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

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

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CN115093483A
CN115093483A CN202210693381.0A CN202210693381A CN115093483A CN 115093483 A CN115093483 A CN 115093483A CN 202210693381 A CN202210693381 A CN 202210693381A CN 115093483 A CN115093483 A CN 115093483A
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pro
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张云涛
王健
阿米尔·阿哈龙尼
李素贞
刘建伟
柳森
郭蓓蕾
刘明扬
闫甲丽
郑秀玉
晁华
祁芳冰
古琼
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China National Biotec Research Institute Co ltd
Valin Biotechnology Ltd
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Valin Biotechnology Ltd
China National Biotec Research Institute Co ltd
National Institute for Biotechnology in the Negev Ltd
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Priority to CN202210693381.0A priority Critical patent/CN115093483A/en
Priority to PCT/CN2022/100748 priority patent/WO2022194311A2/en
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Abstract

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

Description

IL-17RA fusion protein, pharmaceutical composition, injection and application thereof
Technical Field
The invention belongs to the field of medicines, and particularly relates to an IL-17RA fusion protein, a pharmaceutical composition, an injection and application thereof.
Background
Psoriasis is commonly called as psoriasis, is a chronic autoimmune skin disease easy to relapse, generally causes great damage to the physiology and the psychology of patients, is regarded as one of the global major health problems by WHO, has the morbidity accounting for 0.1 to 3 percent of the world population, and has 1.25 hundred million psoriasis patients in the world at present. The total disease rate in China is 0.47%, nearly 650 thousands of patients are clinically registered at present, plaque psoriasis is one of the most common five forms of the disease, and accounts for 80% -90% of all cases, wherein the number of patients with moderate-severe disease accounts for 38%, the number of patients in future reaches 950 thousands of patients in 2030, and the proportion of patients with moderate-severe disease is increased to 40%. The pathogenesis of psoriasis is associated with the dysregulation of the innate and adaptive immune systems, including dendritic cell activation and proinflammatory cytokine secretion, leading to the development of skin inflammation characteristic of psoriasis. In recent years, nearly 13 monoclonal antibodies and antibody-based biologic therapies have been approved for the treatment of psoriasis, about 10 or so monoclonal antibodies in clinical research phase, of which the youth (IL-12/IL-23 antibody, Qiangsheng) antibody, TNF-alpha drug Etanercept (Etanercept, Anin) and adalimumab (Humira, Aiberv) are the most popular drugs. Oral phosphodiesterase 4(PDE4) selective inhibitor drug apremilast (Otezla, Celgene corporation) was also approved in the united states for treatment of refractory psoriasis in 9 months 2014. However, even if these biopharmaceuticals are used as second-line drugs, the therapeutic efficacy and safety remain in great concern, and up to 40% of moderate to severe psoriasis patients fail to be treated with the existing biologicals, a phenomenon known as biofouling.
Recent studies have shown that: the pathogenesis of psoriasis is associated with T17 helper cells (Th17 cells), whereas the pro-inflammatory cytokine IL-17A produced by Th17 cells and innate immune cells has been shown to be the major cytokine in the pathogenesis of psoriasis, with the immune system targeting IL-23 in the IL-17-Th17 pathway, indirectly leading to the development of psoriasis via IL-17A. Theoretically, inhibition of IL-17 may be a safer treatment option than other biologies, as demonstrated by the studies of head-to-head superiority of Nowa marketed for the IL-17A fully human IgG1 monoclonal antibody secukinumab, Aslicon marketed for the IL-17A receptor fully human IgG2 monoclonal antibody Brodalumab, and the present company for the IL-17A fully human IgG4 monoclonal antibody versus Etanercept and Ultezumab (IL-12/IL-23 antibody). Studies have shown that inhibitors developed against the IL-17A target can improve efficacy and safety compared to current therapeutic agents for inflammatory diseases (e.g., TNF- α inhibitors), and are more specific than IL-12 and IL-23 inhibitors in psoriasis patients who have TNF- α inhibitors and IL-12 and IL-23 inhibitors ineffective. It is particularly noteworthy that secukinumab (trade name Cosentyx) marketed at 1 month 2015, which is approved by the European Medicines Agency (EMA) for first line treatment of psoriasis patients in place of other first line systemic therapies with significant side effects.
There is still a need for more innovative drugs with superior efficacy as a therapeutic approach for the choice of patients.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides an IL-17RA fusion protein, a pharmaceutical composition, an injection and application thereof.
Specifically, the present invention provides:
(1) an IL-17RA fusion protein comprising, in operable linkage, in series, a signal peptide, an IL-17RA extracellular domain, and an IgG1 constant region.
(2) The IL-17RA fusion protein according to (1), wherein the amino acid sequence of the IL-17RA extracellular domain is shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No. 3.
(3) The IL-17RA fusion protein of (1), wherein the amino acid sequence of the IgG1 constant region is set forth in SEQ ID No. 4.
(4) The IL-17RA fusion protein of (1), wherein the signal peptide is an IL-17-RA native signal peptide, and the amino acid sequence thereof is shown in SEQ ID NO. 5.
(5) The IL-17RA fusion protein of (1), wherein the IL-17RA extracellular domain is linked to the IgG1 constant region using a linker.
(6) The IL-17RA fusion protein of (1), wherein the amino acid sequence of the IL-17RA fusion protein is shown in SEQ ID No.6, SEQ ID No.7, or SEQ ID No. 8.
(7) An isolated nucleic acid encoding the IL-17RA fusion protein according to any one of (1) - (6).
(8) An expression vector comprising the nucleic acid according to (7) operably linked to a promoter.
(9) A host cell comprising the expression vector according to (8).
(10) The host cell according to (9), wherein the preservation number of the host cell is CGMCC 21011.
(11) A protein dimer formed from the IL-17RA fusion protein of any one of (1) - (6) that is double-stranded from two molecules of the IL-17RA fusion protein bound by cysteines of the IgG1 constant region.
(12) Use of an IL-17RA fusion protein according to any one of (1) to (6) or a protein dimer according to claim 11 in the preparation of a medicament for the treatment of psoriasis, crohn's disease, plaque psoriasis, gastroenteritis, behcet's disease syndrome, arthritis, uveitis, hidradenitis suppurativa, lichen planus, parapsoriasis, asthma, psoriatic arthritis, tendonitis, relapsing-remitting multiple sclerosis, thyroid-associated eye disease, juvenile rheumatoid arthritis, multiple sclerosis, lupus nephritis, spondyloarthritis, ankylosing spondylitis, rheumatoid arthritis, inflammatory bowel disease, non-alcoholic steatohepatitis, giant cell arteritis, non-radioactive axial spine arthritis, 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.
(13) A pharmaceutical composition comprising a therapeutically effective amount of an IL-17RA fusion protein according to any one of (1) to (6) or a protein dimer according to claim 11 as an active ingredient and a pharmaceutically acceptable adjuvant.
(14) The pharmaceutical composition according to (13), wherein the pharmaceutically acceptable adjuvant is selected from one or more of diluents, buffers, protective agents, surfactants, antioxidants.
(15) The pharmaceutical composition of (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, succinic acid buffer, and citric acid buffer.
(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.
(17) The pharmaceutical composition of (14), wherein the surfactant is selected from one or more of tween-20, tween-80, and poloxamer.
(18) The pharmaceutical composition of (13), wherein a single dose of the pharmaceutical composition comprises 5mg/ml to 150mg/ml of the IL-17RA fusion protein or the protein dimer.
(19) The pharmaceutical composition according to (13), wherein the pharmaceutical composition is in the form of a lyophilizate or an injection solution.
(20) An injection for treating psoriasis, crohn's disease, plaque psoriasis, gastroenteritis, behcet's disease syndrome, arthritis, uveitis, hidradenitis suppurativa, lichen planus, psoriasis parapsoriasis, asthma, psoriatic arthritis, tendonitis, relapsing-remitting multiple sclerosis, thyroid-associated eye disease, juvenile rheumatoid arthritis, multiple sclerosis, lupus nephritis, spondyloarthritis, ankylosing spondylitis, rheumatoid arthritis, inflammatory bowel disease, non-alcoholic fatty liver, giant cell arteritis, non-radioactive axial spine arthritis, 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 of any one of (13) to (19).
(21) The injection according to (20), wherein the injection is in the form of a lyophilized powder or in the form of a liquid preparation.
(22) The injection according to (20), wherein the injection is a subcutaneous injection or an intravenous drip.
(23) The injection according to (21), wherein the intravenous formulation comprises 5mg/ml to 150mg/ml of the IL-17RA fusion protein or the protein dimer, 2 to 100mM of Tris-acetic acid, 10 to 250mM of arginine, 50 to 500mM of trehalose, 0.01 to 5% of Tween-20 in the liquid formulation.
(24) The injection according to (21), wherein the liquid preparation is prepared using water for injection, a buffered saline solution, an aqueous glucose solution, an aqueous sodium chloride solution or ringer's lactate.
(25) The injection according to (21), wherein the lyophilized powder is prepared by freeze-drying the liquid preparation.
Compared with the prior art, the invention has the following advantages and positive effects:
the IL-17RA fusion protein provided by the invention is a fully human antibody Fc fusion protein drug. According to the invention, IL-17RA and the antibody Fc segment are fused, so that the half-life period of a drug molecule is prolonged, the longer-acting drug activity is obtained, and the immunogenicity is reduced compared with that of an antibody drug.
In addition, the present inventors have found that selecting the Fc fragment of IgG1 and further appropriately mutating the sequence of the Fc can largely eliminate antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cell-mediated cytotoxicity (CDC), and retain the neonatal Fc receptor (FcRn) -mediated in vivo recycling effect, with less side effects and safety as compared to the IL-17A and IL-17RA antibodies already on the market.
In addition, the invention enables the IL-17RA fusion protein to target multiple targets such as IL-17A, IL-17C, IL-17F, IL-17A/F and the like by selecting and designing the sequence of the IL-17RA, and has high affinity, thereby being capable of selectively blocking the combination of the IL-17A, IL-17C, IL-17F and the IL-17A/F and the receptor thereof, further effectively blocking the biological activity of various proinflammatory IL-17 cytokines and inhibiting inflammatory signal pathways, thereby being capable of more effectively relieving the symptoms of autoimmune diseases, obtaining better treatment benefit than a single IL-17A target antibody medicament and obtaining better safety than an IL-17RA monoclonal antibody. The action mechanism is different from the IL-17 target drugs which are on the market and are researched at present, and is the same type of pioneer drugs all over the world.
The invention comprehensively evaluates the activity and safety of the medicine in terms of binding affinity with IL-17A, biological activity cell experiments, GRO-alpha factor inhibiting capacity, in vitro activity titer, in vivo titer, ADCC/CDC functional activity, FcRn binding affinity, safe pharmacology, pharmacokinetics and toxicology.
Drawings
FIG. 1 shows the amino acid sequence of a recombinant human IL-17RA fusion protein.
FIG. 2 shows a map of the expression vector used in one embodiment, pCHO1.1/NVS 451.
FIG. 3 shows an affinity assay curve and a fitted curve of IL-17RA fusion protein (NVS451) and human IL-17A protein in Experimental example 1.
FIG. 4 shows the affinity assay curves and the fitted curves for secukinumab and human IL-17A protein in Experimental example 1.
FIG. 5 shows an affinity assay curve and a fitted curve of wild-type IL-17RA and human IL-17A protein in Experimental example 1.
FIG. 6 shows affinity assay curves and fitted curves of IL-17RA fusion protein (NVS451) and human IL-17F protein in Experimental example 1.
FIG. 7 shows an affinity assay curve and a fitted curve of IL-17RA fusion protein (NVS451) and human IL-17A/F protein in Experimental example 1.
FIG. 8 shows an affinity assay curve and a fitted curve of IL-17RA fusion protein (NVS451) and human IL-17C protein in Experimental example 1.
FIG. 9 shows the S-curve of GRO- α factor inhibition by IL-17A induction by IL-17RA fusion protein (NVS451) and secukinumab in Experimental example 1.
FIG. 10 is a S-curve showing GRO-. alpha.factor inhibition by IL-17A/F induction by IL-17RA fusion protein (NVS451) in Experimental example 1.
FIG. 11 shows the S-curve of inhibition of GRO-. alpha.factor by IL-17F induction by IL-17RA fusion protein (NVS451) in Experimental example 1.
Fig. 12 shows the appearance of skin after treatment of xenogeneic skin-transplanted SCID mice with different drugs in experimental example 1.
Fig. 13 shows the appearance of skin after treatment of xenogeneic skin-transplanted SCID mice with different drugs in experimental example 1.
