TSG6 polypeptide fragments for dry eye disease

Abstract

The present invention relates to the treatment of dry eye disease, particularly, but not exclusively, to the treatment of dry eye disease with LINK_TSG6 polypeptides.

Description

[Technical Field]

[0001] This application claims priority from GB1910645.9 filed on July 25, 2019, the contents and elements of which are incorporated herein by reference for all purposes.

[0002] The present invention relates to the treatment of ocular surface disorders, particularly, but not exclusively, to the treatment of dry eye disease with LINK_TSG6 polypeptides. [Background technology]

[0003] Tumor necrosis factor (TNF)-stimulated gene 6 (TSG-6) is an approximately 35 kDa secreted product of TNF-stimulated gene-6 that is expressed in response to inflammatory mediators and growth factors.

[0004] While constitutively expressed in several tissues, TSG-6 is generally upregulated wherever inflammation is present. In most cases, TSG-6 exhibits anti-inflammatory and tissue-protective properties, but it has also been implicated in disease pathology, for example, in the lung. While TSG-6 is produced by a wide range of cell types, it has been found to be produced by mesenchymal stem / stromal cells (MSCs) in response to inflammatory signals and mediate many of the immunomodulatory and repair activities, leading to a wealth of publications on the therapeutic effects of this intriguing molecule across a wide range of disease models.

[0005] TSG-6 is a relatively small protein, with a molecular weight of only approximately 35–38 kDa, primarily composed of two modular domains. Given its size, TSG-6 possesses a surprising number of activities, including regulation of immune and stromal cell function and contributions to extracellular matrix formation, mechanics, and remodeling. It is TSG-6's ability to regulate matrix organization and control the association of matrix molecules with cell surface receptors and extracellular signaling factors (e.g., chemokines) that may underlie its diverse functional repertoire. In this regard, TSG-6 interacts with a large array of ligands, including glycosaminoglycans (GAGs), proteoglycan (PG) core proteins, and other matrix components, and directly binds to multiple chemokines and bone morphogenetic proteins (BMPs). One particularly unusual function of TSG-6 is its role as an enzyme catalyzing the covalent modification of the non-sulfated GAG hyaluronic acid (HA) with so-called heavy chains (HCs) from proteoglycans of the α-inhibitor inter-(αI) family. This process, mediated by the full-length TSG6 protein rather than the LINK_TSG6 polypeptide containing only a fragment of TSG-6, leads to the formation of the HC·HA complex, is essential for mammalian ovulation and fertilization, and also occurs in many other situations where HC·HA confers tissue protection or contributes to pathological processes (e.g., inflammation).

[0006] The site and context of TSG-6 expression, its structure and ligand-binding properties, and how these together support diverse biological and therapeutic potential at the molecular level are reviewed in Day & Milner (Matrix Biology (2019) 78-79, 60-83).

[0007] US2015 / 0057229 describes the use of LINK_TSG6 in inhibiting cartilage degradation.

[0008] Dry eye disease (also known as keratoconjunctivitis sicca) is one of the most common ocular disorders, occurring in 7% to 33% of the population worldwide. It is a multifactorial condition of the tear film and ocular surface, involving increased tear film osmolality, ocular surface inflammation, neurotrophic deficiency, and meibomian gland dysfunction.

[0009] Kim et al. (2016) (Cornea 35(4), 536-542) compared topically applied TSG-6, cyclosporine, and prednisolone to treat dry eye. Twelve-week-old NOD.B10.H2b mice were treated with either recombinant TSG-6 (0.1%) four times daily, 0.05% cyclosporine (Restasis) twice daily, or 1% prednisolone (Pred Forte) four times daily for one week. Topical TSG-6 was found to be as effective as cyclosporine eye drops in treating inflammation-mediated dry eye. However, they concluded that the clinical application of TSG-6 is limited by various factors, including difficulties in large-scale production and variable stability of the recombinant protein.

[0010] WO2011 / 139357 describes the use of adult stem / progenitor cells and stem cell proteins for the treatment of ocular injury and disease. They propose a therapy based on the finding that application of MSCs or MSC-conditioned medium to rat corneas reduced inflammation and revascularization after chemical burns. They proposed the use of anti-apoptotic and anti-inflammatory proteins, such as STC-1 and TSG-6, expressed by mesenchymal stem cells. Corneal surface inflammation was induced in rat eyes by application of ethanol and mechanical debridement of the corneal and limbal epithelium. Application of recombinant full-length TSG-6 resulted in reduced corneal opacity and neovascularization compared with PBS controls. The authors conclude that proteins produced by MSCs in response to injury signals can protect the corneal surface from injury by increasing the survival and proliferation of corneal epithelial progenitors and by suppressing corneal surface inflammation.

[0011] US2016 / 0075750 (Prockop et al.) described a method for producing a protein or polypeptide, such as TSG-6 protein, in mammalian cells suspended in a protein-free medium containing at least one agent that inhibits the production of hyaluronic acid, hyaluronan, or salts thereof.

[0012] The present invention has been devised in light of the above considerations. Summary of the Invention

[0013] The present disclosure relates to a LINK_TSG6 polypeptide for use in the treatment or prevention of ocular surface disorders such as dry eye disease or other ocular surface disorders associated with corneal lesions similar to those observed in dry eye disease.The inventors have discovered that this polypeptide can reduce or prevent the signs and symptoms of dry eye disease in a dose-dependent manner.Surprisingly, the inventors have discovered that this polypeptide is more potent in reducing corneal epithelial defects than full-length TSG-6.

[0014] In some cases, the treatment or prevention of dry eye disease described herein includes one or more effects selected from the group consisting of healing corneal epithelial defects, increasing tear production, suppressing inflammation, and increasing or maintaining the number of conjunctival goblet cells. The suppression of inflammation can include reducing the production of one or more proinflammatory cytokines, optionally selected from TNFα, IFNγ, IL-6, and IL-1β, in the cornea, the orbital lacrimal gland, or both the cornea and the orbital lacrimal gland.

[0015] Treatment or prevention of dry eye disease may include healing of corneal epithelial defects, increasing tear production, suppressing inflammation, or increasing or maintaining the number of conjunctival goblet cells compared to the corneal epithelial defects, tear production, inflammation, or number of conjunctival goblet cells before administration of the LINK_TSG6 polypeptide and compared to administration of a PBS vehicle.

[0016] Treatment or prevention of dry eye disease may include healing of corneal epithelial defects, increasing tear production, suppressing inflammation, or increasing or maintaining the number of conjunctival goblet cells compared to the corneal epithelial defects, tear production, inflammation, or number of conjunctival goblet cells in control individuals treated with full-length TSG-6 protein.

[0017] In some embodiments described herein, the treatment comprises topical administration of a LINK_TSG6 polypeptide to the eye. The LINK_TSG6 polypeptide can be formulated as eye drops. The treatment can comprise administering eye drops comprising LINK_TSG6. The LINK_TSG6 polypeptide can be formulated with, or the treatment can involve simultaneous administration of, prednisolone, cyclosporine, Lifitegrast (Xiidra™), artificial tears, or any combination thereof.

[0018] In some embodiments, the treatment comprises administering the LINK_TSG6 polypeptide twice per day. In some embodiments, the treatment comprises administering the LINK_TSG6 polypeptide more than twice per day. In some cases, the treatment comprises administering the LINK_TSG6 polypeptide less than four times per day, or less than three times per day. In some cases, the treatment comprises administering the LINK_TSG6 polypeptide once per day. In some cases, the treatment comprises administering the LINK_TSG6 polypeptide less frequently than once per day, for example, once every two days, once every three days, once per week, or once every two weeks.

[0019] Treatment may involve administration of 10 to 200 μg of LINK_TSG6 per eye, for example, 100 to 200 μg of LINK_TSG6 per eye, 100 to 150 μg of LINK_TSG6 per eye, or 120 to 150 μg of LINK_TSG6 per eye. Preferably, treatment involves administration of about 120 μg to 150 μg of LINK_TSG6 polypeptide per eye, or about 12 to 15 μg of LINK_TSG6 per eye.

[0020] The treatment or prevention of ocular surface disorders such as dry eye disease disclosed herein can be applied to dry eye disease associated with any cause.The individual undergoing treatment may have conditions associated with the increased incidence of dry eye disease, such as Sjogren's syndrome, rheumatoid arthritis or diabetes.In some cases, the individual has type 1 diabetes or type 2 diabetes.The individual may be at risk of developing ocular surface disorders such as dry eye disease due to aging, exposure to air pollution, and / or increased use of visual devices (for example, smartphones, computers, tablets).

[0021] Treatment may involve administration of a LINK_TSG6 polypeptide comprising, consisting of, or consisting essentially of (i) the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:9, or (ii) an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:7 or 9.

[0022] Also disclosed herein is a method for treating or preventing ocular surface disorders, such as dry eye disease. This method may involve administering a therapeutically effective amount of LINK_TSG6 to a patient in need thereof. This method may involve topical administration of LINK_TSG6 to the eye.

[0023] Another aspect disclosed herein is the use of a LINK_TSG6 polypeptide in the manufacture of a medicament for the treatment or prevention of ocular surface disorders, such as dry eye disease. The medicament can be formulated for topical administration to the eye, such as eye drops.

[0024] A further embodiment disclosed herein is a pharmaceutical composition comprising a LINK_TSG6 polypeptide. The pharmaceutical composition may comprise a LINK_TSG6 polypeptide solubilized in saline. The pharmaceutical composition may comprise a LINK_TSG6 polypeptide solubilized in phosphate buffered saline. In some embodiments, the pharmaceutical composition comprises at least 2000 μg / ml, 2100 μg / ml, 2200 μg / ml, 2300 μg / ml, 2400 μg / ml, 2500 μg / ml, 2600 μg / ml, 2700 μg / ml, 2800 μg / ml, 2900 μg / ml, 3000 μg / ml, 3100 μg / ml, 3200 μg / ml, 3300 μg / ml, or more than 3300 μg / ml of LINK_TSG6. In some embodiments, the pharmaceutical composition comprises at least 200 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, 1000 μg / ml, 1200 μg / ml, 1300 μg / ml, 1400 μg / ml, 1500 μg / ml, 1600 μg / ml, 1700 μg / ml, 1800 μg / ml 1900 μg / ml, 2000 μg / ml of LINK_TSG6. In some embodiments, the pharmaceutical composition contains at least 200 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, 1000 μg / ml, 1200 μg / ml, 1300 μg / ml, 1400 μg / ml, 1500 μg / ml, 1600 μg / ml, 1700 μg / ml, 1800 μg / ml, 1900 μg / ml, or 2000 μg / ml of LINK_TSG6. Preferably, the pharmaceutical formulation contains at least 2000 μg / ml of LINK_TSG6. Such formulations are useful for delivering about 120 μg to about 150 μg of LINK_TSG6 per drop.

[0025] The pharmaceutical composition may be an eye drop formulation. The eye drop formulation may contain 1500 μg / ml to 3500 μg / ml, 1500 μg / ml to 3000 μg / ml, 2000 μg / ml to 3000 μg / ml, or 2400 μg / ml to 3000 μg / ml. Preferably, the eye drop formulation contains about 2400 μg / ml to about 3000 μg / ml. In some cases, the eye drop formulation contains at least 2200 μg / ml, at least 2300 μg / ml, at least 2400 μg / ml, at least 2500 μg / ml, at least 2600 μg / ml, at least 2700 μg / ml, at least 2800 μg / ml, at least 2900 μg / ml, at least 3000 μg / ml, or more than 3000 μg / ml. The eye drop formulation may further comprise prednisolone, cyclosporine, Lifitegrast (Xiidra™) or artificial tears. The eye drop formulation may comprise a pharmaceutically acceptable carrier. The eye drop formulation may comprise a LINK_TSG6 polypeptide solubilized in saline. The eye drop formulation may comprise a LINK_TSG6 polypeptide solubilized in phosphate buffered saline.

