Regulation of WNT signaling in corneal disorders

Abstract

Current treatments for corneal disorders, also known as keratopathy, depend on the stage of the keratopathy. In mild cases of keratopathy, supportive care such as saline eye drops may be sufficient to improve symptoms. The present disclosure provides a method for treating corneal disorders with a modulator of the WNT signaling pathway. More specifically, treatments are provided for corneal epithelial and / or endothelial damage, defects, deficiencies, and dystrophy. Dosage methods and pharmaceutical compositions for treating corneal disorders are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 353,968, filed June 21, 2022, and U.S. Provisional Patent Application No. 63 / 469,499, filed May 29, 2023, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing The Sequence Listing XML associated with this application is provided in XML file format and is incorporated herein by reference. The XML file containing the Sequence Listing XML is named SRZN_021_03WO_ST26.xml. The XML file is 21,937 bytes, was created on June 10, 2023, and has been submitted electronically via the USPTO Patent Center.

[0003] FIELD OF THE INVENTION The present disclosure provides WNT signaling modulators for treating various corneal disorders, more specifically, treatments for corneal epithelial and / or endothelial damage, defects, deficiencies, and dystrophies. [Background technology]

[0004] background The vertebrate cornea is an optically transparent, layered tissue that represents the first and strongest refractive medium in the ocular anatomy. (Patel & Tutchenko, Contact Lens & Ant. Eye, 2019 Oct 42(5), 575-80.) The cornea must remain sturdy to contain and protect the eye; be transparent to allow visible light to pass through the eye; and have the proper shape, symmetry, and smoothness to properly refract and direct a sharp image to the image-forming retina at the back of the eye.

[0005] Unfortunately, the cornea is prone to injury and disease that can cause scarring, discoloration, or even worse, severe scarring, significantly impairing vision and quality of life. Current treatment for corneal disorders, also known as keratopathy, depends on the stage of the keratopathy. In mild cases of keratopathy, supportive care such as saline eye drops may be sufficient to improve symptoms. In severe cases of keratopathy, corneal transplantation or cell transplantation may be necessary, as there are currently no non-surgical treatment options available. Corneal transplantation and cell transplantation, i.e., keratoplasty, are expensive, invasive, and carry the risk of tissue rejection. Furthermore, the available supply of donor corneas and cells is often in short supply.

[0006] Promising therapeutic avenues may exist, and signaling of the WNT developmental pathway has been implicated as important in the stratification and layering of the mammalian cornea. (Zhang et al., Development, 2015 142:3383-93.) WNT signaling proteins are among the most important developmental signaling molecules in the animal kingdom. In animals ranging from fruit flies to mice, frogs, nematodes, and flatworms, the involvement of WNT proteins has been demonstrated in body segmentation, central nervous system (CNS) patterning, dorsal / ventral axis formation, and much more. (See, e.g., Wodarz & Nusse, Annu. Rev. Cell Dev. Biol., 1998 14:59-88.)

[0007] Mutations and regulation of WNT proteins are also thought to play an important role in numerous cancers and dystrophies (Nusse, Cell Res., 2005(15), 28-32). In the context of the cornea, it is unclear whether postdevelopmental WNT modulation can improve keratopathy (corneal damage) and injury. WNT signaling promotes the growth and stemness of corneal endothelial and epithelial cells in vitro (Maurizi et al. Sci. Rep., 2020; Nakatsu et al. Invest. Opthalm. Vis. Sci. 2011), suggesting that activation of Wnt signaling may have therapeutic effects in keratopathy, which lacks corneal cells. Wnt signaling is also central to fibrosis, with elevated corneal scarring (Chawla & Ghosh, J. Cell Physiol. 2017), suggesting that antagonizing Wnt signaling may have therapeutic effects in keratopathy, including scarring, fibrosis, EMT, and EnMT. (Kawashima et al. Mol. Vis., 2013). Understanding keratopathy disease progression and the involvement of WNT signaling could lead to new treatment possibilities.

[0008] Furthermore, corneal tissue is one of several rare and valuable organ donor tissues. Corneal transplantation can be used to treat a variety of keratopathy. However, donated corneas may not grow or heal properly in recipient subjects. Therefore, it would be desirable to have a treatment that promotes ex vivo cell growth in donated corneal tissue. Furthermore, treatments can be performed after transplantation to increase the survival rate of the transplanted tissue in the recipient. Understanding the progression of keratopathy disease and the involvement of WNT signaling in this progression can open up new therapeutic possibilities. The present disclosure provides methods for modulating WNT signaling to treat corneal epithelial and endothelial disorders and promote ex vivo cell growth and maintenance of corneal epithelial and endothelial tissues and cells. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Patel & Tutchenko, Contact Lens & Ant. Eye, 2019 Oct 42(5), 575 - 80. [Non-Patent Document 2] Zhang et al., Development, 2015 142:3383 - 93 [Non-Patent Document 3] Wodarz & Nusse, Annu. Rev. Cell Dev. Biol., 1998 14:59 - 88 [Non-Patent Document 4] Nusse, Cell Res., 2005(15), 28 - 32. [Non-Patent Document 5] Maurizi et al. Sci. Rep., 2020; Nakatsu et al Invest. Opthalm. Vis. Sci. 2011 [Non-Patent Document 6] Chawla & Ghosh, J. Cell Physiol. 2017 [Non-Patent Document 7] Refer to Wodarz & Nusse, Annu. Rev. Cell Dev. Biol., 1998 14:59 - 88 [Non-Patent Document 8] Nusse, Cell Res., 2005(15), 28 - 32. [Non-Patent Document 9] Maurizi et al. Sci. Rep., 2020 [Non-Patent Document 10] Nakatsu et al Invest. Opthalm. Vis. Sci. 2011 [Non-Patent Document 11] Chawla & Ghosh, J. Cell Physiol. 2017 [Summary of the Invention]

[0010] Brief Summary The present disclosure relates to WNT signaling modulators for treating various corneal disorders. More specifically, the present disclosure provides treatment for various keratopathy indications, including, for example, corneal epithelial and / or endothelial damage, defects, deficiencies, and dystrophies.

[0011] In some embodiments, the present disclosure provides a method of treating a subject suffering from keratopathy or corneal damage, comprising administering to the subject an engineered WNT signaling modulator. In certain embodiments, the WNT signaling modulator is an engineered WNT agonist or an engineered WNT antagonist. In further embodiments, the engineered WNT agonist and WNT antagonist comprise a binding composition (or region) that binds to one or more Fzd receptors and a binding composition (or region) that binds to one or more LRP receptors. In further embodiments, the engineered WNT agonist binding composition is selected from the group consisting of an Fzd-binding composition, an Lrp5-binding composition, an Lrp6-binding composition, or an LRP5 / 6-binding composition. In particular embodiments, the engineered WNT agonist FZD-binding composition binds to one or more Fzd receptors selected from the group consisting of Fzd1, Fzd2, and Fzd7.

[0012] In some embodiments, the engineered WNT agonist or WNT antagonist is administered independently at the early and / or late stages of keratopathy. In alternative embodiments, the WNT agonist and WNT antagonist are administered sequentially at the early and / or late stages of keratopathy, or the WNT agonist and WNT antagonist are administered simultaneously at the early and / or late stages of keratopathy. In further embodiments, the WNT agonist is administered before or after the WNT antagonist.

[0013] In one aspect, a method for treating keratopathy in a subject is provided, comprising administering a WNT signaling modulator to the subject. In another embodiment, the present disclosure provides a method for administering an isolated polynucleotide encoding a polypeptide of the molecule, wherein the polynucleotide is optionally mRNA, optionally modified mRNA, or an expression vector comprising the isolated polynucleotide.

[0014] In preferred embodiments, the subject is a human patient. In some embodiments, the WNT signaling modulator can be an engineered WNT signaling modulator. In some embodiments, the WNT signaling modulator can be an engineered WNT agonist or an engineered WNT antagonist.

[0015] In a preferred embodiment, the engineered WNT agonist is selected from (i) WNT3a; (ii) WNT mimic; or (iii) R-spondin mimic, or (iv) WNT mimic fused to mutant R-spondin mimic, called super-SWAP or super-agonist. For example, see PCT Application Publication No. WO2021173726, which discloses non-limiting examples of super-SWAP molecules. In a more preferred embodiment, the WNT mimic is a SWAP™ compound, and the R-spondin mimic is a SWEETS™ compound. A molecule that includes SWAP and SWEET in one combined molecule is called super-SWAP or super-agonist. In particular, the WNT signaling modulator can be a canonical WNT modulator or a non-canonical WNT modulator (see, for example, Ackers and Malgor (2017) Diabetes & Vascular Res., 15:3-13).

[0016] In any embodiment of the method, the WNT signalling modulator may be co-administered with R-spondin, or any R-spondin homolog, variant, fragment, mimetic, or any combination thereof.

[0017] In any embodiment of the method, the wnt signaling modulator may be a WNT mimetic fused to a mutant Rspondin mimetic, referred to herein as a super-SWAP or superagonist.

[0018] In preferred embodiments, the WNT signaling modulator is administered to the subject at a therapeutically effective dose. In some embodiments, the WNT signaling modulator can be administered by eye drops, hydrogel, depot, or intraocular injection.

[0019] In any embodiment described herein, the WNT signaling modulator can be suspended in an aqueous solution. The WNT signaling modulator can be suspended in an aqueous solution at a concentration of about 0.01 pM, 0.1 pM, 1 pM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, 100 mM, or up to about 1 M. In a preferred embodiment, the WNT signaling modulator is suspended in an aqueous solution at a concentration ranging from about 0.1 nM to 1 μM. In a more preferred embodiment, the WNT signaling modulator is suspended in an aqueous solution at a concentration ranging from about 1 nM to 100 nM.

[0020] In any embodiment described herein, the subject's keratopathy may be corneal dystrophy. More specifically, in any embodiment of the method, the keratopathy may be bacterial infection, viral infection, fungal infection, protozoan infection, archaeal infection, genetic disorder, brittle cornea syndrome (BCS), corneal endothelial mesenchymal transition (EnMT), corneal epithelial mesenchymal transition (EMT), corneal fibrosis, Cogan's syndrome, corneal ulcer, epithelial basement membrane dystrophy (EBMD), macular corneal dystrophy, Fuchs' dystrophy, gelatinous droplet corneal dystrophy, granular corneal dystrophy type I, granular corneal dystrophy type II, interstitial keratitis, iridocorneal endothelial syndrome (ICE), keratoconjunctivitis sicca, keratoconus, keratomalacia, lattice keratopathy. The corneal disease may be selected from the group consisting of corneal stem cell deficiency syndrome (LSDS), epithelial-mesenchymal transition (EST), endothelial-mesenchymal transition (EST), corneal fibrosis (ESF), corneal fibrosis (ESF), corneal scarring (including post-transplant corneal scarring), corneal fibrosis (ESF ..., corneal fibrosis (ESF), corneal fibrosis, corneal In a still preferred embodiment of the method, the keratopathy is Fuchs' dystrophy.

[0021] Also described herein is a method for increasing the cell yield of mammalian corneal epithelial and / or endothelial cells grown ex vivo before or after corneal transplantation, comprising treating mammalian donor corneas or isolated corneal cells with a WNT signaling modulator. In a preferred embodiment, the mammalian donor cornea is a human cornea. In other embodiments, the isolated corneal cells are limbal cells or corneal endothelial cells. In some embodiments, the WNT signaling modulator can be an engineered WNT signaling modulator. In some embodiments, the WNT signaling modulator can be an engineered WNT agonist.

[0022] In a preferred embodiment of the method for increasing cell yield, the engineered WNT agonist is selected from (i) WNT3a; (ii) a WNT mimetic; (iii) an R-spondin mimetic, or (iv) a WNT mimetic fused to a mutant R-spondin mimetic (referred to as a super-SWAP or super-agonist). In a more preferred embodiment, the WNT mimetic is a SWAP™ compound, and the R-spondin mimetic is a SWEETS™ compound, or a super-SWAP or super-agonist.

[0023] In some embodiments, the present disclosure provides that the WNT signaling modulator is a. WNT3a or any homolog, variant, fragment, or mimetic of WNT3a; b.G211-18R5; c.R2M3-26; d.1SH1-03; e.hp1SH1-03 f.17SB9-03; g. Any of the above, alone or in combination with one or more Rspondins, or another growth factor such as fibroblast growth factor (FGF); h. G211-18R5-Rspo2RA or G211-R2H1-Rspo2RA or other super-SWAP or super-agonist; i. Or any combination of the foregoing.

[0024] In certain embodiments, the engineered WNT agonist comprises a sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in any of SEQ ID NOs: 1-16. In particular embodiments, the engineered WNT agonist comprises one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 1 and one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 2. In particular embodiments, the engineered WNT agonist comprises one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 3, and one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 4. In particular embodiments, the engineered WNT agonist comprises one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 5, and one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 6. In particular embodiments, the engineered WNT agonist comprises two sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:1 and two sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:2.In particular embodiments, the engineered WNT agonist comprises two sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 3 and two sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 4. In particular embodiments, the engineered WNT agonist comprises two sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 5 and two sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 6.

[0025] In some embodiments, the engineered WNT agonist comprises two sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:7 and two sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:8.

[0026] In particular embodiments, the engineered WNT agonist comprises one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 9, and one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 10. In particular embodiments, the engineered WNT agonist comprises one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 11, and one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 12. In particular embodiments, the engineered WNT agonist comprises one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 13, and one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 14. In particular embodiments, the engineered WNT agonist comprises one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 15, and one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 16.

[0027] In any embodiment of the method of increasing cell yield, the WNT signaling modulator may be administered or co-administered with R-spondin, or any R-spondin homolog, variant, fragment, mimetic, or any combination thereof.

[0028] In a preferred embodiment, the WNT signaling modulator is administered to a subject at a therapeutically effective dose. In any of the embodiments described herein, the WNT signaling modulator can be suspended in an aqueous solution. The WNT signaling modulator can be suspended in an aqueous solution at a concentration of about 0.01 pM, 0.1 pM, 1 pM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, 100 mM, or up to about 1 M. In a preferred embodiment, the WNT signaling modulator is suspended in an aqueous solution at a concentration ranging from about 0.1 nM to 1 μM. In a more preferred embodiment, the WNT signaling modulator is suspended in an aqueous solution at a concentration ranging from about 1 nM to 100 nM.

