R-spondin (RSPO) surrogate molecule

Abstract

Provided are R-spondin (RSPO) surrogate compositions and methods for using the same. The RSPO surrogate of the present invention comprises (i) a specific binding domain for RING finger protein 43 (RNF43) or zinc and RING finger protein 3 (ZNRF3), and (ii) a cell-targeting domain, more specifically, the specific binding domain for RNF43 or ZNRF3 is an antibody fragment, and the cell-targeting domain is a cytokine. [Selection diagram] None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 62 / 444,987, filed Jan. 11, 2017, the contents of each of which are incorporated herein by reference. [Background technology]

[0002] The evolutionarily conserved Wnt signaling pathway plays a crucial role in embryonic development and adult tissue homeostasis in all multicellular animals. Wnt proteins are secreted lipoglycoprotein ligands that control cell proliferation, migration, cell fate specification, and polarity formation. The canonical Wnt signaling cascade drives specific gene expression programs through regulation of the stability of the transcriptional cofactor β-catenin. Wnt proteins can also activate the β-catenin-independent planar cell polarity (PCP) pathway to coordinate cell and tissue movements. The Frizzled (FZD) family of seven-transmembrane domain proteins serves as the core receptor for Wnt proteins and is required for both Wnt / β-catenin and Wnt / PCP signaling. Wnt proteins utilize different coreceptors to activate different downstream signaling pathways: Wnt proteins bind to the coreceptors LRP5 / 6 to activate the Wnt / β-catenin pathway, and to the coreceptors ROR1 / 2, RYK, or PTK7 to initiate the Wnt / PCP pathway.

[0003] Ubiquitination-mediated turnover of Wnt receptors has been shown to be a crucial regulatory mechanism in Wnt pathway activity. Cell surface FZD levels are stabilized by UBPY / USP8 and USP6, suggesting that ubiquitination serves as a key regulatory mechanism underlying FZD lysosomal degradation. The cell surface transmembrane E3 ubiquitin ligase zinc and ring finger 3 (ZNRF3) and its functional homologue RING finger protein 43 (RNF43) act as negative feedback regulators of Wnt signaling. ZNRF3 and RNF43 inhibit Wnt / β-catenin signaling by promoting the ubiquitination and subsequent internalization and degradation of Wnt receptors FZD and LRP6. Dishevelled (DVL) acts as a positive regulator of Wnt signaling by directly binding to FZDs, and is an adaptor protein that targets ZNRF3 / RNF43 to FZDs to promote their ubiquitination and degradation.

[0004] R-spondin proteins (RSPO1-4) are secreted proteins that potently sensitize cells to Wnt / β-catenin and Wnt / PCP signaling. All four RSPO proteins have a similar domain structure with two N-terminal furin domains and one C-terminal TSR domain. LGR4, LGR5, and LGR6 are high-affinity receptors for RSPO, and RSPO requires LGR4 / 5 / 6 to activate Wnt signaling but does not activate canonical GPCR signaling downstream of LGR4 / 5 / 6. RSPO enhances Wnt signaling by simultaneously binding to the extracellular domains of ZNRF3 / RNF43 and LGR4 / 5 / 6, inducing autoubiquitination and membrane clearance of ZNRF3 / RNF43, leading to increased cell surface levels of FZD. The regulation of FZD turnover explains how RSPO can control both Wnt / β-catenin and Wnt / PCP signaling. RSPO binds to LGR4 / 5 / 6 via its furin 1 and 2 domains and to ZNRF3 / RNF43 via its furin 1 domain. To function, RSPO must interact with both LGR4 / 5 / 6 and ZNRF3 / RNF43. The Wnt-stimulating activity of various RSPO proteins correlates with the binding affinity of the RSPO proteins to ZNRF3 or RNF43.

[0005] A number of Wnt antagonists are secreted that function by blocking receptor access, sequestration of Wnt ligands, or degradation of Wnt. In contrast, RSPO (Rspo1-4) is the only secreted enhancer of Wnt signaling and has been shown to be a critical regulator of stem cell maintenance in vivo and in vitro. RSPO has been explored for many therapeutic applications ranging from bone regeneration to intestinal recovery after chemotherapy. RSPO is an attractive drug candidate because it amplifies existing Wnt signals while avoiding potential carcinogenic or toxic off-target effects of global Wnt activation. However, RSPO has limited clinical utility due to the requirement for LGR4 / 5 / 6 expression in the target cell type, and the broad tissue expression profile of RNF43 / ZNRF3 and LGR4 / 5 / 6, which may result in RSPO action on many tissues leading to pleiotropic effects and undesirable toxicity.

[0006] Potential barriers to the clinical use of Wnt agonists include off-target effects of RNF43 / ZNRF3-mediated antagonism and toxicity. Wnts are potent morphogens, and overactivation of the Wnt pathway has been linked to the development of several human cancers. Therefore, the development of surrogate molecules that provide RSPO activity that can be delivered in a cell- and tissue-specific manner is of great clinical interest. Summary of the Invention

[0007] Compositions and methods of use of proteins acting as surrogate RSPOs (RSPOs) to enhance Wnt signaling to target cell types are provided. In some embodiments, the RSPO surrogates avoid the requirement for expression of the RSPO cognate receptors LGR4, LGR5, or LGR6 for enhanced Wnt signaling. In other embodiments, the RSPO surrogates specifically target one or more of LGR4, LGR5, or LGR6 for enhanced Wnt signaling, thereby providing enhanced selectivity for RSPO activity. In some other embodiments, the RSPO surrogates specifically target one or more cell surface receptors that do not include LGR4, LGR5, or LGR6 for enhanced Wnt signaling, thereby providing enhanced selectivity for RSPO activity.

[0008] As used herein, a RSPO surrogate comprises (i) a specific binding domain for RNF43 or ZNRF3, and (ii) a cell targeting domain specific for a cell surface receptor expressed on a desired cell type to enable Wnt enhancement specific to the correct cell and tissue. The domains may be directly linked or separated by a linker (e.g., a polypeptide linker, or a non-peptide linker, etc.). The length of the linker, and therefore the spacing between the binding domains, can be used to adjust the length of the signal and can be selected depending on the desired use of the RSPO surrogate. A polypeptide RSPO surrogate can be single chain, dimeric, or higher order multimeric.

[0009] The RNF43 or ZNRF3 binding domain has high affinity, e.g., 1×10 -6 M or less, 1×10 -7 M or less, 1×10 -8 M or less, 1×10 -9 M, or 1×10 -10The specific binding domain for RNF43 or ZNRF3 may be selected from any domain that binds to RNF43 or ZNRF3 with a Kd of M or less. Suitable binding domains include, but are not limited to, de novo designed binding proteins, antibody-derived binding proteins (e.g., scFv, Fab, etc.) and other portions of antibodies that specifically bind to RNF43 or ZNRF3 proteins; nanobody-derived binding proteins; knottin-based engineered scaffolds; Norrin and engineered binding fragments derived therefrom, naturally occurring binding domains, and the like. In some embodiments, the specific binding domain for RNF43 or ZNRF3 is a binding fragment of RSPO, for example, comprising, consisting of, or consisting essentially of the RSPO furin1 domain. The binding domain may be affinity selective to enhance binding to a desired protein or proteins.

[0010] The cell targeting domain or element may be a target of high affinity, e.g., 1×10 -7 M or less, 1×10 -8 M or less, 1×10 -9 M or less, 1×10 -10 K below M D The targeting domain may be selected from any domain that selectively binds to cell surface proteins, carbohydrates, or lipids at the targeting site. Suitable cell targeting domains include, but are not limited to, de novo designed binding proteins, antibody-derived binding proteins (e.g., scFv, Fab, etc.), and other portions of antibodies that specifically bind to cell surface proteins, carbohydrates, or lipids; nanobody-derived binding proteins; knottin-based engineered scaffolds; naturally occurring binding domains or polypeptides, cytokines, growth factors, and the like.

[0011] In some embodiments, the cell targeting domain is a cytokine or growth factor that has a cognate receptor on target cells. In some embodiments, the cytokine or growth factor contains a mutation or mutations that reduce the binding affinity to one or more of its cognate receptors. In some such embodiments, the surrogate RSPO can selectively target cells that express individual receptor subunits of a multi-subunit cytokine or growth factor receptor complex.

[0012] In some embodiments, the targeting domain is a covalent small molecule, carbohydrate, or nucleotide-derived molecule that binds to cell surface proteins, carbohydrates, or lipids.

[0013] In some embodiments, the cell targeting domain is an antibody or an active fragment thereof that has specificity for an antigen present on the target cell surface. In some such embodiments, the antigen is one or more of LGR4, LGR5, or LGR6, for example, an antibody that selectively binds to a single LGR protein (i.e., one of LGR4, LGR5, or LGR6). The LGR binding moiety can be selective for the LGR protein of interest, for example, having at least 10-fold, 25-fold, 50-fold, 100-fold, 200-fold, or more specificity for the desired LGR protein compared to other LGR proteins.

[0014] Contacting a target cell with a RSPO surrogate enhances signaling in the Wnt pathway in the presence of Wnt, for example, activity can be increased by at least about 10%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, and can be about a two-fold, three-fold, four-fold, five-fold, or more increase compared to activity in the absence of the RSPO surrogate.

[0015] In some embodiments, the linker is a rigid linker, and in other embodiments, the linker is a flexible linker. When the linker is a peptide linker, the linker can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 amino acids or more in length, and of sufficient length and amino acid composition to enhance the distance between the binding domains. In some embodiments, the linker comprises or consists of one or more glycine and / or serine residues.

[0016] RSPO surrogates can be multimerized, for example, via the Fc domain, by linkage, coiled coil, polypeptide zipper, biotin / avidin or streptavidin multimerization, etc. RSPO surrogates can also be linked to moieties such as PEG, Fc, etc., as known in the art, to enhance stability in vivo.

[0017] The subject compositions include, but are not limited to, an effective dose of a RSPO surrogate in a pharma- ceutically acceptable excipient. The compositions may also include additional agents, such as adjuvants. RSPO surrogates can be produced synthetically; by a variety of suitable recombinant methods, as known in the art; and the like.

[0018] In some aspects of the present invention, methods are provided for enhancing Wnt signaling in cells. In such methods, cells expressing frizzled receptors are contacted with RSPO surrogates in the presence of active Wnt proteins on the frizzled receptors at a concentration effective to increase signaling, for example, at a concentration effective to increase signaling by 25%, 50%, 75%, 90%, 95% or more compared to signaling in the absence of the RSPO surrogates. Such signaling activation can induce proliferation, differentiation, or a specific gene expression profile in / within target cells (such target cells include, but are not limited to, stem cells), or otherwise enhance the Wnt signaling pathway in target cells. In some methods, the cells are contacted in vitro. In other embodiments, the cells are contacted in vivo. Cells of interest include a wide variety of Fzd receptor expressing cells, such as skin cells, intestinal cells, osteoblasts, liver cells, chondrocytes, hair cells, stem cells, adult stem cells, and the like, as known in the art.

[0019] In some aspects of the invention, a RSPO surrogate is fused or conjugated to a Wnt agonist, a surrogate Wnt agonist, or a native Wnt protein to enhance Wnt signaling activity, hi other embodiments, enhanced Wnt signaling is achieved by co-administering a RSPO surrogate with a Wnt agonist, a surrogate Wnt agonist, or a native Wnt protein.

[0020] In some aspects of the invention, methods are provided for treating or preventing a disease or disorder in a subject in need thereof, comprising providing to the subject an effective amount of a RSPO surrogate. In certain embodiments, the subject has a disease or disorder associated with reduced Wnt signaling or naturally low Wnt signaling. In some aspects of the invention, methods are provided for enhancing wound healing and / or tissue generation in a subject in need thereof, comprising providing to the subject an effective amount of a RSPO surrogate.

[0021] In some aspects of the invention, RSPO surrogates are targeted to selectively regulate the activity of regulatory T cells that are suppressed by Wnt activation. In some such embodiments, the surrogate RSPO comprises IL-2 or an active fragment or derivative thereof as a targeting protein. IL-2 preferentially binds to regulatory T cells (T reg) over effector T cells. In related embodiments, RSPO is targeted to cell type-specific surface markers on cells, including, but not limited to, macrophages, NK cells, dendritic cells, B cells, effector T cells, and the like.

[0022] In some aspects of the invention, methods are provided for surrogate RSPO-mediated intestinal stem cell restoration, a process driven by three protein factors: RSPO, epidermal growth factor (EGF), and Noggin. The surrogate RSPO can comprise an RNF43 / ZNRF3 binding protein fused to EGF, which circumvents the requirement for co-administration of RSPO and EGF by simultaneously activating RSPO and EGF signaling with a single agent. In certain embodiments, for example, the following are provided: (Item 1) A RSPO surrogate composition comprising: (i) a specific binding domain for RNF43 or ZNRF3; and (ii) a cell targeting domain. (Item 2) The composition of claim 1, wherein the specific binding domain for RNF43 or ZNRF3 is a protein or a small molecule. (Item 3) 2. The composition of claim 1, wherein the specific binding domain for RNF43 or ZNRF3 is a fragment of RSPO. (Item 4) The composition of claim 1, wherein the specific binding domain for RNF43 or ZNRF3 is an antibody or a fragment thereof. (Item 5) The composition according to item 4, wherein the specific binding domain for RNF43 or ZNRF3 comprises one to six CDR sequences of the Z6 or R5 binding sequence shown in SEQ ID NOs: 1 to 4. (Item 6) 6. The composition according to any of items 1 to 5, wherein the specific binding domain for RNF43 or ZNRF3 is a single chain Fv (scFv) construct. (Item 7) 7. The composition according to any of items 1 to 6, wherein the cell targeting domain is a cytokine, a growth factor, a small molecule, a nucleotide, a carbohydrate, a hormone, or an antibody or a fragment thereof specific for a cell surface antigen. (Item 8) The composition of claim 7, wherein the antibody is specific to LRG4, 5, or 6 protein. (Item 9) 8. The composition of claim 7, wherein the cell targeting domain is a cytokine. (Item 10) 10. The composition of item 9, wherein the cytokine is IL-2 or IL-4. (Item 11) 11. The composition according to any one of items 1 to 10, wherein the domains are fused via a flexible linker. (Item 12) 11. The composition according to any one of items 1 to 10, wherein the domains are directly linked. (Item 13) 13. The composition according to any of items 1 to 12, formulated with an effective dose of a wnt agent. (Item 14) 13. The composition according to any of items 1 to 12, linked to a wnt agent. (Item 15) 15. A pharmaceutical composition comprising an effective dose of the RSPO surrogate of any one of items 1 to 14 and a pharma- ceutically acceptable excipient. (Item 16) A method for enhancing Wnt signaling, comprising contacting a cell expressing a Frizzled receptor with an effective dose of a RSPO surrogate of any one of items 1 to 15 in the presence of a Wnt agonist. (Item 17) 16. A method for treating or preventing a disease or disorder in a subject in need thereof, the method comprising providing to the subject an effective amount of a RSPO surrogate according to any one of items 1 to 15. (Item 18) The method of claim 17, wherein the subject has a disease or disorder associated with decreased Wnt signaling. (Item 19) 19. The method of item 17 or 18, wherein the subject has a disease or disorder selected from radiation / chemotherapy injury, mucositis, inflammatory bowel disease, short bowel syndrome, hereditary enteropathy, celiac disease, metabolic disease, genetic syndrome, viral infection (e.g., HepB / C), toxic states, alcoholic liver, fatty liver, cirrhosis, infection, pernicious anemia, ulcer, diabetes, diabetic foot ulcer (e.g., refractory diabetic foot ulcer), destruction of islet cells, loss of bone mass (osteoporosis), loss of functional skin, loss of hair, loss of functional lung tissue, loss of kidney tissue (e.g., acute tubular necrosis), loss of sensory cells in the inner ear, joint disorders, osteoporosis and related bone diseases, alopecia, and graft versus host disease. (Item 20) 16. A method for enhancing wound healing and / or tissue generation in a subject in need thereof, the method comprising providing to the subject an effective amount of a RSPO surrogate according to any one of items 1 to 15.

