IL-17RA fusion protein, pharmaceutical composition, injection and their applications

Abstract

The present invention provides an IL-17RA fusion protein, a pharmaceutical composition, an injection and their applications. The IL-17RA fusion protein comprises a signal peptide, an extracellular domain of IL-17RA and an IgG1 constant region which are operably linked and tandemly arranged in sequence. The IL-17RA fusion protein of the present invention has an extended half-life, obtains a longer-lasting drug activity, and reduces immunogenicity compared with antibody drugs. The design of the present invention enables the IL-17RA fusion protein to eliminate ADCC, ADCP and CDC effects, and retains the effect of neonatal Fc receptor (FcRn)-mediated recycling in vivo. Compared with the marketed IL-17A and IL-17RA antibodies, it has lower side effects and is safer.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to an IL-17RA fusion protein, a pharmaceutical composition, an injection, and their applications. Background Art

[0002] Psoriasis, commonly known as psoriasis vulgaris, is a chronic and relapsing autoimmune skin disease that usually causes great damage to the physiology and psychology of patients. The WHO regards it as one of the major global health problems. The incidence rate accounts for 0.1% - 3% of the world's population. Currently, there are 125 million psoriasis patients globally. The total prevalence rate in China is 0.47%, and there are nearly 6.5 million clinically registered patients. Plaque psoriasis is one of the five most common forms of this disease, accounting for 80% - 90% of all cases, among which moderate to severe patients account for 38%. The number of patients will reach 9.5 million in 2030, and the proportion of moderate to severe patients will increase to 40%. The pathogenesis of psoriasis is related to the imbalance of the innate and adaptive immune systems, including dendritic cell activation and the secretion of pro-inflammatory cytokines, which lead to the occurrence of characteristic skin inflammation in psoriasis. In recent years, nearly 13 monoclonal antibodies and antibody-based biotherapies have been approved for the treatment of psoriasis, and there are about 10 monoclonal antibody drugs in the clinical research stage. Among them, ustekinumab (IL-12 / IL-23 antibody, Johnson & Johnson), TNF-α drug Etanercept (Amgen), and Adalimumab (Humira, AbbVie) are the best-selling drugs. 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 biological drugs are used as second-line drugs, the treatment effect and safety are still worrying, and up to 40% of moderate to severe psoriasis patients fail to respond to existing biological agents, a phenomenon known as biologic fatigue.

[0003] The latest research shows that the pathogenesis of psoriasis is related to T17 helper cells (Th17 cells), and the pro-inflammatory cytokine IL-17A produced by Th17 cells and innate immune cells has been confirmed to be the main cytokine in the pathogenesis of psoriasis. The immune system targets IL-23 in the IL-17-TH17 pathway and indirectly causes psoriasis through IL-17A. Theoretically, inhibiting IL-17 may be a safer treatment option compared to other biologics, which has been confirmed in the head-to-head superiority comparison studies of the fully human IgG1 monoclonal antibody secukinumab against IL-17A marketed by Novartis, the fully human IgG2 monoclonal antibody (Brodalumab) against the IL-17A receptor marketed by AstraZeneca, and the fully human IgG4 monoclonal antibody (ixekizumab) against IL-17A of Eli Lilly and etanercept and ustekinumab (IL-12 / IL-23 antibody). The research shows that inhibitors developed against the IL-17A target can improve efficacy and safety compared to current drugs for treating inflammatory diseases (such as TNF-α inhibitors), are effective for psoriasis patients who are ineffective to TNF-α inhibitors and IL-12 and IL-23 inhibitors, and are more specific than IL-12 and IL-23 inhibitors. It is particularly worth mentioning that secukinumab (trade name Cosentyx), which was launched in January 2015, has been approved by the European Medicines Agency (EMA) to replace other first-line systemic therapies with significant side effects for the first-line treatment of psoriasis patients.

[0004] There is still a need for more innovative drugs with excellent efficacy as treatment options for patients. Summary of the Invention

[0005] To solve the problems existing in the above-mentioned prior art, the present invention provides an IL-17RA fusion protein, a pharmaceutical composition, an injection and their applications.

[0006] Specifically, the present invention provides:

[0007] (1) An IL-17RA fusion protein, characterized in that it comprises a signal peptide, an extracellular domain of IL-17RA and an IgG1 constant region which are operably linked and serially connected in sequence.

[0008] (2) The IL-17RA fusion protein according to (1), wherein the amino acid sequence of the extracellular domain of IL-17RA is as shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

[0009] (3) The IL-17RA fusion protein according to (1), wherein the amino acid sequence of the IgG1 constant region is as shown in SEQ ID NO.4.

[0010] (4) The IL-17RA fusion protein according to (1), wherein the signal peptide is the IL-17-RA native signal peptide, and its amino acid sequence is as shown in SEQ ID NO.5.

[0011] (5) The IL-17RA fusion protein according to (1), wherein a linker is used to connect the extracellular domain of IL-17RA and the IgG1 constant region.

[0012] (6) The IL-17RA fusion protein according to (1), wherein the amino acid sequence of the IL-17RA fusion protein is as shown in SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8.

[0013] (7) An isolated nucleic acid encoding the IL-17RA fusion protein according to any one of (1)-(6).

[0014] (8) An expression vector containing the nucleic acid according to (7) operably linked to a promoter.

[0015] (9) A host cell containing the expression vector according to (8).

[0016] (10) The host cell according to (9), wherein the deposit number of the host cell is CGMCC 21011.

[0017] (11) A protein dimer formed by the IL-17RA fusion protein according to any one of (1)-(6), and the dimer is formed by binding two molecules of the IL-17RA fusion protein through the cysteine of the IgG1 constant region to form a double strand.

[0018] (12) Use of the IL-17RA fusion protein according to any one of (1) to (6) or the protein dimer according to claim 11 in the preparation of a medicament for treating 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, non-alcoholic fatty liver disease, giant cell arteritis, non-radiographic 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 and / or other inflammatory diseases.

[0019] (13) A pharmaceutical composition comprising a therapeutically effective amount of the IL-17RA fusion protein according to any one of (1) to (6) or the protein dimer according to claim 11 as an active ingredient and a pharmaceutically acceptable excipient.

[0020] (14) The pharmaceutical composition according to (13), wherein the pharmaceutically acceptable excipient is selected from one or more of a diluent, a buffer, a protective agent, a surfactant, and an antioxidant.

[0021] (15) The pharmaceutical composition according to (14), wherein the buffer is selected from one or more of histidine-acetate buffer, Tris-acetate buffer, hydrochloric acid buffer, phosphate buffer, acetate buffer, histidine buffer, arginine buffer, succinate buffer, and citrate buffer.

[0022] (16) The pharmaceutical composition according to (14), wherein the protective agent is selected from one or more of trehalose, Tween-20, Tween-80, sucrose, amino acids, polyols, disaccharides, and polysaccharides.

[0023] (17) The pharmaceutical composition according to (14), wherein the surfactant is selected from one or more of Tween-20, Tween-80, and poloxamer.

[0024] (18) The pharmaceutical composition according to (13), wherein a single dose of the pharmaceutical composition contains 5 mg / ml-150 mg / ml of the IL-17RA fusion protein or the protein dimer.

[0025] (19) The pharmaceutical composition according to (13), wherein the pharmaceutical composition is in the form of a lyophilized agent or an injection solution.

[0026] (20) An injection for treating psoriasis, Crohn's disease, plaque psoriasis, gastroenteritis, Behçet's syndrome, arthritis, uveitis, hidradenitis suppurativa, lichen planus, parapsoriasis, asthma, psoriatic arthritis, tendinitis, relapsing-remitting multiple sclerosis, thyroid-associated ophthalmopathy, juvenile rheumatoid arthritis, multiple sclerosis, lupus nephritis, spondylarthritis, ankylosing spondylitis, rheumatoid arthritis, inflammatory bowel disease, non-alcoholic fatty liver, giant cell arteritis, non-radiographic 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 and / or other inflammatory diseases, comprising the pharmaceutical composition described in any one of (13)-(19).

[0027] (21) The injection according to (20), wherein the injection is in the form of a lyophilized powder or a liquid preparation.

[0028] (22) The injection according to (20), wherein the injection is a subcutaneous injection or an intravenous drip.

[0029] (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 of Tris-acetate, 10 - 250 mM of arginine, 50 - 500 mM of trehalose, 0.01 - 5% of Tween-20.

[0030] (24) The injection according to (21), wherein the liquid preparation is prepared using water for injection, buffered saline solution, aqueous glucose solution, aqueous sodium chloride solution or lactated Ringer's solution.

[0031] (25) The injection according to (21), wherein the lyophilized powder is prepared by freeze-drying the liquid preparation.

[0032] The present invention has the following advantages and positive effects compared with the prior art:

[0033] The IL-17RA fusion protein provided by the present invention is a fully human antibody Fc fusion protein drug. By fusing IL-17RA with the antibody Fc segment, the present invention prolongs the half-life of the drug molecule, obtains longer-lasting drug activity, and reduces immunogenicity compared with antibody drugs.

[0034] In addition, the present invention discovers that by selecting the Fc segment of IgG1 and further making appropriate mutations to the sequence of this Fc, it is possible to greatly eliminate antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and complement-dependent cell-mediated cytotoxicity (CDC), and retain the function of neonatal Fc receptor (FcRn)-mediated recycling in vivo. Compared with the marketed anti-IL-17A and anti-IL-17RA antibodies, the side effects are low and it is safer.

[0035] In addition, by selecting and designing the sequence of IL-17RA, the present invention enables the IL-17RA fusion protein to target multiple targets such as IL-17A, IL-17C, IL-17F, and IL-17A / F, 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, and further effectively block the biological activities of various pro-inflammatory IL-17 cytokines, inhibit the inflammatory signaling pathway. Therefore, it can more effectively relieve the symptoms of autoimmune diseases, obtain better therapeutic benefits than the antibody drugs targeting a single IL-17A target, and have better safety than anti-IL-17RA monoclonal antibodies. Its mechanism of action is different from the currently marketed and in-development IL-17 target drugs, and it is a globally first-in-class drug.

[0036] The present invention comprehensively evaluates the above-mentioned drug activities and safety aspects from the aspects of binding affinity with IL-17A, biological activity cell experiments, GRO-α factor inhibition ability, in vitro activity titer, in vivo titer, ADCC / CDC functional activity, FcRn binding affinity, safety pharmacology, pharmacokinetics, and toxicology. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Shows the amino acid sequence of recombinant human IL-17RA fusion protein.

[0038] Figure 2 Shows the map of the expression vector pCHO1.1 / NVS451 used in one embodiment.

[0039] Figure 3 Shows the affinity determination curve and fitting curve of IL-17RA fusion protein (NVS451) and human IL-17A protein in Experimental Example 1.

[0040] Figure 4 Shows the affinity determination curve and fitting curve of secukinumab and human IL-17A protein in Experimental Example 1.

[0041] Figure 5 Shows the affinity determination curve and fitting curve of wild-type IL-17RA and human IL-17A protein in Experimental Example 1.

[0042] Figure 6 Show the affinity determination curve and fitting curve of the IL-17RA fusion protein (NVS451) and human IL-17F protein in Experimental Example 1.

[0043] Figure 7 Show the affinity determination curve and fitting curve of the IL-17RA fusion protein (NVS451) and human IL-17A / F protein in Experimental Example 1.

[0044] Figure 8 Show the affinity determination curve and fitting curve of the IL-17RA fusion protein (NVS451) and human IL-17C protein in Experimental Example 1.

[0045] Figure 9 Show the S curve of the inhibitory effect of the IL-17RA fusion protein (NVS451) and secukinumab on the GRO-α factor induced by IL-17A in Experimental Example 1.

[0046] Figure 10 Show the S curve of the inhibitory effect of the IL-17RA fusion protein (NVS451) on the GRO-α factor induced by IL-17A / F in Experimental Example 1.

[0047] Figure 11 Show the S curve of the inhibitory effect of the IL-17RA fusion protein (NVS451) on the GRO-α factor induced by IL-17F in Experimental Example 1.

[0048] Figure 12 Show the skin appearance of SCID mice with xenogeneic skin transplantation after treatment with different drugs in Experimental Example 1.

[0049] Figure 13 Show the skin appearance of SCID mice with xenogeneic skin transplantation after treatment with different drugs in Experimental Example 1.

[0050] Figure 14 Show the skin pathological sections of SCID mice with xenogeneic skin transplantation after treatment with different drugs in Experimental Example 1.

[0051] Figure 15 Show the comparison of the ADCC effects of RitxV301 and rituximab in Experimental Example 1.

[0052] Figure 16 Show the comparison of the CDC effects of RitxV301 and rituximab in Experimental Example 1.

[0053] Figure 17Shows the SPR analysis spectra of different concentrations of NVS451 and human FcRn under acidic conditions (pH 6.0) in Experimental Example 1.

[0054] Figure 18 Shows the SPR analysis spectra of different concentrations of NVS451 and human FcRn under neutral conditions (pH 7.4) in Experimental Example 1.

[0055] Figure 19 Shows the comparison of the affinities of IL-17RA fusion proteins NVS451 (V301), V302, and V303 and IL-17A in Experimental Example 1; the concentrations of V300, V301, V302, V303, V301*, V302*, and V303* in the figure are all 100 μg / ml.

[0056] Biological material preservation information

[0057] The Chinese hamster ovary cells capable of stably expressing the IL-17RA fusion protein of the present invention provided by the present invention were deposited on November 23, 2020 at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (CGMCC), with the deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postcode: 100101, and the deposit number: CGMCC No. 21011. Detailed implementation manners

[0058] The present invention will be further described below through the description of detailed implementation manners 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, they are all within the scope of the present invention.

[0059] In this article, the term "injection" refers to: a sterile solution (including true solution, emulsion, and suspension) made from a drug for injection into the body, as well as a lyophilized powder or concentrated solution for preparing the sterile solution (including true solution, emulsion, and suspension) immediately before use.

[0060] In this article, the term "intravenous drip" refers to: a method of infusing a large amount of liquid containing a drug into the body through a transfusion tube. Also known as "infusion", "drip", "intravenous drip", "hanging water".

[0061] In this article, the term "IL-17A" refers to interleukin 17A.

[0062] In this article, the term "IL-17RA" refers to the receptor of IL-17A.

