Polyvalent fused molecules that activate WNT signaling and their uses

Abstract

This provides a method for activating the Wnt signaling pathway in cells. [Solution] This specification describes a method for influencing the binding of a polyvalent binding molecule to an FZD receptor and a Wnt coreceptor on a cell, wherein the binding of the polyvalent binding molecule to both the FZD receptor and the coreceptor activates the Wnt signaling pathway. Also described this specification are polyvalent binding molecules that activate the Wnt signaling pathway, comprising an FZD receptor binding domain and a Wnt coreceptor binding domain at both ends of an Fc domain, and methods for using them.

Description

Related applications

[0001] This application seeks the benefits under § 119(e) of U.S. Patent Act Provisional Patent Application No. 62 / 630,772, filed February 14, 2018, which is incorporated herein by reference in its entirety. This application includes a sequence listing, which has been electronically submitted in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy thereof, created on 12 February 2019, is named 115773_PA895WO_SL.txt and is 220,360 bytes in size. [Background technology]

[0002] The Wnt signaling pathway is critically important for embryonic development and tissue homeostasis in adults. Wnt ligands are secreted growth factors that regulate various cellular processes, including proliferation, differentiation, survival, and migration. Wnt ligands are universally important for controlling the self-renewal of tissue stem cells and regulating numerous progenitor cell populations. Due to the hydrophobic and sensitive tertiary structure of Wnt proteins, attempts at biochemical purification and its use in vitro and in vivo have been thwarted.

[0003] Humans possess 19 Wnt ligands, which interact with a network of 10 Frizzled cell surface receptors (FZDs) and one of several co-receptors, leading to the selective engagement of different intracellular signaling branches (Wodarz, A. and Nusse, R. Annu. Rev. Cell Dev. Biol. 14, 59-88 (1998); Angers, S and Moon, RT, transduction. Nat. Rev. Mol. Cell Biol. 10, 468-477 (2009)). FZDs have conserved structural features, including seven hydrophobic transmembrane domains and a cysteine-rich ligand-binding domain. FZDs are known for their function in three distinct signaling pathways, known as the Wnt plane cell polarity (PCP) pathway, the classical Wnt / β-catenin pathway, and the Wnt / calcium pathway. Activation of the Wnt signaling pathway also requires the presence of Wnt coreceptors, which dictate the engagement of different intracellular signaling cascades, and these cascades regulate the expression of genes that act on the underlying cellular mechanisms of the cellular processes mentioned above. For example, Wnt ligands bind to the Frizzled receptor and members of the low-density lipoprotein receptor-associated protein 5 and 6 (LRP5 / 6) coreceptor family, which activate the Wnt / β-catenin pathway; or to receptor tyrosine kinase-like orphan receptors 1 and 2 (ROR1 / 2), which are associated with receptor tyrosine kinase (RYK) or protein tyrosine kinase 7 (PTK7) coreceptors, which trigger the Wnt / PCP pathway or alternative β-catenin-independent signaling pathways. The Wnt / β-catenin pathway, sometimes called the classical pathway, reaches its peak during the posttranslational accumulation of the transcription effector β-catenin, which interacts with the T cell factor / lymphocyte enhancer factor (LEF / TCF) family of transcription factors that regulate the expression of context-specific genes. [Overview of the project]

[0004] Wnt requires lipid modification to function (Janda et al., Science. 337, 59-64 (2012); Kadowaki et al., Genes Dev. 10, 3116-3128 (1996)), but their hydrophobic properties complicate biochemical operations, and therefore only small amounts of Wnt are purified (Willert et al., Nature 423, 448-452 (2003)). Furthermore, Wnt inherently exhibits cross-reactivity to multiple receptors, particularly when overexpressed or at high doses (He et al. Science. 275, 1652-1654 (1997); Andres et al. Systematic mapping of Wnt-Frizzled interactions reveals functional selectivity by distinct Wnt-Frizzled pairs. Journal of Biological (2015) (available at http: / / www.jbc.org / content / early / 2015 / 01 / 20 / jbc.M114.12648.short); Holmen et al., J. Biol. Chem. 277, 34727-34735 (2002)). Consequently, selective activation of Frizzled receptor complexes has not been achieved to determine their specific functions in different contexts or to evaluate their therapeutic potential in degenerated states. The polyvalent binding molecules and methods described herein selectively activate pre-selected Frizzled receptor-coreceptor complexes. The administration of the polyvalent binding molecules described herein is expected to treat the degenerative state by activating the appropriate Frizzled coreceptor complex.

[0005] This specification describes a method for influencing the binding of peptides to FZD receptors and Wnt coreceptors on cells, wherein the binding of peptides to both the FZD receptor and coreceptor activates the Wnt signaling pathway.

[0006] Also described herein are polyvalent binding molecules that activate the Wnt signaling pathway and methods for using them. These polyvalent binding molecules bind to both the FZD receptor and the Wnt coreceptor, thereby activating the Wnt signaling pathway. The polyvalent binding molecules of the present invention are also referred to herein as “FZD agonists” or “FZDag”. In specific embodiments in which the molecules of the present invention bind FZD and LRP5 / 6, these molecules may be referred to as “Frizzled and LRP5 / 6 agonists” or “FLAgs”. These polyvalent binding molecules comprise a fragment containing an Fc domain or its CH3 domain, a first binding domain for binding to the FZD receptor, and a second binding domain for binding to the Wnt coreceptor, in which case the FZD binding domain is ligated to one end of the Fc domain and the coreceptor binding domain is ligated to the other end of the Fc domain. Therefore, the binding domain to the FZD receptor and the binding domain to the co-receptor are separated not by direct linkage, but by a fragment containing an Fc domain or a CH3 domain. This configuration of the binding domain results in unexpectedly high levels of Wnt signaling pathway activation. The FZD binding domain may be monovalent and have a single binding site (paratope) to the FZD receptor, or it may be polyvalent and have one or more binding sites to the FZD receptor; for example, this binding domain may be bivalent, trivalent, or tetravalent. The Wnt co-receptor binding domain may be monovalent and have a single binding site (paratope) to the Wnt co-receptor, or it may be polyvalent and have one or more binding sites to the Wnt co-receptor; for example, this binding domain may be bivalent, trivalent, or tetravalent.

[0007] The methods for producing polyvalent conjugated molecules described herein enable the selective and robust activation of any FZD receptor complex in vitro and in vivo. Leveraging a panel of hundreds of synthetic antibodies targeting FZD and their co-receptors, we have generated polyvalent conjugated molecules for the selective and rational activation of one, two, or more FZD receptors. The polyvalent conjugated molecules of the present invention are expected to be highly suitable and readily available for large-scale production and easy purification, possess predictable pharmacokinetics, and exhibit low immunogenicity.

[0008] In one embodiment of the present invention, the binding domain of the polyvalent binding molecule described herein binds to one or more FZD receptors and LRPs, e.g., LRP5 and / or LRP6, and is substituted herein for FLAg. FLAg targeting specific FZDs and their LRP coreceptors improve directional differentiation and cell therapy, sustain tissue organoid growth, mobilize endogenous stem cells in vivo to promote post-wound tissue repair, and restore function following tissue degeneration.

[0009] The Fc domain of the FZD agonist may be the Fc domain of an immunoglobulin. This immunoglobulin may be IgG, for example, IgG1. In one embodiment of the present invention, the polyvalent binding molecule is a peptide dimer, in which case the peptide is dimerized to produce a polyvalent binding domain either through the intrinsic dimerizing ability of the Fc domain or through a knob-in-hole configuration within the Fc that allows for the specific assembly of two different peptides. A method for dimerizing a peptide via a knob-in-hole configuration is described in WO2018 / 026942 by the inventors Van Dyk et al., incorporated herein by reference.

[0010] One or both of the multivalent binding domains of the FZD agonists described herein may be bivalent and monospecific, having two binding sites for the same epitope on their respective receptor targets or co-receptor targets. One or both of the binding domains may be bivalent and bispecific, having two binding sites, each binding to a different epitope on its respective target.

[0011] In one embodiment of the present invention, the FZD-binding domain may include two single-chain variable fragments (scFv) for binding to the same or different epitopes on the FZD receptor. In another embodiment of the present invention, the FZD-binding domain includes one or more heavy-chain variable domain (VH) fragments and / or one or more light-chain variable domain (VL) fragments that bind FZD. In another embodiment of the present invention, the FZD-binding domain consists of one or more single-domain antibody fragments that bind FZD. In another embodiment of the present invention, the FZD-binding domain includes an FZD ligand or a fragment thereof that binds to the FZD receptor. In one embodiment of the present invention, the FZD-binding domain includes synthetic peptides that bind FZD, such as afibody (afibody), ankyrin repeat protein, fibronectin repeat protein, finomer (finomer), and antikalin (antikalin). In one embodiment of the present invention, the FZD-multivalent binding domain does not include scFv. FZD ligands may be, for example, fragments of Wnt proteins that bind to FZD receptors or Norrin, or other native or synthetic peptides that have matured affinity to interact with one or more FZD receptors. Norrin is an FZD4-specific ligand and, in complex with LRP5 and / or LRP6, is associated with the activation of classical Wnt signaling.

[0012] In one embodiment of the present invention, the co-receptor binding domain may comprise two single-chain variable fragments (scFv) for binding to the same or different epitopes on the co-receptor. In another embodiment of the present invention, the Wnt co-receptor binding domain comprises one or more heavy chain variable domain (VH) fragments and / or one or more light chain variable domain (VL) fragments that bind a Wnt co-receptor. In another embodiment of the present invention, the co-receptor binding domain consists of one or more single-domain antibody fragments that bind a co-receptor. In one embodiment of the present invention, the Wnt co-receptor binding domain comprises a peptide that binds a Wnt co-receptor, wherein the peptide is a fragment of a natural ligand that binds a Wnt co-receptor, or is a synthetic peptide that binds a Wnt co-receptor, for example, an affibody, an ankyrin repeat protein, a fibronectin repeat protein, a finomer, or an anticalin. In another embodiment of the present invention, the co-receptor binding domain comprises a co-receptor ligand or a fragment thereof that binds a co-receptor (for example, the ligand Dkk1 for the co-receptor LRP5 / 6), or another natural or synthetic peptide that has undergone affinity maturation to interact with one or more co-receptors.

[0013] In one embodiment of the present invention, the multivalent binding domain of the co-receptor does not comprise an scFv.

[0014] In one embodiment of the present invention, each binding domain of the molecule described herein may be formed by two peptides, each containing a heavy chain variable domain (VH) linked to a light chain variable domain (VL), where the VH and VL from one peptide pair with the VL and VH of the other peptide forming the diabody. In this configuration, the binding domain has two binding sites for its target; that is, the FZD binding domain has two binding sites for the FZD receptor, and the coreceptor binding domain has two binding sites for the coreceptor. Using the knobs-in-hole Fc configuration, peptides containing VH and VL can be engineered, and as a result, these peptides, though not identical, pair to form a bispecific binding domain capable of binding to two different sites on the FZD receptor or coreceptor (see Figure 3A).

[0015] In one embodiment of the present invention, one or both of the multivalent binding domains contain two peptides that form a diabody on each terminus of the Fc domain. Each diabody has two binding sites for one epitope on their respective FZD receptor target or co-receptor target. The diabody may be monospecific, in which case the binding sites bind to the same epitope on the FZD receptor or co-receptor, or the diabody may be bispecific, binding to two different epitopes on the FZD receptor or co-receptor.

[0016] The peptides forming scFv or diabodies may be derived from antibodies that bind to FZD receptors or from antibodies that bind to Wnt co-receptors. Regarding the FZD binding domain, the antibody may be an antibody that binds to one or more FZD receptors to antagonize Wnt signaling or inhibit the binding of Wnt to a given FZD receptor(s), or alternatively, the antibody may be an antibody that binds to one or more FZD receptors without inhibiting the binding of Wnt to FZD receptors. Regarding the co-receptor binding domain, the antibody may be an antibody that binds to the co-receptor to antagonize Wnt signaling or inhibit the binding of Wnt to the co-receptor, or alternatively, the antibody may be an antibody that binds to the co-receptor without inhibiting the binding of Wnt to the co-receptor.

[0017] The FZD binding domain may bind to one or more members of the FZD receptor family, for example, to Frizzled class receptor 1 (FZD1), Frizzled class receptor 2 (FZD2), Frizzled class receptor 3 (FZD3), Frizzled class receptor 4 (FZD4), Frizzled class receptor 5 (FZD5), Frizzled class receptor 6 (FZD6), Frizzled class receptor 7 (FZD7), Frizzled class receptor 8 (FZD8), Frizzled class receptor 9 (FZD9), or Frizzled class receptor 10 (FZD10). The co-receptor binding domain may bind to any Wnt co-receptor, for example, to LRP5 / 6, PTK7, ROR1 / 2, RYK, GPR124, TSPAN12, or CD133. In one embodiment of the present invention, the co-receptor binding domain binds to LRP5 and / or LRP6. In one embodiment of the present invention, the co-receptor binding domain binds to a single epitope on the co-receptor, for example, to an epitope of an LRP protein that binds Wnt1 or Wnt3a. In one embodiment of the present invention, the co-receptor binding domain binds to two epitopes on the co-receptor, for example, one epitope on LRP that binds Wnt1 and one epitope that binds Wnt3a.

[0018] One embodiment of the present invention includes a method for producing induced pluripotent stem (iPS) cells, the method comprising culturing somatic cells under conditions suitable for reprogramming somatic cells in the presence of an effective amount of the polyvalent conjugate molecule described herein. The polyvalent conjugate molecule may be included in an amount that accelerates the generation of iPS cells compared to when iPS cells are produced under the same culture conditions without the polyvalent conjugate molecule.

[0019] Another embodiment of the present invention is a method for directing the differentiation of iPS or other pluripotent stem cells (PSCs) into various lineages by culturing them in the presence of an effective amount of the polyvalent binding molecules described herein.

[0020] One embodiment of the present invention includes a method for generating tissue organoids, the method comprising culturing a tissue sample under conditions suitable for organoid generation in the presence of an effective amount of the polyvalent binding molecule described herein as part of a culture cocktail. In one embodiment or the present invention, the frequency of generation of tissue organoids cultured in a medium containing the polyvalent binding molecule is enhanced compared to organoids cultured in the same medium without the polyvalent binding molecule. In one embodiment or the present invention, tissue organoids are generated more rapidly when cultured in a medium containing the polyvalent binding molecule compared to tissue samples cultured in the same medium without the polyvalent binding molecule. One embodiment of the present invention includes a method for enhancing the maintenance of tissue organoids, the method comprising culturing organoids in the presence of an effective amount of the polyvalent binding molecule described herein as part of a culture cocktail. As described herein, the survival of tissue organoids cultured in a medium containing the polyvalent binding molecule is prolonged compared to organoids cultured in the same medium without the polyvalent binding molecule.

[0021] One aspect of the present invention is a method for producing a polyvalent bond molecule as described herein. In one embodiment of the present invention, the polyvalent bond molecule is a) Select an Fc domain having a C-terminus and an N-terminus. b) Identify antibodies that bind to one or more FZD receptors. c) Identify antibodies that bind to one or more Wnt coreceptors. d) Generating a nucleic acid molecule comprising (i) a nucleotide sequence encoding the Fc domain of step a, (ii) a nucleotide sequence encoding the VL and / or VH of the antibody of step b, or the VL and / or VH derived from the antibody of step b that binds one or more FZDs, and (iii) a nucleotide sequence encoding the VL and VH of the antibody of step c, or the VL and VH derived from the antibody that binds to one or more Wnt receptors of step c. e) Expressing the nucleic acid molecule of (d) to produce a polypeptide, wherein the polypeptide dimerizes to form a polyvalent binding molecule comprising an Fc domain, an FZD binding domain, and a Wnt coreceptor binding domain, the FZD binding domain being composed of VL and VH of or derived from the antibody of step b and linked to one end of the Fc domain, and the Wnt coreceptor binding domain being composed of VL and VH of or derived from the antibody of step c and linked to the other end of the Fc domain, thereby forming a polyspecific binding molecule. It is generated by [the following method / system]. The antibody in step (b) may be an antibody or antibody fragment that binds to one or more FZD receptors and either antagonizes Wnt signaling or inhibits the binding of Wnt to the receptors. The antibody in step (b) may be an antibody or antibody fragment that binds to one or more FZD receptors without antagonizing Wnt signaling or inhibiting the binding of Wnt to the receptors. The antibody in step (c) may be an antibody or antibody fragment that binds to one or more Wnt coreceptors and either antagonizes Wnt signaling or inhibits the binding of Wnt to the coreceptors, or binds to a coreceptor without antagonizing Wnt signaling or inhibiting the binding of Wnt to the coreceptors. The binding domain may be linked to the Fc domain via a linker. Modular embodiments of the present invention allow for the mixing and fitting of the antibody binding domains at the terminals of the Fc domain for any given FZD receptors and coreceptors to generate a multivalent binding molecule capable of engaging multiple Frizzled receptor-coreceptor complexes, or selective engagement of a single Frizzled receptor-coreceptor complex that activates Wnt signaling.

[0022] This polyvalent binding molecule comprises a peptide dimer having an Fc domain, a binding domain for binding one or more FZD receptors, and a second binding domain for binding one or more Wnt coreceptors. In this dimer, the FZD binding domain is ligated to one end of the Fc domain, and the coreceptor binding domain is ligated to the other end of the Fc domain. Each binding domain may be monovalent or polyvalent, for example, divalent, trivalent, or tetravalent.

[0023] Furthermore, one embodiment of the present invention is a method using the multivalent binding molecule for, for example, producing induced pluripotent stem (iPS) cells, for directional differentiation of pluripotent stem cells, and for generating and / or maintaining tissue organoids, or for enhancing tissue regeneration in subjects requiring such organoids.

[0024] An additional embodiment of the present invention is a method for activating the Wnt signaling pathway to mobilize a pool of endogenous stem / progenitor cells for use in regenerative medicine and for disorders or diseases associated with insufficient Wnt signaling. [Brief explanation of the drawing]

[0025] [Figure 1A] This figure shows the binding specificity of five antibodies selected for binding to the extracellular domain (ECD) of human LRP6. LRP6-binding antibodies were selected from a synthetic antibody library by selecting antibodies that bind to the recombinant extracellular domain (ECD) of human LRP6. These antibodies were assayed by ELISA for binding to human LRP6, mouse LRP6, and mouse LRP5. Binding to Fc peptide and bovine serum albumin (BSA) was included as negative controls.

[0026] [Figure 1B] This figure shows that, by monitoring Wnt signaling activation using a luciferase reporter assay, Wnt1 (transient transfection) and Wnt3a (0.5 μg / mL purified protein) had opposite effects on stimulation, demonstrating that IgG 2539 and IgG 2542 (100 μM) bind to different sites on the LRP6 ECD. An anti-MBP antibody served as a control.

[0027] [Figure 2A] This figure shows that typical bispecific IgG (Bi-IgG) and bispecific diabody molecules consist of an FZD-binding domain (5019) and an LRP6-W1 (2942, L61) or -W3 (2539, L63)-binding domain at the same end of the Fc domain.

[0028] [Figure 2B]This figure demonstrates that, as determined by the TOPFlash luciferase reporter assay in HEK293 cells, bispecific IgG (5019-2539Bi-IgG and 5019-2542Bi-IgG) does not activate Wnt signaling, but rather acts as an antagonist to Wnt signaling.

