Novel Wnt agonist antibodies and therapeutic uses thereof

Abstract

Provided herein is an antibody that agonizes Wnt signal transduction, does not compete with Wnt ligand for LRP6 binding, and activates Wnt signal transduction in the presence of inhibitor.Also provided is the method of promoting cell differentiation and tissue regeneration using the antibody of the present disclosure. TIFF2024533616000045.tif48128

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 246,250, filed September 20, 2021, the disclosure of which is incorporated by reference in its entirety for all purposes.

[0002] STATEMENT REGARDING RIGHTS TO INVETIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grants R01 CA118919 and R01 CA171315 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] 2. Background of the Invention The canonical Wnt / β-catenin signaling pathway is involved in a variety of biological processes, including tissue regeneration, stem cell regulation, and cell proliferation and differentiation (Clevers et al., Science 346(6205), 1248012 (2014) (Non-Patent Document 1); Lien & Fuchs, Genes & Development 28(14), 1517-32 (2014) (Non-Patent Document 2); Steinhart & Angers, Development 145 (2018) (Non-Patent Document 3)). Specifically, several studies have demonstrated a key role for canonical Wnt signaling in bone formation, including by analysis of the bone degenerative effects of Wnt signaling inhibitors such as sclerostin or Dickkopf Wnt signaling pathway inhibitor 1 (DKK1) (Markham, Drugs 79:471-476 (2019)); Liu et al., Science Transl. Med. 8 (2016); McDonald et al., Blood 129:3452-64 (2017); Pozzi et al., Bone 53:487-96 (2013)). Specifically, blocking sclerostin has been shown to be clinically effective against osteoporosis, and an anti-sclerostin monoclonal antibody (romosozumab) has been approved for the treatment of osteoporosis. In addition, the anti-DKK1 antibody BHQ880 has been clinically evaluated for the recovery of osteolytic bone loss caused by multiple myeloma (Fulciniti et al., Blood 114:371-79 (2009) (Non-Patent Document 10); Iyer et al., Brit. J. Haematol. 167:366-75 (2014) (Non-Patent Document 11); Munshi & Anderson, Clin. Canc. Res. 19:3337-44 (2013) (Non-Patent Document 12)).

[0004] Although therapies targeting inhibitory ligands have achieved promising results in promoting bone formation, these methods may be less effective if Wnt ligands are absent or below clinical threshold in disease areas.In addition, anti-inhibitor approaches are limited to specific inhibitors that monoclonal antibodies are designed to bind and neutralize.For example, while romosozumab blocks sclerostin, it does not block DKK1, and as a result, blocking the inhibitory activity against Wnt signaling is potentially limited (Joiner et al., TEM 24:31-39 (2013)).

[0005] Alternatively, Wnt signaling can be directly activated using classical Wnt pathway agonists. Classical Wnt signaling is induced by two different Wnt coreceptors, the G protein-coupled receptor Frizzled (Fzd) and low density lipoprotein receptor-related protein 5 or 6 (LRP5 or LRP6). Binding of Wnt ligand leads to the formation of Fzd-Wnt-LRP6 complex, which causes phosphorylation of LRP6 and initiates signaling. Inhibition of classical Wnt signaling by anti-LRP6 antibodies has been reported (Ettenburg et al., PNAS 10:15473-78 (2010)). In addition, ligand surrogate-based Wnt agonists that can activate Wnt signaling and promote bone formation have been reported (Janda et al., Nature 545:234-+ (2017)). This ligand surrogate-based Wnt agonist consists of anti-Fzd scFv and DKK1 LRP6 binding domain, thereby mimicking the mechanism of natural Wnt ligands. Another ligand surrogate-based Wnt agonist consisting of anti-Fzd scFv and anti-LRP6 single domain antibody has also been reported (Fowler et al., Nature Comm. 12:3247 (2021) (Non-Patent Document 16)). Other Wnt ligand surrogates that utilize multivalency and cross-linking to enhance signaling have also been described, a mechanism that may also be used by natural Wnt ligands and coactivators (Chen et al., Cell. Signal. 26:1068-74 (2014) (Non-Patent Document 17); Tao et al., eLife 8 (2019) (Non-Patent Document 18)). However, because all of the ligand surrogate-based Wnt agonists compete with endogenous Wnt ligands for binding to the receptor complex, they are also subject to inhibition by endogenous inhibitors such as DKK1 and sclerostin, which bind to the ligand-binding site. [Prior art documents] [Non-patent literature]

[0006]

Non-licensed literature 1

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

[0007] BRIEF SUMMARY OF THE INVETION In various aspects, the invention disclosed herein can include, but is not necessarily limited to, any one or more of the following embodiments.

[0008] In one aspect, the disclosure provides a monoclonal antibody or antigen-binding portion thereof that agonizes Wnt signaling and does not compete with a Wnt ligand or a Wnt inhibitor.

[0009] In another aspect, the disclosure provides a monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to low density lipoprotein receptor-related protein 6 (LRP6) and agonizes Wnt signaling. In some embodiments of this aspect, the antibody or antigen-binding portion comprises a) a heavy chain variable domain (VH) comprising: (1) the amino acid sequence of SEQ ID NO:2; (2) the amino acid sequence of SEQ ID NO:8; (3) heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs:18, 19, and 21, respectively; (4) heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs:18, 19, and 22, respectively; (5) heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs:18, 19, and 23, respectively; (6) heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs:18, 19, and 24, respectively; or (7) heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs:18, 19, and 25, respectively. In some embodiments, the antibody further comprises b) a light chain variable domain (VL) comprising: (1) the amino acid sequence of SEQ ID NO:15; (2) light chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs:26, 27, and 29, respectively; or (3) light chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs:26, 27, and 30, respectively.

[0010] In some embodiments, the monoclonal antibody or antigen-binding portion thereof of claim 1 comprises a VH comprising the amino acid sequence of SEQ ID NO:3 and a VL comprising the amino acid sequence of SEQ ID NO:15. In some embodiments, the antibody comprises a heavy chain variable region having at least 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO:3. In some embodiments, the antibody comprises a light chain variable region having at least 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO:15.

[0011] In some embodiments, the monoclonal antibody or antigen-binding portion thereof of claim 1 comprises a VH comprising the amino acid sequence of SEQ ID NO:3 and a VL comprising the amino acid sequence of SEQ ID NO:16. In some embodiments, the antibody comprises a heavy chain variable region having at least 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO:3. In some embodiments, the antibody comprises a light chain variable region having at least 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO:16.

[0012] In some embodiments, the monoclonal antibody or antigen-binding portion thereof of claim 1 comprises a VH comprising the amino acid sequence of SEQ ID NO:9 and a VL comprising the amino acid sequence of SEQ ID NO:16. In some embodiments, the antibody comprises a heavy chain variable region having at least 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO:9. In some embodiments, the antibody comprises a light chain variable region having at least 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO:16.

[0013] In some embodiments, the monoclonal antibody or antigen-binding portion thereof of claim 1 comprises a VH comprising the amino acid sequence of SEQ ID NO:2 and a VL comprising the amino acid sequence of SEQ ID NO:15. In some embodiments, the antibody comprises a heavy chain variable region having at least 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO:2. In some embodiments, the antibody comprises a light chain variable region having at least 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO:15.

[0014] In some embodiments, the monoclonal antibody, or antigen-binding portion thereof, comprises: a) a light chain comprising the amino acid sequence of SEQ ID NO:15; and b) a heavy chain comprising (1) HCDR1-3 comprising the amino acid sequences of SEQ ID NOs:18, 19, and 21, respectively; (2) heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs:18, 19, and 22, respectively; (3) heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs:18, 19, and 23, respectively; (4) heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs:18, 19, and 24, respectively; or (5) heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs:18, 19, and 25, respectively. In certain aspects, the monoclonal antibody, or antigen-binding portion thereof, comprises a VH comprising the amino acid sequence of SEQ ID NO:3, 4, 5, 6, or 7, and a VL comprising the amino acid sequence of SEQ ID NO:15. In some embodiments, the antibody comprises a heavy chain variable region having at least 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of any one of SEQ ID NOs:3-7. In some embodiments, the antibody comprises a light chain variable region having at least 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO:15.

[0015] In some embodiments, the monoclonal antibody, or antigen-binding portion thereof, comprises a heavy chain comprising: (1) the amino acid sequence of SEQ ID NO:8; (2) heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs:18, 19, and 21, respectively; (3) heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs:18, 19, and 22, respectively; (4) heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs:18, 19, and 23, respectively; (5) heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs:18, 19, and 24, respectively; or (6) heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs:18, 19, and 25, respectively. In some embodiments, the monoclonal antibody or antigen-binding portion thereof comprises b) a light chain comprising: (1) light chain CDR1-3 comprising the amino acid sequence of SEQ ID NO:26, 27, and 29, respectively; or (2) light chain CDR1-3 comprising the amino acid sequence of SEQ ID NO:26, 27, and 30, respectively. In certain aspects, the monoclonal antibody or antigen-binding portion thereof comprises a VH comprising the amino acid sequence of SEQ ID NO:8, 9, 10, 11, 12, or 13, and a VL comprising the amino acid sequence of SEQ ID NO:16 or 17. In some embodiments, the antibody comprises a heavy chain variable region having at least 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of any one of SEQ ID NO:8-13. In some embodiments, the antibody comprises a light chain variable region having at least 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO:16 or SEQ ID NO:17.

[0016] In some embodiments, the monoclonal antibody or antigen-binding portion thereof comprises a human IgG heavy chain constant region. In some embodiments, the monoclonal antibody or antigen-binding portion thereof is an effector-attenuated IgG1 antibody. In certain embodiments, the effector-attenuated IgG1 antibody is an IgG1 antibody that comprises leucine to alanine substitutions at positions 234 and 235. In some embodiments, the monoclonal antibody or antigen-binding portion thereof is an IgG2 antibody. In some embodiments, the monoclonal antibody or antigen-binding portion thereof is a human monoclonal antibody or antigen-binding portion thereof.

[0017] In some embodiments, the monoclonal antibody or antigen-binding portion thereof specifically binds to an epitope on LRP6 that does not overlap with the binding site for a Wnt ligand or a Wnt inhibitor. The Wnt ligand can be, for example, Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt8a, Wnt8b, Wnt10a, Wnt10b, Wnts2b, or Wnt9b. The Wnt inhibitor can be, for example, Dickkopf Wnt signaling pathway inhibitor 1 (DKK1), Dickkopf Wnt signaling pathway inhibitor 2 (DKK2), Dickkopf Wnt signaling pathway inhibitor 3 (DKK3), Dickkopf Wnt signaling pathway inhibitor 4 (DKK4), Dickkopf-like acrosomal protein 1 (DKKL1), sclerostin (SOST), Wise (SOSTDC1 (sclerostin domain containing 1)), IGFBP-4, or Waif1 / 5T4. In some embodiments, the monoclonal antibody or antigen binding portion binds to a non-linear epitope. In some embodiments, the epitope includes K662 and K684. In some embodiments, the epitope does not include E663, E708, H834, Y875, or M877.

[0018] In another aspect, the disclosure provides a pharmaceutical composition comprising a Wnt agonist antibody, or antigen-binding portion thereof, and a pharma- ceutically acceptable excipient.

[0019] In another aspect, a nucleic acid sequence encoding a Wnt agonist antibody or an antigen-binding portion thereof is provided. The present disclosure also describes a vector and a mammalian host cell comprising the nucleic acid sequence. In some embodiments, the host cell is a CHO, CHO-K1, CHO-S, ExpiCHO, CHO-DG44, CHO-Pro minus, HEK293A, HEK293F cell. In some embodiments, the present disclosure provides a method for producing a monoclonal antibody or an antigen-binding portion thereof, comprising culturing the host cell under conditions to allow the production of the monoclonal antibody or an antigen-binding portion thereof.

[0020] In yet another aspect, the present disclosure provides a method for promoting tissue regeneration comprising adding a Wnt agonist monoclonal antibody, or an antigen-binding portion thereof, described herein to a cell or tissue in vitro or ex vivo.

[0021] In another aspect, the present disclosure provides a method for restoring tissue in an individual in need thereof, comprising administering to the individual a pharmaceutical composition comprising a Wnt agonist antibody or antigen-binding portion thereof described herein. In some embodiments, the tissue is bone tissue, intestinal tissue, liver tissue, or brain tissue. In some embodiments, the individual has a disease or condition characterized by insufficient Wnt signaling. In some embodiments, the individual has age-induced osteoporosis, drug-induced bone loss, osteogenesis imperfecta, inflammatory bowel disease, severe alcoholic hepatitis, diabetic retinopathy, wet age-related macular degeneration (AMD), Fuchs' dystrophy, corneal epithelial stem cell deficiency, atrophic AMD, Sjogren's dry eye, short bowel syndrome, or hearing loss. In some embodiments, the pharmaceutical composition comprising a Wnt agonist antibody or antigen-binding portion thereof described herein is administered by intravenous injection, intraperitoneal injection, or subcutaneous injection.

