), thereby providing an important first-line biological treatment option for the broad psoriasis population. However, the development of anti-IL-17 antibodies with different characteristics, such as different structures, better curative effects, wider indications and the like, has urgent needs and important significance for treating autoimmune-related diseases such as psoriasis, rheumatoid arthritis, ankylosing spondylitis and the like and other diseases related to IL-17.

Disclosure of Invention

The pharmaceutical composition is a high-stability pharmaceutical composition containing a human antibody specifically bound with IL-17A. In particular, the present inventors have found that the combination of arginine hydrochloride and sucrose has the surprising feature of having high stability and low viscosity.

The present invention provides a pharmaceutical composition comprising: (1) a buffer solution; (2) an anti-IL-17A antibody or antigen-binding fragment thereof.

In some embodiments, the HCDR1 of the above anti-IL-17A antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NOs 1, 7, and 13; HCDR2 is selected from 2, 8 and 14; HCDR3 is selected from 3, 9 and 15; LCDR1 is selected from 4, 10 and 16; LCDR2 is selected from 5, 11, and 17; LCDR3 is selected from 6, 12 and 18.

In some embodiments, the VH of the above anti-IL-17A antibody is selected from SEQ ID NOs 19, 21, 23, and 26, and the VL is selected from SEQ ID NOs 20, 22, 24, 25, and 27.

In some embodiments, the HC of the above anti-IL-17A antibody is selected from the group consisting of SEQ ID NOs 28, 30, 32, and 35; LC is selected from SEQ ID NO 29, 31, 33, 34 and 36.

In some embodiments, the above-described anti-IL-17A antibody or antigen-binding fragment thereof has HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO 1, SEQ ID NO 2, and SEQ ID NO 3, respectively, and LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO 4, SEQ ID NO 5, and SEQ ID NO 6, respectively.

In some embodiments, the above-described anti-IL-17A antibody or antigen-binding fragment thereof has HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO 7, SEQ ID NO 8, and SEQ ID NO 9, respectively, and LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO 10, SEQ ID NO 11, and SEQ ID NO 12, respectively.

In some embodiments, the above-described anti-IL-17A antibody or antigen-binding fragment thereof has HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO 13, SEQ ID NO 14, and SEQ ID NO 15, respectively, and LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO 16, SEQ ID NO 17, and SEQ ID NO 18, respectively.

In some embodiments, the anti-IL-17A antibody or antigen-binding fragment thereof is present in the pharmaceutical composition at a concentration of about 1 to about 300mg/mL, preferably about 10 to about 250mg/mL, preferably about 20 to about 200mg/mL, preferably about 60 to about 180mg/mL, more preferably about 80 to about 150 mg/mL; more preferably, the anti-IL-17A antibody or antigen-binding fragment thereof described above has a concentration of about 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL, 110mg/mL, 120mg/mL, 130mg/mL, 140mg/mL, 150mg/mL, 160mg/mL, 170mg/mL, 180mg/mL or 200mg/mL, preferably about 80mg/mL or 150 mg/mL.

In some embodiments, the buffer is selected from one or more of an acetate buffer, a citrate buffer, a succinate buffer, and a histidine buffer.

In some embodiments, the buffer is a histidine buffer, preferably the histidine buffer is selected from a histidine-hydrochloride buffer or a histidine-acetate buffer, preferably a histidine-hydrochloride buffer.

In some embodiments, the histidine-hydrochloride buffer is prepared from histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride. In some embodiments, the histidine buffer is made up of 1-30mM L-histidine and 1-30mM L-histidine monohydrochloride. In some embodiments, the histidine buffer is made up of histidine and histidine hydrochloride in a molar ratio of 1:1 to 1: 4. In some embodiments, the histidine buffer is made up of histidine and histidine hydrochloride in a molar ratio of 1: 1. In some embodiments, the histidine buffer is made up of histidine and histidine hydrochloride in a molar ratio of 1: 3. In some embodiments, the histidine preparation is: histidine buffer pH5.5 was prepared from 4.5mM L-histidine and 15.5mM L-histidine monohydrochloride. In some embodiments, the histidine preparation is: histidine buffer pH5.5 was prepared from 7.5mM L-histidine and 22.5mM L-histidine monohydrochloride. In some embodiments, the histidine preparation is: histidine buffer pH 6.0 was prepared from 15mM histidine and 15mM histidine hydrochloride.

In some embodiments, the histidine buffer is a histidine-acetate buffer, preferably in a molar ratio of 1:1 to 1.5:1, preferably such a buffer has a pH of 5.5 ± 0.3, preferably about 5.5, preferably such a buffer contains 15-20mM histidine and 12-15mM acetic acid.

In some embodiments, the buffer is an acetate buffer, preferably, the acetate buffer is an acetate-sodium acetate buffer or an acetate-potassium acetate buffer, preferably an acetate-sodium acetate buffer.

In some embodiments, the buffer is a citrate buffer, and preferably, the citrate buffer is a citrate-sodium citrate buffer.

In some embodiments, the buffer is a succinic acid buffer, and preferably, the succinic acid buffer is a succinic acid-sodium succinate buffer.

In some embodiments, the buffer has a concentration of about 1 mM to about 200mM, preferably about 5mM to about 200mM, preferably about 10mM to about 50mM, preferably about 10mM to about 30 mM; preferably about 10-20mM, and the above buffer concentration is, by way of non-limiting example, about 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 40mM, 45mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 105mM, 110mM, 115mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, or 180mM, or any two of these ranges, preferably 10mM, 15mM, 20mM, or 30 mM.

In some embodiments, the pH of the buffer is about 5.0 to about 6.5, preferably about 5.0 to about 6.0, preferably about 5.5 to about 6.5, preferably about 5.0 to about 5.5, preferably about 5.5 to about 6.0, preferably about 6.0 to about 6.5, and non-limiting examples of the pH of the buffer are about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, preferably about 5.0, 5.5 or 6.0.

In some embodiments, the pharmaceutical composition further comprises a stabilizer selected from one or more of arginine hydrochloride, proline, glycine, sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol, and trehalose.

In some embodiments, the concentration of the stabilizer is about 10mM to 400mM, preferably 20mM to 300mM, and more preferably 30mM to 200 mM.

In some embodiments, the stabilizing agent is sodium chloride at a concentration of about 30 to 200 mM; or the stabilizer is mannitol at a concentration of about 100 and 300 mM; or the stabilizer is sorbitol at a concentration of about 100 and 300 mM; or the stabilizer is sucrose at a concentration of about 100 and 300 mM; or the stabilizer is trehalose at a concentration of about 100 and 300 mM; or the stabilizer is arginine hydrochloride at a concentration of about 30-200 mM; or the stabilizer is proline at a concentration of about 100 and 300 mM; or the stabilizer is glycine at a concentration of about 100 and 300 mM.

In some embodiments, the stabilizing agent is a combination of about 30-200mM sodium chloride and about 30-200mM mannitol; or the stabilizer is a combination of about 30-200mM sodium chloride and about 30-200mM sorbitol; or the stabilizer is a combination of about 30-200mM sodium chloride and about 30-200mM sucrose; or the stabilizer is a combination of about 30-200mM arginine hydrochloride and about 30-200mM mannitol; or the stabilizer is a combination of about 30-200mM arginine hydrochloride and about 30-200mM sucrose; or the stabilizer is a combination of about 30-200mM arginine hydrochloride and about 30-200mM sorbitol; or the stabilizer is a combination of about 30-200mM arginine hydrochloride and about 30-200mM trehalose.

In some embodiments, the stabilizer is sodium chloride. In some embodiments, the stabilizer is sodium chloride at a concentration of about 30-200mM, preferably at a concentration of about 50-190mM, preferably about 100-180mM, preferably about 120-170mM, preferably about 130-150mM, non-limiting examples of the concentration of sodium chloride are about 100mM, 110mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 170mM, 180mM, 190mM, 200mM, preferably 135mM or 140 mM.

In some embodiments, the stabilizing agent is mannitol. In some embodiments, the stabilizer is mannitol at a concentration of about 100-300mM, preferably about 150-300mM, preferably about 200-280mM, non-limiting examples of which are about 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 240 mM.

In some embodiments, the stabilizer is sorbitol. In some embodiments, the stabilizer is sorbitol at a concentration of about 100-300mM, preferably about 150-300mM, and preferably about 200-280mM, and non-limiting examples of the sorbitol concentration are about 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, and preferably 240 mM.

In some embodiments, the stabilizer is sucrose. In some embodiments, the stabilizer is sucrose at a concentration of about 100-300mM, preferably about 150-300mM, preferably about 200-280mM, and non-limiting examples of the sucrose concentration are about 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 220 mM.

In some embodiments, the stabilizing agent is trehalose. In some embodiments, the stabilizer is trehalose at a concentration of about 100-300mM, preferably about 150-300mM, and preferably about 200-280mM, non-limiting examples of trehalose concentrations are about 180mM, 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, and preferably 220 mM.

In some embodiments, the stabilizing agent is arginine hydrochloride. In some embodiments, the stabilizer is arginine hydrochloride at a concentration of about 30-200mM, preferably about 50-190mM, preferably about 100-180mM, preferably about 120-170mM, preferably about 130-150mM, and non-limiting examples of the arginine hydrochloride concentration are about 100mM, 110mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 170mM, 180mM, 190mM, 200mM, preferably 135mM or 140 mM.

In some embodiments, the stabilizing agent is proline. In some embodiments, the stabilizer is proline at a concentration of about 100-300mM, preferably about 150-300mM, preferably about 200-280mM, and non-limiting examples of the proline concentration are about 180mM, 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 240 mM.

In some embodiments, the stabilizer is glycine. In some embodiments, the stabilizer is glycine at a concentration of about 100-300mM, preferably about 150-300mM, preferably about 200-280mM, non-limiting examples of glycine concentrations are about 180mM, 200mM, 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 260 mM.

