Multi-specific WNT replacement molecules and their uses

Abstract

The present disclosure provides Wnt pathway agonists and related compositions, which can be used in any of a variety of therapeutic methods for treating diseases.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 694,339, filed Jul. 5, 2018; U.S. Provisional Application No. 62 / 782,122, filed Dec. 19, 2018; and U.S. Provisional Application No. 62 / 797,772, filed Jan. 28, 2019, each of which is incorporated herein by reference in its entirety.

[0003] Statement Regarding the Sequence Listing

[0004] The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is SRZN_008_03WO_ST25.txt. The text file is 878 KB, created on Jul. 5, 2019, and was electronically submitted via EFS-Web. Technical Field

[0005] The present disclosure generally relates to Wnt signaling pathway agonist molecules, compositions, and methods of using such Wnt signaling pathway agonist molecules and compositions. Such molecules can be used, for example, to modulate the Wnt signaling pathway. Background Art

[0006] Wnt (“wingless-related integration site” or “wingless and Int-1” or “wingless-Int”) ligands and their signals play a key role in controlling the development, homeostasis, and regeneration of many essential organs and tissues, including bone, liver, skin, stomach, intestine, kidney, central nervous system, mammary gland, taste buds, ovary, cochlea, and many other tissues (e.g., reviewed by Clevers, Loh, and Nusse, 2014; 346:1248012). Modulation of the Wnt signaling pathway has the potential to treat degenerative diseases and tissue damage.

[0007] One of the challenges in using modulation of Wnt signaling as a therapy is the presence of multiple Wnt ligands and Wnt receptors, Frizzled proteins 1-10 (Fzd1-10), many of which are expressed in multiple tissues and with overlapping Fzd. Canonical Wnt signaling also involves low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) or low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) as co-receptors, which, in addition to Fzd, are also widely expressed in various tissues. The ratio of Fzd to the LRP binding moiety has not been previously explored to modulate the level of signaling and confer tissue and / or functional specificity.

[0008] The Wnt signaling pathway is subdivided into the canonical (β-catenin-dependent) and non-canonical (β-catenin-independent) pathways. The non-canonical pathway can be further divided into two distinct branches—the planar cell polarity (PCP) pathway and the Wnt / Ca 2+ pathway. Binding of certain Wnt ligands to certain Frizzled (Fzd) receptors or combinations of Fzd receptors can trigger different pathways and / or confer tissue and functional specificity.

[0009] Accordingly, there is a clear need in the art for binding moieties that specifically bind to one or more of Fzd, LRP5, or LRP6 to modulate different Wnt signaling pathways. There is also a need to generate binding moieties having a certain ratio of co-receptors (e.g., Fzd and LRP receptors) to modulate the level of signal transduction and confer tissue and / or functional specificity. The present disclosure addresses these needs. SUMMARY OF THE INVENTION

[0010] In various embodiments, the present disclosure provides Wnt surrogate molecules and related uses thereof.

[0011] In one aspect, the present disclosure provides a multispecific Wnt surrogate molecule, wherein the Wnt surrogate molecule comprises: (i) a plurality of regions (Fzd-binding regions) each specifically binding to a set of one or more Fzd receptor epitopes, wherein at least two Fzd-binding regions bind to the same or different sets of one or more Fzd receptor epitopes; and (ii) one or more regions (LRP5 / 6-binding regions) specifically binding to low density lipoprotein (LDL) receptor-related protein 5 (LRP5) and / or LDL receptor-related protein 6 (LRP6).

[0012] In some embodiments, at least two Fzd-binding regions bind to different sets of one or more Fzd receptors, different sets of one or more epitopes within the same set of one or more Fzd receptors, or a combination thereof.

[0013] In some embodiments, each Fzd-binding region binds to one or more of the following: Frizzled-1 (Fzd1), Frizzled-2 (Fzd2), Frizzled-3 (Fzd3), Frizzled-4 (Fzd4), Frizzled-5 (Fzd5), Frizzled-6 (Fzd6), Frizzled-7 (Fzd7), Frizzled-8 (Fzd8), Frizzled-9 (Fzd9), and Frizzled-10 (Fzd10).

[0014] In some embodiments, at least one Fzd binding region 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 Fzd10; (ix) Fzd5, Fzd8, and Fzd10; (x) Fzd4, Fzd5, and Fzd8; or (xi) Fzd1, Fzd5, Fzd7, and Fzd8.

[0015] In some embodiments, the plurality of Fzd binding regions includes: (i) a first Fzd binding region that binds to a first set of one or more Fzd receptors; and (ii) a second Fzd binding region that binds to a second, different set of one or more Fzd receptors. In some embodiments, the first Fzd binding region binds to one or more of Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, and Fzd10, and the second Fzd binding region binds to one or more of Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, and Fzd10. In some embodiments, the first Fzd binding region binds to Fzd4, and the second Fzd binding region binds to Fzd9.

[0016] In some embodiments, the plurality of Fzd binding regions includes: (i) a first Fzd binding region that binds to a first set of one or more epitopes within a set of one or more Fzd receptors; and (ii) a second Fzd binding region that binds to a second, different set of one or more epitopes within the same set of one or more Fzd receptors.

[0017] In some embodiments, the Wnt surrogate binds to at least one Fzd receptor that induces non-canonical Wnt signaling; and the second Fzd binding region binds to at least one Fzd receptor that induces canonical Wnt signaling. In additional embodiments, the Wnt surrogate that binds to the first Fzd receptor and the second Fzd receptor elicits canonical Wnt signaling; or non-canonical Wnt signaling.

[0018] In some embodiments, at least one Fzd binding region binds monospecifically to a single Fzd receptor. In some embodiments, the at least one Fzd binding region binds monospecifically to Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, or Fzd10.

[0019] In some embodiments, at least one Fzd binding region binds to a region of the Fzd receptor that: (i) does not include the cysteine-rich domain (CRD) of the Fzd receptor; or (ii) includes less than the entire CRD of the FZD receptor; or (iii) partially overlaps with the CRD of the FZD receptor.

[0020] In some embodiments, the at least one Fzd binding region binds to the hinge region of the Fzd receptor. In some embodiments, the hinge region comprises an amino acid sequence having at least 90% identity, at least 95% identity, or at least 98% identity with any of the sequences shown in SEQ ID NOs: 98 - 107.

[0021] In some embodiments, the at least one Fzd binding region binds to the N-terminal region upstream of the CRD of the Fzd receptor. In some embodiments, the N-terminal region comprises an amino acid sequence having at least 90% identity, at least 95% identity, or at least 98% identity with SEQ ID NO: 108.

[0022] In some embodiments, at least one of the Fzd binding regions comprises one or more antigen-binding fragments of an antibody. In some embodiments, the one or more antigen-binding fragments are selected from the group consisting of IgG, scFv, Fab, and VHH or sdAb. In some embodiments, the one or more antigen-binding fragments are humanized.

[0023] In some embodiments, at least one Fzd binding region comprises an amino acid sequence having at least 90% identity with any of the sequences shown in Table 1A, Table 1B, SEQ ID NOs: 1 - 73 or an antigen-binding fragment thereof.

[0024] In some embodiments, one or more LRP5 / 6 binding regions comprise one or more antigen-binding fragments of an antibody. In some embodiments, the one or more antigen-binding fragments are selected from the group consisting of IgG, scFv, Fab, and VHH or sdAb. In some embodiments, the one or more antigen-binding fragments are humanized.

[0025] In some embodiments, the one or more LRP5 / 6 binding regions comprise an amino acid sequence having at least 90% identity to any of the sequences shown in Table 2A, Table 2B, or SEQ ID NO: 74-97, or an antigen-binding fragment thereof.

[0026] In some embodiments, the Wnt surrogate molecule comprises two or more LRP5 / 6 binding regions.

[0027] In some embodiments, the ratio of the Fzd binding region to the LRP5 / 6 binding region is expressed as Fzd:LRP5 / 6.

[0028] In some embodiments, the ratio of the Fzd binding region to the LRP5 / 6 binding region is Fzd n :LRP5 / 6 n (F n :L n ) where F and L are integers between 1 and 9, inclusive, and n is an integer between 1 and 4, inclusive.

[0029] In some embodiments, the ratio of the Fzd binding region to the LRP5 / 6 binding region, expressed as Fzd:LRP5 / 6, is selected from the group consisting of: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 2:1, 2:3, 2:5, 2:7, 7:2, 5:2, 3:2, 3:4, 3:5, 3:7, 3:8, 8:3, 7:3, 5:3, 4:3, 4:5, 4:7, 4:9, 9:4, 7:4, 5:4, 6:7, 7:6, 1:2, 1:3, 1:4, 1:5, 1:6, 2:1 (in the case of two Fzd binding entities and one LRP binding entity), 1:2 (in the case of one Fzd binding entity and two LRP binding entities), 2:1:1 (in the case of two different LRP binding entities), 1:1:2 (in the case of two different Fzd binding entities), 1:1:1 (in the case of two different Fzd binding entities and one LRP binding entity or one Fzd binding entity and two different LRP binding entities), and 1:1:1:1 (in the case where all Fzd and LRP binding entities are different).

[0030] In some embodiments, the ratio of the Fzd binding region to the LRP5 / 6 binding region (Fzd:LRP5 / 6) comprises two Fzd binding regions and two LRP5 / 6 binding regions; two Fzd binding regions and one LRP5 / 6 binding region; or one Fzd binding region and two LRP5 / 6 binding regions.

[0031] In some embodiments, the ratio of the Fzd binding region to the LRP5 / 6 binding region (Fzd:LRP5 / 6) includes a first Fzd binding region, a second Fzd binding region, and one LRP5 / 6 binding region; or a first Fzd binding region, a second Fzd binding region, a first LRP5 / 6 binding region, and a second LRP5 / 6 binding region. In additional embodiments, the first Fzd binding region and the second Fzd binding region bind to different Fzd receptors or to the same Fzd receptor at different regions / epitopes, and the first LRP5 / 6 binding region and the second LRP5 / 6 binding region bind to different epitopes or different LRP proteins.

[0032] In some embodiments, the LRP binding region includes a first LRP binding region and a second LRP binding region, where the first LRP binding region binds to a first set of one or more LRP receptors and the second LRP binding region binds to a second, different set of one or more LRP receptors.

[0033] In some embodiments, the Wnt surrogate molecule includes a structural form selected from the group consisting of: hetero-Ig, dual-affinity re-targeting (DART), tandem dual-affinity re-targeting (DART), bispecific antibody-Fc, tandem Fab, tandem Fab IgG (FIT-Ig), Fv-IgG, and tandem scFv.

[0034] In some embodiments, the Wnt surrogate molecule includes: (i) a first light chain and a first heavy chain that form a first Fzd binding region; and (ii) a second light chain and a second heavy chain that form a second Fzd binding region, where the first Fzd binding region and the second Fzd binding region bind to different sets of one or more Fzd receptor epitopes.

[0035] In some embodiments, the Wnt surrogate molecule includes a first LRP5 / 6 binding region that is fused to the N-terminus of the first light chain, the C-terminus of the first light chain, the N-terminus of the first heavy chain, or the C-terminus of the first heavy chain. In some embodiments, the Wnt surrogate molecule includes a second LRP5 / 6 binding region that is fused to the N-terminus of the second light chain, the C-terminus of the second light chain, the N-terminus of the second heavy chain, or the C-terminus of the second heavy chain.

[0036] In some embodiments, the first heavy chain and the second heavy chain are connected to each other. In some embodiments, the first heavy chain comprises a first CH3 domain, the second heavy chain comprises a second CH3 domain, and the first CH3 domain and the second CH3 domain are connected to each other. In some embodiments, the first CH3 domain and the second CH3 domain are connected to each other by knobs-into-holes mutations. In some embodiments, (i) the first heavy chain and / or the second heavy chain comprises an amino acid sequence having at least 90% identity, at least 95% identity or at least 98% identity with any one of the sequences shown in SEQ ID NOs: 110, 112, 114, 116, 118, 120 or 122 (or as shown in Table 5 or Table 6A), and (ii) the first light chain and / or the second light chain comprises an amino acid sequence having at least 90% identity with any one of the sequences shown in SEQ ID NOs: 109, 111, 113, 115, 117, 119 or 121 (or as shown in Table 5 or Table 6A). In some embodiments, the Wnt surrogate molecule comprises one or more sequences (e.g., two or three sequences) having at least 90%, at least 95%, at least 98% or at least 99% sequence identity with the sequences disclosed in Table 5 or Table 6A. In certain embodiments, the Wnt surrogate molecule comprises the sequences set forth for any Wnt surrogate molecule in Table 5 or Table 6A, or sequences having at least 90%, at least 95%, at least 98% or at least 99% identity thereto.

[0037] In another aspect, the Wnt surrogate molecule has a structure as shown in Table 6B.

[0038] In some embodiments, the Wnt surrogate molecule modulates the Wnt signaling pathway in cells, optionally mammalian cells. In some embodiments, the Wnt surrogate molecule enhances signal transduction through the Wnt signaling pathway in the cells. In some embodiments, the Wnt signaling pathway is the canonical Wnt signaling pathway. In some embodiments, the Wnt signaling pathway is the non-canonical Wnt signaling pathway.

[0039] In another aspect, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient, diluent or carrier and a Wnt surrogate molecule according to any one of the embodiments herein.

[0040] In another aspect, the present disclosure provides a method for activating the Wnt signaling pathway in a cell, the method comprising contacting the cell with a Wnt surrogate molecule according to any one of the embodiments herein, wherein the Wnt surrogate molecule is an agonist of the Wnt signaling pathway.

[0041] In another aspect, the present disclosure provides a method for treating a subject having a disease or disorder, the method comprising administering to the subject an effective amount of a pharmaceutical composition of any one of the embodiments herein, wherein the Wnt replacement molecule is an agonist of the Wnt signaling pathway.

[0042] In some embodiments, the disease or disorder is associated with reduced or impaired Wnt signaling, and / or wherein the subject would benefit from increased Wnt signaling. In some embodiments, the disease or disorder is selected from the group consisting of: fracture, stress fracture, vertebral compression fracture, osteoporosis, osteoporotic fracture, non-union fracture, delayed union fracture, spinal fusion, preoperative optimization of spinal surgery, osteonecrosis, osseointegration of implants or orthopedic devices, osteogenesis imperfecta, bone graft, tendon repair, tendon-to-bone integration, tooth growth and regeneration, maxillofacial surgery, dental implant, periodontal disease, maxillofacial reconstruction, jaw, hip or femoral head necrosis, avascular necrosis, hair loss, hearing loss, vestibular hypofunction, macular degeneration, age-related macular degeneration (AMD), vitreoretinopathy, retinopathy, diabetic retinopathy, retinal degenerative diseases, Fuchs' dystrophy, corneal diseases, stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, muscular dystrophy, muscle atrophy caused by sarcopenia or cachexia, diseases affecting the blood-brain barrier (BBB), spinal cord injury, spinal cord diseases, oral mucositis, short bowel syndrome, inflammatory bowel disease (IBD), metabolic syndrome, diabetes, dyslipidemia, pancreatitis, pancreatic exocrine insufficiency, wound healing, diabetic foot ulcer, pressure ulcer, venous leg ulcer, epidermolysis bullosa, aplasia cutis, myocardial infarction, coronary artery disease, heart failure, hematopoietic cell disorders, immunodeficiency, graft-versus-host disease, acute kidney injury, chronic kidney disease, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, acute liver failure of all causes, drug-induced acute liver failure, alcoholic liver disease, chronic liver failure of all causes, cirrhosis, liver fibrosis of all causes, portal hypertension, chronic liver insufficiency of all causes, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD) (fatty liver), alcoholic hepatitis, hepatitis C virus-induced liver disease (HCV), hepatitis B virus-induced liver disease (HBV), other viral hepatitis (e.g., hepatitis A virus-induced liver disease (HAV) and hepatitis D virus-induced liver disease (HDV)), primary biliary cirrhosis, autoimmune hepatitis, liver surgery, liver injury, liver transplantation, "small for size" syndrome in liver surgery and transplantation, congenital liver diseases and disorders, any other liver disorder or defect caused by genetic diseases, degeneration, aging, drugs and injury.

[0043] In some embodiments, the disease or disorder is a skeletal disease or disorder. In some embodiments, the Wnt replacement molecule binds to: (i) Fzd1, Fzd2, and Fzd7; or (ii) Fzd1, Fzd2, Fzd5, Fzd7, and Fzd8. Brief Description of the Drawings

[0044] This patent or application document contains at least one color drawing. After a request is made and the necessary fees are paid, a copy of this patent or patent application publication with one or more color drawings will be provided by the patent office.

[0045] Figure 1 .Schematic diagram of illustrative forms of Wnt surrogate molecules. Different VHHs, Fvs or scFvs, bispecific antibodies or Fabs with different VL and VH regions targeting different Fzd receptors and Lrp receptors bind in different ratios. Different colors represent different binding entities (the binding entities can bind to the same target or different targets).

[0046] Figure 2A .Schematic diagram of an Fzd receptor containing a cysteine-rich domain (CRD), a hinge region, and an N-terminal region.

[0047] Figure 2B .Schematic diagram of a Wnt surrogate molecule with binding specificity for the hinge region of the Fzd receptor.

[0048] Figure 2C .Binding kinetics of 1791-3 and 1291-3 Wnt surrogate molecules.

[0049] Figure 2D .In vitro activity of 1791-3 and 1291-3 Wnt surrogate molecules.

[0050] Figure 3A .Schematic diagram of monospecific and multispecific Wnt surrogate molecules.

[0051] Figure 3B .In vitro activity of Wnt surrogate molecules in 293STF cells.

[0052] Figure 3C .In vitro activity of Wnt surrogate molecules in 293STF cells overexpressing Fzd4 (293STF Fzd4OE).

[0053] Figure 3D .In vitro activity of Wnt surrogate molecules in 293STF cells overexpressing Fzd9 (293STF Fzd9OE).

[0054] Figure 3C .In vitro activity of Wnt surrogate molecules in 293STF cells overexpressing Fzd4 and Fzd9 (293STF Fzd4OE+Fzd9OE).

[0055] Figure 4A - 4EShows the sequence alignment of the hinge regions of different Fzds (SEQ ID NO: 2251 - 2260).

[0056] Figure 5A - 5C Shows the structure of heterologous molecules containing different Fzd receptors and soluble ligands of Lrp5, and their in vitro activity in 293STF cells.

[0057] Figure 6A - 6E Shows the structure of Wnt replacement molecules with different ratios of Fzd to Lrp binding entities, and their effect on Wnt3a activation of β - catenin - dependent signaling.

[0058] Figure 7A - 7D Shows the structure of heterodimerization of two different Lrp binding entities and Fzd binding entities and their in vitro activity in 293STF cells.

[0059] Figure 8A - 8J Shows that the 1:1 bivalent bispecific L1 / F1 tandem scFv molecule does not efficiently activate β - catenin - dependent WNT signaling. (A) Diagram of the 1:1 bivalent bispecific L1 / F1 tandem scFv construct. Each circle represents an scFv domain, and the thin black lines at each end of the molecule represent 6xHis tags. (B) Ni - resin - purified tandem scFv molecules separated on a 4 - 15% SDS - PAGE gel. Left panel, from left to right: L1 - F1 tandem scFv with 5 - mer, 10 - mer, and 15 - mer linkers under reducing (R, lanes 1 - 3) or non - reducing (NR, lanes 4 - 6) conditions. Right panel, from left to right, F1 - L1 tandem scFv with 5 - mer, 10 - mer, and 15 - mer linkers under reducing (R, lanes 1 - 3) or non - reducing (NR, lanes 4 - 6) conditions. C) and D) Dose - dependent STF activity of Ni - resin - purified tandem scFv, where L1 is fused to the N - terminus of F1 (C), or F1 is fused to the N - terminus of L1 (D). E) Dose - dependent STF activity of the monomer peak fraction of tandem scFv from size - exclusion column (SEC) purification, where L1 is fused to the N - terminus of F1. F) Dose - dependent STF activity of the monomer peak fraction of tandem scFv from SEC purification, where F1 is fused to the N - terminus of L1. G) and H) STF activity of SEC fractions of different tandem scFvs. The arrow on each panel indicates the position of the monomer tandem scFv protein. I) Elution curve of protein standards on the SEC column, and the thick arrow indicates the expected position of the monomer form of the tandem scFv molecule. J) Addition of anti - His antibody to L1:5:F1 induces significant activation of β - catenin - dependent WNT signaling.

[0060] Figure 9.1:1 divalent bispecific L1 / F1 tandem scFv molecules did not efficiently activate β-catenin-dependent WNT signaling. Dose-dependent STF activity of the tandem scFv, purified alone from Ni column or additionally from SEC column, where L1 is fused to the N-terminus of F1 and F1 is fused to the N-terminus of L1, compared to recombinant WNT3A and surrogate WNT, 18R5-DKK1c. These data are the same as Figure 1 C-1F, in addition to including data on positive control molecules.

[0061] Figure 10A - 10F . Increasing the valency of the L1 and F1 tandem scFv by fusion to the Fc domain significantly increased the activity of Wnt signaling. A) Schematic of the F1 and L1 tandem scFv fused to the Fc domain to generate a 2:2 tetravalent bispecific form. B-C) STF activity of SEC fractions of the tandem scFv-Fc molecule. D) Elution curve of protein standards on the SEC column, with the thick arrow indicating the expected position of the monomeric form of the tandem scFv-Fc molecule. E-F) Dose-dependent STF activity of the tandem scFv-Fc molecule from the protein peak fractions corresponding to the monomeric form of the molecule from the SEC column.

[0062] Figure 11A - 11B . STF activity and Octet binding curves of the 2:2 tetravalent bispecific F1 / L1 molecule. A) Dose-dependent STF activity of the F1 / L1 bivalent tandem scFv molecule from the protein peak fractions corresponding to the monomeric form, compared to recombinant WNT3A and 18R5-DKK1c. These data are the same as Figures 2E and 2F, in addition to including data on positive control molecules. B) Binding affinity of various 2:2 tetravalent bispecific tandem scFv molecules for FZD1 and LRP6E1E2 measured on the Octet.

[0063] Figure 12A - 12E. The 2:2 tetravalent bispecific molecule composed of two F1 and two L2 binding arms is highly potent in inducing Wnt signaling. A) Diagram showing the form of the 2:2 tetravalent bispecific molecule composed of the FZD binder F1 and the LRP6E3E4 binder L2. The STF activities of the SEC fractions of these different 2:2 molecules are shown in the following form diagrams. B-C) Dose-dependent STF activities of the 2:2 tetravalent bispecific molecule composed of F1 and L2 binding arms in two orientations, from the protein peak fractions corresponding to the monomeric form of each molecule from the SEC column. D) The activity of the 2:2 tetravalent bispecific molecule from the combination of F1 and L2 requires the presence of both the FZD and LRP6 binding arms, as substitution of either binding arm with a neutral anti-GFP scFv fragment results in no activity. E) The interaction of the molecule from D) with its corresponding receptor was determined by Octet. The binders L1, L2, and F1 in IgG1 form were also included as comparators.

[0064] Figure 13A - 13B . The 1:1 bivalent bispecific L2 / F1 tandem scFv molecule cannot effectively activate β-catenin-dependent WNT signaling. A) STF activities of the SEC fractions of the 1:1 bivalent bispecific tandem scFv molecule between F1 / L2 in two orientations. The molecular form diagram is also shown at the top. The arrows in each plot region indicate the position of the monomeric form of the protein. B) Dose-dependent STF activities of the molecule shown in A) from the protein peak fractions corresponding to the monomeric form of each molecule from the SEC column.

[0065] Figure 14A - 14I. A 2:2 tetravalent bispecific molecule composed of two F2s and two L1 or L2 binding arms activates Wnt signaling. A) Molecular form diagram of a 2:2 tetravalent bispecific molecule composed of F2 and L1 binding arms, and the STF activity of these different WNT replacement molecules on the entire SEC column. The arrow in each graph area indicates the position of the monomeric form of the protein. B) Dose-dependent STF activity of a 2:2 tetravalent bispecific molecule composed of F2 and L1 binding arms in both orientations, from the protein peak fractions corresponding to the monomeric form of each molecule from the SEC column. Compared with WNT3A, these alternative WNT agonists have higher potency but lower efficacy in activating Wnt signaling. C) and D) 1:1 divalent bispecific molecular form and STF activity of the SEC column fractions of the molecule composed of F2 and L2 combinations in both orientations. In the 1:1 form, the F2-L2 orientation seems inactive, while the reverse L2-F2 orientation seems active. The arrow in each graph area indicates the position of the monomeric form of the protein. E) Dose-dependent STF activity of the 1:1 divalent bispecific L2-F2 molecule from the protein peak fraction corresponding to the monomeric form of each molecule from the SEC column. F) and H) 2:2 tetravalent bispecific molecular form and STF activity of the SEC column fractions of the molecule composed of F2 and L2 combinations in both orientations. The arrow in each graph area indicates the position of the monomeric form of the protein. G) and I) Dose-dependent STF activity of the 2:2 tetravalent bispecific L2 / F2 molecules from F) and H) from the protein peak fractions corresponding to the monomeric form of each molecule from the SEC column.

[0066] Figure 15A - 15B . FZD specificity curves of F1, F2, F3 and STF activity of the 1:1 divalent bispecific F2 / L1 molecule. A) Binding affinity and specificity of F1, F2 and F3 for all 10 FZDs measured on the Octet. B) STF activity of the SEC fractions of the 1:1 divalent bispecific tandem scFv molecule between F2 / L1 in both orientations. The molecular form diagram is also shown at the top. The arrow in each graph area indicates the position of the monomeric form of the protein. The 1:1 divalent bispecific form is ineffective in inducing Wnt / β-catenin signaling.

[0067] Figure 16A - 16D. A 2:2 tetravalent bispecific molecule composed of two F3s and two L1 or L2 binding arms activates Wnt signaling. A) Diagram showing the form of a 2:2 tetravalent bispecific molecule composed of an FZD binder F3 and an LRP6 binder L1 or L2, where the FZD binder is attached to the N-terminus of the LRP binder. The STF activities of the SEC fractions of these two 2:2 molecules are shown in the following form. B) Diagram showing the form of a 2:2 tetravalent bispecific molecule composed of an FZD binder F3 and an LRP6 binder L1 or L2, where the LRP binder is attached to the N-terminus of the FZD binder, and the reverse orientation of the molecule is shown in A). The STF activities of the SEC fractions of these two 2:2 molecules are shown in the following form. C, D) Dose-dependent STF activities of the 2:2 tetravalent bispecific molecules from the protein peak fractions corresponding to the monomeric forms of each molecule from the SEC column. C) and D) correspond to the molecules from A) and B), respectively.

[0068] Figure 17 . The 1:1 bivalent bispecific L1 / F3 or L2 / F3 tandem scFv molecules do not efficiently activate β-catenin-dependent WNT signaling. STF activities of the SEC fractions of the 1:1 bivalent bispecific tandem scFv molecules between F3 / L1 or F3 / L2 in two orientations. The molecular form diagrams are also shown at the top. The arrows in each plot region indicate the positions of the monomeric forms of the proteins. The 1:1 bivalent bispecific form is ineffective in inducing Wnt / β-catenin signaling.

[0069] Figure 18A - 18B . The 2:2 tetravalent bispecific dumbbell form has similar activity to the 2:2 tetravalent bispecific tandem scFv-Fc form. A) Dose-dependent STF activities of the protein peak fractions corresponding to the monomeric forms of each molecule from the SEC column. The alternative WNT agonist tested here is the combination of F1 and L1 in the 2:2 tetravalent bispecific dumbbell form. This form is effective, however, its efficacy is much lower compared to WNT3A. L1 is preferably on the N-terminus of Fc. B) Dose-dependent STF activities of the protein peak fractions corresponding to the monomeric forms of each molecule from the SEC column. The alternative WNT agonist tested here is the combination of F1 and L2 in the 2:2 tetravalent bispecific dumbbell form. L2 is also preferably on the N-terminus of Fc.

[0070] Figure 19A - 19C. Various 1:1 divalent bispecific tandem scFv molecules showed little activity. A) Sequential binding of FZD8, followed by the 1:1 divalent bispecific tandem scFv molecules F3:5:L2 and L2:5:F3, and then addition of LRP6E3E4 on the Octet indicated that the 1:1 tandem scFv molecules could engage both FZD and LRP simultaneously. B) Diagram of various molecules with a 1:1 stoichiometry between the FZD and LRP binders. C) Dose responses of the various molecules in B) showed no induction of the STF signal.

[0071] Figure 20A - 20B . Various 1:1 bispecific scFv molecules showed little activity. Diagram of various combinations of 1:1 divalent bispecific scFv molecules between the F3 / L2 and F2 / L2 binder pairs. B) Dose responses of the various molecules depicted in A, B) showed no induction of the STF signal.

[0072] Figure 21A - 21K . Explore different stoichiometries of the FZD and LRP binders and combine binders or epitopes of different receptor specificities in a 2:2 tetravalent multispecific format. A, D) Diagrams of molecules with different stoichiometries between the FZD and LRP binders, such as 2 FZD binders and 1 LRP binder (2:1) or 1 FZD and 2 LRP binders (1:2). B, C) Dose responses of the molecules in A) in the STF reporter assay. E, F) Dose responses of the molecules in D) in the STF reporter assay. G) Molecular form of a 2:2 tetravalent trispecific molecule where the two FZD binders have different FZD specificities (1:1:2). H) Dose response of the molecule in G) in the STF reporter assay. I) Molecular form of a 2:2 tetravalent trispecific molecule where the two FZD binders have different FZD specificities and only one LRP binder (1:1:1:0). J) Dose response of the molecule in I) in the STF reporter assay. K) Molecular form of a 2:2 tetravalent trispecific molecule where both the two FZD binders and the two LRP binders have different FZD or LRP specificities (1:1:1:1). H) Dose response of the molecule in K) in the STF reporter assay. Detailed Description

[0073] The present disclosure relates to multispecific Wnt surrogate molecules that specifically bind to multiple different Frizzled (Fzd) receptors and epitopes, as well as LRP5 and / or LRP6, in order to modulate the Wnt signaling pathway. In certain embodiments, the Wnt surrogate molecules activate the Wnt signaling pathway or increase signal transduction through the Wnt signaling pathway. In some aspects, the Wnt surrogate molecules of the present disclosure have: (i) multiple domains that each specifically bind to one or more Frizzled (Fzd) receptors and / or a collection of one or more epitopes, herein referred to as "Fzd-binding domains"; and (ii) one or more domains that specifically bind to LRP5 and / or LRP6, herein referred to as "LRP5 / 6-binding domains". Certain embodiments encompass specific structural forms or arrangements of the Fzd-binding domains and the LRP5 / 6-binding domains that are conducive to modulating the Wnt signaling pathway and related biological effects, e.g., for treating diseases and disorders associated with Wnt signaling.

