Molecules modulating WNT signaling pathways and uses thereof

Abstract

The present invention provides WNT pathway surrogate molecules and related compositions, which may be used in any of a variety of therapeutic methods for the treatment of diseases, specifically the treatment of ocular disorders.

Description

Agent’s File Ref. SRZN-033 / 01WO 328202-2278MOLECULES MODULATING WNT SIGNALING PATHWAYS AND USES THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 698,305, filed September 24, 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 033 01WO _ST26.xml. The XML file is 47,393 bytes, and created on September 22, 2025, and is being submitted electronically via USPTO Patent Center.FIELD OF INVENTION

[0003] The present invention relates generally to multivalent polypeptide molecules that bind to one or more LRP5 and / or LRP6 and one or more FZD receptors, and related compositions, and methods of using the same. Such molecules are useful, for example, in modulating WNT signaling pathways. In embodiments, the present invention provides WNT signal modulators for use in treating various ocular 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 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] One of the challenges for modulating WNT signaling as a therapeutic is the existence of multiple WNT ligands and WNT receptors, Frizzled 1-10 (FZD1-10), with many tissues expressing multiple and overlapping FZDs. Canonical WNT signals also involve Low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) or Low-density lipoprotein324806361 1Agent’s File Ref. SRZN-033 / 01WO 328202-2278(LDL) receptor-related protein 6 (LRP6) as co-receptors, which are broadly expressed in various tissues, in addition to FZDs. Accordingly, there is clearly a need in the art for binding moieties that specifically bind to one or more FZD, LRP5, or LRP6 to modulate WNT signaling pathways. In addition, there is a need to reduce or eliminate the effector functions of these molecules, and in certain cases to increase or reduce the systemic elimination of the molecules is also necessary. The present invention addresses this need.

[0006] Specifically, the present invention provides WNT signal modulators for use in treating various ocular disorders. In particular, treatments for vascular diseases of the eye, including retinopathies, are provided.

[0007] The vertebral retina is a thin layer of nerve tissue in the back of the eye. It is responsible for detecting visual stimuli and is the first station for visual information processing. For its proper function, the retinal vasculature is an indispensable source of nutrients and oxygen. The retina is metabolically highly active. Due to the photoreceptors which consume the vast amount of oxygen, a gram of retina shows the highest oxygen consumption rate than any other organs in body. To effectively deliver nutrients and oxygen, the retinal vasculature is positioned in the retina as a stereotyped architecture consisting of three planal vascular plexuses on one side and the choriocapillaries on the other. The inner vascularization initially begins on the vitreal surface of the retina, giving rise to a primary vascular plexus. After the superficial radial expansion of the vascular plexus, vertical penetration of vessels into the retina forms two additional intraretinal capillary plexuses at the inner plexiform layer (IPL) and the outer plexiform layer (OPL). Due to the functional and structural relationship between blood vessels and the retina, aberrant vessel development and / or vascular damage are directly linked to the aberrant or impaired function of the retina, which may result in various types of retinopathy and retinal degeneration.

[0008] WNT signaling has been implicated as an important pathway for vascular development in the retina. Growing genetic evidence from human and rodent studies further supports the importance of WNT signaling in retinal vasculature (Wang et al., 2018, Prog Retin Eye Res. 2018 Dec 1. pii: 81350-9462(18)30046-6). Mutations in human genes encoding various receptors (FZD4, LRP5, TSPAN12) and a ligand (NDP) involved in WNT signaling result in a variety of inherited vitreoretinopathies. Specific genetic mutations in the murine orthologs of these genes (FZD4, LRP5, Tspanl2, Ndp) have also been observed to cause the characteristic phenotypes of aberrant vasculature seen in human retinopathy. This discovery has facilitated researchers to obtain a better understanding of retinopathy disease324806361 2Agent’s File Ref. SRZN-033 / 01WO 328202-2278 progression, as well as opening the possibility of retinopathy treatment through WNT signal modulation.

[0009] Retinopathy is any damage to the retina of the eyes, which may cause vision impairment. Retinopathy encompasses retinal vascular disease, and damage to the retina caused by abnormal blood flow. Age-related macular degeneration is considered a retinopathy but is often discussed as a separate entity. Retinopathy, including diabetic retinopathy, can be divided into early and late stages. In the early stage, also known as nonproliferative retinopathy, there may be a slight deterioration in the small blood vessels of the retina, and portions of the vessels may swell and leak fluid into the surrounding retinal tissue. Late-stage retinopathy involves significant neovascularization as well as microaneurysms and hemorrhages in the retinal area (see, e.g., Grading Diabetic Retinopathy from Stereoscopic Color Fundus Photographs — An Extension of the Modified Airlie House Classification. (1991) Ophthalmology, 98(5), 786-806). Retinopathy may be an ocular manifestation of systemic or metabolic diseases, as seen with diabetes and hypertension. Diabetes is a common cause of retinopathy, and diabetic retinopathy is a leading cause of blindness in working-aged people and has been designated a priority eye disease by the World Health Organization.

[0010] Familial Exudative Vitreoretinopathy (FEVR) is a genetic eye disease characterized by poor formation of intraocular vasculature. Over 50% of FEVR patients show mutations in one of the genes encoding FZD4, LRP5, TSPAN12, NDP. Norrin, a WNT signal ligand, transmits a signal to the endothelial cells through a receptor complex composed of FZD4 / LRP5 / TSPAN12 for normal development of retinal vascularization in the eye. However, in FEVR patients, mutations in one or more of NDP, FZD4, LRP5, or / and TSPAN12 genes result in immature vascular development in the retina. The resulting formation of the avascular region creates a retinal ischemic area, which results in primary damage to the retina. This ischemic condition induces the production of vascular endothelial growth factor (VEGF) and angiopoietin2 (Ang2), leading to neovascularization and vascular tuft formation. The newly generated abnormal blood vessels can be easily broken, leading to secondary damage of the retina due to exudation and hemorrhage. Disease progression of diabetic retinopathy (DR) is similar to that of FEVR and other genetic vascular malformation or insufficiency diseases. Hyperglycemia induces retinal vessel damage, leading to vaso- obliteration, ischemia, neovascularization, and hemorrhage, eventually leading to retinopathy.

[0011] While genetic data has suggested the importance of WNT signaling in establishing the proper vascular structure in the eye, whether activation of WNT signaling post-324806361 3Agent’s File Ref. SRZN-033 / 01WO 328202-2278 developmentally would lead to improvement in vascular structure is unknown. Certain reports have even suggested that antagonizing rather than agonizing WNT signaling would be beneficial in the treatment of retinopathy. Therefore, a greater understanding of retinopathy, disease progression, and WNT signal involvement therein would be beneficial as this potentially could result in new treatments. For effective treatment of retinopathy in particular, there is a need for modulating WNT agonist and antagonist signaling during specific stages of disease. The present invention addresses these needs and provides methods for modulating WNT signaling agonism and antagonism during different stages of retinopathy and disease progression.SUMMARY OF THE INVENTION

[0012] Previous studies determined that multivalent binding to FZD and LRP is important for efficient WNT signaling induction (e.g., PCT application Publication No. WO2019 / 126398; PCT application Publication No. W02020 / 010308; Chen et al, Cell Chemical Biology, 27:598, 2020; and Tao et al, eLife, 8:e46134, 2019). These studies explored molecules based on Fc dimerization for multivalent design. The designs resulted in large molecular weight, and frequently the need for Fc engineering for effector-less function, FcRn mutations to increase elimination, and / or Fc paring technologies for heterodimerization. To solve the challenges associated with Fc-based molecules, the disclosure provides multivalent formats without Fc. This can result in faster elimination in the absence of FcRn binding, and the lack of effector function. In addition, some of these formats allow single chain design and compatibility with more expression systems. In various embodiments, the present invention provides polypeptides modulating WNT signaling pathways and uses thereof.

[0013] In various embodiments, the present invention provides polypeptides modulating WNT signaling pathways and uses thereof.

[0014] In one embodiment, the disclosure provides a multispecific molecule having a structure of(Am-X-Bn)k wherein,A is an antigen binding antibody fragment selected from the group consisting of a Fab, a scFv, a Fv, or a single domain antibody (VHH);B is an antigen binding antibody fragment selected from the group consisting of a Fab, a scFv, a Fv, or a single domain antibody (VHH);324806361 4Agent’s File Ref. SRZN-033 / 01WO 328202-2278X is a peptide linker; m is an integer between 1 and 2; n is an integer between 1 and 2; and k is an integer between 1 and 2.

[0015] In some embodiments, the multispecific molecule has a structure of:(Am-X-Bn)2 wherein,A is an antigen binding antibody fragment that binds FZD receptors or binds LRP5 and / or LRP6 selected from the group consisting of Fv, scFv, or a single domain antibody (VHH);B is an antigen binding antibody fragment that binds one or more FZD receptors or binds LRP5 and / or LRP6 selected from the group consisting of a Fv or a Fab;X is a peptide linker; m=l; n=l; and two repeat structures are connected to each other via a linker.

[0016] In some embodiments, the peptide linker in the WNT surrogate molecule is about 0 amino acid to about 100 amino acids. In particular embodiments, the length of the peptide linker is about 5 amino acids to about 40 amino acids, optionally 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 20, 30, 35, or 40 amino acids. In some embodiments, the peptide linker comprises one or more glycine and / or serine residues; the peptide linker is a peptide selected from the group consisting of a) Glycine X 4-Serine: (G4S) (SEQ ID NO: 32); b) Glycine X 4-Serine: (G4S)2 (SEQ ID NO: 33); c) Glycine X 4-Serine: (G4S)3 (SEQ ID NO: 34); d) Glycine X4-Serine: (G4S)4 (SEQ ID NO: 35); e) Glycine X 4-Serine: (G4S)5 (SEQ ID NO: 36); and f) Glycine X 4-Serine (SEQ ID NO: 37).

