Modulation of WNT signaling in keratopathy

Abstract

The present disclosure provides methods of treating keratopathy with modulators of WNT signaling pathways. More specifically, methods of treating corneal epithelium and / or endothelium injuries, defects, deficiencies and malnutrition are provided. Further provided are methods of administration and pharmaceutical compositions for the treatment of keratopathy.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 353,968 filed on June 21, 2022 and U.S. Provisional Patent Application No. 63 / 469,499 filed on May 29, 2023, the entire contents of which are incorporated herein by reference.

[0003] Sequence Listing

[0004] The sequence listing XML associated with this application is provided in XML file format and is incorporated into the specification by reference. The name of the XML file containing the sequence listing XML is SRZN_021_03WO_ST26.xml. The XML file is 21,937 bytes, created on June 10, 2023, and submitted electronically via the USPTO Patent Center. Technical Field

[0005] The present disclosure provides WNT signaling modulators to treat various corneal diseases. More specifically, treatments for corneal epithelial and / or endothelial damage, defects, deficiencies and malnutrition are provided. Background Art

[0006] The vertebrate cornea is an optically transparent layered tissue that represents the first and most powerful refractive medium of the eye anatomy. (Patel & Tutchenko, Contact Lens & Ant. Eye, 2019 Oct 42(5), 575–80). The cornea must remain strong to contain and protect the eye; transparent to allow visible light to enter the eye; and appropriately shaped, symmetrical, and smooth to correctly refract and direct a clear image to the image-forming retinal tissue at the back of the eye.

[0007] Unfortunately, the cornea is susceptible to injuries and diseases that cause scarring, discoloration, or worse, which can seriously impair vision and quality of life. The current treatment of corneal disease (also referred to as keratopathy) depends on the stage of the corneal disease. For mild corneal disease, supportive treatment such as saline eye drops may be sufficient to relieve symptoms. For severe corneal disease, corneal transplantation or cell transplantation may be necessary because there is currently no available non-surgical treatment option. Corneal and cell transplantation (i.e., keratoplasty) is expensive, invasive, and has a risk of tissue rejection. In addition, the available supply of donor corneas and cells is often lacking.

[0008] Potentially promising therapeutic pathways: Signaling of the WNT developmental pathway plays an important role in the stratification and layering of the mammalian cornea (Zhang et al., Development, 2015 142: 3383–93). Wnt signaling proteins are one of the most important developmental signaling molecules in the animal kingdom. In animals ranging from fruit flies to mice to frogs to nematodes to flatworms, WNT proteins have been shown to be involved in body segmentation, central nervous system (CNS) patterning, dorsal / ventral axis formation, and more (see, e.g., Wodarz & Nusse, Annu. Rev. Cell Dev. Biol., 1998 14: 59–88).

[0009] Mutations and regulation of WNT proteins are also thought to play an important role in many cancers and malnutrition (Nusse, Cell Res., 2005 (15), 28–32). In the case of the cornea, whether post-developmental WNT regulation can lead to improvements in corneal pathology (keratopathy) and damage is unknown. 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 corneal pathologies lacking keratocytes. Wnt signaling is also central to fibrosis and is elevated in corneal scars (Chawla & Ghosh, J. Cell Physiol. 2017), suggesting that antagonizing Wnt signaling may have therapeutic effects in corneal pathologies involving scarring, fibrosis, EMT, and EnMT (Kawashima et al. Mol. Vis., 2013). Understanding the progression of corneal diseases and the involvement of WNT signaling expands the possibilities for new treatments.

[0010] In addition, corneal tissue is one of several rare and valuable organ donor tissues. Corneal transplantation can be used to treat many corneal diseases. However, the donated cornea may not grow or heal normally in the recipient object. Therefore, it is desirable to have a treatment that can promote the growth of cells from donated corneal tissue in vitro. In addition, treatment can also be performed after transplantation to increase the survival of the transplanted tissue in the recipient.

[0011] Understanding corneal disease progression and the involvement of WNT signaling in this progression may expand the possibilities for new treatments.The present disclosure provides methods of modulating WNT signaling to treat corneal epithelial and endothelial diseases and to promote ex vivo cell growth and maintenance of corneal epithelial and endothelial tissues and cells. Summary of the invention

[0012] The present disclosure relates to WNT signaling modulators to treat various corneal diseases. More specifically, the present disclosure provides treatments for various corneal disease indications, including, for example, corneal epithelial and / or endothelial damage, defects, deficiencies and dystrophies.

[0013] In some embodiments, the present disclosure provides a method for treating an object suffering from corneal lesions or corneal damage, the method comprising administering an engineered WNT signaling regulator to the object. In certain embodiments, the WNT signaling regulator is an engineered WNT agonist or an engineered WNT antagonist. In a further embodiment, the engineered WNT agonist and WNT antagonist comprise a binding component (or region) that binds to one or more Fzd receptors and a binding component (or region) that binds to one or more LRP receptors. In a further embodiment, the binding component of the engineered WNT agonist is selected from: a Fzd binding component, a Lrp5 binding component, a Lrp6 binding component, or a LRP5 / 6 binding component. In a specific embodiment, the FZD binding component of the engineered WNT agonist binds to one or more Fzd receptors selected from the following: Fzd1, Fzd2, and Fzd7.

[0014] In some embodiments, the engineered WNT agonist or WNT antagonist is administered independently in the early and / or late stages of corneal lesions. In selectable embodiments, the WNT agonist and WNT antagonist are administered sequentially in the early and / or late stages of corneal lesions, or the WNT agonist and WNT antagonist are administered together in the early and / or late stages of corneal lesions. In further embodiments, the WNT agonist is administered before or after the WNT antagonist.

[0015] In one aspect, the present disclosure provides a method of treating corneal lesions in a subject, the method comprising administering a WNT signaling modulator to the subject. In another embodiment, the present disclosure provides a method comprising administering an isolated polynucleotide encoding a polypeptide of the molecule or administering an expression vector comprising the isolated polynucleotide, wherein the polynucleotide is optionally an mRNA, optionally a modified mRNA.

[0016] In preferred embodiments, the subject is a human patient. In some embodiments, the WNT signaling modulator may be an engineered WNT signaling modulator. In some embodiments, the WNT signaling modulator may be an engineered WNT agonist or an engineered WNT antagonist.

[0017] In preferred embodiments, the engineered WNT agonist is selected from: (i) WNT3a; (ii) a WNT mimetic; or (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. See, e.g., PCT Application Publication No. WO 2021173726, which discloses non-limiting examples of super SWAP molecules. In still preferred embodiments, the WNT mimetic is a SWAP TM Compounds, and R-spondin mimetics are SWEETS TM Compounds; and molecules including SWAP and SWEET in one combined molecule are referred to as super SWAPs or super agonists. In particular, WNT signaling modulators can be classical WNT modulators or non-classical WNT modulators (see, e.g., Ackers and Malgor (2017) Diabetes & Vascular Res., 15: 3-13).

[0018] In any embodiment of the method, a WNT signaling modulator can be co-administered with R-spondin or any R-spondin homolog, mutant, fragment, mimetic, or any combination thereof.

[0019] In any of the embodiments of the method, the wnt signaling modulator can be a WNT mimetic fused to a mutant Rspodin mimetic, referred to herein as a super SWAP or super agonist.

[0020] In preferred embodiments, the WNT signaling modulator is administered to a subject at a therapeutically effective dose. In some embodiments, the WNT signaling modulator may be administered via eye drops, hydrogels, depot formulations, or intraocular injection.

[0021] 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 up to about 1 M. In a preferred embodiment, the WNT signaling modulator is suspended in an aqueous solution at a concentration of 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 of about 1 nM to 100 nM.

[0022] In any embodiment described herein, the corneal lesion of the subject can be corneal dystrophy. More specifically, in any embodiment of the present method, the corneal lesion can be selected from: bacterial infection, viral infection, fungal infection, protozoan infection, archaeal infection, hereditary disease, brittle cornea syndrome (BCS), corneal endothelial-mesenchymal transition (EnMT), corneal epithelial-mesenchymal transition (EMT), corneal fibrosis, Cogon syndrome, corneal ulcer, epithelial basement membrane dystrophy (EBMD), Fleck corneal dystrophy, Fuchs dystrophy, colloid corneal dystrophy, type I granular corneal dystrophy, type II granular corneal dystrophy, interstitial keratitis, iridocorneal endothelial syndrome (ICE), keratoconjunctivitis sicca, Keratoconus, keratomalacia, lattice dystrophy type I, lattice dystrophy type II, Lisch corneal dystrophy, macular corneal dystrophy, Meesmann corneal dystrophy, bullous keratopathy, aniridia, peripheral ulcerative keratitis, herpetic keratoconjunctivitis, posterior polymorphic corneal dystrophy, pterygium, Reis-Bückler corneal dystrophy, Schnyder crystalline corneal dystrophy, Stevens-Hohonson syndrome, superficial punctate keratitis, neurotrophic keratitis, herpetic keratitis, Thiel-Behnke corneal dystrophy, scar, limbal stem cell deficiency, and any combination thereof. In a preferred embodiment of the present method, the corneal lesion is selected from: limbal stem cell deficiency (LSCD), epithelial-mesenchymal transition; endothelial-mesenchymal transition; corneal fibrosis; corneal scarring, including corneal scarring after transplantation; or any combination thereof. In a more preferred embodiment of the method, the corneal lesion is Fuchs' dystrophy.

[0023] Also described herein is a method for increasing the cell yield of ex vivo grown mammalian corneal epithelial and / or endothelial cells before or after corneal transplantation, the method comprising treating a mammalian donor cornea or isolated corneal cells with a WNT signaling regulator. In preferred embodiments, 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 regulator can be an engineered WNT signaling regulator. In some embodiments, the WNT signaling regulator can be an engineered WNT agonist.

[0024] In a preferred embodiment of the method for increasing cell production, 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 Rspondin mimetic, referred to as a super SWAP or super agonist. In a more preferred embodiment, the WNT mimetic is a SWAP TM Compounds, R-spondin mimetics are SWEETS TM Compounds, or super SWAPs or super agonists.

[0025] In some embodiments, the present disclosure provides methods, wherein the WNT signaling regulator is any of the following:

[0026] a. WNT3a or any homologue, mutant, fragment or mimetic of WNT3a;

[0027] b.G211-18R5;

[0028] c.R2M3-26;

[0029] d.1SH1-03;

[0030] e.hp1SH1-03

[0031] f.17SB9-03;

[0032] g. any of the above alone or in combination with one or more Rspondin or another growth factor such as fibroblast growth factor (FGF);

[0033] h. G211-18R5-Rspo2RA or G211-R2H1-Rspo2RA or other super SWAPs or super agonists;

[0034] i. or a combination of any of the above.

[0035] 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 any one of SEQ ID NOs: 1-16. In specific 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 shown 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 shown in SEQ ID NO: 2. In certain 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 shown 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 shown in SEQ ID NO: 4. In certain 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 shown 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 shown in SEQ ID NO: 6. In certain 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 shown 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 shown in SEQ ID NO: 2. In certain 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 shown 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 shown in SEQ ID NO: 4. In certain 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 shown 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 shown in SEQ ID NO:6.

[0036] 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 shown 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 shown in SEQ ID NO:8.

[0037] In certain 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 shown 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 shown in SEQ ID NO: 10. In certain 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 shown 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 shown in SEQ ID NO: 12. In certain 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 shown 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 shown in SEQ ID NO: 14. In certain 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 shown 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 shown in SEQ ID NO: 16.

[0038] In any of the embodiments of the methods of increasing cell yield, a WNT signaling modulator can be administered or co-administered with R-spondin or any R-spondin homolog, mutant, fragment, mimetic, or any combination thereof.

[0039] In a preferred embodiment, the WNT signaling modulator is administered to the 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 0.01pM, 0.1pM, 1pM, 0.01nM, 0.1nM, 1nM, 10nM, 100nM, 1μM, 10μM, 100μM, 1mM, 10mM, 100mM up to about 1M. In a preferred embodiment, the WNT signaling modulator is suspended in an aqueous solution at a concentration of about 0.1nM to 1μM. In a more preferred embodiment, the WNT signaling modulator is suspended in an aqueous solution at a concentration of about 1nM to 100nM.

[0040] In a preferred embodiment of the method for increasing cell yield, the WNT signaling regulator is applied to the mammalian donor cornea by immersing the mammalian donor cornea in vitro in a water bath solution containing a certain dose of the WNT signaling regulator. In other embodiments, the WNT signaling regulator is applied to the limbal stem cells or corneal endothelial cells before or after transplantation.

[0041] In preferred embodiments, the engineered WNT agonists and engineered WNT antagonists comprise a binding component that binds one or more Fzd receptors and a binding component that binds one or more LRP receptors. In a more preferred embodiment of the method of increasing cell yield, the binding component of the engineered WNT agonist is selected from a Fzd binding component, a Lrp5 binding component, a LRP6 binding component, and a LRP5 / 6 binding component. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The patent or application file contains at least one drawing executed in color. Copies of the patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0043] Figure 1A Depicted is a photomicrograph of fluorescently stained donor corneal endothelial tissue in a healthy "normal" eye donor that has been stained using in situ hybridization for the Fzd receptors Fzd 1, Fzd 2, Fzd 4, Fzd 5, Fzd 7, Fzd 8, Fzd 9, and Fzd 10, shown here in green. The expression of Fzd1, Fzd2, and Fzd7 in FECD patients has come into focus, as Fzd1, Fzd2, and Fzd7 are the most prominently expressed.

