Cleavable activators of CXCR3 and methods of use thereof

Abstract

Recombinant C-X-C motif chemokine ligand (CXCL) peptides are described that are modified to introduce proteases, such as cleavage sites of proteases that are activated during inflammatory responses. The CXCL peptide has the ability to activate CXCR3 until cleavage by a protease. Proteolytic cleavage of the CXCL peptides minimizes pro-inflammatory responses and inhibits fibrosis progression.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a divisional application with application number 201980069480.8, filing date October 18, 2019, and entitled “Cleavable activators of CXCR3 and methods of use thereof”.

[0003] This application claims the benefit of U.S. Application No. 62 / 748,711, filed October 22, 2018, the entire contents of which are incorporated herein by reference. Technical Field

[0004] The present disclosure relates to peptide activators of CXC chemokine receptor 3 (CXCR3) that can be cleaved by proteases present at sites of inflammation, and methods of using the same, for example, to inhibit fibrosis without causing inflammation. Background Art

[0005] The chemokine receptor CXCR3 is a G protein-coupled receptor in the CXC chemokine receptor family. CXCR3 is primarily expressed on activated T lymphocytes and natural killer (NK) cells. Ligands for CXCR3 include CXC motif chemokine ligand 4 (CXCL4), CXCL9, CXCL10, and CXCL11. Binding of these ligands to CXCR3 produces pleiotropic effects, including antifibrotic effects on adherent cells, and proinflammatory and fibrotic effects on cells of the innate immune system. Activators of CXCR3 can limit fibrosis and angiogenesis when signaling through CXCR3 receptors expressed on fibroblasts, endothelial cells, and other adherent cells. However, activation of CXCR3 on immune cells can promote inflammatory scarring and fibrosis. Summary of the invention

[0006] Described herein are recombinant CXC motif chemokine ligand (CXCL) peptides that are modified to introduce cleavage sites for proteases (e.g., proteases activated during inflammatory responses). The disclosed peptides have the ability to activate CXCR3 until cleaved by proteases. Proteolytic cleavage of CXCL peptides minimizes proinflammatory responses and inhibits fibrosis development.

[0007] Provided herein are recombinant CXCL peptides modified relative to the wild-type CXCL amino acid sequence to introduce a cleavage site for a protease. In some embodiments, CXCL is a ligand for CXC chemokine receptor 3 (CXCR3), such as CXCL10, CXCL4, CXCL9, or CXCL11. In some embodiments, the protease is a cathepsin, an elastase, or a matrix metalloproteinase (MMP).

[0008] Compositions are also provided, which include a recombinant CXCL peptide disclosed herein. The composition can be formulated, for example, for topical, intranasal, inhaled, intravenous, intravitreal, intramuscular, intradermal or subcutaneous administration. In some embodiments, the composition is in unit dosage form.

[0009] Also provided is a method of inhibiting fibrosis in a subject. In some embodiments, the method comprises administering to the subject a CXCL peptide or composition disclosed herein. In some instances, the subject suffers from trauma, autoimmune disease, inflammatory disease or condition, or iatrogenic disease or condition.

[0010] Further provided are methods of inhibiting angiogenesis in a subject. In some embodiments, the method comprises administering to the subject a CXCL peptide or composition disclosed herein. In some instances, the subject suffers from an ocular angiogenic disease.

[0011] The foregoing and other objects, features and advantages of the present invention will become more apparent from the following detailed description made with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1A-1B : Efficacy of CXCL10-derived cleavable peptides in blocking choroidal neovascularization (CNV). The modified peptide 110 (SEQ ID NO: 2) was tested in a mouse model of CNV. Mice were administered vehicle, peptide 110, positive control peptides (peptides 102, 105, or 107), or the corresponding scrambled peptides as controls. The peptides were administered at 1 μg ( Figure 1A ) or 3 μg ( Figure 1B Exp = experimental peptide; CTRL = scrambled control peptide.

[0013] Sequence Listing

[0014] The nucleic acid and amino acid sequences listed in the attached sequence listing are shown using standard letter abbreviations for nucleotide bases and three letter codes for amino acids (as defined in 37 CFR 1.822). Each nucleic acid sequence shows only one strand, but the complementary strand is understood to include the content of any reference to the displayed strand. The sequence listing is submitted as an ASCII text file, created on October 16, 2019, 13.2KB, which is incorporated herein by reference. In the attached sequence listing:

[0015] SEQ ID NO: 1 is the amino acid sequence of the wild-type CXLC10 peptide.

[0016] SEQ ID NO: 2-7 are the amino acid sequences of modified CXCL10 peptides. The C-terminal proline residue of each peptide is optionally amidated or methylated.

[0017] SEQ ID NO: 8 is the amino acid sequence of human CXCL10.

[0018] SEQ ID NO: 9 is the amino acid sequence of human CXCL4.

[0019] SEQ ID NO: 10 is the amino acid sequence of human CXCL9.

[0020] SEQ ID NO: 11 is the amino acid sequence of human CXCL11.

[0021] SEQ ID NO: 12 is the amino acid sequence of the cathepsin G recognition site.

[0022] SEQ ID NO: 13 is the amino acid sequence of the neutrophil elastase recognition site.

[0023] SEQ ID NOs: 14-16 are the amino acid sequences of wild-type CXLC4 peptides.

[0024] SEQ ID NOs: 17-23 are the amino acid sequences of modified CXCL4 peptides.

[0025] SEQ ID NOs: 24-26 are the amino acid sequences of wild-type CXLC11 peptides.

[0026] SEQ ID NOs: 27-34 are the amino acid sequences of modified CXCL11 peptides.

[0027] SEQ ID NO: 35 is the amino acid sequence of the cathepsin K recognition site.

[0028] SEQ ID NO: 36 is the amino acid sequence of the MMP2 recognition site. DETAILED DESCRIPTION

[0029] I. Abbreviations

[0030] BCG Bacillus Calmette-Guerin

[0031] CNV Choroidal neovascularization

[0032] CXCR3 CXC chemokine receptor 3

[0033] CXCL4 Chemokine (CXC motif) ligand 4

[0034] CXCL9 Chemokine (CXC motif) ligand 9

[0035] CXCL10 Chemokine (CXC motif) ligand 10

[0036] CXCL11 Chemokine (CXC motif) ligand 11

[0037] FITC Fluorescein isothiocyanate

[0038] IP-10 Interferon-γ-induced 10 kDa protein

[0039] IPF Idiopathic Pulmonary Fibrosis

[0040] MMP matrix metalloproteinase

[0041] NV neovascularization

[0042] PLGA Poly(lactic-co-glycolic acid)

[0043] II. Terminology and Methodology

[0044] Unless otherwise indicated, technical terms are used according to conventional usage. Definitions of commonly used terms in molecular biology can be found in Benjamin Lewin, Gene X, published by Jones & Bartlett, 2009; and Meyers et al. (eds.), Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH, Volume 16, 2008; and other similar references.

[0045] As used herein, the singular forms "a", "an", and "the" refer to both the singular and the plural, unless the context clearly indicates otherwise. For example, the term "an antigen" includes single or multiple antigens and can be considered equivalent to the phrase "at least one antigen". As used herein, the term "comprises" means "includes". It should be further understood that, unless otherwise indicated, any and all base sizes or amino acid sizes given for nucleic acids or polypeptides, and all molecular weight or molecular mass values ​​are approximate and are provided for descriptive purposes. Although many methods and materials similar or equivalent to those described herein can be used, specific suitable methods and materials are described herein. In the event of a conflict, the present specification (including term explanations) shall prevail. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. In order to facilitate viewing of various embodiments, the following term explanations are provided:

[0046] Administration: Introducing a composition (e.g., a protein or peptide) into a subject via a chosen route. For example, if the chosen route is intravenous, the composition is administered by introducing it into a vein of the subject. Exemplary routes of administration include, but are not limited to, injection (e.g., intraocular, intravitreal, subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, intratubular, sublingual, transdermal, intranasal, topical, inhalation routes, and via medical implants.