[0022] In another aspect, the disclosure provides a method for identifying a monoclonal antibody, or antigen-binding portion thereof, that agonizes Wnt signaling and does not compete with a Wnt ligand or a Wnt inhibitor, comprising the steps of: a) providing an LRP6 polypeptide, or a portion thereof comprising at least the LRP6 polypeptide P3E3P4E4 domain; b) contacting said LRP6 polypeptide or portion thereof with a library of binding molecules; c) selecting one or more binding molecules from the library that bind to said LRP6 polypeptide or portion thereof; and d) identifying selected binding molecules that do not compete with a Wnt ligand or a Wnt inhibitor for binding to said LRP6 polypeptide or portion thereof. [Brief description of the drawings]

[0023] [Figure 1A] Figures 1A-1H demonstrate the binding of an exemplary Wnt agonist human monoclonal antibody 66 (shown as 6-6 in the figures) to LRP6. Figures 1A and 1B are graphs showing activation of canonical Wnt signaling by 100 nM Wnt agonist antibody in the presence of Wnt1 or Wnt3a (Figure 1A) or in the absence of endogenous Wnt ligands (Figure 1B) (error bars represent SD from n=2. **P<0.01; ***P<0.001.). [Figure 1B] See legend to Figure 1A. [Figure 1C] Figures 1A-1H demonstrate binding of an exemplary Wnt agonist human monoclonal antibody 66 (shown as 6-6 in the figures) to LRP6. Figure 1C is a schematic diagram showing the deletion constructs of LRP6 used in the binding assay (SP: signal peptide; P3 and P4: beta-propeller domains 3 and 4, respectively; E3 and E4: EGF-like domains 3 and 4, respectively; LDLR: low density lipoprotein receptor type A domain; TM: transmembrane domain; Cyto: cytoplasmic domain). [Figure 1D]Figures 1A-1H demonstrate the binding of an exemplary Wnt agonist human monoclonal antibody 66 (shown as 6-6 in the figures) to LRP6. The results of the binding assay are shown in Figure 1D. Each LRP6 truncated plasmid was individually transfected into HEK293 cells along with a GFP expression construct. Binding of 66 IgG in the GFP-positive cell population was analyzed by flow cytometry. [Figure 1E] Figures 1A-1H demonstrate binding of an exemplary Wnt agonist human monoclonal antibody 66 (shown as 6-6 in the figures) to LRP6. Figure 1E is a graph showing the effect of 66 antibody on activation of Wnt signaling in cells expressing truncated LRP6. LRP6 truncated constructs and STF reporter plasmids were transfected into HEK293 cells and cells were incubated in Wnt3aCM with or without 66 IgG. Error bars represent SD of n=2. *P<0.05, ***P<0.001. NS=not significant. [Figure 1F] Figures 1A-1H demonstrate binding of an exemplary Wnt agonist human monoclonal antibody 66 (shown as 6-6 in the figures) to LRP6. Figure 1F is a graph showing fine epitope mapping by alanine scanning of LRP6 and the effect of alanine substitution on 66 antibody binding. Single and double mutants of LRP6 (K662A / K684A) were transfected separately into HEK293 cells. An anti-LRP6 scFv-Fc fusion binding to the LRP6-P1 domain was used as a control to confirm cell surface expression of LRP6. 66 antibody binding was determined by flow cytometry and median fluorescence intensity (MFI) was normalized to the MFI of P1-binding scFv-Fc. [Figure 1G]Figures 1A-1H demonstrate binding of an exemplary Wnt agonist human monoclonal antibody 66 (shown as 6-6 in the figures) to LRP6. Figure 1G is a graph showing the effect of the LRP6 double mutant (K662A / K684A) on binding of the 66 antibody in the presence or absence of Wnt3a ligand. Plasmids encoding wild-type LRP6 (WT) or the K662A / K684A double mutant were transfected into HEK293 cells along with a Wnt3a expression plasmid and an STF reporter expression plasmid, and the cells were incubated with or without 66 IgG. Error bars represent SD of n=2. *P<0.05. [Figure 1H] Figures 1A-1H demonstrate the binding of an exemplary Wnt agonist human monoclonal antibody 66 (shown as 6-6 in the figures) to LRP6. Figure 1F shows the structure of the LRP6 double mutant (K662A / K684A), with residues involved in binding to the Wnt3a ligand (E663, E708, H834, Y875, M877) shown in yellow and residues involved in binding to the 66 antibody (K662A and K684) shown in red. [Figure 2A] Figures 2A and 2B are graphs showing the binding kinetics of an exemplary Wnt agonist human monoclonal antibody 66 (shown as 6-6 in the figures) with the Wnt3a ligand and / or inhibitor DKK1. Recombinant Wnt3a (Figure 2A) or DKK1 (Figure 2B) were bound to a biosensor loaded with LRP6. The biosensor was further immersed in a mixture of Wnt3a and DKK1 (black), a mixture of Wnt3a and 66 Fab (blue in D), or a mixture of DKK1 and 66 Fab (red in Figure 2B). The data show that the 66 antibody binds to LRP6 in the presence of either Wnt3a or DKK1. [Figure 2B] See legend to Figure 2A. [Figure 2C]Figure 2C is a graph showing the effect of the inhibitor DKK1 on the agonist activity of the 66 antibody. STF reporter and Wnt3a expression constructs were transfected into HEK293 cells. Cells were incubated with or without 66 IgG (50 nM) and DKK1 (20 nM). Error bars represent SD of n = 2. [Figure 2D] FIG. 2D is a schematic diagram showing that an exemplary Wnt agonist human monoclonal antibody (66) acts as a novel type of Wnt ligand that has an additive effect to endogenous ligands and activates Wnt signaling in the presence of inhibitors. [Diagram 3]FIG. 3A shows that DKK1 inhibits Wnt3a / RSPO2-induced β-catenin signaling. STF reporter and Wnt3a expression constructs were transfected into HEK293 cells, and HEK293 cells were incubated with RSPO2 alone (5 nM) or with RSPO2 (5 nM) + DKK1 (15 nM). Error bars represent SD of n = 2. The design and results are summarized diagrammatically in the right panel. FIG. 3B shows that DKK1 has no inhibitory effect on 66 / RSPO2-induced Wnt / β-catenin signaling. HEK293 cells transfected with STF reporter constructs were incubated with 66 (100 nM), RSPO2 (5 nM), or DKK1 (15 nM) as indicated. No Wnt ligand was added. Error bars represent SD of n = 2. The design and results are summarized diagrammatically in the right panel. Figure 3C and Figure 3D show that RSPO2-induced Wnt / β-catenin signaling is enhanced by 66 antibody. In Figure 3C, RSPO2 was titrated to HEK293 cells transfected with STF reporter and Wnt3a expression constructs. A constant concentration of 66 IgG (20 nM) was added in the culture medium. In Figure 3D, HEK293 cells were transfected with STF reporter and Wnt3a expression constructs and incubated with different concentrations of 66 IgG in the presence of RSPO2 (5 nM). Error bars represent SD of n = 2. The design and results are summarized diagrammatically in the right panel. [Figure 4A] Figure 4 shows that an exemplary Wnt agonist human monoclonal antibody 66 (shown as 6-6 in the figure) promotes osteoblast differentiation in vitro. Figure 4A is a graph showing cross-species binding of 66 to the extracellular domain of recombinant human or mouse LRP6 in an ELISA assay. Control IgG: non-binding human IgG. Error bars represent SD for n=2. [Figure 4B]FIG. 4 shows that an exemplary Wnt agonist human monoclonal antibody 66 (shown as 6-6 in the figure) promotes osteoblast differentiation in vitro. FIG. 4B and FIG. 4C are graphs showing enhancement of Wnt / β-catenin signaling by 66 IgG in mouse cell lines MC3T3-E1 (FIG. 4B) or C3H / 10T1 / 2 (FIG. 4C). Wnt3a expression and STF reporter constructs were transfected into MC3T3-E1 (FIG. 4B) or C3H / 10T1 / 2 (FIG. 4C) cell lines, and the cell lines were further incubated with or without 66 IgG. Luciferase activity was normalized to the control group transfected with only the reporter construct. Data represent the mean ± SD of n=2. *P<0.05. [Figure 4C] See legend to Figure 4C. [Figure 4D] FIG. 4 shows that an exemplary Wnt agonist human monoclonal antibody 66 (shown as 6-6 in the figure) promotes osteoblast differentiation in vitro. FIG. 4D shows a graph demonstrating the relative mRNA expression levels of osteoblast marker genes (Runx2, BMP2, ALP, and OCN) detected by qRT-PCR. C3H / 10T1 / 2 cells were incubated with Wnt3aCM or 66 IgG for 3 days as indicated. Expression of osteoblast marker genes (RUNX2, BMP2, ALP, and OCN) was assessed by qRT-PCR. Relative mRNA expression levels were calculated using the comparative Ct method and normalized to the GAPDH gene. *P<0.05; **P<0.01. [Figure 4E]Figure 4 shows that an exemplary Wnt agonist human monoclonal antibody 66 (shown as 6-6 in the figure) promotes osteoblast differentiation in vitro. Figure 4E is a graph showing ALP activity induced by Wnt agonist 66 antibody in the presence or absence of Wnt3a conditioned medium. C3H / 10T1 / 2 cells were cultured in Wnt3aCM with or without 66 IgG for 7 days. ALP activity was measured using cell lysates and normalized to the control group without Wnt3aCM and 66 treatment. Error bars represent SD (n=2). *P < 0.05. [Figure 4F] FIG. 4 shows that an exemplary Wnt agonist human monoclonal antibody 66 (denoted as 6-6 in the figure) promotes osteoblast differentiation in vitro. FIG. 4F shows the relative mineralization rate induced by Wnt3a-conditioned medium in the presence or absence of Wnt agonist 66 antibody, either shown graphically (left panel) or shown by Alizarin Red staining (right panel). C3H / 10T1 / 2 cells were cultured for 21 days in osteogenic medium supplemented with Wnt3aCM or 66 IgG, as indicated. Alizarin Red staining assay was applied to quantify mineralization (left), which was normalized to the control (Wnt3a CM- / Ab-). Measurements were shown as mean ± SD (n = 2). *P < 0.05. Representative images are shown (right). Scale bar: 200 μm. [Figure 5A] Figure 5 shows that an exemplary Wnt agonist human monoclonal antibody 66 (shown as 6-6 in the figure) overcomes multiple myeloma-mediated inhibition of Wnt signaling. Figure 5A is a graph showing inhibition of Wnt3a / β-catenin signaling by culture medium from three multiple myeloma cell lines. HEK293 cells transfected with STF reporter and Wnt3a expression constructs were incubated in conditioned medium (CM) from multiple myeloma cell lines and in CM from HEK293 (as a control, Ctrl-CM). Values ​​represent the mean ± SD of n=2. *P < 0.05, **P < 0.01. [Figure 5B]Figure 5 shows that an exemplary Wnt agonist human monoclonal antibody 66 (shown as 6-6 in the figure) overcomes multiple myeloma-mediated inhibition of Wnt signaling. Figure 5B is a graph showing that the 66 antibody overcomes the inhibition of Wnt signaling caused by MM1.S culture medium. STF reporter and Wnt3a expression constructs were transfected into HEK293 cells, and HEK293 cells were incubated with different concentrations of 66 IgG in MM1.S-CM. Values ​​represent the mean ± SD (n = 2). *P < 0.05, **P < 0.01. [Figure 5C] FIG. 5 shows that an exemplary Wnt agonist human monoclonal antibody 66 (shown as 6-6 in the figure) overcomes multiple myeloma-mediated Wnt signaling inhibition. FIG. 5C is a schematic diagram of an animal study in which MM1.S cells were intrafemorally injected into the right femur and allowed to settle for one week, followed by weekly dosing with the 66 antibody. A total of six weekly intraperitoneal injections of PBS or 66 IgG (10 mg / kg) were administered (n=5 / group). One week after the end of dosing, femurs from live mice were scanned by micro-computed tomography. One week after the in vivo scan, mice were sacrificed and serum and femoral tissue were collected for further analysis. [Figure 5D] Figure 5 shows that an exemplary Wnt agonist human monoclonal antibody 66 (shown as 6-6 in the figure) overcomes multiple myeloma-mediated inhibition of Wnt signaling. Figure 5D is a graph showing the results of an ELISA assay evaluating the concentration of human Ig lambda light chain in the serum of MM1.S-implanted mice that were naive or injected with PBS or 66 antibody (naive: MM1.S-implanted mice; PBS: MM1.S-implanted mice injected with PBS (vehicle control); 66: MM1.S-implanted mice injected with 66 IgG. NS indicates not significant; ***P<0.001). [Figure 5E]Figure 5 shows that an exemplary Wnt agonist human monoclonal antibody 66 (shown as 6-6 in the figure) overcomes multiple myeloma-mediated inhibition of Wnt signaling. Figure 5E shows planar and 3D views of whole femurs obtained from microCT of these mice. The microCT images were thresholded to generate sharp planar cross-sections (top) and further reconstructed to obtain stacked 3D views (bottom). [Figure 6A] Figure 6 shows that an exemplary Wnt agonist human monoclonal antibody 66 (shown as 6-6 in the figure) reverses bone loss in the femoral intraosseous MM1.S model. Figure 6A shows a 3D view of the trabecular and cortical bone regions of interest in the femur. Micro-CT images were used to reconstruct 3D data sets of the trabecular and cortical bone regions as shown. [Figure 6B] FIG. 6 shows that an exemplary Wnt agonist human monoclonal antibody 66 (shown as 6-6 in the figure) reverses bone loss in an intrafemoral MM1.S model. FIG. 6B shows representative images of the trabecular ROI in naive mice or mice with MM1.S-implanted femurs (given PBS or 66 antibody). MicroCT images of naive or MM1.S-implanted femurs (PBS and 66 IgG) were used to reconstruct the 3D structure of the trabecular region. Representative images are shown. [Figure 6C] Figure 6 shows that an exemplary Wnt agonist human monoclonal antibody 66 (shown as 6-6 in the figure) reverses bone loss in the intrafemoral MM1.S model. Figures 6C and 6D are graphs showing quantification of trabecular microarchitecture, including bone volume relative to tissue volume (BV / TV; Figure 6C) and trabecular width (Figure 6D). *P<0.05. [Figure 6D] See legend to Figure 6C. [Figure 6E]Figure 6 shows that an exemplary Wnt agonist human monoclonal antibody 66 (shown as 6-6 in the figure) reverses bone loss in the intrafemoral MM1.S model. Figure 6E shows that the 66 antibody enhanced cortical bone formation, as shown visually by microCT images (left panel) and graphically as a measure of cortical bone width (right panel). MicroCT images of cortical bone were reconstructed from the proximal femur region (left). Cortical bone width (Ct.Th) was measured and compared between groups (right). *P<0.05. [Figure 6F] FIG. 6 shows that an exemplary Wnt agonist human monoclonal antibody 66 (shown as 6-6 in the figure) reverses bone loss in an intrafemoral MM1.S model. FIG. 6F shows histological evaluation of osteoblasts in the distal femoral region, with the left panel showing hematoxylin and eosin staining (small panel shown in the left image is enlarged in the right image, yellow triangles identify osteoblasts), and the right panel showing a graph as a measure of the number of osteoblasts on the trabecular bone surface. Femurs with or without antibody treatment were subjected to H&E staining (left). Scale bar = 50 μm. Yellow arrowheads indicate osteoblasts, and the number of osteoblasts on the trabecular bone surface (Ob.N) was counted (right). *P<0.05. [Figure 7A]Figure 7 shows that the 66 antibody (6-6 in the figure) binds to a site on LRP6 that is distinct from previously identified LRP6 binders. LRP6 expression plasmid was transfected into HEK293 cells, and HEK293 cells were incubated with 66 or E34N19 scFv phage for 1 h. E34N19 was previously identified as a Wnt antagonist that binds to the P3E3P4E4 domain (Lee et al., 2018). E34N19 IgG was added simultaneously as a competitor. Bound phage was detected by sequential incubation of mouse anti-fd IgG and PE-labeled anti-mouse IgG. Figure 7A is a graph showing the dissociation curve of the 66 antibody. HEK293 cells were incubated with different concentrations of 66 IgG, and binding was analyzed by flow cytometry. The apparent KD (~5.0 nM) was estimated by curve fitting. Figure 7B is a graph showing, as a measure of mean fluorescence intensity, that E34N19E IgG does not compete with 66 scFv phage in a binding assay. As a positive control, E34N19E IgG competes with E34N19E scFv phage. [Figure 7B] See legend to Figure 7A. [Figure 8]Figure 8A and Figure 8B show modeling of 66 antibody (denoted as 6-6 in the figure) using Rosetta antibody module and its interaction with LRP6-P3E3 domain (PDB:3S8Z and PDB:4A0P in Figure 8A) using structural docking with ZDOCK. (Blue: CDRH of 66 Fv, Light grey: LRP6-P3E3 domain, Purple: Predicted potential binding site (within 4 angstroms) by 66 Fv in S2A (T659, G660, K662, L683, K684, T685, H698, V699, E701, F702, G703, D735, G736, Q737, H738, R739) or Predicted potential binding site (within 4 angstroms) by 66 Fv in S2B. (T659, G660, V661, K662, S682, L683, K684, T685, S687, H698, V699, V700, E701, F702, D735, G736, Q737, H738, R739). Yellow: residues involved in Wnt3a binding (E663, E708, H834, Y875, M877) (Chen et al., 2011). Figure 8C is a graph showing that the inhibitor DKK1 does not inhibit 66-induced Wnt / β-catenin signaling in the absence of Wnt ligand. STF reporter constructs were transfected into HEK293 cells, and HEK293 cells were incubated with 66 (100 nM) with or without DKK1 (15 nM). No Wnt ligand was added. Error bars represent SD of n = 2. *P<0.05. Figure 8D is a graph showing that 66 antibody enhances Wnt / β-catenin signaling in the absence of Wnt ligand. 66 antibody was titrated without Wnt ligand to HEK293 cells transfected with STF reporter construct. EC50 (13.38 nM) was estimated by curve fitting. Error bars represent SD of n = 2. [Figure 9]Figure 9 is a graph demonstrating that 66 antibody (shown as 6-6 in the figure) synergistically enhances Wnt / β-catenin signaling amplification with Wnt ligand and RSPO2. STF reporter and Wnt1 expression constructs were transfected into HEK293 cells, and HEK293 cells were incubated with different concentrations of 66 IgG in the presence of RSPO2 (5 nM). Error bars represent SD of n=2. [Figure 10] FIG. 10 is a photograph showing immunohistochemical staining of distal regions of femoral tissues obtained from mice treated with PBS or 66 antibody using anti-human Ig lambda light chain antibody to mark MM1.S cells. Femoral tissues obtained from PBS or 66 treated mice were fixed in formalin, embedded in paraffin, sectioned at 4 μm, stained with anti-human Ig lambda (λ) light chain antibody to mark MM1.S cells, and counterstained with hematoxylin. The left panel is of femoral tissues without tumor implantation, and the right panel is of femoral tissues injected with MM1.S. Scale bar = 100 μm. [Figure 11] Figure 11 is a schematic diagram of the experiment performed to analyze the effect of exemplary Wnt agonist antibodies (66 and 66-11) in an ovariectomy-induced osteoporosis mouse model system. Starting 4 weeks after ovariectomy, Wnt agonist antibodies (6 mg / kg or 1 mg / kg) or PBS control were delivered intraperitoneally or subcutaneously to ovariectomized mice once a week for 5 weeks as indicated. In vivo micro-CT scans were performed to focus on the distal femur at the indicated time points. Both trabecular and cortical bone were evaluated. [Figure 12]FIG. 12 is a graph showing the results of micro-CT scans of trabecular bone from an experiment performed as shown in the diagram in FIG. 11. Micro-CT scans were performed 4 days after administration of the final dose of Wnt agonist antibody or PBS control. The y-axis indicates trabecular bone volume relative to total volume, and the x-axis indicates dose and route of administration. Six mice were used per test group on the x-axis, and both paws were scanned to generate images for analysis by ImageJ (BoneJ). 66 antibody was administered intraperitoneally at 6 mg / kg. 66-11 antibody was administered intraperitoneally or subcutaneously at 1 mg / kg. *P<0.05;***P<0.001. [Figure 13] Figures 13A and 13B show the results of micro-CT scans of trabecular bone from experiments performed as shown in the diagram in Figure 11. Micro-CT scans were performed 28 days after administration of the final dose of Wnt agonist antibody or PBS control. Figure 13A shows the results in a graph. The y-axis shows trabecular bone volume relative to total volume, and the x-axis shows dose and route of administration. Six mice were used per test group on the x-axis, and both legs were scanned. 66 antibody was administered intraperitoneally at 6 mg / kg. 66-11 antibody was administered intraperitoneally or subcutaneously at 1 mg / kg. ** P < 0.01; *** P < 0.001. Figure 13B shows exemplary images used for analysis of each test group with ImageJ (BoneJ). [Figure 14]FIG. 14A and FIG. 14B show the results of micro-CT scans of trabecular bone from an experiment performed as shown in the diagram in FIG. 11. Micro-CT scans were performed 73 days after administration of the final dose of Wnt agonist antibody or PBS control. FIG. 14A shows the results in a graph. The y-axis shows trabecular bone volume relative to total volume, and the x-axis shows dose and route of administration. Six mice were used per test group on the x-axis, and both legs were scanned. 66 antibody was administered intraperitoneally at 6 mg / kg. 66-11 antibody was administered intraperitoneally or subcutaneously at 1 mg / kg. ** P < 0.01; *** P < 0.001. FIG. 14B shows exemplary images used for analysis by ImageJ (BoneJ) of the PBS control test group and the test group treated subcutaneously with 66-11 antibody. [Figure 15] FIG. 15 is a graph showing the results of a microCT scan of trabecular bone from an experiment performed as shown in the diagram in FIG. 11. MicroCT scans were performed 111 days after administration of the final dose of Wnt agonist antibody or PBS control. The y-axis indicates trabecular bone volume relative to total volume, and the x-axis indicates dose and route of administration. Six mice were used per test group on the x-axis, and both paws were scanned to generate images for analysis by ImageJ (BoneJ). 66 antibody was administered intraperitoneally at 6 mg / kg. 66-11 antibody was administered intraperitoneally or subcutaneously at 1 mg / kg. **P<0.01;****P<0.0001. [Figure 16] FIG. 16 is a graph showing the results of microCT scans of cortical bone from an experiment performed as shown in the diagram in FIG. 11. MicroCT scans were performed 4 days after administration of the final dose of Wnt agonist antibody or PBS control. The y-axis indicates cortical bone width (Ct.Th) in μm and the x-axis indicates the dose and route of administration. Six mice were used per test group on the x-axis, and both paws were scanned to generate images for analysis by ImageJ (BoneJ). 66 antibody was administered intraperitoneally at 6 mg / kg. 66-11 antibody was administered intraperitoneally or subcutaneously at 1 mg / kg. *P < 0.05; ns: not significant. [Figure 17]FIG. 17 is a graph showing the results of microCT scans of cortical bone from an experiment performed as shown in the diagram in FIG. 11. MicroCT scans were performed 28 days after administration of the final dose of Wnt agonist antibody or PBS control. The y-axis indicates cortical bone width (Ct.Th) in μm and the x-axis indicates the dose and route of administration. Six mice were used per test group on the x-axis, and both paws were scanned to generate images for analysis by ImageJ (BoneJ). 66 antibody was administered intraperitoneally at 6 mg / kg. 66-11 antibody was administered intraperitoneally or subcutaneously at 1 mg / kg. *P < 0.05; ns: not significant. [Figure 18] FIG. 18 is a graph showing the results of microCT scans of cortical bone from an experiment performed as shown in the diagram in FIG. 11. MicroCT scans were performed 73 days after administration of the final dose of Wnt agonist antibody or PBS control. The y-axis indicates cortical bone width (Ct.Th) in μm and the x-axis indicates the dose and route of administration. Six mice were used per test group on the x-axis, and both paws were scanned to generate images for analysis by ImageJ (BoneJ). 66 antibody was administered intraperitoneally at 6 mg / kg. 66-11 antibody was administered intraperitoneally or subcutaneously at 1 mg / kg. *** P < 0.001; ns: not significant. [Figure 19] FIG. 19 is a graph showing the results of microCT scans of cortical bone from an experiment performed as shown in the diagram in FIG. 11. MicroCT scans were performed 111 days after administration of the final dose of Wnt agonist antibody or PBS control. The y-axis indicates cortical bone width (Ct.Th) in μm and the x-axis indicates the dose and route of administration. Six mice were used per test group on the x-axis, and both paws were scanned to generate images for analysis by ImageJ (BoneJ). 66 antibody was administered intraperitoneally at 6 mg / kg. 66-11 antibody was administered intraperitoneally or subcutaneously at 1 mg / kg. **P<0.01; ns: not significant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Detailed Description of the Invention I. Introduction Described herein is an antibody that agonizes the classical Wnt pathway but does not act as a surrogate for known Wnt ligands.These novel antibodies can activate classical Wnt signaling in the absence of endogenous Wnt ligands, and activation is further amplified by R-spondin.In addition, the agonist activity of these antibodies is not blocked by endogenous inhibitors such as DKK1 and sclerostin.These novel agonist antibodies can be used to promote tissue regeneration.For example, agonist antibodies can be used to activate classical Wnt / β-catenin signaling to promote cell differentiation or tissue regeneration in vitro or ex vivo, and to treat tissue loss (e.g., bone, intestine, liver, brain tissue) and other degenerative conditions caused by disease or aging.

