Multivalent FZD and WNT binding molecules and uses thereof

Abstract

The present invention relates to multivalent FZD and WNT binding molecules and uses thereof. Described herein are methods of affecting binding of a multivalent binding molecule to a FZD receptor and a Wnt co-receptor on a cell, wherein binding of the multivalent binding molecule to both the FZD receptor and the co-receptor on the cell activates the Wnt signaling pathway. Also described herein are multivalent binding molecules that activate the Wnt signaling pathway, comprising an FZD receptor binding domain and a Wnt co-receptor binding domain on either end of the Fc domain, and methods of using the same.

Description

[0001] This application is a divisional application of the patent application "Multivalent FZD and WNT binding molecules and their uses" with an international application date of June 10, 2020 and application number 202080053985.8 (international application number PCT / IB2020 / 055463). Background Art

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 860,161, filed on June 11, 2019, based on 35 U.S.C. 119(e), the entire contents of which are incorporated herein by reference.

[0003] This application contains a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on June 10, 2020, is named 115773_PC424WO_SL.txt, and is 279,203 bytes in size.

[0004] The Wnt signaling pathway is critical for embryonic development and tissue homeostasis in adults. Wnt ligands are secreted growth factors that regulate various cellular processes, such as proliferation, differentiation, survival, and migration. Wnt ligands are generally important for controlling tissue stem cell self-renewal and regulating many progenitor cell populations. The hydrophobicity and sensitive tertiary structure of Wnt proteins make their biochemical purification challenging and preclude their use in vivo and in vitro.

[0005] There are 19 Wnt ligands in humans that interact with a network of 10 frizzled cell surface receptors (FZDs) and one of several co-receptors that direct 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 to play a role in three different signaling pathways, known as the Wnt planar cell polarity (PCP) pathway, the canonical Wnt / β-catenin pathway, and the Wnt / calcium pathway. Activation of the Wnt signaling pathway also requires the presence of Wnt co-receptors to indicate differential engagement of intracellular signaling cascades, regulating the expression of genes that affect the cellular mechanisms underlying the above-listed cellular processes. For example, Wnt ligands bind to members of the Frizzled receptor and low-density lipoprotein receptor-related protein 5 and 6 (LRP5 / 6) co-receptor families to activate the Wnt / β-catenin pathway, or to receptor tyrosine kinase-like orphan receptor 1 and 2 (ROR1 / 2) involving receptor tyrosine kinase (RYK) or protein tyrosine kinase 7 (PTK7) co-receptors to initiate the Wnt / PCP pathway or alternative β-catenin-independent signaling pathways. The Wnt / β-catenin pathway, sometimes referred to as the canonical pathway, culminates in the post-translational accumulation of the transcriptional effector β-catenin, which interacts with the T-cell factor / lymphoid enhancer factor (LEF / TCF) family of transcription factors to regulate the expression of genes in a specific context. Summary of the invention

[0006] Wnts require lipidation to function (Janda et al., Science. 337, 59-64 (2012); Kadowaki et al., Genes Dev. 10, 3116-3128 (1996)), and their hydrophobicity complicates biochemical manipulation; therefore, only a few Wnts have been purified (Willert et al., Nature 423, 448-452 (2003). In addition, Wnts have inherent cross-reactivity to multiple receptors, especially when overexpressed or applied 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) (obtained from http: / / www.jbc.org / content / early / 2015 / 01 / 20 / jbc.M114.12648.short); Holmen et al., J.Biol.Chem.277, 34727-34735 (2002). ). Therefore, it is not possible to selectively activate the Frizzled receptor complex to determine the specific function of each in different situations or to evaluate its therapeutic potential for degenerative conditions. The multivalent binding molecules and methods described herein selectively activate preselected Frizzled receptor-co-receptor complexes. It is expected that the administration of the multivalent binding molecules described herein will treat degenerative conditions by activating the appropriate Frizzled co-receptor complex.

[0007] Described herein are methods of affecting the binding of a peptide to a FZD receptor and a Wnt co-receptor on a cell, wherein binding of the peptide to both the FZD receptor and the co-receptor activates the Wnt signaling pathway.

[0008] Also described herein are multivalent binding molecules that activate the Wnt signaling pathway and methods of use thereof. The multivalent binding molecules bind to both the FZD receptor and the Wnt co-receptor, thereby activating the Wnt signaling pathway. The multivalent binding molecules of the present invention are also referred to herein as "FZD agonists" or "FZDags". In a specific embodiment in which the molecules of the present invention bind to FZD and LRP5 / 6, the molecule may be referred to as "Frizzled and LRP5 / 6 agonists" or "FLAg". The multivalent binding molecule comprises an Fc domain or a fragment thereof comprising a CH3 domain, and a first binding domain that binds to the FZD receptor and a second binding domain that binds to the Wnt co-receptor, wherein the FZD binding domain is connected to one end of the Fc domain, and the co-receptor binding domain is connected to the other end of the Fc domain. Therefore, the binding domain for the FZD receptor and the binding domain for the co-receptor are not directly connected, but are separated by the Fc domain, or a fragment thereof comprising a CH3 domain. This conformation of the binding domain produces an unexpectedly high level of Wnt signaling pathway activation. The FZD binding domain can be monovalent with a single binding site (paratope) of a FZD receptor, or can be multivalent with more than one binding site of a FZD receptor, for example, the binding domain can be bivalent, trivalent, or tetravalent. The Wnt co-receptor binding domain can be monovalent with a single binding site (paratope) of a Wnt co-receptor, or can be multivalent with more than one binding site of a Wnt co-receptor, for example, the binding domain can be bivalent, trivalent, or tetravalent.

[0009] Methods for producing multivalent binding molecules as described herein enable selective and robust activation of any FZD receptor complex in vitro and in vivo. Using a panel of hundreds of synthetic antibodies targeting FZD and its co-receptors, we generate multivalent binding molecules for selectively and rationally activating one, two or more FZD receptors. The multivalent binding molecules of the present invention are highly stable, suitable for large-scale production and easy purification, have predictable pharmacokinetics, and are expected to exhibit low immunogenicity.

[0010] In one embodiment of the invention, the binding domain of the multivalent binding molecule as described herein binds to more than one FZD receptor and LRP, such as LRP5 and / or LRP6, and may be alternatively referred to herein as FLAg. FLAg targeting specific FZDs and their LRP co-receptors will improve directed differentiation and cell therapy, maintain the growth of tissue organoids, and mobilize endogenous stem cells in vivo, promote tissue repair after injury, and restore function after tissue degeneration.

[0011] The Fc domain of the FZD agonist can be an Fc domain of an immunoglobulin. The immunoglobulin can be an IgG, such as IgG 1In one embodiment of the invention, the multivalent binding molecule is a peptide dimer, wherein the peptide dimerizes through the intrinsic ability of the Fc domain to dimerize or through a knob-in-hole conformation within the Fc, thereby allowing the specific assembly of two different peptides to produce a multivalent binding domain. Methods for dimerizing peptides through a knob-in-hole conformation are described in WO2018 / 026942, inventors Van Dyk et al., incorporated herein by reference.

[0012] One or both of the multivalent binding domains of the FZD agonists as described herein can be bivalent and monospecific, having two binding sites for the same epitope of their respective receptor or co-receptor targets. One or both of the binding domains can be bivalent and bispecific, having two binding sites, wherein each site binds to a different epitope on its respective target.

[0013] In one embodiment of the invention, the FZD binding domain may comprise two single chain variable fragments (scFv) for binding to the same or different epitopes on the FZD receptor. In other embodiments of the invention, the FZD 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 to FZD. In other embodiments of the invention, the FZD binding domain consists of one or more single domain antibody fragments that bind to FZD. In other embodiments of the invention, the FZD binding domain comprises a FZD ligand or a fragment thereof that binds to the FZD receptor. In one embodiment of the invention, the FZD binding domain comprises a synthetic peptide that binds to FZD, for example, an affinity body, an ankyrin repeat protein, a fibronectin repeat protein, a fynomer, or anticalin. In one embodiment of the invention, the FZD multivalent binding domain does not comprise scFv. The FZD ligand may be, for example, a fragment of a Wnt protein that binds to a FZD receptor or a fragment of Norrin, or may be another natural or synthetic peptide that is affinity matured to interact with more than one FZD receptor. Norrin is a FZD4-specific ligand that forms a complex with LRP5 and / or LRP6 and is associated with the activation of canonical Wnt signaling.

[0014] In one embodiment of the 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 other embodiments of the 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 to the Wnt co-receptor. In other embodiments of the invention, the co-receptor binding domain consists of one or more single domain antibody fragments that bind to the co-receptor. In one embodiment of the invention, the Wnt co-receptor binding domain comprises a peptide that binds to the Wnt co-receptor, wherein the peptide is a fragment of a naturally occurring ligand that binds to the Wnt co-receptor or a synthetic peptide that binds to the Wnt co-receptor, such as an affinity body, an ankyrin repeat protein, a fibronectin repeat protein, a fynomer, or anticalin. In another embodiment of the invention, the co-receptor binding domain comprises a co-receptor ligand or a fragment thereof that binds to the co-receptor (e.g., a ligand Dkk1 for the co-receptor LRP5 / 6) or another natural or synthetic peptide that is affinity matured to interact with more than one co-receptor.

[0015] In one embodiment of the invention, the co-receptor multivalent binding domain does not comprise a scFv.

[0016] In one embodiment of the invention, each binding domain of the molecules described herein can be formed from two peptides, each peptide comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL), wherein the VH and VL from one peptide are paired with the VL and VH of the other peptide to form a diabody. In this conformation, the binding domain has two binding sites that bind to its target, i.e., the FZD binding domain has two binding sites for the FZD receptor, and the co-receptor binding domain has two binding sites for the co-receptor. Using the knob-in-hole Fc conformation, peptides comprising the VH and VL can be engineered so that they are different from each other, but still paired to form a bispecific binding domain that is capable of binding to two different sites on the FZD receptor or co-receptor (see Figure 3A ).

[0017] In one embodiment of the invention, one or both of the multivalent binding domains comprises two peptides that form a diabody on each end of the Fc domain. Each diabody has two binding sites for an epitope on its respective FZD receptor or co-receptor target. The diabody can be monospecific in which the binding sites bind to the same epitope on a FZD receptor or co-receptor, or the diabody can bispecifically bind to two different epitopes on a FZD receptor or co-receptor.

[0018] The peptide forming scFv or diabody can be derived from the antibody bound to FZD receptor or from the antibody bound to Wnt co-receptor.For FZD binding domain, antibody can be bound to more than one FZD receptor and antagonize Wnt signal transduction or suppress Wnt and given FZD receptor (more than one) antibody, or antibody can be bound to more than one FZD receptor without suppressing Wnt and FZD receptor binding antibody.For co-receptor binding domain, antibody can be bound to co-receptor and antagonize Wnt signal transduction or suppress Wnt and co-receptor binding antibody or antibody can be bound to co-receptor without suppressing Wnt and co-receptor binding antibody.

[0019] The FZD binding domain can bind to more than one member of the FZD receptor family, for example, Frizzled receptor 1 (FZD1), Frizzled receptor 2 (FZD2), Frizzled receptor 3 (FZD3), Frizzled receptor 4 (FZD4), Frizzled receptor 5 (FZD5), Frizzled receptor 6 (FZD6), Frizzled receptor 7 (FZD7), Frizzled receptor 1 Frizzled receptor 8 (FZD8), Frizzled receptor 9 (FZD9), or Frizzled receptor 10 (FZD10). The co-receptor binding domain can bind to any Wnt co-receptor, for example, LRP5 / 6, PTK7, ROR1 / 2, RYK, GPR124, TSPAN12, or CD133. In one embodiment of the invention, the co-receptor binding domain binds LRP5 and / or LRP6. In one embodiment of the invention, the co-receptor binding domain binds to a single epitope on a co-receptor, e.g., an epitope of an LRP protein that binds to Wnt1 or Wnt3a. In one embodiment of the invention, the co-receptor binding domain binds to two epitopes on a co-receptor, e.g., an epitope on LRP that binds to Wnt1 and an epitope that binds to Wnt3a.

[0020] One embodiment of the present invention includes a method for producing induced pluripotent stem cells (iPS), the method comprising culturing somatic cells under conditions suitable for reprogramming somatic cells in the presence of an effective amount of a multivalent binding molecule as described herein. The multivalent binding molecule may be included in an amount that accelerates the production of iPS cells compared to the production of iPS cells under the same culture conditions without the multivalent binding molecule.

[0021] Another embodiment of the invention is a method of directing differentiation of iPS or other pluripotent stem cells (PSCs) toward various lineages by culturing these cells in the presence of an effective amount of a multivalent binding molecule as described herein.

[0022] One embodiment of the invention includes a method for producing tissue organoids, the method comprising culturing a tissue sample under conditions suitable for producing organoids in the presence of an effective amount of a multivalent binding molecule as described herein as part of a culture mixture. In one embodiment or the invention, the frequency of production of tissue organoids cultured in a culture medium comprising a multivalent binding molecule is increased compared to organoids cultured in the same culture medium without the multivalent binding molecule. In one embodiment or the invention, the production of tissue organoids is faster when cultured in a culture medium comprising a multivalent binding molecule compared to tissue samples cultured in the same culture medium without the multivalent binding molecule.

[0023] One embodiment of the invention includes a method for enhancing the maintenance of tissue organoids, the method comprising culturing the organoids in the presence of an effective amount of a multivalent binding molecule as described herein as part of a culture mixture. As described herein, the survival of tissue organoids cultured in a culture medium comprising a multivalent binding molecule is prolonged compared to organoids cultured in the same culture medium without the multivalent binding molecule.

[0024] One aspect of the invention is a method for preparing a multivalent binding molecule as described herein. In one embodiment of the invention, the multivalent binding molecule is produced by,

[0025] a) selecting an Fc domain having a C-terminus and an N-terminus,

[0026] b) antibodies that recognize antibodies that bind to more than one FZD receptor and

[0027] c) recognizing antibodies that bind to more than one Wnt co-receptor,

[0028] d) producing 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 a nucleotide sequence derived from the VL and / or VH of the antibody of step b that binds to more than one FZD, and (iii) a nucleotide sequence encoding the VL and VH of the antibody of step c, or a nucleotide sequence derived from the VL and VH of the antibody of step c that binds to more than one Wnt receptor of step c,

[0029] e) expressing the nucleic acid molecule of (d) to produce a polypeptide, wherein the polypeptide dimerizes to form a multivalent binding molecule comprising an Fc domain, a FZD binding domain, and a Wnt co-receptor binding domain, wherein the FZD binding domain comprises the VL and VH of the antibody of step b or the antibody derived from step b, and is linked to one end of the Fc domain, and the Wnt co-receptor binding domain comprises the VL and VH of the antibody of step c or the antibody derived from step c, and is linked to the other end of the Fc domain, thereby forming a multispecific binding molecule.

[0030] The antibody in step (b) can be an antibody or antibody fragment that binds to more than one FZD receptor and antagonizes Wnt signaling or inhibits Wnt binding to the receptor. The antibody in step (b) can be an antibody or antibody fragment that binds to more than one FZD receptor without antagonizing Wnt signaling or inhibiting Wnt binding to the receptor. The antibody in step (c) can be an antibody or antibody fragment that binds to more than one Wnt co-receptor and antagonizes Wnt signaling or inhibits Wnt binding to the co-receptor, or binds to the co-receptor without antagonizing Wnt signal transduction or inhibiting Wnt binding to the co-receptor. The binding domain can be connected to the Fc domain by a linker. The modular aspects of the present invention allow the binding domains of antibodies for any given FZD receptor and co-receptor to be mixed and matched at the end of the Fc domain to produce a multivalent binding molecule that can bind to multiple Frizzled protein receptor-co-receptor complexes or selectively bind to a single Frizzled protein receptor-co-receptor complex to activate Wnt signaling.

[0031] The multivalent binding molecule comprises a peptide dimer in a conformation having an Fc domain and a binding domain that binds to more than one FZD receptor and a second binding domain that binds to more than one Wnt co-receptor, wherein the FZD binding domain is connected to one end of the Fc and the co-receptor binding domain is connected to the other end of the Fc. Each binding domain can be monovalent or multivalent, such as bivalent, trivalent or tetravalent.

[0032] Another embodiment of the invention is a method of using the multivalent binding molecules, for example, for generating induced pluripotent stem (iPS) cells, for directed differentiation of pluripotent stem cells, and for generating and / or maintaining organoids, or for enhancing tissue regeneration in a subject in need thereof.

[0033]

[0013] Additional embodiments of the invention are methods of activating the Wnt signaling pathway to mobilize endogenous stem / progenitor cell pools for use in regenerative medicine and for conditions or diseases associated with deficient Wnt signaling. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1AThe binding specificity of five selected antibodies for binding to the extracellular domain (ECD) of human LRP6 is described. LRP6 binding antibodies were selected from a synthetic antibody library by selecting antibodies that bind to the recombinant extracellular domain (ECD) of human LRP6. The antibodies were tested for binding to human LRP6, mouse LRP6, and mouse LRP5 by ELISA. Binding to Fc peptide and bovine serum albumin (BSA) were included as negative controls.

[0035] Figure 1B Depicts the results of a luciferase reporter assay monitoring Wnt signaling activation, demonstrating that IgG2539 and IgG2542 (100 μM) bind to different sites on the LRP6 ECD with opposing effects upon stimulation of Wnt1 (transient transfection) and Wnt3a (0.5 μg / ml purified protein). Anti-MBP antibody was used as a control.

[0036] Figure 2A Representative bispecific IgG (Bi-IgG) and bispecific diabodies (bi-diabodies) comprising a FZD binding domain (5019) and LRP6-W1 (2942, L6 1 ) or W3(2539,L6 3 ) binding domain.

