Compositions containing peptides of lysyl oxidase and uses thereof

Abstract

A method for treating fibrosis 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 fibrosis in the subject, the method not comprising administering D-penicillamine. Polypeptide compositions for use in the treatment are also provided.

Description

Related Applications

[0001] This application claims the benefit of priority to U.S. Patent Application No. 62 / 977,792, filed February 18, 2020, and Israel Patent Application No. 266433, filed May 2, 2019, each of which is incorporated by reference in its entirety.

[0002] An ASCII file entitled 82310SequenceListing.txt, created on April 30, 2020, containing 28,106 bytes, was submitted contemporaneously with the filing of this application and is hereby incorporated by reference. [Technical field]

[0003] In some embodiments, the present invention relates to compositions comprising a propeptide of lysyl oxidase (LOX) and uses thereof. [Background technology]

[0004] DMD is the most common and severe childhood form of a family of muscular dystrophies caused by mutations in the x-linked dystrophin gene. As a result, the anchorage of muscle fibers to the ECM is lost, and repeated muscle contractions lead to mechanical destruction of muscle fibers. DMD causes progressive weakening and loss (atrophy) of skeletal and cardiac muscles [1]. Early signs of DMD may include delayed ability to sit, stand, or walk, and difficulty learning to speak. Muscle weakness is usually noticeable by 3 or 4 years of age, beginning in the hips, pelvic region, lower legs, and shoulders. Damaged fibers induce satellite cell (SC) activation and muscle regeneration [2]. Smooth muscles, such as those that line the GI tract, are also significantly affected by the disease, and patients often suffer from intestinal paresis [3].

[0005] The "muscle regeneration program" is a multi-step process. First, a transient inflammatory response occurs during which growth factors and cytokines are released into the damaged area to allow proliferation and recruitment of macrophages and fibroblasts that dispose of muscle debris and lay down ECM. Muscle fiber formation begins with the activation of SCs, followed by proliferation, differentiation and fusion into new or existing muscle cells [2, 4]. TGFβ1, a potent regulator of tissue wound healing and fibrosis, is upregulated in skeletal muscle after injury and is likely involved in the transient inflammatory response. However, in dystrophic diseases such as DMD, a persistent inflammatory response occurs as a result of the constant muscle damage. High levels of pro-inflammatory 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 is altered. Excessive components of the ECM, such as collagen and fibrinogen, are deposited at the expense of muscle forming highly fibrotic tissues [5]. Notably, such ECM dysregulated processes result in many of the pathological features of DMD that are not directly caused only by the lack of dystrophin, but also due to the complex interactions of muscle fibers and SCs with the ECM. Thus, fibrotic muscle delays muscle repair and regeneration, enhances inflammation, and exacerbates disease progression. Overall, the loss of regenerative capacity and fibrosis, along with muscle dysfunction, are the main causes of the fatal phenotype of DMD and other related muscular dystrophies [6].

[0006] Gene editing and cell transplantation have been suggested as novel approaches to treat DMD. However, these promising approaches rely on early intervention before the wave of necrosis leads to loss of muscle fibers and replacement of tissue with fat and fibrosis, or on multimodality treatments to address the fibrotic response [7, 8]. Furthermore, major side effects such as balance problems and vomiting are associated with these treatment modalities. These approaches would increase dystrophin production and predict improved muscle function, but this has yet to be shown. Thus, the identification of genes associated with physical tissue damage, as well as the development of treatments to address undesired ECM remodeling, outlines an unmet need in DMD.

[0007] In an attempt to identify genes underlying the lethal phenotype of DMD, large-scale gene expression analyses were performed from a large number of human DMD patients and mdx mice. Genes that are significantly up- or down-regulated in mouse model systems and human patients are considered DMD core genes. In these analyses, one of the genes identified as highly overexpressed in DMD is LOX. Its elevated expression was further confirmed in a canine model of DMD, Golden Retriever Muscular Dystrophy (GRMD), using Western blot and immunohistochemistry analyses [9, 10].

[0008] LOX was primarily identified as an extracellular enzyme that initiates the covalent cross-linking of collagen and elastin molecules. LOX is the most abundant member of the family that initiates these post-translational modifications [LOX, LOX-like (LOXL) 1-4]. Its activity is important for maintaining the tensile and elastic properties of connective tissues in the skeletal, pulmonary and cardiovascular systems [11-14]. LOX is synthesized and secreted as a 50 kDa inactive proenzyme, which is processed by proteolytic cleavage of the 18 kDa prodomain by BMP-1 into a functional 32 kDa enzyme (LOX). Thus, Lox mutant mice (Lox - / -) die at birth due to aortic aneurysms and diaphragmatic rupture caused by matrix organization defects [15-17]

[18] . Recent studies have directly linked LOX activity to several human pathologies, including fibrosis, neurodegeneration, and cancer (e.g., [12, 13, 11]). Accumulating evidence suggests that apart from their classical extracellular activities, LOX family members are also found intracellularly in the cytoplasm and nucleus (e.g., [19-21]). These intracellular LOX activities are associated with the regulation of cell motility and migration, gene transcription, and differentiation [12, 22-26]. Taken together, the results demonstrate that LOX plays multiple independent roles both intracellularly and in the ECM. Recently, Lox mutant mice have been shown to exhibit severe skeletal muscle defects

[27] . In these mice, the amount of muscle fibers is significantly reduced, but they show excessive deposition of ECM and fibroblasts. Moreover, these Lox-muscle-associated phenotypes were rescued by inhibition of TGFβ signaling, thus demonstrating that a Lox-TGFβ feedback loop regulates the balance between muscle mass and connective tissue mass.

[0009] Further background art includes the following: Non-patent literature 1, Non-patent literature 2, Non-patent literature 3, Patent Document 1, Patent document 2. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] U.S. Patent Application Publication No. 20080261870 [Patent Document 2] US Patent Application Publication No. 20170360732 [Non-patent literature]

[0011] [Non-Patent Document 1] Thomassin et al.J Biol Chem.2005 Dec 30;280(52):42848-55.Epub 2005 Oct 26. [Non-Patent Document 2] Grimsby et al.J Cell Biochem.Author manuscript;available in PMC 2013 Dec 11. [Non-Patent Document 3] Zhao et al.J Biol Chem.2009 Jan 16;284(3):1385-1393. Summary of the Invention

[0012] According to an aspect of some embodiments of the present invention, there is provided a synthetic polypeptide comprising a propeptide of human lysyl oxidase (LOX), the synthetic polypeptide lacking LOX catalytic activity and comprising a modification that confers enhanced stability to the polypeptide under physiological conditions compared to a native form of the polypeptide that does not comprise the modification. According to some embodiments of the invention, the modification comprises a proteinaceous modification.

[0013] According to some embodiments of the invention, the proteinaceous modification is selected from the group consisting of immunoglobulins, human serum albumin and transferrin.

[0014] According to some embodiments of the invention, the immunoglobulin comprises an Fc domain.

[0015] According to some embodiments of the invention, the polypeptide is a chimeric polypeptide.

[0016] According to some embodiments of the invention, the modification comprises a chemical modification.

[0017] According to some embodiments of the present invention, the chemical modification is a polymer.

[0018] According to some embodiments of the present invention, the polymer is selected from the group consisting of polycationic polymers, non-ionic water soluble polymers, polyether polymers and biocompatible polymers.

[0019] According to an aspect of some embodiments of the present invention there is provided a method of treating fibrosis in a subject in need thereof comprising administering to the subject a polypeptide comprising a propeptide of lysyl oxidase (LOX), wherein the polypeptide lacks LOX catalytic activity, thereby treating fibrosis in the subject, the method not comprising administration of D-penicillamine.

[0020] According to an aspect of some embodiments of the present invention, there is provided a method of 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.

[0021] 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.

[0022] 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.

[0023] According to some embodiments of the invention, the polypeptide is glycosylated at N81, N97 and N144 of SEQ ID NO:1.

[0024] According to some embodiments of the invention, the polypeptide is aglycosylated at N81, N97 and N144 of SEQ ID NO:1.

[0025] According to some embodiments of the invention, the polypeptide is characterized by an EC50 of 10-500 nM as determined by an ELISA assay.

[0026] According to some embodiments of the invention, the polypeptide is characterized by a KD of 10-100 nM as determined by microscale thermophoresis.

[0027] 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).

[0028] 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.

[0029] According to some embodiments of the invention, the polypeptide is capable of altering the orientation of collagen fibers as determined by SHG microscopy.

[0030] According to some embodiments of the invention, the fibrosis is not Ras signaling dependent. According to some embodiments of the invention, the fibrosis is not associated with cancer or bone disease.

[0031] According to some embodiments of the invention, the propeptide of lysyl oxidase (LOX) is that of human LOX.

[0032] According to an aspect of some embodiments of the present invention, there is provided a method for inhibiting the enzymatic activity of LOX, the method comprising contacting LOX with a polypeptide described herein, thereby inhibiting the enzymatic activity of LOX.

[0033] According to some embodiments of the invention, the method is performed in vivo.

[0034] According to some embodiments of the invention, the method is carried out in vitro.

[0035] According to some embodiments of the invention, the method is performed ex-vivo.

[0036] According to an aspect of some embodiments of the present invention there is provided a method of producing a polypeptide as described herein comprising expressing a polynucleotide encoding the polypeptide in a host cell and, optionally, modifying the polypeptide by chemical modification, thereby producing the polypeptide.

[0037] According to some embodiments of the invention, the method further comprises isolating the polypeptide from the host cell.

[0038] According to some embodiments of the invention, the polypeptide is as set forth in SEQ ID NO:1 or 5.

[0039] According to an aspect of some embodiments of the present invention there is provided a polynucleotide encoding a polypeptide described herein.

[0040] According to certain embodiments, the use does not include D-penicillamine.

[0041] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. A simple explanation of the drawings from multiple perspectives

[0042] Certain embodiments of the present invention are described herein, by way of example only, with reference to the accompanying drawings. Referring now specifically to the drawings in detail, it is emphasized that the particulars shown are by way of example for the purpose of an illustrative discussion of embodiments of the present invention. In this regard, from the description considered in conjunction with the drawings, it will become apparent to those skilled in the art how embodiments of the present invention may be practiced. [Brief description of the drawings]