[0063] In this article, 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.

[0064] The present invention provides an IL-17RA fusion protein, which is characterized by comprising a signal peptide, an extracellular domain of IL-17RA, and an IgG1 constant region that are operably linked and tandemly arranged in sequence.

[0065] The main function of the signal peptide is to direct the target protein to be secreted from the cytoplasm of the cell to the extracellular space. Since the fusion protein has an IgG1 constant region, when it is secreted to the extracellular space, two molecules of the fusion protein bind through the cysteines in the constant region to form a double strand and exert activity.

[0066] Preferably, the present invention uses the extracellular domain of human IL-17RA (Gene bank accession number: NP_055154), and performs R108K, D122G, and H155D mutations. The amino acid sequence of this mutant is shown in SEQ ID NO.1. More preferably, the present invention uses the extracellular domain of human IL-17RA and performs L9P, R108K, D122G, and H155D mutations. The amino acid sequence of this mutant is shown in SEQ ID NO.2. The present invention discovers that compared with the one containing the wild type, the IL-17RA fusion protein containing these two mutants has improved thermal stability and improved binding affinity for IL-17A. The amino acid numbering starts from the 1st position of the amino acid sequence of the extracellular domain of the human IL-17RA.

[0067] More preferably, the present invention uses the extracellular domain of human IL-17RA and performs L9P, R108K, D122G, H155D, G243W, and A267V mutations. The amino acid sequence of this mutant is shown in SEQ ID NO.3. The present invention discovers that the IL-17RA fusion protein containing this mutant has improved thermal stability and improved binding affinity for IL-17A, IL-17C, IL-17F, and IL-17A / F compared with the one containing the wild type; and has a higher binding affinity for IL-17A compared with the mutants containing the above three and four mutations.

[0068] The inventors of the present invention selected the constant region (Fc) of human IgG1 (Gene bank number: 3500) to form a fusion protein with IL-17RA. This fusion protein not only retains the biological activity of the functional protein molecule, but also, due to the characteristics of the Fc part being somewhat antibody-like and stable, the fused protein has a longer circulation lifespan and an extended half-life. IgG isotypes include IgG1, IgG2, and IgG4, which can cause different ADCC, ADCP, and CDC effects and can have a significant impact on the toxicity of target and non-target tissues. The constant region of IgG1 has strong ADCC, ADCP, and CDC effects. The present invention found that selecting the constant region of IgG1 and further making appropriate mutations to the sequence of this Fc can reduce the ADCC, ADCP, and CDC effects of the IL-17RA fusion protein.

[0069] In a preferred embodiment, the mutation sites are summarized in Table 1 below. Another mutation is to replace the cysteine residue in the hinge region of IgG1 with a serine residue to avoid unpaired cysteines in the fusion protein sequence.

[0070] Table 1

[0071]

[0072] Note: The amino acid numbers described in Table 1 are counted from the 1st position of the human IgG1 amino acid sequence.

[0073] The amino acid sequence of this mutant of IgG1-Fc is shown in SEQ ID NO.4.

[0074] The signal peptide is one of the main factors affecting yield optimization and product quality. Importantly, the signal peptide cleavage site should be clearly defined by a single residue with clear cleavage and a high cleavage probability. Preferably, the native signal peptide of human IL17RA is used for the fusion protein, and the amino acid sequence of this signal peptide is shown in SEQ ID NO.5.

[0075] Preferably, a linker is used to connect between the extracellular domain of IL-17RA and the constant region of IgG1. The linker can be those commonly used in the art, such as one or more consecutive GSG, one or more consecutive GGGGS, and GSAGSAAGSG.

[0076] 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, and six mutations respectively), and it has 522 amino acid residues.

[0077] For example, the sequence of SEQ ID NO.8 is asFigure 1 As shown, the first 32 amino acids (bold) are the IL-17RA signal peptide, the letters with a light gray background are the amino acid sequence of recombinant human IL-17RA with 6 mutations (bold and underlined letters); the letters with a black background are the linker amino acid sequence; the letters with a dark gray background are the amino acid sequence of the Fc portion with 3 modifications (bold and underlined letters) as described in Table 1, and two bold cysteine residues are responsible for the dimerization of Fc. The asterisk (*) indicates potential glycosylation sites.

[0078] The mechanism of action of the IL-17RA fusion protein provided by the present invention is that a decoy receptor (IL-17RA-Fc) competes with the natural receptor for binding to IL-17 molecules, binds to IL-17A, IL-17C, IL-17F and IL-17A / F with high affinity and selectively blocks the binding of IL-17A, IL-17C, IL-17F and IL-17A / F to their receptors, does not bind to IL-17B, IL-17D, IL-17E, effectively blocks the biological activities of multiple pro-inflammatory IL-17 cytokines, inhibits the inflammatory signaling pathway, thereby more effectively relieving the symptoms of autoimmune diseases, and can obtain better therapeutic benefits than single IL-17A target antibody drugs and better safety than IL-17RA monoclonal antibodies.

[0079] The present invention also provides an isolated nucleic acid encoding the IL-17RA fusion protein according to the present invention.

[0080] In a preferred embodiment, the present invention designed the cDNA sequence of the fusion protein using molecular biology techniques and optimized the codons for expression in CHO cells. The optimized DNA sequences encoding the amino acid sequences SEQ ID NO.1-8 are shown as SEQ ID NO.9-16 respectively.

[0081] The present invention also provides an isolated mRNA transcribed from the DNA encoding the fusion protein according to the present invention.

[0082] The present invention also provides an expression vector containing the nucleic acid according to the present invention operably linked to a promoter.

[0083] The present invention also provides a host cell containing the expression vector according to the present invention.

[0084] In some preferred embodiments, CHO cells suitable for growth in suspension and serum-free medium are used as host cells. Also preferably, the expression vector contains two selectable markers, puromycin and methotrexate, which can facilitate the creation of high-yield and stable cell lines.

[0085] In a more preferred embodiment, the deposit number of the host cell is CGMCC 21011. The host cell was constructed using CHO-S TM Cells, after construction, can stably express the IL-17RA fusion protein of the present invention. In addition, the host cell 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 function; (2) It can grow both on the wall and in suspension culture, and has a high tolerance 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 product extracellular secretion, and rarely secretes its own endogenous proteins, which is convenient for downstream product separation and purification; (5) It can be cultured at high density in suspension culture or in serum-free culture medium. And the culture volume can reach more than 1000L, which can be mass-produced. Therefore, the CHO cell is the preferred system for the production of recombinant glycoproteins. The host cell was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on November 23, 2020. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, and the deposit number is: CGMCC No.21011.

[0086] The present invention also provides a protein dimer formed by the IL-17RA fusion protein according to the present invention, wherein the dimer is formed by two molecules of the IL-17RA fusion protein bound via the cysteine ​​of the IgG1 constant region to form a double chain.

[0087] Since the IL-17RA fusion protein of the present invention has an IgG1 constant region, when secreted outside the cell, two molecules of the fusion protein bind via the cysteine ​​in the constant region to form a double chain and exert activity.

[0088] The present invention also provides the use of the IL-17RA fusion protein according to the present invention or the protein dimer formed by the IL-17RA fusion protein in the preparation of a drug for treating psoriasis, Crohn's disease, plaque psoriasis, gastroenteritis, Behcet's syndrome, arthritis, uveitis, suppurative hidradenitis, lichen planus, parapsoriasis, asthma, psoriatic arthritis, tendinitis, relapsing-remitting multiple sclerosis, thyroid-related eye disease, juvenile rheumatoid arthritis, multiple sclerosis, lupus nephritis, spondylarthritis, ankylosing spondylitis, rheumatoid arthritis, inflammatory bowel disease, non-alcoholic fatty liver disease, giant cell arteritis, non-radiographic 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, etc. and / or other inflammatory diseases.

[0089] The present invention comprehensively evaluated the drug activity and safety of the IL-17AR fusion protein in terms of binding affinity with IL-17A, biological activity cell experiments, GRO-α factor inhibition ability, in vitro activity titer, in vivo titer, ADCC / CDC functional activity, FcRn binding affinity, safety pharmacology, pharmacokinetics, and toxicology. The results demonstrated that the IL-17RA fusion protein can target multiple targets such as IL-17A, IL-17C, IL-17F, and IL-17A / F, and has a high affinity, thereby being able to selectively block the binding of IL-17A, IL-17C, IL-17F, and IL-17A / F to their receptors, and further effectively blocking the biological activities of various pro-inflammatory IL-17 cytokines and inhibiting the inflammatory signaling pathway. Therefore, it can more effectively relieve the symptoms of autoimmune diseases, obtain better therapeutic benefits than single IL-17A target antibody drugs, and achieve better safety than IL-17RA monoclonal antibodies.

[0090] 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 a pharmaceutically acceptable excipient.

[0091] The pharmaceutically acceptable excipient can be selected according to the dosage form used and actual needs.

[0092] The present invention deeply studied and designed the preparation and formulation of the IL-17RA fusion protein from aspects such as packaging materials, buffer systems, excipients, dosages, formulation compositions, and lyophilization processes.

[0093] In some preferred embodiments, the pharmaceutically acceptable excipient of the pharmaceutical composition is selected from one or more of diluents, buffers, protectants, surfactants, and antioxidants.

[0094] Preferably, the buffer is selected from one or more of histidine-acetate buffer, Tris-acetate buffer, hydrochloric acid buffer, phosphate buffer, acetate buffer, histidine buffer, arginine buffer, succinic acid buffer, and citric acid buffer.

[0095] Preferably, the protectant is selected from one or more of trehalose, Tween-20, Tween-80, sucrose, amino acids (such as arginine), polyols, disaccharides, and polysaccharides. Most preferably, it is trehalose and the combination of trehalose + arginine.

[0096] In some embodiments, trehalose exists in the form of trehalose dihydrate.

[0097] Preferably, the surfactant is selected from one or more of Tween-20, Tween-80, and poloxamer.

[0098] In some preferred embodiments, the single-dose drug composition contains 5 mg / ml - 150 mg / ml of the IL-17RA fusion protein or a protein dimer formed by the IL-17RA fusion protein.

[0099] The drug composition of the present invention can be made into a suitable dosage form as needed. In the present invention, the IL-17RA fusion protein is preferably in the form of a freeze-dried preparation or an injection.

[0100] Therefore, the present invention also provides an injection for treating psoriasis, Crohn's disease, plaque psoriasis, gastroenteritis, Behçet's syndrome, arthritis, uveitis, hidradenitis suppurativa, lichen planus, parapsoriasis, asthma, psoriatic arthritis, tendinitis, relapsing-remitting multiple sclerosis, thyroid-associated ophthalmopathy, juvenile rheumatoid arthritis, multiple sclerosis, lupus nephritis, spondylarthritis, ankylosing spondylitis, rheumatoid arthritis, inflammatory bowel disease, non-alcoholic fatty liver, giant cell arteritis, non-radiographic 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, etc. and / or other inflammatory diseases, which contains the drug composition of the present invention.

[0101] The injection can be in the form of a freeze-dried powder or a liquid preparation.

[0102] Preferably, the injection is a subcutaneous injection or an intravenous drip. Most preferably, it is a subcutaneous injection.

[0103] In the liquid preparation, the injection preferably contains the IL-17RA fusion protein, Tris-acetate, arginine, trehalose, Tween-20, and a suitable solvent.

[0104] More preferably, the injection contains 5 mg / ml - 150 mg / ml of the IL-17RA fusion protein or a protein dimer formed by the IL-17RA fusion protein, 2 - 100 mM of Tris-acetate, 10 - 250 mM of arginine, 50 - 500 mM of trehalose, 0.01 - 5% of Tween-20, and a suitable solvent.

[0105] The solvent can be a commonly used solvent for preparing injections. For example, water for injection, buffered saline solution, glucose aqueous solution, sodium chloride aqueous solution, or lactated Ringer's solution, etc.

[0106] The liquid preparation is prepared by using a solvent.

[0107] The liquid preparation can be formulated according to the prescription described in the present invention by using methods commonly used in the pharmaceutical field.

[0108] The lyophilized powder can be prepared by lyophilizing the liquid preparation.

[0109] In a preferred embodiment, the lyophilization process includes pre-freezing, primary drying (sublimation) and secondary drying (desorption). Pre-freezing includes reducing the temperature from 5°C to -40°C and maintaining for an appropriate time; primary drying includes raising the temperature to -5 to 0°C and maintaining for an appropriate time; secondary drying includes raising the temperature to 25°C to 30°C and maintaining for an appropriate time.

[0110] Based on the research on the preparation and formulation of the IL-17RA fusion protein according to the present invention, the present invention has developed a pharmaceutical preparation with excellent drug stability and can be safely administered by injection.

[0111] The following further explains or illustrates the content of the present invention by way of examples, but these examples should not be construed as limiting the scope of protection of the present invention.

[0112] Examples

[0113] Unless otherwise specified, the experimental methods used in the following examples are carried out using conventional experimental procedures, operations, materials and conditions in the fields of bioengineering and pharmacy.

[0114] Unless otherwise specified, the percentage concentration (%) of each reagent refers to the volume percentage concentration (%) (v / v).

[0115] Preparation Example 1: Plasmid construction

[0116] NVS451 (i.e., IL-17RA fusion protein) consists of a signal peptide, rhIL17-RA ECD (i.e., human IL17-RA extracellular domain), a linker peptide segment and an IgG1 Fc domain. The signal peptide used is the native signal peptide of IL17RA. The rhIL17-RA ECD contains six mutation sites, namely L9P, R108L, D122G, H155D, G243W and A267V (amino acid numbering does not include the signal peptide sequence). The linker peptide segment is GSG. The IgG1 Fc domain is a hybrid of IgG1, 2, and 4 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.

[0117] The plasmid was constructed using the pCHO1.0 mammalian cell expression vector from Thermo Scientific. The pCHO1.0 expression vector contains two expression cassettes. Since only one expression cassette is required for the expression of the NVS451 fusion protein, the other expression cassette was removed using the SfiI restriction site. The removed expression cassette contains EcoRV and PacI cloning sites. 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 then transformed into DH5α competent cells. The cells were plated for screening, and the clones containing the plasmid of the correct size were inoculated and amplified. A large amount of the constructed NVS451 fusion protein expression plasmid pCHO1.1-NVS4510 was obtained by plasmid extraction. Gene sequencing verified that the pCHO1.1-NVS451 expression vector was correctly constructed.