[0029] In a preferred embodiment of the method for increasing cell yield, the WNT signaling modulator is administered to the mammalian donor cornea by immersing the mammalian donor cornea in vitro in a water bath solution containing a predetermined dose of the WNT signaling modulator. In other embodiments, the WNT signaling modulator is applied to limbal stem cells or corneal endothelial cells before or after transplantation.

[0030] In preferred embodiments, the engineered WNT agonist and engineered WNT antagonist comprise a binding composition that binds to one or more Fzd receptors and a binding composition that binds to one or more LRP receptors. In a further preferred embodiment of the method for increasing cell yield, the binding composition of the engineered WNT agonist is selected from the group consisting of a Fzd-binding composition, an Lrp5-binding composition, an Lrp6-binding composition, and an LRP5 / 6-binding composition.

[0031] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0032] [Figure 1-1] Figure 1A shows a photomicrograph of fluorescently stained donor corneal endothelial tissue in a healthy "normal" eye donor stained for Fzd receptors, Fzd1, 2, 4, 5, 7, 8, 9, and 10, shown as green in the figure, using in situ hybridization. Because Fzds1, 2, and 7 were most prominently expressed, the focus is on the expression of Fzds1, 2, and 7 in patients with FECD.

[0033] [Figure 1-2] Figure 1B shows photomicrographs of an additional healthy "normal" patient and a patient diagnosed with Fuchs' dystrophy, a keratinization disorder. WNT and R-spondin receptors Lrp5 / 6; Fzd1 / 2 / 7; and the E3 ubiquitin ligase ZNRF3 were stained using in situ hybridization (green in the figure). The corneal endothelial marker Na+K+ATPase was stained using immunofluorescence (red in the figure). DNA was stained with 4',6-diamidino-2-phenylindole (DAPI) (gray-white in the figure).

[0034] [Figure 2-1] Figure 2A shows a graph of the number of Ki67+ (proliferation marker) cells per well in human corneal endothelial cells treated with FGF1, R-spondin-1, the WNT mimetic compound R2M3-26, or the WNT superagonist or super-SWAP G211-18R5-Rspo2RA. The results show that the WNT mimetic and the WNT superagonist or super-SWAP increase the proliferation of human corneal endothelial cells. *p≦0.05. **p≦0.01. ***p≦0.001. Statistical analysis was performed using an unpaired two-tailed t-test.

[0035] Figure 2B shows a graph of the % Ki67+ cells in human corneal endothelial cells treated with the FZD1 / 2 / 5 / 7 / 8+LRP6-binding WNT mimetic R2M3-26 or the FZD1 / 2 / 7+LRP6-binding WNT mimetic 1RC07-26. The results show that WNT mimetics that bind to FZD1 / 2 / 5 / 7 / 8 and LRP6 increase the proliferation of human corneal endothelial cells. *p≦0.05. **p≦0.01. Statistical analysis was performed using an unpaired two-tailed t-test.

[0036] Figure 2C shows a graph of the % Ki67+ cells in human corneal endothelial cells treated with WNT superagonist or super-SWAP, G211-18R5-Rspo2RA, or G211-R2H1-Rspo2RA. The results demonstrate that WNT superagonist or super-SWAP binding to FZD1, 2, 7, and LRP6 is as effective as WNT superagonist or super-SWAP G211-18R5-Rspo2RA (binding to FZD1 / 2 / 5 / 7 / 8 + LRP6) in increasing human corneal endothelial cell proliferation. *p ≤ 0.05. **p ≤ 0.01. Statistical analysis was performed using an unpaired two-tailed t-test.

[0037] [Figure 2-2] Figure 2D shows fluorescence micrographs of untreated primary human corneal endothelial cells and primary human corneal endothelial cells treated with the WNT mimetic G211-18R5+R-spondin-1. The images demonstrate that WNT agonists increase the proliferation of cultured human corneal endothelial cells.

[0038] Figure 2E shows fluorescence micrographs of corneas from human organ donors with Fuchs' dystrophy that were untreated and treated with the WNT mimetic G211-18R5+R-spondin-1. The images demonstrate that WNT agonists increase the proliferation of human corneal endothelial cells in corneal organ cultures.

[0039] [Figure 3-1]FIG. 3A shows changes in Axin2 RNA transcript levels in the corneas of mice injected intracamerally with an anti-GFP isotype control antibody or the wnt mimetic R2M3-26 (61 pmol each).

[0040] Figure 3B shows a photomicrograph of fluorescently stained corneal endothelium from a mouse injected intracamerally with anti-GFP, SWAP, or R2M3-26 (61 pmol each). The WNT target gene Axin2 was stained using in situ hybridization and is shown in green. DNA was stained with 4',6-diamidino-2-phenylindole (DAPI) and is shown in blue.

[0041] [Figure 3-2] Figure 3C shows changes in Axin2 RNA transcript levels in the corneas of mice injected intracamerally (IC) or intravitreally (IVT) with anti-GFP or wnt superagonist or super-SWAP, G211-18R5-Rspo2RA (2.6 mg each).

[0042] Figure 3D shows a photomicrograph of fluorescently stained corneal endothelium from a mouse intracamerally injected with anti-GFP or G211-18R5-Rspo2RA (7 pmol each). The WNT target gene Axin2 was stained using in situ hybridization and is shown in green. DNA was stained with 4',6-diamidino-2-phenylindole (DAPI) and is shown in blue.

[0043] [Figure 3-3] Figure 3E shows central corneal thickness measurements from a rabbit 3 days after ablation of the central 10 mm of corneal endothelium. Figure 3F shows endothelial lesion size measurements from a rabbit 3 days after ablation of the central 10 mm of corneal endothelium. Rabbits were injected intracamerally with anti-GFP or R2M3-26 on day 0.

[0044] [Figure 3-4]Figure 3G shows endothelial lesion size from a rabbit 3 days after ablation of the central 10 mm of corneal endothelium from another study. Figure 3H shows central corneal thickness measurements from a rabbit 3 days after ablation of the central 10 mm of corneal endothelium from another study. Figure 3I shows corneal opacity measurements from a rabbit 3 days after ablation of the central 10 mm of corneal endothelium from another study. Rabbits were intracamerally injected with anti-GFP, WNT superagonist, or Super-SWAP, G211-18R5-Rspo2RA, on day 0. The data in Figures 3G-3I show significant improvement in corneal opacity scores with high doses of WNT superagonist or Super-SWAP, while there appears to be a trend toward decreased corneal thickness in Figure 3H.

[0045] [Figure 4-1] FIG. 4A shows that the wnt superagonist or super-SWAP, G211-18R5-Rspo2RA, reduces corneal thickness. [Figure 4-2] FIG. 4B shows that the wnt superagonist or super-SWAP, G211-18R5-Rspo2RA, improves transparency. [Figure 4-3] Figure 4C shows that the wnt superagonist or super-SWAP, G211-18R5-Rspo2RA, exhibits a pharmacodynamic response (PD) in a mouse model of corneal cryoinjury. Compared to an anti-GFP isotype control, intravitreal (IVT) injection of 5 μg of the wnt superagonist or super-SWAP, G211-18R5-Rspo2RA, demonstrated a significant reduction in central corneal thickness as measured by optical coherence tomography (OCT) (Figure 4A) (a summary of six independent studies) and improved corneal transparency in response to cryoinjury, as measured by brightfield imaging of the anterior chamber of the eye (Figure 4B) (a representative image from one such study). In corneal homogenates from one such study in which G211-18R5-Rspo2RA or anti-GFP antibodies were administered intravitreally to mice after cryoinjury, the Wnt superagonist or super-SWAP showed significant induction of Axin2 mRNA (a Wnt target gene) at day 4 (Figure 4C).

[0046] [Figure 5-1] FIG. 5A shows that 1SH1-03 reduces corneal thickness % and improves corneal transparency in a mouse model of corneal cryoinjury. [Figure 5-2] Figure 5B shows that 1SH1-03 reduces corneal thickness and improves corneal clarity in a corneal cryoinjury mouse model. Figure 5A is a graph showing that 25 μg of intravitreal (IVT) injected 1SH1-03 showed a significant reduction in central corneal thickness (combined from two independent studies) compared to anti-GFP isotype control and 5 μg of 1SH1-03, as measured by optical coherence tomography (OCT). At the 2-day time point, the lines from top to bottom correlate with GFP, 1SH1-03 5 μg, and 1SH1-03 25 μg, respectively. Figure 5B shows a brightfield image (representative image from one such study) demonstrating that 1SH1-03 showed improved corneal clarity in response to cryoinjury, as measured by brightfield imaging of the anterior chamber of the eye.

[0047] [Figure 6] Figure 6 shows that the Wnt mimetic hp1SH1-03 reduced corneal endothelial lesion size in mice after corneal cryoinjury. Intravitreal (IVT) injection of hp1SH1-03 reduced lesion size compared to the anti-GFP isotype control in eyes after cryoinjury. This figure shows corneal flat mounts from eyes from the above treatment groups that were subjected to immunofluorescence, stained with Ki67 (shown in red) and Dapi (shown in blue), and imaged by confocal microscopy. While there is a lesion in the central region in the anti-GFP isotype-treated group, which lacks proliferating Ki67 cells, there appears to be no detectable lesion in the hp1SH1-03-treated eyes; this region is marked by proliferating corneal endothelial cells.

[0048] [Figure 7-1] FIG. 7A shows that hp1SH1-03 induces robust proliferation in injured and uninjured human corneal endothelial cells (HCECs). [Figure 7-2]Figure 7B shows that hp1SH1-03 induces robust proliferation in injured and uninjured human corneal endothelial cells (HCECs). Whole human corneas obtained from an eye bank were cut into quarters and treated with 5 nM hp1SH1-03 for two days. Figure 7A shows the results of ZO-1 detection and the proliferation rate measured by counting the percentage of CECs expressing the proliferation marker Ki67. Figure 7B shows quantitative analysis demonstrating that hp1SH1-03 dramatically promoted HCEC proliferation, with this proliferation being more pronounced in the wound area. Compared to anti-GFP-treated eyes, proliferation was approximately six-fold higher at the edge and three-fold higher in the center.

[0049] [Figure 8-1] Figures 8A-8B show that Wnt superagonist or super-SWAP G211-18R5-Rspo2RA exhibits an increased PD response in a UV-A damage-induced FECD rodent model. Figure 8A shows changes in Axin2 RNA transcript levels in corneas from naive mice or mice treated with 500 J / cm2 of UV-A light in the right eye and mice intravitreally injected with anti-GFP or G211-18R5-Rspo2RA (2.6 μg each). Figure 8B shows central corneal thickness (CCT) in UV-A-treated mice described in Figure 8A and subjected to optical coherence tomography for measurement of CCT as a measure of corneal swelling. At 2 days post-treatment, there is a trend toward a decrease in CCT in response to Wnt superagonist or super-SWAP G211-18R5-Rspo2RA. [Figure 8-2] Same as above.

[0050] [Figure 9-1] Figures 9A-9B show the expression levels of various markers. Figure 9A shows fluorescence micrographs of human corneal samples stained for various WNT receptors, corneal and limbal region markers, and DNA. Images show corneal and limbal epithelium. Figure 9B is a table showing the expression levels of various WNT and R-spondin receptors in corneal epithelium and limbal epithelium. [Figure 9-2] Same as above.

[0051] [Figure 10] Figures 10A-10B show the expression of Axin2 and p63. Figure 10A shows the change in Axin2 RNA transcription level in rabbit limbal epithelial organoids after one week of treatment with 5 nM of the WNT mimetic R2M3-26 or 1 μg / mL of R-spondin-1. *p≦0.05. ***p≦0.001. Statistical analysis was performed using an unpaired two-tailed t-test. Figure 10B shows the change in p63 RNA transcription level in rabbit limbal epithelial organoids after one week of treatment with 5 nM of the WNT mimetic R2M3-26 or 1 μg / mL of R-spondin-1. *p≦0.05. Statistical analysis was performed using an unpaired two-tailed t-test.

[0052] [Figure 11] Figures 11A-11C show data demonstrating increased cell growth and proliferation in human corneal epithelium. Figure 11A shows cell growth data for cells treated with the WNT mimetic G211-18R5 for two days. Cell numbers were measured using the CellTiter-Glo® assay. Figure 11B shows Ki67+ cell proliferation data after treatment with the WNT mimetic R2M3-26. Proliferation was measured by analyzing immunofluorescence micrographs. Figure 11C shows cell growth data for cells treated with R-spondin-1 for two days. Statistical analysis for each figure was performed using an unpaired, two-tailed t-test.

[0053] [Figure 12] Figures 12A-12B show micrographs of an in vitro wound healing assay. Human corneal epithelial cells were plated around a physical barrier. The physical barrier was removed, leaving a gap. The cells were cultured for 2 days in medium without the WNT mimetic R2M3-26 (Figure 12A) and in medium with the WNT mimetic R2M3-26 (Figure 12B). The cells were then stained with DAPI, imaged, and the size of the gap was measured.

[0054] [Figure 13]Figure 13 shows that topical Fc-Rspo2 eye drops reduce corneal epithelial defects in a mouse model of corneal epithelial debridement. Figure 13 shows fluorescent and brightfield micrographs of the central corneal epithelium of naive mice and mice treated with anti-GFP eye drops (5 mg / mL, 3 μL, 4 times daily) or Fc-Rspondin-2 eye drops (5 mg / mL, 3 μL, 4 times daily) 7 days after limbal-limb debridement. The WNT target gene Axin2 was stained using in situ hybridization and is shown in red. DNA was stained with 4',6-diamidino-2-phenylindole (DAPI) and is shown in blue. The progenitor marker p63 was stained using immunofluorescence and is shown in green. Hematoxylin and eosin (H&E) staining is also shown. Data show that R-spondin eye drops activate Wnt signaling, proliferate progenitor cells, thicken the corneal epithelium, and reduce conjunctivalization.

