CN115093483A - IL-17RA fusion protein, pharmaceutical composition, injection and application thereof - Google Patents

Abstract

The invention provides an IL-17RA fusion protein, a pharmaceutical composition, an injection and application thereof. The IL-17RA fusion protein comprises a signal peptide, an IL-17RA extracellular domain and an IgG1 constant region which are operably linked and connected in series. The IL-17RA fusion protein of the invention has prolonged half-life, obtains longer-acting drug activity, and reduces immunogenicity compared with antibody drugs. The design of the present invention allows the IL-17RA fusion protein to eliminate ADCC, ADCP and CDC effects, and retain the neonatal Fc receptor (FcRn) -mediated in vivo recycling effect, with less side effects and greater safety compared to the IL-17A and IL-17RA antibodies that are already on the market.

Description

IL-17RA fusion protein, pharmaceutical composition, injection and application thereof

technical field

The invention belongs to the field of medicine, and specifically relates to an IL-17RA fusion protein, a pharmaceutical composition, an injection and applications thereof.

Background technique

Psoriasis, commonly known as psoriasis, is a chronic autoimmune skin disease that is prone to recurrence. It usually causes great physical and psychological damage to patients. % to 3%, and there are currently 125 million psoriasis patients worldwide. The total prevalence rate in my country is 0.47%, and there are nearly 6.5 million clinically registered patients. Plaque psoriasis is one of the five most common forms of the disease, accounting for 80% to 90% of all cases, with moderate to severe patients. Accounting for 38%, the number of patients in the future will reach 9.5 million in 2030, and the proportion of moderate to severe patients will increase to 40%. The pathogenesis of psoriasis is associated with dysregulation of the innate and adaptive immune systems, including dendritic cell activation and pro-inflammatory cytokine secretion, leading to the development of skin inflammation characteristic of psoriasis. In recent years, nearly 13 kinds of monoclonal antibodies and antibody-based biological therapies have been approved for the treatment of psoriasis, and there are about 10 monoclonal antibody drugs in the clinical research stage, among which ustekinumab (IL- 12/IL-23 antibody, Johnson & Johnson) antibody, TNF-alpha drugs etanercept (Etanercept, Amgen) and adalimumab (Humira, AbbVie) were the top sellers. The oral phosphodiesterase 4 (PDE4) selective inhibitor drug apremilast (Otezla, Celgene) was also approved in the United States in September 2014 for the treatment of refractory psoriasis. However, even when these biologics are used as second-line drugs, the efficacy and safety of the treatment remain worrisome, and up to 40% of patients with moderate-to-severe psoriasis fail treatment with existing biologics, a phenomenon known as Fatigue phenomenon in biological products.

The latest research shows that the pathogenesis of psoriasis is related to T17 helper cells (Th17 cells), and the proinflammatory cytokine IL-17A produced by Th17 cells and innate immune cells has been confirmed to be involved in the pathogenesis of psoriasis. The main cytokine in the mechanism, the immune system targets IL-23 in the IL-17-TH17 pathway, which indirectly contributes to the development of psoriasis through IL-17A. In theory, inhibiting IL-17 may be a safer treatment option than other biologics. The marketed fully human IgG2 monoclonal antibody (Brodalumab) against the IL-17A receptor, and Eli Lilly's fully human IgG4 monoclonal antibody against IL-17A (ixekizumab) and etanercept and ustekin Anti-(IL-12/IL-23 antibody) head-to-head superiority comparison study was confirmed. Studies have shown that inhibitors developed against IL-17A targets can improve efficacy and safety compared with current inflammatory disease treatment drugs (such as TNF-α inhibitors), and TNF-α inhibitors and IL-12 and IL-12. Patients with psoriasis who are refractory to IL-23 inhibitors are effective and more specific than IL-12 and IL-23 inhibitors. In particular, secukinumab (trade name Cosentyx), which was launched in January 2015, was approved by the European Medicines Agency (EMA) to replace other first-line systemic therapies with significant side effects for psoriasis patients. first-line treatment.

At present, more innovative drugs with excellent curative effect are still needed as treatment options for patients.

SUMMARY OF THE INVENTION

In order to solve the problems existing in the above-mentioned prior art, the present invention provides IL-17RA fusion protein, pharmaceutical composition, injection and application thereof.

Specifically, the present invention provides:

(1) An IL-17RA fusion protein, characterized by comprising an operably linked signal peptide, an IL-17RA extracellular domain and an IgG1 constant region in series in series.

(2) The IL-17RA fusion protein according to (1), wherein the amino acid sequence of the extracellular domain of IL-17RA is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

(3) The IL-17RA fusion protein according to (1), wherein the amino acid sequence of the IgG1 constant region is shown in SEQ ID NO.4.

(4) The IL-17RA fusion protein according to (1), wherein the signal peptide is the IL-17-RA natural signal peptide, and its amino acid sequence is shown in SEQ ID NO.5.

(5) The IL-17RA fusion protein according to (1), wherein a linker is used between the IL-17RA extracellular domain and the IgG1 constant region.

(6) The IL-17RA fusion protein according to (1), wherein the amino acid sequence of the IL-17RA fusion protein is shown in SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8.

(7) An isolated nucleic acid encoding the IL-17RA fusion protein according to any one of (1)-(6).

(8) An expression vector containing the nucleic acid according to (7) operably linked to a promoter.

(9) A host cell containing the expression vector according to (8).

(10) The host cell according to (9), wherein the deposit number of the host cell is CGMCC 21011.

(11) A protein dimer formed by the IL-17RA fusion protein according to any one of (1)-(6), the dimer being composed of two molecules of the IL-17RA fusion protein passing through the The cysteines of the IgG1 constant region bind to form a double chain.

(12) The IL-17RA fusion protein according to any one of (1)-(6) or the protein dimer according to claim 11 is prepared for the treatment of psoriasis, Crohn's disease, plaque Mass psoriasis, gastroenteritis, Behçet's syndrome, arthritis, uveitis, hidradenitis suppurativa, lichen planus, parapsoriasis, asthma, psoriatic arthritis, tendonitis, relapsing-remitting Multiple sclerosis, thyroid-related eye disease, juvenile rheumatoid arthritis, multiple sclerosis, lupus nephritis, spondyloarthritis, ankylosing spondylitis, rheumatoid arthritis, inflammatory bowel disease, nonalcoholic fatty liver disease, giant Cellular arteritis, nonradiographic axial spondyloarthritis, acne vulgaris, triple negative breast tumors, multiple myeloma, non-small cell lung cancer, adenocarcinoma, colorectal cancer, prostate cancer, Kaposi's sarcoma, melanoma Cancer, cervical cancer and/or other inflammatory diseases drug application.

(13) A pharmaceutical composition comprising a therapeutically effective amount of the IL-17RA fusion protein according to any one of (1)-(6) or the protein dimer according to claim 11 as an active ingredient and Medicinal excipients.

(14) The pharmaceutical composition according to (13), wherein the pharmaceutically acceptable adjuvant is selected from one or more of diluents, buffers, protective agents, surfactants, and antioxidants.

(15) The pharmaceutical composition according to (14), wherein the buffer is selected from the group consisting of histidine-acetate buffer, Tris-acetate buffer, hydrochloric acid buffer, phosphate buffer, acetate buffer, histidine One or more of buffer, arginine buffer, succinate buffer, citrate buffer.

(16) The pharmaceutical composition according to (14), wherein the protective agent is selected from one of trehalose, Tween-20, Tween-80, sucrose, amino acids, polyols, disaccharides, and polysaccharides or variety.

(17) The pharmaceutical composition according to (14), wherein the surfactant is selected from one or more of Tween-20, Tween-80, and Poloxamers.

(18) The pharmaceutical composition according to (13), wherein a single dose of the pharmaceutical composition comprises 5 mg/ml-150 mg/ml of the IL-17RA fusion protein or the protein dimer.

(19) The pharmaceutical composition according to (13), wherein the pharmaceutical composition is in the form of a lyophilisate or an injection.

(20) A drug for the treatment of psoriasis, Crohn's disease, plaque psoriasis, gastroenteritis, Behcet's syndrome, arthritis, uveitis, hidradenitis suppurativa, lichen planus, parasilt Psoriasis, asthma, psoriatic arthritis, tendonitis, relapsing-remitting multiple sclerosis, thyroid-related eye disease, juvenile rheumatoid arthritis, multiple sclerosis, lupus nephritis, spondyloarthritis, ankylosing spondylitis , rheumatoid arthritis, inflammatory bowel disease, nonalcoholic fatty liver disease, giant cell arteritis, nonradiographic axial spondyloarthritis, acne vulgaris, triple negative breast tumors, multiple myeloma, non-small cell lung cancer , an injection for adenocarcinoma, colorectal cancer, prostate cancer, Kaposi's sarcoma, melanoma, cervical cancer and/or other inflammatory diseases, comprising the pharmaceutical composition described in any one of (13)-(19) .

(21) The injection according to (20), wherein the injection is in the form of a lyophilized powder or a liquid preparation.

(22) The injection according to (20), wherein the injection is a subcutaneous injection or an intravenous drip.

(23) The injection according to (21), wherein in the liquid preparation, the intravenous preparation comprises 5 mg/ml-150 mg/ml of the IL-17RA fusion protein or the protein dimer, 2- 100 mM Tris-acetic acid, 10-250 mM arginine, 50-500 mM trehalose, 0.01-5% Tween-20.

(24) The injection according to (21), wherein the liquid preparation is prepared by using water for injection, buffered saline solution, aqueous dextrose solution, aqueous sodium chloride solution or lactated Ringer's solution.

(25) The injection according to (21), wherein the freeze-dried powder is prepared by freeze-drying the liquid preparation.

Compared with the prior art, the present invention has the following advantages and positive effects:

The IL-17RA fusion protein provided by the present invention is a fully human antibody Fc fusion protein drug. In the present invention, by fusing IL-17RA with the Fc segment of the antibody, the half-life of the drug molecule is prolonged, the longer-acting drug activity is obtained, and the immunogenicity is reduced compared with the antibody drug.

In addition, the present invention finds that selecting the Fc segment of IgG1 and further mutating the Fc sequence appropriately can greatly eliminate antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement Dependent cell-mediated cytotoxicity (CDC), and retains the effect of neonatal Fc receptor (FcRn)-mediated in vivo recycling, with low side effects compared to marketed IL-17A and IL-17RA antibodies and more secure.

In addition, the present invention selects and designs the sequence of IL-17RA, so that the IL-17RA fusion protein can target multiple targets such as IL-17A, IL-17C, IL-17F, IL-17A/F, etc., and has high affinity, Thus, it can selectively block the binding of IL-17A, IL-17C, IL-17F and IL-17A/F to their receptors, thereby effectively blocking the biological activity of various pro-inflammatory IL-17 cytokines, inhibiting Therefore, it can more effectively alleviate the symptoms of autoimmune diseases, obtain better therapeutic benefits than single IL-17A target antibody drugs, and obtain better safety than IL-17RA monoclonal antibodies. Its mechanism of action is different from the IL-17 target drugs currently on the market and under development, and it is the first drug of its kind in the world.

The present invention is based on the binding affinity with IL-17A, biological activity cell experiments, GRO-α factor inhibitory ability, in vitro activity titer, in vivo titer, ADCC/CDC functional activity, FcRn binding affinity, safety pharmacology, pharmacokinetics The activity and safety of the above drugs were comprehensively evaluated from the aspects of science and toxicology.

Description of drawings

Figure 1 shows the amino acid sequence of the recombinant human IL-17RA fusion protein.

Figure 2 shows a map of the expression vector pCHO1.1/NVS451 used in one embodiment.

FIG. 3 shows the affinity measurement curve and fitting curve of IL-17RA fusion protein (NVS451) and human IL-17A protein in Experimental Example 1. FIG.

FIG. 4 shows the affinity measurement curve and fitting curve of secukinumab and human IL-17A protein in Experimental Example 1. FIG.

FIG. 5 shows the affinity measurement curve and fitting curve of wild-type IL-17RA and human IL-17A protein in Experimental Example 1. FIG.

FIG. 6 shows the affinity measurement curve and fitting curve of IL-17RA fusion protein (NVS451) and human IL-17F protein in Experimental Example 1. FIG.

FIG. 7 shows the affinity measurement curve and fitting curve of IL-17RA fusion protein (NVS451) and human IL-17A/F protein in Experimental Example 1. FIG.

FIG. 8 shows the affinity measurement curve and fitting curve of IL-17RA fusion protein (NVS451) and human IL-17C protein in Experimental Example 1. FIG.

FIG. 9 shows the S-curve of the inhibitory effect of IL-17RA fusion protein (NVS451) and secukinumab on GRO-α factor induced by IL-17A in Experimental Example 1. FIG.

FIG. 10 shows the S-curve of the inhibitory effect of IL-17RA fusion protein (NVS451) on GRO-α factor induced by IL-17A/F in Experimental Example 1. FIG.

FIG. 11 shows the S curve of the inhibitory effect of GRO-α factor on the induction of IL-17F by IL-17RA fusion protein (NVS451) in Experimental Example 1. FIG.

FIG. 12 shows the appearance of skin after xenografted SCID mice were treated with different drugs in Experimental Example 1. FIG.

FIG. 13 shows the appearance of skin after xenografted SCID mice were treated with different drugs in Experimental Example 1. FIG.

FIG. 14 shows the skin pathological sections after xenografted skin-grafted SCID mice were treated with different drugs in Experimental Example 1. FIG.

FIG. 15 shows the ADCC effect comparison of RitxV301 and Rituximab in Experimental Example 1. FIG.

FIG. 16 shows the comparison of the CDC effects of RitxV301 and Rituximab in Experimental Example 1. FIG.

FIG. 17 shows the SPR analysis patterns of NVS451 and human FcRn at different concentrations under acidic conditions (pH 6.0) in Experimental Example 1. FIG.

18 shows the SPR analysis patterns of NVS451 and human FcRn at different concentrations under neutral conditions (pH 7.4) in Experimental Example 1.

Figure 19 shows the affinity comparison of IL-17RA fusion proteins NVS451 (V301), V302 and V303 with IL-17A in Experimental Example 1; the concentrations of V300, V301, V302, V303, V301*, V302*, V303* in the figure Both are 100ug/ml.

Biomaterial deposit information

The Chinese hamster ovary cells provided by the present invention capable of stably expressing the IL-17RA fusion protein of the present invention have been deposited in the General Microorganism Center (CGMCC) of the China Microorganism Culture Collection and Management Committee on November 23, 2020, and the deposit address: Chaoyang, Beijing No. 3, No. 1 Yard, Beichen West Road, District, Zip code: 100101, preservation number: CGMCC No.21011.

Detailed ways

The present invention will be further described below through the description of specific embodiments and with reference to the accompanying drawings, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements according to the basic idea of the present invention, but as long as they do not depart from the basic idea of the present invention The basic idea of the present invention is within the scope of the present invention.

As used herein, the term "injectable preparation" refers to sterile solutions (including true solutions, emulsions and suspensions) made of drugs for infusion into the body, as well as for preparation of such sterile solutions just prior to use ( including true solutions, emulsions and suspensions), lyophilized powders or concentrated solutions.