FIG. 14 shows skin pathological sections after treatment of xenogeneic skin-transplanted SCID mice with different drugs in Experimental example 1.
Figure 15 shows a comparison of ADCC effect of RitxV301 and rituximab in experimental example 1.
Fig. 16 shows a comparison of CDC effects of RitxV301 and rituximab in experimental example 1.
Fig. 17 shows SPR analysis profiles of different concentrations of NVS451 to human FcRn under acidic conditions (ph6.0) in experimental example 1.
Fig. 18 shows SPR analysis profiles of different concentrations of NVS451 to human FcRn under neutral conditions (ph7.4) in experimental example 1.
FIG. 19 shows a comparison of affinity of IL-17A for IL-17RA fusion proteins NVS451(V301), V302 and V303 in Experimental example 1; in the figure, the concentrations of V300, V301, V302, V303, V301, V302 and V303 are all 100 ug/ml.
Biological material preservation information
The Chinese hamster ovary cells capable of stably expressing the IL-17RA fusion protein provided by the invention are preserved in China general microbiological culture Collection center (CGMCC) in 11-23.2020, and the preservation addresses are as follows: west way No.1 hospital No.3, north beijing, chaoyang district, zip code: 100101, accession number: CGMCC No. 21011.
Detailed Description
The present invention is further described by the following description of the embodiments with reference to the drawings, which are not intended to limit the invention, but various modifications or improvements can be made by those skilled in the art based on the basic idea of the invention, but the scope of the invention is within the scope of the invention.
Herein, the term "injection" means: sterile solutions (including true solutions, emulsions and suspensions) for infusion into the body, and lyophilized powders or concentrated solutions formulated into such sterile solutions (including true solutions, emulsions and suspensions) just prior to use.
Herein, the term "intravenous drip" refers to: a method of infusing a large amount of liquid containing a drug from a vein into a body through an infusion tube. Also called as infusion, drip, intravenous drip and hanging water.
As used herein, the term "IL-17A" refers to interleukin 17A.
As used herein, the term "IL-17 RA" refers to a receptor for IL-17A.
As used herein, the term "active ingredient" refers to a drug molecule having therapeutic effect on a disease, such as the IL-17RA fusion protein described herein.
The invention provides an IL-17RA fusion protein, which is characterized by comprising a signal peptide, an IL-17RA extracellular domain and an IgG1 constant region which are connected in series in sequence and can be operated.
The signal peptide mainly functions to guide the secretion of the target protein from the cytoplasm of the cell to the outside of the cell. Because the fusion protein has IgG1 constant region, when the fusion protein is secreted to the outside of cells, the two-molecule fusion protein forms double chains through cysteine combination of the constant region and exerts activity.
Preferably, the present invention uses the extracellular domain of human IL-17RA (Gene bank numbering: NP-055154) and carries out the R108K, D122G and H155D mutations, the amino acid sequence of which is shown in SEQ ID NO. 1. Also preferably, the present invention uses the extracellular domain of human IL-17RA and performs the L9P, R108K, D122G and H155D mutations, the amino acid sequence of which is shown in SEQ ID NO. 2. The present inventors have found that IL-17RA fusion proteins comprising these two mutants have improved thermostability, and improved binding affinity for IL-17A, compared to a fusion protein comprising the wild type. The amino acid numbering is from position 1 of the amino acid sequence of the extracellular domain of the human IL-17 RA.
Also 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 which is shown in SEQ ID No. 3. The invention discovers that the IL-17RA fusion protein containing the mutant has improved heat stability compared with the wild type, and improved binding affinity to IL-17A, IL-17C, IL-17F, IL-17A/F; has a higher binding affinity for IL-17A than mutants comprising three and four mutations as described above.
The present invention selects the constant region (Fc) of human IgG1(Gene bank number: 3500) to form a fusion protein with IL-17 RA. The fusion protein not only retains the biological activity of functional protein molecules, but also ensures that the fused protein has longer cycle life and longer half-life because the Fc part has the characteristics of certain antibodies and is stable. IgG isotypes, including IgG1, IgG2, and IgG4, which cause different ADCC, ADCP and CDC effects, 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 inventors have found that selection of the constant region of IgG1, and further appropriate mutation of the sequence of the Fc, can reduce the ADCC, ADCP and CDC effects of the IL-17RA fusion protein.
In preferred embodiments, the mutation sites are summarized in table 1 below. An additional mutation was to replace the cysteine residue in the hinge region of IgG1 with a serine residue to avoid the presence of unpaired cysteines in the fusion protein sequence.
TABLE 1
Figure BDA0003701246140000091
Note: the amino acid numbers shown in Table 1 are counted from position 1 of the amino acid sequence of human IgG 1.
The amino acid sequence of the IgG1-Fc mutant is shown in SEQ ID NO. 4.
Signal peptides are one of the major factors affecting yield optimization and product quality. It is important that the signal peptide cleavage site be well defined by a single residue that is clearly cleaved with a high probability of cleavage. Preferably, the natural signal peptide of human IL17RA is used in the fusion protein, and the amino acid sequence of the signal peptide is shown in SEQ ID NO. 5.
Preferably, the IL-17RA extracellular domain is linked to the IgG1 constant region using a linker. Linkers may be those commonly used in the art, such as one or more contiguous GSGs, one or more contiguous GGGGS, and GSAGSAAGSG.
Preferably, the amino acid sequence of the IL-17RA fusion protein is shown in SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8 (the IL-17RA extracellular domain has three mutations, four mutations and six mutations respectively), and the IL-17RA fusion protein has 522 amino acid residues.
For example, the sequence of SEQ ID NO.8 is shown in FIG. 1, wherein the first 32 amino acids (bold) are the IL-17RA signal peptide and the letters on a light gray background are the amino acid sequence of the recombinant human IL-17RA with 6 mutations (bold underlined letters); black background letters are linker amino acid sequences; the dark grey background letters are the amino acid sequences of the 3 modified (bold underlined letters) Fc portions described in table 1, where two bold cysteine residues are responsible for the dimerization of Fc. Asterisks (, s) show potential glycosylation sites.
The IL-17RA fusion protein provided by the invention has an action mechanism that a decoy receptor (IL-17RA-Fc) and a natural receptor compete to bind to an IL-17 molecule, effectively blocks the biological activity of various proinflammatory IL-17 cytokines and inhibits inflammatory signal paths by binding with IL-17A, IL-17C, IL-17F and IL-17A/F with high affinity, selectively blocking the binding of IL-17A, IL-17C, IL-17F and IL-17A/F with receptors thereof and not binding with IL-17B, IL-17D, IL-17E, thereby more effectively relieving the symptoms of autoimmune diseases, obtaining better treatment benefit than a single IL-17A target antibody medicament, and obtaining better safety than an IL-17RA monoclonal antibody.
The invention also provides an isolated nucleic acid encoding an IL-17RA fusion protein according to the invention.
In a preferred embodiment, the present invention utilizes molecular biology techniques to design the cDNA sequence of a fusion protein and optimize codons for expression in CHO cells. The optimized DNA sequences for coding the amino acid sequences SEQ ID NO.1-8 are respectively shown as SEQ ID NO. 9-16.
The invention also provides an isolated mRNA transcribed from the DNA encoding the fusion protein of the invention.
The invention also provides an expression vector comprising a nucleic acid according to the invention operably linked to a promoter.
The invention also provides a host cell comprising an expression vector according to the invention.
In some preferred embodiments, CHO cells suitable for growth in suspension and serum-free media are used as host cells. Also preferably, the expression vector contains two selectable markers puromycin and methotrexate, which facilitates the creation of high-producing and stable cell lines.
In a more preferred embodiment, the host cell has a accession number of CGMCC 21011. The host cell was constructed using CHO-S from Thermo Scientific TM Cells, which can stably express the IL-17RA fusion proteins of the invention after construction. In addition, the host cell has many advantages: (1) the expressed protein is most close to natural protein molecules in the aspects of molecular structure, physicochemical property and biological function; (2) the culture medium can not only grow in a wall-attached manner, but also be cultured in a suspension manner, and has higher shear force and osmotic pressure resistance; (3) the recombinant protein has the high-efficiency amplification and expression capacity of recombinant genes, and the integration of foreign proteins is stable; (4) the product extracellular secretion function is realized, and endogenous protein of the product is rarely secreted, so that the separation and purification of downstream products are facilitated; (5) can be cultured in suspension culture or in serum-free culture medium to reach high density. And the culture volume can reach more than 1000L, and the large-scale production can be realized. Therefore, the CHO cell is the first choice system for recombinant glycosyl protein production. The host cell is preserved in China general microbiological culture Collection center (CGMCC) at 11/23 of 2020, and the preservation address is as follows: west way No.1 hospital No.3, north beijing, chaoyang district, zip code: 100101, with a deposit number of: CGMCC No. 21011.
The invention also provides a protein dimer formed by the IL-17RA fusion protein according to the invention, wherein the dimer is formed by two molecules of the IL-17RA fusion protein which are combined to form a double chain through cysteine of the IgG1 constant region.
Because the IL-17RA fusion protein has an IgG1 constant region, when the fusion protein is secreted to the outside of cells, the two-molecule fusion protein forms a double chain through cysteine combination of the constant region and exerts activity.
The invention also provides the use of the IL-17RA fusion protein according to the invention or of a protein dimer formed from the IL-17RA fusion protein for the preparation of a medicament for the treatment of psoriasis, Crohn's disease, plaque psoriasis, gastroenteritis, Behcet's disease syndrome, arthritis, uveitis, hidradenitis suppurativa, lichen planus, psoriasis, asthma, psoriatic arthritis, tendonitis, relapsing-remitting multiple sclerosis, thyroid-related eye disease, juvenile rheumatoid arthritis, multiple sclerosis, lupus nephritis, spondyloarthritis, ankylosing spondylitis, rheumatoid arthritis, inflammatory bowel disease, non-alcoholic steatohepatitis, giant cell arteritis, non-radioactive axial spine arthritis, acne vulgaris, triple negative breast tumors, multiple myeloma, non-small cell lung cancer, adenocarcinoma, colorectal cancer, prostate cancer, application of Kaposi's sarcoma, melanoma, cervical cancer and/or other inflammatory diseases in medicine
The invention comprehensively evaluates the pharmaceutical activity and safety of the IL-17AR fusion protein from the aspects of binding affinity with IL-17A, biological activity cell experiments, GRO-alpha factor inhibition capability, in vitro activity titer, in vivo titer, ADCC/CDC functional activity, FcRn binding affinity, safe pharmacology, pharmacokinetics and toxicology. The result proves that the IL-17RA fusion protein can target multiple targets such as IL-17A, IL-17C, IL-17F, IL-17A/F and the like, and has high affinity, so that the combination of IL-17A, IL-17C, IL-17F and IL-17A/F and receptors thereof can be selectively blocked, the biological activities of multiple proinflammatory IL-17 cytokines can be effectively blocked, and inflammatory signal pathways can be inhibited, so that the symptoms of autoimmune diseases can be more effectively relieved, better treatment benefit than a single IL-17A target antibody drug can be obtained, and better safety than an IL-17RA monoclonal antibody can be obtained.
The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the IL-17RA fusion protein according to the invention or a protein dimer formed from the IL-17RA fusion protein as an active ingredient and a pharmaceutically acceptable excipient.
The pharmaceutically acceptable auxiliary materials can be selected according to the dosage form and the actual requirement.
The preparation of the IL-17RA fusion protein and the preparation prescription are deeply researched and designed from the aspects of packaging materials, buffer systems, auxiliary materials, dosage, prescription compositions, freeze-drying processes and the like.
In some preferred embodiments, the pharmaceutically acceptable excipients of the pharmaceutical composition are selected from one or more of diluents, buffers, protective agents, surfactants, antioxidants.
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.
Preferably, the protective agent is selected from one or more of trehalose, tween-20, tween-80, sucrose, amino acids (e.g. arginine), polyols, disaccharides, polysaccharides. Of these, trehalose and combinations of trehalose + arginine are most preferred.
In some embodiments, trehalose is present as trehalose dihydrate.
Preferably, the surfactant is selected from one or more of tween-20, tween-80 and poloxamer (poloxamer).
In some preferred embodiments, a single dose of the pharmaceutical composition comprises 5mg/ml to 150mg/ml of the IL-17RA fusion protein or a protein dimer formed from the IL-17RA fusion protein.
The pharmaceutical composition of the present invention may be formulated into a suitable dosage form as required. In the present invention, the IL-17RA fusion protein is preferably in the form of a lyophilized preparation or an injection solution.
