Regulation of WNT signaling in eye disorders

Abstract

The present invention provides methods for treating ocular disorders using modulators of the WNT signaling pathway. In particular, the ocular disorder is retinopathies. Also provided are methods of administration and pharmaceutical compositions.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 803,835, filed February 11, 2019, the entire contents of which are incorporated herein by reference.

[0002] Description of sequence listing The sequence listing associated with this application is provided in text format rather than paper and is incorporated herein by reference. The name of the text file containing the sequence listing is SRZN_013_01WO_ST25.txt. The text file was created on February 10, 2020, is approximately 27KB, and has been submitted electronically via EFS-Web.

[0003] The present invention provides WNT signaling modulators for the treatment of various ocular disorders, in particular for vascular diseases of the eye, also known as retinopathies. [Background technology]

[0004] The vertebrate retina is a thin layer of nervous tissue at the fundus of the eye. It is responsible for the detection of visual stimuli and is the first station for visual information processing. For its proper function, the retinal vasculature is an essential source of nutrients and oxygen. The retina is highly metabolically active. Due to the photoreceptors consuming vast amounts of oxygen, the retina exhibits the highest oxygen consumption rate per gram than any other organ in the body. To function as an effective source of nutrients and oxygen, the retinal vasculature is located within the retina as a stereotyped structure consisting of three planar vascular plexuses on one side and choriocapillaries on the other. Internal vascularization initially begins at the vitreous surface of the retina, giving rise to the primitive vascular plexus. After radial expansion of the surface of the vascular plexus, the vertical penetration of blood vessels into the retina forms two additional intraretinal capillary plexuses at the inner plexiform layer (IPL) and the outer plexiform layer (OPL). Due to the functional and structural relationship between blood vessels and the retina, aberrant vascular development or vascular disorders are directly related to the function of the retina, which leads to various types of retinopathies and degeneration.

[0005] WNT signaling has been implicated as a key pathway in retinal vascular development. Increasing genetic evidence from human and rodent studies further supports the importance of WNT signaling in retinal vasculature (Wang et al., 2018, Prog Retin Eye Res. 2018 Dec 1. pii:S1350-9462(18)30046-6). Human mutations in genes encoding either the receptor (Fzd4, Lrp5, Tspan12) or ligand (Norlin) involved in WNT signaling result in various hereditary vitreoretinopathy. Individual genetic mutant mice of genes (Fzd4, Lrp5, Tspan12, Norlin) also show typical phenotypes of aberrant vasculature seen in human retinopathies. This not only allowed us to better understand the disease progression of retinopathy but also opened the possibility of treating retinopathy via WNT signaling modulation.

[0006] Retinopathy, especially diabetic retinopathy, can be divided into early and late stages. In the early stage, also known as non-proliferative retinopathy, there may be a slight deterioration of the retinal microvasculature, some of which may expand and leak fluid into the surrounding retinal tissue. Late stage retinopathy involves significant neovascularization in the retinal region as well as microaneurysms and hemorrhages (see, for example, Grading Diabetic Retinopathy from Stereoscopic Color Fundus Photographs-An Extension of the Modified Airlie House Classification. (1991) Ophthalmology, 98(5), 786-806).

[0007] Familial exudative vitreoretinopathy (FEVR) is an inherited eye disease characterized by poor formation of intraocular vasculature. More than 50% of FEVR patients exhibit mutations in one of the genes encoding Fzd4, Lrp5, Tspan12, or norlin. Norlin, a WNT signaling ligand, transmits signals to endothelial cells via a receptor complex composed of Fzd4 / Lrp5 / Tspan12 for normal retinal angiogenesis in the eye. However, in FEVR patients, mutations in one of the genes encoding norlin, Fzd4, Lrp5, or / and Tspan12 cause immature vascular development in the retina. The resulting formation of avascular areas creates retinal ischemic areas, which are the primary injury to the retina. The ischemic condition induces the production of vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2), leading to neovascularization and vascular tuft formation. The newly generated abnormally formed blood vessels can be easily destroyed, leading to secondary retinal damage by exudation and hemorrhage. The disease progression of diabetic retinopathy (DR) is also similar to that of FEVR or other inherited vascular malformations or deficiency diseases. Hyperglycemia induces retinal vascular damage, leading to vascular occlusion, ischemia, neovascularization, and hemorrhage, ultimately resulting in retinopathy. Although genetic data suggest the importance of WNT signaling in establishing proper vasculature in the eye, it is unclear whether activation of WNT signaling after development leads to improved vasculature. Some reports even suggest that antagonizing rather than agonizing WNT signaling is beneficial for retinopathy. Therefore, understanding the involvement of WNT signaling in the disease progression of retinopathy can lead to new therapeutic possibilities. There is a need to control WNT agonist and antagonist signaling depending on the disease stage for proper treatment of retinopathy. The present invention provides a method for controlling the agonism and antagonism of WNT signaling at different stages of retinopathy disease progression. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Wang et al.,2018,Prog Retin Eye Res.2018 Dec 1.pii:S1350-9462(18)30046-6 [Non-Patent Document 2] Grading Diabetic Retinopathy from Stereoscopic Color Fundus Photographs-An Extension of the Modified Airlie House Classification.(1991) Ophthalmology,98(5),786-806 Summary of the Invention [Means for solving the problem]

[0009] The present invention is based, in part, on the use of agonists and antagonists of WNT signaling to regulate aberrant blood vessel formation in retinopathy indications.

[0010] The present invention provides a method for treating a subject suffering from retinopathy, comprising administering to the subject a modified WNT signal regulator. In certain embodiments, the WNT signal regulator is a modified WNT agonist or a modified WNT antagonist. In further embodiments, the modified WNT agonist and the WNT antagonist comprise a binding composition that binds to one or more Fzd receptors and a binding composition that binds to one or more LRP receptors or Tspan12 receptors. In further embodiments, the modified WNT agonist binding composition is selected from the group consisting of a Fzd4 binding composition, an Lrp5 binding composition, an Lrp6 binding composition, an LRP5 / 6 binding composition, and a Tspan12 binding composition.

[0011] In some embodiments, the modified WNT agonist or WNT antagonist is administered independently at early and / or late retinopathy. In alternative embodiments, the WNT agonist and the WNT antagonist are administered sequentially at early and / or late retinopathy, or the WNT agonist and the WNT antagonist are administered simultaneously at early and / or late retinopathy. In further embodiments, the WNT agonist is administered before or after the WNT antagonist.

[0012] In some embodiments, the WNT agonist and / or WNT antagonist is administered with a binding composition specific for either VEGF and / or Ang2. In certain embodiments, the binding composition specific for VEGF or Ang2 is an antagonist of VEGF or Ang2 activity. In further embodiments, the VEGF antagonist is selected from the group consisting of bevacizumab, ranibizumab, aflibercept, ramucirumab, and tanibirumab. In other embodiments, the Ang2 antagonist is selected from the group consisting of nesbacumab, AMG780, and MEDI3617.

[0013] In certain embodiments, the retinopathy is a retinal vascular disease. In some embodiments, the retinal vascular disease is caused by inhibition of vascular development. In alternative embodiments, the retinal vascular disease is caused by excessive angiogenesis. In certain embodiments, the retinal vascular disease is selected from the group consisting of familial exudative vitreoretinopathy (FEVR), exudative vitreoretinopathy, Norrie's disease, diabetic retinopathy (DR), age-related macular degeneration (AMD), retinopathy of prematurity (ROP), osteoporosis-pseudoglioma syndrome (OPPG), retinal vein occlusion, and Coats' disease. [Brief description of the drawings]

[0014] [Figure 1] Figures 1A and 1B provide a description of the WNT surrogate molecule used: Figure 1A shows a diagrammatic representation of the WNT surrogate molecule, and Figure 1B provides the clone name and sequence identifier for each component of the WNT surrogate molecule. [Figure 2-1]Figures 2A-H are graphs showing WNT signaling activity in cells treated with the indicated WNT surrogates and RSPO, as measured by SuperTop Flash (STF) assay. Figures 2A-D show little to no WNT signaling activity in untransfected HEK293 cells treated with various mono-FZD4 WNT surrogates and 20 nM RSPO. In contrast, Figures 2E-H show HEK293 cells transfected with the human FZD4 gene that have WNT signaling activity when treated with various FZD4 WNT surrogates and 20 nM RSPO. [Figure 2-2] Same as above. [Diagram 3] FIG. 3 shows semi-quantitative PCR analysis of FZD4-overexpressing HEK293 cells. [Figure 4-1] Figures 4A-4P show WNT signaling activity (Figures 4A-4D) and Axin2 expression (Figures 4E-4H) in bEnd.3 cells (a mouse brain endothelial cell line used in vascular studies) containing a luciferase gene controlled by a WNT-responsive promoter, or WNT signaling activity (Figures 4I-4L) and Axin2 expression (Figures 4M-4P) in HRMECs (primary human retinal microvascular endothelial cells) containing a luciferase gene controlled by a WNT-responsive promoter. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 4-4] Same as above. [Diagram 5] 5A-5B show semi-quantitative PCR analysis of various WNT receptor gene expression in bEnd.3 cells (FIG. 5A) and HRMECs (FIG. 5B). [Figure 6] Figures 6A-6F show the effect of treatment with various FZD4 WNT surrogates on WNT signaling activity in bEnd.3 cells (Figures 6A-6C) or HRMEC cells (Figures 6D-6F), with or without RSPO addition. [Figure 7A]Figures 7A-7B show the experimental design using various FZD4 WNT surrogates in a rat model of oxygen-induced retinopathy. Figure 7A shows the timeline of the oxygen-induced retinopathy model, and Figure 7B provides details of the study groups. [Figure 7B] Same as above. [Figure 8A] Figures 8A-B show retinal vascular growth and pathological preretinal neovascularization after treatment with either anti-VEGF or FZD4 WNT surrogate molecules. Figure 8A shows fluorescent staining of rat retinal flat mounts. Figure 8B shows quantitative analysis by computer-assisted image analysis of vascular growth and neovascularization. [Figure 8B] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] As used in this specification, including the appended claims, the singular terms "a," "an," and "the" include references to the corresponding plural forms unless the context clearly dictates otherwise.