[0044] Figure 1BDepicted are photomicrographs of an otherwise healthy "normal" patient and a patient diagnosed with Fuchs' dystrophy, a corneal disease. WNT and R-spondin receptors Lrp5 / 6; Fzd1 / 2 / 7; and ZNRF3 (E3 ubiquitin ligase) were stained using in situ hybridization, shown here in green. The corneal endothelial marker Na+K+ATPase was stained using immunofluorescence, shown here in red. DNA was stained with 4',6-diamidino-2-phenylindole (DAPI), shown here in off-white.

[0045] Figure 2A Depicted is a graph of #Ki67+ (proliferation marker) cells per well in human corneal endothelial cells treated with FGF1, R-spondin-1, WNT mimetic compound R2M3-26, or WNT superagonist or super SWAP G211-18R5-Rspo2RA. The results show that WNT mimetic and WNT superagonist or super SWAP increased 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.

[0046] Figure 2B A graph depicting the percentage of Ki67+ cells in human corneal endothelial cells treated with FZD1 / 2 / 5 / 7 / 8+LRP6 combined with WNT mimetic R2M3-26 or FZD1 / 2 / 7+LRP6 combined with WNT mimetic 1RC07-26. The results show that WNT mimetic binding to FZD1 / 2 / 5 / 7 / 8 and LRP6 increased the proliferation of human corneal endothelial cells. *p≤0.05. **p≤0.01. Statistical analysis was performed using an unpaired two-tailed t-test.

[0047] Figure 2C Depicts a graph of the percentage of Ki67+ cells in human corneal endothelial cells treated with WNT superagonists or super SWAPs (G211-18R5-Rspo2RA or G211-R2H1-Rspo2RA). The results show that WNT superagonists or super SWAPs that bind to FZD1, FZD2, FZD7, and LRP6 are equally effective as WNT superagonists or super SWAPs (G211-18R5-Rspo2RA) (which bind to FZD1 / 2 / 5 / 7 / 8+LRP6) in increasing the proliferation of human corneal endothelial cells. *p≤0.05. **p≤0.01. Statistical analysis was performed using an unpaired two-tailed t-test.

[0048] Figure 2DDepicted are fluorescent 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.

[0049] Figure 2E Depicted are fluorescent micrographs of corneas from human organ donors with Fuchs' dystrophy that were untreated and treated with the WNT mimetic G211-18R5+R-spondin-1. Images demonstrate that WNT agonists increase proliferation of human corneal endothelial cells in corneal organ cultures.

[0050] Figure 3A Depicted are changes in Axin2 RNA transcript levels in corneas from mice injected intracamerally with anti-GFP isotype control antibody or the wnt mimetic R2M3-26 (61 pmol each).

[0051] Figure 3B Depicted are micrographs of fluorescently stained corneal endothelium from mice injected intracamerally with anti-GFP or SWAP (R2M3-26) (61 pmol each). The WNT target gene Axin2 was stained using in situ hybridization, shown here in green. DNA was stained with 4',6-diamidino-2-phenylindole (DAPI), shown here in blue.

[0052] Figure 3C Depicted are changes in Axin2 RNA transcript levels in corneas from mice injected intracamerally (IC) or intravitreally (IVT) with anti-GFP or wnt superagonist or super SWAP (G211-18R5-Rspo2RA) (2.6 mg each).

[0053] Figure 3D Depicted are micrographs of fluorescently stained corneal endothelium from mice injected intracamerally with anti-GFP or G211-18R5-Rspo2RA (7 pmol each). The WNT target gene Axin2 was stained using in situ hybridization, shown here in green. DNA was stained with 4',6-diamidino-2-phenylindole (DAPI), shown here in blue.

[0054] FIG. 3E to FIG. 3F Depicted are central corneal thickness measurements from rabbits 3 days after removal of the central 10 mm of corneal endothelium ( Figure 3E ) and endothelial lesion size measurements ( Figure 3F ). Rabbits were injected intracamerally with anti-GFP or R2M3-26 on day 0.

[0055] FIG. 3G to FIG. 3IFrom a separate study, central corneal thickness measurements from rabbits 3 days after ablation of the central 10 mm of corneal endothelium are depicted ( Figure 3H ), size of endothelial lesions ( Figure 3G ) and corneal opacity ( Fig. 3I ) Measurements. Rabbits were injected intracamerally on day 0 with anti-GFP or wnt superagonist or super SWAP G211-18R5-Rspo2RA. FIG. 3G to FIG. 3I The data in the study showed significant improvements in corneal opacity scores with high-dose Wnt superagonist or super-SWAP, whereas Figure 3H A trend of decreasing corneal thickness was shown in Figure 5.

[0056] FIG. 4A to FIG. 4C Wnt superagonist or super SWAP (G211-18R5-Rspo2RA) was shown to reduce corneal thickness in a mouse model of corneal cryoinjury ( Figure 4A ), improve transparency ( Figure 4B ), and showed pharmacodynamic response (PD) ( Figure 4C ). Intravitreal (IVT) injection of 5 μg of superagonist or super SWAP (G211-18R5-Rspo2RA) showed a significant reduction in central corneal thickness measured by optical coherence tomography (OCT) relative to anti-GFP isotype control (pooled from 6 independent studies) ( Figure 4A ), and shows improvement in corneal transparency in response to cryoinjury as measured by brightfield imaging of the anterior chamber of the eye (representative image from one such study) ( Figure 4B Wnt superagonists or super SWAP showed significant induction of Axin2 mRNA (a Wnt target gene) at day 4 in corneal homogenates from a study in which G211-18R5-Rspo2RA or anti-GFP antibody was administered intravitreally to mice following cryoinjury ( Figure 4C ).

[0057] FIG. 5A to FIG. 5B 1SH1-03 was shown to reduce corneal thickness percentage and improve corneal transparency in a corneal cryoinjury mouse model. Figure 5A Is a graph showing that intravitreal (IVT) injection of 25 μg 1SH1-03 showed a significant reduction in central corneal thickness measured by optical coherence tomography (OCT) relative to anti-GFP isotype control and 5 μg of 1SH1-03 (pooled from 2 independent studies). At the 2-day time point, the lines from top to bottom are associated with: GFP, 1SH1-03 5ug, and 1SH1-03 25ug, respectively. Figure 5B Bright field graph showing 1SH1-03 showing improved corneal transparency in response to cryoinjury as measured by bright field imaging of the anterior chamber of the eye (representative image from one such study).

[0058] Figure 6 is an image showing that a Wnt mimetic (hp1SH1-03) shows a reduction in the size of corneal endothelial lesions in mice following corneal cryoinjury. Intravitreal (IVT) injection of hp1SH1-03 shows a reduction in lesion size in eyes following cryoinjury compared to an anti-GFP isotype control. The figure depicts corneal flat mounts from eyes from the above treatment groups that have been subjected to immunofluorescence and stained with Ki67 (in red) and Dapi (in blue) and imaged on a confocal microscope. Although there were lesions in the central region of the anti-GFP isotype treated group without proliferating Ki67 cells, there were no detectable lesions in the hp1SH1-03 treated eyes and this area was marked by proliferating corneal endothelial cells.

[0059] FIG. 7A to FIG. 7B hp1SH1-03 was shown to induce 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 2 days. Fig. 7A Corneal endothelial cells detected by ZO-1 are shown, and the proliferation rate was measured by counting the proportion of CECs expressing the proliferation marker Ki67. Figure 7B Quantitative analysis revealed that hp1SH1-03 significantly promoted HCEC proliferation, and this proliferation was more pronounced in the wound area; approximately 6-fold higher at the edges and approximately 3-fold higher in the middle area compared to anti-GFP treated eyes.

[0060] FIG. 8A to FIG. 8B showed that the Wnt superagonist or super SWAP G211-18R5Rspo2RA displayed enhanced PD response in a UV-A damage-induced FECD rodent model. Fig. 8A Depicted are changes in Axin2 RNA transcript levels in corneas from mice injected intravitreally (IVT) with anti-GFP or G211-18R5-Rspo2RA (2.6 μg each) in naive mice or mice treated with 500 J / cm2 UV-A light in the right eye. Figure 8B Depicted in Fig. 8A Central corneal thickness (CCT) of UV-A treated mice as described in , and optical coherence tomography was performed to measure CCT as a measure of corneal swelling. On day 2 post-treatment, there was a trend toward decreased CCT in response to the Wnt superagonist or the super SWAP G211-18R5-Rspo2RA.

[0061] FIG. 9A to FIG. 9B The expression levels of different markers are shown. Fig. 9ADepicted are fluorescence micrographs of human corneal samples stained for different WNT receptors, corneal and limbal region markers, and DNA. Images are of the cornea and limbal epithelium. Fig. 9B is a table showing the expression levels of various WNT and R-spondin receptors in corneal epithelium and limbal epithelium.

[0062] FIG. 10A to FIG. 10B The expression of Axin2 and p63 is shown. Fig. 10A Depicted are changes in Axin2 RNA transcript levels in rabbit limbal epithelial organoids after 1 week of treatment with 5 nM WNT mimetic R2M3-26 or 1 μg / mL R-spondin-1. *p≤0.05. ***p≤0.001. Statistical analysis was performed using an unpaired two-tailed t-test. Fig. 10B Depicted are changes in p63 RNA transcript levels in rabbit limbal epithelial organoids after 1 week of treatment with 5 nM WNT mimetic R2M3-26 or 1 μg / mL R-spondin-1. *p≤0.05. Statistical analysis was performed using an unpaired two-tailed t-test.

[0063] FIG. 11A to FIG. 11C Data showing enhanced cell growth and proliferation in human corneal epithelium are depicted. Fig.11A Depicted are cell growth data for cells treated with the WNT mimetic G211-18R5 for 2 days. Assays measure cell number. Fig. 11B Depicted are Ki67+ cell proliferation data following treatment with the WNT mimetic R2M3-26. Proliferation was measured by analysis of immunofluorescence micrographs. Fig. 11C Depicted are cell growth data for cells treated with R-spondin-1 for 2 days. In each graph, statistical analysis was performed using an unpaired two-tailed t-test.

[0064] FIG. 12A to FIG. 12B Depicted are micrographs of an in vitro wound healing assay. Human corneal epithelial cells were seeded around a physical barrier. The physical barrier was removed, leaving a gap. The cells were grown in the absence of the WNT mimetic R2M3-26 ( Fig. 12A ) and containing the WNT mimetic R2M3-26 ( Fig. 12B ) medium for 2 days. The cells were then stained with DAPI and imaged, and the size of the gaps was measured.

[0065] Fig.13 Topical Fc-Rspo2 eye drops were shown to reduce corneal epithelial defects in a mouse model of corneal epithelial debridement. Fig.13Depicted are fluorescent and bright field micrographs of the central corneal epithelium from naive mice and mouse eyes treated with anti-GFP eye drops (5 mg / mL, 3uL, 4 times a day) or Fc-Rspondin-2 eye drops (5 mg / mL, 3uL, 4 times a day) 7 days after limbus-to-limbus debridement. The WNT target gene Axin2 was stained using in situ hybridization, shown here in red. DNA was stained with 4',6-diamidino-2-phenylindole (DAPI), shown here in blue. The progenitor cell marker p63 was stained using immunofluorescence, shown here in green. Hematoxylin-eosin (H&E) staining is also depicted. The data show that R-spondin eye drops activate Wnt signaling, expand progenitor cells, thicken the corneal epithelium, and reduce conjunctivalization.

[0066] Fig.14 Depicted is a fluorescent 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 in the central cornea and limbal membranes but not in the conjunctiva, shown here in green. DNA was stained with 4',6-diamidino-2-phenylindole (DAPI), shown here in blue. Fig.14 The data in this article suggest that THBD can be used as a target for the generation of limbal-targeted Rspo mimetics / SWEETs for corneal regeneration. TM molecular.

[0067] Fig.15A Schematic representation of murine conjunctival and corneal epithelial organoids derived from primary tissue is depicted. Expanded cultures maintain markers such as keratin 12 (Ck12) or keratin 19 (Ck19).

[0068] Fig. 15B Depicted are changes in Axin2 mRNA transcript levels in conjunctival or corneal epithelial organoids treated with different WNT mimetics. R2M3-26 is a FZD12578-targeted WNT mimetic. 17SB9 is a FZD8-biased WNT mimetic. Data were normalized to each tissue untreated control. Fig. 15B The data in the present study suggest that 17SB9-03-Rspo mimics can be used as a target for generating corneal epithelial-targeted FZD8-Rspo mimics / SWEETs for corneal regeneration. TM molecular. DETAILED DESCRIPTION

[0069] As used herein, including the appended claims, singular forms such as "a," "an," and "the" include their corresponding plural references unless the context clearly dictates otherwise.

[0070] 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.

[0071] I. Definitions

[0072] 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, regulation of the activity of other molecules, etc. The "activity" of a molecule can also refer to the activity of regulating or maintaining cell-cell interactions (e.g., adhesion), or the activity of maintaining cell structure (e.g., cell membrane or cytoskeleton). "Activity" can also mean a specific activity, for example, [catalytic activity] / [mg protein] or [immune activity] / [mg protein], etc.

[0073] As used herein, the term "administering" or "introducing" or "providing" refers to delivering a composition to a cell, to a cell of a subject, to a tissue of a subject and / or an organ of a subject, or to a subject. Such administration or introduction can be performed in vivo, in vitro or ex vivo.

[0074] As used herein, the term "antibody" means an isolated or recombinant binding agent that contains the necessary variable region sequences to specifically bind to an antigenic epitope. Therefore, an antibody is any form of an antibody or a fragment thereof that exhibits the desired biological activity, for example, binding to a specific target antigen. Therefore, 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, double antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, the antibody fragments including but not limited to scFv, Fab and Fab2, as long as they exhibit the desired biological activity.