[0026] The present invention includes combinations of the described embodiments and preferred features except where such combinations are expressly not permitted or explicitly avoided. [Brief explanation of the drawings]

[0027] BRIEF DESCRIPTION OF THE DRAWINGS Embodiments and experiments illustrating the principles of the present invention will now be described with reference to the accompanying drawings in which: FIG. [Figure 1-1] Sequences relevant to the present disclosure. [Figure 1-2] Sequences relevant to the present disclosure. [Figure 1-3] Sequences relevant to the present disclosure. [Figure 1-4] Sequences relevant to the present disclosure. [Figure 1-5] Sequences relevant to the present disclosure. [Figure 1-6] Sequences relevant to the present disclosure. [Figure 1-7] Sequences relevant to the present disclosure. [Figure 2A]LINK_TSG6 reduces the symptoms of dry eye disease. A. Ocular staining score after Lissamine Green staining; B. Aqueous tear production determined by phenol red thread test; C. Proinflammatory cytokine mRNA levels determined by real-time RT-PCR; D. Conjunctival goblet cell counts in conjunctival sections stained with periodic acid-Schiff staining. For A and B, significance was determined by Wilcoxon paired signed-rank test for comparisons between pre- and post-treatment, and Mann-Whitney U test for comparisons between PBS and LINK_TSG6. ns = not significant, p > 0.05; ** = p < 0.01; *** = p < 0.001. For C and D, significance was determined by one-way analysis of variance and Tukey's multiple comparisons test. ns=not significant, p>0.05, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. [Figure 2B] LINK_TSG6 reduces the symptoms of dry eye disease. A. Ocular staining score after Lissamine Green staining; B. Aqueous tear production determined by phenol red thread test; C. Proinflammatory cytokine mRNA levels determined by real-time RT-PCR; D. Conjunctival goblet cell counts in conjunctival sections stained with periodic acid-Schiff staining. For A and B, significance was determined by Wilcoxon paired signed-rank test for comparisons between pre- and post-treatment, and Mann-Whitney U test for comparisons between PBS and LINK_TSG6. ns = not significant, p > 0.05; ** = p < 0.01; *** = p < 0.001. For C and D, significance was determined by one-way analysis of variance and Tukey's multiple comparisons test. ns=not significant, p>0.05, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. [Figure 2C]LINK_TSG6 reduces the symptoms of dry eye disease. A. Ocular staining score after Lissamine Green staining; B. Aqueous tear production determined by phenol red thread test; C. Proinflammatory cytokine mRNA levels determined by real-time RT-PCR; D. Conjunctival goblet cell counts in conjunctival sections stained with periodic acid-Schiff staining. For A and B, significance was determined by Wilcoxon paired signed-rank test for comparisons between pre- and post-treatment, and Mann-Whitney U test for comparisons between PBS and LINK_TSG6. ns = not significant, p > 0.05; ** = p < 0.01; *** = p < 0.001. For C and D, significance was determined by one-way analysis of variance and Tukey's multiple comparisons test. ns=not significant, p>0.05, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. [Figure 2D] LINK_TSG6 reduces the symptoms of dry eye disease. A. Ocular staining score after Lissamine Green staining; B. Aqueous tear production determined by phenol red thread test; C. Proinflammatory cytokine mRNA levels determined by real-time RT-PCR; D. Conjunctival goblet cell counts in conjunctival sections stained with periodic acid-Schiff staining. For A and B, significance was determined by Wilcoxon paired signed-rank test for comparisons between pre- and post-treatment, and Mann-Whitney U test for comparisons between PBS and LINK_TSG6. ns = not significant, p > 0.05; ** = p < 0.01; *** = p < 0.001. For C and D, significance was determined by one-way analysis of variance and Tukey's multiple comparisons test. ns=not significant, p>0.05, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. [Figure 3A]LINK_TSG6 reduces the symptoms of dry eye disease in a dose-dependent manner. A. Quantification of corneal epithelial defects from Lissamine Green-stained corneas. LINK_TSG6 at 1 μg and 0.1 μg was effective in reducing corneal epithelial defects, but LINK_TSG6 at 0.01 μg did not significantly improve corneal epithelial defects. B. Quantification of aqueous tear production using the phenol red cotton thread test. LINK_TSG6 at 1 μg and 0.1 μg was both effective in increasing tear production, but LINK_TSG6 at 0.01 μg did not significantly improve tear production. C. Quantification of proinflammatory cytokines using real-time RT-PCR analysis. LINK_TSG6 at 1 μg was most effective in suppressing TNF-α expression. In A and B, significance was determined by Wilcoxon paired signed-rank test. *=p<0.05, **=p<0.01, ns=not significant, p>0.05. In C, significance was determined by one-way ANOVA and Tukey's multiple comparison test. ns=not significant, p>0.05, *=p<0.05, **=p<0.01***=p<0.001, ****=p<0.0001. [Figure 3B]LINK_TSG6 reduces the symptoms of dry eye disease in a dose-dependent manner. A. Quantification of corneal epithelial defects from Lissamine Green-stained corneas. LINK_TSG6 at 1 μg and 0.1 μg was effective in reducing corneal epithelial defects, but LINK_TSG6 at 0.01 μg did not significantly improve corneal epithelial defects. B. Quantification of aqueous tear production using the phenol red cotton thread test. LINK_TSG6 at 1 μg and 0.1 μg was both effective in increasing tear production, but LINK_TSG6 at 0.01 μg did not significantly improve tear production. C. Quantification of proinflammatory cytokines using real-time RT-PCR analysis. LINK_TSG6 at 1 μg was most effective in suppressing TNF-α expression. In A and B, significance was determined by Wilcoxon paired signed-rank test. *=p<0.05, **=p<0.01, ns=not significant, p>0.05. In C, significance was determined by one-way ANOVA and Tukey's multiple comparison test. ns=not significant, p>0.05, *=p<0.05, **=p<0.01***=p<0.001, ****=p<0.0001. [Figure 3C]LINK_TSG6 reduces the symptoms of dry eye disease in a dose-dependent manner. A. Quantification of corneal epithelial defects from Lissamine Green-stained corneas. LINK_TSG6 at 1 μg and 0.1 μg was effective in reducing corneal epithelial defects, but LINK_TSG6 at 0.01 μg did not significantly improve corneal epithelial defects. B. Quantification of aqueous tear production using the phenol red cotton thread test. LINK_TSG6 at 1 μg and 0.1 μg was both effective in increasing tear production, but LINK_TSG6 at 0.01 μg did not significantly improve tear production. C. Quantification of proinflammatory cytokines using real-time RT-PCR analysis. LINK_TSG6 at 1 μg was most effective in suppressing TNF-α expression. In A and B, significance was determined by Wilcoxon paired signed-rank test. *=p<0.05, **=p<0.01, ns=not significant, p>0.05. In C, significance was determined by one-way ANOVA and Tukey's multiple comparison test. ns=not significant, p>0.05, *=p<0.05, **=p<0.01***=p<0.001, ****=p<0.0001. [Figure 4A] Dose-response data for LINK_TSG6. A. Quantification of corneal epithelial defects from Lissamine Green-stained corneas by ocular staining. B. Quantification of aqueous tear production by phenol red-thread test. C. Proinflammatory cytokine mRNA levels determined by real-time RT-PCR. D. Conjunctival goblet cell counts in PAS-stained conjunctival sections. Significance determined by one-way ANOVA and Tukey's multiple comparison test; ns = not significant, p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. [Figure 4B]Dose-response data for LINK_TSG6. A. Quantification of corneal epithelial defects from Lissamine Green-stained corneas by ocular staining. B. Quantification of aqueous tear production by phenol red-thread test. C. Proinflammatory cytokine mRNA levels determined by real-time RT-PCR. D. Conjunctival goblet cell counts in PAS-stained conjunctival sections. Significance determined by one-way ANOVA and Tukey's multiple comparison test; ns = not significant, p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. [Figure 4C] Dose-response data for LINK_TSG6. A. Quantification of corneal epithelial defects from Lissamine Green-stained corneas by ocular staining. B. Quantification of aqueous tear production by phenol red-thread test. C. Proinflammatory cytokine mRNA levels determined by real-time RT-PCR. D. Conjunctival goblet cell counts in PAS-stained conjunctival sections. Significance determined by one-way ANOVA and Tukey's multiple comparison test; ns = not significant, p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. [Figure 4D] Dose-response data for LINK_TSG6. A. Quantification of corneal epithelial defects from Lissamine Green-stained corneas by ocular staining. B. Quantification of aqueous tear production by phenol red-thread test. C. Proinflammatory cytokine mRNA levels determined by real-time RT-PCR. D. Conjunctival goblet cell counts in PAS-stained conjunctival sections. Significance determined by one-way ANOVA and Tukey's multiple comparison test; ns = not significant, p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. [Figure 5A]LINK_TSG6 is more effective than full-length human recombinant TSG-6 (FL TSG6) in reducing the symptoms of dry eye disease. A. Ocular staining score after Lissamine Green staining; B. Aqueous tear production determined by phenol red thread test; C. Conjunctival goblet cell count in PAS-stained conjunctival sections; D. Histological analysis of CD3 immunostaining in the lacrimal gland, as represented by the number of foci with CD3 cell infiltration. Equimolar doses (92, 9.2, and 0.92 pmol) corresponding to 1, 0.1, and 0.01 μg of LINK_TSG6 and 3.27, 0.327, and 0.0327 μg of FL TSG-6, respectively, are compared. Significance determined by one-way analysis of variance and Tukey's multiple comparison test. *=p<0.05. [Figure 5B] LINK_TSG6 is more effective than full-length human recombinant TSG-6 (FL TSG6) in reducing the symptoms of dry eye disease. A. Ocular staining score after Lissamine Green staining; B. Aqueous tear production determined by phenol red thread test; C. Conjunctival goblet cell count in PAS-stained conjunctival sections; D. Histological analysis of CD3 immunostaining in the lacrimal gland, as represented by the number of foci with CD3 cell infiltration. Equimolar doses (92, 9.2, and 0.92 pmol) corresponding to 1, 0.1, and 0.01 μg of LINK_TSG6 and 3.27, 0.327, and 0.0327 μg of FL TSG-6, respectively, are compared. Significance determined by one-way analysis of variance and Tukey's multiple comparison test. *=p<0.05. [Figure 5C]LINK_TSG6 is more effective than full-length human recombinant TSG-6 (FL TSG6) in reducing the symptoms of dry eye disease. A. Ocular staining score after Lissamine Green staining; B. Aqueous tear production determined by phenol red thread test; C. Conjunctival goblet cell count in PAS-stained conjunctival sections; D. Histological analysis of CD3 immunostaining in the lacrimal gland, as represented by the number of foci with CD3 cell infiltration. Equimolar doses (92, 9.2, and 0.92 pmol) corresponding to 1, 0.1, and 0.01 μg of LINK_TSG6 and 3.27, 0.327, and 0.0327 μg of FL TSG-6, respectively, are compared. Significance determined by one-way analysis of variance and Tukey's multiple comparison test. *=p<0.05. [Figure 5D] LINK_TSG6 is more effective than full-length human recombinant TSG-6 (FL TSG6) in reducing the symptoms of dry eye disease. A. Ocular staining score after Lissamine Green staining; B. Aqueous tear production determined by phenol red thread test; C. Conjunctival goblet cell count in PAS-stained conjunctival sections; D. Histological analysis of CD3 immunostaining in the lacrimal gland, as represented by the number of foci with CD3 cell infiltration. Equimolar doses (92, 9.2, and 0.92 pmol) corresponding to 1, 0.1, and 0.01 μg of LINK_TSG6 and 3.27, 0.327, and 0.0327 μg of FL TSG-6, respectively, are compared. Significance determined by one-way analysis of variance and Tukey's multiple comparison test. *=p<0.05. [Figure 6A] Evaluation of LINK_TSG6 and comparison with Restasis in a desiccation injury-induced dry eye model. A. Eye staining before desiccation injury induction and before treatment, B. Eye staining after desiccation injury and treatment, C. Tear production before desiccation injury induction and before treatment, D. Tear production after desiccation injury and after treatment, E. Th1 cells in draining cervical lymph nodes, F. Th17 cells in draining cervical lymph nodes, ns=not significant, p>0.05, *=p<0.05, **=p<0.01***=p<0.001, ****=p<0.0001. [Figure 6B]Evaluation of LINK_TSG6 and comparison with Restasis in a desiccation injury-induced dry eye model. A. Eye staining before desiccation injury induction and before treatment, B. Eye staining after desiccation injury and treatment, C. Tear production before desiccation injury induction and before treatment, D. Tear production after desiccation injury and after treatment, E. Th1 cells in draining cervical lymph nodes, F. Th17 cells in draining cervical lymph nodes, ns=not significant, p>0.05, *=p<0.05, **=p<0.01***=p<0.001, ****=p<0.0001. [Figure 6C] Evaluation of LINK_TSG6 and comparison with Restasis in a desiccation injury-induced dry eye model. A. Eye staining before desiccation injury induction and before treatment, B. Eye staining after desiccation injury and treatment, C. Tear production before desiccation injury induction and before treatment, D. Tear production after desiccation injury and after treatment, E. Th1 cells in draining cervical lymph nodes, F. Th17 cells in draining cervical lymph nodes, ns=not significant, p>0.05, *=p<0.05, **=p<0.01***=p<0.001, ****=p<0.0001. [Figure 6D] Evaluation of LINK_TSG6 and comparison with Restasis in a desiccation injury-induced dry eye model. A. Eye staining before desiccation injury induction and before treatment, B. Eye staining after desiccation injury and treatment, C. Tear production before desiccation injury induction and before treatment, D. Tear production after desiccation injury and after treatment, E. Th1 cells in draining cervical lymph nodes, F. Th17 cells in draining cervical lymph nodes, ns=not significant, p>0.05, *=p<0.05, **=p<0.01***=p<0.001, ****=p<0.0001. [Figure 6E]Evaluation of LINK_TSG6 and comparison with Restasis in a desiccation injury-induced dry eye model. A. Eye staining before desiccation injury induction and before treatment, B. Eye staining after desiccation injury and treatment, C. Tear production before desiccation injury induction and before treatment, D. Tear production after desiccation injury and after treatment, E. Th1 cells in draining cervical lymph nodes, F. Th17 cells in draining cervical lymph nodes, ns=not significant, p>0.05, *=p<0.05, **=p<0.01***=p<0.001, ****=p<0.0001. [Figure 6F] Evaluation of LINK_TSG6 and comparison with Restasis in a desiccation injury-induced dry eye model. A. Eye staining before desiccation injury induction and before treatment, B. Eye staining after desiccation injury and treatment, C. Tear production before desiccation injury induction and before treatment, D. Tear production after desiccation injury and after treatment, E. Th1 cells in draining cervical lymph nodes, F. Th17 cells in draining cervical lymph nodes, ns=not significant, p>0.05, *=p<0.05, **=p<0.01***=p<0.001, ****=p<0.0001. [Figure 7A] Evaluation of LINK_TSG6 in pre-dried mice. A. Ocular staining, B. Tear production, C. Conjunctival goblet cell count in PAS-stained conjunctival sections after treatment, D. MMP-9 mRNA levels at the ocular surface. ns=not significant, p>0.05, *=p<0.05, **=p<0.01***=p<0.001, ****=p<0.0001. A and B: Wilcoxon paired signed-rank test for comparisons before and after treatment. C and D: One-way ANOVA and Tukey's multiple comparisons test. [Figure 7B] Evaluation of LINK_TSG6 in pre-dried mice. A. Ocular staining, B. Tear production, C. Conjunctival goblet cell count in PAS-stained conjunctival sections after treatment, D. MMP-9 mRNA levels at the ocular surface. ns=not significant, p>0.05, *=p<0.05, **=p<0.01***=p<0.001, ****=p<0.0001. A and B: Wilcoxon paired signed-rank test for comparisons before and after treatment. C and D: One-way ANOVA and Tukey's multiple comparisons test. [Figure 7C] Evaluation of LINK_TSG6 in pre-dried mice. A. Ocular staining, B. Tear production, C. Conjunctival goblet cell count in PAS-stained conjunctival sections after treatment, D. MMP-9 mRNA levels at the ocular surface. ns=not significant, p>0.05, *=p<0.05, **=p<0.01***=p<0.001, ****=p<0.0001. A and B: Wilcoxon paired signed-rank test for comparisons before and after treatment. C and D: One-way ANOVA and Tukey's multiple comparisons test. [Figure 7D] Evaluation of LINK_TSG6 in pre-dried mice. A. Ocular staining, B. Tear production, C. Conjunctival goblet cell count in PAS-stained conjunctival sections after treatment, D. MMP-9 mRNA levels at the ocular surface. ns=not significant, p>0.05, *=p<0.05, **=p<0.01***=p<0.001, ****=p<0.0001. A and B: Wilcoxon paired signed-rank test for comparisons before and after treatment. C and D: One-way ANOVA and Tukey's multiple comparisons test. [Figure 8A] Comparison of LINK_TSG6 and Restasis in a dry eye disease model. A. Lissamine green staining score before treatment. B. Lissamine green staining score after treatment. C. Quantification of aqueous tear production by phenol red thread test before treatment. D. Quantification of aqueous tear production by phenol red thread test after treatment. E. Conjunctival goblet cell count in PAS-stained conjunctival sections after treatment. F. Histological analysis of CD3 immunostaining in lacrimal glands. ns=not significant, p>0.05, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001, one-way ANOVA and Tukey's multiple comparison test. [Figure 8B]Comparison of LINK_TSG6 and Restasis in a dry eye disease model. A. Lissamine green staining score before treatment. B. Lissamine green staining score after treatment. C. Quantification of aqueous tear production by phenol red thread test before treatment. D. Quantification of aqueous tear production by phenol red thread test after treatment. E. Conjunctival goblet cell count in PAS-stained conjunctival sections after treatment. F. Histological analysis of CD3 immunostaining in lacrimal glands. ns=not significant, p>0.05, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001, one-way ANOVA and Tukey's multiple comparison test. [Figure 8C] Comparison of LINK_TSG6 and Restasis in a dry eye disease model. A. Lissamine green staining score before treatment. B. Lissamine green staining score after treatment. C. Quantification of aqueous tear production by phenol red thread test before treatment. D. Quantification of aqueous tear production by phenol red thread test after treatment. E. Conjunctival goblet cell count in PAS-stained conjunctival sections after treatment. F. Histological analysis of CD3 immunostaining in lacrimal glands. ns=not significant, p>0.05, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001, one-way ANOVA and Tukey's multiple comparison test. [Figure 8D] Comparison of LINK_TSG6 and Restasis in a dry eye disease model. A. Lissamine green staining score before treatment. B. Lissamine green staining score after treatment. C. Quantification of aqueous tear production by phenol red thread test before treatment. D. Quantification of aqueous tear production by phenol red thread test after treatment. E. Conjunctival goblet cell count in PAS-stained conjunctival sections after treatment. F. Histological analysis of CD3 immunostaining in lacrimal glands. ns=not significant, p>0.05, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001, one-way ANOVA and Tukey's multiple comparison test. [Figure 8E]Comparison of LINK_TSG6 and Restasis in a dry eye disease model. A. Lissamine green staining score before treatment. B. Lissamine green staining score after treatment. C. Quantification of aqueous tear production by phenol red thread test before treatment. D. Quantification of aqueous tear production by phenol red thread test after treatment. E. Conjunctival goblet cell count in PAS-stained conjunctival sections after treatment. F. Histological analysis of CD3 immunostaining in lacrimal glands. ns=not significant, p>0.05, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001, one-way ANOVA and Tukey's multiple comparison test. [Figure 8F] Comparison of LINK_TSG6 and Restasis in a dry eye disease model. A. Lissamine green staining score before treatment. B. Lissamine green staining score after treatment. C. Quantification of aqueous tear production by phenol red thread test before treatment. D. Quantification of aqueous tear production by phenol red thread test after treatment. E. Conjunctival goblet cell count in PAS-stained conjunctival sections after treatment. F. Histological analysis of CD3 immunostaining in lacrimal glands. ns=not significant, p>0.05, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001, one-way ANOVA and Tukey's multiple comparison test. DETAILED DESCRIPTION OF THE INVENTION

