Use of il-17rel fusion proteins in the treatment of autoimmune diseases

Abstract

This invention provides an application of the IL-17REL fusion protein in the treatment of autoimmune diseases. Specifically, the IL-17REL fusion protein of this invention can specifically bind to IL-17A, IL-17C, and IL-17F, members of the IL-17 family, and inhibit the expression of downstream inflammatory factors such as IL1β, IL6, CXCL1, and CXCL2, thereby suppressing inflammation and achieving the therapeutic effect on autoimmune diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine. More specifically, the present invention relates to the use of an IL-17REL fusion protein in the treatment of autoimmune diseases. Background Art

[0002] Rheumatoid arthritis is a very common autoimmune disease that occurs in all age groups. Patients with rheumatoid arthritis usually experience stiffness, swelling and pain in multiple joints, and it often occurs in the wrist joints, proximal interphalangeal joints and metacarpophalangeal joints. In severe cases, patients may also experience systemic symptoms such as fatigue, weight loss and low-grade fever. Moreover, rheumatoid arthritis may also induce various diseases such as lung diseases, cardiovascular diseases, malignancies and depression. Through research, it has been found that the occurrence of rheumatoid arthritis is closely related to abnormal immune function caused by genetic factors or environmental factor changes.

[0003] Multiple sclerosis is an autoimmune disease that specifically attacks the myelin sheath of neurons. Research has found that although more than 70% of multiple sclerosis patients can still survive for more than 25 years or even 45 years after the onset of the disease, the limb disabilities, cardiovascular diseases, tumors and mental diseases caused by this disease still make the average life expectancy of patients lower than the expected value by 7 to 14 years. Therefore, early detection and active treatment become extremely important. The causes of multiple sclerosis are multi-faceted, and various factors such as environment and self have played important roles. However, research has found that the abnormal immune function of the body caused by the above factors is the direct cause of the occurrence of multiple sclerosis.

[0004] Psoriasis, also known as psoriasis vulgaris, is a chronic and recurrent autoimmune disease. According to the different locations, scopes and specific symptoms of the disease, psoriasis can be roughly divided into five types clinically, namely: plaque psoriasis, which accounts for 90% of all psoriasis patients, also known as vulgar psoriasis; guttate psoriasis, where the skin plaques are in a drop shape; inverse psoriasis, which occurs in skin folds; pustular psoriasis, which presents scattered dark red macules and white pustules (sterile pustules); and erythrodermic psoriasis, where the whole body shows diffuse flushing, swelling and scales. Therefore, it is particularly important to clarify the pathogenic causes of psoriasis and find effective treatment methods.

[0005] For the above-mentioned autoimmune diseases, immunosuppressive agents such as methotrexate and fumaric acid esters are commonly used clinically for treatment, but the effects are not very significant. And due to the side effects of the above drugs, their clinical applications are restricted. Therefore, it is particularly urgent to develop a more effective and safer drug for the treatment of autoimmune diseases.

[0006] Fusion protein drugs not only have higher pharmacokinetic stability and stronger ability to act on molecular targets, but also have unparalleled advantages in the treatment of autoimmune diseases compared with traditional drugs.

[0007] The IL-17 cytokine family has six members: IL-17A (abbreviated as IL-17), IL-17B, IL-17C, IL-17D, IL-17E (also known as IL-25), and IL-17F. Corresponding to the IL-17 cytokine family is its receptor family. The IL-17 receptor (IL-17R) family has five members: IL-17RA, IL-17RB, IL-17RC, IL-17RD, and IL-17RE. They share the common characteristics of an extracellular FN domain and an intracellular SEFIF sequence. For example, IL-17RA can self-assemble to form a homodimer, or form a heterodimer with IL-17RC, participating in downstream signaling induced by IL-17A and IL-17F.

[0008] With the completion of the human genome sequencing, the sixth member of the IL-17 receptor family, IL-17REL, was discovered in 2011. Based on sequence analysis, IL-17REL was found to be highly similar to IL-17RE, but because it only contains the extracellular region and lacks the transmembrane and intracellular regions, it is called Intelectin-17Receptor E-like (or simply IL-17REL).