As used herein, the term "intravenous drip" refers to a method of infusing a large amount of fluid containing a drug into the body intravenously through an infusion tube. Also known as "infusion", "infusion", "intravenous drip", "hanging water".

As used herein, the term "IL-17A" refers to interleukin 17A.

As used herein, the term "IL-17RA" refers to the receptor for IL-17A.

As used herein, the term "active ingredient" refers to a drug molecule that has a therapeutic effect on a disease, such as the IL-17RA fusion protein described herein.

The present invention provides an IL-17RA fusion protein, which is characterized by comprising an operably linked signal peptide, an IL-17RA extracellular domain and an IgG1 constant region in series.

The role of signal peptides is mainly to guide the secretion of target proteins from the cytoplasm to the outside of the cell. Since this fusion protein has an IgG1 constant region, when it is secreted out of the cell, the two-molecule fusion protein forms a double chain through the combination of cysteines in the constant region and exerts activity.

Preferably, the present invention adopts the extracellular domain of human IL-17RA (Gene bank number: NP_055154), and carries out R108K, D122G and H155D mutations, and the amino acid sequence of the mutant is shown in SEQ ID NO.1. Also preferably, the present invention adopts the extracellular domain of human IL-17RA, and mutates L9P, R108K, D122G and H155D, and the amino acid sequence of the mutant is shown in SEQ ID NO.2. The present inventors found that IL-17RA fusion proteins comprising these two mutants have increased thermostability, and increased binding affinity for IL-17A, compared to those comprising the wild type. The amino acid numbering starts from position 1 of the amino acid sequence of the extracellular domain of the human IL-17RA.

Also preferably, the present invention adopts the extracellular domain of human IL-17RA, and mutates L9P, R108K, D122G, H155D, G243W and A267V, and the amino acid sequence of the mutant is shown in SEQ ID NO.3. The present inventors found that the IL-17RA fusion protein comprising the mutant has improved thermal stability and improved binding affinity to IL-17A, IL-17C, IL-17F, and IL-17A/F compared to the wild type. ; higher binding affinity for IL-17A compared to mutants containing the three and four mutations described above.

The present invention selects the constant region (Fc) of human IgG1 (Gene bank number: 3500) to form a fusion protein with IL-17RA. The fusion protein not only retains the biological activity of the functional protein molecule, but also because the Fc part has certain antibody characteristics and is stable, the fused protein obtains a longer circulation life and prolongs the half-life. IgG isotypes include IgG1, IgG2, and IgG4, which cause distinct ADCC, ADCP, and CDC effects that can have a major impact on toxicity in target and non-target tissues. The constant region of IgG1 has strong ADCC, ADCP and CDC effects. The present invention finds that selecting the constant region of IgG1 and further mutating the Fc sequence appropriately can reduce the ADCC, ADCP and CDC effects of the IL-17RA fusion protein.

In preferred embodiments, the mutation sites are summarized in Table 1 below. An additional mutation was to replace cysteine residues in the hinge region of IgG1 with serine residues to avoid the presence of unpaired cysteines in the fusion protein sequence.

Table 1

Note: The amino acid numbering described in Table 1 is counted from position 1 of the human IgG1 amino acid sequence.

The amino acid sequence of the IgG1-Fc mutant is shown in SEQ ID NO.4.

Signal peptides are one of the main factors affecting yield optimization and product quality. It is important that the signal peptide cleavage site should be well-defined by a single residue with a high probability of cleavage. Preferably, the natural signal peptide of human IL17RA is used for the fusion protein, and the amino acid sequence of the signal peptide is shown in SEQ ID NO.5.

Preferably, a linker is used between the IL-17RA extracellular domain and the IgG1 constant region. Linkers can be those commonly used in the art, such as one or more contiguous GSGs, one or more contiguous GGGGSs, and GSAGSAAGSG.

Preferably, the amino acid sequence of the IL-17RA fusion protein is shown in SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8 (the extracellular domain of IL-17RA has three mutations, four mutations, six mutations), which has 522 amino acid residues.

For example, the sequence of SEQ ID NO. 8 is shown in Figure 1, wherein the first 32 amino acids (bold) are the IL-17RA signal peptide, and the letters on a light gray background are recombination with 6 mutations (bold underlined letters) The amino acid sequence of human IL-17RA; the black background letters are the linker amino acid sequence; the dark gray background letters are the amino acid sequence of the Fc part of the 3 modified (bold and underlined letters) described in Table 1, wherein two bold half Cystine residues are responsible for dimerization of Fc. Asterisks (*) indicate potential glycosylation sites.

The mechanism of action of the IL-17RA fusion protein provided by the present invention is that the decoy receptor (IL-17RA-Fc) is used to compete with the natural receptor for binding to IL-17 molecules, and the IL-17A, IL-17C, IL-17F and IL-17A/F binds with high affinity and selectively blocks the binding of IL-17A, IL-17C, IL-17F and IL-17A/F to their receptors, but not IL-17B, IL-17D, IL- Combined with 17E, it can effectively block the biological activity of a variety of pro-inflammatory IL-17 cytokines and inhibit the inflammatory signaling pathway, thereby more effectively alleviating the symptoms of autoimmune diseases. Good therapeutic benefit and a better safety profile than IL-17RA mAb.

The present invention also provides an isolated nucleic acid encoding the IL-17RA fusion protein according to the present invention.

In a preferred embodiment, the present invention utilizes molecular biology techniques to design the cDNA sequence of the fusion protein, and optimizes the codons to express it in CHO cells. The optimized DNA sequences encoding the amino acid sequences SEQ ID NO. 1-8 are shown in SEQ ID NO. 9-16, respectively.

The present invention also provides an isolated mRNA transcribed from the DNA encoding the fusion protein of the present invention.

The present invention also provides an expression vector containing the nucleic acid according to the present invention operably linked to a promoter.

The present invention also provides a host cell containing the expression vector according to the present invention.

In some preferred embodiments, CHO cells suitable for growth in suspension and serum-free media are used as host cells. Also preferably, the expression vector contains two selectable markers, puromycin and methotrexate, which facilitate the creation of high-yielding and stable cell lines.

In a more preferred embodiment, the host cell has a deposit number of CGMCC 21011. The construction of the host cell uses CHO-S ^TM cells of Thermo Scientific Company, which can stably express the IL-17RA fusion protein of the present invention after construction. In addition, the host cell also has many advantages: (1) It has accurate post-transcriptional modification function, and the expressed protein is closest to the natural protein molecule in terms of molecular structure, physicochemical properties and biological functions; (2) It can grow on the wall. , and can be cultured in suspension, and has high resistance to shear force and osmotic pressure; (3) It has the ability to efficiently amplify and express recombinant genes, and the integration of exogenous proteins is stable; (4) It has the function of extracellular secretion of products , and seldom secrete its own endogenous protein, which is convenient for the separation and purification of downstream products; (5) It can achieve high-density culture in suspension culture or in serum-free medium. And the culture volume can reach more than 1000L, which can be mass-produced. Therefore, this CHO cell is the system of choice for recombinant glycoprotein production. The host cell has been deposited in the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee (CGMCC) on November 23, 2020. The deposit address is: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Zip Code: 100101, and the deposit number is: : CGMCC No.21011.

The present invention also provides a protein dimer formed by the IL-17RA fusion protein according to the present invention, the dimer is composed of two molecules of the IL-17RA fusion protein passing through the cysteine of the IgG1 constant region Acids combine to form double strands.

Since the IL-17RA fusion protein of the present invention has an IgG1 constant region, when it is secreted to the outside of the cell, the two-molecule fusion protein forms a double chain through the combination of cysteines in the constant region and exerts activity.

The present invention also provides the IL-17RA fusion protein according to the present invention or the protein dimer formed by the IL-17RA fusion protein prepared for the treatment of psoriasis, Crohn's disease, plaque psoriasis, Gastroenteritis, Behcet's syndrome, arthritis, uveitis, hidradenitis suppurativa, lichen planus, parapsoriasis, asthma, psoriatic arthritis, tendinitis, relapsing-remitting multiple sclerosis, thyroid related eye disease, juvenile rheumatoid arthritis, multiple sclerosis, lupus nephritis, spondyloarthritis, ankylosing spondylitis, rheumatoid arthritis, inflammatory bowel disease, nonalcoholic fatty liver, giant cell arteritis, Non-radiological axial spondyloarthritis, acne vulgaris, triple negative breast tumor, multiple myeloma, non-small cell lung cancer, adenocarcinoma, colorectal cancer, prostate cancer, Kaposi's sarcoma, melanoma, cervical cancer, etc. and/or other inflammatory disease drug applications

The present invention is based on the binding affinity with IL-17A, biological activity cell experiments, GRO-α factor inhibitory ability, in vitro activity titer, in vivo titer, ADCC/CDC functional activity, FcRn binding affinity, safety pharmacology, pharmacokinetics The pharmacological activity and safety of IL-17AR fusion protein were comprehensively evaluated in terms of science and toxicology. The results show that the IL-17RA fusion protein can target multiple targets such as IL-17A, IL-17C, IL-17F, IL-17A/F, etc., and has high affinity, so that it can selectively block IL-17A , IL-17C, IL-17F and IL-17A/F are combined with their receptors, thereby effectively blocking the biological activity of a variety of pro-inflammatory IL-17 cytokines, inhibiting inflammatory signaling pathways, so it can be more effectively alleviated For the symptoms of autoimmune diseases, better treatment benefits can be obtained than single IL-17A target antibody drugs, and better safety than IL-17RA monoclonal antibodies.

The present invention also provides a pharmaceutical composition, comprising a therapeutically effective amount of the IL-17RA fusion protein according to the present invention or a protein dimer formed by the IL-17RA fusion protein as an active ingredient and pharmaceutically acceptable excipients .

Pharmaceutically acceptable excipients can be selected according to the dosage form used and actual needs.

The present invention thoroughly studies and designs the preparation and preparation prescription of the IL-17RA fusion protein from the aspects of packaging material, buffer system, auxiliary materials, dosage, prescription composition, freeze-drying process and the like.

In some preferred embodiments, the pharmaceutically acceptable adjuvants of the pharmaceutical composition are selected from one or more of diluents, buffers, protective agents, surfactants, and antioxidants.

Preferably, the buffer is selected from histidine-acetate buffer, Tris-acetate buffer, hydrochloric acid buffer, phosphate buffer, acetate buffer, histidine buffer, arginine buffer, succinate buffer, One or more of citrate buffers.

Preferably, the protective agent is selected from one or more of trehalose, Tween-20, Tween-80, sucrose, amino acids (eg, arginine), polyols, disaccharides, and polysaccharides. Among them, trehalose and the combination of trehalose and arginine are most preferred.

In some embodiments, the trehalose is present as trehalose dihydrate.

Preferably, the surfactant is selected from one or more of Tween-20, Tween-80, and poloxamer.

In some preferred embodiments, a single dose of the pharmaceutical composition comprises 5 mg/ml-150 mg/ml of the IL-17RA fusion protein or a protein dimer formed by the IL-17RA fusion protein.

The pharmaceutical composition of the present invention can be prepared into suitable dosage forms as required. In the present invention, the IL-17RA fusion protein is preferably in the form of lyophilized preparation or injection.

Therefore, the present invention also provides a method for the treatment of psoriasis, Crohn's disease, plaque psoriasis, gastroenteritis, Behcet's syndrome, arthritis, uveitis, hidradenitis suppurativa, flat lichen, parapsoriasis, asthma, psoriatic arthritis, tendonitis, relapsing-remitting multiple sclerosis, thyroid-related eye disease, juvenile rheumatoid arthritis, multiple sclerosis, lupus nephritis, spondyloarthritis, Ankylosing spondylitis, rheumatoid arthritis, inflammatory bowel disease, non-alcoholic fatty liver disease, giant cell arteritis, non-radiological axial spondyloarthritis, acne vulgaris, triple negative breast tumors, multiple myeloma, Injections for non-small cell lung cancer, adenocarcinoma, colorectal cancer, prostate cancer, Kaposi's sarcoma, melanoma, cervical cancer, etc. and/or other inflammatory diseases, comprising the pharmaceutical composition of the present invention.

The injection can be in the form of a lyophilized powder or a liquid preparation.

Preferably, the injection is a subcutaneous injection or an intravenous drip. Most preferred are subcutaneous injections.

In a liquid formulation, the injection preferably contains the IL-17RA fusion protein, Tris-acetic acid, arginine, trehalose, Tween-20 and a suitable solvent.

More preferably, the injection comprises 5mg/ml-150mg/ml of the IL-17RA fusion protein or the protein dimer formed by the IL-17RA fusion protein, 2-100mM Tris-acetic acid, 10-250mM Arginine, 50-500 mM trehalose, 0.01-5% Tween-20 and a suitable solvent.

The solvent can be the solvent commonly used in the preparation of injections. For example, water for injection, buffered saline solution, aqueous dextrose solution, aqueous sodium chloride solution or lactated Ringer's solution, etc.

The liquid preparation is prepared by using a solvent.

The liquid preparation can be prepared according to the formulation of the present invention by using methods commonly used in the pharmaceutical field.

Lyophilized powders can be prepared by freeze-drying the liquid formulation.

In a preferred embodiment, the lyophilization process includes pre-freezing, primary drying (sublimation) and secondary drying (decomposition). Pre-freezing includes reducing the temperature from 5°C to -40°C and holding for a suitable time; primary drying includes raising the temperature to -5 to 0°C and holding for a suitable time; secondary drying includes raising the temperature to 25°C to 30°C for a suitable time.

According to the research on the preparation and preparation prescription of the IL-17RA fusion protein in the present invention, the present invention has developed a pharmaceutical preparation with excellent drug stability and safe injection administration.

The following examples are used to further explain or illustrate the content of the present invention, but these examples should not be construed as limiting the protection scope of the present invention.

example

Unless otherwise specified, the experimental methods used in the following examples are all performed using conventional experimental procedures, operations, materials and conditions in the fields of bioengineering and pharmaceuticals.

Below, unless otherwise specified, the percentage concentration (%) of each reagent refers to the volume percentage concentration (% (v/v)) of the reagent.

Preparation Example 1: Plasmid Construction

NVS451 (ie, IL-17RA fusion protein) consists of a signal peptide, rhIL17-RA ECD (ie, human IL17-RA extracellular domain), a linker peptide and an IgG1 Fc domain. The signal peptide adopts the natural signal peptide of IL17RA. rhIL17-RAECD contains six mutation sites, namely L9P, R108L, D122G, H155D, G243W and A267V (the amino acid numbering does not include the signal peptide sequence). The linker peptide is GSG. A hybrid of IgG1, 2, 4 was used for the IgG1 Fc domain to remove potential ADCC, CDC and ADCP effects. The amino acid sequence of NVS451 is shown in SEQ ID NO.8, and the nucleotide sequence is shown in SEQ ID NO.16.