[0075] "Antibody fragments" include a portion of an intact antibody, for example, the antigen binding region or variable region of an 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 antibodies produces two identical antigen-binding fragments, referred to as "Fab" fragments and a residual "Fc" fragment, each of which has a single antigen-binding site, and a residual "Fc" fragment, the name reflecting the ability to crystallize easily. Pepsin treatment produces a F(ab')2 fragment, which has two antigen-binding sites and is still able to cross-link antigens.

[0076] The term "antigen" refers to a molecule or a portion of a molecule that can be bound by a selective binding agent (e.g., an antibody) and that can additionally be used in an animal to produce an antibody that can bind to an epitope of the antigen. In certain embodiments, a binding agent (e.g., a WNT surrogate molecule or its binding region, or a WNT antagonist) is said to specifically bind to an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0077] As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment that contains at least one complementarity determining region (CDR) of an immunoglobulin heavy chain and / or light chain or a VHH / sdAb (single domain antibody) or (Nab), which binds to an antigen of interest, in particular to one or more Fzd receptors, or to LRP5 and / or LRP6. In this regard, the antigen-binding fragment of an antibody described herein may comprise 1, 2, 3, 4, 5 or all 6 CDRs from VH and VL of an antibody that binds to one or more Fzd receptors or LRP5 and / or LRP6.

[0078] As used herein, the terms "biological activity" and "biologically active" refer to the activity attributed to a specific biological element in a cell. For example, the "biological activity" of a WNT agonist or a fragment or variant thereof refers to the ability to simulate or enhance a WNT signal. As another example, the biological activity of a polypeptide or a functional fragment or variant thereof refers to the ability of a polypeptide or a functional fragment or variant thereof to perform its natural function (e.g., 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 a regulatory element or a functional fragment or variant thereof to regulate (i.e., promote, enhance or activate) the expression and translation of a gene to which it is operably connected, respectively.

[0079] As used herein, the term "bifunctional antibody" refers to an antibody comprising a first arm having specificity for one antigenic site and a second arm having specificity for a different antigenic site, ie, the bifunctional antibody has dual specificity.

[0080] "Bispecific antibody" refers to a full-length antibody produced by the following: quadroma technology (see Milstein et al., Nature, 305 (5934): 537-540 (1983)), chemical coupling of two different monoclonal antibodies (see Staerz et al., Nature, 314 (6012): 628-631 (1985)), or knob-into-hole or similar methods to introduce mutations in the Fc region (see Holliger et al., Proc. Natl. Acad. Sci. USA, 90 (14): 6444-6448 (1993)), which produces multiple different immunoglobulin species, only one of which is a functional bispecific antibody. Bispecific antibodies bind to one antigen (or epitope) on one of their two binding arms (a pair of HC / LC) and bind to a different antigen (or epitope) on their second arm (a pair of different HC / LC). By this definition, a bispecific antibody has two distinct antigen-binding arms (different in both specificity and CDR sequences) and is monovalent for each antigen it binds.

[0081] "Comprising" means that the elements described are essential in, for example, a composition, method, kit, etc., but other elements may be included within the scope of the claim to form, for example, a composition, method, kit, etc. For example, an expression cassette "comprising" a gene encoding a therapeutic polypeptide operably linked to a promoter may include other elements in addition to the gene and the promoter, such as polyadenylation sequences, enhancer elements, other genes, linker domains, etc.

[0082] "Substantially consisting of..." means limiting the scope of the described, e.g., composition, method, kit, etc., to specified materials or steps that do not substantially affect the basic and novel features of, e.g., composition, method, kit, etc. For example, an expression cassette "substantially consisting of" a gene encoding a therapeutic polypeptide operably linked to a promoter and a polyadenylation sequence may contain other sequences, such as a linker sequence, as long as they do not substantially affect the transcription or translation of the gene. As another example, a variant or mutant, a polypeptide fragment "substantially consisting of" the sequence has an amino acid sequence of the sequence plus or minus about 10 amino acid residues at the boundary of the sequence of the full-length original polypeptide from which it is derived, e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue less than the boundary amino acid residues, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues more than the boundary amino acid residues.

[0083] "Consisting of" means a composition, method or kit that excludes any element, step or ingredient not specified in the claim. For example, a polypeptide or polypeptide domain that "consists of" a stated sequence comprises only the stated sequence.

[0084] "Control elements" or "control sequences" are nucleotide sequences that participate in molecular interactions that contribute to the regulation of the functions of a polynucleotide, including replication, repetition, transcription, splicing, translation, or degradation of the polynucleotide. Regulation can affect the frequency, speed, or specificity of a process, and can be enhancing or inhibiting in nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a region of DNA that is capable of binding RNA polymerase under certain conditions and initiating transcription of a coding region that is usually located downstream (3' direction) of the promoter.

[0085] As used herein, "cornea" and "corneal" refer to the optically transparent layered tissue covering the iris, pupil, and anterior chamber of the eye. "Corneal edge" or "limbus" refers to the roughly circular border where the cornea connects to the sclera of the eye. It should be readily understood by those of ordinary skill in the art that the boundary between corneal tissue and limbus tissue is not well-defined, and reference to "cornea" should be understood to include the approximate edge. The cornea includes five cell layers: (1) epithelium (outermost, smooth optical surface and barrier to chemicals, water, and microorganisms); (2) Bowman's layer (helps maintain a precise convex shape); (3) stroma (the main mechanical structure and refractive medium of the cornea); (4) Descemet's membrane (resting layer of endothelium); and (5) endothelium (maintains transparency by removing water from the stroma).

[0086] As used herein, "limbal cell deficiency" or "limbal stem cell deficiency" is a hereditary, acquired or idiopathic deficiency or complete loss of limbal stem cells, which are essential for the repopulation of the corneal epithelium. The loss or deficiency of limbal stem cells makes the corneal epithelium unable to repair or renew, leading to corneal conjunctivalization, neovascularization, corneal scarring and chronic inflammation.

[0087] As used herein, "corneal dystrophy" generally refers to a condition characterized by abnormal discoloration, haziness, and / or opacity of any layer or combination of layers of the cornea. Corneal dystrophy may be heritable or non-heritable. They may be caused by abnormal cell growth and / or by the deposition and accumulation of collagen, lipids, cholesterol, and / or other foreign substances.

[0088] "Fuchs' dystrophy" (also known as Fuchs' endothelial dystrophy (FECD), Fuchs' corneal dystrophy (FCD)) is a degenerative disease of the corneal endothelial monolayer characterized by decreased endothelial cell density, guttate cornea, and accumulation of corneal edema, leading to corneal opacity and decreased vision.

[0089] As used herein, the term "engineered" refers to a molecule (eg, a Wnt signaling modulator) that is not naturally occurring, eg, is the result of genetic engineering.

[0090] An "expression vector" is a vector, e.g., a plasmid, a minicircle, a viral vector, a liposome, etc., as discussed herein or as known in the art, which contains a region encoding a gene product of interest and is used to achieve expression of the gene product in an intended target cell. The expression vector also contains control elements, e.g., promoters, enhancers, untranslated regions (UTRs), miRNA targeting sequences, etc., operably linked to the coding region to promote expression of the gene product in the target. The combination of control elements and one or more genes to which they are operably linked for expression is sometimes referred to as an "expression cassette," a large number of which are known and available in the art, or can be readily constructed from components available in the art.

[0091] As used herein, the term "FR set" refers to the four flanking amino acid sequences at the CDR of the CDR set of the heavy or light chain V region that are framed. Some FR residues may contact the bound antigen; however, FR is primarily responsible for folding the V region into an antigen binding site, especially the FR residues directly adjacent to the CDR. In FR, certain amino acid residues and certain structural features are very highly conserved. In this regard, all V region sequences contain an internal disulfide loop of approximately 90 amino acid residues. When the V region is folded into a binding site, the CDR is displayed as a protruding loop motif that forms an antigen binding surface. It is generally recognized that there are conserved structural regions of FR that affect the folding of the CDR loop into certain "classical" structures, regardless of the precise CDR amino acid sequence. In addition, it is known that certain FR residues participate in non-covalent domain contacts to stabilize the interaction of the antibody heavy and light chains.

[0092] 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 apes 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.).

[0093] As used herein, the term "isolated" means that a molecule (e.g., a naturally occurring molecule) is not in its natural environment, or has been separated from one or more components present in its natural environment, or from one or more components present during the manufacture of the molecule.

[0094] As used herein, the terms "keratopathy" and "corneal disease" refer to diseases or conditions of the eye, particularly diseases or conditions of the cornea and / or limbus of the eye. Corneal lesions include, but are not necessarily limited to, bacterial infections, viral infections (e.g., herpes simplex keratitis and herpes zoster ophthalmicus (i.e., herpes zoster)), fungal infections, protozoan infections, archaeal infections, hereditary diseases, fragile cornea syndrome (BCS), corneal endothelial-mesenchymal transition (EnMT), corneal epithelial-mesenchymal transition (EMT), corneal fibrosis, Cochrane syndrome, corneal ulcers, epithelial basement membrane dystrophy (EBMD, ABMD, Map Dot), Fleck corneal dystrophy, Fuchs dystrophy (also called Fuchs corneal endothelial dystrophy (FCED) or Fuchs endothelial dystrophy (FED)), jelly drop 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, Lisch corneal dystrophy, macules Corneal dystrophy, Meesmann corneal dystrophy, bullous keratopathy, aniridia, peripheral ulcerative keratitis, herpetic keratoconjunctivitis, posterior polymorphic corneal dystrophy, pterygium, Reis-Bückler corneal dystrophy, Schnyder crystalline corneal dystrophy, Stevens-Johnson syndrome, superficial punctate keratitis, Thiel-Behnke corneal dystrophy, neurotrophic keratitis, herpetic keratitis, scarring, and limbal stem cell deficiency.

[0095] "Monoclonal antibody" refers to a homogeneous antibody population, wherein the monoclonal antibody is composed of (naturally occurring and non-naturally occurring) amino acids that participate in the selective binding of an epitope. Monoclonal antibodies are highly specific, being directed against a single epitope. The term "monoclonal antibody" includes not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chain (scFv), Variants thereof, fusion proteins comprising antigen-binding fragments of monoclonal antibodies, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configurations of immunoglobulin molecules comprising antigen-binding fragments (epitope recognition sites) with the desired specificity and ability to bind to epitopes, including WNT replacement molecules disclosed herein. It is not intended to limit the source of the antibody or the manner in which it is prepared (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins as well as fragments under the definition of "antibody" above, etc.

[0096] As used herein, the term "natural" or "wild type" refers to a nucleotide sequence present in a wild-type cell, tissue, organ or organism, for example, a gene or gene product, for example, an RNA or protein. As used herein, the term "variant" refers to a mutant of a reference polynucleotide or polypeptide sequence (e.g., a natural polynucleotide or polypeptide sequence), that is, having a sequence identity less than 100% with the reference polynucleotide or polypeptide sequence. In other words, the variant comprises at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) relative to the reference polynucleotide sequence (e.g., a natural polynucleotide or polypeptide sequence). For example, a variant can be a polynucleotide having a sequence identity of 50% or more, 60% or more, or 70% or more with a full-length natural polynucleotide sequence, for example, 75% or 80% or more identity with a full-length natural polynucleotide sequence, for example 85%, 90% or 95% or more, for example, 98% or 99% identity. As another example, a variant can be a polypeptide having 70% or more sequence identity to a full-length native polypeptide sequence, e.g., 75% or 80% or more identity to a full-length native polypeptide sequence, e.g., 85%, 90% or 95% or more, e.g., 98% or 99% identity. A variant can also include a variant fragment of a reference sequence (e.g., a native sequence) that has 70% or more sequence identity to a fragment of a reference sequence (e.g., a native sequence), e.g., 75% or 80% or more identity to a native sequence, e.g., 85%, 90% or 95% or more, e.g., 98% or 99% identity.

[0097] "Operably linked" or "operably linked" refers to the juxtaposition of genetic elements wherein the elements are in a relationship that permits them to operate in the intended manner. For example, a promoter is operably linked to a coding region if the promoter helps initiate transcription of a coding sequence. Intervening residues may exist between the promoter and the coding region as long as this functional relationship is maintained.

[0098] As used herein, the terms "polypeptide", "peptide" and "protein" refer to polymers of amino acids of any length. The term also includes amino acid polymers that have been modified; for example, including disulfide bond formation, glycosylation, lipidation, phosphorylation or conjugation with a labeling component.

[0099] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, the nucleotide structure may be modified before or after polymer assembly. As used herein, the term polynucleotide refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise specified or required, the polynucleotides of any embodiment described herein include each of the double-stranded form and the two complementary single-stranded forms known or predicted to constitute the double-stranded form.

[0100] A polynucleotide or polypeptide has a certain percentage of "sequence identity" with another polynucleotide or polypeptide, which means that when the two sequences are aligned, the percentage of bases or amino acids is the same. As used herein, the terms "identity" and "identical" for polypeptide or polynucleotide sequences refer to the percentage of exact matching residues in the alignment of the "query" sequence with the "object" sequence, such as the alignment generated by the BLAST algorithm. Unless otherwise specified, identity is calculated over the full length of the object sequence. Therefore, if at least x% (rounded down) of the residues in the object sequence are aligned to the corresponding residues in the query sequence when the query sequence is aligned with the object sequence, the query sequence and the object sequence "share at least x% identity". In the case where the object sequence has a variable position (e.g., a residue represented by X), the alignment with any residue in the query sequence is considered a match. Unless otherwise specified, sequence alignments were 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, which can be obtained through the World Wide Web at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, which is available in the Genetics Computing Group (GCG) software package from a wholly owned subsidiary of Oxford Molecular Group, Madison, Wisconsin, USA. Other techniques for alignment are described in METHODS IN ENZYMOLOGY, VOL. 266: COMPUTER METHODS FOR MACROMOLECULAR SEQUENCE ANALYSIS (1996), ed. Doolittle, Academic Press, Harcourt Brace & Co., San Diego, California, USA. Of particular interest are alignment programs that allow gaps in sequences. Smith-Waterman is a class of algorithms that allow gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). In addition, the GAP program using the Needleman and Wunsch alignment method can be used to align sequences. See J. Mol. Biol. 48: 443-453 (1970).