[0118] Preparation Example 2: Screening and Stability Evaluation of Cell Lines

[0119] For the construction and screening of the NVS451 fusion protein cell line, CHO-S TM (a cGMP-banked cell bank that complies with current Good Manufacturing Practice for Pharmaceuticals. This cell line and the supporting expression vector pCHO1.0 are from Thermo Scientific Kit) cells were used as the original cell substrate. 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 using a transfection reagent. The transfection process is as follows: 50 μg of the circular expression plasmid pCHO1.1-NVS451 was diluted to a volume of 1.5 ml with Opti PRO SFM, and 50 μl of Freestyle Max transfection reagent (GIBCO) was diluted to 1.5 ml with Opti PRO SFM. Then, the plasmid dilution and the transfection reagent dilution were mixed in equal volumes and added to 30 ml of a CHO-S cell suspension at 1E6 cells / ml. After culturing the transfected cells for 48 hours, puromycin and MTX were used as selection pressures for two-stage screening of stable transfected cell pools (Pools). Since untransfected CHO-S lacks pac (adenylate cyclase) activity and only has basal DHFR (dihydrofolate reductase) activity, only the CHO-S cells transfected with the pCHO1.1-NVS4510 plasmid and integrated into the genome can survive in CD FortiCHO containing puromycin and MTX TMSurvive and reproduce in the screening medium. The screening was carried out in two stages, and puromycin and MTX were used for screening in each stage. Finally, 4 stable transfection clone cell pools (Pools) were formed, namely T3S1, T3S2, T3S3, and T3S4. The expression level was evaluated by 14-day simple fed-batch culture (SFB). Combining cell-based GRO-α inhibitory activity experiments, affinity ELISA, and SPR analysis methods (using conventional methods, so not elaborated), two stable transfection clone pools, T3S1 and T3S4, were selected for the isolation of monoclonal cells (LDC). The isolation of monoclonal cells was carried out by two rounds of limited dilution to isolate monoclonal cells, and the cell seeding density in each round was less than 1 cell / well (calculated according to the mathematical calculation method of statistics). Finally, 5 candidate monoclonal cells were obtained: T3S4-7E3-6G5; T3S4-7E3-3C3; T3S4-17G2-6E2; T3S4-8F2-6E10; T3S1-18E7-6C5.

[0120] The stability of the 5 candidate monoclonal cells was evaluated for 70 PDL (cell doubling time, equivalent to 70 passages). Based on the stability of expression, gene copy number, and mRNA transcription level, monoclonal cell VAN301-T3S1-18E7-6C5 was finally selected as the master monoclonal cell (deposit number: CGMCC 21011), and VAN301-T3S4-17G2-6E2 was selected as the backup clone.

[0121] The master cell bank (PCB) was established using the monoclonal cell VAN301-T3S1-18E7-6C5, and based on this, the master cell bank (MCB) and working cell bank (WCB) under GMP conditions were established.

[0122] Preparation Example 3: Cell Culture

[0123] The NVS451 fusion protein can be produced by a 14-day fed-batch culture process. Cell resuscitation: Take a vial of cells from the cell bank and resuscitate them in a water bath at 37.0 ± 0.5 °C. Immediately after thawing, transfer the seed solution into a 50 ml centrifuge tube containing 10 ml of pre-warmed Dynamis medium (containing 8 mM L-Gln, 1:100 ACA, and 1 g / L P188). Centrifuge at 300 g for 5 minutes, discard the supernatant, and then add 10 ml of pre-warmed Dynamis medium to resuspend the cells. Transfer the resuspended cell solution to a 125 ml shake flask containing 28.0 ml of Dynamis medium, and the cell density is (0.15 - 0.35) × 10 6 cells / ml, and the cell viability is greater than 90%. Seed passage and amplification: The passage medium is Dynamis, and the initial seeding density of cells in passage culture should be (0.40 ± 0.10) × 10 6cells / mL. After culturing for 3 days, the cell density reaches (2.0 - 5.0)×10 6 cells / mL and then subculture can be carried out. During the subculture process, the cell viability should be ensured to be higher than 90.0%.

[0124] Fed-batch culture in a bioreactor: The initial seeding density of cells is (0.40 ± 0.10)×10 6 cells / mL. Feed 2×Efficient FeedC+ (Cat#: A2503101, Gibco). During the culture process, the glucose and lactate contents are detected daily. On the 3rd / 5th / 7th / 9th / 11th / 13th day, glucose mother liquor with a concentration of 450.0 g / kg is fed to make up to 5.0 g / L respectively.

[0125] Cell culture harvest conditions: Harvest when culturing to the 14th day or when the cell viability is lower than 80.0%, whichever condition is reached first.

[0126] Preparation Example 4: Purification of NVS451 fusion protein

[0127] The cell culture harvest fluid is first clarified by two-stage depth filtration membrane cartridges, then 1% Tween 80 and 0.3% tributyl phosphate are added for virus inactivation. Then, the target protein is captured using MabSelect SuRe affinity chromatography packing material from GE Healthcare. Next, two-step fine purification is carried out using Eshmuno CPX anion exchange chromatography packing material from Millipore and Capto Adhere cation exchange chromatography packing material from GE Healthcare. Then, nanofiltration is carried out using BioEX nanofiltration membrane from ASAHI KASEI, and ultrafiltration diafiltration is carried out using Pellicon 2 ultrafiltration membrane with a molecular weight cut-off of 50KDa and PES material and C-channel from Millipore. Finally, polysorbate 20 is added as an excipient, and the NVS451 bulk solution is obtained after sterile filtration.

[0128] 1. Depth filtration

[0129] Depth filtration is carried out using a series of depth filtration membranes (primary depth filtration membrane D0HC and secondary filter A1HC, from Millipore) to remove impurities such as cells and harvest the clarified solution containing the target protein. When filtering the sample, the inlet flow rate ≤ 100 LMH (based on A1HC), the maximum loading capacity of D0HC is 60 L / m 2 , and the maximum loading capacity of A1HC is 140 L / m 2 . By controlling the pressure, the single-step recovery rate of depth filtration is generally about 90%.

[0130] 2. Detergent virus inactivation

[0131] Add 50 mM Tris-HAc, 150 mM NaCl, 25% PS80, pH 7.4 to the clarified harvest solution so that the final concentration of Tween 80 is 1.0%. Add 100% tributyl phosphate to make the final concentration 0.3%. Incubate at 16 - 26 °C for 360 - 480 minutes, and then perform affinity chromatography.

[0132] 3. Affinity Chromatography

[0133] Use MabSelect SuRe packing material for affinity chromatography to achieve preliminary purification of the product. Use 50 mM Tris-HAc, 150 mM NaCl, pH 7.4 as the equilibration and post-loading wash buffer 1, then use 20 mM Tris, 1 M NaCl, 0.5 M Arg, pH 8.6 for wash 2, and finally elute the target protein with 50 mM glycine, pH 3.5 buffer. The eluate is neutralized to pH 7.8 - 8.2 with 1 M Tris base.

[0134] 4. Cation Exchange Chromatography

[0135] Use Eshmuno CPX from Millipore as the cation exchange packing material. The sample after affinity chromatography neutralization is adjusted to pH 6.3 - 6.7 with 1 M HAc, and the adjusted sample is used as the sample for cation exchange chromatography loading. Through washing and gradient elution, impurities related to the product such as HCP, Protein A, DNA, and some product analog impurities and fragments can be effectively removed.

[0136] Use 20 mM PB, pH 6.5 as the equilibration and post-loading wash buffer, 25 mM PB, 110 mM NaCl, pH 6.5 for wash 2, and for elution, use a gradient from 25 mM PB, 110 mM NaCl, pH 6.5 to 20 mM PB, 1 M NaCl, pH 6.5, 0 - 50% B (10 CV) for gradient elution.

[0137] 5. Anion Exchange Chromatography

[0138] Use Capto Adhere packing material from GE as the anion exchange packing material to remove some impurities related to the product and the process. The eluate from cation exchange chromatography is first adjusted to pH 8.4 - 8.6 and conductivity 19.0 - 23.0 with 1 M Tris base and water for injection, and the sample after adjusting pH and conductivity is used as the sample for anion exchange chromatography loading.

[0139] Use 20 mM Tris, 0.2 M NaCl, pH 8.5 as the equilibration and post-loading wash buffer, 20 mM Tris, 0.2 M NaCl, 60 mM Arg, pH 8.0 as the wash buffer 2, and elute the target protein using a 20 mM Tris, 0.2 M NaCl, 290 mM Arg, pH 7.1 buffer.

[0140] 6. Nanofiltration

[0141] The anion exchange chromatography elution sample is subjected to virus removal filtration using the Asahi Kasei BioEX nanofiltration device.

[0142] The nanofiltration process mainly utilizes the difference in molecular sizes between viruses and protein products. Potential viruses are retained by the nanofiltration membrane, while the target protein passes through, thus achieving the separation of the two. The pre-filter membrane can adsorb impurities such as particles in the loaded sample and increase the processing capacity of the nanofiltration membrane. The nanofiltration membrane can effectively retain potential viruses such as minute viruses. Use A1HC (Millipore) for pre-filtration and BioEX (Asahi Kasei) to remove potential viruses. When filtering the sample, control the pressure difference on the pre-filter membrane ≤ 2 bar and the pressure difference on the nanofiltration membrane ≤ 3 bar, and the loading of the nanofiltration membrane ≤ 600 g / m 2 .

[0143] 7. Ultrafiltration and diafiltration

[0144] The intermediate product after nanofiltration is concentrated using a Pellicon 2 (Millipore) ultrafiltration membrane cassette, and then exchanged into a 20 mM Tris-HAc, 65 mM Arg, 120 mM trehalose, pH 7.5 buffer system. The ultrafiltration membrane cassette is made of PES with a molecular weight cut-off of 50 kDa and a C-channel. The loading of ultrafiltration and diafiltration is ≤ 300 g / m 2 ; during concentration, the inlet flux is 150 - 300 LMH, the transmembrane pressure (TMP) ≤ 1.5 bar, and concentrate to a concentration of 18.0 - 22.0 g / L; then perform buffer exchange, during buffer exchange, the inlet flux is 150 - 300 LMH, TMP ≤ 2 bar, the buffer exchange volume ≥ 5 DV, and during over-concentration, the inlet flux is 150 - 300 LMH, TMP ≤ 2 bar, and over-concentrate to a concentration of 80.0 - 90.0 g / L.

[0145] 8. Excipient addition and final filtration

[0146] Add 10% (w / w) polysorbate 20 stock solution to the sample after ultrafiltration and diafiltration, and then adjust the protein concentration to 70.20 - 150.80 mg / mL with 20 mM Tris-HAc, 65 mM Arg, 120 mM trehalose, pH 7.5 buffer to prepare the stock solution. Then carry out sterile filtration using 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 excipient addition and sterile filtration is generally above 80%.

[0147] 9. Stock solution

[0148] The stock solution after excipient addition and sterile filtration is subjected to quality inspection, and the purity reaches over 97%.

[0149] Store the stock solution in a refrigerator at -40 ± 5°C.

[0150] Experimental example 1: Pharmacodynamic study

[0151] 1. In vitro pharmacodynamics

[0152] The active molecule of NVS451 is a double-stranded fusion protein composed of two parts: the extracellular domain mutant of human IL-17RA and the mutant of human IgG1 Fc, and is obtained by recombinant expression in CHO cells. Therefore, this molecule can exhibit the biological function characteristics of both IL-17RA and Fc molecules. A series of studies on the in vitro biological activity of NVS451 were carried out through analytical techniques such as surface plasmon resonance (SPR) and in vitro target cell killing assay, aiming to clarify its in vitro pharmacodynamic related properties.

[0153] 1.1 Comparative analysis of IL-17A binding affinity

[0154] NVS451 (dosage is 75 μg / ml) can bind to human IL-17A with high affinity. Surface plasmon resonance was carried out with 100 nM - 0.8 nM human IL-17A (Acrobiosystems, Cat: ILA-H5118), and the experimental method is as follows:

[0155] Reagent preparation

[0156] Coating solution: Take 1.06 g of Na 2 CO 3 and 0.84 g of NaHCO 3 Fully dissolve with ultrapure water, adjust its pH value to 9.60 with concentrated hydrochloric acid and then make up the volume to 200 ml, filter with a 0.22 μm filter membrane, and store at 4°C;

[0157] 10×TBS stock solution: Weigh 12.114 g of Tris and 43.83 g of NaCl, dissolve them in ultrapure water completely. Adjust the pH value to 7.55 with hydrochloric acid and then make up the volume to 500 ml. Filter it with a 0.22 μm filter membrane and store it at 4°C.

[0158] 4× Substrate buffer stock solution: Weigh 7.16 g of Na₂HPO₄·12H₂O and 2.1 g of citric acid, dissolve them in ultrapure water completely. Adjust the pH value to 5.5 with NaOH and then make up the volume to 100 ml. Filter it with a 0.22 μm filter membrane and store it at 4°C.

[0159] Experimental operation

[0160] Coating:

[0161] Reconstitute IL-17A with ultrapure water according to the COA, let it stand for about 30 minutes until it is fully dissolved. Dilute IL-17A to 30 nM with the coating buffer equilibrated at room temperature, and then add 100 μl to each well of the ELISA plate. Seal the plate with a sealing film and incubate it at 4°C overnight (about 16 h).

[0162] Washing the plate:

[0163] Prepare the washing solution freshly. Take 100 ml of 10×TBS stock solution, add it to 900 ml of ultrapure water to dilute it to 1×TBS, and then add 2.5 ml of 20% Tween 20. Wash the plate 4 times repeatedly with the prepared washing solution, 300 μl per well, and pat dry.

[0164] Blocking:

[0165] Prepare freshly. Weigh 2.5 g of BSA and dilute it to 50 ml with the washing solution to prepare 5% BSA blocking solution; add 300 μl of the blocking solution to the dried plate and incubate it at 37°C for 1.5 h.

[0166] Washing the plate:

[0167] Repeat the plate washing in step 2.

