WO2018214757A1 - 重组Slit2D2(C386S)-HSA融合蛋白及其在预防和/或治疗肺部炎症中的应用 - Google Patents

重组Slit2D2(C386S)-HSA融合蛋白及其在预防和/或治疗肺部炎症中的应用 Download PDF

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WO2018214757A1
WO2018214757A1 PCT/CN2018/086548 CN2018086548W WO2018214757A1 WO 2018214757 A1 WO2018214757 A1 WO 2018214757A1 CN 2018086548 W CN2018086548 W CN 2018086548W WO 2018214757 A1 WO2018214757 A1 WO 2018214757A1
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fusion protein
slit2d2
hsa
protein
recombinant
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French (fr)
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李华顺
任宝永
刘鹏
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李华顺
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    • C—CHEMISTRY; METALLURGY
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor

Definitions

  • the present invention relates to the field of biomedicine, and in particular to a recombinant Slit2D2 (C386S)-HSA fusion protein and its use in the prevention and/or treatment of pulmonary inflammation.
  • C386S recombinant Slit2D2
  • Acute lung injury (ALI)/Acute respiratory distress syndrome (ARDS) is an acute pulmonary inflammation caused by various pulmonary internal and external factors, acute respiratory distress, non-cardiac Pulmonary edema and a persistent over-inflammatory response characterized by a clinical syndrome.
  • ARDS has now become an important cause of severe trauma, severe infections, and death in elderly patients. The results show that the mortality rate of ARDS is as high as 22%, and the mortality rate of elderly patients over 85 years old is as high as 60%.
  • the recovery rate of young ARDS patients has improved, but their lung function and quality of life can not be restored to normal levels, causing physical and psychological burden on patients with ARDS.
  • asthma is an important global chronic pulmonary inflammation disease.
  • China Asthma Alliance there are as many as 30 million asthma patients in China, and the prevalence of asthma in the population is as high as 1.24%, and in many asthma diseases.
  • 6 million children have a prevalence of 1.97%, which means that 2 out of every 100 children have asthma.
  • the essence of asthma is an airway inflammatory chronic disease caused by allergens.
  • asthma patients Compared with the airway structure of normal people, asthma patients not only have airway inflammation, but also airway remodeling, including goblet cell hyperplasia, subepithelial fiber deposition, smooth muscle atrophy and hypertrophy, and vascular proliferation.
  • airway remodeling is closely related to the severity of the disease.
  • asthma attacks mainly include cough and chest pain, difficulty breathing, and paroxysmal wheezing. Most patients can relieve themselves or be relieved by treatment, but if they are not controlled in time, they can even be life-threatening.
  • the severity and frequency of asthma attacks vary from person to person. Chronic inflammation and remodeling of tissue structure lead to important pathophysiological features of the disease, such as persistent airway hyperresponsiveness.
  • the clinical application of asthma treatment drugs mainly includes two major categories.
  • Drugs include inhaled corticosteroids, leukotriene regulators, long-acting beta 2 receptor agonists, anti-IgE antibodies, and the like.
  • Another type is a relief medication that relieves the symptoms of an acute asthma attack by quickly releasing the bronchospasm.
  • Drugs include rapid-acting inhaled ⁇ 2 receptor agonists, inhaled anticholinergic drugs, etc., mainly through the action of ⁇ 2 receptors on the surface of cell membranes such as airway smooth muscle and mast cells, relaxing airway smooth muscle, reducing mast cells and alkalophilic Granulocyte degranulation and release of inflammatory mediators, reducing microvascular permeability, increasing the swing of airway epithelial cilia, etc., alleviating asthma symptoms.
  • the current drugs are effective in reducing the frequency of asthma attacks and the symptoms at the time of onset, but "treating the symptoms without treating the symptoms" cannot fundamentally cure asthma. Once the medication is stopped, the patient's condition will recur and worsen.
  • Slit is a type of secreted glycoprotein with a molecular weight of about 200kD.
  • Slit1 Slit2
  • Slit3 Slit3 genes cloned in mammals, named Slit1, Slit2 and Slit3. Its structure consists of an N-terminal signal peptide, four leucine-rich repeats (LRRs) and multiple EGF-like repeats (7 in Drosophila and 9 in vertebrates); Studies have shown that the LRRs are the binding regions of the Slit protein and the receptor Robo. The Slit protein functions by binding to the receptor Robo. The extracellular IgG domains of Robos are thought to be required for binding to the ligand Slit.
  • LRRs leucine-rich repeats
  • EGF-like repeats 7 in Drosophila and 9 in vertebrates
  • the longer intracellular region interacts with some important signaling molecules and participates in signal transduction downstream of Slit/Robo, thereby completing the stimulation signal by the cell.
  • the literature has confirmed the mechanism analysis of the protein in the interaction region between Slit2 and Robo, and found that the second domain D2 of Slit2 binds to Ig1 of Robo1 and initiates signal transduction (Morlot, Hemrika et al. 2007, Hohenester 2008, Seiradake, von Philipsborn et al. 2009).