[0009] The possible mechanism of action of the IL-17REL fusion protein is as follows: IL-17REL acts on IL-17A, IL-17C and IL-17F secreted by immune cells or epithelial cells, blocking their binding to their specific receptors IL-7RA, IL-17RC and IL-17RE. This results in a decrease in the phosphorylation of signaling molecules such as JNK and ERK1 / 2, thereby reducing the expression of inflammatory cytokines such as IL-1β, IL-6, CXCL1 and CXCL2, thus achieving the goal of treating autoimmune diseases. Summary of the Invention

[0010] The purpose of this invention is to provide the application of IL-17REL fusion protein in the treatment of autoimmune diseases.

[0011] In a first aspect of the invention, there is provided a use of the IL-17REL fusion protein, characterized in that it is used to prepare a drug or formulation for one or more uses selected from the group consisting of:

[0012] (a) Reduce the expression of downstream genes induced by inflammatory factors IL-17A, IL-17C and / or IL-17F;

[0013] (b) Reduce the expression of inflammatory factors IL1β, IL-6, CXCL1 and / or CXCL2;

[0014] (c) Inhibit the expression and secretion of inflammation-related genes;

[0015] (d) Blocking the binding of IL-17A to IL-17RA;

[0016] (e) Blocking the binding of IL-17C to IL-17RE;

[0017] (f) Blocking the binding of IL-17F to IL-17RC;

[0018] (g) Reduce the degree of phosphorylation of signaling molecules JNK and / or ERK1 / 2;

[0019] (h) Treatment of autoimmune diseases;

[0020] (i) Reduce inflammatory cell infiltration at the lesion site.

[0021] In another preferred embodiment, the nucleotide sequence of the IL-17REL fusion protein is shown in SEQ ID NO.1.

[0022] In another preferred embodiment, the amino acid sequence of the IL-17REL fusion protein is shown in SEQ ID NO.2.

[0023] In another preferred embodiment, the IL-17REL fusion protein further includes a tag protein fused to the N-terminus or C-terminus of the IL-17REL protein.

[0024] In another preferred embodiment, the tag protein is a SUMO tag protein, a His tag protein, a GST tag protein, a MBP tag protein, and / or a NusA tag protein.

[0025] In another preferred embodiment, the drug is a pharmaceutical composition comprising:

[0026] 1) IL-17REL fusion protein; and

[0027] 2) Pharmaceutically acceptable carrier.

[0028] In another preferred embodiment, the administration route of the IL-17REL fusion protein or pharmaceutical composition is selected from the group consisting of: intraperitoneal injection, intravenous injection, subcutaneous injection, intradermal injection, topical application, or intramuscular injection.

[0029] In another preferred embodiment, the drug is a conjugate comprising:

[0030] 1) IL-17REL fusion protein; and

[0031] 2) The conjugated portion of the IL-17REL fusion protein, wherein the conjugated portion is selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

[0032] In another preferred embodiment, the inflammation-related gene is selected from the group consisting of IL1β, IL6, CXCL1, CXCL2, or combinations thereof.

[0033] In another preferred embodiment, the IL-17REL fusion protein is capable of binding to IL-17A, IL-17C, and / or IL-17F.

[0034] In another preferred embodiment, the IL-17REL fusion protein is capable of binding to human IL-17A, IL-17C and / or IL-17F.

[0035] In another preferred embodiment, the IL-17REL fusion protein is capable of binding to mouse-derived IL-17A, IL-17C, and / or IL-17F.

[0036] In another preferred embodiment, the IL-17REL fusion protein does not bind IL-17B, IL-17D, and / or IL-17E.

[0037] In another preferred embodiment, the IL-17REL fusion protein is able to block the binding of IL-17A to IL-17RA, IL-17C to IL-17RE and / or IL-17F to IL-17RC.

[0038] In another preferred embodiment, the IL-17REL fusion protein or a pharmaceutical composition thereof is used to inhibit ear hyperplasia in patients with psoriasis.

[0039] In another preferred embodiment, the IL-17REL fusion protein or a pharmaceutical composition thereof is used to improve lymphocyte infiltration in patients with multiple sclerosis.

[0040] In another preferred embodiment, the IL-17REL fusion protein or a pharmaceutical composition thereof is used to improve lymphocyte infiltration in patients with rheumatoid arthritis.

[0041] In another preferred embodiment, the test subjects of the drug include: humans, non-human primates (rhesus monkeys, cynomolgus monkeys), pigs, dogs, rabbits, or mice (rats, mice, guinea pigs).