[0029] [Figure 2C] This figure shows the binding of the bispecific diabodies, in which case the Fc domain has a knob / hole configuration (K / H). The two diabodies obtained as diabodies, 5019-2539-K / H (FZD / LRP6-W3) and 5019-2542-K / H (FZD / LRP6-W1), retain, albeit very weakly, the original IgG FZD binding profile and LRP6 binding activity. Figure 2C shows the purified FZD-LRP6 diabodies: 5019-2539-K / H and 5019-2542-K / H. [Figure 2D] Figure 2D shows the binding of the bispecific diabodies, in which case the Fc domain is in a knob / hole configuration (K / H). The two diabodies obtained as diabodies, 5019-2539-K / H(FZD / LRP6-W3) and 5019-2542-K / H(FZD / LRP6-W1), retain, albeit very weakly, the FZD binding profile and LRP6 binding activity of the original IgG. Figure 2D shows the FZD receptor binding profiles of the 5019-diabodies to FZD4, FZD5, and FZD7. 5019FZD IgG has previously been characterized as binding to FZD1, 2, 4, 5, 7, and 8. [Figure 2E] This figure shows the binding of the bispecific diabody, in which case the Fc domain has a knob / hole configuration (K / H). The two diabodies obtained as shown, 5019-2539-K / H(FZD / LRP6-W3) and 5019-2542-K / H(FZD / LRP6-W1), retain, albeit very weakly, the FZD binding profile and LRP6 binding activity of the original IgG. Figure 2E shows the FZD receptor binding profile of the bispecific FZD / LRP6 diabody 5019-2539-K / H. [Figure 2F] This figure shows the binding of the bispecific diabody, in which case the Fc domain has a knob / hole configuration (K / H). The two diabodies obtained as shown, 5019-2539-K / H(FZD / LRP6-W3) and 5019-2542-K / H(FZD / LRP6-W1), retain, albeit very weakly, the original IgG FZD binding profile and LRP6 binding activity. Figure 2F shows the FZD receptor binding profile of the bispecific FZD / LRP6 diabody 5019-2542-K / H. [Figure 2G] Figure 2G shows the binding of bispecific diabodies, in which case the Fc domain has a knob / hole configuration (K / H). The two diabodies obtained as shown, 5019-2539-K / H(FZD / LRP6-W3) and 5019-2542-K / H(FZD / LRP6-W1), retain, albeit very weakly, the original IgG FZD binding profile and LRP6 binding activity. Figure 2G demonstrates that homo-diabodies (2539-Fc and 2542-Fc) and hetero-diabodies (5019-2539-Fc and 5019-2542-Fc), which have a binding domain at one end of the Fc domain, interact with the LRP6 extracellular domain. [Figure 2H] This figure shows the binding of a bispecific diabody, in which case the Fc domain has a knob / hole configuration (K / H). The two resulting diabodies, 5019-2539-K / H(FZD / LRP6-W3) and 5019-2542-K / H(FZD / LRP6-W1), retain, albeit very weakly, the original IgG's FZD binding profile and LRP6 binding activity. Figure 2H demonstrates that diabodies 5019-2539-K / H and 5019-2542-K / H co-bind to FZD CRD and LRP6 ECD in solution, as determined by a Bio-Layer Interferometry (BLI) assay.

[0030] [Figure 2I]This figure demonstrates that neither 5019-2539-K / H nor 5019-2542-K / H, whose diabodies forming the binding domains for the FZD receptor and LRP6 receptor are located on the same side of Fc, are FZD agonists that activate the Wnt-mediated pathway. The results, revealed using the TOPFlash luciferase reporter assay in HEK293 cells, demonstrate that the 5019-2539-K / H diabodies (selectively targeting the Wnt3 site on LRP6) completely block the activation of the Wnt3-mediated pathway at 10 nM and 50 nM, while 5019-2542-K / H is less effective.

[0031] [Figure 2J] This figure compares the luciferase activity of tetravalent molecules containing a diabody or scFv-containing binding domain. Molecules containing binding domains with anti-FZDscFv and anti-LRP diabody (FP*+P*-L61+3) exhibited similar activity to molecules containing binding domains with anti-FZD diabody and anti-LRP diabody (FP+P-L61+3). In contrast, molecules containing anti-FZD diabody but also anti-LRP6scFv (FP+P-L61*+3*) or molecules containing scFv at both ends (FP*+P*-L61*+3*) showed significantly reduced activity compared to FP+P-L61+3 molecules.

[0032] [Figure 2K] This figure demonstrates that the difference in activity between tetravalent binding molecules containing binding domains, including diabodies or scFvs, was not due to differences in affinity, as BLI measurements showed equally high affinity binding to LRP6 and FZD isoforms regardless of whether the paratope was presented in the form of a diabody or scFv. [Figure 2L]This figure demonstrates that the difference in activity between tetravalent binding molecules containing binding domains, including diabodies or scFvs, was not due to differences in affinity, as BLI measurements showed equally high affinity binding to LRP6 and FZD isoforms regardless of whether the paratope was presented in the form of a diabody or scFv.

[0033] [Figure 3A] This is a schematic diagram of a tetravalent bond molecule, in which two FZD binding domains, composed of homo (recognizing the same epitope) or hetero (recognizing different epitopes) diabodies, are ligated to one end of the Fc domain, and two LRP6 binding domains, composed of homo or heterodiabodies, are ligated to the other end of the Fc domain.

[0034] [Figure 3B] This figure shows the binding of the polyvalent binding molecules 5019-Fc-2539(FP+P-L63+3) and 5019-Fc-2542(FP+P-L61+1) to ECDs of FZD4, FZD5, and FZD7. Binding to the FZD receptor is detected using the BLI assay.

[0035] [Figure 3C]This figure shows the activation of the Wnt-β-catenin signaling pathway by the tetravalent molecules 5019-Fc-2539(FP+P-L63+3), 5019-Fc-2542(FP+P-L61+1), 5019-K / H-2539-2542(FP+P-L61+3), and purified Wnt3A (0.5 μg / mL). The concentrations of the above molecules are indicated. The above tetravalent molecules are agonists that robustly activate the Wnt-β-catenin pathway in HEK293T cells, as measured using a pBAR luciferase reporter assay. The 5019-Fc-2539 homodiabody binds to multiple FZD receptors (5019:FZD1, 2, 4, 5, 7, 8) and to the Wnt3a site on LRP6 (2539), activating the reporter to levels comparable to purified Wnt ligands. The 5019-K / H-2539:2542 heterodiabody binds to both Wnt binding sites on LRP6 and is even more effective.

[0036] [Figure 3D] This figure shows the activation of the Wnt-β-catenin pathway by a polyvalent binding molecule in which an FZD homodiabody (5019) is linked via Fc to either a monospecific LRP6 homodiabody (5019-Fc-2539, 5019-Fc-2542) or a bispecific LRP6 heterodiabody (5019-K / H-2539-2542, also known as 5019Ag or FP+P-L61+3).

[0037] [Figure 3E]This figure shows the activation of Wnt-β-catenin signaling by molecules containing a monovalent binding domain to either the FZD receptor or the LRP6 coreceptor. Activation of the Wnt-β-catenin pathway was detected using a pBAR luciferase reporter assay performed in HEK293T cells. 5019-MBP-K / H-2539-2542, containing one of the monovalent binding domains to FZD, also activates the Wnt pathway, but shows an 8-fold decrease in efficacy compared to 5019Ag (which contains two FZD binding domains to the same epitope). 5019-K / H-2539-MBP, which retains only one LRP6-W3 binding domain at its C-terminus, exhibits much lower efficacy. Importantly, minimal agonist activity was detected for the two mono-FZD:mono-LRP6 diabodies 5019-MBP-K / H-2539-MBP and 5019-MBP-K / H-MBP-2542, as well as for the diabodies 5019-K / H-MBP-2542 with one LRP6-W1 site.

[0038] [Figure 3F] This figure shows the activation of the Wnt-β-catenin pathway by a tetravalent binding molecule. In the figure, the anti-LRP5 paratope targeting the WNT3A binding site is replaced by the anti-LRP6 paratope targeting the WNT1 binding site, generating a molecule (FP+P-L5 / 63) that can recruit both coreceptors, and similar activity to FP+P-L61+3 was observed (EC50=4nM).

[0039] [Figure 4A]A polyvalent binding molecule (FZD4Ag:5038Ag / 5038-K / H-2539-2542, 5044Ag / 5044-K / H-2539-2542, 5048Ag / 5048-K / H-2539-2542) has a FZD-specific FZD-binding domain (in this case, a homodiabody) on one side of the Fc domain and a co-receptor-binding domain (2539 and 2542) for LRP6 on the other side of the Fc domain. This figure shows the activation of the Wnt-β-catenin pathway in reporter cells that either lack the endogenous FZD4 receptor (-FZD4) or have been modified to express the FZD4 receptor (+FZD4) by 5019Ag (5019-K / H-2539-2542), 5080Ag (50180-K / H-2539-2542), and 5081Ag (5081-K / H-2539-2542). The controls are the multivalent binding molecule 5019Ag (5019-K / H-2539-2542) and Norrin, the endogenous agonist of FZD4. As a result, it has been demonstrated that selective FZD4 agonists can be developed by replacing the 5019FZD binding domain (which recognizes FZD1, 2, 4, 5, 7, and 8) in 5019Ag / 5019-K / H-2539:2542 (a pan-FZD agonist) with a domain that selectively binds to FZD4. HEK293T cells were transfected with pBARL (Wnt-β-catenin luciferase reporter) and Rluc (normalized control), plasmids encoding the listed FZD agonists, with and without FZD4 and LRP6 cDNA. Norrin was used as a positive control for FZD4 activation. HEK293T cells expressed FZD4 at little to no detectable levels, and therefore FZD4 agonists can only activate the reporter gene in the presence of transfected FZD4 cDNA. In contrast, pan-FZDag5019-K / H-2539:2542 robustly activates Wnt-β-catenin signaling in these cells, either in the absence or presence of FZD4, via the activation of other endogenously expressed Frizzled.

[0040] [Figure 4B]This figure shows the activation of the Wnt-β-catenin pathway by a polyvalent binding molecule having a binding domain (homodiabody) specific to FZD2 (2876, 2890), FZD2 / 7 (2886), FZD6 (2747), or FZD9 / 10 (2969, 2974) on one side of the Fc, and an LRP6 heterodiabody formed by antibody fragments 2539 and 2542 on the other side of the Fc. Activation of the Wnt-β-catenin pathway was evaluated in HEK293T cells using the pBARL assay.

[0041] [Figure 4C] This figure illustrates the activation of the Wnt pathway by a polyvalent binding molecule containing an FZD binding domain. This FZD binding domain is pan-specific to FZD and is derived from IgG, which blocks Wnt binding to FZD and Wnt-β-catenin signaling. The LRP6 binding domain within these molecules is located at the C-terminus of Fc and consists of a diabody formed from antibodies 2539 and 2542, which have paratopes that recognize the Wnt3 binding site and the Wnt1 binding site on LRP6, respectively.

[0042] [Figure 4D] This figure illustrates the activation of the Wnt pathway by a polyvalent binding molecule containing an FZD binding domain. This FZD binding domain is pan-specific to FZD, does not block Wnt binding to FZD, and does not antagonize the activation of the Wnt3-inducible pathway, and is derived from IgG. The LRP6 binding domain of these molecules is located at the C-terminus of Fc and consists of a diabody formed by antibodies 2539 and 2542. These antibodies have paratopes that recognize the Wnt3 binding site and the Wnt1 binding site on LRP6, respectively.

[0043] [Figure 5]This figure compares the FZD / LRP6 binding behavior of the three tetravalent bond molecules of the present invention. 5019-Fc-2539, 5019-Fc-2542, and 5019-Fc-2539-2542 bind strongly to FZD but exhibit weak LRP6 interactions (left graph) or FZD / LRP6 cobinding (middle graph). The FZD binding profile of 5019-K / H-2539-2542 (right graph) shows that it recognizes FZD4, FZD5, and FZD7.

[0044] [Figure 6A] Figure 6A illustrates the binding of the two upper propellers (E1-E2) of LRP5 / 6, which are known to mediate binding to Wnt1, and the two lower propellers (E3-E4) of LRP5 / 6, which are located proximal to the plasma membrane and are known to mediate interaction with Wnt3. Figure 6A also illustrates how Wnt1 interacts with LRP5 / 6 and the FZD receptor, and how Wnt3 interacts with LRP5 / 6 and the FZD receptor.

[0045] [Figure 6B] This diagram illustrates the possible interactions between the FZD receptor and the LRP5 / 6 receptor mediated by the polyvalent binding molecules 5019-Fc-2539, 5019-Fc-2542, and 5019-K / H-2539-2542.

[0046] [Figure 6C] This figure demonstrates that the multivalent binding molecule is a robust agonist that activates the Wnt-β-catenin pathway in HEK293T cells, as measured using a pBAR luciferase reporter assay. The 5019-Fc-2539 homodiabody binds to multiple FZD receptors (5019 binds to FZD1, 2, 4, 5, 7, and 8) and to the Wnt3a site (2539) on LRP6, activating the reporter to a level comparable to that of purified Wnt ligands. The 5019-K / H-2539:2542 heterodiabody binds to both the Wnt3a and Wnt1 binding sites on LRP6 and is even more effective.

[0047] [Figure 6D] This figure demonstrates that 5019-K / H-2459:2460 also activates the Wnt-β-catenin pathway in HEK293T cells. This molecule is a tetravalent conjugation molecule that has an Fc domain in a knob-in-hole configuration, a pan-FZD-specific (5019)FZD-binding domain (homodiabody), and bispecific coreceptor-binding domains (heterodiabody) at two sites on LRP5 (2459 binds to the Wnt1 binding site and 2460 binds to the Wnt3 binding site).

[0048] [Figure 7A] This figure demonstrates that a selective FZD5 agonist was generated by replacing the FZD-binding domain in 5019-K / H-2539:2542 (a pan-FZD agonist that recognizes FZD1, 2, 4, 5, 7, and 8) with an FZD-binding domain specific to FZD5 (#2928). HPAF-II cells have been shown to depend on FZD5 signaling for their proliferation. Blocking Wnt-FZD5 signaling with the Wnt secretion inhibitor LGK974 (which targets the acyltransferase porcupine) leads to cell cycle arrest and inhibition of proliferation. Proliferation can be rescued by adding exogenous Wnt3a-conditioned medium or by adding FZD5-selective agonists (2928-K / H-2539:2542) or the pan-FZD agonists (5019-K / H-2539:2542) described herein. The FZD4 selective agonist 5038-K / H-2539:2542 has only moderate rescue capabilities.

[0049] [Figure 7B] This figure demonstrates that stimulation of C3H10T1 / 2 cells with FZD2-specific FLAg results in robust induction of alkaline phosphatase (ALPL), a bone formation marker, to a level similar to that achieved with pan-FZD FLAg, while FZD5-specific FLAg exhibits minimal activity.

[0050] [Figure 8A] This figure shows that the pan-FZDag (FP+P-L61+3) of the present invention completely replaces exogenous Wnt3A-conditioned medium, rescuing inhibited intestinal organoid growth when Wnt secretion is blocked by the porcupine small molecule inhibitor LGK974 (bottom left photo). Intestinal organoids isolated from mice grow in the presence of recombinant R-spongin and require the presence of Wnt ligands secreted by Paneth cells. Figure 8A illustrates that inhibition of Wnt production using LGK974 leads to organoid death (top right photo). Exogenous addition of Wnt3A-conditioned medium (bottom right photo) or FZDag (bottom left photo) rescues organoid growth in the presence of LGK974. The top left photo shows organoids treated with DMSO without LGK974 as a control. [Figure 8B] This figure demonstrates that Wnt production inhibition by LGK974, which leads to organoid death, can be rescued by the addition of Wnt3A-modified medium or FZDag (FP+P-L61+3), as quantified using the CellTiter Glow® assay and Promega.

[0051] [Figure 9A] This figure shows an example of a plasmid encoding a peptide that dimerizes in a knob-into-hole conformation to form pan-FZDag 5019-KH-2539-2542(FP+P-L61+3). Figure 9A illustrates a plasmid encoding a peptide containing an Fc region with a "knob" mutation, VH and VL of pan-FZD antibody #5019, VL of LRP antibody #2542, and VH of LRP antibody #2539. [Figure 9B]Figure 9B illustrates an example plasmid encoding a peptide that dimerizes in a knob-into-hole conformation to form pan-FZDag 5019-KH-2539-2542(FP+P-L61+3). Figure 9B illustrates a plasmid encoding a peptide containing an Fc region with a "hole" mutation and nucleic acids encoding pan-FZD antibody #5019 VH and VL, LRP antibody #2542 VH, and LRP antibody #2539 VL. The peptide encoded by these plasmids forms a heterodimer with a tetravalent binding domain, which includes a homodiabody resulting from the pairing of pan-specific FZD antibody #5019 VH and VL; and a bispecific heterodiabody resulting from the pairing of LRP6 antibody #2539 VL and LRP antibody #2542 VH from one peptide with LRP antibody #2539 VH and LRP antibody #2542 VL from the other peptide.

[0052] [Figure 9C] This is a schematic diagram of the heterodimer 5019-K / H-2539:2542(FP+P-L61+3) with a knob-into-hole configuration. By using the knob-in-hole configuration within the Fc domain, the modularity of the molecule can be increased to up to four different binding sites. For this molecule (5019-K / H-2539:2542), a pan-FZD homodiabody is engineered to create one side of the Fc domain, and a heterodiabody containing Wnt3(2539) and Wnt1(2542)LRP6 binding sites is created on the other side of the Fc domain.

[0053] [Figure 10A] 5019-knob-2539:2542 is an annotation of the domain of the nucleic acid sequence of a polyvalent binding molecule (SEQ ID NO: 21 plus additional 3'TGA, and its complementary sequence). [Figure 10ACont] 5019-knob-2539:2542 is an annotation of the domain of the nucleic acid sequence of a polyvalent binding molecule (SEQ ID NO: 21 plus additional 3'TGA, and its complementary sequence). [Figure 10B]5019-knob-2539:2542 is an annotation of the domain of the nucleic acid sequence of a polyvalent binding molecule (SEQ ID NO: 21 plus additional 3'TGA, and its complementary sequence). [Figure 10BCont] [Figure 10B] 5019-knob-2539:2542 is an annotation of the domain of the nucleic acid sequence of a polyvalent binding molecule (SEQ ID NO: 21 plus additional 3'TGA, and its complementary sequence).

[0054] [Figure 11A] This figure shows the design and validation of a tetravalent ligation molecule that binds to the Wnt1 binding site and the Wnt3 binding site (FLAg) of LRP6 as an activator of the Wnt-β-catenin pathway with FZD. Figure 11A illustrates inhibitory (top) and specific (bottom) activity against FZDFab. [Figure 11B] This figure shows the design and validation of a tetravalent ligation molecule that binds to the Wnt1 binding site and the Wnt3 binding site (FLAg) of FZD and LRP6 as activators of the Wnt-β-catenin pathway. Figure 11B illustrates the inhibition of Wnt1 or Wnt3A signaling by LRP6Ab in the labeled in-diabody-Fc form. [Figure 11C] This figure shows the design and validation of a tetravalent binding molecule that binds to the Wnt1 binding site and the Wnt3 binding site (FLAg) of FZD and LRP6 as an activator of the Wnt-β-catenin pathway. Figure 11C illustrates the molecular structure of tetravalent FLAg. [Figure 11D] This figure shows the design and validation of a tetravalent binding molecule that binds to the Wnt1 and Wnt3 binding sites (FLAg) of FZD and LRP6 as activators of the Wnt-β-catenin pathway. Figure 11D shows dose-response curves for activation of the LEF / TCF reporter gene (y axis) in HEK293T cells using serial dilutions (x axis) of pan-specific FLAg proteins (FP+P-L61+1, FP+P-L63+3, and FP+P-L61+3). [Figure 11E]This figure shows the design and validation of a tetravalent binding molecule that binds to the Wnt1 and Wnt3 binding sites (FLAg) of FZD and LRP6 as an activator of the Wnt-β-catenin pathway. Figure 11E illustrates the levels of β-catenin protein in RKO cells 30 minutes after treatment with the indicated concentrations of pan-FLAg (FP+P-L61+3). [Figure 11F] This figure shows the design and validation of a tetravalent binding molecule that binds to the Wnt1 and Wnt3 binding sites (FLAg) of FZD and LRP6 as an activator of the Wnt-β-catenin pathway. Figure 11F illustrates the time course of β-catenin and phosphorylated Dishevelled-2 (p-Dvl2) protein levels in RKO cells treated with 10 nM pan-FLAg (FP+P-L61+3).