[0101] Of interest is the BestFit program (Adv. Appl. Math. 2: 482-489 (1981) using the Smith-Waterman local homology algorithm to determine sequence identity. The gap creation penalty is typically in the range of 1 to 5, typically 2 to 4, and in many embodiments 3. The gap extension penalty is typically in the range of about 0.01 to 0.20, and in many cases 0.10. The program has default parameters determined by the input sequences to be compared. Preferably, sequence identity is determined using the default parameters determined by the program. The program is also available from the Genetics Computing Group (GCG) software package in Madison, Wisconsin, USA. Another program of interest is the FastDB algorithm. FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. Sequence identity percentages were calculated by FastDB based on the following parameters: mismatch penalty: 1.00; gap penalty: 1.00; gap size penalty: 0.33; and ligation penalty: 30.0.

[0102] As used herein, "promoter" includes a DNA sequence that directs the binding of RNA polymerase to promote RNA synthesis, that is, the minimum sequence sufficient to direct transcription. Promoters and corresponding protein or polypeptide expression can be ubiquitous, meaning that there is strong activity in a wide range of cells, tissues and species, or cell type-specific, tissue-specific or species-specific strong activity. Promoters can be "constitutive", meaning that there is continuous activity, or "inducible", meaning that the promoter can be activated or inactivated by the presence or absence of biological or non-biological factors. The disclosed nucleic acid construct or vector also includes an enhancer sequence, which may be adjacent to the promoter sequence or not. Enhancer sequences affect promoter-dependent gene expression and can be located in the 5' or 3' region of the native gene.

[0103] "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.

[0104] As used herein, “SWAP TM"Surrozen WNT signaling activator protein" refers to a WNT mimetic compound comprising an engineered bispecific full-length immunoglobulin G (IgG) antibody that directly activates the canonical WNT signaling pathway in target tissues (e.g., corneal tissue) like a WNT protein.

[0105] As used herein, “SWEETS TM "(Engineered tissue-specific Surrozen WNT signaling enhancers) refers to antibody-based R-spondin mimetic compounds.

[0106] As used herein, the terms "treat," "treatment," and the like generally mean obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of completely or partially preventing a disease or its symptoms (e.g., reducing the likelihood of a disease or its symptoms occurring in a subject), and / or can be therapeutic in terms of partially or completely curing a disease and / or side effects attributable to a disease.

[0107] As used herein, "treatment" encompasses any treatment of a disease in a mammal and includes: (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., arresting its development; or (c) alleviating the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during, or after the onset of the disease or injury. Treatment of an ongoing disease is of particular interest, where the treatment stabilizes or alleviates undesirable clinical symptoms in the patient. Such treatment is expected to be performed before the affected tissue has completely lost function. The treatment is expected to be administered during and in some cases after the symptomatic stage of the disease.

[0108] 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 scope of those skilled in the art. Such techniques are fully explained in the literature, for example, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (MJ Gait, ed., 1984); "Animal Cell Culture" (RI Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (DM Weir & C.C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (JM Miller & M.P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (FM Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0109] Several aspects of the present disclosure are described below with reference to example applications for illustration. It should be understood that many specific details, relationships and methods are shown to provide a comprehensive understanding of the present disclosure. However, those of ordinary skill in the relevant art will readily recognize that one or more specific details may not be used or other methods may be used to implement the present disclosure. The present disclosure is not limited by the actions or event sequences shown, because some actions may occur in different orders and / or occur simultaneously with other actions or events. In addition, not all actions or events shown are required for realizing methods according to the present disclosure.

[0110] The terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present disclosure. As used herein, the singular forms "a", "an", and "the" are intended to also include the plural forms, unless the context clearly indicates otherwise. In addition, for the terms "comprises", "comprising", "having", "having", "containing" or their variations used in the detailed description and / or claims, such terms are intended to be included in a manner similar to the term "comprising".

[0111] The term "about" or "approximately" means within an acceptable error range for a particular value determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term can mean within an order of magnitude, preferably within 5 times, and more preferably within 2 times of a value. When describing specific values ​​in the application and claims, unless otherwise stated, it should be assumed that the term "about" means within an acceptable error range for the specific value.

[0112] 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. It should be understood that the present disclosure supersedes any disclosure of the incorporated publication in the event of a conflict.

[0113] It should also be noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as antecedent basis for use of exclusive terminology such as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0114] Unless otherwise indicated, all terms used herein have the same meanings as understood by one skilled in the art, and the practice of the present disclosure will employ conventional techniques of microbiology and recombinant DNA technology within the scope known to those skilled in the art.

[0115] II. Overview

[0116] The present disclosure provides methods for modulating WNT signals to treat corneal diseases, including but not limited to scarring, and corneal epithelial and endothelial dystrophy. In particular, the present disclosure provides WNT / β-catenin agonists and / or antagonists to stimulate corneal tissue regeneration and / or prevent corneal fibrosis.

[0117] Wnt ("Wingless-associated integration site" or "Wingless and Int-1" or "Wingless-Int") ligands and their signals play a key role in the control of 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 (e.g., reviewed by Clevers, Loh, and Nusse, 2014; 346: 1248012). Modulation of the WNT signaling pathway has the potential to treat degenerative diseases and tissue damage.

[0118] One of the challenges in modulating WNT signaling as a therapeutic is that there are multiple WNT ligands and WNT receptors (Frizzled 1-10 (Fzd1-10)), and many tissues express multiple overlapping Fzds. In addition to Fzd, 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.

[0119] R-spondin 1-4 is a family of ligands that enhance WNT signaling. Each R-spondin acts through a receptor complex that contains zinc ring finger 3 (ZNRF3) or ring finger protein 43 (RNF43) at one end and G protein-coupled receptor 4-6 (LGR4-6) with leucine-rich repeats at the other end (e.g., Knight & Hankenson 2014, Matrix Biol.; 37:157-161 review). R-spondin can also act through another mechanism of action. ZNRF3 and RNF43 are two membrane-bound E3 ligases that specifically target WNT receptors (FZD1-10 and LRP5 or LRP6) for degradation. The binding of R-spondin to ZNRF3 / RNF43 and LGR4-6 results in the removal or sequestration of the ternary complex, which removes the E3 ligase from the WNT receptor and stabilizes the WNT receptor, resulting in enhanced WNT signaling. Each R-spondin contains two furin domains (1 and 2), of which furin domain 1 binds to ZNRF3 / RNF43 and furin domain 2 binds to LGR4-6. Fragments of R-spondin containing furin domains 1 and 2 are sufficient to enhance WNT signaling. Although the action of R-spondin depends on WNT signaling, the action of R-spondin is not tissue-specific because both LGR4-6 and ZNRF3 / RNF43 are widely expressed in various tissues.

[0120] In some embodiments, WNT / β-catenin signaling antagonists or agonists may include binding agents or epitope binding domains that bind to one or more Fzd receptors and inhibit or enhance WNT signaling. In certain embodiments, the agent or antibody specifically binds to the cysteine-rich domain (CRD) in the human frizzled receptor to which it binds. In addition, antagonistic binding agents containing epitope binding domains for LRP can also be used. In some embodiments, WNT / β-catenin antagonists have binding agents or epitope binding domains that bind to E3 ligase ZNRF3 / RNF43 and one or more FZD receptors or one or more LRP co-receptors to promote the degradation of FZD or LRP receptors, and the molecule can also contain binding domains that bind to cell type-specific epitopes for targeting. E3 ligase agonist antibodies or fragments thereof can be single molecules or combined with other WNT antagonists (e.g., Fzd receptor antagonists, LRP receptor antagonists, etc.).

[0121] As is well known in the art, an antibody is an immunoglobulin molecule that can specifically bind to a target (e.g., carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one epitope binding domain located on the variable region of the immunoglobulin molecule. As used herein, the term includes not only complete polyclonal or monoclonal antibodies, but also fragments thereof (e.g., dAb, Fab, Fab', (F(ab')2, Fv, single chain (scFv), VHH (i.e., ) or single domain antibodies (sdAb), DVD-Igs, synthetic variants thereof, naturally occurring variants, fusion proteins comprising epitope binding domains, humanized antibodies, chimeric antibodies, and any other modified configuration of immunoglobulin molecules comprising antigen binding sites or fragments (epitope recognition sites) of desired specificity. "Diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993), are also specific forms of antibodies considered herein. Minibodies comprising scFvs connected to a CH3 domain are also included herein (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). See, for example, Ward, ES et al., Nature 341, 544-546 (1989); Bird et al., Science, 242, 423-426, 1988; Huston et al. al., Proc.Natl.Acad.Sci.USA, 85, 5879-5883, 1988); PCT / US92 / 09965; WO94 / 13804; P. Holliger et al., Proc.Natl.Acad.Sci.USA 906444–6448, 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. 2016Sept;408(22);5985–6002.

[0122] The proteolytic enzyme papain preferentially cleaves IgG molecules to produce several fragments, two of which (F(ab) fragments) each include a covalent heterodimer comprising a complete antigen binding site. The enzyme pepsin is capable of cleaving IgG molecules to provide several fragments, including a F(ab')2 fragment comprising two antigen binding sites. The Fv fragment used in accordance with certain embodiments of the present disclosure can be produced by preferential proteolytic cleavage of IgM and occasional proteolytic cleavage of IgG or IgA immunoglobulin molecules. However, Fv fragments are more commonly derived from the use of recombinant techniques known in the art. The Fv fragment comprises a non-covalent VH:VL heterodimer comprising an antigen binding site that retains most of the antigen recognition and binding capabilities 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.

[0123] 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 Janusin (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 according to the teachings of the present application on selecting antibodies with desired specificity. In other embodiments, bispecific or chimeric antibodies comprising the ligands disclosed herein can be prepared. For example, chimeric antibodies can comprise CDRs and framework regions from different antibodies, while bispecific antibodies can be produced by specifically binding one or more Fzd receptors through one binding domain and binding to a second molecule through a second binding domain. These antibodies can be produced by recombinant molecular biology techniques or can be physically conjugated together.

[0124] Single-chain Fv (scFv) polypeptides are covalently linked VH:VL heterodimers expressed by gene fusions comprising a VH encoding gene and a VL encoding gene linked by a peptide encoding linker. Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. Many methods have been described to identify chemical structures for converting naturally aggregated but chemically separated light and heavy polypeptide chains from antibody V regions into scFv molecules that will fold into a three-dimensional structure that is substantially similar to the structure of an antigen binding site. See, e.g., U.S. Pat. Nos. 5,091,513 and 132,405 to Huston et al., and U.S. Pat. No. 4,946,778 to Ladner et al.

[0125] In certain embodiments, antibodies as described herein are in the form of diabodies. Diabodies are multimers of polypeptides, each polypeptide comprising a first domain and a second domain, wherein the first domain comprises a binding region for an immunoglobulin light chain, the second domain comprises a binding region for an immunoglobulin heavy chain, the two domains are connected (e.g., by a peptide linker) but cannot be combined with each other to form an antigen binding site, and the antigen binding site is formed by the combination of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94 / 13804).

[0126] The dAb fragment of an antibody consists of the VH domain (Ward, ES et al., Nature 341, 544-546 (1989)).

[0127] When bispecific antibodies are to be used, these antibodies may be conventional bispecific antibodies, which may be prepared in a variety of ways (Holliger, P. & Winter G., Curr. Opin. Biotech. 4, 446-449 (1993)), for example, chemically prepared or prepared from a hybridoma, or may be any of the bispecific antibody fragments mentioned above. Using only variable domains, diabodies and scFvs without Fc regions may be constructed, potentially reducing the impact of anti-idiotypic responses.

[0128] Compared to bispecific complete antibodies, bispecific diabodies can also be particularly useful because they can be easily constructed and expressed in Escherichia coli (E. coli). Phage display (WO94 / 13804) can be used to easily select diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificity from libraries. If one arm of the diabody remains constant, for example, with specificity for antigen X, a library with a variable other arm can be prepared, and antibodies with appropriate specificity can be selected. Bispecific complete antibodies can be prepared by engineering transformation of knobs into sockets (JBBRidgeway et al., Protein Eng., 9, 616-621 (1996)).

[0129] In certain embodiments, the antibodies described herein can be used provided in the form of. is an IgG4 antibody with the hinge region removed (see Genmab Utrecht, The Netherlands; see also, e.g., US2009 / 0226421). This patented antibody technology produces a stable, smaller antibody format with a longer therapeutic window than current small antibody formats are expected to have. IgG4 antibodies are considered 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, obtaining half-molecule fragments with different stability relative to the corresponding intact IgG4 (Genmab, Utrecht). Splitting the IgG4 molecule in half only allows the binding of the cognate antigen (e.g., disease target) to the target region. There is an area left on the Binds monovalently to only one site on the target cell.