Plasmids were constructed using the pCHO1.0 mammalian cell expression vector from Thermo Scientific. The pCHO1.0 expression vector contains two expression cassettes. Since the expression of the NVS451 fusion protein requires only one expression cassette, the other expression cassette was removed using the SfiI restriction site. The removed expression cassette contains EcoRV and PacI clones site. The pCHO1.0 vector after removing one expression cassette was verified by sequence sequencing and named pCHO1.1. The synthesized NVS451 fusion protein gene was inserted into the pCHO1.1 (Kan resistance) expression vector through the AvrII and Bstz17I cloning sites, and transformed into DH5α competent cells, plated and screened, inoculated and amplified the clone containing the correct size plasmid, and the plasmid was extracted to obtain a large number of The constructed NVS451 fusion protein expression plasmid pCHO1.1-NVS4510. Gene sequencing verified that the pCHO1.1-NVS451 expression vector was constructed correctly.

Preparation Example 2: Cell Line Screening and Stability Evaluation

Kit)细胞作为原始细胞基质。NVS451融合蛋白表达质粒pCHO1.1-NVS451含有嘌呤霉素和MTX的筛选标记。pCHO1.1-NVS4510通过转染试剂转入CHO-S细胞。转染过程如下：50μg环状的表达质粒pCHO1.1-NVS451通过Opti PRO SFM稀释至1.5ml体积，50μl Freestyle Max转染试剂(GIBCO)通过Opti PRO SFM稀释至1.5ml，然后质粒稀释液和转染试剂稀释液等体积混合，加入30ml 1E6细胞/ml的CHO-S细胞悬液。转染细胞培养48小时后使用嘌呤霉素和MTX作为筛选压力进行两阶段的稳转克隆细胞池(Pools)筛选。由于未转染的CHO-S缺乏pac(腺苷酸环化酶)活性，仅具有基础DHFR(二氢叶酸还原酶)活性，因此只有被pCHO1.1-NVS4510质粒转染并整合入基因组的CHO-S细胞才能在含有嘌呤霉素和MTX的CD FortiCHO^TM筛选培养基中存活和繁殖。筛选分两个阶段进行，每个阶段都使用嘌呤霉素和MTX进行筛选。最后形成4个稳转克隆细胞池(Pool)，分别为T3S1，T3S2，T3S3，T3S4。通过14天的简单流加批次培养(SFB)来评价表达量。结合基于细胞的GRO-α抑制活性实验、亲和ELISA和SPR分析方法(采用常规方法，故不赘述)，选出T3S1和T3S4两个稳转克隆池用于分离单克隆(LDC)。单克隆的分离采用两轮有限稀释分离单克隆，每轮细胞铺板浓度小于1个细胞/孔(按照统计学的数学计算方法)，最终获得5个候选单克隆：T3S4-7E3-6G5；T3S4-7E3-3C3；T3S4-17G2-6E2；T3S4-8F2-6E10；T3S1-18E7-6C5。The construction and screening of NVS451 fusion protein cell line selected CHO-S ^TM (a cell bank (cGMP-banked) that complies with current drug production management standards), and the cell and the supporting expression vector pCHO1.

Kit) cells as the primitive cell matrix. The NVS451 fusion protein expression plasmid pCHO1.1-NVS451 contains selection markers for puromycin and MTX. pCHO1.1-NVS4510 was transfected into CHO-S cells by transfection reagent. The transfection procedure was as follows: 50 μg circular expression plasmid pCHO1.1-NVS451 was diluted to 1.5 ml volume by Opti PRO SFM, 50 μl Freestyle Max Transfection Reagent (GIBCO) was diluted to 1.5 ml by Opti PRO SFM, and then plasmid dilution and transfection Mix equal volumes of staining reagent diluents and add 30 ml of CHO-S cell suspension of 1E6 cells/ml. After 48 hours of culture of the transfected cells, a two-stage selection of pools of stably transfected clones (Pools) was performed using puromycin and MTX as the selection pressure. Since untransfected CHO-S lacks pac (adenylate cyclase) activity and only has basal DHFR (dihydrofolate reductase) activity, only CHO that was transfected with pCHO1.1-NVS4510 plasmid and integrated into the genome -S cells can survive and propagate in CD FortiCHO ^™ selection medium containing puromycin and MTX. Screening was performed in two phases, each using puromycin and MTX. Finally, four stable cloned cell pools (Pools) were formed, which were T3S1, T3S2, T3S3, and T3S4. Expression levels were evaluated by simple fed-batch culture (SFB) for 14 days. Combined with cell-based GRO-α inhibitory activity assay, affinity ELISA and SPR analysis methods (conventional methods are used, so they are not described in detail), two stably transfected clone pools, T3S1 and T3S4, were selected for the isolation of single clones (LDCs). Isolation of single clones Two rounds of limiting dilution were used to isolate single clones, and the cell plating concentration in each round was less than 1 cell/well (according to statistical mathematical calculation method), and finally 5 candidate clones were obtained: T3S4-7E3-6G5; T3S4- 7E3-3C3; T3S4-17G2-6E2; T3S4-8F2-6E10; T3S1-18E7-6C5.

The stability of 5 candidate monoclones was evaluated at 70PDL (cell doubling time, equivalent to 70 passages). Based on the stability of expression stability, gene copy number, and mRNA transcription level, the final selection of monoclonal VAN301-T3S1- 18E7-6C5 was used as the main single clone (the deposit number was CGMCC 21011), and VAN301-T3S4-17G2-6E2 was selected as the backup clone.

The monoclonal VAN301-T3S1-18E7-6C5 was used to establish PCB (primary cell bank), and on this basis, MCB (master cell bank) and WCB (working cell bank) under GMP conditions were established.

Preparation Example 3: Cell Culture

The NVS451 fusion protein can be produced by a 14-day fed-Batch culture process. Cell recovery: Take a cell from the cell bank, recover it in a water bath at 37.0±0.5°C, transfer the seed solution to 10ml pre-warmed Dynamis (containing 8mM L-Gln, 1:100ACA and 1g/L P188) immediately after thawing for culture Centrifuge at 300g for 5 minutes in a 50ml centrifuge tube, discard the supernatant, add 10ml of pre-warmed Dynamis medium to resuspend the cells, and transfer the resuspended cell solution to a 125ml shake flask containing 28.0ml of Dynamis medium. , the cell density was (0.15～0.35)×10 ⁶ cells/ml, and the cell viability was greater than 90%. Seed passage and expansion: the passage medium is Dynamis, the initial seeding density of cells in subculture should be (0.40±0.10)×10 ⁶ cells/mL, and the cell density reaches (2.0～5.0)×10 ⁶ cells after 3 days of culture /mL can be passaged, and the cell viability rate should be higher than 90.0% during the passage.

Reactor fed batch culture (Fed batch): the initial seeding density of cells was (0.40±0.10)×10 ⁶ cells/mL, fed with 2×Feed C+ (Efficient FeedC+, Cat#: A2503101, Gibco), every day during the culture The contents of glucose and lactate were detected, and 450.0g/kg of glucose mother solution was added to make up to 5.0g/L on the 3rd/5/7/9/11/13 days respectively.

Cell culture harvest conditions: Harvest when cultured to the 14th day or when the cell viability rate is lower than 80.0%, whichever comes first.

Preparation Example 4: Purification of NVS451 Fusion Protein

The cell culture harvest solution was first clarified by a two-stage depth filtration membrane package, then 1% Tween 80, 0.3% tributyl phosphate was added for virus inactivation, and then the target protein was captured using GE's MabSelect SuRe affinity chromatography packing Then use Millipore's Eshmuno CPX anion chromatography packing and GE's Capto Adhere cationic chromatography packing for two-step refinement, then use the BioEX nanofiltration membrane of ASAHI KASEI company for nanofiltration, use Millipore's Pellicon 2, interception The PES material with a molecular weight of 50KDa and the C-channel ultrafiltration membrane are used for ultrafiltration diafiltration, and finally the auxiliary material polysorbate 20 is added, and the NVS451 stock solution is obtained after sterilization and filtration.

1. Deep filtering

Use a series of depth filtration membranes (primary depth filtration membrane DOHC and secondary filter A1HC, Millipore Corporation) to perform depth filtration to remove impurities such as cells, and harvest the clarified solution containing the target protein. When filtering the sample, the inlet flow rate is less than or equal to 100LMH (based on A1HC), the maximum load of DOHC is 60L/m ² , and the maximum load of A1HC is 140L/m ² . The pressure is controlled, and the single-step recovery rate of deep filtration is generally about 90%.

2. Detergent virus inactivation

To the clarified harvest was added 50 mM Tris-HAc, 150 mM NaCl, 25% PS80, pH 7.4 to give a final Tween 80 concentration of 1.0%. 100% tributyl phosphate was added to make a final concentration of 0.3%. Incubate at 16-26°C for 360-480 minutes, followed by affinity chromatography.

3. Affinity chromatography

Preliminary purification of the product was achieved by affinity chromatography using MabSelect SuRe media. Use 50 mM Tris-HAc, 150 mM NaCl, pH 7.4 as equilibration and post-loading rinse 1 buffer, then 20 mM Tris, 1 M NaCl, 0.5 MArg, pH 8.6 for rinse 2, and finally 50 mM glycine, pH 3.5 Buffer to elute the target protein. The eluate was neutralized to pH 7.8-8.2 using 1M Tris base.

4. Cation chromatography

Eshmuno CPX from Millipore Corporation was used as the cation chromatography packing. After neutralization by affinity chromatography, the sample was adjusted to pH 6.3-6.7 with 1M HAc, and the adjusted sample was used as the sample for cation chromatography. Product-related impurities such as HCP, protein A, DNA and some product analog impurities and fragments can be effectively removed after washing and gradient elution.

Use 20 mM PB, pH 6.5 as equilibration and post-load wash buffer, 25 mM PB, 110 mM NaCl, pH 6.5 as wash 2, elute using 25 mM PB, 110 mM NaCl, pH 6.5 pull the gradient to 20 mM PB, Gradient elution was performed with 1 M NaCl, pH 6.5, 0-50% B (10 CV).

5. Anion chromatography

GE's Capto Adere medium was used as an anion chromatography medium to remove some product and process related impurities. The cation chromatography eluent was first adjusted to pH 8.4-8.6 with 1M Tris base and water for injection, and the conductivity was 19.0-23.0. The sample after pH adjustment was used as the sample for anion chromatography.

Use 20 mM Tris, 0.2 M NaCl, pH 8.5 as equilibration and post-loading wash buffer, 20 mM Tris, 0.2 M NaCl, 60 mM Arg, pH 8.0 as wash 2 buffer, use 20 mM Tris, 0.2 M NaCl, 290 mM Arg, pH7.1 buffer to elute the target protein.

6. Nanofiltration

Anion chromatography eluted samples were filtered for virus removal using Asahi Kasei Nanofilter BioEX.

The nanofiltration process mainly utilizes the different molecular sizes of virus and protein products, the potential virus is intercepted by the nanofiltration membrane, and the target protein flows through, thereby realizing the separation of the two. The pre-filtration membrane can adsorb impurities such as particles in the sample, and at the same time increase the processing capacity of the nanofiltration membrane. Nanofiltration membranes can effectively retain potential viruses such as parvoviruses. A1HC (Millipore Corporation) was used for prefiltration and BioEX (Asahi Kasei Corporation) to remove potential viruses. When filtering the sample, control the pressure difference on the prefiltration membrane to be ≤2 bar, the pressure difference on the nanofiltration membrane to be ≤3 bar, and the loading capacity of the nanofiltration membrane to be ≤600 g/m ² .

7. Ultrafiltration Diafiltration

After nanofiltration, the intermediate product was concentrated using a Pellicon 2 (Millipore Company) ultrafiltration membrane package, and then the solution was changed to a buffer system of 20 mM Tris-HAc, 65 mM Arg, 120 mM trehalose, pH 7.5. The ultrafiltration membrane package material is PES, the molecular weight cut off is 50kDa, and the C flow channel. The capacity of ultrafiltration and diafiltration is ≤300g/m ² ; the inlet flux is 150-300LMH, the transmembrane pressure (TMP) is ≤1.5bar, and the concentration is 18.0-22.0g/L; When the inlet flux is 150-300LMH, TMP≤2bar, the liquid exchange volume is ≥5DV, the inlet flux is 150-300LMH, TMP≤2bar when overconcentrated, and the concentration is 80.0-90.0g/L.

8. Excipients addition and final filtration

Add 10% (w/w) polysorbate 20 stock solution to the sample after ultrafiltration diafiltration, and then use 20mM Tris-HAc, 65mM Arg, 120mM Trehalose, pH7.5 buffer to adjust the protein concentration to 70.20～150.80mg/mL , that is, the stock solution is prepared. Then, sterilization filtration was performed, and the sterilization filtration was filtered with a PES material (0.22 μm) filter. The final content of polysorbate 20 in the stock solution is 0.02% (w/v), and the total recovery rate of auxiliary material addition and sterilization filtration is generally above 80%.

9. Stock solution

The quality test of the stock solution after the addition of auxiliary materials and sterilization and filtration shows that the purity is over 97%.

Store the stock solution in a -40±5°C freezer.

Experimental Example 1: Pharmacodynamics Study

1. In vitro pharmacodynamics

The active molecule of NVS451 is a double-chain fusion protein composed of a human IL-17RA extracellular segment mutant and a human IgG1 Fc mutant, which was obtained by recombinant expression in CHO cells. Therefore, the molecule can exhibit the biological functional properties of both IL-17RA and Fc molecules. A series of studies on the in vitro biological activity of NVS451 were carried out through surface plasmon resonance (SPR), in vitro target cell killing assay and other analytical techniques, aiming to clarify its in vitro pharmacodynamic properties.

1.1 Comparative analysis of IL-17A binding affinity

NVS451 (at 75ug/ml) binds human IL-17A with high affinity. Surface plasmon resonance was performed with 100nM-0.8nM human IL-17A (Acrobiosystems, Cat:ILA-H5118), and the experimental method was as follows:

Reagent preparation

Coating solution: take 1.06g of Na ₂ CO ₃ and 0.84g of NaHCO ₃ and fully dissolve them in ultrapure water, adjust the pH to 9.60 with concentrated hydrochloric acid, and then dilute to 200 ml, filter with 0.22 filter membrane, and store at 4°C;

10×TBS mother liquor: Take 12.114g of Tris and 43.83g of NaCl into ultrapure water to fully dissolve, adjust the pH to 7.55 with hydrochloric acid, and then dilute to 500ml, filter with 0.22 membrane, and store at 4°C;

4× Substrate buffer stock solution: Take 7.16g of NaHPO·12HO and 2.1g of citric acid to fully dissolve in ultrapure water, adjust the pH to 5.5 with NaOH, then dilute to 100ml, filter with 0.22 filter membrane, put Store at 4°C.

Experimental operation

Coating:

Reconstitute IL-17A with ultrapure water according to COA, leave it for about 30 minutes to fully dissolve IL-17A, dilute IL-17A to 30 nM with a well-equilibrated coating solution at room temperature, and add 100 μl per well to the microtiter plate. , and the sealing film was placed at 4°C overnight (about 16h).

Wash the board:

The washing solution is prepared as it is. Take 100 ml of 10 × TBS mother solution and add it to 900 ml of ultrapure water to dilute to 1 × TBS, then add 2.5 ml of 20% Tween 20, and wash the plate 4 times with the prepared washing solution. Well 300 μl, pat dry.

closed:

For the current preparation, dilute 2.5 g of BSA with washing solution to 50 ml to prepare a 5% BSA blocking solution; add 300 μl of blocking solution to the patted dry plate, and incubate at 37°C for 1.5 h.