[0055] [Figure 12A] This figure shows the characterization and detailed analysis of the binding and activity of FLAG FP+P-L61+3. Figure 12A illustrates the binding kinetics of FP+P-L61+3 to 9 of 10 human FZD CRDs and human LRP6 ECD. [Figure 12B] This figure shows the characterization and detailed analysis of the binding and activity of FLAG FP+P-L61+3. Figure 12B illustrates the binding kinetics of FP+P-L61+3 to 9 of 10 human FZD CRDs and human LRP6 ECD. [Figure 12C] This figure shows the characterization and detailed analysis of the binding and activity of FLAG FP+P-L61+3. Figure 12C demonstrates that FP+P-L61+3 behaves similarly to conventional IgG and interacts with FcRn in a dose- and pH-dependent manner. [Figure 12D]This figure shows the characterization and detailed analysis of the binding and activity of FLAG FP+P-L61+3. Figure 12D demonstrates that FP+P-L61+3 exhibits similar behavior to IgG in terms of interactions with other Fc effectors, including complement (C1q), natural killer cell marker CD16a, B cell marker CD32a, and monocyte and macrophage marker CD64.

[0056] [Figure 13A] This figure demonstrates that a 3-day treatment with 30 nM FP+P-L61+3for induced robust induction of the mesoderm marker BRACHYURY and a decrease in pluripotency marker OCT4 expression to levels equivalent to treatment with 6 μM GSK3 inhibitor CHIR99021. [Figure 13B] This figure demonstrates that a 3-day treatment with 30 nM FP+P-L61+3for induced robust induction of the mesoderm marker BRACHYURY and a decrease in pluripotency marker OCT4 expression to levels equivalent to treatment with 6 μM GSK3 inhibitor CHIR99021.

[0057] [Figure 14] This figure shows representative fluorescence images of small intestinal sections from LGR5-GFP mice treated with vehicle, C59, or pan-FLAg(FP+P-L61+3)+C59. LGR5-GFP is expressed in stem cells in the lower part of the crypts. Cell nuclei were counterstained with DAPI. [Modes for carrying out the invention]

[0058] Described herein are polyvalent binding molecules comprising an Fc domain, an FZD binding domain, and a Wnt coreceptor binding domain, wherein the binding domain is attached to the opposite end of the Fc domain. The polyvalent binding molecules of the present invention are agonists of the Wnt signaling pathway and are interchangeably referred to herein as FZD agonists or FZDag. Wnt ligands function by promoting clustering of the FZD receptor and coreceptor. While we do not wish to be constrained by theory, it is assumed that the polyspecific molecules described herein simultaneously bind to the FZD receptor and Wnt coreceptor, thereby activating the Wnt signaling pathway.

[0059] The modularity and efficacy of the polyvalent binding molecules for activating the Wnt signaling pathway described herein are in contrast to Wnt surrogates described in prior art, which consist of a monovalent FZD binding ligand and an LRP5 / 6 binding ligand, and do not have a binding ligand attached to the opposite end of the Fc domain. In one embodiment of the present invention, the FZD binding domain includes a binding portion derived from an antibody or polypeptide that specifically binds to one or more FZD receptors, and the coreceptor binding domain includes a binding portion that binds to a coreceptor, such as LRP5 / 6, ROR1 / 2, RYK, or PTK7. In one embodiment of the present invention, the antibody or polypeptide that specifically binds to one or more FZD receptors binds to the cysteine-rich domain (CRD) of one or more FZD receptors.

[0060] The amino acid sequence of the FZD receptor and the nucleotide sequence encoding the FZD receptor, as well as antibodies and libraries of antibodies that conjugate the FZD or Wnt coreceptors LRP5 / 6, ROR1 / 2, RYK, or PTK7, are readily available or can be produced using methods known in the art (e.g., U.S. Patent Application Publication No. 2015 / 0232554 of inventors Gurney et al., and U.S. Patent Application Publication No. 2015 / 0232554 of inventors Sidhu et al.). See U.S. Patent Application Publication No. 2016 / 0194394, and U.S. Patent Application Publication No. 20190040144 by inventors Pan et al.; U.S. Patent Application Publication No. 2017 / 0166636 by inventors Wu et al.; U.S. Patent Application Publication No. 2016 / 0208018 by inventors Chen et al.; U.S. Patent Application Publication No. 2016 / 0053022 by inventors Maceda et al.; and U.S. Patent Application Publication No. 2015 / 031293 by inventors Damelin et al.

[0061] Methods for generating peptides or polypeptides that bind to selected targets are well known in the art; see, for example, Sidhu et al. Methods in Enzymology (2000) 328: 333-336. For example, a library of affibodies that bind to FZD or Wnt coreceptors may be obtained according to protocols known in the art (e.g., U.S. Patent No. 5,831,012 and Lofblom et al., FEBS Letters 584 (2010) 2670-2680); a library of ankyrin repeat proteins used for selecting peptides that bind to FZD or Wnt coreceptors may be obtained according to protocols known in the art (e.g., see WO02 / 020565 by inventors Stumpp et al.); and a library of fibronectin repeat proteins used for selecting peptides that bind to FZD or Wnt coreceptors may also be obtained according to protocols known in the art (e.g., see U.S. Patent No. 9,200,273 by inventors Diem and Jacobs). Furthermore, the peptide that binds to the FZD or Wnt coreceptor may be a finomer, which is a small binding protein derived from the SH3 domain of human Fyn, or an artificial receptor protein based on human apolipoprotein D, "antikalin," may be produced using methods known in the art. See, for example, Silacci et al., J. Biol. Chem (2014) 289(20):14392-8 and Vogt and Skerra, ChemBioChem (2004) 5, 191-199.

[0062] Antibodies suitable as sources of antigen-binding peptides described herein may be isolated by screening a combinatorial library for one or more polypeptides having the desired activity. For example, various methods for generating phage display libraries for antibodies having the desired binding properties and for screening such libraries are known in the art. Such methods are summarized, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and in, for example, McCafferty et al., Nature 348:552-554;Clackson et al., Nature 352: 624-628 (1991);Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003);Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004);Lee et al., J. Mol. Biol. Further details are found in 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004). In certain phage display methods, the gene repertoires of VH and VL can be separately cloned by polymerase chain reaction (PCR), randomly recombined into a phage library, and then this library can be screened for antigen-binding phages, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).Phages typically present antibody fragments as either single-chain Fv(scFv) fragments or Fab fragments. Libraries obtained from immunized sources provide high-affinity antibodies against immunogens without requiring hybridoma construction. Alternatively, naive libraries can be cloned (e.g., from humans) to provide single-source antibodies against a wide range of non-self and self-antigens without performing any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be constructed synthetically, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992), by cloning an unreorganized V gene segment from stem cells and using PCR primers containing random sequences to encode a highly variable CDR3 region and achieve in vitro rearrangement. Examples of patent publications describing human antibody phage libraries include U.S. Patent No. 5,750,373, and U.S. Patent Application Publications 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360. Antibodies or antibody fragments isolated from human antibody libraries are considered to be human antibodies or human antibody fragments as defined herein.

[0063] Therefore, those skilled in the art can easily prepare an Fc domain and mix and fit a polyvalent FZD-binding domain and a Wnt co-receptor-binding domain with the desired specificity onto the N-terminus and C-terminus of the Fc domain to prepare a polyvalent binding molecule that binds the desired FZD receptor and co-receptor and thereby activates a specific Wnt pathway. These specific agonists will serve as powerful tools in enhancing cell proliferation, differentiation, organoid survival and maintenance, and tissue regeneration in vivo. These specific agonists will also serve as powerful tools for profiling the FZD specificity involved in these processes. For example, as described herein, FZD5Ag, rather than FZD4Ag, rescues growth defects in LGK974-treated RNF43 mutant PDAC cell lines, which highlights the importance of FZD5 over the FZD4 receptor in this process.

[0064] One embodiment of the present invention is a method for influencing the binding of a peptide to an FZD receptor and a Wnt coreceptor on a cell, wherein the peptide binding to both the FZD receptor and the coreceptor activates the Wnt signaling pathway in the cell. The method comprises selecting a fragment having a C-terminal and an N-terminal Fc domain or a CH3 domain thereof, ligating a first polyvalent binding domain for binding to the FZD receptor to one end of the Fc domain, ligating a second polyvalent binding domain for binding to the Wnt coreceptor to the other end of the Fc domain, thereby forming a polyvalent binding molecule, and then contacting a cell expressing the FZD receptor and coreceptor under conditions that activate the Wnt signaling pathway.

[0065] In one embodiment of the present invention, the multivalent binding domain may include a single-chain variable fragment (ScFv) that binds to one or more FZD receptors, a ligand for an FZD receptor or coreceptor, or a fragment thereof that binds to an FZD receptor or coreceptor. In another embodiment, the binding domain does not include a single-chain variable fragment (ScFv) that binds to one or more FZD receptors, a ligand for an FZD receptor or coreceptor, or a fragment thereof that binds to an FZD receptor or coreceptor.

[0066] In one embodiment of the present invention, the polyvalent binding domain of at least one FZD or co-receptor comprises a diabody having two peptides, each peptide containing a heavy chain variable domain (VH) linked to a light chain variable domain (VL), in which case the VH and VL from one peptide pair with the VL and VH of the other peptide, resulting in the binding domain having two epitope binding sites. The VH and VL domains may be the VH and VL of an antibody that binds to a Wnt binding site on the FZD receptor or co-receptor. The VH or VL derived from a certain antibody, i.e., the original antibody, may have 50%, 55%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the VH and VL of the original antibody, and still retain binding affinity to the FZD receptor or co-receptor site to which the antibody binds.

[0067] In one embodiment of the present invention, the polyvalent bond molecule of the present invention comprises the polyvalent bond molecules of Table 1 (Table 1 includes Table 1A and Table 1B: Table 1A shows the nucleotide and amino acid sequences of exemplary polyvalent bond molecules of the present invention; Table 1B shows the nucleotide sequences of various domains of exemplary polyvalent bond molecules). In one embodiment of the present invention, the polyvalent bond molecule of the present invention is essentially composed of the polyvalent bond molecules of Table 1. In one embodiment of the present invention, the polyvalent bond molecule of the present invention consists of the polyvalent bond molecules of Table 1.

[0068] In one embodiment of the present invention, the polyvalent binding domain comprises one or more VL domains and VH domains of the molecules in Table 1. In one embodiment of the present invention, the polyvalent binding domain of the polyvalent molecule is essentially composed of one or more VL domains and VH domains of the molecules in Table 1. In one embodiment of the present invention, the polyvalent binding domain of the polyvalent molecule comprises one or more VL domains and VH domains of the molecules in Table 1. In one embodiment of the present invention, the binding domain of the polyvalent molecule described herein comprises VH domains and VL domains that have at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity with the VH and VL of the molecules specified in Table 1, and retain binding affinity to the antigen to which the molecules specified in Table 1 bind.

[0069] In one embodiment of the present invention, the binding domain of the polyvalent molecule described herein includes one or more complementarity-determining regions (CDRs) of the molecules specified in Table 1. In one embodiment of the present invention, the binding domain of the polyvalent molecule described herein includes a CDR that has at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity with the CDRs of the molecules specified in Table 1 and retains binding affinity to the antigen to which the molecules specified in Table 1 bind.

[0070] The FZD receptor to which the polyvalent binding molecule of the present invention binds may be FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, or FZD10. The FZD receptor may be FZD1, FZD2, FZD4, FZD5, FZD7, or FZD8. The polyvalent binding molecule may bind to a single FZD receptor or may pan-specifically bind to more than one FZD receptor. The FZD polyvalent binding domain may bind to, for example, FZD1, FZD2, FZD4, FZD5, FZD7, and FZD8. The FZD polyvalent binding domain may specifically bind to one FZD receptor, for example, FZD2, FZD4, FZD5, or FZD6.

[0071] In one embodiment of the present invention, the FZD-binding domain is monospecific and binds to a single epitope on the FZD receptor. In another embodiment of the present invention, the FZD-binding domain is bispecific and binds to two epitopes on the FZD receptor.

[0072] The coreceptor-binding domain may bind to any Wnt coreceptor, such as LRP5 / 6 or ROR1 / 2. A polyvalent coreceptor-binding domain may bind to, for example, LRP5 / 6, PTK7, ROR1 / 2, RYK, GPR12, TSPAN12, or CD133. In one embodiment of the present invention, the polyvalent coreceptor-binding domain binds to LRP5 or LRP6.

[0073] In one embodiment of the present invention, the coreceptor multivalent binding domain binds to a single epitope on the coreceptor, for example, an epitope on LRP5 / 6 that binds to Wnt1 or Wnt3. In another embodiment of the present invention, the coreceptor multivalent binding domain binds to two epitopes within the coreceptor, for example, an epitope on LRP5 / 6 that binds to Wnt1 and an epitope that binds to Wnt3. The Wnt coreceptor to which the multivalent binding molecule of the present invention binds may be LRP5 or LRP6, PTK7, ROR1, ROR2, RYK, GPR124, TSPAN12, or CD133.

[0074] In one embodiment of the present invention, the polyvalent binding molecule includes an Fc domain, in which case the Fc domain is the Fc domain of an immunoglobulin or a fragment containing its CH3 domain. In one embodiment of the present invention, the immunoglobulin is IgG. In one embodiment of the present invention, the IgG is IgG1.

[0075] One embodiment of the present invention is a method for activating the Wnt signaling pathway in cells, the method comprising contacting cells having an FZD receptor and a Wnt coreceptor with the polyvalent binding molecule of the present invention in an amount effective for activating Wnt signaling.

[0076] In one embodiment of the present invention, at least one polyvalent binding domain includes an scFv that binds to an FZD receptor or coreceptor, or includes a ligand for an FZD receptor or coreceptor, or a fragment of said ligand. In another embodiment of the present invention, at least one polyvalent binding domain does not include an scFv that binds to an FZD receptor or coreceptor, or does not include a ligand for an FZD receptor or coreceptor, or a fragment of said ligand.

[0077] In one embodiment of the present invention, the FZD multivalent binding domain includes an FZD diabody, and the coreceptor multivalent binding domain includes a coreceptor diabody, in which case the diabody includes two peptides, each containing a heavy chain variable domain (VH) linked to a light chain variable domain (VL), and the binding domain is formed by pairing the VH and VL from one peptide with the VH and VL from the other peptide, thereby forming the binding domain.

[0078] The VH and VL domains of the FZD-binding domain may be derived from antibodies that bind to the FZD receptor and either antagonize Wnt signaling or inhibit the binding of Wnt ligand to the FZD receptor. The VH and VL domains of the FZD-binding domain may also be derived from antibodies that bind to the FZD receptor without antagonizing or inhibiting the binding of Wnt ligand to the FZD receptor.

[0079] The VH and VL coreceptor-binding domains may originate from antibodies that bind to the coreceptor and either antagonize Wnt signaling or inhibit the binding of Wnt ligand to the coreceptor. Alternatively, the VH and VL coreceptor-binding domains may originate from antibodies that bind to the coreceptor without antagonizing Wnt signaling or inhibiting the binding of Wnt ligand to the coreceptor.

[0080] In the polyvalent binding molecule of the present invention, one or both of the binding domains may be divalent, and one or both of the divalent binding domains may be bispecific to the FZD receptor or coreceptor. In one embodiment of the present invention, both binding domains are divalent and bispecific, and each binding domain binds to two different epitopes on the FZD receptor or coreceptor, which are their respective targets. For example, the binding molecule may contain a divalent and bispecific FZD binding domain to the FZD receptor (binding to two different epitopes), or the binding molecule may contain a divalent and bispecific coreceptor binding domain to the coreceptor.

[0081] In one embodiment of the present invention, the FZD binding domain is attached to the N-terminus of the Fc domain of the multivalent binding molecule, and the co-receptor binding domain is attached to the C-terminus of the Fc domain.

[0082] Furthermore, one embodiment of the present invention is a nucleic acid molecule encoding a polyvalent binding molecule as described herein, such as an expression cassette and vector containing a nucleic acid molecule encoding a polyvalent binding molecule. This nucleic acid molecule can be inserted into a vector and expressed in a suitable host cell, and the polyvalent binding molecule can then be isolated from that cell using methods well known in the art. As used in the present invention, the term “vector” refers to a nucleic acid delivery vehicle or plasmid that can be engineered to contain a nucleic acid molecule, for example, a nucleic acid sequence encoding a polyvalent binding molecule as described herein. A vector that can express a protein when a polynucleotide is inserted into it is called an expression vector. A vector can be inserted into a host cell by transformation, transfection, or other means, and as a result, the introduced gene material can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages, e.g., lambda phages or M13 phages; and animal viruses. Animal viruses include, but are not limited to, reverse transcriptase viruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), varicella virus, baculovirus, papillomavirus, and papovavirus (e.g., SV40). Vectors may contain multiple components that control the expression of the polyvalent binding molecules described herein, and include, but are not limited to, promoters, e.g., viral or eukaryotic promoters, e.g., CMV promoters; signal peptides, e.g., TRYP2 signal peptides; transcription initiation factors; enhancers; selection elements; and reporter genes. Furthermore, the vector may also contain one or more replication initiation sites.

[0083] In the context of the present invention, the term "host cell" refers to a cell capable of transporting a vector, and such cells include, but are not limited to, prokaryotic cells such as Escherichia coli and Bacillus subtilis; fungal cells such as yeast and Aspergillus; insect cells such as Drosophila S2 cells and Sf9 cells; or human cells, such as animal cells including fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0084] One embodiment of the present invention is a pharmaceutical composition comprising an FZD agonist as described herein and a pharmaceutically acceptable excipient. The pharmaceutical composition may further contain additional agents that activate the Wnt pathway, such as Norrin or R-spondin. The pharmaceutical composition may consist of, or essentially consist of, a polyvalent bond molecule and a pharmaceutically acceptable carrier or excipient as described herein. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. AR Gennaro, Mack Publishing Company, Easton, Pa. 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to make the formulation isotonic. Examples of pharmaceutically acceptable carriers include, but are not limited to, physiological saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Furthermore, the carrier may include a sustained-release preparation such as a semipermeable matrix of a solid hydrophobic polymer containing the antibody, where such a matrix is ​​in the form of a molded article, e.g., a film, liposomes, or microparticles. It will be apparent to those skilled in the art that, for example, certain carriers may be more preferred depending on the administration route and concentration of the FZD agonist being administered.

[0085] Wnt signaling is a ubiquitous pathway that regulates cell and tissue differentiation. For example, in eye development, a specific Wnt pathway, namely the Norrin-FZD4 pathway, has been identified as playing a role in retinal neovascularization. Signaling via the Norrin-FZD4 pathway is necessary for the development and maintenance of retinal vascular structure. Mutations affecting genes in this pathway can result in several childhood vitreoretinopathy conditions, such as Norie's disease, familial exudative vitreoretinopathy (FEVR), and pseudoglioma-osteoporosis syndrome. Furthermore, retinopathy of prematurity (ROP) is associated with mutations in this pathway, and Wnt pathway mutations have been reported in Coats' disease and persistent fetal vascularity (PFV). The Norrin-FZD pathway is also involved in the development of CNS angiogenesis. Genetically removing Norrin, FZD4, Lrp5, and the co-receptor tetraspanin 12 (Tspan-12) results in incomplete angiogenesis and barrier collapse in both retinal and cerebellar blood vessels (Cho et al. (2017) Neuron 95, 1056-1073; Zhou et al., (2014) J Clin Invest 124:3825-3846). The FZD4 agonist of the present invention, specifically FZD4 FLAg containing an FZD4 binding domain at one end of the Fc receptor and LRP5 and / or LRP6 at the other binding domain of the Fc domain, is assumed to enhance barrier function and promote angiogenesis; for example, treatment with FZD4 FLAg is assumed to promote the development and maintenance of retinal vascular structures and / or the blood-retinal barrier (BRB) and blood-brain barrier (BBB). Therefore, an aspect of the present invention is a method for promoting and / or maintaining retinal vascular structure by treating ocular tissue, such as retinal tissue, with an effective amount of FZD4 FLAg via local or systemic administration. Another aspect of the present invention is a method for promoting and / or maintaining BBB vascular structure by treating the BBB with an effective amount of FZD4 FLAg via systemic administration.A further aspect of the present invention is a method for treating a subject having a disorder characterized by reduced retinal or cerebral angiogenesis by administering an effective dose of FZD4 FLAg, the effective dose being sufficient to increase retinal or cerebral angiogenesis in such subject. The subject may be a fetus.