[0028] Aspects and embodiments of the present invention will now be described with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0029] The present invention provides a method for treating or preventing ocular surface disorders, such as dry eye disease, comprising administering a LINK_TSG6 polypeptide to a subject. The inventors have shown that LINK_TSG6 is more potent than recombinant human TSG-6 in reducing corneal epithelial lesions and treating or reducing signs or symptoms of ocular surface disorders, such as dry eye disease. Without wishing to be bound by theory, this may be the result of improved tissue penetration (due to the smaller size of the molecule), differences in biodistribution compared to the full-length protein, or a lack of signaling or enzymatic activity affected by the CUB_C domain. In this regard, full-length TSG-6 binds to HA more effectively than LINK_TSG6, likely due to the cooperative nature of the interaction (presumably involving the CUB_C domain) (Baranova et al., 2011 J. Biol. Chem. 286, 25675-25686). HA has been implicated in several anti-inflammatory effects (see Day and Milner 2019).

[0030] TSG-6 (tumor necrosis factor-stimulated gene-6) TSG-6 is a secreted protein composed of two modular domains. TSG-6 is not normally constitutively expressed in adult tissues but is induced in response to inflammatory mediators. During inflammation, TSG-6 is an endogenous tissue protector. Many of the immunomodulatory and tissue-protective effects of MSCs are mediated by the secretion of TSG-6.

[0031] Recombinant full-length TSG-6 protein has been shown to have anti-inflammatory and tissue-protective effects in a wide range of disease models, including atherosclerosis, myocardial infarction, hypertrophic scarring, colitis, autoimmune diabetes, rheumatoid arthritis, traumatic brain injury, or acute lung injury.

[0032] Full-length TSG-6 is difficult to produce, insoluble, and prone to aggregation. As disclosed herein, these disadvantages are not associated with LINK_TSG6, a short recombinant peptide containing the LINK module of human TSG-6. This short polypeptide is easier to produce, more soluble, and more stable in solution than full-length TSG6.

[0033] The LINK_TSG6 polypeptides disclosed herein comprise only the Link module of human or mammalian TSG-6. In some embodiments, the TSG-6 polypeptide comprises or consists essentially of the amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 5. The Link module corresponds to residues 37-128 of SEQ ID NOs: 2 and 5 and is set forth in SEQ ID NO: 7. In some preferred embodiments, the LINK_TSG6 polypeptides useful in the invention do not include part or all of residues 1-35 of the full-length TSG6 sequence of SEQ ID NO: 2 or 5 at the N-terminus.

[0034] The Link module is responsible for hyaluronan (HA)-binding activity, chondroitin-4-sulfate-binding activity, aggrecan-binding activity, inhibitor-inhibitor (IaI)-binding activity, bikunin-binding activity, versican-binding activity, dermatan sulfate-binding activity, pentraxin-3-binding activity, thrombospondin-1-binding activity, thrombospondin-2-binding activity, fibronectin-binding activity, heparin / heparan sulfate-binding activity, RANKL-binding activity of TSG-6, bone morphogenetic protein (BMP)-2-binding activity, BMP-4-binding activity, BMP-5-binding activity, BMP-6-binding activity, BMP-7-binding activity, BMP-13-binding activity, BMP-14-binding activity, CXCL4-binding activity, CXCL6-binding activity, CXCL8-binding activity, CXCL11-binding activity, CXCL12-binding activity, CCL2-binding activity, CCL5-binding activity, CCL7-binding activity, CCL19-binding activity, CCL21-binding activity, or CCL27-binding activity. .

[0035] LINK_TSG6 has hyaluronan (HA)-binding activity, chondroitin-4-sulfate-binding activity, aggrecan-binding activity, inhibitor-inhibitor (IaI)-binding activity, bikunin-binding activity, versican-binding activity, dermatan sulfate-binding activity, pentraxin-3-binding activity, thrombospondin-1-binding activity, thrombospondin-2-binding activity, fibronectin-binding activity, heparin / heparan sulfate-binding activity, RANKL-binding activity, and bone morphogenetic protein (BMP)-2-binding activity. The fragment may be a fragment of TSG-6 that exhibits one or more of the following binding activities: BMP-4 binding activity, BMP-5 binding activity, BMP-6 binding activity, BMP-7 binding activity, BMP-13 binding activity, BMP-14 binding activity, CXCL4 binding activity, CXCL6 binding activity, CXCL8 binding activity, CXCL11 binding activity, CXCL12 binding activity, CCL2 binding activity, CCL5 binding activity, CCL7 binding activity, CCL19 binding activity, CCL21 binding activity, or CCL27 binding activity.

[0036] The LINK domain of TSG-6 (LINK_TSG6) may be a region of full-length TSG-6 N-terminal to the CUB_C domain, and therefore the LINK_TSG6 protein may lack all or part of the CUB_C domain.

[0037] The LINK domain can comprise the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9. The LINK_TSG6 polypeptide comprises, consists of, or consists essentially of (i) the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9, or (ii) an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 7 or 9.

[0038] LINK_TSG6 is preferably a polypeptide comprising or consisting of (i) the amino acid sequence of SEQ ID NO: 7 or 9, or (ii) an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 7 or 9.

[0039] Thus, the LINK_TSG6 polypeptide may comprise: (a) the amino acid sequence of SEQ ID NO: 7; (b) a variant thereof having at least 50% identity to the amino acid sequence of SEQ ID NO: 7 and having RANKL-binding activity; or (c) A fragment of either (a) or (b) having CXCL4 binding activity, CXCL6 binding activity, CXCL8 binding activity, CXCL11 binding activity, CXCL12 binding activity, CCL2 binding activity, CCL5 binding activity, CCL7 binding activity, CCL19 binding activity, CCL21 binding activity or CCL27 binding activity.