  • Slit2 molecules have anti-inflammatory properties and have potential applications in inflammatory diseases (201510661923.6, PCT/CN2015/092079, 201611110752.9, US20160120940).
  • the present invention provides the following technical solutions.
  • the invention provides a fusion protein comprising a D2 domain portion of a Slit2 protein and an HSA portion, wherein the 386th cysteine corresponding to the Slit2 protein is replaced in the Slit2 protein D2 domain (Slit2D2) portion For serine (C386S).
  • the present invention provides a fusion protein having the structure: Slit2D2(C386S)-HSA; or HSA-Slit2D2 (C386S), wherein "-" represents a chemical bond or a linker.
  • Another aspect of the present invention provides a fusion protein comprising a D2 domain portion of a Slit2 protein and an HSA portion, wherein the 386th cysteine corresponding to the Slit2 protein in the Slit2 protein D2 domain (Slit2D2) portion The acid was replaced with serine (C386S).
  • the fusion protein of the present invention has the structure: Slit2D2 (C386S)-HSA; or HSA-Slit2D2 (C386S), wherein "-" represents a chemical bond or a linker.
  • the fusion protein provided by the present invention can be formed by fusion of a Slit2 protein D2 domain and an HSA protein, wherein the 386th cysteine corresponding to the Slit2 protein in the Slit2 protein D2 domain is replaced with a serine.
  • the Slit2 protein D2 domain comprises or consists of the amino acid sequence set forth in SEQ ID NO : 1.
  • the fusion protein of the invention comprises or consists of the sequence set forth in SEQ ID NO : 2.
  • the invention also provides a nucleotide encoding a fusion protein of the invention, the nucleotide sequence comprising or consisting of the sequence set forth in SEQ ID NO : 3.
  • the invention also provides an expression cassette comprising a nucleotide of the invention.
  • the present invention also provides a vector (for example, a plasmid or a viral vector) comprising a nucleotide encoding the fusion protein of the present invention, or a microorganism (for example, Escherichia coli, Bacillus subtilis, Bacillus megaterium, Corynebacterium, Saccharomyces cerevisiae, Bi Red yeast and yeast, etc.) or recombinant cells (such as plant cells or animal cells).
  • a vector for example, a plasmid or a viral vector
  • a microorganism for example, Escherichia coli, Bacillus subtilis, Bacillus megaterium, Corynebacterium, Saccharomyces cerevisiae, Bi Red yeast and yeast, etc.
  • recombinant cells such as plant cells or animal cells.
  • the invention also provides a method of making a fusion protein of the invention comprising expressing a fusion protein nucleotide encoding the invention.
  • the method for preparing the fusion protein of the present invention comprises the following steps:
  • the recombinant expression vector is preferably a plasmid vector
  • the host cell or microorganism is preferably selected from the group consisting of Escherichia coli, Bacillus subtilis, Bacillus megaterium, Corynebacterium, Saccharomyces cerevisiae, Pichia or mammalian cells.
  • the present invention preferably employs a pCDNA3.4 expression vector; the host cell or microorganism is Escherichia coli TOP10; or the separation and purification of the fusion protein is performed by HSA affinity chromatography and weak anion exchange chromatography.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the fusion protein of the invention and a pharmaceutically acceptable carrier.
  • pulmonary inflammation includes acute chronic pulmonary inflammation diseases and chronic pulmonary inflammation diseases such as acute lung injury or asthma.
  • the present invention uses a genetic engineering method to mutate the cysteine (Cys) at position 386 of the Slit2 sequence to serine (Ser), which unexpectedly increases its stability in vivo, prolongs half-life, and increases
  • the therapeutic effect on lung inflammation is significantly better than that of Slit2D2-HSA recombinant protein.
  • the preparation process is easy to purify and separate, and the purity is as high as 97.48%, which is more conducive to the development and popularization of drugs.
  • the fusion protein provided by the present invention may have at least one of the effects of preventing and/or treating acute lung inflammation by significantly inhibiting immersion of inflammatory cells into the lung; inhibiting the expression of inflammatory factors by inhibiting infiltration of inflammatory cells in the alveoli In addition, it prevents and/or treats chronic lung inflammation, and protects the lungs from respiratory function and has a remarkable effect.