[0025] II. Definition As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "an antibody" optionally includes a combination of two or more such molecules, and the like.

[0026] "Wnt agonist" refers to an agent that increases the canonical Wnt / β-catenin signaling pathway, thereby promoting, for example, tissue regeneration and cell differentiation. See, for example, (Clevers et al., Science 346, 54-+ (2014); Lien & Fuchs, Genes & Development 28, 1517-1532 (2014); Steinhart & Angers, Development 145 (2018)).

[0027] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms encompass both naturally occurring and non-naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the naturally occurring corresponding amino acid.

[0028] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., an α carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to naturally occurring amino acids.

[0029] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by their commonly accepted one-letter symbols.

[0030] The term "recombinant," for example, when used with reference to a cell, or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein, or the alteration of a naturally occurring nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes that are not found in the native (non-recombinant) form of the cell, or expresses naturally occurring genes that are otherwise aberrantly expressed, under-expressed, or not expressed at all.

[0031] An antibody, as described herein, can consist of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as a myriad of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. In some embodiments, the antibody is an IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, or IgE.

[0032] A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains respectively.

[0033] The term "antibody" as used herein includes antibody fragments that retain binding specificity. For example, there are several antibody fragments that have been well characterized. Thus, for example, pepsin digests antibodies C-terminal to the disulfide linkage in the hinge region to produce F(ab)', a dimer of Fab, a light chain itself joined by a disulfide bond to VH-CH1. 2 Generate F(ab)' 2 is reduced under mild conditions to cleave the disulfide linkage in the hinge region, thereby forming (Fab') 2 The dimer can be converted to a Fab' monomer, which is essentially a Fab with a portion of the hinge region (for a more detailed description of other antibody fragments, see Fundamental Immunology, WE Paul, ed., Raven Press, NY (1993)). Although various antibody fragments have been defined with respect to the digestion of an intact antibody, one skilled in the art will appreciate that fragments can be synthesized de novo, either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, includes both antibody fragments produced by the modification of whole antibodies or antibody fragments synthesized using recombinant DNA methodology.

[0034] In antibodies, substitution variants have at least one amino acid residue removed and a different residue inserted in its place.The most interesting sites for substitution mutations include hypervariable regions, but framework modifications are also contemplated.Examples of conservative substitutions are described above.

[0035] Substantial modification of the biological properties of antibodies is achieved by selecting substitutions that differ significantly in their effect on (a) the structure of the polypeptide backbone in the area of ​​the substitution, e.g., β-sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining side chain bulk. Naturally occurring residues are divided into groups based on the following common side chain properties: (1) Nonpolar: Norleucine, Met, Ala, Val, Leu, Ile; (2) polar, uncharged: Cys, Ser, Thr, Asn, Gln; (3) Acidic (negatively charged): Asp, Glu; (4) Basic (positively charged): Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe, His. Non-conservative substitutions are made by exchanging a member of one of these classes for another class.

[0036] One type of substitution that can be made is to change one or more cysteines in an antibody that may be chemically reactive to another residue, such as, but not limited to, alanine or serine. For example, there may be a non-classical cysteine ​​substitution. The substitution may be made in the complementarity determining region (CDR) or framework region of the variable domain, or in the constant region of the antibody. In some embodiments, the cysteine ​​is a classical one (e.g., one that participates in disulfide bond formation). Any cysteine ​​residue that is not involved in maintaining the proper conformation of the antibody may also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine ​​bond(s) may be added to the antibody to improve its stability, especially when the antibody is an antibody fragment, such as an Fv fragment.

[0037] Antibodies include single-chain antibodies (antibodies that exist as a single polypeptide chain), such as single-chain Fv antibodies (sFv or scFv), in which the variable heavy and variable light regions are linked together (directly or via a peptide linker) to form a contiguous polypeptide. H -V L Single chain Fv antibodies include V dimers, either directly linked or linked via a peptide-encoded linker. H Coding sequence and V L A covalently linked V that can be expressed from a nucleic acid containing a coding sequenceH -V L (e.g., Huston, et al. Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). H and V L are linked together as a single polypeptide chain, while V H and V L The domains are non-covalently linked. Alternatively, the antibody can be another fragment. Other fragments can also be generated, for example, using recombinant techniques, as soluble proteins or as fragments obtained from display methods. The antibody can also include diantibodies or mini-antibodies. Wnt agonist antibodies for promoting tissue regeneration and treating tissue loss (e.g., bone, intestine, liver, or brain tissue loss) also include heavy chain dimers, such as antibodies from camelids. In some embodiments, the antibody is a dimer. In other embodiments, the antibody can be a monomeric form with active isotype. In some embodiments, the antibody is a multivalent form, for example, a trivalent or tetravalent form.

[0038] As used herein, the terms "variable region" and "variable domain" refer to portions of the light and heavy chains of an antibody that contain the amino acid sequences of the complementarity determining regions (CDRs, e.g., HCDR1, HCDR2, HCR3, LCDR1, LCDR2, and LCDR3) and framework regions (FRs). The variable regions of the heavy and light chains are generally represented by the V H and V L The variable regions are referred to as Fab, F(ab') and F(ab') as described herein. 2 , Fv, and scFv antibody fragments and are involved in specific antigen recognition.

[0039] As used herein, "complementarity determining region" or "CDR" refers to the three hypervariable regions in each chain that separate the four framework regions established by the light chain variable region and the heavy chain variable region. The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, which are numbered consecutively starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, V H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas V L The CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found.

[0040] The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.

[0041] Unless otherwise specified, CDR1, CDR2, and CDR3 of the heavy chain variable regions and CDR1, CDR2, and CDR3 of the light chain variable regions discussed herein are determined by the North method (see, e.g., North et al., J. Mol. Biol. 406(2):228-256, 2011). In some embodiments, the antibody comprises the CDR1, CDR2, and CDR3 of the heavy and light chain variable regions of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, as determined by the North method. The North method was developed using a data set of antibody structures that was 15 times larger than the set used in developing the Chothia numbering scheme. In defining the CDR boundary positions, the North method selected positions that 1) are nearly free of structural variability between antibodies (the anchors of each CDR loop (residues immediately preceding or following the loop) contain tightly clustered conformations relative to the framework) and 2) are opposite each other in the β-sheet framework (i.e., have equal lengths of spanning into the framework). In North, the CDRs were defined such that they were more or less symmetric between the VH and VL domains.

[0042] In other embodiments, the CDRs of an antibody can be determined using a variety of other definitions well known in the art, such as Kabat, Chothia, the international ImMunoGeneTics database (IMGT), and AbM (see, e.g., Johnson et al., supra; Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992, Structural repertoire of the human VH segments J. Mol. Biol. 227, 799-817; Al-Lazikani et al., J. Mol. Biol 1997, 273(4)).Definitions of antigen-binding sites are also found in: Ruiz et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219-221 (2000); and Lefranc, M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan 1;29(1):207-9 (2001); MacCallum et al, Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); and Martin et al, Proc. Natl Acad. Sci. USA, 86, 9268-9272 (1989); Martin et al., Methods Enzymol., 203, 121-153, (1991); Pedersen et al. al., Immunomethods, 1, 126, (1992); and Rees et al., In Sternberg MJE (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172 1996).

