Compositions containing propeptides of lysyl oxidase and uses thereof

Abstract

A method for treating fibrosis in a subject in need thereof is provided. The method comprises administering to the subject a polypeptide comprising a propeptide of lysyl oxidase (LOX), the polypeptide lacking LOX catalytic activity, thereby treating fibrosis in the subject, wherein the method does not include administration of D-penicillamine. A polypeptide composition for use in therapy is also provided.

Description

[0001] Related Applications

[0002] This application claims the benefit of priority to U.S. Patent Application No. 62 / 977,792, filed on February 18, 2020, and Israel Patent Application No. 266433, filed on May 2, 2019, each of which is incorporated by reference in its entirety.

[0003] Sequence Listing Statement

[0004] An ASCII file entitled 82310SequenceListing.txt, containing 28,106 bytes, created on April 30, 2020, and filed concurrently with the filing of this application is incorporated herein by reference. Technical Field

[0005] In some embodiments thereof, the present invention relates to compositions comprising a propeptide of lysyl oxidase (LOX) and uses thereof. Background Art

[0006] DMD is an X-linked Dystrophin The most common and severe childhood form of muscular dystrophy in families caused by mutations in the gene. As a result, the muscle fibers lose their anchoring to the ECM, and repeated muscle contractions lead to mechanical breakdown of the muscle fibers. DMD causes progressive weakness and loss (atrophy) of skeletal and cardiac muscle [1]. Early signs of DMD may include delayed ability to sit, stand, or walk, and difficulty learning to speak. Muscle weakness is usually evident by age 3 or 4 and begins in the hips, pelvic region, thighs, and shoulders. Damaged fibers induce satellite cell (SC) activation and muscle regeneration [2]. Smooth muscles, such as those lining the GI tract, are also significantly affected in the disease, and patients often suffer from intestinal paralysis [3].

[0007] The 'muscle regeneration program' is a multi-step process. Initially, a transient inflammatory response occurs, during which growth factors and cytokines are released into the injured area to allow the proliferation and recruitment of macrophages and fibroblasts, which process muscle debris and lay down ECM. Myofiber formation begins with the activation of SCs, followed by proliferation, differentiation, and fusion to new or existing myocytes [2,4]. TGFβ1, a powerful regulator of tissue wound healing and fibrosis, is upregulated in skeletal muscle after injury and is presumed to be involved in the transient inflammatory response. However, in malnutrition diseases such as DMD, a persistent inflammatory response occurs as a consequence of sustained muscle damage. High levels of proinflammatory cytokines, growth factors, nitric oxide, and TGFβ produced by inflammatory cells create a stressful environment that affects the balance between SCs and fibroblasts [5]. As a result, the balance between connective tissue (ECM and fibroblasts) and muscle tissue shifts. Excess components of the ECM, such as collagen and fibrinogen, are deposited at the expense of muscle forming highly fibrotic tissue [5]. It is noteworthy that such ECM dysregulation processes lead to many of the pathological features of DMD, which are not only directly caused by the lack of dystrophin, but also due to the complex interaction of myofibers and SCs with the ECM. As a result, fibrotic muscle delays muscle repair and regeneration, enhances inflammation and exacerbates disease progression. In summary, loss of regenerative capacity and fibrosis are the main causes of muscle dysfunction and the lethal phenotype of DMD and other related muscular dystrophies [6].

[0008] Gene editing and cell transplantation have been proposed as novel approaches for treating DMD. However, these promising approaches either rely on early intervention before a wave of necrosis results in loss of muscle fibers, replacement of tissue by fat and fibrosis, or rely on multimodal therapies that address the fibrotic response [7,8]. In addition, major side effects, such as balance problems and vomiting, are associated with these treatment modalities. Although these approaches increase dystrophin production, which would predict improvements in muscle function, this has not yet been demonstrated. Therefore, the identification of genes associated with physical tissue damage, and the development of treatments to address unwanted ECM remodeling, outline unmet needs in DMD.

[0009] In an attempt to identify genes underlying the lethal phenotype of DMD, we performed a series of studies derived from a large number of human DMD patients and mdxLarge-scale gene expression analysis of mice. Genes that are significantly upregulated or downregulated in mouse model systems and human patients are considered DMD core genes. One of the genes identified as highly overexpressed in DMD in these analyses is LOX. Also in golden retriever muscular dystrophy (Golden Retriever Muscular Dystrophy) (GRMD) (a canine model of DMD), its elevated expression was further confirmed using Western blot and immunohistochemical analysis [9,10].

[0010] LOX was originally identified as an extracellular enzyme that initiates covalent cross-linking of collagen and elastin molecules. LOX is the most abundant member of its family [LOX, LOX-like (LOXL) 1-4] that initiates these post-translational modifications. Its activity is essential for maintaining the tensile and elastic properties of connective tissues in the skeleton, lungs, and cardiovascular system [11-14]. LOX is synthesized and secreted as a 50-kDa inactive zymogen that is processed into a functional 32-kDa enzyme (LOX) by proteolytic cleavage of the 18-kDa prodomain via BMP-1. Accordingly, aortic aneurysms and their diaphragmatic ruptures caused by matrix organization defects

[18] [15-17], Lox Mutant mice ( Lox - / - ) die at birth. Recent work has directly linked LOX activity to a number of human pathological conditions, including fibrosis, neurodegeneration, and cancer (e.g., [12, 13, 11]). Accumulating evidence suggests that, in addition to their classical extracellular activities, members of the LOX family are also found within the cytoplasm and nucleus of cells (e.g., [19-21]). These intracellular LOX activities have been associated with the regulation of cell motility and migration, gene transcription, and differentiation [12, 22-26]. In summary, the results demonstrate that LOX plays multiple independent roles both within the cell and in the ECM. Recently, it has been shown that LOX plays multiple independent roles both within the cell and in the ECM. Lox Mutant mice display severe skeletal muscle defects

[27] . In these mice, the mass of myofibers is significantly reduced, and they also display excessive deposition of ECM and fibroblasts. Furthermore, these Lox muscle-related phenotypes are rescued upon inhibition of TGFβ signaling, thus demonstrating that the Lox-TGFβ feedback loop regulates the balance between the mass of muscle relative to the mass of connective tissue.

[0011] Additional background technologies include:

[0012] Thomassin et al. J Biol Chem. 2005 Dec 30;280(52):42848-55. Epub 2005 Oct26.

[0013] Grimsby et al. J Cell Biochem. Author manuscript; available in PMC 2013 Dec 11.

[0014] Zhao et al. J Biol Chem. 2009 Jan 16;284(3): 1385–1393.

[0015] U.S. Publication No. 20080261870.

[0016] U.S. Publication No. 20170360732. Summary of the invention

[0017] According to one aspect of some embodiments of the present invention, there is provided a synthetic polypeptide comprising a propeptide of human lysyl oxidase (LOX), the polypeptide lacking LOX catalytic activity, the synthetic polypeptide comprising a modification, the modification conferring enhanced stability to the polypeptide under physiological conditions compared to a native form of the polypeptide not comprising the modification.

[0018] According to some embodiments of the invention, the modification comprises a protein modification.

[0019] According to some embodiments of the invention, the protein modification is selected from immunoglobulin, human serum albumin and transferrin.

[0020] According to some embodiments of the invention, the immunoglobulin comprises an Fc domain.

[0021] According to some embodiments of the invention, the polypeptide is a chimeric polypeptide.

[0022] According to some embodiments of the invention, the modifying comprises chemical modification.

[0023] According to some embodiments of the invention, the chemical modification is a polymer.

[0024] According to some embodiments of the invention, the polymer is selected from the group consisting of polycationic polymers, nonionic water-soluble polymers, polyether polymers and biocompatible polymers.

[0025] According to an aspect of some embodiments of the present invention, there is provided a method for treating fibrosis in a subject in need thereof, the method comprising administering to the subject a polypeptide comprising a propeptide of lysyl oxidase (LOX), the polypeptide lacking LOX catalytic activity, thereby treating fibrosis in the subject, wherein the method does not include the administration of D-penicillamine.

[0026] According to one aspect of some embodiments of the present invention, there is provided a method for treating Duchenne muscular dystrophy (DMD) in a subject in need thereof, the method comprising administering to the subject a polypeptide comprising a propeptide of lysyl oxidase (LOX), the polypeptide lacking LOX catalytic activity, thereby treating DMD in the subject.

[0027] According to some embodiments of the invention, the polypeptide is glycosylated at at least one of N81, N97 and N144 of SEQ ID NO: 1.

[0028] According to some embodiments of the invention, the polypeptide is glycosylated at at least two of N81, N97 and N144 of SEQ ID NO: 1.

[0029] According to some embodiments of the invention, the polypeptide is glycosylated at N81, N97 and N144 of SEQ ID NO: 1.

[0030] According to some embodiments of the invention, the polypeptide is not glycosylated at N81, N97 and N144 of SEQ ID NO: 1.

[0031] According to some embodiments of the invention, the polypeptide is characterized by an EC50 of 10-500 nM as determined by an ELISA assay.

[0032] According to some embodiments of the invention, the polypeptide is characterized by a KD of 10-100 nM as determined by microthermophoresis.

[0033] According to some embodiments of the invention, the polypeptide is characterized by a transition midpoint of 20 - 70°C as determined by differential scanning fluorimetry (DSF).

[0034] According to some embodiments of the invention, the polypeptide is capable of reducing at least one of fibrillar collagen and cross-linked collagen as determined by SHG microscopy.

[0035] According to some embodiments of the invention, the polypeptide is capable of altering collagen fiber orientation as determined by SHG microscopy.

[0036] According to some embodiments of the invention, fibrosis is not Ras signaling dependent.

[0037] According to some embodiments of the invention, the fibrosis is not related to cancer or bone disease.

[0038] According to some embodiments of the invention, the propeptide of lysyl oxidase (LOX) is the propeptide of human LOX.

[0039] According to an aspect of some embodiments of the present invention there is provided a method of inhibiting the enzymatic activity of LOX, the method comprising contacting LOX with a polypeptide as described herein, thereby inhibiting the enzymatic activity of LOX.

[0040] According to some embodiments of the invention, the method is performed in vivo.

[0041] According to some embodiments of the invention, the method is performed in vitro.

[0042] According to some embodiments of the invention, the method is performed ex vivo.

[0043] According to an aspect of some embodiments of the present invention, there is provided a method for producing a polypeptide as described herein, the method comprising expressing a polynucleotide encoding the polypeptide in a host cell and, if necessary, modifying the polypeptide with a chemical modification, thereby producing the polypeptide.

[0044] According to some embodiments of the invention, the method further comprises isolating the polypeptide from the host cell.

[0045] According to some embodiments of the invention, the polypeptide is as shown in SEQ ID NO: 1 or 5.

[0046] According to an aspect of some embodiments of the present invention, there is provided a polynucleotide encoding a polypeptide as described herein.

[0047] According to a specific embodiment, the use does not contain D-penicillamine.

[0048] Unless otherwise defined, all technical and / or scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs. Although exemplary methods and / or materials are described below, methods and materials similar to or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention. In the event of a conflict, the patent specification shall prevail including definitions. In addition, materials, methods and embodiments are illustrative only and are not intended to be necessarily restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] With reference to the accompanying drawings, some embodiments of the present invention are described herein by way of example only. With specific reference now to the accompanying drawings in detail, it is emphasized that the details shown are by way of example and for the purpose of illustrative discussion of embodiments of the present invention. In this regard, the description in conjunction with the accompanying drawings makes it apparent to those skilled in the art how embodiments of the present invention may be practiced.

[0050] In the attached picture:

[0051] Figure 1A-G shows the design and biochemical properties of the fusion protein Fc-LPD. Figure 1A , 1B , Design and generation of the antibody-like LOX inhibitor Fc-LPD. Figure 1C , Binding affinity of Fc-LPD and Fc-LPD N346, 362, 409Q to the catalytic domain of LOX, EC 50 . Figure 1D , 1E , measurement of the transition midpoint of Fc-LPD, monitoring the intrinsic fluorescence signal of Fc-LPD as a measure of its folding state, K D . Figure 1F , 1G , the ratiometric measurements of the fluorescence signal of Fc-LPD were plotted against increasing temperatures of the chemical denaturant to determine the T of the protein. m .

[0052] Figure 2A -E shows disturbance of ECM assembly as determined by SHG microscopy. Figure 2A , shows a scheme of the experimental setup for monitoring different stages in the assembly of collagen secreted by HDF cells using SHG microscopy, Figure 2B , Representative SHG images of Fc-LPD that disturb ECM alignment. Representative fiber directionality analysis on each SHG image depicts the frequency of fibers in a specific orientation. Figure 2C , Representative SHG images of Fc-LPD and Fc-LPD N346, 362, 409Q that interfere with ECM arrangement compared with Fc. Figure 2C , 2D , average entropy and thickness of the ECM map.

[0053] Figure 3A -E shows the interaction of different Fc-LPD glycosylation forms with ECM proteins released from HDF cells. Figure 3A , semi-separation of 45 kDa Fc-LPD and 60 kDa Fc-LPD. Figure 3B , immunoprecipitation of 45 kDa Fc-LPD and 60 kDa Fc-LPD. Figure 3C , Interaction of the LOX catalytic domain with Fc-LPD after immunoprecipitation (IP). Figure 3D , Binding affinity of Fc-LPD and Fc-LPD N346, 362, 409Q to HSP70, EC 50 . Figure 3E , binding of HSP70 to Lpd by using yeast surface display.

[0054] Figure 4A-H shows early in vivo assessment of DMD diaphragm and gastrocnemius muscle fibrosis following treatment with Fc-LPD and Fc-LPD N346,362,409Q. Figure 4A , Schematic diagram of the experiment. Figure 4B , shows the results of the rotarod running assay. FIG4C , shows H&E and Sirius Red of mouse diaphragm. Figure 4E , Quantification of Sirius Red in mouse diaphragm. Figure 4F, 4G, H&E and Sirius Red in mouse gastrocnemius muscle are shown. Figure 4H , Quantification of Sirius Red in mouse gastrocnemius muscle.