[0040] The LINK_TSG6 polypeptide may consist of or consist essentially of the sequence shown in SEQ ID NO:7.

[0041] SEQ ID NO: 9 shows a recombinant polypeptide comprising the Link module of TSG-6 (LINK_TSG6). Thus, the TSG-6 polypeptide used in the present invention may preferably comprise: (a) the amino acid sequence of SEQ ID NO: 9; (b) a variant thereof having at least 50% identity to the amino acid sequence of SEQ ID NO: 9 and having RANKL-binding activity; or (c) A fragment of either (a) or (b) having CXCL4 binding activity, CXCL6 binding activity, CXCL8 binding activity, CXCL11 binding activity, CXCL12 binding activity, CCL2 binding activity, CCL5 binding activity, CCL7 binding activity, CCL19 binding activity, CCL21 binding activity or CCL27 binding activity.

[0042] The LINK_TSG6 polypeptide preferably consists of or consists essentially of the sequence shown in SEQ ID NO:9.

[0043] In some embodiments described herein, the LINK_TSG6 polypeptide is not conjugated to an active agent, such as an antibody or antigen-binding fragment. For example, in some cases, the LINK_TSG6 polypeptide is not conjugated to an IL-17A antibody or a fragment thereof. In some cases, the LINK_TSG6 polypeptide comprises or consists of a single copy of the LINK_TSG6 polypeptide, such as a single copy of SEQ ID NO:7 or SEQ ID NO:9. In other words, in these cases, the LINK_TSG6 polypeptide does not comprise multiple LINK module sequences, such as multiple copies of SEQ ID NO:7 or SEQ ID NO:9. In some cases, the LINK_TSG6 polypeptide does not comprise a His tag, such as a 6xHIS tag.

[0044] Amino acid identity can be calculated using any suitable algorithm. For example, the UWGCG package provides the BESTFIT program (for example, used with default settings) that can be used to calculate homology (Devereux et al. (1984) Nucleic Acids Research 12,387-395). PILEUP and BLAST algorithms can be used to calculate homology or aligned sequences (for example, as described in Altschul (1993) J. Mol. Evol. 36,290-300, Altschul et al. (1990) J. Mol. Biol. 215,403-10) to identify equivalent or corresponding sequences (usually with default settings).

[0045] Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in a database sequence, match or meet some positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating searches to find HSPs containing them. Word hits are extended in both directions along each sequence for as long as the cumulative alignment score can be increased. Extension of word hits in each direction is stopped when the cumulative alignment score drops by a quantity X from its achieved maximum, when the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89, 10915-10919), alignment (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

[0046] The BLAST algorithm performs statistical analysis of the similarity between two sequences. For example, see Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90, 5873-5787. One measure of similarity provided by the BLAST algorithm is the minimum total probability (P(N)), which provides an indication of the probability that a match between two polynucleotide or amino acid sequences occurs by chance. For example, if the minimum total probability in the comparison of a first sequence with a second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001, a sequence is considered to be similar to another sequence.

[0047] The mutant sequence typically differs by at least 1, 2, 5, 10, 20, 30, 50, or more mutations (which may be amino acid substitutions, deletions, or insertions). For example, 1 to 50, 2 to 30, 3 to 20, or 5 to 10 amino acid substitutions, deletions, or insertions can be made. The modified polypeptide generally retains CXCL4-binding activity, CXCL6-binding activity, CXCL8-binding activity, CXCL11-binding activity, CXCL12-binding activity, CCL2-binding activity, CCL5-binding activity, CCL7-binding activity, CCL19-binding activity, CCL21-binding activity, or CCL27-binding activity, preferably in a dose-dependent manner. Substitutions are preferably conservative substitutions, e.g., according to the table below. Amino acids in the same block in the second column and preferably in the same row in the third column can be substituted for each other. [Table 1]

[0048] The LINK_TSG6 polypeptide used in the present invention is typically at least 10, for example at least 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or more amino acids in length, up to 100, 150, 200 or 250 amino acids in length, so long as it retains the CXCL4-binding activity, CXCL6-binding activity, CXCL8-binding activity, CXCL11-binding activity, CXCL12-binding activity, CCL2-binding activity, CCL5-binding activity, CCL7-binding activity, CCL19-binding activity, CCL21-binding activity or CCL27-binding activity of TSG-6. Preferably, the polypeptide comprises the sequence set forth in SEQ ID NO:7. Fragments of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:9 preferably contain residues shown to be essential for hyaluronan binding in Mahoney et al. (2001) J. Biol. Chem. 276, 22764-22771 and Blundell et al. (2003) J. Biol. Chem. 278, 49261-49270. Fragments of the amino acid sequence of SEQ ID NO:2 or 5 preferably contain residues Lys-46 and / or Tyr-47 and / or Tyr-94 and / or Phe-105 and / or Tyr-113 of SEQ ID NO:2 or 5. Most preferably, fragments of SEQ ID NO:2 or 5 contain each of residues Lys-46, Tyr-47, Tyr-94, Phe-105, and Tyr-113 of SEQ ID NO:2 or 5.

[0049] Fragments of the amino acid sequence of SEQ ID NO: 7 can be used in the present invention. Such fragments preferably contain residues Lys-10 and / or Tyr-11 and / or Tyr-58 and / or Phe-69 and / or Tyr-77 of SEQ ID NO: 7. Most preferably, the fragment of SEQ ID NO: 7 contains each of residues Lys-10, Tyr-11, Tyr-58, Phe-69 and Tyr-77 of SEQ ID NO: 7.

[0050] A fragment of the amino acid sequence of SEQ ID NO:9 preferably comprises residues Lys-11 and / or Tyr-12 and / or Tyr-59 and / or Phe-70 and / or Tyr-78 of SEQ ID NO:9. Most preferably, a fragment of SEQ ID NO:9 comprises each of residues Lys-11, Tyr-12, Tyr-59, Phe-70 and Tyr-78 of SEQ ID NO:9.

[0051] The TSG-6 polypeptides used in the present invention may be chemically modified, for example, post-translationally modified. For example, they may be glycosylated, phosphorylated, or contain modified amino acid residues. They may be modified by adding histidine residues to aid their purification, or by adding transmembrane sequences to facilitate insertion into cell membranes. Such modified polypeptides are within the scope of the term "polypeptide" as used herein.

[0052] Suitable assays for determining the ability of TSG-6 polypeptides to bind to HA, chondroitin-4-sulfate, aggrecan, inter-inhibitor (IaI), bikunin, versican, dermatan sulfate, pentraxin-3, thrombospondin-1, heparin / heparan sulfate, fibronectin, and RANKL are well known in the art (Getting et al. (2002) J. Biol. Chem. 277, 51068-51076; Mahoney et al. (2005) J. Biol. Chem. 280, 27044-27055; Salustri et al. (2004) Development 131, 1577-1586; Parkar et al. (1997) FEBS Lett. 410, 413-417; Parkar et al. (1998) FEBS Lett. 428, 171-176, Mahoney et al. (2001) J. Biol. Chem. 276, 22764-22771, Nentwich et al. (2002) J. Biol Chem. 211, 15354-15362, Kuznetsova et al. al. (2005) J. Biol. Chem. 280, 30899-30908), Dyer et al. (2014) J. Immunol 192, 2177-2185, Dyer et al. (2016) J. Biol. Chem. 291, 12627-12640, and Mahoney et al. al. (2008) J. Biol. Chem 283, 25952-25962.

[0053] The TSG-6 polypeptide for use in the present invention can be in a substantially isolated form. It will be understood that the polypeptide can be mixed with a carrier or diluent that does not interfere with the intended purpose of the polypeptide and still be considered substantially isolated. The polypeptide for use in the present invention can also be in a substantially purified form, which generally comprises the polypeptide in a preparation, and in which more than 50% by weight, for example, 80%, 90%, 95% or more than 99% by weight of the polypeptide in the preparation is the polypeptide of the present invention.

[0054] The LINK_TSG6 polypeptide for use in the present invention can be a naturally occurring or non-naturally occurring polypeptide. The polypeptide can be isolated from any suitable organism that expresses a TSG-6 polypeptide. The TSG-6 polypeptide can be isolated from humans or other suitable mammals, such as primates, rats, or mice. Alternatively, the TSG-6 polypeptide can be isolated from fish or amphibians. The polypeptide for use in the present invention can also be prepared as a fragment of such an isolated polypeptide.

[0055] Furthermore, the LINK_TSG6 polypeptide can also be produced by synthetic or recombinant means. For example, recombinant LINK_TSG6 polypeptide can be produced by transfecting cells in culture with an expression vector containing a nucleotide sequence encoding the polypeptide operably linked to a suitable control sequence, culturing the cells, and extracting and purifying the LINK_TSG6 polypeptide produced by the cells. Methods for recombinant production of polypeptides are well known in the art (e.g., Sambrook et al., 2001, Molecular Cloning: a laboratory manual, 3 rd edition, Cold Harbour Laboratory Press). Preferably, the LINK_TSG6 polypeptide is produced in bacteria such as E. coli. Preferably, the LINK_TSG6 polypeptide is not produced in CHO cells or other mammalian cells. As will be appreciated by those skilled in the art, proteins and polypeptides produced in bacteria such as E. coli are completely or substantially devoid of glycosylation, whereas glycosylation is a common feature of proteins and polypeptides produced in mammalian cells such as CHO cells. In particular, LINK_TSG6 according to the present disclosure may lack N-linked glycosylation on Asn118 (SEQ ID NO: 2 or SEQ ID NO: 5).

[0056] The amino acid sequence of the LINK_TSG6 polypeptide for use in the present invention can be modified to include non-naturally occurring amino acids or to increase the stability of the compound. If the polypeptide is produced by synthetic means, such amino acids can be introduced during production. The polypeptide can also be modified following either synthetic or recombinant production. In some embodiments, the LINK_TSG6 polypeptide described herein does not include a polyhistidine tag, such as a 6His tag. In some embodiments, the LINK_TSG6 polypeptide described herein does not include a polyhistidine tag at the C-terminus of the polypeptide.

[0057] LINK_TSG6 polypeptides for use in the present invention can also be produced using D-amino acids. In such cases, the amino acids are linked in reverse order, in a C to N orientation. This is conventional in the art for producing such polypeptides.

[0058] A number of side chain modifications are known in the art and can be made to the side chains of the LINK_TSG6 polypeptide, provided that the polypeptide retains corneal defect healing activity.

[0059] Ocular surface disorders The present disclosure relates to the treatment or prevention of ocular surface disorders, including dry eye disease (DED), persistent corneal epithelial damage (non-healing epithelial defects) associated with diabetic keratopathy, neurotrophic keratopathy, exposure keratopathy or limbal coloboma, contact lens / eye drop-induced epithelial erosion, ocular graft-versus-host disease (GVHD), Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), ocular surface dysfunction in patients with glaucoma, corneal wounds resulting from glaucoma surgery, recurrent corneal erosion, superficial punctate keratitis, and superior limbal keratoconjunctivitis.

[0060] Dry eye disease The present disclosure relates to the treatment or prevention of dry eye disease. Dry eye disease (also known as dry eye syndrome or keratoconjunctivitis sicca) is a condition characterized by dry eyes that affects 7-33% of the world's population. Dry eye disease occurs when tears are not produced sufficiently (aqueous dry eye disease) or when tears evaporate rapidly (evaporative dry eye disease). Causes include aging (known to cause atrophy and inflammation of the lacrimal glands), infection, exposure to environmental irritants such as smoke, contact lens use, meibomian gland dysfunction, allergies, pregnancy, Sjögren's syndrome, vitamin A deficiency, laser eye treatment, or as a result of medications such as antihistamines, blood pressure medications, hormone replacement therapy, and some antidepressants. In some cases, dry eye disease results from activities associated with a reduced blink rate, such as using screens on computer monitors, smartphones or tablets, television, or driving. Dry eye disease can result in small abrasions on the ocular surface (i.e., defects in the corneal epithelium). Dry eye disease can result in pathological changes in the corneal epithelium, such as squamous metaplasia and goblet cell loss, which, in severe cases, can lead to corneal erosion, ulceration, neovascularization and scarring, or thinning and perforation. Diagnosis of dry eye disease may involve a standardized dry eye questionnaire (Ocular Surface Disease Index, or OSDI), a 12-item scale to assess symptoms associated with dry eye disease and their impact on vision.