[0017] In some embodiments of the WNT surrogate molecule: A is a VHH antigen binding fragment that binds specifically to LRP5 and / or LRP6 receptors, B is a Fab antigen binding fragment that binds specifically to FZD receptors, wherein the VHH is appended to the N-terminus of the variable light chain (VL ) of the Fab antigen binding fragment, the Fabs are linked through C-terminus of the constant heavy chain (CH) to N-terminus of the second variable heavy chain (VH), or the VHH is appended to N-terminus of VH of the Fab antigen binding fragment and Fabs are linked through C-terminus of constant light chain (CL) chain to the N-terminus of the second VL.324806361 5Agent’s File Ref. SRZN-033 / 01WO 328202-2278

[0018] In some embodiments of the WNT surrogate molecule: A is a Fv antigen binding fragment that binds specifically to LRP5 and / or LRP6 receptors, B is a Fab antigen binding fragment that binds specifically to FZD receptors, wherein the Fv is appended on N-terminus of the Fab, and C-terminus CL or CH of the Fab binding fragment is appended on N-terminus of second Fv of VH or VL.

[0019] In some embodiments of the WNT surrogate molecule is a multispecific molecule having a structure of:(Am-X-Bn)l wherein: m=l or 2; n=2;A is a VHH antigen binding fragment that binds specifically to LRP5 and / or LRP6 receptors, B is a Fv and Fab antigen binding fragment that binds specifically to FZD receptors, wherein the VHH (LRP5 / 6 binder) appended at the N-terminus of VH or VL of a Fv binder, and C-terminus of Fv is appended on N-terminus of Fab binder.

[0020] In some embodiments, the WNT surrogate molecule is tetravalent bispecific having two VHHs or Fv that bind to identical LRP5 / or LRP6 receptors, and having two Fabs that bind to identical FZD receptors.

[0021] In some embodiments, the WNT surrogate molecule is tetravalent trispecific having two VHHs or Fv that bind to identical LRP5 / or LRP6 receptors, and having two Fabs that bind to non-identical FZD receptors, or two VHHs or Fv that bind to non-identical LRP5 / or LRP6 receptors, and having two Fabs that bind to identical FZD receptors.

[0022] In some embodiments, the WNT surrogate molecule is tetravalent tetraspecific having two VHHs or Fv that bind to non-identical LRP5 / or LRP6 receptors, and having two Fabs that bind to non-identical FZD receptors.

[0023] In some embodiments of the WNT surrogate molecule, the FZD binding regions bind to FZD 4 receptors.

[0024] In some embodiments of the WNT surrogate molecule, the LRP5 / LRP6 binding regions bind to LRP5 receptors.

[0025] In some embodiments, the WNT surrogate molecule comprises amino acids sequences set forth in: (i) SEQ ID NOs: 10 and 11; (ii) SEQ ID NOs: 10 and 12; (iii) SEQ ID NOs: 13 and 14; (iv) SEQ ID NOs: 13 and 15; (v) SEQ ID NOs: 13 and 16; (vi) SEQ ID NOs: 13 and 17; (vii) SEQ ID NOs: 13 and 18; (viii) SEQ ID NOs: 19 and 20; (ix) SEQ ID NOs:324806361 6Agent’s File Ref. SRZN-033 / 01WO 328202-227821 and 20; (x) SEQ ID NOs: 22, 23, and 24; (xi) SEQ ID NOs: 25, 23, and 24; (xii) SEQ ID NOs: 26, 27, and 28; or (xiii) SEQ ID NOs: 29, 27, and 28.

[0026] In some embodiments, the present disclosure provides a nucleic acid or a combination of nucleic acids encoding the WNT surrogate 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 molecule antibody or antibody fragment of any of embodiments. In particular embodiments, the nucleic acid or combination of nucleic acids comprises: a) a first nucleic acid encoding the first polypeptide LRP5 / 6 binder; and b) a second nucleic acid encoding the second polypeptide FZD binder.

[0027] In some embodiments, the present disclosure provides an expression vector comprising the isolated polynucleotide or nucleic acid or combination of nucleic acids. In further particular embodiments, the present disclosure provides an isolated host cell comprising the expression vector.

[0028] In some embodiments, 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 molecules disclosed herein.

[0029] In some embodiments, the present disclosure provides a method for agonizing a WNT signaling pathway in a cell, comprising contacting the cell any of the WNT surrogate molecules, wherein the WNT surrogate molecule is an agonist of a WNT signaling pathway.

[0030] In some 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 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 (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;324806361 7Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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), or 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.

[0031] In related embodiments, the disclosure provides a method of administering the WNT surrogate 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; (iii) parenterally administering, optionally by injection (optionally intravenous, intramuscular, subcutaneous, intradermal, intrathecal, intra-arterial, intraarticular, intraosseous, or intraperitoneal administration) or by inhalation; or (iv) enterally administering, optionally orally, sublingually, buccally, or rectally.

[0032] In particular embodiments, the FZD binding regions bind to one or more FZD-4 receptors and 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), and 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, or cataract surgery.324806361 8Agent’s File Ref. SRZN-033 / 01WO 328202-2278BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figs. 1A-1D illustrate various formats of the WNT surrogate molecule. Fig. 1A depicts a WNT surrogate molecule comprising one VHH3 LRP binder separated by a short linker G4S to a VH or VL of a Fv polypeptide binder to FZD4, which is linked by (G4S)c to a VH or VL Fab FZD4 binder. Fig. IB depicts a WNT surrogate molecule comprising two VHH3 LRP binders separated by a short linker G4S to a VH or VL of Fv polypeptide binder to FZD4, which is linked by (G4S)c to VH or VL Fab FZD4 binder. Fig. 1C depicts a WNT surrogate molecule comprising a VHH3 LRP binder linked to a FZD4 Fab binder; this structure is duplicated via two Fab linked together by a peptidyl linker (G4S)C. Fig. ID depicts a WNT surrogate molecule comprising a format of (Fv-Fab)2 wherein the two duplicate structures are linked together with a peptidyl linker (G4S)i.

[0034] Figs. 2A-2C illustrate the expression and binding to FZD4 and LRP5 of the VHH3_4SDl-Fv_4SDl-Fab molecule.

[0035] Figs. 3A-3C illustrate the expression and binding to FZD4 and LRP5 of the (VHH3)2_4SDl-Fv_4SDlFab molecule.

[0036] Figs. 4A-4C illustrate the expression and binding to FZD4 and LRP5 of the (VHH3_4SDlFab)2 molecule.

[0037] Fig. 5 illustrates the comparative STF activity of the indicated WNT surrogate molecules VHH3_4SDl-Fv_4SDl-Fab, (VHH3)2_4SDl-Fv_4SDlFab, and (VHH3_4SDlFab)2. The lines from top to bottom at 2 log [Protein] nm correspond to: (VHH3_4SDlFab)2; (VHH3)2_4SDl-Fv_4SDlFab; and VHH3.4SDl-Fv_4SDl-Fab

[0038] Fig. 6 illustrates the comparative STF activity for (VHH3_4SDlFab)2 with various linkers: G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)5, (G4S)6.

[0039] Figs. 7A-7B illustrate the structure and the comparative STF activity of two WNT surrogate molecules 4SD1 9SB2 (Fv-Fab)2_G4S linker and 4SD1 9SB2 (Fv-Fab)2_4xG4S with two different linker length between each Fv Fab

[0040] Figs. 8A-8C show the Ant39 antibody (Fv_Fab)2, that the top and bottom Fv are two different LRP5 binders and purification curves.

[0041] Figs. 9A-9C show the Ant39 antibody (Fv_Fab)2 crossmab, that the top and bottom Fv are two different LRP5 binders and purification curves.

[0042] Fig. 10 shows the synergy data of mini-SWAP in Oxygen-Induced Retinopathy (OIR).

[0043] Figs. 11 A-l 1C illustrate the structure of the mini-SWAP, positive control and the negative control with no binding to LRP5 and FZD4 due to mutations.324806361 9Agent’s File Ref. SRZN-033 / 01WO 328202-2278DETAILED DESCRIPTION

[0044] The present disclosure relates to a multispecific molecule having a structure of: (Am-X-Bn)k wherein,A is an antigen binding antibody fragment selected from the group consisting of a Fab, a scFv, a Fv, and a single domain antibody (VHH);B is an antigen binding antibody fragment selected from the group consisting of a Fab, a scFv, a Fv, and a single domain antibody (VHH);X is a peptide linker; m is an integer between 1 and 2; n is an integer between 1 and 2; and k is an integer between 1 and 2.

[0045] In certain embodiments, the multispecific molecule is a WNT surrogate molecule that binds to one or two FZD receptors and one or two LRP5 or LRP6 receptors and modulates a downstream WNT signaling pathway having a structure of(Am-X-Bn)2 wherein,A is an antigen binding antibody fragment that binds FZD receptors or binds LRP5 and / or LRP6 selected from the group consisting of Fv, scFv, and a single domain antibody (VHH);B is an antigen binding antibody fragment that binds one or more FZD receptors or binds LRP5 and / or LRP6 selected from the group consisting of a Fv and a Fab;X is a peptide linker m=l; n=l; two repeat structures are connected to each other via a linker. In certain embodiments, the linker is peptide linked comprising one or more glycine and / or serine.

[0046] Embodiments of the invention pertain to the use of WNT surrogate molecules for the diagnosis, assessment and treatment of diseases and disorders associated with WNT signaling pathways. In certain embodiments, the subject WNT surrogate molecules are used to modulate a WNT signaling pathway in a cell or tissue. In certain embodiments, the subject WNT surrogate molecules are 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.324806361 10Agent’s File Ref. SRZN-033 / 01WO 328202-2278

[0047] 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.

[0048] 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.

[0049] 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.

[0050] Reference to “an integer between 1 and 2” includes the integers of 1 and 2.

[0051] As used in this specification and the appended claims, the terms “cXGS4” or “(GS4)c” are identical, unless the content clearly dictates otherwise.

[0052] Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.

[0053] 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-324806361 11Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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.

[0054] Embodiments of the present invention relate to polypeptides and antigen-binding fragments thereof that bind to one or more FZD receptor. Sequences of illustrative polypeptides, or antigen-binding fragments, or complementarity determining regions (CDRs) thereof, may be used or present in the WNT surrogate molecules disclosed herein and those described in any of PCT application publication Nos. WO 2019 / 126399, WO 2019 / 126398, WO 2022 / 192445, and WO 2023 / 130055.

