Application of IL1RN-X recombinant protein in treatment of allergic rhinitis and nasosinusitis

Abstract

The invention discloses an application of an IL1RN-X recombinant protein in treatment of allergic rhinitis and nasosinusitis, and particularly relates to a fusion protein (IL1RN-X or IL1RN mut-X) obtained by covalently coupling IL1RN or a mutant thereof with other proteins, and an application of covalent modifiers of the fusion protein in preparation of drugs for treatment of allergic rhinitis and nasosinusitis. The IL1RN recombinant protein is found to be capable of inhibiting inflammatory cells of nasal mucosa from infiltrating into nasal mucosa tissues, so that inflammatory response of the nasal mucosa is inhibited, including invasion of eosinophilic granulocytes and CD3 positive immune cells, so that release of IgE and activation of mast cells caused by the IgE are inhibited, and finally generation of histamine and release of inflammatory factors are inhibited; therefore, anaphylactic reaction is inhibited, and allergic rhinitis and nasosinusitis are inhibited.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine, and in particular to the use of recombinant proteins (IL1RN-X) and mutants (IL1RN mut-X) of interleukin-1 receptor antagonist (IL1RN) covalently coupled with other functional proteins in the preparation of a medicament for treating allergic rhinitis and sinusitis. BACKGROUND

[0002] Allergic rhinitis (AR) is an immunoglobulin E (IgE)-mediated inflammatory disease of the nasal mucosa. The main clinical features are paroxysmal sneezing, watery nasal discharge, nasal itching, and nasal congestion, which seriously affect the quality of life of patients. Epidemiological surveys show that about 10%-40% of adults and 2%-25% of children worldwide are affected by this disease, and the incidence is increasing year by year. Allergic rhinitis can be divided into seasonal and perennial according to the time of onset, and intermittent and persistent according to the course of the disease. More importantly, allergic rhinitis is an independent risk factor for the occurrence of asthma, and about 40% of patients with allergic rhinitis will develop asthma, and 70%-80% of patients with asthma also have allergic rhinitis.

[0003] The pathogenesis of allergic rhinitis is that the external allergen (such as dust mites, pollen) is captured by the nasal mucosa and presented by antigen-presenting cells (such as dendritic cells), which activates Th2 lymphocytes. Activated Th2 cells release IL-4, IL-5, and IL-13 and other cytokines. Among them, IL-4 and IL-13 drive B cells to produce specific IgE, which binds to the high-affinity receptor (FcεRI) on the surface of mast cells and basophils, sensitizing the body. When the same allergen is encountered again, it can trigger mast cell degranulation, rapidly releasing histamine, leukotrienes, prostaglandins, and other inflammatory mediators, leading to early symptoms (such as sneezing and runny nose). Subsequently, IL-5 and other factors recruit inflammatory cells such as eosinophils to the nasal mucosa, infiltrate and release a variety of toxic proteins and inflammatory factors, leading to late symptoms (such as nasal congestion) and persistent inflammation.

[0004] Currently, the clinical treatment of allergic rhinitis mainly relies on symptomatic drug treatment and allergen immunotherapy. Drugs mainly include second-generation antihistamines and nasal corticosteroids, which can effectively relieve symptoms, but have the following limitations: (1) unable to reverse the immunopathological process of the disease, prone to recurrence after drug withdrawal; (2) long-term use of corticosteroids may cause local or systemic side effects; (3) some patients have poor response to existing drugs, and there is drug resistance. Therefore, it is of great clinical significance and urgency to develop new drugs that can target key inflammatory pathways, fundamentally regulate immune imbalance, and have few side effects.

[0005] Rhinosinusitis is closely related to allergic rhinitis and often occurs simultaneously. Allergic factors play an important role in the pathogenesis of chronic rhinosinusitis, and the inflammatory characteristics also include Th2 bias and eosinophil infiltration. The drug treatment of rhinosinusitis is also facing challenges, especially for patients with chronic rhinosinusitis with nasal polyps, the postoperative recurrence rate is high, and new treatment strategies are urgently needed.