[0047] Angiogenesis: The physiological process involving the growth of new blood vessels from pre-existing ones. Angiogenesis is a normal and vital process in growth and development, as well as in wound healing and granulation tissue. However, it is also a fundamental step in the transition of tumors from a dormant to a malignant state, and many other diseases are caused by abnormal angiogenesis. "Abnormal angiogenesis" refers to uncontrolled or pathological angiogenesis present in many different diseases, including ocular diseases, for example, restenosis after glaucoma treatment, wet macular degeneration, diabetic retinopathy, retinopathy of prematurity, or neovascular glaucoma.

[0048] Angiogenic disorder: Any condition, disease or disorder caused by abnormal angiogenesis. Examples of angiogenic disorders include, for example, cancer, diabetic retinopathy, macular degeneration, retinopathy of prematurity, corneal neovascularization, and neovascular glaucoma. The term also includes conditions caused by abnormal pathological angiogenesis resulting from medical intervention, such as restenosis after glaucoma treatment and angiogenesis resulting from corneal transplantation.

[0049] Ocular angiogenic disorders: include any intraocular or extraocular angiogenic disorder. For example, intraocular angiogenic disorders include intraocular disorders such as diabetic retinopathy, wet macular degeneration, retinopathy of prematurity, restenosis after glaucoma treatment, and neovascular glaucoma. Extraocular angiogenic disorders are outside the eye, such as corneal neovascularization.

[0050] Autoimmune disease: A condition in which the immune system produces an immune response (e.g., a B cell or T cell response) to endogenous antigens, resulting in damage to tissues. Autoimmune diseases include, but are not limited to, type 1 diabetes, rheumatoid arthritis, psoriasis, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, and celiac disease.

[0051] Carrier protein: An immunogenic protein that can be linked to another molecule (e.g., a peptide, small molecule, or organic compound) to promote the immunogenicity of that molecule. Examples of carrier proteins include, but are not limited to, bovine serum albumin, ovalbumin, and keyhole limpet hemocyanin.

[0052] Cathepsin: A type of protease. The cathepsin family of proteases includes serine proteases (cathepsin A, cathepsin G), cysteine ​​proteases (cathepsin B, cathepsin C, cathepsin F, cathepsin H, cathepsin K, cathepsin L1, cathepsin L2, cathepsin O, cathepsin S, cathepsin W, cathepsin Z), and aspartyl proteases (cathepsin D, cathepsin E).

[0053] Cathepsin G: A serine protease known to play a role in the elimination of intracellular pathogens and in the breakdown of tissue at sites of inflammation. Cathepsin G is found as stored azurophilic granules in neutrophils and other immune cells.

[0054] Coacervate: A spherical aggregate of colloidal droplets held together by hydrophobic forces. The diameter of the coacervate droplets is typically about 1 to 100 μm.

[0055] Conservative variants: "Conservative" amino acid substitutions are those that do not substantially affect or reduce the activity or antigenicity of a protein or peptide. For example, the peptides disclosed herein may include up to about 1, up to about 2, up to about 3, up to about 4, or up to about 5 conservative substitutions (e.g., 1, 2, 3, 4, or 5 conservative substitutions, and retain biological activity, such as the ability to bind to CXCR3. Specific non-limiting examples of conservative substitutions include the following examples:

[0056]

[0057]

[0058] The term conservative variant also includes the use of substituted amino acids in place of unsubstituted parent amino acids. Non-conservative substitutions are those that reduce activity or antigenicity.

[0059] Corneal neovascularization: Excessive ingrowth of blood vessels from the limbal plexus into the cornea due to insufficient oxygen absorption. One of the most common causes is contact lens wear, especially long-term contact lens wear. Corneal neovascularization is also a common response to eye injury and can also occur after a corneal transplant.

[0060] Chemokine (CXC motif) ligand 4 (CXCL4): A small cytokine that belongs to the CXC chemokine family. CXCL4 is also known as platelet factor 4 (PF4). CXCL4 is a 70 amino acid protein that is released from the α-granules of activated platelets and is highly bound to heparin. Its main physiological role appears to be to neutralize heparin-like molecules on the surface of vascular endothelium, thereby inhibiting local antithrombin III activity and promoting coagulation. As a strong chemoattractant for neutrophils and fibroblasts, CXCL4 is thought to play a role in inflammation and wound repair. CXCL4 is known to bind to the B isoform of CXCR3 (CXCR3-B). The sequence of CXCL4 is publicly available (e.g., see GENBANK). TM Gene ID 5196). An exemplary human CXCL4 sequence is set forth herein as SEQ ID NO:9.

[0061] Chemokine (CXC motif) ligand 9 (CXCL9): A member of the CXC chemokine family. The CXCL9 protein is thought to be involved in T cell trafficking. CXCL9 binds to CXCR3 and is a chemoattractant for lymphocytes but not for neutrophils. The sequence of CXCL4 is publicly available (e.g., see GENBANK TM Gene ID 4283). An exemplary human CXCL4 sequence is set forth herein as SEQ ID NO:10.

[0062] Chemokine (CXC motif) ligand 10 (CXCL10): A ligand for the chemokine of the CXC subfamily and the receptor CXCR3. CXCL10 is also known as interferon-γ-inducible 10 kDa protein (IP-10). Binding of this protein to CXCR3 produces pleiotropic effects, including stimulation of monocyte, natural killer and T cell migration, regulation of adhesion molecule expression and inhibition of angiogenesis. The CXCL10 sequence is publicly available, for example through GENBANK TM (For example, see Gene ID 3627 for human IP-10 sequence; see also GENBANK TM Accession No. P02778). An exemplary human CXCL10 sequence is set forth herein as SEQ ID NO:8.

[0063] Chemokine (CXC motif) ligand 11 (CXCL11): A ligand for the chemokine of the CXC subfamily and the receptor CXCR3. The CXCL11 protein induces a chemotactic response in activated T cells and is the major ligand for CXCR3. The gene encoding this protein includes 4 exons and at least 3 polyadenylation signals, which may reflect cell-specific expression regulation. IFN-γ is a potent inducer of transcription of this gene. The sequence of CXCL11 is publicly available (see, for example, GENBANK TM Gene ID 6373). An exemplary human CXCL11 sequence is set forth herein as SEQ ID NO:11.

[0064] CXCR3 (CXC Chemokine Receptor 3): A G protein-coupled receptor that is selective for the four chemokines CXCL4 / PF4 (platelet factor 4), CXCL9 / Mig (monokine induced by interferon-gamma), CXCL10 / IP-10 (interferon-gamma-induced protein of 10 kDa), and CXCL11 / I-TAC (interferon-inducible T-cell alpha-chemoattractant). Binding of chemokines to this protein induces cellular responses associated with leukocyte trafficking, most notably integrin activation, cytoskeletal changes, and chemotactic migration. Spliced ​​transcript variants encoding different isoforms of this gene have been discovered. One isoform (CXCR3-B) shows high affinity binding to the chemokine CXCL4.

[0065] Diabetic retinopathy: A condition in which the retina is damaged as a complication of diabetes. Proliferative retinopathy usually occurs in the late stages of the disease and is characterized by the abnormal formation of new blood vessels on the surface of the vitreous, extending into the vitreous cavity.

[0066] Elastase: A serine protease that breaks down elastin. Elastases include chymotrypsin-like elastase, chymotrypsin elastase, neutrophil elastase, and macrophage elastase. Neutrophil elastase is able to break down bacterial membrane proteins and virulence factors.

[0067] Fibrosis: A disease associated with the thickening and scarring of connective tissue. Fibrosis usually occurs in response to injury, such as from a disease or condition that damages tissue. Fibrosis is a severe wound healing response that can interfere with normal organ function. Fibrosis can occur in almost any tissue in the body, including the lungs (pulmonary fibrosis, cystic fibrosis, radiation lung injury), liver (cirrhosis, biliary atresia), heart (arterial fibrosis, endomyocardial fibrosis, previous myocardial infarction), brain, skin (scleroderma, sclerosis), kidneys, joints, and intestines (Crohn's disease).

[0068] Glaucoma: An eye condition in which the optic nerve is damaged, permanently impairing vision in the affected eye and, if untreated, progressing to complete blindness. It is usually associated with increased pressure of the fluid in the eye (aqueous humor).