  • 1 is a recombinant plasmid pCDNA3.4-Slit2D2(C386S)-HSA map of a recombinant Slit2D2(C386S)-HSA fusion protein according to an embodiment of the present invention
  • FIG. 2 is a SDS-PAGE diagram showing molecular weight detection of a recombinant Slit2D2 (C386S)-HSA fusion protein according to an embodiment of the present invention
  • Figure 3 is a diagram showing the results of SEC-HPLC detection of purified Slit2D2 (C386S)-HSA fusion protein after purification according to an embodiment of the present invention
  • FIG. 4 is a diagram showing experimental results of inhibiting inflammatory cell migration by a recombinant Slit2D2(C386S)-HSA fusion protein according to an embodiment of the present invention, wherein G1 represents 1 group, G2 represents 2 groups, and so on; other groups and G2 group When compared, *** means p ⁇ 0.001, ** means p ⁇ 0.01, and * means p ⁇ 0.05;
  • Figure 5 is a graph showing the results of inhibition of accumulation of eosinophils in alveolar fluid by a recombinant Slit2D2(C386S)-HSA fusion protein according to an embodiment of the present invention
  • Figure 6 is a graph showing the results of inhibition of accumulation of neutrophils and lymphocytes in alveolar fluid by a recombinant Slit2D2(C386S)-HSA fusion protein according to an embodiment of the present invention
  • FIG. 7 is a graph showing the detection results of an enhanced expiratory pause (Penh) function of an asthma model mouse in an asthma model according to an embodiment of the present invention.
  • FIG. 8 is a graph showing the results of detecting IL-5 cytokine levels in an asthma model mouse alveolar lavage fluid (BALF) according to an embodiment of the present invention.
  • FIG. 9 is a graph showing the results of detecting IL-13 cytokine levels in an asthma model mouse alveolar lavage fluid (BALF) according to an embodiment of the present invention.
  • Slit2D2 (C386S) was designed as shown in SEQ ID NO: 1, and then the Slit2D2 (C386S)-HSA fusion protein was designed.
  • the sequences and the coding genes of Slit2D2(C386S)-HSA are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
  • the Slit2D2 (C386S)-HSA fusion gene fragment was obtained by whole-gene synthesis, and inserted into pCDNA3.4 (brand: Thermo, Cat. No.
  • the HSA map is shown in Figure 1.
  • the recombinant expression vector was transformed into E. coli TOP10 and then transferred to a solid medium containing ampicillin (AMP) for propagation, screening for positive clones, confirmation of successful vector construction and seed conservation by sequencing.
  • AMP ampicillin
  • Toxin plasmid extraction kit was used without the recombinant plasmids in E. coli TOP10, used to transfect cells ExpiCHO-S TM ((Gibco Catalog No.A29127)) cells.
  • Culture ExpiCHO-S TM cells were transfected with the recombinant plasmid (transfection reagent: ExpiFectamine TM CHO Transfection Kit, Gibco Catalog No.A29129) When the cell density reached 4 ⁇ 10 6 -6 ⁇ 10 6 cells / mL when transfected After 10 days of culture, the supernatant was collected, centrifuged at high speed, and passed through HSA affinity chromatography (chromatographic packing: Thermo, Cat. No. 191297050) and weak anion exchange chromatography (brand: Tiandi Renhe, article number: DEAE Beads 6FF, SI005025) The Slit2D2 (C386S)-HSA fusion protein was purified.
  • Figure 2 is a SDS-PAGE of the molecular weight of the recombinant Slit2D2 (C386S)-HSA fusion protein
  • Figure 3 is a SEC-HPLC analysis of the recombinant Slit2D2 (C386S)-HSA fusion protein.
  • the molecular weight of the above-mentioned purified fusion protein was detected by SDS-PAGE method, and the purity of the fusion protein was examined by SEC-HPLC. It can be seen from Fig. 2 and Fig.
  • Acute lung injury (Acutelunginjury, ALI) is a systemic uncontrolled inflammatory response caused by various direct and indirect injury factors, accompanied by alveolar epithelial cells and capillary endothelial cell injury, the pathogenesis of which is still unclear, and there is still no effective treatment. means.
  • Rat airway perfusion of lipopolysaccharide (LPS) is a commonly used animal model. In this study, intratracheal infusion of LPS induced acute lung injury in rats. Compounds were injected intravenously into animals. Four hours later, lung lavage and bronchoalveolar lavage fluid were collected and differential cell counts were measured to represent the efficacy of test compounds in preventing lipopolysaccharide-induced lung injury.
  • Feeder/Supplier Beijing Weitong Lihua Experimental Animal Co., Ltd.
  • PBS phosphate buffer
  • DEX dexamethasone
  • ZD004 slit2D2-HSA
  • ZD018 slit2D2 (C-S)-HSA
  • N/A means Not Applicable.
  • the second group is the LPS model group, and the total number of cells is increased.
  • the positive control dexamethasone can significantly reduce the number of neutrophils and total at 1 mg/kg. Number of cells.
  • the fusion protein Slit2D2(C386S)-HSA significantly inhibited the increase of cell number and the number of neutrophils was significantly controlled compared with the model group.
  • Slit2D2(C386S)-HSA was superior to Slit2D2-HSA recombinant protein in the dose of 5mg/kg.
  • Slit2D2 (C386S)-HSA fusion protein can protect lung tissue structure and function by significantly inhibiting inflammatory cells in the lungs. .
  • Feeder/Supplier Beijing Weitong Lihua Experimental Animal Co., Ltd.
  • ZD018 Slit2D2 (C386S)-HSA
  • sensitization was intraperitoneally injected, and the asthma group was given a sensitization solution containing 20 ⁇ g of ovalbumin and 2 mg of alum suspension.
