Proline / arginine-rich end leucine-rich repeat protein (PRELP) for the treatment of lung disease

Abstract

Proline / arginine-rich end leucine-rich repeat protein (PRELP) or a variant thereof for use in the prevention and / or treatment of lung disease in a subject.

Description

[0001] NOVEL THERAPY

[0002] The present invention relates to the prevention and / or treatment of lung disease, and in particular to the use of proline / arginine-rich end leucine-rich repeat protein (PRELP) or a variant thereof in the prevention and / or treatment of lung disease.

[0003] Lung diseases such as lung fibrosis, also referred to as pulmonary fibrosis, and chronic obstructive pulmonary disease (COPD) are a leading cause of morbidity and mortality worldwide.

[0004] Lung fibrosis is a lung disease that occurs when lung tissue becomes damaged and scarred. The thickened, stiff tissue makes it harder for the lungs to work properly. The scarring that happens in lung fibrosis may be caused by many things. Often the cause of lung fibrosis cannot be found, the condition is then called an interstitial pneumonia (IIP). Idiopathic pulmonary fibrosis (IPL) is an example of an IPP. IPL has a poor prognosis, is associated with aging, and is characterized by the histopathological pattern of usual interstitial pneumonia. Currently the lung damage caused by lung fibrosis cannot be repaired. Medicines and therapies can sometimes help slow down the rate of fibrosis, ease symptoms and improve quality of life.

[0005] Chronic obstructive pulmonary disease (COPD) is a lung disease which results from pathological changes in the bronchioles and the lung parenchyma caused by airway and lung parenchymal inflammation, and has a feature of having obstructive bronchiolitis and pulmonary emphysema (pulmonary parenchymal destruction). Types of chronic obstructive pulmonary disease include chronic obstructive bronchitis, chronic bronchiolitis, emphysema and the like, fibrotic changes in the lungs of COPD patients has also been reported, for example Barnes PJ in Int J Biochem Cell Biol. Nov 2019, vol 116 describes peribronchiolar fibrosis in COPD patients and that in the small airways the fibroblasts may be profibrotic, pro-inflammatory and / or senescent.

[0006] The present invention aims to provide an improved therapy to prevent or treat lung disease. In particular, the present invention aims to provide an improved therapy to prevent or slow down / reduce the progression of lung fibrosis. The present invention offers a therapy which can be administered to patients before the onset of lung fibrosis, to prevent or slow down the progression of lung fibrosis. For example, the therapy could be given to patients who have another condition, a genetic predisposition, or are having another treatment, that increases the risk of the lung fibrosis. The therapy of the present invention may also be used to slow down or reduce the progression of a disease with a lung fibrosis component.

[0007] The therapy of the present invention may also be used to treat, prevent, slow down or reduce the progression of COPD.

[0008] The inventors have demonstrated that PRELP has a therapeutic effect in lung disease. In particular, that it can be used to prevent and / or slow the progression of lung disease, including lung fibrosis and / or COPD. PRELP has previously been shown to have a therapeutic effect in the CNS maintaining the integrity of the blood brain barrier, and also in the treatment and / or prevention of retinoblastoma and bladder cancers, no role has been suggested with respect to conditions affecting the lung.

[0009] Summary of the invention

[0010] According to the invention there is provided proline / arginine-rich end leucine-rich repeat protein (PRELP) or a variant thereof for use in the prevention and / or treatment of lung disease.

[0011] The invention also provides a method of preventing and / or treating lung disease in a subject, comprising administering to the subject a therapeutically effective amount of PRELP or a variant thereof.

[0012] The invention further provides the use of PRELP or a variant thereof in the manufacture of a medicament for preventing and / or treating lung disease.

[0013] The invention also provides the use of PRELP or a variant thereof in the manufacture of a medicament for preventing and / or treating lung disease.

[0014] PRELP or a variant thereof may be administered as a prophylactic to prevent the occurrence lung disease and / or to slow or reduce the progression of lung disease. PRELP or a variant thereof may be administered to a subject already diagnosed with lung disease. The PRELP or a variant thereof may be administered to reduce or slow the progression of lung disease.

[0015] The lung disease may be lung fibrosis. The lung disease may be COPD. A subject to be administered PRELP or a variant thereof may already be diagnosed with lung disease, the subject may already be diagnosed with lung fibrosis and / or COPD.

[0016] The lung disease may be disease with a lung fibrosis component.

[0017] The lung fibrosis may be a form of chronic fibrosing interstitial lung disease, such as idiopathic pulmonary fibrosis; or it may a form of asthma which has lung fibrosis.

[0018] PRELP or a variant thereof may be administered to prevent lung disease or to slow or reduce the progression of lung disease that is induced by another therapeutic agent.