[0069] Hydrogel: A high molecular weight polymer gel composed of a network of cross-linked polymer chains.

[0070] Iatrogenic disease or condition: A disease or condition caused by a medical or diagnostic procedure. Exemplary iatrogenic diseases / conditions include drug-induced (e.g., bleomycin-induced) pulmonary fibrosis, Bacillus Calmette-Guérin (BCG) treatment-induced bladder fibrosis, and chemotherapy-induced bladder fibrosis.

[0071] Inflammatory disease or condition: A disease or condition characterized by inflammation. Examples include, but are not limited to, idiopathic pulmonary fibrosis, allergies, asthma, autoimmune diseases, celiac disease, hepatitis, inflammatory bowel disease, reperfusion injury, and transplant rejection.

[0072] Macular degeneration: A condition that causes the macula to atrophy or degenerate. Age-related macular degeneration is the leading cause of vision loss in the elderly. There are two different forms of macular degeneration, called the dry and wet forms. In atrophic macular degeneration (dry form), there is pigmentation in the macular area, but there is no increase in macular scarring, bleeding or exudation in the macular area. In contrast, in exudative macular degeneration (wet form), a subretinal network of choroidal neovascularization forms.

[0073] Matrix Metalloproteinases (MMPs): Calcium-dependent, zinc-containing endopeptidases. MMPs are capable of breaking down components of the extracellular matrix and are also known to play a role in the cleavage of cell surface receptors, the release of apoptotic ligands (eg, FAS ligands), and the inactivation of chemokines / cytokines.

[0074] Neovascular Glaucoma: A type of glaucoma that is very difficult to treat. This condition is usually caused by proliferative diabetic retinopathy or central retinal vein occlusion. Neovascular glaucoma can also be triggered by other conditions that cause ischemia in the retina or ciliary body. People who have poor blood flow to the eye are most likely to develop this disease. Neovascular glaucoma occurs when new, abnormal blood vessels begin to develop in the corner of the eye and begin to block drainage. People with this condition begin to lose vision very quickly. Sometimes, this disease appears very quickly, especially after cataract surgery.

[0075] Atypical amino acid: An amino acid that is not one of the twenty amino acids directly encoded by a triplet codon in the genetic code. Atypical amino acids are also called "nonstandard" amino acids.

[0076] Peptide or polypeptide: A polymer in which the monomers are amino acid residues linked together by amide bonds. When the amino acid is an α-amino acid, either the L-optical isomer or the D-optical isomer can be used, with the L-isomer being preferred. The terms "polypeptide", "peptide" or "protein" as used herein are intended to encompass any amino acid sequence, and include modified sequences, such as glycoproteins. The terms "polypeptide" and "peptide" are particularly intended to encompass naturally occurring proteins as well as recombinant or synthetically produced proteins. In some embodiments, the length of the peptide is 10 to 200 amino acids, including 10 to 100, 10 to 50, 10 to 30, 15 to 50, 15 to 30 or 18 to 25 amino acids in length. In a specific example, the length of the peptide is about 21 or about 22 amino acids. "Residue" refers to an amino acid or amino acid mimetic incorporated into a polypeptide by an amide bond or an amide bond mimetic.

[0077] Pharmaceutically acceptable carrier: The pharmaceutically acceptable carrier used is conventional. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, PA, 15th Edition, 1975 describes compositions and preparations suitable for drug delivery of the peptides disclosed herein. Generally, the nature of the carrier will depend on the specific mode of administration adopted. For example, parenteral preparations generally include injections that include pharmaceutically and physiologically acceptable fluids, such as water, saline, balanced salt solutions, dextrose aqueous solutions, glycerol, etc. as solvents. For solid compositions (e.g., powders, pills, tablets or capsule forms), conventional non-toxic solid carriers may include, for example, pharmaceutical grade mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may also include a small amount of non-toxic adjuvants, such as wetting agents or emulsifiers, preservatives and pH buffers, such as sodium acetate or sorbitan monolaurate. For topical application to the eye, the agent may be mixed with, for example, artificial tears and other emulsions.

[0078] Poly(lactic-co-glycolic acid) (PLGA): A biodegradable and biocompatible copolymer of glycolic acid and lactic acid.

[0079] Protease: An enzyme that hydrolyzes (breaks down) proteins and peptides.

[0080] Restenosis: The reoccurrence of stenosis, narrowing of the vessel, resulting in restricted blood flow. Stenosis (or restenosis) is a form of response to injury that results in thickening of the walls, narrowing of the lumen, and loss of tissue function provided by a particular channel. Physical injury during interventional procedures (e.g. glaucoma surgery) results in damage to the epithelial lining of the tubes. Tissue repair following physical injury involves both regeneration (replacing damaged cells with cells of the same type) and fibrosis (replacing damaged cells with connective tissue). The fibrotic process involves, among other events, the formation of new blood vessels (angiogenesis).

[0081] Retinopathy of prematurity: An eye disease that affects premature babies. It is thought to be caused by disturbed growth of blood vessels in the retina, which can lead to scarring and retinal detachment. The disease can be mild and resolve spontaneously, but in severe cases it can lead to blindness.

[0082] Sequence identity: The similarity between amino acid sequences is expressed as the similarity between the sequences, also known as sequence identity. Sequence identity is often measured as a percentage identity (or similarity or homology). The higher the percentage, the more similar the two sequences are. Homologues or variants of a particular polypeptide will have a higher degree of sequence identity when aligned using standard methods.

[0083] Methods of sequence alignment for comparison are well known in the art. Various programs and alignment algorithms are described in Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988. In addition, Altschul et al., Nature Genet. 6:119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations.

[0084] The NCBI Base Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from a variety of sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and the Internet, and is used in conjunction with the blastp, blastn, blastx, tblastn, and tblastx sequence analysis programs. Instructions on how to use this program to determine sequence identity are provided on the NCBI website on the Internet.

[0085] Use NCBI Blast 2.0, blastp of the gap is set to default parameters, and the amino acid sequence of polypeptide is compared, and the homologue and variant of polypeptide are usually characterized by having at least about 75% in total length, for example, at least about 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity. In order to compare the amino acid sequence greater than about 30 amino acids, adopt Blast 2 sequence function, use the default BLOSUM62 matrix (gap existence cost is 11, each residue gap cost is 1) set to default parameters. When comparing short peptides (less than 30 amino acids), Blast 2 sequence function should be used to compare, adopt and set PAM30 matrix to default parameters (space 9, extension position 1 penalty). When evaluated by this method, the protein with higher similarity to the reference sequence will show higher and higher identity percentage, for example at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity. When comparing sequence identities of less than the entire sequence, homologues and variants typically have at least 80% sequence identity within a short window of 10-20 amino acids, and may have at least 85% or at least 90% or 95% sequence identity (depending on their similarity to the reference sequence). Methods for determining sequence identity over such short windows can be found on the NCBI website on the Internet. Those skilled in the art will appreciate that these sequence identity ranges are for guidance only. It is entirely possible to obtain very important homologues beyond the ranges provided.

[0086] Subject: Living multicellular vertebrates, including humans and the species of veterinary subjects (including human and non-human mammals).

[0087] Therapeutically effective amount: The amount of a given agent (eg, a CXCL peptide) sufficient to achieve the desired effect in a subject, cell, or culture to be treated with the agent.

[0088] III. Overview of Several Implementation Methods

[0089] Activators of the cell surface G protein-coupled receptor CXCR3 can limit or even reverse fibrosis and angiogenesis through these receptors in fibroblasts, endothelial cells, pericytes, and other adherent cells. Activation of CXCR3 can both block migration through m-calpain inhibition and induce anoikis in endothelial cells through cleavage of β3 integrin by μ-calpain. Both effects can be exploited to limit scarring and prevent or reverse vascular lesions, including those of the eye. The effects of CXCR3 activators are more pronounced than those of promoters of fibrosis and angiogenesis.