In some embodiments, the stabilizing agent is a combination of sodium chloride and mannitol. In some embodiments, the stabilizing agent is a combination of about 30-200mM sodium chloride and about 30-200mM mannitol, preferably a combination of about 40-150mM sodium chloride and about 40-180mM mannitol, preferably a combination of about 40-100mM sodium chloride and about 40-150mM mannitol, non-limiting examples of the stabilizing agent are a combination of about 50mM sodium chloride and about 120mM mannitol, a combination of about 50mM sodium chloride and about 140mM mannitol, and a combination of about 90mM sodium chloride and about 50mM mannitol.

In some embodiments, the stabilizer is a combination of sodium chloride and sucrose. In some embodiments, the stabilizing agent is a combination of about 30-200mM sodium chloride and about 30-200mM sucrose, preferably a combination of about 40-150mM sodium chloride and about 40-180mM sucrose, preferably a combination of about 40-100mM sodium chloride and about 40-150mM sucrose, non-limiting examples of the stabilizing agent are a combination of about 50mM sodium chloride and about 120mM sucrose, and a combination of about 90mM sodium chloride and about 50mM sucrose.

In some embodiments, the stabilizing agent is a combination of arginine hydrochloride and mannitol. In some embodiments, the stabilizing agent is a combination of about 30-200mM arginine hydrochloride and about 30-200mM mannitol, preferably a combination of about 40-150mM arginine hydrochloride and about 40-100mM mannitol, preferably a combination of about 60-120mM arginine hydrochloride and about 40-80mM mannitol, a non-limiting example of the stabilizing agent is a combination of about 90mM arginine hydrochloride and about 50mM mannitol.

In some embodiments, the stabilizer is a combination of arginine hydrochloride and sucrose. In some embodiments, the stabilizing agent is a combination of about 30-200mM arginine hydrochloride and about 30-200mM sucrose, preferably a combination of about 40-150mM arginine hydrochloride and about 40-100mM sucrose, preferably a combination of about 60-120mM arginine hydrochloride and about 40-80mM sucrose, a non-limiting example of the stabilizing agent is a combination of about 90mM arginine hydrochloride and about 50mM sucrose.

In some embodiments, the above pharmaceutical composition further comprises a surfactant selected from polysorbate 80, polysorbate 20, or poloxamer 188.

In some embodiments, the surfactant is selected from polysorbate 80.

In some embodiments, the surfactant is selected from polysorbate 20.

In some embodiments, the surfactant concentration is from about 0.001% to about 0.1%, preferably from about 0.01% to about 0.1%, preferably from about 0.02% to about 0.08%, calculated as w/v; by way of non-limiting example, the concentration of the above surfactant is about 0.02%, 0.04% or 0.08%, preferably 0.02%.

In some embodiments, the anti-IL-17A antibody or antigen-binding fragment thereof described above is selected from the group consisting of a murine antibody, a chimeric antibody, a humanized antibody, preferably a humanized antibody.

In some embodiments, the anti-IL-17A antibody or antigen-binding fragment thereof has the heavy chain variable region shown in SEQ ID NO. 19 and the light chain variable region shown in SEQ ID NO. 20.

In some embodiments, the anti-IL-17A antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:21 and a light chain variable region as set forth in SEQ ID NO: 20.

In some embodiments, the anti-IL-17A antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO. 23 and a light chain variable region as set forth in SEQ ID NO. 24.

In some embodiments, the anti-IL-17A antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:23 and a light chain variable region as set forth in SEQ ID NO: 25.

In some embodiments, the anti-IL-17A antibody or antigen-binding fragment thereof has the heavy chain variable region shown in SEQ ID NO. 26 and the light chain variable region shown in SEQ ID NO. 27.

In some embodiments, the above-described anti-IL-17A antibody or antigen-binding fragment thereof has a heavy chain amino acid sequence as set forth in SEQ ID NO. 28, and a light chain amino acid sequence as set forth in SEQ ID NO. 29.

In some embodiments, the above-described anti-IL-17A antibody or antigen-binding fragment thereof has a heavy chain amino acid sequence as set forth in SEQ ID NO. 30, and a light chain amino acid sequence as set forth in SEQ ID NO. 29.

In some embodiments, the above-described anti-IL-17A antibody or antigen-binding fragment thereof has a heavy chain amino acid sequence as set forth in SEQ ID NO:32, and a light chain amino acid sequence as set forth in SEQ ID NO: 33.

In some embodiments, the above-described anti-IL-17A antibody or antigen-binding fragment thereof has a heavy chain amino acid sequence as set forth in SEQ ID NO. 32, and a light chain amino acid sequence as set forth in SEQ ID NO. 34.

In some embodiments, the anti-IL-17A antibody or antigen-binding fragment thereof has a heavy chain amino acid sequence as set forth in SEQ ID NO. 35 and a light chain amino acid sequence as set forth in SEQ ID NO. 36.

In some embodiments, the above pharmaceutical composition comprises a component according to any one of (1) to (6) below, wherein the anti-IL-17A antibody or antigen-binding fragment thereof is according to any one of the embodiments of the present invention:

In some embodiments, the pharmaceutical composition described above comprises a component as shown in any one of the following (7) to (12):

In some embodiments, the pharmaceutical composition is a liquid formulation or a lyophilized formulation.

In some embodiments, the pharmaceutical composition is a liquid formulation.

In some embodiments, the liquid formulation or lyophilized formulation described above is stable at 2-8 ℃ for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months.

In some embodiments, the aqueous solution or lyophilized formulation is stable at 40 ℃ for at least 7 days, at least 14 days, or at least 28 days.

The invention also provides application of the pharmaceutical composition in preparing a medicament for treating or preventing IL-17A mediated diseases.

In some embodiments, the disease includes inflammatory and autoimmune diseases, such as arthritis, rheumatoid arthritis, ankylosing spondylitis, chronic obstructive pulmonary disease, Systemic Lupus Erythematosus (SLE), lupus nephritis, asthma, multiple sclerosis, cystic fibrosis, psoriasis.

Drawings

FIG. 1: ELISA was performed to detect the binding of the humanized antibody to human IL-17A. The antibodies hu31, hu43, hu44, hu59, hu60 and hu250 have higher specificity in combination with human IL-17A, and the EC50 of the antibodies is 8.13ng/mL, 8.64ng/mL, 6.764ng/mL, 6.102ng/mL, 5.776ng/mL and 6.351ng/mL respectively.

FIG. 2: FACS detection humanized antibodies blocked the binding of human IL-17A to IL-17RA on 293F cells. The antibodies hu31, hu43, hu44, hu59, hu60 and hu250 all have high-efficiency blocking effects, and the IC50 of the antibodies is 867.6ng/mL, 780.8ng/mL, 828.5ng/mL, 467.4ng/mL, 482.8ng/mL and 577.8ng/mL respectively.

FIG. 3: ELISA was performed to detect the effect of the humanized antibody in blocking IL-17A-mediated secretion of CXCL1 from epithelial cells. The antibodies hu31, hu43, hu44, hu59, hu60 and hu250 all inhibit IL-17A from inducing epithelial cells to express CXCL1 with high efficiency, and have stronger blocking effect than that of a control antibody.

FIG. 4: ELISA detects the effect of the humanized antibody on blocking IL-17A-mediated in vivo bioactivity. The antibodies hu31, hu43, hu44, hu60 and hu250 have the effects of efficiently inhibiting IL-17A from inducing mice to express CXCL1, and have stronger blocking effect than a control antibody.

FIG. 5-1: humanized antibody administration had an effect on clinical scores of imiquimod-induced psoriasis mice. Whereas hu31 and hu44 significantly inhibited imiquimod-induced skin scaling, induration, redness, etc. in the psoriasis model in mice, i.e. decreased clinical score (. about.P <0.05 vs. KLH).

FIG. 5-2: effect of humanized antibodies on the degree of imiquimod-induced ear swelling in psoriatic mice. The degree of ear swelling was significantly improved by the administration of hu31 and hu 44. (. P <0.05vs KLH,. P <0.01vs KLH.).

FIG. 6-1: effect of humanized antibody on type II collagen-induced body weight of female cynomolgus monkeys. hu31 and hu59 had some improvement in the weight loss caused by arthritis (. P <0.01,. P <0.0001, compared to "G2: vehicle group"; One-way ANOVA/Dunnett).

FIG. 6-2: effect of humanized antibodies on collagen type II induced female cynomolgus arthritis score. hu31 significantly inhibited the trend of increasing cynomolgus monkey arthritis clinical scores (. about.P <0.001, # P <0.05, and G2: vehicle group; One-way ANOVA/Dunnett).

FIG. 7: effect of humanized antibody on mouse joint swelling induced by NIH3T3-IL17 cells.

FIG. 8: effects of humanized antibodies on NIH3T3-IL17 cell-induced inflammation of mouse air sacs.

Detailed Description

Definitions and explanations

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It is to be understood that this invention is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes a combination of two or more polypeptides and the like.

The term "pharmaceutical composition" or "formulation" means a mixture containing one or more of the antibodies described herein and other components, such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

The term "liquid formulation" refers to a formulation in a liquid state and is not intended to refer to a resuspended lyophilized formulation. The liquid formulations of the present invention are stable upon storage and their stability is independent of lyophilization (or other state change methods, such as spray drying).

The term "aqueous liquid formulation" refers to a liquid formulation that uses water as a solvent. In some embodiments, the aqueous liquid formulation is one that does not require lyophilization, spray drying, and/or freezing to maintain stability (e.g., chemical and/or physical stability and/or biological activity).

The term "excipient" refers to an agent that can be added to a formulation to provide a desired characteristic (e.g., consistency, improved stability) and/or to adjust osmotic pressure. Examples of commonly used excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers.

As used herein, "about" when referring to a measurable value (e.g., amount, duration, etc.) is intended to encompass variations of ± 20% or ± 10% from the particular value, including ± 5%, ± 1% and ± 0.1%, as such variations are suitable for performing the disclosed methods.

The term "buffer pH of about 5.0-6.5" refers to an agent that, through the action of its acid/base conjugate components, renders a solution containing the agent resistant to pH changes. The buffer used in the formulation of the present invention may have a pH in the range of about 5.0 to about 6.5, or a pH in the range of about 5.5 to about 6.5, or a pH in the range of about 5.0 to about 6.0.