[0023] The invention will be best understood by reference to the following detailed description taken in conjunction with the accompanying drawings, in which: It is emphasized that, according to common practice, the various features of the drawings are not drawn to scale. In other respects, dimensions of the various features have been arbitrarily expanded or reduced for clarity. The following figures are included in the drawings: [Brief description of the drawings]

[0024] [Figure 1] Examples of surrogate RSPO proteins. [Diagram 2] Antibody screening. To generate scFv antibody fragments specific for RNF43 or ZNRF3, binders were selected from a yeast display library of naive human scFv constructs. [Diagram 3] A candidate surrogate RSPO was generated by fusing Z6 scFv, which specifically binds ZNRF3, to human IL2. [Figure 4] The Z6-IL2 fusion was expressed in insect cells and purified by nickel and gel filtration chromatography. Z6-IL2 eluted as a monodisperse peak from the gel filtration column, indicating that the protein does not aggregate and has favorable biochemical behavior. [Figure 5-1] Biological effects of surrogate RSPOs. [Figure 5-2] Biological effects of surrogate RSPOs. [Figure 6] Further surrogate RSPO constructs. [Figure 7]R-spondin signaling mechanisms. In the absence of RSPO, ZNRF3 drives membrane clearance of Fzd receptors to negatively regulate Wnt signaling. RSPO-mediated cross-linking of the ECDs of RNF43 and ZNRF3 with LGR4, LGR5, or LGR6 sequesters RNF43 / ZNRF3 and restores Fzd surface levels, thereby enhancing Wnt activity. Surrogate RSPOs mimic the function of wild-type RSPO by cross-linking RNF43 or ZNRF3 with tissue-specific markers known to undergo endocytosis upon ligand binding. [Figure 8A-C] Characterization of scFvs specific for RNF43 and ZNRF3. (A) Flow cytometry dot plots showing binding of yeast-displayed R5 and Z6 scFvs to RNF43 and ZNRF3, respectively. R5- or Z6-expressing yeast was stained with 1 uM concentration of RNF43 or ZNRF3 and surface expression was detected with an antibody against the c-Myc epitope. (B) Construct design of R5-IL2 and Z6-IL2 surrogate RSPO. (C) and (D). Binding of R5-IL2 or Z6-IL2 to RNF43 and ZNRF3, respectively, was measured using SPR. Dissociation constants were obtained from fitting values ​​to a 1:1 binding model. [Figure 8D] Characterization of scFvs specific for RNF43 and ZNRF3. (A) Flow cytometry dot plots showing binding of yeast-displayed R5 and Z6 scFvs to RNF43 and ZNRF3, respectively. R5- or Z6-expressing yeast was stained with 1 uM concentration of RNF43 or ZNRF3 and surface expression was detected with an antibody against the c-Myc epitope. (B) Construct design of R5-IL2 and Z6-IL2 surrogate RSPO. (C) and (D). Binding of R5-IL2 or Z6-IL2 to RNF43 and ZNRF3, respectively, was measured using SPR. Dissociation constants were obtained from fitting values ​​to a 1:1 binding model. [Figure 9A]Enhancement of Wnt signaling by surrogate RSPO. Luciferase reporter assay measuring enhancement of Wnt activity of surrogate RSPO in CD25-expressing cells. HEK STF 293T cells lentivirally transduced with CD25 were incubated with various recombinant proteins in the presence of 20% Wnt3a-conditioned medium. [Figure 9B] Reporter assays were performed using uninfected (CD25-negative) HEK STF 293T cells under the same conditions as in A. [Figure 10] Stimulation of intestinal organoid growth by surrogate RSPO. Surrogate RSPO were tested for their ability to stimulate the growth of LGR5+ human colon organoids transduced by lentivirus to express CD25. RSPO2 lacking medium was supplemented with the indicated proteins at a concentration of 500 nM, and organoid growth was monitored by microscopy (top) and fluorescence (bar graph, bottom). The bar graph on the right is a panel that expands the colored area of ​​the graph on the left. [Figure 11] R5-IL2 and Z6-IL2 proteins eluted as monodisperse peaks from a gel filtration column. UV280 absorbance from gel filtration profiles of R5-IL2 (left) and Z6-IL2 (right) are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Before the methods and compositions of the present invention are described, it is to be understood that this invention is not limited to the particular methods and compositions described, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present specification will be limited only by the appended claims.

[0026] Where a range of values ​​is given, it is understood that each intervening value (to the nearest tenth of the lower limit between the upper and lower limits of that range, unless the context clearly dictates otherwise) is also expressly disclosed. Each subrange between any stated or intervening value and any other stated or intervening value within a stated range is included in the invention. The upper and lower limits of these subranges may be independently included or excluded within the subrange, and each range in which either, neither, or both of the upper and lower limits are included in the invention, subject to any expressly excluded limitations in the stated range. Where a stated range includes one or both of the upper and lower limits, ranges excluding either or both of the included limits are also included in the invention.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In carrying out or testing the present invention, any method and material similar or equivalent to the method and material described herein may be used, but some promising and preferred methods and materials are described below. All publications mentioned in this specification are incorporated herein by reference to disclose and describe the method and / or material related to which these publications are cited. It should be understood that this disclosure supersedes any disclosure of the incorporated publication to the extent that there is a conflict.

[0028] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells, a reference to "the peptide" includes a reference to one or more peptides and equivalents thereof (e.g., polypeptides) known to those skilled in the art, and so forth.

[0029] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0030] "Comprising" means that the recited element is required in the composition / method / kit, although other elements may be included to form the composition / method / kit etc. within the scope of the claims. For example, a composition that comprises a RSPO surrogate is a composition that may include other elements in addition to the RSPO surrogate(s), such as functional moieties, such as polypeptides, small molecules, or nucleic acids, that are attached, e.g., covalently attached, to the RSPO surrogate; agents that promote the stability of the RSPO surrogate composition, agents that promote the solubility of the RSPO surrogate composition, adjuvants, etc., as would be readily understood in the art, with the exception of elements encompassed by any negative qualifications.

[0031] "Consisting essentially of" refers to a limitation of the scope of the composition or method described to specified materials or steps that do not substantially affect the basic novel feature(s) of the subject invention. For example, an RSPO surrogate "consisting essentially of" a disclosed sequence has an amino acid sequence that, relative to the disclosed sequence, plus or minus about 5 amino acid residues at the boundaries of the sequence based on the sequence from which it is derived, e.g., about 5, 4, 3, 2, or about 1 fewer amino acid residues than the recited boundary amino acid residue, or about 1, 2, 3, 4, or 5 more amino acid residues than the recited boundary amino acid residue.

[0032] "Consisting of" means excluding from a composition, method, or kit any element, step, or ingredient not specified in the claim. For example, a RSPO surrogate "consisting of" a disclosed sequence consists only of the disclosed amino acid sequence.

[0033] The term "specific binding" refers to binding that occurs between paired species, such as enzyme / substrate, receptor / ligand, antibody / antigen, and lectin / carbohydrate, which may be mediated by covalent or non-covalent interactions, or a combination of covalent and non-covalent interactions. When the interaction of two species produces a non-covalently bound complex, the resulting binding is typically electrostatic, hydrogen bonded, or the result of lipophilic interactions. Thus, "specific binding" occurs between paired species when there is an interaction between the two species, producing a bound complex that has the characteristics of an antibody / antigen or ligand / receptor interaction. The biological activity of the RSPO surrogate in the composition can be determined by determining the level of activity (e.g., accelerated bone regeneration, enhanced hepatocyte proliferation, etc.; nuclear localization of β-catenin, increased transcription of Wnt-responsive genes, etc.) in a functional assay after in vivo administration.

[0034] "Functional moiety" or "FM" means a polypeptide, small molecule, carbohydrate, or nucleic acid composition that confers functional activity to a composition. Examples of functional moieties include, but are not limited to, therapeutic moieties, binding moieties, and imaging moieties.

[0035] "Therapeutic moiety" or "TM" refers to a polypeptide, small molecule, or nucleic acid composition that confers therapeutic activity to the composition. Examples of therapeutic moieties include cytotoxins, e.g., small molecule compounds, protein toxins, and radiosensitizing moieties that are naturally harmful to cells, i.e., radionuclides, etc.; agents that alter the activity of cells, e.g., small molecules, peptidomimetics, cytokines, chemokines; and moieties that target cells for ADCC or CDC-dependent death, e.g., the Fc component of an immunoglobulin.

[0036] "Imaging moiety" or "IM" means a non-cytotoxic agent that can be used to locate and optionally visualize cells, e.g., cells targeted by a composition of the subject application.

[0037] The terms "treatment", "treating" and the like are used herein broadly to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or its symptoms, and / or therapeutic in terms of partially or completely curing the disease or adverse effects that may result from the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, including (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed as having it, (b) inhibiting the disease, i.e. arresting its development, or (c) alleviating the disease, i.e. causing regression of the disease. Therapeutic agents can be administered before, during, or after the onset of the disease or injury. Treatment of ongoing disease is of particular interest if the treatment stabilizes or reduces undesirable clinical symptoms in the patient. It is desirable for such treatment to be performed before complete loss of function in the affected tissue. The subject therapy can be administered during, and in some cases after, the symptomatic stage of the disease.

[0038] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject for which diagnosis, treatment, or therapy is desired, particularly humans.

[0039] General methods in molecular and cellular biochemistry are described in Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., CSH Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons Genetic engineering techniques, such as those described herein, can be found in standard textbooks, such as The Journal of Molecular Biology (1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors, such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.

[0040] Polypeptides As used herein, "protein" refers to any composition composed of amino acids and recognized by those of skill in the art as a protein. "Protein", "peptide", and polypeptide are used interchangeably herein. Amino acids may be referred to by their full name (e.g., alanine) or by their accepted one-letter (e.g., A) or three-letter (e.g., ala) abbreviations. When a peptide is part of a protein, those of skill in the art will understand the use of the term in context. The term "protein" encompasses the mature form of a protein as well as the pro and prepro forms of the related protein. A prepro form of a protein includes a mature form of a protein having a prosequence operably linked to the amino terminus of the protein and a "pre" or "signal" sequence operably linked to the amino terminus of the prosequence.

[0041] As used herein, "protein of interest" refers to the protein being characterized, identified, and / or modified. In addition to naturally occurring proteins, recombinant proteins, synthetically produced proteins, variant proteins, and derived proteins all find use in the present invention.

[0042] As used herein, functionally similar proteins are considered "related proteins." In some embodiments, such proteins are from different genera and / or species, including differences between classes of organisms (e.g., bacterial proteins and fungal proteins). In additional embodiments, related proteins are provided from the same species. Indeed, it is not intended that the present invention be limited to related proteins from any particular source(s).

[0043] The term "derivative" as used herein refers to a protein derived from a precursor protein by the addition of one or more amino acids to either or both the C-terminus and N-terminus, the substitution of one or more amino acids at multiple different sites in the amino acid sequence, and / or the deletion of one or more amino acids at one or more sites in the amino acid sequence or at either or both termini of the protein, and / or the insertion of one or more amino acids at one or more sites in the amino acid sequence. Preparation of a protein derivative is preferably accomplished by modifying a DNA sequence encoding the native protein, transforming the DNA sequence into a suitable host, and expressing the modified DNA sequence to form the derivative protein.

[0044] One type of related (and derivative) protein is a "variant protein". In a preferred embodiment, variant proteins differ from parent proteins and each other by a small number of amino acid residues. The number of different amino acid residues can be one or more, preferably 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50 or more amino acid residues. In one preferred embodiment, the number of different amino acids between variants is between 1 and 10. In a particularly preferred embodiment, related proteins, particularly variant proteins, comprise at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity. Furthermore, as used herein, a related protein or variant protein refers to a protein that differs from another related protein or parent protein in the number of prominent regions. For example, in some embodiments, the variant protein has 1, 2, 3, 4, 5, or 10 corresponding prominent regions that differ from the parent protein.

[0045] As used herein, "corresponding to" refers to a residue at a recited position in a protein or peptide, or a residue that is similar, homologous, or equivalent to a recited residue in a protein or peptide. As used herein, a "corresponding region" generally refers to a similar position along a related or parent protein.

[0046] The term "analogous sequence" as used herein refers to a sequence in a protein that provides a similar function, tertiary structure, and / or conserved residues to the protein of interest (i.e., typically the original protein of interest). In particularly preferred embodiments, the analogous sequence involves a sequence(s) at or near the epitope. For example, in epitope regions that contain an alpha-helix or beta-sheet structure, the replaced amino acids in the analogous sequence preferably maintain the same specific structure. This term refers to nucleotide sequences as well as amino acid sequences. In some embodiments, the analogous sequence occurs such that the replaced amino acids at or near the epitope exhibit similar functions, tertiary structures, and / or conserved residues as the amino acids in the protein of interest. Thus, if the epitope region contains, for example, an alpha-helix or beta-sheet structure, the replaced amino acids preferably maintain that specific structure.

[0047] As used herein, "homologous protein" refers to a protein that has the same action, structure, antigenicity, and / or immunogenic response as the protein of interest. It is intended that homologs and proteins of interest are not necessarily evolutionarily related. Thus, the term encompasses the same functional proteins obtained from different species.

[0048] As used herein, "wild-type" and "native" proteins refer to proteins found in nature. The terms "wild-type sequence" and "wild-type gene" are used interchangeably herein and refer to a sequence that is native or naturally occurring in a host cell. In some embodiments, a wild-type sequence refers to a sequence of interest that is the starting point for a protein modification project. Genes encoding naturally occurring (i.e., precursor) proteins can be obtained according to common methods known in the art.

[0049] "Wnt gene product" or "Wnt polypeptide" as used herein encompasses native sequence Wnt polypeptides, Wnt polypeptide variants, Wnt polypeptide fragments, and chimeric Wnt polypeptides. In certain embodiments, the Wnt polypeptide is a native human full-length mature Wnt protein.

[0050] For example, human native sequence Wnt proteins of interest in the present application include the following: Wnt-1 (GenBank Accession No. NM_005430); Wnt-2 (GenBank Accession No. NM_003391); Wnt-2B (Wnt-13) (GenBank Accession Nos. NM_004185 (isoform 1), NM_024494.2 (isoform 2)), Wnt-3 (RefSeq.: NM_030753), Wnt3a (GenBank Accession No. NM_033131), Wnt-4 (GenBank Accession No. NM_030761), Wnt-5A (GenBank Accession No. NM_003392), Wnt-5B (GenBank Accession No. NM_032642), Wnt-6 (GenBank Accession No. NM_032642), Wnt-7 (GenBank Accession No. NM_032642), Wnt-8 (GenBank Accession No. NM_032642), Wnt-9 (GenBank Accession No. NM_032642), Wnt-10 (GenBank Accession No. NM_032642), Wnt-11 (GenBank Accession No. NM_032642), Wnt-12 (GenBank Accession No. NM_032642), Wnt-13 (GenBank Accession No. NM_032642), Wnt-14 (GenBank Accession No. NM_032642), Wnt-15 (GenBank Accession No. NM_032642), Wnt-16 (GenBank Accession No. NM_032642), Wnt-17 (GenBank Accession No. NM_032642), Wnt-18 (GenBank Accession No. NM_032642), Wnt-19 (GenBank Access _006522), Wnt-7A (GenBank Accession No. NM_004625), Wnt-7B (GenBank Accession No. NM_058238), Wnt-8A (GenBank Accession No. NM_058244), Wnt-8B (GenBank Accession No. NM_003393), Wnt-9A (Wnt-14) (GenBank Accession No. NM_00339 5), Wnt-9B (Wnt-15) (GenBank Accession No. NM_003396), Wnt-10A (GenBank Accession No. NM_025216), Wnt-10B (GenBank Accession No. NM_003394), Wnt-11 (GenBank Accession No. NM_004626), and Wnt-16 (GenBank Accession No. NM_016087)). Although individual members have varying degrees of sequence identity to the family, all encode small (i.e., 39-46 kD), secreted glycoproteins that are acylated, palmitoylated, and contain 23-24 conserved cysteine ​​residues with highly conserved spacing (McMahon, AP et al., Trends Genet. 1992;8:236-242; Miller, JR. Genome Biol. 2002;3(1):3001.1-3001.15).Other native sequences of the Wnt polypeptide of interest include orthologs of the above from any mammal, including domestic and farm animals, as well as zoo, laboratory, or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, rats, mice, frogs, zebrafish, fruit flies, worms, etc.

[0051] The RSPO family of proteins includes four members (Rspo1-4) that are conserved in vertebrates. The four RSPO proteins share approximately 40-60% pairwise sequence homology and have a common domain architecture consisting of one N-terminal secretory signal peptide sequence, two tandem furin-like cysteine-rich (Fu-CRD) domains, one thrombospondin type I repeat (TSP) domain, and one C-terminal basic amino acid-rich (BR) domain. Among the four subdomains, it has been demonstrated that the two central tandem Fu-CRD domains are essential and sufficient for RSPO stimulation of Wnt signaling. RSPO acts immediately upstream of Wnt proteins. Thus, RSPO-driven Wnt activation is sensitive to the presence of the extracellular Wnt receptor Dkk1.

[0052] The inherent Wnt-enhancing ability of RSPO, together with its dynamic expression pattern in embryonic tissues, predicts important and multifaceted roles for RSPO during embryogenesis. Among other activities, RSPO1 is involved in sex determination. Expression of RSPO-2 has been reported in follicular oocytes. This oocyte-derived RSPO appeared to direct primary follicle development towards the second stage in a paracrine manner. Such observations are consistent with the expression of multiple Wnt ligands and cognate frizzled receptors in the ovary. RSPO-3 plays a predominant role during placental development.