[0055] Figures 5A-I show early in vivo assessment of fibrosis in DMD quadriceps and tibialis anterior muscles after treatment with Fc-LPD and Fc-LPD N346,362,409Q. Figures 5A,B show representative H&E and Sirius Red images in quadriceps muscles of DMD mice after treatment with Fc, Fc-LPD, and Fc-LPD N346,362,409Q. Figure 5C , Quantification of Sirius Red in mouse quadriceps muscle. Figure 5D -F, show representative two-photon SHG images from mouse quadriceps tissue and quadriceps endothelium, respectively. Figure 5E , 5G , Quantification of two-photon SHG images of mouse quadriceps muscle. Figure 5H , 5I , show representative two-photon SHG images from mouse tibialis anterior muscle and quantification of two-photon SHG images of mouse tibialis anterior muscle, respectively.

[0056] 6A-H show in vivo assessment of DMD muscle fibrosis after treatment with Fc-LPD. Fig. 6A , Schematic diagram of the experiment. Figure 6B , shows the results of the rotarod running assay, Figure 6C Results of the hanging test assay are shown. Fig.6D , show representative images of H&E, Sirius Red, and SHG from mouse tibialis anterior muscle. Fig. 6E , 6F , Quantification of Sirius Red and fibers from each region of mouse tibialis anterior (TA) muscle. Figure 6G , shows a semiqualitative histological evaluation analysis of the tibialis anterior muscle tissue. Figure 6H Representative fiber directionality analysis for each SHG image from mouse TA is shown.

[0057] Fig. 7A -E shows in vivo assessment of DMD myofibrosis in the diaphragm and heart after treatment with Fc-LPD. Fig. 7A , 7D, show representative images of H&E and Sirius Red in the diaphragm and heart of DMD mice after Fc and Fc-LPD treatment, respectively. Figure 7B , shows a semiqualitative histological assessment of diaphragmatic tissue. Figure 7C and 7E Quantification of diaphragm and heart Sirius red staining is shown. DETAILED DESCRIPTION

[0058] In some embodiments thereof, the present invention relates to compositions comprising a propeptide of lysyl oxidase (LOX) and uses thereof.

[0059] Before explaining at least one embodiment of the invention in detail, it should be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0060] Embodiments of the present invention relate to the development of protein inhibitors that exploit the extracellular levels of endogenous LOX. Using protein engineering, a stable form of the lysyl oxidase prodomain fused to an Fc antibody fragment (Fc-LPD) was generated that specifically inhibited LOX in vitro and in vivo. Affinity and stability measurements of Fc-LPD were performed for LOX, and the binding affinity (EC 50 = 43nM) and dissociation constant (Kd=32nM). mdx In vivo studies in mice showed improvements in functional tests such as rotarod running and hanging tests, as well as inhibition of fibrosis accumulation and promotion of normal collagen organization. The results herein demonstrate that inhibition of LOX by Fc-LPD is highly specific and effective in minimizing fibrosis and side effects in DMD.

[0061] Therefore, according to one aspect of the present invention, there is provided a method for treating fibrosis in a subject in need thereof, the method comprising administering to the subject a polypeptide comprising a propeptide of lysyl oxidase (LOX), the polypeptide lacking LOX catalytic activity, thereby treating fibrosis in the subject, wherein the method does not include the administration of D-penicillamine.

[0062] According to another aspect, there is provided a polypeptide comprising a propeptide of lysyl oxidase (LOX) for use in treating fibrosis in a subject in need thereof, the polypeptide lacking LOX catalytic activity, wherein the use does not comprise administration of D-penicillamine.

[0063] According to another aspect of the present invention, a method of treating Duchenne muscular dystrophy (DMD) in a subject in need thereof is provided, the method comprising administering to the subject a polypeptide comprising a propeptide of lysyl oxidase (LOX), the polypeptide lacking LOX catalytic activity, thereby treating DMD in the subject.

[0064] As used herein, "fibrosis" refers to the accumulation of extracellular matrix components that occur following trauma, inflammation, tissue repair, immune response, cell proliferation, and neoplasia. Examples of tissue fibrosis include, but are not limited to, pulmonary fibrosis, renal fibrosis, cardiac fibrosis, cirrhosis and liver fibrosis, skin scars and keloids, adhesions, fibromatosis, atherosclerosis, and amyloidosis.

[0065] In some embodiments, the fibrotic condition is primary fibrosis. In some embodiments, the fibrotic condition is idiopathic. In some embodiments, the fibrotic condition is associated with (e.g., secondary to) a disease (e.g., an infectious disease, an inflammatory disease, an autoimmune disease, a malignant or cancerous disease, and / or a connective tissue disease); a toxin; an injury (e.g., an environmental hazard (e.g., asbestos, coal dust, polycyclic aromatic hydrocarbons), smoking, a wound); a medical treatment (e.g., a surgical incision, chemotherapy, or radiation) or a combination thereof.

[0066] In some embodiments, the fibrotic condition is a fibrotic condition of the lung, a fibrotic condition of the liver, a fibrotic condition of the heart or vasculature, a fibrotic condition of the kidney, a fibrotic condition of the skin, a fibrotic condition of the gastrointestinal tract, a fibrotic condition of the bone marrow or hematopoietic tissue, a fibrotic condition of the nervous system, or a combination thereof. In some embodiments, the fibrotic condition affects one or more tissues selected from the group consisting of muscle, tendon, cartilage, skin (e.g., epidermis or endothelium), cardiac tissue, vascular tissue (e.g., artery, vein), pancreatic tissue, lung tissue, liver tissue, kidney tissue, uterine tissue, ovarian tissue, nervous tissue, testicular tissue, peritoneal tissue, colon, small intestine, bile duct, intestine, bone marrow, or hematopoietic tissue.

[0067] In some embodiments, the fibrotic condition is a fibrotic condition of the lung. In some embodiments, the fibrotic condition of the lung is selected from one or more of the following: pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), common interstitial pneumonia (UIP), interstitial lung disease, cryptogenic fibrosing alveolitis (CFA), bronchiolitis obliterans or bronchiectasis. In some embodiments, the fibrosis of the lung is secondary to disease, toxins, injury, medical treatment or a combination thereof. In some embodiments, the fibrosis of the lung is associated with one or more of the following: disease processes, such as asbestosis and silicosis; occupational hazards; environmental pollutants; smoking; autoimmune connective tissue disorders (e.g., rheumatoid arthritis, scleroderma and systemic lupus erythematosus (SLE)); connective tissue disorders, such as sarcoidosis; infectious diseases, such as infection, particularly chronic infection; medical treatment, including but not limited to radiotherapy and drug therapy, such as chemotherapy (e.g., with bleomycin, methotrexate, amiodarone, busulfan and / or nitrofurantoin treatment). In some embodiments, the fibrotic condition of the lung treated with the methods of the invention is associated with (e.g., secondary to) cancer treatment, such as treatment of cancer (e.g., squamous cell carcinoma, testicular cancer, Hodgkin's disease, treatment with bleomycin). In some embodiments, the fibrotic condition is a fibrotic condition of the liver. In certain embodiments, the fibrotic condition of the liver is selected from one or more of the following: fatty liver disease, steatosis (e.g., non-alcoholic steatohepatitis (NASH), cholestatic liver disease (e.g., primary biliary cirrhosis (PBC), cirrhosis, alcohol-induced liver fibrosis, bile duct damage, bile duct fibrosis, cholestasis, or bile duct lesions. In some embodiments, liver fibrosis or liver fibrosis include, but are not limited to, liver fibrosis associated with alcoholism, viral infections such as hepatitis (e.g., hepatitis C, hepatitis B, or hepatitis D), autoimmune hepatitis, non-alcoholic fatty liver disease (NAFLD), progressive massive fibrosis, exposure to toxins or irritants (e.g., alcohol, drugs, and environmental toxins).

[0068] In some embodiments, the fibrotic condition is a fibrotic condition of the heart. In certain embodiments, the fibrotic condition of the heart is myocardial fibrosis (e.g., myocardial fibrosis associated with radiation myocarditis, surgical complications (e.g., myocardial postoperative fibrosis), infectious diseases (e.g., Chagas disease, bacterial, trichinosis, or fungal myocarditis)); granulomatous metabolic storage diseases (e.g., cardiomyopathy, hemochromatosis); developmental disorders (e.g., endocardial fibroelastosis); atherosclerosis, or exposure to toxins or irritants (e.g., drug-induced cardiomyopathy, drug-induced cardiotoxicity, alcoholic cardiomyopathy, cobalt poisoning or exposure). In some embodiments, myocardial fibrosis is associated with an inflammatory disorder of cardiac tissue (e.g., myocardial sarcoidosis).

[0069] In some embodiments, the fibrotic condition is a fibrotic condition of the kidney. In some embodiments, the fibrotic condition of the kidney is selected from one or more of the following: renal fibrosis (e.g., chronic renal fibrosis), renal lesions associated with injury / fibrosis (e.g., chronic renal lesions associated with diabetes (e.g., diabetic nephropathy)), lupus, renal scleroderma, glomerulonephritis, focal segmental glomerulosclerosis, IgA nephropathy, renal fibrosis associated with human chronic kidney disease (CKD), chronic progressive nephropathy (CPN), tubulointerstitial fibrosis, ureteral obstruction, chronic uremia, chronic interstitial nephritis, radiation nephropathy, glomerulosclerosis, progressive glomerulonephritis (PGN), endothelial / thrombotic microangiopathy damage, HIV-related nephropathy, or fibrosis associated with exposure to toxins, irritants, or chemotherapeutic agents.

[0070] In some embodiments, the fibrotic condition is a fibrotic condition of the skin. In some embodiments, the fibrotic condition of the skin is selected from one or more of the following: cutaneous fibrosis, scleroderma, nephrogenic systemic fibrosis (e.g., resulting from exposure to gadolinium, which is frequently used as a contrast agent for MRI in patients with severe renal failure), scarring, and keloids.

[0071] In some embodiments, the fibrotic condition is a fibrotic condition of the gastrointestinal tract. In some embodiments, the fibrotic condition is selected from one or more of the following: fibrosis associated with scleroderma; radiation-induced intestinal fibrosis; fibrosis associated with foregut inflammatory conditions such as Barrett's esophagus and chronic gastritis, and / or fibrosis associated with hindgut inflammatory conditions such as inflammatory bowel disease (IBD), ulcerative colitis, and Crohn's disease.

[0072] In some embodiments, the fibrotic condition is adhesions. In some embodiments, the adhesions are selected from one or more of the following: abdominal adhesions, peritoneal adhesions, pelvic adhesions, pericardial adhesions, epidural adhesions, peritendinous adhesions, or adhesive capsulitis.

[0073] In some embodiments, the fibrotic condition is a fibrotic condition of the eye. In some embodiments, the fibrotic condition of the eye involves diseases of the anterior segment of the eye, such as glaucoma and corneal opacities; in some embodiments, the fibrotic condition of the eye involves diseases of the posterior segment of the eye, such as age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, and neovascular glaucoma; in some embodiments, the fibrotic condition of the eye results from fibrosis following ophthalmic surgery.

[0074] In some embodiments, the fibrotic condition is a fibrotic condition of bone marrow or hematopoietic tissue. In some embodiments, the fibrotic condition of bone marrow is an intrinsic feature of a chronic myeloproliferative tumor of bone marrow, such as primary myelofibrosis (also referred to herein as angiogenic myeloid metaplasia or chronic idiopathic myelofibrosis). In some embodiments, myelofibrosis is associated with a malignant condition or a condition caused by a clonal proliferative disease (e.g., secondary to the condition). In some embodiments, myelofibrosis is associated with a blood disorder (e.g., a blood disorder selected from one or more of the following: polycythemia vera, essential thrombocythemia, myelodysplasia, hairy cell leukemia, lymphoma (e.g., Hodgkin's lymphoma or non-Hodgkin's lymphoma), multiple myeloma, or chronic myeloid leukemia (CIVIL)). In some embodiments, myelofibrosis is associated with (e.g., secondary to) a non-hematological disorder (e.g., a non-hematological disorder selected from the group consisting of: bone marrow metastasis from a solid tumor, an autoimmune disorder (e.g., systemic lupus erythematosus, scleroderma, mixed connective tissue disorder, or polymyositis), an infection (e.g., tuberculosis), or secondary hyperparathyroidism associated with vitamin D deficiency.

[0075] In some embodiments, fibrosis is independent of Ras signaling for cellular transformation.Examples of such conditions include cancers such as colon, breast, lung or prostate cancer, disorders of the kidney, cardiovascular system and immune system, bone diseases such as osteopenic conditions such as osteoporosis.

[0076] According to a specific embodiment, the fibrotic condition is a fibrotic condition of the muscle.

[0077] There are two main types of muscle tissue in animals - striated muscle and smooth muscle.

[0078] Muscles can also be grouped by location or function. In some embodiments, polypeptides can be targeted to tissues of interest, for example, one or more muscles of the face, one or more muscles used for chewing, one or more muscles of the tongue and neck, one or more muscles of the chest, one or more muscles of the shoulder girdle and arm, one or more muscles of the arm and shoulder, one or more ventral and dorsal forearm muscles, one or more muscles of the hand, one or more erector spinae muscles, one or more muscles of the pelvic girdle and leg, and / or one or more muscles of the foreleg and foot.

[0079] In some embodiments, muscles of the face include, but are not limited to, intraocular muscles such as ciliary muscles, iris dilator muscles, iris sphincter muscles, ear muscles such as auricularis muscles, temporoparietis muscles, stapedius muscles, tensor tympani muscles; nasal muscles such as procerus, nasalis muscles, tensor nasalis, depressor septi nasalis, levator labii alaris; mouth muscles, such as levator anguli oris, depressor anguli oris, orbicularis oris, buccinator, zygomaticus major and minor, platysma, levator labii superioris, depressor labii inferioris, risorius, mentalis, and / or corrugator supercilii.