[0090] Cells that mount an immune response also express CXCR3 receptors, but respond in a pro-migratory manner toward these ligands. This creates a potentially confounding situation in which CXCR3 activators may limit immediate scarring through effects on adherent cells (e.g., fibroblasts, endothelial cells, and epithelial cells) while promoting late scarring by attracting and activating immune cells (leukocytes, lymphocytes, macrophages, etc.). The actual outcome depends on the quantitative balance of these two cell populations. In the setting of acute and ongoing inflammation, the risk of a late pro-inflammatory response is highest.

[0091] To eliminate this risk, activators (ligands) of CXCR3 are described herein that can be inactivated by cleavage of extracellular proteases present during active inflammation. Inflammatory scarring is achieved by the production of proteases that degrade the static matrix to replace it with scar matrix. These proteases include a variety of cathepsins and elastases. Using CXCR3 ligands that are sensitive to CXCR3, these ligands are sensitive to cleavage by these proteases, and thus undesirable proinflammatory signals can be eliminated because the ligand is inactivated in the presence of acute inflammation.

[0092] The disclosed peptides can be used to treat fibrotic and angiogenic diseases, including scars, vascular diseases, scleroderma, and autoimmune fibrosis. These diseases and conditions can occur in any organ of the body, such as the skin, lungs, liver, kidneys, heart, and eyes. The diseases also include those caused by trauma, autoimmune pathology, diabetes, and diseases of unknown etiology, such as age-related wet macular degeneration (AMD) and idiopathic pulmonary fibrosis (IPF).

[0093] Provided herein are recombinant CXC motif chemokine ligand (CXCL) peptides that are modified relative to the wild-type CXCL amino acid sequence to introduce a cleavage site for a protease. In some embodiments, CXCL is a ligand for CXC chemokine receptor 3 (CXCR3). In some instances, CXCL is CXCL10, CXCL4, CXCL9, or CXCL11. In a specific non-limiting example, CXCL is CXCL10.

[0094] In some embodiments, the protease is a protease that is activated during an inflammatory response (e.g., an acute inflammatory response). In some instances, the protease is a cathepsin, an elastase, or a matrix metalloproteinase (MMP). In a particular instance, the cathepsin is cathepsin G or cathepsin K. In other particular instances, the MMP is MMP2. In other particular instances, the elastase is a neutrophil elastase.

[0095] In some embodiments, the peptide is about 12 to about 30 amino acids in length, such as about 18 to about 25 amino acids in length, such as about 20 to about 23 amino acids in length, such as about 21 or 22 amino acids in length. In some examples, the peptide is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length.

[0096] In some embodiments, the amino acid sequence of the peptide is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 2-7, 17-23, and 27-34. In some examples, the amino acid sequence of the peptide comprises or consists of any one of SEQ ID NOs: 2-7, 17-23, and 27-34.

[0097] In some embodiments, the peptide includes a C-terminal proline that is amidated or methylated.

[0098] In some embodiments, the peptide includes at least one chemical modification. For example, a chemical modification can be introduced to inhibit the degradation of the peptide and / or increase the half-life of the peptide. In some instances, at least one modification is included in the modification of the N-terminus of the peptide, the modification of the C-terminus of the peptide, or both. In a specific non-limiting example, the modification at the N-terminus includes formylation, acetylation, propionylation, pyroglutamic acid formation, myristoylation, palmitoylation, S-palmitoylation, monomethylation, dimethylation, trimethylation, or any combination thereof. In other specific non-limiting examples, the modification at the C-terminus includes methylation, α-amidation, or a combination thereof. In other embodiments, at least one modification includes non-standard peptide connection.

[0099] In some embodiments, the recombinant CXCL peptide includes at least one D-amino acid. In some examples, the CXCL peptide includes multiple D-amino acids, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 D-amino acids.

[0100] In some embodiments, the recombinant CXCL peptide includes at least one atypical amino acid. In some instances, at least one atypical amino acid is a modified atypical amino acid. The peptide may include a modified atypical amino acid at the N-terminus of the peptide, at the C-terminus of the peptide, or both. In a specific non-limiting example, the peptide includes a modified atypical amino acid at the N-terminus, and the modification includes formylation, acetylation, propionylation, pyroglutamate formation, myristoylation, palmitoylation, S-palmitoylation, monomethylation, dimethylation, trimethylation, or any combination thereof. In other specific non-limiting examples, the peptide includes a modified atypical amino acid at the C-terminus, and the modification includes methylation, α-amidation, or a combination thereof.

[0101] In some embodiments, at least one atypical amino acid is a methylated amino acid, an amino acid conjugated to a polyethylene glycol polymer, an amino acid conjugated to biotin, an amino acid conjugated to fluorescein isothiocyanate (FITC), an amino acid conjugated to a carrier protein, a radioactive isotope labeled amino acid, or any combination thereof. In some instances, the methylated amino acid is a monomethylated amino acid, a dimethylated amino acid, or a trimethylated amino acid. In some instances, the carrier protein is bovine serum albumin, ovalbumin, or keyhole limpet hemocyanin. In some examples, the radioactive isotope is 2 H. 15 N. 13 C or 15 N and 13 C Both

[0102] In some embodiments, the recombinant CXCL peptide is in a sustained release formulation. In some examples, the sustained release formulation includes poly(lactic-co-glycolic acid) (PLGA), a hydrogel, or a coacervate.

[0103] Also provided herein is a composition comprising a recombinant CXCL peptide disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the composition is formulated for topical, intranasal, inhaled, intravenous, intravitreal, intramuscular, intradermal or subcutaneous administration. In some instances, the composition is formulated for delivery to the lungs by atomization or atomizer, for example for the treatment of cystic fibrosis. In other instances, the composition is formulated for delivery to the bladder by catheter and instillation. Fibrosis is a major complication of early bladder cancer treatment and chronic bladder infection, leading to irritable bladder syndrome. Therefore, compositions formulated for delivery to the bladder can be used to treat these conditions.

[0104] In some embodiments, the composition is provided in unit dosage form.

[0105] In some embodiments, the composition may include protease inhibitors, preservatives, tonicity agents, buffers, pH regulators, sterile solvents, or any combination thereof. In some embodiments, protease inhibitors may include but are not limited to TIMP1, odanacatib, calpain, batimastat, ilomastat, or any combination thereof. In some embodiments, tonicity agents may include but are not limited to isotonic buffers, such as sodium chloride. In some embodiments, buffers may include but are not limited to phosphoric acid, acetic acid, citric acid, phosphate buffers, histidine, tromethamine, gluconic acid, lactic acid, tartaric acid, aspartic acid, glutamic acid, tartaric acid, succinic acid, malic acid, fumaric acid, alpha-ketoglutaric acid, or a combination thereof. In some embodiments, pH regulators may include but are not limited to hydrochloric acid. In some embodiments, sterile solvents may include but are not limited to sterile water. In some embodiments, the composition may also include stabilizers, such as but not limited to trehalose. In some embodiments, the composition may also include surfactants, such as polysorbates.

[0106] Further provided herein is a method for inhibiting fibrosis in a subject. In some embodiments, the method includes administering a CXCL peptide or composition disclosed herein to a subject. In some instances, the subject suffers from trauma, autoimmune disease, inflammatory disease or condition, or iatrogenic disease or condition. In a specific instance, the autoimmune disease is diabetes, scleroderma, or autoimmune fibrosis. In other specific instances, the inflammatory disease or condition is idiopathic pulmonary fibrosis (IPF). In other specific instances, the iatrogenic disease or condition is drug-induced (e.g., bleomycin-induced) pulmonary fibrosis, bacillus Calmette-Guérin (BCG) treatment-induced bladder fibrosis, or chemotherapy-induced bladder fibrosis.

[0107] Also provided herein is a method of inhibiting angiogenesis in a subject. In some embodiments, the method comprises administering to a subject a CXCL peptide or composition disclosed herein. In some instances, the subject suffers from an ocular angiogenic disorder. In a specific instance, the ocular angiogenic disorder is wet macular degeneration, diabetic retinopathy, retinopathy of prematurity, restenosis after glaucoma treatment, neovascular glaucoma, or corneal neovascularization.

