WNT surrogate fusion molecule

Abstract

The present invention provides WNT surrogate fusion molecule, wherein the fusion molecule is a WNT agonist and cytokine pathway modulator, which may be used in any of a variety of therapeutic methods for the treatment of diseases.

Description

WNT SURROGATE FUSION MOUECUUECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 686,651, filed August 23, 2024, which is incorporated herein by reference in its entirety.STATEMENT REGARDING SEQUENCE LISTING

[0002] The Sequence Listing XML associated with this application is provided in XML file format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing XML is SRZN 031 01WO _ST26.xml. The XML file is 27,453 bytes, and created on August 19, 2025, and is being submitted electronically via USPTO Patent Center.FIELD OF INVENTION

[0003] The present disclosure relates to WNT surrogate fusion molecules, wherein a WNT agonist comprising two antibodies or antigen-binding fragments is fused to a third antibody or antigen-binding fragment, and treatment methods for various disorders.DESCRIPTION OF THE RELATED ART

[0004] WNT (“Wingless-related integration site” or “Wingless and Int-1” or “Wingless- Inf ’) ligands and their signals play key roles in the control of development, homeostasis and regeneration of many essential organs and tissues, including bone, liver, skin, stomach, intestine, kidney, central nervous system, mammary gland, taste bud, ovary, cochlea, lung, eye, and many other tissues (reviewed, e.g., by Clevers, Loh, and Nusse, 2014;346: 1248012). Modulation of WNT signaling pathways has potential for treatment of degenerative diseases and tissue injuries.

[0005] Cytokines are proteins that regulate the immune system and inflammation. Interleukin-6 (IL-6) is a multifunctional cytokine that plays a central role in host defense due to its wide range of immune and hematopoietic activities and its potent ability to induce the acute phase response. Overexpression of IL-6 has been implicated in the pathology of a number of diseases including multiple myeloma, rheumatoid arthritis, Castleman's disease, psoriasis, and post-menopausal osteoporosis.

[0006] Inflammation has been implicated in the pathogenesis of retinal diseases, and antiinflammatory therapies such as steroids have been effective in treating uveitis and diabeticmacular edema (DME). Detailed studies looking at anti-inflammation and infection in the eye have shown that the pro-inflammatory cytokine, IL-6, is significantly elevated in the ocular fluids of refractory / chronicuveitis patients, and inhibition of IL-6 in animal models inhibits the onset of uveitis. High ocular fluid levels of IL-6 are also found in patients with DME and retinal veinocclusion. Additionally, chronic inflammatory cells have been seen on the surface of the Bruch’s membrane in eyes with neovascular age-related macular degeneration (AMD), patients with AMD have been reported to have increased serum levels of IL-6, and anti -IL-6 pathway antibodies have been explored for treating uveitis and uveitic macular edema.[0007| There is a need in the art for improved therapeutics and methods for treating eye disease, including DME, AMD and DR. The present disclosure meets this need by providing therapeutics and methods for treating these and other diseases.BRIEF SUMMARY

[0008] In various embodiments, the present invention provides WNT surrogate fusion molecules and related uses thereof.

[0009] In one embodiment, the disclosure provides a WNT surrogate fusion molecule, wherein the WNT surrogate fusion molecule comprises: one or more low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or a Low-density lipoprotein (LDL) receptor- related protein 6 (LRP6) or LRP5 / LRP6 binding antibody or antigen-binding fragment that specifically binds to one or more LRP5 / 6 receptor; and one or more frizzled (FZD) binding antibody or antigen-binding fragments that specifically bind to one or more FZD receptor; and one or more cytokine or antibodies or antigen-binding fragments that specifically bind to one or more cytokine receptor and / or ligand.

[0010] In particular embodiment, the WNT surrogate fusion molecule is tetravalent, pentavalent, or hexavalent.

[0011] In some embodiments, the WNT surrogate fusion molecule, wherein the hexavalent surrogate molecule comprises: a) two low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or a Low- density lipoprotein (LDL) receptor-related protein 6 (LRP6) or LRP5 / LRP6) antibody or antigen-binding fragment that specifically binds to one or more LRP5 / 6 receptor; and b) two frizzled (FZD) antibody or antigen-binding fragments that specifically bind to one or more FZD receptor; and c) two cytokine antibodies or antigen-binding fragments that specifically bind to two cytokine receptors and / or ligands.

[0012] In another embodiment, the pentavalent surrogate molecule comprises: a) one low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or a Low- density lipoprotein (LDL) receptor-related protein 6 (LRP6) or LRP5 / LRP6 binding antibody or antigen-binding fragment that specifically binds to one LRP5 / 6 receptor; b) two frizzled (FZD) antibody or antigen-binding fragments that specifically bind to two FZD receptors, and c) two cytokine antibodies or antigen-binding fragments that specifically bind to cytokine receptor and / or ligand.[0013| In related embodiments, the pentavalent surrogate molecule comprises: a) two low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or a Low- density lipoprotein (LDL) receptor-related protein 6 (LRP6) or LRP5 / LRP6 binding antibody or antigen-binding fragments that specifically bind to one LRP5 / 6 receptor; and; b) one frizzled (FZD) antibody or antigen-binding fragment that specifically bind to one FZD receptor, and c) two cytokine antibodies or antigen-binding fragments that specifically bind to cytokine receptor and / or ligand.10014 [ In another embodiment, the pentavalent surrogate molecule comprises: a) two low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or a Low- density lipoprotein (LDL) receptor-related protein 6 (LRP6) or LRP5 / LRP6 binding antibody or antigen-binding fragments that specifically bind to one LRP5 / 6 receptor; and; b) two frizzled (FZD) antibody or antigen-binding fragments that specifically bind to one FZD receptor, and c) one cytokine antibodies or antigen-binding fragments that specifically binds to cytokine receptor and / or ligand.

[0015] In some embodiments, the tetravalent surrogate molecule comprises: a) one low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or a Low- density lipoprotein (LDL) receptor-related protein 6 (LRP6) or two LRP5 / LRP6 antibody or antigen-binding fragments that specifically binds to LRP5 / 6 receptor; and b) two frizzled (FZD) antibody or antigen-binding fragments that specifically bind to one or more FZD receptor or one frizzled (FZD) antibody or antigen-binding fragment that specifically bind to one or more FZD receptor; and c) one cytokine antibodies or antigen-binding fragments that specifically bind to one cytokine receptor and / or ligand.

[0016] In another embodiment, the WNT surrogate fusion molecule’s antigen-binding fragments are selected from the group consisting of: IgG, scFv, Fab, and VHH or single domain antibodies (sdAb), diabody, and Fv.

[0017] In some embodiments, the LRP5 / 6 antigen-binding region comprises a VHH or sdAb or a scFv or a Fab or a Fv; the FZD antigen-binding region comprises a VHH or sdAb or sdAb scFv or Fab or a Fv or a diabody, wherein the cytokine binding region comprises a VHH or sdAb or scFv or Fab or a Fv or a diabody, and wherein the Fab is present within a full immunoglobulin (Ig), optionally an IgG, comprising a light chain and a heavy chain.

[0018] In some embodiments, wherein each of the two LRP5 / 6 binding regions, FZD binding regions, IL6 binding regions are fused or linked to each other via a linker moiety.

[0019] In one embodiment, the one or more FZD binding regions bind to one or more FZD 4 receptors.

[0020] In some embodiment, the cytokine antibodies or antigen-binding fragments that specifically bind to one or more cytokine receptor and / or ligand bind IL6 and / or IL6 receptors. In particular embodiment, IL6 antibodies or antigen-binding fragments that specifically bind to one or more IL6 ligand or IL6 receptors comprise siltuximab, sirukumab, olokizumab, clazakinumab, gerilimzumab, Tocilizumab , Sarilumab, Vamikibart and satralizumab.

[0021] In some embodiments, the disclosure provides a multivalent fusion molecule, wherein the molecule comprises: a) one or more antibody or antigen-binding fragments (anti-antigen2) that specifically bind to one or more antigen 2, wherein the antigen-binding fragment comprises Fab 1; b) one or more antibody or antigen-binding fragments (anti-antigen2) that specifically bind to one or more antigen 2, wherein the antigen-binding fragment comprises Fab 2; and c) one or more antibody or antigen-binding fragments (anti-antigen3) that specifically bind to one or more antigen 3, wherein the antigen-binding fragment comprises Fab 3; wherein the Fab 1 heavy chain (HC) is fused to Fab 3 HC, and Fab2 HC is fused to Fab 3 light chain (LC), or Fab 1 LC is fused to Fab 3 HC and Fab 2 LC is fused to Fab 3 LC, and wherein the Fabs region are fused or linked to each other via a linker moiety, and where in Fabl and Fab2 could have the same or different sequences and could bind to the same or different antigen.

[0022] In a related embodiment of the multivalent fusion molecules: a) one or more anti-antigenl comprises LRP 5 / 6 binding antibody or antigen-binding fragment that specifically bind to one or more LRP5 / 6 receptor;b) one or more anti-antigen2 comprises FZD binding antibody or antigen-binding fragment that specifically bind to one or more FZD receptors; and c) one or more anti-antigen3 that specifically bind to one or more cytokine receptor and / or ligand.

[0023] In a related embodiment, the present disclosure provides a nucleic acid or a combination of nucleic acids encoding the WNT surrogate fusion molecule, optionally wherein the nucleic acid(s) is / are DNA, cDNA, RNA, mRNA, modified mRNA, or a DNA / RNA hybrid. In particular embodiments, an isolated polynucleotide encoding a polypeptide sequence encodes the WNT surrogate fusion molecule antibody or antibody fragment of any of embodiments. In particular embodiment, encodes a) a first nucleic acid encoding the first polypeptide LRP5 / 6 binder; b) a second nucleic acid encoding the second polypeptide FZD binder; and c) a third nucleic acid encoding the third polypeptide cytokine receptor binder.

[0024] In particular embodiments, the present disclosure provides an expression vector comprising the isolated polynucleotide. In further particular embodiments, the present disclosure provides an isolated host cell comprising the expression vector.100251 In a related embodiment, the present disclosure provides a pharmaceutical composition comprising a physiologically acceptable excipient, diluent, or carrier, and a therapeutically effective amount of any of the WNT surrogate fusion molecules disclosed herein.

[0026] In related embodiments, the present disclosure provides a method for agonizing a WNT signaling pathway in a cell, comprising contacting the cell any of the trispecific WNT surrogate fusion molecules, wherein the WNT surrogate molecule is an agonist of a WNT signaling pathway.

[0027] In particular embodiments, the present disclosure provides a method for treating a subject having a disease or disorder associated with reduced WNT signaling, comprising administering to the subject an effective amount of the pharmaceutical composition, wherein the WNT surrogate fusion molecule is an agonist of a WNT signaling pathway. In particular embodiments, the disease or disorder or condition comprises one or more of the following: a) a retinopathy, optionally a retinal vascular disease and / or optionally selected from the group consisting of exudative vitreoretinopathy, familiar exudative vitreoretinopathy (FEVR), retinopathy of prematurity, Norrie disease, diabetic retinopathy (DR), diabetic macular edema, diabetic macular ischemia, age-related macular degeneration (AMD) (including wetAMD and dry AMD), retinopathy of prematurity (ROP), osteoporosis-pseudoglioma syndrome (OPPG), retinal vein occlusion, and Coats disease; b) a vascular disorder, optionally vascular malformation or vascular insufficiency, further optionally associated with ischemia-induced neovascularization; c) a bone disease or bone injury, optionally selected from the group consisting of osteoporosis (including osteoporosis pseudoglioma (OPPG)syndrome and juvenile primary osteoporosis), bone mineral density variability, and cancer-associated osteolysis; d) Uveitis, NIU, UME, ARN acute retinal necrosis, npAIR non parabolic autoimmune retinopathy, cataract surgery; e) a muscle wasting disease, optionally selected from the group consisting of sarcopenia, cachexia, and muscular dystrophies; and / or f) a neural or neurodegenerative disease, optionally selected from the group consisting of stroke, traumatic brain injury, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Friedreich ataxia, Lewy body disease, spinal muscular atrophy, motor neuron disease, multiple sclerosis, Batten disease, and Creutzfeldt-Jakob disease.100281 In related embodiments, the disclosure provides a method of administering the trispecific WNT surrogate fusion molecule (i) to one or more of the subject's eyes, optionally intravitreally or via ocular drops; (ii) locally administering, optionally to the eye, ear, nose (optionally intranasally), skin (optionally transdermally or epicutaneously), mucosa, skin, or vagina, or by inhalation; or (iii) parenterally administering, optionally by injection (optionally intravenous, intramuscular, subcutaneous, intrade Imai, intrathecal, intra-arterial, intraarticular, intraosseous, or intraperitoneal administration) or by inhalation; or (iv) enterally administering, optionally orally, sublingually, buccally, or rectally.

[0029] In particular embodiments, wherein the FZD binding regions bind to one or more FZD- 4 receptors and wherein the disease, disorder, or condition comprises one or more of the following: a retinopathy, optionally a retinal vascular disease and / or optionally selected from the group consisting of exudative vitreoretinopathy, familiar exudative vitreoretinopathy (FEVR), retinopathy of prematurity, Norrie disease, diabetic retinopathy (DR), diabetic macular edema, diabetic macular ischemia, age-related macular degeneration (AMD) (including wet AMD and dry AMD), retinopathy of prematurity (ROP), b) a vascular disorder, optionally vascular malformation or vascular insufficiency, further optionally associated with ischemia-induced neovascularization, c) Uveitis, NIU, UME, ARN acute retinal necrosis, npAIR non parabolic autoimmune retinopathy, cataract surgery.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 provides diagrams of illustrative embodiments of WNT surrogate fusion molecules in the format of VHH-FV-IgG. In particular embodiments, anti-antigen 1 binds to LRP 5 / 6, anti-antigen 2 binds to FZD, and anti-antigen 3 binds a cytokine, cytokine receptor.

[0031] Figure 2 provides diagrams of illustrative embodiments of WNT surrogate fusion molecules in the format of VHH-Fab-Fab. In particular embodiments, anti-antigen 1 binds to LRP 5 / 6, anti-antigen 2 binds to FZD, and anti-antigen 3 binds a cytokine, cytokine receptor.

[0032] Figure 3 provides diagrams of illustrative embodiments of WNT surrogate fusion molecules in the format of VHH-Fab-Fab, chain pairing version Crossmab. In particular embodiments, anti-antigen 1 binds to LRP 5 / 6, anti-antigen 2 binds to FZD and anti-antigen 3 binds a cytokine, cytokine receptor.

[0033] Figure 4 provides diagrams of illustrative embodiments of trispecific WNT surrogate fusion molecules in the format of VHH-Fab-Fab, chain pairing version-charge pairing. In particular embodiments, anti-antigen 1 binds to LRP 5 / 6, anti-antigen 2 binds to FZD, and anti-antigen 3 binds a cytokine, cytokine receptor.

[0034] Figure 5 provides diagrams of illustrative embodiments of WNT surrogate fusion molecules in the format of VHH-ScFv-Fab. In particular embodiments, anti-antigen 1 binds to LRP 5 / 6, anti-antigen 2 binds to FZD, and anti-antigen 3 binds a cytokine, cytokine receptor.

[0035] Figure 6 shows a WNT surrogate anti-IL6 fusion molecule hexavalent with LRP5 / 6 binder as a VHH, and FZD binder as Fv linked to Siltuximab with LALAPG mutation. The protein was isolated with high purity.{0036] Figure 7 shows the sequential binding of FZD4, LRP5 and hIL6 on the WNT surrogate fusion molecule, VHH03-4SD1 -Siltuximab (814-Siltuximab).

[0037] Figure 8 illustrates the STF activity of VHH03 -4 SD1- Siltuximab (814- Siltuximab).

[0038] Figures 9A and 9B describe the activity of VHHO3-4SD1-Siltuximab (814- Siltuximab) on gene expression change of WNT target gene Axin2 and IL6 target gene Socs3. Figure 9C describes the activity of VHH03 -4 SD1- Siltuximab (814-Siltuximab) on STAT3 phosphorylation of IL6 signaling.DETAILED DESCRIPTION10039] The present disclosure relates to WNT surrogate fusion molecules that bind to one or more FZD receptors, one or more LRP5 and / or LRP6 receptors, and one or more cytokinereceptor and / or cytokine, and / or ligand. Such fusion molecules modulate WNT signaling pathways with additive or synergistic effects on anti-inflammatory activity. In particular embodiments, the fusion molecules activate a WNT signaling pathway or increase signaling via a WNT signaling pathway, and synergistically decrease inflammation through antagonism of Interleukin-6 (IL-6), the multifunctional cytokine that plays a central role in host defense due to its wide range of immune and hematopoietic activities and its potent ability to induce the acute phase response. In particular embodiments, the multispecific WNT surrogate fusion molecules disclosed herein comprise: (i) one or more antibodies or antigen-binding fragments thereof that specifically bind to one or more FZD receptors, including antibodies or antigenbinding fragments thereof having particular FZD receptor specificity and / or functional properties; (ii) one or more antibodies or antigen-binding fragments thereof that specifically bind to LRP5 and / or LRP6 (LRP5 / 6), and (iii) one or more antibodies or antigen-binding fragments thereof that specifically bind one or more cytokine receptor and / or ligandincluding antibodies or antigen-binding fragments thereof having particular cytokine receptor specificity and / or functional properties. In some embodiments, cytokine receptor is a receptor for IL6, an interleukin that acts as both a pro-inflammatory cytokine and an antiinflammatory myokine. Certain embodiments encompass specific structural formats or arrangements of the FZD binding region(s), LRP5 / 6 binding region(s), and IL6 binding region(s) of WNT surrogate fusion molecules advantageous in increasing downstream WNT pathway signaling and related biological effects.

[0040] The molecule now called interleukin-6 (IL-6) was originally referred to by such diverse names as interferon-P2 (Weissenbach et al., 1980; Zilberstein et al., 1986), 26K factor (Content et al., 1982; Haegeman et al., 1986), B-cell stimulatory factor 2 (Hirano et al., 1985), hybridoma growth factor (van Snick et al., 1986; Brakenhoff et al., 1987), plasmacytoma growth factor (Nordan et al., 1987), hepatocyte stimulatory factor (Gauldie et al., 1987), a hematopoietic factor (Ikebuchi et al., 1987), and cytotoxic T-cell differentiation factor (Takai et al., 1988), with each name reflecting a different biological activity controlled by the same protein. It is now clear that IL-6 plays a central role in diverse host defense mechanisms such as the immune response, hematopoiesis, and acute-phase reactions (for reviews see van Snick, 1990; Akira et al., 1993; Nard & Kishimoto, 1994). IL-6 plays important roles in both homeostasis as well as an important component of the body’s armory against infection or tissue damage (Akira et al., 1993; Nicola, 1994). In accordance with its functional pleiotropy, IL-6 has been implicated in the pathology of many diseases including multiple myeloma (Kawano et al., 1988; Bataille et al., 1989), rheumatoid arthritis (Hirano etal., 1988), Castleman’s disease (Yoshizaki et al., 1989), AIDS (Nakajima et al., 1989; Poli et al., 1990), mesangial proliferative glomerulonephritis (Horii et al., 1989), psoriasis (Grossman et al., 1989), Kaposi’s sarcoma (Miles et al., 1990), sepsis (Waage et al., 1989), and osteoporosis (Jilka et al., 1992, Poli et al., 1994).

[0041] Inflammation has been implicated in the pathogenesis of retinal diseases, and antiinflammatory therapies such as steroids have been effective in treating uveitis and diabetic macular edema (DME). Detailed studies looking at inflammation and infection in the eye have shown that the pro-inflammatory cytokine, interleukin-6 (IL-6) is significantly elevated in the ocular fluids of refractory / chronic uveitis patients, and inhibition of IL-6 in animal models inhibits the onset of uveitis. High ocular fluid levels of IL-6 are also found in patients with DME and retinal vein occlusion. Additionally, chronic inflammatory cells have been seen on the surface of the Bruch’s membrane in eyes with neovascular AMD, and patients with AMD have been reported to have increased serum levels of IL-6.

[0042] WNT signaling has been implicated as an important pathway for vascular development in the retina. The present WNT surrogate fusion molecules could have a synergistic activity in treating inflammation , including ocular disorders associated with or resulting from inflammation.

[0043] Embodiments of the invention pertain to the use of WNT surrogate fusion molecules for the diagnosis, assessment, and treatment of diseases and disorders associated with WNT signaling pathways and cytokine . In certain embodiments, the subject WNT surrogate fusion molecules are used to modulate a WNT signaling pathway and cytokine in a cell or tissue. In certain embodiments, WNT surrogate fusion molecules may be used in the treatment or prevention of diseases and disorders associated with aberrant or deregulated (e.g., reduced) WNT signaling, or for which modulating, e.g., increasing, WNT signaling would provide a therapeutic benefit.

[0044] The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, N.Y. (2009); Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other like references.

[0045] As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise.

[0046] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0047] Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.

[0048] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques may be used for recombinant technology, molecular biological, microbiological, chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of subjects.

[0049] Embodiments of the present invention relate to antibodies and antigen-binding fragments thereof that bind to one or more FZD receptor. Sequences of illustrative antibodies, or antigen-binding fragments, or complementarity determining regions (CDRs) thereof are provided in International Patent Application WO / 2019 / 126399, and U.S. Patent No.12 / 006,368, titled “Anti-Frizzled antibodies and Methods of Use,” filed 19 December, 2018, each of which is hereby incorporated by reference in its entirety.

[0050] Embodiments of the present invention relate to antibodies and antigen-binding fragments thereof that bind to one or more LRP5 and / or LRP6 receptor. Sequences ofillustrative antibodies, or antigen-binding fragments, or complementarity determining regions (CDRs) thereof are described in International Patent Application WO / 2019 / 126401, and US Patent No. 11 / 746,150 filed on December 19, 2018, titled Anti-LRP5 / 6 Antibodies and Methods of Use, each of which is hereby incorporated by reference in its entirety.

[0051] Embodiments of the present invention relate to antibodies and antigen-binding fragments that bind to one or more cytokine ligand, or one or more cytokine receptor.

[0052] Cytokines are a large family of secreted proteins that act as hormone-like regulatory molecules that control many functions, including the immune response, inflammation, and chemotaxis. Cytokines bind to cytokine receptors, which are integral membrane proteins that transduce signals into the cell. In particular embodiments, the cytokine is IL6 or the cytokine receptor is an IL6 receptor. In particular embodiments, the antibody or antigen-binding fragment thereof that binds IL6 or an IL6 receptor comprises or consists of siltuximab, sirukumab, olokizumab, clazakinumab, gerilimzumab, Tocilizumab, Sarilumab, vamikibart or satralizumab, or an antigen-binding fragment or variant thereof.

[0053] As is well known in the art, an antibody is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one epitope recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as dAb, Fab, Fab', F(ab')2, Fv), single chain (scFv), VHH or sdAb, synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody or an antigen-binding fragment thereof, humanized antibodies, chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen-binding site or fragment (epitope recognition site) of the required specificity. "Diabodies" or 2scFV-Ig antibodies, are multivalent or multispecific fragments constructed by gene fusion (WO94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993) are also a particular form of antibody contemplated herein. Minibodies comprising a scFv joined to a CH3 domain are also included herein (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). See e.g., Ward, E. S. et al., Nature 341, 544-546 (1989); Bird et al., Science, 242, 423-426, 1988; Huston et al., PNAS USA, 85, 5879-5883, 1988); PCT / US92 / 09965; WO94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444- 6448, 1993; Y. Reiter et al., Nature Biotech, 14, 1239-1245, 1996; S. Hu et al., Cancer Res., 56, 3055-3061, 1996.

[0054] The term "antigen-binding fragment" as used herein refers to a polypeptide fragment that contains at least one CDR of an immunoglobulin heavy and / or light chain, or ofa VHH or sdAb, that binds to the antigen of interest, in particular to one or more FZD receptor or LRP5 or LRP6 receptor. In this regard, an antigen-binding fragment of the herein described antibodies may comprise 1, 2, 3, 4, 5, or all 6 CDRs of a VH and VL sequence set forth herein from antibodies that bind one or more FZD receptor or LRP5 and / or LRP6. In particular embodiments, an antigen-binding fragment may comprise all three VH CDRs or all three VL CDRs. Similarly, an antigen binding fragment thereof may comprise all three CDRs of a VHH or sdAb. An antigen-binding fragment of a FZD-specific antibody is capable of binding to a FZD receptor. An antigen-binding fragment of a LRP5 / 6-specific antibody is capable of binding to a LRP5 and / or LRP6 receptor. As used herein, the term encompasses not only isolated fragments but also polypeptides comprising an antigen-binding fragment of an antibody disclosed herein, such as, for example, fusion proteins comprising an antigenbinding fragment of an antibody disclosed herein, such as, e.g., a fusion protein comprising a VHH or sdAb that binds one or more FZD receptors and a VHH or sdAb that binds LRP5 and / or LRP6.