Wash the board:

Repeat step 2 to wash the plate.

Add sample:

The diluent is prepared now. Take 5ml of blocking solution and dilute it to 50ml with washing solution, prepare 0.5% BSA sample diluent, dilute the sample to 10μg/ml by 2-fold gradient, and add the diluted sample to the ELISA plate. 100 μl/well, set background control (no coating + sample + secondary antibody, coating + no sample + secondary antibody, no coating + no sample + no secondary antibody). The plates were sealed with sealing film and incubated at 37°C for 1 h.

Wash the board:

Repeat step 2 to wash the plate.

Secondary Antibodies:

The dilution solution is prepared now, take 5ml of blocking solution and dilute it to 50ml with washing solution, prepare 0.5% BSA antibody dilution solution, and make horseradish peroxidase-labeled AffiniPure goat anti-human IgG and Fcγ fragment specific (min × Bov, Hrs, Ms Sr Prot) were diluted to 1:12000, 100 μl was added to each well, and the plate was sealed with a sealing film and incubated at 37°C for 1 h.

Wash the board:

Repeat step 2 to wash the plate.

Color rendering:

Take 12ml of 4x substrate buffer and add it to 36ml of ultrapure water to dilute to 1x substrate buffer, then add 38.5μl 3% H ₂ O ₂ and 240μl 20mg/mL TMB (dissolved in DMSO), per well Add 200 μl and incubate at 37°C for 20 minutes.

termination:

Add 50 μl of 1 mol/L sulfuric acid to each well of the plate after color development.

reading:

The absorbance (OD value) of each well was measured at a wavelength of 450 nm, and the average value was calculated.

data processing:

The obtained OD values were fitted to the data by four parameters using GraphPad Prism 5, and EC50 was calculated.

(SPR) test (the results are shown in Figure 3), the KD is 1.8×10 ^-12 M, and the affinity is higher than that of secukinumab under the same conditions (the results are shown in Figure 4) (approved for marketing abroad (2015) IL-17A monoclonal antibody drug) and wild-type human IL-17RA (wtIL-17RA, purchased from ACROBIOSYSTEMS) (the results are shown in Figure 5). The results of the comparative analysis of binding affinity to IL-17A are summarized in Table 2.

Table 2. Comparative analysis of binding affinity to IL-17A

The IL-17RA fusion protein (amino acid sequence shown as SEQ ID NO. 6 and shown in SEQ ID NO. 7).

The affinity of V302 and V303 to IL-17A was detected by affinity ELISA. Two kits (R&D, Cat#: 317-ILB-050; Peprotech, Cat#: 900-K84) were used for detection according to the method provided by the manufacturer, and the results showed (Fig. 19) that NVS451 with six mutations and The percentage of IL-17A affinity (776% and 464% for the two kits above) was significantly higher than the affinity of V302 and V303 molecules for IL-17A. The affinity of V302 and V303 molecules to IL-17A was comparable to that of wild-type human IL-17RA.

1.2 Binding affinity analysis of IL-17A from different species

In this study, IL-17A molecules (KINGFISHER, Cat. Nos.: RP0921H-025, RP1031Y-025, RP0355M-025) of 4 species (human, cynomolgus monkey, mouse, rat) were compared under the same conditions; The binding of rat Biolegend, 778704) and NVS451, the results show that NVS451 can bind to 4 different species of IL-17A, and the binding affinity is from high to low: human > cynomolgus > rat > mouse (KD See Table 3).

Table 3. Comparative analysis of binding affinity with different species of IL-17A

1.3 Binding affinity analysis of IL-17A, C, F, AF

In addition to binding to IL-17A molecules of the human IL-17 family, NVS451 also bound to human IL-17C, IL-17F, and IL-17AF (the results are shown in Figures 6-8, respectively). The KD results are summarized in Table 4.

In another study, the affinity of NVS451 and wild-type human IL-17RA to IL-17C, IL-17F (Acrobiosystems, ILC-H52H7, ILF-H4240) was compared, and the results showed that NVS451 had a high affinity for human IL-17C, IL-17F and IL-17F. The affinity of -17F was higher than that of wild-type human IL-17RA (Table 5).

Table 4 Characterization of binding affinity of NVS451 to human IL-17 family

Table 5. Affinity comparison of NVS451 and wild-type IL-17RA with IL-17C, IL-17F

Weak binding: The spectrum has a response signal, but the intensity is low and the readout cannot be quantified.

N.A.: Not applicable

1.4 Biologically active cell experiments

The cell line CCD-1070Sk (human fibroblast cell line, ATCCCRL-2091) used for the in vitro activity assay of NVS451 belongs to the human fibroblast cell line, and there are many kinds of IL-17A, A/F and F on its surface. cytokine receptors. IL-17A can bind to the IL-17A receptor IL-RA on the cell surface to stimulate the cells to produce the cytokine GRO-α, and its content can be accurately detected by ELISA double-antibody sandwich method. When IL-17A and NVS451 were co-incubated with CCD-1070Sk cells, NVS451 was able to inhibit the binding of IL-17A to the IL-17A receptor IL-17RA on the cell surface by competing with IL-17A binding, thereby reducing the cell supernatant The amount of GRO-α secreted in the liquid. And NVS451 can also reduce the secretion of GRO-α in the cell supernatant by combining with IL-17A/F and IL-17F based on the same competitive inhibitory mechanism. Therefore, the in vitro activity of NVS451 on multiple targets including IL-17A, IL-17A/F, and IL-17F can be determined by measuring the secretion of GRO-α in cells.

1.4.1 GRO-α factor inhibitory ability of NVS451 induced by IL-17A

The relative potency of the NVS451 fusion protein in vitro was analyzed by measuring the inhibitory ability of IL-17A-induced GRO-α release from the human fibroblast cell line CCD-1070Sk, and the results were expressed as the median inhibitory concentration _IC50 value (Table 6) .

1. The human fibroblast epithelial cell line CCD-1070Sk cells in the T75 culture flask were digested with 2 ml of 0.05% EDTA trypsin for about 2-5 minutes, and 8 ml of complete medium (EMEM basal medium with 10% FBS) was added to neutralize , then centrifuged at 1000rpm for 5 minutes, resuspended with complete medium to a cell density of 1E5/ml, resuspended in a 96-well plate with 100ul per well, 10,000 cells per well, and cultured for about 24 hours. 95%-100% start dosing experiments.

2. Cell induction and administration: All drugs were diluted in complete medium. First, the NVS451 protein was diluted to an initial concentration of 4ug/ml (final concentration of 1ug/ml), and then a two-fold gradient was diluted to 9 points, and At the last 0ug/ml concentration point, 50ul per well was added to the cell plate, then IL-17A was diluted to a concentration of 40ng/ml (final concentration was 10ng/ml), and 50ul per well was added to each reaction well, and the cells were given The drug was incubated for 24 hours.

3. After 24 hours of incubation, centrifuge at 1500rpm for 5 minutes using a plate centrifuge, and then take 100ul of the supernatant for ELISA test. This step is to mix and dilute the cell supernatant from three replicate wells and add them to the ELISA test. 3 replicate wells of the microtiter plate

4. ELISA analysis

A. Dilute the capture antibody (mouse anti-human GROα antibody, R&D Cat# DY275-05-840255) with PBS x 1 to a concentration of 0.25 μg/ml, immediately add 100 μl to each ELISA microwell, coat the plate and incubate at room temperature Incubate overnight (24°C ± 4).

B. Remove the liquid from each well, and wash the plate 4 times with 300 μl washing buffer per well. A final clean-up with absorbent paper (wash buffer needs to be left at room temperature one day in advance until fully dissolved).

C. Add 300 μl blocking buffer to each well. Incubate at room temperature ((24°C ± 4) for at least 1 hour.

D. Sample preparation:

Centrifuge the 96-well CRL-2091 cell culture plate at 1000 rpm for 5 minutes.

50 μl of the pre-incubated sample (sup) was diluted in 200 μl of sample diluent (samples are now diluted 1:5) ready for loading.

E. Standard preparation:

Prepare serial dilutions of the standard at 1 μg/ml in diluent for a total of 7 point dilutions and one zero point

F. Remove the liquid from each well, and wash the plate 4 times with 300 μl washing buffer per well. Clean it up one last time with absorbent paper.

G. Immediately add 100 μL of standards or samples to each well in triplicate. Incubate for 2 hours at room temperature ((24°C ± 4), 600 rpm.

H. Remove the liquid from each well, and wash the plate 4 times with 300 μl washing buffer per well. Clean it up one last time with absorbent paper.

I. The concentration of the diluted detection antibody (biotinylated goat anti-human GROα antibody, R&D Cat# DY275-05-840256) in the diluent was 1.0 μg/ml (initial concentration 100 μg/ml).

J. Add 100 μl of diluted detection antibody to each well. Incubate for 2 hours at room temperature (24°C ± 4), 600 rpm.

K. Remove the liquid from each well, and wash the plate 4 times with 300 μl of washing buffer per well. Clean it up one last time with absorbent paper.

L. Take 6 μL of biotin-conjugated-HRP conjugate (1:2,000) and add a total volume of 12 ml of sample diluent (calculate one 96-well plate).

M. Add 100 μl to each well. Incubate for 30 minutes at room temperature (24°C ± 4), 600 rpm.

N. Remove the liquid from each well, and wash the plate 4 times with 300 μl washing buffer per well. Clean it up one last time with absorbent paper.

O. Add 100 μl of substrate solution to each well (ABTS substrate was placed at room temperature for 30 minutes in advance, and the amount required for the current experiment was taken out). Incubate each for 20 min at room temperature for color development.

P. Use a microplate reader to monitor the color development at 405nm, and the wavelength correction is set at 650nm.

At the same time, the inhibitory ability of GRO-α of the currently available IL-17A monoclonal antibody secukinumab (Cosentyx) in this in vitro relative titer assay was tested, and the results were expressed as the half-inhibitory concentration IC ₅₀ value (Table 6). The results showed that the NVS451 fusion protein had the ability to inhibit IL-17A-induced release of GRO-α cytokines, and the _IC50 value was lower than that of secukinumab (Cosentyx) (Figure 9).

Table 6 In vitro activity titers of NVS451 and secukinumab: IC ₅₀ (μg/ml) of IL-17A-induced GRO-α production

1.4.2 Comparison of in vitro activity and titer of NVS451 on IL-17A, IL-17A/F and IL-17F ligands

Similar to the above experiments, the human fibroblast cell line CCD-1070Sk induced by IL-17A/F and IL-17F can also produce GRO-α cytokine, and the in vitro titer results of NVS451 fusion protein induced by different ligands Expressed as the median inhibitory concentration _IC50 value (Table 7). The S-curves of inhibition are shown in Figure 10 and Figure 11, respectively.

Table 7 NVS451 inhibits the detection results of human skin fibroblast CCD-1070Sk GRO-a secretion

2. In vivo pharmacodynamics

Psoriasis is a chronic inflammatory skin disease characterized by excessive proliferation and abnormal differentiation of keratinocytes. It is caused by a variety of factors. The pathogenesis has not been fully elucidated. cells, T cells, endothelial cells, etc.), and cytokines are involved in the pathogenesis of psoriasis and the maintenance of the disease state [1]. The existing widely used animal models can be roughly divided into drug-induced acute inflammation models, genetic engineering models and xenograft models.

Imiquimod (IMQ) induction model, IMQ is an agonist of Toll-like receptor (TLR) 7/8, Van der Fits[2] and other studies found that IMQ-induced psoriasis-like in mice The changes in skin lesions are accompanied by changes in the IL-23/IL-17 axis, which have many similarities with the pathological changes of human psoriasis, and have the advantages of simple operation and low cost. Therefore, IMQ was applied to the skin of mice to establish psoriasis. It is a psoriasis model widely used at home and abroad. However, as an acute inflammatory model, its limitations are: (1) non-specific, not only in psoriasis, but also in other diseases such as systemic lupus erythematosus, atopic dermatitis and other skin lesions have similar inflammatory manifestations [ 3], IMQ-induced skin inflammation model has also been used in the study of systemic lupus erythematosus [4]; (2) The treatment window period is short, and topical imiquimod can cause dehydration and weight loss in mice, and continuous topical application exceeds 2 weeks may lead to the death of mice [5]; (3) poor reproducibility, lack of standard operating procedures, etc.

The xenograft skin immunodeficiency mouse (SCID) model is to transplant human skin into SCID mice, and inject the patient's T cells to induce psoriasis symptoms. Its genetic phenotype and pathogenic process are most similar to human psoriasis, and Due to the immunodeficiency of animals, it is not affected by anti-drag antibodies (ADA). Preclinical analysis of psoriasis agents/treatment strategies [6,7,8].

2.1 Efficacy evaluation of NVS451 in xenografted SCID mouse psoriasis model (5, 10, 15 mg/kg NVS451).

The above concentrations of NVS451 protein were formulated into a formulation of 20 mM Tris-acetic acid, 65 mM arginine, 120 mM trehalose, 0.02% Tween 20, pH 7.5.

A total of 60 SCID mice (Envigo, Jerusalem, Israel C.B-17/IcrHsd-scid-bg (beige-SCID) mice) were used in the experiment. After transplanting healthy human skin on the back of the mice, they were randomly divided into 6 groups of 10 in each group. Mice: negative control group (negative control group is using the above formulation to remove NVS451), hormone group (2mg/animal dexamethasone), secukinumab group (60mg/kg) and test article high dose (15mg /kg), middle dose (10mg/kg), low dose (5mg/kg) groups. The negative control group, the test product group and the Sujin group were given subcutaneous injection every other day for a total of 28 days. The hormone group was given topical dexamethasone twice a day for 28 days. At the end of the experiment, the appearance of the skin was observed (see Figure 12 for the results), and the thickness of the skin was measured. The results showed that NVS451 had a certain degree of improvement in redness, plaque and scale, and was positively correlated with the dose; there was no statistical difference in epidermal thickness between the 15 mg/kg dose group and the Sujin group (P>0.05). Similar healing effect to Sukin.

Table 8. Visual inspection results of xenograft skin

Table 9 Histological evaluation and thickness of xenograft skin

*Dexamethasone compared with all treatment groups, -p<0.01

**High dose test article compared to secukinumab, - not significant

***High-dose test article and secukinumab versus low- and mid-dose, -p<0.05, p<0.05

2.2 Efficacy evaluation of NVS451 (formulation formulation described in 2.1) in xenografted SCID mouse psoriasis model (15, 22.5, 30, 45, 60 mg/kg NVS451)

A total of 100 SCID mice (purchased from Envigo) were used in the experiment. After transplanting healthy human skin on the back of the mice, they were randomly divided into 10 groups (see Table 10). At the end of the experiment, the appearance of the skin was observed (see Figure 13 for the results), the grafted skin was taken for psoriasis-related histological evaluation (see Figure 14 for the results), and the thickness of the skin was measured. The results showed that the NVS451 groups all showed therapeutic effects, of which the 15mg/kg twice a week, 30mg/kg twice a week, 22.5mg/kg twice a week and 60mg/kg once a week groups had similar curative effects, all of which were 7/10. The xenograft skin recovered completely with reduced epidermal thickness. However, in the 15 mg/kg twice-weekly group, the remaining 3/10 showed partial recovery compared with the other treatment groups, with a skin thickness of 334±174 μm and a histological score of 1.1 (according to the thickness of the skin lesions, thinning of the epidermis on the papilla, and keratinization). Hyperplasia, parakeratosis, agranulocytosis, Munro microabscess, normal or abnormal reticular crest elongation, vascular tortuosity, mononuclear cell infiltration in the papillary dermis and other indicators).