[0108] IV. Peptide Sequence

[0109] The present disclosure describes modified peptides derived from CXCL proteins that serve as ligands for CXCR3. The disclosed peptides are about 12 to about 30 amino acids in length, such as about 18 to about 25 amino acids in length, such as about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29 or about 30 amino acids in length. The peptide is modified to include a protease cleavage site, thereby inactivating the peptide in the presence of a protease that recognizes the bound cleavage site. In some instances, the cleavage site is a cleavage site for a cathepsin (e.g., cathepsin G or cathepsin K), an elastase (e.g., neutrophil elastase) or a MMP (e.g., MMP2). A person skilled in the art is able to determine appropriate protease cleavage sites, for example with the aid of online tools such as the ExPASy bioinformatics resource portal or the protease specificity prediction server (PROSPER; see also Song et al., PLoS One 7(11):e50300, 2012).

[0110] Those skilled in the art are able to identify and introduce suitable amino acid substitutions that result in protease cleavage sites. For example, it is known that cathepsin G recognizes - / V / L / LHF S / -S / A / V (SEQ ID NO: 12), known cathepsin K recognition - / - / LPV / EA GE / - / - / - (SEQ ID NO: 35), and MMP2 is known to recognize - / P / - / - LI / - / - / - (SEQ ID NO: 36) (Song et al., PLoS One 7(11): e50300, 2012); it is known that neutrophil elastase can recognize FIRW (SEQ ID NO: 13) (Schulenburg et al., Analyst 141(5): 1645-1648, 2016).

[0111] In some embodiments, the peptide is derived from human CXCL10, set forth herein as SEQ ID NO: 8. The peptide may consist of any portion of CXCL10 comprising about 18 to about 25 contiguous amino acids of SEQ ID NO: 8, wherein one or more amino acids are substituted to introduce a cleavage site for a selected protease, wherein the modified peptide (in the absence of the protease) retains the ability to activate CXCR3.

[0112] CXCL10(GENBANK TMAccession No. NP_001565.3; SEQ ID NO: 8)

[0113] MNQTAILICCLIFLTLSGIQGVPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIAT

[0114] MKKKGEKRCLNPESKAIKNLLKAVSKERSKRSP

[0115] In some embodiments herein, the CXCL10 peptide is based on the C-terminal peptide:

[0116] PESKAIKNLLKAVSKERSKRSP(WT; SEQ ID NO:1)

[0117] SEQ ID NO: 1 is modified to introduce a protease cleavage site, such as a cathepsin G, neutrophil cathepsin, or neutrophil elastase cleavage site. Non-limiting examples of modified peptides based on human CXCL10 are provided below.

[0118] Exemplary peptides with cathepsin G and neutrophil elastase cleavage site sequences:

[0119] PESKAIKNLLKAV H KE M SKRSP (peptide 110; SEQ ID NO: 2)

[0120] _ESKAIKNLLKAV H KE M SKRSP (peptide 112; SEQ ID NO: 3)

[0121] PESKAIKNLLKAV H KERSKRSP (peptide 116; SEQ ID NO:4)

[0122] _ESKAIKNLLKAV H KERSKRSP (peptide 117; SEQ ID NO:5)

[0123] Peptide with neutrophil cathepsin cleavage site:

[0124] PESKAIKNLLKAVS EVL SKRSP (peptide 121; SEQ ID NO: 6)

[0125] Peptides with neutrophil elastase cleavage site:

[0126] PES FIRW NLLKAVSKE M SKRSP (peptide 131; SEQ ID NO: 7)

[0127] For the peptides of SEQ ID NOs: 2, 3 and 7, substitution of arginine (R) to methionine (M) was not associated with mutations that introduced a protease cleavage site.

[0128] In some embodiments, the peptide is derived from human CXCL4, set forth herein as SEQ ID NO: 9. The peptide may consist of any portion of CXCL4 that includes about 18 to about 25 contiguous amino acids of SEQ ID NO: 9. One or more amino acids are substituted to introduce a cleavage site for a selected protease, wherein the modified peptide (in the absence of the protease) retains the ability to activate CXCR3.

[0129] CXCL4 (GENBANK TM Accession No. NP_002610.1; SEQ ID NO:9)

[0130] MSSAAGFCASRPGLLFLGLLLLPLVVAFASAEAEEDGDLQCLCVKTTSQVRPRHITSLEVI

[0131] KAGPHCPTAQLIATLKNGRKICLDLQAPLYKKIIKKLLES

[0132] In some embodiments, the modified CXCL4 peptide is based on one of the following CXCL4 peptides:

[0133] DLQCLCVKTTSQVRPRHITSLEVIKAGPH(SEQ ID NO:14)

[0134] PLYKKIIKKLLES (SEQ ID NO: 15)

[0135] LDLQAPLYKKIIKKLLES(SEQ ID NO:16)

[0136] SEQ ID NO: 14, 15 or 16 is modified to introduce a protease cleavage site, such as a cathepsin K or matrix metalloproteinase 2 (MMP2) cleavage site. Non-limiting examples of modified peptides based on human CXCL4 are provided below.

[0137] Exemplary peptides with cathepsin K cleavage site sequences:

[0138] DLQCLCVKTTSQVRPRHITSLE GIKAGPH (SEQ ID NO: 17)

[0139] DLQCLCVKTTSQVRPRHITSLE E IKAGPH (SEQ ID NO: 18)

[0140] PLYKKIIKKL E ES (SEQ ID NO: 19)

[0141] LDLQAPLYKKIIKKL E ES (SEQ ID NO: 20)

[0142] Exemplary peptides with MMP2 cleavage site sequences:

[0143] DLQCLCVKTTSQVRPR L ITSLEVIKAGPH (SEQ ID NO: 21)

[0144] PLY P KIIKKLLES (SEQ ID NO: 22)

[0145] LDLQAPLY P KIIKKLLES (SEQ ID NO: 23)

[0146] In some embodiments, the peptide is derived from human CXCL9, set forth herein as SEQ ID NO: 10. The peptide can consist of any portion of CXCL9 comprising about 18 to about 25 contiguous amino acids of SEQ ID NO: 10, wherein one or more amino acids are substituted to introduce a cleavage site for a selected protease, wherein the modified peptide (in the absence of the protease) retains the ability to activate CXCR3.

[0147] CXCL9 (GENBANK TM Accession No. NP_002407.1; SEQ ID NO: 10)

[0148] MKKSGVLFLLGIILLVLIGVQGTPVVRKGRCSCISTNQGTIHLQSLKDLKQFAPSPSCEKIE

[0149] IIATLKNGVQTCLNPDSADVKELIKKWEKQVSQKKKQKNGKKHQKKKVLKVRKSQRSR

[0150] QKKTT

[0151] In some embodiments, the peptide is derived from human CXCL11, set forth herein as SEQ ID NO: 11. The peptide can consist of any portion of CXCL11 comprising about 18 to about 25 consecutive amino acids of SEQ ID NO: 11, wherein one or more amino acids are substituted to introduce a cleavage site for a selected protease, wherein the modified peptide (in the absence of the protease) retains the ability to activate CXCR3.

[0152] CXCL11 (GENBANK TM Accession No. NP_005400.1; SEQ ID NO: 11)

[0153] MSVKGMAIALAVILCATVVQGFPMFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKI

[0154] EVIITLKENKGQRCLNPKSKQARLIIKKVERKNF

[0155] In some embodiments, the modified CXCL1 peptide is based on one of the following CXCL11 peptides:

[0156] PKSKQARLIIKKVERKNF(SEQ ID NO:24)

[0157] LNPKSKQARLIIKKVERKNF(SEQ ID NO:25)

[0158] DKIEVIITLKENKGQR(SEQ ID NO:26)

[0159] SEQ ID NO: 24, 25 or 26 may be modified to introduce a protease cleavage site, such as a cathepsin K or MMP2 cleavage site. Non-limiting examples of modified peptides based on human CXCL11 are provided below.