[0055] Embodiments of the present invention relate to polypeptides and antigen-binding fragments thereof that bind to LRP5 and / or LRP6. Sequences of illustrative antibodies, or antigen-binding fragments, or complementarity determining regions (CDRs) thereof, may be used or are present in the WNT surrogate molecules disclosed herein and those described in any of PCT application publication Nos. WO 2019 / 126401, WO 2019 / 126398, WO 2022 / 192445, and 2023 / 130055.

[0056] As is well known in the art, therapeutic antibodies comprise fragments such as dAb, Fab, Fab', F(ab')2, Fv), single chain (scFv), VHH or single domain antibody (Nanobodies®), synthetic variants thereof, naturally occurring variants, fusion proteins with configurations that comprise an antigen-binding site or fragment (epitope recognition site) of the required specificity. "Diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993) are also 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.

[0057] 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 that binds to the antigen of interest, in particular to one or more FZD receptor or to an LRP5 and / 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 or from antibodies that bind one or more FZD receptor or LRP5 and / or LRP6. An antigen-binding fragment of a FZD-specific antibody is capable of binding to one or more324806361 12Agent’s File Ref. SRZN-033 / 01WO 328202-2278FZD receptor. An antigen-binding fragment of a LRP5 and / or LRP6-specific antibody is capable of binding to an LRP5 and / or LRP6 receptor. As used herein, the term encompasses not only isolated fragments but also polypeptides comprising an antigen-binding fragment of a polypeptide disclosed herein, such as, for example, fusion proteins comprising an antigenbinding fragment of a polypeptide disclosed herein, such as, e.g., a fusion protein comprising a Nanobody® that binds one or more FZD receptors and a Nanobody® that binds LRP5 and / or LRP6.

[0058] The term "antigen" refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as the polypeptide, and additionally capable of being used in an animal to produce a polypeptide 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 mimetic 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.

[0059] In certain embodiments, WNT surrogate molecules and polypeptides and antigenbinding 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-polypeptide complexes 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.

[0060] 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 region324806361 13Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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.

[0061] 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).

[0062] 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.

[0063] 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 multispecific polypeptides may be made that encompass the ligands of the present disclosure. Bispecific polypeptides 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 polypeptides may be produced through recombinant molecular biological techniques or may be physically conjugated together.

[0064] 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): 5879-5883.324806361 14Agent’s File Ref. SRZN-033 / 01WO 328202-2278A 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.

[0065] In certain embodiments, a WNT surrogate molecule as described herein is in the form of a diabody. Diabodies can be 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). Alternatively, diabodies can form from intramolecular interaction between 2 VH and 2 VL domains, a tandem diabody. The influence of length and amino acid composition of the peptide linkers connecting individual VH and VL domains on the folding efficacy, stability and biological activity of the polypeptide (Fabrice Le Gall, et al., Effect of linker sequences between the antibody variable domains on the formation, stability and biological activity of a bispecific tandem diabody Protein Engineering, Design & Selection vol. 17 no. 4 pp. 357-366, 2004).

[0066] A single domain fragment of an antibody (dAb) consists of a VH or VL domain (Ward, E. S. et al., Nature 341, 544-546 (1989)).

[0067] Diabodies, Fv, Fab, and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti -idiotypic reaction.

[0068] Bispecific diabodies, as opposed to bispecific whole antibodies, may also be particularly useful because they can be readily constructed and expressed in E. coli or other prokaryotic organism. Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (see, e.g., PCT Application Publication No. 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 a polypeptide of appropriate specificity selected.

[0069] In certain embodiments, the polypeptide of the present disclosure may take the form of a Nanobody®. Nanobody® technology was originally developed following the discovery and identification that camelidae (e.g., camels and llamas) possess fully functional324806361 15Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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 “Nanobodies®”. Nanobodies® 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 Nanobodies® have been produced. Nanobodies® may be formulated as a ready-to-use solution having a long shelflife. The Nanoclone® method (see, e.g., WO 06 / 079372) is a proprietary method for generating Nanobodies® against a desired target, based on automated high-throughput selection of B-cells. Nanobodies® are single-domain antigen-binding fragments of camelid-specific heavy-chain only antibodies. Nanobodies®, also referred to as VHH antibodies, typically have a small size of around 15 kDa.

[0070] As stated previously, the variable domains of Fab are responsible for the binding specificity of the whole antibody. Therefore, the smallest unit of Ig with antigen binding activity is the fragment variable or Fv in which the two variable domains (VH and VL) connect with a disulfide bond. ScFv is an engineered form of Fv that, instead of a disulfide bond, the two variable domains are joined together by a flexible linker. The length and amino acid composition of this linker play an important role in correct folding of the protein (Ahmad ZA, et al., ScFv antibody: Principles and clinical application. Clin Dev Immunol. 2012;2012 cited 2020 Aug 31), and it is typically 10-25 amino acid long with Glu Lys stretches to increase the solubility and Gly Ser stretches for the flexibility of the final protein (Alfthan K, et al., Properties of a single-chain antibody containing different linker peptides. Protein Eng Des Sei. 1995; 8(7): 725-731, and Whitlow M, et al. An improved linker for single-chain Fv with reduced aggregation and enhance proteolytic stability. Protein Eng Des Sei. 1993 ;6(8): 989— 95). Within each of the two variable domains of the Fv, or scFv, there are three hypervariable domains or complementary determining regions; significant structural differences, Fv, scFv and VHH (nanobody) display distinct properties in vitro and in vivo.

[0071] Fv, scFv and VHH (Nb) have notable dissimilarity in their size, while Fv, ScFv are almost twice the Nb size by about 30 kDa weight (Bannas P, Hambach J, Koch-Nolte F, Johnson M. Nanobodies and nanobody -based human heavy chain antibodies as antitumor therapeutics. Front Immunol. 2017; 8(NOV): 1603) This smaller size facilitates VHHs genetic324806361 16Agent’s File Ref. SRZN-033 / 01WO 328202-2278 manipulation (Hassani M, Hajari Taheri F, Sharifzadeh Z, Arashkia A, Hadjati J, van Weerden WM, et al. Construction of a chimeric antigen receptor bearing a nanobody against prostate a specific membrane antigen in prostate cancer. J Cell Biochem. 2019; 120(6): 10787- 95), and the presence of only three antigen-binding loops allows for easy enhancement of their intrinsic tendency to antigen binding (Hassanzadeh-Ghassabeh G, et al., Nanobodies and their potential applications, Nanomedicine 2013;8: 1013-26). Due to the renal filtration and degradation, the smaller size of VHHs also results in their short half-life in blood (Kim TY, Park JH, Shim HE, Choi DS, Lee D-E, Song J- J, et al. Prolonged half life of small-sized therapeutic protein using serum albumin-specific protein binder. J Control Release. 2019;315:31-9).[00721 VHHs are more soluble than the Fvs or scFvs with a higher thermodynamic stability of VHHs in comparison to Fvs or scFvs. Therefore, VHHs are more resistant to chemical denaturants and proteolytic enzymes (Hussack G, et al., Characterization of singledomain antibodies with an engineered disulfide bond. Methods Mol Biol. 2012;911 :417-29) and have higher stability under harsh pH or ionic strength (Van Audenhove I, et al., Nanobodies as Versatile Tools to Understand, Diagnose, Visualize and Treat Cancer. EBioMedicine. 2016;8:40-8). This higher conformational stability also stems from the presence of an extra disulfide bond, which lowers the probability of heat-induced aggregation and limits VHHs flexibility. Therefore, although in Fvs scFvs the hydrophobic interface between VL and VH dampens their stability, this two-domain structure make them more flexible and more advantageous for some applications.WNT Surrogate Molecules

[0073] The disclosure provides, in certain aspects, WNT surrogate molecules that bind both one or more FZD receptors and one or both of LRP5 and / or LRP6. WNT surrogate molecules may also be referred to as “WNT mimetics” or “WNT surrogate polypeptides.” In particular embodiments, the WNT surrogate molecules bind one or more human FZD receptors and one or both of a human LRP5 and / or a human LRP6.

[0074] In certain embodiments, a WNT surrogate molecule is capable of modulating or modulates WNT signaling events in a cell contacted with the WNT surrogate molecule. In certain embodiments, the WNT surrogate molecule increases WNT signaling. In certain embodiments, the WNT surrogate molecule binds specifically and modulates the biological activity of a human WNT signaling pathway.324806361 17Agent’s File Ref. SRZN-033 / 01WO 328202-2278

[0075] WNT surrogate molecules of the present invention are biologically active in binding to one or more FZD receptors and to one or more of LRP5 and LRP6, and in activation of WNT signaling, i.e., the WNT surrogate 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 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, including but not limited to those disclosed herein. WNT-signaling 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-signaling 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-signaling agonist, e.g., a WNT surrogate molecule disclosed herein.

[0076] In particular embodiments, WNT surrogate molecules disclosed herein are bispecific, i.e., they specifically bind to two or more different epitopes, e.g., one or more FZD receptor and LRP5 and / or LRP6.

[0077] In particular embodiments, WNT surrogate molecules disclosed herein are multivalent, i.e., they comprise two or more regions that each specifically bind to an epitope, which may be the same of different epitopes, e.g., different epitopes on the same target, or even epitopes different targets. In particular embodiments, WNT surrogate molecules disclosed herein comprise two or more regions that each specifically bind to the same epitope, e.g., two or more regions that bind to an epitope within one or more FZD receptor and / or two or more regions that bind to an epitope within LRP5 and / or LRP6. In particular embodiments, they comprise two or more regions that bind to an epitope within one or more FZD receptor and two or more regions that bind to an epitope within LRP5 and / or LRP6. In certain embodiments, WNT surrogate molecules comprise a ratio of the number of regions that bind one or more FZD receptor to the number of regions that bind LRP5 and / or LRP6 of: 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 1 :2, 1 :3, 1 :4, 1 :5, or 1 :6. In particular embodiments, they comprise two regions that bind to an epitope within one or more FZD receptor and two regions that bind to an epitope within LRP5 and / or LRP6.