[0053] All four RSPOs bind with high affinity to all three LGR proteins.Exemplary RSPO protein sequences are published in Genbank, including, for example, R-spondin-1 isoform 1 precursor [Homo sapiens], accession number: NP_001033722.1; R-spondin-1 isoform 2 [Homo sapiens], accession number: NP_001229838.1; R-spondin-1 isoform 3 precursor [Homo sapiens], accession number: NP_001229839.1; R-spondin-1 isoform X1 [Homo sapiens], accession number: XP_006710646.1; R-spondin-2 isoform X3 [Homo sapiens], accession number: XP_016868884.1; R-spondin-2 isoform 3 [Homo sapiens], accession number: XP_016868884.1; sapiens], Accession: NP_001304871.1; R-spondin-2 isoform X2 [Homo sapiens], Accession: XP_011515321.1; R-spondin-2 isoform X1 [Homo sapiens], Accession: XP_011515320.1; R-spondin-2 isoform 2 precursor [Homo sapiens], Accession: NP_001269792.1; R-spondin-2 isoform 1 precursor [Homo sapiens], Accession: NP_848660.3 GI:222446611; R-spondin-3 precursor [Homo sapiens], Accession: NP_116173.2; R-spondin-4 isoform 1 precursor [Homo sapiens], accession: NP_001025042.2; R-spondin-4 isoform 2 precursor [Homo sapiens], accession: NP_001035096.1.

[0054] Involvement of E3 ubiquitin ligases in Wnt LGR / RSPO signaling. RNF43 and ZNRF3 are two highly homologous Wnt target genes and RING domain E3 ligases. Both proteins show a relationship in basic structure and sequence to Grail (Rnf128), a single-pass transmembrane E3 ligase with an extracellular PA domain. RNF43 and ZNRF3 specifically mediate multiubiquitination of lysines in the cytoplasmic loop of the 7TM domain of Frizzled. This leads to rapid endocytosis of Wnt receptors and destruction in lysosomes. RNF43 and ZNRF3 are encoded by Wnt target genes and therefore may function as negative feedback regulators of Wnt receptor expression. Loss of expression of these two E3 ligases is predicted to result in increased responsiveness to endogenous Wnt signals. Indeed, mutations in RNF43 are found in several human colon cancer cell lines and in a variety of human tumor types affecting the bile duct, pancreas, and ovary. RNF43 / ZNRF3-mediated membrane clearance of Wnt receptors is reversed upon addition of RSPO. The RSPO-LGR complex overrides RNF43 / ZNRF3, allowing persistence of surface frizzled receptors and boosting Wnt signaling strength.

[0055] Exemplary human E3 ubiquitin ligase sequences are published in Genbank and include, for example, E3 ubiquitin-protein ligase RNF43 isoform 1 precursor [Homo sapiens], accession: NP_001292473.1 or NP_060233.3; E3 ubiquitin-protein ligase RNF43 isoform 2 [Homo sapiens], accession: NP_001292474.1; E3 ubiquitin-protein ligase RNF43 isoform X1 [Homo sapiens], accession: XP_016880289.1 or XP_011523257.1; E3 ubiquitin-protein ligase RNF43 isoform X2 [Homo sapiens], accession: XP_016880289.1 or XP_011523257.1; sapiens], accession: XP_011523258.1; E3 ubiquitin-protein ligase ZNRF3 isoform 1 precursor [Homo sapiens], accession: NP_001193927.1; E3 ubiquitin-protein ligase ZNRF3 isoform 2 [Homo sapiens], accession: NP_115549.2.

[0056] Antibodies that specifically bind to RNF43 and ZNRF3 are known in the art, are commercially available, or can be generated de novo. RNF43 and ZNRF3 or fragments thereof can be used as immunogens or in screening assays to develop antibodies, for example, by screening libraries, immunizing animals, etc., as known in the art. Examples of known antibodies include, but are not limited to, the antibodies described herein.

[0057] The RNF43 / ZNRF3 binding domain can be affinity selective to enhance binding to the desired protein. Affinity selection methods for this purpose can optionally utilize one or more rounds of selection, by introducing targeted amino acid changes to generate a library of candidate coding sequences, transforming a cell population, for example yeast cells, with the candidate coding sequences, and selecting for the desired specificity (e.g., using paramagnetic microbeads). Typically, multiple rounds of selection are performed, and the resulting vectors are sequenced and used as the basis for protein modification.

[0058] In certain embodiments, the RNF43 / ZNRF3 binding domain comprises six CDR regions of an scFv antibody, as exemplified herein and shown in FIG.

[0059] In other embodiments, the binding domain comprises variable region sequences, or CDRs thereof, from any of a number of RNF43 / ZNRF3-specific antibodies, which are known in the art, commercially available, or can be generated de novo. RNF43 / ZNRF3 can be used as an immunogen or in screening assays to develop antibodies.

[0060] Group B, leucine-rich repeat G protein-coupled receptors (LGR4, 5, 6) are a unique class of GPCRs characterized by a large extracellular domain (ectodomain) with 17 copies of leucine-rich repeats (LRRs). The LRRs are structural motifs consisting of a conserved 11-residue sequence rich in hydrophobic amino acids, with leucines often at defined positions (LxxLxLxxNxL, where x is any amino acid). The tertiary fold in a series of LRR repeats is known as an α / β horseshoe. The ectodomain links ligand binding to the regulation of downstream LGR intracellular signaling pathways. The 17 LRR repeats in LGR4-6 receptors are flanked by a cysteine-rich LRRNT region at the N-terminus and a cysteine-rich LRRCT region at the C-terminus. The ectodomain mediates ligand binding to regulate downstream intracellular signaling pathways. LGR4-6 share approximately 50% sequence identity, play key roles in stem cell development, and are found on various epithelial stem cells (e.g., hair, skin, intestinal, breast tissue, etc.), while LGR5 is also strongly expressed in ovarian, liver, and lung cancers.

[0061] Exemplary human LGR protein sequences are published in Genbank and include, for example, leucine-rich repeat-containing G protein-coupled receptor 4 isoform 1 precursor [Homo sapiens], Accession: NP_060960.2; leucine-rich repeat-containing G protein-coupled receptor 4 isoform 2 precursor [Homo sapiens], Accession: NP_001333361.1; leucine-rich repeat-containing G protein-coupled receptor 5 isoform 3 precursor [Homo sapiens], Accession: NP_001264156.1; leucine-rich repeat-containing G protein-coupled receptor 5 isoform 2 precursor [Homo sapiens], Accession: NP_001264155.1; leucine-rich repeat-containing G protein-coupled receptor 5 isoform 1 precursor [Homo sapiens], accession: NP_003658.1; leucine-rich repeat-containing G protein-coupled receptor 6 isoform 3 [Homo sapiens], accession: NP_001017404.1; leucine-rich repeat-containing G protein-coupled receptor 6 isoform 2 [Homo sapiens], accession: NP_067649.2; leucine-rich repeat-containing G protein-coupled receptor 6 isoform 1 precursor [Homo sapiens], accession: NP_001017403.1.

[0062] As used herein, "cytokine(s)" refers to a general class of biological molecules that have an effect / influence on cells of the immune system. This definition is intended to include, but is not limited to, biological molecules that can act locally or circulate in the blood and that can target RSPO surrogates to cells of interest when used in the compositions or methods described herein. Exemplary cytokines for use in cell targeting include, but are not limited to, interferon-alpha (IFN-α), interferon-beta (IFN-β), and interferon-gamma (IFN-γ), interleukins (e.g., IL1 through IL29, particularly IL-2, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, and IL-18), tumor necrosis factors (e.g., TNF-alpha and TNF-beta), erythropoietin (EPO), MIP3a, monocyte chemotactic protein (MCP)-1, intracellular adhesion molecules (ICAMs), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). The term is also intended to encompass modified cytokine molecules (i.e., "variant cytokines"), including cytokines having substitutions, deletions, and / or additions to the cytokine receptor amino acid and / or nucleic acid sequence. Thus, the term is intended to encompass wild-type as well as recombinant, synthetically produced, and variant cytokine receptors. As used herein, "cytokine receptor" refers to a receptor molecule that recognizes and binds to a cytokine.

[0063] As an alternative to cytokines, multiple domains or molecules may be used to target RSPO surrogates to cells, including any growth factor with a specific cell surface receptor, a cell surface antigen to which an antibody or analog thereof can be derived, a small molecule that binds to a cell surface receptor, a nucleotide, a hormone, or a carbohydrate, etc.

[0064] The binding domain also includes derivatives, variants, and biologically active fragments of the above-mentioned polypeptides. A "variant" polypeptide refers to a biologically active polypeptide having less than 100% sequence identity to the indicated sequence, as defined below. Such variants include those that contain one or more amino acid modifications (e.g., insertions, deletions, or substitutions) compared to the indicated sequence, such as one or more amino acid residues added to the N-terminus or C-terminus of the native sequence or within the native sequence; polypeptides in which about 1-40 amino acid residues have been deleted and optionally replaced with one or more amino acid residues; and derivatives of the above-mentioned polypeptides in which amino acid residues have been covalently modified such that the resulting product has a non-naturally occurring amino acid. Typically, biologically active variants have an amino acid sequence that has at least about 90%, preferably at least about 95%, more preferably at least about 99% amino acid sequence identity to the native sequence polypeptide.

[0065] A "functional derivative" of a sequence is a compound that has a qualitative biological property in common with the original sequence. "Functional derivatives" include, but are not limited to, fragments of a sequence and derivatives of a sequence, provided that they have a common biological activity. The term "derivative" encompasses both amino acid sequence variants of a polypeptide and covalent modifications thereof.

[0066] RSPO surrogates and methods for use in the subject compositions can be modified using conventional biological and synthetic chemistry techniques to improve resistance to proteolysis, optimize solubility properties, or to make them more suitable as therapeutic agents. Analogs of such polypeptides include those that contain residues other than naturally occurring L-amino acids, such as D-amino acids or non-naturally occurring synthetic amino acids. D-amino acids may be substituted for some or all of the amino acid residues.

[0067] RSPO surrogates can be prepared by in vitro synthesis using conventional methods known in the art. A variety of commercially available synthesis equipment is available, e.g., automated synthesizers from Applied Biosystems, Inc., Beckman, etc. Synthesizers can be used to replace naturally occurring amino acids with artificial amino acids. The particular sequence and mode of preparation is determined by convenience, economics, required purity, etc. If desired, various groups may be introduced into the peptide during synthesis or expression, which allow for conjugation to other molecules or surfaces. Thus, cysteines can be used to make thioethers, histidines for conjugation to metal ion complexes, carboxyl groups for amide or ester formation, amino groups for amide formation, etc.

[0068] Linker. The RNF43 / ZNRF3 binding domain and the cell targeting domain can be separated by a linker (e.g., a polypeptide linker, or a non-peptide linker, etc.). The amino acid linker linking the domains can play an important role in the structure and function of multidomain proteins. There are many examples of proteins that require appropriate linker composition for their catalytic activity. In general, changing the length of the linker connecting the domains has been shown to affect protein stability, folding rate, and interdomain orientation (see George and Hering (2003) Prot. Eng. 15:871-879). The length of the linker in the RSPO surrogate, and therefore the spacing between the binding domains, can be used to adjust the length of the signal of the RSPO surrogate and can be selected depending on the desired use of the RSPO surrogate. The required distance between the binding domains of a RSPO surrogate can vary, but in certain embodiments can be less than about 100 angstroms, less than about 90 angstroms, less than about 80 angstroms, less than about 70 angstroms, less than about 60 angstroms, or less than about 50 angstroms.

[0069] In some embodiments, the linker is a rigid linker, and in other embodiments, the linker is a flexible linker. In some embodiments, the linker moiety is a peptide linker. In some embodiments, the peptide linker comprises 2 to 100 amino acids. In some embodiments, the peptide linker comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 1, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, but not more than 100 amino acids. In some embodiments, the peptide linker comprises between 5-75, 5-50, 5-25, 5-20, 5-15, 5-10, or 5-9 amino acids in length. Exemplary linkers include linear peptides having at least two amino acid residues, such as Gly-Gly, Gly-Ala-Gly, Gly-Pro-Ala, Gly-Gly-Gly-Gly-Ser. Suitable linear peptides include polyglycine, polyserine, polyproline, polyalanine, and oligopeptides consisting of alanyl and / or serinyl and / or prolinyl and / or glycyl amino acid residues.In some embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of Gly9, Glu9, Ser9, Gly5-Cys-Pro2-Cys, (Gly4-Ser)3, Ser-Cys-Val-Pro-Leu-Met-Arg-Cys-Gly-Gly-Cys-Cys-Asn, Pro-Ser-Cys-Val-Pro-Leu-Met-Arg-Cys-Gly-Gly-Cys-Cys-Asn, Gly-Asp-Leu-Ile-Tyr-Arg-Asn-Gln-Lys, and Gly9-Pro-Ser-Cys-Val-Pro-Leu-Met-Arg-Cys-Gly-Gly-Cys-Cys-Asn. In one embodiment, the linker comprises the amino acid sequence GSTSGSGKSSEGKG, or (GGGGS)n (where n is 1, 2, 3, 4, 5, etc.), although many such linkers are known and used in the art and may serve this purpose.

[0070] RSPO surrogates can be provided in a single-chain form, meaning that the binding domains are linked by peptide bonds via a linker peptide. In other embodiments, the binding domains are separate peptides and can be linked via non-peptide linkers.

[0071] Chemical groups used to attach binding domains include carbamates; amides (amine + carboxylic acid); esters (alcohol + carboxylic acid); thioethers (haloalkane + sulfhydryl; maleimide + sulfhydryl), Schiff bases (amine + aldehyde), ureas (amine + isocyanate), thioureas (amine + isothiocyanate), sulfonamides (amine + sulfonyl chloride), disulfides; hydrodrazones, lipids, and the like, as known in the art.

[0072] The linkage between the binding domains can include a spacer (e.g., an alkyl spacer), which can be linear or branched, usually linear, can include one or more unsaturated bonds, and typically has from 1 to about 300 carbon atoms, more usually from about 1 to 25 carbon atoms, and can be from about 3 to 12 carbon atoms. Spacers of this type can also include heteroatoms or functional groups, including amines, ethers, phosphodiesters, and the like. Particular structures of interest include (CH2CHO)n, where n is from 1 to about 12; (CH2CH2NH)n, where n is from 1 to about 12; [(CH2)n(C=O)NH(CH2) m ] z (n and m are from 1 to about 6, and z is from 1 to about 10; [(CH2)nOPO3(CH2) m ] z (n and m are 1 to about 6, and z is 1 to about 10). Such linkers can include polyethylene glycol, which may be linear or branched.

[0073] The binding domains may be linked via homo- or heterobifunctional linkers that have at one end a group capable of forming a stable bond to a hydrophilic head group and at the opposite end a group capable of forming a stable bond to a targeting moiety. Exemplary entities include azidobenzoylhydrazide, N-[4-(p-azidosalicylamino)butyl]-3'-[2'-pyridyldithio]propionamide), bis-sulfosuccinimidyl suberate, dimethyl adipimidate, disuccinimidyl tartrate, N-γ-maleimidobutyryloxysuccinamide ester, N-hydroxysuccinimidyl ... sulfosuccinimidyl-4-azidobenzoate, N-succinimidyl [4-azidophenyl]-1,3'-dithiopropionate, N-succinimidyl [4-iodoacetyl]aminobenzoate, glutaraldehyde, NHS-PEG-MAL; 4-[N-maleimidomethyl]cyclohexane-1-carboxylate succinimidyl; 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP); N,N'-(1,3-phenylene)bismaleimide; N,N'-ethylene-bis-(iodoacetamide); or 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester (SMCC); m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), and the extended chain analog of MBS, 4-(p-maleimidophenyl)butyric acid succinimide (SMPB). The succinimidyl group of these cross-linkers reacts with primary amines and the thiol-reactive maleimide forms a covalent bond with the thiol of a cysteine ​​residue.

[0074] Other reagents useful for this purpose include p,p'-difluoro-m,m'-dinitrodiphenyl sulfone (forms irreversible crosslinks with amino and phenolic groups); dimethyl adipimidate (specific for amino groups); phenol-1,4-disulfonyl chloride (reacts primarily with amino groups); hexamethylene diisocyanate or diisothiocyanate, or azophenyl-p-diisocyanate (reacts primarily with amino groups); disdiazobenzidine (reacts primarily with tyrosine and histidine); O-benzotriazoyloxytetramethyluronium hexafluorophosphate (HATU), dicyclohexylcarbodiimide, bromo-tris(pyrrolidino)phosphonium bromide (PyBroP); N,N-dimethylaminopyridine (DMAP); 4-pyrrolidinopyridine; N-hydroxybenzotriazole, and the like. Homobifunctional cross-linking reagents include bismaleimidohexane ("BMH").

[0075] Antibody: As used herein, the term "antibody" refers to a polypeptide that contains sufficient canonical immunoglobulin sequence elements to confer specific binding to a particular target antigen. As is known in the art, intact antibodies produced in nature are tetrameric agents of approximately 150 kD composed of two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (25 kD each) that associate with each other to form what is commonly referred to as a "Y-shaped" structure. Each heavy chain is composed of at least four domains, each about 110 amino acids long, including an amino-terminal variable (VH) domain (located at the tip of the Y structure) followed by three constant domains: CH1, CH2, and a carboxy-terminal CH3 (located at the base of the stem of the Y). A short region known as the "switch" connects the heavy chain variable and constant regions. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides together in an intact antibody. Each light chain is composed of two domains, an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain, which are separated from each other by another "switch". An intact antibody tetramer is composed of two heavy-light chain dimers, where the heavy and light chains are linked together by one disulfide bond and two other disulfide bonds connect the heavy chain hinge regions together, so that the dimers are linked together to form a tetramer. Naturally occurring antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold", which is formed from two beta sheets (e.g., 3-, 4-, or 5-stranded sheets) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as "complementarity determining regions" (CDR1, CDR2, and CDR3) and four more or less invariant "framework" regions (FR1, FR2, FR3, and FR4).When a native antibody folds, the FR regions form beta sheets that provide the structural framework for the domain, and the CDR loop regions from both the heavy and light chains are packed together in three dimensional space to create a single hypervariable antigen binding site located at the tip of a Y structure.