[0074] In certain embodiments, the Wnt surrogate molecules disclosed herein comprise multiple Fzd-binding domains that have binding specificities for different Fzd receptors and / or epitopes. For example, a Wnt surrogate molecule can comprise at least two Fzd-binding domains that each bind to a different collection of one or more Fzd receptors, a different collection of one or more epitopes within the same collection of one or more Fzd receptors, or a combination thereof. For different Fzd receptor epitopes or multiple Fzd receptor epitopes, each Fzd-binding domain can be monospecific, bispecific, trispecific, etc. Such multispecific Wnt surrogate molecules are capable of selectively activating specific combinations of Fzd receptors while reducing or eliminating the activation of off-target Fzd receptors. Embodiments of the present disclosure are conducive to selectively modulating Wnt signaling in target cell types and / or treating specific diseases or disorders, e.g., by reducing off-target effects.

[0075] Embodiments of the invention relate to the use of Wnt surrogate molecules in the diagnosis, assessment, and treatment of diseases and disorders associated with the Wnt signaling pathway. In certain embodiments, the subject Wnt surrogate molecules are used to modulate the Wnt signaling pathway in cells or tissues. In certain embodiments, the subject Wnt surrogate molecules are used to treat or prevent diseases and disorders associated with abnormal or dysregulated (e.g., reduced) Wnt signaling or diseases and disorders for which modulating (e.g., increasing) Wnt signaling would provide a therapeutic benefit.

[0076] Unless otherwise indicated to the contrary, the practice of the present disclosure will employ conventional methods within the scope of those skilled in the art of virology, immunology, microbiology, molecular biology, and recombinant DNA technology, many of which are described below for illustrative purposes. Such techniques are well explained in the literature. See, for example, Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, (2009); Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., John Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd ed., 2001); Maniatis et al., Molecular Cloning: A Laboratory Manual, (1982); DNA Cloning: A Practical Approach, Volumes I and II (D. Glover ed.); Oligonucleotide Synthesis (N. Gait ed., 1984); Nucleic Acid Hybridization (B. Hames and S. Higgins eds., 1985); Transcription and Translation (B. Hames and S. Higgins eds., 1984); Animal Cell Culture (R. Freshney ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984), and other similar references.

[0077] As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a / an" and "the" include plural referents.

[0078] As used herein, "A and / or B" encompasses one or more of A or B, and combinations thereof, such as A and B.

[0079] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" shall 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.

[0080] Unless otherwise expressly stated, each example in this specification is applicable to all other examples, with necessary modifications.

[0081] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, liposome transfection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as commonly practiced in the art or as described herein. These techniques and procedures, and related techniques and procedures, can generally be performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. Unless otherwise provided with specific definitions, the nomenclature used in conjunction with the molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, and the laboratory procedures and techniques of said molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry, are well known and commonly used nomenclature and laboratory procedures and techniques in the art. Standard techniques can be used for recombinant techniques, molecular biology, microbiology, chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and treatment of subjects.

[0082] Embodiments of the present disclosure relate to antibodies and antigen-binding fragments thereof that bind to one or more Fzd receptors. SEQ ID NOs: 1-73, Tables 1A and 1B, and Table 5 show the sequences of illustrative antibodies or antigen-binding fragments thereof or their complementarity-determining regions (CDRs).

[0083] Embodiments of the present disclosure relate to antibodies and antigen-binding fragments thereof that bind to LRP5 and / or LRP6. SEQ ID NOs: 74-97, Tables 2A and 2B, and Table 5 show the sequences of illustrative antibodies or antigen-binding fragments thereof or their complementarity-determining regions (CDRs).

[0084] As is well known in the art, an antibody is an immunoglobulin molecule capable of specifically binding to a target such as a carbohydrate, polynucleotide, lipid, polypeptide, etc. through at least one epitope recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies but also fragments thereof (e.g., dAb, Fab, Fab', F(ab') 2 , Fv), single-chain (scFv), (Nabs; also known as VHH or single domain antibodies (sdAb)), synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody or antigen-binding fragment thereof, humanized antibodies, chimeric antibodies, and any other modified configurations of immunoglobulin molecules comprising an antigen-binding site or fragment (epitope recognition site) with the desired specificity. "Bifunctional antibodies" (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. Also included herein are minibodies comprising an scFv linked to a CH3 domain (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., Proc. Natl. Acad. Sci. 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.

[0085] As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment containing at least one CDR of an immunoglobulin heavy chain and / or light chain or VHH or sdAb that binds to an antigen of interest, particularly one or more Fzd receptors or LRP5 or LRP6 receptors. In this regard, the antigen-binding fragments of the antibodies described herein can include 1, 2, 3, 4, 5, or all 6 CDRs from the VH and VL sequences of an antibody that binds one or more Fzd receptors or LRP5 and / or LRP6 as shown herein. In certain embodiments, the antigen-binding fragment can include all three VH CDRs or all three VL CDRs. Similarly, the antigen-binding fragment thereof can include all three CDRs of a VHH or sdAb. The antigen-binding fragment of an Fzd-specific antibody is capable of binding to an Fzd receptor. The antigen-binding fragment of an LRP5 / 6-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 the antigen-binding fragments of the antibodies disclosed herein, e.g., fusion proteins comprising the antigen-binding fragments of the antibodies disclosed herein, e.g., a fusion protein comprising a VHH or sdAb that binds one or more Fzd receptors and a VHH or sdAb that binds LRP5 and / or LRP6.

[0086] The term "antigen" refers to a molecule or part of a molecule that can be selectively bound by a binding entity such as an antibody and that can additionally be used in an animal to generate an antibody capable of binding an epitope of the antigen. In certain embodiments, a binding entity (e.g., a Wnt surrogate molecule or its binding region) is considered to specifically bind to an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. In certain embodiments, a Wnt surrogate molecule or its binding region (e.g., an antibody or its antigen-binding fragment) is considered to specifically bind to an antigen when the equilibrium dissociation constant ≤ 10 -7 M or ≤ 10 -8 M. In some embodiments, the equilibrium dissociation constant can be ≤ 10 -9 M or ≤ 10 -10 M.

[0087] In certain embodiments, antibodies and antigen-binding fragments thereof as described herein comprise heavy and light chain CDR sets inserted respectively between heavy and light chain framework region (FR) sets, the heavy and light chain FR sets providing support to the CDRs and defining the spatial relationship of the CDRs relative to each other. As used herein, the term "CDR set" refers to the three hypervariable regions of the heavy or light chain V region. Starting from the N-terminus of the heavy or light chain, these regions are designated "CDR1", "CDR2", and "CDR3", respectively. Thus, the antigen-binding site comprises six CDRs, which include the CDR sets from each of the heavy and light chain V regions. A polypeptide comprising a single CDR (e.g., CDR1, CDR2, or CDR3) is referred to herein as a "molecular recognition unit". Crystallographic analysis of a variety of antigen-antibody complexes has shown that the amino acid residues of the CDRs form extensive contacts with the bound antigen, with the most extensive antigen contacts being with the heavy chain CDR3. Thus, the molecular recognition unit is primarily responsible for the specificity of the antigen-binding site.

[0088] As used herein, the term "FR set" refers to the four flanking amino acid sequences that frame the CDRs of the heavy or light chain V region. Some FR residues can contact the bound antigen; however, the FRs are primarily responsible for folding the V region into the antigen-binding site, particularly the FR residues immediately adjacent to the CDRs. Within the FRs, certain amino acid residues and certain structural features are highly conserved. In this regard, all V region sequences contain an internal disulfide loop consisting of approximately 90 amino acid residues. When the V region folds into the binding site, the CDRs are presented as protruding loop motifs that form the antigen-binding surface. It is generally recognized that there are conserved structural regions of the FRs that shape the folded form of the CDR loops into certain "canonical" structures - regardless of the precise CDR amino acid sequences. Further, certain FR residues are known to participate in non-covalent interdomain contacts that stabilize the interaction between the heavy and light chains of the antibody.

[0089] The structures and positions of immunoglobulin CDRs and variable domains can be determined by reference to Kabat, E.A. et al., "Sequences of Proteins of Immunological Interest". 4th Ed. US Department of Health and Human Services. 1987 and its updates, currently available on the Internet (immuno.bme.nwu.edu). Abgenesis software from Distributed Bio (South San Francisco, CA) was used to map the specificity-determining regions of the antibodies disclosed herein, which specificity-determining regions include the Kabat definition of CDRs. (Padlan et al., "FASEB J." 9, 133-139 (1995)).

[0090] "Monoclonal antibody" refers to a homogeneous population of antibodies, where monoclonal antibodies include the amino acids (naturally occurring and non-naturally occurring) involved in the selective binding of an epitope. Monoclonal antibodies are highly specific, being directed against a single epitope. The term "monoclonal antibody" encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also their fragments (such as Fab, Fab', F(ab') 2 , Fv), single-chain (scFv), VHH or sdAb, their variants, fusion proteins comprising antigen-binding fragments of monoclonal antibodies, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configurations of immunoglobulin molecules comprising an antigen-binding fragment (epitope recognition site) with the desired specificity and the ability to bind an epitope, including the Wnt surrogate molecules disclosed herein. It is not intended to be limiting with respect to the source of the antibody or the manner in which it is formed (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term encompasses the entire immunoglobulin as well as the fragments etc. described above under the definition of "antibody".

[0091] The proteolytic enzyme papain preferentially cleaves IgG molecules to produce several fragments, two of which (F(ab) fragments) each comprise a covalent heterodimer containing a complete antigen-binding site. Pepsin is capable of cleaving IgG molecules to provide several fragments, including the F(ab') 2 fragment comprising two antigen-binding sites. Fv fragments for use in certain embodiments according to the present disclosure can be produced by preferential proteolytic cleavage of IgM and, less frequently, IgG or IgA immunoglobulin molecules. However, Fv fragments are more commonly derived using recombinant techniques known in the art. Fv fragments comprise non-covalent VH ::V L L Heterodimers that contain antigen-binding sites that retain many of the antigen recognition and binding capabilities of native antibody molecules. Inbar et al. (1972) Proc. Natl. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochem. 15:2706-2710; and Ehrlich et al. (1980) Biochemistry 19:4091-4096.

[0092]

[0092] In certain embodiments, single-chain Fv or scFV antibodies are contemplated. For example, diabodies (Ill et al., Prot. Eng. 10:949-57 (1997)); minibodies (Martin et al., EMBO J. 13:5305-9 (1994)); bispecific antibodies (Holliger et al., PNAS 90:6444-8 (1993)); or Janusins (Traunecker et al., EMBO J. 10:3655-59 (1991) and Traunecker et al., Int. J. Cancer Suppl. 7:51-52 (1992)) can be prepared using standard molecular biology techniques according to the teachings in this application regarding selecting antibodies with desired specificities. In other embodiments, bispecific or chimeric antibodies that cover the ligands of this disclosure can be prepared. For example, chimeric antibodies can include CDRs and framework regions from different antibodies, and bispecific antibodies that specifically bind to one or more Fzd receptors through one binding domain and specifically bind to a second molecule through a second binding domain can be generated. These antibodies can be produced by recombinant molecular biology techniques or can be physically conjugated together.

[0093]

[0093] A single-chain Fv (scFv) polypeptide is a covalently linked V H ::V L L Heterodimer that consists of a V H - and a V L- Gene fusion expression of encoding genes. Huston et al. (1988) Proceedings of the National Academy of Sciences of the United States of America 85(16):5879-5883. A variety of methods have been described for identifying chemical structures for converting the naturally aggregated but chemically separated light and heavy polypeptide chains from antibody V regions into scFv molecules that will fold into a three-dimensional structure substantially similar to the structure of the antigen-binding site. See, for example, U.S. Patent Nos. 5,091,513 and 5,132,405 to Huston et al.; and U.S. Patent No. 4,946,778 to Ladner et al.

[0094] In certain embodiments, the antibodies described herein are in the form of bispecific antibodies. A bispecific antibody is a multimer formed from polypeptides, each polypeptide comprising a first domain and a second domain, the first domain comprising a binding domain of an immunoglobulin light chain, the second domain comprising a binding domain of an immunoglobulin heavy chain, the two domains being linked (e.g., by a peptide linker) but not capable of associating with each other to form an antigen-binding site: an antigen-binding site is formed by associating the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94 / 13804).

[0095] The dAb fragment of an antibody consists of the VH domain (Ward, E.S. et al., Nature 341, 544-546 (1989)).

[0096] When bispecific antibodies are to be used, the antibodies can be made in a variety of ways (Holliger, P. and Winter G., Current Opinion Biotechnol. 4, 446-449 (1993)), such as conventional bispecific antibodies prepared chemically or from hybrid hybridomas, or can be any of the bispecific antibody fragments mentioned above. Bifunctional antibodies and scFvs without an Fc region can be constructed using only variable domains, thus potentially reducing the impact of anti-idiotypic responses.

[0097] In contrast to bispecific full antibodies, bispecific bifunctional antibodies can also be particularly useful because they can be readily constructed and expressed in E. coli. Phage display (WO94 / 13804) can be used to readily select bifunctional antibodies (and many other polypeptides, such as antibody fragments) with appropriate binding specificities from libraries. If one arm of the bifunctional antibody is to be kept constant, for example, specific for antigen X, a library can be generated in which the other arm is varied and an antibody with the appropriate specificity selected. Bispecific full antibodies can be generated by the "knobs-into-holes" engineering method (J.B.B. Ridgeway et al., Protein Engineering, 9, 616 - 621 (1996)).

[0098] In certain embodiments, the antibodies described herein can be provided in form. is an IgG4 antibody from which the hinge region has been removed (see GenMab Utrecht, The Netherlands; see also, e.g., US20090226421). This patented antibody technology yields a stable smaller antibody form with a longer therapeutic window than expected for current small antibody formats. IgG4 antibodies are considered inert and thus do not interact with the immune system. A fully human IgG4 antibody can be modified by eliminating the hinge region of the antibody to obtain a half-molecule fragment with different stability properties relative to the corresponding full IgG4 (GenMab Utrecht). Bisecting the IgG4 molecule such that only one region remains on the that can bind to a cognate antigen (e.g., a disease target), and thus binds monovalently to only one site on the target cell.

[0099] In certain embodiments, the antibodies of the present disclosure can be in the form of VHHs or sdAbs. VHH or sdAb technology was initially developed after the discovery and identification of fully functional antibodies in camelids (e.g., camels and llamas) that consist solely of heavy chains and thus lack light chains. These heavy-chain-only antibodies contain a single variable domain (V HH ) and two constant domains (C H 2, C H3). The cloned and isolated single variable domains have full antigen-binding capacity and are highly stable. These single variable domains with their unique structural and functional properties form the basis of "VHH or sdAb". VHH or sdAb are encoded by a single gene and are efficiently produced in almost all prokaryotic and eukaryotic hosts, such as Escherichia coli (see, for example, U.S. Patent No. 6,765,087), molds (e.g., Aspergillus or Trichoderma), and yeasts (e.g., Saccharomyces, Kluyveromyces, Hansenula, or Pichia) (see, for example, U.S. Patent No. 6,838,254). The production process is scalable, and multiple kilograms of VHH or sdAb have been produced. VHH or sdAb can be formulated as ready-to-use solutions with a long shelf life. The method (see, for example, WO 06 / 079372) is a patented method for the automated high-throughput selection of B cells to generate VHH or sdAb against a desired target. VHH or sdAb are single-domain antigen-binding fragments of camelid-specific heavy-chain-only antibodies. VHH or sdAb typically have a small size of approximately 15 kDa.

[0100] In certain embodiments, the antibodies or antigen-binding fragments thereof disclosed herein are humanized. This involves chimeric molecules typically prepared using recombinant techniques, which have antigen-binding sites derived from immunoglobulins from non-human species and the remaining immunoglobulin structure based on the human immunoglobulin structure and / or sequence of the molecule. The antigen-binding site can include a full variable domain fused to a constant domain or only CDRs grafted onto a suitable framework region in the variable domain. The epitope-binding site can be wild-type or can be modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, but there is still a possibility of an immune response to the foreign variable region (LoBuglio, A.F. et al., (1989) Proceedings of the National Academy of Sciences of the United States of America, 86:4220-4224; Queen et al., Proceedings of the National Academy of Sciences of the United States of America (1988) 86:10029-10033; Riechmann et al., Nature, (1988) 332:323-327). Illustrative methods for humanizing the anti-Fzd antibodies disclosed herein include the methods described in U.S. Patent No. 7,462,697.

[0101] Another approach focuses not only on providing a human constant region but also on modifying the variable region to reshape it as much as possible into a human form. It is known that the variable regions of both the heavy and light chains contain three complementarity-determining regions (CDRs) flanked by four framework regions (FRs), the three CDRs vary in response to the epitope in question and determine the binding capacity, and the four FR regions are relatively conserved in a given species and presumably provide a scaffold for the CDRs. When preparing a non-human antibody with respect to a specific epitope, the variable region can be "reshaped" or "humanized" by grafting the CDRs derived from the non-human antibody onto the FRs present in the human antibody to be modified. The application of this method to various antibodies has been reported by: Sato, K. et al., (1993) Cancer Res. 53:851-856; Riechmann, L. et al., (1988) Nature 332:323-327; Verhoeyen, M. et al., (1988) Science 239:1534-1536; Kettleborough, C.A. et al., (1991) Protein Eng. 4:773-3783; Maeda, H. et al., (1991) Human Antibodies Hybridoma 2:124-134; Gorman, S.D. et al., (1991) Proc. Natl. Acad. Sci. USA 88:4181-4185; Tempest, P.R. et al., (1991) Bio / Technology 9:266-271; Co, M.S. et al., (1991) Proc. Natl. Acad. Sci. USA 88:2869-2873; Carter, P. et al., (1992) Proc. Natl. Acad. Sci. USA 89:4285-4289; and Co, M.S. et al., (1992) J Immunol 148:1149-1154. In some embodiments, the humanized antibody retains all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). In other embodiments, the humanized antibody has one or more CDRs (one, two, three, four, five, six) that are altered with respect to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

[0102] In certain embodiments, the antibodies of the present disclosure can be chimeric antibodies. In this regard, a chimeric antibody includes an antigen-binding fragment of an antibody operably linked or otherwise fused to a heterologous Fc portion of a different antibody. In certain embodiments, the heterologous Fc domain is human. In other embodiments, the heterologous Fc domain can be from a different Ig class derived from a parental antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In additional embodiments, the heterologous Fc domain can include CH2 and CH3 domains from one or more of different Ig classes. As described above with respect to humanized antibodies, the antigen-binding fragment of a chimeric antibody can include only one or more of the CDRs of the antibodies described herein (e.g., 1, 2, 3, 4, 5, or 6 CDRs of the antibodies described herein), or can include the entire variable domain (VL, VH, or both).

[0103] Wnt Substitute

[0104] In certain aspects, the present disclosure provides a Wnt surrogate molecule that binds to both: one or more Fzd receptors; and one or both of LRP5 and / or LRP6. The Wnt surrogate molecule can also be referred to as a "Wnt surrogate" or a "Wnt mimetic". In certain embodiments, the Wnt surrogate molecule binds to: one or more human Fzd receptors; and one or both of human LRP5 and / or human LRP6.

[0105] In certain embodiments, the Wnt surrogate molecule is capable of modulating or regulating Wnt signaling events in cells that are in contact with the Wnt surrogate molecule. In certain embodiments, the Wnt surrogate molecule increases Wnt signaling, such as through the canonical Wnt / β-catenin pathway. In certain embodiments, the Wnt surrogate molecule specifically modulates the biological activity of the human Wnt signaling pathway.

[0106] The Wnt surrogate molecules of the present disclosure are biologically active in binding to one or more Fzd receptors and one or more of LRP5 and LRP6 and in activation of Wnt signaling, i.e., the Wnt surrogate molecules are Wnt agonists. The term "Wnt agonist activity" refers to the ability of an agonist to mimic the action or activity of a Wnt protein in binding to an Fzd receptor and / or LRP5 or LRP6. The ability of the Wnt surrogate molecules and other Wnt agonists disclosed herein to mimic Wnt activity can be demonstrated by a variety of assays. Wnt agonists generally elicit responses or activities similar or identical to those elicited by the natural ligand of the receptor. Specifically, the Wnt agonists disclosed herein activate, enhance, or increase the canonical Wnt / β-catenin signaling pathway. As used herein, the term "enhance" refers to a measurable increase in the level of Wnt / β-catenin signaling compared to the level in the absence of a Wnt agonist (e.g., a Wnt surrogate molecule disclosed herein). In certain embodiments, for example, in the same cell type, the increase in the level of Wnt / β-catenin signaling is at least 10%, at least 20%, at least 50%, at least two-fold, at least five-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold compared to the level of Wnt / β-catenin signaling in the absence of a Wnt agonist. Methods for measuring Wnt / β-catenin signaling are known in the art and include the methods described herein. The Wnt surrogate molecules disclosed herein are multispecific, i.e., they specifically bind to two or more different epitopes. At least one epitope is located within one or more Fzd receptors, and at least one epitope binds to LRP5 and / or LRP6. In certain embodiments, the multispecific Wnt surrogate molecule is multispecific for Fzd receptor binding, i.e., it specifically binds to two or more different types of Fzd receptors, two or more different epitopes within a single type of Fzd receptor, or a combination thereof. For example, the multispecific Wnt surrogate molecule can bind to two or more of Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, and Fzd10. In some embodiments, the multispecific Wnt surrogate molecule 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 Fzd10; (ix) Fzd5, Fzd8, and Fzd10; (x) Fzd4, Fzd5, and Fzd8; or (xi) Fzd1, Fzd5, Fzd7, and Fzd8.

[0107] In certain embodiments, a Wnt surrogate molecule that is multispecific for Fzd binding comprises at least one Fzd-binding region that binds to multiple different Fzd receptor epitopes, such as epitopes within different Fzd receptors, different epitopes within the same Fzd receptor, or combinations thereof. For example, the Wnt surrogate molecule can comprise at least one Fzd-binding region that binds to two or more Fzd receptors, such as two or more of Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, and Fzd10. As another example, the Wnt surrogate molecule can comprise at least one Fzd-binding region that 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 Fzd10; (ix) Fzd5, Fzd8, and Fzd10; (x) Fzd4, Fzd5, and Fzd8; or (xi) Fzd1, Fzd5, Fzd7, and Fzd8.

[0108] Alternatively or in combination, in certain embodiments, a Wnt surrogate that is multispecific for Fzd binding comprises at least two Fzd-binding regions that each bind to a different set of one or more Fzd receptor epitopes, such as epitopes within different Fzd receptors, different epitopes within the same Fzd receptor, or combinations thereof. The set of one or more Fzd receptor epitopes can comprise one, two, three, four, five, six, seven, eight, nine, ten, or more Fzd receptor epitopes, such that each Fzd-binding region can be monospecific, bispecific, trispecific, tetraspecific, etc.

[0109] In certain embodiments, a multispecific Wnt surrogate comprises two or more Fzd-binding regions, wherein one or more of these Fzd-binding regions bind only specifically to one Fzd receptor or one receptor epitope. In certain embodiments, two or more, three or more, or four or more Fzd-binding regions in a multispecific Wnt surrogate each bind only specifically to one Fzd receptor or one receptor epitope, wherein at least two or more, at least three or more, or at least four or more Fzd-binding regions bind specifically to different Fzd receptors and / or receptor epitopes.

[0110] In certain embodiments, the Wnt surrogate molecule comprises a first Fzd-binding region that binds to a first set of one or more Fzd receptor epitopes, and a second Fzd-binding region that binds to a second, different set of one or more Fzd receptor epitopes. For example, the first Fzd-binding region can bind to a first set of one or more Fzd receptors, and the second Fzd-binding region can bind to a second, different set of one or more Fzd receptors. Alternatively or in combination, the first Fzd-binding region can bind to a first set of one or more epitopes within a set of one or more Fzd receptors, and the second Fzd-binding region can bind to a second, different set of one or more epitopes within the same set of one or more Fzd receptors. In certain embodiments, the first Fzd-binding region binds to one or more of Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, and Fzd10. In certain embodiments, the second Fzd-binding region binds to one or more of Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, and Fzd10.

[0111] In certain embodiments, the Wnt surrogate molecule comprises a first Fzd-binding region and a second Fzd-binding region, wherein: the first Fzd-binding region binds to Fzd1 and the second Fzd-binding region binds to Fzd2; the first Fzd-binding region binds to Fzd1 and the second Fzd-binding region binds to Fzd3; the first Fzd-binding region binds to Fzd1 and the second Fzd-binding region binds to Fzd4; the first Fzd-binding region binds to Fzd1 and the second Fzd-binding region binds to Fzd5; the first Fzd-binding region binds to Fzd1 and the second Fzd-binding region binds to Fzd6; the first Fzd-binding region binds to Fzd1 and the second Fzd-binding region binds to Fzd7; the first Fzd-binding region binds to Fzd1 and the second Fzd-binding region binds to Fzd8; the first Fzd-binding region binds to Fzd1 and the second Fzd-binding region binds to Fzd9; or the first Fzd-binding region binds to Fzd1 and the second Fzd-binding region binds to Fzd10.

[0112] In certain embodiments, the Wnt surrogate molecule comprises a first Fzd-binding region and a second Fzd-binding region, wherein: the first Fzd-binding region binds to Fzd2 and the second Fzd-binding region binds to Fzd3; the first Fzd-binding region binds to Fzd2 and the second Fzd-binding region binds to Fzd4; the first Fzd-binding region binds to Fzd2 and the second Fzd-binding region binds to Fzd5; the first Fzd-binding region binds to Fzd2 and the second Fzd-binding region binds to Fzd6; the first Fzd-binding region binds to Fzd2 and the second Fzd-binding region binds to Fzd7; the first Fzd-binding region binds to Fzd2 and the second Fzd-binding region binds to Fzd8; the first Fzd-binding region binds to Fzd2 and the second Fzd-binding region binds to Fzd9; or the first Fzd-binding region binds to Fzd2 and the second Fzd-binding region binds to Fzd10.

[0113] In certain embodiments, the Wnt surrogate molecule comprises a first Fzd-binding region and a second Fzd-binding region, wherein: the first Fzd-binding region binds to Fzd3 and the second Fzd-binding region binds to Fzd4; the first Fzd-binding region binds to Fzd3 and the second Fzd-binding region binds to Fzd5; the first Fzd-binding region binds to Fzd3 and the second Fzd-binding region binds to Fzd6; the first Fzd-binding region binds to Fzd3 and the second Fzd-binding region binds to Fzd7; the first Fzd-binding region binds to Fzd3 and the second Fzd-binding region binds to Fzd8; the first Fzd-binding region binds to Fzd3 and the second Fzd-binding region binds to Fzd9; or the first Fzd-binding region binds to Fzd3 and the second Fzd-binding region binds to Fzd10.

[0114] In certain embodiments, the Wnt surrogate molecule comprises a first Fzd-binding region and a second Fzd-binding region, wherein: the first Fzd-binding region binds to Fzd4 and the second Fzd-binding region binds to Fzd5; the first Fzd-binding region binds to Fzd4 and the second Fzd-binding region binds to Fzd6; the first Fzd-binding region binds to Fzd4 and the second Fzd-binding region binds to Fzd7; the first Fzd-binding region binds to Fzd4 and the second Fzd-binding region binds to Fzd8; the first Fzd-binding region binds to Fzd4 and the second Fzd-binding region binds to Fzd9; or the first Fzd-binding region binds to Fzd4 and the second Fzd-binding region binds to Fzd10.

[0115] In certain embodiments, the Wnt surrogate molecule comprises a first Fzd-binding region and a second Fzd-binding region, wherein: the first Fzd-binding region binds to Fzd5 and the second Fzd-binding region binds to Fzd6; the first Fzd-binding region binds to Fzd5 and the second Fzd-binding region binds to Fzd7; the first Fzd-binding region binds to Fzd5 and the second Fzd-binding region binds to Fzd8; the first Fzd-binding region binds to Fzd5 and the second Fzd-binding region binds to Fzd9; or the first Fzd-binding region binds to Fzd5 and the second Fzd-binding region binds to Fzd10.

[0116] In certain embodiments, the Wnt surrogate molecule comprises a first Fzd-binding region and a second Fzd-binding region, wherein: the first Fzd-binding region binds to Fzd6 and the second Fzd-binding region binds to Fzd7; the first Fzd-binding region binds to Fzd6 and the second Fzd-binding region binds to Fzd8; the first Fzd-binding region binds to Fzd6 and the second Fzd-binding region binds to Fzd9; or the first Fzd-binding region binds to Fzd6 and the second Fzd-binding region binds to Fzd10.

[0117] In certain embodiments, the Wnt surrogate molecule comprises a first Fzd-binding region and a second Fzd-binding region, wherein: the first Fzd-binding region binds to Fzd7 and the second Fzd-binding region binds to Fzd8; the first Fzd-binding region binds to Fzd7 and the second Fzd-binding region binds to Fzd9; or the first Fzd-binding region binds to Fzd7 and the second Fzd-binding region binds to Fzd10.

[0118] In certain embodiments, the Wnt surrogate molecule comprises a first Fzd-binding region and a second Fzd-binding region, wherein: the first Fzd-binding region binds to Fzd8 and the second Fzd-binding region binds to Fzd9; or the first Fzd-binding region binds to Fzd8 and the second Fzd-binding region binds to Fzd10. In certain embodiments, the Wnt surrogate molecule comprises a first Fzd-binding region and a second Fzd-binding region, wherein the first Fzd-binding region binds to Fzd9 and the second Fzd-binding region binds to Fzd10.

[0119] For each of the combinations of Fzd receptors disclosed above, it should be understood that in certain embodiments, the first binding region or the second binding region may specifically bind only to the designated Fzd, or it may also bind to additional Fzds. For example, where the first Fzd binding region binds to Fzd1 and the second Fzd binding region binds to Fzd2, the first Fzd binding region may specifically bind only to Fzd1, or it may also bind to one or more other Fzds in addition to Fzd1. Similarly, the second Fzd binding region may specifically bind only to Fzd2, or it may also bind to one or more other Fzds in addition to Fzd2. However, the first Fzd binding region and the second Fzd binding region bind to different sets of Fzd receptors.

[0120] In certain embodiments, the first binding region and the second binding region may specifically bind to different epitopes within the same Fzd receptor. For example, the first binding region may bind to a first epitope in Fzd1, and the second binding region may bind to a different second epitope in Fzd1. The first binding region may bind to a first epitope in Fzd2, and the second binding region may bind to a different second epitope in Fzd2. The first binding region may bind to a first epitope in Fzd3, and the second binding region may bind to a different second epitope in Fzd3. The first binding region may bind to a first epitope in Fzd4, and the second binding region may bind to a different second epitope in Fzd4. The first binding region may bind to a first epitope in Fzd5, and the second binding region may bind to a different second epitope in Fzd5. The first binding region may bind to a first epitope in Fzd6, and the second binding region may bind to a different second epitope in Fzd6. The first binding region may bind to a first epitope in Fzd7, and the second binding region may bind to a different second epitope in Fzd7. The first binding region may bind to a first epitope in Fzd8, and the second binding region may bind to a different second epitope in Fzd8. The first binding region may bind to a first epitope in Fzd9, and the second binding region may bind to a different second epitope in Fzd9. The first binding region may bind to a first epitope in Fzd10, and the second binding region may bind to a different second epitope in Fzd10.

