Use of il-17rel fusion proteins in the treatment of inflammatory bowel disease

Abstract

The application provides application of an IL-17REL fusion protein in treatment of inflammatory bowel disease. Specifically, the IL-17REL fusion protein of the application can specifically bind IL-17A, IL-17C and IL-17F in IL-17 family members, inhibit expression of downstream inflammatory factors such as IL1β, IL6, CXCL1 and CXCL2, thereby inhibiting inflammation and achieving the effect of treating inflammatory bowel disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and more specifically, this invention relates to the application of an IL-17REL fusion protein in the treatment of inflammatory bowel disease. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of chronic autoimmune diseases affecting the intestines, with major types including Crohn's disease and ulcerative colitis. Previously, IBD was more prevalent in developed countries such as Europe and the United States, while its incidence was relatively low in developing countries. However, in recent years, the incidence of IBD has increased significantly in newly industrialized countries. Statistics show that in China, the annual new incidence of IBD has exceeded 1.74 per 100,000 people. IBD primarily affects the colon, while Crohn's disease can even involve the small intestine. Clinical manifestations include diarrhea, abdominal pain, intestinal bleeding, fatigue, and weight loss.

[0003] The exact causes of inflammatory bowel disease are still unclear, but it is generally believed that a variety of factors, including genetic susceptibility, mucosal dysfunction, gut microbiota imbalance, and abnormal activation of the immune system, are involved in the occurrence and development of inflammatory bowel disease.

[0004] For the aforementioned autoimmune diseases, immunosuppressants such as methotrexate and fumarate are commonly used in clinical practice, but their efficacy is not very significant. Furthermore, the side effects of these drugs limit their clinical application. Therefore, developing a more effective and safer drug for treating autoimmune diseases is particularly urgent.

[0005] Fusion protein drugs not only have higher pharmacokinetic stability and stronger ability to act on molecular targets, but also have unparalleled advantages over traditional drugs in the treatment of autoimmune diseases.

[0006] 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.

[0007] 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).

[0008] 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-17RE and IL-17RC. This results in a decrease in the phosphorylation of signaling molecules such as JNK and ERK1 / 2, thereby reducing the expression and secretion of inflammatory cytokines such as IL-1β, IL-6, CXCL1 and CXCL2, thus achieving the goal of treating inflammatory bowel disease. Summary of the Invention

[0009] The purpose of this invention is to provide the application of IL-17REL fusion protein in the treatment of inflammatory bowel disease.

[0010] In a first aspect of the invention, there is provided the use of an IL-17REL fusion protein for the preparation of a drug or formulation, said drug or formulation for one or more uses selected from the group consisting of:

[0011] (a) Reduce the expression of downstream genes induced by inflammatory factors IL-17A, IL-17C and / or IL-17F;

[0012] (b) Reduce the expression of inflammatory factors IL1β, IL6, CXCL1 and / or CXCL2;

[0013] (c) Inhibit the expression and secretion of inflammation-related genes;

[0014] (d) Blocking the binding of IL-17A to IL-17RA;

[0015] (e) Blocking the binding of IL-17C to IL-17RE;

[0016] (f) Blocking the binding of IL-17F to IL-17RC;

[0017] (g) Reduce the degree of phosphorylation of signaling molecules JNK and / or ERK1 / 2;

[0018] (h) Treatment of inflammatory bowel disease;

[0019] (i) Reduce inflammatory cell infiltration at the site of inflammatory bowel disease lesions.

[0020] In another preferred embodiment, the nucleotide sequence of the IL-17REL fusion protein is shown in SEQ ID NO.1.

[0021] In another preferred embodiment, the amino acid sequence of the IL-17REL fusion protein is shown in SEQ ID NO.2.

[0022] 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.

[0023] 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.

[0024] In another preferred embodiment, the drug is a pharmaceutical composition comprising:

[0025] 1) IL-17REL fusion protein; and

[0026] 2) Pharmaceutically acceptable carrier.

[0027] 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.

[0028] In another preferred embodiment, the drug is a drug conjugate, the drug conjugate comprising:

[0029] 1) IL-17REL fusion protein; and

[0030] 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.

[0031] In another preferred embodiment, the inflammation-related gene is selected from the group consisting of IL1β, IL6, CXCL1, CXCL2, or combinations thereof.

[0032] In another preferred embodiment, the IL-17REL fusion protein is capable of binding to IL-17A, IL-17C, and / or IL-17F.

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

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

[0035] In another preferred embodiment, the IL-17REL fusion protein does not bind IL-17B, IL-17D, and / or IL-17E.