[0037] Figure 2B It was shown that the bispecific IgGs (5019-2539Bi-IgG and 5019-2542Bi-IgG) do not activate Wnt signaling, but rather act as antagonists of Wnt signaling as determined in a TOPFlash luciferase reporter assay in HEK293 cells.

[0038] Figure 2C-2G The binding of bispecific diabodies in which the Fc domain is in a knob-and-hole configuration (K / H) is described. The two diabodies thus obtained, 5019-2539-K / H (FZD / LRP6-W3) and 5019-2542-K / H (FZD / LRP6-W1), retain the FZD binding properties of the original IgG as well as the binding activity to LRP6, albeit very weakly. Figure 2C Purified FZD-LRP6 diabodies: 5019-2539-K / H and 5019-2542-K / H are described. Figure 2D Depicting the FZD receptor binding profiles of the 5019 diabody to FZD4, FZD5, and FZD7. The 5019 FZD IgG was previously characterized as binding to FZD1, 2, 4, 5, 7, 8. Figure 2E Depicting the FZD receptor binding curve of the bispecific FZD / LRP6 diabody 5019-2539-K / H. Figure 2F Depicting the FZD receptor binding curve of the bispecific FZD / LRP6 diabody 5019-2542-K / H. Figure 2G The interaction of homodiabodies (2539-Fc and 2542-Fc) and heterodiabodies (5019-2539-Fc and 5019-2542-Fc) having a binding domain on one end of the Fc domain with the extracellular domain of LRP6 was demonstrated. Figure 2H Diabodies 5019-2539-K / H and 5019-2542-K / H were shown to co-bind to FZD CRD and LRP6 ECD in solution as determined by biolayer interferometry (BLI) assays.

[0039] Fig.2I This indicates that the FZD and LRP6 receptor diabodies in which the binding domain is formed are present on the same side of the Fc, and that neither 5019-2539-K / H nor 5019-2542-K / H is a FZD agonist that activates the Wnt-mediated pathway. The results illustrate that, as revealed in the TOPFlash luciferase reporter assay in HEK293 cells, the 5019-2539-K / H diabody (selective for the Wnt3 site on LRP6) completely blocked activation of the Wnt3-mediated pathway at 10 nM and 50 nM, whereas 5019-2542-K / H was less effective.

[0040] Figure 2J Describes a comparison of luciferase activity of tetravalent binding molecules having binding domains comprising a diabody or scFv. P*+P* -L6 1+3 The molecular representation of the binding domain of the antibody is similar to that of the antibody comprising an anti-FZD diabody and an anti-LRP diabody (F P+P -L6 1+3 ) binding domain. P+P -L6 1+3 Compared with the anti-FZD diabody and anti-LRP6 scFv (F P+P -L6 1*+3* ) or contain scFv (F P*+P* -L6 1*+3* ) molecules, the molecular activity was significantly reduced.

[0041] Figure 2K and Figure 2LThis suggests that the differences in activity between tetravalent binding molecules having binding domains comprising diabody or scFv are not due to differences in affinity, as BLI assays showed that binding to LRP6 and FZD isoforms was of comparable high affinity regardless of whether the paratope was presented in diabody or scFv format.

[0042] Figure 3A is a schematic diagram representing a tetravalent binding molecule, wherein two FZD binding domains comprising a homologous (recognizing the same epitope) or heterologous (recognizing different epitopes) diabody are connected to one end of an Fc domain and two LRP6 binding domains comprising a homologous or heterologous diabody are connected to the other end of the Fc domain.

[0043] Figure 3B Describe the multivalent binding molecule 5019-Fc-2539 (F P+P -L6 3+3 ) and 5019-Fc-2542(F P+P -L6 1+1 ) binding to FZD4, FZD5 and FZD7 ECD. Binding to FZD receptors was detected using BLI assay.

[0044] Figure 3C The tetravalent binding molecule 5019-Fc-2539 (F P+P -L6 3+3 ); 5019-Fc-2542(F P+P -L6 1 +1 ); 5019-K / H-2539-2542(F P+P -L6 1+3 ), and purified Wnt3A (0.5 μg / ml). The concentration of the molecules is shown. As determined using the pBAR luciferase reporter gene assay, the tetravalent binding molecule is an agonist that strongly activates the Wnt-β catenin pathway in HEK293T cells. The 5019-Fc-2539 homologous diabody binds to multiple FZD receptors (5019: FZD1, 2, 4, 5, 7, 8) and to the Wnt3a site on LRP6 (2539) and activates the reporter gene to a level comparable to that of purified Wnt ligands. The 5019-K / H-2539:2542 heterologous diabody that binds to two Wnt binding sites on LRP6 is more effective.

[0045] Figure 3DDescribed are Wnt-β catenin pathway activation by multivalent binding molecules having Fc-binding affinity to monospecific LRP6 homodiabodies (5019-Fc-2539, 5019-Fc-2542) or bispecific LRP6 heterodiabodies (5019-K / H-2539-2542, also referred to as 5019Ag or Fc-binding affinity molecules) P+P -L6 1+3 ) either of which is connected to the FZD homologous diabody (5019).

[0046] Figure 3E Activation of Wnt-β catenin signaling by molecules containing monovalent binding domains of either FZD receptor or LRP6 co-receptor is described. 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 contains a monovalent binding domain for FZD and still activates the Wnt pathway, but its efficacy is reduced by 8 times relative to 5019Ag (which contains two FZD binding domains that bind to the same epitope). 5019-K / H-2539-MBP, which retains only one LRP6-W3 binding domain at the C-terminus, exhibits much lower efficacy. Importantly, minimal agonistic activity was detected in two single FZDs: single LRP6 diabodies 5019-MBP-K / H-2539-MBP and 5019-MBP-K / H-MBP-2542, and one LRP6-W1 site diabody 5019-K / H-MBP-2542.

[0047] Figure 3F Describes activation of the Wnt-β-catenin pathway by a tetravalent binding molecule in which the anti-LRP5 paratope targeting the WNT3A binding site is replaced by the anti-LRP6 paratope targeting the WNT1 binding site to generate a WNT3A-binding molecule that can recruit two coreceptors and observes activity comparable to that of F P+P -L6 1+3 Similar (EC 50 =4nM) molecules (F P+P -L5 / 6 3 ).

[0048] Figure 4ADescribed is activation of the Wnt-β catenin pathway in reporter cells that lack endogenous FZD4 receptors (-FZD4) or are modified to express FZD4 receptors (+FZD4) by multivalent binding molecules having a FZD binding domain specific for FZD4 on one side of the Fc domain (in this case a homologous diabody) and a co-receptor binding domain specific for LRP6 (2539 and 2542) on the other side of the Fc domain ( FZD4Ag: 5038Ag / 5038-K / H-2539-2542, 5044Ag / 5044-K / H-2539-2542, 5048Ag / 5048-K / H-2539-2542, 5063Ag / 5063-K / H-2539-2542, 5080Ag / 50180-K / H-2539-2542, 5081Ag / 5081-K / H-2539-2542). Controls were the multivalent binding molecule 5019Ag (5019-K / H-2539-2542) and Norrin, an endogenous agonist of FZD4. The results show that replacing the 5019FZD binding domain (recognizing FZD1, 2, 4, 5, 7, 8) in 5019Ag / 5019-K / H-2539:2542 (pan-FZD agonist) with the selective binding domain of FZD4 enables the development of selective FZD4 agonists. HEK293T cells were transfected with plasmids pBARL (Wnt-β catenin luciferase reporter gene) and Rluc (normalization control) encoding the listed FZD agonists and with or without FZD4 and LRP6 cDNA. Norrin was used as a positive control for the activation of FZD4. HEK293T cells express FZD4 at low to undetectable levels, so FZD4 agonists can only activate the reporter gene in the presence of transfected FZD4 cDNA. In contrast, pan-FZDag 5019-K / H-2539:2542 strongly activated Wnt-β-catenin signaling in the absence or presence of FZD4 through activation of other endogenously expressed Frizzled proteins in these cells.

[0049] Figure 4B Wnt-β catenin pathway activation is shown by multivalent binding molecules with binding domains specific for FZD2 (2876, 2890), FZD2 / 7 (2886) FZD6 (2747) or FZD9 / 10 (2969, 2974) on one side of the Fc (homodiabodies) and heterodiabodies with specificity for LRP6 formed by 2539 and 2542 antibody fragments on the other side of the Fc. Wnt-β catenin pathway activation was assessed using the pBARL assay in HEK293T cells.

[0050] Figure 4C Describes Wnt pathway activation by multivalent binding molecules that have FZD binding domains that are pan-specific for FZDs and are derived from IgG that block Wnt binding to FZDs and Wnt-β-catenin signaling. The LRP6 binding domain in these molecules is located at the c-terminus of the Fc and consists of a diabody formed by antibodies 2539 and 2542 that have paratopes that recognize the Wnt3 and Wnt1 binding sites on LRP6, respectively.

[0051] Figure 4D Wnt pathway activation by multivalent binding molecules having FZD binding domains that are pan-specific for FZDs and are derived from IgG that do not block Wnt binding to FZDs and do not antagonize Wnt3-induced pathway activation are described. The LRP6 binding domain in these molecules is located at the c-terminus of the Fc and consists of diabodies formed by antibodies 2539 and 2542, which have paratopes that recognize the Wnt3 and Wnt1 binding sites on LRP6, respectively.

[0052] Figure 5 Comparison of the FZD / LRP6 binding behavior of three tetravalent binding molecules of the invention is depicted. 5019-Fc-2539, 5019-Fc-2542, 5019-Fc-2539-2542 bind tightly to FZD, but show weaker LRP6 interactions (left) or FZD / LRP6 co-binding (middle). The FZD binding curve of 5019-K / H-2539-2542 (right) shows that it recognizes FZD4, FZD5, and FZD7.

[0053] Fig. 6A Schematic representation of the top two propellers of LRP5 / 6 (E1-E2) known to mediate binding to Wnt1 and the bottom two propellers of LRP5 / 6 (E3-E4) close to the plasma membrane and known to mediate interaction with Wnt3. Fig. 6A It has also been shown that Wnt1 interacts with LRP5 / 6 and FZD receptors and that Wnt3 interacts with LRP5 / 6 and FZD receptors.

[0054] Figure 6B Schematic diagram of the possible interaction of FZD receptor and LRP5 / 6 receptor through the multivalent binding molecules 5019-Fc-2539, 5019-Fc-2542 and 5019-K / H-2539-2542.

[0055] Figure 6CThe multivalent binding molecules were shown to be agonists that strongly activated the Wnt-β catenin pathway in HEK293T cells as determined using a pBAR luciferase reporter assay. The 5019-Fc-2539 homodiabody binds to multiple FZD receptors (5019 binds to FZD1, 2, 4, 5, 7, 8) and to the Wnt3a site on LRP6 (2539), and activated the reporter gene to levels comparable to purified Wnt ligands. The 5019-K / H-2539:2542 heterodiabody, which binds to both the Wnt3a and Wnt1 binding sites on LRP6, was more effective.

[0056] Fig.6D We show that 5019-K / H-2459:2460, a tetravalent binding molecule with an Fc domain in a knob-in-hole conformation and a FZD binding domain (homodiabody) with pan-FZD specificity (5019) and a co-receptor binding domain (heterodiabody) with bispecificity for two sites on LRP5 (2459 binds to the Wnt1 binding site and 2460 binds to the Wnt3 binding site), also activates the Wnt-β-catenin pathway in HEK293T cells.

[0057] Fig. 7A It was shown that replacing the FZD binding domain in 5019-K / H-2539:2542 (a pan-FZD agonist that recognizes FZD1, 2, 4, 5, 7, 8) with a FZD binding domain specific for FZD5 (#2928) produces a selective FZD5 agonist. HPAF-II cell proliferation has been shown to be dependent on FZD5 signaling. Blocking Wnt-FZD5 signaling using the Wnt secretion inhibitor LGK974 (targeting the acyltransferase Porcupine) leads to cell cycle arrest and proliferation inhibition. Proliferation can be rescued by adding exogenous Wnt3a conditioned medium or adding a FZD5 selective agonist (2928-K / H-2539:2542) or a pan-FZD agonist (5019-K / H-2539:2542) as described herein. The FZD4 selective agonist 5038-K / H-2539:2542 had only modest rescue ability.

[0058] Figure 7B It was demonstrated that stimulation of C3H10T1 / 2 cells with FZD2-specific FLag resulted in robust induction of the osteogenic marker alkaline phosphatase (ALPL) to levels similar to those achieved with pan-FZD FLAg, whereas FZD5-specific FLAg exhibited minimal activity.

[0059] Fig. 8A and 8BIt shows that when Wnt secretion is blocked by LGK974, a small molecule inhibitor of Porcupine (lower left figure), the pan-FZDag (F P+P -L6 1+3 ) completely replaced exogenous Wnt3A-conditioned medium to rescue the growth inhibition of intestinal organoids. Intestinal organoids isolated from mice grow in the presence of recombinant R-Spondin and require the presence of Wnt ligands secreted by paneth cells. Fig. 8A The results show that inhibition of Wnt production using LGK974 leads to organoid death (upper right). Exogenous application of Wnt3A conditioned medium (lower right) or FZDag (lower left) rescues organoid growth in the presence of LGK974. The upper left photo shows organoids treated with DMSO without LGK974 as a control. Figure 8B It is shown that using CellTiter Assay, Promega, by applying Wnt3A conditioned medium or FZDag (F P+P -L6 1 +3 ) can rescue organoid death caused by LGK974 inhibition of Wnt production.

[0060] Fig.9A and 9B Describes a gene encoding a protein that dimerizes in a knob-in-hole conformation to form pan-FZDag5019-KH-2539-2542 (F P+P -L6 1+3 ) are examples of plasmids containing peptides. Fig.9A Plasmids encoding peptides comprising an Fc region containing a "knob" mutation, the VH and VL of pan-FZD antibody #5019, and the VL of LRP antibody #2542 and the VH of LRP antibody #2539 are described. Fig. 9B Described are peptide encoding plasmids comprising nucleic acids encoding an Fc region containing a "hole" mutation, the VH and VL of a pan-FZD antibody #5019, and the VH of an LRP antibody #2542 and the VL of an LRP antibody #2539. The peptides encoded by these plasmids form heterodimers with tetravalent binding domains comprising a homodiabody produced by pairing the VH and VL of a pan-specific FZD antibody #5019 and a bispecific heterodiabody produced by pairing the VL of an LRP6 antibody #2539 and the VH of an LRP antibody #2542 from one peptide with the VH of an LRP antibody #2539 and the VL of an LRP antibody #2542 from another peptide.

[0061] Fig. 9C It is a heterodimer knob-hole conformation 5019-K / H-2539:2542(F P+P-L6 1+3 ). Using the knob-in-hole conformation within the Fc, the modularity of the molecule can be increased to 4 different binding sites. For this molecule (5019-K / H-2539:2542), a pan-FZD homodiabody was engineered on one side of the Fc domain and a heterodiabody containing Wnt3 (2539) and Wnt1 (2542) LRP6 binding sites was engineered on the other side of the Fc domain.

[0062] Fig. 10A and 10B It is an annotation of the domains of the nucleic acid sequence of the 5019-knob-2539:2542 multivalent binding molecule (SEQ ID NO:21 plus an additional 3' TGA and its complementary sequence).

[0063] Fig.11A -F describes the design and validation of a tetravalent binding molecule that binds FZD and LRP6 Wnt1 and Wnt3 binding sites (FLAg) as an activator of the Wnt-β-catenin pathway. Fig.11A The inhibitory effect (top) and specific activity (bottom) of anti-FZD Fab are depicted. Fig. 11B Inhibition of Wnt1 or Wnt3A signaling by LRP6Ab shown in diabody-Fc format is depicted. Fig. 11C Describe the molecular architecture of tetravalent FLAg. Fig.11D The pan-specific FLAg protein (F P +P -L6 1+1 ,F P+P -L6 3+3 and F P+P -L6 1+3 ) (x-axis) Dose-response curve for activation of the LEF / TCF reporter gene (y-axis) in HEK293T cells. Fig.11E Describe the use of specified concentrations of pan-FLAg (F P+P -L6 1+3 ) β-catenin levels in RKO cells after 30 min of treatment. Fig.11F Describe the use of 10 nM pan-FLAg (F P+P -L6 1+3 Time course of β-catenin and phosphorylated dishevelled protein-2 (p-Dvl2) protein levels in RKO cells treated with ).

[0064] Figures 12A-12D Description FLAG F P+P -L6 1+3 Characterization and decomposition of binding and activity. Fig. 12A and 12B Display FP+P -L6 1+3 Binding kinetics to 9 of the 10 human FZD CRDs and the human LRP6 ECD. Fig. 12C Indicates F P+P -L6 1+3 Behaves similarly to conventional IgG and interacts with FcRn in a dose- and pH-dependent manner. Fig.12D Indicates F P+P -L6 1+3 It also behaves similarly to IgG in its 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.

[0065] Fig.13A and 13B It is indicated that 30 nM F P+P -L6 1+3 Three days of treatment resulted in a strong induction of the mesoderm marker BRACHYURY, and expression of the pluripotency marker OCT4 was reduced to levels comparable to treatment with the GSK3 inhibitor CHIR99021 at 6 μM.

[0066] Fig.14 Display vehicle, C59 or pan-FLAg (F P+P -L6 1+3 ) Representative fluorescent images of small intestine sections from C59-treated LGR5-GFP mice. LGR5-GFP is expressed in stem cells at the bottom of the crypts. Nuclei were counterstained with DAPI. DETAILED DESCRIPTION

[0067] Described herein is a multivalent binding molecule comprising a binding domain of an Fc domain, a FZD binding domain and a Wnt co-receptor, wherein the binding domain is connected to the opposite ends of the Fc domain. The multivalent binding molecule of the present invention is an agonist of the Wnt signaling pathway, alternatively referred to herein as a FZD agonist or FZDag. The Wnt ligand works by promoting the aggregation of FZD receptors and co-receptors. Without wishing to be bound by theory, it is expected that the multispecific molecules as described herein simultaneously bind to FZD receptors and Wnt co-receptors, thereby activating the Wnt signaling pathway.

