Cleavable activators of CXCR3 and methods of use

Abstract

To provide cleavable activators of CXCR3 and methods of use.SOLUTION: Disclosed is a recombinant C-X-C motif chemokine ligand (CXCL) peptide modified to introduce a cleavage site for a protease (e.g., a protease that is activated during an inflammation response). The CXCL peptide has an ability to activate CXCR3 until the protease cleaves the peptide. The proteolytic cleavage of the CXCL peptide minimizes pro-inflammatory response and inhibits the development of fibrosis. The CXCL may be CXCL10, CXCL4, CXCL9 or CXCL11.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 62 / 748,711, filed October 22, 2018, which is incorporated by reference in its entirety.

[0002] Field 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 their use (eg, to inhibit fibrosis without causing inflammation). [Background technology]

[0003] background 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. CXCR3 ligands 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 profibrotic effects on cells of the innate immune system. CXCR3 activators can limit fibrosis and angiogenesis when signaling through the CXCR3 receptor 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 [Means for solving the problem]

[0004] Abstract Described herein are recombinant C-X-C motif chemokine ligand (CXCL) peptides that are modified to introduce a cleavage site for a protease (a protease activated during an inflammatory response). The disclosed peptides have the ability to activate CXCR3 until they are degraded by the protease. Proteolytic cleavage of the CXCL peptide minimizes the proinflammatory response and inhibits the development of fibrosis.

[0005] Provided herein are recombinant CXCL peptides that are modified relative to the wild-type CXCL amino acid sequence to introduce a protease cleavage site. In some embodiments, the CXCL is a ligand of CXC chemokine receptor 3 (CXCR3) (e.g., CXCL10, CXCL4, CXCL9, or CXCL11). In some embodiments, the protease is a cathepsin, elastase, or matrix metalloproteinase (MMP).

[0006] Also provided are compositions comprising the recombinant CXCL peptides disclosed herein. The compositions can be formulated for, for example, topical, intranasal, inhaled, intravenous, intravitreal, intramuscular, intradermal, or subcutaneous administration. In some embodiments, the compositions are in unit dosage form.

[0007] Also provided are methods for inhibiting fibrosis in a subject. In some embodiments, the methods comprise administering to the subject a CXCL peptide or composition disclosed herein. In some examples, the subject has a wound, an autoimmune disease, an inflammatory disease or disorder, or an iatrogenic disease or disorder.

[0008] Also provided are methods for inhibiting angiogenesis in a subject. In some embodiments, the methods comprise administering to the subject a CXCL peptide or composition disclosed herein. In some examples, the subject has an ocular neovascular disorder.

[0009] The foregoing and other objects, features, and advantages of the present invention will become more apparent from the following detailed description which proceeds with reference to the accompanying drawings. [Brief explanation of the drawings]

[0010] [Figure 1A] 1A-1B: Efficacy of CXCL10-derived cleavable peptides in blocking choroidal neovascularization (CNV). Modified peptide 110 (SEQ ID NO: 2) was tested in a mouse model of CNV. Mice were administered vehicle, peptide 110, a positive control peptide (peptide 102, 105, or 107), or the corresponding scrambled peptide as a control. Peptides were administered at a dose of 1 μg (FIG. 1A) or 3 μg (FIG. 1B). Exp = experimental peptide; CTRL = scrambled control peptide. [Figure 1B] 1A-1B: Efficacy of CXCL10-derived cleavable peptides in blocking choroidal neovascularization (CNV). Modified peptide 110 (SEQ ID NO: 2) was tested in a mouse model of CNV. Mice were administered vehicle, peptide 110, a positive control peptide (peptide 102, 105, or 107), or the corresponding scrambled peptide as a control. Peptides were administered at a dose of 1 μg (FIG. 1A) or 3 μg (FIG. 1B). Exp = experimental peptide; CTRL = scrambled control peptide. DETAILED DESCRIPTION OF THE INVENTION

[0011] Sequence Listing The nucleic acid sequences and amino acids listed in the attached sequence listing are shown using standard abbreviations for nucleotide bases and three-letter codes for amino acids, as specified in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the strand shown. The sequence listing is submitted as an ASCII text file (created October 16, 2019, 13.2 KB, incorporated herein by reference). In the attached sequence listing:

[0012] SEQ ID NO: 1 is the amino acid sequence of the wild-type CXLC10 peptide.

[0013] SEQ ID NOs: 2 to 7 are the amino acid sequences of modified CXCL10 peptides. The C-terminal proline residue of each peptide is amidated or methylated as necessary.

[0014] SEQ ID NO: 8 is the amino acid sequence of human CXCL10.

[0015] SEQ ID NO: 9 is the amino acid sequence of human CXCL4.

[0016] SEQ ID NO: 10 is the amino acid sequence of human CXCL9.

[0017] SEQ ID NO: 11 is the amino acid sequence of human CXCL11.

[0018] SEQ ID NO: 12 is the amino acid sequence of the cathepsin G recognition site.

[0019] SEQ ID NO: 13 is the amino acid sequence of the neutrophil elastase recognition site.

[0020] SEQ ID NOs: 14 to 16 are the amino acid sequences of wild-type CXLC4 peptides.

[0021] SEQ ID NOs: 17 to 23 are the amino acid sequences of modified CXCL4 peptides.

[0022] SEQ ID NOs: 24 to 26 are the amino acid sequences of the wild-type CXLC11 peptide.

[0023] SEQ ID NOs: 27 to 34 are the amino acid sequences of modified CXCL11 peptides.

[0024] SEQ ID NO: 35 is the amino acid sequence of the cathepsin K recognition site.

[0025] SEQ ID NO: 36 is the amino acid sequence of the MMP2 recognition site.

[0026] Detailed Description I. Abbreviations BCG Bacillus Calmette-Guerin CNV choroidal neovascularization CXCR3 CXC chemokine receptor 3 CXCL4 chemokine (CXC motif) ligand 4 CXCL9 chemokine (CXC motif) ligand 9 CXCL10 chemokine (CXC motif) ligand 10 CXCL11 chemokine (CXC motif) ligand 11 FITC Fluorescein isothiocyanate IP-10 Interferon-γ-inducible 10kDa protein IPF Idiopathic Pulmonary Fibrosis MMP matrix metalloproteinase NV neovascularization PLGA Poly(lactic-co-glycolic acid)

[0027] II. Terminology and Methods Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in: Benjamin Lewin, Genes X, published by Jones & Bartlett Publishers, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in 16 volumes, 2008; and other similar references.