  • the normal group did not receive any treatment.
  • mice in groups 2-4 will be stimulated with aerosol, 100 mg OVA dissolved in 10 ml PBS, 5 ⁇ l Triton X-100 added, nebulized The facility (Buxco) was atomized for 30 minutes, then the nebulizer was turned off, the mice were kept in the inhalation box for an additional 7 minutes and then removed. Negative control mice will be exposed to aerosolized PBS for 30 minutes.
  • Group 1 PBS control group
  • Group 2 OVA control group
  • Group 4 ZD018 was administered intraperitoneally 2 hours prior to OVA sensitization on days 28, 29 and 30.
  • Group 5 was administered dexamethasone 2 hours prior to OVA sensitization on days 28, 29 and 30.
  • Penh was detected in mice with a Buxco mouse non-invasive pulmonary function meter.
  • the change of Penh after atomization challenge of 300 ⁇ l doubling concentration of methacholine (Mch) was determined.
  • the excitation concentration was from low to high, and was 0, 0.78, 1.56, 3.125, 6.25, 12.5, 25 and 50 g/L, respectively.
  • the Penh value at each Mch excitation concentration was converted to a percentage of the Penh value at the time of physiological saline challenge (Penh value excited by Mch/Penh value excited by physiological saline ⁇ 100%), expressed as Penh%, as a mouse airway Reactivity index.
  • bronchoalveolar lavage bronchial alveolar lavage fluid (BALF) for cytology.
  • BALF bronchial alveolar lavage fluid
  • IL-13 and IL-5 cytokine levels in alveolar lavage fluid were measured using Elisa Kit (R&D, USA).
  • Eosinophils are the main effector cells in the medical field recognized as asthmatic diseases.
  • the effective inhibition of this eosinophil is the key to evaluating whether a drug is effective.
  • BALF bronchoalveolar lavage fluid
  • Slit2D2 (C386S)-HSA recombinant protein can significantly inhibit eosinophilicity after administration.
  • the granulocytes accumulate in the alveolar fluid, and as shown in Fig. 6, the Slit2D2(C386S)-HSA recombinant protein can also inhibit the accumulation of neutrophils and lymphocytes in the alveolar fluid. This recombinant protein has significant drug efficacy.
  • IL-13 and IL-5 cytokines in alveolar lavage fluid were detected.