Accordingly, the present invention also provides an injection for the treatment of psoriasis, crohn's disease, plaque psoriasis, gastroenteritis, behcet's disease syndrome, arthritis, uveitis, hidradenitis suppurativa, lichen planus, parapsoriasis, asthma, psoriatic arthritis, tendonitis, relapsing-remitting multiple sclerosis, thyroid-associated eye disease, juvenile rheumatoid arthritis, multiple sclerosis, lupus nephritis, spondyloarthritis, ankylosing spondylitis, rheumatoid arthritis, inflammatory bowel disease, non-alcoholic fatty liver, giant cell arteritis, non-radioactive axial spine arthritis, 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 the like and/or other inflammatory diseases, comprising a pharmaceutical composition according to the present invention.
The injection may be in the form of a lyophilized powder or in the form of a liquid preparation.
Preferably, the injection is subcutaneous injection or intravenous drip. Most preferably subcutaneous injections.
In a liquid formulation, the injection preferably comprises the IL-17RA fusion protein, Tris-acetate, arginine, trehalose, Tween-20 and a suitable solvent.
More preferably, the injection comprises 5mg/ml to 150mg/ml of the IL-17RA fusion protein or a protein dimer formed from the IL-17RA fusion protein, 2 to 100mM of Tris-acetic acid, 10 to 250mM of arginine, 50 to 500mM of trehalose, 0.01 to 5% of Tween-20, and a suitable solvent.
The solvent can be a solvent commonly used for preparing injections. Such as water for injection, a buffered saline solution, an aqueous glucose solution, an aqueous sodium chloride solution, or ringer's lactate, etc.
The liquid preparation is prepared by adopting a solvent.
The liquid preparation can be formulated according to the prescription of the present invention by a method commonly used in the pharmaceutical field.
Lyophilized powders can be prepared by freeze-drying the liquid formulation.
In a preferred embodiment, the lyophilization process comprises prefreezing, primary drying (sublimation), and secondary drying (desorption). Pre-freezing comprises reducing the temperature from 5 ℃ to-40 ℃ and keeping for a suitable time; primary drying involves raising the temperature to-5 to 0 ℃ for a suitable time; secondary drying involves raising the temperature to 25 ℃ to 30 ℃ for a suitable time.
According to the research on the preparation of the IL-17RA fusion protein and the preparation prescription, the invention develops the pharmaceutical preparation which has excellent drug stability and can be safely injected and administered.
The present invention will be further explained or illustrated below by way of examples, which should not be construed as limiting the scope of the invention.
Examples of the present invention
Unless otherwise indicated, the experimental procedures used in the examples below were performed using conventional experimental procedures, materials and conditions used in the biotechnology and pharmaceutical fields.
Hereinafter, unless otherwise specified, the percentage concentration (%) of each agent refers to the volume percentage concentration (% (v/v)) of the agent.
Preparation example 1: plasmid construction
NVS451 (i.e., IL-17RA fusion protein) consists of a signal peptide, rhIL17-RA ECD (i.e., human IL17-RA extracellular domain), linker peptide segment, and IgG1 Fc domain. Wherein the signal peptide is the natural signal peptide of IL17 RA. Six mutation sites were included in rhIL17-RA ECD, L9P, R108L, D122G, H155D, G243W, and a267V (amino acid numbering does not include signal peptide sequence). The linker peptide is GSG. IgG1 Fc domain hybrids of IgG1, 2, 4 were used to remove potential ADCC, CDC and ADCP effects. The amino acid sequence of NVS451 is shown as SEQ ID NO.8, and the nucleotide sequence is shown as SEQ ID NO. 16.
Plasmids were constructed using pCHO1.0 mammalian cell expression vectors from Thermo Scientific. The pcho1.0 expression vector contains two expression cassettes, and since only one expression cassette is required for the expression of NVS451 fusion protein, the other expression cassette is removed using the SfiI cleavage site, and the removed expression cassette contains the EcoRV and PacI cloning sites. The pcho1.0 vector after removal of one expression cassette was verified by sequence sequencing and was named pcho 1.1. The synthesized NVS451 fusion protein gene is inserted into a pCHO1.1(Kan resistance) expression vector through AvrII and Bstz17I cloning sites, and is transformed into DH5 alpha competent cells, plate-coating screening is carried out, clone containing plasmids with correct sizes is inoculated and amplified, and a large amount of constructed NVS451 fusion protein expression plasmids pCHO1.1-NVS4510 are obtained by plasmid extraction. Gene sequencing verified that the pCHO1.1-NVS451 expression vector was constructed correctly.
Preparation example 2: cell line screening and stability evaluation
Construction and screening of NVS451 fusion protein cell strain and CHO-S TM (cGMP-banked cell bank meeting current drug manufacturing and management specifications), and the cell and matched expression vector pCHO1.0 are from Thermo Scientific corporation
Figure BDA0003701246140000141
Figure BDA0003701246140000151
Kit) cells as the primary cell matrix. NVS451 fusion protein expression plasmid pCHO1.1-NVS451 contains selection markers for puromycin and MTX. pCHO1.1-NVS4510 was transferred into CHO-S cells by transfection reagents. The transfection procedure was as follows: mu.g of the circular expression plasmid pCHO1.1-NVS451 was diluted to a volume of 1.5ml by Opti PRO SFM, 50. mu.l of Freestyle Max transfection reagent (GIBCO) was diluted to a volume of 1.5ml by Opti PRO SFM, then the plasmid dilution and transfection reagent dilution were mixed in equal volumes, and 30ml of a suspension of CHO-S cells at 1E6 cells/ml was added. Transfected cells were cultured for 48 hours prior to two-stage selection of stable transfectant clonal cell Pools (Pools) using puromycin and MTX as selection pressure. Since untransfected CHO-S lacks pac (adenylate cyclase) activity and has only basal DHFR (dihydrofolate reductase) activity, only CHO-S cells transfected with pCHO1.1-NVS4510 plasmid and integrated into the genome could harbor puromycin and MTX in CD FortCHO cells TM The medium was screened for survival and propagation. The screening was performed in two stages, each using puromycin and MTX. Finally, 4 pools of stably transfected clonal cells (Pool) were formed, T3S1, T3S2, T3S3, and T3S4, respectively. The expression level was evaluated by 14 days of simple fed batch culture (SFB). In combination with cell-based GRO- α inhibitory activity assays, affinity ELISAs and SPR assays (using conventional methods and not described in detail), two pools of T3S1 and T3S4 were selected for isolation of single clones (LDCs). Isolation of monoclonals were isolated using two rounds of limiting dilution, with a cell plating concentration of less than 1 cell/well per round (according to statistical mathematical calculations), and finally 5 candidate monoclonals were obtained: T3S4-7E3-6G 5; T3S4-7E3-3C 3; T3S4-17G2-6E 2; T3S4-8F2-6E 10; T3S1-18E7-6C 5.
Stability evaluation of 70PDL (cell doubling time, equivalent to 70 generations) was performed on 5 candidate clones, and based on expression stability, gene copy number, and stability of mRNA transcription level, one clone VAN301-T3S1-18E7-6C5 was finally selected as a master clone (accession number CGMCC 21011), and VAN301-T3S4-17G2-6E2 was selected as a backup clone.
The monoclonal VAN301-T3S1-18E7-6C5 was used to create PCBs (Primary cell Bank) and on this basis GMP-conditioned MCBs (Master cell Bank) and WCBs (working cell Bank).
Preparation example 3: cell culture
NVS451 fusion protein can be produced by 14 day fed-Batch culture process. Cell recovery: taking a cell from a cell bank, recovering the cell in water bath at 37.0 +/-0.5 ℃, immediately transferring the seed solution into a 50ml centrifuge tube filled with 10ml of preheated Dynamis (containing 8mM L-Gln, 1:100ACA and 1g/L P188) culture medium after thawing, centrifuging for 5 minutes at 300g, removing supernatant, then adding 10ml of preheated Dynamis culture medium to suspend the cell, transferring the resuspended cell solution into a 125ml shake flask filled with 28.0ml Dynamis culture medium, and controlling the cell density to be (0.15-0.35) multiplied by 10 6 Cell/ml, cell viability greater than 90%. Seed passage and amplification: the subculture medium is Dynamis, and the initial seeding density of the cells in subculture should be (0.40 + -0.10) × 10 6 cells/mL, after 3 days of culture, the cell density reaches (2.0-5.0) x 10 6 cells/mL can be passaged, and the cell survival rate is ensured to be higher than 90.0% in the passage process.
Reactor Fed-batch culture (Fed batch): the initial seeding density of the cells is (0.40 +/-0.10) multiplied by 10 6 2 times of supplemented C + (Efficient feed C +, Cat #: A2503101, Gibco) are fed in per mL, the glucose and lactic acid contents are detected every day in the culture process, and 450.0g/kg of glucose mother liquor is fed in per 3/5/7/9/11/13 days to supplement to 5.0 g/L.
Cell culture harvest conditions: when the culture is carried out to the 14 th day or the cell survival rate is lower than 80.0 percent, the harvest is carried out under the condition of first arrival.
Preparation example 4: NVS451 fusion protein purification
The cell culture harvest liquid is firstly clarified by a two-stage deep filtration membrane package, then 1% of Tween 80 and 0.3% of tributyl phosphate are added for virus inactivation, then MabSelect SuRe affinity chromatography packing of GE company is used for capturing target protein, Eshmuno CPX anion chromatography packing of Millipore company and Capto Adhere cation chromatography packing of GE company are used for two-step fine purification, then a BioEX nanofiltration membrane of ASAHI KASEI company is used for nanofiltration, Pellicon 2 of Millipore company, PES material with molecular weight cutoff of 50KDa and C-channel ultrafiltration membrane are used for ultrafiltration, finally, accessory polysorbate 20 is added, and NVS451 stock solution is obtained after sterilization and filtration.
1. Deep filtration
The supernatant containing the target protein was obtained by performing depth filtration using serially connected depth filtration membranes (first-stage depth filtration membrane D0HC and second-stage filtration membrane A1HC, Millipore Co.) to remove impurities such as cells. When the sample is filtered, the inlet flow rate is less than or equal to 100LMH (based on A1HC), and the maximum loading capacity of D0HC is 60L/m 2 A1HC maximum loading of 140L/m 2 The single-step recovery rate of the deep filtration is about 90 percent generally by controlling the pressure.
2. Detergent virus inactivation
To the clarified harvest was added 50mM Tris-HAc,150mM NaCl, 25% PS80, pH7.4 to give a final Tween 80 concentration of 1.0%. 100% tributyl phosphate was added to a final concentration of 0.3%. Incubating for 360-480 minutes at 16-26 ℃, and then performing affinity chromatography.
3. Affinity chromatography
And (3) carrying out affinity chromatography by using Mabselect SuRe packing to realize primary purification of the product. Elution 1 buffer after equilibration and loading with 50mM Tris-HAc,150mM NaCl, pH7.4, followed by elution 2 with 20mM Tris,1M NaCl,0.5M Arg, pH8.6, and finally elution of the protein of interest with 50mM glycine, pH3.5 buffer. The eluate is neutralized to pH7.8-8.2 using 1M Tris base.
4. Cation chromatography
Eshmuno CPX from Millipore corporation was used as the cationic chromatography packing. The neutralized sample after affinity chromatography was adjusted to pH6.3-6.7 with 1M HAc, and the adjusted sample was used as a loading sample for cation chromatography. The HCP, the protein A, DNA, and impurities and fragments of product analogues and other impurities related to the product can be effectively removed through leaching and gradient elution.
Elution was performed with 20mM PB, pH6.5 as equilibration and post-loading wash buffer, 25mM PB,110mM NaCl, pH6.5 as elution 2, using a gradient of 25mM PB,110mM NaCl, pH6.5 to 20mM PB,1M NaCl, pH6.5, 0-50% B (10 CV).
5. Anion chromatography
The Capto Adere packing from GE was used as the anion chromatographic packing to remove some of the product and process related impurities. The cation chromatography eluent is firstly adjusted to pH 8.4-8.6 by 1M Tris alkali and water for injection, the conductance is 19.0-23.0, and the sample after the pH conductance is adjusted is used as an anion chromatography sample.
The target protein was eluted with 20mM Tris,0.2M NaCl, pH8.5 as equilibration and post-loading elution buffer, 20mM Tris,0.2M NaCl,60mM Arg, pH8.0 as elution 2 buffer, and 20mM Tris,0.2M NaCl,290mM Arg, pH7.1 buffer.