[0036] In another preferred embodiment, the IL-17REL fusion protein is capable of blocking the binding of IL-17A to IL-17RA, IL-17C to IL-17RE, and / or IL-17F to IL-17RC / IL-17RA.

[0037] In another preferred embodiment, the drug is applicable to: humans, non-human primates (rhesus monkeys, cynomolgus monkeys), pigs, dogs, rabbits, or mice (rats, mice, guinea pigs).

[0038] In another preferred embodiment, the IL-17REL fusion protein or a composition thereof is used to reduce weight loss in patients with inflammatory bowel disease.

[0039] In another preferred embodiment, the IL-17REL fusion protein or a composition thereof is used to reduce colonic shortening in patients with inflammatory bowel disease.

[0040] In another preferred embodiment, the inflammatory bowel disease is selected from the group consisting of ulcerative colitis (UC), Crohn's disease (CD), indeterminate colitis (IC), acute infectious colitis, lymphocytic colitis, eosinophilic colitis, and collagenous colitis.

[0041] 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 combination thereof to an inflamed tissue site.

[0042] 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.

[0043] In a third aspect of the invention, a treatment method for inflammatory bowel disease is provided, the method comprising: administering the IL-17REL fusion protein of the invention or a composition thereof to a patient suffering from inflammatory bowel disease.

[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 another preferred embodiment, the inflammatory bowel disease is selected from the group consisting of ulcerative colitis (UC), Crohn's disease (CD), indeterminate colitis (IC), acute infectious colitis, lymphocytic colitis, eosinophilic colitis, and collagenous colitis.

[0046] 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

[0047] Figure 1 This is a schematic diagram of the structure of the fusion protein IL-17REL-His;

[0048] Figure 2 Agarose gel electrophoresis results of PCR products of the IL-17REL gene;

[0049] Figure 3 A schematic diagram illustrating the construction of the IL-17REL vector;

[0050] Figure 4 SDS-PAGE analysis of purified IL-17REL protein;

[0051] Figure 5 To detect the binding effect of IL-17REL on human IL-17 ligands using immunoblotting;

[0052] Figure 6 To detect the binding effect of IL-17REL on mouse IL-17 ligands using immunoblotting;

[0053] Figure 7 To detect the competitive binding of IL-17REL to IL-17A using flow cytometry;

[0054] Figure 8 To detect the inhibitory effect of IL-17REL fusion protein on the expression of human inflammation-related genes by qPCR;

[0055] Figure 9 To detect the inhibitory effect of IL-17REL fusion protein on the expression of inflammation-related genes in mice by qPCR;

[0056] Figure 10 The study demonstrated that the IL-17REL fusion protein can effectively alleviate a trinitrobenzenesulfonic acid-induced colitis model. Detailed Implementation

[0057] Through extensive and in-depth research, the inventors have developed an IL-17REL fusion protein for the treatment of inflammatory bowel disease. The inventors unexpectedly discovered that the exogenously prepared IL-17REL fusion protein can specifically bind to inflammatory factors 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 factors, demonstrating excellent efficacy in the treatment of inflammatory bowel disease.

[0058] the term

[0059] 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.

[0060] 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”.

[0061] IL-17REL fusion protein

[0062] 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 a therapeutic function for inflammatory bowel disease.

[0063] 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 of downstream inflammatory factors such as IL1β, IL6, CXCL1, and CXCL2, thereby inhibiting inflammation.

[0064] Composition and application

[0065] The compositions described in this invention include (but are not limited to): pharmaceutical compositions, cosmetic compositions, and / or medical aesthetic compositions.

[0066] 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.)

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] Inflammatory bowel disease

[0073] Inflammatory bowel disease (IBD) is an idiopathic inflammatory bowel disease affecting the ileum, rectum, and colon. Clinical manifestations include diarrhea, abdominal pain, and even bloody stools. IBD includes ulcerative colitis (UC) and Crohn's disease (CD). Ulcerative colitis is a continuous inflammation of the colonic mucosa and submucosa, typically starting in the rectum and gradually spreading to the entire colon. Crohn's disease can affect the entire digestive tract, presenting as a discontinuous, full-thickness inflammation, most commonly affecting the terminal ileum, colon, and perianal area.

[0074] In specific embodiments of the present invention, the IL-17REL fusion protein of the present invention can inhibit inflammation in a mouse colitis model, alleviate weight loss in mice with colitis, and reduce the degree of colonic shortening in mice with colitis, achieving good therapeutic effects on inflammatory bowel disease. Furthermore, 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, making it easy to translate into clinical applications and thus promising for use in clinical patients.