[0042] In another preferred embodiment, the autoimmune disease is selected from the group consisting of: rheumatoid arthritis, multiple sclerosis, psoriasis, systemic lupus erythematosus, sjögren's syndrome, ankylosing spondylitis, scleroderma, polyarteritis nodosa, Wegener's granulomatosis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, pernicious anemia with chronic atrophic gastritis, goodpasture syndrome, primary biliary cirrhosis, and acute idiopathic polyneuritis.

[0043] In a second aspect of the invention, a method for tissue anti-inflammatory treatment is provided, the method comprising: applying the IL-17REL fusion protein of the invention or a pharmaceutical composition thereof to an inflamed tissue site.

[0044] In another preferred embodiment, the method of administration is selected from the group consisting of: intraperitoneal injection, intravenous injection, subcutaneous injection, intradermal injection, topical application, or intramuscular injection.

[0045] In a third aspect of the invention, a treatment method for an autoimmune disease is provided, the method comprising: administering the IL-17REL fusion protein of the invention or a pharmaceutical composition thereof to a patient suffering from an autoimmune disease.

[0046] In another preferred embodiment, the method of administration is selected from the group consisting of: intraperitoneal injection, intravenous injection, subcutaneous injection, intradermal injection, topical application, or intramuscular injection.

[0047] In another preferred embodiment, the autoimmune disease is selected from the group consisting of: rheumatoid arthritis, multiple sclerosis, psoriasis, systemic lupus erythematosus, sjögren's syndrome, ankylosing spondylitis, scleroderma, polyarteritis nodosa, Wegener's granulomatosis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, pernicious anemia with chronic atrophic gastritis, goodpasture syndrome, primary biliary cirrhosis, and acute idiopathic polyneuritis.

[0048] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0049] Figure 1 This is a schematic diagram of the structure of the fusion protein IL-17REL-His;

[0050] Figure 2 Agarose gel electrophoresis results of PCR products of the IL-17REL gene;

[0051] Figure 3 A schematic diagram illustrating the construction of the IL-17REL vector;

[0052] Figure 4 SDS-PAGE analysis of purified IL-17REL protein;

[0053] Figure 5 To detect the binding effect of IL-17REL to human IL-17 ligand using immunoblotting;

[0054] Figure 6 To detect the binding effect of IL-17REL to human and mouse IL-17 ligands using immunoblotting;

[0055] Figure 7 To detect the competitive binding of IL-17REL to IL-17A using flow cytometry;

[0056] Figure 8 To detect the inhibitory effect of IL-17REL fusion protein on the expression of human inflammation-related genes by qPCR;

[0057] Figure 9 To detect the inhibitory effect of IL-17REL fusion protein on the expression of inflammation-related genes in mice by qPCR;

[0058] Figure 10 , 11 Figures 1 and 12 show that the IL-17REL fusion protein can effectively alleviate type II collagen-induced rheumatoid arthritis, experimental autoimmune encephalomyelitis, and imiquimod-induced psoriasis. Detailed Implementation

[0059] Through extensive and in-depth research, the inventors have developed an IL-17REL fusion protein for the treatment of autoimmune diseases. The inventors unexpectedly discovered that the exogenously prepared IL-17REL fusion protein can specifically bind to inflammatory cytokines IL-17A, IL-17C, and IL-17F, thereby blocking the binding of IL-17A, IL-17C, and IL-17F to their receptors and inhibiting the secretion of downstream inflammatory cytokines, demonstrating excellent efficacy in the treatment of autoimmune diseases.

[0060] the term

[0061] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing this invention, it should be understood that it is not limited to the specific methods and experimental conditions described, as such methods and conditions can be varied.

[0062] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0063] IL-17REL fusion protein

[0064] This invention utilizes genetic engineering methods to link the human IL-17REL sequence with 6X histidine (6xHis) and SUMO sequences, and then expresses and purifies the fusion protein in prokaryotic competent cells. This fusion protein is efficiently expressed in prokaryotic competent cells, and the purification process is simple and easy for large-scale production. Simultaneously, it retains the binding properties of the IL-17REL molecule, competitively binding with members of the IL-17 family, thereby blocking IL-17 signaling and downstream gene expression, thus achieving the function of treating autoimmune diseases.