[0130] In certain embodiments, antibodies and antigen-binding fragments thereof as described herein comprise heavy and light chain CDR sets inserted respectively between heavy and light chain framework regions (FR) sets, which provide support for CDRs and define the spatial relationship of CDRs relative to each other. As used herein, the term "CDR set" refers to three hypervariable regions in heavy or light chain V regions. Starting from the N-terminus of the heavy or light chain, these regions are represented as "CDR1", "CDR2" and "CDR3", respectively. Therefore, the antigen binding site comprises six CDRs, each of which comprises a CDR set 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 many antigen-antibody complexes has shown that the amino acid residues of CDRs form extensive contacts with the bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Therefore, the molecular recognition unit is primarily responsible for the specificity of the antigen binding site.

[0131] As used herein, the term "FR set" refers to the four flanking amino acid sequences at the CDR of the CDR set of the heavy chain or light chain V region that are framed. Some FR residues may contact the bound antigen; however, FR is primarily responsible for folding the V region into an antigen binding site, especially the FR residues directly adjacent to the CDR. In FR, certain amino acid residues and certain structural features are very highly conserved. In this regard, all V region sequences contain an internal disulfide loop of approximately 90 amino acid residues. When the V region is folded into a binding site, the CDR is displayed as a protruding loop motif that forms an antigen binding surface. It is generally recognized that there are conserved structural regions of FR that affect the folding of the CDR loop into certain "classical" structures, regardless of the precise CDR amino acid sequence. In addition, it is known that certain FR residues participate in non-covalent domain contacts to stabilize the interaction of the antibody heavy and light chains.

[0132] "Monoclonal antibody" refers to a homogeneous antibody population, wherein the monoclonal antibody is composed of (naturally occurring and non-naturally occurring) amino acids that participate in the selective binding of an epitope. Monoclonal antibodies are highly specific, being directed against a single epitope. The term "monoclonal antibody" includes not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chain (scFv), Variants thereof, fusion proteins comprising antigen-binding fragments of monoclonal antibodies, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configurations of immunoglobulin molecules comprising antigen-binding fragments (epitope recognition sites) with the desired specificity and ability to bind to epitopes, including WNT replacement molecules disclosed herein. It is not intended to limit the source of the antibody or the manner in which it is prepared (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins as well as fragments under the above definition of "antibody", etc.

[0133] In certain embodiments, the antibodies of the present disclosure may be in the form of. The technology was initially developed with the discovery and identification that camelids (e.g., camels, alpacas, and llamas) have fully functional antibodies consisting only of heavy chains and thus lacking light chains. These heavy chain-only antibodies contain a single variable domain (VHH) and two constant regions (CH2, CH3). The cloned single variable domain has full antigen binding capacity and is very stable. These single variable domains with their unique structural and functional properties form basis. It is encoded by a single gene and is efficiently produced in almost all prokaryotic and eukaryotic hosts (e.g., Escherichia coli (see, e.g., U.S. Pat. No. 6,765,087), molds (e.g., Aspergillus or Trichoderma) and yeasts (e.g., Saccharomyces, Kluyvermyces, Hansenula or Pichia) (see, e.g., U.S. Pat. No. 6,838,254). The production process is scalable and several kilogram quantities have been produced. You can Formulated as a ready-to-use solution with a long shelf life. The method (see, e.g., WO06 / 079372) is based on the automated high-throughput selection of B cells to produce antibodies against a desired target. Patented method. It is a single domain antigen-binding fragment of a camelid-specific heavy chain-only antibody. (also called VHH antibodies) usually have a small size of about 15 kDa. See C. Bever et al., Anal Bioanal Chem. 2016 Sept; 408(22); 5985–6002.

[0134] Another antibody fragment under consideration is a dual variable domain immunoglobulin (DVD-Ig), which is an engineered protein that combines the functions and specificity of two monoclonal antibodies in one molecular entity. DVD-Ig is designed as an IgG-like molecule, except that each light chain and heavy chain contain two variable domains in series connected by a short peptide, rather than one variable domain in IgG. The fusion direction of the two variable domains and the selection of the linker sequence are key to the functional activity and efficient expression of the molecule. DVD-Ig can be manufactured and purified as a single species production by conventional mammalian expression systems. DVD-Ig has the specificity of the parent antibody, is stable in vivo, and exhibits IgG-like physicochemical and pharmacokinetic properties. DVD-Ig and its preparation method are described in Wu, C., et al., Nat Biotech, 25: 1290–1297 (2007).

[0135] In certain embodiments, the antibodies or antigen-binding fragments thereof disclosed herein are humanized. This refers to a chimeric molecule that is usually prepared using recombinant technology, which has an antigen binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of a molecule based on the structure and / or sequence of a human immunoglobulin. The antigen binding site may include a complete variable domain fused to a constant region or a CDR that is only transplanted to an appropriate framework region in a 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 a human individual, but maintains the possibility of an immune response to an exogenous variable region (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). Illustrative methods for humanization of the anti-Fzd or LRP antibodies disclosed herein are described in US Pat. No. 7,462,697.

[0136] Another approach focuses not only on providing constant regions of human origin, but also on modifying variable regions so that they are transformed into a form as close to that of humans as possible. It is known that the variable regions of both heavy and light chains contain three complementary determining regions (CDRs), which vary in response to the relevant epitopes and determine the binding capacity, 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 non-human antibodies are prepared for specific epitopes, the variable regions can be "transformed" or "humanized" by transplanting CDRs derived from non-human antibodies to FRs present in the human antibodies to be modified. The following have reported the application of this method to different antibodies: 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, MS, et al., (1991) Proc Natl Acad Sci USA 88:2869-2873; Carter, P., et al., (1992) Proc Natl Acad Sci USA 89:4285-4289; and Co, MS et al., (1992) J Immunol 148:1149-1154. In some embodiments, the humanized antibody retains all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). In other embodiments, the humanized antibody has one or more CDRs (one, two, three, four, five, six) that are changed relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

[0137] In certain embodiments, the antibody of the present disclosure can be a chimeric antibody. In this respect, a chimeric antibody is composed of an antigen-binding fragment of an antibody operably connected or otherwise fused to a heterologous Fc portion of different antibodies. In certain embodiments, a heterologous Fc domain is of human origin. In other embodiments, a heterologous Fc domain can be from an Ig class different from a parent antibody, including IgA (including subclass IgA1 and IgA2), IgD, IgE, IgG (including subclass IgG1, IgG2, IgG3 and IgG4) and IgM. In a further embodiment, a heterologous Fc domain can be composed of CH2 and CH3 domains from one or more different Ig classes. As described above for a humanized antibody, the antigen-binding fragment of a chimeric antibody can only include one or more CDRs (e.g., 1, 2, 3, 4, 5 or 6 CDRs) of an antibody described herein, or can include all variable domains (VL, VH or both).

[0138] The structure and location of immunoglobulin CDRs and variable domains can be determined by reference to Kabat, EA et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services. 1987 and updates thereto, now available on the Internet (Immun.bme.nwu.edu).

[0139] 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. D ). For example, in certain embodiments, a FZD-binding agent or antibody described herein that binds to more than one FZD has a K of about 100 nM or less, about 20 nM or less, or about 10 nM or less. D Binds to those FZDs. In certain embodiments, the 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 20 or less.

[0140] Antibodies or other agents disclosed herein can be determined by any method known in the art for specific binding.Usable immunoassays include, but are not limited to, competitive and non-competitive assay systems using techniques such as biofilm interference technology (BLI) analysis, FACS analysis, immunofluorescence, immunocytochemistry, Western blotting, radioimmunoassay, ELISA, "sandwich" immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescent immunoassay, and protein A immunoassay. Such assays are conventional and well 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, which are incorporated herein by reference in their entirety).

[0141] For example, antibody specific binding to target antigen can be determined using ELISA.ELISA determination includes preparing antigen, coating the hole of 96-well microtiter plates with antigen, adding antibodies or other binding agents coupled with detectable compounds such as enzymatic substrates (e.g., horseradish peroxidase or alkaline phosphatase) into the hole, incubating for a period of time and detecting the presence of antigen. In some embodiments, antibody or agent are not coupled to detectable compounds, but the second coupled antibody identifying the first antibody or agent is added to the hole. In some embodiments, instead of coating the hole with antigen, antibody or agent can be coated to the hole, and the second antibody coupled to the detectable compound can be added after the antigen is added to the coated hole. Those skilled in the art will understand that parameters can be modified to enhance the detected signal and other changes of ELISA 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 at 11.2.1).

[0142] The binding affinity of antibody or other agent to target antigen and the dissociation rate of antibody-antigen interaction can be determined by competitive binding assay.An example of competitive binding assay is radioimmunoassay, which includes incubating labeled antigen (e.g., Fzd, LRP) or its fragment or variant with the target antibody in the presence of an increased amount of unlabeled antigen, and then detecting the antibody bound to the labeled antigen.The affinity of the antibody and the binding dissociation rate can be determined by scatter plot analysis data.In some embodiments, BLI analysis is used to determine the binding rate and dissociation rate of an antibody or agent.BLI kinetic analysis includes analyzing the binding and dissociation of an antibody with a chip having an immobilized antigen on the surface.

[0143] In certain embodiments, the WNT agonist is selected from those disclosed in PCT Publication No. WO2019 / 126398, which is incorporated herein in its entirety. G211 (also known as YW211.31.57) is disclosed in U.S. Pat. No. 8,846,041.

[0144] 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 one 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 one 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 one of the sequences shown in Table 1, and in specific 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 one of SEQ ID NOs: 1-16 or variants thereof, wherein the sequence comprises zero, less than two, less than three, less than four, less than five, less than six, less than seven or less 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 heavy chains (HC) and two of the light chains (LC) 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 one of SEQ ID NOs: 1-16 or variants thereof. In certain embodiments, the Wnt agonist has an Ig form, 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 SWAPs G211-18R5-Rspo2RA and G211-R2H1-Rspo2RA are also listed in Table 1; each of them comprises two of the heavy chains (HC) and two of the light chains (LC) disclosed in Table 1. 17SB9-03 comprises two of the heavy chains (HC) disclosed in Table 1, i.e., one HC of SEQ ID NO: 15 and one heavy chain of SEQ ID NO: 16. In various embodiments, the molecule does not comprise a tag sequence.

[0145] Table 1: Sequences of SWAPTM and Wnt superagonists or super SWAP molecules; variable chains are provided in plain text in the order indicated by the format; when present, VHH is bold (non-italic); constant chain is italic. VL is italic and underlined; VH is italic and bold. The constant region of the λ light chain is highlighted in light gray. The constant region of the κ light chain is bold underlined and highlighted in light gray. CH1 is italic. Fc is bold italic and highlighted in light gray. The Rspo region is bold highlighted (non-italic or non-underlined). The linker is in a smaller font. AA and G in bold, underlined and italic represent substitutions of wild-type LL and P amino acids with AA and G, respectively, at these positions. The tag sequence is dotted underlined.

[0146]

[0147]

[0148]

[0149]

[0150]

[0151] In certain 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 shown 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 shown in SEQ ID NO: 2. In certain 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 shown 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 shown in SEQ ID NO: 4. In certain 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 shown 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 shown in SEQ ID NO: 6. In certain 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 shown 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 shown in SEQ ID NO: 2. In certain 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 shown 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 shown in SEQ ID NO: 4. In certain 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 shown 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 shown in SEQ ID NO: 6.In certain embodiments, each sequence comprises less than one (i.e., zero), less than two, less than three, less than four, less than five, less than six, less than seven, less than eight, less than nine, less than ten, less than eleven, or less than twelve modifications, e.g., substitutions, of amino acids present in 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 one of the sequences of SEQ ID NO: 1-16.

[0152] 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 shown 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 shown in SEQ ID NO: 8. In specific embodiments, each sequence comprises less than one (i.e., zero), less than two, less than three, less than four, less than five, less than six, less than seven, less than eight, less than nine, less than ten, less than eleven, or less than twelve modifications, e.g., substitutions, of amino acids present within the CDRs of the WNT agonist. In certain embodiments, the engineered WNT agonist comprises less than one (i.e., zero), less than two, less than three, less than four, less than five, less than six, less than seven, less than eight, less than nine, less than ten, less than eleven, or less than twelve modifications of amino acids present in the CDRs of the WNT agonist, e.g., less than two, less than three, less than four, less than five, less than six, less than seven, less than eight, less than nine, less than ten, less than eleven, or less than twelve amino acid substitutions in 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 one of the sequences of SEQ ID NOs: 1-16.

[0153] In certain 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 shown 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 shown in SEQ ID NO: 10. In certain 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 shown 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 shown in SEQ ID NO: 12. In certain 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 shown 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 shown in SEQ ID NO: 14. In certain 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 shown 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 shown in SEQ ID NO: 16. In certain embodiments, each sequence comprises less than one (i.e., zero), less than two, less than three, less than four, less than five, less than six, less than seven, less than eight, less than nine, less than ten, less than eleven, or less than twelve modifications of amino acids present in the CDRs of the WNT agonist. In certain embodiments, the engineered WNT agonist comprises less than one (i.e., zero), less than two, less than three, less than four, less than five, less than six, less than seven, less than eight, less than nine, less than ten, less than eleven, or less than twelve modifications of amino acids present in the CDRs of the WNT agonist, e.g., less than two, less than three, less than four, less than five, less than six, less than seven, less than eight, less than nine, less than ten, less than eleven, or less than twelve amino acid substitutions in 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 one of the sequences of SEQ ID NOs: 1-16.

[0154] III. Pharmaceutical Compositions

[0155] Also disclosed are pharmaceutical compositions comprising a WNT antagonist molecule or a 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 collectively to WNT agonist molecules and WNT agonist molecules.