[0068] The modularity and effectiveness of the multivalent binding molecules for activating the Wnt signaling pathway as described herein are in contrast to the Wnt substitutes described in the prior art consisting of monovalent FZD and LRP5 / 6 binding ligands, in which the binding ligands are not connected to the opposite ends of the Fc domain. In one embodiment of the invention, the FZD binding domain comprises a binding portion derived from an antibody or polypeptide that specifically binds to more than one FZD receptor, and the co-receptor binding domain comprises a binding portion that binds to co-receptors such as LRP5 / 6, ROR1 / 2, RYK or PTK7. In one embodiment of the invention, an antibody or polypeptide that specifically binds to more than one FZD receptor binds to a cysteine-rich domain (CRD) of more than one FZD receptor.

[0069] Amino acid sequences of FZD receptors and nucleotide sequences encoding FZD receptors, as well as antibodies and antibody libraries that bind to FZD or Wnt co-receptors LRP5 / 6, ROR1 / 2, RYK or PTK7 are readily available or can be generated using methods well known in the art (see, for example, U.S. Publication No. 2015 / 0232554, inventors Gurney et al. and U.S. Publication No. 2016 / 0194394, inventors Sidhu et al. and U.S. Publication No. 20190040144, inventors Pan et al.; U.S. Publication No.: 2017 / 0166636, inventors Wu et al.; U.S. Publication No. 2016 / 0208018, inventors Chen et al.; U.S. Publication No. 2016 / 0053022, inventors Macheda et al.; U.S. Publication No. 2015 / 031293, inventors Damelin et al.).

[0070] Methods for producing peptides or polypeptides that bind to a selected target are well known in the art, see, for example, Sidhu et al. Methods in Enzymology (2000) 328: 333-336. For example, an affinity library that binds to a FZD or Wnt co-receptor can be obtained according to protocols known in the art (see, for example, U.S. Pat. No. 5,831,012 and Lofblom et al., FEBS Letters 584 (2010) 2670-2680); an ankyrin repeat protein library for selecting peptides that bind to a FZD or Wnt co-receptor can be obtained according to protocols known in the art (see, for example, WO 02 / 020565, inventor Stumpp et al.); a fibronectin repeat protein library for selecting peptides that bind to a FZD or Wnt co-receptor can also be obtained according to protocols known in the art (see, for example, U.S. Pat. No. 9,200,273, inventor Diem and Jacobs. Peptides that bind to a FZD or Wnt co-receptor can also be fynomers, peptides derived from human Fyn Small binding proteins of SH3 domains or artificial receptor proteins "anticalins" based on human apolipoprotein D and can 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).

[0071] Antibodies suitable as a source of antigen-binding peptides as described herein can be isolated by screening a combinatorial library for polypeptides having a desired activity or multiple activities. For example, a variety of methods for generating phage display libraries and screening such libraries for antibodies having desired binding properties are known in the art. Such methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and are further described, for example, in 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, in 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. 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, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be screened for antigen binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phages typically display antibody fragments, either single-chain Fv (scFv) fragments or Fab fragments. Libraries from immune sources can provide high-affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, as described in Griffiths et al., EMBO J, 12: 725-734 (1993), natural repertoires (e.g., from humans) are cloned without any immunization to provide a single source of antibodies to a wide range of non-self and self antigens.Finally, as Hoogenboom and Winter, J.Mol.Biol., 227:381-388 (1992) described, can also be by from stem cell clone the V gene fragment of unrearranged, and use the PCR primer that comprises random sequence to encode the CDR3 district of high variable and complete rearrangement in vitro to synthesize natural library.Description people's antibody phage library patent publication comprises, for example: U.S. Patent number 5,750,373, and U.S. Patent Publication No. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936 and 2009 / 0002360.Antibody or antibody fragment separated from people's antibody library are considered as people's antibody or people's antibody fragment in this article.

[0072] Therefore, those skilled in the art will easily prepare Fc domains and mix and match the multivalent FZD binding domains and Wnt co-receptor binding domains with desired specificity at the N and C-terminal ends of the Fc domains to prepare multivalent binding molecules to bind to the required FZD receptors and co-receptors, thereby activating specific Wnt pathways. These specific agonists will be used as a powerful tool for enhancing cell proliferation, differentiation, organoid survival and maintenance, and tissue regeneration in vivo. These specific agonists are also used as a powerful tool for analyzing the FZD specificity involved in these processes. For example, as shown herein, FZD5Ag, rather than FZD4Ag, rescues the growth defects of the RNF43 mutant PDAC cell line treated with LGK974, highlighting the importance of FZD5 relative to FZD4 receptors in this process.

[0073] One embodiment of the present invention is a method for affecting the binding of a peptide to a FZD receptor and a Wnt co-receptor on a cell, wherein the binding of the peptide to the FZD receptor and the co-receptor activates the Wnt signaling pathway in the cell. The method comprises selecting an Fc domain having a C-terminus and an N-terminus or a fragment thereof comprising a CH3 domain, connecting a first multivalent binding domain that binds to the FZD receptor to one end of the Fc domain, and connecting a second multivalent binding domain that binds to the Wnt co-receptor to the other end of the Fc domain, thereby forming a multivalent binding molecule, and then contacting the multivalent binding molecule with a cell expressing the FZD receptor and the co-receptor under conditions that activate the Wnt signaling pathway.

[0074] In one embodiment of the invention, the multivalent binding domain may comprise a single chain variable fragment (ScFv) that binds to more than one FZD receptor, a ligand of a FZD receptor or a co-receptor, or a fragment thereof that binds to a FZD receptor or a co-receptor. In another embodiment, the binding domain does not comprise a single chain variable fragment (ScFv) that binds to more than one FZD receptor, a ligand of a FZD receptor or a co-receptor, or a fragment thereof that binds to a FZD receptor or a co-receptor.

[0075] In one embodiment of the invention, at least one of the FZD or co-receptor multivalent binding domains comprises a diabody having two peptides, each peptide having a heavy chain variable domain (VH) connected to a light chain variable domain (VL), wherein the VH and VL from one peptide are paired with the VL and VH of another peptide, thereby providing the binding domain with two epitope binding sites. The VH and VL domains can be the VH and VL of an antibody that binds to a Wnt binding site on a FZD receptor or co-receptor. The VH or VL derived from an antibody, i.e., a source antibody, can have 50%, 55%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the VH and VL of the source antibody and still retain binding to the FZD receptor or co-receptor site bound by the antibody.

[0076] In one embodiment of the invention, the multivalent binding molecules of the invention comprise the multivalent binding molecules of Table 1 (Table 1 includes Tables 1A and 1B: Table 1A shows the nucleotide sequences and amino acid sequences of exemplary multivalent binding molecules of the invention; Table 1B shows the nucleotide sequences encoding the various domains of exemplary multivalent binding molecules). In one embodiment of the invention, the multivalent binding molecules of the invention consist essentially of the multivalent binding molecules of Table 1. In one embodiment of the invention, the multivalent binding molecules of the invention consist of the multivalent binding molecules of Table 1. In one embodiment of the invention, the multivalent binding molecules comprise a first polypeptide comprising SEQ ID NO: 77 and a second peptide comprising SEQ ID NO: 79. In one embodiment of the invention, the multivalent binding molecules comprise a first polypeptide comprising SEQ ID NO: 81, or a second peptide comprising 83. In one embodiment of the invention, the multivalent binding molecules consist essentially of a first peptide comprising SEQ ID NO: 77 and a second peptide comprising SEQ ID NO: 79 and bind to FZD2 and LRP 5 / 6. In one embodiment of the invention, the multivalent binding molecule consists essentially of a first peptide comprising SEQ ID NO: 81 and a second peptide comprising SEQ ID NO: 83 and binds to FZD7 and LRP 5 / 6. In one embodiment of the invention, the multivalent binding molecule consists of a first polypeptide consisting of SEQ ID NO: 77 and a second polypeptide consisting of SEQ ID NO: 79. In one embodiment of the invention, the multivalent binding molecule consists of a first polypeptide consisting of SEQ ID NO: 81 and a second polypeptide consisting of SEQ ID NO: 83.

[0077] In one embodiment of the invention, the multivalent binding domain comprises one or more VL and VH domains of a molecule of Table 1. In one embodiment of the invention, the multivalent binding domain of a multivalent molecule consists essentially of one or more VL and VH domains of a molecule of Table 1. In one embodiment of the invention, the multivalent binding domain of a multivalent molecule consists of one or more VL and VH domains of a molecule of Table 1. In one embodiment of the invention, the binding domain of a multivalent molecule as described herein comprises VH and VL domains that are at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the VH and VL of a molecule listed in Table 1, and retains binding to an antigen bound by a molecule listed in Table 1. In one embodiment of the invention, the multivalent binding domain comprises one or more VL and VH domains of SEQ ID NOS: 77 and 79 that bind to FZD2. In one embodiment of the invention, the multivalent binding domain comprises one or more VL and VH domains of SEQ ID NOS: 81 and 83 that bind to FZD7.

[0078] In one embodiment of the invention, the multivalent binding domain of the multivalent molecule consists essentially of the VL and VH domains of one or more SEQ ID NOS: 77 and 79 that bind to FZD2. In one embodiment of the invention, the multivalent binding domain of the multivalent molecule consists essentially of the VL and VH domains of one or more SEQ ID NOS: 81 and 83 that bind to FZD7.

[0079] In one embodiment of the invention, the multivalent binding domain of the multivalent molecule consists of one or more VL and VH domains of SEQ ID NOs: 77 and 79 that bind to FZD2. In one embodiment of the invention, the multivalent binding domain of the multivalent molecule consists of one or more VL and VH domains of SEQ ID NOs: 81 and 83 that bind to FZD7.

[0080] In one embodiment of the invention, the binding domain of the multivalent molecule as described herein comprises VH and VL domains that are at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the VH and VL domains of SEQ ID NOS: 77 and 79, and retains binding to FZD2. In one embodiment of the invention, the binding domain of the multivalent molecule as described herein comprises VH and VL domains that are at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the VH and VL domains of SEQ ID NOS: 81 and 83, and retains binding to FZD7.

[0081] In one embodiment of the invention, the binding domain of a multivalent molecule as described herein comprises one or more complementarity determining regions (CDRs) of a molecule listed in Table 1. In one embodiment of the invention, the binding domain of a multivalent molecule as described herein comprises CDRs that are at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the CDRs of a molecule listed in Table 1, and retains binding to an antigen bound by a molecule as shown in Table 1. In one embodiment of the invention, the binding domain of a multivalent molecule as described herein comprises one or more complementarity determining regions (CDRs) of SEQ ID NO: 77, 79, 81 or 83. In one embodiment of the invention, the binding domain of the multivalent molecule as described herein comprises CDRs that are at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the CDRs of SEQ ID NO:77 or 79, and retains binding to FZD2, or comprises CDRs that are at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the CDRs of SEQ ID NO:81 or 83, and retains binding to FZD7.

[0082] The FZD receptor bound by the multivalent binding molecule of the present invention can be FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, or FZD10. The FZD receptor can be FZD1, FZD2, FZD4, FZD5, FZD7, or FZD8. The multivalent binding molecule can bind only one FZD receptor, or can pan-specifically bind to more than one FZD receptor. The FZD multivalent binding domain can bind, for example, FZD1, FZD2, FZD4, FZD5, FZD7, and FZD8. The FZD multivalent binding domain can specifically bind to one FZD receptor, for example, FZD2, FZD4, FZD5, or FZD6.

[0083] In one embodiment of the invention, the FZD binding domain is monospecific and binds a single epitope on the FZD receptor.In one embodiment of the invention, the FZD binding domain is bispecific and binds two epitopes on the FZD receptor.

[0084] The co-receptor binding domain can bind to any Wnt co-receptor, such as LRP5 / 6, or ROR1 / 2. The multivalent co-receptor binding domain can bind, for example, LRP5 / 6, PTK7, ROR1 / 2, RYK, GPR12, TSPAN12, or CD133. In one embodiment of the invention, the co-receptor multivalent binding domain binds to LRP5 or LRP6.

[0085] In one embodiment of the invention, the co-receptor multivalent binding domain binds to a single epitope on the co-receptor, such as an epitope of LRP5 / 6 that binds to Wnt1 or Wnt3. In one embodiment of the invention, the co-receptor multivalent binding domain binds to two epitopes within the co-receptor, such as an epitope on LRP5 / 6 that binds to Wnt1 and an epitope that binds to Wnt3. The Wnt co-receptor bound by the multivalent binding molecule of the invention can be LRP5 or LRP6, PTK7, ROR1, ROR2, RYK, GPR124, TSPAN12 or CD133.

[0086] In one embodiment of the invention, the multivalent binding molecule comprises an Fc domain, wherein the Fc domain is an Fc domain of an immunoglobulin or a fragment thereof comprising a CH3 domain. In one embodiment of the invention, the immunoglobulin is IgG. In one embodiment of the invention, the IgG is IgG 1 .

[0087] One embodiment of the invention is a method for activating a Wnt signaling pathway in a cell, the method comprising contacting a cell having a FZD receptor and a Wnt co-receptor with a multivalent binding molecule of the invention in an amount effective to activate Wnt signaling.

[0088] In one embodiment of the invention, at least one multivalent binding domain comprises a scFv that binds to a FZD receptor or a co-receptor, or comprises a ligand of a FZD receptor or a co-receptor, or a fragment of said ligand. In one embodiment of the invention, at least one multivalent binding domain does not comprise a scFv that binds to a FZD receptor or a co-receptor, and does not comprise a ligand of a FZD receptor or a co-receptor, or a fragment of said ligand.

[0089] In one embodiment of the invention, the FZD multivalent binding domain comprises a FZD diabody and the co-receptor multivalent binding domain comprises a co-receptor diabody, wherein the diabody comprises two polypeptides, each comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL), wherein the binding domain is formed by pairing the VH and VL from one peptide with the VL and VH of another peptide to form the binding domain.

[0090] The VH and VL of the FZD binding domain can be derived from antibodies that bind to FZD receptors and antagonize Wnt signaling or inhibit binding of Wnt ligands to FZD receptors. The VH and VL of the FZD binding domain can be derived from antibodies that bind to FZD receptors but do not antagonize or inhibit binding of Wnt ligands to FZD receptors.

[0091] The VH and VL of the co-receptor binding domain can be derived from an antibody that binds to a co-receptor and antagonizes Wnt signaling or inhibits binding of a Wnt ligand to a co-receptor. The VH and VL of the co-receptor binding domain can be derived from an antibody that binds to a co-receptor without antagonizing Wnt signaling or inhibiting binding of a Wnt ligand to a co-receptor.

[0092] In the multivalent binding molecules of the present invention, one or both of the binding domains can be bivalent, and one or both of the bivalent binding domains can be bispecific to FZD receptors or co-receptors. In one embodiment of the invention, both binding domains are bivalent and bispecific, and each binding domain binds to two different epitopes on their respective target FZD receptors or co-receptors. For example, a binding molecule can include a FZD binding domain that is bivalent and bispecific (binding to two different epitopes) for a FZD receptor, or a binding molecule can include a co-receptor binding domain that is bivalent and bispecific for a co-receptor.

[0093] In one embodiment of the 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. In one embodiment of the invention, the FZD binding domain is attached to the C-terminus of the Fc domain of the multivalent binding molecule, and the co-receptor binding domain is attached to the N-terminus of the Fc domain.

[0094] Another embodiment of the present invention is a nucleic acid molecule encoding a multivalent binding molecule as described herein, for example, a multivalent binding molecule of Table 1, such as SEQ ID NO: 76 and SEQ ID NO: 78, or SEQ ID NO: 80 and SEQ ID NO: 82, their VH and VL domains (e.g., SEQ ID NO: 84, 85, 86 and 87), and a diabody comprising a VL and VH domain, including an expression cassette and a vector comprising a nucleic acid molecule encoding a multivalent binding molecule, their VH, and Fc domains, and a diabody comprising such VL and VH. The nucleic acid molecule can be inserted into a vector and expressed in a suitable host cell, and then the multivalent binding molecule can be isolated from the 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, such as a nucleic acid sequence encoding a multivalent binding molecule as described herein. A vector that can express a protein when a polynucleotide is inserted is called an expression vector. The vector can be inserted into a host cell by transformation, transduction or transfection, so that the carried genetic material can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or artificial chromosomes (PAC) derived from P1; bacteriophages, such as lambda phage or M13 phage and animal viruses, etc. Animal viruses may include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), varicella virus, baculovirus, papillomavirus, and papovavirus (such as SV40). The vector may include a variety of components that control the expression of multivalent binding molecules as described herein, including but not limited to promoters such as viral or eukaryotic promoters such as CMV promoters, signal peptides such as TRYP2 signal peptides, transcription initiation factors, enhancers, selection elements, and reporter genes. In addition, the vector may also include a replication initiation site.

[0095] As used in the present invention, the term "host cell" refers to a cell into which a vector can be introduced, including but not limited to prokaryotic cells such as Escherichia coli and Bacillus subtilis, fungal cells such as yeast and Aspergillus, insect cells such as S2 Drosophila cells and Sf9, or animal cells, including human cells, such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0096] One embodiment of the present invention is a pharmaceutical composition comprising a FZD agonist as described herein and a pharmaceutically acceptable excipient. The pharmaceutical composition may further comprise an additional agent that activates the Wnt pathway, for example, Norrin or R-Spondin. The pharmaceutical composition may consist of, or consist essentially of, a multivalent binding molecule as described herein and a pharmaceutically acceptable carrier or excipient. Suitable carriers and their preparations are described in Remington: The Science and Practice of Pharmacy (19th Edition) ed. A. R. 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, saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Other carriers include sustained release formulations, such as a semipermeable matrix of a solid hydrophobic polymer containing an antibody, the matrix being in the form of a shaped article, such as a film, liposome, or microparticle. It will be apparent to those skilled in the art that certain carriers may be more preferable depending upon, for example, the route of administration and the concentration of FZD agonist being administered.