[0043] As used herein, a "chimeric antibody" refers to an immunoglobulin molecule in which (a) the constant region or a portion thereof has been modified, replaced or exchanged, such that the antigen binding site (variable region) is linked to a constant region of a different or modified class, effector function and / or species, or to an entirely different molecule that confers new properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc., or (b) the variable region or a portion thereof has been modified, replaced or exchanged with a variable region or portion thereof having a different or modified antigen specificity, or the corresponding sequence from another species or from another antibody class or subclass.

[0044] As used herein, "humanized antibody" refers to an immunoglobulin molecule in which CDRs from a donor antibody are grafted onto human framework sequences. Humanized antibodies may also contain residues of donor origin in the framework sequences. Humanized antibodies may also contain at least a portion of a human immunoglobulin constant region. Humanized antibodies may also contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Humanization can be performed by methods known in the art (e.g., Jones et al., Nature 321:522-525; 1986; Riechmann et al., Nature 332:323-327, 1988: Verhoeyen et al., Science 239:1534-1536, 1988); Presta, Curr. Op. Struct. Biol. 2:593-596, 1997), including techniques such as "superhumanized" antibodies (Tan et al., J. Immunol. 169: 1119, 2002) and "resurfacing" (e.g., Staelens et al., Mol. Immunol. 43: 1243, 2006; and Roguska et al., Proc. Natl. Acad. Sci USA 91: 969, 1994). 1992; U.S. Patent No. 4,816,567).

[0045] The terms "antigen", "immunogen", "antibody target", "target analyte", and similar terms are used herein to refer to a molecule, compound, or complex that can be recognized by an antibody, i.e., specifically bound by an antibody. The terms can refer to any molecule that can be specifically recognized by an antibody, such as a polypeptide, a polynucleotide, a carbohydrate, a lipid, a chemical moiety, or a combination thereof (e.g., a phosphorylated or glycosylated polypeptide, etc.). Those skilled in the art will understand that the terms do not indicate that a molecule is immunogenic in all contexts, but simply that it can be targeted by an antibody.

[0046] An antibody binds to an "epitope" on an antigen. An epitope is a localized site on an antigen that is recognized and bound by an antibody. An epitope may comprise a small number of amino acids or a portion of a small number of amino acids, e.g., 5 or 6 or more, e.g., 20 or more amino acids, or a portion of those amino acids. In some cases, an epitope may comprise a non-protein component, e.g., derived from a carbohydrate, nucleic acid, or lipid. In some cases, an epitope is a three-dimensional portion. Thus, for example, if the target is a protein, an epitope may be composed of contiguous amino acids, or may be composed of amino acids from different parts of the protein that are brought together in close proximity by protein folding (e.g., a non-contiguous epitope). The same applies to other types of target molecules that form three-dimensional structures. An epitope typically comprises at least three, more usually at least five, or 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).

[0047] A "label" or a "detectable moiety" is a diagnostic agent or component detectable by spectroscopic, radiological, photochemical, biochemical, immunochemical, chemical, or other physical means. Exemplary labels include radiolabels (e.g., 111 In, 99m Tc, 131 I, 67 Ga) and other FDA approved imaging agents. Additional labels include: 32 The targeting agent may be a targeting agent that is capable of detecting a targeting agent, such as a nucleic acid, a fluorescent dye, an electron-dense reagent, an enzyme, biotin, digoxigenin, or a hapten, and may be a protein or other entity that can be made detectable by incorporating, for example, a radiolabel into the targeting agent. Any method known in the art for conjugating a nucleic acid or nanocarrier to a label may be used, for example, using the methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.

[0048] A "labeled" or "tagged" antibody or agent is one that is attached to a label, either covalently via a linker or chemical bond, or non-covalently via ionic, van der Waals, electrostatic, or hydrogen bonds, such that the presence of the antibody or agent can be detected by detecting the presence of the label bound to the antibody or agent.

[0049] Techniques for conjugating detectable and therapeutic agents to antibodies are well known (see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery" in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review" in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); and Thorpe et al. al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates," Immunol. Rev., 62:119-58 (1982).

[0050] The terms "specific for", "specifically binds" and similar terms refer to a molecule (e.g., an antibody or antibody fragment) that binds to a target with at least 2-fold higher affinity than a non-target compound, e.g., at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or more affinity for a target compared to an unrelated target when assayed under the same binding affinity assay conditions. For example, an antibody that specifically binds to a target (e.g., human or mouse LRP6) typically binds to the target with at least 2-fold higher affinity than a non-target. Specificity can be determined using standard methods, such as solid-phase ELISA immunoassays (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). In certain aspects, the terms "specific binding," "specifically binds to" a particular target, or "is specific for" a particular target, as used herein, refer to, for example, when a molecule (e.g., an antibody) binds to a target, e.g., at least 10 -2 M Less than, e.g., 10 -3 M , 10 -4 M , 10 -5 M , 10 -6 M , 10 -7 M , 10 -8 M , 10 -9 M , 10 -10 M , 10 -11 M , or 10 -12 M The equilibrium dissociation constant K D In some embodiments, the antibody has a K of less than 100 nM or less than 10 nM. D has.

[0051] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and their polymers in single-stranded or double-stranded form, as well as their complementary strands.The term encompasses synthetic, naturally occurring, and non-naturally occurring nucleic acids that contain known nucleotide analogs or modified backbone residues or linkages, nucleic acids that have similar binding properties as reference nucleic acids, and nucleic acids that are metabolized in a similar manner as reference nucleotides.Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs).

[0052] Unless otherwise specified, a particular nucleic acid sequence implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences in addition to the sequence explicitly indicated.Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0053] The term "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences being the same or having the same amino acid residues or nucleotides in a particular percentage (i.e., about 60% identity over a particular region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity when compared and aligned to maximize correspondence over a comparison window or designated region) as measured using the BLAST 2.0 sequence comparison algorithm with default parameters described below, or by manual alignment and visual inspection (see, e.g., the NCBI website ncbi.nlm.nih.gov / BLAST / or similar). Such sequences are then said to be "substantially identical". As described below, the preferred algorithm can take into account gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 or more amino acids or nucleotides in length.

[0054] For sequence comparison, typically, one sequence serves as the reference sequence to which test sequence is compared.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, partial sequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Preferably, default program parameters can be used, or alternative parameters can be designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence to reference sequence based on program parameters.

[0055] A "comparison window," as used herein, includes reference to any one segment of the number of contiguous positions selected from the group consisting of about 20 to 600, usually about 50 to about 200, more usually about 100 to about 150, over which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods for aligning sequences for comparison are well known in the art.

[0056] Suitable algorithms for determining sequence identity and sequence similarity percentage are BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. To determine the sequence identity percentage of the nucleic acid and protein of the present disclosure, BLAST and BLAST 2.0 are used with the parameters described herein. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm includes first identifying high-scoring sequence pairs (HSPs) by identifying short strings of length W in the query sequence, which either match when aligned with strings of the same length in the database sequence, or meet some positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence as far as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameter M (reward score for a pair of matching residues; always >0) and the parameter N (penalty score for mismatching residues; always <0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. The extension of the word hits in each direction is stopped when: the cumulative alignment score falls by an amount X from its maximum achieved value; the cumulative score falls below 0 due to the accumulation of one or more negatively scored residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)), an alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0057] A "control" sample or value refers to a sample that serves as a reference, usually a known reference, for comparison with a test sample. For example, a test sample may be obtained from a test condition, e.g., in the presence of a test compound, and compared to a sample obtained from a known condition, e.g., in the absence of a test compound (negative control) or in the presence of a known compound (positive control). A control may also represent a mean value or range collected from multiple tests or results. A person skilled in the art will recognize that controls can be designed for the evaluation of any number of parameters. For example, a control can be devised to compare therapeutic benefits based on pharmacological data (e.g., half-life) or treatment measures (e.g., comparison of benefits and / or side effects). A control can be designed for in vitro applications. A person skilled in the art will know which controls are useful in a given situation and can analyze data based on comparison with a control value. Controls are also useful in determining the significance of data. For example, if the value of a given parameter varies significantly in the control, the variation in the test sample is not considered significant.

[0058] The term "therapeutically effective dose", "effective dose" or "therapeutically effective amount" herein refers to a dose that produces the effect for which it is administered. The exact dose and formulation depend on the purpose of treatment and can be ascertained by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro, Editor (2003), and Pickar, Dosage Calculations (1999)). For example, for a given parameter, a therapeutically effective amount shows an increase or decrease in therapeutic effect of at least any of 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, or at least 100%. Therapeutic efficacy may also be expressed as a "fold" of increase or decrease. For example, a therapeutically effective amount may have an effect that is at least 1.2-fold, at least 1.5-fold, at least 2-fold, at least 5-fold, or greater than a control.

[0059] As used herein, the term "pharmaceutical acceptable carrier" refers to a pharmaceutical excipient or diluent in a pharmaceutical composition. A pharmaceutical acceptable carrier must be compatible with other ingredients of the formulation and be harmless to the recipient. In some embodiments, a pharmaceutical acceptable carrier must provide the active ingredient with suitable pharmaceutical stability. The nature of the carrier varies depending on the manner of administration. For example, for intravenous administration, an aqueous solution carrier is generally used; for oral administration, a solid carrier is preferred.

[0060] The terms "agonize", "agonizing" or the like, when used in the context of agonizing the canonical Wnt / β-catenin signaling pathway, refer to any detectable positive change or increase in the amount of a parameter reflecting Wnt signaling, when compared to a standard value obtained under the same conditions but in the absence of an antibody described herein (e.g., a Wnt agonist antibody). The level of this increase after exposure to an antibody described herein is, in some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%.

[0061] The term "compete" as used herein with respect to antibodies means that a first antibody or antigen-binding portion thereof competes for binding with a second antibody or antigen-binding portion thereof, or a ligand or inhibitor, in which case the binding of the first antibody to its relevant epitope is detectably reduced in the presence of the second antibody, ligand, or inhibitor, compared to the binding of the first antibody in the absence of the second antibody, ligand, or inhibitor. Another possibility is that the binding of the second antibody, ligand, or inhibitor to its epitope is also detectably reduced in the presence of the first antibody, but this is not necessarily the case. That is, a first antibody may inhibit the binding of a second antibody, ligand, or inhibitor to its epitope without the second antibody, ligand, or inhibitor inhibiting the binding of the first antibody to its respective epitope. However, if each antibody, ligand, or inhibitor detectably inhibits the binding of the other antibody, ligand, or inhibitor to its associated epitope or ligand, regardless of the degree of inhibition, these antibodies are said to "cross-compete" with each other for the binding of their respective epitope(s). Both competing and cross-competing antibodies are encompassed by the present disclosure. Based on the teachings provided herein, one skilled in the art will understand that, whatever the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or part thereof, and the like), such competing and / or cross-competing antibodies are encompassed and may be useful in the methods disclosed herein.

[0062] Numerous types of competitive binding assays are known, for example: solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see Stahli et al., Methods in Enzymology 9:242-253 (1983)); solid-phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614-3619 (1986)); solid-phase direct labeling assays, solid-phase direct labeling sandwich assays (see Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct labeling RIA using I-125 labels (see Morel et al., Molec. Immunol. 25(1):7-15 (1988)); solid-phase direct biotin-avidin EIA (Cheung et al., Virology 176:546-552 (1990); and direct labeling RIA (Moldenhauer et al., Scand. J. Immunol. 32:77-82 (1990)). Typically, such assays involve the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test immunoglobulin, and a labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Usually, the test immunoglobulin is present in excess. Antibodies identified by competitive assays (competing antibodies) include antibodies that bind to the same epitope as that of the reference antibody and antibodies that bind to adjacent epitopes sufficiently close to the epitope bound by the reference antibody so that steric hindrance occurs. Usually, when a competing antibody is present in excess, it inhibits specific binding of the reference antibody to a common antigen by at least 50 or 75%.

[0063] The terms "treat" and "treatment" are used herein to refer to both therapeutic treatment and prophylactic or preventative measures, the purpose being to prevent or delay undesirable physiological changes or disorders. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, reduced tissue loss, enhanced cell differentiation or tissue regeneration, whether detectable or undetectable, alleviation of symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), slowing or delaying disease progression, amelioration or alleviation of the disease state, and remission (whether partial or complete). "Treatment" can also mean an increase in survival compared to the expected survival in the absence of treatment.

[0064] III. Wnt agonist antibodies Antibodies (including antibody fragments) that agonize the Wnt / β-catenin signaling pathway are provided. Agonist antibodies that specifically bind to LRP6 and can be used to treat or prevent tissue loss are provided. Human LRP6 amino acid sequence can be found at Uniprot Accession No. O75581. Mouse LRP6 amino acid sequence can be found at Uniprot Accession No. O88572.

[0065] In some embodiments, the monoclonal antibody or antigen-binding portion thereof specifically binds to an epitope on LRP6 that does not overlap with binding sites for known Wnt ligands or Wnt inhibitors. The Wnt ligand can be, for example, Wnt1, Wnt2, Wnt2b (Wnt13), Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a (Wnt14), Wnt9b (Wnt14b), Wnt10a, Wnt10b, Wnt11, or Wnt16. In certain embodiments, the Wnt ligand is involved in the canonical signaling pathway (e.g., Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt8a, Wnt8b, Wnt10a, Wnt10b, Wnts2b, and Wnt9b). The Wnt inhibitor can be, for example, Dickkopf Wnt signaling pathway inhibitor 1 (DKK1), Dickkopf Wnt signaling pathway inhibitor 2 (DKK2), Dickkopf Wnt signaling pathway inhibitor 3 (DKK3), Dickkopf Wnt signaling pathway inhibitor 4 (DKK4), Dickkopf-like acrosomal protein 1 (DKKL1), sclerostin (SOST), Wise (SOSTDC1 (sclerostin domain containing 1)), IGFBP-4, or Waif1 / 5T4. In some embodiments, the monoclonal antibody or antigen binding portion binds to a non-linear epitope. In some embodiments, the monoclonal antibody or antigen binding portion binds to the P3 domain of LRP6. In some embodiments, the epitope comprises K662 and K684. In some embodiments, the epitope does not include E663, E708, H834, Y875, or M877.

[0066] In some embodiments, the Wnt agonist antibodies of the disclosure comprise the sequences of heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2, LCDR3, heavy chain variable region (VH), and / or light chain variable region (VL) set forth in Tables 1 and 2. The CDRs set forth in Tables 1 and 2 were determined by the North method (see, e.g., North et al., J. Mol. Biol. 406(2):228-256, 2011).

[0067] [Table 1] TIFF2024533616000003.tif149149

[0068] [Table 2]

[0069] In some embodiments, the antibodies described herein comprise a variable region that specifically binds LRP6, wherein the heavy chain variable region comprises the following: TIFF2024533616000005.tif39149, comprising the CDRs of the entire heavy chain variable sequence or the entire heavy chain variable sequence as follows: In some embodiments, the antibody is a 66-11 antibody, and in some embodiments, the antibody is a 66-11 antibody.