  • IL-13 and IL-5 are important inflammatory factors in the asthma model and play a key role in the occurrence and development of the disease.
  • the results are shown in Figure 8-9.
  • the results showed that the Slit2D2(C386S)-HSA recombinant protein could inhibit the levels of inflammatory cytokines IL-13 and IL-5 in the foaming solution of asthmatic mice.

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Abstract

提供了一种重组融合蛋白，所述融合蛋白由Slit2蛋白D2结构域和HSA蛋白融合形成，所述Slit2蛋白分子的第386位氨基酸为丝氨酸。

Description

重组Slit2D2(C386S)-HSA融合蛋白及其在预防和/或治疗肺部炎症中的应用 技术领域
本发明涉及生物医药领域，具体涉及重组Slit2D2(C386S)-HSA融合蛋白及其在预防和/或治疗肺部炎症中的应用。
背景技术
近年来，随着卫生水平的不断提高，感染性的疾病己不再是危害人类健康的最主要原因。而包括肿瘤、自身免疫病、也血管疾病等在内的严重慢性疾病的危害逐渐成为首要的危害。而以肺部炎症类疾病为代表的相关疾病正严重影响人类的健康，包括急性炎症和慢性炎症。
急性肺损伤(Acute lung injury，ALI)/急性呼吸窘迫综合征(Acute respiratory distress syndrome，ARDS)是一种急性肺部炎症，由各种肺内外因素所致的以急性呼吸窘迫、非心源性肺水肿和持续过度的炎症反应为特点的临床综合征。ARDS目前已成为严重创伤、重症感染以及老年患者死亡的重要原因。研究结果显示，ARDS的病死率高达22％，其中85岁以上老年患者病死率高达60％。近年来，虽然在发病机制和诊治方面有所进展，年轻ARDS患者的康复率有所提高，但其肺功能以及生活质量均不能恢复至正常水平，给ARDS患者生理和心理上造成承重负担。
另外，哮喘是一个重要的全球性慢性肺部炎症类疾病，据中国哮喘联盟发布的一份报告显示，我国哮喘患者多达3000万，人群哮喘患病率高达1.24％，而在众多的哮喘病患者中，儿童就占600万，患病率为1.97％，这就意味着每100名儿童中就有2名患者哮喘。全球范围内哮喘患者约有3.34亿人，COPD患者约有3.28亿人，预计2030年，COPD将成为美国第三大致死性疾病。哮喘其本质是过敏原引起的气道炎症性慢性疾病。与正常人的气道结构相比，哮喘患者不仅存在气道炎症，还发生气道重塑，包括杯状细胞增生、上皮下的纤维沉积、平滑肌増生和肥大、血管增生等。气道重塑的进程与疾病的严重程度密切相关。临床上，哮喘疾病发作主要以咳嗽和胸疼、 呼吸困难、发作性的喘息等为主要表现。多数患者可自行缓解或经治疗缓解，但是若不能及时得到控制，严重的甚至会威胁生命。哮喘发作的严重程度与频率因人而异。慢性炎症与组织结构重塑导致了疾病重要的病理生理学特征，如持续性的气道高反应性。目前临床应用的哮喘治疗药物主要包括两大类。一类是控制药物，需要每天且长期甚至终生的使用，主要是通过抗炎作用控制哮喘发作及其进一步发展。药物包括吸入糖皮质激素、白三稀调节剂、长效β2受体激动剂、抗IgE抗体等。另一类型是缓解药物，通过迅速解除支气管痉挛缓解哮喘急性发作造成的症状。药物包括速效吸入β2受体激动剂、吸入性抗胆碱能药物等，主要通过对气道平滑肌和肥大细胞等细胞膜表面的β2受体的作用，舒张气道平滑肌、减少肥大细胞和嗜碱性粒细胞脱颗粒和炎性介质的释放、降低微血管的通透性、增加气道上皮纤毛的摆动等，缓解哮喘症状。目前的药物对于减少哮喘发作频率和发作时的症状是有效的，但＂治标而不治本＂，不能根本上治愈哮喘。一旦停止用药，患者的病情会反复发作并恶化。如果气道重塑和持续性肺功能损失己经建立，目前医疗手段不能对其逆转。此外，相当一部分患者对现有的抗炎症治疗不敏感，将发展为严重的顽固性哮喘。面对上述肺部类炎症，开发高效的抗炎药物分子具有重要的意义。
Slit是一类分泌性糖蛋白，分子量约为200kD，哺乳类动物中克隆到的Slit基因有三个，分别命名为Slit1，Slit2和Slit3。它的结构由N-端的信号肽，4个富含亮氨酸的重复序列(LRRs)以及多个EGF样的重复序列(在果蝇中是7个，在脊椎动物中是9个)组成；研究表明，其中的LRRs是Slit蛋白和受体Robo的结合区域。Slit蛋白通过结合受体Robo发挥功能。Robos胞外的IgG domains被认为是与配体Slit结合所必需的，较长的胞内区域则和一些重要的信号分子相互作用，参与Slit/Robo下游的信号转导，从而完成刺激信号由细胞外部到内部骨架的传递。目前，已有文献确认Slit2与Robo相互作用区域蛋白质的机构解析，发现Slit2的第二个结构域D2与Robo1的Ig1结合，启动信号传导(Morlot,Hemrika et al.2007,Hohenester 2008,Seiradake,von Philipsborn et al.2009)。Slit2分子具有抑炎的能力，在炎性类疾病中有潜在的应用价值(201510661923.6、PCT/CN2015/092079、201611110752.9、US20160120940)。