[0097] Wnt signaling is a ubiquitous pathway that regulates cell and tissue differentiation. For example, with respect to eye development, a specific Wnt pathway, the Norrin-FZD4 pathway, has been identified to play a role in retinal angiogenesis. Signaling through the Norrin-FZD4 pathway is essential for the development and maintenance of the retinal vasculature. Mutations affecting genes in this pathway can lead to several pediatric vitreoretinal diseases, such as Norrie disease, familial exudative vitreoretinopathy (FEVR), and pseudoglioma and osteoporosis syndrome. In addition, retinopathy of prematurity (ROP) is associated with mutations in this pathway, and Wnt pathway mutations have been reported in Coats disease and persistent fetal vasculature (PFV). The Norrin-FZD pathway is also associated with CNS vascular development. Genetic ablation of Norrin, FZD4, Lrp5 and co-receptor tetraspanin 12 (Tetraspanin-12, Tspan-12) leads to angiogenesis defects and barrier disruption of 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 agonists of the present invention are particularly contemplated herein, particularly FZD4 FLAgs comprising a FZD4 binding domain on one end of the Fc receptor and a binding domain of LRP5 and / or LRP6 on the other side of the Fc domain, which enhance barrier function and promote angiogenesis, for example, treatment with FZD4 FLAgs promotes the development and maintenance of the retinal vasculature and / or blood-retinal barrier (BRB) and blood-brain barrier (BBB). Thus, one aspect of the invention is a method of promoting and / or maintaining retinal vasculature by treating ocular tissue, such as retinal tissue, with an effective amount of FZD4 FLAg, either topically or systemically. Another aspect of the invention is a method of promoting and / or maintaining BBB vasculature by treating the BBB with an effective amount of FZD4 FLAg following systemic administration. Another aspect of the invention is a method of treating a subject having a condition characterized by decreased retinal or cerebral angiogenesis by administering to the subject an effective amount of FZD4 FLAg, wherein the effective amount is an amount sufficient to increase retinal or cerebral angiogenesis in the subject. The subject may be a fetus.

[0098] Pathologically low levels of Wnt signaling are associated with osteoporosis, polycystic kidney disease, and neurodegenerative diseases. Controlled activation of the Wnt pathway 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 (March 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 (January 2005); Clevers H, Cell 127 (3): 469-80 (November 3, 2006). Proof-of-concept experiments have been performed to show the role of Wnt signaling in osteoporosis or mucositis. In addition, it has been proposed that increasing Wnt signaling may be beneficial for the treatment of diabetes and other metabolic diseases. Reduced Wnt signaling is associated with metabolic diseases. Loss of function of LRP6 R611C Mutations lead to early coronary artery disease, metabolic syndrome, and osteoporosis in humans. Main A et al., Science 315: 1278 (2007). "LRP5 loss-of-function mutation is associated with osteoporosis, impaired glucose metabolism and hypercholesterolaemia in human." Saarinnen et al., Clin Endocrinol 72: 481 (2010). In mice lacking both LRP5 and apoE, there is severe hypercholesterolemia, impaired fat tolerance, and advanced atherosclerosis. 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 variant confers 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 is beneficial for treating metabolic diseases. Therefore, administering the multivalent binding molecules of the present invention to subjects with metabolic diseases can be used to treat metabolic diseases in subjects.

[0099] Inflammatory bowel disease (IBP) is a group of inflammatory conditions of the colon and small intestine. The main types of IBD are Crohn's disease and ulcerative colitis. RSPO1 protein has been shown to improve inflammatory bowel disease in animal models. Zhao J et al., Gastroenterology 132: 1331 (2007). Therefore, administering a multivalent binding molecule of the invention, such as a multivalent binding molecule that binds FZD7, such as 12735-K / H-2539-2542, to a subject with IBD is useful for treating IBD in the subject.

[0100] Therefore, one embodiment of the present invention is a method for treating a subject with a condition associated with reduced Wnt signaling, the method comprising administering an effective amount of a FZD agonist of the present invention to a subject in need thereof. The condition may be, for example, osteoporosis, polycystic kidney disease, neurodegenerative disease, mucositis, short bowel syndrome, bacterial translocation in the gastrointestinal mucosa, enterotoxin or enteropathic infectious diarrhea, celiac disease, non-tropical sprue, lactose intolerance, and other conditions in which dietary exposure causes mucosal villi passivation and malabsorption, atrophic gastritis and diabetes, fractures, tissue regeneration such as tissue repair and wound healing, and metabolic diseases such as diabetes, and melanoma, examples of damaged tissues that can be treated using the methods of the present invention include, but are not limited to, intestinal tissue, heart tissue, liver tissue, kidney tissue, skeletal muscle, brain tissue, bone tissue, connective tissue, and skin tissue. The multivalent binding molecules of the present invention may be applied to subjects with a disease or condition characterized by low Wnt signaling. The multivalent binding molecules of the present invention are applied to subjects in an amount that effectively increases Wnt signaling and improves the subject's disease or condition.

[0101] Mucositis is a clinical complication of cancer treatment. Mucositis is caused by the cytotoxic effects of radiation or chemotherapy on rapidly proliferating cells. Mucositis consists of epithelial damage that primarily affects the intestinal and oral mucosae. Clinical symptoms are severe pain in the mouth, nausea, diarrhea, malnutrition, and in severe cases, sepsis and death. These symptoms often lead to dose limitations in cancer treatment. There are currently no treatments for oral or gastrointestinal-mucositis associated with chemotherapy or radiation therapy for solid tumors.

[0102] Oral mucositis is a common and often debilitating complication of cancer treatment. 50% of patients receiving radiation therapy for head and neck cancer and 10-15% of patients receiving 5-FU therapy suffer from grade 3-4 oral mucositis. RSPO1 has been shown to improve oral mucositis in animal models. Zhao J et al., PNAS 106:2331 (2010).

[0103] Short bowel syndrome (SBS) is due to the functional or anatomical loss of a large segment of the small intestine, so digestion and absorption capacity are severely impaired. Every year, many people undergo long segment resection of the small intestine due to various conditions, including trauma, inflammatory bowel disease, malignant tumors, mesenteric ischemia, etc. Various non-surgical procedures such as radiation can cause functional short bowel syndrome. Currently, the treatment of short bowel syndrome includes dietary methods, total parenteral nutrition (TPN), intestinal transplantation, and non-transplant abdominal surgery. Although these treatments help improve the prognosis of SBS patients, they can only partially correct the potential problem of reduced intestinal function. There is currently no therapy that can accelerate the recovery of the remaining small intestine of SBS patients. See Seetharam and Rodrigues, The Saudi Journal of Gastroenterology 17, 229-235 (2011).

[0104] The intestine of adult mammals constitutes one of the most rapidly self-renewing tissues, in which the intestinal mucosa includes a continuous structure folded into hyperplastic crypts and differentiated villi. In response to mucosal damage, the host initiates a healing response, resulting in restoration of mucosal integrity and regeneration of mucosal structure. This process depends largely 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).

[0105] Therefore, factors that regulate the activity of intestinal stem cells play a major role in the host's ability to respond to damage in the intestine. Since Wnt proteins are the most important growth factors that support the proliferation of intestinal stem cells, enhancing Wnt signaling will increase the proliferation of intestinal epithelial cells. This will lead to an increase in the number of intestinal villi and an increase in the absorptive surface area of ​​the mucosa.

[0106] Thus, in one embodiment, the multivalent binding molecules of the invention are administered to a person suffering from short bowel syndrome. In one embodiment of the invention, the multivalent binding molecules of the invention bind to FZD7, e.g., 12735-K / H-2539-2542 as described herein. The multivalent binding molecules are administered in an amount sufficient to increase the absorptive surface area of ​​the gastrointestinal mucosa. The administration of the multivalent binding molecules of the invention results in a successful prognosis as a person suffering from sudden short bowel syndrome adapts to enteral feeding, or as a person suffering from prevalent SBS absorbs nutrients from enteral feeding, or as a person reduces the amount of total parenteral nutrition required daily to maintain their weight.

[0107] Prevention of bacterial translocation. In one embodiment, the antibodies of the present invention are administered to people at risk of septicemia caused by enteric bacteria. The multivalent binding molecules are administered in an amount sufficient to increase the integrity of the gastrointestinal mucosa, thereby preventing enteric bacteria from entering the blood of the person. Decreased integrity of the gastrointestinal mucosa (compared to the integrity of the gastrointestinal mucosa of the normal population) is the main cause of blood infections and sepsis in critically ill patients. When the cases of bacteremia and sepsis observed in intensive care unit (ICU) patients are less than those of patients not administered the multivalent binding molecules of the present invention, the administration of the multivalent binding molecules has a successful prognosis.

[0108] Accelerate recovery during or after enterotoxin or enteropathic infectious diarrhea. Infectious diarrhea is a major pediatric problem. In one embodiment, the multivalent binding molecules of the present invention are administered in an amount sufficient to shorten the time to the end of diarrhea or to normal bowel movements. In addition to the standard of care including oral or parenteral rehydration, sometimes including antibiotics, the multivalent binding molecules of the present invention can also be administered. When it is observed in pediatric patients that the number of hospitalizations is reduced, the length of hospital stay is shortened, or the incidence of dehydration complications and electrolyte abnormalities is reduced compared to pediatric patients who are not administered the multivalent binding molecules of the present invention, the administration of the multivalent binding molecules has a successful prognosis.

[0109] Celiac disease, non-tropical sprue, lactose intolerance, and other conditions in which dietary exposure causes mucosal villi to become blunted and malabsorption. In one embodiment, the multivalent binding molecules of the invention are administered in an amount sufficient to increase the absorptive surface area of ​​the mucosa. The multivalent binding molecules of the invention can be administered in addition to standard care, which is primarily avoidance of offensive foods and sometimes dietary supplements. When a person with celiac disease, non-tropical sprue, lactose intolerance, or other conditions adapts to enteral feeding, or a person with any condition absorbs nutrients from enteral feeding, or when a person reduces the amount of total parenteral nutrition required daily to maintain their weight, administration of the multivalent binding molecules of the invention will result in a successful prognosis.

[0110] Atrophic gastritis, particularly a form known as environmental metaplastic atrophic gastritis. Atrophic gastritis is a common condition in the elderly and is currently treated with vitamin B12 injections. Patients are at increased risk for carcinoid tumors and adenocarcinomas. Medical experts have observed that in the case of carcinoids, administration of multivalent binding molecules has a successful prognosis when the incidence of tumors is reduced by reducing gastrin production by metaplastic G cells. If the medical expert determines that the tumor is activated by an increase in the Wnt pathway, the multivalent binding molecule should not be administered to the subject.

[0111] The FZD agonists of the present invention can be administered, for example, by injection (e.g., subcutaneous, intravenous, intraperitoneal, etc.), topically, or orally. Depending on the route of administration, the active compound can be coated in a material to protect the compound from the effects of acid and other natural conditions that can inactivate the compound. The multivalent binding molecules as described herein can be dissolved or suspended in a pharmaceutically acceptable carrier, preferably an aqueous carrier. In addition, the composition can include excipients, such as buffers, adhesives, propellants (blasting agents), diluents, flavoring agents, lubricants, etc. A broad list of excipients that can be used in such compositions can be, for example, from A. Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). The multivalent binding molecules can also be administered together with immunostimulatory substances such as cytokines.

[0112] One embodiment of the present invention includes a method for producing induced pluripotent stem (iPS) cells, the method including culturing somatic cells under conditions suitable for reprogramming somatic cells, wherein the culture conditions also include a multivalent binding molecule as described herein. Methods for producing 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 celllines derived from human somatic cells. Science 318, 1917-1920; U.S. Patent No. 8,546,140, ​​and; U.S. Patent No. 8,268,620. In one embodiment of the invention, the multivalent binding molecules of the invention are included in the culture medium in an amount sufficient to accelerate the generation of iPS cells.

[0113] One embodiment of the present invention includes a method for directed differentiation of pluripotent or multipotent stem cells (PSC) or induced pluripotent stem cells (iPS), comprising culturing cells under conditions suitable for directed differentiation, wherein the culture conditions further comprise an effective amount of a multivalent binding molecule as described herein. Studies of mouse and human PSCs have identified specific methods for adding growth factors including Wnt that can induce PSCs to differentiate into different lineages. Methods for directed differentiation of PSCs including activation of Wnt signaling are known in the art, see, e.g., Lam et al. (2014) Semin Nephol 34 (4); 445-461; Yucer et al. (September 6, 2017) Scientific Reports 7, article number 10741. It is contemplated that multivalent binding molecules as described herein can be used to affect activation of the Wnt signaling pathway to direct differentiation of PSCs.

[0114] One embodiment of the invention is a method of enhancing tissue regeneration in a subject in need thereof by administering to the subject in need thereof an effective amount of a multivalent binding peptide as described herein to activate Wnt signaling in the subject.

[0115] One embodiment of the present invention includes a method for enhancing bone healing and / or regeneration of a subject in need, such as a subject suffering from osteoporosis or fracture, by administering an effective amount of a multivalent binding molecule as described herein. In a specific embodiment, the multivalent binding molecule of the present invention comprises a binding domain that binds to FZD2 and a binding domain that binds to LRP5 or / and LRP6. The binding domain can be monovalent or multivalent, such as bivalent, trivalent or tetravalent, and is monospecific or multispecific, such as bispecific. In one embodiment of the present invention, a multivalent binding molecule for enhancing bone healing and / or regeneration of a subject in need comprises, for example, 2890-knob-2539-2542 (SEQ ID NO: 77) and 2890-hole-2539-2542 (SEQ ID NO: 79) (together forming 2890-K / H-2539: 2542 or 2890Ag).

[0116] The subject can be any animal (eg, a mammal), including but not limited to humans, non-human primates, horses, cows, dogs, cats, rodents, etc. Typically, the subject is a human.

[0117] The effective dose and schedule for administering a multivalent binding molecule as described herein can be determined empirically, and it is within the skill of the art to make such determinations. It will be appreciated by those skilled in the art that the dose of such FZD agonist that must be administered will vary depending on, for example, the subject to whom the antibody will be administered, the route of administration, the specific type of FZD agonist used, and other drugs being administered. Guidance for selecting a suitable dose of FZD agonist can be found in the literature on the therapeutic use of antibodies, for example, 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 the composition is that which is large enough to produce the desired effect. The dose should not be so large as to cause adverse side effects, such as adverse cross-reactions, allergic reactions, etc. Typically, the dosage will vary with the patient's age, condition, sex, and degree of inflammation, and can be determined by those skilled in the art. In the case of any contraindications, the dosage can be adjusted by the individual physician. The dosage can vary, and can be administered in one or more doses per day for one or more days. Although individual needs vary, it is within the skill of the art to determine the optimal range for an effective amount of the carrier.

[0118] In recent years, methods have been developed for culturing miniature organs called "organoids" that recapitulate the gross anatomy and cell type composition of different tissues. Notably, complete organoids can be generated from a single tissue stem cell, as demonstrated by the first intestinal LGR5+ stem cells isolated from mice. It is known that components in the culture medium that activate the Wnt-β catenin pathway are required for organoid derivation, growth, survival, and maintenance. Therefore, R-spondin and Wnt ligands purified or provided as conditioned medium are generally required for the growth of organoids from different tissues. However, purified Wnt proteins generally have low specific activity and cannot maintain the growth of organoids. Therefore, those skilled in the art rely on the addition of Wnt3A conditioned medium, or on the addition of small molecules such as GSK3 inhibitors to generate organoids. However, the manufacture of Wnt3A conditioned medium is labor intensive, the properties of the conditioned medium are inconsistent, and small molecule GSK3 inhibitors can strongly activate the pathway to toxic levels. The multivalent binding molecules as described herein address these issues because they are easy to make and purify, have consistently reproducible properties, and specifically activate Wnt by selectively binding to the desired combination of FZD receptors and co-receptors.

[0119] One embodiment of the present invention includes a method for producing tissue organoids, the method comprising culturing tissue with an effective amount of a multivalent binding molecule as described herein. Organoids are 3D multicellular in vitro tissue constructs that mimic their corresponding in vivo organs and can therefore be used to study various aspects of the organ in a tissue culture dish. Methods for producing organoids are well known in the art, and, for example, epithelial organoids derived from adult stem cells in various organs of the gastrointestinal tract almost all require Wnt signaling agonists (among other signaling factors, including embedded matrix gel) that can both maintain cells and produce in vivo-like complements of cell types. Wnt signaling also enhances the development of inner ear organoids in 3D culture and has been used for the generation of kidney organoids, see, for example, Natalie de Souza (2018) Nature Methods 15 (1): 23; DeJonge et al. (2016) PLosOne 11 (9), e0162508; Akkerman and Defize, (2017) Bioessays 39, 4, 1600244. The multivalent binding molecules of the invention can be included in the culture medium of the organoid in an amount sufficient to enhance its growth, survival and maintenance in culture. Thus, embodiments of the invention include methods for enhancing the culture of tissue organoids comprising a culture medium containing an effective amount of a multivalent binding molecule as described herein.