[0121] For each of the combinations of Fzd receptor epitopes disclosed above, it should be understood that in certain embodiments, the first binding region or the second binding region may specifically bind only to the designated epitope, or it may also bind to additional epitopes. For example, where the first binding region binds to a first epitope in Fzd1 and the second binding region binds to a different second epitope in Fzd1, the first binding region may specifically bind only to the first epitope in Fzd1, or it may also bind to one or more additional epitopes in Fzd1 or other Fzd receptors. Similarly, the second Fzd binding region may specifically bind only to the second epitope in Fzd1, or it may also bind to one or more additional epitopes in Fzd1 or other Fzd receptors. In embodiments where the first binding region and the second binding region specifically bind to one or more identical Fzd receptors, the first binding region and the second binding region bind to different epitopes within one or more identical receptors.

[0122] In certain embodiments, the multispecific Wnt replacement molecule is multispecific for LRP5 / 6 binding, i.e., it specifically binds to two or more different epitopes within LRP5 and / or LRP6. In some embodiments, the multispecific Wnt replacement molecule comprises a first LRP5 / 6 binding region that binds to a first epitope within LRP5 and / or LRP6, and a second LRP5 / 6 binding region that binds to a different second epitope within LRP5 and / or LRP6.

[0123] For each combination in the combination of LRP receptor epitopes, it should be understood that in certain embodiments, the first binding region or the second binding region can specifically bind only to the designated epitope, or it can also bind to additional epitopes. For example, where the first binding region binds to the first epitope in LRP5E1E2 and the second binding region binds to a different second epitope in LRP5E1E2, the first binding region can specifically bind only to the first epitope in LRP5E1E2, or it can also bind to one or more other epitopes in LRP5E1E2 or other LRP receptors. Similarly, the second LRP binding region can specifically bind only to the second epitope in LRP5E1E2, or it can also bind to one or more other epitopes in LRP5E1E2 or other LRP receptors. In embodiments where the first binding region and the second binding region specifically bind to one or more identical LRP receptors, the first binding region and the second binding region bind to different epitopes within one or more identical receptors. Similar epitope binding can occur for LRP5E3E4, LRP6E1E2, and LRP6E3E4. In certain embodiments, the multispecific Wnt surrogate molecule is multispecific for Fzd binding and LRP5 / 6 binding. For example, the Wnt surrogate molecule can include multiple Fzd binding regions that each bind to a different set of one or more Fzd receptors and multiple LRP5 / 6 binding regions that each specifically bind to different epitopes within LRP5 and / or LRP6. It should be understood that the various embodiments of the Fzd binding regions and LRP5 / 6 binding regions disclosed herein can be combined in many ways to produce a multispecific Wnt surrogate molecule with any desired combination of Fzd and LRP5 / 6 binding specificities.

[0124] In certain embodiments, the Wnt surrogate molecules disclosed herein are multivalent. For example, it includes two or more regions that each specifically bind to the same epitope, such as two or more regions that bind to epitopes within one or more Fzd receptors and / or two or more regions that bind to epitopes within LRP5 and / or LRP6. In certain embodiments, the Wnt surrogate molecules disclosed herein include two or more regions that bind to epitopes within one or more Fzd receptors and two or more regions that bind to epitopes within LRP5 and / or LRP6.

[0125] In certain embodiments, the Wnt surrogate molecule includes an Fzd binding region and an LRP5 / 6 binding region, and the ratio is in terms of Fzd n :LRP5 / 6 n (F n :L n ) where F and L are integers between 1 and 9, inclusive, and n is an integer between 1 and 4, inclusive.

[0126] In certain embodiments, the ratio of the number of regions of a Wnt surrogate molecule that bind one or more Fzd receptors to the number of regions that bind LRP5 and / or LRP6 is or is about: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 2:1, 2:3, 2:5, 2:7, 7:2, 5:2, 3:2, 3:4, 3:5, 3:7, 3:8, 8:3, 7:3, 5:3, 4:3, 4:5, 4:7, 4:9, 9:4, 7:4, 5:4, 6:7, 7:6, 1:2, 1:3, 1:4, 1:5, 1:6, 2:1 (in the case of having two Fzd binders and one LRP binder), 1:2 (in the case of having one Fzd binder and two LRP binders), 2:1:1 (in the case of having two different LRP binders), 1:1:2 (in the case of having two different Fzd binders), 1:1:1 (in the case of having two different Fzd binders and one LRP binder or one Fzd binder and two different LRP binders), and 1:1:1:1 (in the case where all Fzd and LRP binders are different). Altering the ratio of the Fzd-binding portion to the LRP5 / 6-binding portion can confer tissue and / or functional specificity and modulate the level of signal transduction. In certain embodiments, the Wnt surrogate molecule is multispecific and multivalent.

[0127] The Wnt surrogate molecules disclosed herein can have any of a variety of different structural forms or configurations. The Wnt surrogate molecule can include polypeptide and / or non-polypeptide binding portions, such as small molecules. In certain embodiments, the Wnt surrogate molecule includes both a polypeptide region and a non-polypeptide binding portion. In certain embodiments, the Wnt surrogate molecule can include a single polypeptide, or the Wnt surrogate molecule can include two or more, three or more, or four or more polypeptides. In certain embodiments, the Wnt surrogate includes, for example, one, two, three, or four polypeptides that are linked or bound or fused to each other.

[0128] When the Wnt surrogate molecule includes a single polypeptide, it can be a fusion protein that includes one or more Fzd-binding regions (also referred to herein as "Fzd-binding domains") and one or more LRP5 / 6-binding regions (also referred to herein as "LRP5 / 6-binding domains"). The binding regions can be directly fused, or the binding regions are linked by a linker (e.g., a polypeptide or a chemical linker, including but not limited to any of the polypeptides or chemical linkers disclosed herein).

[0129] When the Wnt surrogate molecule comprises two or more polypeptides, the polypeptides can be linked by covalent bonds (e.g., disulfide bonds) and / or non-covalent interactions. For example, the heavy chains of human immunoglobulin IgG interact directly at the level of their CH3 domains, while at the level of their CH2 domains, the heavy chains interact via carbohydrates attached to asparagine (Asn) N84.4 in the DE bend.

[0130] Wnt surrogate polypeptides can be engineered to promote binding between two polypeptides. For example, knob-into-hole amino acid modifications can be introduced into two different polypeptides to promote their binding. Knob-into-hole amino acid (AA) changes are a rational design strategy developed in antibody engineering for heavy chain heterodimerization in the production of bispecific IgG antibodies. The AA changes are engineered such that a knob is created on the CH3 of the heavy chain of a first antibody and a hole is created on the CH3 of the heavy chain of a second antibody. The knob can be represented by tyrosine (Y) belonging to the 'very large' IMGT volume class of AAs, while the hole can be represented by threonine (T) belonging to the'small' IMGT volume class. Other means of introducing modifications into polypeptides to promote their binding are known and available in the art. For example, specific amino acids can be introduced and used for cross-linking, such as introducing cysteine to form intermolecular disulfide bonds.

[0131] In certain embodiments, the Wnt surrogate molecule comprises one or more binding regions derived from an antibody or an antigen-binding fragment thereof (e.g., an antibody heavy chain or an antibody light chain or a fragment thereof). In certain embodiments, one or more of the polypeptides of the Wnt surrogate molecule are an antibody or an antigen-binding fragment thereof. In certain embodiments, the Wnt surrogate comprises two antibodies or antigen-binding fragments thereof, e.g., one antibody or antigen-binding fragment thereof binds to one or more Fzd receptors and another antibody or antigen-binding fragment thereof binds to LRP5 and / or LRP6. In certain embodiments, the Wnt surrogate comprises three antibodies or antigen-binding fragments thereof, e.g., one antibody or antigen-binding fragment thereof binds to a first set of one or more Fzd receptor epitopes, one antibody or antigen-binding fragment thereof binds to a second, different set of one or more Fzd receptor epitopes, and one antibody or antigen-binding fragment thereof binds to LRP5 and / or LRP6. In certain embodiments, the Wnt surrogate comprises four antibodies or antigen-binding fragments thereof, e.g., one antibody or antigen-binding fragment thereof binds to a first set of one or more Fzd receptor epitopes, one antibody or antigen-binding fragment thereof binds to a second, different set of one or more Fzd receptor epitopes, one antibody or antigen-binding fragment thereof binds to a first epitope within LRP5 and / or LRP6, and one antibody or antigen-binding fragment thereof binds to a different second epitope within LRP5 and / or LRP6.

[0132] In certain embodiments, the Wnt surrogate molecule comprises a polypeptide that includes two antibody heavy chain regions (e.g., hinge regions) joined together by one or more disulfide bonds. In certain embodiments, the Wnt surrogate molecule comprises a polypeptide that includes an antibody light chain region (e.g., the C L region) and an antibody heavy chain region (e.g., the C H 1) joined together by one or more disulfide bonds.

[0133] The Wnt surrogate molecule can have various different structural forms, including but not limited to Figure 1 the structural forms shown in

[0134] In one embodiment, the Wnt surrogate molecule comprises an scFv or an antigen-binding fragment thereof fused to a VHH or sdAb or an antigen-binding fragment thereof. In certain embodiments, the scFv specifically binds to one or more Fzd receptor epitopes, and the VHH or sdAb specifically binds to LRP5 and / or LRP6. In certain embodiments, the scFv specifically binds to LRP5 and / or LRP6, and the VHH or sdAb specifically binds to one or more Fzd receptor epitopes. In certain embodiments, the scFv or an antigen-binding fragment thereof is directly fused to the VHH or sdAb or an antigen-binding fragment thereof, while in other embodiments, the two binding regions are fused via a linker moiety. In certain embodiments, the VHH or sdAb is fused to the N-terminus of the scFV, while in other embodiments, the VHH or sdAb is fused to the C-terminus of the scFv. In certain embodiments, the scFv describes or includes any one of the CDR sets described herein. In certain embodiments, the VHH or sdAb describes or includes any one of the CDR sets disclosed herein.

[0135] In various embodiments, including but not limited to Figure 1As depicted, the Wnt surrogate molecules include one or more Fabs or antigen-binding fragments thereof and one or more VHHs or sdAbs or antigen-binding fragments thereof (or alternatively, one or more scFvs or antigen-binding fragments thereof). In certain embodiments, the Wnt surrogate includes two or more Fabs, each of which specifically binds a different set of one or more Fzd receptor epitopes, and a VHH or sdAb (or scFv) that specifically binds LRP5 and / or LRP6. In certain embodiments, the Wnt surrogate includes a Fab that specifically binds LRP5 and / or LRP6 and two or more VHHs or sdAbs (or scFvs) that each specifically bind a different set of one or more Fzd receptor epitopes. In certain embodiments, the VHH or sdAb (or scFv) is fused to the N-terminus of the Fab, while in some embodiments, the VHH or sdAb (or scFv) is fused to the C-terminus of the Fab. In certain embodiments, the Fab is present in the form of a full IgG, and the VHH or sdAb (or scFv) is fused to the N-terminus and / or C-terminus of the IgG light chain. In certain embodiments, the Fab is present in the form of a full IgG, and the VHH or sdAb (or scFv) is fused to the N-terminus and / or C-terminus of the IgG heavy chain. In certain embodiments, two or more VHHs or sdAbs (or scFvs) are fused to the IgG at any combination of these positions, where each of the two or more VHHs or sdAbs (or scFvs) binds a different set of Fzds.

[0136] For example, genetic engineering can be used to generate a fusion polypeptide comprising a Fab fused to an Fc region to convert the Fab into a full IgG form comprising both a Fab and an Fc fragment, i.e., the Fab exists in the full IgG form. The Fc region of the full IgG form can be derived from any of a variety of different Fcs, including but not limited to wild-type or modified IgG1, IgG2, IgG3, IgG4 or other isotypes, such as wild-type or modified human IgG1, human IgG2, human IgG3, human IgG4, human IgG4Pro (including mutations in the core hinge region that prevent the formation of IgG4 half-molecules), human IgA, human IgE, human IgM or a modified IgG1 designated IgG1LALAPG. It has been shown that the L234A, L235A, P329G (LALA-PG) variants eliminate complement binding and fixation and Fc-γ-dependent antibody-dependent cell-mediated cytotoxicity (ADCC) in both murine IgG2a and human IgG1. These LALA-PG substitutions allow for more accurate translation of results generated with "effectorless" antibody framework scaffolds between mice and primates. In certain embodiments of any of the IgGs disclosed herein, the IgG comprises one or more of the following amino acid substitutions: N297G, N297A, N297E, L234A, L235A or P236G.

[0137] Non-limiting examples of bivalent and bispecific Wnt surrogate molecules that are bivalent for one or more Fzd receptor epitopes and LRP5 and / or LRP6 are provided as Figure 1 The first four structures depicted, where the VHH or sdAb or scFv is depicted as a single solid oval in red, blue or yellow, and the Fab or IgG is depicted in blue. As shown, the VHH or sdAb (or scFv) can be fused to the N-terminus of both light chains, the N-terminus of both heavy chains, the C-terminus of both light chains or the C-terminus of both heavy chains. Further envisioned is that, for example, the VHH or sdAb (or scFv) can be fused to the N- and C-termini of the heavy and / or light chains, the N-terminus of the light and heavy chains, the C-terminus of the heavy and light chains, the N-terminus of the heavy chain and the C-terminus of the light chain, or the C-terminus of the heavy chain and the N-terminus of the light chain. In other related embodiments, two or more VHHs or sdAbs (or scFvs) can optionally be fused together by a linker moiety and fused to the Fab or IgG at one or more of these positions. In certain embodiments, two or more VHHs or sdAbs (or scFvs) can each bind a different set of one or more Fzd receptor epitopes.

[0138] In related embodiments, the Wnt surrogate molecule has a hetero-Ig form, while the Fab exists in the form of a half-antibody, and one or more VHHs or sdAbs (or scFvs) are fused to one or more of the N-terminus of the Fc, the N-terminus of the Fab, the C-terminus of the Fc, or the C-terminus of the Fab. In certain embodiments, two or more VHHs or sdAbs (or scFvs) are fused to the N-terminus of the Fc, the N-terminus of the Fab, the C-terminus of the Fc, or the C-terminus of the Fab, wherein each of the VHHs or sdAbs (or scFvs) binds a different set of one or more Fzd receptor epitopes. A bispecific but monovalent version of this form for each receptor is described in Figure 1 C, 1D, 1E, 1F, and the version can be modified to include two or more Fzd-binding regions, wherein at least two of the Fzd-binding regions bind different sets of one or more Fzd receptor epitopes. In some embodiments, the Fab or its antigen-binding fragment (or IgG) is directly fused to the VHH or sdAb (or scFv) or its antigen-binding fragment, while in other embodiments, the binding regions are fused via a linker moiety. In certain embodiments, the Fab describes or includes any one of the CDR sets described herein. In certain embodiments, the VHH or sdAb or scFv describes or includes any one of the CDR sets disclosed herein.

[0139] In various embodiments, including but not limited to Figure 1Those depicted in V, 1W, 1X, 1AA, the Wnt surrogate molecules include one or more Fabs or antigen-binding fragments thereof that bind to one or more Fzd receptor epitopes and one or more Fabs or antigen-binding fragments thereof that bind to LRP5 and / or LRP6. In certain embodiments, the Wnt surrogate molecule includes two Fabs or antigen-binding fragments thereof that bind to different sets of one or more Fzd receptor epitopes and / or two Fabs or antigen-binding fragments thereof that bind to LRP5 and / or LRP6. In particular embodiments, one or more of the Fabs are present in the form of a complete IgG, and in certain embodiments, both Fabs are present in the form of a complete IgG. In certain embodiments, the Fab in the form of a complete IgG specifically binds to one or more Fzd receptor epitopes, while the other Fab specifically binds to LRP5 and / or LRP6. In certain embodiments, the Fab specifically binds to different sets of one or more Fzd receptor epitopes, and the Fab in the form of a complete IgG specifically binds to LRP5 and / or LRP6. In certain embodiments, the Fab specifically binds to LRP5 and / or LRP6, and the Fab in the form of a complete IgG specifically binds to different sets of one or more Fzd receptor epitopes. In certain embodiments, the Fab is optionally fused to the N-terminus of the IgG (e.g., the N-terminus of the heavy or light chain) via a linker. In certain embodiments, the Fab is fused to the N-terminus of the heavy chain of the IgG and not to the light chain. In particular embodiments, the two heavy chains may be fused directly or via a linker. Examples of such bispecific and bivalent to the two receptors are shown in Figure 1 V, 1W, 1X, and 1AA. In other related embodiments, two or more VHHs or sdAbs may optionally be fused together via a linker moiety and fused to a Fab or IgG at one or more of these positions. In related embodiments, the Wnt surrogate molecule has a hetero-IgG form, and one of the Fabs exists in the form of a half-antibody, and the other Fab is fused to one or more of the N-terminus of the Fc, the N-terminus of the Fab, and the C-terminus of the Fc. Figure 1 A bispecific but monovalent version of this form for each receptor is described in D, and the version can be modified to include one or more additional Fabs, where two or more Fabs bind to different sets of Fzd receptor epitopes. In certain embodiments, the Fab or its antigen-binding fragment is directly fused to another Fab or IgG or its antigen-binding fragment, while in other embodiments, the binding regions are fused via a linker moiety. In particular embodiments, one or both of the two Fabs described herein or include any one of the CDR groups described herein.

[0140] In certain embodiments, the Wnt surrogate molecule has the form described in PCT Application Publication No. WO2017 / 136820, e.g., the tandem Fab IgG (FIT-IG) form. Shiyong Gong, Fang Ren, Danqing Wu, Xuan Wu & Chengbin Wu (2017). FIT-IG also encompasses the form disclosed in "Fabs-in-tandem immunoglobulin is a novel and versatile bispecific design for engaging multiple therapeutic targets" Monoclonal Antibodies (mAbs), 9:7, 1118-1128, DOI: 10.1080 / 19420862.2017.1345401. In certain embodiments, FIT-IG combines the functions of two antibodies into one molecule by rearranging the DNA sequences of two parental monoclonal antibodies into two or three constructs and co-expressing them in mammalian cells. In Figure 1 A and 1B and Figure 2A and 2B examples of FIT-IG forms and constructs are provided. In certain embodiments, FIT-IG does not require Fc mutations, and does not require scFv elements, and does not require linkers or peptide linkers. The Fab domains in each arm work "in tandem" to form a tetravalent bispecific antibody with four active and independent antigen-binding sites that retain the biological functions of their parental antibodies. In specific embodiments, the Wnt surrogate includes Fab and IgG. In certain embodiments, the Fab binder LC is fused to the HC of IgG, for example, via linkers of various lengths therebetween. In various embodiments, the Fab binder HC may or may not be fused to the LC of IgG. Variants of this form are referred to as tandem Fab IgG (or FIT-Ig). In certain embodiments, FIT-Ig includes two or more Fzd-binding domains, where at least two Fzd-binding regions bind different sets of one or more Fzd receptor epitopes, e.g., different sets of one or more Fzd receptors or different sets of one or more epitopes within one or more identical Fzd receptors.

[0141] In certain embodiments, the Wnt surrogate molecule comprises two or more VHHs or sdAbs (or scFvs), comprising at least one VHH or sdAb (or scFv) that binds to one or more Fzd receptor epitopes and at least one VHH or sdAb (or scFv) that binds to LRP5 and / or LRP6. In some embodiments, one of the binding regions is a VHH or sdAb and the other binding region is a scFv. In certain embodiments, the Wnt surrogate molecule comprises three or more VHHs or sdAbs (or scFvs), comprising at least two VHHs or sdAbs (or scFvs) that bind to different sets of one or more Fzd receptor epitopes and at least one VHH or sdAb (or scFv) that binds to LRP5 and / or LRP6. The Wnt surrogate molecule comprising two or more VHHs or sdAbs (or scFvs) can be formulated in various configurations, including but not limited to Figure 1 those depicted in K, 1L, 1M, 1N, 1O, 1P, 1Q, 1S, 1T. In certain bispecific bivalent forms, the two or more VHHs or sdAbs (or scFvs) are optionally fused in series or fused to two different ends of the Fc via one or more linkers. In the presence of a linker, the linker and its length can be the same or different between the VHH or sdAb (or scFv) and other VHHs or sdAbs (or scFvs) or between the VHH or sdAb and the Fc. For example, in some embodiments, the VHH or sdAb is fused to the N-terminus and / or C-terminus of the IgG heavy chain. In certain embodiments, the two or more VHHs or sdAbs are fused to the IgG at any combination of these positions. Non-limiting examples of bivalent and bispecific Wnt surrogate molecules of this form are depicted as in Figure 1 the structures depicted in K, 1L, 1M, 1N, 1O, 1P, 1Q, where the first VHH or sdAb is depicted in blue, the Fc or IgG is depicted in blue, and the second VHH or sdAb is depicted in red. In various embodiments, the VHH or sdAb can be fused to the N-terminus of the Fc, the C-terminus of the Fc, or one or more VHHs or sdAbs can be fused to either or both of the N-terminus or C-terminus of the Fc. In related embodiments, the Wnt surrogate molecule has a hetero-IgG form, where one VHH or sdAb is present in the form of a half-antibody and the other VHH or sdAb is fused to the N-terminus or C-terminus of the Fc. Figure 1Depicted in E are bispecific but monovalent versions of this form for each receptor. In certain embodiments, a VHH or sdAb is directly fused to another VHH or sdAb, while in other embodiments, the binding regions are fused via a linker moiety. In particular embodiments, the VHH or sdAb describes or includes any one of the CDR sets described herein. In various embodiments, any one of these forms may include one or more scFvs in place of one or more VHHs or sdAbs.

[0142] In certain embodiments, the Wnt surrogate molecule is formulated as a bifunctional antibody. As Figure 1 shown in R, the binders to Fzd and LRP can also be joined together in a bifunctional antibody (or DART) configuration. The bifunctional antibody can also be in a single-chain configuration. If the bifunctional antibody is fused to Fc, a bivalent bispecific form will result. Without fusion to Fc, this will be a monovalent bispecific form. In certain embodiments, the bifunctional antibody is a non-covalent dimer scFv fragment composed of a heavy-chain variable region (VH) and a light-chain variable region (VL) linked by a small peptide linker. Another form of the bifunctional antibody is single-chain (Fv)2, in which two scFv fragments are covalently linked to each other. In particular embodiments, the bifunctional antibody includes two or more Fzd binding regions, wherein at least two of the Fzd binding regions bind different sets of one or more Fzd receptor epitopes.

[0143] In certain embodiments, two or more bifunctional antibodies, scFvs, and / or VHHs or sdAbs can be tandemly fused in a multivalent form ( Figure 1 A, 1B, 1F, 1U) with or without fusion to Fc. In particular embodiments, at least one of the bifunctional antibody, scFv, and / or VHH or sdAb binds one or more Fzd receptor epitopes, and at least one of the bifunctional antibody, scFv, and / or VHH or sdAb binds LRP5 and / or LRP6.

[0144] In various embodiments, including but not limited to Figure 1Those described in G or 1H, the Wnt surrogate molecules include two or more Fabs or antigen-binding fragments thereof that each bind a different set of one or more Fzd receptor epitopes, and one or more VHHs or sdAbs or antigen-binding fragments thereof (or, alternatively or in combination, one or more scFvs or antigen-binding fragments thereof) that bind, for example, LRP5 / 6. In certain embodiments, a first Fab specifically binds a first set of one or more Fzd receptor epitopes, a second Fab specifically binds a second different set of one or more Fzd receptor epitopes, and the VHH or sdAb (or scFv) specifically binds LRP5 and / or LRP6. In certain embodiments, the VHH or sdAb (or scFv) is fused to the N-terminus of the Fab, while in some embodiments, the VHH or sdAb (or scFv) is fused to the C-terminus of the Fab. In a particular embodiment, the Wnt surrogate molecule has a hetero-Ig form, as Figure 1 depicted in G, 1H, 1AG, wherein the first and second Fabs each exist as half-antibodies, and one or more VHHs or sdAbs (or scFvs) are fused to one or more of the N-terminus of the Fc, the N-terminus of the Fab, the C-terminus of the Fc (e.g., Figure 1 Y) or the C-terminus of the Fab. The first and second Fabs can be linked to each other by a knob-into-hole mutation in their respective Fcs (e.g., within the CH3 domain).

[0145] As discussed, in various embodiments, the Wnt surrogate molecules include one or more antibodies or antigen-binding fragments thereof disclosed herein. Thus, in certain embodiments, the Wnt surrogate includes two polypeptides, each of which includes a VHH or sdAb or scFv that binds LRP5 / 6 and a VHH or sdAb or scFv that binds one or more Fzd receptor epitopes. Optionally, one of the binding domains in the binding domains is an scFv and the other binding domain is a VHH or sdAb. In certain embodiments, each polypeptide includes an Fzd binding region that binds a different set of one or more Fzd receptor epitopes. In some embodiments, the Wnt surrogate includes three polypeptides, wherein the first polypeptide includes an antibody heavy chain and the second polypeptide includes an antibody light chain, wherein the antibody heavy chain and light chain bind LRP5 / 6 or one or more Fzd receptor epitopes, and wherein the third polypeptide includes a VHH or sdAb fused to the Fc region of the heavy chain, wherein the VHH or sdAb binds LRP5 / 6 or one or more Fzd receptor epitopes. In other embodiments, the Wnt polypeptide includes four polypeptides, the four polypeptides comprising two heavy chain polypeptides and two light chain polypeptides, wherein the two heavy chains and two light chains bind LRP5 / 6 or one or more Fzd receptor epitopes; and further includes one or more VHHs or sdAbs or scFvs fused to one or more of the heavy and / or light chains, wherein the VHH or sdAb or scFv binds LRP5 / 6 or one or more Fzd receptor epitopes. In another illustrative embodiment, the Wnt surrogate includes at least four polypeptides, the at least four polypeptides comprising two heavy chain polypeptides and two light chain polypeptides that bind LRP5 / 6 or one or more Fzd receptor epitopes, wherein the Wnt surrogate further includes a Fab that binds LRP5 / 6 or one or more Fzd receptor epitopes. For example, the Fab can include two polypeptides each fused to one of the two heavy chain polypeptides and two polypeptides each fused to one of the two light chain polypeptides, or the Fab can include two polypeptides each fused to one of the two heavy chain polypeptides and two additional polypeptides each binding to one of the two polypeptides fused to the heavy chain polypeptide, thereby forming a second Fab. Other configurations can be used to generate the Wnt surrogates disclosed herein. In certain embodiments of any of these forms, it includes at least two or more Fzd binding regions that each bind a different set of Fzd receptor epitopes.

[0146] In some embodiments, the different ratio (Fzd:LRP) of the Fzd binding region to the LRP binding region is at Figure 1It is represented by AB, 1AC, 1AD, 1AE, 1AF, 1AG, 1AH, 1AI, 1AJ, 1AK, 1AL, 1AM. In certain embodiments, one or more Fabs bind to one or more Fzd receptors or different epitopes in the same Fzd receptor, and one or more VHHs or sdAbs (or scFvs) bind to one or more LRP receptors or different epitopes in the same LRP receptor.

[0147] In certain embodiments, the Wnt surrogate molecule comprises a first light chain and a first heavy chain that form a first Fzd binding region, and a second light chain and a second heavy chain that form a second Fzd binding region, wherein the first Fzd binding region and the second Fzd binding region bind to different sets of Fzd receptor epitopes. In some embodiments, the first and second heavy chains are connected to each other. For example, the first heavy chain may comprise a first CH3 domain, the second heavy chain may comprise a second CH3 domain, and the first CH3 domain and the second CH3 domain may be connected to each other, for example, by a knob-into-hole mutation. In certain embodiments, the first heavy chain and / or the second heavy chain comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% identity to any one of the sequences shown in SEQ ID NOs: 110, 112, 114, 116, 118, 120 and 122. In certain embodiments, the first light chain and / or the second light chain comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% identity to any one of the sequences shown in SEQ ID NOs: 109, 111, 113, 115, 117, 119 and 121. In some embodiments, one or more heavy chains comprise an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% identity to any one of the sequences disclosed in Table 5. In some embodiments, one or more light chains comprise an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% identity to any one of the sequences disclosed in Table 5.

[0148] In certain embodiments, the Wnt surrogate molecule comprises a first LRP5 / 6 binding region and / or a second LRP5 / 6 binding region, and each of the binding regions may be or comprise a Fab or an scFv. The first and second LRP5 / 6 binding regions may bind to the same epitope within LRP5 / 6, or may bind to different epitopes within LRP5 / 6. The first LRP5 / 6 binding region may be fused to the N-terminus of the first light chain, the C-terminus of the first light chain, the N-terminus of the first heavy chain, or the C-terminus of the first heavy chain. The second LRP5 / 6 binding region may be fused to the N-terminus of the second light chain, the C-terminus of the second light chain, the N-terminus of the second heavy chain, or the C-terminus of the second heavy chain.

[0149] In certain embodiments, the Wnt surrogate molecule comprises an Fzd-binding region fused to or conjugated with a polypeptide that specifically binds to one or more Fzd receptors, e.g., an anti-Fzd antibody or an antigen-binding fragment thereof. In certain embodiments, the polypeptide that specifically binds to one or more Fzd receptors is an antibody or an antigen-binding fragment thereof. In some embodiments, it is an antibody or an antigen-binding fragment thereof disclosed in U.S. Provisional Patent Application No. 62 / 607,877, filed on December 19, 2017, entitled "Anti-Frizzled antibodies and Methods of Use", Attorney Docket No. SRZN-004 / 00US, which is incorporated herein by reference in its entirety.

[0150] In certain embodiments, at least one Fzd-binding region of the Wnt surrogate molecule comprises one or more antigen-binding fragments of an antibody. For example, one or more antigen-binding fragments can be or be derived from IgG, scFv, Fab, VHH, or sdAb. In some embodiments, one or more antigen-binding fragments are humanized.

[0151] In certain embodiments, the Fzd-binding region comprises three heavy-chain CDRs and / or three light-chain CDRs disclosed for any of the illustrative antibodies or fragments thereof that bind to one or more Fzd receptors provided in Table 1A. In certain embodiments, the Fzd-binding region comprises three heavy-chain CDRs and / or three light-chain CDRs disclosed for any of the illustrative antibodies or fragments thereof that bind to one or more Fzd receptors provided in Table 1A, wherein the CDRs collectively comprise one, two, three, four, five, six, seven, or eight amino acid modifications, e.g., substitutions, deletions, or additions. In some embodiments, the Fzd-binding region is a VHH or sdAb or is derived from a VHH or sdAb, and thus Table 1A contains only three heavy-chain CDRs. In certain embodiments, the Fzd-binding region comprises the three CDR HC sequences or variants provided in Table 1A, wherein the CDRs collectively comprise one, two, three, four, five, six, seven, or eight amino acid modifications.