[0028] 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 a 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 any and all base or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and, unless otherwise indicated, are provided for illustrative purposes. Although many methods and materials similar or equivalent to those described herein can be used, particularly suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. In order to facilitate review of the various embodiments, the following explanations of terms are provided:

[0029] Administration: The introduction of a composition (e.g., a protein or peptide) into a subject by a selected route. For example, if the selected route is intravenous, the composition is administered by introducing the composition into the subject's vein. Exemplary administration routes include, but are not limited to, injection (e.g., intraocular, intravitreal, subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, intraductal, sublingual, transdermal, intranasal, topical, inhalation, and via medical implant.

[0030] Angiogenesis: The physiological process involving the growth of new blood vessels from pre-existing vessels. 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 disorders result from aberrant angiogenesis. "Aberrant angiogenesis" refers to uncontrolled or pathological angiogenesis that is present in many different diseases, including ocular disorders such as restenosis after glaucoma treatment, wet macular degeneration, diabetic retinopathy, retinopathy of prematurity, or neovascular glaucoma.

[0031] Angiogenic disorder: Any condition, disease, or disorder resulting from abnormal angiogenesis. Examples of angiogenic disorders include, for example, cancer, diabetic retinopathy, macular degeneration, retinopathy of prematurity, corneal neovascularization, and neovascular glaucoma. This term also includes conditions resulting from abnormal pathological angiogenesis resulting from medical intervention (e.g., restenosis after glaucoma treatment and angiogenesis resulting from corneal transplantation).

[0032] Ocular angiogenic disorder: includes any intraocular or extraocular angiogenic disorder. For example, intraocular angiogenic disorders include disorders inside the eye (e.g., diabetic retinopathy, wet macular degeneration, retinopathy of prematurity, restenosis after glaucoma treatment, and neovascular glaucoma). External ocular angiogenic disorders are located outside the eye (e.g., corneal neovascularization).

[0033] Autoimmune disease: A disorder in which the immune system mounts an immune response (e.g., a B cell or T cell response) against 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 (lupus), inflammatory bowel disease, Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, and celiac disease.

[0034] Carrier protein: An immunogenic protein that can be attached to another molecule (e.g., a peptide, small molecule, or organic compound) to enhance the molecule's immunogenicity. Examples of carrier proteins include, but are not limited to, bovine serum albumin, ovalbumin, and keyhole limpet hemocyanin.

[0035] 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).

[0036] Cathepsin G: A serine protease known to play a role in eliminating intracellular pathogens and degrading tissue at sites of inflammation. Cathepsin G is a stored azurophil granule found in neutrophils and other immune cells.

[0037] Coacervate: A spherical aggregate of colloidal droplets held together by hydrophobic forces. Coacervate droplets are typically about 1-100 μm in diameter.

[0038] Conservative variant: "Conservative" amino acid substitutions are those substitutions that do not substantially affect or reduce the activity or antigenicity of a protein or peptide. For example, the peptides disclosed herein may contain at most about 1, at most about 2, at most about 3, at most about 4, or at most about 5 conservative substitutions (e.g., 1, 2, 3, 4, or 5 conservative substitutions) and retain biological activity (e.g., the ability to bind to CXCR3). Specific non-limiting examples of conservative substitutions include the following: [Table 1-1] [Table 1-2]

[0039] The term conservative variant also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid. Non-conservative substitutions are those that reduce activity or antigenicity.

[0040] Corneal neovascularization: Excessive ingrowth of blood vessels from the limbal vascular plexus into the cornea caused by hypoxia. One of the most common causes is contact lens wear, especially prolonged contact lens wear. Corneal neovascularization is also a common response to ocular injury and can occur after corneal transplantation.

[0041] Chemokine (C-X-C motif) ligand 4 (CXCL4): A small cytokine belonging to the C-X-C chemokine family. CXCL4 is also known as platelet factor 4 (PF4). CXCL4 is a 70-amino acid protein released from the alpha granules of activated platelets and binds with high affinity to heparin. Its primary physiological role appears to be the neutralization of heparin-like molecules on the endothelial surface of blood vessels, thereby inhibiting local antithrombin III activity and promoting coagulation. As a potent chemoattractant for neutrophils and fibroblasts, CXCL4 is thought to play a role in inflammation and wound repair. CXCL4 is known to bind the B isoform of CXCR3 (CXCR3-B). The sequence of CXCL4 is publicly available (e.g., GENBANK TM (See Gene ID 5196.) An exemplary human CXCL4 sequence is set forth herein as SEQ ID NO:9.

[0042] Chemokine (C-X-C motif) ligand 9 (CXCL9): A member of the C-X-C 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 neutrophils. The sequence of CXCL4 is publicly available (e.g., GENBANK TM (See Gene ID 4283.) An exemplary human CXCL4 is set forth herein as SEQ ID NO:10.

[0043] Chemokine (C-X-C motif) ligand 10 (CXCL10): A chemokine of the C-X-C subfamily and a ligand for the receptor CXCR3. CXCL10 is also known as interferon-gamma-inducible 10 kDa protein (IP-10). Binding of this protein to CXCR3 produces pleiotropic effects, including stimulation of monocyte, natural killer cell, and T-cell migration, modulation of adhesion molecule expression, and inhibition of angiogenesis. The CXCL10 sequence is publicly available (e.g., GENBANKTM (e.g., see Gene ID 3627 for human IP-10 sequence; GENBANK TM (See also Accession No. P02778.) An exemplary human CXCL10 sequence is set forth herein as SEQ ID NO:8.

[0044] Chemokine (C-X-C motif) ligand 11 (CXCL11): a chemokine of the C-X-C subfamily and a ligand for the receptor CXCR3. The CXCL11 protein induces a chemotactic response in activated T cells and is the predominant ligand for CXCR3. The gene encoding this protein contains four exons and at least three polyadenylation signals that may reflect cell-specific regulation of expression. IFN-γ is a potent inducer of transcription of this gene. The sequence of CXCL11 is publicly available (e.g., GENBANK TM (See Gene ID 6373.) An exemplary human CXCL11 sequence is set forth herein as SEQ ID NO:11.

[0045] CXCR3 (CXC chemokine receptor 3): A G protein-coupled receptor selective for four chemokines: CXCL4 / PF4 (platelet factor 4), CXCL9 / Mig (interferon-γ-induced monokine), CXCL10 / IP-10 (interferon-γ-induced 10 kDa protein), and CXCL11 / I-TAC (interferon-induced T cell α-chemoattractant). Binding of chemokines to this protein induces cellular responses involved in leukocyte trafficking, most notably integrin activation, cytoskeletal changes, and chemotactic migration. Alternatively, spliced ​​transcript variants encoding different isoforms have been found for this gene. One of the isoforms (CXCR3-B) exhibits high affinity binding to the chemokine CXCL4.