[0055] The term "antigen" refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody, and additionally capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen. In certain embodiments, a binding agent (e.g., a WNT surrogate molecule or binding region thereof) is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. In certain embodiments, a WNT surrogate molecule or binding region thereof (e.g., an antibody or antigen-binding fragment thereof) is said to specifically bind an antigen when the equilibrium dissociation constant is <10-7 or <10-8 M. In some embodiments, the equilibrium dissociation constant may be <10-9 M or <10-10 M.

[0056] In certain embodiments, antibodies and antigen-binding fragments thereof as described herein include a heavy chain and a light chain CDR set, respectively interposed between a heavy chain and a light chain framework region (FR) set which provide support to the CDRs and define the spatial relationship of the CDRs relative to each other. As used herein, the term "CDR set" refers to the three hypervariable regions of a heavy or light chain V region. Proceeding from the N terminus of a heavy or light chain, these regions are denoted as "CDR1," "CDR2," and "CDR3" respectively. An antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3) is referred to herein as a "molecular recognition unit." Crystallographic analysis of a number of antigen-antibodycomplexes has demonstrated that the amino acid residues of CDRs form extensive contact with bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the molecular recognition units are primarily responsible for the specificity of an antigen-binding site.

[0057] As used herein, the term "FR set" refers to the four flanking amino acid sequences which frame the CDRs of a CDR set of a heavy or light chain V region. Some FR residues may contact bound antigen; however, FRs are primarily responsible for folding the V region into the antigen-binding site, particularly the FR residues directly adjacent to the CDRs. Within FRs, certain amino residues and certain structural features are very highly conserved. In this regard, all V region sequences contain an internal disulfide loop of around 90 amino acid residues. When the V regions fold into a binding-site, the CDRs are displayed as projecting loop motifs which form an antigen-binding surface. It is generally recognized that there are conserved structural regions of FRs which influence the folded shape of the CDR loops into certain "canonical" structures — regardless of the precise CDR amino acid sequence. Further, certain FR residues are known to participate in non-covalent interdomain contacts which stabilize the interaction of the antibody heavy and light chains.|0058| The structures and locations of immunoglobulin CDRs and variable domains may be determined by reference to Kabat, E. A. et al., Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and updates thereof, now available on the Internet (immuno.bme.nwu.edu). Alternatively, CDRs may be determined by using "IMGT®, the international ImMunoGeneTics information system® available at http: / / www.imgt.org (see, e.g., Lefranc, M.-P. et al. (1999) Nucleic Acids Res., 27:209-212; Ruiz, M. et al. (2000) Nucleic Acids Res., 28:219-221; Lefranc, M.-P. (2001) Nucleic Acids Res., 29:207-209; Lefranc, M.-P. (2003) Nucleic Acids Res., 31 :307-310; Lefranc, M.-P. et al. (2004) In Silico Biol., 5, 0006 [Epub], 5:45-60 (2005)]; Lefranc, M.-P. et al. (2005) Nucleic Acids Res., 33:D593-597; Lefranc, M.-P. et al. (2009) Nucleic Acids Res., 37:D1006-1012; Lefranc, M.-P. et al. (2015) Nucleic Acids Res., 43:D413-422).

[0059] A “monoclonal antibody" refers to a homogeneous antibody population wherein the monoclonal antibody is comprised of amino acids (naturally occurring and non-naturally occurring) that are involved in the selective binding of an epitope. Monoclonal antibodies are highly specific, being directed against a single epitope. The term "monoclonal antibody" encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (scFv), VHH or sdAb, variants thereof, fusion proteins comprising an antigen-binding fragment of a monoclonalantibody, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen-binding fragment (epitope recognition site) of the required specificity and the ability to bind to an epitope, including WNT surrogate molecules disclosed herein. It is not intended to be limited as regards the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins as well as the fragments etc. described above under the definition of "antibody".[00601 The proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the F(ab) fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site. The enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the F(ab')2 fragment which comprises both antigen-binding sites. An Fv fragment for use according to certain embodiments of the present invention can be produced by preferential proteolytic cleavage of an IgM, and on rare occasions of an IgG or IgA immunoglobulin molecule. Fv fragments are, however, more commonly derived using recombinant techniques known in the art. The Fv fragment includes a non-covalent VH::VL heterodimer including an antigen-binding site which retains much of the antigen recognition and binding capabilities of the native antibody molecule. Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochem 15:2706- 2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

[0061] In certain embodiments, single chain Fv or scFV antibodies are contemplated. For example, Kappa bodies (Ill et al., Prot. Eng. 10: 949-57 (1997); minibodies (Martin et al., EMBO J 13: 5305-9 (1994); diabodies (Holliger et al., PNAS 90: 6444-8 (1993); or Janusins (Traunecker et al., EMBO J 10: 3655-59 (1991) and Traunecker et al., Int. J. Cancer Suppl. 7: 51-52 (1992), may be prepared using standard molecular biology techniques following the teachings of the present application with regard to selecting antibodies having the desired specificity. In still other embodiments, bispecific or chimeric antibodies may be made that encompass the ligands of the present disclosure. For example, a chimeric antibody may comprise CDRs and framework regions from different antibodies, while bispecific antibodies may be generated that bind specifically to one or more FZD receptor through one binding domain and to a second molecule through a second binding domain. These antibodies may be produced through recombinant molecular biological techniques or may be physically conjugated together.

[0062] A single chain Fv (scFv) polypeptide is a covalently linked VH: : VL heterodimer which is expressed from a gene fusion including VH- and VL-encoding genes linked by a peptide-encoding linker. Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. A number of methods have been described to discern chemical structures for converting the naturally aggregated — but chemically separated — light and heavy polypeptide chains from an antibody V region into an scFv molecule which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778, to Ladner et al.

[0063] In certain embodiments, an antibody as described herein is in the form of a diabody. Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g. by a peptide linker) but unable to associate with each other to form an antigen binding site: antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94 / 13804).|0064[ A dAb fragment of an antibody consists of a VH domain (Ward, E. S. et al., Nature 341, 544-546 (1989).

[0065] Where WNT surrogate fusion molecules antibodies are to be used, these may be conventional antibodies, which can be manufactured in a variety of ways (Holliger, P. and Winter G. Current Opinion Biotechnol. 4, 446-449 (1993), e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction.

[0066] Diabodies, as opposed to bispecific whole antibodies, may also be particularly useful because they can be readily constructed and expressed in E. coli. Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94 / 13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against antigen X, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected. Trispecific whole antibodies may be made by knobs-into-holes engineering (J. B. B. Ridgeway et al., Protein Eng., 9, 616-621, 1996).

[0067] In certain embodiments, the antibodies described herein may be provided in the form of a UniBody®. A UniBody® is an IgG4 antibody with the hinge region removed (seeGenMab Utrecht, The Netherlands; see also, e.g., US20090226421). This proprietary antibody technology creates a stable, smaller antibody format with an anticipated longer therapeutic window than current small antibody formats. IgG4 antibodies are considered inert and thus do not interact with the immune system. Fully human IgG4 antibodies may be modified by eliminating the hinge region of the antibody to obtain half-molecule fragments having distinct stability properties relative to the corresponding intact IgG4 (GenMab, Utrecht). Halving the IgG4 molecule leaves only one area on the UniBody® that can bind to cognate antigens (e.g., disease targets) and the UniBody® therefore binds univalently to only one site on target cells.

[0068] In certain embodiments, the antibodies of the present disclosure may take the form of a VHH or sdAb. VHH or sdAb technology was originally developed following the discovery and identification that camelidae (e.g., camels and llamas) possess fully functional antibodies that consist of heavy chains only and therefore lack light chains. These heavychain only antibodies contain a single variable domain (VHH) and two constant domains (CH2, CH3). The cloned and isolated single variable domains have full antigen binding capacity and are very stable. These single variable domains, with their unique structural and functional properties, form the basis of “VHH or sdAb”. VHH or sdAb are encoded by single genes and are efficiently produced in almost all prokaryotic and eukaryotic hosts e.g. E. coli (see e.g. U.S. Pat. No. 6,765,087), molds (for example Aspergillus or Trichoderma) and yeast (for example Saccharomyces, Kluyvermyces, Hansenula or Pichia (see e.g. U.S. Pat. No. 6,838,254). The production process is scalable and multi -kilogram quantities of VHH or sdAb have been produced. VHH or sdAb may be formulated as a ready-to-use solution having a long shelf life. The VHH or sdAb method (see, e.g., WO 06 / 079372) is a proprietary method for generating VHH or sdAb against a desired target, based on automated high-throughput selection of B-cells. VHH or sdAb are single-domain antigen-binding fragments of camelid-specific heavy-chain only antibodies. VHH antibodies or sdAb, typically have a small size of around 15 kDa.

[0069] In certain embodiments, the antibodies or antigen-binding fragments thereof as disclosed herein are humanized. This refers to a chimeric molecule, generally prepared using recombinant techniques, having an antigen-binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based upon the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise either complete variable domains fused onto constant domains or only the CDRs grafted onto appropriate framework regions in the variable domains. Epitope bindingsites may be wild type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, but the possibility of an immune response to the foreign variable region remains (LoBuglio, A. F. et al., (1989) Proc Natl Acad Sci USA 86:4220-4224; Queen et al., PNAS (1988) 86: 10029-10033; Riechmann et al., Nature (1988) 332:323-327). Illustrative methods for humanization of the anti-FZD antibodies disclosed herein include the methods described in U.S. patent no. 7,462,697.

[0070] Another approach focuses not only on providing human-derived constant regions, but modifying the variable regions as well so as to reshape them as closely as possible to human form. It is known that the variable regions of both heavy and light chains contain three complementarity-determining regions (CDRs) which vary in response to the epitopes in question and determine binding capability, flanked by four framework regions (FRs) which are relatively conserved in a given species and which putatively provide a scaffolding for the CDRs. When nonhuman antibodies are prepared with respect to a particular epitope, the variable regions can be "reshaped" or "humanized" by grafting CDRs derived from nonhuman antibody on the FRs present in the human antibody to be modified. Application of this approach to various antibodies has been reported by Sato, K., et al., (1993) Cancer Res 53:851-856. Riechmann, L., et al., (1988) Nature 332:323-327; Verhoeyen, M., et al., (1988) Science 239: 1534-1536; Kettleborough, C. A., et al., (1991) Protein Engineering 4:773-3783; Maeda, H., et al., (1991) Human Antibodies Hybridoma 2: 124-134; Gorman, S. D., et al., (1991) Proc Natl Acad Sci USA 88:4181-4185; Tempest, P. R., et al., (1991) Bio / Technology 9:266-271; Co, M. S., et al., (1991) Proc Natl Acad Sci USA 88:2869-2873; Carter, P., et al., (1992) Proc Natl Acad Sci USA 89:4285-4289; and Co, M. S. et al., (1992) J Immunol 148: 1149-1154. In some embodiments, humanized antibodies preserve all CDR sequences (for example, a humanized mouse antibody which contains all six CDRs from the mouse antibodies). In other embodiments, humanized antibodies have one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs "derived from" one or more CDRs from the original antibody.[0071 [ In certain embodiments, the antibodies of the present disclosure may be chimeric antibodies. In this regard, a chimeric antibody is comprised of an antigen-binding fragment of an antibody operably linked or otherwise fused to a heterologous Fc portion of a different antibody. In certain embodiments, the heterologous Fc domain is of human origin. In other embodiments, the heterologous Fc domain may be from a different Ig class from the parent antibody, including IgA (including subclasses IgAl and IgA2), IgD, IgE, IgG (including subclasses IgGl, IgG2, IgG3, and IgG4), and IgM. In further embodiments, the heterologousFc domain may be comprised of CH2 and CH3 domains from one or more of the different Ig classes. As noted above with regard to humanized antibodies, the antigen-binding fragment of a chimeric antibody may comprise only one or more of the CDRs of the antibodies described herein (e.g., 1, 2, 3, 4, 5, or 6 CDRs of the antibodies described herein), or may comprise an entire variable domain (VL, VH or both).WNT Surrogates Fusion Molecules

[0072] The disclosure provides, in certain aspects, WNT surrogates fusion molecules that bind one or more FZD receptors, LRP5 and / or LRP6, and one or more cytokine, cytokine receptor. As used herein, “LRP5 and / or LRP6” may be referred to as “LRP5 / 6.” Cytokines may be referred to herein as “cytokines” or “cytokine ligands.” WNT surrogate fusion molecules may also be referred to as “fusion molecules” or “WNT surrogate.”

[0073] In particular embodiments, WNT surrogate fusion molecules bind one or more human FZD receptors, one or both of a human LRP5 and / or a human LRP6, and one or more human IL6 ligands or human IL6 receptors.

[0074] In certain embodiments, a WNT surrogate fusion molecule is capable of modulating or modulates WNT signaling events in a cell contacted with the WNT surrogate fusion molecule. In certain embodiments, the molecule increases WNT signaling, e.g., via the canonical WNT / p-catenin pathway. In certain embodiments, the WNT surrogate fusion molecule specifically modulates the biological activity of a human WNT signaling pathway and modulates cytokine.

[0075] WNT surrogate fusion molecules of the present invention are biologically active in binding to one or more FZD receptor and to one or more of LRP5 and LRP6, in activation of WNT signaling, i.e., the WNT surrogate fusion molecule is a WNT agonist. The term "WNT agonist activity" refers to the ability of an agonist to mimic the effect or activity of a WNT protein binding to a frizzled protein and / or LRP5 or LRP6. The ability of the WNT surrogate molecules and other WNT agonists disclosed herein to mimic the activity of WNT can be confirmed by a number of assays. WNT agonists typically initiate a reaction or activity that is similar to or the same as that initiated by the receptor's natural ligand. In particular, the WNT agonists disclosed herein activate, enhance or increase the canonical WNT / p-catenin signaling pathway. As used herein, the term "enhances" refers to a measurable increase in the level of WNT / p-catenin signaling compared with the level in the absence of a WNT agonist, e.g., a WNT surrogate molecule disclosed herein. In particular embodiments, the increase in the level of WNT / p-catenin signaling is at least 10%, at least20%, at least 50%, at least two-fold, at least five-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold as compared to the level of WNT / p-catenin signaling in the absence of the WNT agonist, e.g., in the same cell type. Methods of measuring WNT / p- catenin signaling are known in the art and include those described herein or in publications cited herein.

[0076] In particular embodiments, WNT surrogate fusion molecules disclosed herein specifically bind to two or more different epitopes, e.g., one or more FZD receptor, LRP5 and / or LRP6, and one or more different cytokine ligand or receptors. In particular embodiments, where the molecule comprises two antibodies or fragments that bind LRP5 / 6, each may bind different or the same LRP5 / 6. In particular embodiments, where the molecule comprises two antibodies or fragments that bind a FZD, each may bind different or the same FZD(s). In particular embodiments, where the molecule comprises two antibodies or fragments that bind a cytokine or cytokine receptor, each may bind different or the same cytokine or cytokine receptor. In embodiments, “antigen-binding fragments” include fragments of an antibody that bind the antigen bound by the antibody (e.g., a FZD, LRP5 / 6, cytokine, or cytokine receptor antigen), and polypeptides that bind the antigen, including, e.g., fragments and variants of polypeptides that bind the antigen.

[0077] The WNT surrogate fusion molecules also bind one or more cytokine, cytokine receptor.

[0078] In certain embodiments, WNT surrogate fusion molecules disclosed herein are tetravalent, pentavalent, or hexavalent.

[0079] In some embodiments, hexavalent WNT surrogate fusion molecules comprise: i) two low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or a Low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) (LRP5 / LRP6) binding antibodies or antigen-binding fragments that specifically binds to two LRP5 / 6 receptors; ii) two frizzled (FZD) binding antibodies or antigen-binding fragments that specifically bind to two FZD receptors; and iii) two cytokine and / or cytokine receptor antigen-binding fragments that specifically bind to two cytokines and / or cytokine receptors.

[0080] In some embodiment, the pentavalent WNT surrogate fusion molecules disclosed herein comprise: i) one low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or a Low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) (LRP5 / LRP6) binding antibody or antigen-binding fragment that specifically binds to one LRP5 / 6 receptor;ii) two frizzled (FZD) binding antibodies or antigen-binding fragments that specifically bind to two FZD receptors; and iii) two cytokine, cytokine receptor, or antigen-binding fragments that specifically bind to two cytokines, or cytokine receptors.

[0081] In some embodiments, the pentavalent WNT surrogate fusion molecules disclosed herein comprise: i) two low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or a Low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) (LRP5 / LRP6) binding antibodies or antigen-binding fragments that specifically bind to two LRP5 / LRP6 receptors; ii) one frizzled (FZD) binding antibody or antigen-binding fragment that specifically binds to one FZD receptor; and iii) two cytokine, cytokine receptor, or antigen-binding fragments that specifically bind to two.

[0082] In some embodiments, the pentavalent WNT surrogate fusion molecules disclosed herein comprise: i) two low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or a Low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) (LRP5 / LRP6) binding antibodies or antigen-binding fragments that specifically bind to two LRP5 / LRP6; ii) two frizzled (FZD) binding antibody or antigen-binding fragment that specifically bind to two FZD receptor; and iii) one cytokine, cytokine receptoror antigen-binding fragment that specifically binds to a cytokine, cytokine receptor.

[0083] In another embodiment, the tetraval ent surrogate molecule fusion has a format of: i) one low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or a Low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) (LRP5 / LRP6) binding antibody or antigen-binding fragment that specifically binds to one LRP5 / 6 receptor; ii) two frizzled (FZD) binding antibody or antigen-binding fragments that specifically bind to one or more FZD receptor; and iii) one cytokine, cytokine receptor, or antigen-binding fragment that specifically binds to one cytokine, cytokine receptor.

[0084] WNT surrogate fusion molecules disclosed herein may have any of a variety of different structural formats or configurations, including but not limited to any of those described herein or in the attached figures. WNT surrogate fusion molecules may comprise polypeptides and / or non-polypeptide binding moieties, e.g., small molecules. In particularembodiments, WNT surrogate fusion molecules may comprise both a polypeptide region and a non-polypeptide binding moiety. In certain embodiments, WNT surrogate fusion molecules may comprise a single polypeptide, or they may comprise two or more, three or more, four or more polypeptides, five or more polypeptide, or six or more polypeptides. In certain embodiments, one or more polypeptides of a WNT surrogate fusion molecule are antibodies or antigen-binding fragments thereof. In certain embodiments, fusion molecule comprises two antibodies or antigen binding fragments thereof, one that binds one or more FZD one or more that binds LRP5 and / or LRP6. , and one or more two cytokine receptors and / or ligand.

[0085] In certain embodiments, the WNT surrogates comprises one, two, three, four, five, or six polypeptides e.g., linked or bound to each other or fused to each other.

[0086] When the WNT fusion molecules comprise a single polypeptide, they may be a fusion protein comprising one or more FZD binding domain, one or more LRP5 / 6 binding domain, and two cytokine receptors and / or ligand. The binding domains may be directly fused or they may be connected via a linker, e.g., a polypeptide linker, including but not limited to any of those disclosed herein.

[0087] When the WNT surrogate fusion molecules comprise two or more polypeptides, the polypeptides may be linked via covalent bonds, such as, e.g., disulfide bonds, and / or noncovalent interactions. For example, heavy chains of human immunoglobulin IgG interact at the level of their CH3 domains directly, whereas, at the level of their CH2 domains, they interact via the carbohydrate attached to the asparagine (Asn) N84.4 in the DE turn. In particular embodiments, the WNT surrogate fusion molecules comprise one or more regions derived from an antibody or antigen-binding fragment thereof, e.g., antibody heavy chains or antibody light chains or fragments thereof. In certain embodiments, a trispecific polypeptide comprises two antibody heavy chain regions (e.g., hinge regions) bound together via one or more disulfide bond. In certain embodiments, a WNT surrogate fusion molecule comprises an antibody light chain region (e.g., a CL region) and an antibody heavy chain region (e.g., a CHI region) bound together via one or more disulfide bond.

[0088] WNT surrogate fusion molecules may be engineered to facilitate binding between two polypeptides. For example, Knob-into-holes amino acid modifications may be introduced into two different polypeptides to facilitate their binding. Knobs-into-holes amino acid (AA) changes is a rational design strategy developed in antibody engineering, used for heterodimerization of the heavy chains, in the production of bispecific IgG antibodies. AA changes are engineered in order to create a knob on the CH3 of the heavy chains from a first antibody and a hole on the CH3 of the heavy chains of a second antibody. The knob may berepresented by a tyrosine (Y) that belongs to the 'very large' IMGT volume class of AA, whereas the hole may be represented by a threonine (T) that belongs to the 'small' IMGT volume class. Other means of introducing modifications into polypeptides to facilitate their binding are known and available in the art. For example, specific amino acids may be introduced and used for cross-linking, such as Cysteine to form an intermolecular disulfide bond.

[0089] WNT surrogate fusion molecules may have a variety of different structural formats, including but not limited to those shown in Figure 1, wherein the one or more LRP5 / 6 antigen-binding fragment thereof (a-antigen 1) that binds one or more LRP 5 / 6 receptors is VHH, the one or more FZD antigen-binding fragment thereof (a-antigen 2) that binds one or more FZD receptors is Fv, and the one or more cytokine, cytokine receptoror antigen-binding fragments thereof (a-antigen 3) that binds one or more cytokine receptor and / or ligandis IgG.(0090] In particular embodiments, the LRP5 / 6 antigen-binding fragment is fused or linked to variable light chain or variable heavy chain of FZD antigen-binding fragment that is linked or fused to heavy or light chain of IgG cytokine, cytokine receptorantibodies or antigen-binding fragments.

[0091] In other embodiments, the LRP5 / 6 antigen-binding fragment is fused or linked to fc domain of IgG IL6 cytokine, IL6 receptorantibodies or antigen-binding fragments and variable light chain or variable heavy chain of FZD antigen-binding fragment that is linked or fused to heavy or light chain of IgG antibody cytokine, cytokine receptorantibodies or antigen-binding fragments.

[0092] In other embodiments, the LRP5 / 6 antigen-binding fragment is fused or linked to variable heavy and light chain of cytokine antibodies or antigen-binding fragments and variable light chain or variable heavy chain of FZD antigen binding fragment that is linked or fused to heavy or light chain of IgG cytokine antibodies or antigen-binding fragments.

[0093] In particular embodiments, the Fv sequences of illustrative antigen-binding fragments, or complementarity determining regions (CDRs) are described in PCT Application Publication No. WO 2019 / 126399 filed on December 19, 2018 titled Anti- Frizzled Antibodies and Methods of Use, and (CDRs) thereof, and / or Table 1 of the present disclosure. In another embodiment, sequences of illustrative VHH antigen-binding fragments thereof that bind to one or more LRP5 and / or LRP6 receptors, antigen-binding fragments, or complementarity determining regions (CDRs) are described in PCT Application Publication No. WO 2019 / 126401 filed on December 19, 2018 titled Anti-LRP5 / 6 Antibodies and 1Methods of Use, and (CDRs) thereof, and Table 2 of the present disclosure. In some embodiments, the present invention relates to antibodies and antigen-binding fragments thereof that bind to one or more cytokine ligand. In particular embodiments, the cytokine antibodies or antigen-binding fragments bind to IL6 ligand or IL6 receptor, and illustrative examples of such antibodies include but are not limited to: siltuximab, sirukumab, olokizumab, clazakinumab, gerilimzumab, Tocilizumab, Sarilumab, Vamikibart and Satralizumab. In various embodiments, including but not limited to those depicted in Figure 2, a WNT surrogate fusion molecule comprises one or more VHH antigen binding fragment LRP 5 / 6 (a-antigen 1) that binds to one or more LRP 5 / 6 receptors, one or more Fab antigen binding fragment (a-antigen 2) FZD that binds to one or more FZD receptors, and one or more Fab antigen binding fragment (a-antigen 3) that binds to cytokine or cytokine receptors. In some embodiments, the one or more LRP5 / 6 antigen-binding fragment thereof (a-antigen1) that binds one or more LRP 5 / 6 receptors VHH, the one or more FZD antigen-binding fragment thereof (a-antigen 2) that binds one or more LRP are fused to N-terminus or C- terminus of the one or more variable heavy or variable light chain of antibody of cytokine (a- antigen 3) that binds to cytokine or cytokine ligand. In another embodiment, the one or more LRP5 / 6 antigen-binding fragment thereof (a-antigen 1) that binds one or more LRP 5 / 6 receptors VHH, fused to the one or more FZD antigen-binding fragment thereof (a-antigen2) Fab is fused to N-terminus or C-terminus of the one or more variable heavy or variable light chain of antibody of cytokine (a-antigen 3) that binds to cytokine or cytokine ligand, or to Fc domain of antibody of cytokine (a-antigen 3) that binds to cytokine receptor or cytokine ligand. In particular embodiments, the one or more LRP5 / 6 antigen-binding fragment thereof (a-antigen 1) that binds one or more LRP 5 / 6 receptors VHH is fused or linked to variable heavy or variable light chain of antibody or antigen binding fragment to cytokine (a-antigen 3) that binds to cytokine receptor or cytokine ligand, and the one or more FZD antigen-binding fragment thereof (a-antigen 2) that binds one or more LRP 5 / 6 receptors VHH is fused or linked to antibody of antigen binding fragment to cytokine (a-antigen 3) that binds to cytokine receptor or cytokine ligand.