[0070] In some embodiments, the antibodies described herein comprise a variable region that specifically binds LRP6, wherein the heavy chain variable region comprises the following: TIFF2024533616000007.tif39149, comprising the CDRs of the entire heavy chain variable sequence or the entire heavy chain variable sequence as follows: It is combined with a light chain variable region including the CDRs or the entire light chain variable sequence shown in TIFF2024533616000008.tif34149.

[0071] In some embodiments, the antibodies described herein comprise a variable region that specifically binds LRP6, wherein the heavy chain variable region comprises the following: TIFF2024533616000009.tif39149, comprising the CDRs of the entire heavy chain variable sequence or the entire heavy chain variable sequence as set forth below: It is combined with a light chain variable region including the CDRs of the entire light chain variable sequence shown in TIFF2024533616000010.tif34149 or the entire light chain variable sequence.

[0072] In some embodiments, the antibodies described herein comprise a variable region that specifically binds LRP6, wherein the heavy chain variable region comprises the following: TIFF2024533616000011.tif39149, comprising the CDRs of the entire heavy chain variable sequence or the entire heavy chain variable sequence as set forth below: It is combined with a light chain variable region including the CDRs of the entire light chain variable sequence shown in TIFF2024533616000012.tif34149 or the entire light chain variable sequence.

[0073] In some embodiments, the antibodies described herein comprise a variable region that specifically binds LRP6, wherein the heavy chain variable region comprises the following: TIFF2024533616000013.tif39149, comprising the CDRs of the entire heavy chain variable sequence or the entire heavy chain variable sequence as set forth below: It is combined with a light chain variable region including the CDRs of the entire light chain variable sequence shown in TIFF2024533616000014.tif34149 or the entire light chain variable sequence.

[0074] In some embodiments, the antibodies described herein comprise a variable region that specifically binds LRP6, wherein the heavy chain variable region comprises the following: TIFF2024533616000015.tif39149, comprising the CDRs of the entire heavy chain variable sequence or the entire heavy chain variable sequence as follows: It is combined with a light chain variable region including the CDRs or the entire light chain variable sequence shown in TIFF2024533616000016.tif34149.

[0075] In some embodiments, the antibodies described herein comprise a variable region that specifically binds LRP6, wherein the heavy chain variable region comprises the following: TIFF2024533616000017.tif39149, comprising the CDRs of the entire heavy chain variable sequence or the entire heavy chain variable sequence as follows: It is combined with a light chain variable region including the CDRs of the entire light chain variable sequence shown in TIFF2024533616000018.tif34149 or the entire light chain variable sequence.

[0076] In some embodiments, the antibodies described herein comprise a variable region that specifically binds LRP6, wherein the heavy chain variable region comprises the following: TIFF2024533616000019.tif39149, comprising the CDRs of the entire heavy chain variable sequence or the entire heavy chain variable sequence as follows: It is combined with a light chain variable region including the CDRs or the entire light chain variable sequence shown in TIFF2024533616000020.tif34149.

[0077] In some embodiments, the antibodies described herein comprise a variable region that specifically binds LRP6, wherein the heavy chain variable region comprises the following: TIFF2024533616000021.tif39149, comprising the CDRs of the entire heavy chain variable sequence or the entire heavy chain variable sequence as set forth below: It is combined with a light chain variable region including the CDRs of the entire light chain variable sequence shown in TIFF2024533616000022.tif34149 or the entire light chain variable sequence.

[0078] In some embodiments, the antibodies described herein comprise a variable region that specifically binds LRP6, wherein the heavy chain variable region comprises the following: TIFF2024533616000023.tif39149, comprising the CDRs of the entire heavy chain variable sequence or the entire heavy chain variable sequence as follows: It is combined with a light chain variable region including the CDRs of the entire light chain variable sequence shown in TIFF2024533616000024.tif34149 or the entire light chain variable sequence.

[0079] In some embodiments, the antibodies described herein comprise a variable region that specifically binds LRP6, wherein the heavy chain variable region comprises the following: TIFF2024533616000025.tif39149, comprising the CDRs of the entire heavy chain variable sequence or the entire heavy chain variable sequence as set forth below: It is combined with a light chain variable region including the CDRs of the entire light chain variable sequence shown in TIFF2024533616000026.tif34149 or the entire light chain variable sequence.

[0080] In some embodiments, the antibodies described herein comprise a variable region that specifically binds LRP6, wherein the heavy chain variable region comprises the following: TIFF2024533616000027.tif39149, comprising the CDRs of the entire heavy chain variable sequence or the entire heavy chain variable sequence as follows: It is combined with a light chain variable region including the CDRs or the entire light chain variable sequence shown in TIFF2024533616000028.tif34149.

[0081] In some embodiments, the antibodies described herein comprise a variable region that specifically binds LRP6, wherein the heavy chain variable region comprises the following: TIFF2024533616000029.tif39149, comprising the CDRs of the entire heavy chain variable sequence or the entire heavy chain variable sequence as set forth below: It is combined with a light chain variable region including the CDRs of the entire light chain variable sequence shown in TIFF2024533616000030.tif34149 or the entire light chain variable sequence.

[0082] In some embodiments, the antibodies described herein comprise a variable region that specifically binds LRP6, wherein the heavy chain variable region comprises the following: TIFF2024533616000031.tif39149, comprising the CDRs of the entire heavy chain variable sequence or the entire heavy chain variable sequence as set forth below: It is combined with a light chain variable region including the CDRs of the entire light chain variable sequence shown in TIFF2024533616000032.tif34149 or the entire light chain variable sequence.

[0083] In some embodiments, the antibodies described herein comprise a variable region that specifically binds LRP6, wherein the heavy chain variable region comprises the following: TIFF2024533616000033.tif39149, comprising the CDRs of the entire heavy chain variable sequence or the entire heavy chain variable sequence as set forth below: It is combined with a light chain variable region including the CDRs of the entire light chain variable sequence shown in TIFF2024533616000034.tif34149 or the entire light chain variable sequence.

[0084] In some embodiments, the antibodies described herein comprise a variable region that specifically binds LRP6, wherein the heavy chain variable region comprises the following: TIFF2024533616000035.tif39149, comprising the CDRs of the entire heavy chain variable sequence or the entire heavy chain variable sequence as set forth below: It is combined with a light chain variable region including the CDRs of the entire light chain variable sequence shown in TIFF2024533616000036.tif34149 or the entire light chain variable sequence.

[0085] In some embodiments, the antibodies described herein comprise a variable region that specifically binds LRP6, wherein the heavy chain variable region comprises the following: TIFF2024533616000037.tif39149, comprising the CDRs of the entire heavy chain variable sequence or the entire heavy chain variable sequence as set forth below: It is combined with a light chain variable region including the CDRs of the entire light chain variable sequence shown in TIFF2024533616000038.tif34149 or the entire light chain variable sequence.

[0086] In some embodiments, the antibodies described herein comprise a variable region that specifically binds LRP6, wherein the heavy chain variable region comprises the following: TIFF2024533616000039.tif39149, comprising the CDRs of the entire heavy chain variable sequence or the entire heavy chain variable sequence as set forth below: It is combined with a light chain variable region including the CDRs of the entire light chain variable sequence shown in TIFF2024533616000040.tif34149 or the entire light chain variable sequence.

[0087] In some embodiments, the antibodies described herein comprise a variable region that specifically binds LRP6, wherein the heavy chain variable region comprises the following: TIFF2024533616000041.tif39149, comprising the CDRs of the entire heavy chain variable sequence or the entire heavy chain variable sequence as follows: It is combined with a light chain variable region including the CDRs of the entire light chain variable sequence shown in TIFF2024533616000042.tif34149 or the entire light chain variable sequence.

[0088] In some embodiments, the antibody comprises a heavy chain variable region having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of any one of SEQ ID NOs:3-7. In some embodiments, the antibody comprises a light chain variable region having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of SEQ ID NO:15.

[0089] In some embodiments, the antibody comprises a heavy chain variable region having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of any one of SEQ ID NOs:8-13. In some embodiments, the antibody comprises a light chain variable region having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of SEQ ID NO:16 or SEQ ID NO:17.

[0090] Any of the Wnt agonist antibodies described herein may contain one or more human framework regions (e.g., one, two, three, or four FRs). In some embodiments, the one or more human framework regions contain at least one back mutation.

[0091] In further embodiments, the Wnt agonist antibody described herein can cross-react with mouse LRP6.In certain embodiments, the Wnt agonist antibody agonizes the classical Wnt / β-catenin signaling pathway.In addition, the Wnt agonist antibody does not compete with Wnt ligand or Wnt inhibitor for LRP6 binding.In addition, the Wnt agonist antibody can activate the classical Wnt pathway in the presence of Wnt inhibitor and / or in the absence of Wnt ligand, and can amplify the signaling Wnt / β-catenin signaling in the presence of RSPO2.

[0092] In some embodiments, modifications can be optionally introduced into the antibody (e.g., either within the polypeptide chain or at the N-terminus or C-terminus), such as PEGylation or incorporation of long chain polyethylene glycol polymers (PEG), for example, to increase in vivo half-life. The introduction of PEG or long chain polymers of PEG increases the effective molecular weight of the polypeptide, for example, to prevent rapid filtration into urine. In some embodiments, lysine residues in the sequence are conjugated to PEG, either directly or via a linker. Such linkers can be, for example, Glu residues or acyl residues that contain a thiol functional group for linking to an appropriately modified PEG chain. An alternative method for introducing a PEG chain is to first introduce a Cys residue at the C-terminus or at a solvent-exposed residue, such as a substitution for an Arg or Lys residue. This Cys residue is then site-specifically added to a PEG chain that contains, for example, a maleimide functional group. Methods for introducing PEG or long chain polymers of PEG are known in the art (e.g., as described in Veronese, FM, et al., Drug Disc. Today 10: 1451-8 (2005); Greenwald, RB, et al., Adv. Drug Deliv. Rev. 55: 217-50 (2003); Roberts, MJ, et al., Adv. Drug Deliv. Rev., 54: 459-76 (2002)), the contents of which are incorporated herein by reference.

[0093] In certain embodiments, specific mutations of the antibody may be made to alter the glycosylation of the polypeptide. Such mutations may be selected to introduce or eliminate one or more glycosylation sites, including, but not limited to, O-linked or N-linked glycosylation sites. In certain embodiments, the protein has unaltered glycosylation sites and patterns compared to the naturally occurring protein. In certain embodiments, protein variants include glycosylation variants, in which the number and / or type of glycosylation sites are altered compared to the naturally occurring protein. In certain embodiments, polypeptide variants include more or fewer N-linked glycosylation sites compared to the naturally occurring polypeptide. N-linked glycosylation sites are characterized by the sequence: Asn-X-Ser or Asn-X-Thr, in which the amino acid residue designated X can be any amino acid residue. Substitution of amino acid residues to create this sequence provides a new potential site for the addition of an N-linked glycan. Alternatively, substitution to eliminate this sequence removes existing N-linked glycans. In certain embodiments, a rearrangement of the N-linked glycan is performed to eliminate one or more N-linked glycosylation sites (typically those that are naturally occurring) and to create one or more new N-linked sites.

[0094] Monoclonal antibodies, as well as chimeric and especially humanized antibodies, are particularly useful for human therapeutic use of the antibodies described herein. Monoclonal antibodies can be obtained by a variety of techniques well known to those skilled in the art. Briefly, spleen cells from an animal immunized with a desired antigen are immortalized, typically by fusion with a myeloma cell (see, e.g., Kohler & Milstein, Eur. J. Immunol. 6: 511-519 (1976)). Alternative methods of immortalization include transformation with Epstein-Barr virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from a single immortalized cell are screened for production of antibodies of the desired specificity and affinity to the antigen, and the yield of monoclonal antibodies produced by such cells can be enhanced by a variety of techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, DNA sequences encoding monoclonal antibodies or binding fragments thereof can be isolated by screening a DNA library derived from human B cells following the general protocol outlined by Huse et al., Science 246: 1275-1281 (1989).

[0095] Further, monoclonal antibodies can be collected and titrated against LRP6 polypeptides in an immunoassay, e.g., a solid-phase immunoassay using a ligand immobilized on a solid support. In some embodiments, the monoclonal antibodies have a K of at least about 0.1 mM, e.g., at least about 1 μM, e.g., at least about 0.1 μM or better, e.g., 0.01 μM or less. d can be bonded with

[0096] The immunoglobulins of the present disclosure, including their binding fragments and other derivatives, can be readily produced by a variety of recombinant DNA techniques, including by expression in transfected cells (e.g., immortalized eukaryotic cells such as myeloma or hybridoma cells) or in mice, rats, rabbits, or other vertebrates capable of producing antibodies by well-known methods. In one aspect, a nucleic acid sequence encoding a Wnt agonist antibody or an antigen-binding portion thereof is provided. The present disclosure also describes vectors and mammalian host cells comprising the nucleic acid sequence. In some embodiments, the mammalian host cells are CHO, CHO-K1, CHO-S, ExpiCHO, CHO-DG44, CHO-Pro minus, HEK293A, HEK293F cells. In some embodiments, the present disclosure provides a method for producing a monoclonal antibody or an antigen-binding portion thereof, comprising culturing the host cells under conditions to permit production of the monoclonal antibody or an antigen-binding portion thereof. Suitable source cells for DNA sequences, as well as host cells for immunoglobulin expression and secretion, can be obtained from several sources, such as the American Type Culture Collection (Catalogue of Cell Lines and Hybridomas, Fifth edition (1985) Rockville, Md.).

[0097] In some embodiments, the antibody is a Fab, F(ab') 2The antibody fragment is an antibody fragment such as a Fv, Fv, or scFv. Antibody fragments can be produced using any means known in the art, including chemical digestion (e.g., papain or pepsin) and recombinant methods. Methods for isolating and preparing recombinant nucleic acids are known to those skilled in the art (see Sambrook et al., Molecular Cloning. A Laboratory Manual (2d ed. 1989); Ausubel et al., Current Protocols in Molecular Biology (1995)). Antibodies can be expressed in a variety of host cells, including E. coli, other bacterial hosts, yeast, and a variety of higher eukaryotic cells, such as COS, CHO, and HeLa cell lines and myeloma cell lines.

[0098] Competitive binding assays can be used to identify antibodies that compete with the antibodies described herein for specific binding to LRP6. Competition between two antibodies for the same antigen can be measured using any of several competitive binding assays known in the art. Briefly, different antibodies can be tested for their ability to inhibit the binding of another antibody. For example, antibodies can be differentiated by the epitopes they bind using a sandwich ELISA assay. This can be done by using a capture antibody to coat the surface of a well. A subsaturating concentration of tagged antigen can then be added to the capture surface. This protein can bind to the antibody through a specific antibody:epitope interaction. After washing, a second antibody that is covalently linked to a detectable moiety (e.g., HRP, the labeled antibody is defined as the detection antibody) can be added to the ELISA. If this antibody recognizes the same epitope as the capture antibody, it will not be able to bind to the target protein, since that particular epitope will no longer be available for binding. On the other hand, if this second antibody recognizes a different epitope on the target protein, it will be able to bind, and this binding can be detected by quantifying the level of activity (and thus bound antibody) using a related substrate. The background can be defined using a single antibody as both capture and detection antibody, while the maximum signal can be established by capturing with an antigen-specific antibody and detecting with an antibody against a tag on the antigen. The antibodies can be evaluated in a pair-wise manner by using the background and maximum signals as references to determine epitope specificity. In some embodiments, a first antibody is considered to competitively inhibit the binding of a second antibody if the binding of the second antibody to the antigen is reduced in the presence of the first antibody by at least 30%, usually at least about 40%, 50%, 60%, or 75%, and often at least about 90% using any of the above assays.