[0168] Adding samples:

[0169] Prepare the dilution solution freshly. Take 5 ml of the blocking solution and dilute it to 50 ml with the washing solution to prepare 0.5% BSA sample dilution solution. Dilute the sample by 2-fold serial dilution to 10 μg / ml, and add the diluted sample to the ELISA plate, 100 μl per well. Set up background controls (not coated + sample + secondary antibody, coated + no sample + secondary antibody, not coated + no sample + no secondary antibody). Seal the plate with a sealing film and incubate it at 37°C for 1 h.

[0170] Washing the plate:

[0171] Repeat the plate washing in step 2.

[0172] Secondary antibody:

[0173] The diluent is prepared immediately before use. Take 5 ml of the blocking solution and dilute it to 50 ml with the washing solution to prepare a 0.5% BSA antibody diluent. Dilute the AffiniPure Goat Anti-Human IgG and Fcγ Fragment Specific (min X Bov, Hrs, Ms Sr Prot) labeled with horseradish peroxidase to 1:12,000, add 100 μl to each well, and incubate for 1 h at 37 °C with the plate sealed with a sealing film.

[0174] Washing the plate:

[0175] Repeat step 2 to wash the plate.

[0176] Color development:

[0177] Take 12 ml of 4× substrate buffer, add it to 36 ml of ultrapure water to dilute it to 1× substrate buffer, and then add 38.5 μl of 3% H 2 O 2 and 240 μl of 20 mg / mL TMB (dissolved in DMSO), add 200 μl to each well, and incubate for 20 minutes at 37 °C.

[0178] Termination:

[0179] Add 50 μl of 1 mol / L sulfuric acid to each well in the plate after color development ends.

[0180] Reading:

[0181] Measure the absorbance (OD value) of each well at a wavelength of 450 nm and calculate its average value.

[0182] Data processing:

[0183] Use GraphPad Prism 5 to perform four-parameter fitting on the obtained OD values and calculate the EC50.

[0184] (SPR) test (the results are as Figure 3 shown), the KD is 1.8×10 -12 M, and the affinity is higher than that of secukinumab under the same conditions (the results are as Figure 4 shown) (an IL-17A monoclonal antibody drug approved for marketing abroad in 2015) and wild-type human IL-17RA (wtIL-17RA, purchased from ACROBIOSYSTEMS) (the results are as Figure 5 shown). The results of the comparative analysis of the binding affinity with IL-17A are summarized in Table 2.

[0185] Table 2. Comparative analysis of the binding affinity with IL-17A

[0186]

[0187] The IL-17RA fusion proteins with 3 mutations (V302) and 4 mutations (V303) in the extracellular domain of IL-17RA were prepared by the same method as in Preparation Examples 1-4 (the amino acid sequences are shown in SEQ ID NO.6 and SEQ ID NO.7 respectively).

[0188] The affinity of V302 and V303 for 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 methods provided by the manufacturers. The results showed ( Figure 19 ), the percentage of the affinity of NVS451 with six mutations for IL-17A (776% and 464% for the above two kits respectively) was significantly higher than that of V302 and V303 molecules for IL-17A. The affinity of V302 and V303 molecules for IL-17A was comparable to that of wild-type human IL-17RA.

[0189] 1.2 Analysis of the binding affinity of IL-17A from different species

[0190] In this study, the binding of IL-17A molecules of 4 species (human, cynomolgus monkey, mouse, rat) (KINGFISHER, catalog numbers are: RP0921H-025, RP1031Y-025, RP0355M-025; rat Biolegend, 778704) to NVS451 was compared under the same conditions. The results showed that NVS451 could bind to IL-17A of 4 different species, and the binding affinity from high to low was: human > cynomolgus monkey > rat > mouse (KD is shown in Table 3).

[0191] Table 3. Comparative analysis of the binding affinity with IL-17A from different species

[0192]

[0193] 1.3 Analysis of the binding affinity of IL-17A, C, F, AF

[0194] In addition to binding to the IL-17A molecule of the human IL-17 family, NVS451 also binds to human IL-17C, IL-17F, IL-17AF (the results are shown in Figures 6 - 8 respectively). The KD results are summarized in Table 4.

[0195] In another study, the affinities of NVS451 and wild-type human IL-17RA for IL-17C and IL-17F (Acrobiosystems, ILC-H52H7, ILF-H4240) were compared, and the results showed that the affinity of NVS451 for human IL-17C and IL-17F was higher than that of wild-type human IL-17RA (Table 5).

[0196] Table 4 Characterization of the binding affinity of NVS451 to the human IL-17 family

[0197]

[0198] Table 5 Comparison of the affinities of NVS451 and wild-type IL-17RA for IL-17C and IL-17F

[0199]

[0200] Weak binding: There is a response signal in the spectrum, but the intensity is low and the reading value cannot be quantified.

[0201] N.A.: Not applicable

[0202] 1.4 Biological activity cell experiment

[0203] The cell line CCD-1070Sk (human fibroblast epithelial cell line, ATCC CRL-2091) used for the in vitro activity detection of NVS451 belongs to the human fibroblast epithelial cell line, and there are various cytokine receptors including IL-17A, A / F, and F on its surface. IL-17A can bind to the IL-17A receptor IL-RA on the cell surface, stimulate the cell to produce the cytokine GRO-α, and its content can be accurately detected by the ELISA sandwich method. When IL-17A and NVS451 are co-incubated with CCD-1070Sk cells, NVS451 can competitively inhibit the binding of IL-17A to the IL-17A receptor IL-17RA on the cell surface by binding to IL-17A, thereby reducing the secretion of GRO-α in the cell supernatant. And NVS451 can also reduce the secretion of GRO-α in the cell supernatant by binding to IL-17A / F and IL-17F based on the same competitive inhibition mechanism. Therefore, the in vitro activity of NVS451 against multiple targets including IL-17A, IL-17A / F, and IL-17F can be determined by measuring the secretion of GRO-α by the cells.

[0204] 1.4.1 Inhibitory ability of NVS451 on GRO-α factor induced by IL-17A

[0205] The in vitro relative potency of the NVS451 fusion protein was analyzed by measuring its inhibitory ability to release GRO-α induced by IL-17A from the human fibroblast epithelial cell line CCD-1070Sk, and the results were expressed as the half inhibitory concentration IC 50 value (Table 6).

[0206] 1. The human fibroblast epithelial cell line CCD-1070Sk cells in a T75 culture flask were digested with 2 ml of trypsin containing 0.05% EDTA for about 2 - 5 minutes, then 8 ml of complete medium (EMEM basal medium with 10% FBS) was added to neutralize. After that, the cells were centrifuged at 1000 rpm for 5 minutes, resuspended with complete medium to a cell density of 1E5 / ml, and resuspended in a 96-well plate at 100 ul per well and 10000 cells per well. The cells were cultured for about 24 hours, and the drug administration experiment was started when the cell confluence reached 95% - 100%.

[0207] 2. Cell induction and drug administration: All drugs were diluted using complete medium. First, the NVS451 protein was diluted to an initial concentration of 4 ug / ml (final concentration of 1 ug / ml), then serially diluted two-fold at 9 points, and a final 0 ug / ml concentration point. 50 ul per well was added to the cell plate, and then IL-17A was diluted to a concentration of 40 ng / ml (final concentration of 10 ng / ml) and 50 ul per well was added to each reaction well. The cells were cultured with the drugs for 24 hours.

[0208] 3. After incubation for 24 hours, the plate was centrifuged at 1500 rpm for 5 minutes using a plate centrifuge, and then 100 ul of the supernatant was taken for ELISA assay. In this step, the cell supernatants of 3 replicate wells were mixed and diluted, and then added to 3 replicate wells of the ELISA plate respectively.

[0209] 4. ELISA analysis

[0210] 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, and immediately add 100 μl to each ELISA microplate well, coat the plate and incubate overnight at room temperature (24℃ ± 4).

[0211] B. Remove the liquid from each well, wash the plate 4 times with 300 μl of washing buffer per well. The last time, clean it thoroughly with absorbent paper (the washing buffer needs to be placed at room temperature one day in advance until completely dissolved).

[0212] C. Add 300 μl of blocking buffer to each well. Incubate at room temperature ((24℃ ± 4) for at least 1 hour.

[0213] D. Sample preparation:

[0214] Centrifuge the 96-well CRL-2091 cell culture plate at 1000 rpm for 5 minutes.

[0215] Add 50 μl of the pre-incubated sample (sup) to 200 μl of sample diluent (the sample is now diluted 1:5) and prepare for loading after dilution.

[0216] E. Standard preparation:

[0217] Prepare a series of standard dilutions of 1 μg / ml in diluent, with a total of 7 dilution points and a zero point.

[0218] F. Remove the liquid from each well and wash the plate 4 times with 300 μl of wash buffer per well. Thoroughly clean it with absorbent paper for the last time.

[0219] G. Immediately add 100 μL of standard or sample to each well, in triplicate. Incubate at room temperature ((24°C ± 4), 600 rpm) for 2 hours.

[0220] H. Remove the liquid from each well and wash the plate 4 times with 300 μl of wash buffer per well. Thoroughly clean it with absorbent paper for the last time.

[0221] I. The concentration of the detection antibody (biotinylated goat anti-human GROα antibody, R&D Cat#DY275-05-840256) in diluent is 1.0 μg / ml (initial concentration 100 μg / ml).

[0222] J. Add 100 μl of the diluted detection antibody to each well. Incubate at room temperature (24°C ± 4), 600 rpm) for 2 hours.

[0223] K. Remove the liquid from each well and wash the plate 4 times with 300 μl of wash buffer per well. Thoroughly clean it with absorbent paper for the last time.

[0224] L. Take 6 μL of biotin conjugate-HRP conjugate (1:2,000) and add it to a total volume of 12 ml of sample diluent (calculated for 1 96-well plate).

[0225] M. Add 100 μl to each well. Incubate at room temperature (24°C ± 4), 600 rpm) for 30 minutes.

[0226] N. Remove the liquid from each well and wash the plate 4 times with 300 μl of wash buffer per well. Thoroughly clean it with absorbent paper for the last time.

[0227] O. Add 100 μl of substrate solution to each well (the ABTS substrate was placed at room temperature 30 minutes in advance, and take out the amount needed for the current experiment). Incubate at room temperature for 20 minutes for color development.

[0228] P. Monitor the color development at 405 nm using a microplate reader, and set the wavelength calibration at 650 nm.

[0229] At the same time, detect the inhibitory ability of the currently commercially available anti-IL-17A monoclonal antibody secukinumab (Cosentyx) on GRO-α under this in vitro relative potency detection method, and the results are expressed as the half inhibitory concentration IC 50 value (Table 6). The results show that the NVS451 fusion protein has the ability to inhibit the release of GRO-α cytokine induced by IL-17A, and the IC 50 value is lower than that of secukinumab (Cosentyx) Figure 9 ).

[0230] Table 6 In vitro activity titers of NVS451 and secukinumab: IC 50 (μg / ml)

[0231]

[0232]

[0233] 1.4.2 Comparison of in vitro activity titers of NVS451 against various ligands of IL-17A, IL-17A / F, and IL-17F

[0234] Similar to the above experiment, IL-17A / F and IL-17F-induced human fibroblast epithelial cell line CCD-1070Sk can also produce GRO-α cytokine. The in vitro titer results of the NVS451 fusion protein under the induction of different ligands are expressed as the half inhibitory concentration IC 50 value (Table 7). The S curves of the inhibitory effects are shown in Figure 10 and Figure 11 .

[0235] Table 7 Detection results of NVS451 inhibiting the secretion of GRO-α by human skin fibroblasts CCD-1070Sk

[0236]

[0237] 2. In vivo pharmacodynamics

[0238] Psoriasis is a chronic inflammatory skin disease characterized by excessive proliferation and abnormal differentiation of keratinocytes. It is caused by multiple factors, and its pathogenesis has not been fully elucidated. Currently, it is believed that many immune cells and immune-related cells (including dendritic cells, T cells, endothelial cells, etc.), as well as cytokines, are involved in the pathogenesis and maintenance of the disease state of psoriasis [1]. The existing widely used animal models can be roughly divided into drug-induced acute inflammation models, genetic engineering models, and allograft models.

[0239] Imiquimod (IMQ)-induced model: IMQ is an agonist of Toll-like receptor (TLR) 7 / 8. Studies by Van der Fits [2] et al. found that the IMQ-induced changes in psoriasiform skin lesions in mice were accompanied by changes in the IL-23 / IL-17 axis, which had many similarities with the pathological changes of human psoriasis. Moreover, it had the advantages of simple operation and low cost. Therefore, establishing psoriasis in mice by applying IMQ to the skin was a psoriasis model widely used at home and abroad. However, as an acute inflammatory model, its limitations were as follows: (1) Non-specificity: Similar inflammatory manifestations were not only found in psoriasis but also in the skin lesions of other diseases such as systemic lupus erythematosus and atopic dermatitis [3]. The IMQ-induced skin inflammation model was also used in the study of systemic lupus erythematosus [4]; (2) Short treatment window period: Topical application of imiquimod could cause dehydration and weight loss in mice, and continuous topical application for more than 2 weeks might lead to mouse death [5]; (3) Poor reproducibility: Lack of standard operating procedures, etc.

[0240] Xenogeneic skin transplantation immunodeficient mouse (SCID) model: Human skin was transplanted onto SCID mice, and the T cells of the patients were injected to induce psoriasis symptoms. Its genetic phenotype and pathogenic process were closest to human psoriasis. Moreover, due to the immunodeficiency of the animals, it was not affected by anti-drug antibodies (ADA), making it one of the most suitable tools for studying the pathogenesis of psoriasis and drug development, and was most suitable for preclinical analysis of exploring new anti-psoriasis agents / therapeutic strategies before starting clinical trials [6, 7, 8].

[0241] 2.1 Pharmacodynamic evaluation of NVS451 in the xenogeneic skin transplantation SCID mouse psoriasis model (5, 10, 15 mg / kg NVS451).

[0242] The NVS451 protein at the above concentrations was formulated into a preparation prescription of 20 mM Tris-acetate, 65 mM arginine, 120 mM trehalose, 0.02% Tween 20, pH 7.5.