6. Nanofiltration
The anion chromatography elution sample was subjected to virus removal filtration using a Nafilter BioEX (Asahi Kasei Co., Ltd.).
The nanofiltration process mainly utilizes the difference of molecular sizes of viruses and protein products, potential viruses are intercepted by the nanofiltration membrane, and target proteins flow through, so that the separation of the viruses and the protein products is realized. The pre-filtering membrane can adsorb impurities such as particles in a sample, and the treatment capacity of the nano-filtering membrane is increased. The nanofiltration membrane can effectively intercept potential viruses such as parvovirus and the like. A1HC (Millipore) was used for prefiltration and BioEX (Asahi Chemicals) for potential virus removal. When a sample is filtered, the pressure difference on the pre-filtering membrane is controlled to be less than or equal to 2bar, the pressure difference on the nanofiltration membrane is controlled to be less than or equal to 3bar, and the loading capacity of the nanofiltration membrane is controlled to be less than or equal to 600g/m 2 。
7. Ultrafiltration diafiltration
After nanofiltration the intermediate was concentrated using a Pellicon 2 (Millipore) ultrafiltration membrane cartridge and then re-pooled into a 20mM Tris-HAc,65mM Arg,120mM trehalose, pH7.5 buffer system. The ultrafiltration membrane is made of PES with a molecular weight cutoff of 50kDa and a C flow channel. The loading capacity of ultrafiltration and diafiltration is less than or equal to 300g/m 2 (ii) a When in concentration, the inlet flux is 150-300LMH, the transmembrane pressure (TMP) is less than or equal to 1.5bar, and the concentration is concentrated to 18.0-22.0 g/L; then, the liquid is changed, and the flux of the inlet is changed when the liquid is changed150-300LMH, TMP less than or equal to 2bar, the liquid change volume more than or equal to 5DV, the inlet flux of 150-300LMH and TMP less than or equal to 2bar during over-concentration, and the over-concentration is carried out until the concentration is 80.0-90.0 g/L.
8. Addition of adjuvants and final filtration
And adding 10% (w/w) polysorbate 20 mother liquor into the sample subjected to ultrafiltration diafiltration, and regulating the protein concentration to 70.20-150.80 mg/mL by using 20mM Tris-HAc,65mM Arg,120mM Trehalose and pH7.5 buffer solution to prepare a stock solution. Then, sterile filtration was performed using PES (0.22 μm) filter. The final content of the original liquid polysorbate 20 is 0.02% (w/v), and the total recovery rate of the auxiliary material addition and the sterilization filtration is generally more than 80%.
9. Stock solution
And (4) performing quality detection on the stock solution after adding the auxiliary materials and sterilizing and filtering, wherein the purity is over 97 percent.
The stock solution was stored in a-40 + -5 deg.C refrigerator.
Experimental example 1: study of pharmacodynamics
1. In vitro pharmacodynamics
The active molecule of NVS451 is a double-chain fusion protein consisting of a human IL-17RA extracellular domain mutant and a human IgG1 Fc mutant, and is obtained by CHO cell recombinant expression. Thus, the molecule may exhibit both the biological functional properties of the IL-17RA and Fc molecules. A series of researches on NVS451 in vitro biological activity are carried out by analyzing technologies such as Surface Plasmon Resonance (SPR) and in vitro target cell killing measurement, and the like, and aims to clarify the related attributes of in vitro drug effects.
1.1 IL-17A binding affinity comparison assay
NVS451 (used at 75ug/ml) binds to human IL-17A with high affinity. Surface plasmon resonance was performed with 100nM to 0.8nM human IL-17A (Acrobiosystems, Cat: ILA-H5118) as follows:
reagent preparation
Coating liquid: 1.06g of Na was taken 2 CO 3 And 0.84g NaHCO 3 Fully dissolving with ultrapure water, adjusting pH to 9.60 with concentrated hydrochloric acid, diluting to 200ml, filtering with 0.22 filter membrane, and storing at 4 deg.C;
10 × TBS mother liquor: adding 12.114g of Tris and 43.83g of NaCl into ultrapure water for full dissolution, adjusting the pH value to 7.55 by using hydrochloric acid, metering the volume to 500ml, filtering by using a 0.22 filter membrane, and storing at 4 ℃;
4 × substrate buffer mother liquor: 7.16g of NaHPO12H O and 2.1g of citric acid were dissolved sufficiently in ultrapure water, adjusted to pH 5.5 with NaOH and made to volume 100ml, filtered through a 0.22 filter, and stored at 4 ℃.
Experimental procedures
Coating:
IL-17A was reconstituted with ultrapure water according to COA, left to stand for about 30 minutes to dissolve it sufficiently, and IL-17A was diluted to 30nM with a coating solution equilibrated at room temperature, and then added to an ELISA plate at 100. mu.l per well, and the plate was left overnight at 4 ℃ (about 16 hours).
Washing the plate:
the washing solution is prepared at present, 100ml of 10 xTBS mother liquor is added into 900ml of ultrapure water to be diluted into 1 xTBS, 2.5ml of 20% Tween 20 is added, and the plate is repeatedly washed 4 times by the prepared washing solution, wherein each hole is 300 mu l, and the plate is patted dry.
And (3) sealing:
preparing the BSA solution on site, namely diluting 2.5g of BSA with a washing solution to 50ml to prepare a 5% BSA blocking solution; mu.l of blocking solution was added to the patted plate and incubated at 37 ℃ for 1.5 h.
Washing the plate:
and (5) repeating the step (2) to wash the plate.
Adding a sample:
and (2) preparing a diluent at present when the diluent is used, diluting 5ml of blocking solution to 50ml by using a washing solution to prepare 0.5% BSA sample diluent, diluting the sample to 10 mu g/ml by using a 2-fold gradient, adding the diluted sample into an enzyme label plate at a concentration of 100 mu l/hole, and setting background controls (non-coating + sample + secondary antibody, coating + non-adding sample + secondary antibody, non-coating + non-adding sample + non-adding secondary antibody). The plate-sealing membrane plate is incubated for 1h at 37 ℃.
Washing the plate:
and (5) repeating the step 2 to wash the plate.
Secondary antibody:
the diluent is prepared as before, 5ml of confining liquid is diluted to 50ml by washing liquor to prepare 0.5% BSA antibody diluent, horse radish peroxidase-labeled Affinipure goat anti-human IgG and Fc gamma fragment specificity (min X Bov, Hrs, Ms Sr Prot) are diluted to 1:12000, 100 mu l of the antibody diluent is added into each hole, and the plate is incubated for 1h at 37 ℃ in a plate sealing membrane sealing plate.
Washing the plate:
and (5) repeating the step 2 to wash the plate.
Color development:
12ml of 4 Xsubstrate buffer was added to 36ml of ultrapure water and diluted to 1 Xsubstrate buffer, and 38.5. mu.l of 3% H was added 2 O 2 And 240. mu.l of 20mg/mL TMB (dissolved in DMSO), 200. mu.l per well, and incubated at 37 ℃ for 20 minutes.
And (4) terminating:
50. mu.l of 1mol/L sulfuric acid was added to each well of the plate after completion of color development.
Reading:
the absorbance (OD value) of each well was measured at a wavelength of 450nm, and the average value was obtained.
Data processing:
the OD values obtained were fitted to the data with GraphPad Prism 5 to obtain EC 50.
(SPR) test (results are shown in FIG. 3) with a KD of 1.8X 10 -12 M, higher affinity than secukinumab under equivalent conditions (results are shown in FIG. 4) (IL-17A mab drug approved to market abroad (2015)) and wild-type human IL-17RA (wtIL-17RA, available from ACROBIOSSYSTEMS corporation) (results are shown in FIG. 5). The results of the binding affinity comparison assay with IL-17A are summarized in Table 2.
TABLE 2 binding affinity comparison assay for IL-17A
Figure BDA0003701246140000211
IL-17RA fusion proteins (amino acid sequences shown in SEQ ID NO.6 and SEQ ID NO.7, respectively) having 3 mutations (V302) and 4 mutations (V303) in the extracellular domain of IL-17RA were prepared in the same manner as in preparation examples 1 to 4.
The affinity of V302 and V303 to IL-17A was determined by an affinity ELISA method. Two kits (R & D, Cat #: 317-ILB-050; Peprotech, Cat #:900-K84) were used and tested according to the manufacturer's protocol, and the results showed (FIG. 19) that the percentage of affinity of NVS451 to IL-17A with six mutations (776% and 464% for the two kits above) was significantly higher than the affinity of the V302 and V303 molecules to IL-17A. Whereas the affinity of the V302 and V303 molecules to IL-17A is comparable to that of wild-type human IL-17 RA.
1.2 binding affinity analysis of IL-17A from different species
In this study, comparison of IL-17A molecules (KINGFISHER, cat # RP0921H-025, RP1031Y-025, RP 0355M-025; rat Biolegend, 778704) from 4 species (human, cynomolgus monkey, mouse, rat) under the same conditions with NVS451 revealed that NVS451 binds to IL-17A from 4 different species with high to low affinity: human > cynomolgus monkey > rat > mouse (KD see table 3).
TABLE 3 comparative analysis of binding affinity to IL-17A of different species
Figure BDA0003701246140000221
1.3 binding affinity analysis of IL-17A, C, F, AF
In addition to binding to the IL-17A molecules of the human IL-17 family, NVS451 also binds to human IL-17C, IL-17F, IL-17AF (see FIGS. 6-8, respectively). KD results are summarized in table 4.
In another study, NVS451 and wild-type human IL-17RA were compared for their affinities for IL-17C, IL-17F (Acrobiosystems, ILC-H52H7, ILF-H4240), and it was shown that NVS451 had a higher affinity for human IL-17C, IL-17F than for wild-type human IL-17RA (Table 5).
TABLE 4 NVS451 binding affinity characterization for the human IL-17 family
Figure BDA0003701246140000222
TABLE 5 comparison of the affinity of NVS451 and wild-type IL-17RA to IL-17C, IL-17F
Figure BDA0003701246140000231
Weak bonding: the map had a response signal, but the intensity was low and the reading could not be quantified.
N.A.: not applicable to
1.4 biologically active cell assay
The cell line CCD-1070Sk (human fibroblast epithelial cell line, ATCC CRL-2091) used for the NVS451 in vitro activity assay belongs to a human fibroblast epithelial cell line, and a plurality of cytokine receptors including IL-17A, A/F and F exist on the surface of the cell line. IL-17A can bind to IL-17A receptor IL-RA on the cell surface, and stimulate the cell to produce the cytokine GRO-alpha, and the content can be accurately detected by ELISA double antibody sandwich method. When IL-17A and NVS451 are co-incubated with CCD-1070Sk cells, NVS451 is able to inhibit the binding of IL-17A to the IL-17A receptor IL-17RA on the cell surface by competing with the binding of IL-17A, thereby reducing the amount of GRO-. alpha.secreted in the cell supernatant. And NVS451 can also reduce the secretion of GRO-alpha in cell supernatant by binding to IL-17A/F, IL-17F based on the same mechanism of competitive inhibition. Thus, the in vitro activity of NVS451 on multiple targets, including IL-17A, IL-17A/F, IL-17F, can be determined by measuring cellular GRO- α secretion.
1.4.1 GRO-alpha factor inhibitory ability of NVS451 under IL-17A induction
The relative potency of NVS451 fusion protein in vitro was analyzed by measuring the IL-17A-induced inhibitory ability of GRO-alpha release from the human fibroblast epithelial cell line CCD-1070Sk, and the results were expressed as half inhibitory concentration IC 50 Values are shown (Table 6).
1. Human fibroblast epithelial cell line CCD-1070Sk cells in a T75 flask were digested with 2ml of trypsin with 0.05% EDTA for about 2-5 minutes, neutralized by 8ml of complete medium (10% FBS EMEM basal medium), centrifuged at 1000rpm for 5 minutes, resuspended in complete medium to a cell density of 1E5/ml, resuspended in a 96-well plate at 100ul per well and 10000 cells per well, cultured for about 24 hours, and the dosing experiment was started until the confluency of cells reached 95% -100%.
2. Cell induction and administration: all drug dilutions used complete medium, NVS451 protein was first diluted to an initial concentration of 4ug/ml (final concentration of 1ug/ml), then diluted in a two-fold gradient for 9 spots, and finally added to the cell plate at 0ug/ml concentration, 50ul per well, then IL-17A was diluted to a concentration of 40ng/ml (final concentration of 10ng/ml), added to each reaction well at 50ul per well, and cells were dosed for 24 hours for incubation.