[0016] All references cited in this specification 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.

[0017] I. Definition The "activity" of a molecule may describe or refer to the binding of the molecule to a ligand or receptor, to catalytic activity, to the ability to stimulate gene expression, to antigenic activity, to the modulation of the activity of other molecules, and the like. The "activity" of a molecule may also refer to activity that modulates or maintains cell-cell interactions, e.g., adhesion, or to activity that maintains the structure of a cell, e.g., a cell membrane or cytoskeleton. "Activity" may also refer to specific activity, e.g., [catalytic activity] / [mg protein], or [immune activity] / [mg protein], and the like.

[0018] As used herein, the terms "administering" or "introducing" or "providing" refer to the delivery of a composition to a cell(s), tissue, and / or organ of a subject or to a subject. Such administration or introduction can occur in vivo, in vitro, or ex vivo.

[0019] As used herein, the term "antibody" refers to an isolated or recombinant binding agent that contains the necessary variable region sequence to specifically bind to an antigen epitope. Thus, an antibody is any form of antibody or fragment thereof that exhibits the desired biological activity, such as binding to a specific target antigen. It is therefore used in the broadest sense, specifically covering monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, nanobodies, diabodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, including but not limited to scFv, Fab, and Fab2, so long as they exhibit the desired biological activity.

[0020] An "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding 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 an antibody produces two identical antigen-binding fragments, i.e., "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a designation that reflects the ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0021] The term "antigen" refers to a molecule or portion of a molecule capable of being bound by a selective binding agent, such as an antibody,30 and can additionally be used in an animal to generate antibodies capable of binding to an epitope of that antigen. In certain embodiments, a binding agent (e.g., a WNT surrogate molecule or binding region thereof, or a WNT antagonist) is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0022] As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment containing at least one CDR of an immunoglobulin heavy and / or light chain, or a VHH / sdAb (single domain antibody) or Nanobody® (Nab), that binds to an antigen of interest, particularly one or more Fzd receptors, or LRP5 and / or LRP6. In this regard, an antigen-binding fragment of an antibody described herein can contain one, two, three, four, five, or all six CDRs of the VH and VL from an antibody that binds to one or more Fzd receptors, or LRP5 and / or LRP6.

[0023] As used herein, the terms "biological activity" and "biologically active" refer to the activity attributed to a particular biological element in a cell. For example, the "biological activity" of a WNT agonist or its fragment or variant refers to its ability to mimic or enhance WNT signal. As another example, the biological activity of a polypeptide or its functional fragment or variant refers to the ability of the polypeptide or its functional fragment or variant to achieve its original function, such as binding, enzymatic activity, etc. As a third example, the biological activity of a gene regulatory element, such as a promoter, enhancer, Kozak sequence, etc., refers to the ability of the regulatory element or its functional fragment or variant to regulate, i.e., promote, enhance, or activate, respectively, the translation of the expression of the gene to which it is operably linked.

[0024] As used herein, the term "bifunctional antibody" refers to an antibody that contains a first arm that has specificity for one antigenic site and a second arm that has specificity for a different antigenic site, i.e., a bifunctional antibody has dual specificity.

[0025] As used herein, "bispecific antibodies" refers to full-length antibodies generated by quadroma technology (see Milstein et al., Nature, 305(5934):537-540 (1983)), by chemical conjugation of two different monoclonal antibodies (see Staerz et al., Nature, 314(6012):628-631 (1985)), or by knob-into-hole or similar approaches to introduce mutations into the Fc region (see Holliger et al., Proc. Natl. Acad. Sci. USA, 90(14):6444-6448 (1993)), resulting in a number of different immunoglobulin species, only one of which is a functional bispecific antibody. A bispecific antibody binds one antigen (or epitope) with one of its two binding arms (a pair of HC / LC) and a different antigen (or epitope) with its second arm (a different pair of HC / LC). By this definition, a bispecific antibody has two distinct antigen-binding arms (both specificity and CDR sequences) and is monovalent for each antigen to which it binds.

[0026] "Comprising" means that the recited elements are required, e.g., in a composition, method, kit, etc., but other elements may be included, e.g., to form a composition, method, kit, etc. within the scope of the claim. For example, an expression cassette "comprising" a gene encoding a therapeutic polypeptide operably linked to a promoter is an expression cassette that may include other elements in addition to the gene and promoter, e.g., polyadenylation sequences, enhancer elements, other genes, linker domains, etc.

[0027] "Consisting essentially of" refers to a limitation in scope of a composition, method, kit, etc. described in the specified materials or steps that does not substantially affect the basic and novel properties of the composition, method, kit, etc. For example, an expression cassette "consisting essentially of" a gene encoding a therapeutic polypeptide operably linked to a promoter and a polyadenylation sequence may include additional sequences, such as linker sequences, so long as they do not substantially affect the transcription or translation of the gene. As another example, a variant or mutant polypeptide fragment "consisting essentially of" a recited sequence has an amino acid sequence that is plus or minus about 10 amino acid residues at the boundaries of the recited sequence based on the full-length non-sensitized polypeptide from which it is derived, such as 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 fewer residues than the recited boundary amino acid residues, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 more residues than the recited boundary amino acid residues.

[0028] "Consisting of" refers to the exclusion from a composition, method, or kit of any element, step, or ingredient not specified in the claim. For example, a polypeptide or polypeptide domain "consisting of" a recited sequence contains only the recited sequence.

[0029] A "control element" or "control sequence" is a nucleotide sequence involved in molecular interactions that contribute to the functional regulation of a polynucleotide, including the replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. Regulation may affect the frequency, rate, or specificity of a process, and may in effect enhance or repress. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region that has the ability, under certain conditions, to bind RNA polymerase and initiate transcription of a coding region that is usually located downstream (3' direction) of the promoter.

[0030] An "expression vector" is a vector, such as a plasmid, minicircle, viral vector, liposome, etc., described herein or known in the art, that contains a region encoding a gene product of interest and is used to affect expression of the gene product in an intended target cell. An expression vector also contains control elements, such as promoters, enhancers, 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 operably linked gene(s) for expression is sometimes referred to as an "expression cassette," many of which are known and available in the art or can be readily constructed from components available in the art.

[0031] As used herein, the term "FR set" refers to four adjacent amino acid sequences that frame the CDRs of a CDR set of a heavy chain V region or a light chain V region. Although some FR residues may contact the bound antigen, FRs are primarily responsible for folding the V region into the antigen binding site, especially the FR residues directly adjacent to the CDRs. Within the FRs, certain amino acid residues and certain structural features are highly conserved. In this regard, all V region sequences contain an internal disulfide loop of about 90 amino acid residues. When the V region folds into the binding site, the CDRs are displayed as protruding loop motifs that form the antigen binding surface. Regardless of the exact CDR amino acid sequence, it is generally recognized that there are structural regions of the FRs that are conserved in certain "canonical" structures that affect the folding shape of the CDR loops. In addition, certain FR residues are known to participate in non-covalent interdomain contacts that stabilize the interaction of the heavy and light chains of an antibody.

[0032] 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), domestic mammalian animals (e.g., sheep, goats, etc.), pet mammals (dogs, cats, etc.), and rodents (e.g., mice, rats, etc.).

[0033] "Monoclonal antibody" refers to a homogenous antibody population that contains amino acids (natural and non-naturally occurring) involved in selective binding of an epitope. Monoclonal antibodies are highly specific and directed against a single epitope. The term "monoclonal antibody" encompasses intact and full-length monoclonal antibodies, but also fragments thereof (Fab, Fab', F(ab')2, Fv), single chain (scFv), Nanobodies®, variants thereof, fusion proteins containing antigen-binding fragments of monoclonal antibodies, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of immunoglobulin molecules that contain an antigen-binding fragment (epitope recognition site) of the requisite specificity and ability to bind to an epitope, including the WNT surrogate molecules disclosed herein. This is not intended to be limited with respect to the source of the antibody or the manner in which the antibody is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes not only whole immunoglobulins, but also fragments such as those described under the definition of "antibody" above.

[0034] As used herein, the term "native" or "wild type" refers to a nucleotide sequence (e.g., a gene) or a gene product (e.g., an RNA or protein) present in a wild-type cell, tissue, organ, or organism. As used herein, the term "variant" refers to a mutant of a reference polynucleotide or polypeptide sequence, e.g., a native polynucleotide or polypeptide sequence, i.e., having less than 100% sequence identity with the reference polynucleotide or polypeptide sequence. In other words, a variant contains at least one amino acid difference (e.g., an amino acid substitution, an amino acid insertion, an amino acid deletion) with respect to a reference polynucleotide sequence, e.g., a native polynucleotide or polypeptide sequence. For example, a variant may be a polynucleotide having 50% or more, 60% or more, or 70% or more sequence identity with a full-length native polynucleotide sequence, e.g., 75% or 80% or more identity (e.g., 85%, 90%, or 95% or more), e.g., 98% or 99% identity with a full-length native polynucleotide sequence. As another example, a variant may be a polypeptide having 70% or more sequence identity with a full-length native polypeptide sequence, e.g., 75% or 80% or more identity (such as 85%, 90%, or 95% or more), e.g., 98% or 99% identity, to the full-length native polypeptide sequence. Variants may also include variant fragments of a reference (e.g., native) sequence that share 70% or more sequence identity with a fragment of the reference (e.g., native) sequence, e.g., 75% or 80% or more identity (such as 85%, 90%, or 95% or more), e.g., 98% or 99% identity, to the native sequence.