[0055] [Figure 14] Figure 14 shows a fluorescence micrograph of a cornea from a human organ donor stained for THBD (thrombomodulin / CD141 / BDCA-3, a high-affinity thrombin receptor present on endothelial cell membranes), showing specific staining (shown in green) in the central corneal and limbal membranes, but not in the conjunctiva. DNA was stained with 4',6-diamidino-2-phenylindole (DAPI), shown in blue. The data in Figure 14 suggest that THBD can be used to generate limbal-targeted Rspo mimics / SWEETs™ molecules for corneal regeneration.

[0056] [Figure 15-1] Figure 15A shows a schematic diagram of mouse conjunctival and corneal epithelial organoids derived from primary tissue. Expansion cultures maintain markers such as keratin 12 (Ck12) or keratin 19 (Ck19).

[0057] [Figure 15-2]Figure 15B shows the changes in Axin2 mRNA transcription levels in conjunctival or corneal epithelial organoids treated with different WNT mimetics. R2M3-26 is a WNT mimetic targeting FZD12578. 17SB9 is a WNT mimetic biased towards FZD8. Data are normalized to untreated controls for each tissue. The data in Figure 15B suggest that 17SB9-03-Rspo mimetic can be used to generate corneal epithelial-targeted Fzd8-Rspo mimetic / SWEETs™ molecules for corneal regeneration. DETAILED DESCRIPTION OF THE INVENTION

[0058] Detailed Description As used in this specification, including the appended claims, the singular forms of words such as "a," "an," and "the" include their corresponding plural references unless the context clearly dictates otherwise.

[0059] All references cited herein are incorporated by reference to the same extent as if each individual publication, patent application, or patent was specifically and individually indicated to be incorporated by reference. I. Definition

[0060] The "activity" of a molecule can describe or refer to the binding of the molecule to a ligand or receptor, catalytic activity, ability to stimulate gene expression, antigenic activity, modulation of the activity of other molecules, etc. The "activity" of a molecule can also refer to activity that modulates or maintains cell-cell interactions, e.g., adhesion, or activity that maintains the structure of a cell, e.g., a cell membrane or cytoskeleton. "Activity" can also mean specific activity, e.g., [catalytic activity] / [mg protein] or [immunological activity] / [mg protein], etc.

[0061] As used herein, the terms "administering" or "introducing" or "providing" refer to the delivery of a composition to a cell, tissue, and / or organ of a subject, or to a subject. Such administration or introduction can occur in vivo, in vitro, or ex vivo.

[0062] As used herein, the term "antibody" refers to an isolated or recombinant binding agent that contains the necessary variable region sequence for specific binding to an antigen epitope. Thus, an antibody is any form of antibody or fragment thereof that exhibits the desired biological activity, e.g., binds to a specific target antigen. Thus, it is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, nanobodies, diabodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, including, but not limited to, scFv, Fab, and Fab2, so long as they exhibit the desired biological activity.

[0063] An "antibody fragment" comprises a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (e.g., Zapata et al., Protein Eng. 8(10):1057-1062(1995)); single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a designation reflecting its ability to readily crystallize. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0064] The term "antigen" refers to a molecule or portion of a molecule that can be bound by a selective binding agent, such as an antibody, and that can be used in an animal to generate antibodies capable of binding to an epitope of that antigen. In certain embodiments, a binding agent (e.g., a WNT surrogate molecule or binding region thereof, or a WNT antagonist) is said to specifically bind to an antigen if it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0065] As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment comprising at least one complementarity-determining region (CDR) of an immunoglobulin heavy and / or light chain, or a VHH / sdAb (single domain antibody) or Nanobody® (Nab), that binds to an antigen of interest, particularly one or more Fzd receptors, or LRP5 and / or LRP6. In this regard, the antigen-binding fragment of an antibody described herein can comprise one, two, three, four, five, or all six CDRs of the VH and VL from an antibody that binds to one or more Fzd receptors, or LRP5 and / or LRP6.

[0066] As used herein, the terms "biological activity" and "biologically active" refer to the activity resulting from a specific biological element in a cell. For example, the "biological activity" of a WNT agonist or a fragment or variant thereof refers to its ability to mimic or enhance WNT signaling. As another example, the biological activity of a polypeptide or a functional fragment or variant thereof refers to the ability of the polypeptide or a functional fragment or variant thereof to perform its natural function, such as binding, enzymatic activity, etc. As a third example, the biological activity of a gene regulatory element (e.g., a promoter, an enhancer, a Kozak sequence, etc.) refers to the ability of the regulatory element or a functional fragment or variant thereof to regulate the expression of the gene to which it is operably linked, i.e., to promote, enhance, or activate the translation of its expression, respectively.

[0067] As used herein, the term "bifunctional antibody" refers to an antibody that contains a first arm with specificity for one antigenic site and a second arm with specificity for a different antigenic site, i.e., a bifunctional antibody has dual specificities.

[0068] "Bispecific antibodies" refer to full-length antibodies generated by quadroma technology (see Milstein et al., Nature, 305(5934):537-540 (1983)), by chemical conjugation of two different monoclonal antibodies (see Staerz et al., Nature, 314(6012):628-631 (1985)), or by knob-into-hole or similar approaches, which introduce mutations into the Fc region (see Holliger et al., Proc. Natl. Acad. Sci. USA, 90(14):6444-6448 (1993)), resulting in multiple different immunoglobulin species, only one of which is the functional bispecific antibody. A bispecific antibody binds to one antigen (or epitope) with one of its two binding arms (one HC / LC pair) and to a different antigen (or epitope) with its second arm (a different HC / LC pair). By this definition, a bispecific antibody has two distinct antigen-binding arms (both in specificity and CDR sequence) and is monovalent for each antigen to which it binds.

[0069] "Comprising" means that the recited elements are required, e.g., by a composition, method, kit, etc., although other elements may be included within the claims to form, e.g., a composition, method, kit, etc. For example, an expression cassette that "comprises" a gene encoding a therapeutic polypeptide operably linked to a promoter is an expression cassette that may include other elements in addition to the gene and promoter, e.g., polyadenylation sequences, enhancer elements, other genes, linker domains, etc.

[0070] "Consisting essentially of" refers to, for example, limiting the scope of the described composition, method, kit, etc. to specific materials or steps that do not substantially affect the basic and novel characteristic(s) of the composition, method, kit, etc. For example, an expression cassette "consisting essentially of" a gene encoding a therapeutic polypeptide operably linked to a promoter and polyadenylation sequence may include additional sequences, e.g., linker sequences, so long as they do not substantially affect transcription or translation of the gene. As another example, a variant or mutant polypeptide fragment "consisting essentially of" a recited sequence has the amino acid sequence of the recited sequence plus or minus about 10 amino acid residues at the boundary of the sequence based on the full-length native polypeptide from which it is derived, e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue less than the recited boundary amino acid residue, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues more than the recited boundary amino acid residue.

[0071] "Consisting of" means excluding from a composition, method, or kit any element, step, or ingredient not specified in the claim. For example, a polypeptide or polypeptide domain "consisting of" a recited sequence includes only the recited sequence.

[0072] A "control element" or "control sequence" is a nucleotide sequence involved in a molecular interaction that contributes to the functional regulation of a polynucleotide, including the replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. Regulation can affect the frequency, rate, or specificity of the process and can be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region that, under certain conditions, is capable of binding RNA polymerase and initiating transcription of a coding region typically located downstream (3' direction) of the promoter.

[0073] As used herein, "cornea" and "cornea" refer to the optically transparent layered tissue that covers the iris, pupil, and anterior chamber of the eye. "Corneal limbus" or "limbus" refers to the approximately circular boundary where the cornea meets the sclera of the eye. Those skilled in the art will readily understand that the boundary between corneal and limbal tissue is imprecise, and references to the "cornea" should be understood to encompass approximate margins. The cornea comprises five cellular layers: (1) the epithelium (outermost layer, smooth optical surface, and barrier to chemicals, water, and microorganisms); (2) Bowman's layer (helps maintain its precise convex shape); (3) the stroma (the cornea's main mechanical structure and refractive medium); (4) Descemet's membrane (stationary layer of endothelium); and (5) the endothelium (maintains transparency by removing water from the stroma).

[0074] As used herein, "limbal cell deficiency" or "corneal epithelial stem cell deficiency" refers to a genetic, acquired, or idiopathic deficiency or complete failure of stem cells in the limbus, which are essential for corneal epithelial repopulation. The loss or failure of limbal stem cells results in the inability to repair or regenerate the corneal epithelium, leading to keratoconjunctivalization, neovascularization, corneal scarring, and chronic inflammation.

[0075] As used herein, "corneal dystrophies" generally refer to disorders characterized by abnormal discoloration, opacity, and / or clouding of any layer or combination of layers of the cornea. Corneal dystrophies can be hereditary or non-hereditary. They can be caused by abnormal cell growth and / or by the deposition and accumulation of collagen, lipids, cholesterol, and / or other foreign substances.

[0076] Fuchs dystrophy (also known as Fuchs endothelial cell dystrophy (FECD) or Fuchs corneal dystrophy (FCD)) is a class of degenerative disorders of the corneal endothelial monolayer, characterized by a decrease in endothelial cell density, corneal grooves, and the accumulation of corneal edema, leading to corneal opacity and decreased vision.

[0077] As used herein, the term "engineered" indicates that a molecule, e.g., a Wnt signalling modulator, does not occur in nature, e.g., is the result of genetic engineering.

[0078] An "expression vector" is a vector discussed herein or known in the art, e.g., a plasmid, minicircle, viral vector, liposome, etc., that contains a region encoding a gene product of interest and is used to effect expression of the gene product in an intended target cell. An expression vector also contains control elements, e.g., a promoter, enhancer, untranslated region (UTR), miRNA targeting sequence, etc., operably linked to the coding region to promote expression of the gene product in the target. The combination of control elements and the gene(s) to which they are operably linked for expression is sometimes referred to as an "expression cassette," many of which are known and available in the art or can be readily constructed from components available in the art.

[0079] As used herein, the term "FR set" refers to the four adjacent amino acid sequences that constitute the CDRs of a CDR set of a heavy or light chain V region. While some FR residues may contact a bound antigen, FRs primarily serve to fold the V region into the antigen-binding site, particularly the FR residues directly adjacent to the CDRs. Within FRs, certain amino acid residues and certain structural features are highly conserved. In this regard, all V region sequences contain an internal disulfide loop of approximately 90 amino acid residues. When the V region folds into the binding site, the CDRs are displayed as protruding loop motifs that form the antigen-binding surface. Regardless of the exact CDR amino acid sequence, it is generally recognized that there are conserved structural regions of FRs that influence the folding of the CDR loops into specific "canonical" structures. Furthermore, certain FR residues are known to participate in noncovalent interdomain contacts that stabilize the interaction between the heavy and light chains of an antibody.

[0080] The terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, humans and non-human primates, including monkeys and humans; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0081] As used herein, the term "isolated" indicates that a molecule, e.g., a naturally occurring molecule, is not in its natural environment or is separated from one or more components present in its natural environment or present during the production of the molecule.

[0082] As used herein, the terms "keratopathy" and "keratopathy" refer to diseases or disorders of the eye, particularly the cornea and / or limbus of the eye. Keratopathy includes, but is not necessarily limited to, bacterial infections, viral infections (e.g., herpes simplex keratitis and ophthalmic varicella (i.e., shingles)), fungal infections, protozoal infections, archaeal infections, genetic disorders, brittle cornea syndrome (BCS), corneal endothelial-mesenchymal transition (EnMT), corneal epithelial-mesenchymal transition (EMT), corneal fibrosis, Cogan's syndrome, corneal ulcers, epithelial basement membrane dystrophy (EBMD, ABMD, Map), and others. Dot), macular corneal dystrophy, Fuchs' dystrophy (also called Fuchs' corneal endothelial dystrophy (FCED) or Fuchs' endothelial dystrophy (FED)), colloidal droplet corneal dystrophy, granular corneal dystrophy type I, granular corneal dystrophy type II, interstitial keratitis, iridocorneal endothelial syndrome (ICE), keratoconjunctivitis sicca, keratoconus, keratomalacia, lattice dystrophy type I, lattice dystrophy type II, Risch corneal dystrophy These include macular corneal dystrophy, Miesmann corneal dystrophy, bullous keratopathy, aniridia, marginal ulcerative keratitis, phlyctenular keratoconjunctivitis, posterior polymorphous corneal dystrophy, pterygium, Reiss-Bückler corneal dystrophy, Schneider crystalline corneal dystrophy, Stevens-Johnson syndrome, superficial punctate keratitis, Thiel-Behnke corneal dystrophy, neurotrophic keratitis, corneal herpes, scarring, and corneal epithelial stem cell deficiency.

[0083] "Monoclonal antibody" refers to a homogeneous antibody population; monoclonal antibodies are composed of amino acids (naturally occurring and non-naturally occurring) involved in selective binding of an epitope. Monoclonal antibodies are highly specific and directed against a single epitope. The term "monoclonal antibody" encompasses not only intact and full-length monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv), nanobodies®, variants thereof, fusion proteins comprising an antigen-binding fragment of a monoclonal antibody, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of an immunoglobulin molecule containing an antigen-binding fragment (epitope recognition site) with the requisite specificity and ability to bind to the epitope, including the WNT surrogate molecules disclosed herein. It is not intended to be limited as to the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins as well as fragments such as those described above under the definition of "antibody."

[0084] As used herein, the term "natural" or "wild-type" refers to a nucleotide sequence, such as a gene or gene product, such as RNA or protein, present in a wild-type cell, tissue, organ, or organism. As used herein, the term "variant" refers to a variant having less than 100% sequence identity with a reference polynucleotide or polypeptide sequence, such as a naturally occurring polynucleotide or polypeptide sequence. In other words, a variant contains at least one amino acid difference (e.g., an amino acid substitution, an amino acid insertion, an amino acid deletion) compared to a reference polynucleotide sequence, such as a naturally occurring polynucleotide sequence or polypeptide sequence. For example, a variant can be a polynucleotide having 50% or more, 60% or more, or 70% or more sequence identity with a full-length naturally occurring polynucleotide sequence, such as 75% or 80% or more, for example 85%, 90%, or 95% or more, for example 98% or 99% identity with a full-length naturally occurring polynucleotide sequence. As another example, a variant can be a polypeptide having 70% or more sequence identity to a full-length naturally occurring polypeptide sequence, for example 75% or 80% or more, for example 85%, 90%, or 95% or more, for example 98% or 99% identity to a full-length naturally occurring polypeptide sequence. Variants can also include fragments of a reference, e.g., a variant fragment of a reference sequence, e.g., a naturally occurring sequence, that share 70% or more sequence identity with the naturally occurring sequence (e.g., 75% or 80% or more, for example 85%, 90%, or 95% or more, for example 98% or 99% identity with the naturally occurring sequence).