[0043] [Figure 1A] 1 shows the design and biochemical characterization of the fusion protein Fc-LPD. Design and construction of the antibody-like LOX inhibitor Fc-LPD. [Figure 1B] 1 shows the design and biochemical characterization of the fusion protein Fc-LPD. Design and construction of the antibody-like LOX inhibitor Fc-LPD. [Figure 1C] Figure 1 shows the design and biochemical properties of the fusion protein Fc-LPD. Binding affinity and EC50 of Fc-LPD and Fc-LPD N346, 362, 409Q to the LOX catalytic domain. [Figure 1D] Design and biochemical characterization of the fusion protein Fc-LPD. Measurement of the transition midpoint KD of Fc-LPD monitoring the intrinsic fluorescence signal of Fc-LPD as a measure of its folding state. [Figure 1E] Design and biochemical characterization of the fusion protein Fc-LPD. Measurement of the transition midpoint KD of Fc-LPD monitoring the intrinsic fluorescence signal of Fc-LPD as a measure of its folding state. [Figure 1F] Figure 1 shows the design and biochemical characterization of the fusion protein Fc-LPD. Ratiometric measurements of the fluorescence signal of Fc-LPD are plotted against increasing temperatures of the chemical denaturant to determine the Tm of the protein. [Figure 1G] Figure 1 shows the design and biochemical characterization of the fusion protein Fc-LPD. Ratiometric measurements of the fluorescence signal of Fc-LPD are plotted against increasing temperatures of the chemical denaturant to determine the Tm of the protein. [Figure 2A]Figure 1 shows the disruption of ECM assembly as determined by SHG microscopy.Scheme illustrating the experimental set-up for monitoring different stages of collagen assembly secreted by HDF cells using HG microscopy. [Figure 2B] Figure 1. Disruption of ECM assembly as determined by SHG microscopy. Representative SHG images of Fc-LPD disrupting ECM alignment. Representative fiber orientation analysis for each SHG image showing the frequency of fibers in a particular orientation. [Figure 2C] Figure 1 shows disruption of ECM assembly as determined by SHG microscopy. Representative SHG images of Fc-LPD & Fc-LPD N346, 362, 409Q disrupting ECM alignment compared to Fc. Average entropy and thickness of ECM graphs. [Figure 2D] Disruption of ECM assembly as determined by SHG microscopy. Mean entropy and thickness of ECM graphs. [Figure 2E] 1 shows disruption of ECM assembly as determined by SHG microscopy. [Figure 3A] Figure 2 shows the interaction of different Fc-LPD glycosylation forms with ECM proteins released from HDF cells. Semi-separation of 45 kDa Fc-LPD and 60 kDa Fc-LPD. [Figure 3B] Figure 2 shows the interaction of different Fc-LPD glycosylation forms with ECM proteins released from HDF cells. Immunoprecipitation of 45 kDa Fc-LPD and 60 kDa Fc-LPD. [Figure 3C] Figure 2 shows the interaction of different Fc-LPD glycosylation forms with ECM proteins released from HDF cells. Interaction of the LOX catalytic domain with Fc-LPD after immunoprecipitation (IP). [Figure 3D] 1 shows the interaction of different Fc-LPD glycosylation forms with ECM proteins released from HDF cells. Binding affinity, EC50, of Fc-LPD and Fc-LPD N346, 362, 409Q to HSP70. [Figure 3E]Figure 2 shows the interaction of different Fc-LPD glycosylation forms with ECM proteins released from HDF cells. Binding of HSP70 to LPD by using yeast surface display. [Figure 4A] 1 shows an in vivo assessment of early stages of fibrosis in DMD diaphragm and gastrocnemius muscles following treatment with Fc-LPD and Fc-LPD N346, 362, 409Q. [Figure 4B] Figure 1 shows an in vivo assessment of early stages of fibrosis in DMD diaphragm and gastrocnemius muscles following treatment with Fc-LPD and Fc-LPD N346, 362, 409Q.Figure 2 shows the results of a rotarod running assay. [Figure 4C] Figure 1 shows an in vivo assessment of early stages of fibrosis in DMD diaphragm and gastrocnemius muscle following treatment with Fc-LPD and Fc-LPD N346, 362, 409Q. H&E and Sirius Red of mouse diaphragm. [Figure 4D] FIG. 1 shows in vivo assessment of early stages of fibrosis in DMD diaphragm and gastrocnemius muscles following treatment with Fc-LPD and Fc-LPD N346, 362, 409Q. [Figure 4E] Figure 1 shows in vivo assessment of early stages of fibrosis in DMD diaphragm and gastrocnemius muscle following treatment with Fc-LPD and Fc-LPD N346, 362, 409Q. Quantification of Sirius Red in mouse diaphragm. [Figure 4F] 1 shows an in vivo assessment of early stages of fibrosis in DMD diaphragm and gastrocnemius muscles following treatment with Fc-LPD and Fc-LPD N346, 362, 409Q. H&E and Sirius Red of mouse gastrocnemius muscles are shown. [Figure 4G] 1 shows an in vivo assessment of early stages of fibrosis in DMD diaphragm and gastrocnemius muscles following treatment with Fc-LPD and Fc-LPD N346, 362, 409Q. H&E and Sirius Red of mouse gastrocnemius muscles are shown. [Figure 4H] Figure 1 shows an in vivo assessment of early stages of fibrosis in DMD diaphragm and gastrocnemius muscles following treatment with Fc-LPD and Fc-LPD N346, 362, 409Q. Quantification of Sirius red in mouse gastrocnemius muscles. [Figure 5A] Figure 1 shows in vivo assessment of early stages of fibrosis in DMD quadriceps and tibialis anterior muscles after treatment with Fc-LPD and Fc-LPD N346, 362, 409Q. Representative images of H&E and Sirius Red in quadriceps muscles of DMD mice after treatment with Fc, Fc-LPD and Fc-LPD N346, 362, 409Q are shown. [Figure 5B] Figure 1 shows in vivo assessment of early stages of fibrosis in DMD quadriceps and tibialis anterior muscles after treatment with Fc-LPD and Fc-LPD N346, 362, 409Q. Representative images of H&E and Sirius Red in quadriceps muscles of DMD mice after treatment with Fc, Fc-LPD and Fc-LPD N346, 362, 409Q are shown. [Figure 5C] 1 shows in vivo assessment of early stages of fibrosis in DMD quadriceps and tibialis anterior muscles following treatment with Fc-LPD and Fc-LPD N346, 362, 409Q. Quantification of Sirius Red in mouse quadriceps muscles. [Figure 5D] Figure 1 shows in vivo assessment of early stages of fibrosis in DMD quadriceps and tibialis anterior muscles following treatment with Fc-LPD and Fc-LPD N346, 362, 409Q. Representative images of two-photon SHG from mouse quadriceps tissue and quadriceps endothelium, respectively. [Figure 5E] Figure 1 shows in vivo assessment of early stages of fibrosis in DMD quadriceps and tibialis anterior muscles following treatment with Fc-LPD and Fc-LPD N346, 362, 409Q. Representative images of 2-photon SHG from mouse quadriceps tissue and quadriceps endothelium, respectively. Quantification of 2-photon SHG images of mouse quadriceps. [Figure 5F] Figure 1 shows in vivo assessment of early stages of fibrosis in DMD quadriceps and tibialis anterior muscles following treatment with Fc-LPD and Fc-LPD N346, 362, 409Q. Representative images of two-photon SHG from mouse quadriceps tissue and quadriceps endothelium, respectively. [Figure 5G] 4 shows in vivo assessment of early stages of fibrosis in DMD quadriceps and tibialis anterior muscles following treatment with Fc-LPD and Fc-LPD N346, 362, 409Q. Quantification of two-photon SHG images of mouse quadriceps muscles. [Figure 5H]Figure 1 shows in vivo assessment of early stages of fibrosis in DMD quadriceps and tibialis anterior muscles following treatment with Fc-LPD and Fc-LPD N346, 362, 409Q. Representative images of two-photon SHG of mouse tibialis anterior muscles and quantification of two-photon SHG images of mouse tibialis anterior muscles are shown. [Figure 5I] Figure 1 shows in vivo assessment of early stages of fibrosis in DMD quadriceps and tibialis anterior muscles following treatment with Fc-LPD and Fc-LPD N346, 362, 409Q. Representative images of two-photon SHG of mouse tibialis anterior muscles and quantification of two-photon SHG images of mouse tibialis anterior muscles are shown. [Figure 6A] 1 shows in vivo assessment of DMD muscle fibrosis following treatment with Fc-LPD. Schematic of the experiment. [Figure 6B] Figure 1 shows in vivo assessment of DMD muscle fibrosis following treatment with Fc-LPD.Figure 2 shows the results of a rotarod running assay. [Figure 6C] 1 shows in vivo assessment of DMD muscle fibrosis following treatment with Fc-LPD. 1 shows the results of a hanging test assay. [Figure 6D] Figure 1 shows in vivo assessment of DMD muscle fibrosis after treatment with Fc-LPD.Representative images of H&E, Sirius Red and SHG from mouse tibialis anterior muscles are shown. [Figure 6E] Figure 1 shows in vivo assessment of DMD muscle fibrosis following treatment with Fc-LPD. Quantification of Sirius red and fibers per area from mouse tibialis anterior (TA) muscles. [Figure 6F] Figure 1 shows in vivo assessment of DMD muscle fibrosis following treatment with Fc-LPD. Quantification of Sirius red and fibers per area from mouse tibialis anterior (TA) muscles. [Figure 6G] Figure 1 shows an in vivo assessment of DMD muscle fibrosis following treatment with Fc-LPD.Figure 2 shows a semi-qualitative histological assessment analysis of tibialis anterior muscle tissue. [Figure 6H] Figure 1 shows in vivo assessment of DMD muscle fibrosis following treatment with Fc-LPD.Representative fiber orientation analysis for each SHG image from mouse TA is shown. [Figure 7A]Figure 1 shows in vivo assessment of DMD muscle fibrosis in the diaphragm and heart after treatment with Fc-LPD.Representative images of H&E and Sirius Red in the diaphragm and heart of DMD mice after Fc and Fc-LPD treatment are shown. [Figure 7B] Figure 1 shows in vivo assessment of DMD muscle fibrosis in the diaphragm and heart following treatment with Fc-LPD. Figure 2 shows a semi-qualitative histological assessment of diaphragm tissue. [Figure 7C] Figure 1 shows in vivo assessment of DMD muscle fibrosis following treatment with Fc-LPD in the diaphragm and heart. Quantification of Sirius red staining in the diaphragm and heart is shown. [Figure 7D] Figure 1 shows in vivo assessment of DMD muscle fibrosis in the diaphragm and heart after treatment with Fc-LPD.Representative images of H&E and Sirius Red in the diaphragm and heart of DMD mice after Fc and Fc-LPD treatment are shown. [Figure 7E] Figure 1 shows in vivo assessment of DMD muscle fibrosis following treatment with Fc-LPD in the diaphragm and heart. Quantification of Sirius red staining in the diaphragm and heart is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] In some embodiments, the present invention relates to compositions comprising a propeptide of lysyl oxidase (LOX) and uses thereof.

[0045] Before describing at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by way of examples, as the invention is capable of other embodiments or of being practiced or carried out in various ways.

[0046] Embodiments of the present invention relate to the development of protein inhibitors that exploit extracellular levels of endogenous LOX. Protein engineering was used to generate a stable form of lysyl oxidase prodomain fused to an Fc antibody fragment (Fc-LPD) that specifically inhibits LOX in vitro and in vivo. Fc-LPD affinity and stability measurements were performed against LOX, revealing binding affinity (EC 50 The affinity of LOX to LOX (Kd = 43 nM) and the dissociation constant (Kd = 32 nM) were determined. In vivo studies in mdx mice showed improvement in functional tests such as rotarod running and hanging test, as well as inhibition of fibrotic accumulation and promotion of normal collagen organization. The results demonstrate that inhibition of LOX by Fc-LPD is highly specific and effectively minimizes fibrosis and side effects of DMD.

[0047] Thus, according to an aspect of the present invention, there is provided a method of treating fibrosis in a subject in need thereof, comprising administering to the subject a polypeptide comprising a propeptide of lysyl oxidase (LOX), wherein the polypeptide lacks LOX catalytic activity, thereby treating fibrosis in the subject, the method not comprising administration of D-penicillamine.

[0048] 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, wherein the polypeptide lacks LOX catalytic activity, and wherein the use does not include administration of D-penicillamine.

[0049] According to another aspect of the present invention, there is provided a method of 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.

[0050] As used herein, "fibrosis" refers to the accumulation of extracellular matrix components following trauma, inflammation, tissue repair, immunological response, cellular hyperplasia, and neoplasia. Examples of tissue fibrosis include, but are not limited to, pulmonary fibrosis, renal fibrosis, cardiac fibrosis, cirrhosis and fibrosis of the liver, skin scars and keloids, adhesions, fibromatosis, atherosclerosis, and amyloidosis.

[0051] 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 disease); a toxin; an injury (e.g., an environmental hazard (e.g., asbestos, coal dust, polycyclic aromatic hydrocarbons), smoking, a wound); a medical procedure (e.g., a surgical incision, chemotherapy, or radiation), or a combination thereof.

[0052] In some embodiments, the fibrotic condition is a pulmonary fibrotic condition, a hepatic fibrotic condition, a cardiac or vascular fibrotic condition, a renal fibrotic condition, a skin fibrotic condition, a gastrointestinal fibrotic condition, a bone marrow or hematopoietic tissue fibrotic condition, a nervous system fibrotic condition, or a combination thereof. In some embodiments, the fibrotic condition affects a tissue selected from one or more of muscle, tendon, cartilage, skin (e.g., skin epidermis or endothelium), cardiac tissue, vascular tissue (e.g., arteries, veins), pancreatic tissue, lung tissue, liver tissue, kidney tissue, uterine tissue, ovarian tissue, nervous tissue, testicular tissue, peritoneal tissue, colon, small intestine, biliary tract, intestine, bone marrow, or hematopoietic tissue.