Examples of "buffers" that control the pH within this range herein include acetates (e.g., sodium acetate), succinates (e.g., sodium succinate), gluconic acid, histidine hydrochloride, methionine, citrates, phosphates, citrate/phosphates, imidazole, acetic acid, acetates, citrates, combinations thereof, and other organic acid buffers.

"histidine buffer" is a buffer comprising histidine ions. Examples of histidine buffers include salts of histidine and histidine, such as histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate, and the like, such as histidine buffer containing histidine and histidine hydrochloride; the histidine buffer of the present invention also includes histidine buffers comprising histidine and an acetate salt (e.g., sodium or potassium salt).

A "citrate buffer" is a buffer that includes citrate ions. Examples of citrate buffers include sodium citrate-citrate, potassium citrate-citrate, calcium citrate-citrate, magnesium citrate-citrate, and the like. The preferred citrate buffer is a citric acid-sodium citrate buffer.

An "acetate buffer" is a buffer that includes acetate ions. Examples of acetate buffers include sodium acetate, potassium acetate, calcium acetate, magnesium acetate, and the like. The preferred acetate buffer is acetate-sodium acetate buffer.

A "succinate buffer" is a buffer that includes succinate ions. Examples of the succinate buffer include sodium succinate-succinate, potassium succinate-succinate, calcium succinate-succinate, magnesium succinate-succinate, and the like. The preferred succinate buffer is sodium succinate-succinate buffer.

The term "stabilizer" means a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and/or formulation from chemical and/or physical degradation during manufacture, storage and use. Stabilizers include, but are not limited to, sugars, amino acids, salts, polyols and their metabolites such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine or salts thereof (e.g., arginine hydrochloride), glycine, alanine (α -alanine, β -alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, γ -aminobutyric acid (GABA), octopine (opines), alanine, octopine (strombine), and the N-oxide of Trimethylamine (TMAO), human serum albumin (hsa), albumin (bsa), α -bovine serum albumin, globulin, α -lactalbumin, GABA, lysozyme, myoglobin, ovalbumin, and rnasea. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, etc., may also act to control osmotic pressure. The stabilizer used in the present invention is one or more selected from the group consisting of a polyol, an amino acid, a salt and a sugar. Preferred salts are sodium chloride, preferred sugars are sucrose and trehalose, and preferred polyols are sorbitol and mannitol. Preferred amino acids are arginine or its salts (e.g. arginine hydrochloride), glycine, proline. Preferred stabilizers are sodium chloride, mannitol, sorbitol, sucrose, trehalose, arginine hydrochloride, glycine, proline, sodium chloride-sorbitol, sodium chloride-mannitol, sodium chloride-sucrose, sodium chloride-trehalose, arginine hydrochloride-mannitol, arginine hydrochloride-sucrose, more preferably arginine hydrochloride, sodium chloride-sucrose, arginine hydrochloride-mannitol, arginine hydrochloride-sucrose, more preferably arginine hydrochloride-sucrose.

The term "viscosity" as used herein may be "kinematic viscosity" or "absolute viscosity". ' YunKinematic viscosity "is a measure of the resistance of a fluid to flow under the influence of gravity. "absolute viscosity", sometimes referred to as dynamic viscosity or simple viscosity, is the product of kinematic viscosity and fluid density (absolute viscosity ═ kinematic viscosity X density). Kinematic viscosity is measured as L2/T, where L is the length and T is the time. Typically, kinematic viscosities are expressed in centistokes (cSt). The international unit system unit of kinematic viscosity is mm²I.e., lcSt. Absolute viscosity is expressed in units of centipoise (cP). The international unit system of absolute viscosity is millipascal-seconds (mPa-s), where 1cP is lmPa · s.

For the pharmaceutical compositions of the present invention, the term "low level viscosity" as used herein means an absolute viscosity of less than about 15 centipoise (cP). For example, a pharmaceutical composition of the present invention will be considered to have a "low viscosity" if the formulation exhibits an absolute viscosity of about 15cP, about 14cP, about 13cP, about 12cP, about 11cP, about 10cP, about 9cP, about 8cP, about 7cP, about 6cP, about 5cP, about 4cP, about 3cP, about 2cP, about 1cP, or less, when measured using standard viscosity measurement techniques. For the pharmaceutical compositions of the present invention, the term "intermediate level viscosity" as used herein will mean an absolute viscosity of between about 35cP and about 15 cP. For example, a pharmaceutical composition of the invention will be considered to have a "medium viscosity" if the formulation exhibits an absolute viscosity of about 34cP, about 33cP, about 32cP, about 31cP, about 30cP, about 29cP, about 28cP, about 27cP, about 26cP, about 25cP, about 24cP, about 23cP, about 22cP, about 21cP, about 20cP, about 19cP, 18cP, about 17cP, about 16cP, or about 15.lcP, when measured using standard viscosity measurement techniques. The pharmaceutical compositions of the present invention may, in certain embodiments, exhibit low levels of viscosity. In some embodiments, a comparison of the viscosities of different excipients reveals that arginine or a salt thereof can achieve a significantly lower viscosity level than other excipients. In some embodiments, the viscosity level of the histidine buffer system is lower than other buffer systems, as compared to the buffer system.

The term "surfactant" generally includes agents that protect proteins, such as antibodies, from air/solution interface-induced stress, solution/surface-induced stress to reduce aggregation of the antibodies or minimize the formation of particulate matter in the formulation. Exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycols, polyoxyethylene-stearates, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenylpolyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamers, pluronics), Sodium Dodecyl Sulfate (SDS).

The term "isotonic" means that the formulation has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured using an osmometer of the vapor pressure or subfreezing type.

The term "stable" formulation is one in which the antibody substantially retains its physical and/or chemical stability and/or biological activity during the manufacturing process and/or upon storage. The pharmaceutical preparation may be stable even if the contained antibody fails to maintain 100% of its chemical structure or biological function after storage over a certain period of time. In certain instances, an antibody structure or function that maintains about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% after storage over a period of time may also be considered "stable". Various analytical techniques for measuring Protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery 247-: 29-90 (both incorporated by reference).

After storage of the formulation at a temperature and for a period of time, its stability can be measured by determining the percentage of native antibody remaining therein (among other methods). The percentage of native antibody can be measured by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [ SEC-HPLC ]), among other methods, "native" referring to unaggregated and undegraded. In some embodiments, the stability of a protein is determined as a percentage of monomeric protein in a solution having a low percentage of degraded (e.g., fragmented) and/or aggregated protein. In some embodiments, the formulation is stable for storage at room temperature, about 25-30 ℃, or 40 ℃ for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer, up to no more than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody in aggregated form.

Stability can be measured by determining (among other methods) the percentage of antibody that migrates ("acidic form") in the fraction where this main fraction of antibody ("predominantly charged form") is acidic during ion exchange, where stability is inversely proportional to the percentage of acidic form antibody. The percentage of "acidified" antibody can be measured by, among other methods, ion exchange chromatography (e.g., cation exchange high performance liquid chromatography [ CEX-HPLC ]). In some embodiments, an acceptable degree of stability means that no more than about 49%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody in acidic form can be detected in the formulation after storage of the formulation at a temperature and for a time. The certain time period of storage prior to measuring stability can be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer. When evaluating stability, a temperature that allows for storage of the pharmaceutical formulation can be any temperature in the range of about-80 ℃ to about 45 ℃, e.g., storage at about-80 ℃, about-30 ℃, about-20 ℃, about 0 ℃, about 2-8 ℃, about 5 ℃, about 25 ℃, or about 40 ℃.

An antibody "retains its physical stability" in the pharmaceutical composition if it shows substantially no signs of, for example, aggregation, precipitation and/or denaturation when visually inspected for color and/or clarity or measured by UV light scattering or by pore size exclusion chromatography. Aggregation is the process by which individual molecules or complexes associate, covalently or non-covalently, to form aggregates. Aggregation may proceed to the extent that a visible precipitate is formed.

Stability, e.g., physical stability, of a formulation can be assessed by methods well known in the art, including measuring the apparent extinction (absorbance or optical density) of a sample. Such extinction measurements correlate with the turbidity of the formulation. Turbidity of a formulation is, in part, an inherent property of proteins dissolved in solution and is typically measured by nephelometry and is measured in Nephelometric Turbidity Units (NTU).

The level of turbidity which varies with, for example, the concentration of one or more components in the solution (e.g., protein and/or salt concentration) is also referred to as the "opacification" or "opacified appearance" of the formulation. The turbidity level can be calculated with reference to a standard curve generated using suspensions of known turbidity. Reference standards for determining the turbidity level of a pharmaceutical composition may be based on the "European Pharmacopoeia" standards (European Pharmacopoeia), fourth edition, "European commission for Quality of Medicine instructions" (EDQM), Strasbourg, France). According to the european pharmacopoeia standard, a clear solution is defined as a solution having a turbidity lower than or equal to that of a reference suspension according to the european pharmacopoeia standard having a turbidity of about 3. Nephelometric turbidity measurements can detect rayleigh scattering in the absence of association or non-ideal effects, which typically varies linearly with concentration. Other methods for assessing physical stability are well known in the art.

An antibody "retains its chemical stability" in a pharmaceutical composition if its chemical stability at a given point in time is such that the antibody is considered to still retain its biological activity as defined hereinafter. Chemical stability can be assessed, for example, by detecting or quantifying chemically altered forms of the antibody. Chemical changes may include size changes (e.g., clipping), which may be assessed using, for example, pore size exclusion chromatography, SDS-PAGE, and/or matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI/TOF MS). Other types of chemical changes include charge changes (e.g., occurring as a result of deamidation or oxidation), which can be assessed by, for example, ion exchange chromatography.

An antibody in a pharmaceutical composition "retains its biological activity" if it is biologically active for its intended purpose. For example, a formulation of the invention may be considered stable if, after storage of the formulation for a certain period of time (e.g., 1 to 12 months) at a temperature, e.g., 5 ℃, 25 ℃, 45 ℃, etc., the formulation comprises an anti-IL-17A antibody that binds IL-17A with an affinity that is at least 90%, 95% or more of the binding affinity of the antibody prior to said storage. Binding affinity can also be determined using, for example, ELISA or plasmon resonance techniques.