[0076] The Fc region of a naturally occurring antibody binds to elements of the complement system and also binds to receptors on effector cells (including, for example, effector cells that mediate cytotoxicity). As is known in the art, the affinity and / or other binding attributes of the Fc region for the Fc receptor can be modulated via glycosylation or other modifications. In some embodiments, antibodies produced and / or utilized in accordance with the present invention include a glycosylated Fc domain, including Fc domains with such glycosylation that have been modified or altered.

[0077] Any polypeptide or polypeptide complex that contains sufficient immunoglobulin domain sequences as found in a natural antibody may be referred to and / or used as an "antibody," whether such polypeptide is produced naturally (e.g., produced by an organism in response to an antigen) or produced by recombinant modification, chemical synthesis, or other artificial systems or methodologies. In some embodiments, antibody sequence elements are humanized, primatized, chimerized, etc., as known in the art.

[0078] Moreover, "antibody," as used herein, can refer, in appropriate embodiments (unless otherwise specified or clear from the context), to any of the constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative presentations. For example, in embodiments, antibodies utilized in accordance with the present invention are in a format selected from, but not limited to, intact IgG, IgE, and IgM, bi- or multispecific antibodies (such as, for example, Zybodies®), single chain Fv, Fab, Small Modular ImmunoPharmaceuticals ("SMIPs" (trademark)), single chain or tandem diabodies (TandAb®), VHH, Anticalins®, Nanobodies®, minibodies, BiTEs®, ankyrin repeat proteins or DARPINs®, Avimers®, DART, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProtein, Fynomers®, Centyrins®, and KALBITOR®. In some embodiments, the antibody may lack a covalent modification (e.g., glycan attachment) that it would have if it were naturally produced. In some embodiments, the antibody may contain a covalent modification (e.g., glycan attachment), a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group (e.g., poly-ethylene glycol, etc.).

[0079] In many embodiments, an antibody agent is or comprises a polypeptide having an amino acid sequence that includes one or more structural elements recognized by those of skill in the art as a complementarity determining region (CDR). In some embodiments, an antibody agent is or comprises a polypeptide having an amino acid sequence that includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to a CDR found in a reference antibody. In some embodiments, the included CDRs are substantially identical to the reference CDRs by being identical in sequence or containing between one and five amino acid substitutions compared to the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs by exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs by exhibiting at least 96%, 97%, 98%, 99%, or 100% sequence identity to the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs by having at least one amino acid in the included CDR deleted, added, or substituted compared to the reference CDR, but the included CDRs otherwise have the same amino acid sequence as the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs by having one to five amino acids in the included CDR deleted, added, or substituted compared to the reference CDR, but the included CDRs otherwise have the same amino acid sequence as the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs by having at least one amino acid in the included CDR replaced compared to the reference CDR, but the included CDRs otherwise have the same amino acid sequence as the reference CDR.In some embodiments, the included CDRs are substantially identical to the reference CDRs by having one to five amino acids deleted, added, or substituted in the included CDRs compared to the reference CDRs, but the included CDRs otherwise have the same amino acid sequence as the reference CDRs. In some embodiments, the antibody agent is or comprises a polypeptide having an amino acid sequence that includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain.

[0080] Expression constructs: In the methods of the invention, RSPO surrogates can be produced by recombinant methods. Amino acid sequence variants are prepared by introducing appropriate nucleotide changes into the DNA coding sequence. Such variants represent insertions, substitutions, and / or designated deletions of residues within the amino acid sequence or at one or both ends of the amino acid sequence. Any combination of insertions, substitutions, and / or designated deletions can be made to arrive at the final construct, provided that the final construct has the desired biological activity as defined herein. Amino acid changes can also alter post-translational processing of the polypeptide, for example, by inserting, deleting, or otherwise affecting the leader sequence of the polypeptide, thereby changing the number or location of glycosylation sites, altering the membrane anchoring properties, and / or altering the cellular location.

[0081] The nucleic acid encoding the surrogate can be inserted into a replicable vector for expression. Many such vectors are available. In general, the vector components include one or more of the following, but are not limited to: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0082] An expression vector will contain a promoter that is recognized by the host organism and operably linked to a surrogate coding sequence. A promoter is a non-translated sequence located upstream (5') of the start codon of a structural gene (generally within about 100-1000 bp) that controls the transcription and translation of a particular nucleic acid sequence to which it is operably linked. Such promoters are typically divided into two classes: inducible and constitutive. An inducible promoter is one that initiates increased levels of transcription from DNA under its control in response to some change in culture conditions (e.g., the presence or absence of a nutrient, or a change in temperature).

[0083] Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) can also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' (and occasionally 3') untranslated regions of eukaryotic or viral DNAs or cDNAs.

[0084] Construction of suitable vectors containing one or more of the above components uses standard techniques. Isolated plasmids or DNA fragments can be cleaved, tailored, and religated in the desired configuration to generate the required plasmid. For analysis to confirm the correct sequences in the constructed plasmid, the ligation mixture is used to transform host cells and successful transformants are selected by ampicillin or tetracycline resistance. Plasmids from the transformants are prepared, analyzed by restriction endonuclease digestion, and / or sequenced.

[0085] Suitable host cells for cloning or expressing DNA in the vectors herein are prokaryotes, yeast, or higher eukaryotic cells as mentioned above. Suitable prokaryotes for this purpose include eubacteria, such as gram-negative or gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, for example, E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example, Salmonella typhimurium, Serratia, for example, Serratia marcescens, and Shigella, as well as Bacilli, for example, B. subtilis and B. licheniformis, Pseudomonas, for example, P. aeruginosa, and Streptomyces. These examples are illustrative and not limiting.

[0086] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable expression hosts. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, several other genera, species, and strains, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis, K. fragilis, etc.; Pichia pastoris; Candida; Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger, are also commonly available and useful herein.

[0087] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can be used as hosts. Typically, plant cells are transfected by incubating with certain strains of the bacterium Agrobacterium tumefaciens. During such incubation of the plant cell cultures, the DNA coding sequence is transferred to the plant cell host, so that the plant cell host is transfected and expresses the DNA under appropriate conditions. In addition, control and signal sequences compatible with plant cells are available, such as the nopaline synthase promoter and polyadenylation signal sequence.

[0088] Host cells are transfected with the expression vectors described above for RSPO surrogate production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. Mammalian host cells can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM), Sigma), RPMI 1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing host cells. Any of these media can be supplemented as necessary with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphates), buffers (e.g., HEPES), nucleosides (e.g., adenosine and thymidine), antibiotics, trace elements, and glucose or an equivalent energy source. Any other necessary supplements can also be included at concentrations that would be known to those of skill in the art. Culture conditions such as temperature, pH, and the like will be those previously used for the host selected for expression, and such conditions will be apparent to one of skill in the art.

[0089] Small Molecule Compositions The RSPO surrogates of the invention can also include organic molecules, preferably small organic compounds having a molecular weight of more than 50 Daltons and less than about 20,000 Daltons. Useful surrogates are identified, for example, by screening assays in which molecules are assayed for high affinity binding to RNF43 / ZNRF3 and then linked to a cell targeting domain. Molecules can comprise a binding moiety that is linked to another binding moiety, or a binding domain as described above for polypeptide agents.

[0090] Candidate surrogates contain functional groups necessary for structural interactions with proteins, particularly hydrogen bonding, and typically contain at least one amine, carbonyl, hydroxyl, or carboxyl group, preferably at least two of these functional chemical groups. Candidate surrogates often contain cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules, including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.

[0091] Candidate surrogates can be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or are readily produced. In addition, natural or synthetically produced libraries and compounds can be easily modified through conventional chemical, physical, and biochemical means and used to produce combinatorial libraries. Known pharmacological agents can be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidation, etc., to produce structural analogs. Test agents can be obtained from libraries, such as natural product libraries or combinatorial libraries. Several different types of combinatorial libraries and methods for preparing such libraries have been described, including, for example, PCT publications WO93 / 06121, WO95 / 12608, WO95 / 35503, WO94 / 08051, and WO95 / 30642, each of which is incorporated herein by reference.

[0092] When the screening assay is a binding assay, one or more molecules may be linked to a label, where the label directly or indirectly provides a detectable signal. Various labels include radioisotopes, fluorescers, chemiluminescers, enzymes, specific binding molecules, particles (e.g., magnetic particles), and the like. Specific binding molecules include pairs such as biotin and streptavidin, digoxin and antidigoxin. For specific binding members, the complementary member will usually be labeled with a molecule that provides detection according to known procedures.

[0093] A variety of other reagents can be included in the screening assay. Such reagents include reagents such as salts, neutral proteins, e.g., albumin, detergents, etc., which are used to facilitate optimal protein-protein binding and / or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay may be used, e.g., protease inhibitors, nuclease inhibitors, antimicrobial agents, etc. The mixture of components is added in any order that results in the requisite binding. Incubation is performed at any suitable temperature, typically between 4 and 40° C. Incubation periods are selected for optimal activity, but may also be optimized to facilitate rapid high-throughput screening. Typically, between 0.1 and 1 hour is considered sufficient.

[0094] Preliminary screening can be performed by screening for compounds capable of binding to the RNF43 / ZNRF3 polypeptide. Binding assays typically involve contacting the RNF43 / ZNRF3 polypeptide with one or more test compounds and allowing sufficient time for the protein and the test compounds to form a binding complex. Any binding complexes formed can be detected using any of several established analytical techniques. Protein binding assays include, but are not limited to, methods that measure co-precipitation, co-migration on non-denaturing SDS-polyacrylamide gels, and co-migration on Western blots (see, for example, Bennett, JP and Yamamura, HI (1985) "Neurotransmitter, Hormone or Drug Receptor Binding Methods" in Neurotransmitter Receptor Binding (Yamamura, HI, et al., eds.), pp. 61-89).

[0095] Certain screening methods involve screening for compounds that enhance Wnt signaling activity. Such methods involve performing cell-based assays in which a test compound is contacted with one or more cells expressing Fzd, followed by detecting increased expression of Wnt-responsive genes, detecting nuclear localization of β-catenin, etc.

[0096] The expression or activity level can be compared to a baseline value. As shown above, the baseline value can be the value of a control sample, or a statistical value representative of the expression level of a control population. The expression level can also be determined for cells that do not express Wnt receptors as a negative control. Generally, such cells are otherwise substantially genetically identical to the test cells. Various control methods can be performed to confirm that the observed activity is genuine, including performing parallel reactions with cells that lack the reporter construct, or by not contacting the cells with the reporter construct with the test compound. Compounds can also be further verified as described below.

[0097] Compounds initially identified by any of the above screening methods can be further tested to verify the apparent activity. A basic form of such a method involves administering the lead compound identified during the initial screening to an animal or to a cell culture model that serves as a model for humans. Typically, the animal models utilized in validation experiments are mammals. Specific examples of suitable animals include, but are not limited to, primates, mice, and rats.

[0098] Active test agents identified by the screening methods described herein can serve as lead compounds for the synthesis of analog compounds. Typically, analog compounds are synthesized to have similar electronic configurations and molecular conformations as the lead compounds. Identification of analog compounds can be performed through the use of techniques such as self-consistent field (SCF) analysis, configuration interaction (CI) analysis, and normal mode dynamics analysis. Computer programs that implement such techniques are available. See, for example, Rein et al., (1989) Computer-Assisted Modeling of Receptor-Ligand Interactions (Alan Liss, New York).

[0099] RSPO surrogates and Wnt signaling RSPO surrogate compositions and methods for using same are provided. These and other objects, advantages, and features of the present invention will become apparent to those skilled in the art upon reading the details of the compositions and methods that are more fully described below.

[0100] RSPO surrogate molecules are defined by their physical and biological properties. The sequence of the surrogate is different from the native RSPO protein. As used herein, a RSPO surrogate comprises (i) a specific binding domain for RNF43 or ZNRF3 and (ii) a cell targeting domain. The domains may be directly linked or separated by a linker (e.g., a polypeptide linker, or a non-peptide linker, etc.). The length of the linker, and therefore the spacing between the binding domains, can be used to adjust the length of the signal and can be selected depending on the desired use of the RSPO surrogate. Polypeptide RSPO surrogates can be single chain, dimers, or higher order multimers. The main feature of the surrogates is the enhancement of Wnt signaling in the canonical β-catenin Wnt signaling cascade and the β-catenin-independent planar cell polarity (PCP) pathway in cells. In some embodiments, the cell is a mammalian cell, e.g., a human cell.

[0101] The term "Wnt enhancing activity" refers to the ability of the RSPO surrogate to enhance the effect or activity of the Wnt protein binding to the frizzled protein. The ability of the surrogate of the present invention to enhance the activity of Wnt can be confirmed by multiple assays. As used herein, the term "enhance" refers to a measurable increase in the level of Wnt / β-catenin signaling or Wnt / PCP signaling compared to the level in the absence of the surrogate of the present invention.

[0102] In some embodiments, the RSPO surrogate circumvents the requirement for expression of the RSPO cognate receptors LGR4, LGR5, or LGR6 for enhanced Wnt signaling, hi other embodiments, the RSPO surrogate specifically targets one or more of LGR4, LGR5, or LGR6 for enhanced Wnt signaling, thereby providing enhanced selectivity for RSPO activity.

[0103] The RNF43 or ZNRF3 binding domain has high affinity, e.g., 1×10 -6 M or less, 1×10 -7 M or less, 1×10 -8 M or less, 1×10 -9 M, or 1×10 -10The specific binding domain for RNF43 or ZNRF3 may be selected from any domain that binds to RNF43 or ZNRF3 with a Kd of M or less. Suitable binding domains include, but are not limited to, de novo designed binding proteins, antibody-derived binding proteins (e.g., scFv, Fab, etc.) and other portions of antibodies that specifically bind to RNF43 or ZNRF3 proteins; nanobody-derived binding proteins; knottin-based engineered scaffolds; Norrin and engineered binding fragments derived therefrom, naturally occurring binding domains, and the like. In some embodiments, the specific binding domain for RNF43 or ZNRF3 is a binding fragment of RSPO, for example, comprising, consisting of, or consisting essentially of the RSPO furin1 domain. The binding domain may be affinity selective to enhance binding to a desired protein or proteins.

[0104] The cell targeting domain or element may be a target of high affinity, e.g., 1×10 -7 M or less, 1×10 -8 M or less, 1×10 -9 M or less, 1×10 -10 K below M DThe cell targeting domain may be selected from any domain that selectively binds to a cell surface protein at a specific site. Suitable cell targeting domains include, but are not limited to, de novo designed binding proteins, antibody-derived binding proteins (e.g., scFv, Fab, etc.), and other portions of antibodies that specifically bind to cell surface proteins; nanobody-derived binding proteins; knottin-based engineered scaffolds; naturally occurring binding domains or polypeptides, cytokines, growth factors, and the like. In some embodiments, the cell targeting domain is a cytokine or growth factor that has a cognate receptor on the target cell. In some embodiments, the cell targeting domain is an antibody or active fragment thereof that has specificity for an antigen present on the target cell surface. In some such embodiments, the antigen is one or more of LGR4, LGR5, or LGR6, e.g., an antibody that selectively binds to a single LGR protein (i.e., one of LGR4, LGR5, or LGR6). The LGR binding portion can be selective for the LGR protein of interest, eg, having at least 10-fold, 25-fold, 50-fold, 100-fold, 200-fold, or more specificity for the desired LGR protein over other LGR proteins.

[0105] Specific embodiments of RSPO surrogates include, but are not limited to, binding domains (e.g., scFvs) derived from antibodies specific for human RNF43 or human ZNRF3 linked to a cytokine (e.g., IL-2 or IL-4) as set forth in the exemplary proteins described herein, such as SEQ ID NOs: 1-4, where SEQ ID NO: 1 represents a surrogate specific for ZNRF3 linked to IL-2, SEQ ID NO: 2 represents a surrogate specific for ZNRF3 linked to IL-4, SEQ ID NO: 3 represents a surrogate specific for RNF43 linked to IL-2, and SEQ ID NO: 4 represents a surrogate specific for RNF43 linked to IL-4.

[0106] Alternative specific embodiments include, but are not limited to, binding domains (e.g., scFvs) derived from antibodies specific for human RNF43 or human ZNRF3 linked to a growth factor (e.g., EGF, NGF, etc.). Alternative specific embodiments include, but are not limited to, binding domains (e.g., scFvs) derived from antibodies specific for human RNF43 or human ZNRF3 linked to an antibody or fragment thereof specific for one of human LGR4, LGR5, or LGR6.