[0075] Compared with the prior art, the advantages of the present invention are as follows:

[0076] 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;

[0077] 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;

[0078] 3. The IL-17REL fusion protein or its pharmaceutical composition of the present invention has the effect of treating inflammatory bowel disease;

[0079] 4. The IL-17REL fusion protein or its pharmaceutical composition of the present invention is readily mass-produced in vitro;

[0080] 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 applications.

[0081] 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.

[0082] Example 1. Preparation of the fusion protein IL-17REL

[0083] 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:

[0084] SEQ ID NO.1:

[0085] atgtccaggtcagtcctggaggcctgacgtcctccactgccatgcagtgtgtcccctctgacggctgcgcgatgctcct

[0086] gcgtgtacgcgcctccatcaccctgcatgagcgcctgcggggcctggaggcctgtgccatgagcctggacacccagg

[0087] agacgcagtgtcagagcgtgtgggtggccagggcctcccaccggcagcagggggggcagcagctccaagtgcactt

[0088] tggctgctttgcggtgagcgtggcccagcacctctatgtcaccctgaggaccatccctcatttctgcggggtccagctgg

[0089] accagaggcacctcgtggaagcggggaagctcagctactgggtggaccggaggcgcaaggcgattctggtgcaagt

[0090] gcccagggcctccgggagccccgactactacctgcggctctgcctcaagcggttcacctgcgaggacgccggcgcc

[0091] cctgtgcgagtgaccgccaacagcgtctcccaggccgtcttcctgccctacagccaggagctgccgtgcctgtgcctg

[0092] gagggctggtctgcgacccctgacgcggtgcggatccagatctgcccctttgaaaacgacactgaggcactggaggt

[0093] gctgtgggacacggtctactaccacccggagagccagacactgagctgggagcccgcctgccctgtgagtggccatg

[0094] tgagcctgtgctggcgcccggggccgggggccggctgccgtaagctgcagcaatccagccagctggtgcatcgcag

[0095] agtgcagtacccgctggtggacacccagccccagctctgcctgaagttctctaccagttgggggtcctgggtgcggtgc

[0096] cctttcgaacagcgtcgcttcccaaccccgcccacttccaggtgcacctgtgtcacaggaggaagtcacagctccctgc

[0097] ctgccaacgcacactccaggccagcccgctcccttcagcctcaggtgacctggcagccgcccctgcttttgccttcctag;

[0098] The amino acid sequence of the IL-7REL fusion protein is shown in SEQ ID NO.2.

[0099] SEQ ID NO.2:

[0100] MSRSVLEALTSSTAMQCVPSDGCAMLLRVRASITLHERLRGLEACAMSLDTQETQCQSVWVARASHRQQGGQQLQVHFGCFAVSVAQHLYVTLRTIPHFCGVQLDQRHLVEAGKLSYWVDRRRKAILVQVPRASGSPDYYLRLCLKRFTCEDAGAPVRVTANSVSQAV FLPYSQELPCLCLEGWSATPDAVRIQICPFENDTEALEVLWDTVYYHPESQTLSWEPACPVSGHVSLCWRPGPGAGCRKLQQSSQLVHRRVQYPLVDTQPQLCLKFSTSWGSWVRCPFEQRRFPTPPTSRCTCVTGGSHSSLPANAHSRPARSLQPQVTWQPPLLLPS;

[0101] 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.

[0102] Example 2. Specific binding effect of IL-17REL fusion protein to IL-17 ligand

[0103] 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 (plasmid map shown) was expressed. 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.

[0104] The results are as follows Figure 5 As 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.

[0105] Example 3. Biological binding activity of the IL-17REL fusion protein

[0106] 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.

[0107] Test method:

[0108] HEK293 cells overexpressing IL-17RA were centrifuged at 300g for 5 min at 4°C, the supernatant was discarded, and the cells were resuspended in 10 ml of PBS. This process was repeated once, followed by resuspending the HEK293 cells in PBS to achieve a final concentration of 1*102. 7 / ml;

[0109] Take 50 μL of cell suspension and add a mixture of IL-17 and IL-17REL fusion protein to make the final concentration of IL-17A reach 1 μg / ml, while the concentration of IL-17REL fusion protein is 0.1, 1, 10.50 μg / ml. Mix well and incubate at 4°C for 30 min.

[0110] The cells were resuspended in PBS, washed, and then centrifuged.