[0094] In some embodiments, not limited to those depicted in Figure 3, chain pairing formats of VHH antigen binding fragment LRP 5 / 6 that binds to one or more LRP 5 / 6 receptors, one or more Fab antigen binding fragment FZD that binds to one or more FZD receptors, and one or more Fab antigen binding fragment that binds to cytokine ligand or IL6 receptors using CrossMab technology that enforces correct light chain association based on the domain crossover of immunoglobulin domains in the Fab region of the bispecificantibody adapted to the trispecific molecules disclosed herein. This can be achieved by exchange of different domains within the Fab-fragment. Either the Fab domains (in the CrossMabFab format), or only the variable VH-VL domains (CrossMabVH-VL format) or the constant CH1-CL domains (CrossMabCHl-CL format) within the Fab-fragment can be exchanged for this purpose. Indeed, for the CrossMab CHI -CL format the respective original light chain and the novel VL-CH1 light chain do not result in undesired interactions with the respective original and VH-CL containing heavy chains, and no theoretical side products can be formed, in the case of the CrossMabFab format a non-functional monovalent antibody (MoAb) as well as a non-functional Fab-fragment can be formed. These side products can be removed by chromatographic techniques. In the case of the CrossMabVH-VL format, an undesired side product with a VL-CH1 / VL-CL domain association known from Bence-Jones proteins can occur between the VL-CH1 containing heavy chain and the original unmodified VL-CL light chain. The introduction of repulsive charge pairs based on existing conserved charge pairs in the wildtype antibody framework into the constant CHI and CL domains of the wildtype non-crossed Fab-fragment can overcome the formation of this Bence-Jones-like side product in the CrossMab VH-VL+ / - format. In some embodiments, Figure 4 comprises charge pairing in chain pairing formats of VHH antigen binding fragment LRP 5 / 6 that binds to one or more LRP 5 / 6 receptors, one or more Fab antigen binding fragment FZD that binds to one or more FZD receptors, and one or more Fab antigen binding fragment that binds to cytokine ligand or cytokine receptors.

[0095] Fabs may be converted into a full IgG format that includes both the Fab and Fc fragments, for example, using genetic engineering to generate a fusion polypeptide comprising the Fab fused to an Fc region, i.e., the Fab is present in a full IgG format. The Fc region for the full IgG format may be derived from any of a variety of different Fes, including but not limited to, a wild-type or modified IgGl, IgG2, IgG3, IgG4 or other isotype, e.g., wild-type or modified human IgGl, human IgG2, human IgG3, human IgG4, human IgG4Pro (comprising a mutation in core hinge region that prevents the formation of IgG4 half molecules), human IgA, human IgE, human IgM, or the modified IgGl referred to as IgGl LALAPG. The L234A, L235A, P329G (LALA-PG) variant has been shown to eliminate complement binding and fixation as well as Fc-y dependent antibody-dependent cell- mediated cytotoxity (ADCC) in both murine IgG2a and human IgGl . These LALA-PG substitutions allow a more accurate translation of results generated with an “effectorless” antibody framework scaffold between mice and primates. In particular embodiments of any of the IgG disclosed herein, the IgG comprises one or more of the following amino acidsubstitutions: N297G, N297A, N297E, L234A, L235A, or P329G. In related embodiments, the fusion molecule comprises an Fc region of an immunoglobulin molecule, wherein said Fc region has at least one amino acid modification resulting in reduced affinity to the neonatal Fc receptor (FcRn), comprising I253A, H310A and H435Q amino acid substitutions or 1253 A, H310A and H435A substitutions, as numbered according to the EU index.

[0096] In some embodiments, not limited to those depicted in Figure 5, formats of VHH antigen binding fragment LRP 5 / 6 that binds to one or more LRP 5 / 6 receptors, one or more scFv antigen binding fragment FZD that binds to one or more FZD receptors, and one or more Fab antigen binding fragment that binds to cytokine ligand or cytokine receptors.

[0097] In certain embodiments, a WNT surrogate molecule is formatted as a diabody. The binders against FZD, LRP, and cytokine ligand or cytokine receptors can also be linked together in a diabody (or DART) configuration. The diabody can also be in a single chain configuration. If the diabody is fused to an Fc, this will create a trispecific format. Without fusion to Fc, this would be a monovalent bispecific format. In certain embodiments, a diabody is a noncovalent dimer scFv fragment that consists of the heavy-chain variable (VH) and light-chain variable (VL) regions connected by a small peptide linker. Another form of diabody is a single-chain (Fv)2 in which two scFv fragments are covalently linked to each other.

[0098] In particular embodiments, a WNT surrogate fusion molecule comprises a FZD binding region, e.g., an anti-FZD antibody, or antigen-binding fragment thereof, fused or bound to a polypeptide that specifically binds to one or more FZD receptor. In particular embodiments, the polypeptide that specifically binds to one or more FZD receptor is an antibody or antigen-binding fragment thereof. If certain embodiments, it is an antibody or antigen-binding fragment thereof disclosed herein or in the published International Patent Application WO / 2019 / 126399, U.S. Patent No. 12 / 006,368, titled, “Anti-Frizzled antibodies and Methods of Use,” filed 19 December, 2018 which is incorporated herein by reference in its entirety. In particular embodiments, the FZD binding domain comprises the three heavy chain CDRs and / or the three light chain CDRs disclosed for any of the illustrative antibodies or fragments thereof that bind to one or more FZD receptor provided in Tables of in the published International Patent Application WO / 2019 / 126401 ,the U.S. Patent No.12 / 006,368 filed 19 December, 2018 . In particular embodiments, the FZD binding domain comprises the three heavy chain CDRs and / or the three light chain CDRs disclosed for any of the illustrative antibodies or fragments thereof that bind to one or more FZD receptor wherein the CDRs collectively comprise one, two, three, four, five, six, seven, or eight amino acidmodifications, e.g., substitutions, deletions, or additions, or less than one, less than two, less than three, less than four, less than five, less than six, less than seven, or less than eight amino acid modifications, e.g., substitutions, deletions, or additions. In particular embodiments, the FZD binding domain comprises the three CDR HC sequences provided in the CDRs collectively comprise one, two, three, four, five, six, seven or eight amino acid modifications. In particular embodiments, the FZD 4 binding domain comprises the heavy chain fragment and / or light chain fragment of any of the illustrative antibodies or fragments thereof that bind to one or more FZD 4 receptor provided in (or an antigen-binding fragment or variant of either). In certain embodiments, the FZD 4 binding domain is an Fab or was derived from an Fab, so the heavy chain includes VH and CHI sequence, but not CH2 or CH3 sequences. In certain embodiments, the FZD binding domain is a VHH or sdAb or was derived from a VHH or sdAb, so includes the VHH domain. In certain embodiments, the FZD binding region is a polypeptide, e.g., an antibody or antigen-binding fragment thereof, that competes with any of these antibodies for binding to one or more FZD 4 receptors.

[0099] In certain embodiment, the FZD binding domain may be selected from any binding domain that binds a FZD with an affinity of, e.g., a KD of at least about 1 x 10-4 M, at least about 1 x 10-5 M, at least about 1 x 10-6 M, at least about 1 x 10-7 M, at least about 1 x 10-8 M, at least about 1 x 10-9 M, or at least about 1 x 10-10 M. In certain embodiment, the FZD binding domain may be selected from any binding domain that binds one or more FZD receptor at high affinity, e.g., a KD of less than about 1 x 10-7 M, less than about 1 x 10-8 M, less than about 1 x 10-9 M, or less than about 1 x 10-10 M. In certain embodiment, the FZD binding domain may be selected from any binding domain that binds FZD at high affinity, e.g. a KD of less than or equal to about 1 x 10-4 M, less than or equal to about 1 x 10-5 M, less than or equal to about 1 x 10-6 M, less than or equal to about 1 x 10-7 M, less than or equal to about 1 x 10-8 M, less than or equal to about 1 x 10-9 M, or at least about 1 x 10-10 M in the context of a WNT surrogate molecule.

[0100] Suitable FZD binding domains include, without limitation, de novo designed FZD binding proteins, antibody derived binding proteins, e.g. scFv, Fab, etc. and other portions of antibodies that specifically bind to one or more FZD proteins; VHH or single domain antibody derived binding domains; knottin-based engineered scaffolds; norrin and engineered binding fragments derived therefrom, naturally occurring FZD binding domains, and the like. A FZD binding domain may be affinity selected to enhance binding to a desired FZD protein or plurality of FZD proteins, e.g. to provide tissue selectivity.

[0101] In some embodiments, the FZD binding domain binds to one, two, three, four, five or more different frizzled proteins, e.g. one or more of human frizzled proteins FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10. In some embodiments, the FZD binding domain binds to FZD1, FZD2, and FZD 7. In some embodiments, the FZD binding domain binds to FZD1, FZD2, FZD5, FZD7 and FZD8. In other embodiments the FZD binding domain is selective for one or more frizzled protein of interest, e.g. having a specificity for the one or more desired frizzled protein of at least 10-fold, 25-fold, 50-fold, 100-fold, 200-fold or more relative to other frizzled proteins.

[0102] In particular embodiments, the FZD binding domain is an antibody or derivative thereof, including without limitation scFv, minibodies, VHH or single domain antibodies (sdAb) and various antibody mimetics comprising any of these CDR sequences. In certain embodiments, these CDR sequences comprise one or more amino acid modifications.

[0103] In other embodiments, the FZD binding domain comprises a variable region sequence, or the CDRs thereof, from any of a number of frizzled specific antibodies, which are known in the art and are commercially available, or can be generated de novo. Any of the frizzled polypeptides can be used as an immunogen or in screening assays to develop an antibody. Non-limiting examples of frizzled binding domains include antibodies available from Biolegend, e.g. Clone CH3 A4A7 specific for human frizzled 4 (CD344); Clone W3C4E11 specific for human FZD9 (CD349); antibodies available from Abeam, e.g. ab64636 specific for FZD7; ab83042 specific for human FZD4; ab77379 specific for human FZD7; ab75235 specific for human FZD8; abl02956 specific for human FZD9; and the like. Other examples of suitable antibodies are described in, inter alia, US Patent application 20140105917; US Patent application 20130230521; US Patent application 20080267955; US Patent application 20080038272; US Patent application 20030044409; etc., each herein specifically incorporated by reference.10104] The FZD binding region of a WNT surrogate molecule may be an engineered protein that is selected for structural homology to the frizzled binding region of a WNT protein. Such proteins can be identified by screening a structure database for homologies. The initial protein thus identified, for example the microbial Bhl478 protein. The native protein is then engineered to provide amino acid substitutions that increase affinity and may further be selected by affinity maturation for increased affinity and selectivity in binding to the desired frizzled protein. Non-limiting examples of frizzled binding moieties include the Fz27 and Fz27-B12 proteins.

[0105] In particular embodiments, a WNT surrogate molecule comprises an LRP5 / 6 binding domain, e.g., an anti-LRP5 / 6 antibody, or antigen-binding fragment thereof, which may be fused to a polypeptide that specifically binds to one or more FZD receptors. In particular embodiments, the polypeptide that specifically binds to LRP5 / 6 is an antibody or antigen-binding fragment thereof. If certain embodiments, it is an antibody or antigenbinding fragment thereof disclosed in the U.S. Patent No. 11 / 746,150, titled, “Anti-LR5 / 6 Antibodies and Methods of Use,” filed on December 19, 2017, which is incorporated herein by reference in its entirety. In particular embodiments, the LRP5 / 6 binding domain comprises the three heavy chain CDRs and / or the three light chain CDRs disclosed for any of the illustrative antibodies or fragments thereof that bind to LRP5 and / or LRP6 provided in the present disclosure. In particular embodiments, the LRP5 / 6 binding domain comprises the three heavy chain CDRs and / or the three light chain CDRs disclosed for any of the illustrative antibodies or fragments thereof that bind to one or more FZD receptor provided in , wherein the CDRs collectively comprise one, two, three, four, five, six, seven, or eight amino acid modifications, e.g., substitutions, deletions, or additions or less than one, less than two, less than three, less than four, less than five, less than six, less than seven, or less than eight amino acid modifications, e.g., substitutions, deletions, or additions. In certain embodiments, the LRP5 / 6 binding domain is a VHH or sdAb or was derived from a VHH or sdAb, so only includes the three heavy chain CDRs. In particular embodiments, the LRP5 / 6 binding domain comprises the heavy chain fragment and / or light chain fragment of any of the illustrative antibodies or fragments thereof that bind to LRP5 and / or LRP6 provided in. In certain embodiments, the LRP5 / 6 binding domain is an Fab or was derived from an Fab, so includes VH and CHI sequence, but not CH2 or CH3 sequences. In certain embodiments, the LRP5 / 6 binding domain is a VHH or sdAb or was derived from a VHH or sdAb, so includes the VHH domain. In certain embodiments, the LRP5 / 6 binding region is a polypeptide, e.g., an antibody or antigen-binding fragment thereof, that competes with one of these antibodies for binding to LRP5 and / or LRP6.

[0106] In certain embodiment, the LRP5 / 6 binding domain may be selected from any binding domain that binds LRP5 or LRP6 with a KD of less than or equal to about 1 x 10-4 M, less than or equal to about 1 x 10-5 M, less than or equal to about 1 x 10-6 M, less than or equal to about 1 x 10-7 M, less than or equal to about 1 x 10-8 M, less than or equal to about 1 x 10-9 M, or less than or equal to about 1 x 10-10 M in the context of a WNT surrogate molecule. In certain embodiment, the LRP5 / 6 binding domain may be selected from any binding domain that binds LRP5 or LRP6 with a KD of greater than or equal to about 1 x 10-4 M, greater than or equal to about 1 x 10-5 M, greater than or equal to about 1 x 10-6 M, greater than or equal to about 1 x 10-7 M, greater than or equal to about 1 x 10-8 M, greater than or equal to about 1 x 10-9 M, or greater than about 1 x 10-10 M in the context of a WNT surrogate molecule. In certain embodiment, the LRP5 / 6 binding domain may be selected from any binding domain that binds LRP5 or LRP6 at high affinity, e.g. a KD of less than about 1 x 10-7 M, less than about 1 x 10-8 M, less than about 1 x 10-9 M, or less than about 1 x 10-10 M.[01071 Other suitable LRP5 / 6 binding domains include, without limitation, de novo designed LRP5 / 6 binding proteins, antibody derived binding proteins, e.g. scFv, Fab, etc. and other portions of antibodies that specifically bind to one or more FZD proteins; VHH or sdAb derived binding domains; knottin-based engineered scaffolds; naturally occurring LRP5 / 6, including without limitation, DKK1, DKK2, DKK3, DKK4, sclerostin; Wise; fusions proteins comprising any of the above; derivatives of any of the above; variants of any of the above; and biologically active fragments of any of the above, and the like. A LRP5 / 6 binding domain may be affinity selected to enhance binding.

[0108] Antibodies that specifically bind to LRP5 or LRP6 are known in the art and are commercially available or can be generated de novo. LRP5, LRP6 or fragments thereof can be used as an immunogen or in screening assays to develop an antibody. Examples of known antibodies include, without limitation, those described in Gong et al. (2010) PLoS One. 5(9):el2682; Ettenberg et al. (2010) Proc Natl Acad Sci U S A. 107(35): 15473-8; and those commercially available from, for example Santa Cruz biotechnology antibody clone 1 A12, which was raised against synthetic LRP5 / 6 of human origin and binds to both the full length and proteolytic fragment of LRP6 and LRP5 of mouse and human origin; the monoclonal antibody 2B11; Cell Signaling Technology antibody specific for LRP5 (D80F2), catalog number 5731; etc.10.1.091 In particular embodiments, a WNT surrogate molecule comprises a cytokine ligand or cytokine receptor binding domain, e.g., an anti-IL6 antibody or antigen-binding fragment thereof, which optionally may be fused to a polypeptide that specifically binds to one or more FZD receptors or LRP5 / 6. In particular embodiments, the cytokine ligand or cytokine receptor binding domain comprises the three heavy chain CDRs and / or the three light chain CDRs disclosed for any of the illustrative antibodies or fragments thereof that bind to a cytokine or cytokine receptor provided in the present disclosure. In particular embodiments, the cytokine or cytokine receptor binding domain comprises the three heavy chain CDRs and / or the three light chain CDRs disclosed for any of the illustrative antibodiesor fragments thereof that bind to a cytokine or cytokine receptor provided herein, wherein the CDRs collectively comprise zero, one, two, three, four, five, six, seven, or eight amino acid modifications, e.g., substitutions, deletions, or additions, or less than one, less than two, less than three, less than four, less than five, less than six, less than seven, or less than eight amino acid modifications, e.g., substitutions, deletions, or additions. In certain embodiments, the cytokine or cytokine receptor binding domain is a VHH or sdAb or was derived from a VHH or sdAb, so only includes the three heavy chain CDRs. In particular embodiments, the cytokine or cytokine receptor binding domain comprises the heavy chain fragment and / or light chain fragment of any of the illustrative antibodies or fragments thereof that bind to a cytokine or cytokine receptor provided herein. In certain embodiments, the cytokine or cytokine receptor binding domain is an Fab or was derived from an Fab, so includes VH and CHI sequence, but not CH2 or CH3 sequences. In certain embodiments, the cytokine or cytokine receptor binding domain is a VHH or sdAb or was derived from a VHH or sdAb, so includes the VHH domain. In certain embodiments, the cytokine or cytokine receptor binding region is a polypeptide, e.g., an antibody or antigen-binding fragment thereof, that competes with one of these antibodies for binding to the cytokine or cytokine receptor.|0110| In certain embodiment, the cytokine or cytokine receptor binding domain may be selected from any binding domain that binds the cytokine or cytokine receptor with a KD of less than or equal to about 1 x 10-4 M, less than or equal to about 1 x 10-5 M, less than or equal to about 1 x 10-6 M, less than or equal to about 1 x 10-7 M, less than or equal to about 1 x 10-8 M, less than or equal to about 1 x 10-9 M, or less than or equal to about 1 x 10-10 M in the context of a WNT surrogate molecule. In certain embodiment, the cytokine or cytokine receptor binding domain may be selected from any binding domain that binds the cytokine or cytokine receptor with a KD of greater than or equal to about 1 x 10-4 M, greater than or equal to about 1 x 10-5 M, greater than or equal to about 1 x 10-6 M, greater than or equal to about 1 x 10-7 M, greater than or equal to about 1 x 10-8 M, greater than or equal to about 1 x 10-9 M, or greater than about 1 x 10-10 M in the context of a WNT surrogate molecule. In certain embodiment, the cytokine or cytokine receptor binding domain may be selected from any binding domain that binds the cytokine or cytokine receptor at high affinity, e.g. a KD of less than about 1 x 10-7 M, less than about 1 x 10-8 M, less than about 1 x 10-9 M, or less than about 1 x 10-10 M.

[0111] Other suitable cytokine or cytokine receptor binding domains include, without limitation, polypeptides that bind a cytokine or cytokine receptor, de novo designed cytokine or cytokine receptor binding proteins, antibody derived binding proteins, e.g. scFv, Fab, etc.and other portions of antibodies that specifically bind to one or more cytokine or cytokine receptor; VHH or sdAb derived binding domains; knottin-based engineered scaffolds; derivatives of any of the above; variants of any of the above; and biologically active fragments of any of the above, and the like. A cytokine or cytokine receptor binding domain may be affinity selected to enhance binding.

[0112] Antibodies that specifically bind to a cytokine or cytokine receptor are known in the art and are commercially available or can be generated de novo. A cytokine or cytokine receptor, or fragments thereof, can be used as an immunogen or in screening assays to develop an antibody. In certain embodiments the antibody or fragment thereof that binds to a cytokine or cytokine receptor binds to IL6 or an IL6 receptor, and is optionally selected from siltuximab, olokizumab, clazakizumab, sirukamab, vanikibart, or tocolizumab, or antigenbinding variants or fragments thereof.

[0113] In certain embodiments, WNT surrogate fusion molecules disclosed herein comprise one or more polypeptides comprising two or more binding regions. For example, the two or more binding regions may be two or more FZD binding regions or two or more LRP5 / 6 binding regions, or they may comprise one or more FZD binding region, one or more LRP5 / 6 binding region, and one or more IL6 ligands or receptors binding region. The binding regions may be directly joined or contiguous, or may be separated by a linker, e.g. a polypeptide linker, or a non-peptidic linker, etc. The length of the linker, and therefore the spacing between the binding domains can be used to modulate the signal strength, and can be selected depending on the desired use of the WNT surrogate molecule. The enforced distance between binding domains can vary, but in certain embodiments may 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. In some embodiments the linker is a rigid linker, in other embodiments the linker is a flexible linker. In certain embodiments where the linker is a peptide linker, it may be from 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 or more amino acids in length, and is of sufficient length and amino acid composition to enforce the distance between binding domains. In some embodiments, the linker comprises or consists of one or more glycine and / or serine residues.

[0114] In particular embodiments, the disclosure provides fusion polypeptide sequences comprising a sequence having at least 80%, at least 85%, at least 90%, at least 98%, at least 99%, at least 99%, or 100% identity to any one of SEQ ID NOs: 1-19 or an antigen-binding fragment thereof. In particular embodiments, wherein the fusion polypeptides comprise oneor more CDR, the CDR sequences are conserved. In particular embodiments, wherein the fusion polypeptides comprise one or more CDR, the CDR sequences collectively comprise 12 or less, 10 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, 1 or less, or no amino acid modifications, e.g., substitution or deletion. The disclosure further provides nucleic acid sequences that encode any of these fusion polypeptides or variants thereof having one of the recited percent identities to any one of SEQ ID NOs: 1-19 or antigen-binding fragments thereof.|0115| In particular embodiments, a WNT surrogate anti-IL6 fusion molecule, not limited to the structure described in Figure 6, comprises a polypeptide sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to a polypeptide sequence disclosed in any of SEQ ID NOs: 2-19, or an antigen-binding fragment thereof.

[0116] In particular embodiments, the WNT surrogate anti-IL6 fusion molecule comprises or consists of a combination of polypeptides selected from the following combinations: SEQ ID NOs: 2 and 3; 4 and 5; 6 and 7; 8 and 9; 10 and 11; 2 and 12; 4 and 13; 6 and 14; 8 and 15; 10 and 16; or 17 and 18; or 17 and 19, or antigen-binding fragments thereof. In certain embodiments, any of the polypeptides may be a variant having at least 90%, at least 95%, at least 98%, at least 99% identity to the recited sequences.

[0117] In particular embodiments, the WNT surrogate anti-IL6 fusion molecule comprises or consists of a combination comprising one or more polypeptide variant, each combination comprising or consisting of polypeptides having at least 90%, at least 95%, at least 98% or at least 99% identity to a polypeptide sequence disclosed in any combination of: SEQ ID NOs: 2 and 3; 4 and 5; 6 and 7; 8 and 9; 10 and 11; 2 and 12; 4 and 13; 6 and 14; 8 and 15; 10 and 16; 17 and 18; or 17 and 19, or antigen-binding fragments thereof.(0118] In certain embodiments, the trispecific fusion WNT surrogate molecules comprises or consists of a combination of polypeptide sequences set forth in any of SEQ ID NOs: 2 and 3; 4 and 5; 6 and 7; 8 and 9; 10 and 11; 2 and 12; 4 and 13; 6 and 14; 8 and 15; 10 and 16; or 17 and 18; or 17 and 19, or an antigen-binding fragment thereof.

[0119] In particular embodiments, the WNT surrogate anti-IL6 fusion protein comprises one or two of each of the indicated polypeptides present in any combination. In embodiments, one or more the polypeptides present in the WNT surrogate anti-IL6 fusion protein are bound to each other, e.g., through one or more covalent bonds, hydrogen bonds, ionic bonds, or hydrophobic interactions. In embodiments, one or more polypeptides present in the WNT surrogate anti-IL6 fusion protein are fused to each other, e.g., directly or via a linker moiety, e.g., a peptide linker.

[0120] In particular embodiments, the antigen-binding fragment binds one or more FZD receptors, binds one or more LRP5 and / or LRP6, and binds one or more IL6 ligand or IL6 receptor.10121] Table 1 shows the polypeptides present in the indicated illustrative fusions comprising an anti-IL6 or anti-IL6R antibody or fragment thereof fused to a WNT surrogate molecule.Table 1.Table 2 shows the structure of illustrative WNT surrogate fusion molecules, including the polypeptide sequences.Table 2: Sequences of WNT surrogate fusion molecules. Sequence in bold and highlighted in light gray is VHH, underlined is LC, Bold is HC, bold and underlined is Fd, in Bold, underlined and italic is VH, underlined and italic is VL, Bold and waved underlined is CHI. Elbow regions of CrossMab Fabs are bold italic highlighted in the light dark, AAA mutation are bold, Italic, underline and gray highlight.