In the context of the present invention, a "therapeutically effective amount" or "effective amount" of an antibody, in a pharmacological sense, refers to an amount effective in the prevention or treatment or alleviation of the symptoms of the disorder that the antibody is effective to treat. In the present invention, a "therapeutically effective amount" or "therapeutically effective dose" of a drug is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease or promotes disease regression as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic phases of the disease, or the prevention of injury or disability resulting from the affliction of the disease. The ability of a drug to promote disease regression can be evaluated using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems that predict human efficacy, or by assaying the activity of the agent in an in vitro assay. A therapeutically effective amount of a drug includes a "prophylactically effective amount," i.e., any amount of a drug that inhibits the development or recurrence of a disease when administered to a subject at risk of developing a disease or a subject with a relapse of a disease, either alone or as combined with other therapeutic drugs.

The term "subject" or "patient" is intended to include mammalian organisms. Examples of subjects/patients include human and non-human mammals, such as non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In a particular embodiment of the invention, the subject is a human.

The terms "administering," "administering," and "treating" refer to introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods or delivery systems known to those of skill in the art. Routes of administration of anti-PD-1 antibodies include intravenous, intramuscular, subcutaneous, peritoneal, spinal or other parenteral routes of administration, such as injection or infusion. "parenteral administration" refers to modes of administration other than enteral or topical administration, typically by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraframe, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and via in vivo electroporation.

anti-IL-17A antibodies

The term "antibody" as used herein is to be understood as including whole antibody molecules and antigen-binding fragments thereof. The term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to human IL-17A (interleukin 17A)) or an epitope thereof.

The term "full length antibody" or "whole antibody molecule" as used herein refers to an immunoglobulin molecule comprising four peptide chains, two heavy (H) chains (about 50-70kDa in length) and two light (L) chains (about 25kDa in length) linked to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of 3 domains, CH1, CH2, and CH 3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into Complementarity Determining Regions (CDRs) with high variability and regions that are spaced apart to be more conserved, called Framework Regions (FRs). Each VH or VL region is formed by, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 are composed of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

As used herein, the term "CDR" refers to complementarity determining regions within an antibody variable sequence. There are 3 CDRs in each variable region of the heavy and light chains, designated HCDR1, HCDR2 and HCDR3 or LCDR1, LCDR2 and LCDR3 for each heavy and light chain variable region. The exact boundaries of these CDRs are defined differently for different systems. The system described by Kabat (supra) provides not only a clear residue numbering system applicable to any variable region of an antibody, but also provides an accurate residue boundary defining 3 CDRs. These CDRs may be referred to as Kabat CDRs. Chothia et al found that certain subsections within the Kabat CDRs adopt nearly identical peptide backbone configurations despite large diversity at the amino acid sequence level (Chothia et al, (1987) mol.biol.196: 901-883; Chothia et al, (1989) Nature 342: 877-883). Other boundaries defining CDRs which overlap with the Kabat CDRs have been described by Padlan (1995) FASEB J.9: 133-. Other CDR boundary definitions may not strictly follow one of the systems described herein, but still overlap with the Kabat CDRs, although they may be shortened or lengthened, since specific residues or groups of residues or even the entire CDR are not found to significantly affect antigen binding according to prediction or experimentation. The methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use Kabat or Chothia defined CDRs.

As used herein, an "antigen-binding fragment" includes a fragment of an antibody or derivative thereof, typically including at least one fragment of an antigen-binding region or variable region (e.g., one or more CDRs) of a parent antibody that retains at least some of the binding specificity of the parent antibody. Examples of antigen binding fragments include, but are not limited to, Fab ', F (ab')2, and Fv fragments; a diabody; a linear antibody; single chain antibody molecules, such as sc-Fv; nanobodies (nanobodies) and multispecific antibodies formed from antibody fragments. When the binding activity of an antibody is expressed on a molar concentration basis, the binding fragment or derivative thereof typically retains at least 10% of the antigen binding activity of the parent antibody. Preferably, the binding fragment or derivative thereof retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody. It is also contemplated that antigen-binding fragments of an antibody may include conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "functionally conservative variants" of the antibody) that do not significantly alter its biological activity.

The anti-IL-17A antibodies or antigen-binding fragments thereof described herein include any of the anti-IL-17A antibodies described in application No. PCT/CN2019/124486, the entire disclosure of which is incorporated herein by reference. In some embodiments, the CDR sequences of the antibodies used in the methods and compositions of the invention comprise CDR sequences from antibody hu31 described in PCT/CN 2019/124486.

Non-limiting, exemplary antibodies used in the examples herein are selected from the humanized antibodies hu31, hu43, hu44, hu59, hu60 and hu250 described in CN201811515045.7, the variable regions thereof and the amino acid CDR sequences thereof are shown in table 1, and the light/heavy chain amino acid full length sequences thereof are shown in table 2.

Table 1: humanized antibody variable regions and CDR sequences

Antibodies

hu31

hu43

hu44

hu59

hu60

hu250

HCDR1

SEQ ID NO:1

SEQ ID NO:7

SEQ ID NO:13

HCDR2

SEQ ID NO:2

SEQ ID NO:8

SEQ ID NO:14

HCDR3

SEQ ID NO:3

SEQ ID NO:9

SEQ ID NO:15

LCDR1

SEQ ID NO:4

SEQ ID NO:10

SEQ ID NO:16

LCDR2

SEQ ID NO:5

SEQ ID NO:11

SEQ ID NO:17

LCDR3

SEQ ID NO:6

SEQ ID NO:12

SEQ ID NO:18

SEQ ID NO:19

SEQ ID NO:21

SEQ ID NO:23

SEQ ID NO:26

SEQ ID NO:20

SEQ ID NO:22

SEQ ID NO:24

SEQ ID NO:25

SEQ ID NO:27

Table 2: humanized antibody amino acid full-length sequence

Humanized antibodies	Heavy Chain (HC)	Light Chain (LC)
			hu31	SEQ ID NO:28	SEQ ID NO:29
hu43	SEQ ID NO:30	SEQ ID NO:29
			hu44	SEQ ID NO:30	SEQ ID NO:31
hu59	SEQ ID NO:32	SEQ ID NO:33
			hu60	SEQ ID NO:32	SEQ ID NO:34
hu250	SEQ ID NO:35	SEQ ID NO:36

Pharmaceutical preparation

The pharmaceutical composition of the present invention is a highly stable pharmaceutical composition comprising a humanized antibody that specifically binds to IL-17A. The invention finds that the combination of arginine salt (such as arginine hydrochloride) and sucrose can obviously improve the stability of the pharmaceutical composition.

The pharmaceutical composition of the present invention is a liquid formulation containing a high concentration of active antibody and having high stability and low viscosity. In particular, the present inventors have found that formulations containing arginine salts have significantly lower viscosities than formulations containing other adjuvants alone. In addition, the viscosity level of histidine buffer systems is significantly lower than other buffer systems.

The present invention provides a pharmaceutical composition comprising: (1) a buffer solution; (2) an anti-IL-17A antibody or antigen-binding fragment thereof.

The antibody in the pharmaceutical composition of the present invention may be a murine antibody, a chimeric antibody, a humanized antibody, preferably a humanized antibody, and may have HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3, respectively, and LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, respectively. Preferably, the antibody in the pharmaceutical composition of the present invention has the heavy chain variable region as shown in SEQ ID NO. 19 and the light chain variable region as shown in SEQ ID NO. 20, or has the heavy chain variable region as shown in SEQ ID NO. 21 and the light chain variable region as shown in SEQ ID NO. 22; more preferably, the antibody in the pharmaceutical composition of the present invention has a heavy chain amino acid sequence as shown in SEQ ID NO. 28 and a light chain amino acid sequence as shown in SEQ ID NO. 29, respectively, or the antibody in the pharmaceutical composition of the present invention has a heavy chain amino acid sequence as shown in SEQ ID NO. 30 and a light chain amino acid sequence as shown in SEQ ID NO. 31, respectively; more preferably, the antibody in the pharmaceutical composition of the present invention has a heavy chain amino acid sequence as shown in SEQ ID NO. 28 and a light chain amino acid sequence as shown in SEQ ID NO. 29, respectively.

The humanized antibody in the pharmaceutical composition of the present invention may preferably have HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9, respectively, and LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12, respectively. Preferably, the antibody in the pharmaceutical composition of the present invention has the heavy chain variable region as shown in SEQ ID NO. 23 and the light chain variable region as shown in SEQ ID NO. 24, or has the heavy chain variable region as shown in SEQ ID NO. 23 and the light chain variable region as shown in SEQ ID NO. 25; more preferably, the antibody in the pharmaceutical composition of the present invention has a heavy chain amino acid sequence as shown in SEQ ID NO. 32 and a light chain amino acid sequence as shown in SEQ ID NO. 33, respectively, or has a heavy chain amino acid sequence as shown in SEQ ID NO. 32 and a light chain amino acid sequence as shown in SEQ ID NO. 34, respectively.

The humanized antibody in the pharmaceutical composition of the present invention may preferably have HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO 13, SEQ ID NO 14 and SEQ ID NO 15, respectively, and LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 18, respectively. Preferably, the antibody in the pharmaceutical composition of the present invention has a heavy chain variable region as shown in SEQ ID NO. 26 and a light chain variable region as shown in SEQ ID NO. 27; more preferably, the antibody in the pharmaceutical composition of the present invention has a heavy chain amino acid sequence as shown in SEQ ID NO. 35 and a light chain amino acid sequence as shown in SEQ ID NO. 36, respectively.

In the pharmaceutical compositions of the invention, the concentration of the anti-IL-17A antibody or antigen-binding fragment thereof is about 1-300mg/mL, preferably about 10-250mg/mL, preferably about 20-200mg/mL, more preferably 60-180mg/mL, more preferably 80-150 mg/mL. As non-limiting examples; the anti-IL-17A antibody or antigen-binding fragment thereof is present at a concentration of about 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL, 110mg/mL, 120mg/mL, 130mg/mL, 140mg/mL, 150mg/mL, 160mg/mL, 170mg/mL, 180mg/mL, or 200mg/mL, preferably about 80mg/mL or 150 mg/mL.