[0086] Pathologically low levels of Wnt signaling are associated with osteoporosis, polycystic kidney disease, and neurodegenerative diseases. Controlling Wnt pathway activation has been shown to promote regenerative processes such as tissue repair and wound healing. (Zhao J, Kim KA, and Abo A, Trends Biotechnol. 27(3):131-6 (Mar. 2009). See also Logan CY and Nusse R, Annu. Rev. Cell. Dev. Biol. 20:781-810 (2004); Nusse R., Cell Res. 15(1):28-32 (Jan. 2005); Clevers H, Cell 127(3):469-80 (3 Nov. 2006).) Proof-of-concept experiments have demonstrated the role of Wnt signaling in osteoporosis or mucositis. Furthermore, increased Wnt signaling has been suggested to be beneficial in the treatment of diabetes and other metabolic diseases. Decreased Wnt signaling is associated with metabolic diseases. Loss of function LRP6 R611CMutations lead to early coronary artery disease, metabolic syndromes, and osteoporosis in humans. Main A et al, Science 315:1278 (2007). "Loss-of-function mutations in LRP5 are associated with osteoporosis, glucose metabolism deficiency, and hypercholesterolemia in humans." Saarinnen et al., Clin Endocrinol 72:481 (2010). Severe hypercholesterolemia, impaired fat tolerance, and progression of atherosclerosis in mice lacking both LRP5 and apoE. Magoori K. et al., JBC 1 1331 (2003). LRP5 is essential for normal cholesterol metabolism and glucose-induced insulin secretion in mice. Fujino et al., PNAS 100:229 (2003). TCF7L2 variants pose a risk of type 2 diabetes. Grant et al., Nat Genet 38:320 (2006); Florez et al., N Engl J Med 355:241 (2006). Increased Wnt signaling may be beneficial for treating metabolic diseases. Therefore, administering the polyvalent conjugated molecule of the present invention to subjects suffering from metabolic diseases may be useful for treating the metabolic diseases of those subjects.

[0087] Inflammatory bowel disease (IBD) is a group of inflammatory conditions of the colon and small intestine. The main types of IBD are Crohn's disease and ulcerative colitis. The RSP01 protein has been shown to induce remission of inflammatory bowel disease in animal models. Zhao J et al., Gastroenterology 132:1331 (2007). Therefore, administering the polyvalent conjugated molecule of the present invention to subjects suffering from IBD is useful for treating IBD in those subjects.

[0088] Therefore, one embodiment of the present invention is a method for treating subjects having a condition associated with reduced Wnt signaling, the method comprising administering an effective amount of the FZD agonist of the present invention to a subject in need thereof. The above conditions may include, for example, osteoporosis, polycystic kidney disease, neurodegenerative diseases, mucositis, short bowel syndrome, bacterial migration of the gastrointestinal mucosa, enterotoxigenic or enteric infectious diarrhea, celiac disease, non-tropical sprue, lactose intolerance, and other conditions in which exposure to food causes smoothing of mucociliary slicks and malabsorption, atrophic gastritis and diabetes mellitus, fractures, tissue regeneration, such as tissue repair and wound healing, as well as metabolic diseases such as diabetes mellitus, and melanoma. Examples of damaged tissues that can be treated using the method of the present invention include, but are not limited to, intestinal tissue, cardiac tissue, liver tissue, kidney tissue, skeletal muscle, brain tissue, bone tissue, connective tissue, and skin tissue. The polyvalent binding molecules of the present invention can be administered to subjects suffering from a disease or condition characterized by low Wnt signaling. The polyvalent conjugated molecule of the present invention is administered to a subject in an amount effective in increasing Wnt signaling and achieving remission of a disease or condition within the subject.

[0089] Mucositis is a clinical complication of cancer therapy. It is caused by the cytotoxic effects of radiation or chemotherapy on rapidly proliferating cells. Mucositis consists primarily of epithelial damage affecting the mucosa of the intestines and oral cavity. Clinical signs include severe oral pain, nausea, diarrhea, and malnutrition, and in severe cases, sepsis and death. These symptoms often lead to dose limitations in cancer therapy. Currently, there are no available treatments for oral or gastrointestinal mucositis associated with chemotherapy or radiation therapy for solid tumors.

[0090] Oral mucositis is a common and often debilitating complication of cancer treatment. Fifty percent of patients receiving radiation therapy for head and neck cancer and 10–15 percent of patients treated with 5-FU develop grade 3–4 oral mucositis. RSP01 has been shown to induce remission of oral mucositis in animal models. Zhao J et al., PNAS 106:2331 (2010).

[0091] Short bowel syndrome (SBS) results from the functional or anatomical loss of a large segment of the small intestine, and therefore severely impairs digestive and absorptive capacity. Every year, many people undergo resection of a long segment of the small intestine for a variety of reasons, including trauma, inflammatory bowel disease, malignant lesions, and mesenteric ischemia. Various non-surgical procedures, such as radiation therapy, can cause functional short bowel syndrome. Current treatments for short bowel syndrome include food approaches, total parenteral nutrition (TPN), bowel transplantation, and non-transplant abdominal surgery. While these treatments contribute to improved outcomes in SBS patients, they only partially address the underlying problem of reduced small bowel function. There are no current therapies that can accelerate the recovery of the remaining small intestine in SBS patients. See Seetharam and Rodrigues, The Saudi Journal of Gastroenterology 17, 229-235 (2011).

[0092] The digestive tract of adult mammals constitutes one of the most rapidly self-regenerating tissues, where the small intestinal mucosa contains continuous structures folded within proliferative crypts and differentiated villi. In response to mucosal breakdown, the host initiates a healing response, resulting in the restoration of mucosal integrity and the regeneration of mucosal structures. This process is highly dependent on the proliferation of intestinal stem cells. Neal et al., Journal of Surgical Research 167, 1-8 (2010); van der Flier and Clevers, Annual Review of Physiology 71, 241-261 (2009).

[0093] Therefore, factors that regulate the activity of intestinal stem cells play a dominant role in the host's ability to respond to wounds within the intestinal tract. Wnt protein is the most important growth factor supporting the proliferation of intestinal stem cells, and by enhancing Wnt signaling, it leads to increased proliferation of the intestinal epithelium. This results in an increase in the number of small intestinal villi and an increase in the mucosal absorptive surface area.

[0094] Therefore, in one embodiment, the polyvalent conjugated molecule of the present invention is administered to a person suffering from short bowel syndrome. The polyvalent conjugated molecule is administered in an amount sufficient to increase the surface area of ​​gastrointestinal mucosal absorption. Administration of the polyvalent conjugated molecule of the present invention has successful outcomes when a person with accidental short bowel syndrome adapts to enteral nutrition, or when a person with common SBS absorbs nutrients from enteral nutrition, or when the total amount of parenteral nutrition required daily to maintain body weight decreases for that person.

[0095] Prevention of bacterial translocation. In one embodiment, the antibody of the present invention is administered to individuals at risk of developing sepsis due to enterobacteria. The polyvalent conjugated molecule is administered in an amount sufficient to increase the integrity of the gastrointestinal mucosa, and therefore prevent enterobacteria from passing through the individual's bloodstream. Reduced gastrointestinal mucosal integrity (compared to the integrity of the gastrointestinal mucosa that is normal in the human population) is a major source of bloodstream infections and sepsis in critically ill patients. Administration of the polyvalent conjugated molecule has a successful outcome when the number of cases of bacteremia and sepsis observed in intensive care unit (ICU) patients is lower than in patients who have not been administered the polyvalent conjugated molecule of the present invention.

[0096] Accelerated recovery during or after enterotoxogenic or enteropathy-related infectious diarrhea. Infectious diarrhea is a major problem in children. In one embodiment, the polyvalent conjugated molecule of the present invention is administered in an amount sufficient to shorten the time to the end of diarrhea or the time to normal bowel motility. The polyvalent conjugated molecule of the present invention can be administered in addition to standard treatment, which includes oral or parenteral rehydration and, optionally, antibiotics. Administration of the polyvalent conjugated molecule of the present invention has a successful outcome when a reduction in hospitalization, a reduction in length of hospital stay, or a reduction in the incidence of complications such as dehydration and electrolyte abnormalities is observed in pediatric patients compared to pediatric patients who have not been administered the polyvalent conjugated molecule of the present invention.

[0097] Celiac disease, non-tropical sprue, lactose intolerance, and other conditions in which exposure to food causes mucociliary blunting and malabsorption. In one embodiment, the polyvalent conjugated molecule of the present invention is administered in an amount sufficient to increase the surface area of ​​mucosal absorption. The polyvalent conjugated molecule of the present invention can be administered in addition to standard treatment, which mainly involves avoiding harmful foods and, if applicable, nutritional supplements. Administration of the polyvalent conjugated molecule of the present invention has a successful outcome when a person suffering from celiac disease, non-tropical sprue, lactose intolerance, or other conditions resists enteral nutrition, or when a person suffering from any of the above conditions absorbs nutrients from enteral nutrition, or when the total amount of parenteral nutrition required daily to maintain body weight for that person decreases.

[0098] Atrophic gastritis, specifically a form called environmentally induced dysplastic atrophic gastritis. Atrophic gastritis is a common condition in middle-aged and elderly individuals and is currently treated with vitamin B12 injections. Patients have an increased risk of carcinoid tumors and adenocarcinomas. In the case of carcinoid tumors, administration of this multivalent conjugated molecule has a successful outcome when medical professionals have observed a reduction in tumor incidence by decreasing gastrin production from dysplastic G cells. This multivalent conjugated molecule should not be administered when medical professionals determine that the tumor is activated by enhancement of the Wnt pathway.

[0099] The FZD agonists of the present invention may be administered topically or orally, for example by injection (e.g., subcutaneously, intravenously, intraperitoneally, etc.). Depending on the route of administration, the active compound may be coated onto a material to protect it from the action of acids and other natural conditions that inactivate the compound. The polyvalent conjugated molecules described herein may be dissolved or suspended in a pharmaceutically acceptable, preferably aqueous, carrier. Furthermore, the composition may contain excipients, such as buffers, binders, propellants, diluents, fragrances, lubricants, etc. A broad list of excipients that can be used in such compositions can be cited, for example, from Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). The polyvalent conjugated molecules may also be administered together with immunostimulants, such as cytokines.

[0100] One embodiment of the present invention includes a method for producing induced pluripotent stem (iPS) cells, the method comprising culturing somatic cells under conditions suitable for reprogramming somatic cells, wherein the culture conditions further include the polyvalent binding molecules described herein. Methods for generating pluripotent stem cells are well known in the art; see, for example, Takahashi and Yamanaka, (2006), Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors, Cell 126, 663-676; Takahashi et al. (2007) Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors, Cell 131, 861-872; Yu et al. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920; U.S. Patents 8,546,140 and 8,268,620. In one embodiment of the present invention, the polyvalent binding molecule of the present invention is included in the culture medium in an amount sufficient to accelerate the generation of iPS cells.

[0101] One embodiment of the present invention includes a method for directing the differentiation of pluripotent stem cells (PSCs) or induced pluripotent stem (iPS) cells, the method comprising culturing cells under conditions suitable for oriented differentiation, wherein the culture conditions further include an effective amount of polyvalent binding molecules as described herein. Studies of mouse and human PSCs have identified specific approaches for adding growth factors, such as Wnt, which can induce differentiation of PSCs into various lineages. Methods for oriented differentiation of PSCs, including activation of Wnt signaling, are known in the art; see, for example, Lam et al. (2014) Semin Nephol 34(4); 445-461; Yucer et al. (September 6, 2017) Scientific Reports 7, Article number 10741. It is assumed that the Wnt signaling pathway can be activated using the polyvalent binding molecules described herein, thereby directing the differentiation of PSCs.

[0102] One embodiment of the present invention is a method for enhancing tissue regeneration in a subject in need thereof, the method by activating Wnt signaling in such subject by administering an effective amount of the polyvalent conjugated peptide described herein to such subject.

[0103] One embodiment of the present invention includes a method for enhancing bone healing and / or regeneration in subjects in need, for example, subjects with osteoporosis or fractures, the method comprising administering an effective amount of the polyvalent binding molecule described herein. In a particular embodiment, the polyvalent binding molecule of the present invention comprises a binding domain that binds to FZD2 and a binding domain that binds to LRP5 and / or LRP6. These binding domains may be monovalent or polyvalent, for example, divalent, trivalent, or tetravalent, and may be monospecific or polyspecific, for example, bispecific.

[0104] The subject can be any animal (e.g., mammal), including, but not limited to, humans, non-human primates, horses, cattle, dogs, cats, rodents, etc. Typically, the subject is humans.

[0105] Effective dosages and schedules for administering the polyvalent conjugated molecules described herein may be determined empirically, and making such determinations is within the scope of the art. Those skilled in the art will recognize that the dosage of such an FZD agonist to be administered will vary depending, for example, the recipient of the antibody, the route of administration, the specific type of FZD agonist used, and other drugs being administered. Guidance on selecting an appropriate dose of an FZD agonist can be found in the literature on the therapeutic use of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone, eds., Noges Publications, Park Ridge, NJ, (1985) ch. 22 and pp. 303-357; Smith, Antibodies in Human Diagnosis and Therapy, Haber, eds., Raven Press, New York (1977) pp. 365-389. The dosage range for administering this composition is sufficient to produce the desired effect. The dosage should not be so high that it causes adverse side effects, such as unwanted cross-reactions or anaphylactic reactions. Generally, the dosage will vary depending on age, condition, sex, and the degree of inflammation in the patient, and can be determined by those skilled in the art. The dosage may be adjusted by the individual physician in the event of any contraindications. The dosage may vary and may be administered daily, once or multiple times, over a day or several days. Determining the optimal range of effective doses of the vector, as individual needs vary, is within the skill of the art.

[0106] In recent years, methods have been developed for culturing organelles called "organoids," which are compositions of macroscopic anatomical and cellular types from different tissues. Surprisingly, complete organoids can be generated from a single tissue stem cell, as was first demonstrated using intestinal LGR5+ stem cells isolated from mice. Components in the culture medium that activate the Wnt-β-catenin pathway are known to be necessary for organoid induction, growth, survival, and maintenance. Therefore, purified or prepared R-spondin and Wnt ligands are universally required to grow organoids from different tissues as conditioning media. However, purified Wnt proteins generally have low specific activity and cannot sustain organoid growth. Hence, those skilled in the art rely on the addition of Wnt3A conditioning medium or small molecules, such as GSK3 inhibitors, to generate organoids. However, the production of Wnt3A conditioning medium is labor-intensive, the characteristics of the conditioning medium are inconsistent, and small molecules like GSK3 inhibitors can firmly activate the pathway to toxic levels. The polyvalent binding molecules described herein are easy to produce and purify, possess consistent and reproducible characteristics, and specifically activate Wnt by selectively engaging with desired combinations of FZD receptors and co-receptors, thereby solving the above-mentioned problems.

[0107] One embodiment of the present invention includes a method for generating tissue organoids, the method comprising culturing a tissue in an effective amount of a polyvalent binding molecule described herein. An organoid is a 3D multicellular in vitro tissue construct that mimics its corresponding in vivo organ, and can therefore be used to study the appearance of that organ in a tissue culture dish. Methods for generating organoids are well known in the art, and almost all epithelial organoids derived from adult stem cells in various organs, such as the gastrointestinal tract, require Wnt signaling agonists (including embedding in Matrigel, among other signaling factors) to both maintain the cells and generate in vivo-like complements of the cell type. Wnt signaling has also been used to enhance the development of inner ear organoids in 3D culture and for the generation of kidney organoids; see, for example, Natalie de Souza (2018) Nature Methods s 15(1): 23; DeJonge et al. (2016) PLosOne 11(9), e0162508; Akkerman and Defize, (2017) Bioessays 39, 4, 1600244. The polyvalent conjugated molecules of the present invention can be included in the culture medium of organoids in an amount sufficient to enhance their growth, survival, and maintenance in culture. Thus, one embodiment of the present invention comprises a method for enhancing the culture of tissue organoids, the method comprising a culture medium containing an effective amount of the polyvalent conjugated molecules described herein.

[0108] Furthermore, one aspect of the present invention is a method for producing a polyvalent bond molecule as described herein. In one embodiment of the present invention, the polyvalent bond molecule is a) Select an Fc domain having a C-terminus and an N-terminus. b) Identifying antibodies that bind to one or more FZD receptors, c) Identifying antibodies that bind to one or more Wnt coreceptors, d) Generating a nucleic acid molecule comprising (i) a nucleotide sequence encoding the Fc domain of step a, (ii) a nucleotide sequence encoding the peptide of step b, or a nucleotide sequence encoding the VL and / or VH of the antibody of step b, or a nucleotide sequence encoding the VL and / or VH derived from the antibody of step b that binds one or more FZD receptors, and (iii) a nucleotide sequence encoding the peptide of step c, or a nucleotide sequence encoding the VL and / or VH of the antibody of step c, or a nucleotide sequence encoding the VL and / or VH derived from the antibody of step c that binds one or more Wnt coreceptors. e) Expressing the nucleic acid molecule of (d) to produce a polypeptide, wherein the polypeptide dimerizes to form a tetravalent binding molecule comprising (i) an Fc domain, (ii) an FZD binding domain, and (iii) a Wnt coreceptor binding domain, the FZD binding domain comprising the peptide of step b or the VL and / or VH of step b and ligated to one end of the Fc domain, and the Wnt coreceptor binding domain comprising the peptide of step c or the VL and / or VH of step c and ligated to the other end of the Fc domain, thereby forming a polyspecific binding molecule. It is generated by [the following method / system].

[0109] The peptides that bind to one or more of the above-mentioned FZD receptors may be synthetic polypeptides, such as synthetic peptides, aphibodies, ankyrin repeat proteins, fibronectin repeat proteins, finomers, or anticarin, or peptides of native proteins that bind to FZD receptors. Native proteins may be, for example, Wnt, and may include Wnt-1, Wnt-2, Wnt-2b, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7a / b, Wnt-7b, Wnt-8a, Wnt-8b, Wnt-9a, Wnt-9b, Wnt-10a, Wnt-10b, Wnt-11, or Wnt-16b. The peptide in step b may be polyvalent and bind to more than one site on the FZD, for example, it may be divalent, trivalent, or tetravalent, and may be monospecific and bind to a single epitope, or it may be polyspecific and bind to more than one epitope on the FZD.

[0110] The peptide that binds to one or more Wnt coreceptors may be a synthetic peptide, such as an afibody, ankyrin repeat protein, fibronectin repeat protein, finomer, or anticharin, or it may be a peptide of a native protein that binds to the Wnt coreceptor. The native protein may be, for example, Wnt, and may be Wnt-1, Wnt-2, Wnt-2b, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7a / b, Wnt-7b, Wnt-8a, Wnt-8b, Wnt-9a, Wnt-9b, Wnt-10a, Wnt-10b, Wnt-11, or Wnt-16b, or Dickkopf-1.

[0111] The peptide in step c may be polyvalent and bind to more than one epitope on the Wnt coreceptor, for example, it may be bivalent, trivalent, or tetravalent, and may be monospecific and bind to a single epitope, or it may be polyspecific and bind to more than one epitope on the Wnt coreceptor.

[0112] The native protein that binds to the FZD receptor and the native protein that binds to the Wnt coreceptor may be the same protein.