[0085] "Operably linked" or "operably linked" refers to a juxtaposition of genetic elements, the elements being in a relationship permitting them to function in the expected manner. For example, a promoter is operably linked to a coding region if it helps initiate transcription of the coding sequence. There can be intervening residues between the promoter and the coding region, so long as this functional relationship is maintained.

[0086] As used herein, the terms "polypeptide," "peptide," and "protein" refer to polymers of amino acids of any length. The terms also encompass modified amino acid polymers, including, for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.

[0087] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. As used herein, the term polynucleotide refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment described herein that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute the double-stranded form.

[0088] A polynucleotide or polypeptide has a certain percentage of "sequence identity" with another polynucleotide or polypeptide, meaning that when aligned, that percentage of bases or amino acids are the same when comparing the two sequences. As used herein, the terms "identity" and "identical," with respect to a polypeptide or polynucleotide sequence, refer to the percentage of exactly matching residues in an alignment between the "query" sequence and the "subject" sequence, such as an alignment generated by the BLAST algorithm. Identity is calculated over the entire length of the subject sequence unless otherwise specified. Thus, if at least x% (truncated) of the residues in the subject sequence align as exact matches with the corresponding residues in the query sequence when the query sequence is aligned with the subject sequence, the query sequence "shares at least x% identity" with the subject sequence. If the subject sequence has variable positions (e.g., residues designated by X), alignment to any residue in the query sequence is counted as a match. Unless otherwise indicated, sequence alignment is performed using the NCBI Blast service (BLAST+ version 2.12.0). To determine sequence identity, sequences can be aligned using methods and computer programs, including BLAST, available on the World Wide Web at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc. (a division of Harcourt Brace & Co., San Diego, California, USA). Of particular interest are alignment programs that allow gaps in the sequence.The Smith-Waterman is one type of algorithm that allows for gaps in sequence alignments. See Meth. Mol. Biol. 70:173-187 (1997). Sequences can also be aligned using the GAP program using the Needleman and Wunsch alignment method. See J. Mol. Biol. 48:443-453 (1970).

[0089] Of note is the BestFit program, which uses the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482-489 (1981)) to determine sequence identity. The gap creation penalty generally ranges from 1 to 5, usually from 2 to 4, and in many embodiments is 3. The gap extension penalty generally ranges from about 0.01 to 0.20, and is often 0.10. The program has default parameters determined by the sequence input to be compared. Preferably, sequence identity is determined using the default parameters determined by the program. This program is also available from the Genetics Computing Group (GCG) package, Madison, Wisconsin, USA. Another program of interest is the FastDB algorithm. FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecules Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, by Alan R. Liss, Inc. Percent sequence identity is calculated by FastDB based on the following parameters: mismatch penalty: 1.00; gap penalty: 1.00; gap size penalty: 0.33; joining penalty: 30.0.

[0090] As used herein, "promoter" encompasses a DNA sequence that directs the binding of RNA polymerase, thereby promoting RNA synthesis, i.e., a minimal sequence sufficient to direct transcription. Expression of a promoter and the corresponding protein or polypeptide can be ubiquitous, meaning broad cell, tissue, and species-specific, or cell-type, tissue-, or species-specific. A promoter can be "constitutive," meaning continuously active, or "inducible," meaning that the promoter can be activated or inactivated by the presence or absence of a biotic or abiotic factor. The disclosed nucleic acid constructs or vectors also include enhancer sequences, which may or may not be contiguous with the promoter sequence. Enhancer sequences affect promoter-dependent gene expression and can be located in the 5' or 3' regions of the native gene.

[0091] "Recombinant" as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in constructs that differ from polynucleotides found in nature.

[0092] As used herein, "SWAP™" (Surrozen WNT signal activating protein) refers to a WNT mimetic compound comprising an engineered bispecific full-length immunoglobulin G (IgG) antibody that, like WNT protein, directly activates the canonical WNT signaling pathway in target tissue, e.g., corneal tissue.

[0093] As used herein, "SWEETS™" (Surrozen WNT-signal enhancers engineered for tissue specificity) refers to antibody-based Rspondin mimetic compounds.

[0094] The terms "treatment," "treating," and the like are used herein generally to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that it completely or partially prevents a disease or its symptoms, e.g., reduces the likelihood that a disease or its symptoms will occur in a subject, and / or it may be therapeutic, in that it partially or completely cures a disease and / or adverse effects resulting from a disease.

[0095] As used herein, "treatment" encompasses any treatment of disease in a mammal, including (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with it; (b) inhibiting the disease, i.e., halting its development; or (c) alleviating the disease, i.e., causing regression of the disease. Therapeutic agents can be administered before, during, or after the onset of disease or injury. Treatment of ongoing disease is particularly interesting if the treatment stabilizes or alleviates undesirable clinical symptoms in the patient. Such treatment desirably occurs before complete loss of function in the affected tissue. The subject therapies are desirably administered during, and optionally after, the symptomatic phase of the disease.

[0096] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology, microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are described in the literature, e.g., "Molecular Cloning: A Laboratory Manual," 2nd edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M.J. Gait, ed., 1984); "Animal Cell Culture" (R.I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D.M. Weir & C.C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller & M.P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F.M. Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction" (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (J.E. Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0097] Some aspects of the present disclosure are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are described to provide a thorough understanding of the present disclosure. However, one skilled in the art will readily recognize that the present disclosure can be implemented without one or more of the specific details or using other methods. The present disclosure is not limited by the illustrated order of acts or events, as some acts may occur in different orders and / or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the present disclosure.

[0098] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the present disclosure. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Furthermore, to the extent the terms "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be as inclusive as the term "comprising."

[0099] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, in accordance with practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. When particular values are described in this application and claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable error range of the particular value.

[0100] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited, and it is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a conflict.

[0101] It is further noted that the claims may be drafted to exclude any element. Accordingly, this statement is intended to serve as a predicate for use of exclusive terminology, such as "solely," "only," and the like, in connection with the recitation of claim elements or the use of a "negative" limitation.

[0102] Unless otherwise indicated, all terms used herein have the same meaning as they would to one of ordinary skill in the art, and the practice of the present disclosure employs conventional techniques of microbiology and recombinant DNA techniques within the knowledge of those skilled in the art. II.General

[0103] The present disclosure provides methods for modulating WNT signaling to treat corneal disorders, including but not limited to scarring and corneal epithelial and endothelial dystrophies. In particular, the present disclosure provides WNT / β-catenin agonists and / or antagonists for stimulating corneal tissue regeneration and / or preventing corneal fibrosis.

[0104] WNT ("Wingless-related integration site" or "Wingless and Int-1" or "Wingless-Int") ligands and their signals play important roles in regulating the development, homeostasis, and regeneration of many essential organs and tissues, including bone, liver, skin, stomach, intestine, kidney, central nervous system, mammary gland, taste buds, ovary, cochlea, lung, and many other tissues (reviewed, e.g., by Clevers, Loh, and Nusse, 2014;346:1248012). Modulation of the WNT signaling pathway has potential for the treatment of degenerative diseases and tissue injury.

[0105] One of the challenges for modulating WNT signaling therapeutically is the existence of numerous WNT ligands and WNT receptors, Frizzled 1–10 (Fzd1–10), and many tissues express numerous overlapping Fzds. Canonical WNT signaling also involves low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) or low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) as co-receptors, which are widely expressed in various tissues in addition to Fzds.

[0106] R-spondins 1-4 are a family of ligands that amplify WNT signaling. Each R-spondin acts through a receptor complex containing zinc and ring finger 3 (ZNRF3) or ring finger protein 43 (RNF43) at one end and leucine-rich repeat-containing G protein-coupled receptor 4-6 (LGR4-6) at the other end (e.g., reviewed by Knight & Hankenson 2014, Matrix Biol.; 37:157-161). R-spondins can also act through additional mechanisms of action. ZNRF3 and RNF43 are two membrane-bound E3 ligases that specifically target WNT receptors (Fzd1-10 and LRP5 or LRP6) for degradation. Binding of R-spondin to ZNRF3 / RNF43 and LGR4-6 leads to clearance or sequestration of the ternary complex, which removes the E3 ligase from the WNT receptor, stabilizing the WNT receptor, and enhancing WNT signaling. Each R-spondin contains two Furin domains (1 and 2); Furin domain 1 binds to ZNRF3 / RNF43, and Furin domain 2 binds to LGR4-6. A fragment of R-spondin containing Furin domains 1 and 2 is sufficient to amplify WNT signaling. Although the effects of R-spondin depend on WNT signaling, they are not tissue-specific because both LGR4-6 and ZNRF3 / RNF43 are widely expressed in various tissues.

[0107] In some embodiments, the WNT / β-catenin signaling antagonist or agonist may contain a binding agent or epitope-binding domain that binds to one or more Fzd receptors and inhibits or enhances WNT signaling. In certain embodiments, the agent or antibody specifically binds to the cysteine-rich domain (CRD) within the human Frizzled receptor(s) to which it binds. In addition, antagonist binding agents containing an epitope-binding domain for LRP may also be used. In some embodiments, the WNT / β-catenin antagonist has a binding agent or epitope-binding domain that binds to the E3 ligase ZNRF3 / RNF43 and one or more FZD receptors or one or more LRP co-receptors to promote degradation of the FZD or LRP receptor, and the molecule may also contain a binding domain that binds to a cell-type-specific epitope for targeting. The E3 ligase agonist antibody or fragment thereof may be a single molecule or may be combined with other WNT antagonists, such as Fzd receptor antagonists, LRP receptor antagonists, and the like.

[0108] As is well known in the art, an antibody is an immunoglobulin molecule that can specifically bind to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., via at least one epitope-binding domain located on the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof comprising the epitope-binding domain (e.g., dAb, Fab, Fab', (F(ab')2, Fv, single chain (scFv), VHH (i.e., Nanobody®) or single domain antibody (sdAb), DVD-Ig, synthetic variants thereof, naturally occurring variants, fusion proteins comprising the epitope-binding domain and humanized antibodies, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule comprising an antigen-binding site or fragment (epitope recognition site) of the required specificity, gene fusions (WO 94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 "Diabodies," which are multivalent or multispecific fragments constructed by the method of S. Hu et al., Cancer Res., 56, 3055-3061, 1993, are also specific forms of antibodies contemplated herein. Minibodies comprising scFvs linked to CH3 domains are also included herein (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). See, e.g., Ward, E.S. et al., Nature, 341, 544-546 (1989); Bird et al., Science, 242, 423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA, 85, 5879-5883, 1988); PCT / US92 / 09965; WO 94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA, 90 6444-6448, 1993; Y. Reiter et al., Nature Biotech, 14, 1239-1245, 1996; S. Hu et al., Cancer Res., 56, 3055-3061, 1996; C. Bever et al., Anal Bioanal Chem. 2016 Sept; 408(22); 5985-6002.

[0109] The proteolytic enzyme papain preferentially cleaves IgG molecules to generate several fragments, two of which (F(ab) fragments) each contain a covalently linked heterodimer with an intact antigen-binding site. The enzyme pepsin can cleave IgG molecules to provide several fragments, including the F(ab')2 fragment, which contains both antigen-binding sites. Fv fragments for use according to certain embodiments of the present disclosure can be generated by preferential proteolytic cleavage of IgM, and rarely IgG or IgA, immunoglobulin molecules. However, Fv fragments are more commonly derived using recombinant techniques known in the art. Fv fragments contain a noncovalently linked VH:VL heterodimer containing an antigen-binding site that retains much of the antigen recognition and binding ability of a native antibody molecule. Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

[0110] In certain embodiments, single-chain Fv (scFv) antibodies are contemplated. For example, kappa bodies (Ill et al., Prot. Eng. 10:949-57 (1997)); minibodies (Martin et al., EMBO J 13:5305-9 (1994)); diabodies (Holliger et al., Proc. Nat. Acad. Sci. 90:6444-8 (1993)); or Janusins (Traunecker et al., EMBO J 10:3655-59 (1991) and Traunecker et al., Int. J. Cancer Suppl. 7:51-52 (1992)) can be prepared using standard molecular biology techniques following the teachings of the present application regarding the selection of antibodies with desired specificity. In yet other embodiments, bispecific or chimeric antibodies can be made that incorporate the ligands of the present disclosure. For example, a chimeric antibody may comprise CDRs and framework regions from different antibodies, but bispecific antibodies may be generated that specifically bind to one or more Fzd receptors through one binding domain and to a second molecule through a second binding domain. These antibodies may be produced by recombinant molecular biology techniques or may be physically conjugated to one another.

[0111] Single-chain Fv (scFv) polypeptides are covalently linked VH:VL heterodimers expressed from gene fusions containing VH-encoding and VL-encoding genes linked by a peptide-encoding linker. Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. Several methods have been described for identifying chemical structures for converting naturally aggregated (chemically separated) light and heavy polypeptide chains from antibody V regions into scFv molecules that fold into a three-dimensional structure substantially similar to the structure of an antigen-binding site. See, for example, U.S. Patent Nos. 5,091,513 and 5,132,405 by Huston et al. and U.S. Patent No. 4,946,778 by Ladner et al.

[0112] In certain embodiments, the antibodies described herein are in the form of diabodies. Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising an immunoglobulin light chain binding region and a second domain comprising an immunoglobulin heavy chain binding region, the two domains being linked (e.g., by a peptide linker) but unable to associate with each other to form an antigen-binding site, the antigen-binding site being formed by association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO 94 / 13804).

[0113] A dAb fragment of an antibody consists of the VH domain (Ward, ES et al., Nature 341, 544-546 (1989)).