The buffer in the pharmaceutical composition of the present invention may be selected from the group consisting of an acetate buffer, a citrate buffer, a succinate buffer and a histidine buffer to provide a pH of 5.0 to 6.5, preferably 5.0 to 6.0, more preferably 5.5 ± 0.3, more preferably about 5.5 to the pharmaceutical composition of the present invention. In another aspect, the buffer used in the pharmaceutical composition of the present invention may have a pH of 5.0 to 6.5, preferably 5.0 to 6.0, more preferably 5.5. + -. 0.3, more preferably about 5.5.

Particularly preferred buffers in the pharmaceutical composition of the invention are histidine buffers, including histidine-hydrochloride buffers or histidine-acetate buffers, preferably histidine-hydrochloride buffers. More preferably, the histidine-hydrochloride buffer is made of histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride. In some embodiments, the histidine buffer is made up of 1-20mM L-histidine and 1-20mM L-histidine monohydrochloride. In some embodiments, the histidine buffer is made up of histidine and histidine hydrochloride in a molar ratio of 1:1 to 1: 4. In some embodiments, the histidine buffer is made up of histidine and histidine hydrochloride in a molar ratio of 1: 1. In some embodiments, the histidine buffer is made up of histidine and histidine hydrochloride in a molar ratio of 1: 3. In some embodiments, the histidine preparation is: histidine buffer with a pH of 5.5. + -. 0.3, preferably about 5.5, prepared from 4.5mM L-histidine and 15.5mM L-histidine monohydrochloride. In some embodiments, the histidine preparation is: histidine buffer with a pH of 5.5. + -. 0.3, preferably about 5.5, prepared from 7.5mM L-histidine and 22.5mM L-histidine monohydrochloride. In some embodiments, the histidine preparation is: histidine buffer with a pH of 6.0. + -. 0.3, preferably about 6.0, prepared from 15mM histidine and 15mM histidine hydrochloride.

The histidine buffer in the pharmaceutical composition of the invention may be a histidine-acetate buffer, preferably in a molar ratio of 1:1 to 1.5:1, preferably such a buffer has a pH of 5.5 ± 0.3, preferably about 5.5, preferably such a buffer contains 15-20mM histidine and 12-15mM acetic acid.

Accordingly, the pharmaceutical composition of the present invention may contain: a histidine-histidine hydrochloride buffer at a pH of 5.0 to 6.0, in a concentration of 10 to 30mM in the pharmaceutical composition; and 60-180mg/mL of an anti-IL-17A antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, particularly hu31, hu43, hu44, hu59, hu60 and hu250 or antigen-binding fragment thereof as described herein.

In some embodiments, the pharmaceutical compositions of the present invention further comprise a stabilizer. Preferably, the stabilizer is selected from one or more of arginine hydrochloride, proline, glycine, sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol and trehalose. Preferably, the stabilizer in the pharmaceutical composition at least contains arginine hydrochloride, and optionally contains one or more of mannitol, sorbitol, sucrose and trehalose. For example, the pharmaceutical composition may comprise arginine hydrochloride and mannitol, arginine hydrochloride and sorbitol, arginine hydrochloride and sucrose, or arginine hydrochloride and trehalose. The concentration of the stabilizer in the pharmaceutical composition of the present invention is about 10mM-400mM, preferably 20mM-300mM, more preferably 30mM-200 mM. In some embodiments, the stabilizing agent is sodium chloride at a concentration of about 30-200 mM; or the stabilizer is mannitol at a concentration of about 100-300mM, preferably 200-300 mM; or the stabilizer is sorbitol at a concentration of about 100-300mM, preferably 200-300 mM; or the stabilizer is sucrose at a concentration of about 100-300mM, preferably 50-250 mM; or the stabilizer is trehalose at a concentration of about 100-300mM, preferably 200-300 mM; or the stabilizer is arginine hydrochloride at a concentration of about 30-200mM, preferably about 60-150 mM; or the stabilizer is proline at a concentration of about 100 and 300 mM; or the stabilizer is glycine at a concentration of about 100 and 300 mM. In some embodiments, the stabilizing agent is a combination of about 30-200mM sodium chloride and about 30-200mM mannitol; or the stabilizer is a combination of about 30-200mM sodium chloride and about 30-200mM sucrose; or the stabilizer is a combination of about 30-200mM arginine hydrochloride and about 30-200mM mannitol; or the stabilizer is a combination of about 30-200mM, preferably about 60-120mM, arginine hydrochloride and about 30-200mM, preferably about 40-80mM, sucrose.

Thus, in some embodiments, the pharmaceutical compositions of the present invention comprise: a histidine-histidine hydrochloride buffer at a pH of 5.0 to 6.0, in a concentration of 10 to 30mM in the pharmaceutical composition; 60-180mg/mL of an anti-IL-17A antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, in particular hu31, hu43, hu44, hu59, hu60 and hu250 or antigen-binding fragment thereof as described herein; and 20mM-300mM of a stabilizer, preferably, the stabilizer at least comprises arginine hydrochloride, and optionally comprises one of mannitol, sorbitol, sucrose and trehalose, preferably 30-200mM of arginine hydrochloride, or 30-200mM of arginine hydrochloride and 30-200mM of mannitol, or 30-200mM of arginine hydrochloride and 30-200mM of sucrose. In some embodiments, the stabilizer is 30-200mM arginine hydrochloride with 30-200mM sucrose. In some embodiments, the stabilizer is 60-120mM arginine hydrochloride with 40-80mM sucrose.

In some embodiments, the pharmaceutical compositions of the present invention further comprise a surfactant. Preferred surfactants are selected from polysorbate 80, polysorbate 20 and poloxamer 188. The most preferred surfactant is polysorbate 20. The concentration of the surfactant in the pharmaceutical composition of the present invention is about 0.001% to about 0.1%, preferably about 0.02% to about 0.08%, preferably about 0.02% to about 0.04%, in w/v. By way of non-limiting example, the concentration of surfactant in the pharmaceutical composition of the present invention is about 0.02%, 0.04% or 0.08%, preferably 0.02%.

Thus, in some embodiments, the pharmaceutical compositions of the present invention comprise: a histidine-histidine hydrochloride buffer at a pH of 5.0 to 6.0, in a concentration of 10 to 30mM in the pharmaceutical composition; 60-180mg/mL of an anti-IL-17A antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, in particular hu31, hu43, hu44, hu59, hu60 and hu250 or antigen-binding fragment thereof as described herein; 20mM-300mM of stabilizer, preferably, the stabilizer at least comprises arginine hydrochloride, optionally comprises one of mannitol, sorbitol, sucrose and trehalose, preferably 100-180mM of arginine hydrochloride, or 60-120mM of arginine hydrochloride and 40-80mM of mannitol, or 60-120mM of arginine hydrochloride and 40-80mM of sucrose; and 0.02% -0.04% polysorbate 20 by w/v.

The pharmaceutical composition of the present invention may be a liquid formulation, or a lyophilized formulation.

Medical use and method

The invention also provides a pharmaceutical composition according to any embodiment of the invention for use in the treatment or prevention of an IL-17A mediated disease, the use of a pharmaceutical composition according to any embodiment of the invention for the manufacture of a medicament for the treatment or prevention of an IL-17A mediated disease, and a method of administering to a subject or patient in need thereof a therapeutically effective amount of a pharmaceutical composition according to any embodiment of the invention for the treatment or prevention of an IL-17A mediated disease.

In the present invention, IL-17A mediated diseases refer to diseases in which IL-17A is involved in the onset and progression of diseases, including but not limited to inflammatory diseases and autoimmune diseases. In the present invention, diseases suitable for treatment and prevention by the pharmaceutical composition of the present invention include, but are not limited to, arthritis, rheumatoid arthritis, ankylosing spondylitis, chronic obstructive pulmonary disease, Systemic Lupus Erythematosus (SLE), lupus nephritis, asthma, multiple sclerosis, cystic fibrosis, psoriasis.

The present invention will be illustrated below by way of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the present invention. The methods and materials used in the examples are, unless otherwise indicated, conventional in the art.

Example 1: buffer system and pH screening assay

In the liquid type pharmaceutical composition, the buffer system and pH closely affect the stability of the antibody, and each antibody having unique physicochemical properties has the optimum type and pH of the buffer. This example is directed to screening an optimal buffer system and pH for optimal stability of the anti-IL-17A antibodies disclosed herein for clinical use.

This example was performed with antibody hu31 at concentrations of about 80mg/mL and 150 mg/mL. The sample was prepared using Millipore Pellicon 30.11m²And (3) carrying out ultrafiltration concentration and liquid change on the membrane, placing the sample in a corresponding prescription after the liquid change, and placing the sample in a sealed centrifugal tube for buffer liquid screening. Acetate, succinate and histidine buffers were selected at a pH of from 5.0 to 6.5 (as shown in table 3). Mixing the sampleThe sample was placed at 40. + -. 2 ℃ and taken out at week 0, week 2 and week 4, respectively, for analysis and detection. The major pathways for protein degradation are the formation of aggregates, cleavage products and charged variants. The percentage of the native form (protein monomers) to the aggregated form was determined by size exclusion chromatography (SEC-HPLC) and the percentage of the acidic and basic forms of the antibody was determined by cation exchange chromatography (CEX-HPLC). The effect of different buffer systems and pH on antibody hu31 antibody stability was examined by fitting a straight line and calculating the slope of decline (%/week) with SEC-HPLC monomer content and CEX-HPLC main peak content for four weeks (4W) of standing and the results are summarized in tables 4 and 5.

Table 3: buffer system and prescription information in pH screening experiments

Table 4: buffer system and SEC-HPLC monomer content degradation rate in pH screening experiment

Table 5: buffer system and rate of degradation of CEX-HPLC charge heteroplasmon content in pH screening experiments

As shown in tables 4 and 5, in the SEC-HPLC experimental detection, after being placed at a high temperature of 40 +/-2 ℃ for 4 weeks, the monomer content decreases faster along with the increase of the protein concentration, but the monomer content decreases at lower rates under different protein concentrations under a histidine buffer system with a pH of 5.5, and the average decrease rate is 0.24%/week; in the CEX-HPLC experimental detection, the content degradation rate of the main peak of the CEX-HPLC is related to the pH value to a certain extent, the higher the pH value is, the faster the content degradation rate of the main peak is, and the relatively lower the degradation rate of the content of the main peak is in a buffer system with the pH value of 5.0-5.5.