[0099] The antibody described herein may comprise an Fc polypeptide. The Fc polypeptide may be a wild-type Fc polypeptide, for example, a human IgG1 Fc polypeptide. In some embodiments, the Fc polypeptide in the antibody described herein may comprise an amino acid substitution that modulates effector function.

[0100] IV. METHODS FOR PROMOTING TISSUE REGENERATION The antibody (including antibody fragment) described herein agonizes the Wnt / β-catenin signaling pathway and promotes tissue regeneration.Various conditions and diseases can cause degeneration or loss of tissue, such as bone tissue, intestinal tissue, liver tissue, or brain tissue.The Wnt agonist antibody described herein can be used to regenerate such tissue by agonizing the Wnt / β-catenin signaling pathway in the cells of the tissue.

[0101] In some embodiments, a Wnt agonist antibody may be used to promote cell differentiation or tissue regeneration in vitro by adding the antibody, or an antigen-binding portion thereof, to cells in vitro.

[0102] In other embodiments, the antibody or antigen-binding portion thereof is administered ex vivo. In such embodiments, a portion of a cell or tissue is removed from the tissue of an individual in need of regeneration, and the antibody or antigen-binding portion thereof is administered to the cell or tissue ex vivo. The treated cell or tissue is then returned to the individual.

[0103] In other embodiments, Wnt agonist antibody can be used to restore tissue in individuals who need it.In certain embodiments, the individual can have a disease or condition that involves the degeneration or loss of tissue, such as bone tissue, intestinal tissue, liver tissue, or brain tissue.In some embodiments, the individual has a disease or condition in which insufficient Wnt signaling contributes to the disease or condition and / or its progression. In some embodiments, the individual has age-induced osteoporosis, drug-induced bone loss, osteogenesis imperfecta, microgravity-induced bone loss, inflammatory bowel disease, Crohn's disease, ulcerative colitis, celiac disease, rheumatoid arthritis, diabetes, chronic kidney disease, juvenile arthritis, dementia, Alzheimer's disease, stroke, cirrhosis, hepatitis, chronic alcoholism, severe alcoholic hepatitis, diabetic retinopathy, wet age-related macular degeneration (AMD), Fuchs' dystrophy, corneal epithelial stem cell deficiency, atrophic AMD, Sjogren's dry eye, short bowel syndrome, hearing loss, and / or an autoimmune disease affecting one or more of bone, intestinal, liver, or brain tissues (e.g., primary biliary cholangitis). In some embodiments, an antibody or antigen-binding portion thereof is administered to the individual. In some embodiments, the pharmaceutical composition comprising the Wnt agonist antibody or antigen-binding portion thereof described herein is administered by intravenous, intraperitoneal, or subcutaneous injection. In other embodiments, any one of a variety of other administration means known in the art may be used.

[0104] In some embodiments, the antibody or antigen-binding portion thereof may be used to treat in combination with other treatments to prevent tissue loss or promote tissue regeneration in an individual in need thereof. For example, in some aspects, the antibody or antigen-binding portion thereof is used in addition to bisphosphonates; calcitonin; hormone therapy; parathyroid hormone (PTH) analogs; parathyroid hormone-related protein (PTHrp) analogs; RANK ligand (RANKL) inhibitors; romosozumab; or combinations thereof to treat patients experiencing bone loss.

[0105] V. Pharmaceutical Compositions The Wnt agonist antibody for promoting tissue regeneration and treating tissue loss (e.g., bone tissue, intestinal tissue, liver tissue, or brain tissue loss) can be provided in a pharmaceutical composition. The pharmaceutical composition can include a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is determined in part by the specific composition being administered and the specific method used to administer the composition. Thus, there are a wide variety of suitable formulations of the pharmaceutical composition of the present disclosure (see, for example, Remington's Pharmaceutical Sciences, 17th ed., 1989).

[0106] The preparation suitable for administration includes aqueous and non-aqueous solutions, isotonic sterile solutions that may contain antioxidants, buffers, bacterial growth inhibitors, and solutes that make the preparation isotonic, and aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.The compound preparations may be in unit-dose or multi-dose sealed containers such as ampoules and vials.Solutions and suspensions may be prepared from the above-mentioned types of sterile powders, granules, and tablets.The regulator may also be administered as part of the food or drug that is prepared.

[0107] In certain embodiments, the pharmaceutical composition may be selected for parenteral delivery. Preparation of such pharma-ceutically acceptable compositions is within the skill of one of ordinary skill in the art. In certain embodiments, the formulation components are present in concentrations that are acceptable to the site of administration. In certain embodiments, a buffer is used to maintain the composition at physiological pH or slightly lower, typically within a pH range of about 5 to about 8.

[0108] In certain embodiments, when parenteral administration is intended, the therapeutic composition can be in the form of a pyrogen-free parenterally acceptable aqueous solution containing Wnt agonist antibody in a pharma- ceutically acceptable vehicle.In certain embodiments, the vehicle for parenteral injection is sterile distilled water, in which Wnt agonist antibody is formulated as a sterile isotonic solution, and is appropriately preserved.In certain embodiments, preparation can include the formulation of the desired molecule with agents such as injectable microspheres, biodegradable particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, which can provide controlled or sustained release of the product that can be delivered later via depot injection.In certain embodiments, hyaluronic acid can also be used, which can have the effect of promoting survival time in circulation.In certain embodiments, implantable drug delivery devices can be used to deliver the desired molecule.

[0109] The dose administered to a patient should be sufficient to induce beneficial responses in the subject over time.The optimal dose level for any patient depends on various factors, including the potency of the antibody used, the age, weight, physical activity, and diet of the patient, as well as possible combinations with other drugs.The dose is also determined by the existence, nature, and extent of any adverse side effects associated with the administration of a particular compound or vector in a particular subject.

[0110] In determining the effective amount of agonist antibody to be administered, the physician may evaluate the circulating plasma level of agonist antibody and the toxicity of agonist antibody. In general, the dose equivalent of agonist antibody is about 1 ng / kg to 10 mg / kg for a typical subject. In some embodiments, the dose range for subcutaneous or iv administration is 0.1 to 20 mg / kg, for example, 0.3 to 10 mg / kg.

[0111] For administration, Wnt agonist antibodies are administered at the EC 50and at a frequency determined by the side effects of the agonist at various concentrations, which is adjusted according to the subject's weight and general health. Administration can be achieved by a single dose or divided doses.

[0112] Compositions for treating or preventing tissue loss can be administered periodically (e.g., weekly) for a period of time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 months, or 1-3 years or more).

[0113] VI. Methods for Identifying Wnt Agonist Antibodies The present disclosure also provides a method for identifying a monoclonal antibody or antigen-binding portion thereof that agonizes Wnt signaling and does not compete with Wnt ligand or Wnt inhibitor.In some embodiments, the method includes the steps of: a) providing an LRP6 polypeptide or a portion thereof that comprises at least the LRP6 polypeptide P3E3P4E4 domain; b) contacting the LRP6 polypeptide or a portion thereof with a library of binding molecules; c) selecting one or more binding molecules from the library that bind to the LRP6 polypeptide or a portion thereof; and d) identifying selected binding molecules that do not compete with Wnt ligand or Wnt inhibitor for binding to the LRP6 polypeptide or a portion thereof.In certain embodiments, the binding molecule is an antibody.In other embodiments, the binding molecule can be an aptamer, a ligand, a peptide, or a small molecule. EXAMPLES

[0114] The following examples are offered to illustrate, but not to limit, the claimed invention.

[0115] Example 1 – Materials and Methods cell line Human embryonic kidney (HEK) 293A and 293, multiple myeloma H929, MM1.S, MM1.R, and RPMI8226 cell lines, mouse L Wnt-3a cell line, mouse preosteoblastic MC3T3-E1 cell line, and mouse mesenchymal C3H / 10T1 / 2 cell line were obtained from the American Type Culture Collection (ATCC). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2 in DMEM, RPMI1640, or α-MEM supplemented with 10% FBS (Fisher Scientific) and 100 μg / ml penicillin / streptomycin (Axenia BioLogix). Conditioned medium (CM) from myeloma cell lines was collected by centrifugation of cell cultures at 70–80% confluency. All cell lines were used within six passages and were not confirmed by short tandem repeat profiling. Cells were tested negative for mycoplasma using a PCR mycoplasma detection kit (abm, Canada) (last tested in September 2020).

[0116] Selection of Wnt agonist antibodies from a phage display library Recombinant LRP6 P3E3P4E4 domain was produced as an Fc fusion protein and purified on a protein A column as previously described (Lee et al., 2018). A naive phage antibody display library was selected against biotin-labeled LRP6-P3E3P4E4 fragment as previously described (Lee et al., 2018). After three rounds of selection, monoclonal phages were arrayed in 96-well plates and tested by flow cytometry for binding to LRP6-transfected HEK293 cells. Unique scFv antibodies were sequenced and identified from LRP6-binding phages, and individual phage clones were amplified and purified for further characterization.

[0117] Plasmids, cloning, and site-directed mutagenesis Full-length human LRP6 was cloned into pCMV-Entry (Origene) and used for subcloning, point mutation, or transfection. LRP6 ectodomain truncated constructs were cloned into pCMV-Entry and used for transient expression. Alanine mutants of the LRP6 ectodomain were constructed using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) according to the manufacturer's protocol. pFUSE-hIgG1-Fc2 (InvivoGen) was used for cloning Fc fusion constructs. Antibody genes were cloned into Abvec Ig-γ and Ig-κ plasmids kindly provided by Dr. Patrick Wilson, University of Chicago, modified from Smith et al., Nature Protocols 4:372-84 (2009) (Lee et al., 2018). For Wnt / β-catenin-responsive reporter assays, the TCF / LEF luciferase reporter SuperTopFlash (STF) and control pRL-SV40 Renilla luciferase constructs (Addgene) were used. Wnt ligands were supplied by transient co-transfection of pcDNA-Wnt1 or -Wnt3a expression plasmids (Addgene).

[0118] Recombinant protein and antibody production To construct Fc fusions, LRP6-P3E3P4E4 or scFv genes were cloned into pFUSE-hIgG1-Fc2 plasmid. To construct IgG, the variable heavy (VH) and variable kappa light (Vκ) chain genes were subcloned into original or modified Abvec as previously described (Lee et al., 2018). For Fab constructs, CH2-CH3 were deleted from Ig-γ Abvec and a 6X His tag was introduced at the C-terminus of CH1. For transient transfections, plasmid DNA was resuspended in Opti-MEM (Life Technologies), mixed with polyethylenimine, and added to HEK293A cells. After 24 h of transfection, the medium was changed to Freestyle 293 expression medium (Gibco) and the cells were cultured for another 6–8 days. Proteins secreted into the supernatant were collected, filtered, and purified on protein A agarose (Thermo Scientific) for Fc fusions and on IgG or Ni-NTA resin (Thermo Scientific) for Fabs according to the manufacturer's protocol.

[0119] SuperTopFlash (STF) luciferase reporter assay Cells cultured in 24- or 96-well plates were transiently transfected with the STF luciferase reporter and pRL-SV40 plasmids with or without Wnt1 or Wnt3a expression constructs using TransIT-2020 (Mirus Bio). To express truncated and alanine mutants of LRP6, plasmid DNA encoding the constructs was co-transfected with the reporter plasmid into HEK293 cells. Antibodies diluted in culture medium were added to the transfected cells along with recombinant DKK1 or RSPO2 (R&D Systems) and further incubated for 16 h. Firefly luciferase (FL) and Renilla luciferase (RL) activities were detected and normalized using the Dual-Luciferase Reporter Assay System (Promega) as previously described (Lee et al., 2018). Data were expressed as fold increase relative to the control group transfected with only the reporter construct.

[0120] Apparent K D Decision Apparent K of antibody D were analyzed by FACS as described (Lee et al., 2018). Briefly, cells were trypsinized, washed, and resuspended in FACS buffer (PBS, 1% FBS). Antibodies were serially diluted in FACS buffer and incubated with target cells (3 x 10 5 The cells were incubated with 100 μg / tube (100 μg / tube) at 4° C. overnight. The cells were washed and incubated with Alexa Fluor® 647-labeled goat anti-human IgG (Jackson ImmunoResearch). After 1 h of incubation, the cells were washed in PBS and analyzed using a BD Accuri C6 flow cytometer (BD Biosciences). Affinity was determined by analyzing median fluorescence intensity (MFI) by curve fitting (GraphPad).

[0121] Biolayer Interferometry Competitive binding activity between anti-LRP6 Fab and recombinant DKK1 or Wnt3a against the LRP6 ectodomain was estimated by biolayer interferometry (BLI) using a BLitz (ForteBio) instrument. A Protein A biosensor (ForteBio) was loaded with human LRP6-ECD-Fc (R&D Systems) for 120 s, and the Protein A biosensor was immersed in recombinant DKK1 or Wnt3a for 75 s, followed by immersion in a mixture of DKK1 + Wnt3a, DKK1 + anti-LRP6 Fab, or Wnt3a + anti-LRP6 Fab for 75 s. Baselines were determined for 30 s before and after the loading step according to the manufacturer's instructions. K D For measurements, a streptavidin biosensor (ForteBio) was loaded with biotinylated LRP6-P3E3P4E4-Fc for 120 seconds, and the streptavidin biosensor was soaked in IgG or Fab for 120 seconds, followed by dissociation in PBS for 120 seconds.

[0122] Antibody-receptor docking analysis Antibody variable fragments (Fv) consisting of anti-LRP6 VH and Vκ sequences were generated by homology modeling using Rossetta antibody (Weitzner et al., Nature Protocols 12:401-16 (2017)). Docking models between Fv and LRP6-P3E3 domains obtained from 3S8Z (Cheng et al., Nature Structural & Mol. Biol. 18:1204-U1244 (2011)) or 4A0P (Chen et al., 2011) were generated using ZDOCK (Pierce et al., Bioinformatics (2014)). Wnt3a-binding or Fv-binding residues were analyzed in the docking models and visualized using PyMOL Molecular Graphics System (Schrodinger, LLC).