[0156] In further embodiments, a pharmaceutical composition comprising a polynucleotide and one or more pharmaceutically acceptable diluents, carriers or excipients is also disclosed, wherein the polynucleotide comprises a nucleic acid sequence encoding a WNT antagonist / agonist molecule as described herein. In certain embodiments, the polynucleotide is a DNA or mRNA, for example, a modified mRNA. In a specific embodiment, the polynucleotide is a modified mRNA further comprising a 5' cap sequence and / or a 3' tail sequence (e.g., a polyA tail). In other embodiments, the polynucleotide is an expression cassette comprising a promoter operably connected to a coding sequence.

[0157] In some embodiments, WNT antagonists / agonists are engineered recombinant polypeptides comprising various epitope binding fragments that bind to various molecules in the WNT signaling pathway. For example, a WNT antagonist can be an antibody or fragment thereof that binds to a FZD receptor and / or a LRP receptor and inhibits WNT signaling. FZD and LRP antibody fragments (e.g., Fab, scFv, VHH / sdAb, etc.) can be directly linked together on one molecule or linked together with linkers of various sizes.

[0158] In contrast, engineered WNT agonists / antagonists can also be recombinant polypeptides comprising 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 a Fzd receptor and / or a LRP receptor and enhances WNT signaling. Fzd and LRP antibody fragments (e.g., Fab, scFv, VHH / sdAb, etc.) can be directly linked together on a molecule or linked together with linkers of various sizes.

[0159] In a further embodiment, a pharmaceutical composition comprising an expression vector (e.g., a viral vector) and one or more pharmaceutically acceptable diluents, carriers or excipients is also disclosed, wherein the expression vector comprises a polynucleotide comprising a nucleic acid sequence encoding a WNT antagonist / agonist molecule described herein. 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).

[0160] The present disclosure further contemplates a pharmaceutical composition comprising a cell and one or more pharmaceutically acceptable diluents, carriers or excipients, wherein the cell comprises an expression vector, wherein the expression vector comprises a polynucleotide, wherein the polynucleotide comprises a promoter operably connected to a nucleic acid encoding a WNT antagonist / agonist molecule. In a specific embodiment, the pharmaceutical composition further comprises a cell, wherein the cell comprises an expression vector, wherein the expression vector comprises a polynucleotide, wherein the polynucleotide comprises a promoter operably connected 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., expression cassette) and / or the same cell. In a specific embodiment, the cell is a heterologous cell, or an autologous cell obtained from an object to be treated.

[0161] In certain embodiments, the cell is a stem cell, for example, a stem cell of adipose origin or a hematopoietic stem cell. The present disclosure contemplates a pharmaceutical composition comprising a first molecule for delivering a WNT antagonist molecule as a first active agent and a WNT agonist as a second molecule. The first molecule and the second molecule can be molecules of the same type or molecules of different types. For example, in certain embodiments, the first molecule and the second molecule can be molecules independently selected from the following types: polypeptides, small organic molecules, nucleic acids encoding the first active agent or the second active agent (optionally DNA or mRNA, optionally modified RNA), vectors (optionally expression vectors or viral vectors) encoding nucleic acid sequences of the first active agent or the second active agent, and cells comprising nucleic acid sequences (optionally expression cassettes) encoding the first active agent or the second active agent.

[0162] The molecules can be combined, alone or in combination, with generally safe, nontoxic and desirable pharmaceutically acceptable carriers, diluents, excipients and agents useful in preparing formulations, including excipients suitable for use in mammals (e.g., humans or primates). Such excipients can be solid, liquid, semisolid, or in the case of aerosol compositions, can be gases. 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. Solutions or suspensions for formulations may include sterile diluents, such as water for injection, saline solutions, fixed oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; antibacterial compounds, such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating compounds, 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 osmotic pressure, such as sodium chloride or dextrose. pH may be adjusted with an acid or base (e.g., hydrochloric acid or sodium hydroxide). In a particular embodiment, the pharmaceutical composition is sterile.

[0163] The pharmaceutical composition may further include a sterile aqueous solution or a dispersant and a sterile powder for the temporary preparation of a sterile injectable solution or a dispersant. For intravenous administration, suitable carriers include physiological saline, antibacterial water or phosphate buffered saline (PBS). In some cases, the composition is sterile and should be a fluid so that it can be sucked into a syringe or delivered to an object from a syringe. In certain embodiments, it is stable under manufacturing and storage conditions, and prevents the contamination of microorganisms such as bacteria and fungi. The carrier may be, for example, a solvent or a dispersion medium containing such as water, ethanol, a polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and a suitable mixture thereof. For example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersant, and by using a surfactant, suitable fluidity can be maintained. Prevention of microbial effects can be achieved by various antibacterial and antifungal agents (for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it is preferred to include an isotonic agent in the composition, for example, sugar, a polyol (for example mannitol, sorbitol), sodium chloride. Prolonged absorption of the internal ingredients can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0164] Sterile solutions can be prepared by: the desired amount of WNT antagonist / agonist antibody or its antigen binding fragment (or encoding polynucleotide or comprising polynucleotide) and a combination of ingredients listed above or ingredients as required in an appropriate solvent, and then filtered sterilization. Typically, a dispersant is prepared by incorporating the active compound into a sterile vehicle, the sterile vehicle comprising a basic dispersion medium and other required ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method is vacuum drying and freeze drying, producing a powder of the active ingredient plus any additional required ingredients from a previously sterile filtered solution.

[0165] In one embodiment, the pharmaceutical composition is prepared with a carrier that will protect the antibody or its antigen-binding fragment from rapid elimination from the body, such as a controlled release formulation, including implants and microcapsule delivery systems. Biodegradable biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such preparations will be apparent to those skilled in the art. These materials can also be 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.

[0166] For ease of administration and uniformity of dosage, it may be advantageous to formulate the pharmaceutical composition in dosage unit form. As used herein, dosage unit form refers to physically discrete units suitable as single dosages for the subject to be treated; each unit contains a predetermined amount of active antibody or antigen-binding fragment thereof calculated to produce the desired therapeutic effect associated with the required pharmaceutical carrier. The specifications for the dosage unit form are determined by and directly dependent on the unique characteristics of the antibody or antigen-binding fragment thereof and the specific therapeutic effect to be achieved, as well as the limitations inherent in the art of compounding such active antibody or antibody-binding fragment thereof for treating individuals.

[0167] The pharmaceutical compositions may be included in a container, pack, or dispenser (eg, a syringe or eye dropper, eg, a prefilled syringe or eye dropper) together with instructions for administration.

[0168] The pharmaceutical compositions of the present disclosure may be delivered to a subject in the form of a pill, capsule, cream, ointment, syrup, skin patch, suppository, intravenous drip, aqueous solution, non-aqueous solution, eye wash, or any combination thereof.

[0169] The pharmaceutical compositions of the present disclosure may be delivered to a subject by direct ophthalmic application, intramuscular injection, intravenous injection, peritoneal injection, nasally, orally, rectally, or any combination thereof.

[0170] The pharmaceutical compositions of the present disclosure include any pharmaceutically acceptable salt, ester or salt of such ester, or any other compound that is capable of providing (directly or indirectly) a biologically active antibody or antigen-binding fragment thereof when administered to an animal, including a human.

[0171] 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: a salt that retains the desired biological activity of the parent compound and does not produce undesirable toxicological effects thereon. Various pharmaceutically acceptable salts are known in the art and are described in, for example, "Remington's Pharmaceutical Sciences", 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and updated versions thereof), "Encyclopedia of Pharmaceutical Technology", 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and J. Pharm. Sci. 66: 2 (1977). Furthermore, for a review of suitable salts, see Stahland Wermuth (Wiley-VCH, 2002) “Handbook of Pharmaceutical Salts: Properties, Selection, and Use”. Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines.

[0172] 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). Base addition salts of the acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in a conventional manner. The free acid form can be regenerated by contacting the salt form with an acid and separating the free acid in a conventional manner. The free acid forms differ from their respective salt forms in certain physical properties, such as solubility in polar solvents, but for the purposes of this disclosure, these salts are otherwise equivalent to their respective free acids.

[0173] In some embodiments, the pharmaceutical compositions provided herein include a therapeutically effective amount of a WNT antagonist / agonist molecule or a pharmaceutically acceptable salt thereof mixed 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 are octylphenoxy polyethoxyethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitol 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, Hank's solution, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinyl pyrrolidone, polyethylene and glycol. Preferably, the formulation is stable at 4°C for at least 6 months.

[0174] 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 of ordinary skill, such as those described by Good et al. (1966) Biochemistry 5: 467. The pH of the buffer may be 6.5 to 7.75, preferably 7 to 7.5, and most preferably 7.2 to 7.4.

[0175] IV. How to use

[0176] The present disclosure also provides methods of using WNT antagonist / agonist molecules (e.g., modulating WNT signaling pathways to, for example, increase or decrease WNT signaling), and methods of administering WNT antagonist / agonist molecules in various therapeutic settings. Provided herein are methods of treatment using WNT antagonist / agonist molecules. In one embodiment, a WNT antagonist / agonist molecule is provided to a subject suffering from a disease involving inappropriate or dysregulated WNT signaling.

[0177] In certain embodiments, WNT antagonist / agonist molecules can be used to block or enhance WNT signaling pathways in tissues or cells. Antagonizing WNT signaling pathways can include reducing or inhibiting WNT signaling in cells or tissues. Exciting WNT signaling pathways can include, for example, increasing or enhancing WNT signaling in tissues or cells. Therefore, in some aspects, the present disclosure provides a method for antagonizing / exciting a WNT signaling pathway in a cell, 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, contact occurs in vitro, ex vivo, or in vivo. In specific embodiments, the cell is a cultured cell, and contact occurs in vitro.

[0178] WNT antagonist / agonist molecules can be used to treat corneal diseases. In particular, experiments have shown that activation of WNT signaling regulates corneal cell growth and stratification patterns (Zhang et al., Development, 2015 142:3383–93). Conditional knockout of β-catenin or Lrp5 / 6 leads to mis-stratification, especially epithelial mis-stratification. Recent in vitro experiments have shown that WNT agonists promote cell growth in homogenous corneal cell cultures. See, for example, FIG. 2A to FIG. 2E and FIG. 7A to FIG. 7B Therefore, controlled administration of WNT agonists and / or antagonists is proposed to promote epithelial and endothelial cell growth and reverse the pathology of various corneal lesions (including dystrophy and scarring). In certain embodiments, WNT agonists / antagonists are administered to a subject at an early or late stage of corneal lesion disease progression.

[0179] Both WNT agonists and antagonists can be administered separately as monotherapy or sequentially.In certain embodiments, the methods comprise administering a WNT agonist at an early stage of disease progression followed by administration of a WNT antagonist at a later stage of disease where significant scarring or fibrosis is present.

[0180] The present disclosure also provides combined treatments with known corneal disease treatments. Examples of therapies that can be administered with WNT agonists or antagonists include, but are not limited to, Rho kinase inhibitors (e.g., Ripasudil or Fasudil), anti-inflammatory drugs (e.g., steroids and non-steroids), and immunosuppressants.

[0181] Corneal lesions that may be treated include, but are not limited to, bacterial infections, viral infections (e.g., herpes simplex keratitis and herpes zoster ophthalmicus (i.e., shingles)), fungal infections, protozoan infections, archaean infections, genetic diseases, brittle cornea syndrome (BCS), corneal endothelial-mesenchymal transition (EnMT), corneal epithelial-mesenchymal transition (EMT), corneal fibrosis, Cochrane syndrome, corneal ulcers, epithelial basement membrane dystrophy (EBMD, ABMD, MapDot), Fleck corneal dystrophy, Fuchs dystrophy (also known as Fuchs endothelial corneal dystrophy (FCED) or Fuchs endothelial dystrophy (FED) or Fuchs endothelial cell dystrophy (FECD)), jelly drop corneal dystrophy, I 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, Lisch corneal dystrophy, macular corneal dystrophy, Meesmann corneal dystrophy, peripheral ulcerative keratitis, herpetic keratitis, posterior polymorphic corneal dystrophy, pterygium, Reis-Bückler corneal dystrophy, Schnyder crystalline corneal dystrophy, Stevens-Johnson syndrome, superficial punctate keratitis, Thiel-Behnke corneal dystrophy, neurotrophic keratitis, herpetic keratitis, and scarring.

[0182] In another embodiment, the WNT antagonist and / or WNT agonist molecule may further comprise a tissue targeting moiety, for example, an antibody or fragment thereof that recognizes a corneal tissue or cell type specific receptor or cell surface molecule.

[0183] Dosage and dosing regimen can depend on various factors that can be easily determined by the doctor, such as the nature of the disease or condition, the characteristics of the subject, and the medical history of the subject. In a specific embodiment, the amount of the Wnt signaling molecule administered or provided to the subject is within the range of 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 a specific embodiment, the amount of the engineered WNT signaling regulator administered or provided to the subject is within the range of 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 method disclosed herein, the WNT signaling regulator is administered intravenously (e.g., bolus injection) or subcutaneously to the subject, e.g., a mammal. In a specific embodiment, the WNT signaling regulator is administered at least once a week. In certain embodiments, the subject is administered about 0.5 mg / kg to about 100 mg / kg of WNT signaling modulator, or about 2 mg / kg to about 50 mg / kg of WNT agonist, for example, 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, the subject is administered about 25 mg, about 75 mg, about 250 mg, about 750 mg, about 1500 mg, or about 2250 mg of WNT signaling modulator. In certain embodiments, about 3 mg / kg to about 30 mg / kg of body weight is administered intravenously or subcutaneously to the subject at least once a week.