[0114] When bispecific antibodies are used, they can be conventional bispecific antibodies, which can be produced in a variety of ways (Holliger, P. & Winter G., Curr. Opin. Biotech. 4, 446-449 (1993)), for example, chemically or from hybrid hybridomas, or any of the bispecific antibody fragments described above. Diabodies and scFvs can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction.

[0115] In contrast to bispecific whole antibodies, bispecific diabodies can be particularly useful because they can be easily constructed and expressed in Escherichia coli (E. coli). Diabodies (and many other polypeptides, such as antibody fragments) with appropriate binding specificities can be easily selected from libraries using phage display (WO 94 / 13804). If one arm of the diabody is held constant, e.g., specificity for antigen X, the other arm can be varied to create a library from which antibodies of appropriate specificity can be selected. Bispecific whole antibodies can be generated by knobs-into-hole engineering (J.B.B. Ridgeway et al., Protein Eng., 9, 616-621 (1996)).

[0116] In certain embodiments, the antibodies described herein may be provided in the form of a UniBody®. A UniBody® is an IgG4 antibody with the hinge region removed (see GenMab Utrecht, The Netherlands; see also, e.g., U.S. Patent Application Publication No. 2009 / 0226421). This proprietary antibody technology creates a stable, small antibody format with an expected longer therapeutic window than current small antibody formats. IgG4 antibodies are thought to be inert and therefore do not interact with the immune system. Fully human IgG4 antibodies can be modified by eliminating the hinge region of the antibody to obtain half-molecule fragments with different stability compared to the corresponding intact IgG4 (GenMab, Utrecht). Halving the IgG4 molecule leaves only one region on the UniBody® that can bind to the cognate antigen (e.g., disease target); therefore, the UniBody® binds monovalently to only one site on the target cell.

[0117] In certain embodiments, the antibodies and antigen-binding fragments thereof described herein comprise a set of heavy and light chain CDRs sandwiched between a set of heavy and light chain framework regions (FRs), respectively, which support the CDRs and define the spatial relationship of the CDRs to each other. As used herein, the term "CDR set" refers to the three hypervariable regions of the heavy or light chain V region. Proceeding from the N-terminus of the heavy or light chain, these regions are designated "CDR1," "CDR2," and "CDR3," respectively. Thus, an antigen-binding site comprises six CDRs, comprising the CDR sets derived from each of the heavy and light chain V regions. A polypeptide comprising a single CDR (e.g., CDR1, CDR2, or CDR3) is referred to herein as a "molecular recognition unit." Crystallographic analysis of several antigen-antibody complexes has demonstrated that amino acid residues of CDRs form extensive contacts with the bound antigen, with the most extensive antigen contact being with the heavy chain CDR3. Thus, the molecular recognition unit is primarily responsible for the specificity of the antigen-binding site.

[0118] As used herein, the term "FR set" refers to the four adjacent amino acid sequences that constitute the CDRs of a CDR set of a heavy or light chain V region. While some FR residues may contact a bound antigen, FRs primarily serve to fold the V region into the antigen-binding site, particularly the FR residues directly adjacent to the CDRs. Within FRs, certain amino acid residues and certain structural features are highly conserved. In this regard, all V region sequences contain an internal disulfide loop of approximately 90 amino acid residues. When the V region folds into the binding site, the CDRs are displayed as protruding loop motifs that form the antigen-binding surface. Regardless of the exact CDR amino acid sequence, it is generally recognized that there are conserved structural regions of FRs that influence the folding of the CDR loops into specific "canonical" structures. Furthermore, certain FR residues are known to participate in noncovalent interdomain contacts that stabilize the interaction between the heavy and light chains of an antibody.

[0119] "Monoclonal antibody" refers to a homogeneous antibody population; monoclonal antibodies are composed of amino acids (naturally occurring and non-naturally occurring) involved in selective binding of an epitope. Monoclonal antibodies are highly specific and directed against a single epitope. The term "monoclonal antibody" encompasses not only intact and full-length monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv), nanobodies®, variants thereof, fusion proteins comprising an antigen-binding fragment of a monoclonal antibody, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of an immunoglobulin molecule containing an antigen-binding fragment (epitope recognition site) with the requisite specificity and ability to bind to the epitope, including the WNT surrogate molecules disclosed herein. It is not intended to be limited as to the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins as well as fragments such as those described above under the definition of "antibody."

[0120] In certain embodiments, the antibodies of the present disclosure may take the form of Nanobodies®. Nanobody® technology was initially developed after the discovery and identification of camelids (e.g., camels, alpacas, and llamas) as having fully functional antibodies composed only of heavy chains and thus lacking light chains. These heavy-chain-only antibodies contain a single variable domain (VHH) and two constant domains (CH2, CH3). Cloned and isolated single variable domains have full antigen-binding capacity and are highly stable. These single variable domains, together with their unique structural and functional properties, form the basis of "Nanobodies®." Nanobodies® are encoded by a single gene and are efficiently produced in almost all prokaryotic and eukaryotic hosts, such as E. coli (e.g., No. 6,765,087), molds (e.g., Aspergillus or Trichoderma), and yeasts (e.g., Saccharomyces, Kluyvermyces, Hansenula, or Pichia (e.g., No. 6,838,254)). The manufacturing process is scalable, allowing production of nanobodies in multi-kilogram quantities. Nanobodies® are manufactured in various antibody-specific antibody domains. Nanobodies® can be formulated as ready-to-use solutions with a long shelf life. The Nanoclone® method (see, e.g., WO 06 / 079372) is a proprietary method for generating Nanobodies® against desired targets based on automated, high-throughput selection of B cells. Nanobodies® are single-domain antigen-binding fragments of camelid-specific heavy chain-only antibodies. Nanobodies®, also called VHH antibodies, typically have a small size of approximately 15 kDa. See Bever et al., Anal Bioanal Chem. 2016 Sept;408(22);5985-6002.

[0121] Another contemplated antibody fragment is the dual variable domain immunoglobulin (DVD-Ig), an engineered protein that combines the functions and specificities of two monoclonal antibodies in a single molecular entity. DVD-Igs are designed as IgG-like molecules, but each light and heavy chain contains two variable domains in tandem via a short peptide bond, instead of the single variable domain found in IgG. The fusion orientation of the two variable domains and the selection of the linker sequence are important for the functional activity and efficient expression of the molecule. DVD-Igs can be produced as a single species for production and purification using conventional mammalian expression systems. DVD-Igs retain the specificity of the parent antibody, are stable in vivo, and exhibit IgG-like physicochemical and pharmacokinetic properties. DVD-Igs and methods for their production are described in Wu, C. et al., Nat Biotech, 25:1290-1297 (2007).

[0122] In certain embodiments, the antibodies or antigen-binding fragments disclosed herein are humanized. This refers to chimeric molecules, generally prepared using recombinant technology, that have an antigen-binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise a complete variable domain fused to a constant domain, or only CDRs grafted into appropriate framework regions in the variable domain. The epitope-binding site may be wild-type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, although the possibility of an immune response to the foreign variable region remains (LoBuglio, AF et al., (1989) Proc Natl Acad Sci USA 86:4220-4224; Queen et al., PNAS (1988) 86:10029-10033; Riechmann et al., Nature (1988) 332:323-327). Exemplary methods for humanizing the anti-Fzd or LRP antibodies disclosed herein include those described in US Pat. No. 7,462,697.

[0123] Another approach focuses not only on providing constant regions of human origin, but also on modifying the variable regions to reshape them as closely as possible to human forms. Both heavy and light chain variable regions contain three complementarity-determining regions (CDRs), which vary in response to the epitope in question and determine binding ability, and are flanked by four framework regions (FRs), which are relatively conserved in a given species and are presumed to provide a scaffold for the CDRs. When a non-human antibody is prepared for a specific epitope, the variable region can be "reshaped" or "humanized" by grafting CDRs from the non-human antibody onto the FRs present in the modified human antibody. Application of this approach to various antibodies has been reported in Sato, K., et al., (1993) Cancer Res 53:851-856; Riechmann, L., et al., (1988) Nature 332:323-327; Verhoeyen, M., et al., (1988) Science 239:1534-1536; Kettleborough, CA, et al., (1991) Protein Engineering 4:773-3783; Maeda, H., et al., (1991) Human Antibodies Hybridoma 2:124-134; Gorman, SD, et al., (1991) Proc Natl Acad Sci USA 88:4181-4185; Tempest, PR, et al., (1991) Bio / Technology 9:266-271; Co, and Co, MS et al., (1992) J Immunol 148:1149-1154. In some embodiments, the humanized antibody preserves all CDR sequences (e.g., a humanized mouse antibody that contains all six CDRs from the mouse antibodies).In other embodiments, a humanized antibody has one or more CDRs (1, 2, 3, 4, 5, 6) that are altered relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

[0124] In certain embodiments, an antibody of the present disclosure may be a chimeric antibody. In this regard, a chimeric antibody is composed of an antigen-binding fragment of an antibody operatively linked or fused to a heterologous Fc portion of a different antibody. In certain embodiments, the heterologous Fc domain is of human origin. In other embodiments, the heterologous Fc domain may be derived from an Ig class different from that of the parent antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In further embodiments, the heterologous Fc domain may be composed of CH2 and CH3 domains from one or more different Ig classes. As described above with respect to humanized antibodies, the antigen-binding fragment of a chimeric antibody may include only one or more CDRs of an antibody described herein (e.g., one, two, three, four, five, or six CDRs of an antibody described herein) or may include the entire variable domain (VL, VH, or both).

[0125] The structures and locations of immunoglobulin CDRs and variable domains can be determined by reference to Kabat, EA et al., Sequences of Proteins of Immunological Interest, 4th ed., US Department of Health and Human Services. 1987 and updates thereto (now available on the internet) (immuno.bme.nwu.edu).

[0126] In certain embodiments, the antagonist or agonist binding agent has a dissociation constant (K) of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, or about 10 nM or less. DFor example, in certain embodiments, FZD-binding agents or antibodies described herein that bind to more than one FZD have a K of about 100 nM or less, about 20 nM or less, or about 10 nM or less. D In certain embodiments, a binding agent binds to one or more of its target antigens with an EC50 of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, or about 1 nM or less.

[0127] The antibody or other agent of the present disclosure can be assayed for specific binding by any method known in the art.The immunoassays that can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as biolayer interferometry (BLI) analysis, FACS analysis, immunofluorescence, immunocytochemistry, Western blot, radioimmunoassay, ELISA, "sandwich" immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescence immunoassay, and protein A immunoassay.Such assays are routine and well known in the art (see, for example, Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated herein by reference in its entirety).

[0128] For example, the specific binding of an antibody to a target antigen can be determined using ELISA. An ELISA assay involves preparing an antigen, coating the wells of a 96-well microtiter plate with the antigen, adding an antibody or other binding agent conjugated to a detectable compound, such as an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase), to the wells, incubating for a period of time, and detecting the presence of the antigen. In some embodiments, the antibody or agent is not conjugated to a detectable compound; instead, a second conjugated antibody that recognizes the first antibody or agent is added to the well. In some embodiments, instead of coating the wells with the antigen, the antibody or agent can be coated to the well, and the second antibody conjugated to a detectable compound can be added after the antigen is added to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1).

[0129] The binding affinity of an antibody or other agent to a target antigen and the dissociation rate of the antibody-antigen interaction can be determined by a competitive binding assay. One example of a competitive binding assay is a radioimmunoassay, which involves incubating a labeled antigen (e.g., Fzd, LRP) or a fragment or variant thereof with the antibody of interest in the presence of increasing amounts of unlabeled antigen, followed by detecting the antibody bound to the labeled antigen. The affinity and dissociation rate of the antibody can be determined from the data by scatter plot analysis. In some embodiments, BLI analysis is used to determine the binding on-rate and binding off-rate of an antibody or agent. BLI kinetic analysis involves analyzing the binding and dissociation of an antibody from a chip with an antigen immobilized on its surface.

[0130] In certain embodiments, the WNT agonist is selected from those disclosed in PCT Publication No. WO2019 / 126398, the entirety of which is incorporated herein. G211 (also known as YW 211.31.57) is disclosed in U.S. Patent No. 8,846,041.

[0131] In some embodiments, the WNT agonist comprises a sequence having at least 90% identity (e.g., 95%, 98%, 99%, or 100% identity) to a sequence disclosed in any of the sequences shown in Table 1. In some embodiments, the WNT agonist comprises two sequences, each having at least 90% identity (e.g., 95%, 98%, 99%, or 100% identity) to a sequence disclosed in any of the sequences shown in Table 1. In some embodiments, the WNT agonist comprises four sequences, each having at least 90% identity (e.g., 95%, 98%, 99%, or 100% identity) to a sequence disclosed in any of the sequences shown in Table 1, and in certain embodiments, two of the sequences are light chain sequences and two of the sequences are heavy chain sequences. In some embodiments, the WNT agonist comprises two heavy chain sequences. In certain embodiments, the WNT agonist comprises a light chain sequence and / or a heavy chain sequence, each independently comprising at least one, at least two, or all three CDR sequences disclosed in any of SEQ ID NOS: 1-16, or variants thereof, wherein the sequences comprise zero, fewer than two, fewer than three, fewer than four, fewer than five, fewer than six, fewer than seven, or fewer than eight amino acid modifications, e.g., substitutions, within the CDRs. G211-18R5, R2M3-26, 1RCO7-26, 1SH1-03, and hp1SH1-03 comprise two of the indicated heavy chains (HC) and two of the indicated light chains (LC), as disclosed in Table 1. In certain embodiments, the WNT agonist comprises two light chain sequences and two heavy chain sequences, each independently comprising at least one, at least two, or all three CDR sequences disclosed in any of SEQ ID NOS: 1-16, or variants thereof. In certain embodiments, the Wnt agonist has an Ig format, and in certain embodiments, the Wnt agonist comprises two heavy chains bound to each other and two light chains, each bound to a different one of the two heavy chains. Wnt superagonists or super-SWAP G211-18R5-Rspo2RA and G211-R2H1-Rspo2RA are also listed in Table 1, and each comprises two of the indicated heavy chains (HC) and two of the indicated light chains (LC) disclosed in Table 1.17SB9-03 contains two of the indicated heavy chains (HC) disclosed in Table 1, namely one HC of SEQ ID NO: 15 and one heavy chain of SEQ ID NO: 16. In various embodiments, the molecule does not contain a tag sequence.