[0061] Dry eye disease (DED) is a condition resulting from dry injury to the ocular surface or inflammatory damage to the lacrimal gland. It is clinically distinct from other trauma-induced ocular disorders, such as chemical burns or injuries resulting from direct exposure of the ocular surface to chemicals (e.g., alcohol), physical or chemical debridement, blunt trauma to the eye, penetrating eye injury, or other ocular wounds. However, these trauma-induced ocular conditions can lead to the subsequent development of dry eye disease.

[0062] Signs and symptoms of dry eye disease include irritation, redness, discharge, eye fatigue, and blurred vision. Signs and symptoms range from mild and occasional to severe and persistent, and if left untreated, can lead to corneal scarring.

[0063] As disclosed herein, the peptide LINK_TSG6 can be used to treat or prevent dry eye disease. Administration of LINK_TSG6 can result in healing of corneal epithelial defects, increased tear production, suppression of inflammation, and / or increased / maintained number of conjunctival goblet cells. Healing of corneal epithelial defects can result in a reduction in the number or size of corneal epithelial defects. Preferably, healing of corneal epithelial defects results in a reduction in the proportion of the corneal surface containing corneal epithelial defects.

[0064] Corneal epithelial defects are areas of epithelial loss (the outermost corneal layer) that can result from mechanical trauma, corneal dryness, neurotrophic corneal edema, postoperative changes, or any other variety of etiologies. LINK_TSG6 can be used to reduce or repair corneal epithelial defects. The presence or absence of a corneal epithelial defect can be determined by scoring. Corneal epithelial defects can be visualized by staining, for example, with lissamine green or fluorescein dye. By applying the stain to the cornea and staining the area of ​​the epithelial defect, the defect can be visualized.

[0065] Increased tear production can be determined by the Schirmer test or the phenol red thread test.

[0066] The number of conjunctival goblet cells can be determined by impression cytology. Cellulose acetate filter paper is pressed against the conjunctival surface to collect the superficial layer, which is then stained with PAS to stain for mucin-secreting goblet cells. The number of goblet cells is calculated on the PAS-stained slide.

[0067] The presence or amount of ocular surface inflammation can be determined by observing conjunctival redness or conjunctival epithelial defects. Inflammation can lead to destruction of the corneal and conjunctival epithelium.

[0068] Inflammatory cytokine levels can be determined in a tear sample from a patient. The tear sample can be obtained using Schirmer tear test strips. Nucleic acids and / or proteins can be extracted from the tear strip by incubating the strip in ammonium bicarbonate and acetone. Inflammatory cytokine levels can be quantified or qualified by RT-PCR or ELISA. Inflammatory cytokines can be selected from IFN-γ, TNF-α, IL-1β, and IL-6. In some cases, inflammatory cytokines can be quantified or qualified by immunoassay. In some cases, MMP9 levels can be determined semiquantitatively (positive, trace, or negative) using the "InflammaDry" kit in human eyes.

[0069] The cornea is the transparent front part of the eye that covers the iris, pupil, and anterior chamber.

[0070] The cornea, with its anterior chamber and lens, refracts light and accounts for approximately two-thirds of the eye's total refractive power. While the cornea contributes most of the eye's focusing power, its focus is fixed. The cornea contains unmyelinated nerve endings that are sensitive to touch, temperature, and chemicals, and touching the cornea causes the eyelids to close involuntary. Because transparency is paramount, a healthy cornea does not require or have blood vessels within it. Instead, oxygen dissolves in tears and then diffuses throughout the cornea, maintaining its health. Similarly, nutrients are transported from tears by diffusion through the outer surface, and aqueous humor is transported through the inner surface. Nutrients also arrive via neurotrophic pathways, supplied by corneal nerves. In humans, the cornea is approximately 11.5 mm in diameter and 0.5–0.6 mm thick at the center and 0.6–0.8 mm thick at the periphery. Its transparency, avascularity, the presence of immature resident immune cells, and immunological privilege make it a highly specialized tissue.

[0071] The methods disclosed herein relate specifically to the corneal epithelium and damage thereto. The corneal epithelium is a very thin (approximately 50 pm) multicellular epithelial tissue layer (non-keratinized stratified squamous epithelium) of rapidly proliferating and regenerating cells that is kept moist by tears. The corneal epithelium is composed of epithelial cells and covers the anterior surface of the cornea. It serves as the first line of defense against the cornea, resisting the free flow of fluid from tears and preventing bacterial invasion into the cornea and the interior of the eye. Irregularities or defects in the corneal epithelium disrupt the smoothness of the air / tear film interface, the most important component of the eye's total refractive power, thereby reducing vision. It is continuous with the conjunctival epithelium and is composed of approximately six layers of cells that are constantly shed in the exposed layer and regenerated by proliferation in the basal layer. Corneal epithelium damage is common in dry eye disease.

[0072] Some methods increase tear production or the thickness of the tear film covering the eye. In some cases, tear retention decreases, causing tears to evaporate quickly from the eye. Tear shedding (also known as lacrimation or epiphora) is the reflex secretion of tears in response to external or internal irritants. Tears are a bodily fluid that may help clean and lubricate the eye in response to irritation. In healthy mammalian eyes, the cornea is continuously kept moist and nourished by basal tears, which help lubricate the eye and keep out dust. Tears contain water, mucin, lipids, lysozyme, lactoferrin, lipocalin, lacritin, immunoglobulins, glucose, urea, sodium, and potassium. Some substances in tears, such as lysozyme, fight bacterial infections as part of the immune system. Lysozyme does this by dissolving the outer layer of the peptidoglycan coating of certain bacteria. Tears are a typical bodily fluid with a salt content similar to that of plasma. Typically, 0.75 to 1.1 grams (0.03 to 0.04 ounces) of tears are secreted over a 24-hour period, and this rate slows with age. Basal tears are composed of antioxidants such as ascorbate, urate, cysteine, glutathione, and tyrosine. Ascorbate and urate make up half of the tear fluid.

[0073] The second type of tearing results from ocular irritation by a foreign body or the presence of irritants in the ocular environment, including the cornea, conjunctiva, or nasal mucosa, such as onion vapor, perfumes and other fragrances, tear gas, or pepper spray, which trigger TRP channels in the ocular nerve. It can also occur in response to bright light and hot or peppery stimuli on the tongue and mouth. It is also associated with vomiting, coughing, and yawning. These reflex tears attempt to wash away any irritants that may have come into contact with the eye.

[0074] The methods disclosed herein may relate to maintaining or improving tear production, such as basal or reflex tear production.

[0075] Patient selection According to the methods of the present invention, the methods may additionally comprise the step of selecting a subject for treatment with a therapeutically effective amount of a polypeptide comprising or consisting of LINK_TSG6.

[0076] The method may include evaluating a subject or patient for evidence of or susceptibility to ocular surface disorders such as dry eye disease, such as corneal damage (e.g., the presence of corneal epithelial lesions), insufficient tear production, and inflammation (e.g., redness or swelling, or the presence of one or more biological markers of inflammation, such as one or more of the pro-inflammatory cytokines TNFα, IL-1β, IFN-γ, or IL-6 and MMP9).

[0077] The method may involve a comprehensive eye examination, which may include an evaluation of the patient's medical history to determine their signs and symptoms and note any systemic health issues, medications, or environmental factors that may be contributing to the dry eye problem, an external examination of the eye, including eyelid structure and blink dynamics, evaluation of the eyelid and cornea using bright light and magnification, or measurement of tear quantity and quality for any abnormalities.

[0078] An individual can be determined to have a corneal epithelial defect, for example, by staining with Lissamine Green, Rose Bengal, or fluorescein dyes.

[0079] An individual may be determined to have insufficient tear production, for example, by using the phenol red thread test or the Schirmer test.

[0080] In some cases described herein, the treated individual has a condition associated with an increased incidence of ocular surface disorders, such as dry eye disease. In some cases, the condition is an autoimmune condition. The autoimmune condition may be Sjögren's syndrome. The individual may have previously been diagnosed with Sjögren's syndrome. Sjögren's syndrome causes one of the most severe forms of dry eye disease, characterized by inflammatory destruction of the lacrimal gland and ocular surface. Inflammation of the lacrimal gland and ocular surface is a key feature of dry eye disease and plays an important role in the pathogenesis of dry eye disease and Sjögren's syndrome. In some cases, the autoimmune condition is rheumatoid arthritis or diabetes. In some cases, the individual has type 1 or type 2 diabetes.

[0081] In some cases, ocular surface disorders such as dry eye disease are not associated with inflammation. Non-inflammatory dry eye disease may be related to tear film instability and / or rapid tear evaporation.

[0082] treatment Disclosed herein is the method for treating or preventing ocular surface disorders such as dry eye disease.This method can involve reducing or eliminating one or more visual signs and symptoms of ocular surface disorders such as dry eye disease, such as reducing corneal epithelial defect or lesion, increasing tear production, increasing or maintaining the number of goblet cells, reducing the expression of one or more inflammatory cytokines, or reducing redness or burning sensation, pain or discomfort, or improving the visual signs and symptoms such as vision loss or blurred vision.

[0083] The method may involve treating or preventing corneal epithelial defects or lesions. The method may result in a reduction in the number and / or extent of corneal epithelial lesions. The method may result in a reduction in the number and / or extent of corneal epithelial lesions compared to the number and / or extent of corneal epithelial lesions before treatment. The method may result in a reduction in the number and / or extent of corneal epithelial lesions compared to the number and / or extent of corneal epithelial lesions in an untreated control or a control treated with full-length TSG-6, such as TSG-6 comprising or consisting of SEQ ID NO: 3 or SEQ ID NO: 5. The extent of corneal epithelial lesions may be reduced by at least 25%, at least 50%, at least 75%, or at least 100% compared to the control. Treatment may result in corneal epithelial lesions affecting 0%, less than 10%, less than 20%, less than 30%, less than 40%, or less than 50% of the corneal surface.

[0084] The method may involve treating or preventing insufficient tear production. The method may result in increased tear production, such as increased tear production. The method may result in an increase in the amount of tear produced compared to the amount of tear produced before treatment. The method may result in an increase in the amount of tear produced compared to the amount of tear in an untreated control or a control treated with full-length TSG-6, such as TSG-6 comprising or consisting of SEQ ID NO: 3 or SEQ ID NO: 5. The amount of tear may be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more than 100% compared to the control.

[0085] The method may involve increasing or maintaining the number of goblet cells. In this context, increasing or maintaining the number of goblet cells means that the number of goblet cells after treatment with LINK_TSG6 is not reduced to the same extent as that observed without treatment, such as in an untreated control. In some cases, the number of goblet cells is increased or unchanged compared to the number of goblet cells before treatment.

[0086] This method may involve reducing the level of one or more inflammatory cytokines. For example, this method may result in a reduction in the level of nucleic acids corresponding to one or more inflammatory cytokines. In some cases, this method results in a reduction in the level of inflammatory cytokine proteins. The inflammatory cytokines may be selected from TNFα, IL6, IFN-γ, and IL-1β. The levels may be reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% compared to pre-treatment levels or compared to untreated controls.

[0087] Treatment can result in complete resolution of signs and symptoms of ocular surface disorders such as dry eye disease.

[0088] Prevention can mean that signs and symptoms of an ocular surface disorder, such as dry eye disease, do not appear, or that signs and symptoms of an ocular surface disorder, such as dry eye disease, occur less frequently than they would without treatment.

[0089] The method described herein can involve local administration of LINK_TSG6. LINK_TSG6 can be locally administered to the eye (ocular delivery), preferably to the cornea, for example, the surface of the cornea. In some cases, LINK_TSG6 is administered as eye drops. LINK_TSG6 can be formulated as a topical liquid preparation, such as eye drops. LINK_TSG6 can be formulated as a suspension or emulsion.

[0090] Administration is preferably carried out in a therapeutically effective amount.The therapeutically effective amount of LINK_TSG6 can be determined according to various parameters, particularly according to the polypeptide, the age, weight and condition of the patient to be treated, the route of administration and the required regimen.In this case, a doctor can determine the route of administration and the dosage required for a specific patient.The therapeutically effective amount of LINK_TSG6 is an amount that is effective for improving one or more signs and symptoms of ocular surface disorders such as dry eye disease, for example, reducing the level of corneal damage or corneal lesions, increasing lacrimal gland function or increasing the tear production of lacrimal gland, or reducing ocular inflammation such as corneal inflammation.