[0152] In certain embodiments, the Fzd binding region comprises a heavy chain fragment and / or a light chain fragment (or an antigen-binding fragment or variant of either) of any one of the illustrative antibodies or fragments thereof provided in Table 1B or SEQ ID NOs: 1-73 that bind to one or more Fzd receptors. In some embodiments, the Fzd binding region is a Fab or derived from a Fab, such that the heavy chain of Table 1B contains the VH and CH1 sequences but not the CH2 or CH3 sequences. In some embodiments, the Fzd binding region is a VHH or sdAb or derived from a VHH or sdAb, such that Table 1B contains a VHH domain. In certain embodiments, the Fzd binding region is a polypeptide that competes with any one of these antibodies for binding to one or more Fzd receptors, e.g., an antibody or an antigen-binding fragment thereof.

[0153] In certain embodiments, the Fzd binding region comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99% identity to any one of the sequences shown in Table 1A, Table 1B, or SEQ ID NOs: 1-73 or an antigen-binding fragment thereof. The binding characteristics of the clones listed in Table 1B were determined and are shown in Table 1B. The heavy chain CDRs are designated CDRH1, CDRH2, and CDRH3, and the light chain CDRs are designated CDRL1, CDRL2, and CDRL3.

[0154] Table 1A: Anti-Fzd Antibody Clone IDs and CDR Sequences

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167] Table 1A, continued

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178] Table 1B: Anti-Fzd antibody clone IDs, heavy chain (HC) and light chain (LC) Seq ID Nos, and binding characteristics

[0179]

[0180]

[0181]

[0182]

[0183] In certain embodiments, the Fzd binding region can be selected from any binding domain that binds to an Fzd receptor epitope with an affinity of, for example, at least about 1×10 D M, at least about 1×10 -4 M, at least about 1×10 -5 M, at least about 1×10 -6 M, at least about 1×10 -7 M, at least about 1×10 -8 M, at least about 1×10 -9 M, at least about 1×10 -10 M, at least about 1×10 -11 M, at least about 1×10 -12 M, at least about 1×10 -13 M, at least about 1×10 -14 M or at least about 1×10 -15M. In certain embodiments, the Fzd binding region can be selected from any binding domain that binds one or more Fzd receptor epitopes with high affinity, such as, for example, a K of D less than about 1 × 10 -7 M, less than about 1 × 10 -8 M, less than about 1 × 10 -9 M, less than about 1 × 10 -10 M, less than about 1 × 10 -11 M, less than about 1 × 10 - 12 M, less than about 1 × 10 -13 M, less than about 1 × 10 -14 M, or less than about 1 × 10 -15 M. In certain embodiments, in the context of a Wnt surrogate molecule, the Fzd binding region can be selected from any binding domain that binds an Fzd receptor epitope with high affinity, such as, for example, a K of D less than or equal to about 1 × 10 -4 M, less than or equal to about 1 × 10 -5 M, less than or equal to about 1 × 10 -6 M, less than or equal to about 1 × 10 -7 M, less than or equal to about 1 × 10 -8 M, less than or equal to about 1 × 10 -9 M, less than or equal to about 1 × 10 -10 M, less than or equal to about 1 × 10 -11 M, less than or equal to about 1 × 10 -12 M, less than or equal to about 1 × 10 -13 M, less than or equal to about 1 × 10 -14 M, or less than or equal to about 1 × 10 -15 M.

[0184] Suitable Fzd binding regions include, but are not limited to, de novo designed Fzd binding proteins, antibody-derived binding proteins (e.g., scFv, Fab, etc.), and other portions of antibodies that specifically bind one or more Fzd proteins, VHH- or sdAb-derived binding domains, knottin-based engineered scaffolds, norrin, and engineered binding fragments derived therefrom, naturally occurring Fzd binding domains, etc. The Fzd binding domain can be affinity selected to enhance binding to a desired Fzd protein or multiple Fzd proteins to provide, for example, tissue selectivity.

[0185] In some embodiments, the Fzd binding region binds to one, two, three, four, five or more different Frizzled proteins, such as one or more human Frizzled proteins among human Frizzled proteins Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9 and Fzd10. In some embodiments, the Fzd binding region binds to Fzd1, Fzd2 and Fzd7. In some embodiments, the Fzd binding region binds to Fzd1, Fzd2, Fzd5, Fzd7 and Fzd8. In other embodiments, the Fzd binding region is selective for one or more Frizzled proteins of interest, for example having at least 10-fold, 25-fold, 50-fold, 100-fold, 200-fold or more specificity for one or more desired Frizzled proteins relative to other Frizzled proteins.

[0186] In certain embodiments, the Fzd binding region comprises six CDR regions of the pan-specific Frizzled antibody OMP-18R5 (vantictumab). In certain embodiments, the Fzd binding region is a scFv comprising six CDR regions of the pan-specific Frizzled antibody OMP-18R5 (vantictumab). See, e.g., U.S. Patent No. 8,507,442, which is specifically incorporated herein by reference. For example, the CDR sequences of OMP-18R5 comprise: (i) a heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO:270), a heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO:677), and a heavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO:1033); and (ii) a light chain CDR1 comprising SGDKLGKKYAS (SEQ ID NO:1152) or SGDNIGSFYVH (SEQ ID NO:1153), a light chain CDR2 comprising EKDNRPSG (SEQ ID NO:1200) or DKSNRPSG (SEQ ID NO:1201), and a light chain CDR3 comprising SSFAGNSLE (SEQ ID NO:1435) or QSYANTLSL (SEQ ID NO:1436). In certain embodiments, the Fzd binding region is an antibody or a derivative thereof, including but not limited to scFv, minibody, VHH or sdAb, and various antibody mimetics comprising any of these CDR sequences. In certain embodiments, these CDR sequences comprise one or more amino acid modifications.

[0187] In certain embodiments, the Fzd binding region comprises six CDR regions of anti-FZD7-1791 or anti-FZD7-1291. Anti-FZD7-1791 and anti-FZD7-1291 are antibodies that bind to different epitopes within the hinge region of Fzd7, as described in PCT patent publications WO2016 / 205551 and WO2016 / 205566, which are specifically incorporated herein by reference. In certain embodiments, the Fzd binding region is a scFv comprising six CDR regions of anti-FZD7-1791 or anti-FZD7-1291. For example, the CDR sequences of anti-FZD7-1791 comprise: (i) a heavy chain CDR1 comprising TYAMH (SEQ ID NO:2190), a heavy chain CDR2 comprising RIRSKSNNYAKNYDDSVKD (SEQ ID NO:2193), and a heavy chain CDR3 comprising ENYGGRFDY (SEQ ID NO:2196); and (ii) a light chain CDR1 comprising KASENVLNYVS (SEQ ID NO:2199), a light chain CDR2 comprising GASNRYT (SEQ ID NO:2202), and a light chain CDR3 comprising GQSYRYP (SEQ ID NO:2205). The heavy chain sequence of anti-FZD7-1791 comprises SEQ ID NO:70, and the light chain sequence of anti-FZD7-1791 comprises SEQ ID NO:71. As another example, the CDR sequences of anti-FZD7-1291 comprise (i) a heavy chain CDR1 comprising SYAMS (SEQ ID NO:2191), a heavy chain CDR2 comprising TISDGGSYTRYPDKLKG (SEQ ID NO:2194), and a heavy chain CDR3 comprising VGGRRDYFDY (SEQ ID NO:2197), and (ii) a light chain CDR1 comprising KSSQSLLYSSNQKNYLAW (SEQ ID NO:2200), a light chain CDR2 comprising WASTRES (SEQ ID NO:2203), and a light chain CDR3 comprising QQYYSYP (SEQ ID NO:2206). The heavy chain sequence of anti-FZD7-1291 comprises SEQ ID NO:72, and the light chain sequence of anti-FZD7-1291 comprises SEQ ID NO:73. In some embodiments, in certain embodiments, the Fzd binding region is an antibody or a derivative thereof, including but not limited to scFv, minibody, VHH, or sdAb, and various antibody mimetics comprising any of these CDR sequences. In certain embodiments, these CDR sequences include one or more amino acid modifications.

[0188] In other embodiments, the Fzd binding region comprises the variable region sequence or CDRs thereof of any of a plurality of Frizzled-specific antibodies, the plurality of Frizzled-specific antibodies being known in the art and commercially available or can be generated de novo. Any of the Frizzled polypeptides in the Frizzled polypeptide can be used as an immunogen or in screening assays for the development of antibodies. Non-limiting examples of Frizzled binding domains include: antibodies available from Biolegend, e.g., clone CH3A4A7 (CD344) specific for human Frizzled 4, clone W3C4E11 (CD349) specific for human Fzd9; antibodies available from Abcam, e.g., ab64636 specific for Fzd7; ab83042 specific for human Fzd4; ab77379 specific for human Fzd7; ab75235 specific for human Fzd8; ab102956 specific for human Fzd9; etc. Other examples of suitable antibodies are particularly described in the following: U.S. Patent Application No. 20140105917; U.S. Patent Application No. 20130230521; U.S. Patent Application No. 20080267955; U.S. Patent Application No. 20080038272; U.S. Patent Application No. 20030044409; etc., each of which is specifically incorporated herein by reference.

[0189] The Fzd binding region of the Wnt surrogate molecule can be an engineered protein selected to be structurally homologous to the Frizzled binding region of the Wnt protein. Such proteins can be identified by screening a structural database for homology. An initial protein, such as the microbial Bh1478 protein, is thus identified. The native protein is then engineered to provide amino acid substitutions that increase affinity, and further selection can be carried out by affinity maturation to have increased affinity and selectivity when binding to the desired Frizzled. Non-limiting examples of the Frizzled binding moiety include the Fz27 and Fz27-B12 proteins.

[0190] In certain embodiments, the Wnt surrogate molecule comprises an LRP5 / 6 binding region fused to a polypeptide that specifically binds to one or more epitopes of an Fzd receptor, e.g., an anti-LRP5 / 6 antibody or an antigen-binding fragment thereof. In certain embodiments, the polypeptide that specifically binds to LRP5 / 6 is an antibody or an antigen-binding fragment thereof. In some embodiments, it is the antibody or an antigen-binding fragment thereof disclosed in U.S. Provisional Patent Application No. 62 / 607,879, entitled "Anti-LR5 / 6 Antibodies and Methods of Use," filed on December 19, 2017, with Attorney Docket No. SRZN-005 / 00US, which is incorporated herein by reference in its entirety.

[0191] In certain embodiments, at least one LRP5 / 6 binding region of the Wnt surrogate molecule comprises one or more antigen-binding fragments of an antibody. For example, the one or more antigen-binding fragments can be or be derived from IgG, scFv, Fab, VHH, or sdAb. In some embodiments, the one or more antigen-binding fragments are humanized.

[0192] In certain embodiments, the LRP5 / 6 binding region comprises three heavy chain CDRs and / or three light chain CDRs disclosed for any one of the illustrative antibodies or fragments thereof provided in Table 2A that bind to LRP5 and / or LRP6. In certain embodiments, the LRP5 / 6 binding region comprises three heavy chain CDRs and / or three light chain CDRs disclosed for any one of the illustrative antibodies or fragments thereof provided in Table 2A that bind to LRP5 and / or LRP6, wherein the CDRs together comprise one, two, three, four, five, six, seven, or eight amino acid modifications, e.g., substitutions, deletions, or additions. In certain embodiments, the LRP5 / 6 binding region is a VHH or sdAb or is derived from a VHH or sdAb, and thus Table 2A contains only three heavy chain CDRs. In certain embodiments, the LRP5 / 6 binding region comprises the three heavy chain CDRs or variants shown in Table 2A, wherein the CDRs together comprise one, two, three, four, five, six, seven, or eight amino acid modifications. In certain embodiments, the LRP5 / 6 binding region comprises the heavy chain fragment and / or light chain fragment (or antigen-binding fragment or variant of either) of any one of the illustrative antibodies or fragments thereof provided in Table 2B or SEQ ID NOs: 74-97 that bind to LRP5 and / or LRP6. In certain embodiments, the LRP5 / 6 binding region is a Fab or is derived from a Fab, and thus Table 2B contains VH and CH1 sequences but does not contain CH2 or CH3 sequences. In certain embodiments, the LRP5 / 6 binding region is a VHH or sdAb or is derived from a VHH or sdAb, and thus Table 2B contains the VHH domain. In certain embodiments, the LRP5 / 6 binding region is a polypeptide that competes with one of these antibodies for binding to LRP5 and / or LRP6, e.g., an antibody or antigen-binding fragment thereof.

[0193] In certain embodiments, the LRP5 / 6 binding region comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity to any one of the sequences shown in Table 2A, Table 2B, or SEQ ID NOs: 74-97 or an antigen-binding fragment thereof. The binding characteristics of the clones listed in Table 2B were determined and are shown in Table 2B.

[0194] Table 2A: Anti-LRP5 / 6 antibody clone IDs and CDR sequences.

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203] Table 2A, continued

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210] Table 2B. Anti-LRP5 / 6 antibody clone IDs, heavy chain (HC) Seq ID Nos, and binding characteristics.

[0211]

[0212]

[0213] In certain embodiments, in the context of a Wnt surrogate molecule, the LRP5 / 6 binding region can be selected from a K that is D any binding domain that binds to LRP5 or LRP6: less than or equal to about 1×10 -4 M, less than or equal to about 1×10 -5 M, less than or equal to about 1×10 -6 M, less than or equal to about 1×10 -7 M, less than or equal to about 1×10 -8 M, less than or equal to about 1×10 - 9 M, less than or equal to about 1×10 -10 M, less than or equal to about 1×10 -11 M, less than or equal to about 1×10 -12 M, less than or equal to about 1×10 -13 M, less than or equal to about 1×10 -14 M, or less than or equal to 1x10 -15M. In certain embodiments, in the context of a Wnt surrogate molecule, the LRP5 / 6 binding region can be selected from, for example, a K D any binding domain that binds to LRP5 or LRP6: greater than or equal to approximately 1×10 -4 M, greater than or equal to approximately 1×10 -5 M, greater than or equal to approximately 1×10 -6 M, greater than or equal to approximately 1×10 -7 M, greater than or equal to approximately 1×10 -8 M, greater than or equal to approximately 1×10 -9 M, greater than approximately 1×10 -10 M, greater than or equal to approximately 1×10 -11 M, greater than or equal to approximately 1×10 -12 M, greater than or equal to approximately 1×10 -13 M, greater than or equal to approximately 1×10 -14 M, or greater than or equal to 1×10 -15 M. In certain embodiments, the LRP5 / 6 binding region can be selected from, for example, a K D any binding domain that binds to LRP5 or LRP6 with high affinity as follows: less than approximately 1×10 -7 M, less than approximately 1×10 -8 M, less than approximately 1×10 -9 M or less than approximately 1×10 -10 M.

[0214] Other suitable LRP5 / 6 binding regions include but are not limited to: redesigned LRP5 / 6 binding proteins, antibody-derived binding proteins (such as scFv, Fab, etc.), and other portions of antibodies that specifically bind to one or more Fzd proteins; VHH- or sdAb-derived binding domains; knottin-based engineered scaffolds; naturally occurring LRP5 / 6, including but not limited to DKK1, DKK2, DKK3, DKK4, sclerostin; Wise; fusion proteins comprising any of the foregoing; derivatives of any of the foregoing; variants of any of the foregoing; and bioactive fragments of any of the foregoing, etc. The LRP5 / 6 binding region can be affinity selected to enhance binding.

[0215] 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, as well as the DKK-3-related protein Soggy (Sgy). hDKK 1-4 contain two distinct cysteine-rich domains, with the positions of 10 cysteine residues being highly conserved among family members. Exemplary sequences of human Dkk genes and proteins are publicly available, such as Genbank accession numbers: 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 present disclosure, the LRP6-binding portion is a DKK1 peptide, including but not limited to the C-terminal domain of human DKK1. The C-terminal domain may include the following sequence: KMYHTKGQEGSVCLRSSDCASGLCCARHFWSKICKPVLKEGQVCTKHRRKGSHGLEIFQRCYCGEGLSCRIQKDHHQASNSSRLHTCQRH (SEQ ID NO:2249) (see Genbank accession number NP_036374) or a bioactive fragment thereof.

[0216] The binding of DKK proteins to LRP5 / 6 is discussed, for example, in Brott and Sokol Mol. Cell. Biol. 22(17), 6100-6110 (2002); and Li et al. J. Biol. Chem. 277(8), 5977-5981 (2002), each of which is hereby expressly incorporated by reference. The corresponding region of human DKK2 (Genbank reference NP_055236) may include the following sequence: KMSHIKGHEGDPCLRSSDCIEGFCCARHFWTKICKPVLHQGEVCTKQRKKGSHGLEIFQRCDCAKGLSCKVWKDATYSSKARLHVCQK (SEQ ID NO:2250) or a bioactive fragment thereof.

[0217] 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 immunogens or in screening assays to develop antibodies. Examples of known antibodies include, but are not limited to, those described in Gong et al. (2010) PLoS One. 5(9):e12682; Marvin et al. (2010) Proc Natl Acad Sci USA 107(35):15473-8; and those commercially available from, for example, Santa Cruz biotechnology antibody clone 1A12, which was generated against synthetic LRP5 / 6 of human origin and binds to both full-length and proteolytic fragments of LRP6 and LRP5 of mouse and human origin; said monoclonal antibody 2B11; Cell Signaling Technology antibody specific for LRP5 (D80F2) catalog number 5731; etc.

[0218] In certain embodiments, the Wnt surrogate molecules disclosed herein include one or more polypeptides, and the one or more polypeptides include two or more binding regions. For example, the two or more binding regions can be two or more Fzd binding regions or two or more LRP5 / 6 binding regions, or the two or more binding regions can include one or more Fzd binding regions and one or more LRP5 / 6 binding regions. The binding regions can be directly linked or adjacent or can be separated by a linker (e.g., a polypeptide linker or a non-peptide linker, etc.). The length of the linker and thus the spacing between the binding domains can be used to modulate the signal strength and can be selected according to the desired use of the Wnt surrogate molecule. The spacing distance between the binding domains can vary, but in certain embodiments can be less than about 100 angstroms, less than about 90 angstroms, less than about 80 angstroms, less than about 70 angstroms, less than about 60 angstroms, or less than about 50 angstroms. In some embodiments, the linker is a rigid linker, and in other embodiments, the linker is a flexible linker. In certain embodiments, where the linker is a peptide linker, the linker can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids in length and has sufficient length and amino acid composition to space the binding domains. In some embodiments, the linker includes or consists of one or more glycine and / or serine residues.

[0219] In certain embodiments, the Wnt surrogate molecule comprises a polypeptide sequence having at least 90%, at least 95%, at least 98% or at least 99% identity to the polypeptide sequence disclosed in any of SEQ ID NOs: 109-124 or 125-157 or having at least 90%, at least 95%, at least 98% or at least 99% identity to an antigen-binding fragment of the polypeptide sequence disclosed in any of SEQ ID NOs: 109-124 or 125-157. In some embodiments, the Wnt surrogate molecule comprises or consists of the polypeptide sequence shown in any of SEQ ID NOs: 109-124 or 125-147 or an antigen-binding fragment thereof. In certain embodiments, the antigen-binding fragment binds one or more Fzd receptors and also binds LRP5 and / or LRP6.

[0220] The Wnt surrogate molecule can be polymerized, for example, via an Fc domain, by means of a cascade, coiled coil, polypeptide zipper, biotin / avidin or streptavidin multimerization, etc. The Wnt surrogate molecule can also be linked to moieties known in the art, such as PEG, Fc, etc., to enhance in vivo stability.

[0221] In some embodiments, the Wnt surrogate molecule directly activates the canonical Wnt signaling by binding to one or more Fzd proteins and LRP5 / 6, particularly by binding to these proteins on the cell surface (e.g., the surface of human cells). The direct activation of Wnt signaling by the Wnt surrogate molecule is contrary to the potentiation of Wnt signaling, which only enhances the activity in the presence of native Wnt protein.

[0222] The Wnt surrogate molecule activates Wnt signaling, for example, by mimicking the action or activity of Wnt protein binding to frizzled proteins. The ability of the Wnt surrogate molecules of the present disclosure to mimic Wnt activity can be demonstrated by a variety of assays. The Wnt surrogate molecule generally elicits a reaction or activity similar or identical to that elicited by the native ligand of the receptor. Specifically, the Wnt surrogate molecules of the present disclosure enhance the canonical Wnt / β-catenin signaling pathway. As used herein, the term "enhance" refers to a measurable increase in the level of Wnt / β-catenin signaling as compared to the level in the absence of the Wnt surrogate molecules of the present disclosure.

[0223] In the art, a variety of methods are known for measuring the levels of canonical Wnt / β-catenin signaling. These methods include, but are not limited to, assays that measure: Wnt / β-catenin target gene expression; TCF reporter gene expression; β-catenin stabilization; LRP phosphorylation; Axin translocation from the cytoplasm to the cell membrane and binding to LRP. The canonical Wnt / β-catenin signaling pathway ultimately causes changes in gene expression through the transcription factors TCF7, TCF7L1, TCF7L2, and LEF. Transcriptional responses to Wnt activation have been characterized in many cells and tissues. Thus, global transcriptional profiling by methods well known in the art can be used to assess Wnt / β-catenin signaling activation or inhibition.

[0224] Changes in Wnt-responsive gene expression are generally mediated by TCF and LEF transcription factors. The TCF reporter gene assay assesses transcriptional changes in TCF / LEF-controlled genes to determine the level of Wnt / β-catenin signaling. The TCF reporter gene assay was first described by Korinek, V. et al., in 1997. This method is also known as TOP / FOP and involves the use of three copies of the optimal TCF motif CCTTTGATC or three copies of the mutant motif CCTTTGGCC (pTOPFI_ASH and pFOPFI_ASH, respectively) located upstream of a minimal c-Fos promoter driving luciferase expression to determine the transactivation activity of endogenous p-catenin / TCF4. A higher ratio of the activities of these two reporter genes (TOP / FOP) indicates higher β-catenin / TCF4 activity, while a lower ratio of the activities of these two reporter genes indicates lower β-catenin / TCF4 activity.

[0225] A variety of other reporter gene transgenes that respond to Wnt signals are intact in animals and thus effectively reflect endogenous Wnt signaling. These reporter genes are based on multimerized TCF binding sites that drive the expression of LacZ or GFP, which can be easily detected by methods known in the art. These reporter genes include: TOP-GAL, BAT-GAL, ins-TOPEGFP, ins-TOPGAL, LEF-EGFP, Axin2-LacZ, Axin2-d2EGFP, Lgr5tm1(cre / ERT2), TOPdGFP.

[0226] In some cases, the recruitment of dephosphorylated β-catenin to the membrane, the stabilization and phosphorylation status of β-catenin, and the translocation of β-catenin to the nucleus mediated by the formation of a complex with TCF transcription factors and TNIK (Klapholz-Brown Z et al., PLoS ONE 2(9):e945, 2007) are key steps in the Wnt signaling pathway. Stabilization is mediated by Disheveled family proteins that inhibit the "destruction" complex, thus reducing the degradation of intracellular β-catenin, and thereafter β-catenin translocates to the nucleus. Thus, measuring the level and location of β-catenin in cells provides a good reflection of the level of Wnt / β-catenin signaling. A non-limiting example of such an assay is the "Biolmage β-catenin Redistribution Assay" (Thermo Scientific), which provides recombinant U20S cells stably expressing human β-catenin fused to the C-terminus of enhanced green fluorescent protein (EGFP). Imaging and analysis are performed using a fluorescence microscope or an HCS platform, thus allowing visualization of the level and distribution of EGFP-β-catenin.

[0227] Another way to inhibit the destruction complex is to remove Axin by recruiting Axin to the cytoplasmic tail region of the Wnt co-receptor LRP. Axin has been shown to preferentially bind to the phosphorylated form of the LRP tail region. Thus, visualizing Axin translocation, for example with a GFP-Axin fusion protein, is another method for assessing the level of Wnt / β-catenin signaling.

[0228] In certain embodiments, as measured in the above assays, for example as measured in the TOPFLASH assay, a Wnt surrogate molecule will enhance or increase canonical Wnt pathway signaling (e.g., β-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% compared to neutral-matter-induced β-catenin signaling or a negative control. A negative control can be included in these assays. In certain embodiments, when measured in the above assays, for example when measured in the TOPFLASH assay or any one of the other assays mentioned herein, a Wnt surrogate molecule can enhance β-catenin signaling by 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10000-fold or more compared to the activity in the absence of the Wnt surrogate molecule.

[0229] As used herein, "Wnt gene product" or "Wnt polypeptide" encompasses native sequence Wnt polypeptides, Wnt polypeptide variants, Wnt polypeptide fragments, and chimeric Wnt polypeptides. In certain embodiments, the Wnt polypeptide is a native human full-length mature Wnt protein.

[0230] For example, the naturally occurring Wnt proteins of interest in the present application include the following: Wnt-1 (GenBank accession number NM_005430); Wnt-2 (GenBank accession number NM_003391); Wnt-2B (Wnt-13) (GenBank accession numbers NM_004185 (isoform 1), NM_024494.2 (isoform 2)), Wnt-3 (RefSeq.: NM_030753), Wnt3a (GenBank accession number NM_033131), Wnt-4 (GenBank accession number NM_030761), Wnt-5A (GenBank accession number NM_003392), Wnt-5B (GenBank accession number NM_032642), Wnt-6 (GenBank accession number NM_006522), Wnt-7A (GenBank accession number NM_004625), Wnt-7B (GenBank accession number NM_058238), Wnt-8A (GenBank accession number NM_058244), Wnt-8B (GenBank accession number NM_003393), Wnt-9A (Wnt-14) (GenBank accession number NM_003395), Wnt-9B (Wnt-15) (GenBank accession number NM_003396), Wnt-10A (GenBank accession number NM_025216), Wnt-10B (GenBank accession number NM_003394), Wnt-11 (GenBank accession number NM_004626), Wnt-16 (GenBank accession number NM_016087)). Although each member has a different degree of sequence identity with the family, all members encode a smaller (i.e., 39-46 kD), acylated, palmitoylated, secreted glycoprotein containing a highly conserved spacing of 23-24 conserved cysteine residues (McMahon, A P et al., Trends Genet. 1992; 8:236-242; Miller, JR., Genome Biol. 2002; 3(1):3001.1-3001.15). Other naturally occurring Wnt polypeptides of interest include orthologs of the above from any mammal, including domestic and farm animals, as well as zoo, laboratory or pet animals such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, rats, mice, frogs, zebrafish, fruit flies, worms, etc.

[0231] As used herein, "Wnt pathway signaling" or "Wnt signaling" refers to the mechanism by which bioactive Wnt exerts its effects on cells to regulate cell activity. Wnt proteins regulate cell activity by binding to Wnt receptors, which include proteins from the Frizzled (Fzd) family of proteins, proteins from the ROR family of proteins, the LRP family proteins LRP5, LRP6, the protein FRL1 / crypto, and the protein Derailed / Ryk. Once activated by Wnt binding, one or more Wnt receptors 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 / Ca 2+ ) pathway (Giles, RH et al. (2003) Biochim Biophys Acta 1653, 1-24; Peifer, M. et al. (1994) Development 120:369-380; Papkoff, J. et al. (1996) Mol. Cell Biol. 16:2128-2134; Veeman, MT et al. (2003) Dev. Cell 5:367-377); and other Wnt signaling pathways well known in the art.

[0232] For example, activation of the canonical Wnt signaling pathway causes inhibition of phosphorylation of the intracellular protein β-catenin, leading to accumulation of β-catenin in the cytosol and subsequent translocation to the nucleus, where β-catenin interacts with transcription factors (e.g., TCF / LEF) to activate target genes. Activation of the Wnt / PCP pathway activates the RhoA, c-Jun N-terminal kinase (JNK), and nemo-like kinase (NLK) signaling cascades to control biological processes such as tissue polarity and cell movement. Activation of Wnt / Ca by binding of Wnt-4, Wnt-5A, or Wnt-11 2+ causes release of intracellular calcium ions, which activates calcium-sensitive enzymes such as protein kinase C (PKC), calcium-calmodulin-dependent kinase II (CamKII), or calcineurin (CaCN). By assaying the activity of the above signaling pathways, the biological activity of an antibody or its antigen-binding fragment (e.g., a Wnt surrogate molecule) can be readily determined.

[0233] In certain embodiments, the functional properties of a Wnt surrogate molecule can be assessed using a variety of methods known to those of skill in the art, the variety of methods including, for example, affinity / binding assays (e.g., surface plasmon resonance, competitive inhibition assays) responsive to Wnt, cancer cell, and / or tumor growth inhibition using in vitro or in vivo models (including but not limited to any of the in vitro or in vivo models described herein), cytotoxicity assays, cell viability assays, cell proliferation or differentiation assays. The effects of the Wnt surrogate molecules described herein on Fzd internalization, in vitro and in vivo efficacy, etc. can also be tested. Such assays can be performed using well-recognized protocols or commercially available kits known to those of skill in the art (see, for example, Current Protocols in Molecular Biology (Greene Publ. Assoc. Inc. and John Wiley & Sons, Inc., New York, 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, Inc., New York, NY); or commercially available kits).

[0234] In certain embodiments, the Fzd binding region of a Wnt surrogate molecule (e.g., an antigen-binding fragment of an anti-Fzd antibody) includes one or more of the CDRs of the anti-Fzd antibodies described herein. In certain embodiments, the LRP5 / 6 binding region of a Wnt surrogate molecule (e.g., an antigen-binding fragment of an anti-LRP5 / 6 antibody) includes one or more of the CDRs of the anti-LRP5 / 6 antibodies described herein. In this regard, it has been shown that in some cases, it is possible to transfer only the VH CDR3 of an antibody while still retaining the desired specific binding (Barbas et al., Proc. Natl. Acad. Sci. USA (1995) 92:2529-2533). See also McLane et al., Proc. Natl. Acad. Sci. USA (1995) 92:5214-5218, Barbas et al., J. Am. Chem. Soc. (1994) 116:2161-2162.

[0235] The present invention also discloses a method for obtaining an antibody or antigen-binding domain that is specific for an Fzd receptor, the method comprising providing a VH domain as shown herein or a VH domain that is an amino acid sequence variant of the VH domain by addition, deletion, substitution, or insertion of one or more amino acids in the amino acid sequence; optionally combining the thus-provided VH domain with one or more VL domains; and testing the VH domain or one or more VH / VL combinations to identify a specific binding member or an antibody antigen-binding domain that is specific for one or more Fzd receptors and optionally has one or more desired properties. The VL domain can have an amino acid sequence substantially as shown herein. A similar method can be employed, wherein one or more sequence variants of the VL domain disclosed herein are combined with one or more VH domains.