[0184] Therapeutic agents (e.g., WNT antagonists / agonists) can be administered before, during, or after the onset of a disease or injury. Treatment of an ongoing disease is of particular interest, wherein the treatment stabilizes or reduces the patient's undesirable clinical symptoms. Such treatment is preferably performed before the affected tissue completely loses function. The therapy is expected to be administered during the symptomatic stage of the disease, and in some cases, after the symptomatic stage of the disease. In some embodiments, the method produces a therapeutic benefit, for example, preventing the development of a disease, stopping the progression of a disease, reversing the progression of a disease, etc. In some embodiments, the method includes a step of detecting that a therapeutic benefit has been achieved. One of ordinary skill will recognize that measurements of such therapeutic efficacy will be modified for specific diseases, and will recognize appropriate detection methods for measuring therapeutic efficacy.

[0185] All of the above-mentioned U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to in this specification and / or listed in the Application Data Sheet are incorporated herein by reference in their entirety.

[0186] From the foregoing, it can be understood that although specific embodiments of the present disclosure have been described herein for illustrative purposes, various modifications may be made without departing from the spirit and scope of the present disclosure. Therefore, the present disclosure is not to be limited except by the appended claims.

[0187] The broad scope of the present disclosure is best understood by reference to the following examples, which are not intended to limit the disclosure to specific embodiments.

[0188] Example

[0189] Example I. General Methods

[0190] Describes standard methods in molecular biology. 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 also appear 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), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0191] Protein purification methods are described, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization. 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 glycosylation of proteins are described. See, e.g., 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) Bio Directory, 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 for characterizing ligand / receptor interactions are available. See, e.g., Coligan et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York.

[0192] Includes fluorescence activated cell sorting detection system Methods for flow cytometry are available. See, for example, 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 (e.g., for use as diagnostic reagents). Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.

[0193] Standard methods for immunohistochemistry 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.

[0194] Software packages and databases are available for determining, for example, antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments. See, for example, GenBank, Vector Suite (Informax, Inc., Bethesda, Md.); GCG Wisconsin Package (Accelrys, Inc., San Diego, Calif.); (TimeLogic Corp., Crystal Bay, Nev.); Menne et al. (2000) Bioinformatics 16:741-742; Menne et al. (2000) Bioinformatics Applications Note 16: 741-742; Wren et al. (2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne (1983) Eur. J. Biochem. 133: 17-21; von Heijne (1986) Nucleic Acids Res. 14: 4683-4690.

[0195] Example II. Construction of WNT mimetics

[0196] Wnt mimetics R2M3-26, G211-18R5, 1RC07-26, 1SH1-03 and hp1SH1-03 were constructed as described in WO 2019 / 126398A1, WO 2020 / 010308A1 and WO 2020132365 A1, WO 2021173726, which are incorporated herein by reference in their entirety. All molecules are shown in Table 1. Unless otherwise stated, all recombinant proteins were produced by transient transfection in Expi293F TM Cells (Thermo Fisher Scientific) were produced. All IgG-based and Fc-containing constructs were first purified with protein-A resin and eluted with 0.1 M glycine at pH 3.5. All proteins were then refined in 2x HBS buffer (40 mM HEPES, 300 mM NaCl at pH 7.4) by size exclusion column.

[0197] Example III. WNT agonist treatment in primary human corneal endothelial cell cultures

[0198] Human corneal endothelial cells with attached Desmedes membrane were received from Eversight (Ann Arbor, MI). Multiple donor samples were pooled and digested in MEM-α+15% FBS+20 μg / mL gentamicin sulfate containing 2 mg / mL collagenase A at 37°C for 3 hours with occasional grinding. The samples were then washed with MEM-α+15% FBS+20 μg / mL gentamicin and then resuspended and plated in human endothelial SFM+5% FBS+2ng / mL EGF+20 μg / mL gentamicin sulfate.

[0199] Endothelial cells were treated with test proteins (5nM R2M3-26, 1μg / mL hR-spondin-1, 5ng / mL FGF1, 5nM G211-18R5-Rspo2RA, 5nM G211-R2H1-Rspo2RA, or 5nM 1RC07-26) or kept untreated in human endothelial SFM + 5% FBS + 2ng / mL EGF + 20μg / 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, e.g., the discussion in Aung TN et al., Mod Pathol (2021), https: / / doi.org / 10.1038 / s41379-021-00745-6. Samples were imaged on a Leica Dmi8 microscope. A custom Python script was used to quantify the percentage of Ki67+ cells in the resulting images (Ki67+ cell # / total cell #). The results are depicted in Figure 2A , Figure 2B , Figure 2C , Figure 2D , Figure 2E middle.

[0200] Example IV. WNT agonist treatment in human corneal organ cultures

[0201] Human donor corneas with Fuchs' dystrophy were obtained from the Eversight Eye Bank. Tissues were sliced ​​and cultured in human endothelial SFM + 5% FBS + antimycotic / antibiotic for 3 days, either untreated or treated with a Wnt agonist (5nM G211-18R5 + 1μg / mL R-spondin-1). 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, :100). The results are depicted in Figure 2D middle.

[0202] Example V. WNT agonist treatment in primary human corneal epithelial cell cultures

[0203] American Type Culture Collection Primary human corneal epithelial cells (PCS-700-010 TM, batch #80915170) (cell product information sheets available upon application at https: / / www.atcc.org / products / all / PCS-700-010.aspx, incorporated herein by reference in its entirety). Recommended Culture Conditions (available at the time of application at https: / / www.atcc.org / products / all / PCS-700-010.aspx#cultureconditions, incorporated herein by reference in its entirety.)

[0204] Cells were treated with WNT agonist (1 nM of R2M3-26) or left as untreated negative control for 2 days. Next, cells were fixed and immunofluorescence was performed for Ki67 cell proliferation marker (Abcam #ab16667, 1:100) before nuclear staining with DAPI. Samples were imaged on a Leica Dmi8 microscope. A custom Python script was used to quantify #Ki67 per field of view in the resulting images. + cell. Fig. 11B .

[0205] Cells were treated with Wnt agonists (5 nM G211-18R5 or 1 μg / mL R-spondin-1) or left untreated for 2 days. Afterwards, cells were assayed using the ELISA kit according to the manufacturer's instructions (available at https: / / www.promega.com / products / cell-health-assays / cell-viability-and-cytotoxicity-assays / celltiter glo-luminescent-cell-viability-assay / ?catNum=G7570 at the time of filing, incorporated herein by reference in its entirety). Cell number was measured by viability assay (Promega). The results are depicted in Fig.11A and Fig. 11C middle.

[0206] The wound healing assay kit (Abcam, ab242285) was used according to the manufacturer's instructions. Cells were either left untreated or treated with a Wnt agonist (1 nM R2M3-26). The results are depicted in Fig. 12A and Fig. 12B middle.

[0207] Example VI. Wnt agonist treatment in rabbit limbal epithelial organoid cultures

[0208] Rabbit eyes were received from Innovative Research. Limbal regions were excised and epithelial cells were dissociated by incubation in organoid basal medium (Advanced DMEM / F12+1x N-2+1x B-27+2mM GlutaMAX+10mM HEPES+1mM N-acetylcysteine+1x penicillin-streptomycin+1x normocin) containing 2.5U / mL dispase II. Epithelial cells were embedded in matrigel and incubated with organoid basal medium containing WNT agonist (5nM R2M3-26) or 1μg / mL R-spondin-1.

[0209] RNA was collected from each well using RNeasy kits including the RNeasy Plus Mini kit (Qiagen) and analyzed using SuperScript TM IV VILO TM cDNA was synthesized using the cDNA Synthesis Kit (ThermoFisher) (product information sheet available upon submission to 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, incorporated herein by reference in its entirety). TM Gene expression assay reagent (ThermoFisher) was used in CFX96 TM qPCR of the Wnt target gene Axin2 and the progenitor cell marker p63 was performed on a thermal cycler (BioRad). β-actin was used for normalization. The results are depicted in Fig. 10A and Fig. 10B middle.

[0210] Example VII. Lrp5 / 6, Fzd and Znrf3 / Rnf43 RNA expression in human corneal epithelial and endothelial tissues

[0211] Whole eyes from a 47-year-old Caucasian female donor with no history of ocular disease were received from Eversight (Ann Arbor, MI). Corneas were dissected, fixed in 10% formalin, and embedded in paraffin.

[0212] Fuchs dystrophy tissue sample corneas delivered from Eversight (Ann Arbor, MI) were obtained from 73 years old (Fuchs #1) and 96 years old (Fuchs #2). Fuchs #1 was posthumously diagnosed with Fuchs dystrophy based on low endothelial cell counts and the presence of severe guttate. Fuchs #2 was diagnosed antemortem with Fuchs dystrophy. Corneas were dissected, fixed in 10% formalin, and embedded in paraffin.

[0213] Sections of paraffin-embedded tissue were prepared and analyzed using the RNAscope Multiplex Fluorescence Reagent Kit v2 (ACDBio) according to the manufacturer's instructions. Kits were used to measure Lrp5 / 6, Fzd, and Znrf3 / Rnf43 RNA levels. Corneal epithelial marker CK12 (Abcam #ab185627, 1:100), limbal progenitor cell marker p63 (Biocare #4A4, 1:100), and corneal endothelial marker Na + K + Immunofluorescence of ATPase (ThermoFisher #05-369-MI, 1:100) was used to confirm tissue identity. The stained sections were mounted in mounting medium with DAPI and imaged on a Leica DM18A epifluorescence microscope. The results are depicted in Figures 1 and 9.

[0214] Example VIII. WNT Agonist Treatment in the Mouse Limbal-to-Limbal Debridement Model

[0215] Mechanical debridement model: Female C57B1 / 6 mice aged 6 to 12 weeks were anesthetized by placing the animals in an induction chamber connected to an oxygen source and an isoflurane vaporizer with an oxygen flow rate of 0.9 L / min and an isoflurane vaporizer set to 3% to 4%. After the mice became unresponsive and their breathing became shallow, they were transferred to an anesthesia platform 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 was injured per mouse, and the contralateral side was used as a control. One drop of Proparacaine was placed on the cornea for 30 seconds. Using weck-cel TMA cellulose eye spear was used to dry the cornea by gently sweeping it over the cornea once and cushioning both corners of the eye. Periocular pressure was applied to the mouse eye with one hand. The right cornea was marked with a 3 mm trephine and the corneal epithelium was scraped with an Algerbrush (Algerbrush II with 0.5 mm Burr (Katena, catalog number: K 2-4900)), taking care to avoid the limbal vessels. After scraping, the eyes were treated with erythromycin eye ointment to minimize inflammation and keep the ocular surface moist while the mice were 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, 4X / day / 7 days). On day 7, the mice were humanely euthanized and the eyeballs were completely removed from each animal, collected in Davidson's fixative followed by 10% phosphate-buffered formalin, and fixed as described above. Fig.13 The indicated treatments were subjected to histological analysis of Axin2, p63 and H&E. The data suggest that R-spondin-2 eye drops activate Wnt signaling, expand progenitor cells, thicken the corneal epithelium and reduce conjunctivalization.

[0216] Example IX. Treatment of naive mice by intracameral and intravitreal injection of WNT agonists

[0217] Female Balb / C mice aged 6 to 12 weeks were anesthetized by placing the animals in an induction chamber connected to an oxygen source and an isoflurane vaporizer with an oxygen flow rate of 0.9 liters / minute and an isoflurane vaporizer set to 3% to 4%. After the mice became unresponsive and their breathing became shallow, they were transferred to an anesthesia platform with a nose cone. The head was positioned so that the eye to be injected faced upward toward the microscope objective. Only one eye was injected per mouse, and the contralateral side was used as a control. One drop of proparacaine was placed on the cornea for 30 seconds. The cornea was punctured just in front of the iridocorneal angle. The fluid flowing out of the anterior chamber was wiped off with a weck-cell, and then 1 microliter of the wnt agonist R2M3-26 or superagonist G211-18R5-Rspo2RA or anti-GFP antibody (2.6 μg) was injected into the anterior chamber (anterior chamber injection) or posterior chamber (intravitreal injection) using a 33-gauge needle connected to a Hamilton syringe. The mice were allowed to recover from anesthesia. The next day, the mice were removed, the eyeballs were removed, and the corneas were dissected, fixed in 10% formalin, and embedded in paraffin. For half of the eyeball samples, paraffin-embedded tissue sections were prepared and analyzed using the RNAscope Multiplex Fluorescence Reagent Kit v2 (ACDBio) according to the manufacturer's instructions. For the remaining eye samples, Axin2 levels were measured using the TaqMan TM Gene expression assay reagent (ThermoFisher) was used in CFX96 TMRT-qPCR of the Wnt target gene Axin2 was performed on a thermal cycler (BioRad). β-actin was used for normalization. The results of this study are described in FIG. 3A to FIG. 3D middle.

[0218] Example X. Wnt agonist treatment in the rabbit endothelial ablation model

[0219] The purpose of these studies was to evaluate the recovery of endogenous corneal endothelial cells (CECs) in the central cornea after removal by surgical scraping of CECs. Wnt agonists or controls were injected intracamerally after the excision surgery. Two independent studies were performed to evaluate the ability of the WNT mimetic R2M3-26 and the WNT superagonist G211-18R5-Rspo2RA in the rabbit endothelial excision model. In the first study, two different doses (1 μM and 8.3 μM) of R2M3-26 or anti-GFP antibody (8.3 μM) were administered on day 0 after endothelial cell scraping to form a 10 mm lesion. The eyes of the rabbits were examined at different time points, including baseline and day 3 postoperatively, and corneal thickness was examined by optical coherence tomography, corneal opacity was assessed by eye examination, and lesion size was assessed by performing corneal tiling and histological staining for corneal endothelial markers. On day 3, treatment with lower doses of R2M3-26 tended to reduce corneal thickness, lesion size, and increase cell density ( Figure 3E and Figure 3F ). The second study was performed with two concentrations (1 uM and 5 uM) of the WNT superagonist G211-18R5-Rspo2RA and anti-GFP (5 uM). On day 3, a significant reduction in corneal opacity was observed in response to 5 μM wnt superagonist, but there was a trend toward reduced corneal swelling / thickness and reduced lesion size in response to wnt superagonist ( FIG. 3G to FIG. 3I ).