[0078] WNT surrogate molecules disclosed herein may have any of a variety of different structural formats or configurations. WNT mimetics may comprise polypeptides and / or nonpolypeptide binding moi eties, e.g., small molecules. In particular embodiments, WNT surrogate molecules comprise both a polypeptide region and a non-polypeptide binding324806361 18Agent’s File Ref. SRZN-033 / 01WO 328202-2278 moiety. In certain embodiments, WNT surrogate molecules may comprise a single polypeptide, or they may comprise two or more, three or more, or four or more polypeptides.

[0079] When a WNT surrogate molecule comprise a single polypeptide, it may be a fusion protein comprising one or more FZD binding domains and one or more LRP5 / 6 binding domains. 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.

[0080] WNT surrogate polypeptides may be engineered to facilitate binding between two polypeptides. 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.

[0081] WNT surrogate molecules described herein may have a variety of different structural formats, including but not limited to those shown in Figures 1 to 6. It is understood that the disclosed WNT surrogate molecules are not limited to the structural formats depicted in Figures 1-6, which are illustrative of certain embodiments of various structural formats. For example, the linkers present in the illustrative formats may be different or have different sequences than shown in Figures 1 to 6.

[0082] In one embodiment, a WNT surrogate molecule comprises an scFv, Fv or Fab antigen-binding fragment thereof fused to a VHH (Nanobody®) or antigen-binding fragment thereof. In certain embodiments, the scFv, Fv, or Fab specifically binds one or more FZD receptor, and the VHH specifically binds LRP5 and / or LRP6.

[0083] In particular embodiments, the WNT surrogate molecule form a multivalent multispecific binding composition including but not limited to Figs. 1 A-1D. In particular embodiments, Fig. 1 A, the single chain polypeptide comprises a VHH with specificity to LRP5 and / or LRP6 and one identical or different Fv and Fab from intramolecular VH / VL interaction, that specifically binds one or more FZD receptors to form a trivalent bispecific or trispecific binding molecule. In certain embodiments, the single chain polypeptide comprises two VHH with specificity to LRP5 and / or LRP6 and one identical or different Fv and Fab from intramolecular VH / VL interaction, that specifically binds one or more FZD receptors to form a tetravalent bispecific, trispecific, or tetraspecific binding molecule Fig. IB. In particular embodiments, binding VHH is fused or linked to N-terminus of the VL or VH of Fv via peptide linker, and C-terminus of VL or VH of Fv is fused or linked to VH or VL of Fab polypetide.

[0084] In particular embodiment, the molecule comprises structure of (VHH3)i or 2 _4SDl-Fv_4SDl-Fab, wherein the VHH3 are antigen binding fragments that bind324806361 19Agent’s File Ref SRZN-033 / 01WO 328202-2278 specifically to LRP5, and comprise or consist of CDRs described in Table 1 or have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NOs: 1-3, optionally according to IMGT or Kabat; and the 4SD1-Fv and 4SD1-Fab are antigen binding fragments that bind specifically to FZD4 receptors, and comprise or consist of CDRs described in Table 2 or have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NOs: 4-10, optionally according to IMGT or Kabat.Table 1 : Anti-LRP5 VHH3 CDR sequencesTable 2: Anti-FZD4 4SD1 CDR sequences

[0085] In certain embodiments, the molecule has a formula (Am-X-Bn)2, wherein,A is an antigen binding antibody fragment that binds FZD receptors or binds LRP5 and / or LRP6 selected from the group consisting of Fv, scFv, or a single domain antibody (VHH);B is an antigen binding antibody fragment that binds one or more FZD receptors or binds LRP5 and / or LRP6 selected from the group consisting of a Fv or a Fab; X is a peptide linker, m=l;324806361 20Agent’s File Ref. SRZN-033 / 01WO 328202-2278 n=l; two repeat structures are connected to each other via a linker, e.g., Figs. 1C and ID. In particular embodiments, A is an antigen binding antibody fragment that binds LRP5 and / or LRP6 selected from the group consisting of Fv, or a single domain antibody (VHH); B is an antigen binding antibody fragment that binds one or more FZD receptors, e.g., a Fv or a Fab. In particular embodiments, the two A are identical and the two B are identical, thus creating tetravalent bispecific molecules, including but not limited to those shown in Figs. 1C and ID. In other embodiments, the two A are nonidentical and the two B are identical, creating tetravalent trispecific WNT surrogate molecules. In particular embodiments, the two A are nonidentical and the two B are identical, creating tetravalent tetraspecific WNT surrogate molecules, including but not limited to those shown in Figs. 1C and ID.

[0086] In some embodiments, VHH is appended to N-terminus of variable light chain (VL) of Fab antigen binding fragment, Fabs are linked through C-terminus of constant heavy chain (CH) to N-terminus of the second variable heavy chain (VH), or VHH is appended to N-terminus of VH of Fab antigen binding fragment and Fabs are linked through C-terminus of constant light chain (CL) chain to the N-terminus of the second VL.

[0087] In particular embodiments, VL or VH of Fv is appended to N-terminus of variable light chain (VL )of Fab antigen binding fragment, Fabs are linked through C-terminus of constant heavy chain (CH) to N-terminus of the second variable heavy chain (VH), or VL or VH of Fv is appended to N-terminus of VH of Fab antigen binding fragment and Fabs are linked through C-terminus of constant light chain (CL) chain to the N-terminus of the second VL.

[0088] In some embodiments, VL and VH of Fv is appended to N-terminus of variable light chain (VL ) and heavy chain (VH) of Fab antigen binding fragment, Fabs are linked through C-terminus of constant heavy chain (CH) to N-terminus of the second variable heavy chain (VH), or VL or VH of Fv is appended to N-terminus of VH and VL of Fab antigen binding fragment and Fabs are linked through C-terminus of constant light chain (CL) and constant heavy chain (CH) Figs. 7A, 8A, and 9A (CrossMab).

[0089] WNT surrogate molecules, in various embodiments, comprise one or more antigen-binding fragments thereof disclosed herein. In certain embodiments, the WNT surrogate molecules comprise two or more, three or more, or four or more antigen-binding fragments thereof disclosed herein.

[0090] In particular embodiments, a WNT surrogate molecule comprises a FZD binding region, e.g., an anti-FZD antibody or antigen-binding fragment thereof, or an LRP5 and / or LRP6 binding region, e.g., an anti-LRP5 and / orLRP6 antibody or antigen-binding fragment324806361 21Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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 a polypeptide antigen-binding fragment thereof. In certain embodiments, an antigen-binding fragment is disclosed herein or in the PCT application titled, “Anti-Frizzled antibodies and Methods of Use,” WO 2019 / 126399 filed on December 19, 2017, 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. In certain embodiments, the FZD binding domain may be selected from any binding domain that binds 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 embodiments, 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 embodiments, 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 10'4M, less than or equal to about 1 x 10'5M, less than or equal to about 1 x 10'6M, less than or equal to about 1 x 10-7 M, less than or equal to about 1 x 10'8M, less than or equal to about 1 x 10'9M, or at least about 1 x 10'10M in the context of a WNT mimetic.

[0091] Suitable FZD binding domains include, without limitation, de novo designed FZD binding proteins, derived binding proteins, e.g., scFv, Fab, diabody, VHH etc. that specifically bind to one or more FZD proteins; nanobody 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.

[0092] 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, 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.324806361 22Agent’s File Ref. SRZN-033 / 01WO 328202-2278

[0093] In other embodiments, the FZD binding domain comprises a variable region sequence, or the CDRs thereof, from any of a number of FZD-specific antibodies, which are known in the art and are commercially available, or can be generated de novo. Any of the FZD polypeptides can be used as an immunogen or in screening assays to develop a WNT surrogate molecule. Non-limiting examples of frizzled binding domains include antibodies available from Biolegend, e.g., Clone CH3A4A7 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.

[0094] The FZD binding region of a WNT mimetic may be an engineered protein that is selected for structural homology to the FZD-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 be further 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.

[0095] 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, 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 antigen-binding fragment. In certain embodiments, it is an antigen-binding fragment thereof disclosed in the PCT application titled, “Anti-LR5 / 6 Antibodies and Methods of Use,” WO 2019 / 126401, 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. 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.

[0096] In certain embodiments, 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 10-4324806361 23Agent’s File Ref. SRZN-033 / 01WO 328202-2278M, 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 mimetic. In certain embodiments, 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 mimetic. 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.

[0097] Other suitable LRP5 / 6 binding domains include, without limitation, de novo designed LRP5 / 6 binding proteins, e.g. Fv, scFv, Fab, VHH etc. and other portions of antibodies that specifically bind to one or more LRP proteins; nanobody derived binding domains; knottin-based engineered scaffolds; naturally occurring LRP5 / 6 binders, including without limitation, DKK1, DKK2, DKK3, DKK4, sclerostin; Wise; fusion 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.

[0098] Members of the Dickkopf (DKK) gene family (see Krupnik et al. (1999) Gene 238(2):301-13) include DKK-1, DKK-2, DKK-3, and DKK-4, and the DKK-3 related protein Soggy (Sgy). hDKKs 1-4 contain two distinct cysteine-rich domains in which the positions of 10 cysteine residues are highly conserved between family members. Exemplary sequences of human Dkk genes and proteins are publicly available, e.g. Genbank accession number NM_014419 (soggy-1); NM_014420 (DKK4); AF177394 (DKK-1); AF177395 (DKK-2); NM_015881 (DKK3); and NM_014421 (DKK2). In some embodiments of the invention, the LRP6 binding moiety is a DKK1 peptide, including without limitation the C-terminal domain of human DKK1. The C-terminal domain may comprise the sequence (SEQ ID NO: 30): KMYHTKGQEGSVCLRSSDCASGLCCARHFWSKICKPVLKEGQVCTKHRRKGSHGLE IFQRCYCGEGLSCRIQKDHHQASNSSRLHTCQRH (see Genbank accession number NP_036374) or a biologically active fragment thereof.

[0099] Binding of DKK proteins to LRP5 / 6 are discussed, for example in Brott and SokolMol. Cell. Biol. 22 (17), 6100-6110 (2002); and Li et al. J. Biol. Chem. 277 (8), 5977-5981 (2002), each herein specifically incorporated by reference. The corresponding region of324806361 24Agent’s File Ref. SRZN-033 / 01WO 328202-2278 human DKK2 (Genbank reference NP 055236) may comprise the sequence (SEQ ID NO: 31): KMSHIKGHEGDPCLRSSDCIEGFCCARHFWTKICKPVLHQGEVCTKQRKKGSHGLEI FQRCDCAKGLSCKVWKDATYSSKARLHVCQK or a biologically active fragment thereof.