[0122] In particular embodiments, a WNT surrogate fusion molecule comprises any of the combinations of polypeptides set forth for any of the WNT surrogate fusion molecules shown in Table 2, or variants thereof having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity thereto. In certain embodiments, a WNT surrogate fusion polypeptide comprises a combination of polypeptides selected from the following: i) a polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2 and a polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3;ii) a polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 4 and a polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 5; iii) a polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 6 and a polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 7; iv) a polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 8and a polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 9; v) a polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 10 and a polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 11; vi) a polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2 and a polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 12; vii) a polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 4 and a polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 13; viii) a polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 6 and a polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 14; ix) a polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 8 and a polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 15; x) a polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 10 and a polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 16; xi) a polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 17 and a polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 18; xii) a polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 17 and a polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 19; or antigen-binding fragments thereof. In certain embodiments, the CDRS within a WNT surrogate fusion molecule collectivelycomprise 12 or less, 10 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, 1 or less, or no amino acid modifications, e.g., substitution or deletions. In particular embodiments, the WNT surrogate fusion molecule comprises one or more of each of the polypeptides of the various combinations, e.g., in some embodiments, consistent with illustrative structures depicted herein. In particular embodiments, the WNT surrogate fusion molecule comprises two of each of the polypeptides of the various combinations. In some embodiments, the WNT surrogate fusion molecule has a structure or format consistent with illustrative structures depicted herein.

[0123] The WNT surrogate fusion molecules disclosed herein could be applied to the WNT modulators selected from those disclosed in any of the following: PCT Application Publication No. WO 2019 / 126399, PCT Application Publication No. WO 2019 / 126401, PCT Application Publication No. WO 2019 / 126398, PCT Application Publication No. WO 2020 / 010308, PCT Application Publication No. WO 2020 / 167848, PCT Application Publication No. WO 2019 / 159084, PCT Application Publication No. WO 2022 / 130341, PCT Application Publication No. WO 2022 / 130342, PCT Application Publication No. WO 2023 / 250402, and US Patent No. 12 / 297,278 of which are incorporated by reference. It will be readily understood to those skilled in the art that the above constructs may be altered and expressed as various homologs and isoforms, may be edited at non-binding-domain sequences, and may be expressed using various synonymous nucleotide sequences using various suitable expression vector systems.10124] WNT surrogate molecule can be multimerized, e.g. through an Fc domain, by concatenation, coiled coils, polypeptide zippers, biotin / avidin or streptavidin multimerization, and the like. The fusion molecules can also be joined to a moiety such as PEG, Fc, etc., as known in the art to enhance stability in vivo.

[0125] In certain embodiments, a WNT surrogate molecule directly activates canonical WNT signaling through binding to one or more FZD proteins, to LRP5 / 6, and IL6 ligands and IL6 receptors or ligand particularly by binding to these proteins on a cell surface, e.g., the surface of a human cell. The direct activation of WNT signaling by a WNT surrogate molecule is in contrast to potentiation of WNT signaling, which enhances activity only when native WNT proteins are present and inhibit the IL6 activity.|O126] WNT surrogate fusion molecules may activate WNT signaling, e.g., by mimicking the effect or activity of a WNT protein binding to a frizzled protein. The ability of the WNT surrogate molecules of the invention to mimic the activity of WNT can be confirmed by a number of assays. The WNT surrogate molecules typically initiate a reaction or activity thatis similar to or the same as that initiated by the receptor's natural ligand. In particular, the WNT surrogate molecules of the invention enhance the canonical WNT / p-catenin signaling pathway. As used herein, the term "enhances" refers to a measurable increase in the level of WNT / p-catenin signaling compared with the level in the absence of a WNT surrogate molecule of the invention.

[0127] Various methods are known in the art for measuring the level of canonical WNT / p-catenin signaling. These include, but are not limited to assays that measure: WNT / p- catenin target gene expression; TCF reporter gene expression; P-catenin stabilization; LRP phosphorylation; Axin translocation from cytoplasm to cell membrane and binding to LRP. The canonical WNT / p-catenin signaling pathway ultimately leads to changes in gene expression through the transcription factors TCF7, TCF7L1, TCF7L2 (a.k.a. TCF4), and LEF. The transcriptional response to WNT activation has been characterized in a number of cells and tissues. As such, global transcriptional profiling by methods well known in the art can be used to assess WNT / p-catenin signaling activation or inhibition.

[0128] Changes in WNT -responsive gene expression are generally mediated by TCF and LEF transcription factors. A TCF reporter assay assesses changes in the transcription of TCF / LEF controlled genes to determine the level of WNT / p-catenin signaling. A TCF reporter assay was first described by Korinek, V. et al., 1997. Also known as TOP / FOP this method involves the use of 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 (pTOPFI ASH and pFOPFI ASH, respectively) to determine the transactivational activity of endogenous p-catenin / TCF4. A higher ratio of these two reporter activities (TOP / FOP) indicates higher P-catenin / TCF4 activity, whereas a lower ratio of these two reporter activities indicates lower P-catenin / TCF4 activity.

[0129] Various other reporter transgenes that respond to WNT signals exist intact in animals and therefore, effectively reflect endogenous WNT signaling. These reporters are based on a multimerized TCF binding site, which drives expression of LacZ or GFP, which are readily detectable by methods known in the art. These reporter genes include: TOP-GAL, BAT-GAL, ins-TOPEGFP, ins-TOPGAL, LEF-EGFP, Axin2-LacZ, Axin2-d2EGFP, Lgr5tml (cre / ERT2), TOPdGFP.[01301 The recruitment of dephosphorylated P-catenin to the membrane, stabilization and phosphorylation status of P-catenin, and translocation of P-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 Disheveled family proteins that inhibit the "destruction" complex so that degradation of intracellular P-catenin is reduced, and translocation of P-catenin to the nucleus follows thereafter. Therefore, measuring the level and location of P-catenin in a cell is a good reflection of the level of WNT / p-catenin signaling. A non-limiting example of such an assay is the "BioImage P-Catenin Redistribution Assay" (Thermo Scientific) which provides recombinant U20S cells that stably express human P-catenin fused to the C-terminus of enhanced green fluorescent protein (EGFP). Imaging and analysis is performed with a fluorescence microscope or HCS platform allowing the levels and distribution of EGFP-P- catenin to be visualized.

[0131] Another way, in which the destruction complex is inhibited, is by removal of Axin by recruitment of Axin to the cytoplasmic tail of the WNT co-receptor LRP. Axin has been shown to bind preferentially to a phosphorylated form of the LRP tail. Visualization of Axin translocation, for example with a GFP-Axin fusion protein, is therefore another method for assessing levels of WNT / p-catenin signaling.

[0132] In certain embodiments, a WNT surrogate molecule enhances or increases canonical WNT pathway signaling, e.g., P-catenin 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 the P-catenin signaling induced by a neutral substance or negative control as measured in an assay described above, for example as measured in the TOPFIash assay. A negative control may be included in these assays. In particular embodiments, WNT surrogate fusion molecules may enhance P-catenin signaling by a factor of 2x, 5x, lOx, lOOx, lOOOx, lOOOOx or more as compared to the activity in the absence of the WNT surrogate molecule when measured in an assay described above, for example when measured in the TOPFIash assay, or any of the other assays mentioned herein.

[0133] "WNT gene product" or "WNT polypeptide" when used herein encompass native sequence WNT polypeptides, WNT polypeptide variants, WNT polypeptide fragments and chimeric WNT polypeptides. In particular embodiments, a WNT polypeptide is a native human full length mature WNT protein.

[0134] 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 No. 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 (GenBankAccession No. NM_032642), WNT-6 (GenBank Accession No. NM_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_003395), WNT-9B (WNT-15) (GenBank Accession No. NM_003396), WNT-1 OA (GenBank Accession No. NM_025216), WNT-1 OB (GenBank Accession No. NM_003394), WNT-11 (GenBank Accession No. NM_004626), WNT- 16 (GenBank Accession No. NM_016087)). Although each member has varying degrees of sequence identity with the family, all encode small (i.e., 39-46 kD), acylated, palmitoylated, secreted glycoproteins that contain 23-24 conserved cysteine residues whose spacing is highly conserved (McMahon, A P et al., Trends Genet. 1992; 8: 236-242; Miller, J R. Genome Biol. 2002; 3(1): 3001.1-3001.15). Other native sequence WNT polypeptides of interest include orthologs of the above from any mammal, including domestic and farm animals, and zoo, laboratory or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, rats, mice, frogs, zebra fish, fruit fly, worm, etc.

[0135] "WNT pathway signaling" or "WNT signaling" is used herein to refer to the mechanism by which a biologically active WNT exerts its effects upon a cell to modulate a cell's activity. WNT proteins modulate cell activity by binding to WNT receptors, including proteins from the Frizzled (FZD) family of proteins, proteins from the ROR family of proteins, the proteins LRP5, LRP6 from the LRP family of proteins, the protein FRLl / crypto, and the protein Derailed / Ryk. Once activated by WNT binding, the WNT receptor(s) will activate one or more intracellular signaling cascades. These include the canonical WNT signaling pathway; the WNT / planar cell polarity (WNT / PCP) pathway; the WNT-calcium (WNT / Ca2+) 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, M. T. et al. (2003) Dev. Cell 5: 367-377); and other WNT signaling pathways as is well known in the art.

[0136] For example, activation of the canonical WNT signaling pathway results in the inhibition of phosphorylation of the intracellular protein P-catenin, leading to an accumulation of P- catenin in the cytosol and its 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 such biological processes as tissue polarity and cell movement. Activation of the WNT / Ca2+ by, for example, binding of WNT- 4, WNT-5A or WNT-11, elicits an intracellular release of calcium ions, which activatescalcium sensitive enzymes like protein kinase C (PKC), calcium-calmodulin dependent kinase II (CamKII) or calcineurin (CaCN). By assaying for activity of the above signaling pathways, the biological activity of an antibody or antigen-binding fragment thereof, e.g., a WNT surrogate molecule, can be readily determined.

[0137] In certain embodiments, functional properties of WNT surrogate fusion molecules may be assessed using a variety of methods known to the skilled person, including e.g., affinity / binding assays (for example, surface plasmon resonance, competitive inhibition assays), cytotoxicity assays, cell viability assays, cell proliferation or differentiation assays in response to a WNT, cancer cell and / or tumor growth inhibition using in vitro or in vivo models, including but not limited to any described herein. The WNT surrogate molecules described herein may also be tested for effects on FZD receptor internalization, in vitro and in vivo efficacy, etc. Such assays may be performed using well-established protocols known to the skilled person (see e.g., Current Protocols in Molecular Biology (Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., NY, NY); Current Protocols in Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober 2001 John Wiley & Sons, NY, NY); or commercially available kits.10.1.381 In certain embodiments, a FZD-binding region of a WNT surrogate fusion molecule (e.g., an antigen-binding fragment of an anti-FZD antibody) comprises one or more of the CDRs of the anti-FZD antibodies described in the International Publication No. WO 2019 / 12639, US Patent No 12 / 006,368 filed 19 December, 2018. In certain embodiments, a LRP5 / 6-binding region of a WNT surrogate fusion molecule (e.g., an antigen-binding fragment of an anti-LRP5 / 6 antibody) comprises one or more of the CDRs described in the International Publication No. WO 2019 / 126401, Us Patent No. 11,746,150 filed 19 December 2018, or the anti-LRP5 / 6 antibodies described herein, and IL6 binding region comprises siltuximab, sirukumab, olokizumab, clazakinumab, gerilimzumab, tocilizumab, Sarilumab, Vamikibart or Satralizumab.

[0139] Also disclosed herein is a method for obtaining an antibody or antigen binding domain specific for a FZD receptor, the method comprising providing by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a VH domain set out herein or a VH domain which is an amino acid sequence variant of the VH domain, optionally combining the VH domain thus provided with one or more VL domains, and testing the VH domain or VH / VL combination or combinations to identify a specific binding member or an antibody antigen binding domain specific for one or more FZD receptor and optionally with one or more desired properties. The VL domains may havean amino acid sequence which is substantially as set out herein. An analogous method may be employed in which one or more sequence variants of a VL domain disclosed herein are combined with one or more VH domains.

[0140] In particular embodiments, WNT surrogate molecules are water soluble. By "water soluble" it is meant a composition that is soluble in aqueous buffers in the absence of detergent, usually soluble at a concentration that provides a biologically effective dose of the polypeptide. Compositions that are water soluble form a substantially homogenous composition that has a specific activity that is at least about 5% that of the starting material from which it was purified, usually at least about 10%, 20%, or 30% that of the starting material, more usually about 40%, 50%, or 60% that of the starting material, and may be about 50%, about 90% or greater. WNT surrogate molecules disclosed herein typically form a substantially homogeneous aqueous solution at concentrations of at least 25 pM and higher, e.g. at least 25 pM, 40 pM, or 50 pM, usually at least 60 pM, 70 pM, 80 pM, or 90 pM, sometimes as much as 100 pM, 120 pM, or 150 pM. In other words, WNT surrogate molecules disclosed herein typically form a substantially homogeneous aqueous solution at concentrations of about 0.1 mg / ml, about 0.5 mg / ml, of about 1 mg / ml or more.10141] An antigen or epitope that "specifically binds" or "preferentially binds" (used interchangeably herein) to an antibody or antigen-binding fragment thereof is a term well understood in the art, and methods to determine such specific or preferential binding are also well known in the art. A molecule, e.g., a WNT surrogate fusion molecule, is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, with greater duration and / or with greater affinity with a particular cell or substance than it does with alternative cells or substances. A molecule or binding region thereof, e.g., a WNT surrogate fusion molecule or binding region thereof, "specifically binds" or "preferentially binds" to a target antigen, e.g., a FZD receptor, if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. For example, a WNT surrogate fusion molecule or binding region thereof that specifically or preferentially binds to the FZD1 receptor is an antibody that binds to the FZD1 receptor with greater affinity, avidity, more readily, and / or with greater duration than it binds to other FZD receptors or non-FZD proteins. It is also understood by reading this definition that, for example, a WNT surrogate fusion molecule or binding region thereof that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, "specific binding" or "preferential binding" does not necessarilyrequire (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.101421 In some embodiments, any of the one or more FZD binding region of a WNT surrogate fusion molecule binds to one, two, three, four, five or more different frizzled proteins, e.g., one or more of human frizzled proteins FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD 10. In some embodiments, any of the FZD binding regions binds to FZD1, FZD2, FZD5, FZD7 and FZD8. In various embodiments, any of the FZD binding regions binds to: (i) FZD1, FZD2, FZD7 and FZD9; (ii) FZD1, FZD2 and FZD7; (iii) FZD5 and FZD8; (iv) FZD5, FZD7 and FZD8; (v) FZD1, FZD4, FZD5 and FZD8; (vi) FZD1, FZD2, FZD5, FZD7 and FZD8; (vii) FZD4 and FZD9; (viii) FZD9 and FZD 10; (ix) FZD5, FZD8 and FZD10; or (x) FZD4, FZD5 and FZD8; FZD1, FZD5, FZD7 and FZD8. In some embodiments, the FZD binding region is selective for one or more FZD protein of interest, e.g. having a specificity for the one or more desired FZD protein of at least 10-fold, 25-fold, 50-fold, 100-fold, 200-fold or more relative to other FZD proteins. In some embodiments, any of the one or more FZD binding region of a WNT surrogate molecule is monospecific and binds or specifically binds to only one of FZD 1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, or FZDIO.

[0143] In some embodiments, any of the one or more LRP5 / 6 binding region of a WNT surrogate fusion molecule binds to one or both of LRP5 / 6. For convenience, the term “LRP5 / 6” is used to refer collectively to either or both of LRP5 and / or LRP6.10144] In some embodiments, any of the one or more IL6 binding region of a WNT surrogate fusion molecule binds to one or both of IL6 ligand or IL6 receptor. For convenience, the term “IL6 ligand or IL6 receptor” is used to refer collectively to either or both of IL6 binder.

[0145] In some embodiments, any of the one or more IL6 binding region of a Trispecific WNT surrogate fusion molecule binds to one or both of IL6 ligand or IL6 receptor. Immunological binding generally refers to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific, for example by way of illustration and not limitation, as a result of electrostatic, ionic, hydrophilic and / or hydrophobic attractions or repulsion, steric forces, hydrogen bonding, van der Waals forces, and other interactions. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, wherein a smaller Kd represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known inthe art. One such method entails measuring the rates of antigen-binding site / antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and on geometric parameters that equally influence the rate in both directions. Thus, both the "on rate constant" (Kon) and the "off rate constant" (Koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. The ratio of Koff / Kon enables cancellation of all parameters not related to affinity, and is thus equal to the dissociation constant Kd. See, generally, Davies et al. (1990) Annual Rev. Biochem. 59:439-473.

[0146] In certain embodiments, the WNT surrogate fusion molecules or binding regions thereof described herein have an affinity of less than about 10,000, less than about 1000, less than about 100, less than about 10, less than about 1 or less than about 0.1 nM, and in some embodiments, the antibodies may have even higher affinity for one or more FZD receptor or LRP5 or LRP6 receptor.

[0147] The constant regions of immunoglobulins show less sequence diversity than the variable regions, and are responsible for binding a number of natural proteins to elicit important biochemical events. In humans, there are five different classes of antibodies including IgA (which includes subclasses IgAl and IgA2), IgD, IgE, IgG (which includes subclasses IgGl, IgG2, IgG3, and IgG4), and IgM. The distinguishing features between these antibody classes are their constant regions, although subtler differences may exist in the V region.

[0148] The Fc region of an antibody interacts with a number of Fc receptors and ligands, imparting an array of important functional capabilities referred to as effector functions. For IgG, the Fc region comprises Ig domains CH2 and CH3 and the N-terminal hinge leading into CH2. An important family of Fc receptors for the IgG class are the Fc gamma receptors (FcyRs). These receptors mediate communication between antibodies and the cellular arm of the immune system (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12: 181-220; Ravetch et al., 2001, Annu Rev Immunol 19:275-290). In humans this protein family includes FcyRI (CD64), including isoforms FcyRIa, FcyRIb, and FcyRIc; FcyRII (CD32), including isoforms FcyRIIa (including allotypes H131 and R131), FcyRIIb (including FcyRIIb-1 and FcyRIIb-2), and FcyRIIc; and FcyRIII (CD16), including isoforms FcyRIIIa (including allotypes V158 and F158) and FcyRIIIb (including allotypes FcyRIIIb-NAl and FcyRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65). These receptors typically have an extracellular domain that mediates binding to Fc, a membrane spanning region, and an intracellular domain that may mediate some signaling event within the cell. These receptors are expressed in a varietyof immune cells including monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans' cells, natural killer (NK) cells, and T cells. Formation of the Fc / FcyR complex recruits these effector cells to sites of bound antigen, typically resulting in signaling events within the cells and important subsequent immune responses such as release of inflammation mediators, B cell activation, endocytosis, phagocytosis, and cytotoxic attack.

[0149] The ability to mediate cytotoxic and phagocytic effector functions is a potential mechanism by which antibodies destroy targeted cells. The cell-mediated reaction wherein nonspecific cytotoxic cells that express FcyRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell is referred to as antibody dependent cell-mediated cytotoxicity (ADCC) (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12: 181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766; Ravetch et al., 2001, Annu Rev Immunol 19:275- 290). The cell-mediated reaction wherein nonspecific cytotoxic cells that express FcyRs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell is referred to as antibody dependent cell-mediated phagocytosis (ADCP). All FcyRs bind the same region on Fc, at the N-terminal end of the Cg2 (CH2) domain and the preceding hinge. This interaction is well characterized structurally (Sondermann et al., 2001, J Mol Biol 309:737-749), and several structures of the human Fc bound to the extracellular domain of human FcyRIIIb have been solved (pdb accession code 1E4K) (Sondermann et al., 2000, Nature 406:267-273.) (pdb accession codes 1IIS and 1IIX) (Radaev et al., 2001, J Biol Chem 276: 16469-16477.)

[0150] The different IgG subclasses have different affinities for the FcyRs, with IgGl and IgG3 typically binding substantially better to the receptors than IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett 82:57-65). All FcyRs bind the same region on IgG Fc, yet with different affinities: the high affinity binder FcyRI has a Kd for IgGl of 10-8 M-l, whereas the low affinity receptors FcyRII and FcyRIII generally bind at 10-6 and 10-5 respectively. The extracellular domains of FcyRIIIa and FcyRIIIb are 96% identical; however, FcyRIIIb does not have an intracellular signaling domain. Furthermore, whereas FcyRI, FcyRIIa / c, and FcyRIIIa are positive regulators of immune complex-triggered activation, characterized by having an intracellular domain that has an immunoreceptor tyrosine-based activation motif (IT AM), FcyRIIb has an immunoreceptor tyrosine-based inhibition motif (ITIM) and is therefore inhibitory. Thus the former are referred to as activation receptors, and FcyRIIb is referred to as an inhibitory receptor. The receptors also differ in expression pattern and levels on different immune cells. Yet another level of complexity is the existence of a number ofFcyR polymorphisms in the human proteome. A particularly relevant polymorphism with clinical significance is V158 / F158 FcyRIIIa. Human IgGl binds with greater affinity to the VI 58 allotype than to the Fl 58 allotype. This difference in affinity, and presumably its effect on ADCC and / or ADCP, has been shown to be a significant determinant of the efficacy of the anti-CD20 antibody rituximab (Rituxan®, a registered trademark of IDEC Pharmaceuticals Corporation). Subjects with the VI 58 allotype respond favorably to rituximab treatment; however, subjects with the lower affinity Fl 58 allotype respond poorly (Cartron et al., 2002, Blood 99:754-758). Approximately 10-20% of humans are V158 / V158 homozygous, 45% are V158 / F158 heterozygous, and 35-45% of humans are F158 / F158 homozygous (Lehrnbecher et al., 1999, Blood 94:4220-4232; Cartron et al., 2002, Blood 99:754-758). Thus 80-90% of humans are poor responders, that is, they have at least one allele of the F158 FcyRIIIa.

[0151] The Fc region is also involved in activation of the complement cascade. In the classical complement pathway, Cl binds with its Clq subunits to Fc fragments of IgG or IgM, which has formed a complex with antigen(s). In certain embodiments of the invention, modifications to the Fc region comprise modifications that alter (either enhance or decrease) the ability of a FZD-specific antibody as described herein to activate the complement system (see e.g., U.S. Patent 7,740,847). To assess complement activation, a complement-dependent cytotoxicity (CDC) assay may be performed (See, e.g., Gazzano- Santoro et al., I. Immunol. Methods, 202: 163 (1996)).

[0152] Thus in certain embodiments, the present invention provides anti-FZD antibodies having a modified Fc region with altered functional properties, such as reduced or enhanced CDC, ADCC, or ADCP activity, or enhanced binding affinity for a specific FcyR or increased serum half-life. Other modified Fc regions contemplated herein are described, for example, in issued U.S. Patents 7,317,091; 7,657,380; 7,662,925; 6,538,124; 6,528,624; 7,297,775; 7,364,731; Published U.S. Applications US2009092599; US20080131435; US20080138344; and published International Applications W02006 / 105338; W02004 / 063351; W02006 / 088494; W02007 / 024249.

[0153] In certain embodiments, WNT surrogate fusion molecules comprise antibody variable domains with the desired binding specificities fused to immunoglobulin constant domain sequences. In certain embodiments, the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. In particular embodiments, the first heavy-chain constant region (CHI) containing the site necessary for light chain bonding, present in at least one of the fusions. DNAs encoding theimmunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired bispecific antibody. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios have no significant effect on the yield of the desired chain combination.

[0154] WNT surrogate fusion molecules disclosed herein may also be modified to include an epitope tag or label, e.g., for use in purification or diagnostic applications. There are many linking groups known in the art for making antibody conjugates, including, for example, those disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 Bl, and Chari et al., Cancer Research 52: 127-131 (1992). The linking groups include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups, as disclosed in the above-identified patents, disulfide and thioether groups being preferred.

[0155] In certain embodiments, anti-LRP5 / 6 antibodies and antigen-binding fragments thereof and / or anti-FZD antibodies and antigen-binding fragments thereof present within a WNT surrogate fusion molecule are monoclonal. In certain embodiments, they are humanized.

[0156] The present invention further provides in certain embodiments an isolated nucleic acid encoding a polypeptide present in a WNT surrogate fusion molecule disclosed herein. Nucleic acids include DNA and RNA. These and related embodiments may include polynucleotides encoding antibody fragments that bind one or more FZD receptors and / or LRP5 or LRP6 as described herein. The term "isolated polynucleotide" as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, which by virtue of its origin, the isolated polynucleotide: (1) is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature; (2) is linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence. An isolated polynucleotide may include naturally occurring and / or artificial sequences.

[0157] The term "operably linked" means that the components to which the term is applied are in a relationship that allows them to carry out their inherent functions undersuitable conditions. For example, a transcription control sequence "operably linked" to a protein coding sequence is ligated thereto so that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences.