[0053] In some embodiments, the fibrotic condition is a pulmonary fibrotic condition. In some embodiments, the pulmonary fibrotic condition is selected from one or more of pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), common interstitial pneumonia (UIP), interstitial lung disease, fibrosing alveolitis of unknown etiology (CFA), bronchiolitis obliterans, or bronchiectasis. In some embodiments, the pulmonary fibrosis occurs following a disease, a toxin, an injury, a medical treatment, or a combination thereof. In some embodiments, the pulmonary fibrosis is associated with one or more of the following medical treatments, including, but not limited to, 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 infections, particularly chronic infections; radiation therapy and drug therapy, such as chemotherapy (e.g., treatment with bleomycin, methotrexate, amiodarone, busulfan, and / or nitrofurantoin). In some embodiments, the pulmonary fibrotic condition treated by the methods of the invention is associated with (e.g., secondary to) a cancer treatment, such as treatment for cancer (e.g., squamous cell carcinoma, testicular cancer, Hodgkin's disease with bleomycin). In some embodiments, the fibrotic condition is a liver fibrotic condition. In certain embodiments, the fibrotic condition of the liver is selected from one or more of fatty liver disease, fatty liver (e.g., nonalcoholic steatohepatitis (NASH)), cholestatic liver disease (e.g., primary biliary cirrhosis (PBC)), cirrhosis, alcohol-induced liver fibrosis, bile duct injury, bile duct fibrosis, cholestasis, or cholangiopathy. In some embodiments, liver fibrosis or liver fibrosis includes, but is not limited to, liver fibrosis associated with alcoholism, viral infections, such as hepatitis (e.g., hepatitis C, B, or D), autoimmune hepatitis, nonalcoholic fatty liver disease (NAFLD), progressive massive fibrosis, exposure to toxins or irritants (e.g., alcohol, pharmaceuticals, and environmental toxins).

[0054] In some embodiments, the fibrotic condition is a cardiac fibrotic condition. In certain embodiments, the cardiac fibrotic condition is myocardial fibrosis (e.g., myocardial fibrosis associated with radiation myocarditis, surgical procedure complications (e.g., myocardial postoperative fibrosis), infectious diseases (e.g., Chagas disease, bacterial, trichinellosis, or fungal myocarditis)); granulomatous metabolic storage disorders (e.g., cardiomyopathy, hemochromatosis); developmental disorders (e.g., endocardial fibroelastosis); arteriosclerosis, or exposure to toxins or irritants (e.g., drug-induced cardiomyopathy, drug-induced cardiotoxicity, alcoholic cardiomyopathy, cobalt poisoning or exposure). In some embodiments, the myocardial fibrosis is associated with an inflammatory disorder of cardiac tissue (e.g., myocardial sarcoidosis).

[0055] In some embodiments, the fibrotic condition is a renal fibrotic condition. In some embodiments, the renal fibrotic condition is selected from one or more of the following: renal fibrosis (e.g., chronic renal fibrosis), injury / fibrosis-related nephropathy (e.g., chronic nephropathy associated with diabetes (e.g., diabetic nephropathy)), lupus, renal scleroderma, glomerulonephritis, focal segmental glomerulosclerosis, IgA nephropathy associated with human chronic kidney disease (CKD), renal fibrosis, chronic progressive nephropathy (CPN), tubulointerstitial fibrosis, ureteral obstruction, chronic uremia, chronic interstitial nephritis, radiation nephropathy, glomerulosclerosis, progressive glomerulonephropathy (PGN), injury of endothelial / thrombotic microangiopathy, HIV-associated nephropathy, or fibrosis associated with exposure to toxins, irritants, or chemotherapeutic agents.

[0056] In some embodiments, the fibrotic condition is a fibrotic condition of the skin, hi some embodiments, the fibrotic condition of the skin is selected from one or more of dermal fibrosis, scleroderma, nephrogenic systemic fibrosis (e.g., occurring after exposure to gadolinium, which is frequently used as a contrast agent for MRI in patients with severe renal failure), scarring, and keloids.

[0057] In some embodiments, the fibrotic condition is a fibrotic condition of the gastrointestinal tract, hi some embodiments, the fibrotic condition is selected from one or more of fibrosis associated with scleroderma, radiation-induced intestinal fibrosis, fibrosis associated with anterior gut inflammatory disorders such as Barrett's esophagus and chronic gastritis, and / or fibrosis associated with posterior gut inflammatory disorders such as inflammatory bowel disease (IBD), ulcerative colitis, and Crohn's disease.

[0058] In some embodiments, the fibrotic condition is an adhesion, hi some embodiments, the adhesion is selected from one or more of abdominal adhesions, peritoneal adhesions, pelvic adhesions, pericardial adhesions, epidural adhesions, peritendinous adhesions, or adhesive capsulitis.

[0059] In some embodiments, the fibrotic condition is an ocular fibrotic condition, in some embodiments, the ocular fibrotic condition comprises diseases of the anterior segment of the eye, such as glaucoma and corneal opacities, in some embodiments, the ocular fibrotic condition comprises 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 ocular fibrotic condition results from fibrosis following ocular surgery.

[0060] 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 inherent feature of a chronic myeloproliferative neoplasm of bone marrow, such as primary myelofibrosis (also referred to herein as angiogenic myeloid metaplasia or chronic idiopathic myelofibrosis). In some aspects, the myelofibrosis is associated with (e.g., secondary to) a malignant condition or a condition caused by a clonal proliferative disease. In some embodiments, the myelofibrosis is associated with a hematological disorder (e.g., a hematological disorder selected from one or more of polycythemia vera, essential thrombocythemia, myelodysplasia, hairy cell leukemia, lymphoma (e.g., Hodgkin's lymphoma or non-Hodgkin's lymphoma), multiple myeloma, or chronic myelogenous leukemia (CIVIL)). In some embodiments, the myelofibrosis is associated with (e.g., secondary to) a non-hematological disorder (e.g., a non-hematological disorder selected from solid tumor metastasis to the bone marrow, 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.

[0061] In some embodiments, the fibrosis is not associated with Ras signaling for cell transformation. Examples of such conditions include cancer, such as colon, breast, lung or prostate cancer, disorders of the kidney, cardiovascular and immune systems, bone diseases, such as osteopenic conditions, such as osteoporosis.

[0062] According to certain embodiments, the fibrotic condition is of the muscle.

[0063] There are two main types of muscle tissue in animals: striated and smooth muscle.

[0064] Muscles may also be grouped by location or function. In some embodiments, the polypeptide may target a tissue of a subject of interest, such as one or more muscles of the face, one or more muscles for chewing, one or more muscles of the tongue and neck, one or more muscles of the rib cage, one or more muscles of the pectoral girdle and arms, one or more muscles of the arms and shoulders, one or more muscles of the ventral and dorsal forearm, one or more muscles of the hand, one or more muscles of the erector spinae, one or more muscles of the pelvic girdle and legs, and / or one or more muscles of the forelimbs and legs.

[0065] In some embodiments, the facial muscles include, but are not limited to, intraocular muscles such as the ciliary body, iris dilator, and sphincter iris; ear muscles such as the auricle, temporalis, stapedius, and tensor tympani; nose muscles such as the root of the nose, nasal muscles, dilator naris, depressor septum nasalis, and levator labii ala naris; mouth muscles such as the levator anguli oris, depressor anguli oris, orbicularis oris, buccinator, zygomaticus major, and zygomaticus minor, platysma, levator labii superioris, depressor labii inferioris, mirth muscles, mentalis, and / or corrugator supercilii.

[0066] In some embodiments, the masticatory muscles include, but are not limited to, the masseter, temporalis, medial pterygoid, and lateral pterygoid. In some embodiments, the tongue and neck muscles include, but are not limited to, the genioglossus, styloglossus, palatoglossus, hyoglossus, digastric, styloglossus, mylohyoid, genioglossus, omohyoid, sternohyoid, sternothyroid, thyrohyoid, sternocleidomastoid, anterior scalene, middle scalene, and / or posterior scalene.

[0067] In some embodiments, muscles of the rib cage, pectoral girdle, and arms include, but are not limited to, the subclavius ​​pectoralis major, pectoralis minor, rectus abdominis, external oblique, internal oblique, transverse abdominis, diaphragm, external intercostal, internal intercostal, serratus anterior, trapezius, levator scapulae, rhomboid major, rhomboid minor, latissimus dorsi, deltoid, subscapularis, supraspinatus, infraspinatus, teres major, teres minor, and / or coracobrachialis.

[0068] In some embodiments, the muscles of the arm and shoulder include, but are not limited to, the long head of the biceps, the short head of the biceps, the long head of the triceps, the lateral head of the triceps, the medial head of the triceps, the elbow, the pronator teres, the supinator, and / or the brachialis.

[0069] In some embodiments, muscles of the ventral and dorsal forearm include, but are not limited to, brachioradialis, flexor carpi radialis, flexor carpi ulnaris, palmaris longus, extensor carpi ulnaris, extensor carpi radialis, extensor carpi radialis brevis, extensor digitorum, and extensor digitorum minimi.

[0070] In some embodiments, the intrinsic muscles of the hand include, but are not limited to, the thenar, abductor pollicis brevis, flexor pollicis brevis, opponens pollicis, thenar, abductor digiti minimi, flexor digiti brevis, opponens digiti minimi, palmar interosseous, dorsal interosseous, and / or medius.

[0071] In some embodiments, the erector spinae muscles include, but are not limited to, the cervical muscles, spinalis, longissimus, and / or iliocostalis.

[0072] In some embodiments, muscles of the pelvic girdle and legs include, but are not limited to, psoas major, iliacus, quadriceps, adductor longus, adductor brevis, adductor magnus, gracilis, sartorius, quadriceps such as rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gastrocnemius, peroneal 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, pubic and / or tibialis anterior.

[0073] In some embodiments, muscles of the forelimbs and legs include, but are not limited to, extensor digitorum longus, extensor pollicis longus, peroneus brevis, plantaris, tibialis posterior, flexor hallucis longus, extensor digitorum brevis, extensor pollicis brevis, abductor pollicis brevis, flexor digitorum minimi, flexor digitorum brevis, opponens digiti minimi, extensor digitorum brevis, lumbrical muscles of the foot, quadratus plantae or flexor accessorius, flexor digitorum brevis, dorsal interosseous, and / or plantar interosseous muscles.

[0074] Muscular dystrophies are a group of genetic disorders that cause muscle degeneration, resulting in reduced and impaired movement. The central feature of all muscular dystrophies is that they are progressive in nature. Muscular dystrophies include, but are not limited to, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, and myotonic dystrophy types 1 and 2, including the congenital form myotonic dystrophy type 1. Symptoms may vary depending on the type of muscular dystrophy, whether some or all muscles are affected. Exemplary symptoms of muscular dystrophy include delayed development of muscle motor skills, difficulty using one or more muscle groups, difficulty swallowing, speaking, or feeding, tearing, ptosis, frequent falls, loss of strength in an adult muscle or muscle group, loss of muscle size, problems walking due to physical weakness or changes in biomechanics, muscle hypertrophy, muscle pseudohypertrophy, fatty infiltration of muscle, replacement of muscle with non-contractile tissue (e.g., muscle fibrosis), muscle necrosis, and / or cognitive or behavioral disorders / mental retardation.

[0075] There is no known cure for muscular dystrophies, but several supportive treatments, including both symptomatic and disease-modifying treatments, are used. Corticosteroids, physical therapy, orthopedic appliances, wheelchairs, or other assistive medical devices for ADL and pulmonary function are commonly used in muscular dystrophies. Cardiac pacemakers are used to prevent sudden death due to cardiac arrhythmia in myotonic dystrophies. Anti-myotonic drugs that improve symptoms of myotonia (inability to relax) include mexilitine, and sometimes phenytoin, procainamide, and quinine. These can be combined with treatment with the polypeptide of some embodiments of the present invention.

[0076] Duchenne muscular dystrophy (DMD) is characterized by proximal muscle weakness, abnormal gait, pseudohypertrophy of the gastrocnemius (calf) muscles, and elevated creatine kinase (CK). Many DMD patients are typically diagnosed around age 5 when symptoms / signs become more evident. Affected individuals typically stop walking around age 10-13 and die in the mid- or late 20s due to cardiopulmonary insufficiency.

[0077] The disorder DMD is caused by mutations in the dystrophin gene, located on the human X chromosome, which codes for the protein dystrophin, a key structural component in muscle tissue that provides structural stability to the dystroglycan complex (DGC) of the cell membrane. Dystrophin connects the internal cytoplasmic actin filament network with the extracellular matrix, providing physical strength to muscle fibers. Thus, alterations or absence of dystrophin leads to abnormal sarcolemmal rupture and necrosis of muscle fibers. Both genders can carry the mutation, but women rarely show severe symptoms of the disease.