[0019] PRELP or a variant thereof may be administered orally, nasally, intratracheally, or intravenously. Preferably PRELP or a variant thereof is administered intratracheally.

[0020] The subject may be a mammal. The mammal may be a human, a cat, a dog, a cow, a sheep or a horse, preferably a human, a cat or a dog, preferably a human.

[0021] PRELP

[0022] Proline / arginine-rich end leucine-rich repeat protein (PRELP) is a class II member of the small leucine-rich proteoglycan (SLRP) family. SLRP family members bind various extracellular proteins such as TGF-P, BMP, EGF, IGF, Wnt, and collagens, and can regulate multiple signalling pathways in context dependent manners. They are known to be involved in various biological processes such as cancer, inflammation and development, including in regulating the integrity of the blood brain barrier.

[0023] The amino acid sequence of human PRELP is SEQ ID NO 1 :

[0024] QPTRRPRPGTGPGRRPRPRPRPTPSFPQPDEPAEPTDLPPPLPPGPPSIFPDCPRECY CPPDFPSALYCDSRNLRKVPVIPPRIHYLYLQNNFITELPVESFQNATGLRWINLD NNRIRKIDQRVLEKLPGLVFLYMEKNQLEEVPSALPRNLEQLRLSQNHISRIPPGV FSKLENLLLLDLQHNRLSDGVFKPDTFHGLKNLMQLNLAHNILRKMPPRVPTAIH QLYLDSNKIETIPNGYFKSFPNLAFIRLNYNKLTDRGLPKNSFNISNLLVLHLSHNR ISSVPAINNRLEHLYLNNNSIEKINGTQICPNDLVAFHDFSSDLENVPHLRYLRLDG NYLKPPIPLDLMMCFRLLQSVVI

[0025] The protein may have an sp l-2 signal peptide (MRVLVLLACLAAASNA) at the start, connected to the PRELP peptide by a GS linker. This peptide may help secretion of the recombinant protein during protein production in a baculovirus expression vector system (BEVS). The signal peptide may be removed before the protein is used.

[0026] The protein may have a purification tag. The tag may be a FLAG-tag and the sequence of this may be AAADYKDDDDK. This tag may help with protein purification.

[0027] A variant of PRELP includes variants of SEQ ID NO: 1 which retain the biological activity of the protein of SEQ ID NO: 1, preferably at least 50, 60, 70, 80, 90% or more of the biological activity of a PRELP protein with SEQ ID No: 1 is retained in a variant of PRELP. Variants may include fragments of PRELP, truncated versions of PRELP, PRELP homologues, PRELP orthologues, PRELP expressed from mutated human genes, tagged PRELP, and PRELP with a signal sequence. A variant of PRELP may also include PRELP or a variant thereof as part of a fusion protein.

[0028] A variant of PRELP may be a protein at least about 75%, 80%, 85%, 90%, 95% or more homologous to SEQ ID NO: 1 or at least a portion of SEQ ID NO: 1. In some embodiments the homology will be as high as 94 to 96, 98 or 99%. Homology in this context means sequence similarity or identity, with identity being preferred. To determine whether a candidate peptide region has the requisite percentage similarity or identity to a reference polypeptide or peptide oligomer, the candidate amino acid sequence and the reference amino acid sequence are first aligned using a standard computer programme such as are commercially available and widely used by those skilled in the art. In a preferred embodiment the NCBI BLAST method is used (http: / / www.ncbi.nlm.nih.gov / BLAST / ). Once the two sequences have been aligned, a percent similarity score may be calculated. PRELP or a variant thereof may be administered as a protein. The protein may be full length PRELP or a variant thereof.

[0029] PRELP may be provided in a pharmaceutical composition comprising PRELP or a variant thereof and one or more suitable pharmaceutical excipient according to the route of administration. Suitable excipients may include: acidifiers, acidity regulators, anticaking agents, antioxidants, bulking agents, firming agents, gelling agents, coating agents, modified starches, sequestrants, thickeners, sweeteners, diluents, disintegrants, glidants, colourings, binders, lubricants, stabilisers, adsorbents, preservatives, humectants, flavourings, filmogenic substances, emulsifiers, wetting agents, release retardants and mixtures thereof.