[0113] In one embodiment, the peptide or antibody in step b may bind to FZD2, the peptide in step c may be a Wnt5a peptide, and the antibody in step c may be an antibody that binds to a site on the coreceptor that binds to Wnt5a.

[0114] In one embodiment, the peptide or antibody in step b may bind to FZD4, the peptide in step c may be one or more peptides selected from Norrin, Wnt1, Wnt8, or Wnt5a, and the antibody in step c may be an antibody that binds to a site on a coreceptor that binds to Norrin, Wnt1, Wnt8, or Wnt5a.

[0115] In one embodiment, the peptide or antibody in step b may bind to FZD5, the peptide in step c may be one or more peptides from among Wnt7a, Wnt5a, Wnt10b, or Wnt2, and the antibody in step c may be an antibody that binds to a site on the coreceptor, in which case this site binds to one or more of Wnt7a, Wnt5a, Wnt10b, or Wnt2.

[0116] In one embodiment, the peptide or antibody in step c may bind to LRP6 and / or LRP5, for example, this peptide may be a peptide of Norrin, Wnt1 and / or Wnt3a, and the antibody in step c may be an antibody that binds to a site on LRP6 / LRP5, in which case this site binds to Norrin, Wnt1 and / or Wnt3a.

[0117] In one embodiment, the peptide or antibody in step c may bind to LRP6, for example, this peptide may be Wnt1 or Wnt3a, or both, and the antibody may be an antibody that binds to a site on LRP6 that binds to Wnt1 or Wnt3a.

[0118] In one embodiment, the peptide or antibody in step c binds to ROR1 and / or ROR2.

[0119] In one embodiment, the peptide or antibody in step c may bind to RYK.

[0120] In one embodiment, the peptide or antibody in step c may be conjugated to PTK7.

[0121] In one embodiment, the peptide or antibody in step (b) may be a peptide or antibody that binds to one or more FZD receptors and antagonizes Wnt signaling or inhibits the binding of Wnt to the receptors. In one embodiment, the peptide or antibody in step (b) may be a peptide or antibody that binds to one or more FZD receptors without antagonizing Wnt signaling or inhibiting the binding of Wnt to the receptors. In one embodiment, the peptide or antibody in step (c) may be a peptide or antibody that binds to one or more Wnt coreceptors and antagonizes Wnt signaling or inhibits the binding of Wnt to the coreceptors. In one embodiment, the peptide or antibody in step (c) may be a peptide or antibody that binds to a Wnt coreceptor without antagonizing Wnt signaling or inhibiting the binding of Wnt to the coreceptor. The binding domain may be linked to the Fc domain via a linker. According to the modular aspects of the present invention, it is possible to mix or fit the VH and VL of a peptide or antibody that binds to any given FZD receptor and Wnt coreceptor at the opposite end of the Fc domain to generate a multivalent binding molecule that can engage multiple Frizzled receptor-coreceptor complexes, or to selectively engage a single Frizzled receptor-coreceptor complex to activate Wnt signaling.

[0122] One embodiment of the present invention is a method for producing a polyvalent bond molecule that activates the Wnt signaling pathway, and this method is a) Select an Fc domain that has a C-terminus and an N-terminus. For example, selecting the Fc domain of an immunoglobulin containing the CH3 domain, such as IgG, for example, IgG1. b) Identify antibodies that have binding specificity to 1 or more FZD receptors. c) Identify antibodies that have binding specificity to Wnt coreceptors. d) (i) A nucleotide sequence encoding the selected Fc domain, (ii) Nucleotide sequences encoding VL and / or VH derived from the antibody in step b, (iii) Nucleotide sequences encoding VL and / or VH derived from the antibody in step c and To produce nucleic acid molecules containing, d) Expressing the nucleic acid molecule of (d) to produce a polypeptide, the polypeptide dimerizing via an Fc domain to form a polyvalent binding molecule comprising (i) an Fc domain, (ii) an FZD binding domain, and (iii) a Wnt coreceptor binding domain, wherein the FZD binding domain is ligated to one end of the Fc domain and the Wnt coreceptor binding domain is ligated to the other end of the Fc domain, thereby forming a polyspecific binding molecule. In a preferred embodiment, the polyvalent binding molecule is a dimer of two polypeptides encoding a nucleic acid molecule in which the Fc domain has a knob-in-hole configuration. One or both of the binding domains may be polyvalent binding domains. The antibody of step b may be an antibody fragment that binds to the FZD receptor. The VH and / or VL of step d)(ii) may be identical to the VH and / or VL of the antibody of step b). The antibody of step c may be an antibody fragment that binds to the Wnt coreceptor. The VH and / or VL values ​​in step d)(iii) may be the same as the VH and / or VL values ​​of the antibody in step c).

[0123] The polyvalent molecules of the present invention may be generated by dimerizing two polypeptides in a "knob-in-hole" configuration. The knob-in-hole configuration increases the modularity of the present invention by promoting the association of peptides containing binding sites that bind to different epitopes on FZD receptors or coreceptors, or to different members of the same FZD receptor or coreceptor family; see, for example, Figure 3A. Methods for the engineering of Fc molecules through knobs-in-hole design are well known in the art; see, for example, WO2018 / 026942 by the inventors Van Dyk et al., Carter P. (2001) J. Immunol. Methods 248, 7-15; Ridgway et al. (1996) Protein Eng. 9, 617-621; Merchant AM, et al. (1998) Nat. Biotechnol. 16, 677-681 and et al., (1997) J. Mol. Biol. 270, 26-35.

[0124] Another embodiment of the present invention is a method for promoting interaction between an FZD receptor and a co-receptor on a cell, thereby activating the Wnt signaling pathway in the cell, the method comprising: a) selecting an Fc domain having a C-terminus and an N-terminus, or a fragment thereof containing a CH3 domain; b) ligating a first polyvalent binding domain for binding to the FZD receptor to one end of the Fc domain and a second binding domain for binding to the Wnt co-receptor to the other end of the Fc domain, thereby forming a binding molecule; c) contacting the polyvalent binding molecule with a cell expressing the FZD receptor and the Wnt co-receptor under certain conditions, so that both the FZD receptor and the co-receptor bind to the polyvalent binding molecule, thereby activating the Wnt signaling pathway. One or both of the binding domains may be monovalent or polyvalent, for example, divalent, trivalent, or tetravalent. The FZD-binding domain may include a peptide of a native protein that binds FZD; a synthetic peptide that binds FZD, such as an aphibody, ankyrin repeat protein, fibronectin repeat protein, finomer, or antikalin; a VH fragment and / or VL fragment that binds FZD; an scFV that binds FZD; or a diabody that binds FZD. The Wnt coreceptor-binding domain may include a peptide of a native protein that binds Wnt coreceptors; a synthetic peptide that binds Wnt coreceptors, such as an aphibody, ankyrin repeat protein, fibronectin repeat protein, finomer, or antikalin; a VH fragment and / or VL fragment that binds Wnt coreceptors; an scFV that binds Wnt coreceptors; or a diabody that binds Wnt coreceptors.

[0125] One embodiment of the present invention is a molecule comprising an Fc domain and two binding domains, wherein the first domain binds to an FZD receptor and the second domain binds to a Wnt coreceptor, and these two parts are linked together by a fragment comprising the Fc domain or its CH3 domain, in which case one domain is linked to the N-terminus of the Fc receptor and the other domain is linked to the C-terminus of the Fc receptor. The binding domains may be linked to the Fc receptor directly or via a peptide linker, such as a polypeptide linker or a non-peptidic linker. Suitable linkers are well known in the art, such as the XTEN linker (see WO2013120683 by inventors Schellenberger et al.).

[0126] One embodiment of the present invention is a method for activating the Wnt signaling pathway, comprising contacting cells expressing the FZD receptor and its co-receptor with an effective amount of the polyvalent molecule of the present invention. While not intended to be theoretically bound, it is assumed that the polyvalent molecule described herein binds to both the FZD receptor and its co-receptor, thereby forming a complex that mimics the binding of a Wnt molecule to the FZD receptor and co-receptor(s), and subsequently activates the Wnt signaling pathway.

[0127] The polyvalent bond molecules of the present invention may be prepared by recombination, for example by Gibson assembly (see Gibson et al. (2009). Nature Methods. 6 (5): 343-345 and Gibson DG. (2011). Methods in Enzymology. 498: 349-361), or by synthesis, for example using commercially available synthesis equipment, such as automated synthesizers from Applied Biosystems, Inc., Beckman, etc. By using a synthesizer, natural amino acids may be substituted with unnatural amino acids. The specific sequence and method of preparation will be determined by convenience, cost-effectiveness, required purity, etc. If necessary, various groups may be introduced into the peptide during synthesis or expression, and these groups will enable linkage to other molecules or surfaces.

[0128] In some embodiments, the binding domain is attached to the Fc domain via a peptide linker, such as the XTEN linker. In some embodiments, the peptide linker is at least 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, 25 amino acids, 26 amino acids, 27 amino acids, 28 amino acids, 29 amino acids, 30 amino acids, 31 amino acids, 32 amino acids, 33 amino acids, 34 amino acids, 35 amino acids, 36 amino acids, 37 amino acids, 38 amino acids, 39 amino acids, 40 amino acids, 41 amino acids, 42 amino acids, 43 amino acids, 44 amino acids, 45 amino acids, 46 amino acids, 47 amino acids, 48 ​​amino acids, 49 amino acids, 50 amino acids, 5 Contains 1 amino acid, 52 amino acids, 53 amino acids, 54 amino acids, 55 amino acids, 56 amino acids, 57 amino acids, 58 amino acids, 59 amino acids, 60 amino acids, 61 amino acids, 62 amino acids, 63 amino acids, 64 amino acids, 65 amino acids, 66 amino acids, 67 amino acids, 68 amino acids, 69 amino acids, 70 amino acids, 71 amino acids, 72 amino acids, 73 amino acids, 74 amino acids, 75 amino acids, 76 amino acids, 77 amino acids, 78 amino acids, 79 amino acids, 80 amino acids, 81 amino acids, 82 amino acids, 83 amino acids, 84 amino acids, 85 amino acids, 86 amino acids, 87 amino acids, 88 amino acids, 89 amino acids, 90 amino acids, 91 amino acids, 92 amino acids, 93 amino acids, 94 amino acids, 95 amino acids, 96 amino acids, 97 amino acids, 98 amino acids, 99 amino acids, or at least 100 amino acids. Depending on the embodiment, the peptide linker may be between 5 and 75 amino acids, between 5 and 50 amino acids, between 5 and 25 amino acids, between 5 and 20 amino acids, between 5 and 15 amino acids, or between 5 and 10 amino acids in length. The Fc domain, with or without a linker, has the length and flexibility to enable the polyvalent binding molecule to bind to both the FZD receptor and its co-receptor, thereby activating the Wnt signaling pathway.In one embodiment of the present invention, the Fc domain with or without a linker, or the fragment containing its CH3 domain, is greater than 100 amino acids, greater than 125 amino acids, greater than 150 amino acids, greater than 175 amino acids, or greater than 200 amino acids.

[0129] In this specification and in the appended claims, it should be noted that the singular forms “a,” “an,” and “the” include plural references unless the context otherwise explicitly states. For example, “a cell” includes multiple such cells, and “the peptide” includes one or more peptides and their equivalents, such as polypeptides known to those skilled in the art.

[0130] "Affinity-matured" antibody or "antibody maturation" refers to an antibody that has one or more modifications within one or more hypervariable regions (HVRs) compared to a parent or source antibody without such modifications, and such modifications result in improved antibody affinity to an antigen or other desirable properties of the molecule.

[0131] "Includes" means that the mentioned elements are necessary for the composition / method / kit, but other elements may be included to form the composition / method / kit, etc., within the scope of the claims. For example, a composition containing a polyvalent bond molecule is a composition that, in addition to the polyvalent bond molecule, may include other elements, such as functional parts such as polypeptides, small molecules, or nucleic acids, which are covalently bonded to the polyvalent bond molecule; an agent that promotes the stability of the polyvalent bond molecule composition; an agent that promotes the solubility of the polyvalent bond molecule composition; an adjuvant, etc., except for elements that are included by any negative conditions, as readily understood in the art.

[0132] "Essentially derived from" means a limitation of the scope of the described composition or method to a specified substance or step that does not materially affect the basic and novel features(s) of the subject invention. For example, a polyvalent bond molecule "essentially derived from" the disclosed sequence has the amino acid sequence of the disclosed sequence plus or minus about 5 amino acid residues at the sequence boundary based on the sequence from which it originated, for example, about 5, 4, 3, 2, or about 1 residue fewer than the amino acid residues at the boundary mentioned, or about 1, 2, 3, 4, or 5 residues more than the amino acid residues at the boundary mentioned.

[0133] "Consists of" means excluding any element, step, or component not specified in the claims from the composition, method, or kit. For example, a polyvalent bond molecule "consists of" the disclosed sequence consists only of that disclosed amino acid sequence.

[0134] Where a range of values ​​is indicated, it is understood that each intervening value is also specifically disclosed between the upper and lower limits of that range, up to one-tenth of the lower limit unit, unless the context otherwise explicitly states. Each small range between a described value or intervening value within a described range and any other described value or intervening value within a described range is included within the present invention. The upper and lower limits of these small ranges are independently included in or excluded from the above range, and each range that includes either or both of the limit values, or neither, is also included within the present invention according to any specifically excluded limit values ​​within the described range. If a described range includes one or both of the limit values, the range excluding either or both of those included limit values ​​is also included within the present invention.

[0135] The basic structural unit of an antibody is known to include a tetramer. Each tetramer consists of a pair of two identical polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminus of each chain contains a variable region of approximately 100 to 110 amino acids or more, primarily involved in antigen recognition. The carboxyl-terminus of each chain defines a constant region, primarily involved in effector function. Generally, antibody molecules obtained from humans belong to one of the classes IgG, IgM, IgA, IgE, and IgD, which differ from one another in the properties of the heavy chain present in the molecule. Certain classes have further subclasses, such as IgG1, IgG2, and others. Furthermore, in humans, the light chain can be a kappa chain or a lambda chain.

[0136] Three highly diverse stretches within each of the heavy-chain variable domain VH and light-chain variable domain VL, referred to as complementarity-determining regions (CDRs), are interposed between further conserved lateral stretches known as “framework regions” or “FRs.” Therefore, the term “FR” refers to naturally occurring amino acid sequences between or adjacent to the CDRs of immunoglobulins. The VH domain typically has four FRs, which are referred herein to as VH framework region 1 (FR1), VH framework region 2 (FR2), VH framework region 3 (FR3), and VH framework region 4 (FR4). Similarly, the VL domain typically has four FRs, which are referred herein to as VL framework region 1 (FR1), VL framework region 2 (FR2), VL framework region 3 (FR3), and VL framework region 4 (FR4). In an antibody molecule, three CDRs (CDR-L1, CDR-L2, and CDR-L3) in the VL domain and three CDRs (CDR-H1, CDR-H2, and CDR-H3) in the VH domain are arranged relative to each other in three-dimensional space to form an antigen-binding site within the antibody's variable region. The surface of the antigen-binding site is complementary to the three-dimensional plane of the bound antigen. The amino acid sequences of the VL and VH domains may be numbered, and the CDRs and FRs within them may be identified / defined according to the Kabat numbering system (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.) or the International Immunogenetic Information System (IMGT numbering system; Lefranc et al., 2003, Development and Comparative Immunology 27:55-77). A person skilled in the art will have the knowledge to number the amino acid residues of the VL domain and VH domain, and to identify the CDR and FR within them according to a standardized numbering system such as the IMGT numbering system or the Kabat numbering system.

[0137] The terms “antigen-binding moiety” or “antigen-binding fragment” (or simply “antibody moiety” or “antibody fragment”) of an antibody, as used herein, refer to one or more fragments, portions, or domains of an antibody that retain the ability to specifically bind to an antigen. Fragments of full-length antibodies have been shown to be capable of performing the antigen-binding function of the antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) the Fab fragment, a monovalent fragment consisting of a VL domain, a VH domain, a CL1 domain, and a CH1 domain; (ii) the F(ab')2 fragment, a bivalent fragment containing two F(ab)' fragments linked by disulfide crosslinks in the hinge region; (iii) the Fd fragment, consisting of a VH domain and a CH1 domain; (iv) the Fv fragment, consisting of the VL domain and VH domain of a single arm of the antibody; (v) the dAb fragment, consisting of a VH domain (Ward et al. (1989) Nature 241:544-546); and (vi) isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked using recombination with a synthetic linker, which allows for their creation as a single continuous chain. In this case, the VL and VH regions pair up to form a monovalent molecule (known as single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Other forms of single-chain antibodies, such as diabodies, are also included (see, e.g., Holliger et al. (1993) PNAS. USA 90:6444-6448).

[0138] Affibodies are small, single-domain proteins that have been engineered to mimic monoclonal antibodies and bind with high affinity to a wide range of target proteins or peptides. They consist of a bundle of three helices based on a single scaffold of the IgG-binding domain of Staphylococcus aureus protein A. This scaffold domain consists of 58 amino acids, 13 of which are randomized to generate an affibo library containing numerous ligand variants. See, for example, U.S. Patent No. 5,831,012 and Lofblom et al. FEBS Letters 584 (2010) 2670-2680. Antibodies mimicking affibo molecules have a molecular weight of approximately 6 kDa.

[0139] As used herein, "diabody" refers to a dimeric antibody fragment. Each polypeptide in the heavy chain variable domain (VH) is linked to the light chain variable domain (VL), but unlike single-chain Fv fragments, the linker between VL and VH is too short for intramolecular pairing. Therefore, each antigen-binding site is formed by the pairing of the VH and VL of one polypeptide with the VH and VL of the other polypeptide; see, for example, Figure 3A. Therefore, the diabody has two antigen-binding sites and can be monospecific or bispecific (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123; Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5)).

[0140] As used herein, “effective amount” of an agent, such as a polyvalent molecule or a pharmaceutical composition containing such molecule, means an amount effective in achieving the desired result in the required dosage and over a period of time. Depending on the embodiment, the therapeutically effective amount is one that reduces the incidence and / or severity of one or more symptoms of a disease, disorder, and / or condition, stabilizes one or more characteristics, and / or delays the onset.

[0141] As used herein, the term “epitope” refers to any protein determinant that can specifically bind to an immunoglobulin or a fragment thereof or a T cell receptor. The term “epitope” includes any protein determinant that can specifically bind to an immunoglobulin or a T cell receptor. Epitope determinants typically consist of a chemically active surface group of a molecule, such as an amino acid side chain or a sugar side chain, and usually possess a specific three-dimensional structure as well as specific charge characteristics. An antibody is said to bind specifically to an antigen when its dissociation constant is ≤10 μM, for example ≤100 nM, preferably ≤10 nM, and more preferably ≤1 nM.

[0142] The constant region of an immunoglobulin molecule is also called the fragment crystallizable region, the "Fc region," or the "Fc domain." The Fc domain consists of two identical protein fragments, which originate from the second and third constant domains of the antibody's two heavy chains. The Fc domain of IgG carries a highly conserved N-linked glycosylation site. Glycanization of the Fc fragments is mediated by the Fc receptor. It is essential for activity. In one embodiment of the present invention, the Fc domain of the multivalent molecule is engineered to not target cells that bind the multivalent molecule toward ADCC or CDC-dependent cell death. In one embodiment of the present invention, the Fc domain of the multivalent binding molecule is a peptide dimer in a knob-in-hole configuration. This peptide dimer may be a heterodimer.

[0143] The terms “individual,” “subject,” “host,” and “patient” are used interchangeably herein and refer to any mammalian subject, specifically human, for whom diagnosis, treatment, or therapy is desired.

[0144] "LRP," "LRP protein," and "LRP receptor" are used herein to refer to members of the low-density lipoprotein receptor-related protein family. These receptors are single-pass transmembrane proteins that bind and internalize ligands in the process of receptor-mediated endocytosis. The LRP proteins LRP5 (GenBank acceptance number NM002335.2) and LRP6 (GenBank acceptance number NM002336.2) are contained within the Wnt receptor complex, which is required for activation on the Wnt-β-catenin signaling pathway.