[0236] In certain embodiments, the Wnt surrogate molecule is water-soluble. "Water-soluble" means soluble in an aqueous buffer in the absence of a detergent, typically a composition soluble at a concentration that provides a biologically effective dose of the polypeptide. The water-soluble composition forms a substantially homogeneous composition, and the specific activity of the substantially homogeneous composition is at least about 5% of the specific activity of the starting material from which the substantially homogeneous composition is purified, typically at least about 10%, 20%, or 30% of the specific activity of the starting material, more typically about 40%, 50%, or 60% of the specific activity of the starting material, and can be about 50%, about 90%, or greater. The Wnt surrogate molecules disclosed herein typically form a substantially homogeneous aqueous solution having a concentration of at least 25 μM or higher, e.g., at least 25 μM, 40 μM, or 50 μM, typically at least 60 μM, 70 μM, 80 μM, or 90 μM, and sometimes up to 100 μM, 120 μM, or 150 μM. In other words, the Wnt surrogate molecules disclosed herein typically form a substantially homogeneous aqueous solution having a concentration of about 0.1 mg / ml, about 0.5 mg / ml, about 1 mg / ml, or greater.

[0237] An antigen or epitope that "specifically binds" or "preferentially binds" (used interchangeably herein) to an antibody or an antigen-binding fragment thereof is a term well known in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule (e.g., a Wnt surrogate molecule) is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular cell or substance more frequently, more rapidly, for a longer duration, and / or with greater affinity than it does with alternative cells or substances. A molecule or its binding region (e.g., a Wnt surrogate molecule or its binding region) "specifically binds" or "preferentially binds" to a target antigen (e.g., an Fzd receptor) if it binds with greater affinity, higher avidity, more readily, and / or for a longer duration than it does to other substances. For example, a Wnt surrogate molecule or its binding region that specifically binds or preferentially binds to the Fzd1 receptor is an antibody that binds to the Fzd1 receptor with greater affinity, higher avidity, more readily, and / or for a longer duration than it binds to other Fzd receptors or non-Fzd proteins. It should also be understood from this definition that, for example, a Wnt surrogate molecule or its binding region that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.

[0238] In some embodiments, any one of the one or more Fzd-binding regions of a Wnt surrogate molecule binds to one, two, three, four, five, or more different Frizzled receptors, such as one or more of the human Frizzled receptors Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, Fzd10. In some embodiments, any one of the Fzd-binding regions binds to Fzd1, Fzd2, Fzd5, Fzd7, and Fzd8. In various embodiments, any one of the Fzd-binding regions binds to the following: (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 Fzd10; (ix) Fzd5, Fzd8, and Fzd10; (x) Fzd4, Fzd5, and Fzd8; or (xi) Fzd1, Fzd5, Fzd7, and Fzd8.

[0239] In some embodiments, the Fzd binding region is selective for one or more Fzd receptors of interest, e.g., having at least 10-fold, 25-fold, 50-fold, 100-fold, 200-fold or more specificity for one or more desired Fzd receptors relative to other Fzd receptors. In some embodiments, any one of the one or more Fzd binding regions of the Wnt surrogate molecule is multispecific and binds or specifically binds to multiple Fzd receptors (e.g., two or more of Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9 or Fzd10). For example, any one of the one or more Fzd binding regions can be bispecific, trispecific, tetraspecific, etc. In some embodiments, any one of the one or more Fzd binding regions of the Wnt surrogate molecule is monospecific and binds or specifically binds to a single Fzd receptor, e.g., binds or specifically binds to only one of the following: Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9 or Fzd10.

[0240] In some embodiments, the monospecific Fzd binding region binds to a region of the Fzd receptor that: does not include the cysteine-rich domain (CRD) of the Fzd receptor; or includes less than the entire CRD of the FZD receptor. As Figure 2A shown, the sequences within the CRD show strong homology among the 10 Fzd receptors, with even higher homology among members of the subfamily. Thus, certain embodiments of the monospecific Fzd binding regions disclosed herein do not bind the CRD, or bind only a subset of the CRD.

[0241] In some embodiments, the Fzd binding region (e.g., the monospecific Fzd binding region) binds to an epitope that includes at least a portion of the extracellular domain following the CRD, which is referred to herein as the "hinge region" of the Fzd receptor (see Figure 2A ). In certain embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the epitope is present within the hinge region of the Fzd receptor. As Figure 4A - 4E shown, the hinge region of the extracellular domain of the Fzd receptor shows highly divergent sequences. The sequences of illustrative Fzd receptor hinge regions are shown in SEQ ID NOs: 98 - 107 and Table 3 below. In certain embodiments, the hinge region includes an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% identity to any one of the sequences shown in SEQ ID NOs: 98 - 107.

[0242] Table 3: Fzd hinge region sequences

[0243]

[0244] In some embodiments, the monospecific Fzd binding region binds to an epitope that comprises at least a portion of the N-terminal region upstream of the CRD of the Fzd receptor. Figure 2A ) In certain embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the epitope is present within the N-terminal region of the Fzd receptor. SEQ ID NO:108 and Table 4 below show the sequences of illustrative N-terminal regions. In certain embodiments, the N-terminal region comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO:108.

[0245] Table 4: Fzd N-terminal region sequences

[0246]

[0247] In some embodiments, any one of the one or more LRP5 / 6 binding regions of the Wnt surrogate molecule binds to one or both of LRP5 and LRP6. For convenience, the term "LRP5 / 6" is used to refer collectively to either or both of LRP5 and / or LRP6.

[0248] Immunobinding generally refers to the type of non-covalent interaction that occurs, for example but not limited to, between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific due to electrostatic, ionic, hydrophilic and / or hydrophobic attractions or repulsions, steric hindrance, hydrogen bonding, van der Waals forces and other interactions. The strength or affinity of an immunobinding interaction can be expressed in terms of the dissociation constant (K d ) of the interaction, where a smaller K d represents a greater affinity. The immunobinding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, where those rates depend on the concentrations of the complex partners, the affinity of the interaction and on geometric parameters that affect the rates equally in both directions. Thus, the "association rate constant" (K on ) and the "dissociation rate constant" (K off ) can be determined by calculating the concentrations and actual rates of association and dissociation. The ratio of K off / K on achieves the elimination of all parameters that are independent of affinity and thus is equal to the dissociation constant K d。See generally Davies et al. (1990) Annual Rev. Biochem. 59:439-473.

[0249] In certain embodiments, the Wnt surrogate molecules or binding regions thereof described herein have an affinity of less than about 10,000 nM, less than about 1000 nM, less than about 100 nM, less than about 10 nM, less than about 1 nM, less than about 0.1 nM, less than about 0.01 nM, less than about 0.001 nM, less than about 0.0001 nM, less than about 0.00001 nM, or less than about 0.000001 nM, and in some embodiments, the antibodies can have an even higher affinity for one or more Fzd receptor epitopes or the LRP5 or LRP6 receptors.

[0250] The constant regions of immunoglobulins exhibit less sequence diversity than the variable regions and are responsible for binding to many natural proteins to initiate important biochemical events. In humans, there are five different classes of antibodies, including IgA (which includes the subclasses IgA1 and IgA2), IgD, IgE, IgG (which includes the subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. The distinguishing feature between these antibody classes is their constant regions, although there can be subtle differences in the V regions.

[0251] The Fc region of an antibody interacts with a number of Fc receptors and ligands, thereby conferring a range of important functional capabilities known as effector functions. For IgG, the Fc region includes the Ig domains CH2 and CH3 and the N-terminal hinge leading to CH2. An important family of Fc receptors for the IgG class is the Fcγ receptors (FcγR). These receptors mediate communication between antibodies and the cellular arm of the immune system (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ravetch et al., 2001, Annu Rev Immunol 19:275-290). In humans, this protein family includes: FcγRI (CD64), which includes the isotypes FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes the isotypes FcγRIIa (including the allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes the isotypes FcγRIIIa (including the allotypes V158 and F158) and FcγRIIIb (including the allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65). These receptors generally have an extracellular domain that mediates binding to Fc, a transmembrane region, and an intracellular domain that can mediate some signal transduction events within the cell. These receptors are expressed on a variety of immune cells, including monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and T cells. Formation of the Fc / FcγR complex recruits these effector cells to the site of antigen binding, thereby generally generating intracellular signal transduction events and important subsequent immune responses, such as the release of inflammatory mediators, B cell activation, endocytosis, phagocytosis, and cytotoxic attack.

[0252] The ability to mediate cytotoxic and phagocytic effector functions is a potential mechanism by which antibodies destroy target cells. A cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR recognize bound antibody on the target cell and subsequently cause lysis of the target cell is called antibody-dependent cell-mediated cytotoxicity (ADCC) (Raghavan et al., 1996, Annual Review of Cell and Developmental Biology 12:181-220; Ghetie et al., 2000, Annual Review of Immunology 18:739-766; Ravetch et al., 2001, Annual Review of Immunology 19:275-290). A cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR recognize bound antibody on the target cell and subsequently cause phagocytosis of the target cell is called antibody-dependent cell-mediated phagocytosis (ADCP). All FcγRs bind to the same region on Fc at the N-terminal end of the Cg2 (CH2) domain and the preceding hinge. This interaction has been well characterized structurally (Sondermann et al., 2001, Journal of Molecular Biology 309:737-749), and several structures of human Fc in complex with the extracellular domain of human FcγRIIIb have been solved (pdb accession code 1E4K) (Sondermann et al., 2000, Nature 406:267-273). (pdb accession codes 1IIS and 1IIX) (Radaev et al., 2001, Journal of Biological Chemistry 276:16469-16477).

[0253] Different IgG subclasses have different affinities for FcγRs, and IgG1 and IgG3 generally bind the receptors significantly better than IgG2 and IgG4 (Jefferis et al., 2002, Immunology Letters 82:57-65). All FcγRs bind to the same region on IgG Fc, but with different affinities: the high-affinity binder FcγRI has a K d for IgG1 of 10 -8 M -1 , while the low-affinity receptors FcγRII and FcγRIII generally have Kd values of 10 -6 and 10 -5Association. The extracellular domains of FcγRIIIa and FcγRIIIb are 96% identical; however, FcγRIIIb does not have an intracellular signaling domain. In addition, while FcγRI, FcγRIIa / c, and FcγRIIIa are positive regulators of immune complex-triggered activation (the positive regulators are characterized by having an intracellular domain with an immunoreceptor tyrosine-based activation motif (ITAM)), FcγRIIb has an immunoreceptor tyrosine-based inhibitory motif (ITIM) and is thus inhibitory. Therefore, the former are called activating receptors and FcγRIIb is called an inhibitory receptor. The expression patterns and levels of the receptors also vary on different immune cells. Another level of complexity is the presence of many FcγR polymorphisms in the human proteome. A particularly relevant polymorphism with clinical significance is V158 / F158 FcγRIIIa. The binding affinity of human IgG1 to the V158 allotype is greater than that to the F158 allotype. This difference in affinity and presumably its effect on ADCC and / or ADCP have been shown to be important determinants of the efficacy of the anti-CD20 antibody rituximab ( a registered trademark of IDEC Pharmaceuticals Corporation). Subjects with the V158 allotype respond well to rituximab treatment; however, subjects with the lower-affinity F158 allotype respond poorly (Cartron et al., 2002, Blood 99:754-758). Approximately 10-20% of humans are V158 / V158 homozygotes, 45% are V158 / F158 heterozygotes, and 35-45% are F158 / F158 homozygotes (Lehrnbecher et al., 1999, Blood 94:4220-4232; Cartron et al., 2002, Blood 99:754-758). Thus, 80-90% of humans are poor responders, that is, they have at least one allele of F158 FcγRIIIa.

[0254] The Fc region is also involved in the activation of the complement cascade. In the classical complement pathway, C1 uses its C1q subunit to bind to the Fc fragment of IgG or IgM that has formed a complex with one or more antigens. In certain embodiments of the present disclosure, modifications to the Fc region include modifications that alter (enhance or reduce) the ability of the Fzd-specific antibodies described herein to activate the complement system (see, e.g., U.S. Patent No. 7,740,847). To assess complement activation, a complement-dependent cytotoxicity (CDC) assay can be performed (see, e.g., Gazzano-Santoro et al., Journal of Immunological Methods, 202:163 (1996)).

[0255] Accordingly, in certain embodiments, the present disclosure provides anti-Fzd antibodies having a modified Fc region, which have altered functional properties such as reduced or enhanced CDC, ADCC, or ADCP activity or enhanced binding affinity for specific FcγRs or increased serum half-life. Other modified Fc regions contemplated herein are described, for example, in issued U.S. Patent Nos. 7,317,091; 7,657,380; 7,662,925; 6,538,124; 6,528,624; 7,297,775; 7,364,731; published U.S. Applications Nos. US2009092599; US20080131435; US20080138344; and published International Applications Nos. WO2006 / 105338; WO2004 / 063351; WO2006 / 088494; WO2007 / 024249.

[0256] In certain embodiments, the Wnt surrogate molecules include antibody variable domains fused to immunoglobulin constant domain sequences having a desired binding specificity. In certain embodiments, the fusion is with an Ig heavy chain constant domain comprising at least a portion of the hinge region, C H 2 and C H 3 regions. In a particular embodiment, a first heavy chain constant region (C H1) In at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host cell. This provides greater flexibility in adjusting the relative proportions of the three polypeptide fragments in the regulatory examples when unequal ratios of the three polypeptide chains used in the construction provide the optimal yield of the desired bispecific antibody. However, when high yields are obtained with equal ratios of at least two polypeptide chains or when the ratio has no significant effect on the yield of the desired chain combination, the coding sequences for two or all three polypeptide chains can be inserted into a single expression vector.

[0257] The Wnt surrogate molecules disclosed herein can also be modified to include, for example, epitope tags or labels for purification or diagnostic applications. There are many linker groups known in the art for preparing antibody conjugates, including, for example, those disclosed in U.S. Patent No. 5,208,020 or European Patent 0 425 235 B1 and Chari et al., Cancer Research 52:127-131 (1992). As disclosed in the above patents, the linker groups include disulfide, thioether, acid-labile, photo-labile, peptidase-labile, or esterase-labile groups, preferably disulfide and thioether groups.

[0258] In certain embodiments, the anti-LRP5 / 6 antibodies and antigen-binding fragments thereof and / or anti-Fzd antibodies and antigen-binding fragments thereof present within the Wnt surrogate molecule are monoclonal. In certain embodiments, they are humanized.

[0259] In certain embodiments, the present disclosure further provides an isolated nucleic acid encoding a polypeptide present in a Wnt surrogate molecule disclosed herein. The nucleic acid includes DNA and RNA. These and related embodiments can include polynucleotides encoding antibody fragments that bind to one or more of the Fzd receptors and / or LRP5 or LRP6 described herein. As used herein, the term "isolated polynucleotide" 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 with 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 as part of a larger sequence in nature. Isolated polynucleotides can include naturally occurring sequences and / or artificial sequences.

[0260] The term "operably linked" means that the components to which the term applies are in a relationship that permits them to perform their inherent functions under appropriate conditions. For example, a transcriptional control sequence that is "operably linked" to a protein coding sequence is joined thereto such that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequence.

[0261] As used herein, the term "control sequence" means a polynucleotide sequence that can affect the expression, processing, or intracellular localization of a coding sequence to which it is ligated or operably linked. The nature of such control sequences can depend on the host organism. In certain embodiments, transcriptional control sequences for prokaryotes can include a promoter, a ribosome binding site, and a transcription termination sequence. In other certain embodiments, transcriptional control sequences for eukaryotes can include a promoter that includes one or more recognition sites for transcription factors, transcriptional enhancer sequences, a transcription termination sequence, and a polyadenylation sequence. In some embodiments, a "control sequence" can include a leader sequence and / or a fusion partner sequence.

[0262] As used herein, the term "polynucleotide" means a single-stranded or double-stranded nucleic acid polymer. In certain embodiments, the nucleotides that comprise the polynucleotide can be ribonucleotides or deoxyribonucleotides or modified forms of either nucleotide type. Such modifications include base modifications such as bromouridine, ribose modifications such as arabinose and 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, dithiophosphate, selenophosphate, diselenophosphate, phosphorothioanilidate, phosphoraniladate, and phosphoroamidate. The term "polynucleotide" specifically includes single-stranded and double-stranded forms of DNA.

[0263] The term "naturally occurring nucleotide" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotide" includes nucleotides having modified or substituted sugar moieties, etc. The term "oligonucleotide linkage" includes oligonucleotide linkages such as phosphorothioate, dithiophosphate, selenophosphate, diselenophosphate, phosphorothioanilide, anilide phosphate, amino phosphate, etc. See, e.g., LaPlanche et al., 1986, Nucleic Acids Res., 14:9081; Stec et al., 1984, J. Am. Chem. Soc., 106:6077; Stein et al., 1988, Nucleic Acids Res., 16:3209; Zon et al., 1991, Anti-Cancer Drug Design, 6:539; Zon et al., 1991, Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein ed.), Oxford University Press, Oxford, UK; Stec et al., U.S. Patent No. 5,151,510; Uhlmann and Peyman, 1990, Chemical Reviews, 90:543, the disclosures of which are incorporated herein by reference for any purpose. Oligonucleotides can contain detectable labels to enable detection of the oligonucleotide or its hybridization.

[0264] 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 transforming a host cell and contains nucleic acid sequences that direct and / or control the expression of an inserted heterologous nucleic acid sequence. If introns are present, expression includes, but is not limited to, processes such as transcription, translation and RNA splicing.

[0265] As will be understood by those skilled in the art, polynucleotides can contain genomic sequences, episomal and plasmid-encoded sequences, and smaller engineered gene segments that express or are adapted to express proteins, polypeptides, peptides, etc. Such segments can be naturally isolated or synthetically modified by those skilled in the art.

[0266] As will also be appreciated by those skilled in the art, polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules can include HnRNA molecules that contain introns and correspond to DNA molecules in a one-to-one manner, as well as mRNA molecules that do not contain introns. Additional coding or non-coding sequences can, but need not, be present within the polynucleotides according to the present disclosure, and the polynucleotides can, but need not, be linked to other molecules and / or support materials. Polynucleotides can include native sequences or can include sequences encoding variants or derivatives of such sequences.

[0267] Those of ordinary skill in the art will understand that due to the degeneracy of the genetic code, there are many nucleotide sequences that encode the antibodies described herein. Some of these polynucleotides have minimal sequence identity to the nucleotide sequences of the native or original polynucleotide sequences that encode polypeptides within the Wnt replacement molecule. However, the present disclosure clearly contemplates polynucleotides that vary due to differences in codon usage. In certain embodiments, sequences that have been codon-optimized for mammalian expression are specifically contemplated.

[0268] Thus, in another embodiment of the present disclosure, mutagenesis methods such as site-directed mutagenesis can be used to prepare variants and / or derivatives of the polypeptides described herein. By this method, specific modifications of the polypeptide sequence can be made by mutating the underlying polynucleotide that encodes it. These techniques provide a direct method for preparing and testing sequence variants (e.g., including one or more of the above considerations) by introducing one or more nucleotide sequence changes into the polynucleotide.

[0269] Site-directed mutagenesis allows mutants to be generated by using a specific oligonucleotide sequence that encodes the desired mutated DNA sequence and a sufficient number of adjacent nucleotides to provide a primer sequence of sufficient size and sequence complexity to form stable duplexes on both sides of the traversed deletion junction. Mutations can be introduced into the selected polynucleotide sequence to improve, alter, reduce, modify or otherwise change the properties of the polynucleotide itself and / or change the properties, activity, composition, stability or primary sequence of the encoded polypeptide.

[0270] In certain embodiments, the inventors contemplate mutagenizing polynucleotide sequences encoding polypeptides present in Wnt surrogate molecules to alter one or more properties of the encoded polypeptides, such as binding affinity or the function of a particular Fc region or the affinity of the Fc region for a particular FcγR. Site-directed mutagenesis techniques are well known in the art and are widely used to generate variants of polypeptides and polynucleotides. For example, site-directed mutagenesis is commonly used to alter specific portions of DNA molecules. In such embodiments, primers that are typically about 14 to about 25 nucleotides in length are employed, wherein about 5 to about 10 residues on either side of the junction of the sequences are altered.

[0271] As will be appreciated by those skilled in the art, site-directed mutagenesis techniques typically employ phage vectors that exist in both single-stranded and double-stranded forms. Typical vectors that can be used for site-directed mutagenesis include vectors such as M13 phage. These phages are readily commercially available and their uses are generally well known to those skilled in the art. Double-stranded plasmids are also commonly used for site-directed mutagenesis that eliminates the step of transferring the gene of interest from the plasmid to the phage.

[0272] Using site-directed mutagenesis to prepare sequence variants of selected DNA segments encoding peptides provides a means of generating potentially useful species, but is not meant to be limiting as there are other methods by which sequence variants of peptides and the DNA sequences encoding them can be obtained. For example, a recombinant vector encoding a desired peptide sequence can be treated with a mutagen such as hydroxylamine to obtain sequence variants. Specific details regarding these methods and protocols are found in the following teachings: Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994; and Maniatis et al., 1982, each of which is incorporated herein by reference for the purposes stated.

[0273] In many embodiments, one or more nucleic acids encoding polypeptides of Wnt surrogate molecules are directly introduced into host cells and the cells are cultured under conditions sufficient to induce expression of the encoded polypeptides. The Wnt surrogate polypeptides of the present disclosure can be prepared using standard techniques well known to those skilled in the art in combination with the polypeptide and nucleic acid sequences provided herein. The polypeptide sequences can be used to determine the appropriate nucleic acid sequences encoding the particular polypeptides disclosed herein. The nucleic acid sequences can be optimized to reflect the particular codon "preferences" of various expression systems according to standard methods well known to those skilled in the art.

[0274] According to certain related embodiments, a recombinant host cell is provided, the recombinant host cell comprising one or more constructs as described herein, e.g., a vector comprising a nucleic acid encoding a Wnt surrogate molecule or a polypeptide thereof; and a method of producing an encoded product, the method comprising expression from its encoding nucleic acid. Expression can be conveniently achieved by culturing the recombinant host cell containing the nucleic acid under appropriate conditions. After production by expression, the antibody or an antigen-binding fragment thereof can be isolated and / or purified using any suitable technique and then used as desired.

[0275] The polypeptide, as well as the encoding nucleic acid molecule and vector, can be isolated and / or purified, for example, from its natural environment in a substantially pure or homogeneous form, or, in the case of the nucleic acid, free or substantially free of the original nucleic acid or gene other than the sequence encoding the polypeptide having the desired function. The nucleic acid can comprise DNA or RNA and can be wholly or partially synthetic. Unless the context otherwise requires, reference to a nucleotide sequence shown herein encompasses a DNA molecule having the specified sequence and encompasses an RNA molecule having the specified sequence in which U has replaced T.

[0276] Systems for cloning and expressing polypeptides 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 expressing heterologous polypeptides include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, etc. A commonly preferred bacterial host is Escherichia coli.

[0277] Expression of polypeptides (e.g., antibodies and antigen-binding fragments thereof) in prokaryotic cells such as E. coli has been well established in the art. For a review, see, e.g., Pluckthun, Biotechnology 9:545 - 551 (1991). Expression in cultured eukaryotic cells is also available to those skilled in the art as an option for producing antibodies or antigen-binding fragments thereof, see recent reviews, e.g., Ref, M.E. (1993) Curr. Opinion Biotech. 4:573 - 576; Trill J.J. et al. (1995) Curr. Opinion Biotech. 6:553 - 560.

[0278] When appropriate, a suitable vector containing appropriate regulatory sequences (including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes) and other sequences can be selected or constructed. The vector can be a plasmid, virus, such as a phage or phagemid, as needed. For further details, see, for example, Molecular Cloning: A Laboratory Manual, 2nd ed., Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Current Protocols in Molecular Biology, 2nd ed., Ausubel et al. eds., John Wiley & Sons, 1992 or subsequent updates describe in detail many known techniques and protocols for manipulating nucleic acids, for example, in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells, and gene expression and protein analysis.

[0279] The term "host cell" is used to refer to a cell into which a nucleic acid sequence encoding one or more of the polypeptides described herein has been or can be introduced, and which further expresses or is capable of expressing the selected gene of interest, such as a gene encoding any of the polypeptides described herein. The term includes progeny of the parent cell, whether or not the progeny are identical in morphology or genetic constitution to the original parent, so long as the selected gene is present. Thus, methods for introducing such nucleic acids into host cells are also contemplated. The introduction can employ any available technique. For eukaryotic cells, suitable techniques can include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses (e.g., vaccinia or viruses for insect cells, baculovirus). For bacterial cells, suitable techniques can include calcium chloride transformation, electroporation, and transfection using phages. After introduction, expression from the nucleic acid can be induced or allowed, for example, by culturing the host cell under conditions that express the gene. In one embodiment, the nucleic acid is integrated into the genome (e.g., chromosome) of the host cell. Integration can be facilitated by including sequences that promote recombination with the genome, according to standard techniques.

[0280] In certain embodiments, the present disclosure also provides a method that includes using the construct as described above in an expression system to express a specific polypeptide, such as a Wnt mimetic molecule as described herein. The term "transduction" is used to refer to the transfer of genes from one bacterium to another, typically by a bacteriophage. "Transduction" also refers to the acquisition and transfer of eukaryotic cell sequences by retroviruses. The term "transfection" is used to refer to the uptake of foreign or exogenous DNA by a cell, and when the exogenous DNA has been introduced inside the cell membrane, the cell has been "transfected". Many transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory; 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 a suitable host cell.

[0281] As used herein, the term "transformation" refers to a change in the genetic characteristics of a cell, and when the cell has been modified to contain new DNA, the cell has been transformed. For example, in the case of genetically modifying a cell from its natural state, the cell is transformed. After transfection or transduction, the transforming DNA can recombine with the cell's DNA by physically integrating into the cell's chromosome, or can be maintained transiently as an episomal element without replication, or can replicate independently as a plasmid. A cell is considered to be stably transformed when the DNA replicates with cell division. When used in conjunction with biological materials such as nucleic acid molecules, polypeptides, host cells, etc., the term "naturally occurring" or "natural" refers to materials found in nature and not manipulated by humans. Similarly, as used herein, "non-naturally occurring" or "non-natural" refers to materials not found in nature or that have been structurally modified or synthesized by humans.

[0282] The terms "polypeptide", "protein" and "peptide" and "glycoprotein" are used interchangeably and mean a polymer of amino acids that is not limited to any particular length. The terms do not exclude modifications such as myristoylation, sulfation, glycosylation, phosphorylation, and addition or deletion of signal sequences. The term "polypeptide" or "protein" means one or more amino acid chains, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein the polypeptide or protein may include multiple chains non-covalently and / or covalently linked by peptide bonds and having a natural protein sequence, i.e., a protein produced by naturally occurring and, in particular, non-recombinant cells, or genetically engineered or recombinant cells, and includes molecules having the amino acid sequence of a natural protein, or molecules having one or more amino acids deleted, added and / or substituted from the natural sequence. The terms "polypeptide" and "protein" specifically encompass Wnt replacement molecules that bind to the Fzd receptor or LRP5 or LRP6 receptor disclosed herein, their Fzd binding regions, their LRP5 / 6 binding regions, antibodies and antigen-binding fragments thereof, or sequences having one or more amino acids deleted, added and / or substituted from any of these polypeptides. Thus, a "polypeptide" or "protein" can be comprised of one (termed a "monomer") or multiple (termed a "polymer") amino acid chains.

[0283] The terms "isolated protein," "isolated Wnt surrogate molecule," or "isolated antibody" as referred to herein mean that the subject protein, Wnt surrogate molecule, or antibody: (1) is free of at least some other proteins normally found in nature; (2) is substantially free of other proteins from the same source, such as from the same species; (3) is expressed by cells from a different species; (4) has been separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other substances with which it is associated in nature; (5) is not associated with a portion of the protein with which the "isolated protein" is associated in nature (by covalent or non-covalent interactions); (6) is operably associated with a polypeptide with which it is not associated in nature (by covalent or non-covalent interactions); or (7) does not occur in nature. Such isolated proteins may 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 include naturally occurring polypeptide sequences and / or artificial polypeptide sequences. In certain embodiments, an isolated protein is substantially free of proteins or polypeptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research, or otherwise).

[0284] One or more amino acid sequence modifications of any of the polypeptides described herein (e.g., a Wnt surrogate molecule or its Fzd-binding region or LRP5 / 6-binding region) are envisioned. For example, it may be desirable to improve the binding affinity and / or other biological properties of a Wnt surrogate molecule. For example, amino acid sequence variants of a Wnt surrogate molecule can be prepared by introducing appropriate nucleotide changes into the polynucleotide encoding the antibody or its chain or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to obtain the final Wnt surrogate molecule, provided that the final construct has the desired properties (e.g., high affinity for binding to one or more Fzd and / or LRP5 / 6 receptors). Amino acid changes can also alter the post-translational processes of the antibody, such as changing the number or location of glycosylation sites. Any of the variants and modifications of the polypeptides of the present disclosure described above can be included in the antibodies of the present disclosure.

[0285] The present disclosure provides variants of any of the polypeptides disclosed herein (e.g., a Wnt surrogate molecule or its Fzd-binding region or LRP5 / 6-binding region, or an antibody or its antigen-binding fragment). In certain embodiments, the variant has at least 90%, at least 95%, at least 98%, or at least 99% identity to the polypeptide disclosed herein. In certain embodiments, the binding of such variant polypeptides to one or more Fzd receptors and / or one or more LRP5 / 6 receptors is at least about 50%, at least about 70%, and in certain embodiments at least about 90% relative to the binding of the Wnt surrogate molecule to one or more Fzd receptors and / or one or more LRP5 / 6 receptors specifically shown herein. In additional embodiments, such variant Wnt surrogate molecules bind to one or more Fzd receptors and / or one or more LRP5 / 6 receptors with a greater affinity than the Wnt surrogate molecule shown herein, e.g., its binding is at least about 105%, 106%, 107%, 108%, 109%, or 110% quantitatively relative to the binding of the antibody sequence specifically shown herein.

[0286] In certain embodiments, a Wnt surrogate molecule or a binding region thereof, such as a Fab, scFv, or VHH or sdAb, can include: a) a heavy chain variable region that includes: i. a CDR1 region having an amino acid sequence identical to the heavy chain CDR1 region of a selected antibody described herein; ii. a CDR2 region having an amino acid sequence identical to the heavy chain CDR2 region of the selected antibody; and iii. a CDR3 region having an amino acid sequence identical to the heavy chain CDR3 region of the selected antibody; and / or b) a light chain variable domain that includes: i. a CDR1 region having an amino acid sequence identical to the light chain CDR1 region of the selected antibody; ii. a CDR2 region having an amino acid sequence identical to the light chain CDR2 region of the selected antibody; and iii. a CDR3 region having an amino acid sequence identical to the light chain CDR3 region of the selected antibody; wherein the antibody specifically binds to a selected target (e.g., one or more Fzd receptor epitopes or LRP5 or LRP6 receptors). In additional embodiments, the antibody or an antigen-binding fragment thereof is a variant antibody or an antigen-binding fragment thereof, wherein the variant includes the same heavy and light chains as the selected antibody, except for up to 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, 1, 2, 3, 4, 5, 6, 7, 8 amino acid substitutions may be present in the CDR regions of the selected antibody, or in certain embodiments, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions. Substitutions can be in the CDRs of the VH and / or VL regions. (See, e.g., Muller, 1998, Structure 6:1153-1167).