[0065] The IL-17REL fusion protein of the present invention specifically binds to IL-17A, IL-17C, and IL-17F, members of the IL-17 family, but not to IL-17B, IL-17D, and IL-17E; and can inhibit the expression and secretion of downstream inflammatory factors such as IL1β, IL6, CXCL1, and CXCL2, thereby exerting an anti-inflammatory effect.

[0066] Composition and application

[0067] The compositions described in this invention include (but are not limited to): pharmaceutical compositions, cosmetic compositions, and / or medical aesthetic compositions.

[0068] The compositions of the present invention may further include pharmaceutically, cosmetically, or medically acceptable carriers. "Pharmaceutically, cosmetically, or medically acceptable carriers" refers to one or more compatible solid or liquid fillers or gelling substances suitable for human use and possessing sufficient purity and low toxicity. "Compatibility" here refers to the ability of the components in the composition to interact with and incorporate the active ingredients of the present invention without significantly reducing the efficacy of the active ingredients. Some examples of pharmaceutically, cosmetically, or medically acceptable carriers include cellulose and its derivatives, gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as... (e.g., wetting agents, colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, dispersants, humectants, UV stabilizers, film-forming agents, oil-soluble gelling agents, organically modified clay minerals, antibacterial agents, fragrances, salts, pH adjusters, chelating agents, cooling agents, anti-inflammatory agents, skin beautifying ingredients (whitening agents, cell activators, skin roughness improvers, blood circulation promoters, skin astringents, anti-seborrheic agents, etc.), vitamins, amino acids, etc.)

[0069] The composition of the present invention is preferably an injectable formulation, and representative administration methods include (but are not limited to): parenteral (intravenous, intramuscular, intraperitoneal, subcutaneous) injection and local application.

[0070] Buffers may also be included in capsules, tablets, and pills. Solid dosage forms such as tablets, sugar pills, capsules, pills, and granules may be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents.

[0071] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0072] Dosage forms of the compositions of the present invention for topical application or administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as needed.

[0073] When administering the composition, a safe and effective amount of the composition of the present invention is applied to the human or non-human animal (such as rat, mouse, dog, cat, cow, sheep, chicken, duck, etc.) requiring treatment, wherein the dosage at the time of administration is a pharmaceutically, food-, or health-product-acceptable effective dosage. As used herein, the term "safe and effective amount" refers to an amount that produces a function or activity in humans and / or animals and is acceptable to humans and / or animals. Those skilled in the art will understand that the "safe and effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and whether it is used in combination with other drugs. For example, for a person weighing 60 kg, the daily dosage is typically 0.1-1000 mg, preferably 1-600 mg, and more preferably 2-300 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are within the scope of the skill of a skilled physician.

[0074] Autoimmune diseases

[0075] Autoimmune diseases are diseases caused by damage to the body's own tissues due to an immune response to its own antigens. Autoimmune diseases are further divided into organ-specific autoimmune diseases and systemic autoimmune diseases.

[0076] Systemic autoimmune diseases, also known as collagen diseases or connective tissue diseases, are caused by widespread deposition of antigen-antibody complexes in the blood vessel walls and other factors, leading to damage to multiple organs throughout the body. These are caused by immune damage resulting in fibrinoid necrotizing inflammation of the blood vessel walls and interstitial tissue, followed by collagen fiber proliferation in multiple organs. Common systemic autoimmune diseases include: systemic lupus erythematosus, Sjögren's syndrome, rheumatoid arthritis, ankylosing spondylitis, scleroderma, polyarteritis nodosa, and Wegener's granulomatosis.

[0077] Organ-specific autoimmune diseases mainly include psoriasis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, pernicious anemia with chronic atrophic gastritis, goodpasture syndrome, primary biliary cirrhosis, multiple cerebral sclerosis, and acute idiopathic polyneuritis.

[0078] In specific embodiments of the present invention, the IL-17REL fusion protein of the present invention reduced the clinical scores and improved inflammatory symptoms in mouse models of multiple sclerosis and rheumatoid arthritis after administration; after administration to a mouse model of psoriasis, it significantly reduced the degree of ear hyperplasia or swelling, indicating that the IL-17REL fusion protein has the effect of inhibiting disease development in the above three autoimmune disease models; and the IL-17REL fusion protein of the present invention can specifically bind to human IL-17A, IL-17C, and / or IL-17F in vitro, as well as mouse IL-17A, IL-17C, and / or IL-17F, which is easy to translate into clinical applications and therefore has the potential to be used in clinical patients.