[0111] Carefully remove the supernatant, resuspend the cells in PBS, add sufficient GST antibody, and incubate at 4°C for 30 min;

[0112] The cells were resuspended in PBS, washed, and then centrifuged.

[0113] 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;

[0114] Carefully remove the supernatant, resuspend the cells in PBS solution containing 0.4% paraformaldehyde, and detect the fluorescence intensity by flow cytometry.

[0115] The results are as follows Figure 7 As shown, the addition of IL-17REL protein inhibited the binding of IL-17A to IL-17RA. The binding amount of IL-17 to IL-17RA decreased with increasing IL-17REL levels.

[0116] Example 4. Assay of IL-17REL fusion protein inhibitory activity

[0117] The purpose of this embodiment is to evaluate the anti-inflammatory effect of the IL-17REL fusion protein. Specific methods and results are as follows:

[0118] 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. An equal volume of PBS was used as a negative control for IL-17REL protein. 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 for the Real-time PCR experiment are shown in the table below.

[0119]

[0120]

[0121] 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.

[0122] Example 4. Animal model of IL-17REL fusion protein inhibiting colitis

[0123] The purpose of this embodiment is to evaluate the therapeutic effect of IL-17REL fusion protein on colitis model animals by blocking IL-17 signaling and inhibiting the production of downstream inflammatory factors after being introduced into the colitis model.

[0124] Trinitrobenzenesulfonic acid-induced mouse colitis model:

[0125] Wild-type mice aged 8-10 weeks were selected. Animal modeling was initiated the day before the experiment, with all groups fasted but allowed free access to water for 24 hours. Mice were anesthetized by intraperitoneal injection of tribromoethanol. An enema needle was inserted through the anus into the colon, and a TNBS solution containing 50% ethanol was slowly injected at a dose of 100 mg / kg. The control group received an enema of the same volume of 50% ethanol solution. The enema needle was removed, and the mice were left to stand with their tails facing upwards for 5 minutes to allow for complete absorption of the TNBS. Mouse weight was recorded for three consecutive days. On the third day, the mice were euthanized, and the colon length was recorded.

[0126] Specific groups:

[0127] 1. Control group: 5 C57BL / 6 mice, 8-10 weeks old, were perfused with 50% ethanol solution in the colon and rectum;

[0128] 2. Model group: C57BL / 6 mice, 5 mice, 8-10 weeks old, were instilled with 50% ethanol TNBS solution in the colon and rectum. The drug was administered once a day by intraperitoneal injection, starting 1 day after modeling.

[0129] 3. IL-17REL group: 5 C57BL / 6 mice, 8-10 weeks old, were instilled with 50% ethanol TNBS solution in the colon and rectum and administered IL-7REL 10mg / kg once a day by intraperitoneal injection starting 1 day after modeling.

[0130] The results are as follows Figure 10 As shown, in a mouse model of colitis, the introduction of IL-17REL protein significantly increased the body weight of mice compared to the model group, and the degree of colonic shortening was significantly reduced. Figure 10 A); Hematoxylin-eosin staining of mouse colon tissue revealed that mice inoculated with IL-17REL protein showed significantly reduced mucosal damage at the lesion site, accompanied by a decrease in inflammatory cell infiltration. Figure 10B). Furthermore, RNA was extracted from colonic lesions in mice, reverse transcribed into cDNA, and then subjected to Real-time PCR. This revealed that the expression levels of IL-1β, IL-6, CXCL1, and CXCL2, inflammation-related factors, were significantly reduced in the colonic region of mice inoculated with IL-17REL protein. Figure 10 C).

[0131] 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. The use of an IL-17REL protein in the preparation of a medicament for treating Crohn's disease, characterized in that, The amino acid sequence of the IL-17REL protein is shown in SEQ ID NO.

2.

2. The use as described in claim 1, characterized in that, The drug works through the following pathways: (a) Reduce the expression of inflammatory factors IL1β, IL-6, CXCL1 and / or CXCL2; (b) Reduce the expression of downstream genes induced by inflammatory factors IL-17A, IL-17C and / or IL-17F; (c) Blocking the binding of IL-17A to IL-17RA; (d) Blocking the binding of IL-17C to IL-17RE; (e) Block the binding of IL-17F to IL-17RC; (f) Reduce inflammatory cell infiltration at the site of inflammatory bowel disease lesions.

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

1.

4. 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.

5. The use as described in claim 4, 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, external application, or intramuscular injection.

6. 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.

7. 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.

8. 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.

9. 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.

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

11. The use as described in claim 9, characterized in that, The term "mouse" includes: rats, mice, and guinea pigs.

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