3. After 24 hours of incubation, the cells are centrifuged for 5 minutes at 1500rpm by using a plate centrifuge, and then 100ul of supernatant is taken to prepare for detection in an ELISA experiment, wherein the cell supernatant of 3 multiple wells is mixed and diluted, and then 3 multiple wells of an ELISA plate are respectively added into the ELISA experiment
4. ELISA assay
A. The capture antibody (mouse anti-human GRO. alpha. antibody, R & D Cat # DY275-05-840255) was diluted with PBS X1 to a concentration of 0.25. mu.g/ml, 100. mu.l was immediately added to each ELISA well, the plate was coated and incubated overnight (24 ℃. + -. 4) at room temperature.
B. The wells were cleared of liquid and the plates were washed 4 times with 300 μ l wash buffer per well. The last time was thoroughly cleaned with absorbent paper (wash buffer required one day earlier room temperature until complete dissolution).
C. Add 300. mu.l blocking buffer per well. Incubate at room temperature ((24 ℃. + -. 4) for at least 1 hour.
D. Sample preparation:
the 96-well CRL-2091 cell culture plates were centrifuged at 1000rpm for 5 minutes.
The sample was prepared by adding 50. mu.l of the pre-incubated sample (sup) to 200. mu.l of the sample diluent (the sample was now diluted at 1: 5).
E. Preparing a standard substance:
a1. mu.g/ml dilution series of standards was prepared in diluent, for a total of 7 point dilutions and one zero point
F. The wells were cleared of liquid and the plates were washed 4 times with 300 μ l of wash buffer per well. And finally, thoroughly cleaning the paper by using absorbent paper.
G. 100 μ L of standard or sample was immediately added to each well in triplicate. Incubate at room temperature ((24 ℃. + -. 4), 600rpm, for 2 hours.
H. The wells were cleared of liquid and the plates were washed 4 times with 300 μ l of wash buffer per well. And finally, thoroughly cleaning the paper by using absorbent paper.
I. The concentration of the diluted detection antibody (biotinylated goat anti-human GRO. alpha. antibody, R & D Cat # DY275-05-840256) in the diluent was 1.0. mu.g/ml (initial concentration 100. mu.g/ml).
J. 100 μ l of diluted detection antibody was added to each well. Incubation was carried out at room temperature (24 ℃ C.. + -. 4), 600rpm, for 2 hours.
K. The wells were cleared of liquid and the plates were washed 4 times with 300 μ l of wash buffer per well. And finally, thoroughly cleaning the paper by using absorbent paper.
L, 6. mu.L of biotin-conjugated-HRP conjugate (1: 2,000) was added to a total volume of 12ml of sample dilution (1 96 well plate was calculated).
M, 100. mu.l per well. Incubate at room temperature (24 ℃. + -. 4) for 30 minutes at 600 rpm.
N, clear each well of liquid and wash the plate 4 times with 300 μ l wash buffer per well. And finally, thoroughly cleaning the paper by using absorbent paper.
Add 100 μ l substrate solution per well (ABTS substrate 30 minutes in advance, take out the required amount for this experiment). The cells were incubated at room temperature for 20 minutes to develop color.
P, color development was monitored at 405nm using a microplate reader, and the wavelength was calibrated at 650 nm.
Simultaneously detecting the GRO-alpha inhibition capability of the current commercial IL-17A monoclonal antibody secukinumab (Cosentyx) under the in-vitro relative titer detection method, and using the half inhibition concentration IC as the result 50 Values are shown (Table 6). The results show that NVS451 fusion protein has the capability of inhibiting IL-17A-induced release of GRO-alpha cytokines and is better than the IC of secukinumab (Cosentyx) 50 The value is lower (fig. 9).
Table 6 in vitro activity titers of NVS451 and secukinumab: IC for induction of GRO-alpha by IL-17A 50 (μg/ml)
Figure BDA0003701246140000251
Figure BDA0003701246140000261
1.4.2 comparison of potency of NVS451 on multiple ligands of IL-17A, IL-17A/F, IL-17F in vitro
Similar to the above experiment, IL-17A/F and IL-17F induced human fibroblast epithelial cell line CCD-1070Sk were also able to produce GRO-alpha cytokine, and results of in vitro potency of NVS451 fusion protein under different ligand induction were used as half inhibitory concentration IC 50 Values are shown (Table 7). The inhibition S-curves are shown in fig. 10 and fig. 11, respectively.
TABLE 7NVS451 inhibition of human dermal fibroblast CCD-1070Sk GRO-a secretion assay results
Figure BDA0003701246140000262
2. Pharmacodynamics in vivo
Psoriasis is a chronic inflammatory skin disease, which is characterized by excessive proliferation and abnormal differentiation of keratinocytes, is caused by various factors, the pathogenesis is not completely clarified, a plurality of immune cells, immune-related cells (including dendritic cells, T cells, endothelial cells and the like) and cytokines are considered to participate in the pathogenesis of psoriasis and the maintenance of disease states at present [1], and the existing animal models with wide application can be roughly divided into a drug-induced acute inflammation model, a genetic engineering model and a xenotransplantation model.
Research on IMQ (Imiquimod, IMQ) induced model, wherein IMQ is an agonist of Toll-like receptor (TLR) 7/8, Van der Fits [2] and the like discovers that the change of psoriasis-like skin lesions of mice induced by IMQ is accompanied by the change of IL-23/IL-17 axis, has many similarities with the pathological change of human psoriasis, and has the advantages of simple and convenient operation, low cost and the like, so the method for smearing the skin of the mice with the IMQ to establish the psoriasis is the psoriasis model widely applied at home and abroad at present. However, as an acute inflammatory model, the limitations are: (1) non-specific, not only in psoriasis, but also in other diseases such as systemic lupus erythematosus, atopic dermatitis and other skin lesions [3], IMQ-induced skin inflammation model was also used for systemic lupus erythematosus research [4 ]; (2) short treatment window, topical application of imiquimod causes dehydration and loss of body mass in mice, and continuous topical application for more than 2 weeks may lead to death of mice [5 ]; (3) poor repeatability, lack of standard operating procedures, etc.
A xenogeneic skin graft immunodeficient mouse (SCID) model, in which human skin is transplanted into SCID mice, and T cells injected into patients induce psoriasis symptoms, the genetic phenotype and pathogenic process of which are closest to those of human psoriasis, and are not affected by anti-drug antibodies (ADA) due to animal immunodeficiency, are one of the most suitable tools for studying the pathogenesis of psoriasis and drug development, and are most suitable for preclinical analyses for exploring new anti-psoriasis agents/treatment strategies prior to the start of clinical trials [6,7,8 ].
2.1 evaluation of the efficacy of NVS451 in the xenograft skin-transplanted SCID mouse psoriasis model (5, 10, 15mg/kg NVS 451).
NVS451 protein at the above concentrations was formulated in 20mM Tris-acetate, 65mM arginine, 120mM trehalose, 0.02% Tween 20, pH 7.5.
A total of 60 SCID mice (Envigo, yasuan, israel c.b-17/IcrHsd-SCID-bg (beige-SCID) mice) were used for the experiment, and healthy skin was transplanted onto the back of the mice and randomly divided into 6 groups of 10 mice each: negative control group (negative control group is prepared by using the preparation formula to remove NVS451), hormone group (2 mg/animal dexamethasone), secukinumab group (60mg/kg), test sample high dose group (15mg/kg), middle dose group (10mg/kg) and low dose group (5 mg/kg). The negative control group, the test group and the Sujin group were administered by subcutaneous injection for 28 days. Dexamethasone was administered topically 2 times daily for 28 days. At the end of the experiment, the skin appearance was observed (see figure 12 for results), and the skin thickness was measured. The results show that: NVS451 has a certain improvement effect on red swelling, plaques and scales, and is positively correlated with the dosage; the 15mg/kg dose group had no statistical difference in skin thickness (P > 0.05) from the sujin group, and NVS451 in this model had similar therapeutic effects to sujin.
TABLE 8 visual examination of xenograft skin results
Figure BDA0003701246140000281
TABLE 9 histological evaluation and thickness of xenograft skin
Figure BDA0003701246140000282
Dexamethasone vs total treatment, -p < 0.01
High dose test product compared to secukinumab, -not significant
Comparison of high dose test article and secukinumab to low and medium dose, -p < 0.05, p < 0.05
2.2 evaluation of the efficacy of NVS451 (formulation as described in 2.1) in the xenograft skin-grafting SCID mouse psoriasis model (15, 22.5, 30, 45, 60mg/kg NVS451)
A total of 100 SCID mice (purchased from Envigo) were used in the experiment and were randomly divided into 10 groups after healthy skin was transplanted onto the backs of the mice (see Table 10). At the end of the experiment, the appearance of the skin was observed (see results in fig. 13), the transplanted skin was taken for the histological evaluation related to psoriasis (see results in fig. 14), and the skin thickness was measured. The results show that the NVS451 groups all showed therapeutic effects, with the 15mg/kg twice a week, 30mg/kg twice a week, 22.5mg/kg twice a week and 60mg/kg once a week groups having similar efficacy, with the xenograft skin all 7/10 fully recovered and the epidermis thickness decreased. However, 15mg/kg twice weekly, the remaining 3/10 showed partial recovery with a skin thickness of 334 ± 174 μm and a histological score of 1.1 (overall evaluation based on skin lesion epidermal thickness, thinning of the epidermis on the nipple, hyperkeratosis, parakeratosis, agranulocytosis, Munro micro-abscesses, elongation of normal or abnormal reticulum crest, vascular tortuosity, dermal papillary layer mononuclear cell infiltration, etc.).
TABLE 10 study groups and dosages (adjuvant control group formulation minus NVS451)
Figure BDA0003701246140000291
TABLE 11 histological evaluation and thickness of xenograft skin
Figure BDA0003701246140000301
3. Secondary pharmacodynamics
3.1 ADCC/CDC Effect-related receptor binding affinity assay
The mechanism of action of NVS451 to exert drug effect is independent of ADCC/CDC activity, and the cytotoxicity ADCC/CDC of Fc may bring unnecessary immune-related adverse events (iraE), with potential safety risk [9]]. To avoid this effect, the Fc portion of NVS451 was mutated at the relevant active site. In the affinity assay study, 7 CD molecules associated with ADCC and C1q molecules associated with CDC were evaluated. The results indicate that NVS451(75ug/ml) has a non-binding or low level (KD greater than 10) of binding affinity to ADCC-associated CD molecules -5 M order of magnitude, see table 12). In another study, NVS451 had significantly reduced affinity for ADCC/CDC-associated Fc receptors compared to an otherwise partially identical IL-17AR fusion protein (V301-wtFc) (75ug/ml) but with native IgG1 Fc (see Table 13).
The amino acid sequence of V301-wtFc is shown in SEQ ID NO. 17.
TABLE 12 affinity assay of NVS451 for ADCC and CDC Effect-associated receptors
Figure BDA0003701246140000311
TABLE 13 NVS451 and V301-wtFc affinity assays for ADCC and CDC Effect-related receptors
Figure BDA0003701246140000312
Figure BDA0003701246140000321
3.2 evaluation of ADCC/CDC functional Activity of Fc fragment region
The Fab region of IgG1 and IgG3 antibody subtypes bind to target cells, and the Fc portion of the Fab region binds to Fc receptors (e.g., Fc γ RIII) on CTL cells such as NK cells, and ADCC is induced. Cell models lacking membrane-expressed IL-17 are not effective in verifying in vitro the development of ADCC. Thus, the Fab fragment of Rituximab (Rituximab), which stimulates ADCC, was fused with the Fc fragment of NVS451 to construct RitxV301 (i.e., RitxNV 451) verification 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). The removal of ADCC/CDC effect was verified (experimental data were analyzed using GraphPad Prism 5 analysis software, logarithm of antibody concentration was used as X-axis, corresponding calculated killing value was used as y-axis, four-parameter equation regression simulation was used to fit the antibody' S measured effect curve, the data were analyzed using GraphPad Prism 5 software, positive control antibody (rituximab) was fit to the four-parameter equation y ═ D + (a-D)/[1+ (X/C) B ], typical sigmoidal curve on semi-logarithmic axis, R2 ≧ 0.95, EC50 was calculated, ADCC and CDC effect were judged by EC50 values)). The results of the study showed (fig. 15 and 16): rituximab (roche (switzerland) shows strong ADCC and CDC killing effects on Raji cells, while RitxV301 shows no ADCC and CDC effects, indicating that the design of the Fc region of NVS451 is such that RitxV301 cannot induce NK-92MI CD16a cells to kill Raji cells, and the Fc region of NVS451 cannot bind to C1q molecules in complement and cannot initiate the complement cascade.