According to the screening results, the clinical dosage, the product specification and the target quality attributes (polymer content and charge variation content level) of the product are comprehensively considered, and a histidine buffer system with the protein concentration of 150mg/ml and the pH value of 5.5 is selected for subsequent prescription screening.

Example 2: screening experiment of stabilizer (adjuvant)

2.1 examination of the Effect of adjuvants on viscosity

In order to further explore the influence of different auxiliary materials on the stability and viscosity of the antibody, a preparation of one of sodium chloride, sucrose, arginine hydrochloride, proline, glycine, trehalose, sorbitol or mannitol is selected for comparative testing. The effect of the different excipients on the viscosity was examined under the conditions of the same osmotic pressure and pH5.5, the concentration of monoclonal antibody hu31 of 80mg/ml and 150mg/ml for the combination of 20mM histidine buffer system and other buffer systems, and the specific prescription information is shown in Table 6. The preparations of each prescription are subpackaged, placed at 40 +/-2 ℃, taken out at the 4 th week, analyzed and detected according to a standard method to obtain the viscosity of the preparation, and the results are shown in a table 7.

Table 6: prescription information for investigating viscosity of different auxiliary materials

Note: "-" indicates no addition.

Table 7: viscosity measurement data of different auxiliary materials

As can be seen from Table 7, the viscosity and the protein concentration are in a direct proportion, and the viscosity becomes larger as the protein concentration increases, and compared with the viscosity of different excipients (formula 7-formula 14), the viscosity of arginine hydrochloride is obviously lower than that of other excipients, and the viscosity data is only about 65% of that of mannitol.

In comparison with the buffer systems (recipe 15 to recipe 18), the viscosity of the histidine buffer system is lower than that of the other buffer systems. On the basis, the subsequent prescription design and screening are carried out.

2.2 examination of the Effect of auxiliary materials on stability

In order to further explore the influence of different auxiliary materials on the stability of the antibody, a preparation of one or a combination of sodium chloride, sucrose, arginine hydrochloride, trehalose, sorbitol and mannitol is selected for comparative testing. Namely, the above-mentioned various adjuvants or combinations thereof were added to 30mM histidine buffer containing about 150mg/mL of antibody hu31, respectively, and the specific formulation information is shown in Table 8. The preparations of each prescription are subpackaged and placed at 40 +/-2 ℃, and are taken out at the 0 th week, the 2 nd week and the 4 th week respectively for analysis and detection. The content change of the monomer of the antibody hu31 is detected by a molecular exclusion high performance liquid chromatography (SEC-HPLC), and the content of the main charge peak of the antibody hu31 is detected by a weak cation high performance liquid chromatography (CEX-HPLC). The results are shown in Table 9.

According to the stability investigation result, after the preparation prescription samples of different auxiliary materials are placed for 4 weeks at the high temperature of 40 +/-2 ℃, the antibody has stronger thermal stability.

Through the comprehensive analysis of various data, the comparison of the prescription 19 to the prescription 24 shows that the single auxiliary material containing arginine hydrochloride is obviously superior to the single auxiliary materials containing sodium chloride, mannitol, sorbitol, sucrose and trehalose, and the degradation rate of the monomer content of SEC-HPLC and the main peak content of CEX-HPLC is lower. Comparing the prescription 25 to the prescription 30, the combination of arginine hydrochloride and sucrose containing auxiliary materials is the most stable, the monomer content reduction rate of SEC-HPLC is as low as 0.2%/week, the combination is obviously superior to that of the single auxiliary material, and the degradation rate of the main peak content of CEX-HPLC has no obvious difference.

Therefore, the auxiliary material combination of arginine hydrochloride and sucrose is more favorable for the stability of the product.

Table 8: prescription information in adjuvant screening experiments

Note: "-" indicates no addition.

Table 9: summary of findings from the adjuvant screening experiment

And through the analysis of all data, the preparation which is prepared by combining the auxiliary materials of arginine hydrochloride and sucrose is selected, and the antibody has the strongest stability and the lowest viscosity. Specifically, after being placed at a high temperature of 40 +/-2 ℃ for 4 weeks, the preparation group containing the combination of arginine hydrochloride and sucrose: (1) for the stability of the antibody structure, the reduction rate of the monomer purity of the antibody is obviously lowest, as low as 0.2%/week, about 40% of the highest mannitol group, and the monomer purity of the antibody is as high as 99.2%; (2) for stability of antibody charge, the rate of decrease of antibody main charge is low, as low as 6.00%/week, main charge is as high as 86.0%; (3) with respect to the viscosity of the formulation, arginine hydrochloride significantly reduced the viscosity of the formulation, and the viscosity of the formulation containing arginine hydrochloride alone was lower than 6cP (table 7), which is significantly lower than that of the other formulations, especially the formulation containing mannitol alone.

High concentration antibody solutions are generally more likely to cause antibody aggregation, precipitation, etc., resulting in decreased antibody stability, and increased solution viscosity leading to difficulty in administration by injection (particularly subcutaneous or intramuscular injection). By combining the experiments, the arginine salt in the liquid preparation can ensure the stability of the antibody and can obviously reduce the liquid viscosity. In particular, antibody stability and solution viscosity effects are best when the buffer is histidine at pH5.5, a stabilizer comprising arginine hydrochloride and sucrose.

Example 3: surfactant screening experiments

The addition of surfactants to liquid formulations is commonly used as an agent to protect proteins such as antibodies from air/solution interface-induced stress, solution/surface-induced stress during storage to reduce aggregation of the antibodies or minimize the formation of particulate matter in the formulation, which facilitates the stabilization of the physicochemical properties of the antibodies. Polysorbate 20 or polysorbate 80 was added to a preparation containing 30mM histidine buffer (histidine-histidine hydrochloride, pH 5.5) and 150mg/ml antibody hu31 (adjuvant 1 and adjuvant 2 were added according to the recipe 30) at different concentrations, and the mixture was left at 40. + -. 2 ℃ for 2 weeks and analyzed and examined after 4 weeks. The results are shown in Table 10.

Comprehensive analysis shows that the results of surfactant screening tests (prescription 31-36) show that the addition of polysorbate 80 or polysorbate 20 with different concentrations has no obvious influence on the monomer content of SEC-HPLC.

Table 10: results of surfactant screening

Example 4: prescription dilution stability and prescription confirmation of formulations

A prescription preparation No. 30 shown in Table 8 was prepared by selecting an antibody stock solution, and the stability of the sample was examined by diluting the sample with physiological saline, glucose injection and different prescriptions to different concentrations and by examining the stability of the sample under repeated freeze-thaw conditions, and the prescription conditions were confirmed (Table 11). The screening results are shown in Table 12.

Table 11: investigation information (prescription 30)

Repeated freeze thawing	Freeze thawing at-40 + -5 deg.C for 5 times
		Dilution with physiological saline	Diluting to 10mg/ml
Glucose injection	Diluting to 10mg/ml
		Prescription 30 buffer solution	Diluting to 2mg/ml and 10mg/ml

Table 12: prescription stability screening results

Note: "/" indicates no measurement or addition.

The samples are repeatedly frozen and thawed at 25 +/-2 ℃ under the prescription condition, and the glucose injection, the normal saline and the prescription 30 buffer solution (namely 30mM histidine buffer solution) dilute the samples to different concentrations without great influence on the monomer content, so that the good stability of the prescription is shown.

In conclusion, the stability of the recombinant humanized anti-IL-17A monoclonal antibody hu31 is researched and researched by examining different buffer systems, different pH conditions, different antibody concentrations and different auxiliary materials and surfactant compositions, and the optimal liquid preparation formula is determined. Antibody hu31 histidine buffer was chosen to adjust pH, arginine hydrochloride and sucrose to adjust osmotic pressure and viscosity of the formulation, and polysorbate 20 was added to increase formulation solubility.

Example 5: binding specificity of humanized antibodies to human IL-17A

The binding specificity of the different humanized antibodies (formulated according to formula 31) to human IL-17A was determined using conventional ELISA detection methods. Namely, 0.5 mu g/ml of human IL-17A-mFc is coated on a 96-well enzyme label plate and incubated for 60-90 minutes at the constant temperature of 37 ℃. The well solution was then discarded, washed 3 times with wash buffer, and blocked for 60 minutes by adding 2% BSA in PBS. Washing with washing buffer for 3 times, adding humanized antibody diluents with different concentrations, incubating at 37 ℃ for 60 minutes, washing with washing buffer for 3 times, and adding 1: biotin-anti-IgG 4 diluted 10000 times was incubated at 37 ℃ for 1 hour, washed three times with wash buffer, and added with wash buffer at a rate of 1: 10000 times diluted HPR-Strep, incubated at room temperature for 1 hour, washed 3 times with washing buffer, added with 100. mu.L of TMB substrate solution for color development, reacted at room temperature for 30 minutes, then quenched with 100. mu.L of 2M hydrochloric acid solution and the absorbance read at 450 nm. As shown in FIG. 1, humanized antibodies hu31, hu43, hu44, hu59, hu60 and hu250 all specifically bind to IL-17A. The EC50 was 8.13ng/mL, 8.64ng/mL, 6.76ng/mL, 6.10ng/mL, 5.78ng/mL and 6.35ng/mL, respectively.