[0035] "Operably linked" or "operably linked" refers to the juxtaposition of genetic elements, where the elements are in a relationship that permits them to operate in their expected manner. For example, a promoter is operably linked to a coding region if it helps initiate transcription of the coding sequence. There can be intervening residues between the promoter and the coding region so long as this functional relationship is maintained.

[0036] As used herein, the terms "polypeptide," "peptide," and "protein" refer to polymers of amino acids of any length. The terms also encompass modified amino acid polymers, including, for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling moiety.

[0037] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute a double-stranded form.

[0038] A polynucleotide or polypeptide has a certain percentage of "sequence identity" to another polynucleotide or polypeptide, meaning that the percentage of its bases or amino acids are the same when the two sequences are compared and aligned. Sequence similarity can be determined in many different ways. To determine sequence identity, sequences can be aligned using methods and computer programs including BLAST, available on the World Wide Web at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package (a wholly owned subsidiary of Oxford Molecular Group, Inc., 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, Inc. (a division of Harcourt Brace & Co., San Diego, Calif., USA). Of particular interest are alignment programs that allow for gaps in the sequence. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70:173-187 (1997). Sequences can also be aligned using the GAP program using the Needleman and Wunsch alignment method. See J. Mol. Biol. 48:443-453 (1970).

[0039] Of interest is the BestFit program, which uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2:482-489 (1981)) to determine sequence identity. Gap creation penalties generally range from 1 to 5, usually from 2 to 4, and in many embodiments are 3. Gap extension penalties generally range from about 0.01 to 0.20, and in many cases are 0.10. The program has default parameters determined by the sequences entered for comparison. Sequence identity is preferably determined using the default parameters determined by the program. This program is also available from the Genetics Computing Group (GCG) package (Madison, Wis., USA).

[0040] 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. The percentage of sequence identity is calculated by FastDB based on the following parameters: mismatch penalty: 1.00, gap penalty: 1.00, gap size penalty: 0.33, and joining penalty: 30.0.

[0041] As used herein, a "promoter" encompasses a DNA sequence that directs the binding of RNA polymerase, thereby facilitating RNA synthesis, i.e., a minimal sequence sufficient to direct transcription. The promoter and the expression of the corresponding protein or polypeptide may be ubiquitous, meaning that it is strongly active in a wide range of cells, tissues and species, or cell type-specific, tissue-specific, or species-specific. The promoter may be "constitutive," meaning continuously active, or "inducible," meaning that the promoter can be activated or deactivated by the presence or absence of a biotic or abiotic factor. The nucleic acid construct or vector of the invention also includes enhancer sequences, which may or may not be contiguous with the promoter sequence. Enhancer sequences affect promoter-dependent gene expression and may be located in the 5' or 3' region of the native gene.

[0042] "Recombinant" when 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 a construct that differs from polynucleotides found in nature.

[0043] The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that it completely or partially prevents the disease or its symptoms, e.g., reduces the likelihood that the disease or its symptoms will occur in a subject, and / or it may be therapeutic, in that it partially or completely cures the disease and / or the deleterious effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, including (a) preventing the onset of the disease in a subject who may be susceptible to the disease but has not yet been diagnosed as suffering from it, (b) inhibiting the disease, i.e., arresting its progression, or (c) relieving the disease, i.e., causing regression of the disease. Therapeutic agents may be administered before, during, or after the onset of the disease or injury. Of particular interest is the treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient. It is desirable that such treatment is performed before there is a complete loss of function in the affected tissue. The subject therapies are desirably administered during, and in some cases after, the symptomatic stage of the disease.

[0044] As used herein, the phrase "retinal vascular disease" refers to a disease of the eye, particularly the retina, caused by aberrant vasculature formation. In some aspects, aberrant vasculature is caused by inhibition of vasculature development, while in other aspects, aberrant vasculature is caused by excessive neovascularization.

[0045] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology techniques, microbiology, biochemistry and immunology, which are within the skill of the art. These techniques are described in “Molecular Cloning: A Laboratory Manual,” second edition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (MJ Gait, ed., 1984); “Animal Cell Culture” (RIFreshney, ed., 1987); “Gene Transfer Vectors for Mammalian Cells” (JMMiller & MPCalos, eds., 1987); “Current Protocols in Molecular Biology” (FMAusubel et al., eds., 1987); “PCR: The Polymerase Chain Reaction”, (Mullis et al., eds., 1994); Immunology” (JEColigan et al. al., eds., 1991), each of which is expressly incorporated herein by reference.

[0046] Some aspects of the present invention are described below with reference to illustrative applications. It should be understood that numerous specific details, relationships, and methods are set forth in order to provide a thorough understanding of the present invention. However, one skilled in the art will readily recognize that the present invention can be practiced without one or more of the specific details, or with other methods. The present invention is not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and / or simultaneously with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the present invention.

[0047] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including", "includes", "having", "has", "with" or variations thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising".

[0048] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on the method by which the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice of 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 even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. When a particular value is described in the specification and claims, the term "about" meaning within an acceptable error range for the particular value should be assumed unless otherwise stated.

[0049] All publications mentioned herein are incorporated by reference herein to disclose and describe the methods and / or materials for which the publications are cited, and it is understood that the present disclosure supersedes any disclosure of the incorporated publications to the extent of any inconsistencies.

[0050] It is further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a priori basis for using exclusive terminology, such as "solely," "only," and the like in connection with the recitation of claim elements, or the use of a "negative" limitation.

[0051] Unless otherwise indicated, all terms used herein have the same meaning as they would be used by one of ordinary skill in the art, and the practice of the present invention employs conventional techniques of microbiology and recombinant DNA technology that are within the knowledge of those skilled in the art.

[0052] II.General The present invention provides methods of modulating WNT signaling to treat retinopathies, including but not limited to FEVR and other genetic disorders, DR, and AMD. In particular, the present invention provides WNT / b-catenin agonists and / or antagonists to inhibit aberrant neovascularization in the progression of retinopathy.

[0053] WNT ("Wingless-associated integration site", or "Wingless and Int-1", or "Wingless-Int") ligands and their signals play important roles in controlling the development, homeostasis, and regeneration of numerous vital organs and tissues, including bone, liver, skin, stomach, intestine, kidney, central nervous system, mammary gland, taste buds, ovary, cochlea, lung, and many other tissues (reviewed, for example, by Clevers, Loh, and Nusse, 2014;346:1248012). Modulation of the WNT signaling pathway has therapeutic potential for degenerative diseases and tissue injury.

[0054] One of the challenges to modulating WNT signaling therapeutically is the existence of multiple WNT ligands and WNT receptors, Frizzled 1-10 (Fzd1-10), and many tissues express multiple overlapping Fzds. In addition to Fzds, canonical WNT signaling also involves low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) or low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) as co-receptors that are widely expressed in various tissues.

[0055] R-spondins 1-4 are a family of ligands that amplify WNT signals. Each of the R-spondins acts through a receptor complex that contains zinc and ring finger 3 (ZNRF3) or ring finger protein 43 (RNF43) at one end and G protein-coupled receptor 4-6 (LGR4-6) that contains leucine-rich repeats at the other end (e.g., reviewed by Knight and Hankenson 2014, Matrix Biology; 37:157-161). R-spondins may also act through additional mechanisms of action. ZNRF3 and RNF43 are two membrane-bound E3 ligases that specifically target WNT receptors (Fzd1-10 and LRP5 or LRP6) for degradation. Binding of R-spondin to ZNRF3 / RNF43 and LGR4-6 leads to the clearance 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), with Furin domain 1 binding to ZNRF3 / RNF43 and Furin domain 2 binding to LGR4-6. A fragment of R-spondin containing Furin domains 1 and 2 is sufficient to amplify WNT signaling. Although R-spondin effects are dependent on WNT signaling, the effects of R-spondin are not tissue specific, as both LGR4-6 and ZNRF3 / RNF43 are widely expressed in various tissues.

[0056] In some embodiments, the WNT / β-catenin signaling antagonist or agonist can include a binding agent or epitope binding domain that binds one or more Fzd receptors and inhibits or enhances WNT signaling. In certain embodiments, the agent or antibody specifically binds to a cysteine-rich domain (CRD) in the human frizzled receptor(s) to which it binds. In addition, antagonist binding agents containing an epitope binding domain for LRP can also be used. In some embodiments, the WNT / β-catenin antagonist possesses a binding agent or epitope binding domain that binds E3 ligase ZNRF3 / RNF43 and one or more FZD receptors or one or more LRP co-receptors, promoting the degradation of the FZD receptor or LRP receptor. The molecule can also contain a binding domain that binds a cell type specific epitope for targeting. The E3 ligase agonist antibody or fragment thereof can be a single molecule or can be combined with other WNT antagonists, such as Fzd receptor antagonists, LRP receptor antagonists, etc.

[0057] As is well known in the art, an antibody is an immunoglobulin molecule that has the ability to specifically bind to a target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, via at least one epitope-binding domain located on the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal or monoclonal antibodies, as well as fragments thereof that contain an epitope-binding domain (e.g., dAb, Fab, Fab', (F(ab')). 2Also included herein are fragments including Fv, single chain (scFv), VHH or single domain antibodies (sdAb), DVD-Ig, synthetic variants thereof, naturally occurring variants, fusion proteins containing epitope binding domains, humanized antibodies, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule that contains an antigen binding site or fragment (epitope recognition site) of the required specificity. "Diabodies", multivalent or multispecific fragments constructed by gene fusion (International Patent Application No. 94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993), are also specific forms of antibodies contemplated herein. Minibodies containing scFvs linked to CH3 domains are also included herein (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). See, e.g., Ward, E. S. et al., Nature 341, 544-546 (1989); Bird et al., Science, 242, 423-426, 1988; Huston et al., PNAS USA, 85, 5879-5883, 1988); PCT Application No. US92 / 09965; International Patent Application No. 94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993; Y. Reiter et al., Nature Biotech, 14, 1239-1245, 1996; S. Hu et al., Cancer Res., 56, 3055-3061, 1996.