3.3 FcRn binding affinity assay
Fc fusion proteins generally improve the half-life of the protein drug of interest by FcRn-mediated circulation of Fc [10]. The magnitude of binding affinity for FcRn correlates with the half-life of the drug in vivo [11]. NVS451 binds to FcRn (enantiomers FCM-H5286, FCM-C5284, FCM-R5287) in human, monkey, and rat. Under the same conditions, the affinity of the protein is similar to that of the thuringiensis antibody and wild type IgG1 Fc (namely V301)wtFc) are all at the same level (KD of the order of 10 -8 M, see table 14), the experimental method is as follows: .
Reagent preparation
Running the reagent: containing 2mM KH 2 PO 4 ,10mM Na 2 HPO 4 137mM NaCl,2.7mM KCl, 0.05% Tween-20 (Tween-20), pH adjusted to 6.0;
his capture kit (cat # 28-9950-56, GE) comprising: mouse anti-His antibody (1mg/mL), immobilized reagent (10mM sodium acetate, pH 4.5), regenerated reagent (glycine hydrochloric acid, pH 1.5);
an amino coupling kit (cat # BR100050, GE) comprising: 115mg of N-hydroxysuccinimide (NHS), 750mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 10.5mL of 1M ethanolamine (pH 8.5). 10mL of deionized water is respectively added into each tube of EDC and NHS, and the mixture is subpackaged and stored at a temperature of-18 ℃ to a lower temperature and has a shelf life of two months. (see GE amino coupling instruction Manual 22-0510-62 AG).
IL-17RA-wtFc (i.e. V301-wtFc) protein, FcRn protein of different species, was solubilized according to the product COA and dispensed at a size of greater than 10 μ g per tube. Repeated freeze thawing is avoided.
Protein desalination
Secukinumab and IL-17RA-wtFc protein were desalted using desalting column and Running Buffer (Running Buffer). The concentration of the desalted protein was measured by UV-V. The desalted protein is packaged in a size of more than 10 mug per tube, and repeated freeze thawing is avoided.
Chip preparation
The mouse anti-His antibody was diluted to 50. mu.g/mL with the immobilized reagent (10mM sodium acetate, pH 4.5), using about 100. mu.L of mouse anti-His antibody per channel on the chip, and adding 10. mu.L of mouse anti-His antibody using about 190. mu.L of the immobilized reagent for both channels. First, the surface of a CM5 chip (biocore sensor chip, GE) was activated for 420 seconds with 400mM EDC and 100mM NHS at a flow rate of 10. mu.L/min. Next, 50. mu.g/mL of mouse anti-His antibody was injected into the experimental channel (FC4) at a flow rate of 10. mu.L/min for about 420s, at a fixed amount of about 9000 to 14000 RU. Finally, the chip was blocked with 1M ethanolamine at 10. mu.L/min for 420 s. The reference channel (FC3) was subjected to the same procedure as the test channel (FC 4). (refer to the His Capture kit instruction manual of GE, 28-9974-71 AB).
Capture ligands
An FcRn stock sample was diluted to 0.5 μ g/mL with running reagent and injected into the experimental channel (FC4) at a flow rate of 10 μ L/min for approximately 40 RU. The reference channel (FC3) did not require capture of the ligand.
Analyte multicycle analysis
NVS451, secukinumab and IL-17RA-wtFc samples with different concentrations are diluted by corresponding running reagents, and the diluted samples are injected into an experimental channel and a reference channel at a flow rate of 30 mu L/min according to corresponding binding time and dissociation time in sequence. After each concentration analysis, the chip was regenerated for 60s with glycine HCl pH 1.5 at a flow rate of 30 μ L/min, washing off the ligand and undissociated analyte. For the next concentration analysis, the experimental channel needs to recapture the same amount of ligand.
Others
All the operation steps are carried out in the running reagent, and the analytical reagent of SPR needs to be filtered and degassed for use.
Results
And (3) data analysis:
KD values for each antibody were calculated using Biacore 8K analysis software. The reference channel (FC3) was used for background subtraction.
NVS451 (concentration: 25nM-1.5625nM) binds to FcRn from different species of human, cynomolgus monkey, rat 3 in 4 independent test studies at pH6.0 under acidic conditions (FIG. 17); and no binding was observed under neutral conditions (NVS451 concentration 100nM to 1.5625nM) at pH7.4 (FIG. 18).
TABLE 14 FcRn receptor affinity comparison of human, rat, and cynomolgus monkey
Figure BDA0003701246140000351
Experimental example 2: evaluation of safety
1. Safe pharmacology
1.1 Macaca fascicularis safety pharmacological test
The safe pharmacological test of cynomolgus monkeys (purchased from Beijing Zhongkoiling Biotechnology Ltd.) was carried out with long toxicity by subcutaneous injection of the formulation in section 2.1 of Experimental example 1 at doses of 15mg/kg, 50mg/kg and 150mg/kg of NVS451(2 times weekly, 4 consecutive weeks, 9 times in total, i.e., D1, D4, D8, D11, D15, D18, D22, D25 and D29, respectively). In the test, an auxiliary material control group is set, NVS451 is removed by using a prescription in section 2.1 of experimental example 1, and safety pharmacological indexes are detected by combining toxicity index observation, so that the mental state, behavior and activity and the like of animals in each group are not abnormal; the animals breathe smoothly without tachypnea or slowdown, and without abnormal chest or abdominal breathing. Respiratory index and electrocardiographic examination of each group of animals were performed at different time points (before the first dose (D-2), and 4h (+ -20 min), 24h (+ -20 min), 48h (+ -20 min), 72h (+ -20 min) after the first dose), and no administration-related TV and RR were observed, and no regular changes in heart rate, P-R interval, QT interval, QRS time limit, QRS voltage, STe, and QTcB values were observed, and electrocardiographic waveforms were not abnormal. Performing electrocardiogram limb II lead measurement 3-5 hours (D1) after the first dose (D-2), 3-5 hours (D15) after the 5-time dose (D1), 3-5 hours (D29) after the last dose and before the recovery period (D56), observing the heart rate, electrocardiogram waveform, P-R interval, QRS time limit and Q-T interval, wherein no abnormality is found, and no obvious abnormal change is found in blood pressure detection including MBP, DBP and SBP detection at each time point. Therefore, the test sample is considered to have no obvious influence on the central nervous system, the cardiovascular system and the respiratory system of the cynomolgus monkey by subcutaneous injection at the dosage of 15, 30 and 150 mg/kg.
1.2 central nervous System test in rats
The effect of a single subcutaneous administration of NVS451 on the central nervous system function of SD rats (purchased from the laboratory animal technology company Limited of Wei Tony, Beijing (animal production permit: SCXK (Jing)) at doses of 30mg/kg (low dose), 100mg/kg (medium dose) and 300mg/kg (high dose) under GLP (good laboratory practice) conditions, respectively, male animals in the high-dose group of the test article showed an increase in body temperature 2h after administration (the adjuvant control group (formulation prescription minus NVS451) vs high-dose group: 37.72 + -0.29 ℃ vs 38.40 + -0.14 ℃), the animals in the high-, medium-and low-dose groups showed an increase in body temperature 24h after administration, and the adjuvant control group: 37.60 + -0.28 ℃ vs 38.80 + -0.23, 38.72 + -0.44, 38.54 + -0.28 ℃) were evaluated by the functional combination of observations (FOB). The above-mentioned changes in body temperature were not observed in male-female concordance and no regular change, and were considered to be irrelevant to administration. In the cage observation, hand grasping observation, open environment observation and irritation reaction observation processes, no abnormal expression related to administration of the animals appears; no changes in the animal erection count, fecal mass count, forelimb grasping power, and body temperature were observed in relation to the test article. The result shows that NVS451 has no influence on the central nervous system function of rats under the test condition.
2. Pharmacokinetics
2.1 in vivo kinetics of cynomolgus monkeys
A total of 24 cynomolgus monkeys were used in the experiment and divided into 4 groups (6 animals per group, male and female halves). Single subcutaneous and intravenous administration, groups 1 to 4 were given NVS451 at 5 (subcutaneous), 15 (subcutaneous), 50 (subcutaneous), 15 (intravenous) mg/kg, respectively, at a dose capacity of 1 mL/kg.
Test A drug-containing blood sample (about 1mL) was collected from the non-administration site of the subcutaneous vein of the hind limb of the animal into a tube without anticoagulant, and the blood sampling time points of 1-3 groups of animals were: pre-drug (previous day), 1h, 2h, 4h, 8h, 24h (D2), 32h (D2), 48h (D3), 56h (D3), 72h (D4), 96h (D5) after the start of administration; the blood sampling time points of the animals in group 4 were: before drug administration (day before), 3 minutes after the start of drug administration, 1h, 2h, 6h, 24h (D2), 32h (D2), 48h (D3), 56h (D3), 72h (D4) and 96h (D5). Blood samples were used to prepare serum samples and for pharmacokinetic analysis (table 15).
TABLE 15 pharmacokinetic parameters of NVS451 in cynomolgus monkeys
Figure BDA0003701246140000371
Figure BDA0003701246140000381
C max Ratio is C max Medium and high dose mean/C max Low dose mean;
AUC ratio AUC last Medium and high dose mean/AUC last Low dose mean;
F％＝(AUC last dose (SC))/(AUC) last Dose (IV)). 100%.
The test result shows that: within the dosage range of 5-50 mg/kg, after a single subcutaneous injection is given to the cynomolgus monkey, the blood concentration of NVS451 in the cynomolgus monkey body is increased along with the increase of the dosage; no significant difference between sexes was observed after a single subcutaneous and intravenous administration to cynomolgus monkeys.
Average C of NVS451 after single subcutaneous injection of NVS451 at 5, 15, 50mg/kg in cynomolgus monkeys max And AUC last The growth rate is higher than the dose growth rate. C of NVS451 given by single subcutaneous injection to 5, 15, 50mg/kg dose group animals max The ratios were 1:6.12:38.06 (male) and 1:5.46:34.29 (female), respectively; AUC last The ratios were 1:4.83:25.52 (male) and 1:4.76:20.88 (female), respectively.
Independent sample t test shows that the metabolic kinetic parameters of subcutaneous injection and intravenous injection have no statistical difference between sexes, and the metabolic characteristics of NVS451 in animals have no obvious sex difference basically.
NVS 451T in cynomolgus monkeys at 5-50 mg/kg dose 1/2 Mean T of Male and female animals in each dose group 1/2 Between 27.20 and 39.90; subcutaneous injection of T for each dose group max Basically consistent, and the drug concentration reaches the peak within 4-8 h.
2.2 rat in vivo kinetics
The test shared 48 SD rats divided into 4 groups (12 animals per group, male and female halves). Single subcutaneous and intravenous administration, groups 1 to 4 were given 10 (subcutaneous), 30 (subcutaneous), 100 (subcutaneous), 30 (intravenous) mg/kg NVS451, respectively, at a dose capacity of 2 mL/kg.
Test A drug-based blood sample (about 0.4mL) was collected from the jugular vein of an animal into a tube without anticoagulant, and the blood sampling time points of 1-3 groups of animals were: before administration (D-1), 1h, 2h, 4h, 8h, 24h, 32h, 48h, 56h, 72h and 96h after the start of administration; the blood sampling time points of the animals in the 4 th group are as follows: before administration (D-1), 3min, 1h, 2h, 6h, 24h, 32h, 48h, 56h, 72h and 96h after the start of administration. Blood samples were used to prepare serum samples and for pharmacokinetic analysis (table 16).
TABLE 16 pharmacokinetic parameters of NVS451 in SD rats
Figure BDA0003701246140000391
C max Ratio is C max Medium and high dose mean/C max Low dose mean;
AUC ratio AUC last Medium and high dose mean/AUC last Low dose mean;
F％＝(AUC last dose (SC))/(AUC) last Dose (IV)). 100%.
The test result shows that: within the dose range of 10-100 mg/kg, after a single subcutaneous injection is given to SD rats, the blood concentration of NVS451 in SD rats is increased along with the increase of the dose; no significant difference between sexes was observed after single subcutaneous and intravenous administration to SD rats.
After a single subcutaneous injection of NVS451 at a dose of 10, 30, 100mg/kg to SD rats, the mean Cmax and AUClast increase rates for NVS451 were lower than the dose increase rate. The Cmax ratio of NVS451 for the single subcutaneous injection group at 10, 30, 100mg/kg dose was 1: 2.03: 6.56 (Male) and 1: 1.62: 6.04 (female); the AUClast ratios are 1: 1.92: 5.29 (Male) and 1: 1.85: 5.85 (female).