[0078] The main symptom of DMD is muscle weakness associated with muscle wasting, typically affecting voluntary muscles first, particularly those of the hips, pelvic region, thighs, shoulders, and calves. Weakness can also occur in the arms, neck, and other areas. The calves are often enlarged. Signs and symptoms usually appear before age 6 and can appear as early as infancy. Other physical symptoms include, but are not limited to, delayed ability to walk independently, progressive difficulty walking, stepping or running, and eventual loss of ability to walk (usually by age 15 years); frequent falls; fatigue; difficulty with motor skills (running, hopping, jumping); increased lumbar lordosis leading to shortening of the hip flexors; contractures of the Achilles tendon and hamstrings that impair function as muscle fibers shorten and fibrosis occurs in the connective tissue; deformed muscle fibers; pseudohypertrophy (enlargement) of the tongue and calf muscles caused by replacement of muscle tissue with fat and connective tissue; higher risk of neurobehavioral disorders (e.g., ADHD), learning disabilities (dyslexia), and non-progressive decline in certain cognitive skills (especially short-term memory); skeletal deformities (including scoliosis in some cases).

[0079] As used herein, "treating" refers to negating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating the clinical or cosmetic symptoms of a condition, or substantially preventing the appearance of clinical or cosmetic symptoms of a condition.

[0080] As used herein, "subject" refers to a subject diagnosed with or at risk for fibrosis.

[0081] As used herein, "synthetic polypeptide" refers to a protein that does not exist in nature because it has been isolated from its natural environment, e.g., the human or animal body, or because it has been mutated relative to a wild-type form, or modified by attachment to a heterologous moiety, e.g., a protein or chemical.

[0082] 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 pre-proprotein (pre-proLOX) (UniProtKB-P28300, which undergoes several post-translational modifications in the endoplasmic reticulum (ER), such as 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 C-terminal sequence (also referred to herein as the portion containing LOX catalytic activity) that comprises the 249 amino acid residue mature protein characteristically folds to obtain at least three disulfide bonds. Copper is a functional catalytic cofactor and is incorporated into the nascent enzyme in the ER. The enzyme also contains a peptidyl-organic cofactor, lysyltyrosine quinone (LTQ), which is generated by an intramolecular cross-link between lysine 320 and the copper-dependent oxidation product of tyrosine 355.

[0083] "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, following secretion of proLOX into the extracellular space, is cleaved by procollagen-C-proteinase (BMP-1) or BMP-1-associated metalloproteinase to generate the free propeptide (LPD) and catalytically active LOX.

[0084] An important intracellular function of LOX-PP (LPD) is probably the maintenance of inactive lysyl oxidase in the secretory pathway. The propeptide may also function as an intramolecular chaperone to facilitate the correct folding and targeting of these proteins to their final destination. Indeed, mass spectrometry analysis shown in Tables 1 and 2 below indicates that LPD co-immunoprecipitates with chaperone proteins, such as heat shock proteins.

[0085] LOX-PP contains three consensus N-glycosylation sites at residues 81, 97 and 144 of the LOX protein sequence.

[0086] The present teachings contemplate, in one embodiment, a glycosylated LPD.

[0087] 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 a glycosylation site, the indicated residue is the corresponding residue in SEQ ID NO: 1.

[0088] According to one embodiment, the polypeptide is glycosylated at one glycosylation site in the LPD, ie, N81 of SEQ ID NO:1.

[0089] According to one embodiment, the polypeptide is glycosylated at one glycosylation site in the LPD, ie, N97 of SEQ ID NO:1.

[0090] According to one embodiment, the polypeptide is glycosylated at one glycosylation site in the LPD, ie, N144 of SEQ ID NO:1.

[0091] According to one embodiment, the polypeptide is glycosylated at at least two of N81, N97 and N144 of SEQ ID NO:1.

[0092] According to one embodiment, the polypeptide is glycosylated at two glycosylation sites in the 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.

[0093] According to one embodiment, the polypeptide is glycosylated at N81, N97 and N144 of SEQ ID NO:1.

[0094] According to another embodiment, the polypeptide is not glycosylated on N81, N97 and N144 of SEQ ID NO:1.

[0095] Altered glycosylation can be accomplished by methods well known in the art, such as by selecting an appropriate expression system (e.g., prokaryotic versus eukaryotic), separating glycosylated from non-glycosylated forms, site-directed mutagenesis at glycosylation sites, and / or using enzymatic modification.

[0096] The inventors have shown that the glycosylated and non-glycosylated forms are endowed with different properties (see Example 5). For example, the non-glycosylated form has a higher binding ratio to HSP70 as determined by immunoprecipitation experiments followed by mass spectrometry.

[0097] The polypeptides of some embodiments of the present invention have some biological activity.

[0098] According to a specific embodiment, the protein inhibits LOX in vitro and in vivo. Fc-LPD affinity and stability measurements were performed for LOX, and binding affinity (EC 50 The affinity of LOX to LOX (Kd = 43 nM) and the dissociation constant (Kd = 32 nM) were determined. In vivo studies in mdx mice showed improvement in functional tests such as rotarod running and hanging test, as well as inhibition of fibrotic accumulation and promotion of normal collagen organization. The results demonstrate that inhibition of LOX by Fc-LPD is highly specific and effectively minimizes fibrosis and side effects of DMD.

[0099] According to a specific embodiment, the polypeptide is characterized by an EC50 of about 10-2000 nM, about 10-500 nM, for example about 43 nM, as determined by an ELISA assay (eg, according to the assay conditions of FIG. 1C).

[0100] According to certain embodiments, the polypeptide has a K of about 10-100 nM, e.g., about 32 nM, as determined by microscale thermophoresis (e.g., according to the assay conditions of FIG. 1D). D It is characterized by:

[0101] According to certain embodiments, the polypeptides can downregulate collagen cross-linking in an in vitro assay system, which is further described in the Examples section herein below. The polypeptides of some embodiments of the present invention can disrupt collagen cross-linking, thereby altering the structure of the extracellular matrix (ECM), relaxing collagen fibers and changing them from an ordered orientation to a more random orientation. Thus, the polypeptides of some embodiments of the present invention alter collagen fibrillation (and therefore collagen strength) and affect collagen thickness.

[0102] Methods for analyzing collagen structure are known in the art and include, for example, second harmonic generation (SHG) microscopy (see, for example, Figures 2A-B-C-D), electron microscopy imaging, and Raman microscopy.

[0103] Thus, according to embodiments of the invention, the polypeptides are capable of reducing at least one of fibrillar collagen (eg, type I) and cross-linked collagen as determined by SHG microscopy.

[0104] By "reduce" is intended to mean that the amount of fibrillar / crosslinked 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 compared to the amount in the absence of the polypeptide.

[0105] According to an embodiment of the invention, the polypeptide is capable of altering the orientation of collagen fibers as determined by SHG microscopy.

[0106] According to one embodiment, the LPD is derived from (taken or originating from) human LOX, although other mammalian sequences of LOX are also contemplated.

[0107] The mRNA and amino acid sequences of Homo sapiens LOX can be found under GenBank accession numbers P28300.2 (protein accession number), AF039291.1 (mRNA accession number), and NC_000005 (genome accession).

[0108] As stated, the polypeptide lacks LOX catalytic activity.

[0109] As used herein, "LOX catalytic activity" refers to the lysyl oxidase activity of the enzyme, typically attributable to a 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 shown in SEQ ID NOs: 9 (amino acid sequence) and 10 (nucleic acid sequence).

[0110] The catalytic activity of LOX can be determined in vitro by SHG microscopy (described in detail in the Examples section below).

[0111] Functional equivalents of LPDs that have about the same or even greater activity / stability than SEQ ID NO:1 or 5 are also contemplated.

[0112] According to certain embodiments, 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, such as 100%, identical to the LPD sequence set forth 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 a peptide and a protein of larger size, the homology is measured only in the corresponding region, i.e. the protein is considered to have only the same general length as the peptide, allowing for gaps and insertions.

[0113] Homologs may also refer to deletion, insertion or substitution variants (including amino acid substitutions thereof), and biologically active polypeptide fragments thereof.

[0114] Further modifications and changes can be made in the structure of the LPD portion of the polypeptide of the presently disclosed subject matter and still obtain a molecule capable of inhibiting LOX. For example, certain amino acids can be substituted with other amino acids in the sequence without significant loss of peptide activity. Since it is the interaction ability and properties of a polypeptide that define its biological functional activity, certain amino acid sequence substitutions can be made in the polypeptide sequence (or, of course, the underlying DNA coding sequence) and still obtain a polypeptide with similar properties.

[0115] In making such changes, the hydropathic index of an amino acid may be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide having similar biological activity. Each amino acid is 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); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0116] The relative hydropathic properties of amino acids are believed to determine the secondary structure of the resulting polypeptide, which in turn defines the interaction of the polypeptide with other molecules (e.g., 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 to obtain a functionally equivalent polypeptide. In such changes, substitution of amino acids whose hydropathic index is within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0117] Substitutions of like amino acids can also be made on the basis of hydrophilicity, particularly where the biologically functional equivalent polypeptide or peptide thereby produced is intended for use in immunological embodiments. U.S. Patent No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a polypeptide, as governed by the hydrophilicity of adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., a biological property of the polypeptide. As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0.+-.1); glutamate (+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 is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular an immunologically equivalent, polypeptide. In such changes, substitution of amino acids whose hydrophilicity values ​​are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0118] Thus, as outlined above, amino acid substitutions are 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 the various aforementioned characteristics are well known to those of skill in the art and include arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; valine, leucine and isoleucine. Thus, the subject matter of the present disclosure contemplates functional or biological equivalents of the above-described polypeptides or LPD portions thereof.

[0119] Biological or functional equivalents of the polypeptides can be prepared using site-directed mutagenesis according to procedures well known in the art. Thus, amino acid residues can be added or deleted from the LPDs of the presently disclosed subject matter using standard molecular biology techniques without altering the functionality of the peptide.

[0120] According to one embodiment, the amino acid sequence of the LPD is modified to increase its stability, bioavailability and / or pharmacological effectiveness.

[0121] Thus, according to an aspect of the present invention there is provided a synthetic polypeptide comprising a propeptide of human lysyl oxidase (LOX), wherein the polypeptide lacks LOX catalytic activity and comprises a modification that confers enhanced stability to the polypeptide under physiological conditions compared to a native form of the polypeptide that does not comprise the modification.

[0122] Methods for determining stability are well known in the art.

[0123] As used herein, "stability" refers to at least thermal stability. The method is based on measuring ultra-high resolution protein stability using endogenous tryptophan or tyrosine fluorescence.

[0124] As used herein, "enhanced" or "increased" refers to an increase of at least 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90% or more, e.g., 100%, over native LPD.

[0125] According to a specific embodiment, the polypeptide is characterized by a transition midpoint between 20 and 95° C. (eg, a Tm of about 70° C.) as determined by differential scanning fluorimetry (DSF).

[0126] Thus, to improve the stability / activity of the LPD, the polypeptide is modified.

[0127] According to certain embodiments, the modification comprises a proteinaceous modification.

[0128] The proteinaceous modifications can be attached to the polypeptide by chemical conjugation (fusion polypeptides, such as by use of linkers and / or active groups) or by recombinant DNA techniques, the synthetic polypeptide being a chimeric polypeptide.

[0129] 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 proteinaceous modification. The fusion / chimera (or collectively "fusion") can have an N-to-C or C-to-N orientation of the LPD relative to the proteinaceous portion (also referred to herein as a "heterologous polypeptide"). The fusion protein can include a myc, HA, or His6 tag. The fusion protein further comprises an LPD fused to the Fc domain of human IgG (referred to herein in one embodiment as Fc-LPD). In a particular aspect, the immunoglobulin fusion comprises the hinge, CH2, and CH3, or hinge, CH1, CH2, and CH3 regions of an IgG1 molecule. According to a specific embodiment, the Fc is as shown in SEQ ID NO: 7. For the generation of immunoglobulin fusions, see also U.S. Pat. No. 5,428,130. The Fc portion is a fusion protein that is fused to a murine IgG1 or human IgG2 M 4. Human IgG2 M4 (see US Patent Application Publication Nos. 20070148167 and 20060228349) is an IgG2-derived antibody with mutations such that the antibody maintains a normal pharmacokinetic profile but does not have any known effector function.

[0130] Exemplary amino acid sequences of an LPD fused to an Fc domain are shown in SEQ ID NO:5 (amino acid) and SEQ ID NO:6 (nucleic acid).

[0131] The fusion protein further comprises an LPD fused to human serum albumin, transferrin, or an antibody.

[0132] In a further embodiment, the LPD is conjugated to a carrier protein, such as human serum albumin, transferrin, or an antibody molecule.