[0058] The proteolytic enzyme papain preferentially cleaves IgG molecules to produce several fragments, two of which (F(ab) fragments) each contain a covalently linked heterodimer containing an intact antigen binding site. The enzyme pepsin can cleave IgG molecules to provide several fragments, including the F(ab')2 fragment, which contains both antigen binding sites. Fv fragments for use according to certain embodiments of the present disclosure can be generated by preferential proteolytic cleavage of IgM immunoglobulin molecules, and rarely IgG or IgA immunoglobulin molecules. However, Fv fragments are more commonly derived using recombinant techniques known in the art. Fv fragments contain a non-covalently linked VH::VL heterodimer containing an antigen binding site that retains much of the antigen recognition and binding ability of the 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.

[0059] In certain embodiments, single chain Fv or 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., PNAS 90:6444-8 (1993)); or Janusins ​​(Traunecker et al., EMBO J 10:3655-59 (1991) and Traunecker et al., Int. J. Cancer Suppl. 7:51-52 (1992)) can be prepared using standard molecular biology techniques following the teachings herein for the selection of antibodies with desired specificity. In yet other embodiments, bispecific or chimeric antibodies can be made that include the ligands of the present disclosure. For example, chimeric antibodies may comprise CDRs and framework regions from different antibodies, while bispecific antibodies may be created which specifically bind to one or more Fzd receptors through one binding domain and specifically bind to a second molecule through a second binding domain. These antibodies may be generated through recombinant molecular biology techniques or may be physically conjugated together.

[0060] Single chain Fv (scFv) polypeptides are covalently linked VH:VL heterodimers, which are expressed from gene fusions that include 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. Numerous methods have been described to identify chemical structures for converting naturally aggregated but chemically separated light and heavy polypeptide chains derived from antibody V regions into scFv molecules that fold into a three-dimensional structure substantially similar to the structure of an antigen-binding site. See, for example, U.S. Patent Nos. 5,091,513 and 5,132,405 to Huston et al., and U.S. Patent No. 4,946,778 to Ladner et al.

[0061] In certain embodiments, the antibodies described herein are in the form of diabodies. Diabodies are multimers of polypeptides, each of which comprises a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g., by a peptide linker) but unable to associate with each other to form an antigen-binding site. The antigen-binding site is formed by the association of a first domain of one polypeptide in the multimer with a second domain of another polypeptide in the multimer (International Patent Application No. 94 / 13804).

[0062] A dAb fragment of an antibody consists of the VH domain (Ward, ES et al., Nature 341, 544-546 (1989)).

[0063] Where bispecific antibodies are used, they may be conventional bispecific antibodies, which can be produced in a variety of ways (Holliger, P. and Winter G., Current Opinion Biotechnol. 4, 446-449 (1993)), e.g., prepared chemically or from mixed hybridomas, or any of the bispecific antibody fragments described above. Diabodies and scFvs can be constructed without an Fc region, using only the variable domains, potentially reducing the effects of anti-idiotypic reaction.

[0064] Bispecific diabodies can be particularly useful, as they can be easily constructed and expressed in E. coli, as opposed to bispecific whole antibodies. Diabodies (and many other polypeptides, such as antibody fragments) of appropriate binding specificity can be easily selected from libraries using phage display (International Patent Application No. 94 / 13804). Libraries can be made in which one arm of the diabody is held constant, for example with specificity directed against antigen X, while the other arm is varied, and antibodies of appropriate specificity are selected. Bispecific whole antibodies can be made by knobs-into-holes engineering (JBB Ridgeway et al., Protein Eng., 9, 616-621 (1996)).

[0065] In certain embodiments, the antibodies described herein may be provided in the form of a UniBody®. A UniBody® is an IgG4 antibody with the hinge region removed (see GenMab Utrecht, The Netherlands, see also, e.g., US Patent Publication No. 20090226421). This proprietary antibody technology creates a stable smaller antibody format with a longer expected therapeutic window than current small antibody formats. IgG4 antibodies are considered inert and therefore do not interact with the immune system. Fully human IgG4 antibodies may be modified by removing the hinge region of the antibody to obtain half-molecule fragments with distinct stability characteristics relative to the corresponding intact IgG4 (GenMab, Utrecht). Halving the IgG4 molecule leaves only one region on the UniBody® that can bind to the cognate antigen (e.g., disease target), and thus the UniBody® binds monovalently to only one site on the target cell.

[0066] In certain embodiments, the antibodies and antigen-binding fragments thereof described herein comprise a set of heavy and light chain CDRs, each interposed between a set of heavy and light chain framework regions (FRs), which provide support for the CDRs and define the spatial relationship of the CDRs to each other. As used herein, the term "CDR set" refers to the three hypervariable regions of a heavy or light chain V region. These regions originating from the N-terminus of the heavy or light chain are: They are designated as "CDR1", "CDR2" and "CDR3", respectively. Thus, an antigen-binding site comprises six CDRs, including a set of CDRs from each of the heavy chain V region and the light chain V region. A polypeptide comprising a single CDR (e.g., CDR1, CDR2 or CDR3) is referred to herein as a "molecular recognition unit". Crystallographic analysis of numerous antigen-antibody complexes has shown that the amino acid residues of the CDRs form extensive contacts with the bound antigen, with the most extensive antigen contact being with the heavy chain CDR3. Thus, the molecular recognition unit is primarily responsible for the specificity of the antigen-binding site.

[0067] As used herein, the term "FR set" refers to four adjacent amino acid sequences that frame the CDRs of a CDR set of a heavy chain V region or a light chain V region. Although some FR residues may contact the bound antigen, FRs are primarily responsible for folding the V region into the antigen binding site, especially the FR residues directly adjacent to the CDRs. Within the FRs, certain amino acid residues and certain structural features are highly conserved. In this regard, all V region sequences contain an internal disulfide loop of about 90 amino acid residues. When the V region folds into the binding site, the CDRs are displayed as protruding loop motifs that form the antigen binding surface. Regardless of the exact CDR amino acid sequence, it is generally recognized that there are structural regions of the FRs that are conserved in certain "canonical" structures that affect the folding shape of the CDR loops. In addition, certain FR residues are known to participate in non-covalent interdomain contacts that stabilize the interaction of the heavy and light chains of an antibody.

[0068] "Monoclonal antibody" refers to a homogenous antibody population that contains amino acids (natural and non-naturally occurring) involved in selective binding of an epitope. Monoclonal antibodies are highly specific and directed against a single epitope. The term "monoclonal antibody" encompasses intact and full-length monoclonal antibodies, but also fragments thereof (Fab, Fab', F(ab')2, Fv), single chain (scFv), Nanobodies®, variants thereof, fusion proteins containing antigen-binding fragments of monoclonal antibodies, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of immunoglobulin molecules that contain an antigen-binding fragment (epitope recognition site) of the requisite specificity and ability to bind to an epitope, including the WNT surrogate molecules disclosed herein. This is not intended to be limited with respect to the source of the antibody or the manner in which the antibody is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes not only whole immunoglobulins, but also fragments such as those described under the definition of "antibody" above.

[0069] In certain embodiments, the antibodies of the present disclosure may take the form of Nanobodies®. Nanobody® technology was initially developed following the discovery and identification that Camelidae (e.g., camels and llamas) possess fully functional antibodies consisting of only heavy chains and therefore lacking light chains. These heavy chain-only antibodies contain a single variable domain (VHH) and two constant domains (CH2, CH3). The cloned and isolated single variable domains have full antigen-binding capacity and are highly stable. These single variable domains form the basis of "Nanobodies®" due to their unique structural and functional properties. Nanobodies® are encoded by a single gene and are efficiently produced in nearly all prokaryotic and eukaryotic hosts, such as, for example, E. coli (see, e.g., U.S. Pat. No. 6,765,087), molds (e.g., Aspergillus or Trichoderma), and yeasts (e.g., Saccharomyces, Kluyveromyces, Hansenula, or Pichia (see, e.g., U.S. Pat. No. 6,838,254). The production process is scalable and multi-kilogram quantities of Nanobodies® have been produced. Nanobodies® can be formulated as a ready-to-use solution with a long shelf life. The Nanoclone® method (see, for example, International Patent Application No. WO 06 / 079372) is a proprietary method for generating Nanobodies® against desired targets based on automated high-throughput selection of B cells. Nanobodies® are single domain antigen-binding fragments of camelid-specific heavy chain-only antibodies. Nanobodies®, also called VHH antibodies, typically have a small size of about 15 kDa.

[0070] Another contemplated antibody fragment is the dual variable domain immunoglobulin (DVD-Ig), an engineered protein that combines the functions and specificities of two monoclonal antibodies into one molecular entity. DVD-Ig is designed as an IgG-like molecule, except that each light and heavy chain contains two variable domains tandemly aligned via a short peptide bond, rather than one variable domain in IgG. The orientation of the fusion of the two variable domains and the selection of the linker sequence are important for the functional activity and efficient expression of the molecule. DVD-Ig can be produced by conventional mammalian expression systems as a single species for manufacturing and purification. DVD-Ig has the specificity of the parent antibody, is stable in vivo, and exhibits IgG-like physicochemical and pharmacokinetic properties. DVD-Ig and methods for its production are described in Wu, C., et al., Nature Biotechnology, 25:1290-1297 (2007).

[0071] In certain embodiments, the antibodies or antigen-binding fragments thereof disclosed herein are humanized. This refers to chimeric molecules, generally prepared using recombinant techniques, that have an antigen-binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise either a complete variable domain fused onto a constant domain, or only the CDRs grafted onto appropriate framework regions in the variable domain. The epitope-binding site may be wild-type or modified by one or more amino acid substitutions. This removes the constant region as an immunogen in human individuals, but leaves the possibility of an immune response against the foreign 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). Disclosed herein Exemplary methods for humanization of anti-Fzd or LRP antibodies include those described in US Pat. No. 7,462,697.