[0243] A total of 60 SCID mice (Envigo, C.B-17 / IcrHsd-scid-bg (beige-SCID) mice, Jerusalem, Israel) were used in the experiment. After transplanting healthy human skin onto the backs of the mice, they were randomly divided into 6 groups, with 10 mice in each group: a negative control group (the negative control group used the above-mentioned preparation prescription without NVS451), a hormone group (dexamethasone at 2 mg / animal), a secukinumab group (60 mg / kg), and high-dose (15 mg / kg), medium-dose (10 mg / kg), and low-dose (5 mg / kg) groups of the test article. The negative control group, the test article group, and the secukin group were administered subcutaneously every other day for a total of 28 days. The hormone group was topically applied with dexamethasone twice a day for a total of 28 days. At the end of the experiment, the skin appearance was observed (the results are shown in Figure 12 ), and the skin thickness was measured. The results showed that NVS451 had a certain degree of improvement effect on redness, plaques, and scales, and was positively correlated with the dose; there was no significant difference in the epidermal thickness between the 15 mg / kg dose group and the secukin group (P > 0.05). In this model, NVS451 had a treatment effect similar to that of secukin.

[0244] Table 8. Visual inspection results of xenografted skin

[0245]

[0246] Table 9. Histological evaluation and thickness of xenografted skin

[0247]

[0248] * Compared with all treatment groups, -p < 0.01 for dexamethasone

[0249] ** Compared with secukinumab, -not significant for the high-dose test article

[0250] *** Compared with the low-dose and medium-dose groups, -p < 0.05, p < 0.05 for the high-dose test article and secukinumab

[0251] 2.2 Pharmacodynamic evaluation of NVS451 (the preparation prescription described in 2.1) in a psoriasis model of SCID mice with xenogeneic skin transplantation (15, 22.5, 30, 45, 60 mg / kg NVS451)

[0252] A total of 100 SCID mice (purchased from Envigo) were used in the experiment. After transplanting healthy human skin onto the backs of the mice, they were randomly divided into 10 groups (see Table 10). At the end of the experiment, the skin appearance was observed (the results are shown in Figure 13 ), and the transplanted skin was taken for histological evaluation related to psoriasis (the results are shown in Figure 14), and the skin thickness was measured. The results showed that all groups in the NVS451 group exhibited therapeutic effects. Among them, the groups with 15 mg / kg twice a week, 30 mg / kg twice a week, 22.5 mg / kg twice a week, and 60 mg / kg once a week had similar efficacy, with 7 / 10 of the xenografted skin completely recovered and the epidermal thickness reduced. However, in the group with 15 mg / kg twice a week, the remaining 3 / 10 showed partial recovery compared with other treatment groups, with a skin thickness of 334 ± 174 μm and a histological score of 1.1 (comprehensively evaluated based on indicators such as the epidermal thickness of the skin lesion, thinning of the supra-papillary epidermis, hyperkeratosis, parakeratosis, granulocytopenia, Munro microabscess, normal or abnormal elongation of the rete ridges, vascular tortuosity, and mononuclear cell infiltration in the dermal papillary layer).

[0253] Table 10. Study groups and doses (the excipient control group is the preparation formula without NVS451)

[0254]

[0255] Table 11. Histological evaluation and thickness of xenografted skin

[0256]

[0257] 3. Secondary pharmacodynamics

[0258] 3.1 Analysis of binding affinity of ADCC / CDC effector-related receptors

[0259] The mechanism of action of NVS451 to exert its pharmacodynamic effects does not depend on ADCC / CDC activity, and the cytotoxic effects of Fc ADCC / CDC may bring unnecessary immune-related adverse events (irAE), presenting potential safety risks [9]. To avoid this effect, the relevant active sites of the Fc part of NVS451 were mutated. In the affinity analysis study, 7 CD molecules related to ADCC and the C1q molecule related to CDC were evaluated. The results showed that the binding affinity of NVS451 (75 μg / ml) with the CD molecules related to ADCC was at a non-binding or low level (KD greater than 10 -5 M order of magnitude, see Table 12). In another study, compared with the IL-17AR fusion protein with natural IgG1 Fc (V301-wtFc) (75 μg / ml) which was the same in other parts, the affinity of NVS451 with the Fc receptors related to ADCC / CDC was significantly reduced (see Table 13).

[0260] The amino acid sequence of V301-wtFc is shown in SEQ ID NO.17.

[0261] Table 12. Affinity analysis of NVS451 with ADCC and CDC effector-related receptors

[0262]

[0263] Table 13 Analysis of the Affinities of NVS451 and V301-wtFc with Receptors Related to ADCC and CDC Effects

[0264]

[0265]

[0266] 3.2 Evaluation of the ADCC / CDC Functional Activities of the Fc Fragment Region

[0267] The Fab regions of IgG1 and IgG3 subtype antibodies first bind to target cells, and then their Fc parts bind to Fc receptors (such as FcγRIII) on CTL cells such as NK, and then ADCC can be induced. A cell model lacking membrane-expressed IL-17 cannot effectively verify whether ADCC is produced in vitro. Therefore, the Fab segment of rituximab, which can stimulate ADCC, was fused with the Fc segment of NVS451 to construct RitxV301 (i.e., RitxNVS451) to verify the chimeric molecule (the amino acid sequence of the light chain is shown in SEQ ID NO.18, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.19). This was used to verify the removal of ADCC / CDC effects (the experimental data was analyzed using GraphPad Prism 5 analysis software. The logarithm of the antibody concentration was used as the x-axis, and the corresponding calculated killing rate value was used as the y-axis. A four-parameter equation regression simulation was selected to fit the dose-effect curve of the antibody. The obtained data was analyzed using GraphPad Prism5 software. The positive control antibody (rituximab) conforms to the four-parameter equation y = D + (A - D) / [1 + (X / C)^B], showing a typical S-shaped curve on the semi-logarithmic coordinate axis, with R2≥0.95. The EC50 was calculated, and the ADCC and CDC effects were judged based on the EC50 value)). The research results show ( Figure 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 design of the Fc region of NVS451 makes RitxV301 unable to induce NK-92MI CD16a cells to produce killing of Raji cells, and the Fc region of NVS451 cannot bind to the C1q molecule in complement and cannot initiate the complement cascade reaction.

[0268] 3.3 Analysis of the Binding Affinity of FcRn

[0269] Fc fusion proteins generally increase the half-life of target protein drugs and improve pharmacokinetic characteristics through FcRn-mediated recycling of Fc

[10] . The binding affinity to FcRn is related to the in vivo half-life of the drug

[11] . NVS451 can bind to FcRn of human, monkey, and rat (acrobiosystems FCM-H5286, FCM-C5284, FCM-R5287). Under the same conditions, its affinity is at the same level as that of secukinumab and wild-type IgG1 Fc (i.e., V301-wtFc) (the order of magnitude of KD reaches 10 -8 M, see Table 14), and the experimental method is as follows:.

[0270] Reagent preparation

[0271] 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, pH adjusted to 6.0;

[0272] His capture kit (product number: 28-9950-56, GE), which includes: mouse anti-His antibody (1 mg / mL), immobilization reagent (10 mM sodium acetate, pH 4.5), regeneration reagent (glycine hydrochloride, pH 1.5);

[0273] Amino coupling kit (product number: BR100050, GE), which includes: 115 mg N-hydroxysuccinimide (NHS), 750 mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and 10.5 mL 1 M ethanolamine (pH 8.5). Add each tube of EDC and NHS to 10 mL of deionized water respectively, aliquot and store at -18 °C or lower temperature, with a shelf life of two months. (Refer to the GE amino coupling instruction manual "22-0510-62AG").

[0274] Dissolve the IL-17RA-wtFc (i.e., V301-wtFc) protein and FcRn proteins of different species according to the product COA, and aliquot in specifications of more than 10 μg per tube. Avoid repeated freezing and thawing.

[0275] Protein desalting

[0276] Use a desalting column and running buffer to desalt secukinumab and IL-17RA-wtFc proteins. The concentration of the desalted protein is measured by UV-V. The desalted protein is aliquoted in specifications of more than 10 μg per tube and avoid repeated freezing and thawing.

[0277] Chip Preparation

[0278] Dilute the mouse anti-His antibody with a fixation reagent (10 mM sodium acetate, pH 4.5) to 50 μg / mL. Approximately 100 μL of the mouse anti-His antibody is used for each channel of the chip, and about 190 μL of the fixation reagent is added to 10 μL of the mouse anti-His antibody for fixing two channels. First, the surface of the CM5 chip (biocore sensor chip, GE) is activated with 400 mM EDC and 100 mM NHS at a flow rate of 10 μL / min for 420 s. Second, the 50 μg / mL mouse anti-His antibody is injected into the experimental channel (FC4) at a flow rate of 10 μL / min for about 420 s, and the fixed amount is about 9000 to 14000 RU. Finally, the chip is blocked with 1 M ethanolamine at a flow rate of 10 μL / min for 420 s. The reference channel (FC3) is operated in the same way as the test channel (FC4). (Refer to the instruction manual of the His Capture Kit of GE, 《28-9974-71AB》).

[0279] Capture Ligand

[0280] Dilute the FcRn stock solution sample with the running reagent to 0.5 μg / mL and inject it into the experimental channel (FC4) at a flow rate of 10 μL / min for about 40 RU. The reference channel (FC3) does not need to capture the ligand.

[0281] Multi-Cycle Analysis of Analytes

[0282] Dilute the samples of different concentrations of NVS451, secukinumab, and IL-17RA-wtFc with the corresponding running reagents, and inject the diluted samples into the experimental channel and the reference channel at a flow rate of 30 μL / min in sequence according to the corresponding binding time and dissociation time. After analyzing each concentration, the chip needs to be regenerated with glycine hydrochloride with a pH of 1.5 at a flow rate of 30 μL / min for 60 s to wash away the ligand and the undissociated analyte. When performing the next concentration analysis, the experimental channel needs to recapture the same amount of ligand.

[0283] Others

[0284] All operation steps are carried out in the running reagent, and all SPR analysis reagents need to be filtered and degassed before use.

[0285] Results

[0286] Data Analysis:

[0287] Use the Biacore 8K analysis software to calculate the KD value of each antibody. The reference channel (FC3) is used for background subtraction.

[0288] In 4 batches of independent test studies, NVS451 (concentration: 25 nM - 1.5625 nM) can bind to FcRn of three different species, namely human, cynomolgus monkey, and rat, under acidic conditions with a pH of 6.0 ( Figure 17 ); and it does not bind under neutral conditions with a pH of 7.4 (NVS451 concentration: 100 nM - 1.5625 nM) ( Figure 18 ).

[0289] Table 14 Comparison of FcRn receptor affinities among human, rat, and cynomolgus monkey

[0290]

[0291] Experimental Example 2: Safety evaluation

[0292] 1. Safety pharmacology

[0293] 1.1 Safety pharmacology test in cynomolgus monkeys

[0294] The safety pharmacology test in cynomolgus monkeys (purchased from Beijing Zhongke Lingrui Biotechnology Co., Ltd.) was carried out concomitantly with the long-term toxicity test. The prescription in Section 2.1 of Experimental Example 1 was subcutaneously injected, and the doses of NVS451 were 15 mg / kg, 50 mg / kg, and 150 mg / kg (administered twice a week for 4 consecutive weeks, a total of 9 administrations, i.e., administered on D1, D4, D8, D11, D15, D18, D22, D25, and D29 respectively). In this test, a vehicle control group was set up, using the prescription in Section 2.1 of Experimental Example 1 without NVS451, and the safety pharmacology indicators were detected in combination with the observation of toxicity indicators. The mental state and behavioral activities of the animals in each group were normal; the animals breathed smoothly, without rapid or slow breathing, and no abnormal thoracic or abdominal breathing was observed. Respiratory indicators and electrocardiograms of the animals in each group were examined at different time points (before the first administration (D - 2), and 4 h (±20 minutes), 24 h (±20 minutes), 48 h (±20 minutes), 72 h (±20 minutes) after the first administration). No TV and RR related to drug administration, as well as regular changes in heart rate, P - R interval, QT interval, QRS duration, QRS voltage, STe, and QTcB values, were observed, and the electrocardiogram waveform was normal. Electrocardiogram limb lead II was measured before the first dose (D - 2), 3 - 5 hours after the first dose (D1), 3 - 5 hours after the fifth dose (D15), 3 - 5 hours after the last dose (D29), and before the end of the recovery period (D56) to observe heart rate, electrocardiogram waveform, P - R interval, QRS duration, and Q - T interval, and no abnormalities were found. Blood pressure measurements at each time point, including MBP, DBP, and SBP, showed no obvious abnormal changes. Therefore, it is considered that subcutaneous injection of the test article at doses of 15, 30, and 150 mg / kg has no obvious effect on the central nervous system, cardiovascular system, and respiratory system of cynomolgus monkeys.

[0295] 1.2 Central Nervous System Tests in Rats

[0296] Under GLP (Good Laboratory Practice) conditions, the effects of a single subcutaneous injection of NVS451 on the central nervous system function of SD rats (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Animal Production License: SCXK (Beijing))) were evaluated by the Functional Observation Battery (FOB) method at doses of 30 mg / kg (low dose), 100 mg / kg (medium dose), and 300 mg / kg (high dose). An increase in body temperature was observed in male animals in the high-dose group of the test article 2 hours after dosing (vehicle control group (i.e., the formulation prescription without NVS451) vs high-dose group: 37.72 ± 0.29°C vs 38.40 ± 0.14°C); an increase in body temperature was observed in female animals in the high-, medium-, and low-dose groups of the test article 24 hours after dosing, vehicle control group vs high-, medium-, and low-dose groups: 37.60 ± 0.28°C vs 38.80 ± 0.23, 38.72 ± 0.44, 38.54 ± 0.28°C). The changes in body temperature were not consistent between males and females and showed no regular pattern, and it was considered not related to drug administration. No abnormal manifestations related to drug administration were observed in the animals during the in-cage observation, hand-holding observation, open-field observation, and irritability response observation; no changes in the number of animals standing upright, number of fecal pellets, forelimb grip strength, and body temperature related to the test article were observed. The results showed that under the conditions of this test, NVS451 had no effect on the central nervous system function of rats.