[0079] Compared with the prior art, the advantages of the present invention are as follows:

[0080] 1. The IL-17REL fusion protein of the present invention or its pharmaceutical composition specifically binds to IL-17A, IL-17C, and / or IL-17F;

[0081] 2. The IL-17REL fusion protein or its pharmaceutical composition of the present invention inhibits the expression and secretion of downstream inflammatory factors of IL-17;

[0082] 3. The IL-17REL fusion protein or its pharmaceutical composition of the present invention has the effect of treating autoimmune diseases;

[0083] 4. The IL-17REL fusion protein or its pharmaceutical composition of the present invention is readily mass-produced in vitro;

[0084] 5. The IL-17REL fusion protein of the present invention has species homology and can produce anti-inflammatory effects in different species, making it easy to translate into clinical practice.

[0085] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0086] Example 1. Preparation of the fusion protein IL-17REL

[0087] In this embodiment, the IL-17REL fusion protein was prepared by Huaan Biotechnology. The specific structure of the fusion protein is shown in [link to specific details]. Figure 1 The gene sequence of the IL-7REL fusion protein is shown in SEQ ID NO.1:

[0088] SEQ ID NO.1:

[0089] atgtccaggtcagtcctggaggcctgacgtcctccactgccatgcagtgtgtcccctctgacggctgcgcgatgctcct

[0090] gcgtgtacgcgcctccatcaccctgcatgagcgcctgcggggcctggaggcctgtgccatgagcctggacacccagg

[0091] agacgcagtgtcagagcgtgtgggtggccagggcctcccaccggcagcagggggggcagcagctccaagtgcactt

[0092] tggctgctttgcggtgagcgtggcccagcacctctatgtcaccctgaggaccatccctcatttctgcggggtccagctgg

[0093] accagaggcacctcgtggaagcggggaagctcagctactgggtggaccggaggcgcaaggcgattctggtgcaagt

[0094] gcccagggcctccgggagccccgactactacctgcggctctgcctcaagcggttcacctgcgaggacgccggcgcc

[0095] cctgtgcgagtgaccgccaacagcgtctcccaggccgtcttcctgccctacagccaggagctgccgtgcctgtgcctg

[0096] gagggctggtctgcgacccctgacgcggtgcggatccagatctgcccctttgaaaacgacactgaggcactggaggt

[0097] gctgtgggacacggtctactaccacccggagagccagacactgagctgggagcccgcctgccctgtgagtggccatg

[0098] tgagcctgtgctggcgcccggggccgggggccggctgccgtaagctgcagcaatccagccagctggtgcatcgcag

[0099] agtgcagtacccgctggtggacacccagccccagctctgcctgaagttctctaccagttgggggtcctgggtgcggtgc

[0100] cctttcgaacagcgtcgcttcccaaccccgcccacttccaggtgcacctgtgtcacaggaggaagtcacagctccctgc

[0101] ctgccaacgcacactccaggccagcccgctcccttcagcctcaggtgacctggcagccgcccctgcttttgccttcctag;

[0102] [[ID=2,7]]The amino acid sequence of the IL-7REL fusion protein is shown in SEQ ID NO.2,

[0103] SEQ ID NO.2:

[0104] MSRSVLEALTSSTAMQCVPSDGCAMLLRVRASITLHERLRGLEACAMSLDTQETQCQSVWVARASHRQQGGQQLQVHFGCFAVSVAQHLYVTLRTIPHFCGVQLDQRHLVEAGKLSYWVDRRRKAILVQVPRASGSPDYYLRLCLKRFTCEDAGAPVRVTANSVSQAV FLPYSQELPCLCLEGWSATPDAVRIQICPFENDTEALEVLWDTVYYHPESQTLSWEPACPVSGHVSLCWRPGPGAGCRKLQQSSQLVHRRVQYPLVDTQPQLCLKFSTSWGSWVRCPFEQRRFPTPPTSRCTCVTGGSHSSLPANAHSRPARSLQPQVTWQPPLLLPS;

[0105] PCR identification results of IL-7REL fusion protein are as follows: Figure 2 As shown, the results of Western blot analysis are as follows: Figure 3 As shown, the results indicate that the separable IL-7REL fusion protein was successfully prepared.