Example 6: humanized antibodies block the binding of human IL-17A to IL-17RA

Different humanized antibodies (formulated according to formula 31) were tested to block the binding of IL-17A to IL-17RA on cells using a competitive cell-based flow cytometry assay (FACS). Briefly, different concentrations of humanized antibody dilutions (initial 10ug/ml, 3-fold titration) were mixed with pre-biotin-labeled human IL-17A-mFC (3ug/ml) obtained from example 1 and incubated for 30 minutes at room temperature. The mixture was then mixed with a cell suspension (293F IL-17RA stable cell line obtained in example 2, 1.5X 10⁵Individual cells/well) were incubated at 37 ℃ for 15 minutes, and after 3 times elution with PBS, 5 μ g/ml of anti-mouse IgG was added and incubated at room temperature for 30 minutes. After 3 elution with PBS, the humanized antibody was examined by flow cytometry for its inhibitory effect on the binding of IL-17A to IL-17RA on the surface of 293F cells. As shown in FIG. 2, humanized antibodies hu31, hu43, hu44, hu59, hu60 and hu250 all significantly inhibited specific binding of IL-17A to IL-17RA on cells. Its IC50 was 867.6ng/mL, 780.8ng/mL, 828.5ng/mL, 467.4ng/mL, 482.8ng/mL and 577.8ng/mL, respectively.

Example 7: humanized antibody antagonizes IL-17A to induce epithelial cell to express CXCL1

IL-17A can stimulate the expression and release of a plurality of epithelial cells and other cell secretion cytokines CXCL1, and the change of the expression level of CXCL1 in cell supernatant can be quantitatively detected by ELISA, so that the influence of different humanized antibodies (which are configured according to the formula of the formula 31) on the biological activity mediated by IL-17A in the cells can be judged.

HT-29 cells (human colorectal adenocarcinoma epithelial cells, ATCC) were maintained in culture/assay medium in tissue culture treated flasks using standard techniques. HT-29 were grown in tissue culture flasks until they reached 50-80% confluence on the day of the assay. On the day of assay, cells were washed with PBS and detached from the flask with trypsin + EDTA and made into cell suspensions. Humanized antibodies hu31, hu43, hu44, hu59, hu60, hu250 or a dilution of a reference antibody (Secukinumab, nova) (initial concentration 55ug/ml, 3-fold concentration gradient dilution) were mixed with human IL-17A (1ug/ml), plated in 96-well plates, and incubated for 1 h. To each well was added 100ul (2X 104) of HT-29 cell (ATCC, human colorectal adenocarcinoma epithelial cells) suspension at 37 ℃ with 7% CO₂Culturing for 48 h. The culture supernatant was transferred to a new 96-well plate by centrifugation, and CXCL1 expression was detected using an ELISA kit.

As shown in fig. 3, the humanized antibodies hu31, hu43, hu44, hu59, hu60 and hu250 had a stronger antagonistic effect on IL-17A stimulation of CXCL1 release from epithelial cells than the reference antibody Secukinumab.

Example 8: humanized antibody antagonizes IL-17A to induce mice to express CXCL1

The influence of different humanized antibodies (formulated according to formula 31) on IL-17A mediated biological activity in vivo is judged by detecting the change of mouse serum CXCL1 level. Briefly, 40 female Balb/c mice, 10 weeks old, were selected and divided into 8 groups of 5 mice each. 4 days before administration, serum was collected and the amount of CXCL1 expression was measured as a base value. On the day of administration, candidate antibodies (humanized antibodies hu31, hu43, hu44, hu60 and hu250) and an IgG4 isotype control (hIgG) were injected intracardially at 1mg/kg, respectively; 1 hour after administration, human IL-17A was injected subcutaneously at a dose of 150 ug/kg; at 2 hours after the injection of human IL-17A, serum was collected to measure the concentration of CXCL1 in blood, and compared with the basal value, and the fold change (mean. + -. standard error) of the concentration of CXCL1 before and after the administration was calculated for each group. Comparative analysis of candidate antibodies to IgG4 isotype controls using Student's-t test, P <0.05 was considered significantly different, # P <0.05, # P <0.01, # P < 0.001.

As shown in figure 4, the candidate humanized antibodies hu31, hu43, hu44, hu60 and hu250 had a stronger antagonistic effect on the release of CXCL1 in IL-17A-stimulated mice compared to the IgG4 isotype control.

Example 9: therapeutic effect study of humanized antibody on improvement of imiquimod-induced mouse psoriasis model

The psoriasis mouse model can be constructed by applying imiquimod on the skin of the ear back of a mouse to induce psoriasis-like pathological features, namely keratinocyte hyper-proliferation, inflammatory cell aggregation, dermal papilla vascular hyperplasia and the like. The treatment effect of the medicament on psoriasis mice is judged by taking clinical scores, ear swelling degrees and the like as indexes.

1. Experimental methods

48C 57BL/6 female mice (purchased at model animal institute of Nanjing university, animal license number 201605578) were harvested at 6-8 weeks for back depilation and sensitization two days later except in the sham group ("sham"). Two days prior to sensitization, were randomly divided into 6 groups (8 per group): group I is a sham operation group; group II was PBS group, group III was KLH control group (isotype IgG), KLH was given; group IV is hu31 group; group V is hu43 administration group; group VI is a hu44 administration group, and the administration dose of each group is 50 mg/kg; the above groups were administered with the drug intraperitoneally 1 time on day 0 and day 3. On the day of sensitization (day 1), group II-VI mice were coated with approximately 62.5mg of imiquimod cream (idamole, 5%, 3M Health Care Limited) on the right ear and back skin for 4 consecutive days. The humanized antibody was formulated as formula 31.

2. Evaluation method

The thickness of the right ear of the mouse was measured with a micrometer every day from the day of sensitization, and the value of the ear swelling thickness of the mouse was calculated with the thickness of the right ear on day 1 as a control. Meanwhile, weighing the mice every day, observing the skin scales, induration and erythema, and grading by adopting a 4-grade grading method: 0 point, no disease occurs; 1 point, slight; 2 points, medium; grade 3, severe; 4 points, very serious. Results are expressed as mean ± standard error, using one-way analysis of variance (ANOVA) first, and comparisons between the two groups after the difference using Student's-t test, P <0.05 was considered to be a significant difference.

As shown in FIG. 5-1, the humanized antibody of the present invention can significantly inhibit the phenomena of psoriasis model skin scales, induration, redness, etc. induced by imiquimod, i.e. the score value is small.

As shown in FIG. 5-2, when imiquimod cream was applied to the mouse right ear from the day of sensitization, severe swelling occurred in the right ear and the ear thickness increased, while the humanized antibody of the present invention significantly improved the degree of ear swelling.

In an imiquimod-induced mouse psoriasis model, the humanized antibody can obviously resist the morbidity of a mouse, and the phenotype is the reduction of the clinical score and the ear swelling degree of the mouse.

Example 10: therapeutic effect study of humanized antibody on improvement of II type collagen-induced female cynomolgus monkey arthritis

Type II collagen-induced arthritis is an animal model widely used in the study of Rheumatoid Arthritis (RA), has the same histopathological features as human RA, and is characterized by inflammation of the facet joints and progressive erosion of cartilage and bone. The human/humanized biological macromolecule including antibody has better cross reaction with antigen in the cynomolgus monkey, so the cynomolgus monkey arthritis model is an effective system for detecting the anti-rheumatism effect of the humanized antibody IL-17A in the invention. The experiment evaluates the drug effect of the candidate antibody on a cynomolgus monkey type arthritis model.

1. Experimental methods

Bovine collagen type II (CII, Sichuan university) was dissolved in acetic acid (cat # 10000218; national drug; Shanghai; China) and placed in a refrigerator at 4 ℃ for overnight stirring, and then collagen was emulsified with an equal volume of complete Freund's adjuvant (cat # F5881, Sigma-Aldrich, USA) to a final emulsion collagen concentration of 2 mg/ml. On day 0, animals were anesthetized with sutai (1.5-5mg/kg, i.m), multiple injections of collagen emulsion immunization were performed on the back and tail roots, and anesthesia was maintained with 1.5% -5% isoflurane as needed. Collagen was re-injected 3 weeks later (day 21) as before. The experiment is divided into 4 groups, G1 is a normal animal group (negative), and arthritis induction is not carried out; g2 is vehicle control ("vehicle"); g3 is antibody hu31 administration group; g4 is antibody hu59 administration group. When the clinical score of a certain animal reaches 5 percent (192 multiplied by 5 percent approximately equals to 10) of the maximum value of the clinical score, the animals are sequentially divided into experimental groups, the animals which reach the score are firstly divided into groups, and the process is circulated until all the animals which meet the conditions are sequentially divided into the groups. After the group administration, the administration was started once a week at 7.5mg/kg each time, and the administration was continued for 5 weeks by continuous pumping from an infusion pump (infusion pump) for 30 minutes. The humanized antibody was formulated as formula 31.

2. Evaluation method

And (3) measuring the body weight: animal body weights were measured the day before immunization, and weekly thereafter until the end of the experiment.

And (3) arthritis scoring: monkeys were scored for inflammation on days 0 and 21 and weekly after 21 days until the end of the experiment (if an early onset occurred, then the arthritis score was correspondingly advanced by once a week). Scoring criteria for arthritis: score 0, normal; score 1, mild arthritis, slight onset but clearly distinguishable; 2 points, moderate swelling; score 3, severe arthritis, severe swelling or marked joint deformity. The following 15 joints per paw were scored: 5 metacarpophalangeal joints (MCP), 4 proximal phalangeal joints (PIP), 5 distal phalangeal joints (DIP), 1 wrist joint or ankle joint. It is also desirable to assess the extent of knee/elbow joint morbidity in the extremities. The sum of the individual joint scores was the arthritis score for this animal, with a maximum score of 192(16 × 3 × 4).

Experimental data are expressed as mean ± standard error (mean ± s.e.m). Statistical analysis of differences between groups for vehicle control, reference drug and test drug group parameters, p <0.05 was considered statistically different (One-way ANOVA/Dunnett).

As shown in FIG. 6-1, normal cynomolgus monkeys (G1) were stable in weight; the average weight of the cynomolgus monkeys after the induction of arthritis, the vehicle-treated group (G2) continued to decrease, compared to the test antibodies hu31 and hu59, which controlled the decrease. Thus, under the conditions of this experiment, hu31 and hu59 had some improvement in the weight loss due to arthritis (. about.P <0.01,. about.P <0.0001, as compared to the "G2: vehicle group"; One-way ANOVA/Dunnett).