[0030] The excipients may be one or more of potassium sorbate, sodium benzoate, e-polylysine, sucralose, maltodextrin, citric acid, sodium carbonate, calcium carbonate, magnesium carbonate, magnesium stearate, stearic acid, polyethylene glycol, natural starch, partially hydrolysed starch, modified starch, maize starch, potato starch, lactose, lactose monohydrate, calcium phosphate, calcium carbonate, calcium sulphate, polyvinylpyrrolidone, silica, colloidal silica, precipitated silica, magnesium silicates, aluminium silicates, sodium lauryl sulphate, magnesium lauryl sulphate, methacrylate copolymers, sodium dehydroacetate, xanthan gum, guar gum, tara gum, locust bean gum, fenugreek gum, gum arabic, alginic acid, sodium alginate, propylene glycol alginate, sodium croscarmellose, polyvinylpolypyrrolidone, polysorbate, glyceryl behenate, titanium dioxide, indigo carmine, cellulose, modified cellulose, calcium carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, ethylcellulose, gelatine, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, polydextrose, carrageenan, methylcellulose, saccharose, saccharose esters, sorbitol, xylitol, dextrose, fructose, maltitol, tragacanth gum, pectin, agar-agar, carboxypolymethylene, hydroxypropylmethylcellulose, tragacanth, mannitol, or a mixture thereof.

[0031] The pharmaceutical composition may be prepared by using methods known to a person skilled in the art.

[0032] The skilled person will be able to determine the appropriate dose of PRELP to be administered to a subject. In one embodiment, PRELP may be administered at a dose of about 20mg / day for one week for acute phase of a disease and about 5mg / day for chronic phase of a disease.

[0033] PRELP may be provided by upregulating the expression of PRELP in the subject. This may be achieved by providing a suitable vector comprising a promoter and a nucleotide sequence which encodes PRELP, or a variant thereof, wherein once administered to a subject the PRELP or a variant thereof will be expressed. The promoter may be tissue- or cell-specific to target the expression of PRELP or a variant thereof. The viral vector for nucleic acid expression of PRELP or a variant thereof may be any suitable viral vector that can be used to deliver genetic material to the cells of interest for expression of the PRELP or variant thereof. The viral vector may be an adeno-associated virus (AAV) vector.

[0034] Lung Fibrosis

[0035] Lung fibrosis results from abnormal tissue repair and is associated with persistent and / or severe tissue damage and cellular stress. Failure to adequately contain or eliminate factors triggering fibrosis can exacerbate inflammation and chronic wound healing responses, resulting in continued tissue damage and inadequate regeneration and, ultimately, fibrosis.

[0036] Although differing in aetiology and causative mechanisms, lung fibrotic diseases all have abnormal and exaggerated accumulation of extracellular matrix (ECM) components, mainly fibrillar collagens. The resulting lung fibrosis disturbs the normal architecture of the lungs, which ultimately leads to their dysfunction and failure.

[0037] Lung diseases with lung fibrosis may include IPF, giant cell interstitial pneumonia, sarcoidosis, cystic fibrosis, respiratory distress syndrome, drug-induced lung fibrosis, granulomatosis, silicosis, asbestosis, systemic scleroderma, virally induced hepatic cirrhosis selected from hepatitis C induced hepatic cirrhosis, sarcoidosis, systemic lupus erythematosus, and diseases of the skin with a lung fibrotic component such as scleroderma. Preferably the invention relates to the treatment or prevention of chronic fibrosing interstitial lung disease, even more preferably to the treatment of IPF. The compounds of the invention may also be used to treat other diseases with a lung fibrosis component including rheumatoid arthritis, acute lung injury, radiation induced lung fibrosis, pneumonitis, chronic hypersensitivity pneumonitis, systemic sclerosis, Sjogren's syndrome, interstitial lung diseases, or pulmonary arterial hypertension (PAH).

[0038] Other lung diseases such as asthma, pulmonary infection, and / or chronic obstructive pulmonary disease (COPD), may be associated with “airway epithelial damage” and fibrotic changes.

[0039] The compounds of the invention may be used to treat and / or prevent lung fibrosis, and / or to treat diseases with lung fibrosis, and / or to treat lung fibrosis associated with a pulmonary infection.

[0040] Lung fibrosis may be drug induced (e.g. as a result of exposure to amiodarone, nitrofurantoin, chemotherapy, methotrexate, cytotoxic anticancer drugs, molecular targeted drugs, immune checkpoint inhibitors, antirheumatic drugs, interferons, antiinflammatory analgesic, antibiotics and antiarrhythmic drugs etc), radiation induced, environmental induced (hypersensitivity pneumonitis) (e.g. exposure to allergens), autoimmune induced (connective tissue disease-associated interstitial lung disease - CTD-ILD), occupational induced (pneumoconiosis) (e.g. exposure to dust, fibers, fumes, asbestos, coal, silica).

[0041] Definitions

[0042] Prevention of lung disease according to the invention may include preventing the onset or preventing the progression of lung disease, such as lung fibrosis and / or COPD. The term prevention of lung disease may include where the invention includes achieving a prophylactic benefit. For prophylactic benefit, the compositions are optionally administered to an individual at risk of developing a particular lung disease, to an individual reporting one or more of the physiological symptoms of a lung disease, or to an individual at risk of reoccurrence of a lung disease.