[0123] osteogenic differentiation C3H10T1 / 2 cells were induced to differentiate as previously described (Zhong et al., 1481:119-25 (2016)). Briefly, C3H10T1 / 2 cells were cultured in regular growth medium (α-MEM, 10% FBS, 100 μg / ml penicillin / streptomycin) at a confluency of 70–80% in 24-well culture plates. The next day, the culture medium was changed to osteogenic medium (growth medium supplemented with 50 μg / ml ascorbic acid, 10 mM β-glycerol phosphate) and medium was replaced with osteogenic medium every 2–3 days. Cells were either cultured in osteogenic medium alone or treated with a combination of antibodies and / or 30% L-cell Wnt3a conditioned medium (Wnt3aCM) for 21 days. To determine matrix mineralization, cells were stained using Alizarin Red S (ARS) Staining Quantification Assay (ScienCell Research Laboratories) and images were taken with a BIOREVO BZ-9000 microscope (Keyence). ARS dye was extracted from stained cells and quantified according to the manufacturer's instructions.

[0124] Quantitative real-time PCR (qRT-PCR) Osteodifferentiation of C3H / 10T1 / 2 cells was induced for 3 days as described above. Total RNA was isolated from cells using TRIzol™ Reagent (Invitrogen) and used to generate cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's protocol. qRT-PCR was performed on 15 ng of cDNA using Power SYBR Green PCR Master Mix (Applied Biosystems) on an ABI 7300 Real-Time PCR System (Applied Biosystems). All reactions were performed in duplicate and target gene transcript copy numbers were normalized to GAPDH. Data are presented as relative mRNA expression in antibody-treated cells versus control cells. Specific primer sets for each target gene are as follows: TIFF2024533616000043.tif71128.

[0125] Alkaline phosphatase (ALP) activity assay Cells were incubated with antibodies and Wnt3aCM (30%) in osteogenic medium as described above for 7 days, washed, harvested, and lysed by repeated freeze-thaw cycles in NP-40 buffer (150 mM NaCl, 1.0% NP-40, 50 mM Tris, pH 8.0) supplemented with protease inhibitors (Cell Signaling Technology). ALP activity was measured in cell lysates using p-nitrophenyl phosphate (Sigma-Aldrich) according to the manufacturer's instructions. ALP activity was normalized to the control group without antibody and Wnt3a treatment.

[0126] In vivo micro-CT scanning A micro x-ray computed tomography (microCT), a component of the VECTor4 / CT (MILabs BV, Utrecht, The Netherlands) preclinical imaging system, was used for the study. To visualize the femur and its joints, the field of view of the microCT was set around the femur using the built-in optical camera, followed by CT imaging with x-ray tube settings of 50 kVp and 0.24 mA. A total of 1,440 projection images spanning 360 degrees were acquired in step-and-shoot mode with an x-ray exposure time of 75 ms at each step. No data binning was applied during acquisition (i.e., 1 × 1 binning). The animals were kept under anesthesia using isoflurane (approximately 2% isoflurane mixed with medical-grade oxygen) during CT data acquisition. Image reconstruction after projection image acquisition was performed using a vendor-supplied cone-beam filtered backprojection algorithm. The reconstructed image volume had a voxel size of 0.02 mm × 0.02 mm × 0.02 mm. The volume matrix size was due to the field of view selected during the reconstruction process, focusing only on the distal femur. After reconstruction, the image volumes were processed to present a common orientation by reorienting the isotropic volumes using PMOD (PMOD Technologies, Zurich, Switzerland).

[0127] In vivo bone formation test NOD / SCID / IL-2Rγ - / - (NSG) 2 x 10 mice were injected into the right femur of female mice. 5Mice were transplanted with MM1.S cells. One week later, mice were randomized (n=5 / group) and treated intraperitoneally once a week with vehicle (PBS) or 10 mg / kg 66 IgG for a total of six injections. One week after treatment, mice were anesthetized and scanned by micro-CT. One week after CT scanning, blood and femurs were collected from mice and free human Ig lambda light chains in serum were assessed using the Human Lambda ELISA Kit (Bethyl Laboratories) according to the manufacturer's instructions. All mouse studies were performed according to protocols approved by the UCSF Institutional Animal Care and Use Committee.

[0128] Trabecular and cortical bone image analysis CT data files were utilized to perform 3D reconstruction or planar image generation of the entire, distal, and proximal regions of the femur using the BoneJ2 plugin operated by Fiji software as previously described (Doube et al., Bone 47:1076-79 (2010); Schindelin et al., Nature Methods 9:676-82 (2012)). Stacked 3D bone images were used to analyze trabecular and cortical microarchitecture parameters to obtain bone volume to tissue volume (BV / TV), trabecular width (Tb.Th), and cortical width (Ct.Th).

[0129] immunohistochemistry Femurs were dissected to remove soft tissues, fixed in 10% neutral buffered formalin, and decalcified in 14% EDTA for 4 weeks. Tissues were embedded in paraffin and cut into 4 mm pieces. Tissue sections were stained with hematoxylin and eosin (H&E) or anti-human Ig lambda light chain antibody (Abcam) as previously described (Su et al., JCI Insight 3 (2018)). Images of stained sections were taken using a BIOREVO BZ-9000 microscope (Keyence).

[0130] statistical analysis All statistical analyses to determine P values ​​were performed using two-tailed Student's T-test, and P<0.05 was used to reject the null hypothesis. For multiple between-group comparisons, one-way ANOVA with Tukey's test was used.

[0131] Example 2 –Identification of novel human Wnt agonist monoclonal antibodies To identify novel LRP6-binding Wnt pathway agonist antibodies, recombinant fragments of the extracellular domain of LRP6, specifically, the P3E3P4E4 domain, were generated. Recombinant LRP6 P3E3P4E4 domain was produced as an Fc fusion protein and purified on a protein A column as previously described (Lee et al., Scientific Reports 8 (2018)). To construct Fc fusions, LRP6-P3E3P4E4 or scFv genes were cloned into the pFUSE-hIgG1-Fc2 plasmid. To construct IgG, the variable heavy (VH) chain gene and the variable kappa light (Vκ) chain gene were subcloned into the original Abvec or modified Abvec as previously described (Lee et al. (2018)). For Fab constructs, CH2-CH3 were deleted from Ig-γ Abvec and a 6X His tag was introduced at the C-terminus of CH1. For transient transfections, plasmid DNA was resuspended in Opti-MEM (Life Technologies), mixed with polyethyleneimine, and added to HEK293A cells. After 24 h of transfection, the medium was changed to Freestyle 293 Expression Medium (Gibco) and cells were cultured for an additional 6–8 days. Proteins secreted into the supernatant were collected, filtered, and purified on Protein A agarose (Thermo Scientific) for Fc fusions and on IgG or Ni-NTA resin (Thermo Scientific) for Fabs according to the manufacturer's protocol.

[0132] A naive phage antibody display library was selected against this biotin-labeled LRP6 fragment to identify binding clones. After three rounds of selection, monoclonal phages were arrayed in 96-well plates and tested by flow cytometry for binding to LRP6-transfected HEK293 cells. These LRP6-binding clones were tested for agonistic effects on canonical Wnt signaling using the SuperTopFlash (STF) reporter assay. Cells cultured in 24- or 96-well plates were transiently transfected with the STF luciferase reporter and pRL-SV40 plasmid using TransIT-2020 (Mirus Bio) with or without Wnt1 or Wnt3a expression constructs. To express truncated and alanine mutants of LRP6, plasmid DNA encoding the constructs was cotransfected with the reporter plasmid into HEK293 cells. Antibodies diluted in culture medium were added to transfected cells together with recombinant DKK1 or RSPO2 (R&D Systems) and further incubated for 16 h. Firefly luciferase (FL) and Renilla luciferase (RL) activities were detected and normalized using the Dual-Luciferase Reporter Assay System (Promega) as previously described (Lee et al. (2018)). Data were expressed as fold increase relative to the control group transfected with only the reporter construct.

[0133] Unique scFv antibodies were sequenced and identified from LRP6-binding phages, and individual phage clones were amplified and purified for further characterization. One antibody, 66, was identified as having agonistic activity, first as a phage-displayed scFv and then as an IgG (Figure 1A). HEK293 cells transfected with STF reporter and different Wnt ligand expression constructs were incubated with or without 66 IgG (100 nM). Error bars represent SD of n=2. **P < 0.01;***P < 0.001. Wnt agonistic activity of 66 IgG was tested in the absence of endogenous Wnt ligand. HEK293 cells transfected with STF reporter construct were incubated with or without 66 IgG (100 nM) and luciferase activity was normalized to the no antibody treatment control. Interestingly, 66 IgG showed induction of Wnt / β-catenin signaling even in the absence of exogenously added Wnt ligand (Figure 1B), indicating that this antibody has Wnt ligand-like properties. The apparent affinity of 66 IgG for LRP6 was measured in HEK293 cells and found to be approximately 5 nM (Figure 7A).

[0134] This novel canonical Wnt pathway agonist 66 antibody does not compete with previously identified LRP6 P3E3 binders that are antagonistic to Wnt / β-catenin signaling (Lee et al., 2018) (Figure 7B). LRP6 expression plasmid was transfected into HEK293 cells, and the HEK293 cells were incubated with 66 IgG or E34N19 scFv phage for 1 h. E34N19 was previously identified as a Wnt antagonist that binds to the P3E3P4E4 domain (Lee et al., 2018). E34N19 IgG was added simultaneously as a competitor. Bound phage was detected by sequential incubation of mouse anti-fd IgG and PE-labeled anti-mouse IgG. The results show that agonist 66 IgG binds to different sites on LRP6.

[0135] Example 3 - Novel mechanism of action: 66 agonist antibodies do not function as ligand surrogates To map where the 66 antibody binds to LRP6, a series of truncated mutants of the LRP6 P3E3P4E4 domain were generated (Figure 1C; SP: signal peptide; P3 and P4: beta-propeller domains 3 and 4, respectively; E3 and E4: EGF-like domains 3 and 4, respectively; LDLR: low density lipoprotein receptor type A domain; TM: transmembrane domain; Cyto: cytoplasmic domain). Full-length human LRP6 was cloned into pCMV-Entry (Origene) and utilized for subcloning, point mutation, or transfection. Truncated constructs of the LRP6 ectodomain were cloned into pCMV-Entry and used for transient expression in HEK293 cells. Binding of the truncated constructs to the 66 antibody was analyzed by flow cytometry (Figure 1D). The 66 antibody was found to bind to the P3 domain of LRP6. The 66 agonist activity against the LRP6 truncated mutants was further examined using an STF reporter assay (Figure 1E). 66 IgG activated canonical Wnt signaling in cells expressing LRP6 full-length and the LRP6 P3E3P4E4 construct, but not in cells expressing other variants lacking the P3 domain, consistent with results from cell binding studies showing that the 66 antibody binds to the P3 domain.

[0136] To further map the binding site, the binding of the 66 antibody to LRP6 was modeled using the homology modeling predicted structures for the 66 Fv and two known crystal structures of LRP6 (3S8Z and 4A0P; Figures 8A and 8B, respectively). Several potential 66 contact sites were identified on LRP6. Alanine scanning mutagenesis was performed at those sites. Alanine mutants of the LRP6 ectodomain were constructed using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) according to the manufacturer's protocol. Binding of 66 IgG to these LRP6 mutants was analyzed by flow cytometry. The K662A single mutation and the K684A single mutation caused a marked loss of 66 binding (Figure 1F). A double mutant (K662A / K684A) was produced and tested, which showed an almost complete loss of binding to the 66 antibody (Figure 1F), confirming that K662 and K684 are the critical contact sites. Consistent with the binding results, 66-induced Wnt signaling activity was significantly reduced in HEK293 cells transfected with the double mutant (K662A / K684A) compared to wild-type (WT) controls (Figure 1G). These two sites (residues marked in red) are spatially distinct from the known Wnt3a binding site (residues marked in yellow, Figure 1H), suggesting that the 66 antibody does not compete with ligand binding to LRP6.

[0137] The binding characteristics of the 66 antibody were further analyzed using biolayer interferometry. The competitive binding activity between anti-LRP6 Fab and recombinant DKK1 or Wnt3a against the LRP6 ectodomain was estimated by biolayer interferometry (BLI) using a BLitz (ForteBio) instrument. A Protein A biosensor (ForteBio) was loaded with human LRP6-ECD-Fc (R&D Systems) for 120 s, and the Protein A biosensor was immersed in recombinant DKK1 or Wnt3a for 75 s, followed by immersion in a mixture of DKK1 + Wnt3a, DKK1 + anti-LRP6 Fab (66 Fab), or Wnt3a + anti-LRP6 Fab for 75 s. Baselines were determined for 30 s before and after the loading step according to the manufacturer's instructions.

[0138] As expected by the epitope mapping results, 66 Fab bound simultaneously to the LRP6-Wnt3a complex, while a mixture of Wnt3a and DKK1 failed to bind additionally to the complex (Fig. 2A). This additive binding of 66 Fab was also observed when it was added to the LRP6-DKK1 complex (Fig. 2B), confirming that 66 antibody does not compete with Wnt3a or DKK1 for LRP6 binding. This non-competitive binding of 66 antibody was further examined by STF reporter assay. Even in the presence of the inhibitor DKK1, 66 IgG was still able to significantly enhance signaling activity (Fig. 2C). These results suggest that 66 antibody does not function as a ligand surrogate. Instead, it acts in parallel with Wnt ligands and is not inhibited by endogenous Wnt inhibitors (Fig. 2D).

[0139] Example 4 – Amplification of canonical Wnt pathway activation Since 66 antibody does not bind to known Wnt ligand binding sites, we tested the agonist effect of 66 antibody to determine whether it can be amplified by Wnt signaling enhancer R-spondin (RSPO). As a control, we first showed that Wnt3a-induced Wnt / β-catenin signaling was greatly enhanced in the presence of RSPO2, and the addition of DKK1 significantly reduced the signaling enhancement (Figure 3A). We then examined 66 antibody in a similar manner in the context of RSPO2. Interestingly, in the absence of exogenously supplied Wnt ligands (either Wnt3a or Wnt1), the agonist effect of 66 IgG could be dramatically increased by the addition of RSPO2, and this signaling enhancement was not affected by the addition of DKK1 (Figure 3B). Thus, 66 antibody acts like a new type of Wnt ligand whose agonist activity is independent of known Wnt ligands and amplified by R-spondin. Unlike known Wnt ligands, 66 agonist activity is not inhibited by endogenous inhibitors.

[0140] Next, the effect of increasing RSPO2 concentration on the agonist activity of 66 antibody in the presence of known Wnt ligands was evaluated. As shown in Figure 3C, supplying 66 antibody (20 nM) increased the maximum Wnt ligand signaling activity amplified by RSPO2 (528-fold with 66 vs. 308-fold without 66). This effect was further examined across a range of 66 concentrations. In the presence of a constant concentration of Wnt ligand (Wnt3a for Figure 3D and Wnt1 for Figure 9) and RSPO2, 66 antibody showed dose-dependent agonist activity, with EC50s of 4.8 nM and 1.8 nM for Wnt3a- and Wnt1-mediated β-catenin signaling, respectively. Thus, these data further support that 66 antibody behaves like a new type of Wnt ligand that acts additively, but not competitively, with known Wnt ligands and responds to RSPO2-mediated enhanced signaling with or without known Wnt ligands.