[0072] Another approach focuses not only on providing constant regions of human origin, but also on modifying variable regions to reshape them as close as possible to human form. Both heavy and light chain variable regions are known to contain three complementarity determining regions (CDRs) that change in response to the epitope in question, and are adjacent to four framework regions (FRs) that are relatively conserved in a given species and presumably provide the anchorage of the CDRs, determining their binding ability. When a non-human antibody is prepared for a specific epitope, the variable region can be "rearranged" or "humanized" by grafting the CDRs from the non-human antibody onto the FRs present in the modified human antibody. Application of this approach to various antibodies has been reviewed in Sato, K., et al., (1993) Cancer Res 53:851-856; Riechmann, L., et al., (1988) Nature 332:323-327; Verhoeyen, M., et al., (1988) Science 239:1534-1536; Kettleborough, CA, et al., (1991) Protein Engineering 4:773-3783; Maeda, H., et al., (1991) Human Antibodies Hybridoma 2:124-134; Gorman, SD, et al., (1991) Proc Natl Acad Sci USA 88:4181-4185; Tempest, PR, et al., (1991) Bio / Technology 9:266-271; Co, 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 preserves all CDR sequences (e.g., a humanized mouse antibody that contains all six CDRs from the mouse antibodies).In other embodiments, a humanized antibody has one or more CDRs (one, two, three, four, five, six) that are altered with respect to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

[0073] In certain embodiments, the antibody of the present disclosure may be a chimeric antibody. In this regard, a chimeric antibody comprises an antigen-binding fragment of an antibody operatively linked or otherwise fused to a heterologous Fc portion of a different antibody. In certain embodiments, the heterologous Fc domain is of human origin. In other embodiments, the heterologous Fc domain may be derived from an Ig class different from the parent antibody, including IgA (including IgA1 and IgA2 subclasses), IgD, IgE, IgG (including IgG1, IgG2, IgG3, and IgG4 subclasses), and IgM. In further embodiments, the heterologous Fc domain may comprise a CH2 domain and a CH3 domain from one or more of the different Ig classes. As described above with respect to humanized antibodies, the antigen-binding fragment of a chimeric antibody may comprise only one or more of the CDRs of an antibody described herein (e.g., one, two, three, four, five, or six CDRs of an antibody described herein) or may comprise the entire variable domain (VL, VH, or both).

[0074] The structures and locations of immunoglobulin CDRs and variable domains can be determined by Kabat, EA et al., Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and updates thereto, available on the Internet at immuno.bme.nwu.edu.

[0075] In certain embodiments, antagonist or agonist binding agents have a dissociation constant (K DFor example, in certain embodiments, an FZD-binding agent or antibody described herein that binds to multiple FZDs has a K of about 100 nM or less, about 20 nM or less, or about 10 nM or less. D In certain embodiments, the binding agents bind to one or more of their target antigens with an EC50 of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, or about 1 nM or less.

[0076] The antibody or other agent of the present invention can be tested for specific binding by any method known in the art. Immunoassays that can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as biolayer interferometry (BLI) analysis, FACS analysis, immunofluorescence, immunocytochemistry, Western blot, radioimmunoassay, ELISA, "sandwich" immunoassay, immunoprecipitation, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescent immunoassay, and protein A immunoassay. Such assays are routine and well known in the art (see, for example, Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York, the entire text of which is incorporated herein by reference).

[0077] For example, specific binding of an antibody to a target antigen may be determined using an ELISA assay. An ELISA assay involves preparing an antigen, coating the wells of a 96-well microtiter plate with the antigen, adding an antibody or other binding agent (conjugated to a detectable compound such as an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase)) to the wells, incubating for a period of time, and detecting the presence of the antigen. In some embodiments, the antibody or agent is not conjugated to a detectable compound, and instead a second conjugated antibody that recognizes the first antibody or agent is added to the well. In some embodiments, instead of coating the wells with the antigen, the wells can be coated with the antibody or agent and a second antibody conjugated to a detectable compound can be added after the antigen has been added to the coated wells. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISA methods known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1).

[0078] The binding affinity of an antibody or other agent to a target antigen and the off-rate of antibody-antigen interaction can be determined by competitive binding assay. One example of competitive binding assay is radioimmunoassay, which involves incubation of a labeled antigen (e.g., Fzd, LRP) or a fragment or variant thereof with an antibody of interest in the presence of increasing amounts of unlabeled antigen, followed by detection of the antibody bound to the labeled antigen. The affinity and binding off-rate of the antibody can be determined from the data by Scatchard plot analysis. In some embodiments, BLI analysis is used to determine the binding on-rate and off-rate of an antibody or agent. BLI kinetic analysis involves analysis of the binding and dissociation of an antibody from a chip with an antigen immobilized on its surface.

[0079] In certain embodiments, the WNT agonist is selected from those disclosed in PCT Patent Publication No. WO2019126398, which is incorporated herein in its entirety. In certain embodiments, the WNT agonist has a structure depicted in FIG. 1A and / or comprises a sequence disclosed for any of the WNT agonists disclosed in FIG. 1B. In some embodiments, the WNT agonist comprises a sequence having at least 90% identity (e.g., 95%, 98%, or 100% identity) to a sequence disclosed in any of SEQ ID NOs: 1-8, in which the leader sequence is italicized, the linker sequence is underlined, and the VHH / sdAb sequence or the VH sequence or the VL sequence is bolded. [ka] [ka]

[0080] III. Pharmaceutical Compositions Also disclosed are pharmaceutical compositions comprising a WNT antagonist or agonist molecule described herein and one or more pharma- ceutically acceptable diluents, carriers, or excipients.

[0081] In further embodiments, pharmaceutical compositions are also disclosed that include a polynucleotide comprising a nucleic acid sequence encoding a WNT antagonist / agonist molecule as described herein and one or more pharma- ceutically acceptable diluents, carriers, or excipients.In certain embodiments, the polynucleotide is DNA or mRNA, e.g., modified mRNA.In certain embodiments, the polynucleotide is modified mRNA that further comprises a 5' cap sequence and / or a 3' tailing sequence, e.g., a polyA tail.In other embodiments, the polynucleotide is an expression cassette that comprises a promoter operably linked to the coding sequence.

[0082] In some embodiments, the WNT antagonist / agonist is a modified recombinant polypeptide incorporating various epitope-binding fragments that bind to various molecules in the WNT signaling pathway. For example, the WNT antagonist may be an antibody or fragment thereof that binds to the Fzd4 receptor and / or the LRP receptor and inhibits WNT signaling. Fzd4 and LRP antibody fragments (e.g., Fab, scFv, VHH / sdAbs, etc.) may be linked together on one molecule, directly or with linkers of various sizes.

[0083] Conversely, modified WNT agonists / antagonists can be recombinant polypeptides incorporating epitope-binding fragments that bind to various molecules of the WNT signaling pathway and enhance WNT signaling. For example, WNT agonists can be antibodies or fragments thereof that bind to Fzd and / or LRP receptors and enhance WNT signaling. Fzd and LRP antibody fragments (e.g., Fab, scFv, VHH / sdAbs, etc.) can be linked together on one molecule, either directly or with linkers of various sizes.

[0084] In further embodiments, pharmaceutical compositions are also disclosed that include an expression vector, e.g., a viral vector, that includes a polynucleotide that includes a nucleic acid sequence encoding a WNT antagonist / agonist molecule described herein and one or more pharma- ceutically acceptable diluents, carriers, or excipients. In certain embodiments, the nucleic acid sequence encoding the WNT antagonist molecule and the nucleic acid sequence encoding the WNT agonist are in the same polynucleotide, e.g., an expression cassette.

[0085] The present disclosure further contemplates a pharmaceutical composition comprising a cell comprising an expression vector comprising a polynucleotide, the expression vector comprising a promoter operably linked to a nucleic acid encoding a WNT antagonist / agonist molecule, and one or more pharma- ceutically acceptable diluents, carriers, or excipients. In certain embodiments, the pharmaceutical composition further comprises a cell comprising an expression vector comprising a polynucleotide, the promoter operably linked to a nucleic acid sequence encoding a WNT antagonist and a WNT agonist. In certain embodiments, the nucleic acid sequence encoding a WNT antagonist molecule and the nucleic acid sequence encoding a WNT agonist molecule are present in the same polynucleotide, e.g., expression cassette, and / or in the same cell. In certain embodiments, the cell is a heterologous or autologous cell obtained from the subject to be treated.

[0086] In certain embodiments, the cell is a stem cell, for example, an adipose-derived stem cell or a hematopoietic stem cell. The present disclosure contemplates a pharmaceutical composition comprising a first molecule for delivery of a WNT antagonist molecule as a first active agent, and a WNT agonist as a second molecule. The first molecule and the second molecule may be the same type of molecule, or may be different types of molecules. For example, in certain embodiments, the first and second molecules may each be independently selected from the following types of molecules: a polypeptide, an organic small molecule, a nucleic acid encoding a first or second active agent (optionally DNA or mRNA, optionally modified RNA), a vector comprising a nucleic acid sequence encoding a first or second active agent (optionally an expression vector or viral vector), a cell comprising a nucleic acid sequence encoding a first or second active agent (optionally an expression cassette).

[0087] The subject molecules can be combined with pharma- ceutically acceptable carriers, diluents, excipients, and reagents, either alone or in combination, that are generally safe, non-toxic, and useful for preparing desirable formulations, including excipients acceptable for use by mammals, e.g., humans or primates. Such excipients can be solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous. Examples of such carriers, diluents, and excipients include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Supplementary active compounds can also be incorporated into the formulation. The solution or suspension used in the formulation can contain a sterile diluent (such as water for injection, saline, fixed oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvent), an antibacterial compound (such as benzyl alcohol or methylparaben), an antioxidant (such as ascorbic acid or sodium bisulfite), a chelating compound (such as ethylenediaminetetraacetic acid (EDTA)), a buffer (such as acetate, citrate or phosphate), a detergent (such as Tween 20 to prevent aggregation), and a compound for adjusting tonicity (such as sodium chloride or dextrose). The pH can be adjusted using an acid or base such as hydrochloric acid or sodium hydroxide. In certain embodiments, the pharmaceutical composition is sterile.