[0107] In some such embodiments, a binding domain (e.g., scFv) derived from an antibody specific for human RNF43 or human ZNRF3 comprises one, two, three, four, five, six CDR sequences from the antibody-derived portion of SEQ ID NOs: 1-4. One of skill in the art will appreciate that multiple definitions of CDRs are commonly used, including the Kabat definition (see, "Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions." Mol Immunol. 2010;47:694-700). The Kabat definition is based on sequence variability and is the most commonly used. The Chothia definition is based on the location of structural loop regions (Chothia et al. "Conformations of immunoglobulin hypervariable regions." Nature. 1989;342:877-883). Alternative CDR definitions of interest include, but are not limited to, Honegger, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool.” J Mol Biol. 2001;309:657-670; Ofran et al. “Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes.” J Immunol. 2008;181:6230-6235; Almagro “Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires.” J Mol Recognit. 2004;17:132-143; and Padlan et al. “Identification of specificity-determining residues in antibodies.” Faseb J. 1995;9:133-139, each of which is specifically incorporated herein by reference.

[0108] "Wnt protein signaling" or "Wnt signaling" is used herein to refer to the mechanism by which biologically active Wnt exerts its effect on cells to regulate cellular activity. Wnt proteins regulate cellular activity by binding to Wnt receptors, including proteins from the Frizzled (Fz) family of proteins, proteins from the ROR family of proteins, proteins LRP5, LRP6, proteins FRL1 / crypto, and proteins Derailed / Ryk from the LRP family of proteins. Once activated by Wnt binding, the Wnt receptor(s) activate one or more intracellular signaling cascades. Such signaling cascades include the canonical Wnt signaling pathway; the Wnt / planar cell polarity (Wnt / PCP) pathway; the Wnt-calcium (Wnt / Ca 2+ ) pathway (Giles, RH et al. (2003) Biochim Biophys Acta 1653,1-24; Peifer, M. et al. (1994) Development 120:369-380; Papkoff, J. et al (1996) Mol. Cell Biol. 16:2128-2134; Veeman, MT et al. (2003) Dev. Cell 5:367-377); and other Wnt signaling pathways as known in the art.

[0109] For example, activation of the canonical Wnt signaling pathway results in the inhibition of phosphorylation of the intracellular protein β-catenin, leading to its accumulation in the cytosol and subsequent translocation to the nucleus, where it interacts with transcription factors (e.g., TCF / LEF) to activate target genes. Activation of the Wnt / PCP pathway activates RhoA, c-Jun N-terminal kinase (JNK), and nemo-like kinase (NLK) signaling cascades to control biological processes such as tissue polarity and cell motility. For example, Wnt / Ca2+ signaling by binding Wnt-4, Wnt-5A, or Wnt-11 is involved in the activation of the cascades. 2+Activation of RSPO surrogate induces intracellular release of calcium ions, which activates calcium-sensitive enzymes such as protein kinase C (PKC), calcium-calmodulin-dependent kinase II (CamKII), or calcineurin (CaCN). By assaying the activity of the above signaling pathways, the biological activity of the Wnt composition can be easily determined. A "biologically active RSPO surrogate" is a RSPO surrogate composition that can specifically bind to an Fzd receptor and activate Wnt signaling when provided to a cell in vitro or in vivo (i.e., when administered to an animal, e.g., a mammal).

[0110] In certain embodiments, a RSPO surrogate of the invention increases Wnt pathway signaling by at least about 50%, about 75%, about 100%, about 150%, about 200%, about 300%, about 400%, about 5-fold, about 10-fold relative to the level of Wnt signaling in the absence of the surrogate, and optionally increases signaling by 50-fold, 100-fold, 500-fold, or more.

[0111] Various methods for measuring the level of Wnt signaling are known in the art. Such methods include, but are not limited to, assays that measure Wnt / β-catenin target gene expression; TCF reporter gene expression; beta-catenin stabilization; LRP phosphorylation; translocation of axin from the cytoplasm to the cell membrane and binding to LRP. The canonical Wnt / β-catenin signaling pathway ultimately leads to changes in gene expression through transcription factors TCF7, TCF7L1, TCF7L2, and LEF. The transcriptional response to Wnt activation has been characterized in multiple cells and tissues. Therefore, global transcriptional profiling by methods known in the art can be used to evaluate Wnt / β-catenin signaling activation.

[0112] Changes in Wnt-responsive gene expression are generally mediated by TCF and LEF transcription factors. The TCF reporter assay assesses changes in transcription of TCF / LEF control genes to determine the level of Wnt / beta-catenin signaling. The TCF reporter assay was first described by Korinek, V. et al., 1997. Also known as TOP / FOP, this method involves determining the transactivation activity of endogenous β-catenin / TCF4 using three copies of the optimal TCF motif CCTTTGATC or three copies of the mutant motif CCTTTGGCC upstream of a minimal c-Fos promoter driving luciferase expression (pTOPFLASH and pFOPFLASH, respectively). A higher ratio of these two reporter activities (TOP / FOP) indicates higher β-catenin / TCF4 activity.

[0113] A variety of other reporter transgenes that respond to Wnt signals are present in animals in an intact state, and therefore effectively reflect endogenous Wnt signaling. Such reporters are based on the multimerized TCF binding sites that drive the expression of LacZ or GFP, and can be easily detected by methods known in the art. Such reporter genes include TOP-GAL, BAT-GAL, ins-TOPEGFP, ins-TOPGAL, LEF-EGFP, Axin2-LacZ, Axin2-d2EGFP, LGR5tm1(cre / ERT2), and TOPdGFP.

[0114] The recruitment of dephosphorylated β-catenin to the membrane, the stabilization and phosphorylation state of β-catenin, and the translocation of β-catenin to the nucleus (Klapholz-Brown Z et al., PLoS One.2(9) e945,2007), in some cases mediated by complex formation with TCF transcription factors and TNIK, are key steps in the Wnt signaling pathway. Stabilization is mediated by Dishevelled family proteins that inhibit the "destruction" complex, resulting in reduced degradation of intracellular β-catenin and subsequent translocation of β-catenin to the nucleus. Therefore, measuring the level and location of β-catenin in cells is a good reflection of the level of Wnt / β-catenin signaling. One non-limiting example of such an assay is the "BioImage β-Catenin Redistribution Assay" (Thermo Scientific), which provides recombinant U2OS cells stably expressing human β-catenin fused to the C-terminus of enhanced green fluorescent protein (EGFP). Imaging and analysis are performed using a fluorescent microscope or HCS platform that allows visualization of the levels and distribution of EGFP-β-catenin.

[0115] Another way to inhibit the destruction complex is by axin removal by recruiting it to the cytoplasmic tail of the Wnt co-receptor LRP. Axin has been shown to preferentially bind to the phosphorylated form of the LRP tail. Therefore, visualization of axin translocation (e.g., with a GFP-axin fusion protein) is another way to assess the level of Wnt / β-catenin signaling.

[0116] In certain embodiments, the surrogates of the present invention can enhance Wnt signaling by at least 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 150%, 200%, 250%, 300%, 400%, or 500% as compared to signaling induced by a neutral substance or a negative control, as measured by the above assays, for example, by TOPFlash assay. A negative control may be included in such assays. In certain embodiments, the surrogates of the present invention can enhance β-catenin signaling by 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, or more as compared to activity in the absence of agonist, as measured by the above assays, for example, by TOPFlash assay, or any of the other assays mentioned herein.

[0117] Alternatively, the activity of the surrogate can be determined by measuring ubiquitination and / or destruction of Frizzled receptors on target cells, where an active RSPO surrogate results in a number of Frizzled proteins remaining on the cell surface and not modified by ubiquitination, e.g., an increase of at least about 5%, at least about 10%, at least about 20%, at least about 50%, or more.

[0118] RSPO surrogates may be fused or conjugated to additional polypeptide sequences. Examples include immunoadhesins, which combine a surrogate with immunoglobulin sequences (particularly Fc sequences), and epitope tagged polypeptides that comprise a native inhibitor polypeptide, or a portion thereof, fused to a "tag polypeptide." The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with biological activity of the native inhibitor polypeptide. In general, suitable tag polypeptides have at least six amino acid residues and typically have between about 6-60 amino acid residues.

[0119] Pharmaceutical Compositions For therapeutic uses, RSPO surrogates are administered to mammals, including humans, in a physiologically acceptable dosage form, including dosage forms that can be administered to humans as a bolus or by continuous infusion over a period of time. Alternative routes of administration include topical, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. RSPO surrogates are also suitably administered by intratumoral, peritumoral, intralesional, or perilesional routes, or to the lymphatics to exert local and systemic therapeutic effects.

[0120] Pharmaceutical compositions can also include combinations of molecules of the invention with cells (including stem cells, progenitor cells, etc.). In some embodiments, compositions include combinations of molecules of the invention with regenerative somatic stem cells, e.g., epithelial stem cells, neural stem cells, hepatic stem cells, hematopoietic stem cells, osteoblasts, muscle stem cells, mesenchymal stem cells, pancreatic stem cells, etc. In such combinations, the cells may be pre-treated with the molecules of the invention (e.g., ex vivo treatment of the cells with RSPO surrogates), the cells may be administered simultaneously with the molecules of the invention in separate or combined formulations, the cells may be provided to an individual prior to treatment with the molecules of the invention, etc.

[0121] The term "stem cell" as used herein refers to a cell that has self-renewal properties and the developmental potential to differentiate into multiple cell types. A stem cell can proliferate and give rise to further such stem cells while maintaining its developmental potential. A stem cell can divide asymmetrically, with one daughter cell retaining the developmental potential of the parent stem cell and the other daughter cell expressing some other specific function, phenotype, and / or developmental potential that is somewhat different from the parent cell. The daughter cell itself can be induced to proliferate and then produce progeny that differentiate into one or more mature cell types while also retaining one or more cells with the developmental potential of the parent. Differentiated cells may be derived from pluripotent cells, which themselves are derived from pluripotent cells, and so on. Each such pluripotent cell may be considered a stem cell, but the range of cell types that each stem cell can give rise to, i.e., the developmental potential, may vary considerably. Alternatively, a portion of the stem cells within a population may divide symmetrically into two stem cells (known as stochastic differentiation), thus retaining some stem cells in the population as a whole, while other cells in the population give rise to only differentiated progeny. Thus, the term "stem cells" refers to any subset of cells that, under certain circumstances, have the developmental potential to differentiate into a further specialized or differentiated phenotype, and, under certain circumstances, retain the ability to proliferate without substantial differentiation.

[0122] The term "somatic stem cell" is used herein to refer to any multipotent or pluripotent stem cell derived from non-embryonic tissues, including fetal, juvenile, and adult tissues. Natural somatic stem cells have been isolated from a wide variety of adult tissues, including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle. The term "progenitor cell" is used herein to refer to a cell that is at an early stage along a developmental pathway or progression, in contrast to a cell that can be generated by differentiation. Often, progenitor cells have significant or very high proliferation potential. Progenitor cells may give rise to multiple different differentiated cell types or to a single differentiated cell type, depending on the developmental pathway and the environment in which the cell develops and differentiates.

[0123] Depending on the desired formulation, the pharmaceutical composition may contain a non-toxic carrier of a pharma- ceutically acceptable diluent, which is defined as a vehicle commonly used in formulating pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the formulation. Examples of such diluents include distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may contain other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, excipients, etc. The composition may also contain additional substances for appropriate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents, and detergents.

[0124] The composition may include any of a variety of stabilizers, such as antioxidants. When the pharmaceutical composition includes a polypeptide, the polypeptide may be complexed with a variety of well-known compounds that enhance the in vivo stability of the polypeptide or enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce toxicity, enhance solubility or uptake). Such modifiers or complexing agents include sulfates, gluconates, citrates, and phosphates. The polypeptide of the composition may be complexed with molecules that enhance its in vivo attributes. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.

[0125] Further guidance regarding suitable formulations for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of drug delivery methods, see Langer, Science 249:1527-1533 (1990).

[0126] The pharmaceutical compositions can be administered for prophylactic and / or therapeutic treatments. Toxicity and therapeutic efficacy of the active ingredients can be determined by standard pharmaceutical procedures in cell cultures and / or experimental animals (e.g., LD 50 Determination of the dose (lethal to 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be determined according to the LD ratio. 50 / ED 50 Compounds that exhibit large therapeutic indices are preferred.

[0127] The data obtained from cell culture and / or animal studies can be used in formulating a range of dosages for human use. The dosage of the active ingredient is typically chosen to be within the range of ED 50 The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

[0128] For oral administration, the active ingredient can be administered in solid dosage forms such as capsules, tablets, and powders, or in liquid dosage forms such as elixirs, syrups, and suspensions. The active ingredient(s) can be encapsulated in gelatin capsules with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talc, magnesium carbonate. Examples of additional inactive ingredients that can be added to provide desirable color, taste, stability, buffering capacity, dispersion, or other known desirable characteristics are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, and edible white ink. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide sustained release of the drug over a period of several hours. Compressed tablets can be coated with sugar or film to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the digestive tract. Liquid dosage forms for oral administration may contain coloring and flavoring to increase patient acceptance.

[0129] The active ingredient, alone or in combination with other suitable components, can be made into an aerosol formulation (i.e., "nebulized") and administered via inhalation. The aerosol formulation can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen.

[0130] Formulations suitable for parenteral administration (e.g., parenteral administration by intra-articular (into a joint), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes) include aqueous and non-aqueous isotonic sterile injection solutions (which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient) and aqueous and non-aqueous sterile suspensions (which may contain suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives).

[0131] Components used to formulate pharmaceutical compositions are preferably of high purity and substantially free of potentially harmful impurities (e.g., at least National Food (NF) grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Furthermore, compositions intended for in vivo use are usually sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic agents, particularly any endotoxins that may be present during the synthesis or purification process. Compositions intended for parenteral administration are furthermore sterile, substantially isotonic, and manufactured under GMP conditions.

[0132] The effective amount of the therapeutic composition administered to a particular patient will depend on a variety of factors, some of which will vary from patient to patient. The formulation may be provided, for example, in a unit dose. A skilled clinician will be able to determine the effective amount of the therapeutic agent to administer to the patient. The dosage of the surrogate will depend on the treatment, the route of administration, the nature of the treatment, the susceptibility of the disease to the therapeutic agent, etc. The clinician will determine the LD 50 Using animal data and other available data, the maximum safe dose for an individual can be determined depending on the route of administration. Compositions that are rapidly cleared from the body may be administered at higher doses or in repeated doses to maintain therapeutic concentrations. A competent clinician would be able to use routine skill to optimize the dosage of a particular therapeutic or imaging composition during the course of routine clinical trials. Typically, dosages will be between 0.001 and 100 milligrams of agent per kilogram of subject body weight.

[0133] The composition may be administered to the subject in more than one series of doses. For therapeutic compositions, regular periodic administration (e.g., once every 2-3 days) may sometimes be required or desirable to reduce toxicity. For therapeutic compositions utilized in repeated dose regimens, moieties that do not induce an immune response are preferred.

[0134] In another embodiment of the present invention, an article of manufacture is provided that contains materials useful for treating the conditions described herein. The article of manufacture includes a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials, such as glass or plastic. The container holds a composition that is effective for treating the condition and can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). The active agent in the composition is an RSPO surrogate. A label on or associated with the container indicates that the composition is used to treat the selected condition. Additional container(s) holding a pharma- ceutically acceptable buffer, such as, for example, phosphate buffered saline, Ringer's solution, or dextrose solution, may be provided with the article of manufacture. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts containing instructions for use.

[0135] The term "therapeutically effective amount" as used herein means an amount that is sufficient to treat a disease, disorder, and / or condition when administered to a population suffering from or susceptible to the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of, stabilizes one or more characteristics of, and / or delays the onset of, one or more symptoms of the disease, disorder, and / or condition. Those skilled in the art will appreciate that the term "therapeutically effective amount" does not actually require the achievement of successful treatment in a particular individual. Rather, a therapeutically effective amount is considered to be an amount that, when administered to a patient in need of such treatment, produces a particular desired pharmacological response in a meaningful number of subjects.

[0136] For example, in some embodiments, the term "therapeutically effective amount," in the context of the therapy of the present invention, refers to an amount that, when administered to an individual in need thereof, blocks, stabilizes, attenuates, or reverses a disease process occurring in said individual.

[0137] How to use RSPO surrogates are useful for both preventative and therapeutic purposes. Thus, the term "treating" as used herein is used to refer to both the prevention of disease and the treatment of existing conditions. In certain cases, prevention refers to inhibiting or delaying the onset of a disease or condition in a patient who has been identified as being at risk for developing a disease or condition. Treatment of ongoing disease to stabilize or improve the clinical symptoms of the patient is a particularly important benefit provided by the present invention. Such treatment is desirably performed before loss of function in the affected tissue, so that the preventative therapeutic benefit provided by the present invention is also important. The basis of therapeutic efficacy can be any reduction in the severity of the disease. The therapeutic efficacy can be measured in terms of clinical results or determined by immunological or biochemical tests. The patient to be treated can be a mammal (e.g., a primate, including a human) or a laboratory animal (e.g., rabbits, rats, mice, etc.), particularly for evaluation of therapies, horses, dogs, cats, livestock, etc.