[0091] In some cases, LINK_TSG6 is administered at 12 μg to 15 μg per eye, for example, 10 μg to 20 μg, or 12 to 15 μg per eye. In some cases, LINK_TSG6 is administered at about 8 μg per eye, about 9 μg per eye, about 10 μg per eye, about 11 μg per eye, about 12 μg per eye, about 13 μg per eye, about 14 μg per eye, or about 15 μg per eye.

[0092] In some cases, a higher dose is desired. In such cases, LINK_TSG6 is administered at approximately 120 μg to 150 μg per eye. In some cases, the dose of LINK_TSG6 is at least 80 μg, at least 90 μg, at least 100 μg, at least 110 μg, at least 120 μg, at least 130 μg, or at least 140 μg per eye. In some cases, the dose of LINK_TSG6 is less than 170 μg, less than 160 μg, less than 150 μg, less than 140 μg, less than 130 μg, or less than 120 μg per eye.

[0093] In some cases, even higher doses are desirable. For example, the dosage of LINK_TSG6 may be at least 170 μg, at least 180 μg, at least 190 μg, at least 200 μg, at least 210 μg, at least 220 μg, at least 230 μg, at least 240 μg, at least 250 μg, at least 260 μg, at least 270 μg, at least 280 μg, at least 290 μg, at least 300 μg, at least 320 μg, at least 340 μg, at least 360 μg, at least 380 μg, at least 400 μg , at least 420 μg, at least 440 μg, at least 460 μg, at least 480 μg, at least 500 μg, at least 550 μg, at least 600 μg, at least 650 μg, at least 700 μg, at least 750 μg, at least 800 μg, at least 900 μg, at least 1 mg, at least 1.1 mg, at least 1.2 mg, at least 1.3 mg, at least 1.4 mg, at least 1.5 mg, at least 1.6 mg, or at least 1.7 mg.

[0094] Administration can be once a day, twice a day, three times a day, four times a day, five times a day, six times a day, seven times a day, eight times a day, nine times a day, ten times a day, or more than ten times a day. Administration is preferably twice a day. In some preferred cases, administration is more than twice a day, for example, three or four times a day. In some preferred cases, administration is less than four times a day, or less than three times a day, or twice a day or once a day. In particularly preferred cases, administration is twice a day. In some cases, treatment involves administering the LINK_TSG6 polypeptide once a day. In some cases, treatment involves administering the LINK_TSG6 polypeptide less frequently than once a day, for example, once every two days, once every three days, once a week, or once every two weeks.

[0095] The dose, schedule, mode or time course of administration of the polypeptide of the present invention can be modified according to the response to therapy.For example, if the response to therapy is not optimal, the dose and / or frequency of administration and / or time course of administration can be increased.Conversely, if the response to therapy is better than expected, the dose and / or frequency of administration and / or time course of administration can be reduced.

[0096] In some cases, treatment involves co-administration of LINK_TSG6 with artificial tears, prednisolone, cyclosporine, Lifitegrast (Xiidra™), or any combination thereof. Administration may be sequential or simultaneous. Preferably, when LINK_TSG6 is co-administered with one or more of prednisolone, cyclosporine, Lifitegrast (Xiidra™), or artificial tears, administration is simultaneous or substantially simultaneous. Preferably, the co-administered agents are administered via the same route of administration, for example, by topical administration to the eye.

[0097] The subject's assessment of ocular surface disorders, such as dry eye disease, can occur at any time before, during, or after the administration of a therapeutically effective amount of LINK_TSG6.In some embodiments, the method of the present invention comprises starting administration, assessing the subject as described above, and continuing, changing, or discontinuing further administration based on the assessment.In some embodiments, changing administration comprises increasing or decreasing the dose and / or frequency and / or time course of administration.

[0098] formulation Formulations suitable for ocular administration include eye drops in which the active compound is dissolved or suspended in a suitable carrier, particularly an aqueous solvent for the active compound. In a preferred formulation, LINK_TSG6 is solubilized in saline. In some formulations, LINK_TSG6 is solubilized in PBS (phosphate buffered saline). As described herein, the inventors have discovered that LINK_TSG6 polypeptide has higher solubility in saline compared to full-length TSG6. Described herein are compositions, particularly pharmaceutical compositions, containing high concentrations of LINK_TSG6.

[0099] The pharmaceutical composition may comprise a LINK_TSG6 polypeptide solubilized in saline. The pharmaceutical composition may comprise a LINK_TSG6 polypeptide solubilized in phosphate buffered saline. In some embodiments, the pharmaceutical composition comprises at least 2000 μg / ml, 2100 μg / ml, 2200 μg / ml, 2300 μg / ml, 2400 μg / ml, 2500 μg / ml, 2600 μg / ml, 2700 μg / ml, 2800 μg / ml, 2900 μg / ml, 3000 μg / ml, 3100 μg / ml, 3200 μg / ml, 3300 μg / ml, or more than 3300 μg / ml of LINK_TSG6. Preferably, the pharmaceutical formulation comprises at least 2000 μg / ml of LINK_TSG6.

[0100] The eye drop formulations disclosed herein may further comprise one or more of a preservative, an antioxidant, a stabilizer, a tonicity adjuster, a viscosity adjuster, or a buffer. Preferably, the eye drop formulation is a sterile eye drop formulation. In some cases, the eye drop formulation contains about 240 μg / ml to about 300 μg / ml of LINK_TSG6. Such a formulation is useful for delivering about 12 μg to about 15 μg of LINK_TSG6 per drop. Each drop may be about 50 μl. The eye drop formulation may contain 200 μg / ml to 350 μg / ml, 200 μg / ml to 320 μg / ml, 220 μg / ml to 320 μg / ml, or 240 μg / ml to 300 μg / ml. Preferably, the eye drop formulation contains about 240 μg / ml to about 300 μg / ml. The eye drop formulation may contain at least 200 μg / ml, at least 220 μg / ml, at least 230 μg / ml, at least 240 μg / ml, at least 250 μg / ml, at least 260 μg / ml, at least 270 μg / ml, at least 280 μg / ml, at least 290 μg / ml, at least 300 μg / ml, at least 310 μg / ml, at least 320 μg / ml, or more than 320 μg / ml of the LINK_TSG6 polypeptide.

[0101] In some cases, higher dose formulations are desired. Such formulations may contain 2400 to 3000 μg / ml of LINK_TSG6. Such formulations are useful for delivering about 120 μg to about 150 μg of LINK_TSG6 per drop. Eye drop formulations may contain 2000 μg / ml to 3500 μg / ml, 2000 μg / ml to 3200 μg / ml, 2200 μg / ml to 3200 μg / ml, or 2400 μg / ml to 3000 μg / ml. Preferably, the eye drop formulation contains about 2400 μg / ml to about 3000 μg / ml. The eye drop formulation may contain at least 2000 μg / ml, at least 2200 μg / ml, at least 2300 μg / ml, at least 2400 μg / ml, at least 2500 μg / ml, at least 2600 μg / ml, at least 2700 μg / ml, at least 2800 μg / ml, at least 2900 μg / ml, at least 3000 μg / ml, at least 3100 μg / ml, at least 3200 μg / ml, or more than 3200 μg / ml of the LINK_TSG6 polypeptide.

[0102] In some cases, the eye drop formulation may further comprise artificial tears.Artificial tears are lubricating eye drops.Artificial tears may contain one or more agents selected from carboxymethylcellulose, polyvinyl alcohol, hydroxypropylmethylcellulose, hydroxypropylcellulose, hyaluronic acid, water, salt, and polymers such as polyethylene glycol or polypropylene glycol.

[0103] Optionally, the eye drop formulation may further comprise a combination of prednisolone, cyclosporine, Lifitegrast (Xiidra™), or prednisolone and cyclosporine, prednisolone and Lifitegrast (Xiidra™), cyclosporine and Lifitegrast (Xiidra™), or prednisolone, cyclosporine and Lifitegrast (Xiidra™).

[0104] Pharmaceutical compositions can be prepared using pharmaceutically acceptable "carriers" composed of materials deemed safe and effective. "Pharmaceutically acceptable" refers to molecular entities and compositions that are "generally regarded as safe," e.g., physiologically tolerable when administered to humans and typically do not produce allergic or similar adverse reactions, such as stomach upset, loss or alteration of taste (dysgeusia). In some embodiments, the term refers to molecular entities and compositions that have been approved by a U.S. federal or state regulatory agency as GRAS listed under Sections 204(s) and 409 of the Federal Food, Drug, and Cosmetic Act, subject to premarket review and approval by the FDA or a similar listing, the U.S. Pharmacopeia, or another generally recognized pharmacopeia for use in animals, and more specifically, in humans.

[0105] The term "carrier" refers to diluents, binders, lubricants, and disintegrants. Those skilled in the art are familiar with such pharmaceutical carriers and how to formulate pharmaceutical compositions using such carriers.

[0106] The pharmaceutical compositions provided herein may contain one or more excipients, such as solvents, solubility enhancers, suspending agents, buffers, isotonicity agents, antioxidants, or antimicrobial preservatives. When used, the excipients of the composition will not adversely affect the stability, bioavailability, safety, and / or efficacy of the active ingredient, i.e., LINK_TSG6, used in the composition. Therefore, those skilled in the art will understand that a composition is provided that is not incompatible with any of the components of the dosage form. The excipients may be selected from the group consisting of buffers, solubilizers, tonicity agents, chelating agents, antioxidants, antimicrobial agents, and preservatives. Ointments are usually prepared from an active compound and a paraffinic or water-miscible ointment base.

[0107] Creams are usually prepared from an active compound and an oil-in-water cream base. Optionally, the aqueous phase of the cream base may contain, for example, at least about 30% w / w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups, such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol, and polyethylene glycol, and mixtures thereof. Topical formulations may desirably contain a compound that enhances the absorption or penetration of the active compound through the skin or other affected areas. Examples of such skin penetration enhancers include dimethyl sulfoxide and related analogs.

[0108] Emulsions are usually prepared from an active compound and an oil phase, which may optionally contain only an emulsifier (also known as an emulsifier), or may contain a mixture of at least one emulsifier with a fat or oil, or both a fat and an oil.Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier that acts as a stabilizer.It is also preferable to include both an oil and a fat.Together, the emulsifier, with or without a stabilizer, constitutes the so-called emulsifying wax, and the wax together with the oil and / or fat constitutes the so-called emulsifying ointment base, which forms the oily dispersed phase of a cream formulation.

[0109] Suitable emulsifiers and emulsion stabilizers include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate, and sodium lauryl sulfate. Because the solubility of active compounds in most oils likely to be used in pharmaceutical emulsion formulations can be very low, the selection of suitable oils or fats for formulation is based on achieving the desired cosmetic properties. Therefore, creams are preferably non-greasy, non-staining, and washable products with a suitable consistency to avoid leakage from tubes or other containers. Mono- or di-basic alkyl esters such as linear or branched diisoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acid, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate, or a blend of branched esters known as Crodamol CAP can be used, the last three being preferred. These can also be used alone or in combination, depending on the desired properties. Alternatively, high melting point lipids such as white soft paraffin and / or liquid paraffin or other mineral oils can be used.

[0110] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, and appropriately expressed in their specific form, or in terms of means for performing a disclosed function, or a method or process for obtaining a disclosed result, can be utilized separately or in any combination of such features to realize the invention in diverse forms thereof.

[0111] While the present invention has been described in conjunction with the above exemplary embodiments, many equivalent modifications and variations will be apparent to those skilled in the art given this disclosure. Accordingly, the above exemplary embodiments of the invention are considered to be illustrative and not limiting. Various changes can be made to the described embodiments without departing from the spirit and scope of the invention.

[0112] For the avoidance of doubt, the theoretical explanations provided herein are provided for the purpose of enhancing the understanding of the reader, and the inventors do not wish to be bound by any of these theoretical explanations.

[0113] Any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0114] Throughout this specification, including the claims which follow, unless the context requires otherwise, the words "comprise" and "include," and variations such as "comprises," "comprising," and "including," are understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0115] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges can be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. The term "about" in connection with numerical values ​​is optional and means, for example, + / - 10%. [Example]

[0116] Example 1: LINK TSG6 is more soluble than full-length TSG-6 The solubility and aggregation state of full-length TSG-6 and LINK_TSG6 in PBS were compared at various concentrations using UV spectrophotometry and dynamic light scattering (DLS). LINK_TSG6 was completely soluble at 2 mg / ml and showed no aggregation at 0.4, 0.8, 1.6, or 3.2 mg / ml. The full-length protein had much lower solubility, with less than 40% of the protein remaining in solution at 0.4, 0.8, 1.6, and 3.2 mg / ml. The full-length protein was also highly aggregated at 0.2, 0.4, 0.8, 1.6, and 3.2 mg / ml. DLS measurements indicated that aggregates exceeding 25 million Da had formed at 1.6 and 3.2 mg / ml, outside the range of full-length TSG-6.