[0106] Example 2. Specific binding effect of IL-17REL fusion protein to IL-17 ligand

[0107] First, using a prokaryotic expression system, the GEX-4T1 plasmid expressing human IL-17A-IL-17F and mouse IL-17A, IL-17C, and IL-17F proteins was expressed (plasmid map shown). Figure 4 (As shown) The cells were transferred into BL21 competent cells and cultured at 37℃ and 250 rpm until the OD value of the competent cells reached 0.4-0.6. Then, IPTG was added to a final concentration of 0.5 mM, and induction was continued at 20℃ and 200 rpm for 18 h. The competent cells were then collected. Lysozyme (0.5 mg / ml), DNase1 (10 u / ml), and PMSF (0.5 mM) were added to dissociate the competent cells at 4℃ for 30 min. The cell lysate was then collected by sonication and high-speed centrifugation for purification of GST-Beads. The purified IL-17 protein-GST bead complex was then bound to IL-17REL, and Western blot was used to determine which IL-17 receptor family member could bind to IL-17REL.

[0108] The results are as follows Figure 5As shown, the human IL-17REL fusion protein can bind to human IL-17A, IL-17C, and IL-17F proteins, but not to human IL-17B, IL-17D, and IL-17E. Figure 6 As shown, the human IL-17REL fusion protein can bind to human or mouse IL-17A, IL-17C, and IL-17F proteins.

[0109] Example 3. Biological binding activity of the IL-17REL fusion protein

[0110] Flow cytometry was used to inoculate HEK293 cells overexpressing IL-17RA with IL-17A and simultaneously add the IL-17REL fusion protein to detect the inhibitory effect of the IL-17REL fusion protein on the binding of IL-17A to IL-17RA. This method is an indirect immunofluorescence assay. IL-17RA on HEK293 / IL-17RA cells can bind to the added IL-17. Anti-GST antibody was used to bind to IL-17A on the cell surface, followed by the addition of a fluorescein-conjugated anti-mouse F(ab) antibody. ， ) 2 Secondary antibody for the fragment. The fluorescence intensity reflects the inhibitory effect of IL-17REL protein on the binding of IL-17 and IL-17RA.

[0111] Test method:

[0112] HEK293 cells overexpressing IL-17RA were centrifuged at 300g for 5 min at 4°C, the supernatant was removed, and the cells were resuspended in 10 ml of PBS. This process was repeated once, and then HEK293 cells were resuspended in PBS to achieve a final concentration of 1*107 / ml.

[0113] Take 50 μL of cell suspension and add a mixture of IL-17 and IL-17REL fusion protein to achieve a final IL-17 concentration of 1 μg / ml and an IL-17REL fusion protein concentration of 0.1, 1, 10.50 μg / ml. Mix well and incubate at 4°C for 30 min.

[0114] The cells were resuspended in PBS, washed, and then centrifuged.

[0115] Carefully remove the supernatant, resuspend the cells in PBS, add sufficient GST antibody, and incubate at 4°C for 30 min;

[0116] The cells were resuspended in PBS, washed, and then centrifuged.

[0117] Carefully remove the supernatant, resuspend the cells in PBS, add sufficient secondary antibody conjugated with fluorescein-labeled anti-mouse F(ab')2 fragment, and incubate at 4°C for 30 min;

[0118] Carefully remove the supernatant, resuspend the cells in PBS solution containing 0.4% paraformaldehyde, and detect the fluorescence intensity by flow cytometry.

[0119] The results are as follows Figure 7 As shown, the addition of IL-17REL protein inhibited the binding of IL-17 to IL-17RA. The binding amount of IL-17 to IL-17RA decreased with increasing IL-17REL levels.