[0046] Diabetic retinopathy: A disorder in which damage to the retina occurs due to complications of diabetes. Proliferative retinopathy, which generally occurs in advanced stages of the disease, is characterized by the abnormal formation of new blood vessels at the vitreous surface that extend into the vitreous cavity.

[0047] Elastase: A serine protease that degrades elastin. Elastases include chymotrypsin-like elastase, chymotrypsin elastase, neutrophil elastase, and macrophage elastase. Neutrophil elastase can degrade bacterial membrane proteins and virulence factors.

[0048] Fibrosis: A condition associated with thickening and scarring of connective tissue. Often, fibrosis occurs in response to injury (e.g., from a disease or condition that damages tissue). Fibrosis is an exaggerated wound-healing response that can, in severe cases, interfere with normal organ function. Fibrosis can occur in almost any tissue in the body, including in the lungs (pulmonary fibrosis, cystic fibrosis, radiation-induced 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).

[0049] Glaucoma: An eye disorder in which the optic nerve is damaged, permanently impairing vision in the affected eye and progressing to complete blindness if left untreated. It is generally associated with increased pressure of fluid (aqueous humor) within the eye.

[0050] Hydrogel: A polymeric gel of large molecules composed of a network of cross-linked polymer chains.

[0051] Iatrogenic disease or disorder: A disease or disorder caused by a medical treatment or diagnostic procedure. Exemplary iatrogenic diseases / disorders include drug-induced (e.g., bleomycin-induced) pulmonary fibrosis, Bacillus Calmette-Guerin (BCG) treatment-induced bladder fibrosis, and chemotherapy-induced bladder fibrosis.

[0052] Inflammatory disease or disorder: A disease or disorder 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.

[0053] Macular degeneration: A condition that results in atrophy or degeneration of the macula. Age-related macular degeneration is the leading cause of vision loss in older adults. There are two different types of macular degeneration, termed dry and wet. In dry macular degeneration (dry), there is pigmentary abnormality in the macular region, but there is no elevated macular scar or hemorrhage or exudate in the macular region. In contrast, in exudative macular degeneration (wet), there is the formation of a subretinal network of choroidal neovascularization.

[0054] Matrix metalloproteinase (MMP): Calcium-dependent, zinc-containing endopeptidase. MMPs can degrade components of the extracellular matrix and are also known to play a role in cleaving cell surface receptors, releasing apoptotic ligands (e.g., FAS ligand), and inactivating chemokines / cytokines.

[0055] Neovascular glaucoma: A type of glaucoma that is very difficult to treat. This condition is often caused by proliferative diabetic retinopathy or central retinal vein occlusion. Neovascular glaucoma can also be triggered by other conditions that result in ischemia of the retina or ciliary body. Individuals with insufficient blood flow to the eye are at high risk for this condition. Neovascular glaucoma occurs when new, abnormal blood vessels begin to form in the angle of the eye, blocking drainage. Patients with this condition begin to lose their vision rapidly. Sometimes the disease appears very suddenly, especially after cataract surgery.

[0056] Non-canonical amino acid: An amino acid that is not one of the 20 amino acids directly encoded by a codon triplet in the genetic code. A non-canonical amino acid is also called a "non-standard" amino acid.

[0057] Peptide or polypeptide: A polymer in which the monomers are amino acid residues linked together through amide bonds. When the amino acids are α-amino acids, 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 (e.g., glycoproteins). The terms "polypeptide" and "peptide" are specifically intended to encompass naturally occurring proteins as well as those produced recombinantly or synthetically. In some embodiments, peptides are between 10 and 200 amino acids in length, including lengths of 10 to 100, 10 to 50, 10 to 30, 15 to 50, 15 to 30, or 18 to 25 amino acids. In particular examples, the peptides are about 21 or about 22 amino acids in length. "Residue" refers to an amino acid or amino acid mimetic incorporated into a polypeptide by an amide bond or amide bond mimetic.

[0058] Pharmaceutically acceptable carriers: Useful pharmaceutically acceptable carriers are conventional. Remington's Pharmaceutical Sciences, 15th Edition, 1975, by EW Martin, Mack Publishing Co., Easton, PA, describes compositions and formulations suitable for pharmaceutical delivery of the peptides disclosed herein. Generally, the nature of the carrier will depend on the particular mode of administration used. For example, parenteral formulations usually contain injectable fluids containing pharmaceutically and physiologically acceptable fluids (e.g., water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, etc.) as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances (e.g., wetting or emulsifying agents, preservatives, pH buffering agents, and the like), 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.

[0059] Poly(lactic-co-glycolic acid) (PLGA): a biodegradable and biocompatible copolymer of glycolic acid and lactic acid.

[0060] Protease: An enzyme that hydrolyzes (breaks down) proteins and peptides.

[0061] Restenosis: The recurrence of stenosis (narrowing of a blood vessel), resulting in restricted blood flow. Stenosis (or restenosis) is a form of response to injury that results in wall thickening, lumen narrowing, and loss of function of the tissue supplied by a particular pathway. Physical injury during an interventional procedure (e.g., glaucoma surgery) causes damage to the epithelial lining of the vessel. Repair of tissue after physical injury involves regeneration (replacement of damaged cells by cells of the same type) and fibrosis (replacement of damaged cells by connective tissue). The process of fibrosis involves, among other events, the formation of new blood vessels (angiogenesis).

[0062] Retinopathy of prematurity: An eye disease that affects infants born prematurely. It is thought to be caused by unregulated growth of retinal blood vessels, which can result in scarring and retinal detachment. The disease can be mild and resolve spontaneously, but in severe cases can lead to blindness.

[0063] Sequence identity: The similarity between amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is often measured in terms of the percentage of identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a particular polypeptide have a relatively high degree of sequence identity when aligned using standard methods.

[0064] Methods for aligning sequences 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. Additionally, Altschul et al., Nature Genet. 6:119, 1994, presents detailed considerations of sequence alignment methods and homology calculations.

[0065] NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, The NCBI (1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD), and on the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Details on how to determine sequence identity using this program are available on the Internet at the NCBI website.

[0066] Homologs and variants of a polypeptide are typically characterized by having at least about 75%, e.g., at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, as counted over a full-length alignment with the amino acid sequence of the polypeptide, using NCBI Blast 2.0, gapped blastp, set to default parameters. For comparison of amino acid sequences of more than about 30 amino acids, default parameters (gap existence cost 11, and per residue gap cost 11) are used. The Blast 2 alignment function is used with the default BLOSUM62 matrix set to 1 (gap cost). When aligning short peptides (fewer than approximately 30 amino acids), alignments should be performed using the Blast 2 alignment function with the PAM30 matrix set to default parameters (open gap penalty of 9, extension gap penalty of 1). Proteins with even greater similarity to a reference sequence will exhibit increasing percentage identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity) when assessed by this method. When less than the entire sequence is being compared for sequence identity, homologs and variants typically have at least 80% sequence identity over 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 are available on the Internet at the NCBI website. Those skilled in the art will recognize that these sequence identity ranges are provided for guidance only; it is entirely possible that strong and significant homologs can be obtained that fall outside the ranges provided.