[0088] The pharmaceutical compositions may further include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In some cases, the composition should be sterile and fluid so that it can be drawn into a syringe or delivered from a syringe to a subject. In certain embodiments, it is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents in the composition, such as sugars, polyalcohols (such as mannitol, sorbitol, etc.), sodium chloride, etc. Prolonged absorption of the internal composition can be achieved by including in the composition an agent that delays absorption (such as aluminum monostearate and gelatin, etc.).

[0089] Sterile solutions can be prepared by incorporating the WNT antagonist / agonist antibody or antigen-binding fragment thereof (or those encoding the polynucleotide or cells containing the polynucleotide) in the required amount in a suitable solvent with one or a combination of the ingredients listed above, as needed, followed by sterilization by filtration. In general, dispersions are prepared by incorporating the active compound into a sterile medium containing a basic dispersion medium and other ingredients required 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, which yields a powder of the active ingredient plus any additional desired ingredients from the solution previously sterile-filtered.

[0090] In one embodiment, the pharmaceutical composition is prepared with a carrier that protects the antibody or its antigen-binding fragment from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Methods for preparing such formulations will be clear to those skilled in the art. Materials can also be commercially obtained. Liposomal suspensions can also be used as pharma-ceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

[0091] For ease of administration and uniformity of dosage, it may be advantageous to formulate pharmaceutical compositions in unit dosage form.As used herein, unit dosage form refers to a physically separate unit suitable as a unitary dose for the subject to be treated, each unit containing a predetermined amount of active antibody or its antigen-binding fragment calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier.The specification of unit dosage form is determined by and directly depends on the unique characteristics of antibody or its antigen-binding fragment, and the specific therapeutic effect to be achieved, as well as the inherent limitations of the art of compounding such active antibody or its antigen-binding fragment for the treatment of an individual.

[0092] The pharmaceutical compositions can be included in a container, pack, or dispenser (eg, a syringe, eg, a prefilled syringe) together with instructions for administration.

[0093] The pharmaceutical compositions of the present disclosure include any pharma- ceutically acceptable salts, esters, or salts of such esters, or any other compounds that, upon administration to an animal, including a human, can provide (directly or indirectly) a biologically active antibody or antigen-binding fragment thereof.

[0094] The present disclosure includes pharma- ceutically acceptable salts of the WNT antagonist / agonist molecules described herein. The term "pharma- ceutically acceptable salts" refers to physiologically and pharma- ceutical acceptable salts of the compounds of the present disclosure, i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. Various pharma- ceutical acceptable salts are known in the art and are described, for example, in "Remington's Pharmaceutical Sciences", 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and its recent editions), "Encyclopaedia of Pharmaceutical Technology", 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and J.Pharm.Sci.66:2 (1977). Also, for a review of suitable salts, see "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, 2002). Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines.

[0095] 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, for example, Berge et al., "Pharmaceutical Salts," J. Pharma Sci., 1977, 66, 119). The base addition salts of the aforementioned 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 may be regenerated by contacting the salt form with an acid and isolating the free acid in a conventional manner. The free acid forms differ somewhat from their respective salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acids for purposes of this disclosure.

[0096] In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of a WNT antagonist / agonist molecule or a pharma- ceutically acceptable salt thereof mixed with a pharma- ceutically acceptable carrier, diluent, and / or excipient, such as saline, phosphate buffered saline, phosphates, and amino acids, polymers, polyols, sugars, buffers, preservatives, and other proteins. Exemplary amino acids, polymers, sugars, and the like are octylphenoxypolyethoxyethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, Ringer's and Hank's solutions, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and glycols. The formulation is preferably stable at 4° C. for at least 6 months.

[0097] 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 skill in the art, such as those described in Good et al. (1966) Biochemistry 5: 467. The pH of the buffer may be in the range of 6.5 to 7.75, preferably 7 to 7.5, and most preferably 7.2 to 7.4.

[0098] IV.How to use The present disclosure also provides methods for modulating, for example, WNT signaling pathway, for example, increasing or decreasing WNT signaling, using WNT antagonist / agonist molecules, and methods for administering WNT antagonist / agonist molecules in various therapeutic settings.Herein, a method of treatment using WNT antagonist / agonist molecules is provided.In one embodiment, the WNT antagonist / agonist molecule is provided to a subject having a disease involving inappropriate or uncontrolled WNT signaling.

[0099] In certain embodiments, WNT antagonist / agonist molecules can be used to block or enhance WNT signaling pathway in tissues or cells. Antagonism of WNT signaling pathway can include reduction or inhibition of WNT signaling in cells or tissues. Agonism of WNT signaling pathway can include, for example, increase of WNT signaling or enhancement of WNT signaling in tissues or cells. Thus, in some aspects, the present disclosure provides a method of antagonizing / agonizing WNT signaling pathway in cells, which comprises contacting tissues or cells with an effective amount of a WNT antagonist / agonist molecule disclosed herein or a pharma- ceutically acceptable salt thereof, wherein the WNT antagonist / agonist molecule is a WNT signaling pathway antagonist / agonist. In some embodiments, the contacting occurs in vitro, ex vivo, or in vivo. In certain embodiments, the cells are cultured cells and the contacting occurs in vitro.

[0100] WNT antagonist / agonist molecules can be used to treat retinopathy. In particular, activation of WNT signaling is necessary for retinal neovascularization during ocular vascular development. Genetic deletion of Norlin, Fzd4, Lrp5, or Tspan12 significantly regresses not only vascular development on the superficial retinal surface, but also vascular penetration into the deep layers of the retina. In addition, the avascular area generated by immature neovascularization leads to ischemia-induced neovascularization. Therefore, timely and controlled administration of WNT agonist or / and antagonist will not only regress retinopathy disease progression, but also lead to disease improvement. In certain embodiments, WNT agonist / antagonist is administered either early or late in the disease progression of retinopathy in a subject.

[0101] Both WNT agonists and antagonists may be administered alone as monotherapy or sequentially. Administration of the agonist early in the development of the disease, which shows avascular areas of the retina, will stimulate / stabilize angiogenesis and protect the vessels from avascular factors. On the other hand, administration of the antagonist at a later stage, which shows neovascularization, may inhibit aberrant revascularization of the retina. Thus, sequential treatment of both agonists and antagonists is one possible option to modulate the disease. In a typical administration schedule, the agonist is first administered in the avascularization phase, followed by the antagonist in the neovascularization phase. To test the reverse role, the WNT agonist and antagonist are administered to subjects in the reverse sequence order. However, considering the possible effect of WNT on stabilizing the vascular structure, administration of the agonist in the neovascularization phase is also considered.

[0102] Retinal vascular diseases may include, but are not limited to, familial exudative vitreoretinopathy (FEVR), exudative vitreoretinopathy, Norrie's disease, diabetic retinopathy (DR), age-related macular degeneration (AMD), retinopathy of prematurity (ROP), osteoporosis-pseudoglioma syndrome (OPPG), retinal vein occlusion, and Coats' disease.

[0103] The present invention also provides combination therapy with known treatments for FEVR and / or DR. For example, WNT antagonists / agonists can be administered in combination with current therapies for retinopathy, including but not limited to anti-VEGF antibodies. In some embodiments, anti-Ang2 antibodies are also administered to subjects in combination with WNT agonists / antagonists. Hypoxia-induced VEGF and Ang2 expression are important clues for pathological neovascularization, and in fact antagonist Ang2 antibodies are being investigated for the treatment of retinopathy patients (Gadkar et al., Invest Ophthalmol Vis Sci. 2015 Aug; 56(9): 5390-400). Anti-VEGF antibodies or anti-Ang2 antibodies can be administered sequentially or simultaneously with the molecules of the present invention. VEGF antagonists include, but are not limited to, bevacizumab, ranibizumab, aflibercept, ramucirumab, and tanibirumab, and Ang2 antagonists include, but are not limited to, nesbacumab, AMG780, and MEDI3617.

[0104] In further embodiments, the antagonist and / or agonist molecules may also incorporate a tissue targeting moiety, such as an antibody or fragment thereof, that recognizes a retinal tissue-specific receptor or cell surface molecule.

[0105] Therapeutic agents (e.g., WNT antagonists / agonists) may be administered before, during, or after the onset of disease or injury. Of particular interest is the treatment of ongoing disease, where the treatment stabilizes or reduces undesirable clinical symptoms in the patient. Such treatment is desirably performed before complete loss of function in affected tissues. The subject therapies are desirably administered during, and in some cases after, the symptomatic phase of disease. In some embodiments, the subject methods provide a therapeutic benefit, such as preventing the onset of a disorder, halting the progression of a disorder, reversing the progression of a disorder, and the like. In some embodiments, the subject methods include a step of detecting that a therapeutic benefit has been achieved. Those skilled in the art will understand that such measures of therapeutic efficacy are applicable to the particular disease to be modified, and will recognize the appropriate detection methods to use to measure therapeutic efficacy.

[0106] All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent literature referenced herein and / or listed in the Application Data Sheets are hereby incorporated by reference in their entireties.

[0107] From the foregoing, it will be understood that, although specific embodiments of the present disclosure have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, the disclosure is not limited except as by the appended claims.

[0108] The broad scope of this invention is best understood with reference to the following examples, which are not intended to limit the inventions to the specific embodiments. EXAMPLES

[0109] Example 1 I. General method Standard methods in molecular biology are described in 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 rded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif. Standard methods are also described in Ausbel et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describe cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugate and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0110] Methods for protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization are described. 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) BioDirectory, Piscataway, NJ, pp. 384-391. Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described. Coligan et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra. Standard techniques are available for characterizing ligand / receptor interactions. See, e.g., Coligan et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York.