Table 10. Study grouping and dosage

Table 11. Histological Evaluation and Thickness of Xenograft Skin

3. Secondary Pharmacodynamics

3.1 ADCC/CDC effect related receptor binding affinity analysis

The mechanism of NVS451 exerting its efficacy is not dependent on ADCC/CDC activity, and the cytotoxic effect of Fc ADCC/CDC may bring unnecessary immune-related adverse events (irAEs), posing potential safety risks [9]. To avoid this effect, the relevant active site was mutated in the Fc portion of NVS451. In the affinity analysis study, seven CD molecules associated with ADCC and C1q molecules associated with CDC were evaluated. The results showed that the binding affinity of NVS451 (75ug/ml) to ADCC-related CD molecules was at non-binding or low levels (KD greater than the order of ^10-5 M, see Table 12). In another study, the affinity of NVS451 for ADCC/CDC-related Fc receptors was significantly reduced compared to the other partially identical IL-17AR fusion protein (V301-wtFc) (75ug/ml) with native IgG1 Fc (see Table 13).

The amino acid sequence of V301-wtFc is shown in SEQ ID NO.17.

Table 12. Affinity analysis of NVS451 and ADCC, CDC effector related receptors

Table 13 Affinity analysis of NVS451 and V301-wtFc and ADCC, CDC effect related receptors

3.2 Evaluation of ADCC/CDC functional activity of Fc fragment region

The Fab regions of IgG1 and IgG3 subtype antibodies bind to target cells first, and then their Fc part binds to Fc receptors (such as FcγRIII) on CTL cells such as NK, and then can induce ADCC. Cell models lacking membrane-expressed IL-17 cannot be validated in vitro for ADCC production. Therefore, the Fab segment of Rituximab, which can stimulate ADCC, and the Fc segment of NVS451 were fused to construct RitxV301 (that is, RitxNVS451) to verify the chimeric molecule (the amino acid sequence of the light chain is shown in SEQ ID NO.18, The amino acid sequence of the heavy chain is shown in SEQ ID NO. 19). In this way, the removal of ADCC/CDC effect was verified (analyze the experimental data with GraphPad Prism 5 analysis software, take the logarithm of the concentration of the antibody as the x-axis, and the corresponding calculated killing rate value as the y-axis, select a four-parameter equation regression simulation, fit The measurement effect curve of the combined antibody. The obtained data were analyzed by GraphPad Prism5 software, and the positive control antibody (rituximab) conformed to the four-parameter equation y=D+(A-D)/[1+(X/C)B], in the half pair A typical S-shaped curve is formed on the number axis, R2≥0.95, EC50 is calculated, and the ADCC and CDC effects are judged by the EC50 value)). The results showed (Figures 15 and 16): Rituximab (Roche (Switzerland)) showed strong ADCC and CDC killing effects on Raji cells, while RitxV301 did not show ADCC and CDC effects, indicating that the Fc region of NVS451 The design of RitxV301 is such that RitxV301 cannot induce NK-92MI CD16a cells to kill Raji cells, and the Fc region of NVS451 cannot bind to the C1q molecule in complement and cannot initiate the complement cascade.

3.3 FcRn binding affinity analysis

Fc fusion proteins generally increase the half-life of target protein drugs and improve their pharmacokinetic characteristics through FcRn-mediated circulation of Fc [10]. The binding affinity to FcRn is related to the half-life of the drug in vivo [11]. NVS451 can bind to human, monkey and rat FcRn (acrobiosystems FCM-H5286, FCM-C5284, FCM-R5287). Under the same conditions, its affinity is at the same level as secukin antibody and wild-type IgG1 Fc (ie V301-wtFc) (KD is in the order of 10 ^-8 M, see Table 14). The experimental method is as follows: .

Reagent preparation

Running reagent: containing 2 mM KH ₂ PO ₄ , 10 mM Na ₂ HPO ₄ , 137 mM NaCl, 2.7 mM KCl, 0.05% Tween-20 (Tween-20), pH adjusted to 6.0;

His Capture Kit (Cat. No. 28-9950-56, GE), including: mouse anti-His antibody (1 mg/mL), immobilization reagent (10 mM sodium acetate, pH 4.5), regeneration reagent (glycine hydrochloride, pH 1.5);

Amino Coupling Kit (Cat. No. BR100050, GE), including: 115mg N-hydroxysuccinimide (NHS), 750mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide salt acid (EDC) and 10.5 mL of 1 M ethanolamine (pH 8.5). Add 10 mL of deionized water to each tube of EDC and NHS, respectively, and store in aliquots at -18°C to a lower temperature. The shelf life is two months. (Refer to GE Amino Coupling Instruction Manual "22-0510-62AG").

Dissolve IL-17RA-wtFc (ie V301-wtFc) protein and FcRn proteins of different species according to the product COA, and dispense with the specification of more than 10 μg per tube. Avoid repeated freezing and thawing.

protein desalting

The secukinumab and IL-17RA-wtFc proteins were desalted using a desalting column and Running Buffer. The concentration of the desalted protein was determined by UV-V. The desalted protein should be aliquoted at a size greater than 10 μg per tube, and repeated freezing and thawing should be avoided.

Chip preparation

The mouse anti-His antibody was diluted to 50 μg/mL with immobilization reagent (10 mM sodium acetate, pH 4.5), about 100 μL of mouse anti-His antibody was used for each channel of the chip, and about 190 μL of immobilization reagent was used to fix two channels and 10 μL of mouse anti-His antibody was added. First, the surface of a CM5 chip (biocore sensor chip, GE) was activated with 400 mM EDC and 100 mM NHS at a flow rate of 10 μL/min for 420 s. Next, 50 μg/mL of mouse anti-His antibody was injected into the experimental channel (FC4) at a flow rate of 10 μL/min for about 420 s, and the immobilized amount was about 9000 to 14000 RU. Finally, the chip was blocked with 1 M ethanolamine at 10 μL/min for 420 s. The reference channel (FC3) performs the same operation as the test channel (FC4). (Refer to GE's His Capture Kit Instruction Manual "28-9974-71AB").

capture ligand

A sample of the FcRn stock solution was diluted to 0.5 μg/mL with running reagent and injected into the experimental channel (FC4) for approximately 40 RU at a flow rate of 10 μL/min. The reference channel (FC3) does not require ligand capture.

Analyte Multicycle Analysis

Dilute different concentrations of NVS451, secukinumab and IL-17RA-wtFc samples with corresponding running reagents, and inject the diluted samples into the experimental channel and reference at a flow rate of 30 μL/min according to the corresponding binding time and dissociation time. aisle. After each concentration analysis, the chip needs to be regenerated with glycine hydrochloride pH 1.5 at a flow rate of 30 μL/min for 60 s to wash away ligands and undissociated analytes. For the next concentration analysis, the experimental channel needs to recapture the same amount of ligand.

other

All operation steps are carried out in the running reagent, and the analytical reagents of SPR need to be filtered and degassed.

result

data analysis:

KD values for each antibody were calculated using Biacore 8K analysis software. The reference channel (FC3) is used for background subtraction.

In 4 independent testing studies, NVS451 (concentration: 25nM-1.5625nM) can bind to FcRn of 3 different species of human, cynomolgus monkey and rat under acidic conditions of pH6.0 (Figure 17); and at pH7.4 None of them bound under neutral conditions (NVS451 concentration 100 nM-1.5625 nM) (Figure 18).

Table 14 Comparison of FcRn receptor affinity of human, rat and cynomolgus monkey

Experimental Example 2: Safety Evaluation

1. Safety Pharmacology

1.1 Safety pharmacology test in cynomolgus monkeys

Cynomolgus monkeys (purchased from Beijing Zhongke Lingrui Biotechnology Co., Ltd.) safety pharmacology test was carried out with long-term toxicity, subcutaneous injection of the prescription in Section 2.1 of Experimental Example 1, NVS451 doses of 15mg/kg, 50mg/kg and 150mg/ kg (administered twice a week for 4 consecutive weeks, a total of 9 doses, ie, administered on D1, D4, D8, D11, D15, D18, D22, D25 and D29, respectively). In this experiment, an excipient control group was set up, and NVS451 was removed by using the prescription in Section 2.1 of Experimental Example 1. The safety and pharmacological indicators were detected in combination with the observation of toxicity indicators. There was no abnormality in the mental state and behavior of animals in each group; Breathing was steady, no tachypnea or slowing, and no abnormal chest or abdominal breathing. At different time points (before the first administration (D-2), and 4h (±20 minutes), 24h (±20 minutes), 48h (±20 minutes), 72h (±20 minutes) after the first administration) There were no drug-related TV and RR, as well as regular changes in heart rate, P-R interval, QT interval, QRS duration, QRS voltage, STe and QTcB values in the respiratory index and electrocardiogram examination of the animals in the group. See exception. Before the first dose (D-2), 3 to 5 hours after the first dose (D1), 3 to 5 hours after 5 doses (D15), 3 to 5 hours after the last dose (D29) and before the end of the recovery period (D56) ) to conduct ECG limb II lead measurement, observe heart rate, ECG waveform, P-R interval, QRS time limit, Q-T interval, no abnormality was found, and blood pressure detection at each time point, including MBP, DBP and SBP, showed no obvious abnormal changes. Therefore, it is considered that subcutaneous injection at doses of 15, 30 and 150 mg/kg, the test substance has no obvious effect on the central nervous system, cardiovascular system and respiratory system of cynomolgus monkeys.

1.2 Rat central nervous system test

Under GLP (Good Laboratory Practice) conditions, the effect of a single subcutaneous injection of NVS451 on SD rats (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. (animal production license: The effect of SCXK (Jing)) on the central nervous system function, the doses were 30mg/kg (low dose), 100mg/kg (medium dose) and 300mg/kg (high dose). Body temperature can be increased in 2 hours (excipient control group (that is, NVS451 removed from the formulation) vs high dose group: 37.72±0.29℃ vs 38.40±0.14℃); female animals in high, medium and low dose groups of the test product can see body temperature 24 hours after the drug increased, excipient control group vs high, medium and low dose groups: 37.60 ± 0.28 ℃ vs 38.80 ± 0.23, 38.72 ± 0.44, 38.54 ± 0.28 ℃). There was no male and female consistency in the above changes in body temperature, and no regular changes were found, which was considered to be irrelevant to drug administration. During the observation in the cage, hand grasping, open environment, and irritant reaction, no abnormal performance related to drug administration was found in animals; no animals were found to stand upright, number of fecal blocks, and forelimb grasping related to the test article. Changes in strength and body temperature. The results showed that under the experimental conditions, NVS451 had no effect on the central nervous system function of rats.

2. Pharmacokinetics

2.1 In vivo kinetics in cynomolgus monkeys

A total of 24 cynomolgus monkeys were used in the experiment, and they were divided into 4 groups (6 animals in each group, half male and half female). Single subcutaneous and intravenous administration, group 1 to group 4 were given 5 (subcutaneous injection), 15 (subcutaneous injection), 50 (subcutaneous injection), 15 (intravenous injection) mg/kg of NVS451, administration volume 1mL/ kg.

Pharmacokinetic blood samples (about 1 mL) were collected from the non-administration site of the subcutaneous vein of the hind limbs of the animals to the tube without anticoagulant. 2h, 4h, 8h, 24h(D2), 32h(D2), 48h(D3), 56h(D3), 72h(D4), 96h(D5); the blood collection time points of the fourth group of animals were: before the drug (the day before ), 3 minutes, 1h, 2h, 6h, 24h(D2), 32h(D2), 48h(D3), 56h(D3), 72h(D4), 96h(D5) after the start of administration. Blood samples were used to prepare serum samples and for pharmacokinetic analysis (Table 15).

Table 15. Pharmacokinetic parameters of NVS451 in cynomolgus monkeys

The ratio of C _max = the mean value of C _max medium and high doses / the mean value of C _max low dose;

AUC ratio = AUC _last medium and high dose mean / AUC _last low dose mean;

F%=(AUC _last /dose (SC))/(AUC _last /dose (IV))*100%.

The test results showed that within the dose range of 5-50 mg/kg, after a single subcutaneous injection was administered to cynomolgus monkeys, the plasma concentration of NVS451 in cynomolgus monkeys increased with the dose; single subcutaneous injection and intravenous injection were administered to cynomolgus monkeys. After monkeys, no significant differences were observed between the sexes.

Following single subcutaneous injection of NVS451 to cynomolgus monkeys at doses of 5, 15, and 50 mg/kg, the mean _Cmax and AUC _last increased proportionally to NVS451 compared to dose proportionality. The _Cmax ratios of NVS451 in the single subcutaneous injection groups of 5, 15, and 50 mg/kg dose groups were 1:6.12:38.06 (male) and 1:5.46:34.29 (female), respectively; the AUC _last ratios were 1:4.83:25.52, respectively (male) and 1:4.76:20.88 (female).

The independent samples t test showed that there was no statistical difference in the metabolic kinetic parameters between the subcutaneous injection and intravenous injection groups between the genders, and there was basically no significant gender difference in the metabolic characteristics of NVS451 in animals.

Within the dose range of 5-50 mg/kg, the T _1/2 of _NVS451 in cynomolgus monkeys, the average T _1/2 of male and female animals in each dose group was between 27.20 and 39.90; The drug concentration peaked in 4-8h.

2.2 In vivo kinetics in rats

A total of 48 SD rats were used in the experiment and were divided into 4 groups (12 animals in each group, half male and half female). Single subcutaneous and intravenous administration, group 1 to group 4 were given 10 (subcutaneous injection), 30 (subcutaneous injection), 100 (subcutaneous injection), 30 (intravenous injection) mg/kg of NVS451, administration volume 2mL/ kg.

Pharmacokinetic blood samples (about 0.4 mL) were collected from the animal's jugular vein in the experiment and placed in a tube without anticoagulant. The blood collection time points of animals in groups 1-3 were: before administration (D-1), 1h, 2h, 4h, 8h, 24h, 32h, 48h, 56h, 72h, 96h; the blood collection time points of animals in group 4 were: before administration (D-1), 3min, 1h, 2h, 6h, 24h, 32h, 48h, 56h, 72h, 96h. Blood samples were used to prepare serum samples and for pharmacokinetic analysis (Table 16).

Table 16. Pharmacokinetic parameters of NVS451 in SD rats

The ratio of C _max = the mean value of C _max medium and high doses / the mean value of C _max low dose;

AUC ratio = AUC _last medium and high dose mean / AUC _last low dose mean;

F%=(AUC _last /dose (SC))/(AUC _last /dose (IV))*100%.