[0120] Example 4. Assay of IL-17REL fusion protein inhibitory activity

[0121] The purpose of this embodiment is to evaluate the anti-inflammatory effect of the IL-17REL fusion protein. The specific methods and results are as follows:

[0122] Methods: Human colon cancer cell line HT29 and mouse colon cancer cell line MC38 were used. hIL-17A, IL-17REL, and hIL-17A+IL-17REL, or mIL-17A, IL-17REL, and mIL-17A+IL-17REL, were added, respectively. After 6 hours, cells were collected, RNA was extracted, and reverse transcribed into cDNA. The relative expression levels of IL-1β, IL-6, CXCL1, and CXCL2 in the cells were detected by Real-time PCR. The primers used in the Real-time PCR experiment are shown in the table below:

[0123]

[0124] The results are as follows Figure 8 and Figure 9 As shown, the addition of IL-17REL protein significantly inhibited the expression of inflammation-related genes in both human and mouse cells.

[0125] Example 5. Animal model of autoimmune disease inhibited by IL-17REL fusion protein

[0126] The purpose of this embodiment is to evaluate the therapeutic effect of IL-17REL fusion protein on autoimmune disease models by blocking IL-17 signaling and inhibiting the production of downstream inflammatory factors after being introduced into these models.

[0127] Experimental autoimmune encephalomyelitis model:

[0128] Wild-type mice aged 8-10 weeks were selected to establish an experimental self-reactive encephalomyelitis (EAE) model. Mice were housed and tested in our SPF-grade animal facility. On day 0, Mog35-55 was dissolved in PBS to a stock solution concentration of 10 mg / ml. 30 μL of the Mog35-55 solution was diluted to 100 μL in PBS and thoroughly mixed with 100 μL of Freund's complete adjuvant (H37Ra was added to Freund's incomplete adjuvant to a final concentration of 5 mg / ml). 100 μL of the emulsion was then injected subcutaneously into both sides of the mouse's back. Simultaneously, 200 ng of pertussis toxin was injected intraperitoneally. On day 2, the same dose of pertussis toxin was repeated to promote the development of the model. Over the next 20 days, changes in the mice's activity levels were observed to determine the severity of the disease model.

[0129] The severity of this encephalomyelitis model can be scored according to the following criteria:

[0130]

[0131] *A score of 0.5 can be set for intermediate states.

[0132] Specific groups:

[0133] 1. Control group: 5 C57BL / 6 mice, 7-8 weeks old, not injected with Mog35-55 and pertussis toxin for modeling, and fed normally;

[0134] 2. Model group: C57BL / 6 mice, 5 mice, 7-8 weeks old, were injected with Mog35-55 and pertussis toxin to establish the model. The drug was physiological saline. After the mice reached a clinical score of 2, they were injected intravenously once every 3 days.

[0135] 3. IL-17REL group: 5 C57BL / 6 mice, 7-8 weeks old, were injected with Mog35-55 and pertussis toxin to establish the model. The mice were then given IL-7REL 10mg / kg. The mice were given IL-7REL once every 3 days after their clinical score reached 2 points.

[0136] like Figure 10 As shown, the clinical scores of mice in the IL-17REL group were significantly lower than those in the model group. Hematoxylin-eosin staining of the spinal cord of the model mice revealed less inflammatory cell infiltration in the spinal cord of the IL-17REL group, indicating that the encephalopathy and spinal cord inflammation of the mice were significantly improved after administration of the IL-7REL fusion protein.

[0137] Collagen-induced rheumatoid arthritis model:

[0138] Male C57BL / 6 mice aged 8-10 weeks were housed and tested in our SPF-grade animal facility. Chodrex chicken type II collagen was used at an initial concentration of 2 mg / ml. The type II collagen solution was mixed with an equal volume of Freund's complete adjuvant and thoroughly emulsified to a final concentration of 1 mg / ml. An intradermal injection of 100 μL per mouse was administered 2-3 cm from the base of the tail, and this day was designated Day 0. On Day 21, the mice were injected again with type II collagen emulsified in Freund's incomplete adjuvant. The severity of joint disease, paw thickness, and clinical scores were observed at 5-7 day intervals using the following scoring criteria:

[0139]

[0140] Specific groups:

[0141] 1. Control group: C57BL / 6 mice, 5 mice, 8-10 weeks old, without injection of type II collagen for modeling;

[0142] 2. Model group: C57BL / 6 mice, 5 mice, 8-10 weeks old, injected with type II collagen to establish the model, drug: physiological saline, once every 3 days after the mice reached a clinical score of 1, intra-articular injection;

[0143] 3. IL-17REL group: 5 C57BL / 6 mice, 8-10 weeks old, were injected with type II collagen to establish the model. The mice were given IL-7REL 10mg / kg. The mice were injected intra-articularly once every 3 days after their clinical score reached 1 point.