[0120] Another aspect of the invention is a method for preparing a multivalent binding molecule as described herein. In one embodiment of the invention, the multivalent binding molecule is produced by:

[0121] a) Select an Fc domain with a C-terminus and an N-terminus

[0122] b) recognizing a peptide that binds to more than one FZD receptor, or recognizing an antibody that binds to more than one FZD receptor, and

[0123] c) recognizing a peptide that binds to more than one Wnt co-receptor or recognizing an antibody that binds to more than one Wnt co-receptor,

[0124] d) producing a nucleic acid molecule comprising the following: (i) a nucleotide sequence encoding the Fc domain of step a, (ii) a nucleotide sequence encoding the peptide of step b that binds to more than one FZD receptor, or a nucleotide sequence encoding the VL and / or VH of the antibody of step b that binds to more than one FZD receptor, or a nucleotide sequence encoding the VL and / or VH derived from the antibody of step b that binds to more than one FZD receptor, and (iii) a nucleotide sequence encoding the peptide of step c that binds to more than one Wnt co-receptor, or a nucleotide sequence encoding the VL and / or VH of the antibody of step c that binds to more than one Wnt co-receptor, or a nucleotide sequence encoding the VL and / or VH derived from the antibody of step c that binds to more than one Wnt co-receptor.

[0125] e) expressing the nucleic acid molecule of (d) to produce a polypeptide, wherein the polypeptide dimerizes to form a tetravalent binding molecule, the tetravalent binding molecule comprising (i) an Fc domain, (ii) a FZD binding domain, and (iii) a Wnt co-receptor binding domain, wherein the FZD binding domain comprises the peptide of step b, or the VL and / or VH of step b, and is linked to one end of the Fc domain, and the Wnt co-receptor binding domain comprises the peptide of step c, or the VL and / or VH of step c, and is linked to the other end of the Fc domain, thereby forming a multispecific binding molecule.

[0126] The peptides that bind to more than one FZD receptor can be synthetic polypeptides, such as synthetic peptides, affimers, ankyrin repeat proteins, fibronectin repeat proteins, fynomers, or anticalins or peptides of naturally occurring proteins that bind to FZD receptors. Naturally occurring proteins can be, for example, Wnt, such as 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, Wnt-16b. The peptides of step b can be multivalent, bind to more than one site on FZD, such as divalent, trivalent or tetravalent, and can be monospecific that binds to a single epitope on FZD or multispecific that binds to more than one epitope on FZD.

[0127] The peptide that binds to one or more Wnt co-receptors can be a synthetic peptide, such as an affibody, an ankyrin repeat protein, a fibronectin repeat protein, a fynomer, or an anticalin, or a peptide that binds a naturally occurring protein that binds a Wnt co-receptor. The naturally occurring protein can be, for example, a Wnt, such as 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.

[0128] The peptide of step c can be multivalent, binding to more than one epitope on the Wnt co-receptor, such as bivalent, trivalent or tetravalent, and can be monospecific, binding to a single epitope on the Wnt co-receptor, or multispecific, binding to more than one epitope on the Wnt co-receptor.

[0129] The naturally occurring protein that binds a FZD receptor and the naturally occurring protein that binds a Wnt co-receptor may be the same protein.

[0130] In one embodiment, the peptide or antibody of step b may bind to FZD2, and the peptide of step c may be a peptide of Wnt5a, and the antibody of step c may be an antibody that binds to a site on a co-receptor that binds to Wnt5a.

[0131] In one embodiment, the peptide or antibody of step b can bind to FZD4, and the peptide of step c can be one or more peptides of Norrin, Wnt1, Wnt8 or Wnt5a, and the antibody of step c can be an antibody that binds to a site on a co-receptor that binds to Norrin, Wnt1, Wnt8, or Wnt5a.

[0132] In one embodiment, the peptide or antibody of step b can bind to FZD5, and the peptide of step c can be one or more peptides of Wnt7a, Wnt5a, Wnt10b, or Wnt2, and the antibody of step c can be an antibody that binds to a site on a co-receptor that binds to one or more of Wnt7a, Wnt5a, Wnt10b, or Wnt2.

[0133] In one embodiment, the peptide or antibody of step c binds to LRP6 and / or LRP5, for example, the peptide can be a peptide of Norrin, Wnt1 and / or Wnt3a, and the antibody of step c can be an antibody that binds to a site on LRP6 / LRP5 that binds to Norrin, Wnt1 and / or Wnt3a.

[0134] In one embodiment, the peptide or antibody of step c can bind to LRP6, for example, the peptide can be a peptide of Wnt1 or Wnt3a or both, and the antibody can be an antibody that binds to a site on LRP6 that binds to Wnt1 or Wnt3a.

[0135] In one embodiment, the peptide or antibody of step c binds to ROR1 and / or ROR2.

[0136] In one embodiment, the peptide or antibody of step c can bind to RYK.

[0137] In one embodiment, the peptide or antibody of step c can bind to PTK7.

[0138] In one embodiment, the peptide or antibody in step (b) can be a peptide or antibody that is combined with more than one FZD receptor and antagonizes Wnt signal transduction or suppresses Wnt from binding to a receptor. In one embodiment, the peptide or antibody in step (b) can be a peptide or antibody that is combined with more than one FZD receptor and does not antagonize Wnt signal transduction or suppresses Wnt from binding to a receptor. In one embodiment, the peptide or antibody in step (c) can be a peptide or antibody that is combined with more than one Wnt co-receptor and antagonizes Wnt signal transduction or suppresses Wnt from binding to a co-receptor. In one embodiment, the peptide or antibody in step (c) can be a peptide or antibody that is combined with Wnt co-receptors without antagonizing Wnt signal transduction or suppressing Wnt from binding to a co-receptor. The binding domain can be connected to the Fc domain by a linker. The modular aspects of the invention allow for mixing and matching peptide or antibody VH and VL that bind to any given FZD receptor and Wnt co-receptor on opposite ends of the Fc domain to create multivalent binding molecules that can bind to multiple Frizzled receptor-co-receptor complexes, or selectively bind to a single Frizzled receptor-co-receptor complex to activate Wnt signaling.

[0139] One embodiment of the present invention is a method for preparing a multivalent binding molecule that activates the Wnt signaling pathway, comprising:

[0140] a) selecting an Fc domain having a C-terminus and an N-terminus, such as an Fc domain of an immunoglobulin, such as IgG, such as IgG1, comprising a CH3 domain,

[0141] b) identifying antibodies with binding specificity for more than one FZD receptor and

[0142] c) identifying antibodies with binding specificity for Wnt co-receptors;

[0143] d) producing a nucleic acid molecule comprising

[0144] (i) a nucleotide sequence encoding a selected Fc domain,

[0145] (ii) a nucleotide sequence encoding the VL and / or VH of the antibody derived from step b, and

[0146] (iii) a nucleotide sequence encoding the VL and / or VH of the antibody derived from step c,

[0147] d) expressing the nucleic acid molecule of (d) to produce a polypeptide that forms a multivalent binding molecule by dimerization of the Fc domain, the multivalent binding molecule comprising (i) an Fc domain, (ii) a FZD binding domain and (iii) a Wnt co-receptor binding domain, such that the FZD binding domain is connected to one end of the Fc domain, and the Wnt co-receptor binding domain is connected to the other end of the Fc domain, thereby forming a multivalent binding molecule. In a preferred embodiment, the multivalent binding molecule is a dimer of two polypeptides encoded by the nucleic acid molecule, wherein the Fc domain is in a knob-and-hole conformation. One or both of the binding domains can be a multivalent binding domain. The antibody of step b can be an antibody fragment that binds to a FZD receptor. The VH and / or VL in step d)(ii) can be the same as the VH and / or VL of the antibody of step b). The antibody of step c can be an antibody fragment that binds to a Wnt co-receptor. The VH and / or VL in step d)(iii) can be the same as the VH and / or VL of the antibody of step c).

[0148] The multivalent molecules of the invention can be generated by dimerizing two polypeptides in a "knob-in-hole" conformation. The knob-in-hole conformation increases the modularity of the invention by promoting the association of peptides comprising binding moieties that bind to different epitopes on FZD receptors or co-receptors or different members of the same FZD receptor or co-receptor family, see e.g. Figure 3A Methods for engineering Fc molecules by knob-and-hole design are well known in the art, see, for example, WO2018 / 026942, 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.

[0149] Another embodiment of the present invention is a method for promoting the interaction between a FZD receptor and a co-receptor on a cell to activate the Wnt signaling pathway in the cell, comprising: a) selecting an Fc domain having a C-terminus and an N-terminus or a fragment thereof comprising a CH3 domain, b) connecting a first multivalent binding domain that binds to a FZD receptor at one end of the Fc domain, and connecting a second binding domain that binds to a Wnt co-receptor at the other end of the Fc domain, thereby forming a binding molecule; c) contacting the multivalent binding molecule with a cell expressing the FZD receptor and the Wnt co-receptor under conditions where both the FZD receptor and the co-receptor bind to the multivalent binding molecule, thereby activating the Wnt signaling pathway. One or both of the binding domains may be monovalent or multivalent, such as bivalent, trivalent, or tetravalent. The FZD binding domain may comprise a peptide of a naturally occurring protein that binds to FZD, a synthetic peptide that binds to FZD, such as an affibody, an ankyrin repeat protein, a fibronectin repeat protein, a fynomer, or an anticalin, a VH and / or VL fragment that binds to FZD, a scFV that binds to FZD, or a diabody that binds to FZD. The Wnt co-receptor binding domain may comprise a peptide of a naturally occurring protein that binds to a Wnt co-receptor, a synthetic peptide that binds to a Wnt co-receptor, such as an affibody, an ankyrin repeat protein, a fibronectin repeat protein, a fynomer, or an anticalin, a VH and / or VL fragment that binds to a Wnt co-receptor, a scFV that binds to a Wnt co-receptor, or a diabody that binds to a Wnt co-receptor.

[0150] One embodiment of the present invention is a molecule comprising an Fc domain and two binding domains, the first domain binds to a FZD receptor, the second domain binds to a Wnt co-receptor, and the two parts are connected together by an Fc domain or a fragment thereof comprising a CH3 domain, wherein one domain is connected to the N-terminus of the Fc receptor, and the other domain is connected to the C-terminus of the Fc receptor. The binding domain can be connected to the Fc receptor directly or by a peptide linker or a non-peptide linker such as a polypeptide linker. Suitable linkers are well known in the art, such as XTEN linkers (see WO2013120683, inventor Schellenberger et al.).

[0151] One embodiment of the present invention is a method for activating the Wnt signaling pathway, the method comprising contacting a cell expressing a FZD receptor and its co-receptor with an effective amount of a multivalent molecule of the present invention. Without wishing to be bound by theory, it is expected that the multivalent molecules as described herein bind to both the FZD receptor and its co-receptor, thereby forming a complex that mimics the binding of the Wnt molecule to the FZD receptor and co-receptor, which in turn activates the Wnt signaling pathway.

[0152] The multivalent binding molecules of the present invention can be prepared recombinantly, 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 these molecules can be prepared synthetically, for example, using commercial synthesis equipment, such as automatic synthesizers from Applied Biosystems, Inc., Beckman, etc. By using a synthesizer, naturally occurring amino acids can be replaced by non-natural amino acids. The specific order and preparation method will be determined by convenience, economy, required purity, etc. If desired, various groups can be introduced into the peptide during synthesis or during expression to allow connection to other molecules or surfaces.

[0153] In some embodiments, the binding domain is linked to the Fc domain via a peptide linker, such as an XTEN linker. In some embodiments, the peptide linker comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 1, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or at least 100 amino acids. In some embodiments, the length of the peptide linker is between 5 to 75, 5 to 50, 5 to 25, 5 to 20, 5 to 15, or 5 to 10 amino acids. The length and flexibility of the Fc domain with or without a linker allows the multivalent binding molecule to bind to the FZD receptor and its co-receptor, thereby activating the Wnt signaling pathway. In one embodiment of the invention, the Fc domain or a fragment thereof comprising the CH3 domain, with or without a linker, 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.

[0154] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "a peptide" includes reference to more than one peptide and equivalents thereof, such as polypeptides known to those skilled in the art, etc.

[0155] "Affinity matured" antibodies or "antibody maturity" refer to antibodies with one or more alterations in one or more hypervariable regions (HVRs) which result in an improvement in the affinity of the antibody for the antigen or in other desired properties of the molecule, compared to a parent or source antibody which does not possess such alteration.

[0156] "Comprising" means that the elements listed are required in the composition / method / kit, but other elements may be included within the scope of the claim to form the composition / method / kit, etc. For example, as will be readily understood in the art, a composition comprising a multivalent binding molecule is a composition that may include other elements in addition to the multivalent binding molecule, in addition to the elements covered by any negative condition, such as a functional portion such as a polypeptide, small molecule, or nucleic acid that is bound, e.g., covalently bound, to the multivalent binding molecule; an agent that promotes the stability of the multivalent binding molecule composition, an agent that promotes the solubility of the multivalent binding molecule composition, an adjuvant, etc.

[0157] "Consisting essentially of" means limiting the scope of the described composition or method to specified materials or steps that do not materially affect the basic and novel feature(s) of the invention. For example, a multivalent binding molecule that "consists essentially of" a disclosed sequence has an amino acid sequence of the disclosed sequence plus or minus about 5 amino acid residues at the boundaries of the sequence based on the sequence from which the sequence is derived, such as about 5 residues, 4 residues, 3 residues, 2 residues, or about 1 residue less than the recited binding amino acid residues, or about 1 residue, 2 residues, 3 residues, 4 residues, or 5 residues more than the recited binding amino acid residues.

[0158] "Consisting of" means excluding any elements, steps or ingredients not specified in the claims from the composition, method or kit. For example, a multivalent binding molecule "consisting of" a disclosed sequence consists only of the disclosed amino acid sequence.

[0159] In the case of providing a range of values, it should be understood that, unless the context clearly indicates otherwise, each intermediate value between the upper and lower limits of the range is also specifically disclosed to one tenth of the lower limit unit. Each smaller range between any specified value or intermediate value in the specified range and any other specified value or intermediate value in the specified range is included in the present invention. The upper and lower limits of these smaller ranges can be independently included in the range or excluded in the range, and each range wherein includes any one of the upper and lower limits, does not include the upper and lower limits, or includes both of the upper and lower limits in a smaller range is also included in the present invention, subject to any specific exclusion in the specified range. In the case where the range includes one or two of the upper and lower limits, the scope excluding one or two of those included upper and lower limits is also included in the present invention.

[0160] It is known that the basic antibody structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region, which is primarily responsible for effector function. In general, antibody molecules obtained from humans are related to any of IgG, IgM, IgA, IgE, and IgD, which differ from each other in the nature of the heavy chain present in the molecule. Certain classes also have subclasses, such as IgG 1 IgG 2 Etc. Furthermore, in humans, the light chain can be a kappa chain or a lambda chain.

[0161] Three highly divergent segments within each of the heavy chain variable domain VH and the light chain variable domain VL, referred to as the complementary determining regions (CDRs), are inserted between more conservative flanking segments referred to as "framework regions" or "FRs". Therefore, the term "FR" refers to the amino acid sequence naturally present between and adjacent to the CDRs of immunoglobulins. The VH domain typically has four FRs, referred to herein 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, referred to herein 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, the three CDRs (CDR-L1, CDR-L2, and CDR-L3) of the VL domain and the three CDRs (CDR-H1, CDR-H2, and CDR-H3) of the VH domain are interlaced relative to each other in three-dimensional space, thereby forming an antigen binding site within the antibody variable region. The surface of the antigen binding site is complementary to the three-dimensional surface of the bound antigen. According to the Kabat numbering system (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition 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), the amino acid sequences of the VL and VH domains can be numbered and the CDRs and FRs therein can be identified / defined. According to commonly used numbering systems, such as the IMGT numbering system, the Kabat numbering system, and the like, one of ordinary skill in the art will have the knowledge to number the amino acid residues of the VL and VH domains and to identify the CDRs and FRs therein.

[0162] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment") refers to one or more fragments, parts or domains of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of a full-length antibody can perform the antigen-binding function of an antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) Fab fragments, which are monovalent fragments consisting of the VL, VH, CL1 and CH1 domains; (ii) F(ab') 2Fragment, is a bivalent fragment comprising two F(ab)' fragments connected by a disulfide bond at the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) Fv fragment consisting of VL and VH domains of a single arm of an antibody; (v) dAb fragment consisting of VH domain (Ward et al. (1989) Nature 241:544-546); and (vi) isolated complementarity determining region (CDR). In addition, although the two domains VL and VH of the Fv fragment are encoded by their own genes, they can be connected into a continuous chain by a synthetic linker using recombinant methods, wherein the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv); see, for example, 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 (see, e.g., Holliger et al. (1993) PNAS. USA 90: 6444-6448) are also encompassed.

[0163] "Affibodies" are small single-domain proteins that are engineered to mimic the binding of monoclonal antibodies to a large number of target proteins or peptides with high affinity. They consist of a three-helix bundle based on a scaffold of one of the IgG binding domains of Staphylococcal protein A. The scaffold domain consists of 58 amino acids, 13 of which are randomized to generate affibody libraries with a large number of ligand variants. See, e.g., U.S. Patent No. 5,831,012 and Lofblom et al. FEBS Letters 584 (2010) 2670-2680. Affibody molecules mimic the molecular weight of antibodies of about 6 kDa.

[0164] As used herein, "diabodies" are dimeric antibody fragments. In each polypeptide of a diabody, a heavy chain variable domain (VH) is linked to a light chain variable domain (VL), but unlike a single-chain Fv fragment, the linker between VL and VH is too short for intramolecular pairing, so each antigen binding site is formed by pairing the VH and VL of one polypeptide with the VH and VL of another polypeptide, see e.g. Figure 3ADiabodies thus have two antigen-binding sites and can be monospecific or bispecific. (See, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123; Kontermann and Dubeleds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).

[0165] As used herein, an "effective amount" of an agent, such as a multivalent binding molecule or a pharmaceutical composition comprising the molecule, refers to an amount effective to achieve the desired result at the desired dosage and time period. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of one or more symptoms of a disease, disorder, and / or condition, stabilizes one or more characteristics of one or more symptoms of a disease, disorder, and / or condition, and / or delays the onset of one or more symptoms of a disease, disorder, and / or condition.