The test results showed that: within the dose range of 10-100 mg/kg, after a single subcutaneous injection was administered to SD rats, the plasma concentration of NVS451 in SD rats increased with the increase of the dose; single subcutaneous injection and intravenous injection of SD rats After mice, no significant differences were observed between the sexes.

Following single subcutaneous injection of NVS451 to SD rats at doses of 10, 30, and 100 mg/kg, the mean Cmax and AUClast of NVS451 increased proportionally less than the dose increased. The ratios of Cmax of NVS451 in single subcutaneous injection of 10, 30 and 100 mg/kg groups were 1:2.03:6.56 (male) and 1:1.62:6.04 (female), respectively; the ratio of AUClast was 1:1.92:5.29 (male), respectively ) and 1:1.85:5.85 (female).

There was no significant gender difference in the metabolic kinetic parameters between subcutaneous injection and intravenous injection groups, and there was basically no significant gender difference in the metabolic characteristics of NVS451 in animals.

Within the dose range of 10-100 mg/kg, the T1/2 of NVS451 in SD rats and the T1/2 of male and female animals in each dose group were between 51.46 and 69.48; the Tmax of each dose group by subcutaneous injection was basically the same, and the drug concentration was between 8 ~ 24h peak.

3. Distribution and excretion

In this experiment, the TCA-precipitated protein method combined with SHPLC (size exclusion high performance chromatography) was used to investigate the tissue distribution and excretion characteristics of the isotope (125I)-labeled NVS451 fusion protein for injection (NVS451) after subcutaneous injection into SD rats. Research.

The experiment consisted of 36 animals, divided into 6 groups, with 6 animals in each group, half male and half female. The test article was administered as a single subcutaneous injection on the back of the neck at a dose of 30 mg/kg. The radiochemical purity of the test product after 125I labeling was 99.03%, and the specific activity was 0.09KBq/μg. The thyroid, heart, lung, liver, spleen, kidney, bladder, gonad (ovary, testis), stomach, small intestine, fat, muscle, brain, Back skin (non-administration site), intestinal contents, serum, urine. The feces and urine of the animals in the 5 groups were collected once a day for 5 days. The 6 groups of animals collected bile every hour after injection for a total of 8 hours.

The results showed that:

1) After subcutaneous injection of 125I-NVS451 to rats, the drug is mainly distributed in intestinal contents, urine, bladder, back skin and other tissues/organs, but less in muscle, fat, and brain. The peak time of the drug in most tissues is 4h to 24h, and then the concentration gradually decreases with the prolongation of time.

The drug has some distribution on the back skin (non-administration site). The peak time is 24h, and the drug content of 4h and 120h is roughly equal. Compared to other tissues/organs, the concentration of the drug decreases more slowly in the back skin.

2) After subcutaneous injection of 125I-NVS451 in rats, the radioactivity was mainly excreted through the kidneys, and more than 90% of the injected drug was excreted after 5 days; the bile excretion was less.

4. Metabolism

According to the "ICH-S6(R1) Preclinical Safety Evaluation of Biologics" guideline, NVS451 is a macromolecular protein drug that is expected to be degraded into peptides and amino acids in vivo and then excreted or re-used for in vivo protein or peptide synthesis , so the metabolism of NVS451 was not assessed.

5. Toxicology

Cynomolgus monkeys and SD rats were selected for toxicological studies, and NVS451 cross-reacted with cynomolgus monkeys and rat IL-17A, which were related species. The trials were all conducted under GLP conditions, following the current Food and Drug Administration Good Laboratory Practice (21CFR Part 58), the State Drug Administration (formerly the State Food and Drug Administration) "Quality Management Practice for Nonclinical Drug Research" ” (Order No. 34 of the Bureau, September 2017).

6. Single-dose toxicity

6.1 Cynomolgus monkey toxicity study

The purpose is to evaluate the acute toxicity that may occur within 14 days after a single subcutaneous injection of NVS451 in cynomolgus monkeys, and set three dose groups of 45mg/kg, 150mg/kg and 450mg/kg. During the experiment, no animals in each group were found to be dead or dying. There was no abnormality in the clinical observation of animals in each administration group. Compared with its own pre-drug value and the excipient control group (with NVS451 removed from the formulation prescription), the body weight, body temperature, ECG parameters, blood cell count, coagulation index, blood biochemistry and urine examination of animals in each administration group showed no drug-related abnormalities. Change. At the end of the observation period (D15), no abnormality was found in the gross anatomy of all animals. Conclusion: Under the conditions of this experiment, no death or dying was observed in all animals, and no abnormal changes related to administration were observed. The maximum tolerated dose (MTD) was greater than or equal to 450 mg/kg.

6.2 Toxicity studies in rats

The purpose is to evaluate the acute toxicity that may occur within 14 days after single subcutaneous injection of NVS451 into SD rats, and set three dose groups of 90 mg/kg, 300 mg/kg and 900 mg/kg. During the experiment, no animals in each group were found to be dead or dying, and at the end of the observation period (D15), there was no abnormal change in the general observation of the animals in each group. Conclusion: Under the conditions of this experiment, all animals were not found dead or dying, and the maximum tolerated dose (MTD) of animals was greater than or equal to 900 mg/kg.

7. Repeated Dosing Toxicity

7.1 Repeated administration toxicity study in cynomolgus monkeys

Using 40 cynomolgus monkeys (20/sex), they were randomly divided into 4 groups (5/sex/group) according to gender, which were the excipient control group and the test product low-dose, medium-dose and high-dose groups. Animals in excipient control group were given excipient control substance, 2mL/kg, and the low-dose, medium-dose and high-dose groups of test substance were given NVS451, the doses were 15, 50, and 150mg/kg, and the doses were 0.2, 0.67, and 2mL/kg, respectively. kg, and the administration concentration was 75 mg/mL. All animals were subcutaneously injected into the hind limbs, twice a week for 4 consecutive weeks, 9 times in total, namely D1, D4, D8, D11, D15, D18, D22, D25 and D29. During the experiment, the animals were subjected to clinical observation, body weight, body temperature, electrocardiogram, respiration (respiratory frequency and tidal volume), blood pressure, ophthalmological examination, blood cell count, coagulation function, blood biochemistry, urinalysis and immunological indicators (T lymphocyte subgroups). group, cytokines, C-reactive protein, serum immunoglobulin, complement, anti-drug antibody) indexes; before and after the first and the eighth administration, blood was collected from animals in each group for determination of blood drug concentration. Some animals (3 animals/sex/group) were euthanized on the day after the last drug (D30), and the remaining animals were euthanized at the end of the 4-week recovery period (D57). , Weigh the main organs, calculate the relative organ weight (visceral-body ratio and visceral-brain ratio), and conduct histopathological examination of more than 40 kinds of tissues and organs.

RESULTS: During the experiment, there was no death or dying in each group of animals. Clinical observation, body weight, body temperature, ophthalmological examination, urine examination, blood cell count, coagulation function (except FIB), blood biochemical indexes, lymphocyte subgroups of animals in each group were observed. Population, complement, cytokines (except IL-17A) and immunoglobulins showed no abnormal changes related to administration. There were no abnormal changes related to drug administration in the animals' ECG, respiratory and blood pressure parameters. There were no abnormal reactions such as erythema, edema, induration, and ulceration at the administration site.

During the test period, compared with the same-sex excipient control group at the same period, the CRP increased (193.0%) in male animals in the high-dose group of the test product on the next day (D16) after 5 doses; the next day after the last dose (D30), Male animals in the low-dose, medium- and high-dose groups of the test article showed increased CRP (70.5%, 218.5%, 246.9%), and male animals in the high-dose group of the test article also increased FIB (56.4%), and the difference was statistically significant ( P≤0.05). After 4 weeks of drug withdrawal, the above changes were fully recovered.

7.1.1 Histopathological examination:

No animal death or moribund situation was found during the experiment.

After the last drug (D30), the injection sites and inguinal lymph nodes of the animals in the 15, 50 and 150 mg/kg dose groups showed changes related to the test article, and the injection sites showed mild to moderate mononuclear cells in the dermis/subcutaneous/muscular layer Inflammation, which is an irritant reaction caused by the test product; mild to moderate increase in the number of lymphocytes in the paracortical area/medulla cord of the inguinal lymph nodes, which is related to the local mononuclear cell inflammation of the injection, and no obvious clinical pathological indicators. Changes are considered non-adverse reactions.

At the end of the 4-week recovery period (D57), the changes related to the injection site and the test article in the 15 and 50 mg/kg dose groups have completely recovered, and the changes related to the injection site and the test article in the 150 mg/kg dose group have basically recovered.

7.2 Repeated administration toxicity study in rats

192 SD rats, half male and half male, were randomly divided into 8 groups according to body weight in gender segments. 120 rats in the main experimental group from groups 1 to 4 were used for toxicology studies (15 rats/sex/group), 5 Seventy-two rats in ~8 satellite groups were used for serum antibody and toxicokinetic testing (6-10 rats/sex/group). During the test period, the animals in the excipient control group were given NVS451 white excipient solution (4mL/kg); the low-dose, medium-dose and high-dose groups of the test product were given NVS451, respectively, at a dose of 30, 100, and 300 mg/kg, and the administration volume was 0.4, 1.33, 4 mL/kg. All animals were administered subcutaneously via the back of the neck, twice a week, for 4 consecutive weeks, for a total of 9 doses (ie, D1, D4, D8, D11, D15, D18, D22, D25 and D29 doses). ), a 4-week recovery period.

During the experiment, the animals in the main experimental group were mainly subjected to clinical observation, and the body weight, food intake, body temperature, blood cell count, coagulation function, blood biochemistry, ophthalmological examination, urine examination, T lymphocyte subsets and cytokines were detected; the animals in the satellite group were examined. Blood was collected before and after the first and eighth administrations for toxicokinetic testing, and at different time points (before the first administration (D-1), before the 15th administration (D14), before the last administration (D28) and the end of the recovery period (D56)) blood was collected for antibody determination. The first 10 animals/sex/group in the main experimental group were euthanized on the next day after the last dose (D30), and the remaining animals were euthanized after the 4-week recovery period (D57). All animals in the main experimental group were subjected to gross anatomical observation, the main organs were weighed, and the relative organ weight was calculated; more than 40 kinds of tissues and organs were injected into the animals in the control group and high-dose group, and the animals in the low- and medium-dose groups were injected with local and tissue. Pathological examination.

Results: During the test period, animals in each group were not found dead or dying, and no abnormality was found in clinical observation. No erythema, hyperemia, swelling, ulcer and induration were found in the administration site. There were no abnormalities in the ophthalmological examination and urine examination of the animals in each group; compared with the control group of the same sex in the same period, the body weight, body temperature, food intake, blood cell count, coagulation function, blood biochemistry, and T lymphocyte subsets of the animals in each group were not found. Abnormal changes associated with administration.

7.2.1 Pathological examination:

No animal died during this experiment.

The general observation of euthanized animals showed no changes related to the test article.

At the end of administration (D30), the animals were euthanized. The 30, 100 and 300 mg/kg dose groups showed local irritation related to the test article, manifested as mild to moderate mononuclear cell inflammation in the local subcutaneous and/or dermis injected. By the end of the 4-week recovery period, the local inflammation of the injection was basically completely recovered.

8. Locally tolerated toxicity

The local tolerance toxicity test was carried out with the long-term toxicity. Both cynomolgus monkeys and SD rats were administered twice a week (30, 100 and 300 mg/kg in three dose groups for rats, 15, 50 mg/kg in three dose groups for cynomolgus monkeys). and 150 mg/kg) for 4 consecutive weeks, a total of 9 doses (ie D1, D4, D8, D11, D15, D18, D22, D25 and D29 doses). During the test, no abnormal reactions such as erythema, edema, induration, and ulceration were found in the local observation of animals in each group.

9. Other toxicity

In vitro test tube method was used to observe the effect of 75mg/mL NVS451 on hemolysis and aggregation of human erythrocytes (experimental conditions were conventional). From 15 minutes after incubation in the incubator to the end of 3 hours of observation, the upper liquid of the test tube was colorless and clear, and the red blood cells at the bottom of the tube sank. Under the conditions of this test, 75mg/mL NVS451 has no hemolysis effect on human erythrocytes in vitro, and does not cause human erythrocyte aggregation.

in conclusion

1. Comprehensive evaluation and conclusion of pharmacodynamics

In vitro pharmacodynamics: Comparative analysis of binding affinity with IL-17A showed that NVS451 could bind to human IL-17A with high affinity, which was higher than that of secukinumab and wild-type human IL-17RA under the same conditions. In addition to binding to IL-17A molecules of the human IL-17 family, NVS451 also binds to IL-17C, IL-17F, and IL-17AF. The affinity of NVS451 to human IL-17C and IL-17F was higher than that of wild-type human IL-17RA. Biologically active cell experiments showed that NVS451 fusion protein showed inhibitory ability to IL-17A, IL-17A/F, IL-17F-induced GRO-α cytokines, and inhibited IL-17A-induced GRO-α cytokines. The inhibitory ability is lower than the IC ₅₀ value of secukinumab (Cosentyx).

Based on these in vitro potency results, NVS451 primarily acts by inhibiting the activities of IL-17A and IL-17C, IL-17F, and IL-17AF. The interactions shown by the SPR experiments suggest that in vivo, the recommended dose of NVS451 exerts clinical activity mainly inhibiting IL-17A, supplemented by inhibition of IL-17C, IL-17F, and IL-17AF molecules in patients with psoriasis.

Affinity analysis studies showed that the binding affinity of NVS451 to ADCC-related CD molecules was at no binding or low level. Compared with native IgG1 Fc, its affinity for ADCC/CDC-related Fc receptors was significantly reduced, proving that the ADCC binding site of NVS451 was mutated remove. Binding affinity analysis to FcRn showed that NVS451 could bind to human, monkey and rat FcRn. Under the same conditions, its affinity is at the same level as that of secukinumab and wild-type IgG1 Fc. NVS451 can bind to FcRn of three different species of human, cynomolgus monkey and rat under acidic conditions of pH6.0, and at pH7. 4 are not bound under neutral conditions. NVS451 is a soluble antibody Fc fusion protein carrying the human IgG1 Fc domain, so it can theoretically interact with Fc receptors, but the mechanism of NVS451's drug efficacy does not depend on ADCC/CDC activity, and the cytotoxic effect of Fc ADCC/CDC may bring unnecessary immune-related side effects and have potential safety risks. To avoid this effect, the relevant active site was mutated in the Fc portion of NVS451. The study confirmed that compared with native IgG1 Fc, its affinity for ADCC/CDC/ADCP-related Fc receptors was significantly reduced, and target cell killing experiments (shown in Figures 15 and 16) confirmed that NVS451 had no ADCC/CDC activity.