[0145] As used herein, the term "polypeptide fragment" refers to a polypeptide having an amino-terminus and / or carboxyl-terminus deletion, in which case the remaining amino acid sequence corresponds to the corresponding position in the native sequence, for example, as inferred from the full-length cDNA sequence.

[0146] As used herein, the term "paratope" includes an antigen-binding site within the variable region of an antibody that binds to an epitope.

[0147] The terms “treatment,” “to treat,” etc., are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. This effect may be prophylactic in that it completely or partially prevents the disease or symptoms, and / or therapeutic in that it partially or completely cures the disease and / or adverse effects resulting from the disease. As used herein, “treatment” covers any treatment of a disease in a mammal and includes: (a) preventing the disease from occurring in a subject that is susceptible to the disease but has not yet been diagnosed with it; (b) inhibiting the disease, i.e., preventing its development; or (c) reducing the disease, i.e., causing regression of the disease. Therapeutic agents may be administered before, during, or after a disease or wound. Treatment of a disease in progress is of particular interest if the treatment stabilizes or reduces the patient’s undesirable clinical symptoms. Such treatment is preferably carried out before complete loss of function in the affected tissue. Therapies of the subject may be administered during the symptomatic stage of the disease, and possibly after the symptomatic stage of the disease.

[0148] The ability of the polyvalent conjugated molecules of the present invention to activate Wnt signaling can be confirmed by multiple assays. The polyvalent conjugated molecules of the present invention typically initiate a reaction or activity similar to or identical to that initiated by the innate ligand of the FZD receptor. The polyvalent conjugated molecules of the present invention activate the Wnt signaling pathway, e.g., the classical Wnt-β-catenin signaling pathway. As used herein, the term “activate” means that the intracellular levels of the Wnt signaling pathway, e.g., the Wnt-β-catenin signaling pathway, are measurably increased compared to the levels in the absence of the FZD agonist of the present invention.

[0149] Various methods for measuring the level of Wnt-β-catenin activation are known in the art. These include, but are not limited to, assays that measure: expression of Wnt-β-catenin target genes; expression of LEF / TCF reporter genes (e.g., TopFLASH, superTopFLASH, pBAR, etc.); stabilization of β-catenin; phosphorylation of LRP5 / 6; transposition of axins from the cytoplasm to the cell membrane and binding to LRP5 / 6. The classical Wnt-β-catenin signaling pathway ultimately results in changes in gene expression via the transcription factors TCF1, TCF7L1, TCF7L2, and LEF. The transcriptional response to Wnt activation has been characterized in multiple cells and tissues. Therefore, Wnt-β-catenin signaling activation can be evaluated using comprehensive transcriptional profiling by methods known in the art.

[0150] Changes in the expression of Wnt-responsive genes are generally mediated by the transcription factors TCF and LEF. The TCF reporter assay assesses transcriptional changes in TCF / LEF regulatory genes to determine the level of Wnt-β-catenin signaling. The TCF reporter assay was first described by Korinek, V. et al., 1997. Also known as TOP / FOP, this method involves determining the transactivation activity of endogenous β-catenin / TCF using three copies of the optimal TCF motif CCTTTGATC or three copies of the mutant motif CCTTTGGCC (pTOPFLASH and pFOPFLASH, respectively) upstream of the minimal c-Fos promoter that drives luciferase expression. A high ratio of the activities of these two reporters (TOP / FOP) indicates high β-catenin / TCF activity. A newer, more sensitive version of this reporter is called pBAR and contains 12 repeats of the TCF motif (Biechele and Moon, Methods s Mol Biol. 2008;468:99-110, PMID: 19099249).

[0151] Common methods in molecular and cellular biochemistry can be found in standard textbooks such as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., CSH Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998).

[0152] A "single-chain Fv" or "scFv" antibody fragment contains the VH and VL domains of the antibody, in which case these domains are present on a single polypeptide chain. Generally, Fv polypeptides further contain a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding. For an overview of scFv and other antibody fragments, see James D. Marks, Antibody Engineering, Chapter 2, Oxford University Press (1995) (Carl K. Borrebaeck, Ed.).

[0153] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include plural terms, and plural terms shall include singular terms. In general, the terminology and techniques used in connection with cell and tissue culture, molecular biology, and the chemistry and hybridization of proteins and oligonucleotides or polynucleotides described herein are well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, or as commonly performed in the art, or as described herein. The aforementioned techniques and procedures are generally carried out according to conventional methods well known in the art, and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). The terminology, laboratory procedures, and techniques used in relation to analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein are well known and commonly used in the art. Standard techniques are used in chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, as well as in patient treatment. [Examples] Example I 1. Development of polyvalent FZD agonists

[0154] To create a multivalent binding molecule having a first binding domain containing an FZD diabody and a second binding domain containing a co-receptor diabody, we identified FZD-specific antibodies from a synthetic Fab phage library (Library F; see U.S. Patent Application Publication No. 2016 / 0194394 by inventors Sidhu et al.) by selecting those bound to the cysteine-rich domain (CRD) of the FZD receptor using conventional phage display techniques. Affinity or specificity maturation was performed as needed. For example, using FZD4 CRD as an antigen, we matured pan-FZD-binding antibody #5019 (recognizing FZD1, 2, 4, 5, 7, and 8) from an antibody derived from FZD7. In our previous research, we identified several antibodies that are perfectly specific to FZD4 (5038, 5044, 5048, 5062, 5063, 5080, 5081) or FZD5 (2928) (see, for example, U.S. Patent Application Publication No. 20160194394 by inventor Sidhu et al., and WO2017127933A1 by inventor Pan et al.).

[0155] These FZD antibodies were used to construct FZD-specific diabodies. Diabodies are antibody forms similar to single-chain variable fragments (scFv), but are dimers of two peptides, each encoding VL and VH, respectively. However, unlike scFv, the linker between VH and VL within the polypeptide is very short, making it impossible to confer intramolecular complementarity between the VH and VL domains. Therefore, to functionally reconstitute two antigen-binding paratopes, the VH-VL fragment of one polypeptide is dimerized with the VH-VL fragment of another polypeptide. Diabodies with identical or non-identical paratopes were generated by forming dimers of polypeptides having the same VL and VH, thereby forming homodiabodies, or by forming dimers from two polypeptides having different VL and VH domains, thereby forming diabodies.

[0156] LRP6 antibodies were also selected from a synthetic antibody library by selecting antibodies conjugated with the recombinant extracellular domain (ECD) of human LRP6. Five Fabs with unique CDR regions were identified. After conversion to IgG, all of them exhibited human LRP6 binding and mouse LRP6 binding. No LRP5 binding was detected via ELISA, demonstrating that these antibodies are LRP6 specific (Figure 1A). The LRP6 ECD contains four β-propeller motifs alternating with four epidermal growth factor (EGF)-like repeats. The first two β-propeller motifs are thought to be involved in Wnt1 binding, and the remaining two are thought to be involved in Wnt3 binding, thus creating two potential epitopes for antibody binding. See Figure 6A. The epitope binding results suggest that these five antibodies bind to two separate sites on LRP6, and that they can be divided into two groups: antibodies 2538, 2542, and 2543 that bind to the Wnt1 binding site on LRP6, and antibodies 2539 and 2540 that bind to the Wnt3 binding site. Generally speaking, antibodies that bind to the LRP6-Wnt1 site are expected to block the activation of the Wnt pathway induced by Wnt1.

[0157] To prepare the N-terminal binding domain of Fc containing a homodiabody specific to FZD, VH and VL fragments of selected FZD antibodies, VH-1, VH-2, VL-1, and VL-2, were amplified by PCR from the corresponding phagemide template and isolated. The isolated fragments (VH-1 and VL-2) were then introduced into an EcoRI / XhoI-cleaved vector (pSCST skeleton) containing the Fc-knob region using Gibson assembly (see Gibson et al. (2009). Nature Methods. 6 (5): 343-345 and Gibson DG. (2011) Methods in Enzymology. 498: 349-361). Similarly, the fragments (VH-2 and VL-1) were also introduced into an EcoRI / XhoI-cleaved vector containing the Fc-hole region using Gibson assembly. The correct assembly was confirmed using DNA sequencing. Next, the second binding domain was introduced to the C-terminus of the Fc domain using the two plasmids described above (one pair, Fc-knob and Fc-hole).

[0158] The conformation of the Fc-knob and Fc-hole is necessary to generate a multivalent binding domain, in which case one of the binding domains is a heterodiabody. However, the conformation of the Fc-knob and Fc-hole is not necessary to prepare a binding molecule containing homodiabodies at both the N-terminus and C-terminus of the Fc domain; therefore, for such binding molecules, VH and VL were ligated to the wild-type Fc region, and a VH-VL-containing polypeptide was generated using only one plasmid to form a homodimer. Optionally, a linker, such as a peptide linker or a non-peptide linker, may be present between the binding domain and the Fc domain.

[0159] To generate the C-terminal binding domain, an LRP5 / 6 antibody was identified, and the LRP5 / 6 diabody was generated according to the same protocol as above for generating the FZD diabody. The C-terminal binding domain was generated by PCR amplification of the VH-3, VH-4, VL-3, and VL-4 fragments from the corresponding phagemide template for the LRP antibody, followed by isolation of the amplified fragments. As described above, the VH-3 and VL-4 fragments were then introduced into the PpuMI / BamHI site of the Fc-knob plasmid using Gibson assembly. The other VH-4 and VL-3 fragments were inserted into the PpuMI / BamHI cleavage site of the Fc-hole plasmid using Gibson assembly.

[0160] Two plasmids (a pair, Fc-knob and Fc-hole) containing different VL and VH sequences were used to generate bispecific FZD-binding domains or co-receptor-binding domains capable of binding to two different sites. Since the knob-into-hole configuration was not necessary to generate dimers with single-specific binding domains, only a single plasmid containing the wild-type Fc sequence was used when each binding domain was single-specific.

[0161] Figure 9A illustrates a plasmid encoding a peptide containing an Fc region with a "knob" mutation, VH and VL of pan-FZD antibody #5019, VL of LRP antibody #2542, and VH of LRP antibody #2539. Figure 9B illustrates a plasmid encoding a peptide containing an Fc region with a "hole" mutation, and nucleic acids encoding VH and VL of pan-FZD antibody #5019, VH of LRP antibody #2542, and VL of LRP antibody #2539. The peptides encoded by these plasmids form heterodimers having a polyvalent binding site containing a homodiabody derived from pan-specific FZD antibody #5019; and a polyvalent binding site containing a bispecific heterodiabody produced by the pairing of LRP antibody #2539 VL and LRP antibody #2542 VH, derived from one peptide having LRP antibody #2539 VH and the other peptide having LRP antibody #2542 VL.

[0162] The resulting plasmids were then sequenced, and the sequenced plasmids were prepared according to the manufacturer's instructions using the PureLink HiPure Plasmid Filter Maxiprep Kit (Invitrogen). The plasmids were then transfected into Expi293F cells (Thermo Fisher Scientific), and antibodies were expressed using FectoPRO Reagent (Polyplus) according to the manufacturer's instructions. Typically, 200 mL of cells were used for small-batch antibody production.

[0163] Typically, 80 hours after transfection, the culture medium of Expi293F cells was collected by centrifugation to pellet the cells and cell fragments. The supernatant was transferred to a clean bottle and buffered with 10×PBS buffer. After incubation for 1 hour with an appropriate amount of Protein A bed (GE Healthcare), the bed was washed and the binding molecules were eluted according to the manufacturer's instructions. Finally, the buffer was replaced with PBS. 2. Polyvalent bond molecules of heterodimers

[0164] Using the method described above, we also generated tetravalent heterodimer molecules containing intact bispecific diabodies fused to the N-terminus and C-terminus of the Fc domain (Knob / Hole), respectively (Figures 2A and 3A). Specifically, we generated tetravalent conjugated molecules with an FZD-conjugated homodiabody from antibody 5019 at the N-terminus of the Fc domain and a homodiabody from LRP6-W1 antibody 2542 (5019-Fc-2542) or LRP6-W3 antibody 2539 (5019-Fc-2539) at the C-terminus of the Fc domain. Surprisingly, both tetravalent molecules activated the Wnt pathway, but 5019-Fc-2542 was far less potent (Figure 3C). While we do not wish to be constrained by theory, this difference appears to reflect a difference in the ability of LRP6-W1 and LRP6-W3 binding to activate Wnt signaling. Wnt binding at the LRP6-W3 site has been observed to be more effective than Wnt binding at the LRP6-W1 site in activating Wnt signaling.

[0165] We also generated a tetravalent, triplicate binding molecule (referred to as 5019-K / H-2539-2542, 5019Ag) with an FZD-binding homodiabody derived from antibody 5019 at the N-terminus of the Fc domain and an LRP heterodiabody derived from LRP6-W1 antibody 2542 and LRP6-W3 antibody 2539 at the C-terminus of the Fc domain (Figure 5). 5019Ag is unexpectedly more effective in activating Wnt signaling than molecules with a single-specific LRP6 homodiabody (Figure 3C). All three nanomolar forms activate Wnt signaling as determined by the pBAR luciferase reporter assay (Figure 3D), indicating that they are effective Wnt mimetic molecules. While we do not wish to be constrained by theory, it is hypothesized that engaging a strong Wnt3A site with a weak Wnt1 site together is more effective than engaging two strong Wnt3A sites. The two best polyvalent binding molecules, possessing an FZD binding domain and an LRP binding domain, i.e., "FLAg", have a single-digit nanomolar titer (EC2). 50It had a titer of ~5 nM, which was nearly identical to that of purified Wnt3A, and presented a bell-shaped dose-response profile (Figure 11D). We interpreted this as indicating that polyvalent binding of FLAg is required for maximum stimulation, and that the decrease in efficacy at high concentrations may be due to monovalent binding with either FZD or LRP6. We used RKO cells expressing low levels of β-catenin (Major et al. Science. 316, 1043-1046 (2007)) in F P+P -L6 1+3 Treatment with this method resulted in dose-dependent and time-dependent increases in β-catenin protein levels and phosphorylation of DVL2, which is a marker of activation of the Wnt-FZD pathway (Figures 11E and 11F). Thus, tetravalent FLAg is a modular, engineerable human Ab modality that functions as a synthetic agonist for FZD and LRP6.

[0166] Optimal FLAg F P+P -L6 1+3 To confirm the engineered affinity and specificity of FLAg, we used biolayer interferometry (BLI) to measure its binding kinetics with nine of ten human FZD CRDs and human LRP6 ECD (Figures 12A and 12B). FLAg bound to six FZDs recognized by the FZD diabodies derived from the parent pan-FZD paratope (Pavlovic et al. 2018) with affinities ranging from picomolars (KD = 10 to 800 pM), but did not bind to the other three FZDs to a detectable degree. Furthermore, its affinity for LRP6 was in the nanomolar range (KD = 12 nM) (Figure 12B). Next, we used BLI to evaluate the binding of FLAg to various Fc receptors.

[0167] FLAg exhibited behavior similar to conventional IgG, interacting with FcRn in a dose-dependent and pH-dependent manner (Figure 12C). Natural IgG binds to FcRn at pH 6 but not at pH 7.4, which in vivo results in reuse during action and consequently a long half-life. FLAg also exhibited behavior similar to IgG in interactions with other Fc effectors, including complement (C1q), the natural killer cell marker CD16a, the B cell marker CD32a, and the monocyte and macrophage marker CD64 (Figure 12D). We concluded that FLAg contains a functional Fc moiety that should confer effector function and a long half-life in vivo.

[0168] tetravalent F P+P -L6 1+3Through the modular design of FLAg, we have been able to dissect the contribution of each of the four paratopes to the intrinsic agonist activity, which was achieved by replacing each paratope with a null paratope that binds to the irrelevant antigen maltose-binding protein (MBP). We generated a "monovalent" molecule comprising an Fc domain, a FZD binding domain appended to one Fc domain terminus, and an LRP binding domain appended to the other Fc domain terminus, wherein rather than having two binding sites for FZD or LRP in the diabody, the binding domains have only a single binding site or a single binding site plus one control maltose-binding protein binding site "MBP". Five monovalent molecules were generated by introducing one MBP binding site into at least one binding domain of said molecule. 5019-MBP-K / H-2539-2542, which contains one FZD binding site and one MBP binding site at the N-terminus, still activates the Wnt pathway, but has an 8-fold reduction in potency compared to 5019Ag (Figure 3E). Similarly, 5019-K / H-2539-MBP, which retains only one LRP6-W3 site at the C-terminus, exhibits much less Wnt activation compared to 5019Ag (Figure 3E). Minimal agonist activity was detected for the two MBP-FZD / MBP-LRP6 molecules 5019-MBP-K / H-2539-MBP and 5019-MBP-K / H-MBP-2542, as well as 5019-K / H-MBP-2542, a molecule having one LRP6-W1 diabody (Figure 3E). The results of these β-catenin signaling assays showed that maximal stimulation was greatly reduced by disabling one anti-FZD paratope or one anti-LRP6 paratope directed against the WNT1 binding site, and was completely abolished by disabling the anti-LRP6 paratope directed against the WNT3A binding site, or by simultaneously disabling one anti-FZD paratope and either anti-LRP6 paratope. We also replaced the anti-LRP5 paratope targeting the WNT3A binding site with the anti-LRP6 paratope targeting the WNT1 binding site, resulting in the molecule (F P+P -L5 / 6 3 ), which is capable of recruiting both co-receptors, and the observed activity was FP+P -L6 1+3 It was similar (Figure 3F, EC) 50 (=4nM). In summary, these data show that optimal agonist activity is achieved by a molecule that can recruit two FZDs via a common epitope and LRP6 via two distinct epitopes, but the activity can be adjusted to a moderate level by neutralizing either the anti-FZD paratope or the anti-LRP6 paratope. Furthermore, a molecule capable of recruiting FZDs to two different coreceptors was generated by combining two anti-FZD paratopes into one paratope for LRP5 and one for LRP6, respectively.

[0169] We also explored the requirements regarding the geometric and spatial constraints imposed by the intermolecular diabody form by substituting pairs of diabody pairs with pairs of less constrained intramolecular single-chain variable fragments (scFv) (Figure 2J). P+P -L6 1+3 Compared to anti-FZD scFv(F P*+P* -L6 1+3 FLAg containing ) exhibited similar activity, whereas anti-LRP6 scFv(F P+P -L6 1*+3* ) contains or has scFv(F at both ends P*+P* -L6 1*+3*For FLAg containing ), activity was significantly reduced. These differences in activity were not due to differences in affinity, but rather because BLI measurements showed equivalent high-affinity binding to LRP6 and FZD isoforms regardless of whether the paratope was presented in diabody or scFv form (Figures 2K and 2L). In summary, these results indicate that the optimal assembly of the FZD / LRP6 signaling complex requires a specific stoichiometric and geometric configuration, and that the constraints are particularly clear for LRP6, which requires engagement of two distinct epotopes in a specific geometric configuration defined by the diabody form. Notably, the loose constraints on FZD engagement allow for significant activation by a single anti-FZD paratope (Figure 2D), which opens the door to further enhancing specificity or altering signaling by recruiting different cell surface proteins via additional paratopes linked to the N-terminal anti-FZD paratope of heterodimer Fc. 3. Forms of other bispecific antibodies

[0170] We constructed bispecific molecules containing the FZD-binding domain of antibody #5019 and the LRP6-W1-binding domain of antibody #2942 (5019 / 2942) or the LRP6-W3-binding domain of antibody #2539 (5019 / 2539) on the same end of the Fc domain, purified the corresponding proteins (Figure 2A), and assayed for Wnt signaling activation using a pBAR luciferase reporter assay. These molecules failed to activate Wnt signaling. Notably, both bispecific molecules antagonized the activity of Wnt ligands (Figure 2B). While we do not wish to be constrained by theory, the distance and flexibility between the two paratopes of these bispecific molecules may prevent the recruitment of FZD and LRP6 receptors in a geometric configuration suitable for activation.