[0067] Subject: Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects (including human and non-human mammals).

[0068] Therapeutically effective amount: A quantity of a specified agent (e.g., a CXCL peptide) sufficient to achieve a desired effect in a subject, cell, or culture being treated with that agent.

[0069] III. Overview of Some Embodiments Activators of the cell surface G protein-coupled receptor CXCR3 can limit and even ameliorate fibrosis and angiogenesis through these receptors on fibroblasts, endothelial cells, pericytes, and other adherent cells. CXCR3 activation also halts migration through m-calpain inhibition and induces anoikis in endothelial cells through μ-calpain cleavage of β3 integrin. These two actions can be used to limit scarring and prevent or ameliorate vascular disorders, including those of the eye. The effects of CXCR3 activators are superior to those of fibrosis and angiogenesis promoters.

[0070] Cells of the immune response also express the CXCR3 receptor but respond in a pro-migratory manner, chemotactically toward these ligands. This creates a potentially perplexing situation in which CXCR3 activators can limit immediate scarring through their effects on adherent cells (e.g., fibroblasts, endothelial cells, and epithelial cells) while promoting late scarring by attracting and activating immune cells (e.g., leukocytes, lymphocytes, macrophages, etc.). The actual outcome is determined by the quantitative balance of these two cell populations. The risk of a late pro-inflammatory response is highest in situations of acute and ongoing inflammation.

[0071] To eliminate this risk, this paper describes a CXCR3 activator (ligand) that can be inactivated by cleavage by extracellular proteases present during active inflammation.Inflammatory scarring is achieved by the production of proteases that degrade resting matrix and replace it with scar matrix.These proteases include many cathepsins and elastases.Using a CXCR3 ligand that is sensitive to cleavage by these proteases allows unnecessary pro-inflammatory signaling to disappear as the ligand is inactivated under the presence of acute inflammation.

[0072] The disclosed peptides can be used to treat fibrotic and angiogenic diseases, including scarring, vascular disorders, scleroderma, and autoimmune fibrosis. These diseases and disorders can occur in any organ of the body (e.g., skin, lungs, liver, kidneys, heart, and eyes). The diseases also include those caused by wounds, autoimmune pathologies, diabetes, and those of unknown etiology (e.g., wet age-related macular degeneration (AMD) and idiopathic pulmonary fibrosis (IPF)).

[0073] Provided herein is a recombinant CXC motif chemokine ligand (CXCL) peptide that is modified relative to the wild-type CXCL amino acid sequence to introduce a protease cleavage site. In some embodiments, the CXCL is a ligand for CXC chemokine receptor 3 (CXCR3). In some examples, the CXCL is CXCL10, CXCL4, CXCL9, or CXCL11. In a specific, non-limiting example, the CXCL is CXCL10.

[0074] In some embodiments, the protease is a protease that is activated during an inflammatory response (e.g., an acute inflammatory response). In some examples, the protease is a cathepsin, elastase, or matrix metalloproteinase (MMP). In specific examples, the cathepsin is cathepsin G or cathepsin K. In other specific examples, the MMP is MMP2. In other specific examples, the elastase is neutrophil elastase.

[0075] In some embodiments, the peptide is about 12 to about 30 amino acids in length (e.g., about 18 to about 25 amino acids in length, e.g., about 20 to about 23 amino acids in length, e.g., about 21 or 22 amino acids in length), or 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length.

[0076] 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.

[0077] In some embodiments, the peptide comprises a C-terminal proline that is amidated or methylated.

[0078] In some embodiments, the peptide comprises at least one chemical modification. For example, chemical modifications may be introduced to inhibit degradation of the peptide and / or increase its half-life. In some examples, the at least one modification comprises a modification at the N-terminus of the peptide, a modification at the C-terminus of the peptide, or both. In specific, non-limiting examples, the modification at the N-terminus comprises formylation, acetylation, propionylation, pyroglutamate formation, myristoylation, palmitylation, S-palmitoylation, monomethylation, dimethylation, trimethylation, or any combination thereof. In other specific, non-limiting examples, the modification at the C-terminus comprises acetylation, α-amidation, or a combination thereof. In other embodiments, the at least one modification comprises a non-standard peptide bond.

[0079] In some embodiments, the recombinant CXCL peptide comprises at least one D-amino acid. In some examples, the CXCL peptide comprises a plurality of D-amino acids (e.g., 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).

[0080] In some embodiments, the recombinant CXCL peptide comprises at least one non-standard amino acid. In some examples, the at least one non-standard amino acid is a modified non-standard amino acid. The peptide may comprise a modified non-standard 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 comprises a modified non-standard amino acid at the N-terminus, wherein the modification comprises formylation, acetylation, propionylation, pyroglutamate formation, myristoylation, palmitylation, S-palmitoylation, monomethylation, di-methylation, tri-methylation, or any combination thereof. In another specific, non-limiting example, the peptide comprises a modified non-standard amino acid at the C-terminus, wherein the modification comprises methylation, α-amidation, or a combination thereof.

[0081] In some embodiments, the at least one non-standard 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 radioactive isotope, or any combination thereof. In some examples, the methylated amino acid is a monomethylated amino acid, a dimethylated amino acid, or a trimethylated amino acid. In some examples, 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 Both C and C.

[0082] In some embodiments, the recombinant CXCL peptide is in a sustained release formulation. In some examples, the sustained release formulation comprises poly(lactic-co-glycolic acid) (PLGA), a hydrogel, or a coacervate.

[0083] Also provided herein are compositions comprising the recombinant CXCL peptides disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the compositions are formulated for topical, intranasal, inhalation, intravenous, intravitreal, intramuscular, intradermal, or subcutaneous administration. In some examples, the compositions are formulated for delivery to the lungs by aerosolization or nebulizer (e.g., for the treatment of cystic fibrosis). In other examples, the compositions are 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 overactive bladder syndrome. Therefore, compositions formulated for delivery to the bladder can be used to treat these conditions.

[0084] In some embodiments, the composition is provided in a unit dosage form.