[0133] As used herein, the term "polypeptide" refers to a polymer of natural or synthetic amino acids and includes natural peptides (either cleavage products, synthetically synthesized polypeptides, or recombinant polypeptides) and peptidomimetics (typically synthetically synthesized peptides), as well as polypeptide analogs such as peptoids and semipeptoids, which may have modifications that, for example, make the polypeptide more stable while in the body or more cell-friendly.

[0134] 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 described, 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 set forth herein. Further details in this regard are provided below.

[0135] The polypeptide bond (-CO-NH-) in a polypeptide may be replaced with, for example, an N-methylated bond (-N(CH3)-CO-), an ester bond (-C(R)HCOOC(R)-N-), a ketomethylene bond (-CO-CH2-), an α-aza bond (-NH-N(R)-CO-), where R is any alkyl, e.g., methyl, a carba bond (-CH2-NH-), a hydroxyethylene bond (-CH(OH)-CH2-), a thioamide bond (-CS-NH-), an olefinic double bond (-CH=CH-), a retroamide bond (-NH-CO-), a polypeptide derivative (-N(R)-CH2-CO-), and R is a "normal" side chain naturally occurring on a carbon atom.

[0136] These modifications can occur at any of the bonds along the polypeptide chain, or even at several (2-3) simultaneously.

[0137] The natural aromatic amino acids Trp, Tyr and Phe may be substituted with synthetic unnatural acids such as phenylglycine, TIC, naphthylalanine (Nol), ring methylated derivatives of Phe, halogenated derivatives of Phe, or o-methyl-Tyr.

[0138] In addition to the above, the polypeptides of the present invention may also contain one or more modified amino acids or one or more non-amino acid monomers (eg, fatty acids, complex glycosaccharides, etc.).

[0139] As used herein and in the claims section that follows, the term "amino acid" or "amino acids" is understood to include the 20 naturally occurring amino acids; amino acids that are frequently post-translationally modified in vivo, including, for example, hydroxyproline, phosphoserine, and phosphothreonine; as well as other unusual amino acids, including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine, and ornithine. Additionally, the term "amino acid" includes both D- and L-amino acids (stereoisomers).

[0140] The following Tables A and B list naturally occurring amino acids (Table A) and non-conventional or modified amino acids (Table B) that can be used with the present invention.

[0141] JPEG0007602486000001.jpg124129

[0142] JPEG0007602486000002.jpg193125JPEG0007602486000003.jpg192125JPEG0007602486000004.jpg82166

[0143] Recombinant techniques are typically used to produce the polypeptides of the invention (or just the LPD portion thereof). Such recombinant techniques are described in 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 VIII, pp 421-463.

[0144] To produce a polypeptide of the invention using recombinant techniques, a polynucleotide encoding a polypeptide of the invention, e.g., SEQ ID NO: 2 or 6, is ligated into a nucleic acid expression vector comprising a polynucleotide sequence under the transcriptional control of a cis-regulatory sequence (e.g., a promoter sequence) suitable for directing constitutive, tissue-specific or inducible transcription of the polypeptide of the invention in a host cell.

[0145] 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).

[0146] As used herein, the expression "complementary polynucleotide sequence" refers to a sequence resulting from reverse transcription of messenger RNA using reverse transcriptase or any other RNA-dependent DNA polymerase. Such a sequence can then be amplified in vivo or in vitro using a DNA-dependent DNA polymerase.

[0147] As used herein, the phrase "genomic polynucleotide sequence" refers to a sequence derived (isolated) from a chromosome, and thus, it represents a contiguous portion of a chromosome.

[0148] 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 contain some exon sequences necessary to encode a polypeptide of the invention, as well as some intron sequences intervening therebetween. The intron sequences may be of any source, including other genes, and typically contain conserved splicing signal sequences. Such intron sequences may further include cis-acting expression regulatory elements.

[0149] As described herein above, the polynucleotide sequence of the present invention is inserted into an expression vector (i.e., a nucleic acid construct) to allow expression of the recombinant polypeptide. The expression vector of the present invention may contain additional sequences (e.g., shuttle vectors) that make the vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both. Typical cloning vectors include transcription and translation initiation sequences (e.g., promoters, enhancers) and transcription and translation terminators (e.g., polyadenylation signals). It will be understood that the expression vector may also contain polynucleotide sequences encoding other polypeptides transcriptionally linked to the nuclear targeting peptide of the present invention. Such polypeptides are further described herein below.

[0150] Promoters used in expression vectors can be constitutive or inducible. Tissue-specific promoters are also contemplated.

[0151] A variety of prokaryotic or eukaryotic cells (e.g., HEK293-6E) can be used as host expression systems to express the peptides of the 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 the polypeptide coding sequence, yeast transformed with recombinant yeast expression vectors containing the polypeptide coding sequence, plant cell lines infected with recombinant viral expression vectors (e.g., Cauliflower Mosaic Virus, CaMV; Tobacco Mosaic Virus, TMV), or plant cell lines transformed with recombinant plasmid expression vectors, such as Ti plasmids, containing the polypeptide coding sequence.

[0152] According to this embodiment of this aspect of the invention, the nucleic acid sequence encoding the polypeptide of the invention may be altered to further improve expression levels in an expression system. Thus, the polynucleotide sequence encoding the polypeptide may be modified according to preferred codon usage for bacterial or particular mammalian expression.

[0153] The phrase "codon optimization" refers to the selection of appropriate DNA nucleotides for use within a structural gene or a fragment thereof that approximates the codon usage in the relevant system.

[0154] Examples of polynucleotide sequences that can be used to express a polypeptide of the present invention are provided in SEQ ID NOs:2 and 6.

[0155] It will be understood that the polynucleotides of the invention can also be expressed directly in the subject (i.e., in vivo gene therapy) or expressed ex vivo in cell lines (autologous or non-autologous) and then administered to the subject.

[0156] In addition to containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding a polypeptide), the expression constructs of the invention may also contain sequences engineered to optimize the stability, production, purification, yield, or activity of the expressed peptide.

[0157] A variety of methods can be used to introduce the expression vectors of the present invention into host cell systems. 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.

[0158] The transformed cells are cultured under effective conditions that allow for expression of large amounts of the recombinant polypeptide. Effective culture conditions include, but are not limited to, effective media, bioreactors, temperature, pH and oxygen conditions that allow for protein production. Effective media refers to any medium in which cells are cultured to produce the recombinant polypeptide of the invention. Such media typically include aqueous solutions having assimilable carbon, nitrogen and phosphate sources, as well as other nutrients such as appropriate salts, minerals, metals and vitamins. The cells of the invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri dishes. Cultivation can be carried out at temperatures, pH and oxygen content suitable for the recombinant cells. Such culture conditions are within the expertise of one of ordinary skill in the art.

[0159] Depending on the vector and host system used for production, the resulting polypeptides of the invention may remain within the recombinant cell, be secreted into the fermentation medium, be secreted into the space between two cell membranes, e.g., the periplasmic space in E. coli, or be retained on the outer surface of a cellular or viral membrane.

[0160] After a certain time in culture, the recovery of the recombinant polypeptide is affected.

[0161] As used herein, the phrase "recovery of recombinant polypeptide" refers to collecting the entire fermentation medium containing the polypeptide and does not necessarily imply additional steps of separation or purification.

[0162] Recovery is also encompassed by the terms "isolating" or "purifying", which may be from a host cell.

[0163] Thus, the polypeptides of the present invention can be purified using a variety of standard protein purification techniques, including, 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 solubilization.

[0164] To facilitate recovery, the expressed coding sequence can be engineered to encode a polypeptide of the invention and a fusion cleavable moiety. Such fusion proteins can be designed so that the polypeptide can be easily isolated by affinity chromatography, for example, by immobilization on a column specific for the cleavable moiety. If a cleavage site is engineered between the polypeptide and the cleavable moiety, the polypeptide can be released from the chromatography column by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at this site [see, e.g., Booth et al., Immunol. Lett. 19:65-70 (1988); and Gardella et al., J. Biol. Chem. 265:15854-15859 (1990)].

[0165] Exemplary purification tags for 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].

[0166] The polypeptides of the present invention are preferably recovered in "substantially pure" form.

[0167] As used herein, the phrase "substantially pure" refers to a purity that allows for effective use of the protein in the applications described herein.

[0168] The LPD or polypeptides (eg, including an Fc portion) of some embodiments of the invention may be chemically modified by chemical modification after expression to enhance bioavailability.

[0169] Thus, for example, the present invention contemplates modifications in which a polypeptide is linked to a polymer. The polymer selected is usually 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 in the final product preparation, the polymer is pharma- ceutically acceptable.

[0170] The polymer or mixtures thereof may be selected from the group consisting of, for example, polyethylene glycol (PEG), monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate-based polymers, poly-(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), and polyvinyl alcohol.

[0171] In further embodiments, the polypeptide is modified by PEGylation, HES-CTP (C-terminal peptide), cross-linking to albumin, encapsulation, modification with polysaccharides and alteration of polysaccharides. Modifications can be to any amino acid residue in the polypeptide.

[0172] According to one embodiment, the modification is to the N- or C-terminal amino acid of the LPD. This can be effected directly or by coupling with the thiol group of a cysteine ​​residue added to the N- or C-terminus or a linker, such as Ttds, added to the N- or C-terminus. In a further embodiment, the N- or C-terminus of the polypeptide comprises a cysteine ​​residue with a protecting group attached to the N-terminal amino group of the cysteine ​​residue, and the cysteine ​​thiolate group is derivatized with a functional group such as N-ethylmaleimide, a PEG group, or HES-CTP.

[0173] 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, thereby slowing its clearance by renal filtration (see, e.g., Clark et al., J. Biol. Chem. 271:21969-21977 (1996)). It is therefore envisioned that core peptide residues can be PEGylated to enhance therapeutic benefits, such as, for example, increased efficacy by extending in vivo half-life. Thus, PEGylation of a polypeptide will improve the pharmacokinetics and pharmacodynamics of the propeptide domain of the polypeptide.

[0174] 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. al., Protein Expr. Purif. 12:45-52 (1998). Polyethylene glycol or PEG is meant to encompass any form of PEG used to derivatize other proteins, including, but not limited to, mono-(C.sub.1-10) alkoxy or aryloxy-polyethylene glycols. 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)maleimide-propionamide (Dow, Midland, Mich.); 31 kDa alpha-methyl-w-(3-oxopropoxy), polyoxyethylene (NOF Corporation, Tokyo); mPEG.sub.2-NHS-40k (Nektar); mPEG2-MAL-40k (Nektar), SUNBRIGHT GL2-400MA((PEG).sub.240 kDa (NOF Corporation, Tokyo), SUNBRIGHT ME-200MA(PEG20 kDa) (NOF Corporation, Tokyo).PEG groups are generally attached to the LPD polypeptide via acylation, amidation, thioetherification, or reductive alkylation of a reactive group on the polypeptide (e.g., an aldehyde, amino, carboxyl, or thiol group) by a reactive group on the PEG moiety (e.g., an aldehyde, amino, carboxyl, or thiol group).

[0175] The PEG molecule may 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. Optionally, also amino acids with carboxylic acid side chains. The polypeptides described herein can be PEGylated directly to any amino acid at the N-terminus via the N-terminal amino group. To facilitate PEGylation, a "linker arm" can be added to the polypeptide. PEGylation at the thiol side chain of cysteine ​​has been widely reported (see, for example, Caliceti & Veronese, Adv. Drug Deliv. Rev. 55:1261-77 (2003)). If no cysteine ​​residue is present in the polypeptide, a cysteine ​​residue can be introduced by substitution or by adding cysteine ​​to the N-terminal amino acid. Other options include reagents that add thiols to polypeptides, such as Traut's reagent and SATA.

[0176] In certain embodiments, the PEG molecule is branched, while in other embodiments, the PEG molecule may be linear. In certain embodiments, the PEG molecule has a molecular weight between 1 kDa and 150 kDa. More specifically, the PEG molecule has a molecular weight between 1 kDa and 100 kDa. In further embodiments, the PEG molecule is selected from 5, 10, 20, 30, 40, 50, and 60 kDa.

[0177] A useful strategy for PEGylation of polypeptides consists of combining a peptide and a PEG moiety, each having a specific functionality reactive with the other, by forming a conjugate bond in solution. Polypeptides can be readily prepared by recombinant means as described above.

[0178] In one embodiment, PEG is "pre-activated" prior to conjugation to a polypeptide, for example, carboxyl-terminal PEG can be converted to an NHS ester for activation, making it more reactive towards lysines and the N-terminus.