[0166] As used herein, the term "epitope" includes 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 are usually composed of chemically active surface groups of molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics. When the dissociation constant is ≤10 μM, for example, ≤100 nM, preferably ≤10 nM, and more preferably ≤1 nM, it is considered that the antibody specifically binds to the antigen.

[0167] The constant region of an immunoglobulin molecule is also referred to as a fragment crystallizable region, "Fc region" or "Fc domain". The Fc domain consists of two identical protein fragments, the second and third constant domains from the two heavy chains of an antibody, respectively, and the Fc domain of IgG has a highly conserved N-glycosylation site. Glycosylation of the Fc fragment is essential for Fc receptor-mediated activity. In one embodiment of the present invention, the Fc domain of a multivalent molecule is engineered so that it does not target cells that bind to the multivalent molecule for ADCC or CDC-dependent death. In one embodiment of the present invention, the Fc domain of a multivalent binding molecule is a peptide dimer in a knob-and-hole conformation. The peptide dimer can be a heterodimer.

[0168] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, for whom diagnosis, treatment, or therapy is desired.

[0169] As used herein, "LRP," "LRP protein," and "LRP receptor" 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 a receptor-mediated endocytosis. The LRP proteins LRP5 (GenBank Accession No. NM 002335.2) and LRP6 (GenBank Accession No. NM 002336.2) are included in the Wnt receptor complex required for activation of the Wnt-β-catenin signaling pathway.

[0170] As used herein, the term "polypeptide fragment" refers to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion, but wherein the remaining amino acid sequence is identical to the corresponding positions in the naturally occurring sequence deduced, for example, from the full-length cDNA sequence.

[0171] As used herein, the term "paratope" includes the antigen binding site in the variable region of an antibody that binds to an epitope.

[0172] The term "treatment" (treatment and treating, etc.) as used herein generally refers to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of the disease and / or side effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its development; or (c) alleviating the disease, i.e., causing the disease to regress. The therapeutic agent may be administered before, during, or after the onset of the disease or injury. Of particular interest is the treatment of an ongoing disease, where the treatment stabilizes or alleviates the patient's adverse clinical symptoms. It is desirable to perform such treatment before complete loss of function in the affected tissue. The treatment may be administered during the symptomatic stage of the disease and in some cases after the symptomatic stage of the disease.

[0173] The ability of the multivalent binding molecules of the present invention to activate Wnt signaling can be confirmed by a variety of assays. The multivalent binding molecules of the present invention generally induce a reaction or activity similar or identical to that induced by the natural ligands of the FZD receptor. The multivalent binding molecules of the present invention activate Wnt signaling pathways, such as the typical Wnt-β catenin signaling pathway. As used herein, the term "activation" refers to a measurable increase in the intracellular level of a Wnt signaling pathway, such as the Wnt-β catenin signaling pathway, compared to the level in the absence of the FZD agonist of the present invention.

[0174] Various methods for measuring Wnt-β catenin activation levels are known in the art. These include, but are not limited to, assays for: Wnt-β catenin target gene expression; LEF / TCF reporter gene expression (e.g., TopFLASH, superTopFLASH, pBAR); β catenin stabilization; LRP5 / 6 phosphorylation; Axin translocation from the cytoplasm to the cell membrane and binding to LRP5 / 6. The typical Wnt-β catenin signaling pathway ultimately leads to changes in gene expression through transcription factors TCF1, TCF7L1, TCF7L2, and LEF. The transcriptional response to Wnt activation has been characterized in many cells and tissues. In this way, the global transcription profile performed by methods well known in the art can be used to assess the activation of Wnt-β catenin signaling.

[0175] Changes in Wnt response gene expression are usually mediated by TCF and LEF transcription factors. The TCF reporter gene assay evaluates transcriptional changes of TCF / LEF controlled genes to determine the level of Wnt-β-catenin signaling. The TCF reporter gene assay was first described by Korinek, V. et al., 1997. The method, also known as TOP / FOP, involves using three copies of the optimal TCF motif CCTTTGATC or three copies of the mutant motif CCTTTGGCC, and a minimal c-Fos promoter located upstream to drive luciferase expression (pTOPFLASH and pFOPFLASH, respectively) to determine the transactivation activity of endogenous β-catenin / TCF. The higher the ratio of these two reporter gene activities (TOP / FOP), the higher the activity of β-catenin / TCF. A newer and more sensitive version of this reporter gene is called pBAR and contains 12 repeats of the TCF motif (Biechele and Moon, Methods Mol Biol. 2008; 468:99-110, PMID: 19099249).

[0176] General methods in molecular and cellular biochemistry can be found in standard textbooks such as Molecular Cloning: A Laboratory Manual, 3rd Edition. (Sambrook et al., CSH Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Edition. (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 Celland Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998).

[0177] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in one polypeptide chain. Typically, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form a desired antigen binding structure. For a review of scFv and other antibody fragments, see James D. Marks, Antibody Engineering, Chapter 2, Oxford University Press (1995) (Carl K. Borrebaeck, Ed.).

[0178] Unless otherwise defined, the scientific and technical terms used in conjunction with the present invention should have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms should include plural, and plural terms should include singular. Generally, the terms used in conjunction with cell and tissue culture, molecular biology, protein and oligonucleotide or polynucleotide chemistry and hybridization as described herein and the technology of the above content 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 instructions or as usually completed in the art or as described herein. The aforementioned techniques and procedures are usually carried out according to conventional methods well-known in the art, and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and pharmacy and medicinal chemistry as described herein and the laboratory procedures and techniques of the foregoing are well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, delivery, and treatment of patients.

[0179] Embodiment 1

[0180] 1. Development of multivalent FZD agonists

[0181] In order to prepare a multivalent binding molecule having a first binding domain comprising a FZD diabody and a second binding domain comprising a co-receptor diabody, we identified FZD-specific antibodies from a synthetic Fab phage library (library F; see U.S. Publication No. 2016 / 0194394, inventor Sidhu et al.) by selecting those that bind to the cysteine-rich domain (CRD) of the FZD receptor using conventional phage display technology. Affinity or specificity maturation is performed as required. For example, using FZD4 CRD as an antigen, a pan-FZD binding antibody #5019 (recognizing FZD1, 2, 4, 5, 7, and 8) was matured from an antibody derived from FZD7. Our previous work also identified several antibodies that were completely specific for FZD4 (5038, 5044, 5048, 5062, 5063, 5080, 5081) or for FZD5 (2928) (see, e.g., US20160194394, inventors Sidhu et al. and WO2017127933A1, inventors Pan et al.).

[0182] These FZD antibodies are used to prepare FZD-specific diabodies. Diabodies are antibody formats similar to single-chain variable fragments (scFv), but they are dimers of two peptides, each encoding a VL and a VH, but, unlike scFv, the linker between the VH and VL in the polypeptide is too short to allow intramolecular complementation between the VH and VL domains. Therefore, the VH-VL fragment of one polypeptide dimerizes with the VH-VL fragment of another polypeptide, thereby functionally reconstructing two antigen-binding paratopes. Diabodies with identical or different paratopes are produced by forming dimers of polypeptides with identical VL and VH to form homologous diabodies, or by forming dimers of two polypeptides with different VL and VH to form heterologous diabodies.

[0183] LRP6 antibodies were also selected from the synthetic antibody library by selecting those that bound to the recombinant extracellular domain (ECD) of human LRP6. Five Fabs with unique CDR regions were identified. After conversion to IgG format, they all showed human LRP6 binding as well as mouse LRP6 binding. No LRP5 binding was detected by ELISA, indicating that these antibodies are LRP6 specific ( Figure 1A ). The LRP6 ECD contains four β-propeller motifs that alternate with four epidermal growth factor (EGF)-like repeats. The first two β-propeller motifs are thought to be involved in Wnt1 binding, while the second two β-propeller motifs are thought to be involved in Wnt3 binding, resulting in two potential antibody binding epitopes. See Fig. 6A The epitope binding results showed that the five antibodies bind to two independent sites on LRP6 and can be divided into two groups, where antibodies 2538, 2542, and 2543 bind to the Wnt1 binding site on LRP6, and 2539 and 2540 bind to the Wnt3 binding site on LRP6. In general, antibodies that bind to the LRP6-Wnt1 site are expected to prevent Wnt1-induced Wnt pathway activation.

[0184] To prepare the Fc N-terminal binding domain containing homologous diabodies specific for FZD, the VH and VL fragments of the selected FZD antibodies, VH-1, VH-2, VL-1 and VL-2, were amplified and isolated from the corresponding phagemid templates by PCR. The isolated fragments (VH-1 and VL-2) were then introduced into an EcoRI / XhoI pre-cut vector containing the Fc knob region (pSCST backbone) using Gibson assembly (see Gibson et al. (2009). Nature Methods. 6 (5): 343-345 and Gibson DG. (2011) Methods in Enzymology. 498: 349-361). Gibson assembly was also used to introduce the fragments (VH-2 and VL-1) into an EcoRI / XhoI pre-cut vector containing the Fc groin region. DNA sequencing was used to verify the correct assembly. Two plasmids (a pair, Fc knob and Fc hole) were then used to introduce the second binding domain at the C-terminus of the Fc domain.

[0185] The Fc knob and Fc hole conformation is required to generate multivalent binding domains, wherein one of the binding domains is a heterodiabody. However, the Fc knob and Fc hole conformation is not required to prepare binding molecules comprising homodiabodies at both the N- and C-termini of the Fc domain, and thus for such binding molecules, the VH and VL are linked to a wild-type Fc region, and only one plasmid is used to generate a VH-VL containing polypeptide 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.

[0186] To generate the C-terminal binding domain, the LRP5 / 6 antibody was recognized and the LRP5 / 6 diabody was generated according to the same protocol as described above for generating the FZD diabody. The C-terminal binding domain was generated by amplifying the VH-3, VH-4, VL-3 and VL-4 fragments by PCR from the corresponding LRP antibody phagemid template and then isolating the amplified fragments. As described above, the VH-3 and VL-4 fragments were then introduced into the PpuMI / BamHI site of the above-mentioned knob-in-hole plasmid using Gibson assembly. The other VH-4 and VL-3 fragments were inserted into the PpuMI / BamHI cut site of the knob-in-hole plasmid using Gibson assembly.

[0187] Two plasmids (a pair, Fc knob and Fc hole) with different VL and VH sequences are used to generate bispecific, i.e., FZD or co-receptor binding domains capable of binding to two different sites. Because the generation of the knob-hole conformation is not required to generate dimers with monospecific binding domains, if each binding domain is monospecific, only a single plasmid containing the wild-type Fc sequence is used.

[0188] Fig.9ADescribed are plasmids encoding peptides comprising an Fc region with a "knob" mutation, the VH and VL of pan-FZD antibody #5019, and the VL of LRP antibody #2542 and the VH of LRP antibody #2539. Fig. 9B Described are plasmids encoding peptides comprising nucleic acids encoding an Fc region having a "hole" mutation, VH and VL of a pan-FZD antibody #5019, and VH of an LRP antibody #2542 and VL of an LRP antibody #2539. The peptides encoded by these plasmids form heterodimers having a multivalent binding site comprising a homodiabody derived from a pan-specific FZD antibody #5019, and a bispecific heterodiabody produced by pairing the VL of an LRP antibody #2539 and the VH of an LRP antibody #2542 from one peptide with the VH of an LRP antibody #2539 and the VL of an LRP antibody #2542 from another peptide.

[0189] The resulting plasmids were then sequenced and sequence-verified plasmids were prepared using the PureLink HiPure PlasmidFilter Maxiprep Kit (Invitrogen) according to the manufacturer's instructions. The plasmids were then transfected into Expi293F cells (Thermo Fisher Scientific) and FectoPRO reagent (Polyplus) was used for antibody expression according to the manufacturer's instructions. Typically, small batch antibody production was performed using a 200 ml cell scale.

[0190] Typically, 80 h after transfection, the culture medium of Expi 293F cells was collected by centrifugation to precipitate cells and cell debris. The supernatant was transferred to a clean bottle and buffered with 10x PBS buffer. After incubation with an appropriate amount of protein A magnetic beads (GE Healthcare) for 1 h, the magnetic beads were washed and the bound molecules were eluted according to the manufacturer's instructions. Finally, the buffer was replaced with PBS.

[0191] 2. Heterodimer multivalent binding molecules

[0192] Using the above method, we also generated tetravalent heterodimeric molecules comprising a complete bispecific diabody fused to each of the N-terminus and C-terminus of the Fc domain (knob / hole), Figure 2A and 3AIn particular, we generated tetravalent binding molecules having a homodiabody that binds FZD derived from antibody 5019 on the N-terminus of the Fc domain and a homodiabody derived from either LRP6-W1 antibody 2542 (5019-Fc-2542) or LRP6-W3 antibody 2539 (5019-Fc-2539) on the C-terminus of the Fc domain. Surprisingly, both tetravalent molecules activated the Wnt pathway, but 5019-Fc-2542 was much less efficient ( Figure 3C Without wishing to be bound by theory, this difference may reflect a difference in the ability of LRP6-W1 and LRP6-W3 to bind to activate Wnt signaling. It has been observed that the LRP6-W3 site is more effective in activating Wnt signaling than Wnt binding to the LRP6-W1 site.

[0193] We also generated a tetravalent trispecific binding molecule having a FZD-binding homodiabody derived from antibody 5019 on the N-terminus of the Fc domain and an LRP heterodiabody derived from LRP6-W1 antibody 2542 and LRP6-W3 antibody 2539 on the C-terminus of the Fc domain (5019-K / H-2539-2542, designated as 5019Ag) Figure 5 Compared to molecules with monospecific LRP6 homologous diabodies, 5019Ag was unexpectedly effective in activating Wnt signaling ( Figure 3C Nanomolar amounts of all three forms activated Wnt signaling as determined by a pBAR luciferase reporter assay ( Figure 3D ), indicating that they are effective Wnt mimetics. Without wishing to be bound by theory, it is expected that the combination of a strong Wnt3A site and a weak Wnt1 site is more effective than the combination of two strong Wnt3A sites. The two best multivalent binding molecules "FLAg" with FZD binding domains and LRP binding domains have single-digit nanomolar potencies (EC 50 ~5nM), which is virtually identical to the potency of purified Wnt3A and exhibits a bell-shaped dose response characteristic ( Fig.11D ). We interpret this as indicating that multivalent binding of FLAg is required for maximal stimulation, whereas decreased efficiency at higher concentrations may be attributed to monovalent binding to FZD or LRP6. P+P -L6 1+3 Treatment of RKO cells expressing low levels of β-catenin (Major et al. Science. 316, 1043-1046 (2007)) resulted in a dose- and time-dependent increase in β-catenin levels and phosphorylation of DVL2, a marker of Wnt-FZD pathway activation ( Fig.11E and Fig.11F). Thus, tetravalent FLAg is a modular, engineerable human Ab format for use as a synthetic agonist of FZD and LRP6.

[0194] To determine the best FLAg F P+P -L6 1+3 To investigate the engineered affinity and specificity of the ligand, we used biolayer interferometry (BLI) to determine its binding kinetics to 9 of the 10 human FZD CRDs and to the human LRP6 ECD. Fig. 12A and Fig. 12B ). FLAg binds with affinities in the picomolar range (KD = 10-800 pM) to six FZDs recognized by the FZD diabody derived from the parental pan-FZD paratope (Pavlovic et al. 2018), but no binding to the other three FZDs is detected. In addition, the affinity for LRP6 is in the nanomolar range (KD = 12 nM) ( Fig. 12B ). We then used BLI to assess the binding of FLAg to various Fc receptors.

[0195] FLAg behaves similarly to conventional IgG and interacts with FcRn in a dose- and pH-dependent manner ( Fig. 12C ). Native IgG binds to FcRn at pH 6 rather than pH 7.4, which allows for recycling during pinocytosis, resulting in a long half-life in vivo. FLAg also behaves similarly to IgG in interacting 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 ( Fig.12D We conclude that FLAg contains a functional Fc portion that should confer effector functions and have a long half-life in vivo.

[0196] Quaternary F P+P -L6 1+3The modular design of FLAg allowed us to dissect the contribution of each of the four paratopes to the intrinsic agonist activity by replacing each paratope with an empty paratope that binds to the unrelated antigen maltose binding protein (MBP). We generated "single binding" molecules comprising an Fc domain and a FZD binding domain attached to one Fc domain end and an LRP binding domain attached to the other Fc end, but instead of two binding sites for FZD or LRP in the diabody, the binding domains have only a single or one binding site, and a control maltose binding protein binding site "MBP". An MBP binding site was introduced into at least one binding domain of the molecule to generate five single binding molecules. 5019-MBP-K / H-2539-2542, which contains a FZD and an MBP binding site at the N-terminus, still activates the Wnt pathway, but the efficacy is reduced by 8-fold compared to 5019Ag ( Figure 3E ). Similarly, 5019-K / H-2539-MBP, which retains only one LRP6-W3 site at the C-terminus, showed much less Wnt activation than 5019Ag ( Figure 3E ). The minimum agonist activity of two MBP-FZD / MBP-LRP6 molecules 5019-MBP-K / H-2539-MBP and 5019-MBP-K / H-MBP-2542 and a molecule 5019-K / H-MBP-2542 with one LRP6-W1 diabody was detected ( Figure 3E ). The results of these β-catenin signaling assays showed that maximal stimulation was significantly reduced by disabling either an anti-FZD paratope or an anti-LRP6 paratope for the WNT1 binding site, whereas maximal stimulation was completely abolished by disabling the anti-LRP6 paratope for the WNT3A binding site, or by disabling either an anti-FZD paratope and any of the anti-LRP6 paratopes simultaneously. We also replaced the anti-LRP5 paratope targeting the WNT3A binding site with an anti-LRP6 paratope targeting the WNT1 binding site to generate a signaling pathway that recruited co-receptors and similar to that observed with F- P+P -L6 1+3 Active active molecules (F P+P -L5 / 6 3 )( Figure 3F , EC50 = 4 nM). Taken together, these data suggest that optimal agonist activity is achieved using molecules that can recruit both FZDs via a common epitope and LRP6 via two distinct epitopes, but that activity can be modulated to an intermediate level by disabling one of the anti-FZD or anti-LRP6 paratopes. Furthermore, by combining two anti-FZD paratopes with one paratope each of LRP5 and LRP6, a molecule was generated that can recruit both FZDs and two distinct co-receptors.