[0080] In some embodiments, the muscles of mastication include but are not limited to the masseter, temporalis, medial pterygoid, lateral pterygoid. In some embodiments, the muscles of the tongue and neck include but are not limited to the genioglossus, styloglossus, palatoglossus, hyoglossus, digastric, stylohyoid, mylohyoid, geniohyoid, omohyoid, sternohyoid, sternothyroid, thyrohyoid, sternocleidomastoid, anterior scalene, middle scalene and / or posterior scalene.

[0081] In some embodiments, muscles of the chest, shoulder girdle, and arms include, but are not limited to, the subclavius, pectoralis major, pectoralis minor, rectus abdominis, external obliques, internal obliques, transverse abdominis, diaphragm, external intercostal muscles, internal intercostal muscles, serratus anterior, trapezius, levator scapulae, rhomboids major, rhomboids minor, latissimus dorsi, deltoids, subscapularis, supraspinatus, infraspinatus, teres major, teres minor, and / or coracobrachialis.

[0082] In some embodiments, muscles of the arm and shoulder include, but are not limited to, the biceps brachii - long head, the biceps brachii - short head, the triceps brachii - long head, the triceps brachii - lateral head, the triceps brachii - medial head, the anconeus, the pronator teres, the supinator, and / or the brachialis.

[0083] In some embodiments, muscles of the ventral and dorsal forearm include, but are not limited to, the brachioradialis, flexor carpi radialis, flexor carpi ulnaris, palmaris longus, extensor carpi ulnaris, extensor carpi radialis longus, extensor carpi radialis brevis, extensor digitorum, and extensor digiti minimi.

[0084] In some embodiments, the muscles of the hand include, but are not limited to, the intrinsic muscles of the hand, such as the thenar muscles, abductor pollicis brevis, flexor pollicis brevis, opponens pollicis, hypothenar muscles, abductor digiti minimi, flexor digiti minimi brevis, opponens digiti minimi, palmaris interosseus, dorsal interosseus, and / or lumbrical muscles.

[0085] In some embodiments, erector spinae muscles include, but are not limited to, the cervical, spinalis, longissimus, and / or iliocostalis muscles.

[0086] In some embodiments, the muscles of the pelvic girdle and legs include but are not limited to

[0087] Psoas major, iliacus, quadratus femoris, adductor longus, adductor brevis, adductor magnus, gracilis, sartorius, quadriceps femoris such as rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gastrocnemius, Fibularis (Peroneus) longus, soleus, gluteus maximus, gluteus medius, gluteus minimus, hamstrings: biceps femoris: long head, hamstrings: biceps femoris: short head, hamstrings: semitendinosus, hamstrings, semimembranosus, tensor fasciae latae, pectineus and / or tibialis anterior.

[0088] In some embodiments, muscles of the foreleg and foot include but are not limited to the extensor digitorum longus, extensor hallucis longus, peroneus brevis, plantaris, tibialis posterior, flexor hallucis longus, extensor digitorum brevis, extensor hallucis brevis, abductor hallucis, flexor hallucis brevis, abductor digiti minimi, flexor digiti minimi, opponens digiti minimi, extensor digitorum brevis, lumbricalis, quadratus plantaris or accessory flexor digitorum brevis, flexor digitorum brevis, dorsal interosseous muscles, and / or plantar interosseous muscles.

[0089] Muscular dystrophy is a group of hereditary disorders that cause muscle degeneration, leading to weak and impaired movement. The central feature of all muscular dystrophies is that they are progressive in nature. Muscular dystrophy includes, but is not limited to, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, and myotonic muscular dystrophy type 1 and type 2, including congenital forms of myotonic muscular dystrophy type 1. Symptoms may vary depending on the type of muscular dystrophy, with some or all of the muscles affected. Exemplary symptoms of muscular dystrophy include delayed development of motor skills, difficulty using one or more muscle groups, difficulty swallowing, speaking or eating, drooling, droopy eyelids, frequent falls, loss of strength in a muscle or muscle group in adulthood, loss of muscle size, walking problems due to weakness or altered biomechanics of the body, muscle hypertrophy, muscle pseudohypertrophy, fatty infiltration of muscle, replacement of muscle with non-contractile tissue (e.g., myofibrosis), muscle necrosis, and / or cognitive or behavioral impairment / mental retardation.

[0090] Although there is no known cure for muscular dystrophy, several supportive treatments are used, including both symptomatic and disease-modifying therapies. Corticosteroids, physical therapy, orthotic devices, wheelchairs, or other auxiliary medical devices for ADL and lung function are commonly used in muscular dystrophy. Pacemakers are used to prevent sudden death due to arrhythmias in myotonic dystrophy. Antimyotonic agents that improve symptoms of myotonia (inability to relax) include mexiletine, and in some cases phenytoin, procainamide, and quinine. These can be combined with treatment with the polypeptides of some embodiments of the present invention.

[0091] Duchenne muscular dystrophy (DMD) is characterized by proximal muscle weakness, gait abnormalities, pseudohypertrophy in the gastrocnemius (calf) muscle, and elevated creatine kinase (CK). Many DMD patients are diagnosed around age 5, when symptoms / signs typically become more apparent. Affected individuals typically stop walking around age 10-13 and die by their mid- to late 20s or earlier due to cardiopulmonary dysfunction.

[0092] The disease DMD is caused by a mutation in the dystrophin gene located on human chromosome X, which encodes the protein dystrophin, an important structural component in muscle tissue that provides structural stability to the dystrophin glycan complex (DGC) of the cell membrane. Dystrophin links the internal cytoplasmic actin filament network and the extracellular matrix, providing physical strength to the muscle fiber. Accordingly, changes or absence of dystrophin lead to abnormal sarcolemma tearing and necrosis of muscle fibers. Although individuals of both sexes can carry this mutation, women rarely show severe signs of the disease.

[0093] The main symptom of DMD is muscle weakness associated with muscular atrophy, in which voluntary muscles are usually affected first, especially affecting the buttocks, pelvic area, thigh, shoulder muscles and calf muscles. Muscle weakness also occurs in arms, neck and other areas. The calf often becomes larger. Signs and symptoms usually occur before the age of 6, and may occur as early as infancy. Other physical symptoms include but are not limited to delayed ability to walk independently, progressive difficulties in walking, stepping or running, and the final loss of walking ability (usually to 15 years old); frequent falls; fatigue; difficulty in motor skills (running, jumping, jumping); increased lumbar lordosis, resulting in shortening of hip flexors; functional Achilles tendon and hamstring contracture that impairs function, because muscle fibers are shortened, and fibrosis occurs in connective tissue; muscle fiber deformity; pseudohypertrophy (enlargement) of tongue and calf muscles caused by replacement of muscle tissue by fat and connective tissue; high risk of non-progressive weakness of neurobehavioral disorders (such as ADHD), learning disabilities (dyslexia) and specific cognitive skills (especially short-term language memory); skeletal deformity (in some cases, including scoliosis).

[0094] As used herein, "treating" means abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetic symptoms of a condition, or substantially preventing the appearance of clinical or aesthetic symptoms of a condition.

[0095] As used herein, a "subject" refers to a subject diagnosed with or at risk of fibrosis.

[0096] As used herein, a "synthetic polypeptide" refers to a protein that does not occur in nature, either because it is isolated from its natural environment, such as the human or animal body, or because it is a mutation with respect to its wild-type form, or because it is modified, such as by attachment to a heterologous moiety, such as a protein or chemical.

[0097] As used herein, "lysyl oxidase" or "LOX" refers to the protein product of the LOX gene. In humans, the LOX gene is located on chromosome 5q23.3-31.2. The primary sequence of the LOX protein is highly conserved in mammals. Human LOX is synthesized as a 417 amino acid preproprotein (prepro-LOX) (UniProtKB-P28300, which undergoes many post-translational modifications in the endoplasmic reticulum (ER) and after the ER, for example, glycosylation as described below. After cleavage of the 21 amino acid signal sequence, the N-terminal propeptide containing 147 amino acid residues is N-glycosylated, and the mature protein C-terminal sequence containing 249 amino acid residues, which is also referred to herein as the portion containing the catalytic activity of LOX, is uniquely folded to obtain at least three disulfide bonds. Copper is a cofactor for the functional catalyst and is incorporated into the nascent enzyme in the ER. The enzyme also contains a peptidyl organic cofactor, lysyltyrosine quinone (LTQ) generated by intramolecular cross-linking between the copper-dependent oxidation products of lysine 320 and tyrosine 355.

[0098] "LOX-propeptide" (LOX-PP) or "LOX-prodomain" (LPD) is the N-terminal propeptide corresponding to amino acid residues 22-168 of SEQ ID NO: 3, which is cleaved by procollagen-C-proteinase (BMP-1) or BMP-1-related metalloproteinase after secretion of proLOX into the extracellular space to generate free propeptide (LPD) and catalytically active LOX.

[0099] The key intracellular function of LOX-PP (LPD) is likely to be to maintain lysyl oxidases in an inactive state within the secretory pathway. The propeptide may also act as an intramolecular chaperone to facilitate the correct folding and ultimate targeting of these proteins to their destination. Indeed, mass spectrometry, shown in Tables 1 and 2 below, shows that LPD co-immunoprecipitates with chaperones such as heat shock proteins.

[0100] LOX-PP contains three consensus N-glycosylation sites at residues 81, 97 and 144 of the LOX protein sequence.

[0101] In one embodiment, the present teachings contemplate a glycosylated LPD.

[0102] According to one embodiment, the polypeptide is glycosylated at at least one of N81, N97 and N144 of SEQ ID NO: 1. When referring to glycosylation sites, the indicated residues are those corresponding to SEQ ID NO: 1.

[0103] According to one embodiment, the polypeptide is glycosylated at one glycosylation site of LPD, namely N81 of SEQ ID NO: 1.

[0104] According to one embodiment, the polypeptide is glycosylated at one glycosylation site of LPD, namely N97 of SEQ ID NO: 1.

[0105] According to one embodiment, the polypeptide is glycosylated at one glycosylation site of LPD, namely N144 of SEQ ID NO: 1.

[0106] According to one embodiment, the polypeptide is glycosylated at at least two of N81, N97 and N144 of SEQ ID NO: 1.

[0107] According to one embodiment, the polypeptide is glycosylated at two glycosylation sites of LPD, such as N81+N97 of SEQ ID NO: 1, such as N81+N144 of SEQ ID NO: 1, such as N97+N144 of SEQ ID NO: 1.

[0108] According to one embodiment, the polypeptide is glycosylated at N81, N97 and N144 of SEQ ID NO: 1.

[0109] According to an alternative embodiment, the polypeptide is not glycosylated at N81, N97 and N144 of SEQ ID NO: 1.

[0110] Alteration of glycosylation can be achieved by methods well known in the art, such as selection of an appropriate expression system (e.g., prokaryotic versus eukaryotic), separation of glycosylated from non-glycosylated forms, site-directed mutagenesis at glycosylation sites, and / or use of any of enzymatic modifications.

[0111] The inventors have shown that glycosylated and non-glycosylated forms are endowed with different properties, see Example 5. For example, the non-glycosylated form has a higher binding rate to HSP70, as determined by immunoprecipitation experiments followed by mass spectrometry.

[0112] The polypeptides of some embodiments of the present invention are endowed with multiple biological activities.

[0113] According to a specific embodiment, the protein inhibits LOX in vitro and in vivo. Fc-LPD affinity and stability measurements were performed against LOX to determine the binding affinity (EC 50 = 43nM) and dissociation constant (Kd=32nM). mdx In vivo studies in mice showed improvements in functional tests such as rotarod running and hanging tests, as well as inhibition of fibrosis accumulation and promotion of normal collagen organization. The results herein demonstrate that inhibition of LOX by Fc-LPD is highly specific and effective in minimizing fibrosis and side effects of DMD.

[0114] According to a specific embodiment, the polypeptide is characterized by an EC50 of about 10-2000 nM, about 10-500 nM, such as about 43 nM, as determined by an ELISA assay (e.g., according to Figure 1C The assay conditions are determined by

[0115] According to a specific embodiment, the polypeptide is characterized by a K of about 10-100 nM, such as about 32 nM. D , such as by microthermophoresis (e.g., according to Figure 1D The assay conditions are determined by

[0116] According to a specific embodiment, the polypeptide can down-regulate the cross-linking of collagen in an in vitro assay system, as further described in the Examples section below. Since the polypeptide of some embodiments of the present invention can interfere with collagen cross-linking, it changes the structure of the extracellular matrix (ECM), relaxes the collagen fibers, and changes them from an ordered orientation to a more random orientation. Therefore, the polypeptide of some embodiments of the present invention changes the fibrillation of collagen (and accordingly the strength of collagen) and affects the thickness of collagen.

[0117] Methods for analyzing collagen architecture are known in the art and include, for example, second harmonic generation (SHG) microscopy (see, e.g., Figure 2A -BCD), electron microscopy imaging, and Raman microscopy.

[0118] Thus, according to one embodiment of the present invention, the polypeptide is capable of reducing at least one of fibrillar collagen (eg, type I) and cross-linked collagen as determined by SHG microscopy.

[0119] "Reduced" is intended to mean that the amount of fibril / cross-linked collagen is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% or 98% less relative to the amount in the absence of the polypeptide.

[0120] According to one embodiment of the invention, the polypeptide is capable of altering collagen fiber orientation as determined by SHG microscopy.

[0121] According to one embodiment, the LPD is derived from (taken from or derived from) human LOX, although other mammalian sequences of LOX are also contemplated.

[0122] The mRNA and amino acid sequences for Homo sapiens LOX can be found under GenBank Accession Nos. P28300.2 (protein accession number), AF039291.1 (mRNA accession number), NC_000005 (genome accession number).

[0123] As mentioned, this polypeptide lacks LOX catalytic activity.

[0124] As used herein, "LOX catalytic activity" refers to the lysyl oxidase activity of an enzyme, which is generally attributed to the domain between amino acid coordinates 169-417 of SEQ ID NO: 3 (e.g., SEQ ID NO: 9). According to a specific embodiment, the catalytic activity is set forth in SEQ ID NO: 9 (amino acid sequence) and 10 (nucleic acid sequence).

[0125] The catalytic activity of LOX can be determined in vitro by SHG microscopy (described in detail in the Examples section below).