[0006] Interleukin-1 receptor antagonist (IL1RN) is a natural inhibitor of IL-1, which can competitively bind to IL-1 receptor I (IL-1RI) without triggering receptor activation, thereby blocking the strong pro-inflammatory signal of IL-1α and IL-1β. IL-1 is an upstream core factor of inflammatory response, which can promote the expression of various pro-inflammatory cytokines (such as TNF-α, IL-6), chemokines and adhesion molecules. Studies have shown that IL-1β expression is increased in the nasal mucosa of patients with allergic rhinitis and can activate mast cells to release histamine. In animal models, IL-1 can enhance antigen-induced Th2 cell expansion. These evidences suggest that IL1RN may have therapeutic potential for Th2-type inflammation of allergic rhinitis and rhinosinusitis by antagonizing IL-1.

[0007] However, the natural IL1RN has problems such as short half-life and poor in vivo stability, which limits its clinical application. Protein engineering is a common strategy to improve the properties of protein drugs. Through point mutation, the thermodynamic stability of the protein can be enhanced; by covalent coupling with other functional proteins (such as cytokines with anti-inflammatory or immunomodulatory effects), not only the pharmacokinetic properties can be improved, but also a synergistic therapeutic effect can be produced. Currently, there is no report on the coupling of IL1RN with other proteins for the treatment of allergic rhinitis or rhinosinusitis. SUMMARY

[0008] In order to solve the technical problems of the existing drugs for treating allergic rhinitis and improving the stability of protein drugs, the present application provides a novel IL1RN-X recombinant protein drug, which is covalently coupled with IL1RN and a specific functional protein, enhances its stability, half-life and therapeutic efficacy, and is used for efficient treatment of allergic rhinitis and rhinosinusitis.

[0009] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows: The present application provides an application of a recombinant protein in the preparation of a drug for treating allergic rhinitis and / or rhinosinusitis, wherein the recombinant protein is IL1RN-X or IL1RN mut-X. IL1RN-X is a recombinant protein composed of IL1RN and a functional protein covalently coupled; IL1RN mut-X is a recombinant protein composed of IL1RN mutant and functional protein by covalent coupling; The functional protein is selected from the group consisting of cytokines with immunomodulatory or anti-inflammatory functions, including but not limited to: IL-4, IL-13, TGF-β or IL-10, and this coupled functional protein can increase the biological activity and stability of IL1RN.

[0010] Experiments have shown that IL1RN-TGFβ fusion protein has better therapeutic effect than IL1RN in the mouse model of allergic rhinitis, and can better reduce the number of scratching and sneezing, and also inhibit inflammation.

[0011] The IL1RN-X or IL1RN mut-X recombinant protein drug in the application can inhibit the infiltration of inflammatory cells in the nasal mucosa, thereby inhibiting the inflammatory response of the nasal mucosa, including inhibiting the invasion of eosinophils and CD3-positive immune cells, inhibiting the release of inflammatory factors, and inhibiting the production of IgE and histamine, thereby achieving a therapeutic effect.

[0012] The plasmid construction of IL1RN-X or IL1RN mut-X is as follows: CMV-promotor-IL-1RN CDS sequence (coding sequence without terminator) or single-base mutant IL-1RN CDS sequence is connected to 4 to 8 lysine base sequences, -(connection) X gene (such as IL-4 or IL-13 or TGF-β or IL-10, etc.) CDS (without terminator)-His Tag, as shown in the following formula. Figure 1 The design of this fusion protein does not change the tertiary structure of IL1RN or X protein, and the covalent coupling of IL1RN and X protein can increase the activity and stability of IL-1RN.

[0013] Further, the IL1RN is a human IL1RN protein, which is a recombinant, non-glycosylated form of protein expressed in 293T cells, with a molecular weight of 21.15KD, and is purchased from MedChemExpress, MCE company. The amino acid sequence is shown in SEQ ID NO: 1.