[0100] 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 and / 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 1A12, 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.

[0101] In certain embodiments, WNT surrogate 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 one or more LRP5 / 6 binding regions, or they may comprise one or more FZD binding region and one or more LRP5 / 6 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 mimetic. 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 0,1, 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. In one embodiment, a peptide linker is peptide consisting of four sequential glycines and one serine (G4S) (SEQ ID NO: 32). In another embodiment, the peptide linker herein is three sequential G4S peptides (G4S)3 (SEQ324806361 25Agent’s File Ref. SRZN-033 / 01WO 328202-2278ID NO: 34). In another embodiments the the peptide linker herein is three sequential G4S peptides (G4S)4 (SEQ ID NO: 35), In another embodiment, the peptide linker herein is three sequential G4S peptides (G4S)5 (SEQ ID NO: 36), In another embodiment, the peptide linker herein is three sequential G4S peptides (G4S)6 (SEQ ID NO: 37). In some embodiments a peptide linker is a ASTKG (Ala-Ser- Thr-Lys-Gly) (SEQ ID NO: 41) or DKTHT (Asp-Lys- Thr-His-Thr) (SEQ ID NO: 42). In particular embodiments, ASTKG (SEQ ID NO: 41) or DKTHT (SEQ ID NO: 42) are combined with G4S (SEQ ID NO: 32). (Clarissa G. Jakob, et al., Structure reveals function of the dual variable domain immunoglobulin (DVD-Ig™) molecule, mAbs 5:3, 358-363; 2013; Joshua S. Klein, et al., Design and characterization of structured protein linkers with differing flexibilities; Protein Engineering, Design & Selection vol. 27 no. 10 pp. 325-330, 2014; Weizao Chen, et al.„ Improving the CH1-CK heterodimerization and pharmacokinetics of 4Dm2m, a novel potent CD4 antibody fusion protein against HIV-1, MABS, 2016, VOL. 8, NO. 4, 761-774.).

[0102] In particular embodiments, a WNT surrogate molecule comprises one or more linkers. In certain embodiments, the linker is a linear, peptide linker comprising from about 0 to about 100 amino acids in length. In some WNT surrogate molecules comprising a linker, the linker is a linear, peptide linker comprising from about 5 to about 40 amino acids in length. In one embodiment, the linker is one, or two, or three, or four, or five or six sequential peptides, each peptide consisting of four sequential glycines and one serine G4S.

[0103] In particular embodiments, the linker within VHH to Fv or Fab is a short linker. In some embodiments, the linker between two Fab is a G4S (SEQ ID NO: 32), or (G4S)2 (SEQ ID NO: 33), or (G4S)3 (SEQ ID NO: 34), or (G4S)4 (SEQ ID NO: 35), or (G4S)5 (SEQ ID NO: 36), (G4S)6 (SEQ ID NO: 37), or (G4S)7 (SEQ ID NO: 38), or (G4S)8 (SEQ ID NO: 39), or (G4S)9 (SEQ ID NO: 40), etc.

[0104] In particular embodiments, a WNT surrogate molecule comprises a polypeptide sequence having at least 90%, at least 95%, at least 98% or at least 99% identity to a polypeptide sequence disclosed in any of SEQ ID NOs: 1-13, or having at least 90%, at least 95%, at least 98% or at least 99% identity to an antigen-binding fragment of a polypeptide sequence disclosed in any of SEQ ID NOs: 1-13. In certain embodiments, the WNT surrogate molecule comprises or consists of a polypeptide sequence set forth in any of SEQ ID NOs: 1- 13, or an antigen-binding fragment thereof. Table 3 shows polypeptides comprising fusions of anti-LRP5 and FZD4 binding domains.324806361 26Agent’s File Ref. SRZN-033 / 01WO 328202-2278Table 3. Illustrative fusions of anti-LRP5 and FZD4 binding domains324806361 27Agent’s File Ref. SRZN-033 / 01WO 328202-2278Table 4 shows sequences of illustrative FZD4 and LRP5 fusion WNT surrogate molecules: VHH03: bold, grey highlighted, 4SD1 VL: Italic, underline, 4SD1 VH: bold, underlined, italic, hK: double line underlined, hlgG CHI : bold, His-tag: Italic.Table 4. Sequences of illustrative FZD4 and LRP5 fusion WNT surrogate molecules324806361 28Agent’s File Ref. SRZN-033 / 01WO 328202-2278324806361 29Agent’s File Ref. SRZN-033 / 01WO 328202-2278324806361 30Agent’s File Ref. SRZN-033 / 01WO 328202-2278Table 5 shows sequences of WNT surrogate molecules (Fv_Fab)2: 4SD1 VL: Italic, underline ,4SD1 VH: bold, underlined, italic, 9SB2 VL: bold, underline, 9SB2 VH: italic, double line underlined, hK: double line underlined, hlgG CHI: bold, His-tag: ItalicTable 5. Sequences of illustrative WNT surrogate molecules324806361 31Agent’s File Ref SRZN-033 / 01WO 328202-2278Table 6 shows the sequences of ANT39 LRP5-1 & LRP5-2 VL: Italic, underline, ANT39 L5- 1 & LRP5-2 VH: bold, underlined, italic, ANT39 FZD4 VL: bold, underline, ANT39 FZD4 VH: italic, double line underlined, hK: double line underlined, hlgG CHI: bold, His-tag: ItalicTable 6. Sequences of illustrative molecules324806361 32Agent’s File Ref. SRZN-033 / 01WO 328202-2278324806361 33Agent’s File Ref. SRZN-033 / 01WO 328202-2278324806361 34Agent’s File Ref. SRZN-033 / 01WO 328202-2278324806361 35Agent’s File Ref. SRZN-033 / 01WO 328202-2278[01051 The WNT surrogate 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 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.

[0106] In certain embodiments, a WNT surrogate molecule directly activates canonical WNT signaling through binding to one or more FZD proteins and to LRP5 and / or 6, 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 to324806361 36Agent’s File Ref. SRZN-033 / 01WO 328202-2278 potentiation of WNT signaling, which enhances activity only when native WNT proteins are present.

[0107] WNT surrogate molecules may activate WNT signaling, e.g., by mimicking the effect or activity of a WNT protein binding to a FZD protein. The ability of the WNT surrogate molecule of the disclosure to mimic the activity of WNT can be confirmed by a number of assays. The WNT surrogate molecules 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 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.

[0108] 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 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.

[0109] Changes in WNT-responsive gene expression are generally mediated by TCF and LEF transcription factors. A TCF reporter assay assesses changes in the transcription and translation 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 (pTOPFlash and pFOPFlash, respectively) to determine the transactivation 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.[OHO] 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,324806361 37Agent’s File Ref. SRZN-033 / 01WO 328202-2278BAT-GAL, ins-TOP-EGFP, ins-TOPGAL, LEF-EGFP, Axin2-LacZ, Axin2-d2EGFP, Lgr5tml (cre / ERT2), TOPdGFP.

[0111] 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 high conten screening (HCS) platform allowing the levels and distribution of EGFP-P-catenin to be visualized.

[0112] 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.

[0113] 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 mimetics 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.

[0114] "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.324806361 38Agent’s File Ref. SRZN-033 / 01WO 328202-2278

[0115] 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 (GenBank Accession 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-10B (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.

[0116] "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.

[0117] For example, activation of the canonical WNT signaling pathway results in the inhibition of phosphorylation of the intracellular protein P-catenin, leading to an324806361 39Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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 (INK), and nemo-like kinase (NLK) signaling cascades to control such biological processes as tissue polarity and cell movement. Activation of the WNT / Ca2+ pathway by, for example, binding of WNT-4, WNT-5A or WNT-11, elicits an intracellular release of calcium ions, which activates calcium 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 mimetic, can be readily determined.

[0118] In certain embodiments, functional properties of WNT surrogate 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 surrogate molecule, 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 molecule 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.

[0119] In certain embodiments, a FZD-binding region of a WNT surrogate 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 herein. In certain embodiments, a LRP5 / 6- binding region of a WNT mimetic (e.g., an antigen-binding fragment of an anti-LRP5 / 6 antibody) comprises one or more of the CDRs of the anti-LRP5 / 6 antibodies described herein. In this regard, it has been shown in some cases that the transfer of only the VHCDR3 of an antibody can be performed while still retaining desired specific binding (Barbas et al., PNAS (1995) 92: 2529-2533). See also, McLane et al., PNAS (1995) 92:5214-5218, Barbas et al., J. Am. Chem. Soc. (1994) 116:2161-2162.

[0120] Also disclosed herein is a method for obtaining a polypeptide or antigen binding domain specific for a FZD receptor, the method comprising by way of addition, deletion,324806361 40Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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 a polypeptide antigen binding domain specific for one or more FZD receptor and optionally with one or more desired properties. The VL domains may have an 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.

[0121] 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 homogeneous 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 mimetics disclosed herein typically form a substantially homogeneous aqueous solution at concentrations of at least 5 pM and higher, e.g. at least 5 pM, 20 pM, or 40 pM, usually at least 60 pM, 70 pM, 80 pM, or 90 pM, sometimes as much as 100 pM, 150 pM, or 250 pM. In other words, WNT mimetics disclosed herein typically form a substantially homogeneous aqueous solution at concentrations of about 0.4 mg / ml, about 1.0 mg / ml, of about 20 mg / ml or more.

[0122] In some embodiments, any of the one or more FZD binding region of a WNT surrogate 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 FZD 10; or (x) FZD4, FZD5 and FZD8; FZD1, FZD5, FZD7 and FZD8; or (xi) FZD4. 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 some324806361 41Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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.

[0123] In some embodiments, any of the one or more LRP5 / 6 binding regions of a WNT surrogate molecule binds to one or both of LRP5 / 6. For convenience, the term “LRP5 / 6” is used to refer collectively to either or both LRP5 and / or LRP6.