[0158] The term "control sequence" as used herein refers to polynucleotide sequences that can affect expression, processing or intracellular localization of coding sequences to which they are ligated or operably linked. The nature of such control sequences may depend upon the host organism. In particular embodiments, transcription control sequences for prokaryotes may include a promoter, ribosomal binding site, and transcription termination sequence. In other particular embodiments, transcription control sequences for eukaryotes may include promoters comprising one or a plurality of recognition sites for transcription factors, transcription enhancer sequences, transcription termination sequences and polyadenylation sequences. In certain embodiments, "control sequences" can include leader sequences and / or fusion partner sequences.(0159] The term "polynucleotide" as referred to herein means single-stranded or doublestranded nucleic acid polymers. In certain embodiments, the nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include base modifications such as bromouridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose and intemucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate. The term "polynucleotide" specifically includes single and double stranded forms of DNA.(0160] The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" includes nucleotides with modified or substituted sugar groups and the like. The term "oligonucleotide linkages" includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the like. See, e.g., LaPlanche et al., 1986, Nucl. Acids Res., 14:9081; Stec et al., 1984, J. Am. Chem. Soc., 106:6077; Stein et al., 1988, Nucl. Acids Res., 16:3209; Zon et al., 1991, AntiCancer Drug Design, 6:539; Zon et al., 1991, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, pp. 87-108 (F. Eckstein, Ed ), Oxford University Press, Oxford England; Stec et al., U.S. Pat. No. 5,151,510; Uhlmann and Peyman, 1990, Chemical Reviews, 90:543, the disclosures of which are hereby incorporated by reference for anypurpose. An oligonucleotide can include a detectable label to enable detection of the oligonucleotide or hybridization thereof.|0l6l| The term "vector" is used to refer to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information to a host cell. The term "expression vector" refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and / or control expression of inserted heterologous nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing, if introns are present.[0162| As will be understood by those skilled in the art, polynucleotides may include genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, peptides and the like. Such segments may be naturally isolated, or modified synthetically by the skilled person.

[0163] As will be also recognized by the skilled artisan, polynucleotides may be singlestranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules may include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide according to the present disclosure, and a polynucleotide may, but need not, be linked to other molecules and / or support materials. Polynucleotides may comprise a native sequence or may comprise a sequence that encodes a variant or derivative of such a sequence.

[0164] It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encodes an antibody as described herein. Some of these polynucleotides bear minimal sequence identity to the nucleotide sequence of the native or original polynucleotide sequence encoding a polypeptide within a WNT surrogate fusion molecule. Nonetheless, polynucleotides that vary due to differences in codon usage are expressly contemplated by the present disclosure. In certain embodiments, sequences that have been codon-optimized for mammalian expression are specifically contemplated.

[0165] Therefore, in another embodiment of the invention, a mutagenesis approach, such as site-specific mutagenesis, may be employed for the preparation of variants and / or derivatives of the polypeptides described herein. By this approach, specific modifications in a polypeptide sequence can be made through mutagenesis of the underlying polynucleotidesthat encode them. These techniques provide a straightforward approach to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the polynucleotide.

[0166] Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Mutations may be employed in a selected polynucleotide sequence to improve, alter, decrease, modify, or otherwise change the properties of the polynucleotide itself, and / or alter the properties, activity, composition, stability, or primary sequence of the encoded polypeptide.10167] In certain embodiments, the inventors contemplate the mutagenesis of the polynucleotide sequences that encode a polypeptide present in a WNT surrogate fusion molecule, to alter one or more properties of the encoded polypeptide, such as the binding affinity, or the function of a particular Fc region, or the affinity of the Fc region for a particular FcyR. The techniques of site-specific mutagenesis are well-known in the art, and are widely used to create variants of both polypeptides and polynucleotides. For example, site-specific mutagenesis is often used to alter a specific portion of a DNA molecule. In such embodiments, a primer comprising typically about 14 to about 25 nucleotides or so in length is employed, with about 5 to about 10 residues on both sides of the junction of the sequence being altered.

[0168] As will be appreciated by those of skill in the art, site-specific mutagenesis techniques have often employed a phage vector that exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phages are readily commercially available and their use is generally well-known to those skilled in the art. Double-stranded plasmids are also routinely employed in site directed mutagenesis that eliminates the step of transferring the gene of interest from a plasmid to a phage.

[0019] The preparation of sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis provides a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained. For example, recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants. Specific details regarding these methods andprotocols are found in the teachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994; and Maniatis et al., 1982, each incorporated herein by reference, for that purpose.

[0170] In many embodiments, one or more nucleic acids encoding a polypeptide of a WNT surrogate fusion molecule are introduced directly into a host cell, and the cell incubated under conditions sufficient to induce expression of the encoded polypeptides. The WNT surrogate polypeptides of this disclosure may be prepared using standard techniques well known to those of skill in the art in combination with the polypeptide and nucleic acid sequences provided herein. The polypeptide sequences may be used to determine appropriate nucleic acid sequences encoding the particular polypeptide disclosed thereby. The nucleic acid sequence may be optimized to reflect particular codon "preferences" for various expression systems according to standard methods well known to those of skill in the art.

[0171] According to certain related embodiments there is provided a recombinant host cell which comprises one or more constructs as described herein, e.g., a vector comprising a nucleic acid encoding a WNT surrogate fusion molecule or polypeptide thereof; and a method of production of the encoded product, which method comprises expression from encoding nucleic acid therefor. Expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression, an antibody or antigen-binding fragment thereof, may be isolated and / or purified using any suitable technique, and then used as desired.

[0172] Polypeptides, and encoding nucleic acid molecules and vectors, may be isolated and / or purified, e.g. from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes of origin other than the sequence encoding a polypeptide with the desired function. Nucleic acid may comprise DNA or RNA and may be wholly or partially synthetic. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise.

[0173] Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells and many others. A common, preferred bacterial host is E. coli.

[0174] The expression of polypeptides, e.g., antibodies and antigen-binding fragments thereof, in prokaryotic cells such as E. coli is well established in the art. For a review, see for example Pluckthun, A. Bio / Technology 9: 545-551 (1991). Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production of antibodies or antigen-binding fragments thereof, see recent reviews, for example Ref, M. E. (1993) Curr. Opinion Biotech. 4: 573-576; Trill J. J. et al. (1995) Curr. Opinion Biotech 6: 553-560.

[0175] Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992, or subsequent updates thereto.10.176] The term "host cell" is used to refer to a cell into which has been introduced, or which is capable of having introduced into it, a nucleic acid sequence encoding one or more of the herein described polypeptides, and which further expresses or is capable of expressing a selected gene of interest, such as a gene encoding any herein described polypeptide. The term includes the progeny of the parent cell, whether or not the progeny are identical in morphology or in genetic make-up to the original parent, so long as the selected gene is present. Accordingly there is also contemplated a method comprising introducing such nucleic acid into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE- Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene. In one embodiment, the nucleic acid is integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance-with standard techniques.

[0177] The present invention also provides, in certain embodiments, a method which comprises using a construct as stated above in an expression system in order to express a particular polypeptide such as a Wnyt mimetic molecule as described herein. The term "transduction" is used to refer to the transfer of genes from one bacterium to another, usually by a phage. "Transduction" also refers to the acquisition and transfer of eukaryotic cellular sequences by retroviruses. The term "transfection" is used to refer to the uptake of foreign or exogenous DNA by a cell, and a cell has been "transfected" when the exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Laboratories; Davis et al., 1986, BASIC METHODS IN MOLECULAR BIOLOGY, Elsevier; and Chu et al., 1981, Gene 13: 197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.

[0178] The term "transformation" as used herein refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain a new DNA. For example, a cell is transformed where it is genetically modified from its native state. Following transfection or transduction, the transforming DNA may recombine with that of the cell by physically integrating into a chromosome of the cell, or may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid. A cell is considered to have been stably transformed when the DNA is replicated with the division of the cell. The term "naturally occurring" or "native" when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, refers to materials which are found in nature and are not manipulated by a human. Similarly, "non-naturally occurring" or "non-native" as used herein refers to a material that is not found in nature or that has been structurally modified or synthesized by a human.

[0179] The terms "polypeptide" "protein" and "peptide" and "glycoprotein" are used interchangeably and mean a polymer of amino acids not limited to any particular length. The term does not exclude modifications such as myristylation, sulfation, glycosylation, phosphorylation and addition or deletion of signal sequences. The terms "polypeptide" or "protein" means one or more chains of amino acids, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein said polypeptide or protein can comprise a plurality of chains non-covalently and / or covalently linked together by peptide bonds, having the sequence of native proteins, that is, proteins produced by naturally-occurring and specifically non-recombinant cells, or genetically-engineered or recombinant cells, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and / or substitutions of one or more amino acids of the native sequence. The terms "polypeptide" and "protein" specifically encompass WNT surrogate fusion molecules, FZD binding regions thereof, LRP5 / 6 binding regions thereof, antibodies and antigen-binding fragments thereof that bind to a FZD receptor or a LRP5 or LRP6 receptor disclosed herein, or sequences that have deletions from, additions to, and / or substitutions of one or more amino acid of any of these polypeptides. Thus, a "polypeptide" or a "protein" can comprise one (termed "a monomer") or a plurality (termed "a multimer") of amino acid chains.

[0180] The term "isolated protein,” “isolated WNT surrogate fusion molecule or “isolated antibody” referred to herein means that a subject protein, WNT surrogate fusion molecule, or antibody: (1) is free of at least some other proteins with which it would typically be found in nature; (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species; (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature; (5) is not associated (by covalent or noncovalent interaction) with portions of a protein with which the "isolated protein" is associated in nature; (6) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature; or (7) does not occur in nature. Such an isolated protein can be encoded by genomic DNA, cDNA, mRNA or other RNA, or may be of synthetic origin, or any combination thereof. In certain embodiments, an isolated protein may comprise naturally- occurring and / or artificial polypeptide sequences. In certain embodiments, the isolated protein is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).

[0181] Amino acid sequence modification(s) of any of the polypeptides (e.g., WNT surrogate fusion molecules or FZD binding regions or LRP5 / 6 binding regions thereof or IL6 binding regions) described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the WNT surrogate fusion molecule. For example, amino acid sequence variants of a WNT surrogate fusion molecule may be prepared by introducing appropriate nucleotide changes into a polynucleotide that encodes the antibody, or a chain thereof, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of, residues within theamino acid sequences of the antibody. Any combination of deletion, insertion, and substitution may be made to arrive at the final WNT surrogate fusion molecule, provided that the final construct possesses the desired characteristics (e.g., high affinity binding to one or more FZD and / or LRP5 / 6 receptor or IL6 binding regions). The amino acid changes also may alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites. Any of the variations and modifications described above for polypeptides of the present invention may be included in antibodies of the present invention.

[0182] The present disclosure provides variants of any of the polypeptides (e.g., WNT surrogate fusion molecules or FZD binding regions, LRP5 / 6 binding regions, or cytokine receptor or cytokine ligand binding regions thereof, or antibodies or antigen-binding fragments thereof) disclosed herein. In certain embodiments, a variant has at least 90%, at least 95%, at least 98%, or at least 99% identity to a polypeptide disclosed herein. In certain embodiments, such variant polypeptides bind to one or more FZD receptor, and / or to one or more LRP5 / 6 receptor, and / or cytokine receptor or cytokine ligand, at least about 50%, at least about 70%, and in certain embodiments, at least about 90% as well as a WNT surrogate fusion molecule specifically set forth herein. In further embodiments, such variant WNT surrogate fusion molecules bind to one or more FZD receptor, and / or to one or more LRP5 / 6 receptor and / or IL6 or IL6 receptor, with greater affinity than the WNT surrogate fusion molecules set forth herein, for example, that bind quantitatively at least about 105%, 106%, 107%, 108%, 109%, or 110% as well as an antibody sequence specifically set forth herein.10183] In particular embodiments, the WNT surrogate fusion molecule or a binding region thereof, e.g., a Fab, scFv, or VHH or sdAb may comprise: a) a heavy chain variable region comprising: i. a CDR1 region that is identical in amino acid sequence to the heavy chain CDR1 region of a selected antibody described herein; ii. a CDR2 region that is identical in amino acid sequence to the heavy chain CDR2 region of the selected antibody; and iii. a CDR3 region that is identical in amino acid sequence to the heavy chain CDR3 region of the selected antibody; and / or b) a light chain variable domain comprising: i. a CDR1 region that is identical in amino acid sequence to the light chain CDR1 region of the selected antibody; ii. a CDR2 region that is identical in amino acid sequence to the light chain CDR2 region of the selected antibody; and iii. a CDR3 region that is identical in amino acid sequence to the light chain CDR3 region of the selected antibody; wherein the antibody specifically binds a selected target (e.g., one or more FZD receptors or LRP5 or LRP6 receptors).

[0184] In particular embodiments, the WNT surrogate fusion molecule or a binding region thereof, e.g., a Fab, scFv, or VHH or sdAb may have: a) a heavy chain variable regionhaving an amino acid sequence that is at least 80% identical, at least 95% identical, at least 90%, at least 95% or at least 98% or 99% identical, to the heavy chain variable region of an antibody or antigen-binding fragments thereof described herein; and / or b) a light chain variable region having an amino acid sequence that is at least 80% identical, at least 85%, at least 90%, at least 95% or at least 98% or 99% identical, to the light chain variable region of an antibody or antigen-binding fragments thereof described herein.

[0185] A polypeptide has a certain percent "sequence identity" to another polypeptide, meaning that, when aligned, that percentage of amino acids are the same when comparing the two sequences. As used herein, the terms “identity” and “identical” refer, with respect to a polypeptide or polynucleotide sequence, to the percentage of exact matching residues in an alignment of that “query” sequence to a “subject” sequence, such as an alignment generated by the BLAST algorithm. Identity is calculated, unless specified otherwise, across the full length of the subject sequence. Thus, a query sequence “shares at least x% identity to” a subject sequence if, when the query sequence is aligned to the subject sequence, at least x% (rounded down) of the residues in the subject sequence are aligned as an exact match to a corresponding residue in the query sequence. Where the subject sequence has variable positions (e.g., residues denoted X), an alignment to any residue in the query sequence is counted as a match. Sequence alignments may be performed using the NCBI Blast service (BLAST+ version 2.12.0).

[0186] Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA. Of particular interest are alignment programs that permit gaps in the sequence. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970).

[0187] Of interest is the BestFit program using the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2: 482-489 (1981) to determine sequenceidentity. The gap generation penalty will generally range from 1 to 5, usually 2 to 4 and in many embodiments will be 3. The gap extension penalty will generally range from about 0.01 to 0.20 and in many instances will be 0.10. The program has default parameters determined by the sequences inputted to be compared. Preferably, the sequence identity is determined using the default parameters determined by the program. This program is available also from Genetics Computing Group (GCG) package, from Madison, Wis., USA.

[0188] Another program of interest is the FastDB algorithm. FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. Percent sequence identity is calculated by FastDB based upon the following parameters: Mismatch Penalty: 1.00; Gap Penalty: 1.00; Gap Size Penalty: 0.33; and Joining Penalty: 30.0.10189] In particular embodiments, the WNT surrogate fusion molecule or a binding region thereof, e.g., a Fab, scFv, or VHH or sdAb may comprises: a) a heavy chain variable region comprising: i. a CDR1 region that is identical in amino acid sequence to the heavy chain CDR1 region of a selected antibody described herein; ii. a CDR2 region that is identical in amino acid sequence to the heavy chain CDR2 region of the selected antibody; and iii. a CDR3 region that is identical in amino acid sequence to the heavy chain CDR3 region of the selected antibody; and b) a light chain variable domain comprising: i. a CDR1 region that is identical in amino acid sequence to the light chain CDR1 region of the selected antibody; ii. a CDR2 region that is identical in amino acid sequence to the light chain CDR2 region of the selected antibody; and iii. a CDR3 region that is identical in amino acid sequence to the light chain CDR3 region of the selected antibody; wherein the antibody specifically binds a selected target (e.g., a FZD receptor, such as FZD1.

[0190] Determination of the three-dimensional structures of representative polypeptides (e.g., variant FZD binding regions or LRP5 / 6 binding regions of WNT surrogate fusion molecules as provided herein) may be made through routine methodologies such that substitution, addition, deletion or insertion of one or more amino acids with selected natural or non-natural amino acids can be virtually modeled for purposes of determining whether a so derived structural variant retains the space-filling properties of presently disclosed species. See, for instance, Donate et al., 1994 Prot. Sci. 3:2378; Bradley et al., Science 309: 1868- 1871 (2005); Schueler-Furman et al., Science 310:638 (2005); Dietz et al., Proc. Nat. Acad. Sci. USA 103: 1244 (2006); Dodson et al., Nature 450: 176 (2007); Qian et al., Nature 450:259 (2007); Raman et al. Science 327: 1014-1018 (2010). Some additional non-limiting examples of computer algorithms that may be used for these and related embodiments, suchas for rational design of binding regions include VMD which is a molecular visualization program for displaying, animating, and analyzing large biomolecular systems using 3-D graphics and built-in scripting (see the website for the Theoretical and Computational Biophysics Group, University of Illinois at Urbana-Champagne, at ks.uiuc.edu / Research / vmd / . Many other computer programs are known in the art and available to the skilled person and which allow for determining atomic dimensions from space-filling models (van der Waals radii) of energy-minimized conformations; GRID, which seeks to determine regions of high affinity for different chemical groups, thereby enhancing binding, Monte Carlo searches, which calculate mathematical alignment, and CHARMM (Brooks et al. (1983) J. Comput. Chem. 4: 187-217) and AMBER (Weiner et al (1981) J. Comput. Chem. 106: 765), which assess force field calculations, and analysis (see also, Eisenfield et al. (1991) Am. J. Physiol. 261 :C376-386; Lybrand (1991) J. Pharm. Belg. 46:49-54; Froimowitz (1990) Biotechniques 8:640-644; Burbam et al. (1990) Proteins 7:99- 111; Pedersen (1985) Environ. Health Perspect. 61 : 185-190; and Kini et al. (1991) J. Biomol. Struct. Dyn. 9:475-488). A variety of appropriate computational computer programs are also commercially available, such as from Schrodinger (Munich, Germany).Compositions101.91] Pharmaceutical compositions comprising a WNT surrogate fusion molecule described herein and one or more pharmaceutically acceptable diluent, carrier, or excipient are also disclosed. In particular embodiments, the pharmaceutical composition further comprises one or more WNT polypeptides or Norrin polypeptides.

[0192] In further embodiments, pharmaceutical compositions comprising a polynucleotide comprising a nucleic acid sequence encoding a WNT surrogate fusion molecule described herein and one or more pharmaceutically acceptable diluent, carrier, or excipient are also disclosed. In particular embodiments, the pharmaceutical composition further comprises one or more polynucleotides comprising a nucleic acid sequence encoding a WNT polypeptide or Norrin polypeptide. In certain embodiments, the polynucleotides are DNA or mRNA, e.g., a modified mRNA. In particular embodiments, the polynucleotides are modified mRNAs further comprising a 5’ cap sequence and / or a 3’ tailing sequence, e.g., a polyA tail. In other embodiments, the polynucleotides are expression cassettes comprising a promoter operatively linked to the coding sequences. In certain embodiments, the nucleic acid sequence encoding the WNT surrogate fusion molecule and the nucleic acid sequence encoding the WNT polypeptide or Norrin polypeptide are present in the same polynucleotide.

[0193] In further embodiments, pharmaceutical compositions comprising an expression vector, e.g., a viral vector, comprising a polynucleotide comprising a nucleic acid sequence encoding a WNT surrogate fusion molecule described herein and one or more pharmaceutically acceptable diluent, carrier, or excipient are also disclosed. In particular embodiments, the pharmaceutical composition further comprises an expression vector, e.g., a viral vector, comprising a polynucleotide comprising a nucleic acid sequence encoding a WNT polypeptide or Norrin polypeptide. In certain embodiments, the nucleic acid sequence encoding the WNT surrogate fusion molecule and the nucleic acid sequence encoding the WNT polypeptide or Norrin polypeptide are present in the same polynucleotide, e.g., expression cassette.

[0014] The present invention further contemplates a pharmaceutical composition comprising a cell comprising an expression vector comprising a polynucleotide comprising a promoter operatively linked to a nucleic acid encoding a WNT surrogate fusion molecule and one or more pharmaceutically acceptable diluent, carrier, or excipient. In particular embodiments, the pharmaceutical composition further comprises a cell comprising an expression vector comprising a polynucleotide comprising a promoter operatively linked to a nucleic acid sequence encoding a WNT polypeptide or a Norrin polypeptide. In certain embodiments, the nucleic acid sequence encoding the WNT surrogate fusion molecule and the nucleic acid sequence encoding the WNT polypeptide or Norrin polypeptide are present in the same polynucleotide, e.g., expression cassette and / or in the same cell. In particular embodiments, the cell is a heterologous cell or an autologous cell obtained from the subject to be treated. In particular embodiments, the cell is a stem cell, e.g., an adipose-derived stem cell or a hematopoietic stem cell.

[0195] The present disclosure contemplates pharmaceutical compositions comprising a first molecule for delivery of a WNT surrogate fusion molecule as a first active agent and a second molecule for delivery of a WNT polypeptide or Norrin polypeptide. The first and second molecule may be the same type of molecule or different types of molecules. For example, in certain embodiments, the first and second molecule may each be independently selected from the following types of molecules: polypeptides, small organic molecules, nucleic acids encoding the first or second active agent (optionally DNA or mRNA, optionally modified RNA), vectors comprising a nucleic acid sequence encoding the first or second active agent (optionally expression vectors or viral vectors), and cells comprising a nucleic acid sequence encoding the first or second active agent (optionally an expression cassette).

[0196] The subject molecules, alone or in combination, can be combined with pharmaceutically-acceptable carriers, diluents, excipients and reagents useful in preparing a formulation that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for mammalian, e.g., human or primate, use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous. Examples of such carriers, diluents and excipients include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Supplementary active compounds can also be incorporated into the formulations. Solutions or suspensions used for the formulations can include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; detergents such as Tween 20 to prevent aggregation; and compounds for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. In particular embodiments, the pharmaceutical compositions are sterile.101971 Pharmaceutical compositions may further include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In some cases, the composition is sterile and should be fluid such that it can be drawn into a syringe or delivered to a subject from a syringe. In certain embodiments, it is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, e.g., a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the internal compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0198] Sterile solutions can be prepared by incorporating the anti-FZD antibody or antigen-binding fragment thereof (or encoding polynucleotide or cell comprising the same) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0019] In one embodiment, the pharmaceutical compositions are prepared with carriers that will protect the antibody or antigen-binding fragment thereof against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

[0200] It may be advantageous to formulate the pharmaceutical compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active antibody or antigen-binding fragment thereof calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms are dictated by and directly dependent on the unique characteristics of the antibody or antigen-binding fragment thereof and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active antibody or antigen-binding fragment thereof for the treatment of individuals.

[0201] The pharmaceutical compositions can be included in a container, pack, or dispenser, e.g. syringe, e.g. a prefilled syringe, together with instructions for administration.

[0202] The pharmaceutical compositions of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal comprising a human, is capable of providing (directly or indirectly) the biologically active antibody or antigen-binding fragment thereof.

[0203] The present invention includes pharmaceutically acceptable salts of a WNT surrogate fusion molecule described herein. The term “pharmaceutically acceptable salt” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. A variety of pharmaceutically acceptable salts are known in the art and described, e.g., in “Remington’s Pharmaceutical Sciences”, 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and more recent editions thereof), in the “Encyclopaedia of Pharmaceutical Technology”, 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and in J. Pharm. Sci. 66: 2 (1977). Also, for a review on suitable salts, see “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” by Stahl and Wermuth (Wiley-VCH, 2002).

[0204] Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Metals used as cations comprise sodium, potassium, magnesium, calcium, and the like. Amines comprise N-N’- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-m ethylglucamine, and procaine (see, for example, Berge et al., “Pharmaceutical Salts,” J. Pharma Sci., 1977, 66, 119). The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention.

[0205] In some embodiments, the pharmaceutical composition provided herein comprise a therapeutically effective amount of a WNT surrogate fusion molecule or pharmaceutically acceptable salt thereof in admixture with a pharmaceutically acceptable carrier, diluent and / or excipient, for example saline, phosphate buffered saline, phosphate and amino acids, polymers, polyols, sugar, buffers, preservatives and other proteins. Exemplary amino acids, polymers and sugars and the like are octylphenoxy polyethoxy ethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, Ringer's and Hank's solutions, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine,polyvinylpyrrolidone, polyethylene and glycol. Preferably, this formulation is stable for at least six months at 4° C.10206] In some embodiments, the pharmaceutical composition provided herein comprises a buffer, such as phosphate buffered saline (PBS) or sodium phosphate / sodium sulfate, tris buffer, glycine buffer, sterile water and other buffers known to the ordinarily skilled artisan such as those described by Good et al. (1966) Biochemistry 5:467. The pH of the buffer may be in the range of 6.5 to 7.75, preferably 7 to 7.5, and most preferably 7.2 to 7.4.Methods of Use

[0207] The present disclosure also provides methods for using the WNT surrogate fusion molecules disclosed herein, e.g., to modulate a WNT signaling pathway, e.g., to increase WNT signaling, and the administration of a WNT surrogate fusion molecule disclosed herein in a variety of therapeutic settings. Provided herein are methods of treatment using a WNT surrogate fusion molecule. In one embodiment, a WNT surrogate fusion molecule is provided to a subject having a disease involving inappropriate or deregulated WNT signaling, e.g., reduced WNT signaling.