[0085] In some embodiments, the composition may include a protease inhibitor, a preservative, a tonicity agent, a buffering agent, a pH adjusting agent, a sterile solvent, or any combination thereof. In some embodiments, the protease inhibitor may include, but is not limited to, TIMP1, odanacatib, calpeptin, batimastat, ilomastat, or any combination thereof. In some embodiments, the tonicity agent may include, but is not limited to, an isotonic buffering agent (e.g., sodium chloride). In some embodiments, the buffering agent may include, but is not limited to, phosphoric acid, acetic acid, citric acid, a phosphate buffer, histidine, tromethamine, gluconic acid, lactic acid, tartaric acid, aspartic acid, glutamic acid, tartaric acid, succinic acid, malic acid, fumaric acid, α-ketoglutaric acid, or a combination thereof. In some embodiments, the pH adjusting agent may include, but is not limited to, hydrochloric acid. In some embodiments, the sterile solvent may include, but is not limited to, sterile water. In some embodiments, the composition may also include a stabilizer, such as, but not limited to, trehalose. In some embodiments, the composition may also include a surfactant, such as, polysorbate.

[0086] Further provided herein are methods for inhibiting fibrosis in a subject. In some embodiments, the method comprises administering to the subject a CXCL peptide or composition disclosed herein. In some examples, the subject has a wound, an autoimmune disease, an inflammatory disease or disorder, or an iatrogenic disease or disorder. In specific examples, the autoimmune disease is diabetes, scleroderma, or autoimmune fibrosis. In other specific examples, the inflammatory disease or disorder is idiopathic pulmonary fibrosis (IPF). In other specific examples, the iatrogenic disease or disorder is drug-induced (e.g., bleomycin-induced) pulmonary fibrosis, Bacillus Calmette-Guerin (BCG) treatment-induced bladder fibrosis, or chemotherapy-induced bladder fibrosis.

[0087] Also provided herein is a method for inhibiting angiogenesis in a subject. In some embodiments, the method comprises administering to the subject a CXCL peptide or composition disclosed herein. In some examples, the subject has an ocular neovascular disorder. In certain examples, the ocular neovascular disorder is wet macular degeneration, diabetic retinopathy, retinopathy of prematurity, restenosis after glaucoma treatment, neovascular glaucoma, or corneal neovascularization.

[0088] IV. Peptide Sequence The present disclosure describes modified peptides derived from CXCL proteins that function as ligands for CXCR3. The disclosed peptides are about 12 to about 30 amino acids in length, e.g., about 18 to about 25 amino acids in length, e.g., 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 peptides are modified to contain a protease cleavage site, allowing inactivation of the peptide in the presence of a protease that recognizes the incorporated cleavage site. In some cases, the cleavage site is a cleavage site for a cathepsin (e.g., cathepsin G or cathepsin K), an elastase (e.g., neutrophil elastase), or an MMP (e.g., MMP2). Those skilled in the art can determine suitable protease cleavage sites, for example, with the aid of online tools (e.g., ExPASy Bioinformatics Resource Portal or Protease Specificity Prediction Server (PROSPER; see also Song et al., PLoS One 7(11):e50300, 2012)).

[0089] Those skilled in the art can identify and introduce appropriate amino acid substitutions that create protease cleavage sites. For example, cathepsin G is known to recognize - / V / L / LHF†S / -S / A / V (SEQ ID NO: 12), cathepsin K is known to recognize - / - / LPV / EA†GE / - / - / - (SEQ ID NO: 35), MMP2 is known to recognize - / P / - / -†LI / - / - / - (SEQ ID NO: 36) (Song et al., PLoS One 7(11):e50300, 2012); and neutrophil elastase is known to recognize FIRW (SEQ ID NO: 13) (Schulenburg et al., Analyst 141(5):1645-1648, 2016).

[0090] In some embodiments, the peptide is derived from human CXCL10, set forth herein as SEQ ID NO: 8. The peptide can consist of any portion of CXCL10 comprising about 18 to about 25 contiguous amino acids of SEQ ID NO: 8, where one or more amino acids are substituted to introduce a cleavage site for a selected protease, and where the modified peptide retains the ability to activate CXCR3 (in the absence of the protease). [ka]

[0091] In some embodiments herein, the CXCL10 peptide is based on the C-terminal peptide: [ka]

[0092] SEQ ID NO: 1 can be modified to introduce a protease cleavage site (e.g., the cleavage site for cathepsin G, neutrophil cathepsin, or neutrophil elastase). Non-limiting examples of modified peptides based on human CXCL10 are provided below.

[0093] Exemplary peptides with cathepsin G and neutrophil elastase cleavage site sequences: [ka]

[0094] Peptides containing neutrophil cathepsin cleavage sites: [ka]

[0095] Peptides containing a neutrophil elastase cleavage site: [ka]

[0096] For the peptides of SEQ ID NOs: 2, 3 and 7, the substitution of arginine (R) with methionine (M) does not involve a mutation that introduces a protease cleavage site.

[0097] In some embodiments, the peptide is derived from human CXCL4, set forth herein as SEQ ID NO: 9. The peptide can consist of any portion of CXCL4 comprising about 18 to about 25 contiguous amino acids of SEQ ID NO: 9, where one or more amino acids are substituted to introduce a cleavage site for a selected protease, and where the modified peptide retains the ability to activate CXCR3 (in the absence of the protease). [ka]

[0098] In some embodiments, the modified CXCL4 peptide is based on one of the following CXCL4 peptides: [ka] [ka]

[0099] SEQ ID NO: 14, 15, or 16 can be modified to introduce a protease cleavage site (e.g., a cleavage site for cathepsin K or matrix metalloproteinase 2 (MMP2)). Non-limiting examples of modified peptides based on human CXCL4 are provided below.

[0100] Exemplary peptides with cathepsin K cleavage site sequences: [ka]

[0101] Exemplary peptides with MMP2 cleavage site sequences: [ka]

[0102] 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, where one or more amino acids are substituted to introduce a cleavage site for a selected protease, and where the modified peptide retains the ability to activate CXCR3 (in the absence of the protease). [ka]

[0103] In some embodiments, the peptide is derived from human CXCL11, shown herein as SEQ ID NO: 11. The peptide can consist of any portion of CXCL11 comprising about 18 to about 25 contiguous amino acids of SEQ ID NO: 11, where one or more amino acids are substituted to introduce a cleavage site for a selected protease, and where the modified peptide retains the ability to activate CXCR3 (in the absence of the protease). [ka]

[0104] In some embodiments, the modified CXCL1 peptide is based on one of the following CXCL11 peptides: [ka]

[0105] SEQ ID NO: 24, 25 or 26 can be modified to introduce a protease cleavage site (e.g., a cleavage site for cathepsin K or MMP2). Non-limiting examples of modified peptides based on human CXCL11 are provided below.