[0124] 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 in the 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.

[0125] In certain embodiments, the WNT surrogate molecule 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.

[0126] WNT surrogate 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 labile324806361 42Agent’s File Ref. SRZN-033 / 01WO 328202-2278 groups, as disclosed in the above-identified patents, disulfide and thioether groups being preferred.

[0127] In certain embodiments, anti-LRP5 / 6 antigen-binding fragments thereof and / or anti-FZD antigen-binding fragments thereof present within a WNT surrogate molecule are monoclonal. In certain embodiments, they are humanized.

[0128] The present invention further provides in certain embodiments an isolated nucleic acid encoding a polypeptide present in a WNT surrogate 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.

[0129] 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 under suitable 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.

[0130] 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.

[0131] The term "polynucleotide" as referred to herein means single-stranded or doublestranded nucleic acid polymers. In certain embodiments, the nucleotides comprising the324806361 43Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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.[01321 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 any purpose. An oligonucleotide can include a detectable label to enable detection of the oligonucleotide or hybridization thereof.[01331 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.

[0134] 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.

[0135] 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 mRNA324806361 44Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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.

[0136] 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 a polypeptide 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 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.

[0137] 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 polynucleotides that 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.

[0138] 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.

[0139] In certain embodiments, the disclosure contemplates the mutagenesis of the polynucleotide sequences that encode a polypeptide present in a WNT surrogate molecule, to alter one or more properties of the encoded polypeptide, such as the binding affinity, or the function. 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, sitespecific mutagenesis is often used to alter a specific portion of a DNA molecule. In such324806361 45Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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.

[0140] 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.

[0141] 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 and protocols 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.

[0142] In many embodiments, one or more nucleic acids encoding a polypeptide of a WNT surrogate polypeptide 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.

[0143] 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 mimetic 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, an324806361 46Agent’s File Ref. SRZN-033 / 01WO 328202-2278 antibody or antigen-binding fragment thereof, may be isolated and / or purified using any suitable technique, and then used as desired.

[0144] 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.

[0145] 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 HEK293 cells, Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells and many others. Expression in prokaryotes include, but is not limited to the bacterial host, E. coli.

[0146] The expression of polypeptides, e.g., 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 antigenbinding 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.

[0147] 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.

[0148] 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 more324806361 47Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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.

[0149] 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 WNT surrogate 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.

[0150] 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 independently324806361 48Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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.

[0151] 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 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.

[0152] The term "isolated protein,” “isolated WNT surrogate polypeptide or “isolated polypeptide” referred to herein means that a subject protein, WNT mimetic,: (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)324806361 49Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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). [01531 Amino acid sequence modification(s) of any of the polypeptides (e.g., WNT surrogate polypeptides or FZD binding regions or LRP5 / 6 binding regions thereof) described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the WNT surrogate molecule. For example, amino acid sequence variants of a WNT mimetic 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 the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution may be made to arrive at the final WNT surrogate molecule, provided that the final construct possesses the desired characteristics (e.g., high affinity binding to one or more FZD and LRP5 / 6 receptor). 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.

[0154] The present disclosure provides variants of any of the polypeptides (e.g., WNT surrogate molecule or FZD binding regions or LRP5 / 6 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, at least about 50%, at least about 70%, and in certain embodiments, at least about 90% as well as a WNT surrogate molecule specifically set forth herein. In further embodiments, such variant WNT surrogate molecule bind to one or more FZD receptor, and / or to one or more LRP5 / 6 receptor, with greater affinity than the WNT surrogate molecule 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.324806361 50Agent’s File Ref. SRZN-033 / 01WO 328202-2278

[0155] In particular embodiments, the WNT surrogate molecule or a binding region thereof, e.g., a Fab, Fv, scFv, or VHH 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). In a further embodiment, the polypeptide, or antigen-binding fragment is a variant antigen-binding fragment thereof wherein the variant comprises a heavy and light chain identical to the selected antibody except for up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions in the CDR regions of the VH and VL regions. In this regard, there may be 1, 2, 3, 4, 5, 6, 7, 8, or in certain embodiments, 9, 10, 11, 12, 13, 14, 15 more amino acid substitutions in the CDR regions of the selected antibody. Substitutions may be in CDRs either in the VH and / or the VL regions. (See e.g., Muller, 1998, Structure 6: 1153- 1167).

[0156] In particular embodiments, the WNT surrogate molecule or a binding region thereof, e.g., a Fab, Fv, scFv, or VHH, may have: a) a heavy chain variable region having 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.

[0157] 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. 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 Genetics324806361 51Agent’s File Ref. SRZN-033 / 01WO 328202-2278Computing 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).

[0158] 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 sequence identity. 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.

[0159] 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:

[0160] Mismatch Penalty: 1.00; Gap Penalty: 1.00; Gap Size Penalty: 0.33; and Joining Penalty: 30.0.

[0161] Determination of the three-dimensional structures of representative polypeptides (e.g., variant FZD binding regions or LRP5 / 6 binding regions of WNT surrogate moleculs 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, such as for rational324806361 52Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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).Compositions

[0162] Pharmaceutical compositions comprising a WNT surrogate 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.

[0163] In further embodiments, pharmaceutical compositions comprising a polynucleotide comprising a nucleic acid sequence encoding a WNT surrogate 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 mimetic and the nucleic acid sequence encoding the WNT polypeptide or Norrin polypeptide are present in the same polynucleotide.324806361 53Agent’s File Ref. SRZN-033 / 01WO 328202-2278

[0164] 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 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 mimetic and the nucleic acid sequence encoding the WNT polypeptide or Norrin polypeptide are present in the same polynucleotide, e.g., expression cassette.

[0165] 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 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 mimetic 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.

[0166] The present disclosure contemplates pharmaceutical compositions comprising a first molecule for delivery of a WNT surrogate 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, eukaryotic or prokaryotic, comprising a nucleic acid sequence encoding the first or second active agent (optionally an expression cassette).324806361 54Agent’s File Ref. SRZN-033 / 01WO 328202-2278

[0167] In some embodiments, the present disclosure contemplates pharmaceutical compositions comprising a first molecule for delivery of a WNT surrogate molecule as a first active agent and a second molecule for delivery angiogenic factor binding antibodies or antigen-binding fragments that specifically bind to angiogenic receptor or ligand. In particular embodiments, the anti-VEGF molecule is covalently linked to WNT surrogate molecule of the present invention. In another embodiment, the anti-VEGF molecule is formulated or administrated with the molecule of the present disclosure. In some embodiments, the angiogenic antibodies or antigen-binding fragments that specifically bind to one or more angiogenic receptor and / or ligand comprising VEGF or VEGF receptors. In particular embodiment, VEGF receptor and / or ligand binders comprises ranibizumab, aflibercept (Eylea), bevacizumab, brolucizumab, faricimab-svoa.

[0168] 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.

[0169] 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 microorganisms324806361 55Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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.

[0170] Sterile solutions can be prepared by incorporating the WNT surrogate molecule 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.

[0171] 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.

[0172] 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 the324806361 56Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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.

[0173] 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.

[0174] 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.

[0175] The present invention includes pharmaceutically acceptable salts of a WNT surrogate 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).

[0176] 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-methylglucamine, 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.324806361 57Agent’s File Ref. SRZN-033 / 01WO 328202-2278

[0177] In some embodiments, the pharmaceutical composition provided herein comprise a therapeutically effective amount of a WNT surrogate 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.

[0178] 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

[0179] The present disclosure also provides methods for using the WNT surrogate molecules disclosed herein, e.g., to modulate a WNT signaling pathway, e.g., to increase WNT signaling, and the administration of a WNT mimetic disclosed herein in a variety of therapeutic settings. Provided herein are methods of treatment using a WNT mimetic. In one embodiment, a WNT mimetic is provided to a subject having a disease involving inappropriate or deregulated WNT signaling, e.g., reduced WNT signaling.

[0180] Increasing WNT Pathway Signaling and Related Therapeutic Methods. In certain embodiments, a WNT surrogate 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 molecule or pharmaceutically acceptable salt thereof disclosed herein, wherein the a WNT surrogate 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. In324806361 58Agent’s File Ref. SRZN-033 / 01WO 328202-2278 certain embodiments, the method comprises further contacting the tissue or cell with one or more WNT polypeptides or Norrin polypeptides.

[0181] 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 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 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 molecule and the nucleic acid sequence encoding the WNT polypeptide or Norrin polypeptide are present in the same polynucleotide.

[0182] 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 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 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.

[0183] 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 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 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 molecule or the324806361 59Agent’s File Ref. SRZN-033 / 01WO 328202-2278WNT 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.

[0184] WNT surrogate 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 pharmaceutical composition comprising any of: a WNT surrogate molecule; a polynucleotide comprising a nucleic acid sequence encoding a WNT surrogate molecule, e.g., a DNA or mRNA, optionally a modified mRNA; a vector comprising a nucleic acid sequence encoding a WNT surrogate molecule, e.g., an expression vector or viral vector; or a cell comprising a nucleic acid sequence encoding a WNT surrogate molecule, e.g., a cell transduced with an expression vector or viral vector encoding a WNT surrogate 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.

[0185] 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 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).324806361 60Agent’s File Ref. SRZN-033 / 01WO 328202-2278

[0186] 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 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 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 molecule and the nucleic acid sequence encoding the WNT polypeptide or Norrin polypeptide are present in the same polynucleotide.

[0187] 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 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 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.

[0188] 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 molecule. In certain embodiments, the subject is also contacted with a cell comprising a nucleic acid sequence324806361 61Agent’s File Ref. SRZN-033 / 01WO 328202-2278 that encodes a WNT polypeptide or a Norrin polypeptide. In certain embodiments, the nucleic acid sequence encoding the WNT surrogate 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 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.

[0189] 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 most tissues after injuries and diseases. WNT surrogate 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.

[0190] 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 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.; treatment324806361 62Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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 is beneficial; 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.

[0191] 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 molecule disclosed herein. Methods for regeneration of bone tissues benefit from administration of the WNT surrogate 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.324806361 63Agent’s File Ref. SRZN-033 / 01WO 328202-2278

[0192] 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, 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.

[0193] 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 molecule and an antiresorptive agent. In certain embodiments, the methods are used for the treatment of osteoporosis, optionally post-menopausal osteoporosis.