[0208] Increasing WNT Pathway Signaling and Related Therapeutic Methods (0209] In certain embodiments, a WNT surrogate fusion molecule may be used to increase WNT signaling in a tissue or cell. Thus, in some aspects, the present invention provides a method for increasing WNT signaling or enhancing WNT signaling in a tissue or cell, comprising contacting the tissue or cell with an effective amount of a WNT surrogate fusion molecule or pharmaceutically acceptable salt thereof disclosed herein, wherein the a WNT surrogate fusion molecule is a WNT signaling pathway agonist. In some embodiments, contacting occurs in vitro, ex vivo, or in vivo. In particular embodiments, the cell is a cultured cell, and the contacting occurs in vitro. In certain embodiments, the method comprises further contacting the tissue or cell with one or more WNT polypeptides or Norrin polypeptides.102.10] In related aspects, the present invention provides a method for increasing WNT signaling in a tissue or cell, comprising contacting the tissue or cell with an effective amount of a polynucleotide comprising a WNT surrogate fusion molecule disclosed herein. In certain embodiments, the target tissue or cell is also contacted with a polynucleotide comprising a nucleic acid sequence that encodes a WNT polypeptide or a Norrin polypeptide. In certain embodiments, the polynucleotides are DNA or mRNA, e.g., a modified mRNA. In particular embodiments, the polynucleotides are modified mRNAs further comprising a 5’ cap sequenceand / or a 3’ tailing sequence, e.g., a polyA tail. In other embodiments, the polynucleotides are expression cassettes comprising a promoter operatively linked to the coding sequences. In certain embodiments, the nucleic acid sequence encoding the WNT surrogate fusion molecule and the nucleic acid sequence encoding the WNT polypeptide or Norrin polypeptide are present in the same polynucleotide.

[0211] In related aspects, the present invention provides a method for increasing WNT signaling in a tissue or cell, comprising contacting the tissue or cell with an effective amount of a vector comprising a nucleic acid sequence encoding a WNT surrogate fusion molecule. In certain embodiments, the tissue or cell is also contacted with a vector comprising a nucleic acid sequence that encodes a WNT polypeptide or a Norrin polypeptide. In certain embodiments, the vector is an expression vector, and may comprise a promoter operatively linked to the nucleic acid sequence. In particular embodiments, the vector is a viral vector. In certain embodiments, the nucleic acid sequence encoding a WNT surrogate fusion molecule and the nucleic acid sequence encoding the WNT polypeptide or Norrin polypeptide are present in the same vector, e.g., in the same expression cassette.

[0212] In related aspects, the present invention provides a method for increasing WNT signaling in a tissue, comprising contacting the tissue with an effective amount of a cell comprising a nucleic acid sequence encoding a WNT surrogate fusion molecule of the present invention. In certain embodiments, the tissue is also contacted with a cell comprising a nucleic acid sequence that encodes a WNT polypeptide or Norrin polypeptide. In certain embodiments, the nucleic acid sequence encoding the WNT surrogate fusion molecule and the nucleic acid sequence encoding the WNT polypeptide or Norrin polypeptide are present in the same cell. In particular embodiments, the cell is a heterologous cell or an autologous cell obtained from the subject to be treated. In certain embodiments, the cell was transduced with a vector comprising an expression cassette encoding the WNT surrogate fusion molecule or the WNT polypeptide or Norrin polypeptide. In particular embodiments, the cell is a stem cell, e.g., an adipose-derived stem cell or a hematopoietic stem cell.

[0213] WNT surrogate fusion molecules disclosed herein may be used in to treat a disease, disorder or condition, for example, by increasing WNT signaling in a targeted cell, tissue or organ. Thus, in some aspects, the present invention provides a method for treating a disease or condition in a subject in need thereof, e.g., a disease or disorder associated with reduced WNT signaling, or for which increased WNT signaling would provide a therapeutic benefit, comprising contacting the subject with an effective amount of a composition of the present disclosure. In particular embodiments, the composition is a pharmaceuticalcomposition comprising any of: a WNT surrogate fusion molecule; a polynucleotide comprising a nucleic acid sequence encoding a WNT surrogate fusion molecule, e.g., a DNA or mRNA, optionally a modified mRNA; a vector comprising a nucleic acid sequence encoding a WNT surrogate fusion molecule, e.g., an expression vector or viral vector; or a cell comprising a nucleic acid sequence encoding a WNT surrogate fusion molecule, e.g., a cell transduced with an expression vector or viral vector encoding a WNT surrogate fusion molecule. In particular embodiments, the disease or condition is a pathological disease or disorder, or an injury, e.g., an injury resulting from a wound. In certain embodiments, the wound may be the result of another therapeutic treatment. In certain embodiments, the disease or condition comprises impaired tissue repair, healing or regeneration, or would benefit from increased tissue repair, healing or regeneration. In some embodiments, contacting occurs in vivo, i.e., the subject composition is administered to a subject.

[0214] In certain embodiments, the method comprises further contacting the subject with a pharmaceutical composition comprising one or more WNT polypeptides or Norrin polypeptides. The present disclosure contemplates contacting a subject with a first molecule for delivery of a WNT surrogate fusion molecule as a first active agent and a second molecule for delivery of a WNT polypeptide or Norrin polypeptide. The first and second molecule may be the same type of molecule or different types of molecules. For example, in certain embodiments, the first and second molecule may each be independently selected from the following types of molecules: polypeptides, small organic molecules, nucleic acids encoding the first or second active agent (optionally DNA or mRNA, optionally modified RNA), vectors comprising a nucleic acid sequence encoding the first or second active agent (optionally expression vectors or viral vectors), and cells comprising a nucleic acid sequence encoding the first or second active agent (optionally an expression cassette).

[0215] In related aspects, the present invention provides a method for treating a disease or condition, e.g., a disease or disorder associated with reduced WNT signaling, or for which increased WNT signaling would provide a therapeutic benefit, comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a WNT surrogate fusion molecule disclosed herein. In certain embodiments, the subject is also contacted with a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence that encodes a WNT polypeptide or a Norrin polypeptide. In certain embodiments, the polynucleotides are DNA or mRNA, e.g., a modified mRNA. In particular embodiments, the polynucleotides are modified mRNAs further comprising a 5’ cap sequenceand / or a 3’ tailing sequence, e.g., a polyA tail. In other embodiments, the polynucleotides are expression cassettes comprising a promoter operatively linked to the coding sequences. In certain embodiments, the nucleic acid sequence encoding the WNT surrogate fusion molecule and the nucleic acid sequence encoding the WNT polypeptide or Norrin polypeptide are present in the same polynucleotide.

[0216] In related aspects, the present invention provides a method for treating a disease or condition, e.g., a disease or disorder associated with reduced WNT signaling, or for which increased WNT signaling would provide a therapeutic benefit, comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of a vector comprising a nucleic acid sequence encoding a WNT surrogate fusion molecule. In certain embodiments, the subject is also contacted with a pharmaceutical composition comprising an effective amount of a vector comprising a nucleic acid sequence that encodes a WNT polypeptide or a Norrin polypeptide. In certain embodiments, the vector is an expression vector, and may comprise a promoter operatively linked to the nucleic acid sequence. In particular embodiments, the vector is a viral vector. In certain embodiments, the nucleic acid sequence encoding the WNT surrogate fusion molecule and the nucleic acid sequence encoding the WNT polypeptide or Norrin polypeptide are present in the same vector, e.g., in the same expression cassette.

[0217] In related aspects, the present invention provides a method for treating a disease or condition, e.g., a disease or disorder associated with reduced WNT signaling, or for which increased WNT signaling would provide a therapeutic benefit, comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of a cell comprising a nucleic acid sequence encoding a WNT surrogate fusion molecule. In certain embodiments, the subject is also contacted with a cell comprising a nucleic acid sequence that encodes a WNT polypeptide or a Norrin polypeptide. In certain embodiments, the nucleic acid sequence encoding the WNT surrogate fusion molecule and the nucleic acid sequence encoding the WNT polypeptide or Norrin polypeptide are present in the same cell. In particular embodiments, the cell is a heterologous cell or an autologous cell obtained from the subject to be treated. In certain embodiments, the cell was transduced with a vector comprising an expression cassette encoding the WNT surrogate fusion molecule or the WNT polypeptide or Norrin polypeptide. In particular embodiments, the cell is a stem cell, e.g., an adipose-derived stem cell or a hematopoietic stem cell.

[0218] WNT signaling plays key roles in the developmental process and maintenance of stem cells. Reactivation of WNT signals is associated with regeneration and repair of mosttissues after injuries and diseases. WNT surrogate fusion molecule molecules are expected to provide benefit of healing and tissue repair in response to injuries and diseases. Causes of tissue damage and loss include but are not limited to aging, degeneration, hereditary conditions, infection and inflammation, traumatic injuries, toxins / metabolic-induced toxi cities, or other pathological conditions. WNT signals and enhancers of WNT signals have been shown to activate adult, tissue-resident stem cells. In some embodiments, the compounds of the invention are administered for use in treating diseased or damaged tissue, for use in tissue regeneration and for use in cell growth and proliferation, and / or for use in tissue engineering.

[0219] Human diseases associated with mutations of the WNT pathway provide strong evidence for enhancement of WNT signals in the treatment and prevention of diseases. Preclinical in vivo and in vitro studies provide additional evidence of involvement of WNT signals in many disease conditions and further support utilization of a WNT surrogate fusion molecule in various human diseases. For example, compositions of the present invention may be used to promote or increase bone growth or regeneration, bone grafting, healing of bone fractures, treatment of osteoporosis and osteoporotic fractures, spinal fusion, spinal cord injuries, including vertebral compression fractures, pre-operative spinal surgery optimization, osseointegration of orthopedic devices, tendon-bone integration, tooth growth and regeneration, dental implantation, periodontal diseases, maxillofacial reconstruction, and osteonecrosis of the jaw. They may also be used in the treatment of alopecia; enhancing regeneration of sensory organs, e.g. treatment of hearing loss, including regeneration of inner and outer auditory hair cells treatment of vestibular hypofunction, treatment of macular degeneration, treatment of retinopathies, including vitreoretinopathy, diabetic retinopathy, other diseases of retinal degeneration, Fuchs’ dystrophy, other cornea disease, etc.; treatment of stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, multiple dystrophy, muscle atrophy as a result of sarcopenia or cachexia, and other conditions affecting the degeneration or integrity of the blood brain barrier; . The compositions of this invention may also be used in treatment of oral mucositis, treatment of short bowel syndrome, inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), in particular CD with fistula formation, other gastrointestinal disorders; treatment of metabolic syndrome, dyslipidemia, treatment of diabetes, treatment of pancreatitis, conditions where exocrine or endocrine pancreas tissues are damaged; conditions where enhanced epidermal regeneration is desired, e.g., epidermal wound healing, treatment of diabetic foot ulcers, syndromes involving tooth, nail, or dermal hypoplasia, etc., conditions where angiogenesis isbeneficial; treatment of myocardial infarction, coronary artery disease, heart failure; enhanced growth of hematopoietic cells, e.g. enhancement of hematopoietic stem cell transplants from bone marrow, mobilized peripheral blood, treatment of immunodeficiencies, graft versus host diseases, etc.; treatment of acute kidney injuries, chronic kidney diseases; treatment of lung diseases, chronic obstructive pulmonary diseases (COPD), pulmonary fibrosis, including idiopathic pulmonary fibrosis, enhanced regeneration of lung tissues. The compositions of the present invention may also be used in enhanced regeneration of liver cells, e.g. liver regeneration, treatment of cirrhosis, enhancement of liver transplantations, treatment of acute liver failure, treatment of chronic liver diseases with hepatitis C or B virus infection or post-antiviral drug therapies, alcoholic liver diseases, including alcoholic hepatitis, non-alcoholic liver diseases with steatosis or steatohepatitis, and the like. The compositions of this invention may treat diseases and disorders including, without limitation, conditions in which regenerative cell growth is desired.

[0220] Human genetics involving loss-of-function or gain-of-function mutations in WNT signaling components show strong evidence supporting enhancing WNT signals for bone growth. Conditions in which enhanced bone growth is desired may include, without limitation, fractures, grafts, ingrowth around prosthetic devices, osteoporosis, osteoporotic fractures, spinal fusion, vertebral compression fractures, pre-operative optimization for spinal surgeries, osteonecrosis of the jaw, dental implantation, periodontal diseases, maxillofacial reconstruction, and the like. WNT surrogate fusion molecules enhance and promotes WNT signals which are critical in promoting bone regeneration. Methods for regeneration of bone tissues benefit from administration of the compounds of the invention, which can be systemic or localized. In some embodiments, bone marrow cells are exposed to molecules of the invention, such that stem cells within that marrow become activated.

[0221] In some embodiments, bone regeneration is enhanced by contacting a responsive cell population, e.g. bone marrow, bone progenitor cells, bone stem cells, etc. with an effective dose of a WNT surrogate fusion molecule disclosed herein. Methods for regeneration of bone tissues benefit from administration of the WNT surrogate fusion molecule which can be systemic or localized. In some such embodiments, the contacting is performed in vivo. In other such embodiments, the contacting is performed ex vivo. The molecule may be localized to the site of action, e.g. by loading onto a matrix, which is optionally biodegradable, and optionally provides for a sustained release of the active agent. Matrix carriers include, without limitation, absorbable collagen sponges, ceramics, hydrogels, polymeric microspheres, nanoparticles, bone cements, and the like.

[0222] In particular embodiments, compositions and methods disclosed herein may be used to a retinopathy, optionally a retinal vascular disease (optionally caused by inhibition of vascular development or excessive angiogenesis) and / or optionally selected from the group consisting of exudative vitreoretinopathy, familiar exudative vitreoretinopathy (FEVR), retinopathy of prematurity, Norrie disease, diabetic retinopathy (DR), diabetic macular edema, diabetic macular ischemia, age-related macular degeneration (AMD) (including wet AMD and dry AMD), retinopathy of prematurity (ROP), osteoporosis-pseudoglioma syndrome (OPPG), retinal vein occlusion, and Coats disease; a vascular disorder, optionally vascular malformation or vascular insufficiency, further optionally associated with ischemia- induced neovascularization; a bone disease or bone injury, optionally selected from the group consisting of osteoporosis (including osteoporosis pseudoglioma (OPPG) syndrome and juvenile primary osteoporosis), bone mineral density variability, and cancer-associated osteolysis; Uveitis, NIU, UME, ARN acute retinal necrosis, non-paraneoplastic autoimmune retinopathy (npAIR), non parabolic autoimmune retinopathy, cataract surgery a muscle wasting disease, optionally selected from the group consisting of sarcopenia, cachexia, and muscular dystrophies; and / or a neural or neurodegenerative disease, optionally selected from the group consisting of stroke, traumatic brain injury, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Friedreich ataxia, Lewy body disease, spinal muscular atrophy, motor neuron disease, multiple sclerosis, Batten disease, and Creutzfeldt-Jakob disease.10223] Methods disclosed herein, including methods for treating or preventing a bone disease or disorder include methods that comprise providing to a subject in need thereof both a WNT surrogate fusion molecule and an antiresorptive agent. In certain embodiments, the methods are used for the treatment of osteoporosis, optionally post-menopausal osteoporosis.

[0224] The disclosure also provides a method for inhibiting or reducing bone resorption in a subject in need thereof, comprising providing to the subject an effective amount of a WNT surrogate fusion molecule, wherein the WNT surrogate fusion molecule is an agonist of a WNT signaling pathway. In certain embodiments, the method further comprises providing to the subject an antiresorptive agent. In certain embodiments, the subject has been diagnosed with or is at risk for osteoporosis, optionally postmenopausal osteoporosis. A variety of antiresorptive agents are known in the art and include, but are not limited to, those disclosed herein.

[0225] When a WNT surrogate fusion molecule is provide to the subject in combination with another therapeutic agent, such as an antiresorptive agent, the two agent may beprovided in the same or different pharmaceutical compositions. They may be provided to the subject at the same time, at different times, e.g., simultaneously, consecutively, or during overlapping or non-overlapping time periods. In certain embodiments, the two agents are therapeutically active in the subject during an overlapping time period.

[0226] Compositions comprising one or more WNT surrogate fusion molecule disclosed herein (or a polynucleotide encoding a WNT surrogate fusion molecule, or a vector or cell comprising a polynucleotide encoding a WNT surrogate fusion molecule) can be used for the in vivo treatment of skeletal tissue deficiencies. By "skeletal tissue deficiency", it is meant a deficiency in bone or other skeletal connective tissue at any site where it is desired to restore the bone or connective tissue, no matter how the deficiency originated, e.g. whether as a result of surgical intervention, removal of tumor, ulceration, implant, fracture, or other traumatic or degenerative conditions. The compositions of the present invention can be used as part of a regimen for restoring cartilage function to a connective tissue, for the repair of defects or lesions in cartilage tissue such as degenerative wear and arthritis, trauma to the tissue, displacement of torn meniscus, meniscectomy, a luxation of a joint by a torn ligament, malalignment of joints, bone fracture, or by hereditary disease.102271 A WNT surrogate fusion molecule may also be used for treatment of periodontal diseases. Periodontal diseases are a leading cause of tooth loss and are linked to multiple systemic conditions. In some embodiments, tooth or underlying bone regeneration is enhanced by contacting a responsive cell population. In some such embodiments, the contacting is performed in vivo. In other such embodiments, the contacting is performed ex vivo, with subsequent implantation of the activated stem or progenitor cells. The molecule may be localized to the site of action, e.g. by loading onto a matrix, which is optionally biodegradable, and optionally provides for a sustained release of the active agent. Matrix carriers include, without limitation, absorbable collagen sponges, ceramics, hydrogels, bone cements, polymeric microspheres, nanoparticles, and the like.

[0228] Studies have shown that biology of WNT signaling and R-spondins are capable of promoting sensory hair cell regeneration in the inner ear following injuries, aging, or degeneration. Loss of sensory hair cells in the inner ear involved in hearing loss or vestibular hypofunction may also benefit from the compositions of the invention. In the inner ear, the auditory organ houses mechanosensitive hair cells required for translating sound vibration to electric impulses. The vestibular organs, comprised of the semicircular canals (SSCs), the utricle, and the saccule, also contain sensory hair cells in order to detect head position and motion. Compositions of the present invention can be used, for example, in an infusion; in amatrix or other depot system; or other topical application to the ear for enhancement of auditory regeneration.102291 A WNT surrogate fusion molecule may also be used in regeneration of retinal tissue. In the adult mammalian retina, Muller glia cells are capable of regenerating retinal cells, including photoreceptors, for example after neurotoxic injury in vivo. WNT signaling and enhancers of WNT signals can promote proliferation of Muller glia-derived retinal progenitors after damage or during degeneration. The compositions of the invention may also be used in the regeneration of tissues and other cell types in the eye. For examples age-related macular degeneration (AMD), other retina degenerative diseases, cornea diseases, Fuchs’ dystrophy, vitreoretinopathy, hereditary diseases, etc. can benefit from the compositions of the present inventions. AMD is characterized by progressively decreased central vision and visual acuity. Fuchs’ dystrophy is characterized by progressive loss of cornea endothelial cells. WNT signal and enhancing of WNT signal can promote regeneration of cornea endothelium, retina epithelium, etc. in the eye tissue. In other embodiments, compositions of the present invention can be used, for example, in an infusion; in a matrix or other depot system; or other topical application to the eye for retinal regeneration and treatment of macular degeneration.[0230| Specific populations of proliferating cells for homeostatic renewal of hepatocytes have been identified through lineage tracing studies, for example Axin2-positive cells in pericentral region. Lineage tracing studies also identified additional potential liver progenitor cells, including but not limited to Lgr-positive cells. The self-renewing liver cells and other populations of potential progenitor cells, including Lgr5-positive and Axin2-positive cells, are identified to be capable of regeneration responding to WNT signals and / or R-spondins following injuries. Numerous preclinical models of acute liver injury and failure and chronic liver diseases showed recovery and regeneration of hepatocytes benefit from enhancing WNT signals.[0231 | In certain embodiments, compositions comprising a WNT surrogate fusion molecule disclosed herein (or a polynucleotide encoding a WNT surrogate fusion molecule, or a vector or cell comprising a polynucleotide encoding a WNT surrogate fusion molecule) are used to promote liver regeneration, reduce fibrosis, and / or improve liver function. In certain embodiments, compositions and methods disclosed herein are used to: increase liver weight, increase the liver to body weight ratio, increase the number of PCNA and pH3 positive nuclei in liver, increase expression of Ki67 and / or Cyclin DI in liver, increase livercell proliferation and / or mitosis, decrease fibrosis following chronic liver injury, or increase hepatocyte function.102321 In particular embodiments, the compositions of this invention may be used in treatment of acute liver failure, acute alcoholic liver injuries, treatment of chronic liver diseases with hepatitis C or B virus infection or post-antiviral drug therapies, chronic alcoholic liver diseases, alcoholic hepatitis, non-alcoholic fatty liver diseases and nonalcoholic steatohepatitis (NASH), treatment of cirrhosis and severe chronic liver diseases of all causes, and enhanced regeneration of liver cells. Methods for regeneration of liver tissue benefit from administration of the compounds of the invention, which can be systemic or localized. These include, but are not limited to, methods of systemic administration and methods of localized administration e.g. by injection into the liver tissue, by injection into veins or blood vessels leading into the liver, by implantation of a sustained release formulation, and the like.

[0233] In particular embodiments, compositions comprising a WNT surrogate fusion molecule disclosed herein (or a polynucleotide encoding a WNT surrogate fusion molecule, or a vector or cell comprising a polynucleotide encoding a WNT surrogate fusion molecule) are used to treat or prevent a liver disease or disorder, including but not limited to, or to treat or prevent a liver injury or disorder resulting from any of the following: acute liver failure (all causes), chronic liver failure (all causes), cirrhosis, liver fibrosis (all causes), portal hypertension, alcoholic liver diseases including alcoholic hepatitis, nonalcoholic steatohepatisis (NASH), nonalcoholic fatty liver disease (NAFLD) (fatty liver), alcoholic hepatitis, hepatitis C virus-induced liver diseases (HCV), hepatitis B virus-induced liver diseases (HBV), other viral hepatitis (e.g., hepatitis A virus-induced liver diseases (HAV) and hepatitis D virus-induced liver diseases (HDV)), primary biliary cirrhosis, autoimmune hepatitis, livery surgery, liver injury, liver transplantation, “small for size” syndrome in liver surgery and transplantation, congenital liver disease and disorders, any other liver disorder or detect resulting from genetic diseases, degeneration, aging, drugs, or injuries.

[0234] WNT signals play an important role in regeneration of various epithelial tissues. Various epidermal conditions benefit from treatment with the compounds of the present invention. Mucositis occurs when there is a breakdown of the rapidly divided epithelial cells lining the gastro-intestinal tract, leaving the mucosal tissue open to ulceration and infection. The part of the epithelial lining that covers the mouth, called the oral mucosa, is one of the most sensitive parts of the body and is particularly vulnerable to chemotherapy and radiation. Oral mucositis is probably the most common, debilitating complication of cancer treatments,particularly chemotherapy and radiation. In addition, the compositions of the invention may also benefit treatment of short bowel syndrome, inflammatory bowel diseases (IBD), or other gastrointestinal disorders. Other epidermal conditions include epidermal wound healing, diabetic foot ulcers, syndromes involving tooth, nail, or dermal hypoplasia, and the like. Molecules of the present invention may be used in all these conditions, where regenerative cells are contacted with compounds of the invention. Methods for regeneration of epithelial tissues benefit from administration of the compounds of the invention, which can be systemic or localized. Contacting can be, for example, topical, including intradermal, subdermal, in a gel, lotion, cream etc. applied at targeted site, etc.

[0235] In addition to skin and gastrointestinal tract, WNT signals and enhancement and promotion of WNT signals also play an important role in repair and regeneration of tissues including pancreas, kidney, and lung in preclinical models. A WNT surrogate fusion molecule may benefit various disease conditions involving exocrine and endocrine pancreas, kidney, or lung. The WNT surrogate fusion molecules may be used in treatment of metabolic syndrome; treatment of diabetes, treatment of acute or chronic pancreatitis, exocrine pancreatic insufficiency, treatment of acute kidney injuries, chronic kidney diseases, treatment of lung diseases, including but not limited to chronic obstructive pulmonary diseases (COPD), pulmonary fibrosis, in particular idiopathic pulmonary fibrosis (IPF), and other conditions that cause loss of lung epithelial tissues. Methods for regeneration of these tissues benefit from administration of the compounds of the invention, which can be systemic or localized.

[0236] Epidermal WNT signaling, in coordination with signaling via other development factors, is critical for adult hair follicle regeneration. Hair loss is a common problem, and androgenetic alopecia, often called male pattern baldness, is the most common form of hair loss in men. In some embodiments, hair follicle regeneration is enhanced by contacting a responsive cell population with a molecule of the present invention. In some such embodiments, the contacting is performed in vivo. In other such embodiments, the contacting is performed ex vivo. The molecule may be localized to the site of action, e.g. topical lotions, gels, creams and the like.