[0014] Further, the IL1RN mutant is a point mutant, specifically, a mutant obtained by substituting, deleting or inserting one amino acid of IL1RN (SEQ ID NO: 1). The point mutation is located in the non-active center region of SEQ ID NO: 1, and is intended to improve the stability of the protein without affecting the tertiary structure and antagonistic activity of IL1RN.

[0015] Further, the recombinant protein comprises a purification tag, which is a His-tag.

[0016] Further, the recombinant protein is expressed and purified in E. coli, 293 engineered cells or CHO engineered cells.

[0017] Further, the allergic rhinitis is seasonal allergic rhinitis, perennial allergic rhinitis, intermittent allergic rhinitis or drug-induced allergic rhinitis.

[0018] Further, the sinusitis is acute sinusitis, chronic sinusitis, fungal sinusitis, allergic sinusitis or odontogenic sinusitis.

[0019] Further, the effective component of the drug is IL1RN-X or IL1RN mut-X, the concentration is 0.01-2000 ng / mL, and the solvent is physiological saline.

[0020] The application also provides a pharmaceutical composition for treating allergic rhinitis and / or sinusitis, comprising the above-mentioned recombinant protein and a pharmaceutically acceptable carrier.

[0021] Further, the carrier is selected from at least one of isotonic agents, bacteriostatic agents, stabilizers, viscosity enhancers, solubilizers, protein protectants, and pharmaceutical carriers.

[0022] The dosage form of the pharmaceutical composition is preferably a topical administration dosage form, such as a nasal spray, a nasal drop, a nasal gel or a nasal ointment.

[0023] Compared with the prior art, the application has the following beneficial effects: (1) The application first proposes and covalently couples IL1RN or IL1RN mutant with specific functional proteins (such as IL-4, IL-13, TGF-β or IL-10), and confirms that the fusion protein has excellent effects in treating allergic rhinitis and sinusitis.

[0024] (2) The IL1RN-X or IL1RN mut-X fusion protein of the application does not simply add functions, experiments show that IL1RN-TGFβ is significantly better than IL1RN alone in relieving nasal symptoms, inhibiting inflammatory cell infiltration and reducing the level of pro-inflammatory factors, producing a synergistic effect of "1+1>2".

[0025] (3) By point mutation (IL1RN mut) of IL1RN and then fusing it with X protein, the thermal stability and chemical stability of the recombinant protein are significantly improved, the half-life and shelf life of the drug are prolonged, and the cold chain requirements for storage and transportation are reduced.

[0026] (4) The recombinant protein is expressed by 293, CHO and other mammalian cell expression systems, which can realize large-scale production, ensure the correct folding and glycosylation modification of the protein, and the product quality is stable, which can be popularized in a large number of patients.

[0027] (5) The drug of the present application plays a role through double routes, the treatment mechanism is clear, and the targeting is strong: the IL1RN component effectively blocks the IL-1 signal pathway, and inhibits inflammation from the upstream; the X protein (such as TGF-β) further plays the immune suppression and anti-inflammatory effect, and jointly regulates the Th1 / Th2 immune balance, and fundamentally interferes with the disease process, rather than only relieving symptoms.

[0028] (6) The local nasal cavity administration has low systemic exposure, small potential side effects, good safety, the drug component is a human protein or a reasonable mutation / fusion form, and the immunogenicity risk is low. BRIEF DESCRIPTION OF DRAWINGS

[0029] The present application will be further described in detail below in combination with the drawings and specific embodiments.