发明内容
有鉴于此，本发明提供了如下的技术方案。
本发明一方面提供了一种融合蛋白，其包含Slit2蛋白D2结构域部分和HSA部分，其中在所述Slit2蛋白D2结构域(Slit2D2)部分中对应于Slit2蛋白的386位半胱氨酸被替换为丝氨酸(C386S)。
示例性地，本发明提供了具有如下结构的融合蛋白：Slit2D2(C386S)-HSA；或HSA-Slit2D2(C386S)，其中“-”代表化学键或者连接子。
本发明另一方面提供了一种融合蛋白，其由Slit2蛋白D2结构域部分和HSA部分组成，其中在所述Slit2蛋白D2结构域(Slit2D2)部分中对应于Slit2蛋白的第386位半胱氨酸被替换为丝氨酸(C386S)。示例性地，本发明所述的融合蛋白具有如下结构：Slit2D2(C386S)-HSA；或HSA-Slit2D2(C386S)，其中“-”代表化学键或者连接子。
优选地，本发明所提供的融合蛋白可由Slit2蛋白D2结构域和HSA蛋白融合形成，其中在所述Slit2蛋白D2结构域中对应于Slit2蛋白的第386位半胱氨酸被替换为丝氨酸。
示例性地，Slit2蛋白D2结构域包含或者由SEQ ID NO ：1所示的氨基酸序列组成。
优选地，本发明所述融合蛋白包含或者由如SEQ ID NO ：2所示的序列组成。
本发明还提供一种编码本发明所述的融合蛋白的核苷酸，所述核苷酸序列包含或由如SEQ ID NO ：3所示的序列组成。
本发明还提供了一种表达盒，其包含本发明所述的核苷酸。
本发明还提供了包含编码本发明所述的融合蛋白的核苷酸的载体(例如质粒或病毒载体)、或微生物(例如大肠杆菌、枯草芽孢杆菌、巨大芽孢杆菌、棒状杆菌、酿酒酵母、毕赤酵母和酵母等)或重组细胞(例如植物细胞或动物细胞)。
本发明还提供了本发明所述的融合蛋白的制备方法，包括表达编码本发明所述的融合蛋白核苷酸。
任选的，本发明所述的融合蛋白的制备方法包括如下步骤：
(1)构建编码本发明所述的融合蛋白核苷酸的重组表达载体；
(2)将所制得的重组表达载体转化宿主细胞或微生物并使编码本发明所述的融合蛋白核苷酸得以表达；和
(3)融合蛋白的分离和纯化。
在本发明中，所述重组表达载体优选为质粒载体，所述宿主细胞或者微生物优选选自大肠杆菌、枯草芽孢杆菌、巨大芽孢杆菌、棒状杆菌、酿酒酵母、毕赤酵母或哺乳动物细胞。具体地，本发明优选采用pCDNA3.4表达载体；所述宿主细胞或微生物为大肠杆菌TOP10；或融合蛋白的分离纯化采用HSA亲和层析和弱阴离子交换层析。
本发明还提供了一种药物组合物，包括本发明所述的融合蛋白以及药学上可接受的载体。
本发明还提供了本发明所述的融合蛋白在制备用于预防和/或治疗肺部炎症的药物组合物中的用途。在本发明中，肺部炎症包括急性慢性肺部炎症类疾病和慢性肺部炎症类疾病，例如急性肺损伤或哮喘等。
本发明采用基因工程手段，将Slit2序列的第386位的半胱氨酸(Cys)突变为丝氨酸(Ser)，这一替换出乎意料地增加了其在体内的稳定性，延长了半衰期，提高了对肺部炎症的治疗效果，其效果明显优于Slit2D2-HSA重组蛋白的效果。且其制备过程易于纯化和分离，纯度高达97.48％，更有利于药物的开发及推广应用。
本发明提供的融合蛋白可以至少具有如下功效之一：通过显著抑制炎性细胞浸入肺部进而预防和/或治疗急性肺部炎症；通过抑制肺泡中炎性细胞的浸润、抑制炎性因子的表达进而预防和/或治疗慢性肺部炎症，保护肺部呼吸功能且效果显著。
附图说明
图1所示为本发明实施例提供的重组Slit2D2(C386S)-HSA融合蛋白的重组质粒pCDNA3.4-Slit2D2(C386S)-HSA图谱；
图2所示为本发明实施例提供的重组Slit2D2(C386S)-HSA融合蛋白分子量检测的SDS-PAGE图；
图3所示为本发明实施例提供的重组Slit2D2(C386S)-HSA融合蛋白纯化后的SEC-HPLC检测结果图；
图4所示为本发明实施例提供的重组Slit2D2(C386S)-HSA融合蛋白抑制炎性细胞迁移的实验结果图，其中，G1表示1组，G2表示2组，依次类推；其他组与G2组相比时，***表示p＜0.001，**表示p＜0.01，*表示p＜0.05；
图5所示为本发明实施例提供的重组Slit2D2(C386S)-HSA融合蛋白抑制嗜酸性粒细胞在肺泡液中堆积的实验结果图；
图6所示为本发明实施例提供的重组Slit2D2(C386S)-HSA融合蛋白抑制中性粒细胞和淋巴细胞在肺泡液中堆积的实验结果图；
图7所示为本发明实施例提供的哮喘模型小鼠肺功能增强呼气间歇(enhanced pause，Penh)功能的检测结果图；
图8所示为本发明实施例提供的哮喘模型小鼠肺泡灌洗液(BALF)中的IL-5细胞因子含量检测结果图；
图9所示为本发明实施例提供的哮喘模型小鼠肺泡灌洗液(BALF)中的IL-13细胞因子含量检测结果图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1 融合蛋白Slit2D2(C386S)-HSA的制备