The metabolic kinetic parameters of subcutaneous injection and intravenous injection have no obvious sex difference, and the metabolic characteristics of NVS451 in animals have no obvious sex difference basically.
In the dose range of 10-100 mg/kg, NVS451 is T1/2 in SD rats, and T1/2 of male and female animals in each dose group is 51.46-69.48; tmax of each dose group injected subcutaneously is basically consistent, and the drug concentration reaches the peak within 8-24 h.
3. Distribution and drainage
In the experiment, the tissue distribution and excretion characteristics of SD rats after the isotope (125I) -labeled NVS451 fusion protein (NVS451) for injection is subcutaneously injected are researched mainly by adopting a TCA protein precipitation method in combination with SHPLC (molecular exclusion high performance chromatography).
The test was carried out with 36 animals in total, divided into 6 groups of 6 animals each, half male and half female. The test article was administered as a single cervical and dorsal subcutaneous injection at a dose of 30 mg/kg. The 125I-labeled sample had an assay purity of 99.03% and a specific activity of 0.09 KBq/. mu.g. Animals of groups 1-4 were collected at 4h, 24h, 96h and 120h after administration, thyroid, heart, lung, liver, spleen, kidney, bladder, gonad (ovary, testis), stomach, small intestine, fat, muscle, brain, back skin (non-administration site), intestinal contents, serum, urine, respectively. Feces and urine were collected once a day for 5 days after administration to 5 groups of animals. The bile drained from 6 groups of animals was collected 1 time per hour after injection for a total of 8 hours.
The test result shows that:
1) after the rats are injected with 125I-NVS451 by subcutaneous injection, the medicine is mainly distributed in tissues/organs such as intestinal contents, urine, bladder, back skin and the like, and is less distributed in muscle, fat and brain. The peak time of the drug in most tissues is 4-24 h, and then the concentration of the drug gradually decreases with the time.
The drug has a certain distribution in the dorsal skin (non-administration site). The peak reaching time is 24 hours, and the drug content in 4 hours is approximately equal to that in 120 hours. The drug concentration in the skin of the back decreases more slowly than in other tissues/organs.
2) After the rats are injected subcutaneously and administered with 125I-NVS451, radioactivity is mainly excreted by kidneys, and more than 90% of the administered drug is excreted after 5 days; bile excretion is less.
4. Metabolism
According to the guidelines for "preclinical safety assessment of ICH-S6(R1) biologics," NVS451 is a macromolecular protein drug that is expected to degrade into peptides and amino acids in vivo, and then be excreted or reused for protein or peptide synthesis in vivo, and thus the metabolism of NVS451 was not assessed.
5. Toxicology
The cynomolgus monkey and SD rat were selected for toxicological studies, and NVS451 cross-reacts with cynomolgus monkey and rat IL-17A, which is a related species. The tests were all performed under GLP conditions, following the current good laboratory practice of the food and drug administration (21CFR Part 58), the "quality control practice for non-clinical research on drugs" of the national drug administration (former national food and drug administration (ministry of administration for supervision and administration for drug and drug administration) (title 34, 9 months 2017).
6. Single dose toxicity
6.1 toxicity study of Macaca fascicularis
In order to evaluate the possibility of acute toxicity after a single subcutaneous injection of NVS451 to a cynomolgus monkey within 14 days, three dose groups of 45mg/kg, 150mg/kg and 450mg/kg were set. No mortality or moribundity was observed in any of the groups during the test. No abnormality is found in clinical observation of animals in each administration group. Compared with the prior-drug value and the auxiliary material control group (NVS451 is removed from the preparation prescription), the weight, the body temperature, the electrocardiogram parameters, the blood cell count, the blood coagulation index, the blood biochemistry and the urine examination of animals in each administration group have no administration related abnormal change. At the end of the observation period (D15), all animals had no gross anatomical abnormalities. And (4) conclusion: under the conditions of this test, no mortality or moribundness, no administration-related abnormal changes, and a Maximum Tolerated Dose (MTD) of greater than or equal to 450mg/kg were observed in all animals.
6.2 rat toxicity study
In order to evaluate the possible acute toxicity reaction condition in 14 days after a single subcutaneous injection of NVS451 to SD rats, three dose groups of 90mg/kg, 300mg/kg and 900mg/kg are set. During the test period, no death or moribund of each animal group was observed, the observation period was over (D15), and no abnormal change was observed in each animal group. And (4) conclusion: under the test conditions, all animals are not dead or dying, and the Maximum Tolerated Dose (MTD) of the animals is greater than or equal to 900 mg/kg.
7. Repeated administration toxicity
7.1 toxicity study on repeated dosing of cynomolgus monkeys
40 cynomolgus monkeys (20/sex) were used, and randomly divided into 4 groups (5/sex/group) by sex, which were an adjuvant control group and low, medium, and high dose groups of the test article, respectively. The animals in the adjuvant control group are administered with 2mL/kg adjuvant control, and the low, medium and high dosage groups of the test sample are administered with NVS451 at doses of 15, 50 and 150mg/kg, respectively, at doses of 0.2, 0.67 and 2mL/kg, respectively, and at a dose concentration of 75 mg/mL. All animals were administered via hind limb subcutaneous injection 2 times a week for 4 weeks for 9 times, i.e., D1, D4, D8, D11, D15, D18, D22, D25 and D29. During the test period, the animals are subjected to clinical observation, weight, body temperature, electrocardiogram, respiration (respiratory frequency and tidal volume), blood pressure, ophthalmologic examination, blood cell count, blood coagulation function, blood biochemistry, urine analysis and detection of immunological indexes (T lymphocyte subpopulation, cell factor, C-reactive protein, serum immunoglobulin, complement, anti-drug antibody) indexes; blood concentration was measured in each group of animals before and after the first and 8 th administrations. Part of the animals (3/sex/group) were euthanized the next day after the last dose (D30), the remaining animals were euthanized at the end of the 4-week recovery period (D57); all animals were bone marrow smeared and read, gross anatomical observations were made, the major organs were weighed, the relative organ weights (visceral volume ratio and visceral brain ratio) were calculated, and histopathological examination was performed on over 40 histo-organs.
As a result: during the test, no death or dying was observed in each group of animals, and no abnormal change associated with administration was observed in each group of animals in clinical observation, body weight, body temperature, ophthalmic examination, urine examination, blood cell count, blood coagulation function (excluding FIB), biochemical indices of blood, lymphocyte subpopulation, complement, cytokines (excluding IL-17A), and immunoglobulin. No abnormal changes related to drug administration were observed in the electrocardiogram, respiration and blood pressure parameters of the animals. Abnormal reactions such as erythema, edema, induration, ulceration and the like are not seen when the medicine is locally and visually observed.
During the test period, the test high dose group males observed an increase in CRP (193.0%) 5 days after dosing (D16) compared to the same cohort adjuvanted control group; on the next day after the last dose (D30), CRP (70.5%, 218.5%, 246.9%) was observed in the low, medium and high dose test group males, and FIB (56.4%) was also observed in the high dose test group males, with statistical significance of the difference (P ≦ 0.05). After 4 weeks of withdrawal, all the changes were completely recovered.
7.1.1 histopathological examination:
no death or dying condition of the animals is found in the test process.
After the last dose (D30), the regional injection and inguinal lymph nodes of the animals of 15, 50 and 150mg/kg dose groups are changed related to the test sample, wherein the regional injection shows slight to moderate mononuclear cell inflammation of dermis/subcutaneous/muscular layer and is the irritant reaction caused by the test sample; the minor to moderate accessory cortex area/medullary cord lymphocyte of the inguinal lymph node is increased in number, is related to local mononuclear cell inflammation by injection, is not obviously changed by combining with relevant indexes of clinical pathology, and is considered as non-adverse reaction.
At the end of the 4-week recovery period (D57), the changes associated with the test article in the 15 and 50mg/kg dose groups were completely recovered and the changes associated with the test article in the 150mg/kg dose groups were substantially recovered.
7.2 repeated dose toxicity Studies in rats
192 SD rats, male and female, were used, and divided into 8 groups by sex section at random according to body weight, 120 rats of 1-4 main test groups were used for toxicology study (15 rats/sex/group), and 72 rats of 5-8 satellite groups were used for serum antibody and pharmacokinetic test (6-10 rats/sex/group). During the test period, the vehicle control group animals were given NVS451 white vehicle solution (4 mL/kg); the low, medium and high dose fractions of the test sample were administered to NVS451 at doses of 30, 100, 300mg/kg, respectively, and at doses of 0.4, 1.33, 4mL/kg, respectively. All animals were given a subcutaneous injection at the back of the neck 2 times a week for 4 weeks for 9 total doses (i.e., D1, D4, D8, D11, D15, D18, D22, D25, and D29) for a 4-week recovery period.
During the test period, the animals in the main test group are mainly clinically observed and detected for weight, food intake, body temperature, blood cell count, blood coagulation function, blood biochemistry, ophthalmology examination, urine examination, T lymphocyte subpopulation and cell factors; the animals of the satellite group were bled before and after the first and 8 th administrations for pharmacokinetic testing and antibody measurements at different time points (before the first administration (D-1), before the 15 th administration (D14), before the last administration (D28) and at the end of the recovery period (D56)). The first 10 animals/sex/group of the main test group were euthanized the next day after the last dose (D30), and the remaining animals were euthanized at the end of the 4-week recovery period (D57). All animals in the main test group were subjected to gross anatomical observation, the main organs were weighed, and the relative organ weights were calculated; and injecting the auxiliary material into more than 40 tissues and organs of the animals in the control group and the high-dosage group and the animals in the low-dosage and medium-dosage groups for local injection and histopathological examination.
As a result: during the test period, no death or dying of each group of animals is observed, and no abnormality is observed in clinical observation. No erythema, congestion, swelling, ulceration and induration were observed visually on the dosed area. The ophthalmic examination and the urine examination of each group of animals are not abnormal; compared with the control group with the same-period auxiliary materials, the animal weight, body temperature, food consumption, blood cell count, blood coagulation function, blood biochemistry and T lymphocyte subpopulation of each group have no abnormal change related to administration.
7.2.1 pathological examination:
no animals died during this test.
The animals were euthanized and observed for gross observation, and no changes were observed in relation to the test article.
Animals were euthanized at the end of dosing (D30), and the groups at 30, 100 and 300mg/kg doses had local visible irritative reactions associated with the test article, manifested by a slight to moderate mononuclear cell inflammation of the local subcutaneous and/or dermal injection. By the end of the 4-week recovery period, the local inflammation was substantially completely recovered by injection.
8. Local tolerance to toxicity
Local tolerability toxicity test was carried out with prolonged toxicity, and cynomolgus monkey and SD rat were dosed twice a week (three rat dose groups 30, 100 and 300mg/kg, three cynomolgus monkey dose groups 15, 50 and 150mg/kg) for 4 weeks (i.e. D1, D4, D8, D11, D15, D18, D22, D25 and D29) for 9 consecutive doses. During the test period, abnormal reactions such as erythema, edema, induration, ulceration and the like are not observed in the local administration of each group of animals, the local tolerance is good, and toxicity is not seen.
9. Other toxicity
Using an in vitro test tube method, the effect of 75mg/mL NVS451 on hemolysis and aggregation of human erythrocytes was observed (experimental conditions are conventional). From 15 minutes after the incubation in the incubator to the end of 3 hours of observation, the upper liquid of the test tube can be seen in the test tube, colorless and clear, the red blood cells at the bottom of the test tube sink, and are uniformly dispersed after shaking, so that hemolysis and condensation do not occur. Under the test conditions, 75mg/mL of NVS451 did not cause hemolysis or aggregation of human erythrocytes in vitro.
Conclusion
1. Pharmacodynamic comprehensive assessment and conclusion
In vitro pharmacodynamics: the analysis of binding affinity with IL-17A showed that NVS451 binds human IL-17A with high affinity, 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 binds to IL-17C, IL-17F, IL-17 AF. NVS451 had a higher affinity for human IL-17C, IL-17F than wild-type human IL-17 RA. The cell experiment of biological activity shows that NVS451 fusion protein shows the inhibition ability to IL-17A, IL-17A/F, IL-17F-induced GRO-alpha cytokine, and the inhibition ability to IL-17A-induced GRO-alpha cytokine is better than the IC of Sujin monoclonal antibody (Cosentyx) 50 The value is lower.
Based on these in vitro potency results, NVS451 acts primarily by inhibiting IL-17A and IL-17C, IL-17F, IL-17AF activity. The interactions shown by the SPR experiments suggest that in vivo, the recommended dose of NVS451 exerts a primary inhibition of IL-17A in psoriasis patients, supplemented by the clinical activity of the IL-17C, IL-17F, IL-17AF molecule.