[0043] Treatment according to the invention may result in a reduction in the progression of lung disease compared to in an untreated subject. The treatment may slow down or stop the progression of lung disease, for example slow down or stop the progression of lung fibrosis and / or COPD. The treatment may prevent, reduce or slow down deterioration in lung function caused by lung disease. Treatment may also encompass where the invention includes achieving a prophylactic benefit. The level of lung disease, for example lung fibrosis, may be quantified by high resolution computed tomography.

[0044] A reduction or slow down in the progression of a lung disease may refer to a decrease in the rate of disease progression as measured by any symptom of the disease (not just lung fibrosis) and may result in an improved quality of life.

[0045] A reduction or slow down in the progression of lung fibrosis may refer to a decrease in the rate of lung fibrosis progression by a statistically significant amount. Preferably a reduction by at least 10% as compared to a reference level (e.g. the level in the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about

[0046] 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. In some embodiments, reducing progression of lung fibrosis may encompass preventing lung fibrosis occurring at all or preventing any further lung fibrosis.

[0047] The skilled person will appreciate that preferred features of any one embodiment and / or aspect of the invention may be applied to all other embodiments and / or aspects of the invention.

[0048] Brief Description of the Drawings

[0049] Figure 1 - shows the results of histological studies of lungs from PRELP knockout mice. Figures la and lb show hematoxylin and eosin staining (a) and Elastica van Gieson staining (b) of lung tissue obtained from wild type mice and PRELP knock out (KO) mice with and without the addition of bleomycin (BLM). BLM-induced fibrotic change was worsened in PRELP knockout lungs. Figure 1c shows that Aschroft scores (a measure of the extent and severity of fibrosis) were significantly increased in BLM- treated PRELP knockout lungs compared to BLM-treated wild-type lungs. Figure Id shows that the amount of collagen contained in whole lungs was also significantly increased in BLM-treated PRELP knockout lungs.

[0050] Figure 2 - shows the results of physiological studies and Bronchoalveolar Lavage Fluid (BALF) analysis on BLM-treated mice. Figure 2a shows that lung compliance was significantly decreased in BLM-treated PRELP KO mice compared to BLM-treated wild type mice, which means BLM-treated PRELP KO mice are harder to inflate. Figure 2b shows that numbers of total cells were significantly increased in BALF of BLM-treated PRELP KO mice compared to BLM-treated wild-type mice.

[0051] Figures 1 and 2 demonstrate that PRELP knockout (KO) causes severe lung fibrosis in a BLM-induced lung fibrosis mouse model.

[0052] Figure 3 - shows the results of the analysis of BLM-induced lung fibrosis in mice with or without pretreatment using recombinant PRELP. Figures 3a show hematoxylin and eosin staining and Elastica van Gieson staining of lung tissue obtained from mice that received intratracheal pretreatment with lOmg or lOOmg of recombinant PRELP before the addition of BLM. The results demonstrate that pre-treatment with recombinant PRELP reduced BLM-induced fibrotic change in the mouse lungs. Figures 3b shows that the Aschroft scores were decreased in BLM-treated lungs with administration of recombinant PRELP. Figure 3c shows that administration of recombinant PRELP increased significantly lung compliance in BLM-treated lungs.

[0053] Figure 3 demonstrates that intratracheal administration of recombinant PRELP reduces lung fibrosis in BLM-treated mouse lungs.

[0054] Figure 4 - shows the results of Western Blot analysis of Vimentin and E-cadherin to see TGFp-induced epithelial-mesenchymal transition (EMT) in A549 cells (human pulmonary epithelial-derived cells). Figure 4a shows that using siRNA to knock down PRELP expression enhanced EMT in A549 cells. Figure 4b shows the administration of recombinant PRELP reduced EMT in them.

[0055] EMT is known to be associated with lung fibrosis. Figure 4 demonstrates that PRELP reduced TGFp-induced EMT. Figure 5 - shows immunostaining of A549 cells using anti zo-1 and cav-1 antibodies. Figures 5a and 5b show that the expression of zo-1 was reduced in A549 cells by PRELP knockdown using siRNA. Figures 5c and 5d illustrate that the expression of cav-1 was also reduced by PRELP knockdown. On the other hand, Figures 5e and 5f show that recombinant PRELP treatment increased the expression of zo-1 in A549 cells. Figures 5g and 5h show that the expression of cav-1 was also increased by addition of recombinant PRELP.

[0056] Zo-1 and cav-1 are cell-adhesion factors, which are known to be involved in EMT. The increased expression caused by PRELP supports the underlying mechanism of reduced EMT.

[0057] Figure 6 - shows cell migration assays of HFL1 (Human fetal lung fibroblast 1) cells. Figures 6a and 6b show that PRELP knock down using siRNA increased chemotactic activity of HFL1 cells. Figures 6c and 6d shows that recombinant PRELP decreased chemotactic activity of HFL1 cells.