根据已知的Slit2序列[GenBank:EAW92793.1]分析设计构建Slit2的第二个结构域Slit2D2，设计Slit2D2(C386S)如SEQ ID NO:1所示，进而设计Slit2D2(C386S)-HSA融合蛋白的序列及Slit2D2(C386S)-HSA的编码基因分别如SEQ ID NO:2和SEQ ID NO:3所示。通过全基因合成得到Slit2D2(C386S)-HSA融合基因片段，利用T/A克隆将其插入到pCDNA3.4(品牌：Thermo，货号：A14697)表达载体，重组载体pCDNA3.4-Slit2D2(C386S)-HSA图谱如图1所示。将上述重组表达载体转化到大肠杆菌TOP10后转种到含有氨苄青霉素(AMP)的固体培养基中进行繁殖、筛选阳性克隆、通过测序确认载体构建成功及保种。
用无内毒素质粒提取试剂盒提取大肠杆菌TOP10中的重组质粒，用于转染ExpiCHO-S TM细胞((Gibco Catalog No.A29127))细胞。培养ExpiCHO-S TM细胞，当细胞密度达到4×10 6-6×10 6细胞/毫升时进行重组质粒的转染(转染试剂：ExpiFectamine TM CHO Transfection Kit，Gibco Catalog No.A29129)，转染后培养10天，收集上清，高速离心，通过HSA亲和层析(层析填料：Thermo，货号：191297050)和弱阴离子交换层析(品牌：天地人 和，货号：DEAE Beads 6FF，SI005025)纯化Slit2D2(C386S)-HSA融合蛋白。
图2为重组Slit2D2(C386S)-HSA融合蛋白分子量检测的SDS-PAGE图，图3为重组Slit2D2(C386S)-HSA融合蛋白纯化后的SEC-HPLC检测结果图。通过SDS-PAGE方法检测上述纯化得到的融合蛋白的分子量，并通过SEC-HPLC检测融合蛋白纯度。由图2和图3可知，已经成功构建表达融合蛋白Slit2D2((C386S))-HSA的重组表达载体，并实现了在宿主细胞中表达和纯化融合蛋白Slit2D2((C386S))-HSA，其纯度可高达97.48％。
实施例2 融合蛋白Slit2D2(C386S)-HSA在大鼠急性肺损伤模型中的药效学检测
急性肺损伤(Acutelunginjury，ALI)是各种直接和间接致伤因素导致的全身失控性炎症反应，伴有肺泡上皮细胞及毛细血管内皮细胞损伤，其发病机制尚不清楚，目前尚缺乏有效的治疗手段。大鼠气道内灌注脂多糖(LPS)是一种常用的动物模型，在本研究中，气管内灌注LPS诱导大鼠急性肺损伤。化合物尾静脉注射到动物，四小时后，收集肺灌洗和支气管肺泡灌洗液，差分细胞计数测量，以代表测试化合物在预防脂多糖致肺损伤的疗效。
2.1 实验动物
动物：Wistar大鼠
治疗史：无
性别：雄性
体重：220-250克
饲养者/供应商：北京维通利华实验动物有限公司
测试设施：上海澎立生物公司
适应：不少于7天
房间：SPF房间
室温：20-26℃
房间相对湿度：40-70％
光周期：12小时光照(08:00-20:00)和12小时黑暗
动物饲养：治疗组3～4只/笼
2.2 动物分组及给药方案
动物分组的实验设计及给药方案如表1所示。
表1 动物分组及给药方案
Figure PCTCN2018086548-appb-000001
注：PBS：磷酸缓冲液；DEX：地塞米松；ZD004：slit2D2-HSA；ZD018：slit2D2(C-S)-HSA；N/A表示Not Applicable。
2.3 动物模型建立
除1组外，其他各组动物均接受脂多糖肺部灌注，将动物用3-5％异氟烷麻醉，用微型喷雾器插管将100μlLPS溶液(1mg/ml)导入大鼠气管，4h后用水合氯醛(750mg/kg)处死动物。用PBS+1％白蛋白4毫升原位轻轻灌洗肺部三次。灌洗后支气管肺泡灌洗液BALF被保存在冰中，之后进行嗜酸性粒细胞(EOS)、巨噬细胞(Mac)、中性粒细胞(Neu)、淋巴细胞(Lym)和细胞总量的计数。
2.4 实验结果
在这项研究中，药效结果如图4，第2组为LPS模型组，其细胞总数升高，阳性对照地塞米松在1mg/kg的剂量条件下可显著降低中性粒细胞数目和总细胞数目。融合蛋白Slit2D2(C386S)-HSA在5mg/kg、1mg/kg和0.2mg/kg测试剂量下，与模型组相比，均可以显著抑制细胞数量的增加，中性粒细胞数目也得到明显的控制。同时，Slit2D2(C386S)-HSA在5mg/kg给药剂量下，其效果优于Slit2D2-HSA重组蛋白药效。
急性肺损伤模型中，炎性细胞的浸润是造成组织损伤的最主要因素，因此上述结果说明Slit2D2(C386S)-HSA融合蛋白可通过显著抑制炎性细胞在肺部浸入进而保护肺脏组织结构和功能。
实施例3 融合蛋白Slit2D2(C386S)-HSA在小鼠哮喘模型上的药效评价
3.1 材料、环境与设施：
动物：BALB/c
治疗史：无
性别：雌性
年龄：6-7周
饲养者/供应商：北京维通利华实验动物有限公司
测试设施：上海澎立生物公司
适应：不少于7天
房间：SPF房间
室温：20-26℃
房间相对湿度：40-70％
光周期：12小时光照(08:00-20:00)和12小时黑暗
动物饲养：治疗组3～4只/笼
3.2 动物分组及给药方案