In vivo pharmacodynamics: Functionally in vivo, the efficacy of NVS451 against psoriasis was primarily evaluated in a human T cell-driven psoriasis model. Studies have confirmed that the human skin xenograft model is best suited for preclinical analysis exploring novel anti-psoriatic agents/therapeutic strategies prior to initiating clinical trials, as assessed by histological evaluation of human skin including scoring and epidermal thickness in a xenograft SCID mouse model , showing the efficacy of NVS451 in the treatment of humanized psoriasis models in xenografted SCID mouse models of psoriasis, after 28 days of NVS451 administration at doses of 5 mg/kg, 10 mg/kg and 15 mg/kg every other day, the results The results showed that NVS451 had a certain degree of improvement in redness, plaque and scale, and it was positively correlated with the dose; there was no significant difference in epidermal thickness between the 15 mg/kg dose group and the Sujin group (P>0.05), and the highest dose of NVS451 was used every week. (60mg/kg) once treatment was as effective as twice weekly low dose treatment and most importantly, twice weekly treatment with NVS451 or once weekly high dose treatment showed positive results with secukinumab Comparison of similar treatment effects. Secukinumab is considered to be one of the most promising drugs for the treatment of psoriasis, and this study provides strong clinical evidence support for the effectiveness of clinical treatment.

Safety pharmacology: Under GLP conditions, NVS451 was administered with a single subcutaneous injection of 30 mg/kg, 100 mg/kg and 300 mg/kg. The results showed that NVS451 had no effect on the central nervous system function of rats. The safety pharmacology test of cynomolgus monkeys was carried out with long poison: subcutaneous injection at doses of 15, 30 and 150 mg/kg, the test substance had no obvious effect on the central nervous system, cardiovascular system and respiratory system of cynomolgus monkeys.

2. Comprehensive evaluation and conclusion of pharmacokinetics and toxicology

Since NVS451 cross-reacts with cynomolgus monkey and rodent IL-17A, cynomolgus monkey and rat were selected as relevant species for toxicity evaluation. NVS451 was repeatedly subcutaneously injected into cynomolgus monkeys at doses of 15, 50 and 150 mg/kg, 2 times a week, for 4 consecutive weeks, for a total of 9 doses. There was no death or moribund in each group of animals. Obvious systemic toxicity, the no-adverse-effect dose (NOAEL) in this trial was considered to be 150 mg/kg. The NVS451 fusion protein for injection was administered subcutaneously to SD rats at doses of 30, 100 and 300 mg/kg, 2 times a week, for 4 consecutive weeks, for a total of 9 times, and no obvious systemic toxicity was found in the animals. , the no-adverse reaction dose (NOAEL) was considered to be 300 mg/kg. Cynomolgus monkey and rat toxicokinetics showed no accumulation of NVS451 after repeated administration.

Single-dose non-GLP pharmacokinetic studies were performed in cynomolgus monkeys and rats to observe the basic pharmacokinetics of NVS451 administered by intravenous and subcutaneous routes. The absorption pharmacokinetics of NVS451 in male and female cynomolgus monkeys were evaluated after a single subcutaneous administration, and its pharmacokinetic behavior was typical of low serum clearance and half-life of immunoglobulin molecules. Effective drugs were detected 96 hours after administration. In a single pharmacokinetic test in cynomolgus monkeys and SD rats, there was no significant gender difference in metabolic data. Within the dose range of 5-50 mg/kg, the T1/2 of NVS451 in cynomolgus monkeys, the male and female of each dose group The average T1/2 of animals was between 27.20 and 39.90h. The T1/2 of NVS451 in SD rats ranged from 10 to 100 mg/kg, and the T1/2 of male and female animals in each dose group ranged from 51.46 to 69.48 h.

3. Non-clinical conclusions:

NVS451 is a high-affinity antibody Fc fusion protein that selectively targets IL-17A, IL-17C, IL-17F, and IL-17AF heterodimeric cytokines. The target has a high affinity.

In conclusion, the present inventors conducted a summary of the non-clinical data of the project, which provided evidence of target specificity and mode of action, and treatment with NVS451 twice weekly or once weekly with high doses showed a The anti-positive control had a similar treatment effect. NVS451 was well tolerated in comprehensive toxicology studies. All non-clinical evaluation studies provided strong evidence support for the efficacy and safety of NVS451 clinical treatment.

references:

[1] Yang Jianqiang, Li Wei, Zheng Min. Research progress on animal models of psoriasis [J]. Chinese Journal of Leprosy and Dermatology, 2017, 33(009): 560-562.

[2] L. Van der Fits, S. Mourits, J.S.A. Voerman, et al. Imiquimod-Induced Psoriasis-Like Skin Inflammation in Mice Is Mediated via the IL-23/IL-17Axis. J Immunol, 2009, 182:5836-5845 .

[3] Zhang Tongmei, Wang Gang. Research progress on animal models of psoriasis [J]. Journal of Practical Dermatology, 2018,11(05):42-46.

[4] Yokogawa M, Takaishi M, Nakajima K, et al. Epicutaneous application of Toll-like receptor 7agonists leads to systemic autoimmunity in wild-typemice: A new model of systemic lupuserythematosus [J]. Arthritis Rheumatol, 2014, 66(3): 694-706.

[5] Liu Aimin, Li Weiling, Wu Panhong, et al. Research progress of imiquimod-induced psoriasis mouse model [J]. China Journal of Integrated Traditional Chinese and Western Medicine Dermatology, 2018, 17(4): 380-382

[6] Nousbeck J, Ishida-Yamamoto A, Bidder M, Fuchs D, Eckl K, Hennies HC, Sagiv N, Gat A, Gini M, Filip I, Matz H, Goldberg I, Enk CD, Sarig O, Meilik B, Aberdam D, Gilhar A, Sprecher E. IGFBP7 as a potential therapeutic target inpsoriasis. J Invest Dermatol. 2011;131:1767-1770.

[7] Schafer PH, Parton A, Gandhi AK, Capone L, Adams M, Wu L, Bartlett JB, Loveland MA, Gilhar A, Cheung YF, Baillie GS, Houslay MD, Man HW, Muller GW, Stirling DI. Apremilast, a cAMP phosphodiesterase-4 inhibitor, demonstrates anti-inflammatory activity in vitro and in a model of psoriasis. Br J Pharmacol. 2010;159:842-855

[8] Wrone-Smith T, Nickoloff BJ. Dermal injection of immunocytes inducespsoriasis. J Clin Invest. 1996;98:1878-1887.

[9] Zhang P, Xiong X, Rolfo C, et al. Mechanism-and Immune Landscape-Based Ranking of Therapeutic Responsiveness of 22Major Human Cancers to NextGeneration Anti-CTLA-4 Antibodies[J]. Cancers, 2020, 12(2).

[10] Roopenian D C, Akilesh S. FcRn: the neonatal Fc receptor comes ofage [J]. Nature Reviews Immunology, 2007, 7(9):715–725.

[11] Levin D, Golding B, Strome S E, et al. Fc fusion as a platformtechnology: potential for modulating immunogenicity [J]. Trends in Biotechnology, 2015, 33(1): 27–34.

SEQUENCE LISTING

<110> Sinopharm Zhongsheng Biotechnology Research Institute Co., Ltd.

Valin Biotechnology Ltd.

Negev National Institute of Biotechnology

<120> IL-17RA fusion protein, pharmaceutical composition, injection and application thereof

<130> FI-215323-59:52

<160> 19

<170> PatentIn version 3.5

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Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

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Leu Arg Leu Leu Asp His Arg Ala Pro Val Cys Ser Gln Pro Gly Leu

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His Phe Ala His Thr Gln Gln Gly Asp Leu Phe Pro Val Ala His Ile

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Glu Trp Thr Leu Gln Thr Asp Ala Ser Ile Leu Tyr Leu Glu Gly Ala

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Val His His Leu Pro Lys Pro Ile Pro Asp Gly Asp Pro Asn His Gln

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Ser Cys Leu Asn Asp Cys Leu Arg His Ser Val Thr Val Ser Cys Pro

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Glu Met Pro Asp Thr Pro Glu Pro Ile Pro Asp Tyr Met Pro Leu Trp

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Gly Ser Gly Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro

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

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

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Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

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

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

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

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

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

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Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

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Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

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ctgctgacct ccttccccca catggaaaac cacagctgct tcgagcacat gcaccacatc 660

cctgcccctc ggcccgagga attccaccag cggtccaacg tgaccctgac cctgcggaac 720

ctgaagggct gctgccggca ccaggtgcag attcagccct tcttctcctc ttgcctgaac 780

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cccaaggacc tgcagatcca gctgcacttc gcccacaccc agcagggcga cctgttcccc 180

gtggcccaca tcgagtggac cctgcagacc gacgcctcca tcctgtacct ggaaggcgcc 240

gagctgtccg tgctgcagct gaacaccaac gagcggctgt gcgtgcgctt cgagttcctg 300

tccaagctgc ggcaccacca caagcggtgg cggttcacct tctcccactt cgtggtggac 360

cccggccagg aatacgaagt gaccgtgcac catctgccca agcccatccc cgacggcgac 420

cccaaccacc agtccaagaa ctttctggtg cccgactgcg aggacgcccg gatgaaggtc 480

acaaccccct gcatgtcctc cggctccctg tgggacccca acatcaccgt ggaaaccctg 540

gaagcccacc agctgcgggt gtccttcacc ctgtggaacg agtccaccca ctaccagatc 600

ctgctgacct ccttccccca catggaaaac cacagctgct tcgagcacat gcaccacatc 660

cctgcccctc ggcccgagga attccaccag cggtccaacg tgaccctgac cctgcggaac 720

ctgaagggct gctgccggca ccaggtgcag attcagccct tcttctcctc ttgcctgaac 780

gactgcctgc ggcactccgc caccgtgtcc tgccctgaga tgcccgacac ccccgagccc 840

atccctgact acatgcctct gtgg 864

<210> 11

<211> 864

<212> DNA

<213> Artificial sequence

<400> 11

ctgagactgc tggaccacag agcccccgtc tgctcccagc ccggcctgaa ctgcaccgtg 60

aagaactcta cctgcctgga cgactcctgg atccaccccc ggaacctgac cccctccagc 120

cccaaggacc tgcagatcca gctgcacttc gcccacaccc agcagggcga cctgttcccc 180

gtggcccaca tcgagtggac cctgcagacc gacgcctcca tcctgtacct ggaaggcgcc 240

gagctgtccg tgctgcagct gaacaccaac gagcggctgt gcgtgcgctt cgagttcctg 300

tccaagctgc ggcaccacca caagcggtgg cggttcacct tctcccactt cgtggtggac 360

cccggccagg aatacgaagt gaccgtgcac catctgccca agcccatccc cgacggcgac 420

cccaaccacc agtccaagaa ctttctggtg cccgactgcg aggacgcccg gatgaaggtc 480

acaaccccct gcatgtcctc cggctccctg tgggacccca acatcaccgt ggaaaccctg 540

gaagcccacc agctgcgggt gtccttcacc ctgtggaacg agtccaccca ctaccagatc 600

ctgctgacct ccttccccca catggaaaac cacagctgct tcgagcacat gcaccacatc 660

cctgcccctc ggcccgagga attccaccag cggtccaacg tgaccctgac cctgcggaac 720

ctgaagtggt gctgccggca ccaggtgcag attcagccct tcttctcctc ttgcctgaac 780

gactgcctgc ggcactccgt gaccgtgtcc tgccctgaga tgcccgacac ccccgagccc 840

atccctgact acatgcctct gtgg 864

<210> 12

<211> 693

<212> DNA

<213> Artificial sequence

<400> 12

gagcccaagt cctccgacaa gacccacacc tgtcccccct gccctgctcc tcctgtggct 60

gggccctccg tgttcctgtt ccccccaaag cccaaggaca ccctgatgat ctcccggacc 120

cccgaagtga cctgcgtggt ggtggacgtg tcccacgagg accctgaagt gaagttcaat 180

tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcccagaga ggaacagtac 240

aactccacct accgggtggt gtctgtgctg acagtgctgc accaggactg gctgaacggc 300

aaagagtaca agtgcaaggt gtccaacaag ggcctgccct ccagcatcga aaagaccatc 360

tccaaggcca agggccagcc ccgcgagccc caggtgtaca ccctgccccc tagccgggac 420

gagctgacca agaaccaggt gtccctgacc tgcctggtga aaggcttcta cccctccgat 480

atcgccgtgg aatgggagtc caacggccag cccgagaaca actacaagac caccccccct 540

gtgctggact ccgacggctc attcttcctg tactccaagc tgaccgtgga caagtcccgg 600

tggcagcagg gcaacgtgtt ctcctgctcc gtgatgcacg agggcctgca caaccactac 660

acccagaagt ccctgtccct gagccccggg aaa 693

<210> 13

<211> 96

<212> DNA

<213> People (Homo sapiens)