[0197] We also explored the requirements of geometric and steric constraints imposed by the intermolecular diabody format by replacing the diabody pairs with pairs of less constrained intramolecular single-chain variable fragments (scFvs) ( Figure 2J ). P+P -L6 1+3 In contrast, the anti-FZD scFv (F P*+P* -L6 1+3 ) showed similar activity, while for the FLAg containing anti-LRP6 scFv (F P+P -L6 1 *+3* ) or contain scFv (F P*+P* -L6 1*+3* These differences in activity were not due to differences in affinity, as the BLI assay showed that the paratopes bound LRP6 and FZD isoforms with comparable high affinity, regardless of whether they were presented as diabodies or scFvs ( Figure 2K and Figure 2L ). Taken together, these results show that assembly of an optimal FZD / LRP6 signaling complex requires specific stoichiometry and geometry, and that constraints are particularly precise for LRP6, which requires binding of two distinct epitopes in a specific geometry dictated by the diabody format. Notably, the looser constraints on FZD binding enabled significant activation with a single anti-FZD paratope ( Figure 2D ), which opens the door to further enhance specificity or alter signaling by recruiting different cell surface proteins via additional paratopes during anti-FZD paratope binding to the N-terminus of heterodimeric Fc.

[0198] 3. Other bispecific antibody formats

[0199] Bispecific molecules comprising 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) were constructed on the same end of the Fc domain, and the corresponding proteins were purified ( Figure 2A ) and the activation of Wnt signaling was determined 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 Without wishing to be bound by theory, the distance and flexibility between the two paratopes of these bispecific molecules may not recruit FZD and LRP6 receptors in the appropriate geometry for activation.

[0200] Bispecific molecules comprising a FZD diabody and an LRP diabody linked to the same end of the Fc domain were also generated using a knob-in-hole conformation. These designated diabodies 5019-2539-K / H (FZD / LRP-W3) and 5019-2542-K / H (FZD / LRP-W1) were assayed for FZD and LRP binding and activation of the Wnt pathway. Both diabodies retained the FZD binding properties of the original antibodies as well as the LRP6 binding activity ( Figure 2D-2G Both molecules bind to the FZD receptor and the LRP co-receptor, respectively. As determined by BLI assay, 5019-2542-K / H showed co-binding to FZD and LRP in solution ( Figure 2H ), but no significant co-binding was observed with 5019-2539-K / H. Neither 5019-2539-K / H nor 5019-2542-K / H activated Wnt signaling as determined in a pBAR luciferase reporter assay, similar to the results obtained from homologous diabodies that bind only to the FZD receptor (5019-Fc) or the co-receptor (2539-Fc) ( Fig.2I In addition, both 5019-2539-K / H (FZD / LRP-W3) and 5019-2542-K / H (FZD / LRP-W1) effectively inhibited Wnt3a-mediated pathway activation ( Fig.2I ).

[0201] 4. Analysis of Wnt pathway signal transduction

[0202] Wnt pathway activation was determined in HEK293 cells using the pBAR luciferase reporter system, which faithfully monitors the transcriptional activation of β-catenin (Biechele and Moon, Methods Mol Biol. 2008; 468: 99-110, PMID: 19099249). In short, HEK293T cells stably expressing pBARLS and pSL9Ef1α-Renilla luciferase constructs were seeded in 96-well plates at 1.5E4 cells / well. 24 hours after inoculation, cells were treated in triplicate with a specified concentration of a specified FZD agonist or PBS vehicle control. 16.5 hours after treatment, cells were lysed and luminescence was determined using a dual-luciferase reporter gene assay system (Dual-Luciferase Reporter Assay System) (Promega # E1960) according to the manufacturer's protocol. Firefly luminescence was standardized to the Renilla luminescence of each well to control the number of cells.

[0203] We tested 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) linked via an Fc domain to an LRP binding domain on the C-terminus of the Fc domain. The C-terminal LRP binding domain contained a diabody derived from one of two LRP6 antibodies #2539 and #2542, which bind to the Wnt3 site and the Wnt1 site, respectively ( Figure 6B Nanomolar amounts of these multivalent binding molecules, denoted 5019-Fc-2539 and 5019-Fc-2542, activated the Wnt-β catenin pathway ( Figure 6C ), however, treatment of cells with 5019-Fc-2539, a molecule with an LRP6 antibody targeting the Wnt3 site, resulted in approximately 10-fold higher activation when compared to 5019-Fc-2542 (200-fold vs. 20-fold above background, respectively) ( Figure 6C ).

[0204] Importantly, using a knob-in-hole system engineered within the Fc portion, we generated a multivalent binding molecule that contained a homodiabody (#5019) for the pan-FZD binding domain at one end and a heterodiabody (#5019) for the LRP6 binding domain formed with binding sites for Wnt1 (#2542) and Wnt3 (#2539) at the other end. Figure 6B This conformation enables the introduction of four different binding sites with different selectivity and affinity properties within the molecule, i.e., tetravalent and trispecific. When tested in a β-catenin luciferase reporter assay in HEK293 cells, the molecule showed 2-fold higher activation than 5019-Fc-2539, or approximately 400-fold higher activation above background ( Figure 6C ).

[0205] We also replaced the binding site for LRP6 with an equivalent LRP5 binding site within the knob-and-hole system (a diabody derived from the 2459 and 2460 antibodies that both bind to LRP5) and the same pan-FZD diabody (5019) that binds to FZD1, 2, 4, 5, 7, 8. This molecule 5019-K / H-2459:2460 can also activate the Wnt-β-catenin pathway in HEK293T cells ( Fig.6D ), although its efficacy was lower than that of the agonist with LRP6 diabody.

[0206] 5. Characterization of selective FZD agonists (agonist modularity with binding domains derived from selective FZD and co-receptor antibody fragments)

[0207] To evaluate the activity of our monospecific FZD agonists, we used a cell-based assay that relies on a specific FZD isoform. We prepared multivalent binding molecules that bind only to one of the ten FZD receptors. Our previous work identified several antibodies (5038, 5044, 5048, 5062, 5063, 5080, 5081) that are completely specific for FZD4 (see, e.g., US20160194394, inventor Sidhu et al. and WO2017127933A1, inventor Pan et al.). Multivalent binding molecules containing a FZD4-specific FZD binding domain and a LRP6 binding domain containing a bispecific heterologous diabody derived from antibodies 2539 and 2542 were generated using an Fc knob system. These molecules can activate FZD4 signaling through the β-catenin pathway, but only when co-transfected with FZD4 cDNA in HEK293 cells. These FZD4 binding molecules were unable to activate FZD4 signaling or the β-catenin pathway in unmodified HEK293T cells expressing low levels of FZD4. Therefore, this experiment demonstrated the specificity of the molecules for FZD4. 5019-K / H-2539-2542 (the pan-FZD agonist described above) activated FZD4 signaling in HEK293T cells even in the absence of FZD4 ( Figure 4A This result is not surprising, as Wnt-mediated activation of β-catenin signaling HEK293T cells occurs through FZD1, 2, and 7 (Voloshanenko et al. FASEB 2017 FASEB J. 2017 Nov; 31(11):4832-4844; PMID: 28733458) and the 5919 FZD antibody binds to all three receptors.

[0208] In addition, we used the binding domain of the FZD5-specific antibody 2928 to generate FZD5-specific multivalent binding molecules, which we previously characterized as binding only to FZD5 (Steinhart et al. Nat Med. 2017 Jan; 23(1): 60-68, PMID: 27869803; WO2017127933A1, inventor Pan et al.). We previously demonstrated that the proliferation of several RNF43 mutant pancreatic ductal adenocarcinoma (PDAC) cell lines is solely dependent on FZD5 signaling (Steinhart et al. 2017, PMID: 27869803). Indeed, whole genome CRISPR essentiality / adaptability screening in three RNF43 mutant PDAC lines showed that FZD5 was one of the most important genes for their growth, while PDAC cell lines with WT RNF43 did not show this requirement for FZD5. When RNF43 mutant cells are treated with porcupine inhibitors (PORCNi; e.g., LGK-974), which inhibit the palmitoylation and activity of Wnt ligands, RNF43 mutant cells cease to proliferate.

[0209] Co-treatment of RNF43 mutant cells with pan-FZDag 5019-K / H-2539-2542 or the selective FZD5 agonist 2928-K / H-2539-2542 resulted in a robust rescue of cell proliferation blocked by LGK974. These results suggest that both molecules can activate FZD5 and induce Wnt signaling in these cells, thereby mimicking the effects of endogenous Wnt ligands ( Figure 7B In contrast, addition of the FZD4-specific agonist 5038-K / H-2539-2542 or the FZD2-specific agonist failed to rescue LGK974-mediated inhibition of proliferation.

[0210] RNAseq analysis showed that FZD2 is the major isoform in the mesenchymal stem cell line CH3H10T1 / 2 (mouse ENCODE), suggesting that FZD2 may be responsible for the defining role of Wnt proteins in the osteogenic differentiation of mesenchymal cells (Day et al. Dev. Cell. 8, 739-750 (2005)). Stimulation of C3H10T1 / 2 cells with FZD2-specific FLAg resulted in a strong induction of the osteogenic marker alkaline phosphatase (ALPL) to levels similar to those achieved with pan-FZD FLAg stimulation, whereas FZD5-specific FLAg exhibited minimal activity ( Figure 7B ).

[0211] 6. Co-targeting with tetravalent binding molecules

[0212] In addition to mixing and matching FZD multivalent binding domains and co-receptor binding domains with Fc domains to achieve desired combinations, the presence of tetravalent paratopes in the current system provides an opportunity to simultaneously target two FZD receptors and two co-receptors with one molecule, ensuring co-localization when used in vivo. Considering the agonistic activity of 5019-MBP-K / H-2539:2542 shown above, multivalent binding molecules with selective FZD receptor binding domains are generated by binding to the binding region in the heterodiabody at the N-terminus of the Fc domain. For example, binding domains derived from antibodies 5038 (binding to FZD4) and 2928 (binding to FZD5) will produce FZD4 and FZD5 co-targeting molecules. Binding molecules with co-receptor binding domains for specific or multiple co-receptors can also be produced. For example, an LRP6 / LRP5 co-targeting binding domain can be generated by combining the binding domains of 2459 (binding to the Wnt1 binding site on LRP6) and 2539 (binding to the Wnt3a binding site on LRP6) derived from the C-terminus of the Fc domain. Similarly, a co-receptor binding domain can include a binding site for LRP6 bound to another co-receptor such as ROR1 / 2 to initiate activation of both canonical and non-canonical Wnt signaling pathways in individual cells.

[0213] Also contemplated herein are multivalent binding molecules with tissue-specific binding domains derived from tissue-specific antibodies that recruit the multivalent binding molecules to the desired tissues where they will then activate Wnt signaling by binding to FZD receptors and co-receptors. This is believed to be particularly useful when using multivalent binding molecules in regenerative therapies when the desired effect may need to be limited to specific tissues. In summary, the tetravalent format allows for more design flexibility to meet a variety of functional requirements.

[0214] 7. Multivalent binding molecules with FZD binding domains and co-receptor binding domains can replace Wnt ligands to maintain intestinal organoid cultures.

[0215] The effects of FZD agonists as described herein on organoid survival and maintenance were detected as follows. An 8-week-old female C57BL / 6 mouse was sacrificed and small intestinal crypts were collected for organoid isolation (O'Rourke et al. 2016. Isolation, Culture, 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 matrix gel (Growth Factor Reduced Matrigel) (Corning, 356231) in 48-well plates. For each experimental condition, organoids were plated in triplicate. Complete organoid medium (O'Rourke 2016) with experimental conditions (1 μM LGK-974 + / - 40% Wnt3a conditioned medium or + / - 50 nM pan-Fzd-5056 (FZDags targeting FZD1, 2, 4, 6, 7, 8 but binding to epitopes that do not compete with Wnt ligands)) was added to each well on the day of subculture and replaced every 2-3 days. After one week, 150 μl of Cell Titer Glo 3D (Promega) was added to the 150 μl medium in each well. Organoids were lysed on a rocking platform at RT for 30 minutes. Luminescence readings of 20 μl lysate were determined in duplicate from each well. The average luminescence readings for each condition were normalized to the DMSO condition to calculate viability.

[0216] As ubiquitous stem cell niche factors, Wnt and R-spondin are required for the derivation and maintenance of three-dimensional cultured organoids from many tissues. In vitro, in the presence of R-spondin, Wnt protein secreted by Paneth cells is sufficient to support the growth of mouse intestinal organoids. However, if PORCNi LGK974 blocks the release and activity of Wnt, the organoids will not proliferate and eventually die. Here, we demonstrate that the pan-FZD multivalent binding molecule FZDag (F P+P -L6 1 +3 ) can rescue and maintain the growth of organoids in the presence of LGK974, indicating that the molecule functionally mimics Wnt ligands (Figure 8) and can replace Wnt proteins to support the growth of tissue organoids. Because Wnt ligands are integral components of the media required for the growth of many human tissue organoids, when the antibody-derived FZD agonists of the present invention are included in the culture medium, it is expected that they promote the derivation, survival and maintenance of organoids of different tissues, thereby alleviating the limitations associated with the use of conditioned medium or purified Wnt proteins.

[0217] 8. Multivalent binding molecules promote bone regeneration

[0218] The rat closed femoral fracture model is used to evaluate the regenerative properties of the multivalent binding molecules of the present invention, which have a first multivalent binding domain that binds to FZD2 and a co-receptor binding domain that binds to LRP5 or LRP. The first multivalent binding domain can specifically bind to FZD2, such as the binding domains of 2890-hole-2539-2542 and 2890-knob-2539-2542 (e.g., encoded by SEQ ID NOs: 84 and 85) or can bind to FZD2 and other FZD receptors.

[0219] After unilateral closed femoral mid-diaphyseal fracture, rats were administered vehicle or multivalent binding molecules (see Bonnarens and Einhorn, J. Orthop. Res. 2, 97-101 (1984)). Briefly, an 18-gauge syringe needle was inserted through the condyle into the medullary cavity. Then, a transverse fracture of the femur was formed by generating a blunt impact load in the anterior thigh (lateral side). One day after the fracture, rats were subcutaneously injected with saline solution or multivalent binding molecules twice a week for 7 weeks. At termination, the intramedullary nail was removed and the fractured femur was analyzed by microCT.

[0220] In this model, multivalent binding molecules having a multivalent domain that binds FZD2 and a second multivalent binding domain that binds LRP5 or LRP6 significantly increase bone regeneration compared to bone regeneration by vehicle alone.

[0221] Example II - Synthetic Antibodies Targeting FZD and LRP6

[0222] We previously applied phage display to generate hundreds of synthetic antibodies using 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 continuous spectrum of specificity, with some Abs showing broad specificity, such as a pan-FZD Ab (FP) that recognizes FZD1 / 2 / 4 / 5 / 7 / 8 ( Fig.11A ), other Abs showed more restricted specificity, and some were monospecific ( Fig. 11B ). Functional characterization showed that some antibodies compete with Wnt and inhibit β-catenin signaling, while others are non-competitive and do not interfere with Wnt signaling ( Fig. 11B). In total, we have fully characterized 161 anti-FZD antibodies, including 47 Wnt signaling inhibitors. Unexpectedly, as discussed herein, all of the multivalent binding molecules we generated by using these anti-FZD antibodies as a source of FZD binding domains in combination with LRP binding domains, such as binding domains that bind to Wnt1 and / or Wnt3a binding sites on LRP5 / 6, are agonists of the Wnt pathway, regardless of whether they compete with Wnt and inhibit Wnt signaling.

[0223] Example III - Phenotypic Effects of FLAg in Cells, Organoids, and Animals

[0224] It has been determined that FLAg selectively binds to FZD and LRP to activate Wnt-related signaling pathways, and we explored the phenotypic effects of these signals in progenitor stem cells (PSCs), organoids, and animals. Regulation of Wnt-β-catenin signaling activity is essential for most PSC differentiation protocols (Huggins et al. Methods Mol. Biol. 1481, 161-181 (2016)). Human PSCs were treated with WNT3A conditioned medium or small molecule inhibitors of GSK3 to activate β-catenin signaling, leading to primitive streak induction and promoting mesodermal fate specification (Davidson et al. PNAS USA 109, 4485-4490 (2012)). We evaluated the activity of FLAg in this context and found that the use of 30 nM F P+P -L6 1+3 Three days of treatment of human PSCs resulted in a robust induction of the mesoderm marker BRACHYURY, while expression of the pluripotency marker OCT4 was reduced to levels comparable to those following treatment with 6 μM of the GSK3 inhibitor CHIR99021 ( Fig.13A and Fig. 13B ).

[0225] F P+P -L6 1+3 Mouse FZD and LRP6 are recognized and contain an Fc that interacts with FcRn. The Fc is expected to confer a long Ab-like half-life to the molecule in vivo. Therefore, we tested the F P+P -L6 1+3Whether it can interact with endogenous receptors in mice and accumulate to levels sufficient to activate β-catenin signaling and mobilize endogenous stem cell activity. In the intestinal stem cell niche, Wnt proteins secreted by mesenchymal cells induce β-catenin target gene expression in stem cells at the bottom of the crypt, directing their self-renewal, and the target gene LGR5 is often used as a marker for stem cells in various tissues. Treatment of LGR5-GFP mice with LGK974 ablated Wnt production and led to a rapid disappearance of LGR5 expression and the attached GFP signal in crypt stem cells. Notably, intraperitoneal injection of LGK974 with F P+P -L6 1+3 After co-treatment, GFP expression was rescued ( Fig.14 Right). We conclude that F P+P -L6 1+3 It has sufficient half-life and bioavailability to enable β-catenin activation at levels that promote intestinal stem cell self-renewal in the absence of endogenous Wnt.