[0297] 2. Pharmacokinetics

[0298] 2.1 Pharmacokinetics in Cynomolgus Monkeys

[0299] A total of 24 cynomolgus monkeys were used in the experiment and divided into 4 groups (6 animals in each group, with an equal number of males and females). A single subcutaneous and intravenous injection was administered, and groups 1 to 4 were given 5 (subcutaneous injection), 15 (subcutaneous injection), 50 (subcutaneous injection), and 15 (intravenous injection) mg / kg of NVS451, respectively, with a dosing volume of 1 mL / kg.

[0300] Pharmacokinetic blood samples (about 1 mL) were collected from the non-dosing site of the subcutaneous vein in the hind limb of the animals into tubes without anticoagulant. The blood sampling time points for animals in groups 1 - 3 were: before dosing (the previous day), 1 h, 2 h, 4 h, 8 h, 24 h (D2), 32 h (D2), 48 h (D3), 56 h (D3), 72 h (D4), 96 h (D5) after the start of dosing; the blood sampling time points for animals in group 4 were: before dosing (the previous day), 3 minutes, 1 h, 2 h, 6 h, 24 h (D2), 32 h (D2), 48 h (D3), 56 h (D3), 72 h (D4), 96 h (D5) after the start of dosing. The blood samples were used to prepare serum samples and for pharmacokinetic analysis (Table 15).

[0301] Table 15. Pharmacokinetic Parameters of NVS451 in Cynomolgus Monkeys

[0302]

[0303]

[0304] C max Ratio = C max Mean of Medium and High Doses / C max Mean of Low Dose;

[0305] AUC Ratio = AUC last Mean of Medium and High Doses / AUC last Mean of Low Dose;

[0306] F% = (AUC last / Dose (SC)) / (AUC last / Dose (IV)) * 100%.

[0307] The test results showed that: in the dose range of 5 - 50 mg / kg, after a single subcutaneous injection to cynomolgus monkeys, the blood drug concentration of NVS451 in cynomolgus monkeys increased with the increase of the dose; after a single subcutaneous injection and intravenous injection to cynomolgus monkeys, no significant difference was observed between genders.

[0308] After NVS451 was given to cynomolgus monkeys by single subcutaneous injection at doses of 5, 15, and 50 mg / kg, the average C max and AUC last increased at a higher proportion than the dose increase. The C max ratios of NVS451 in the animals of the 5, 15, and 50 mg / kg dose groups given by single subcutaneous injection were 1:6.12:38.06 (male) and 1:5.46:34.29 (female); the AUC last ratios were 1:4.83:25.52 (male) and 1:4.76:20.88 (female), respectively.

[0309] Independent sample t - test showed that there were no statistically significant differences in the metabolic kinetic parameters between genders in each subcutaneous injection and intravenous injection group, and there were basically no obvious gender differences in the metabolic characteristics of NVS451 in animals.

[0310] In 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 T max of each subcutaneous injection dose group was basically the same, and the drug concentration reached the peak at 4 - 8 h.

[0311] 2.2 Kinetics in Rats

[0312] A total of 48 Sprague-Dawley rats were used in the experiment and divided into 4 groups (12 animals in each group, with an equal number of males and females). Single subcutaneous and intravenous injections were administered, and groups 1 to 4 were given NVS451 at doses of 10 (subcutaneous injection), 30 (subcutaneous injection), 100 (subcutaneous injection), and 30 (intravenous injection) mg / kg, respectively, with a dosing volume of 2 mL / kg.

[0313] In the experiment, pharmacokinetic blood samples (about 0.4 mL) were collected from the jugular vein of the animals into tubes without anticoagulant. The blood sampling time points for the animals in groups 1 - 3 were: before dosing (D - 1), 1 h, 2 h, 4 h, 8 h, 24 h, 32 h, 48 h, 56 h, 72 h, and 96 h after the start of dosing; the blood sampling time points for the animals in group 4 were: before dosing (D - 1), 3 min, 1 h, 2 h, 6 h, 24 h, 32 h, 48 h, 56 h, 72 h, and 96 h after the start of dosing. The blood samples were used to prepare serum samples and for pharmacokinetic analysis (Table 16).

[0314] Table 16. Pharmacokinetic parameter table of NVS451 in Sprague-Dawley rats

[0315]

[0316] C max Ratio = C max Mean of medium and high doses / C max Mean of low dose;

[0317] AUC ratio = AUC last Mean of medium and high doses / AUC last Mean of low dose;

[0318] F% = (AUC last / dose (SC)) / (AUC last / dose (IV)) * 100%.

[0319] The experimental results showed that: within the dose range of 10 - 100 mg / kg, after single subcutaneous injection in Sprague-Dawley rats, the blood drug concentration of NVS451 in Sprague-Dawley rats increased with the increase of the dose; after single subcutaneous and intravenous injections in Sprague-Dawley rats, no significant differences were observed between genders.

[0320] After single subcutaneous injection of NVS451 to SD rats at doses of 10, 30, and 100 mg / kg, the growth ratios of the average Cmax and AUClast of NVS451 were lower than the dose growth ratio. The Cmax ratios of NVS451 in the animals of the 10, 30, and 100 mg / kg dose groups after single subcutaneous injection were 1:2.03:6.56 (male) and 1:1.62:6.04 (female); the AUClast ratios were 1:1.92:5.29 (male) and 1:1.85:5.85 (female).

[0321] There were no significant differences in the pharmacokinetic parameters between genders in the subcutaneous injection and intravenous injection groups, and there were basically no significant gender differences in the metabolic characteristics of NVS451 in animals.

[0322] In the dose range of 10 - 100 mg / kg, the T1 / 2 of NVS451 in SD rats was between 51.46 - 69.48 for male and female animals in each dose group; the Tmax of each subcutaneous injection dose group was basically the same, and the drug concentration reached the peak at 8 - 24 h.

[0323] 3. Distribution and Excretion

[0324] In this experiment, the tissue distribution and excretion characteristics of NVS451 fusion protein (NVS451) for injection labeled with isotope (125I) after subcutaneous injection to SD rats were studied mainly by the method of TCA precipitation of proteins combined with SHPLC (molecular exclusion high performance chromatography).

[0325] A total of 36 animals were used in the experiment, divided into 6 groups with 6 animals in each group, with half male and half female. The test article was administered by single subcutaneous injection at the nape of the neck at a dose of 30 mg / kg. The radiochemical purity of the test article after 125I labeling was 99.03%, and the specific activity was 0.09 KBq / μg. Animals in groups 1 - 4 were sacrificed at 4 h, 24 h, 96 h, and 120 h after dosing to collect thyroid, heart, lung, liver, spleen, kidney, bladder, gonads (ovary, testis), stomach, small intestine, fat, muscle, brain, back skin (non-dosing site), intestinal contents, serum, and urine. Animals in group 5 collected feces and urine once a day after dosing for a total of 5 days. Animals in group 6 collected bile excreted once per hour after injection for a total of 8 h.

[0326] The test results showed that:

[0327] 1) After subcutaneous injection of 125I-NVS451 to rats, the drug was mainly distributed in tissues / organs such as intestinal contents, urine, bladder, and back skin, and less distributed in muscle, fat, and brain. The peak time of the drug in most tissues was between 4 h and 24 h, and then the concentration gradually decreased with the extension of time.

[0328] The drug was distributed to a certain extent in the back skin (non-dosing site). The peak time was 24 h, and the drug contents at 4 h and 120 h were approximately equal. Compared with other tissues / organs, the drug concentration in the back skin decreased relatively slowly.

[0329] 2) After subcutaneous injection of 125I-NVS451 in rats, the radioactivity was mainly excreted through the kidneys, and more than 90% of the administered drug was excreted after 5 days; the biliary excretion was less.

[0330] 4. Metabolism

[0331] According to the guiding principle of "ICH-S6(R1) Preclinical Safety Evaluation of Biologics", NVS451 is a macromolecular protein drug, and it is expected to be degraded into peptides and amino acids in vivo and then excreted or reused for the synthesis of proteins or peptides in the body. Therefore, the metabolism of NVS451 was not evaluated.

[0332] 5. Toxicology

[0333] Cynomolgus monkeys and SD rats were selected for toxicology studies. NVS451 had a cross-reaction with cynomolgus monkey and rat IL-17A, which were related species. The tests were all carried out under GLP conditions, following the current Good Laboratory Practice of the Food and Drug Administration (21 CFR Part 58) and the "Good Laboratory Practice for Non-clinical Research of Drugs" (Order No. 34 of the Bureau, September 2017) of the National Medical Products Administration (formerly the China Food and Drug Administration).

[0334] 6. Single-dose Toxicity

[0335] 6.1 Toxicity Study in Cynomolgus Monkeys

[0336] The purpose was to evaluate the possible acute toxicity reactions after single subcutaneous injection of NVS451 in cynomolgus monkeys within 14 days of observation, and three dose groups of 45 mg / kg, 150 mg / kg, and 450 mg / kg were set. During the test period, no death or near death was observed in each group of animals. No abnormalities were observed in the clinical observation of each dosing group of animals. Compared with their own pre-drug values and the excipient control group (the formulation prescription without NVS451), no drug-related abnormal changes were found in the body weight, body temperature, electrocardiogram parameters, blood cell count, coagulation index, blood biochemistry, and urine examination of each dosing group of animals. At the end of the observation period (D15), no abnormalities were found in the gross anatomy of all animals. Conclusion: Under the conditions of this test, no death or near death was observed in all animals, no drug-related abnormal changes were found, and the maximum tolerated dose (MTD) was greater than or equal to 450 mg / kg.

[0337] 6.2 Toxicity Study in Rats

[0338] The purpose was to evaluate the acute toxicity reactions that might occur within 14 days after a single subcutaneous injection of NVS451 to SD rats, and three dose groups of 90 mg / kg, 300 mg / kg, and 900 mg / kg were set up. During the test period, no death or near death was observed in the animals of each group. At the end of the observation period (D15), no abnormal changes were observed in the general observation of the animals in each group. Conclusion: Under the conditions of this test, no death or near death was observed in all animals, and the maximum tolerated dose (MTD) of the animals was greater than or equal to 900 mg / kg.

[0339] 7. Repeated-dose toxicity

[0340] 7.1 Repeated-dose toxicity study in cynomolgus monkeys

[0341] Forty cynomolgus monkeys (20 of each sex) were used and randomly divided into 4 groups (5 of each sex / group) according to sex, namely the excipient control group and the low, medium, and high dose groups of the test article. The animals in the excipient control group were given the excipient control product, 2 mL / kg, and the low, medium, and high dose groups of the test article were given NVS451. The dosing doses were 15, 50, and 150 mg / kg respectively, the dosing volumes were 0.2, 0.67, and 2 mL / kg respectively, and the dosing concentration was 75 mg / mL. All animals were administered by subcutaneous injection into the hind limb, dosed 2 times a week for 4 consecutive weeks, a total of 9 times, that is, on D1, D4, D8, D11, D15, D18, D22, D25, and D29. During the test period, the animals were subjected to clinical observation, body weight, body temperature, electrocardiogram, respiration (respiratory rate and tidal volume), blood pressure, ophthalmic examination, blood cell count, coagulation function, blood biochemistry, urine analysis, and immunological index (T lymphocyte subsets, cytokines, C-reactive protein, serum immunoglobulins, complement, drug antibodies) index detection; before and after the first and 8th dosing, blood samples were collected from the animals in each group for blood drug concentration determination. On the day after the last dose (D30), euthanasia was performed on some animals (3 of each sex / group), and on the 4th week of the recovery period (D57), euthanasia was performed on the remaining animals; all animals were subjected to bone marrow smear and reading, gross dissection observation, weighing of major organs, calculation of relative organ weights (viscerosomatic ratio and viscerobrain ratio), and histopathological examination of more than 40 kinds of tissues and organs.

[0342] Results: During the test period, no death or near death was observed in the animals of each group, and no abnormal changes related to dosing were observed in the clinical observation, body weight, body temperature, ophthalmic examination, urine examination, blood cell count, coagulation function (except FIB), blood biochemical indexes, lymphocyte subsets, complement, cytokines (except IL-17A), and immunoglobulins of the animals in each group. No abnormal changes related to dosing were observed in the electrocardiogram, respiration, and blood pressure parameters of the animals. No abnormal reactions such as erythema, edema, induration, and ulceration were observed in the local area of administration by naked eye observation.

[0343] During the test period, compared with the same-sex vehicle control group in the same period, on the day after the fifth dose (D16), an increase in CRP (193.0%) was observed in male animals in the high-dose group of the test article; on the day after the last dose (D30), an increase in CRP (70.5%, 218.5%, 246.9%) was observed in male animals in the low-, medium-, and high-dose groups of the test article, and an increase in FIB (56.4%) was also observed in male animals in the high-dose group of the test article. The differences were statistically significant (P≤0.05). After 4 weeks of drug withdrawal, the above changes all fully recovered.

[0344] 7.1.1 Histopathological examination:

[0345] No animal deaths or near-death conditions were found during the test process.

[0346] After the last dose (D30), test article-related changes were observed in the injection site and inguinal lymph nodes of animals in the 15, 50, and 150 mg / kg dose groups. Among them, the injection site showed mild to moderate mononuclear cell inflammation in the dermis / subcutaneous / muscular layer, which was an irritant reaction caused by the test article; there was a mild to moderate increase in the number of lymphocytes in the paracortical area / medullary cord of the inguinal lymph nodes, which was related to the mononuclear cell inflammation at the injection site. Considering that there were no obvious changes in the relevant clinical pathology indicators, it was considered a non-adverse reaction.

[0347] At the end of the 4-week recovery period (D57), the test article-related changes at the injection site in the 15 and 50 mg / kg dose groups had fully recovered, and the test article-related changes at the injection site in the 150 mg / kg dose group had basically recovered.

[0348] 7.2 Repeated-dose toxicity study in rats

[0349] 192 SD rats, half male and half female, were randomly divided into 8 groups according to body weight in gender segments. 120 rats in the main test groups of groups 1-4 were used for toxicological studies (15 rats / sex / group), and 72 rats in the satellite groups of groups 5-8 were used for serum antibody and toxicokinetic detection (6-10 rats / sex / group). During the test period, animals in the vehicle control group were given the NVS451 white vehicle solution (4 mL / kg); the low-, medium-, and high-dose groups of the test article were given NVS451 at doses of 30, 100, and 300 mg / kg, respectively, and the dosing volumes were 0.4, 1.33, and 4 mL / kg, respectively. All animals were administered by subcutaneous injection in the nape of the neck, dosed twice a week for 4 consecutive weeks, for a total of 9 doses (i.e., dosed on D1, D4, D8, D11, D15, D18, D22, D25, and D29), and a 4-week recovery period.