[0132] Table 1: Sequences of SWAP™ and Wnt superagonists or Super-SWAP molecules; variable chains are provided in plain text in the order indicated by the format. Where present, VHHs are in bold (not italicized), and constant chains are in italics. VLs are italicized and underlined, and VHs are italicized and bolded. The constant region of the lambda light chain is highlighted in light gray. The constant region of the kappa light chain is underlined, bolded, and highlighted in light gray. CH1 is in italics. Fc is italicized, bolded, and highlighted in light gray. The Rspo region is highlighted and in bold (not italicized or underlined). The linker is in smaller font. Bold, underlined, and italicized AA and G represent substitutions of AA and G for the wild-type LL and P amino acids at these positions, respectively. The tag sequence is underlined with a dotted line. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0133] In particular embodiments, the engineered WNT agonist comprises one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 1, and one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 2. In particular embodiments, the engineered WNT agonist comprises one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 3, and one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 4. In particular embodiments, the engineered WNT agonist comprises one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 5, and one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 6. In particular embodiments, the engineered WNT agonist comprises two sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 1, and two sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 2. In particular embodiments, the engineered WNT agonist comprises two sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:3 and two sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:4.In particular embodiments, the engineered WNT agonist comprises two sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 5 and two sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 6. In particular embodiments, each sequence comprises fewer than 1 (i.e., 0), fewer than 2, fewer than 3, fewer than 4, fewer than 5, fewer than 6, fewer than 7, fewer than 8, fewer than 9, fewer than 10, fewer than 11, or fewer than 12 modifications, e.g., substitutions, of amino acids present within the CDRs of the WNT agonist. In particular embodiments, the engineered WNT agonist comprises fewer than 1 (i.e., 0), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid modifications present within the CDRs of the WNT agonist. In certain embodiments, the engineered Wnt agonist comprises an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises at least two, at least three, at least four, at least five, or at least six CDRs of any of the sequences of SEQ ID NOs: 1-16.

[0134] In some embodiments, the engineered WNT agonist comprises two sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 7 and two sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 8. In particular embodiments, each sequence comprises fewer than 1 (i.e., 0), fewer than 2, fewer than 3, fewer than 4, fewer than 5, fewer than 6, fewer than 7, fewer than 8, fewer than 9, fewer than 10, fewer than 11, or fewer than 12 modifications, e.g., substitutions, of amino acids present within the CDRs of the WNT agonist. In particular embodiments, the engineered WNT agonist comprises fewer than 1 (i.e., 0), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid modifications present within the CDRs of the WNT agonist, e.g., fewer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions within the CDRs of the WNT agonist. In certain embodiments, the engineered Wnt agonist comprises an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises at least two, at least three, at least four, at least five, or at least six CDRs of any of the sequences of SEQ ID NOs: 1-16.

[0135] In particular embodiments, the engineered WNT agonist comprises one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 9, and one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 10. In particular embodiments, the engineered WNT agonist comprises one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 11, and one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 12. In particular embodiments, the engineered WNT agonist comprises one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 13, and one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 14. In particular embodiments, the engineered WNT agonist comprises one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 15, and one or more, e.g., two, sequences having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO: 16. In particular embodiments, each sequence contains fewer than 1 (i.e., 0), fewer than 2, fewer than 3, fewer than 4, fewer than 5, fewer than 6, fewer than 7, fewer than 8, fewer than 9, fewer than 10, fewer than 11, or fewer than 12 modifications of amino acids present within the CDRs of the WNT agonist.In particular embodiments, the engineered WNT agonist comprises fewer than 1 (i.e., 0), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid modifications present within the CDRs of the WNT agonist, e.g., fewer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions within the CDRs of the WNT agonist. In certain embodiments, the engineered Wnt agonist comprises an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises at least two, at least three, at least four, at least five, or at least six CDRs of any of the sequences of SEQ ID NOs: 1-16. III. Pharmaceutical Compositions

[0136] Also disclosed are pharmaceutical compositions comprising a WNT antagonist or WNT agonist molecule (WNT antagonist / agonist molecule) described herein and one or more pharmaceutically acceptable diluents, carriers, or excipients. The term "WNT antagonist / agonist molecule" refers generically to both WNT agonist molecules and WNT agonist molecules.

[0137] In further embodiments, pharmaceutical compositions are also disclosed that include a polynucleotide comprising a nucleic acid sequence encoding a WNT antagonist / agonist molecule described herein and one or more pharmaceutically acceptable diluents, carriers, or excipients. In certain embodiments, the polynucleotide is DNA or mRNA, e.g., a modified mRNA. In particular embodiments, the polynucleotide is a modified mRNA that further comprises a 5' cap sequence and / or a 3' tail sequence, e.g., a polyA tail. In other embodiments, the polynucleotide is an expression cassette that includes a promoter operably linked to the coding sequence.

[0138] In some embodiments, the WNT antagonist / agonist is an engineered recombinant polypeptide incorporating various epitope-binding fragments that bind to various molecules in the WNT signaling pathway. For example, the WNT antagonist can be an antibody or fragment thereof that binds to the FZD receptor and / or the LRP receptor and inhibits WNT signaling. FZD and LRP antibody fragments (e.g., Fab, scFv, VHH / sdAb, etc.) can be linked together on a single molecule, either directly or via linkers of various sizes.

[0139] Conversely, engineered WNT agonists / antagonists can also be recombinant polypeptides incorporating epitope-binding fragments that bind to various molecules in the WNT signaling pathway and enhance WNT signaling. For example, a WNT agonist can be an antibody or fragment thereof that binds to an Fzd receptor and / or an LRP receptor and enhances WNT signaling. Fzd and LRP antibody fragments (e.g., Fab, scFv, VHH / sdAb, etc.) can be linked together on a single molecule, either directly or via linkers of various sizes.

[0140] In further embodiments, pharmaceutical compositions are also disclosed that include an expression vector, e.g., a viral vector, comprising a polynucleotide that includes a nucleic acid sequence encoding a WNT antagonist / agonist molecule described herein, and one or more pharmaceutically acceptable diluents, carriers, or excipients. In certain embodiments, the nucleic acid sequence encoding the WNT antagonist molecule and the nucleic acid sequence encoding the WNT agonist are in the same polynucleotide, e.g., an expression cassette.

[0141] The present disclosure further contemplates pharmaceutical compositions comprising cells comprising an expression vector comprising a polynucleotide comprising a promoter operably linked to a nucleic acid encoding a WNT antagonist / agonist molecule, and one or more pharmaceutically acceptable diluents, carriers, or excipients. In certain embodiments, the pharmaceutical composition further comprises cells comprising an expression vector comprising a polynucleotide comprising a promoter operably linked to a nucleic acid sequence encoding a WNT antagonist and a WNT agonist. In certain embodiments, the nucleic acid sequence encoding the WNT antagonist molecule and the nucleic acid sequence encoding the WNT agonist molecule are present in the same polynucleotide, e.g., an expression cassette, and / or the same cell. In certain embodiments, the cells are heterologous cells or autologous cells obtained from the subject to be treated.

[0142] In certain embodiments, the cells are stem cells, such as adipose-derived stem cells or hematopoietic stem cells. The present disclosure contemplates a pharmaceutical composition comprising a first molecule for delivery of a WNT antagonist molecule as a first active agent and a WNT agonist as a second molecule. The first and second molecules may be the same type of molecule or different types of molecules. For example, in certain embodiments, the first and second molecules may each independently be selected from the following types of molecules: polypeptides, small organic molecules, nucleic acids (optionally DNA or mRNA, optionally modified RNA) encoding the first or second active agent, vectors (optionally expression vectors or viral vectors) containing nucleic acid sequences encoding the first or second active agent, and cells containing nucleic acid sequences (optionally expression cassettes) encoding the first or second active agent.

[0143] The subject molecules, alone or in combination, can be combined with pharmaceutically acceptable carriers, diluents, excipients, and generally safe, non-toxic, and useful reagents for preparing desired formulations, including excipients that are acceptable for use in mammals, such as humans or primates. Such excipients can be solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous. Examples of such carriers, diluents, and excipients include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Supplementary active compounds can also be incorporated into the formulation. The solutions or suspensions used in the formulations may contain sterile diluents such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates; detergents such as Tween® 20 to prevent aggregation; penetration enhancers such as sodium caprate, viscosity enhancers such as hydroxypropylmethylcellulose, particularly for eye drop formulations; and compounds for adjusting isotonicity, such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. In certain embodiments, the pharmaceutical compositions are sterile.

[0144] Pharmaceutical compositions may further include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate-buffered saline (PBS). In some cases, the composition must be sterile and fluid so that it can be drawn into a syringe or delivered to a subject from a syringe. In certain embodiments, it is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the internal composition can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0145] Sterile solution can be prepared by incorporating the WNT antagonist / agonist antibody or its antigen-binding fragment (or a polynucleotide or cell encoding it) in the required amount in a suitable solvent containing one or a combination of the above-listed ingredients as needed, followed by filtration sterilization.Generally, dispersion is prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other necessary ingredients listed above.For the preparation of sterile powder for sterile injection, the preparation method is vacuum drying and freeze-drying, which allows the powder of the active ingredient and any additional desired ingredients to be obtained from its previously sterile-filtered solution.

[0146] In one embodiment, the pharmaceutical composition is prepared using a carrier that protects the antibody or its antigen-binding fragment from rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be clear to those skilled in the art. These materials are also commercially available. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

[0147] For ease of administration and uniformity of dosage, it may be advantageous to formulate pharmaceutical compositions in dosage unit form.Dosage unit form as used herein refers to a physically separate unit suitable as a unit dosage for the subject to be treated, each unit containing a predetermined amount of active antibody or its antigen-binding fragment calculated to be associated with the required pharmaceutical carrier to produce the desired therapeutic effect.The specifications of the dosage unit are determined by and directly depend on the inherent characteristics of the antibody or its antigen-binding fragment and the specific therapeutic effect to be achieved, as well as the limitations inherent in the technical field of compounding such active antibody or its antigen-binding fragment for the treatment of individuals.

[0148] The pharmaceutical compositions can be included in a container, pack, or dispenser, such as a syringe or eye dropper, eg, a prefilled syringe or eye dropper, together with instructions for administration.

[0149] The pharmaceutical compositions of the present disclosure may be delivered to a subject in the form of a pill, capsule, cream, salve, syrup, skin patch, suppository, intravenous infusion, aqueous solution, non-aqueous solution, eyewash, or any combination thereof.

[0150] The pharmaceutical compositions of the present disclosure may be delivered to a subject by direct ophthalmic application, intramuscular injection, intravenous injection, peritoneal injection, nasal, oral, rectal, or any combination thereof.

[0151] The pharmaceutical compositions of the present disclosure include any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compounds that, upon administration to an animal, including a human, can provide (directly or indirectly) a biologically active antibody or antigen-binding fragment thereof.

[0152] The present disclosure includes pharmaceutically acceptable salts of the WNT antagonist / agonist molecules described herein. The term "pharmaceutically acceptable salt" refers to a physiologically and pharmaceutically acceptable salt of a compound of the present disclosure, i.e., a salt that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects. Various pharmaceutically acceptable salts are known in the art and are described, for example, in "Remington's Pharmaceutical Sciences," 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and more recent editions), in the "Encyclopedia of Pharmaceutical Technology," 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and in J.Pharm.Sci. 66:2 (1977). Also, for a review of suitable salts, see "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, 2002). Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines.

[0153] Metals used as cations include sodium, potassium, magnesium, calcium, and the like. Amines include N-N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, e.g., Berge et al., "Pharmaceutical Salts," J. Pharma Sci., 1977, 66, 119). The base-addition salts of such acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form can be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ somewhat from their respective salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acids for purposes of this disclosure.

[0154] In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of a WNT antagonist / agonist molecule or a pharmaceutically acceptable salt thereof in admixture with a pharmaceutically acceptable carrier, diluent, and / or excipient, such as saline, phosphate-buffered saline, phosphate, and amino acids, polymers, polyols, sugars, buffers, preservatives, and other proteins. Exemplary amino acids, polymers, and sugars include octylphenoxypolyethoxyethanol compounds, monostearate polyethylene glycol compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, Ringer's solution and Hank's solution, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and glycol. Preferably, the formulation is stable at 4°C for at least 6 months.

[0155] In some embodiments, the pharmaceutical compositions provided herein comprise a buffer such as phosphate-buffered saline (PBS) or sodium phosphate / sodium sulfate, Tris buffer, glycine buffer, sterile water, and other buffers known to those skilled in the art, such as those described in Good et al. (1966) Biochemistry 5:467. The pH of the buffer can range from 6.5 to 7.75, preferably 7 to 7.5, and most preferably 7.2 to 7.4. IV.How to use

[0156] The present disclosure also provides the method of using WNT antagonist / agonist molecule to regulate, for example, WNT signaling pathway, for example, increase or decrease WNT signaling, and the administration of WNT antagonist / agonist molecule in various therapeutic situations.Provided herein is the method of treatment using WNT antagonist / agonist molecule.In one embodiment, WNT antagonist / agonist molecule is provided to the subject with disease that is associated with inappropriate or deregulated WNT signaling.

[0157] In certain embodiments, WNT antagonist / agonist molecules can be used to block or enhance the WNT signaling pathway in tissues or cells. Antagonizing the WNT signaling pathway can include reducing or inhibiting WNT signaling in cells or tissues. Agonizing the WNT signaling pathway can include, for example, increasing WNT signaling or enhancing WNT signaling in tissues or cells. Thus, in some aspects, the present disclosure provides a method for antagonizing / agonizing the WNT signaling pathway in cells, comprising contacting a tissue or cell with an effective amount of a WNT antagonist / agonist molecule disclosed herein or a pharmaceutically acceptable salt thereof, wherein the WNT antagonist / agonist molecule is a WNT signaling pathway antagonist / agonist. In some embodiments, the contacting is performed in vitro, ex vivo, or in vivo. In certain embodiments, the cell is a cultured cell, and the contacting is performed in vitro.