[0138] The dosage of the therapeutic formulation (e.g., pharmaceutical composition) will vary widely depending on the nature of the condition, frequency of administration, mode of administration, clearance of the agent from the host, etc. In certain embodiments, a larger initial dose may be administered followed by smaller maintenance doses. In certain embodiments, the dose may be administered infrequently, such as once per week or once every two weeks, or more frequently, divided into smaller doses and administered once per day, twice per week, or as frequently as needed to maintain an effective dosage level.

[0139] In some embodiments of the present invention, the administration of the composition or formulation comprising the RSPO surrogate is performed by local administration. Local administration, as used herein, may refer to topical administration, but also refers to injection or other introduction into the body at the treatment site. Examples of such administration include intramuscular injection, subcutaneous injection, intraperitoneal injection, and the like. In other embodiments, the composition or formulation comprising the RSPO surrogate is administered systemically, for example, orally or intravenously. In one embodiment, the composition of the formulation comprising the RSPO surrogate is administered by infusion, for example, by continuous infusion over a period of time, for example, 10 minutes, 20 minutes, 3 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or more.

[0140] In some embodiments of the invention, the compositions or formulations are administered on a short-term basis to obtain a rapid and meaningful increase in activity, e.g., a single dose or a series of doses administered over, e.g., 1, 2, 3 or more days to 1 or 2 weeks. The size of the dose administered must be determined by the physician and will depend on several factors, such as the nature and severity of the disease, the age and health of the patient, and the patient's tolerance to the drug itself.

[0141] In certain embodiments of the invention, an effective amount of a composition comprising a RSPO surrogate is provided to a cell, e.g., by contacting the cell with an effective amount of the composition to achieve a desired effect (e.g., enhancing Wnt signaling, proliferation, etc.). In certain embodiments, the contacting occurs in vitro, ex vivo, or in vivo. In certain embodiments, the cell is derived from or present within a subject in need of increased Wnt signaling.

[0142] In some methods of the present invention, the subject composition is provided in an effective amount for enhancing Wnt signaling in cells.Biochemically speaking, an effective amount or effective dose of RSPO surrogate is an amount that increases Wnt signaling in cells by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% compared to signaling in the absence of said surrogate.The amount of cellular activity modulation can be determined by several methods known to those skilled in the art of Wnt biology.

[0143] In clinical terms, an effective dose of a RSPO surrogate composition is one that, when administered to a subject for a suitable period of time, for example, at least about one week, and in some cases about two or more weeks, up to about four, eight or more weeks, demonstrates a change in symptoms associated with lack of Wnt signaling. In some embodiments, an effective dose can not only slow or halt the progression of a disease state, but can also induce a reversal of the condition. Those skilled in the art will understand that an initial dose can be administered for such a period of time, followed by a maintenance dose, which in some cases is at a reduced dosage.

[0144] Calculation of an effective amount or dose of a RSPO surrogate composition to be administered is within the skill of one in the art and is believed to be routine for such a person of ordinary skill in the art. Of course, the final amount to be administered will depend on the route of administration and the nature of the disorder or condition to be treated.

[0145] Suitable cells for use in the subject method are generally cells that contain one or more Fzd receptors, where ubiquitination of the Fzd receptor is reduced by administration of a RSPO surrogate. The cells to be contacted can be in vitro, i.e., in culture, or in vivo, i.e., in a subject. The cells can be from / in any organism, but are preferably mammals, including humans, domestic and farm animals, as well as zoo, laboratory, or pet animals, such as dogs, cats, cows, horses, sheep, goats, rabbits, rats, mice, frogs, zebrafish, fruit flies, (parasitic) worms, etc. Preferably, the mammal is a human. The cells can be from any tissue. The cells can be frozen or fresh. The cells can be primary cells or cell lines. Often, the cells are primary cells used in vivo, or treated ex vivo before being introduced into the recipient.

[0146] Cells in vitro can be contacted with a composition comprising a RSPO surrogate by any of several methods known in the art. For example, the protein composition can be provided to the cells in the medium in which the subject cells are cultured. A nucleic acid encoding a RSPO surrogate can be provided to the subject cells on a vector or to cells co-cultured with the subject cells under conditions that promote uptake (e.g., electroporation, calcium chloride transfection, and lipofection) known in the art. Alternatively, a nucleic acid encoding a RSPO surrogate can be provided to the subject cells or to cells co-cultured with the subject cells via a virus, i.e., the cells are contacted with a viral particle that includes a nucleic acid encoding a Wnt peptide surrogate polypeptide. Retroviruses (e.g., lentiviruses) are particularly suitable for the methods of the present invention, since they can be used to transfect non-dividing cells (see, e.g., Uchida et al. (1998) PNAS95(20):11939-44). Commonly used retroviral vectors are "defective," i.e., unable to produce viral proteins required for productive infection. Conversely, replication of the vector requires growth in the packaging cell line.

[0147] Similarly, cells in vivo can be contacted with a subject RSPO surrogate composition by any of a number of methods known in the art for administration of proteins, peptides, small molecules, or nucleic acids to a subject. The RSPO surrogate compositions can be incorporated into a variety of formulations or pharmaceutical compositions, and in some embodiments, such formulations or pharmaceutical compositions are formulated in the absence of detergents, liposomes, etc., as described for formulating full-length Wnt proteins.

[0148] WNT signaling is required for the healing of almost any tissue in the human body. For example, WNT has been shown to activate adult tissue-resident stem cells. These stem cells self-renew and divide, and in so doing generate progeny cells, which then mature in the target tissue. The molecules of the present invention enhance WNT activity in a pharmacologically acceptable manner.

[0149] In some embodiments, the compounds of the invention are administered for use in the treatment of diseased or damaged tissue, for use in tissue regeneration, and for use in cell growth and proliferation, and / or for use in tissue modification. In particular, the invention provides RSPO surrogates, or compositions comprising one or more surrogates according to the invention, for use in the treatment of tissue loss or damage due to aging, trauma, infection, or other pathological conditions.

[0150] The target conditions to be treated with the composition of the present invention include, but are not limited to, a number of conditions where regenerative cell growth is desired. Such conditions may include, for example, enhanced bone growth or regeneration, such as in bone regeneration, bone grafts, fracture healing, etc.; treatment of alopecia; enhanced sensory organ regeneration, such as hearing loss treatment, macular degeneration treatment, etc.; tooth growth, tooth regeneration, stroke treatment, traumatic brain injury, Alzheimer's disease, multiple sclerosis, and other conditions that affect the blood-brain barrier; treatment of oral mucositis, conditions where enhanced epidermal regeneration is desired, such as epidermal wound healing, diabetic foot ulcer treatment, etc.; enhanced hematopoietic cell growth, such as bone marrow, enhanced hematopoietic stem cell graft from mobilized peripheral blood, treatment of immunodeficiency, etc.; enhanced liver cell regeneration, such as liver regeneration, treatment of liver cirrhosis, enhanced liver transplantation, etc.

[0151] Conditions in which enhanced bone growth is desired may include, but are not limited to, fractures, implants, ingrowth around prostheses, etc. WNT proteins are crucial regulators of bone turnover, and ample scientific data support the role of WNT proteins in promoting bone regeneration. In some embodiments, bone marrow cells are exposed to the molecules of the present invention, resulting in activation of stem cells within the bone marrow cells. Such activated cells may remain in situ for the benefit of the individual, or may be used in bone grafting procedures.

[0152] In some embodiments, bone regeneration is enhanced by contacting responsive cell populations (e.g., bone marrow, osteoprogenitor cells, osteoprogenitor cells, etc.) with an effective dose of a molecule of the invention. In some such embodiments, the contacting is performed in vivo. In other such embodiments, the contacting is performed ex vivo. The molecule can be localized to the site of action, for example, by loading onto a matrix that is optionally biodegradable and optionally provides sustained release of the active agent. Matrix carriers include, but are not limited to, absorbable collagen sponges, ceramics, hydrogels, bone cements, and the like.

[0153] Compositions comprising one or more of the molecules of the present invention can be used in the in vitro generation of bone tissue (e.g., from skeletal stem cells) and in the in vivo treatment of bone tissue defects. The subject compounds can be used to control the rate of chondrogenesis and / or bone formation. By "bone tissue defect" is meant a defect in bone or other bone connective tissue at any site where restoration of bone or connective tissue is desired, regardless of how the defect was created, for example, as a result of surgical intervention, tumor removal, ulcer, transplant, fracture, or other traumatic or degenerative condition. For example, the compositions of the present invention can be used as part of a regimen to restore cartilage function to connective tissue. Such methods are useful, for example, in repairing defects or lesions of cartilage tissue that are the result of degenerative wear (e.g., wear that leads to arthritis), as well as in repairing other mechanical disturbances that may be caused by trauma to tissue (e.g., replacement of torn meniscal tissue, meniscal resection, joint loosening due to torn ligaments, joint malalignment, fracture) or by genetic disease.

[0154] The compositions of the present invention can also be used for regeneration of intraocular tissue. Age-related macular degeneration (AMD) is characterized by progressive loss of central vision and visual acuity, and remains the leading cause of vision loss and blindness in elderly Americans. Currently, the standard treatment for AMD is intravitreal vascular endothelial growth factor (VEGF) inhibitors. AMD is a multifactorial disease with multiple pathogenic factors contributing to angiogenesis, inflammation, fibrosis, and oxidative stress in AMD, such as VEGF, platelet-derived growth factor (PDGF), intracellular adhesion molecule-1 (ICAM-1), tumor necrosis factor-alpha (TNF-α), cyclooxygenase-2 (Cox-2), connective tissue growth factor (CTGF), and fibronectin (FN). The compositions of the present invention can be used, for example, by injection, matrix or other depot system, or other topical application to the eye for the treatment of macular degeneration.

[0155] In another embodiment, the composition of the present invention is used in the regeneration of retinal tissue. In adult mammalian retina, Müller glia dedifferentiate and produce retinal cells, including photoreceptors, for example, after in vivo neurotoxic injury. However, newly generated retinal neurons are very limited. However, Wnt signaling can promote proliferation and neural regeneration of Müller glia-derived retinal progenitor cells after injury or during degeneration. The composition of the present invention can be used, for example, in injection, matrix or other depot system, or other topical application to the eye, for enhancing retinal regeneration.

[0156] Other sensory organs, such as cells involved in hearing loss, can also benefit from the compositions of the present invention. In the inner ear, the hearing organ contains the mechanosensitive hair cells required to convert sound vibrations into electrical impulses. The vestibular organ, consisting of the semicircular canals (SSC), utricle, and saccule, also contains sensory hair cells to detect head position and movement. Sound and vestibular signals are then centrally relayed via spiral and vestibular ganglion neurons, allowing for the perception of sound and balance. Numerous studies have characterized the multiple roles of the Wnt signaling pathway during cochlear development and in promoting hair cell regeneration. Mature mammalian hearing and vestibular organs do not spontaneously initiate a proliferative response after hair cell degeneration. However, active Wnt / β-catenin signaling can promote hair cell proliferation, where LGR5-positive supporting cells can act as hair cell progenitors. LGR5 positive supporting cells can mitotically regenerate hair cells, and signaling increases both the mitotic response and the extent of hair cell regeneration.Wnt signaling can also induce ectopic hair cell formation.The compositions of the present invention can be used, for example, by injection, matrix or other depot system, or other local application to the ear, for enhancing hearing regeneration.

[0157] Periodontal disease is the leading cause of tooth loss and is associated with multiple systemic conditions. Reconstruction of support and function of affected tooth-supporting tissues represents an important therapeutic endpoint in periodontal regenerative medicine. Improved understanding of periodontal biology coupled with recent advances in scaffold matrices provides treatments providing compositions of the present invention, optionally in combination with regenerative cell delivery for predictable tissue regeneration of supporting alveolar bone, periodontal ligament, and cementum. In some embodiments, tooth or underlying bone regeneration is enhanced by contacting a responsive cell population with an effective dose of a molecule of the present invention. In some such embodiments, contacting is performed in vivo. In other such embodiments, contacting is performed ex vivo, followed by transplantation of activated stem or progenitor cells. Molecules can be localized to the site of action, for example, by loading onto a matrix that is optionally biodegradable and optionally provides sustained release of the active agent. Matrix carriers include, but are not limited to, absorbable collagen sponges, ceramics, hydrogels, bone cements, and the like.

[0158] Hair loss is a common problem with multiple causes ranging from hormone sensitivity to autoimmunity. Androgenetic alopecia, often called male pattern baldness, is the most common form of hair loss in men, affecting as many as 50% of men with age. In androgenetic alopecia, hair loss is caused by the sensitivity of hair follicles in the upper scalp to the androgen 5α-dihydrotestosterone (DHT). DHT causes these hair follicles to progressively shrink to the point where they no longer produce clinically evident hair shafts. The cells affected by DHT are dermal papilla cells, which cease to grow and lose the ability to induce hair growth. Wnt signaling in the epidermis is crucial for adult hair follicle regeneration. In some embodiments, hair follicle regeneration is enhanced by contacting a responsive cell population with an effective dose of the molecule of the present invention. In some such embodiments, contacting is performed in vivo. In other such embodiments, the contacting is performed ex vivo, followed by transplantation of the activated stem or progenitor cells (e.g., follicular cells).The molecule can be localized to the site of action, for example, with a topical lotion, gel, cream, etc.

[0159] A variety of epidermal conditions can benefit from treatment with the compounds of the present invention. Mucositis occurs when the rapid division of epithelial cells lining the gastrointestinal tract is disrupted, leaving the mucosal tissue vulnerable to ulceration and infection. Mucosal tissue, also known as mucosa or mucous membrane, lines all body passages that communicate with the air (e.g., the respiratory tract and digestive tract) and contains mucus-secreting cells and associated glands. This membrane portion that lines the inside of the mouth, called the oral mucosa, is one of the most sensitive parts of the body and is particularly vulnerable to chemotherapy and radiation. The oral cavity is the most common location of mucositis. Oral mucositis is perhaps the most common and debilitating complication of cancer treatment, especially chemotherapy and radiation. Oral mucositis can result in several problems, including pain, nutritional problems as a result of inability to eat, and increased risk of infection due to open wounds in the mucosa. Oral mucositis has a significant impact on the patient's quality of life and can be dose-limiting (i.e., requiring a reduction in subsequent chemotherapy doses). Other epidermal conditions include epidermal wound healing, diabetic foot ulcers, etc. The molecules of the present invention can be used in such conditions where regenerative cells are contacted with the compounds of the present invention. Contacting can be, for example, topical (including intradermal, subcutaneous), applied to the target site, etc. in gels, lotions, creams, etc.

[0160] The liver has regenerative capacity, and regenerative capacity can be enhanced by Wnt signaling. Adult hepatic progenitor (oval) cells are facultative stem cells in the liver. Active Wnt / β-catenin signaling occurs preferentially in the oval cell population, and Wnt signaling promotes the proliferation of the oval cell population in the regenerated liver. Methods for regenerating liver tissue benefit from the administration of the compounds of the present invention, which may be systemic or localized, for example, by injection into liver tissue, injection into a vein leading to the liver, implantation of a sustained release formulation, etc. Liver damage may be associated with infection, alcohol abuse, etc.

[0161] Stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, and other conditions that affect the blood-brain barrier. Angiogenesis is crucial to ensure the supply of oxygen and nutrients to many tissues throughout the body, and is particularly important to the central nervous system because nervous tissue is sensitive to hypoxia and ischemia. Blood vessels in the brain form a specialized structure called the blood-brain barrier (BBB), which limits the flow of molecules and ions from the blood to the brain. The BBB is crucial to maintaining brain homeostasis and protecting the central nervous system from toxins and pathogens. CNS endothelial cells that form the BBB differ from endothelial cells in non-neuronal tissues in that they are highly polarized cells held together by tight junctions that limit the paracellular flow of molecules and ions. In addition, CNS endothelial cells also express specific transporters that provide selective transport of essential nutrients across the BBB into the brain and to efflux potential toxins from the brain. Wnt signaling specifically regulates angiogenesis and / or function in the central nervous system. Conditions in which the BBB is compromised may benefit from administration of the compounds of the present invention, for example, by direct injection, intrathecal administration, implantation of sustained release formulations, etc.

[0162] The patient may be any animal (e.g., mammal), including, but not limited to, humans, non-human primates, rodents, and the like. Typically, the patient is a human. The treatment methods and medical uses of the surrogates of the present invention or compounds or compositions comprising the surrogates of the present invention promote tissue regeneration. The term "tissue" refers to a part of an organism consisting of cells or an aggregate of cells, optionally having similar structure, function, and / or origin. Examples of tissues include, but are not limited to, epithelial tissue, such as skin tissue, the lining of the stomach, the lining of the pancreas, the liver; connective tissue, such as the inner layer of the skin, tendons, ligaments, cartilage, bone, fat, hair, blood; muscle tissue; and nervous tissue, such as glial cells and neurons. The loss or damage can be any event that reduces the number of cells. For example, an accident, an autoimmune disorder, a side effect of a treatment, or a disease state would constitute trauma. Specific examples of conditions that may reduce cell numbers include, but are not limited to, radiation / chemotherapy, mucositis, IBD, short bowel syndrome, hereditary enteropathy, celiac disease, metabolic disease, genetic syndromes, (viral) infections (HepB / C), toxic conditions, alcoholic liver, fatty liver, cirrhosis, infections, pernicious anemia, ulcers, diabetes, diabetic foot ulcers (e.g., refractory diabetic foot ulcers), destruction of islet cells, loss of bone mass (osteoporosis), loss of functional skin, loss of hair, loss of functional lung tissue, loss of kidney tissue (e.g., acute tubular necrosis), loss of sensory cells in the inner ear. Tissue regeneration increases the number of cells in the tissue, preferably allowing connections between the cells of the tissue to be re-established, and more preferably allowing the functionality of the tissue to be restored.