[0287] In certain embodiments, a Wnt surrogate molecule or a binding region thereof, such as a Fab, scFv, or VHH or sdAb, can 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 an antigen-binding fragment 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% identical, at least 90%, at least 95% or at least 98% or 99% identical to the light chain variable region of an antibody or an antigen-binding fragment thereof described herein. Amino acid sequences of illustrative antigen-binding fragments are shown in SEQ ID NOs: 1-97 and 109-157.

[0288] A polypeptide has a certain percentage of "sequence identity" with another polypeptide, which means that when the two sequences are aligned, the percentage of amino acids that are the same. Sequence similarity can be determined in a variety of different ways. To determine sequence identity, methods and computer programs including BLAST, which is available via the World Wide Web at ncbi.nlm.nih.gov / BLAST / , can be used to align sequences. Another alignment algorithm is FASTA, which is available from the Genetics Computing Group (GCG) software package, a wholly owned subsidiary of Oxford Molecular Group, Inc., Madison, Wis., USA. Other techniques for alignment are described in the following: Methods in Enzymology, Volume 266: Computer Methods for Macromolecular Sequence Analysis (1996), edited by Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA. Particular attention is paid to alignment programs that allow gaps in the sequences. Smith-Waterman is one type of algorithm that allows gaps in sequence alignment. See Meth. Mol. Biol. 70:173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be used to align sequences. See J. Mol. Biol. 48:443-453 (1970).

[0289] Attention is paid to the BestFit program that uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2:482-489 (1981)) to determine sequence identity. The range of the gap generation penalty will generally be from 1 to 5, often from 2 to 4 and in many embodiments will be 3. The range of the gap extension penalty will generally be from 0.01 to 0.20 and in many cases will be 0.10. The program has default parameters determined by the sequences input for comparison. Preferably, sequence identity is determined using the default parameters determined by the program. This program is also available from the Genetics Computing Group (GCG) software package, Madison, Wis., USA.

[0290] 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, pages 127-149, 1988, Alan R. Liss, Inc. The percent sequence identity is calculated by FastDB based on the following parameters:

[0291] Mismatch penalty: 1.00; gap penalty: 1.00; gap size penalty: 0.33; and join penalty: 30.0.

[0292] In certain embodiments, a Wnt surrogate molecule or a binding region thereof, such as a Fab, scFv, or VHH or sdAb, can include: a) a heavy chain variable region that includes: i. a CDR1 region having an amino acid sequence identical to the amino acid sequence of the heavy chain CDR1 region of a selected antibody described herein; ii. a CDR2 region having an amino acid sequence identical to the amino acid sequence of the heavy chain CDR2 region of the selected antibody; and iii. a CDR3 region having an amino acid sequence identical to the amino acid sequence of the heavy chain CDR3 region of the selected antibody; and b) a light chain variable domain that includes: i. a CDR1 region having an amino acid sequence identical to the amino acid sequence of the light chain CDR1 region of the selected antibody; ii. a CDR2 region having an amino acid sequence identical to the amino acid sequence of the light chain CDR2 region of the selected antibody; and iii. a CDR3 region having an amino acid sequence identical to the amino acid sequence of the light chain CDR3 region of the selected antibody; wherein the antibody specifically binds to a selected target (e.g., an Fzd receptor, such as Fzd1). In additional embodiments, the antibody or an antigen-binding fragment thereof is a variant antibody, wherein the variant includes the same heavy and light chains as the selected antibody, except for up to 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, 1, 2, 3, 4, 5, 6, 7, 8 amino acid substitutions may be present in the CDR regions of the selected antibody, or in certain embodiments, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions. Substitutions can be in the CDRs in the VH and / or VL regions. (See, e.g., Muller, 1998, Structure 6:1153-1167).

[0293] The determination of the three-dimensional structure of a representative polypeptide (e.g., a variant Fzd-binding region or LRP5 / 6-binding region of a Wnt surrogate molecule provided herein) can be carried out by conventional methods such that, for example, one or more amino acid substitutions, additions, deletions, or insertions made with selected natural or unnatural amino acids can be virtually modeled for the purpose of determining whether the space-filling properties of the species so derived are retained. See, e.g., Donate et al., 1994, Prot. Sci. 3:2378; Bird et al., Science 309:1868-1871 (2005); Schueler-Furman et al., Science 310:638 (2005); Dietz et al., Proc. Natl. 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 can be used for these and related embodiments (such as for the rational design of binding regions) include VMD, which is a molecular visualization program for displaying, animating, and analyzing large biomolecular systems using 3-D graphics and built-in scripting (see the website of the Theoretical and Computational Biophysics Group, University of Illinois at Urbana-Champagne, ks.uiuc.edu / Research / vmd / ).Many other computer programs are known in the art and available to the skilled person and allow determination of the atomic scale according to space filling models (van der Waals radii) of energy minimized conformations; GRID which attempts to determine high affinity regions for different chemical groups to enhance binding, Monte Carlo searches for computational 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 evaluate 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). Various suitable computational computer programs can also be obtained commercially from, for example. (Munich, Germany).

[0294] Composition

[0295] There is also disclosed a pharmaceutical composition comprising a Wnt replacement molecule as described herein and one or more pharmaceutically acceptable diluents, carriers or excipients. In certain embodiments, the pharmaceutical composition further comprises one or more Wnt polypeptides or Norrin polypeptides.

[0296] In a further embodiment, a pharmaceutical composition is also disclosed that comprises a polynucleotide and one or more pharmaceutically acceptable diluents, carriers, or excipients, wherein the polynucleotide comprises a nucleic acid sequence encoding a Wnt surrogate molecule as described herein. In certain embodiments, the pharmaceutical composition further comprises one or more polynucleotides, wherein the one or more polynucleotides comprise a nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide. In some embodiments, the polynucleotide is DNA or mRNA, such as modified mRNA. In certain embodiments, the polynucleotide is a modified mRNA that further comprises a 5' cap sequence and / or a 3' trailing sequence (such as a polyA tail). In other embodiments, the polynucleotide is an expression cassette that comprises a promoter operably linked to a coding sequence. In some embodiments, the nucleic acid sequence encoding a Wnt surrogate molecule and the nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide are present in the same polynucleotide.

[0297] In a further embodiment, a pharmaceutical composition is also disclosed that comprises an expression vector, such as a viral vector, and one or more pharmaceutically acceptable diluents, carriers, or excipients, wherein the vector comprises a polynucleotide that comprises a nucleic acid sequence encoding a Wnt surrogate molecule as described herein. In certain embodiments, the pharmaceutical composition further comprises an expression vector (e.g., a viral vector) that comprises a polynucleotide that comprises a nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide. In some embodiments, the nucleic acid sequence encoding a Wnt surrogate molecule and the nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide are present in the same polynucleotide (e.g., an expression cassette).

[0298] The present disclosure further contemplates a pharmaceutical composition that comprises a cell and one or more pharmaceutically acceptable diluents, carriers, or excipients, wherein the cell comprises an expression vector that comprises a polynucleotide that comprises a promoter operably linked to a nucleic acid encoding a Wnt surrogate molecule. In certain embodiments, the pharmaceutical composition further comprises a cell that comprises an expression vector that comprises a polynucleotide that comprises a promoter operably linked to a nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide. In some embodiments, the nucleic acid sequence encoding a Wnt surrogate molecule and the nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide are present in the same polynucleotide (e.g., an expression cassette) and / or the same cell. In certain embodiments, the cell is a heterologous cell or an autologous cell obtained from a subject to be treated. In certain embodiments, the cell is a stem cell, such as an adipose-derived stem cell or a hematopoietic stem cell.

[0299] The present disclosure contemplates pharmaceutical compositions that include a first molecule for delivering a Wnt surrogate molecule as a first active agent and a second molecule for delivering a Wnt polypeptide or a Norrin polypeptide. The first molecule and the second molecule can be the same type of molecule or different types of molecules. For example, in certain embodiments, the first molecule and the second molecule can each independently be selected from the group consisting of molecules of the following types: polypeptides, organic small molecules, nucleic acids encoding the first active agent or the second active agent (optionally, DNA or mRNA, optionally, modified RNA), vectors including a nucleic acid sequence encoding the first active agent or the second active agent (optionally, expression vectors or viral vectors), and cells including a nucleic acid sequence encoding the first active agent or the second active agent (optionally, expression cassettes).

[0300] The subject molecules, alone or in combination, can be combined with pharmaceutically acceptable carriers, diluents, excipients, and reagents useful in preparing generally safe, non-toxic, and desirable formulations, and include excipients acceptable for use in mammals such as humans or primates. Such excipients can be solid, liquid, semi-solid, or gaseous in the case of aerosol compositions. Examples of such carriers, diluents, and excipients include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Supplementary active compounds can also be incorporated into the formulations. Solutions or suspensions for use in the formulations can include: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate, or phosphate; detergents such as Tween 20 used to prevent polymerization; and compounds for adjusting the osmotic pressure such as sodium chloride or dextrose. The pH can be adjusted with an acid or a base such as hydrochloric acid or sodium hydroxide. In certain embodiments, the pharmaceutical composition is sterile.

[0301] The pharmaceutical composition may further comprise a sterile aqueous solution or dispersion and a sterile powder for the extemporaneous preparation of a sterile injectable solution or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In certain cases, the composition is sterile and should be fluid so that it can be drawn into a syringe and delivered to a subject from the syringe. In certain embodiments, the composition is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using surfactants. The action of preventing microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it is preferred to include isotonic agents (e.g., sugars, polyols such as mannitol, sorbitol, etc., sodium chloride) in the composition. Prolonged absorption of the internal composition can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0302] The sterile solution can be prepared by incorporating the required amount of the anti-Fzd antibody or its antigen-binding fragment (or the encoding polynucleotide or cells comprising the same) into a suitable solvent, along with one or a combination of the ingredients listed above, as needed, followed by filtration sterilization. Generally, the dispersion is prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other required ingredients from those listed above. In the case of a sterile powder for the preparation of a sterile injectable solution, the methods of preparation are vacuum drying and freeze drying, which yield a powder of the active ingredient plus any additional desired ingredients from its previously sterile filtered solution.

[0303] In one embodiment, the pharmaceutical composition is prepared with a carrier that protects the antibody or its antigen-binding fragment from rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The methods for preparing such formulations are apparent to those skilled in the art. The materials are also commercially available. Liposome suspensions can also be used as pharmaceutically acceptable carriers. These liposome suspensions can be prepared according to methods known to those skilled in the art.

[0304] It can be advantageous to formulate the pharmaceutical composition in dosage unit form for ease of administration and to achieve dosage uniformity. As used herein, a dosage unit form refers to a physically discrete unit suitable as a single dosage for a subject to be treated; each unit contains a predetermined amount of the active antibody or antigen-binding fragment thereof calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification of the dosage unit form is determined by and directly depends on the unique characteristics of the antibody or antigen-binding fragment thereof and the particular therapeutic effect to be achieved, as well as the limitations inherent in the art of compounding such active antibody or antigen-binding fragment for the treatment of individuals.

[0305] The pharmaceutical composition can be contained in a container, package, or dispenser (e.g., a syringe, such as a drug-loaded syringe) together with instructions for administration.

[0306] The pharmaceutical compositions of the present disclosure encompass any pharmaceutically acceptable salt, ester, or salt of such an ester, or any other compound that is capable of (directly or indirectly) providing a bioactive antibody or antigen-binding fragment thereof when administered to an animal, including a human.

[0307] The present disclosure includes pharmaceutically acceptable salts of the Wnt surrogate molecules described herein. The term "pharmaceutically acceptable salts" refers to physiologically acceptable and pharmaceutically acceptable salts of the compounds of the present disclosure: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects to the parent compound. A variety of pharmaceutically acceptable salts are known in the art and are described, for example, in Remington's Pharmaceutical Sciences, 17th Edition, Alfonso R. Gennaro (ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and its more recent editions); Encyclopaedia of Pharmaceutical Technology, 3rd Edition, James Swarbrick (ed.), Informa Healthcare USA (Inc.), NY, USA, 2007; and J. Pharm. Sci. 66:2 (1977). Moreover, for a review of suitable salts, see Stahl and Wermuth, "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002).

[0308] 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 include sodium, potassium, magnesium, calcium, etc. Amines include N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, e.g., Berge et al., "Pharmaceutical Salts", J. Pharma Sci., 1977, 66, 119). The base addition salts of acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in a conventional manner. The free acid form can be regenerated by contacting the salt form with an acid and separating the free acid in a conventional manner. The free acid form differs slightly in certain physical properties such as solubility in polar solvents from its respective salt form, but for the purposes of this disclosure, the salt is equivalent to its respective free acid.

[0309] In some embodiments, the pharmaceutical compositions provided herein include a therapeutically effective amount of a Wnt replacement molecule or a pharmaceutically acceptable salt thereof, mixed with a pharmaceutically acceptable carrier, diluent, and / or excipient, such as saline, phosphate buffered saline, phosphates and amino acids, polymers, polyols, sugars, buffers, preservatives, and other proteins. Exemplary amino acids, polymers, and sugars, etc. are octylphenoxypolyethoxyethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, Ringer's and Hank's solutions, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and ethylene glycol. Preferably, this formulation is stable at 4 °C for at least six months.

[0310] In some embodiments, the pharmaceutical compositions provided herein include a buffer, such as phosphate buffered saline (PBS) or sodium phosphate / sodium sulfate, tris buffer, glycine buffer, sterile water, and other buffers known to those of ordinary skill in the art, such as those described by Good et al. (1966) Biochemistry 5:467. The pH value of the buffer can be in the range of 6.5 to 7.75, preferably 7 to 7.5, and most preferably 7.2 to 7.4.

[0311] Method of Use

[0312] The present disclosure also provides methods for using the Wnt replacement molecules disclosed herein, e.g., to modulate the Wnt signaling pathway (e.g., increase Wnt signaling), and methods for administering the Wnt replacement molecules disclosed herein in a variety of therapeutic settings. Therapeutic methods using Wnt replacement molecules are provided herein. In one embodiment, a Wnt replacement molecule is provided to a subject having a disease involving inappropriate or dysregulated Wnt signaling (e.g., reduced Wnt signaling).

[0313] Activating Wnt Pathway Signaling and Related Therapeutic Methods

[0314] In certain embodiments, the Wnt replacement molecules can be used to activate the Wnt signaling pathway in a tissue or cell. Activating the Wnt signaling pathway can include, e.g., increasing Wnt signaling or enhancing Wnt signaling in the tissue or cell. Accordingly, in some aspects, the present disclosure provides a method for activating the Wnt signaling pathway in a cell, the method comprising contacting the tissue or cell with an effective amount of a Wnt replacement molecule disclosed herein or a pharmaceutically acceptable salt thereof, wherein the Wnt replacement molecule is a Wnt signaling pathway agonist. In some embodiments, the contacting occurs in vitro, ex vivo, or in vivo. In certain embodiments, the cell is a cultured cell and the contacting occurs in vitro. In certain embodiments, the method further comprises contacting the tissue or cell with one or more Wnt polypeptides or Norrin polypeptides.

[0315] In a related aspect, the present disclosure provides a method for activating Wnt signaling in a tissue or cell, the method comprising contacting the tissue or cell with an effective amount of a polynucleotide comprising a Wnt replacement molecule disclosed herein. In certain embodiments, the target tissue or target cell is further contacted with a polynucleotide comprising a nucleic acid sequence encoding a Wnt polypeptide or Norrin polypeptide. In certain embodiments, the polynucleotide is DNA or mRNA, e.g., modified mRNA. In a particular embodiment, the polynucleotide is a modified mRNA further comprising a 5' cap sequence and / or a 3' trailing sequence (e.g., polyA tail). In other embodiments, the polynucleotide is an expression cassette comprising a promoter operably linked to a coding sequence. In certain embodiments, the nucleic acid sequence encoding the Wnt replacement molecule and the nucleic acid sequence encoding the Wnt polypeptide or Norrin polypeptide are present in the same polynucleotide.

[0316] In a related aspect, the present disclosure provides a method for activating Wnt signaling in a tissue or cell, the method 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 further contacted with a vector comprising a nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the vector is an expression vector and may comprise a promoter sequence operably linked to the nucleic acid sequence. In a particular embodiment, 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., the same expression cassette).

[0317] In a related aspect, the present disclosure provides a method for activating Wnt signaling in a tissue, the method comprising contacting the tissue with an effective amount of a cell comprising a nucleic acid sequence encoding a Wnt surrogate molecule of the present disclosure. In certain embodiments, the tissue is further contacted with a cell comprising a nucleic acid sequence encoding 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 a particular embodiment, the cell is an allogeneic cell or an autologous cell obtained from a subject to be treated. In certain embodiments, the cell is transduced with a vector comprising an expression cassette encoding a Wnt surrogate molecule or a Wnt polypeptide or a Norrin polypeptide. In a particular embodiment, the cell is a stem cell, such as an adipose-derived stem cell or a hematopoietic stem cell.

[0318] The Wnt surrogate molecules disclosed herein can be used, for example, to treat diseases, disorders, or conditions by activating (e.g., increasing) Wnt signaling in targeted cells, tissues, or organs. Thus, in some aspects, the present disclosure provides a method for treating a disease or condition in a subject in need thereof (e.g., a disease or disorder associated with reduced or impaired Wnt signaling and / or a disease or disorder for which increasing Wnt signaling would provide a therapeutic benefit), the method comprising contacting the subject with an effective amount of a composition of the present disclosure. In certain embodiments, the composition is a pharmaceutical composition, the pharmaceutical composition comprising any of the following: a Wnt surrogate molecule; a polynucleotide comprising a nucleic acid sequence encoding a Wnt surrogate molecule, such as DNA or mRNA, optionally modified mRNA; a vector comprising a nucleic acid sequence encoding a Wnt surrogate molecule, such as an expression vector or a viral vector; or a cell comprising a nucleic acid sequence encoding a Wnt surrogate molecule, such as a cell transduced with an expression vector or a viral vector encoding a Wnt surrogate molecule. In certain embodiments, the disease or condition is a pathological disease or disorder or an injury, such as an injury caused by a wound. In certain embodiments, the wound may be the result of another therapeutic treatment. In certain embodiments, the disease or condition includes impaired tissue repair, healing, or regeneration, or would benefit from increased tissue repair, healing, or regeneration. In some embodiments, the contacting occurs in vivo, i.e., the subject composition is administered to the subject.

[0319] In certain embodiments, the method further comprises contacting the subject with a pharmaceutical composition comprising one or more Wnt polypeptides or Norrin polypeptides. The present disclosure contemplates contacting the subject with a first molecule for delivering a Wnt surrogate molecule as a first active agent and a second molecule for delivering a Wnt polypeptide or Norrin polypeptide. The first molecule and the second molecule can be of the same type or different types of molecules. For example, in certain embodiments, the first molecule and the second molecule can each independently be selected from the group consisting of: polypeptides, organic small molecules, nucleic acids encoding the first active agent or the second active agent (optionally, DNA or mRNA, optionally, modified RNA), vectors comprising a nucleic acid sequence encoding the first active agent or the second active agent (optionally, expression vectors or viral vectors), and cells comprising a nucleic acid sequence encoding the first active agent or the second active agent (optionally, expression cassettes).

[0320] In a related aspect, the present disclosure provides a method for treating a disease or condition (e.g., a disease or disorder associated with reduced Wnt signaling or a disease or disorder for which increasing Wnt signaling would provide a therapeutic benefit), the method 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 further contacted with a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the polynucleotide is DNA or mRNA, such as modified mRNA. In a particular embodiment, the polynucleotide is a modified mRNA further comprising a 5' cap sequence and / or a 3' trailing sequence (e.g., a polyA tail). In other embodiments, the polynucleotide is an expression cassette comprising a promoter operably linked to a coding sequence. In certain embodiments, the nucleic acid sequence encoding a Wnt surrogate molecule and the nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide are present in the same polynucleotide.

[0321] In a related aspect, the present disclosure provides a method for treating a disease or condition (e.g., a disease or disorder associated with reduced Wnt signaling or a disease or disorder for which increasing Wnt signaling would provide a therapeutic benefit), the method 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 further contacted with a pharmaceutical composition comprising an effective amount of a vector comprising a nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the vector is an expression vector and may comprise a promoter sequence operably linked to the nucleic acid sequence. In a particular embodiment, the vector is a viral vector. In certain embodiments, the nucleic acid sequence encoding a Wnt surrogate molecule and the nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide are present in the same vector (e.g., the same expression cassette).

[0322] In relevant aspects, the present disclosure provides a method for treating a disease or condition (e.g., a disease or disorder associated with reduced Wnt signaling or a disease or disorder for which increasing Wnt signaling would provide a therapeutic benefit), the method comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of cells, the cells comprising a nucleic acid sequence encoding a Wnt surrogate molecule. In certain embodiments, the subject is also contacted with cells comprising a nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the nucleic acid sequence encoding a Wnt surrogate molecule and the nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide are present in the same cell. In certain embodiments, the cells are allogeneic or autologous cells obtained from the subject to be treated. In certain embodiments, the cells are transduced with a vector comprising an expression cassette encoding a Wnt surrogate molecule or a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the cells are stem cells, such as adipose-derived stem cells or hematopoietic stem cells.

[0323] Wnt signaling plays a key role in the development and maintenance of stem cells. Reactivation of Wnt signaling is associated with the regeneration and repair of most tissues following injury and disease. A Wnt surrogate molecule is expected to provide the benefits of healing and tissue repair in response to injury and disease. Causes of tissue damage and loss include, but are not limited to, aging, degeneration, genetic conditions, infection and inflammation, traumatic injury, toxin / metabolism-induced toxicity, or other pathological conditions. It has been shown that Wnt signaling and enhancers of Wnt signaling activate adult tissue-resident stem cells. In some embodiments, the compounds of the present disclosure are administered for treating diseased or damaged tissue, for tissue regeneration, and for cell growth and proliferation and / or for tissue engineering.

[0324] Human diseases associated with mutations in the Wnt pathway provide strong evidence for the enhancement of Wnt signaling in the treatment and prevention of diseases. Preclinical in vivo and in vitro studies provide additional evidence of the involvement of Wnt signaling in many disease conditions and further support the use of Wnt replacement molecules in various human diseases. For example, the compositions of the present disclosure can be used to promote or increase bone growth or regeneration, bone grafting, fracture healing, stress fractures, vertebral compression fractures, the treatment of osteoporosis and osteoporotic fractures, spinal fusion, osseointegration of orthopedic devices, tendon-to-bone integration, tooth growth and regeneration, dental implants, periodontal diseases, maxillofacial reconstruction, and jaw necrosis. The compositions of the present invention can also be used for: treating hair loss; enhancing the regeneration of sensory organs, such as treating hearing loss, treating vestibular hypofunction, treating macular degeneration, treating vitreoretinopathy, other retinal degenerative diseases, Fuchs' dystrophy, other corneal diseases, etc.; treating stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, muscular dystrophy, muscle atrophy caused by sarcopenia or cachexia, and other conditions affecting the blood-brain barrier; treating spinal cord injury, other spinal cord diseases. The compositions of the present disclosure can also be used for: treating oral mucositis, treating short bowel syndrome, inflammatory bowel disease (IBD), other gastrointestinal disorders; treating metabolic syndrome; treating diabetes, dyslipidemia, treating pancreatitis, conditions in which exocrine or endocrine pancreatic tissue is damaged, conditions in which enhanced epidermal regeneration, such as epidermal wound healing, is desired; treating diabetic foot ulcers, syndromes involving dental, nail, or dermal hypoplasia, etc., conditions in which angiogenesis is beneficial; treating myocardial infarction, coronary artery disease, heart failure, enhancing the growth of hematopoietic cells, such as enhancing hematopoietic stem cell transplantation from bone marrow, mobilizing peripheral blood; treating immunodeficiency, graft-versus-host disease, etc.; treating acute kidney injury, chronic kidney disease; treating lung diseases, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF); enhancing the regeneration of lung tissue. The compositions of the present disclosure can also be used to enhance hepatocyte regeneration (NASH), such as liver regeneration, treating cirrhosis, enhancing liver transplantation, treating acute liver failure, treating chronic liver diseases that have experienced hepatitis C or hepatitis B virus infection or antiviral post-drug therapy, alcoholic liver disease, alcoholic hepatitis, non-alcoholic liver disease with steatosis or steatohepatitis (NASH), etc. The compositions of the present disclosure can treat diseases and disorders, including but not limited to conditions in which regenerative cell growth is desired.

[0325] Human genetics involving loss-of-function or gain-of-function mutations in Wnt signaling components shows strong evidence in support of enhanced Wnt signaling for bone growth. Conditions in which enhanced bone growth is desired can include but are not limited to fractures, grafts, ingrowth around prosthetic devices, osteoporosis, osteoporotic fractures, spinal fusion, osteonecrosis of the jaw, dental implants, periodontal disease, maxillofacial reconstruction, and the like. Wnt replacement molecules enhance and promote Wnt signaling that is crucial for bone regeneration. Methods for regenerating bone tissue benefit from administration of the compounds of the present disclosure, which administration can be systemic or localized. In some embodiments, bone marrow cells are exposed to the molecules of the present disclosure, thereby activating stem cells within the bone marrow.

[0326] 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 replacement molecule disclosed herein. Methods for regenerating bone tissue benefit from administration of a Wnt replacement molecule, which administration 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 can be localized to the site of action, for example, by loading onto an optionally biodegradable matrix, and optionally provide a sustained release of the active agent. Matrix carriers include but are not limited to absorbable collagen sponges, ceramics, hydrogels, polymeric microspheres, nanoparticles, bone cements, and the like.

[0327] In certain embodiments, a composition comprising one or more Wnt replacement molecules disclosed herein (or a polynucleotide encoding a Wnt replacement molecule, or a vector or cell comprising a polynucleotide encoding a Wnt replacement molecule) is used for treating or preventing a skeletal disease or disorder including but not limited to any of the following, or treating or preventing an injury associated with but not limited to any of the following: osteoporosis, osteoporotic fracture, fracture, unhealed fracture, delayed union fracture, spinal fusion, osteonecrosis, necrosis of the jaw, hip, or femoral head, etc., osseointegration of an implant (e.g., for accelerating recovery after partial or total knee or hip replacement), osteogenesis imperfecta, bone graft, tendon repair, maxillofacial surgery, dental implant, any other bone disorder or defect caused by a genetic disease, degeneration, aging, drug, or injury. In one embodiment, a Wnt replacement molecule that binds to Fzd1, Fzd2, and Fzd7 and LRP5 and / or LRP6 is used for treating or preventing any skeletal disease or disorder. In one embodiment, a Wnt replacement molecule that binds to Fzd1, Fzd2, Fzd5, Fzd7, and Fzd8 and LRP5 and / or LRP6 is used for treating or preventing any skeletal disease or disorder.

[0328] In certain embodiments, the compositions and methods disclosed herein can be used to: increase the bone mineral density of a subject, increase the bone volume of the subject (e.g., tibial and / or femoral bone volume), increase the cortical thickness of the subject (e.g., in the trabecular region or in the midshaft of the femur), increase the mineral apposition rate of the subject, increase the number of osteoblasts (e.g., in bone) and / or decrease the number of osteoclasts (e.g., in bone) of the subject, increase the bone stiffness of the subject, increase the ultimate load at the fracture point of the subject, improve the resistance of the subject's bone to fracture, reduce the bone loss associated with osteoporosis in the subject, or increase the biochemical strength of the subject's bone. In one embodiment, a Wnt surrogate molecule that binds to Fzd1, Fzd2, and Fzd7 is used for any one of these specified uses. In one embodiment, a Wnt surrogate molecule that binds to Fzd1, Fzd2, Fzd5, Fzd7, and Fzd8 is used for any one of these specified uses.

[0329] Compositions comprising one or more Wnt surrogate molecules (or polynucleotides encoding Wnt surrogate molecules, or vectors or cells comprising polynucleotides encoding Wnt surrogate molecules) disclosed herein can be used for in vivo treatment of skeletal tissue defects. "Skeletal tissue defect" means a defect in bone or other skeletal connective tissue at any site where restoration of bone or connective tissue is desired, regardless of how the defect was caused, e.g., whether as a result of surgical intervention, tumor resection, ulceration, implantation, fracture, or other traumatic or degenerative condition. The compositions of the present disclosure can be used as part of a protocol for restoring the chondral function of connective tissue and for repairing defects or injuries in cartilage tissue, such as degenerative wear and arthritis, tissue trauma, meniscal tear displacement, meniscectomy, joint dislocation caused by ligament tear, joint malalignment, fracture, or genetic disease.

[0330] Wnt surrogate molecules can also be used to treat periodontal disease. Periodontal disease is a major cause of tooth loss and is associated with a variety of systemic conditions. In some embodiments, tooth or underlying bone regeneration is enhanced by contacting responsive cell populations. In some such embodiments, the contacting is performed in vivo. In other such embodiments, the contacting is performed ex vivo, followed by implantation of activated stem cells or progenitor cells. The molecule can be localized to the site of action, for example, by loading onto an optionally biodegradable matrix, and optionally provide a sustained release of the active agent. Matrix carriers include, but are not limited to, absorbable collagen sponges, ceramics, hydrogels, bone cements, polymer microspheres, nanoparticles, and the like.

[0331] Studies have shown that the biology of Wnt signaling and R-spondin can promote the regeneration of sensory hair cells in the inner ear after injury, aging, or degeneration. The loss of sensory hair cells in the inner ear involved in hearing loss or vestibular hypofunction can also benefit from the compositions of the present disclosure. In the inner ear, the auditory organ houses the mechanosensitive hair cells required to convert sound vibrations into electrical impulses. The vestibular organs, including the semicircular canals (SSCs), utricle, and saccule, also contain sensory hair cells to detect head position and head movement. The compositions of the present disclosure can be used, for example, for infusion; matrix or other depot systems; or other topical applications to the ear to enhance auditory regeneration.

[0332] Wnt replacement molecules can also be used for the regeneration of retinal tissue. In the adult mammalian retina, Muller glia cells are capable of regenerating retinal cells, including photoreceptors, for example, after in vivo neurotoxic injury. Wnt signaling and enhancers of the Wnt signal can promote the proliferation of Muller glia-derived retinal progenitor cells after injury or during degeneration. The compositions of the present disclosure can also be used to regenerate tissues and other cell types in the eye. For example, age-related macular degeneration (AMD), other retinal degenerative diseases, corneal diseases, Fuchs' dystrophy, vitreoretinopathy, genetic diseases, etc. can benefit from the compositions of the present disclosure. AMD is characterized by progressive loss of central vision and visual acuity. Fuchs' dystrophy is characterized by a gradual loss of corneal endothelial cells. Wnt signaling and enhancement of the Wnt signal can promote the regeneration of corneal endothelium, retinal epithelium, etc. in ocular tissues. In other embodiments, the compositions of the present disclosure can be used, for example, for infusion; matrix or other depot systems; or other topical applications to the eye to regenerate the retina and treat macular degeneration.