[0111] Methods for flow cytometry, including fluorescence-activated cell sorting detection systems (FACS®), are available. See, e.g., Owens et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2 nd’ ed.; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ. Fluorescent reagents suitable for modifying nucleic acids for use, for example, as diagnostic reagents, are available, including nucleic acid primers and probes, polypeptides, and antibodies. Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.

[0112] Standard methods for immune system histology are described. See, e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY; Hiatt, et al. (2000) Color Atlas of Histology, Lippincott, Williams, and Wilkins, Phila, Pa.; Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY.

[0113] For example, software packages and databases are available for determining antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments. For example, GenBank, Vector NTI® Suite (Informax, Inc, Bethesda, Md.); GCG Wisconsin Package (Accelrys, Inc., San Diego, Calif.); DeCypher® (TimeLogic Corp., Crystal Bay, Nev.); Menne et al. (2000) Bioinformatics 16:741-742; Menne et al. al.(2000)Bioinformatics Applications Note16:741-742;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 See Res.14:4683-4690.

[0114] II.FZD4 WNT surrogate Monospecific FZD4 WNT surrogates (3SD10-3, 3SD10-26, 3SD10-36, 4SD1-3, 4SD1-26, and 4SD1-36) were constructed as described in PCT Patent Publication No. 2019126398. FIG. 1A provides a diagrammatic representation of the structure of the WNT surrogate molecule used, and FIG. 1B is a table showing the Fzd and LRP binding domains present in the WNT surrogates and providing the sequences present in the indicated WNT surrogates. FZD4 receptor specificity was tested as described below.

[0115] WNT signaling activity was measured using a HEK293 cell line (293STF) containing a luciferase gene controlled by a WNT-responsive promoter (293STF) as previously reported (see, e.g., Janda et al. (2017) Nature 545:234-237). Briefly, 293STF cells were seeded at a density of 10,000 cells per well in 96-well plates 24 hours prior to treatment and then treated with 3SD10-3, 3SD10-26, 4SD1-3, or 4SD1-26 together with 20 nM Rspo. Cells were lysed with luciferase cell culture lysis reagent (Promega) and activity was measured with a luciferase assay system (Promega) using the procedure suggested by the supplier. Data were plotted as the mean ± standard deviation of triplicates and fitted by nonlinear regression using Prism (GraphPad Software). For overexpression of FZD4, cells were transiently transfected with a plasmid containing the human FZD4 gene under the CMV promoter (GenScript OHu21807) and then split into 96-well plates for STF assays 24 hours after transfection. Figures 2A-D show that there is no WNT signaling activity in non-transfected 293STF cells. In contrast, cells transiently transfected with the FZD4 receptor showed WNT signaling (Figures 2E-H).

[0116] RNA from parental or FZD4-overexpressing 293STF cells was extracted using Qiagen RNeasy Micro Kit (Qiagen, 74004). cDNA was produced using SuperScript™ VILO™ cDNA Synthesis Kit (ThermoFisher, 11754050). Human FZD4 expression was measured using TaqMan® Fast Advanced Master Mix (ThermoFisher, 4444963) and Hs00201853_m1 FZD4 probe (ThermoFisher, 4331182). Values ​​were normalized to constitutive ACTIN B gene expression using Hs01060665_m1 probe (ThermoFisher, 4331182). Figure 3 illustrates gene expression levels of FZD4 transiently transfected cells relative to the expression of FZD4.

[0117] III. WNT activity in additional cell lines WNT signaling activity was measured using bEnd.3 (a mouse brain endothelial cell line used in vascular studies) or HRMEC (Primary Human Retinal Microvascular Endothelial Cells) cells containing a luciferase gene controlled by a WNT-responsive promoter. Cells were transiently transfected with an STF plasmid encoding a firefly luciferase reporter under the control of a minimal promoter and a concatemer of seven LEF / TCF binding sites. Transfected cells were seeded at a density of 10,000 cells per well in 96-well plates 24 hours prior to treatment and then treated with 3SD10-3, 3SD10-26, 4SD1-3, 4SD1-26, or WNT3a. Cells were lysed with Luciferase Cell Culture Lysis Reagent (Promega) and activity was measured with a Luciferase Assay System (Promega) using the procedure suggested by the supplier. Data were plotted as the mean ± standard deviation of triplicates and fitted by nonlinear regression using Prism (GraphPad Software). Figures 4A-H show increased WNT signaling activity and Axin2 expression in bEnd.3 cells treated with the monoFZD4 WNT surrogate. Figures 4I-4P show similar WNT signaling and increased Axin2 expression in HRMEC cells.

[0118] RNA from bEnd.3 and HRMEC cells was extracted using the Qiagen RNeasy Micro Kit (Qiagen, 74004). cDNA was generated using the SuperScript™ VILO™ cDNA Synthesis Kit (ThermoFisher, 11754050). Human gene expression shown in HRMEC was analyzed using TaqMan® Fast Advanced Master Mix (ThermoFisher, 4444963) and the following PCR products: Hs00268943_s1 FZD1, Hs00361432_s1 FZD2, Hs00184043_m1 FZD3, Hs00201853_m1 FZD4, Hs00258278_s1 FZD5, Hs00171574_m1 FZD6, Hs00275833_s1 FZD7, Hs00259040_s1 FZD8, Hs00268954_s1 FZD9, Hs00273077_s1 FZD10, Hs00182031_m1 LRP5, Hs00233945_m1 LRP6, Hs00610344_m1 AXIN2 probe (ThermoFisher, 4331182) was used to measure expression of the ACTIN B gene. Values ​​were normalized to constitutive expression of the ACTIN B gene using the Hs01060665_m1 probe (ThermoFisher, 4331182). Expression of the indicated mouse genes in bEnd.3 cells was analyzed using TaqMan® Fast Advanced Master Mix (ThermoFisher, 4444963) and Mm00445405_s1 Fzd1, Mm02524776_s1 Fzd2, Mm00445423_m1 Fzd3, Mm00433382_m1 Fzd4, Mm00445623_s1 Fzd5, Mm00433387_m1 Fzd6, Mm00433409_s1 Fzd7, Mm01234717_s1 Fzd8, Mm01206511_s1 Fzd9, Mm00558396_s1 Fzd10, Mm01227476_m1 Fzd11, Mm01227476_m1 Fzd12, Mm01227476_m1 Fzd13, Mm01227476_m1 Fzd14, Mm01227476_m1 Fzd15, Mm01227476_m1 Fzd16, Mm01227476_m1 Fzd17, Mm01227476_m1 Fzd18, Mm01227476_m1 Fzd19, Mm01227476_m1 Fzd20, Mm01227476_m1 Fzd21, Mm01227476_m1 Fzd22, Mm01227476_m1 Fzd31, Mm01227476_m1 Fzd42, Mm01227476_m1 Fzd53, Mm01 Lrp5, Mm00999795_m1 Lrp6, Mm00443610_m1 were measured using the Axin2 probe (ThermoFisher, 4331182).Values ​​were normalized to constitutive Actin B gene expression using the Mm02619580_g1 probe (ThermoFisher, 4331182). Data for Axin2 expression were plotted as mean ± standard deviation of triplicates and fitted by nonlinear regression using Prism (GraphPad Software). Figure 5A and Figure 5B show the expression of WNT receptors in bEnd.3 cells and HRMEC, respectively.

[0119] IV. Effect of RSPO on FZD4 WNT surrogate activity WNT signaling activity was measured using bEnd.3 or HRMEC cells containing a luciferase gene controlled by a WNT-responsive promoter. Cells were transiently transfected with an STF plasmid encoding a firefly luciferase reporter under the control of a minimal promoter and a concatemer of seven LEF / TCF binding sites. Transfected cells were seeded at a density of 10,000 per well in 96-well plates 24 hours before treatment, and then treated with R2M3-3, R2M3-26, 3SD10-3, 3SD10-26, 4SD1-3, 4SD1-26 (see, for example, International Patent Publication No. 2019126398) with or without 20 nM Rspo. Cells were lysed with Luciferase Cell Culture Lysis Reagent (Promega) and activity was measured with a Luciferase Assay System (Promega) using the procedure proposed by the supplier. Data were plotted as the mean ± standard deviation of triplicates and fitted by nonlinear regression using Prism (GraphPad Software). Figures 6A-F show that addition of RSPO with different FZD4WNT surrogates in both types of endothelial cells had little significant effect on WNT signaling activity.

[0120] V. Oxygen-induced retinopathy Within 8 hours of birth, Sprague-Dawley rat litters of pups and their mothers were transferred to an oxygen exposure chamber where they were subjected to alternating 50% and 10% oxygen at 24-hour intervals for 14 days (i.e., P1-P14). On postnatal day 14, i.e., P14(0), the oxygen-exposed rats were returned to room air. They remained in room air for an additional 6 days, from P14(1) to P14(6). Age-matched rat littermates were also maintained in room air (RA) to serve as controls. Each eye of the three groups of rats received an intravitreal injection of 3ug of anti-EGFP Ab, 0.3ug of 4SD1-03, or 3ug of 4SD1-03 at P7, while another group of rats received an intravitreal injection of anti-VEGF treatment at P14(0) (see, for example, the experimental design shown in Figures 7A and 7B).

[0121] Following treatment, all rats were sacrificed on P14 (6), at which time both normal intraretinal vessel growth and pathological preretinal neovascularization (NV) were assessed in isolectin B4 stained retinal flat mounts using computer-assisted image analysis of high-resolution digital images. TA: total area. Figures 8A-B show that 0.3 μg of 4SD1-3 inhibits neovascular tuft formation to the same extent as anti-VEGF treatment. This demonstrates that FZD4 WNT surrogate treatment has comparable efficacy to anti-VEGF treatment in this model of retinopathy.