[0126] Functional equivalents of LPD having about the same or even higher activity / stability than SEQ ID NO: 1 or 5 are also contemplated.

[0127] According to a specific embodiment, the LPD sequence is at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95% or more, say 100%, identical to the LPD sequence described in SEQ ID NO: 1 or 5, as determined using the BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters, and is capable of any of the above-mentioned biological functions. When measuring homology between peptides and proteins of larger size, homology is measured only in the corresponding regions; that is, the protein is considered to have only the same general length as the peptide, allowing for gaps and insertions.

[0128] Homologs may also refer to deletion, insertion or substitution variants thereof, including amino acid substitutions thereof, and biologically active polypeptide fragments thereof.

[0129] Additional modifications and changes can be made in the structure of the LPD portion of the polypeptides of the subject matter disclosed herein and still obtain a molecule capable of inhibiting LOX. For example, certain amino acids can be substituted for other amino acids in the sequence without significant loss of peptide activity. Because the ability and properties of a polypeptide to interact determine the biological functional activity of the polypeptide, certain amino acid sequence substitutions can be made in a polypeptide sequence (or, of course, its underlying DNA coding sequence) and still obtain a polypeptide with similar properties.

[0130] When making such changes, the hydropathic index of the amino acid may be considered. The importance of the hydropathic amino acid index in conferring interactive biological function to a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids with similar hydropathic indexes or scores and still result in polypeptides with similar biological activity. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics. These indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

[0131] It is believed that the relative hydropathicity of amino acids determines the secondary structure of the resulting polypeptide, which in turn determines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, etc. It is known in the art that an amino acid can be substituted with another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, substitution of amino acids whose hydropathic index is within ±2 is preferred, substitution of amino acids within ±1 is particularly preferred, and substitution of amino acids within ±0.5 is even more particularly preferred.

[0132] Substitution of similar amino acids can also be made based on hydrophilicity, particularly where the resulting biologically functionally equivalent polypeptides or peptides are intended for use in immunological embodiments. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a polypeptide, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with the biological properties of the polypeptide. As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values ​​have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0.+−.1); glutamic acid (+3.0.+−.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (−0.5.+−.1); threonine (−0.4); alanine (−0.5); histidine (−0.5); cysteine ​​(−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It will be appreciated that an amino acid may be substituted with another amino acid having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, immunologically equivalent, polypeptide. In such changes, substitution of amino acids whose hydrophilicity values ​​are within ±2 is preferred, substitution of amino acids within ±1 is particularly preferred, and substitution of amino acids within ±0.5 is even more particularly preferred.

[0133] As outlined above, amino acid substitutions are therefore generally based on the relative similarity of the amino acid side chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account various of the aforementioned properties are well known to those skilled in the art, and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine. Thus, the subject matter disclosed herein contemplates functional equivalents or biological equivalents of the polypeptides or their LPD portions as set forth above.

[0134] Biological or functional equivalents of polypeptides can be prepared using site-directed mutagenesis according to procedures well known in the art. Accordingly, amino acid residues can be added to or deleted from the LPD of the subject matter disclosed herein without changing the functionality of the peptide by using standard molecular biology techniques.

[0135] According to one embodiment, the amino acid sequence of LPD is modified in such a way as to increase its stability, bioavailability and / or pharmacological efficacy.

[0136] Thus, according to one aspect of the present invention, there is provided a synthetic polypeptide comprising a propeptide of human lysyl oxidase (LOX), said polypeptide lacking LOX catalytic activity, said synthetic polypeptide comprising a modification which confers enhanced stability to said polypeptide under physiological conditions compared to the native form of said polypeptide not comprising said modification.

[0137] Methods for determining stability are well known in the art.

[0138] As used herein, "stability" refers to at least thermal stability. The method is based on the use of intrinsic tryptophan or tyrosine fluorescence to measure protein stability at ultra-high resolution.

[0139] As used herein, "enhanced" or "increased" refers to an increase of at least 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90% or more, such as 100%, with respect to native LPD.

[0140] According to a specific embodiment, the polypeptide is characterized by a transition midpoint of 20 - 95°C, eg, a Tm of about 70°C, as determined by differential scanning fluorimetry (DSF).

[0141] Therefore, in order to improve the stability / activity of LPD, the polypeptide is modified.

[0142] According to a specific embodiment, the modification comprises a protein modification.

[0143] Protein modifications can be attached to the polypeptide by chemical attachment (fusion polypeptide, for example, by using linkers and / or reactive groups) or by recombinant DNA technology, whereby the synthetic polypeptide is a chimeric polypeptide.

[0144] Thus, the polypeptide has a first portion that is an LPD, and a second portion that is a heterologous peptide or protein (i.e., a protein modification). The fusion / chimera (or collectively referred to as a "fusion") can be an N to C or C to N orientation of the LPD relative to the protein portion (also referred to herein as a "heterologous polypeptide"). The fusion protein may include a myc, HA or His6 tag. The fusion protein further includes an LPD fused to the Fc domain of human IgG (such as the Fc-LPD mentioned herein in one embodiment). In a specific aspect, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of the IgG1 molecule. According to a specific embodiment, Fc is as shown in SEQ ID NO: 7. For the production of immunoglobulin fusions, see also U.S. Patent No. 5,428,130. The Fc portion can be derived from mouse IgG1 or human IgG2 M 4. Human IgG2 M4 (see US Published Application No. 20070148167 and US Published Application No. 20060228349) is an antibody from IgG2 with mutations that maintain a normal pharmacokinetic profile but do not have any known effector functions.

[0145] Exemplary amino acid sequences of LPD fused to an Fc domain are set forth in SEQ ID NO: 5 (amino acid) and SEQ ID NO: 6 (nucleic acid).

[0146] Fusion proteins further include LPD fused to human serum albumin, transferrin, or antibodies.

[0147] In a further aspect, LPD is conjugated to a carrier protein such as human serum albumin, transferrin, or an antibody molecule.

[0148] As used herein, the term "polypeptide" refers to a polymer of natural or synthetic amino acids, including natural peptides (truncated products, synthetically synthesized polypeptides or recombinant polypeptides) and peptidomimetics (usually synthetically synthesized peptides), as well as peptoids and hemi-peptoids that are polypeptide analogs that may have modifications that, for example, render the polypeptide even more stable or more capable of penetrating cells when in vivo.

[0149] Such modifications include, but are not limited to, N-terminal modifications, C-terminal modifications, polypeptide bond modifications (including, but not limited to, CH2-NH, CH2-S, CH2-S=O, O=C-NH, CH2-O, CH2-CH2, S=C-NH, CH=CH or CF=CH), backbone modifications, and residue modifications. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, CA Ramsden Gd. Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully described herein. Further details in this regard are provided below.

[0150] The polypeptide bond (-CO-NH-) within the polypeptide can be replaced by, for example, the following: N-methylated bonds (-N(CH3)-CO-), ester bonds (-C(R)HCOOC(R)-N-), ketomethylene bonds (-CO-CH2-), α-aza bonds (-NH-N(R)-CO-) where R is any alkyl group such as methyl, carba bonds (-CH2-NH-), hydroxyethylene bonds (-CH(OH)-CH2-), thioamide bonds (-CS-NH-), olefin double bonds (-CH=CH-), reverse amide bonds (-NH-CO-), polypeptide derivatives (-N(R)-CH2-CO-), where R is a "normal" side chain naturally occurring on a carbon atom.

[0151] These modifications can occur at any bond along the polypeptide chain, and even at several (2-3) bonds simultaneously.

[0152] Natural aromatic amino acids Trp, Tyr and Phe can replace synthetic non-natural acids such as phenylglycine, TIC, naphthylalanine (Nol), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.

[0153] In addition to the above, the polypeptide of the present invention may also include one or more modified amino acids, or one or more non-amino acid monomers (eg, fatty acids, complex carbohydrates, etc.).

[0154] As used herein in the specification and in the appended claims, the term "amino acid" or "amino acid" is understood to include the 20 naturally occurring amino acids; those amino acids that are frequently post-translationally modified in vivo, including, for example, hydroxyproline, phosphoserine, and phosphothreonine; and other rare amino acids, including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, norvaline, norleucine, and ornithine. In addition, the term "amino acid" includes both D-amino acids and L-amino acids (stereoisomers).

[0155] Tables A and B below list naturally occurring amino acids (Table A) and unconventional or modified amino acids (Table B) that may be used in the present invention.

[0156] Table A

[0157] Amino Acids Three letter abbreviation Single letter symbols Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gln Q Glutamate Glu E Glycine Gly G Histidine His H Isoleucine Iie I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V Any amino acid as above XA X

[0158] Table B

[0159] Unconventional amino acids Code Unconventional amino acids Code α-Aminobutyric acid Abu LN-methylalanine Nmala α-Amino-α-butyric acid methyl ester Mgabu LN-methylarginine Nmarg Aminocyclopropane- Cpro LN-methylasparagine Nm Carboxylates LN-methylaspartate Nmasp GABA Aib LN-methylcysteine Nmcys Amino norbornyl- Norb LN-methylglutamine Nmgin Carboxylates LN-methylglutamate Nmglu Cyclohexylalanine Chexa LN-methylhistidine Nmhis Cyclopentylalanine Cpen LN-methylisoleucine Nmile D-Alanine Dal LN-methylleucine Nmleu D-Arginine Darg LN-methyllysine Nmlys D-Aspartic Acid Dasp LN-methylmethionine Nmmet D-Cysteine Dcys LN-methylnorleucine Nmn D-Glutamine Dg LN-methylnorvaline Nmv D-Glutamic Acid Dglu LN-methylornithine Nmorn D-Histidine Dhis LN-methylphenylalanine Nm D-Isoleucine Dile LN-methylproline Nmpro D-Leucine Dleu LN-methylserine Nmser D-Lysine Dlys LN-methylthreonine Nmthr D-Methionine Dmet LN-methyltryptophan Nmtrp D-Ornithine Dorn LN-methyltyrosine Nmt D-Phenylalanine Dphe LN-methylvaline Nmval D-Proline Dpro LN-Methylethylglycine N D-serine Dser LN-methyl-tert-butylglycine Nmtbug D-Threonine Dthr L-Norleucine Nle D-Tryptophan Dt L-Norvaline Nv D-Tyrosine Dtyr α-Methyl-aminoisobutyric acid Maib D-Valine Dval α-Methyl-γ-aminobutyric acid Mgabu D- alpha-methylalanine Dmala α-Ethylcyclohexylalanine Mchexa D- α -methylarginine Dmarg α-Methylcyclopentylalanine Mcpen D- α -methylasparagine Dma α-Methyl-α-naphthylalanine Manap D- α -methylaspartic acid Dmasp α-Methylpenicillamine Mpen D- α -methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu D- α -methylglutamate Dmgln N-(2-aminoethyl)glycine Naeg D- α -methylhistidine Dmhis N-(3-aminopropyl)glycine Norn D- α -methylisoleucine Dmile N-Amino-α-methylbutyric acid Nmaabu D- α -methylleucine Dmlu α-naphthylalanine Anap D- α -methyllysine Dmlys N-Benzylglycine Nphe D- α -methylmethionine Dmmet N-(2-Carbamoylethyl)glycine Ngl D- α -methylornithine Dmorn N-(Carbamoylmethyl)glycine Nasn D- α -methylphenylalanine Dmphe N-(2-Carboxyethyl)glycine Nglu D- α -methylproline Dmpro N-(Carboxymethyl)glycine Nasp D- α -methylserine Dmser N-cyclobutylglycine Ncbut D- α -methylthreonine Dmth N-Cycloheptylglycine Nchep D- α-methyltryptophan Dmtrp N-Cyclohexylglycine Nchex D- α -methyltyrosine DmT N-Cyclodecylglycine Ncdec D- α -methylvaline Dmval N-Cyclododecylglycine Ncdot D- alpha-methylalanine Dnmala N-Cyclooctylglycine Ncoct D- α -methylarginine Dnmarg N-Cyclopropylglycine Ncpro D- α -methylasparagine DnMasn N-Cycloundecylglycine Ncund D- α -methylaspartic acid Dnmasp N-(2,2-Diphenylethyl)glycine Nbh D- α -methylcysteine Dnmcys N-(3,3-Diphenylpropyl)glycine Nb DN-methylleucine Dnmleu N-(3-Indoleethyl)glycine Nh DN-methyllysine Dnmlys N-methyl-γ-aminobutyric acid Nmgabu N-Methylcyclohexylalanine Nmchexa DN-methylmethionine Dnmmet DN-methylornithine Dnmorn N-Methylcyclopentylalanine Nmcpen N-methylglycine Nala DN-methylphenylalanine Dnmphe N-Methylaminoisobutyric acid Nmaib DN-methylproline Dnmpro N-(1-Methylpropyl)glycine Nile DN-methylserine Dnmser N-(2-Methylpropyl)glycine Nile DN-methylserine Dnmser N-(2-Methylpropyl)glycine Nleu DN-methylthreonine Dnmthr DN-methyltryptophan Dnmtrp N-(1-methylethyl)glycine Nv DN-methyltyrosine Dnmtyr N-methyl a-naphthylalanine Nmanap DN-methylvaline Dnmval N-methylpenicillamine Nmpen GABA Gabu N-(p-Hydroxyphenyl)glycine Nhtyr L-tert-Butylglycine Tbug N-(Methylthio)glycine Nc L-Ethylglycine Etg Penicillamine Pen L-homophenylalanine Hphe L- alpha-methylalanine Mala L- alpha-methylarginine Marg L-α-methylasparagine Masn L- α -methylaspartic acid Masp L-α-Methyl-tert-butylglycine Mtbug L- α -methylcysteine Mcys L-Methylethylglycine Met L-α-methylglutamine (thylglutamine) Mgl L- α -methylglutamate Mglu L- α -methylhistidine Mhis L- α -methylhomophenylalanine Mhphe L- α -methylisoleucine Mile N-(2-Methylthioethyl)glycine Nmet DN-Methylglutamine Dnmgl N-(3-guanidinopropyl)glycine Narg DN-methylglutamate Dnmglu N-(1-Hydroxyethyl)glycine Nthr DN-methylhistidine Dnmhis N-(Hydroxyethyl)glycine Nser DN-methylisoleucine Dnmile N-(Imidazolylethyl)glycine Nhis DN-methylleucine Dnmleu N-(3-Indolylethyl)glycine Nh DN-methyllysine Dnmlys N-methyl-γ-aminobutyric acid Nmgabu N-Methylcyclohexylalanine Nmchexa DN-methylmethionine Dnmmet DN-methylornithine Dnmorn N-Methylcyclopentylalanine Nmcpen N-methylglycine Nala DN-methylphenylalanine Dnmphe N-Methylaminoisobutyric acid Nmaib DN-methylproline Dnmpro N-(1-Methylpropyl)glycine Nile DN-methylserine Dnmser N-(2-Methylpropyl)glycine Nleu DN-methylthreonine Dnmthr DN-methyltryptophan Dnmtrp N-(1-methylethyl)glycine Nval DN-methyltyrosine Dnmtyr N-methyl a-naphthylalanine Nmanap DN-methylvaline Dnmval N-methylpenicillamine Nmpen GABA Gabu N-(p-Hydroxyphenyl)glycine Nhtyr L-tert-Butylglycine Tbug N-(Methylthio)glycine Nc L-Ethylglycine Etg Penicillamine Pen L-homophenylalanine Hphe L- alpha-methylalanine Mala L- alpha-methylarginine Marg L-α-methylasparagine Masn L- α -methylaspartic acid Masp L-α-Methyl-tert-butylglycine Mtbug L- α -methylcysteine Mcys L-Methylethylglycine Met L- α -methylglutamine Mgl L- α -methylglutamate Mglu L-α-ethylhistidine Mhis L- α -methylhomophenylalanine Mhphe L- alpha methylisoleucine (thylisoleucine) Mile N-(2-Methylthioethyl)glycine Nmet L- α -methylleucine Mleu L- α-methyl lysine Mlys L- alpha-methylmethionine Mmet L-α-methylnorleucine Mn L- α -methylnorvaline MnV L-α-methylornithine Morn L- alpha -methylphenylalanine Mphe L- α -methylproline Mpro L- α -methylserine Mser L-α-methylthreonine Mthr L-α-ethyl valine Mtrp L-α-Methyltyrosine Mtyr L- α -methylleucine Mval Nnbhm LN-methylhomophenylalanine Nmhphe N-(N-(2,2-Diphenylethyl) N-(N-(3,3-diphenylpropyl) Carbamoylmethylglycine Nnbhm Carbamoylmethyl(1)glycine Nnb 1-Carboxy-1-(2,2-diphenylethylamino)cyclopropane Nmbc