[0160] Exemplary peptides with cathepsin K cleavage site sequences:

[0161] PKSKQARLIIKKVEEKNF(SEQ ID NO:27)

[0162] PKSKQARLIIKKVEGKNF(SEQ ID NO:28)

[0163] LNPKSKQARLIIKKVEEKNF(SEQ ID NO:29)

[0164] LNPKSKQARLIIKKVEGKNF(SEQ ID NO:30)

[0165] DKIEVIITLEENKGQR(SEQ ID NO:31)

[0166] Exemplary peptides with MMP2 cleavage site sequences:

[0167] PKSKPARLIIKKVERKNF(SEQ ID NO:32)

[0168] LNPKSKPARLIIKKVERKNF(SEQ ID NO:33)

[0169] DKPEVIITLKENKGQR(SEQ ID NO:34)

[0170] V. Exemplary Embodiments

[0171] 1. A recombinant CXC motif chemokine ligand (CXCL) peptide, wherein the peptide is modified relative to the wild-type CXCL amino acid sequence to introduce a protease cleavage site.

[0172] 2. The recombinant CXCL peptide according to embodiment 1, wherein the CXCL is a ligand of CXC chemokine receptor 3 (CXCR3).

[0173] 3. The recombinant CXCL peptide according to embodiment 1 or embodiment 2, wherein the CXCL is CXCL10, CXCL4, CXCL9 or CXCL11.

[0174] 4. The recombinant CXCL peptide according to any one of embodiments 1-3, wherein the protease is a cathepsin, an elastase or a matrix metalloproteinase (MMP).

[0175] 5. The recombinant CXCL peptide according to embodiment 4, wherein the cathepsin is cathepsin G.

[0176] 6. The recombinant CXCL peptide according to embodiment 4, wherein the cathepsin is cathepsin K.

[0177] 7. The recombinant CXCL peptide according to embodiment 4, wherein the elastase is neutrophil elastase.

[0178] 8. The recombinant CXCL peptide according to embodiment 4, wherein the MMP is MMP2.

[0179] 9. The recombinant CXCL peptide of any one of embodiments 1-8, wherein the peptide is about 12 to about 30 amino acids in length.

[0180] 10. The recombinant CXCL peptide of any one of embodiments 1-9, wherein the peptide is about 18 to about 25 amino acids in length.

[0181] 11. The recombinant CXCL peptide of any one of embodiments 1-10, wherein the peptide is 21 or 22 amino acids in length.

[0182] 12. The recombinant CXCL peptide according to any one of embodiments 1-11, wherein the amino acid sequence of the peptide is at least 90% identical to any one of SEQ ID NOs: 2-7, 17-23 and 27-34.

[0183] 13. The recombinant CXCL peptide according to any one of embodiments 1-11, wherein the amino acid sequence of the peptide is at least 95% identical to any one of SEQ ID NOs: 2-7, 17-23 and 27-34.

[0184] 14. The recombinant CXCL peptide according to any one of embodiments 1-11, wherein the amino acid sequence of the peptide comprises or consists of any one of SEQ ID NOs: 2-7, 17-23 and 27-34.

[0185] 15. The recombinant CXCL peptide according to any one of embodiments 1-14, wherein the amino acid sequence of the peptide comprises or consists of SEQ ID NO: 2.

[0186] 16. The recombinant CXCL peptide of any one of embodiments 1-15, wherein the peptide comprises at least one chemical modification.

[0187] 17. The recombinant CXCL peptide according to embodiment 16, wherein the at least one chemical modification comprises a modification at the N-terminus of the peptide, a modification at the C-terminus of the peptide, or both.

[0188] 18. The recombinant CXCL peptide of embodiment 17, wherein the modification at the N-terminus comprises formylation, acetylation, propionylation, pyroglutamate formation, myristoylation, palmitoylation, S-palmitoylation, monomethylation, dimethylation, trimethylation, or any combination thereof.

[0189] 19. The recombinant CXCL peptide according to embodiment 17 or embodiment 18, wherein the modification at the C-terminus comprises methylation, α-amidation or a combination thereof.

[0190] 20. The recombinant CXCL peptide of embodiment 16, wherein the at least one chemical modification comprises a non-standard peptide linkage.

[0191] 21. The recombinant CXCL peptide of any one of embodiments 1-20, comprising at least one D-amino acid.

[0192] 22. The recombinant CXCL peptide of any one of embodiments 1-21, comprising at least one atypical amino acid.

[0193] 23. The recombinant CXCL peptide of embodiment 22, wherein the at least one atypical amino acid is a modified atypical amino acid.

[0194] 24. The recombinant CXCL peptide of embodiment 23, wherein the peptide comprises a modified atypical amino acid at the N-terminus of the peptide, at the C-terminus of the peptide, or both.

[0195] 25. The recombinant CXCL peptide of embodiment 24, wherein the peptide comprises a modified atypical amino acid at the N-terminus, and the modification comprises formylation, acetylation, propionylation, pyroglutamate formation, myristoylation, palmitoylation, S-palmitoylation, monomethylation, dimethylation, trimethylation, or any combination thereof.

[0196] 26. The recombinant CXCL peptide of embodiment 24 or embodiment 25, wherein the peptide comprises a modified atypical amino acid at the C-terminus, and the modification comprises methylation, α-amidation, or a combination thereof.

[0197] 27. The recombinant CXCL peptide of embodiment 22, wherein the at least one atypical amino acid is a methylated amino acid, an amino acid conjugated to a polyethylene glycol polymer, an amino acid conjugated to biotin, an amino acid conjugated to fluorescein isothiocyanate (FITC), an amino acid conjugated to a carrier protein, an amino acid labeled with a radioisotope, or any combination thereof.

[0198] 28. The recombinant CXCL peptide according to any one of embodiments 1-27 in a sustained release formulation.

[0199] 29. The recombinant CXCL peptide of embodiment 28, wherein the sustained release formulation comprises poly(lactic-co-glycolic acid) (PLGA), a hydrogel, or a coacervate.

[0200] 30. A composition comprising the recombinant CXCL peptide of any one of embodiments 1-29 and a pharmaceutically acceptable carrier.

[0201] 31. The composition of embodiment 30, formulated for topical, intranasal, inhaled, intravenous, intravitreal, intramuscular, intradermal or subcutaneous administration.

[0202] 32. The composition of embodiment 30, formulated for delivery to the lungs by atomization or nebulizer.

[0203] 33. The composition of embodiment 30, formulated for delivery to the bladder via catheter and instillation.

[0204] 34. The composition of any one of embodiments 30-33, which is in unit dosage form.

[0205] 35. A method of inhibiting fibrosis in a subject, the method comprising administering to the subject the CXCL peptide of any one of embodiments 1-29 or the composition of any one of embodiments 30-34.

[0206] 36. The method of embodiment 35, wherein the subject suffers from trauma, an autoimmune disease, an inflammatory disease or condition, or an iatrogenic disease or condition.

[0207] 37. The method of embodiment 36, wherein the autoimmune disease is diabetes, scleroderma, or autoimmune fibrosis.

[0208] 38. The method of embodiment 36, wherein the inflammatory disease or condition is idiopathic pulmonary fibrosis (IPF).

[0209] 39. The method of embodiment 36, wherein the iatrogenic disease or condition is drug-induced pulmonary fibrosis, Bacillus Calmette-Guérin (BCG) treatment-induced bladder fibrosis, or chemotherapy-induced bladder fibrosis.

[0210] 40. A method of inhibiting angiogenesis in a subject, the method comprising administering to the subject a CXCL peptide of any one of embodiments 1-29 or a composition of any one of embodiments 30-34.

[0211] 41. The method of embodiment 40, wherein the subject suffers from an ocular angiogenic disorder.

[0212] 42. The method of embodiment 41, wherein the ocular angiogenic disorder is wet macular degeneration, diabetic retinopathy, retinopathy of prematurity, restenosis following glaucoma treatment, neovascular glaucoma, or corneal neovascularization.

[0213] In addition, the present application also relates to the implementation methods of the following items.

[0214] 1. A recombinant CXC motif chemokine ligand (CXCL) peptide, wherein the peptide is modified relative to the wild-type CXCL amino acid sequence to introduce a protease cleavage site.

[0215] 2. The recombinant CXCL peptide according to item 1, wherein the CXCL is a ligand of CXC chemokine receptor 3 (CXCR3).

[0216] 3. The recombinant CXCL peptide according to item 1, wherein the CXCL is CXCL10, CXCL4, CXCL9 or CXCL11.