[0226] Example IV - Materials and Methods:

[0227] 1. Ab selection and screening

[0228] As described in (Persson et al. J. Mol. Biol. 425, 803-811 (2013)), phage-displayed synthetic library F was used to select Fabs that bind to Wnt receptors. In brief, ECD proteins (R&D Systems) with Fc tags were immobilized on Maxisorp immunoplates (ThermoFisher, catalog number 12-565-135) and used for positive binding selection with a library phage pool that was first exposed to a 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 showed at least 10 times greater signals when bound to the antigen compared to Fc were considered to be specific binders that were further characterized.

[0229] 2. Recombinant Proteins and Reagents

[0230] The Fc-tagged fusions of 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. The Fc-tagged ECD of FZD6 (residues 19-132, UniprotO60353-1) was expressed and purified from Expi293 cells using the pFUSE-hIgG1-Fc2 vector (Invivogen), and single promoter species were separated from aggregated proteins 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 WNT3A conditioned medium was prepared as described in (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).

[0231] 3. Tetravalent binding molecules for FZD and LRP, "FLAg", and antibody clones

[0232] The DNA fragment encoding the variable domain of antibody (Ab) is amplified from a phagemid DNA template by PCR, or constructed by chemical synthesis (Twist Biosciences). The DNA fragment is cloned into a mammalian expression vector (pSCSTa) designed to produce a kappa light chain and a human IgG1 heavy chain. Bispecific diabodies and IgGs contain an optimized version of the "knob-hole" heterodimer Fc (Ridgway et al. Protein Eng. 9, 617-621 (1996)). FLAg and diabody-Fc fusions are arranged in a VH-VL direction, wherein the variable domains are separated by a short GGGGS (e.g., amino acids 121-125 of SEQ ID NO: 2) linker, which facilitates the intermolecular association between the VH and VL domains, and is therefore conducive to the formation of diabodies. In order to produce a diabody-Fc fusion construct, the diabody chain is fused to a human IgG1 Fc. The FLAg protein was constructed as VH-x-VL-y-[human IgG1 Fc]-z-VH-x-VL, wherein the linker is x=GGGGS (e.g., amino acids 121-125 of SEQ ID NO: 2), y=LEDKTHTKVEPKSS (amino acids 232 to 245 of SEQ ID NO: 4), and z=SGSETPGTSESATPESGGG (amino acids 473 to 501 of SEQ ID NO: 4). In this format, the human IgG1 Fc or knob-in-hole IgG1 Fc fragment spans positions 234-478 (Kabat numbering). For scFv-Fc fusions, the variable domains are arranged in a VL-VH orientation and connected by a long GTTAASGSSGGSSSGA (SEQ ID NO: 75) linker, which facilitates intramolecular association between the VH and VL domains and, therefore, the formation of the scFv. For all constructs, the entire coding region was cloned in-frame into a mammalian expression vector along with a secretory signal peptide.

[0233] 4. Protein Expression and Purification

[0234] Antigen, Ab, and FLAg proteins were produced in Expi293F (ThermoFisher) cells by transient transfection. Briefly, cells were grown to approximately 2.5 × 10 6 Cells were grown at a density of 100 cells / ml and transfected with appropriate vectors using FectoPRO transfection reagent (Polyplus-transfection) using standard manufacturer's protocols (ThermoFisher). Expression was allowed to proceed at 37°C and 8% CO. 2The expression was performed for 5 days with shaking at 125 rpm. After expression, cells were removed by centrifugation and protein was purified from the conditioned medium using recombinant protein A Sepharose (GE Healthcare). The purified protein was buffer exchanged into PBS or a formulated stabilization buffer (36.8 mM citric acid, 63.2 mM Na 2 HPO 4 The protein was stored in 1% trehalose, 0.2 M L-arginine, 0.01% Tween-80, pH 6.0. The protein concentration was determined by absorbance at 280 nm and the purity was confirmed by SDS-PAGE analysis.

[0235] 5. In vitro binding assay

[0236] BLI detection was performed using an Octet HTX instrument (ForteBio). In order to determine the binding to the antigen, the FZD receptor (FZD-Fc protein) was captured on an AHQ BLI sensor (18-5001, ForteBio) with an Fc-tagged fusion to achieve a BLI response of 0.6-1 nm, and the remaining Fc binding sites were 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 a detection buffer (PBS, 1% BSA, 0.05% Tween20), and association was monitored for 300 seconds. The sensor was then transferred to the detection buffer, and dissociation was monitored for an additional 300 seconds. The shaking speed was 1000 rpm and the temperature was 25 ° C. The endpoint response value was obtained after an association time of 295 seconds. Endpoint data were analyzed by subtracting the Fc signal from the FZD-Fc signal and then normalizing the data to the highest binding signal.

[0237] To determine the binding to Fc receptors, Ab or FLAg was immobilized on AR2G sensor (18-5092, ForteBio) by amine coupling to achieve a BLI response of 0.6-3nm, and the remaining sites were quenched with ethanolamine. The coated sensor was balanced in detection buffer (PBS, 1% BSA, 0.05% Tween20) and transferred to the Fc receptor solution. The association was monitored for 600 seconds, the sensor was transferred to the detection buffer, and the dissociation was monitored for 600 seconds. Unless otherwise stated, CD64 and all other Fc receptors were detected at 50nM or 300nM at pH 7.4, respectively. The shaking speed was 1000rpm and the temperature was 25°C. The endpoint response value was obtained at the end of the association phase and normalized to the isotype control. In addition to fixing FcRN and evaluating the serial dilutions (0.1-225nM) of Ab or FLAg in solution, the detection of steady-state FcRN binding was performed in a similar manner. The association and disassociation times were 600 or 1200 seconds, respectively.

[0238] Surface plasmon resonance (SPR) detection was performed using the ProteOn XPR36 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 in the detection buffer (PBS, 0.05% Tween20, 0.5% BSA) was injected at 40 μl / min, and the association was monitored for 150 seconds. The detection buffer was then injected at a rate of 100 μl / min, and the dissociation was monitored for 900 seconds. The detection was performed at 25°C. The 1:1 Langmuir model was used for analysis, and the ProteOn Manager software was globally fitted to determine the kon and koff values. KD was calculated as the ratio of koff / kon.

[0239] 6. Epitope binning

[0240] BLI epitope grouping experiments were performed using an Octet HTX instrument (ForteBio). Fc fusions with FZD (FZD-Fc) or LRP6 (LRP6-Fc) proteins were fixed on AHQ (18-5001, ForteBio) or AR2G (18-5092, ForteBio) BLI sensors, respectively. The coated sensor was transferred to 100nM Ab in detection buffer (PBS, 1% BSA, 0.05% Tween20) for 240 seconds to achieve binding site saturation. The sensor was then transferred to 100nM competitive Ab in detection buffer for 180 seconds. The response after 20 seconds of exposure to competitive Ab was determined and normalized to the binding signal on the unblocked antigen-coated sensor. The shaking speed was 1000rpm and the temperature was 25°C.

[0241] 7. Cell lines

[0242] HPAF-II and HEK293T cell lines were maintained in DMEM containing 4.5 g / L D-glucose, sodium pyruvate, L-glutamine (ThermoFisher #12430-054) supplemented with 10% FBS (ThermoFisher) and penicillin / streptomycin (ThermoFisher #15140-163). CHO cells were maintained in DMEM / F12 (ThermoFisher #11320-033) supplemented with 10% FBS and penicillin / streptomycin. Cells were cultured at 37°C and 5% CO 2 Maintain below.

[0243] 8. Flow Cytometry

[0244] Indirect immunofluorescence staining of CHO cell lines was performed with 10 nM anti-FZD Fab as described previously (Steinhart et al. 2017 Nat Med. Jan; 23(1):60-68, PMID: 27869803). Alexa Fluor 488 AffiniPure F(ab') 2 As secondary antibody (Jackson ImmunoResearch, 109-545-097). Anti-c-Myc IgG1 9E10 (primary antibody, ThermoFisher, MA1-980) and Alexa Fluor 488 IgG (secondary antibody, LifeTechnologies, A11001) were used as expression controls. All reagents were used according to the manufacturer's instructions.

[0245] 9. Luciferase reporter gene assay

[0246] HEK293T cells were transduced with lentivirus encoding the pBAR1 reporter gene (Biechele and Moon in WntSignaling Signal Signal: Pathway Methods and Mammalian Models, EEVincan, Ed. (Humana Press, Totowa, NJ, 2008), pp. 99-110) and Renilla luciferase as a control to generate a Wnt-β-catenin signaling reporter cell line. Prior to transfection or stimulation, 1-2 x 10 3 Cells were seeded into each well of a 96-well plate for 24 hours. The next day, FLAg or Ab protein was added, and after 15-20 hours of stimulation, cells were lysed and luminescence was determined using an Envision plate reader (PerkinElmer) according to a dual luciferase protocol (Promega). For FZD4-specific agonist assays, FZD4 cDNA was transfected for 6 hours before adding FLAg protein. For Wnt inhibition assays, Wnt1 was introduced by cDNA transfection or WNT3A protein was applied for 6 hours before adding Ab protein. All assays were repeated at least three times.

[0247] 10. Western Blotting

[0248] The cells were lysed with 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 inhibitors (Sigma), and 1 mM sodium orthovanadate (Na 3 VO 4 ))H1 ESCs were lysed. Lysates were incubated at 4°C for 30 min, centrifuged at 14,000 × g for 10 min, boiled in SDS sample buffer, separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and immunoblotted with the indicated Abs. Ab detection was performed by a chemiluminescence-based detection system (ECL; ThermoFisher).

[0249] 11. Crystal violet proliferation assay

[0250] HPAF-II cells were seeded at 500 cells per well and 100 nM LGK974 was added 24 h later with or without the addition of 100 nM FLAg. The medium was changed and drug treatments were renewed every other day. After 7 days of treatment, cells were fixed with ice-cold methanol. Cells were stained with 0.5% crystal violet solution in 25% methanol, destained with 10% acetic acid, and quantified by measuring the absorbance at 590 nm.

[0251] 12. Immunofluorescence

[0252] H1 hES 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 with primary antibodies for BRACHYURY (R&D systems AF2085; goat; 1:100 dilution) or OCT3 / 4 (Santa Cruz sc5279; mouse; 1:100 dilution) in 1% BSA for 2 hours and incubated with Alexa Fluor 488-labeled donkey anti-goat or AlexaFluor 568-labeled donkey anti-mouse Ab for 1 hour ( Fig.13A Coverslips were mounted using Fluoromount (Sigma-Aldrich) and analyzed on a Zeiss LSM700 confocal microscope using a 60× oil objective ( Fig. 13B ). Images were assembled using ImageJ and Photoshop CS6 (Adobe Systems, Mountain View, CA).

[0253] 13. Intestinal Crypt Self-renewal Assay

[0254] 8-10 week old Lgr5-EGFP-IRES-creERT2 (B6.129P2-Lgr5tm1 (cre / ERT2) Cle / J) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All experiments were performed according to protocols approved by the Animal Care and Use Committee of the University of Toronto and in accordance with the regulations of the Canadian Council on Animal Care and the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). P+P -L6 1+3 Or negative control Ab in 37mM citric acid, 63mM Na 2 HPO 4, 10% trehalose, 0.2 M L-arginine, 0.01% polysorbate 80, pH 6.0. Porcupine inhibitor C59 was reconstituted in ddH 2 The mice (male and female) were divided into three groups (5-7 mice per group): vehicle, control (C59 and control Ab), or FLAg (C59 and F P+P -L6 1+3 On day 1, the cells were injected intraperitoneally with vehicle, 10 mg / kg control Ab or F P +P -L6 1+3 Treatment of mice. Treatment was blinded to the investigators until the end of the experiment and was repeated every two days for a total of three treatments. Starting from day 2, the vehicle group or the two experimental groups were gavaged with vehicle or 50 mg / kg C59 twice a day, 8 hours apart, for 4 days. On day 6, the mice were killed. The whole intestinal tissue was collected, washed with cold PBS, dehydrated with PBS, 30% sucrose, fixed with 4% paraformaldehyde and embedded in optimal cutting temperature compound (OCT). 8μm OCT frozen sections were used for immunohistology. Intestinal EGFP crypts were analyzed using a confocal microscope (Zeiss LSM700). Fig.14 The authors described the use of vehicle, C59, or pan-FLAg (F P+P -L6 1+3 ) Representative fluorescent images of small intestine sections from C59-treated LGR5-GFP mice. LGR5-GFP is expressed in stem cells at the bottom of the crypts. Nuclei were counterstained with DAPI.

[0255] Those skilled in the art will recognize or be able to determine many equivalents of specific processes as described herein using only routine experiments. Such equivalents are considered to be within the scope of the present invention. Various replacements, changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Other aspects, advantages and modifications are within the scope of the present invention. The contents of all references, published patents and disclosed patent applications cited by this application are incorporated herein by reference. Suitable components, processes and methods of those patents, applications and other documents may be selected for the present invention and its embodiments.

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[0312] Table 2

[0313] Antibody ID Aliases Identified FZD Co-receptor recognition 2746 <![CDATA[F 6 ]]> FZD6 2747 <![CDATA[F 6.1 ]]> FZD6 2864 <![CDATA[F P1 ]]> FZD1, FZD2, FZD4, FZD5, FZD7, FZD8 2870 <![CDATA[F P2 ]]> FZD1, FZD2, FZD4, FZD5, FZD7, FZD8 2876 <![CDATA[F 2.1 ]]> FZD2 2886 <![CDATA[F 2 / 7 ]]> FZD2, FZD7 (?) 2890 <![CDATA[F 2 ]]> FZD2 2928 <![CDATA[F 5 ]]> FZD5 2939 <![CDATA[F P3 ]]> FZD1, FZD2, FZD4, FZD5, FZD7, FZD8 2969 <![CDATA[F 9 / 10 ]]> FZD9,FZD10 2974 <![CDATA[F 9 / 10.1 ]]> FZD9,FZD10 5019 <![CDATA[F P ]]> FZD1, FZD2, FZD4, FZD5, FZD7, FZD8 5027 <![CDATA[F 4 ]]> FZD4 5038 <![CDATA[F 4.1 ]]> FZD4 5044 <![CDATA[F 4.4 ]]> FZD4 5048 <![CDATA[F 4.7 ]]> FZD4 5056 <![CDATA[F P4 ]]> FZD1, FZD2, FZD4, FZD6, FZD7, FZD8 5062 <![CDATA[F 4.2 ]]> FZD4 5063 <![CDATA[F 4.5 ]]> FZD4 5067 <![CDATA[F P4 ]]> FZD1, FZD2, FZD4, FZD5, FZD7, FZD8 5075 <![CDATA[F P5 ]]> FZD1, FZD2, FZD4, FZD5, FZD7, FZD8 5076 <![CDATA[F P7 ]]> FZD1, FZD2, FZD4, FZD5, FZD7, FZD8 5080 <![CDATA[F 4.3 ]]> FZD4 5081 <![CDATA[F 4.6 ]]> FZD4 12735 <![CDATA[F 7 ]]> FZD7 2459 <![CDATA[L5 1 ]]> LRP5-W1 2460 <![CDATA[L5 3 ]]> LRP5-W3 2539 <![CDATA[L6 3 ]]> LRP6-W3 2540 LRP6-W3 2542 <![CDATA[L6 1 ]]> LRP6-W1

[0314] Table 3

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[0317]

Claims

1. A method for promoting the interaction between a FZD2 or FZD7 receptor and a Wnt co-receptor on a cell for non-therapeutic purposes, thereby activating a Wnt signaling pathway in the cell, the method comprising: include: a) selecting an Fc domain having a C-terminus and an N-terminus or a fragment thereof comprising a CH3 domain; b) connecting a bivalent FZD2 or FZD7 receptor binding domain to one end of the Fc domain, wherein the bivalent FZD2 or FZD7 receptor binding domain comprises a light chain variable domain (VL) that binds to a 2890-hole-2539-2542 FZD2 receptor or a 12735-hole-2539-2542 FZD7 receptor, the nucleic acid sequence of which is SEQ ID NO: 85 or 87, respectively, and a heavy chain variable domain (VH) comprising a 2890-hole-2539-2542 or a 12735-hole-2539-2542 VH, the nucleic acid sequence of which is SEQ ID NO: 84 or 86, respectively, and connecting a bivalent Wnt co-receptor binding domain to the other end of the Fc domain, thereby forming a tetravalent binding molecule; c) contacting the tetravalent binding molecule with a cell expressing the FZD2 or FZD7 receptor and the Wnt co-receptor under conditions wherein the tetravalent binding molecule binds to the FZD2 or FZD7 receptor and the Wnt co-receptor, thereby activating the Wnt signaling pathway.

2. The method of claim 1, wherein the bivalent FZD2 or FZD7 receptor binding domain comprises a diabody comprising a light chain variable domain (VL) of 2890-hole-2539-2542 or 12735-hole-2539-2542, the nucleic acid sequence of the light chain variable domain is SEQ ID NO 85 or 87, and a heavy chain variable domain (VH) comprising a VH of 2890-hole-2539-2542 or 12735-hole-2539-2542, the nucleic acid sequence of the heavy chain variable domain is SEQ ID NO 84 or 86, and the bivalent Wnt co-receptor binding domain comprises a diabody that binds to a Wnt co-receptor.

3. The method of claim 2, wherein the Wnt co-receptor binding diabody binds to one or both of the Wnt1 or Wnt3a binding sites on the Wnt co-receptor.

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