[0106] Exemplary peptides with cathepsin K cleavage site sequences: [ka]

[0107] Exemplary peptides with MMP2 cleavage site sequences: [ka]

[0108] V. Illustrative Embodiments 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. 2. The recombinant CXCL peptide of embodiment 1, wherein said CXCL is a ligand for CXC chemokine receptor 3 (CXCR3). 3. The recombinant CXCL peptide of embodiment 1 or 2, wherein the CXCL is CXCL10, CXCL4, CXCL9 or CXCL11. 4. A recombinant CXCL peptide according to any one of embodiments 1 to 3, wherein the protease is a cathepsin, elastase or matrix metalloproteinase (MMP). 5. The recombinant CXCL peptide of embodiment 4, wherein the cathepsin is cathepsin G. 6. The recombinant CXCL peptide of embodiment 4, wherein the cathepsin is cathepsin K. 7. The recombinant CXCL peptide of embodiment 4, wherein the elastase is neutrophil elastase. 8. The recombinant CXCL peptide of embodiment 4, wherein the MMP is MMP2. 9. The recombinant CXCL peptide of any one of embodiments 1 to 8, wherein the peptide is about 12 to about 30 amino acids in length. 10. A recombinant CXCL peptide according to any one of embodiments 1 to 9, wherein the peptide is about 18 to about 25 amino acids in length. 11. A recombinant CXCL peptide described in any one of embodiments 1 to 10, wherein the peptide is 21 or 22 amino acids in length. 12. A recombinant CXCL peptide described in any one of embodiments 1 to 11, wherein the amino acid sequence of the peptide is at least 90% identical to any one of SEQ ID NOs: 2 to 7. 13. A recombinant CXCL peptide described in any one of embodiments 1 to 11, wherein the amino acid sequence of the peptide is at least 95% identical to any one of SEQ ID NOs: 2 to 7. 14. A recombinant CXCL peptide according to any one of embodiments 1 to 11, wherein the amino acid sequence of the peptide comprises or consists of any one of SEQ ID NOs: 2 to 7. 15. A recombinant CXCL peptide described in any one of embodiments 1 to 14, wherein the amino acid sequence of the peptide comprises or consists of SEQ ID NO:2. 16. A recombinant CXCL peptide described in any one of embodiments 1 to 15, wherein the peptide comprises at least one modification. 17. The recombinant CXCL peptide of embodiment 16, wherein the at least one modification comprises a modification at the N-terminus of the peptide, a modification at the C-terminus of the peptide, or both. 18. The recombinant CXCL peptide of embodiment 17, wherein the modification at the N-terminus comprises formylation, acetylation, propionylation, pyroglutamate formation, myristoylation, palmitylation, S-palmitoylation, monomethylation, dimethylation, trimethylation, or any combination thereof. 19. A recombinant CXCL peptide described in embodiment 17 or embodiment 18, wherein the modification at the C-terminus comprises methylation, α-amidation, or a combination thereof. 20. The recombinant CXCL peptide of embodiment 16, wherein the at least one modification comprises a non-canonical peptide bond. 21. A recombinant CXCL peptide according to any one of embodiments 1 to 20, comprising at least one D-amino acid. 22. A recombinant CXCL peptide according to any one of embodiments 1 to 21, comprising at least one non-standard amino acid. 23. The recombinant CXCL peptide of embodiment 22, wherein the at least one non-standard amino acid is a modified non-standard amino acid. 24. A recombinant CXCL peptide described in embodiment 23, wherein the peptide comprises a modified non-standard amino acid at the N-terminus of the peptide, the C-terminus of the peptide, or both. 25. The recombinant CXCL peptide of embodiment 24, wherein the peptide comprises a modified non-standard amino acid at the N-terminus, and the modification comprises formylation, acetylation, propionylation, pyroglutamate formation, myristoylation, palmitylation, S-palmitoylation, monomethylation, dimethylation, trimethylation, or any combination thereof. 26. A recombinant CXCL peptide described in embodiment 24 or embodiment 25, wherein the peptide comprises a modified non-standard amino acid at the C-terminus, and all modifications include methylation, α-amidation, or a combination thereof. 27. The recombinant CXCL peptide of embodiment 22, wherein the at least one non-standard 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 radioactive isotope, or any combination thereof. 28. A recombinant CXCL peptide according to any one of embodiments 1 to 27, in a sustained release formulation. 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. 30. A composition comprising a recombinant CXCL peptide according to any one of embodiments 1 to 29 and a pharmaceutically acceptable carrier. 31. The composition of embodiment 30, formulated for topical, intranasal, inhaled, intravenous, intravitreal, intramuscular, intradermal, or subcutaneous administration. 32. The composition of embodiment 30, which is formulated for delivery to the lungs by aerosolization or nebulizer. 33. The composition of embodiment 30, formulated for delivery to the bladder via catheter and instillation. 34. A composition described in any one of embodiments 30 to 33, in unit dosage form. 35. A method for inhibiting fibrosis in a subject, the method comprising administering to the subject a CXCL peptide described in any one of embodiments 1 to 29 or a composition described in any one of embodiments 30 to 34. 36. The method of embodiment 35, wherein the subject has a wound, an autoimmune disease, an inflammatory disease or disorder, or an iatrogenic disease or disorder. 37. The method of embodiment 36, wherein the autoimmune disease is diabetes, scleroderma, or autoimmune fibrosis. 38. The method of embodiment 36, wherein the inflammatory disease or disorder is idiopathic pulmonary fibrosis (IPF). 39. The method of embodiment 36, wherein the iatrogenic disease or disorder is drug-induced pulmonary fibrosis, bacillus Calmette-Guerin (BCG) treatment-induced bladder fibrosis, or chemotherapy-induced bladder fibrosis. 40. A method for inhibiting angiogenesis in a subject, the method comprising administering to the subject a CXCL peptide described in any one of embodiments 1 to 29 or a composition described in any one of embodiments 30 to 34. 41. The method of embodiment 40, wherein the subject has an ocular neovascular disorder. 42. The method of embodiment 41, wherein the ocular neovascular disorder is wet macular degeneration, diabetic retinopathy, retinopathy of prematurity, restenosis after glaucoma treatment, neovascular glaucoma, or corneal neovascularization.

[0109] The following examples are provided to illustrate certain particular features and / or embodiments, and should not be construed as limiting the disclosure to the particular features or embodiments described. [Example]

[0110] Example 1: Modified peptides susceptible to protease cleavage This example describes peptides engineered to be susceptible to cleavage by extracellular proteases of the acute inflammatory response.

[0111] Previous studies have shown that linear peptide fragments of CXCR3's natural ligand (e.g., CXCL10) can bind to and activate CXCR3 (see U.S. Patent Nos. 9,180,167; 9,452,200; and 9,872,889, the contents of which are incorporated by reference in their entireties). Chemokines that signal through CXCR3 can induce antifibrotic effects on adherent cells and can also promote inflammation and fibrotic effects on cells of the innate immune system. To limit the fibrotic effects of CXCR3 activators during an inflammatory response, peptides were designed that are susceptible to cleavage by proteases present during acute inflammatory responses. Thus, upon induction of an inflammatory response, the engineered peptides are cleaved, thereby limiting their profibrotic effects.