[0194] 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 molecule, wherein the WNT surrogate 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.

[0195] When a WNT surrogate molecule is provide to the subject in combination with another therapeutic agent, such as an antiresorptive agent, the two agent may be provided in the same or different pharmaceutical compositions. They may be provided to the subject at324806361 64Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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.

[0196] Compositions comprising one or more WNT surrogate molecule disclosed herein (or a polynucleotide encoding a WNT surrogate molecule, or a vector or cell comprising a polynucleotide encoding a WNT surrogate 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 tom ligament, malalignment of joints, bone fracture, or by hereditary disease.

[0197] A WNT surrogate 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.

[0198] 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 a324806361 65Agent’s File Ref. SRZN-033 / 01WO 328202-2278 matrix or other depot system; or other topical application to the ear for enhancement of auditory regeneration.

[0199] A WNT surrogate 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.

[0200] 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.

[0201] In certain embodiments, compositions comprising a WNT surrogate molecule disclosed herein (or a polynucleotide encoding a WNT surrogate molecule, or a vector or cell comprising a polynucleotide encoding a WNT surrogate 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 liver cell proliferation and / or mitosis, decrease fibrosis following chronic liver injury, or increase hepatocyte function.324806361 66Agent’s File Ref. SRZN-033 / 01WO 328202-2278

[0202] 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.

[0203] In particular embodiments, compositions comprising a WNT surrogate molecule disclosed herein (or a polynucleotide encoding a WNT surrogate molecule, or a vector or cell comprising a polynucleotide encoding a WNT surrogate 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.

[0204] 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 other324806361 67Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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.

[0205] 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 molecule may benefit various disease conditions involving exocrine and endocrine pancreas, kidney, or lung. The WNT surrogate 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.

[0206] 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.

[0207] 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 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 the BBB 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 benefit324806361 68Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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.[02081 WNT signals also play a role in angiogenesis. A WNT surrogate 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.

[0209] 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.

[0210] 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.

[0211] 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 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.

[0212] 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 molecule administered or provided to the subject is in the range of about 0.01324806361 69Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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.

[0213] 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 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.

[0214] Other embodiments relate, in part, to the use of the WNT surrogate 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 a subject. They may also be used to generate cells, tissue or organoids for research use. The WNT surrogate molecules have widespread applications in non-therapeutic methods, for example in vitro research methods.324806361 70Agent’s File Ref. SRZN-033 / 01WO 328202-2278

[0215] The invention provides a method for tissue regeneration of damaged tissue, such as the tissues discussed above, comprising administering a WNT surrogate molecule to cells. The WNT surrogate 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 molecule is administered to ex vivo cells, these cells may be transplanted into a subject before, after or during administration of the WNT surrogate molecule.

[0216] 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 molecules disclosed herein are suitable alternatives to Rspondin for use in these stem cell culture media, or may be combined with Rspondin.

[0217] 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 molecules disclosed herein. In one embodiment, the disclosure provides a cell culture medium comprising one or more WNT surrogate 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 molecule(s) disclosed herein. For example, the culture medium may be as described in as described in W02010 / 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.

[0218] 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 and of 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 stem324806361 71Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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 molecule disclosed herein, e.g., optionally in combination with one or more of the growth factors or combinations thereof described herein.

[0219] 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).

[0220] In some embodiments, the WNT surrogate 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.

[0221] 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 molecule.

[0222] 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 portion or 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 a324806361 72Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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.

[0223] In various embodiments, the WNT surrogate 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 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).

[0224] 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.

[0225] 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

[0226] requiring addition of a secondary binding agent or antibody include enzymatic detectable binding agents and hapten detectable binding agents or antibodies.

[0227] 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 other324806361 73Agent’s File Ref. SRZN-033 / 01WO 328202-2278 embodiments, the detectable label is conjugated to an antibody which comprises the first binding agent (e.g., in an IHC process).

[0228] Examples of detectable labels which may be conjugated to WNT surrogate 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.

[0229] 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.

[0230] 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.

[0231] 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).

[0232] 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-324806361 74Agent’s File Ref. SRZN-033 / 01WO 328202-2278 phosphate / fast red TR (NAMP / FR), Naphthol-AS-Bl-phosphate / new fuschin (NABP / NF), bromochloroindolyl phosphate / nitroblue tetrazo burn (BCIP / NBT), 5-Bromo-4-chloro-3- indolyl-b— d-galactopyranoside (BCIG).

[0233] 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.

[0234] 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.).

[0235] 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.

[0236] All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and / or listed in the Application Data Sheet, are incorporated herein by reference, in their entirety.

[0237] From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications324806361 75Agent’s File Ref. SRZN-033 / 01WO 328202-2278 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.EXAMPLESReagents and Materials

[0238] The proteins were produced in Expi293F cells (Thermo Fisher Scientific) by transient transfection. The protein was first purified using cOmplete™ His-Tag Purification Column (Roche) on AKTA pure (Cytiva). The column was washed with 5 column volumes of Buffer A (40 mM HEPES pH 7.4, 10 mM imidazole, 300 mM NaCl) after protein binding; the protein was eluted with Buffer B (40 mM HEPES pH 7.4, 500 mM imidazole, 300 mM NaCl). The protein was further polished with HiLoad 16 / 600 Superdex 200 pg (Cytiva) sizeexclusion 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). The protein was examined by SDS-polyacrylamide electrophoresis and SEC-HPLC (Agilent).

[0239] Binding kinetics of VHH3 4SD1 Fv_4SDl Fab, (VHH3)2_4SD1 Fv_4SDl Fab, and (VHH3)2_(4SD1 Fab)2 to FZD4 or LRP5 were determined by BLI using an Octet Red instrument at 30°C and 1000 rpm with SA or AMC biosensors (Sartorius), respectively. Biotinylated hFZD4 CRD or mouse Fc-tagged hLRP5 LDLRA were diluted to 50 nM in the running buffer and captured by the SA or AMC biosensors, followed by dipping into wells containing the VHH3 4SD1 Fv_4SDl Fab, (VHH3)2_4SD1 Fv_4SDl Fab, and (VHH3)2_(4SD1 Fab)2 at different concentrations in a running buffer or into a well with only the running buffer as a reference channel. The dissociation of the interaction was 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.Super Top Flash (STF) Assay

[0240] WNT signaling activity was measured using bEnd.3 cells containing a luciferasereporter gene controlled by a WNT-responsive promoter (Super Top Flash reporter assay, STF) as previously reported (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 proteins were added to the cells and then incubated for another 18-24 hours. Cells were lysed with Luciferase Cell Culture Lysis324806361 76Agent’s File Ref. SRZN-033 / 01WO 328202-2278Reagent (Promega) and luciferase activity was measured using the Luciferase Assay System (Promega) following vendor suggested procedures.Example 1VHH3 4SDl-Fv_4SDlFab

[0241] Fig. 1 A is an illustrative example of a trivalent bispecific WNT Surrogate molecule, wherein one VHH-3 antigen binding fragment that binds to LRP5, is linked with G4S peptide linker to VL of 4SDl_Fv FZD4 binder, the C-terminal VH of 4SD14_Fv is linked to N-terminus VH of 4SDl_Fab FZD4 binder via a (G4S )4 binder (SEQ ID NO: 35).

[0242] Fig. 2A illustrates the protein expression; Figs 2B and 2C show the binding data of the molecule to FZD4 and LRP5 respectively.Example 2(VHH3)2 4SDl-Fv_4SDlFab

[0243] Fig. IB is an illustrative example of a tetravalent bispecific the WNT Surrogate molecule, wherein two VHH-3 antigen binding fragments that bind to LRP5, are linked with G4S peptide linker to VL of 4SDl_Fv FZD4 binder, the C-terminal VH of 4SD14_Fv is linked to N-terminus VH of 4SDl_Fab FZD4 binder via a (G4S )4 binder (SEQ ID NO: 35).

[0244] Fig. 3 A illustrates the protein expression; Figs 3B and 3C show the binding data of the molecule to FZD4 and LRP5 respectively.Example 3(VHH3 4SDlFab)2

[0245] Fig. 1C is an illustrative example of a tetraval ent bispecific WNT Surrogate molecule, wherein two VHH-3 antigen binding fragments that bind to LRP5, are linked with G4S peptide linker to VL of 4SDl_Fab FZD4 binder, the C-terminal VH of first 4SD14_Fab is linked to N-terminus VH of second 4SDl_Fab FZD4 binder via a different length of (G4S), such as (G4S)2 (SEQ ID NO: 33), (G4S)3 (SEQ ID NO: 34), (G4S)4 (SEQ ID NO: 35), and (G4S)6 (SEQ ID NO: 37), binder.

[0246] Fig. 4A illustrates the protein expression; Figs. 4B and 4C show the binding data of the molecule to FZD4 and LRP5 respectively.324806361 77Agent’s File Ref. SRZN-033 / 01WO 328202-2278Example 4Super Top Flash (STF) Assay

[0001] Fig. 5 illustrates comparative activity of VHH3_4SDl-Fv_4SDlFab, (VHH3)2_4SDl-Fv_4SDlFab, and (VHH3_4SDlFab)2 in the STF assay.