[0122] The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent literature referenced herein and / or listed in the application data sheet are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified as necessary to adopt concepts from various patents, applications, and publications to provide still further embodiments. These and other changes can be made to the embodiments in light of the description detailed above.

[0123] In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but rather to include all possible embodiments, along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by this disclosure. In certain embodiments, for example, the following items are provided: (Item 1) A method of treating retinopathy in a subject, comprising administering to the subject a modified WNT signal regulator. (Item 2) The method of item 1, wherein the WNT signal regulator is a modified WNT agonist or a modified WNT antagonist. (Item 3) The method of claim 2, wherein the modified WNT agonist and modified WNT antagonist comprise a binding composition that binds to one or more Fzd receptors and a binding composition that binds to one or more LRP receptors or Tspan12 receptors. (Item 4) The method of claim 3, wherein the binding composition of the modified WNT agonist is selected from the group consisting of a Fzd4 binding composition, a Lrp5 binding composition, a Lrp6 binding composition, a LRP5 / 6 binding composition, and a Tspan12 binding composition. (Item 5) 2. The method of claim 1, comprising administering a modified WNT agonist and a modified WNT antagonist, wherein the modified WNT agonist and the modified WNT antagonist are administered independently at early and / or late stages of the retinopathy. (Item 6) 2. The method of claim 1, comprising administering a modified WNT agonist and a modified WNT antagonist, wherein the modified WNT agonist and the modified WNT antagonist are administered sequentially at early and / or late stages of the retinopathy. (Item 7) 2. The method of claim 1, comprising administering a modified WNT agonist and a modified WNT antagonist, wherein the modified WNT agonist and the modified WNT antagonist are administered simultaneously at early and / or late stages of the retinopathy. (Item 8) 7. The method of claim 6, wherein the WNT agonist is administered before or after the WNT antagonist. (Item 9) 8. The method of any of items 1 to 7, comprising administering a modified WNT agonist and a modified WNT antagonist, wherein the WNT agonist and / or the WNT antagonist are administered together with a binding composition specific for either VEGF and / or Ang2. (Item 10) 10. The method of claim 9, wherein the binding composition specific for VEGF or Ang2 is an antagonist of VEGF or Ang2 activity. (Item 11) 11. The method of claim 10, wherein the VEGF antagonist is selected from the group consisting of bevacizumab, ranibizumab, aflibercept, ramucirumab, and tanibirumab. (Item 12) 11. The method of item 10, wherein the Ang2 antagonist is selected from the group consisting of nesbacumab, AMG780, and MEDI3617. (Item 13) 13. The method according to any one of items 1 to 12, wherein the retinopathy is a retinal vascular disease. (Item 14) Item 14. The method of item 13, wherein the retinal vascular disease is caused by inhibition of vascular development. (Item 15) 14. The method of claim 13, wherein the retinopathy is caused by excessive neovascularization. (Item 16) Item 15. The method according to item 13 or item 14, wherein the retinal vascular disease is selected from the group consisting of familial exudative vitreoretinopathy (FEVR), exudative vitreoretinopathy, Norrie's disease, diabetic retinopathy (DR), age-related macular degeneration (AMD), retinopathy of prematurity (ROP), osteoporosis-pseudoglioma syndrome (OPPG), retinal vein occlusion, and Coats' disease.

Claims

1. A composition comprising a modified WNT agonist for treating retinopathy in a subject, wherein the modified WNT agonist comprises a sequence shown in SEQ ID NO:5 and a sequence shown in SEQ ID NO:6, and the modified WNT agonist is a bispecific antibody comprising an anti-Fzd epitope binding domain and an anti-LRP5 epitope binding domain.

2. The composition of claim 1, wherein the anti-Fzd epitope binding domain is specific for FZD4 and the anti-LRP5 epitope binding domain is specific for LRP5.

3. A combination comprising a modified WNT agonist and an anti-VEGF antibody, an anti-Ang2 antibody, or R-spondin for treating retinopathy in a subject, wherein the modified WNT agonist comprises the sequence shown in SEQ ID NO:5 and the sequence shown in SEQ ID NO:6, and the modified WNT agonist is a bispecific antibody comprising an anti-Fzd epitope binding domain and an anti-LRP5 epitope binding domain, and the modified WNT agonist and the anti-VEGF antibody, anti-Ang2 antibody, or R-spondin are administered independently at the early and / or late stages of the retinopathy.

4. A combination comprising a modified WNT agonist and an anti-VEGF antibody, an anti-Ang2 antibody, or an R-spondin for treating retinopathy in a subject, wherein the modified WNT agonist comprises the sequence shown in SEQ ID NO:5 and the sequence shown in SEQ ID NO:6, and the modified WNT agonist is a bispecific antibody comprising an anti-Fzd epitope binding domain and an anti-LRP5 epitope binding domain, and the modified WNT agonist and the anti-VEGF antibody, anti-Ang2 antibody, or R-spondin are administered sequentially at the early and / or late stages of the retinopathy.

5. A combination comprising a modified WNT agonist and an anti-VEGF antibody, an anti-Ang2 antibody, or R-spondin for treating retinopathy in a subject, wherein the modified WNT agonist comprises the sequence shown in SEQ ID NO:5 and the sequence shown in SEQ ID NO:6, and the modified WNT agonist is a bispecific antibody comprising an anti-Fzd epitope binding domain and an anti-LRP5 epitope binding domain, and the modified WNT agonist and the anti-VEGF antibody, anti-Ang2 antibody, or R-spondin are co-administered at early and / or late stages of the retinopathy.

6. The combination of claim 4, wherein the WNT agonist is administered before or after the anti-VEGF antibody, anti-Ang2 antibody, or R-spondin.

7. A combination comprising a modified WNT agonist and an antibody specific for either VEGF and / or Ang2 for treating retinopathy in a subject, wherein the modified WNT agonist comprises a sequence shown in SEQ ID NO:5 and a sequence shown in SEQ ID NO:6, and the modified WNT agonist is a bispecific antibody comprising an anti-Fzd epitope binding domain and an anti-LRP5 epitope binding domain.

8. The combination of claim 7, wherein said antibody specific for VEGF or Ang2 is an antagonist of VEGF or Ang2 activity.

9. 9. The combination of claim 8, wherein the VEGF antagonist is selected from the group consisting of bevacizumab, ranibizumab, aflibercept, ramucirumab, and tanibirumab.

10. 9. The combination of claim 8, wherein the Ang2 antagonist is selected from the group consisting of nesbacumab, AMG780, and MEDI3617.

11. A combination comprising a modified WNT agonist and R-spondin for treating retinopathy in a subject, wherein the modified WNT agonist comprises a sequence shown in SEQ ID NO:5 and a sequence shown in SEQ ID NO:6, and the modified WNT agonist is a bispecific antibody comprising an anti-Fzd epitope binding domain and an anti-LRP5 epitope binding domain.

12. The composition according to claim 1 or claim 2, or the combination according to any one of claims 3 to 11, wherein the retinopathy is a retinal vascular disease.

13. The composition or combination of claim 12, wherein the retinal vascular disease is caused by inhibition of vascular development.

14. The composition or combination of claim 12, wherein the retinopathy is caused by excessive neovascularization.

15. 14. The composition or combination of claim 12 or claim 13, wherein the retinal vascular disease is selected from the group consisting of familial exudative vitreoretinopathy (FEVR), exudative vitreoretinopathy, Norrie's disease, diabetic retinopathy (DR), age-related macular degeneration (AMD), retinopathy of prematurity (ROP), osteoporosis pseudoglioma syndrome (OPPG), retinal vein occlusion, and Coats' disease.

16. A composition comprising a modified WNT agonist for treating retinopathy in a subject, wherein the modified WNT agonist comprises the sequence shown in SEQ ID NO:5 and the sequence shown in SEQ ID NO:6, and the modified WNT agonist is a bispecific antibody comprising an anti-Fzd epitope binding domain and an anti-LRP5 epitope binding domain, and the composition is administered in combination with an anti-VEGF antibody, an anti-Ang2 antibody, or an R-spondin, and the composition and the anti-VEGF antibody, anti-Ang2 antibody, or R-spondin are administered independently at the early and / or late stages of the retinopathy.

17. A composition comprising a modified WNT agonist for treating retinopathy in a subject, wherein the modified WNT agonist comprises the sequence shown in SEQ ID NO:5 and the sequence shown in SEQ ID NO:6, and the modified WNT agonist is a bispecific antibody comprising an anti-Fzd epitope binding domain and an anti-LRP5 epitope binding domain, and the composition is administered in combination with an anti-VEGF antibody, an anti-Ang2 antibody, or an R-spondin, wherein the composition and the anti-VEGF antibody, the anti-Ang2 antibody, or an R-spondin are administered sequentially at the early and / or late stages of the retinopathy.

18. A composition comprising a modified WNT agonist for treating retinopathy in a subject, wherein the modified WNT agonist comprises the sequence shown in SEQ ID NO:5 and the sequence shown in SEQ ID NO:6, and the modified WNT agonist is a bispecific antibody comprising an anti-Fzd epitope binding domain and an anti-LRP5 epitope binding domain, and the composition is administered in combination with an anti-VEGF antibody, an anti-Ang2 antibody, or an R-spondin, wherein the composition and the anti-VEGF antibody, anti-Ang2 antibody, or R-spondin are administered simultaneously at early and / or late stages of the retinopathy.

19. A composition comprising a modified WNT agonist for treating retinopathy in a subject, wherein the modified WNT agonist comprises the sequence shown in SEQ ID NO:5 and the sequence shown in SEQ ID NO:6, and the modified WNT agonist is a bispecific antibody comprising an anti-Fzd epitope binding domain and an anti-LRP5 epitope binding domain, and the composition is administered in combination with an antibody specific for either VEGF and / or Ang2.

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