[0058] In lung fibrosis, activated fibroblasts gather at a fibrosis site. Figure 6 proves that PRELP inhibits chemotactic activity of fibroblasts, resulting in decreased fibrosis.

[0059] Figure 7 - shows the results of qRT-PCR of CTGF and collagenlal mRNA in A549 cells. Figures 7a and 7b show that administration of recombinant PRELP decreased TGFb-induced expression of these genes in A549 cells.

[0060] Figure 8 - shows the results of Western Blot analysis of phosphorylated smad2 / 3 and CTGF in A549 cells. Figures 8a and 8b show that administration of recombinant PRELP decreased phosphorylation of smad2 / 3. Figures 8c and 8d show that recombinant PRELP decreased CTGF expression.

[0061] Figure 9 - shows the results of qRT-PCR of CTGF and collagenlal mRNA in mouse lungs. Figures 9a and 9b show that PRELP knockout lungs had increased mRNA expressions of CTGF and colalgenlal . Figure 9c shows that CTGF(Ccn2) mRNA expression is significantly decreased by administration of recombinant PRELP in BLM- induced lung fibrosis. Figures 7, 8 and 9 demonstrate that PRELP inhibits TGFp / smad signals and decreases the expression of target genes including CTGF and collagenlal .

[0062] Figure 10 - Figures 10a to lOd shows the results of Western Blot analysis of phosphorylated FAK and YAP in A549 cells. Figures 10a and 10b show that administration of recombinant PRELP decreased phosphorylation of FAK. Figures 10c and lOd show that recombinant PRELP increased YAP phosphorylation, which means it decreased YAP activity. Figures lOe to lOh shows the results of Western Blot analysis of DIAPH1 and phosphorylated YAP in A549 cells. Figures 10a and 10b show that recombinant PRELP decreased DIAPH1 expression. Figures 10c and lOd show that recombinant PRELP increased YAP phosphorylation, which means it decreased YAP activity.

[0063] Figure 11 - Figures I la to 11c shows the results of Western Blot analysis of YAP in mouse lungs. Figures 1 la, 1 lb and 11c show that PRELP knockout lungs had increased total YAP expressions. Figures 1 Id to 1 If shows the results of Western Blot analysis of DIAPH1 and YAP in mouse lungs. Figures l id and l ie show that recombinant PRELP decreased DIAPH1 expression in mouse lungs. Figures l id and I lf show that recombinant PRELP increased YAP phosphorylation in mouse lungs.

[0064] Integrin / FAK signals is known to activate YAP, another key molecule of lung fibrosis. Figures 10 and 11 demonstrate that that PRELP inhibits Integrin / FAK signals and YAP activity.

[0065] RAGE / DIAPH1 signals are also known to activate YAP, a key molecule of TGFbeta / smads signals and lung fibrosis. Figure 10 and 11 prove that that PRELP inhibits TGFbeta / smads signals via RAGE / DIAPH1YAP cascade.

[0066] Figure 12 - details the schedule for administering recombinant PRELP intratracheally to C57BL / 6J mice, one day before BLM treatment.

[0067] Figure 13A - shows a schematic model of how PRELP reduces progression / prevents lung fibrosis through inhibition of TGFp / Smad and Integrin / FAK signaling. Figure 13B - shows a schematic model of how PRELP reduces progression / prevents lung fibrosis through inhibition of TGFp / Smad and RAGE / DIAPH1 signaling.

[0068] Figure 14 - shows the results of histological studies of lungs from aged PRELP knockout mice. Figures 14a shows hematoxylin and eosin staining of lung tissue obtained from wild type mice and PRELP knock out (KO) mice at 6month, 12month and 18month years old. 18month-year old KO mice have obviously larger airspaces. Figure 14b shows mean linear intercept (MLI), a measure of airspace enlargement, which is larger in KO mice than in wild type mice at 12month and 18month years old.

[0069] Figure 15 - shows the results of physiological studies and Bronchoalveolar Lavage Fluid (BALF) analysis on PRELP KO mice. Figure 15a shows that lung compliance was significantly increased in KO mice at 12 months and 18 months old, which means they are easier to inflate. Figure 2b shows that numbers of total cells were significantly increased in BALF of 12month-year old PRELP KO mice compared to wild-type mice.

[0070] Figures 13 and 14 demonstrate that PRELP knockout mice develop COPD-like lung phenotypes after 12 months.