<400> 13

atgggcgctg ccagatctcc cccttctgct gtgcctggac ctctgctggg cctccttctg 60

ctgctgctgg gagtgctggc tcctggcggc gctagc 96

<210> 14

<211> 1662

<212> DNA

<213> Artificial sequence

<400> 14

atgggcgctg ccagatctcc cccttctgct gtgcctggac ctctgctggg cctccttctg 60

ctgctgctgg gagtgctggc tcctggcggc gctagcctga gactgctgga ccacagagcc 120

ctggtctgct cccagcccgg cctgaactgc accgtgaaga actctacctg cctggacgac 180

tcctggatcc acccccggaa cctgaccccc tccagcccca aggacctgca gatccagctg 240

cacttcgccc acacccagca gggcgacctg ttccccgtgg cccacatcga gtggaccctg 300

cagaccgacg cctccatcct gtacctggaa ggcgccgagc tgtccgtgct gcagctgaac 360

accaacgagc ggctgtgcgt gcgcttcgag ttcctgtcca agctgcggca ccaccacaag 420

cggtggcggt tcaccttctc ccacttcgtg gtggaccccg gccaggaata cgaagtgacc 480

gtgcaccatc tgcccaagcc catccccgac ggcgacccca accaccagtc caagaacttt 540

ctggtgcccg actgcgagga cgcccggatg aaggtcacaa ccccctgcat gtcctccggc 600

tccctgtggg accccaacat caccgtggaa accctggaag cccaccagct gcgggtgtcc 660

ttcaccctgt ggaacgagtc cacccactac cagatcctgc tgacctcctt cccccacatg 720

gaaaaccaca gctgcttcga gcacatgcac cacatccctg cccctcggcc cgaggaattc 780

caccagcggt ccaacgtgac cctgaccctg cggaacctga agggctgctg ccggcaccag 840

gtgcagattc agcccttctt ctcctcttgc ctgaacgact gcctgcggca ctccgccacc 900

gtgtcctgcc ctgagatgcc cgacaccccc gagcccatcc ctgactacat gcctctgtgg 960

ggctccggcg agcccaagtc ctccgacaag acccacacct gtcccccctg ccctgctcct 1020

cctgtggctg ggccctccgt gttcctgttc cccccaaagc ccaaggacac cctgatgatc 1080

tcccggaccc ccgaagtgac ctgcgtggtg gtggacgtgt cccacgagga ccctgaagtg 1140

aagttcaatt ggtacgtgga cggcgtggaa gtgcacaacg ccaagaccaa gcccagagag 1200

gaacagtaca actccaccta ccgggtggtg tctgtgctga cagtgctgca ccaggactgg 1260

ctgaacggca aagagtacaa gtgcaaggtg tccaacaagg gcctgccctc cagcatcgaa 1320

aagaccatct ccaaggccaa gggccagccc cgcgagcccc aggtgtacac cctgccccct 1380

agccgggacg agctgaccaa gaaccaggtg tccctgacct gcctggtgaa aggcttctac 1440

ccctccgata tcgccgtgga atgggagtcc aacggccagc ccgagaacaa ctacaagacc 1500

accccccctg tgctggactc cgacggctca ttcttcctgt actccaagct gaccgtggac 1560

aagtcccggt ggcagcaggg caacgtgttc tcctgctccg tgatgcacga gggcctgcac 1620

aaccactaca cccagaagtc cctgtccctg agccccggga aa 1662

<210> 15

<211> 1662

<212> DNA

<213> Artificial sequence

<400> 15

atgggcgctg ccagatctcc cccttctgct gtgcctggac ctctgctggg cctccttctg 60

ctgctgctgg gagtgctggc tcctggcggc gctagcctga gactgctgga ccacagagcc 120

cccgtctgct cccagcccgg cctgaactgc accgtgaaga actctacctg cctggacgac 180

tcctggatcc acccccggaa cctgaccccc tccagcccca aggacctgca gatccagctg 240

cacttcgccc acacccagca gggcgacctg ttccccgtgg cccacatcga gtggaccctg 300

cagaccgacg cctccatcct gtacctggaa ggcgccgagc tgtccgtgct gcagctgaac 360

accaacgagc ggctgtgcgt gcgcttcgag ttcctgtcca agctgcggca ccaccacaag 420

cggtggcggt tcaccttctc ccacttcgtg gtggaccccg gccaggaata cgaagtgacc 480

gtgcaccatc tgcccaagcc catccccgac ggcgacccca accaccagtc caagaacttt 540

ctggtgcccg actgcgagga cgcccggatg aaggtcacaa ccccctgcat gtcctccggc 600

tccctgtggg accccaacat caccgtggaa accctggaag cccaccagct gcgggtgtcc 660

ttcaccctgt ggaacgagtc cacccactac cagatcctgc tgacctcctt cccccacatg 720

gaaaaccaca gctgcttcga gcacatgcac cacatccctg cccctcggcc cgaggaattc 780

caccagcggt ccaacgtgac cctgaccctg cggaacctga agggctgctg ccggcaccag 840

gtgcagattc agcccttctt ctcctcttgc ctgaacgact gcctgcggca ctccgccacc 900

gtgtcctgcc ctgagatgcc cgacaccccc gagcccatcc ctgactacat gcctctgtgg 960

ggctccggcg agcccaagtc ctccgacaag acccacacct gtcccccctg ccctgctcct 1020

cctgtggctg ggccctccgt gttcctgttc cccccaaagc ccaaggacac cctgatgatc 1080

tcccggaccc ccgaagtgac ctgcgtggtg gtggacgtgt cccacgagga ccctgaagtg 1140

aagttcaatt ggtacgtgga cggcgtggaa gtgcacaacg ccaagaccaa gcccagagag 1200

gaacagtaca actccaccta ccgggtggtg tctgtgctga cagtgctgca ccaggactgg 1260

ctgaacggca aagagtacaa gtgcaaggtg tccaacaagg gcctgccctc cagcatcgaa 1320

aagaccatct ccaaggccaa gggccagccc cgcgagcccc aggtgtacac cctgccccct 1380

agccgggacg agctgaccaa gaaccaggtg tccctgacct gcctggtgaa aggcttctac 1440

ccctccgata tcgccgtgga atgggagtcc aacggccagc ccgagaacaa ctacaagacc 1500

accccccctg tgctggactc cgacggctca ttcttcctgt actccaagct gaccgtggac 1560

aagtcccggt ggcagcaggg caacgtgttc tcctgctccg tgatgcacga gggcctgcac 1620

aaccactaca cccagaagtc cctgtccctg agccccggga aa 1662

<210> 16

<211> 1662

<212> DNA

<213> Artificial sequence

<400> 16

atgggcgctg ccagatctcc cccttctgct gtgcctggac ctctgctggg cctccttctg 60

ctgctgctgg gagtgctggc tcctggcggc gctagcctga gactgctgga ccacagagcc 120

cccgtctgct cccagcccgg cctgaactgc accgtgaaga actctacctg cctggacgac 180

tcctggatcc acccccggaa cctgaccccc tccagcccca aggacctgca gatccagctg 240

cacttcgccc acacccagca gggcgacctg ttccccgtgg cccacatcga gtggaccctg 300

cagaccgacg cctccatcct gtacctggaa ggcgccgagc tgtccgtgct gcagctgaac 360

accaacgagc ggctgtgcgt gcgcttcgag ttcctgtcca agctgcggca ccaccacaag 420

cggtggcggt tcaccttctc ccacttcgtg gtggaccccg gccaggaata cgaagtgacc 480

gtgcaccatc tgcccaagcc catccccgac ggcgacccca accaccagtc caagaacttt 540

ctggtgcccg actgcgagga cgcccggatg aaggtcacaa ccccctgcat gtcctccggc 600

tccctgtggg accccaacat caccgtggaa accctggaag cccaccagct gcgggtgtcc 660

ttcaccctgt ggaacgagtc cacccactac cagatcctgc tgacctcctt cccccacatg 720

gaaaaccaca gctgcttcga gcacatgcac cacatccctg cccctcggcc cgaggaattc 780

caccagcggt ccaacgtgac cctgaccctg cggaacctga agtggtgctg ccggcaccag 840

gtgcagattc agcccttctt ctcctcttgc ctgaacgact gcctgcggca ctccgtgacc 900

gtgtcctgcc ctgagatgcc cgacaccccc gagcccatcc ctgactacat gcctctgtgg 960

ggctccggcg agcccaagtc ctccgacaag acccacacct gtcccccctg ccctgctcct 1020

cctgtggctg ggccctccgt gttcctgttc cccccaaagc ccaaggacac cctgatgatc 1080

tcccggaccc ccgaagtgac ctgcgtggtg gtggacgtgt cccacgagga ccctgaagtg 1140

aagttcaatt ggtacgtgga cggcgtggaa gtgcacaacg ccaagaccaa gcccagagag 1200

gaacagtaca actccaccta ccgggtggtg tctgtgctga cagtgctgca ccaggactgg 1260

ctgaacggca aagagtacaa gtgcaaggtg tccaacaagg gcctgccctc cagcatcgaa 1320

aagaccatct ccaaggccaa gggccagccc cgcgagcccc aggtgtacac cctgccccct 1380

agccgggacg agctgaccaa gaaccaggtg tccctgacct gcctggtgaa aggcttctac 1440

ccctccgata tcgccgtgga atgggagtcc aacggccagc ccgagaacaa ctacaagacc 1500

accccccctg tgctggactc cgacggctca ttcttcctgt actccaagct gaccgtggac 1560

aagtcccggt ggcagcaggg caacgtgttc tcctgctccg tgatgcacga gggcctgcac 1620

aaccactaca cccagaagtc cctgtccctg agccccggga aa 1662

<210> 17

<211> 523

<212> PRT

<213> Artificial sequence

<400> 17

Leu Arg Leu Leu Asp His Arg Ala Pro Val Cys Ser Gln Pro Gly Leu

1 5 10 15

Asn Cys Thr Val Lys Asn Ser Thr Cys Leu Asp Asp Ser Trp Ile His

20 25 30

Pro Arg Asn Leu Thr Pro Ser Ser Pro Lys Asp Leu Gln Ile Gln Leu

35 40 45

His Phe Ala His Thr Gln Gln Gly Asp Leu Phe Pro Val Ala His Ile

50 55 60

Glu Trp Thr Leu Gln Thr Asp Ala Ser Ile Leu Tyr Leu Glu Gly Ala

65 70 75 80

Glu Leu Ser Val Leu Gln Leu Asn Thr Asn Glu Arg Leu Cys Val Arg

85 90 95

Phe Glu Phe Leu Ser Lys Leu Arg His His His Lys Arg Trp Arg Phe

100 105 110

Thr Phe Ser His Phe Val Val Asp Pro Gly Gln Glu Tyr Glu Val Thr

115 120 125

Val His His Leu Pro Lys Pro Ile Pro Asp Gly Asp Pro Asn His Gln

130 135 140

Ser Lys Asn Phe Leu Val Pro Asp Cys Glu Asp Ala Arg Met Lys Val

145 150 155 160

Thr Thr Pro Cys Met Ser Ser Gly Ser Leu Trp Asp Pro Asn Ile Thr

165 170 175

Val Glu Thr Leu Glu Ala His Gln Leu Arg Val Ser Phe Thr Leu Trp

180 185 190

Asn Glu Ser Thr His Tyr Gln Ile Leu Leu Thr Ser Phe Pro His Met

195 200 205

Glu Asn His Ser Cys Phe Glu His Met His His Ile Pro Ala Pro Arg

210 215 220

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

225 230 235 240

Leu Lys Trp Cys Cys Arg His Gln Val Gln Ile Gln Pro Phe Phe Ser

245 250 255

Ser Cys Leu Asn Asp Cys Leu Arg His Ser Val Thr Val Ser Cys Pro

260 265 270

Glu Met Pro Asp Thr Pro Glu Pro Ile Pro Asp Tyr Met Pro Leu Trp

275 280 285

Gly Ser Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro

290 295 300

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

305 310 315 320

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

325 330 335

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

515 520

<210> 18

<211> 213

<212> PRT

<213> Artificial sequence

<400> 18

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

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

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

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 19

<211> 450

<212> PRT

<213> Artificial sequence

<400> 19

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

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

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

115 120 125

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

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Ser

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

325 330 335

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

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Gly Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

Claims (25)

What is claimed is: 1. An IL-17RA fusion protein, characterized in that it comprises an operably linked signal peptide, an IL-17RA extracellular domain and an IgG1 constant region in series in series. 2 . The IL-17RA fusion protein according to claim 1 , wherein the amino acid sequence of the extracellular domain of IL-17RA is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3. 3 . 3. The IL-17RA fusion protein according to claim 1, wherein the amino acid sequence of the IgG1 constant region is shown in SEQ ID NO.4. 4. The IL-17RA fusion protein according to claim 1, wherein the signal peptide is IL-17-RA natural signal peptide, and its amino acid sequence is shown in SEQ ID NO.5. 5. The IL-17RA fusion protein of claim 1, wherein a linker is used between the IL-17RA extracellular domain and the IgGl constant region. 6. The IL-17RA fusion protein according to claim 1, wherein the amino acid sequence of the IL-17RA fusion protein is shown in SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8. 7. An isolated nucleic acid encoding the IL-17RA fusion protein of any one of claims 1-6. 8. An expression vector comprising the nucleic acid of claim 7 operably linked to a promoter. 9. A host cell comprising the expression vector of claim 8. 10. The host cell according to claim 9, wherein the deposit number of the host cell is CGMCC 21011. 11. A protein dimer formed by the IL-17RA fusion protein according to any one of claims 1-6, the dimer consisting of two molecules of the IL-17RA fusion protein through the IgG1 constant region cysteines combine to form a double chain. 12. The IL-17RA fusion protein according to any one of claims 1-6 or the protein dimer according to claim 11 is prepared for the treatment of psoriasis, Crohn's disease, plaque psoriasis disease, gastroenteritis, Behçet's syndrome, arthritis, uveitis, hidradenitis suppurativa, lichen planus, parapsoriasis, asthma, psoriatic arthritis, tendinitis, relapsing-remitting multiple sclerosis , thyroid-related eye disease, juvenile rheumatoid arthritis, multiple sclerosis, lupus nephritis, spondyloarthritis, ankylosing spondylitis, rheumatoid arthritis, inflammatory bowel disease, nonalcoholic fatty liver disease, giant cell arterial Inflammation, non-radiological axial spondyloarthritis, acne vulgaris, triple negative breast tumors, multiple myeloma, non-small cell lung cancer, adenocarcinoma, colorectal cancer, prostate cancer, Kaposi's sarcoma, melanoma, cervical cancer The use of drugs in cancer and/or other inflammatory diseases. 13. A pharmaceutical composition comprising a therapeutically effective amount of the IL-17RA fusion protein according to any one of claims 1-6 or the protein dimer according to claim 11 as an active ingredient and pharmaceutically acceptable of accessories. 14. The pharmaceutical composition according to claim 13, wherein the pharmaceutically acceptable adjuvant is selected from one or more of diluents, buffers, protective agents, surfactants, and antioxidants. 15. The pharmaceutical composition according to claim 14, wherein the buffer is selected from the group consisting of histidine-acetate buffer, Tris-acetate buffer, hydrochloric acid buffer, phosphate buffer, acetate buffer, histidine buffer One or more of arginine buffer, succinate buffer, and citrate buffer. 16. The pharmaceutical composition according to claim 14, wherein the protective agent is selected from one or more of trehalose, Tween-20, Tween-80, sucrose, amino acids, polyols, disaccharides, and polysaccharides kind. 17. The pharmaceutical composition of claim 14, wherein the surfactant is selected from one or more of Tween-20, Tween-80, and Poloxamers. 18. The pharmaceutical composition of claim 13, wherein a single dose of the pharmaceutical composition comprises 5 mg/ml-150 mg/ml of the IL-17RA fusion protein or the protein dimer. 19. The pharmaceutical composition according to claim 13, wherein the pharmaceutical composition is in the form of a lyophilisate or an injection. 20. A preparation for the treatment of psoriasis, Crohn's disease, plaque psoriasis, gastroenteritis, Behcet's syndrome, arthritis, uveitis, hidradenitis suppurativa, lichen planus, parapsoriasis disease, asthma, psoriatic arthritis, tendonitis, relapsing-remitting multiple sclerosis, thyroid-related eye disease, juvenile rheumatoid arthritis, multiple sclerosis, lupus nephritis, spondyloarthritis, ankylosing spondylitis, Rheumatoid Arthritis, Inflammatory Bowel Disease, Nonalcoholic Fatty Liver, Giant Cell Arteritis, Nonradiographic Axial Spondyloarthritis, Acne Vulgaris, Triple Negative Breast Tumors, Multiple Myeloma, Non-Small Cell Lung Cancer, An injection for adenocarcinoma, colorectal cancer, prostate cancer, Kaposi's sarcoma, melanoma, cervical cancer and/or other inflammatory diseases, comprising the pharmaceutical composition of any one of claims 13-19. 21. The injection according to claim 20, wherein the injection is in the form of a lyophilized powder or a liquid preparation. 22. The injection according to claim 20, wherein the injection is a subcutaneous injection or an intravenous drip. 23. The injection according to claim 21, wherein in the liquid formulation, the intravenous formulation comprises the IL-17RA fusion protein or the protein dimer, 2-100 mM of 5 mg/ml-150 mg/ml Tris-acetic acid, 10-250 mM arginine, 50-500 mM trehalose, 0.01-5% Tween-20. 24. The injection according to claim 21, wherein the liquid preparation is prepared by using water for injection, buffered saline solution, aqueous dextrose solution, aqueous sodium chloride solution or lactated Ringer's solution. 25. The injection according to claim 21, wherein the freeze-dried powder is prepared by freeze-drying the liquid preparation.

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