[0333] A specific population of proliferating cells for the homeostatic renewal of hepatocytes, such as Axin2-positive cells in the periportal area, has been identified through lineage tracing studies. Lineage tracing studies have also identified additional potential liver progenitor cells, including but not limited to Lgr-positive cells. Self-renewing hepatocytes and other potential progenitor cell populations, including Lgr5-positive cells and Axin2-positive cells, have been identified as being capable of regenerating in response to Wnt signaling and / or R-spondin after injury. Many preclinical models of acute liver injury and failure, as well as chronic liver diseases, have shown that the recovery and regeneration of hepatocytes benefit from enhanced Wnt signaling.

[0334] In certain embodiments, a composition 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) is used to promote liver regeneration, reduce fibrosis, and / or improve liver function. In certain embodiments, the compositions and methods disclosed herein are used to: increase liver weight, increase the ratio of liver weight to body weight, increase the number of PCNA- and pH3-positive cell nuclei in the liver, increase the expression of Ki67 and / or cyclin D1 in the liver, increase hepatocyte proliferation and / or mitosis, reduce fibrosis after chronic liver injury, or increase hepatocyte function.

[0335] In certain embodiments, the compositions of the present disclosure can be used to: treat acute liver failure, acute alcoholic liver injury; treat chronic liver diseases, chronic alcoholic liver disease, non-alcoholic fatty liver disease, and non-alcoholic steatohepatitis (NASH) that have experienced hepatitis C or hepatitis B virus infection or post-antiviral drug therapy; treat cirrhosis and severe chronic liver diseases of all causes; and enhance the regeneration of hepatocytes. Methods for regenerating liver tissue benefit from the administration of the compounds of the present disclosure, which administration can be systemic or localized. These methods include, but are not limited to, systemic administration methods and localized administration methods, such as by injection into liver tissue, by injection into veins or blood vessels leading to the liver, by implantation of a sustained-release formulation, and the like.

[0336] In certain embodiments, a composition 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) is used to treat or prevent liver diseases or disorders, including but not limited to treating or preventing liver injury or disorders caused by any of the following: acute liver failure (all causes), chronic liver failure (all causes), cirrhosis, liver fibrosis (all causes), portal hypertension, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD) (fatty liver), alcoholic hepatitis, hepatitis C virus-induced liver disease (HCV), hepatitis B virus-induced liver disease (HBV), other viral hepatitis (e.g., hepatitis A virus-induced liver disease (HAV) and hepatitis D virus-induced liver disease (HDV)), primary biliary cirrhosis, autoimmune hepatitis, liver surgery, liver injury, liver transplantation, "small-for-size" syndrome in liver surgery and transplantation, congenital liver diseases and disorders, and any other liver disorders or defects caused by genetic diseases, degeneration, aging, drugs, and injury.

[0337] Wnt signaling plays an important role in the regeneration of various epithelial tissues. A variety of epidermal conditions benefit from treatment with the compounds of the present disclosure. Mucositis occurs when rapidly dividing epithelial cells attached to the gastrointestinal tract rupture, making the mucosal tissue prone to ulceration and infection. The portion of the epithelial lining that covers the oral cavity, known as the oral mucosa, is one of the most sensitive parts of the body and is particularly vulnerable to the damage of chemotherapy and radiation. Oral mucositis can be the most common debilitating complication in cancer treatment, especially chemotherapy and radiation. Additionally, the compositions of the present disclosure can also be beneficial for treating short bowel syndrome, inflammatory bowel disease (IBD), or other gastrointestinal disorders. Other epidermal conditions include epidermal wound healing, diabetic foot ulcers, syndromes involving dental, nail, or skin hypoplasia, etc. The molecules of the present disclosure can be used in all these conditions where regenerative cells are contacted with the compounds of the present disclosure. Methods for regenerating epithelial tissues benefit from the administration of the compounds of the present disclosure, which can be systemic or localized. The contact can be, for example, local, including intradermal, subcutaneous, in the form of gels, lotions, creams, etc. applied at the targeted site, etc.

[0338] In addition to the skin and gastrointestinal tract, Wnt signaling, as well as the enhancement and promotion of Wnt signaling, also play important roles in the repair and regeneration of tissues including the pancreas, kidney, and lung in preclinical models. Wnt replacement molecules can be beneficial for various disease conditions involving the exocrine and endocrine pancreas, kidney, or lung. Wnt replacement molecules can be used to: treat metabolic syndrome; treat diabetes; treat acute or chronic pancreatitis, exocrine pancreatic insufficiency; treat acute kidney injury, chronic kidney disease; treat conditions including but not limited to chronic obstructive pulmonary disease (COPD), other conditions resulting in the loss of lung epithelial tissue. Methods for regenerating these tissues benefit from the administration of the compounds of the present disclosure, which can be systemic or localized.

[0339] Epidermal Wnt signaling, coordinated with signaling via other developmental factors, is crucial for adult hair follicle regeneration. Hair loss is a common problem, and androgenetic alopecia, commonly known as 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 the molecules of the present disclosure. In some such embodiments, the contact is performed in vivo. In other such embodiments, the contact is performed ex vivo. The molecules can be localized at the site of action, such as topical lotions, gels, creams, etc.

[0340] Wnt replacement molecules can be used to treat stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, and other conditions that affect the blood-brain barrier (BBB). Angiogenesis is crucial for ensuring the supply of oxygen and nutrients to many tissues throughout the body, and is particularly important for the CNS, as neural tissue is extremely sensitive to hypoxia and ischemia. CNS endothelial cells that form the BBB differ from endothelial cells in non-neural tissues in that CNS endothelial cells are highly polarized cells held together by tight junctions and express specific transport proteins. Wnt signaling regulates CNS angiogenesis and / or function. Conditions with impaired BBB can benefit from administration of the compounds of the present disclosure, which can be systemic or localized, such as by direct injection, intrathecal administration, implantation of a sustained-release formulation, etc. Additionally, Wnt signaling is actively involved in neurogenesis and plays a neuroprotective role after injury. The compositions of the present disclosure can also be used to treat spinal cord injury, other spinal cord diseases, stroke, traumatic brain injury, etc.

[0341] Wnt signaling also plays a role in angiogenesis. Wnt replacement molecules can be beneficial for conditions where angiogenesis is advantageous, treating myocardial infarction, coronary artery disease, heart failure, etc., as well as conditions resulting from genetic diseases. Methods for regenerating these tissues benefit from administration of the compounds of the present disclosure, which can be systemic or localized.

[0342] In certain embodiments, the methods of the present disclosure promote tissue regeneration, such as in tissues that have undergone damage or tissue or cell reduction or depletion. The depletion or damage can be anything that results in a decrease in cell number, including disease or injury. For example, an accident, an autoimmune disorder, a treatment side effect, or a disease state may constitute a trauma. Tissue regeneration increases the number of cells within the tissue and preferably enables the reestablishment of connections between the cells of the tissue, and more preferably enables the tissue to regain function.

[0343] As used herein, the terms "administer" or "introduce" or "provide" refer to delivering a composition to a cell, to the cells, tissues, and / or organs of a subject, or to a subject. Such administration or introduction can occur in vivo, in vitro, or ex vivo.

[0344] In certain embodiments, the pharmaceutical composition is administered parenterally, e.g., intravenously, orally, rectally, or by injection. In some embodiments, the pharmaceutical composition is administered locally, e.g., topically or intramuscularly. In some embodiments, the composition is administered to a target tissue, e.g., bone, joint, ear tissue, eye tissue, gastrointestinal tract, skin, wound site, or spinal cord. The methods of the present disclosure can be practiced in vivo or ex vivo. In some embodiments, the contacting of the target cells or target tissue with the Wnt replacement molecule is performed ex vivo, followed by implantation of the cells or tissue, e.g., activated stem cells or progenitor cells, into a subject. One of ordinary skill in the art can determine the appropriate site of administration and route of administration based on the disease or disorder being treated.

[0345] The dosage and dosage regimen can depend on various factors readily determined by the physician, such as the nature of the disease or disorder, the characteristics of the subject, and the medical history of the subject. In certain embodiments, the amount of the Wnt replacement molecule administered or provided to the subject ranges from about 0.01 mg / kg to about 50 mg / kg, 0.1 mg / kg to about 500 mg / kg, or about 0.1 mg / kg to about 50 mg / kg of the subject's body weight.

[0346] The terms “treatment,” “treating,” etc. are used herein generally to mean obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of completely or partially preventing a disease or its symptoms, e.g., reducing the likelihood of occurrence of a disease or its symptoms in a subject, and / or therapeutic in terms of partially or completely curing a disease and / or an adverse reaction caused by the disease. As used herein, “treatment” covers any treatment of a disease in a mammal and includes: (a) preventing the disease from occurring in a subject who may be predisposed to having the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., arresting the development of the disease; or (c) relieving the disease, i.e., causing regression of the disease. A therapeutic agent (e.g., a Wnt replacement molecule) can be administered before, during, or after the onset of a disease or injury. Particular attention is paid to the treatment of a developing disease, wherein the treatment stabilizes or reduces the undesired clinical symptoms of the patient. Desirably, such treatment is carried out before complete loss of function of the affected tissue. Desirably, the subject therapy will be administered during the symptomatic phase of the disease and in some cases after the symptomatic phase 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 includes the step of detecting that a therapeutic benefit has been achieved. One of ordinary skill in the art will appreciate that such measures of therapeutic efficacy will be applicable to the particular disease being modified and will recognize the appropriate assays for measuring therapeutic efficacy.

[0347] Other embodiments relate to the use of the Wnt surrogate molecules disclosed herein for promoting or enhancing the growth or proliferation of cells, tissues, and organoids, for example, by contacting the cells or tissues with one or more Wnt surrogates optionally in combination with Norrin or Rspondin polypeptides. In certain embodiments, the cells or tissues are contacted ex vivo, in vitro, or in vivo. Such methods can be used to generate cells, tissues, or organoids for therapeutic use, for example, for implantation or transplantation into a subject. They can also be used to generate cells, tissues, or organoids for research use. Wnt surrogate molecules have a wide range of applications in non-therapeutic methods, such as in vitro research methods.

[0348] The present disclosure provides a method for tissue regeneration of damaged tissues (such as the above-mentioned tissues), the method comprising administering a Wnt surrogate molecule to cells. The Wnt surrogate molecule can be administered directly in vivo to the cells, orally, intravenously, or by other methods known in the art to a subject or to isolated cells. In some embodiments where the Wnt surrogate molecule is administered to isolated cells, these cells can be transplanted into a subject before, after, or during the administration of the Wnt surrogate molecule.

[0349] Wnt signaling is a key component of stem cell culture. For example, in WO2010 / 090513, WO2012 / 014076, Sato et al., 2011 (GASTROENTEROLOGY 2011; 141:1762 - 1772), and Sato et al., 2009 (Nature 459, 262 - 5). The Wnt surrogate molecules disclosed herein are suitable alternatives to Rspondin for these stem cell culture media or can be combined with Rspondin.

[0350] Thus, in one embodiment, the present disclosure provides a method for enhancing stem cell proliferation, the method comprising contacting the stem cells with one or more Wnt surrogate molecules disclosed herein. In one embodiment, the present disclosure provides a cell culture medium comprising one or more Wnt surrogate molecules disclosed herein. In some embodiments, the cell culture medium can be any cell culture medium known in the art that typically comprises Wnt or Rspondin, but wherein the Wnt or Rspondin is replaced or supplemented (in whole or in part) with one or more Wnt surrogate molecules disclosed herein. For example, the culture medium can be as described in WO2010 / 090513, WO2012 / 014076, Sato et al., 2011 (GASTROENTEROLOGY 2011; 141:1762 - 1772), and Sato et al., 2009 (Nature 459, 262 - 5), which are hereby incorporated by reference in their entirety.

[0351] Stem cell culture media typically include additional growth factors. Thus, this method can additionally include supplying growth factors to the stem cells. Growth factors commonly used in cell culture media include epidermal growth factor (EGF, (Peprotech)), transforming growth factor-α (TGF-α, 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 various cultured ectodermal and mesodermal cells and has profound effects on the differentiation of specific cells in vivo and in vitro as well as certain fibroblasts in cell culture. The EGF precursor exists as a membrane-bound molecule that is proteolytically cleaved to produce the 53-amino acid peptide hormone that stimulates cells. Thus, EGF or other mitogenic growth factors can be supplied to the stem cells. During stem cell culture, mitogenic growth factors can be added to the culture media every two days while the culture media is preferably renewed every four days. Generally, the mitogenic factors are selected from the group consisting of: i) EGF, TGF-α, and KGF; ii) EGF, TGF-α, and FGF7; iii) EGF, TGF-α, and FGF; iv) EGF and KGF; v) EGF and FGF7; vi) EGF and FGF; vii) TGF-α and KGF; viii) TGF-α and FGF7; ix) or from TGF-α and FGF. In certain embodiments, the present disclosure encompasses a stem cell culture media that includes, for example, a Wnt surrogate molecule disclosed herein optionally in combination with one or more of the growth factors or combinations thereof described herein.

[0352] These methods of enhancing stem cell proliferation can be used to grow new organoids and tissues from stem cells, such as, for example, in WO2010 / 090513, WO2012 / 014076, Sato et al., 2011 (Gastroenterology 2011;141:1762-1772), and Sato et al., 2009 (Nature 459, 262-5).

[0353] In some embodiments, Wnt surrogate molecules are used to enhance stem cell regeneration. Exemplary stem cells of interest include, but are not limited to: muscle satellite cells; hematopoietic stem cells and progenitor cells derived therefrom (U.S. Patent No. 5,061,620); neural stem cells (see Morrison et al. (1999) Cell 96:737-749); embryonic stem cells; mesenchymal stem cells; mesodermal stem cells; hepatic stem cells; adipose tissue-derived stem cells, etc.

[0354] Other embodiments of the present disclosure relate in part to diagnostic applications for detecting the presence of cells or tissues expressing one or more Frizzled (Fzd) receptors or LRP5 or LRP6 receptors. Accordingly, the present disclosure provides methods for detecting one or more Fzd receptors or LRP5 or LRP6 receptors in a sample (such as detecting cells or tissues expressing Fzd1). Such methods can be applied in a variety of known detection formats, including but not limited to immunohistochemistry (IHC), immunocytochemistry (ICC), in situ hybridization (ISH), whole mount in situ hybridization (WISH), fluorescence in situ hybridization (FISH), flow cytometry, enzyme immunoassay (EIA), and enzyme-linked immunosorbent assay (ELISA), for example, by detecting the binding of a Wnt surrogate molecule.

[0355] ISH is a type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., the primary binder) to localize a specific DNA or RNA sequence in a part or section of a cell or tissue (in situ), or, if the tissue is small enough, in the whole tissue (whole mount ISH). One of ordinary skill in the art will recognize that this is different from immunohistochemistry, which uses an antibody as the primary binder to localize a protein in a tissue section. DNA ISH can be directed against genomic DNA to determine chromosomal structure. Fluorescence in situ hybridization (FISH) can be used, for example, in medical diagnostics to assess chromosomal integrity. RNA ISH (hybridization histochemistry) is used to measure and localize mRNA and other transcripts in tissue sections or whole mounts.

[0356] In various embodiments, the Wnt surrogate molecules described herein are conjugated to a detectable label that can be detected directly or indirectly. In this regard, an antibody “conjugate” refers to a Wnt surrogate molecule covalently linked to a detectable label. In the present disclosure, DNA probes, RNA probes, monoclonal antibodies, antigen-binding fragments thereof, and antibody derivatives thereof, such as single-chain variable fragment antibodies or epitope-tagged antibodies, can all be covalently linked to a detectable label. In “direct detection”, only one detectable antibody is used, i.e., the first detectable antibody. Thus, direct detection means that an antibody conjugated to a detectable label per se can be detected without the addition of a second antibody (secondary antibody).

[0357] A “detectable label” is a molecule or material that can produce a detectable (such as visually, electronically, or otherwise) signal indicating the presence and / or concentration of the label in a sample. When conjugated to an antibody, a detectable label can be used to localize and / or quantify the target against which the specific antibody is directed. Thus, 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 antibodies of different specificities can be used in combination to detect one or more targets.

[0358] Examples of directly detectable detectable labels include fluorescent dyes, radioactive materials, and metallic particles. In contrast, indirect detection requires the application of one or more additional antibodies, i.e., secondary antibodies, after the application of the first antibody. Thus, the detection is carried out by detecting the binding of the secondary antibody or binding agent to the first detectable antibody. Examples of first detectable binding agents or antibodies that require the addition of a secondary binding agent or antibody include enzyme-detectable binding agents and hapten-detectable binding agents or antibodies.

[0359] In some embodiments, the detectable label is conjugated to a nucleic acid polymer comprising a first binding agent (e.g., in an ISH, WISH, or FISH procedure). In other embodiments, the detectable label is conjugated to an antibody comprising a first binding agent (e.g., in an IHC procedure).

[0360] Examples of detectable labels that can be conjugated to the 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, metallic particles, haptens, and dyes.

[0361] Examples of fluorescent labels include 5-(and 6)-carboxyfluorescein, 5- or 6-carboxyfluorescein, 6-(fluorescein)-5-(and 6)-carboxamidocaproic acid, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, and dyes (such as Cy2, Cy3, and Cy5), optionally substituted coumarins (including AMCA, PerCP), phycobiliproteins (including R-phycoerythrin (RPE) and allophycocyanin (APC)), Texas Red, Princeton Red, green fluorescent protein (GFP) and its analogs, and conjugates of R-phycoerythrin or allophycocyanin, inorganic fluorescent labels such as particles based on semiconductor materials, such as coated CdSe nanocrystals.

[0362] Examples of polymer particle labels include microparticles or latex particles of polystyrene, PMMA, or silica that can be embedded with fluorescent dyes or polymer micelles or capsules containing dyes, enzymes, or substrates.

[0363] Examples of metal particle labels include gold particles that can be converted by silver staining and coated gold particles. Examples of haptens include DNP, fluorescein isothiocyanate (FITC), biotin, and digoxin. Examples of enzyme labels include horseradish peroxidase (HRP), alkaline phosphatase (ALP or AP), β-galactosidase (GAL), glucose-6-phosphate dehydrogenase, β-N-acetylglucosaminidase, β-glucuronidase, invertase, xanthine oxidase, firefly luciferase, and glucose oxidase (GO). Examples of common substrates for horseradish peroxidase include 3,3'-diaminobenzidine (DAB), diaminobenzidine with nickel enhancement, 3-amino-9-ethylcarbazole (AEC), benzidine dihydrochloride (BDHC), Hanker-Yates reagent (HYR), indigo blue (IB), tetramethylbenzidine (TMB), 4-chloro-1-naphthol (CN), α-naphthol pyronin (α-NP), o-dianisidine (OD), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), nitroblue tetrazolium (NBT), 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT), tetranitroblue tetrazolium (TNBT), 5-bromo-4-chloro-3-indolyl-β-D-galactoside / ferric-ferrocyanide (BCIG / FF).

[0364] Examples of common substrates for alkaline phosphatase include naphthol-AS-B1-phosphate / fast red TR (NABP / FR), naphthol-AS-MX-phosphate / fast red TR (NAMP / FR), naphthol-AS-B1-phosphate / -fast red TR (NABP / FR), naphthol-AS-MX-phosphate / fast TR (NAMP / FR), naphthol-AS-B1-phosphate / new fuchsin (NABP / NF), bromochloroindolyl phosphate / nitroblue tetrazolium (BCIP / NBT), 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (BCIG).

[0365] 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 iodine, cobalt, selenium, tritium, carbon, sulfur, and phosphorus.

[0366] The detectable label can be linked to the antibodies described herein or any other molecule that specifically binds to a biomarker of interest, such as an antibody, nucleic acid probe, or polymer. In addition, those of ordinary skill in the art will understand that the detectable label can also be conjugated to a second and / or third and / or fourth and / or fifth binder or antibody, etc. In addition, those skilled in the art will understand that each other binder or antibody used to characterize the target biological marker can be used as a signal amplification step. The biological marker can be visually detected using, for example, an optical microscope, a fluorescence microscope, or an electron microscope, where the detectable substance is, for example, a dye, colloidal gold particles, or a luminescent reagent. The visually detectable substance bound to the biomarker can also be detected using a spectrophotometer. When the detectable substance is a radioisotope, it can be visually detected by autoradiography or using a scintillation counter. See, for example, 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.).

[0367] The present disclosure further provides a kit for detecting one or more Fzd or LRP5 / 6 receptors or cells or tissues expressing one or more Fzd or LRP5 / 6 receptors in a sample, wherein the kit contains at least one antibody, polypeptide, polynucleotide, vector, or host cell described herein. In certain embodiments, the kit can include a buffer, an enzyme, a label, a substrate, beads, or other surfaces, etc. associated with the antibodies of the present disclosure, as well as instructions for use.

[0368] All of the above-mentioned U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification and / or listed in the application data sheet are hereby incorporated by reference in their entirety.

[0369] In view of the foregoing, it should be understood that although specific embodiments of the present disclosure have been described for purposes of illustration, various modifications can be made without departing from the spirit or scope of the present disclosure. Accordingly, the present disclosure is not limited except as restricted by the appended claims.

[0370] Examples

[0371] Example 1

[0372] Multispecific Wnt replacement molecules

[0373] As further described in the following examples, a number of multispecific Wnt surrogate molecules representing different configurations were generated. These include the Wnt surrogate molecules disclosed in Table 5 below, which include the sequences shown in SEQ ID NOs: 109 - 157. Tables 1A and 1B and 2A and 2B list the specific Fzd and LRP binding elements presented in these examples for the Wnt surrogate molecules.

[0374] Table 5. Wnt surrogate molecule sequences

[0375] Lrp VHH or sdAb = italic

[0376] Anti - Fzd light chain = underlined

[0377] Anti - Fzd heavy chain = bold

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397] Example 2

[0398] Generation of Wnt surrogate molecules with binding specificity for the Fzd receptor hinge region

[0399] Active Wnt surrogate molecules were generated, the active Wnt surrogate molecules comprising various combinations of an Fzd binder that binds to the Fzd receptor hinge region (see Figure 2A ). Two antibodies that bind to the hinge region of Fzd7, anti-FZD7-1791 (SEQ ID NO:70-71) and anti-FZD7-1291 (SEQ ID NO:72-73), are described in WO2016 / 205551 and WO2016 / 205566. These two antibodies were used to demonstrate that binders to the Fzd hinge region produce active Wnt surrogate molecules.

[0400] Both anti-FZD7-1791 and anti-FZD7-1291 were cloned into the human IgG1 framework with a LALA-PG mutation in the Fc to reduce effector function. The LRP5 binder #3 (008S-D01; SEQ ID NO:97) was cloned into the N-terminus of the light chains of the two antibodies in the framework, as Figure 2BDepicted. Two recombinant attached IgG proteins, named 1791-3 (SEQ ID NO:113-114) and 1291-3 (SEQ ID NO:111-112) respectively, were prepared by transfecting the corresponding expression vectors into Expi293F cells (Thermo Fisher Scientific, Waltham, MA). Briefly, four days after transfection, the cell culture medium was collected after slowing the rotation of the cell pellet. The medium was incubated with protein A resin (Repligen, Waltham, MA) to collect the protein containing the human IgG-Fc portion. The protein was eluted from the protein A resin with 10 mM glycine at pH 3.5. Subsequently, the protein eluate was fractionated and further purified by size exclusion chromatography (SEC). SEC was performed using Superdex 200 Increase 10 / 300 GL (GE Healthcare, Pittsburgh, PA) in HBS buffer (10 mM HEPES, 150 mM NaCl, pH 7.4) by fast protein liquid chromatography. The peak fractions were further analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm the content.

[0401] Antibodies that bind to the hinge regions of Fzd1 and Fzd2 were used to demonstrate that, in addition to the Fzd7 hinge, other Fzd hinge regions also produce active Wnt surrogate molecules. Recombinant Fab fragments of these antibodies were generated from Expi293F cells (Thermo Fisher Scientific, Waltham, MA) by transient transfection. The Fab was purified from the culture medium using nickel resin and further refined by size exclusion chromatography (SEC).

[0402] Both the anti-FZD1 hinge and anti-FZD2 hinge antibodies were cloned into the human IgG1 framework with a LALA-PG mutation in the Fc to reduce effector function. LRP5 binder #3 (008S-D01; SEQ ID NO:97) or LRP5 / 6 binder #36 (013S-D05; SEQ ID NO:91 and 96) was cloned into the N-terminus of the light chain of the corresponding antibody in the framework, as Figure 2BDepicted. According to the manufacturer's instructions, recombinant attached IgG proteins were prepared by transfecting the corresponding expression vectors into Expi293F cells (Thermo Fisher Scientific, Waltham, MA). Briefly, four days after transfection, the cell culture medium was collected after slowing down the rotation of the cell pellet. The medium was incubated with protein A resin (Repligen, Waltham, MA) to collect the protein containing the human IgG-Fc portion. The protein was eluted from the protein A resin with 10 mM glycine at pH 3.5. Subsequently, the protein eluate was fractionated and further purified by size exclusion chromatography (SEC). SEC was performed using Superdex 200 Increase 10 / 300 GL (GE Healthcare, Pittsburgh, PA) in HBS buffer (10 mM HEPES, 150 mM NaCl, pH 7.4) by fast protein liquid chromatography. The peak fractions were further analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm the content. The results indicated that these antibodies also produced active Wnt surrogate molecules.

[0403] Example 3

[0404] Binding kinetics of hinge region-specific Wnt surrogate molecules

[0405] The binding kinetics of 1791-3 and 1291-3 to Fzd7 CRD or Fzd7 CRD with an extracellular hinge region sequence (Fzd7 CRD + hinge) were determined by biolayer interferometry (BLI) using an Octet Red 96 instrument (PALL ForteBio, Fremont, CA) at 30 °C and 1000 rpm with a streptavidin (SA) biosensor. N-terminally biotinylated Fzd7 CRD and Fzd7 CRD + hinge proteins were captured on the SA biosensor. After capturing biotinylated Fzd7, the SA biosensor with captured biotinylated Fzd7 was dipped into wells containing the relevant antibodies at 7 different concentrations in running buffer and a well with only running buffer as a reference channel. KD was determined by global fitting. As Figure 2C shown, these two antibody fusion proteins only bind to the Fzd7 protein with a hinge region and not to the Fzd7 CRD domain alone.

[0406] Example 4

[0407] In vitro activity of hinge region-specific Wnt surrogate molecules

[0408] The ability of 1791-3 and 1291-3 to activate Wnt signaling was evaluated in the 293STF cell line, which stably integrated the β-catenin luciferase reporter plasmid Super TOP Flash (STF). As Figure 2D shown, both 1791-3 and 1291-3 activated Wnt signaling, as judged by induction of the luciferase reporter in these cells in the presence of 20 nM R-spondin 2 (RPSO). Although 1291-3 had a higher affinity for the Fzd7 CRD+hinge, 1791-3 also showed higher activity than 1291-3, suggesting that potential differences in the geometry between the two antibodies could cause differences in activity. Another Wnt surrogate molecule, R2M3-3 (Fzd binder 001S-A04; Lrp binder 008S-D01; SEQ ID NO:109-110), which can engage Fzd1, Fzd2, Fzd7, Fzd5, and Fzd8, was also tested. As Figure 2D shown, the maximal effect from 1791-3 was close to that of the multifamily-specific R2M3-3.

[0409] The ability of Wnt surrogates, including antibodies that bind to the hinge region of Fzd1 or Fzd2, to activate Wnt signaling was evaluated in 293STF cell lines overexpressing Fzd1 or Fzd2, which stably integrated the β-catenin luciferase reporter plasmid Super TOP Flash (STF). For the luciferase reporter assay, 1 million cells were seeded in each 96-well plate, and IWP2 (a Wnt signaling inhibitor) was added at a final concentration of 3 μM. At 26 hours after seeding, the compounds were added to the 96-well plates in triplicate, with 10-fold serial dilutions starting from 100 nM and a maximum concentration of 500 nM. After 18 hours, the cells were lysed with 100 μl of lysis buffer. From the above lysed cells, 20 μl of the sample was transferred to an opaque 96-well plate. 10 μl of luciferase substrate was added to each well. The plate was immediately placed in a molecular device, the Lum96 plate reader, and the luciferase luminescence signal was collected. The data were processed with Prism7. These antibodies activated Wnt signaling, as judged by induction of the luciferase reporter in these cells with Fzd1 or Fzd 2 overexpression in the presence of 20 nM R-spondin 2 (RPSO). These results indicate that Fzd hinge-binding antibodies can induce Wnt signaling activation when assembled with an Lrp binder.

[0410] Example 5

[0411] Generation of Multispecific Wnt Surrogate Molecules

[0412] The sequences from monospecific Wnt surrogate molecules are combined to generate a multispecific Wnt surrogate molecule having the desired combination of Fzd binding specificities. As Figure 3A depicted, two monospecific Wnt surrogate molecules: 4SD1-3 that binds Fzd4 (Fzd binder 004S-D01; Lrp binder 008S-D01; SEQ ID NO: 115-116) and 14SB6-3 that binds Fzd9 (Fzd binder 014S-B06; Lrp binder 008S-D01; SEQ ID NO: 117-118), are combined into a heterologous Ig Wnt surrogate molecule heterologous Ig 4SD1-3+14SB6-3 (SEQ ID NO: 120 and 122) using a mortar and pestle technique. One half of the heterologous Ig molecule is from 4SD1-3, in which a "mortar" mutation is introduced in the CH3 domain (SEQ ID NO: 120). This half of the molecule contains one binding arm to Fzd4 and one binding arm to LRP5. The other half of the heterologous Ig molecule is from 14SB6-3, in which a "pestle" mutation is introduced in the CH3 domain (SEQ ID NO: 122). This half of the molecule contains one binding arm to Fzd9 and one binding arm to LRP5. The final Wnt surrogate (heterologous Ig 4SD1-3+14SB6-3) is a tetravalent trispecific molecule that contains one binding arm to Fzd4, one binding arm to Fzd9, and two binding arms to LRP5.

[0413] Two control molecules are also generated: 4SD1-3 monovalent bispecific (SEQ ID NO: 120 and 123) and 14SB6-3 monovalent bispecific( Figure 3A )(SEQ ID NO: 122 and 124). The 4SD1-3 monovalent bispecific molecule contains the 4SD1-3 half of the heterologous Ig with the "mortar" mutation (SEQ ID NO: 120), which is paired with an Fc having a "pestle" mutation (SEQ ID NO: 123), but does not have Fab arms; thus, this molecule is bispecific but monovalent for Fzd4 and LRP5. The 14SB6-3 monovalent bispecific contains the 14SB6-3 half of the heterologous Ig with the "pestle" mutation (SEQ ID NO: 122), which is paired with an Fc having a "mortar" mutation (SEQ ID NO: 124), but does not have Fab arms; thus, this molecule is bispecific but monovalent for Fzd9 and LRP5.