[0217] 4. The recombinant CXCL peptide according to item 1, wherein the protease is a cathepsin, an elastase or a matrix metalloproteinase (MMP).

[0218] 5. The recombinant CXCL peptide according to item 4, wherein the cathepsin is cathepsin G.

[0219] 6. The recombinant CXCL peptide according to item 4, wherein the cathepsin is cathepsin K.

[0220] 7. The recombinant CXCL peptide according to item 4, wherein the elastase is neutrophil elastase.

[0221] 8. The recombinant CXCL peptide according to item 4, wherein the MMP is MMP2.

[0222] 9. The recombinant CXCL peptide according to item 1, wherein the peptide has a length of about 12 to about 30 amino acids.

[0223] 10. The recombinant CXCL peptide according to item 1, wherein the peptide is about 18 to about 25 amino acids in length.

[0224] 11. The recombinant CXCL peptide according to item 1, wherein the peptide is 21 or 22 amino acids in length.

[0225] 12. The recombinant CXCL peptide according to item 1, wherein the amino acid sequence of the peptide is at least 90% identical to any one of SEQ ID NOs: 2-7, 17-23 and 27-34.

[0226] 13. The recombinant CXCL peptide according to item 1, wherein the amino acid sequence of the peptide is at least 95% identical to any one of SEQ ID NOs: 2-7, 17-23 and 27-34.

[0227] 14. The recombinant CXCL peptide according to item 1, wherein the amino acid sequence of the peptide comprises or consists of any one of SEQ ID NOs: 2-7, 17-23 and 27-34.

[0228] 15. The recombinant CXCL peptide according to item 1, wherein the amino acid sequence of the peptide comprises or consists of SEQ ID NO: 2.

[0229] 16. The recombinant CXCL peptide according to item 1, wherein the peptide comprises at least one chemical modification.

[0230] 17. The recombinant CXCL peptide according to item 16, wherein the at least one chemical modification comprises a modification at the N-terminus of the peptide, a modification at the C-terminus of the peptide, or both.

[0231] 18. The recombinant CXCL peptide of claim 17, wherein the modification at the N-terminus comprises formylation, acetylation, propionylation, pyroglutamate formation, myristoylation, palmitoylation, S-palmitoylation, monomethylation, dimethylation, trimethylation or any combination thereof.

[0232] 19. The recombinant CXCL peptide according to item 17, wherein the modification at the C-terminus comprises methylation, α-amidation or a combination thereof.

[0233] 20. The recombinant CXCL peptide of claim 16, wherein the at least one chemical modification comprises a non-standard peptide linkage.

[0234] 21. The recombinant CXCL peptide according to item 1, comprising at least one D-amino acid.

[0235] 22. The recombinant CXCL peptide according to item 1, comprising at least one atypical amino acid.

[0236] 23. The recombinant CXCL peptide according to item 22, wherein the at least one atypical amino acid is a modified atypical amino acid.

[0237] 24. A recombinant CXCL peptide according to item 23, wherein the peptide comprises a modified atypical amino acid at the N-terminus of the peptide, at the C-terminus of the peptide, or both.

[0238] 25. A recombinant CXCL peptide according to claim 24, wherein the peptide comprises a modified atypical amino acid at the N-terminus, and the modification comprises formylation, acetylation, propionylation, pyroglutamate formation, myristoylation, palmitoylation, S-palmitoylation, monomethylation, dimethylation, trimethylation or any combination thereof.

[0239] 26. A recombinant CXCL peptide according to item 24, wherein the peptide comprises a modified atypical amino acid at the C-terminus, and the modification comprises methylation, α-amidation or a combination thereof.

[0240] 27. A recombinant CXCL peptide according to claim 22, wherein the at least one atypical amino acid is a methylated amino acid, an amino acid conjugated to a polyethylene glycol polymer, an amino acid conjugated to biotin, an amino acid conjugated to fluorescein isothiocyanate (FITC), an amino acid conjugated to a carrier protein, an amino acid labeled with a radioisotope, or any combination thereof.

[0241] 28. The recombinant CXCL peptide according to item 1 in a sustained release formulation.

[0242] 29. The recombinant CXCL peptide according to item 28, wherein the sustained release formulation comprises poly(lactic-co-glycolic acid) (PLGA), a hydrogel or a coacervate.

[0243] 30. A composition comprising the recombinant CXCL peptide of item 1 and a pharmaceutically acceptable carrier.

[0244] 31. A composition according to item 30, which is formulated for topical, intranasal, inhalation, intravenous, intravitreal, intramuscular, intradermal or subcutaneous administration.

[0245] 32. A composition according to item 30, formulated for delivery to the lungs by atomization or nebulizer.

[0246] 33. A composition according to item 30, formulated for delivery to the bladder via catheter and instillation.

[0247] 34. The composition according to item 30, which is in unit dosage form.

[0248] 35. A method of inhibiting fibrosis in a subject, the method comprising administering the CXCL peptide of item 1 to the subject.

[0249] 36. A method according to claim 35, wherein the subject suffers from trauma, an autoimmune disease, an inflammatory disease or condition, or an iatrogenic disease or condition.

[0250] 37. A method according to claim 36, wherein the autoimmune disease is diabetes, scleroderma or autoimmune fibrosis.

[0251] 38. A method according to claim 36, wherein the inflammatory disease or condition is idiopathic pulmonary fibrosis (IPF).

[0252] 39. A method according to claim 36, wherein the iatrogenic disease or condition is drug-induced pulmonary fibrosis, Bacillus Calmette-Guérin (BCG) treatment-induced bladder fibrosis, or chemotherapy-induced bladder fibrosis.

[0253] 40. A method of inhibiting angiogenesis in a subject, the method comprising administering the CXCL peptide of item 1 to the subject.

[0254] 41. A method according to claim 40, wherein the subject suffers from an ocular angiogenic disorder.

[0255] 42. The method of claim 41, wherein the ocular angiogenic disorder is wet macular degeneration, diabetic retinopathy, retinopathy of prematurity, restenosis following glaucoma treatment, neovascular glaucoma, or corneal neovascularization.

[0256] The following examples are provided to illustrate certain specific features and / or embodiments. These examples should not be construed as limiting the disclosure to the specific features or embodiments described.

[0257] Example

[0258] Example 1: Modified peptides susceptible to protease cleavage

[0259] This example describes peptides engineered to be susceptible to cleavage by extracellular proteases of acute inflammatory responses.

[0260] Previous studies have shown that linear peptide fragments of natural ligands of CXCR3 (e.g., CXCL10) are able to bind and activate CXCR3 (see U.S. Pat. Nos. 9,180,167, 9,452,200, and 9,872,889, the entire contents of which are incorporated herein by reference). Chemokines that signal through CXCR3 can induce both anti-fibrotic effects on adherent cells and pro-inflammatory and fibrotic effects on cells of the innate immune system. In order to limit the fibrotic effects of CXCR3 activators during inflammatory responses, peptides that are easily cleaved by proteases during acute inflammatory responses were designed. Therefore, when an inflammatory response is induced, the engineered peptides are cleaved, thereby limiting their pro-fibrotic effects.

[0261] Peptide 110 (SEQ ID NO: 2) is a modified fragment of human CXCL10 (also known as IP-10). Compared to the wild-type human sequence (SEQ ID NO: 1), peptide 110 contains a substitution of arginine for methionine and a substitution of serine residues with histidine. The latter substitution results in the introduction of cathepsin G and neutrophil elastase cleavage sites. The substituted residues are indicated in bold underline:

[0262] Human sequence: PESKAIKNLLKAVSKERSKRSP (SEQ ID NO: 1)

[0263] Peptide 110: PESKAIKNLLKAV H KE M SKRSP (SEQ ID NO:2)

[0264] Since the substitution of histidine for serine is a non-conservative amino acid change, experiments were performed to confirm that the engineered peptides retained the ability to block angiogenesis and fibrosis in a mouse model of choroidal neovascularization (CNV).