[0117] Example 2: LINK TSG6 ameliorates signs and symptoms of dry eye disease in a mouse model Topical TSG-6 has previously been shown to be effective in inflammation-mediated dry eye disease, similar to cyclosporine eye drops (Kim et al., 2016). To test whether the LINK_TSG6 polypeptide is useful for this indication, we investigated its effects in the NOD.B10 mouse model of primary ocular Sjögren's syndrome (spontaneous dry eye disease without diabetes).

[0118] NOD.B10.H2, a model of primary ocular Sjögren's syndrome (non-diabetic dry eye disease) b Mice (12 weeks old, Jackson Lab) were treated with topical administration of LINK_TSG6 for 7 days. C57BL / 6 mice were used as a negative control because they do not develop spontaneous dry eye.

[0119] Mice were treated with topical application of 1 μg of LINK_TSG6 in 10 μl of PBS four times per day (QID) for 7 days. Mice were anesthetized with an intraperitoneal injection of zolazepam-tiletamine (Zoletil®, Virbac, Carros, France) and administered 10 μl of either LINK_TSG6 or PBS using a pipette. Mice were randomly assigned to treatment groups as follows: 1) Group 1 (negative control): C57BL / 6 mice, 12 weeks old (n = 6, 12 eyes), untreated 2) Group 2 (positive control): NOD.B10, 12 weeks old (n=6, 6 eyes) + PBS 10 μl QID 3) Group 3 (experimental group): NOD.B10, 12 weeks old (n=8, 8 eyes) + LINK_TSG6 1 μg (in 10 μl PBS) QID.

[0120] To evaluate the efficacy of treatments for dry eye disease, the following assays were performed. 1) Corneal epithelial defects by vital staining (with Lissamine Green) and scoring of the defect site. After applying one drop of 3% Lissamine Green B to the inferior lateral conjunctival sac of mice, the dye staining of the corneal surface was graded in a blinded manner by two ophthalmologists according to the following ocular staining scoring system: score 0 for no punctate staining, score 1 for staining of less than one-third of the corneal surface, score 2 for staining of two-thirds or less, and score 3 for staining of more than two-thirds. 2) Tear production in the lacrimal gland as assessed by the phenol red cotton thread test; 3) Expression of inflammatory cytokines by real-time RT PCR in the cornea and orbital glands, and 4) Number of PAS-stained conjunctival goblet cells in the fornix. The number of PAS-stained cells per 100 μm was counted in four different sections from the same animal's eye, and the average number for each eye was determined as the goblet cell density.

[0121] As shown in Figure 2A, corneal epithelial defects were significantly reduced after LINK_TSG6 treatment (p<0.01). There was a significant improvement in corneal epithelial defects between the PBS- and Link_TSG6-treated groups (p<0.001).

[0122] As shown in Figure 2B, tear production was significantly increased by LINK_TSG6 treatment (p<0.01). There was a significant improvement in tear production between the PBS- and LINK_TSG6-treated groups (p<0.001).

[0123] Figure 2C shows that LINK_TSG6 treatment significantly suppressed the mRNA levels of IFN-γ, TNF-α, and IL-1β in the cornea and orbital glands. Eyes treated with LINK_TSG6 had cytokine levels similar to those of control C57BL / 6 mice.

[0124] 2D shows that LINK_TSG6 treatment significantly increased the number of conjunctival goblet cells (these cells produce mucin components in tears).

[0125] Overall, these results demonstrate that LINK_TSG6 significantly suppressed inflammation, reduced corneal epithelial defects, increased tear production, and increased conjunctival goblet cells in NOD.B10 dry eye mice.

[0126] Example 3: The effect of LINK TSG6 on dry eye disease is dose-dependent We were interested in understanding whether the effects observed in Example 1 were dose-dependent. To this end, 1, 0.1, or 0.01 μg of LINK_TSG6 (in 5 μl of PBS) was applied topically QID for 7 days to 12-week-old NOD.B10.H2b mice (Jackson Lab).

[0127] Mice were randomly assigned to the following treatment groups: 1) Group 1 (negative control): C57BL / 6 mice, 12 weeks old (n = 6, 12 eyes), untreated 2) Group 2 (positive control): NOD.B10 (n=8, 8 eyes) + PBS 5 μl QID 3) Group 3 (experimental group): NOD.B10 (n = 8, 8 eyes) + LINK_TSG6 1 μg (in 5 μl PBS) QID 4) Group 4 (experimental group): NOD.B10 (n = 8, 8 eyes) + LINK_TSG6 0.1 μg QID 5) Group 5 (experimental group): NOD.B10 (n = 8, 8 eyes) + LINK_TSG6 0.01 μg QID

[0128] The effect of the treatment was determined according to Example 1.

[0129] As shown in Figure 3A, corneal epithelial defects were quantified in corneas stained with Lissamine Green. LINK_TSG6 at 1 μg and 0.1 μg was effective in reducing corneal epithelial defects, but LINK_TSG6 at 0.01 μg did not significantly improve corneal epithelial defects.

[0130] In FIG. 3B, the phenol red cotton thread test showed that LINK_TSG6 at 1 μg and 0.1 μg was effective in increasing tear production, but LINK_TSG6 at 0.01 μg did not significantly improve tear production.

[0131] As shown in Figures 3C and D, the highest dose of LINK_TSG6 tested, 1 μg, was most effective in suppressing TNF-α expression.

[0132] Overall, these results demonstrate a dose-dependent improvement in dry eye parameters with topically applied LINK_TSG6 polypeptide (1 μg being most effective and 0.01 μg being least effective).

[0133] These effects were also observed with higher doses of LINK_TSG6. As shown in Figure 4A, higher doses (1 μg and 10 μg) of LINK_TSG6 resulted in significantly less corneal epithelial damage compared to PBS-treated mice. At the highest dose, most of the LINK_TSG6-treated eyes were free of corneal lesions (score 0), the same as untreated negative control C57BL / 6 mice. This indicates that LINK_TSG6 can promote the healing or repair of epithelial lesions.

[0134] Dose-dependent responses were also observed with respect to tear production (Figure 4B - note that even the lowest dose of LINK_TSG6 tested significantly affected tear production), inflammation (Figure 4C), and conjunctival goblet cell count (Figure 4D).

[0135] Example 4: Comparison of the effects of full-length TSG-6 protein and LINK TSG6 on dry eye disease Our experiments demonstrated that the LINK_TSG6 polypeptide is effective in treating dry eye disease. We were interested in understanding how this compares to full-length (FL) TSG-6 (FL TSG6). Equimolar doses of FL TSG6 and LINK_TSG6 administered twice daily (BID) were compared.

[0136] Twelve-week-old NOD.B10 and C57BL / 6 mice were randomly assigned to treatment groups as follows: 1) Group 1: NOD.B10 + PBS 5 μl BID for 7 days (positive control); 2) Group 2: NOD.B10 + full-length TSG-6 (FL TSG6) (R&D Systems) 5 μl (3.27, 0.327, 0.0327 μg) BID for 7 days. 3) Group 3: NOD.B10 + LINK_TSG6 5 μl (1, 0.1, 0.01 μg) BID for 7 days, and 4) Group 4 C57BL / 6 mice (negative control, no dry eye).

[0137] Doses of 0.01, 0.1, and 1.0 micrograms of LINK_TSG6 were selected according to Example 2. Using mass spectrometry, the molecular weight of recombinant human TSG-6 (R&D Systems) was determined to be 35.7 kDa. Based on this determination, equimolar doses of 0.037, 0.37, and 3.7 micrograms were calculated for full-length TSG-6 (FL TSG6).

[0138] Signs and symptoms of dry eye disease were determined according to the previous example, i.e. 1) Lissamine Green staining and corneal epithelial damage score. After applying one drop of 3% Lissamine Green B to the inferior lateral conjunctival sac of mice, the dye staining of the corneal surface was graded in a blinded manner by two ophthalmologists according to the following ocular staining scoring system: score 0 for no punctate staining, score 0.5 for trace staining, score 1 for staining of less than one-third of the corneal surface, score 2 for staining of two-thirds or less, and score 3 for staining of more than two-thirds. 2) Phenol red cotton thread test for lacrimal gland tear secretion 3) Histology (conjunctival PAS staining, lacrimal gland CD3 immunostaining)

[0139] As shown in Figure 5A, LINK_TSG6 resulted in a significant reduction in corneal epithelial lesions compared to full-length TSG-6 at equimolar concentrations.

[0140] Figures 5B, 5C, and 5D show that 1 μg of LINK_TSG6, the highest dose tested, caused increased tear production, preservation of goblet cells, and a reduction in the number of CD3-stained inflammatory foci in the orbital glands compared to an equimolar dose (3.27 μg) of full-length TSG-6.

[0141] Example 5: Evaluation of LINK TSG6 in a dry injury-induced dry eye model and comparison with Restasis. Having demonstrated efficacy of LINK_TSG6 in the NOD.B10 mouse model of primary ocular Sjögren's syndrome (spontaneous dry eye disease without diabetes), we were interested in understanding whether LINK_TSG6 would also be effective in treating individuals with the prevalent environmental evaporative dry eye disorder.

[0142] We used a dryness model that better emulates evaporative dry eye, a more prevalent form of dry eye compared to less common conditions such as Sjögren's syndrome. Seven-week-old C57BL / 6 mice were kept in a dryness chamber and intraperitoneally injected with scopolamine three times daily for 10 days to induce dryness injury. Airflow from a fan was allowed through the screen in the cage 24 hours a day, maintaining humidity at 30-35% within the cage. Mice were divided into treatment groups as follows: 1) No dry eye injury (negative control, 2 mice, 4 eyes) 2) Group 1 (5 mice, 10 eyes): Dry injury + 5 μl PBS BID for 10 days (positive control) 3) Group 2 (5 mice, 10 eyes): Dry injury + LINK_TSG6 0.1 μg (in 5 μl PBS) BID for 10 days 4) Group 3 (5 mice, 10 eyes): Dry injury + LINK_TSG6 1 μg (in 5 μl PBS) BID for 10 days 5) Group 4 (5 mice, 10 eyes): Dry injury + LINK_TSG6 10 μg (in 5 μl PBS) BID for 10 days 6) Group 5 (5 mice, 10 eyes): Dry injury + Restasis (0.05% cyclosporine A) 5 μl BID for 10 days

[0143] After 10 days, signs and symptoms of dry eye were assessed as follows: 1) Fluorescein staining and scoring of corneal epithelial damage (C57BL / 6 mice are black, and fluorescein staining provides better visualization than lissamine green). 2) Phenol red cotton thread test for lacrimal gland tear secretion 3) Histology (conjunctival PAS staining, lacrimal gland CD3 immunostaining) 4) Molecular assays of the ocular surface and lacrimal gland (real-time RT-PCR for inflammatory cytokines)

[0144] As shown in Figure 6A (before desiccation and treatment, indicating that animals selected for treatment had no corneal lesions at the start of the study) and 6B (after desiccation and treatment), desiccation injury significantly induced corneal epithelial defects in C57BL / 6 mice (p<0.01), and LINK_TSG6 1µg was effective in reducing corneal epithelial defects under desiccation (p<0.001). Interestingly, Restasis did not significantly reduce corneal epithelial defects in this model, and LINK_TSG6 1µg was superior to Restasis in reducing corneal epithelial defects (p<0.05).

[0145] As shown in Figures 6C (before desiccation and treatment) and 6D (after desiccation and treatment), desiccation injury significantly reduced tear production in C57BL / 6 mice (p<0.0001). Similar to the mouse model of primary ocular Sjögren's syndrome, LINK_TSG6 at 1 μg and 10 μg was effective in maintaining tear production under desiccation (p<0.0001), and even 0.1 μg of LINK_TSG6 maintained tear production (p=0.0552). Interestingly, although Restasis was effective in maintaining tear production under desiccation (p<0.05), LINK_TSG6 at 1 μg was superior to Restasis in maintaining tear production (p<0.001).

[0146] It is well known that Th1 and Th17 cells increase in draining cervical lymph nodes (DLN) under desiccation stress, and these cells are involved in the induction of dry eye. Therefore, we performed FACS analysis on cervical draining lymph nodes to investigate the expression of Th1 (IFN-γ) cells in response to various treatments. + CD4 + cells) and Th17 cells (IL17A + CD4 + As shown in Figures 6E and 6F, LINK_TSG6 was also effective in suppressing Th1 and Th17 cells in cervical lymph nodes. Restasis had no such inhibitory effect.