[0160] Table B continued

[0161] Recombinant techniques are generally used to produce the polypeptides of the invention (or just the LPD portion thereof). Such recombinant techniques are described by Bitter et al. (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al. (1984) Science 224:838-843, Gurley et al. (1986) Mol. Cell. Biol. 6:559-565, and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section 5.10. VIII, pp. 421-463.

[0162] In order to produce the polypeptide of the present invention using recombinant technology, a polynucleotide encoding the polypeptide of the present invention, such as SEQ ID NO: 2 or 6, is ligated into a nucleic acid expression vector, which contains a polynucleotide sequence under the transcriptional control of a cis-regulatory sequence (e.g., a promoter sequence), which is suitable for directing constitutive, tissue-specific or inducible transcription of the polypeptide of the present invention in a host cell.

[0163] The phrase "isolated polynucleotide" refers to a single-stranded or double-stranded nucleic acid sequence that is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence, and / or a composite polynucleotide sequence (e.g., a combination of the above).

[0164] As used herein, the phrase "complementary polynucleotide sequence" refers to a sequence that results from reverse transcription of a messenger RNA using a reverse transcriptase or any other RNA-dependent DNA polymerase. Such sequences can then be amplified in vivo or in vitro using a DNA-dependent DNA polymerase.

[0165] As used herein, the phrase "genomic polynucleotide sequence" refers to a sequence derived from (isolated from) a chromosome, and thus it represents a contiguous portion of a chromosome.

[0166] As used herein, the phrase "composite polynucleotide sequence" refers to a sequence that is at least partially complementary and at least partially genomic. A composite sequence may include some exon sequences required to encode a polypeptide of the present invention, as well as some intron sequences inserted therein. Intron sequences may have any origin, including other genes, and typically include conserved splicing signal sequences. Such intron sequences may further include cis-acting expression control elements.

[0167] As mentioned above, the polynucleotide sequence of the present invention is inserted into an expression vector (i.e., a nucleic acid construct) to allow expression of a recombinant polypeptide. The expression vector of the present invention may include additional sequences that render the vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). Common cloning vectors contain transcription and translation initiation sequences (e.g., promoters, enhancers), and transcription and translation terminators (e.g., polyadenylation signals). It should be understood that the expression vector may also include polynucleotide sequences encoding other polypeptides, which are connected to the nuclear targeting peptide of the present invention through transcription. Such polypeptides are further described herein below.

[0168] The promoter used in the expression vector can be constitutive or inducible. Tissue-specific promoters are also contemplated.

[0169] Various prokaryotic or eukaryotic cells (e.g., HEK293-6E) can be used as host expression systems to express the peptides of the present invention. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing polypeptide coding sequences; yeast transformed with recombinant yeast expression vectors containing polypeptide coding sequences; plant cell systems infected with recombinant viral expression vectors containing polypeptide coding sequences (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV), or transformed with recombinant plasmid expression vectors containing polypeptide coding sequences (e.g., Ti plasmids).

[0170] According to this embodiment of this aspect of the present invention, the nucleic acid sequence encoding the polypeptide of the present invention can be changed to further improve the expression level in the expression system. Therefore, the polynucleotide sequence encoding the polypeptide can be modified according to the preferred codons used for bacterial or some mammalian expression.

[0171] The phrase "codon optimization" refers to the selection of appropriate DNA nucleotides for use within a structural gene or fragment thereof that approximate the codon usage within the relevant system.

[0172] Examples of polynucleotide sequences that can be used to express the polypeptides of the present invention are provided in SEQ ID NOs: 2 and 6.

[0173] It will be appreciated that the polynucleotides of the invention may also be expressed directly in a subject (ie, in vivo gene therapy), or may be expressed ex vivo in a cell system (autologous or non-autologous) and then administered to a subject.

[0174] In addition to containing the necessary elements for transcription and translation of the inserted coding sequence (encoding a polypeptide), the expression constructs of the present invention may also include sequences engineered to optimize the stability, production, purification, yield or activity of the expressed peptide.

[0175] Various methods can be used to introduce the expression vector of the present invention into the host cell system. Such methods are generally described in: Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et al. [Biotechniques 4 (6): 504-512, 1986], and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. Additionally, see US Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0176] The transformed cells are cultured under effective conditions that allow a large amount of recombinant polypeptide expression. Effective culture conditions include, but are not limited to, effective culture medium, bioreactor, temperature, pH and oxygen conditions that allow protein production. Effective culture medium refers to any culture medium in which cells are cultured to produce the recombinant polypeptide of the present invention. Such culture medium generally includes an aqueous solution having assimilable carbon sources, nitrogen sources and phosphate sources, as well as suitable salts, minerals, metals and other nutrients such as vitamins. The cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter plates and culture dishes. Cultivation can be carried out at a temperature, pH and oxygen content suitable for recombinant cells. Such culture conditions are within the expertise of those of ordinary skill in the art.

[0177] Depending on the vector and host system used for production, the resulting polypeptide of the invention may be retained within the recombinant cell, secreted into the fermentation medium, secreted into the space between two cell membranes, such as the periplasmic space in E. coli; or retained on the outer surface of a cell or viral membrane.

[0178] After a predetermined time in culture, recovery of the recombinant polypeptide is achieved.

[0179] As used herein, the phrase "recovering the recombinant polypeptide" refers to collecting the entire fermentation medium containing the polypeptide and does not necessarily imply additional isolation or purification steps.

[0180] Recovery is also encompassed by the terms "isolation" or "purification", which may also be from a host cell.

[0181] Thus, the polypeptides of the present invention may be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, Concanavalin A chromatography, chromatofocusing, and differential solubility.

[0182] To facilitate recovery, the expressed coding sequence can be modified to encode the polypeptide of the present invention and the fused cleavable portion. Such fusion proteins can be designed so that the polypeptide can be easily separated by affinity chromatography; for example, by being fixed on a column specific for the cleavable portion. When a cleavage site is engineered between the polypeptide and the cleavable portion, the polypeptide can be released from the chromatography column by treatment with an appropriate enzyme or reagent that specifically cuts the fusion protein at that site [e.g., see Booth et al., Immunol. Lett. 19:65-70 (1988); and Gardella et al., J. Biol. Chem. 265:15854-15859 (1990)].

[0183] Exemplary purification tags for the purposes of the present invention include, but are not limited to, polyhistidine, V5, myc, protein A, glutathione-S-transferase, maltose binding protein (MBP), and cellulose binding domain (CBD) [Sassenfeld, 1990, TIBTECH, 8, 88-9].

[0184] The polypeptides of the present invention are preferably recovered in a "substantially pure" form.

[0185] As used herein, the phrase "substantially pure" refers to a degree of purity that permits effective use of the protein in the applications described herein.

[0186] The LPD or polypeptides of some embodiments of the invention (including, for example, an Fc portion) can be chemically modified after expression for increased bioavailability.

[0187] Thus, for example, the present invention contemplates modifications in which a polypeptide is linked to a polymer. The selected polymer is typically modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of modification can be controlled. Included within the scope of polymers are mixtures of polymers. Preferably, for therapeutic use of the final product formulation, the polymer will be pharmaceutically acceptable.

[0188] The polymer or mixture thereof may be selected from, for example, polyethylene glycol (PEG), monomethoxypolyethylene glycol, dextran, cellulose or other carbohydrate-based polymers, poly(N-vinylpyrrolidone)polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), and polyvinyl alcohol.

[0189] In further embodiments, the polypeptide is modified by PEGylation, HESylation of CTP (C-terminal peptide), cross-linking with albumin, encapsulation, modification with polysaccharides, and polysaccharide alteration. The modification can be directed to any amino acid residue in the polypeptide.

[0190] According to one embodiment, the modification is to the N- or C-terminal amino acid of LPD. This can be achieved directly or by coupling with: the addition of a thiol group of a cysteine ​​residue at the N- or C-terminus, or the addition of a linker such as Ttds at the N- or C-terminus. In a further embodiment, the N- or C-terminus of the polypeptide comprises a cysteine ​​residue, a protecting group is coupled to the N-terminal amino group of the cysteine ​​residue, and the cysteine ​​thiol group is derivatized with a functional group such as N-ethylmaleimide, a PEG group, HESylated CTP.

[0191] It is well known that the properties of certain proteins can be modulated by the attachment of polyethylene glycol (PEG) polymers, which increase the hydrodynamic volume of the protein and thereby slow its clearance by renal filtration. (See, e.g., Clark et al., J. Biol. Chem. 271: 21969-21977 (1996). Thus, it is contemplated that core peptide residues may be PEGylated to provide enhanced therapeutic benefits, e.g., increased efficacy by extending half-life in vivo. Thus, PEGylating a polypeptide will improve the pharmacokinetics and pharmacodynamics of the propeptide domain of the polypeptide.

[0192] PEGylation methods are well known in the literature and are described in the following references, each of which is incorporated herein by reference: Lu et al., Int. J. Pept. Protein Res. 43: 127-38 (1994); Lu et al., Pept. Res. 6: 140-6 (1993); Felix et al., Int. J. Pept. Protein Res. 46: 253-64 (1995); Gaertner et al., Bioconjug. Chem. 7: 38-44 (1996); Tsutsumi et al., Thromb. Haemost. 77: 168-73 (1997); Francis et al., Int. J. Hematol. 68: 1-18 (1998); Roberts et al., J. Pharm. Sci. 87: 1440-45 (1998); and Tan et al., Protein Expr. Purif. 12: 45-52 (1998). Polyethylene glycol or PEG is intended to include any form of PEG that has been used to derivatize other proteins, including but not limited to mono-(C 1-10) alkoxy or aryloxy-polyethylene glycol. Suitable PEG moieties include, for example, 40 kDa methoxypoly(ethylene glycol) propionaldehyde (Dow, Midland, Mich.); 60 kDa methoxypoly(ethylene glycol) propionaldehyde (Dow, Midland, Mich.); 40 kDa methoxypoly(ethylene glycol) maleimido-propionamide (Dow, Midland, Mich.); 31 kDa α-methyl-w-(3-oxopropoxy), polyoxyethylene (NOF Corporation, Tokyo); mPEG2-NHS-40k (Nektar); mPEG2-MAL-40k (Nektar), SUNBRIGHT GL2-400MA ((PEG)2240 kDa) (NOFCorporation, Tokyo), SUNBRIGHT ME-200MA (PEG20 kDa) (NOF Corporation, Tokyo). The PEG group is generally attached to the LPD polypeptide by acylation, amidation, thioetherification, or reductive alkylation of a reactive group (e.g., an aldehyde, amino, carboxyl, or thiol group) on the PEG moiety with a reactive group (e.g., an aldehyde, amino, carboxyl, or thiol group) on the LPD polypeptide.

[0193] PEG molecules can be covalently attached to any Lys or Cys residue at any position in the polypeptide. Other amino acids that can be used are Tyr and His. Optional amino acids with carboxyl side chains are also available. The polypeptides described herein can be PEGylated directly to any amino acid at the N-terminal via the N-terminal amino group. A "joint arm" can be added to the polypeptide to promote PEGylation. PEGylation at the thiol side chain of cysteine ​​has been widely reported (see, e.g., Caliceti & Veronese, Adv. Drug Deliv. Rev. 55: 1261-77 (2003)). If there is no cysteine ​​residue in the polypeptide, a cysteine ​​residue can be introduced via substitution or by adding cysteine ​​to the N-terminal amino acid. Other options include reagents that add thiols to the polypeptide, such as Traut's reagent and SATA.