[0071] Figure 16 - shows the results of the analysis of PPE and LPS-induced COPD model in PRELP KO mice. Figure 16a shows the schedule for administering Porcine Pancreatic Elastase (PPE) and 25pg of LPS intratracheally to create a COPD model. Intratracheal administration of PPE followed by LPS produced COPD model mice. Figures 16b and 16c shows the total cell count in the BALF from PRELP knockout mouse (PKO 25) is increased compared to control wild type mouse (W 25) > Figure 16b is for one mouse whereas Figure 16c demonstrate the significance of the data with more mice. Figure 16d reveals that TNFa mRNA expression of the BALF are increased in PRELP knockout mice (PKO 25) compared to wild type mice (W 25) and heterozygous mouse (HE 25). The data presented in Figure 16 demonstrates the prophylactic effect of PRELP and thus its therapeutic efficacy in the treatment of COPD and lung fibrosis where repeated exacerbations worsen the condition. Being able to prevent exacerbation is a good strategy in COPD therapy.

[0072] Figure 17 - shows the results of the analysis of PPE and LPS-induced COPD model using wild-type mice with or without recombinant PRELP administration. Figure 17a shows the schedule for administering PPE and LPS intratracheally to them. Recombinant PRELP (25 pg) is administered intratracheally one day before LPS stimulation. Figure 17b and 17c show the results of BALF analysis, Figure 17c includes more mice in the analysis and Figure 17b. Total cell count is decreased by recombinant PRELP treatment.

[0073] Figure 18 - shows the results of qRT-PCR of IL-6, IL-8 and TNFa mRNA in A549 cells and B2B cells (human bronchial epithelial cell line). Figure 18a, 18b and 18c show that LPS-induced IL-6, IL-8 and TNFa mRNA expressions are decreased by 1 pg of recombinant PRELP treatment in A549 cells. Figure 18d, 18e and 18f show that LPS- induced IL-6, IL-8 and TNFa mRNA expressions are decreased by 1 pg of recombinant PRELP treatment inB2B cells. Figure 18g shows the results of qRT-PCR of TNFa mRNA in B2B cells (human bronchial epithelial cell line). LPS-induced TNFa mRNA expression is decreased by 2 pg or 20 pg of recombinant PRELP treatment (PRELP 2 and PRELP 20, respectively).

[0074] Figures 16, 17 and 18 demonstrate that PRELP has an essential role in COPD pathogenesis, especially in terms of inflammation, and that recombinant PRELP reduces the inflammation in vivo and in vitro studies.

[0075] Materials and Methods

[0076] Genetically modified mice

[0077] PRELP knockout mice were generated by the Takeda Pharmaceutical Company. All animal experiments were approved by the University of Tokyo Ethics Committee for Animal Experiments.

[0078] Bleomycin-treated mice

[0079] Twelve-week-old male mice were intratracheally injected with 25 pg / body bleomycin (Nihon Kayaku, Tokyo, Japan) or 50 pL PBS (Wako, Osaka, Japan) using a sprayer. Fourteen days after bleomycin administration, the mice were sacrificed and analyzed.

[0080] Lung analysis

[0081] To obtain bronchoalveolar lavage fluid (BALF), the lungs were washed with 1 mL of sterile saline three times. The isolated BALF was centrifuged at 2000 rpm for 3 min. The pellets were dissolved in 1 mL PBS, and the cells were counted. To assess lung function of the mice, tracheostomy was performed and an 18G intravenous catheter was inserted to connect to FlexiVent (Scireq, Montreal, QC, Canada). Analysis was performed according to manufacturer’s instructions.

[0082] For histological analysis, mice lungs were fixed in 4% formaldehyde (Wako, Osaka, Japan). The lung tissue slides were stained with hematoxylin-eosin (HE) and Elastica van Gieson (EVG). To histopathologically assess the severity of pulmonary fibrosis, lung sections were stained with EVG and the Ashcroft score was measured as previously described.

[0083] Cell lines and cell culture

[0084] A549 cells and HFL1 cells were maintained in continuous cell culture at 37°C and 5% CO2 cultured in Dulbecco’s modified Eagle’s medium (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (Biowest, France) and 1% penicillinstreptomycin (Wako).

[0085] Cell migration analysis

[0086] Cell migration was determined by a scratch wound healing assay. HFL-1 cells were cultured at confluency, and wounds were generated in the central area of each well. Photographs were taken after the scratch and after 24 h. The distance of cell migration was measured under phase-contrast microscopy.