[0350] During the experiment, the animals in the main experimental group were mainly under clinical observation, and their 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 bled before the first and the 8th drug administrations for toxicokinetics detection, and were bled at different time points (before the first drug administration (D-1), before the 15th drug administration (D14), before the last drug administration (D28), and at the end of the recovery period (D56)) for antibody determination. On the day after the last drug administration (D30), the first 10 animals / sex / group in the main experimental group were euthanized, and the remaining animals were euthanized at the end of 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 weights were calculated; and more than 40 kinds of tissues and organs of the animals in the excipient control group and the high-dose group, and the injection sites of the animals in the low- and medium-dose groups were subjected to histopathological examination.

[0351] Results: During the experiment, no death or near-death was observed in the animals of each group, and no abnormality was found in the clinical observation. No erythema, congestion, swelling, ulcer and induration were observed by naked-eye observation at the drug administration site. No abnormality was found in the ophthalmological examination and urine examination of the animals in each group; compared with the excipient control group of the same sex in the same period, no drug-related abnormal changes were found in the body weight, body temperature, food intake, blood cell count, coagulation function, blood biochemistry, and T lymphocyte subsets of the animals in each group.

[0352] 7.2.1 Histopathological examination:

[0353] No death occurred in the animals during this experiment.

[0354] No change related to the test article was found in the gross observation of the euthanized animals.

[0355] In the animals euthanized at the end of drug administration (D30), irritant reactions related to the test article were observed at the injection sites in the 30, 100 and 300 mg / kg dose groups, manifested as mild to moderate mononuclear cell inflammation in the subcutaneous and / or dermis at the injection sites. By the end of the 4-week recovery period, the inflammation at the injection sites had basically completely recovered.

[0356] 8. Local tolerance toxicity

[0357] The local tolerance toxicity test was carried out concomitantly with the long-term toxicity test. Both cynomolgus monkeys and SD rats were administered the drug twice a week (three dose groups for rats: 30, 100 and 300 mg / kg, three dose groups for cynomolgus monkeys: 15, 50 and 150 mg / kg), and the drug was administered continuously for 4 weeks, a total of 9 times (i.e., administered on D1, D4, D8, D11, D15, D18, D22, D25 and D29). During the experiment, no abnormal reactions such as erythema, edema, induration, ulceration, etc. were observed at the drug administration sites of the animals in each group, and the local tolerance was good and no toxicity was found.

[0358] 9. Other toxicities

[0359] Using the in vitro test tube method, the effects of 75 mg / mL NVS451 on human red blood cell hemolysis and aggregation were observed (the experimental conditions were conventional conditions). From 15 minutes after incubation in the incubator until the end of the 3-hour observation, the upper layer of the test tube in the test sample tube was colorless and clear, and the red blood cells settled to the bottom of the tube. After shaking, they were evenly dispersed, and no hemolysis or aggregation occurred. Under the conditions of this experiment, 75 mg / mL of NVS451 had no hemolytic effect on human red blood cells in vitro and did not cause human red blood cell aggregation.

[0360] Conclusion

[0361] 1. Comprehensive evaluation and conclusion of pharmacodynamics

[0362] In vitro pharmacodynamics: Comparative analysis of the binding affinity with IL-17A showed that NVS451 could bind to human IL-17A with high affinity, and the affinity was higher than that of secukinumab and wild-type human IL-17RA under the same conditions. In addition to binding to the IL-17A molecule of the human IL-17 family, NVS451 also bound to IL-17C, IL-17F, and IL-17AF. The affinity of NVS451 for human IL-17C and IL-17F was higher than that of wild-type human IL-17RA. Biological activity cell experiments showed that the NVS451 fusion protein showed the ability to inhibit the GRO-α cytokines induced by IL-17A, IL-17A / F, and IL-17F, and the inhibitory ability of the GRO-α cytokine induced by IL-17A was lower than the IC 50 value of secukinumab (Cosentyx).

[0363] Based on these in vitro potency results, NVS451 mainly exerts its effect by inhibiting the activities of IL-17A and IL-17C, IL-17F, and IL-17AF. The interactions shown by SPR experiments suggest that in vivo, the recommended dose of NVS451 mainly inhibits IL-17A in psoriasis patients, supplemented by inhibiting the clinical activities of IL-17C, IL-17F, and IL-17AF molecules.

[0364] Affinity analysis studies have shown that the binding affinity of NVS451 to CD molecules related to ADCC is at a non-binding or low level. Compared with natural IgG1 Fc, its binding affinity to Fc receptors related to ADCC / CDC is significantly reduced, demonstrating that the ADCC binding site of NVS451 has been mutated and removed. The binding affinity analysis with FcRn shows that NVS451 can bind to FcRn of human, monkey, and rat. Under the same conditions, its affinity is at the same level as that of secukinumab and wild-type IgG1 Fc. Under acidic conditions of pH 6.0, NVS451 can bind to FcRn of three different species, namely human, cynomolgus monkey, and rat, and does not bind under neutral conditions of pH 7.4. NVS451 is a soluble antibody Fc fusion protein carrying the human IgG1 Fc domain. Therefore, theoretically, it can interact with Fc receptors. However, the mechanism of action of NVS451 to exert its pharmacological effects does not depend on ADCC / CDC activity, and the cytotoxic effects of Fc, ADCC / CDC, may bring unnecessary immune-related side effects, presenting potential safety risks. To avoid this effect, the relevant active sites of the Fc part of NVS451 have been mutated. Studies have confirmed that compared with natural IgG1 Fc, its binding affinity to Fc receptors related to ADCC / CDC / ADCP is significantly reduced, and the target cell killing experiment (such as Figure 15 and 16 shown) confirmed that NVS451 has no ADCC / CDC activity.

[0365] In vivo pharmacodynamics: In terms of in vivo function, the efficacy of NVS451 against psoriasis was mainly evaluated in a human T cell-driven psoriasis model. Studies have confirmed that the human skin xenograft model is most suitable for preclinical analysis of exploring new anti-psoriatic agents / therapeutic strategies before the start of clinical trials. Through histological evaluation of human skin in the xenograft SCID mouse model, including scoring and epidermal thickness, the efficacy of NVS451 in treating the humanized psoriasis model was shown. In the psoriasis model of xenogeneic skin transplantation in SCID mice, after NVS451 was administered at doses of 5 mg / kg, 10 mg / kg, and 15 mg / kg every other day for 28 days, the results showed that NVS451 had a certain degree of improvement effect on erythema, plaques, and scales, and was positively correlated with the dose; there was no significant difference in epidermal thickness between the 15 mg / kg dose group and the secukinumab group (P>0.05). Treating once a week with the highest dose of NVS451 (60 mg / kg) was as effective as treating twice a week with a low dose. Most importantly, treating twice a week with NVS451 or treating once a week with a high dose showed a therapeutic effect similar to the positive control of secukinumab. Secukinumab is considered to be one of the most promising drugs for treating psoriasis. This study provides strong clinical evidence support for the effectiveness of clinical treatment.

[0366] Safety Pharmacology: Under GLP conditions, NVS451 was administered subcutaneously at single doses of 30 mg / kg, 100 mg / kg, and 300 mg / kg, and the results showed that NVS451 had no effect on the central nervous system function of rats. The safety pharmacology test in cynomolgus monkeys was conducted concomitantly with the long-term toxicity test: The test article was administered subcutaneously at doses of 15, 30, and 150 mg / kg, and no obvious effects on the central nervous system, cardiovascular system, and respiratory system of cynomolgus monkeys were observed.

[0367] 2. Comprehensive Evaluation and Conclusion of Pharmacokinetics and Toxicology

[0368] Since NVS451 cross-reacts with cynomolgus monkey and rodent IL-17A, cynomolgus monkeys and rats were selected as relevant species for toxicity evaluation. NVS451 was repeatedly administered subcutaneously to cynomolgus monkeys at doses of 15, 50, and 150 mg / kg, twice a week for 4 weeks, for a total of 9 administrations. No deaths or near-deaths were observed in each group of animals, and no obvious systemic toxic reactions were seen. The no-observed-adverse-effect level (NOAEL) of this test was considered to be 150 mg / kg. The NVS451 fusion protein for injection was repeatedly administered subcutaneously to SD rats at doses of 30, 100, and 300 mg / kg, twice a week for 4 weeks, for a total of 9 administrations. No obvious systemic toxic reactions were observed in the animals, and the no-observed-adverse-effect level (NOAEL) was considered to be 300 mg / kg. The toxicokinetics of cynomolgus monkeys and rats showed that there was no accumulation of NVS451 after repeated administration.

[0369] Single-dose non-GLP pharmacokinetic tests were conducted in cynomolgus monkeys and rats to observe the basic pharmacokinetics of NVS451 after intravenous and subcutaneous administration. The absorption pharmacokinetics of NVS451 in male and female cynomolgus monkeys after single subcutaneous administration were evaluated. Its pharmacokinetic behavior had the low serum clearance rate and half-life typical of immunoglobulin molecules, and effective drugs could be detected in each dose group 96 hours after subcutaneous administration. In the single-dose pharmacokinetic tests of cynomolgus monkeys and SD rats, there were no obvious gender differences in the metabolic data. In the dose range of 5 - 50 mg / kg, the T1 / 2 of NVS451 in cynomolgus monkeys, the average T1 / 2 of male and female animals in each dose group was between 27.20 - 39.90 h. The T1 / 2 of NVS451 in SD rats, in the dose range of 10 - 100 mg / kg, the T1 / 2 of male and female animals in each dose group was between 51.46 - 69.48 h.

[0370] 3. Non-clinical Conclusion:

[0371] NVS451 is a high-affinity antibody Fc fusion protein that selectively targets the heterodimeric cytokines IL-17A, IL-17C, IL-17F, and IL-17AF. Binding data indicate that NVS451 has a high affinity for the intended targets in humans and cynomolgus monkeys.

[0372] In summary, this invention presents a non-clinical data summary of the project. This data provides evidence of target specificity and mode of action. Treating twice weekly with NVS451 or once weekly with a high dose showed therapeutic effects similar to the positive control of secukinumab. NVS451 was well tolerated in comprehensive toxicology studies. All non-clinical evaluation studies provide strong evidence to support the effectiveness and safety of NVS451 in clinical treatment.

[0373] References:

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[0375] [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-17 Axis. J Immunol, 2009, 182: 5836 - 5845.

[0376] [3] Zhang Tongmei, Wang Gang. Research progress of psoriasis animal models [J]. Journal of Practical Dermatology, 2018, 11(05): 42 - 46.

[0377] [4] Yokogawa M, Takaishi M, Nakajima K, et al. Epicutaneous application of Toll-like receptor 7 agonists leads to systemic autoimmunity in wild-type mice: A new model of systemic lupus erythematosus [J]. Arthritis Rheumatol, 2014, 66(3): 694 - 706.

[0378] [5] Liu Aimin, Li Weiling, Wu Panhong, et al. Research progress on imiquimod-induced psoriasis mouse model [J]. Chinese Journal of Dermatovenereology of Integrated Traditional and Western Medicine, 2018, 17(4): 380-382

[0379] [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 in psoriasis. J Invest Dermatol. 2011;131:1767-1770.

[0380] [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

[0381] [8] Wrone-Smith T, Nickoloff BJ. Dermal injection of immunocytes induces psoriasis. J Clin Invest. 1996;98:1878-1887.