[0171] Bispecific molecules containing FZD and LRP diabodies attached to the same end of the Fc domain were also generated using the knob-in-hole configuration. These diabodies, named 5019-2539-K / H (FZD / LRP-W3) and 5019-2542-K / H (FZD / LRP-W1), were assayed for binding to FZD and LRP, as well as activation of the Wnt pathway. Both diabodies retained the FZD binding profile and LRP6 binding activity of the original antibody (Figures 2D-2G). Both molecules individually bound to the FZD receptor and LRP coreceptor. 5019-2542-K / H exhibited cobinding to both FZD and LRP in solution as determined by the BLI assay (Figure 2H), while no significant cobinding was observed in 5019-2539-K / H. Neither 5019-2539-K / H nor 5019-2542-K / H activated Wnt signaling as determined by the pBAR luciferase reporter assay, and the results were similar to those obtained with homodiabodies binding to the FZD receptor (5019-Fc) or co-receptor (2539-Fc) (Figure 2I). Furthermore, 5019-2539-K / H (FZD / LRP-W3) and 5019-2542-K / H (FZD / LRP-W1) effectively inhibited the activation of the Wnt3a-mediated pathway (Figure 2I). 4. Wnt pathway signaling assay

[0172] Activation of the Wnt pathway was assayed in HEK293 cells using a pBAR luciferase reporter system (Biechele and Moon, Metods Mol Biol. 2008; 468:99-110, PMID: 19099249) that faithfully monitors β-catenin transcriptional activation. In short, HEK293T cells stably expressing pBARLS and pSL9 Ef1α-sea oyster luciferase constructs were seeded in 96-well plates at 1.5E4 cells / well. 24 hours after seeding, cells were treated with labeled FZD agonists at the indicated concentrations in triplicates or with a PBS vehicle control. 16.5 hours after treatment, cells were lysed and luminescence was measured using a dual luciferase reporter assay system (Promega#E1960) according to the manufacturer's protocol. For each well, the firefly bioluminescence was normalized to that of sea cucumber bioluminescence, taking into account the number of cells.

[0173] We assayed the agonist activity of a multivalent molecule containing an N-terminal FZD diabody derived from an antibody fragment (antibody #5019) that recognizes several FZD receptors (FZD1, 2, 4, 5, 7, and 8), with the LRP-binding domain on the C-terminus of the Fc domain conjugated via the Fc domain. The C-terminal LRP-binding domain consists of a diabody derived from one of two LRP6 antibodies, #2539 and #2542, which bind to the Wnt3 and Wnt1 sites, respectively (Figure 6B). These polyvalent conjugated molecules, named 5019-Fc-2539 and 5019-Fc-2542, in nanomolar amounts activated the Wnt-β-catenin pathway (Figure 6C). However, treating cells with a molecule carrying the LRP6 antibody 5019-Fc-2539, which targets the Wnt3 site, resulted in approximately 10 times higher activation compared to 5019-Fc-2542 (200 times higher than background vs. 20 times, respectively) (Figure 6C).

[0174] Importantly, using an engineered knob-hole system within the Fc moiety, we generated a multivalent binding molecule (Figure 1C) (Figure 6B) containing a (#5019) homodiabody for the pan-FZD binding domain at one end and a heterodiabody 5019-K / H-2539:2542 at the other end, forming an LRP6 binding domain with binding sites for Wnt1 (#2542) and Wnt3 (#2539). This configuration allowed for the incorporation of four different binding sites within a molecule with different selectivity and affinity profiles, i.e., tetravalent and triplicate. When subjected to a β-catenin luciferase reporter assay in HEK293 cells, this molecule exhibited twice the activation of 5019-Fc-2539, or approximately 400 times the background (Figure 6C).

[0175] We also replaced the binding site for LRP6 in a knob-in-hole system that also possesses the same pan-FZD diabody (5019) that binds FZD1, 2, 4, 5, 7, and 8 with an equivalent LRP5 binding site (diabodies derived from the 2459 and 2460 antibodies, both of which bind LRP5). This molecule, 5019-K / H-2459:2460, was also able to activate the Wnt-β-catenin pathway in HEK293T cells, although with lower efficacy than agonists carrying the LRP6 diabody (Figure 6D). 5. Selective FZD agonists with binding domains (characterization of agonist modularity derived from selective FZD antibody fragments and co-receptor antibody fragments)

[0176] To evaluate the activity of our monospecific FZD agonists, we used cell-based assays dependent on specific FZD isoforms. We prepared polyvalent binding molecules that bind to only one of 10 FZD receptors. In our previous studies, we identified several antibodies (5038, 5044, 5048, 5062, 5063, 5080, 5081) that are completely specific to FZD4 (see, e.g., U.S. Patent Application Publication No. 20160194394 by inventor Sidhu et al., and WO2017127933A1 by inventor Pan et al.). Polyvalent binding molecules consisting of an FZD4-specific FZD-binding domain and an LRP6-binding domain containing bispecific heterodiabodies derived from antibodies 2539 and 2542 were generated using a knob-in-hole system of Fc. These molecules were able to activate FZD4 signaling via the β-catenin pathway, but only when co-transfected with FZD4 cDNA into HEK293 cells. These FZD4-binding molecules failed to activate FZD4 signaling or the β-catenin pathway in unmodified HEK293T cells expressing low levels of FZD4. Therefore, this experiment demonstrates the specificity of the molecules to FZD4. 5019-K / H-2539-2542 (the pan-FZD agonist described above) can activate signaling in HEK293T cells even in the absence of FZD4 (Figure 4A). Given that Wnt-mediated activation of β-catenin signaling in HEK293T cells occurs via FZD1, 2, and 7 (Voloshanenko et al. FASEB 2017 FASEB J. 2017 Nov;31(11):4832-4844;PMID:28733458), and that the 5919 FZD antibody binds to all three receptors, the above results are not surprising.

[0177] Furthermore, we generated an FZD5-specific polyvalent binding molecule using the binding domain of the FZD5-specific antibody 2928 (Steinhart et al. Nat Med. 2017 Jan;23(1):60-68, PMID:27869803; WO2017127933A1 by inventors Pan et al.), which we previously demonstrated binds exclusively to FZD5. We previously demonstrated that several RNF43-mutated pancreatic ductal adenocarcinoma (PDAC) cell lines depend solely on FZD5 signaling for their growth (Steinhart et al. 2017, PMID:27869803). In fact, genome-wide CRISPR essentiality / adaptability screening in three RNF43-mutated PDAC cell lines showed that FZD5 was one of the most essential genes for its growth, whereas PDAC cell lines with WT RNF43 did not show this requirement for FZD5. When RNF43 mutant cells are treated with a porcupine inhibitor (PORCNi; e.g., LGK-974) that inhibits palmitoylation and Wnt ligand activity, the RNF43 mutant cells cease to proliferate.

[0178] Co-treatment of RNF43 mutant cells with pan-FZDag 5019-K / H-2539-2542 or with the selective FZD5 agonist 2928-K / H-2539-2542 resulted in robust rescue of LGK974-blocked cell proliferation. These results demonstrate that these two molecules can activate FZD5, inducing Wnt signaling in these cells and thereby mimicking the action of endogenous Wnt ligands (Figure 7B). In contrast, the addition of the FZD4-specific agonist 5038-K / H-2539-2542 or the FZD2-specific agonist failed to rescue the inhibition of LGK974 proliferation in the culture medium ted by.

[0179] RNA sequencing analysis revealed that FZD2 is the dominant isoform in the mesenchymal stem cell line CH3H10T1 / 2 (mouse ENCODE), suggesting that FZD2 may be involved in the established role of Wnt proteins during the osteogenic differentiation of mesenchymal cells (Day et al. Dev. Cell. 8, 739-750 (2005)). Stimulation of C3H10T1 / 2 cells with an FZD2-specific flag resulted in robust induction of the osteogenic marker alkaline phosphatase (ALPL) to levels similar to those achieved with pan-FZDFLAg, whereas FZD5-specific FLAg showed minimal activity (Figure 7B). 6. Co-targeting using tetravalent bond molecules

[0180] In addition to achieving the desired combination by mixing and fitting the FZD multivalent binding domain and coreceptor binding domain to the Fc domain, the presence of a tetravalent paratope in the current system provides an opportunity to simultaneously target two FZD receptors and two coreceptors with a single molecule and ensure reliable co-localization when applied in vivo. Considering the agonist activity of 5019-MBP-K / H-2539:2542 as described above, combining the binding domain within the heterodiabody at the N-terminus of the Fc domain allows for the generation of a multivalent binding molecule with a binding domain for a selective FZD receptor. For example, binding domains derived from antibodies 5038 (binding FZD4) and 2928 (binding FZD5) would produce a molecule that targets both FZD4 and FZD5. This binding molecule can also be generated to have coreceptor binding domains for specific or multiple coreceptors. For example, a binding domain that targets both LRP6 and LRP5 could be constructed by combining binding domains derived from antibodies 2459 (which binds to the Wnt1 binding site of LRP6) and 2539 (which binds to the Wnt3a binding site of LRP6) at the C-terminus of the Fc domain. Similarly, coreceptor binding domains may contain a binding site for LRP6 in combination with another coreceptor, e.g., ROR1 / 2, to initiate activation of both classical and non-classical Wnt signaling pathways in a single cell.

[0181] Furthermore, this specification envisions a polyvalent conjugated molecule having a tissue-specific binding domain derived from a tissue-specific antibody, which recruits the polyvalent conjugated molecule to a desired tissue, where the molecule then activates Wnt signaling by binding to the FZD receptor and co-receptor. This is expected to be particularly useful when using polyvalent conjugated molecules in regenerative medicine, where it is necessary to limit the desired effect to a specific tissue. In summary, the tetravalent form provides further design flexibility to meet the functional requirements of versatility. 7. A polyvalent binding molecule having an FZD-binding domain and a co-receptor-binding domain can replace Wnt ligands and sustain intestinal organoid culture.

[0182] The effects of the FZD agonists described herein on organoid survival and maintenance were assayed as follows: Eight-week-old female C57BL / 6 mice were sacrificed, and small intestinal crypts were collected for organoid isolation (O'Rourke et al. 2016. Isolation, Cuture, and Maintenance of Mouse Intestinal Stem Cells. Bio Protoc. 20:4). Organoid cultures were passed through mechanical dissociation (O'Rourke 2016) and embedded in 25 μL of growth factor-reduced Matrigel (Corning, 356231) in 48-well plates. Organoids were seeded in triplicates for each experimental condition. Complete organoid medium (O'Rourke 2016) containing experimental conditions (1 μM LGK-974 + / - 40% Wnt3a condition medium or + / - 50 nM Pan Fzd-5056 (FZDag that targets FZD1, 2, 4, 6, 7, 8 but binds to epitopes that do not compete with Wnt ligands)) was added to each well on the subculturing day and replaced every 2-3 days. After one week, 150 μL of Cell Titer Glo 3D (Promega) was added to 150 μL of medium in each well. The organoids were lysed on an oscillating stirrer at RT for 30 minutes. Luminescence readings were measured in two sets for 20 μL of lysate from each well. The average of the luminescence readings from each condition was normalized to the DMSO condition to calculate viability.

[0183] As broad stem cell niche factors, Wnt and R-spongin are required for the derivatization and maintenance of three-dimensional cultured organoids derived from many tissues. In vitro, Wnt protein secreted by Paneth cells is sufficient to support the growth of mouse small intestinal organoids in the presence of R-spongin. However, when Wnt is released and its activity is shielded by PORCNi LGK974, the organoids are unable to proliferate and eventually die. Hereinafter, we introduce FZDag(F), a pan-FZD polyvalent binding molecule of the present invention. P+P -L6 1+3 The study demonstrated that LGK974 can rescue and sustain organoid growth, suggesting that this molecule functionally mimics Wnt ligand (Figure 8) and can substitute for Wnt protein to support tissue organoid growth. Since Wnt ligand is an essential component of the culture medium required to grow many human tissue organoids, it is expected that the antibody-derived FZD agonist of the present invention, when included in culture medium, will promote the derivatization, survival, and maintenance of organoids from different tissues, thereby mitigating the limitations associated with the use of conditional media or purified Wnt protein. 8. Polyvalent bond molecules that promote bone regeneration

[0184] The regeneration properties of the polyvalent binding molecule of the present invention, which has a first polyvalent binding domain that binds to FZD2 and a coreceptor binding domain that binds to LRP5 or LRP6, are evaluated using a rat non-surgical femoral fracture model. The first polyvalent binding domain may specifically bind to FZD2 or may bind FZD2 to other FZD receptors.

[0185] Following a unilateral, non-surgical mid-diaphysis fracture of the femur, rats are administered either a vehicle or a polyvalent conjugate molecule (see Bonnarens, and Einhorn, J. Orthop. Res. 2, 97-101 (1984)). In short, an 18-gauge syringe needle is inserted into the medullary canal through the bone condyle. A transverse fracture of the femur is then created by applying a blunt force to the anterior (lateral) surface of the femur. One day after the fracture, rats are subcutaneously injected with either a saline vehicle or a polyvalent conjugate molecule twice a week for seven weeks. At the end of the treatment, the intramedullary pin is removed, and the fractured femur is analyzed by micro-CT.

[0186] A polyvalent binding molecule having a polyvalent domain that binds to FZD2 and a second polyvalent binding domain that binds to LRP5 or LRP6 significantly increases bone regeneration in this model compared to bone regeneration by vehicle alone. Example II - Synthetic antibody targeting FZD and LRP6

[0187] We have already obtained hundreds of synthetic Abs by applying phage display to nine recombinant FZD CRDs as antigens (FZD3 CRD could not be purified) (Steinhart et al. Nat. Med. 23, 60 (2016); Pavlovic et al. MAbs (2018), doi: 10.1080 / 19420862.2018.1515565). Systematic characterization revealed a continuity in the specificity profiles, in which some Abs exhibited broad specificity, exemplified by pan-FZD Abs (FPs) that recognized FZD1 / 2 / 4 / 5 / 7 / 8 (Figure 11A), others exhibited more limited specificity, and some were single-specific (Figure 11B). Functional characterization revealed that some antibodies compete with Wnt and inhibit β-ka signaling, while others are non-competitive and do not interfere with Wnt signaling (Figure 11B). Overall, we fully characterized 161 anti-FZD antibodies, including 47 Wnt signaling inhibitors. Unexpectedly, as discussed herein, all of the polyvalent binding molecules we generated by using these anti-FZD antibodies as a source of FZD binding domains in conjunction with binding domains that bind to LRP binding domains, e.g., the Wnt1 binding site and / or Wnt3a binding site on LRP5 / 6, were agonists of the Wnt pathway, regardless of whether they compete with Wnt and inhibit Wnt signaling. Example III - Phenotypic effects of FLAg in cells, organoids, and animals

[0188] Given that FLAg selectively engages FZD and LRP to activate Wnt-related signaling pathways, we explored the phenotypic effects of these signals in progenitor stem cells (PSCs), organoids, and animals. Modulation of Wnt-β-catenin signaling activity is essential to the differentiation protocol of most PSCs (Huggins et al. Methods Mol. Biol. 1481, 161-181 (2016)). Treatment of human PSCs with WNT3A-conditioned medium or a small molecule inhibitor of GSK3 activates β-catenin signaling, leading to the induction of primitive streaks and promoting mesoderm fate determination (Davidson et al. PNAS USA 109, 4485-4490 (2012)). We evaluated FLAg activity in this context and conditioned human PSCs at 30 nM F P+P -L6 1+3 We found that treatment with this method for 3 days induced robust induction of the mesoderm marker BRACHYURY to a level equivalent to that of treatment with the GSK3 inhibitor CHIR99021 at 6 μM, and reduced the expression of the pluripotency marker OCT4 (Figures 13A and 13B).

[0189] F P+P -L6 1+3 It contains Fc, which recognizes mouse FZD and LRP6 and interacts with FcRn. It is hypothesized that Fc confers an Ab-like long half-life to this molecule in vivo. Therefore, we have developed F P+P -L6 1+3We tested whether it could interact with endogenous receptors in mice, accumulate to a level sufficient to activate β-catenin signaling, and mobilize endogenous stem cell activity. Within the intestinal stem cell niche, the Wnt protein secreted by mesenchymal cells induces the expression of β-catenin target genes in lower crypt stem cells, directing their self-renewal; therefore, the target gene LGR5 is often used as a stem cell marker in various tissues. Treatment of LGR5-GFP mice with LGK974 quenched Wnt production in crypt stem cells, leading to a rapid decay of LGR5 expression and linked GFP signaling. Notably, intraperitoneal injection of F P+P -L6 1+3 Co-treatment with F rescued GFP expression (Figure 14, right panel). P+P -L6 1+3 However, we concluded that it possesses sufficient half-life and bioavailability to enable β-catenin activation at a level that promotes the self-regeneration of intestinal stem cells in the absence of endogenous Wnt. Example IV - Materials and Methods: 1. Selection and screening of Ab

[0190] As described (Persson et al. J. Mol. Biol. 425, 803-811 (2013)), phage display synthetic library F was used to select Fabs that bind to the Wnt receptor. In short, Fc-tagged ECD protein (R&D Systems) was immobilized on Maxisorp immunoplates (Thermo Fisher, catalog no. 12-565-135) and used for positive binding selection by library phage pooling. The pool was first exposed to similarly immobilized Fc protein to deplete nonspecific binders. After four rounds of binding selection, clonal phages were prepared and evaluated by phage ELISA (Birtalan et al. J. Mol. Biol. 377, 1518-1528 (2008)). Clones that exhibited at least 10 times stronger signals regarding antigen binding compared to Fc were designated as specific binders for further characterization. 2. Recombinant proteins and reagents

[0191] The Fc tag fusion units FZD1 (5988-FZ-050), FZD2 (1307-FZ-050), FZD4 (5847-FZ-050), FZD5 (1617-FZ-050), FZD7 (6178-FZ-050), FZD8 (6129-FZ-050), FZD9 (9175-FZ-050), and FZD10 (3459-FZ-050) were purchased from R&D Systems. Fc-tagged ECD of FZD6 (residues 19-132, UniprotO60353-1) was expressed and purified in Expi293 cells using a pFUSE-hIgG1-Fc2 vector (Invitrogen), and a single protomer species was isolated from the aggregated protein by size exclusion chromatography on a Superdex200 (10 / 300) column (GE Healthcare). Fc-tagged ECD fusion proteins of human (1505-LR-025) and mouse (2960-LR-025) LRP6 and mouse LRP5 (7344-LR-025 / CF) were purchased from R&D Systems. WNT1 (SRP4754-10ug), WNT2b (3900-WN-010 / CF), WNT5a (645-WN-010 / CF), and WNT3A (5036-WN-010 / CF) were purchased from R&D Systems, and the WNT3A condition medium was prepared as described (PMID:12717451). Other proteins and chemicals were purchased from the following suppliers: FcRN (R&D, 8693-FC), C1q (Sigma, C1740), CD16a (R&D, 4325-FC), CD32a (R&D, 1330-CD / CF), CD64 (R&D, 1257-FC), LGK974 (Cayman Chemicals), porcupine inhibitor C59 (Dalriada Therapeutics), and CHIR99021 (Sigma-Aldrich). 3. Cloning of the tetravalent conjugate molecule "FLAg" and antibodies against FZD and LRP.