[0179] According to another embodiment, the polypeptide is "preactivated" with a suitable functional group at a specific site. The conjugation of the polypeptide with PEG can be performed in aqueous phase or organic co-solvent and can be easily monitored by SDS-PAGE, isoelectric focusing (IEF), SEC and mass spectrometry. The PEGylated polypeptide is then purified. Small PEGs can be removed by ultrafiltration. Larger PEGs are typically purified using anion, cation or affinity chromatography. The characterization of the PEGylated polypeptide can be performed by analytical HPLC, amino acid analysis, IEF, enzyme activity analysis, electrophoresis, PEG:protein ratio analysis, laser desorption mass spectrometry and electrospray mass spectrometry.

[0180] Removal of excess free PEG can be performed by packing a column (Tricorn Empty High-Performance Columns, GE Healthcare) with POROS 50 HQ support (Applied Biosystems) and then equilibrating the column with equilibration buffer (25 mM Tris-HCl buffer, pH 8.2). The PEGylated polypeptide is loaded onto the equilibrated column, and the column is then washed with 5 CV of equilibration buffer. Under these conditions, the polypeptide binds to the column. The PEGylated polypeptide is eluted in the next step by elution buffer and stored at 2-8 °C for short-term storage or frozen at -20 °C for long-term storage.

[0181] The resulting polypeptide can be anywhere between 40-100 KDa, depending on whether modifications (as above) have been added and on the glycosylation state (as above).

[0182] For example, for Fc fusions: According to a specific embodiment, the polypeptide is between 40 and 70 KDa. According to a specific embodiment, the polypeptide is between 45 and 65 KDa. According to a specific embodiment, the polypeptide is between 50 and 70 KDa. According to a specific embodiment, the polypeptide is 45 KDa. According to a specific embodiment, the polypeptide is 65 KDa.

[0183] According to a specific embodiment, the polypeptide domain of LOX lacking LOX catalytic activity is less than 400 amino acids, for example, 100-200, 100-150, 100-300 amino acids in length.

[0184] The synthetic polypeptides can be used in any method that inhibits the catalytic activity (enzymatic activity) of LOX by simply contacting the LOX with a synthetic polypeptide that contains the modification. Such methods can be performed in vitro, in vivo, or ex vivo.

[0185] The polypeptides of some embodiments of the present invention may be used in treating fibrosis or fibrotic conditions as referred to hereinabove.

[0186] According to specific embodiments, the polypeptides of some embodiments of the present invention are not used (or are not administered) as part of a regimen that includes treatment with D-penicillamine.

[0187] The polypeptide of the present invention or a polynucleotide encoding it can be provided to the treated subject (i.e., mammal) by itself (e.g., purified or directly as part of an expression system) or in a pharmaceutical composition comprising the polypeptide of the present invention. As used herein, a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0188] As used herein, the term "active ingredient" refers to a polypeptide of the invention (or a polynucleotide encoding same) that is accountable for a biological effect.

[0189] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmacologically acceptable carrier", which may be used interchangeably, refer to a carrier or diluent that does not cause significant irritation to an organism and does not abolish the biological activity and properties of the administered compound. These phrases include adjuvants.

[0190] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0191] Techniques for drug formulation and administration can be found in the latest edition of "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., which is incorporated herein by reference.

[0192] Suitable peripheral administration routes may include, for example, oral, rectal, transmucosal, particularly nasal, intraintestinal or parenteral delivery (including intramuscular, subcutaneous and intravenous, intraperitoneal, intranasal or intraocular injection).

[0193] The pharmaceutical compositions of the present invention may be manufactured by processes that are well known in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0194] Thus, pharmaceutical compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and auxiliaries that facilitate processing of the active ingredient into a pharma- ceutically usable preparation. Appropriate formulations will vary depending on the route of administration chosen.

[0195] For injection, the active ingredients of the pharmaceutical composition can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0196] For oral administration, pharmaceutical compositions can be easily formulated by combining the active compounds with pharma- ceutical acceptable carriers well known in the art. Such carriers allow the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by the patient. Pharmacological preparations for oral use can be made using solid excipients, optionally milling the resulting mixture and processing the mixture of granules to obtain tablets or dragee cores, after adding suitable auxiliaries as necessary. Suitable excipients are, in particular, sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carbomethylcellulose, etc.; and / or fillers, such as physiologically acceptable polymers, such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0197] The dragee core is provided with a suitable coating.For this purpose, gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solution, and concentrated sugar solution, which may optionally contain suitable organic solvent or solvent mixture, can be used.Dyes or pigments can be added to the tablet or dragee coating for identification or to characterize different combinations of doses of active compound.

[0198] Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Push-fit capsules may 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 optionally a stabilizer. In soft capsules, the active ingredient may be dissolved or suspended in a suitable liquid such as fatty oils, liquid paraffin, or liquid polyethylene glycol. Additionally, stabilizers may be added. All formulations for oral administration should be in a dosage suitable for the chosen route of administration.

[0199] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0200] For administration by nasal inhalation, the active ingredient for use according to the invention is conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer using a suitable propellant, for example, 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, for example of gelatin, for use in a dispenser can be formulated to contain a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0201] The pharmaceutical compositions described herein can be formulated for parenteral administration, for example, by bolus injection or continuous infusion. Preparations for injection can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, optionally containing preservatives. The compositions can be suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulating agents such as suspending, stabilizing and / or dispersing agents.

[0202] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active ingredient in water-soluble form. Furthermore, suspensions of the active ingredient can be prepared as suitable oil-based or water-based injection suspensions. 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 may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. If necessary, the suspension may also contain suitable stabilizers or agents that increase the solubility of the active ingredient to allow for the preparation of highly concentrated solutions.

[0203] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, a sterile pyrogen-free water-based solution, before use.

[0204] Pharmaceutical compositions of the present 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.

[0205] 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 its intended purpose. More specifically, a therapeutically effective amount means an amount of the active ingredient (nucleic acid construct) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., ischemia) or prolong survival of the subject being treated.

[0206] 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.

[0207] For any preparation used in the methods of the present invention, the therapeutically effective amount or dose can be initially estimated from in vitro and cell culture assays. For example, a dose can be formulated in an animal model to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0208] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures, or in experimental animals. Data obtained from these in vitro and cell culture assays, as well as animal studies, can be used in formulating a range of dosages for use in humans. Dosages can vary depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition. (See, for example, Fingl, et al., 1975, ''The Pharmacological Basis of Therapeutics'', Ch.1 p.1).

[0209] The Examples section below details animal models of DMD and standard tests for determining efficacy.

[0210] Animal models of other fibrotic conditions are known in the art, for example a liver fibrosis model is described in Delire et al. J Clin Transl Hepatol. 2015 Mar;3(1):53-66, Moore et al describe an animal model of fibrotic lung disease, and Rai et al. Mol Cell Biochem. 2017 Jan;424(1-2):123-145. doi:10.1007 / s11010-016-2849-0. describe an animal model of myocardial fibrosis.

[0211] Dosage and intervals can be adjusted individually to provide plasma or brain levels of the active ingredient sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC varies from preparation to preparation but can be estimated from in vitro data. The dosage required to reach the MEC varies with individual characteristics and route. Detection assays can be used to determine plasma concentrations.

[0212] Depending on the severity and responsiveness of the condition to be treated, dosing may be a single or multiple administration, with a course of treatment lasting from several days to several weeks, or until a cure is effected or a diminution of the disease condition is achieved.

[0213] The amount of a composition administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0214] The compositions of the present invention may be presented in a pack or dispenser device, such as a U.S. Food and Drug Administration (FDA) approved kit, which may contain one or more unit dosage forms containing the active ingredient, if desired. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by administration instructions. The pack or dispenser may also be adapted with a notice associated with the container in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects the agency's approval of the composition or form of administration for human or animal use. Such notice may, for example, be on a label approved by the U.S. Food and Drug Administration (FDA) for prescription drugs, or an approved product insert. Compositions comprising the preparations of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of a designated condition, as further detailed above.

[0215] As used herein, the term "about" refers to ±10%.

[0216] The terms "comprises," "comprising," "includes," "including," "having," and combinations thereof mean "including but not limited to."

[0217] The term "consisting of" means "including and limited to."

[0218] The term "consisting essentially of" means that a composition, method, or structure may include additional ingredients, steps, and / or components, but only if the additional ingredients, steps, and / or components do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0219] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include a plurality of compounds, including mixtures thereof.

[0220] Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to specifically disclose all possible subranges as well as each individual number within that range. For example, description of a range such as 1 to 6 should be considered to specifically disclose each individual number within that range, e.g., subranges 1, 2, 3, 4, 5, and 6, as well as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc. This applies regardless of the breadth of the range.

[0221] Whenever a numerical range is given herein, it is meant to include the cited numbers (fractions or integers) within the stated range. The phrases "range between" a first and a second designated number and "range from" a first designated number to a second designated number are used interchangeably herein and are meant to include the first and second designated numbers and all fractions and integers therebetween.

[0222] As used herein, the term "method" refers to manners, means, techniques, and procedures for accomplishing a given task, and includes, but is not limited to, those manners, means, techniques, and procedures known to or readily developed by those skilled in the art of chemistry, pharmacology, biology, biochemistry, and medicine from known manners, means, techniques, and procedures.

[0223] As used herein, the term "treating" includes negating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating the clinical or cosmetic symptoms of a condition, or substantially preventing the appearance of clinical or cosmetic symptoms of a condition.

[0224] When reference is made to a particular sequence listing, such reference shall be understood to also encompass sequences that substantially correspond to the complementary sequence, including minor sequence variations due to, for example, sequencing errors, cloning errors, or other alterations resulting in base substitutions, deletions, or 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.

[0225] It is understood that any sequence identification number (SEQ ID NO:) disclosed in this application can refer to either a DNA sequence or an RNA sequence, depending on the context in which the SEQ ID NO: is referenced, even if the SEQ ID NO: is expressed only in that format.

[0226] It will be understood that certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the invention that are described in the context of a single embodiment may also be provided separately or in any suitable subcombination or as appropriate with any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0227] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims below find experimental support in the following examples. Working Example

[0228] 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.

[0229] In general, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are fully described in the literature, e.g., "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (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. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York. New York (1998); methodologies described in U.S. Patents 4,666,828, 4,683,202, 4,801,531, 5,192,659, and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, JE, ed. (1994); "Culture of Animal Cells-A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), Third Edition; "Current Protocols in Immunology" Volumes 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'', WHFreeman and Co., New York (1980); available immunoassays are described extensively in the patent and scientific literature, e.g., in 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; 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);''A Practical 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.See, "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. Materials and Methods Expression and purification of Fc-LPD

[0230] 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 HEK 293-6E cells in the exponential growth phase using polyethylenimine (PEI) transfection reagent with a DNA-PEI ratio of 1:2. After 120 h 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 elution was performed by 100 mmol / L citrate buffer (pH 3-4.5). Expression and purification of the LOX catalytic domain

[0231] The catalytic domain of human LOX(169-417) of SEQ ID NO:3, i.e. SEQ ID NO:10, was cloned into pET28 expression vector and transformed into competent E. coli BL21 SHuffle strain. A single colony was resuspended in 10 ml of liquid medium supplemented with antibiotics. 10 ml of culture was used to inoculate 1 L of liquid medium. OD 600The culture was incubated at 30 °C until the NA reached 0.4-0.8, after which 0.4 mM IPTG inducer was added. The culture was incubated overnight at 16 °C and 250 rpm. The medium was centrifuged at 4000 rpm for 15 min. The pelleted fraction was incubated on ice with lysis buffer containing 50 mM Tris-HCl (pH 8), 200 mM NaCl, 40 mM imidazole, lysozyme, protein inhibitors and DNase, and then frozen for 30 min. The solution was sonicated and centrifuged at 24,000 rpm for 40 min. The suspension was purified through a 0.22 μM filter and loaded onto a HisTrap column (GE Healthcare) pre-equilibrated with the following buffer: 50 mM Tris-HCl pH 8, 200 mM NaCl, 40 mM imidazole. The 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 a HiLoad 16 / 60 Superdex 75 (Amersham Biosciences) and eluted with 50 mM Tris-HCl pH 8, 200 mM NaCl. Collagen assembly assay

[0232] HDF cells were seeded in 35 mm dishes in DMEM 10% FCS medium. The next day, the cell culture medium was supplemented with the following factors 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) in the second experiment. ECM formation was monitored for 1-2 weeks using two-photon microscopy and second harmonic generation (Figure 2B-C) (see below). ELISA binding assay