动物分组的实验设计及给药方案参见表2。
表2 动物分组及给药方案
Figure PCTCN2018086548-appb-000002
注：ZD018：Slit2D2(C386S)-HSA
3.3 动物模型建立
3.3.1 哮喘模型致敏
实验第1天、第14天腹腔注射致敏，哮喘组每次给予致敏溶液含20微克卵清蛋白和2毫克明矾混悬液。正常组不予任何处理。
3.3.2 在第28，29和30天，组2-4中的小鼠将用气溶胶进行刺激，100毫克OVA溶解在10毫升PBS中，加入5微升Triton X-100，用雾化吸入设 施(Buxco)雾化30分钟，然后雾化器将被关闭，小鼠保持在吸入箱额外7分钟，然后取出。负对照小鼠将暴露于雾化PBS中30分钟。
3.3.3 给药
组1(PBS对照组)在第28，29和30天PBS致敏前2小时腹膜内注射给药PBS。组2(OVA对照组)在第28，29和30天OVA致敏前2小时腹膜内注射给药PBS。组4，在第28，29和30天OVA致敏前2小时腹膜内注射给药ZD018。组5在第28，29和30天OVA致敏前2小时灌胃给药地塞米松。
3.4 增强呼气间歇(enhanced pause，Penh)的检测
在第31天(最后一次挑战后24小时)，用Buxco小鼠无创肺功能仪检测小鼠的Penh。测定300μl倍增浓度的乙酰甲胆碱(Mch)雾化激发后Penh的变化，激发浓度由低到高，依次为0、0.78、1.56、3.125、6.25、12.5、25和50g/L，记录各浓度级Mch激发下的Penh平均值。将每个Mch激发浓度下的Penh值转换为与生理盐水激发时的Penh值的百分比(Mch激发的Penh值/生理盐水激发的Penh值×100％)，以Penh％表示，作为小鼠气道反应性的评价指标。
3.5 气道炎症的观察：支气管肺泡灌洗，支气管肺泡灌洗液(BALF)行细胞学检查。
3.6 肺泡灌洗液(BALF)中的IL-13和IL-5细胞因子含量用Elisa Kit(R&D，USA)进行检测。
3.7 实验结果
嗜酸性粒细胞是医学界公认的哮喘疾病中主要效应细胞，能否有效抑制该嗜酸性粒细胞是评价一个药物是否有效的关键。在小鼠哮喘模型中，通过实验后收集支气管肺泡灌洗液(BALF)，对其炎性细胞进行计数，结果如图5，Slit2D2(C386S)-HSA重组蛋白给药后，可以显著抑制嗜酸性粒细胞在肺泡液中堆积，同时如图6结果，该Slit2D2(C386S)-HSA重组蛋白也可以抑制中性粒细胞和淋巴细胞在在肺泡液中堆积。说明，该重组蛋白具有显著的药物疗效。
通过增强呼气间歇(enhanced pause，Penh)功能的检测，结果如图7显示，Slit2D2(C386S)-HSA重组蛋白可以有效抑制乙酰甲胆碱(Mch)激发下的pench指标，说明：Slit2D2(C386S)-HSA重组蛋白治疗哮喘后，与对照治疗G2组比，其肺功能表现良好。
检测肺泡灌洗液(BALF)中的IL-13和IL-5细胞因子含量，IL-13和IL-5是哮喘模型中的重要炎性因子，对疾病的发生和发展具有关键作用。结果如图8-9，结果显示，Slit2D2(C386S)-HSA重组蛋白给药后，可以抑制哮喘模型小鼠泡灌洗液中炎性细胞因子IL-13和IL-5的含量。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换等，均应包含在本发明的保护范围之内。

Claims (12)

  1. 融合蛋白，其包含Slit2蛋白D2结构域部分和HSA部分，其中在所述Slit2蛋白D2结构域(Slit2D2)部分中对应于Slit2蛋白的第386位半胱氨酸被替换为丝氨酸(C386S)。
  2. 根据权利要求1所述的融合蛋白，其具有如下结构：Slit2D2(C386S)-HSA；或HSA-Slit2D2(C386S)，其中“-”代表化学键或者连接子。
  3. 根据权利要求1或2所述的融合蛋白，其由Slit2蛋白D2结构域部分和HSA部分组成。
  4. 根据权利要求1或2所述的融合蛋白，其中所述Slit2蛋白D2结构域的序列包含或者由SEQ ID NO：1所示的序列组成。
  5. 根据权利要求1或2所述的融合蛋白，其包含或者由SEQ ID NO：2所示的序列组成。
  6. 编码权利要求1-5中任一项所述的融合蛋白的核苷酸。
  7. 根据权利要求6所述的核苷酸，其包括或者由SEQ ID NO：3所示的序列组成。
  8. 重组载体或表达盒，其包含权利要求6或7的核苷酸。
  9. 包含权利要求6或7的核苷酸、或权利要求8所述的重组载体或表达盒的微生物或宿主细胞。
  10. 权利要求1-5中任一项所述的融合蛋白的制备方法，其包括表达权利要求6或7中的核苷酸。
  11. 药物组合物，其包含权利要求1-5中任一项所述的融合蛋白。
  12. 权利要求1-5中任一项所述的融合蛋白在制备预防和/或治疗肺部炎症(例如急性肺损伤或哮喘)的药物中的用途。
PCT/CN2018/086548 2017-05-26 2018-05-11 重组Slit2D2(C386S)-HSA融合蛋白及其在预防和/或治疗肺部炎症中的应用 WO2018214757A1 (zh)

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