[0087] Expression and purification of recombinant PRELP

[0088] DNA encoding the human PRELP with a FLAG tag at the C terminus was subcloned into the pFASTBacl vector (Invitrogen). Bacmids were prepared according to the manufacturer's protocol (Bac-to-Bac Baculovirus Expression System, Invitrogen). Sf9 insect cells (Thermo Fisher Scientific) were transfected with PRELP bacmids, followed by incubation at 27 °C for 4 days; the supernatant was passage 1 (Pl) virus. Sf9 cells (1.0 x 106 cells / ml) were infected with Pl virus (1 :25, v / v) and incubated with shaking at 120 rpm at 27 °C for 2 days; the supernatant was passage 2 (P2) virus. The above procedure was repeated to prepare passage 3 (P3) virus. For rPRELP expression, 1.8 x 106 cells / ml of Mimic Sf9 cells (Thermo Fisher Scientific) suspended in Sf900II serum- free medium (Thermo Fisher Scientific) containing 10% (v / v) fetal bovine serum were infected with P3 virus (1 :50, v / v) and incubated with shaking at 120 rpm at 27 °C for 4-5 days. rPRELP was purified from the supernatant using DDDDK-tagged Protein Purification Gel (MBL). The gel was washed with PBS (pH 7.4), and protein was eluted with 1 M arginine-HCl (pH 4.4). The eluate was immediately neutralized with 2 M Tris- HC1 (pH 8.0). The eluted fraction was treated with Benzonase nuclease (Millipore) as per the manufacturer’s protocol, and a second purification using DDDDK-tagged Protein Purification Gel was carried out. The eluate was purified by size-exclusion chromatography using a HiLoad 26 / 600 Superdex 200 pg column (GE Healthcare) equilibrated with 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 400 mM arginine-HCl.

[0089] Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR)

[0090] Total RNA was extracted from cells or lung tissue using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA), and cDNA was synthesized using SuperScript III reverse transcriptase (Thermo Fisher Scientific). cDNA quantification was performed using TB Green Fast qPCR Mix (Takara, Shiga, Japan) using Thermal Cycler Dice Real Time System III (Takara). Relative expression was calculated using the AACt method. Individual data were normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase.

[0091] Western blotting

[0092] Cells or lung tissues samples were lysed in RIPA buffer (20 mM Tris-Hcl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% Na-deoxycholate, and 0.1% SDS); Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA), and Halt phosphatase inhibitor cocktail (Thermo Fisher Scientific), followed by SDS gelelectrophoresis and transferred to PVDF membranes (Millipore, Burlington, MA, USA). The membrane was blocked using TBS-T buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, and 0.1% Tween-20) and 2% ECL Prime Blocking Reagent (GE Healthcare, Little Chalfont, UK) for 1 h, followed by incubation with primary antibodies for CTGF (Abeam, Cambridge, MA, USA); pSMAD2 / 3 (Cell Signaling Technology, Beverly, MA, USA); SAMD2 / 3 (Cell Signaling Technology); pFAK (Cell Signaling Technology); FAK (Cell Signaling Technology); pYAP (Cell Signaling Technology); YAP (Cell Signaling Technology); Vimentin (Cell Signaling Technology); E-cadherin (Cell Signaling Technology); DIAPHl(ab l29167; Abeam, Cambridge, UK) or -actin (Abeam) at 4°C overnight. After incubation with secondary anti-mouse antibody (1 : 10000, Abeam) or anti-rabbit antibody (1 : 10000, Abeam), immunodetection was performed using the ECL Prime Western Blotting Detection Kit (GE Healthcare). Pictures were taken using EZ-Capture MG (ATTO, Tokyo, Japan). The density of the band was measured via densitometry.

[0093] Immunofluorescence staining

[0094] A549 cells were fixed using 4% formalin solution (Wako, Osaka, Japan) and blocked with Blocking One (Nacalai Tesque, Kyoto, Japan) for 1 h. After incubation with primary antibodies for Caveolin-1 (1 :400, Cell Signaling Technology) or ZO-1 (1 : 100, Invitrogen, Carlsbad, CA, USA) at 4°C overnight, the cells were incubated with anti- rabbit fluorescent secondary antibody (1 :300, BD Pharmingen, San Diego, CA, USA) or anti -mouse fluorescent secondary antibody (1 :200, Invitrogen). VECTASHIEED mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA) was used to mount cells on to a slide. Immunofluorescence intensity was measured using Image J in four representative fields. Immunofluorescence intensity per individual cell was calculated.

[0095] Statistics

[0096] Statistical analyses were performed using GraphPad Prism 5 software. Unpaired t-test was used for analysis. P values < 0.05 were considered statistically significant.

[0097] Examples

[0098] The examples below demonstrate, using PRELP knock out mice and in vitro cell lines, that PRELP can prevent and / or treat lung fibrosis and COPD.

[0099] Example 1 - the absence PRELP results in an increase in lung fibrosis induced by bleomycin

[0100] Figures 1 and 2 demonstrate that when bleomycin (BLM) is administered to induce lung fibrosis in mice the lung fibrosis is worse in PRELP knock out mice than in WT mice. Thereby demonstrating that the presence of PRELP helps to prevent / reduce lung fibrosis.