[0233] 96-well plates (Nunc) were coated with 10 μg / mL LOX catalytic domain, HSP70 or BSA. After blocking with 2% BSA in PBS, the plates were incubated with Fc and Fc-LPD for 1 h at 37° C. Bound antibodies were detected by peroxidase-conjugated antibody goat anti-human (Jackson ImmunoResearch). EC 50 was calculated using GraphPad Prism from Find ECanything curve fitting analysis. Microscale Thermophoresis (MST)

[0234] LOX (200 nM) (SEQ ID NO: 9) was labeled with NT-647 dye (RED-tris-NTA) using Monolith His-tag Labeling Kit and incubated for 30 min. Increasing concentrations of Fc and Fc-LPD were measured using a Monolith NT.115 (Nanotemper, Germany) with 50% Laser-power, laser on-time 35 sec and 40% LED-power. Differential Scanning Fluorometry (DSF)

[0235] The NanoDSF Prometheus NT.48 measures ultra-high resolution LOX protein stability using endogenous tryptophan or tyrosine fluorescence by continuously heating samples at an adjustable heating rate of 1 °C / min in the range of 20-95 °C, and simultaneously reads both fluorescence and reflectance signals. Immunoprecipitation

[0236] Fc-LPD was covalently coated onto Protein G beads (GE) according to the manufacturer's instructions (GE IP beads). The coated beads were then transferred to 10 cm of HDF cells. 2The beads were incubated overnight at 4°C with concentrated supernatant from dish cultures. Pellet beads were obtained by centrifugation (approximately 500×g, 5 min, 4°C) and the beads were washed with PBS at least 5 times the initial bead volume. The bead pellet was then placed in a collection column, which was inserted into an Eppendorf tube containing 10 ul of 1 M Tris-HCl pH 8.90 μl of elution buffer (Thermo-Scientific) and elution was performed by centrifugation after 5 min of incubation. The last step was repeated 4 times. Mass spectrometry for characterization of Fc-LPD

[0237] Gel bands were subjected to in-gel trypsin digestion followed by a desalting step. The resulting peptides were analyzed using nanoflow liquid chromatography (nanoAcquity) coupled to high-resolution, high-mass accuracy mass spectrometry (Fusion Lumos) using EThcD fragmentation to generate fragmentation of both glycans and peptides. Each sample was analyzed separately in discovery mode in random order. Data were processed with Byonic v2.15.89, a proprietary software capable of processing such data and identifying glycans. Searches were performed against the Uniprot human database, allowing the following modifications: carbamidomethylation of C as fixed modifications, carbamidomethylation of N-terminus, H or K, deamidation of N or Q, Glu or Gln to pyro-Glu, ammonia loss, N-terminal acetylation, formylation of STK, dehydration of ST and N-glycosylation as variable. Data were filtered to allow a maximum false discovery rate (FDR) of 1%. Mass spectrometry to identify gel bands from IP of Fc-LPD

[0238] Gel bands were subjected to in-gel trypsin digestion followed by a desalting step. The resulting peptides were analyzed using nanoflow liquid chromatography (nanoAcquity) coupled to high-resolution, high-mass accuracy mass spectrometry (Q Exactive Plus). Each sample was analyzed on the instrument separately in random order in discovery mode. Data were processed using Proteome Discoverer version 2.2.0.388 searched against the Uniprot human protein database and supplemented with a list of common laboratory contaminants. Searches were performed using two search algorithms, namely SequestHT and Mascot. Two-photon microscopy and second harmonic generation

[0239] Images of the samples were taken using a 2PM:Zeiss LSM 510 META NLO microscope equipped with a broadband Mai Tai-HP-femtosecond single box tunable Ti-sapphire oscillator, using an 855 nm wavelength (detection at 400 nm) with 20x and 25x objectives and automated broadband wavelength tuning from 700 nm to 1,020 nm from Spectra-Physics. ECM thickness was calculated based on the z-stack (um). Image analysis

[0240] Image analysis was performed with the Fiji package

[28] . A Fourier component analysis of directionality was performed on the data using the Fiji plugin "Directionality" written by Jean-Yves Tinevez (pacificdotmpi-cbgdotde / wiki / indexdotphp / Directionality) following the instructions in Fiji. For entropy calculation, the Fiji macro CDS.ijm was used to create the orientation analysis file, after which the average entropy was calculated with a matlab script developed by O. Golani and G. Molodij and modified by E. Shimshoni. Animal testing

[0241] For the initial experiments, male Mdx mice (8 mice / group) approximately 8 weeks old were used. One group was treated with 5 mg / kg Fc, a second group was treated with 5 mg / kg Fc-LPD, and a third group was treated with 5 mg / kg Fc-LPD N346, 362, 409Q by intraperitoneal injection once a week. Every 2 weeks, mice were tested in the rotarod assay. Mice were sacrificed after 3 months and the heart, diaphragm, tibialis anterior, quadriceps, and gastrocnemius muscles were immediately fixed or frozen in OTC.

[0242] For later experiments, male Mdx mice (4 per group) aged approximately 23-25 ​​weeks were used. One group was treated with 5 mg / kg Fc and the other group was treated with 5 mg / kg Fc-LPD by intraperitoneal injection once a week. Every 2 weeks, mice were tested in the rotarod assay and every other week in the limb hanging test. Mice were sacrificed after 3 months and the heart, diaphragm, tibialis anterior and gastrocnemius muscles were either immediately fixed or frozen in OTC. Rotarod running

[0243] Muscle strength, coordination, balance, and condition were determined for the rotarod running assay. Mice were placed on the rotarod tube when the rotarod was rotating at a slow steady speed of 5 rpm. After the mouse was positioned, the speed was accelerated from 5 to 45 rpm during the first 15 seconds, and then the speed was maintained. The running time was continuously recorded by the software and was automatically stopped when the mouse fell off the tube. If the mouse faced the opposite direction on the tube during running, it was repositioned without stopping the rotation of the tube. Hanging test

[0244] The hanging test allows the assessment of balance, coordination and muscle condition. This test was based on the knowledge that the mouse would like to remain hanging from a wire or grid until exhaustion. Specifically, a large cage lid for rats was used. The mouse was approached to the grid by tail handling. The mouse was allowed to grasp the grid with all four paws. The grid was inverted so that the mouse could hang, and a timer was started directly. After the mouse fell off the wire, the timer was stopped and a record of the time spent hanging was marked. histochemistry

[0245] Two different tissue preparation protocols (paraffin embedding and microtome frozen sections) were applied as previously described (Rolls et al., 2008). Slides were exposed to Hoechst stain (1:4,000; Invitrogen Probes) for 1 min. Sirius red staining was performed to label collagen types I and III, reticulin staining for collagen III, and Masson's trichrome staining to distinguish cells from the surrounding connective tissue. A Nikon light microscope (Eclipse E800) equipped with a Nikon digital camera (DS-Ri1) was used for microscopic analysis. Yeast surface display expression

[0246] The construct was amplified and cloned into the pETCON(LPD) plasmid in frame with the AGA2 gene, an N-terminal HA tag, and a C-terminal Myc tag. The construct was then transformed into EBY100 (Saccharomyces cerevisiae) strain and plated on SD-Trp plates for selection. After 48 h of incubation at 30°C, single colonies were subjected to colony PCR. Fragments meeting the expected band sizes were PCR purified and sequenced.

[0247] Construct SD-CAA 143 At 30°C in medium, up to 0.8 OD 600 At that point the cells were pelleted and fresh SG-CAA was added. 143The cells were induced in culture for 38 hours at 15° C. Displayed protein expression was measured by two complementary analyses: flow cytometry and activity assays (for the wt construct).

[0248] 3×10 7 Fully induced cells were pelleted and washed with two successive washes, each consisting of resuspension in 0.8ml PBS+0.5%BSA buffer (wash buffer), centrifugation for 30 seconds and 13kRPM. Washed cells were incubated with α-c-Myc antibody (Santa Cruz 9e10, 1:50 dilution) in 200μl wash buffer for 45 minutes at room temperature and washed twice. Cells were labeled with secondary goat α-mouse IgG (Fc specific)-FITC antibody (Sigma F4143, 1:50 dilution) for an additional 30 minutes in 200μl at 4°C.

[0249] After expression and α-c-Myc / FITC labeling, 3.3×10 6 EBY100 cells were washed twice, supplemented with 200 μl of wash buffer containing biotinylated peptides (30–100 μM) and incubated for 2 h at room temperature. After incubation, cells were washed twice with wash buffer and incubated for another 30 min on ice with 1:100 diluted streptavidin-APC (allophycocyanin-streptavidin, 0.5 mg / ml, Jackson ImmunoResearch) in 100 μl of wash buffer. For LPD-HSP70 interaction, cells were incubated with HSP70 labeled with Atto 633 dye (Sigma 51253) for 30 min at RT. Finally, cells were washed twice, filtered through a 70 μm mesh nylon strainer and diluted with 1.6 ml of wash buffer. These were followed by flow cytometry analysis and data were analyzed by Flow Jo software. statistical analysis

[0250] All analyses were performed at least in triplicate. Statistical analysis was performed using GraphPad Prism 7.

[0251] Materials and Methods for Glycosylation Mutants. Example 1 Expression and characterization of Fc-LPD

[0252] To analyze extracellular LOX activity in DMD and reveal fibrosis during disease progression, we developed protein engineered inhibitors using endogenous inhibitors of LOX. Specifically, an 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 prodomains are not stable and their production faces difficulties, an LPD fused to an Fc tag (SEQ ID NO: 7, encoded by the nucleotide sequence shown in SEQ ID NO: 8) was created to enhance functionality. Importantly, Fc-LPD (SEQ ID NO: 5, encoded by the nucleotide sequence shown in SEQ ID NO: 6) was expressed in dimerization, creating a minimized antibody-like inhibitor with a molecular size of approximately 95 kDa (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 starting the evaluation of functional assays.

[0253] The binding affinity and inhibitory activity of Fc-LPD were evaluated using EC 50 and the dissociation constant (K D The binding affinity was estimated by measuring the EC 50 was measured by ELISA (Figure 1C) in the range of 10 nM to 2000 nM against the LOX catalytic domain (EC 50 = 43 nmol / L). The dissociation constant (K D ) was determined by microscale thermophoresis (Figure 1D), but no interactions were detected with the Fc region of the inhibitor. The ultrahigh-resolution stability of Fc-LPD was measured by differential scanning fluorimetry (DSF) and the transition midpoint was identified at 70 °C, indicating that Fc-LPD is stable in solution (Figures 1F, 1G).

[0254] After purification of Fc-LPD, SDS-page analysis of samples under reduced or non-reduced conditions showed a mixture population. Since LPD is known for three different N-glycosylation sites and expression was performed in a mammalian expression system, the different size bands (45 kDa and 60 kDa) were analyzed by MS. The results showed that N81-glycosylation (N346 in the construct of the present invention) was detected in both bands with multiple glycoforms ranging from 203 to 2790 Da in the 45 kDa Fc-LPD and the 60 kDa band (Figure 1A), but more frequent 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 to 3809 Da. The N97-glycosylation site (N362 in the construct) was detected in both the 45 and 60 kDa bands only after digestion with α-lytic protease (Sigma A6362) ranging from 203 to 2499 Da in the 45 kDa Fc-LPD and the 60 kDa band. Finally, another glycosylation site in the Fc region of both bands was identified, with several glycoforms ranging in size from 1300 to 1800 Da. In Figure 1A, the amino acid positions shift from N81, 97, 144 to N346, 362, 409 due to the presence of Fc (SEQ ID NO: 5). Example 2 Identifying the ability of Fc-LPD to disrupt collagen fibril assembly

[0255] The effect of Fc-LPD on the enzymatic activity of LOX was investigated in an in vitro system using activated human dermal fibroblasts (HDFs). In detail, we developed a screening system to monitor specific events and stages of collagen cross-linking reaction by advanced microscopy tools (Figure 2A). HDF cells were cultured with activation medium containing EGF, insulin and L-ascorbic acid to induce the expression of ECM molecules, mainly collagen. Monitoring the assembly of fibrillar collagen by two-photon SHG microscopy with or without Fc-LPD identified that the newly designed Fc-LPD targets the advanced stages of collagen cross-linking and assembly. These results demonstrate that inhibition of LOX-mediated collagen cross-linking by Fc-LPD alters the orientation of normal collagen fibril alignment in vitro in native fibroblast-derived 3D matrix scaffolds (Figure 2B).