[0030] Figure 1 The flow chart for plasmid construction of IL1RN-X or IL1RN mut-X in the present application is shown in the figure; Figure 2 The statistical graph of the number of sneezing (left) and scratching nose (right) of mice in 10 minutes in different treatment groups is shown in the figure; Figure 3 The HE staining graph of the nasal mucosa tissue of different treatment groups is shown in the figure; Figure 4 The HE staining graph of eosinophils in the nasal mucosa tissue and the immunohistochemical graph of infiltrated immune cells of different treatment groups are shown in the figure; A. The HE staining graph of eosinophils in the nasal mucosa tissue, the arrow indicates the multinuclear eosinophils; B. The immunohistochemical graph of infiltrated immune cells (CD3e positive) in the nasal mucosa tissue; C. is the quantitative analysis of B figure, which is the number of CD3e positive cells in each field, at least 6 fields are counted and statistically analyzed; Figure 5 The mRNA level of inflammatory factors in the HNEpC cells of the nasal mucosa epithelial cells of different treatment groups is shown in the figure. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the protection scope of the present application.

[0032] Example 1 The plasmid construction and expression purification of IL1RN-TGFβ recombinant protein are as follows: 1. Plasmid design: use molecular biology techniques to construct an expression vector. The structure is as follows: CMV promoter-IL1RNCDS (without stop codon)-(Lys)4 linker-TGF-β CDS (without stop codon)-His-tag.

[0033] 2. Protein expression: transfected the correctly constructed plasmid into human embryonic kidney 293T cells for transient expression, or screened stable expression cell lines in CHO-K1 cells.

[0034] 3. Protein purification: collect the cell supernatant, purify the His-tagged fusion protein by nickel column affinity chromatography. Use ultrafiltration tubes for concentration and liquid exchange (to PBS buffer). Verify the protein molecular weight and specificity by SDS-PAGE and Western Bloting.

[0035] Example 2 Verify the therapeutic effect of IL1RN and IL1RN-TGFβ on allergic rhinitis through animal models: Establishment of mouse allergic rhinitis model: select 6-week-old C57BL / 6 mice, male and female, use chicken egg white protein (OVA) to establish an allergic rhinitis mouse model.

[0036] The 0th to 14th day is the sensitization period, on the 0th, 7th, and 14th day, each mouse is injected intraperitoneally with 100 mg OVA dissolved in 100 mL phosphate buffer solution (PBS) and added with 50 mL aluminum hydroxide Al(OH)3 agent, and shaken into a suspension.

[0037] The normal control group is injected with PBS.

[0038] Challenge period: from the 21st to the 28th day, 10 mL OVA is dissolved in PBS and dropped into the mouse nasal cavity on both sides, and the normal control group is given PBS.

[0039] The method of nasal drops is as follows: 10 mL of IL1RN or IL1RN-TGFβ or dexamethasone (DEX) nasal drops is used once 30 minutes before OVA challenge, then OVA is used for nasal challenge, and the allergic reaction is detected 20 minutes later.

[0040] Grouping and Drug Administration: Mice were randomly divided into 5 groups (n=8): Normal control group (PBS), AR model group (OVA), positive control group (OVA + dexamethasone, 50 μg / mL), IL1RN treatment group (OVA + IL1RN, 100 ng / mL), and IL1RN-TGFβ treatment group (OVA + IL1RN-TGFβ, 100 ng / mL). Thirty minutes before each OVA challenge, each group received either the corresponding drug via intranasal instillation or PBS (10 μL).

[0041] 1. Behavioral assessment Within 20 minutes after the last challenge, observe and record the number of sneezes and nose scratches in each mouse. Test results are as follows: Figure 1 As shown, the AR model group had significant symptoms, with a marked increase in the number of sneezes and nose scratching. The IL1RN treatment group and the IL1RN-TGFβ treatment group, like the dexamethasone treatment group (positive control group), effectively relieved the symptoms and significantly reduced the number of sneezes and nose scratching in the AR model group mice. Moreover, the treatment effect of the IL1RN-TGFβ treatment group was significantly better than that of the IL1RN treatment group alone.