[0163] Other conditions that may be treated using a surrogate of the invention or a composition comprising one or more surrogates include, but are not limited to, joint disorders, osteoporosis and related bone diseases, alopecia, and graft-versus-host disease.

[0164] The surrogates of the present invention or compositions comprising one or more surrogates may also be used in wound healing and the generation of smooth muscle tissue in many organs (eg, airways, aorta, uterus).

[0165] In some embodiments, the present invention provides methods of treatment and medical uses, as described above, in which two or more surrogates of the present invention or compounds or compositions comprising a surrogate of the present invention are administered simultaneously, sequentially, or separately to an animal or patient. Also, the surrogate(s) may be administered simultaneously, sequentially, or separately from the Wnt protein or a surrogate thereof.

[0166] In some embodiments, the present invention provides methods of treatment and medical uses, as described above, in which one or more surrogates of the present invention or compounds or compositions comprising a surrogate of the present invention are administered to an animal or patient in combination with one or more additional compounds or drugs, wherein said surrogates of the present invention or compounds or compositions comprising a surrogate of the present invention and said additional compounds or drugs are administered simultaneously, sequentially or separately.

[0167] The surrogates of the invention also have broad application in non-therapeutic methods, such as in vitro research methods.

[0168] The present invention provides methods for tissue regeneration of damaged tissue (e.g., tissues discussed in the medical uses section above), comprising administering a surrogate of the present invention. The surrogate may be administered directly to cells in vivo, or administered to a patient orally, intravenously, or by other methods known in the art, or to ex vivo cells. In some embodiments in which a surrogate of the present invention is administered to ex vivo cells, such cells may be transplanted into a patient before, after, or during administration of an agonist of the present invention.

[0169] The present invention also provides methods for enhancing proliferation of a cell, comprising providing to the cell a surrogate of the present invention. Such methods may be carried out in vivo, ex vivo, or in vitro.

[0170] Wnt signaling is a key component of stem cell culture. For example, stem cell culture media as described in WO2010 / 090513, WO2012 / 014076, Sato et al., 2011 (GASTROENTEROLOGY 2011;141:1762-1772), and Sato et al., 2009 (Nature 459,262-5). The surrogate of the present invention is a suitable RSPO substitute for use in such stem cell culture media, or can be combined with RSPO.

[0171] Thus, in one embodiment, the present invention provides a method for enhancing the proliferation of stem cells, comprising providing a surrogate of the present invention in combination with a Wnt protein or surrogate thereof to stem cells. In one embodiment, the present invention provides a cell culture medium comprising one or more proteins of the present invention. In some embodiments, the cell culture medium can be any cell culture medium already known in the art that normally contains a Wnt or RSPO, but where the Wnt or RSPO is replaced or provided (fully or partially) by a surrogate of the present invention. For example, the culture medium can be as described in WO2010 / 090513, WO2012 / 014076, Sato et al., 2011 (GASTROENTEROLOGY 2011;141:1762-1772), and Sato et al., 2009 (Nature 459,262-5), which are incorporated herein by reference in their entirety.

[0172] Stem cell culture media often contain additional growth factors. Thus, the method may additionally include supplying the stem cells with growth factors. Growth factors commonly used in cell culture media include epidermal growth factor (EGF, (Peprotech)), transforming growth factor (TGF-alpha, Peprotech), basic fibroblast growth factor (bFGF, Peprotech), brain-derived neurotrophic factor (BDNF, R&D Systems), human growth factor (HGF), and keratinocyte growth factor (KGF, Peprotech, also known as FGF7). EGF is a potent mitogen for a variety of cultured ectodermal and mesodermal cells, and has a profound effect on the differentiation of certain cells in vivo and in vitro, and on the differentiation of some fibroblasts in cell culture. The EGF precursor exists as a membrane-bound molecule that is proteolytically cleaved to generate a 53 amino acid peptide hormone that stimulates the cells. EGF or other mitogenic growth factors can be supplied to the stem cells in this way. During the culture of stem cells, mitogenic growth factors may be added to the medium every 2 days, while the medium is preferably changed every 4 days. Typically, the mitogenic factors are selected from the group consisting of: i) EGF, TGF-alpha, and KGF; ii) EGF, TGF-alpha, and FGF7; iii) EGF, TGF-alpha, and FGF; iv) EGF and KGF; v) EGF and FGF7; vi) EGF and FGF; vii) TGF-α and KGF; viii) TGF-alpha and FGF7; or ix) TGFα and FGF.

[0173] These methods of enhancing stem cell proliferation can be used to grow new organoids and tissues from stem cells, for example, as described in WO2010 / 090513, WO2012 / 014076, Sato et al., 2011 (GASTROENTEROLOGY 2011;141:1762-1772), and Sato et al., 2009 (Nature 459,262-5).

[0174] Several clinically significant conditions are characterized by an inability to regenerate tissue, in which enhanced Wnt signaling is desirable.

[0175] In some embodiments, RSPO surrogates are used to enhance stem cell regeneration. Stem cells of interest include muscle satellite cells, hematopoietic stem cells and progenitor cells derived therefrom (U.S. Patent No. 5,061,620), neural stem cells (see Morrison et al. (1999) Cell 96:737-749), embryonic stem cells, mesenchymal stem cells, mesodermal stem cells, hepatic stem cells, and the like.

[0176] RSPO surrogates are used in enhancing bone healing. In many clinical situations, bone healing conditions are not ideal due to reduced activity of bone-forming cells, for example, in the elderly, after injury, in cases of osteogenesis imperfecta, etc. A variety of bone and cartilage disorders affect older individuals. Such tissues are normally regenerated by mesenchymal stem cells. Such conditions include osteoarthritis. Osteoarthritis occurs as a manifestation of "wear and tear" on the joints of the body. Thus, athletes or overweight individuals develop osteoarthritis in large joints (knees, shoulders, hips) due to loss or damage of cartilage. This hard, smooth cushion that covers the bony joint surfaces is composed mainly of collagen, a structural protein in the body that forms a mesh to provide support and flexibility to the joints. When cartilage is damaged and lost, the bone surfaces undergo abnormal changes. Some inflammation occurs, but not to the same extent as seen in other types of arthritis. Nevertheless, osteoarthritis causes considerable pain and disability in the elderly.

[0177] In a method for accelerating bone repair, the pharmaceutical composition of the present invention is administered to a patient suffering from injury to bone (e.g., after injury). The formulation is preferably administered at or near the site of injury after injury requiring bone regeneration. The Wnt formulation is preferably administered for a short period of time and at a dose effective to increase the number of osteoprogenitor cells present at the injury site. In some embodiments, the Wnt is administered within about 2 days, usually within about 1 day, of injury, and is provided for about 2 weeks or less, about 1 week or less, about 5 days or less, about 3 days or less, etc.

[0178] In an alternative method, a patient suffering from damage to bone is provided with a composition comprising bone marrow cells, e.g., a composition comprising mesenchymal stem cells capable of differentiating into osteoblasts, bone marrow cells, etc. The bone marrow cells can be treated ex vivo with a pharmaceutical composition comprising a dose of Wnt protein(s) sufficient to enhance regeneration, or the cell composition can be administered to the patient along with a Wnt formulation of the invention.

[0179] array An scFv fragment recognizing ZNRF3 is fused to human IL-2 in an exemplary construct as follows.

[0180] Z6 scFv-human IL2 (SEQ ID NO: 1): [ka]

[0181] The elements of the array are: SEQ ID NO:1, residues 1-255 scFv sequence specific for human ZNRF3. Exemplary CDR sequences are underlined. SEQ ID NO:1, residues 256-265 is a linker. SEQ ID NO:1, residues 266-398 is a human IL-2 sequence. SEQ ID NO:1, residues 399-408 is a histidine tag. [ka] Thus, the CDR sequences of an exemplary antibody are as follows: [ka]

[0182] A similar construct containing the Z6 antibody was made using an IL-4 fusion as follows. Z6-IL4 (SEQ ID NO:2) (residues 266-394 are human IL-4 protein) [ka]

[0183] An scFv fragment recognizing RNF43 is fused to human IL-2 in an exemplary construct as follows.

[0184] R5 scFv-human IL2 (SEQ ID NO:3): [ka]

[0185] The elements of the array are: SEQ ID NO:1, residues 1-258 scFv sequence specific for human RNF43. Exemplary CDR sequences are underlined. SEQ ID NO:1, residues 258-268 is a linker. SEQ ID NO:1, residues 268-403 is a human IL-2 sequence. SEQ ID NO:1, residues 404-414 is a histidine tag. [ka] Thus, the CDR sequences of an exemplary antibody are as follows: [ka]

[0186] A similar construct containing the R5 antibody was made using an IL-4 fusion as follows. R5-IL4 (SEQ ID NO: 4) (residues 268-396 are human IL-4 protein) R5IL4 (SEQ ID NO: 4) [ka]

[0187] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is near or near atmospheric pressure. EXAMPLES

[0188] Example 1 To generate scFv antibody fragments that recognize RNF43 and anti-ZNRF3, the inventors used a yeast display library of naive human scFvs to select for binders to recombinant RNF43 and ZNRF3.

[0189] A candidate surrogate RSPO was generated by fusing Z6 scFv with human IL2 (SEQ ID NO:1).

[0190] The Z6-IL2 fusion was expressed in insect cells and purified by nickel and gel filtration chromatography. Z6-IL2 eluted as a monodisperse peak from the gel filtration column, indicating that the protein does not aggregate and has favorable biochemical behavior.

[0191] We used two different reporter cell lines, HEK293T SuperTOPflash (STF) cells and CD25+ HEK293T STF cells, to test the signaling activity of surrogate RSPOs. HEK293 STF cells harbor a stably integrated firefly luciferase driven by 8x TCF / LEF binding sites (Wnt-responsive promoter) and a constitutively expressed Renilla luciferase control reporter. CD25+ HEK293T STF cells were generated using a retroviral expression system to transduce HEK293T STF cells with human CD25 (IL-2 receptor).

[0192] To monitor the enhancement of Wnt activity, we treated STF or CD25+ STF cells with either Wnt3a conditioned medium or a surrogate Wnt agonist in the presence or absence of the surrogate RSPO Z6-IL2 or a protein of interest, then lysed the cells and performed luciferase assays.

[0193] Surrogate RSPO (Z6-IL2) has a moderate effect on non-transfected STF 293 cells treated with Wnt3a conditioned medium. Z6-IL2, negative control scFv, IL2 have no effect in the absence of Wnt agonist. Surrogate RSPO (Z6-IL2) has a moderate effect on non-transfected STF 293 cells treated with surrogate Wnt (scFv+Dkk1). Z6-IL2, negative control scFv, IL2 have no effect in the absence of Wnt agonist. Surrogate RSPO (Z6-IL2) has a strong effect on CD25+ cells treated with surrogate Wnt (scFv+Dkk1), indicating selectivity for the target cell type. Z6-IL2, negative control scFv, IL2 have no effect in the absence of Wnt agonist.

[0194] The inventors also generated fusions of the Z6 scFv with IL4, and fusions of the RNF43-binding scFv, R5, with IL2 and IL4.

[0195] Example 2 Secreted R-spondin 1-4 proteins (RSPO1-4) orchestrate intestinal stem cell renewal and tissue homeostasis by enhancing canonical Wnt signaling. RSPO functions by connecting the extracellular domains (ECDs) of negative Wnt regulators RNF43 or ZNRF3 to their co-receptors LGR4, LGR5, or LGR6, which induces membrane clearance of the complex. Although RSPO is an emerging candidate for regenerative medicine applications, the sparse tissue distribution of LGR4 / 5 / 6 proteins limits the overall biomedical utility of RSPO.

[0196] Here, the inventors provide a dual-specific ligand that enhances Wnt activity independent of LGR4 / 5 / 6. Such "surrogate RSPO" consists of a single-chain antibody variable fragment (scFv) specific for RNF43 or ZNRF3 connected to the immunocytokine IL-2 via a flexible linker. The surrogate RSPO mimics the function of native RSPO by crosslinking the ECD of RNF43 or ZNRF3 with the IL-2 receptor CD25, resulting in highly selective Wnt signaling amplification on CD25-expressing cells. Furthermore, the surrogate RSPO was able to functionally replace wild-type RSPO and stimulate the growth of CD25+ human colon organoids. These results demonstrate engineered ligands that mimic RSPO function on a broad range of cell or tissue types, opening new avenues for the development of RSPO-based therapeutics.

[0197] The Wnt signaling pathway controls stem cell development and tissue homeostasis in all metazoans. Wnt activity is tightly regulated by several host-encoded proteins, which can function to enhance or attenuate signaling. R-spondin proteins (RSPO1-4 in mammals) function by antagonizing negative regulators of the Wnt pathway, and coadministration of Wnt and RSPO can result in signaling output hundreds of times greater than Wnt alone. RSPO-mediated enhancement of Wnt activity occurs through an indirect mechanism that greatly increases the levels of the Wnt receptors frizzled (Fzd) and LRP5 or LRP6 on the cell surface. In the absence of RSPO, Wnt receptors are persistently downregulated by the transmembrane E3 ligases (TMULs) RNF43 and ZNRF3, which mediate ubiquitination of the intracellular domain of Fzd, targeting both Fzd and the associated LRP5 / 6 for internalization and degradation (Figure 7A). RSPO inactivates RNF43 / ZNRF3 by simultaneously binding its extracellular protease-associated (PA) domain and the extracellular domain (ECD) of a second receptor, leucine-rich repeat-containing G protein-coupled receptor 4, 5, or 6 (LGR4-6). Cross-linking of RNF43 / ZNRF3 with LGR4 / 5 / 6 by RSPO induces membrane clearance of the ternary complex, which sequesters RNF43 / ZNRF3 from Fzd and thus enhances Wnt signaling (Figure 7B).

[0198] Biomedical applications of recombinant RSPO have been widely explored in the field of regenerative medicine, particularly in the context of the small intestine, where RSPO functions to promote stem cell renewal at the base of the crypts. For example, recombinant RSPO protects animals from both experimentally induced colitis and lethal doses of chemoradiotherapy by stimulating intestinal regeneration. Furthermore, exogenous RSPO is crucial for intestinal organoid cultures, an in vitro model system used to study the intestinal epithelium. It has recently been reported that Wnt and RSPO play non-equivalent roles in intestinal stem cell (ISC) self-renewal through a feedback loop in which Wnt drives LGR5 expression and RSPO induces ISC proliferation. This raises the possibility that targeting pathways individually or in concert may be associated with unique therapeutic outcomes.

[0199] Although RSPOs are promising candidates to selectively enhance innate Wnt activity and avoid off-target effects, the biomedical utility of RSPOs is limited by the restrictive expression of LGR4 / 5 / 6 in only a small subset of cell types. To overcome the limitations of naturally encoded RSPOs, we designed synthetic ligands that phenocopy RSPO-mediated Wnt signal enhancement through an LGR4 / 5 / 6-independent mechanism. Such "surrogate RSPOs" function by crosslinking RNF43 or ZNRF3 ECD with a cell surface marker (CD25) known to mimic RSPO-mediated sequestration of RNF43 / ZNRF3 by undergoing internalization upon ligand binding (Figure 7C). Surrogate RSPOs drive receptor-specific amplification of Wnt signaling in reporter cells and human colonic organoids, and their modular design allows them to be adapted to target virtually any cell type, a feature with major implications for the development of Wnt-based therapeutics.

[0200] result Generation of scFv specific for RNF43 and ZNRF3 Our surrogate RSPO design utilized the fusion of two components: (i) an RNF43 or ZNRF3 binding protein, and (ii) a tissue-specific targeting protein that induces endocytosis of the receptor. Administration of the surrogate RSPO protein mimics the function of the native RSPO by cross-linking the ECD of RNF43 or ZNRF3 with the modular cognate receptor, promoting membrane clearance of the complex. The inventors used approximately 1×10 9 RNF43 or ZNRF3 binding protein components for the surrogate RSPO were isolated by selecting single-chain variable fragments (scFv) from a yeast display library of published sequences. scFvs were obtained by performing two parallel sets of selections on the RNF43 ECD or ZNRF3 ECD using magnetic activated cell sorting (MACS) or fluorescence activated cell sorting (FACS). After several rounds of selection, a panel of different scFvs specific for RNF43 or ZNRF3 were isolated (Figure 11), and clones "R5" and "Z6" were chosen for incorporation into the surrogate RSPO cassette based on their ability to robustly bind RNF43 and ZNRF3, respectively, in a fluorescence-based assay (Figure 8A).