[0158] WNT antagonist / agonist molecules can be used to treat keratopathy. In particular, experiments have shown that activation of WNT signaling regulates corneal cell growth and stratification (Zhang et al., Development, 2015, 142:3383-93). Conditional knockout of either β-catenin or Lrp5 / 6 results in malstratification of the epithelium, among other things. Recent in vitro experiments have shown that WNT agonists promote cell growth in homogenized keratocyte cultures (see, for example, Figures 2A-E and 7A-B). Therefore, controlled administration of WNT agonists or / and antagonists has been proposed to promote epithelial and endothelial cell growth and reverse various keratopathy pathologies, including dystrophy and scarring. In certain embodiments, WNT agonists / antagonists are administered either early or late in the progression of keratopathy in a subject.

[0159] Both the WNT agonist and antagonist can be administered individually or sequentially as monotherapy. In certain embodiments, the method comprises administering a WNT agonist at an early stage of disease development, followed by administering a WNT antagonist at a later stage of disease when significant scarring or fibrosis is present.

[0160] The present disclosure also provides a combination treatment with known treatment for keratopathy.Examples of therapies that can be administered together with WNT agonists or antagonists include but are not limited to Rho kinase inhibitors (e.g., ripasudil or fasudil), anti-inflammatory drugs (e.g., steroidal and non-steroidal) and immunosuppressants.

[0161] Keratopathy that may be treated includes, but is not necessarily limited to, bacterial infections, viral infections (e.g., herpes simplex keratitis and ophthalmic varicella (i.e., shingles)), fungal infections, protozoal infections, archaeal infections, genetic disorders, brittle cornea syndrome (BCS), corneal endothelial-mesenchymal transition (EnMT), corneal epithelial-mesenchymal transition (EMT), corneal fibrosis, Cogan's syndrome, corneal ulcers, epithelial basement membrane dystrophy (EBMD, ABMD, Map), and others. Dot), macular corneal dystrophy, Fuchs' dystrophy (also called Fuchs' corneal endothelial dystrophy (FCED) or Fuchs' endothelial dystrophy (FED) or Fuchs' endothelial cell dystrophy (FECD)), colloidal droplet corneal dystrophy, granular corneal dystrophy type I, granular corneal dystrophy type II, interstitial keratitis, iridocorneal endothelial syndrome (ICE), keratoconjunctivitis sicca, keratoconus, keratomalacia, lattice dystrophy type I, lattice Corneal dystrophy type II, Risch corneal dystrophy, macular corneal dystrophy, Miesmann corneal dystrophy, marginal ulcerative keratitis, phlyctenular keratoconjunctivitis, posterior polymorphous corneal dystrophy, pterygium, Reiss-Bückler corneal dystrophy, Schneider crystalline corneal dystrophy, Stevens-Johnson syndrome, superficial punctate keratitis, Thiel-Behnke corneal dystrophy, neurotrophic keratitis, corneal herpes, and scarring.

[0162] In further embodiments, the WNT antagonist and / or WNT agonist molecules may also incorporate a tissue targeting moiety, such as an antibody or fragment thereof that recognizes a corneal tissue or cell type specific receptor or cell surface molecule.

[0163] Dosage and administration regimens can depend on a variety of factors, such as the nature of the disease or disorder, the characteristics of the subject, and the subject's medical history, which can be readily determined by a physician. In certain embodiments, the amount of Wnt signaling molecule administered or provided to a subject ranges from about 0.01 mg / kg to about 50 mg / kg, 0.1 mg / kg to about 500 mg / kg, or about 0.1 mg / kg to about 50 mg / kg of the subject's body weight. In certain embodiments, the amount of engineered WNT signaling modulator administered or provided to a subject ranges from about 0.01 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 500 mg / kg, or about 0.1 mg / kg to about 50 mg / kg of the subject's body weight. In certain embodiments of any of the methods disclosed herein, the WNT signaling modulator is administered to a subject, e.g., a mammal, intravenously, e.g., as a bolus injection, or subcutaneously. In certain embodiments, the WNT signaling modulator is administered at least once a week. In certain embodiments, a subject is administered about 0.5 to about 100 mg / kg body weight of a WNT signaling modulator or about 2 to about 50 mg / kg body weight of a WNT agonist, e.g., about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, or about 50 mg / kg. In certain embodiments, a subject is administered about 25 mg, about 75 mg, about 250 mg, about 750 mg, about 1500 mg, or about 2250 mg of a WNT signaling modulator. In certain embodiments, a subject is administered about 3 to about 30 mg / kg body weight intravenously or subcutaneously at least once a week.

[0164] Therapeutic agents (e.g., WNT antagonists / agonists) can be administered before, during, or after the onset of disease or injury. Treatment of ongoing disease is particularly interesting if the treatment stabilizes or alleviates undesirable clinical symptoms in the patient. Such treatment desirably occurs before complete loss of function in the affected tissue. The subject therapies desirably are administered during, or in some cases after, the symptomatic phase of the disease. In some embodiments, the subject methods provide a therapeutic benefit, such as preventing the onset of a disorder, halting the progression of a disorder, reversing the progression of a disorder, etc. In some embodiments, the subject methods include detecting that a therapeutic benefit has been achieved. Those skilled in the art will understand that such measures of therapeutic efficacy are applicable to the particular disease in which they are altered, and will recognize appropriate detection methods to use to measure therapeutic efficacy.

[0165] All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to herein and / or listed in the Application Data Sheet are hereby incorporated by reference in their entirety.

[0166] From the foregoing, it will be understood that, although specific embodiments of the present disclosure have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, the disclosure is not limited except as by the appended claims.

[0167] The broad scope of this disclosure is best understood with reference to the following examples, which are not intended to limit the disclosure to the specific embodiments. [Example]

[0168] Example Example I. General Methods Standard methods in molecular biology include those described by Maniatis et al. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3 rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif. Standard methods are also set forth in Ausbel et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugate and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0169] Methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, are described in Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York. Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and protein glycosylation are also described. See, for example, Coligan et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, NJ, pp. 384-391. The production, purification, and fragmentation of polyclonal and monoclonal antibodies are described. Coligan et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra. Standard techniques are available for characterizing ligand / receptor interactions.See, for example, Coligan et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York.

[0170] Methods for flow cytometry, including fluorescence-activated cell sorting and detection systems (FACS®), are available. See, e.g., Owens et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2 nd ed.; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ. Fluorescent reagents suitable for modifying nucleic acids are available, including nucleic acid primers and probes, polypeptides, and antibodies, for use as diagnostic reagents, for example. Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, MO.

[0171] Standard methods for immune system histology are described (see, e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY; Hiatt, et al. (2000) Color Atlas of Histology, Lippincott, Williams, and Wilkins, Phila, Pa.; Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY).

[0172] For example, software packages and databases are available for determining antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments. For example, GenBank, Vector NTI(R) Suite (Informax, Inc, Bethesda, Md.); GCG Wisconsin Package (Accelrys, Inc., San Diego, Calif.); DeCypher(R) (TimeLogic Corp., Crystal Bay, Nev.); Menne et al. (2000) Bioinformatics 16: 741-742; Menne et al. (2000) Bioinformatics Applications Note See von Heijne (1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res. 14:4683-4690. Example II. Construction of WNT Mimetics

[0173] WNT mimetics R2M3-26, G211-18R5, 1RC07-26, 1SH1-03, and hp1SH1-03 were constructed as described in WO 2019 / 126398 A1, WO 2020 / 010308 A1, WO 2020132365 A1, and WO 2021173726, which are incorporated by reference in their entireties. All molecules are listed in Table 1. Unless otherwise noted, all recombinant proteins were produced in Expi293F™ cells (Thermo Fisher Scientific) by transient transfection. All IgG-based and Fc-containing constructs were initially purified on Protein A resin and eluted with 0.1 M glycine pH 3.5. All proteins were then purified by size exclusion column in 2x HBS buffer (40 mM HEPES pH 7.4, 300 mM NaCl). Example III. WNT Agonist Treatment in Primary Human Corneal Endothelial Cell Cultures

[0174] Human corneal endothelial cells with attached Descemet's membrane were obtained from Eversight (Ann Arbor, MI). Multiple donor samples were pooled and digested in 2 mg / mL collagenase A in MEM-α + 15% FBS + 20 μg / mL gentamicin sulfate at 37°C for 3 hours with occasional trituration. Samples were then washed in MEM-α + 15% FBS + 20 μg / mL gentamicin before being resuspended and seeded in human endothelial SFM + 5% FBS + 2 ng / mL EGF + 20 μg / mL gentamicin sulfate.

[0175] Endothelial cells were treated with test proteins (5 nM R2M3-26, 1 μg / mL hR-spondin-1, 5 ng / mL FGF1, 5 nM G211-18R5-Rspo2RA, 5 nM G211-R2H1-Rspo2RA, or 5 nM 1RC07-26) or left untreated in human endothelial SFM + 5% FBS + 2 ng / mL EGF + 20 μg / mL gentamicin sulfate. After 2 days, cells were fixed and immunofluorescence was performed for Ki67 (Abcam #ab16667, 1:100) and Na+K+ATPase (Sigma #05-369, 1:100) before nuclear staining with DAPI. Ki67 is a well-known marker for cell proliferation. See, for example, the discussion in Aung TN et al., Mod Pathol (2021), https: / / doi.org / 10.1038 / s41379-021-00745-6. Samples were imaged with a Leica Dmi8 microscope. The percentage of Ki67+ cells in the resulting images (number of Ki67+ cells / total number of cells) was quantified using a custom Python script. The results are shown in Figures 2A, 2B, 2C, 2D, and 2E. Example IV. WNT agonist treatment in human corneal organ cultures

[0176] Human donor corneas with Fuchs' dystrophy were obtained from Eversight Eye Bank. The tissues were cut into small pieces and cultured in human endothelial SFM + 5% FBS + antimycotic / antibiotic for 3 days with or without treatment or with Wnt agonist (5 nM G211-18R5 + 1 mg / mL R-spondin-1). The corneas were then fixed and immunofluorescence was performed for the proliferation marker Ki67 (Cell Signaling Technology #9449S, 1:400) and the corneal endothelial marker ZO1 (Abcam #ab216880, 1:100). The results are shown in Figure 2D. Example V. WNT Agonist Treatment in Primary Human Corneal Epithelial Cell Cultures

[0177] Primary human corneal epithelial cells (PCS-700-010™, Lot No. 80915170) were obtained from the American Type Culture Collection (ATCC®). (Cell product information sheet available at the time of filing at https: / / www.atcc.org / products / all / PCS-700-010.aspx, which is incorporated herein by reference in its entirety.) Cells were cultured according to ATCC® recommendations. (Available as of the time of filing at https: / / www.atcc.org / products / all / PCS-700-010.aspx#cultureconditions, which is incorporated herein by reference in its entirety.)

[0178] Cells were treated with a WNT agonist (1 nM R2M3-26) or left untreated as a negative control for 2 days. Cells were then fixed and immunofluorescence was performed for Ki67 cell proliferation marker (Abcam #ab16667, 1:100) before nuclear staining with DAPI. Samples were imaged with a Leica Dmi8 microscope. #Ki67 per field of view of the resulting images. + Cells were quantified using a custom Python script.

[0179] The cells were treated with Wnt agonists (5 nM G211-18R5 or 1 mg / mL R-spondin-1) for 2 days or left untreated. After this, cell number was measured using the CellTiter-Glo® Viability Assay (Promega) according to the manufacturer's instructions. (Available at the time of filing at https: / / www.promega.com / products / cell-health-assays / cell-viability-and-cytotoxicity-assays / celltiter glo-luminescent-cell-viability-assay / ?catNum=G7570, which is incorporated herein by reference in its entirety.) The results are shown in Figure 11A and Figure 11C.

[0180] A wound healing assay kit (Abcam, ab242285) was used according to the manufacturer's instructions. Cells were left untreated or treated with a Wnt agonist (1 nM R2M3-26). The results are shown in Figures 12A and 12B. Example VI. WNT agonist treatment in rabbit limbal epithelial organoid cultures

[0181] Rabbit eyes were obtained from Innovative Research. The limbal region was dissected, and epithelial cells were dissociated by incubation in 2.5 U / mL dispase II in organoid basal medium (Advanced DMEM / F12 + 1x N-2 + 1x B-27 + 2 mM GlutaMAX + 10 mM HEPES + 1 mM N-acetylcysteine + 1x penicillin-streptomycin + 1x normocin). The epithelial cells were embedded in Matrigel and incubated with organoid basal medium containing WNT agonist (5 nM R2M3-26) or 1 μg / mL R-spondin-1.

[0182] RNA was recovered from individual wells using an RNEasy Kit containing the RNeasy Plus Mini Kit (Qiagen), and cDNA was synthesized using the SuperScript™ IV VILO™ cDNA Synthesis Kit (ThermoFisher). (The product information sheet, available at the time of filing at https: / / www.thermofisher.com / document-connect / document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FsuperscriptIV_VILO_master_mix_UG.pdf&title=VXNlciBHdWlkZTogU3VwZXJTY3JpcHQgSVYgVklMTyBNYXN0ZXIgTWl4, is incorporated herein by reference in its entirety.) qPCR for the Wnt target gene Axin2 and precursor marker p63 was performed using TaqMan™ Gene Expression Assay reagents (ThermoFisher) on a CFX96™ thermocycler (BioRad). β-actin was used for normalization. Results are shown in Figures 10A and 10B. Example VII. Lrp5 / 6, Fzd, and Znrf3 / Rnf43 RNA Expression in Human Corneal Epithelial and Endothelial Tissues

[0183] Whole eyes from a 47-year-old Caucasian female donor with no history of ocular disorders were received from Eversight (Ann Arbor, Mich.) Corneas were dissected, fixed in 10% formalin, and embedded in paraffin.

[0184] Fuchs' dystrophy tissue specimen corneas were obtained from a 73-year-old (Fuchs #1) and a 96-year-old (Fuchs #2) and delivered by Eversight (Ann Arbor, MI). Fuchs #1 received a postmortem diagnosis of Fuchs' dystrophy based on low endothelial cell count and the presence of severe droplets. Fuchs #2 received a lifetime diagnosis of Fuchs' dystrophy. Corneas were dissected, fixed in 10% formalin, and embedded in paraffin.