[0042] 2. Histological analysis: Mice were euthanized, and nasal mucosal tissue was collected, fixed in formalin, embedded in paraffin, sectioned, and subjected to HE staining and immunohistochemical staining to detect changes in immune cells. Figure 2 The results showed that the nasal mucosal epithelium of mice in the AR model group was significantly damaged and structurally disordered (indicated by arrows), while the nasal mucosal epithelium of mice in the IL1RN treatment group, IL1RN-TGFβ treatment group, and dexamethasone treatment group (AR+DEX) was significantly repaired and arranged in a tight and regular manner.

[0043] like Figure 4 The results showed that the AR group had a significant increase in eosinophils, and the AR model group also had a significant increase in infiltrating immune cells. In contrast, the IL-1RN treatment group, IL-1RN-TGFβ treatment group, and dexamethasone (AR+DEX) treatment group all showed a significant decrease in eosinophils and infiltrating immune cells in the nasal mucosa epithelium of mice. The IL-1RN-TGFβ treatment group exhibited the most significant inhibitory effect.

[0044] 3. Cell culture and treatment: The experiment was conducted using human nasal mucosal epithelial cells (HNEpC). The following groups were established: a control group, an HDM (house dust mite, 10 μg / mL, treated for 24 hours) stimulation group, an HDM+IL1RN (100 ng / mL) pretreatment group, and an HDM+IL1RN-TGFβ (100 ng / mL) pretreatment group.

[0045] Pretreatment and stimulation: Cells were first pretreated with IL1RN or IL1RN-TGFβ for 6 hours, and then co-cultured with HDM for 24 hours.

[0046] mRNA level detection: Total RNA was extracted from cells, reverse transcribed into cDNA, and the mRNA expression levels of inflammatory factors TNF-α, IL-1β, IL-4, IL-13, and IL-33 were detected by real-time quantitative PCR (qPCR).

[0047] Test results are as follows Figure 4 As shown, HDM stimulation significantly induced the expression of all detected factors. IL1RN pretreatment partially inhibited this induction, while IL1RN-TGFβ pretreatment exhibited a stronger inhibitory effect, restoring the expression of almost all factors to near-normal levels, demonstrating its superior anti-inflammatory activity and synergistic effect.

[0048] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The use of IL1RN-X recombinant protein in the treatment of allergic rhinitis and sinusitis, characterized in that, The recombinant protein is IL1RN-X or IL1RN mut-X; Among them: IL1RN-X is a recombinant protein composed of IL1RN and a functional protein covalently coupled; IL1RN mut-X is a recombinant protein composed of an IL1RN mutant and a functional protein covalently coupled. The functional proteins are IL-4, IL-13, TGF-β, or IL-10.

2. The use according to claim 1, characterized in that, The IL1RN is a human IL1RN protein, and its amino acid sequence is shown in SEQ ID NO:

1.

3. The use according to claim 1, characterized in that, The IL1RN mutant is a point mutant, wherein the point mutation is located in the inactive center region of SEQ ID NO:1, and the mutation does not affect the tertiary spatial structure of IL1RN.

4. The use according to claim 1, characterized in that, The recombinant protein contains a purification tag, which is a His-tag.

5. The use according to claim 4, characterized in that, The recombinant protein was expressed and purified in Escherichia coli, 293 engineered cells, or CHO engineered cells.

6. The use according to claim 1, characterized in that, The allergic rhinitis referred to is seasonal allergic rhinitis, perennial allergic rhinitis, intermittent allergic rhinitis, or drug-induced allergic rhinitis.

7. The use according to claim 1, characterized in that, The sinusitis referred to includes acute sinusitis, chronic sinusitis, fungal sinusitis, allergic sinusitis, or odontogenic sinusitis.

8. The use according to claim 1, characterized in that, The active ingredient of the drug is IL1RN-X or IL1RNmut-X, and the concentration used is 0.01-2000 ng / mL.

9. A pharmaceutical composition for treating allergic rhinitis and / or sinusitis, characterized in that, It comprises the recombinant protein as described in any one of claims 1-8, and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, characterized in that, The carrier is selected from at least one of isotonic agents, antibacterial agents, stabilizers, thickeners, solubilizers, protein protectants, and pharmaceutical carriers.

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