[0002] Fig. 6 illustrates comparative activity with different length of the linker between the two Fab moiety of (VHH3_4SDlFab)2.Example 5 4SD1 9SB2 (Fv_Fab)2

[0247] Fig. 7A is an example of a tetravalent bispecific WNT surrogate molecule, wherein the VH and VL of anti FZD4 Fv binders are linked to with G4S peptide linker to N- terminus of VL and VH of 9SB2_Fab LRP6 binder, the C-terminal of constant domainof anti-LRP6 Fab binder via a different length of (G4S), such as (G4S)2 (SEQ ID NO: 33), (G4S)3 (SEQ ID NO: 34), (G4S)4 (SEQ ID NO: 35), and (G4S)6 (SEQ ID NO: 37), binder is fused yo N-terminus of second anti-FZD4 (4SD1) Fv binder, and Fig. 7B illustrates the activity of STF 4SD1 9SB2 mini SWAP (Fv-Fab)2 with G4S (SEQ ID NO: 32) and 4xG4S linker (SEQ ID NO: 35).Example 6 ANT39 (Fv_Fab)2

[0248] Fig. 8A an illustrative example of a tetravalent bispecific WNT Surrogate molecule, wherein two Fv antigen binding fragments that bind to LRP5, are linked with G4S peptide linker to VL and VH of 4SDl_Fab FZD4 binder, the C-terminal constant domain of first 4SD14_Fab is linked to N-terminus VL second LRP5 binder linked of second 4SDl_Fab FZD4 binder via a different length of (G4S), such as (G4S)2 (SEQ ID NO: 33), (G4S)3 (SEQ ID NO: 34), (G4S)4 (SEQ ID NO: 35), and (G4S)6 (SEQ ID NO: 37), binder; Figs. 8B and 8C show the purification curve of Fig. 8 A. Fig. 9A is the CrossMab structure of Fig. 8 A.Example 7Mouse Model of Oxygen-Induced Retinopathy

[0249] Newborn pups at postnatal day 7 (P7) were exposed to a hyperoxic environment with a 75% inspired oxygen level for 5 days. As a result, the pups developed avascular (AV) retina from vaso-obliteration of newly formed capillaries in the center retina from P7 to P12. After the pups were moved to room air at Pl 2, the AV central retina became hypoxic and324806361 78Agent’s File Ref. SRZN-033 / 01WO 328202-2278 intravitreal neovascular (NV) tufts formed at the junctions of the vascular and AV retina from P12 to P18. For treatment, P12 pups received an intravitreal injection of indicated proteins at 4 pM in the vitreal space. To evaluate the efficacy of test articles, whole-mount retinas from pups at Pl 8 were stained with isolectin B4 and imaged. AV and NV tuft areas were manually measured and presented as a percentage of the AV or NV tuft area over total retinal area (Fig. 10), and Fig. 11 showing structures of compounds used in this study.324806361 79

Claims

Agent’s File Ref. SRZN-033 / 01WO 328202-2278Claims:

1. A multispecific molecule having a structure of:( Am-X-B n)k wherein,A is an antigen binding antibody fragment selected from the group consisting of a Fab, a scFv, a Fv, or a single domain antibody (VHH);B is an antigen binding antibody fragment selected from the group consisting of a Fab, a scFv, a Fv, or a single domain antibody (VHH);X is a peptide linker; m is an integer between 1 and 2; n is an integer between 1 and 2; and k is an integer between 1 and 2.

2. The multispecific molecule of claim 1, wherein the molecule is a WNT surrogate molecule having a structure of(Am-X-Bn)2 wherein,A is an antigen binding antibody fragment that binds FZD receptors or binds LRP5 and / or LRP6 selected from the group consisting of Fv, scFv, or a single domain antibody (VHH);B is an antigen binding antibody fragment that binds one or more FZD receptors or binds LRP5 and / or LRP6 selected from the group consisting of a Fv or a Fab;X is a peptide linker m=l; n=l; wherein the two repeat structures are connected to each other via a linker.

3. The WNT surrogate molecule of claim 2, wherein peptide linkers are independently about 0 amino acid to about 100 amino acids.

4. The WNT surrogate molecule of claim 3, wherein the length of the peptide linker is about 5 amino acids to about 40 amino acids, optionally 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 20, 30, 35, or 40 amino acids.324806361 80Agent’s File Ref. SRZN-033 / 01WO 328202-22785. The WNT surrogate molecule of claim 4, wherein the peptide linker comprises one or more glycine and / or serine residues.

6. The WNT surrogate molecule of claim 5, wherein the peptide linker is a peptide selected from the group consisting of: a) Glycine X 4-Serine: (G4S) (SEQ ID NO: 32); b) Glycine X 4-Serine: (G4S)2 (SEQID NO: 33); c) Glycine X 4-Serine: (G4S)3 (SEQ ID NO: 34); d) Glycine X4-Serine: (G4S)4 (SEQ ID NO: 35); e) Glycine X 4-Serine: (G4S)5 (SEQ ID NO: 36); and f) Glycine X 4-Serine: (G4S)6 (SEQ ID NO: 37).

7. The WNT surrogate molecule of claim 2, whereinA is a VHH antigen binding fragment that binds specifically to LRP5 and / or LRP6 receptors,B is a Fab antigen binding fragment that binds specifically to FZD receptors, wherein VHH is appended to N-terminus of variable light chain (VL) of Fab antigen binding fragment, Fabs are linked through C-terminus of constant heavy chain (CH) to N- terminus of the second variable heavy chain (VH), or VHH is appended to N-terminus of VH of Fab antigen binding fragment and Fabs are linked through C-terminus of constant light chain (CL) chain to the N-terminus of the second VL.

8. The WNT surrogate molecule of claim 2, whereinA is a Fv antigen binding fragment that binds specifically to LRP5 and / or LRP6 receptors,B is a Fab antigen binding fragment that binds specifically to FZD receptors, wherein Fv is appended on N-terminus of Fab, and C-terminus CL or CH of Fab binding fragment is appended on N-terminus of second Fv of VH or VL.

9. The molecule of claim 1, wherein the molecule is a WNT surrogate molecule, m=l or 2, n=2, k=l,A is a VHH antigen binding fragment that binds specifically to LRP5 and / or LRP6 receptors,324806361 81Agent’s File Ref. SRZN-033 / 01WO 328202-2278B is a Fv and Fab antigen binding fragment that binds specifically to FZD receptors, wherein the VHH (LRP5 / 6 binder) is appended at the N-terminus of VH or VL of a Fv binder, and C-terminus of Fv is appended on N-terminus of Fab binder.

10. The molecule of claim 2, wherein the WNT surrogate molecule is tetraval ent bispecific having two VHHs or Fv that bind to identical LRP5 / or LRP6 receptors, and having two Fabs that bind to identical FZD receptors.

11. The WNT surrogate molecule of claim 2, wherein the molecule is tetravalent trispecific having two VHHs or Fv that bind to identical LRP5 / or LRP6 receptors, and having two Fabs that bind to non-identical FZD receptors, or two VHHs or Fv that bind to nonidentical LRP5 / or LRP6 receptors, and having two Fabs that bind to identical FZD receptors.

12. The WNT surrogate molecule of claim 2, wherein the molecule is tetravalent tetraspecific having two VHHs or Fv that bind to non-identical LRP5 / or LRP6 receptors, and having two Fabs that bind to non-identical FZD receptors.

13. The WNT surrogate molecule of any of claims 2-11, wherein the FZD binding regions bind to FZD 4 receptors.

14. The WNT surrogate molecule of any of claims 2-11, wherein the LRP5 / LRP6 binding regions bind to LRP5 receptors.

15. The WNT surrogate molecule of any of claims 1-14, wherein the molecule comprises amino acids sequences set forth in: i. SEQ ID NOs: lO and 11; or ii. SEQ ID NOs: 10 and 12; or iii. SEQ ID NOs: 13 and 14; or iv. SEQ ID NOs: 13 and 15; or v. SEQ ID NOs: 13 and 16; or vi. SEQ ID NOs: 13 and 17; or vii. SEQ ID NOs: 13 and 18; or viii. SEQ ID NOs: 19 and 20; or324806361 82Agent’s File Ref. SRZN-033 / 01WO 328202-2278 ix. SEQ ID NOs: 21 and 20; or x. SEQ ID NOs: 22, 23, and 24; or xi. SEQ ID NOs: 25, 23, and 24; or xii. SEQ ID NOs: 26, 27, and 28; or xiii. SEQ ID NOs: 29, 27, and 28.

16. A nucleic acid or a combination of nucleic acids encoding the WNT surrogate molecule of any one of claims 1-15, optionally wherein the nucleic acid(s) is / are DNA, cDNA, RNA, mRNA, modified mRNA, or a DNA / RNA hybrid.

17. The nucleic acid or a combination of nucleic acids of claim 16, which encodes the WNT surrogate molecule of any one of claims 1-15 and comprises: a) a first nucleic acid encoding the first polypeptide LRP5 / 6 binder; b) a second nucleic acid encoding the second polypeptide FZD binder.

18. A vector or a combination of vectors which comprises the nucleic acid or the combination of nucleic acids of claim 16 or 17, 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 adeno-associated viral, adenoviral, lentiviral, or retroviral), 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.

19. A host cell, which comprises: a) the WNT surrogate molecule of any one of claims 1-18; b) the nucleic acid or the combination of nucleic acids of claim 16 or 17; and / or c) the vector or the combination of vectors of claim 18, optionally wherein the host cell is:(i) mammalian, optionally human, non-human primate, monkey, rabbit, rodent, hamster, rat, or mouse; or324806361 83Agent’s File Ref. SRZN-033 / 01WO 328202-2278(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.

20. A pharmaceutical composition, which comprises:(I) (A) the WNT surrogate molecule of any one of claims 1-15;(B) the nucleic acid or the combination of nucleic acids of claim 16 or 17;(C) the vector or the combination of vectors of claim 16; and / or(D) the host cell of claim 19; and(II) a pharmaceutically acceptable carrier and / or excipient.

21. 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 ofA) the WNT surrogate molecule of any one of claimsl-15;B) the nucleic acid or the combination of nucleic acids of claim 16 or 17;C) the vector of claim 18;D) the host cell of claim 19 or the population of cells of claim 19; and / orE) the pharmaceutical composition of claim 20, optionally wherein:(a) the subject is a mammal, optionally a human, a non-human primate;(b) the subject comprises or has a risk of developing a disease, disorder, or a condition; and / or(c) 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 / or324806361 84Agent’s File Ref. SRZN-033 / 01WO 328202-2278(ii) the method promotes, stimulates, enhances, and / or supports is recruitment of multiple receptor complexes each comprising FZD; LRP5 / 6.

22. The method of claim 21, 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.

23. The method of any one of claims 21 - 22, wherein the administering is:324806361 85Agent’s File Ref. SRZN-033 / 01WO 328202-2278(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, intradermal, intrathecal, intra-arterial, intraarticular, intraosseous, or intraperitoneal administration) or by inhalation; or(iv) enterally administering, optionally orally, sublingually, buccally, or rectally.

24. The method of any claims of 21-23, 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.324806361 86

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