The affinity analysis study showed that the binding affinity of NVS451 to ADCC-associated CD molecules was at a non-binding or low level and that its affinity to ADCC/CDC-associated Fc receptors was significantly reduced compared to native IgG1 Fc, demonstrating that the ADCC binding site of NVS451 was removed by mutation. Binding affinity analysis to FcRn showed that NVS451 can bind to FcRn in human, monkey, rat. Under the same conditions, the affinity of the protein is the same as that of secukinumab and wild type IgG1 Fc, NVS451 can be combined with FcRn of different species of human, cynomolgus monkey and rat 3 under the acidic condition of pH6.0, and is not combined under the neutral condition of pH 7.4. NVS451 is a soluble antibody Fc fusion protein carrying the Fc domain of human IgG1 and is therefore theoretically capable of interacting with Fc receptors, but the mechanism of action of NVS451 to exert its pharmacological effect is independent of ADCC/CDC activity, and the cytotoxicity of Fc, ADCC/CDC, may bring unwanted immune-related side effects, with potential safety risks. To avoid this effect, the Fc portion of NVS451 was mutated at the relevant active site. Studies demonstrated a significant decrease in ADCC/CDC/ADCP-related Fc receptor affinity compared to native IgG1 Fc, and target cell killing experiments (as shown in figures 15 and 16) demonstrated that NVS451 had no ADCC/CDC activity.
In vivo pharmacodynamics: functionally in vivo, NVS451 was evaluated for efficacy on psoriasis, primarily in a human T cell driven psoriasis model. Studies have demonstrated that human skin xenograft models are best suited for preclinical analysis before the start of clinical trials exploring new anti-psoriasis agents/treatment strategies, showing the efficacy of NVS451 in treating humanized psoriasis models by xenograft SCID mouse model skin histology assessment including score and epidermal thickness, in xenograft SCID mouse psoriasis models NVS451 is administered at 5mg/kg, 10mg/kg and 15mg/kg doses every other day for 28 days, showing a certain degree of improvement in NVS451 on redness, plaques and scales, and positively correlated with dose; the 15mg/kg dose group had no statistical difference in skin thickness from the secukins group (P > 0.05), and treatment with the highest dose of NVS451(60mg/kg) once a week was as effective as treatment with the low dose twice a week, most importantly, treatment with NVS451 twice a week or treatment with the high dose once a week showed similar therapeutic effects to the positive control of secukinumab. Secukinumab is considered one of the most promising drugs for the treatment of psoriasis, and this study provides strong clinical evidence support for the effectiveness of clinical treatments.
Safe pharmacology: under GLP conditions, NVS451 was administered at 30mg/kg, 100mg/kg and 300mg/kg by single subcutaneous injection, and the results showed that NVS451 had no effect on rat central nervous system function. The cynomolgus monkey safety pharmacological test is carried out along with long toxicity: the test article was injected subcutaneously at doses of 15, 30 and 150mg/kg, and showed no significant effect on the central nervous system, cardiovascular system and respiratory system of cynomolgus monkeys.
2. Comprehensive evaluation and conclusion of pharmacology and toxicology
Since NVS451 cross-reacts with cynomolgus monkeys and rodent IL-17A, cynomolgus monkeys and rats were selected as the relevant species for toxicity evaluation. NVS451 was administered to cynomolgus monkeys by repeated subcutaneous injections at 15, 50, and 150mg/kg doses, 2 times per week for 4 consecutive weeks for 9 total doses, and no death or moribund of each group of animals was observed, and no significant systemic toxicity was observed, and the non-adverse effect dose (NOAEL) in this test was considered to be 150 mg/kg. The NVS451 fusion protein for injection is repeatedly injected subcutaneously into SD rats at the dose of 30, 100 and 300mg/kg for 2 times per week, continuously administered for 4 weeks, and administered for 9 times, and the animals have no obvious systemic toxicity reaction and are considered to have no adverse reaction dose (NOAEL) of 300 mg/kg. Cynomolgus monkey and rat pharmacokinetics showed no accumulation of NVS451 after repeated dosing.
Single dose non-GLP pharmacokinetic experiments were performed in cynomolgus monkeys and rats to observe the pharmacokinetic basic of NVS451 administered via intravenous and subcutaneous routes. The absorption pharmacokinetics of NVS451 in male and female cynomolgus monkeys after a single subcutaneous administration was evaluated, and its pharmacokinetic behavior had a low serum clearance and half-life of typical immunoglobulin molecules, and effective drugs were detected after 96 hours of subcutaneous administration in each dose group. In a single pharmacokinetic experiment of a cynomolgus monkey and an SD rat, the metabolic data have no obvious sex difference, the NVS451 is T1/2 in the cynomolgus monkey in a dose range of 5-50 mg/kg, and the average T1/2 of males and females in each dose group is between 27.20-39.90 h. NVS451 has T1/2 in SD rats within the dose range of 10-100 mg/kg, and T1/2 in male and female animals of each dose group is between 51.46-69.48 h.
3. Non-clinical conclusions:
NVS451 is a high affinity antibody Fc fusion protein that selectively targets IL-17A, IL-17C, IL-17F and IL-17AF heterodimeric cytokines, binding data indicating that NVS451 has high affinity for a predetermined target in humans and cynomolgus monkeys.
In summary, the present invention provides a summary of non-clinical data for a program that provides evidence of target specificity and mode of action, showing similar therapeutic effects to the positive control of secukinumab using NVS451 twice a week or high dose once a week. NVS451 was well tolerated in a comprehensive toxicology study. All non-clinical evaluation studies provide strong evidence support for the effectiveness and safety of clinical treatment of NVS 451.
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SEQUENCE LISTING
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Valin Biotechnology Ltd
The National Institute for Biotechnology in the Negev Ltd.
<120> IL-17RA fusion protein, pharmaceutical composition, injection and application thereof
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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 (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 (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 (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 (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 (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 (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> human (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 (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 (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 (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 (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 (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 (artificial sequence)
<400> 19
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile
35 40 45
Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly
100 105 110
Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Ser
210 215 220
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Gly Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450

Claims (25)

1. An IL-17RA fusion protein comprising, in operable linkage, in series, a signal peptide, an IL-17RA extracellular domain, and an IgG1 constant region.
2. The IL-17RA fusion protein of claim 1, wherein the amino acid sequence of the IL-17RA extracellular domain is set forth in SEQ ID No.1, SEQ ID No.2, or SEQ ID No. 3.
3. The IL-17RA fusion protein of claim 1, wherein the amino acid sequence of the IgG1 constant region is set forth in SEQ ID No. 4.
4. The IL-17RA fusion protein of claim 1, wherein the signal peptide is a native IL-17-RA signal peptide having an amino acid sequence as set forth in SEQ ID No. 5.
5. The IL-17RA fusion protein of claim 1, wherein the IL-17RA extracellular domain is linked to the IgG1 constant region with a linker.
6. The IL-17RA fusion protein of claim 1, wherein the amino acid sequence of the IL-17RA fusion protein is set forth in SEQ ID No.6, SEQ ID No.7, or SEQ ID No. 8.
7. An isolated nucleic acid encoding the IL-17RA fusion protein of any one of claims 1-6.
8. An expression vector comprising the nucleic acid of claim 7 operably linked to a promoter.
9. A host cell comprising the expression vector of claim 8.
10. The host cell according to claim 9, wherein the host cell has a accession number of CGMCC 21011.
11. A protein dimer formed from the IL-17RA fusion protein of any one of claims 1-6, which dimer is double-stranded by two molecules of the IL-17RA fusion protein bound by cysteines of the IgG1 constant region.
12. Use of an IL-17RA fusion protein according to any one of claims 1-6 or a protein dimer according to claim 11 in the preparation of a medicament for the treatment of psoriasis, Crohn's disease, plaque psoriasis, gastroenteritis, Behcet's disease syndrome, arthritis, uveitis, hidradenitis suppurativa, lichen planus, parapsoriasis, asthma, psoriatic arthritis, tendonitis, relapsing-remitting multiple sclerosis, thyroid-associated eye disease, juvenile rheumatoid arthritis, multiple sclerosis, lupus nephritis, spondyloarthritis, ankylosing spondylitis, rheumatoid arthritis, inflammatory bowel disease, non-alcoholic fatty liver, giant cell arteritis, non-radioactive 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.
13. A pharmaceutical composition comprising a therapeutically effective amount of an IL-17RA fusion protein according to any one of claims 1-6 or a protein dimer according to claim 11 as an active ingredient and a pharmaceutically acceptable excipient.
14. The pharmaceutical composition according to claim 13, wherein the pharmaceutically acceptable adjuvant is selected from one or more of diluents, buffers, protective agents, surfactants, antioxidants.
15. The pharmaceutical composition according to claim 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, succinic acid buffer, citric acid buffer.
16. The pharmaceutical composition according to claim 14, wherein the protective agent is selected from one or more of trehalose, tween-20, tween-80, sucrose, amino acids, polyols, disaccharides, polysaccharides.
17. The pharmaceutical composition according to claim 14, wherein the surfactant is selected from one or more of tween-20, tween-80, poloxamer.
18. The pharmaceutical composition of claim 13, wherein a single dose of the pharmaceutical composition comprises 5mg/ml to 150mg/ml of the IL-17RA fusion protein or the protein dimer.
19. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition is in the form of a lyophilizate or an injection solution.
20. An injection for the treatment of psoriasis, crohn's disease, plaque psoriasis, gastroenteritis, behcet's disease syndrome, arthritis, uveitis, hidradenitis suppurativa, lichen planus, psoriasis, asthma, psoriatic arthritis, tendonitis, relapsing-remitting multiple sclerosis, thyroid-associated eye disease, juvenile rheumatoid arthritis, multiple sclerosis, lupus nephritis, spondyloarthritis, ankylosing spondylitis, rheumatoid arthritis, inflammatory bowel disease, non-alcoholic fatty liver, giant cell arteritis, non-radioactive axial spine arthritis, 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 a pharmaceutical composition according to any one of claims 13-19.
21. The injection according to claim 20, wherein the injection is in the form of a lyophilized powder or in the form of a liquid formulation.
22. The injection according to claim 20, wherein the injection is a subcutaneous injection or an intravenous drip.
23. The injection of claim 21, wherein the intravenous formulation comprises 5-150 mg/ml of the IL-17RA fusion protein or the protein dimer, 2-100mM Tris-acetate, 10-250mM arginine, 50-500mM trehalose, 0.01-5% tween-20 in the liquid formulation.
24. The injection preparation according to claim 21, wherein the liquid preparation is prepared using water for injection, a buffered saline solution, an aqueous glucose solution, an aqueous sodium chloride solution or ringer's lactate.
25. The injection according to claim 21, wherein the lyophilized powder is prepared by freeze-drying the liquid preparation.
CN202210693381.0A 2022-06-17 2022-06-17 IL-17RA fusion protein, pharmaceutical composition, injection and application thereof Pending CN115093483A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022194311A3 (en) * 2022-06-17 2023-03-02 国药中生生物技术研究院有限公司 Il-17ra antibody fc fusion protein, and use thereof
CN116593718A (en) * 2023-07-18 2023-08-15 军科正源（北京）药物研究有限责任公司 Reagents for detection of antibodies against secukinumab

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UA117218C2 (en) * 2011-05-05 2018-07-10 Мерк Патент Гмбх Amino acid sequences directed against il-17a, il-17f and/or il17-a/f and polypeptides comprising the same
EP2734541B1 (en) * 2011-07-19 2017-09-06 The National Institute for Biotechnology in the Negev, Ltd. Novel il-17r-ecd mutants
CN109628406B (en) * 2019-01-03 2021-02-26 北京贝来生物科技有限公司 Mesenchymal stem cell for treating autoimmune disease and preparation method and application thereof
CN115093483A (en) * 2022-06-17 2022-09-23 国药中生生物技术研究院有限公司 IL-17RA fusion protein, pharmaceutical composition, injection and application thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022194311A3 (en) * 2022-06-17 2023-03-02 国药中生生物技术研究院有限公司 Il-17ra antibody fc fusion protein, and use thereof
CN116593718A (en) * 2023-07-18 2023-08-15 军科正源（北京）药物研究有限责任公司 Reagents for detection of antibodies against secukinumab
CN116593718B (en) * 2023-07-18 2023-09-15 军科正源（北京）药物研究有限责任公司 Reagents for detection of antibodies against secukinumab

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