[0192] The DNA fragment encoding the antibody (Ab) variable domain is either amplified by PCR from a phagemide DNA template or constructed by chemosynthesis (Twist Bioscience). The DNA fragment was cloned into a mammalian expression vector (pSCSTa) designed to produce kappa light chains and human IgG single heavy chains. The bispecific diabody and IgG contained an optimized version of the "knobs-in-holes" heterodimer Fc (Ridgway et al. Protein Eng. 9, 617-621 (1996)). The FLAg and diabody-Fc fusions were sequenced in a VH-VL orientation, and the variable domain was separated by a short GGGGS linker (e.g., amino acids 121-125 of SEQ ID NO: 2), which prefers intermolecular association between the VH and VL domains and therefore prefers diabody formation. To produce a diabody-Fc fusion construct, the diabody chain was fused to human IgG1 Fc. The FLAg protein was constructed as VH-x-VL-y-[human IgG1 Fc]-z-VH-x-VL, where the linkers were x=GGGGS (e.g., amino acids 121-125 of SEQ ID NO: 2), y=LEDKTHTKVEPKSS (amino acids 232-245 of SEQ ID NO: 4), and z=SGSETPGTSESATPESGGG (amino acids 473-501 of SEQ ID NO: 4). In this configuration, the human IgG1 Fc or knob-in-hole IgG1 Fc fragment extended from position 234 to position 478 (Kabat-numbered rule). For the scFv-Fc fusion, the variable domain was arranged in a VL-VH orientation and linked by a long GTTAASGSSGGSSSGA (SEQ ID NO: 75) linker. This linker prefers intramolecular association between the VH and VL domains, and therefore prefers scFv formation. For all constructs, the entire coding region was cloned in-frame into mammalian expression vectors along with the secretory signal peptide. 4. Protein expression and purification

[0193] The antigen, Ab, and FLAg proteins were produced by transient transfection in Expi293F (Thermo Fisher) cells. In short, the cells were transfected in baffled cell culture flasks in Expi293 expression medium (Gibco) at a rate of approximately 2.5 × 10⁶ 6 Cells were grown to a density of cells / mL and transfected with an appropriate vector using FectoPRO transfection reagent (Polyplus Transfection) according to a standard manufacturing protocol (Thermo Fisher). Expression was carried out for 5 days at 37°C and 8% CO2 with stirring at 125 rpm. After expression, cells were removed by centrifugation, and the protein was purified from the medium using r-protein A Sepharose (GE Healthcare). The purified protein was buffer-changed with either PBS or a storage-specific stabilization buffer (36.8 mM citrate, 63.2 mM Na2HPO4, 10% trehalose, 0.2 M L-arginine, 0.01% Tween-80, pH 6.0). Protein concentration was determined by absorbance at 280 nm, and purity was confirmed by SDS-PAGE analysis. 5. In vitro binding assay

[0194] The BLI assay was performed using the Octet HTX instrument (ForteBio). To measure binding to the antigen, the FZD receptor Fc-tagged fusion (FZD-Fc protein) was captured on the AHQBLI sensor (18-5001, ForteBio) to reach a BLI response of 0.6-1 nm, and the remaining Fc binding site was saturated with human Fc (009-000-008, Jackson ImmunoResearch). The FZD-coated or control (Fc-coated) sensor was transferred to 100 nM Ab or FLAg in assay buffer (PBS, 1% BSA, 0.05% Tween20), and association was monitored for 300 seconds. The sensor was then transferred to assay buffer, and dissociation was monitored for another 300 seconds. The stirring speed was set to 1000 rpm and the temperature to 25°C. The endpoint response value was obtained at the association time after 295 seconds. The endpoint data was analyzed by subtracting the Fc signal from the FZD-Fc signal, and then normalizing the data to the highest combined signal.

[0195] To measure binding to Fc receptors, Abs or FLAg was immobilized on an AR2G sensor (18-5092, Forte Bio) by amine coupling to reach a BLI response of 0.6–3 nm, and the remaining portion was eliminated with ethanolamine. The coated sensor was equilibrated in assay buffer (PBS, 1% BSA, 0.05% Tween 20) and transferred to the Fc receptor solution. Association was monitored for 600 seconds, and the sensor was transferred to assay buffer and dissociation was monitored for 600 seconds. CD64 and all other Fc receptors were assayed at 50 nM or 300 nM, respectively, at pH 7.4, unless otherwise indicated. The stirring speed was set to 1000 rpm and the temperature to 25°C. The endpoint response value was obtained at the end of the association phase and normalized to the isotype control. The steady-state FcRN binding assay was performed similarly, except that FcRN was immobilized and a dilution series of Ab or FLAg (0.1–225 nM) was evaluated in solution. The association time and dissociation time were set to 600 seconds or 1200 seconds, respectively.

[0196] Surface plasmon resonance (SPR) assays were performed using the ProteOnXPR36 system (Bio-Rad). FZD-Fc or LRP-Fc proteins were immobilized on the GLC sensor surface (176-5011) using standard amine coupling chemistry. Ab or FLAg was injected at 40 μL / min in assay buffer (PBS, 0.05% Tween 20, 0.5% BSA), and association was monitored for 150 seconds. Subsequently, assay buffer was injected at 100 μL / min, and dissociation was monitored for 900 seconds. The assay was performed at 25°C. Analysis was performed using a 1:1 Langmuir model and global fit, and kon and koff values ​​were determined using ProteOn Manager software. KD was calculated as the koff / kon ratio. 6. Epitope Binning

[0197] BLI epitope binning experiments were performed using the Octet HTX instrument (ForteBio). Fc fusions with FZD (FZD-Fc) or LRP6 (LRP6-Fc) proteins were immobilized onto AHQ (18-5001, ForteBio) or AR2G (18-5092, ForteBio) BLI sensors, respectively. The coated sensors were transferred to 100 nM Ab in assay buffer (PBS, 1% BSA, 0.05% Tween20) for 240 seconds to achieve binding site saturation. Subsequently, the sensors were transferred to 100 nM competitive Ab in assay buffer for 180 seconds. The response 20 seconds after exposure to competitive Ab was measured and normalized to the binding signal on the non-blocking antigen-coated sensor. The stirring speed was 1000 rpm and the temperature was 25°C. 7. Cell lines

[0198] HPAF-II and HEK293T cell lines were maintained in DMEM supplemented with 4.5 g / L D-glucose, sodium pyruvate, and L-glutamine (Thermo Fisher #12430-054), along with 10% FBS (Thermo Fisher) and penicillin / streptomycin (Thermo Fisher #15140-163). CHO cells were maintained in DMEM / F12 (Thermo Fisher #11320-033) supplemented with 10% FBS and penicillin / streptomycin. Cells were maintained at 37°C and 5% CO2. 8. Flow cytometry

[0199] Indirect immunofluorescence staining of cells was performed on CHO cell lines using 10 nM anti-FZD Fab, as previously described (Steinhart et al. 2017 Nat Med. Jan;23(1):60-68, PMID: 27869803). Alexa Fluor 488 AffiniPure F(ab')2 was used as the secondary antibody (Jackson ImmunoResearch, 109-545-097). Anti-c-Myc IgG1 9E10 (primary antibody, Thermo Fisher, MA1-980) and Alexa Fluor 488 IgG (secondary antibody, Life Technologies, A11001) were used as expression controls. All reagents were used according to the manufacturer's instructions. 9. Luciferase Reporter Assay

[0200] A lentivirus encoding the pBARls reporter (Biechele and Moon in Wnt Signalling: Pathway Methods and Mammalian Models, E. Vincan, Ed. (Humana Press, Totowa, NJ, 2008), pp. 99-110) and sea urchin luciferase as a control were transfused into HEK293T cells to generate a Wnt-β-catenin signaling reporter cell line. Before transfection or stimulation, 1–2 × 10⁶ units were added in 120 μL. 3 Cells were seeded in each well of a 96-well plate for 24 hours. The following day, FLAg or Ab protein was added, and 15–20 hours after stimulation, the cells were lysed and luminescence was measured using an Envision plate reader (PerkinElmer) according to the dual luciferase protocol (Promega). For the FZD4-specific agonist assay, FZD4 cDNA was transfected for 6 hours, followed by the addition of FLAg protein. For the Wnt inhibition assay, Wnt1 was introduced by cDNA transfection, or WNT3A protein was applied for 6 hours, followed by the addition of Ab protein. All assays were repeated at least three times. 10. Western blot assay

[0201] H1 ESC was solubilized in a lysis buffer (1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 0.1% deoxycholic acid, 50 mM Tris (pH 7.4), 0.1 mM EGTA, 0.1 mM EDTA, 20 mM sodium fluoride (NaF), 1:500 protease inhibitor (Sigma), and 1 mM sodium orthovanadate (Na3VO4)). The lysate was incubated at 4°C for 30 minutes, centrifuged at 14,000 × g for 10 minutes, boiled in SDS sample buffer, separated by SDS polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and subjected to Western blotting using the indicated Ab. Ab detection was performed using a chemiluminescence-based detection system (ECL; Thermo Fisher). 11. Crystal Violet Growth Assay

[0202] HPAF-II cells were seeded at a rate of 500 cells per well, and after 24 hours, 100 nM LGK974 was added in the presence or absence of 100 nM FLAg. The medium was changed, and the drug treatment was renewed every other day. After 7 days of treatment, the cells were fixed in ice-cold methanol. The cells were stained with 0.5% crystal violet solution in 25% methanol, destained in 10% acetic acid, and quantified by measuring the absorbance at 590 nm. 12. Immunofluorescence

[0203] H1 hES cells treated with FLAg and CHIR99021 for 3 days were washed with cold PBS and fixed with 4% PFA for 20 minutes. The fixed cells were rinsed with PBS, permeabilized with 0.3% triton for 10 minutes, and blocked with 1% BSA for 1 hour. The cells were incubated in 1% BSA with either a primary Ab against brachyury (R&D Systems AF2085; goat; dilution 1:100) or OCT3 / 4 (Santa Cruz sc5279; mouse; dilution 1:100) for 2 hours, and with either an Alexa Fluor488-labeled donkey anti-goat or Alexa Fluor568-labeled donkey anti-mouse Ab for 1 hour (Figure 13A). Coverslips were mounted using Fluoromount (Sigma-Aldrich) and analyzed on a Zeiss LSM700 confocal microscope using a 60× oil immersion objective lens (Figure 13B). Images were assembled using ImageJ and Photoshop CS6 (Adobe Systems, Mountain View, California). 13. Intestinal crypt autoregeneration assay

[0204] Lgr5-EGFP-IRES-creERT2(B6.129P2-Lgr5tm1(cre / ERT2)Cle / J) mice aged 8-10 weeks were purchased from Jackson Laboratory (Bar Harbor, Maine). All experiments were conducted according to protocols approved by the University of Toronto's Animal Experimentation Committee and complied with the regulations and ARRIVE guidelines (Reporting of Animal Experiments: In Vivo Experiments) of the Canadian Animal Protection Council. P+P -L6 1+3 Alternatively, the negative control Ab was reconstituted in 37 mM citrate, 63 mM Na2HPO4, 10% trehalose, 0.2 ml arginine, 0.01% polysorbate 80, pH 6.0. The porcupine inhibitor C59 was reconstituted using 0.5% methylcellulose mixed with 0.1% Tween 80 in ddH2O. Mice (male and female) were divided into the following three groups (5-7 mice / group): vehicle, control (C59 and control Ab), or FLAg (C59 and F P+P -L6 1+3On day 1, the vehicle or control Ab or F at 10 mg / kg. P+P -L6 1+3 Mice were treated by intraperitoneal injection. This treatment was blinded to the researchers until the end of the experiment and was repeated every two days for a total of three treatments. Starting on day 2, either the vehicle group or 50 mg / kg of C59 was administered via gastric tube to the vehicle group or the two experimental groups, twice daily at 8-hour intervals for 4 days. Mice were sacrificed on day 6. Whole intestinal tissue was collected, cleaned with cold PBS, dehydrated with PBS and 30% sucrose, fixed with 4% paraformaldehyde, and embedded in an optimal cutting temperature compound (OCT). 8 μm OCT frozen sections were used for immunohistochemical analysis. EGFP crypts of the intestine were analyzed using a confocal microscope (Zeiss LSM700). Vehicle, C59, or pan-FLAg (F P+P -L6 1+3 Representative fluorescence images of small intestinal sections from LGR5-GFP mice treated with )+C59 are shown in Figure 14. LGR5-GFP is expressed in stem cells in the lower part of the crypts. Cell nuclei were counterstained with DAPI.

[0205] Those skilled in the art will be able to recognize or verify a great many equivalents of the specific procedures described herein without using anything beyond ordinary experimentation. Such equivalents are considered to be within the scope of the invention. Various substitutions, changes, and modifications may be made to the invention without departing from the spirit and scope of this application. Other aspects, advantages, and modifications are within the scope of the invention. The contents of all references, published patents, and published patent applications cited throughout this application are incorporated herein by reference. Appropriate components, processes, and methods of those patents, applications, and other documents may be selected for the invention and its embodiments.

[0206] [Table 1A] JPEG2026139669000003.jpg216153JPEG2026139669000004.jpg216153JPEG2026139669000005.jpg216153JPEG2026139669000006.jpg216153JPEG2026139669000007.jpg216153JPEG2026139669000008.jpg216153JPEG2026139669000009.jpg216153JPEG2026139669000010.jpg216153JPEG2026139669000011.jpg216153JPEG2026139669000012.jpg216153JPEG2026139669000013.jpg216153JPEG2026139669000014.jpg216153JPEG2026139669000015.jpg216153JPEG2026139669000016.jpg216153JPEG2026139669000017.jpg216153JPEG2026139669000018.jpg216153JPEG2026139669000019.jpg216153JPEG2026139669000020.jpg216153JPEG2026139669000021.jpg216153JPEG2026139669000022.jpg216153JPEG2026139669000023.jpg216153

[0207]

Table 1B

[0208] [Table 2] JPEG2026139669000046.jpg101153

[0209] [Table 3] JPEG2026139669000048.jpg233153JPEG2026139669000049.jpg227153JPEG2026139669000050.jpg226153JPEG2026139669000051.jpg204153

Claims

1. A method for activating the Wnt signaling pathway in cells, comprising contacting cells having a Frizzled (FZD) receptor and a Wnt coreceptor with a polyvalent binding molecule, the polyvalent binding molecule is (a) A fragment having a C-terminus and an N-terminus, an Fc domain, or a fragment containing its CH3 domain, (b) An FZD binding domain having at least two binding sites, wherein at least one binding site binds to the FZD receptor, and (c) A Wnt coreceptor domain having at least two binding sites, wherein at least one binding site binds to the Wnt coreceptor. Includes, A method wherein the FZD binding domain is attached to one end of the Fc domain or one end of a fragment thereof, and the Wnt coreceptor binding domain is attached to the other end of the Fc domain or the other end of a fragment thereof.

2. The FZD binding domain is (a) A diabody that binds the FZD receptor, comprising two peptides, each containing a heavy chain variable domain (VH) linked to a light chain variable domain (VL), wherein the VH and VL from one peptide pair with the VL and VH of the other peptide to form the diabody, or (b) scFv including a VL region and a VH region that bind to the FZD receptor, (c) Endogenous ligand of the FZD receptor Includes, The Wnt coreceptor-binding domain is (d) A diabody that binds the Wnt coreceptor, comprising two peptides, each containing a heavy chain variable domain (VH) linked to a light chain variable domain (VL), wherein the VH and VL from one peptide pair with the VL and VH of the other peptide to form the diabody, or (e) scFv including a VL region and a VH region that bind the coreceptor, (f) an endogenous ligand of the coreceptor, or a fragment of such ligand that binds to the coreceptor. The method according to claim 1, including the method described in claim 1.

3. The method according to claim 2, wherein the FZD binding domain binds to the Wnt ligand binding site of the FZD receptor, or the Wnt coreceptor binding domain binds to the Wnt ligand binding site of the Wnt coreceptor.

4. The method according to claim 3, wherein the Wnt coreceptor-binding domain binds to the Wnt3 binding site and / or the Wnt1 binding site.

5. The method according to claim 3, wherein VH and VL are derived from the polyvalent bond molecules listed in Table 1.

6. The method according to claim 1, wherein the FZD-binding domain binds one or more FZD receptors.

7. The method according to claim 1, wherein the Fc domain is an IgG Fc domain.

8. The method according to any one of the above claims, wherein the FZD receptor is FZD1, FZD2, FZD4, FZD5, FZD7, or FZD8, and the Wnt coreceptor is LRP5, LRP6, ROR1, ROR2, RYK, PTK7, GPR124, or TSPAN12.

9. (a) A fragment having a C-terminus and an N-terminus, an Fc domain, or a fragment containing its CH3 domain, (b) An FZD binding domain having at least two binding sites, wherein at least one binding site binds to the FZD receptor, and (c) A Wnt coreceptor domain having at least two binding sites, wherein at least one binding site binds to the Wnt coreceptor. Includes, A polyvalent binding molecule in which the FZD binding domain is attached to one end of the Fc domain, and the Wnt coreceptor binding domain is attached to the other end of the Fc domain.

10. The FZD binding domain is (a) A diabody that binds the FZD receptor, comprising two peptides, each containing a heavy chain variable domain (VH) linked to a light chain variable domain (VL), wherein the VH and VL from one peptide pair with the VL and VH of the other peptide to form the diabody, or (b) scFv including a VL region and a VH region that bind to the FZD receptor, (c) Endogenous ligand of the FZD receptor Includes, The Wnt coreceptor-binding domain is (d) A diabody that binds the Wnt coreceptor, comprising two peptides, each containing a heavy chain variable domain (VH) linked to a light chain variable domain (VL), wherein the VH and VL from one peptide pair with the VL and VH of the other peptide to form the diabody, or (e) scFv including a VL region and a VH region that bind the coreceptor, (f) an endogenous ligand of the Wnt coreceptor, or a fragment of such ligand that binds to the Wnt coreceptor. A polyvalent bond molecule according to claim 9, comprising:

11. The polyvalent binding molecule according to claim 9, wherein at least one of the binding domains is bispecific.

12. The polyvalent binding molecule according to claim 10, wherein VH and VL are derived from an antibody that binds to the FZD receptor and selectively inhibit the binding of Wnt ligand to the FZD receptor, or VH and VL are derived from an antibody that binds to the Wnt coreceptor and selectively inhibit the binding of Wnt ligand to the coreceptor.

13. The polyvalent bond molecule according to claim 10, wherein VH and VL are derived from the polyvalent bond molecules listed in Table 1.

14. (a) The FZD-binding domain binds to one or more of the FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, or FZD10 receptors, or (b) The Wnt coreceptor-binding domain binds one or more of LRP5, LRP6, ROR1, ROR2, RYK, PTK7, GPR124, or TSPAN12, or (c) The FZD-binding domain binds one or more of the FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, or FZD10 receptors, and the Wnt coreceptor-binding domain binds one or more of the LRP5, LRP6, ROR1, ROR2, RYK, PTK7, GPR124, or TSPAN12. A polyvalent bond molecule according to any one of claims 9 to 13.

15. A pharmaceutical composition comprising a polyvalent bond molecule according to any one of claims 9 to 14 and a pharmaceutically acceptable carrier.

16. A method for treating a subject having a condition associated with reduced Wnt signaling, which involves administering to the subject a polyvalent binding molecule according to any one of claims 9 to 14 in an amount sufficient to enhance tissue regeneration or alleviate symptoms associated with the condition.

17. A method for promoting the interaction between FZD receptors and Wnt coreceptors on a cell, thereby activating the Wnt signaling pathway in the cell, a) Select a fragment having a C-terminus and an N-terminus, and containing an Fc domain or its CH3 domain. b) Linking a divalent FZD receptor-binding domain to one end of the Fc domain and a divalent Wnt coreceptor-binding domain to the other end of the Fc domain, thereby forming a tetravalent binding molecule; c) Contacting the cells expressing the FZD receptor and the Wnt coreceptor with the tetravalent conjugation molecule under conditions in which the tetravalent conjugation molecule binds to the FZD receptor and the Wnt coreceptor, thereby activating the Wnt signaling pathway. A method that includes this.

18. The method according to claim 17, wherein the divalent FZD receptor-binding domain includes a diabody that binds to an FZD receptor, and the divalent Wnt receptor-binding domain includes a diabody that binds to a Wnt coreceptor.

19. The method according to claim 18, wherein the diabody that binds the Wnt coreceptor binds to one or both of the Wnt1 binding site or the Wnt3a binding site on the Wnt coreceptor.

Images