[0144] like Figure 11 As shown, the clinical scores of mice in the IL-17REL group were significantly lower than those in the model group. After the experiment reached its endpoint, hematoxylin-eosin staining was performed on the ankle joint sections of the mice. It was found that the joint boundaries of the mice in the IL-17REL group were clear, the synovial structure was intact, and there was less lymphocyte infiltration, indicating that the rheumatoid arthritis of the mice was significantly improved after administration of IL-7REL fusion protein.

[0145] Imiquimod-induced mouse psoriasis model:

[0146] Wild-type mice aged 8-10 weeks and IL-17REL gene knock-in mice were selected to induce a mouse psoriasis model using imiquimod cream. Mice were housed and tested in our SPF-grade animal facility. On day -2, mice were anesthetized, and the hair on their backs was removed using a razor and depilatory cream. On day 0, after anesthesia, 5% imiquimod cream was applied to the skin on the back and ears. The same amount of imiquimod cream was applied continuously for 5 days, and the morbidity and ear thickening were statistically analyzed.

[0147] Specific groups:

[0148] 1. Control group: C57BL / 6 mice, 5 mice, 8-10 weeks old, without imiquimod induction of modeling;

[0149] 2. Model group: C57BL / 6 mice, 5 mice, 8-10 weeks old, modeled by imiquimod, administered with saline, once a day via intraperitoneal injection after the end of administration;

[0150] 3. IL-17REL group: 5 C57BL / 6 mice, 8-10 weeks old, were induced to develop the model using imiquimod and administered IL-7REL 10mg / kg once a day via intraperitoneal injection after the end of the administration period.

[0151] The results are as follows Figure 12 As shown, compared with the model group, the mice in the IL-17REL group had significantly reduced ear thickness hyperplasia or swelling. By staining mouse ear sections with hematoxylin and eosin and counting the epidermal thickness, it was found that the hyperplasia of the stratum corneum in mice injected with IL-17REL protein was significantly reduced and the lymphocyte infiltration was weakened. This indicates that after introducing IL-17REL protein into the mice, the development of psoriasis was significantly inhibited, and a therapeutic effect was achieved.

[0152] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Use of an IL-17REL protein for the manufacture of a medicament for the treatment of an autoimmune disease, characterized in that, The autoimmune diseases mentioned are selected from: autoreactive encephalomyelitis, rheumatoid arthritis, psoriasis, scleroderma, and myasthenia gravis; The amino acid sequence of the IL-17REL protein is shown in SEQ ID NO. 2; the treatment of autoimmune diseases works through the following pathways: (a) Reduce the expression of inflammatory factors IL1β, IL-6, CXCL1 and / or CXCL2; (b) Blocking the binding of IL-17A to IL-17RA; (c) Reduce inflammatory cell infiltration at the lesion site.

2. The use as described in claim 1, characterized in that, The nucleotide sequence encoding the IL-17REL protein is shown in SEQ ID NO.

1.

3. The use as described in claim 1, characterized in that, The drug is a pharmaceutical composition, and the pharmaceutical composition comprises: 1) IL-17REL protein; and 2) Pharmaceutically acceptable carrier.

4. The use as described in claim 3, characterized in that, The administration method of the pharmaceutical composition is selected from the group consisting of: intraperitoneal injection, intravenous injection, subcutaneous injection, intradermal injection, or intramuscular injection.

5. The use as described in claim 1, characterized in that, The IL-17REL protein can bind to human IL-17A, IL-17C and / or IL-17F.

6. The use as described in claim 1, characterized in that, The IL-17REL protein can bind to murine IL-17A, IL-17C and / or IL-17F.

7. The use as described in claim 1, characterized in that, The IL-17REL protein does not bind to IL-17B, IL-17D, and / or IL-17E.

8. The use as described in claim 1, characterized in that, The drugs are suitable for use on humans, non-human primates, pigs, dogs, rabbits, or mice.

9. The use as described in claim 8, characterized in that, The non-human primates mentioned include: rhesus monkeys and cynomolgus monkeys.

10. The use as described in claim 8, characterized in that, The term "mouse" includes: rats, mice, and guinea pigs.

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