[0256] Furthermore, HDF cells were cultured with ECM activation medium in the presence of Fc, Fc-LPD and Fc-LPD N346, 362, 409Q (5 uM). After 7 days of culture, collagen fibers were monitored by two-photon SHG microscopy and disruption of fiber orientation was observed (Figure 2C). A significant increase in entropy in the system containing Fc-LPD N346, 362, 409Q was demonstrated (Figure 2D). Furthermore, measurements of ECM thickness (z-stacks) highlighted a significant decrease in ECM production in Fc-LPD- and Fc-LPD N346, 362, 409Q-treated cells (Figure 2E). Example 3 Interaction of various Fc-LPD glycosylation forms

[0257] The interaction of Fc-LPD with proteins secreted by HDF was examined by immunoprecipitation experiments using supernatants from HDF cells. For this purpose, HDF cells were cultured at 60 cm 2The cells were cultured in dishes for 3 days. The medium was collected, concentrated using a 10 kDa cutoff vivaspin filter, and incubated separately with 45 kDa Fc-LPD and 60 kDa Fc-LPD, which had previously been semi-separated by gel filtration and incubated with protein G beads (Figure 3A). The samples were analyzed by SDS-page and Western blot. In the SDS-page analysis of the two samples, two high molecular weight bands were detected, which were analyzed by MS (Figure 3B), whereas in the Western blot experiment, LOX (30 kDa) was detected, highlighting the ability of Fc-LPD to bind LOX in physiological conditions in vitro (Figure 3C). Analysis of the MS data showed interactions of both Fc-LPD forms with ECM molecules that are either part of the core matrisome or matrisome-associated. Specifically, among the co-immunoprecipitated proteins were ECM glycoproteins (fibronectin and thrombospondin-1), ECM regulators (serpin B12, A2-macroglobulin), secreted factors (filaggrin-2, hornerin), extracellular domain or secreted proteins (HSP70, a2-HS-glycoprotein, etc.) and other proteins (Table 1 below). These interactions are direct interactions with Fc-LPD. HSP70 was identified as a key interactor, and thus, EC 50 The binding affinity of Fc-LPD and Fc-LPD N346, 362, 409Q was evaluated by estimating the binding affinity by measuring the EC 50 was measured by ELISA (Figure 3D) in the range of 10–2000 nM against HSP70 (EC 50 = 41 nmol / L). Furthermore, the direct binding and high affinity of this interaction was demonstrated by yeast surface display. Yeast cells expressed LPD on the cell surface and labeled HSP70 was added to the solution. After 30 min of incubation at RT, the interaction was measured by flow cytometry, and a significant population of LPD-positive cells was positive for binding to HSP70 (Figure 3E).

[0258] JPEG0007602486000005.jpg123166 Example 4 In vivo assessment of DMD muscle fibrosis following treatment with Fc-LPD and Fc-LPD N346, 362, 409Q

[0259] To test whether a LOX inhibitor can attenuate muscle fibrosis in vivo in a DMD background and therefore whether it could be used as a novel route to treat DMD or other fibrotic diseases, two experiments were performed using mdx mice.

[0260] In the early stage model, 2-month-old male mdx mice were injected once a week with 5 mg / kg of LOX inhibitors (Fc-LPD & Fc-LPD N346, 362, 409Q) or control antibody (Fc) (n=8 per group) (Figure 4A). During treatment, functional muscle assays such as rotarod running were performed, but no significant improvement was observed after treatment with Fc-LPD and Fc-LPD N346, 362, 409Q (Figure 4B). Three months after the start of treatment (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 to monitor native collagen fibers.

[0261] Specifically, H&E staining of diaphragm tissues showed a significant amelioration of the centrally nucleated pathological phenotype observed in DMD pathological tissues after treatment with Fc-LPD and Fc-LPD N346, 362, 409Q (Figure 4C). Furthermore, Sirius Red staining of the diaphragm showed a significant reduction of fibrillar collagen deposition by approximately 15% and approximately 20% in Fc-LPD and Fc-LPD N346, 362, 409Q mice, respectively (Figure 4D-E). Interestingly, in the case of gastrocnemius muscle, significantly fewer centrally nucleated muscle fibers were observed (Figure 4F). Collagen deposition after staining with Sirius Red showed a reduction of approximately 45% in the case of Fc-LPD & Fc-LPD N346, 362, 409Q compared to Fc control (Figure 4G-H). By examining quadriceps muscle tissue, no significant reduction in collagen deposition was observed between anti-LOX and Fc-treated mice (Figure 5A, 5B, 5C). However, further monitoring of fibrillar collagen in quadriceps muscle tissue by two-photon SHG microscopy after treatment with Fc-LPD N346, 362, 409Q showed a significant reduction in collagen fiber signal (about 26%). The reduction in collagen fibers in Fc-LPD N346, 362, 409Q-treated mice was significant (about 34%) when the analysis was performed only in the endomysium of the quadriceps muscle. The last tissue was analyzed in the early stage model by two-photon SHG analysis of the tibialis anterior muscle. Collagen fibers were reduced by about 53% in Fc-LPD-treated mice and about 76% in Fc-LPD N346, 362, 409Q-treated mice compared to control Fc.

[0262] At the later stage, 6-month-old male 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) (Figure 6A). During treatment, functional muscle assays such as rotarod running and hanging test were performed, and significant improvements were observed after Fc-LPD treatment (Figure 6B, 6C). Two months after the start of treatment (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 to monitor native collagen fibers. Finally, histopathological analysis of muscle tissue sections was performed to monitor inflammation, necrosis and mean fiber diameter as a readout of muscle fiber maturity.

[0263] Specifically, Sirius red staining of the tibialis anterior (TA) muscle demonstrated a 50% reduction in fibrillar collagen deposition in LOX-inhibited mice (Figure 6D). iii、iv , 6E, 6G). This staining further revealed that connective tissue (mainly collagen fibers) was diffuse in the control muscle. In contrast, those treated with Fc-LPD were sharper, more defined, and the muscle was better organized as monitored by the circularity ratio (Fig. 6F). Histological evaluation of anterior tibial tissue from mdx mice showed a reduction in inflammation grade (about 25%) and fibrosis score (about 50%). When the assembly of fibrillar collagen in its native unlabeled state was monitored by two-photon SHG microscopy, fewer collagen fiber signals with a more linear orientation were observed after treatment with Fc-LPD. To quantify the changes in directional fiber orientation, Fourier component analysis for directionality was applied to representative images obtained by SHG (Fig. 6D). v、vi , 6H). Furthermore, two-photon SHG images showed thick fibrous bundles in Fc-LPD-treated tissues that were absent in Fc-treated tissues. Collectively, these results demonstrate that treatment of mice with this anti-LOX molecule inhibits the accumulation of fibrosis in a DMD background.

[0264] By examining cardiac tissue, a significant ~50% reduction in cardiac collagen deposition was observed in Fc-LPD treated mice when compared to mice treated with Fc (Figure 7D, 7E). At the start of the experiment (6-7 month old mdx mice), the myocardium and TA muscle were not highly fibrotic, whereas the diaphragm was significantly affected, thus serving as a model to test whether anti-LOX treatment could promote the relaxation of already fibrotic tissue. Sirius red staining of the diaphragm of harvested mice showed a similar pattern to that observed in the myocardium and TA muscle, with a reduction in the amount of collagen deposition observed (Figure 7A, 7C). Similarly, the pathologist report performed suggested a ~20% reduction in fibrosis score (25% in Fc treatment vs. 21% in Fc-LPD treatment). (Figure 7B). Example 5 Analysis of the significance of glycosylation sites for LPD activity Cloning and characterization

[0265] Using the pYD5-Fc-LPD plasmid as a template, point mutations were made at three N-glycosylation sites N81, 97, 144 of LPD (these are N346, 362, 409 in our Fc-LPD construct with QSEQ ID NO: 11). Mutations were performed using site-directed mutagenesis according to the kit manufacturer's instructions. EC of the generated Fc-LPD N346, 362, 409Q inhibitors 50 was measured by ELISA (Figure 1C) in the range of 10–2000 nM against the LOX catalytic domain (EC 50 =58 nmol / L). Immunoprecipitation

[0266] Fc-LPD was coated onto magnetic protein G beads (GE) according to the manufacturer's instructions. The coated beads were then transferred to 10 cm of HDF cells. 2The beads were incubated overnight at 4° C. with concentrated supernatant from dish cultures. Pellet beads were obtained by centrifugation (approximately 500 g, 5 min, 4° C.) and the beads were washed with PBS at least 5 times the initial bead volume. The bead pellet was then placed in a collection column, which was inserted into an Eppendorf tube containing 10 ul of 1 M Tris-HCl pH 8.90 ul of elution buffer (Thermo-Scientific) and elution was performed by centrifugation after 5 min of incubation. The last step was repeated 4 times. Mass spectrometry to identify gel bands from IP of Fc-LPD

[0267] Samples were eluted with 5% SDS and trypsin digested using S-trap. Resulting peptides were analyzed using nanoflow liquid chromatography (nanoAcquity) coupled to high-resolution, high-mass accuracy mass spectrometry (Q Exactive HFX). Each sample was processed with MaxQuant v1.6.0.16. Data were searched using the Andromeda search engine against the human proteome database with common experimental protein contaminants and the following modifications added: carbamidomethylation of C as fixed modification and oxidation of M and protein N-terminal acetylation as variable modifications. LFQ intensities (label-free quantification) were calculated and used for further calculations using Perseus v1.6.07. Decoy hits, as well as proteins identified based only on modified peptides, were excluded. LFQ intensities were log-transformed and only proteins with at least two valid values ​​in at least one experimental group were retained. GO annotations were added. result

[0268] 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 demonstrate the interaction of LPD with ECM proteins in the supernatant of HDF cells, as in Example 3. For this purpose, HDF cells were cultured at 60 cm in 10 ml of serum-free and phenol red-free medium. 2The cells were cultured in dishes for 3 days. The medium was collected, concentrated using a 10 kDa cutoff vivaspin filter, and incubated with Fc, Fc-LPD and Fc-LPD-N346, 362, 409Q, which were preincubated in magnetic protein G beads. The immunoprecipitated eluates were analyzed by mass spectrometry, and analysis of the MS data showed interactions of both Fc-LPD forms with ECM molecules that are either part of the core matrisome or matrisome-associated. Specifically, among the co-immunoprecipitated proteins were ECM glycoproteins (nidogen-1), secreted factors (filaggrin-2, hornerin), extracellular domains or secreted proteins (HSP70, HSP90, HSP60). It is noteworthy that the quantified ratio between Fc-LPD and Fc-LPD-N346, 362, 409Q distinguished between different proteins. For example, the ratio of the unglycosylated form to the Fc control was significantly higher for Nidogen-1 and HSP70 compared to the ratio of glycosylated Fc-LPD to Fc (Table 2 below): JPEG0007602486000006.jpg59159

[0269] While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the present invention is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0270] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. Furthermore, citation or identification of a reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. To the extent section headings are used, they should not be construed as necessarily limiting. Additionally, the priority document(s) of this application are incorporated herein by reference in their entirety. References (Other references are cited throughout this application.) REFERENCES (other references are cited in the application) 1. 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Claims

1. 1. Use of a polypeptide comprising a propeptide of lysyl oxidase (LOX) for the manufacture of a medicament for the treatment of fibrosis in a subject in need thereof, said polypeptide lacking LOX catalytic activity, said polypeptide being as set forth in SEQ ID NO:1 or at least 95% identical thereto, said polypeptide being capable of inhibiting LOX-mediated collagen cross-linking, without administering D-penicillamine to said subject.

2. 1. Use of a polypeptide comprising a propeptide of lysyl oxidase (LOX) for the manufacture of a medicament for the treatment of Duchenne muscular dystrophy (DMD) in a subject in need thereof, wherein the polypeptide lacks LOX catalytic activity, the polypeptide is as set forth in SEQ ID NO:1 or is at least 95% identical thereto, and the polypeptide is capable of inhibiting LOX-mediated collagen cross-linking.

3. The use according to any one of claims 1 to 2, wherein the polypeptide is glycosylated at at least one or two or all of N81, N97 and N144 of SEQ ID NO:

1.

4. The use according to any one of claims 1 to 2, wherein the polypeptide is aglycosylated at N81, N97 and N144 of SEQ ID NO:

1.

5. The use according to claim 1 , wherein the fibrosis is not Ras signaling dependent and / or is not associated with cancer or bone disease.

6. The use according to any one of claims 1 to 5, wherein the polypeptide is as shown in SEQ ID NO: 1 or 5.

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