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[11] Levin D, Golding B, Strome S E, et al. Fc fusion as a platform technology: potential for modulating immunogenicity[J]. Trends in Biotechnology, 2015, 33(1): 27–34. SEQUENCE LISTING <110> China National Biotec Group Research Institute Co., Ltd. Valin Biotechnology Co., Ltd. The National Institute for Biotechnology in the Negev <120> IL-17RA Fusion Protein, Pharmaceutical Composition, Injection and Their Applications <130> FI-215323-59:52 <160> 19 <170> PatentIn version 3.5 <210> 1 <211> 288 <212> PRT <213> artificial sequence <400> 1 Leu Arg Leu Leu Asp His Arg Ala Leu 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 Gly 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 Ala 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 <210> 2 <211> 288 <212> PRT <213> artificial sequence <400> 2 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 Gly 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 Ala 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 <210> 3 <211> 288 <212> PRT <213> artificial sequence <400> 3 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 <210> 4 <211> 231 <212> PRT <213> artificial sequence <400> 4 Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 20 25 30 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 35 40 45 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 50 55 60 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 65 70 75 80 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 85 90 95 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 100 105 110 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 115 120 125 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 130 135 140 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 145 150 155 160 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 165 170 175 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 180 185 190 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 195 200 205 Cys Ser Val Met His Glu Gly Leu His Asn His Tyr Thr Gln Lys Ser 210 215 220 Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 5 <211> 32 <212> PRT <213> Homo sapiens <400> 5 Met Gly Ala Ala Arg Ser Pro Pro Ser Ala Val Pro Gly Pro Leu Leu 1 5 10 15 Gly Leu Leu Leu Leu Leu Leu Gly Val Leu Ala Pro Gly Gly Ala Ser 20 25 30 <210> 6 <211> 554 <212> PRT <213> artificial sequence <400> 6 Met Gly Ala Ala Arg Ser Pro Pro Ser Ala Val Pro Gly Pro Leu Leu 1 5 10 15 Gly Leu Leu Leu Leu Leu Leu Gly Val Leu Ala Pro Gly Gly Ala Ser 20 25 30 Leu Arg Leu Leu Asp His Arg Ala Leu Val Cys Ser Gln Pro Gly Leu 35 40 45 Asn Cys Thr Val Lys Asn Ser Thr Cys Leu Asp Asp Ser Trp Ile His 50 55 60 Pro Arg Asn Leu Thr Pro Ser Ser Pro Lys Asp Leu Gln Ile Gln Leu 65 70 75 80 His Phe Ala His Thr Gln Gln Gly Asp Leu Phe Pro Val Ala His Ile 85 90 95 Glu Trp Thr Leu Gln Thr Asp Ala Ser Ile Leu Tyr Leu Glu Gly Ala 100 105 110 Glu Leu Ser Val Leu Gln Leu Asn Thr Asn Glu Arg Leu Cys Val Arg 115 120 125 Phe Glu Phe Leu Ser Lys Leu Arg His His His Lys Arg Trp Arg Phe 130 135 140 Thr Phe Ser His Phe Val Val Asp Pro Gly Gln Glu Tyr Glu Val Thr 145 150 155 160 Val His His Leu Pro Lys Pro Ile Pro Asp Gly Asp Pro Asn His Gln 165 170 175 Ser Lys Asn Phe Leu Val Pro Asp Cys Glu Asp Ala Arg Met Lys Val 180 185 190 Thr Thr Pro Cys Met Ser Ser Gly Ser Leu Trp Asp Pro Asn Ile Thr 195 200 205 Val Glu Thr Leu Glu Ala His Gln Leu Arg Val Ser Phe Thr Leu Trp 210 215 220 Asn Glu Ser Thr His Tyr Gln Ile Leu Leu Thr Ser Phe Pro His Met 225 230 235 240 Glu Asn His Ser Cys Phe Glu His Met His His Ile Pro Ala Pro Arg 245 250 255 Pro Glu Glu Phe His Gln Arg Ser Asn Val Thr Leu Thr Leu Arg Asn 260 265 270 Leu Lys Gly Cys Cys Arg His Gln Val Gln Ile Gln Pro Phe Phe Ser 275 280 285 Ser Cys Leu Asn Asp Cys Leu Arg His Ser Ala Thr Val Ser Cys Pro 290 295 300 Glu Met Pro Asp Thr Pro Glu Pro Ile Pro Asp Tyr Met Pro Leu Trp 305 310 315 320 Gly Ser Gly Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro 325 330 335 Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro 340 345 350 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 355 360 365 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 370 375 380 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 385 390 395 400 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 405 410 415 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 420 425 430 Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 435 440 445 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 450 455 460 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 465 470 475 480 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 485 490 495 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 500 505 510 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 515 520 525 Val Phe Ser Cys Ser Val Met His Glu Gly Leu His Asn His Tyr Thr 530 535 540 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 545 550 <210> 7 <211> 554 <212> PRT <213> artificial sequence <400> 7 Met Gly Ala Ala Arg Ser Pro Pro Ser Ala Val Pro Gly Pro Leu Leu 1 5 10 15 Gly Leu Leu Leu Leu Leu Leu Gly Val Leu Ala Pro Gly Gly Ala Ser 20 25 30 Leu Arg Leu Leu Asp His Arg Ala Pro Val Cys Ser Gln Pro Gly Leu 35 40 45 Asn Cys Thr Val Lys Asn Ser Thr Cys Leu Asp Asp Ser Trp Ile His 50 55 60 Pro Arg Asn Leu Thr Pro Ser Ser Pro Lys Asp Leu Gln Ile Gln Leu 65 70 75 80 His Phe Ala His Thr Gln Gln Gly Asp Leu Phe Pro Val Ala His Ile 85 90 95 Glu Trp Thr Leu Gln Thr Asp Ala Ser Ile Leu Tyr Leu Glu Gly Ala 100 105 110 Glu Leu Ser Val Leu Gln Leu Asn Thr Asn Glu Arg Leu Cys Val Arg 115 120 125 Phe Glu Phe Leu Ser Lys Leu Arg His His His Lys Arg Trp Arg Phe 130 135 140 Thr Phe Ser His Phe Val Val Asp Pro Gly Gln Glu Tyr Glu Val Thr 145 150 155 160 Val His His Leu Pro Lys Pro Ile Pro Asp Gly Asp Pro Asn His Gln 165 170 175 Ser Lys Asn Phe Leu Val Pro Asp Cys Glu Asp Ala Arg Met Lys Val 180 185 190 Thr Thr Pro Cys Met Ser Ser Gly Ser Leu Trp Asp Pro Asn Ile Thr 195 200 205 Val Glu Thr Leu Glu Ala His Gln Leu Arg Val Ser Phe Thr Leu Trp 210 215 220 Asn Glu Ser Thr His Tyr Gln Ile Leu Leu Thr Ser Phe Pro His Met 225 230 235 240 Glu Asn His Ser Cys Phe Glu His Met His His Ile Pro Ala Pro Arg 245 250 255 Pro Glu Glu Phe His Gln Arg Ser Asn Val Thr Leu Thr Leu Arg Asn 260 265 270 Leu Lys Gly Cys Cys Arg His Gln Val Gln Ile Gln Pro Phe Phe Ser 275 280 285 Ser Cys Leu Asn Asp Cys Leu Arg His Ser Ala Thr Val Ser Cys Pro 290 295 300 Glu Met Pro Asp Thr Pro Glu Pro Ile Pro Asp Tyr Met Pro Leu Trp 305 310 315 320 Gly Ser Gly Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro 325 330 335 Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro 340 345 350 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 355 360 365 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 370 375 380 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 385 390 395 400 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 405 410 415 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 420 425 430 Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 435 440 445 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 450 455 460 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 465 470 475 480 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 485 490 495 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 500 505 510 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 515 520 525 Val Phe Ser Cys Ser Val Met His Glu Gly Leu His Asn His Tyr Thr 530 535 540 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 545 550 <210> 8 <211> 554 <212> PRT <213> artificial sequence <400> 8 Met Gly Ala Ala Arg Ser Pro Pro Ser Ala Val Pro Gly Pro Leu Leu 1 5 10 15 Gly Leu Leu Leu Leu Leu Leu Gly Val Leu Ala Pro Gly Gly Ala Ser 20 25 30 Leu Arg Leu Leu Asp His Arg Ala Pro Val Cys Ser Gln Pro Gly Leu 35 40 45 Asn Cys Thr Val Lys Asn Ser Thr Cys Leu Asp Asp Ser Trp Ile His 50 55 60 Pro Arg Asn Leu Thr Pro Ser Ser Pro Lys Asp Leu Gln Ile Gln Leu 65 70 75 80 His Phe Ala His Thr Gln Gln Gly Asp Leu Phe Pro Val Ala His Ile 85 90 95 Glu Trp Thr Leu Gln Thr Asp Ala Ser Ile Leu Tyr Leu Glu Gly Ala 100 105 110 Glu Leu Ser Val Leu Gln Leu Asn Thr Asn Glu Arg Leu Cys Val Arg 115 120 125 Phe Glu Phe Leu Ser Lys Leu Arg His His His Lys Arg Trp Arg Phe 130 135 140 Thr Phe Ser His Phe Val Val Asp Pro Gly Gln Glu Tyr Glu Val Thr 145 150 155 160 Val His His Leu Pro Lys Pro Ile Pro Asp Gly Asp Pro Asn His Gln 165 170 175 Ser Lys Asn Phe Leu Val Pro Asp Cys Glu Asp Ala Arg Met Lys Val 180 185 190 Thr Thr Pro Cys Met Ser Ser Gly Ser Leu Trp Asp Pro Asn Ile Thr 195 200 205 Val Glu Thr Leu Glu Ala His Gln Leu Arg Val Ser Phe Thr Leu Trp 210 215 220 Asn Glu Ser Thr His Tyr Gln Ile Leu Leu Thr Ser Phe Pro His Met 225 230 235 240 Glu Asn His Ser Cys Phe Glu His Met His His Ile Pro Ala Pro Arg 245 250 255 Pro Glu Glu Phe His Gln Arg Ser Asn Val Thr Leu Thr Leu Arg Asn 260 265 270 Leu Lys Trp Cys Cys Arg His Gln Val Gln Ile Gln Pro Phe Phe Ser 275 280 285 Ser Cys Leu Asn Asp Cys Leu Arg His Ser Val Thr Val Ser Cys Pro 290 295 300 Glu Met Pro Asp Thr Pro Glu Pro Ile Pro Asp Tyr Met Pro Leu Trp 305 310 315 320 Gly Ser Gly Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro 325 330 335 Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro 340 345 350 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 355 360 365 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 370 375 380 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 385 390 395 400 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 405 410 415 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 420 425 430 Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 435 440 445 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 450 455 460 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 465 470 475 480 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 485 490 495 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 500 505 510 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 515 520 525 Val Phe Ser Cys Ser Val Met His Glu Gly Leu His Asn His Tyr Thr 530 535 540 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 545 550 <210> 9 <211> 864 <212> DNA <213> artificial sequence <400> 9 ctgagactgc tggaccacag agccctggtc 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 ctgaagggct gctgccggca ccaggtgcag attcagccct tcttctcctc ttgcctgaac 780 gactgcctgc ggcactccgc caccgtgtcc tgccctgaga tgcccgacac ccccgagccc 840 atccctgact acatgcctct gtgg 864 <210> 10 <211> 864 <212> DNA <213> Artificial Sequence <400> 10 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 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> 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

1. An IL-17RA fusion protein, characterized in that it comprises a signal peptide, an extracellular domain of IL-17RA, and an IgG1 constant region that are operably linked and serially connected in sequence; wherein the amino acid sequence of the extracellular domain of IL-17RA is as shown in SEQ ID NO.3, and the amino acid sequence of the IgG1 constant region is as shown in SEQ ID NO.

4.

2. The IL-17RA fusion protein according to claim 1, wherein the signal peptide is the natural signal peptide of IL-17-RA, and its amino acid sequence is as shown in SEQ ID NO.

5.

3. The IL-17RA fusion protein according to claim 1, wherein a linker is used to connect between the extracellular domain of IL-17RA and the IgG1 constant region.

4. The IL-17RA fusion protein according to claim 1, wherein the amino acid sequence of the IL-17RA fusion protein is as shown in SEQ ID NO.

8.

5. An isolated nucleic acid encoding the IL-17RA fusion protein according to any one of claims 1-4.

6. An expression vector containing the nucleic acid according to claim 5 that is effectively linked to a promoter.

7. A host cell containing the expression vector according to claim 6.

8. The host cell according to claim 7, wherein the host cell is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 21011.

9. A protein dimer formed by the IL-17RA fusion protein according to any one of claims 1-4, and the dimer is formed by two molecules of the IL-17RA fusion protein binding through cysteine in the IgG1 constant region to form a double strand.

10. Use of the IL-17RA fusion protein according to any one of claims 1-4 or the protein dimer according to claim 9 in the preparation of a drug for treating psoriasis, arthritis, uveitis, hidradenitis suppurativa, asthma, tendinitis, thyroid-associated ophthalmopathy, multiple sclerosis, lupus nephritis, spondylarthritis, ankylosing spondylitis, rheumatoid arthritis, inflammatory bowel disease, non-alcoholic fatty liver, giant cell arteritis, non-radiographic axial spondyloarthritis, triple-negative breast tumor, multiple myeloma, non-small cell lung cancer, adenocarcinoma, colorectal cancer, prostate cancer, Kaposi's sarcoma, melanoma or cervical cancer.

11. The use according to claim 10, wherein the psoriasis is plaque psoriasis, parapsoriasis or psoriatic arthritis.

12. The use according to claim 10, wherein the multiple sclerosis is relapsing-remitting multiple sclerosis.

13. The use according to claim 10, wherein the rheumatoid arthritis is juvenile rheumatoid arthritis.

14. A pharmaceutical composition comprising a therapeutically effective amount of the IL-17RA fusion protein according to any one of claims 1-4 or the protein dimer according to claim 9 as an active ingredient and a pharmaceutically acceptable excipient.

15. The pharmaceutical composition according to claim 14, wherein the pharmaceutically acceptable excipient is selected from one or more of a diluent, a buffer, a protectant, a surfactant, and an antioxidant.

16. The pharmaceutical composition according to claim 15, wherein the buffer is selected from one or more of a histidine-acetate buffer, a Tris-acetate buffer, a hydrochloric acid buffer, a phosphate buffer, an acetate buffer, a histidine buffer, an arginine buffer, a succinic acid buffer, and a citric acid buffer.

17. The pharmaceutical composition according to claim 15, wherein the protectant is selected from one or more of Tween-20, Tween-80, amino acids, polyols, disaccharides, and polysaccharides.

18. The pharmaceutical composition according to claim 17, wherein the disaccharide is selected from trehalose and sucrose.

19. The pharmaceutical composition according to claim 15, wherein the surfactant is selected from one or more of Tween-20, Tween-80, and poloxamer.

20. The pharmaceutical composition according to claim 14, wherein a single dose of the pharmaceutical composition contains 5 mg / ml - 150 mg / ml of the IL-17RA fusion protein or the protein dimer.

21. The pharmaceutical composition according to claim 14, wherein the pharmaceutical composition is in the form of a lyophilized product or an injection.

22. An injection for treating psoriasis, arthritis, uveitis, hidradenitis suppurativa, asthma, tendinitis, thyroid-associated ophthalmopathy, multiple sclerosis, lupus nephritis, spondylarthritis, ankylosing spondylitis, rheumatoid arthritis, inflammatory bowel disease, non-alcoholic fatty liver, giant cell arteritis, non-radiographic axial spondyloarthritis, triple-negative breast tumor, multiple myeloma, non-small cell lung cancer, adenocarcinoma, colorectal cancer, prostate cancer, Kaposi's sarcoma, melanoma, or cervical cancer, comprising the pharmaceutical composition according to any one of claims 14-21.

23. The injection according to claim 22, wherein the psoriasis is plaque psoriasis, parapsoriasis, or psoriatic arthritis.

24. The application according to claim 22, wherein the multiple sclerosis is relapsing-remitting multiple sclerosis.

25. The application according to claim 22, wherein the rheumatoid arthritis is juvenile rheumatoid arthritis.

26. The injection according to claim 22, wherein the injection is in the form of a freeze-dried powder or a liquid preparation.

27. The injection according to claim 22, wherein the injection is a subcutaneous injection or an intravenous drip.

28. The injection according to claim 26, 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 of Tris-acetate, 10 - 250 mM of arginine, 50 - 500 mM of trehalose, and 0.01 - 5% of Tween-20.

29. The injection according to claim 26, wherein the liquid preparation is prepared by using water for injection, buffered saline solution, aqueous glucose solution, aqueous sodium chloride solution, or lactated Ringer's solution.

30. The injection according to claim 26, wherein the lyophilized powder is prepared by lyophilizing the liquid preparation.

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