As shown in fig. 6-2, the arthritis clinical score remained 0 in normal control animals (G1) after animals were enrolled; the arthritis score of the animals in the vehicle control group (G2) is increased gradually, and the test antibodies hu31 and hu59 obviously inhibit the increase trend of the arthritis clinical score of the animals. Therefore, the test antibodies hu31 and hu59 have the function of inhibiting the progressive development of arthritis disease, and the test antibody hu31 significantly inhibits the increasing trend of the clinical scores of the arthritis of the cynomolgus monkey (. about.P <0.001, # P <0.05, compared with the vehicle control group; One-way ANOVA/Dunnett).

Example 11: effect of humanized antibody on NIH3T3-IL17 cell-induced joint swelling in mice

1. Animals: c57BL/6, female, 6-8 weeks, Beijing Wittiulihua laboratory animals technologies, Inc.

2. Cell: NIH3T3 cell, NIH3T3 cell expressing human IL-17.

3. Grouping and dosing regimens:

NIH3T3 group;

NIH3T3-IL-17+ control IgG antibody group (30 mg/kg);

NIH3T3-IL-17+ test antibody high dose group (antibody hu31,3 mg/kg);

NIH3T3-IL-17+ test antibody medium dose group (antibody hu31,10 mg/kg);

NIH3T3-IL-17+ test antibody low dose group (antibody hu31,30 mg/kg);

NIH3T3-IL-17+ positive drug administration group (cosentyx,10 mg/kg).

The humanized antibody was formulated as formula 31.

4. Molding and administration:

NIH3T3-IL-17 cells and NIH3T3 control cells (2.5X 10)⁵One/one, each injection volume of 25uL) was injected into the joint cavity of the right ankle joint of each group of mice, respectively.

The influence of the injection solution of the antibody hu31 on the mouse arthritis model was examined 1 time every 3 days after the intraperitoneal injection administration of the antibody hu31(3, 10, 30mg/kg) and cosentyx (10mg/kg) was started 1 day before the model selection.

5. And (3) detection:

and (5) measuring the thickness of the ankle joint of the mouse by using a vernier caliper, and calculating the swelling degree.

6. The experimental results are as follows:

as shown in FIG. 7, by injecting NIH3T3 cells, which stably transfer hIL-17, into the stomp cavity of a mouse, severe swelling of the stomp of the mouse was observed the next day.

The swelling inhibition rate is calculated according to the thickness of the ankle joint of the mouse, and the calculation formula is as follows: the inhibition ratio (%) - (NIH3T3-ILI7 group ankle thickness-administration group ankle thickness)/(NIH 3-ILl7 fine ankle thickness-NIH 3T3 group ankle thickness) × 100. The results showed that on day 2 after administration, inhibition of ankle swelling in mice was observed for each drug group at each dose until the end of the experiment, and maximum inhibition efficiency was reached on day 6 after administration. On day 10, the inhibition rates of antibody hu31(3, 10, 30mg/kg) were 49.4%, 65.9%, and 74.1%, respectively. The swelling inhibition rate of cosentyx (10mg/kg) was 67.1%. The average inhibition rates were calculated for the different days in each group, and the results showed that the average inhibition rates for antibody hu31 in each group (3, 10 and 30mg/kg) were 45.2%, 57.0% and 73.9%, respectively. The inhibition rate of cosentyx (10mg/kg) swelling was 60.4%. The experimental results suggest that: the antibody hu31 was able to dose-dependently inhibit IL-17-induced ankle swelling in mice with an effect of 10mg/kg comparable to the positive control drug cosentyx (10 mg/kg). The effect of 30mg/kg is better than that of the positive control drug cosentyx (10 mg/kg).

Example 12: effect of humanized antibody on NIH3T3-IL17 cell-induced mouse air sac inflammation

1. Animals: c57BL/6, male, 6-8 weeks, purchased from Experimental animals technologies, Inc., Viton, Beijing).

2. Cell: NIH3T3 cell, NIH3T3 cell expressing human IL-17.

3. Reagent: gr1-FITC antibody, Biolegend.

4. Grouping and dosing regimens:

NIH3T3 cell group

NIH3T3-IL-17 cell group + control IgG antibody group (30 mg/kg);

NIH3T3-IL-17 cell group + test antibody high dose administration group (antibody hu31,30 mg/kg);

NIH3T3-IL-17 cell group + test antibody medium dose group (antibody hu31,10 mg/kg);

NIH3T3-IL-17 cell group + test antibody low dose administration group (antibody hu31,3 mg/kg);

the administration route is as follows: intraperitoneal injection, and the humanized antibody is prepared according to the prescription 31.

5. Molding:

an air bag: mice were injected with 2.5ml of air at the back on days 0 and 3, respectively. Injection of cells into the air cells began on day 5. The number of cells injected per mouse was 2X 10⁵One/500. mu.l PBS. Each group had 8 mice.

6. And (3) detection:

migration of white blood cells to the air sac, calculating the total number of cells of the lavage fluid, flow-measuring the Gr1+ cell proportion, and calculating the number of neutrophils; the number of neutrophils is equal to the total number of the cells multiplied by the proportion of Gr1+ cells.

7. Results of the experiment

As shown in figure 8, when the NIH-3T3 cells stably transformed with hlL-17A are injected into the air sac at the back of a mouse, the number of the white blood cells infiltrated into the air sac of the NIH3T3-IL-17 cell group is remarkably increased, the proportion and the number of Grl + cells are also remarkably increased and the model selection is successful compared with the NIH3T3 cell group from the total number of the infiltrated cells. On the day of modeling, the antibody hu31 (the dose is 3, l0 and 30mg/kg respectively) is given by intraperitoneal injection for intervention administration. Calculating the inhibition rate according to the total number of infiltrated cells and the number of Grl + cells, wherein the calculation formula is as follows: the inhibition ratio (%) × 100 (NIH3T3-ILI7-lgG group cell number-administered group cell number)/(NIH 3T3-ILl7-IgG group cell number-NIH 3T3 cell number). The results showed that the total inhibition rate of infiltrating cells of antibody hu31 groups (3, 10, 30mg/kg) was 50.0%, 56.7% and 78.3%, respectively. The inhibition rate of the Grl + cell number is calculated, and the result shows that: the average Grl + cell inhibition rates of each group (3, 10 and 30mg/kg) of the antibody hu31 are 59.1%, 54.5% and 81.8%, respectively, and the antibody hu31 can inhibit IL-17-induced mouse total cell infiltration and inflammatory cell infiltration in a dose-dependent manner.

Sequence listing

<110> Shanghai Junshi biomedical science and technology Co., Ltd

SUZHOU JUNMENG BIOSCIENCES Co.,Ltd.

SHANGHAI JUNSHI BIOENGINEERING Co.,Ltd.

<120> anti-IL-17A antibody pharmaceutical composition and use thereof

<130> 203520

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Gly Tyr Ile Phe Thr Asn Tyr Gly

1 5

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Ile Asn Asp His Thr Gly Glu Pro

1 5

<210> 3

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Ala Asn Tyr Gly Phe Gly Tyr Phe Asp Tyr

1 5 10

<210> 4

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Gln Ser Leu Val His Ser Asn Gly Asn Ser Tyr

1 5 10

<210> 5

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Lys Val Ser

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Ser Gln Ser Thr His Val Pro Tyr Thr

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Ser Asp Tyr Ala Trp

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<213> Artificial Sequence (Artificial Sequence)

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Phe Ile Ser Tyr Ser Gly Pro Thr Ser Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 9

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Gly Gly Asp Gly Asp Ser Phe Asp Tyr

1 5

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Asp

1 5 10 15

<210> 11

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Lys Val Ser Asn Arg Phe Ser

1 5

<210> 12

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Phe Gln Gly Ser His Phe Pro Thr

1 5

<210> 13

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Gly Tyr Ser Ile Thr Ser Asp Tyr Ala

1 5

<210> 14

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Ile Ser Tyr Ser Gly Pro Thr

1 5

<210> 15

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Ala Arg Gly Gly Asp Gly Asp Ser Phe Asp Tyr

1 5 10

<210> 16

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr

1 5 10

<210> 17

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Lys Val Ser

<210> 18

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Phe Gln Gly Ser His Phe Pro Thr

1 5

<210> 19

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Asp His Thr Gly Glu Pro Thr Tyr Ala Asp Lys Phe

50 55 60

Gln Gly Arg Val Thr Phe Thr Leu Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Asn Tyr Gly Phe Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 20

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Ser Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser

85 90 95

Thr His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 21

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Gln Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Asp His Thr Gly Glu Pro Thr Tyr Ala Asp Lys Phe

50 55 60

Gln Gly Arg Val Thr Phe Thr Leu Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Asn Tyr Gly Phe Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 22

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Ser Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 23

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Ser Ser Asp

20 25 30

Tyr Ala Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Phe Ile Ser Tyr Ser Gly Pro Thr Ser Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Asp Gly Asp Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 24

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Lys Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Phe Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 25

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Phe Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 26

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Phe Ile Ser Tyr Ser Gly Pro Thr Ser Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Leu Lys Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Asp Gly Asp Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 27

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Phe Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 28

<211> 444

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 28

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Asp His Thr Gly Glu Pro Thr Tyr Ala Asp Lys Phe

50 55 60

Gln Gly Arg Val Thr Phe Thr Leu Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Asn Tyr Gly Phe Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 29

<211> 218

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 29

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Ser Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser

85 90 95

Thr His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu

210 215

<210> 30

<211> 444

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

Gln Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Met Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Asp His Thr Gly Glu Pro Thr Tyr Ala Asp Lys Phe

50 55 60

Gln Gly Arg Val Thr Phe Thr Leu Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Asn Tyr Gly Phe Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 31

<211> 219

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 31

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Ser Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 32

<211> 445

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Ser Ser Asp

20 25 30

Tyr Ala Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Phe Ile Ser Tyr Ser Gly Pro Thr Ser Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Asp Gly Asp Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 33

<211> 218

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 33

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Lys Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Phe Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 34

<211> 218

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 34

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Phe Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 35

<211> 445

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 35

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Phe Ile Ser Tyr Ser Gly Pro Thr Ser Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Leu Lys Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Asp Gly Asp Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 36

<211> 218

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 36

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Phe Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

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anti-IL-17A antibody pharmaceutical composition and application thereof

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