[0201] Design, expression, and purification of surrogate RSPO. It has been reported that membrane clearance of the RSPO signaling complex requires internalization of the receptors LGR4-6. Therefore, we selected the immunocytokine IL-2 to act as a surrogate for RSPO-LGR binding in our surrogate RSPO constructs based on its ability to induce endocytosis of its cognate receptor, CD25. Two RSPO constructs were generated, one targeted to RNF43 by fusing R5 to IL-2 (R5-IL2) and one targeted to ZNRF3 by fusing Z6 to IL-2 (Z6-IL2). Single-chain constructs were designed such that R5 or Z6 was connected to the N-terminus of IL-2 via a flexible 5x(Gly-Ser) linker, with the intention that R5 / Z6 and IL-2 could bind independently to their respective targets. R5-IL2 and Z6-IL2 were expressed in insect cells using baculovirus and both proteins eluted as monodisperse peaks from a gel filtration column, indicating favorable biochemical behavior (Figure 11).

[0202] Biophysical characterization of surrogate RSPO-receptor interactions. We used surface plasmon resonance (SPR) to measure the binding affinity of R5-IL2 and Z6-IL2 to RNF43 and ZNRF3. We found that R5-IL2 had a dissociation constant (K d ) binds to RNF43 and Z6-IL2 has a K of 80 nM d We determined that R5-IL2 bound to ZNRF3 at 1 kDa. R5-IL2 did not cross-react with ZNRF3, and Z6-IL2 did not cross-react with RNF43. We also used SPR to further demonstrate that R5-IL2 and Z6-IL2 bound to ZNRF3 at 1 kDa. dWe determined that R5 and Z6 bind to CD25 with nearly identical Kd values ​​to those of RNF43 / ZNRF3. In summary, our SPR experiments demonstrate that R5 and Z6 bind to their respective targets with similar affinities, that the affinities of the surrogate RSPOs fall within the range of affinities reported between wild-type RSPO and RNF43 / ZNRF3 (approximately 5-10,000 nM), and that IL-2 binding to CD25 is retained in the surrogate RSPO format.

[0203] Surrogate RSPO-mediated Wnt signaling enhancement. We performed luciferase assays to test the ability of R5-IL2 and Z6-IL2 to selectively enhance Wnt signaling. We compared the activity of surrogate RSPOs in HEK293 Super Top Flash (STF) Wnt reporter cells or HEK293 STF cells transduced with CD25 by lentivirus. In CD25-expressing cells, R5-IL2 increased Wnt3a reporter activity by 9.5-fold, and Z6-IL2 increased Wnt3a activity by 41-fold (Figure 9A). The greater activity of Z6-IL2 than that of R5-IL2 is consistent with previous reports that found ZNRF3 to be a predominantly expressed homolog (vs. RNF43) in HEK293 cells. Given that native RSPOs are cross-reactive and that the potent activity of RSPO2 may require both RNF43 and ZNRF3, we hypothesized that the combination of R5-IL2 and Z6-IL2 would have a synergistic effect. Therefore, we incubated cells with a 1:1 mixture of R5-IL2 and Z6-IL2 and found that this combination increased Wnt3a activity by 148-fold (Figure 9A). In non-transduced cells, the addition of R5-IL2, Z6-IL2, and R5-IL2+Z6-IL2 resulted in only a 2.7-fold, 3.9-fold, and 5.8-fold increase in Wnt3a activity, respectively (Figure 9B), indicating that the surrogate RSPO is highly selective for cells expressing CD25.

[0204] In both CD25-positive and CD25-negative cells, we found that a mixture of Wnt3a-conditioned medium and the receptor-binding fragment of RSPO2 (furin domains 1 and 2) elicited a response that was 286-304 times greater than Wnt3a alone, similar to the level of RSPO-mediated enhancement observed previously. As a negative control, we treated cells with Wnt3a-conditioned medium supplemented with R5, Z6, or IL-2. R5 scFv and IL2 cytokines did not substantially enhance Wnt3a signaling (Figure 9B). However, Z6 scFv displayed mild agonistic activity, resulting in a 10- and 12-fold increase in HEK293 STF and CD25-expressing cells, respectively (Figures 9A, 9B).

[0205] Surrogate RSPO-mediated stimulation of intestinal organoid growth. Differentiation of stem cells into human colonic organoids requires the addition of exogenous RSPO along with Wnt, EGF, Noggin, and TGF-β inhibitors. To determine whether R5-IL2 or Z6-IL2 can substitute for RSPO in human colonic organoid culture, the inventors performed an organoid growth assay using LGR5+ intestinal stem cells transduced by lentivirus to express CD25. In this assay, CD25+ stem cells were cultured in the presence of RSPO, IL-2, R5 scFv, Z6 scFv, R5-IL2, Z6-IL2, or a 1:1 mixture of R5-IL2 and Z6-IL2, and organoid growth was monitored by fluorescence. R5-IL2, Z6-IL2, or a mixture of R5-IL2 and Z6-IL2 each resulted in a significant increase in organoid growth compared to the negative control protein, IL-2 (Figure 10). In contrast, the addition of either R5 scFv or Z6 scFv did not result in a significant increase. Fluorescence increased 7.3-fold in R5-IL2-treated cultures, 9.4-fold in Z6-IL2-treated cultures, and 11-fold in cultures treated with a mixture of R5-IL2 and Z6-IL2. The positive control RSPO2 stimulated growth 73-fold, a more potent effect than the mixture of R5-IL2 and Z6-IL2. This is similar to the results we observed in the luciferase assay. Importantly, neither R5-IL2 nor Z6-IL2 stimulated the growth of wild-type organoids (Figure 10). This indicates that the surrogate RSPO exerts its effect specifically on cells expressing the CD25 target receptor.

[0206] The development of surrogate RSPO proteins represents a new method to facilitate tissue-specific enhancement of Wnt activity and expands our understanding of the RSPO signaling mechanism. Here, we show that surrogate RSPOs can mimic the activity of endogenous RSPOs in the absence of the cognate LGR4 / 5 / 6 receptors by utilizing the IL-2 ligand, known to induce endocytosis of the CD25 receptor. This finding supports the current model that RSPOs function by promoting endocytosis of RNF43 / ZNRF3 and opens new avenues for engineered surrogate RSPOs acting on a broad spectrum of endocytic receptors. Of note, the most robust Wnt signal enhancement in 293 cells was achieved by coadministration of R5-IL2 and Z6-IL2 surrogate RSPOs (Figure 9). This indicates that the inherent cross-reactivity of native RSPOs allows overcoming both RNF43- and ZNRF3-mediated inhibition. Thus, optimization of surrogate RSPOs can be achieved by incorporating cross-reactive RNF43 / ZNRF3 binding modules (e.g., RSPO FU1 domains) into the design, while the ability of existing surrogate RSPOs to target a given E3 ligase (RNF43 vs. ZNRF3) confers an additional degree of selectivity that may be beneficial in certain contexts.

[0207] Materials and Methods Protein expression and purification. Human RNF43 extracellular domain (ECD) (amino acids 24-197) and ZNRF3 ECD (amino acids 56-219) were cloned into pAcGp67A vector with a C-terminal biotin acceptor peptide tag (SEQ ID NO: 5, GLNDIFEAQKIEW) followed by a 6X His tag. Unless otherwise stated, RNF43 / ZNRF3 in the methods section refers to their ECDs only. Selected RNF43 and ZNRF3 high affinity human antibody scFv fragments were cloned into pAcGp67A with a C-terminal 6X His tag. These human antibody scFv fragments were further cloned in frame into pAcGp67A with a (GS)5 linker and human interleukin-2 (amino acids 21-153) followed by a 6X His tag. All proteins were expressed in Hi-Five cells (Invitrogen) from Trichoplusia ni infected with baculovirus. Cultures were harvested 60 hours after infection. Proteins were purified by nickel affinity chromatography followed by size exclusion chromatography in 1× HBS buffer (10 mM HEPES pH 7.2, 150 mM sodium chloride). Prior to size exclusion chromatography, RNF43 and ZNRF3 were site-specifically biotinylated at the C-terminal biotin acceptor peptide by BirA ligase. This size exclusion chromatography is performed to remove free biotin in the buffer. All proteins were used immediately after purification or flash frozen in liquid nitrogen with 20% glycerol.

[0208] Selection of RNF43 / ZNRF3-binding scFv fragments. A non-immune yeast surface display library of human antibody scFv fragments was kindly provided by the Wittrup group (Feldhaus et al. Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Nature biotechnology 21, 163 (2003)). A time-course selection strategy with magnetic bead selection followed by flow cytometric sorting was used as previously reported (Luca et al. Structural basis for Notch1 engagement of Delta-like 4. Science 347, 847-853 (2015)). Selection round 1 was performed by first mixing 250 uL of magnetic streptavidin microbeads (Militenyi) with 400 nM biotinylated RNF43 / ZNRF3. These microbeads were loaded with 1 × 10 mAbs of the scFv fragment library. 10 The yeast was then passed through a magnetic activated cell sorting (MACS) LS separation column (Militenyi) to collect ZNRF3 / RNF43 binders. Round 2 was performed by collecting 1 × 10 8 Round 1 was repeated with yeast. In round 3, yeast were preincubated with 200 nM biotinylated RNF43 / ZNRF3 and then incubated with Alexafluor-647 dye (SA-647, Life Technologies). ZNRF3 / RNF43 binders were enriched by MACS using anti-647 microbeads (Militenyi). In round 4, yeast were preincubated with 5 nM biotinylated RNF43 or 10 nM biotinylated ZNRF3. Yeast were further stained with SA-647 and an Alexa Fluor 488-conjugated antibody against the c-Myc epitope (Myc-488, Cell Signaling). High affinity RNF43 / ZNRF3 binders were isolated by fluorescence-activated cell sorting (FACS).

[0209] Plasmids from the final round of selection were isolated and sequenced using Zymoprep Yeast Plasmid Miniprep Kit (Zymo Research). The plasmids were transferred into S. cerevisiae EBY100 yeast by electroporation, cured in SDCAA selection medium, and then induced in SGCAA induction medium. Individual scFv-transformed yeast were incubated with increasing concentrations of biotinylated RNF43 / ZNRF3. The yeast were stained with SA-647 and Myc-488, and fluorescence was monitored by flow cytometry. Data were analyzed with GraphPad Prism 7 to identify the highest affinity clones for further experiments.

[0210] Binding of RNF43 and ZNR3 to yeast surface-displayed scFv. Yeast cells expressing either the R5 or Z6 scFv were stained with recombinant RNF43 or ZNR3 ECD at 1 μM concentration (in PBS + 0.1% BSA, respectively). Cells were then washed, incubated with SA-647 and Myc-488, washed again, and then analyzed by flow cytometry.

[0211] Surface Plasmon Resonance. All binding measurements were performed using a BIAcore T100 instrument (GE Healthcare). Biotinylated RNF43, ZNRF3, and CD25 were bound at low density onto a SA sensor chip (GE Healthcare). An irrelevant biotinylated protein was captured at a binding density equivalent to the control flow cell. Increasing concentrations of R5-IL2 and Z6-IL2 were injected at 30 μl / mL onto the chip in HBS-P (GE Healthcare). Resonance units were calculated by subtracting the resonance units observed on the flow cells containing RNF43, ZNRF3, or CD25 from those of the control flow cell. Curves were fitted to a 1:1 binding model using Biaevaluation software (Biacore / GE Healthcare).

[0212] Luciferase signaling assay. HEK293 cells were stably transfected with a firefly luciferase reporter and a Renilla luciferase reporter under the control of a concatemer of seven LEF / TCF binding sites by lentivirus as previously described in Janda et al. Surrogate Wnt agonists that phenocopy canonical Wnt and β-catenin signalling. Nature 545,234-237 (2017). These cells were also stably transfected with human CD25 by retrovirus. These cells co-express YFP. Transfected HEK293 cells were FACS sorted for YFP expression. CD25 surface expression was further confirmed by staining transfected HEK293 cells with Brilliant Violet 605 antibody (BioLegend, Clone BC96, #302632). Fluorescence was monitored by flow cytometry and compared to non-transfected HEK293 cells. Dual luciferase assays were performed using the Dual Luciferase Assay kit (Promega) as directed. Briefly, cells were plated in 96-well plates at a density of 10,000 cells per well 24 h prior to stimulation. Cells were stimulated with 20% Wnt3A-conditioned medium (ATCC) supplemented with RSPO2 protein or surrogate RSPO. Cells were cultured in the presence of stimulating reagents for an additional 24 h, after which cells were washed and lysed as per the Dual Luciferase Assay kit (Promega) manual. Brightness signals were recorded using a SpectraMax Paradigm and analyzed with GraphPad Prism 7.

[0213] Organoid growth assay. Fresh colon samples were collected at Stanford Hospital with informed consent. Cultures were passaged several times in WENR medium (Wnt3a, R-spondin, EGF, Noggin) to ensure robust growth of organoids. To assess surrogate activity, organoids were transduced with lentivirus expressing CD25 and FACS sorted for YFP+ (YFP co-expressed with CD25) to enrich for the CD25+ population. CD25+ cells were subcultured in medium conditioned as in Figure 10. Organoid morphology was observed and phase contrast photographs were taken using a Nikon TS100. To quantify cell growth, organoids are dissociated into single cells and plated at 10,000 cells per well in a 96-well format. Three days after plating, cell viability assays were performed using alamarBlue cell viability reagent (Thermo Fisher).

[0214] The foregoing merely illustrates the principles of the present invention. It will be appreciated that those skilled in the art can devise various arrangements that embody the principles of the present invention and are within the spirit and scope of the present invention, even if not explicitly described or shown herein. Furthermore, all examples and conditional language given herein are intended primarily to aid the reader in understanding the principles of the present invention and the concepts that the inventors contribute to the advancement of the art, and should not be construed as being limited to such specifically listed examples and conditions. Furthermore, all statements herein that recite principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. In addition, such equivalents are intended to include both currently known equivalents and equivalents developed in the future (i.e., any elements developed that perform the same function, regardless of structure). Thus, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

Claims

1. (i) a polypeptide comprising a fragment of RSPO that contains a furin1 domain, the fragment of RSPO specifically binding to RNF43 or ZNRF3; (ii) a cytokine that specifically binds to a cell surface receptor, or a polypeptide comprising an antibody or antigen-binding fragment thereof that specifically binds to a cell surface antigen, which does not target LGR4, LGR5, or LGR6; A molecule that enhances Wnt signaling activity, comprising:

2. (i) a polypeptide comprising said fragment of RSPO; (ii) the antibody or antigen-binding fragment thereof that specifically binds to the cell surface antigen; The molecule of claim 1 , comprising:

3. (i) a polypeptide comprising said fragment of RSPO; (ii) the cytokine that specifically binds to the cell surface receptor; The molecule of claim 1 , comprising:

4. The molecule of claim 3, wherein the cytokine is IL-2 or IL-4.

5. (i) The molecule described in any one of claims 1 to 2, wherein the fragment of RSPO is fused via a linker to the antibody or antigen-binding fragment thereof that specifically binds to the cell surface antigen.

6. (i) The molecule of any one of claims 1 to 2, wherein the fragment of RSPO is fused directly to the antibody or antigen-binding fragment thereof that specifically binds to the cell surface antigen.

7. The molecule of any one of claims 1 to 6, linked to a Wnt agonist or a Wnt polypeptide.

8. A pharmaceutical composition comprising a molecule according to any one of claims 1 to 7 and a pharma- ceutically acceptable excipient.

9. 10. A composition for use in a method of enhancing Wnt signaling, the composition comprising a molecule according to any one of claims 1 to 7, the method comprising contacting a cell expressing a Frizzled receptor with the molecule in the presence of a Wnt agonist or a Wnt polypeptide.

10. A composition comprising a molecule according to any one of claims 1 to 7 for treating or preventing a disease or disorder in a subject in need thereof.

11. A composition for treating a disease or disorder in a subject in need thereof, wherein the subject has a disease or disorder associated with decreased Wnt signaling, the composition comprising: (i) a fragment of RSPO that contains a furin1 domain, the fragment of RSPO that specifically binds to RNF43 or ZNR3, or a polypeptide that contains an antibody or antigen-binding fragment thereof that specifically binds to RNF43 or ZNR3; (ii) a cytokine that specifically binds to a cell surface receptor, or a polypeptide comprising an antibody or antigen-binding fragment thereof that specifically binds to a cell surface antigen, which does not target LGR4, LGR5, or LGR6; Molecules that enhance Wnt signaling activity, including A composition comprising:

12. 12. The composition of claim 10 or claim 11, wherein the subject has a disease or disorder selected from radiation / chemotherapy injury, mucositis, inflammatory bowel disease, short bowel syndrome, inherited enteropathy, celiac disease, metabolic disease, genetic syndrome, viral infection (e.g., HepB / C), toxic states, alcoholic liver, fatty liver, cirrhosis, infection, pernicious anemia, ulcers, diabetes, diabetic foot ulcers (e.g., refractory diabetic foot ulcers), destruction of islet cells, loss of bone mass (osteoporosis), loss of functional skin, loss of hair, loss of functional lung tissue, loss of kidney tissue (e.g., acute tubular necrosis), loss of sensory cells in the inner ear, joint disorders, osteoporosis and related bone diseases, alopecia, and graft versus host disease.

13. A composition comprising a molecule according to any one of claims 1 to 7 for enhancing wound healing and / or tissue generation in a subject in need thereof.

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