[0194] In certain aspects, the PEG molecule is branched, while in other aspects, the PEG molecule can be linear. In certain aspects, the molecular weight of the PEG molecule is from 1 kDa to 150 kDa. More particularly, the molecular weight of the PEG molecule is from 1 kDa to 100 kDa. In further aspects, the PEG molecule is selected from 5, 10, 20, 30, 40, 50 and 60 kDa.

[0195] A useful strategy for PEGylation of polypeptides consists of combining the peptide and the PEG moiety, each with specific functionality for mutual reaction with the other, by forming the conjugated linkage in solution.Polypeptides can be readily prepared by recombinant means as described above.

[0196] According to one embodiment, PEG is "pre-activated" prior to attachment to a polypeptide. For example, carboxyl-terminated PEGs can be converted to NHS esters for activation, making them more reactive toward lysine and the N-terminus.

[0197] According to another embodiment, the polypeptide is "preactivated" with an appropriate functional group at a specific site. The conjugation of polypeptide to PEG can be carried out in an aqueous phase or an organic cosolvent, and can be easily monitored by SDS-PAGE, isoelectric focusing (IEF), SEC and mass spectrometry. The PEGylated polypeptide is then purified. Small PEG can be removed by ultrafiltration. Larger PEGs are usually purified using anion chromatography, cation chromatography or affinity chromatography. The characterization of PEGylated polypeptides can be carried out by analytical HPLC, amino acid analysis, IEF, analysis of enzymatic activity, electrophoresis, PEG: protein ratio analysis, laser desorption mass spectrometry and electrospray mass spectrometry.

[0198] The removal of excess free PEG can be performed by filling a column (Tricorn Empty High-Performance Columns, GE Healthcare) with POROS 50 HQ support (Applied Biosystems), followed by balancing the column with an equilibrium buffer (25 mM Tris-HCl buffer, pH 8.2). The PEGylated polypeptide is loaded onto the equilibrium column, and thereafter the column is washed with 5 CV of equilibrium buffer. Under these conditions, the polypeptide binds to the column. The PEGylated polypeptide is eluted in the next step by an elution buffer and used for short-term storage at 2-8°C, or frozen at -20°C for long-term storage.

[0199] The resulting polypeptide may be anywhere in size between 40-100 KDa, depending on whether modifications have been added (as described above) and the glycosylation state (as described above).

[0200] For example, in the case of an Fc fusion:

[0201] According to a specific embodiment, the polypeptide is 40-70 KDa.

[0202] According to a specific embodiment, the polypeptide is 45-65 KDa.

[0203] According to a specific embodiment, the polypeptide is 50-70 KDa.

[0204] According to a specific embodiment, the polypeptide is 45 KDa.

[0205] According to a specific embodiment, the polypeptide is 65 KDa.

[0206] According to a specific embodiment, the polypeptide domain of LOX lacking LOX catalytic activity is less than 400 amino acids, such as 100-200, 100-150, 100-300 amino acids in length.

[0207] The synthetic polypeptide can be used in any method of inhibiting the catalysis (enzymatic activity) of LOX by merely contacting LOX with the synthetic polypeptide comprising the modification. Such methods can be performed in vitro, in vivo or ex vivo.

[0208] The polypeptides of some embodiments of the present invention may be used to treat the fibrosis or fibrotic conditions mentioned above.

[0209] According to a specific embodiment, the polypeptides of some embodiments of the invention are not used (or administered) as part of a regimen that includes treatment with D-penicillamine.

[0210] The polypeptide of the present invention or the polynucleotide encoding it can be provided to the subject (i.e., mammal) to be treated by itself (e.g., purified or directly as part of an expression system), or can be provided in a pharmaceutical composition comprising the polypeptide of the present invention. As used herein, "pharmaceutical composition" refers to a preparation of one or more active ingredients described herein with other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate the administration of a compound to an organism.

[0211] As used herein, the term "active ingredient" refers to a polypeptide of the present invention (or a polynucleotide encoding the same) that can cause a biological effect.

[0212] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" may be used interchangeably to refer to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. Adjuvants are included under these phrases.

[0213] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of an active ingredient. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various types of sugars and starches, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0214] Techniques regarding drug formulation and administration may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, most recent edition, which reference is incorporated herein by reference.

[0215] Suitable peripheral administration routes may, for example, include oral, rectal, transmucosal, especially nasal, enteral or parenteral delivery, including intramuscular, subcutaneous and intravenous, intraperitoneal, intranasal or intraocular injection.

[0216] The pharmaceutical compositions of the present invention can be manufactured by processes well known in the art, for example, by means of conventional mixing, dissolving, granulating, dragee-making, hydrogelling, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0217] Thus, pharmaceutical compositions for use according to the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active ingredients into preparations which can be used pharmaceutically. Appropriate formulation depends on the route of administration chosen.

[0218] For injection, the active ingredients of the pharmaceutical composition can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0219] For oral administration, the pharmaceutical composition can be easily prepared by combining the active compound with a pharmaceutically acceptable carrier well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by patients. Pharmaceutical preparations for oral use can be prepared by: using solid excipients, optionally grinding the resulting mixture, and after adding suitable adjuvants when necessary, processing the mixture of particles to obtain tablets or dragee cores. Suitable excipients are particularly fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose; and / or physiologically acceptable polymers, such as polyvinyl pyrrolidone (PVP). When necessary, disintegrants such as cross-linked polyvinyl pyrrolidone, agar or alginic acid or its salts such as sodium alginate can be added.

[0220] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablets or dragee coatings for identification or characterization of different combinations of active compound dosages.

[0221] Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin, and sealed soft capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Push-fit capsules can contain the active ingredient mixed with a filler such as lactose, a binder such as starch, a lubricant such as talc or magnesium stearate, and an optional stabilizer. In the soft capsule, the active ingredient can be dissolved or suspended in a suitable liquid, such as a fatty oil, liquid paraffin, or a liquid polyethylene glycol. In addition, a stabilizer can be added. All formulations for oral administration should be in a dosage suitable for the selected route of administration.

[0222] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0223] For administration by nasal inhalation, the active ingredient used according to the invention is conveniently delivered in the form of an aerosol spray from a pressurized package or a nebulizer, using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges such as gelatin for use in a dispenser can be formulated containing a powder mixture of the compound and a suitable powder base such as lactose or starch.

[0224] The pharmaceutical compositions described herein can be formulated for parenteral administration, for example by bolus injection or continuous infusion. Formulations for injection can be in unit dosage form, for example in ampoules or multidose containers, with an optional preservative added. The composition can be a suspension, solution or emulsion in an oily or aqueous vehicle, and can contain formulation agents such as suspending agents, stabilizers and / or dispersants.

[0225] Pharmaceutical compositions for parenteral administration include aqueous solutions of active agents in water-soluble form. In addition, the suspension of active ingredient can be prepared as an injection suspension suitably based on oil or water. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions can contain materials that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension can also contain a suitable stabilizer or an agent that increases the solubility of the active ingredient to allow the preparation of a highly concentrated solution.

[0226] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, such as a sterile, pyrogen-free water based solution, before use.

[0227] The pharmaceutical compositions of this invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, eg, conventional suppository bases such as cocoa butter or other glycerides.

[0228] Pharmaceutical compositions suitable for use in the context of the present invention include compositions in which the active ingredient is contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of the active ingredient (e.g., a nucleic acid construct) that is effective to prevent, alleviate or ameliorate the symptoms of a disorder (e.g., ischemia), or to prolong the survival of the subject to be treated.

[0229] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0230] For any formulation used in the methods of the invention, the therapeutically effective amount or dosage can be estimated initially from in vitro and cell culture assays. For example, a dosage can be formulated in an animal model to achieve a desired concentration or titer. Such information can be used to more accurately determine useful dosages in humans.

[0231] The toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, cell culture or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage can vary depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration and dosage can be selected by each physician taking into account the patient's condition. (See, e.g., Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics", Chapter 1, page 1).

[0232] The Examples section below describes in detail animal models for DMD and standard tests for determining efficacy.

[0233] Animal models for other fibrotic conditions are well known in the art, see, for example, the liver fibrosis model described in Delire et al. J Clin Transl Hepatol. 2015 Mar;3(1):53–66; the animal model for fibrotic lung disease described in Moore et al.; and the animal model for cardiac fibrosis described in Rai et al. Mol Cell Biochem. 2017 Jan;424(1-2):123-145. doi: 10.1007 / s11010-016-2849-0.

[0234] The amount and interval of dosage can be adjusted individually to provide plasma or brain levels of active ingredient that are sufficient to induce or suppress biological effects (minimum effective concentration, MEC). MEC is different for each formulation, but can be estimated from in vitro data. The dosage necessary to reach MEC depends on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0235] Depending on the severity and responsiveness of the condition to be treated, dosing can be a single administration or multiple administrations, with the course of treatment continuing for several days to several weeks, or until a cure is effected or a diminution of the disease state is achieved.

[0236] The amount of composition to be administered will, of course, depend on the subject to be treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and the like.

[0237] When necessary, the composition of the present invention may be present in a package or dispenser device, such as a kit approved by the U.S. Food and Drug Administration (US Food and Drug Administration) (FDA), which may include one or more unit dosage forms containing an active ingredient. The package may, for example, include a metal foil or plastic foil, such as a blister package. The package or dispenser device may be accompanied by instructions for use. The package or dispenser may also accommodate a notice accompanying the container, which is in the form specified by a government agency that manages the manufacture, use or sale of the drug, and which reflects the approval of the agency for the form of the composition or human or veterinary administration. For example, such notices may be labels approved by the U.S. Food and Drug Administration (FDA) for prescription drugs, or product inserts approved. As further described above, the composition of the preparation of the present invention prepared in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for the treatment of the indicated condition.

[0238] As used herein, the term "about" refers to ± 10%.

[0239] The terms "comprises," "comprising," "includes," "including," "having" and their conjugations mean "including but not limited to."

[0240] The term "consisting of" means "including and limited to."

[0241] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially change the basic and novel characteristics of the claimed composition, method or structure.

[0242] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0243] Throughout this application, various embodiments of the present invention can be presented in the form of ranges. It should be understood that the description in the form of ranges is only for convenience and brevity, and should not be interpreted as a hard limit to the scope of the present invention. Accordingly, the description of the range should be deemed to have specifically disclosed all possible sub-ranges and each numerical value within the range. For example, a description of a range such as from 1 to 6 should be deemed to have specifically disclosed a sub-range, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and each number within the range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the scope, this applies.

[0244] Whenever a numerical range is indicated herein, it is intended to include any cited numeral (fractional or integer) within the indicated range. The phrases "ranging from a first indicated number to a second indicated number / ranging between a first indicated number and a second indicated number", and "ranging from a first indicated number to a second indicated number / ranging from a first indicated number to a second indicated number" are used interchangeably herein and are intended to include the first indicated number and the second indicated number, and all fractions and integers therebetween.

[0245] As used herein, the term "method" refers to manners, means, techniques and procedures for accomplishing a given task, including but not limited to those manners, means, techniques and procedures known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine, or readily developable from known manners, means, techniques and procedures.

[0246] As used herein, the term "treating" includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetic symptoms of a condition, or substantially preventing the appearance of clinical or aesthetic symptoms of a condition.

[0247] When reference is made to a particular sequence listing, such reference should be understood to also include sequences that substantially correspond to the complementary sequence thereof, including minor sequence variations resulting from, for example, sequencing errors, cloning errors or other changes leading to base substitutions, base deletions or base additions, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

[0248] It should be understood that any sequence identification number (SEQ ID NO) disclosed in this application may refer to a DNA sequence or an RNA sequence, depending on the context in which the SEQ ID NO is mentioned, even if the SEQ ID NO is only expressed in DNA sequence form or RNA sequence form.

[0249] It should be understood that certain features of the present invention described in the context of separate embodiments for the sake of clarity may also be provided in combination in a single embodiment. Conversely, various features of the present invention described in the context of a single embodiment for the sake of brevity may also be provided separately or in any suitable sub-combination or as suitably provided in any other described embodiment of the present invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiment is inoperable without those elements.

[0250]

[00136] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. Example

[0251] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

[0252] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are explained in detail in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM Editor (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (Editors) "Genome Analysis: A Laboratory Manual Series", Volumes 1-4, Cold Spring Harbor Laboratory Press, New York (1998); as in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Vols. I-III Cellis, JE ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), 3rd edition; "Current Protocols in Immunology", Vols. I-III Coligan JE ed. (1994); Stites et al. (eds.), "Basic and Clinical Immunology" (8th edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds.), "Selected Methods in Cellular Immunology", WH Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, MJ ed. (1984); "Nucleic Acid Hybridization" Hames, BD and Higgins SJ eds. (1985); "Transcription and Translation" Hames, BD and Higgins SJ, eds. (1984); "Animal Cell Culture" Freshney, RI, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "APractical Guide to Molecular Cloning" Perbal, B., (1984), and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All information contained therein is incorporated herein by reference.

[0253] Materials and Methods

[0254] Fc-LPD expression and purification

[0255] The prodomain of human LOX (22–168) SEQ ID NO: 1 was cloned into the pYD5 mammalian expression vector and the plasmid was transformed into competent HEK293-6E cells in exponential growth phase using polyethyleneimine (PEI) transfection reagent at a DNA-PEI ratio of 1: 2. After 120 hours of incubation, the cells were harvested and the supernatant was purified on a HiTrap protein A column, equilibrated with 100 mmol / L phosphate buffer (pH 8), and eluted by 100 mmol / L citrate buffer (pH 3-4.5).