[0237] Stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis and other conditions affecting the blood brain barrier (BBB) may be treated with a WNT surrogate fusion molecule. Angiogenesis is critical to ensure the supply of oxygen and nutrients to many tissues throughout the body, and is especially important for the CNS as the neural tissue is extremely sensitive to hypoxia and ischemia. CNS endothelial cells which form theBBB differ from endothelial cells in non-neural tissue, in that they are highly polarized cells held together by tight junctions and express specific transporters. WNT signaling regulates CNS vessel formation and / or function. Conditions in which the BBB is compromised can benefit from administration of the compounds of the invention, which can be systemic or localized e.g. by direct injection, intrathecal administration, implantation of sustained release formulations, and the like. In addition, WNT signal is actively involved in neurogenesis and plays a role of neuroprotection following injury. The compositions of the present invention may also be used in treatment of spinal cord injuries, other spinal cord diseases, stroke, traumatic brain injuries, etc.

[0238] WNT signals also play a role in angiogenesis. A WNT surrogate fusion molecule may benefit conditions where angiogenesis is beneficial, treatment of myocardial infarction, coronary artery disease, heart failure, diabetic retinopathy, etc., and conditions from hereditary diseases. Methods for regeneration of these tissues benefit from administration of the compounds of the invention, which can be systemic or localized.

[0239] In certain embodiments, methods of the present invention promote tissue regeneration, e.g., in a tissue subjected to damage or tissue or cell reduction or loss. The loss or damage can be anything which causes the cell number to diminish, including diseases or injuries. For example, an accident, an autoimmune disorder, a therapeutic side-effect or a disease state could constitute trauma. Tissue regeneration increases the cell number within the tissue and preferably enables connections between cells of the tissue to be re-established, and more preferably the functionality of the tissue to be regained.

[0240] The terms "administering" or "introducing" or “providing”, as used herein, refer to delivery of a composition to a cell, to cells, tissues and / or organs of a subject, or to a subject. Such administering or introducing may take place in vivo, in vitro or ex vivo.

[0241] In particular embodiments, a pharmaceutical composition is administered parenterally, e.g., intravenously, orally, rectally, or by injection. In some embodiments, it is administered locally, e.g., topically or intramuscularly. In some embodiments, a composition is administered to target tissues, e.g., to bone, joints, ear tissue, eye tissue, gastrointestinal tract, skin, a wound site or spinal cord. Methods of the invention may be practiced in vivo or ex vivo. In some embodiments, the contacting of a target cell or tissue with a WNT surrogate fusion molecule is performed ex vivo, with subsequent implantation of the cells or tissues, e.g., activated stem or progenitor cells, into the subject. The skilled artisan can determine an appropriate site of and route of administration based on the disease or disorder being treated.

[0242] The dose and dosage regimen may depend upon a variety of factors readily determined by a physician, such as the nature of the disease or disorder, the characteristics of the subject, and the subject's history. In particular embodiments, the amount of a WNT surrogate fusion molecule administered or provided to the subject is in the range of about 0.01 mg / kg to about 50 mg / kg, 0.1 mg / kg to about 500 mg / kg, or about 0.1 mg / kg to about 50 mg / kg of the subject’s body weight.

[0243] The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof, e.g. reducing the likelihood that the disease or symptom thereof occurs in the subject, and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease. The therapeutic agent (e.g., a WNT surrogate fusion molecule) may be administered before, during or after the onset of disease or injury. The treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues. The subject therapy will desirably be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease. In some embodiments, the subject method results in a therapeutic benefit, e.g., preventing the development of a disorder, halting the progression of a disorder, reversing the progression of a disorder, etc. In some embodiments, the subject method comprises the step of detecting that a therapeutic benefit has been achieved. The ordinarily skilled artisan will appreciate that such measures of therapeutic efficacy will be applicable to the particular disease being modified, and will recognize the appropriate detection methods to use to measure therapeutic efficacy.

[0244] Other embodiments relate, in part, to the use of the WNT surrogate fusion molecules disclosed herein to promote or enhance the growth or proliferation of cells, tissues and organoids, for example, by contacting cells or tissue with one or more WNT surrogate, optionally in combination with a Norrin or Rspondin polypeptide. In certain embodiments, the cells or tissue are contacted ex vivo, in vitro, or in vivo. Such methods may be used to generate cells, tissue or organoids for therapeutic use, e.g., to be transplanted or grafted into asubject. They may also be used to generate cells, tissue or organoids for research use. The WNT surrogate fusion molecules have widespread applications in non-therapeutic methods, for example in vitro research methods.

[0245] The invention provides a method for tissue regeneration of damaged tissue, such as the tissues discussed above, comprising administering a WNT surrogate fusion molecule to cells. The WNT surrogate fusion molecule may be administered directly to the cells in vivo, administered to a subject orally, intravenously, or by other methods known in the art, or administered to ex vivo cells. In some embodiments where the WNT surrogate fusion molecule is administered to ex vivo cells, these cells may be transplanted into a subject before, after or during administration of the WNT surrogate fusion molecule.

[0246] WNT signaling is a key component of stem cell culture. For example, the stem cell culture media as described in W02010 / 090513, W02012 / 014076, Sato et al., 2011 (GASTROENTEROLOGY 201 1; 141 : 1762-1772) and Sato et al., 2009 (Nature 459, 262- 5). The WNT surrogate fusion molecules disclosed herein are suitable alternatives to Rspondin for use in these stem cell culture media, or may be combined with Rspondin.

[0247] Accordingly, in one embodiment, the disclosure provides a method for enhancing the proliferation of stem cells comprising contacting stem cells with one or more WNT surrogate fusion molecules disclosed herein. In one embodiment, the disclosure provides a cell culture medium comprising one or more WNT surrogate fusion molecules disclosed herein. In some embodiments, the cell culture medium may be any cell culture medium already known in the art that normally comprises WNT or Rspondin, but wherein the WNT or Rspondin is replaced (wholly or partially) or supplemented by WNT surrogate fusion molecule(s) disclosed herein. For example, the culture medium may be as described in as described in WO2010 / 090513, WO2012 / 014076, Sato et al., 2011 (GASTROENTEROLOGY 201 1; 141 : 1762-1772) and Sato et al., 2009 (Nature 459, 262- 5), which are hereby incorporated by reference in their entirety.

[0248] Stem cell culture media often comprise additional growth factors. This method may thus additionally comprise supplying the stem cells with a growth factor. Growth factors commonly used in cell culture medium include epidermal growth factor (EGF, (Peprotech), Transforming Growth Factor-alpha (TGF-alpha, Peprotech), basic Fibroblast Growth Factor (bFGF, Peprotech), brain-derived neurotrophic factor (BDNF, R&D Systems), Hepatocyte Growth Factor (HGF) and Keratinocyte Growth Factor (KGF, Peprotech, also known as FGF7). EGF is a potent mitogenic factor for a variety of cultured ectodermal and mesodermal cells and has a profound effect on the differentiation of specific cells in vivo and in vitro andof some fibroblasts in cell culture. The EGF precursor exists as a membrane-bound molecule which is proteolytically cleaved to generate the 53-amino acid peptide hormone that stimulates cells. EGF or other mitogenic growth factors may thus be supplied to the stem cells. During culturing of stem cells, the mitogenic growth factor may be added to the culture medium every second day, while the culture medium is refreshed preferably every fourth day. In general, a mitogenic factor is selected from the groups 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 a FGF; vii) TGF-alpha and KGF; viii) TGF-alpha, and FGF7; ix) or from TGF-alpha and a FGF. In certain embodiments, the disclosure includes a stem cell culture media comprising a WNT surrogate fusion molecule disclosed herein, e.g., optionally in combination with one or more of the growth factors or combinations thereof described herein.

[0249] These methods of enhancing proliferation of stem cells can be used to grow new organoids and tissues from stem cells, as for example described in WO2010 / 090513 WO20 12 / 014076, Sato et al., 201 1 (GASTROENTEROLOGY 2011; 141 : 1762-1772) and Sato et al., 2009 (Nature 459, 262-5).

[0250] In some embodiments, the WNT surrogate fusion molecules are used to enhance stem cell regeneration. Illustrative stem cells of interest include but are not limited to: muscle satellite cells; hematopoietic stem cells and progenitor cells derived therefrom (U.S. Pat. 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; liver stem cells; adipose-tissue derived stem cells, etc.

[0251] Other embodiments of the present invention relate, in part, to diagnostic applications for detecting the presence of cells or tissues expressing one or more FZD receptors or LRP5 or LRP6 receptors. Thus, the present disclosure provides methods of detecting one or more FZD receptor or LRP5 or LRP6 receptor in a sample, such as detection of cells or tissues expressing FZD1. Such methods can be applied in a variety of known detection formats, including, but not limited to immunohistochemistry (H4C), immunocytochemistry (ICC), in situ hybridization (ISH), whole-mount in situ hybridization (WISH), fluorescent DNA in situ hybridization (FISH), flow cytometry, enzyme immunoassay (EIA), and enzyme linked immuno-assay (ELISA), e.g., by detecting binding of a WNT surrogate fusion molecule.

[0252] ISH is a type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., primary binding agent) to localize a specific DNA or RNA sequence in a portionor section of a cell or tissue (in situ), or if the tissue is small enough, the entire tissue (whole mount ISH). One having ordinary skill in the art would appreciate that this is distinct from immunohistochemistry, which localizes proteins in tissue sections using an antibody as a primary binding agent. DNA ISH can be used on genomic DNA to determine the structure of chromosomes. Fluorescent DNA ISH (FISH) can, for example, be used in medical diagnostics to assess chromosomal integrity. RNA ISH (hybridization histochemistry) is used to measure and localize mRNAs and other transcripts within tissue sections or whole mounts.[02531 In various embodiments, the WNT surrogate fusion molecules described herein are conjugated to a detectable label that may be detected directly or indirectly. In this regard, an antibody "conjugate" refers to a WNT surrogate fusion molecule that is covalently linked to a detectable label. In the present invention, DNA probes, RNA probes, monoclonal antibodies, antigen-binding fragments thereof, and antibody derivatives thereof, such as a single-chain-variable-fragment antibody or an epitope tagged antibody, may all be covalently linked to a detectable label. In “direct detection”, only one detectable antibody is used, i.e., a primary detectable antibody. Thus, direct detection means that the antibody that is conjugated to a detectable label may be detected, per se, without the need for the addition of a second antibody (secondary antibody).

[0254] A "detectable label" is a molecule or material that can produce a detectable (such as visually, electronically or otherwise) signal that indicates the presence and / or concentration of the label in a sample. When conjugated to an antibody, the detectable label can be used to locate and / or quantify the target to which the specific antibody is directed. Thereby, the presence and / or concentration of the target in a sample can be detected by detecting the signal produced by the detectable label. A detectable label can be detected directly or indirectly, and several different detectable labels conjugated to different specificantibodies can be used in combination to detect one or more targets.

[0255] Examples of detectable labels, which may be detected directly, include fluorescent dyes and radioactive substances and metal particles. In contrast, indirect detection requires the application of one or more additional antibodies, i.e., secondary antibodies, after application of the primary antibody. Thus, the detection is performed by the detection of the binding of the secondary antibody or binding agent to the primary detectable antibody. Examples of primary detectable binding agents or antibodies requiring addition of a secondary binding agent or antibody include enzymatic detectable binding agents and hapten detectable binding agents or antibodies.

[0256] In some embodiments, the detectable label is conjugated to a nucleic acid polymer which comprises the first binding agent (e.g., in an ISH, WISH, or FISH process). In other embodiments, the detectable label is conjugated to an antibody which comprises the first binding agent (e.g., in an IHC process).

[0257] Examples of detectable labels which may be conjugated to WNT surrogate fusion molecules used in the methods of the present disclosure include fluorescent labels, enzyme labels, radioisotopes, chemiluminescent labels, electrochemiluminescent labels, bioluminescent labels, polymers, polymer particles, metal particles, haptens, and dyes.

[0258] Examples of fluorescent labels include 5-(and 6)-carboxyfluorescein, 5- or 6- carboxyfluorescein, 6-(fluorescein)-5-(and 6)-carboxamido hexanoic acid, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, and dyes such as Cy2, Cy3, and Cy5, optionally substituted coumarin including AMCA, PerCP, phycobiliproteins including R- phycoerythrin (RPE) and allophycoerythrin (APC), Texas Red, Princeton Red, green fluorescent protein (GFP) and analogues thereof, and conjugates of R-phycoerythrin or allophycoerythrin, inorganic fluorescent labels such as particles based on semiconductor material like coated CdSe nanocrystallites.10259] Examples of polymer particle labels include micro particles or latex particles of polystyrene, PMMA or silica, which can be embedded with fluorescent dyes, or polymer micelles or capsules which contain dyes, enzymes or substrates.

[0260] Examples of metal particle labels include gold particles and coated gold particles, which can be converted by silver stains. Examples of haptens include DNP, fluorescein isothiocyanate (FITC), biotin, and digoxigenin. Examples of enzymatic labels include horseradish peroxidase (HRP), alkaline phosphatase (ALP or AP), P-galactosidase (GAL), glucose-6-phosphate dehydrogenase, P-N-acetylglucosamimidase, P-glucuronidase, invertase, Xanthine Oxidase, firefly luciferase and glucose oxidase (GO). Examples of commonly used substrates for horseradishperoxidase include 3, 3 '-diaminobenzidine (DAB), diaminobenzidine with nickel enhancement, 3-amino-9-ethylcarbazole (AEC), Benzidine dihydrochloride (BDHC), Hanker- Yates reagent (HYR), Indophane blue (IB), tetramethylbenzidine (TMB), 4-chloro-l-naphtol (CN), . alpha. -naphtol pyronin (.alpha.-NP), o-dianisidine (OD), 5-bromo-4-chloro-3-indolylphosp- hate (BCIP), Nitro blue tetrazolium (NBT), 2-(p-iodophenyl)-3-p-nitropheny- 1-5-phenyl tetrazolium chloride (INT), tetranitro blue tetrazolium (TNBT), 5-bromo-4-chloro-3-indoxyl-beta-D-galactoside / ferro-ferricyanide (BCIG / FF).

[0261] Examples of commonly used substrates for Alkaline Phosphatase include Naphthol- AS-B 1-phosphate / fast red TR (NABP / FR), Naphthol -AS-MX-phosphate / fast red TR (NAMP / FR), Naphthol-AS-Bl -phosphate / - fast red TR (NABP / FR), Naphthol-AS-MX- phosphate / fast red TR (NAMP / FR), Naphthol-AS-Bl -phosphate / new fuschin (NABP / NF), bromochloroindolyl phosphate / nitroblue tetrazo lium (BCIP / NBT), 5-Bromo-4-chloro-3- indolyl-b— d-galactopyranoside (BCIG).

[0262] Examples of luminescent labels include luminol, isoluminol, acridinium esters, 1,2-dioxetanes and pyridopyridazines. Examples of electrochemiluminescent labels include ruthenium derivatives. Examples of radioactive labels include radioactive isotopes of iodide, cobalt, selenium, tritium, carbon, sulfur and phosphorous.

[0263] Detectable labels may be linked to the antibodies described herein or to any other molecule that specifically binds to a biological marker of interest, e.g., an antibody, a nucleic acid probe, or a polymer. Furthermore, one of ordinary skill in the art would appreciate that detectable labels can also be conjugated to second, and / or third, and / or fourth, and / or fifth binding agents or antibodies, etc. Moreover, the skilled artisan would appreciate that each additional binding agent or antibody used to characterize a biological marker of interest may serve as a signal amplification step. The biological marker may be detected visually using, e.g., light microscopy, fluorescent microscopy, electron microscopy where the detectable substance is for example a dye, a colloidal gold particle, a luminescent reagent. Visually detectable substances bound to a biological marker may also be detected using a spectrophotometer. Where the detectable substance is a radioactive isotope detection can be visually by autoradiography, or non-visually using a scintillation counter. See, e.g., Larsson, 1988, Immunocytochemistry: Theory and Practice, (CRC Press, Boca Raton, Fla.); Methods in Molecular Biology, vol. 80 1998, John D. Pound (ed.) (Humana Press, Totowa, N.J.).

[0264] The invention further provides kits for detecting one or more FZD or LRP5 / 6 receptor or cells or tissues expressing one or more FZD or LRP5 / 6 receptors in a sample, wherein the kits contain at least one antibody, polypeptide, polynucleotide, vector or host cell as described herein. In certain embodiments, a kit may comprise buffers, enzymes, labels, substrates, beads or other surfaces to which the antibodies of the invention are attached, and the like, and instructions for use.EXAMPLESExample 1 Illustrative WNT Surrogate Fusion Formats

[0265] WNT surrogate fusion molecules having a variety of different formats disclosed herein were made. These included the following illustrative formats, each comprising a binding domain (“binder”) that binds to one or more FZD receptor, binding domain (“binder”) that binds to an LRP5 and / or LRP6 receptor, and binding domain (“binder”) that binds to an cytokine ligand and / or cytokine receptor .

[0266] Figure 1 shows illustrative formats of WNT surrogate fusion molecules, wherein LRP 5 / 6 binders are VHH (anti-antigen 1), FZD binders are Fv (anti-antigen 2), and cytokine binders are IgG (anti-antigen 3). In certain instances, the LRP 5 / 6 binding domains are linked to the FZD binding domains; in other instances, the VHH binding domains are linked to the IgG binding domains, and the VHH binder can be fused to any of the 4 available termini of the IgG. For example, the VHH can be fused to the N-terminus of the IgG light chain (LC), N-terminus of the IgG heavy chain (HC), the C-terminus of LC, and / or the C- terminus of HC. The linkers and the length of the linkers between the IgG and the VHH can be varied. Fv binding domains may be linked to the IgG binding domains by N-terminus of HC or LC, C-terminus HC or LC, or the Fc region of IgG. These formats are trispecific. Figure 2 shows illustrative formats of WNT surrogate fusion molecules wherein LRP 5 / 6 binder(s) are VHH (anti-antigen 1), FZD binders are Fab (anti-antigen 2), and cytokine binder(s) are Fab (anti-antigen 3). In certain instances, the LRP 5 / 6 binding domains are linked to the FZD binding domains, and in other instances the VHH binding domains are linked to the Fab IL6 binding domains. The format may be trispecific hexavalent, and in some embodiments, trispecific pentavalent, with one LRP 5 / 6 binder, two FZD binders, and two IL6 binders. Alternatively, the WNT surrogate fusion molecules may comprise two LRP 5 / 6 binders, one FZD binder, and two cytokine binders, or alternatively, two LRP 5 / 6 binders, two FZD binders, and one cytokine factor binder. In some cases, the format is trispecific tetravalent with one LRP 5 / 6 binder, one cytokine binder, and two FZD binding domains.

[0267] Figure 5 describes illustrative WNT surrogate fusion molecules wherein the LRP 5 / 6 binder(s) are VHH (anti-antigen 1), the FZD binders are ScFv (anti-antigen 2), and the cytokine binder(s) are Fab (anti-antigen 3). In certain instances, the LRP5 / 6 binding domains are linked to the FZD binding domains; in other instances, the VHH binding domains are linked to the Fab cytokine binding domains.

[0268] A WNT surrogate IL6 fusion molecule is described in Figure 6, wherein two LRP 5 binders are linked to two N-termini of Fv FZD 4 binders, and the Fv FZD 4 binders are linked to two anti-IL6 IgG binders (Siltuximab) with LALAPG mutation, represented in SEQ 2 and 3 of Table 2.Example 2 Characterization of WNT Surrogate Fusion Molecule, 814-Siltuximab

[0269] The 814-Siltuximab molecule consists of the LRP5 binder VHH-03, the FZD 4 binder 4SD1 and the IL6 binder Siltuximab. Figure 6 shows the protein produced in Expi293Fcells(Thermo Fisher Scientific) by transient transfection. The fusion molecule was first purified using a Mab Select SuRe Protein A affinity column (Cytiva) on AKTA pure (Cytiva). The column was washed with 10 column volumes of 2xHBS buffer (40 mM HEPES pH 7.4, 300 mM NaCl) after protein binding; and the protein was eluted with elution buffer (100 mM glycine pH 3, 300 mM NaCl). The protein was further polished with HiLoad 16 / 600 Superdex 200 pg (Cytiva) size-exclusion chromatography (SEC) using 2xHBS buffer (40 mM HEPES pH 7.4, 300 mM NaCl) on AKTA pure (Cytiva). The protein was buffer exchanged into storage buffer (10 mM Histidine pH 6).Example 3Sequential Bindings of FZD4, LRP5 and hIL6 on the Trispecific 814-Siltuximab

[0270] The 814-Siltuximab molecule consists of the LRP5 binder VHH-03, the FZD 4 binder 4SD1, and the IL6 binder Siltuximab. Figure 7 shows sequential binding of FZD4, LRP5, and hIL6. The step-binding assay was performed with the bio-layer interferometry (BLI) using the Octet Red 96 instrument (PALL ForteBio) at 30°C and 1000 rpm with SA biosensors. Biotin-labeled FZD4 CRD proteins were diluted to 50 nM in the running buffer (PBS, 0.05% Tween-20, 0.5% BSA, 2mM CaC12, pH 7.2) and captured by the SA biosensor, followed by dipping into wells containing 50 nM 814-Siltuximab. The sensor chips next moved sequentially into 200 nM LRP5 LDLRA protein and 500 nM hIL6 (Sino Biological). Binding kinetics of hIL6 to 814-Siltuximab were determined by BLI using an Octet Red instrument at 30°C and 1000 rpm with AHC biosensors (Sartorius). 814-Siltuximab was diluted to 50 nM in the running buffer and captured by the AHC biosensor, followed by dipping into wells containing the hIL6 at different concentrations in a running buffer or into a well with only the running buffer as a reference channel. The dissociation of the interactionwas followed with the running buffer. The monovalent KD for each binder was calculated using Octet System software, based on fitting to a 1 : 1 binding model.Example 4STF and Gene Activity of 814-Siltuximab Fusion Molecules

[0271] Figure 8 illustrates the WNT signaling activity of 814-Siltuximab (814-aIL6) measured using bEnd.3 cells containing a luciferase-reporter gene controlled by a WNT- responsive promoter (Super Top Flash reporter assay, STF) (Chen at al., (2020) STAR Protocols, 1 : 100043). Cells were seeded at a density of 10,000 cells per well in 96-well plates and in the presence of 3 pM IWP2, then incubated for 24 hours. The recombinant protein was added to the cells and then incubated for another 18-24 hours. Cells were lysed with Luciferase Cell Culture Lysis Reagent (Promega), and luciferase activity was measured using the Luciferase Assay System (Promega) following vendor suggested procedures.