[0141] Example 5 – Wnt agonist antibodies induce osteoblast differentiation One of the biological consequences of classical Wnt signaling activation is the induction of osteoblast differentiation and bone formation. We analyzed the Wnt agonist effect of 66 IgG on mouse preosteoblastic MC3T3-E1 and bone marrow-derived mesenchymal C3H / 10T1 / 2 cell lines. First, the cross-reactive binding of 66 IgG to human and mouse LRP6 was analyzed. 66 IgG bound specifically to both human and mouse LRP6 ectodomains (Figure 4A). Then, the effect of 66 IgG on Wnt / β-catenin signaling activation by STF reporter assay was analyzed using the aforementioned mouse-derived cell lines. Wnt3a induced reporter activity, and the addition of 66 IgG significantly enhanced signaling in both MC3T3-E1 (Figure 4B) and CH3 / 10T1 / 2 (Figure 4C) cell lines.

[0142] Next, induction of osteoblast differentiation of C3H / 10T1 / 2 cells was examined by measuring the expression of osteoblast marker genes (RUNX2, BMP2, ALP, and OCN) by qRT-PCR. Osteoblast differentiation of C3H / 10T1 / 2 cells was induced for 3 days as described above. Total RNA was isolated from cells using TRIzol™ Reagent (Invitrogen) and used to generate cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's protocol. qRT-PCR was performed with 15 ng of cDNA using Power SYBR Green PCR Master Mix (Applied Biosystems) on an ABI 7300 Real-Time PCR System (Applied Biosystems). All reactions were performed in duplicate, and copy numbers of target gene transcripts were normalized to GAPDH. Data are presented as relative mRNA expression in antibody-treated cells versus control cells.

[0143] The specific primer sets for each target gene are as follows: TIFF2024533616000044.tif64128.

[0144] As shown in Figure 4D, Wnt agonist 66 induced the expression of Wnt downstream genes involved in osteoblast differentiation, and 66 combined with Wnt3a conditioned medium (Wnt3aCM) was more potent than Wnt3aCM alone. In addition to mRNA expression, we also measured alkaline phosphatase activity (ALP) in C3H / 10T1 / 2 cells incubated with 66. As shown in Figure 4E, Wnt3aCM upregulated ALP activity, and the addition of 66 further increased ALP activity in the presence of Wnt3aCM.

[0145] To directly assess osteogenic commitment of C3H / 10T1 / 2 cells, an in vitro mineralization assay (Gregory et al., Analyt. Biochem. 329:777-84 (2004)) was performed in the presence of 66 antibody and Wnt3aCM. As shown in FIG. 4F, Wnt3aCM increased mineralization, which was further enhanced by the addition of 66. These data suggest that the Wnt agonist antibody 66 promotes osteoblast differentiation and acts additively with the natural Wnt ligand.

[0146] Example 6 – Wnt agonist antibodies induce bone formation Certain types of primary and metastatic cancers are located in bone and cause extensive bone remodeling in patients. Multiple myeloma is known to reside in bone marrow and induce significant bone loss caused by the secretion of inhibitors of classical Wnt signaling (Edwards, Blood 112:216-17 (2008); Glass et al., NEJM 349:2479-80 (2003)). The novel Wnt agonist antibody 66 does not compete with known Wnt inhibitors for LRP6 binding, so the 66 antibody can effectively counteract the Wnt inhibitors produced by myeloma cells. To test this hypothesis, we screened a panel of multiple myeloma cell lines for the presence of Wnt inhibitors in the conditioned medium with HEK293 cells co-transfected with STF reporter and Wnt3a expression constructs. As shown in Figure 5A, conditioned medium from MM1.S (MM1.S-CM) showed a significant inhibitory effect compared to the control (no conditioned medium) or other conditioned medium from different multiple myeloma cell lines. Therefore, MM1.S cells were selected for further testing. Using HEK293 and STF reporter assays again in the presence of both Wnt3a and MM1.S-CM, 66 IgG restored the signal inhibited by MM1.S-CM, and increasing concentrations of 66 treatment stimulated signaling even more (Figure 5B).

[0147] To ascertain the effect of 66 antibody on bone remodeling in vivo, a femoral intraosteolytic model was established by implantation of MM1.S cells into the right femur of NSG mice. As outlined in Figure 5C, antibody treatment was initiated 1 week after implantation and continued for 6 weeks by weekly intraperitoneal (ip) injection. Live mice were scanned by micro-CT to evaluate changes in femoral bone structure. To confirm tumor establishment, we first analyzed human Ig lambda light chain levels in serum by ELISA. As shown in Figure 5D, the concentration of human Ig lambda light chain in the MM1.S implanted groups (PBS and 66) was significantly higher than that of the group without MM1.S injection (naive). Although the light chain level in the 66 group was lower than that in the PBS group, there was no statistically significant difference between the two groups (Figure 5D). By immunohistochemistry, MM1.S myeloma cells established in the right femur were also detected by anti-human Ig lambda antibody (Figure 10). The osteogenic effect of 66 IgG was evaluated by micro-CT analysis. Whole femurs were analyzed to generate planar and 3D images from CT scan data. As shown in Figure 5E, MM1.S implantation caused osteolysis, especially in the trabecular bone area (naive vs. PBS). Remarkably, treatment with 66 IgG reversed femoral bone loss compared to controls (66 IgG vs. PBS), indicating that the Wnt agonist antibody 66 promotes bone formation in vivo (Figure 5E).

[0148] The quantitative bone formation effect of 66 IgG was further investigated. Regions of interest (ROIs) for evaluating bone structure were designated as shown in Figure 6A, and micro-CT images were reconstructed for 3D view and quantification of bone microstructure. By analyzing trabecular bone ROIs, compared with the naive group without myeloma implantation, the trabeculae of the PBS group were degraded by implanted MM1.S cells secreting Wnt / β-catenin signaling inhibitors (Figure 6B). Meanwhile, consistent with the whole femur image analysis, 66 IgG treatment showed trabecular anabolic activity (Figure 6B). 66 IgG treatment resulted in a significant increase in bone volume relative to tissue volume (BV / TV) and trabecular width (Tb.Th) (Figures 6C and 6D, respectively). In addition to the distal femur, we analyzed the cortical bone in the proximal femur region. By reconstructing cortical bone ROIs and measuring cortical bone width (Ct.Th), we found that 66 IgG significantly increased cortical bone formation compared to the control group (PBS) (Figure 6E), suggesting that the 66 Wnt agonist effect stimulates both trabecular and cortical bone formation. To evaluate the 66 IgG effect on osteoblast differentiation, we analyzed the number of bone surface osteoblasts, which was found to be increased by 66 IgG treatment. Representative H&E staining images of femurs are shown in Figure 6F, which shows a significant increase in the number of osteoblasts on the trabecular surface layer in the 66 treatment group compared to the PBS group. Taken together, the novel Wnt agonist antibody 66 reverses bone loss in the femoral intraosseous MM1.S myeloma model, demonstrating its potential in treating osteolytic disease.

[0149] Example 7 – Wnt agonist antibodies promote bone formation in vivo in a mouse model of osteoporosis Experiments were performed to examine the effect of Wnt agonist antibodies on bone tissue in an ovariectomy-induced osteoporosis mouse model (Figure 11). The experiments evaluated two variables: (1) the antibody, and more specifically, the affinity of the antibody (66 antibody vs. its higher affinity variant 66-11 antibody (at 1 / 6 the dose of 66)), and (2) the route of delivery (intraperitoneal (ip) vs. subcutaneous (subcu) injection). For readouts, both trabecular and cortical bone were evaluated.

[0150] Eight-week-old female C57BL / 6j mice were ovariectomized by Jackson Laboratory (JAX). Four weeks after ovariectomy, mice were treated with Wnt agonist antibodies (66 or 66-11) or PBS control. 66 antibody was administered intraperitoneally at 6 mg / kg, and 66-11 antibody was administered intraperitoneally or subcutaneously at 1 mg / kg. Six mice were treated and analyzed per study group. After five weekly doses, mice were scanned by in vivo microCT (focused on the distal femur) at the indicated time points (4, 28, 73, and 111 days after the last dose). For each mouse, both legs were scanned to generate images for analysis with ImageJ (BoneJ).

[0151] Results of trabecular bone volume analysis are shown in Figure 12 (4 days after final dose), Figures 13A and 13B (28 days after final dose), Figures 14A and 14B (73 days after final dose), and Figure 15 (111 days after final dose). Compared to the PBS control, the 66 antibody showed significant bone-promoting activity at the first two time points analyzed. The 66-11 antibody showed significant bone-promoting activity at all time points analyzed. Both the intraperitoneal and subcutaneous delivery routes produced significant bone-promoting activity.

[0152] The results of the cortical bone width analysis are shown in Figure 16 (4 days after the last dose), Figure 17 (28 days after the last dose), Figure 18 (73 days after the last dose), and Figure 19 (111 days after the last dose). Compared to the PBS control, the 66-11 antibody showed significant bone-promoting activity at three time points when administered intraperitoneally, or at all time points when administered subcutaneously.

[0153] These data indicate that Wnt agonist antibodies are highly active in promoting bone formation in vivo in ovariectomized mice, with the higher affinity antibody 66-11 exhibiting more potent activity even at 1 / 6 the dose of parent antibody 66. Both intraperitoneal and subcutaneous delivery routes resulted in potent bone-promoting activity, with long-lasting effects. No weight loss or any overt toxicity was observed during the duration of the experiment (139 days).

[0154] The above examples are provided to illustrate the present disclosure, and are not intended to limit its scope.Other variations of the present disclosure are readily apparent to those skilled in the art and are encompassed by the appended claims.All publications, databases, Internet sources, patents, patent applications, and accession numbers cited herein are incorporated herein by reference in their entirety for all purposes.

Claims

1. A monoclonal antibody or an antigen-binding portion thereof that specifically binds to low-density lipoprotein receptor-related protein 6 (LRP6) and agonizes Wnt signaling, a) (1) heavy chain CDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 18, 19, and 21, respectively; (2) the amino acid sequence of SEQ ID NO:2; (3) the amino acid sequence of SEQ ID NO:8; (4) heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 18, 19, and 22, respectively; (5) heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 18, 19, and 23, respectively; (6) heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 18, 19, and 24, respectively; or (7) Heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 18, 19, and 25, respectively. a heavy chain variable domain (VH) comprising: b) (1) the amino acid sequence of SEQ ID NO:15; (2) light chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 26, 27, and 29, respectively; or (3) light chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 26, 27, and 30, respectively; a light chain variable domain (VL) comprising The monoclonal antibody or antigen-binding portion thereof, comprising:

2. a) the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:3 and a VL comprising the amino acid sequence of SEQ ID NO:15; b) the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:3 and a VL comprising the amino acid sequence of SEQ ID NO:16; c) the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:9 and a VL comprising the amino acid sequence of SEQ ID NO:16; or d) the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:2 and a VL comprising the amino acid sequence of SEQ ID NO:15; The monoclonal antibody or antigen-binding portion thereof of claim 1.

3. The antibody a) (1) a heavy chain comprising HCDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 18, 19, and 21, respectively; (2) heavy chain CDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 18, 19, and 22, respectively; (3) heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 18, 19, and 23, respectively; (4) heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 18, 19, and 24, respectively; or (5) Heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 18, 19, and 25, respectively. and a heavy chain comprising b) a light chain comprising the amino acid sequence of SEQ ID NO:15; 2. The monoclonal antibody or antigen-binding portion thereof of claim 1, comprising:

4. The monoclonal antibody or antigen-binding portion thereof of claim 3, wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 7 and a VL comprising the amino acid sequence of SEQ ID NO:

15.

5. The antibody a) (1) the amino acid sequence of SEQ ID NO:8; (2) heavy chain CDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 18, 19, and 21, respectively; (3) heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 18, 19, and 22, respectively; (4) heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 18, 19, and 23, respectively; (5) heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 18, 19, and 24, respectively; or (6) Heavy chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 18, 19, and 25, respectively. and a heavy chain comprising b) (1) light chain CDRs 1-3 comprising the amino acid sequences of SEQ ID NOs: 26, 27, and 29, respectively; or (2) light chain CDR1-3 comprising the amino acid sequences of SEQ ID NOs: 26, 27, and 30, respectively; and a light chain comprising 2. The monoclonal antibody or antigen-binding portion thereof of claim 1, comprising:

6. The monoclonal antibody or antigen-binding portion thereof of claim 5, wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:8, 9, 10, 11, 12, or 13 and a VL comprising the amino acid sequence of SEQ ID NO:16 or 17.

7. 2. The monoclonal antibody or antigen-binding portion thereof of claim 1, wherein the antibody comprises a human IgG heavy chain constant region.

8. The monoclonal antibody or antigen-binding portion thereof according to claim 7, wherein the antibody is an effector-attenuated IgG1 antibody.

9. 9. The monoclonal antibody or antigen-binding portion thereof of claim 8, wherein the antibody is an IgG1 antibody comprising leucine to alanine substitutions at positions 234 and 235.

10. The monoclonal antibody or antigen-binding portion thereof according to claim 7, wherein the antibody is an IgG2 antibody.

11. The monoclonal antibody or antigen-binding portion thereof of claim 1, wherein the antibody is a human antibody.

12. A monoclonal antibody or antigen-binding portion thereof that agonizes Wnt signaling and does not compete with Wnt ligands or Wnt inhibitors.

13. A pharmaceutical composition comprising the monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 12 and a pharmaceutically acceptable excipient.

14. A nucleic acid sequence encoding the monoclonal antibody or antigen-binding portion thereof of any one of claims 1 to 12.

15. A vector comprising the nucleic acid sequence of claim 14.

16. 15. A mammalian host cell comprising the nucleic acid sequence of claim 14.

17. 17. A method for producing a monoclonal antibody or antigen-binding portion thereof, comprising culturing the host cell of claim 16 under conditions to allow production of the monoclonal antibody or antigen-binding portion thereof.

18. A method for promoting tissue regeneration, comprising adding the monoclonal antibody or antigen-binding portion thereof described in claim 1 to cells in vitro or ex vivo.

19. 14. The pharmaceutical composition of claim 13, for use in a method for restoring tissue in an individual in need thereof.

20. 20. The pharmaceutical composition of claim 19, wherein the tissue is bone tissue, intestinal tissue, liver tissue, or brain tissue.

21. 20. The pharmaceutical composition of claim 19, wherein the individual has a disease or condition selected from the group consisting of age-induced osteoporosis, drug-induced bone loss, osteogenesis imperfecta, inflammatory bowel disease, severe alcoholic hepatitis, diabetic retinopathy, wet age-related macular degeneration (AMD), Fuchs' dystrophy, corneal epithelial stem cell deficiency, dry AMD, Sjogren's dry eye, short bowel syndrome, and hearing loss.

22. 1. A method for identifying a monoclonal antibody or antigen-binding portion thereof that agonizes Wnt signaling and does not compete with a Wnt ligand or a Wnt inhibitor, comprising: a) providing an LRP6 polypeptide, or a portion thereof comprising at least the LRP6 polypeptide P3E3P4E4 domain; b) contacting said LRP6 polypeptide or a portion thereof with a library of binding molecules; c) selecting one or more binding molecules from said library that bind to said LRP6 polypeptide or a portion thereof; and d) identifying a selective binding molecule that does not compete with a Wnt ligand or a Wnt inhibitor for binding to said LRP6 polypeptide or portion thereof. The method comprising:

Images