[0265] Laser-induced CNV

[0266] As previously described, mouse CNV was induced by laser photocoagulation-induced rupture of Bruch's membrane. Briefly, 7- to 8-week-old female C57BL / 6J mice were anesthetized with ketamine hydrochloride (100 mg / kg body weight) and the pupils were dilated with 1% tropicamide. Three burns of 532-nm diode laser photocoagulation (spot size, 75 mm; duration, 0.1 sec; power, 120 mW) were performed on each retina using a slit-lamp delivery system of the OcuLight GL diode laser (Iridex, Mountain View, CA), and the retina was viewed using a cover glass as a contact lens. Burns were performed at the 9, 12, and 3 o'clock positions of the posterior pole of the retina. The generation of bubbles during laser application (indicating rupture of Bruch's membrane) is an important factor in the acquisition of choroidal neovascularization (NV), therefore, only burns that produced bubbles were included in this study.

[0267] Immediately after laser treatment, 1 μg ( Figure 1A ) or 3 μg ( Figure 1B ) of peptide 110 (SEQ ID NO: 2) or one of three positive control peptides (peptide 102, peptide 105 or peptide 107), and the contralateral eye was injected with scrambled peptide as a control. Other control mice were injected with only the vehicle. After injection, the cornea was protected with antibiotic ointment. Seven days after laser treatment, the mice were euthanized. The eyes were removed and fixed in 10% PBS-buffered formalin for 3 hours. The choroid was dissected and placed in a 1.5 ml Eppendorf tube and stained with FITC-conjugated GSA-lectin IB4 (1:150, Vector, Frederick, MD) and rotated overnight at 4°C. After washing 3 times with PBST, the choroid was fixed on a slide and examined by fluorescence microscopy. Images were digitized using a three-color CCD camera and an image acquisition card. The total area of ​​choroidal NV at each rupture site was measured using image analysis software (Image-Pro Plus; MediaCybernetics, Silver Spring, MD).

[0268] like Figure 1A-1B As shown, peptide 110 retained its anti-angiogenic activity as evidenced by the significant reduction in choroidal neovascularization at both doses of the peptide.

[0269] Example 2: CXCR3 Activating Peptides with Engineered Protease Cleavage Sites

[0270] This example describes additional peptides based on the sequence of human CXCL10, human CXCL4, or human CXCL11 that are designed to contain one or more cleavage sites for proteases that are activated during acute inflammation.

[0271] CXCL10-derived peptides with cathepsin G and neutrophil elastase cleavage sites:

[0272] PESKAIKNLLKAV H KEMSKRSP (peptide 110; SEQ ID NO: 2)

[0273] _ESKAIKNLLKAV H KEMSKRSP (peptide 112; SEQ ID NO: 3)

[0274] PESKAIKNLLKAV H KERSKRSP (peptide 116; SEQ ID NO:4)

[0275] _ESKAIKNLLKAV H KERSKRSP (peptide 117; SEQ ID NO:5)

[0276] CXCL10-derived peptides with a neutrophil cathepsin cleavage site:

[0277] PESKAIKNLLKAVS EVL SKRSP (peptide 121; SEQ ID NO: 6)

[0278] CXCL10-derived peptides with a neutrophil elastase cleavage site:

[0279] PES FIRW NLLKAVSKEMSKRSP (peptide 131; SEQ ID NO:7)

[0280] Peptide CXCL4-derived peptide with cathepsin K cleavage site sequence:

[0281] DLQCLCVKTTSQVRPRHITSLE G IKAGPH (SEQ ID NO: 17)

[0282] DLQCLCVKTTSQVRPRHITSLE E IKAGPH (SEQ ID NO: 18)

[0283] PLYKKIIKKL E ES (SEQ ID NO: 19)

[0284] LDLQAPLYKKIIKKL E ES (SEQ ID NO: 20)

[0285] CXCL4-derived peptide with MMP2 cleavage site sequence:

[0286] DLQCLCVKTTSQVRPR L ITSLEVIKAGPH (SEQ ID NO: 21)

[0287] PLY P KIIKKLLES (SEQ ID NO: 22)

[0288] LDLQAPLY P KIIKKLLES (SEQ ID NO: 23)

[0289] CXCL11-derived peptide with cathepsin K cleavage site sequence:

[0290] PKSKQARLIIKKVE E KNF (SEQ ID NO: 27)

[0291] PKSKQARLIIKKVE G KNF (SEQ ID NO: 28)

[0292] LNPKSKQARLIIKKVE E KNF (SEQ ID NO: 29)

[0293] LNPKSKQARLIIKKVE G KNF (SEQ ID NO:30)

[0294] DKIEVIITL E ENKGQR (SEQ ID NO:31)

[0295] CXCL11-derived peptide with MMP2 cleavage site sequence:

[0296] PKSK P ARLIIKKVERKNF (SEQ ID NO:32)

[0297] LNPKSK P ARLIIKKVERKNF (SEQ ID NO:33)

[0298] DKP EVIITLKENKGQR (SEQ ID NO: 34)

[0299] Given that the principles of the disclosed subject matter can be applied to many possible embodiments, it should be recognized that the illustrated embodiments are merely preferred examples of the disclosure and should not be considered to limit the scope of the disclosure. Instead, the scope of the disclosure is defined by the following claims. We therefore claim as our invention all that falls within the scope and spirit of these claims.

Claims

1. Use of a recombinant CXC motif chemokine ligand (CXCL) peptide in the preparation of a medicament for inhibiting fibrosis in a subject or inhibiting angiogenesis in a subject, wherein the peptide comprises a modification relative to the wild-type CXCL amino acid sequence, and wherein the modification is a cleavage site for a protease.

2. The use according to claim 1, wherein the use is for inhibiting fibrosis wherein the subject suffers from trauma, an autoimmune disease, an inflammatory disease or condition, or an iatrogenic disease or condition.

3. The use according to claim 2, in: The autoimmune disease is diabetes, scleroderma or autoimmune fibrosis; The inflammatory disease or disorder is idiopathic pulmonary fibrosis (IPF); or The iatrogenic disease or condition is drug-induced pulmonary fibrosis, Bacillus Calmette-Guérin (BCG) treatment-induced bladder fibrosis, or chemotherapy-induced bladder fibrosis.

4. The use according to claim 1, wherein the use is for inhibiting angiogenesis and the subject suffers from an ocular angiogenic disorder.

5. The use according to claim 4, wherein the ocular angiogenic disorder is wet macular degeneration, diabetic retinopathy, retinopathy of prematurity, restenosis after glaucoma treatment, neovascular glaucoma or corneal neovascularization.

6. Use according to any one of claims 1 to 5, wherein the protease is a cathepsin, an elastase or a matrix metalloproteinase (MMP). The use according to claim 6 , wherein the cathepsin is cathepsin G or cathepsin K.

8. The use according to claim 6, wherein the elastase is neutrophil elastase.

9. The use according to claim 6, wherein the MMP is MMP2.

10. The use according to any one of claims 1 to 5, wherein the peptide is about 12 to about 30 amino acids in length.

11. Use according to claim 10, wherein the peptide has a length of about 18 to about 25 amino acids.

12. The use according to claim 10, wherein the peptide is 21 or 22 amino acids in length.

13. The use according to any one of claims 1 to 5, wherein the amino acid sequence of the peptide is at least 90% identical to any one of SEQ ID NOs: 2-7, 17-23 and 27-34.

14. The use according to any one of claims 1 to 5, wherein the amino acid sequence of the peptide comprises or consists of any one of SEQ ID NOs: 2-7, 17-23 and 27-34.

15. The use according to any one of claims 1 to 5, wherein the amino acid sequence of the peptide comprises or consists of SEQ ID NO:

2.

16. The use according to any one of claims 1 to 5, wherein the peptide comprises at least one chemical modification.

17. The use according to claim 16, wherein the at least one chemical modification comprises a modification at the N-terminus of the peptide, a modification at the C-terminus of the peptide, or both.

18. The use according to claim 17, wherein the modification at the N-terminus comprises formylation, acetylation, propionylation, pyroglutamate formation, myristoylation, palmitoylation, S-palmitoylation, monomethylation, dimethylation, trimethylation or any combination thereof.

19. The use according to claim 17, wherein the modification at the C-terminus comprises methylation, α-amidation or a combination thereof.

20. The use according to any one of claims 1 to 5, wherein the CXCL peptide is a sustained-release preparation.

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