[0185] Paraffin-embedded tissues were sectioned and Lrp5 / 6, Fzd, and Znrf3 / Rnf43 RNA levels were measured using the RNAscope® kit containing the RNAscope Multiplex Fluorescent Reagent Kit v2 (ACDBio) according to the manufacturer's instructions. The corneal epithelial marker CK12 (Abcam #ab185627, 1:100), limbal progenitor marker p63 (Biocare #4A4, 1:100), and corneal endothelial marker Na + K + Immunofluorescence for ATPase (ThermoFisher #05-369-MI, 1:100) was subsequently performed to confirm tissue identity. Stained sections were mounted in VECTASHIELD® mounting medium with DAPI and imaged with a Leica DMi8A epifluorescence microscope. The results are shown in Figures 1 and 9. Example VIII. WNT agonist treatment in a mouse limbal-limbal debridement model

[0186] Mechanical debridement model: Six- to 12-week-old female C57Bl / 6 mice were anesthetized by placing them in an induction chamber connected to an oxygen source and an isoflurane vaporizer. The oxygen flow rate was adjusted to 0.9 L / min and the isoflurane vaporizer to 3-4%. As soon as the mouse became unresponsive and began shallow breathing, it was transferred to an anesthesia platform equipped with a nose cone and placed on a heating pad. The head was positioned so that the eye to be injured faced upward toward the microscope objective. Only one eye per mouse was injured, and the contralateral eye served as a control. A drop of proparacaine was placed on the cornea for 30 seconds. The cornea was gently wiped once using a weck-cel™ cellulose eye spear, and the cornea was dried by tapping both corners of the eye. The mouse's eye was proptose by applying periocular pressure with one hand. The right cornea was marked with a 3 mm trephine and scraped with an Algerbrush II (Katena, Catalog No. K2-4900) containing a 0.5 mm burr to remove the corneal epithelium, taking care to avoid limbal vessels. After scraping, the eyes were treated with erythromycin ointment to minimize inflammation and keep the ocular surface moist while the mouse was under anesthesia. After surgery, the affected eyes of the mice were treated with topical Fc-R-spondin-2 or anti-GFP antibody (5 mg / ml, 3 μL, 4 times daily for 7 days). On day 7, the mice were humanely euthanized, and the eyes were completely enucleated from each animal and collected in Davidson's fixative followed by 10% phosphate-buffered formalin. They were then processed for histological analysis of Axin2, p63, and H&E as described in Figure 13. Data show that R-spondin-2 eye drops activate Wnt signaling, proliferate progenitor cells, thicken the corneal epithelium, and reduce conjunctivalization. Example IX. WNT Agonist Treatment in Naive Mice by Intracameral and Intravitreal Injection

[0187] Female Balb / C mice, 6-12 weeks old, were anesthetized by placing them in an induction chamber connected to an oxygen source and an isoflurane vaporizer. The oxygen flow rate was set to 0.9 L / min and the isoflurane vaporizer was set to 3-4%. As soon as the mouse became unresponsive and began shallow breathing, it was transferred to an anesthesia platform equipped with a nose cone. The head was positioned so that the eye to be injected was facing upward toward the microscope objective. Only one eye per mouse was injected, and the contralateral eye served as a control. A drop of proparacaine was placed on the cornea for 30 seconds. The cornea was punctured just anterior to the iridocorneal angle. After wiping the fluid emanating from the anterior chamber with a Wecksel, 1 microliter of the wnt agonist R2M3-26, the superagonist G211-18R5-Rspo2RA, or an anti-GFP antibody (2.6 μg) was injected into the anterior chamber (intracameral) or posterior chamber (intravitreal) using a 33-gauge needle attached to a Hamilton syringe. The mice were allowed to recover from anesthesia. The next day, the mice were removed, the eyes were harvested, the corneas were dissected, fixed in 10% formalin, and embedded in paraffin. Paraffin-embedded tissue sections were prepared for half of the eye samples, and Axin2 levels were measured using the RNAscope® kit with the RNAscope Multiplex Fluorescent Reagent Kit v2 (ACDBio) according to the manufacturer's instructions. For the remaining eye samples, RT-qPCR for the Wnt target gene Axin2 was performed using TaqMan™ Gene Expression Assay reagents (Thermo Fisher Scientific) in a CFX96™ thermocycler (BioRad). β-actin was used for normalization. The results of these studies are presented in Figures 3A-3D. Example X. WNT agonist treatment in a rabbit endothelial ablation model

[0188] The purpose of these studies was to evaluate corneal endothelial cell (CEC) recovery in the central cornea after surgical scraping removal of endogenous CECs. WNT agonists or controls were injected intracamerally after the ablation surgery. Two separate studies were conducted to evaluate the performance of the WNT mimetic R2M3-26 and the WNT superagonist G211-18R5-Rspo2RA in a rabbit endothelial ablation model. In the first study, two different doses (1 and 8.3 μM) of R2M3-26 or an anti-GFP antibody (8.3 μM) were administered on day 0 after endothelial scraping to create a 10 mm lesion. Rabbit eyes were examined at various time points, including baseline and 3 days postoperatively, for corneal thickness by optical coherence tomography, ocular examination to assess corneal opacity, and lesion size by performing corneal flat mounts and histological staining for corneal endothelial markers. On day 3, low-dose R2M3-26 treatment tended toward decreased corneal thickness, increased lesion size, and increased cell density (Figure 3E and Figure 3F). A second study was performed using two concentrations (1 and 5 μM) of the WNT superagonist G211-18R5-Rspo2RA and anti-GFP (5 μM). A significant decrease in corneal opacity was observed in response to the 5 μM WNT superagonist, while there was a trend toward decreased corneal swelling / thickness and decreased lesion size in response to the WNT superagonist on day 3 (Figure 3G-3I). Example XI. UV-A Sunburn Model for Corneal Endothelial Damage (Liu et al., 2019, PNAS)

[0189] C57BL / 6 wild-type mice (7-15 weeks old; The Jackson Laboratory) were used in this study. Mice were anesthetized with isoflurane. Light emitted at a peak of 365 nm, a bandwidth of 9 nm (FWHM), and 398 mW / cm. 2 A UVA LED light source (M365LP1; Thorlabs) with an irradiance of 500 J / cm was focused to illuminate a 4 mm diameter spot on the mouse cornea. A laser sensor (model L49 [150A]; Ophir) was used to measure the energy, and the UVA exposure time was adjusted to provide the appropriate fluence (500 J / cm). 2The treatment was completed in 20 minutes and 57 seconds. The right eye (OD) was irradiated while the contralateral eye (OS) was covered with a thermal drape (SpaceDrapes, Inc.) and served as the untreated control. Drugs were injected intravenously several hours before UVA sunburn. Corneas were imaged and photographed daily, and OCT and CCT were measured on day 2 (Figures 8A and 8B). Example XII. Cold injury model

[0190] Mice were randomly divided into two groups, and two cycles of cryoinjury were applied to the right eye of each mouse (Han et al., 2013). Under isoflurane anesthesia, transcorneal freezing was initiated by gently placing a stainless steel cryoprobe (2.5 mm inner diameter; flat tip; ERBE Elektromedizin GmbH, Tübingen, Germany) pre-cooled in liquid nitrogen for 1 minute on the central cornea. A 2.5 mm diameter cryoprobe was chosen because it is similar to the corneal diameter of C57BL / 6 mice (2.6 mm). No pressure was applied to avoid damage to adjacent tissues, including the lens and trabecular meshwork. The cryoprobe was held on the corneal surface until an ice ball formed on the cornea and covered the entire corneal surface. This corresponds to a duration of 3 seconds, as evidenced by an endothelial defect of the same size as the ice ball. This injury was repeated after a 1-minute interval. Immediately after freezing, the cryoprobe was washed with balanced salt solution to remove it from the corneal surface, allowing the cornea to spontaneously thaw. Only the right eye was treated with the cryoprobe, and the left eye served as an untreated control. Corneal recovery was typically followed over 4-5 days by subsequent corneal thickness measurements by optical coherence tomography (OCT) and corneal clarity measurements by brightfield imaging (both on a Phoenix Micron IV microscope) (Figure 4, and Figures 5A and 5B). Example XIII. Methods for Corneal and Conjunctival Organoids

[0191] An average of three mice (six eyes) were used per experiment. Eyes were enucleated, and the cornea and conjunctiva were separated under a dissecting scope as shown in Figure 15A. Corneal epithelium was digested to a single-cell suspension in 1x TrypLE Express. Conjunctiva was digested for 1 hour in 2.5 mg / mL collagenase A in HBSS. The vicinity of the single-cell suspension was resuspended in Matrigel and plated in 40 μL droplets and allowed to solidify. The basal medium contained Advanced DMEM, 10 mM HEPES, 1x pen / strep, 100 mg / mL Normocin, 2 mM GlutaMAX, 1x B27, 1 mM N-acetylcysteine, 100 ng / mL KGF, and 10 μM Y-27632. Cells were grown for 5–7 days before 48 hours of treatment with the indicated WNT mimic. The results are shown in Figure 15B.

[0192] The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or listed in the Application Data Sheet are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified, if necessary, to employ concepts from various patents, applications, and publications to provide further embodiments. These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments along with the full range of equivalents to which such claims are entitled. Accordingly, the scope of the claims is not limited by the present disclosure. References: [ka]

Claims

1. A composition for treating keratopathy in a subject, comprising a WNT signaling modulator, wherein the subject is a human patient.

2. A composition for increasing the cell yield of mammalian corneal epithelium and / or endothelial cells grown ex vivo before or after corneal or corneal cell transplantation, comprising a WNT signaling modulator, wherein a mammalian donor cornea or isolated mammalian corneal cells are treated with the composition.

3. The composition according to claim 1 or claim 2, wherein the WNT signaling modulator is an engineered WNT signaling modulator.

4. The composition according to claim 1 or claim 2, wherein the WNT signaling modulator comprises an engineered WNT agonist.

5. The composition according to claim 4, wherein the manipulated WNT agonist is selected from the group consisting of (i) a WNT mimetic, (ii) an R-spongin mimetic, and (iii) a super SWAP / super agonist.

6. The composition according to claim 5, wherein the WNT imitation is a SWAP (trademark) compound, and the R-spongin imitation is a SWEETS (trademark) compound or a WNT enhancer super SWAP compound.

7. The WNT signaling modulator, WNT3a, or any homolog, variant, fragment, or mimetic of WNT3a; G211-18R5; R2M3-26; 1SH1-03 hp1SH1-03; 17SB9-03; and Super SWAP or Super Agonist A composition according to claim 1 or claim 2, selected from the group consisting of any combination thereof.

8. The composition according to claim 1, characterized in that the WNT signaling modulator is administered simultaneously with R-spongin-1, or any R-spongin-1 homolog, mutant, fragment, or mimetic, or any combination thereof.

9. The composition according to claim 1, characterized in that the WNT signaling modulator is administered to the subject in a therapeutically effective dose.

10. The composition according to claim 1, characterized in that the WNT signaling modulator is administered by eye drops, intrachorporeal injection, intravitreous injection, intrastromal injection, or subconjunctival injection.

11. The composition according to claim 1, characterized in that the WNT signaling modulator is suspended in an aqueous solution.

12. The composition according to claim 11, characterized in that the WNT signaling modulator is suspended in an aqueous solution at a concentration of 0.1 nM to 10 mM.

13. The composition according to claim 11, characterized in that the WNT signaling modulator is suspended in an aqueous solution at a concentration of 1 nM to 100 nM.

14. The composition according to claim 4, wherein the manipulated WNT agonist comprises a binding composition that binds to one or more Fzd receptors and / or a binding composition that binds to one or more LRP receptors.

15. The composition according to claim 14, wherein the binding composition of the manipulated WNT agonist is selected from the group consisting of a Fzd binding composition, an Lrp5 binding composition, an Lrp6 binding composition, and an LRP5 / 6 binding composition.

16. The composition according to claim 14, wherein the Fzd binding composition of the manipulated WNT agonist is selected from the group consisting of Fzd1, Fzd2, and Fzd7.

17. The composition according to claim 1, characterized in that an isolated polynucleotide encoding the polypeptide of the Wnt signaling modulator is administered, wherein the polynucleotide is optionally mRNA, and optionally modified mRNA.

18. The composition according to claim 17, characterized in that an expression vector containing the isolated polynucleotide is administered.

19. The composition according to claim 1, wherein the corneal disease is corneal dystrophy.

20. The aforementioned keratitis includes bacterial infections, viral infections, fungal infections, protozoan infections, archaeal infections, genetic disorders, brittle keratitis syndrome (BCS), corneal endothelial-mesenchymal transition (EnMT), corneal epithelial-mesenchymal transition (EMT), corneal fibrosis, Cogan syndrome, corneal ulcers, epithelial-basement membrane dystrophy (EBMD), maculoplasty, Fuchs dystrophy, post-corneal transplant scarring, gelatinous drop-like corneal dystrophy, granular corneal dystrophy type I, granular corneal dystrophy type II, interstitial keratitis, iris-corneal endothelial syndrome (ICE), keratoconjunctivitis sicca, keratoconus, keratomalacia, lattice dystrophy type I, lattice dystrophy The composition according to claim 1, selected from the group consisting of strophy type II, Risch corneal dystrophy, maculoplasty, Miesmann corneal dystrophy, bullous keratopathy, aniridia, marginal ulcerative keratitis, phlyctenular keratoconjunctivitis, posterior polymorphic corneal dystrophy, pterygium, Reiss-Bückler corneal dystrophy, Schneider crystalline corneal dystrophy, Stevens-Johnson syndrome, punctate superficial keratitis, Thiel-Benkhe corneal dystrophy, neurotrophic keratitis, corneal herpes, scarring, corneal epithelial stem cell deficiency, and corneal scarring or any combination thereof.

21. The aforementioned corneal disease, Epithelial-mesenchymal transition; Endodermal-mesenchymal transition; Corneal fibrosis; and Corneal scarring; or The composition according to claim 20, selected from the group consisting of any combination thereof.

22. The composition according to claim 20, wherein the keratopathy is Fuchs dystrophy or corneal epithelial stem cell deficiency.

23. The composition according to claim 20, wherein the keratopathy is post-corneal transplantation scarring.

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