[0147] Thus, these data indicate that LINK_TSG6 is also effective in treating dry eye disease, including in patients with prevalent environmental evaporative dry eye.

[0148] Example 6: Evaluation of LINK TSG6 in established sicca injury-induced dry eye Because LINK_TSG6 has proven effective in treating dry eye disease, including individuals with prevalent environmental evaporative dry eye, there was interest in understanding whether LINK_TSG6 would also be effective in treating individuals with established dry eye and reversing the effects of the disease.

[0149] The same dry eye model as in Example 5 was used, but this time dry eye disease was established for one week before treatment with LINK_TSG6. After dry eye injury was caused to the ocular surface, LINK_TSG6 was applied to the ocular surface. The mice were then kept in a dry chamber with scopolamine injection for another 10 days, during which treatment was administered.

[0150] Seven-week-old C57BL / 6 mice were kept in a dry chamber and given intraperitoneal injections of scopolamine three times daily for seven days to establish dry eye. Mice were divided into treatment groups as follows: 1) No dry eye injury (negative control, 2 mice, 4 eyes) 2) Group 1 (5 mice, 10 eyes): Dry injury + 5 μl PBS BID for 10 days (positive control) 3) Group 2 (5 mice, 10 eyes): Dry injury + LINK_TSG6 0.1 μg (in 5 μl of PBS) BID for 10 days 4) Group 3 (5 mice, 10 eyes): Dry injury + LINK_TSG6 1 μg (in 5 μl PBS) BID for 10 days 5) Group 4 (5 mice, 10 eyes): dry injury + LINK_TSG6 10 μg (in 5 μl PBS) BID for 10 days.

[0151] Signs and symptoms of dry eye disease were determined according to the previous example, i.e. 1) Fluorescein staining and corneal epithelial damage score (B6 mice are black, and fluorescein staining is better for visualization than lissamine green), 2) the phenol red cotton thread test for lacrimal gland tear secretion, and 3) Histology (conjunctival PAS staining, lacrimal gland CD3 immunostaining).

[0152] As shown in Figure 7A, LINK_TSG6 was effective in reducing corneal epithelial defects at all concentrations tested. As shown in Figure 7B, LINK_TSG6 was effective in increasing tear production at all concentrations tested. Significant changes were observed even at the lowest concentration tested.

[0153] FIG. 7C shows that 1 μg and 10 μg of LINK_TSG6 resulted in an increase in goblet cell number compared to the control, restoring it to levels comparable to those observed in the absence of desiccation.

[0154] Furthermore, as shown in Figure 7D, treatment with LINK_TSG6 was effective in suppressing the levels of MMP-9 mRNA on the ocular surface.

[0155] Thus, these data demonstrate that LINK_TSG6 is effective in treating established dry eye disease in addition to reducing the severity of dry eye lesions. These data support the use of LINK_TSG6 for the treatment of individuals with dry eye disorders, including those with prevalent environmental evaporative dry eye.

[0156] Example 7: Evaluation of LINK TSG6 in a dry eye model and comparison with Restasis. Having demonstrated that LINK_TSG6 is superior to Restasis in treating evaporative dry eye, we were interested in comparing LINK_TSG6 with Restasis in the NOD.B10 mouse model of primary ocular Sjogren's syndrome (spontaneous dry eye disease without diabetes), used in Example 4.

[0157] Twelve-week-old NOD.B10 and C57BL / 6 mice were randomly assigned to treatment groups as follows: 1) Group 1 C57BL / 6 mice (negative control, no dry eye / xerosis). 2) Group 2: NOD.B10 + 5 μl PBS BID for 7 days (positive control); 3) Group 3: NOD.B10 + LINK_TSG6 5 μl (0.1, 1, 10 μg / 5 μl) BID for 7 days, and 4) Group 4: NOD.B10+Restasis 5 μl BID for 7 days.

[0158] Signs and symptoms of dry eye disease were determined according to the previous example, i.e. 1) Lissamine green staining and corneal epithelial damage score. 2) Phenol red cotton thread test for lacrimal gland tear secretion 3) Histology (conjunctival PAS staining, lacrimal gland CD3 immunostaining)

[0159] As shown in Figures 8B and D, and consistent with the studies in Example 4, LINK_TSG LINK_TSG6 significantly reduced corneal epithelial lesions and increased tear production in a NOD.B10 mouse model of primary ocular Sjögren's syndrome. Furthermore, LINK_TSG6 promoted healing to a greater extent than Restasis at both the 1 μg and 10 μg doses, resulting in reduced corneal lesions, maintained goblet cell numbers, and reduced the number of CD3-stained inflammatory foci in the orbital glands compared with Restasis. This suggests that even at the lowest dose tested, LINK_TSG6 was superior to Restasis in treating dry eye in a NOD.B10 mouse model of primary ocular Sjögren's syndrome (spontaneous dry eye disease without diabetes), as previously observed in preventing or treating established evaporative dry eye disease. These data further support the use of LINK_TSG6 for the treatment of individuals with dry eye disorders. The present invention includes, but is not limited to, the following aspects. [Aspect 1] A LINK_TSG6 polypeptide for use in the treatment or prevention of ocular surface disorders in dry eye disease 1-15. [Aspect 2] The treatment or prevention of an ocular surface disorder such as dry eye disease, Healing of corneal epithelial defects, increased tear production, Suppression of inflammation, and 2. The LINK_TSG6 polypeptide for use according to embodiment 1, comprising one or more effects selected from the group consisting of: a greater number of conjunctival goblet cells. [Aspect 3] 3. The LINK_TSG6 polypeptide for use according to embodiment 1 or 2, wherein said treatment comprises topical administration of the LINK_TSG6 polypeptide to the eye. [Aspect 4] 4. The LINK_TSG6 polypeptide for use according to any one of embodiments 1 to 3, wherein said treatment comprises administering the LINK_TSG6 polypeptide twice per day. [Aspect 5] 4. The LINK_TSG6 polypeptide for use according to any one of aspects 1 to 3, wherein said treatment comprises administering the LINK_TSG6 polypeptide less than four times per day. [Aspect 6] 4. The LINK_TSG6 polypeptide for use according to embodiment 3, wherein said LINK_TSG6 polypeptide is formulated as eye drops. [Aspect 7] LINK_TSG6 polypeptide for use according to any one of the preceding aspects, wherein LINK_TSG6 is co-administered with one or more of prednisolone, cyclosporine, Lifitegrast (Xiidra™) or artificial tears. [Aspect 8] LINK_TSG6 polypeptide for use according to any one of the preceding aspects, wherein about 10 μg to 200 μg of LINK_TSG6 polypeptide is administered per eye, preferably 120 μg to 150 μg of LINK_TSG6 polypeptide is administered per eye. [Aspect 9] 3. The LINK_TSG6 polypeptide for use according to aspect 2, wherein said suppression of inflammation comprises a reduction in production of one or more pro-inflammatory cytokines in the cornea or intraorbital lacrimal gland, wherein said pro-inflammatory cytokines are optionally selected from TNF-α, IL-6, IFN-γ and IL-1β. [Aspect 10] The LINK_TSG6 polypeptide for use according to aspect 2, wherein the treatment or prevention of dry eye disease comprises healing of corneal epithelial defects, increased tear production, suppression of inflammation or an increase in the number of conjunctival goblet cells compared to the corneal epithelial defects, tear production, inflammation or number of conjunctival goblet cells before administration of the LINK_TSG6 polypeptide. [Aspect 11] The LINK_TSG6 polypeptide for use according to aspect 2, wherein the treatment or prevention of dry eye disease comprises healing of the corneal epithelial defect, increased tear production, suppression of inflammation or an increase in the number of conjunctival goblet cells compared to the corneal epithelial defect, tear production, inflammation or number of conjunctival goblet cells compared to a control individual treated with full-length TSG-6 protein. [Aspect 12]

[0023] The method of any one of the preceding aspects, wherein the individual being treated has Sjogren's syndrome. LINK_TSG6 polypeptide for use as described above. [Aspect 13] 20. The LINK_TSG6 polypeptide for use according to any one of the preceding aspects, wherein said treatment comprises said administering eye drops comprising LINK_TSG6. [Aspect 14] 10. The LINK_TSG6 polypeptide for use according to any one of the preceding aspects, wherein said LINK_TSG6 polypeptide comprises, consists of, or consists essentially of (i) the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9, or (ii) an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 7 or 9. [Aspect 15] An eye drop formulation comprising a LINK_TSG6 polypeptide. [Aspect 16] 16. The eye drop formulation according to aspect 15, comprising 240 μg / ml to 3000 μg / ml, preferably 2400 μg / ml to 3000 μg / ml. [Aspect 17] 17. The ophthalmic formulation of embodiment 15 or 16, further comprising prednisolone, cyclosporine, Lifitegrast (Xiidra™), or artificial tears.

Claims

1. A pharmaceutical composition comprising a LINK_TSG6 polypeptide for use in the treatment or prevention of dry eye disease, the LINK_TSG6 polypeptide consists of (i) the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9, or (ii) an amino acid sequence of up to 100 amino acids in length having at least 90% identity to the amino acid sequence of SEQ ID NO: 7 or 9; and wherein the LINK_TSG6 polypeptide has an activity to treat or prevent dry eye disease. The pharmaceutical composition.

2. The treatment or prevention of dry eye disease is Healing of corneal epithelial defects, increased tear production, Suppression of inflammation, and Increased number of conjunctival goblet cells and one or more effects selected from the group consisting of: The pharmaceutical composition of claim 1.

3. The pharmaceutical composition of claim 1 or 2, wherein the treatment comprises topical administration of a LINK_TSG6 polypeptide to the eye.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the treatment comprises administering the LINK_TSG6 polypeptide twice per day.

5. The pharmaceutical composition of any one of claims 1 to 3, wherein the treatment comprises administering the LINK_TSG6 polypeptide less than four times per day.

6. The pharmaceutical composition of claim 3, wherein the LINK_TSG6 polypeptide is formulated as an eye drop.

7. 7. The pharmaceutical composition of any one of claims 1 to 6, wherein LINK_TSG6 is co-administered with one or more of prednisolone, cyclosporine, Lifitegrast (Xiidra™), or artificial tears.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein 10 μg to 200 μg of the LINK_TSG6 polypeptide is administered per eye.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein 120 μg to 150 μg of the LINK_TSG6 polypeptide is administered per eye.

10. 3. The pharmaceutical composition of claim 2, wherein the suppression of inflammation comprises a reduction in the production of one or more pro-inflammatory cytokines in the cornea or intraorbital lacrimal gland.

11. 11. The pharmaceutical composition of claim 10, wherein the proinflammatory cytokine is selected from TNF-α, IL-6, IFN-γ, and IL-1β.

12. The pharmaceutical composition of claim 2, wherein the treatment or prevention of dry eye disease includes healing of corneal epithelial defects, increased tear production, suppression of inflammation, or increased number of conjunctival goblet cells compared to the corneal epithelial defects, tear production, inflammation, or number of conjunctival goblet cells before administration of the LINK_TSG6 polypeptide.

13. The pharmaceutical composition of claim 2, wherein the treatment or prevention of dry eye disease comprises healing of corneal epithelial defects, increased tear production, suppression of inflammation, or increased number of conjunctival goblet cells compared to corneal epithelial defects, tear production, inflammation, or number of conjunctival goblet cells compared to a control individual treated with full-length TSG-6 protein.

14. The pharmaceutical composition of any one of claims 1 to 13, wherein the individual to be treated has Sjogren's syndrome.

15. The pharmaceutical composition of any one of claims 1 to 14, wherein the treatment comprises administering eye drops comprising LINK_TSG6.

16. An eye drop formulation comprising a LINK_TSG6 polypeptide for use in the treatment or prevention of dry eye disease, comprising: the LINK_TSG6 polypeptide consists of (i) the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9, or (ii) an amino acid sequence of up to 100 amino acids in length having at least 90% identity to the amino acid sequence of SEQ ID NO: 7 or 9; and wherein the LINK_TSG6 polypeptide has an activity to treat or prevent dry eye disease. The eye drop preparation.

17. The eye drop formulation of claim 16, comprising 240 μg / ml to 3000 μg / ml of the LINK_TSG6 polypeptide.

18. 18. The eye drop formulation of claim 17, comprising 2400 μg / ml to 3000 μg / ml of the LINK_TSG6 polypeptide.

19. The eye drop formulation of any one of claims 16 to 18, further comprising prednisolone, cyclosporine, Lifitegrast (Xiidra™) or artificial tears.

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