[0220] Example XI. UV-A burn corneal endothelial injury model (Liu et al., 2019, PNAS)

[0221] C57BL / 6 wild-type mice (7 to 15 weeks old; The Jackson Laboratory) were used for this study. The mice were anesthetized with isoflurane. The emission peak was 365 nm, with a bandwidth of 9 nm (FWHM) and 398 mW / cm 2 A UVA LED light source (M365LP1; Thorlabs) with an irradiance of 1.5 Å was focused to illuminate a 4 mm diameter spot on the mouse cornea. The energy was measured using a laser sensor (Model L49 [150A]; Ophir), and the duration of UVA exposure was adjusted to deliver the appropriate fluence (for 500 J / cm 2The right eye (OD) was irradiated, while the contralateral eye (OS) was covered with a thermal blanket (SpaceDrape, Inc.) and used as an untreated control. IVT injection of drugs was performed several hours before UV A burn. The cornea was imaged and photographed daily, and CCT was measured by OCT on the second day ( Fig. 8A and Figure 8B ).

[0222] Example XII. Cryogenic Injury Model

[0223] Mice were randomly divided into two groups, and 2 cycles of cryotherapy were applied to the right eye of each mouse (Han et al., 2013). Under isoflurane anesthesia, corneal freezing was started by gently placing a cryoprobe made of stainless steel (2.5 mm diameter; flat head; ERBE Elektromedizin GmbH, Tubingen, Germany) on the central cornea and precooled in liquid nitrogen for one minute. A cryoprobe with a diameter of 2.5 mm was selected 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 kept on the corneal surface until an ice ball was formed on the cornea and covered the entire corneal surface, which corresponded to a duration of 3 s because the defects on the endothelium appeared to be the same size as the ice ball. The injury was repeated after a 1 min interval. Immediately after freezing, the cryoprobe was released from the corneal surface by rinsing with a balanced salt solution, and the cornea was allowed to thaw naturally. Only the right eye was treated with a cryoprobe, and the left eye was used as an untreated control. The cornea usually recovered within 4 to 5 days, and corneal thickness was followed by OCT and corneal transparency by bright field imaging (Phoenix Micron IV) (Figures 4 and Figure 5A and Figure 5B ).

[0224] Example XIII. Methods for Corneal and Conjunctival Organoids

[0225] An average of three mice (six eyeballs) were used for each experiment. Fig.15AEyeballs were removed and corneas and conjunctivas were isolated under the indicated dissecting scope. Corneal epithelium was digested into single cell suspensions in 1x TrypLE Express. Conjunctivae were digested in HBSS containing 2.5mg / mL collagenase A for one hour. Nearly single cell suspensions were resuspended in Matrigel and plated in 40uL drops to solidify. Basal medium contained Advanced DMEM, 10mM HEPES, 1x pen / strep, 100mg / mL Normocin, 2mM GlutaMAX, 1x B27, 1mM N-acetylcysteine, 100ng / mL KGF, and 10uM Y-27632. Cells were expanded for 5 to 7 days before treatment with the indicated WNT mimetics for 48h. Results are shown in Fig. 15B middle.

[0226] The various embodiments described above can be combined to provide further embodiments.All U.S. patents, U.S. patent application disclosures, U.S. patent applications, foreign patents, foreign patent applications and non-patent disclosures mentioned in this specification and / or listed in the application data sheet are incorporated by reference into the entire article. If necessary, the various aspects of the embodiment can be modified to provide further embodiments using the concepts of various patents, applications and publications. These and other changes can be made to the embodiment according to the above detailed description. Generally, in the following claims, the terms used should not be interpreted as limiting the claims to the specific embodiments disclosed in the specification and claims, but should be interpreted as including the full range of equivalents given by all possible embodiments and such claims. Therefore, the claims are not limited by the present disclosure.

[0227] References:

[0228] 1)Sudi Patel,Larysa Tutchenko.The refractive index of the humancornea:A review.Cont Lens Anterior Eye 2019Oct;42(5):575-580.

[0229] 2)Zhang Y,Yeh LK,Zhang S,Call M,Yuan Y,Yasunaga M,Kao WW,Liu CY.Wnt / beta-catenin signaling modulates corneal epithelium stratification viainhibition of Bmp4 during mouse development.Development.2015;142:3383–3393.

[0230] 3)A Wodarz,R Nusse.Mechanisms of Wnt signaling in development.AnnuRev Cell Dev Biol 1998;14:59-88.

[0231] 4)Roel Nusse Wnt signaling in disease and in development CellRes.2005Jan;15(1):28-32.

[0232] 5)Eleonora Maurizi,Davide Schiroli,Roberta Zini,Anna Limongelli,Raffaela Mistò,Claudio Macaluso,Graziella Pellegrini.A fine-tunedβ-cateninregulation during proliferation of corneal endothelial cells revealed usingproteomics analysis.Sci Rep 2020 Aug 14;10(1):13841.

[0233] 6)Martin N.Nakatsu,Zhenhua Ding,Madelena Y.Ng,Thuy T.Truong,Fei Yu,and Sophie X.Deng Wnt / β-Catenin Signaling Regulates Proliferation of HumanCornea Epithelial Stem / Progenitor Cells.Invest Ophthalmol Vis Sci.2011 Jun;52(7):4734–4741.

[0234] 7)Shikha Chawla,Sourabh Ghosh.Establishment of in vitro model ofcorneal scar pathophysiology.Journal of cellular Physiology.28 June2017.https: / / doi.org / 10.1002 / jcp.26071

Claims

1. A method of treating a corneal lesion in a subject comprising administering to the subject a WNT signaling modulator.

2. The method of claim 1, wherein the WNT signaling regulator is an engineered WNT signaling regulator.

3. The method of claim 1, wherein the WNT signaling regulator is an engineered WNT agonist or an engineered WNT antagonist.

4. The method of claim 3, wherein the engineered WNT agonist is selected from: (i) a WNT mimetic; or (ii) an R-spondin mimetic; or (iii) a super SWAP / super agonist.

5. The method of claim 4, wherein the WNT mimetic is SWAP TM Compound, and wherein the R-spondin mimetic is SWEETS TM Compounds or WNT enhancer super SWAP compounds. 6 . The method of claim 1 , wherein the WNT signaling regulator is Wnt3a or any homolog, mutant, fragment, mimetic of Wnt3a, or any combination thereof.

7. The method of any one of claims 1 to 6, wherein the WNT signaling regulator is co-administered with R-spondin-1 or any R-spondin-1 homolog, mutant, fragment, mimetic, or any combination thereof.

8. The method of claim 1, wherein the WNT signaling regulator is administered to the subject at a therapeutically effective dose.

9. The method of claim 1, wherein the subject is a human patient.

10. The method of claim 1, wherein the WNT signaling modulator is administered by eye drops, intracameral injection, intravitreal injection, intrastromal or subconjunctival injection.

11. The method of claim 1, wherein the WNT signaling modulator is suspended in an aqueous solution.

12. The method of claim 11, wherein the WNT signaling regulator is suspended in an aqueous solution at a concentration of 0.1 nM to 10 mM.

13. The method of claim 11, wherein the WNT signaling regulator is suspended in an aqueous solution at a concentration of 1 nM to 100 nM.

14. The method of claim 3, wherein the engineered WNT agonist and engineered WNT antagonist comprises a binding component that binds one or more Fzd receptors and / or a binding component that binds one or more LRP receptors.

15. The method of claim 14, wherein the binding component of the engineered WNT agonist is selected from the group consisting of a Fzd binding component, a Lrp5 binding component, a LRP6 binding component, and a LRP5 / 6 binding component.

16. The method of claim 14, wherein the Fzd binding component of the engineered WNT agonist is selected from the group consisting of Fzd1, Fzd2, and Fzd7.

17. The method of claim 1, comprising administering an engineered WNT agonist and an engineered WNT antagonist, wherein the engineered WNT agonist and engineered WNT antagonist are administered independently at an early and / or advanced stage of corneal disease.

18. The method of claim 1, comprising administering an engineered WNT agonist and an engineered WNT antagonist, wherein the engineered WNT agonist and the engineered WNT antagonist are administered sequentially at an early and / or advanced stage of corneal disease.

19. The method of claim 1, comprising administering an engineered WNT agonist and an engineered WNT antagonist, wherein the engineered WNT agonist and engineered WNT antagonist are co-administered at an early and / or advanced stage of corneal disease.

20. The method of any one of claims 1 to 19, comprising administering an isolated polynucleotide encoding a polypeptide of the Wnt signaling regulator, optionally wherein the polynucleotide is mRNA, optionally a modified mRNA.

21. The method of claim 20, comprising administering an expression vector comprising the isolated polynucleotide.

22. The method according to any one of claims 1 to 21, wherein the corneal lesion is a corneal dystrophy.

23. The method according to any one of claims 1 to 21, wherein the corneal lesion is selected from the group consisting of: bacterial infection, viral infection, fungal infection, protozoan infection, archaeal infection, hereditary disease, brittle cornea syndrome (BCS), corneal endothelial-mesenchymal transition (EnMT), corneal epithelial-mesenchymal transition (EMT), corneal fibrosis, Cochrane syndrome, corneal ulcer, epithelial basement membrane dystrophy (EBMD), Fleck corneal dystrophy, Fuchs dystrophy, post-keratoplasty scar, colloid corneal dystrophy, type I granular corneal dystrophy, type II granular corneal dystrophy, stromal keratitis, iridocorneal endothelial syndrome (ICE), keratoconus sicca Conjunctivitis, keratoconus, keratomalacia, lattice dystrophy type I, lattice dystrophy type II, Lisch corneal dystrophy, macular corneal dystrophy, Meesmann corneal dystrophy, bullous keratopathy, aniridia, peripheral ulcerative keratitis, herpetic keratoconjunctivitis, posterior polymorphic corneal dystrophy, pterygium, Reis-Bückler corneal dystrophy, Schnyder crystalline corneal dystrophy, Stevens-Johnson syndrome, superficial punctate keratitis, Thiel-Behnke corneal dystrophy, neurotrophic keratitis, herpetic keratitis, scarring, limbal stem cell deficiency, and any combination thereof.

24. The method of claim 23, wherein the corneal lesion is selected from: Epithelial-mesenchymal transition; Endothelial-mesenchymal transition; Corneal fibrosis; Corneal scarring; or Any combination thereof.

25. The method of claim 23, wherein the corneal lesion is Fuchs' dystrophy or limbal stem cell deficiency.

26. The method of claim 23, wherein the corneal lesion is a post-keratoplasty scar.

27. A method for increasing the cell yield of mammalian corneal epithelial and / or endothelial cells grown ex vivo prior to or following corneal or corneal cell transplantation, comprising: Mammalian donor corneas or isolated mammalian corneal cells are treated with a WNT signaling modulator.

28. The method of claim 27, wherein the WNT signaling regulator is an engineered WNT signaling regulator.

29. The method of claim 27, wherein the WNT signaling regulator is an engineered WNT agonist or an engineered WNT antagonist.

30. The method of claim 29, wherein 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 Rspondin mimetic, which is referred to as a super SWAP or super agonist.

31. The method of claim 30, wherein the WNT mimetic is SWAP TM Compound, and wherein the R-spondin mimetic is SWEETS TM Compound.

32. The method of claim 27, wherein the WNT signaling regulator is any of: WNT3a or any homologue, mutant, fragment or mimetic of WNT3a; G211-18R5; R2M3-26; 1SH1-03; hp1SH1-03 17SB9-03; Super SWAP or super agonist; or Its combination.

33. The method of any one of claims 27 to 32, wherein the WNT signaling regulator is co-administered with R-spondin-1 or any R-spondin-1 homolog, mutant, fragment, mimetic, or any combination thereof.

34. The method of claim 27, wherein the WNT signaling regulator is administered to the subject at a therapeutically effective dose.

35. The method of claim 27, wherein the subject is a human patient.

36. The method of claim 27, wherein the WNT signaling modulator is administered via eye drops, intracameral injection, subconjunctival injection, intrastromal or intravitreal injection.

37. The method of claim 27, wherein the WNT signaling modulator is suspended in an aqueous solution.

38. A method according to claim 37, wherein the WNT signaling regulator is administered to the mammalian donor cornea by immersing the mammalian donor cornea in vitro in a water bath solution containing a certain dose of the WNT signaling regulator, or a polynucleotide encoding the polypeptide, or an expression vector comprising the isolated polynucleotide, wherein the polynucleotide is mRNA or optionally modified mRNA.

39. The method of claim 37, wherein the WNT signaling regulator is suspended in an aqueous solution at a concentration of 0.1 nM to 10 mM.

40. The method of claim 39, wherein the WNT signaling modulator is suspended in an aqueous solution at a concentration of 1 nM to 100 nM.

41. The method of claim 27, wherein the engineered WNT agonist and engineered WNT antagonist comprise a binding component that binds one or more Fzd receptors and a binding component that binds one or more LRP receptors.

42. The method of claim 41, wherein the binding component of the engineered WNT agonist is selected from the group consisting of a Fzd binding component, a Lrp5 binding component, a LRP6 binding component, and a LRP5 / 6 binding component.

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