[0272] Figures 9A and 9B show the effect of 814-Siltuximab on gene expression of the WNT target gene, Axin2, and the IL6 target gene, Socs3. Mouse bEnd3 cells were treated for 24h with hIL6 (20 ng / mL) and / or a combination of SWAP WNT agonist molecules (10 nM). Controls consisted of medium only, L6-F4-2 is a FZD4, LRP5 / 6 SWAP WNT agonist. 814- aIL6 consists of anti-LRP5, anti-FZD4, and anti-IL6 (Siltuximab) antibodies. Figure 9C describes the role of 814-aIL6 on STAT3 phosporylation of IL6 binding. In short, 814-aIL6 retains WNT activating capacity, evident by Axin2 elevation, while blocking the effects of hIL6.Enumerated Embodiments

[0273] The disclosure includes, but is not limited to, the following enumerated embodiments.1. A WNT surrogate fusion molecule, wherein the WNT surrogate fusion molecule comprises: a) one or more low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or Low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) (LRP5 / LRP6) binding antibody or antigen-binding fragment that specifically binds to one or more LRP5 / 6 receptor (LRP5 / 6 antigen-binding region OR LRP5 / 6 binding region OR LRP5 / 6 binder), optionally wherein the LRP5 / 6 binding antibody or antigen binding fragment is an antigen-binding fragment of the LRP5 / 6 binding antibody; andb) one or more frizzled (FZD) binding antibody or antigen-binding fragment that specifically bind to one or more FZD receptor (FZD antigen-binding region or FZD binding region OR FZD binder), optionally wherein the FZD binding antibody or antigen binding fragment is an antigen-binding fragment of the FZD binding antibody; and c) one or more antibody or antigen-binding fragment that specifically bind to one or more cytokine receptor or cytokine ligand (cytokine ligand and / or cytokine receptor antigenbinding region OR cytokine factor binding region OR cytokine binder or cytokine receptor binder), optionally wherein the cytokine factor binding antibody or antigen binding fragment is an antigen-binding fragment of the cytokine factor binding antibody.2. The WNT surrogate fusion molecule of embodiment 1, wherein the WNT surrogate fusion molecule is tetravalent, pentavalent, or hexavalent.3. The WNT surrogate fusion molecule of embodiment 2, wherein the hexavalent surrogate molecule comprises: a) two low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or a Low- density lipoprotein (LDL) receptor-related protein 6 (LRP6) or LRP5 / LRP6 binding antibodies or antigen-binding fragments that specifically bind to two LRP5 / 6 receptors; and b) two frizzled (FZD) binding antibodies or antigen-binding fragments that specifically bind to two FZD receptors; and c) two cytokine binding antibodies or antigen-binding fragments that specifically bind to two cytokine receptors and / or cytokine ligands.4. The WNT surrogate fusion molecule of embodiment 2, wherein the pentavalent surrogate molecule comprises: a) one low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or a Low- density lipoprotein (LDL) receptor-related protein 6 (LRP6) or LRP5 / LRP6 binding antibody or antigen-binding fragment that specifically binds to one LRP5 / 6 receptor or two low- density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or a Low-density lipoprotein (LDL) receptors related protein 6 (LRP6) or LRP5 / LRP6 receptors; and / or b) two frizzled (FZD) binding antibodies or antigen-binding fragments that specifically bind to two FZD receptors; and c) two cytokine antibodies or antigen-binding fragments that specifically bind to two cytokine receptors and / or cytokine ligands.5. The WNT surrogate fusion molecule of embodiment 2, wherein the pentavalent surrogate molecule comprises: a) two low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or a Low- density lipoprotein (LDL) receptor-related protein 6 (LRP6) or LRP5 / LRP6 binding antibodies or antigen-binding fragments that specifically bind to two LRP5 / 6 receptors; and / or b) one frizzled (FZD) binding antibody or antigen-binding fragment that specifically binds to one FZD receptor; and c) two cytokine antibodies or antigen-binding fragments that specifically bind to two cytokine receptors and / or cytokine ligands.6. The WNT surrogate fusion molecule of embodiment 2, wherein the pentavalent surrogate molecule comprises: a) two low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or a Low- density lipoprotein (LDL) receptor-related protein 6 (LRP6) or LRP5 / LRP6 binding antibodies or antigen-binding fragments that specifically bind to two LRP5 / 6 receptors; and / or b) two frizzled (FZD) binding antibodies or antigen-binding fragments that specifically bind to two FZD receptors; and c) one cytokine antibody or antigen-binding fragment that specifically binds to a cytokine receptor and / or cytokine ligand.7. The WNT surrogate fusion molecule of embodiment 2, wherein the tetravalent surrogate molecule comprises: a) one low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or a Low- density lipoprotein (LDL) receptor-related protein 6 (LRP6) or LRP5 / LRP6 binding antibody or antigen-binding fragment that specifically binds to one LRP5 / 6 receptor; and b) two frizzled (FZD) binding antibodies or antigen-binding fragments that specifically bind to one or more FZD receptor; and c) one cytokine antibody or antigen-binding fragment that specifically binds to one cytokine receptor and / or cytokine ligand.8. The WNT surrogate fusion molecule of any of embodiments 1-7, wherein the antigenbinding fragments are selected from the group consisting of: IgG, scFv, Fab, VHH or single domain antibodies (sdAb), diabody, and Fv.9. The WNT surrogate fusion molecule of any of embodiments 1-7, wherein the LRP5 / 6 antigen-binding region comprises a VHH or sdAb, a scFv, a Fab, a Fv, or a diabody.10. The WNT surrogate fusion molecule of any of embodiments 1-7, wherein the FZD antigen-binding region comprises a VHH or sdAb, a scFv, a Fab, a Fv, or a diabody.11. The WNT surrogate fusion molecule of any of embodiments 1-7, wherein the cytokine receptor or cytokine antigen-binding region comprises a VHH or sdAb, a scFv, a Fab, a Fv, or a diabody.12. The WNT surrogate fusion molecule of embodiments 8-11, wherein the Fab is present within a full immunoglobulin (Ig), optionally an IgG, comprising a light chain and a heavy chain.13. The WNT surrogate fusion molecule of any of embodiments 1-12, wherein each of the LRP5 / 6 binding regions, FZD binding regions, and cytokine receptor and / or ligand is fused or linked to another binding region via a linker moiety.14. The WNT surrogate fusion molecule of any of embodiments 1-13, wherein the one or more FZD binding regions bind to one or more FZD 4 receptors.15. The WNT surrogate fusion molecule of any of embodiments 1-14, wherein the cytokine antibodies or antigen-binding fragments specifically bind to an interleukin 6 (IL6) IL6 or an IL6 receptor.16. The WNT surrogate fusion molecule of embodiment 15, wherein the IL6 receptor and / or IL6 ligand binding domain comprises siltuximab, sirukumab, olokizumab,clazakinumab, gerilimzumab, Tocilizumab, Sarilumab, Vamikibart, or Satralizumab, or a variant or fragment thereof that specifically binds the IL6 receptor and / or IL6 ligand.17. A multivalent fusion molecule, wherein the molecule comprises: a) a first binding domain that comprises one or more antibody or antigen-binding fragment thereof that specifically binds to one or more first antigen, wherein the antigen-binding fragment comprises a first Fab; b) a second binding domain that comprises one or more antibody or antigen-binding fragment thereof that specifically binds to one or more second antigen, wherein the antigen-binding fragment comprises a second Fab; and c) a third binding domain that comprises one or more antibody or antigen-binding fragment thereof that specifically bind to one or more third antigen, wherein the antigen-binding fragment comprises a third Fab; and wherein the first Fab heavy chain (HC) is fused to the second Fab HC, and the third Fab HC is fused to the second Fab light chain (LC), or wherein the first Fab LC is fused to the second Fab HC, and the third Fab LC is fused to the second Fab LC; and wherein the Fabs are fused or linked to each other via a linker moiety.18. The multivalent fusion molecule of embodiments 17, wherein: a) the first binding domain comprises one or more LRP 5 / 6 binding antibody or antigenbinding fragment thereof that specifically binds to one or more LRP5 and / or LRP6 receptor; and b) the second binding domain comprises one or more FZD binding antibody or antigenbinding fragment thereof that specifically bind to one or more FZD receptors; and c) the third binding domain comprises one or more antibody or antigen-binding fragment thereof that specifically binds to one or more cytokine receptor and / or cytokine ligand.19. A nucleic acid or a combination of nucleic acids encoding the fusion molecule of any one of embodimentss 1-18, optionally wherein the nucleic acid(s) is / are DNA, cDNA, RNA, mRNA, modified mRNA, or a DNA / RNA hybrid.20. The nucleic acid or combination of nucleic acids of embodiment 19, which encode the the fusion molecule of any one of embodiments 1-18 and comprises: a) a first nucleic acid encoding the LRP5 / LRP6 binding antibody or antigen-binding fragment; b) a second nucleic acid encoding the FZD binding antibody or antigen-binding fragment; and c) a third nucleic acid encoding the antibody or antigen-binding fragment that specifically binds to one or more cytokine receptor or cytokine ligand.21. A vector or a combination of vectors, which comprises the nucleic acid or the combination of nucleic acids of embodiments 19 or 20, optionally wherein: a) the vector(s) comprise(s) one or more promoters operably linked to the nucleic acid(s); b) the vector(s) is / are an expression vector; and / or c) the vector(s) comprise(s) a plasmid, a viral vector (optionally an adeno-associated virus, adenovirus, lentivirus, or retrovirus vector), a lipid-based vector, a self-replicating RNA vector, a virus-like particle, a polymer-based vector, and / or a nanoparticle, optionally a lipid- based nanoparticle.22. A host cell, which comprises: a) the WNT surrogate fusion molecule of any one of embodiments 1-18; b) the nucleic acid or the combination of nucleic acids of embodiments 19 or 20; and / or c) the vector or the combination of vectors of embodiment 21, optionally wherein the host cell is:(i) mammalian, optionally human, non-human primate, monkey, rabbit, rodent, hamster, rat, or mouse; or(ii) non-mammalian, optionally plant, bacterial, fungal, yeast, protozoa, or insect, and optionally wherein the host cell is:(i) a human embryonal kidney (HEK) cell, optionally a HEK293 cell, or a variant thereof, further optionally Expi293F™ cell;(ii) a CHO (Chinese Hamster Ovary) cell;(iii) an immune cell or(iv) a hybridoma.23. A pharmaceutical composition, which comprises: a) the fusion molecule of any one of embodiments 1-18; the nucleic acid or the combination of nucleic acids of embodiments 19 or 20; the vector or the combination of vectors of embodiment 21; and / or the host cell of embodiment 22; and b) a pharmaceutically acceptable carrier and / or excipient.24. A method for agonizing, inducing, promoting, stimulating, enhancing, and / or supporting a WNT signaling in a target cell of a subject, comprising administering to the subject an effective amount of a) the WNT surrogate fusion molecule of any one of embodiments 1-18; b) the nucleic acid or the combination of nucleic acids of embodiments 19 or 20; c) the vector of embodiment 21; d) the host cell of embodiment 20 or the population of cells of embodiment 22; and / or e) the pharmaceutical composition of embodiment 23, optionally wherein:(i) the subject is a mammal, optionally a human, a non-human primate;(ii) the subject comprises or has a risk of developing a disease, disorder, or a condition; and / or(iii) the method further comprises administering to the subject an additional agent, optionally an adjuvant or a therapeutic agent, and optionally wherein:(i) the WNT signaling is or comprises WNT / beta-catenin signaling; and / or(ii) the method promotes, stimulates, enhances, and / or supports recruitment of multiple receptor complexes each comprising FZD; LRP5 / 6; and cytokine receptor or ligand.25. The method of embodiment 24, wherein the disease, disorder, or condition comprises one or more of the following: a) a retinopathy, optionally a retinal vascular disease (optionally caused by inhibition of vascular development or excessive angiogenesis) and / or optionally selected from the group consisting of exudative vitreoretinopathy, familiar exudative vitreoretinopathy(FEVR), retinopathy of prematurity, Norrie disease, diabetic retinopathy (DR), diabetic macular edema, diabetic macular ischemia, age-related macular degeneration (AMD) (including wet AMD and dry AMD), retinopathy of prematurity (ROP), osteoporosis-pseudoglioma syndrome (OPPG), retinal vein occlusion, and Coats disease; b) a vascular disorder, optionally vascular malformation or vascular insufficiency, further optionally associated with ischemia-induced neovascularization; c) a bone disease or bone injury, optionally selected from the group consisting of osteoporosis (including osteoporosis pseudoglioma (OPPG)syndrome and juvenile primary osteoporosis), bone mineral density variability, and cancer-associated osteolysis; d) uveitis, NIU, UME, ARN acute retinal necrosis, npAIR non parabolic autoimmune retinopathy, cataract surgery; e) a muscle wasting disease, optionally selected from the group consisting of sarcopenia, cachexia, and muscular dystrophies; and / or f) a neural or neurodegenerative disease, optionally selected from the group consisting of stroke, traumatic brain injury, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Friedreich ataxia, Lewy body disease, spinal muscular atrophy, motor neuron disease, multiple sclerosis, Batten disease, and Creutzfeldt-Jakob disease.26. The method of any one of embodiments 24 and 25, wherein the administering is:(i) to one or more of the subject's eyes, optionally intravitreally or via ocular drops;(ii) locally administering, optionally to the eye, ear, nose (optionally intranasally), skin (optionally transdermally or epicutaneously), mucosa, skin, or vagina, or by inhalation; or(iii) parenterally administering, optionally by injection (optionally intravenous, intramuscular, subcutaneous, intrade Imai, intrathecal, intra-arterial, intraarticular, intraosseous, or intraperitoneal administration) or by inhalation; or(iv) enterally administering, optionally orally, sublingually, buccally, or rectally.27. The method of any embodiments 24-25, wherein the FZD binding regions bind toFZD 4 receptors.28. The method of any embodiments 24-27, wherein the disease, disorder, or condition comprises one or more of the following:(i) a retinopathy, optionally a retinal vascular disease (optionally caused by inhibition of vascular development or excessive angiogenesis) and / or optionally selected from the group consisting of exudative vitreoretinopathy, familiar exudative vitreoretinopathy (FEVR), retinopathy of prematurity, Norrie disease, diabetic retinopathy (DR), diabetic macular edema, diabetic macular ischemia, age-related macular degeneration (AMD) (including wet AMD and dry AMD), retinopathy of prematurity (ROP), b) a vascular disorder, optionally vascular malformation or vascular insufficiency, further optionally associated with ischemia- induced neovascularization, c) Uveitis, NIU, UME, ARN acute retinal necrosis, npAIR non parabolic autoimmune retinopathy, cataract surgery.

[0274] All of U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and / or listed in the Application Data Sheet, are incorporated herein by reference, in their entirety.

[0275] From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

Claims1. A WNT surrogate fusion molecule, wherein the WNT surrogate fusion molecule comprises: a) one or more low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or Low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) (LRP5 / LRP6) binding antibody or antigen-binding fragment that specifically binds to one or more LRP5 / 6 receptor; and b) one or more frizzled (FZD) binding antibody or antigen-binding fragment that specifically bind to one or more FZD receptor; and c) one or more antibodies or antigen-binding fragment that specifically bind to one or more cytokine receptor or cytokine ligand.

2. The WNT surrogate fusion molecule of claim 1, wherein the WNT surrogate fusion molecule is tetravalent, pentavalent, or hexavalent.

3. The WNT surrogate fusion molecule of claim 2, wherein the hexavalent surrogate molecule comprises: a) two low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or a Low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) or LRP5 / LRP6 binding antibodies or antigen-binding fragments that specifically bind to two LRP5 / 6 receptors; and b) two frizzled (FZD) binding antibodies or antigen-binding fragments that specifically bind to two FZD receptors; and c) two cytokine binding antibodies or antigen-binding fragments that specifically bind to two cytokine receptors and / or cytokine ligands.

4. The WNT surrogate fusion molecule of claim 2, wherein the pentavalent surrogate molecule comprises: a) one low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or a Low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) or LRP5 / LRP6 binding antibody or antigen-binding fragment that specifically binds to one LRP5 / 6 receptor or two low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or a Low-density lipoprotein (LDL) receptors related protein 6 (LRP6) or LRP5 / LRP6 receptors; and / orb) two frizzled (FZD) binding antibodies or antigen-binding fragments that specifically bind to two FZD receptors; and c) two cytokine antibodies or antigen-binding fragments that specifically bind to two cytokine receptors and / or cytokine ligands.

5. The WNT surrogate fusion molecule of claim 2, wherein the pentavalent surrogate molecule comprises: a) two low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or a Low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) or LRP5 / LRP6 binding antibodies or antigen-binding fragments that specifically bind to two LRP5 / 6 receptors; and / or b) one frizzled (FZD) binding antibody or antigen-binding fragment that specifically binds to one FZD receptor; and c) two cytokine antibodies or antigen-binding fragments that specifically bind to two cytokine receptors and / or cytokine ligands.

6. The WNT surrogate fusion molecule of claim 2, wherein the pentavalent surrogate molecule comprises: a) two low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or a Low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) or LRP5 / LRP6 binding antibodies or antigen-binding fragments that specifically bind to two LRP5 / 6 receptors; and / or b) two frizzled (FZD) binding antibodies or antigen-binding fragments that specifically bind to two FZD receptors; and c) one cytokine antibody or antigen-binding fragment that specifically binds to a cytokine receptor and / or cytokine ligand.

7. The WNT surrogate fusion molecule of claim 2, wherein the tetravalent surrogate molecule comprises: a) one low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or a Low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) or LRP5 / LRP6 binding antibody or antigen-binding fragment that specifically binds to one LRP5 / 6 receptor; and b) two frizzled (FZD) binding antibodies or antigen-binding fragments that specifically bind to one or more FZD receptor; andc) one cytokine antibody or antigen-binding fragment that specifically binds to one cytokine receptor and / or cytokine ligand.

8. The WNT surrogate fusion molecule of any of claims 1-7, wherein the antigenbinding fragments are selected from the group consisting of: IgG, scFv, Fab, VHH or single domain antibodies (sdAb), diabody, and Fv.

9. The WNT surrogate fusion molecule of any of claims 1-7, wherein the LRP5 / 6 antigen-binding region comprises a VHH or sdAb, a scFv, a Fab, a Fv, or a diabody.

10. The WNT surrogate fusion molecule of any of claims 1-7, wherein the FZD antigenbinding region comprises a VHH or sdAb, a scFv, a Fab, a Fv, or a diabody.

11. The WNT surrogate fusion molecule of any of claims 1-7, wherein the cytokine receptor or cytokine antigen-binding region comprises a VHH or sdAb, a scFv, a Fab, a Fv, or a diabody.

12. The WNT surrogate fusion molecule of claims 8-11, wherein the Fab is present within a full immunoglobulin (Ig), optionally an IgG, comprising a light chain and a heavy chain.

13. The WNT surrogate fusion molecule of any of claims 1-12, wherein each of the LRP5 / 6 binding regions, FZD binding regions, and cytokine receptor and / or ligand is fused or linked to another binding region via a linker moiety.

14. The WNT surrogate fusion molecule of any of claims 1-13, wherein the one or more FZD binding regions bind to one or more FZD 4 receptors.

15. The WNT surrogate fusion molecule of any of claims 1-14, wherein the cytokine antibodies or antigen-binding fragments specifically bind to an interleukin 6 (IL6) IL6 or an IL6 receptor.

16. The WNT surrogate fusion molecule of claim 15, wherein the IL6 receptor and / or IL6 ligand binding domain comprises siltuximab, sirukumab, olokizumab, clazakinumab, gerilimzumab, Tocilizumab, Sarilumab, Vamikibart, or Satralizumab, or a variant or fragment thereof that specifically binds the IL6 receptor and / or IL6 ligand.

17. A multivalent fusion molecule, wherein the molecule comprises: a) a first binding domain that comprises one or more antibody or antigen-binding fragment thereof that specifically binds to one or more first antigen, wherein the antigenbinding fragment comprises a first Fab; b) a second binding domain that comprises one or more antibody or antigen-binding fragment thereof that specifically binds to one or more second antigen, wherein the antigenbinding fragment comprises a second Fab; and c) a third binding domain that comprises one or more antibody or antigen-binding fragment thereof that specifically bind to one or more third antigen, wherein the antigenbinding fragment comprises a third Fab; and wherein the first Fab heavy chain (HC) is fused to the second Fab HC, and the third Fab HC is fused to the second Fab light chain (LC), or wherein the first Fab LC is fused to the second Fab HC, and the third Fab LC is fused to the second Fab LC; and wherein the Fabs are fused or linked to each other via a linker moiety.

18. The multivalent fusion molecule of claim 17, wherein: a) the first binding domain comprises one or more LRP 5 / 6 binding antibody or antigen-binding fragment thereof that specifically binds to one or more LRP5 and / or LRP6 receptor; and b) the second binding domain comprises one or more FZD binding antibody or antigen-binding fragment thereof that specifically bind to one or more FZD receptors; and c) the third binding domain comprises one or more antibody or antigen-binding fragment thereof that specifically binds to one or more cytokine receptor and / or cytokine ligand.

19. A nucleic acid or a combination of nucleic acids encoding the fusion molecule of any one of claims 1-18, optionally wherein the nucleic acid(s) is / are DNA, cDNA, RNA, mRNA, modified mRNA, or a DNA / RNA hybrid.

20. The nucleic acid or combination of nucleic acids of claim 19, which encode the the fusion molecule of any one of claims 1-18 and comprises: a) a first nucleic acid encoding the LRP5 / LRP6 binding antibody or antigen-binding fragment;b) a second nucleic acid encoding the FZD binding antibody or antigen-binding fragment; and c) a third nucleic acid encoding the antibody or antigen-binding fragment that specifically binds to one or more cytokine receptor or cytokine ligand.

21. A vector or a combination of vectors, which comprises the nucleic acid or the combination of nucleic acids of claim 19 or 20, optionally wherein: a) the vector(s) comprise(s) one or more promoters operably linked to the nucleic acid(s); b) the vector(s) is / are an expression vector; and / or c) the vector(s) comprise(s) a plasmid, a viral vector (optionally an adeno-associated virus, adenovirus, lentivirus, or retrovirus vector), a lipid-based vector, a self-replicating RNA vector, a virus-like particle, a polymer-based vector, and / or a nanoparticle, optionally a lipid-based nanoparticle.

22. A host cell, which comprises: a) the WNT surrogate fusion molecule of any one of claims 1-18; b) the nucleic acid or the combination of nucleic acids of claim 19 or 20; and / or c) the vector or the combination of vectors of claim 21, optionally wherein the host cell is:(i) mammalian, optionally human, non-human primate, monkey, rabbit, rodent, hamster, rat, or mouse; or(ii) non-mammalian, optionally plant, bacterial, fungal, yeast, protozoa, or insect, and optionally wherein the host cell is:(i) a human embryonal kidney (HEK) cell, optionally a HEK293 cell, or a variant thereof, further optionally Expi293F™ cell;(ii) a CHO (Chinese Hamster Ovary) cell;(iii) an immune cell or(iv) a hybridoma.

23. A pharmaceutical composition, which comprises: a) the fusion molecule of any one of claims 1-18;the nucleic acid or the combination of nucleic acids of claim 19 or 20; the vector or the combination of vectors of claim 21; and / or the host cell of claim 22; and b) a pharmaceutically acceptable carrier and / or excipient.

24. A method for agonizing, inducing, promoting, stimulating, enhancing, and / or supporting a WNT signaling in a target cell of a subject, comprising administering to the subject an effective amount of a) the WNT surrogate fusion molecule of any one of claims 1-18; b) the nucleic acid or the combination of nucleic acids of claim 19 or 20; c) the vector of claim 21; d) the host cell of claim 20 or the population of cells of claim 22; and / or e) the pharmaceutical composition of claim 23, optionally wherein:(i) the subject is a mammal, optionally a human, a non-human primate;(ii) the subject comprises or has a risk of developing a disease, disorder, or a condition; and / or(iii) the method further comprises administering to the subject an additional agent, optionally an adjuvant or a therapeutic agent, and optionally wherein:(i) the WNT signaling is or comprises WNT / beta-catenin signaling; and / or(ii) the method promotes, stimulates, enhances, and / or supports recruitment of multiple receptor complexes each comprising FZD; LRP5 / 6; and cytokine receptor or ligand.

25. The method of claim 24, wherein the disease, disorder, or condition comprises one or more of the following: a) a retinopathy, optionally a retinal vascular disease (optionally caused by inhibition of vascular development or excessive angiogenesis) and / or optionally selected from the group consisting of exudative vitreoretinopathy, familiar exudative vitreoretinopathy (FEVR), retinopathy of prematurity, Norrie disease, diabetic retinopathy (DR),diabetic macular edema, diabetic macular ischemia, age-related macular degeneration (AMD) (including wet AMD and dry AMD), retinopathy of prematurity (ROP), osteoporosis-pseudoglioma syndrome (OPPG), retinal vein occlusion, and Coats disease; b) a vascular disorder, optionally vascular malformation or vascular insufficiency, further optionally associated with ischemia-induced neovascularization; c) a bone disease or bone injury, optionally selected from the group consisting of osteoporosis (including osteoporosis pseudoglioma (OPPG)syndrome and juvenile primary osteoporosis), bone mineral density variability, and cancer-associated osteolysis; d) uveitis, NIU, UME, ARN acute retinal necrosis, npAIR non parabolic autoimmune retinopathy, cataract surgery; e) a muscle wasting disease, optionally selected from the group consisting of sarcopenia, cachexia, and muscular dystrophies; and / or f) a neural or neurodegenerative disease, optionally selected from the group consisting of stroke, traumatic brain injury, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Friedreich ataxia, Lewy body disease, spinal muscular atrophy, motor neuron disease, multiple sclerosis, Batten disease, and Creutzfeldt-Jakob disease.

26. The method of any one of claims 24 and 25, wherein the administering is:(i) to one or more of the subject's eyes, optionally intravitreally or via ocular drops;(ii) locally administering, optionally to the eye, ear, nose (optionally intranasally), skin (optionally transdermally or epicutaneously), mucosa, skin, or vagina, or by inhalation; or(iii) parenterally administering, optionally by injection (optionally intravenous, intramuscular, subcutaneous, intradelmal, intrathecal, intra-arterial, intraarticular, intraosseous, or intraperitoneal administration) or by inhalation; or(iv) enterally administering, optionally orally, sublingually, buccally, or rectally.

27. The method of any claims of 24-25, wherein the FZD binding regions bind to FZD 4 receptors.

28. The method of any claims of 24-27, wherein the disease, disorder, or condition comprises one or more of the following: a retinopathy, optionally a retinal vascular disease (optionally caused by inhibition of vascular development or excessive angiogenesis) and / or optionally selected from the group consisting of exudative vitreoretinopathy, familiar exudative vitreoretinopathy (FEVR), retinopathy of prematurity, Norrie disease, diabetic retinopathy (DR), diabetic macular edema, diabetic macular ischemia, age-related macular degeneration (AMD) (including wet AMD and dry AMD), retinopathy of prematurity (ROP), b) a vascular disorder, optionally vascular malformation or vascular insufficiency, further optionally associated with ischemia-induced neovascularization, c) Uveitis, NIU, UME, ARN acute retinal necrosis, npAIR non parabolic autoimmune retinopathy, cataract surgery.

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