[0112] 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 an arginine to methionine substitution and replaces a serine residue with a histidine. The latter substitution results in the introduction of cleavage sites for cathepsin G and neutrophil elastase. The substituted residues are shown in bold and underlined: [ka]

[0113] Because the histidine to serine substitution is a non-conservative amino acid change, experiments were performed to confirm that the engineered peptide retained the ability to block angiogenesis and fibrosis in a mouse model of choroidal neovascularization (CNV).

[0114] Laser-induced CNV Murine CNV was induced by laser photocoagulation-induced rupture of Bruch's membrane as previously described. Briefly, 7-8 week-old female C57BL / 6J mice were anesthetized with ketamine hydrochloride (100 mg / kg body weight) and mydriasis was performed with 1% tropicamide. Three diode laser photocoagulation burns (spot size, 75 mm; duration, 0.1 s; power, 120 mW) were delivered to each retina with the slit lamp delivery system of an OcuLight GL diode laser (Iridex, Mountain View, CA), using a coverslip as a contact lens to visualize the retina. Burns were placed at the 9, 12, and 3 o'clock positions on the posterior pole of the retina. Air bubble formation during laser irradiation, indicative of rupture of Bruch's membrane, is an important factor in obtaining choroidal neovascularization (NV); therefore, only burns that produced air bubbles were included in the study.

[0115] Immediately after laser treatment, mice were injected with either 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) in a volume of 1 μl into one eye, and a scrambled peptide was injected into the contralateral eye as a control. Other control mice were injected with vehicle alone. After injection, the cornea was protected with antibiotic ointment. Seven days after laser treatment, the mice were euthanized. Eyes were enucleated and fixed in 10% PBS-buffered formalin for 3 hours. Choroids were dissected, placed in 1.5 ml Eppendorf tubes, and stained with FITC-conjugated GSA-Lectin IB4 (1:150, Vector, Frederick, MD) overnight at 4°C. After three washes with PBST, the choroids were mounted on glass slides and examined by fluorescence microscopy. Images were digitized with a three-color CCD video camera and frame grabber. The total area of ​​choroidal NV at each rupture site was measured using image analysis software (Image-Pro Plus; MediaCybernetics, Silver Spring, MD).

[0116] As shown in Figures 1A-1B, peptide 110 retained its anti-angiogenic activity as evidenced by a significant reduction in choroidal neovascularization at both doses of peptide.

[0117] Example 2: CXCR3-activating peptides with engineered protease cleavage sites This example describes additional peptides based on the sequences of human CXCL10, human CXCL4 or human CXCL11 that are designed to contain more than one cleavage site for proteases activated during acute inflammation.

[0118] CXCL10-derived peptides containing cathepsin G and neutrophil elastase cleavage sites: [ka]

[0119] CXCL10-derived peptides containing neutrophil cathepsin cleavage sites: [ka]

[0120] CXCL10-derived peptide with a neutrophil elastase cleavage site: [ka]

[0121] CXCL4-derived peptide with cathepsin K cleavage site sequence: [ka]

[0122] CXCL4-derived peptide with MMP2 cleavage site sequence: [ka]

[0123] CXCL11-derived peptide with cathepsin K cleavage site sequence: [ka]

[0124] CXCL11-derived peptide with MMP2 cleavage site sequence: [ka]

[0125] In view of the many possible embodiments to which the principles of the disclosed subject matter may be applied, it should be recognized that the illustrated embodiments are merely preferred examples of the present disclosure and should not be construed as limiting the scope of the present disclosure. Rather, the scope of the present disclosure is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims. In certain embodiments, for example, the following items are provided: (Item 1) A recombinant CXC motif chemokine ligand (CXCL) peptide, wherein said peptide is modified relative to the wild-type CXCL amino acid sequence to introduce a cleavage site for a protease. (Item 2) 2. The recombinant CXCL peptide according to item 1, wherein the CXCL is a ligand for CXC chemokine receptor 3 (CXCR3). (Item 3) 2. The recombinant CXCL peptide of item 1, wherein the CXCL is CXCL10, CXCL4, CXCL9 or CXCL11. (Item 4) 2. The recombinant CXCL peptide according to item 1, wherein the protease is a cathepsin, an elastase or a matrix metalloproteinase (MMP). (Item 5) 5. The recombinant CXCL peptide according to item 4, wherein the cathepsin is cathepsin G. (Item 6) 5. The recombinant CXCL peptide according to item 4, wherein the cathepsin is cathepsin K. (Item 7) 5. The recombinant CXCL peptide according to item 4, wherein the elastase is neutrophil elastase. (Item 8) 5. The recombinant CXCL peptide according to item 4, wherein the MMP is MMP2. (Item 9) 2. The recombinant CXCL peptide according to item 1, wherein the peptide is about 12 to about 30 amino acids in length. (Item 10) 2. The recombinant CXCL peptide according to item 1, wherein the peptide is about 18 to about 25 amino acids in length. (Item 11) 2. The recombinant CXCL peptide of item 1, wherein the peptide is 21 or 22 amino acids in length. (Item 12) 2. 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 to 7, 17 to 23 and 27 to 34. (Item 13) 2. 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 to 7, 17 to 23 and 27 to 34. (Item 14) 2. 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 to 7, 17 to 23, and 27 to 34. (Item 15) 2. The recombinant CXCL peptide of item 1, wherein the amino acid sequence of the peptide comprises or consists of SEQ ID NO:2. (Item 16) 2. The recombinant CXCL peptide according to item 1, wherein the peptide comprises at least one chemical modification. (Item 17) 18. The recombinant CXCL peptide of 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 recombinant CXCL peptide of item 17, wherein the modification at the N-terminus comprises formylation, acetylation, propionylation, pyroglutamate formation, myristoylation, palmitylation, S-palmitoylation, monomethylation, dimethylation, trimethylation, or any combination thereof. (Item 19) 18. The recombinant CXCL peptide according to item 17, wherein the modification at the C-terminus comprises methylation, α-amidation, or a combination thereof. (Item 20) 17. The recombinant CXCL peptide of item 16, wherein the at least one chemical modification comprises a non-canonical peptide bond. (Item 21) 2. The recombinant CXCL peptide according to item 1, comprising at least one D-amino acid. (Item 22) 2. The recombinant CXCL peptide according to item 1, comprising at least one non-standard amino acid. (Item 23) 23. The recombinant CXCL peptide of item 22, wherein the at least one non-standard amino acid is a modified non-standard amino acid. (Item 24) 24. The recombinant CXCL peptide of claim 23, wherein the peptide comprises a modified non-standard amino acid at the N-terminus of the peptide, the C-terminus of the peptide, or both. (Item 25) 25. The recombinant CXCL peptide of claim 24, wherein the peptide comprises a modified non-standard amino acid at the N-terminus, the modification comprising formylation, acetylation, propionylation, pyroglutamate formation, myristoylation, palmitylation, S-palmitoylation, monomethylation, di-methylation, tri-methylation, or any combination thereof. (Item 26) 25. The recombinant CXCL peptide of claim 24, wherein the peptide comprises a modified non-standard amino acid at the C-terminus, the modification comprising methylation, α-amidation, or a combination thereof. (Item 27) 23. The recombinant CXCL peptide of claim 22, wherein the at least one non-standard 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. (Item 28) 2. The recombinant CXCL peptide according to item 1, in a sustained release formulation. (Item 29) 29. The recombinant CXCL peptide of item 28, wherein the sustained release formulation comprises poly(lactic-co-glycolic acid) (PLGA), a hydrogel, or a coacervate. (Item 30) A composition comprising the recombinant CXCL peptide of item 1 and a pharmaceutically acceptable carrier. (Item 31) 31. The composition of item 30, formulated for topical, intranasal, inhaled, intravenous, intravitreal, intramuscular, intradermal, or subcutaneous administration. (Item 32) 31. The composition of item 30, formulated for delivery to the lungs by aerosolization or nebulizer. (Item 33) 31. The composition of item 30, formulated for delivery to the bladder by catheter and instillation. (Item 34) 31. The composition of item 30 in unit dosage form. (Item 35) A method for inhibiting fibrosis in a subject, the method comprising administering to the subject the CXCL peptide of item 1. (Item 36) 36. The method of claim 35, wherein the subject has a wound, an autoimmune disease, an inflammatory disease or disorder, or an iatrogenic disease or disorder. (Item 37) 37. The method of claim 36, wherein the autoimmune disease is diabetes, scleroderma, or autoimmune fibrosis. (Item 38) 37. The method of claim 36, wherein the inflammatory disease or disorder is idiopathic pulmonary fibrosis (IPF). (Item 39) 37. The method of claim 36, wherein the iatrogenic disease or disorder is drug-induced pulmonary fibrosis, bacillus Calmette-Guerin (BCG) treatment-induced bladder fibrosis, or chemotherapy-induced bladder fibrosis. (Item 40) A method for inhibiting angiogenesis in a subject, the method comprising administering to the subject the CXCL peptide described in item 1. (Item 41) 41. The method of claim 40, wherein the subject has an ocular neovascular disorder. (Item 42) 42. The method of claim 41, wherein the ocular neovascular disorder is wet macular degeneration, diabetic retinopathy, retinopathy of prematurity, restenosis after glaucoma treatment, neovascular glaucoma, or corneal neovascularization.