[0256] LOX catalytic domain expression and purification

[0257] The catalytic domain of human LOX (169-417) of SEQ ID NO: 3, i.e., SEQ ID NO: 10, was cloned into the pET28 expression vector and transformed into competent E. coli BL21 SHuffle strain. A single colony was resuspended in 10 ml liquid medium supplemented with antibiotics. 10 ml of culture was used to inoculate 1 L of liquid medium. The culture was incubated at 30°C until OD 600 The protein was incubated at 16 °C, 250 rpm overnight. The culture was centrifuged at 4000 rpm for 15 minutes. After incubation on ice with a lysis buffer containing 50 mM Tris-HCl (pH 8), 200 mM NaCl, 40 mM imidazole, lysozyme, protein inhibitors and DNA enzyme, the precipitated fractions were frozen for 30 minutes. The solution was ultrasonically treated and centrifuged at 24,000 rpm for 40 minutes. The suspension was purified with a 0.22 μM filter and loaded onto a HisTrap column (GE Healthcare), which was pre-balanced with the following buffer: 50 mMTris-HCl pH 8, 200 mM NaCl, 40 mM imidazole. Protein was eluted with 50 mM Tris-HCl pH 8, 200 mM NaCl, 400 mM imidazole. The enzyme was purified by size exclusion chromatography using HiLoad 16 / 60 Superdex 75 (Amersham Biosciences) and eluted with 50 mM Tris-HCl pH 8, 200 mM NaCl.

[0258] Collagen assembly assay

[0259] HDF cells were plated in DMEM 10% FCS medium in 35 mm dishes. On the second day in cell culture medium, the following factors were added for induction of extracellular matrix expression: 5 ng / mL EGF, 5 ug / mL insulin and 150 ug / mL L-ascorbic acid, in the presence (1 uM and 10 uM) or absence of Fc-LPD in the first experiment, and in the presence of Fc, Fc-LPD and Fc-LPD N346, 362, 409Q (5 uM) for the second experiment. Two-photon microscopy and second harmonic generation ( Figure 2B -C) (see below), monitoring ECM formation from 1 to 2 weeks.

[0260] ELISA binding assay

[0261] 96-well plates (Nunc) were coated with LOX catalytic domain, HSP70 or BSA at 10 μg / mL. After blocking with 2% BSA in PBS, the plates were incubated with Fc and Fc-LPD for 1 hour at 37°C. Bound antibodies were detected by peroxidase-conjugated goat anti-human antibodies (Jackson ImmunoResearch). EC were calculated from FindECanything curve fitting analysis using GraphPad Prism. 50 .

[0262] Microscale Thermophoresis (MST)

[0263] LOX (200 nM) (SEQ ID NO: 9) was labeled with NT-647 dye (RED-tris-NTA) using the Monolith His-tag Labeling Kit and incubated for 30 minutes. The concentrations of Fc and Fc-LPD were increased and the samples were measured using Monolith NT.115 (Nanotemper, Germany) at 50% laser power, 35 seconds laser on time and 40% LED power.

[0264] Differential Scanning Fluorescence (DSF)

[0265] The NanoDSF Prometheus NT.48 measures LOX protein stability at ultra-high resolution using intrinsic tryptophan or tyrosine fluorescence by continuously heating the sample in the range of 20 - 95 °C with an adjustable heating rate of 1 °C / min, and reading both the fluorescence signal and the back-reflected signal simultaneously.

[0266] Immunoprecipitation

[0267] Fc-LPD was covalently coated onto protein G beads (GE) according to the manufacturer's instructions (GE IP beads). The coated beads were incubated with 10 cm 2 The concentrated supernatant of the plate culture was incubated overnight at 4°C. The precipitated beads were obtained by centrifugation (~500x g, 5 minutes, 4°C) and washed with PBS at least 5 times the volume of the initial beads. The bead pellet was then placed in a collection column, which was inserted into an eppendorf tube containing 10ul 1M Tris-HCl pH 8. 90 µl elution buffer (Thermo-Scientific) was added to the beads and, after incubation for 5 minutes, elution was performed by centrifuge. The last step was repeated four times.

[0268] Mass spectrometry for characterization of Fc-LPD

[0269] Gel bands were subjected to in-gel trypsin digestion, followed by a desalting step. Nano-liquid chromatography (nanoAcquity) coupled with high resolution, high mass accuracy mass spectrometry (Fusion Lumos) was used to analyze the resulting peptides, using EThcD fragmentation to generate fragmentation of both polysaccharides and peptides. In discovery mode, each sample was analyzed in random order. Data were processed in Byonic v2.15.89. This is unique software that can process such data and identify polysaccharides. Searches were completed for the uniprot database, and the following modifications were allowed: carbamidomethylation of C as fixed modification, carbamidomethylation of N-terminal, H or K, deamidation of N or Q, Glu or Gln to pyro-Glu, ammonia loss, dehydration of N-terminal acetylation, formylation of STK, ST and N-glycosylation as variable modifications. Filter data to allow a maximum false discovery rate (FDR) of 1%.

[0270] Mass spectrometry for identification of gel bands from IP of Fc-LPD

[0271] The gel bands were subjected to in-gel trypsin digestion followed by a desalting step. Nano-liquid chromatography (nanoAcquity) coupled with high resolution, high mass accuracy mass spectrometry (Q Exactive Plus) was used to analyze the resulting peptides. In discovery mode, each sample was analyzed on the instrument in a random order. Data were processed using ProteomeDiscoverer version 2.2.0.388, which was searched for the Uniprot human protein database, in which a list of common laboratory contaminants was added. The search was completed with two search algorithms - SequestHT and Mascot.

[0272] Two-photon microscopy and second harmonic generation

[0273] Images of the samples were acquired using a 2PM: Zeiss LSM 510 META NLO microscope equipped with a broadband Mai Tai-HP-femtosecond single-box tunable Ti-sapphire oscillator with broadband wavelength auto-tuning from 700 to 1,020 nm using an 855-nm wavelength (detected at 400 nm) from Spectra-Physics, and 20x and 25x objectives. The thickness of the ECM was calculated in μm based on the z-stack.

[0274] Image analysis

[0275] The imaging analysis was done with the Fiji package

[28] . Fourier component analysis for directionality was performed on the data using the Fiji plugin “Directionality” created by Jean-Yves Tinevez (pacificdotmpi-cbgdotde / wiki / indexdotphp / Directionality) and following the Fiji instructions. For entropy calculations, Fijimacro CDS.ijm was used to create the orientation analysis file, and the mean entropy was then calculated using a matlab script developed by O. Golani & G. Molodij and modified by E. Shimshoni.

[0276] Animal experiments

[0277] For early experiments, male Mdx mice (8 per group) of approximately 8 weeks old were used. One group was treated with 5 mg / kg Fc, the second group was treated with 5 mg / kg Fc-LPD, and the third group was treated with 5 mg / kg Fc-LPD N346, 362, 409Q, once a week, using intraperitoneal injection. Every two weeks, mice were tested in the rotarod assay. Mice were killed after 3 months, and the heart, diaphragm, tibialis anterior, quadriceps femoris and gastrocnemius were immediately fixed or frozen in OTC.

[0278] For late experiments, male Mdx mice (4 per group) of about 23-25 ​​weeks of age were used. One group was treated with 5mg / kg Fc, and the other group was treated with 5mg / kg Fc-LPD, once a week, using intraperitoneal injection. Every two weeks, mice were tested in the rotarod assay, and every other week, they were tested with the four-limb hanging test. Mice were put to death after 3 months, and the heart, diaphragm, tibialis anterior and gastrocnemius were immediately fixed or frozen in OTC.

[0279] Rotating stick running

[0280] For the rotarod running assay, muscle strength, coordination, balance and condition were determined. The mice were placed on the tube of the rotarod while the rotarod was rotating at a slow steady speed of 5 rpm. After the mice were positioned, the speed of the first 15 seconds was accelerated from 5 to 45 rpm, and then maintained at this speed. The running time was continuously recorded by the software and stopped automatically when the mice fell from the tube. If the mice turned to face the opposite direction on the tube when running, repositioning was performed without stopping the rotation of the tube.

[0281] Suspension Test

[0282] Using the hanging test, balance, coordination and muscle condition can be evaluated. This test is based on the understanding that mice are eager to keep hanging on a line or grid until exhaustion. Specifically, the lid of a large cage for rats is used. The mice treated by the tail are brought near the grid. The mice are allowed to grab the grid with four paws. The grid is turned upside down so that the mice are hanging and the timer is started directly. After the mouse falls from the line, the timer is stopped and the record of the hanging time is marked.

[0283] Histochemistry

[0284] As previously described (Rolls et al., 2008), two different tissue preparation protocols (paraffin embedding and microtomed frozen sections) were applied. Slides were exposed to Hoechst stain (1:4,000; Invitrogen Probes) for 1 min. Sirius red staining to label type I and type III collagen, reticulin staining for type III collagen, and Masson's trichrome staining to distinguish cells from surrounding connective tissue were performed. For microscopic analysis, a Nikon optical microscope (Eclipse E800) equipped with a Nikon digital camera (DS-Ri1) was used.

[0285] Yeast surface display expression

[0286] The construct was amplified and cloned into the pETCON (LPD) plasmid and AGA2 The gene, N-terminal HA tag and C-terminal Myc tag were in frame. The construct was then transformed into EBY100 (Saccharomyces cerevisiae ( Saccharomyces cerevisiae )) strain and plated on SD-Trp plates for selection. After incubation at 30°C for 48 hours, individual colonies were subjected to colony PCR. Fragments that met the expected band size were PCR purified and sequenced.

[0287] Constructs in SD-CAA 143 Culture in the culture medium at 30°C until the OD reaches 0.8 600 At this time, the cells were pelleted and incubated in fresh SG-CAA 143The expression of displayed proteins was measured by two complementary analyses: flow cytometry and activity assays (for wt construct).

[0288] Makes 3 x 10 7 The fully induced cells were pelleted and washed twice in succession. Each wash consisted of resuspension in 0.8 ml PBS + 0.5% BSA buffer (wash buffer), centrifugation for 30 seconds and 13kRPM. The washed cells were incubated with α-c-Myc antibody (Santa Cruz 9e10, 1:50 dilution) in 200μl wash buffer at RT for 45 minutes and then washed twice. The cells were further labeled with a secondary goat α mouse-IgG (Fc specific)-FITC antibody (Sigma F4143, 1:50 dilution) in 200µl at 4°C for another 30 minutes.

[0289] After expression and α-c-Myc / FITC labeling, 3.3 x 10 6 EBY100 cells were washed twice in 200 μl washing buffer, supplemented with biotinylated peptide (30-100 μM), and incubated for 2 hours at RT. After incubation, cells were washed twice with washing buffer, and incubated for another 30 minutes on ice with streptavidin-APC (allophycocyanin-streptavidin, 0.5 mg / ml, Jackson ImmunoResearch) diluted 1:100 in 100 μl washing buffer. For LPD-HSP70 interactions, cells were incubated for 30 minutes at RT with HSP70 labeled with Atto 633 dye (Sigma 51253). Finally, cells were washed twice, filtered by 70 μm mesh nylon filter, and diluted in 1.6 ml washing buffer. These were subsequently analyzed by flow cytometry, and data were analyzed by Flow Jo software.

[0290] Statistical analysis

[0291] All analyses were performed at least three times. Statistical analyses were performed using GraphPad Prism 7.

[0292] Materials and methods for glycosylation mutants.

[0293] Example 1

[0294] Expression and characterization of Fc-LPD

[0295] To dissect extracellular LOX activity in DMD and elucidate fibrosis during disease progression, protein inhibitors engineered with endogenous inhibitors of LOX were developed. Specifically, the autoinhibitory LPD (SEQ ID NO: 1, encoded by the nucleotide sequence shown in SEQ ID NO: 2) was selected to inhibit the activated form of LOX ( Figure 1A Based on the fact that the prodomains are unstable and their production faces difficulties, LPD fused to an Fc-tag (SEQ ID NO: 7 encoded by the nucleotide sequence shown in SEQ ID NO: 8) was generated in order to increase functionality. Importantly, Fc-LPD (SEQ ID NO: 5 encoded by the nucleotide sequence shown in SEQ ID NO: 6) is expressed in a dimer form, resulting in a minimized antibody-like inhibitor of approximately 95 kDa molecular size ( Figure 1B ). After expression in mammalian cells HEK293-6E, the cell culture medium was purified by protein A column and the pure Fc-LPD was dialyzed in PBS before evaluation in functional assays.

[0296] By measuring EC 50 and dissociation constant (K D ) to estimate binding affinity to perform an assessment of the binding affinity and inhibitory activity of Fc-LPD. The EC values ​​of the generated Fc-LPD inhibitors are 50 By ELISA ( Figure 1C ) were measured in the range of 10 to 2000 nM (EC 50 =43 nmol / L). The dissociation constant (K) of Fc-LPD (27 nmol / L) D ) was determined by microthermophoresis ( Figure 1D ), while no interaction was detected with the Fc region of the inhibitor. The ultra-high resolution stability of Fc-LPD was measured using differential scanning fluorimetry (DSF), and the transition midpoint was identified as 70°C, indicating that Fc-LPD is stable in solution ( Figure 1F , 1G ).

[0297] After purification of Fc-LPD, SDS-page analysis of samples under both reducing and non-reducing conditions indicated a mixed population. LPD is known to have three different N-glycosylation sites, and since the expression was performed in a mammalian expression system, bands of different sizes (45 kDa and 60 kDa) were analyzed by MS. The results showed that N81-glycosylation was detected in both bands (N346 in the construct herein), with multiple glycoforms ranging from 203-2790Da in the 45 kDa Fc-LPD and 60 kDa bands ( Figure 1A ), but with a higher frequency in the case of the 60 kDa band. The N144-glycosylation (N409 in the construct) site was again detected in both the 45 and 60 kDa Fc-LPD, with glycans ranging from 203-3809 Da. Only after digestion with α-lytic protease (Sigma A6362) was the N97-glycosylation site (N362 in the construct) detected in both the 45 and 60 kDa bands, ranging from 203-2499 Da in the 45 kDa Fc-LPD and 60 kDa bands. Finally, yet another glycosylation site in the Fc region of both bands was identified, bearing a minority of glycoforms with a size of 1300-1800 Da. Figure 1A In the present invention, due to the presence of Fc (SEQ ID NO: 5), the amino acid positions have been switched, N81, 97, 144 are now N346, 362, 409.