[0101] Example 2 - the addition of PRELP reduces / prevents lung fibrosis induced by bleomycin

[0102] Figures 3 demonstrates the efficacy of PRELP in preventing or reducing the progression of lung fibrosis. The data presented shows that when recombinant PRELP was administered to WT mice before the administration of BLM, the level of lung fibrosis induced by the BLM was reduced.

[0103] Example 3 - demonstrates in vitro in human cells that PRELP prevents / reduce lung fibrosis

[0104] Figures 4 to 11 provide in vitro data to support the mouse data in Figures 1 to 3. Using cell lines derived from lung tissue, the results demonstrate that PRELP reduces EMT and fibroblast migration and enhances cell adhesion. These findings support that PRELP has a protective role in the pathogenesis of lung fibrosis. The additional results show that PRELP inhibits TGFb / smad signals as well as integrin / FAK / YAP signals, which are known to be essential in the formation of lung fibrosis.

[0105] Example 4 - demonstrates that PRELP can be used to prevent / reduce COPD

[0106] Figures 14 and 15 demonstrate that PRELP knockout mice develop a COPD-like lung phenotype after 12 months, indicating the role of PRELP in the pathogenesis of COPD.

[0107] Figures 16 to 18 demonstrate model PRELP can attenuate inflammation and TNF pathway in the lung in an LPS-induced mouse model of COPD, thus reducing COPD.

Claims

CLAIMS1. Proline / arginine-rich end leucine-rich repeat protein (PRELP) or a variant thereof for use in the prevention and / or treatment of lung disease in a subject.

2. A method of preventing or treating lung disease, in a subject, comprising administering to the subject a therapeutically effective amount of PRELP or a variant thereof.

3. PRELP or a variant thereof for the use of claim 1, or the method of claim 2, wherein the subject is already diagnosed with a lung disease.

4. PRELP or a variant thereof for the use of claim 1 or 3, or the method of claims 2 or 3, wherein the PRELP or a variant thereof is administered to prevent lung disease or to slow or reduce the progression of lung disease, wherein the lung disease is induced by another therapeutic agent.

5. PRELP or a variant thereof for the use of claim 4, or the method of claim 4, wherein the other therapeutic agent is one or more of amiodarone, nitrofurantoin, chemotherapy, methotrexate, a cytotoxic anticancer drug, a molecular targeted drug, an immune checkpoint inhibitor, an antirheumatic drug, interferon, an anti-inflammatory analgesic, an antibiotic, and an anti arrhythmic drug.

6. PRELP or a variant thereof for the use of claim 1 or 3, or the method of claim 2 or 3, wherein the PRELP or a variant thereof is administered to prevent lung disease or to slow or reduce the progression of lung disease, wherein the lung disease is induced by one or more of radiation, the environment (e.g. exposure to allergens), an autoimmune condition (such as connective tissue disease-associated interstitial lung disease - CTD-ILD), and an occupational condition (pneumoconiosis) (such as exposure to dust, fibers, fumes, asbestos, coal, or silica).

7. PRELP or a variant thereof for the use of any of claims 1 or 3 to 6, or the method of any of claims 2 to 6, wherein the lung disease is lung fibrosis or a disease with a lung fibrosis component.

8. PRELP or a variant thereof for the use of any of claims 1 or 3 to 7, or the method of any of claims 2 to 7, wherein the subject has a form of chronic fibrosing interstitial lung disease, such as idiopathic pulmonary fibrosis, or a form of asthma which has lung fibrosis.

9. PRELP or a variant thereof for the use of any of claims 1 or 3 to 6, or the method of any of claims 2 to 6, wherein the lung disease is COPD.

10. PRELP or a variant thereof for the use of any of claims 1 or 3 to 6, or the method of any of claims 2 to 6, wherein the lung disease is selected from the group comprising idiopathic pulmonary fibrosis, giant cell interstitial pneumonia, sarcoidosis, cystic fibrosis, respiratory distress syndrome, drug-induced lung fibrosis, granulomatosis, silicosis, asbestosis, systemic scleroderma, virally induced hepatic cirrhosis selected from hepatitis C induced hepatic cirrhosis, sarcoidosis, systemic lupus erythematosus, diseases of the skin with a lung fibrotic component such as scleroderma, rheumatoid arthritis, acute lung injury, radiation induced lung fibrosis, pneumonitis, chronic hypersensitivity pneumonitis, systemic sclerosis, Sjogren's syndrome, interstitial lung diseases, pulmonary arterial hypertension (PAH), asthma, pulmonary fibrosis, and chronic obstructive pulmonary disease (COPD).

11. PRELP or a variant thereof for the use of any of claims 1 or 3 to 10, or the method of any of claims 2 to 10, wherein the PRELP or a variant thereof is administered intratracheally.

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