Claims

1. A composition for inhibiting fibrosis in a subject, wherein the composition comprises a recombinant C-X-C motif chemokine ligand (CXCL) peptide having an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 2 to 5, wherein the recombinant CXCL peptide comprises a serine-to-histidine substitution at the position corresponding to position 14 of SEQ ID NO: 1 for cathepsin and / or elastase cleavage, the recombinant CXCL peptide retains the ability to inhibit fibrosis, and the recombinant CXCL peptide is about 12 to about 30 amino acids in length.

2. The composition according to claim 1, wherein the subject has a wound, autoimmune disease, inflammatory disease or disorder, or iatrogenic disease or disorder.

3. The aforementioned autoimmune diseases are diabetes, scleroderma, or autoimmune fibrosis; The aforementioned inflammatory disease or disorder is idiopathic pulmonary fibrosis (IPF); or The composition according to claim 2, wherein the iatrogenic disease or disorder is drug-induced pulmonary fibrosis, Bacillus calmette-guéran treatment-induced bladder fibrosis, or chemotherapy-induced bladder fibrosis.

4. The composition according to claim 1, wherein the cathepsin is cathepsin G.

5. The composition according to claim 1, wherein the elastase is neutrophil elastase.

6. The composition according to claim 1, wherein the peptide has a length of 21 or 22 amino acids.

7. The composition according to claim 1, wherein the amino acid sequence of the peptide is at least 95% identical to any one of sequence numbers 2 to 5.

8. The composition according to claim 1, wherein the amino acid sequence of the peptide comprises one of sequence numbers 2 to 5.

9. The composition according to claim 1, wherein the amino acid sequence of the peptide consists of any one of SEQ ID NOs: 2 to 5.

10. The composition according to claim 1, wherein the peptide comprises at least one chemical modification.

11. The composition according to claim 10, wherein the at least one chemical modification includes a modification at the N-terminus of the peptide, a modification at the C-terminus of the peptide, or both.

12. The modifications at the N-terminus include formylation, acetylation, propionylation, pyroglutamate formation, myristoylation, palmitylation, S-palmitoylation, monomethylation, dimethylation, trimethylation, or any combination thereof; and / or The composition according to claim 11, wherein the modification at the C-terminus includes methylation, α-amidation, or a combination thereof.

13. The peptide contains non-standard peptide bonds; The peptide comprises at least one D-amino acid; and / or The composition according to claim 1, wherein the peptide comprises at least one non-standard amino acid.

14. The composition according to claim 13, wherein the at least one non-standard amino acid is a modified non-standard amino acid.

15. The composition according to claim 14, wherein the peptide comprises a non-standard amino acid modified at the N-terminus, C-terminus, or both of the peptide.

16. The peptide comprises a non-standard amino acid modified at the N-terminus, the modification including formylation, acetylation, propionylation, pyroglutamate formation, myristoylation, palmitylation, S-palmitoylation, monomethylation, dimethylation, trimethylation, or any combination thereof; and / or The composition according to claim 15, wherein the peptide comprises a non-standard amino acid modified at the C-terminus, the modification comprising methylation, α-amidation, or a combination thereof.

17. The composition according to claim 13, wherein the at least one non-standard 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.

18. A composition according to any one of claims 1 to 17, contained in a sustained-release formulation.

19. The composition according to claim 18, wherein the sustained-release formulation comprises poly(lactic acid-coglycolic acid) (PLGA), hydrogel, or coacervate.

20. A composition for inhibiting fibrosis in a subject, comprising a recombinant CXCL peptide as described in any one of claims 1 to 19 and a pharmaceutically acceptable carrier.

21. The composition according to claim 20, wherein the composition is formulated for local administration, intranasal administration, inhalation, intravenous administration, intravitreous administration, intramuscular administration, intradermal administration, or subcutaneous administration.

Images