[0298] Example 2

[0299] Identification of the ability of Fc-LPD to interfere with collagen fiber assembly

[0300] The effect of Fc-LPD on the enzymatic activity of LOX was examined in an in vitro system using activated human dermal fibroblasts (HDFs). In detail, a screening system was developed to monitor specific events and stages of the collagen cross-linking reaction by advanced microscopy tools ( Figure 2A ). HDF cells were cultured with activation medium containing EGF, insulin and L-ascorbic acid, and the expression of ECM molecules (mainly collagen) was induced. The assembly of fibrillar collagen was monitored by two-photon SHG microscopy, with and without Fc-LPD, and the newly designed Fc-LPD was identified to target the late stages of collagen cross-linking and assembly. These results confirm that inhibition of LOX-mediated collagen cross-linking by Fc-LPD changes the orientation of normal collagen fibril arrangement in 3D matrix scaffolds derived from natural fibroblasts in vitro ( Figure 2B ).

[0301] In addition, HDF cells were cultured with ECM activation medium in the presence of Fc, Fc-LPD and Fc-LPD N346, 362, 409Q (5uM). After 7 days in culture, collagen fibers were monitored by two-photon SHG microscopy and disruption of fiber orientation was observed ( Figure 2C ). A significant entropy increase was confirmed in the system containing Fc-LPD N346, 362, 409Q ( Figure 2D Furthermore, measurements of ECM thickness (z-stacks) highlighted a significant reduction in ECM production in Fc-LPD and Fc-LPD N346, 362, 409Q treated cells ( Figure 2E ).

[0302] Example 3

[0303] Interactions of different Fc-LPD glycosylation forms

[0304] The interaction of Fc-LPD with proteins secreted by HDFs was examined by immunoprecipitation experiments using supernatants from HDF cells. To this end, HDF cells were incubated at 60 cm 2 The cells were cultured in 10 ml serum-free and phenol red-free medium in a 10-well plate for 3 days. The culture medium was collected and concentrated using a 10 kDa cut-off vivaspin filter and incubated with 45 kDa Fc-LPD and 60 kDa Fc-LPD, respectively, which had been semi-fractionated by gel filtration and incubated with protein G beads ( Figure 3A ). The samples were analyzed by SDS-page and Western blotting. In the SDS-page analysis of both samples, two high molecular weight bands were detected and analyzed by MS ( Figure 3B ), while in Western blot experiments, LOX (30 kDa) was detected, highlighting the ability of Fc-LPD to bind LOX under physiological conditions in vitro ( Figure 3C ). Analysis of the MS data showed interactions of the two Fc-LPD forms with ECM molecules that are either part of the core matrix group (matrisome) or associated with the matrix group. Specifically, among the immunoprecipitated proteins were ECM glycoproteins (fibronectin and thrombospondin-1), ECM regulators (SerpinB12, A2-macroglobulin), secretory factors (filament aggrecan-2, keratin (hornerin)), extracellular domain or secretory proteins (HSP70, a2-HS-glycoprotein, etc.) and other proteins (Table 1 below). These interactions are direct interactions with Fc-LPD. Since HSP70 was identified as an important interactor, by measuring the ECM 50Estimation of binding affinity was performed to evaluate the binding affinity of Fc-LPD and Fc-LPD N346, 362, 409Q. The EC values ​​of the generated Fc-LPD and Fc-LPD N346, 362, 409Q inhibitors were 50 By ELISA ( Figure 3D ) were measured in the range of 10 to 2000 nM (EC 50 =41 nmol / L). In addition, direct binding and high affinity of this interaction were demonstrated using yeast surface display. Yeast cells expressed LPD on the cell surface, and labeled HSP70 was added to the solution. After incubation at RT for 30 minutes, the interaction was measured by flow cytometry, and a significant population of LPD-positive cells was positive for binding to HSP70 ( Figure 3E ).

[0305] Table 1. Summary of proteins identified by mass spectrometry

[0306]

[0307] Example 4

[0308] In vivo assessment of DMD muscle fibrosis after treatment with Fc-LPD and Fc-LPD N346, 362, 409Q

[0309] To test whether LOX inhibition could attenuate muscle fibrosis in vivo in the context of DMD, and therefore whether it could be used as a new approach to treat DMD or other fibrotic diseases, two experiments were performed using mdx mice.

[0310] For early models, 2-month-old males mdx Mice were injected once a week with 5 mg / kg of LOX inhibitors (Fc-LPD and Fc-LPDN346, 362, 409Q) or control antibody (Fc) (n=8 for each group). Figure 4A Functional muscle tests such as rotarod running were performed during treatment, but no significant improvements were observed after treatment with Fc-LPD and Fc-LPD N346, 362, 409Q ( Figure 4B Three months after treatment had begun (a total of 11 anti-LOX injections), mice were harvested and histological analysis of muscle fibrosis (heart, quadriceps, gastrocnemius, tibialis anterior, and diaphragm) was performed using Sirius red staining and two-photon second harmonic generation microscopy, which monitors native collagen fibers.

[0311] Specifically, H&E staining in diaphragm tissues after treatment with Fc-LPD and Fc-LPD N346,362,409Q showed significant improvement in the central nuclear pathological phenotype observed in DMD pathological tissues (Figure 4C). In addition, Sirius red staining of the diaphragm showed a significant reduction of -15% and -20% in fibrillar collagen deposition in Fc-LPD and Fc-LPD N346,362,409Q mice, respectively (Figure 4C). Figure 4D -E). Interestingly, in the case of the gastrocnemius muscle, significantly fewer centrally nucleated myofibers were observed (Fig. 4F). Collagen deposition after staining with Sirius red showed a reduction of approximately 45% in the case of Fc-LPD and Fc-LPD N346,362,409Q compared to the Fc control (Fig. Figure 4G -H). By examining the quadriceps tissue, no significant reduction in the amount of collagen deposition between anti-LOX treated mice and Fc treated mice was observed (Figures 5A, 5B, 5C). However, further monitoring of the fibrillar collagen of the quadriceps tissue by two-photon SHG microscopy after treatment with Fc-LPD N346,362,409Q showed a significant reduction in the collagen fiber signal (~26%). When only the endomysium of the quadriceps was analyzed, the reduction in collagen fibers in the Fc-LPD N346,362,409Q treated mice was significant (~34%). The last tissue analyzed by two-photon SHG analysis in the early model was the tibialis anterior muscle. Compared to the control Fc, the collagen fibers were reduced by ~53% for Fc-LPD treated mice and by ~76% for Fc-LPD N346,362,409Q treated mice.

[0312] For late, 6-month-old males mdx Mice were injected once a week with 5 mg / kg of LOX inhibitor (Fc-LPD) or control antibody (Fc) (n=4 for each group). Fig. 6A Functional myometry, such as rotarod running and hanging tests, was performed during the treatment period, and significant improvements were observed after Fc-LPD treatment ( Figure 6B , 6C Two months after treatment had begun (a total of 8 anti-LOX injections), mice were harvested and histological analysis of muscle fibrosis (heart, tibialis anterior, and diaphragm) was performed using Sirius red staining and two-photon second harmonic generation microscopy (which monitors native collagen fibers). Finally, histopathological analysis of muscle tissue sections was performed to monitor inflammation, necrosis, and mean fiber diameter as a readout of myofiber maturity.

[0313] Specifically, Sirius red staining of the tibialis anterior (TA) muscle demonstrated a 50% reduction in fibrillar collagen deposition in LOX-inhibited mice ( Fig.6D iii,iv , 6E, 6G). This staining further revealed diffuse connective tissue (primarily collagen fibers) in control muscles. In contrast, the tissue of Fc-LPD-treated muscles was more distinct and distinct, and the muscles were better organized, as monitored by the circular ratio ( Fig. 6F ). Histological evaluation of tibialis anterior muscle tissue from mdx mice showed a reduction in inflammation grade (~25%) and fibrosis score (~50%). The assembly of fibrillar collagen in its native, unlabeled state was monitored by two-photon SHG microscopy, and fewer collagen fiber signals with more linear orientation were observed after treatment with Fc-LPD. To quantify changes in directional fiber orientation, Fourier component analysis of the directionality of representative images obtained by SHG was applied ( Fig.6D v，vi , 6H). In addition, two-photon SHG images showed thick fiber bundles in Fc-treated tissues that were absent in Fc-LPD-treated tissues. Taken together, these results confirm that treatment of mice with this anti-LOX molecule inhibits the accumulation of fibrosis in a DMD background.

[0314] By examining cardiac tissue, a significant reduction in cardiac collagen deposition of ~50% was observed in Fc-LPD-treated mice when compared to mice treated with Fc ( Fig.7D , 7E Although at the beginning of the experiment (6-7 months old mdx In 1984 mice, the heart and TA muscle were not highly fibrotic, but the diaphragm was significantly affected, thus serving as a model for testing whether anti-LOX treatment could promote the reduction of already fibrotic tissue. Sirius red staining of the diaphragms harvested from the mice confirmed a similar pattern to that observed in the heart and TA muscle, where reduced collagen deposition was observed ( Fig. 7A , 7C Likewise, the pathologist report performed suggested a ~20% reduction in fibrosis score (25% in Fc treatment vs. 21% in Fc-LPD treatment; ( Figure 7B ).

[0315] Example 5

[0316] Analysis of the importance of glycosylation sites for Lpd activity

[0317] Cloning and characterization

[0318] The pYD5-Fc-LPD plasmid was used as a template to generate point mutations in three N-glycosylation sites in LPD, N81, 97, 144 (which are N346, 362, 409 in our Fc-LPD construct with Q SEQ ID NO: 11). Mutations were performed using site-directed mutagenesis according to the kit manufacturer's instructions. The EC50 of the generated Fc-LPD N346, 362, 409Q inhibitors was measured by EC 50 ( Figure 1C ) were measured in the range of 10 to 2000 nM (EC 50 =58 nmol / L).

[0319] Immunoprecipitation

[0320] Fc-LPD was coated onto magnetic protein G beads (GE) according to the manufacturer's instructions. The coated beads were incubated with 10 cm 2 The concentrated supernatant of the plate culture was incubated overnight at 4°C. The precipitated beads were obtained by centrifugation (~500g, 5 minutes, 4°C) and washed with PBS at least 5 times the volume of the initial beads. The bead pellet was then placed in a collection column, which was inserted into an eppendorf tube containing 10ul 1M Tris-HCl pH 8. 90µl elution buffer (Thermo-Scientific) was added to the beads and after incubation for 5 minutes, elution was performed by centrifuge. The last step was repeated four times.

[0321] Mass spectrometry for identification of gel bands from IP of Fc-LPD

[0322] Samples were eluted with 5% SDS and subjected to trypsin digestion using S-trap. Nano-liquid chromatography (nanoAcquity) coupled with high resolution, high mass accuracy mass spectrometry (Q Exactive HFX) was used to analyze the resulting peptides. Each sample was processed with MaxQuant v1.6.0.16. Data were searched for the human proteome database with the Andromeda search engine, and the database was attached with common laboratory protein contaminants and the following modifications: carbamidomethylation of C as fixed modification, and oxidation of M and protein N-terminal acetylation as variable modification. LFQ intensity (label-free quantification) was calculated using Perseus v1.6.07 and used for further calculations. Bait hits were filtered out, as well as proteins identified only based on modified peptides. LFQ intensity was logarithmically transformed, and only proteins with at least 2 valid values ​​were retained in at least one experimental group. GO annotations were added.

[0323] result

[0324] Fc-LPD has three glycosylation sites, and to investigate the importance of glycosylation, a new construct Fc-LPD-N346,362,409Q was generated. These two proteins, including Fc (as a control), were used to elucidate the interaction of LPD with ECM proteins in the supernatant of HDF cells, as in Example 3. To this end, HDF cells were cultured at 60 cm 2 10 ml serum-free and phenol red-free medium in a dish were cultured for 3 days. The culture medium was collected and concentrated using a 10 kDa cut-off viaspin filter, and incubated together with Fc, Fc-LPD and Fc-LPD-N346, 362, 409Q (which were pre-incubated in magnetic protein G beads). Immunoprecipitation was eluted by mass spectrometry, and the analysis of MS data showed the interaction of two Fc-LPD forms with ECM molecules, which were either part of the core matrix group or associated with the matrix group. Specifically, there were ECM glycoproteins (nestin-1), secretory factors (filament aggrecan-2, keratin), extracellular regions or secretory proteins (HSP70, HSP90, HSP60) in the immunocoprecipitated proteins. It is worth mentioning that the quantitative ratio between Fc-LPD and Fc-LPD-N346, 362, 409Q was different in different proteins. For example, the ratio between the non-glycosylated form and the Fc control was significantly higher for nidogen-1 and HSP70 compared to the ratio of glycosylated Fc-LPD to Fc. (Table 2 below):

[0325]

[0326] Although the present invention has been described in conjunction with its specific embodiments, it is apparent that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to cover all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0327] All publications, patents and patent applications mentioned in this specification are incorporated herein as a reference in their entirety, to the same extent as each individual publication, patent or patent application specifically and individually indicates that it is incorporated herein as a reference. In addition, the citation or identification of any reference in this application should not be construed as admitting that such reference can be used as the prior art of the present invention. With regard to the use of section headings, they should not be construed as necessary restrictions. In addition, any priority document of the application is incorporated herein by reference in its entirety.

[0328] References

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Claims

1. Use of a polypeptide comprising a propeptide of lysyl oxidase (LOX) in the preparation of a medicament for treating Duchenne muscular dystrophy (DMD), wherein the polypeptide consists of the amino acid sequence shown in SEQ ID NO: 5 and lacks LOX catalytic activity.

2. The use of claim 1, wherein the polypeptide is glycosylated at at least one of N346, N362 and N409 of SEQ ID NO:

5.

3. The use of claim 1 or 2, wherein the polypeptide is glycosylated at at least two of N346, N362 and N409 of SEQ ID NO:

5.

4. The use of claim 1 or 2, wherein the polypeptide is glycosylated at N346, N362 and N409 of SEQ ID NO:

5.

5. The method according to claim 1 or 2, characterized in that The EC of the polypeptide 50 100-500nM.

6. The method according to claim 1 or 2, characterized in that The K of the polypeptide d 10-100nM.

7. The method according to claim 1 or 2, characterized in that The midpoint of the transition of the polypeptide is 20–70°C.

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