[0414] The hetero-Ig 4SD1-3+14SB6-3, 4SD1-3 monovalent bispecific, and 14SB6-3 monovalent bispecific were purified by a 4-step purification process. Since the Fc containing the "knob" mutation also contained a FLAG tag, and the Fc containing the "hole" mutation also contained a His tag, these proteins were first purified by protein A and Ni-NTA affinity purification steps, followed by a SEC step; anti-FLAG M2 beads were used as the final purification step.

[0415] Example 6

[0416] Characterization of multispecific Wnt surrogate molecules

[0417] The ability of hetero-Ig 4SD1-3+14SB6-3, 4SD1-3 monovalent bispecific, and 14SB6-3 monovalent bispecific to activate Wnt signaling was assessed in Wnt-responsive 293STF reporter cells in the presence or absence of 20 nM RPSO ( Figure 3B - 3E Only data in the presence of RPSO are shown). R2M3-3, which can bind Fzd1, Fzd2, Fzd7, Fzd5, and Fzd8, was also tested.

[0418] The 293STF cell line expressed low levels of Fzd4, and no Fzd9 expression was detected by QPCR. All molecules except R2M3-3 showed very little or no activity in parental 293STF cells ( Figure 3B ).

[0419] A retroviral-based Fzd4 overexpressing 293STF stable cell line (293STF Fzd4OE) was generated. In this cell line, both R2M3-3 and 4SD1-3 (Fzd4 monospecific) potently activated Wnt signaling, while the hetero-Ig 4SD1-3+14SB6-3 showed very weak activity, and the Fzd9 monospecific molecule 14SB6-3 was inactive ( Figure 3C ).

[0420] Fzd9 was introduced into parental 293STF cells by transient transfection to generate the 293STF Fzd9OE cell line. In this cell line, both R2M3-3 and 14SB6-3 (Fzd9 monospecific) were able to activate Wnt signaling, while the hetero-Ig 4SD1-3+14SB6-3 showed very weak activity, and the Fzd4 monospecific molecule 4SD1-3 was inactive ( Figure 3D ).

[0421] The combination of retroviral Fzd4 delivery and transient transfection of Fzd9 was used to co-introduce Fzd4 and Fzd9 into 293STF cells to generate the 293STF Fzd4OE+Fzd9OE cell line. In this cell line, R2M3-3, 4SD1-3, 14SB6-3, and the hybrid Ig 4SD1-3+14SB6-3 are all fully active( Figure 3E )

[0422] Two monovalent bispecific molecules, the 4SD1-3 monovalent bispecific and the 14SB6-3 monovalent bispecific, are inactive in all cells( Figure 3B - 3E ). These results indicate that for the two monospecific binders, the monovalent bispecific in combination with the LRP5 binder cannot induce or cannot effectively induce Wnt signaling. Thus, the observed activity of the hybrid Ig 4SD1-3+14SB6-3 is indeed the result of Fzd4 and Fzd9 proteins and LRP engagement. These results further suggest that this method can be applied to any combination of two or more Fzd binders of different specificities, enabling the generation of any desired Fzd specificity combination by this process.

[0423] This example shows that two Fzd binders of different specificities can be combined in a hybrid Ig form to generate Wnt surrogate molecules of different specificities. This concept can also be extended to using two Fzd binders for the same Fzd at two different epitopes. The resulting hybrid Ig Wnt surrogate molecules will be bispecific molecules, which can be advantageous for further receptor aggregation and activation of Wnt signaling.

[0424] Example 7

[0425] Heterodimerization of canonical and non-canonical signaling Fzd receptors and LRP receptors binding leads to activation of β-catenin-dependent signaling

[0426] Wnt signaling can be mediated through β-catenin-dependent (canonical) and β-catenin-independent (non-canonical) pathways. It has been reported that there are Fzd receptors that activate β-catenin-independent signaling, such as Fzd6, while other Fzd receptors activate β-catenin-dependent signaling. It has not been previously considered whether forced dimerization of non-canonical Fzd receptors with Lrp or heterodimerization of canonical and non-canonical Fzd receptors with Lrp can lead to β-catenin-dependent or β-catenin-independent signaling. To test these ideas, the following examples were generated.

[0427] First, a multivalent molecule consisting of a bivalent binding arm (004S-C10) against the non-canonical receptor Fzd6 was combined with a bivalent binding arm (3; 004S-D01) against Lrp5. The molecule was named 4SC10-3( Figure 5A upper panel region). When the Fzd binder targets a canonical Fzd receptor, this format has been shown to activate β-catenin-dependent signaling. However, as Figure 5A shown in the lower panel region of

[0428] Figure 5B , 4SC10-3 was unable to efficiently activate β-catenin-dependent signaling in 293STF reporter cells (in the presence of 20 nM R-spondin) compared to the positive control molecule R2M3-26 (Fzd binder 001S-A04; Lrp binder 009S-E04) that engages Fzd1, 2, 7, 5, 8. Figure 5B upper panel region), and it activated β-catenin-dependent signaling in 293STF reporter cells. To rule out the possibility that the 1RC07-3 arm alone could activate signaling, the Figure 5C monovalent bispecific molecule 1RC07-3 shown in the upper panel region was also tested. As Figure 5C shown, the monovalent bispecific molecule 1RC07-3 was unable to efficiently activate signaling. These results demonstrate for the first time that combining canonical and non-canonical Fzd receptors with Lrp can elicit β-catenin-dependent signaling.

[0429] Example 8

[0430] Explore the effect of different ratios of Fzd to Lrp binders in Wnt surrogate molecules on β-catenin-dependent signaling

[0431] The multivalent format gave rise to potent and highly effective activators of Wnt signaling. Experiments were designed to test the effect of the format on endogenous Wnt signaling. To understand the effect of bivalent / multivalent Lrp binding arms on endogenous Wnt ligands, inactive surrogate molecules were generated with the null mutants of R2M3, R2M3mut(Y9A), and fused with Lrp binders 3 or 26, and named R2M3mut-3 or R2M3mut-26, respectively. As Figure 6AAs shown, although the wild-type R2M3 substitutes R2M3-3 and R2M3-26 are fully active in the 293STF Wnt reporter cell line in the presence of 20 nM R-spondin, R2M3mut-3 and R2M3mut-26 are inactive. To test the effect of the bivalent Lrp arms in the substitute molecules on endogenous Wnt signaling, these two inactive substitute molecules were tested in the 293STF Wnt reporter cell line in the presence of 20% conditioned medium containing Wnt3A. As Figure 6B shown, although R2M3mut-3 and R2M3mut-26 are inactive by themselves, R2M3mut-3 synergizes with Wnt3a in a dose-responsive manner, and R2M3mut-26 antagonizes Wnt3a in a dose-responsive manner. This suggests that the multivalent form may have the potential to interact with endogenous Wnt ligands.

[0432] To understand the form that reduces potential interaction with endogenous Wnt and to further understand the number of receptor binders required for active signaling, different numbers and ratios of Fzd and Lrp binders in the multivalent substitute molecules were explored.

[0433] Since the bivalency of the Lrp binding arms may lead to synergy with endogenous Wnt, removal of one Lrp binding arm was tested, so that there are two Fzd binding arms and one Lrp binder in the final molecule; this molecule is called 2:1. The original molecular form was 2:2 because there were two Fzd binders and two Lrp binding arms. Compared with the 2:2 form, an example of the 2:1 molecule, 1RC07-3(2:1), is shown in Figure 6C the left panel. As Figure 6C shown in the right panel, 1RC07-3(2:1) is active and able to induce Wnt signaling in 293STF reporter cells. In principle, this form should lack the ability to synergize with endogenous Wnt ligands through cross-linking of the Lrp coreceptor. Reverse ratios were also constructed, one Fzd binder and two Lrp binders, as exemplified by several forms in Figure 6D which. Generally, these molecules showed at most modest signal transduction induction ( Figure 6D ).

[0434] Since the heterodimeric Fzd of the 2:2 form can induce Wnt signaling (as shown in Figures 3 and 5), molecules that heterodimerize two different Fzd binders with one Lrp binder (1:1:1) form were constructed, and the ability of the molecules to confer Wnt signaling was tested. As Figure 6EAs shown, several examples of 1:1:1 molecules activated Wnt signaling in 293STF reporter cells. The Fzd binders tested in this construct included R2M3 (001S-A04), 1RC07 (001S-B03), R2M13 (004S-G06), and 5S-H5 (005S-H05).

[0435] Example 9

[0436] Heterodimerization of Lrp binders and Fzd binding activates Wnt signaling

[0437] In addition to Fzd receptor binders with different heterodimerization / polymerization, the effects of Lrp coreceptor binders with different heterodimerization / polymerization on Wnt signaling were also tested. As shown above, the two Fzd: one Lrp form reduced the synergy with endogenous Wnt ligands. To determine whether heterodimerized / polymerized Lrp binders targeting two different regions on Lrp could also reduce the synergistic effect of the bivalent Lrp binder form on endogenous Wnt ligands, tests were conducted. As Figure 7A shown, several examples were constructed in a 2:1:1 ratio, where two different Lrp binders (one binding to Lrp5E1E2 and the other binding to Lrp6E3E4) were each fused to the N-terminus of the light chain of the Fzd binder 1RC07 as 1RC07-3-26NL (2:1:1); or to the N-terminus of the heavy chain of 1RC07 as 1RC07-3-26NH (2:1:1). Both of these molecules were highly active in inducing Wnt signaling, as detected in 293STF Wnt-responsive reporter cells ( Figure 7B ).

[0438] Finally, multivalent molecules with two different Fzd binding arms and two different Lrp binding arms were generated, which were designated as the 1:1:1:1 form, as Figure 7C shown. Both the 1RC07-26+4SD1-3 (1:1:1:1) and R2M9-26+1RC07-3 (1:1:1:1) combinations caused strong induction of Wnt signaling ( Figure 7D ). The R2M9 construct contains the 003S-E07 Fzd binding element.

[0439] The examples shown in Figures 5-7 illustrate various constructs for inducing canonical or non-canonical Wnt signaling (or both), as well as the ability to synergize with, antagonize, or leave unaffected the signaling by endogenous Wnt ligands within the target tissue. This general collection of soluble Wnt surrogate ligands can be used in therapies with customized Wnt signaling, which can allow for maximum therapeutic effects and minimum side effects.

[0440] Example 10

[0441] Structural and functional analysis of Wnt surrogate molecules containing Fzd and LRP binders in different stoichiometric ratios

[0442] A surrogate WNT agonist generated by linking an FZD binder (the 18R5 antibody in scFv form) and the C-terminal portion of Dickkopf (DKK1c) into a single polypeptide chain (18R5-DKK1c) exhibits the ability to activate WNT / β-catenin signaling (Janda et al., 2017, Nature, 545(7653):234–237). Based on this concept, various combinations of FZD- and LRP-binding antibody fragments have been generated.

[0443] The sequences of the Fzd-binding scFvs (designated F1, F2, and F3) and the LRP scFvs (designated L1 and L2) and the linker sequences used to combine them are shown in Table 6A. Since these molecules have monovalent binding arms to each target, this form is referred to as the bivalent bispecific form (designated 1:1 to denote the stoichiometry of binding one FZD and one LRP molecule).

[0444] Additional stoichiometries of the tandem scFv constructs were tested. Table 6B shows a summary of the combined Wnt surrogate constructs used for this analysis.

[0445] Table 6A: Wnt surrogate components for stoichiometry analysis

[0446]

[0447]

[0448]

[0449]

[0450] Table 6B: Combined Wnt surrogate constructs

[0451]

[0452]

[0453]

[0454] The resulting molecular collection replaced the DKK1c component in the 18R5-DKK1c molecule with an LRP-binding antibody fragment in scFv form (Janda et al., 2017, Nature, 545(7653):234–237) to generate tandem scFv molecules, asFigure 8A depicted. The LRP conjugate is an LRP6 E1E2 domain conjugate, 1115.3 (US 8,715,941B2), which is referred to herein as L1. To test the effect of geometry on activity, L1 was fused to the N-terminus or C-terminus of 18R5 scFv (referred to herein as F1) with a 5, 10, or 15 amino acid linker in between. This set of six proteins was purified to near homogeneity by Ni affinity column ( Figure 8B ) and tested in WNT-responsive 293STF reporter cells. Although linker length did not appear to significantly affect activity, fusing the LRP6 conjugate to the N-terminus of the FZD conjugate caused much higher activity than other orientations (compare Figure 8C and Figure 8D , note the difference in the y-axis). Compared to WNT3A and 18R5-DKK1c tested in parallel, all of these new alternative molecules showed lower Emax (maximum response) ( Figure 9 ).

[0455] Since scFv fragments have a tendency to form higher molecular weight aggregates, to confirm whether the alternative WNT activity was from the monomeric form of the tandem scFv molecules, these six proteins were purified on size exclusion chromatography (SEC) and the peak fractions corresponding to the monomeric form were tested in 293STF reporter cells. Compared to the Ni-purified material before SEC, the SEC-purified proteins significantly lost activity (compare Figure 8E and 8F with 8C and 8D). The fractions across the SEC run were assayed in 293STF reporter cells. The peak activity of the column fractions was identified; however, the STF activity peak did not coincide with the protein peak of the monomeric molecules. Instead, in all cases, the STF activity peaked in the higher molecular weight fractions ( Figure 8G and 8H , Figure 8I shown SEC molecular weight standards), indicating that 1:1 tandem scFv molecules were ineffective in inducing β-catenin-dependent signaling. These results suggest that effective activation of WNT signaling requires multimerization of the receptor complex. Tandem scFv molecules containing a 6xHis tag were artificially crosslinked with anti-His antibodies and tested in the 293STF reporter assay. As Figure 8J shown, although in the presence of an isotype control (anti-GFP) antibody, L1:5:F1 from the monomer fraction alone could not effectively activate β-catenin-dependent signaling, the addition of anti-His antibody significantly induced signaling, thus supporting the view that signaling may require multimerization of the receptor complex.

[0456] As Figure 10ADepicted is the generation of multivalent molecules in a more defined and facile form by attaching the L1 and F1 tandem scFv fusion to the N-terminus of the Fc fragment to generate a tetravalent bispecific form. In this form, there are two binding sites for each receptor target (represented as 2:2 in the following sections, indicating the stoichiometric ratio of two FZD and two LRP). These proteins are purified by a protein A affinity step followed by SEC. As Figure 10B and 10C shown, the activity peak from the 293STF assay of the SEC fraction corresponds to the protein peak of the monomeric species of the Figure 10A shown molecule. The SEC molecular weight standards are as Figure 10D shown. These new configurations demonstrate the correlation between STF and the major protein peak. Dose-response curves of the peak fractions were also performed. Although the linker length did not appear to significantly affect the activity, the relative orientation of the two binders did affect the activity. Fusing L1 to the N-terminus of F1 is the preferred orientation for the combination of these two binders (compare Figure 10E and 10F y-axis). Compared to WNT3A and 18R5-DKK1c, the relative Emax of the activity of these multivalent bispecific FZD / LRP-binding molecules is similar to that of the pre-SEC material shown in Figure 8 (Figure 11). In this tetravalent bispecific form, the affinity of the binding arms for their respective receptors was determined using biolayer interferometry on an Octet instrument. As Figure 11B shown, the relative orientation and linker length had no effect on the affinity of the corresponding arms for their target receptors, indicating that the effect of orientation on reporter gene activity is not due to the effect of the form on binding, but rather is caused by the geometry of the receptors assembled by the alternative molecules.

[0457] DKK1c mainly binds to the LRP E3E4 domain (Ahn et al., 2011, Cheng et al., 2011). To understand the effect of the LRP epitope on activity, the fusion of F1 with the LRP6E3E4 binder YW211.31.57 (US 8,846,041 B2) (referred to herein as L2) in a 1:1 tandem scFv form was tested, where L2 was fused to the N-terminus or C-terminus of the F1 scFv with a 5-, 10-, or 15-amino acid linker in between. Although these fusion proteins performed poorly and were expressed at lower levels compared to the L1 / F1 fusion protein ( Figure 13A ), similar to the L1 / F1 fusion protein shown in Figure 8, the 1:1 tandem scFv of L2 / F1 was also ineffective in inducing β-catenin-dependent signaling in 293STF reporter gene cells, as shown in the activity test across the SEC column ( Figure 13A ) and the titration of the monomer peak fraction from the SEC column ( Figure 13B)as seen in. Fusion of the tandem scFv L2 / F1 molecule with the Fc fragment to generate a 2:2 tetravalent bispecific molecule potently activates canonical WNT signaling. As Figure 12A shown, the peak from the 293STF assay of the SEC fraction corresponded to the protein peak of the monomeric species. These L2 / F1 fusion molecules had higher activity than the L1 / F1 fusion molecules with an Emax range similar to WNT3A, and significantly improved the potency over WNT3A and 18R5-DKK1c( Figure 12B ). The L2 and F1 combination was less sensitive to orientation; both orientations produced similar Emax, while having L2 at the N-terminus of F1 in the case of the 15-mer linker produced higher potency( Figure 12B and 12C ).

[0458] As a control to verify that activation of WNT signaling by the 2:2 tetravalent bispecific molecule requires binding to both FZD and LRP receptors, the FZD binder (F1) or the LRP binder (L1 or L2) was replaced with an scFv fragment that binds GFP in a 2:2 tetravalent bispecific form. These molecules are referred to as tetravalent monospecific or 2:0 or 0:2 forms. As Figure 12D and 12E shown, although all these molecules bound to their respective receptors, none of these tetravalent monospecific molecules were active, indicating that the ability to activate WNT signaling requires engagement of both FZD and LRP.

[0459] To generate WNT surrogate molecules with different FZD specificities and to understand whether the multivalency requirement for WNT signaling is a general requirement beyond the FZD binder F1 (which binds FZD1, 2, 7, 5, 8, Gurney et al., 2012), in the case of the two LRP6 binders L1 and L2, fusion proteins were generated between an additional FZD binder R2H1 (US 2016 / 0194394 A1) (which binds to FZD1, 2, 7, referred to herein as F2) and 2919 (WO 2017 / 127933 A1) (which binds to FZD5, 8, referred to herein as F3). The specificities of F2 and F3 were verified in an Octet binding assay( Figure 15A )

[0460] The F2 / L1 combination in a 1:1 tandem scFv form was constructed with 5-mer and 15-mer linkers. Similar to the F1 fusion data in Figure 8, the STF activity of the peaks across the SEC column was widely distributed in the higher molecular weight fractions and did not coincide with the peaks of the monomeric form of the molecule( Figure 15B ). In contrast, when the tandem scFv was fused with the Fc fragment in a 2:2 tetravalent bispecific form, the STF peak activity across the SEC column coincided with the monomeric formFigure 14A ). A second STF activity peak on the SEC column was also observed in the higher molecular weight fraction, which was not observed in the F1 fusion molecule ( Figure 14A ). The dose-response curve of the monomer SEC peak fraction from 293STF reporter cells showed a slight preference for the N-terminal fusion of L1 with F2 to obtain higher activity similar to that of the fusion with F1 (Figure 10). Figure 14B ).

[0461] Fusions between F2 and L2 were also constructed in various forms (Table 6B). Although on the SEC column, the peak STF activity did not coincide with the monomeric form of F2 fused to the N-terminus of L2 in a 1:1 form ( Figure 14C ), it seems that the reverse-oriented monomeric 1:1 form of the N-terminal fusion of L2 with F2 is active ( Figure 14D ). The dose-response of the peak fraction in this orientation showed weaker activity compared to WNT3A with a preferred pentameric linker ( Figure 14E ). The 2:2 tetravalent bispecific form resulting from the N-terminal fusion of these tandem scFvs with Fc produced highly potent molecules. On the SEC column, the peak STF activity coincided with the protein peak, which was consistent with the monomeric form ( Figure 14F and 14H ), and the dose-response in 293STF reporter cells showed a potent and effective molecule that exceeded the activity of the F1 / L2 fusion shown in Figure 3 ( Figure 14G and 14I ).

[0462] 1:1 divalent bispecific tandem scFv forms and 2:2 tetravalent bispecific Fc fusions were also constructed between F3 and two LRP binders. Similar to the observations for the F1 fusion shown in Figure 8, no robust activity was observed for the 1:1 tandem scFv fusion molecules between F3 and L1 or L2 ( Figure 17 ). In contrast, the 293STF reporter gene activity of the fusion molecules between F3, LRP binder, and Fc in the 2:2 tetravalent bispecific form from the SEC column fraction coincided with the protein peak of the monomeric form of the molecule ( Figure 16A and 16B ). Notably, similar to the fusions with F1 and F2, the dose-response of the SEC peak fraction showed that the L2-F3 fusion was highly active in both orientations, while the L1-F3 fusion was less active and showed a preference for L1 on the N-terminus of F3 ( Figure 16C and 16D ). All these results together indicate that the multivalency of FZD and LRP binding and the stoichiometry of two FZDs and two LRPs are required for the consistent and effective activation of β-catenin-dependent signaling, at least among the binders tested.

[0463] After establishing the multivalency requirement for efficient induction of canonical WNT signaling, alternative 2:2 tetravalent bispecific formats (termed dumbbell formats) were explored, in which, as Figure 18A and 18B shown, two scFv binding entities are attached to the two termini of the Fc fragment. In reporter gene assays, the fusion of F1 with L1 in this dumbbell format minimally activated WNT signaling ( Figure 18A ). Interestingly, there was also a preference for the orientation of the L1 molecule, where its attachment to the C-terminus of the Fc affected activity. The fusion of F1 and L2 in this format also activated WNT signaling, with a similar preference in the orientation of the LRP binding arm ( Figure 18B ). The activity of these molecules across an SEC column was also tested, and as Figure 18C shown, the peak STF reporter gene activity was consistent with the protein peak corresponding to the monovalent form of the protein.

[0464] To understand whether the activity from the 2:2 tetravalent bispecific format is due to a single alternative molecule simultaneously binding multiple FZD and LRP, or due to a unique geometry provided by the format that does not require multivalent binding to the receptors, Octet binding studies were performed to assess whether the 1:1 tandem scFv format allows simultaneous binding of FZD and LRP. As Figure 19A shown, by sequentially adding FZD8 CRD to the sensor surface, followed by the 1:1 tandem scFv L2:5:F3 or F3:5:L2, and then LRP6 E3E4, a stepwise increase in the binding signal was observed. This indicates that the 1:1 tandem scFv molecule is capable of binding both receptors simultaneously, and thus, the lack of activity of these molecules to induce WNT signaling is not due to an inability to crosslink the FZD and LRP receptors.

[0465] However, to determine whether the 2:2 tetravalent bispecific format still engages the receptors in a 1:1 manner through a unique geometry that allows signaling not provided by the 1:1 tandem scFv format. As Figure 19B and depicted in Figure 20, four different variants of a possible 1:1 geometry were constructed on the framework of the 2:2 tetravalent bispecific molecule. None of these four molecules induced WNT signaling ( Figure 19C and Figure 20A ), thus indicating that efficient signaling requires multiple copies of receptor crosslinking.

[0466] To further understand the required ratio of FZD receptor to LRP receptor, as Figure 21A and 21DDepicted are combinations of 2 FZD binders with 1 LRP binder (2:1) or 1 FZD binder with 2 LRP binders (1:2). As shown in Figure 21, removal of 1 FZD binder or 1 LRP binder from the 2:2 form still activates β-catenin-dependent signaling. Since the 1:1 form shown in Figure 19 did not activate signaling, these results further support the concept that simultaneous engagement of multiple FZDs and LRPs is necessary for efficient signaling. Since removal of 1 LRP binder has a much smaller effect on activity than removal of 1 FZD binder, these results also suggest that dimerization of FZD receptors is more important for the formation of the signaling complex.

[0467] A multivalency requirement for efficient induction of canonical WNT signaling has been established. However, since each of the FZD-binding arms in the alternative molecules is specific for several different FZD receptors, it is not clear whether signaling can arise from heterodimerization of different FZD receptors. To address this question, two FZD binders, F2 and F3, with non-overlapping specificities were combined into one molecule, as Figure 21G depicted. Although F2 or F3 in the 1:2 form (1 FZD binder combined with 2 copies of the LRP binder, Figure 21B and 21C ) showed low to very low activity in 293STF reporter cells, respectively, combining F2 and F3 and 2 copies of the LRP binder L2 into one molecule (this is referred to as the 1:1:2 form, where the stoichiometry is one FZD plus a different FZD plus two LRPs) produced a highly potent and effective alternative molecule ( Figure 21H ). To further confirm this, a molecule with one copy of F2 and F3 and the LRP binder L2 (1:1:1:0 form, where the stoichiometry is one FZD plus a different FZD plus one LRP binder, Table 6B and Figure 21I ) was constructed. This molecule was also highly active ( Figure 21J ), even though F2 or F3 alone with one copy of L2 did not show activity (Figure 20). Since F2 and F3 bind different FZDs, these results suggest that heterodimerization of FZDs leads to β-catenin-dependent signaling.

[0468] As a final configuration, alternative molecules with two different FZD-binding arms and two different LRP-binding arms (1:1:1:1 form) were also constructed, as Figure 21Kdepicted. It is noted that, although the alternative molecule containing the LRP binder L1 always produces a much lower Emax compared to WNT3A, it produces a highly potent and effective molecule when combined with L2 in the context of a 1:1:1:1 format ( Figure 21L ).

[0469] All of the observations together demonstrate that the new alternative WNT platform is flexible, can generate any FZD / LRP specificity combination, and can generate highly potent and active molecules.

[0470] The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification and / or listed in the application data sheet are hereby incorporated by reference in their entirety. Aspects of the embodiments may be modified, if necessary, to employ the concepts of the various patents, applications, and publications to provide yet further embodiments. These and other changes may be made to the embodiments in light of the above detailed description.

[0471] In summary, in the following claims, the terms used should not be construed as limiting the claims to the specific embodiments disclosed in this specification and the claims, but should be construed to include the entire scope of all possible embodiments together with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the present disclosure.

Claims

1. A multispecific Wnt surrogate molecule, wherein said Wnt surrogate molecule comprises: two Fzd-binding regions each specifically binding to a set of one or more Frizzled (Fzd) receptor epitopes, wherein said two Fzd-binding regions bind to different sets of one or more Fzd receptor epitopes, wherein said Wnt surrogate molecule comprises a first light chain and a first heavy chain forming the first Fzd-binding region, and a second light chain and a second heavy chain forming the second Fzd-binding region, and two LRP5 / 6-binding regions each specifically binding to low density lipoprotein (LDL) receptor-related protein LRP5 and / or LRP6, wherein said Wnt surrogate molecule comprises a first LRP5 / 6-binding region fused to the N-terminus of the first light chain or the first heavy chain, and a second LRP5 / 6-binding region fused to the N-terminus of the second light chain or the second heavy chain, wherein: the pair of the first heavy chain and the first light chain and / or the pair of the second heavy chain and the second light chain are selected from: SEQ ID NO: 110 and 109, 112 and 111, 114 and 113, 116 and 115, 118 and 117, 120 and 119, 122 and 121, 130 and 129, 137 and 148, 150 and 149, 151 and 149, 154 and 153, 156 and 155, 130 and 152, and 130 and 157.

2. The Wnt surrogate molecule according to claim 1, wherein said two Fzd-binding regions bind to different sets of one or more FzD receptors, different sets of one or more epitopes within the same set of one or more FzD receptors, or a combination thereof.

3. The Wnt surrogate molecule according to claim 2, wherein each Fzd-binding region binds to one or more of the following: Frizzled Fzd1, Frizzled Fzd2, Frizzled Fzd3, Frizzled Fzd4, Frizzled Fzd5, Frizzled Fzd6, Frizzled Fzd7, Frizzled Fzd8, Frizzled Fzd9, and Frizzled Fzd10.

4. The Wnt surrogate molecule according to claim 3, wherein at least one Fzd-binding region 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 Fzd10; (ix) Fzd5, Fzd8, and Fzd10; (x) Fzd4, Fzd5, and Fzd8; or (xi) Fzd1, Fzd5, Fzd7, and Fzd8.

5. The Wnt surrogate molecule according to claim 4, wherein: (i) The first Fzd binding region binds to at least one of Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, and Fzd10, and (ii) The second Fzd binding region binds to at least one of Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, and Fzd10.

6. The Wnt surrogate molecule according to claim 5, wherein the plurality of Fzd binding regions comprises: (i) a first Fzd binding region that binds to a first set of one or more epitopes within a collection of one or more Fzd receptors, and (ii) a second Fzd binding region that binds to a second, different set of one or more epitopes within the same collection of one or more Fzd receptors.

7. The Wnt surrogate according to claim 6, wherein: a) The first Fzd binding region binds to at least one Fzd receptor that induces non-canonical Wnt signaling; and b) The second Fzd binding region binds to at least one Fzd receptor that induces canonical Wnt signaling, and wherein the binding of the first Fzd receptor to the second Fzd receptor results in canonical Wnt signaling or non-canonical Wnt signaling.

8. The Wnt surrogate molecule according to claim 7, wherein the at least one Fzd binding region binds monospecifically to Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, or Fzd10, and wherein the at least one Fzd binding region binds to a region of the Fzd receptor that: (i) does not contain the cysteine-rich domain (CRD) of the Fzd receptor; or (ii) contains less than the entire CRD of the FZD receptor; or (iii) overlaps with the CRD of the Fzd receptor.

9. The Wnt surrogate molecule according to claim 8, wherein the at least one Fzd binding region binds to the hinge region of the Fzd receptor shown in SEQ ID NOs: 98 - 107.

10. The Wnt surrogate molecule according to claim 8, wherein the at least one Fzd binding region binds to the N-terminal region upstream of the CRD of the Fzd receptor, and wherein the N-terminal region comprises the amino acid sequence shown in SEQ ID NO:

108.

11. The Wnt surrogate molecule according to claim 9, wherein the ratio of the Fzd binding region to the LRP5 / 6 binding region, expressed as Fzd:LRP5 / 6, is selected from the group consisting of: 2:1:1 in the case of having two different LRP binders, 1:1:2 in the case of having two different Fzd binders, 2:2 in the case of having the same Fzd and the same LRP binder, and 1:1:1:1 in the case of having all different Fzd and LRP binders.

12. The Wnt surrogate molecule according to claim 1, wherein the first heavy chain comprises a first CH3 domain, the second heavy chain comprises a second CH3 domain, and the first CH3 domain and the second CH3 domain are linked / interact with each other.

13. The Wnt surrogate molecule according to claim 12, wherein the first CH3 domain and the second CH3 domain are linked to each other by a knob-into-hole mutation.

14. The Wnt surrogate molecule according to claim 1, which regulates the Wnt signaling pathway in cells.

15. The Wnt surrogate molecule according to claim 1, which regulates the Wnt signaling pathway in mammalian cells.

16. A pharmaceutical composition comprising a pharmaceutically acceptable excipient or carrier and the Wnt surrogate molecule according to any one of claims 1 to 15.

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